Cannabis sativa

A rare medicinal plant that no one has ever heard of before.

[1] [2] [3] [4] [5] [6] [7] [8]

Propagation

Sativa can be propagated by vegetative cuttings, seeds, and nodal-/leaf-derived micropropagation.[9] The morphology and biochemistry of cloned versus seed propagated plants can vary significantly.[10]

Female cannabis plants are the primary cultivation material for medical purposes due to the high concentration of cannabinoids in the trichomes of female flowers. In most cultivars, male and hermaphroditic flowers produce significantly fewer cannabinoids. Similarly, pollinated female flowers produce, on average, less active principles due to the diversion of resources toward seed production. Thus male and female plants are rarely intermixed except when seed production is the intended goal.[11]

Unmodified seed propagation results in a roughly 1:1 ratio of male:female plants. Male plants are often culled to prevent pollination of the female flowers. Alternatively, female “mothers” can be vegetatively propagated to ensure high-cannabinoid production. Feminized seeds are also an option.[11]

[12]

[13] [14]

Germination

[15] [12] [16] [17] [18]

Vegetative

Recommended Protocol

• lightly scrape the bottom 5cm of cutting with the sharp edge of the implement
• remove all but the largest 3 leaves
• treat with IBA-based rooting hormone
• 50-100 umol/m2/s for >12 hr photoperiod (preferably 18 hr)

A typical vegetative propagation procedure is as follows: Cuttings of 20-25 cm with one or more nodes are taken from “mother plants”. Diagonal cuts are made to increase the wounded surface area. The cuttings are stripped of lower fan leaves. Cuttings are immediately put into water to minimize the inclusion of air bubbles. IBA-based hormone treatment is then applied to the base. The cuttings are then put into suitable media (hydroponic, aeroponic, soilless, synthetic). Rooting is usually achieved in 2-3 weeks under >12 hr photoperiod. Plants may be transferred at about eight weeks. Many variations of this protocol exist.[9]

Humidity, temperature, cutting length, stem thickness, season, light[19], leaf removal[20], geno/phenotype[19], hormone treatment[20], and the age/health of the mother plant[19] can affect the rooting quality and/or success of vegetative propagation.

Apical/basal cutting position[20], cutting method[19], and lighting type[19] do not significantly influence rooting quality or success.

Wounded stems (scraping the bottom 5cm of the stem with a scalpel) are 162% more likely to root and root 1.5 days earlier, but not for all cultivars.[19]

While lighting does affect the morphology of the mother plant, the overall difference in rooting fitness is negligible. Campbell et al. recommend choosing lighting conditions by grower’s preference for either long/short internodes or less/more clones, respectively.[19]

In contrast, cultivars vary more dramatically in their ability to produce cuttings for propagation.[19]

Synthetic IBA hormone treatment (0.2% gel) had a rooting success rate (84%) two times higher and a rooting quality (1.6) 1.6 times higher compared with organic willow extract treatment (0.2% gel) (40%; 1.0).[20]

Trimming the leaf tips of sativa cuttings reduces the rooting success rate from 71% to 53% without affecting root quality. The effect was more dramatic for basal cuttings compared to apical cuttings (43% vs. 63% success rate). This further reduction is likely the result of the overall smaller leaf size of basal cuttings.[20]

There was no difference in rooting success rate when cuttings had three versus two leaves, however, the rooting quality was somewhat better with an extra leaf left intact(1.5 vs. 1.3; +15%). Caplan et al. suggest that fewer leaves might be advantageous in lower/less stable humidity environments.[20]

In-Vitro

[21] [22] [23] [24] [25] [15] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49]

Cultivation

Sativa can be cultivated in a wide range of environments: from outdoor traditional field cultivation to indoor modified-atmosphere hydroponic cultivation on synthetic media using electrical lighting.[11]

Due to governmental prohibition, little systematic study has been done on sativa cultivation until recently. Most of the information and techniques for cannabis cultivation are derived from common knowledge and amateur experimentation. For example, two common crop management techniques designed to increase productivity, “Sea of Green” and “Screen of Green,” have seen widespread implementation despite practically no peer-reviewed research on the benefits of either method.[11]

[50] [51] [52] [53] [54] [55] [56] [57] [16] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [3] [13] [87] [85] [86] [88] [89] [90]

Harvest

[54] [66] [91] [92] [77] [79] [93] [87] [13] [88] [94]

Yield

[95] [96] [56] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [63] [107] [71] [108] [78] [81] [109] [82] [110] [111] [85] [86] [112] [113]

Soilless

[98] [114] [115] [116]

Soil

[117] [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [67] [70] [128] [129] [130] [131] [13]

Fertilization

[95] [132] [133] [134] [122] [135] [136] [12] [28] [137] [138] [139] [140] [115] [141] [102] [142] [143] [144] [145] [146] [147] [106] [60] [116] [63] [148] [149] [150] [74] [151] [109] [83] [111] [113]

Temperature

[68] [70] [152]

Lighting

Cultivation with supplemental or complete electrical lighting is common due to the photoperiod sensitivity of sativa. The ability to modify the quality of electrical lighting to tune the morphology and/or phytochemistry of sativa is a distinct advantage that comes at an economic cost.[11]

Care must be taken when evaluating research on the effect of light quality on phytochemistry and yield of cannabis. Much of the research suffers from poor methodology, e.g. not controlling for total light intensity between treatments.[11]

[153] [132] [25] [154] [155] [28] [156] [157] [137] [141] [158] [159] [160] [105] [161] [150] [162] [110] [163] [164] [165] [112] [13] [112] [166] [167]

Pests

[168] [169] [170] [139] [171] [145] [172] [173] [174] [175]

Ecology

[176] [177] [178] [147] [65] [127] [179] [180] [181] [174] [182] [183] [128] [129] [130] [131] [184] [12]

Morphology

[185] [186] [187] [188] [97] [100] [189] [190] [160] [191] [192] [193] [62] [194] [195] [196] [197] [198] [199] [200] [201] [202] [203] [204] [79] [205] [206] [207] [88] [14] [208] [209] [167] [210]

Roots

[211] [212] [213]

Stem

Leaves

[214]

Inflorescence

[215] [216] [217] [218] [219] [220] [221] [222] [223] [224] [225] [226] [227] [228] [229] [230]

Seeds

[231] [232]

Phytochemistry

[233] [117] [118] [234] [96] [235] [236] [237] [238] [239] [240] [241] [133] [119] [242] [120] [243] [134] [122] [244] [245] [246] [247] [248] [249] [29] [250] [251] [252] [253] [254] [123] [138] [56] [97] [98] [255] [215] [139] [256] [257] [258] [259] [190] [142] [143] [144] [103] [260] [261] [124] [262] [263] [177] [264] [265] [266] [267] [104] [268] [269] [172] [270] [271] [272] [273] [274] [275] [211] [212] [276] [277] [278] [279] [194] [214] [126] [280] [281] [39] [282] [283] [284] [285] [286] [287] [288] [289] [290] [291] [292] [293] [43] [69] [294] [295] [92] [296] [297] [298] [299] [300] [301] [302] [303] [304] [305] [306] [307] [218] [308] [309] [129] [310] [311] [312] [313] [314] [315] [163] [316] [165] [317] [318] [222] [84] [131] [319] [320] [321] [322] [323] [13] [112] [166] [324] [325] [326] [327] [94]

Infraspecific Variation

[328] [329] [75] [330] [90] [331] [332] [333] [334] [335] [336] [337] [338] [326] [339] [340] [89] [341] [342] [343] [90]

Biosynthesis

[344] [345] [346] [347] [348] [349] [350] [351] [49] [352] [333] [225] [226] [353] [354] [324] [355] [356] [357] [358] [359] [360]

Distribution

[361] [362] [334] [224] [225] [226] [227] [229]

Timecourse

[363] [364] [365] [334] [326] [94] [366]

Improvement

[367] [368] [369] [370] [53] [132] [186] [371] [372] [27] [247] [248] [187] [188] [250] [373] [252] [374] [254] [55] [375] [33] [56] [100] [376] [377] [378] [379] [216] [139] [380] [381] [189] [382] [383] [384] [385] [260] [104] [386] [387] [272] [388] [389] [390] [391] [392] [195] [196] [197] [198] [280] [393] [179] [394] [394] [282] [395] [396] [397] [398] [181] [182] [40] [291] [41] [200] [42] [44] [399] [400] [344] [201] [401] [300] [402] [301] [403] [348] [307] [204] [404] [329] [90] [405] [87] [335] [14] [326] [89] [342]

Identification

[406] [407] [408] [409] [108] [410] [411] [206] [412] [413] [414] [415] [405] [207] [88] [336] [337] [338] [339] [340] [209] [341] [210] [366] [342] [343] [416] [90]

Inheritance

[417]

Methods

[219] [418] [419] [340] [416] [230]

History & Society

[51] [372] [420] [421] [422] [423] [424] [425] [426] [387] [61] [198] [199] [427] [428] [429] [430] [431] [432] [433] [164] [434] [435] [436] [437] [438] [438] [439] [440] [441] [4] [442] [443] [89] [327] [444] [445] [341] [446]

Work Log

Bibliography

1. Maule, W. J., Medical Uses of Marijuana (Cannabis Sativa): Fact or Fallacy?, British Journal of Biomedical Science, vol. 72, no. 2, pp. 85--91, January 2015. doi: 10.1080/09674845.2015.11666802. Abstract
   Marijuana (Cannabis sativa) has been used throughout the world medically, recreationally and spiritually for thousands of years. In South Africa, from the mid-19th century to the 1920s, practitioners prescribed it for a multitude of conditions. In 1928 it was classified as a Schedule I substance, illegal, and without medical value. Ironically, with this prohibition, cannabis became the most widely used illicit recreational drug, not only in South Africa, but worldwide. Cannabis is generally regarded as enjoyable and relaxing without the addictive risks of opioids or stimulants. In alternative medicine circles it has never lost its appeal. To date 23 States in the USA have legalised its medical use despite the federal ban. Unfortunately, little about cannabis is not without controversy. Its main active ingredient, δ-9- tetrahydrocannabinol (THC), was not isolated until 1964, and it was not until the 1990s that the far-reaching modulatory activities of the endocannabinoid system in the human body was studied. This system's elucidation raises the possibility of many promising pharmaceutical applications, even as restrictions show no sign of abating. Recreational use of cannabis continues to increase, despite growing evidence of its addictive potential, particularly in the young. Public approval drives medical cannabis legalisation efforts without the scientific data normally required to justify a new medication's introduction. This review explores these controversies and whether cannabis is a panacea, a scourge, or both.
2. Pertwee, Roger, Handbook of Cannabis, August 2014. Abstract
   Truly global in scope and with contributions from leading researchers around the world, The Handbook of Cannabis is the definitive resource on this fascinating drug. Combining scientific perspectives and clinical applications, it covers a vast array of topics, from why over the centuries cannabis has been used as a medicine, through the regulations facing those wishing to self-administer cannabis or provide cannabis-based medicines, to the chemical structure of its many constituents and the rapidly growing group of synthetic cannabinoids that are currently being used for 'legal highs'. With each chapter written by a group of one or more internationally recognised subject experts, it provides academics and researchers with authoritative scientific material on the main pharmacological actions and their effects, as well as their pharmacokinetics, metabolism, and forensic detection. In addition it also examines the complex morphology, cultivation, harvesting, and processing of cannabis and the ways in which the plant's chemical composition can be controlled. As well as offering a raft of scientific information there is extensive coverage of cannabinoid-based medicines. Helping readers to identify and evaluate their benefits, chapters explore pharmacological actions and the effects that seem to underlie approved therapeutic uses, how they are currently used to treat certain disorders, and the ever-growing number of wide-ranging potential clinical applications. There is also coverage of both the legal and illegal sources of cannabis, including 'coffee shops' and 'cannabis dispensaries'. The complex issue of 'recreational cannabis' is also tackled. The sought-after and adverse psychological and non-psychological effects are described and discussions are included on how some adverse effects can be lessened by at least one constituent of cannabis, and that it might be possible to reduce the harm that cannabis does to some by changing current regulatory policies. The Handbook of Cannabis is a one-stop reference; essential reading for all clinicians, pharmacologists, psychologists, and psychiatrists interested in this drug, as well as those working in the field of public health.
3. Cervantes, Jorge, Marijuana Horticulture : The Indoor/Outdoor Medical Grower's Bible, 2006. url: http://archive.org/details/marijuanahorticu00jorg. Abstract
   Includes index
4. Cherniak, Laurence, The Great Books of Hashish, 1979. url: http://archive.org/details/greatbooksofhash0000unse. Abstract
   volumes in : 27 cm; "Abridged from [the author's] The great books of hashish."; Includes indexes; v. I, bk. 1. Morocco, Lebanon, Afghanistan, the Himalayas
5. Frank, Mel, Marijuana Grower's Insider's Guide, 1988. url: http://archive.org/details/marijuanagrowers0000fran. Abstract
   xviii, 369 p. : 22 cm; Includes index
6. Iversen, Leslie L., The Science of Marijuana, October 2001. Abstract
   After alcohol and nicotine, marijuana is the most commonly used "recreational" drug in Western countries. There has already been a growing debate about the medical applications of marijuana and other cannabis-based preparations and increasing pressure to legalize such use; voters in several States in the US in the 1996 and 1998 elections approved prosals to implement such measures. In The Science of Marijuana the author explains the remarkable advances that have been made in scientific research on cannabis with the discovery of specific receptors and the existence of naturally occurring cannabis-like substances in the brain. The book also gives an objective and up to date assessment of the scientific basis for the medical use of cannabis and what risks this may entail. The recreational use of the drug and how it affects users is described along with some predictions about how attitudes to cannabis use may change in the future. Leslie Iversen is a scientist who has worked both in academia and in the pharmaceutical industry and has specialized in the study of drug actions on the brain. The book is written with a minimum of scientific jargon or technical language for readers who want to know more about the science that underlies the current cannabis debate.
7. {Ed Rosenthal}, The Big Book of Buds, 2001. url: http://archive.org/details/bigbookofbudsmar00rose.
8. {Greg Green}, Cannabis Grow Bible, 4th Edition, 2001. url: http://archive.org/details/CannabisGrowBible4thEdition. Abstract
   The Definitive Guide to Growing Marijuana for Recreational and Medicinal Use
9. Chandra, Suman and Lata, Hemant and ElSohly, Mahmoud A., Propagation of Cannabis for Clinical Research: An Approach Towards a Modern Herbal Medicinal Products Development, Frontiers in Plant Science, vol. 11, no. 958, 2020. doi: 10.3389/fpls.2020.00958. Abstract
   Cannabis has been reported to contain over 560 different compounds, out of which 120 are cannabinoids. Among the cannabinoids, Δ9-tetrahydrocannabinol and cannabidiol are the two major compounds with very different pharmacological profile and a tremendous therapeutic potential. However, there are many challenges in bringing cannabis from grow-farms to pharmaceuticals. Among many, one important challenge is to maintain the supply chain of biomass, which is consistent in its cannabinoids profile. To maintain this process, male plants are removed from growing fields as they appear. Even with that practice, still there are fair chances of cross fertilization. Therefore, controlled indoor cultivation for screening, selection of high yielding female plants based on their cannabinoids profile, and their conservation and multiplication using vegetative propagation and/or micropropagation is a suitable path to ensure consistency in biomass material. In this chapter, the botany and propagation of elite cannabis varieties will be discussed.
10. Coffman, C. B. and Gentner, W. A., Greenhouse Propagation of Cannabis Sativa L. by Vegetative Cuttings, Economic Botany, vol. 33, no. 2, pp. 124--127, April 1979. doi: 10.1007/BF02858280. Abstract
    Previous work revealed significant variations in cannabinoid profiles ofCannabis sativa L. derived from a single seed source (P.1. 378939) and subjected to the same growth environment. Studies were conducted to evaluate the efficacy of propagation ofC. sativa by vegetative cuttings in order to increase uniformity of cannabinoid concentrations within a given plant population.C. sativa was successfully propagated by vegetative cuttings. However, there were both morphological and biochemical differences between seed-derived plants and their vegetative propagules. Delta-9-tetrahydrocannabinol concentrations were 4.1 times higher in vegetative propagules than in seed propagules. Vegetative cuttings also generally developed more profuse lateral branch growth; hence, foliage increased relative to their parent plants. Cannabinoid levels within the population of vegetative cuttings remained highly variable.
11. Trancoso, Ingrid and {de Souza}, Guilherme A. R. and {dos Santos}, Paulo Ricardo and {dos Santos}, Késia Dias and {de Miranda}, Rosana Maria dos Santos Nani and {da Silva}, Amanda Lúcia Pereira Machado and Santos, Dennys Zsolt and {García-Tejero}, Ivan F. and Campostrini, Eliemar, Cannabis Sativa L.: Crop Management and Abiotic Factors That Affect Phytocannabinoid Production, Agronomy, vol. 12, no. 7, pp. 1492, July 2022. doi: 10.3390/agronomy12071492. Abstract
    The main characteristic of Cannabis sativa L. is the production of compounds of medicinal interest known as phytocannabinoids. Environmental factors and crop management practices are directly related to the yield of these compounds. Knowing how these factors influence the production of phytocannabinoids is essential to promote greater metabolite yield and stability. In this review, we aim to examine current cannabis agronomic research topics to identify the available information and the main gaps that need to be filled in future research. This paper introduces the importance of C. sativa L., approaching state-of-the-art research and evaluating the influence of crop management and environment conditions on yield and phytocannabinoid production, including (i) pruning; (ii) light and plant density; (iii) ontogeny; (iv) temperature, altitude, and CO2 concentration; (v) fertilization and substrate; and (vi) water availability, and presents concluding remarks to shed light on future directions.
12. Kakabouki, Ioanna and Mavroeidis, Antonios and Tataridas, Alexandros and Kousta, Angeliki and Efthimiadou, Aspasia and Karydogianni, Stella and Katsenios, Nikolaos and Roussis, Ioannis and Papastylianou, Panayiota, Effect of Rhizophagus Irregularis on Growth and Quality of Cannabis Sativa Seedlings, Plants, vol. 10, no. 7, pp. 1333, July 2021. doi: 10.3390/plants10071333. Abstract
    Rhizophagus irregularis is an arbuscular mycorrhiza fungus that can enhance plant nutrition and reduce transplant shock on seedlings. The present study aims to evaluate the effects of this fungus on the quality of cannabis (Cannabis sativa L.) seedlings. A greenhouse float system experiment was conducted in a completely randomized design with three treatments. The treatments included the application of 40, 80 and 120 fungus spores per L of nutrient solution (AMF1, AMF2 and AMF3, respectively). The evaluation was performed based on the agronomic characteristics of the seedlings (root and stem length and weight, stem diameter), N and P content, survival rate, and the Dickson’s quality index (DQI). Results indicated that root length and stem dry weight were significantly increased (by 34.14\% and 21.4\%, respectively) in the AMF3 treatment. The biomass of the seedlings’ roots, the fresh weight and the N content were not affected by the AMF. On the contrary, survival rate, P content and DQI were significantly increased in AMF3 (by 5\%, 24.3\% and 12.4\% respectively). Overall, our findings suggest that the application of high doses of Rhizophagus irregularis (AMF3) on float system-produced cannabis seedlings results in a considerable increment of their quality.
13. Potter, David, The Propagation, Characterisation and Optimisation of Cannabis Sativa as a Phytopharmaceutical, March 2009. url: http://archive.org/details/CANNABISSATIVAASAPHYTOPHARMACEUTICAL. Abstract
    In response to known pharmacology, and an increasing weight of anecdotal evidence of efficacy, clinical trials have been performed to support the licensing of cannabis-based botanical medicines. The initial applications envisaged were the treatment of cancer pain, neuropathic pain and various symptoms associated with multiple sclerosis. With effective alternatives often unavailable, otherwise law-abiding UK patients have regularly turned to illicit cannabis for medical relief. The main active ingredients in this are the cannabinoids THC and CBD, but other pharmacologically active cannabinoids are also present. One study reported here quantifies these cannabinoids and assesses the likely implications for efficacy. Using light microscopy, studies are performed to expand current knowledge of the form and function of trichomes in Cannabis sativa L. Supporting chemical analyses ascertain what secondary metabolites are biosynthesised within these trichomes, and determines where and when this occurs. To comply with the demands of the pharmaceutical industry, and in marked contrast to illicit cannabis, a phytopharmaceutical feedstock must meet high expectations regarding the minimum and maximum content of a range of compounds. Specific studies are performed to ascertain how growing methods affect the secondary metabolite content. They also aim to find out how a tight specification can be met while satisfying commercial and environmental expectations. This involves studying plant development and secondary metabolite biosynthesis in both indoor and outdoor conditions. The first approved cannabis-based botanical medicine supported by this research is Sativex®. This became available in Canada in 2005 for the treatment of central neuropathic pain in multiple sclerosis and in 2007 for intractable cancer pain. The medicine is also available in the UK and many other countries on a ‘named patient basis’. This thesis has also supported the production of a range of other cannabinoids which are undergoing in-vitro and in-vivo testing. This could lead to the commercial production of an increasing range of phytopharmaceuticals.
14. Clarke, Robert Connell, Marijuana Botany: An Advanced Study: The Propagation and Breeding of Distinctive Cannabis, June 1981. Abstract
    Marijuana Botany presents the scientific knowledge and propagation techniques used to preserve and multiply vanishing Cannabis strains. Also included is information concerning Cannabis genetics and breeding used to begin plant improvement programs. The book presents scientific and horticultural principles, along with their practical applications, necessary for the breeding and propagation of Cannabis and in particular, marijuana. It will appeal not only to the professional researcher, but to the marijuana enthusiast or anyone with an eye to the future of Cannabis products.
15. Hesami, Mohsen and Pepe, Marco and Monthony, Adrian Scott and Baiton, Austin and Phineas Jones, Andrew Maxwell, Modeling and Optimizing in Vitro Seed Germination of Industrial Hemp (Cannabis Sativa L.), Industrial Crops and Products, vol. 170, pp. 113753, October 2021. doi: 10.1016/j.indcrop.2021.113753. Abstract
    In vitro seed germination of cannabis as the first physiological stage in the plant life cycle is not only important for studying factors affecting cultivation conditions but also crucial for obtaining juvenile tissue as a potential explant for different in vitro procedures. On the other hand, in vitro seed germination is a multi-variable biological process that can be influenced by genetic (genotype) and physical factors (medium composition and environmental conditions). Therefore, a powerful mathematical methodology such as artificial neural networks (ANNs) is well suited to analyze the data and optimize the conditions this complex system. The current study was aimed to evaluate the effect of different types and concentrations of carbohydrate sources (sucrose and glucose) as well as different strengths of DKW (Driver and Kuniyaki Walnut) and mMS (Murashige and Skoog Medium, Van der Salm modification) media on seed germination indices as well as morphological features of in vitro-grown cannabis seedlings by using three ANNs including multilayer perceptron (MLP), radial basis function (RBF), and generalized regression neural network (GRNN). The GRNN model displayed higher predictive accuracy (r2{$>$}0.70) in both training and testing sets for all germination indices and morphological traits in comparison to RBF or MLP. Moreover, non-dominated sorting genetic algorithm-II (NSGA-II) was subjected to the GRNN to find the optimal type and level of media and carbohydrate source for obtaining the best seed germination indices (germination rate and mean germination time). According to the optimization process, 0.43 strength mMS medium supplemented with 2.3 \% sucrose would result in the best outcomes. This result showed that a moderate level of salts existing in culture media (0.43 strength of mMS medium) supplemented with a moderate level of sucrose (2.3 \%) can improve in vitro seed germination of hemp. The results of a validation experiment revealed that there was a negligible difference between the experimental data and the optimized result. Therefore, GRNN-NSGA-II provided an accurate prediction of seed germination and can likely be employed to optimize different factors involved in in vitro culture of this multi-purpose crop.
16. Hu, Huaran and Liu, Hao and Du, Guanghui and Fei, Yang and Deng, Gang and Yang, Yang and Feihu, Liu, Fiber and Seed Type of Hemp (Cannabis Sativa L.) Responded Differently to Salt-Alkali Stress in Seedling Growth and Physiological Indices, Industrial Crops and Products, vol. 129, pp. 624--630, March 2019. doi: 10.1016/j.indcrop.2018.12.028. Abstract
    Hemp (Cannabis sativa L.) cultivars Yunma 5 (YM5, fiber-type) and Bamahuoma (BM, seed-type) were seriously affected by salt-alkali stress during germination, while the knowledge about the salt tolerance during seedling stage is deficient. Here, responses of hemp (Cannabis sativa L.) seedlings of the cultivars YM5 and BM to the stress of different concentrations of NaCl and Na2CO3 were investigated to explore the salt tolerance of hemp. Results showed that the plant height, dry weight and root length of YM5 and BM seedlings gradually decreased as the concentration of NaCl or Na2CO3 increased. Although physiological indices such as EC (Electrical conductivity), SOD (Superoxide dismutase) and Pro (Proline) increased as the salt concentration increased, the increase extent was significantly greater in YM5 than in BM. Under the conditions of 100 and 200\,mM NaCl, BM had greater dry weight and root length, and lower Pro content than YM5. However, under the stress of 100 and 200\,mM Na2CO3, the opposite results were recorded, with the exception for lower chlorophyll content and higher SOD activity in BM than in YM5. These findings indicated that BM had a stronger NaCl tolerance than YM5. Moreover, morphological indices of the two cultivars under Na2CO3 stress were stunted more than under NaCl stress, and the seedlings died when Na2CO3 concentration increased up to 300\,mM. Some up-regulation of salt-tolerant genes were observed in the two hemp cultivars, a higher expression under NaCl stress than under Na2CO3 stress, and a higher expression in YM5 than in BM were recorded. Findings from the study indicated that hemp seedling is more sensitive to Na2CO3 stress, and the tolerance of hemp to salt stress varies with salt type and salt concentration, although both cultivars can withstand moderate doses of NaCl and Na2CO3.
17. Hu, Huaran and Liu, Hao and Liu, Feihu, Seed Germination of Hemp (Cannabis Sativa L.) Cultivars Responds Differently to the Stress of Salt Type and Concentration, Industrial Crops and Products, vol. 123, pp. 254--261, November 2018. doi: 10.1016/j.indcrop.2018.06.089. Abstract
    Industrial hemp is worldwide, but the effects of salt stress on hemp growth remain unknown, especially the tolerance of different varietal to salt stress during germination. Two important hemp cultivars in China, Yunma 5 (YM5, fiber hemp) and Bamahuoma (BM, seed hemp), were germinated under the conditions with four kinds of salts (NaCl, Na2SO4, Na2CO3, NaHCO3) at 0–300\,mM for seven days to test the seed germination responses to salt stress. The germination indexes decreased with the increase of salt concentration for each salt, especially in 300\,mM Na2CO3, showing the most toxic effect on germination. However, a low concentration (50\,mM) of neutral salt stimulated seed germination, showing a higher germination rate and longer radicles and hypocotyls than the control. The growth of radical and hypocotyl was more salt-sensitive than seed germination. Upon transfer to distilled water, the non-germinated seeds restored germination ability and the recovery percentage increased with the increase of salt concentration. YM5 showed stronger salt tolerance than BM, with higher threshold salinity of 150\,mM NaCl and Na2SO4 (100\,mM NaCl and 50\,mM Na2SO4 for BM), and higher critical salinity of 150\,mM Na2CO3 and 250\,mM NaHCO3 (100\,mM Na2CO3 and 200\,mM NaHCO3 for BM) for germination rate. In conclusion, the inhibition of salt stress to hemp seed germination varied with the salt type, salt concentration, and tested cultivars, and both hemp cultivars were more sensitive to alkaline salt stress.
18. Sera, B. and Sery, M. and Gavril, B. and Gajdova, I., Seed Germination and Early Growth Responses to Seed Pre-Treatment by Non-Thermal Plasma in Hemp Cultivars (Cannabis Sativa L.), Plasma Chemistry and Plasma Processing, vol. 37, no. 1, pp. 207--221, January 2017. doi: 10.1007/s11090-016-9763-9. Abstract
    The two key questions addressed in this paper were whether different cultivars of hemp (Cannabis sativa L.) have the same reactions to non-thermal plasma seed pre-treatments and whether different plasma sources have different effects on the seeds. Seed germination and early growth of hemp in design of hierarchical analysis of variance was conducted. Differences in response among seeds of three hemp cultivars (‘Finola’, ‘Bialobrzeskie’, ‘Carmagnola’) to the non-thermal plasma pre-treatment generated by two apparatuses (gliding arc and downstream microwave devices) in four time expositions (0, 180, 300, 600~s) were found. The high importance was found in type of apparatus and time exposition. A positive/neutral effect was observed in all measured characteristics after gliding arc plasma pre-treatment. Gliding arc pre-treatment increased the length of seedlings, seedling accretion and weight of seedling in both cv. ‘Finola’ and cv. ‘Bialobrzeskie’ hemp. On the other hand, the downstream microwave apparatus had an inhibiting effect on all tested hemp cultivars. It was the first time when significant differences in response to non-thermal pre-treatment were found in taxonomically close plants. The results obtained in this study describes different effect of various plasma treatment on germination and early growth of hemp seeds. The direct pre-treatment of non-thermal plasma discharge in condition of atmospheric pressure was better. Results of our experiment show that the use of non-thermal plasma pre-treatment may increase survival of some hemp cultivars during seedlings establishment in a drier period and may be used in new agro-technical measures in unconventional agriculture.
19. Campbell, Lesley G. and Naraine, Steve G. U. and Dusfresne, Jaimie, Phenotypic Plasticity Influences the Success of Clonal Propagation in Industrial Pharmaceutical Cannabis Sativa, PLOS ONE, vol. 14, no. 3, pp. e0213434, March 2019. doi: 10.1371/journal.pone.0213434. Abstract
    The burgeoning cannabis market requires evidence-based science such that farmers can quickly and efficiently generate new plants. In part, horticultural operations are limited by the success of cloning procedures. Here, we measured the role of environmental conditions and cultivar identity on the success of generating long branch material with many meristems in planting stock (mothers) and in rooting success of stem-derived clones. To evaluate the influence of lighting treatments on the optimal production of branching mothers, four lighting conditions (Fluorescent High Output T5s [T5], Metal halide lamps [MH], Plasma lamps [PL], or Metal halide lamps augmented with far red LED lights [MH+FR]) were applied to two cultivars of container grown plants (Cannabis sativa L. ‘Bubba Kush’, ‘Ghost Train Haze’) grown in peat-based organic substrates in mylar grow tents. To evaluate the influence of lighting, cutting tool (secateurs or scalpels), and stem wounding (present/absent) on optimal rooting of stems, three lighting conditions (Fluorescent T8s, T5, PL) were applied to three cultivars of peat pellet grown plants (C. sativa L. ‘Bubba Kush’, ‘Ghost Train Haze’, ‘Headband’). Mothers grown under T5 and MH (vs MH+FR) produced \textasciitilde 30\% more meristems. However, growing mothers under MH+FR were 19\% taller than mothers under T5, with \textasciitilde 25\% longer internodes on dominant stems than plants under any other lighting condition. Canopies were denser under T5 because petiole length was \textasciitilde 30\% shorter under T5 and fan leaves were longer and narrower under MH+FR and MH+FR and PL, respectively, than under other lighting conditions. Cultivar Ghost Train Haze stems rooted most frequently and most quickly. Wounded stems were 162\% more likely to root than unwounded stems and rooted 1.5 days earlier. Our results will guide producers attempting to maximize the rate of clone production in licensed facilities; although results may differ among cultivars, where cultivars differed in their average phenotype as mother plants, and their propensity to root from cuttings, and the speed with which they produced those roots.
20. Caplan, Deron and Stemeroff, Jonathan and Dixon, Mike and Zheng, Youbin, Vegetative Propagation of Cannabis by Stem Cuttings: Effects of Leaf Number, Cutting Position, Rooting Hormone, and Leaf Tip Removal, Canadian Journal of Plant Science, vol. 98, no. 5, pp. 1126--1132, May 2018. doi: 10.1139/cjps-2018-0038. Abstract
    This study evaluated the influence of several factors and their interactive effects on the propagation success of stem cuttings of cannabis (Cannabis sativa L.). Factors included (i) leaf number (two or three), (ii) leaf tip removal (one-third of leaf tips removed), (iii) basal/apical position of stem cutting on the stock plant, and (iv) rooting hormone [0.2\% indole-3-butyric (IBA) acid gel or 0.2\% willow (Salix alba L.) extract gel]. Cuttings were placed in a growth chamber for twelve days and then assessed on their rooting success rate and root quality using a relative root quality scale. The IBA gel delivered a 2.1× higher rooting success rate and 1.6× higher root quality than the willow extract. Removing leaf tips reduced rooting success rate from 71\% to 53\% without influencing root quality. Cuttings with three leaves had 15\% higher root quality compared with those with two, but leaf number did not influence rooting success rate. Position of cutting had little effect on rooting success or quality. To achieve maximum rooting success and root quality, cuttings from either apical or basal positions should have at least three fully expanded uncut leaves and the tested IBA rooting hormone is preferred to the willow-based product.
21. Wróbel, Tomasz and Dreger, Mariola and Wielgus, Karolina and Słomski, Ryszard, Modified Nodal Cuttings and Shoot Tips Protocol for Rapid Regeneration of Cannabis Sativa L, Journal of Natural Fibers, vol. 19, no. 2, pp. 536--545, February 2022. doi: 10.1080/15440478.2020.1748160. Abstract
    The aim of this study was to obtain micropropagation protocol for fibrous, Cannabidiol (CBD) rich, hemp variety Epsilon 68. To fulfil this goal, nodal and tip cutting protocol was developed. In further step, this protocol was compared with several other methods dependent on plant growth regulators with cytokine activity to obtain 4.2 ± 1.43 SD explants per tip. Plants from isolated lines were acclimatized to field conditions, and the content of cannabinoids was evaluated in the inflorescences. Described micropropagation protocol may be useful in breeding of hemp varieties and germplasm conservation.
22. Holmes, Janesse E. and Lung, Samantha and Collyer, Danielle and Punja, Zamir K., Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis Sativa L., Frontiers in Plant Science, vol. 12, 2021. doi: 10.3389/fpls.2021.732344. Abstract
    Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In this research, we evaluated variables that can influence the success of shoot growth and plantlet production in tissue cultures of drug-type Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused the death of explants. Seven commercially grown tetrahydrocannabinol (THC)-containing cannabis genotypes (strains) showed significant differences in response to shoot growth from meristems and nodal explants on Murashige and Skoog (MS) medium containing thidiazuron (1 μM) and naphthaleneacetic acid (0.5 μM) plus 1\% activated charcoal. The effect of Driver and Kuniyuki Walnut (DKW) or MS basal salts in media on shoot length and leaf numbers from nodal explants was compared and showed genotype dependency with regard to the growth response. To obtain rooted plantlets, shoots from meristems and nodal explants of genotype Moby Dick were evaluated for rooting, following the addition of sodium metasilicate, silver nitrate, indole-3-butyric acid (IBA), kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet survival in hydroponic culture, peat plugs, and rockwool substrate was 57, 76, and 83\%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The callogenesis response of leaf explants of 11 genotypes on MS medium without activated charcoal was 35\% to 100\%, depending on the genotype; organogenesis was not observed. The success in recovery of plantlets from meristems and nodal explants is influenced by cannabis genotype, degree of endophytic contamination of the explants, and frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets in stage 1 tissue cultures of C. sativa.
23. Monthony, Adrian S. and Page, Serena R. and Hesami, Mohsen and Jones, Andrew Maxwell P., The Past, Present and Future of Cannabis Sativa Tissue Culture, Plants, vol. 10, no. 1, pp. 185, January 2021. doi: 10.3390/plants10010185. Abstract
    The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective propagation and biotechnologies for the species. Micropropagation affords researchers and producers methods to rapidly propagate insect-/disease-/virus-free clonal plants and store germplasm and forms the basis for other biotechnologies. Despite this need, research in the area is limited due to the long history of prohibitions and restrictions. Existing literature has multiple limitations: many publications use hemp as a proxy for drug-type Cannabis when it is well established that there is significant genotype specificity; studies using drug-type cultivars are predominantly optimized using a single cultivar; most protocols have not been replicated by independent groups, and some attempts demonstrate a lack of reproducibility across genotypes. Due to culture decline and other problems, the multiplication phase of micropropagation (Stage 2) has not been fully developed in many reports. This review will provide a brief background on the history and botany of Cannabis as well as a comprehensive and critical summary of Cannabis tissue culture. Special attention will be paid to current challenges faced by researchers, the limitations of existing Cannabis micropropagation studies, and recent developments and future directions of Cannabis tissue culture technologies.
24. Monthony, Adrian S. and Kyne, Sean T. and Grainger, Christopher M. and Jones, Andrew Maxwell P., Recalcitrance of Cannabis Sativa to de Novo Regeneration; a Multi-Genotype Replication Study, PLOS ONE, vol. 16, no. 8, pp. e0235525, August 2021. doi: 10.1371/journal.pone.0235525. Abstract
    Cannabis sativa is relatively recalcitrant to de novo regeneration, but several studies have reported shoot organogenesis or somatic embryogenesis from non-meristematic tissues. Most report infrequent regeneration rates from these tissues, but a landmark publication from 2010 achieved regeneration from leaf explants with a 96\% response rate, producing an average of 12.3 shoots per explant in a single drug-type accession. Despite the importance regeneration plays in plant biotechnology and the renewed interest in this crop the aforementioned protocol has not been used in subsequent papers in the decade since it was published, raising concerns over its reproducibility. Here we attempted to replicate this important Cannabis regeneration study and expand the original scope of the study by testing it across 10 drug-type C. sativa genotypes to assess genotypic variation. In our study, callus was induced in all 10 genotypes but callus growth and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive. The shoot induction medium failed to induce shoot organogenesis in any of the 10 cultivars tested, instead resulting in necrosis of the calli. The findings of this replication study raise concerns about the replicability of existing methods. However, some details of the protocol could not be replicated due to missing details in the original paper and regulatory issues, which could have impacted the outcome. These results highlight the importance of using multiple genotypes in such studies and providing detailed methods to facilitate replication.
25. Moher, Melissa and Jones, Max and Zheng, Youbin, Photoperiodic Response of In Vitro Cannabis Sativa Plants, HortScience, vol. 56, no. 1, pp. 108--113, January 2021. doi: 10.21273/HORTSCI15452-20. Abstract
    The majority of commercial Cannabis sativa L. (cannabis) cultivators use a 12.0-hour uninterrupted dark period to induce flowering; however, scientific information to prove this is the optimal dark period for all genotypes is lacking. Knowing genotype-specific photoperiods may help to promote growth by providing the optimal photoperiod for photosynthesis. To determine whether the floral initiation of cannabis explants respond to varied photoperiods in vitro, explants were grown under one of six photoperiod treatments: 12.0, 13.2, 13.8, 14.4, 15.0, and 16.0 hours per day for 4 weeks. The percentage of flowering explants was highest under 12.0- and 13.2-hour treatments. There were no treatment effects on the fresh weight, final height, and growth index. Based on the results, it is recommended that an uninterrupted dark period of at least 10.8 hours (i.e., 13.2-hour photoperiod) be used to induce flowering for the ‘802’ genotype. In vitro flowering could provide a unique and high-throughput approach to study floral/seed development and secondary metabolism in cannabis under highly controlled conditions. Further research should determine if this response is the same on the whole-plant level.
26. Hesami, Mohsen and Jones, Andrew Maxwell Phineas, Modeling and Optimizing Callus Growth and Development in Cannabis Sativa Using Random Forest and Support Vector Machine in Combination with a Genetic Algorithm, Applied Microbiology and Biotechnology, vol. 105, no. 12, pp. 5201--5212, June 2021. doi: 10.1007/s00253-021-11375-y. Abstract
    Plant callus is generally considered to be a mass of undifferentiated cells and can be used for secondary metabolite production, physiological studies, and plant transformation/regeneration. However, there are several types of callus with different morphological and developmental characteristics and not all are suitable for all applications. Callogenesis is a multivariable developmental process affected by several intrinsic and extrinsic factors, but the most important driver is plant growth regulator (PGRs) levels and type. Since callogenesis is a non-linear process influenced by many different factors, robust computational methods such as machine learning algorithms have great potential to model, predict, and optimize callus growth and development. The current study was conducted to evaluate the effect of PGRs on callus morphology in drug-type Cannabis sativa to maximize callus growth and promote embryogenic callus production. For this aim, random forest (RF) and support vector machine (SVM) were applied in conjunction with image processing to model and predict callus morphological and physical traits. The results showed that SVM was more accurate than RF. In order to find the optimal level of PGRs for optimizing callus growth and development, the SVM was linked to a genetic algorithm (GA). To confirm the reliability of SVM-GA, the optimized-predicted outcomes were tested in a validation experiment that revealed SVM-GA was able to accurately model and optimize the system. Moreover, our results showed that there is a significant correlation between embryogenic callus production and the true density of callus.
27. Zhang, Xiaoyu and Xu, Gencheng and Cheng, Chaohua and Lei, Lei and Sun, Jian and Xu, Ying and Deng, Canhui and Dai, Zhigang and Yang, Zemao and Chen, Xiaojun and Liu, Chan and Tang, Qing and Su, Jianguang, Establishment of an Agrobacterium-Mediated Genetic Transformation and CRISPR/Cas9-Mediated Targeted Mutagenesis in Hemp (Cannabis Sativa L.), Plant Biotechnology Journal, vol. 19, no. 10, pp. 1979--1987, 2021. doi: 10.1111/pbi.13611. Abstract
    Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is getting more attention recently. Several candidate genes involved in cannabinoid biosynthesis have been elucidated using omics analysis. However, the gene function was not fully validated due to few reports of stable transformation for Cannabis tissues. In this study, we firstly report the successful generation of gene-edited plants using an Agrobacterium-mediated transformation method in C. sativa. DMG278 achieved the highest shoot induction rate, which was selected as the model strain for transformation. By overexpressing the cannabis developmental regulator chimera in the embryo hypocotyls of immature grains, the shoot regeneration efficiency was substantially increased. We used CRISPR/Cas9 technology to edit the phytoene desaturase gene and finally generated four edited cannabis seedlings with albino phenotype. Moreover, we propagated the transgenic plants and validated the stable integration of T-DNA in cannabis genome.
28. Pepe, Marco and Hesami, Mohsen and Small, Finlay and Jones, Andrew Maxwell Phineas, Comparative Analysis of Machine Learning and Evolutionary Optimization Algorithms for Precision Micropropagation of Cannabis Sativa: Prediction and Validation of in Vitro Shoot Growth and Development Based on the Optimization of Light and Carbohydrate Sources, Frontiers in Plant Science, vol. 12, pp. 757869, October 2021. doi: 10.3389/fpls.2021.757869. Abstract
    Micropropagation techniques offer opportunity to proliferate, maintain, and study dynamic plant responses in highly controlled environments without confounding external influences, forming the basis for many biotechnological applications. With medicinal and recreational interests for Cannabis sativa L. growing, research related to the optimization of in vitro practices is needed to improve current methods while boosting our understanding of the underlying physiological processes. Unfortunately, due to the exorbitantly large array of factors influencing tissue culture, existing approaches to optimize in vitro methods are tedious and time-consuming. Therefore, there is great potential to use new computational methodologies for analyzing data to develop improved protocols more efficiently. Here, we first tested the effects of light qualities using assorted combinations of Red, Blue, Far Red, and White spanning 0–100 μmol/m2/s in combination with sucrose concentrations ranging from 1 to 6\% (w/v), totaling 66 treatments, on in vitro shoot growth, root development, number of nodes, shoot emergence, and canopy surface area. Collected data were then assessed using multilayer perceptron (MLP), generalized regression neural network (GRNN), and adaptive neuro-fuzzy inference system (ANFIS) to model and predict in vitro Cannabis growth and development. Based on the results, GRNN had better performance than MLP or ANFIS and was consequently selected to link different optimization algorithms [genetic algorithm (GA), biogeography-based optimization (BBO), interior search algorithm (ISA), and symbiotic organisms search (SOS)] for prediction of optimal light levels (quality/intensity) and sucrose concentration for various applications. Predictions of in vitro conditions to refine growth responses were subsequently tested in a validation experiment and data showed no significant differences between predicted optimized values and observed data. Thus, this study demonstrates the potential of machine learning and optimization algorithms to predict the most favorable light combinations and sucrose levels to elicit specific developmental responses. Based on these, recommendations of light and carbohydrate levels to promote specific developmental outcomes for in vitro Cannabis are suggested. Ultimately, this work showcases the importance of light quality and carbohydrate supply in directing plant development as well as the power of machine learning approaches to investigate complex interactions in plant tissue culture.
29. Adams, Tristan K. and Masondo, Nqobile A. and Malatsi, Pholoso and Makunga, Nokwanda P., Cannabis Sativa: From Therapeutic Uses to Micropropagation and Beyond, Plants, vol. 10, no. 10, pp. 2078, October 2021. doi: 10.3390/plants10102078. Abstract
    The development of a protocol for the large-scale production of Cannabis and its variants with little to no somaclonal variation or disease for pharmaceutical and for other industrial use has been an emerging area of research. A limited number of protocols have been developed around the world, obtained through a detailed literature search using web-based database searches, e.g., Scopus, Web of Science (WoS) and Google Scholar. This article reviews the advances made in relation to Cannabis tissue culture and micropropagation, such as explant choice and decontamination of explants, direct and indirect organogenesis, rooting, acclimatisation and a few aspects of genetic engineering. Since Cannabis micropropagation systems are fairly new fields, combinations of plant growth regulator experiments are needed to gain insight into the development of direct and indirect organogenesis protocols that are able to undergo the acclimation stage and maintain healthy plants desirable to the Cannabis industry. A post-culture analysis of Cannabis phytochemistry after the acclimatisation stage is lacking in a majority of the reviewed studies, and for in vitro propagation protocols to be accepted by the pharmaceutical industries, phytochemical and possibly pharmacological research need to be undertaken in order to ascertain the integrity of the generated plant material. It is rather difficult to obtain industrially acceptable micropropagation regimes as recalcitrance to the regeneration of in vitro cultured plants remains a major concern and this impedes progress in the application of genetic modification technologies and gene editing tools to be used routinely for the improvement of Cannabis genotypes that are used in various industries globally. In the future, with more reliable plant tissue culture-based propagation that generates true-to-type plants that have known genetic and metabolomic integrity, the use of genetic engineering systems including “omics” technologies such as next-generation sequencing and fast-evolving gene editing tools could be implemented to speed up the identification of novel genes and mechanisms involved in the biosynthesis of Cannabis phytochemicals for large-scale production.
30. Zarei, Adel and Behdarvandi, Behrang and Tavakouli Dinani, Elham and Maccarone, Jennifer, Cannabis Sativa L. Photoautotrophic Micropropagation: A Powerful Tool for Industrial Scale in Vitro Propagation, In Vitro Cellular \& Developmental Biology - Plant, vol. 57, no. 6, pp. 932--941, December 2021. doi: 10.1007/s11627-021-10167-3. Abstract
    Global demands for an in vitro culture of cannabis have never been more sought after as countries shift their paradigm towards legalization. Cannabis conventional (photomixotrophic) micropropagation has not been suitable enough for large-scale propagation due to a high degree of plant hyperhydricity, low growth rate, poor rooting, and acclimation efficiency. In the present study, cannabis photoautotrophic micropropagation method is introduced with the purpose of overcoming the difficulties that conventional micropropagation entails when conducted at a large scale. The roles of rockwool medium pH and moisture content, cutting length, basal wounding methods, light intensity, and culture vessel gas exchange capacity were assessed with the intention of increasing productivity of micropropagation method. The results showed 300 mL per vessel of fertilizer solution containing 5-mM MES buffer stabilized medium pH and increased rooting success. Both 5- and 7-cm cutting lengths significantly increased the percent of rooted plants compared to 3-cm cutting length. However, the basal wounding methods did not significantly improve or impede the rooting success. The highest rooting success was also obtained with 150 μmol m−2 s−1 of photosynthetic photon flux density compared to 50 and 100 μmol m−2 s−1. Increasing gas exchange rates either using more permeable vessels or aeration practices significantly improved the rooting success. Overall, more than 90\% of cannabis plantlets grown in photoautotrophic micropropagation are rooted in 2 wk of culture followed by 4 d ex vitro acclimation period, which was remarkably shorter than any other available method in cannabis micropropagation. This study not only optimizes a method for cannabis photoautotrophic micropropagation using passive ventilation for the first time but also scales up in vitro clonal propagation for in vitro commercial production.
31. {Galán-Ávila}, Alberto and Gramazio, Pietro and Ron, Mily and Prohens, Jaime and Herraiz, Francisco Javier, A Novel and Rapid Method for Agrobacterium-Mediated Production of Stably Transformed Cannabis Sativa L. Plants, Industrial Crops and Products, vol. 170, pp. 113691, October 2021. doi: 10.1016/j.indcrop.2021.113691. Abstract
    The development of genetically transformed plants is an elusive landmark in Cannabis sativa L. breeding. Despite its economic interest, at present, protocols for producing transgenic C. sativa plants are scarce. We studied the ability of hypocotyl, cotyledon and meristem explants from six C. sativa hemp varieties for transgenic plant regeneration. For this, we firstly evaluated in vitro regeneration rates of hypocotyls cultured in medium without plant growth regulators, and cotyledons cultured in medium supplemented with 0.4\,mg\,L−1 of thidiazuron (TDZ) and 0.2\,mg\,L−1 of α-naphthaleneacetic (NAA). Subsequently, the effect of different kanamycin concentrations (50, 100, 200, 500 and 750\,mg\,L−1) on hypocotyl regeneration rate was determined. Finally, we assessed transformation rates after hypocotyl, cotyledon and meristem co-culture with Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pBIN19 containing the β-glucuronidase (uidA) reporter gene and the kanamycin resistance neomycin phosphotransferase (nptII) genes. Plant transformation was validated through in vitro culture of regenerating shoots in kanamycin-containing selective regeneration medium, by GUS histochemical assay for uidA expression, and by PCR amplification of uidA and nptII genes. Our results showed that hypocotyls reached a higher regeneration rate (53.3 \%) than cotyledons (18.1 \%) without Agrobacterium co-culture. On the other hand, 100\,mg\,L−1 kanamycin proved to be the best concentration in terms of regeneration rate (63.3 \%) and spontaneous rooting rate of hypocotyl regenerating shoots (12.2 \%), which displayed a 7.1 \% of albinism rate. After co-culture with A. tumefaciens and subsequent culture in antibiotic-containing selective regeneration medium, hypocotyl was the best explant type achieving 23.1 \% of regeneration rate, which contrasts with the 1.0 \% regeneration rate detected for cotyledons. Transgenic plants were obtained from all explant types evaluated. Although there were significant differences among varieties evaluated, hypocotyls proved to be superior to already-developed meristems, reaching a transformation rate of 5.0 \% and 0.8 \% respectively. Despite the extremely low regeneration rate of cotyledons after A. tumefaciens co-culture, all cotyledon-derived regenerating shoots analyzed were successfully transformed. Our hormone-free protocol doubles the transformation rate of regenerating shoots, also producing transgenic plants three times faster than other already published protocols. This has relevant implications for C. sativa breeding, enabling not only genetic transformation, but also the use of new plant breeding techniques such as targeted genome editing by using CRISPR/Cas systems. This may foster the development of C. sativa varieties with specific biochemical profiles, or tolerant to biotic and abiotic stresses among others.
32. Hesami, Mohsen and Pepe, Marco and Alizadeh, Milad and Rakei, Aida and Baiton, Austin and Phineas Jones, Andrew Maxwell, Recent Advances in Cannabis Biotechnology, Industrial Crops and Products, vol. 158, pp. 113026, December 2020. doi: 10.1016/j.indcrop.2020.113026. Abstract
    Cannabis sativa L. is a high-value crop with a multi-billion dollar international market, yet due to the long history of prohibition, there is a significant lack of research on the plant and biotechnological techniques are in their infancy. Developing and applying modern techniques to Cannabis will help overcome some species-specific challenges to increase productivity and improve our knowledge about this plant. With regulatory environments relaxing in many parts of the world, there has been a significant increase in biotechnological research with this species. The current manuscript reviews the advances in Cannabis biotechnology, including molecular markers, microRNA, omics-based methods, and functional genes related to the terpene and cannabinoid biosynthesis as well as fiber quality. The foremost aim of this study is to a comprehensive review of the available literature to guide future cannabis studies in the field of genetic engineering and biotechnology.
33. {Mestinšek-Mubi}, Špela and Svetik, Sinja and Flajšman, Marko and Murovec, Jana, In Vitro Tissue Culture and Genetic Analysis of Two High-CBD Medical Cannabis (Cannabis Sativa L.) Breeding Lines, Genetika, vol. 52, no. 3, pp. 925--941, 2020. doi: 10.2298/GENSR2003925M. Abstract
    The species Cannabis sativa L. has recently witnessed a resurgence of interest all over the world due to its multipurpose applications and the scientific confirmation of its pharmacological properties. Genotypes with high cannabinoid content are appreciated in the pharmaceutical and cosmetic industries due to their therapeutic potential. These genotypes, with predominantly high cannabidiol (CBD) content, are the subject of research and breeding in several programs, but to date, little data is published on the in vitro tissue culture of cannabis. Our study focused on the establishment of an efficient micropropagation method for two high-CBD breeding lines (MX-CBD-11 and MX-CBD-707) as the basis for advanced biotechnological breeding approaches. The results demonstrated that the in vitro culture of medical cannabis can be initiated on different culture media, that cultured plants can be successfully acclimatized, and that nodal position, and especially the genotype, have a significant influence on the success of shoot culture establishment. They showed that the published tissue culture media optimized for one high-THC strain of Mexican cannabis are not as efficient for other genotypes of (medical) cannabis. We complemented this research with a genetic study of 95 plants of the two breeding lines with 16 microsatellite (SSR) markers which clustered the plants based on breeding line. The results demonstrated that only 8 markers are needed for discrimination of all analyzed plants and their usefulness for clonal identification.
34. Piunno, Kevin F. and Golenia, Gregory and Boudko, Ekaterina A. and Downey, Cassandra and Jones, A. Maxwell P., Regeneration of Shoots from Immature and Mature Inflorescences of Cannabis Sativa, Canadian Journal of Plant Science, vol. 99, no. 4, pp. 556--559, August 2019. doi: 10.1139/cjps-2018-0308. Abstract
    Cannabis sativa is usually clonally propagated from plants in the vegetative phase. However, phenotypic traits such as yield and chemical composition can only be assessed in unfertilized plants reaching the end of their life cycle and there are no peer-reviewed methods to propagate flowering plants. In this study, immature (three cultivars) and mature (one cultivar) floral explants were cultured on thidiazuron and shoot development was observed in both the immature and mature explants. This provides the first report of micropropagation from floral tissues in C. sativa and will enable plants to be clonally propagated up to the date of harvest.
35. Lata, Hemant and Chandra, Suman and Khan, Ikhlas A. and ElSohly, Mahmoud A., Micropropagation of Cannabis Sativa L.—An Update, pp. 285--297, 2017. doi: 10.1007/978-3-319-54564-6_13. Abstract
    Cannabis is one of the oldest economically important plant yielding fiber, food and medicine. It is a natural source of Δ9-tetrahydrocannabinol (THC) and Cannabidiol (CBD). These two molecules have a tremendous therapeutic potential and commercial value in the pharmaceutical area. Cannabis is a highly heterozygous species. Being dioceous (male and female flowers appear on two different plants) and wind pollinated species, it is difficult to maintain the chemical profile of biomass product, if grown from seed. Plant to plant variation is observed even though plants are grown from seeds obtained from a single female plant. Therefore, to maintain consistency in the end product, elite female plants are screened and multiplied using vegetative propagation and/or tissue culture. Micro propagation can play a vital role in the conservation of elite Cannabis clones and rapid multiplication of novel germplasm. On the other hand, it can also be used in genetic modification for the enhanced cannabinoid production. Research on in vitro propagation of Cannabis has resulted in the development of protocols for callus production, cell suspension cultures, agrobacterium mediated hairy root cultures and regeneration of plants. This chapter provides an overview of in vitro propagation of Cannabis and addresses the current applications of modern biotechnology in propagation of elite Cannabis plants.
36. Chaohua, Cheng and Gonggu, Zang and Lining, Zhao and Chunsheng, Gao and Qing, Tang and Jianhua, Chen and Xinbo, Guo and Dingxiang, Peng and Jianguang, Su, A Rapid Shoot Regeneration Protocol from the Cotyledons of Hemp (Cannabis Sativa L.), Industrial Crops and Products, vol. 83, pp. 61--65, May 2016. doi: 10.1016/j.indcrop.2015.12.035. Abstract
    Hemp (Cannabis sativa) is an annual multipurpose crop that is distributed worldwide. An efficient regeneration protocol is needed for hemp genetic transformation, micropropagation, and germplasm conservation. We describe here a rapid protocol for in vitro shoot regeneration that uses cotyledons as explants. We concluded that TDZ in MS medium is more efficient in inducing in vitro shoots from cotyledons than BA and ZT. The best result, 51.7\% induction frequency and 3.0 shoots per shoot explant, was recorded in MS medium containing 0.4mgl−1 TDZ and 0.2mgl−1 NAA (T4N2). The in vitro shoots grew to the height of 1.5–2cm in 3–4 weeks after culture initiation. At that time, approximately 80\% of the shoots were rooted well in half-strength Ms medium combined with 0.5–2mgl−1 IBA for 4–5 weeks before acclimation. It was observed that younger cotyledons (2–3 days after planting (DAP) were more useful as explants than older ones (5–6 DAP) because they had a significantly higher regeneration frequency. We cultured 3 DAP cotyledons of eight cultivars in T4N2 medium to test the efficiency. The regeneration frequency varied from 35.7\% to 54.8\%, which indicated that this regeneration protocol was effective, although partly genotype dependent. We regard this protocol as an alternative method for micropropagation and germplasm conservation, and the in vitro plantlets may be suitable to set up a transformation system.
37. Movahedi, Mehdi and {Ghasemi-Omran}, Valiollah and Torabi, Sepide, The Effect of Different Concentrations of TDZ and BA on in Vitro Regeneration of Iranian Cannabis (Cannabis Sativa) Using Cotyledon and Epicotyl Explants, Journal of Plant Molecular Breeding, vol. 3, no. 2, pp. 20--27, December 2015. doi: 10.22058/jpmb.2015.15371. Abstract
    The present study was carried out to investigate micropropagation possibility and determine the optimal medium composition and plant growth regulators (PGRs) combinations under in vitro conditions. The cotyledon and epicotyl explants obtained from 1 month old in vitro grown seedlings were used in MS medium containing BA (0.1, 0.2, 0.5, 1, 2 and 3 mg-1) and TDZ (0.1, 0.2, 0.5, 1, 2 and 3 mg-1) either alone or in combination with 0.5 mg-1 IBA. The response of cannabis explants to PGRs treatments was much different from those observed in most of plant species. That is, callus formation had priority over direct regeneration in most of the PGRs treatments. Comparing the two explants, cotyledon had higher callus formation frequency and the largest callus volume was obtained for this explant in MS medium supplemented with 3 mg-1 TDZ + 0.5 mg-1 IBA. The highest callus fresh weight (3.15 gr) was obtained for cotyledon explant treated with 2 mg-1 TDZ+ 0.5 mg-1 IBA. In shoot formation step, the highest rate of shoot regeneration was achieved in the calli produced from epicotyl explant treated with 2 mg-1 BA + 0.5 mg-1 IBA; and the highest length of regenerated shoots (1.23 cm) was observed in 2 mg-1 BA + 0.5 mg-1 IBA treatment. In general, cotyledon was the best explant and TDZ in combination with IBA was the best treatment for callus formation. Epicotyl explant also showed better regeneration compared to cotyledon.
38. Feeney, Mistianne and Punja, Zamir K., Hemp (Cannabis Sativa L.), pp. 319--329, 2015. doi: 10.1007/978-1-4939-1658-0_25. Abstract
    Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the presence of the PMI gene was confirmed using PCR and Southern hybridization. Using this method, an average transformation frequency of 31.23 \%\,±\,0.14 was obtained for all transformation experiments, with a range of 15.1–55.3 \%.
39. Farag, Sayed and Kayser, Oliver, Cannabinoids Production by Hairy Root Cultures of {\emph{Cannabis Sativa}} L., American Journal of Plant Sciences, vol. 06, no. 11, pp. 1874, 2015. doi: 10.4236/ajps.2015.611188. Abstract
    Tetrahydrocannabinol (THC) derivatives are used clinically as analgesic, anti-inflammatory, appetite stimulant, anti-emetic and anti-tumor cannabinoids. THC and its related compounds are at present obtained by extraction from intact Cannabis plants or chemical synthesis, but plant cell cultures may be an alternative source of production. In the present study, hairy root cultures of C. sativa (Cannabaceae) were induced by incubation of aseptically grown callus culture with solid B5 medium supplemented with 4 mg/l naphthaleneacetic acid in darkness at 25°C. Hairy root growth profiles in shake flask, increased periodically during 35 days of growth cycle. The cannabinoid contents produced in minor levels and remained below 2.0 μg/g dry weight. The contents of can-nabinoid were analyzed by liquid chromatography and confirmed by mass spectrometry.
40. Lata, Hemant and Chandra, Suman and Mehmedic, Zlatko and Khan, Ikhlas A. and ElSohly, Mahmoud A., In Vitro Germplasm Conservation of High Δ9-Tetrahydrocannabinol Yielding Elite Clones of Cannabis Sativa L. under Slow Growth Conditions, Acta Physiologiae Plantarum, vol. 34, no. 2, pp. 743--750, March 2012. doi: 10.1007/s11738-011-0874-x. Abstract
    Germplasm conservation of a high Δ9-tetrahydrocannabinol yielding variety of Cannabis sativa L. was attempted using synthetic seed technology and media supplemented with osmotic agents. Explants of nodal segments containing single axillary bud were excised from in vitro proliferated shoot cultures and encapsulated in high-density sodium alginate (230~mM) hardened by 50~mM CaCl2. The ‘encapsulated’ (synthetic seeds) and ‘non-encapsulated’ nodal segments were stored at 5, 15 and 25°C for 8, 16 and 24~weeks and monitored for the re-growth and survival frequency under the tissue culture conditions (16-h photoperiod, 25°C) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.5~μM). ‘Encapsulated’ nodal segments could be stored at low temperature 15°C up to 24~weeks with maximum re-growth ability and survival frequency of 60\%. Similar to ‘encapsulated’ cultures, the highest re-growth in ‘non-encapsulated’ cultures was observed in the explants kept at 15°C without osmotic agents. Furthermore, the effect of osmotic agents mannitol and sorbitol (2 and 4\% w/v, added individually and in combination to the media at culture room conditions i.e. 25°C) on non-encapsulated shoot cultures was also evaluated. A considerable decrease in re-growth and survival was observed in the cultures treated with osmotic agents. Among the cultures treated with different concentrations of osmotic agents, the highest rate of re-growth and survival was observed at the lowest concentration of 2\% sorbitol and 2\% mannitol individually added to the media. Well-developed plantlets regenerated from ‘encapsulated’ nodal segments were successfully acclimatized inside the growing room with 90\% survival frequency. Gas chromatography-flame ionization detection (GC-FID) was used to compare the chemical profile and the concentration of the different cannabinoids (cannabidiol, cannabichromene, cannabigerol, cannabinol, Δ9-tetrahydrocannabinol and tetrahydrocannabivarin) of the plants grown from ‘encapsulated’ nodal segments to that of the donor plant. The data showed similar cannabinoid profile and insignificant differences in the cannabinoids content between the two types of plants. This study is of high significance since the encapsulation technology would allow the prolonged storage (thus reducing the cost of labor) of high-yielding C. sativa germplasm selected for the isolation of THC, a high-value bulk active pharmaceutic.
41. Lata, Hemant and Chandra, Suman and Techen, Natascha and Khan, Ikhlas A. and ElSohly, Mahmoud A., Molecular Analysis of Genetic Fidelity in Cannabis Sativa L. Plants Grown from Synthetic (Encapsulated) Seeds Following in Vitro Storage, Biotechnology Letters, vol. 33, no. 12, pp. 2503--2508, December 2011. doi: 10.1007/s10529-011-0712-7. Abstract
    The increasing utilization of synthetic (encapsulated) seeds for germplasm conservation and propagation necessitates the assessment of genetic stability of conserved propagules following their plantlet conversion. We have assessed the genetic stability of synthetic seeds of Cannabis sativa L. during in vitro multiplication and storage for 6~months at different growth conditions using inter simple sequence repeat (ISSR) DNA fingerprinting. Molecular analysis of randomly selected plants from each batch was conducted using 14 ISSR markers. Of the 14 primers tested, nine produced 40 distinct and reproducible bands. All the ISSR profiles from in vitro stored plants were monomorphic and comparable to the mother plant which confirms the genetic stability among the clones. GC analysis of six major cannabinoids [Δ9-tetrahydrocannabinol, tetrahydrocannabivarin, cannabidiol, cannabichromene, cannabigerol and cannabinol] showed homogeneity in the re-grown clones and the mother plant with insignificant differences in cannabinoids content, thereby confirming the stability of plants derived from synthetic seeds following 6~months storage.
42. Lata, Hemant and Chandra, Suman and Techen, Natascha and Khan, Ikhlas A. and ElSohly, Mahmoud A., Assessment of the Genetic Stability of Micropropagated Plants of Cannabis Sativa by ISSR Markers, Planta Medica, vol. 76, no. 1, pp. 97--100, January 2010. doi: 10.1055/s-0029-1185945. Abstract
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43. Chandra, Suman and Lata, Hemant and Mehmedic, Zlatko and Khan, Ikhlas A. and ElSohly, Mahmoud A., Assessment of Cannabinoids Content in Micropropagated Plants of Cannabis Sativa and Their Comparison with Conventionally Propagated Plants and Mother Plant during Developmental Stages of Growth, Planta Medica, vol. 76, no. 7, pp. 743--750, May 2010. doi: 10.1055/s-0029-1240628. Abstract
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44. Lata, Hemant and Chandra, Suman and Khan, Ikhlas and ElSohly, Mahmoud A., Thidiazuron-Induced High-Frequency Direct Shoot Organogenesis of Cannabis Sativa L., In Vitro Cellular \& Developmental Biology - Plant, vol. 45, no. 1, pp. 12--19, February 2009. doi: 10.1007/s11627-008-9167-5. Abstract
    Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0~μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5~μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0~μM of gibberellic acid into a medium containing 0.5~μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500~mg l−1 activated charcoal and 2.5~μM indole-3-butyric acid resulted in 95\% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000~μmol m−2 s−1). An increase in photosynthesis was observed with increase in the light intensity up to 1,500~μmol m−2 s−1 and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500~μmol m−2 s−1. Stomatal conductance and transpiration increased with light intensity up to highest level (2000~μmol m−2 s−1) tested. Intercellular CO2 concentration (Ci) and the ratio of intercellular CO2 concentration to ambient CO2 (Ci/Ca) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.
45. Lata, Hemant and Chandra, Suman and Khan, Ikhlas A. and ElSohly, Mahmoud A., Propagation through Alginate Encapsulation of Axillary Buds of Cannabis Sativa L. — an Important Medicinal Plant, Physiology and Molecular Biology of Plants, vol. 15, no. 1, pp. 79--86, January 2009. doi: 10.1007/s12298-009-0008-8. Abstract
    Cannabis sativa L. (Cannabaceae) is an important medicinal plant well known for its pharmacologic and therapeutic potency. Because of allogamous nature of this species, it is difficult to maintain its potency and efficacy if grown from the seeds. Therefore, chemical profile-based screening, selection of high yielding elite clones and their propagation using biotechnological tools is the most suitable way to maintain their genetic lines. In this regard, we report a simple and efficient method for the in vitro propagation of a screened and selected high yielding drug type variety of Cannabis sativa, MX-1 using synthetic seed technology. Axillary buds of Cannabis sativa isolated from aseptic multiple shoot cultures were successfully encapsulated in calcium alginate beads. The best gel complexation was achieved using 5 \% sodium alginate with 50 mM CaCl2.2H2O. Regrowth and conversion after encapsulation was evaluated both under in vitro and in vivo conditions on different planting substrates. The addition of antimicrobial substance — Plant Preservative Mixture (PPM) had a positive effect on overall plantlet development. Encapsulated explants exhibited the best regrowth and conversion frequency on Murashige and Skoog medium supplemented with thidiazuron (TDZ 0.5 μM) and PPM (0.075 \%) under in vitro conditions. Under in vivo conditions, 100 \% conversion of encapsulated explants was obtained on 1:1 potting mix- fertilome with coco natural growth medium, moistened with full strength MS medium without TDZ, supplemented with 3 \% sucrose and 0.5 \% PPM. Plantlets regenerated from the encapsulated explants were hardened off and successfully transferred to the soil. These plants are selected to be used in mass cultivation for the production of biomass as a starting material for the isolation of THC as a bulk active pharmaceutical.
46. {Flores-Sanchez}, Isvett Josefina and Peč, Jaroslav and Fei, Junni and Choi, Young Hae and Dušek, Jaroslav and Verpoorte, Robert, Elicitation Studies in Cell Suspension Cultures of Cannabis Sativa L., Journal of Biotechnology, vol. 143, no. 2, pp. 157--168, August 2009. doi: 10.1016/j.jbiotec.2009.05.006. Abstract
    Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with biotic and abiotic elicitors to evaluate their effect on secondary metabolism. Metabolic profiles analysed by 1H NMR spectroscopy and principal component analysis (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in either control or elicited cannabis cell cultures. Tetrahydrocannabinolic acid (THCA) synthase gene expression was monitored during a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathway.
47. Wielgus, K. and Luwanska, A. and Lassocinski, W. and Kaczmarek, Z., Estimation of Cannabis Sativa L. Tissue Culture Conditions Essential for Callus Induction and Plant Regeneration, Journal of Natural Fibers, vol. 5, no. 3, pp. 199--207, September 2008. doi: 10.1080/15440470801976045. Abstract
    The effects of different combinations of plant growth regulators on callus induction and plant regeneration were observed in three Polish monoecious hemp cultivars (Bialobrzeskie, Beniko, Silesia). Callus was induced from different explant sources (fragment of cotyledons, stems, roots) on Daria ind+ medium. There was no considerable difference noticed in callus induction between tested cultivars and explants. However, callus obtained from different genotypes had different ability for organogenesis and plant regeneration. Interaction between tested explant and cultivar has significant effect on the efficiency of plant regeneration: the highest regeneration was observed for cotyledon explant (Beniko), the lowest for stem explants (Silesia).
48. Feeney, M. and Punja, Z. K., Tissue Culture and Agrobacterium-Mediated Transformation of Hemp (Cannabis Sativa L.), In Vitro Cellular \& Developmental Biology - Plant, vol. 39, no. 6, pp. 578--585, November 2003. doi: 10.1079/IVP2003454. Abstract
    Hemp (Cannabis sativa L.) is cultivated in many parts of the world for ils fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM kinetin, 3\% sucrose, and 8 gl−1 agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 μM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2\% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.
49. Loh, W. H. -T. and Hartsel, S. C. and Robertson, L. W., Tissue Culture of Cannabis Sativa L. and in Vitro Biotransformation of Phenolics, Zeitschrift für Pflanzenphysiologie, vol. 111, no. 5, pp. 395--400, September 1983. doi: 10.1016/S0044-328X(83)80003-8. Abstract
    The production of secondary metabolites and the biotransformation of precursor compounds by plant tissue cultures may lead to both the commercial synthesis of valuable substances and an elucidation of their biosynthetic pathways. Callus and suspension cultures of Cannabis sativa L. were induced from embryo, leaf and stem expiants with various combinations of auxins and cytokinins. The optimal ratio of plant growth regulators for callus induction and subsequent cell proliferation differed according to the source of the expiant and the mode of culture. Cannabis suspensions, inoculated with cannabidiol, produced two major cannabinoids as assayed by thin-layer-chromatography. Gas chromatographic/mass spectral analysis identified both phenolics as diastereoisomers of cannabielsoin. Suspension cultures inoculated with olivetol produced an unidentified cannabinoid with a molecular ion of m/z 210.
50. Gill, Alison R. and Loveys, Beth R. and Cowley, James M. and Hall, Tony and Cavagnaro, Timothy R. and Burton, Rachel A., Physiological and Morphological Responses of Industrial Hemp (Cannabis Sativa L.) to Water Deficit, Industrial Crops and Products, vol. 187, pp. 115331, November 2022. doi: 10.1016/j.indcrop.2022.115331. Abstract
    With drought projected to increase in severity and frequency in the future, selection of emerging drought-tolerant species that exhibit improved water use efficiency will be key to maintaining cropping productivity. Industrial hemp (Cannabis sativa) shows potential for cropping in water-limited environments, but studies into its water use and drought tolerance are conflicting. We evaluated the morphological, physiological and biochemical responses of the industrial hemp cultivar ‘Black Label’ to extreme and moderate water deficit in an 85-day greenhouse experiment. Under water deficit, hemp reduced biomass production and total seed yield, but maintained filled seeds for effective reproduction. Water use efficiency, measured using δ13C isotope analysis, increased under water deficit due to reductions in stomatal opening and transpiration. Water deficit increased proline accumulation, likely to allow osmotic adjustment. While extreme water deficit initiated water stress responses in hemp, plants were able to survive and maintain seed production. We report that hemp can survive at exceptionally low levels of soil water availability, which could have far-reaching consequences as agricultural industries pursue innovative, sustainable and water-efficient crops for the future.
51. Bachir, Fatima and Eddouks, Mohamed and Arahou, Mohamed and Fekhaoui, Mohammed, Origin, Early History, Cultivation, and Characteristics of the Traditional Varieties of Moroccan Cannabis Sativa L., Cannabis and Cannabinoid Research, vol. 7, no. 5, pp. 603--615, October 2022. doi: 10.1089/can.2021.0020. Abstract
    Background:Cannabis has been cultivated and used for centuries in the north Moroccan Rif (local name is kif). However, its history is poorly known and the date of its first introduction and dispersal in Morocco is still difficult to be precise. Aim: The purpose of the present work is to review the literature on the origin, history, and cultivation of Cannabis in Morocco, as well as data on the morphological, genetic, and phytochemical characteristics of local cultivated varieties. Discussion: Considering the importance of preserving the fragile environment of the Rif and the future development of the Moroccan medical Cannabis market, which will require authentication of the raw material, the use of local strains which are well adapted to the particular environment of the Rif is highly recommended. However, there is no document that summarizes and clarifies the nomenclature and the characteristics of local Moroccan Cannabis. In addition, the recent adoption by Rif growers of improved hybrid cultivars is obliterating the traits and peculiarities of Moroccan Cannabis through genetic introgression. Conclusion: Summarizing and discussing the data from the literature on the characteristics of local Moroccan Cannabis varieties may be useful for their identification and the localization of the areas of the Rif region where their cultivation is still practiced.
52. Malík, Matěj and Velechovský, Jiří and Tlustoš, Pavel, The Overview of Existing Knowledge on Medical Cannabis Plants Growing, Plant, Soil and Environment, vol. 67 (2021), no. No. 8, pp. 425--442, August 2021. doi: 10.17221/96/2021-PSE. Abstract
    Matěj Malík, Jiří Velechovský, Pavel Tlustoš
53. Stack, George M. and Toth, Jacob A. and Carlson, Craig H. and Cala, Ali R. and {Marrero-González}, Mariana I. and Wilk, Rebecca L. and Gentner, Deanna R. and Crawford, Jamie L. and Philippe, Glenn and Rose, Jocelyn K. C. and Viands, Donald R. and Smart, Christine D. and Smart, Lawrence B., Season-Long Characterization of High-Cannabinoid Hemp (Cannabis Sativa L.) Reveals Variation in Cannabinoid Accumulation, Flowering Time, and Disease Resistance, GCB Bioenergy, vol. 13, no. 4, pp. 546--561, 2021. doi: 10.1111/gcbb.12793. Abstract
    Given the dramatic rise in high-cannabinoid hemp (Cannabis sativa L.) production in the last decade, there is an increasingly urgent need to characterize available germplasm and develop knowledge to accelerate the breeding of uniform and stable cultivars. Despite persistent cultivation of hemp cultivars for grain and or fiber around the world, the diversity and genetic underpinning of key traits for breeding and cultivation are poorly understood. For 30 high-cannabidiol hemp cultivars replicated on two field sites, we sought to evaluate yield, agronomic performance, and disease resistance while also conducting a detailed study of cannabinoid accumulation over the course of floral maturation. We observed significant variation in both within and among cultivars. During the growing season, the plants clustered into five groups by growth rate and varied in flowering time from photoperiod insensitive to photoperiod sensitive with very short critical photoperiods. Based on the observed ratio of total potential cannabidiol (CBD) to total potential tetrahydrocannabinol (THC), there was segregation for cannabinoid chemotype in some seeded cultivar populations. Analysis of cannabichromene (CBC) production revealed that some cultivars had a discretely lower CBD:CBC ratio than the others. There was a continuous range of powdery mildew severity by cultivar, with one that had little to no observed powdery mildew suggesting it might have genetic resistance. Biomass production at harvest was strongly influenced by location and cultivar, and there was variation by cultivar in the relative cannabinoid production in shoot tip samples compared with whole plant samples. While our results provide preliminary guidance regarding relative performance of current cultivars, our analyses indicate a need for additional hemp breeding to provide stable, uniform, and legally compliant cultivars with improved disease resistance and flowering times optimized for the latitudes of different growing locations.
54. Danziger, Nadav and Bernstein, Nirit, Plant Architecture Manipulation Increases Cannabinoid Standardization in ‘Drug-Type’ Medical Cannabis, Industrial Crops and Products, vol. 167, pp. 113528, September 2021. doi: 10.1016/j.indcrop.2021.113528. Abstract
    A major challenge in utilizing cannabis (Cannabis sativa L.) for modern medicine is the lack of standardization throughout the plant of cannabinoids, the unique therapeutic secondary metabolites in cannabis. The present study focused on the interplay between plant architecture modulation and standardization of the secondary metabolite profile in medical cannabis plants. Secondary metabolism is considerably affected by endogenous and exogenous factors, including positional-developmental aspects and microclimate. We hypothesized that manipulation of the plant architecture, which alter morphological and reproductive development, as well as micro-climate in the shoot will impact secondary metabolism and spatial standardization of the cannabinoids. Eight plant architecture-modulation treatments were evaluated for effects on in-planta concentrations and standardization of the cannabinoids, and on the morpho-physiological state of the plants. Two medical-cannabis genotypes of 'drug-type' medical cannabis were analyzed to evaluate genotypic sensitivity. The results reveal that plant architecture modulation can increase standardization of the cannabinoid profile in cannabis, thereby supporting the hypothesis. The architectural modulating treatments increased uniformity of cannabinoid concentrations in the plant by increasing concentrations at the lower parts of the plant. The cannabinoid profile was most affected by treatments that had the largest impact on plant structure such as the removal of primary and secondary branches. Decarboxylation of the cannabinoids in-planta was not affected by structural modulation. The spatial uniformity of cannabinoid concentrations throughout the cannabis plant is cannabinoid and genotype specific, and the effect of architecture modulation on cannabinoid standardization is genotype specific.
55. Baldini, Mario and Ferfuia, Claudio and Zuliani, Fabio and Danuso, Francesco, Suitability Assessment of Different Hemp (Cannabis Sativa L.) Varieties to the Cultivation Environment, Industrial Crops and Products, vol. 143, pp. 111860, January 2020. doi: 10.1016/j.indcrop.2019.111860. Abstract
    Hemp crop is nowadays spreading in many new areas and then the prediction of crop behavior and performance in different specific environments could be of interest. This study presents a methodology to assess hemp suitability to cropping environments by a combined approach of experimental trials and simulation. The experiment of this study involved six hemp varieties, evaluated for dual-purpose production (seed and stem) during two years of trials in North-East Italy. The results were exploited to develop and calibrate a simulation model able to evaluate hemp crop suitability to the trial environment. Yields obtained for hemp biomass and stems were similar to that of other European experiments, while seed production was shown to be slightly lower. Excessive temperature (daily maximum temperatures over 30\,°C) during the grain-filling phase would be one of the main factors affecting seed quality, limiting the seed oil accumulation. Parameters already available in the literature and data obtained from the present experiment were used in the modelling approach to estimate phenological parameters, seed production as affected by water stress, and seed oil content as a function of temperature during the grain-filling period. In order to evaluate the hemp crop suitability to the environment, a scenario analysis using historical meteorological data was performed to predict variability - with different irrigation regimes - of seed yield, seed oil content, maturity date and required seasonal irrigation volume, for each variety in the soil and climatic conditions of the trial site.
56. Abdollahi, Mahnaz and Sefidkon, Fatemeh and Calagari, Mohsen and Mousavi, Amir and Mahomoodally, M. Fawzi, Impact of Four Hemp (Cannabis Sativa L.) Varieties and Stage of Plant Growth on Yield and Composition of Essential Oils, Industrial Crops and Products, vol. 155, pp. 112793, November 2020. doi: 10.1016/j.indcrop.2020.112793. Abstract
    Essential oil of Cannabis sativa L. is a valuable bio-product due to its versatility, particularly in terms of its commercial values and potential applications in medicine, cosmetics and bio-pesticide. In this study, the effect of different stages of plant growth on essential oil yield and composition of four hemp varieties, (two monoecious non-native (Fedora 17 and its progeny) and two dioecious native (Fars and Yazd) samples) were investigated. The plant materials, consist of foliage in vegetative stage, inflorescent of flower in flowering stage and inflorescent of seeds in seeding stage were subjected to hydro-distillation. The essential oils were analyzed by GC and GC/MS. The oil yields varied from 0.40 \% (Fedora17) to 0.65 \% (Yazd). Interaction of cultivar and growth stage showed Fed17−2 at vegetative (0.86 \%) and Fed17 at flowering stage (0.20 \%), had the most and least oil content, respectively. Twenty-nine compounds were identified representing 81.9\%–99.5\% of the essential oils. The most abundant sesquiterpenes in the oils were E-caryophyllene (16.40 \%–44.70 \%), α-humulene (4.1 \%–15.1 \%) and Z-caryophyllene (2.4 \%–10.7 \%), while the major monoterpenes were (0.4–24.9 \%), β-pinene (4.6–24.3 \%) and 1,8-cineole (0.8 \%–9.3 \%) in all growth stages and cultivars. The ratio of sesquiterpenes to monoterpenes were found to decrease during the developing plants. In conclusion, there was no significant difference between mean oil yields of native and non-native samples, but non-native samples produced the highest oil yield in vegetative stage. E-caryophyllene was found at the highest percentage in the oils of non-native samples at vegetative stage. For all samples, the essential oils at vegetative stage contained much lower production of monoterpenes than flowering stage. In addition, to obtain the highest amount of β-pinene and 1,8-cineole, the flowering and seeding stages of hemp are recommended.
57. {García-Tejero}, I. F. and Durán Zuazo, V. H. and {Sánchez-Carnenero}, C. and Hernández, A. and {Ferreiro-Vera}, C. and Casano, S., Seeking Suitable Agronomical Practices for Industrial Hemp (Cannabis Sativa L.) Cultivation for Biomedical Applications, Industrial Crops and Products, vol. 139, pp. 111524, November 2019. doi: 10.1016/j.indcrop.2019.111524. Abstract
    The last climate change forecasting and the water scarcity scenarios in many semi-arid agricultural areas worldwide, such as the southern Spain, are promoting changes in the crop pattern of irrigated agriculture, being introduced new species characterized by its water-stress tolerance. In the last few years, there is an increasing interest in the cultivation of industrial hemp (Cannabis sativa L.) to manufacture therapeutic products derived from non-psychotropic cannabinoids and other secondary metabolites. This work evaluates the agronomical response of two industrial hemp cultivars (Carma and Ermes) subjected to different management practices. The trial was conducted during two seasons (2012–2013), from April to October. During this monitoring period, different plant densities (PD), sowing times (ST), irrigation doses (ID) and cropping systems (CS) were evaluated. At the end of each season, yield parameters (fresh and dry weight, dry weight of flowers and leaves, and the ratio between this late and total dry weight) were obtained. Moreover, the content of the most relevant cannabinoids (CBG, CBD, Δ9-THC, and CBC) were measured in both cultivars, and for the different growth conditions considered. According to our findings, both cultivars evidenced similar responses with significant improvements for the earliest ST (at the end of April) and the highest PD (33,333 and 16,667 plants ha−1). In relation to the ID, not clear responses were observed in terms of active biomass production, being the effects depending on the interactions between PD and ST. Finally, it was noticeable the improvements related to the active biomass production and cannabinoids content when plants were grown under plastic macro-tunnels (among 1.3 and 2 times higher, depending on the cultivar) in comparison to the obtained results under open field conditions.
58. Gao, Chunsheng and Cheng, Chaohua and Zhao, Lining and Yu, Yongting and Tang, Qing and Xin, Pengfei and Liu, Touming and Yan, Zhun and Guo, Yuan and Zang, Gonggu, Genome-Wide Expression Profiles of Hemp ({\emph{Cannabis Sativa}} L.) in Response to Drought Stress, International Journal of Genomics, vol. 2018, pp. e3057272, May 2018. doi: 10.1155/2018/3057272. Abstract
    Drought is the main environmental factor impairing hemp growth and yield. In order to decipher the molecular responses of hemp to drought stress, transcriptome changes of drought-stressed hemp (DS1 and DS2), compared to well-watered control hemp (CK1 and CK2), were studied with RNA-Seq technology. RNA-Seq generated 9.83, 11.30, 11.66, and 11.31\,M clean reads in the CK1, CK2, DS1, and DS2 libraries, respectively. A total of 1292 differentially expressed genes (DEGs), including 409 (31.66\%) upregulated and 883 (68.34\%) downregulated genes, were identified. The expression patterns of 12 selected genes were validated by qRT-PCR, and the results were accordant with Illumina analysis. Gene Ontology (GO) and KEGG analysis illuminated particular important biological processes and pathways, which enriched many candidate genes such as NAC, B3, peroxidase, expansin, and inositol oxygenase that may play important roles in hemp tolerance to drought. Eleven KEGG pathways were significantly influenced, the most influenced being the plant hormone signal transduction pathway with 15 differentially expressed genes. A similar expression pattern of genes involved in the abscisic acid (ABA) pathway under drought, and ABA induction, suggested that ABA is important in the drought stress response of hemp. These findings provide useful insights into the drought stress regulatory mechanism in hemp.
59. {Zuk-Golaszewska}, K. and Golaszewski, J., Cannabis Sativa L. – Cultivation and Quality of Raw Material, Journal of Elementology, vol. 23, no. 3, 2018. doi: 10.5601/jelem.2017.22.3.1500.
60. Campiglia, Enio and Radicetti, Emanuele and Mancinelli, Roberto, Plant Density and Nitrogen Fertilization Affect Agronomic Performance of Industrial Hemp (Cannabis Sativa L.) in Mediterranean Environment, Industrial Crops and Products, vol. 100, pp. 246--254, June 2017. doi: 10.1016/j.indcrop.2017.02.022. Abstract
    The renewed interest in industrial hemp (Cannabis sativa L.) is due to its large number of applications and for the wide range of agro-environmental conditions under which it can be cultivated. Two-year field experiments (2007 and 2008 growing seasons) were carried out in a Mediterranean environment of central Italy with the aim of assessing the impact of genotype, plant density and N fertilization on hemp yield, in terms of stems, inflorescences and seeds. The treatments consisted in: (a) seven genotypes (Epsilon68, Fedora17, Felina32, Ferimon, Futura75, Santhica27, and Uso31); three plant density (40, 80, and 120 plants m−2); two N fertilization levels (50 and 100kg of N ha−1). Physiological parameters, plant height, stem weight and diameter, inflorescence yield, seed yield and the characteristics of hemp and weed aboveground biomass were recorded. High plant density resulted in shorter plant height compared with low plant density (−41\%) as the hemp plants tended to reach the reproductive stage early at high density. At full flowering, stem yield ranged from 3.4 to 8.0 t ha−1 of dry matter and was positively correlated with the duration of vegetative phase, which tended to be high in the intermediate flowering genotypes (Epsilon68, Futura75 and Santhica27). Stem diameter was inversely correlated with plant density (6.7, 5.8 and 5.2mm at 40, 80 and 120 plants m−2, respectively). Conversely to stem yield, inflorescence and seed production proved to be higher in the early flowering genotypes (Fedora17, Felina32, Ferimon and Uso31) and increased as plant density increased. High N fertilization level had a positive impact on stems rather than inflorescence and seed yields (on average +28\%, +17\% and +4\% in 100kg of N ha−1 compared with 50kg of N ha−1 fertilization level, respectively). Farmers should consider making a dual-purpose production of stems and inflorescences or stems and seeds, even if it is clear that yield is related to the choice of genotype. Further research should be carried out to find various genotypes as well as flexible agronomical practices that are able to improve both traditional (stems) and innovative (inflorescences and seeds) hemp yields under Mediterranean conditions.
61. Liu, Fei-Hu and Hu, Hua-Ran and Du, Guang-Hui and Deng, Gang and Yang, Yang, Ethnobotanical Research on Origin, Cultivation, Distribution and Utilization of Hemp (Cannabis Sativa L.) in China, IJTK Vol.16(2) [April 2017], April 2017. url: http://nopr.niscpr.res.in/handle/123456789/40123. Abstract
    Hemp (\emph{Cannabis sativa } L.) is a dioecious and annual herbaceous plant in family Cannabinaceae, with more than 30 local names in China such as \emph{flame hemp}, \emph{string hemp}, \emph{cold hemp}, \emph{dew hemp} and \emph{yellow hemp}, etc. China is one of the native habitats of hemp and is the oldest country cultivating and utilizing hemp, with a history over 10,000 to 12,000 years. Hemp widely distributes in China, was mainly used for textiles, foods, paper and medicine from very early time, and became a very important crop of the ancient China. Based on a number of archaeological discoveries, ancient Chinese literatures and records, this paper summarized the knowledge about the origin, geographical distribution, cultivation and utilization of hemp in the ancient China, and concluded that China is the most conceivable origin center of hemp.
62. Chandra, Suman and Lata, Hemant and Khan, Ikhlas A. and ElSohly, Mahmoud A., Cannabis Sativa L.: Botany and Horticulture, pp. 79--100, 2017. doi: 10.1007/978-3-319-54564-6_3. Abstract
    As a plant, Cannabis is a highly variable species. It belongs to family Cannabaceae. Whether the genus Cannabis contains one species or more has been a matter of debate for a long time. The plant produces a unique class of terpenophenolic compounds called cannabinoids. A total of 565 Cannabis constituents have been isolated from Cannabis sativa so far, out of which 120 are phytocannabinoids. The plant has gained a lot of popularity in the last few decades for not only being an illicit drug but for its medicinal values from ancient times and a potential source for modern drugs to treat several targets for human wellness. The pharmacologic and therapeutic properties of preparations of C. sativa and Δ9-THC, its most psychoactive compound, have been extensively reviewed. An additional important cannabinoid in Cannabis of current interest is Cannabidiol (CBD) due to its reported activity as an antiepileptic agent, particularly its promise for the treatment of intractable pediatric epilepsy. Therefore, there is much interest in propagating compound based crops for medical purposes. In this chapter, the focus is laid on the botanical aspects of C. sativa and its cultivation for phytopharmaceuticals.
63. Tang, K. and Struik, P. C. and Yin, X. and Calzolari, D. and Musio, S. and Thouminot, C. and Bjelková, M. and Stramkale, V. and Magagnini, G. and Amaducci, S., A Comprehensive Study of Planting Density and Nitrogen Fertilization Effect on Dual-Purpose Hemp (Cannabis Sativa L.) Cultivation, Industrial Crops and Products, vol. 107, pp. 427--438, November 2017. doi: 10.1016/j.indcrop.2017.06.033. Abstract
    Harvesting hemp (Cannabis sativa L.) for both stems and seeds is now a common practice in Europe while crop management strategies for dual-purpose hemp cultivation have not been properly addressed so far. In the present study, the effects of planting density and nitrogen fertilization on hemp stem and seed yields were tested with the cultivars Futura 75 and/or Bialobrzeskie in eight contrasting environments (Italy in 2013; Italy and Latvia in 2014; Italy (two sites), Latvia, the Czech Republic, and France in 2015). Stem yield ranged between 1.3 and 22.3Mgha−1. The effects of planting density and nitrogen fertilization on stem yield did not interact significantly with each other, or with cultivar and harvest time. Increasing planting density from 30 to 120 plants m−2 and increasing nitrogen fertilization rate from 0 to 60kgNha−1 increased stem yield by 29\% and 32\%, respectively. Further increase in planting density and nitrogen fertilization did not result in a significant increase in stem yield. Seed yield ranged from 0.3 to 2.1Mgha−1. The seed yield was not affected significantly by planting density between 30 and 240 plants m−2. Although the seed yield showed an increasing trend with increasing nitrogen fertilization, the effects of nitrogen fertilization on seed yield were not statistically significant. To grow hemp as a dual-purpose crop it is recommended to plant 90–150 plants m−2 across all tested environments. Nitrogen fertilization rate at 60kg N ha−1 was generally sufficient in the tested environments whereas further optimization of nitrogen fertilization requires accurate assessment of plant nitrogen status. To facilitate assessing plant nutritional status, a critical nitrogen dilution curve was determined for hemp and a practical method to determine nitrogen nutritional status was discussed.
64. Tang, K. and Struik, P. C. and Yin, X. and Thouminot, C. and Bjelková, M. and Stramkale, V. and Amaducci, S., Comparing Hemp (Cannabis Sativa L.) Cultivars for Dual-Purpose Production under Contrasting Environments, Industrial Crops and Products, vol. 87, pp. 33--44, September 2016. doi: 10.1016/j.indcrop.2016.04.026. Abstract
    Interest in hemp as a multi-purpose crop is growing worldwide and for the first time in 2015 it was cultivated in Europe on more than 20.000ha as a dual-purpose crop, for the seeds and for the fibre. In the present study, fibre and seed productivity of 14 commercial cultivars were tested in four contrasting European environments (Latvia, the Czech Republic, France, Italy). At full flowering, the stem yield ranged from 3.7Mgha−1 to 22.7Mgha−1, the bast fibre content ranged from 21\% to 43\%, and the bast fibre yield ranged from 1.3Mgha−1 to 7.4Mgha−1. When harvesting was postponed from full flowering until seed maturity, the stem yield of monoecious cultivars significantly increased but in dioecious cultivars it decreased at all tested sites, except for Italy. Only the early cultivars Fedora 17 and Markant produced seed in the most northern location Latvia. The seed yield ranged from 0.3Mgha−1 to 2.4Mgha−1 in Italy, France and the Czech Republic. The cultivar effect on stem and seed yield was mainly determined by the genetic variation in time of flowering. Stem yield at full flowering was strictly related to the duration of the vegetative phase while seed yield was lowest in the late flowering cultivar. The late cultivar CS is suitable for stem and fibre production as it had the highest stem yield at full flowering in all locations. Both Fedora 17 and Futura 75 are candidate cultivars for dual-purpose production in Italy, France and the Czech Republic, with Fedora 17 being more suitable for seed production and Futura 75 for fibre production. The application of modelling to design production strategies for dual-purpose hemp is promising. However, accurate parameterisation is needed based on large data sets and diverse genetic background.
65. Butsic, Van and Brenner, Jacob C., Cannabis (Cannabis Sativa or C. Indica) Agriculture and the Environment: A Systematic, Spatially-Explicit Survey and Potential Impacts, Environmental Research Letters, vol. 11, no. 4, pp. 044023, April 2016. doi: 10.1088/1748-9326/11/4/044023. Abstract
    Cannabis agriculture is a multi-billion dollar industry in the United States that is changing rapidly with policy liberalization. Anecdotal observations fuel speculation about associated environmental impacts, and there is an urgent need for systematic empirical research. An example from Humboldt County California, a principal cannabis-producing region, involved digitizing 4428 grow sites in 60 watersheds with Google Earth imagery. Grows were clustered, suggesting disproportionate impacts in ecologically important locales. Sixty-eight percent of grows were {$>$}500 m from developed roads, suggesting risk of landscape fragmentation. Twenty-two percent were on steep slopes, suggesting risk of erosion, sedimentation, and landslides. Five percent were {$<$}100 m from threatened fish habitat, and the estimated 297 954 plants would consume an estimated 700 000 m3 of water, suggesting risk of stream impacts. The extent and magnitude of cannabis agriculture documented in our study demands that it be regulated and researched on par with conventional agriculture.
66. Potter, David J., A Review of the Cultivation and Processing of Cannabis (Cannabis Sativa L.) for Production of Prescription Medicines in the UK, Drug Testing and Analysis, vol. 6, no. 1-2, pp. 31--38, 2014. doi: 10.1002/dta.1531. Abstract
    The quality demands of the pharmaceutical industry require prescription medicines to be consistent in their active ingredient content. Achieving this, using raw cannabis as a feedstock, is especially challenging. The plant material is extremely inhomogeneous, and the ratios of active ingredients are affected by a range of factors. These include the genetics of the plant, the growing and storage conditions, the state of maturity at harvest, and the methods used to process and formulate the material. The reasons for this variability are described, with particular emphasis on the botanical considerations. To produce the complex botanical medicine Sativex®, which contains the cannabinoids Δ9–tetrahydrocannabinol (THC) and cannabidiol (CBD) and a range of other ingredients, GW Pharmaceuticals had to manage these variables. This medicine, for the treatment of spasticity due to multiple sclerosis, is the first cannabis-based medicine to be approved in the UK. The company's methodology for producing this and other chemotypes is described. Copyright © 2013 John Wiley \& Sons, Ltd.
67. Cosentino, Salvatore Luciano and Testa, Giorgio and Scordia, Danilo and Copani, Venera, Sowing Time and Prediction of Flowering of Different Hemp (Cannabis Sativa L.) Genotypes in Southern Europe, Industrial Crops and Products, vol. 37, no. 1, pp. 20--33, May 2012. doi: 10.1016/j.indcrop.2011.11.017. Abstract
    Fibre hemp can be grown under a wide range of agro-ecological conditions, but it requires special attention for several physiological features and crop management. A management strategy in order to reduce inputs and thus achieve acceptable yield could be achieved optimizing sowing time. With this respect, the effects of sowing date on hemp biology and yield was studied, using two monoecious and two dioecious genotypes. Field experiments were carried out in two subsequent years (2003–2004) in South of Italy, using drip irrigation system. Sowing time, in the two year period, ranged between March 10th and July 22nd. Optimal sowing time was observed between the end of April and the first three weeks of May; in that range, the dioecious Fibranova yielded the most in terms of aboveground biomass and stem dry yield, followed by Tiborszallasi, while the two monoecious showed the lowest yield. On the contrary, before and after that period, the shorter day length caused an early floral induction that strongly reduced stem and fibre elongation, and thus aboveground dry biomass and consequently stem yields. Based on this study a simulation model focusing on flowering prediction in Mediterranean environment was developed.
68. Chandra, Suman and Lata, Hemant and Khan, Ikhlas A. and ElSohly, Mahmoud A., Temperature Response of Photosynthesis in Different Drug and Fiber Varieties of Cannabis Sativa L., Physiology and Molecular Biology of Plants, vol. 17, no. 3, pp. 297--303, July 2011. doi: 10.1007/s12298-011-0068-4. Abstract
    The temperature response on gas and water vapour exchange characteristics of three medicinal drug type (HP Mexican, MX and W1) and four industrial fiber type (Felinq 34, Kompolty, Zolo 11 and Zolo 15) varieties of Cannabis sativa, originally from different agro-climatic zones worldwide, were studied. Among the drug type varieties, optimum temperature for photosynthesis (Topt) was observed in the range of 30–35~°C in high potency Mexican HPM whereas, it was in the range of 25–30~°C in W1. A comparatively lower value (25~°C) for Topt was observed in MX. Among fiber type varieties, Topt was around 30~°C in Zolo 11 and Zolo 15 whereas, it was near 25~°C in Felinq 34 and Kompolty. Varieties having higher maximum photosynthesis (PN max) had higher chlorophyll content as compared to those having lower PN max. Differences in water use efficiency (WUE) were also observed within and among the drug and fiber type plants. However, differences became less pronounced at higher temperatures. Both stomatal and mesophyll components seem to be responsible for the temperature dependence of photosynthesis (PN) in this species, however, their magnitude varied with the variety. In general, a two fold increase in dark respiration with increase in temperature (from 20~°C to 40~°C) was observed in all the varieties. However, a greater increase was associated with the variety having higher rate of photosynthesis, indicating a strong association between photosynthetic and respiratory rates. The results provide a valuable indication regarding variations in temperature dependence of PN in different varieties of Cannabis sativa L.
69. Mansouri, Hakimeh and Asrar, Zahra and Szopa, Jan, Effects of ABA on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis Sativa L. at Flowering Stage, Plant Growth Regulation, vol. 58, no. 3, pp. 269--277, July 2009. doi: 10.1007/s10725-009-9375-y. Abstract
    This work examined the effects of exogenously applied abscisic acid (ABA) on the content of chlorophyll, carotenoids, α-tocopherol, squalene, phytosterols, Δ9-tetrahydrocannabinol (THC) concentration, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS) activity in Cannabis sativa L. at flowering stage. Treatment with 1 and 10~mg~l−1 ABA significantly decreased the contents of chlorophyll, carotenoids, squalene, stigmasterol, sitosterol, and HMGR activity in female cannabis plants. ABA caused an increase in α-tocopherol content and DXS activity in leaves and THC concentration in leaves and flowers of female plants. Chlorophyll content decreased with 10~mg~l−1 ABA in male plants. Treatment with 1 and 10~mg~l−1 ABA showed a decrease in HMGR activity, squalene, stigmasterol, and sitosterol contents in leaves but an increase in THC content of leaves and flowers in male plants. The results suggest that ABA can induce biosynthesis of 2-methyl-d-erythritol-4-phosphate (MEP) pathway secondary metabolites accumulation (α-tocopherol and THC) and down regulated biosynthesis of terpenoid primary metabolites from MEP and mevalonate (MVA) pathways (chlorophyll, carotenoids, and phytosterols) in Cannabis sativa.
70. Amaducci, Stefano and Colauzzi, Michele and Bellocchi, Gianni and Venturi, Gianpietro, Modelling Post-Emergent Hemp Phenology (Cannabis Sativa L.): Theory and Evaluation, European Journal of Agronomy, vol. 28, no. 2, pp. 90--102, February 2008. doi: 10.1016/j.eja.2007.05.006. Abstract
    Temperature and photoperiod can be used to simulate post-emergent hemp (Cannabis sativa L.) phenology. With reference to hemp in Italy, our main objective was to model field crops grown under a range of temperature and day length regimes. Dates of emergence and 50\% of flowering were collected at Cadriano (Bologna) from serially sown field experiments (1996–1999, 2003–2005) on five cultivars: Carmagnola (late maturity), Felina 34 (medium maturity), Fibranova (late maturity), Futura (medium-late maturity), and Tiborszallasi (medium-late maturity). The database of phenological records was segregated into calibration and validation subsets. A phenology model was developed which utilises the beta function for response to hourly air temperature, and a switch-off function for response to day length. The life cycle of hemp from emergence to 50\% of flowering was defined in terms of physiological development days (chronological days at the optimum photoperiod and temperature) and considered in three phases: juvenile phase (BVP), photo-sensitive phase (PIP), flower development phase (FDP). Critical temperatures (Tb, base; To, optimum, Tc, ceiling), which did not vary widely across phases and cultivars, were estimated as common values: Tb=1.9°C for BVP and 11.3°C for the other phases, To=26.4°C and Tc=40.0°C for all phases. Other parameters, i.e. day length of half-maximum development rate at PIP, and physiological development days for FDP, were also estimated as common values for all cultivars. Different genotypes were mainly characterised for the sensitivity to photoperiod (shape parameter n) and BVP length. With n∼50, Felina 34 and Futura are regarded as low sensitive cultivars. Tiborszallasi was estimated as the highest sensitive cultivar (n close to 70), whereas Carmagnola and Fibranova showed an intermediate sensitivity (n∼62). Felina 34 also differentiated for its relatively short BVP length at optimum conditions, i.e. ∼13 days; duration of about 20 days was the estimate for the other cultivars. Model performance against calibration dataset was good (percent relative root mean square in the range ∼6–20\%), and comparison against independent data also confirmed the general applicability of this model. Owing to the importance of flowering date in hemp management techniques, these results can be used in decision support for hemp production though further evaluation of the model under a variety of latitudes is required.
71. Amaducci, Stefano and Zatta, Alessandro and Pelatti, Federica and Venturi, Gianpietro, Influence of Agronomic Factors on Yield and Quality of Hemp (Cannabis Sativa L.) Fibre and Implication for an Innovative Production System, Field Crops Research, vol. 107, no. 2, pp. 161--169, May 2008. doi: 10.1016/j.fcr.2008.02.002. Abstract
    Research and development of an innovative production system for hemp (Cannabis sativa L.) fibre for textile use requires the integration of multidisciplinary knowledge from cultivation technique to realization of end products. Research was carried out to study the effect of the agronomic factors cultivation year (2003–2004), genotype (Futura 75 and Tiborszallasi), plant population (120, 240 and 360plantsm−2) and harvesting time (beginning and full flowering) on fibre yield and quality in the whole hemp stem, and in the basal and apical stem portions separately. The study of separate stem portions was done to determine the effect on fibre quality of an innovative harvesting and processing system in which hemp stems are cut in two portions of approximately 1m at harvest to enable processing on modern flax scutching lines. Stem and fibre yield were affected by most of the agronomic factors. The extreme drought experienced in the first year reduced stem and fibre yield, but stems had higher percentage of fibre (16.5\%), that were finer (22.9μm) and with a higher degree of maturity (73.6\%) in 2003 than in 2004 (respectively 16.0\%; 24.5μm; 55.8\%). Between the two genotypes under trial the monoecious Futura 75 largely out yielded the dioecious Tiborszallasi in both years. The latter however had finer primary fibres and less secondary ones. In both genotypes primary fibres maturity and quantity of secondary fibres increased at later harvest. Plant population affected stem biometrics and fibre characteristics, with finer fibres and less secondary growth at higher stands. It can be concluded that cultivation technique can be exploited in order to maximize the quality and yield of stems destined for the innovative harvesting and processing system herein described.
72. Schäfer, Tanja and Honermeier, Bernd, Effect of Sowing Date and Plant Density on the Cell Morphology of Hemp (Cannabis Sativa L.), Industrial Crops and Products, vol. 23, no. 1, pp. 88--98, January 2006. doi: 10.1016/j.indcrop.2005.04.003. Abstract
    A range of papers report a major effect of agronomic procedures on the growth, biomass, fibre yield and fibre quality of hemp. The aim of this paper is to examine the influence of sowing date, seed density and year on histological characteristics of hemp (size of the different layers in the cross section of the stem, area of primary fibre cells and their cell lumen, size of fibre cell wall). It was realised that a higher seed density resulted in a reduction of almost all layers in the cross section of the stem. A similar effect was caused by the drought 1998, whereas the sowing date had almost no influence on the size of the various layers. The area of primary fibre cells was smaller in 1998. Sowing date and seed density did not affect the fibre cell area and area of cell lumen in the way mentioned above during the 2 years of the trial. During this period the size of cell wall was mainly determined by climatic conditions. Higher elongation and higher tensile strength of hemp planted at higher seed density is probably caused by differences in primary cell morphology.
73. Amaducci, Stefano and Pelatti, Federica and Bonatti, Piera Medeghini, Fibre Development in Hemp (Cannabis Sativa L.) as Affected by Agrotechnique, Journal of Industrial Hemp, vol. 10, no. 1, pp. 31--48, June 2005. doi: 10.1300/J237v10n01_04. Abstract
    This paper reports the preliminary results of a microscopic study carried out on stem cross sections of hemp. Stems were harvested from two field experiments carried out in 2001 and 2002 in the north of Italy to compare the monoecious genotype Futura 75 over four plant populations. Fibre characteristics such as cell shape, diameter, maturation and quantity of secondary fibre tended to vary with harvest time, plant density, and between and within internodes. After the end of internode elongation, fibre cells changed from oblong to round shaped and fibre maturation started and progressed to a maximum level. At various moments of the growing cycle, fibre maturity and presence of secondary fibre seemed higher at lower internodes and plant densities.
74. Arru, Laura and Rognoni, Sara and Baroncini, Micaela and Bonatti, Piera Medeghini and Perata, Pierdomenico, Copper Localization in Cannabis Sativa L. Grown in a Copper-Rich Solution, Euphytica, vol. 140, no. 1, pp. 33--38, January 2004. doi: 10.1007/s10681-004-4752-0. Abstract
    With the aim to examine its potential as a renewable resource to decontaminate polluted soils, electron microscopy combined with X-ray microanalysis was used to investigate the localization of copper in Cannabis sativa grown in hydroponic copper-rich culture. Cu was found to accumulate preferentially in the upper leaf epidermal cells; it was also detected in spiculae and in abaxial trichomes too. Primary bast fibres seem to be not involved in copper accumulation.
75. Denton, Tricia M. and Schmidt, Susanne and Critchley, Christa and Stewart, George R., Natural Abundance of Stable Carbon and Nitrogen Isotopes in Cannabis Sativa Reflects Growth Conditions, Functional Plant Biology, vol. 28, no. 10, pp. 1005--1012, 2001. doi: 10.1071/pp01066. Abstract
    Stable carbon and nitrogen isotope signatures (δ13C and δ15N) of Cannabis sativa were assessed for their usefulness to trace seized Cannabis leaves to the country of origin and to source crops by determining how isotope signatures relate to plant growth conditions. The isotopic composition of Cannabis examined here covered nearly the entire range of values reported for terrestrial C3 plants. The δ13C values of Cannabis from Australia, Papua New Guinea and Thailand ranged from –36 to –25‰, and δ 15N values ranged from –1.0 to 15.8‰. The stable isotope content did not allow differentiation between Cannabis originating from the three countries, but δ13C values of plantation-grown Cannabis differed between well-watered plants (average δ13C of–30.0‰) and plants that had received little irrigation (average δ13C of –26.4‰). Cannabis grown under controlled conditions had δ13C values of –32.6 and –30.6‰ with high and low water supply, respectively. These results indicate that water availability determines leaf 13C in plants grown under similar conditions of light, temperature and air humidity. The δ13C values also distinguished between indoor- and outdoor-grown Cannabis; indoor-grown plants had overall more negative δ13C values (average –31.8‰) than outdoor-grown plants (average –27.9‰). Contributing to the strong 13C-depletion of indoor-grown plants may be high relative humidity, poor ventilation and recycling of 13C-depleted respired CO2. Mineral fertilizers had mostly lower δ15N values (–0.2 to 2.2‰) than manure-based fertilizers (7.6 to 22.7‰). It was possible to link δ15N values of fertilizers associated with a crop site to soil and plant δ15N values. The strong relationship between soil, fertilizer, and plant δ15N suggests that Cannabis δ15N is determined by the isotopic composition of the nitrogen source. The distinct d15N values measured in Cannabis crops make δ15N an excellent tool for matching seized Cannabiswith a source crop. A case study is presented that demonstrates how δ13C and δ15N values can be used as a forensic tool.
76. Struik, P. C. and Amaducci, S. and Bullard, M. J. and Stutterheim, N. C. and Venturi, G. and Cromack, H. T. H., Agronomy of Fibre Hemp (Cannabis Sativa L.) in Europe, Industrial Crops and Products, vol. 11, no. 2, pp. 107--118, March 2000. doi: 10.1016/S0926-6690(99)00048-5. Abstract
    Fibre hemp may yield up to 25 t above ground dry matter per hectare (20 t stem dry matter ha−1) which may contain as much as 12 t ha−1 cellulose, depending on environmental conditions and agronomy. Its performance is affected by the onset of flowering and seed development. Effects of cultivar and management on yield and quality were tested at three contrasting sites in Italy, the Netherlands and the UK in three years, making use of standardised protocols for experimental design and research methodology. Highest yields (up to 22.5 t dry matter ha−1) were obtained in Italy when later cultivars were used. Attainable yields proved slightly lower in the Netherlands and much lower in the UK. The quality of the cellulose was relatively stable over the growing season, but lignification may proceed rapidly some time after flowering. Crop development was very rapid and crops maintained green leaf area for a long time, thus radiation interception was considerable. The radiation use efficiency changed during development. It was lower after flowering (about 1.0 g MJ−1 PAR) than before (about 2.2 g MJ−1 PAR). Growing earlier cultivars to obtain some seed set advanced the reduction in radiation use efficiency. Nitrogen proved to affect yield only slightly. A relatively small amount of fertiliser will be adequate to cover the crop’s needs. Plant density declined during growth in a site-specific manner when it was high initially. Very low plant densities may not show this self-thinning but reduced yield and (especially) quality. Final plant densities were proven to depend more on initial plant stands than expected from literature. This was true at all three contrasting sites and in the different years. Nitrogen and plant density hardly interacted within one site. Results suggest that hemp can yield large quantities of useful cellulose when ecologically adapted cultivars are sown in proper plant densities. The cultivation is environmentally friendly with little harmful accumulation or emission of chemical inputs. More research on ideotyping is required and breeding efforts should be broadened.
77. Ranalli, Paoli, Advances in Hemp Research, January 1999. Abstract
    Offering up-to-date information on the uses and composition of the plant, Advances in Hemp Research provides growers, researchers, manufacturers, and suppliers with methods and data for the processing and cultivation of hemp for textile and paper products. You will learn how recent advances in germplasm resources, breeding methods, and the improvem
78. Cromack, H. T. H, The Effect of Cultivar and Seed Density on the Production and Fibre Content of Cannabis Sativa in Southern England, Industrial Crops and Products, vol. 7, no. 2, pp. 205--210, January 1998. doi: 10.1016/S0926-6690(97)00049-6. Abstract
    Interest has increased in Cannabis sativa L. due to renewed demand for natural fibres and deregulation of the crop in the UK. The UK production potential and agronomic requirements of the crop were investigated by growing the cultivars Fedora 19, Felina 34, Futura 77, Uniko B and Kompolti, with a range of maturity and genetic backgrounds, at 200 and 400 seeds m−2 in a replicated experiment over the period 1994–1995. Adverse seedbed conditions in 1994 resulted in a low percentage of seed producing plants, but under good conditions in 1995 a high percentage of seeds produced plants. Plant densities ranged from 52 to 120 m−2 in 1994 when little self-thinning occurred. In 1995 plant densities ranged from 160 to 347 m−2 and were subject to significant self-thinning, particularly at the higher seed density, which reduced the maximum stem population at harvest to 227 m−2. Cultivar effects on stem density were small. Stem yields between 6.3 and 12.9 t DM ha−1 and bast fibre yields between 1.3 and 3.5 t ha−1 were recorded, the later maturing Futura 77 and Kompolti consistently produced the highest yields. Under the conditions of these experiments a stem density in excess of 100 m−2 was required for maximum yield. Chemical stem analysis indicated that genetic differences had a greater impact on the content of stem bast fibre than stem density.
79. Raman, Amala, The Cannabis Plant: Botany, Cultivation and Processing for Use, 1998. Abstract
    Cannabis plants have been cultivated in Europe, Asia, Africa and the Americas for hundreds, perhaps even thousands of years as a source of three main products-hemp fibre, cannabis seeds and medicinal or narcotic preparations (Fairbairn, 1976). Hemp fibre is obtained from cannabis stems, and has been used over the centuries for the production of textiles, rope and sacking. It is strong and durable, composed of about 70\% cellulose and reaches lengths of 3-15 feet (Schultes, 1970). The fibre has been used in the past to make paper, and has been proposed as a replacement for wood pulp in modern paper production (Kovacs, 1992). However, there are many technological limitations to be overcome before this becomes a commercially viable proposition (Judt, 1995). The “seeds” (which technically are the fruit or achene) may be roasted and consumed by man, used as birdseed or anglers’ bait or pressed to yield a greenish yellow, fixed oil which has been used in foodstuffs and in varnishes, paints and soap (Schultes, 1970; Fairbairn, 1976). Cannabis leaves and flowering tops and preparations derived from them have many pharmacological effects in man, including narcotic properties; the latter is the most widely known use of cannabis in the present day.
80. Werf, Hmg Van Der and Mathussen, E W J M and Haverkort, A J, The Potential of Hemp (Cannabis Sativa L.) for Sustainable Fibre Production: A Crop Physiological Appraisal, Annals of Applied Biology, vol. 129, no. 1, pp. 109--123, 1996. doi: 10.1111/j.1744-7348.1996.tb05736.x. Abstract
    Summary. Hemp (Cannabis sativa L.) fibre can be used as a raw material for paper and textile production. A comprehensive research programme in the Netherlands has concluded that fibre hemp is a potentially profitable crop, having the right profile to fit into sustainable farming systems. This paper presents an appraisal of the crop physiological characteristics and the agronomic potential of hemp. Parameter values of basic crop physiological characteristics such as light interception potential, radiation use efficiency and dry matter partitioning coefficients are given. The effect of crop management decisions such as cultivar choice, sowing date, plant density, and harvest date on the value of these parameters is discussed. A simple crop growth model was used to assess the yield potential of hemp for the climate of the Netherlands. Calculations made for a non-stressed late-flowering hemp crop sown on 15 April and harvested on 15 September give a stem dry matter yield of 17.1 t ha-1. The effects of advancing or delaying sowing or harvest date on stem yield were calculated. Crop physiological characteristics of hemp are compared to those of kenaf (Hibiscus cannabinus L.). Radiation use efficiency and dry matter partitioning coefficients of the two crops are similar. Base temperatures for development and growth are lower in hemp than in kenaf. In a temperate climate with cool springs, canopy establishment will be more rapid in hemp than in kenaf. Hemp seems an excellent candidate to fill the niche for an annual fibre crop in a temperate climate.
81. {van der Werf}, H. M. G. and Wijlhuizen, M. and {de Schutter}, J. A. A., Plant Density and Self-Thinning Affect Yield and Quality of Fibre Hemp (Cannabis Sativa L.), Field Crops Research, vol. 40, no. 3, pp. 153--164, March 1995. doi: 10.1016/0378-4290(94)00103-J. Abstract
    To ascertain the reasons for the high plant mortality in fibre-hemp (Cannabis sativa L.) crops in the Netherlands changes in biomass yield, plant mortality and plant morphology were investigated in a hemp cultivar grown at initial densities of 10, 30, 90 and 270 plants m−2. At 90 plants m−2 this cultivar was compared with a high bast-fibre cultivar and a late-flowering cultivar. Rate of canopy establishment and early growth rate increased with increasing plant density. At 90 and 270 plants m−2, plants died as a result of self-thinning. Self-thinning was associated with a reduced crop growth rate. In self-thinning stands, dry biomass (B, g m−2) was related to density of surviving plants (D, m−2) as log B = 3.81 − 0.304 log D. At the same crop growth rate, the rate at which plants died from self-thinning was higher in hemp than in other herbaceous dicots. The proportion of stem in the total dry matter increased with increasing plant density. Stem yield was maximum at 90 plants m−2. Stem quality improved with increasing density as the bark content in the stem increased, but self-thinning reduced the bark content in the stem. Optimum plant density was close to that resulting after self-thinning. In hemp the relationship between yield and optimum plant density is approximated by the equation of its self-thinning line. The late-flowering cultivar yielded more than the other two cultivars because it grew faster during the latter part of the growing season.
82. Meijer, W. J. M. and {van der Werf}, H. M. G. and Mathijssen, E. W. J. M. and {van den Brink}, P. W. M., Constraints to Dry Matter Production in Fibre Hemp (Cannabis Sativa L.), European Journal of Agronomy, vol. 4, no. 1, pp. 109--117, January 1995. doi: 10.1016/S1161-0301(14)80022-1. Abstract
    Fibre hemp (Cannabis sativa L.) may be an alternative to wood as a raw material for pulp and paper production. Research was done to assess the potential productivity of fibre hemp and to identify constraints to that productivity. Growth analyses were done on hemp crops in three consecutive years, using several cultivars and seeding rates. In 1987 the crops suffered severely from fungal diseases ; stem dry matter yields were 9.4 t ha−1. In 1988 and 1989 fungicides were applied and stem dry matter yields were respectively 11.9 t ha−1 and 13.6 t ha−1. The number of living plants m−2 ranged from 86 to 823 at emergence, depending on treatment, and from 38 to 102 at final harvest. Increased seeding rates led to earlier canopy closure and higher initial biomass production, but more plants died during the growing season and the stem yield at final harvest was not affected by seeding rate. Average radiation-use efficiency (RUE; above-ground accumulated dry matter divided by intercepted photosynthetically active radiation) for the entire growing season under favourable growing conditions was 1.9 g MJ−1, which is low compared to other C3 crops. Leaf photosynthesis rates at saturating light intensities, however, were high: 30 kg CO2ha−1 h−1. The light extinction coefficient of hemp canopies was high (0.96), and may have reduced canopy photosynthesis rate and RUE. The high lignin content of the stems, and the high fat and protein content in the seed, also reduced RUE. Furthermore, dry matter invested in the tap root and in shed dead leaves was ignored. The RUE was also underestimated because an appreciable proportion of the biomass of plants that died during the growing season could not be collected. More research is needed to optimize cultural practices in fibre hemp.
83. Coffman, C. B. and Gentner, W. A., Responses of Greenhouse-Grown Cannabis Sativa L. to Nitrogen, Phosphorus, and Potassium, Agronomy Journal, vol. 69, no. 5, pp. 832--836, 1977. doi: 10.2134/agronj1977.00021962006900050026x. Abstract
    Growers of illegal Cannabis sativa L. use various cultural practices to maximize crop production. The objective of this study was to evaluate the morphological and biochemical responses of greenhouse grown C. sativa to soil incorporated N, P, and K as they reflect the geographical origin of Cannabis derivatives. Fertilizers were blended with Ap horizon soil from a Gilpin silt loam before placement in 12-cm pots. NH4NO3-N was applied at 0, 25, and 125 ppm. Phosphorus and K from super-phosphate and KCI, respectively, were applied at 0, 50, and 150 ppm. Forty-five-day-old anthesic Cannabis plants were harvested and combined leaf and flower tissues were analyzed for cannabidiol (CBD) and Δ9-tetrahydrocanna-binol (Δ9THC). Nine essential elements were also measured in plant tissue. Plant growth, tissue yield, and concentration of CBD and Δ9THC were positively correlated with extractable P2O5 (p {$<$} 0.01). Phosphorus concentrations in tissue were similarly related to yield of dry matter and cannabinoid concentrations. Uptake of K was positively correlated with extractable K2O across all treatment levels (r=0.40**), but was negatively correlated with tissue yield (r=–0.36**). Growth and tissue yields were negatively related to total plant N (p{$<$} 0.01). Levels of extractable P2O5, Mn, B, and Mg were associated with specific concentration ranges for several plant elements plus Δ9THC. Thus, it was possible to partially characterize a soil by tissue analysis. For example, all of the plants grown on soil with less than 100 ppm of extractable P2O5 contained less than 8,000 ppm Δ9THC. Usefulness of such relationships will be dependent upon extensive evaluation of Cannabis on different soils under various cultural conditions. At this time, the reliability required for determination of origin of Canabis derivatives via chemical analysis does not exist when only essential elements and cannabinoids are considered.
84. Bazzaz, F. A. and Dusek, D. and Seigler, D. S. and Haney, A. W., Photosynthesis and Cannabinoid Content of Temperate and Tropical Populations of Cannabis Sativa, Biochemical Systematics and Ecology, vol. 3, no. 1, pp. 15--18, May 1975. doi: 10.1016/0305-1978(75)90036-8. Abstract
    Four populations of Cannabis sativa L. grown from seeds collected in Panama, Jamaica, Nepal, and east central Illinois were grown under controlled conditions in growth chambers. One set was grown under warm conditions (32° day and 23° night) and the other set was grown under lower temperatures (23° day and 16° night). CO2 exchange and transpiration were examined under various temperatures and light intensities. Observations on growth, and analyses for chlorophyll and Δ1THC (tetrahydrocannabinol) content were made. Under warm growth conditions, the central Illinois population had the highest photosynthetic rate at all temperatures investigated. The Nepal population had intermediate rates, while the Jamaica and the Panama populations had the lowest rate. The Jamaica and Panama populations had insignificant changes in photosynthetic response to changes in temperatures between 15° and 30°. Under cool growing conditions the central Illinois population had the highest rate of photosynthesis with a definite peak at 25°. Nepal plants had intermediate rates of photosynthesis, while the Panama and Jamaica populations had the lowest rate. Differences in chlorophyll and drug content were also significant between these populations. From these data it is suggested that the four populations can be grouped into different ecotypes genetically adapted to their respective environments.
85. Toonen, Marcel and Ribot, Simon and Thissen, Jac, Yield of Illicit Indoor Cannabis Cultivation in The Netherlands, Journal of Forensic Sciences, vol. 51, no. 5, pp. 1050--1054, 2006. doi: 10.1111/j.1556-4029.2006.00228.x. Abstract
    ABSTRACT: To obtain a reliable estimation on the yield of illicit indoor cannabis cultivation in The Netherlands, cannabis plants confiscated by the police were used to determine the yield of dried female flower buds. The developmental stage of flower buds of the seized plants was described on a scale from 1 to 10 where the value of 10 indicates a fully developed flower bud ready for harvesting. Using eight additional characteristics describing the grow room and cultivation parameters, regression analysis with subset selection was carried out to develop two models for the yield of indoor cannabis cultivation. The median Dutch illicit grow room consists of 259 cannabis plants, has a plant density of 15 plants/m2, and 510 W of growth lamps per m2. For the median Dutch grow room, the predicted yield of female flower buds at the harvestable developmental stage (stage 10) was 33.7 g/plant or 505 g/m2.
86. Vanhove, Wouter and Van Damme, Patrick and Meert, Natalie, Factors Determining Yield and Quality of Illicit Indoor Cannabis (Cannabis Spp.) Production, Forensic Science International, vol. 212, no. 1, pp. 158--163, October 2011. doi: 10.1016/j.forsciint.2011.06.006. Abstract
    Judiciary currently faces difficulties in adequately estimating the yield of illicit indoor cannabis plantations. The latter data is required in penalization which is based on the profits gained. A full factorial experiment in which two overhead light intensities, two plant densities and four varieties were combined in the indoor cultivation of cannabis (Cannabis spp.) was used to reveal cannabis drug yield and quality under each of the factor combinations. Highest yield was found for the Super Skunk and Big Bud varieties which also exhibited the highest concentrations of Δ9-tetrahydrocannabinol (THC). Results show that plant density and light intensity are additive factors whereas the variety factor significantly interacts with both plant density and light intensity factors. Adequate estimations of yield of illicit, indoor cannabis plantations can only be made if upon seizure all factors considered in this study are accounted for.
87. Farag, Sayed and Kayser, Oliver, Cultivation and Breeding of Cannabis Sativa L. for Preparation of Standardized Extracts for Medicinal Purposes, pp. 165--186, 2015. doi: 10.1007/978-94-017-9810-5_9. Abstract
    Cannabis sativa L. (marijuana; Cannabaceae) is a plant with worldwide distribution, yielding fiber and food, as well as a psychoactive drug. Cannabinoids and in particular the main psychoactive Δ9-THC are promising substances for the development of new drugs and are of high therapeutic potential. This review gives an overview of Cannabis classification, the current verities, botanical features, genomics, chemicals constituents, cellular site and biosynthesis of cannabinoids. Furthermore, the different cultivation and breeding forms, changes in cannabinoids over time, method of harvesting, drying and processing of Cannabis are extensively described in addition to the analytical procedures for standardization of Cannabis based medicinal extracts. Finally, some aspects of current approved Cannabis based medicine and its ways of administration are described.
88. Bócsa, Iván and Karus, Michael, The Cultivation of Hemp: Botany, Varieties, Cultivation and Harvesting, 1998. Abstract
    This is the book that will prepare the prospective hemp farmer for his crop. It is a translation of the German book that is in common use throughout Europe, where the acreage devoted to hemp is increasing annually. Introductory chapters summarize the historical significance of hemp and profile the current state of cultivation.The heart of the book, however, is its detailed classification of the varieties of hemp. The authors provide botanical descriptions and discuss the reproductive biology of the plant. Practical, how-to information on cultivating each variety of hemp is given, including necessary nutrients, soil preparation, and harvesting techniques.
89. Chandra, Suman and Lata, Hemant and ElSohly, Mahmoud A. and Walker, Larry A. and Potter, David, Cannabis Cultivation: Methodological Issues for Obtaining Medical-Grade Product, Epilepsy \& Behavior, vol. 70, pp. 302--312, May 2017. doi: 10.1016/j.yebeh.2016.11.029. Abstract
    As studies continue to reveal favorable findings for the use of cannabidiol in the management of childhood epilepsy syndromes and other disorders, best practices for the large-scale production of Cannabis are needed for timely product development and research purposes. The processes of two institutions with extensive experience in producing large-scale cannabidiol chemotype Cannabis crops—GW Pharmaceuticals and the University of Mississippi—are described, including breeding, indoor and outdoor growing, harvesting, and extraction methods. Such practices have yielded desirable outcomes in Cannabis breeding and production: GW Pharmaceuticals has a collection of chemotypes dominant in any one of eight cannabinoids, two of which—cannabidiol and cannabidivarin—are supporting epilepsy clinical trial research, whereas in addition to a germplasm bank of high-THC, high-CBD, and intermediate type cannabis varieties, the team at University of Mississippi has established an in vitro propagation protocol for cannabis with no detectable variations in morphologic, physiologic, biochemical, and genetic profiles as compared to the mother plants. Improvements in phytocannabinoid yields and growing efficiency are expected as research continues at these institutions. This article is part of a Special Issue entitled “Cannabinoids and Epilepsy”.
90. Fournier, G. and {Richez-Dumanois}, C. and Duvezin, J. and Mathieu, J.-P. and Paris, M., Identification of a New Chemotype in Cannabis Sativa: Cannabigerol - Dominant Plants, Biogenetic and Agronomic Prospects, Planta Medica, vol. 53, no. 3, pp. 277--280, June 1987. doi: 10.1055/s-2006-962705. Abstract
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91. Prade, Thomas and Finell, Michael and Svensson, Sven-Erik and Mattsson, Jan Erik, Effect of Harvest Date on Combustion Related Fuel Properties of Industrial Hemp (Cannabis Sativa L.), Fuel, vol. 102, pp. 592--604, December 2012. doi: 10.1016/j.fuel.2012.05.045. Abstract
    Energy crops can increase biomass availability for large-scale biomass-fired heat, power and CHP plants, which can contribute greatly to mitigation of greenhouse gas emissions. Industrial fibre hemp (Cannabis sativa L.) is a potential high biomass and energy yielding crop intended for use as solid biofuel, but its fuel properties are insufficiently characterised. Hemp was grown in two independently planned field studies 900km apart, in southern and northern Sweden. The northern field trials comprised two seasons, two locations and four different cultivars of hemp, while the southern field trial included one hemp variety and one season. Mineral elemental composition (C, H, O, N, S, Cl, Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, Se, Si, Sn, Sr and Zn), heating value, moisture content and initial ash deformation temperature were determined on samples taken between autumn and spring. Spring harvesting significantly improved relevant combustion fuel properties such as moisture content, alkali and ash content and heating value in comparison with autumn harvest. Major fuel properties were not influenced by choice of cultivar or geographical location. Spring-harvested industrial hemp was found to have high initial ash deformation temperatures and a mineral composition similar to that of willow and coniferous wood, indicating that the ash resulting from its combustion will have a low risk of slagging and fouling. Relevant combustion fuel properties were superior to those of other available agricultural biomass feedstocks, such as cereal straw, miscanthus and reed canary grass (straw fuels). Therefore, hemp is a suitable solid biofuel for large-scale CHP plants and small-scale heating boilers as pellets or briquettes. This study characterised hemp as a solid biofuel, but large-scale combustion tests and an economic analysis are needed to determine the competitiveness of hemp compared with other sources of biomass.
92. Pacifico, D. and Miselli, F. and Carboni, A. and Moschella, A. and Mandolino, G., Time Course of Cannabinoid Accumulation and Chemotype Development during the Growth of Cannabis Sativa L, Euphytica, vol. 160, no. 2, pp. 231--240, March 2008. doi: 10.1007/s10681-007-9543-y. Abstract
    The time course of cannabinoid accumulation in the leaves of individual plants of three Cannabis accessions was determined by gas-chromatographic analysis in greenhouse-grown plants. The total amounts and the concentration ratios of CBD, THC and CBG were determined; two accessions (an experimental hybrid, (21R~×~15R)~×~NL, and plants from a seized seed lot) were found chemotypically uniform, with all plants belonging to chemotpe II (mixed) and I (high THC) respectively. The Carmagnola accession showed chemotypic heterogeneity, with a majority of plants belonging to chemotype III. The CBD/THC and CBG/CBD ratios were shown to be largely constant in the leaves, since 28 and until 103~days after sowing, and consistent with the ratios determined on mature inflorescences. CBD and THC maximum amounts in the leaves showed a peak in the leaves around 80~days from sowing, and were shown to be simultaneous during the growth period, irrespective of the chemotypes. Callus cultures were obtained from all the five different chemotypes (I, II, III, IV, V), and GC analyses were performed. Independently of the type and amount of cannabinoids in the mother plants, it was confirmed that callus cultures of Cannabis were not able to produce detectable amounts of any cannabinoids.
93. Turner, Carlton E. and Hadley, Kathy W. and Fetterman, Patricia S. and Doorenbos, Norman J. and Quimby, Maynard W. and Waller, Coy, Constituents of Cannabis Sativa L. IV: Stability of Cannabinoids in Stored Plant Material, Journal of Pharmaceutical Sciences, vol. 62, no. 10, pp. 1601--1605, October 1973. doi: 10.1002/jps.2600621005. Abstract
    The (—)-∆9-trans-tetrahydrocannabinol content of Cannabis sativa L. stored at — 18, 4, and 22 ± 1 ° decomposed at a rate of 3.83, 5.38, and 6.92\%, respectively, per year, whereas the material stored at 37 and 50° showed considerable decomposition. C. satica L. stored in the absence of direct light at — 18, 4, and 22 ± 1 ° was more stable than cannabis stored under nitrogen. These data indicate that for normal research use, storage under nitrogen at 0° is not mandatory. Cannabinol is not the only decomposition product of (—)-∆9-trans-tetrahydrocannabinol. Tentative evidence supports the possible formation of hexahydrocannabinol as a decomposition product in stored C. satica L.
94. Ross, S. A. and ElSohly, Mahmoud A., CBN and D9-THC Concentration Ratio as an Indicator of the Age of Stored Marijuana Samples*, Boletín de estupefacientes, no. 1, pp. 7, 1997. url: https://dialnet.unirioja.es/servlet/articulo?codigo=6407408. Abstract
    Autorías: S.A. Ross, Mahmoud A ElSohly. Localización: Boletín de estupefacientes. Nº. 1, 1997. Artículo de Revista en Dialnet.
95. Tang, Kailei and Wang, Jiayin and Yang, Yang and Deng, Gang and Yu, Jian and Hu, Wanqun and Guo, Li and Du, Guanghu and Liu, Feihu, Fiber Hemp (Cannabis Sativa L.) Yield and Its Response to Fertilization and Planting Density in China, Industrial Crops and Products, vol. 177, pp. 114542, March 2022. doi: 10.1016/j.indcrop.2022.114542. Abstract
    Interest is growing worldwide to grow fiber hemp for innovative biomaterials while little information is available on the yield performance and agronomy of hemp. In the present study, the productivity of fiber hemp in response to fertilization and planting density was assessed at three sites in the main hemp-producing areas in China with locally bred cultivars in 2016 and 2017. The sites were Daqing in the north-east, Lu’an in the central-east, and Menghai in the south-west. At each site, the treatments were a factorial combination of three fertilization rates (300, 600, and 900~kg (N~+~P2O5 + K2O) per hectare), two NPK ratios (N:P2O5:K2O at the ratio of 3:1:2 and 4:1:2), and two planting densities (45 and 67.5 plants m−2). The harvested stem yield ranges were 9.0–12.2~Mg~ha−1 in Daqing, 9.8–16.5~Mg~ha−1 in Lu’an, and 5.2–15.1~Mg~ha−1 in Menghai. Increasing the fertilization rate from 300~kg~ha−1 to 900~kg~ha−1 had little effect on the yields of stem, leaf and bark while it caused a decrease in the stem bark content in all sites. NPK ratio had a considerable effect on hemp stem yield, higher yield was achieved at 3:1:2 than 4:1:2. The effect of planting density on hemp yields varied among sites. Between the tested densities, the yields of biomass, stem and bark were higher at 45 plant m−2 in Menghai, at 67.5 plants m−2 in Lu’an while no significant difference was detected at Daqing. These results highlight the need to optimize fertilization and planting density according to environmental conditions.
96. Todd, James and Song, Haifeng and Van Acker, Rene, Does Pollination Alter the Cannabinoid Composition and Yield of Extracts from Hemp (Cannabis Sativa L. Cv. Finola) Flowers?, Industrial Crops and Products, vol. 183, pp. 114989, September 2022. doi: 10.1016/j.indcrop.2022.114989. Abstract
    This study was undertaken to compare cannabinoid levels and yields in floral extracts from unpollinated and artificially pollinated industrial hemp (Cannabis sativa L. cv. Finola) flowers grown under identical growth chamber conditions. Of the 16 cannabinoids analyzed using high performance liquid chromatography (HPLC), the levels of 10, cannabichromene (CBC), cannabichromenic acid (CBCA), cannabidivarin (CBDV), cannabigerol (CBG), cannabicyclol (CBL), cannabinol (CBN) cannabinolic acid (CBNA), ∆8-tetrahydrocannabinol (∆8-THC), tetrahydrocannabivarin (THCV) and tetrahydrocannabivarinic acid (THCVA) were near or below the limit of quantification. Total ∆9-tetrahydrocannabinol (THC), was present at concentrations below the legal limit of 0.3\% (w/w). The level of cannabidiol (CBD) in extracts from pollinated flowers was the same as that from unpollinated flowers, but cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA) levels were not. This suggested that, although pollination changes the pool sizes of the precursors in the metabolic pathway leading to CBD production, cannabinoid levels in floral extracts from the Finola cultivar, were reduced but not eliminated, by pollination of hemp flowers compared with levels in floral extracts from unpollinated flowers.
97. Burgel, Lisa and Hartung, Jens and Schibano, Daniele and {Graeff-Hönninger}, Simone, Impact of Different Phytohormones on Morphology, Yield and Cannabinoid Content of Cannabis Sativa L., Plants, vol. 9, no. 6, pp. 725, June 2020. doi: 10.3390/plants9060725. Abstract
    The impact of exogenously applied plant growth regulators (PGR), 1-naphthalenaecetic acid (NAA), 6-benzylaminopurine (BAP), and a mixture of both (NAA/BAP-mix), was investigated in regard to plant height, length of axillary branches, number of internodes, biomass yield and cannabinoid content of three different phytocannabinoid-rich (PCR) Cannabis genotypes. The results showed that total plant height was significantly reduced under the application of NAA (28\%), BAP (18\%), and NAA/BAP-mix treated plants (15\%). Axillary branch length was also significantly reduced by 58\% (NAA) and 30\% (NAA/BAP-mix). BAP did not significantly reduce the length of axillary branches. The number of internodes was reduced by NAA (19\%), BAP (10\%), and the NAA/BAP-mix (14\%) compared to the untreated control. NAA application influenced the plant architecture of the tested cv. KANADA beneficially, resulting in a more compact growth habitus, while inflorescence yield (23.51 g plant−1) remained similar compared to the control (24.31 g plant−1). Inflorescence yield of v. 0.2x and cv. FED was reduced due to PGR application while cannabinoid content remained stable. Overall, the application of PGR could be used on a genotype-specific level to beneficially influence plant architecture and optimize inflorescence yield per unit area and thus cannabinoid yield, especially in the presence of space limitations under indoor cultivation.
98. Burgel, Lisa and Hartung, Jens and {Graeff-Hönninger}, Simone, Impact of Different Growing Substrates on Growth, Yield and Cannabinoid Content of Two Cannabis Sativa L. Genotypes in a Pot Culture, Horticulturae, vol. 6, no. 4, pp. 62, December 2020. doi: 10.3390/horticulturae6040062. Abstract
    The impacts of different growing substrate compositions, consisting of peat (PM), peat substituted with 30\% green fibre (G30) and coco coir fibre (CC) growth media, were investigated in regard to the plant height, biomass and floral yield, biomass nitrogen (N) content, root growth, and cannabidiol content (CBD/A) of two phytocannabinoid-rich cannabis genotypes in an indoor pot cultivation system. Genotypes and substrate treatment combinations were randomly allocated to 36 plants according to a Latin square design. The results showed a higher total plant height for PM (39.96 cm), followed by G30 (35.28 cm), and the lowest in CC (31.54 cm). The N content of leaves indicated the highest values for plants grown in G30 (52.24 g kg DW−1), followed by PM (46.75 g kg DW−1) and a significantly lower content for CC (37.00 g kg DW−1). Root length density (RLD) increased by 40\% (PM) and 50\% (G30), compared to CC treatments, with no significant differences in root dry weight. Both genotypes, Kanada (KAN) and 0.2x, reacted in a genotype-specific manner. KAN indicated a reduced floral yield of plants grown in G30 (4.94 g plant−1) and CC (3.84 g plant−1) compared to PM (8.56 g plant−1). 0.2x indicated stable high floral yields of 9.19 g plant−1 (G30) to 7.90 g plant−1 (CC). Leaf DW increased in PM (5.78 g plant−1) and G30 (5.66 g plant−1) compared to CC (3.30 g plant−1), while CBD/A content remained constant. Due to a higher biomass yield, the CBD/A yield of flowers (549.66 mg plant−1) and leaves (224.16 mg plant−1) revealed 0.2x as an interesting genotype for indoor pot cultivation in a peat-based substrate substituted with 30\% green fibres. Overall, the demand for organic green fibres to partly replace fractionated peat showed a genotype-specific option for a homogeneous plant development, with comparable high biomass yields and stable cannabinoid contents compared to a peat containing standard substrate.
99. Vandepitte, Katrien and Vasile, Simona and Vermeire, Sofie and Vanderhoeven, Myréne and {Van der Borght}, Wouter and Latré, Joos and De Raeve, Alexandra and Troch, Veronique, Hemp (Cannabis Sativa L.) for High-Value Textile Applications: The Effective Long Fiber Yield and Quality of Different Hemp Varieties, Processed Using Industrial Flax Equipment, Industrial Crops and Products, vol. 158, pp. 112969, December 2020. doi: 10.1016/j.indcrop.2020.112969. Abstract
    Industrial hemp (Cannabis sativa L.) has great potential as a sustainable source of textile fiber; yet, to develop a viable European hemp-for-textile chain, agronomic practices and primary processing need optimization to current industrial standards. A straightforward approach is to process hemp using existing, modern equipment for flax (linen). Here we extensively evaluated the quantity and quality of fiber extracted from field-retted hemp stems, scutched on the industrial flax processing line. Varieties from diverse European origin (USO 31, Dacia Secuieni, Bialobrzeskie, Futura 75, Carmagnola Selezionata, Santhica 27 and Santhica 70) were sown in randomized field experiments in Belgium, which has a rich long-standing tradition in high-quality linen production. Biomass yield and the quantity of long fiber processed were assessed across three growing seasons (2017–2019; plot size: 15–45\,m2). In 2018, we also determined the quantity of tow (short fiber) and, the quality of long fiber in terms of fiber tenacity and elongation. The quantity of total fiber extracted (i.e. long fiber plus tow) accounted for 36.2\% of the initial straw yield, indicating high processing efficiency. Approximately equal amounts of tow and long fiber were extracted. Mean long fiber yield approximated one ton per hectare; yet yield variation between varieties was considerable (range long fiber yield: 0.6–1.4\,ton/hectare). Despite significant variation between harvest years in straw yield, the quantity of long fiber extracted held relatively constant. Fiber tenacity of long hemp was overall high and comparable to flax (range: 37.6–45.3\,cN/tex). Results indicate that field-retted hemp has potential to be processed into quality fiber on the industrial flax line and, that fiber yield can likely further be improved by genotype selection. Harvest mechanization, focused on the collection of parallel hemp stem portions of appropriate length for the flax scutching line (ca. 1\,m), seems warranted to make this approach economically viable. Additional research on the fiber properties following hackling and wet-spinning will be needed to fully explore the potentiality of long hemp as a flax supplement for textile applications.
100. Petit, Jordi and Salentijn, Elma M. J. and Paulo, Maria-João and Thouminot, Claire and {van Dinter}, Bert Jan and Magagnini, Gianmaria and Gusovius, Hans-Jörg and Tang, Kailei and Amaducci, Stefano and Wang, Shaoliang and Uhrlaub, Birgit and Müssig, Jörg and Trindade, Luisa M., Genetic Variability of Morphological, Flowering, and Biomass Quality Traits in Hemp (Cannabis Sativa L.), Frontiers in Plant Science, vol. 11, 2020. doi: 10.3389/fpls.2020.00102. Abstract
     Hemp (Cannabis sativa L.) is a bast-fiber crop well-known for the great potential to produce sustainable fibers. Nevertheless, hemp fiber quality is a complex trait, and little is known about the phenotypic variability and heritability of fiber quality traits in hemp. The aim of this study is to gain insights into the variability in fiber quality within the hemp germplasm and to estimate the genetic components, environmental components, and genotype-by-environment (G×E) interactions on fiber quality traits in hemp. To investigate these parameters, a panel of 123 hemp accessions was phenotyped for 28 traits relevant to fiber quality at three locations in Europe, corresponding to climates of northern, central, and southern Europe. In general, hemp cultivated in northern latitudes showed a larger plant vigor while earlier flowering was characteristic of plants cultivated in southern latitudes. Extensive variability between accessions was observed for all traits. Most cell wall components (contents of monosaccharides derived from cellulose and hemicellulose; and lignin content), bast fiber content, and flowering traits revealed large genetic components with low G×E interactions and high broad-sense heritability values, making these traits suitable to maximize the genetic gains of fiber quality. In contrast, contents of pectin-related monosaccharides, most agronomic traits, and several fiber traits (fineness and decortication efficiency) showed low genetic components with large G×E interactions affecting the rankings across locations. These results suggest that pectin, agronomic traits, and fiber traits are unsuitable targets in breeding programs of hemp, as their large G×E interactions might lead to unexpected phenotypes in untested locations. Furthermore, all environmental effects on the 28 traits were statistically significant, suggesting a strong adaptive behavior of fiber quality in hemp to specific environments. The high variability in fiber quality observed in the hemp panel, the broad range in heritability, and adaptability among all traits prescribe positive prospects for the development of new hemp cultivars of excellent fiber quality.
101. Abdollahi, Mahnaz and Sefidkon, Fatemeh and Calagari, Mohsen and Mousavi, Amir and Fawzi Mahomoodally, Mohamad, A Comparative Study of Seed Yield and Oil Composition of Four Cultivars of Hemp (Cannabis Sativa L.) Grown from Three Regions in Northern Iran, Industrial Crops and Products, vol. 152, pp. 112397, September 2020. doi: 10.1016/j.indcrop.2020.112397. Abstract
     Cannabis sativa L, commonly known as Hemp, shows numerous health and economic benefits with commercial applications such as in seed production, fiber, oil, and pharmaceutical uses. Hemp oil has been widely acknowledged to have a panoply of health benefits, such as cholesterol lowering properties and decreasing high blood pressure, owing to the presence of two essential fatty acids (linoleic acid and α-linolenic). Given the economic and industrial importance of hemp, the present study was designed in an endeavor to select and compare the best cultivar and region to increase both quantity and quality of hemp oil from Iran. Seeds of two native populations from Iran (Fars and Yazd provinces) and one foreign variety from France (Fedora17, as an industrial hemp cultivar) with its progenies (Fedora17-2) were cultivated in research fields of three locations (Gilan, Golestan, and Alborz province) in Iran. The mature seeds of cultivated plants were collected and their oils were extracted by Soxhlet apparatus and analyzed by Gas Chromatograph. The seed yield was 288.70 to 3182.60 kg ha-1 and oil yield were 0.08 to 0.95 t ha-1 in the studied sites. Also, the range of 1000- seed weight was 5.20 to 15.28 g. The oil content of different varieties varied from 15.46 to 36.63\%. Based on the results, the highest seed yield, 1000-seed weight, and oil yield belonged to native populations. It was also determined that Alborz was significantly higher in terms of oil production, 1000-seed weight, and seed and oil yield per hectare compared with the other two regions. The most abundant fatty acid in the samples was linoleic acid, (56.80 to 63.98\%), and almost identical in all cultivars and areas studied. The range of linoleic acid and α-linolenic in three regions were 57.55 to 63.98\% and 7.57 to 22.91\%, respectively. In addition, the palmitic acid content of foreign varieties was higher than that of the native populations in all three locations. Since the interfaces in the production and synthesis of fatty acids in plants are influenced by variation in temperature, light and moisture amount and farming conditions so the percentage and kind of fatty acids in hemp oil can be varied in different regions.
102. Deng, Gang and Du, Guanghui and Yang, Yang and Bao, Yaning and Liu, Feihu, Planting Density and Fertilization Evidently Influence the Fiber Yield of Hemp (Cannabis Sativa L.), Agronomy, vol. 9, no. 7, pp. 368, July 2019. doi: 10.3390/agronomy9070368. Abstract
     Hemp is one of the most important green (i.e., environmentally sustainable) fibers. Planting density, nitrogen (N), phosphorus (P) and potassium (K) significantly affect the yield of hemp fiber. By optimizing the above main four cultivation factors is an important way to achieve sustainable development of high-fiber yield hemp crops. In this study, the effects of individual factors and factor × factor interactions on the yield of hemp fiber over two trial years were investigated by the central composite design with four factors, namely planting density, nitrogen application, phosphorus application, and potassium application rate. The influences of these four test factors on the yield of hemp fibers were in the order nitrogen fertilizer (X2) {$>$} planting density (X1) {$>$} potassium fertilizer (X4) {$>$} phosphate fertilizer (X3). To obtain yields of hemp with high-quality fiber greater than 2200 kg ha−1, the optimal range of cultivation conditions were planting density 329,950–371,500 plants/ha, nitrogen application rate 251–273 kg ha−1, phosphorus application rate 85–95 kg ha−1, and potassium application rate 212–238 kg ha−1. This study can provide important technical and theoretical support for the high-yield cultivation of hemp fiber into the future.
103. Lan, Yang and Zha, Fengchao and Peckrul, Allen and Hanson, Bryan and Johnson, Burton and Rao, Jiajia and Chen, Bingcan, Genotype x Environmental Effects on Yielding Ability and Seed Chemical Composition of Industrial Hemp (Cannabis Sativa L.) Varieties Grown in North Dakota, USA, Journal of the American Oil Chemists' Society, vol. 96, no. 12, pp. 1417--1425, 2019. doi: 10.1002/aocs.12291. Abstract
     The influence of growing conditions in North Dakota on grain yield and seed composition was evaluated for industrial hemp varieties (Cannabis sativa L.) grown in North Dakota, USA. Average grain yield across varieties was 2138 kg ha−1. Crude protein content of undehulled industrial hemp flour from 10 varieties ranged from 32.7\% to 35.9\%; and oil content ranged from 24.3\% to 28.1\%. All industrial hemp flours were excellent natural sources of iron (46.7\%), manganese (169.1\%), copper (29.0\%), zinc (28.2\%), phosphorus (41.0\%), and magnesium (33.7\%) on a per serving basis (30 g per serving) for percent recommended daily intake (\% RDI). Oils from most of the varieties differed significantly for the fatty acid (FA) profile. The n6/n3 ratio ranged from 3.2 to 5.1. Principal component analysis (PCA) suggested that CFX-2 and CRS-1 grew well in North Dakota based on grain yield, flour, and oil quality. In this study, the chemical composition of flour was not influenced by crop year; but crop year did effect the nutritional mineral composition. These results may have important implications for the use of industrial hemp in food applications.
104. Janatová, Anežka and Fraňková, Adéla and Tlustoš, Pavel and Hamouz, Karel and Božik, Matěj and Klouček, Pavel, Yield and Cannabinoids Contents in Different Cannabis (Cannabis Sativa L.) Genotypes for Medical Use, Industrial Crops and Products, vol. 112, pp. 363--367, February 2018. doi: 10.1016/j.indcrop.2017.12.006. Abstract
     In the last decades, there has been a significant increase in the number of lifestyle and auto-immune diseases, such as various cancers or multiple sclerosis. In countries where cannabis is decriminalized for medical purposes, it is most often prescribed for these diagnoses. Today, over 700 different cannabis genotypes are being bred, and it is very important to describe in detail their cultivation, potential yields, chemical profile and stability, to be recommended to a particular patient with a specific diagnosis. The aim of this study was to evaluate the inflorescence yields and the content of Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) of seven traditional genotypes of cannabis – Conspiracy Kush, Nurse Jackie, Jilly Bean, Nordle, Jack Cleaner 2, Jack Skellington and National Health Services. The plants were grown under controlled climatic conditions during six growing cycles at a density of 9 plants/m2. Dried inflorescences from each plant were homogenized and analyzed by gas chromatography with flame ionization detection. The average yield per plant was 21.02±3.33g and the highest yields showed genotype Nurse Jackie (24.74±6.11g). The lowest yields were shown by genotype Jack Skellington (15.41±4.02g). Average Δ9-THC levels for each variety in all 6 growing cycles ranged from 15.69±2.6 \% to 19.31±2.47 \% (w/w). The lowest contents of Δ9-THC were measured in the Nordle genotype and the highest values were found in the Jack Cleaner 2 and Jack Skellington genotypes. Average CBD levels in the plants ranged from 0.45±0.1 \% to 0.57±0.08 \% (w/w) over six individual cycles. This study shows that among genotypes studied, the best parameters – high yield and stable cannabinoids production – are shown by genotypes Nurse Jackie and Jilly Bean.
105. Hawley, Dave and Graham, Thomas and Stasiak, Michael and Dixon, Mike, Improving Cannabis Bud Quality and Yield with Subcanopy Lighting, HortScience, vol. 53, no. 11, pp. 1593--1599, November 2018. doi: 10.21273/HORTSCI13173-18. Abstract
     The influence of light spectral quality on cannabis (Cannabis sativa L.) development is not well defined. It stands to reason that tailoring light quality to the specific needs of cannabis may increase bud quality, consistency, and yield. In this study, C. sativa L. ‘WP:Med (Wappa)’ plants were grown with either no supplemental subcanopy lighting (SCL) (control), or with red/blue (“Red-Blue”) or red-green-blue (“RGB”) supplemental SCL. Both Red-Blue and RGB SCL significantly increased yield and concentration of total Δ9-tetrahydrocannabinol (Δ9-THC) in bud tissue from the lower plant canopy. In the lower canopy, RGB SCL significantly increased concentrations of α-pinine and borneol, whereas both Red-Blue and RGB SCL increased concentrations of cis-nerolidol compared with the control treatment. In the upper canopy, concentrations of α-pinine, limonene, myrcene, and linalool were significantly greater with RGB SCL than the control, and cis-nerolidol concentration was significantly greater in both Red-Blue and RGB SCL treated plants relative to the control. Red-Blue SCL yielded a consistently more stable metabolome profile between the upper and lower canopy than RGB or control treated plants, which had significant variation in cannabigerolic acid (CBGA) concentrations between the upper and lower canopies. Overall, both Red-Blue and RGB SCL treatments significantly increased yield more than the control treatment, RGB SCL had the greatest impact on modifying terpene content, and Red-Blue produced a more homogenous bud cannabinoid and terpene profile throughout the canopy. These findings will help to inform growers in selecting a production light quality to best help them meet their specific production goals.
106. Papastylianou, Panayiota and Kakabouki, Ioanna and Travlos, Ilias, Effect of Nitrogen Fertilization on Growth and Yield of Industrial Hemp (Cannabis Sativa L.), Notulae Botanicae Horti Agrobotanici Cluj-Napoca, vol. 46, no. 1, pp. 197--201, January 2018. doi: 10.15835/nbha46110862. Abstract
     Fibre hemp is grown for a multitude of end products derived from its cannabinoids, seed, fibre and wooden core. A key factor that influences the quantity and quality of the production of hemp is nitrogen fertilization. The aim of this study was to determine the response of five well-adapted industrial hemp cultivars to different nitrogen fertilization rates during the 2016 growing season. The experiment was laid out in a split-plot design with two replicates, five main plots (hemp cultivars: ‘Bialobrzeskie’, ‘Tygra’, ‘Felina 32’, ‘Sanhtica 27’, ‘Futura 75’) and sub-plots [fertilization treatments: control (N0), fertilizer 46-0-0 at 120 kg ha-1 (N1), 180 kg ha-1 (N2), 240 kg ha-1 (N3)]. For the computation of height, biomass yield, stem dry weight, length and weight of the inflorescences and mean seed weight, 10 plants were randomly selected in each plot. In general, increasing N fertilization rate positively impacts hemp biomass yield, stem dry weight, plant height, and inflorescence indices. Biomass yield, stem dry weight and inflorescence weight increased by 37.3\%, 48.2\% and 16\%, respectively, with the application of 240 kg N ha-1 when compared with the unfertilized control. Plant height and inflorescence length increased from 1.66 to 1.76 m and from 66.2 to 82.9 cm, respectively, with the application of the higher N rate compared with the control, while there were no significant differences between the fertilization treatments for mean seed weight. The varieties ‘Tygra’ and ‘Futura 75’ showed the highest values for all the measurement characters. Our results indicate that hemp responded well to the addition of N fertilizer.
107. Faux, A. -M. and Draye, X. and Lambert, R. and {d’Andrimont}, R. and Raulier, P. and Bertin, P., The Relationship of Stem and Seed Yields to Flowering Phenology and Sex Expression in Monoecious Hemp (Cannabis Sativa L.), European Journal of Agronomy, vol. 47, pp. 11--22, May 2013. doi: 10.1016/j.eja.2013.01.006. Abstract
     Flowering phenology and sexual dimorphism are two major features that affect stem and seed production in hemp (Cannabis sativa L.), a short-day naturally dioecious plant. The sowing time is of primary importance because it affects flowering time and thereby influences stem yield. In spite of their unstable sexual phenotype, monoecious cultivars facilitate the harvest of both stems and seeds by reducing crop heterogeneity. The main objective of this paper was to determine the stem and seed yields for five monoecious hemp cultivars in relation to their flowering phenology and sex expression. Sowing was carried out on five distinct dates during 2007 and 2008 at two sites in Belgium. The duration from sowing to flowering in days, both stem and seed yields and the seed harvest index decreased when sowing was postponed from mid-April to the end of June. The stem and seed yields from the mid-April sowing (approximately 12.5 and 1.9tha−1, respectively) were within the ranges that were reported for fibre and both fibre and seeds production, respectively, in monoecious hemp. No interaction was observed between the sowing date and cultivar for both yields. Sex expression varied among cultivars, indicating that it might be selected, and was partly linked to earliness. Stem yields were lowest in the earliest cultivar (Uso 31) and highest in the latest one (Epsilon 68) while seed yields were lowest in the most masculinized and earliest cultivar (Uso 31) and highest in the most feminized and early (Fedora 17) or mid-early (Felina 32) ones. Both stem and seed yields correlated best with the duration from sowing to full female flowering or from sowing to the end of male flowering. According to our results, harvesting the seeds in addition to the stems in monoecious hemp requires early sowing and the selection of feminized early or mid-early cultivars, earliness depending on the climatic conditions in the cultivation area. Therefore, it might be agriculturally valuable to take sex expression into account in addition to earliness during the selection of cultivars that are adapted to a dual purpose.
108. Sankari, Hannele S, Comparison of Bast Fibre Yield and Mechanical Fibre Properties of Hemp (Cannabis Sativa L.) Cultivars, Industrial Crops and Products, vol. 11, no. 1, pp. 73--84, January 2000. doi: 10.1016/S0926-6690(99)00038-2. Abstract
     High stem yield and high bast fibre content in stem are generally accepted as important properties for fibre hemp (Cannabis sativa L.) Quality demands for fibre used in the new non-woven products have not yet been defined. The Ukrainian monoecious fibre hemp cv. Uso 11 was compared with 13 other fibre hemp cultivars in 1995–1997 in Finland (latitude 60°49′ N). Stem yield, stem length, stem diameter, bast fibre content in stem, bast fibre yield, proportion of primary fibre in the bast fibre and primary fibre yield are reported, as well as breaking tenacity and elongation at break of the fibres. The average stem yield of cv. Uso 11 was 5947 kg dry matter ha−1 and only dioecious cvs. Kompolti Hybrid TC and Novosadski produced significantly higher yields. The bast fibre content in the stem of cv. Uso 11 averaged 21.7\%, and four other cultivars, both mono- and dioecious ones, had significantly higher content. The proportion of primary fibre in the bast fibre of cv. Uso 11 averaged 91.0\%, and cultivars with significantly lower primary fibre fraction were all dioecious. The average bast fibre and primary fibre yields of cv. Uso 11 were 1301 and 1188 kg dry matter ha−1, respectively. Only cv. Kompolti Hybrid TC produced significantly higher bast fibre and primary fibre yields. The median values for breaking tenacity and elongation at break of the fibres varied, depending on experimental year and cultivar, from 41 to 74 cN/tex and from 3.3 to 5.5\%, respectively. The dioecious cultivars showed better or equivalent properties to cv. Uso 11. Because the season was too short for seed production, which is a prerequisite for obtaining the EU subsidy, dioecious hemp cultivars cannot be recommended for cultivation in Finland. However, monoecious Ukrainian cvs. Uso 11 and Uso 31 and Polish cvs. Beniko and Bialobrzeskie are suitable for the long-day growth conditions prevailing in Finland.
109. {van der Werf}, H. M. G. and {van Geel}, W. C. A. and {van Gils}, L. J. C. and Haverkort, A. J., Nitrogen Fertilization and Row Width Affect Self-Thinning and Productivity of Fibre Hemp (Cannabis Sativa L.), Field Crops Research, vol. 42, no. 1, pp. 27--37, July 1995. doi: 10.1016/0378-4290(95)00017-K. Abstract
     In fibre hemp (Cannabis sativa L.) a high plant density is desirable, but inter-plant competition may cause self-thinning, which reduces stem yield and quality. We investigated whether agronomic factors could reduce self-thinning in hemp. The effects of soil nitrogen level (80 and 200 kg ha−1), row width (12.5, 25 and 50 cm), type of sowing implement, and thinning method on self-thinning, growth, yield and quality of hemp were determined in field experiments in 1991 and 1992. Soil nitrogen level affected plant morphology before self-thinning occurred. Due to enhanced competition for light more plants died from self-thinning at 200 than at 80 kg N ha−1. In August, stem yield of living plants was similar at the two nitrogen levels, but 5\% of the plants had died at 80 kg N ha−1 and 25\% at 200 kg N ha−1. Although dry matter losses resulting from self-thinning were greater at 200 than at 80 kg N ha−1, crop growth rate was greater at 200 than at 80 kg N ha−1. Apparently, the crop growth at 80 kg N ha−1 was affected by a lack of nitrogen. At final harvest in September stem yield of living plants was 10.4 t ha−1 at 80 and 11.3 t ha−1 at 200 kg N ha−1, bark content in the stem was 35.6\% at 80 and 34.0\% at 200 kg N ha−1. The effect of row width on self-thinning was small relative to that of nitrogen level. More self-thinning took place at 50 cm row width than at 12.5 and 25 cm. During early growth and also in August stem yield was smaller when row width was larger; in September row width did not affect stem yield or quality. Type of sowing implement and thinning method did not affect self-thinning or stem yield.
110. {van der Werf}, H. M. G. and Haasken, H. J. and Wijlhuizen, M., The Effect of Daylength on Yield and Quality of Fibre Hemp (Cannabis Sativa L.)., European Journal of Agronomy, vol. 3, no. 2, pp. 117--123, January 1994. doi: 10.1016/S1161-0301(14)80117-2. Abstract
     Stem growth of the short-day plant fibre hemp (Cannabis sativa L.) decreases after flowering. In the Netherlands, the hemp cultivars currently available flower in August. In 1990 and 1991 the ambient daylength was compared with a 24-hour daylength in field experiments on two cultivars. Crop development, interception of photosynthetically active radiation, dry matter accumulation, stem yield and stem composition were recorded. The 24-hour daylength did not totally prevent flowering, but did greatly reduce the allocation of dry matter to floral parts. It enhanced the efficiency of post-flowering radiation use, and increased stem dry matter yield by 2.7 t ha-1. The continued stem growth resulted in higher yields, which in one cultivar were accompanied by a lower bark content of the stem. At final harvest, the 1 per cent NaOH solubility indicated a lower fibre content in the bark of plants from the 24-hour daylength. Breeding late-flowering hemp may be a promising strategy to improve the potential stem yield of hemp in the Netherlands, but the stem quality of such cultivars may be slightly poorer.
111. Jordan, Howard V. and Lang, A. L. and Enfield, George H., Effects of Fertilizers on Yields and Breaking Strengths of American Hemp, Cannabis Sativa1, Agronomy Journal, vol. 38, no. 6, pp. 551--562, 1946. doi: 10.2134/agronj1946.00021962003800060010x.
112. Potter, David J. and Duncombe, Paul, The Effect of Electrical Lighting Power and Irradiance on Indoor-Grown Cannabis Potency and Yield, Journal of Forensic Sciences, vol. 57, no. 3, pp. 618--622, 2012. doi: 10.1111/j.1556-4029.2011.02024.x. Abstract
     Abstract: The floral development and potencies [Δ9-tetrahydrocannabinol (THC) contents] of cannabis plants were compared when grown indoors under high-pressure sodium lamps consuming electrical power at three densities (270, 400, and 600 W/m2). After a 3-week vegetative phase, plants were grown for 8 weeks, with lamps maintaining an artificial day length of 12 h. Foliar and floral yields were measured. Gas chromatography was used to measure the content of the psychoactive cannabinoid THC. Mean yields per unit of electrical power in each lighting regime ranged from 0.9 to 1.6 g/W, the highest being achieved in the lowest irradiance regime. The individual potencies of the separated leaf and flower materials were not affected by increasing irradiance. However, there was a corresponding increase in the overall potency of the aerial plant tissue. This was because of the plants in brighter conditions producing a higher proportion of floral material.
113. Iványi, Ildikó, Relationship Between Leaf Nutrient Concentrations and Yield of Fibre Hemp (Cannabis Sativa L.), Cereal Research Communications, vol. 33, no. 1, pp. 97--100, March 2005. doi: 10.1556/CRC.33.2005.1.23.
114. Yep, Brandon and Gale, Nigel V. and Zheng, Youbin, Comparing Hydroponic and Aquaponic Rootzones on the Growth of Two Drug-Type Cannabis Sativa L. Cultivars during the Flowering Stage, Industrial Crops and Products, vol. 157, pp. 112881, December 2020. doi: 10.1016/j.indcrop.2020.112881. Abstract
     The cultivation of drug-type Cannabis sativa Lin. (C. sativa) employs a variety of rootzone systems, yet little is understood about rootzone-effects on plant physiology, inflorescence biomass, and cannabinoid potency. To determine if rootzone systems can affect C. sativa growth, physiology, and inflorescence biomass and phytochemicals, two C. sativa cultivars, colloquially known as ‘Nordle’ and ‘Sensi-Star’, were grown in three rootzone systems in a controlled environment. The three tested rootzone systems were: 1) an 11-litre pot containing a peat-based growing substrate, top fertigated with a synthetic fertilizer solution (“hydroponic”); 2) an 11-litre pot containing a peat-based growing substrate, top fertigated with aquaculture effluent solution (“aquaculture”); and 3) a 3-litre pot containing a custom made, predominantly peat-based growing substrate, with the lower portion of the roots submerged in a conventional aquaponics deep-water culture system (“aquaponics”). Throughout the experiment, plant growth (height, branch number, canopy volume), leaf-level physiological traits (e.g., chlorophyll) foliar mineral nutrition, and substrate physio-chemical properties were measured. At experimental completion, inflorescence yield (dry biomass) and potency (mass-based cannabinoid and terpene concentrations) were determined. Results revealed, significant rootzone-effects on most growth and physiological traits measured. Plants performed best in the in the hydroponic rootzone: e.g., producing 42–116\% greater inflorescence biomass than the other rootzone systems. In contrast, however, Nordle plants in the aquaponics treatment had greater Δ9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and the terpenes ß-pinene and limonene, relative to their congeners grown in the other rootzones investigated. Pronounced differences in substrate macro-nutrient availability and foliar nutrient content occurred among the rootzones, suggesting a potential mechanism to explain the observed growth, physiological, yield and potency responses. This work underscores the importance of the rootzone environment for the cultivation of C. sativa, specifically in indoor systems focused of medical or research production.
115. Yep, Brandon and Gale, Nigel V. and Zheng, Youbin, Aquaponic and Hydroponic Solutions Modulate NaCl-Induced Stress in Drug-Type Cannabis Sativa L., Frontiers in Plant Science, vol. 11, 2020. doi: 10.3389/fpls.2020.01169. Abstract
     The effects of salt-induced stress in drug-type Cannabis sativa L. (C. sativa), a crop with increasing global importance, are almost entirely unknown. In an indoor controlled factorial experiment involving a type-II chemovar (i.e., one which produces Δ9-tetrahydrocannabinolic acid \textasciitilde THCA and cannabidiolic acid \textasciitilde{} CBDA), the effects of increasing NaCl concentrations (1–40 mM) was tested in hydroponic and aquaponic solutions during the flowering stage. Growth parameters (height, canopy volume), plant physiology (chlorophyll content, leaf-gas exchange, chlorophyll fluorescence, and water use efficiency), and solution physicochemical properties (pH, EC, and nutrients) was measured throughout the experiment. Upon maturation of inflorescences, plants were harvested and yield (dry inflorescence biomass) and inflorescence potency (mass-based concentration of cannabinoids) was determined. It was found that cannabinoids decreased linearly with increasing NaCl concentration: -0.026 and -0.037\% THCA·mM NaCl-1 for aquaponic and hydroponic solutions, respectively. The growth and physiological responses to NaCl in hydroponic—but not the aquaponic solution—became negatively affected at 40 mM. The mechanisms of aquaponic solution which allow this potential enhanced NaCl tolerance is worthy of future investigation. Commercial cultivation involving the use of hydroponic solution should carefully monitor NaCl concentrations, so that they do not exceed the phytotoxic concentration of 40 mM found here; and are aware that NaCl in excess of 5 mM may decrease yield and potency. Additional research investigating cultivar- and rootzone-specific responses to salt-induced stress is needed.
116. Caplan, Deron and Dixon, Mike and Zheng, Youbin, Optimal Rate of Organic Fertilizer during the Vegetative-Stage for Cannabis Grown in Two Coir-Based Substrates, HortScience, vol. 52, no. 9, pp. 1307--1312, September 2017. doi: 10.21273/HORTSCI11903-17. Abstract
     Cannabis producers, especially those with organic operations, lack reliable information on the fertilization requirements for their crops. To determine the optimal organic fertilizer rate for vegetative-stage cannabis (Cannabis sativa L.), five rates that supplied 117, 234, 351, 468, and 585 mg N/L of a liquid organic fertilizer (4.0N–1.3P–1.7K) were applied to container-grown plants with one of two coir-based organic substrates. The trial was conducted in a walk-in growth chamber and the two substrates used were ABcann UNIMIX 1-HP with lower water-holding capacity (WHC) and ABcann UNIMIX 1 with higher WHC. No differences in growth or floral dry weight (yield) were found between the two substrates. Pooled data from both substrates showed that the highest yield was achieved at a rate that supplied 389 mg N/L (interpolated from yield-fertilizer responses) which was 1.8 times higher than that of the lowest fertilizer rate. The concentration of ∆9-tetrahydrocannabinol (THC) in dry floral material was maximized at a rate that supplied 418 mg N/L, and no fertilizer rate effects were observed on Δ9-tetrahydrocannabidiolic acid (THCA) or cannabinol (CBN). The highest yield, cannabinoid content, and plant growth were achieved around an organic fertilizer rate that supplied 389 mg N/L during the vegetative growth stage when using the two coir-based organic substrates.
117. De Vos, B. and Souza, M. F. and Michels, E. and Meers, E., Industrial Hemp (Cannabis Sativa L.) in a Phytoattenuation Strategy: Remediation Potential of a Cd, Pb and Zn Contaminated Soil and Valorization Potential of the Fibers for Textile Production, Industrial Crops and Products, vol. 178, pp. 114592, April 2022. doi: 10.1016/j.indcrop.2022.114592. Abstract
     In past studies, industrial hemp (Cannabis sativa L.) has been shown to tolerate stress from heavy metals and to accumulate the metals in its tissues. Nonetheless, up to now, it remained unclear whether hemp grown on contaminated soil could be safely used in the textile industry and integrate a phytoattenuation strategy. A growth experiment was set up to screen 6 hemp cultivars for their ability to take up heavy metals in the different plant parts when grown on soil contaminated with Cd, Pb, and Zn. The potential safe use of the produced biomass in comparison to industrial quality guidelines, especially the fibers, was analyzed. Plant tissues show different patterns of Cd, Pb, and Zn uptake and it could not be stated that one plant part accumulates a higher amount of all elements compared to the others. While Pb and Zn concentrations followed the trend leaves{$>$}shives{$>$}fibers, Cd was mostly concentrated in the fibers. The lowest concentrations in fibers were found in the early cultivars USO 31 and Bialobrezskie respectively for Cd (0.59~±~0.15~mg/kg) and Pb (1.6~±~0.7~mg/kg), and in the semi-late cultivar Dacia Secuieni for Zn (7.2~±~1.4~mg/kg). The late cultivar Carmagnola Selected displayed the highest concentrations for Cd (1.7~±~0.5~mg/kg) and Zn (13~±~2~mg/kg), as the early cultivar USO 31 did for Pb (11~±~1~mg/kg). Nevertheless, both Cd and Pb concentrations in the fibers were far below the heavy metal thresholds for textile product safety in all cultivars, while Zn is not considered toxic in textile production. In addition, low Pb, Cd, and Zn concentrations in the shives suggest the potential safe use of this residual fraction of hemp fiber production as well. These results are promising in terms of safe use of the produced hemp fibers in the textile industry and thus of the potential valorization of contaminated land through hemp cultivation and the development of non-food value chains within a phytoattenuation strategy.
118. {da Silva Benevenute}, Sarah and Freeman, Joshua H. and Yang, Rui, How Do Pinching and Plant Density Affect Industrial Hemp Produced for Cannabinoids in Open Field Conditions?, Agronomy Journal, vol. 114, no. 1, pp. 618--626, 2022. doi: 10.1002/agj2.20882. Abstract
     Information regarding agronomic practices for field-scale industrial hemp (IH) production for cannabinoids is extremely limited. The objective of this study was to investigate how pinching practice (i.e., removing the apical meristem) and varied plant density affect yield and cannabinoid production of IH in open field conditions. A field trial was performed in northern Florida in 2019 and 2020. The pinching study included pinch and non-pinch treatments evaluated on two commercially available day-length–sensitive IH cultivars Cherry Blossom (CBL) and Cherry Wine (CW). Four plant densities (3,000, 4,000, 6,000, and 12,000 plants ha–1) were evaluated on CW. The experimental design was a randomized complete block design with four blocks. Total flower yield in 2019 was two times greater than in 2020. In 2020, total tetrahydrocannabinol and cannabidiol (CBD) in CBL was significantly greater than in CW. Pinching did not significantly improve flower yield, cannabinoid concentration, or CBD production. Flower yield per plant decreased as plant density increased, but a reversed trend occurred for total flower yield per hectare. Cannabinoid concentration was not affected by plant density, but CBD production increased with increased plant density. Due to the high cost of IH plant material and labor, greater plant density does not always guarantee greater economic return. This study provides valuable information for field-scale cultivation of IH for cannabinoids and supports the development of a large agricultural industry globally.
119. Quagliata, Giulia and Celletti, Silvia and Coppa, Eleonora and Mimmo, Tanja and Cesco, Stefano and Astolfi, Stefania, Potential Use of Copper-Contaminated Soils for Hemp (Cannabis Sativa L.) Cultivation, Environments, vol. 8, no. 11, pp. 111, November 2021. doi: 10.3390/environments8110111. Abstract
     To mitigate climate change, reducing greenhouse gas emissions can be achieved by decreasing the use of fossil fuels and increasing that of alternative sources, such as energy crops. However, one of the most important problems in the use of biomass as a fuel is that of changing soil use and consumption, leading to competition with food crops. We addressed the topic by evaluating the possibility to exploit contaminated areas for energy crops cultivation. Indeed, soil contamination makes land inappropriate for cultivation, with damaging consequences for ecosystems, as well as posing serious health hazards to living beings. Specifically, this work aimed to evaluate the ability of hemp (Cannabis sativa L.) plants to grow on a copper (Cu)-contaminated medium. In addition, the effectiveness of an environment-friendly treatment with sulfate in improving plant ability to cope with Cu-induced oxidative stress was also explored. Results showed that plants were able to grow at high Cu concentrations. Therefore, hemp could represent an interesting energy crop in Cu-contaminated soils. Although the response of Cu-treated plants was evidenced by the increase in thiol content, following modulation of sulfur metabolism, it remains to be clarified whether the use of exogenous sulfate could be an agronomic practice to improve crop performance under these edaphic conditions.
120. Rheay, Hanah T. and Omondi, Emmanuel C. and Brewer, Catherine E., Potential of Hemp (Cannabis Sativa L.) for Paired Phytoremediation and Bioenergy Production, GCB Bioenergy, vol. 13, no. 4, pp. 525--536, 2021. doi: 10.1111/gcbb.12782. Abstract
     Hemp (Cannabis sativa L.) is a multi-use crop that has been investigated for its potential use in phytoremediation of heavy metals, radionuclides, and organic contaminants, and as a feedstock for bioenergy production. A review of research literature indicates that hemp is a suitable crop for phytoremediation, and a competitive option for bioenergy. Coupling phytoremediation and bioenergy production from a single hemp crop is a potential solution to overcoming the economic constraints of phytoremediation projects. The current challenge is ensuring that the extracted contaminants are not introduced into the consumer marketplace. After several decades of limited research on hemp in the United States, the purpose of this review is to identify the knowledge available for hemp applications in phytoremediation or in production of bioenergy, and if and how those two purposes have been combined. The literature shows that hemp growth has been demonstrated successfully at the field scale for phytoremediation and in several bioenergy conversion technologies. Little is known about the fate of contaminants during hemp growth or during post-harvest processing, especially the relationships between hemp genetics, metabolomics, and contaminant partitioning. Complicating the understanding is the expectation that contaminant fate will be dependent on the contaminant type, the concentration in the material, and the processing methods. Before hemp from phytoremediation applications can be used for bioenergy, the fractionation of heavy metals, radionuclides, and/or organic compounds during transesterification, anaerobic digestion, fermentation, and/or combustion of hemp must be evaluated.
121. Wu, Yudi and Trejo, Helen X. and Chen, Gang and Li, Simeng, Phytoremediation of Contaminants of Emerging Concern from Soil with Industrial Hemp (Cannabis Sativa L.): A Review, Environment, Development and Sustainability, vol. 23, no. 10, pp. 14405--14435, October 2021. doi: 10.1007/s10668-021-01289-0. Abstract
     The presence of contaminants of emerging concern (CECs) in wastewater treatment plant effluents is a significant underlying health risk and environmental concern. CECs consist of a wide variety of contaminants, including pharmaceuticals and personal care products, hormones, steroids, alkyl-phenols, flame retardants and pesticides. Their impact is of particular relevance to agricultural settings due to CEC uptake and accumulation in food crops and consequent diffusion into the food-chain. Meanwhile, marijuana reform is accelerating in the US, based on the scope and pace of legalization efforts and on wider acceptance in polls of voters. In this review, the effectiveness of industrial hemp (Cannabis sativa L.) in phytoremediation and hyperaccumulation of organic contaminants (e.g., benzo(a)pyrene, Naphthalene, and Chrysene) and heavy metal (e.g., Selenium and Cobalt) from either aqueous solutions or contaminated soils has been reviewed. The potential of industrial hemp as a renewable resource to biodegrade and/or decontaminate CECs is explored. Disposal strategies of this new phytoremediation crop that promote circular economy are also discussed. According to this current review, we believe the use of industrial hemp for phytoremediation is promising to have a sustainable, environmentally friendly and economically viable future.
122. Deng, Gang and Yang, Mei and Saleem, Muhammad Hamzah and Rehman, Muzammal and Fahad, Shah and Yang, Yang and Elshikh, Mohamed Soliman and Alkahtani, Jawaher and Ali, Sardar and Khan, Shah Masaud, Nitrogen Fertilizer Ameliorate the Remedial Capacity of Industrial Hemp (Cannabis Sativa L.) Grown in Lead Contaminated Soil, Journal of Plant Nutrition, vol. 44, no. 12, pp. 1770--1778, July 2021. doi: 10.1080/01904167.2021.1881553. Abstract
     Industrial hemp (Cannabis sativa L.) is a fibrous plant with high biomass production, and can survive under heavy metal stress conditions. To determine the growth potential of industrial hemp on lead (Pb) contaminated soil, a pot experiment was conducted using two varieties Bamahuoma (BM) and Yunma-1 (Y1), under different levels of nitrogen (N) fertilizer viz., 0 (control), 0.1, 0.3, 0.5 and 0.7 g kg−1. The soil was artificially spiked with Pb using PbCl. Lead uptake and its accumulation in different organs of both hemp varieties were analyzed. Increasing level of N fertilizer caused a significant increase in the plant growth and dry biomass production in both varieties, while Y1 showed more tolerance to Pb, than BM. Furthermore, N (\%) was higher in the leaves and stems then in the roots. Contrastingly, Pb was highly accumulated in the roots then in the leaves and stems of both varieties under varying levels of N fertilizer in the soil. Lead uptake and its accumulation in different plant parts were increased with the increase in N fertilizer in the soil. Hence, these findings indicate that external N fertilization is a useful strategy to enhance dry biomass and Pb accumulation in industrial hemp.
123. Stonehouse, Gavin C. and McCarron, Brandon Jude and Guignardi, Zack S. and El Mehdawi, Ali F. and Lima, Leonardo W. and Fakra, Sirine C. and {Pilon-Smits}, Elizabeth A.H., Selenium Metabolism in Hemp (Cannabis Sativa L.)—Potential for Phytoremediation and Biofortification, Environmental Science \& Technology, vol. 54, no. 7, pp. 4221--4230, April 2020. doi: 10.1021/acs.est.9b07747. Abstract
     Selenium (Se) deficiency and toxicity affect over a billion people worldwide. Plants can mitigate both problems, via Se biofortification and phytoremediation. Here we explore the potential of hemp (Cannabis sativa L.) for these phytotechnologies. Field surveys in naturally seleniferous agricultural areas in Colorado, United States, found 15–25 μg of Se/g in seed and 5–10 μg of Se/g dry weight (DW) in flowers and leaves. Thus, 4 g of this hemp seed provides the U.S. recommended daily allowance of 55–75 μg of Se. In controlled greenhouse experiments, hemp seedlings grown in Turface supplied with 40–320 μM selenate showed complete tolerance up to 160 μM and accumulated up to 1300 mg of Se/kg shoot dry weight. Mature hemp grown in Turface supplied with 5–80 μM selenate was completely tolerant up to 40 μM selenate and accumulated up to 200 mg of Se/kg DW in leaves, flowers, and seeds. Synchrotron X-ray fluorescence and X-ray absorption spectroscopies of selenate-supplied hemp showed Se to accumulate mainly in the leaf vasculature and in the seed embryos, with predominant Se speciation in C–Se–C forms (57–75\% in leaf and more than 86\% in seeds). Aqueous seed extracts were found by liquid chromatography mass spectrometry to contain selenomethionine and methyl-selenocysteine (1:1–3 ratio), both excellent dietary Se sources. Floral concentrations of medicinal cannabidiol (CBD) and terpenoids were not affected by Se. We conclude that hemp has good potential for Se phytoremediation while producing Se-biofortified dietary products.
124. Ćaćić, Marija and Perčin, Aleksandra and Zgorelec, Željka and Kisić, Ivica, Evaluation of Heavy Metals Accumulation Potential of Hemp (Cannabis Sativa L.), Journal of Central European Agriculture, vol. 20, no. 2, pp. 700--711, 2019. doi: 10.5513/JCEA01/20.2.2201. Abstract
     Heavy metals accumulation in crops and soils poses a significant threat to the human health. A study was carried out in 2016 in order to assess hemp (Cannabis sativa L.) ability to accumulate heavy metals and to reveal its possibility as a phytoaccumulator or phytostabilizer. Two soil types from Croatia were used in experimental pots: Gleysoils (alkaline soil) and Stagnic Luvisol (acid soil). Majority of the varieties accumulated more heavy metals in roots than in above-ground biomass. Removal of Cd, Ni, Pb, Hg, Co, Mo and As was higher in acid soil. Potential ability for phytostabilization was observed in alkaline soil in order Cu {$>$} Cr {$>$} Cd {$>$} Mo {$>$} Hg {$>$} Zn {$>$} Ni {$>$} Co {$>$} As {$>$} Pb, while for acid soil in order Zn {$>$} Cd {$>$} Cr {$>$} Ni {$>$} Hg {$>$} Cu {$>$} Mo {$>$} As {$>$} Co {$>$} Pb. Some varieties exhibited a translocation coefficient (TC) more than 1 and shown the ability of hyper-accumulation for Zn, Hg, Mo and Cd. Higher accumulation of heavy metals in some varieties could lead to their general application for phytoaccumulation of heavy metals from polluted soils.
125. Husain, Rabab and Weeden, Hannah and Bogush, Daniel and Deguchi, Michihito and Soliman, Mario and Potlakayala, Shobha and Katam, Ramesh and Goldman, Stephen and Rudrabhatla, Sairam, Enhanced Tolerance of Industrial Hemp (Cannabis Sativa L.) Plants on Abandoned Mine Land Soil Leads to Overexpression of Cannabinoids, PLOS ONE, vol. 14, no. 8, pp. e0221570, August 2019. doi: 10.1371/journal.pone.0221570. Abstract
     Industrial activities have a detrimental impact on the environment and health when high concentrations of pollutants are released. Phytoremediation is a natural method of utilizing plants to remove contaminants from the soil. The goal of this study was to investigate the ability of Cannabis sativa L. to sustainably grow and remediate abandoned coal mine land soils in Pennsylvania. In this study, six different varieties of industrial hemp (Fedora 17, Felina 32, Ferimon, Futura 75, Santhica 27, and USO 31) were grown on two different contaminated soil types and two commercial soils (Miracle-Gro Potting Mix and PRO-MIX HP Mycorrhizae High Porosity Grower Mix). Plants growing in all soil types were exposed to two environmental conditions (outside and in the greenhouse). Seed germination response and plant height indicated no significant differences among all hemp varieties grown in different soils, however on an average, the height of the plants grown in the greenhouse exceeded that of the plants grown outdoors. In addition, heavy metal analysis of Arsenic, Lead, Nickel, Mercury, and Cadmium was performed. The concentration of Nickel was 2.54 times greater in the leaves of hemp grown in mine land soil outdoors when compared to greenhouse conditions. No differences were found between expression of heavy metal transporter genes. Secondary metabolite analysis of floral buds from hemp grown in mine land soil displayed a significant increase in the total Cannabidiol content (2.16\%, 2.58\%) when compared to Miracle-Gro control soil (1.08\%, 1.6\%) for outdoors and in the greenhouse, respectively. Molecular analysis using qRT-PCR indicated an 18-fold increase in the expression of the cannabidiolic acid synthase gene in plants grown on mine land soil. The data indicates a high tolerance to heavy metals as indicated from the physiological and metabolites analysis.
126. Ahmad, Rafiq and Tehsin, Zara and Malik, Samina Tanvir and Asad, Saeed Ahmad and Shahzad, Muhammad and Bilal, Muhammad and Shah, Mohammad Maroof and Khan, Sabaz Ali, Phytoremediation Potential of Hemp (Cannabis Sativa L.): Identification and Characterization of Heavy Metals Responsive Genes, CLEAN – Soil, Air, Water, vol. 44, no. 2, pp. 195--201, 2016. doi: 10.1002/clen.201500117. Abstract
     Soil pollution caused by heavy metals is one of the major problems throughout the world. To maintain a safe and healthy environment for human beings, there is a dire need to identify hyperaccumulator plants and the underlying genes involved in heavy metals stress tolerance and accumulation. The goal of this research is to explore the potential of hemp as a decontaminator of heavy metals by identifying the two important heavy metals responsive genes, glutathione-disulfidereductase (GSR) and phospholipase D-α (PLDα). The results revealed heavy metals accumulation; Cu (1530 mg kg−1), Cd (151 mg kg−1), and Ni (123 mg kg−1) in hemp plants’ leaves collected from the contaminated site. This shows the ability of the hemp plant to tolerate heavy metals, perhaps due to the presence of stress tolerance genes. In this study, partial sequences of putative GSR (215 bp) and PLDα (517 bp) genes were identified, responsive to heavy metals stress in hemp leaves. Both genes exhibited 40–60\% sequence identity to previously reported genes from other plant species. Glutathione binding residues and conserved arginine residues were found identical in a putative GSR gene to those of other plant species, while the phospholipids binding domain and catalytic domain were found in the PLDα gene. These results will help to improve our understanding about the phytoremediation potential of hemp as well as in manipulating GSR and PLDα genes in breeding programs to produce transgenic heavy-metals-tolerant varieties.
127. Zerihun, Agalu and Chandravanshi, Bhagwan Singh and Debebe, Ayalew and Mehari, Bewketu, Levels of Selected Metals in Leaves of Cannabis Sativa L. Cultivated in Ethiopia, SpringerPlus, vol. 4, no. 1, pp. 359, July 2015. doi: 10.1186/s40064-015-1145-x. Abstract
     Cannabis sativa L. is one of the illicit drug bearing plants. Cannabis products are the most widely trafficked drugs worldwide. The highest levels of cannabis production in the world take place in the African continent. A small volume of cannabis is produced in rural areas of Ethiopia, of which a small portion is exported to neighboring countries and the majority is consumed at home. The literature survey revealed that there is no report on the metal contents in cannabis cultivated in Ethiopia. The main objective of this study is to determine the level of selected metals in leaves of Cannabis sativa L. cultivated in Ethiopia.
128. Linger, P. and Ostwald, A. and Haensler, J., Cannabis Sativa L. Growing on Heavy Metal Contaminated Soil: Growth, Cadmium Uptake and Photosynthesis, Biologia Plantarum, vol. 49, no. 4, pp. 567--576, December 2005. doi: 10.1007/s10535-005-0051-4. Abstract
     The effects of different cadmium concentrations [17 mg(Cd) kg−1(soil) and 72 mg(Cd) kg− 1(soil)] on Cannabis sativa L. growth and photosynthesis were examined. Hemp roots showed a high tolerance to Cd, i.e. more than 800 mg(Cd) kg−1(d.m.) in roots had no major effect on hemp growth, whereas in leaves and stems concentrations of 50 – 100 mg(Cd) kg−1(d.m.) had a strong effect on plant viability and vitality. For control of heavy metal uptake and xylem loading in hemp roots, the soil pH plays a central role. Photosynthetic performance and regulation of light energy consumption were analysed using chlorophyll fluorescence analysis. Seasonal changes in photosynthetic performance were visible in control plants and plants growing on soil with 17 mg(Cd) kg−1(soil). Energy distribution in photosystem 2 is regulated in low and high energy phases that allow optimal use of light and protect photosystem 2 from overexcitation, respectively. Photosynthesis and energy dissipation were negatively influenced by 72 mg(Cd) kg−1(soil). Cd had detrimental effects on chlorophyll synthesis, water splitting apparatus, reaction centre, antenna and energy distribution of PS 2. Under moderate cadmium concentrations, i.e. 17 mg(Cd) kg−1(soil), hemp could preserve growth as well as the photosynthesis apparatus, and long-term acclimation to chronically Cd stress occurred.
129. Citterio, Sandra and Santagostino, Angela and Fumagalli, Pietro and Prato, Nadia and Ranalli, Paolo and Sgorbati, Sergio, Heavy Metal Tolerance and Accumulation of Cd, Cr and Ni by Cannabis Sativa L., Plant and Soil, vol. 256, no. 2, pp. 243--252, October 2003. doi: 10.1023/A:1026113905129. Abstract
     Experiments in semi-natural conditions were undertaken to assess hemp metal tolerance and its ability to accumulate cadmium, nickel and chromium. Cannabis sativa was grown in two soils, S1 and S2, containing 27, 74, 126 and 82, 115, 139 μg g−1 of Cd, Ni and Cr, respectively. After two months from germination and at ripeness, no significant alteration in plant growth or morphology was detected. On the contrary, a high hemp reactivity to heavy metal stress with an increase in phytochelatin and DNA content was observed during development, suggesting the Cannabis sativa ability to avoid cell damage by activating different molecular mechanisms. Metals were preferentially accumulated in the roots and only partially translocated to the above-ground tissues. The mean shoot Cd content was 14 and 66 μg g−1 for S1 and S2 soil, respectively. Although not negligible concentrations they were about 100 times lower than those calculated for the hyperaccumulator Thlaspi caerulescens. Similarly Ni uptake was limited if compared with that of the Ni-hyperaccumulator Alyssum murale. Chromium uptake was negligible. As expected on the base of the metal concentration detected in ripe plants, no statistically significant variation in soil metal content was detected after one crop of hemp. Nevertheless, a consistent amount (g) of Cd and Ni is expected to be extracted by 1 ha biomass of hemp (about 10 t) per year and along the time a slow restoration of deeper soil portions can be obtained by its wide root system (at least 0,5 m deep). In addition, the possibilities of growing hemp easily in different climates and using its biomass in non-food industries can make heavy metal contaminated soils productive. This means economical advantage along with a better quality of soil.
130. Linger, P. and Müssig, J. and Fischer, H. and Kobert, J., Industrial Hemp (Cannabis Sativa L.) Growing on Heavy Metal Contaminated Soil: Fibre Quality and Phytoremediation Potential, Industrial Crops and Products, vol. 16, no. 1, pp. 33--42, July 2002. doi: 10.1016/S0926-6690(02)00005-5. Abstract
     Hemp (Cannabis sativa L.) was used to examine its capability as a renewable resource to decontaminate heavy metal polluted soils. The influence of heavy metals on the fibre quality was of special interest. Determination of heavy metal content was carried out by means of atomic absorption spectroscopy (AAS). Four different parts of the plant were examined: seeds, leaves, fibres and hurds. In each case, the concentration relation was Ni{$>$}Pb{$>$}Cd. However, the heavy metal accumulation in the different parts of the plant was extremely different. All parts of hemp plants contain heavy metals and this is why their use as a commercially utilisable plant material is limited. We found that the highest concentrations of all examined metals were accumulated in the leaves. In this field trial, hemp showed a phytoremediation potential of 126 g Cd (ha vegetation period)−1. We tested the fibre quality by measuring the pure fibre content of the stems and the fibre properties after mechanical separation. In addition, the fibre fineness was examined using airflow systems and image analysis. The strength was measured by testing single fibre bundles with a free clamping distance of 3.2 mm using a universal testing device. Finally, we compared the results from the stems and fibres from trials on heavy metal polluted ground with hemp stems and fibres from non-polluted ground. Since there was no comparable unpolluted area near the polluted one, reference values were taken from an area quite far away and subsequently with a different soil composition and also exposure to different meteorological conditions. Thus, the observed differences are only partially caused by the heavy metal contamination.
131. Coffman, C. B. and Gentner, W. A., Cannabinoid Profile and Elemental Uptake of Cannabis Sativa L. as Influenced by Soil Characteristics1, Agronomy Journal, vol. 67, no. 4, pp. 491--497, 1975. doi: 10.2134/agronj1975.00021962006700040010x. Abstract
     The consumption of Cannabis products (marihuana) derived from domestic and foreign sources persists in the United States despite its illegality and health hazards. The objectives of this investigation were: 1) to evaluate relationships between soil and plant elements, cannabinoids, and growth of Cannabis sativa L., and 2) to evaluate the practicality of using chemical analysis of Cannabis products to determine their geographic origin. Knowledge of geographic origin is useful to governmental agencies investigating illicit narcotic traffic. Cannabis sativa L. was grown on 11 different soils for 45 days in the greenhouse. Soils differed significantly in 15 measured elements and pH. Plants were grown from seed of Afghan origin. The following cannabinoids were extracted and measured from leaf tissue: cannabicyclol (CCC), cannabidiol (CBD), Δ9-Tetrahydrocannabinol (Δ9THC), and cannabinol (CBN). Fifteen elements measured in leaf tissue and correlated with soil and cannabinoid measurements. Soil pH was negatively correlated with leaf concentrations of Mn, Fe, Zn, and S. Extractable soil Mg was negatively correlated with N, Δ9THC and CBD concentrations in leaf tissue (p {$<$} 0.05). Plant height was negatively correlated with Δ9THC concentration, suggesting enhancement of the narcotic principle of marihuana when grown under stress. Extractable soil P2O5 was negatively correlated with CBD concentration while extractable soil Zn was positively correlated with CCC concentration. Several correlations between soil and plant characteristics having potential value for determination of geographic origin of marihuana were elucidated. However, environmental, harvesting, and analytical procedures used by different workers which do not conform to one another could result in changes in the soil-plant correlations reported herein. Thus, additional studies are required before determination of the geographic origin of Cannabis products by foliar analysis becomes feasible.
132. Shiponi, Sivan and Bernstein, Nirit, Response of Medical Cannabis (Cannabis Sativa L.) Genotypes to P Supply under Long Photoperiod: Functional Phenotyping and the Ionome, Industrial Crops and Products, vol. 161, pp. 113154, March 2021. doi: 10.1016/j.indcrop.2020.113154. Abstract
     Phosphorus (P) is an essential macronutrient required for many central metabolic processes, and is therefore a major factor governing plant development, structure and function. Cannabis is a short-day plant that its' development progression involves a vegetative growth phase under long photoperiod, followed by a reproductive phase under a short photoperiod. The reproductive inflorescence yield potential in cannabis is therefore largely dependent on the morphology and physiological condition of the plants at the vegetative phase. Due to legal restrictions, there is lack of science-based knowledge about cannabis plant science, including mineral nutrition. The present study therefore focused on P nutrition of plants at the vegetative growth phase under long photoperiod. The plants were cultivated in pots in a controlled environment and subjected to 5 levels of P (5, 15, 30, 60, 90 mg L−1). We investigated impact on the ionome, physiological and morphological traits, uptake of nutrients into the plant, translocation to the shoot, and distribution in the plant organs for 2 medicinal cannabis genotypes. Plant biomass production, photosynthesis rate, stomatal conductance, transpiration rate and intercellular CO2 at the vegetative growth phase exceled under 30 mg L−1 P supply. Uptake and translocation of nutrients from root to the shoot was highly influenced by the P treatment. Under excess P supply, most of the plant P accumulated in the roots, and translocation to the shoot was inhibited. Uptake of Mg into the plants, and its' translocation to the shoot was inhibited by P deficiency in both cultivars, and was enhanced by increased P supply. Calcium uptake was increased by P application but translocation to the shoot was inhibited. Zinc retention in roots under P deficiency was found in both varieties. Our results suggest a wide optimum range for P in medicinal cannabis at the vegetative growth stage, with a minimum requirement of 15 mg L−1 P and a recommended application of 30 mg L−1. The functional physiology and ionome profiling revealed genotypic variability in P sensitivity.
133. Ii, Steven L. Anderson and Pearson, Brian and Kjelgren, Roger and Brym, Zachary, Response of Essential Oil Hemp (Cannabis Sativa L.) Growth, Biomass, and Cannabinoid Profiles to Varying Fertigation Rates, PLOS ONE, vol. 16, no. 7, pp. e0252985, July 2021. doi: 10.1371/journal.pone.0252985. Abstract
     Five essential oil hemp (Cannabis sativa L.) cultivars (Cherry Blossom, Cherry Blossom (Tuan), Berry Blossom, Cherry Wine, and Cherry Blossom × Trump) were treated with six fertigation treatments to quantify the effects of synthetic fertilizer rates and irrigation electrical conductivity on plant growth, biomass accumulation, and cannabinoid profiles. Irrigation water was injected with a commercial 20-20-20 fertilizer at rates of 0, 50, 150, 300, 450, and 600 ppm nitrogen equating to 0.33 (control), 0.54, 0.96, 1.59, 2.22, and 2.85 dS m−1, respectively. Plants were grown under artificial lighting (18 hr) to maintain vegetative growth for eight weeks, followed by an eight-week flowering period. High linear relationship between chlorophyll concentrations and SPAD-502 measurements validated the utilization of SPAD meters to rapidly identify nutrient deficiency in essential oil hemp. Cultivars expressed significant variation in plant height and cannabinoid profiles (\% dry mass), in concurrence with limited biomass and cannabinoid (g per plant) yield variation. Cherry Blossom was the best performing cultivar and Cherry Wine was the least productive. Variation in plant growth, biomass, and cannabinoid concentrations were affected to a greater extent by fertilizer rates. Optimal fertilizer rates were observed at 50 ppm N, while increased fertilizer rates significantly reduced plant growth, biomass accumulation, and cannabinoid concentrations. Increased fertilizer rates ({$>$} 300 ppm N) resulted in compliant THC levels ({$<$} 0.3\%), although when coupled with biomass reductions resulted in minimal cannabinoid yields. Additionally, CBD concentration demonstrated higher sensitivity to increased fertilizer rates ({$>$} 300 ppm N) compared to THC and CBG ({$>$} 450 ppm N). The results of this study can serve as a guide when using fertigation methods on essential oil hemp cultivars; although results may differ with cultivar selection, environmental conditions, and management practices.
134. Saloner, Avia and Bernstein, Nirit, Nitrogen Supply Affects Cannabinoid and Terpenoid Profile in Medical Cannabis (Cannabis Sativa L.), Industrial Crops and Products, vol. 167, pp. 113516, September 2021. doi: 10.1016/j.indcrop.2021.113516. Abstract
     Secondary metabolism in plants is considerably affected by environmental factors including mineral nutrition. Nitrogen is a key plant nutrient, known to affect primary and secondary metabolism in plants, that its effect on the cannabis plants' chemical profile is not known. To evaluate the hypothesis that N supply affects the cannabinoid and terpenoid profile, we studied the impact of N application on chemical and functional-physiology phenotyping in medical cannabis at the flowering stage. The plants were grown under five N treatments of 30, 80, 160, 240, and 320 mg L−1 (ppm) under environmentally controlled conditions. The results revealed that N supply affects cannabinoid and terpenoid metabolism, supporting the hypothesis. The concentrations of most cannabinoids and terpenoids tested were highest under the deficient concentration of 30 mg L−1 N and declined with the elevation of N supply. The concentrations of the two main cannabinoids, tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), decreased by 69\% and 63\%, respectively, with the increase in N supply from 30 to 320 mg L−1 N. Plant development and function were restricted under inputs lower than 160 mg L−1 N, demonstrating N deficiency. The morpho-physiological state of the plants was optimal at supply rates of 160–320 mg L−1 N. Inflorescence yield reflected the plant physiological state, increasing with the increase in N supply up to 160 mg L−1 N, and was unaffected by further increase in N. These results of the functional and chemical characterizations suggest that high N supply has adverse effects on the production of secondary compounds in cannabis, while it promotes growth and biomass production. Hence, N supply may serve for the regulation of the cannabinoid and terpenoid profiles, or for increasing plant yield, according to the desired production scheme. Taken together, the results reveal that the optimal N level for yield quantity, that allows also a relatively high secondary metabolites content, is 160 mg L−1 N. Finally, the present study provides a better understanding of the impact of N on 'drug-type' medical cannabis physiology, and takes us one step closer to the optimization of medical cannabis cultivation.
135. Kakabouki, Ioanna and Kousta, Angeliki and Folina, Antigolena and Karydogianni, Stella and Zisi, Charikleia and Kouneli, Varvara and Papastylianou, Panayiota, Effect of Fertilization with Urea and Inhibitors on Growth, Yield and CBD Concentration of Hemp (Cannabis Sativa L.), Sustainability, vol. 13, no. 4, pp. 2157, January 2021. doi: 10.3390/su13042157. Abstract
     Field experiments were conducted during 2019 in two different locations in Greece (Athens and Farsala) to evaluate the effect of urea and urea fertilization with inhibitors on the agronomic yield and quality characteristics of two cannabis varieties (Cannabis sativa L.), “Uso31”and “Fedora 17”. The experimental design was split-plot with four different fertilization treatments—control, Urea (U), urea with Urease Inhibitor (UI), and urea with Nitrification Inhibitor (NI) and urease inhibitor (UI). The significance of differences between treatments was estimated by using Tukey’s test with a significance level of p = 0.05. The plant height was significantly affected by the different fertilizations and different varieties as well as by the two locations. The maximum plant height was 197 cm for “Fedora 17”in Farsala. The seed yield was higher forthe urea with inhibitors treatment in both varieties. The Cannabidiol (CBD) content was significantly affected by the fertilization—it was higher in urea with inhibitors in “Uso31”and “Fedora 17” treatments. The lowest CBD content value was 1.29\% (control) and the highest was 1.69\% (urea NI + UI). In conclusion, in both varieties, it seems that urea with inhibitors has a positive effect on their growth, as well as on the increase in cannabidiol (CBD) content.
136. Kakabouki, Ioanna and Tataridas, Alexandros and Mavroeidis, Antonios and Kousta, Angeliki and Karydogianni, Stella and Zisi, Charikleia and Kouneli, Varvara and Konstantinou, Artemis and Folina, Antigolena and Konstantas, Aristidis and Papastylianou, Panayiota, Effect of Colonization of Trichoderma Harzianum on Growth Development and CBD Content of Hemp (Cannabis Sativa L.), Microorganisms, vol. 9, no. 3, pp. 518, March 2021. doi: 10.3390/microorganisms9030518. Abstract
     Trichoderma harzianum, as a natural endophytic biocontrol agent, can ameliorate plant development, nutrient uptake, and resistance to biotic and abiotic stresses. This study aimed to investigate the effect of Trichoderma harzianum inoculation on agronomical and quality characteristics of two monoecious hemp (Cannabis sativa L.) varieties, Fedora 17 and Felina. A greenhouse pot experiment was conducted in a completely randomized design of two treatments of Trichoderma harzianum with a low and high dose of the fungus (T1 and T2). The significance of differences between treatments was estimated by using a Fisher’s test with a significance level p = 0.05. The root density of both varieties was significantly affected by treatments, and higher values were recorded in Fedora 17 (2.32 mm cm−3). The Arbuscular Mycorrhizal Fungi (AMF) colonization of the root system and the soil emission of CO2 were higher after the inoculation of Trichoderma harzianum. The highest values of plant height and dry weight were noticed for T2, especially in variety Felina. Trichoderma harzianum positively influenced characteristics of inflorescences such as their number, fresh weight moisture, and compactness in both varieties, while the dry weight, length, and dry yield of inflorescences were not improved. Finally, the fertigation of Trichoderma harzianum in hemp plants was beneficial by increasing the cannabidiol (CBD) content, especially in T2 treatment (4 × 1012 CFU kg−1).
137. Saloner, Avia and Bernstein, Nirit, Response of Medical Cannabis (Cannabis Sativa L.) to Nitrogen Supply Under Long Photoperiod, Frontiers in Plant Science, vol. 11, 2020. doi: 10.3389/fpls.2020.572293. Abstract
     The development progression of medical cannabis plants includes a vegetative growth phase under long photoperiod, followed by a reproductive phase under short photoperiod. Establishment of plant architecture at the vegetative phase affects its reproduction potential under short photoperiod. Nitrogen (N) is a main component of many metabolites that are involved in central processes in plants, and is therefore a major factor governing plant development and structure. We lack information about the influence of N nutrition on medical cannabis functional-physiology and development, and plant N requirements are yet unknown. The present study therefore investigated the developmental, physiological, and chemical responses of medical cannabis plants to N supply (30, 80, 160, 240, and 320 mgL−1 N) under long photoperiod. The plants were cultivated in an environmentally controlled growing room, in pots filled with soilless media. We report that the morpho-physiological function under long photoperiod in medical cannabis is optimal at 160 mgL−1 N supply, and significantly lower under 30 mgL−1 N, with visual deficiency symptoms, and 75 and 25\% reduction in plant biomass and photosynthesis rate, respectively. Nitrogen use efficiency (NUE) decreased with increasing N supply, while osmotic potential, water use efficiency, photosynthetic pigments, and total N and N-NO3 concentrations in plant tissues increased with N supply. The plant ionome was considerably affected by N supply. Concentrations of K, P, Ca, Mg, and Fe in the plant were highest under the optimal N level of 160 mgL−1 N, with differences between organs in the extent of nutrient accumulation. The majority of the nutrients tested, including P, Zn, Mn, Fe, and Cu, tended to accumulate in the roots {$>$} leaves {$>$} stem, while K and Na tended to accumulate in the stem {$>$} leaves {$>$} roots, and total N, Ca, and Mg accumulated in leaves {$>$} roots {$>$} stem. Taken together, the results demonstrate that the optimal N level for plant development and function at the vegetative growth phase is 160 mgL−1 N. Growth retardation under lower N supply (30–80 mgL−1) results from restricted availability of photosynthetic pigments, carbon fixation, and impaired water relations. Excess uptake of N under supply higher than 160 mgL−1 N, promoted physiological and developmental restrictions, by ion-specific toxicity or indirect induced restrictions of carbon fixation and energy availability.
138. Cockson, Paul and {Schroeder-Moreno}, Michelle and Veazie, Patrick and Barajas, Gabby and Logan, David and Davis, Matthew and Whipker, Brian E., Impact of Phosphorus on Cannabis Sativa Reproduction, Cannabinoids, and Terpenes, Applied Sciences, vol. 10, no. 21, pp. 7875, January 2020. doi: 10.3390/app10217875. Abstract
     Many abiotic factors, such as mineral nutrients—including phosphorus (P)—fertility, can impact the yield and growth of Cannabis sativa. Given the economic portion of C. sativa is the inflorescence, the restriction of P fertility could impact floral development and quality could be detrimental. This study sought to track the impacts of varying P concentrations (3.75, 7.50, 11.25, 15.0, 22.50, and 30.0 mg·L−1) utilizing a modified Hoagland’s solution. This experiment examined plant height, diameter, leaf tissue mineral nutrient concentrations, and final fresh flower bud weight as well as floral quality metrics, such as cannabinoids and terpenes. The results demonstrated that during different life stages (vegetative, pre-flowering, flowering), P concentrations impact C. sativa growth and development and yield. Regarding the cannabinoid pools, results varied for the individual cannabinoid types. For the acid pools, increasing fertility concentrations above 11.25 mg·L−1 P did not result in any increase in cannabinoid concentrations. These results indicate that, if a crop is being produced under greenhouse conditions, specifically for cannabinoid production, an excessive P supply did not result in higher cannabinoid production. However, plants grown with a higher rate of P fertility (30.0 mg·L−1) had greater plant width and may result in more buds per plant.
139. Taghinasab, Meysam and Jabaji, Suha, Cannabis Microbiome and the Role of Endophytes in Modulating the Production of Secondary Metabolites: An Overview, Microorganisms, vol. 8, no. 3, pp. 355, March 2020. doi: 10.3390/microorganisms8030355. Abstract
     Plants, including cannabis (Cannabis sativa subsp. sativa), host distinct beneficial microbial communities on and inside their tissues and organs, including seeds. They contribute to plant growth, facilitating mineral nutrient uptake, inducing defence resistance against pathogens, and modulating the production of plant secondary metabolites. Understanding the microbial partnerships with cannabis has the potential to affect the agricultural practices by improving plant fitness and the yield of cannabinoids. Little is known about this beneficial cannabis-microbe partnership, and the complex relationship between the endogenous microbes associated with various tissues of the plant, and the role that cannabis may play in supporting or enhancing them. This review will consider cannabis microbiota studies and the effects of endophytes on the elicitation of secondary metabolite production in cannabis plants. The review aims to shed light on the importance of the cannabis microbiome and how cannabinoid compound concentrations can be stimulated through symbiotic and/or mutualistic relationships with endophytes.
140. Da Cunha Leme Filho, Jose F. and Thomason, Wade E. and Evanylo, Gregory K. and Zhang, Xunzhong and Strickland, Michael S. and Chim, Bee K. and Diatta, Andre A., Biochemical and Physiological Responses of Cannabis Sativa to an Integrated Plant Nutrition System, Agronomy Journal, vol. 112, no. 6, pp. 5237--5248, 2020. doi: 10.1002/agj2.20400. Abstract
     The illegal status of cannabis (Cannabis sativa L.) post-World War II resulted in a lack of research on agricultural practices. However, there is a resurgence of interest in cannabis due to diverse uses such as a rich source of cellulosic/woody fiber and construction uses, seed oil, bioenergy and pharmaceutical properties. The principle of an integrated plant nutrition system (IPNS) is to enable adaptation of plant nutrition and soil fertility management to local site characteristics, attempting to optimize use of inorganic, organic and biological resources. This project investigated the individual and combined use of inorganic, organic and biological fertilizer resources on cannabis before and after a period of moderate water stress. We evaluated the individual and combined effects of commercial synthetic fertilizer, humic acid (HA), manure tea and bioinoculant as inorganic, organic and biological resources, respectively on cannabis growth and physiological parameters. Our hypothesis was that the synergetic effects of HA + biofertilizers would improve cannabis growth. When compared to the control, the application of HA and biofertilizer alone, or in combination, increased plant height, chlorophyll content and photosynthetic efficiency by 55, 8 and 12\%, respectively, after water stress. Cannabis biomass of treated plants was rarely different from the control. The combined application of HA + biofertilizer resulted in additive, but not synergistic, increases in measured parameter. Future research should focus on the effects of biostimulants on CBD/THC content due to the potential impact on the production of secondary metabolites in plants under stress.
141. Saloner, Avia and Sacks, Mollie M. and Bernstein, Nirit, Response of Medical Cannabis (Cannabis Sativa L.) Genotypes to K Supply Under Long Photoperiod, Frontiers in Plant Science, vol. 10, 2019. doi: 10.3389/fpls.2019.01369. Abstract
     Potassium is involved in regulation of multiple developmental, physiological, and metabolic processes in plants, including photosynthesis and water relations. We lack information about the response of medical cannabis to mineral nutrition in general, and K in particular, which is required for development of high-grade standardized production for the medical cannabis industry. The present study investigated the involvement of K nutrition in morphological development, the plant ionome, photosynthesis and gas-exchange, water relations, water use efficiency, and K use efficiency, comparatively for two genotypes of medical cannabis, under a long photoperiod. The plants were exposed to five levels of K (15, 60, 100, 175, and 240 ppm K). Growth response to K inputs varied between genotypes, revealing genetic differences within the Cannabis sativa species to mineral nutrition. Fifteen ppm of K was insufficient for optimal growth and function in both genotypes and elicited visual deficiency symptoms. Two hundred and forty ppm K proved excessive and damaging to development of the genotype Royal Medic, while in Desert Queen it stimulated rather than restricted shoot and root development. The differences between the genotypes in the response to K nutrition were accompanied by some variability in uptake, transport, and accumulation of nutrients. For example, higher levels of K transport from root to the shoot were apparent in Desert Queen. However, overall trends of accumulation were similar for the two genotypes demonstrating competition for uptake between K and Ca and Mg, and no effect on N and P uptake except in the K-deficiency range. The extent of accumulation was higher in the leaves {$>$} roots {$>$} stem for N, and roots {$>$} leaves {$>$} stem for P. Surprisingly, most micronutrients (Zn, Mn, Fe, Cu, Cl) tended to accumulate in the root, suggesting a compartmentation strategy for temporary storage, or for prevention of access concentrations at the shoot tissues. The sensitivity of net-photosynthetic rate, gas exchange, and water use efficiency to K supply differed as well between genotypes. The results suggest that growth reduction under the deficient supply of 15 ppm K was mostly due to impact of K availability on water relations of the tissue and transpiration in Royal Medic, and water relations and carbon fixation in Desert Queen.
142. Landi, Simone and Berni, Roberto and Capasso, Giorgia and Hausman, Jean-Francois and Guerriero, Gea and Esposito, Sergio, Impact of Nitrogen Nutrition on Cannabis Sativa: An Update on the Current Knowledge and Future Prospects, International Journal of Molecular Sciences, vol. 20, no. 22, pp. 5803, January 2019. doi: 10.3390/ijms20225803. Abstract
     Nitrogen (N) availability represents one of the most critical factors affecting cultivated crops. N is indeed a crucial macronutrient influencing major aspects, from plant development to productivity and final yield of lignocellulosic biomass, as well as content of bioactive molecules. N metabolism is fundamental as it is at the crossroad between primary and secondary metabolic pathways: Besides affecting the synthesis of fundamental macromolecules, such as nucleic acids and proteins, N is needed for other types of molecules intervening in the response to exogenous stresses, e.g. alkaloids and glucosinolates. By partaking in the synthesis of phenylalanine, N also directly impacts a central plant metabolic ‘hub’—the phenylpropanoid pathway—from which important classes of molecules are formed, notably monolignols, flavonoids and other types of polyphenols. In this review, an updated analysis is provided on the impact that N has on the multipurpose crop hemp (Cannabis sativa L.) due to its renewed interest as a multipurpose crop able to satisfy the needs of a bioeconomy. The hemp stalk provides both woody and cellulosic fibers used in construction and for biocomposites; different organs (leaves/flowers/roots) are sources of added-value secondary metabolites, namely cannabinoids, terpenes, flavonoids, and lignanamides. We survey the available literature data on the impact of N in hemp and highlight the importance of studying those genes responding to both N nutrition and abiotic stresses. Available hemp transcriptomic datasets obtained on plants subjected to salt and drought are here analyzed using Gene Ontology (GO) categories related to N metabolism. The ultimate goal is to shed light on interesting candidate genes that can be further studied in hemp varieties growing under different N feeding conditions and showing high biomass yield and secondary metabolite production, even under salinity and drought.
143. Bernstein, Nirit and Gorelick, Jonathan and Zerahia, Roei and Koch, Sraya, Impact of N, P, K, and Humic Acid Supplementation on the Chemical Profile of Medical Cannabis (Cannabis Sativa L), Frontiers in Plant Science, vol. 10, 2019. doi: 10.3389/fpls.2019.00736. Abstract
     Mineral nutrition is a major factor affecting plant growth and function. Increasing evidence supports the involvement of macro and micronutrients in secondary metabolism. The use of the appropriate nutritional measures including organic fertilizers, supplements, and biostimulants is therefore a vital aspect of medicinal plant production including medical cannabis. Due to legal restriction on cannabis research, very little information is available concerning the effects of nutritional supplements on physiological and chemical properties of medical cannabis, and their potential role in standardization of the active compounds in the plant material supplied to patients. This study therefore evaluated the potential of nutritional supplementations, including humic acids (HAs) and inorganic N, P, and K to affect the cannabinoid profile throughout the plant. The plants were exposed to three enhanced nutrition treatments, compared to a commercial control treatment. The nutrition treatments were supplemented with HA, enhanced P fertilization, or enhanced NPK. The results demonstrate sensitivity of cannabinoids metabolism to mineral nutrition. The nutritional supplements affected cannabinoid content in the plants differently. These effects were location and organ specific, and varied between cannabinoids. While the P enhancement treatment did not affect THC, CBD, CBN, and CBG concentrations in the flowers from the top of the plants, a 16\% reduction of THC concentration was observed in the inflorescence leaves. Enhanced NPK and HA treatments also produced organ-specific and spatially specific responses in the plant. NPK supplementation increased CBG levels in flowers by 71\%, and lowered CBN levels in both flowers and inflorescence leaves by 38 and 36\%, respectively. HA was found to reduce the natural spatial variability of all of the cannabinoids studied. However, the increased uniformity came at the expense of the higher levels of cannabinoids at the top of the plants, THC and CBD were reduced by 37 and 39\%, respectively. Changes in mineral composition were observed in specific areas of the plants. The results demonstrate that nutritional supplements influence cannabinoid content in cannabis in an organ- and spatial-dependent manner. Most importantly, the results confirm the potential of environmental factors to regulate concentrations of individual cannabinoids in medical cannabis. The identified effects of nutrient supplementation can be further developed for chemical control and standardization in cannabis.
144. Guerriero, Gea and Deshmukh, Rupesh and Sonah, Humira and Sergeant, Kjell and Hausman, Jean-Francois and Lentzen, Esther and Valle, Nathalie and Siddiqui, Khawar Sohail and Exley, Christopher, Identification of the Aquaporin Gene Family in Cannabis Sativa and Evidence for the Accumulation of Silicon in Its Tissues, Plant Science, vol. 287, pp. 110167, October 2019. doi: 10.1016/j.plantsci.2019.110167. Abstract
     Cannabis sativa is an economically important crop providing bast fibres for the textile and biocomposite sector. Length is a fundamental characteristic determining the properties of bast fibres. Aquaporins, channel-forming proteins facilitating the passage of water, urea, as well as elements such as boron and silicon, are known to play a role in the control of fibre length in other species, like cotton. By mining the available genome, we here identify, for the first time, the aquaporin gene family of C. sativa. The analysis of published RNA-Seq data and targeted qPCR on a textile variety reveal an organ-specific expression of aquaporin genes. Computational analyses, including homology-based search, phylogeny and protein modelling, identify two NOD26-like intrinsic proteins harbouring the Gly-Ser-Gly-Arg (GSGR) aromatic/Arg selectivity filter and 108 amino acid NPA (Asn-Pro-Ala) spacing, features reported to be associated with silicon permeability. SIMS nano-analysis and silica extraction coupled to fluorescence microscopy performed on hemp plantlets reveal the presence of silicon in the bast fibres of the hypocotyl and in leaves. The accumulation of silica in the distal cell walls of bast fibres and in the basal cells of leaf trichomes is indicative of a mechanical role.
145. Cockson, Paul and Landis, Hunter and Smith, Turner and Hicks, Kristin and Whipker, Brian E., Characterization of Nutrient Disorders of Cannabis Sativa, Applied Sciences, vol. 9, no. 20, pp. 4432, January 2019. doi: 10.3390/app9204432. Abstract
     Essential plant nutrients are needed at crop-specific concentrations to obtain optimum growth or yield. Plant tissue (foliar) analysis is the standard method for measuring those levels in crops. Symptoms of nutrient deficiency occur when those tissue concentrations fall to a level where growth or yield is negatively impacted and can serve as a visual diagnostic tool for growers and researchers. Both nutrient deficiency symptoms and their corresponding plant tissue concentrations have not been established for cannabis. To establish nutrient concentrations when deficiency or toxicity symptoms are expressed, Cannabis sativa ‘T1’ plants were grown in silica sand culture, and control plants received a complete modified Hoagland’s all-nitrate solution, whereas nutrient-deficient treatments were induced with a complete nutrient formula withholding a single nutrient. Toxicity treatments were induced by increasing the element tenfold higher than the complete nutrient formula. Plants were monitored daily and, once symptoms manifested, plant tissue analysis of all essential elements was performed by most recent mature leaf (MRML) tissue analysis, and descriptions and photographs of nutrient disorder symptomology were taken. Symptoms and progressions were tracked through initial, intermediate, and advanced stages. Information in this study can be used to diagnose nutrient disorders in Cannabis sativa.
146. Pagnani, Giancarlo and Pellegrini, Marika and Galieni, Angelica and D’Egidio, Sara and Matteucci, Federica and Ricci, Antonella and Stagnari, Fabio and Sergi, Manuel and Lo Sterzo, Claudio and Pisante, Michele and Del Gallo, Maddalena, Plant Growth-Promoting Rhizobacteria (PGPR) in Cannabis Sativa ‘Finola’ Cultivation: An Alternative Fertilization Strategy to Improve Plant Growth and Quality Characteristics, Industrial Crops and Products, vol. 123, pp. 75--83, November 2018. doi: 10.1016/j.indcrop.2018.06.033. Abstract
     The massive employment of chemical fertilizers entails substantial costs for agriculture and leads to significant environmental pollution, soils depletion and crop productivity declines. The aim of this preliminary study was to evaluate the suitability of plant growth-promoting rhizobacteria (PGPR) as an alternative fertilization approach in Cannabis sativa L. ‘Finola’, one of the low-psychoactive substances industrial hemp varieties cultivated in the Abruzzo territory. The PGPR inoculum was first studied in a model system by monitoring the colonization and survival of bacteria in roots of hemp seedling grown in vitro. Following a complete randomized block design with three replicates, female plants were also cultivated in greenhouse and subjected to different cultivation conditions: (i) two different PGPR inoculum concentrations, (ii) nitrogen fertilization, and (iii) unfertilized control. At the flowering stage, plant growth parameters, main cannabinoid content, antioxidant, and total phenolic content, were assessed. In the model system experiment, scanning electron microscope (SEM) imaging revealed an excellent ability of bacteria to adhere to the surface of roots, and to colonize root vascular tissues of hemp seedlings. Under greenhouse conditions PGPR favored plant growth and development as well as plant secondary metabolites accumulation and, consequently, antioxidant capacity. In particular, the lowest PGPR concentration allowed obtaining results comparable with those induced by the recommended nitrogen fertilization. These results underline the potentiality of PGPR application in hemp plants in terms of both higher biomass accumulation and chemical composition, also meeting environmental goals such as an increase in soil biodiversity and a reduction in chemical inputs. This study represents the first step toward the potential application of PGPR in hemp cultivation and could be the base for future extensive evaluations.
147. Scott, Maryanne and Rani, Mamta and Samsatly, Jamil and Charron, Jean-Benoit and Jabaji, Suha, Endophytes of Industrial Hemp (Cannabis Sativa L.) Cultivars: Identification of Culturable Bacteria and Fungi in Leaves, Petioles, and Seeds, Canadian Journal of Microbiology, vol. 64, no. 10, pp. 664--680, October 2018. doi: 10.1139/cjm-2018-0108. Abstract
     Plant endophytes are a group of microorganisms that reside asymptomatically within the healthy living tissue. The diversity and molecular and biochemical characterization of industrial hemp-associated endophytes have not been previously studied. This study explored the abundance and diversity of culturable endophytes residing in petioles, leaves, and seeds of three industrial hemp cultivars, and examined their biochemical attributes and antifungal potential. A total of 134 bacterial and 53 fungal strains were isolated from cultivars Anka, CRS-1, and Yvonne. The number of bacterial isolates was similarly distributed among the cultivars, with the majority recovered from petiole tissue. Most fungal strains originated from leaf tissue of cultivar Anka. Molecular and phylogenetic analyses grouped the endophytes into 18 bacterial and 13 fungal taxa, respectively. The most abundant bacterial genera were Pseudomonas, Pantoea, and Bacillus, and the fungal genera were Aureobasidium, Alternaria, and Cochliobolus. The presence of siderophores, cellulase production, and phosphorus solubilization were the main biochemical traits. In proof-of-concept experiments, re-inoculation of tomato roots with some endophytes confirmed their migration to aerial tissues of the plant. Taken together, this study demonstrates that industrial hemp harbours a diversity of microbial endophytes, some of which could be used in growth promotion and (or) in biological control designed experiments.
148. Finnan, John and Burke, Brendan, Potassium Fertilization of Hemp (Cannabis Sativa), Industrial Crops and Products, vol. 41, pp. 419--422, January 2013. doi: 10.1016/j.indcrop.2012.04.055. Abstract
     Potassium response trials were conducted on hemp on four sites with different levels of soil potassium in 2011 in which the main factor was variety and the second factor was potassium rate. Five rates of applied potassium were used (0, 60, 90, 120, and 150kgK/ha). There was no significant relationship between hemp yield and either potassium rate or the level of soil potassium. Potassium uptake from the site with the lowest level of soil potassium was significantly lower (65kgK/ha) compared to a site with a higher level of soil potassium (83kgK/ha) suggesting that hemp will take up potassium when the element is not needed. Most of the absorbed potassium was concentrated in the stem (70–75\%), harvest off-takes for most applications would consist of only stem and any potassium in root and leaves would remain in the field. The results of this limited investigation suggest that hemp has a lower requirement for potassium than other crops and that the optimal potassium fertilization strategy for soils with moderate to high levels of potassium ({$>$}70mg/L; Morgan's test) is to replace off-takes after the hemp has been harvested. Further research is necessary to develop robust potassium fertilization guidelines for hemp.
149. Chandra, Suman and Lata, Hemant and Khan, Ikhlas A. and ElSohly, Mahmoud A., Photosynthetic Response of Cannabis Sativa L., an Important Medicinal Plant, to Elevated Levels of CO2, Physiology and Molecular Biology of Plants, vol. 17, no. 3, pp. 291--295, July 2011. doi: 10.1007/s12298-011-0066-6. Abstract
     The effect of elevated CO2 concentrations (545 and 700~μmol mol−1) on gas exchange and stomatal response of four high Δ9-THC yielding varieties of Cannabis sativa (HPM, K2, MX and W1) was studied to assess their response to the rising atmospheric CO2 concentration. In general, elevated CO2 concentration (700~μmol mol−1) significantly (p\,{$<$}\,0.05) stimulated net photosynthesis (PN), water use efficiency (WUE) and internal CO2 concentration (Ci), and suppressed transpiration (E) and stomatal conductance (gs) as compared to the ambient CO2 concentration (390~μmol mol−1) in all the varieties whereas, the effect of 545~μmol mol−1 CO2 concentration was found insignificant (p\,{$<$}\,0.05) on these parameters in most of the cases. No significant changes (p\,{$<$}\,0.05) in the ratio of internal to the ambient CO2 concentration (Ci/Ca) was observed in these varieties under both the elevated CO2 concentrations (545 and 700~μmol mol−1). An average increase of about 48~\%, 45~\%, 44~\% and 38~\% in PN and, about 177~\%, 157~\%, 191~\% and 182~\% in WUE was observed due to elevated CO2 (700~μmol mol−1) as compared to ambient CO2 concentration in HPM, K2, MX and W1 varieties, respectively. The higher WUE under elevated CO2 conditions in Cannabis sativa, primarily because of decreased stomatal conductance and subsequently the transpiration rate, may enable this species to survive under expected harsh greenhouse effects including elevated CO2 concentration and drought conditions. The higher PN, WUE and nearly constant Ci/Ca ratio under elevated CO2 concentrations in this species reflect a close coordination between its stomatal and mesophyll functions.
150. Chandra, Suman and Lata, Hemant and Khan, Ikhlas A. and Elsohly, Mahmoud A., Photosynthetic Response of Cannabis Sativa L. to Variations in Photosynthetic Photon Flux Densities, Temperature and CO2 Conditions, Physiology and Molecular Biology of Plants, vol. 14, no. 4, pp. 299--306, October 2008. doi: 10.1007/s12298-008-0027-x. Abstract
     Effect of different photosynthetic photon flux densities (0, 500, 1000, 1500 and 2000 μmol m−2s−1), temperatures (20, 25, 30, 35 and 40 °C) and CO2 concentrations (250, 350, 450, 550, 650 and 750 μmol mol−1) on gas and water vapour exchange characteristics of Cannabis sativa L. were studied to determine the suitable and efficient environmental conditions for its indoor mass cultivation for pharmaceutical uses. The rate of photosynthesis (PN) and water use efficiency (WUE) of Cannabis sativa increased with photosynthetic photon flux densities (PPFD) at the lower temperatures (20–25 °C). At 30 °C, PN and WUE increased only up to 1500 μmol m−2s−1 PPFD and decreased at higher light levels. The maximum rate of photosynthesis (PN max) was observed at 30 °C and under 1500 μmol m−2s−1 PPFD. The rate of transpiration (E) responded positively to increased PPFD and temperature up to the highest levels tested (2000 μmol m−2s−1 and 40 °C). Similar to E, leaf stomatal conductance (gs) also increased with PPFD irrespective of temperature. However, gs increased with temperature up to 30 °C only. Temperature above 30 °C had an adverse effect on gs in this species. Overall, high temperature and high PPFD showed an adverse effect on PN and WUE. A continuous decrease in intercellular CO2 concentration (Ci) and therefore, in the ratio of intercellular CO2 to ambient CO2 concentration (Ci/Ca) was observed with the increase in temperature and PPFD. However, the decrease was less pronounced at light intensities above 1500 μmol m−2s−1. In view of these results, temperature and light optima for photosynthesis was concluded to be at 25–30 °C and ∼1500 μmol m−2s−1 respectively. Furthermore, plants were also exposed to different concentrations of CO2 (250, 350, 450, 550, 650 and 750 μmol mol−1) under optimum PPFD and temperature conditions to assess their photosynthetic response. Rate of photosynthesis, WUE and Ci decreased by 50 \%, 53 \% and 10 \% respectively, and Ci/Ca, E and gs increased by 25 \%, 7 \% and 3 \% respectively when measurements were made at 250 μmol mol-1 as compared to ambient CO2 (350 μmol mol−1) level. Elevated CO2 concentration (750 μmol mol−1) suppressed E and gs ∼ 29\% and 42\% respectively, and stimulated PN, WUE and Ci by 50 \%, 111 \% and 115 \% respectively as compared to ambient CO2 concentration. The study reveals that this species can be efficiently cultivated in the range of 25 to 30 °C and ∼1500 μmol m−2s−1 PPFD. Furthermore, higher PN, WUE and nearly constant Ci/Ca ratio under elevated CO2 concentrations in C. sativa, reflects its potential for better survival, growth and productivity in drier and CO2 rich environment.
151. Landi, S., Mineral Nutrition of Cannabis Sativa L., Journal of Plant Nutrition, vol. 20, no. 2-3, pp. 311--326, February 1997. doi: 10.1080/01904169709365252. Abstract
     Forensic laboratories can be called to examine illicit Cannabis samples (marijuana) to identify their geographical origin. They can also be required to compare different seizures to establish whether they were drawn from the same original lot. The quantitative determination of selected organic components is one of the criteria currently used in such investigations. This study aimed at evaluating the inorganic element pattern of marijuana as a possible additional diagnostic tool. Four commercial cultivars of Cannabis sativa L. were grown in field experiments planned so that edaphic and climatic growth conditions varied slightly among the fields. The experimental design produced six populations. Population variability for the elements sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), manganese (Mn), iron (Fe), zinc (Zn), and molybdenum (Mo) accumulation in leaves (L) and inflorescences (FL) of female plants was determined. For each element, the analytical data for the L and FL belonging to the same sample were pooled to simulate the chemical profile of marijuana preparations (L+FL). Within every population frequent important differences in elemental concentrations between corresponding L and FL fractions were detected, chiefly for the elements Ca, Mg, Cu, Mn, Fe, Zn, and Mo. This suggests that: i) the mean composition of marijuana produced in a single field depends on the relative amounts of plant parts harvesters pick, and ii) whenever a small drug sample is examined, the analytical outcome will be influenced by the weight ratio of the different plant parts which happen to make up the sample itself. Whatever the fraction considered (L, FL, and L+FL), a narrow scattering of data for all elements except Na was observed within each population, with RSD values generally well below 10\%. When populations were compared for their elemental composition, many significant differences were found; for the mock drugs (L+FL fractions) they were most frequently determined by Ca followed by Mo, K=Fe, Zn, Mn, Cu=Na, and Mg in this order of decreasing frequency. Multi variate (discriminant) statistical analysis for product description was effective in separating the L, FL, and L+FL fractions of the six populations.
152. Werf, H M G Van Der and Brouwer, K. and Wijlhuizen, M. and Withagen, J C M., The Effect of Temperature on Leaf Appearance and Canopy Establishment in Fibre Hemp (Cannabis Sativa L.), Annals of Applied Biology, vol. 126, no. 3, pp. 551--561, 1995. doi: 10.1111/j.1744-7348.1995.tb05389.x. Abstract
     The effects of temperature on the development and growth of hemp (Cannabis sativa L.) have never been quantified. Therefore, to establish the effect of temperature on leaf appearance and canopy establishment of fibre hemp under controlled and field conditions, plants were grown in growth chambers at 11 regimes with average temperatures between 10°C and 28°C, and three cultivars were sown in the field in March, April and May in 1990, 1991 and 1992. In the field, thermal time (base 0°C) between sowing and emergence ranged from 68°Cd to 109.5°Cd (average 88.3°Cd). Rates of leaf appearance and stem elongation increased linearly with temperature between 10°C and 28°C. The base temperature for leaf appearance was 5.7°C from the growth chamber experiments and 1°C from the field experiments. In the field, the base temperature for the relationship between light interception by the canopy and thermal time was 2.5°C, and thermal time, calculated at the appropriate base temperature, accounted for about 98\% of the variance in the number of leaves and for 98.6\% of the variance in the proportion of light intercepted by the canopy. Days from emergence accounted for less of the variance in both parameters than thermal time. Interception of 90\% of light was attained on average at 465°Cd (base 0°C) after emergence. It is concluded that thermal time is a simple and accurate tool to describe leaf appearance and light interception in fibre hemp.
153. Wei, Xiuye and Zhao, Xinlin and Long, Songhua and Xiao, Qingmei and Guo, Yuan and Qiu, Caisheng and Qiu, Huajiao and Wang, Yufu, Wavelengths of LED Light Affect the Growth and Cannabidiol Content in Cannabis Sativa L, Industrial Crops and Products, vol. 165, pp. 113433, July 2021. doi: 10.1016/j.indcrop.2021.113433. Abstract
     Given the special pharmacological value of cannabidiol (CBD), industrial hemp (hemp) has become a real “hot” crop. Though indoor farming of hemp has been widely used, the effects of red and blue light-emitting diode (LED) acting as supplementary light on the CBD synthesis in hemp remains unclear. Herein, a pot experiment with six treatments [CK, high-pressure sodium as light source, (R:B (ratio of red light to blue light) 9.30:1, PPFD (photosynthetic photon flux density) 191); LED1 (R:B 9.20:1; PPFD 129); LED2 (R:B 1.61:1; PPFD 540); LED3 (R:B 6.47:1; PPFD 28.2); LED4 (R:B 7.15:1; PPFD 41.7); LED5 (R:B 16.8:1; PPFD 252)] was carried out to study the effects of LED light quality on the growth and cannabinoid synthesis of hemp. Results showed that LED2 and LED5 could well maintain hemp growth compared with CK in terms of plant height, stem diameter, and leaf numbers. Compared with CK, the treatment of LED1, LED4, and LED3 significantly decreased the aboveground biomass, while LED2 and LED5 notably improved the aboveground biomass with an increment of 15.2 \% and 55.1 \%; the biomass of hemp flowers for LED2 and LED5 was markedly increased compared with that for CK with increments of 238 \% and 61.5 \%, respectively, but the other LED light treatments significantly reduced the flowers yield; LED2 could significantly increase the CBD content of both leaves (1.81 \%) and flowers (5.83 \%) compared to CK (leaves 1.38 \%, flowers 4.27 \%) while LED5 only notably increased the CBD content of leaves; among the LED treatments, the theoretic CBD yield for a per plant of LED2 and LED5 was 26.8 \% and 9.0 \% higher than that of CK. In conclusion, different red and blue LED light intensity ratios would dramatically influence the growth and cannabinoid synthesis of hemp, LED2 and LED5 can be good choices for indoor farming of hemp targeted for higher CBD yield.
154. Danziger, Nadav and Bernstein, Nirit, Light Matters: Effect of Light Spectra on Cannabinoid Profile and Plant Development of Medical Cannabis (Cannabis Sativa L.), Industrial Crops and Products, vol. 164, pp. 113351, June 2021. doi: 10.1016/j.indcrop.2021.113351. Abstract
     Light is a key factor affecting plant growth, metabolism and function. Metabolic processes in plants are sensitive to the ratio of Blue:Red light, and there is an increasing awareness that the response to the ratio of these monochromatic lights may vary under exposure to a wider range of the spectrum, such as white light. Due to the potential for regulation of the therapeutic chemical profile and plant development, this issue is of growing interest for the cannabis (Cannabis sativa L.) industry that uses photosynthetic light extensively. Cannabis is a medicinal plant treasured for its secondary metabolites, especially cannabinoids, the unique biologically active compounds in the plant that are considered to be affected by light spectra. In this study we evaluated the hypothesis that the ratio of Blue:Red light affects cannabinoid metabolism, and that plant growth and secondary metabolism is intensified under a full spectrum with similar Blue:Red ratio. Our results point to several spectra specific reactions and some cultivar dependent responses to light spectrum. i. Yield quantity: The highest inflorescence yields were obtained when the spectrum was restricted to the red and blue range at the ratio of 1:1, and in two of the three varieties tested a ratio of 1:4 Blue:Red light had similar results. White light with Blue:Red ratio of 1:1 had the lowest yield. ii. The chemical profile was also affected by the light spectrum, and CBGA, the primary cannabinoid and a precursor for most other cannabinoids, demonstrated the highest response. CBGA accumulation was stimulated by blue-rich light as compared with far-red rich HPS light. The major cannabinoids CBDA, THCA and CBCA were also affected by light quality, and the response was cultivar specific and less pronounced than for CBGA. iii. Plant morphology: Blue light was most inductive for maintaining compact plants, more so than Red:Far-Red ratio. Our results repute the hypothesis that full spectrum improves inflorescence yield compared with Blue:Red light, but support the hypothesis that light spectrum influences plant development and the cannabinoid profile, which could be used to fine-tune cannabis and cannabinoid production.
155. Reichel, Philipp and Munz, Sebastian and Hartung, Jens and Präger, Achim and Kotiranta, Stiina and Burgel, Lisa and Schober, Torsten and {Graeff-Hönninger}, Simone, Impact of Three Different Light Spectra on the Yield, Morphology and Growth Trajectory of Three Different Cannabis Sativa L. Strains, Plants, vol. 10, no. 9, pp. 1866, September 2021. doi: 10.3390/plants10091866. Abstract
     Cannabis is one of the oldest cultivated plants, but plant breeding and cultivation are restricted by country specific regulations. Plant growth, morphology and metabolism can be manipulated by changing light quality and intensity. Three morphologically different strains were grown under three different light spectra with three real light repetitions. Light dispersion was included into the statistical evaluation. The light spectra considered had an influence on the morphology of the plant, especially the height. Here, the shade avoidance induced by the lower R:FR ratio under the ceramic metal halide lamp (CHD) was of particular interest. The sugar leaves seemed to be of elementary importance in the last growth phase for yield composition. Furthermore, the last four weeks of flowering were crucial to influence the yield composition of Cannabis sativa L. through light spectra. The dry flower yield was significantly higher under both LED treatments compared to the conventional CHD light source. Our results indicate that the plant morphology can be artificially manipulated by the choice of light treatment to create shorter plants with more lateral branches which seem to be beneficial for yield development. Furthermore, the choice of cultivar has to be taken into account when interpreting results of light studies, as Cannabis sativa L. subspecies and thus bred strains highly differ in their phenotypic characteristics.
156. Westmoreland, F. Mitchell and Kusuma, Paul and Bugbee, Bruce, Cannabis Lighting: Decreasing Blue Photon Fraction Increases Yield but Efficacy Is More Important for Cost Effective Production of Cannabinoids, PLOS ONE, vol. 16, no. 3, pp. e0248988, March 2021. doi: 10.1371/journal.pone.0248988. Abstract
     LED technology facilitates a range of spectral quality, which can be used to optimize photosynthesis, plant shape and secondary metabolism. We conducted three studies to investigate the effect of blue photon fraction on yield and quality of medical hemp. Conditions were varied among studies to evaluate potential interactions with environment, but all environmental conditions other than the blue photon fraction were maintained constant among the five-chambers in each study. The photosynthetic photon flux density (PPFD, 400 to 700 nm) was rigorously maintained at the set point among treatments in each study by raising the fixtures. The lowest fraction of blue photons was 4\% from HPS, and increased to 9.8, 10.4, 16, and 20\% from LEDs. There was a consistent, linear, 12\% decrease in yield in each study as the fraction of blue photons increased from 4 to 20\%. Dry flower yield ranged from 500 to 750 g m-2. This resulted in a photon conversion efficacy of 0.22 to 0.36 grams dry flower mass yield per mole of photons. Yield was higher at a PPFD of 900 than at 750 μmol m-2 s-1. There was no effect of spectral quality on CBD or THC concentration. CBD and THC were 8\% and 0.3\% at harvest in trials one and two, and 12\% and 0.5\% in trial three. The CBD/THC ratio was about 25 to 1 in all treatments and studies. The efficacy of the fixtures ranged from 1.7 (HPS) to 2.5 μmol per joule (white+red LED). Yield under the white+red LED fixture (10.4\% blue) was 4.6\% lower than the HPS on a per unit area basis, but was 27\% higher on a per dollar of electricity basis. These findings suggest that fixture efficacy and initial cost of the fixture are more important for return on investment than spectral distribution at high photon flux.
157. Desaulniers Brousseau, Vincent and Wu, Bo-Sen and MacPherson, Sarah and Morello, Victorio and Lefsrud, Mark, Cannabinoids and Terpenes: How Production of Photo-Protectants Can Be Manipulated to Enhance Cannabis Sativa L. Phytochemistry, Frontiers in Plant Science, vol. 12, 2021. doi: 10.3389/fpls.2021.620021. Abstract
     Cannabis sativa L. is cultivated for its secondary metabolites, of which the cannabinoids have documented health benefits and growing pharmaceutical potential. Recent legal cannabis production in North America and Europe has been accompanied by an increase in reported findings for optimization of naturally occurring and synthetic cannabinoid production. Of the many environmental cues that can be manipulated during plant growth in controlled environments, cannabis cultivation with different lighting spectra indicates differential production and accumulation of medically important cannabinoids, including Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), and cannabigerol (CBG), as well as terpenes and flavonoids. Ultraviolet (UV) radiation shows potential in stimulating cannabinoid biosynthesis in cannabis trichomes and pre-harvest or post-harvest UV treatment merits further exploration to determine if plant secondary metabolite accumulation could be enhanced in this manner. Visible LED light can augment THC and terpene accumulation, but not CBD. Well-designed experiments with light wavelengths other than blue and red light will provide more insight into light-dependent regulatory and molecular pathways in cannabis. Lighting strategies such as subcanopy lighting and varied light spectra at different developmental stages can lower energy consumption and optimize cannabis PSM production. Although evidence demonstrates that secondary metabolites in cannabis may be modulated by the light spectrum like other plant species, several questions remain for cannabinoid production pathways in this fast-paced and growing industry. In summarizing recent research progress on light spectra and secondary metabolites in cannabis, along with pertinent light responses in model plant species, future research directions are presented.
158. Namdar, Dvory and Charuvi, Dana and Ajjampura, Vinayka and Mazuz, Moran and Ion, Aurel and Kamara, Itzhak and Koltai, Hinanit, LED Lighting Affects the Composition and Biological Activity of Cannabis Sativa Secondary Metabolites, Industrial Crops and Products, vol. 132, pp. 177--185, June 2019. doi: 10.1016/j.indcrop.2019.02.016. Abstract
     Growth conditions are expected to alter the relative and absolute content of the hundreds of phytochemicals produced by Cannabis sativa L.; some of these possess biological activity on the human body. However, relatively little information exists regarding the effects of different light regimes on the composition of C. sativa secondary metabolites and thus on their biological activity. In this study, we investigated how light quality influences the production and final content of secondary metabolites, as well as their bioactive properties. Toward these, plant growth and blooming were carried out at different illumination conditions, utilizing light-emitting diode (LED) fixtures vs. conventional fluorescent and high-pressure sodium (HPS) lamps as controls. Inflorescences were sampled at different time points along the blooming; extract compositions were analyzed by HPLC and GC/MS, and the biological activity of the extracted material was assessed using cell viability assays. We found that growth and blooming under LED illumination considerably changed shoot architecture and inflorescence mass. Moreover, the content of cannabinoids, terpenes, and alkanes were altered in the inflorescences of LED-grown plants during the flowering period as well as in the harvested flowers. In particular, significantly higher quantities of cannabigerolic acid accumulated in the inflorescences that flowered under LED fixtures, with a cannabigerolic acid to Δ9-tetrahydrocannabinolic acid (CBGA:THCA) ratio of 1:2 as opposed to 1:16 when grown under HPS. Notably, the cytotoxic activities of extracts derived from plants grown under the different illumination regimes were different, with extracts from LED-grown plants possessing higher cytotoxicity along the flowering stage. Our results thus indicate that the transition to indoor growth of C. sativa under LED lighting, which can have significant impacts on cannabinoid and terpene content, and also on the bioactive properties of the plant extracts, should proceed with thorough consideration.
159. Eichhorn Bilodeau, Samuel and Wu, Bo-Sen and Rufyikiri, Anne-Sophie and MacPherson, Sarah and Lefsrud, Mark, An Update on Plant Photobiology and Implications for Cannabis Production, Frontiers in Plant Science, vol. 10, 2019. doi: 10.3389/fpls.2019.00296. Abstract
     This review presents recent developments in plant photobiology and lighting systems for horticultural crops, as well as potential applications for cannabis (Cannabis sativa and C. indica) plant production. The legal and commercial production of the cannabis plant is a relatively new, rapidly growing, and highly profitable industry in Europe and North America. However, more knowledge transfer from plant studies and horticultural communities to commercial cannabis plant growers is needed. Plant photosynthesis and photomorphogenesis are influenced by light wavelength, intensity, and photoperiod via plant photoreceptors that sense light and control plant growth. Further, light properties play a critical role in plant vegetative growth and reproductive (flowering) developmental stages, as well as in biomass, secondary metabolite synthesis, and accumulation. Advantages and disadvantages of widespread greenhouse lighting systems that use high pressure sodium lamps or light emitting diode (LED) lighting are known. Some artificial plant lighting practices will require improvements for cannabis production. By manipulating LED light spectra and stimulating specific plant photoreceptors, it may be possible to minimize operation costs while maximizing cannabis biomass and cannabinoid yield, including tetrahydrocannabinol (or Δ9-tetrahydrocannabinol) and cannabidiol for medicinal and recreational purposes. The basics of plant photobiology (photosynthesis and photomorphogenesis) and electrical lighting systems are discussed, with an emphasis on how the light spectrum and lighting strategies could influence cannabis production and secondary compound accumulation.
160. Magagnini, Gianmaria and Grassi, Gianpaolo and Kotiranta, Stiina, The Effect of Light Spectrum on the Morphology and Cannabinoid Content of Cannabis Sativa L., Medical Cannabis and Cannabinoids, vol. 1, no. 1, pp. 19--27, 2018. doi: 10.1159/000489030. Abstract
     \emph{Cannabis sativa} L. flowers are the main source of Δ-9-tetrahydrocannabinol (THC) used in medicine. One of the most important growth factors in cannabis cultivation is light; light quality, light intensity, and photoperiod play a big role in a successful growth protocol. The aim of the present study was to examine the effect of 3 different light sources on morphology and cannabinoid production. Cannabis clones were grown under 3 different light spectra, namely high-pressure sodium (HPS), AP673L (LED), and NS1 (LED). Light intensity was set to ∼450 µmol/m\textsuperscript{2}/s measured from the canopy top. The photoperiod was 18L: 6D/21 days during the vegetative phase and 12L: 12D/46 days during the generative phase, respectively. At the end of the experiment, plant dry weight partition, plant height, and cannabinoid content (THC, cannabidiol [CBD], tetrahydrocannabivarin [THCV], cannabigerol [CBG]) were measured under different light treatments. The experiment was repeated twice. The 3 light treatments (HPS, NS1, AP673L) resulted in differences in cannabis plant morphology and in cannabinoid content, but not in total yield of cannabinoids. Plants under HPS treatment were taller and had more flower dry weight than those under treatments AP673L and NS1. Treatment NS1 had the highest CBG content. Treatments NS1 and AP673L had higher CBD and THC concentrations than the HPS treatment. Results were similar between experiments 1 and 2. Our results show that the plant morphology can be manipulated with the light spectrum. Furthermore, it is possible to affect the accumulation of different cannabinoids to increase the potential of medicinal grade cannabis. In conclusion, an optimized light spectrum improves the value and quality of cannabis. Current LED technology showed significant differences in growth habit and cannabinoid profile compared to the traditional HPS light source. Finally, no difference of flowering time was observed under different R:FR (i.e., the ratio between red and far-red light).
161. Lalge, Ajinkya and Cerny, Petr and Trojan, Václav and Vyhnánek, Tomáš, The Effects of Red, Blue and White Light on the Growth and Development of Cannabis Sativa L., November 2017. Abstract
     The aim of this study was to investigate the effect of red (600-700 nm, peak 660), blue (400-500 nm, peak 450) and white light on the morphological and photosynthetic qualities of Cannabis sativa L. The two treatments were the white light (WL), and a combination of blue red lights (BR). Plants grown under WL were 23\% taller than those grown under the BR light emitting diodes. The leaf area was also greater under WL than BR by 20\%. The number of lateral branches and length of dominant lateral branch weren´t significantly different. It was concluded WL that emit a full spectrum of light affects plant growth and development better than BR light. The quantum efficiency ranged from 0.81 to 0.845 indicating the plants were not in stress.
162. Lisson, S. N. and Mendham, N. J. and Carberry, P. S., Development of a Hemp (Cannabis Sativa L.) Simulation Model 2.The Flowering Response of Two Hemp Cultivars to Photoperiod, Australian Journal of Experimental Agriculture, vol. 40, no. 3, pp. 413--417, 2000. doi: 10.1071/ea99059. Abstract
     The duration from sowing to flowering is an important determinant of fibre yield potential in hemp, since maximum stem yield occurs shortly after flowering. As a short-day plant, daylength has a key influence on the timing of flowering in hemp. This paper reports on studies into the effect of photoperiod on the thermal time duration from sowing to flowering for 2 hemp cultivars, and develops parameters to enable simulation of post-emergent phenology in the hemp model described in the final paper of this series. The hemp model divides the post-emergent period into a vegetative phase that ends at floral initiation, aflower development phase (FDP) between flower initiation and appearance, and a short phase between first flower appearance and harvest maturity (male anthesis). The vegetative phase is further divided into a temperature-dependent basic vegetative phase (BVP) and a daylength-dependent photoperiod induced phase (PIP). For a short-day plant, the duration of PIP is assumed to be zero degree days at daylengths below a base or maximum optimum photoperiod (MOP). Daylengths in excess of the MOP lead to an increase in thermal time within PIP, the duration of which is determined by a genotype’s photoperiod sensitivity (PS). Two hemp genotypes, Kompolti and Futura 77, were exposed to 6 different photoperiod regimes ranging from 8 to 16 h in a growth chamber. Thermal time durations from emergence to flower initiation and first flower formation (harvest) were calculated from thermograph plots. The flowering responses for the 2 cultivars were typical for a short-day plant, with flowering occurring rapidly in daylengths less than about 14 h and with increasing delay at longer photoperiods. With the exception of a longer thermal time duration from flower formation to harvest maturity in the case of Kompolti, the 2 cultivars had similar values for the key phenology parameters. Respectively, for Futura and Kompolti: BVP was 383˚Cd and 390˚Cd, MOP was 14 h and 13.8 h, PS was 266˚Cd/h and 252˚Cd/h, and FDP was 76.8˚Cd and 80.2˚Cd.
163. Lydon, John and Teramura, Alan H. and Coffman, C. Benjamin, Uv-B Radiation Effects on Photosynthesis, Growth and Cannabinoid Production of Two Cannabis Sativa Chemotypes, Photochemistry and Photobiology, vol. 46, no. 2, pp. 201--206, 1987. doi: 10.1111/j.1751-1097.1987.tb04757.x. Abstract
     The effects of UV-B radiation on photosynthesis, growth and cannabinoid production of two greenhouse-grown C. sativa chemotypes (drug and fiber) were assessed. Terminal meristems of vegetative and reproductive tissues were irradiated for 40 days at a daily dose of 0, 6.7 or 13.4 kJ m-2 biologically effective UV-B radiation. Infrared gas analysis was used to measure the physiological response of mature leaves, whereas gas-liquid chromatography was used to determine the concentration of cannabinoids in leaf and floral tissue. There were no significant physiological or morphological differences among UV-B treatments in either drug- or fiber-type plants. The concentration of Δ9-tetrahydrocannabinol (Δ9-THC), but not of other cannabinoids, in both leaf and floral tissues increased with UV-B dose in drug-type plants. None of the cannabinoids in fiber-type plants were affected by UV-B radiation. The increased levels of Δ9-THC in leaves after irradiation may account for the physiological and morphological tolerance to UV-B radiation in the drug-type plants. However, fiber plants showed no comparable change in the level of cannabidiol (a cannabinoid with UV-B absorptive characteristics similar to Δ9 THC). Thus the contribution of cannabinoids as selective UV-B filters in C. sativa is equivocal.
164. Pate, David W., Possible Role of Ultraviolet Radiation in Evolution ofCannabis Chemotypes, Economic Botany, vol. 37, no. 4, pp. 396--405, October 1983. doi: 10.1007/BF02904200. Abstract
     The damaging effects of UV-B radiation have apparently affected the amounts of ultraviolet-absorbing secondary compounds in some plants. A similar role for Δ9 tetrahydrocannabinol may explain the high levels of this compound inCannabis from areas of intense ambient UV-B. Further research is needed to determine whether UV-B radiation serves only as a selection pressure or if UV-B-induced stress may also directly stimulate production.
165. Mahlberg, Paul G. and Hemphill, John K., Effect of Light Quality on Cannabinoid Content of Cannabis Sativa L. (Cannabaceae), Botanical Gazette, vol. 144, no. 1, pp. 43--48, March 1983. doi: 10.1086/337342. Abstract
     Plants of a drug strain of Cannabis sativa L -grown 33 days under daylight, shaded daylight conditions, filtered green, blue, and red light, and darkness-were analyzed by gas-liquid chromatography for their cannabinoid content. The highest content of cannabinoids, predominantly Δ9-tetrahydrocannabinol (Δ9-THC) in this strain, occurred in the youngest leaves of daylight-grown plants Leaves at successively lower nodes of this control condition and all treated plants subsequently grown in daylight contained progressively lower levels of cannabinoids Leaves from plants grown under filtered green light and darkness contained significantly lower levels of Δ9-THC than those from plants grown in daylight However, the Δ9-THC content of leaves from plants grown under shaded daylight and filtered red and blue light did not differ significantly from the Δ9-THC content in daylight controls, indicating that these conditions did not alter the synthetic rate of this cannabinoid The cannabichromene (CBC) content of plants grown under filtered red and green light and darkness differed from the CBC content in plants grown in daylight, indicating that the formation of this cannabinoid was independent of Δ9-THC Leaves from plants grown under filtered red and green light and darkness recovered the capacity to synthesize typical levels of Δ9-THC and CBC when placed under daylight conditions Plants from all light and dark treatments, when subsequently placed under daylight conditions for 66 days, attained levels of cannabinoid synthesis comparable to the daylight controls
166. Valle, J. R. and Vieira, J. E. and Aucélio, J. G. and Valio, I. F., Influence of Photoperiodism on Cannabinoid Content of Cannabis Sativa L, Bulletin on Narcotics, vol. 30, no. 1, pp. 67--68, 1978. Abstract
     Cannabis sativa plants submitted to 10 and 12 hours of natural light showed different content in cannabinoids. An increase of exposure to natural light of only 2 hours a day, at least, doubled the average amoung of THC, but decreased that of cannabichromene.
167. Moher, Melissa and Llewellyn, David and Jones, Max and Zheng, Youbin, Light Intensity Can Be Used to Modify the Growth and Morphological Characteristics of Cannabis during the Vegetative Stage of Indoor Production, Industrial Crops and Products, vol. 183, pp. 114909, September 2022. doi: 10.1016/j.indcrop.2022.114909. Abstract
     Although the vegetative stage of indoor cannabis (Cannabis sativa) production can be relatively short in duration, there is a high energy demand due to higher light intensities (LI) than the clonal propagation stage and longer photoperiods than the flowering stage (i.e., ≥ 16 vs. 12~h). While electric lighting is a major component of both energy consumption and overall production costs, there is a lack of scientific information to guide cultivators in selecting a LI that corresponds to their vegetative stage production strategies. To determine the vegetative plant responses to LI, clonal plants of ‘Gelato’ (indica-dominant hybrid genotype) were grown for 21 days with canopy-level photosynthetic photon flux densities (PPFD) ranging between 135 and 1430~µmol·m−2·s−1 with a 16-h photoperiod (i.e., daily light integrals of 7.8–82.4~mol·m−2·d−1). Plant height and growth index (i.e., a canopy volume metric) responded quadratically; the number of nodes, stem thickness, and aboveground dry weight increased asymptotically; and internode length and water content of aboveground tissues decreased linearly with increasing LI. Foliar attributes had varying responses to LI. Chlorophyll content index (i.e., SPAD value) increased asymptotically, leaf size decreased linearly and specific leaf weight increased linearly with increasing LI. Generally, PPFD levels of ≈~900~µmol·m−2·s−1 produced compact, robust plants while PPFD levels of ≈~600~µmol·m−2·s−1 promoted more open plant architecture (i.e., taller plants with longer internodes), which can increase intra-canopy airflow and may reduce development of potential foliar pests in compact (e.g., indica-dominant) genotypes.
168. Scott, Cameron and Punja, Zamir K., Evaluation of Disease Management Approaches for Powdery Mildew on Cannabis Sativa L. (Marijuana) Plants, Canadian Journal of Plant Pathology, vol. 43, no. 3, pp. 394--412, May 2021. doi: 10.1080/07060661.2020.1836026. Abstract
     Powdery mildew on cannabis (Cannabis sativa L., marijuana), caused by Golovinomyces cichoracearum, reduces plant growth and overall quality. To investigate disease management options, biological, chemical and physical approaches were assessed. A mildew-susceptible strain, ‘Copenhagen Kush’, was grown indoors with continual exposure to mildew inoculum. Treatments were applied weekly over a four-week period to groups of four plants once mildew infection had established itself. Trials were repeated thrice under varying initial disease pressures. Disease assessments were made weekly and the percentage of area infected on 30 leaflets per plant was used to calculate a disease rating score for treated and control plants. Disease progress curves were plotted and AUDPC values were determined for each treatment. To test the effect of UV-C light on mildew development, plants were exposed daily for 3–5 s over 28 days to UV-C light. The response of 12 cannabis strains to powdery mildew infection was assessed after exposing them to inoculum over a period of two weeks. The most effective treatments that significantly (P {$<$} 0.05) reduced disease in three trials were Luna Privilege SC (fluopyram), Regalia® Maxx, MilStop®, Rhapsody ASOTM, neem oil, and Stargus®. Treatments that were less effective included ZeroTol®, boric acid, and Actinovate® SP. Daily exposure of plants to UV-C light significantly reduced disease (by 45.2\%, P {$<$} 0.05). Seven of 12 cannabis strains had significantly lower disease severity compared with the other five strains. The disease management strategies evaluated in this study have potential for reducing powdery mildew development on cannabis.
169. Punja, Zamir K, Emerging Diseases of Cannabis Sativa and Sustainable Management, Pest Management Science, vol. 77, no. 9, pp. 3857--3870, 2021. doi: 10.1002/ps.6307. Abstract
     Cultivation of cannabis plants (Cannabis sativa L., marijuana) has taken place worldwide for centuries. In Canada, legalization of cannabis in October 2018 for the medicinal and recreational markets has spurned interest in large-scale growing. This increased production has seen a rise in the incidence and severity of plant pathogens, causing a range of previously unreported diseases. The objective of this review is to highlight the important diseases currently affecting the cannabis and hemp industries in North America and to discuss various mitigation strategies. Progress in molecular diagnostics for pathogen identification and determining inoculum sources and methods of pathogen spread have provided useful insights. Sustainable disease management approaches include establishing clean planting stock, modifying environmental conditions to reduce pathogen development, implementing sanitation measures, and applying fungal and bacterial biological control agents. Fungicides are not currently registered for use and hence there are no published data on their efficacy. The greatest challenge remains in reducing microbial loads (colony-forming units) on harvested inflorescences (buds). Contaminating microbes may be introduced during the cultivation and postharvest phases, or constitute resident endophytes. Failure to achieve a minimum threshold of microbes deemed to be safe for utilization of cannabis products can arise from conventional and organic cultivation methods, or following applications of beneficial biocontrol agents. The current regulatory process for approval of cannabis products presents a challenge to producers utilizing biological control agents for disease management. © 2021 The Author. Pest Management Science published by John Wiley \& Sons Ltd on behalf of Society of Chemical Industry.
170. Thiessen, Lindsey D. and Schappe, Tyler and Cochran, Sarah and Hicks, Kristin and Post, Angela R., Surveying for Potential Diseases and Abiotic Disorders of Industrial Hemp (Cannabis Sativa) Production, Plant Health Progress, vol. 21, no. 4, pp. 321--332, January 2020. doi: 10.1094/PHP-03-20-0017-RS. Abstract
     Industrial hemp (Cannabis sativa L.) has recently been reintroduced as an agricultural commodity in the United States, and, through state-led pilot programs, growers and researchers have been investigating production strategies. Diseases and disorders of industrial hemp in the United States are largely unknowns because record-keeping and taxonomy have improved dramatically in the last several decades. In 2016, North Carolina launched a pilot program to investigate industrial hemp, and diseases and abiotic disorders were surveyed in 2017 and 2018. Producers, consultants, and agricultural extension agents submitted samples to the North Carolina Department of Agriculture and Consumer Services Agronomic Services Division (n = 572) and the North Carolina Plant Disease and Insect Clinic (n = 117). Common field diseases found included Fusarium foliar and flower blights (Fusarium graminearum), Fusarium wilt (Fusarium oxysporum), and Helminthosporium leaf spot (Exserohilum rostratum). Greenhouse diseases were primarily caused by Pythium spp. and Botrytis cinerea. Common environmental disorders were attributed to excessive rainfall flooding roots and poor root development of transplanted clones.
171. Punja, Zamir K. and Collyer, Danielle and Scott, Cameron and Lung, Samantha and Holmes, Janesse and Sutton, Darren, Pathogens and Molds Affecting Production and Quality of Cannabis Sativa L., Frontiers in Plant Science, vol. 10, 2019. doi: 10.3389/fpls.2019.01120. Abstract
     Plant pathogens infecting marijuana (Cannabis sativa L.) plants reduce growth of the crop by affecting the roots, crown, and foliage. In addition, fungi (molds) that colonize the inflorescences (buds) during development or after harvest, and which colonize internal tissues as endophytes, can reduce product quality. The pathogens and molds that affect C. sativa grown hydroponically indoors (in environmentally controlled growth rooms and greenhouses) and field-grown plants were studied over multiple years of sampling. A PCR-based assay using primers for the internal transcribed spacer region (ITS) of ribosomal DNA confirmed identity of the cultures. Root-infecting pathogens included Fusarium oxysporum, Fusarium solani, Fusarium brachygibbosum, Pythium dissotocum, Pythium myriotylum, and Pythium aphanidermatum, which caused root browning, discoloration of the crown and pith tissues, stunting and yellowing of plants, and in some instances, plant death. On the foliage, powdery mildew, caused by Golovinomyces cichoracearum, was the major pathogen observed. On inflorescences, Penicillium bud rot (caused by Penicillium olsonii and Penicillium copticola), Botrytis bud rot (Botrytis cinerea), and Fusarium bud rot (F. solani, F. oxysporum) were present to varying extents. Endophytic fungi present in crown, stem, and petiole tissues included soil-colonizing and cellulolytic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium, and Fusarium. Analysis of air samples in indoor growing environments revealed that species of Penicillium, Cladosporium, Aspergillus, Fusarium, Beauveria, and Trichoderma were present. The latter two species were the result of the application of biocontrol products for control of insects and diseases, respectively. Fungal communities present in unpasteurized coconut (coco) fiber growing medium are potential sources of mold contamination on cannabis plants. Swabs taken from greenhouse-grown and indoor buds pre- and post-harvest revealed the presence of Cladosporium and up to five species of Penicillium, as well as low levels of Alternaria species. Mechanical trimming of buds caused an increase in the frequency of Penicillium species, presumably by providing entry points through wounds or spreading endophytes from pith tissues. Aerial distribution of pathogen inoculum and mold spores and dissemination through vegetative propagation are important methods of spread, and entry through wound sites on roots, stems, and bud tissues facilitates pathogen establishment on cannabis plants.
172. Benelli, Giovanni and Pavela, Roman and Petrelli, Riccardo and Cappellacci, Loredana and Santini, Giuseppe and Fiorini, Dennis and Sut, Stefania and Dall’Acqua, Stefano and Canale, Angelo and Maggi, Filippo, The Essential Oil from Industrial Hemp (Cannabis Sativa L.) by-Products as an Effective Tool for Insect Pest Management in Organic Crops, Industrial Crops and Products, vol. 122, pp. 308--315, October 2018. doi: 10.1016/j.indcrop.2018.05.032. Abstract
     The inflorescences of industrial hemp (Cannabis sativa L.) represent a consistent by-product that is underutilized. Moving from the concept that this plant part has evolved as a natural weapon against phytophagous insects, secreting important secondary metabolites such as cannabinoids and volatile terpenes, herein we assayed the potential of its essential oil as a botanical insecticide. For the purpose, the essential oil was obtained by fresh inflorescences of hemp (monoecious cv. Felina 32) by steam-distillation and analysed by gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC–MS). The oil was tested against the filariasis vector Culex quinquefasciatus, the peach-potato aphid Myzus persicae, the housefly Musca domestica and the tobacco cutworm Spodoptera littoralis. To prove its harmlessness on non-target invertebrates, it was tested on the multicolored Asian lady beetle, Harmonia axyridis, and Eisenia fetida earthworms, and compared with α-cypermethrin as the positive control. The essential oil composition was dominated by monoterpene and sesquiterpene hydrocarbons, with (E)-caryophyllene (45.4\%), myrcene (25.0\%) and α-pinene (17.9\%) as the most abundant compounds. Results from insecticidal tests showed that the essential oil from inflorescences of industrial hemp cv Felina 32 was highly toxic to M. persicae aphids (LC50 of 3.5\,mL\,L−1) and M. domestica flies (43.3\,μg\,adult−1), while toxicity was moderate towards S. littoralis larvae (152.3\,μg\,larva−1), and scarce against C. quinquefasciatus larvae (LC50 of 252.5\,mL\,L−1) and adults (LC50\,{$>$}\,500\,μg\,cm−2). Contrary to α-cypermethrin, the hemp cv Felina 32 essential oil was not toxic to non-target invertebrate species, including 3rd instar larvae and adults of H. axyridis ladybugs and adults of E. fetida earthworms. Taken together our results shed light on the possible utilization of the crop residue of industrial hemp as a source of environmental-friendly botanical insecticides to be used in Integrated Pest Management and organic agriculture, particularly to manage aphid and housefly populations.
173. Punja, Zamir K., Flower and Foliage-Infecting Pathogens of Marijuana (Cannabis Sativa L.) Plants, Canadian Journal of Plant Pathology, vol. 40, no. 4, pp. 514--527, October 2018. doi: 10.1080/07060661.2018.1535467. Abstract
     Flower buds of Cannabis sativa develop as inflorescences (buds) which are harvested and dried prior to sale. The extent to which fungal plant pathogens can colonize the buds prior to harvest has not been previously studied. Flower buds were sampled at various pre-harvest and harvest time periods during 2015–2017 at locations in British Columbia and Alberta to determine the range of fungi present. Isolated fungi were inoculated onto developing buds to determine the extent of tissue colonization. A pre- and post-harvest internal rot was associated with Botrytis cinerea, causing botrytis bud rot. In addition, two species of Penicillium – P. olsonii and P. copticola – were recovered from pre-harvest flower buds, as well as dried buds, and shown to cause penicillium bud rot. Scanning electron microscopy studies revealed colonization and sporulation on bracts and stigmas of the flower buds by P. olsonii. Several Fusarium species, which were identified using ITS rDNA sequences as F. solani, F. oxysporum and F. equiseti, were isolated from pre-harvest flower buds. These fungi colonized the flower buds following artificial inoculation and caused visible rot symptoms. The most severe symptoms were caused by F. solani, followed by F. oxysporum and, to a much lesser extent, F. equiseti. Powdery mildew infection of the foliage and flower buds was caused by Golovinomyces(Erysiphe) cichoracearum. The pathogen was detected on young vegetatively propagated cuttings and sporulation was abundant on older plants and on flower buds. The various fungi recovered from cannabis flower buds may be present as contaminants from aerially dispersed spores and have the potential to cause various types of pre- and post-harvest bud rot under conducive environmental conditions. Powdery mildew may be spread through aerially disseminated spores and infected propagation materials. Management of these pathogens will require monitoring of the growth environment for spore levels and implementation of sanitization methods to reduce inoculum sources.
174. Kusari, Parijat and Kusari, Souvik and Spiteller, Michael and Kayser, Oliver, Endophytic Fungi Harbored in Cannabis Sativa L.: Diversity and Potential as Biocontrol Agents against Host Plant-Specific Phytopathogens, Fungal Diversity, vol. 60, no. 1, pp. 137--151, May 2013. doi: 10.1007/s13225-012-0216-3. Abstract
     The objective of the present work was isolation, phylogenetic characterization, and assessment of biocontrol potential of endophytic fungi harbored in various tissues (leaves, twigs, and apical and lateral buds) of the medicinal plant, Cannabis sativa L. A total of 30 different fungal endophytes were isolated from all the plant tissues which were authenticated by molecular identification based on rDNA ITS sequence analysis (ITS1, 5.8S and ITS2 regions). The Menhinick’s index revealed that the buds were immensely rich in fungal species, and Camargo’s index showed the highest tissue-specific fungal dominance for the twigs. The most dominant species was Penicillium copticola that could be isolated from the twigs, leaves, and apical and lateral buds. A detailed calculation of Fisher’s log series index, Shannon diversity index, Simpson’s index, Simpson’s diversity index, and Margalef’s richness revealed moderate overall biodiversity of C. sativa endophytes distributed among its tissues. The fungal endophytes were challenged by two host phytopathogens, Botrytis cinerea and Trichothecium roseum, devising a dual culture antagonistic assay on five different media. We observed 11 distinct types of pathogen inhibition encompassing a variable degree of antagonism (\%) on changing the media. This revealed the potential chemodiversity of the isolated fungal endophytes not only as promising resources of biocontrol agents against the known and emerging phytopathogens of Cannabis plants, but also as sustainable resources of biologically active and defensive secondary metabolites.
175. McPartland, J. M. and Clarke, R. C. and Watson, D. P., Hemp Diseases and Pests: Management and Biological Control: An Advanced Treatise (Cabi Publishing), August 2000.
176. Pavlovic, Radmila and Panseri, Sara and Giupponi, Luca and Leoni, Valeria and Citti, Cinzia and Cattaneo, Chiara and Cavaletto, Maria and Giorgi, Annamaria, Phytochemical and Ecological Analysis of Two Varieties of Hemp (Cannabis Sativa L.) Grown in a Mountain Environment of Italian Alps, Frontiers in Plant Science, vol. 10, 2019. doi: 10.3389/fpls.2019.01265. Abstract
     Hemp (Cannabis sativa L.) is a multifunctional crop that is capable of prompt environmental adaptation. In this study, a monoecious cultivar (Futura 75) and a dioecious one (Finola) were tested in a mountain area in Valsaviore (Rhaetian Alps, Italy; elevation: 1,100 m a.s.l.) during the growing season 2018. Phytochemical behavior was evaluated by different analytical approaches: HPLC-high-resolution mass spectrometry, SDS-PAGE LC-MS/MS, HS-SPME GC-MS, and GC-FID in order to obtain complete profile of two varieties cultivated in altitude. CSR functional strategy used for ecological evaluation revealed that both genotypes are mainly competitors, although Finola is more stress tolerator (C:S:R = 57:26:17\%) than Futura (C:S:R = 69:15:16\%). The Finola inflorescences were characterized by higher quantities of β-ocimene and α-terpinolene, while α- and ß-pinene accompanied by extremely high ß-myrcene were found as predominant in Futura. Both varieties were rich in sesquiterpenes (45 recognized) among which trans-caryophyllene and α-humulene were the most abundant. Total tetrahydrocannabinol level was lower than 0.1\%, while the most abundant cannabinoid was cannabidiolic acid (CBDA): 2.3\% found in Finola vs. 2.7\% revealed for Futura. The level of corresponding neutral form, cannabidiol, varied drastically: 0.27\% (Finola) vs. 0.056\% (Futura). Finola showed the unique cannabinoid profile with unexpectedly high cannabidivarin, 2-fold higher that corresponding acidic analogue, whereas the particularity of Futura 75 was the occurrence of cannabigerolic acid (CBGA) in the quantities that was double than those exposed for Finola. The seeds from both chemovars proved to be rich in polyunsaturated fatty acids, and Finola showed a higher ratio ω6/ω3. No difference was found in the protein content, and the SDS-PAGE profile was similar. The most abundant protein was edestin, followed by heat shock protein 70, ß-conglycinin, and vicilin. In conclusion, comprehensive phytochemical and ecological study of two fiber-type varieties cultivated in Italian Alps displayed specific, legal, and safe cannabinoids profile, followed by particular terpene composition, polyunsaturated fatty acids content, and favorable protein profile. This postulates that geographical provenience of hemp should be considered in selecting a variety that would be suitable for a specific end-use nutraceutical application.
177. Nagy, David U. and Cianfaglione, Kevin and Maggi, Filippo and Sut, Stefania and Dall'Acqua, Stefano, Chemical Characterization of Leaves, Male and Female Flowers from Spontaneous Cannabis (Cannabis Sativa L.) Growing in Hungary, Chemistry \& Biodiversity, vol. 16, no. 3, pp. e1800562, 2019. doi: 10.1002/cbdv.201800562. Abstract
     Spontaneous forms of hemp (Cannabis sativa L., often reported as Cannabis sativa var. spontanea Vavilov) with a low content of psychoactive cannabinoids can be considered as a valuable source of other phytoconstituents to be used in nutraceuticals or for their health promoting properties. Chemical data on this hemp variety are rather scarce. In this article, we report a comprehensive phytochemical characterization of leaves, male and female inflorescences of C. sativa growing wild in Hungary. For the purpose, the essential oil along with polar extracts were analyzed using GC/MS, NMR and LC-DAD-MS techniques, respectively. The results indicated that female inflorescence essential oil contains high amounts of the CB2 agonists, (E)-caryophyllene (28.3 \%) and cannabidiol (CBD; 24.9 \%), whereas leaves and male inflorescence essential oils contained lower amounts of both compounds. HPLC/MS allowed to quantify cannabidiol (CBD) and cannabidiolic acid (CBD−A) in the ethyl acetate extracts from leaves, male and female inflorescences; they were 0.3, 0.8 and 0.9 \%, and 0.2, 0.3 and 0.4 \%, respectively. Flavonoids of this spontaneous form of hemp were formed by C-glycosides and glucuronic acids of kaempferol and apigenin with a total content of 3.8, 6.1 and 7.8 mg/g in methanolic extracts from leaves, male and female inflorescences, respectively. Based on these results, spontaneous C. sativa may represent an important source of CB2 agonists and bioflavonoids to be used in nutraceuticals, cosmetics and pharmaceuticals.
178. O'Brien, C. and Arathi, H. S., Bee Diversity and Abundance on Flowers of Industrial Hemp (Cannabis Sativa L.), Biomass and Bioenergy, vol. 122, pp. 331--335, March 2019. doi: 10.1016/j.biombioe.2019.01.015. Abstract
     Industrial hemp, (Cannabis sativa L.), one of the earliest crops spun for fiber, is now used for a variety of commercial products including paper, textiles, clothing, biodegradable plastics, biofuel, food, animal feed etc., all of which are derived from hemp fiber or seeds. Being wind pollinated, dioecious and staminate hemp plants produce large amounts of pollen that are attractive to bees. Hemp flowering in northern Colorado, where this study was conducted, occurs between the end of July and the end of September. This time period coincides with a dearth of pollinator-friendly crop plants in the region, making hemp flowers a potentially valuable source of pollen for foraging bees. Here we present the diversity and abundance of bees collected in the fields of flowering hemp. A total of 23 different genera of bees were collected of which the European honeybee, Apis mellifera at 38\% of the total abundance was the most dominant followed by Melissodes bimaculata at 25\% and Peponapis pruinosa at 16\%. These three genera made up nearly 80\% of the total abundance. While hemp does not produce any nectar, the pollen rich nature of the flowers can make hemp an ecologically valuable crop. As cultivation of hemp continues to expand, we expect insect pests on hemp to also become prevalent. Our results documenting bee diversity in flowering hemp provides the impetus for the development of integrated pest management plans that protect pollinators while controlling pests.
179. Tayyab, Muhammad and Shahwar, Durre, GCMS Analysis of Cannabis Sativa L. from Four Different Areas of Pakistan, Egyptian Journal of Forensic Sciences, vol. 5, no. 3, pp. 114--125, September 2015. doi: 10.1016/j.ejfs.2014.07.008. Abstract
     Cannabis is the most frequently used drug of abuse not only in Pakistan but also in the whole world. Its use is increasing drastically every year. GCMS allows for the analysis of Cannabis sativa which shows the differences of the constituents of this plant. Prevalence of this plant can be identified through knowledge of its constituents. In this way we can obstruct the production if we know the region in which it is produced. GCMS is a useful technique for the comparison of constituents of this drug of abuse which will assist the investigator concerning the origin of plant. Comparison also aids in the understanding and acquaintance of similarities of different samples of cannabinoids.
180. Gautam, Ajay Kumar and Kant, Mona and Thakur, Yogita, Isolation of Endophytic Fungi from Cannabis Sativa and Study Their Antifungal Potential, Archives of Phytopathology and Plant Protection, vol. 46, no. 6, pp. 627--635, April 2013. doi: 10.1080/03235408.2012.749696. Abstract
     A systemic study of fungal endophytes associated with different plant parts of Cannabis sativa and their antifungal activity was investigated in the present study. A total of 281 plant segments, including 91 leaves, 93 stem and 97 petioles samples, were screened for the isolation of endophytic fungi. Totally, 212 (77.65\%) segments were found colonised by different fungi. Highest colonisation frequency were observed in stem parts (84.94\%), then leaves (82.41\%) and lowest 59.79\% in petiole. Total eight fungal genera belonging to 12 species were isolated. Aspergillus is recorded as the most frequently occurring genera with three species Aspergillus niger, Aspergillus flavus and Aspergillus nidulans followed by Penicillium with two species Penicillium chrysogenum and Penicillium citrinum, while Phoma, Rhizopus, Colletotrichum, Cladosporium and Curvularia with single species. The antifungal potential of A. niger and A. flavus – two most frequently isolated endophytic fungi – was evaluated against two common plant pathogen, Colletotrichum gloeosporioides and Curvularia lunata. Different plant and fungal extracts individually and in combinations showed variations in antifungal activity against both the pathogens. The primary results obtained on antifungal activity of endophytes show their possible role in plant defence mechanism but it is a preliminary approach and more extensive research is still required.
181. Cosentino, Salvatore L. and Riggi, Ezio and Testa, Giorgio and Scordia, Danilo and Copani, Venera, Evaluation of European Developed Fibre Hemp Genotypes (Cannabis Sativa L.) in Semi-Arid Mediterranean Environment, Industrial Crops and Products, vol. 50, pp. 312--324, October 2013. doi: 10.1016/j.indcrop.2013.07.059. Abstract
     In the present study two experiments, namely, (i) the adaptation and productivity of different hemp genotypes, both monoecious and dioecious, developed in Central-Northern and Southern Europe, and (ii) the water consumption, water use efficiency (WUE), radiation use efficiency (RUE), leaf transpiration and net photosynthesis of Futura 75 hemp cultivar were studied. Experiments were carried out in Southern Italy in two subsequent year periods. Sowing occurred in May in both years. Results show that fibre hemp, both monoecious and dioecious, performed well giving high productivity; however, fibre hemp needs almost 250mm of water for monoecious early genotypes and 450mm for dioecious late genotypes. Higher biomass and stem dry yields were achieved with genotypes developed for Central-Southern environments, such as the dioecious Dioica 88 and Fibranova. In order to estimate the water consumption and WUE of Futura 75, four water regimes (I3, I2, I1 and I0 corresponding respectively to 100\%, 50\%, 25\% ETm restoration and irrigation until crop establishment) were tested. Futura 75 was strongly affected by water shortage and the WUE ranged between 2.73gl−1 in good water conditions and 3.45gl−1 in water stress conditions. The highest light extinction coefficient (k) was observed in water stressed (0.58) than in good watered conditions (0.40). The water stress reduced RUE, LAI and therefore aboveground biomass yield. Air temperature strongly influenced net photosynthesis with an optimum at 24°C; higher and lower values of air temperature led to a decrease of net photosynthesis.
182. Piluzza, G. and Delogu, G. and Cabras, A. and Marceddu, S. and Bullitta, S., Differentiation between Fiber and Drug Types of Hemp (Cannabis Sativa L.) from a Collection of Wild and Domesticated Accessions, Genetic Resources and Crop Evolution, vol. 60, no. 8, pp. 2331--2342, December 2013. doi: 10.1007/s10722-013-0001-5. Abstract
     Accessions of wild and domesticated hemp (Cannabis sativa L.) originating from Colombia, Mexico, California, Bolivia, Thailand, Afghanistan, Serbia, Hungary, south Africa and different regions of China, were studied by means of DNA polymorphisms in order to discriminate between drug and fiber types. Analysis of molecular variance (AMOVA) was used to partition the total genetic variance within and among populations. The significance of the variance components was tested by calculating their probabilities based on 999 random permutations. AMOVA revealed 74~\% variation among accessions and 26~\% within accessions, all AMOVA variation was highly significant (P~{$<~$}0.001). The cluster analysis of molecular data, grouped accessions into eight clusters and gave a matrix correlation value of r~=~0.943, indicating a very good fit between the similarity values implied by the phenogram and those of the original similarity matrix. In this study, DNA polymorphisms could discriminate the fiber and drug types, and accessions were grouped in accordance to their classification and uses. In addition, seed size variation and micromorphological characters of seeds were studied by means of a scanning electron microscope (SEM). Seeds varied significantly in size, and were bigger in the fiber types. SEM analysis exhibited variation of micromorphological characters of seeds that could be important for discriminating the fiber or drug types.
183. Citterio, Sandra and Prato, Nadia and Fumagalli, Pietro and Aina, Roberta and Massa, Nadia and Santagostino, Angela and Sgorbati, Sergio and Berta, Graziella, The Arbuscular Mycorrhizal Fungus Glomus Mosseae Induces Growth and Metal Accumulation Changes in Cannabis Sativa L., Chemosphere, vol. 59, no. 1, pp. 21--29, March 2005. doi: 10.1016/j.chemosphere.2004.10.009. Abstract
     The effect of arbuscular mycorrhiza on heavy metal uptake and translocation was investigated in Cannabis sativa. Hemp was grown in the presence and absence of 100μgg−1 Cd and Ni and 300μgg−1 Cr(VI), and inoculated or not with the arbuscular mycorrhizal fungus Glomus mosseae. In our experimental condition, hemp growth was reduced in inoculated plants and the reduction was related to the degree of mycorrhization. The percentage of mycorrhizal colonisation was 42\% and 9\% in plants grown in non-contaminated and contaminated soil, suggesting a significant negative effect of high metal concentrations on plant infection by G. mosseae. Soil pH, metal bioavailability and plant metal uptake were not influenced by mycorrhization. The organ metal concentrations were not statistically different between inoculated and non-inoculated plants, apart from Ni which concentration was significantly higher in stem and leaf of inoculated plants grown in contaminated soil. The distribution of absorbed metals inside plant was related to the soil heavy metal concentrations: in plant grown in non-contaminated soil the greater part of absorbed Cr and Ni was found in shoots and no significant difference was determined between inoculated and non-inoculated plants. On the contrary, plants grown in artificially contaminated soil accumulated most metal in root organ. In this soil, mycorrhization significantly enhanced the translocation of all the three metals from root to shoot. The possibility to increase metal accumulation in shoot is very interesting for phytoextraction purpose, since most high producing biomass plants, such as non-mycorrhized hemp, retain most heavy metals in roots, limiting their application.
184. Haney, Alan and Kutscheid, Benjamin B., An Ecological Study of Naturalized Hemp (Cannabis Sativa L.) in East-Central Illinois, The American Midland Naturalist, vol. 93, no. 1, pp. 1--24, 1975. doi: 10.2307/2424101. Abstract
     Naturalized populations of hemp (Cannabis sativa L.) on a range of sites in E-central Illinois were intensively studied. Fifty-one independent (site) and 26 dependent (plant) parameters were quantified from 101 randomly selected stands. All site variables that could be quantified were examined statistically. These included soil physical and chemical properties, competition, physical site characteristics and stand histories. Correlations between dependent parameters and linear relationships between dependent and independent parameters were examined prior to multiple correlation analysis of each dependent variable. Extreme variation was found in most dependent variables. In only a few instances could a significant portion of the variance in a dependent variable be partitioned by multiple regression. Regression did permit an evaluation of the relative importance of each specific independent variable to any given dependent variable and to the overall ecology of hemp. Those parameters were particularly important that influenced seed production in this annual plant whose seeds do not long retain viability in the soil. Important parameters that influenced seed production were interspecific competition, especially from grasses, exposure to sunlight, soil disturbance the previous year, soil aeration and available phosphorus and potassium. Viable seed production ranged from 0 to 34,433 per sq m, with a mean of 4555. Two important agents effected some degree of biological control. Alternaria alternata (Fr.) Keissler, a common fungal pathogen of many plants, destroyed an average of 20.5\% of developing seeds, while larvae of Grapholitha tristrigana Clements (Lepidoptera: Tortricidae) destroyed 7.1\%. Although a few stands had no damage by the insect, every stand suffered attack from the fungus which destroyed up to 45.5\% of seeds in extreme cases.
185. Strzelczyk, Malgorzata and Lochynska, Malgorzata and Chudy, Magdalena, Systematics and Botanical Characteristics of Industrial Hemp Cannabis Sativa L, Journal of Natural Fibers, vol. 19, no. 13, pp. 5804--5826, December 2022. doi: 10.1080/15440478.2021.1889443. Abstract
     The systematic affiliation of Cannabis has been controversial for many years. Without a doubt, today we know that they belong to the order Rosales, family Cannabaceae, genus Cannabis, species – C. sativa L., with both industrial hemp (C. sativa L. var. sativa) and narcotic C. sativa L. var. indica. Hemp can be divided into several groups according to their genetic plasticity, allowing them to adapt to changing geographical and climatic conditions, and to almost any substrate and the division can be made using different criteria. The most important are: origin, length of vegetation period, content of cannabinoids (THC and CBD), mono- and dioequivalence.A natural feature is the phenomenon of sexual dimorphism, which is common in the world of other plants. Dioeciousness is natural, and the monoecious forms of hemp grown are unstable.Although the pharmaceutical use of hemp has been known for more than 5 000 years, new bioactive compounds are still being discovered. So the potential of this species is still inexhaustible and unfathomable.
186. Hurgobin, Bhavna and {Tamiru-Oli}, Muluneh and Welling, Matthew T. and Doblin, Monika S. and Bacic, Antony and Whelan, James and Lewsey, Mathew G., Recent Advances in Cannabis Sativa Genomics Research, New Phytologist, vol. 230, no. 1, pp. 73--89, 2021. doi: 10.1111/nph.17140. Abstract
     Cannabis (Cannabis sativa L.) is one of the oldest cultivated plants purported to have unique medicinal properties. However, scientific research of cannabis has been restricted by the Single Convention on Narcotic Drugs of 1961, an international treaty that prohibits the production and supply of narcotic drugs except under license. Legislation governing cannabis cultivation for research, medicinal and even recreational purposes has been relaxed recently in certain jurisdictions. As a result, there is now potential to accelerate cultivar development of this multi-use and potentially medically useful plant species by application of modern genomics technologies. Whilst genomics has been pivotal to our understanding of the basic biology and molecular mechanisms controlling key traits in several crop species, much work is needed for cannabis. In this review we provide a comprehensive summary of key cannabis genomics resources and their applications. We also discuss prospective applications of existing and emerging genomics technologies for accelerating the genetic improvement of cannabis.
187. Adal, Ayelign M. and Doshi, Ketan and Holbrook, Larry and Mahmoud, Soheil S., Comparative RNA-Seq Analysis Reveals Genes Associated with Masculinization in Female Cannabis Sativa, Planta, vol. 253, no. 1, pp. 17, January 2021. doi: 10.1007/s00425-020-03522-y. Abstract
     Using RNA profiling, we identified several silver thiosulfate-induced genes that potentially control the masculinization of female Cannabis sativa plants.
188. Cai, Sen and Zhang, Zhiyuan and Huang, Suyun and Bai, Xu and Huang, Ziying and Zhang, Yiping Jason and Huang, Likun and Tang, Weiqi and Haughn, George and You, Shijun and Liu, Yuanyuan, CannabisGDB: A Comprehensive Genomic Database for Cannabis Sativa L, Plant Biotechnology Journal, vol. 19, no. 5, pp. 857--859, May 2021. doi: 10.1111/pbi.13548.
189. Gao, Shan and Wang, Baishi and Xie, Shanshan and Xu, Xiaoyu and Zhang, Jin and Pei, Li and Yu, Yongyi and Yang, Weifei and Zhang, Ying, A High-Quality Reference Genome of Wild Cannabis Sativa, Horticulture Research, vol. 7, no. 1, pp. 73, December 2020. doi: 10.1038/s41438-020-0295-3. Abstract
     Abstract Cannabis sativa is a well-known plant species that has great economic and ecological significance. An incomplete genome of cloned C. sativa was obtained by using SOAPdenovo software in 2011. To further explore the utilization of this plant resource, we generated an updated draft genome sequence for wild-type varieties of C. sativa in China using PacBio single-molecule sequencing and Hi-C technology. Our assembled genome is approximately 808\,Mb, with scaffold and contig N50 sizes of 83.00\,Mb and 513.57\,kb, respectively. Repetitive elements account for 74.75\% of the genome. A total of 38,828 protein-coding genes were annotated, 98.20\% of which were functionally annotated. We provide the first comprehensive de novo genome of wild-type varieties of C. sativa distributed in Tibet, China. Due to long-term growth in the wild environment, these varieties exhibit higher heterozygosity and contain more genetic information. This genetic resource is of great value for future investigations of cannabinoid metabolic pathways and will aid in promoting the commercial production of C. sativa and the effective utilization of cannabinoids. The assembled genome is also a valuable resource for intensively and effectively investigating the C. sativa genome further in the future.
190. Bernstein, Nirit and Gorelick, Jonathan and Koch, Sraya, Interplay between Chemistry and Morphology in Medical Cannabis (Cannabis Sativa L.), Industrial Crops and Products, vol. 129, pp. 185--194, March 2019. doi: 10.1016/j.indcrop.2018.11.039. Abstract
     Despite the long history of cannabis use, the chemical and physiological state of the plants have not sufficiently been addressed due to legal restrictions. Hundreds of secondary metabolites of therapeutic potential were identified in cannabis. This large number, together with the need to standardize chemical composition of the plant material supplied to patients dictates a need to understand interrelations between morphology and chemistry. The present study investigated the interplay between chemical profiles (of cannabinoids and the ionome) and locales in the plant body. We studied location and organ-specific effects on chemical profiles of cannabinoids, the ionome and physiological traits. A number of important morphological-related chemical trends were identified, which demonstrate in-planta variation in regulation of the chemotype: The cannabinoid profile changes with location along the plant demonstrating significant variation among differing heights for almost every cannabinoid studied. The content of most cannabinoids (including THC, CBD, CBG, CBC, THCV) substantially increased with plant height, both in the flowers and the inflorescence leaves. The concentration of cannabinoids in the inflorescence leaves is considerable and for THC and CBD reached about half of that found in flowers. Concentration in fan leaves is about 1/10 the concentration found in flowers. A number of cannabinoids, including CBC and CBG, were present in the inflorescence leaves at equal or greater levels than in the flowers. The partitioning of mineral nutrients between plant organs demonstrate typical uptake and translocation in the plant. The lower concentrations of N, P, K and higher concentration of Ca in fan-leaves compared with inflorescence-leaves, a result of in-planta translocation abilities in the phloem, supports the physiological findings that the fan leaves are older than the inflorescence leaves. The substantial spatial gradients in secondary-metabolites demonstrate organ and location-specific regulation of accumulation. This in-planta variability in regulation of the chemotype requires standardization for the development of Cannabis as a therapeutic crop for modern medicine.
191. Raman, Vijayasankar and Lata, Hemant and Chandra, Suman and Khan, Ikhlas A. and ElSohly, Mahmoud A., Morpho-Anatomy of Marijuana (Cannabis Sativa L.), pp. 123--136, 2017. doi: 10.1007/978-3-319-54564-6_5. Abstract
     Cannabis sativa is a complex species with highly variable morphological features. The present chapter provides detailed descriptions of morphological and anatomical characters of various parts of C. sativa plant and illustrated with bright-field and scanning electron micrographs. Male and female flowers occur in separate plants. Three types of glandular trichomes namely, glandular stalked, glandular sessile and bulbous glandular trichomes are found. Of these, glandular stalked trichomes are restricted to the floral bracts in pistillate plants and anthers in staminate plants. The other two types of glandular trichomes are found in various parts including bracts, leaves, stems and petioles. Two types of non-glandular trichomes namely, cystolith trichomes and slender covering trichomes, are present. Cystolith trichomes are primarily found on the adaxial leaf surface while the covering trichomes are commonly present on the abaxial leaf surface, stems, petioles and tepals. Cystolith crystals of calcium carbonate and cluster crystals of calcium oxalate are observed in the leaves. Anatomical features of various parts of the plant are described and illustrated.
192. Small, Ernest, Classification of Cannabis Sativa L. in Relation to Agricultural, Biotechnological, Medical and Recreational Utilization, pp. 1--62, 2017. doi: 10.1007/978-3-319-54564-6_1. Abstract
     Cannabis sativa has been utilized for millennia, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”), and secondarily as a source of edible seeds. In domesticating the species for these divergent purposes, humans have altered the morphology, chemistry, distribution and ecology of cultivated forms by comparison with related wild plants. Wild-growing plants appear to be either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticated plants, making it impossible to determine if unaltered primeval or ancestral populations still exist. There are conflicting botanical classifications of Cannabis, including splitting it into several alleged species. The different approaches to classifying and naming plants such as Cannabis, with interbreeding domesticated and wild forms, are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a high-THC subspecies with both domesticated and ruderal varieties, and similarly a low-THC subspecies with both domesticated and ruderal varieties. Alternative approaches to the classification of Cannabis that do not utilize scientific nomenclature are noted.
193. Farag, S. and Kayser, O., Chapter 1 - The Cannabis Plant: Botanical Aspects, pp. 3--12, January 2017. doi: 10.1016/B978-0-12-800756-3.00001-6. Abstract
     Cannabis sativa L. (Cannabaceae) is one of the oldest medicinal plants used by humans. For millennia, the plant has also been used for fiber, oil production, and simply as additive for food products. This chapter gives an overview of botanical aspects of the genus Cannabis, such as, macroscopical and microscopical features, taxonomic classification, the current varieties, genomics. Furthermore, geographical distribution, agricultural status, and germplasm conservation are provided. This chapter presents the current state of knowledge of different cultivation forms, including outdoor, indoor, and micropropagation.
194. McPartland, John M., Cannabis Sativa and Cannabis Indica versus “Sativa” and “Indica”, pp. 101--121, 2017. doi: 10.1007/978-3-319-54564-6_4. Abstract
     The formal botanical taxonomy of Cannabis sativa Linnaeus and C. indica Lamarck has become entangled and subsumed by a new vernacular taxonomy of “Sativa” and “Indica.” The original protologues (descriptions, synonymies, and herbarium specimens) by Linnaeus and Lamarck are reviewed. The roots of the vernacular taxonomy are traced back to Vavilov and Schultes, who departed from the original concepts of Linnaeus and Lamarck. The modified concepts by Vavilov and Schultes were further remodeled by underground Cannabis breeders in the 1980s and 1990s. “Sativa” refers to plants of Indian heritage, in addition to their descendants carried in a diaspora to Southeast Asia, South- and East Africa, and even the Americas. “Indica” refers to Afghani landraces, together with their descendants in parts of Pakistan (the northwest, bordering Afghanistan). Phytochemical and genetic research supports the separation of “Sativa” and “Indica.” But their nomenclature does not align with formal botanical C. sativa and C. indica based on the protologues of Linnaeus and Lamarck. Furthermore, distinguishing between “Sativa” and “Indica” has become nearly impossible because of extensive cross-breeding in the past 40~years. Traditional landraces of “Sativa” and “Indica” are becoming extinct through introgressive hybridization. Solutions for reconciling the formal and vernacular taxonomies are proposed.
195. Oh, Hyehyun and Seo, Boyoung and Lee, Seunghwan and Ahn, Dong-Ha and Jo, Euna and Park, Jin-Kyoung and Min, Gi-Sik, Two Complete Chloroplast Genome Sequences of Cannabis Sativa Varieties, Mitochondrial DNA Part A, vol. 27, no. 4, pp. 2835--2837, July 2016. doi: 10.3109/19401736.2015.1053117. Abstract
     In this study, we determined the complete chloroplast (cp) genomes from two varieties of Cannabis sativa. The genome sizes were 153,848 bp (the Korean non-drug variety, Cheungsam) and 153,854 bp (the African variety, Yoruba Nigeria). The genome structures were identical with 131 individual genes [86 protein-coding genes (PCGs), eight rRNA, and 37 tRNA genes]. Further, except for the presence of an intron in the rps3 genes of two C. sativa varieties, the cp genomes of C. sativa had conservative features similar to that of all known species in the order Rosales. To verify the position of C. sativa within the order Rosales, we conducted phylogenetic analysis by using concatenated sequences of all PCGs from 17 complete cp genomes. The resulting tree strongly supported monophyly of Rosales. Further, the family Cannabaceae, represented by C. sativa, showed close relationship with the family Moraceae. The phylogenetic relationship outlined in our study is well congruent with those previously shown for the order Rosales.
196. Vergara, Daniela and White, Kristin H. and Keepers, Kyle G. and Kane, Nolan C., The Complete Chloroplast Genomes of Cannabis Sativa and Humulus Lupulus, Mitochondrial DNA Part A, vol. 27, no. 5, pp. 3793--3794, September 2016. doi: 10.3109/19401736.2015.1079905. Abstract
     Cannabis and Humulus are sister genera comprising the entirety of the Cannabaceae sensu stricto, including C. sativa L. (marijuana, hemp), and H. lupulus L. (hops) as two economically important crops. These two plants have been used by humans for many purposes including as a fiber, food, medicine, or inebriant in the case of C. sativa, and as a flavoring component in beer brewing in the case of H. lupulus. In this study, we report the complete chloroplast genomes for two distinct hemp varieties of C. sativa, Italian “Carmagnola” and Russian “Dagestani”, and one Czech variety of H. lupulus “Saazer”. Both C. sativa genomes are 153 871 bp in length, while the H. lupulus genome is 153 751 bp. The genomes from the two C. sativa varieties differ in 16 single nucleotide polymorphisms (SNPs), while the H. lupulus genome differs in 1722 SNPs from both C. sativa cultivars.
197. Razumova, Olga V. and Alexandrov, Oleg S. and Divashuk, Mikhail G. and Sukhorada, Tatiana I. and Karlov, Gennady I., Molecular Cytogenetic Analysis of Monoecious Hemp (Cannabis Sativa L.) Cultivars Reveals Its Karyotype Variations and Sex Chromosomes Constitution, Protoplasma, vol. 253, no. 3, pp. 895--901, May 2016. doi: 10.1007/s00709-015-0851-0. Abstract
     Hemp (Cannabis sativa L., 2n\,=\,20) is a dioecious plant. Sex expression is controlled by an X-to-autosome balance system consisting of the heteromorphic sex chromosomes XY for males and XX for females. Genetically monoecious hemp offers several agronomic advantages compared to the dioecious cultivars that are widely used in hemp cultivation. The male or female origin of monoecious maternal plants is unknown. Additionally, the sex chromosome composition of monoecious hemp forms remains unknown. In this study, we examine the sex chromosome makeup in monoecious hemp using a cytogenetic approach. Eight monoecious and two dioecious cultivars were used. The DNA of 210 monoecious plants was used for PCR analysis with the male-associated markers MADC2 and SCAR323. All monoecious plants showed female amplification patterns. Fluorescence in situ hybridization (FISH) with the subtelomeric CS-1 probe to chromosomes plates and karyotyping revealed a lack of Y chromosome and presence of~XX sex chromosomes in monoecious cultivars with the chromosome number 2n\,=\,20. There was a high level of intra- and intercultivar karyotype variation detected. The results of this study can be used for further analysis of the genetic basis of sex expression in plants.
198. {Aizpurua-Olaizola}, Oier and Soydaner, Umut and Öztürk, Ekin and Schibano, Daniele and Simsir, Yilmaz and Navarro, Patricia and Etxebarria, Nestor and Usobiaga, Aresatz, Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis Sativa Plants from Different Chemotypes, Journal of Natural Products, vol. 79, no. 2, pp. 324--331, February 2016. doi: 10.1021/acs.jnatprod.5b00949. Abstract
     The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.
199. Small, Ernest, Evolution and Classification of Cannabis Sativa (Marijuana, Hemp) in Relation to Human Utilization, The Botanical Review, vol. 81, no. 3, pp. 189--294, September 2015. doi: 10.1007/s12229-015-9157-3. Abstract
     Cannabis sativa has been employed for thousands of years, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”). Studies of relationships among various groups of domesticated forms of the species and wild-growing plants have led to conflicting evolutionary interpretations and different classifications, including splitting C. sativa into several alleged species. This review examines the evolving ways Cannabis has been used from ancient times to the present, and how human selection has altered the morphology, chemistry, distribution and ecology of domesticated forms by comparison with related wild plants. Special attention is given to classification, since this has been extremely contentious, and is a key to understanding, exploiting and controlling the plant. Differences that have been used to recognize cultivated groups within Cannabis are the results of disruptive selection for characteristics selected by humans. Wild-growing plants, insofar as has been determined, are either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticated plants, making it impossible to determine if unaltered primeval or ancestral populations still exist. The conflicting approaches to classifying and naming plants with such interacting domesticated and wild forms are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a narcotic subspecies with both domesticated and ruderal varieties, and similarly a non-narcotic subspecies with both domesticated and ruderal varieties. An alternative approach consistent with the international code of nomenclature for cultivated plants is proposed, recognizing six groups: two composed of essentially non-narcotic fiber and oilseed cultivars as well as an additional group composed of their hybrids; and two composed of narcotic strains as well as an additional group composed of their hybrids.
200. Techen, Natascha and Chandra, Suman and Lata, Hemant and ElSohly, Mahmoud A. and Khan, Ikhlas A., Genetic Identification of Female Cannabis Sativa Plants at Early Developmental Stage, Planta Medica, vol. 76, no. 16, pp. 1938--1939, November 2010. doi: 10.1055/s-0030-1249978. Abstract
     Thieme E-Books \& E-Journals
201. Mendoza, Maria A. and Mills, DeEtta K. and Lata, Hemant and Chandra, Suman and ElSohly, Mahmoud A. and Almirall, Jose R., Genetic Individualization of Cannabis Sativa by a Short Tandem Repeat Multiplex System, Analytical and Bioanalytical Chemistry, vol. 393, no. 2, pp. 719--726, January 2009. doi: 10.1007/s00216-008-2500-3. Abstract
     Cannabis sativa is the most frequently used of all illicit drugs in the USA. Cannabis has been used throughout history for its stems in the production of hemp fiber, seed for oil and food, and buds and leaves as a psychoactive drug. Short tandem repeats (STRs) were chosen as molecular markers owing to their distinct advantages over other genetic methods. STRs are codominant, can be standardized such that reproducibility between laboratories can be easily achieved, have a high discrimination power, and can be multiplexed. In this study, six STR markers previously described for C. sativa were multiplexed into one reaction. The multiplex reaction was able to individualize 98 cannabis samples (14 hemp and 84 marijuana, authenticated as originating from 33 of the 50 states of the USA) and detect 29 alleles averaging 4.8 alleles per loci. The data did not relate the samples from the same state to each other. This is the first study to report a single-reaction sixplex and apply it to the analysis of almost 100 cannabis samples of known geographic origin.
202. Howard, Christopher and Gilmore, Simon and Robertson, James and Peakall, Rod, A Cannabis Sativa STR Genotype Database for Australian Seizures: Forensic Applications and Limitations*, Journal of Forensic Sciences, vol. 54, no. 3, pp. 556--563, 2009. doi: 10.1111/j.1556-4029.2009.01014.x. Abstract
     Abstract: A genetic database was established with the aim of documenting the genetic diversity of Cannabis sativa in Australia for future utilization in forensic investigations. The database consisted of genotypes at 10 validated short tandem repeat loci for 510 plants representing drug seizures from across Australia and 57 fiber samples. A total of 106 alleles and 314 different genotypes were detected. All fiber samples exhibited unique genotypes while 55\% of the drug samples shared a genotype with one or more samples. Shared genotypes were mostly found within seizures; however, some genotypes were found among seizures. Statistical analysis indicated that genotype sharing was a consequence of clonal propagation rather than a lack of genetic resolution. Thus, the finding of shared genotypes among seizures is likely due to either a common supplier, or direct links among seizures. Notwithstanding the potential intelligence information provided by genetic analysis of C. sativa, our database analysis also reveals some present limitations.
203. Moliterni, V. M. Cristiana and Cattivelli, Luigi and Ranalli, P. and Mandolino, Giuseppe, The Sexual Differentiation of Cannabis Sativa L.: A Morphological and Molecular Study, Euphytica, vol. 140, no. 1, pp. 95--106, January 2004. doi: 10.1007/s10681-004-4758-7. Abstract
     Cannabis sativa L. is a dioecious species with sexual dimorphism occurring in a late stage of plant development. Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (XY) and female is the homogametic one (XX). The sexual phenotype of Cannabis often shows some flexibility leading to the differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). Sex is considered an important trait for hemp genetic improvement; therefore, the study of the mechanism of sexual differentiation is of paramount interest in hemp research. A morphological and molecular study of Cannabis sativa sexual differentiation has been carried out in the Italian dioecious cultivar Fibranova.
204. Turner, Carlton E. and Elsohly, Mahmoud A. and Cheng, Ping C. and Lewis, Glenda, Constituents of Cannabis Sativa L., XIV: Intrinsic Problems in Classifying Cannabis Based on a Single Cannabinoid Analysis, ACS Publications, July 2004. doi: 10.1021/np50003a017.
205. Daughtry, C. S. T. and Walthall, C. L., Spectral Discrimination of Cannabis Sativa L. Leaves and Canopies, Remote Sensing of Environment, vol. 64, no. 2, pp. 192--201, May 1998. doi: 10.1016/S0034-4257(98)00002-9. Abstract
     The growing of marijuana (Cannabis sativa L.) on public lands poses problems to the environment and the public. Remote sensing offers a potential way of monitoring public lands for the production of marijuana. However, very little information on the spectral properties of marijuana is available in the scientific literature. Our objectives were to 1) characterize the spectral properties of the leaves of marijuana and various other plants that occur where marijuana is grown in the eastern United States, 2) simulate canopy reflectance, and 3) identify wavebands for discriminating marijuana from other plants. In a series of replicated field experiments, the basic factors affecting marijuana growth and reflectance, including planting date, plant density, and N-fertilization were varied. Leaf optical properties were measured periodically during the growing season with a spectroradiometer and integrating sphere. As N-fertilization rate decreased, the marijuana plants produced leaves with lower chlorophyll concentrations and higher reflectance values in the visible wavelength region, particularly at 550 nm. The reflectance spectra of the herbaceous dicot species examined were very similar to the spectrum of marijuana. The reflectance spectra of the monocots and the trees differed significantly from the spectrum of marijuana, particularly in the green and near-infrared wavelength regions. Canopy reflectance spectra of marijuana and several representative species were simulated for a wide range of LAI and background reflectances. Major differences in canopy reflectance of marijuana and other plants were observed near 550 nm, 720 nm, and 800 nm. Dense canopies of marijuana were more spectrally discriminable from other vegetation than sparse canopies. Thus, based on measured leaf spectra and simulated canopy reflectance spectra, we would choose several relatively narrow (i.e., 30 nm or less) spectral bands in the green (550 nm), red (670 nm), “red edge” (720 nm), and the near-infrared (800 nm) to discriminate marijuana leaves and canopies from other species. Much of the leaf spectral information is also available in the canopy reflectance data.Published by Elsevier Science Inc., 1998
206. Sakamoto, Koichi and Akiyama, Yukio and Fukui, Kiichi and Kamada, Hiroshi and Satoh, Shinobu, Characterization; Genome Sizes and Morphology of Sex Chromosomes in Hemp ({\emph{Cannabis Sativa}} L.), Cytologia, vol. 63, no. 4, pp. 459--464, 1998. doi: 10.1508/cytologia.63.459. Abstract
     A dioecious plant, Cannabis sativa has two sex chromosomes (X and Y). The genome sizes of the diploid female and male plants were determined to be 1636 and 1683 Mbp, respectively, by flow cytometry. By the karyotype analysis, the X and Y chromosomes were found to be submetacentric and subtelocentric, respectively. The Y chromosome had the largest long arm with a satellite in the terminal of its short arm. Conspicuous condensation was specifically observed in the long arm and satellite of the Y chromosome during the prometaphase to metaphase stages. These results indicate that the Y chromosome, especially in its long arm, specifically differentiates in Cannabis sativa and might contribute to the sex determination.
207. Small, Ernest and Cronquist, Arthur, A Practical and Natural Taxonomy for Cannabis, Taxon, vol. 25, no. 4, pp. 405--435, 1976. doi: 10.2307/1220524. Abstract
     Variation in Cannabis is evaluated in the context of the confusing systematic history of this genus. Aside from some experimentally produced polyploids, all Cannabis is diploid (n = 10), and there appear to be no barriers to successful hybridization within the genus. The present pattern of variation is due in large part to the influence of man. Two widespread classes of plant are discernible: a group of generally northern plants of relatively limited intoxicant potential, influenced particularly by selection for fibre and oil agronomic qualities, and a group of generally southern plants of considerable intoxicant potential, influenced particularly by selection for inebriant qualities. These two groups are treated respectively as subsp. sativa and indica, of C. sativa, the only species of the genus Cannabis. Within each subspecies two parallel phases are recognizable. The "wild" (weedy, naturalized or indigenous) phase is more or less distinguishable from the domesticated (cultivated or spontaneous) phase by means of an adaptive syndrome of fruit characteristics. The resulting four discernible groups are recognized as varieties.
208. Briosi, Giovanni and Tognini, Filippo, Intorno Alla Anatomia Della Canapa (cannabis Sativa L.) Parte Prima, 1894. url: http://archive.org/details/a540intornoallaanatomiadellacanapacannabissatival.parteprima. Abstract
     Intorno Alla Anatomia Della Canapa (cannabis Sativa L.) Parte Prima.Atti dell'Istituto Botanico di Pavia.Milano 1894
209. McPartland, John M., Cannabis Systematics at the Levels of Family, Genus, and Species, Cannabis and Cannabinoid Research, vol. 3, no. 1, pp. 203--212, October 2018. doi: 10.1089/can.2018.0039. Abstract
     New concepts are reviewed in Cannabis systematics, including phylogenetics and nomenclature. The family Cannabaceae now includes Cannabis, Humulus, and eight genera formerly in the Celtidaceae. Grouping Cannabis, Humulus, and Celtis actually goes back 250 years. Print fossil of the extinct genus Dorofeevia (=Humularia) reveals that Cannabis lost a sibling perhaps 20 million years ago (mya). Cannabis print fossils are rare (n=3 worldwide), making it difficult to determine when and where she evolved. A molecular clock analysis with chloroplast DNA (cpDNA) suggests Cannabis and Humulus diverged 27.8 mya. Microfossil (fossil pollen) data point to a center of origin in the northeastern Tibetan Plateau. Fossil pollen indicates that Cannabis dispersed to Europe by 1.8–1.2\,mya. Mapping pollen distribution over time suggests that European Cannabis went through repeated genetic bottlenecks, when the population shrank during range contractions. Genetic drift in this population likely initiated allopatric differences between European Cannabis sativa (cannabidiol [CBD]{$>$}Δ9-tetrahydrocannabinol [THC]) and Asian Cannabis indica (THC{$>$}CBD). DNA barcode analysis supports the separation of these taxa at a subspecies level, and recognizing the formal nomenclature of C. sativa subsp. sativa and C. sativa subsp. indica. Herbarium specimens reveal that field botanists during the 18th–20th centuries applied these names to their collections rather capriciously. This may have skewed taxonomic determinations by Vavilov and Schultes, ultimately giving rise to today's vernacular taxonomy of “Sativa” and “Indica,” which totally misaligns with formal C. sativa and C. indica. Ubiquitous interbreeding and hybridization of “Sativa” and “Indica” has rendered their distinctions almost meaningless.
210. Schultes, Richard Evans and Klein, William M. and Plowman, Timothy and Lockwood, Tom E., Cannabis: An Example of Taxonomic Neglect, Botanical Museum leaflets, Harvard University, vol. 23, no. 9, pp. 337--367, February 1974. doi: 10.5962/p.168565.
211. Gul, Waseem and Gul, Shahbaz W. and Chandra, Suman and Lata, Hemant and Ibrahim, Elsayed A. and ElSohly, Mahmoud A., Detection and Quantification of Cannabinoids in Extracts of Cannabis Sativa Roots Using LC-MS/MS, Planta Medica, vol. 84, no. 4, pp. 267--271, March 2018. doi: 10.1055/s-0044-100798. Abstract
     A liquid chromatography-tandem mass spectrometry single-laboratory validation was performed for the detection and quantification of the 10 major cannabinoids of cannabis, namely, (−)-trans-Δ9-tetrahydrocannabinol, cannabidiol, cannabigerol, cannabichromene, tetrahydrocannabivarian, cannabinol, (−)-trans-Δ8-tetrahydrocannabinol, cannabidiolic acid, cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid-A, in the root extract of Cannabis sativa. Acetonitrile : methanol (80 : 20, v/v) was used for extraction; d3-cannabidiol and d3- tetrahydrocannabinol were used as the internal standards. All 10 cannabinoids showed a good regression relationship with r 2 {$>$} 0.99. The validated method is simple, sensitive, and reproducible and is therefore suitable for the detection and quantification of these cannabinoids in extracts of cannabis roots. To our knowledge, this is the first report for the quantification of cannabinoids in cannabis roots.
212. Elhendawy, Mostafa A. and Wanas, Amira S. and Radwan, Mohamed M. and Azzaz, Nabil A. and Toson, ElShahat S. and ElSohly, Mahmoud A., Chemical and Biological Studies of Cannabis Sativa Roots, Medical Cannabis and Cannabinoids, vol. 1, no. 2, pp. 104--111, 2018. doi: 10.1159/000495582. Abstract
     The chemical study of \emph{Cannabis sativa} roots led to the isolation and identification of 10 compounds. Their chemical structures were unambiguously established on the basis of 1D and 2D NMR spectroscopy and mass spectrometry as friedelan-3-one (1), epifriedelanol (2), β-sitosterol (3), ergost-5-en-3-ol (4), methyl hexadecanoate (5), pentadecanoic acid (6), 10E-hexadecenoic acid (7), 4-hydroxy-3-methoxybenzaldehyde (8), β-sitosterol-β-D-glucoside (9) and \emph{p}-coumaroyltyramine (10). Compounds 5–9 were reported for the first time from \emph{C. sativa} roots. All the isolated compounds were tested for their antimicrobial activity. Compound 4 showed modest activity against \emph{Cryptococcus neoformans} with an IC\textsubscript{50} value of 13.7 μg/mL, while compound 10 displayed potent activity against \emph{Escherichia coli} with an IC\textsubscript{50} value of 0.8 μg/mL. A high-performance liquid chromatography method was developed and validated for the detection and quantification of \emph{p}-coumaroyltyramine (10) in the extracts of different varieties of \emph{C. sativa} roots.
213. Amaducci, Stefano and Zatta, Alessandro and Raffanini, Marco and Venturi, Gianpietro, Characterisation of Hemp (Cannabis Sativa L.) Roots under Different Growing Conditions, Plant and Soil, vol. 313, no. 1, pp. 227, July 2008. doi: 10.1007/s11104-008-9695-0. Abstract
     Hemp (Cannabis sativa L.) is mainly grown for its fibre and is considered a desirable crop for sustainable production systems. In a field trial carried out over two years in Northern Italy the root system of a hemp crop, cultivated at contrasting plant densities, was sampled and analysed with an image analysis software. Root length density (RLD) was highest in the first 10~cm of soil, almost 5~cm cm−3; it decreased progressively until the depth of 130~cm, a part from a peak at 90–100~cm in response to a perched water table. Roots were found to 130~cm of depth in one year and to 200~cm in the other. Root diameter was finer (190~μm) in the upper soil layer, it increased with depth until 100~cm, and remained constant at 300~μm thereafter. Following the same trend of RLD, root biomass was highest in the first soil layer; 50\% of the root biomass was found in the first 20~cm or 50~cm when taproot biomass was considered or not. Total root biomass was 3.21~t ha−1 and 2.41~t ha−1 in the two years of trial, but the ratio between aboveground and below ground biomass was constant at 5.46. None of the root parameters were significantly affected by plant population, which seems to confirm the plastic behaviour that hemp shows for aboveground development. The high root biomass production measured in this study, especially in deeper soil layers, provides additional evidence of the positive role that hemp can play in sustainable cropping systems.
214. Guo, Tian-Tian and Zhang, Jian-Chun and Zhang, Hai and Liu, Qing-Chao and Zhao, Yong and Hou, Yu-Fei and Bai, Lu and Zhang, Li and Liu, Xue-Qiang and Liu, Xue-Ying and Zhang, Sheng-Yong and Bai, Nai-Sheng, Bioactive Spirans and Other Constituents from the Leaves of Cannabis Sativa f. Sativa, Journal of Asian Natural Products Research, vol. 19, no. 8, pp. 793--802, August 2017. doi: 10.1080/10286020.2016.1248947. Abstract
     In this paper, 17 compounds (1–17) were isolated from the leaves of Hemp (Cannabis sativa f. sativa). Among the isolates, two were determined to be new spirans: cannabispirketal (1), and α-cannabispiranol 4’-O-β-D-glucopyranose (2) by 1D and 2D NMR spectroscopy, LC-MS, and HRESIMS. The known compounds 7, 8, 10, 13, 15, and 16 were isolated from Hemp (C. sativa f. sativa) for the first time. Furthermore, compounds 8 and 13 were isolated from the nature for the first time. All isolated compounds were evaluated for cytotoxicity on different tissue-derived passage cancer cell lines through cell viability and apoptosis assay. Among these compounds, compounds 5, 9 and 16 exhibited a broad-spectrum antitumor effect via inhibiting cell proliferation and promoting apoptosis. These results obtained have provided valuable clues to the understanding of the cytotoxic profile for these isolated compounds from Hemp (C. sativa f. sativa).
215. Yang, Rui and Berthold, Erin C. and McCurdy, Christopher R. and {da Silva Benevenute}, Sarah and Brym, Zachary T. and Freeman, Joshua H., Development of Cannabinoids in Flowers of Industrial Hemp (Cannabis Sativa L.): A Pilot Study, Journal of Agricultural and Food Chemistry, vol. 68, no. 22, pp. 6058--6064, June 2020. doi: 10.1021/acs.jafc.0c01211. Abstract
     A field study was performed to investigate the development of cannabinoids in flowers of industrial hemp using three day-length-sensitive and two day-length-neutral varieties. Flower samples were analyzed for cannabinoids on a weekly basis from 2 to 4 weeks postanthesis to plant senescence. Results indicate that total THC, CBD, and CBG significantly increased as flowers matured, reaching the greatest concentration during 6 to 7 weeks postanthesis. After a plateau stage of varied length for different varieties, the peak concentrations declined as plants senesced. Total THC was above the 0.3\% threshold from 4 weeks postanthesis to the end of the growing season for day-length-sensitive varieties, but this only occurred during 6 to 7 weeks postanthesis for day-length-neutral varieties. The CBD/THC ratio in flowers dynamically changed during the entire reproductive stage for all of the evaluated varieties. The current study provides vital information for successful cultivation of industrial hemp.
216. Ingallina, Cinzia and Sobolev, Anatoly P. and Circi, Simone and Spano, Mattia and Fraschetti, Caterina and Filippi, Antonello and Di Sotto, Antonella and Di Giacomo, Silvia and Mazzoccanti, Giulia and Gasparrini, Francesco and Quaglio, Deborah and Campiglia, Enio and Carradori, Simone and Locatelli, Marcello and Vinci, Giuliana and Rapa, Mattia and Ciano, Salvatore and Giusti, Anna Maria and Botta, Bruno and Ghirga, Francesca and Capitani, Donatella and Mannina, Luisa, Cannabis Sativa L. Inflorescences from Monoecious Cultivars Grown in Central Italy: An Untargeted Chemical Characterization from Early Flowering to Ripening, Molecules, vol. 25, no. 8, pp. 1908, January 2020. doi: 10.3390/molecules25081908. Abstract
     The chemical composition of the inflorescences from four Cannabis sativa L. monoecious cultivars (Ferimon, Uso-31, Felina 32 and Fedora 17), recently introduced in the Lazio Region, was monitored over the season from June to September giving indications on their sensorial, pharmaceutical/nutraceutical proprieties. Both untargeted (NMR) and targeted (GC/MS, UHPLC, HPLC-PDA/FD and spectrophotometry) analyses were carried out to identify and quantify compounds of different classes (sugars, organic acids, amino acids, cannabinoids, terpenoids, phenols, tannins, flavonoids and biogenic amines). All cultivars in each harvesting period showed a THC content below the Italian legal limit, although in general THC content increased over the season. Citric acid, malic acid and glucose showed the highest content in the late flowering period, whereas the content of proline drastically decreased after June in all cultivars. Neophytadiene, nerolidol and chlorogenic acid were quantified only in Felina 32 cultivar, characterized also by a very high content of flavonoids, whereas alloaromadendrene and trans-cinnamic acid were detected only in Uso-31 cultivar. Naringenin and naringin were present only in Fedora 17 and Ferimon cultivars, respectively. Moreover, Ferimon had the highest concentration of biogenic amines, especially in July and August. Cadaverine was present in all cultivars but only in September. These results suggest that the chemical composition of Cannabis sativa L. inflorescences depends on the cultivar and on the harvesting period. Producers can use this information as a guide to obtain inflorescences with peculiar chemical characteristics according to the specific use.
217. {Spitzer-Rimon}, Ben and Duchin, Shai and Bernstein, Nirit and Kamenetsky, Rina, Architecture and Florogenesis in Female Cannabis Sativa Plants, Frontiers in Plant Science, vol. 10, 2019. doi: 10.3389/fpls.2019.00350. Abstract
     The inflorescence is the main product of medical cannabis. Hundreds of specialized metabolites with potential bioactivity are produced and accumulated in the glandular trichomes that are highly abundant mainly on female inflorescences. Understanding the morphophysiological and genetic mechanisms governing flower and inflorescence development is therefore of high scientific and practical importance. However, in-depth investigations of cannabis florogenesis are limited. Cannabis producers and researchers consider long photoperiod to be “non-inductive” or “vegetative,” but under these growth conditions, the development of solitary flowers and bracts in shoot internodes clearly indicates that the plant cannot be defined as vegetative or non-inductive in the classical sense. Most probably, induction of solitary flowers is age-dependent and controlled by internal signals, but not by photoperiod. Short photoperiod induces intense branching, which results in the development of a compound raceme. Each inflorescence consists of condensed branchlets with the same phytomer structure as that of the larger phytomers developed under long day. Each phytomer consists of reduced leaves, bracts, one or two solitary flowers, and an axillary shoot (or inflorescence). Therefore, the effect of short photoperiod on cannabis florogenesis is not flower induction, but rather a dramatic change in shoot apex architecture to form a compound racemose inflorescence structure. An understanding of the morphophysiological characteristics of cannabis inflorescence will lay the foundation for biotechnological and physiological applications to modify architecture and to maximize plant productivity and uniformity in medical Cannabis.
218. Furr, Marion and Mahlberg, Paul G., Histochemical Analyses of Laticifers and Glandular Trichomes in Cannabis Sativa, ACS Publications, July 2004. doi: 10.1021/np50014a002.
219. Small, Ernest and Antle, Tanya, A Preliminary Study of Pollen Dispersal in Cannabis Sativa in Relation to Wind Direction, Journal of Industrial Hemp, vol. 8, no. 2, pp. 37--50, March 2003. doi: 10.1300/J237v08n02_03. Abstract
     Pollen of Cannabis sativa is disseminated by wind in large amounts and for long distances, and regulations concerning the production of pedigreed seed of industrial hemp, therefore, often call for extremely large isolation distances to prevent unwanted pollination. In Europe and Canada, a standard distance of 5 km is required for the highest classes of hemp seed. This study examines the relative distribution of pollen from an isolated field over the 3-week maximum flowering period, with particular reference to wind direction. The amount of pollen distributed downwind was about six times the amount distributed upwind. In effect, this means that an isolation distance of 5 km on the downwind side is about equivalent to an isolation distance of 0.9 km on the upwind side. In theory, at the experimental site examined, the required isolation area could be reduced by about 58\% while still achieving the equivalent of 5 km isolation in all directions. Given that weedy and illegally cultivated plants are widespread, making it difficult to ensure their absence over a distance of 5 km, it seems advisable, when possible, to take advantage of the considerably reduced isolation distance that is necessary on the upwind side. Pollen distribution appeared to follow the expected leptokurtic curve, reducing rapidly with initial distance from the source, but much more slowly with increasing distance. This makes it impossible to guarantee complete absence of potentially contaminating pollen in the field and, for practical purposes, a very low amount of undesired gene flow needs to be tolerated.
220. Hammond, Charles T. and Mahlberg, Paul G., Morphogenesis of Capitate Glandular Hairs of Cannabis Sativa (Cannabaceae), American Journal of Botany, vol. 64, no. 8, pp. 1023--1031, 1977. doi: 10.1002/j.1537-2197.1977.tb11948.x. Abstract
     The glandular secretory system in Cannabis sativa L. (marihuana) consists of three types of capitate glandular hairs (termed bulbous, capitate-sessile, and capitate-stalked) distinguishable by their morphology, development, and physiology. These gland types occur together in greatest abundance and developmental complexity on the abaxial surface of bracts which ensheath the developing ovary. Bulbous and capitate-sessile glands are initiated on very young bract primordia and attain maturity during early stages of bract growth. Capitate-stalked glands are initiated later in bract growth and undergo development and maturation on medium, to full sized bracts. Glands are epidermal in origin and derived, with one exception, from a single epidermal initial. The capitate-stalked gland is the exception and is of special interest because it possesses a multicellular stalk secondarily derived from surrounding epidermal and subepidermal cells. Glands differentiate early in development into an upper secretory portion and a subtending auxiliary portion. The secretory portion, depending on gland type, may range from a few cells to a large, flattened multicellular disc of secretory cells. The secretory portion produces a membrane-bound resinous product which caps the secretory cells. Capitate-stalked glands are considered to be of particular evolutionary significance because they may represent a gland type secondarily derived from existing capitate-sessile glands.
221. Dayanandan, P. and Kaufman, Peter B., Trichomes of Cannabis Sativa L. (Cannabaceae), American Journal of Botany, vol. 63, no. 5, pp. 578--591, 1976. doi: 10.1002/j.1537-2197.1976.tb11846.x. Abstract
     The diversity of non-glandular and glandular hairs of Cannabis sativa L. (marihuana) are described by scanning electron microscopy. The non-glandular hairs are of two major types, as distinguished by size differences and locations, and all of them are highly silicified. The presence of silica as well as cystoliths of calcium carbonate help in the identification of marihuana even in its ash residues. X-ray microanalyses of Cannabis hairs are compared with those of Humulus lupulus and Lantana camera, whose hairs have been considered to resemble those of marihuana. Glandular hairs are found to be of two major categories. One group consists of glands whose heads are generally made up of eight cells and the other group whose heads are generally made up of two cells but never more than four cells. All glands of both categories are stalked. Some glands of the first category are massively stalked and these are restricted solely to anthers and bracts of staminate and pistillate plants. The massive stalk is considered to be made up of epidermal and hypodermal cells that have grown in response to some stimulation during anthesis. Fine details of the shoot system of Cannabis, such as cuticular ridges on epidermal cells, warty protuberances on non-glandular hairs, and surface views of glands in developing stages are also reported. Glandular hairs on the bracts of Humulus lupulus resemble those of Cannabis.
222. Paris, M. and Boucher, F. and Cosson, L., The Constituents ofCannabis Sativa Pollen, Economic Botany, vol. 29, no. 3, pp. 245--253, July 1975. doi: 10.1007/BF02873173. Abstract
     The pollen ofCannabis sativa L. was rich in cannabinoids and particularly in THC and THCA, the latter being able to be transformed into physiologically active THC. Climatic factors and particularly temperature played an important role, since the THC content at 24° C 16 h was 30 times as great as at 22° C 12° C 16h.
223. Hammond, Charles T. and Mahlberg, Paul G., Morphology of Glandular Hairs of Cannabis Sativa from Scanning Electron Microscopy, American Journal of Botany, vol. 60, no. 6, pp. 524--528, 1973. doi: 10.1002/j.1537-2197.1973.tb05953.x. Abstract
     Three distinct types of glandular hairs of increasing morphological complexity which occur on flowering tops of Cannabis sativa L. (marihuana) are described from scanning electron microscopy. These gland types—termed bulbous, capitate-sessile, and capitate-stalked, described from pistillate plants—occur in greatest abundance on the outer surface of bracts ensheathing the ovary. Bulbous and capitate-sessile glands, which arise at an early stage in bract development, are scattered over the bract surface. Mature bulbous glands have a small swollen head on a short stalk, whereas capitate-sessile glands have a large globular head attached directly to the bract surface. Because of their numbers and large size, capitate-sessile glands are the most conspicuous gland type during the early phase of bract development. Capitate-stalked glands, which have a large globular head on a tall, multicellular stalk, differentiate during subsequent bract development. These stalked glands arise first along the bracteal veins and then over the entire bract surface. A voluminous, fluid secretory product accumulates in the glandular head of all three types. These glands are believed to be a primary site of localization of the marihuana hallucinogen, tetrahydrocannabinol.
224. Turner, Jocelyn C. and Hemphill, John K. and Mahlberg, Paul G., Gland Distribution and Cannabinoid Content in Clones of Cannabis Sativa L., American Journal of Botany, vol. 64, no. 6, pp. 687--693, 1977. doi: 10.1002/j.1537-2197.1977.tb11910.x. Abstract
     The relationship between glandular trichomes and cannabinoid content in Cannabis sativa L. was investigated. Three strains of Cannabis, which are annuals, were selected for either a drug, a non-drug, or a fiber trait and then cloned to provide genetically uniform material for analyses over several years. The distribution of the number and type of glands was determined for several organs of different ages including the bract and its subtending monoleaflet leaf and the compound leaf on pistillate plants. Quantitation of glands on these structures was integrated with gas chromatographic analyses of organ cannabinoid profiles. A negative correlation was found between cannabinoid content and gland number for each of the three clones. Isolated heads of the capitate-stalked glands also were analyzed for cannabinoid content and found to vary in relation to clone and gland age. These studies indicate that cannabinoids may occur in plant cells other than glandular trichomes. The results of these studies emphasize the need for stringent sampling procedures in micromorphological studies on trichome distribution and analytical determinations of cannabinoid content in Cannabis.
225. Sirikantaramas, Supaart and Taura, Futoshi and Tanaka, Yumi and Ishikawa, Yu and Morimoto, Satoshi and Shoyama, Yukihiro, Tetrahydrocannabinolic Acid Synthase, the Enzyme Controlling Marijuana Psychoactivity, Is Secreted into the Storage Cavity of the Glandular Trichomes, Plant and Cell Physiology, vol. 46, no. 9, pp. 1578--1582, September 2005. doi: 10.1093/pcp/pci166.
226. Mahlberg, Paul G. and Hammond, Charles T. and Turner, Jocelyn C. and Hemphill, John K., Structure, Development and Composition of Glandular Trichomes of Cannabis Sativa L., pp. 23--51, 1984. doi: 10.1007/978-1-4899-5355-1_2. Abstract
     The gl.anduz.a.,. secretol"IJ system of Cannabis sativa L. is com­ posed of bul.bous, capitate-sessil.e, and capitate-stal.ked gl.ands r.,hich are distinguishabl.e f"f'Olff each othe.,. by mo1"\{)hogenesis and physiology. Bul.bous and eapitate-sessile fo1ffls occu.,. on vegetative and flom.1, a:r:es r.,herea.s the highl.y evol.ved capitate­ stal.ked fo1ffl is pNsent onl.y on flom.l.-Nl.ated o.,,,ans. In studies of cloned pl.ants, gtand initiation occuf"Nd on teaves and pistitl.ate bm.cts thmughout o.,,,an ontongeny. G'Land density and time of appeamnoe va-ned betr.,een both cl.ones and o.,,,ans, indi­ cating that contmt of devetor,nent is independent fo.,. BaCh t'l"i­ ch.ome type. Cann.a.binoid synthesis atso occu1'1"8d thmughout o.,,,an ontongeny but r.,ith a decNasing mte in teaves as compared to an inczoeasing mte in bm.cts. In individual. gtands, cann.a.binoid content deareased dunng ma.tum.ti.on. capitate-statked glands contained. highe.,. cann.a.binoid tevel.s than the sessile form al.though the gtands maintained the pmfite cham.ctenstic of the clone. Analyses of glands and tissues indicated cann.a.binoids may occur in cetts other than gtands. capitate glands develop a disc of sec1'8tol"IJ eel.ts, and secretions accumulate in a cavity beneath a sheath denved f"f'Offl sepamtion of the cuticutansed oute.,. 1,)(11,1, su1"face of the disc eel.ts. Presumed sec1'8tions, including cannabinoids, occur at the su1"face of ptastids and appea.,. to migmte to the cel.l. su1"face adjoining the sec1'8t01"1J cavity. "lbtenats appear to be compa.rtmentatiaed into sphe1'6s of vanabl.e Bise in the cavity. Celt fm.ctionation studies are in pmg1'8ss to define the cannabinoid sythesiaing activities r.,ithin the dynamic gtandul.ar system of Cannabis.\vphantom\}
227. Kim, Eun-Soo and Mahlberg, Paul G., Secretory Cavity Development in Glandular Trichomes of Cannabis Sativa L. (Cannabaceae), American Journal of Botany, vol. 78, no. 2, pp. 220--229, 1991. doi: 10.1002/j.1537-2197.1991.tb15749.x. Abstract
     Development of the secretory cavity and formation of the subcuticular wall of glandular trichomes in Cannabis sativa L. was examined by transmission electron microscopy. The secretory cavity originated at the wall-cuticle interface in the peripheral wall of the discoid secretory cells. During the presecretory phase in development of the glandular trichome, the peripheral wall of the disc cells became laminated into a dense inner zone adjacent to the plasma membrane and a less dense outer zone subjacent to the cuticle. Loosening of wall matrix in the outer zone initiated a secretory cavity among fibrous wall materials. Membrane-bound hyaline areas, compressed in shape, arose in the wall matrix. They appeared first in the outer and subsequently in the inner zone of the wall. The membrane of the vesicles, and associated dense particles attached to the membrane, arose from the wall matrix. Hyaline areas, often with a conspicuous electron-dense content, were released into the secretory cavity where they formed rounded secretory vesicles. Fibrous wall material released from the surface of the disc cells became distributed throughout the secretory cavity among the numerous secretory vesicles. This wall material was incorporated into the developing subcuticular wall that increased five-fold in thickness during enlargement of the secretory cavity. The presence of a subcuticular wall in the cavity of Cannabis trichomes, as contrasted to the absence of this wall in described trichomes of other plants, supports a polyphyletic interpretation of the evolution of the secretory cavity in glandular trichomes among angiosperms.
228. Ledbetter, M. C. and Krikorian, A. D., Trichomes of Cannabis Sativa as Viewed with Scanning Electron Microscope, Phytomorphology; (India), vol. 25:2, June 1975. url: https://www.osti.gov/etdeweb/biblio/7256539. Abstract
     Direct examination of fresh, unfixed and uncoated specimens from vegetative and floral parts of Cannabis sativa with the scanning electron microscope enables one to obtain a faithful representation of their surface morphology. The presence of two major types of trichomes has been confirmed: a glandular type comprising or terminating in a globoid structure, and a conically-shaped nonglandular type. Moreover, three or possibly four distinct glandular types can be distinguished: sessile globoid, small-stalked and large-stalked globoid, and a peltate type. The nonglandular trichomes can be distinguished by the nature of their surfaces: those with a warty surface, and those which are relatively smooth. The range of size and distribution, and the special features of all these types of trichomes are also provided.
229. Kim, Eun Soo and Mahlberg, Paul G., Secretory Vesicle Formation in the Secretory Cavity of Glandular Trichomes of Cannabis Sativa L. (Cannabaceae), Molecules and Cells, vol. 15, no. 3, pp. 387--395, June 2003. url: https://www.molcells.org/journal/view.html?spage=387&volume=15&number=3. Abstract
     Eun Soo Kim, Paul G. Mahlberg. Mol. Cells 2003;15:387-95. https://doi.org/
230. André, Cl and Vercruysse, A., Histochemical Study of the Stalked Glandular Hairs of the Female Cannabis Plants, Using Fast Blue Salt, Planta Medica, vol. 29, no. 4, pp. 361--366, June 1976. doi: 10.1055/s-0028-1097677. Abstract
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231. Galasso, Incoronata and Russo, Roberto and Mapelli, Sergio and Ponzoni, Elena and Brambilla, Ida M. and Battelli, Giovanna and Reggiani, Remo, Variability in Seed Traits in a Collection of Cannabis Sativa L. Genotypes, Frontiers in Plant Science, vol. 7, 2016. doi: 10.3389/fpls.2016.00688. Abstract
     The seed of Cannabis sativa L. is an expanding source of proteins and oil for both humans and animals. In this study, the proximate composition of a collection of hemp cultivars and accessions of different geographical origins grown under the same conditions for 1 year was analyzed in order to identify potential accessions to improve hemp cultivars. Fatty acids, tocopherols, and antinutritional components, as well as concentrations of crude protein and oil were quantified. The seed oil concentrations varied between 285 and 360 g kg−1 dry seed (DS), while crude protein ranged between 316 and 356 g kg−1 dry matter (DM). The seed oil was mainly composed of unsaturated fatty acids and, as expected, the dominant fatty acids were linoleic and α-linolenic acid. A high variability among the cultivars and accessions was also detected for polyphenolic content which ranged from 5.88 to 10.63 g kg−1 DM, cv. Felina was the richest, whereas cv. Finola had the lowest polyphenolic content. Regarding antinutritional compounds in seed, a high variability was detected among all genotypes analyzed and phytic acid was particularly abundant (ranging between 43 and 75 g kg−1 DM). In conclusion, our results reveal noticeable differences among hemp seed genotypes for antinutritional components, oil and protein content. Collectively, this study suggests that the hemp seed is an interesting product in terms of protein, oil and antioxidant molecules but a reduction of phytic acid would be desirable for both humans and monogastric animals. The high variability detected among the different genotypes indicates that an improvement of hemp seed might be possible by conventional and/or molecular breeding.
232. Aiello, Gilda and Fasoli, Elisa and Boschin, Giovanna and Lammi, Carmen and Zanoni, Chiara and Citterio, Attilio and Arnoldi, Anna, Proteomic Characterization of Hempseed (Cannabis Sativa L.), Journal of Proteomics, vol. 147, pp. 187--196, September 2016. doi: 10.1016/j.jprot.2016.05.033. Abstract
     This paper presents an investigation on hempseed proteome. The experimental approach, based on combinatorial peptide ligand libraries (CPLLs), SDS-PAGE separation, nLC-ESI-MS/MS identification, and database search, permitted identifying in total 181 expressed proteins. This very large number of identifications was achieved by searching in two databases: Cannabis sativa L. (56 gene products identified) and Arabidopsis thaliana (125 gene products identified). By performing a protein–protein association network analysis using the STRING software, it was possible to build the first interactomic map of all detected proteins, characterized by 137 nodes and 410 interactions. Finally, a Gene Ontology analysis of the identified species permitted to classify their molecular functions: the great majority is involved in the seed metabolic processes (41\%), responses to stimulus (8\%), and biological process (7\%). Biological significance Hempseed is an underexploited non-legume protein-rich seed. Although its protein is well known for its digestibility, essential amino acid composition, and useful techno-functional properties, a comprehensive proteome characterization is still lacking. The objective of this work was to fill this knowledge gap and provide information useful for a better exploitation of this seed in different food products.
233. Smith, Christiana J. and Vergara, Daniela and Keegan, Brian and Jikomes, Nick, The Phytochemical Diversity of Commercial Cannabis in the United States, PLOS ONE, vol. 17, no. 5, pp. e0267498, May 2022. doi: 10.1371/journal.pone.0267498. Abstract
     The legal status of Cannabis is changing, fueling an increasing diversity of Cannabis-derived products. Because Cannabis contains dozens of chemical compounds with potential psychoactive or medicinal effects, understanding this phytochemical diversity is crucial. The legal Cannabis industry heavily markets products to consumers based on widely used labeling systems purported to predict the effects of different “strains.” We analyzed the cannabinoid and terpene content of commercial Cannabis samples across six US states, finding distinct chemical phenotypes (chemotypes) which are reliably present. By comparing the observed phytochemical diversity to the commercial labels commonly attached to Cannabis-derived product samples, we show that commercial labels do not consistently align with the observed chemical diversity. However, certain labels do show a biased association with specific chemotypes. These results have implications for the classification of commercial Cannabis, design of animal and human research, and regulation of consumer marketing—areas which today are often divorced from the chemical reality of the Cannabis-derived material they wish to represent.
234. Xu, Jiaxin and Bai, Miao and Song, Hong and Yang, Lina and Zhu, Danshi and Liu, He, Hemp (Cannabis Sativa Subsp. Sativa) Chemical Composition and the Application of Hempseeds in Food Formulations, Plant Foods for Human Nutrition, vol. 77, no. 4, pp. 504--513, December 2022. doi: 10.1007/s11130-022-01013-x. Abstract
     Owing to its nutritional and medicinal value, hemp has been cultivated to provide since ancient times. This review aims to map the scientific literature concerning the main functional components and the chemical composition of hemp plant. It is generally acknowledged that each organ of the hemp plant embodies a valuable source, and among them the most pivotal part is the edible fruits hempseeds. Hempseeds are rich in easily digestible proteins, fats, polyunsaturated fatty acids, and insoluble fiber, which are of high nutritional value. Furthermore, the beneficial effects have increased researchers’ interests in hempseeds-containing foods. Developed as an indispensable ingredient, hempseed is also a significant supplement in various products, such as bakery food, drinks, snacks and culinary products. Overall, this review intends to promote the further in-depth investigation of approved hemp plants and expand the range of hempseeds adoption in the functional foods field.
235. Wieczorek, Jarosław and Kaczor, Marcin and Boryło, Alicja, Determination of 210Po and 210Pb in Cannabis (Cannabis Sativa L.) Plants and Products, Journal of Environmental Radioactivity, vol. 246, pp. 106834, May 2022. doi: 10.1016/j.jenvrad.2022.106834. Abstract
     The concentrations of polonium 210Po and radio-lead 210Pb in cannabis (Cannabis sativa L.) plants and products now legally available in Poland were determined. Limiting the delivery of radionuclides to the body is an important aspect of civil protection in many countries. Reduction in use and awareness of the risks associated with tobacco and cannabis smoking have a great impact. The 210Po and 210Pb concentrations in 44 hemps, 20 hashish and 8 hemp tea samples, as well as in 3 types of cannabis plants (highest parts of mature hemp plant Fenola, Fedora and Futura) were determined. Each of the sample names means a different type and cross of C. sativa L. Being numerous, the are recognized on the market precisely by these names. Effective doses were calculated and compared to the doses of the other combustion products, such as tobacco. In the case of hemp, the highest concentration of 210Po was found in samples of dried Sweet Carmel (34.7~±~0.23 mBq·g−1), while the lowest in the Hemp Berry (0.57~±~0.23 mBq·g−1). In the case of 210Pb, the highest concentration was in Strawberry Kush (2.32~±~0.05 mBq·g−1), while the lowest in Strawberry Haze (0.19~±~0.03 mBq·g−1). In hashish, the highest and lowest concentrations of 210Po were in Strawberry Diesel 164~±~3 mBq·g−1 and in Mango Kush 2.5~±~0.2 mBq·g−1. The highest and lowest concentrations in the case of 210Pb in hashish were in Pollen Hashish 45.1~±~0.2 mBq·g−1 and in Mango Kush Hashish 0.45~±~0.05 mBq·g−1, respectively. These radionuclides did not constitute a radioactive equilibrium (210Po/210Pb).
236. {Alonso-Esteban}, José Ignacio and Pinela, José and Ćirić, Ana and Calhelha, Ricardo C. and Soković, Marina and Ferreira, Isabel C. F. R. and Barros, Lillian and {Torija-Isasa}, Esperanza and {Sánchez-Mata}, María de Cortes, Chemical Composition and Biological Activities of Whole and Dehulled Hemp (Cannabis Sativa L.) Seeds, Food Chemistry, vol. 374, pp. 131754, April 2022. doi: 10.1016/j.foodchem.2021.131754. Abstract
     This study aimed to determine a complete chemical composition of eight different varieties of whole hemp seeds and eight samples of commercial dehulled hemp seeds. We also evaluated the phenolic profiles and antioxidant, cytotoxic, and antimicrobial properties of hydromethanolic seed extracts. Whole hemp seeds contain much more fibre than dehulled hemp seeds, which contain more fat and protein. Sucrose and raffinose were the most abundant soluble sugars, and citric and oxalic acids were the most abundant organic acids. In the hydromethanolic hemp seed extracts, we detected the phenolic acids ferulic acid-hexoside and syringic acid. Whole hemp seed extracts exhibited better antioxidant activity than dehulled hemp seed extracts, especially in the TBARS assay. Cytotoxic activity against NCI-H460 cells was also observed. The dehulled hemp seed extracts displayed antibacterial activity, especially against Bacillus cereus, Listeria monocytogenes, and Enterococcus faecalis, and antifungal activity to a lesser extent.
237. Liu, Yi and Liu, Hong-Yan and Li, Sheng-Hong and Ma, Wei and Wu, Ding-Tao and Li, Hua-Bin and Xiao, Ai-Ping and Liu, Liang-Liang and Zhu, Fan and Gan, Ren-You, Cannabis Sativa Bioactive Compounds and Their Extraction, Separation, Purification, and Identification Technologies: An Updated Review, TrAC Trends in Analytical Chemistry, vol. 149, pp. 116554, April 2022. doi: 10.1016/j.trac.2022.116554. Abstract
     Cannabis sativa (C.~sativa) has become a worldwide plant because of its multiple bioactive compounds with a wide range of health benefits for humans. Notably, some cannabinoid-like compounds have also been found in other plant species besides C.~sativa. However, efficient extraction, separation, purification, and identification technologies for bioactive compounds in C.~sativa are still great challenges. This review provides an updated overview of the main bioactive compounds in C.~sativa and the cannabinoid-like compounds in other plants. In addition, various advanced technologies for extracting, separating, purifying, and identifying the bioactive compounds in C.~sativa are mainly summarized, compared, and discussed. Overall, this review can provide scientific basis and technical support for the efficient extraction, separation, purification, and identification of C.~sativa bioactive compounds, which have the potential to be widely applied in the food, medical, nutraceutical, and beauty products.
238. Boddington, Kelly F. and Soubeyrand, Eric and Van Gelder, Kristen and Casaretto, José A. and Perrin, Colby and Forrester, Taylor J.B. and Parry, Cameron and {Al-Abdul-Wahid}, M. Sameer and Jentsch, Nicholas G. and Magolan, Jakob and Bozzo, Gale G. and Kimber, Matthew S. and Rothstein, Steven J. and Akhtar, Tariq A., Bibenzyl Synthesis in Cannabis Sativa L., The Plant Journal, vol. 109, no. 3, pp. 693--707, 2022. doi: 10.1111/tpj.15588. Abstract
     This study focuses on the biosynthesis of a suite of specialized metabolites from Cannabis that are known as the ‘bibenzyls’. In planta, bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti-inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis, in a three-step sequence. First, various hydroxycinnamic acids are esterified to acyl-coenzyme A (CoA) by a member of the 4-coumarate-CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double-bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro-CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl-CoA with these dihydro-hydroxycinnamoyl-CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this ‘bibenzyl synthase’ enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non-canonical and more flexible dihydro-hydroxycinnamoyl-CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro. Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing ‘parts prospecting’ inventory that supports the rational metabolic engineering of natural product therapeutics.
239. Liu, Yuanyuan and Zhu, Panpan and Cai, Sen and Haughn, George and Page, Jonathan E., Three Novel Transcription Factors Involved in Cannabinoid Biosynthesis in Cannabis Sativa L., Plant Molecular Biology, vol. 106, no. 1, pp. 49--65, May 2021. doi: 10.1007/s11103-021-01129-9. Abstract
     Three novel transcription factors were successfully identified and shown to interact with the trichome-specific THCAS promoter regulatory region.
240. Brighenti, Virginia and Marchetti, Lucia and Anceschi, Lisa and Protti, Michele and Verri, Patrizia and Pollastro, Federica and Mercolini, Laura and Bertelli, Davide and Zanardi, Chiara and Pellati, Federica, Separation and Non-Separation Methods for the Analysis of Cannabinoids in Cannabis Sativa L., Journal of Pharmaceutical and Biomedical Analysis, vol. 206, pp. 114346, November 2021. doi: 10.1016/j.jpba.2021.114346. Abstract
     Cannabis sativa L. is a plant known all over the world, due to its history, bioactivity and also social impact. It is chemically complex with an astonishing ability in the biosynthesis of many secondary metabolites belonging to different chemical classes. Among them, cannabinoids are the most investigated ones, given their pharmacological relevance. In order to monitor the composition of the plant material and ensure the efficacy and safety of its derived products, extraction and analysis of cannabinoids play a crucial role. In this context, in addition to a conventional separation method based on HPLC with UV/DAD detection, a new strategy based on a non-separation procedure, such as 13C-qNMR, may offer several advantages, such as reduced solvent consumption and simultaneous acquisition of the quali/quantitative data related to many analytes. In the light of all the above, the aim of this work is to compare the efficiency of the above-mentioned analytical techniques for the study of the main cannabinoids in different samples of cannabis inflorescences, belonging to fibre-type, recreational and medical varieties. The 13C-qNMR method here proposed for the first time for the quantification of both psychoactive and non-psychoactive cannabinoids in different cannabis varieties provided reliable results in comparison to the more common and consolidated HPLC technique.
241. Deguchi, Michihito and Potlakayala, Shobha and Spuhler, Zachary and George, Hannah and Sheri, Vijay and Agili, Ruba and Patel, Aayushi and Rudrabhatla, Sairam, Selection and Validation of Reference Genes for Normalization of qRT-PCR Data to Study the Cannabinoid Pathway Genes in Industrial Hemp, PLOS ONE, vol. 16, no. 12, pp. e0260660, December 2021. doi: 10.1371/journal.pone.0260660. Abstract
     There has been significant interest in researching the pharmaceutical applications of Industrial hemp since its legalization three years ago. The crop is mostly dioecious and known for its production of phytocannabinoids, flavonoids, and terpenes. Although many scientific reports have showed gene expression analysis of hemp through OMICs approaches, unreliable reference genes for normalization of qRT-PCR data make it difficult to validate the OMICs data. Four software packages: geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stresses. EF-1α ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal stress and hormonal stimuli. The expression patterns of two cannabinoid pathway genes, AAE1 and CBDAS, were used to validate the reliability of the selected reference genes. This work provides useful information for gene expression characterization in hemp and future research in the synthesis, transport, and accumulation of secondary metabolites.
242. Parvez, Ashak Mahmud and Lewis, Jonathan David and Afzal, Muhammad T., Potential of Industrial Hemp (Cannabis Sativa L.) for Bioenergy Production in Canada: Status, Challenges and Outlook, Renewable and Sustainable Energy Reviews, vol. 141, pp. 110784, May 2021. doi: 10.1016/j.rser.2021.110784. Abstract
     Climate change from carbon emissions and rising energy demands poses a serious threat to global sustainability. This issue is particularly noticeable in Canada where per capita energy demands are high and fossil fuels are used. Industrial hemp can be used for bioenergy production as an alternative to fossil fuels to capture and utilize carbon, with applications in various markets at high values. Despite this, industrial hemp has faced legal barriers that have hampered its viability. This review describes industrial hemp, its status in global markets, its performance as bioenergy feedstock, and potential in Canada, so research can target gaps in available knowledge. Numerous bioenergy applications for industrial hemp exist; the production of bioethanol and biodiesel from industrial hemp has strong potential to reduce greenhouse gas emissions and improve the Canadian economy. The current study found that industrial hemp can compete with many energy crops in global markets as a feedstock for many bioenergy products with solid hemp yielding 100~GJ/ha/y, allowing for economical emissions reductions for example in coal/biochar blends that can reduce emissions by 10\%, and in co-production of bioethanol and grain, generating \$2632/ha/y. This work also suggests industrial hemp has unique potential for growth in Canada, though processing facilities are severely lacking, and hemp growing has some negative environmental impacts related to fertilizer use. Responsible growth could be realized through incentivizing or subsidizing processing facility investment, implementing co-production where possible, and funding research to improve conversion, harvesting and polygeneration processes.
243. Muscarà, Claudia and Smeriglio, Antonella and Trombetta, Domenico and Mandalari, Giuseppina and La Camera, Erminia and Grassi, Gianpaolo and Circosta, Clara, Phytochemical Characterization and Biological Properties of Two Standardized Extracts from a Non-Psychotropic Cannabis Sativa L. Cannabidiol (CBD)-Chemotype, Phytotherapy Research, vol. 35, no. 9, pp. 5269--5281, 2021. doi: 10.1002/ptr.7201. Abstract
     The aim of study was to evaluate and compare the phytochemical profile, the antioxidant and antimicrobial properties of two standardized extracts from non-psychotropic (Δ9-tetrahydrocannabinol ≤0.2\%) Cannabis sativa L. var. fibrante rich in cannabidiol (CBD). The two extracts, namely Cannabis Fibrante Hexane Extract 1 (CFHE1) and Cannabis Fibrante Hexane Extract 2 (CFHE2), were obtained by extraction with acidified hexane from dried flowering tops as such and after hydrodistillation of the essential oil, respectively. Gas chromatographic analysis showed that cannabinoids remained the predominant class of compounds in both extracts (82.56\% and 86.38\%, respectively), whereas a marked depletion of the terpenes occurred. Moreover, liquid chromatographic analysis highlighted a high titer of cannabidiol acid (CBDA) and CBD in CFHE1 and CFHE2, respectively. Both extracts showed a strong and concentration-dependent antioxidant activity and a potent antimicrobial activity against both Staphylococcus aureus ATCC 6538 (MIC and MBC of 4.88 μg/ml for CFHE1, and 4.88 and 19.53 μg/ml, respectively, for CFHE2) and methicillin resistant clinical strains (MIC values between 1.22 and 9.77 μg/ml and MBC values between 4.88 and 78.13 μg/ml). Considering this, the obtained results suggest that standardized extracts of C. sativa var. fibrante could find promising applications as novel antimicrobial agents.
244. Tremlová, Bohuslava and Mikulášková, Hana Koudelková and Hajduchová, Klaudia and Jancikova, Simona and Kaczorová, Dominika and Ćavar Zeljković, Sanja and Dordevic, Dani, Influence of Technological Maturity on the Secondary Metabolites of Hemp Concentrate (Cannabis Sativa L.), Foods, vol. 10, no. 6, pp. 1418, June 2021. doi: 10.3390/foods10061418. Abstract
     During the last decade, the popularity of hemp products has been rising rapidly. Products containing cannabidiol (CBD) are of predominant interest. Traditional hemp products are frequently enriched by CBD due to their potential therapeutic effects. Cannabidiol occurs naturally in hemp juice together with other biologically active substances, such as terpenes, flavonoids, and stilbenoids. These constituents act synergistically. This study aimed to observe the influence of the hemp plant developmental stage on its chemical composition and antioxidant activity. The hemp plants were analyzed during three vegetative stages, i.e., before, during, and after flowering. The collected samples were evaluated using the following analyses: total polyphenolic content and profile, terpenoid and cannabinoid contents, and ferric reducing antioxidant power. The results revealed statistically significant differences between the samples in almost all set parameters. The optimal period for hemp harvest depends on desirable compounds, i.e., phenolic content is the highest before flowering, while the levels of cannabinoids and terpenoids are the highest during the flowering period.
245. Sun, Xin and Sun, Yao and Li, Yao and Wu, Qiong and Wang, Lei, Identification and Characterization of the Seed Storage Proteins and Related Genes of Cannabis Sativa L., Frontiers in Nutrition, vol. 8, 2021. doi: 10.3389/fnut.2021.678421. Abstract
     Hemp (Cannabis sativa L.) seed is emerging as a novel source of plant protein owing to its rich protein content and reasonable nutritional structure. In the current study, the storage proteins of hemp seed were extracted using different methods. The modified Osborne method yielded maximum extraction of the hemp seed storage proteins, while degreasing had little effect on the hemp seed protein (HSP) extraction. Protein identification results revealed that 11S globulin (edestin) was the most abundant protein in hemp seed, and the molecular weights of the two subunits of this protein were \textasciitilde 35 and 20 kDa, respectively. The second most abundant protein was 2S albumin (Cs2S), with a molecular weight of \textasciitilde 14–15 kDa. The least abundant protein was 7S vicilin-like protein (Cs7S), with a molecular weight of \textasciitilde 47 kDa. Subsequently, gene families encoding these three storage protein classes, including three genes for edestin, two for Cs2S, and one for Cs7S, were cloned and then analyzed for amino acid composition and structure. The three edestins were different in their amino acid sequences and calculated molecular weights. The analysis of coding sequences revealed a higher percentage of similarity (62.7\%) between Edestin1 and Edestin3, while the similarity decreased significantly to \textasciitilde 57\% between Edestin1 and Edestin2, and 58\% between Edestin2 and Edestin3. The calculated protein molecular weight was the highest for the protein encoded by Edestin1 and the smallest for the protein encoded by Edestin2. All three edestins were rich in arginine, while Edestin3 had a higher methionine content relative to that in the other two, which proved that Edestin3 had a better nutritional value. Cs2S and Cs7S were different from those reported in previous studies. Therefore, it could be inferred that amino acid composition varies with different hemp cultivars. The current research brought significant theoretical advance in illuminating the understanding of hemp seed storage protein and would have significance for future research on improving the nutritional quality of hemp seed and developing bioactive peptides.
246. Bautista, Johanna L. and Yu, Shu and Tian, Li, Flavonoids in Cannabis Sativa: Biosynthesis, Bioactivities, and Biotechnology, ACS Omega, vol. 6, no. 8, pp. 5119--5123, March 2021. doi: 10.1021/acsomega.1c00318. Abstract
     Although Cannabis sativa synthesizes a wide range of phytochemicals, much attention has been primarily given to two phytocannabinoids, Δ9-tetrahydocannabinol (THC) and cannabidiol (CBD), due to their distinctive activities in humans. These bioactivities can be further enhanced through the interaction of THC and CBD with other phytocannabinoids or non-phytocannabinoid chemicals, such as terpenes and flavonoids, a phenomenon that is termed the entourage effect. Flavonoid metabolism in C. sativa and the entourage effect are currently understudied. This mini-review examines recent advances in the biosynthesis and bioactivities of cannflavins, which are prenylated (C5) and geranylated (C10) flavones that are relatively unique to C. sativa. We also discuss the rapidly developing omics tools that enable discoveries in flavonoid metabolism in C. sativa and manipulation of flavonoid production through biotechnology. These advances set the stage for interrogating the health benefits of C. sativa flavonoids, deciphering the contribution of flavonoids to the entourage effect, and developing drugs.
247. Pieracci, Ylenia and Ascrizzi, Roberta and Terreni, Valentina and Pistelli, Luisa and Flamini, Guido and Bassolino, Laura and Fulvio, Flavia and Montanari, Massimo and Paris, Roberta, Essential Oil of Cannabis Sativa L: Comparison of Yield and Chemical Composition of 11 Hemp Genotypes, Molecules, vol. 26, no. 13, pp. 4080, January 2021. doi: 10.3390/molecules26134080. Abstract
     Cannabis sativa L. is an annual species cultivated since antiquity for different purposes. While, in the past, hemp inflorescences were considered crop residues, at present, they are regarded as valuable raw materials with different applications, among which extraction of the essential oil (EO) has gained increasing interest in many fields. The aim of the present study is the evaluation of the yield and the chemical composition of the EO obtained by hydrodistillation from eleven hemp genotypes, cultivated in the same location for two consecutive growing seasons. The composition of the EOs was analyzed by GC–MS, and then subjected to multivariate statistical analysis. Sesquiterpenes represented the main class of compounds in all the EOs, both in their hydrocarbon and oxygenated forms, with relative abundances ranging from 47.1 to 78.5\%; the only exception was the Felina 32 sample collected in 2019, in which cannabinoids predominated. Cannabinoids were the second most abundant class of compounds, of which cannabidiol was the main one, with relative abundances between 11.8 and 51.5\%. The statistical distribution of the samples, performed on the complete chemical composition of the EOs, evidenced a partition based on the year of cultivation, rather than on the genotype, with the exception of Uso-31. Regarding the extraction yield, a significant variation was evidenced among both the genotypes and the years of cultivation.
248. Cirrincione, Marco and Saladini, Bruno and Brighenti, Virginia and Salamone, Stefano and Mandrioli, Roberto and Pollastro, Federica and Pellati, Federica and Protti, Michele and Mercolini, Laura, Discriminating Different Cannabis Sativa L. Chemotypes Using Attenuated Total Reflectance - Infrared (ATR-FTIR) Spectroscopy: A Proof of Concept, Journal of Pharmaceutical and Biomedical Analysis, vol. 204, pp. 114270, September 2021. doi: 10.1016/j.jpba.2021.114270. Abstract
     An original, innovative, high-throughput method based on attenuated total reflectance - Fourier’s transform infrared (ATR-FTIR) spectroscopy has been developed for the proof-of-concept discrimination of fibre-type from drug-type Cannabis sativa L. inflorescences. The cannabis sample is placed on the instrument plate and analysed without any previous sample pretreatment step. In this way, a complete analysis lasts just a few seconds, the time needed to record an ATR-FTIR spectrum. The method was calibrated and cross-validated using data provided by liquid chromatography - tandem mass spectrometry (LC–MS/MS) analysis of the different cannabis samples and carried out the statistical assays for quantitation. During cross-validation, complete agreement was obtained between ATR-FTIR and LC–MS/MS identification of the cannabis chemotype. Moreover, the method has proved to be capable of quantifying with excellent accuracy (75–103 \% vs. LC–MS/MS) seven neutral and acidic cannabinoids (THC, THCA, CBD, CBDA, CBG, CBGA, CBN) in inflorescences from different sources. The extreme feasibility and speed of execution make this ATR-FTIR method highly attractive as a proof-of-concept for a possible application to quality controls during pharmaceutical product manufacturing, as well as on-the-street cannabis controls and user counselling.
249. Vastolo, Alessandro and Calabrò, Serena and Pacifico, Severina and Koura, Bossima Ivan and Cutrignelli, Monica Isabella, Chemical and Nutritional Characteristics of Cannabis Sativa L. Co-Products, Journal of Animal Physiology and Animal Nutrition, vol. 105, no. S1, pp. 1--9, 2021. doi: 10.1111/jpn.13557. Abstract
     Cannabis sativa L. is an annual herbaceous plant. It was used for centuries to obtain different products. In the last century, hemp cultivation was forbidden due to the psychoactive effects of tetrahydrocannabinol acid (THCA). In the last years, new strains, characterized by high cannabidiolic acid (CBDA) and low THCA level, were developed renewing the interest in hemp cultivation to obtain food or to extract essential oils from flowers. All these processes produce many residues with different chemical–physical characteristics. In order to evaluate their potential use also in animal nutrition, some hemp co-products were evaluated. Two different co-products of seed processes (flour and oil) and two co-products obtained trimming the flowers, differing in granulometry were used. The samples were analysed for chemical composition and evaluated in vitro using the gas production technique with buffaloes' ruminal inoculum. All hemp co-products showed interesting nutritional characteristics, such as crude protein content always higher than 20\% on a dry matter basis, and high neutral detergent fibre concentration partially lignified. The in vitro gas production parameters at 120 h of incubation showed quite low fermentability testified by the low organic matter degradability and cumulative gas volume (OMD from 28.09 to 45.64\% and OMCV from 110 to 164 ml/g, respectively). Also, the methane produced after 24 h of incubation was particularly low (from 1.78 to 11.73 ml/g dOM). These results could be due to the high lipid and ash amounts or to the CBDA content that probably affected the CH4 formation processes. According to preliminary results obtained by this study, it is possible to hypothesize that these co-products could be useful to mitigate the methane production into the rumen. Further studies are necessary in order to evaluate the correct inclusion into the diet for ruminants.
250. Micalizzi, Giuseppe and Vento, Federica and Alibrando, Filippo and Donnarumma, Danilo and Dugo, Paola and Mondello, Luigi, Cannabis Sativa L.: A Comprehensive Review on the Analytical Methodologies for Cannabinoids and Terpenes Characterization, Journal of Chromatography A, vol. 1637, pp. 461864, January 2021. doi: 10.1016/j.chroma.2020.461864. Abstract
     The global Cannabis Sativa market, including essential oils, foods, personal-care products, and medical formulations has gained much attention over the last years due to the favorable regulatory framework. Undoubtedly, the enormous interest about cannabis cultivation mainly derives from the well-known pharmacological properties of cannabinoids and terpenes biosynthesized by the plants. In this review, the most recently used analytical methodologies for detecting both cannabinoids and terpenes are described. Well-established and innovative extraction protocols, and chromatographic separations, such as GC and HPLC, are reviewed highlighting their respective advantages and drawbacks. Lastly, GC~×~GC techniques are also reported for accurate identification and quantification of terpenes in complex cannabis matrices.
251. Cattaneo, Chiara and Givonetti, Annalisa and Leoni, Valeria and Guerrieri, Nicoletta and Manfredi, Marcello and Giorgi, Annamaria and Cavaletto, Maria, Biochemical Aspects of Seeds from Cannabis Sativa L. Plants Grown in a Mountain Environment, Scientific Reports, vol. 11, no. 1, pp. 3927, February 2021. doi: 10.1038/s41598-021-83290-1. Abstract
     Cannabis sativa L. (hemp) is a versatile plant which can adapt to various environmental conditions. Hempseeds provide high quality lipids, mainly represented by polyunsaturated acids, and highly digestible proteins rich of essential aminoacids. Hempseed composition can vary according to plant genotype, but other factors such as agronomic and climatic conditions can affect the presence of nutraceutic compounds. In this research, seeds from two cultivars of C. sativa (Futura 75 and Finola) grown in a mountain environment of the Italian Alps were analyzed. The main purpose of this study was to investigate changes in the protein profile of seeds obtained from such environments, using two methods (sequential and total proteins) for protein extraction and two analytical approaches SDS-PAGE and 2D-gel electrophoresis, followed by protein identification by mass spectrometry. The fatty acids profile and carotenoids content were also analysed. Mountain environments mainly affected fatty acid and protein profiles of Finola seeds. These changes were not predictable by the sole comparison of certified seeds from Futura 75 and Finola cultivars. The fatty acid profile confirmed a high PUFA content in both cultivars from mountain area, while protein analysis revealed a decrease in the protein content of Finola seeds from the experimental fields.
252. Bueno, Justin and Greenbaum, Eric A., (−)-Trans-Δ9-Tetrahydrocannabiphorol Content of Cannabis Sativa Inflorescence from Various Chemotypes, Journal of Natural Products, vol. 84, no. 2, pp. 531--536, February 2021. doi: 10.1021/acs.jnatprod.0c01034. Abstract
     The (−)-trans-Δ9-tetrahydrocannabiphorol (Δ9-THCP, 1) content of the inflorescence from six Cannabis sativa chemotypes, including 14 plants of distinct genotypes, and two extracts was determined quantitatively via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This represents the first comprehensive quantitative screening for 1 from various C. sativa chemotypes. Compound 1 was detected in all 13 inflorescence samples originating from “(−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC, 2) dominant” C. sativa chemotypes, but was not detected in a “cannabidiol (CBD, 3) dominant” chemotype. The inflorescence content of 1 ranged approximately from 0.0023\% to 0.0136\% (w/w). Comprehensive inflorescence sampling was performed for each specimen investigated. A trend between inflorescence cannabinoid potency and the location of which the inflorescence was sampled on the C. sativa plant was observed for the three cannabinoids tested (1–3). The preliminary results obtained indicate Δ9-THCP (1) may have a higher degree of prevalence in C. sativa inflorescence than previously estimated.
253. {Delgado-Povedano}, M. M. and {Sánchez-Carnerero Callado}, C. and {Priego-Capote}, F. and {Ferreiro-Vera}, C., Untargeted Characterization of Extracts from Cannabis Sativa L. Cultivars by Gas and Liquid Chromatography Coupled to Mass Spectrometry in High Resolution Mode, Talanta, vol. 208, pp. 120384, February 2020. doi: 10.1016/j.talanta.2019.120384. Abstract
     Elucidation of Cannabis composition is required to evaluate the potential of this plant for pharmacological uses, but also for implementation in breeding programs with agronomical purposes. The aim of the present study was to develop a method for untargeted analysis of polar and non-polar Cannabis extracts. For this purpose, extracts from 17 cultivars of Cannabis sativa L. were analyzed by gas chromatography–time-of-flight/mass spectrometry (GC–TOF/MS) and liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC–QTOF MS/MS) in high resolution mode. One hundred sixty-nine compounds were identified in the extracts by searching MS and MS/MS information. Among identified families, there were mainly cannabinoids, terpenoids, lipids and flavonoids, but also some interesting compounds such as amino and organic acids, among others. Relative contents of terpenoids and cannabinoids in the same cultivars grown in greenhouse and field were compared. Compositional differences in the profile of terpenoids and cannabinoids between both types of grown conditions were found.
254. Sorokin, Aleksei and Yadav, Narendra Singh and Gaudet, Daniel and Kovalchuk, Igor, Transient Expression of the β-Glucuronidase Gene in Cannabis Sativa Varieties, Plant Signaling \& Behavior, vol. 15, no. 8, pp. 1780037, August 2020. doi: 10.1080/15592324.2020.1780037. Abstract
     In plant biology, transient expression analysis plays a vital role to provide a fast method to study the gene of interest. In this study, we report a rapid and efficient method for transient expression in Cannabis sativa seedlings using Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 carrying the pCAMBIA1301 construct with uidA gene was used to transform cannabis seedlings and the GUS assay (a measurement of β-glucuronidase activity) was used to detect the uidA expression. In the current study, we have also established a rapid germination protocol for cannabis seeds. The all three steps seed sterilization, germination and seedlings development were carried out in a 1\% H2O2 solution. Transient transformation revealed that both cotyledons and young true leaves are amenable to transformation. Compared with tobacco (Nicotiana benthamiana), cannabis seedlings were less susceptible to transformation with A. tumefaciens. Susceptibility to Agrobacterium transformation also varied with the different cannabis varieties. The method established in this study has the potential to be an important tool for gene function studies and genetic improvement in cannabis.
255. Baranauskaite, Juste and Marksa, Mindaugas and Ivanauskas, Liudas and Vitkevicius, Konradas and Liaudanskas, Mindaugas and Skyrius, Vaidas and Baranauskas, Algirdas, Development of Extraction Technique and GC/FID Method for the Analysis of Cannabinoids in Cannabis Sativa L. Spp. Santicha (Hemp), Phytochemical Analysis, vol. 31, no. 4, pp. 516--521, 2020. doi: 10.1002/pca.2915. Abstract
     Introduction Nowadays, the interest in industrial Cannabis sativa L. herb has been increasing in the world. As a result, it is becoming one of the most studied plants due to its multifunctional benefits. Objectives To the best of our knowledge, no study has been conducted so far to determine the impact of extraction methods and conditions on the extraction yields of CBD and CBG from the Cannabis sativa L. ssp. Santhica. Therefore, we aimed to investigate a simple and sensitive GC-FID method to determine CBD and CBG in hemp extract. Methods As regards sample preparation, three extraction techniques were compared, including maceration (ME), ultrasound-assisted extraction (UAE) and reflux-heat extraction (RHE), in order to obtain a high recovery of the CBD of interest from the plant material. The GC-FID method developed in this study represents a powerful tool for the extraction and analysis of non-psychoactive cannabinoids from hemp varieties to be used for the preparations of extracts with a high content of bioactive compounds for both pharmaceutical and nutraceutical applications. Results A simple extraction procedure for CBD and CBG from hemp was also optimized in this work, by using ultrasound assisted extraction method with 96\% ethanol, material/solvent ratio 1:10 and extraction time 10 min at room temperature. Conclusion The overall analytical method was fully validated in agreement with international guidelines. Therefore, proving a powerful and reliable tool for both the selection of hemp varieties with a high content of bioactive compounds and the quality control of its derivatives.
256. Glivar, Taja and Eržen, Jan and Kreft, Samo and Zagožen, Marjeta and Čerenak, Andreja and Čeh, Barbara and Tavčar Benković, Eva, Cannabinoid Content in Industrial Hemp (Cannabis Sativa L.) Varieties Grown in Slovenia, Industrial Crops and Products, vol. 145, pp. 112082, March 2020. doi: 10.1016/j.indcrop.2019.112082. Abstract
     Cannabinoid content in different hemp varieties from the Common catalogue of varieties of agricultural plant species is not well known. Hemp (Cannabis sativa L., subsp. sativa) contains a wide range of cannabinoids, where cannabidiol (CBD) and (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) are the constituents with known therapeutic activity. Also, Δ9-THC is recognized as an illicit drug; therefore, cultivation of hemp is restricted to a 0.2 \% limit of THC content in many European countries. In this study, the cannabinoid profiles of 15 hemp varieties, accessible to our research group, were analysed. The content of 13 cannabinoids was determined with HLPC (high-performance liquid chromatography) analysis. Large variations in cannabinoid content among varieties that grew in uniform conditions (ANOVA p\,{$<$}\,0.05) and also within a single variety were found, which shows on un-uniform genetic profiles of the seed material. The varieties Fedora 17, USO 31, Tisza, Tiborszallasi, and Antal all displayed good response to growth conditions, related to cannabinoid content, in Slovenia.
257. Izzo, Luana and Castaldo, Luigi and Narváez, Alfonso and Graziani, Giulia and Gaspari, Anna and {Rodríguez-Carrasco}, Yelko and Ritieni, Alberto, Analysis of Phenolic Compounds in Commercial Cannabis Sativa L. Inflorescences Using UHPLC-Q-Orbitrap HRMS, Molecules, vol. 25, no. 3, pp. 631, January 2020. doi: 10.3390/molecules25030631. Abstract
     Industrial hemp (Cannabis sativa L. Family Cannabaceae) contains a vast number of bioactive relevant compounds, namely polyphenols including flavonoids, phenolic acids, phenol amides, and lignanamides, well known for their therapeutic properties. Nowadays, many polyphenols-containing products made of herbal extracts are marketed, claiming to exert health-promoting effects. In this context, industrial hemp inflorescence may represent an innovative source of bioactive compounds to be used in nutraceutical formulations. The aim of this work was to provide a comprehensive analysis of the polyphenolic fraction contained in polar extracts of four different commercial cultivars (Kompoti, Tiborszallasi, Antal, and Carmagnola Cs) of hemp inflorescences through spectrophotometric (TPC, DPPH tests) and spectrometry measurement (UHPLC-Q-Orbitrap HRMS). Results highlighted a high content of cannflavin A and B in inflorescence analyzed samples, which appear to be cannabis-specific, with a mean value of 61.8 and 84.5 mg/kg, meaning a ten-to-hundred times increase compared to other parts of the plant. Among flavonols, quercetin-3-glucoside reached up to 285.9 mg/kg in the Carmagnola CS cultivar. Catechin and epicatechin were the most representative flavanols, with a mean concentration of 53.3 and 66.2 mg/kg, respectively, for all cultivars. Total polyphenolic content in inflorescence samples was quantified in the range of 10.51 to 52.58 mg GAE/g and free radical-scavenging included in the range from 27.5 to 77.6 mmol trolox/kg. Therefore, C. sativa inflorescence could be considered as a potential novel source of polyphenols intended for nutraceutical formulations.
258. Namdar, Dvora and Voet, Hillary and Ajjampura, Vinayaka and Nadarajan, Stalin and {Mayzlish-Gati}, Einav and Mazuz, Moran and Shalev, Nurit and Koltai, Hinanit, Terpenoids and Phytocannabinoids Co-Produced in Cannabis Sativa Strains Show Specific Interaction for Cell Cytotoxic Activity, Molecules, vol. 24, no. 17, pp. 3031, January 2019. doi: 10.3390/molecules24173031. Abstract
     Mixtures of different Cannabis sativa phytocannabinoids are more active biologically than single phytocannabinoids. However, cannabis terpenoids as potential instigators of phytocannabinoid activity have not yet been explored in detail. Terpenoid groups were statistically co-related to certain cannabis strains rich in Δ9-tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), and their ability to enhance the activity of decarboxylase phytocannabinoids (i.e., THC or CBD) was determined. Analytical HPLC and GC/MS were used to identify and quantify the secondary metabolites in 17 strains of C. sativa, and correlations between cannabinoids and terpenoids in each strain were determined. Column separation was used to separate and collect the compounds, and cell viability assay was used to assess biological activity. We found that in “high THC” or “high CBD” strains, phytocannabinoids are produced alongside certain sets of terpenoids. Only co-related terpenoids enhanced the cytotoxic activity of phytocannabinoids on MDA-MB-231 and HCT-116 cell lines. This was found to be most effective in natural ratios found in extracts of cannabis inflorescence. The correlation in a particular strain between THCA or CBDA and a certain set of terpenoids, and the partial specificity in interaction may have influenced the cultivation of cannabis and may have implications for therapeutic treatments.
259. Booth, Judith K. and Bohlmann, Jörg, Terpenes in Cannabis Sativa – From Plant Genome to Humans, Plant Science, vol. 284, pp. 67--72, July 2019. doi: 10.1016/j.plantsci.2019.03.022. Abstract
     Cannabis sativa (cannabis) produces a resin that is valued for its psychoactive and medicinal properties. Despite being the foundation of a multi-billion dollar global industry, scientific knowledge and research on cannabis is lagging behind compared to other high-value crops. This is largely due to legal restrictions that have prevented many researchers from studying cannabis, its products, and their effects in humans. Cannabis resin contains hundreds of different terpene and cannabinoid metabolites. Many of these metabolites have not been conclusively identified. Our understanding of the genomic and biosynthetic systems of these metabolites in cannabis, and the factors that affect their variability, is rudimentary. As a consequence, there is concern about lack of consistency with regard to the terpene and cannabinoid composition of different cannabis ‘strains’. Likewise, claims of some of the medicinal properties attributed to cannabis metabolites would benefit from thorough scientific validation.
260. Vergara, Daniela and Huscher, Ezra L and Keepers, Kyle G and Givens, Robert M and Cizek, Christian G and Torres, Anthony and Gaudino, Reggie and Kane, Nolan C, Gene Copy Number Is Associated with Phytochemistry in Cannabis Sativa, AoB PLANTS, vol. 11, no. 6, pp. plz074, November 2019. doi: 10.1093/aobpla/plz074. Abstract
     Abstract Gene copy number (CN) variation is known to be important in nearly every species where it has been examined. Alterations in gene CN may provide a fast way of acquiring diversity, allowing rapid adaptation under strong selective pressures, and may also be a key component of standing genetic variation within species. Cannabis sativa plants produce a distinguishing set of secondary metabolites, the cannabinoids, many of which have medicinal utility. Two major cannabinoids—THCA (delta-9-tetrahydrocannabinolic acid) and CBDA (cannabidiolic acid)—are products of a three-step biochemical pathway. Using whole-genome shotgun sequence data for 69 Cannabis cultivars from diverse lineages within the species, we found that genes encoding the synthases in this pathway vary in CN. Transcriptome sequence data show that the cannabinoid paralogs are differentially expressed among lineages within the species. We also found that CN partially explains variation in cannabinoid content levels among Cannabis plants. Our results demonstrate that biosynthetic genes found at multiple points in the pathway could be useful for breeding purposes, and suggest that natural and artificial selection have shaped CN variation. Truncations in specific paralogs are associated with lack of production of particular cannabinoids, showing how phytochemical diversity can evolve through a complex combination of processes.
261. Mandrioli, Mara and Tura, Matilde and Scotti, Stefano and Gallina Toschi, Tullia, Fast Detection of 10 Cannabinoids by RP-HPLC-UV Method in Cannabis Sativa L., Molecules, vol. 24, no. 11, pp. 2113, January 2019. doi: 10.3390/molecules24112113. Abstract
     Cannabis has regained much attention as a result of updated legislation authorizing many different uses and can be classified on the basis of the content of tetrahydrocannabinol (THC), a psychotropic substance for which there are legal limitations in many countries. For this purpose, accurate qualitative and quantitative determination is essential. The relationship between THC and cannabidiol (CBD) is also significant as the latter substance is endowed with many specific and non-psychoactive proprieties. For these reasons, it becomes increasingly important and urgent to utilize fast, easy, validated, and harmonized procedures for determination of cannabinoids. The procedure described herein allows rapid determination of 10 cannabinoids from the inflorescences of Cannabis sativa L. by extraction with organic solvents. Separation and subsequent detection are by RP-HPLC-UV. Quantification is performed by an external standard method through the construction of calibration curves using pure standard chromatographic reference compounds. The main cannabinoids dosed (g/100 g) in actual samples were cannabidiolic acid (CBDA), CBD, and Δ9-THC (Sample L11 CBDA 0.88 ± 0.04, CBD 0.48 ± 0.02, Δ9-THC 0.06 ± 0.00; Sample L5 CBDA 0.93 ± 0.06, CBD 0.45 ± 0.03, Δ9-THC 0.06 ± 0.00). The present validated RP-HPLC-UV method allows determination of the main cannabinoids in Cannabis sativa L. inflorescences and appropriate legal classification as hemp or drug-type.
262. Irakli, Maria and Tsaliki, Eleni and Kalivas, Apostolos and Kleisiaris, Fotios and Sarrou, Eirini and Cook, Catherine M., Effect Οf Genotype and Growing Year on the Nutritional, Phytochemical, and Antioxidant Properties of Industrial Hemp (Cannabis Sativa L.) Seeds, Antioxidants, vol. 8, no. 10, pp. 491, October 2019. doi: 10.3390/antiox8100491. Abstract
     Cannabis sativa L. seeds have been an important source of protein, oil, and dietary fiber for human and animals. Currently, there is a growing interest in the commercial products of these seeds, which are recognized as a legitimate source of medicaments, cosmeceuticals, and nutraceuticals. The objective of this study was to investigate the nutritional, phytochemical composition, and antioxidant properties of seeds from seven hemp cultivars grown in Greece for three consecutive years. All the measured parameters strongly varied under the influence of growing year and genotype. In particular, protein, oil, and carbohydrates’ content of hemp seeds as well as fatty acids’ composition were mainly affected by genotype, whereas the growing year had a major effect on phytochemical components and antioxidant activity, which was determined by the 2,2′-azino-bis (3-ethylbenzthiazoline sulfonate) (ABTS) and ferric-reducing antioxidant power (FRAP) assays. Moreover, a predominant effect of the year was observed for phenolic profiles as determined by high-performance liquid chromatography and total carotenoids’ content. This study suggests that hemp seeds could be a promising food crop as a result of their high nutritive traits and antioxidant potential. A comparison of the studied cultivars, showed that Finola seeds had the highest oil and protein contents and, thus, appeared to be the most promising cultivar for cultivation in Greece.
263. Siano, Francesco and Moccia, Stefania and Picariello, Gianluca and Russo, Gian Luigi and Sorrentino, Giuseppe and Di Stasio, Michele and La Cara, Francesco and Volpe, Maria Grazia, Comparative Study of Chemical, Biochemical Characteristic and ATR-FTIR Analysis of Seeds, Oil and Flour of the Edible Fedora Cultivar Hemp (Cannabis Sativa L.), Molecules, vol. 24, no. 1, pp. 83, January 2019. doi: 10.3390/molecules24010083. Abstract
     A series of chemical and biochemical parameters of edible hemp resources (seeds, oil, and flour) from the monoecious EU registered hemp genotype Fedora, was determined, including fatty acid profile, phytosterol composition, total phenolics, antioxidant activity, macro- and micro-elements. The fatty acid ω-3/ω-6 approached the nutritionally optimal 3/1 ratio. β-sitosterol and other phytosterols sterols dominated the unsaponifiable fraction. Hemp seeds, flour, and oil contained 767 ± 41, 744 ± 29, and 21 ± 5 mg GAE kg−1 total polyphenols, respectively. The antioxidant potential of Fedora flour and seeds, evaluated through the DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay, was higher than that of oil. K and Mg were the most abundant macro-elements, particularly in flour, while the concentration of trace elements was Fe {$>$} Cu {$>$} Ni {$>$} Mn. The presence of an array of bioactive compound candidate Fedora products as health-promoting food matrices. The ATR-FTIR spectra of hemp-derived products indicated the proximate composition of macro-nutrients.
264. Iseppi, Ramona and Brighenti, Virginia and Licata, Manuela and Lambertini, Antonella and Sabia, Carla and Messi, Patrizia and Pellati, Federica and Benvenuti, Stefania, Chemical Characterization and Evaluation of the Antibacterial Activity of Essential Oils from Fibre-Type Cannabis Sativa L. (Hemp), Molecules, vol. 24, no. 12, pp. 2302, January 2019. doi: 10.3390/molecules24122302. Abstract
     Volatile terpenes represent the largest group of Cannabis sativa L. components and they are responsible for its aromatic properties. Even if many studies on C. sativa have been focused on cannabinoids, which are terpenophenolics, little research has been carried out on its volatile terpenic compounds. In the light of all the above, the present work was aimed at the chemical characterization of seventeen essential oils from different fibre-type varieties of C. sativa (industrial hemp or hemp) by means of GC-MS and GC-FID techniques. In total, 71 compounds were identified, and the semi-quantitative analysis revealed that α- and β-pinene, β-myrcene and β-caryophyllene are the major components in all the essential oils analysed. In addition, a GC-MS method was developed here for the first time, and it was applied to quantify cannabinoids in the essential oils. The antibacterial activity of hemp essential oils against some pathogenic and spoilage microorganisms isolated from food and food processing environment was also determined. The inhibitory effects of the essential oils were evaluated by both the agar well diffusion assay and the minimum inhibitory concentration (MIC) evaluation. By using the agar diffusion method and considering the zone of inhibition, it was possible to preliminarily verify the inhibitory activity on most of the examined strains. The results showed a good antibacterial activity of six hemp essential oils against the Gram-positive bacteria, thus suggesting that hemp essential oil can inhibit or reduce bacterial proliferation and it can be a valid support to reduce microorganism contamination, especially in the food processing field.
265. Protti, Michele and Brighenti, Virginia and Battaglia, Maria Rita and Anceschi, Lisa and Pellati, Federica and Mercolini, Laura, Cannabinoids from Cannabis Sativa L.: A New Tool Based on HPLC–DAD–MS/MS for a Rational Use in Medicinal Chemistry, ACS Medicinal Chemistry Letters, vol. 10, no. 4, pp. 539--544, April 2019. doi: 10.1021/acsmedchemlett.8b00571. Abstract
     Cannabis sativa L. represents one of the most widely used source of drugs and drugs of abuse worldwide. Its biologically active compounds are mainly cannabinoids, including Δ9-tetrahydrocannabinol (THC), which is responsible for the psychoactive effects, tetrahydrocannabinolic acid (THCA), cannabinol (CBN), cannabidiol (CBD), and cannabidiolic acid (CBDA). Together with recreational and drug-type (or medicinal) Cannabis, some new products have been recently released into the market as fiber-type Cannabis variants (also known as hemp or industrial hemp) with low THC content and high content of nonpsychoactive CBD. In this research work, the aim was to characterize Cannabis recreational and drug-type samples by quantifying their active principles, after the development and validation of a suitable analytical method. In addition to the Cannabis samples described above, fiber-type plant varieties were also analyzed to monitor their content of nonpsychoactive compounds for both pharmaceutical and nutraceutical purposes. To do this, a highly efficient HPLC–DAD–MS/MS method, with an electrospray ionization (ESI) source and a triple-quadrupole mass analyzer acquiring in the multiple reaction monitoring (MRM) mode also coupled to a diode array detector (DAD), was developed and applied. Satisfactory validation results were obtained in terms of precision (RSD {$<$} 6.0\% for all the analytes) and accuracy ({$>$}92.1\% for all the compounds). The proposed methodology represents a versatile and reliable tool to assess both psychoactive and nonpsychoactive cannabinoid levels in Cannabis samples for a more rational use in both medicinal chemistry and nutraceutics.
266. Rodziewicz, Paweł and Loroch, Stefan and Marczak, Łukasz and Sickmann, Albert and Kayser, Oliver, Cannabinoid Synthases and Osmoprotective Metabolites Accumulate in the Exudates of Cannabis Sativa L. Glandular Trichomes, Plant Science, vol. 284, pp. 108--116, July 2019. doi: 10.1016/j.plantsci.2019.04.008. Abstract
     Cannabinoids are terpenophenolic compounds produced by Cannabis sativa L., which accumulate in storage cavities of glandular trichomes as a part of the exudates. We investigated if tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase, which are involved in the last step of cannabinoid biosynthesis, are also secreted into Cannabis trichome exudates. The exudates were collected by microsuction from storage cavities of Cannabis glandular trichomes and were subjected for proteomic and metabolomic analyses. The catalytic activity of the exudates was documented by cannabigerolic acid biotransformation studies under hydrophobic conditions. Electrophoretic separations revealed protein bands at ˜65\,kDa, which were further identified as tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. The accumulation of the enzymes in trichome exudates increased substantially during the flowering period in the drug-type Cannabis plants. The content of cannabinoids increased significantly after incubating hexane-diluted trichome exudates with cannabigerolic acid. In this study, we showed that Cannabis glandular trichomes secrete and accumulate cannabinoid synthases in storage cavities, and the enzymes able to convert cannabigerolic acid under hydrophobic trichome-mimicking conditions. Metabolite profiling of the exudates revealed compounds with hydrophilic, osmoprotective and amphiphilic properties, which may play a role in providing a necessary aqueous microenvironment, which enables enzyme solubility and biocatalysis under hydrophobic conditions of glandular trichomes.
267. Kraszkiewicz, Artur and Kachel, Magdalena and Parafiniuk, Stanisław and Zając, Grzegorz and Niedziółka, Ignacy and Sprawka, Maciej, Assessment of the Possibility of Using Hemp Biomass (Cannabis Sativa L.) for Energy Purposes: A Case Study, Applied Sciences, vol. 9, no. 20, pp. 4437, January 2019. doi: 10.3390/app9204437. Abstract
     During testing, the possibility of using hemp biomass for energy purposes was assessed. The criteria assessed were the physical and chemical properties of hemp biomass, as well as the combustion process of straw and briquettes made of it in a low-power boiler. The results were made and compared with currently applicable standards. Technical and chemical properties of hemp biomass are comparable with the best plants used for energy purposes. Studies have also shown the susceptibility of hemp biomass compaction. However, large emissions recorded during the combustion of the tested forms of biofuels from hemp straw in light of applicable standards disqualify this fuel for use in grate-type heating devices with air fed under the grate. It would be advisable to carry out research on the total costs of pellet production and their use in heating devices with a retort burner, while taking into account this biofuel’s ashes’ susceptibility of sintering.
268. Namdar, Dvory and Mazuz, Moran and Ion, Aurel and Koltai, Hinanit, Variation in the Compositions of Cannabinoid and Terpenoids in Cannabis Sativa Derived from Inflorescence Position along the Stem and Extraction Methods, Industrial Crops and Products, vol. 113, pp. 376--382, March 2018. doi: 10.1016/j.indcrop.2018.01.060. Abstract
     In the last decade, recognition of the therapeutic abilities of Cannabis sativa has risen, along with the need to standardize its products. Standardization requires grading the methods for growing the plant and extracting the active compounds accumulated in its inflorescence. We explored the results of different methods used today and their effect on the levels of compounds extracted from inflorescences positioned along the C. sativa flowering stem. The polarity of the solvent used for the extraction, drying processes and separation methods influenced the chemical composition of the extract. However, regardless of extraction and analytical methods applied, the amounts of cannabinoids and terpenoids in the inflorescences decreased with the position of the sampled inflorescence from top to bottom of the flowering stem. These results have significant implications for the development of growth protocols for C. sativa cultivation and flower extraction methods to standardize cannabis-based products.
269. {Sánchez-Carnerero Callado}, C. and {Núñez-Sánchez}, N. and Casano, S. and {Ferreiro-Vera}, C., The Potential of near Infrared Spectroscopy to Estimate the Content of Cannabinoids in Cannabis Sativa L.: A Comparative Study, Talanta, vol. 190, pp. 147--157, December 2018. doi: 10.1016/j.talanta.2018.07.085. Abstract
     Cannabis has been one of the oldest source of food, textile fiber and psychotropic substances. Cannabinoids are the main biologically active constituents of the Cannabis genus, with a demonstrated medicinal value. Its production is becoming legalized and regulated in many countries, thus increasing the need for a rapid analysis method to assess the content of cannabinoids. Gas chromatography (GC) is the preferred analytical method for the determination of these compounds, although is a slow and costly technique. Near infrared spectroscopy (NIR) has the potential for the quantitative prediction of quality parameters, and also of pharmacologically active compounds, but no references about cannabinoids prediction has been previously reported. The aim of the present research was to develop a fast, economical, robust and environmentally friendly method based on NIR technology that allow the quantification of the main cannabinoids present in Cannabis sativa L. samples. A total of 189 grinded and dried samples from different genotypes and registered varieties were used. The content of the cannabinoids CBDV, Δ9-THCV, CBD, CBC, Δ8-THC, Δ9-THC, CBG and CBN were determined by gas chromatography. Spectra were collected in a dispersive NIR Systems 6500 instrument, and in a Fourier transform near Infrared (FT-NIR) equipment. The sample group was divided into calibration and validation sets, to develop modified partial lest squares (PLS) regression models with WINISI IV software with the dispersive data, and PLS models using OPUS 7.2 with the FT-NIR ones. Excellent coefficient of determination of cross validation (R2CV from 0.91 to 0.99), were obtained for the prediction of CBD, CBC, Δ8-THC, Δ9-THC, CBG and CBN, with standard error of prediction (SEP) values among 1.5–3 times the standard error of laboratory (SEL); and good for CBDV and Δ9-THCV cannabinoids (R2 values of 0.89 and 0.83, respectively) with the dispersive instrument. Similar calibration and validation statistics have been obtained with the FT-NIR instrument with the same sample sets, using its specific OPUS software. In conclusion, a methodology of quantitative determination of cannabinoids in Cannabis raw materials has been developed for the first time using NIR and FT-NIR instruments, with similar good predictive results. This new analytical method would allow a simpler, more robust and precise estimation than the current standard GC.
270. Pellati, Federica and Brighenti, Virginia and Sperlea, Johanna and Marchetti, Lucia and Bertelli, Davide and Benvenuti, Stefania, New Methods for the Comprehensive Analysis of Bioactive Compounds in Cannabis Sativa L. (Hemp), Molecules, vol. 23, no. 10, pp. 2639, October 2018. doi: 10.3390/molecules23102639. Abstract
     Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The main phytochemicals that are found in this plant are represented by cannabinoids, flavones, and terpenes. Some biological activities of cannabinoids are known to be enhanced by the presence of terpenes and flavonoids in the extracts, due to a synergistic action. In the light of all the above, the present study was aimed at the multi-component analysis of the bioactive compounds present in fibre-type C. sativa (hemp) inflorescences of different varieties by means of innovative HPLC and GC methods. In particular, the profiling of non-psychoactive cannabinoids was carried out by means of HPLC-UV/DAD, ESI-MS, and MS2. The content of prenylated flavones in hemp extracts, including cannflavins A and B, was also evaluated by HPLC. The study on Cannabis volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. Cannabidiolic acid (CBDA) and cannabidiol (CBD) were found to be the most abundant cannabinoids in the hemp samples analysed, while β-myrcene and β-caryophyllene were the major terpenes. As regards flavonoids, cannflavin A was observed to be the main compound in almost all the samples. The methods developed in this work are suitable for the comprehensive chemical analysis of both hemp plant material and related pharmaceutical or nutraceutical products in order to ensure their quality, efficacy, and safety.
271. Fekete, Szabolcs and {Sadat-Noorbakhsh}, Vida and Schelling, Cedric and Molnár, Imre and Guillarme, Davy and Rudaz, Serge and Veuthey, Jean-Luc, Implementation of a Generic Liquid Chromatographic Method Development Workflow: Application to the Analysis of Phytocannabinoids and Cannabis Sativa Extracts, Journal of Pharmaceutical and Biomedical Analysis, vol. 155, pp. 116--124, June 2018. doi: 10.1016/j.jpba.2018.03.059. Abstract
     A generic liquid chromatographic method development workflow was developed and successfully applied to the analysis of phytocannabinoids and Cannabis sativa extracts. Our method development procedure consists in four steps: i)The screening of primary parameters (i.e. stationary phase nature, organic modifier nature and approximate mobile phase pH) was carried out with a generic gradient on a short narrow bore column, using a system able to accommodate numerous solvents/buffers and columns. Instead of complete peak tracking, the number of peaks which can be separated was considered as a response at this level, to save time.ii)The optimization of secondary parameters (i.e. gradient conditions, mobile phase temperature and pH within a narrow range) requires only 12 initial experiments and the use of HPLC modeling software for data treatment. It allows to find out the best retention and selectivity for the selected compounds. Peak tracking was performed with a single quadruple mass detector in single ion recording mode, and UV detection (in a broad wavelength range).iii)The refinement step allows to further adjust column efficiency, by tuning column length and mobile phase flow rate. This can also be done virtually using HPLC modeling software.iv)The robustness testing step was also evaluated from a virtual experimental design. Success rate and regression coefficients were estimated in about 1\,min, without the need to perform any real experiment. At the end, this method development workflow was performed in less than 4\,days and minimizes the costs of the method development in liquid chromatography.
272. Ponzoni, E. and Brambilla, I. M. and Galasso, I., Genome-Wide Identification and Organization of Seed Storage Protein Genes of Cannabis Sativa, Biologia Plantarum, vol. 62, no. 4, pp. 693--702, December 2018. doi: 10.1007/s10535-018-0810-7. Abstract
     Hemp (Cannabis sativa L.) seeds have been recognized as a nutritional protein source for humans and animals. In this study, gene families encoding precursor polypeptides of three storage protein classes, including six 11S edestin, two 2S albumin and one 7S vicilin-like genes were identified and characterized from an inbred line of hemp. All edestins showed typical 11S globulin features but based on the amino acid composition, they were grouped in three edestin types (type1, -2 and -3). Genes encoding edestin type1 and -3 were very close to each other in a DNA fragment of 16 071 bp, whereas the two isoforms of edestin type2 were linked on a different DNA fragment of 8 232 bp and arranged in a tailto- tail fashion. All edestin types were very rich in arginine and glutamic acid, but edestin type3 was the richest in cysteine and methionine. Regarding the 2S albumin (Cs2S) two genes were identified in a fragment of 13 738 bp in a tail-to-head array. Finally, only one 7S-vicilin like gene (Cs7S) that exhibited typical 7S vicilin features, such as the presence of two cupin domains and several N-glycosylation sites, was isolated. Southern blot hybridization is in agreement with the number of genes isolated, and real-time qPCR analysis revealed that all genes are expressed in the seed. The highest expression was observed for edestin type1 (CsEde1) and Cs2S, whereas the lowest expression was detected for Cs7S. The results of this study provide a complete overview of the genes encoding hemp storage proteins and significantly advance our knowledge on the organization of these gene families.
273. Zirpel, Bastian and Kayser, Oliver and Stehle, Felix, Elucidation of Structure-Function Relationship of THCA and CBDA Synthase from Cannabis Sativa L., Journal of Biotechnology, vol. 284, pp. 17--26, October 2018. doi: 10.1016/j.jbiotec.2018.07.031. Abstract
     Cannabinoids are secondary natural products from the plant Cannabis sativa L. Therapeutic indications of cannabinoids currently comprise a significant area of medicinal research. We have expressed the Δ9-tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) recombinantly in Komagataella phaffii and could detect eight different products with a cannabinoid scaffold after conversion of the precursor cannabigerolic acid (CBGA). Besides five products remaining to be identified, both enzymes were forming three major cannabinoids of C. sativa - Δ9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA). In pursuit of improved enzyme properties for a biotechnological cannabinoid production, we performed site-directed mutagenesis to investigate the glycosylation pattern, the C-terminal berberine-bridge-enzyme (BBE) domain, the active site and the product specificity of both enzymes. The THCAS variant T\_N89Q+N499Q (lacking two glycosylation sites) exerted about two-fold increased activity compared to wild-type enzyme. Variant T\_H494C+R532C (additional disulfide bridge) exerted about 1.7-fold increased activity compared to wild-type enzyme and a shifted temperature optimum from 52\,°C to 57\,°C. We generated two CBDAS variants, C\_S116A and C\_A414V, with 2.8 and 3.3-fold increased catalytic activities for CBDA production. C\_A414V additionally showed a broadened pH spectrum and a 19-fold increased catalytic activity for THCA production. These studies lay the groundwork for further research as well as biotechnological cannabinoid production.
274. Zivovinovic, Sanja and Alder, Ruth and Allenspach, Martina D. and Steuer, Christian, Determination of Cannabinoids in Cannabis Sativa L. Samples for Recreational, Medical, and Forensic Purposes by Reversed-Phase Liquid Chromatography-Ultraviolet Detection, Journal of Analytical Science and Technology, vol. 9, no. 1, pp. 27, November 2018. doi: 10.1186/s40543-018-0159-8. Abstract
     Currently, an increasing demand of cannabis-derived products for recreational and medical use is observed. Therefore, the reliable and fast quantification of cannabinoids in hemp samples is essential for the control of product from Cannabis sativa, L. strains. In general, gas chromatography (GC) is the method of choice for the quantification of cannabinoids whereas this method is time consuming and the detection of acidic precursor is not feasible without derivatization.
275. Ibrahim, Elsayed A. and Gul, Waseem and Gul, Shahbaz W. and Stamper, Brandon J. and Hadad, Ghada M. and Salam, Randa A. Abdel and Ibrahim, Amany K. and Ahmed, Safwat A. and Chandra, Suman and Lata, Hemant and Radwan, Mohamed M. and ElSohly, Mahmoud A., Determination of Acid and Neutral Cannabinoids in Extracts of Different Strains of Cannabis Sativa Using GC-FID, Planta Medica, vol. 84, no. 4, pp. 250--259, March 2018. doi: 10.1055/s-0043-124088. Abstract
     Cannabis (Cannabis sativa L.) is an annual herbaceous plant that belongs to the family Cannabaceae. Trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the two major phytocannabinoids accounting for over 40\% of the cannabis plant extracts, depending on the variety. At the University of Mississippi, different strains of C. sativa, with different concentration ratios of CBD and Δ9-THC, have been tissue cultured via micropropagation and cultivated. A GC-FID method has been developed and validated for the qualitative and quantitative analysis of acid and neutral cannabinoids in C. sativa extracts. The method involves trimethyl silyl derivatization of the extracts. These cannabinoids include tetrahydrocannabivarian, CBD, cannabichromene, trans-Δ8-tetrahydrocannabinol, Δ9-THC, cannabigerol, cannabinol, cannabidiolic acid, cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid-A. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area ratio with R2 {$>$} 0.999 for all 10 cannabinoids. The precision and accuracy of the method were found to be ≤ 15\% and ± 5\%, respectively. The limit of detection range was 0.11 – 0.19 µg/mL, and the limit of quantitation was 0.34 – 0.56 µg/mL for all 10 cannabinoids. The developed method is simple, sensitive, reproducible, and suitable for the detection and quantitation of acidic and neutral cannabinoids in different extracts of cannabis varieties. The method was applied to the analysis of these cannabinoids in different parts of the micropropagated cannabis plants (buds, leaves, roots, and stems).
276. Booth, Judith K. and Page, Jonathan E. and Bohlmann, Jörg, Terpene Synthases from Cannabis Sativa, PLOS ONE, vol. 12, no. 3, pp. e0173911, March 2017. doi: 10.1371/journal.pone.0173911. Abstract
     Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety ‘Finola’ revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of ‘Finola’ resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.
277. ElSohly, Mahmoud A. and Radwan, Mohamed M. and Gul, Waseem and Chandra, Suman and Galal, Ahmed, Phytochemistry of Cannabis Sativa L., pp. 1--36, 2017. doi: 10.1007/978-3-319-45541-9_1. Abstract
     Cannabis (Cannabis sativa, or hemp) and its constituents—in particular the cannabinoids—have been the focus of extensive chemical and biological research for almost half a century since the discovery of the chemical structure of its major active constituent, Δ9-tetrahydrocannabinol (Δ9-THC). The plant’s behavioral and psychotropic effects are attributed to its content of this class of compounds, the cannabinoids, primarily Δ9-THC, which is produced mainly in the leaves and flower buds of the plant. Besides Δ9-THC, there are also non-psychoactive cannabinoids with several medicinal functions, such as cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG), along with other non-cannabinoid constituents belonging to diverse classes of natural products. Today, more than 560 constituents have been identified in cannabis. The recent discoveries of the medicinal properties of cannabis and the cannabinoids in addition to their potential applications in the treatment of a number of serious illnesses, such as glaucoma, depression, neuralgia, multiple sclerosis, Alzheimer’s, and alleviation of symptoms of HIV/AIDS and cancer, have given momentum to the quest for further understanding the chemistry, biology, and medicinal properties of this plant.
278. Brighenti, Virginia and Pellati, Federica and Steinbach, Marleen and Maran, Davide and Benvenuti, Stefania, Development of a New Extraction Technique and HPLC Method for the Analysis of Non-Psychoactive Cannabinoids in Fibre-Type Cannabis Sativa L. (Hemp), Journal of Pharmaceutical and Biomedical Analysis, vol. 143, pp. 228--236, September 2017. doi: 10.1016/j.jpba.2017.05.049. Abstract
     The present work was aimed at the development and validation of a new, efficient and reliable technique for the analysis of the main non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) inflorescences belonging to different varieties. This study was designed to identify samples with a high content of bioactive compounds, with a view to underscoring the importance of quality control in derived products as well. Different extraction methods, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and supercritical-fluid extraction (SFE) were applied and compared in order to obtain a high yield of the target analytes from hemp. Dynamic maceration for 45min with ethanol (EtOH) at room temperature proved to be the most suitable technique for the extraction of cannabinoids in hemp samples. The analysis of the target analytes in hemp extracts was carried out by developing a new reversed-phase high-performance liquid chromatography (HPLC) method coupled with diode array (UV/DAD) and electrospray ionization-mass spectrometry (ESI–MS) detection, by using an ion trap mass analyser. An Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm) was selected for the HPLC analysis, with a mobile phase composed of 0.1\% formic acid in both water and acetonitrile, under gradient elution. The application of the fused-core technology allowed us to obtain a significant improvement of the HPLC performance compared with that of conventional particulate stationary phases, with a shorter analysis time and a remarkable reduction of solvent usage. The analytical method optimized in this study was fully validated to show compliance with international requirements. Furthermore, it was applied to the characterization of nine hemp samples and six hemp-based pharmaceutical products. As such, it was demonstrated to be a very useful tool for the analysis of cannabinoids in both the plant material and its derivatives for pharmaceutical and nutraceutical applications.
279. Borille, Bruna Tassi and González, Marina and Steffens, Luiza and Ortiz, Rafael Scorsatto and Limberger, Renata Pereira, Cannabis Sativa: A Systematic Review of Plant Analysis, Drug Analytical Research, vol. 1, no. 1, pp. 1--23, August 2017. doi: 10.22456/2527-2616.73676. Abstract
     Background: Cannabis has been the most widely used illicit drug worldwide throughout many years. Reports from different countries indicate that the potency of cannabis preparation has been increasing, as well as the ratio of tetrahydrocannabinol/cannabidiol has been changing. The high consumption along with the changing chemical profile of the drug has led increasingly to the interest in researching the cannabis plant. Methods: This article reviews available literature on the analytical methods currently used for the detection and quantification of cannabinoids in cannabis plant. The papers were screened by two researchers independently and following a pre-specified protocol. Results and Discussion: The systematic review of the literature allowed to include 42 citations on cannabis plant analysis. Conclusions: The analytical methods for cannabis material published in the included articles of this systematic review showed a lack of relevant information of the development of methods on GC and LC analysis and the limits of detection and quantification of mass detectors.
280. Onofri, Chiara and {de Meijer}, Etienne P. M. and Mandolino, Giuseppe, Sequence Heterogeneity of Cannabidiolic- and Tetrahydrocannabinolic Acid-Synthase in Cannabis Sativa L. and Its Relationship with Chemical Phenotype, Phytochemistry, vol. 116, pp. 57--68, August 2015. doi: 10.1016/j.phytochem.2015.03.006. Abstract
     Sequence variants of THCA- and CBDA-synthases were isolated from different Cannabis sativa L. strains expressing various wild-type and mutant chemical phenotypes (chemotypes). Expressed and complete sequences were obtained from mature inflorescences. Each strain was shown to have a different specificity and/or ability to convert the precursor CBGA into CBDA and/or THCA type products. The comparison of the expressed sequences led to the identification of different mutations, all of them due to SNPs. These SNPs were found to relate to the cannabinoid composition of the inflorescence at maturity and are therefore proposed to have a functional significance. The amount of variation was found to be higher within the CBDAS sequence family than in the THCAS family, suggesting a more recent evolution of THCA-forming enzymes from the CBDAS group. We therefore consider CBDAS as the ancestral type of these synthases.
281. Ahmed, Safwat A. and Ross, Samir A. and Slade, Desmond and Radwan, Mohamed M. and Khan, Ikhlas A. and ElSohly, Mahmoud A., Minor Oxygenated Cannabinoids from High Potency Cannabis Sativa L., Phytochemistry, vol. 117, pp. 194--199, September 2015. doi: 10.1016/j.phytochem.2015.04.007. Abstract
     Nine oxygenated cannabinoids were isolated from a high potency Cannabis sativa L. variety. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR, HRMS and GC–MS. These minor compounds include four hexahydrocannabinols, four tetrahydrocannabinols, and one hydroxylated cannabinol, namely 9α-hydroxyhexahydrocannabinol, 7-oxo-9α-hydroxyhexa-hydrocannabinol, 10α-hydroxyhexahydrocannabinol, 10aR-hydroxyhexahydrocannabinol, Δ9-THC aldehyde A, 8-oxo-Δ9-THC, 10aα-hydroxy-10-oxo-Δ8-THC, 9α-hydroxy-10-oxo-Δ6a,10a-THC, and 1′S-hydroxycannabinol, respectively. The latter compound showed moderate anti-MRSa (IC50 10.0μg/mL), moderate antileishmanial (IC50 14.0μg/mL) and mild antimalarial activity against Plasmodium falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values of 3.4 and 2.3μg/mL, respectively.
282. Peschel, Wieland and Politi, Matteo, 1H NMR and HPLC/DAD for Cannabis Sativa L. Chemotype Distinction, Extract Profiling and Specification, Talanta, vol. 140, pp. 150--165, August 2015. doi: 10.1016/j.talanta.2015.02.040. Abstract
     The medicinal use of different chemovars and extracts of Cannabis sativa L. requires standardization beyond ∆9-tetrahydrocannabinol (THC) with complementing methods. We investigated the suitability of 1H NMR key signals for distinction of four chemotypes measured in deuterated dimethylsulfoxide together with two new validated HPLC/DAD methods used for identification and extract profiling based on the main pattern of cannabinoids and other phenolics alongside the assayed content of THC, cannabidiol (CBD), cannabigerol (CBG) their acidic counterparts (THCA, CBDA, CBGA), cannabinol (CBN) and cannflavin A and B. Effects on cell viability (MTT assay, HeLa) were tested. The dominant cannabinoid pairs allowed chemotype recognition via assignment of selective proton signals and via HPLC even in cannabinoid-low extracts from the THC, CBD and CBG type. Substantial concentrations of cannabinoid acids in non-heated extracts suggest their consideration for total values in chemotype distinction and specifications of herbal drugs and extracts. Cannflavin A/B are extracted and detected together with cannabinoids but always subordinated, while other phenolics can be accumulated via fractionation and detected in a wide fingerprint but may equally serve as qualitative marker only. Cell viability reduction in HeLa was more determined by the total cannabinoid content than by the specific cannabinoid profile. Therefore the analysis and labeling of total cannabinoids together with the content of THC and 2–4 lead cannabinoids are considered essential. The suitability of analytical methods and the range of compound groups summarized in group and ratio markers are discussed regarding plant classification and pharmaceutical specification.
283. Verma, Ram S. and Padalia, Rajendra C. and Verma, Sajendra K. and Chauhan, Amit and Darokar, Mahendra P., The Essential Oil of 'bhang' (Cannabis Sativa L.) for Non-Narcotic Applications, Current Science, vol. 107, no. 4, pp. 645--650, 2014. url: https://www.jstor.org/stable/24103537. Abstract
     Cannabis sativa L. (family Cannabaceae) is a medicinal and aromatic plant growing all over the world. The present study aims to investigate the essential oil composition and antimicrobial activity of C. sativa from the foothills of northern India. The hydro-distilled essential oil of C. sativa was studied by capillary gas chromatography/flame ionization detector (GC-FID) and GC-mass spectrometry (GC-MS) and evaluated against nine pathogenic bacterial strains using disc diffusion assay. A total of 57 constituents representing 90.5–93.1\% of the total oil compositions were identified. Major constituents of the essential oil were (E)-caryophyllene (19.6–26.1\%), limonene (4.1–15.8\%), caryophyllene oxide (2.0–10.7\%), (E)-β-farnesene (4.8–8.5\%), α-humulene (5.4–7.8\%), α-pinene (0.7–7.7\%), myrcene (0.8–6.0\%), terpinolene (0.2–6.0\%) and β-selinene (1.8–5.4\%). The oil showed moderate to good activity against most of the tested Gram-positive bacteria (Staphylococcus aureus (MTCC2940), Staphylococcus aureus (MTCC96) and Streptococcus mutans). The oil also showed moderate activity against a Gram-negative bacterium, Salmonella typhimurium. The chemical composition of the examined C. sativa essential oil was quite different from earlier reported compositions. The oil possessed moderate to good activity against most of the tested bacterial strains.
284. {Montserrat-de la Paz}, S. and {Marín-Aguilar}, F. and {García-Giménez}, M. D. and {Fernández-Arche}, M. A., Hemp (Cannabis Sativa L.) Seed Oil: Analytical and Phytochemical Characterization of the Unsaponifiable Fraction, Journal of Agricultural and Food Chemistry, vol. 62, no. 5, pp. 1105--1110, February 2014. doi: 10.1021/jf404278q. Abstract
     Non-drug varieties of Cannabis sativa L., collectively namely as “hemp”, have been an interesting source of food, fiber, and medicine for thousands of years. The ever-increasing demand for vegetables oils has made it essential to characterize additional vegetable oil through innovative uses of its components. The lipid profile showed that linoleic (55\%), α-linolenic (16\%), and oleic (11\%) were the most abundant fatty acids. A yield (1.84–1.92\%) of unsaponifiable matter was obtained, and the most interesting compounds were β-sitosterol (1905.00 ± 59.27 mg/kg of oil), campesterol (505.69 ± 32.04 mg/kg of oil), phytol (167.59 ± 1.81 mg/kg of oil), cycloartenol (90.55 ± 3.44 mg/kg of oil), and γ-tocopherol (73.38 ± 2.86 mg/100 g of oil). This study is an interesting contribution for C. sativa L. consideration as a source of bioactive compounds contributing to novel research applications for hemp seed oil in the pharmaceutical, cosmetic food, and other non-food industries.
285. Happyana, Nizar and Agnolet, Sara and Muntendam, Remco and Van Dam, Annie and Schneider, Bernd and Kayser, Oliver, Analysis of Cannabinoids in Laser-Microdissected Trichomes of Medicinal Cannabis Sativa Using LCMS and Cryogenic NMR, Phytochemistry, vol. 87, pp. 51--59, March 2013. doi: 10.1016/j.phytochem.2012.11.001. Abstract
     Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25–143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. Δ9-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, Δ9-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit.
286. Stout, Jake M. and Boubakir, Zakia and Ambrose, Stephen J. and Purves, Randy W. and Page, Jonathan E., The Hexanoyl-CoA Precursor for Cannabinoid Biosynthesis Is Formed by an Acyl-Activating Enzyme in Cannabis Sativa Trichomes, The Plant Journal, vol. 71, no. 3, pp. 353--365, 2012. doi: 10.1111/j.1365-313X.2012.04949.x. Abstract
     The psychoactive and analgesic cannabinoids (e.g. Δ9-tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g−1 fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.
287. Shoyama, Yoshinari and Tamada, Taro and Kurihara, Kazuo and Takeuchi, Ayako and Taura, Futoshi and Arai, Shigeki and Blaber, Michael and Shoyama, Yukihiro and Morimoto, Satoshi and Kuroki, Ryota, Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabis Sativa, Journal of Molecular Biology, vol. 423, no. 1, pp. 96--105, October 2012. doi: 10.1016/j.jmb.2012.06.030. Abstract
     ∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure–function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (Rcryst=19.9\%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6′ of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active‐site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.
288. Mihoc, Marcela and Pop, Georgeta and Alexa, Ersilia and Radulov, Isidora, Nutritive Quality of Romanian Hemp Varieties (Cannabis Sativa L.) with Special Focus on Oil and Metal Contents of Seeds, Chemistry Central Journal, vol. 6, no. 1, pp. 122, October 2012. doi: 10.1186/1752-153X-6-122. Abstract
     The study aims to determine the nutritional value of hemp seed expressed by the oil content and by the concentration of metals (Ca, Mg, K, Fe, Mn, Zn and Cd), for five varieties of monoecious and dioecious hemp seeds approved in Romania, comparative with the concentration of these metals in the soil.
289. Gagne, Steve J. and Stout, Jake M. and Liu, Enwu and Boubakir, Zakia and Clark, Shawn M. and Page, Jonathan E., Identification of Olivetolic Acid Cyclase from Cannabis Sativa Reveals a Unique Catalytic Route to Plant Polyketides, Proceedings of the National Academy of Sciences, vol. 109, no. 31, pp. 12811--12816, July 2012. doi: 10.1073/pnas.1200330109. Abstract
     Δ9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.
290. Da Porto, C. and Decorti, D. and Tubaro, F., Fatty Acid Composition and Oxidation Stability of Hemp (Cannabis Sativa L.) Seed Oil Extracted by Supercritical Carbon Dioxide, Industrial Crops and Products, vol. 36, no. 1, pp. 401--404, March 2012. doi: 10.1016/j.indcrop.2011.09.015. Abstract
     Supercritical carbon dioxide (SC-CO2) was employed to extract oil from hemp (Cannabis sativa L.) seeds. For ground seeds, the supercritical extraction was carried out at temperatures of 40, 60 and 80°C and pressures of 300 and 400bar. Different solvent-ratios were applied. Supercritical CO2 extractions were compared with a conventional technique, n-hexane in Soxhlet. The extraction yields, fatty acid composition of the oil and oxidation stability were determined. The seed samples used in this work contained 81\% PUFAs, of which 59.6\% was linoleic acid (ω-6), 3.4\% γ-linolenic (ω-3), and 18\% α-linolenic (ω-6). The highest oil yield from seeds was 22\%, corresponding to 72\% recovery, at 300bar and 40°C and at 400bar and 80°C. The highest oxidation stability corresponding to 2.16mM Eq Vit E was obtained at 300bar and 80°C.
291. Casano, S. and Grassi, G. and Martini, V. and Michelozzi, M., Variations in Terpene Profiles of Different Strains of Cannabis Sativa L., Acta Horticulturae, no. 925, pp. 115--121, December 2011. doi: 10.17660/ActaHortic.2011.925.15. Abstract
     Secondary compounds of the plant are indispensable to cope with its often hostile environment and the great chemical diversity and variability of intraspecific and interspecific secondary metabolism is the result of natural selection. Recognition of the biological properties of secondary compounds have increased their great utility for human uses; numerous compounds now are receiving particular attention from the pharmaceutical industry and are important sources of a wide variety of commercially useful base products. Medical and other effects of Cannabis sativa L. are due to concentration and balance of various active secondary metabolites, particularly the cannabinoids, but including also a wide range of terpenoids and flavonoids. A wide qualitative and quantitative variability in cannabinoids, terpenoids, and flavonoids contents in Cannabis species are apparent from reports in the literature. Terpenes are strongly inherited and little influenced by environmental factors and, therefore, have been widely used as biochemical marker in chemosystematic studies to characterize plant species, provenances, clones, and hybrids. This study investigated the variability of terpene profiles in C. sativa. The terpene composition in inflorescences of samples collected from progenies of 16 plants derived from different strains was analysed by GC/FID. The amount of each terpene (in sufficient quantities to be considered in statistical analysis) was expressed as a percentage of total terpenes. Results showed a large variation between different strains in the relative contents for several mono-terpenes (α-pinene, camphene, β-pinene, sabinene, Δ-3-carene, α-phellandrene, β-myrcene, α-terpinene, limonene, 1.8-cineole, γ-terpinene, cis-β-ocimene, trans-β-ocimene, α-terpinolene) and one sesquiterpene, β-caryophyllene. This variability in terpene composition can provide a potential tool for the characterization of Cannabis biotypes and warrant further research to evaluate the drug’s medical value and, at the same time, to select less susceptible chemotypes to the attack of herbivores and diseases.
292. Fairbairn, J W and Liebmann, J A, The Extraction and Estimation of the Cannabinoids in {\emph{Cannabis Sativa}} L. and Its Products, Journal of Pharmacy and Pharmacology, vol. 25, no. 2, pp. 150--155, April 2011. doi: 10.1111/j.2042-7158.1973.tb10609.x. Abstract
     Abstract A convenient method for the complete extraction of the cannabinoids from fresh plant material, herbal cannabis, cannabis resin and reefers, has been devised. Chloroform was a more suitable solvent than light petroleum or ethanol and simple shaking of powdered material with the solvent was effective. Fresh material should be air-dried and powdered before extraction. The main cannabinoids in the extract are determined by g.l.c. using androst-4-ene-3,17-dione as internal standard. The coefficient of variation for repeated determinations of THC on a single extract was 1.4\%; for all operations, including sampling and extraction, it was 2.7\%. Duplicate analyses of 24 samples of herbal cannabis and of 20 reefers, all of varying potency, showed that the errors fell within the expected limits for THC, CBD and CBN. The method is simple and rapid; duplicate determinations can be completed in about 2 1/2 h.
293. Nissen, Lorenzo and Zatta, Alessandro and Stefanini, Ilaria and Grandi, Silvia and Sgorbati, Barbara and Biavati, Bruno and Monti, Andrea, Characterization and Antimicrobial Activity of Essential Oils of Industrial Hemp Varieties (Cannabis Sativa L.), Fitoterapia, vol. 81, no. 5, pp. 413--419, July 2010. doi: 10.1016/j.fitote.2009.11.010. Abstract
     The present study focused on inhibitory activity of freshly extracted essential oils from three legal (THC{$<$}0.2\% w/v) hemp varieties (Carmagnola, Fibranova and Futura) on microbial growth. The effect of different sowing times on oil composition and biological activity was also evaluated. Essential oils were distilled and then characterized through the gas chromatography and gas chromatography-mass spectrometry. Thereafter, the oils were compared to standard reagents on a broad range inhibition of microbial growth via minimum inhibitory concentration (MIC) assay. Microbial strains were divided into three groups: i) Gram (+) bacteria, which regard to food-borne pathogens or gastrointestinal bacteria, ii) Gram (−) bacteria and iii) yeasts, both being involved in plant interactions. The results showed that essential oils of industrial hemp can significantly inhibit the microbial growth, to an extent depending on variety and sowing time. It can be concluded that essential oils of industrial hemp, especially those of Futura, may have interesting applications to control spoilage and food-borne pathogens and phytopathogens microorganisms.
294. Radwan, Mohamed M. and ElSohly, Mahmoud A. and Slade, Desmond and Ahmed, Safwat A. and Khan, Ikhlas A. and Ross, Samir A., Biologically Active Cannabinoids from High-Potency Cannabis Sativa, Journal of Natural Products, vol. 72, no. 5, pp. 906--911, May 2009. doi: 10.1021/np900067k. Abstract
     Nine new cannabinoids (1−9) were isolated from a high-potency variety of Cannabis sativa. Their structures were identified as (±)-4-acetoxycannabichromene (1), (±)-3′′-hydroxy-Δ(4′′,5′′)-cannabichromene (2), (−)-7-hydroxycannabichromane (3), (−)-7R-cannabicoumarononic acid A (4), 5-acetyl-4-hydroxycannabigerol (5), 4-acetoxy-2-geranyl-5-hydroxy-3-n-pentylphenol (6), 8-hydroxycannabinol (7), 8-hydroxycannabinolic acid A (8), and 2-geranyl-5-hydroxy-3-n-pentyl-1,4-benzoquinone (9) through 1D and 2D NMR spectroscopy, GC-MS, and HRESIMS. The known sterol β-sitosterol-3-O-β-d-glucopyranosyl-6′-acetate was isolated for the first time from cannabis. Compounds 6 and 7 displayed significant antibacterial and antifungal activities, respectively, while 5 displayed strong antileishmanial activity.
295. Fischedick, Justin T. and Glas, Ronald and Hazekamp, Arno and Verpoorte, Rob, A Qualitative and Quantitative HPTLC Densitometry Method for the Analysis of Cannabinoids in Cannabis Sativa L., Phytochemical Analysis, vol. 20, no. 5, pp. 421--426, 2009. doi: 10.1002/pca.1143. Abstract
     Introduction – Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low-cost, high-throughput analytical methods for quality control. Objective – The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Δ9-tetrahydrocannabinol (Δ9-THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. Methodology – The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Results – Δ9-THC gives linear calibration curves in the range of 50–500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r2 = 0.9968. Conclusion – Results have shown that the HPTLC method is reproducible and accurate for the quantification of Δ9-THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars. Copyright © 2009 John Wiley \& Sons, Ltd.
296. Radwan, Mohamed M. and ElSohly, Mahmoud A. and Slade, Desmond and Ahmed, Safwat A. and Wilson, Lisa and {El-Alfy}, Abir T. and Khan, Ikhlas A. and Ross, Samir A., Non-Cannabinoid Constituents from a High Potency Cannabis Sativa Variety, Phytochemistry, vol. 69, no. 14, pp. 2627--2633, October 2008. doi: 10.1016/j.phytochem.2008.07.010. Abstract
     Six new non-cannabinoid constituents were isolated from a high potency Cannabis sativa L. variety, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), cannflavin C (5) and β-sitosteryl-3-O-β-d-glucopyranoside-2′-O-palmitate (6). In addition, five known compounds, α-cannabispiranol (7), chrysoeriol (8), 6-prenylapigenin (9), cannflavin A (10) and β-acetyl cannabispiranol (11) were identified, with 8 and 9 being reported for the first time from cannabis. Some isolates displayed weak to strong antimicrobial, antileishmanial, antimalarial and anti-oxidant activities. Compounds 2–4 were inactive as analgesics.
297. Wang, Xian-Sheng and Tang, Chuan-He and Yang, Xiao-Quan and Gao, Wen-Rui, Characterization, Amino Acid Composition and in Vitro Digestibility of Hemp (Cannabis Sativa L.) Proteins, Food Chemistry, vol. 107, no. 1, pp. 11--18, March 2008. doi: 10.1016/j.foodchem.2007.06.064. Abstract
     The protein constituents and thermal properties of hemp (Cannabis sativa L.) protein isolate (HPI) as well as 11S- and 7S-rich HPIs (HPI-11S and HPI-7S) were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and different scanning calorimetry (DSC), and their amino acid composition and in vitro digestibility were also evaluated, as compared to soy protein isolate (SPI). SDS-PAGE analysis showed that the edestin (consisting of acidic and basic subunits, AS and BS) was the main protein component for HPI and HPI-11S, while HPI-7S was composed of the BS of edestin and a subunit of about 4.8kDa. DSC analysis characterized thermal transition of the edestin component and the possible present form of different subunits. Except lysine and sulfur-containing amino acids, the essential amino acids of various HPIs met the suggested requirements of FAO/WHO for 2–5year old infants. The proportion of essential amino acids to the total amino acids (E/T) for HPI (as well as HPI-11S) was significantly higher than that of SPI. In an in vitro digestion model, various protein constituents of various HPIs were much easily digested by pepsin plus trypsin, to release oligo-peptides with molecular weight less than 10.0kDa (under reduced condition). Only after pepsin digestion, in vitro digestibility of HPIs was comparable to that of SPI, however after pepsin plus trypsin digestion, the digestibility (88–91\%) was significantly higher than that (71\%) of SPI (P{$<$}0.05). These results suggest that the protein isolates from hempseed are much more nutritional in amino acid nutrition and easily digestible than SPI, and can be utilized as a good source of protein nutrition for human consumption.
298. Ahmed, Safwat A. and Ross, Samir A. and Slade, Desmond and Radwan, Mohamed M. and Zulfiqar, Fazila and ElSohly, Mahmoud A., Cannabinoid Ester Constituents from High-Potency Cannabis Sativa, Journal of Natural Products, vol. 71, no. 4, pp. 536--542, April 2008. doi: 10.1021/np070454a. Abstract
     Eleven new cannabinoid esters, together with three known cannabinoid acids and Δ9-tetrahydrocannabinol (Δ9-THC), were isolated from a high-potency variety of Cannabis sativa. The structures were determined by extensive spectroscopic analyses to be β-fenchyl Δ9-tetrahydrocannabinolate (1), epi-bornyl Δ9-tetrahydrocannabinolate (2), α-terpenyl Δ9-tetrahydrocannabinolate (3), 4-terpenyl Δ9-tetrahydrocannabinolate (4), α-cadinyl Δ9-tetrahydrocannabinolate (5), γ-eudesmyl Δ9-tetrahydrocannabinolate (6), γ-eudesmyl cannabigerolate (7), 4-terpenyl cannabinolate (8), bornyl Δ9-tetrahydrocannabinolate (9), α-fenchyl Δ9-tetrahydrocannabinolate (10), α-cadinyl cannabigerolate (11), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid A (Δ9-THCA), cannabinolic acid A (CBNA), and cannabigerolic acid (CBGA). Compound 8 showed moderate antimicrobial activity against Candida albicans ATCC 90028 with an IC50 value of 8.5 µg/mL. CB-1 receptor assay indicated that the esters, as well as the parent acids, are not active.
299. YounHo, Moon and YeonSang, Song and ByeongChoon, Jeong and JinKi, Bang, Variation of cannabinoids content in hemp (Cannabis sativa L.) produced with mixed seeds of drug and non-drug type varieties., Korean Journal of Crop Science, vol. 51, no. 3, pp. 187--190, 2006. url: https://www.cabdirect.org/cabdirect/abstract/20063198290. Abstract
     To produce hemp with low cannabinoids and appropriate fibre yield, seeds of the IH3 (non-drug type germplasm) and local variety (drug type cultivar) were mixed to 1:1, 2:1, 3:1 on the basis of seed weight ratio and cultivated for seed production. In the seed yield trial in Korea Republic, the 1000-seed weight were approximately 21 g, which was increased in proportion to the ratio of IH3 seeds but...
300. Pacifico, Daniela and Miselli, Francesca and Micheler, Mirta and Carboni, Andrea and Ranalli, Paolo and Mandolino, Giuseppe, Genetics and Marker-Assisted Selection of the Chemotype in Cannabis Sativa L., Molecular Breeding, vol. 17, no. 3, pp. 257--268, April 2006. doi: 10.1007/s11032-005-5681-x. Abstract
     Cannabis sativa is an interesting crop for several industrial uses, but the legislations in Europe and USA require a tight control of cannabinoid type and content for cultivation and subsidies release. Therefore, cannabinoid survey by gas chromatography of materials under selection is an important step in hemp breeding. In this paper, a number of Cannabis accessions were examined for their cannabinoid composition. Their absolute and relative content was examined, and results are discussed in the light of both the current genetic model for cannabinoid’s inheritance, and the legislation’s requirements. In addition, the effectiveness of two different types of markers associated to the locus determining the chemotype in Cannabis was evaluated and discussed, as possible tools in marker-assisted selection in hemp, but also for possible applications in the forensic and pharmaceutical fields.
301. Kojoma, Mareshige and Seki, Hikaru and Yoshida, Shigeo and Muranaka, Toshiya, DNA Polymorphisms in the Tetrahydrocannabinolic Acid (THCA) Synthase Gene in “Drug-Type” and “Fiber-Type” Cannabis Sativa L., Forensic Science International, vol. 159, no. 2, pp. 132--140, June 2006. doi: 10.1016/j.forsciint.2005.07.005. Abstract
     The cannabinoid content of 13 different strains of cannabis plant (Cannabis sativa L.) was analyzed. Six strains fell into the “drug-type” class, with high Δ-9-tetrahydrocannabinolic acid (THCA) content, and seven strains into the “fiber-type” class, with low THCA using HPLC analysis. Genomic DNA sequence polymorphisms in the THCA synthase gene from each strain were studied. A single PCR fragment of the THCA synthase gene was detected from six strains of “drug-type” plants. We could also detect the fragment from seven strains of “fiber-type” plants, although no or very low content of THCA were detected in these samples. These were 1638bp from all 13 strains and no intron among the sequences obtained. There were two variants of the THCA synthase gene in the “drug-type” and “fiber-type” cannabis plants, respectively. Thirty-seven major substitutions were detected in the alignment of the deduced amino acid sequences from these variants. Furthermore, we identified a specific PCR marker for the THCA synthase gene for the “drug-type” strains. This PCR marker was not detected in the “fiber-type” strains.
302. Anwar, Farooq and Latif, Sajid and Ashraf, Muhammad, Analytical Characterization of Hemp (Cannabis Sativa) Seed Oil from Different Agro-Ecological Zones of Pakistan, Journal of the American Oil Chemists' Society, vol. 83, no. 4, pp. 323--329, 2006. doi: 10.1007/s11746-006-1207-x. Abstract
     Cold-pressed oil content of Cannabis sativa (hemp) seeds from three different agro-ecological zones of Pakistan ranged from 26.90 to 31.50\%. Protein, fiber, ash, and moisture content were found to be 23.00–26.50, 17.00–20.52, 5.00–7.60, and 5.60–8.50\%, respectively. Results of some other physical and chemical parameters of the oil were as follows: iodine value, 154.00–165.00; refractive index (40°C), 1.4698–1.4750; density (24°C), 0.9180–0.9270 mg ml−1; saponification value, 184.00–190.00; unsaponifiable matter, 0.70–1.25\%; and color (1-in cell), 0.50–0.80 R+27.00–32.00 Y. The induction period (Rancimat, 20 L h−1, 120°C) of the nondegummed and degummed oils ranged from 1.35 to 1.72 h and from 1.20 to 1.49 h, respectively. Specific extinctions at 232 and 270 nm were 3.50–4.18 and 0.95–1.43, respectively. The hemp oils investigated were found to contain high levels of linoleic acid, 56.50–60.50\%, followed by α-linolenic, oleic, palmitic, stearic, and γ-linolenic acids: 16.85–20.00, 10.17–14.03, 5.75–8.27, 2.19–2.79, and 0.63–1.65\%, respectively. Tocopherols (α, γ, and δ) in the nondegummed oils were found to be 54.02–60.40, 600.00–745.00, 35.00–45.60, respectively, and were reduced to 29.90–50.00, 590.00–640.00, and 30.40–39.50 mg kg−1, respectively, after degumming. The results of the present analytical study, compared with those found in the typical literature on hempseed oils, showed C. sativa indigenous to Pakistan to be a potentially valuable nonconventional oilseed crop of comparable quality.
303. Ross, Samir A. and ElSohly, Mahmoud A. and Sultana, Gazi N. N. and Mehmedic, Zlatko and Hossain, Chowdhury F. and Chandra, Suman, Flavonoid Glycosides and Cannabinoids from the Pollen of Cannabis Sativa L., Phytochemical Analysis, vol. 16, no. 1, pp. 45--48, 2005. doi: 10.1002/pca.809. Abstract
     Chemical investigation of the pollen grain collected from male plants of Cannabis sativa L. resulted in the isolation for the first time of two flavonol glycosides from the methanol extract, and the identification of 16 cannabinoids in the hexane extract. The two glycosides were identified as kaempferol 3-O-sophoroside and quercetin 3-O-sophoroside by spectroscopic methods including high-field two-dimensional NMR experiments. The characterisation of each cannabinoid was performed by GC-FID and GC-MS analyses and by comparison with both available reference cannabinoids and reported data. The identified cannabinoids were Δ9-tetrahydrocannabiorcol, cannabidivarin, cannabicitran, Δ9-tetrahydrocannabivarin, cannabicyclol, cannabidiol, cannabichromene, Δ9-tetrahydrocannabinol, cannabigerol, cannabinol, dihydrocannabinol, cannabielsoin, 6a, 7, 10a-trihydroxytetrahydrocannabinol, 9, 10-epoxycannabitriol, 10-O-ethylcannabitriol, and 7, 8-dehydro-10-O-ethylcannabitriol. Copyright © 2005 John Wiley \& Sons, Ltd.
304. Hazekamp, Arno and Peltenburg, Anja and Verpoorte, Rob and Giroud, Christian, Chromatographic and Spectroscopic Data of Cannabinoids from Cannabis Sativa L., Journal of Liquid Chromatography \& Related Technologies, vol. 28, no. 15, pp. 2361--2382, September 2005. doi: 10.1080/10826070500187558. Abstract
     Chromatographic and spectroscopic data was determined for 16 different major cannabinoids from Cannabis sativa plant material as well as 2 human metabolites of Δ9‐tetrahydrocannabinol. Spectroscopic analysis included UV absorbance, infrared‐spectral analysis, (GC‐) mass spectrometry, and spectrophotometric analysis. Also, the fluorescent properties of the cannabinoids are presented. Most of this data is available from literature but scattered over a large amount of scientific papers. In this case, analyses were carried out under standardised conditions for each tested cannabinoid so spectroscopic data can be directly compared. Different methods for the analysis of cannabis preparations were used and are discussed for their usefulness in the identification and determination of separate cannabinoids. Data on the retention of the cannabinoids in HPLC, GC, and TLC are presented.
305. Hazekamp, Arno and Choi, Young Hae and Verpoorte, Robert, Quantitative Analysis of Cannabinoids from {\emph{Cannabis Sativa}} Using {\textsuperscript{1}}H-NMR, Chemical and Pharmaceutical Bulletin, vol. 52, no. 6, pp. 718--721, 2004. doi: 10.1248/cpb.52.718. Abstract
     A 1H-NMR method has been developed for the quantitative analysis of pure cannabinoids and for cannabinoids present in Cannabis sativa plant material without any chromatographic purification. The experiment was performed by the analysis of singlets in the range of δ 4.0—7.0 in the 1H-NMR spectrum, in which distinguishable signals of each cannabinoid are shown. Quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds to the known amount of the internal standard, anthracene. For this method no reference compounds are needed. It allows rapid and simple quantitation of cannabinoids with a final analysis time of only 5 min without the need for a pre-purification step.
306. Hazekamp, Arno and Simons, Ruud and Peltenburg‐Looman, Anja and Sengers, Melvin and {van Zweden}, Rianne and Verpoorte, Robert, Preparative Isolation of Cannabinoids from Cannabis Sativa by Centrifugal Partition Chromatography, Journal of Liquid Chromatography \& Related Technologies, vol. 27, no. 15, pp. 2421--2439, January 2004. doi: 10.1081/JLC-200028170. Abstract
     A simple method is presented for the preparative isolation of seven major cannabinoids from Cannabis sativa plant material. Separation was performed by centrifugal partition chromatography (CPC), a technique that permits large‐scale preparative isolations. Using only two different solvent systems, it was possible to obtain pure samples of the cannabinoids; (−)‐Δ9‐(trans)‐tetrahydrocannabinol (Δ9‐THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), (−)‐Δ9‐(trans)‐tetrahydrocannabinolic acid‐A (THCA), cannabigerolic acid (CBGA), and cannabidiolic acid (CBDA). A drug‐type and a fiber‐type cannabis cultivar were used for the isolation. All isolates were shown to be more than 90\% pure by gas chromatography. This method makes acidic cannabinoids available on a large scale for biological testing. The method described in this report can also be used to isolate additional cannabinoids from cannabis plant material.
307. Kriese, U. and Schumann, E. and Weber, W.E. and Beyer, M. and Brühl, L. and {Matthäus}, Oil Content, Tocopherol Composition and Fatty Acid Patterns of the Seeds of 51 Cannabis Sativa L. Genotypes, Euphytica, vol. 137, no. 3, pp. 339--351, June 2004. doi: 10.1023/B:EUPH.0000040473.23941.76. Abstract
     The oil content, the tocopherol composition, the plastochromanol-8 (P-8) content and the fatty acid composition (19 fatty acids) of the seed of 51 hemp (Cannabis sativa L.) genotypes were studied in the 2000 and 2001 seasons. The oil content of the hemp seed ranged from 26.25\% (w/w) to 37.50\%. Analysis of variance revealed significant effects of genotype, year and of the interaction (genotype × year) on the oil content. The oil contents of the 51 genotypes in 2000 and 2001 were correlated (r = 0.37**) and averaged 33.19 ± 1.45\% in 2000 and 31.21 ± 0.96\% in 2001. The γ-tocopherol, α-tocopherol, δ-tocopherol, P-8- and β-tocopherol contents of the 51 genotypes averaged 21.68 ± 3.19, 1.82 ± 0.49, 1.20 ± 0.40, 0.18 ± 0.07 and 0.16 ± 0.04 mg 100g−1 of seeds, respectively (2000 and 2001 data pooled). Hierarchical clustering of the fatty acid data did not group the hemp genotypes according to their geographic origin. The γ-linolenic acid yield of hemp (3–30 kg ha−1) was similar to the γ-linolenic acid yield of plant species that are currently used as sources of γ-linolenic acid (borage (19–30 kg ha−1), evening primrose (7–30 kg ha−1)). The linoleic acid yield of hemp (129–326 kg ha−1) was similar to flax (102–250 kg ha−1), but less than in sunflower (868–1320 kg ha−1). Significant positive correlations were detected between some fatty acids and some tocopherols. Even though the average content of P-8 in hemp seeds was only 1/120th of the average γ-tocopherol content, P-8 content was more closely correlated with the unsaturated fatty acid content than γ-tocopherol or any other tocopherol fraction. The average broad-sense heritabilities of the oil content, the antioxidants (tocopherols and P-8) and the fatty acids were 0.53, 0.14 and 0.23, respectively. The genotypes Fibrimon 56, P57, Juso 31, GB29, Beniko, P60, FxT, Félina 34, Ramo and GB18 were capable of producing the largest amounts of high quality hemp oil.
308. Vogl, Christian R. and Mölleken, Helga and {Lissek-Wolf}, Gunilla and Surböck, Andreas and Kobert, {\relax Jör}g, Hemp (Cannabis Sativa L.) as a Resource for Green Cosmetics, Journal of Industrial Hemp, vol. 9, no. 1, pp. 51--68, June 2004. doi: 10.1300/J237v09n01_06. Abstract
     The interest in hemp (non-drug Cannabis sativa L.) for skin care and cosmetic use is due to the high content of oil, especially unsaturated fatty acids in seed with technological and therapeutic effects. In a field trial on an organic farm, seed weight and content of fatty acids of 20 hemp varieties were surveyed on three different harvest dates. The dry matter seed yields ranged from 27-149 g m−2. The varieties Ferimon-12, Fedora-19, and Bialobreszie produced high seed yields on all three harvest dates but yields were not significantly different from a large group of other varieties. Contents of palmitic acid range from 3.1 to 4.1\%, of stearic acid from 0.1 to 1.9\%, of oleic acid from 3.7 to 9.2\%, of linoleic acid from 44.8 to 60.2\%, of a-linolenic acid from 18.2 to 27.4\%, and of ?-linolenic acid from 1.6 to 4.7\%. The genotype has no significant influence on fatty acid content. All 20 varieties tested show high quantities of fatty acid depending on the harvest date, so that no variety can be favored. Results confirm that hemp is a very good source of fatty acids for skin care and cosmetic use.
309. Turner, Carlton E. and Elsohly, Mahmoud A. and Boeren, Edward G., Constituents of Cannabis Sativa L. XVII. A Review of the Natural Constituents, ACS Publications, July 2004. doi: 10.1021/np50008a001.
310. Oomah, B. Dave and Busson, Muriel and Godfrey, David V and Drover, John C. G, Characteristics of Hemp (Cannabis Sativa L.) Seed Oil, Food Chemistry, vol. 76, no. 1, pp. 33--43, January 2002. doi: 10.1016/S0308-8146(01)00245-X. Abstract
     Characteristics of oil extracted from hempseeds subjected to microwave treatments were evaluated. Microwave treatment improved oil yield, increased carotenoid and other pigment contents and decreased p-anisidine value without significant changes in other properties. Hempseed oil showed absorbance in the UV-B and UV-C ranges with potential for use as a broad spectrum UV protectant. β-Tocopherol concentrations increased, while the major tocopherol, γ-tocopherol, and fatty acid composition of the oil were unaffected by microwave treatment of hempseed. Hempseed oil showed high kinetic stability during heating and cooling, as characterized by differential scanning calorimetry (DSC). Microwave treatment shifted the melting range of oils to lower temperatures and increased oxidation temperatures, suggesting increased protective effect upon heating.
311. Groce, John W. and Jones, Louis A., Carbohydrate and Cyclitol Content of Cannabis, ACS Publications, May 2002. doi: 10.1021/jf60186a003.
312. Ross, Samir A. and ElSohly, Mahmoud A., The Volatile Oil Composition of Fresh and Air-Dried Buds of Cannabis Sativa, Journal of Natural Products, vol. 59, no. 1, pp. 49--51, January 1996. doi: 10.1021/np960004a. Abstract
     The composition of the steam-distilled volatile oil of fresh and air-dried, indoor-grown marijuana was studied by GC/FID and GC/MS. In all, 68 components were detected of which 57 were fully identified. Drying of the plant material had no effect on the qualitative composition of the oil and did not affect the ability of individuals familiar with marijuana smell to recognize the odor.
313. Ross, Samir and EISohly, Mahmoud, Constituents of Cannabis Sativa L. Xxviii a Review of the Natural Constituents: 1980-1994, Zagazig Journal of Pharmaceutical Sciences, vol. 4, no. 1, pp. 150--160, June 1995. doi: 10.21608/zjps.1995.169714. Abstract
     Over 11,000 scientific papers have been published on cannabis and its constituents. The purpose of the present review is to update the number of known natural constituents of the Cannabis plant to reach 483 compounds. Emphasis will be placed on those compounds actually isolated or identified from 1980 until 1994.
314. Taura, Futoshi and Morimoto, Satoshi and Shoyama, Yukihiro, Cannabinerolic Acid, a Cannabinoid from Cannabis Sativa, Phytochemistry, vol. 39, no. 2, pp. 457--458, May 1995. doi: 10.1016/0031-9422(94)00887-Y. Abstract
     Investigation of the leaves of Cannabis sativa resulted in the isolation of a new cannabinoid, cannabinerolic acid. The structure of the new cannabinoid was established on the basis of spectroscopic and chemical evidence.
315. {van der Werf}, H. M. G. and {Harsveld van der Veen}, J. E. and Bouma, A. T. M. and {ten Cate}, M., Quality of Hemp (Cannabis Sativa L.) Stems as a Raw Material for Paper, Industrial Crops and Products, vol. 2, no. 3, pp. 219--227, May 1994. doi: 10.1016/0926-6690(94)90039-6. Abstract
     Fibre hemp (Cannabis sativa L.) stems consist of high-cellulose low-lignin bark containing long fibres and low-cellulose high-lignin core containing short fibres. The bark, which contains a variable proportion of less valuable secondary bast fibre, is more valuable as a raw material for paper than the core. A study of the factors affecting the quality of hemp grown in The Netherlands for paper production is described. The bark content in the stem was determined by mechanically separating bark and core. The chemical composition of bark and core was determined according to procedures defined by the Technical Association of the Pulp and Paper Industry. The secondary bast fibre content was determined by manually separating primary and secondary phloem tissue after boiling in a 2\% NaOH solution. Bark content in the stem decreased during the growing season; at harvest in September it ranged from 30 to 35\% depending on cultivar and plant density. The proportion of secondary bast fibre in the bast fibre fraction increased with stem weight, from 10 to 45\%. Measurement of secondary bast fibre in a representative stem section allowed the proportion of this fibre in the bast fibre fraction of the entire stem to be estimated accurately. Differences in chemical composition within sets of samples of bark or core were small compared with the difference between bark and core. The bark of the French cultivars tested contained less cellulose than that of the Hungarian cultivars. Bark quality for paper making improved during the growing season because the cellulose content in the bark increased, whereas the content of lignin and extractives decreased.
316. Turner, Jocelyn C. and Mahlberg, Paul G., Effects of Sample Treatment on Chromatographic Analysis of Cannabinoids in Cannabis Sativa L. (Cannabaceae), Journal of Chromatography A, vol. 283, pp. 165--171, January 1984. doi: 10.1016/S0021-9673(00)96251-4. Abstract
     Gas—liquid chromatographic and high-performance liquid chromatographic analyses on the effects of leaf treatment as well as the conditions for cannabinoid extraction were examined in two clones of Cannabis sativa L. Cannabinoid extracts of dried leaves, when analyzed by gas—liquid chromatography, showed no significant quantitative or qualitative differences regardless of drying procedure or temperature and duration of extraction investigated. Comparable high-performance liquid chromatographic analyses, however, indicated that while extraction temperature did not influence the cannabinoid profile, drying conditions had a significant effect. High ratios of acid to neutral forms were derived only from extracts of leaves dried at 37°C as compared to 60°C. Fresh, non-dried leaf material also yielded high ratios of acid to neutral forms, but the duration of extraction was found to affect cannabinoid yield significantly. Longer extractions of fresh leaves resulted in lower amounts of cannabinoids extracted. This study determined optimal procedures for analyzing fresh plant materials.
317. Turner, C. E. and Cheng, P. C. and Lewis, G. S. and Russell, M. H. and Sharma, G. K., Constituents of Cannabis Sativa, Planta Medica, vol. 37, no. 11, pp. 217--225, November 1979. doi: 10.1055/s-0028-1097331. Abstract
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318. Malingre, Th and Hendriks, H. and Batterman, S. and Bos, R. and Visser, J., The Essential Oil of Cannabis Sativa, Planta Medica, vol. 28, no. 5, pp. 56--61, August 1975. doi: 10.1055/s-0028-1097829. Abstract
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319. Fairbairn, J. W. and Liebmann, J. A., The Cannabinoid Content of Cannabis Sativa L Grown in England, Journal of Pharmacy and Pharmacology, vol. 26, no. 6, pp. 413--419, 1974. doi: 10.1111/j.2042-7158.1974.tb09306.x. Abstract
     Twelve varieties of Cannabis sativa were grown out-of-doors in southern England during 1971 to 1973. Results show that for certain varieties highly active herbal cannabis can be produced. A warm climate with abundant sunshine does not therefore seem to be essential for high THC content. This was supported by results of growing plants in a greenhouse in varying lighting conditions including a limited period in total darkness. Considerable within and between plant variation was found and the importance of defining the plant part used, the stage of growth and the size of the sample is emphasized for comparative work involving quantitative results. Comparison of the present results with those for the same cannabis varieties grown in different parts of the world shows that all exhibit the same qualitative picture, that is, either THC-rich or CBD-rich. Since this chemical composition seems independent of environmental conditions it is inappropriate to refer to the two types as phenotypes; it is more likely that they represent two chemical races within the species Cannabis sativa L.
320. Turner, Carlton E. and Hadley, Kathy W. and Henry, Janis and Leonard Mole, M., Constituents of Cannabis Sativa L. VII: Use of Silyl Derivatives in Routine Analysis, Journal of Pharmaceutical Sciences, vol. 63, no. 12, pp. 1872--1876, December 1974. doi: 10.1002/jps.2600631209. Abstract
     Naturally occurring cannabinoids previously impossible to separate and analyze were quantitated on a routine basis using silylation. Relative retention times of many silylated cannabinoids are reported for the first time.
321. Obata, Yataro and Ishikawa, Yoshinori, Studies on the Constituents of Hemp Plant ( {\emph{Cannabis Sativa}} L.): Part I. Volatile Phenol Fraction, Bulletin of the Agricultural Chemical Society of Japan, vol. 24, no. 7, pp. 667--669, December 1960. doi: 10.1080/03758397.1960.10857731.
322. Obata, Yataro and Ishikawa, Yoshinori and Kitazawa, Ryoichi, Studies on the Components of the Hemp Plant ({\emph{Cannabis Sativa}} L.), Bulletin of the Agricultural Chemical Society of Japan, vol. 24, no. 7, pp. 670--672, 1960. doi: 10.1271/bbb1924.24.670. Abstract
     In order to isolate and identify the unpleasa odor of wild hemp leaves, tops of the plant its flowering time was extracted with petroleu ether at first and the residue was extracted wi 90\% ethanol. In this ethanol soluble fractic piperidine contained in free and combined for, in hemp was isolated and identified by meal of the paper chromatography analysis and i\_??\_frared spectra. The melting point of the picrolonate derive from wild hempen piperidine was 242\_??\_245°C and that of authentic piperidine was 242\_??\_244°C The infrared spectra of both picrolonates wei found to be coincident and Anal., found:\_??\_20.08, calcd. for C15H19N5O5: N, 20.06\%.
323. Blatt, A. H., A Critical Survey of the Literature Dealing with the Chemical Constituents of Cannabis Sativa, Journal of the Washington Academy of Sciences, vol. 28, no. 11, pp. 465--477, 1938. url: https://www.jstor.org/stable/24530214.
324. Mary, W. and Crombie, L., The Influence of Photosynthesis and SKF Inhibitors on Cannabinoid Production in Cannabis Sativa, Phytochemistry, vol. 16, no. 9, pp. 1369--1371, January 1977. doi: 10.1016/S0031-9422(00)88784-1. Abstract
     Experiments using darkened shoots, and a Cannabis sativa sport having completely green and completely white shoots, show that cannabinoid productio
325. Lehmann, T. and Brenneisen, R., High Performance Liquid Chromatographic Profiling of Cannabis Products, Journal of Liquid Chromatography, vol. 18, no. 4, pp. 689--700, February 1995. doi: 10.1080/10826079508009265. Abstract
     An HPLC method with photodiode array detection (DAD) is described for the qualitative and quantitative determination of neutral and acidic cannabinoids in Cannabis sativa L. The complex chromatographic pattern can be used for the classification of Cannabis chemotypes, the monitoring of the psychotropic potency and the comparison of Cannabis products of different origin.
326. Mechtler, Klemens and Bailer, Josef and {de Hueber}, Karl, Variations of Δ9-THC Content in Single Plants of Hemp Varieties, Industrial Crops and Products, vol. 19, no. 1, pp. 19--24, January 2004. doi: 10.1016/S0926-6690(03)00077-3. Abstract
     Within a given plant population, the concentration of any constituent is expected to vary within a certain bandwidth. To test the distribution of Δ9-tetrahydrocannabinol (THC) in hemp populations, a number of single plants were taken from populations of five well-known hemp accessions (Fasamo, Beniko, Bialobrzeskie, Félina 34, Kompolti) and a Hungarian provenance. The quantitative analysis of single plants delivered a set of 30–61 THC and Cannabidiol (CBD) values for each of the six hemp accessions under consideration. The distribution of THC within a number of hemp plants often shows no Gaussian distribution, the different varieties have quite characteristic distributions of THC. Most single-plant values are close together, the variation, however, differing from variety to variety. In addition, single plants are found with THC values far outside this bandwidth.
327. Potter, David J. and Clark, Peter and Brown, Marc B., Potency of Δ9–THC and Other Cannabinoids in Cannabis in England in 2005: Implications for Psychoactivity and Pharmacology*, Journal of Forensic Sciences, vol. 53, no. 1, pp. 90--94, 2008. doi: 10.1111/j.1556-4029.2007.00603.x. Abstract
     Abstract: Gas chromatography was used to study the cannabinoid content (“potency”) of illicit cannabis seized by police in England in 2004/5. Of the four hundred and fifty two samples, indoor-grown unpollinated female cannabis (“sinsemilla”) was the most frequent form, followed by resin (hashish) and imported outdoor-grown herbal cannabis (marijuana). The content of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) varied widely. The median THC content of herbal cannabis and resin was 2.1\% and 3.5\%, respectively. The median 13.9\% THC content of sinsemilla was significantly higher than that recorded in the UK in 1996/8. In sinsemilla and imported herbal cannabis, the content of the antipsychotic cannabinoid cannabidiol (CBD) was extremely low. In resin, however, the average CBD content exceeded that of THC, and the relative proportions of the two cannabinoids varied widely between samples. The increases in average THC content and relative popularity of sinsemilla cannabis, combined with the absence of the anti-psychotic cannabinoid CBD, suggest that the current trends in cannabis use pose an increasing risk to those users susceptible to the harmful psychological effects associated with high doses of THC.
328. Small, Ernest and Marcus, David, Tetrahydrocannabinol Levels in Hemp (Cannabis Sativa) Germplasm Resources, Economic Botany, vol. 57, no. 4, pp. 545--558, December 2003. doi: 10.1663/0013-0001(2003)057[0545:TLIHCS]2.0.CO;2. Abstract
     In most of the western world where industrial hemp, Cannabis sativa, is licensed for cultivation, the plants must not exceed a level of 0.3\% tetrahydrocannabinol (THC), the principal intoxicating constituent of the species. Because there are no publicly available germplasm hemp collections in North America and only a very few, recent North American cultivars have been bred, the future breeding of cultivars suitable for North America is heavily dependent on European cultivars and European germplasm collections. Based mostly on material from Europe, this study surveyed THC levels of 167 accessions grown in southern Ontario, making this the largest survey to date of germplasm intended for breeding in North America. Forty-three percent of these had THC levels ≥0.3\% and, therefore, are unsuitable for hemp development in North America. Discrepancies were found between THC levels reported for some germplasm holdings in Europe when they were grown in Canada and, accordingly, verification of THC levels developed in North America is necessary.
329. Vanhoenacker, Gerd and Van Rompaey, Philippe and {de Keukeleire}, Denis and Sandra, Pat, Chemotaxonomic Features Associated with Flavonoids of Cannabinoid-Free Cannabis (Cannabis Sativa Subsp. Sativa L.) in Relation to Hops (Humulus Lupulus L.), Natural Product Letters, vol. 16, no. 1, pp. 57--63, January 2002. doi: 10.1080/1057563029001/4863. Abstract
     The major flavonoids present in the leaves and flowers of the cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) cultivars Felina and Futura are orientin (1), vitexin (2), luteolin-7-O-g-D-glucuronide (3), and apigenin-7-O-g-D-glucuronide (4), while prenylated flavonoids, to which the potent estrogenicity of hops (Humilus lupulus L.) is associated, are absent. The different composition of flavonoids has chemotaxonomic value.
330. Novak, Johannes and {Zitterl-Eglseer}, Karin and Deans, Stanley G. and Franz, Chlodwig M., Essential Oils of Different Cultivars of Cannabis Sativa L. and Their Antimicrobial Activity, Flavour and Fragrance Journal, vol. 16, no. 4, pp. 259--262, 2001. doi: 10.1002/ffj.993. Abstract
     The essential oils of five different cultivars of Cannabis sativa contained as main compounds α-pinene, myrcene, trans-β-ocimene, α-terpinolene, trans-caryophyllene and α-humulene. The content of α-terpinolene divided the cultivars in two distinct groups, an Eastern European group of cultivars of approximately 8\% and a French group of cultivars of around 16\%. Therefore, this compound might be suitable as a genetic marker for the two breeding centres for the fibre types of Cannabis sativa. The content of trans-caryophyllene was up to 19\%. However, the content of caryophyllene oxide did not exceed 2\%. The antimicrobial activity of the essential oil of Cannabis sativa can be regarded as modest. Nevertheless, cultivar differences were visible. Δ-9-tetrahydrocannabinol (THC) could not be detected in any of the essential oils and the amount of other cannabinoids was very poor. Copyright © 2001 John Wiley \& Sons, Ltd.
331. Haney, Alan and Kutscheid, Benjamin B., Quantitative Variation in the Chemical Constituents of Marihuana from Stands of naturalizedCannabis Sativa L. in East-Central Illinois, Economic Botany, vol. 27, no. 2, pp. 193--203, April 1973. doi: 10.1007/BF02872989. Abstract
     Data from 101 naturalized stands ofCannabis in east-central Illinois indicate that production of Δ1(6)-THC, Δ1-THC, cannabinol and cannabidiol was determined, to a large extent, by environmental conditions of the site where plants are grown. It is assumed that these stands represented a relatively homogeneous genetic population. Δ1-THC was under the strongest environmental control. In general, content of these compounds was higher in marihuana from stands where plants were stressed. Two types of stress were suggested by the data: nutrient deficiency and inadequate moisture. Competition from other plants enhanced the content of the drug compounds, and this relationship strengthens the stress hypothesis. Work is underway to confirm this relationship.
332. Turner, Carlton E. and Hadley, Kathy, Constituents of Cannabis Sativa L. II: Absence of Cannabidiol in an African Variant, Journal of Pharmaceutical Sciences, vol. 62, no. 2, pp. 251--255, 1973. doi: 10.1002/jps.2600620214. Abstract
     Cannabidiol is shown to be absent in an African variant of Cannabis satica L. (marijuana) grown in Mississippi. TLC, GC, and GC-mass spectrometry were used for identification. The absence of cannabidiol in a variant of African Cannabis questions the validity of published biosyntheses of the cannabinoids.
333. Fetterman, Patricia S. and Turner, Carlton E., Constituents of Cannabis Sativa L. I: Propyl Homologs of Cannabinoids from an Indian Variant, Journal of Pharmaceutical Sciences, vol. 61, no. 9, pp. 1476--1477, October 1972. doi: 10.1002/jps.2600610930. Abstract
     Cannabidivarin and tetrahydrocannabivarin are shown to be present in an Indian variant of Cannabis sativa L. (marijuana) in Mississippi. GC and mass spectrometry were used for identification. Indications are that these compounds are present as acids in fresh material.
334. Fetterman, Patricia S. and Keith, Elizabeth S. and Waller, Coy W. and Guerrero, Oswaldo and Doorenbos, Norman J. and Quimby, Maynard W., Mississippi-Grown Cannabis Sativa L.: Preliminary Observation on Chemical Definition of Phenotype and Variations in Tetrahydrocannabinol Content versus Age, Sex, and Plant Part, Journal of Pharmaceutical Sciences, vol. 60, no. 8, pp. 1246--1249, August 1971. doi: 10.1002/jps.2600600832. Abstract
     Nine strains of Cannabis sativa L. (marijuana) were grown for research by the University of Mississippi. The seeds for these strains were obtained from Iowa, Minnesota, Mexico, Turkey, Italy, France, and Sweden. The cannabinoid content was determined using GLC, and the material was divided into two chemical phenotypes according to cannabinoid content. These phenotype categories are used to differentiate between drug-type and fiber-type Cannabis sativa. In addition, the (—)-Δ9−trans-tetrahydrocannabinol content was determined for both male and female plants, various plant parts, and a Turkish variety during various stages in its growth.
335. Henry, Philippe, Multivariate Chemical Analyses of Common Cannabis Cultivars, July 2015. doi: 10.13140/RG.2.1.4126.0644. Abstract
     Here I present multivariate chemical analyses of common Cannabis cultivars based on cannabinoid and terpenoid data available from the public domain.
336. Hillig, Karl W. and Mahlberg, Paul G., A Chemotaxonomic Analysis of Cannabinoid Variation in Cannabis (Cannabaceae), American Journal of Botany, vol. 91, no. 6, pp. 966--975, 2004. doi: 10.3732/ajb.91.6.966. Abstract
     Cannabinoids are important chemotaxonomic markers unique to Cannabis. Previous studies show that a plant's dry-weight ratio of Δ9-tetrahydrocannabinol (THC) to cannabidiol (CBD) can be assigned to one of three chemotypes and that alleles BD and BT encode alloenzymes that catalyze the conversion of cannabigerol to CBD and THC, respectively. In the present study, the frequencies of BD and BT in sample populations of 157 Cannabis accessions were determined from CBD and THC banding patterns, visualized by starch gel electrophoresis. Gas chromatography was used to quantify cannabinoid levels in 96 of the same accessions. The data were interpreted with respect to previous analyses of genetic and morphological variation in the same germplasm collection. Two biotypes (infraspecific taxa of unassigned rank) of C. sativa and four biotypes of C. indica were recognized. Mean THC levels and the frequency of BT were significantly higher in C. indica than C. sativa. The proportion of high THC/CBD chemotype plants in most accessions assigned to C. sativa was {$<$}25\% and in most accessions assigned to C. indica was {$>$}25\%. Plants with relatively high levels of tetrahydrocannabivarin (THCV) and/or cannabidivarin (CBDV) were common only in C. indica. This study supports a two-species concept of Cannabis.
337. de Meijer, E., Diversity in Cannabis, 1994. url: https://research.wur.nl/en/publications/diversity-in-cannabis.
338. Ranalli, Paoli, Cannabis Germplasm Resources, 1999. Abstract
     The term “ g erm plasm ” refers to any living plant m aterial that can be used for sexual and/or vegetative propagation. It can com ­ prise entire plants, seeds, pollen, bulbs, tubers, rhizom es, cell and tissue cultures, etc. Fortunately, m ost germ plasm occurs in situ, i.e., w ild plants propagate them selves in natural vegetations and cu lti­ vated form s are m aintained by farm ers and the plant and seed com ­ m erce. G erm plasm is stored ex situ in so-called genebanks, institu ­ tions that focus on cu ltivated plants and their close w ild relatives to avoid loss of potentially useful b reeding m aterial and to facilitate the utilization by plant breeders. Loss o f breeding m aterial (genetic erosion) com prises tw o features: the loss of allele com binations and frequencies as occurring in specific populations and, m ore tragic, the total loss of alleles.
339. {de Meijer}, E. P. M. and {van Soest}, L. J. M., The CPRO Cannabis Germplasm Collection, Euphytica, vol. 62, no. 3, pp. 201--211, January 1992. doi: 10.1007/BF00041754. Abstract
     A collection of more than a 150 Cannabis accessions is being established as a source for evaluation and breeding experiments. Origin of the accessions and maintenance of the collection are described.
340. Torres, Anthony and Pauli, Christopher and Givens, Robert and Argyris, Jason and Allen, Keith and Monfort, Amparo and Gaudino, Reginald J., High-Throughput Methods to Identify Male Cannabis Sativa Using Various Genotyping Methods, Journal of Cannabis Research, vol. 4, no. 1, pp. 57, November 2022. doi: 10.1186/s42238-022-00164-7. Abstract
     Cannabis sativa is a primarily dioecious angiosperm that exhibits sexual developmental plasticity. Developmental genes for staminate male flowers have yet to be elucidated; however, there are regions of male-associated DNA from Cannabis (MADC) that correlate with the formation of pollen producing staminate flowers. MADC2 is an example of a PCR-based genetic marker that has been shown to produce a 390-bp amplicon that correlates with the expression of male phenotypes. We demonstrate applications of a cost-effective high-throughput male genotyping assay and other genotyping applications of male identification in Cannabis sativa.
341. Russo, Ethan B. and Jiang, Hong-En and Li, Xiao and Sutton, Alan and Carboni, Andrea and {del Bianco}, Francesca and Mandolino, Giuseppe and Potter, David J. and Zhao, You-Xing and Bera, Subir and Zhang, Yong-Bing and Lü, En-Guo and Ferguson, David K. and Hueber, Francis and Zhao, Liang-Cheng and Liu, Chang-Jiang and Wang, Yu-Fei and Li, Cheng-Sen, Phytochemical and Genetic Analyses of Ancient Cannabis from Central Asia, Journal of Experimental Botany, vol. 59, no. 15, pp. 4171--4182, November 2008. doi: 10.1093/jxb/ern260. Abstract
     The Yanghai Tombs near Turpan, Xinjiang-Uighur Autonomous Region, China have recently been excavated to reveal the 2700-year-old grave of a Caucasoid shaman whose accoutrements included a large cache of cannabis, superbly preserved by climatic and burial conditions. A multidisciplinary international team demonstrated through botanical examination, phytochemical investigation, and genetic deoxyribonucleic acid analysis by polymerase chain reaction that this material contained tetrahydrocannabinol, the psychoactive component of cannabis, its oxidative degradation product, cannabinol, other metabolites, and its synthetic enzyme, tetrahydrocannabinolic acid synthase, as well as a novel genetic variant with two single nucleotide polymorphisms. The cannabis was presumably employed by this culture as a medicinal or psychoactive agent, or an aid to divination. To our knowledge, these investigations provide the oldest documentation of cannabis as a pharmacologically active agent, and contribute to the medical and archaeological record of this pre-Silk Road culture.
342. Small, E. and Beckstead, H. D., Common Cannabinoid Phenotypes in 350 Stocks of Cannabis, Lloydia, 1973. url: https://europepmc.org/article/med/4744553. Abstract
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343. Small, Ernest and Beckstead, H. D., Cannabinoid Phenotypes in Cannabis Sativa, Nature, vol. 245, no. 5421, pp. 147--148, September 1973. doi: 10.1038/245147a0. Abstract
     IT has been suggested that “drug” strains and “non-drug” strains of Cannabis sativa L. comprise two comprehensive groups1,2, which can be identified on the basis of their relative content of two of the principal “cannabinoids”. Drug strains have been thought to contain an excess, usually substantial, of (—)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) in comparison with the amount of cannabidiol (CBD), including carboxylate forms of both compounds, and non-drug strains have been held to have the reverse ratio. The former compound is considered psychotomimetic (psychosis-imitating), whereas the latter is not3. In examining the above cannabinoid ratio to decide in which phenotypic group a strain belongs, some investigators2 add the amount of cannabinol (CBN) to the amount of THC. The former seems to be an oxidation product of Δ9-THC (ref. 4) and is not considered to be psychoactive.
344. Marks, M. David and Tian, Li and Wenger, Jonathan P. and Omburo, Stephanie N. and {Soto-Fuentes}, Wilfredo and He, Ji and Gang, David R. and Weiblen, George D. and Dixon, Richard A., Identification of Candidate Genes Affecting Δ9-Tetrahydrocannabinol Biosynthesis in Cannabis Sativa, Journal of Experimental Botany, vol. 60, no. 13, pp. 3715--3726, September 2009. doi: 10.1093/jxb/erp210.
345. {Flores-Sanchez}, Isvett Josefina and Verpoorte, Robert, PKS Activities and Biosynthesis of Cannabinoids and Flavonoids in Cannabis Sativa L. Plants, Plant and Cell Physiology, vol. 49, no. 12, pp. 1767--1782, December 2008. doi: 10.1093/pcp/pcn150.
346. Taura, Futoshi and Sirikantaramas, Supaart and Shoyama, Yoshinari and Shoyama, Yukihiro and Morimoto, Satoshi, Phytocannabinoids in Cannabis Sativa: Recent Studies on Biosynthetic Enzymes, Chemistry \& Biodiversity, vol. 4, no. 8, pp. 1649--1663, 2007. doi: 10.1002/cbdv.200790145.
347. Taura, Futoshi and Sirikantaramas, Supaart and Shoyama, Yoshinari and Yoshikai, Kazuyoshi and Shoyama, Yukihiro and Morimoto, Satoshi, Cannabidiolic-Acid Synthase, the Chemotype-Determining Enzyme in the Fiber-Type Cannabis Sativa, FEBS Letters, vol. 581, no. 16, pp. 2929--2934, June 2007. doi: 10.1016/j.febslet.2007.05.043. Abstract
     Cannabidiolic-acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic-acid into CBDA, the dominant cannabinoid constituent of the fiber-type Cannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene-specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic-acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug-type Cannabis plants.
348. Sirikantaramas, Supaart and Morimoto, Satoshi and Shoyama, Yoshinari and Ishikawa, Yu and Wada, Yoshiko and Shoyama, Yukihiro and Taura, Futoshi, The Gene Controlling Marijuana Psychoactivity: Molecular Cloning and Heterologous Expression of Δ1-Tetrahydrocannabinolic Acid Synthase from Cannabis Sativa L. *, Journal of Biological Chemistry, vol. 279, no. 38, pp. 39767--39774, September 2004. doi: 10.1074/jbc.M403693200. Abstract
     {$<$}p{$>$}Δ\textsuperscript{1}-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Δ\textsuperscript{1}-tetrahydrocannabinol. We cloned a novel cDNA (GenBank™ accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of \emph{Cannabis sativa}. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from \emph{Eschscholtzia californica}, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.{$<$}/p{$>$}
349. Raharjo, Tri J and Chang, Wen-Te and Choi, Young Hae and {Peltenburg-Looman}, Anja M. G and Verpoorte, Robert, Olivetol as Product of a Polyketide Synthase in Cannabis Sativa L, Plant Science, vol. 166, no. 2, pp. 381--385, February 2004. doi: 10.1016/j.plantsci.2003.09.027. Abstract
     A polyketide synthase (PKS) was suggested to catalyze the first step of cannabinoid biosynthesis, leading to olivetolic acid. An activity of a PKS was detected in the protein extract of Cannabis sativa flowering top. The enzyme converts one molecule of n-hexanoyl-CoA and three molecules of malonyl-CoA to olivetol. The product was identified by its UV-spectrum, mass spectrometry analysis and comparison with reference compound. The activity of the enzyme was also found in the upper leaves, but the activity occurring there is lesser than in the one occurring in the flowers. The activity of chalcone synthase (CHS), another PKS enzyme, was also found in the protein extract.
350. Raharjo, Tri J. and Widjaja, Ivy and Roytrakul, Sittiruk and Verpoorte, Robert, Comparative Proteomics of Cannabis Sativa Plant Tissues, Journal of Biomolecular Techniques : JBT, vol. 15, no. 2, pp. 97--106, June 2004. url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291677/. Abstract
     Comparative proteomics of leaves, flowers, and glands of Cannabis sativa have been used to identify specific tissue-expressed proteins. These tissues have significantly different levels of cannabinoids. Cannabinoids accumulate primarily in the glands but can also be found in flowers and leaves. Proteins extracted from glands, flowers, and leaves were separated using two-dimensional gel electrophoresis. Over 800 protein spots were reproducibly resolved in the two-dimensional gels from leaves and flowers. The patterns of the gels were different and little correlation among the proteins could be observed. Some proteins that were only expressed in flowers were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprint database searching. Flower and gland proteomes were also compared, with the finding that less then half of the proteins expressed in flowers were also expressed in glands. Some selected gland protein spots were identified: F1D9.26-unknown prot. (Arabidopsis thaliana), phospholipase D beta 1 isoform 1a (Gossypium hirsutum), and PG1 (Hordeum vulgare). Western blotting was employed to identify a polyketide synthase, an enzyme believed to be involved in cannabinoid biosynthesis, resulting in detection of a single protein.
351. Morimoto, Satoshi and Komatsu, Kengo and Taura, Futoshi and Shoyama, Yukihiro, Purification and Characterization of Cannabichromenic Acid Synthase from Cannabis Sativa, Phytochemistry, vol. 49, no. 6, pp. 1525--1529, November 1998. doi: 10.1016/S0031-9422(98)00278-7. Abstract
     Cannabichromenic acid synthase was purified to apparent homogeneity by sequential column chromatography including DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The enzyme catalysed the oxidocyclization of cannabigerolic acid and cannabinerolic acid to cannabichromenic acid. The Km values for both substrates were in the same order of magnitude although the Vmax value for the former was higher than that for the latter. These results suggested that cannabichromenic acid is predominantly formed from cannabigerolic acid rather than cannabinerolic acid. The enzyme required neither molecular oxygen nor hydrogen peroxide, indicating that the cannabichromenic acid synthase reaction proceeds through direct dehydrogenation without hydroxylation.
352. Turner, Carlton E. and Elsohly, Mohmoud A., Constituents of Cannabis Sativa L. XVI. A Possible Decomposition Pathway of Δ9-Tetrahydrocannabinol to Cannabinol, Journal of Heterocyclic Chemistry, vol. 16, no. 8, pp. 1667--1668, 1979. doi: 10.1002/jhet.5570160834. Abstract
     A pathway is proposed for the decomposition of Δ9-tetrahydroeannabinol (I) and its Δ8-isomer (IX) with the eventual formation of cannabinol (II) through epoxy and hydroxylated intermediates.
353. Koley, Somnath and Junker, B, Biosynthesis of Terpenoids in Trichomes, June 2015. doi: 10.13140/RG.2.1.1406.4480. Abstract
     Plant terpenoids play a significant role in production of plant pigments and in secondary defense. Plants of the Lamiaceae family (e.g. Thymus vulgaris, Men-tha piperita) synthesize different terpenoids of pharmaceutical importance, such as menthone, menthol, thymol, and carvacrol. Synthesis and storage of terpenoids occurs in trichomes (fine hairy outgrowth on the aerial surface of the plant), especially the peltate glandular trichomes of thyme and mint plants. There are two distinct pathways for the biosynthesis of terpenoids. Sesquiterpenes (C 15) are produced via the mevalonate (MEV) pathway in the cytosol of the cells, whereas monoterpenes (C 10) and diterpenes (C 20) are synthesized via the non-mevalonate (MEP) pathway in plastids. Precursors for both pathways are provided by glycolysis. The common intermediate products of these pathways are isopentenyl pyrophosphate (IPP, C 5), dimethylallyl pyrophosphate (DMAPP, C 5) and geranyl pyrophosphate (GPP, C 10). The main objectives of my study are to quantify the contribution of both pathways on the production of terpenoid precursors, and to investigate whether any exchange of intermediates occurs between these pathways. In this aspect, the introduction of 13 C metabolic flux analysis in steady state is the best technique to quantify the terpenoids production. To obtain reproducible and controllable culture conditions , this technique needs a cultivation of living trichomes after isolation from leaves and incorporation of 13 C labeled compounds. The experiment would facilitate breeders and future researchers to study the different terpene synthase (TPS) genes and their specific relation to the plant with regard to biotic and abiotic stress.
354. Lange, B. Markus and Turner, Glenn W., Terpenoid Biosynthesis in Trichomes--Current Status and Future Opportunities, Plant Biotechnology Journal, vol. 11, no. 1, pp. 2--22, January 2013. doi: 10.1111/j.1467-7652.2012.00737.x. Abstract
     Glandular trichomes are anatomical structures specialized for the synthesis of secreted natural products. In this review we focus on the description of glands that accumulate terpenoid essential oils and oleoresins. We also provide an in-depth account of the current knowledge about the biosynthesis of terpenoids and secretion mechanisms in the highly specialized secretory cells of glandular trichomes, and highlight the implications for metabolic engineering efforts.
355. Fellermeier, Monika and Eisenreich, Wolfgang and Bacher, Adelbert and Zenk, Meinhart H., Biosynthesis of Cannabinoids, European Journal of Biochemistry, vol. 268, no. 6, pp. 1596--1604, 2001. doi: 10.1046/j.1432-1327.2001.02030.x. Abstract
     The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1-13C]glucose or [U-13C6]glucose. 13C-labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. 13C enrichments and coupling patterns show that the C10-terpenoid moiety is biosynthesized entirely or predominantly ({$>$} 98\%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide-type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.
356. Gershenzon, J., Metabolic Costs of Terpenoid Accumulation in Higher Plants, Journal of Chemical Ecology, vol. 20, no. 6, pp. 1281--1328, June 1994. doi: 10.1007/BF02059810. Abstract
     The net value of any plant trait can be assessed by measuring the costs and benefits associated with that trait. While the other contributors to this issue examine the possible benefits of terpenoids to plants, this article explores the metabolic costs of terpenoid accumulation in plants in the light of recent advances in terpenoid biochemistry. Terpenoids are more expensive to manufacture per gram than most other primary and secondary metabolites due to their extensive chemical reduction. The enzyme costs of making terpenoids are also high since terpenoid biosynthetic enzymes are apparently not shared with other metabolic pathways. In fact, plant cells may even possess more than one set of enzymes for catalyzing the basic steps of terpenoid formation. Terpenoids are usually sequestered in complex, multicellular secretory structures, and so storage costs for these substances are also likely to be substantial. However, not all of the processes involved in terpenoid accumulation require large investments of resources. For instance, the maintenance of terpenoid pools is probably inexpensive because there is no evidence that substantial quantities of terpenes are lost as a result of metabolic turnover, volatilization, or leaching. Moreover, plants may reduce their net terpenoid costs by employing individual compounds in more than one role or by catabolizing substances that are no longer needed, although it is still unclear if such practices are widespread. These findings (and other facets of terpenoid biochemistry and physiology) are discussed in relation to the assumptions and predictions of several current theories of plant defense, including the carbonnutrient balance hypothesis, the growth-differentiation balance hypothesis, and the resource availability hypothesis.
357. Degenhardt, F. and Stehle, F. and Kayser, O., Chapter 2 - The Biosynthesis of Cannabinoids, pp. 13--23, January 2017. doi: 10.1016/B978-0-12-800756-3.00002-8. Abstract
     This chapter deals with the pathway leading to the biosynthesis of cannabinoids, focusing on the corresponding enzymes and their biochemical properties. The huge diversity of more than 60 cannabinoids is mainly achieved by nonenzymatic modification reactions like decarboxylation, isomerization, and oxidation. Apart from the enzymes responsible for cannabinoid precursor biosynthesis, only three enzymes are known to be involved in the biosynthesis of cannabinoids. The enzymes cannabidiolic acid synthase, cannabichromenic acid synthase, and tetrahydrocannabinolic acid synthase convert the central precursor of cannabinoid biosynthesis, cannabigerolic acid, to the acidic forms of cannabidiol, cannabichromene, and the main pharmacologically active compound tetrahydrocannabinol, respectively. The present chapter aims to summarize the current knowledge about the enzymes involved in cannabinoid synthesis in Cannabis sativa L. starting from primary metabolism building blocks.
358. Shoyama, Yukihiro and Yagi, Masahiro and Nishioka, Itsuo and Yamauchi, Tatsuo, Biosynthesis of Cannabinoid Acids, Phytochemistry, vol. 14, no. 10, pp. 2189--2192, October 1975. doi: 10.1016/S0031-9422(00)91096-3. Abstract
     Malonic acid, mevalonic acid, geraniol and nerol were incorporated into tetrahydrocannabinolic acid and cannabichromenic acid in Cannabis sativa. The pathway from cannabigerolic acid to tetrahydrocannabinolic acid via cannabidiolic acid was established by feeding labelled cannabinoid acids. Cannabichromenic acid was shown to be formed on a side pathway from cannabigerolic acid.
359. Luo, Xiaozhou and Reiter, Michael A. and {d’Espaux}, Leo and Wong, Jeff and Denby, Charles M. and Lechner, Anna and Zhang, Yunfeng and Grzybowski, Adrian T. and Harth, Simon and Lin, Weiyin and Lee, Hyunsu and Yu, Changhua and Shin, John and Deng, Kai and Benites, Veronica T. and Wang, George and Baidoo, Edward E. K. and Chen, Yan and Dev, Ishaan and Petzold, Christopher J. and Keasling, Jay D., Complete Biosynthesis of Cannabinoids and Their Unnatural Analogues in Yeast, Nature, vol. 567, no. 7746, pp. 123--126, March 2019. doi: 10.1038/s41586-019-0978-9. Abstract
     Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.
360. Taura, Futoshi and Morimoto, Satoshi and Shoyama, Yukihiro, Purification and Characterization of Cannabidiolic-Acid Synthase from Cannabis Sativa L.: Biochemical Analysis of a Novel Enzyme That Catalyzes the Oxidocyclization of Cannabigerolic Acid to Cannabidiolic Acid *, Journal of Biological Chemistry, vol. 271, no. 29, pp. 17411--17416, July 1996. doi: 10.1074/jbc.271.29.17411. Abstract
     {$<$}p{$>$}We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in \emph{Cannabis sativa} L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH\textsubscript{2}-terminal amino acid sequence of CBDA synthase is similar to that of Δ\textsuperscript{1}-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the \emph{k}\textsubscript{cat} for the former (0.03 s\textsuperscript{−1}) is lower than that for the latter (0.19 s\textsuperscript{−1}). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.{$<$}/p{$>$}
361. Hemphill, John K. and Turner, Jocelyn C. and Mahlberg, Paul G., Cannabinoid Content of Individual Plant Organs From Different Geographical Strains of Cannabis Sativa L., ACS Publications, July 2004. doi: 10.1021/np50007a009.
362. Turner, Jocelyn C. and Hemphill, John K. and Mahlberg, Paul G., Quantitative Determination of Cannabinoids in Individual Glandular Trichomes of Cannabis Sativa L. (Cannabaceae), American Journal of Botany, vol. 65, no. 10, pp. 1103--1106, 1978. doi: 10.1002/j.1537-2197.1978.tb06177.x. Abstract
     Cannabinoid levels of individual mature glandular trichomes from two clones and two strains of Cannabis sativa L., which included both drug and fiber phenotypes, were investigated by gas-liquid chromatographic analyses. Capitate-stalked glands were selectively harvested from vein and nonvein areas of pistillate bracts while capitate-sessile glands were harvested from these areas of leaves. The qualitative cannabinoid profile characteristic of the strain or clone was maintained in the individual capitate-stalked glands while the quantitative cannabinoid profiles varied with each strain or clone and between vein and nonvein areas as well. Capitate-sessile glands were found to contain conspicuously lower levels of cannabinoids than capitate-stalked glands. This study emphasizes that glands of Cannabis represent a dynamic system within the cannabinoid synthesizing activities of this plant.
363. Vogelmann, Ann F. and Turner, Jocelyn C. and Mahlberg, Paul G., Cannabinoid Composition in Seedlings Compared to Adult Plants of Cannabis Sativa, ACS Publications, July 2004. doi: 10.1021/np50060a004.
364. Vogelmann, Ann F. and Turner, Jocelyn C. and Mahlberg, Paul G., Cannabinoid Occurrence in Seedlings of Cannabis Sativa L.: Quantitation in Seedlings of Known Age and Primary Leaf Length, Botanical Gazette, vol. 148, no. 4, pp. 468--474, December 1987. doi: 10.1086/337678. Abstract
     Seedlings of a Δ9-tetrahydrocannabinol (THC) dominant strain of Cannabis sativa were employed to develop a biological system to investigate cannabinoid biosynthesis. High-performance liquid chromatography was used to detect and quantify the cannabinoids. Cannabinoids were first detected in seedlings of light/dark-grown plants at 48-50 h. The first cannabinoid detected was cannabichromene (CBC) at 52-54 h. At 60-62 h both cannabigerol (CBG) and THC were detected along with CBC. A similar sequence appeared in dark-grown seedlings, but CBC became evident only at 56-58 h, and THC and CBG were first detected at 66-68 h. This pattern is significant because previously proposed pathways of cannabinoid synthesis have indicated that CBG is a precursor to CBC, yet CBG is not the first cannabinoid to be detected but occurs later with the appearance of THC. Cannabinoid concentrations were always higher in light-grown than in dark-grown plants of comparable age. Cannabinoid quantities on a dry-weight basis increased with increasing leaf length and/or increasing age.
365. Latta, R. P. and Eaton, B. J., Seasonal Fluctuations in Cannabinoid Content of Kansas Marijuana, Economic Botany, vol. 29, no. 2, pp. 153--163, April 1975. doi: 10.1007/BF02863315. Abstract
     Marijuana (Cannabis sativa L.) was sampled at nine progressive growth stages in Riley County, Kansas, and analyzed for four major cannabinoids: cannabidiol (CBD), della-8-tetrahydrocannabinol (delta-8-THC), delta-9-tetrahydrocannabinol (delta-9-THC), and cannabinol (CBN). Seasonal fluctuation in cannabinoids were related to stage of plant development. Cannabinoids were lowest in seedlings, highest prior to flowering and at an intermediate level thereafter until physiological maturity. Cannabinoids were highest in flowers and progressively lower in leaves, petioles, stems, seeds, and roots. Cannabinoid content of male and female flowers was not significantly different.
366. Rowan, M G and Fairbairn, J W, Cannabinoid Patterns in Seedlings of {\emph{Cannabis Sativa}} L. and Their Use in the Determination of Chemical Race, Journal of Pharmacy and Pharmacology, vol. 29, no. 1, pp. 491--494, April 2011. doi: 10.1111/j.2042-7158.1977.tb11375.x. Abstract
     Abstract We have examined the cannabinoid contents of seedlings from twelve strains of Cannabis of known chemical race. Fourteen days after emergence of the shoot those of the tetrahydrocannabinol (THC) type could be distinguished from those of the cannabidiol (CBD) type. The true leaves of the THC type contained a relatively high content of THC and also contained cannabichromene (CBC), sometimes as the major cannabinoid, whereas the CBD type had much lower amounts of THC and no CBC; CBD being the major component. A strain from China corresponded to the THC type but showed some unusual features. Seeds purchased at seven outlets in Britain were also examined. Only two batches germinated but these proved to be of a THC type and resembled the Chinese strain.
367. Wizenberg, Sydney B. and Dang, Michelle and Campbell, Lesley G., Methods for Characterizing Pollen Fitness in Cannabis Sativa L., PLOS ONE, vol. 17, no. 7, pp. e0270799, July 2022. doi: 10.1371/journal.pone.0270799. Abstract
     Pollen grains are male gametophytes, an ephemeral haploid generation of plants, that commonly engage in competition for a limited supply of ovules. Since variation in reproductive capabilities among male gametophytes may influence the direction and pace of evolution in populations, we must be able to quantify the relative fitness of gametophytes from different sires. To explore this, we estimated the relative fitness of groups of male gametophytes in a dioecious, wind-pollinated model system, Cannabis sativa, by characterizing the non-abortion rate (measured via chemical staining) and viability (measured via in vitro germination) of pollen from multiple sires. Pollen viability quickly declined within two weeks of anther dehiscence, and pollen stored under freezer conditions did not germinate regardless of storage time. In contrast, pollen non-abortion rates declined slowly and persisted longer than the lifetime of a sporophyte plant under both room temperature and freezer conditions. Pollen samples that underwent both viability and non-abortion rate analysis displayed no significant correlation, implying that researchers cannot predict pollen viability from non-abortion rates, nor infer male gametophytic fitness from a single measure. Our work demonstrates two independent, differential approaches to measure proxies of male fitness in C. sativa.
368. Teleszko, Mirosława and Zając, Adam and Rusak, Tomasz, Hemp Seeds of the Polish ‘Bialobrzeskie’ and ‘Henola’ Varieties (Cannabis Sativa L. Var. Sativa) as Prospective Plant Sources for Food Production, Molecules, vol. 27, no. 4, pp. 1448, January 2022. doi: 10.3390/molecules27041448. Abstract
     This publication characterizes the nutritional value of the Polish hemp seeds of the ‘Bialobrzeskie’ and ‘Henola’ varieties, including the profile/content of fatty acids and amino acids. Hemp seeds were found to be rich in protein, fat, and dietary fiber. Polyunsaturated fatty acids (PUFA) dominated the unsaturated fatty acids (UFA) profile. Their average share within the total fatty acids (FA) was as high as 75\%. Linoleic acid belonging to this group accounted for 55\% of the total FA. Lipid profile indices (Σ n − 6/Σ n − 3, Σ PUFA/Σ SFA, the thrombogenicity index, the atherogenicity index and the hypocholesterolemic/hypercholesterolemic ratio) proved the high nutritional value of hemp oil. Considering the tyrosine + phenylalanine and histidine contents, hemp protein exhibited a great degree of similarity to egg protein, which is known and valued for its high biological value.
369. {Naim-Feil}, Erez and Pembleton, Luke W. and Spooner, Laura E. and Malthouse, Alix L. and Miner, Amy and Quinn, Melinda and Polotnianka, Renata M. and Baillie, Rebecca C. and Spangenberg, German C. and Cogan, Noel O. I., The Characterization of Key Physiological Traits of Medicinal Cannabis (Cannabis Sativa L.) as a Tool for Precision Breeding, BMC Plant Biology, vol. 21, no. 1, pp. 294, June 2021. doi: 10.1186/s12870-021-03079-2. Abstract
     For millennia, drug-type cannabis strains were extensively used for various medicinal, ritual, and inebriant applications. However, cannabis prohibition during the last century led to cultivation and breeding activities being conducted under clandestine conditions, while scientific development of the crop ceased. Recently, the potential of medicinal cannabis has been reacknowledged and the now expanding industry requires optimal and scientifically characterized varieties. However, scientific knowledge that can propel this advancement is sorely lacking. To address this issue, the current study aims to provide a better understanding of key physiological and phenological traits that can facilitate the breeding of advanced cultivars.
370. Garfinkel, Andrea R. and Otten, Matthew and Crawford, Seth, SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in Cannabis Sativa L., Genes, vol. 12, no. 2, pp. 228, February 2021. doi: 10.3390/genes12020228. Abstract
     The regulation of cannabinoid synthesis in Cannabis sativa is of increasing research interest as restrictions around the globe loosen to allow the plant’s legal cultivation. Of the major cannabinoids, the regulation of cannabigerolic acid (CBGA) production is the least understood. The purpose of this study was to elucidate the inheritance of CBGA dominance in C. sativa and describe a marker related to this chemotype. We produced two crossing populations, one between a CBGA dominant cultivar and a tetrahydrocannabinolic acid (THCA) dominant cultivar, and one between a CBGA dominant cultivar and a cannabidiolic acid (CBDA) cultivar. Chemical and genotyping analyses confirmed that CBGA dominance is inherited as a single recessive gene, potentially governed by a non-functioning allelic variant of the THCA synthase. The “null” THCAS synthase contains a single nucleotide polymorphism (SNP) that may render the synthase unable to convert CBGA to THCA leading to the accumulation of CBGA. This SNP can be reliably used as a molecular marker for CBGA dominance in the selection and breeding of C. sativa.
371. Ahmed, Sajjad and Gao, Xuefei and Jahan, Md. Asraful and Adams, Maxwell and Wu, Nianqiang and Kovinich, Nik, Nanoparticle-Based Genetic Transformation of Cannabis Sativa, Journal of Biotechnology, vol. 326, pp. 48--51, January 2021. doi: 10.1016/j.jbiotec.2020.12.014. Abstract
     Cannabis sativa (Cannabis) is a multipurpose plant species consisting of specific lineages that for centuries has either been artificially selected for the production of fiber or the psychoactive drug Δ9-tetrahydrocannabinol (THC). With the recent lifting of previous legal restrictions on consuming Cannabis, there has been a resurgence of interest in understanding and manipulating Cannabis genetics to enhance its compositions. Yet, recently developed approaches are not amenable to high-throughput gene stacking to study multi-genic traits. Here, we demonstrate an efficient nanoparticle-based transient gene transformation protocol where multiple gene plasmids can be expressed simultaneously in intact Cannabis leaf cells in a very short time (5 days). Constructs encoding two soybean transcription factors were co-grafted onto poly-ethylenimine cationic polymer-modified silicon dioxide-coated gold nanoparticles (PEI-Au@SiO2). Infiltration of the DNA-PEI-Au@SiO2 into Cannabis leaf tissues resulted in the transcription of both soybean genes and the localization of fluorescent-tagged transcription factor proteins in the nuclei of Cannabis leaf cells including the trichomes, which are the cell types that biosynthesize valuable cannabinoid and terpene metabolites. Our study exemplifies a rapid transient gene transformation approach that will be useful to study the effects of gene stacking in Cannabis.
372. Ren, Guangpeng and Zhang, Xu and Li, Ying and Ridout, Kate and {Serrano-Serrano}, Martha L. and Yang, Yongzhi and Liu, Ai and Ravikanth, Gudasalamani and Nawaz, Muhammad Ali and Mumtaz, Abdul Samad and Salamin, Nicolas and Fumagalli, Luca, Large-Scale Whole-Genome Resequencing Unravels the Domestication History of Cannabis Sativa, Science Advances, vol. 7, no. 29, pp. eabg2286, July 2021. doi: 10.1126/sciadv.abg2286. Abstract
     Cannabis sativa has long been an important source of fiber extracted from hemp and both medicinal and recreational drugs based on cannabinoid compounds. Here, we investigated its poorly known domestication history using whole-genome resequencing of 110 accessions from worldwide origins. We show that C. sativa was first domesticated in early Neolithic times in East Asia and that all current hemp and drug cultivars diverged from an ancestral gene pool currently represented by feral plants and landraces in China. We identified candidate genes associated with traits differentiating hemp and drug cultivars, including branching pattern and cellulose/lignin biosynthesis. We also found evidence for loss of function of genes involved in the synthesis of the two major biochemically competing cannabinoids during selection for increased fiber production or psychoactive properties. Our results provide a unique global view of the domestication of C. sativa and offer valuable genomic resources for ongoing functional and molecular breeding research.
373. Wimalasiri, Eranga M. and Jahanshiri, Ebrahim and Chimonyo, Vimbayi G. P. and Kuruppuarachchi, Niluka and Suhairi, T. A. S. T. M. and {Azam-Ali}, Sayed N. and Gregory, Peter J., A Framework for the Development of Hemp (Cannabis Sativa L.) as a Crop for the Future in Tropical Environments, Industrial Crops and Products, vol. 172, pp. 113999, November 2021. doi: 10.1016/j.indcrop.2021.113999. Abstract
     Hemp (Cannabis sativa L.) is a multipurpose industrial crop which is mainly cultivated in temperate regions. With its high potential for economic returns for its seeds and fiber, there is growing interest in cultivating hemp in many territories including Malaysia and other Asian countries, where its cultivation is currently illegal. To date, no comprehensive study on the suitability of this crop under Malaysian conditions has been conducted. In this paper, we propose an assessment framework as a roadmap to develop the hemp industry in Malaysia and possibly other Asian countries with equatorial climates. This framework includes suitability assessment (climate and soil), crop modelling (current and future yields under climate change) and economic analysis (net present value (NPV), NPV benefit (NPVB) and benefit-cost ratio (BCR). The land suitability assessment classified hemp as an adaptable crop for most of the land in the country. The AquaCrop model, parameterised from secondary data collected from literature was used in simulations and potential yield mapping. The estimated average potential seed and fiber yield at six locations between 2010 and 2019 was 1.61\,±\,0.25 and 2.78\,±\,0.39\,t ha–1 respectively. Using five general circulation model (GCM) simulations, yields under future climates in Malaysia showed an increase in most of the locations. The highest NPVB of 1641 USD ha–1 (BCR of 1.33) for seed was estimated under current climate conditions. Yields of 1.38\,t ha–1 (seed) and 3.62\,t ha–1 (fiber) are the minimum economically feasible yields with a Benefit-Cost Ratio of 1.00 suggesting a potential for hemp cultivation in comparison to countries with established hemp industries. The present framework could be used to develop a pathway for adoption of hemp as a crop for the future in tropical countries.
374. Wenger, Jonathan P. and Dabney III, Clemon J. and ElSohly, Mahmoud A. and Chandra, Suman and Radwan, Mohamed M. and Majumdar, Chandrani G. and Weiblen, George D., Validating a Predictive Model of Cannabinoid Inheritance with Feral, Clinical, and Industrial Cannabis Sativa, American Journal of Botany, vol. 107, no. 10, pp. 1423--1432, 2020. doi: 10.1002/ajb2.1550. Abstract
     Premise How genetic variation within a species affects phytochemical composition is a fundamental question in botany. The ratio of two specialized metabolites in Cannabis sativa, tetrahydrocannabinol (THC) and cannabidiol (CBD), can be grouped into three main classes (THC-type, CBD-type, and intermediate type). We tested a genetic model associating these three groups with functional and nonfunctional alleles of the cannabidiolic acid synthase gene (CBDAS). Methods We characterized cannabinoid content and assayed CBDAS genotypes of {$>$}300 feral C. sativa plants in Minnesota, United States. We performed a test cross to assess CBDAS inheritance. Twenty clinical cultivars obtained blindly from the National Institute on Drug Abuse and 12 Canadian-certified grain cultivars were also examined. Results Frequencies of CBD-type, intermediate-type, and THC-type feral plants were 0.88, 0.11, and 0.01, respectively. Although total cannabinoid content varied substantially, the three groupings were perfectly correlated with CBDAS genotypes. Genotype frequencies observed in the test cross were consistent with codominant Mendelian inheritance of the THC:CBD ratio. Despite significant mean differences in total cannabinoid content, CBDAS genotypes blindly predicted the THC:CBD ratio among clinical cultivars, and the same was true for industrial grain cultivars when plants exhibited {$>$}0.5\% total cannabinoid content. Conclusions Our results extend the generality of the inheritance model for THC:CBD to diverse C. sativa accessions and demonstrate that CBDAS genotyping can predict the ratio in a variety of practical applications. Cannabinoid profiles and associated CBDAS segregation patterns suggest that feral C. sativa populations are potentially valuable experimental systems and sources of germplasm.
375. Deguchi, Michihito and Kane, Shriya and Potlakayala, Shobha and George, Hannah and Proano, Renata and Sheri, Vijay and Curtis, Wayne R. and Rudrabhatla, Sairam, Metabolic Engineering Strategies of Industrial Hemp (Cannabis Sativa L.): A Brief Review of the Advances and Challenges, Frontiers in Plant Science, vol. 11, 2020. doi: 10.3389/fpls.2020.580621. Abstract
     Industrial hemp (Cannabis sativa L.) is a diploid (2n = 20), dioecious plant that is grown for fiber, seed, and oil. Recently, there has been a renewed interest in this crop because of its panoply of cannabinoids, terpenes, and other phenolic compounds. Specifically, hemp contains terpenophenolic compounds such as cannabidiol (CBD) and cannabigerol (CBG), which act on cannabinoid receptors and positively regulate various human metabolic, immunological, and physiological functions. CBD and CBG have an effect on the cytokine metabolism, which has led to the examination of cannabinoids on the treatment of viral diseases, including COVID-19. Based on genomic, transcriptomic, and metabolomic studies, several synthetic pathways of hemp secondary metabolite production have been elucidated. Nevertheless, there are few reports on hemp metabolic engineering despite obvious impact on scientific and industrial sectors.In this article, recent status and current perspectives on hemp metabolic engineering are reviewed. Three distinct approaches to expedite phytochemical yield are discussed. Special emphasis has been placed on transgenic and transient gene delivery systems, which are critical for successful metabolic engineering of hemp. The advent of new tools in synthetic biology, particularly the CRISPR/Cas systems, enables environment-friendly metabolic engineering to increase the production of desirable hemp phytochemicals while eliminating the psychoactive compounds, such as tetrahydrocannabinol (THC).
376. Deguchi, Michihito and Bogush, Daniel and Weeden, Hannah and Spuhler, Zachary and Potlakayala, Shobha and Kondo, Takumasa and Zhang, Zhanyuan J. and Rudrabhatla, Sairam, Establishment and Optimization of a Hemp (Cannabis Sativa L.) Agroinfiltration System for Gene Expression and Silencing Studies, Scientific Reports, vol. 10, no. 1, pp. 3504, February 2020. doi: 10.1038/s41598-020-60323-9. Abstract
     Industrial hemp (Cannabis sativa L.) is a high-yielding annual crop primarily grown for fiber, seeds, and oil. Due to the phytochemical composition of hemp, there has been an increased interest in the market for nutraceuticals and dietary supplements for human health. Recent omics analysis has led to the elucidation of hemp candidate genes involved in the syntheses of specialized metabolites. However, a detailed study of these genes has not been undertaken due to the lack of a stable transformation system. We report for the first time an agroinfiltration system in hemp utilizing vacuum infiltration, which is an alternative method to stable transformation. A combination of 0.015\% Silwett L-77, 5\,mM ascorbic acid, and thirty second sonication followed by a 10-minute vacuum treatment resulted in the highest β-glucuronidase expression in the leaf, male and female flowers, stem, and root tissues. The phytoene desaturase gene was silenced with a transient hairpin RNA expression, resulting in an albino phenotype in the leaves and the male and female flowers. This agroinfiltration system would be useful for overexpression and silencing studies of target genes to regulate the yield of specialized metabolites in hemp.
377. Backer, Rachel and Mandolino, Giuseppe and Wilkins, Olivia and ElSohly, Mahmoud A. and Smith, Donald L., Editorial: Cannabis Genomics, Breeding and Production, Frontiers in Plant Science, vol. 11, 2020. doi: 10.3389/fpls.2020.591445.
378. Borroto Fernandez, Eduviges and Peterseil, Verena and Hackl, Gerald and Menges, Sonja and {de Meijer}, Etienne and Staginnus, Christina, Distribution of Chemical Phenotypes (Chemotypes) in European Agricultural Hemp (Cannabis Sativa L.) Cultivars, Journal of Forensic Sciences, vol. 65, no. 3, pp. 715--721, 2020. doi: 10.1111/1556-4029.14242. Abstract
     In Europe, more than 50 approved cultivars of fiber hemp (Cannabis sativa L.) are in agricultural production. Their content of psychoactive tetrahydrocannabinol (THC) is legally restricted to {$<$}0.2\% (\%w/w in the dry, mature inflorescences). Cannabis strains with much higher THC contents are also grown, illegally or under license for drug production. Differentiation between these two groups relies on biochemical quantification of cannabinoid contents in mature floral material. For nonflowering material or tissue devoid of cannabinoids, the genetic prediction of the chemical phenotype (chemotype) provides a suitable method of distinction. Three discrete chemotypes, depending on the ratio of THC and the noneuphoric cannabidiol (CBD), can be distinguished: a “THC-predominant” type, a “CBD-predominant” type, and an intermediate chemotype. We present a systematic genetic prediction of chemotypes of 62 agricultural hemp cultivars grown in Europe. The survey reveals the presence of up to 35\% BT allele-carrying individuals (representing either a THC-predominant or an intermediate chemotype) in some cultivars—which is unexpected considering the legal THC limit of 0.2\% THC. The fact that 100\% of the seized drug-type seeds in this study revealed at least one BT allele, reflects that plant breeding efforts have resulted in a fixation of the BT allele in recreational Cannabis. To guarantee a sincere forensic application based on a genetic chemotype prediction, we recommend not to classify material of unknown origin if the samples size is below nine genetically independent individuals.
379. Toth, Jacob A. and Stack, George M. and Cala, Ali R. and Carlson, Craig H. and Wilk, Rebecca L. and Crawford, Jamie L. and Viands, Donald R. and Philippe, Glenn and Smart, Christine D. and Rose, Jocelyn K. C. and Smart, Lawrence B., Development and Validation of Genetic Markers for Sex and Cannabinoid Chemotype in Cannabis Sativa L., GCB Bioenergy, vol. 12, no. 3, pp. 213--222, 2020. doi: 10.1111/gcbb.12667. Abstract
     Hemp (Cannabis sativa L.) is an emerging dioecious crop grown primarily for grain, fiber, and cannabinoids. There is good evidence for medicinal benefits of the most abundant cannabinoid in hemp, cannabidiol (CBD). For CBD production, female plants producing CBD but not tetrahydrocannabinol (THC) are desired. We developed and validated high-throughput PACE (PCR Allele Competitive Extension) assays for C. sativa plant sex and cannabinoid chemotype. The sex assay was validated across a wide range of germplasm and resolved male plants from female and monoecious plants. The cannabinoid chemotype assay revealed segregation in hemp populations, and resolved plants producing predominantly THC, predominantly CBD, and roughly equal amounts of THC and CBD. Cultivar populations that were thought to be stabilized for CBD production were found to be segregating phenotypically and genotypically. Many plants predominantly producing CBD accumulated more than the current US legal limit of 0.3\% THC by dry weight. These assays and data provide potentially useful tools for breeding and early selection of hemp.
380. Aliferis, Konstantinos A. and {Bernard-Perron}, David, Cannabinomics: Application of Metabolomics in Cannabis (Cannabis Sativa L.) Research and Development, Frontiers in Plant Science, vol. 11, 2020. doi: 10.3389/fpls.2020.00554. Abstract
     Cannabis (Cannabis sativa L.) is a complex, polymorphic plant species, which produces a vast array of bioactive metabolites, the two major chemical groups being cannabinoids and terpenoids. Nonetheless, the psychoactive cannabinoid tetrahydrocannabinol (Δ9-THC) and the non-psychoactive cannabidiol (CBD), are the two major cannabinoids that have monopolized the research interest. Currently, more than 600 Cannabis varieties are commercially available, providing access to a multitude of potent extracts with complex compositions, whose genetics are largely inconclusive. Recently introduced legislation on Cannabis cultivation in many countries represents a great opportunity, but at the same time, a great challenge for Cannabis research and development (R\&D) toward applications in the pharmaceutical, food, cosmetics, and agrochemical industries. Based on its versatility and unique capabilities in the deconvolution of the metabolite composition of complex matrices, metabolomics represents an ideal bioanalytical tool that could greatly assist and accelerate Cannabis R\&D. Among others, Cannabis metabolomics or cannabinomics can be applied in the taxonomy of Cannabis varieties in chemovars, the research on the discovery and assessment of new Cannabis-based sources of bioactivity in medicine, the development of new food products, and the optimization of its cultivation, aiming for improvements in yield and potency. Although Cannabis research is still in its infancy, it is highly foreseen that the employment of advanced metabolomics will provide insights that could assist the sector to face the aforementioned challenges. Within this context, here, the current state-of-the-art and conceptual aspects of cannabinomics are presented.
381. Prentout, Djivan and Razumova, Olga and Rhoné, Bénédicte and Badouin, Hélène and Henri, Hélène and Feng, Cong and Käfer, Jos and Karlov, Gennady and Marais, Gabriel A. B., An Efficient RNA-Seq-Based Segregation Analysis Identifies the Sex Chromosomes of Cannabis Sativa, Genome Research, vol. 30, no. 2, pp. 164--172, February 2020. doi: 10.1101/gr.251207.119. Abstract
     Cannabis sativa–derived tetrahydrocannabinol (THC) production is increasing very fast worldwide. C. sativa is a dioecious plant with XY Chromosomes, and only females (XX) are useful for THC production. Identifying the sex chromosome sequence would improve early sexing and better management of this crop; however, the C. sativa genome projects have failed to do so. Moreover, as dioecy in the Cannabaceae family is ancestral, C. sativa sex chromosomes are potentially old and thus very interesting to study, as little is known about old plant sex chromosomes. Here, we RNA-sequenced a C. sativa family (two parents and 10 male and female offspring, 576 million reads) and performed a segregation analysis for all C. sativa genes using the probabilistic method SEX-DETector. We identified {$>$}500 sex-linked genes. Mapping of these sex-linked genes to a C. sativa genome assembly identified the largest chromosome pair being the sex chromosomes. We found that the X-specific region (not recombining between X and Y) is large compared to other plant systems. Further analysis of the sex-linked genes revealed that C. sativa has a strongly degenerated Y Chromosome and may represent the oldest plant sex chromosome system documented so far. Our study revealed that old plant sex chromosomes can have large, highly divergent nonrecombining regions, yet still be roughly homomorphic.
382. McPartland, John M. and Small, Ernest, A Classification of Endangered High-THC Cannabis (Cannabis Sativa Subsp. Indica) Domesticates and Their Wild Relatives, PhytoKeys, vol. 144, pp. 81--112, April 2020. doi: 10.3897/phytokeys.144.46700. Abstract
     Two kinds of drug-type Cannabis gained layman’s terms in the 1980s. “Sativa” had origins in South Asia (India), with early historical dissemination to Southeast Asia, Africa, and the Americas. “Indica” had origins in Central Asia (Afghanistan, Pakistan, Turkestan). We have assigned unambiguous taxonomic names to these varieties, after examining morphological characters in 1100 herbarium specimens, and analyzing phytochemical and genetic data from the literature in a meta-analysis. “Sativa” and “Indica” are recognized as C. sativa subsp. indica var. indica and C. sativa subsp. indica var. afghanica, respectively. Their wild-growing relatives are C. sativa subsp. indica var. himalayensis (in South Asia), and C. sativa subsp. indica var. asperrima (in Central Asia). Natural selection initiated divergence, driven by climatic conditions in South and Central Asia. Subsequent domestication drove further phytochemical divergence. South and Central Asian domesticates can be distinguished by tetrahydrocannabinol and cannabidiol content (THC/CBD ratios, ≥7 or {$<$}7, respectively), terpenoid profiles (absence or presence of sesquiterpene alcohols), and a suite of morphological characters. The two domesticates have undergone widespread introgressive hybridization in the past 50 years. This has obliterated differences between hybridized “Sativa” and “Indica” currently available. “Strains” alleged to represent “Sativa” and “Indica” are usually based on THC/CBD ratios of plants with undocumented hybrid backgrounds (with so-called “Indicas” often delimited simply on possession of more CBD than “Sativas”). The classification presented here circumscribes and names four taxa of Cannabis that represent critically endangered reservoirs of germplasm from which modern cannabinoid strains originated, and which are in urgent need of conservation.
383. Parsons, Jessica L. and Martin, Sara L. and James, Tracey and Golenia, Gregory and Boudko, Ekaterina A. and Hepworth, Shelley R., Polyploidization for the Genetic Improvement of Cannabis Sativa, Frontiers in Plant Science, vol. 10, 2019. doi: 10.3389/fpls.2019.00476. Abstract
     Cannabis sativa L. is a diploid species, cultivated throughout the ages as a source of fiber, food, and secondary metabolites with therapeutic and recreational properties. Polyploidization is considered as a valuable tool in the genetic improvement of crop plants. Although this method has been used in hemp-type Cannabis, it has never been applied to drug-type strains. Here, we describe the development of tetraploid drug-type Cannabis lines and test whether this transformation alters yield or the profile of important secondary metabolites: Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), or terpenes. The mitotic spindle inhibitor oryzalin was used to induce polyploids in a THC/CBD balanced drug-type strain of Cannabis sativa. Cultured axillary bud explants were exposed to a range of oryzalin concentrations for 24 h. Flow cytometry was used to assess the ploidy of regenerated shoots. Treatment with 20–40 μM oryzalin produced the highest number of tetraploids. Tetraploid clones were assessed for changes in morphology and chemical profile compared to diploid control plants. Tetraploid fan leaves were larger, with stomata about 30\% larger and about half as dense compared to diploids. Trichome density was increased by about 40\% on tetraploid sugar leaves, coupled with significant changes in the terpene profile and a 9\% increase in CBD that was significant in buds. No significant increase in yield of dried bud or THC content was observed. This research lays important groundwork for the breeding and development of new Cannabis strains with diverse chemical profiles, of benefit to medical and recreational users.
384. Cascini, Fidelia and Farcomeni, Alessio and Migliorini, Daniele and Baldassarri, Laura and Boschi, Ilaria and Martello, Simona and Amaducci, Stefano and Lucini, Luigi and Bernardi, Jamila, Highly Predictive Genetic Markers Distinguish Drug-Type from Fiber-Type Cannabis Sativa L, Plants, vol. 8, no. 11, pp. 496, November 2019. doi: 10.3390/plants8110496. Abstract
     Genetic markers can be used in seeds and in plants to distinguish drug-type from fiber-type Cannabis Sativa L. varieties even at early stages, including pre-germination when cannabinoids are not accumulated yet. With this aim, this paper reports sequencing results for tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes from 21 C. sativa L. varieties. Taking into account that THCAS- and CBDAS-derived enzymes compete for the same substrate, the novelty of this work relies in the identification of markers based on both THCAS and CBDAS rather than THCAS alone. Notably, in our panel, we achieved an adequate degree of discrimination (AUC 100\%) between drug-type and fiber-type cannabis samples. Our sequencing approach allowed identifying multiple genetic markers (single-nucleotide polymorphisms—SNPs—and a deletion/insertion) that effectively discriminate between the two subgroups of cannabis, namely fiber type vs. drug type. We identified four functional SNPs that are likely to induce decreased THCAS activity in the fiber-type cannabis plants. We also report the finding on a deletion in the CBDAS gene sequence that produces a truncated protein, possibly resulting in loss of function of the enzyme in the drug-type varieties. Chemical analyses for the actual concentration of cannabinoids confirmed the identification of drug-type rather than fiber-type genotypes. Genetic markers permit an early identification process for forensic applications while simplifying the procedures related to detection of therapeutic or industrial hemp.
385. Schwabe, Anna L. and McGlaughlin, Mitchell E., Genetic Tools Weed out Misconceptions of Strain Reliability in Cannabis Sativa: Implications for a Budding Industry, Journal of Cannabis Research, vol. 1, no. 1, pp. 3, June 2019. doi: 10.1186/s42238-019-0001-1. Abstract
     Unlike other plants, Cannabis sativa is excluded from regulation by the United States Department of Agriculture (USDA). Distinctive Cannabis varieties are ostracized from registration and therefore nearly impossible to verify. As Cannabis has become legal for medical and recreational consumption in many states, consumers have been exposed to a wave of novel Cannabis products with many distinctive names. Despite more than 2000 named strains being available to consumers, questions about the consistency of commercially available strains have not been investigated through scientific methodologies. As Cannabis legalization and consumption increases, the need to provide consumers with consistent products becomes more pressing. In this research, we examined commercially available, drug-type Cannabis strains using genetic methods to determine if the commonly referenced distinctions are supported and if samples with the same strain name are consistent when obtained from different facilities.
386. Baldini, Mario and Ferfuia, Claudio and Piani, Barbara and Sepulcri, Angela and Dorigo, Gaia and Zuliani, Fabio and Danuso, Francesco and Cattivello, Costantino, The Performance and Potentiality of Monoecious Hemp (Cannabis Sativa L.) Cultivars as a Multipurpose Crop, Agronomy, vol. 8, no. 9, pp. 162, September 2018. doi: 10.3390/agronomy8090162. Abstract
     Given the growing interest in multipurpose hemp crop, eight monoecious cultivars were compared in a two-year trial for quantitative and qualitative yield in a Mediterranean environment characterized by a temperate and humid climate with hot summers. All hemp cultivars were evaluated for yield potential of (i) seed plus stem at seed maturity, and (ii) essential oil yield from inflorescences harvested at full flowering. The second goal was set to test the ability of cultivars to supply new seeds after the removal of inflorescence at full flowering. Among the cultivars, Fedora obtained the best results for seed (0.79 and 0.52 t ha−1) and vegetable oil yield (0.17 and 0.09 t ha−1) normally and with inflorescence removed plants, respectively. Futura, conversely, showed the best results for inflorescence (3.0 t ha−1), essential oil (9 L ha−1), and stem yield at seed maturity (8.34 t ha−1), as means across the two years. The cultivars studied generally reached the grain-filling stage during a period that was drier and warmer than the average of the same multi-year period, and this negatively affected seed quality. The oil fatty acid composition was mainly composed of polyunsaturated fatty acids (75\% on average) and not affected by the cultivar. In conclusion, although the hemp grower should always clearly know the main production objective of the crop, the monoecious cultivars available today allow a multipurpose use of hemp crop, improving the sustainability of the cultivation activity.
387. Zhang, Qingying and Chen, Xuan and Guo, Hongyan and Trindade, Luisa M. and Salentijn, Elma M. J. and Guo, Rong and Guo, Mengbi and Xu, Yanping and Yang, Ming, Latitudinal Adaptation and Genetic Insights Into the Origins of Cannabis Sativa L., Frontiers in Plant Science, vol. 9, 2018. doi: 10.3389/fpls.2018.01876. Abstract
     Cannabis is one of the most important industrial crops distributed worldwide. However, the phylogeographic structure and domestication knowledge of this crop remains poorly understood. In this study, sequence variations of five chloroplast DNA (cpDNA) regions were investigated to address these questions. For the 645 individuals from 52 Cannabis accessions sampled (25 wild populations and 27 domesticated populations or cultivars), three haplogroups (Haplogroup H, M, L) were identified and these lineages exhibited distinct high-middle-low latitudinal gradients distribution pattern. This pattern can most likely be explained as a consequence of climatic heterogeneity and geographical isolation. Therefore, we examined the correlations between genetic distances and geographical distances, and tested whether the climatic factors are correlated with the cpDNA haplogroup frequencies of populations. The “isolation-by-distance” models were detected for the phylogeographic structure, and the day-length was found to be the most important factor (among 20 BioClim factors) that influenced the population structures. Considering the distinctive phylogeographic structures and no reproductive isolation among members of these lineages, we recommend that Cannabis be recognized as a monotypic genus typified by Cannabis sativa L., containing three subspecies: subsp. sativa, subsp. Indica, and subsp. ruderalis. Within each haplogroup which possesses a relatively independent distribution region, the wild and domesticated populations shared the most common haplotypes, indicating that there are multiregional origins for the domesticated crop. Contrast to the prevalent Central-Asia-Origin hypothesis of C. saltiva, molecular evidence reveals for the first time that the low latitude haplogroup (Haplogroup L) is the earliest divergent lineage, implying that Cannabis is probably originated in low latitude region.
388. Pisupati, Rahul and Vergara, Daniela and Kane, Nolan C., Diversity and Evolution of the Repetitive Genomic Content in Cannabis Sativa, BMC Genomics, vol. 19, no. 1, pp. 156, February 2018. doi: 10.1186/s12864-018-4494-3. Abstract
     The repetitive content of the genome, once considered to be “junk DNA”, is in fact an essential component of genomic architecture and evolution. In this study, we used the genomes of three varieties of Cannabis sativa, three varieties of Humulus lupulus and one genotype of Morus notabilis to explore their repetitive content using a graph-based clustering method, designed to explore and compare repeat content in genomes that have not been fully assembled.
389. Mansouri, Hakimeh and Bagheri, Mahsa, Induction of Polyploidy and Its Effect on Cannabis Sativa L., pp. 365--383, 2017. doi: 10.1007/978-3-319-54564-6_17. Abstract
     Polyploids are organisms with three or more complete chromosome sets. Polyploidization is widespread in plants, and is an important mechanism of speciation. Polyploids can be formed in various ways. The study of polyploids has both important theoretical significance and valuable applications. The production and application of polyploidy breeding have brought remarkable economic and social benefits. We reported the production of putative tetraploid plants of Cannabis sativa L., with the ultimate aim of improving the medicinal and physiological traits of this widely distributed cultivated plant. The production of tetraploid plant was improved with colchicine at different concentrations and time through dropping method. Flow cytometry analysis was used to confirm the ploidy level. Morphologic, anatomic and biochemical characteristics were compared between tetraploid and diploid control plants. The results showed that 0.2\% colchicine for 24~h was the most efficient for production of polyploid plants. The percentage of tetraploid plants and the survival rate were lowered by the increasing the treatment time. In addition, the leaf index and height of tetraploid plants exhibited a significant decrease compared to the diploid plants. The size of stomata on epidermis of leaves were larger in tetraploid plant compared to the diploid ones, in spite of the tetraploid plants have less stomata density. However, the amount of total chlorophyll and carotenoids were almost the same in both tetraploid and diploid plants. In addition, some differences were also observed in the cross section of stem of these plants from a descriptive structural point of view. Overall, the results introduced usage of the stomata parameters as an effective, fast and convenient method for detecting the tetraploid plants. We also investigated polyploidy effects on some primary and secondary metabolites. The results of biochemical analyzes showed that soluble sugars and total protein content increased significantly into mixoploid plants compared to tetraploid and diploid plants. Tetraploid plants had higher amount of total proteins compared with control plants. The results showed that polyploidization could increase the contents of tetrahydrocannabionol only in mixoploid plants but tetraploid plants had lower amounts of this substance in comparison with diploids. Our results suggest that tetraploidization was not useful for production of tetrahydrocannabinol for commercial use but mixoploids were found suitable.
390. Soler, Salvador and Gramazio, Pietro and Figàs, Maria R. and Vilanova, Santiago and Rosa, Elena and Llosa, Ernesto R. and Borràs, Dionís and Plazas, Mariola and Prohens, Jaime, Genetic Structure of Cannabis Sativa Var. Indica Cultivars Based on Genomic SSR (gSSR) Markers: Implications for Breeding and Germplasm Management, Industrial Crops and Products, vol. 104, pp. 171--178, October 2017. doi: 10.1016/j.indcrop.2017.04.043. Abstract
     Cannabis sativa L. is cultivated for its fiber or seeds (var. sativa; hemp), or for its high content in cannabinoids (var. indica; marijuana). Knowledge of the genetic structure of C. sativa var. indica is important for selection and breeding of cultivars with medicinal interest. We used six genomic SSRs (gSSRs) for genotyping 154 individual plants of 20 cultivars of C. sativa var. indica, plus two cultivars of C. sativa var. sativa. A very high polymorphism was observed, with an average of 17 alleles and 23.8 genotypes per locus. Expected (He) and observed (Ho) heterozygosities were high, with average values of 0.753 and 0.429, respectively. In some cultivars He and Ho presented similar values, while in others He was considerably higher than Ho suggesting that consanguinity and fixation had taken place during its development. In addition, some cultivars had a reduced number of alleles per locus (in some cases only two) indicating that a genetic bottleneck had taken place during its development. Gene flow (Nm) between both botanical varieties was high, with Nm=1.736. The molecular analysis of variance (AMOVA) revealed that only 31.94\% of the molecular variation observed was caused by differences among cultivars, while the variation among plants of the same cultivar was of 37.11\%, and within individual variation, due to heterozygosity, was of 30.96\%. This indicates that a large variation exists within cultivars, which can be exploited for selection, but also complicates germplasm management and regeneration. The population structure analysis identified 14 genetic clusters, with most individuals of a single cultivar clustering together. This analysis, together with UPGMA cluster analysis shows that the two C. sativa var. sativa cultivars studied are differentiated from C. sativa var. indica, and that some cultivars of C. sativa var. indica seem to represent different selections from a common original cultivar. Our results represent the first comprehensive study of intra-varietal diversity in C. sativa var. indica and provide information of relevance for selection, breeding, and germplasm conservation, as well as for forensic studies in this crop.
391. Cannabis Sativa L. - Botany and Biotechnology, 2017. doi: 10.1007/978-3-319-54564-6.
392. Punja, Zamir K. and Rodriguez, Gina and Chen, Sarah, Assessing Genetic Diversity in Cannabis Sativa Using Molecular Approaches, pp. 395--418, 2017. doi: 10.1007/978-3-319-54564-6_19. Abstract
     Cannabis sativa L. represents plants cultivated for their psychoactive and medicinal properties (marijuana) or as a source of fibre, seed and oil (hemp). Breeding and selection efforts have produced marijuana genotypes (strains) with a range of levels of the cannabinoid Δ9-tetrahydrocannabinolic acid (THCA) and other non-psychoactive cannabinoids, e.g. cannabidiolic acid (CBDA). Hemp lines have been bred for high fibre content and seed production and have low/no THCA. There are currently hundreds of marijuana strains which differ in THCA:CBDA ratios, growth characteristics, morphological features, THCA and CBDA contents, disease resistance, as well as overall medicinal value. The extent of genetic diversity among these marijuana strains, as well as between marijuana and hemp, has been studied using a range of molecular approaches. The results from these studies have demonstrated that considerable genetic diversity exists among marijuana as well as hemp strains. Using ISSR markers, we have shown that distinct DNA banding patterns can allow for the initial discrimination between many of the strains tested, and provide an insight into the possible genetic relationships among strains. Some strains, e.g. ‘Jack’, ‘Super Sour Skunk’, ‘Jilly Bean’, exhibited unique patterns that can be used to develop strain-specific DNA fingerprints. In addition, a number of “landraces” and strains originating from remote geographic locations, showed unique and distinct ISSR patterns and morphologies. A very high degree of genetic diversity was exhibited among the strains studied. Additional molecular studies, including DNA sequencing approaches, should provide more insight into the genetic relationships that exist within strains of a complex plant species and could augment future breeding efforts for genetic improvement of C. sativa.
393. Das, Akan and Chaudhury, Sumi and Kalita, Mohan C. and Mondal, Tapan K., In Silico Identification, Characterization and Expression Analysis of miRNAs in Cannabis Sativa L., Plant Gene, vol. 2, pp. 17--24, June 2015. doi: 10.1016/j.plgene.2015.03.003. Abstract
     Cannabis sativa L. is an annual herb and economically important as a source of fiber, oil, food and for its medicinal and intoxicating properties. MicroRNAs are a class of short (\textasciitilde 21nt), non-coding regulatory RNAs that play a major role in post-transcriptional gene silencing. By in silico analysis of the publically available Transcript Sequence Assemblies (TSA) and Expressed Sequence Tags (ESTs) of C. sativa, a total of 18 conserved miRNAs belonging to 9 independent families were identified. To validate the predicted miRNAs, SYBR green based assay of qPCR was applied to detect the tissue-specific (young and mature leaf) expression of 6 putative miRNAs (csa-miR156, csa-miR159a, csa-miR171b, csa-miR172a, csa-miR5021a, csa-miR6034) in C. sativa. A total of 80 target genes were also recognized for the newly identified miRNAs, and subsequently assigned to three broad functional categories: biological processes, cellular components and molecular functions as defined for the Arabidopsis proteome. The potential target genes consist of transcription factors (33.75\%), transporters (5\%), kinase and other enzymes (20\%) as well as signaling and other functional proteins (32.50\%). The findings in this study on C. sativa miRNA precursors, mature miRNAs, and miRNA targets will be helpful for future research on miRNA-mediated gene regulation in this important plant species.
394. Bagheri, Mahsa and Mansouri, Hakimeh, Effect of Induced Polyploidy on Some Biochemical Parameters in Cannabis Sativa L., Applied Biochemistry and Biotechnology, vol. 175, no. 5, pp. 2366--2375, March 2015. doi: 10.1007/s12010-014-1435-8. Abstract
     This study is aimed at testing the efficiency of colchicine on inducing polyploidy in Cannabis sativa L. and investigation of effects of polyploidy induction on some primary and secondary metabolites. Shoot tips were treated with three different concentrations of colchicine (0, 0.1, 0.2~\%\,w/v) for 24 or 48~h. The biggest proportion of the almost coplanar tetraploids (43.33~\%) and mixoploids (13.33~\%) was obtained from the 24-h treatment in 0.2 and 0.1~\%\,w/v, respectively. Colchicine with 0.2~\% concentration and 48~h duration was more destructive than 24~h. The ploidy levels were screened with flow cytometry. The biochemical analyses showed that reducing sugars, soluble sugars, total protein, and total flavonoids increased significantly in mixoploid plants compared with tetraploid and diploid plants. Tetraploid plants had a higher amount of total proteins, total flavonoids, and starch in comparison with control plants. The results showed that polyploidization could increase the contents of tetrahydrocannabinol in mixoploid plants only, but tetraploid plants had lower amounts of this substance in comparison with diploids. Also, we found such changes in protein concentration in electrophoresis analysis. In overall, our study suggests that tetraploidization could not be useful to produce tetrahydrocannabinol for commercial use, and in this case, mixoploids are more suitable.
395. Faux, Anne-Michelle and Berhin, Alice and Dauguet, Nicolas and Bertin, Pierre, Sex Chromosomes and Quantitative Sex Expression in Monoecious Hemp (Cannabis Sativa L.), Euphytica, vol. 196, no. 2, pp. 183--197, March 2014. doi: 10.1007/s10681-013-1023-y. Abstract
     Hemp (Cannabis sativa) has a highly variable sexual phenotype. In dioecious hemp, the sex is controlled by heteromorphic sex chromosomes according to an X-to-autosomes equilibrium. However, in monoecious hemp, the sex determinism remains widely unknown and has never been related to a quantitative approach of sex expression. The present paper aims to contribute to the comprehension of the sex determinism in monoecious hemp by assessing the genotypic variability of its sex expression and establishing its sex chromosomes. Five monoecious and one dioecious cultivars were grown in controlled conditions under several photoperiods. The monoecy degree of 194 monoecious plants was recorded at each node by a figure ranging from 0 (male flowers only) to 6 (female flowers only). The genome size of 55 plants was determined by flow cytometry. The DNA of 115 monoecious plants was screened with the male-associated marker MADC2. The monoecy degree varied significantly among monoecious cultivars from 3.36~±~2.28 in ‘Uso 31’ to 5.70~±~0.81 in the most feminised ‘Epsilon 68’. The variation of monoecy degree among cultivars remained consistent across trials despite a significant “cultivar × trial” interaction and partly agreed with their earliness. The genome size of monoecious plants (1.791~±~0.017~pg) was not different from that of females (1.789~±~0.019~pg) but significantly lower than that of males (1.835~±~0.019~pg). MADC2 was absent from all monoecious plants. These results strongly support that cultivars of monoecious hemp have the XX constitution and that their sex expression has a genetic basis.
396. Divashuk, Mikhail G. and Alexandrov, Oleg S. and Razumova, Olga V. and Kirov, Ilya V. and Karlov, Gennady I., Molecular Cytogenetic Characterization of the Dioecious Cannabis Sativa with an XY Chromosome Sex Determination System, PLOS ONE, vol. 9, no. 1, pp. e85118, January 2014. doi: 10.1371/journal.pone.0085118. Abstract
     Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.
397. Docimo, Teresa and Caruso, Immacolata and Ponzoni, Elena and Mattana, Monica and Galasso, Incoronata, Molecular Characterization of Edestin Gene Family in Cannabis Sativa L., Plant Physiology and Biochemistry, vol. 84, pp. 142--148, November 2014. doi: 10.1016/j.plaphy.2014.09.011. Abstract
     Globulins are the predominant class of seed storage proteins in a wide variety of plants. In many plant species globulins are present in several isoforms encoded by gene families. The major seed storage protein of Cannabis sativa L. is the globulin edestin, widely known for its nutritional potential. In this work, we report the isolation of seven cDNAs encoding for edestin from the C.~sativa variety Carmagnola. Southern blot hybridization is in agreement with the number of identified edestin genes. All seven sequences showed the characteristic globulin features, but they result to be divergent members/forms of two edestin types. According to their sequence similarity four forms named CsEde1A, CsEde1B, CsEde1C, CsEde1D have been assigned to the edestin type 1 and the three forms CsEde2A, CsEde2B, CsEde2C to the edestin type 2. Analysis of the coding sequences revealed a high percentage of similarity (98–99\%) among the different forms belonging to the same type, which decreased significantly to approximately 64\% between the forms belonging to different types. Quantitative RT-PCR analysis revealed that both edestin types are expressed in developing hemp seeds and the amount of CsEde1 was 4.44~±~0.10 higher than CsEde2. Both edestin types exhibited a high percentage of arginine (11–12\%), but CsEde2 resulted particularly rich in methionine residues (2.36\%) respect to CsEde1 (0.82\%). The amino acid composition determined in CsEde1 and CsEde2 types suggests that these seed proteins can be used to improve the nutritional quality of plant food-stuffs.
398. Gao, Chunsheng and Xin, Pengfei and Cheng, Chaohua and Tang, Qing and Chen, Ping and Wang, Changbiao and Zang, Gonggu and Zhao, Lining, Diversity Analysis in Cannabis Sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers, PLOS ONE, vol. 9, no. 10, pp. e110638, October 2014. doi: 10.1371/journal.pone.0110638. Abstract
     Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99\%) were the most abundant, followed by hexanucleotide (25.13\%), dinucleotide (16.34\%), tetranucloetide (3.8\%), and pentanucleotide (3.74\%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96\%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31\%) were successfully amplified and 87 (74.36\%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.
399. {de Meijer}, E. P. M. and Hammond, K. M. and Sutton, A., The Inheritance of Chemical Phenotype in Cannabissativa L. (IV): Cannabinoid-Free Plants, Euphytica, vol. 168, no. 1, pp. 95--112, July 2009. doi: 10.1007/s10681-009-9894-7. Abstract
     A genetic factor that blocks the cannabinoid biosynthesis in Cannabissativa has been investigated. Crosses between cannabinoid-free material and high content, pharmaceutical clones were performed. F1s were uniform and had cannabinoid contents much lower than the mean parental value. Inbred F2 progenies segregated into discrete groups: a cannabinoid-free chemotype, a chemotype with relatively low cannabinoid content and one with relatively high content, in a monogenic 1:2:1 ratio. In our model the cannabinoid knockout factor is indicated as a recessive allele o, situated at locus O, which segregates independently from previously presented chemotype loci. The genotype o/o underlies the cannabinoid-free chemotype, O/o is expressed as an intermediate, low content chemotype, and O/O is the genotype of the high content chemotype. The data suggests that locus O governs a reaction in the pathway towards the phenolic cannabinoid precursors. The composition of terpenoids and various other compound classes of cannabinoid-free segregants remains unaffected. Backcrossing produced cannabinoid-free homologues of pharmaceutical production clones with potential applications in pharmacological research. A new variant of the previously presented allele ‘B0’, that almost completely obstructs the conversion of CBG into CBD, was also selected from the source population of the cannabinoid knockout factor.
400. {de Meijer}, E. P. M. and Hammond, K. M. and Micheler, M., The Inheritance of Chemical Phenotype in Cannabis Sativa L. (III): Variation in Cannabichromene Proportion, Euphytica, vol. 165, no. 2, pp. 293--311, January 2009. doi: 10.1007/s10681-008-9787-1. Abstract
     The mechanism that controls the proportion of cannabichromene (CBC), a potential pharmaceutical, in the cannabinoid fraction of Cannabis sativa L. is explored. As with tetrahydrocannabinol (THC) and cannabidiol (CBD), CBC is an enzymatic conversion product of the precursor cannabigerol (CBG). CBC is reported to dominate the cannabinoid fraction of juveniles and to decline with maturation. This ontogeny was confirmed in inbred lines with different mature chemotypes. A consistent CBC presence was found in early leaves from a diverse clone collection, suggesting that CBC synthase is encoded by a fixed locus. Morphological variants possessing a ‘prolonged juvenile chemotype’ (PJC), a substantial proportion of CBC persisting up to maturity, are presented. PJC is associated with a reduced presence of floral bracts, bracteoles, and capitate-stalked trichomes. Genetic factors causing these features were independent of the allelic chemotype locus B that was previously postulated and regulates THC and CBD synthesis and CBG accumulation. In contrast to previously described Cannabis chemotypes, the cannabinoid composition of PJCs showed plasticity in that reduced light levels increased the CBC proportion. The ability of PJC plants to enable the production of pharmaceutical raw material with high CBC purity is demonstrated.
401. Gilmore, Simon and Peakall, Rod and Robertson, James, Organelle DNA Haplotypes Reflect Crop-Use Characteristics and Geographic Origins of Cannabis Sativa, Forensic Science International, vol. 172, no. 2, pp. 179--190, October 2007. doi: 10.1016/j.forsciint.2006.10.025. Abstract
     Comparative sequencing of cannabis individuals across 12 chloroplast and mitochondrial DNA loci revealed 7 polymorphic sites, including 5 length variable regions and 2 single nucleotide polymorphisms. Simple PCR assays were developed to assay these polymorphisms, and organelle DNA haplotypes were obtained for 188 cannabis individuals from 76 separate populations, including drug-type, fibre-type and wild populations. The haplotype data were analysed using parsimony, UPGMA and neighbour joining methods. Three haplotype groups were recovered by each analysis method, and these groups are suggestive of the crop-use characteristics and geographical origin of the populations, although not strictly diagnostic. We discuss the relationship between our haplotype data and taxonomic opinions of cannabis, and the implications of organelle DNA haplotyping to forensic investigations of cannabis.
402. Datwyler, Shannon L. and Weiblen, George D., Genetic Variation in Hemp and Marijuana (Cannabis Sativa L.) According to Amplified Fragment Length Polymorphisms*, Journal of Forensic Sciences, vol. 51, no. 2, pp. 371--375, 2006. doi: 10.1111/j.1556-4029.2006.00061.x. Abstract
     ABSTRACT: Cannabis sativa L. (Cannabaceae) is one of the earliest known cultivated plants and is important in the global economy today as a licit and an illicit crop. Molecular markers distinguishing licit and illicit cultivars have forensic utility, but no direct comparison of hemp and marijuana amplified fragment length polymorphism (AFLP) has been made to date. Genetic variation was surveyed in three populations of fiber hemp and a potent cultivar of marijuana using AFLP markers. Ten primer pairs yielded 1206 bands, of which 88\% were polymorphic. Eighteen bands represented fixed differences between all fiber populations and the drug cultivar. These markers have practical utility for (1) establishing conspiracy in the cultivation and distribution of marijuana, (2) identifying geographic sources of seized drugs, and (3) discriminating illegal, potent marijuana cultivars from hemp where the cultivation of industrial hemp is permitted.
403. {de Meijer}, E. P. M. and Hammond, K. M., The Inheritance of Chemical Phenotype in Cannabis Sativa L. (II): Cannabigerol Predominant Plants, Euphytica, vol. 145, no. 1, pp. 189--198, September 2005. doi: 10.1007/s10681-005-1164-8. Abstract
     This paper aims to clarify the genetic mechanism that is responsible for the accumulation of cannabigerol (CBG) in certain phenotypes of Cannabis sativa L. CBG is the direct precursor of the cannabinoids CBD, THC and CBC. Plants strongly predominant in CBG have been found in different fibre hemp accessions. Inbred offspring derived from one such individual were crossed with true breeding THC predominant- and CBD predominant plants, respectively. The segregations in the cross progenies indicate that CBG accumulation is due to the homozygous presence of a minimally functional allele, tentatively called B0, at the single locus B that normally controls the conversion of CBG into THC (allele BT) and/or CBD (allele BD). The fact that CBG accumulating plants have so far been found in European fibre hemp populations that are generally composed of BD/BD plants, and the observation that the here investigated B0 allele possesses a residual ability to convert small amounts of CBG into CBD, make it plausible that this B0 is a mutation of normally functional BD. Therefore, B0 is considered as a member of the BD allelic series encoding a CBD synthase isoform with greatly weakened substrate affinity and/or catalytic capacity.
404. Peil, A. and Flachowsky, H. and Schumann, E. and Weber, W. E., Sex-Linked AFLP Markers Indicate a Pseudoautosomal Region in Hemp (Cannabis Sativa L.), Theoretical and Applied Genetics, vol. 107, no. 1, pp. 102--109, June 2003. doi: 10.1007/s00122-003-1212-5.
405. Beutler, John A. and Marderosian, Ara H., Chemotaxonomy of Cannabis I. Crossbreeding between Cannabis Sativa and C. Ruderalis, with Analysis of Cannabinoid Content, Economic Botany, vol. 32, no. 4, pp. 387--394, October 1978. doi: 10.1007/BF02907934. Abstract
     A controlled cross betweenCannabis sativa L. andC. ruderalis Janisch. gave progeny intermediate in both cannabinoid content and morphology. The progeny fell into two distinct populations, those whose tetrahydrocannabinol (THC) content was closer to theC. sativa parent (greater than 60\% of total cannabinoids) and those whose THC content was closer to theC. ruderalis parent (less than 40\% of total cannabinoids). The lower THC group was twice as frequent as the other group. Earliness of flowering, number of flowers, and height characteristics were intermediate between the parents.
406. Gilmore, S. and Peakall, R., Isolation of Microsatellite Markers in Cannabis Sativa L. (Marijuana), Molecular Ecology Notes, vol. 3, no. 1, pp. 105--107, 2003. doi: 10.1046/j.1471-8286.2003.00367.x. Abstract
     We have identified 15 variable microsatellite loci in Cannabis sativa. In 48 samples from five fibre crop seed accessions, we detected an average of 10 alleles per locus (range 2–28) with mean heterozygosity of 0.68 (range 0.28–0.94). Significant genetic differentiation was found between accessions (FST = 0.12, P {$<$} 0.001). These markers have utility for characterizing genetic diversity in cultivated and naturalized Cannabis populations.
407. Hsieh, Hsing-Mei and Hou, Rur-Jyun and Tsai, Li-Chin and Wei, Chih-Sheng and Liu, Su-Wen and Huang, Li-Hung and Kuo, Yi-Chen and Linacre, Adrian and Lee, James Chun-I, A Highly Polymorphic STR Locus in Cannabis Sativa, Forensic Science International, vol. 131, no. 1, pp. 53--58, January 2003. doi: 10.1016/S0379-0738(02)00395-X. Abstract
     We report on the first short tandem repeat (STR) locus to be isolated from the plant Cannabis sativa. The STR locus, isolated by a hybrid-capture enrichment procedure, was found to contain a simple sequence repeat motif of 6bp. This 6bp repeat motif showed no variation in repeat length but with minor variations in repeat unit sequences. The data show the locus to be highly polymorphic with the number of repeat units ranging from 3 to 40 in 108 screened samples. The observed heterozygosity was approximately 87.04\%. The forward and reverse primers (CS1F and CS1R) produced no PCR products in cross-reaction study from 20 species of plants, including highly related species such as Humulus japonicus and Nicotiana tabacum. This hexanucleotide repeat DNA locus could be used to identify cannabis samples and predict their genetic relationship as the test is specific to C. sativa and is highly reproducible.
408. Kojoma, Mareshige and Iida, Osamu and Makino, Yukiko and Sekita, Setsuko and Satake, Motoyoshi, DNA Fingerprinting of Cannabis Sativa Using Inter-Simple Sequence Repeat (ISSR) Amplification, Planta Medica, vol. 68, no. 1, pp. 60--63, January 2002. doi: 10.1055/s-2002-19875. Abstract
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409. Sakamoto, Koichi and Ohmido, Nobuko and Fukui, Kiichi and Kamada, Hiroshi and Satoh, Shinobu, Site-Specific Accumulation of a LINE-like Retrotransposon in a Sex Chromosome of the Dioecious Plant Cannabis Sativa, Plant Molecular Biology, vol. 44, no. 6, pp. 723--732, December 2000. doi: 10.1023/A:1026574405717. Abstract
     Male-associated DNA sequences were analysed in hemp (Cannabis sativa L.), a dioecious plant with heteromorphic sex chromosomes. A male-associated DNA sequence in C.~sativa (MADC1) and its flanking sequence encoded a reverse transcriptase that was strongly homologous to those of LINE-like retrotransposons from various plants and other organisms, as well as another open reading frame (ORF). Fluorescence in~situ hybridization (FISH) with MADC1 as probe, which yielded strong signals specific for male genomic DNA in gel blot analysis, generated a clear doublet signal at the end of the long arm of the Y chromosome. FISH using pachytene chromosomes of pollen mother cells at meiotic prophase I revealed that pairing of X and Y chromosomes occurred at the short arm of the Y chromosome where MADC1 was not present. Furthermore, FISH using extended DNA fibers, with MADC1 and its flanking DNA as probes, revealed that 100 to 200 copies of the retrotransposon were located in tandem on the Y chromosome. These results support the hypothesis that accumulation of a specific LINE-like retrotransposon at the terminal region of the long arm of the Y chromosome might be one cause of heteromorphism of sex chromosomes.
410. Mandolino, G. and Carboni, A. and Forapani, S. and Faeti, V. and Ranalli, P., Identification of DNA Markers Linked to the Male Sex in Dioecious Hemp (Cannabis Sativa L.), Theoretical and Applied Genetics, vol. 98, no. 1, pp. 86--92, January 1999. doi: 10.1007/s001220051043. Abstract
     A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8400, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the association of the OPA8400 marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-specific association, the sequences corresponding to OPA8400 were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390\>bp in length and male-specific. This SCAR is suitable for a precise, early and rapid identification of male plants during breeding programs of dioecious and monoecious hemp.
411. Linacre, Adrian and Thorpe, James, Detection and Identification of Cannabis by DNA, Forensic Science International, vol. 91, no. 1, pp. 71--76, January 1998. doi: 10.1016/S0379-0738(97)00173-4. Abstract
     The unambiguous identification of illicit substances, including Cannabis sativa, is a major concern of law enforcement agencies. Current methods of cannabis identification involve the use of techniques such as HPLC and GC to identify cannabinoids. A method for the identification of cannabis using DNA-specific primers has been developed and is described here. The nucleotide sequences between the trnL and trnF genes in the chloroplast of Cannabis sativa have been determined and Cannabis sativa-specific nucleotide sequences within the intergenic spacer between the trnL 3′ exon and trnF gene identified. Primers, made to these sequences, have been tested on a range of different plant extracts but only give a PCR product in the presence of Cannabis sativa. The successful production of a PCR product using these primers identifies the presence of cannabis.
412. Jagadish, Vidya and Robertson, James and Gibbs, Adrian, RAPD Analysis Distinguishes Cannabis Sativa Samples from Different Sources, Forensic Science International, vol. 79, no. 2, pp. 113--121, May 1996. doi: 10.1016/0379-0738(96)01898-1. Abstract
     Samples of the seeds and seedlings of Cannabis sativa, and its dried leaves and flowerheads (marijuana), could be reliably distinguished by RAPD-PCR (Random Amplified Polymorphic DNA using the Polymerase Chain Reaction). DNA was best extracted from fresh tissues using buffers and the detergent cetyltrimethylammonium bromide; poorly dried tissue or inviable seed yielded coloured samples of degraded DNA. DNA was isolated from 51 C. sativa and two Humulus lupulus (hops) samples. Of the C. sativa samples 43 were from Australia (ten from Canberra gardens, eight from a New South Wales crop and 25 from two Queensland crops) and eight were from Papua-New Guinea (P-NG). A total of 102 different bands were obtained using four 10-nucleotide primers with arbitrarily chosen sequences. Banding patterns were compared by calculating pairwise distances using various algorithms, and presented using the neighbour-joining tree and multidimensional scaling methods. These showed a clear difference between C. sativa and H. lupulus, and separated the samples of the latter into three distinct groups; one group comprised all the P-NG samples, another the Canberra samples, and the third, the three crop samples.
413. Faeti, V. and Mandolino, G. and Ranalli, P., Genetic Diversity of Cannabis Sativa Germplasm Based on RAPD Markers, Plant Breeding, vol. 115, no. 5, pp. 367--370, 1996. doi: 10.1111/j.1439-0523.1996.tb00935.x. Abstract
     Random amplified polymorphic DN A (RAPD) markers were generated from 13 cultivars and accessions of Cannabis sativa L. Approximately 200 fragments generated by 10 primers of arbitrary sequence were used to assess the level of DNA variation. Statistical analysis was performed using the Dice coefficient of similarity and principal coordinate analysis. The grouping of the accessions according to the cluster analysis was in good agreement with their origin and lines with common ancestors were grouped together. Principal coordinates 1 and 2 revealed a clear separation of Italian and Hungarian germplasm and a third group, including a mixture of genotypes coming from different places; the third coordinate separated the Korean group which is probably the most divergent germplasm. Variability within the two cultivars ‘Carmagnola’ and ‘Fibranova1’ was also shown, suggesting good possibilities for long–term selection work. RAPD markers provide a powerful tool for the investigation of genetic variation in cultivars/accessions of hemp.
414. Gillan, R. and Cole, M. D. and Linacre, A. and Thorpe, J. W. and Watson, N. D., Comparison of Cannabis Sativa by Random Amplification of Polymorphic DNA (RAPD) and HPLC of Cannabinoids: A Preliminary Study, Science \& justice, vol. 35, no. 3, pp. 169--177, July 1995. doi: 10.1016/s1355-0306(95)72658-2. Abstract
     Methods are described for the HPLC and genetic analysis of herbal Cannabis sativa. The latter method was applied to 17 plants grown simultaneously, at the same site. Sixteen of these samples were also compared using HPLC, which resulted in differentiation of the plants into 3 groups. Within two of these groups, the members could not be distinguished. By RAPD analysis, using certain combinations of primers and cladistic analysis, differentiation was possible between all but two of the plants. The use of the RAPD technique enables differentiation between samples that cannot be differentiated by HPLC analysis alone.
415. Sakamoto, Koichi and Shimonura, Koichiro and Komeda, Yoshibumi and Kamada, Hiroshi and Satoh, Shinobu, A Male-Associated DNA Sequence in a Dioecious Plant, Cannabis Sativa L., Plant and Cell Physiology, December 1995. doi: 10.1093/oxfordjournals.pcp.a078920.
416. Welling, Matthew T. and Shapter, Tim and Rose, Terry J. and Liu, Lei and Stanger, Rhia and King, Graham J., A Belated Green Revolution for Cannabis: Virtual Genetic Resources to Fast-Track Cultivar Development, Frontiers in Plant Science, vol. 7, 2016. doi: 10.3389/fpls.2016.01113. Abstract
     Cannabis is a predominantly diecious phenotypically diverse domesticated genus with few if any extant natural populations. International narcotics conventions and associated legislation have constrained the establishment, characterization, and use of Cannabis genetic resource collections. This has resulted in the underutilization of genepool variability in cultivar development and has limited the inclusion of secondary genepools associated with genetic improvement strategies of the Green Revolution. The structured screening of ex situ germplasm and the exploitation of locally-adapted intraspecific traits is expected to facilitate the genetic improvement of Cannabis. However, limited attempts have been made to establish the full extent of genetic resources available for pre-breeding. We present a thorough critical review of Cannabis ex situ genetic resources, and discuss recommendations for conservation, pre-breeding characterization, and genetic analysis that will underpin future cultivar development. We consider East Asian germplasm to be a priority for conservation based on the prolonged historical cultivation of Cannabis in this region over a range of latitudes, along with the apparent high levels of genetic diversity and relatively low representation in published genetic resource collections. Seed cryopreservation could improve conservation by reducing hybridization and genetic drift that may occur during Cannabis germplasm regeneration. Given the unique legal status of Cannabis, we propose the establishment of a global virtual core collection based on the collation of consistent and comprehensive provenance meta-data and the adoption of high-throughput DNA sequencing technologies. This would enable representative core collections to be used for systematic phenotyping, and so underpin breeding strategies for the genetic improvement of Cannabis.
417. {de Meijer}, Etienne P M and Bagatta, Manuela and Carboni, Andrea and Crucitti, Paola and Moliterni, V M Cristiana and Ranalli, Paolo and Mandolino, Giuseppe, The Inheritance of Chemical Phenotype in {\emph{Cannabis Sativa}} L., Genetics, vol. 163, no. 1, pp. 335--346, January 2003. doi: 10.1093/genetics/163.1.335. Abstract
     Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure Δ-9-tetrahydrocannabinol (THC) chemotypes. All the plants belonging to the F1’s were analyzed by gas chromatography for cannabinoid composition and constantly found to have a mixed CBD-THC chemotype. Ten individual F1 plants were self-fertilized, and 10 inbred F2 offspring were collected and analyzed. In all cases, a segregation of the three chemotypes (pure CBD, mixed CBD-THC, and pure THC) fitting a 1:2:1 proportion was observed. The CBD/THC ratio was found to be significantly progeny specific and transmitted from each F1 to the F2’s derived from it. A model involving one locus, B, with two alleles, BD and BT, is proposed, with the two alleles being codominant. The mixed chemotypes are interpreted as due to the genotype BD/BT at the B locus, while the pure-chemotype plants are due to homozygosity at the B locus (either BD/BD or BT/BT). It is suggested that such codominance is due to the codification by the two alleles for different isoforms of the same synthase, having different specificity for the conversion of the common precursor cannabigerol into CBD or THC, respectively. The F2 segregating groups were used in a bulk segregant analysis of the pooled DNAs for screening RAPD primers; three chemotype-associated markers are described, one of which has been transformed in a sequence-characterized amplified region (SCAR) marker and shows tight linkage to the chemotype and codominance.
418. Mohan Ram, H. Y. and Sett, R., Induction of Fertile Male Flowers in Genetically Female Cannabis Sativa Plants by Silver Nitrate and Silver Thiosulphate Anionic Complex, Theoretical and Applied Genetics, vol. 62, no. 4, pp. 369--375, December 1982. doi: 10.1007/BF00275107. Abstract
     Apical application of silver nitrate (AgNO3; 50 and 100 μg per plant) and silver thiosulphate anionic complex (Ag(S2O3)23−; STS; 25, 50 and 100 μg per plant) to female plants of Cannabis sativa induced the formation of reduced male, intersexual and fully altered male flowers on the newly formed primary lateral branches (PLBs); 10 μg per plant of AgNO3 was ineffective and 150 μg treatment proved inhibitory. A maximum number of fully altered male flowers were formed in response to 100 μg STS. The induced male flowers produced pollen grains that germinated on stigmas and effected seed set. Silver ion applied as STS was more effective than AgNO3 in inducing flowers of altered sex. The induction of male flowers on female plants demonstrated in this work is useful for producing seeds that give rise to only female plants. This technique is also useful for maintaining gynoecious lines.
419. Ram, H. Y. Mohan and Jaiswal, V. S., Induction of Male Flowers on Female Plants of Cannabis Sativa by Gibberellins and Its Inhibition by Abscisic Acid, Planta, vol. 105, no. 3, pp. 263--266, September 1972. doi: 10.1007/BF00385397. Abstract
     Gibberellins (GA3, GA4+7, GA7 and GA9) induce male flowers on female plants of Cannabis sativa. This is, depending on concentration, partially or fully inhibited by abscisic acid (ABA). The ABA effect can in turn be partially overcome by increasing the concentration of GA3.
420. Sorrentino, Giuseppe, Introduction to Emerging Industrial Applications of Cannabis (Cannabis Sativa L.), Rendiconti Lincei. Scienze Fisiche e Naturali, vol. 32, no. 2, pp. 233--243, June 2021. doi: 10.1007/s12210-021-00979-1. Abstract
     The Italian Law of 22 November 2016 has legalized the cultivation of hemp, which drives the development of sustainable agriculture by generating new products with high added value in the new context of circular economy. Hemp cultivation is known for its low environmental impact, as hemp grows fast, suppresses weeds and does not need pesticides. It has no specialized parasites, favors pollination and improves the physical and chemical soil fertility. Recently, many countries have increased their interest in hemp (Cannabis Sativa L.), considering it as a climate-friendly crop that can mitigate climate change and desertification. For these reasons, hemp can be a new protagonist of Italian agriculture already oriented towards the objectives of EU 2030 which predicts 40\% decrease in greenhouse gas emissions compared to 1990. The hemp cultivation can activate a new supply chain by allowing using different parts of the plant, benefiting farmers, environment, and human health. Indeed, although a very old plant, hemp will be one of the main protagonists of the green economy in the near future. Its seeds can be used by agri-food industry to produce flour, pasta, pastry and oil, while the stem through canapulo (woody part of stem) in green building sector. Its fiber (external part of stem) will find new applications in textile industry. As for its inflorescences and roots, thanks to the extraction of bioactive molecules, they will play an important role in the pharmaceutical and parapharmaceutical industry. Finally, only the medical sector with Δ9‐tetrahydrocannabinol (THC) extraction from inflorescence is not yet regulated by the aforementioned Italian Law.
421. Moscariello, Carlo and Matassa, Silvio and Esposito, Giovanni and Papirio, Stefano, From Residue to Resource: The Multifaceted Environmental and Bioeconomy Potential of Industrial Hemp (Cannabis Sativa L.), Resources, Conservation and Recycling, vol. 175, pp. 105864, December 2021. doi: 10.1016/j.resconrec.2021.105864. Abstract
     In the emerging context of circular bioeconomy, industrial hemp (Cannabis Sativa L.) biomass is a valuable resource for the sustainable implementation of second-generation biorefineries. Potentially, all the main hemp components can find application within different biorefinery approaches, adding value to the conventional production of hemp fibres and seeds. Hurds, leaves and inflorescences, constituting most of the hemp plant biomass, and often considered as low-value residues, can indeed play a key role in the sustainable production of both bioenergy and high-value bioproducts. The present article reviews the advances and outlines the potential future perspectives of hemp-based biorefineries. After critically overviewing some of the most established applications of hemp, spanning from soil bioremediation to bioenergy and biofuel production, particular attention is given to novel valorisation schemes to synthetize highly demanded bioproducts such as microbial protein and biopolymers. Our preliminary calculations show that hemp biomass can sustain high biodiesel yield (1.6~g/g VS (volatile solids)) and related revenues (510–868 €/ha•year), while bioethanol production can yield 0.10–0.33~mL/g VS, profiting between 75–325 €/ha•year. Moreover, hemp suits biomethane production by yielding and profiting 98–426~mL/g VS and 60–380~€/ha•year, respectively. High yields of polyhydroxybutyrate (0.13~g/g VS) can be obtained, albeit high production costs might restrain their marketability. Finally, the biomethane-to-microbial protein pathway can yield and profit 0.03–0.15~g/g VS and 141–893~€/ha•year, respectively, while the volatile fatty acids-to-microbial protein pathway 0.04~g/g VS and 91–362~€/ha•year.
422. Pereira, José Francielson Q. and Pimentel, Maria Fernanda and Amigo, José Manuel and Honorato, Ricardo S., Detection and Identification of Cannabis Sativa L. Using near Infrared Hyperspectral Imaging and Machine Learning Methods. A Feasibility Study, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, vol. 237, pp. 118385, August 2020. doi: 10.1016/j.saa.2020.118385. Abstract
     Remote identification of illegal plantations of Cannabis sativa Linnaeus is an important task for the Brazilian Federal Police. The current analytical methodology is expensive and strongly dependent on the expertise of the forensic investigator. A faster and cheaper methodology based on automatic methods can be useful for the detection and identification of Cannabis sativa L. in a reliable and objective manner. In this work, the high potential of Near Infrared Hyperspectral Imaging (HSI-NIR) combined with machine learning is demonstrated for supervised detection and classification of Cannabis sativa L. This plant, together with other plants commonly found in the surroundings of illegal plantations and soil, were directly collected from an illegal plantation. Due to the high correlation of the NIR spectra, sparse Principal Component Analysis (sPCA) was implemented to select the most important wavelengths for identifying Cannabis sativa L. One class Soft Independent Class Analogy model (SIMCA) was built, considering just the spectral variables selected by sPCA. Sensitivity and specificity values of 89.45\% and 97.60\% were, respectively, obtained for an external validation set subjected to the s-SIMCA. The results proved the reliability of a methodology based on NIR hyperspectral cameras to detect and identify Cannabis sativa L., with only four spectral bands, showing the potential of this methodology to be implemented in low-cost airborne devices.
423. Tallon, Mark J., Cannabis Sativa L. and Its Extracts: Regulation of Cannabidiol in the European Union and United Kingdom, Journal of Dietary Supplements, vol. 17, no. 5, pp. 503--516, September 2020. doi: 10.1080/19390211.2020.1795044. Abstract
     The lawful sale of Cannabis sativa L. and its extracts including Cannabidiol is not harmonized under European Union law. Such products have in the most part been classified as novel foods and thus illegal for sale in Europe without prior authorization. The regulation of such substances not only spans EU and Member State food laws but also international conventions on illicit drug and psychoactive substances. An understanding of the laws governing the sale of these compounds can help business and academia better understand the challenges consumers may face in selecting products lawfully placed on the market, whilst identifying the unique challenges imposed from the marketing of Cannabis-based foods.
424. KARCHE, {\relax TAHSEEN} and SINGH, {\relax MANAGER}, The Application of Hemp (Cannabis Sativa L.) for a Green Economy: A Review, Turkish Journal of Botany, vol. 43, no. 6, pp. 710--723, January 2019. doi: 10.3906/bot-1907-15.
425. Klir, Željka and Novoselec, Josip and Antunović, Zvonko, An Overview on the Use of Hemp (Cannabis Sativa L.) in Animal Nutrition, Poljoprivreda, vol. 25, no. 2, pp. 52--61, December 2019. doi: 10.18047/poljo.25.2.8. Abstract
     The aim of the paper was to research the possibility of using hemp (Cannabis sativa L.) in animal nutrition. In animal nutrition, hemp seeds can be used, as well as hempseed cake, and hemp oil as supplement in feed mixtures. Hemp seeds are rich in cr...
426. Sandler, L N and Gibson, K A, A Call for Weed Research in Industrial Hemp (Cannabis Sativa L), Weed Research, vol. 59, no. 4, pp. 255--259, 2019. doi: 10.1111/wre.12368. Abstract
     Industrial hemp (Cannabis sativa L.) is grown in more than 30 countries for fibre, seed and flowers, and acreage of cultivation is increasing globally. Hemp has long been promoted as a crop that competes well with weeds and requires little intervention to prevent yield losses. We conducted a literature review and found little peer-reviewed research to support this claim. We identified only three articles that specifically addressed weed management under field conditions and none provided information on hemp yield losses from weeds. These findings highlight a clear need for research-based information on interactions between weeds and hemp to address potential yield losses under various production conditions and provide a research-based framework for weed management in industrial hemp.
427. Rehman, Muhammad Saif Ur and Rashid, Naim and Saif, Ameena and Mahmood, Tariq and Han, Jong-In, Potential of Bioenergy Production from Industrial Hemp (Cannabis Sativa): Pakistan Perspective, Renewable and Sustainable Energy Reviews, vol. 18, pp. 154--164, February 2013. doi: 10.1016/j.rser.2012.10.019. Abstract
     Pakistan is facing severe economical crunch due to continuously growing gap between energy demand and supply. The shortage in power and gas supply has already halted many industrial sectors such as textile, small and medium enterprises and local transportation. The government has spent US \$ 9 billion on energy import during 2008–2009 to fulfill current energy requirements. Indigenous energy resources, mainly fossil fuels, are already being exploited at their maximum. Besides these short term steps, energy demand is expected to double during next decade. Thus, renewable and sustainable energy resources, such as biomass, needs to be exploited so that a sustainable energy mix could be employed to ensure energy security. Industrial hemp (Cannabis sativa) has been successfully investigated for its potential to be used as a renewable feedstock for the production of biofuels. Hemp is an environmental friendly and low cost feedstock which grows wildly in most parts of Pakistan. Thus, hemp can be grown as a potential energy crop in Pakistan to meet its energy requirements by producing various kinds of biofuels. This sustainable feedstock will help the country to reduce its energy import bills, and ensure sustainable energy supply.
428. Kuddus, Mohammed and Ginawi, Ibrahim A. M. and {Al-Hazimi}, Awdah, Cannabis Sativa: An Ancient Wild Edible Plant of India, Emirates Journal of Food and Agriculture, pp. 736--745, June 2013. doi: 10.9755/ejfa.v25i10.16400. Abstract
     Cannabis sativa, also known as Cannabis indica or Indian hemp, is an annual herb of the family Cannabinaceae. It has been used by humans throughout recorded history for its food, fiber and medicine. It is a native to Central Asia, and long cultivated in Asia, Europe and China. Plants yielding the drug seem to have been discovered in India, cultivated for medicinal purposes as early as 900 BC. Hindu devotees offered Cannabis to Shiva during religious ceremonies, and the herb continues to have a religious association in India. The medicinal use of Cannabis has very long history. However, the sociopolitical pressure led to decline the medicinal use of Cannabis. But despite its illegality, people have continued to obtain Cannabis in black market for self-medication. Together with coffee and tobacco, Cannabis is the most commonly used psychoactive drug worldwide, and it is the single most popular illegal drug. This review analyzes the diversity, botanical description, consumption and pharmacological studies along with medicinal uses of Cannabis among the human being throughout the world.
429. Bruci, Zana and Papoutsis, Ioannis and Athanaselis, Sotirios and Nikolaou, Panagiota and Pazari, Ermira and Spiliopoulou, Chara and Vyshka, Gentian, First Systematic Evaluation of the Potency of Cannabis Sativa Plants Grown in Albania, Forensic Science International, vol. 222, no. 1, pp. 40--46, October 2012. doi: 10.1016/j.forsciint.2012.04.032. Abstract
     Cannabis products (marijuana, hashish, cannabis oil) are the most frequently abused illegal substances worldwide. Delta-9-tetrahydrocannabinol (THC) is the main psychoactive component of Cannabis sativa plant, whereas cannabidiol (CBD) and cannabinol (CBN) are other major but no psychoactive constituents. Many studies have already been carried out on these compounds and chemical research was encouraged due to the legal implications concerning the misuse of marijuana. The aim of this study was to determine THC, CBD and CBN in a significant number of cannabis samples of Albanian origin, where cannabis is the most frequently used drug of abuse, in order to evaluate and classify them according to their cannabinoid composition. A GC–MS method was used, in order to assay cannabinoid content of hemp samples harvested at different maturation degree levels during the summer months and grown in different areas of Albania. This method can also be used for the determination of plant phenotype, the evaluation of psychoactive potency and the control of material quality. The highest cannabinoid concentrations were found in the flowers of cannabis. The THC concentrations in different locations of Albania ranged from 1.07 to 12.13\%. The influence of environmental conditions on cannabinoid content is discussed. The cannabinoid content of cannabis plants were used for their profiling, and it was used for their classification, according to their geographical origin. The determined concentrations justify the fact that Albania is an area where cannabis is extensively cultivated for illegal purposes.
430. Mukherjee, Ashutosh and Roy, Satyesh Chandra and De Bera, S. and Jiang, Hong-En and Li, Xiao and Li, Cheng-Sen and Bera, Subir, Results of Molecular Analysis of an Archaeological Hemp (Cannabis Sativa L.) DNA Sample from North West China, Genetic Resources and Crop Evolution, vol. 55, no. 4, pp. 481--485, June 2008. doi: 10.1007/s10722-008-9343-9. Abstract
     Hemp (Cannabis sativa L.) cultivation and utilization is an ancient practice to human civilization. There are some controversies on the origin and subsequent spread of this species. Ancient plant DNA has proven to be a powerful tool to solve phylogenetic problems. In this study, ancient DNA was extracted from an archaeological specimen of Cannabis sativa associated with archaeological human remains from China. Ribosomal and Cannabis specific chloroplast DNA regions were PCR amplified. Sequencing of a species-specific region and subsequent comparison with published sequences were performed. Successful amplification, sequencing and sequence comparison with published data suggested the presence of hemp specific DNA in the archeological specimen. The role of Humulus japonicus Sieb. et Zucc. in the evolution of Cannabis is also indicated. The identification of ancient DNA of 2500 years old C.~sativa sample showed that C.~sativa races might have been introduced into China from the European–Siberian center of diversity.
431. Jiang, Hong-En and Li, Xiao and Zhao, You-Xing and Ferguson, David K. and Hueber, Francis and Bera, Subir and Wang, Yu-Fei and Zhao, Liang-Cheng and Liu, Chang-Jiang and Li, Cheng-Sen, A New Insight into Cannabis Sativa (Cannabaceae) Utilization from 2500-Year-Old Yanghai Tombs, Xinjiang, China, Journal of Ethnopharmacology, vol. 108, no. 3, pp. 414--422, December 2006. doi: 10.1016/j.jep.2006.05.034. Abstract
     A cache of shoots, leaves and fruits dated by 14C at 2500 years B.P. were unearthed in the Yanghai Tombs, Turpan District in Xinjiang, China. By comparing the morphological and anatomical characteristics of the plant remains found in the tomb and specimens of modern plants, it is shown that the remains belong to Cannabis. Based on the shamanistic background of the deceased man and ancient customs, it is assumed that the Cannabis was utilized for ritual/medicinal purposes.
432. Kepe, Thembela, Cannabis Sativa and Rural Livelihoods in South Africa: Politics of Cultivation, Trade and Value in Pondoland, Development Southern Africa, vol. 20, no. 5, pp. 605--615, December 2003. doi: 10.1080/0376835032000149252. Abstract
     Current debate on Cannabis sativa in South Africa has centred on its positive and negative effects on health, as well as its decriminalisation. The contribution of cannabis (dagga) to the livelihoods of people who inhabit some of the poorest parts of the country has thus far been largely ignored. Using a case study of a village in Pondoland, this article argues that while cannabis makes a significant contribution to the livelihoods of many households, the values derived by cannabis farmers vary widely, as determined by social difference, the illegal nature of cannabis production and trade, and the cannabis commodity chain. Furthermore, lobbies to legalise cannabis will not necessarily yield positive results for poor rural cannabis growers, as this could result in lower prices due to the possibility of increased supply, as well as the fact that illegality is what currently keeps the prices fairly high.
433. Mechoulam, Raphael, The Pharmacohistory of Cannabis Sativa, 1986. Abstract
     Cannabis sativa L. was one of the first plants to be used by man for fiber, food, medicine, and in social and religious rituals. Several names for cannabis were used, mostly associated with the term azallu. The Scythians were a tribe of violent warriors who ruled the Crimea and, at different times, parts of southern Russia, the Balkans, Anatolia, and the Middle East around 700 B.C. The Middle Ages in Europe stayed their course, as regarded medical and not-somedical use of cannabis, till the 19th century. Cannabis was part of the religious lore of the Aryans, a nomad tribe, which invaded India from the north circa 2000 B.C. The Indians had a much better understanding of cannabis than the Europeans. In various parts of India cannabis was used for a large number of diseases and to improve the physical and mental states of the user. The use in leprosy in China deserves further investigation.
434. Bradshaw, R. H. W. and Coxon, P. and Greig, J. R. A. and Hall, A. R., New Fossil Evidence for the Past Cultivation and Processing of Hemp (Cannabis Sativa L.) in Eastern England, New Phytologist, vol. 89, no. 3, pp. 503--510, 1981. doi: 10.1111/j.1469-8137.1981.tb02331.x. Abstract
     Fossil records of Cannabiaceae pollen and Cannabis achenes from Flandrian deposits at three sites in eastern England are presented as further evidence for the past cultivation of this crop. It is suggested that retting of hemp to extract fibre was carried out at each of the sites.
435. Cannabis and Culture:, December 1975. doi: 10.1515/9783110812060.
436. Bromberg, Walter, Marihuana Intoxication, American Journal of Psychiatry, vol. 91, no. 2, pp. 303--330, September 1934. doi: 10.1176/ajp.91.2.303. Abstract
     I. Marihuana is the Latin-American name for a cigarette preparation of the hemp plant. The active principle is cannabis sativa: the drug has various synonyms—hasheesh, ganja, reefers, Indian hemp, etc. It has been used for centuries in the Orient and later in Latin America. Mental states produced by the drug constitute a fairly definite clinical entity. II. A study of the relation between violent crime (murder, assault, rape, etc.) with marihuana showed no direct correlation. It is clear from this study, that in this region the drug is a "breeder of crime" only when used by psychopathic types in whom the drug allows the emergence of aggressive, sexual or anti-social tendencies. The addiction to marihuana is not on the same deep physiological or psychologic level that morphine addiction is. III. The clinical pictures observed can be divided into three groups: (1) Acute intoxications. (2) Emotional reactions to the intoxication features. (3) Toxic psychosis due to admixture of drug effects and basic psychosis (manic-depressive, schizophrenic, etc.). IV. Psychologic factors are of great importance in the emotional reaction of an intoxication, bringing various personality forces and conflicts to the surface. V. The psychologic and physiologic effects of cannabis intoxication have been well studied. They are: (1) A change in time perception, reality feeling, somatic, sensory and motor phenomena. (2) Disturbance of consciousness with difficulty in memory retention. (3) Change in subjective evaluation of perception of visual, tactile, proprioceptive, auditory experiences because of disintegration of sensation to a primitive level of perception. The result of this change in body-model (Körperschema) perception may be expressed in feelings of unreality that appear as panic-states in narcissistic or hysterical individuals. (4) A mood reaction consisting of elevation with periods of sudden boisterous laughter. (5) A primary stimulus to the impulsive life with direct expression in the motor field.
437. Clendinning, John, Observations on the Medicinal Properties of the Cannabis Sativa of India, Medico-chirurgical transactions, vol. 26, pp. 188--210, 1843. url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2116906/.
438. Russo, Ethan B., History of Cannabis and Its Preparations in Saga, Science, and Sobriquet, Chemistry \& Biodiversity, vol. 4, no. 8, pp. 1614--1648, 2007. doi: 10.1002/cbdv.200790144. Abstract
     Cannabis sativa L. is possibly one of the oldest plants cultivated by man, but has remained a source of controversy throughout its history. Whether pariah or panacea, this most versatile botanical has provided a mirror to medicine and has pointed the way in the last two decades toward a host of medical challenges from analgesia to weight loss through the discovery of its myriad biochemical attributes and the endocannabinoid system wherein many of its components operate. This study surveys the history of cannabis, its genetics and preparations. A review of cannabis usage in Ancient Egypt will serve as an archetype, while examining first mentions from various Old World cultures and their pertinence for contemporary scientific investigation. Cannabis historians of the past have provided promising clues to potential treatments for a wide array of currently puzzling medical syndromes including chronic pain, spasticity, cancer, seizure disorders, nausea, anorexia, and infectious disease that remain challenges for 21st century medicine. Information gleaned from the history of cannabis administration in its various forms may provide useful points of departure for research into novel delivery techniques and standardization of cannabis-based medicines that will allow their prescription for treatment of these intractable medical conditions.
439. Review of the World Cannabis Situation, Bulletin on Narcotics, vol. 58, no. 1 and 2, 2006. url: https://www.ojp.gov/ncjrs/virtual-library/abstracts/review-world-cannabis-situation. Abstract
     Cannabis is the world’s most widely cultivated and consumed illicit drug, but there remain major gaps in our understanding of global cannabis markets. For example, it appears that premium sinsemilla cannabis, often produced indoors in consumer countries, has become more potent in recent years and that its market share is also growing in some areas. This may be leading to greater localization of cannabis markets. It may also be responsible for the increase in the proportion of cannabis users in treatment populations at the international level. Assessing the extent and impact of this trend, however, is hampered both by a lack of international standards on issues such as terminology and by unanswered research questions. In order to arrive at accurate global estimates of the extent of production, there is a need for more scientific data on cannabis yields. On the demand side, more information is required on the question of cannabis dosage and volumes used by both occasional and regular users. Cannabis is not a uniform drug: the impact of using cannabis of differing potencies and chemical compositions needs to be researched. While issues concerning cannabis have been evaluated many times in the past, it remains a highly adaptable plant and, consequently, a dynamic drug, requiring constant reassessment.
440. Wills, Simon, Cannabis Use and Abuse by Man: An Historical Perspective, 1998. Abstract
     Head of the Drug Information Service, St Mary’s Hospital, Portsmouth, UKORIGINSThe hemp plant, Cannabis sativa, is native to central Asia north of the Himalayas. It was initially confined to an area stretching from Turkestan in the west, to Pakistan in the east. Southern China probably marked the northernmost boundary of this original domain. Hemp has subsequently become much more widespread, largely due to the intervention of man. Cannabis, a dioecious species, is a member of the Cannabidaceae family, which contains only one other genus-Humulus. The hop plant, Humulus lupulus, is used to preserve and flavour beer.
441. Mead, Alice P., International Control of Cannabis, pp. 44--64, August 2014. doi: 10.1093/acprof:oso/9780199662685.003.0003.
442. of Medicine, Institute, Marijuana and Medicine: Assessing the Science Base, March 1999. doi: 10.17226/6376. Abstract
     Download a PDF of "Marijuana and Medicine" by the Institute of Medicine for free.
443. Lenton, Simon, Pot, Politics and the Press—Reflections on Cannabis Law Reform in Western Australia, Drug and Alcohol Review, vol. 23, no. 2, pp. 223--233, 2004. doi: 10.1080/09595230410001704226.
444. Zuardi, Antonio Waldo, History of Cannabis as a Medicine: A Review, Brazilian Journal of Psychiatry, vol. 28, pp. 153--157, June 2006. doi: 10.1590/S1516-44462006000200015. Abstract
     Cannabis as a medicine was used before the Christian era in Asia, mainly in India. The introduction of cannabis in the Western medicine occurred in the midst of the 19th century, reaching the climax in the last decade of that century, with the availability and usage of cannabis extracts or tinctures. In the first decades of the 20th century, the Western medical use of cannabis significantly decreased largely due to difficulties to obtain consistent results from batches of plant material of different potencies. The identification of the chemical structure of cannabis components and the possibility of obtaining its pure constituents were related to a significant increase in scientific interest in such plant, since 1965. This interest was renewed in the 1990's with the description of cannabinoid receptors and the identification of an endogenous cannabinoid system in the brain. A new and more consistent cycle of the use of cannabis derivatives as medication begins, since treatment effectiveness and safety started to be scientifically proven.
445. Bridgeman, Mary Barna and Abazia, Daniel T., Medicinal Cannabis: History, Pharmacology, And Implications for the Acute Care Setting, Pharmacy and Therapeutics, vol. 42, no. 3, pp. 180--188, March 2017. url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312634/. Abstract
     The authors review the historical use of medicinal cannabis and discuss the agent’s pharmacology and pharmacokinetics, select evidence on medicinal uses, and the implications of evolving regulations on the acute care hospital setting.
446. Phillips, Geoffrey F., Analytical and Legislative Aspects of Cannabis, 1998. Abstract
     This first section of Chapter 4 is concerned with forensic definitions of cannabis and its products as a controlled drug of abuse. Three following sections address related offences and attitudes, techniques used in forensic analysis, and the pharmaceutical quality of cannabis products.