Isatis tinctoria

Propagation

Tinctoria usually dies after flowering. Preflowering (i.e. bolting) has been selected against due to adverse indigo production. The same cannot be said for seed producers.[1]

Self-pollinated plants produce fewer siliques (7.1 vs 44.1), fewer seeds with lower weight, poorer germination (8.2% vs. 46%), and subsequent low-vigor plants compared to cross-pollinated plants. Some self-pollinated plants do not produce seeds at all. This is consistent with an outbreeding reproductive system.[2]

Germination

Tinctoria germination is inhibited by a water-soluble compound in the silique (fruit). Washing the seeds with running tap water (no details given) for 48 hours or more markedly increases germination. Manually removing the silique (threshing) also increases germination drastically.[4]

The compound also inhibits the germination of several competitor species. It is denatured by boiling but not freezing.[4]

Germination rate is identical between 10 and 25°C (88-100%) in threshed seeds, though the seedling length is slightly reduced at 10°C.[4]

Tinctoria seed germination does not vary over 10 months of storage in laboratory conditions.[3]

Vegetative

In-Vitro

Tinctoria can be multiplied in-vitro using 1.0 mg/L BAP on Gamborg B5 media with 20 g/L sucrose. No significant differences were noted with MS media. BAP was more effective at inducing more replication while kinetin increased the internode number.[5]

Tinctoria seeds germinate on MS media.[6]

Cultivation

Predominantly a biennial but can be a monocarpic perennial.[3]

A ruderal plant that can grow in hot spots and nitrogen-rich limestone soils.[7] Greater indigo production is usually seen with fertile clay soils.[8]

Transplanting increases total indigo yield compared to direct seeding at the same time.[9]

Tinctoria will begin flowering when exposed to cold weather/short photoperiod, those this varies widely by accession (<10% to >80%).[1] Plants that are transplanted too early in the growing season will tend to flower and produce seeds. However, this behavior varies with the cultivar.[9]

Tinctoria is not a high water-demanding crop.[9]

Tinctoria survives sub-zero (°C) winter temperatures even at high altitudes.[1]

Harvest

Harvested leaves are prone to mildew so it is best to harvest on dry days after the morning dew has evaporated.[8]

Leaves produced during the first year are preferable for indigo production because flowering adversely affects biomass quality and accumulation.[11]

Post-Harvest Processing

The post-harvest processing of tinctoria greatly affects the quality of the resulting dye.[7]

Indigo is not present in the leaves of tinctoria, rather it is produced by a post-harvest process of moiety cleavage and condensation reactions. These reactions occur spontaneously with sufficient oxygen and moisture.[12]

Isatan A and B almost entirely disappear in the conventional drying process.[13]

The traditional method of producing “woad balls” is inefficient, slow, and produces a putrid odor.[12]

Indirubin, a red dye with limited use, the formation must be minimized during the post-harvest processing of tinctoria.[12]

The waxy coating of tinctoria leaves interferes with proper indigo precursor extraction. Maceration, traditionally used in the first step of tinctoria processing, immediately begins the indigo formation process and results in an inferior product.[12]

1. 1 part freshly harvested woad leaves are transferred to mesh bag(s)
2. bags are added to a large tank (~5x the size v/w)
3. 2 parts (w/w) 60°C water is poured over the bag(s)
4. Cold water is added until 38°C is reached
5. Adjust pH with sulfuric acid to reach 3.5
6. Submerge the leaves, seal, and steep for 24 hours
7. Remove leaves and rinse
8. Adjust pH to 9 or 10 using ammonia (ca 0.67 ml/l)
9. Aerate for 2-4 hours
10. Allow to settle for 24 hours
11. Decant/siphon (optional: rinse again to remove water-soluble impurities)
12. ultrafilter and dry at room temperature
13. Neutralize the wastewater with sulfuric acid (forming ammonium sulfate fertilizer)
14. Compost the woad leaves[12]

Alternatively, tinctoria can be used for dyeing immediately after harvest, though this presents logistical difficulties.[14]

Soil and other foreign particles reduces the yield of pure indigo, possibly by inducing side reactions.[15]

john_production_nodate

Yield

Typical field yields are 1-2 g/kg fresh weight of isatan B, 0.3-0.7 g/kg fresh weight of indican, and 11-22 t/ha of leaves.[10]

Indigotica, an alternative indigo crop, has a higher concentration of indigo precursors though it is less stable than tinctoria, prone to bolting and disease.[10]

Greater total biomass was achieved with 10 plants/m2 than with 5 plants/m2 without a reduction of indigo content. Twenty plants/m2 was not different from ten, though this may depend on local soil fertility and water availability. [9]

Similarly, total biomass was increased with N fertilization but indigo content remained unchanged. High-nitrogen application rates were no different from standard application rates for total biomass.[9]

Seed production is unaffected by the sowing date.[9]

Tinctoria leaves are harvested four to six times per year depending on growing conditions.[8]

Soilless

Soil

Fertilization

Pests

Some tinctoria plants are susceptible to a form of rust.[16]

Ecology

Considered an invasive/noxious species in many western US states. Seeds and plants remain in plantations where indigo dye was cultivated in the past.[17][7][18]

Tinctoria often self-sows.[19]

Requires vernalization to flower.[16]

Morphology

Herbaceous biennial or short-lived perennial.[7][3]

Grows up to 120 cm tall.[7][8]

Tetraploid (2n=4x=28).[1]

Wild populations of tinctoria have excellent genetic diversity. USA populations are more closely related, genetically, to those of Switzerland, Ukraine, and Germany compared to populations from Turkey, Iran, and Kazakhstan. UK populations are a possibility though they were not accessed.[17]

I. littoralis may be a subspecies of I. tinctoria.[17]

[18]

Roots

Primarily a tap-rooted plant with some lateral roots in the upper 30cm of soil. No difference in length was noted between vegetative or flowering plants. However, a greater portion of lateral branching occurred during the second year of growth, though this might be environmentally determined.[3]

The tap root must be removed for physical control of the plant. Various herbicides are also effective.[16][18]

Stem

Tinctoria stems are an average of 7.8 ± 0.43 mm (7.3-8.4 mm) in diameter with 16.8 ± 7.56 branches per plant (8-26) at flowering.[20]

Leaves

Tinctoria leaves are covered in a water-repellent waxy layer that can prevent indigo extraction.[12][8]

Leaf width does not vary with genotype or sowing density.[11]

Inflorescence

Small yellow flowers with four petals typical of the Brassicaceae family.[8]

Fruit

Tinctoria silique contains an autotoxic, water-soluble germination/root elongation inhibitor.[4]

Seeds

The average 1000-seed weight is 5.3 ± 0.316 g (5.0-5.8 g). Seed width is 4.03 ± 0.564 mm (3.65-5.03 mm) and length is 15.83 ± 0.903 mm (14.6-17.2 mm).[20]

Phytochemistry

Many phenolics, glucosinolates, carotenoids, monolingnols, and oligolignols are also present.[7]

The main volatile compound (40% of the leaf volatiles; 82% of the root) is 3-butenyl isothiocyanate.[7]

Infraspecific Variation

The concentration and total production of precursor compounds and indigo depend highly on the cultivar, cultivation, harvest protocol, and post-processing.[7][22]

Biosynthesis

Distribution

Leaves contain isatan A/B and indican, both of which serve as indigo precursors. They also contain other pigments indigotin, isoindigotin, isoindirubin, indigo brown, cis-indigo, and many biosynthetic intermediates.[7]

The leaves also contain the alkaloid tryptanthrin at 0.034-0.33% subject to variation in the postharvest process, cultivar, and environmental conditions.[23][7]

Timecourse

Isatan A is the dominant indigo precursor at harvest time in tinctoria.[13]

The ratio between isatan A and isatan B can vary across a season from 1-3:1 to 5-10:1 from later harvests. The ratio between isatan B to indican varied little across a season at approximately 2-3:1.[13]

Young leaves have a higher concentration of indigo precursors than old.[22]

Eighteen months is required from anthesis to seed maturation.[24]

Improvement

Identification

Inheritance

Methods

Spectrophotometric determination of indigo purity is subject to error because of the aggregates formed and the inclusion of water. Indigo purity can instead be determined by weight lost after extraction by solvents. Acetone selectively dissolves indirubin and NMP dissolves indigo.[15]

History & Society

[25]

Consumption

Not considered edible, though historically the flower buds have been consumed.[7]

Dye

One optimized dyeing procedure for cotton is 10 minutes at 70°C using 4 g/L indigo, 12 g/L NaOH, 20 g/L glucose or 10 g/L sodium dithionite (50:1 bath:material ratio).[26] Other fabrics require a longer soaking time (50 min) with similar dyeing parameters (10-12 g/L glucose, 4-6 g/L indigo) using calcium hydroxide (4-8 g/L) instead.[27]

Work Log

13 Jun 2023

Bagged group 2 seeds that were dried outdoors but protected from the rain.

Germination tests:

1. 50 seeds from each harvest group
2. 50 light-colored seeds
3. 50 threshed seeds
4. 50 leached seeds 24 hours (10 volumes?)
5. 50 leached seeds 48 hours (10 volumes?)
6. 50 seeds from each: 4°C storage, -18°C storage, silica dried, sat. CaCl2 dried batches

08 Jun 2023

Harvested the rest of the seeds. Some by the method used on 06 Jun. The rest by cutting the flower stems.

06 Jun 2023

Oops. Need to update this page.

Harvested seeds today by gently combing through the stems with my fingers allowing the seeds to drop onto some fabric for collection. Left about half to one-third on the plants. 266.6g total in 6 trays placed in the dehydrator without heat.

10 Oct 2022

Removed bags and squeezed. Rinsed with water. Squeezed again. Adjusted pH to 9-10 with dilute ammonia. Oxidized with sparger for 4 hours. Let settle for 24 hours. Decanted. Rinsed. Decanted.

A pair of my jeans weighs about 600 grams. That should require about 10 grams of indigo.

09 Oct 2022

The second harvest of tinctoria. ~650 grams wet weight harvested from 5 plants.

Rinsed with cold running water. Repacked into mesh bags. Poured 1300g 60°C water over bags. Added cold/warm tap water until completely submerged bags in 40°C water. Adjusted to a pH of 2-3 with ~2ml concentrated sulfuric acid. Let sit for 24 hours.

05 Sep 2022

Final yield: 2.5 g (3.7 mg/g FW). This is in line with the research (~2mg/g FW). The result is likely 20-40% pure indigo, so about 1.1 mg/g FW pure indigo recovery from the plant.[12]

31 Aug 2022

Attempting an indigo extraction. Harvested 680 grams of woad leaves by removing the lowest leaves of the rosette from 5 plants. Rinsed under running water while removing dead leaves. Heated 700ml of water to 60C (should have used 1400ml). Poured over in large SS pot. Added enough warm water (~40C) water to cover. Let sit for 22 hours (should be 24).

Bibliography

1. Spataro, Giorgia and Negri, Valeria, Adaptability and Variation in Isatis Tinctoria L.: A New Crop for Europe, Euphytica, vol. 163, no. 1, pp. 89--102, September 2008. doi: 10.1007/s10681-007-9604-2. Abstract
   Isatis tinctoria L. was cultivated until the 19th century to produce indigo, a natural blue pigment used principally for dyestuffs. The current search for alternative crops and interest in natural products has led to reconsidering I. tinctoria as a crop to be grown in marginal areas to produce natural indigo. To reintroduce I. tinctoria into cultivation, its behaviour under different climatic conditions as well as its morpho-physiological and genetic diversity must be assessed in order to evaluate the possibilities of future breeding work. To do this, a Eurasian collection of 15 accessions was studied in a 2-year experiment. The study was carried out in four locations in order to assess plant performance at altitudes ranging from 380 to 1,700~m a.s.l. A second experiment evaluated the morpho-physiological diversity of several traits (some related to agronomic performances) of the collection. In a third experiment the genetic traits of the collection were characterised by using eight AFLP and eight SAMPL markers. The species showed a wide adaptability to different mountainous conditions and the populations showed high morphologic and genetic variability and differed according to their origins. Both morpho-physiological and molecular characterisation allowed the accessions to be distinguished into groups of European and Asian origin. Future breeding work is recommended because some accessions have good agronomic potential.
2. Spataro, G. and Negri, V., Assessment of the Reproductive System of Isatis~Tinctoria L., Euphytica, vol. 159, no. 1, pp. 229--231, January 2008. doi: 10.1007/s10681-007-9479-2. Abstract
   Isatis tinctoria L. (woad) is a dye plant whose cultivation is increasing because of its adaptability to marginal conditions and increasing demand for natural products. Suitable breeding schemes need to be set up in order to obtain woad varieties for each proposed environment. Presently, no data about the reproductive system are available. The effects of selfing and crossing on setting and progeny vigour were assessed. Results showed the existence of an outcrossing system in I.~tinctoria. Obligate self-pollinated plants produced fewer siliques (7.1~g per plant) with lower weight (6.0~mg) and lower seed germinability (8.2\%) than outcrossing plants (44.1~g, 8.0~mg and 46.0\% for each character, respectively). Self-pollinated progenies also generally showed lower vigour than outcrossing progenies.
3. Farah, Kassim O. and Tanaka, Ann F. and West, Neil E., Autecology and Population Biology of Dyers Woad (Isatis Tinctoria), Weed Science, vol. 36, no. 2, pp. 186--193, March 1988. doi: 10.1017/S0043174500074695. Abstract
   Dyers woad (Isatis tinctoria L. \# ISATI) has greatly expanded on rangelands in the Intermountain region. Herbicidal and tillage controls are not feasible on rangelands. Better knowledge of the biology of this species could help in the development of biological controls. We examined characteristics that could assist in this effort. Seed viability remained high and relatively stable, but germination decreased over a 10-month period. The seed dispersal pattern of dyers woad was best described by a negative exponential model (log10 y = 1.92-0.02x; r2=0.60), where y equals seeds/m2 and x = distance from mother plant (cm). The root system of dyers woad is dominated by a taproot with some laterals in the upper 30 cm of the soil profile. Survivorship of experimentally established populations monitored over 2 yr showed constriction at two stages: 1) germination and establishment, and 2) young rosette. The latter stage should be targeted for biological control.
4. Young, James A. and Evans, Raymond A., Germination of Dyers Woad, Weed Science, vol. 19, no. 1, pp. 76--78, January 1971. doi: 10.1017/S0043174500048323. Abstract
   Seeds of dyers woad (Isatis tinctoria L.) readily germinated at temperatures from 3 to 25 C. Dyers woad seeds in their fruits, from which they do not normally dehisce, had greatly depressed germination compared to that of threshed seeds. The presence of dyers woad fruits in the same Petri dish markedly depressed germination and/or root elongation of threshed dyers woad seeds and the seeds of some competing broadleaf and grass species. The depression was especially evident on seeds of other species of Cruciferae. Similar reductions in germination or root elongation were obtained when water used to soak dyers woad fruits was used to wet germination substrates.
5. Leal, F. and Cipriano, J. and Carnide, V. and {Pinto-Carnide}, O., In Vitro Culture Establishment of Woad (Isatis Tinctoria L.), Acta Horticulturae, no. 812, pp. 121--124, February 2009. doi: 10.17660/ActaHortic.2009.812.10. Abstract
   Woad (Isatis tinctoria L.) was widely cultivated in Europe to produce indigo, a natural blue dye. This plant, besides its importance as a dye, reveals anti-inflammatory and anticancer medicinal properties. Our goal was to evaluate the best culture medium to establish woad plants in vitro. Nodal segments of young plants were cultured in Murashige and Skoog (MS) and Gamborg (B5) media supplemented with BAP or KIN, with or without NAA. Number and length of shoots and number of internodes were evaluated in two woad populations. In the Bristol population higher values for all the parameters were observed as compared with Coimbra population. Considering the two basal media B5 was more effective to induce multiplication. The best multiplication rates were obtained in B5 medium supplemented with 1.0 mg/L BAP (3.4 shoots per explant). The addition of auxin to the culture media did not increase multiplication. The addition of activated charcoal showed a positive effect in the length of shoots and in the number of internodes, reaching the highest values when MS medium was supplemented with 1.0 mg/L KIN.
6. Miceli, Natalizia and Kwiecień, Inga and Nicosia, Noemi and Speranza, Jasmine and Ragusa, Salvatore and Cavò, Emilia and Davì, Federica and Taviano, Maria Fernanda and Ekiert, Halina, Improvement in the Biosynthesis of Antioxidant-Active Metabolites in In Vitro Cultures of Isatis Tinctoria (Brassicaceae) by Biotechnological Methods/Elicitation and Precursor Feeding, Antioxidants, vol. 12, no. 5, pp. 1111, May 2023. doi: 10.3390/antiox12051111. Abstract
   This study aimed to establish the in vitro shoot culture of Isatis tinctoria L. and its ability to produce antioxidant bioactive compounds. The Murashige and Skoog (MS) medium variants, containing different concentrations (0.1–2.0 mg/L) of benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) were tested. Their influence on the growth of biomass, accumulation of phenolic compounds, and antioxidant potential was evaluated. To improve the phenolic content, agitated cultures (MS 1.0/1.0 mg/L BAP/NAA) were treated with different elicitors, including the following: Methyl Jasmonate, CaCl2, AgNO3, and yeast, as well as with L-Phenylalanine and L-Tyrosine—precursors of phenolic metabolites. The total phenolic content (TPC) of hydroalcoholic extracts (MeOH 70\%) obtained from the biomass grown in vitro was determined spectrophotometrically; phenolic acids and flavonoids were quantified by RP-HPLC. Moreover, the antioxidant potential of extracts was examined through the DPPH test, the reducing power, and the Fe2+ chelating assays. The biomass extracts obtained after 72 h of supplementation with Tyr (2 g/L), as well as after 120 and 168 h with Tyr (1 g/L), were found to be the richest in TPC (49.37 ± 0.93, 58.65 ± 0.91, and 60.36 ± 4.97 mg GAE/g extract, respectively). Whereas among the elicitors, the highest TPC achieved was with CaCl2 (20 and 50 mM 24 h), followed by MeJa (50 and 100 µM, 120 h). The HPLC of the extracts led to the identification of six flavonoids and nine phenolic acids, with vicenin-2, isovitexin, syringic, and caffeic acids being the most abundant compounds. Notably, the amount of all flavonoids and phenolic acids detected in the elicited/precursor feeding biomass was higher than that of the leaves of the parental plant. The best chelating activity was found with the extract of biomass fed with Tyrosine 2 g/L, 72 h (IC50 0.27 ± 0.01 mg/mL), the strongest radical scavenging (DPPH test) for the extract obtained from biomass elicited with CaCl2 50 mM, after 24 h of incubation (25.14 ± 0.35 mg Trolox equivalents (TE)/g extract). In conclusion, the in vitro shoot culture of I. tinctoria supplemented with Tyrosine, as well as MeJa and/or CaCl2, could represent a biotechnological source of compounds with antioxidant properties.
7. Speranza, Jasmine and Miceli, Natalizia and Taviano, Maria Fernanda and Ragusa, Salvatore and Kwiecień, Inga and Szopa, Agnieszka and Ekiert, Halina, Isatis Tinctoria L. (Woad): A Review of Its Botany, Ethnobotanical Uses, Phytochemistry, Biological Activities, and Biotechnological Studies, Plants, vol. 9, no. 3, pp. 298, March 2020. doi: 10.3390/plants9030298. Abstract
   Isatis tinctoria L. (Brassicaceae), which is commonly known as woad, is a species with an ancient and well-documented history as an indigo dye and medicinal plant. Currently, I. tinctoria is utilized more often as medicinal remedy and also as a cosmetic ingredient. In 2011, I. tinctoria root was accepted in the official European phytotherapy by introducing its monograph in the European Pharmacopoeia. The biological properties of raw material have been known from Traditional Chinese Medicine (TCM). Over recent decades, I. tinctoria has been investigated both from a phytochemical and a biological point of view. The modern in vitro and in vivo scientific studies proved anti-inflammatory, anti-tumour, antimicrobial, antiviral, analgesic, and antioxidant activities. The phytochemical composition of I. tinctoria has been thoroughly investigated and the plant was proven to contain many valuable biologically active compounds, including several alkaloids, among which tryptanthrin, indirubin, indolinone, phenolic compounds, and polysaccharides as well as glucosinolates, carotenoids, volatile constituents, and fatty acids. This article provides a general botanical and ethnobotanical overview that summarizes the up-to-date knowledge on the phytochemistry and biological properties of this valuable plant in order to support its therapeutic potential. Moreover, the biotechnological studies on I. tinctoria, which mainly focused on hairy root cultures for the enhanced production of flavonoids and alkaloids as well as on the establishment of shoot cultures and micropropagation protocols, were reviewed. They provide input for future research prospects.
8. Guarino, Carmine and Casoria, Paolo and Menale, Bruno, Cultivation and Use of Isatis Tinctoria L. (Brassicaceae) in Southern Italy, Economic Botany, vol. 54, no. 3, pp. 395--400, July 2000. doi: 10.1007/BF02864789. Abstract
   Isatis tinctoria L. (Brassicaceae), commonly known as wood, is a biennial species with erect stem, hastate leaves, and yellow flowers clustered in racemes. Fruits are pendulous siliques. This species, probably indigenous of southeastern Asia, was used for the extraction of a dyeing agent called “indigo.” Wood was introduced in ancient times in Italy and the first records of its cultivation date back to the Roman period. For many centuries, wood cultivation remained stable, but grew dramatically in the eighteenth century. In that century, the Societá Economiche established by Bourbons encouraged the cultivation of it in Southern Italy. Near Caserta, in Campania region (Italy), a factory for the extraction of dyeing agents was established and the dye was used in textile production in San Leucio (Caserta). The cultivation of I. tinctoria is abandoned today, although this species grows spontaneously as a weed in Italy. The authors discuss the history of wood and some ancient extractive and dyeing methods.
9. Sales, Ester and Kanhonou, Rodolphe and Baixauli, Carlos and Giner, Alfonso and Cooke, David and Gilbert, Kerry and Arrillaga, Isabel and Segura, Juan and Ros, Roc, Sowing Date, Transplanting, Plant Density and Nitrogen Fertilization Affect Indigo Production from Isatis Species in a Mediterranean Region of Spain, Industrial Crops and Products, vol. 23, no. 1, pp. 29--39, January 2006. doi: 10.1016/j.indcrop.2005.03.002. Abstract
   The increasing interest in natural products from a renewable source has encouraged growers to reintroduce indigo-producing crops into the European agriculture. We studied agronomic conditions (sowing date, plant density, nitrogen fertilization, irrigation rate, seedling transplanting) influencing production of the blue pigment indigo, from Isatis tinctoria and I. indigotica crops in a Mediterranean region of Spain (Valencia). I. tinctoria was more suitable for cultivation in our climate conditions than I. indigotica. Indigo yield from Spanish I. tinctoria trials was greater than in Northern and Central Europe. Furthermore, indigo production was maintained when water and nitrogen supplies were significantly restricted, showing that I. tinctoria is not a high-demanding crop.
10. Angelini, Luciana G. and Tozzi, Sabrina and {Nassi o Di Nasso}, Nicoletta, Differences in Leaf Yield and Indigo Precursors Production in Woad (Isatis Tinctoria L.) and Chinese Woad (Isatis Indigotica Fort.) Genotypes, Field Crops Research, vol. 101, no. 3, pp. 285--295, March 2007. doi: 10.1016/j.fcr.2006.12.004. Abstract
    Isatis tinctoria L. (woad) is one of the earliest known sources of indigo in Europe where it was cultivated since the Middle Ages. Isatis indigotica Fort. (Chinese woad), widely distributed in China, had been used from ancient times as indigo-producing plant and medicinal plant. Both species produce indigo precursors indican (indoxyl β-d glucoside) and isatan B (indoxyl ketogluconate) in their leaves. In order to identify new suitable crops for indigo production in Italy, 17 woad lines were studied under field conditions in Central Italy (Pisa, 43°40′N, 10°19′E) from 2001 to 2003. We analyzed the effects of year, genotype, and harvest times together with their reciprocal interactions on leaf yield and indigo precursors production. Woad lines were then compared with seven I. indigotica lines in a field crop experiment set up in 2003. Extraction and quantification of indigo precursors were accomplished by HPLC-ELSD. Isatan B and indican content, as well as equivalent indigo and fresh/dry leaf yield, were compared between species and among genotypes. In I. tinctoria wide variations in phytochemical and agronomic traits were observed among genotypes, with significant differences in isatan B (1–2gkg−1FW), indican (0.3–0.7gkg−1FW) and leaf yield per harvest (11–22tFWha−1). In I. indigotica significant differences were observed in indican (0.3–0.6gkg−1FW) and fresh leaf yield per harvest (10–20tFWha−1). Chinese woad showed higher isatan B than woad (4.9 and 1.5gkg−1FW, respectively). In both species isatan B represented the major precursor, particularly in I. indigotica. The ratio indican:isatan B recorded was 1:5 in woad against 1:14 in Chinese woad, leading to significantly higher +55\% equivalent indigo in the latter. Interestingly, I. tinctoria showed good adaptation to Mediterranean climate conditions with high re-growth capacity after harvest and elevated biomass production. Conversely, I. indigotica, although its higher indigo precursors content/leaf weigh, appeared to be more affected by climate conditions and produced −25\% leaf yield per hectare per season. The present work identified high indigo yielding genotypes that may be used for genetic improvement in order to re-introduce Isatis species in the agricultural systems of Mediterranean regions.
11. Kizil, S. and Arslan, N. and Khawar, K., Effect of Different Sowing Densities on Some Characteristics of Isatis Tinctoria L. and Isatis Constricta Davis and on the Recovery of Indican, Acta Agronomica Hungarica, vol. 55, no. 2, pp. 251--260, June 2007. doi: 10.1556/AAgr.55.2007.2.13. Abstract
    The study reports the effects of four sowing densities (40 × 10, 40 × 20, 60 × 10 and 60 × 20 cm) on the agronomic characteristics of Isatis tinctoria and I. constricta under the rainfed conditions of South Eastern Anatolia. Wide row spacings of 60 × 10 or 60 × 20 cm were effective in obtaining maximum number of leaves per plant, leaf length, leaf width, petiole length, stem diameter, fruit length, 1000 fruit weight and 1000-seed weight. However, narrow row spacing (40 × 10 or 40 × 20 cm) led to maximum values of fresh and dry leaf yield 10 m −2 , plant height, fruit yield and fruit length, minimum hull content, and the highest indican percentage and indican yield m −2 . This information will be helpful for the economical cultivation of these plants under the rainfed conditions of South Eastern Anatolia.
12. Stoker, Kerry G. and Cooke, David T. and Hill, David J., An Improved Method for the Large-Scale Processing of Woad (Isatis Tinctoria) for Possible Commercial Production of Woad Indigo, Journal of Agricultural Engineering Research, vol. 71, no. 4, pp. 315--320, December 1998. doi: 10.1006/jaer.1998.0329. Abstract
    The increasing use of alternative crops has meant the introduction of new technologies to process their products. In this work, an alternative method is presented for the extraction of natural indigo from woad (Isatis tinctoria) based on a technique used to extract indigo fromIndigoferaspp. This method does not rely on the old fermentation procedure used throughout Northern Europe and is cheap, clean and efficient. Evolved from laboratory-based tests, it involves steeping the leaves at low pH in warm water and extracting the indigo at a higher pH, followed by ultra-filtration of the product, which is then left to air-dry. Problems encountered during the development of the technique and how they were overcome are discussed.
13. Oberthür, Christine and Graf, Heidemarie and Hamburger, Matthias, The Content of Indigo Precursors in Isatis Tinctoria Leaves — a Comparative Study of Selected Accessions and Post-Harvest Treatments, Phytochemistry, vol. 65, no. 24, pp. 3261--3268, December 2004. doi: 10.1016/j.phytochem.2004.10.014. Abstract
    We recently clarified the nature of indigo precursors in woad (Isatis tinctoria L.), by identifying the major indoxyl glycoside as isatan A (indoxyl-3-O-(6′-O-malonyl-β-D-ribohexo-3-ulopyranoside)), and by correcting the structure of the related isatan B (indoxyl-3-O-β-d-ribohexo-3-ulopyranoside). A quantitative densitometric assay for isatans A and B, and indican, was established and validated. HPTLC separation on silica gel was followed by densitometric analysis of indigoid pigments formed after treatment with dilute acid or base. The seasonal variation of indoxyl glycosides in woad leaves was investigated with first-year plants (rosette stage) of five defined I. tinctoria L. and one I. indigotica L. accessions. Isatan A content reached up to 7.6\% of dry weight in I. tinctoria, and up to 21.8\% in I. indigotica. The influence of various post-harvest treatments was studied. High concentrations of isatans A and B were found in freeze-dried leaf samples, whereas the content of indican was lowest. Conventional drying at ambient or 40 °C led to complete disappearance of isatans A and B. The concentration of indican, in contrast, was 3- to 5-fold higher in leaf samples submitted to drying at ambient and 40 °C, respectively.
14. {Hook\&Light}, Naturally Dye with Woad, August 2020. url: https://www.youtube.com/watch?v=HKQK39ST_oM. Abstract
    The latest video in our Seed to Sweater series. Today we will be looking at how to dye wool or fabric using the plant Woad. We will be posting more videos on natural dyeing using your Seed to Sweater plants soon, until then check out our blog on www.hookandlight.com for more info. Remember to share your story with us on Instagram using \#seedtosweater and tagging @hookandlight .
15. {Garcia-Macias}, Paulina and John, Philip, Formation of Natural Indigo Derived from Woad (Isatis Tinctoria L.) in Relation to Product Purity, Journal of Agricultural and Food Chemistry, vol. 52, no. 26, pp. 7891--7896, December 2004. doi: 10.1021/jf0486803. Abstract
    There is an increasing commercial demand for naturally sourced indigo that meets the purity standards set by the synthetic product. This study concerns the indigo made from leaves of woad (Isatis tinctoria L.), and in particular its interaction with particulate impurities arising from soil and plant materials. Also, a more reliable method using N-methyl-2-pyrrolidone has been developed for the spectrophotometric determination of indigo. In a novel application of fluorescence spectroscopy, indoxyl intermediates in indigo formation are shown to be stable for minutes. The main indigo precursor from woad can be adsorbed onto Amberlite XAD16 in conformity with a Langmuir isotherm, but indigo precursors break down on this and other resin beads to yield indigo and red compounds. Indigo made from indoxyl acetate aggregates into particles, the size distribution of which can be modified by the inclusion of a fine dispersion of calcium hydroxide. Bright field microscopy of indigo products made under defined conditions and scanning electron microscopy combined with energy-dispersive X-ray analysis reveal the relationship of indigo with particulate materials. A model illustrating the interaction of indigo with particulate contaminants is developed on the basis of the results obtained, and recommendations are made for improving the purity of natural indigo. Keywords: Fluorescence spectroscopy; dispersive X-ray analysis; indigo extraction; N-methyl-2-pyrrolidone; scanning electron microscopy; soil; woad (Isatis tinctoria L.)
16. {Utah State University Extension}, How to Control Dyers Woad Weed, May 2018. url: https://www.youtube.com/watch?v=_X8NvUY40UY. Abstract
    The following links will take you to documents that discuss specific herbicides on dyer's woad. Always read and follow the label. http://wric.ucdavis.edu/information/n... https://www.fs.usda.gov/Internet/FSE\_...
17. Gaskin, John F and Schwarzländer, Mark and Gibson, Robert D and Simpson, Heather and Marshall, Diane L and Gerber, Esther and Hinz, Hariet, Geographic Population Structure in an Outcrossing Plant Invasion after Centuries of Cultivation and Recent Founding Events, AoB Plants, vol. 10, no. 2, pp. ply020, March 2018. doi: 10.1093/aobpla/ply020. Abstract
    We investigated the genetic diversity and origins of a long-term cultivar. Dyer’s woad has been used as a dye source for at least eight centuries in Eurasia. It was introduced to eastern USA in the 1600s, and is now considered invasive in the western USA. Our analysis of plants from the USA and Eurasia suggests that there are two distinct invasions in western USA that most likely originate from Switzerland, Ukraine and Germany. This information assists in finding effective biological control agents, and continued combination of ecological and molecular data helps bring us closer to sustainable management of plant invasions., Population structure and genetic diversity of invasions are the result of evolutionary processes such as natural selection, drift and founding events. Some invasions are also molded by specific human activities such as selection for cultivars and intentional introduction of desired phenotypes, which can lead to low genetic diversity in the resulting invasion. We investigated the population structure, diversity and origins of a species with both accidental and intentional introduction histories, as well as long-term selection as a cultivar. Dyer’s woad (Isatis tinctoria; Brassicaceae) has been used as a dye source for at least eight centuries in Eurasia, was introduced to eastern USA in the 1600s, and is now considered invasive in the western USA. Our analyses of amplified fragment length polymorphisms (AFLPs) from 645 plants from the USA and Eurasia did not find significantly lower gene diversity (Hj) in the invaded compared to the native range. This suggests that even though the species was under cultivation for many centuries, human selection of plants may not have had a strong influence on diversity in the invasion. We did find significantly lower genetic differentiation (Fst) in the invasive range but our results still suggested that there are two distinct invasions in the western USA. Our data suggest that these invasions most likely originated from Switzerland, Ukraine and Germany, which correlates with initial biological control agent survey findings. Genetic information on population structure, diversity and origins assists in efforts to control invasive species, and continued combination of ecological and molecular analyses will help bring us closer to sustainable management of plant invasions.
18. {Biological Weed Control at the University of Idaho}, Dyer's Woad, November 2021. url: https://www.youtube.com/watch?v=gKqqwONdG8w. Abstract
    Short video describing the history, distribution and impact of dyer's woad in North America, and detailed footage and descriptions for accurately identifying this species in the field.
19. {Mountain Gardens}, Isatis Tinctoria (Woad), July 2016. url: https://www.youtube.com/watch?v=1TKLE62U1CQ.
20. Kizil, Süleyman, Morphological and Agronomical Characteristics of Some Wild and Cultivated Isatis Species, Journal of Central European Agriculture, vol. 7, no. 3, pp. 479--484, December 2006. url: https://hrcak.srce.hr/17387. Abstract
    The study evaluated Isatis tinctoria, I. constricta, I. glauca, I. cochlearis, I. aucheri and I. demiriziana during 2002-03 and 2003-04 growing seasons for different agronomic characteristics affecting the percentage of dye in them. The results showe...
21. Comlekcioglu, Nazan and Efe, Lale and Karaman, Sengul, Extraction of Indigo from Some Isatis Species and Dyeing Standardization Using Low-Technology Methods, Brazilian Archives of Biology and Technology, vol. 58, pp. 96--102, 2015-Jan-Feb. doi: 10.1590/S1516-8913201502658. Abstract
    Fresh leaves of four Isatis species culture form of I. tinctoria L and wild forms of I. buschiana Schischkin, I. candolleana Boiss. (endemic) and I. tinctoria L. subsp. corymbosa. (Boiss.) were used for indigo production. Dyes were extracted by fermentation and hot water application. The extracted dyes were optimized with different pH and reducing agents. Results showed that the dye from hot water application produced the desired dying quality at pH 11. Reducing agent concentrations had no significant effect on color quality. Dark blue and blue colors were obtained from I. tinctoria and I. candolleana extracts although I. tinctoria subsp. corymbosa and I. buschiana produced mostly yellow-gray colors. Light, dry and wet rubbing fastness values varied between 3 and 3/4 while washing fastness was between 2 and 4/5. The highest indigo amounts were determined spectrophotometrically as 4.19 mg/g and 2.53 mg/g in I. tinctoria and I. candolleana, respectively. Results also showed that harvesting season was important for indigo production and the highest indigo amount was observed in mid-June.
22. Maugard, Thierry and Enaud, Estelle and Choisy, Patrick and Legoy, Marie Dominique, Identification of an Indigo Precursor from Leaves of Isatis Tinctoria (Woad), Phytochemistry, vol. 58, no. 6, pp. 897--904, November 2001. doi: 10.1016/S0031-9422(01)00335-1. Abstract
    Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-β-d-glucoside). We suggest the name “isatan C” for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.
23. Oberthür, Christine and Hamburger, Matthias, Tryptanthrin Content in Isatis Tinctoria Leaves - A Comparative Study of Selected Strains and Post-Harvest Treatments, Planta Medica, vol. 70, no. 7, pp. 642--645, July 2004. doi: 10.1055/s-2004-827188. Abstract
    Thieme E-Books \& E-Journals
24. Bhattacharya, Amita and Nagar, P. K. and Ahuja, P. S., Seed Development in Camellia Sinensis (L.) O. Kuntze, Seed Science Research, vol. 12, no. 1, pp. 39--46, March 2002. doi: 10.1079/SSR200196. Abstract
    Seed development of tea was studied to identify the maturity index and the optimal time of seed collection. After harvest, the moisture content (28–30\%fresh weight basis) of mature seeds, which germinated 100\%, declined progressively (19\% moisture content) after shedding, with a decrease in seed germination and viability. However, this viability loss could be prevented to some extent by storing seeds within intact fruits. The maximum rate of seed dry matter accumulation coincided with the accumulation of starch in the embryos and seeds at stage 8, the embryo maturation phase. Although the embryo abscisic acid (ABA) content was highest at stage 8, free ABA declined in the tea embryos throughout the remainder of the seed maturation cycle.
25. Mocquard, Julia and Le Lamer, Anne-Cécile and Fabre, Paul-Louis and Mathieu, Céline and Chastrette, Clément and Vitrai, Adrien and Vandenbossche, Virginie, Indigo Dyeing from Isatis Tinctoria L.: From Medieval to Modern Use, Dyes and Pigments, pp. 110675, August 2022. doi: 10.1016/j.dyepig.2022.110675. Abstract
    Since ancient times, indigo has been one of the most widely used natural pigments for textile dyeing. In Europe, the only source of indigo dye was from woad (Isatis tinctoria). Woad leaves were processed to obtain an insoluble indigo pigment, which had to be reduced to leuco-indigo to dye textiles. Today, most indigo comes from the chemical industry, the production of which raises public health and ecological problems. For the past few years, renewed interest in natural pigments has led to the revival of I. tinctoria cultivation for indigo pigment production. However, the woad blue is still obtained with uncontrolled and inconsistent yields. The aim of the following paper is to provide an overview of what is known about the production of the woad blue pigment, from the leaves of I. tinctoria to its use as a dye, from medieval times to the present day. Despite numerous studies, the behaviour of the woad indigogenic precursors and the mechanisms leading to indigo formation remain unclear.
26. Saikhao, Laksanawadee and Setthayanond, Jantip and Karpkird, Thitinun and Suwanruji, Potjanart, Comparison of Sodium Dithionite and Glucose as a Reducing Agent for Natural Indigo Dyeing on Cotton Fabrics, MATEC Web of Conferences, vol. 108, pp. 03001, 2017. doi: 10.1051/matecconf/201710803001. Abstract
    A traditional reducing agent in an indigo dyeing process with cotton fabrics is sodium dithionite (Na\textsubscript{2{$<$}sub/{$>$}S\textsubscript{2{$<$}sub/{$>$}O\textsubscript{4{$<$}sub/{$>$}) which is environmentally unfavorable because the resulting by-products cause various problems to the disposal wastewaters. In this research, glucose was used as a possible replacement of Na\textsubscript{2{$<$}sub/{$>$}S\textsubscript{2{$<$}sub/{$>$}O\textsubscript{4{$<$}sub/{$>$} in indigo dyeing. The comparison of reduction power of Na\textsubscript{2{$<$}sub/{$>$}S\textsubscript{2{$<$}sub/{$>$}O\textsubscript{4{$<$}sub/{$>$} and glucose for natural indigo dyeing on cotton fabrics based on reduction potential was analyzed. The optimum reduction temperature for natural indigo dye of both reducing agents was at 70°C. The reduction time did not have a significant effect on the reduction potential under the condition studied. Na\textsubscript{2{$<$}sub/{$>$}S\textsubscript{2{$<$}sub/{$>$}O\textsubscript{4{$<$}sub/{$>$} could give higher color strength than glucose. However, wash fastness of the fabric samples from a glucose reduction was slightly better than Na\textsubscript{2{$<$}sub/{$>$}S\textsubscript{2{$<$}sub/{$>$}O\textsubscript{4{$<$}sub/{$>$} ones. Hence, glucose virtually has a potential to be used as a green reducing agent in natural indigo dyeing.}}}}}}}}}}}}}}}
27. Shin, Youn-Sook and Cho, A.-Rang and Yoo, Dong-Il, Natural Indigo Dyeing by Using Glucose Reduction, Textile Coloration and Finishing, vol. 21, no. 3, pp. 10--18, 2009. doi: 10.5764/TCF.2009.21.3.010. Abstract
    Dyeing process of the natural indigo powder onto ramie and silk fabrics was investigated by using glucose and calcium hydroxide as a reducing system. Effect of reduction and dyeing conditions such as temperature and time of reduction/dyeing, and concentrations of glucose and calcium hydroxide on the dyeing process were explored. Indigo powder was obtained by drying the conventional niram paste in an oven at {$<$}TEX{$>\$$}50\textasciicircum\{\textbackslash circ\}C\${$<$}/TEX{$>$}. Color strength of the dyed fabrics was evaluated by K/S value measured at the wavelength of maximum absorption({$<$}TEX{$>\$$}\{\textbackslash lamda\}\${$<$}/TEX{$>$}max). Munsell color coordinates(H V/C) were used to compare fabric colors of ramie and silk. Ramie fabric showed purple-blue color for all the temperature and time. On the contrary, silk fabric showed wide range of color including brown, brown-green, green at the different temperature. With the increase of K/S value, the coordinate of value(lightness) decreased for both of ramie and silk fabrics. The coordinate of hue(shade) changed drastically with the increase of K/S value for silk fabric, compared with that of ramie fabric which showed nearly constant value at the whole range of K/S value. Optimum concentrations of calcium hydroxide were for 6 g/L for ramie and 4 g/L for silk at {$<$}TEX{$>\$$}60\textasciicircum\{\textbackslash circ\}C\${$<$}/TEX{$>$} and 50 min. K/S value increased with the indigo concentration. Maximum K/S value was shown at {$<$}TEX{$>\$$}10\{\textbackslash sim\}12\${$<$}/TEX{$>$} g/L of glucose concentration. For both of ramie and silk fabrics, the colorfastness of washing and light was lower than that of rubbing. All the colorfastness values were improved with the increase of color strength.