Abstract
Background
Baccharoides anthelmintica (L.) Moench, commonly known as Aranya Jeeraka in Sanskrit, is a pharmaceutically important medicinal plant used in various traditional healthcare systems, including the Indian System of Medicine. As the majority of the raw material of this species comes from wild/ natural stands, promoting cultivation is a viable means for meeting its current and future requirements.
Results
During the present investigation, three studies were conducted to standardize nursery techniques in this species. In the laboratory study with eleven pre-sowing seed treatments, significantly highest germination (58.00%) was obtained with GA3 (500 ppm), when compared with untreated control (31.33%). This treatment also promoted seedling growth characteristics and seedling vigour index. In the second study, the effect of thirteen pre-sowing seed treatments was studied under a protected structure, in which significantly superior germination (46.33%) was observed in the GA3 (500 ppm) treatment, when compared to 32.33% in the untreated control. In the third trial, use of vermicompost as a substrate was found to support seed germination, germination characteristics and seedling vigour.
Conclusions
Soaking of seeds in GA3 (500 ppm) for 24 h could improve the seed germination and seedling growth under in vitro as well as ex vitro conditions. Use of vermicompost as substrate was found to be ideal for raising quality seedlings in this species. This study could help raise quality planting material to promote systematic cultivation of Aranya Jeeraka, thereby ensuring an undisturbed and sustainable supply of raw material to the relevant industries.
Keywords: Asteraceae, Bitter cumin, Gibberellins, Seedling vigour index, Substrate
Background
Bitter cumin or Aranya Jeeraka (Sanskrit), scientifically known as Baccharoides anthelmintica (L.) Moench (syn. Centratherum anthelminticum (L.) Gamble; Vernonia anthelmintica Willd), is an annual plant of the Asteraceae family having multifarious uses. The species is believed to be originated in India and Pakistan [1] and has been reported to occur in Afghanistan, Botswana, China, Kenya, Laos, Malawi, Mozambique, Myanmar, Namibia, Nepal, Sri Lanka, Tanzania, Uganda, Vietnam, Zambia, Zaïre and Zimbabwe [2].
The species has traditionally been used in the folklore medicinal systems in India, Pakistan, China, Sri Lanka and Bangladesh [3]. In India, it is used in the treatment of spermatorrhoea and for stopping menstruation [4], skin diseases, mainly leprosy [5] and was also administered orally as a blood purifier in scabies and eczema apart from topical application [6]. Root juice has been reported to protect from postpartum obesity in Kerala, India [7]. Further, it has been used for curing ailments of central nervous system, renal function and gastro-intestine [3]. The species is part of various Ayurvedic and Siddha formulations used for the treatment of asthma, kidney troubles, cough, leukoderma, psoriasis and skin infections [8].
In the recent past, several clinical trials have been conducted in the species. In the review by Dogra et al. [3], a number of pharmacological activities have been described from crude or purified extracts of the species, and the prominent ones include anti-vitiligo, anti-diabetic, anti-proliferative, neuro-protective, hepatoprotective, analgesic, antipyretic, immunomodulatory, etc. It was found to have potential in the treatment of stress-induced hair loss [9], which is one of the common issues in the recent past. Fatima et al. [10] reported significant antidiabetic and antihyperlipidemic property without apparent toxic effects on the tested animals. In a non-blinded controlled trial conducted in 40 T2DM patients, B. anthelmintica seed powder (500 mg) was effective as an anti-diabetic and anti-lipidemic [11]. Vernodalin, a compound isolated from the species, has been shown to have cytotoxic and apoptotic activities against human breast cancer cells; thereby opening avenues for its potential as an anti-cancer drug [12]. Kumar et al. [13], through biochemical, proteomics and molecular simulation approaches, elucidated the excellent impact of the seed extract against human lymphatic filarial parasites even at extremely low doses.
Apart from the medicinal properties, seeds are known to be the source of fatty acids, mainly vernolic acid, which has industrial applications in the preparation of toughened epoxy resins, lubricants, surface coatings, plastics and oligoethylene glycols [3, 14]. There were 20 patent applications filed for products containing B. anthelmintica in various countries, which are used in both medical and non-medical applications, including skin diseases, hair disorders, diabetes, immunomodulation, as a fungicide and many more [3].
Data pertaining to the production and trade are scarce for this species as most of the raw material is sourced from the wild [15]. As per the data on high-demand medicinal plants available with National Medicinal Plant Board [16], New Delhi, India, the annual trade volume of Aranya Jeeraka lies between 200 and 500 tonnes. The state of Chhattisgarh, India, reportedly harvested ca. 680 tonnes of Aranya Jeeraka seeds from the wild in a single year [17], while the estimated produce from the Madhya Pradesh State was ca. 261 tonnes [18], revealing the huge dependence on wild habitats for its sourcing. There is good scope for its cultivation to meet the growing demand of the species in commercial and pharmaceutical industries, and standardization of agro-techniques would be a prerequisite. Thus, studies were initiated to develop nursery techniques and the present report concerned the effect of various seed treatments and substrates on germination characteristics.
Methods
The present set of experiments was carried out in the Division of Horticulture and Crop Improvement of ICAR-Central Island Agricultural Research Institute and CCRAS-Regional Ayurveda Research Institute, both located in Sri Vijaya Puram, Andaman and Nicobar Islands, India.
Seed material and authentication
Fully mature seeds of Baccharoides anthelmintica (L.) Moench were procured from the Directorate of Medicinal and Aromatic Plants Research, Boriavi, Gujarat, India. The procured seeds were then authenticated from the Siddha Central Research Institute, Chennai, India (Central Council for Research in Siddha, Chennai, India). A voucher specimen of the seeds was deposited in the Regional Ayurveda Research Institute, Sri Vijaya Puram Herbarium (RARISVPH). The seeds were cleaned and sorted to remove underdeveloped/ immature seeds. Parameters such as seed length and seed thickness were recorded using a digital Vernier caliper with 20 seeds and weight of 100 seeds was recorded using digital analytical balance (Sartorius, Germany) in triplicate. Three independent experiments were conducted as given below.
Experiment 1: effect of seed treatments under laboratory conditions
Mature seeds were surface-sterilized by immersion in 5% sodium hypochlorite solution for 10 min, followed by thorough rinsing with sterile distilled water and air-drying under sterile conditions. The sterilized seeds were soaked in different chemicals namely GA3 (100, 250 and 500 ppm), Thiourea (0.5, 1.0 and 2.0%), 65% HNO3 (30, 60 and 90 s) and water, while untreated seeds served as control. Seed soaking duration was 24 h (except for HNO3), and seeds were sown in Petri plates lined with absorbent cotton and a circle of filter paper moistened with distilled water in triplicate. Temperature and relative humidity were recorded using digital data logger (Metravi, India), which suggested mean temperature of 28.0 °C and mean relative humidity of 88.1% during the study period. After 77 days of sowing, 15 representative seedlings from the five successful treatments were used for recording the observations on seedling growth parameters. Seedling vigour index (SVI) was calculated as: SVI = seedling length × germination (%) [19]. Seed germination was evaluated over a six-month period under laboratory conditions (28 ± 2 °C), with assessments conducted at three-month intervals. Seeds were stored in screw cap air tight polypropylene bottles, and germination percentage of seeds sown in Petri plates lined with moistened absorbent cotton and filter paper was recorded in triplicate.
Leaf samples from treatment groups with sufficient biomass were washed with deionized water and surface-dried. For pigment extraction, 0.125 g of leaf tissue was incubated in 6.25 ml of dimethyl sulfoxide (DMSO) at 60 °C for 3 h in aluminum foil-wrapped tubes to prevent light degradation. The absorbance of the supernatant was measured at 480, 649, and 665 nm using Biospectrometer (Eppendorf, Germany). Chlorophyll a, chlorophyll b, and total chlorophyll contents were calculated [20] and expressed as µg/g.
Experiment 2: effect of seed treatments under nursery condition
Mature seeds were used to study the influence of various treatments under naturally ventilated polyhouse with insect proof net on all the sides and polycarbonate sheet on the top. Temperature and relative humidity were recorded using digital data logger (Metravi, India), which suggested mean temperature of 33.1 °C and mean relative humidity of 64.9% during the study period. Unsterilized seeds were soaked for 24 h as per treatment in water, KNO3 (0.1% and 0.2%), GA3 (100, 250 and 500 ppm), NAA (100, 250 and 500 ppm) or IAA (100, 250 and 500 ppm) along with untreated control. After the treatment, the solution was discarded and seeds were sown in protrays (50 cavities, 5 cm depth) filled with soil + farmyard manure (1:1, v: v) as substrate. Immediately after sowing, trays were drenched with Carbendazim (0.2%) for 15 min to provide protection against soil-borne pathogens. Number of seeds per replication was 100 and each treatment was repeated thrice. Seven weeks after sowing (WAS), seedlings were carefully uprooted from the trays and randomly selected 15 seedlings were used from each treatment for recording morphological parameters. Shoot length (cm) was measured using a measuring scale as the distance between collar region and growing tip, whereas root length (cm) was measured from collar region to tip of the tap root. Collar thickness was measured using a digital vernier calliper (mm) and number of leaves per seedling were counted manually in each seedling. Seedling vigour index was calculated as mentioned above.
Experiment 3: effect of substrates under nursery condition
In this experiment, untreated seeds were sown in protrays (2.5 cm depth, 98 cavities) filled with sand, soil, vermicompost or coir pith compost as a substrate. The experiment was replicated twice and each replication consisted of 98 seeds. The trays were irrigated regularly and emergence of seedlings was recorded on daily basis. Temperature and relative humidity were recorded using digital data logger (Metravi, India), which suggested mean temperature of 29.6 °C and mean relative humidity of 83.4% during the study period. After 55 days of sowing, twelve representative seedlings from each treatment were used for recording plant morphological parameters as given above apart from calculation of SVI.
Statistical analysis
The collected data on various morphological observations described above was subjected to Analysis of Variance (ANOVA) using Web Agri Statistical Package (WASP 2.0, ICAR-CCARI, Goa, India). Mean separation was done using least significant difference at 5% level of significance. In case of Experiment 1, arc sin transformation was adopted and the values were shown in parentheses after the actual values. In cases wherein ANOVA was not feasible, data was presented as mean ± standard error of mean.
Results and discussion
In the standardization of herbal drugs, variability in the biological activity is commonly observed in the raw material derived from heterogeneous sources [8] thereby increasing the chances of non-uniform batches of drugs. Cultivation could assist in the large-scale production of raw material of near-uniform potency, and the availability of quality planting material is a key step in the process. In order to improve the seed germination process and seedling characteristics, three experiments were conducted in bitter cumin. Seeds used for the experiment had a mean length of 5.0 ± 0.05 mm, mean thickness of 1.0 ± 0.04 mm, while 100 seed weight was 0.52 ± 0.015 g. Bhatia et al. [8] reported the seeds to be 4.5 to 6.0 mm long, while weight of 1000 seeds was 4.6 to 5.2 g, when grown under conditions of China [1]. It has been suggested that in the species, the germination rate is low just after harvesting and seeds require some period of storage before they can be sown [1, 21]. However, in order to know the effect of different treatments, the present studies were conducted using fresh seeds.
Experiment 1: effect of seed treatments under laboratory conditions
Various priming techniques are adopted in crops to alleviate seed dormancy, improve germination rates, enhance seedling vigour and seedling capacity to grow and establish across diverse environmental conditions [22]. In this study, the effect of eleven treatments was studied to identify suitable treatments for obtaining seedlings of Aranya Jeeraka in large numbers. A total of 31.33% seeds could germinate in untreated control (Table 1). Interestingly, this percentage is much higher than the earlier reports which suggested that fresh seeds show poor germination [21]. Water soaking of seeds, which is one of the most common methods of improving germination, resulted in 40.67% germination. Gibberellic acid soaking showed concentration-dependent success and significantly highest germination of 58.00% was obtained with the highest dose of GA3 studied i.e. 500 ppm. Dose-dependent variations in the germination as well as seedling growth characteristics as observed in the present study, have also been reported earlier [23].
Table 1.
Seed germination (%) in B. anthelmintica germinated with different seed treatments under Petri plate conditions
| Treatment | Germination (%) |
|---|---|
| GA3 (100 ppm) | 13.33 (21.37) c |
| GA3 (250 ppm) | 16.67 (23.94) c |
| GA3 (500 ppm) | 58.00 (49.70) a |
| Thiourea (0.5%) | 10.67 (18.49) c |
| Thiourea (1.0%) | 4.67 (12.03) d |
| Thiourea (2.0%) | 0.67 (2.98) e |
| HNO3 65% for 30 s | 0.00 (0.405) e |
| HNO3 65% for 60 s | 0.00 (0.405) e |
| HNO3 65% for 90 s | 0.00 (0.405) e |
| Water | 40.67 (39.62) b |
| Untreated | 31.33 (34.01) b |
Values in parenthesis are arc sin transformed values. Value followed by similar alphabet in a column do not differ significantly at 5% level of significance using least significant difference
Acid scarification and thiourea treatment have been regarded as useful treatments for improving the seed germination in various species [24, 25]. However, in the present study, treatment of seeds with nitric acid resulted in complete failure of germination as no response was obtained in any of the treatments studied. Also, use of thiourea was detrimental as treatment with it at 0.5% concentration could induce germination in a mere 10.67% seeds and the germination was further reduced to 0.67% with an increase in concentration of thiourea to 2.0%.
Seedling growth parameters and seedling vigour index were determined to understand the effect of different treatments on these attributes (Table 2). Due to poor germination response in some treatments, only five treatments with sufficient number of replications were used for the analysis. Analysis of variance suggested that use of GA3 (500 ppm), which supported maximum germination, was significantly effective in improving the shoot length (5.31 cm), root length (1.97 cm) and collar thickness (0.91 mm), while all the treatments remained on par in terms of number of leaves produced per seedling. Seedling vigour index, which is a product of seedling length and germination (%), was also significantly highest (422.24) in GA3 (500 ppm) treatment. The effect of storage on seed germinability was evaluated over the period of six months to find that it substantially decreased from 31.33% to 14.66% in 3 months during storage and ceased germination entirely upon storage for 6 months (Fig. 1).
Table 2.
Seedling morphological parameters and seedling vigour index in B. anthelmintica germinated with different seed treatments under Petri plate conditions
| Treatment | Shoot length (cm) | Root length (cm) | Collar thickness (mm) | No. of leaves | Seedling Vigour Index |
|---|---|---|---|---|---|
| GA3 (100 ppm) | 3.11 d | 1.42 b | 0.79 b | 2 a | 60.44 d |
| GA3 (250 ppm) | 3.12 d | 1.54 b | 0.73 c | 2 a | 77.68 d |
| GA3 (500 ppm) | 5.31 a | 1.97 a | 0.91 a | 2 a | 422.24 a |
| Water | 4.75 b | 1.54 b | 0.74 bc | 2 a | 255.95 b |
| Untreated | 4.13 c | 1.34 b | 0.77 bc | 2 a | 171.28 c |
Values followed by similar alphabet in a column do not differ significantly at 5% level of significance using least significant difference
Fig. 1.
Germination percentage in B. anthelmintica as influenced by seed storage under laboratory conditions
The photosynthetic pigments, including Chlorophyll a, Chlorophyll b, total chlorophyll content and ratio of Chl a: Chl b were also studied in the regenerated seedlings from the five treatments (Table 3). It was observed that the highest total chlorophyll content of 3061.4 ± 159.97 µg/g were observed in the untreated control, while the highest dose of GA3 showed drastic reduction in the greenness of the plants. In general, the GA3 treated seeds started germinating earlier and the in vitro Petri plates are devoid of the required nutrients for subsequent growth. Thus, the seedlings start turning pale, whereas the untreated seeds germinated slower and thus the pigments were retained for longer. Decrease in photosynthetic pigments in over-grown seedlings has been reported in earlier reports as well [26].
Table 3.
Photosynthetic pigments content in B. anthelmintica germinated with different seed treatments under Petri plate conditions
| Treatment | Chlorophyll a (µg/g) | Chlorophyll b (µg/g) | Total chlorophyll (µg/g) | Chlorophyll a: Chlorophyll b |
|---|---|---|---|---|
| GA3 (100 ppm) | 1879.6 ± 306.94 | 532.9 ± 69.81 | 2142.5 ± 376.21 | 3.53 |
| GA3 (250 ppm) | 2021.1 ± 196.17 | 559.5 ± 56.45 | 2580.6 ± 252.36 | 3.61 |
| GA3 (500 ppm) | 815.6 ± 59.92 | 221.6 ± 14.73 | 1037.1 ± 74.53 | 3.68 |
| Water | 1546.5 ± 118.75 | 418.7 ± 17.06 | 1965.2 ± 135.15 | 3.69 |
| Untreated | 2368.3 ± 116.39 | 693.2 ± 49.03 | 3061.4 ± 159.97 | 3.42 |
Experiment 2: effect of seed treatments under protected structure
An independent experiment was conducted under naturally ventilated polyhouse condition to know the response of 12 treatments on germination characteristics along with untreated control (Fig. 2). In this study, 32.33% germination was noticed in the untreated control, and soaking of seeds in water remained statistically similar to untreated seeds unlike the laboratory trial. Here also, GA3 was the only chemical which could statistically improve the germination in this species, though its success varied with concentration as also witnessed in the previous trial. Significantly highest germination of 46.33% was observed in GA3 (500 ppm) treatment in this trial. As an experiment in Petri plates is under much controlled conditions than the trial under polyhouse conditions, reduced germination in the latter case is expected. Interestingly, none of the studied concentrations of auxins, i.e. NAA and IAA, could improve the germination process, and a mere 4.00 to 16.00% germination was reported in these treatments.
Fig. 2.
Germination percentage in B. anthelmintica as influenced by seed treatments 7 WAS (Weeks after sowing) under protected structure (Bars with similar alphabet do not differ significantly at 5% level of significance using least significant difference)
Nine of the thirteen treatments with a sufficient number of replications were used to compare the seedling growth parameters and seedling vigour index (Table 4). Shoot length was significantly the poorest in auxin-treated seeds, i.e. 4.94 cm (NAA 100 ppm) and 6.47 cm (IAA 100 ppm). Shoot length in both levels of KNO3 and all the levels of GA3 was statistically superior but remained on par with the untreated control. Similar results were noticed for number of leaves per seedling in which except for auxin treatments, all other treatments could produce significantly highest number of leaves per seedling i.e. 5.53–6.13. Root length varied between 3.16 and 4.19 cm but the differences remained statistically non-significant. A number of treatments also supported thickening of the collar region. Though untreated seeds could result in germination of seeds and growth of seedlings, the seedling vigour index in it was 395.14; whereas, use of GA3 (500 ppm) resulted in the highest SVI of 567.64.
Table 4.
Seedling growth characteristics in B. anthelmintica as influenced by seed treatments at 7 WAS (Weeks after sowing) under protected structure
| Treatment | Shoot length (cm) | Root length (cm) | No. of leaves/ seedling | Collar thickness (mm) | Seedling Vigour Index |
|---|---|---|---|---|---|
| Untreated | 8.12 ab | 4.11 a | 6.00 a | 0.86 abc | 395.14 c |
| Water soaking | 7.19 bc | 4.15 a | 5.53 a | 1.01 a | 291.44 d |
| KNO3 (0.1%) | 8.10 ab | 3.79 a | 6.00 a | 0.87 abc | 246.05 e |
| KNO3 (0.2%) | 7.95 ab | 3.16 a | 6.00 a | 0.71 c | 222.13 e |
| GA3 (100 ppm) | 8.03 ab | 3.56 a | 6.13 a | 0.97 a | 486.64 b |
| GA3 (250 ppm) | 8.64 a | 3.89 a | 6.07 a | 1.02 a | 379.56 c |
| GA3 (500 ppm) | 8.07 ab | 4.19 a | 5.60 a | 0.88 abc | 567.64 a |
| NAA (100 ppm) | 4.94 d | 3.89 a | 4.07 b | 0.93 ab | 85.68 g |
| IAA (100 ppm) | 6.47 c | 4.10 a | 4.67 b | 0.76 bc | 169.17 f |
Values followed by similar alphabet in a column do not differ significantly at 5% level of significance using least significant difference
GA3 is known to facilitate the embryonic growth activation and mobilization of seed reserves thereby promoting seed germination apart from improving seedling vigour [23]. This chemical has been reported to significantly improve seed germination and seedling characteristics in a number of Asteraceae members. In Helichrysum aureonitens Sch.Bip, pre-soaking of seeds in 500 mg/L GA3 substantially increased seed germination in the light and dark compared to other treatments [27]. Similarly, seeds of Parthenium argentatum A.Gray, a commercially important plant employed for high-quality, low-allergenic rubber production meaningfully responded to GA3 and exhibited 91% germination, totally replacing the need for light and/or overcoming the inhibitory effect of the seed coat [28]. In Musk Yarrow (Achillea erba-rotta subsp. moschata (Wulfen)), GA3 application resulted in an augmented seed germination of 90% [29]. Application of GA3 (200 ppm) has been reported to improve growth characters, including shoot length, root length, leaf length, leaf width, fresh and dry weight of seedlings of Callistephus chinensis L. Nees [30]. The current findings in bitter cumin are in line with the earlier observations of the positive effect of Gibberellic acid in supporting superior germination and seedling growth characteristics in Asteraceae members.
Experiment 3: effect of substrates under protected structure
In order to identify the most suitable substrate for germination of bitter cumin, four substrates, namely, soil, sand, vermicompost and coir pith compost, were evaluated (Fig. 3). Results suggested the significant influence of substrate on the germination process. After 15 days of sowing, the highest germination of 15.3% was noticed in sand, which is considered as a popular substrate for nursery purposes, while the substrates soil and coir pith compost did not support the germination at this stage. After 30 DAS, influence of vermicompost became prominent, though it remained on par with sand at 30 and 45 DAS. However, at the end of 55 days, vermicompost was significantly superior substrate with highest germination of 38.8%, whereas the increments noticed in other treatments including sand remained nominal. A dramatic increase in germination in the vermicompost substrate over time has been reported in tropical Haematocarpus validus (Miers) Bakh.f. ex Forman by earlier researchers [25].
Fig. 3.
Germination percentage over the period of time in B. anthelmintica as influenced by substrates under protected structure
Seedling growth characteristics of plants obtained from different substrates showed significant differences (Table 5). As the seeds sown in the substrates- sand and vermicompost germinated faster, the growth parameters such as shoot length (12.39 and 13.26 cm), root length (5.48 and 5.41 cm) and number of leaves per seedling (8.67 and 7.58) observed in these treatments were significantly superior over the other two substrates studied. Significantly superior collar thickness of 2.40 mm was noticed in vermicompost treatment. As the seedlings grew luxuriantly and higher germination was noticed in vermicompost substrate, SVI was the highest (724.0 ± 19.04) in this treatment. Vermicompost, being an important source of nutrients during nursery raising, facilitates better growth of regenerated seedlings, as also seen in seed- fertile wild banana species [26].
Table 5.
Seedling growth characteristics in B. anthelmintica as influenced by substrates under protected structure
| Substrate | Shoot length (cm) | Root length (cm) | Number of Leaves | Collar thickness (mm) | Seedling Vigour Index |
|---|---|---|---|---|---|
| Soil | 8.05 b | 3.28 b | 6.42 bc | 1.03 c | 202.4 ± 5.78 |
| Sand | 12.39 a | 5.48 a | 8.67 a | 1.76 b | 364.9 ± 36.48 |
| Vermicompost | 13.26 a | 5.41 a | 7.58 ab | 2.40 a | 724.0 ± 19.04 |
| Coir pith compost | 6.78 b | 3.38 b | 6.08 c | 0.78 c | 134.6 ± 10.40 |
Values followed by similar alphabet in a column do not differ significantly at 5% level of significance using least significant difference
In case of other members of Asteraceae such as marigold (Tagetes erecta L.), early initiation of seed germination and overall increase in growth parameters were observed with use of vermicompost [31]. The increase in plant vigour due to the application of vermicompost is primarily attributed to the continuous availability of macro and micronutrients and biological effects related to enzymatic activities and plant hormones [32]. Soaking of the seeds in vermicompost and joint application of vermicompost along with vermicompost leachates were reported to significantly improve the biochemical characteristics, including photosynthetic rate, total chlorophyll, and transpiration rate in another medicinal plant Withania somnifera (L.) Dunal [33]. The positive response of bitter cumin seeds to vermicompost on germination and SVI corroborates the earlier reports of increased germination and growth in other plants on application of vermicompost. The combined use of GA3 (500 ppm) seed priming and vermicompost as a nursery substrate provides a practical nursery protocol that can readily be adopted by growers to raise vigorous B. anthelmintica seedlings at scale under protected conditions. Given the current reliance on wild stands to meet commercial demand and the documented harvests of several hundred tonnes of Aranya Jeeraka seeds from central Indian states, the availability of robust, standardized nursery techniques is an essential first step towards organized cultivation and reduced pressure on natural populations. Integration of these protocols into on-farm nurseries and contract-farming schemes could support a more reliable supply of quality raw material for Ayurvedic and Siddha formulations and for industrial uses.
Conclusion
Soaking of seeds in GA3 (500 ppm) for 24 h could improve the seed germination and seedling growth under in vitro as well as ex vitro conditions. Use of vermicompost as substrate was found to be ideal for raising quality seedlings in this species. Standardized seed priming and nursery substrate protocols developed in this study provide a foundation for large-scale cultivation of Aranya Jeeraka to supplement/ replace wild collection in commercial supply chains.
Acknowledgements
Authors are thankful to the Director, ICAR-CIARI, Sri Vijaya Puram & Director General, CCRAS, New Delhi for providing necessary facilities for conducting the experiment. Authors are thankful to Dr. Pooja Bohra, Senior Scientist, ICAR-CIARI, Sri Vijaya Puram for her help throughout the study.
Authors’ contributions
**Ajit Arun Waman: ** Conceptualization, methodology in polyhouse trials **,** statistical analysis, writing of first draft. **Jishin Prakash T.S.:** Conceptualization, methodology and data collection in laboratory trials, revision of draft **. Riyah Raj: ** Methodology in polyhouse trials, data collection. **Ganesh N. Khadke: ** Maintenance of mother plants and provision of seed material for trials. **Saroj Kumar Debnath: ** Supervision and data collection in laboratory trial **. Akashlal M: ** Methodology and data collection in laboratory trial **. Shiddamallayya Nagayya: ** Fund acquisition and progress monitoring. **Nartunai Govindarajan: ** **Fund acquisition and progress monitoring.**.
Funding
Financial assistance from Central Council for Research in Ayurvedic Sciences (Office Order No. 422/2023-24 dated 01.08.2023), Ministry of Ayush, Government of India is gratefully acknowledged.
Data availability
All the data generated in the study has been presented in the manuscript.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Ajit Arun Waman and Jishin Prakash T.S. contributed equally to this work.
References
- 1.Aidarhan N, Yili A, Huojiaaihemaiti H, Chunfang LU, Aisa HA. Study on cultivation techniques for Vernonia anthelmintica Willd. Med Plant. 2020;11(4):64–5. [Google Scholar]
- 2.POWO. Baccharoides anthelmintica (L.) Moench. Plants of the World Online. Kew: Royal Botanic Gardens. 2024. Available from: https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:183904-1. Cited 13 Oct 2024.
- 3.Dogra NK, Kumar S, Kumar D. Vernonia anthelmintica (L.) Willd.: An ethnomedicinal, phytochemical, pharmacological and toxicological review. J Ethnopharmacol. 2020;256:112777. 10.1016/j.jep.2020.112777. [DOI] [PubMed] [Google Scholar]
- 4.Modak BK, Gorai P, Dhan R, Mukherjee A, Dey A. Tradition in treating taboo: Folkloric medicinal wisdom of the aboriginals of Purulia district, West Bengal, India against sexual, gynaecological and related disorders. J Ethnopharmacol. 2015;169:370–86. 10.1016/j.jep.2015.04.020. [DOI] [PubMed] [Google Scholar]
- 5.Saikia AP, Ryakala AK, Sharma P, Goswami P, Bora U. Ethnobotany of medicinal plants used by Assamese people for various skin ailments and cosmetics. J Ethnopharmacol. 2006;106:149–57. 10.1016/j.jep.2005.11.033. [DOI] [PubMed] [Google Scholar]
- 6.Harsha VH, Hebbar SS, Shripathi V, Hegde GR. Ethnomedicobotany of Uttara Kannada District in Karnataka, India- plants in treatment of skin diseases. J Ethnopharmacol. 2003;84:37–40. [DOI] [PubMed] [Google Scholar]
- 7.Surendran S, Prasannan P, Jeyaram Y, Palanivel V, Pandian A, Ramasubbu R. Knowledge on ethnogynaecology of Indian Tribes-a comprehensive review. J Ethnopharmacol. 2023;303:115880. 10.1016/j.jep.2022.115880. [DOI] [PubMed] [Google Scholar]
- 8.Bhatia D, Gupta MK, Gupta A, Singh M, Kaithwas G. Pharmacognostical studies on seeds Centratherum anthelminticum Kuntze. Nat Prod Rad. 2008;7(4):326–9. [Google Scholar]
- 9.Wang Q, Wang Y, Pang S, Zhou J, Cai J, Shang J. Alcohol extract from Vernonia anthelmintica Willd (L.) seed counteracts stress-induced murine hair follicle growth inhibition. BMC Complement Altern Med. 2019;19:37. 10.1186/s12906-019-2744-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Fatima SS, Rajasekhar MD, Kumar KV, Kumar MTS, Babu KR, Rao CA. Antidiabetic and antihyperlipidemic activity of ethyl acetate: Isopropanol (1:1) fraction of Vernonia anthelmintica seeds in Streptozotocin induced diabetic rats. Food Chem Toxicol. 2010;48:495–501. 10.1016/j.fct.2009.10.048. [DOI] [PubMed] [Google Scholar]
- 11.Mudassir HA, Arshad SMT, Naheed M, Anwer S. Effect of Centratherum anthelminticum seeds on glycated hemoglobin, cardiovascular disease risks and dyslipidemia in Type-2 diabetic patients in Karachi: A non-blind randomized controlled trial. Pak J Med Sci. 2025;41(1):257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Looi CY, Arya A, Cheah FK, Muharram B, Leong KH, Khalit M, et al. Induction of apoptosis in human breast cancer cells via caspase pathway by vernodalin isolated from Centratherum anthelminticum (L.) seeds. PLoS ONE. 2013;8(2):e56643. 10.1371/journal.pone.0056643. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Kumar S, Mishra A, Singh SP, Singh A. Anti-filarial efficacy of Centratherum anthelminticum: unravelling the underlying mechanisms through biochemical, HRAMS proteomics and MD simulation approaches. RSC Adv. 2024;14(35):25198–220. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Singh S, Mahajan S, Kaur H. Synthesis of tetrahydrofuran ring containing oligoethylene glycol ethers from the seed oil of Vernonia anthelmintica. J Am Oil Chem Soc. 1999;76:103–7. [Google Scholar]
- 15.Bhatnagar P, Shukla PK, Barman R. Value chain analysis of some important medicinal plants in Madhya Pradesh, India. J Non Timber Prod. 2021;28(3):115–20. 10.54207/bsmps2000-2022-G38264. [DOI] [Google Scholar]
- 16.NMPB. Medicinal plant list. New Delhi: National Medicinal Plants Board. 2024. Available from: https://nmpb.nic.in/medicinal_list. Cited 12 Oct 2024.
- 17.Anonymous. Chhattisgarh state minor forest produce market survey. Raipur: State MFP (Trading and development) Federation Ltd.; 2006. [Google Scholar]
- 18.Bhatnagar P, Barman R. Assessment of demand and supply of some important medicinal plants in Madhya Pradesh. J Non Timber Prod. 2020;27(2):113–6. 10.54207/bsmps2000-2020-4TEU2I. [DOI] [Google Scholar]
- 19.Abdul-Baki AA, Anderson JD. Vigor determination in soybean seed by multiple criteria. Crop Sci. 1973;13(6):630–3. [Google Scholar]
- 20.Wellburn AR. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol. 1994;144(3):307–13. [Google Scholar]
- 21.Tiwari RKS, Das K. Impact of differential storage conditions on seed germination and viability of some medicinal plants. Afr J Agric Res. 2014;9(20):1578–85. [Google Scholar]
- 22.Sher A, Sarwar T, Nawaz A, Ijaz M, Sattar A, Ahmad S. Methods of Seed Priming. In: Hasanuzzaman M, Fotopoulos V, editors. Priming and Pretreatment of Seeds and Seedlings. Singapore: Springer; 2019. pp. 1–10. 10.1007/978-981-13-8625-1_1. [DOI] [Google Scholar]
- 23.Ma HY, Zhao DD, Ning QR, Wei JP, Li Y, Wang MM, et al. A multi-year beneficial effect of seed priming with gibberellic acid-3 (GA3) on plant growth and production in a perennial grass, Leymus chinensis. Sci Rep. 2018;8(1):13214. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Satti JM, Kumar MK, Pola S, Sandeep BV. Effect of chemical treatments on germination Acacia nilotica L. of seeds. Int J Biotechnol. 2016;115:456–62. [Google Scholar]
- 25.Bohra P, Waman AA, Basantia D, Devi HL, Reang E. Domestication and conservation efforts in Haematocarpus validus (Miers.) Bakh. f. ex Forman (Menispermaceae): an underutilized fruit species and natural colourant. Curr Sci. 2018;115:1098–105. [Google Scholar]
- 26.Bohra P, Waman AA, Jerard BA. Seed germination and storage studies in seed-fertile Musa indandamanensis and its conservation. S Afr J Bot. 2020;128:161–6. 10.1016/j.sajb.2019.09.022. [DOI] [Google Scholar]
- 27.Afolayan AJ, Meyer JJM, Leeuwner DV. Germination in Helichrysum aureonitens (Asteraceae): effects of temperature, light, gibberellic acid, scarification and smoke extract. S Afr J Bot. 1997;63(1):22–4. [Google Scholar]
- 28.Dissanayake P, George DL, Gupta ML. Effect of light, gibberellic acid and abscisic acid on germination of guayule (Parthenium argentatum Gray) seed. Ind Crops Prod. 2010;32(2):111–7. [Google Scholar]
- 29.Aiello N, Lombardo G, Giannì S, Scartezzini F, Fusani P. The effect of cold stratification and of gibberellic acid on the seed germination of wild musk yarrow [Achillea erba-rotta subsp. moschata (Wulfen) I. Richardson] populations. J Appl Res Med Arom Plants. 2017;7:108–12. [Google Scholar]
- 30.Sidana G, Hembrom R, Sisodia A, Padhi M, Singh AK. Effect of seed priming on seedling growth in China aster cv Kamini. J Pharmacogn Phytochem. 2019;8(5):372–5. [Google Scholar]
- 31.Shafique I, Saiqa A, Malik SA, Farrukh N, Shaukat A, et al. Efficiency of cow dung based vermi-compost on seed germination and plant growth parameters of Tagetes erectus (Marigold). Heliyon. 2021;7(1):e05895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Subler S, Edwards C, Metzger J. Comparing vermicomposts and composts. Biocycle. 1998;39(7):63–6. [Google Scholar]
- 33.Kaur A, Pati PK, Ohri P, Kaur A. Effects of Vermicompost and vermicompost leachate on the biochemical and physiological response of Withania somnifera (L.) Dunal. J Soil Sci Plant Nutr. 2022;22(3):3228–42. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All the data generated in the study has been presented in the manuscript.