Indigofera tinctoria

Source of the blue dye *indigo*.

Propagation

Germination

Tinctoria seeds show physical dormancy. Unless scarified, they exhibit very low/slow germination.[1][2]

In one study, optimized scarification by rubbing lightly with sandpaper for 5 minutes resulted in the highest germination (67%) compared to soaking in hot water (20%) or cold water (27%). Soaking in sulfuric acid for 3 and 6 minutes resulted in 3% germination and no seeds germination when soaked for 9 minutes.[3]

Scarification by soaking in 95% ethanol for 15 minutes resulted in 65% germination. Either 10 minutes or 20 minutes was significantly worse at 15% and 40%, respectively. Soaking in 98% sulfuric acid for 15 minutes has the highest germination at 80%. Either 10 minutes or 20 minutes was worse at 45% and 25%, respectively. Twenty-five minutes resulted in zero germinations, likely due to damage to the seed. Seed without pretreatment had a 6.7% germination rate. Other treatments like IAA, GA3, cow’s urine, cow’s milk, and coconut water had variable to mild improvements to germination rate compared to the control.[1]

In a later study, five and ten-minute soaks in concentrated sulfuric acid had 1% and 0% germination. A “quick dip” in the same acid resulted in 76% germination, the highest noted in the study. Mechanical and thermal scarification as well as lower concentrations of sulfuric acid had worse germination rates, with a maximum of 10%, 28%, and 61%, respectively. No significance or deviation values were given, however.[2]

Yet another study found 87-95% germination in tinctoria seeds scarified with 95% sulfuric acid treatment with no statistically significant variation between 15 and 60 minutes. Similar germination (93%; NS difference) was seen with physical scarification by removal of a small section of integument with a scalpel.[4]

Other Indigofera species respond heterogeneously to physical scarification (rubbing with sandpaper) and boiling water immersion (put into boiling water and allowed to cool naturally). Cryptantha, brevicalyx, arrecta, spicata (2 accessions), vohemarensis, and trita generally respond favorably to either treatment. Cryptantha and spicata respond best to physical scarification while the remainder perform best with boiling water treatment.[5]

Similarly, in glandulosa, 75% germination was noted after soaking in concentrated sulfuric acid for 10 minutes compared to 0% in control seeds without pretreatment.[6]

Zollingeriana seeds soaked overnight in 100°C water resulted in an average of about 60% germination with only moderate variation between seed pod harvest colors.[7]

The optimal germination temperature for tinctoria lies somewhere between 25-35°C (nonlinear correlation) with the highest rate (53%) at 35°C.[4]

Vegetative

In-Vitro

Cultivation

Greater shading at 50%, 75%, and 90% reduced root mass in tinctoria. Non-significant heterogeneity was preset in many parameters. Leaf area increased with decreasing light levels.[8] ¹

Similarly, the number of leaves, the number of leaf nodes, and the fresh weight were maximized at 100% light levels (63,200 lux, max), compared to 50% and 25%. Indigo concentration was inversely correlated with light intensity, reaching a maximum of 6.52 mg/l² at 25% intensity though total indigo extract weight remained remarkably unchanged between light intensities.[9]

Harvest

Yield

Soilless

Soil

Fertilization

Tinctoria responds well to mineral fertilizers. Dry mass (leaf, stem, root, total) and wet mass (leaf, stem, root, total) dose-dependently increased with NPK fertilization.[10] ³

Similarly, organic fertilization in the range of 200-400 grams with 10 grams of mycorrhizal inoculant per plant was able to increase root/shoot fresh weight, root biomass, and the number of leaves significantly. The organic fertilizer was made (details not shown) from dye waste tinctoria.[11]

The optimal NPK ratio for maximizing dye yield in tinctoria is 43:23:34 (mol %). Leaf yield is more sensitive to NPK variation than dye concentration.[12]⁴ In the more traditional weight % base of N:P2O5:K2O this would be about 42:43:16.

Temperature

Lighting

Pests

Ecology

Morphology

Indigofera is divided into four clades: paleotropical, pantropical, cape, and tethyan. Tinctoria is in the pantropical clade along with arrecta, astragalina, austalis, decora, heterantha, hirsuta, suffruticosa, zollingeriana, and about 300 other species. All species in the pantropical clade produce the indican glycoside though that is not exclusive to the clade.[13][14]

Roots

Stem

Leaves

Inflorescence

Seeds

Phytochemistry

Infraspecific Variation

Biosynthesis

Distribution

Timecourse

Improvement

Identification

Inheritance

Methods

History & Society

Work Log

18 Mar 2022

The first germination from trial 1 has progressed to hairy-root development.

17 Mar 2022

The first germination is from the control group.

14 Mar 2022

Started 3 groups of 12 seeds (WSS) each: 15-minute immersion in boiling water, 15-minute treatment with 95% ethanol, and a 15-minute soak in water (control).

I think the heat treatment was a little excessive. The seeds were expanded immediately upon treatment and the water was darkened. I should try a 1-5 minute immersion if this trial is unsuccessful.

11 Mar 2022

Indigo testing service

Bibliography

1. Hari, N and Warrier, K C S and Gopalakrishnan, P K, Pre-Sowing Treatments to Promote Seed Germination in Indigofera Tinctoria Linn., pp. 11, January 2002. url: https://www.researchgate.net/publication/281405050_Pre-sowing_Treatments_to_Promote_Seed_Germination_in_Indigofera_tinctoria_Linn. Abstract
   Thirty-four pre-sowing treatments were attempted to break seed dormancy and improve the germination parameters in Indigofera tinctoria Linn. Seed treatments were carried out with ethanol, sulphuric acid, indole 3 acetic acid, gibberellic acid, cow’s urine, cow’s milk and tender coconut water. Scarifying the seeds with concentrated sulphuric acid for 15 minutes recorded the maximum values for germination percentage (80.00) and vigour parameters where germination percentage in the control was only 6.7. However, acid scarification for longer duration had a negative influence on germination. Pre-treatment with ethanol (95\%) for 15 minutes also helped in improving the germination and was found to be on par with the acid scarification for 15 minutes.
2. Neema, M. and Aparna, V. and Krishna, Prakash and Reghunath, B. R., Pre-Sowing Seed Treatments for Indian Indigo (Indigofera Tinctoria), International Journal of Innovative Horticulture, vol. 7, no. 1, pp. 54--55, 2018. url: https://krishi.icar.gov.in/jspui/bitstream/123456789/14021/1/Pre%20Sowing%20seed%20treatments%20for%20Indian%20Indigo.pdf. Abstract
   Indian indigo (Indigofera tinctoria) is a dye yielding plant with medicinal properties. The seeds of the plant are very small and possess a hard seed coat. Different kinds of scarification treatments i.e. thermal, mechanical and chemical were carried out to break the dormancy of the seeds. Best result was obtained when the seeds were subjected to chemical scarification. About 76\% of the seeds germinated when they were given a pre-sown treatment of quick dip in 100\% concentrated sulfuric acid.
3. Barker, J. K. and Abdi, A. W., The Influence of Three Scarification Treatments on Indigofera Tinctoria L. Seed Germination, East African Agricultural and Forestry Journal, vol. 52, no. 3, pp. 208--210, January 1987. doi: 10.1080/00128325.1987.11663517.
4. Sy, A. and Grouzis, M. and Danthu, P., Seed Germination of Seven Sahelian Legume Species, Journal of Arid Environments, vol. 49, no. 4, pp. 875--882, December 2001. doi: 10.1006/jare.2001.0818. Abstract
   As part of the restoration of degraded land south of the Sahara, an experimental study has been carried out on the germinative properties of the seeds of seven Sahelian leguminous species (Cassia obtusifolia, Cassia occidentalis, Indigofera astragalina, Indigofera senegalensis, Indigofera tinctoria, Sesbania pachycarpa and Tephrosia purpurea). Analysis of the effects of temperature, pretreatment and water potential has enabled definition of their optimum germination conditions. For the temperature range studied (20–40°C), germination capacity was significantly greater between 30 and 35°C for all species. All species except Cassia obtusifolia developed a very strong integumental inhibition which was easily eliminated by mechanical scarification or immersion in concentrated sulphuric acid (H2SO4, 95\%). Study of the influence of water potential on germination showed that these species are able to germinate at relatively low water potentials. Different patterns of response to water stress are highlighted and explained by the different behaviours of these species in the semi-arid conditions of the Sahelian environment.
5. Hassen, Abubeker, Characterization and Evaluation of Indigofera Species as Potential Forage and Cover Crops for Semi-Arid and Arid Ecosystems, February 2007. url: https://repository.up.ac.za/handle/2263/23566. Abstract
   The potential of Indigofera species as forage and/or cover crops for semi-arid and arid environments was investigated in several experiments conducted on the Hatfield Experimental Farm in Pretoria, South Africa. Dormancy associated with hard seededness is the main constraint for uniform germination and large-scale propagation of these species. In this study, pretreatment increased germination in most accessions with scarification being more effective than boiling water treatment in six accessions, but not in the case of I. vohemarensis 8730. In five accessions (I. cryptantha 7067, I. brevicalyx 7517, I. arrecta 7524, I. spicata 8254 and I. vohemarensis 8730), scarification improved the total germination percentage, though it simultaneously resulted in higher seed mortality of I. brevicalyx 7517, I. arrecta 7524 and I. vohemarensis 8730 than in the control. In four accessions (I. brevicalyx 7517, I. arrecta 7524, I. vohemarensis 8730 and I. trita 10297), boiling water treatment improved germination percentage without causing any significant risk of seed mortality in the latter three species. In a field study, 41 Indigofera accessions were characterized in terms of morphological and agronomic parameters, using multivariate techniques to describe their phenotypic variability. Eight morpho-agronomic groups with various potentials were identified along with eight determinant characteristics that can be regarded as the core attributes for future Indigofera germplasm characterisation. Further evaluation of promising accessions revealed remarkable differences, both between and within species, in terms of plant height, canopy spread diameter, forage biomass, crude protein content, in vitro organic matter digestibility and indospicine level of the forage. These suggest the possibility of directly selecting accessions with forage potential for subsequent evaluation with target animals. The response of four selected Indigofera accessions under simulated moisture deficit stress and non-stress conditions exhibited significant variation. I. amorphoides was relatively sensitive while I. vicioides was able to maintain growth under water stress conditions, while the response of the two I. arrecta accessions were intermediate. The influence of season and species on forage quality was also studied. Spring growth had a significantly higher (P{$<$} 0.05) CP content than autumn growth in all species. In vitro digestibility of dry material also tended to decrease from the spring of 2004 to the autumn of 2004. Higher levels of Ca, P, Mg, Zn and Cu concentration were revealed in the leaf meal of the first harvest than in the re-growth harvest. All of the species had Ca, Mg, Zn and Mn concentration levels that could support the requirements of ruminants. P and Cu were slightly deficient for some of the species in the autumn suggesting the need to supplement P and Cu from other sources. Compared to Leucaena forage, Indigofera forage had higher apparent organic matter and dry matter digestibility coefficients and higher crude protein and neutral detergent fibre digestibility coefficients. The difference between Indigofera and Leucaena forage in terms of DM intake per unit of metabolic body weight (DMI g BW-0.75 day-1) was not significant (P{$>$} 0.05). The digestible organic matter intake (DOMI) and digestible crude protein intake (DCPI) of the sheep on Indigofera forage was similar to that of sheep fed Leucaena. In this study, lack of differences between Indigofera and Leucaena forage in terms of DOMI, DCPI and DNDFI means that Indigofera forage would likely support similar weight gains as that of Leucaena, but lower than that of M. sativa forage.
6. Ghane, Savaliram Goga and Lokhande, Vinayak Haribhau and Ahire, Mahendra Laxman and Nikam, Tukaram Dayaram, Indigofera Glandulosa Wendl. (Barbada) a Potential Source of Nutritious Food: Underutilized and Neglected Legume in India, Genetic Resources and Crop Evolution, vol. 57, no. 1, pp. 147--153, January 2010. doi: 10.1007/s10722-009-9496-1. Abstract
   Indigofera glandulosa Wendl., (Barbada) belongs to the family Leguminosae, subfamily—Papilionoidae and tribe Indigoferae is widely distributed as weed in India, Indonesia and North Australia. It is an annual herb or sub-shrub growing along roadside and open grassland areas. The plant produce seeds rich in valuable food ingredients such as proteins, carbohydrates, essential amino acids and vitamins. The plant is described as nourishing food for human beings and is believed to possess the qualities of a tonic in Indian medicine. It is highly palatable forage legume; green plants are generally appreciated by domestic animals. Environmentally, it is utilized for the nitrogen enrichment in degraded soil, as the roots produce nodules fixing atmospheric nitrogen. It can be grown in dry regions, therefore appears to drought resistant and at low cost. The plant species remains unexploited although it has high forage and nutritious value. The meagre information on I. glandulosa lead us to explore this neglected and underutilized species to utilize it as food for human beings, forage for animals and for nitrogen enrichment of the soil. The seed viability and seed germination data revealed seed dormancy associated with the hard and impermeable seed coat and it could be overcome by treating the seeds with concentrated sulphuric acid for 10–15~min thus improving the seed germination percentage up to 75\%. The result of the present investigation provides preliminary information on agronomical and morphological traits related to yield and biomass production of I. glandulosa from its natural habitat. In addition the detailed survey about taxonomic characters, distribution, cultivation and utilization of I. glandulosa has been documented.
7. Hutasoit, Rijanto and goat research Station, Indonesian and Riyadi and goat research Station, Indonesian and Sirait, Juniar and goat research Station, Indonesian, The Relationship of Pod Colour with the Quality of Indigofera Zollingeriana, Jurnal Ilmu Ternak dan Veteriner; Vol 24, No 1 (2019): MARCH 2019; 22-28, October 2019. doi: 10.14334/jitv.v24i1.1923. Abstract
   Indigofera zollingeriana (Indigofera) plant is potential feed ingredients. The propagation of this plant is through seed. The low quality of seed is a problem in its development. This study was aimed to evaluate the relationship of pod colour with quality of Indigofera seeds. The study was designed in a complete randomized design consisting of four pod colours and four replications, namely: P1= green, P2= brownish green, P3= brown, and P4= black. The parameters observed were: characteristic and morphology of pods and seeds of Indigofera, the growth of sprouts, and the growth of fungus on Indigofera seed. Results showed that the number of pests was fewest found in P2, brownish green pod (14\%). The highest number of seeds was in P1, green pod (5173) and P2, brownish green pod (4944). The highest germination (62\%) was detected in P2 (brownish green). The heaviest sprout was in P2, in brownish green pod (0.035g), highest sprout (2.68 cm) in P4, black pod colour. Based on fungus observation, the black pod (P4) provided the fewest result (6.63\%), however most fungus grew very well in P1, the green pod (47.88\%). It could be concluded that the brownish green pod colour was the best phase for harvesting good quality I. zolligeriana seed.
8. Budiastuti, M. T. S. and Purnomo, D. and {Supriyono} and Pujiasmanto, B. and Setyaningrum, D. and Manurung, I. R., Indigofera Tinctoria L. Growth at Various Light Intensities and Shading Time Intervals, IOP Conference Series: Earth and Environmental Science, vol. 824, no. 1, pp. 012070, July 2021. doi: 10.1088/1755-1315/824/1/012070. Abstract
   Plant production and changes in physiological aspects during the growing season can be influenced by climate change, one of which is the light factor in Indigofera tictoria. The production of secondary metabolites Indigofera tinctoria as a source of natural dyes is responsive to light. This study examines the effect of shading time intervals and light intensity on the growth of Indigofera tinctoria L. The study used a Randomized Complete Block Design with a split-plot design consisting of 2 treatment factors, namely: the shading time interval as the main plot with five levels, namely 1-4, 1-8, 1-12, 8-12 and 4-12 weeks after planting. Light intensity as a subplot with three levels, namely the light intensity of 50\%, 25\%, and 10\%. The results showed that combination shading time interval and light intensity significantly affected the number of nodia, leaf area of 8 WAP root biomass. Shade time of 1-4 weeks with a light intensity of 50\% showed the highest number of nodia was 45.67 nodia, root fresh weight was 137.00 g, and root biomass was 60.10 g. The shading time interval had a significant effect on the net assimilation rate of the vegetative phase and root fresh weight. The vegetative phase’s net assimilation rate in the 8-12 WAP shading time treatment was 0.029 g.cm2.day−1. The longer the shading time interval with the lower the light intensity can increase the area index and decrease plant growth.
9. Budiastuti, M. T. S. and Purnomo, D. and {Supriyono} and Pujiasmanto, B. and Setyaningrum, D., Effect of Light Intensity on Growth, Yield and Indigo Content of Indigofera Tinctoria L., IOP Conference Series: Earth and Environmental Science, vol. 724, no. 1, pp. 012085, April 2021. doi: 10.1088/1755-1315/724/1/012085. Abstract
   Synthetic dyes can increase the amount of pollutants that become a serious problem in the environment. The use of synthetic dyes can be replaced with dyes from natural ingredients, namely Indigofera tinctoria. These plants are a source of blue natural dyes because it contains indigo. The content of indigo is very responsive to light. The impact of climate change is a serious threat to the supply of natural dyes. So, judge the suitability of the environment and indigo content under climate change are essential for the sustainable production of natural dyes Indigofera tinctoria. The research aimed to examine the role of light on the growth, yield, and indigo content in Indigofera tinctoria. The study was conducted using a randomized complete design with one factor, namely light intensity with 3 levels namely light intensity 100\%, 50\%, and 25\%, with 9 replications. Light intensity affected the number of leaves, nodes, fresh weight, and indigo content. The highest number of leaves, nodes, and fresh weight were at 100\% light intensity, while the highest indigo content was at 25\% light intensity. The fresh weight with indigo paste is positively correlated. The higher the fresh weight of the plant, the more paste will be produced. However, the content of indigo was negatively correlated with indigo paste.
10. Ayasov, Kh G. and Akhmedov, E. and Khidirov, S., Effects of Certain Mineral Fertilizers on the Biological Mass of Indigofera Tinctoria and Impatiens Balsamina Plants, IOP Conference Series: Earth and Environmental Science, vol. 939, no. 1, pp. 012082, December 2021. doi: 10.1088/1755-1315/939/1/012082. Abstract
    The article describes the effect of mineral fertilizers on the cultivation of dyed Indigofera (Indigofera tinctoria L.) and henna (Impatiens balsamina L), the amount of their application and the ratio of basic nutrients (nitrogen, phosphorus and potassium) related. It has been established that the adequate development of Indigofera and henna plants depends on the ratio of mineral fertilizers used.
11. Budiastuti, M. T. S. and {Supriyono} and Manurung, I. R. and Setyaningrum, D. and Nurmalasari, A. I. and Arista, N. I. D., The Role of Organic Fertilizer from Natural Dye Waste and Mycorrhizal Inoculation on the Growth of Indigofera Tinctoria L., IOP Conference Series: Earth and Environmental Science, vol. 905, no. 1, pp. 012011, November 2021. doi: 10.1088/1755-1315/905/1/012011. Abstract
    Management of Indigofera tinctoria as a natural dye produces organic waste that has not been utilized. One of the proper managements of organic waste is to process it into organic fertilizer. This study examines the role of organic fertilizer waste and mycorrhizae on the growth and yield of Indigofera tinctoria. The study used a completely randomized block design with two factors: organic waste fertilizer and mycorrhizae. The results showed that organic fertilizers and mycorrhizae did not affect the net assimilation and leaf area indexes. The combination of organic fertilizers with mycorrhizae supports leaf growth. The combination of 400 g.plant−1 organic fertilizer and 10 g.plant−1 mycorrhizal fertilizer increased the number of leaves by 257\%. Organic fertilizer has a significant effect on the fresh weight of the crown, fresh weight and root biomass. Organic fertilizer dose of 200 g.plant−1 increased fresh root weight, root biomass and fresh crown weight by 68.5\%, 68.29\% and 63.27\% respectively. Mycorrhizae 10 g.plant−1 increased root length by 23.54\%. Leaf growth correlated with length, fresh weight and root biomass. Organic fertilizer from the extraction of Indigofera tinctoria is an effort to achieve zero waste to support plant growth.
12. Mairapetyan, S.K. and Tadevosyan, A.H., Optimisation of Lawsonia Inermis L. and Indigofera Articulata Gouan. Nutrient Solution in Open-Air Hydroponics, Acta Horticulturae, no. 481, pp. 321--326, January 1999. doi: 10.17660/ActaHortic.1999.481.36. Abstract
    Research was carried out to determine the effect of major nutrients, e.g., Nitrogen (N), Phosphorus (P) and Potassium (K) ratio on the leaf productivity and the leaf dye content of Henna (Lawsonia inermis L.) and Indigo (Indigofera articulata Gouan.). Trials were conducted under open-air soilless conditions and the mathematical relationships between these parameters were described by the regression equations: for Henna for indigo Y=4.54-0.037X; r=0.03(1) Y=0.52-0.0005X; r=0.0005(2) Y=3.82-0.028X; r=0.028(3) Y=1.24-0.011X; r=0.11(4) where Y (1,2) is the leaf productivity of plant (g/plant), Y (3,4) is the dye content in leaves (\%), X is the difference between experimental N:P:K: and the optimal N:P:K: for each index of each plant (atom \%). When X=0, experimental ratio us optimal and maximal leaf productivity for henna and indigo are 4.54 and 0.52 g/plant respectively, while the highest content of dye in leaves are 3.82 and 1.24\% respectively. When N,P,K, ratio in nutrient solution declines from the optimal one on 1 atom \% (X=1), the leaf productivity anf dye content decreased by 0.037 g/plant and 0.028\% for Henna and 0.005 g/plant and 0.011\% for Indigo. For N,P,K, ratio in nutrient solution the following data were obtained for Henna and Indigo respectively: 36:28:36, 43:23:34 atom\% for leaf productivity and 37:33:30, 25:33:42 atom\% for dye content in leaves.
13. Schrire, Brian, A Review of Tribe Indigofereae (Leguminosae–Papilionoideae) in Southern Africa (Including South Africa, Lesotho, Swaziland \& Namibia; Excluding Botswana), South African Journal of Botany, vol. 89, pp. 281--283, November 2013. doi: 10.1016/j.sajb.2013.06.014. Abstract
    A review is given of the legume tribe Indigofereae for the region of southern Africa including Namibia. The overall distribution, taxon statistics, phylogenetic relationships, uses, bibliography and a checklist of taxa in the tribe are provided covering the c. 340 taxa of Indigofereae within the region.
14. Gerometta, Elise and Grondin, Isabelle and Smadja, Jacqueline and Frederich, Michel and {Gauvin-Bialecki}, Anne, A Review of Traditional Uses, Phytochemistry and Pharmacology of the Genus Indigofera, Journal of Ethnopharmacology, vol. 253, pp. 112608, May 2020. doi: 10.1016/j.jep.2020.112608. Abstract
    Ethnopharmacological relevance Indigofera is the third-largest genus in the family of Fabaceae, with approximately 750 species. It is distributed across all tropical regions. Indigofera species are widely employed in traditional medicine all around the world, against many ailments. Thus, based on these medicinal properties, various investigations have been undertaken in order to appraise the pharmacological activities and the chemical composition of these species. A recent paper provides a summary of the phytochemistry and pharmacology of the genus Indigofera. Consequently, this review is a continuation of this previous study by updating some data and adding information about the phylogeny and traditional uses of the genus. Aim of the study To provide an overview of the phylogeny, traditional uses, phytochemistry, pharmacology and toxicity of the genus Indigofera, and to identify the remaining gaps and thus supply a basis for further investigations. Materials and methods A review of the literature was performed by consulting scientific databases such as ‘ScienceDirect’, ‘PubMed’, ‘Google Scholar’ and ‘SpringerLink’ and using the keyword Indigofera. Results Over 60 Indigofera species are reported in traditional medicine. The uses depend on the country and the species, but similarities have been noticed. Indeed, treatments of gastrointestinal disorders, inflammatory conditions and pain, skin ailments, and respiratory and infectious diseases are recurring. Phytochemical studies have led to the identification of more than 200 compounds, notably flavonoids and terpenoids. Many pharmacological activities have been demonstrated, particularly antimicrobial, cytotoxic and anti-inflammatory activities, and thus allowed to assert most of the traditional uses of the genus. Some active compounds have been isolated and have shown remarkable therapeutic potential, like the alkaloid indirubin, which is currently being clinically trialed. Conclusions The data on the genus Indigofera are extensive, but gaps still remain. Indeed, some promising species have not been assessed for their phytochemistry and/or pharmacology and thus remain unexplored. Moreover, relatively few active compounds have been isolated and tested for their biological activity, and studies to explain their mechanism of action are nearly inexistent. Furthermore, some pharmacological studies have inappropriate methodologies that make the results difficult to interpret. Consequently, further in-depth and relevant research is required to supplement the knowledge on this wide-ranging genus and to confirm its reported therapeutic potential.