Callus production and Micropropagation and Phytochemical Investigation of Corallocarpus epigaeus (Arn.) Cl. – A Potential Antidiabetic Medicinal Plant

Callus production and Micropropagation and Phytochemical Investigation of Corallocarpus epigaeus (Arn.) 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Cl. – A Potential Antidiabetic Medicinal Plant Kaushik H Nakum, Vipul B Vaja, Kalpesh B Ishnava This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6739131/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present study deals with the in vitro callus induction, micropropagation and phytochemical analysis of Corallocarpus epigaeus (Arn.) Cl. The tuber, node and leaf were used as explants and cultured on MS medium with NAA, 2,4-D, BAP and KN alone and different combinations for callus induction and micropropagation. The best callus formation of tuber explants was obtained on BAP: NAA after 20 days of inoculation while the nodal explants show best callus formation on BA: NAA after 10 days. Leaf explants not show callus induction in any combination. The nodal explants were selected for shoot multiplication and inoculated on MS media supplemented with the combination (BAP and IBA) of PGR shows the positive results of shoot multiplication and maximum shoot obtained after 15 days of inoculation. NAA of PGR shows the positive results of root production and maximum root and length obtained after 25 days of inoculation. After 25-day callus of tuber explants was collected and subjected to phytochemical investigation which indicates the presence of alkaloids, flavonoids, Phenols, steroids and saponins. The yield of alkaloids, flavonoids and Phenols are 4.71%, 0.23% and 0.013% respectively. Further study required for the large-scale production of secondary metabolites through callus production and also large-scale production of propagules required the standardization. Biological sciences/Biotechnology Biological sciences/Plant sciences Corallocarpus epigaeus Antidiabetic MS medium Callus Micropropagation Phytochemical analysis Figures Figure 1 Figure 2 Introduction The proof about utilization of therapeutic plants for human wellbeing can be seen in numerous old records, for instance records in ayurveda reports have numerous treatment methodology for various sicknesses 1 , 2 , 3 . These days there are many notable plant-based prescriptions are accessible in the market which has capability of relieving savage infections are famous all around the globe. As per world wellbeing association (WHO), around 80% of total populace relies upon the restorative plants for essential social insurance 4 . The plant-based medicines are growing nowadays worldwide, 25% peoples of United Kingdom (UK) use the plant-based medicines for curing the diseases 4 . The substance amalgamation of some pharmaceutical mixes is beyond the realm of imagination or not financially practical in this way around 40% compound utilized in assembling of prescriptions are gotten from plants 5 , 6 , 7 . Plant tissue culture is viewed as the most proficient innovation and it has a few favorable circumstances over conventional strategy for spread like uniting, through seed and cutting. Micropropagation method for the most part utilized and through this prevalent nature of plant is produce which has better ailment opposition and stress resilience limits, micropropagation system is utilized for the protection and duplication of yield plant like organic products, vegetables, grains, restorative, fancy and woodland, has made new open doors for ranchers, nursery proprietors, in worldwide exchanging and furthermore supportive for rustic advancement 8 , 9 . Additionally the micropropagation techniques are employed for culturing the medicinal plants to produce important compounds for herbal and pharmaceutical industries. Micropropagation techniques are also used for conservation of threatened medicinal plants 10 . The Corallocarpus epigaeus (Arn.) Cl. is a rare important medicinal plant which belongs to the family Cucurbitaceae. It is commonly known as aakashguddah, jangalee suran, rakashguddah etc. It is a monoecious, deciduous and perennial tendril climber with a large turnip- shaped root and distributed in tropical Africa, Persian Gulf region and India. Monoecious, climber with tuberous root. Stem angular-sulcate, glabrous to finely pubescent. Leaves sinuately 3 lobed, hairy on both surfaces. Male peduncle 4–6 cm long, 5–15 flowered. Calyx lobes lanceolate. Corolla greenish yellow color and ovate. Female flowers often solitary; pedicel 1–5 mm long, thickened in fruit; calyx-tube campanulate, petals 1.5–2.5 mm long, 1-1.5 mm broad, reflexed. Fruit ovoid or ellipsoid, beaked, glabrous, smooth, red except for the greenish base and portion of beak which is 2.5-5 mm long. Seeds asymmetrically pyriform, smooth, yellowish, turgid. Flowers and fruiting in June to October 11 , 12 . Phytochemical screening of tubers reveals the presence of phytonutrients and medicinally valuable secondary metabolites including flavonoids, alkaloids, phenolics, tannins, saponins and steroids 13 , 14 . Phytochemicals perhaps impart potent pharmacological properties to the great rhizomes that used in the controlling the diabetic disease 15 , 16 , 17 . Because of exploitative collecting of tubers for exchange and denudation of backwoods, the normal populace of Corallocarpus epigaeus has declined to such a degree, that it is presently viewed as uncommon and undermined in its common natural surroundings. A few creators put it under imperiled class since normal recovery of this species is very poor by virtue of long seed lethargy, failed seeds, amazingly poor germination, and deficient accessibility of tuberous roots as propagules attributable to exploitative gathering. Consequently, they unequivocally anticipate misgiving of its annihilation soon if sufficient preservation measures are not embraced earnestly. Arrangement lies in mediations of biotechnological approaches for in vitro recovery which offers quick and boundless accessibility of planting materials consistently. Biotechnological mediations for recovery utilizing in vitro culture method have been effectively utilized for some yields and a few reports are likewise accessible for comparable wild Cucurbitaceae taxa. There has been no report accessible so far for in vitro callus induction and micropropagation and support of in vitro biodiversity for conservation of the germplasm for the specie Corallocarpus. Hence, into account the rising biological basic for restoration of species of Corallocarpus epigaeus for conservation for human benefit. Material and Methods 2.1 Collection of seeds and explants The seed, tuber and whole plant of Corallocarpus epigaeus were collected from surrounding area of Jalaram temple, Virpur, Gujarat during the month of June 2023 (Figure1). The plant specimen was identified by Dr. Kalpesh Ishnava (Plant Taxonomist) at Sardar Patel University, Vallabh Vidyanagar, Gujarat. The voucher specimen (SPU-2023-02) were deposited in the herbarium of P. G. Department of Biosciences, Sardar Patel University. 2.2 Seed germination study 2.2.1 In vivo seed germination (Effect of GA3 on seed germination) Petriplates was taken and the same size of filter papers was placed into it. Then 2 to 3ml of different stock solutions of GA 3 was added to each plate and 1 seed per each plate was inoculated. Control was also prepared by inoculating 1 seed in distilled water. All the petridishes provided the normal light (16hr) and proper nutrition for growth and development. Seed germination rate was observed after 5 days. 2.2.2 In vitro seed germination and effect of GA3 on seed germination The viability of the seed was determined by soaking the seeds in distilled water for 24 h. The non-viable seeds were observed at the surface of water and were discarded immediately. The surface sterilization of seeds was carried out in 3 different steps. Seeds were immersed in lab wash containing beaker under running tap water for about 30 minutes. Then immerse into surface sterilization 0.1% HgCl2 solution for approximately 3 min 18 . 2.2.3 Culture medium Seeds were then washed with sterile distilled water 3 times and inoculated on MS medium 19 with sucrose (3%) and pH 5.7-5.8 and also use of different concentration of hormones of GA3 (0.1ppm to 0.5ppm). After transfers the seed in semisolid medium inoculated and all tube transfers in the culture room in temperature 25 0 C. Before the inoculation the Laminar Air Flow was subjected for UV light transmission for about 30 – 45 minutes. The inoculation of seeds was carried out under aseptic conditions under Horizontal Laminar Air Flow. 2.2.4 Surface sterilization The surface sterilization of seed was carried out in different steps. The seeds were placed in different beaker and covered with net and washed for 30 minutes under running tap water to remove all the adhering dust particles and microbes from the surface, then surface sterilized by immersion in 75% alcohol for 30 seconds, followed by one rinse of 3 min in sterile distilled water, additionally immersed in 0.1% mercuric chloride (HgCl 2 ) solution for 4 min followed by five times rinses of 5 min each in distilled water. The surface sterilized different seeds were blotted dry on sterile filter paper. 2.2.5 Inoculation of seeds All the experimental manipulations were carried out under strictly aseptic conditions in laminar air flow bench. The chamber was then sterilized with U.V. rays continuously on for one hour. Hands and arms which were to be used inside the inoculation chamber were scrubbed with alcohol before inoculation. The rims of the test tubes and the sides of the plugs were flame sterilized. Instruments (like forceps, scalpels, spatula etc.) were all sterilized by dipping in the alcohol and flaming a number of times. Care was taken to cool the instruments before putting into operation. After sterilization of seeds, seed were inoculated in culture tubes aseptically. For inoculation explants were transferred to large sterile glass petriplate or glass plate with the help of sterile forceps under strict aseptic conditions. Some Seeds transferred to culture tubes containing MS medium and some in to MS media with GA3 (0.1 % to 0.5%). After inoculating the seed in culture tube, the mouth of tube is quick flamed and tubes are tightly closed by cotton plug. After proper labeling clearly mentioning media, date of inoculation etc. the tubes were transferred to growth room. 2.2.6 Culture Condition The tubes were shifted to culture room with controlled facility of diffused light (2000 lux) for 8 hrs. and rest of the period dark condition daily at 28 ± 2°C temperatures and 50 to 60% relative humidity. 2.3 Callus induction study 2.3.1 Preparation of culture medium MS medium was used for callus of Corallocarpus epigaeus . Murashige and Skoog (MS) medium supplemented with different concentrations of PGRs such as α-naphthalene acetic acid (NAA), 2,4-Dichlorophenoxy acetic acid (2,4-D), benzyl amino purine (BAP) alone and BAP in combinations with NAA, Benzyl adenine (BA) in combinations with NAA, 2,4-D in combinations with Kinetin (KN) for callus induction. The medium was supplemented with 3% sucrose, gelled with 0.8% agar and the pH was adjusted to 5.7-5.8. Around 20ml of medium was used for each culture tubes. Then media was autoclaved at 121°C and 15 psi pressure for 15 minutes. 2.3.2 Callus induction All the experimental manipulations were carried out under strictly aseptic conditions in laminar air flow bench. After sterilization of explants (Nodal, tuber and leaf), explants were inoculated in culture tubes aseptically. For inoculation explants as per standard protocol. The culture tubes were transferred to growth room 20 . 2.3.3 Culture Condition The tubes and bottles were shifted to culture room with controlled facility of diffused light (3000 lux) for 10 hrs. and rest of time dark condition daily at 28 ± 2°C temperatures and 50 to 60% relative humidity. 2.3.4 Collection of calluses The calluses were collected after 25 days. Calluses were washed with distilled water to remove all adhering particles. After that calluses were allowed to dry at room temperature. This callus was use for different analysis for study. Good quality callus was sub cultured and again collected after more 30 days and subjected to further analysis. 2.4 Micropropagation 2.4.1 Preparation of culture medium MS medium was used for micropropagation of Corallocarpus epigaeus . Murashige and Skoog (MS) medium supplemented with different concentrations of PGRs such as α-naphthalene acetic acid (NAA), 2,4-Dichlorophenoxy acetic acid (2,4-D), benzyl amino purine (BAP) alone and 2,4-D in combinations with Kinetin (KN), IBA in combination with BAP for shooting And IBA in combination with BAP for rooting. The medium was supplemented with 3% sucrose, gelled with 0.8% agar and the pH was adjusted to 5.7-5.8. Around 40ml of medium was used for each culture bottles. Then media was autoclaved at 121°C and 15 Psi pressure for 15 minutes. 2.4.2 Explants sterilization The surface sterilization of nodal explants was carried out in different steps. First off, all cut the leaves of isolated nodal segment. Explants were further soaked in fungicide (1gm/lit Bavistin) for 5 min and then antibiotic Streptomycin (50mg/lit) for 3 min then surface sterilized by immersion in 75% alcohol for 30 seconds, followed by one rinse of 3 min in sterile distilled water, additionally immersed in 0.1% mercuric chloride (HgCl 2 ) solution for 3 min followed by five rinses of 5 min each in distilled water. The surface sterilized nodal explants were blotted dry on sterile filter paper. 2.4.3 Inoculation of explants All the experimental manipulations were carried out under strictly aseptic conditions in laminar air flow bench. After sterilization of explants, explants were inoculated in culture tubes aseptically. For inoculation explants were transferred to large sterile glass petriplate or glass plate with the help of sterile forceps under strict aseptic conditions. Here the explants were further trimmed and extra outer leaves were removed to make them in suitable sizes. Trimming was removed with sterile scalpel blade. After cutting explants into suitable size (for Node 1-2 cm), explants are transferred to culture tubes and bottles containing MS medium with different hormone concentration. After vertically inoculating the explants in culture tube the mouth of tubes is quick flamed and tubes are tightly capped with cotton plug. After proper labeling clearly mentioning media, date of inoculation etc. the bottles were transferred to growth room. 2.4.4 Multiplications of shoot stage After 15 days of inoculation culture showing spouting were transferred to full length MS media supplemented with different combination of growth hormones concentration for multiplication of shoot regeneration. 2.4.5 Rooting stage Newly formed shoots measuring 2.0-3.0 cm in length were excised individually from the parents and explants and transferred to new MS medium with different concentration of IAA and NAA hormone. 2.4.6 Culture Condition The tubes and bottles were shifted to culture room with controlled facility of diffused light (3000 lux) for 10 hrs. and rest of time dark condition daily at 28 ± 2°C temperatures and 50 to 60% relative humidity. 2.4.7 Hardening In vitro rooted plantlets with 2 to 3 nodes and having at least 2 to 4 roots of 2 to 4 cm length were washed carefully with water to remove traces of agar and then transferred to the pots containing different sterile soil mixtures viz. Cocopeat + Sand + Soil (1:1:1). The pots were covered with tight plastic covers (With holes) to prevent desiccation and to avoid rapid changes in environment and acclimatized in the mist house at 25-30 °C temperature and 14 hrs. illumination. During the hardening procedure, plastic covers were gradually perforated after one week and after second week they were removed and the plants were maintained in the mist house conditions for 15 more days. 2.5 Phytochemical analysis of callus 2.5.1 Extraction of callus Dried sample of callus from in vitro grown plant were grinded with mortar and pestle. The dry sample was extracted using 20 ml of methanol in Erlenmeyer flask placed on shaker at 100 rpm for overnight at room temperature. The crude extract filtered with filter paper (Whatman No. 1). The filtrate was collected and allowed to solvent evaporation. After evaporation the remaining material was collected and different stocks were prepared by dissolving in methanol. 2.5.2 Phytochemical screening Qualitative assay for the presence of plant phytoconstituents such as Alkaloids, Glycosides, Flavonoids, Phenol, Tannins and Saponins and quantitative assay were carried out on alkaloids, total phenol and total flavonoidsfollowing standard procedure 20, 21 . Results and Discussion Due to exploitative harvesting of tubers for trade and denudation of forests, the natural population of Corallocarpus epigaeus has declined to such an extent that it is now considered rare and threatened in its natural habitats. Hence, they explicitly predict apprehension of its extinction in the near future if adequate conservation measures are not adopted urgently. So here in the present study we have initiated and developed the callus and micropropagation of the leaf, node and tuber of Corallocarpus epigaeus and then evaluated the phytochemical analysis of methanolic extract of tuber callus. 3.1 Seed germination study ( In vivo and in vitro ) In vivo seeds germination of Corallocarpus epigaeus was done usingGA 3 at different concentration (1ppm to 5ppm). There is no germination was observed in any concentration of GA 3 after 25 days. The seed germination of this plant is very poor because of seeds having long dormant period. Seed coat of this plant is very hard and also the embryo abortion observed during seed germination. In vitro seeds germination attempt of Corallocarpus epigaeus was made byusing MS media supplemented with different concentrations of GA 3 (1ppm to 5ppm). No seed germination was observed in MS medium ( in vitro ). 3.2 Callus Production Callus induction from leaf explants of Corallocarpus epigaeus using MS basal medium supplemented with different plant growth regulators such as NAA, 2,4-D and BAP alone and KN in combination with 2,4-D is mentioned in Table 1. The NAA, 2,4-D and BAP alone do not show any response in the leaf explants. The leaf explants inoculated in MS media supplemented with KN (0.3mg/lit) + 2,4-D (3mg/lit) and KN (0.5mg/lit) + 2,4-D (5mg/lit) shows the callus induction after 6 days of inoculation. The callus produced is green and yellow respectively. Callus induction from nodal explants of Corallocarpus epigaeus using MS basal medium supplemented with different concentrations of BAP and NAA is mentioned in Table 1 and Figure 2A. Brownish callus was produced after 6 days of incubation on MS media supplemented with BAP (4ppm). Brownish, White and Green callus was produced in the nodal explants inoculated in MS media supplemented with NAA (1ppm to 5pmm) after 6-7 days of inoculation (Figure 2B). Callus induction from nodal explants of Corallocarpus epigaeus using MS basal medium supplemented with different concentrations of 2,4-D is mentioned in Table 1. Brownish callus was produced in the nodal explants inoculated in MS media supplemented with 2,4-D (0.2 mg/lit.), 2,4-D (1.5 mg/lit.) and 2,4-D (5 mg/lit.) after 15 and 6 days of inoculation respectively. Callus induction in the nodal explants was observed in the different combinations of NAA and BAP (Table 1). Brownish callus was produced in the nodal explants inoculated in MS media supplemented with different combinations of hormones BAP (0.5mg/lit) + NAA (5mg/lit), BAP (1mg/lit) + NAA (4.5mg/lit) and BAP (3.5mg/lit) + NAA (2mg/lit) after 6-8 days of inoculation (Figure 2B). Mehul and Kalpesh 2015 reported that a combination of BAP and NAA was more suitable combination for callus induction of M. dioica 22 . Hoque et al., (1995) found that a combination of 1.5mg/l BAP and 0.1 mg/l NAA was more suitable combination for adventitious multiple shoots formation of M. dioica 23 . Where present investigated BAP + NAA combination is most suitable for callus induction. Devendra et al. (2012) reported that the callus initiation started from nodal base on 10th day 2.0 mg/l NAA, 2.0 mg/l BAP auxin or cytokinin either alone or in combinations were efficient for the induction of callus depending on the varied concentrations of the growth regulators 24 . The different combinations of KN and 2,4-D shows callus induction in the nodal explants (Table 1). Off white and brownish callus was produced in the nodal explants inoculated in MS media supplemented with different combinations of hormones KN(1mg/lit) + 2,4-D(1mg/lit), KN(0.1mg/lit) + 2,4-D(1mg/lit), KN(2mg/lit) + 2,4-D(0.2mg/lit) and KN(0.2mg/lit) + 2,4-D(2mg/lit) after 4-5 days of inoculation (Figure 2B). Salvador et al., (2009) reported the Alternanthera tenella , an amaranthaceae family, MS basal medium was supplemented with 1.0 mg/lit 2, 4-D and 1.0 mg/lit KN and 2.5 mg/lit NAA and 1.0 mg/lit BA for callus induction 25 . The different combinations of NAA and BA show callus induction in the nodal explants (Table 1). Green callus was produced in the nodal explants inoculated in MS media supplemented with different combinations of hormones BA (2mg/lit) + NAA (1mg/lit), BA (1mg/lit) + NAA (1mg/lit), BA (0.5mg/lit) + NAA (1mg/lit) after 4-5 days of inoculation. Callus induction from tuber explants of Corallocarpus epigaeus using MS basal medium supplemented with different concentrations of BAP in combination with NAA is mentioned in Table 1 and Figure 2F. The different combinations of NAA and BAP show callus induction in the tuber explants. Brownish callus was produced in the tuber explants inoculated in MS media supplemented with different combinations of hormones BAP (0.5mg/lit) + NAA (5mg/lit), BAP (1mg/lit) + NAA (4.5mg/lit) and BAP (1.5mg/lit) + NAA (4.0mg/lit) after 7 days of inoculation (Figure 2I, 2J, 2K, 2L). Castillo et al ., (1998) reported that auxin 2,4-D by itself or in combination with cytokinin has been widely used to enhance callus induction and maintenance 26 . Moreover, many researchers observed 2,4-D as the best auxin for callus induction as common as in monocot and even in dicot 27, 28, 29, 30, 31 . This combination is more suitable for more production of the biomass for secondary metabolites. Callus induction from tuber explants of C. epigaeus using MS basal medium supplemented with different concentrations of BAP is mentioned in Table 1. Brownish, green and yellow callus was produced after 6 days of incubation on MS media supplemented with BAP (1 to 5 mg/lit). Callus induction from tuber explants of C. epigaeus using MS basal medium supplemented with different concentrations of NAA is mentioned in Table 1. Brownish, yellow and green callus was produced in the tuber explants inoculated in MS media supplemented with NAA (3 and 5 mg/lit) after 9 days of inoculation (Figure 2H). Callus induction from tuber explants of C. epigaeus using MS basal medium supplemented with different concentrations of 2,4-D is mentioned in Table 1. Brownish callus was produced in the tuber explants inoculated in MS media supplemented with 2,4-D (0.1mg/lit to 5mg/lit) after 9 days of inoculation (Figure 2H). Callus induction from tuber explants of C. epigaeus using MS basal medium supplemented with different concentrations of BAP in combination of NAA is mentioned in Table 1. The different combinations of NAA and BAP show callus induction in the tuber explants. Cream white callus was produced in the nodal explants inoculated in MS media supplemented with different combinations of hormone KN (2mg/lit) + 2,4-D (0.2mg/lit) after 4-5 days of inoculation (Figure 2G). No callus was produced in the tuber explants inoculated in MS media supplemented with different combinations of hormones BA in combination with NAA after 40 days of inoculation. 3.3 Micropropagation MS media supplemented with higher concentration of cytokinin along with lower concentration of auxins were used for the shoot multiplication. The nodal explants were selected for shoot multiplication and inoculated on MS media supplemented with higher concentration of BAP and lower concentration of IBA. The combination (BAP 1ppm : IBA 0.01ppm) of PGR shows the positive results of shoot multiplication and maximum shoot (3 shoot) obtained after 15 days of inoculation (Table 2 and Figure 2C). The in vitro raised shoots were inoculated on to MS supplemented with various combinations of auxin and cytokinin for root formation. Root formed in all combination and concentration of hormone but highest frequencies of rooting were observed on MS fortified with 1.0 mg/lit of IAA and 0.5 mg/lit NAA after 15 days of inoculation (Figure 2D). But prior to the root initiation callus was taking place (Figure 2B). The reduced rooting may be due to the imbalance between the endogenous auxin and exogenous auxin, IAA. In the present study, initially the rooting percentage was very low, these results are in accordance with those on Withania somnifera 32 , when IAA was tested for rooting, there was not only decrease in the rooting response but also enhanced basal callusing from the in vitro raised shoots were observed. These results conform to those of Anand et al., 1997 in Kaempfera rotunda 33 and Ankita and Kalpesh, (2015) in Coccinia grandis (L.) Voigt. 34 . After shoot and root production transfer to primary and secondary hardening of the plants. After 3 week plants transfer to natural condition (Figure 2E). 3.4 Phytochemical analysis of callus 3.4.1 Qualitative analysis of callus from tuber The callus obtained in different combination of growth hormones MS + BAP (0.5mg/lit) + NAA (5mg/lit), MS + BAP (0.5mg/lit) + NAA (5mg/lit) and MS + BAP (0.5mg/lit) + NAA (5mg/lit) was extracted by methanol. Methanolic extract of various calluses was subjected to qualitative analysis. The analysis shows the presence of alkaloids, flavonoids, saponins, phenols and steroids (Table 3). Kattamanchi et al., (2013) reported the ethanolic extract of Corallocarpus epigaeus rhizomes was found the presence of phenolic compounds, Flavonoids, terpenoids and phytosterols 15 . 3.4.2 Quantitative analysis of callus Estimation of alkaloids in vitro callus obtained in the medium supplemented different combinations of PGRs (BAP-0.5mg/lit + NAA-5mg/lit; BAP-1mg/lit + NAA-4.5mg/lit; BAP-1.5mg/lit + NAA-4mg/lit) was done by Harborne method. The evaluation alkaloids of callus were done after 25 days of inoculation. Then remaining callus was subjected to sub culture on same medium and again analysis of alkaloids was done after sub culture. The percent yield of alkaloids in callus was calculated by back calculation and mentioned in Table 4. After 25 days maximum percentage yield present in BAP-1mg/lit + NAA-4.5mg/lit and after sub culture percentage yield of alkaloids was obtained in same concentration. Estimation of phenols in vitro callus obtained in the medium supplemented different combination of PGRs (BAP-0.5mg/lit + NAA-5mg/lit; BAP-1mg/lit + NAA-4.5mg/lit; BAP-1.5mg/lit + NAA-4mg/lit) was done by Folin’s method. The evaluation alkaloids of callus were done after 25 days of inoculation. Then remaining callus was subjected to sub culture on same medium and again analysis of alkaloids was done after sub culture. The percent yield of phenols in callus was calculated by back calculation and mentioned in Table 4. Estimation of flavonoids in vitro callus obtained in the medium supplemented different combination of PGRs (BAP-0.5mg/lit + NAA-5mg/lit; BAP-1mg/lit + NAA-4.5mg/lit; BAP-1.5mg/lit + NAA-4mg/lit) was done by aluminum chloride method. The evaluation flavonoids of callus were done after 25 days of inoculation. Then remaining callus was subjected to sub culture on same medium and again analysis of flavonoids was done after sub culture. The percent yield of flavonoids in callus was calculated by back calculation and mentioned in Table 4. Kattamanchi et al., (2013) reported the ethanolic extract of Corallocarpus epigaeus rhizomes possesses the antidiabetic action which is comparable with that of the standard Glibenclamide drug employed 15 . This work supports the traditional claim of the rhizomes for their use in diabetes. This ethanolic extract of Corallocarpus epigaeus presents the flavonoids. In our study show the in vitro callus induction more production of theflavonoids compares to the normal is less produce. This work supports the traditional claim of the rhizomes for secondary metabolites production through callus induction increases the biomass. Conclusion Callus initiation of nodal explants took place after 4-6 days of inoculation while the tuber explants shows callus initiation after 7-9 days of inoculation. The best degree for callus formation of tuber explants was obtained on MS medium supplemented with BAP:NAA (0.5mg/lit:5.0mg/lit, 1.0mg/lit:4.5mg/lit and 1.5mg/lit:4mg/lit) while the nodal explants shows highest degree for callus formation on MS medium supplemented with BA (2mg/lit) in combination with NAA (1mg/lit). The nodal explants were selected for shoot multiplication and inoculated on MS media supplemented with higher concentration of BAP and lower concentration of IBA. The combination (BAP 1ppm: IBA 0.01ppm) of PGR shows the positive results of shoot multiplication and maximum shoot (3 shoot) obtained after 15 days of inoculation. NAA (3ppm) of PGR shows the positive results of root production and maximum root (1 root) and length (4cm) obtained after 25 days of inoculation. The present study showed successfully micropropagation of Corallocarpus epigaeus. Phytochemical investigation the tuber callus indicated the presence of alkaloids, flavonoids, phenols, steroids and saponins. The yield of alkaloids, flavonoids and phenols are 4.71%, 0.23% and 0.013% respectively in 25-day old callus of tuber explants. Further study required for the large-scale production of secondary metabolites through callus production and also large-scale production of propagules required the standardization. Declarations Funding The author(s) received the financial support from its institutional research grant of P. G. Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India for the research work. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Data availability All data include in the manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest about this manuscript and research. Acknowledgments Authors are thankful to P. G. Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India for providing necessary support for research and laboratory facility. Author Contribution Kaushik Nakum and Vipul Vaja design the methodology and perform the experiments. Dr. Kalpesh Ishnava interpretation of experiment results and preparing the manuscript. References Stafford, A., Morris, P. & Fowler, M. W. Fowler plant cell biotechnology: A perspective. Enzym. Microb. Technol. 8 , 578–587 (1986). Soumya, P., Rout, K. A., Choudary, D. M., Kar, L., Das, A. & Jain Plants in traditional medicinal system - Future source of new drugs. Int. J. Pharm. Pharm. Sci. 1 (1), 1–23 (2009). Farnsworth, N. R. Ethnopharmacology and drug development. Ciba Foundation Symposium 185:42–59. (1994). Cowan, M. M. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12 (4), 564–582 (1999). Singh, P. et al. 2009 Somatic embryogenesis and in vitro regeneration of an endangered medicinal plant sarpgandha ( Rauvolfia serpentina L). Life Sci. J. 6 (3):57–62 . Ghani, A. Medicinal plants of Bangladesh - Chemical constituents and uses 2nd edn pp 337–338 (Asiatic Society of Bangladesh, 1998). Krishnan, P. N. & Seeni, S. Rapid micropropagation of Woodfordia fruticosa (L.) Kurz (Lythraceae), A rare medicinal plant. Plant Cell Rep. 14 , 55–58 (1994). Brown, D. C. W. & Thorpe, T. A. Crop improvement through tissue culture. World J. Microbiol. Biotechnol. 11 , 409–415 (1995). Akin-Idowu, P. E., Ibitoye, D. O. & Ademoyegun, O. T. Tissue culture as a plant production technique for horticultural crops. Afr. J. Biotechnol. 8 (16), 3782–3788 (2009). Yaadwinder & Sidhu In vitro micropropagation of medicinal plants by tissue culture. Plymouth Student Sci. 4 (1), 432–449 (2010). Kumar, V., Cotran, R. S., Robbins, S. L. & Saunders, W. B. Basic pathology 5th edn pp 25–40 (Harcourt, Brace & Company, 1992). Shah, G. L. Flora of Gujarat State (University press, Sardar Patel University, Vallabh Vidyanagar, 1978). Kirtikar, K. R. & Basu, B. D. Indian Medicinal Plants Vol. IIpp 1166–1167 (International Book Distributors, 1918). Pandey, C. N., Raval, B. R., Mali, S. & Salvi, H. Medicinal plants of Gujarat pp 1–5 (Gujarat Ecological Education and Research (GEER) Foundation, 2005). Kattamanchi Gnananath, K. R., Reddy, G. P., Kumar, B. & Krishna Karka Srinivas Reddy, Avvari Sanjeeva Kumar, 2013 Evaluation of antidiabetic activity in Corallocarpus epigaeus Rhizomes. Int. Curr. Pharm. J. 2 (3):53–56 . Kapesh Ishnava, R., Kotadia, S. & Patel Nutritional properties and chemical composition of Corallocarpus epigaeus (Arn.) Cl: As remedy to control diabetes mellitus. Chiang Mai J. Sci. 42 (4), 806–815 (2015). Tejas, P. Genetic Transformation of Medicinal Plant, Withania somnifera (L.) Dunnal by Agrobecterium tumefaciens (MTCC-431). Dissertation, Sardar Patel University. (2010). Murashige, T. & Skoog, F. Revised medium for rapid growth and bioassays with Tobacco tissue cultures. Physiol. Plant. 15 , 473–499 (1962). Meghana In vitro study of Tinospora cordifolia (Wild.) Miers (Menispermaceae) - ‘A multipurpose plant’ by using different plant bark extracts for secondary metabolite production. Dissertation, Sardar Patel University. (2013). Harborne, J. B. Phytochemical methods - A guide to modern techniques of plant analysis (Chapman and Hall, 1973). Hossain, M. A., AL-Raqmi, K. A., AL-Mijizy, Z. H., Weli, A. M. & Al-Riyami, Q. Study of total phenol, flavonoids contents and phytochemical screening of various leaves crude extracts of locally grown Thymus vulgaris. Asian Pacific. J. Trop. Biomed. 3 (9), 705–710 (2013). Patel, M. G. & Ishnava, K. B. Momordica dioica Roxb. (Spine Gourd): Multiple shoot induction from nodal cultures and its antidiabetic activity. Journal of Medicinal Plants Studies 3(6):82–88. (2015). Hoque, A., Islam, R. & Joarder, O. I. vitro plantlets differentiation in kakrol ( Momordica dioica Roxb). Plant. Tissue Cult. 2 , 119–124 (1995 In). Devendra, N. K., Everaldo, G. A., Raghunandan, D. & Seetharam, Y. N. vitro production of Cucurbitacins from Trichosanthes cucumerina L. var. cucumerina Adv. Life Sci. 2 (4), 108–111 (2012 In). Salvador, M. J. et al. Bioactive chemical constituents and comparative antimicrobial activity of callus culture and adult plant extracts from Alternanthera tenella . Z. für Naturforschung C . 64 (5–6), 373–381 (2009). Castillo, A. M., Egana, B., Sanz, J. M. & Cistue, L. Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain. Plant. Cell. Rep. 17 , 902–906 (1998). Evans, D. A., Sharp, W. R. & Bravo, J. E. Cell culture methods for crop improvement. Hand Book of Plant Cell Culture. Vol.2. Macmillan Publishing Company, New York. (1984). Ho, W. J. & Vasil, I. K. Somatic embryogenesis in sugarcane ( Saccharum officinarum L.) I. the morphology and physiology of callus formation and the ontogeny of somatic embryos. Protoplasma 118 , 169–180 (1983). Jaiswal, V. S. & Naryan, P. Regeneration of plantlets from the callus of stem segment of adult plants of Fucus religiosa L. Plant Cell Rep. 4 , 256–258 (1985). Chee, P. P. High frequency of somatic embryogenesis and recover of fertile cucumber plants. HortScience 25 (7), 792–793 (1990). Mamun, A. N., Islam, R., Reza, M. A. & Joadar, O. I. vitro differentiation of plantlet of tissue culture of Samonea saman . Plant. Tissue Cult. 6 , 1–5 (1996 In). Manickam, V. S., Mathavan, R. E. & Antonisamy, R. Regeneration of Indian ginseng plantlets from stem callus. Plant. Cell. Tissue Organ. Cult. 62 , 181–185 (2000). Anand, P. H. M., Harikrishnan, K. N., Martin, K. P. & Hariharan, M. vitro propagation of Kaempferia rotunda Linn. Indian crocus - a medicinal plant. Phytomorphology 47 (3), 281–286 (1997 In). Patel, A. & Ishnava, K. vitro shoot multiplication from nodal explants of Coccinia grandis (L.) Voigt. and it’s antidiabetic and antioxidant activity. Asian J. Biol. Sci. 8 (2), 57–71 (2015 In). Tables Table 1. Callus induction from tuber and nodal explants of Corallocarpus epigaeus Nutrient media Tuber Explants Nodal explants MS- media + Growth hormones (mg/L) % of explants showing callus Duration of callus production Responses % of explants showing callus Duration of callus production Responses BAP (1) 50 9 days Light yellow - - - BAP (2) 100 11 days Brownish - - - BAP (3) 50 6-7 days Green - - - BAP (4) - - No change 100 6 days Brownish BAP (5) 100 15 days Brown green - - - NAA (1) - - - 100 6-7 days Brownish NAA (2) - - - 100 6-7 days Brownish NAA (3) 50 9 days Light yellow 100 6-7 days Brownish NAA (4) - - No change 100 5-6 days White NAA (5) 100 9 days Brown green 100 6-7 days Green 2,4-D (0.1) 50 9 days Off white - - - 2,4-D (0.2) - - No change 100 15 days Brownish 2,4-D (0.5) 50 9 days Off white - - No change 2,4-D (0.7) 50 22 days Off white - - No change 2,4-D (1) 100 15 days Brownish - - - 2,4-D (1.5) 100 22 days Off white 100 6 days Brownish 2,4-D (2) 50 22 days Off white - - - 2,4-D (3) 50 15 days Off white - - - 2,4-D (4) 50 9 days Off white - - No change 2,4-D (5) 50 15 days Off white 100 6 days Brownish BAP (0.5) + NAA (5) 100 7 days Light brown 100 6-7 days Cream brown BAP (1) + NAA (4.5) 100 7 days Light brown 100 6-7 days brown BAP (1.5) + NAA (4) 100 7days Light brown - - No change BAP (2) + NAA (3.5) 50 15 days Light brown - - - BAP (2.5) + NAA (3) 50 15 days Light brown - - No change BAP (3) + NAA (2.5) 50 11 days Light brown - - No change BAP (3.5) + NAA (2) 50 9 days Light green 100 8 days Brownish BAP (4) + NAA (1.5) - - No change - - No change BAP (4.5) + NAA (1) - - No change - - - BAP (5) + NAA (0.5) - - - - - - BAP (3) + NAA (3) 100 9 days Light green - - - KN (1) + 2,4-D (1) - - No change 100 4-5 days Off white KN (0.1) + 2,4-D (1) - - No change 100 4-5 days Off white KN (2) + 2,4-D (0.2) 50 15 days Cream white 100 4-5 days Off white KN (2) + 2,4-D (2) - - No change - - - KN (0.2) + 2,4-D (2) - - No change 100 4-5 days Brown BA (1) + NAA (0.5) - - No change 100 4-5 days Green BA (1) + NAA (1) - - No change - - - BA (0.5) + NAA (1) - - No change 100 4-5 days Brown green BA (2) + NAA (1) - - No change 100 4-5 days Light Green BA (1) + NAA (0.5) - - No change 100 4-5 days Green Table 2. Effect of hormones on shoot multiplication and root production Sr. No. MS medium + BAP : IBA No. of shoots (in Days) Length of shoot (cm) No. of Roots (in Days) Length of Root (cm) 5 th 10 th 15 th 5 th 10 th 15 th 5 th 10 th 15 th 5 th 10 th 15 th 1 1 ppm : 0.1 ppm - - - -- - - 2 1 ppm : 0.01 ppm 1 3 3 1±0.2 4±0.5 6±0.4 - - - -- - - 3 2 ppm : 0.2 ppm - - - -- - - - - - -- - - 4 2 ppm : 0.02 ppm - - - -- - - - - - -- - - 5 3 ppm NAA - - - -- - - 1 3 3 1±0.5 3±0.4 5±1 Table 3: Qualitative analysis of Corallocarpus epigaeus callus from tuber Secondary Metabolite MS + BAP (0.5mg/l) + NAA (5mg/l) MS + BAP (1mg/l) + NAA (4.5mg/l) MS + BAP (1.5mg/l) + NAA (4mg/l) Alkaloids + + + Flavonoids + + + Saponins + + + Terpenoids - - - Phenols + + + Tannins - - - Glycosides - - - Steroids + + + Table 4: Quantity analysis of Corallocarpus epigaeus callus from tuber Callus inoculated on MS-media + PGRs (mg/L) Alkaloids Phenols Flavonoids Wt. of callus (gm) Wt. of alkaloids (mg) % yield Wt. of callus (gm) Wt. of Phenols (mg) % yield Wt. of callus (gm) Wt. of flavonoids (mg) % yield BAP (0.5) + NAA (5) 1.511 68 4.50 1.511 199.8 0.013 1.511 1.34 0.088 BAP (1) + NAA (4.5) 1.528 72 4.71 1.528 201.6 0.013 1.528 3.45 0.225 BAP (1.5) + NAA (4) 1.541 57 3.69 1.541 219.6 0.014 1.541 1.50 0.097 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6739131","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":496024365,"identity":"b497813c-2d9a-457e-8ce1-5f08def21d40","order_by":0,"name":"Kaushik H Nakum","email":"","orcid":"","institution":"Sardar Patel University","correspondingAuthor":false,"prefix":"","firstName":"Kaushik","middleName":"H","lastName":"Nakum","suffix":""},{"id":496024368,"identity":"4c8435a0-f9a2-4401-988d-b6009ee44593","order_by":1,"name":"Vipul B Vaja","email":"","orcid":"","institution":"Sardar Patel University","correspondingAuthor":false,"prefix":"","firstName":"Vipul","middleName":"B","lastName":"Vaja","suffix":""},{"id":496024369,"identity":"053839d9-e65e-499f-b228-937b8561c834","order_by":2,"name":"Kalpesh B Ishnava","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIie3PPWsCMRjA8econMtTuuZIrV8hcqAO4n0VQ+CmGzp26HBTbqnOkX4JV7c7Ao66HuigCJ0rhdLCYRtB2goN1a1D/kNeCD+SALhc/zACF6mZcjSrfd2vEy+1Eu+IxKcTOBD9NwmyQq6eq+V143FQvOD9PGqTpEVvoVsf578TijxrjuQTsuVMUJwu+EQZoiAObeQGuKSXqUZGEhYof9FnZSxCBM2t5GotaVVpbKgkfFe7WXQgH1ZCibkFfI1QJi2ylbk3LkWxQcitJFDrrDmQ5mGGdLZDwScPG0OYCEcWQuZiunqrdLR/WNl/7UXtGjdfu+vVhxZi8o+37Md4FnG5XC7Xd5+BD2JVbE8iUwAAAABJRU5ErkJggg==","orcid":"","institution":"Sardar Patel University","correspondingAuthor":true,"prefix":"","firstName":"Kalpesh","middleName":"B","lastName":"Ishnava","suffix":""}],"badges":[],"createdAt":"2025-05-24 12:43:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6739131/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6739131/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88470243,"identity":"7fee9661-aa11-4100-a479-a2a96b56f78a","added_by":"auto","created_at":"2025-08-06 18:51:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExplants (Leaf, Nodal and Tuber) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCorallocarpus epigaeus\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6739131/v1/c6717799d3143a992e25393a.jpg"},{"id":88470705,"identity":"2878a00a-f699-46b6-8f42-72f273852d1f","added_by":"auto","created_at":"2025-08-06 18:59:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":107314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCallus induction and micropropagation of Corallocarpus\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e epigaeus\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA: Inoculation of nodal explants; B: Nodal explants produce callus; C: Shoot production; D: Shoot and root production; E: Harding the plants; F: Inoculation of tuber; G: Tuber explants produce white callus; H: Tuber explants produce brown callus; I: Tuber explants produce brownish green callus; J: Tuber explants produce green callus; K: Callus production after 25 days; L: Callus production after 35 days\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6739131/v1/0999c0e7d73793ca67dd60a9.jpg"},{"id":106678490,"identity":"6e4c4481-ebbc-4594-8b7f-7d5283318a8c","added_by":"auto","created_at":"2026-04-11 13:26:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1952715,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6739131/v1/fbe553a7-8aed-4d47-aae4-268b52afa871.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Callus production and Micropropagation and Phytochemical Investigation of Corallocarpus epigaeus (Arn.) Cl. – A Potential Antidiabetic Medicinal Plant","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe proof about utilization of therapeutic plants for human wellbeing can be seen in numerous old records, for instance records in ayurveda reports have numerous treatment methodology for various sicknesses \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. These days there are many notable plant-based prescriptions are accessible in the market which has capability of relieving savage infections are famous all around the globe. As per world wellbeing association (WHO), around 80% of total populace relies upon the restorative plants for essential social insurance \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. The plant-based medicines are growing nowadays worldwide, 25% peoples of United Kingdom (UK) use the plant-based medicines for curing the diseases\u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. The substance amalgamation of some pharmaceutical mixes is beyond the realm of imagination or not financially practical in this way around 40% compound utilized in assembling of prescriptions are gotten from plants \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePlant tissue culture is viewed as the most proficient innovation and it has a few favorable circumstances over conventional strategy for spread like uniting, through seed and cutting. Micropropagation method for the most part utilized and through this prevalent nature of plant is produce which has better ailment opposition and stress resilience limits, micropropagation system is utilized for the protection and duplication of yield plant like organic products, vegetables, grains, restorative, fancy and woodland, has made new open doors for ranchers, nursery proprietors, in worldwide exchanging and furthermore supportive for rustic advancement \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Additionally the micropropagation techniques are employed for culturing the medicinal plants to produce important compounds for herbal and pharmaceutical industries. Micropropagation techniques are also used for conservation of threatened medicinal plants \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e (Arn.) Cl. is a rare important medicinal plant which belongs to the family Cucurbitaceae. It is commonly known as aakashguddah, jangalee suran, rakashguddah etc. It is a monoecious, deciduous and perennial tendril climber with a large turnip- shaped root and distributed in tropical Africa, Persian Gulf region and India. Monoecious, climber with tuberous root. Stem angular-sulcate, glabrous to finely pubescent. Leaves sinuately 3 lobed, hairy on both surfaces. Male peduncle 4\u0026ndash;6 cm long, 5\u0026ndash;15 flowered. Calyx lobes lanceolate. Corolla greenish yellow color and ovate. Female flowers often solitary; pedicel 1\u0026ndash;5 mm long, thickened in fruit; calyx-tube campanulate, petals 1.5\u0026ndash;2.5 mm long, 1-1.5 mm broad, reflexed. Fruit ovoid or ellipsoid, beaked, glabrous, smooth, red except for the greenish base and portion of beak which is 2.5-5 mm long. Seeds asymmetrically pyriform, smooth, yellowish, turgid. Flowers and fruiting in June to October \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePhytochemical screening of tubers reveals the presence of phytonutrients and medicinally valuable secondary metabolites including flavonoids, alkaloids, phenolics, tannins, saponins and steroids \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e. Phytochemicals perhaps impart potent pharmacological properties to the great rhizomes that used in the controlling the diabetic disease \u003csup\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBecause of exploitative collecting of tubers for exchange and denudation of backwoods, the normal populace of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e has declined to such a degree, that it is presently viewed as uncommon and undermined in its common natural surroundings. A few creators put it under imperiled class since normal recovery of this species is very poor by virtue of long seed lethargy, failed seeds, amazingly poor germination, and deficient accessibility of tuberous roots as propagules attributable to exploitative gathering. Consequently, they unequivocally anticipate misgiving of its annihilation soon if sufficient preservation measures are not embraced earnestly.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eArrangement lies in mediations of biotechnological approaches for \u003cem\u003ein vitro\u003c/em\u003e recovery which offers quick and boundless accessibility of planting materials consistently. Biotechnological mediations for recovery utilizing \u003cem\u003ein vitro\u003c/em\u003e culture method have been effectively utilized for some yields and a few reports are likewise accessible for comparable wild Cucurbitaceae taxa. There has been no report accessible so far for \u003cem\u003ein vitro\u003c/em\u003e callus induction and micropropagation and support of \u003cem\u003ein vitro\u003c/em\u003e biodiversity for conservation of the germplasm for the specie \u003cem\u003eCorallocarpus.\u003c/em\u003e Hence, into account the rising biological basic for restoration of species of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e for conservation for human benefit.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Collection of seeds and explants\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The seed, tuber and whole plant of \u003cem\u003eCorallocarpus epigaeus\u0026nbsp;\u003c/em\u003ewere collected from surrounding area of Jalaram temple, Virpur, Gujarat during the month of June 2023 (Figure1). The plant specimen was identified by Dr. Kalpesh Ishnava (Plant Taxonomist) at\u0026nbsp;Sardar Patel University, Vallabh Vidyanagar, Gujarat. The voucher specimen (SPU-2023-02) were deposited in the herbarium of P. G. Department of Biosciences, Sardar Patel University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Seed germination study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 \u003cem\u003eIn vivo\u0026nbsp;\u003c/em\u003eseed germination (Effect of GA3 on seed germination)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Petriplates was taken and the same size of filter papers was placed into it. Then 2 to 3ml of different stock solutions of GA\u003csub\u003e3\u003c/sub\u003e was added to each plate and 1 seed per each plate was inoculated. Control was also prepared by inoculating 1 seed in distilled water. All the petridishes provided the normal light (16hr) and proper nutrition for growth and development. Seed germination rate was observed after 5 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2 \u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003eseed germination and effect of GA3 on seed germination\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The viability of the seed was determined by soaking the seeds in distilled water for 24 h. The non-viable seeds were observed at the surface of water and were discarded immediately. The surface sterilization of seeds was carried out in 3 different steps. Seeds were immersed in lab wash containing beaker under running tap water for about 30 minutes. Then immerse into surface sterilization 0.1% HgCl2 solution for approximately 3 min \u003cu\u003e\u003csup\u003e18\u003c/sup\u003e\u003c/u\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3 Culture medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Seeds were then washed with sterile distilled water 3 times and inoculated on MS medium\u003cu\u003e\u003csup\u003e19\u003c/sup\u003e\u003c/u\u003e with sucrose (3%) and pH 5.7-5.8 and also use of different concentration of hormones of GA3 (0.1ppm to 0.5ppm). After transfers the seed in semisolid medium inoculated and all tube transfers in the culture room in temperature 25\u003csup\u003e0\u003c/sup\u003eC. Before the inoculation the Laminar Air Flow was subjected for UV light transmission for about 30 – 45 minutes. The inoculation of seeds was carried out under aseptic conditions under Horizontal Laminar Air Flow.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.4 Surface sterilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The surface sterilization of seed was carried out in different steps. The seeds were placed in different beaker and covered with net and washed for 30 minutes under running tap water to remove all the adhering dust particles and microbes from the surface, then surface sterilized by immersion in 75% alcohol for 30 seconds, followed by one rinse of 3 min in sterile distilled water, additionally immersed in 0.1% mercuric chloride (HgCl\u003csub\u003e2\u003c/sub\u003e) solution for 4 min followed by five times rinses of 5 min each in distilled water. The surface sterilized different seeds were blotted dry on sterile filter paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.5 Inoculation of seeds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;All the experimental manipulations were carried out under strictly aseptic conditions in laminar air flow bench. The chamber was then sterilized with U.V. rays continuously on for one hour. Hands and arms which were to be used inside the inoculation chamber were scrubbed with alcohol before inoculation. The rims of the test tubes and the sides of the plugs were flame sterilized. Instruments (like forceps, scalpels, spatula etc.) were all sterilized by dipping in the alcohol and flaming a number of times. Care was taken to cool the instruments before putting into operation. After sterilization of seeds, seed were inoculated in culture tubes aseptically. For inoculation explants were transferred to large sterile glass petriplate or glass plate with the help of sterile forceps under strict aseptic conditions. Some Seeds transferred to culture tubes containing MS medium and some in to MS media with GA3 (0.1 % to 0.5%). After inoculating the seed in culture tube, the mouth of tube is quick flamed and tubes are tightly closed by cotton plug. After proper labeling clearly mentioning media, date of inoculation etc. the tubes were transferred to growth room.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.6 Culture Condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The tubes were shifted to culture room with controlled facility of diffused light (2000 lux) for 8 hrs. and rest of the period dark condition daily at 28 ± 2°C temperatures and 50 to 60% relative humidity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Callus induction study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1 Preparation of culture medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;MS medium was used for callus of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e. Murashige and Skoog (MS) medium supplemented with different concentrations of PGRs such as α-naphthalene acetic acid (NAA), 2,4-Dichlorophenoxy acetic acid (2,4-D), benzyl amino purine (BAP) alone and BAP in combinations with NAA, Benzyl adenine (BA) in combinations with NAA, 2,4-D in combinations with Kinetin (KN) for callus induction. The medium was supplemented with 3% sucrose, gelled with 0.8% agar and the pH was adjusted to 5.7-5.8. Around 20ml of medium was used for each culture tubes. Then media was autoclaved at 121°C and 15 psi pressure for 15 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2 Callus induction \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;All the experimental manipulations were carried out under strictly aseptic conditions in laminar air flow bench. After sterilization of explants (Nodal, tuber and leaf), explants were inoculated in culture tubes aseptically. For inoculation explants as per standard protocol. The culture tubes were transferred to growth room \u003cu\u003e\u003csup\u003e20\u003c/sup\u003e\u003c/u\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.3 Culture Condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The tubes and bottles were shifted to culture room with controlled facility of diffused light (3000 lux) for 10 hrs. and rest of time dark condition daily at 28 ± 2°C temperatures and 50 to 60% relative humidity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.4 Collection of calluses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The calluses were collected after 25 days. Calluses were washed with distilled water to remove all adhering particles. After that calluses were allowed to dry at room temperature. This callus was use for different analysis for study. Good quality callus was sub cultured and again collected after more 30 days and subjected to further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Micropropagation \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.1 Preparation of culture medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;MS medium was used for micropropagation of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e. Murashige and Skoog (MS) medium supplemented with different concentrations of PGRs such as α-naphthalene acetic acid (NAA), 2,4-Dichlorophenoxy acetic acid (2,4-D), benzyl amino purine (BAP) alone and 2,4-D in combinations with Kinetin (KN), IBA in combination with BAP for shooting And IBA in combination with BAP for rooting. The medium was supplemented with 3% sucrose, gelled with 0.8% agar and the pH was adjusted to 5.7-5.8. Around 40ml of medium was used for each culture bottles. Then media was autoclaved at 121°C and 15 Psi pressure for 15 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.2 Explants sterilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The surface sterilization of nodal explants was carried out in different steps. First off, all cut the leaves of isolated nodal segment. \u0026nbsp;Explants were further soaked in fungicide (1gm/lit Bavistin) for 5 min and then antibiotic Streptomycin (50mg/lit) for 3 min then surface sterilized by immersion in 75% alcohol for 30 seconds, followed by one rinse of 3 min in sterile distilled water, additionally immersed in 0.1% mercuric chloride (HgCl\u003csub\u003e2\u003c/sub\u003e) solution for 3 min followed by five rinses of 5 min each in distilled water. The surface sterilized nodal explants were blotted dry on sterile filter paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.3 Inoculation of explants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;All the experimental manipulations were carried out under strictly aseptic conditions in laminar air flow bench. After sterilization of explants, explants were inoculated in culture tubes aseptically. For inoculation explants were transferred to large sterile glass petriplate or glass plate with the help of sterile forceps under strict aseptic conditions. Here the explants were further trimmed and extra outer leaves were removed to make them in suitable sizes. Trimming was removed with sterile scalpel blade. After cutting explants into suitable size (for Node 1-2 cm), explants are transferred to culture tubes and bottles containing MS medium with different hormone concentration. After vertically inoculating the explants in culture tube the mouth of tubes is quick flamed and tubes are tightly capped with cotton plug. After proper labeling clearly mentioning media, date of inoculation etc. the bottles were transferred to growth room.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.4 Multiplications of shoot stage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;After 15 days of inoculation culture showing spouting were transferred to full length MS media supplemented with different combination of growth hormones concentration for multiplication of shoot regeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.5 Rooting stage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Newly formed shoots measuring 2.0-3.0 cm in length were excised individually from the parents and explants and transferred to new MS medium with different concentration of IAA and NAA hormone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.6 Culture Condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The tubes and bottles were shifted to culture room with controlled facility of diffused light (3000 lux) for 10 hrs. and rest of time dark condition daily at 28 ± 2°C temperatures and 50 to 60% relative humidity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.7 Hardening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;In vitro\u0026nbsp;\u003c/em\u003erooted plantlets with 2 to 3 nodes and having at least 2 to 4 roots of 2 to 4 cm length were washed carefully with water to remove traces of agar and then transferred to the pots containing different sterile soil mixtures viz. Cocopeat + Sand + Soil (1:1:1). The pots were covered with tight plastic covers (With holes) to prevent desiccation and to avoid rapid changes in environment and acclimatized in the mist house at 25-30 °C temperature and 14 hrs. illumination. During the hardening procedure, plastic covers were gradually perforated after one week and after second week they were removed and the plants were maintained in the mist house conditions for 15 more days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Phytochemical analysis of callus\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.1 Extraction of callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Dried sample of callus from \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003egrown plant were grinded with mortar and pestle. The dry sample was extracted using 20 ml of methanol in Erlenmeyer flask placed on shaker at 100 rpm for overnight at room temperature. The crude extract filtered with filter paper (Whatman No. 1). The filtrate was collected and allowed to solvent evaporation. After evaporation the remaining material was collected and different stocks were prepared by dissolving in methanol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.2 Phytochemical screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQualitative assay for the presence of plant phytoconstituents such as Alkaloids, Glycosides, Flavonoids, Phenol, Tannins and Saponins and quantitative assay were carried out on alkaloids, total phenol and total flavonoidsfollowing standard procedure \u003cu\u003e\u003csup\u003e20, 21\u003c/sup\u003e\u003c/u\u003e.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eDue\u0026nbsp;to\u0026nbsp;exploitative\u0026nbsp;harvesting\u0026nbsp;of\u0026nbsp;tubers\u0026nbsp;for trade and denudation of forests, the natural population of \u003cem\u003eCorallocarpus epigaeus\u0026nbsp;\u003c/em\u003ehas declined to such an extent that it is now considered rare and threatened in its natural habitats. Hence, they explicitly predict apprehension of its extinction in the near future if adequate conservation measures are not adopted urgently. So here in the present study we have initiated and developed the callus and micropropagation of the leaf, node and tuber of\u0026nbsp;\u003cem\u003eCorallocarpus\u0026nbsp;\u003c/em\u003e\u003cem\u003eepigaeus\u003c/em\u003e and then evaluated the phytochemical analysis of methanolic extract of tuber callus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;3.1 Seed germination study (\u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e seeds germination of\u003cem\u003e\u0026nbsp;Corallocarpus epigaeus\u0026nbsp;\u003c/em\u003ewas done usingGA\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eat different concentration (1ppm to 5ppm). There is no germination was observed in any concentration of GA\u003csub\u003e3\u003c/sub\u003e after 25 days. The seed germination of this plant is very poor because of seeds having long dormant period. Seed coat of this plant is very hard and also the embryo abortion observed during seed germination. \u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003eseeds germination attempt of\u003cem\u003e\u0026nbsp;Corallocarpus epigaeus\u003c/em\u003e was made byusing MS media supplemented with different concentrations of GA\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(1ppm to 5ppm). No seed germination was observed in MS medium (\u003cem\u003ein vitro\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Callus Production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Callus induction from leaf explants of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e using MS basal medium supplemented with different plant growth regulators such as NAA, 2,4-D and BAP alone and KN in combination with 2,4-D is mentioned in Table 1. The NAA, 2,4-D and BAP alone do not show any response in the leaf explants. The leaf explants inoculated in MS media supplemented with KN (0.3mg/lit) + 2,4-D (3mg/lit) and KN (0.5mg/lit) + 2,4-D (5mg/lit) shows the callus induction after 6 days of inoculation. The callus produced is green and yellow respectively.\u003c/p\u003e\n\u003cp\u003eCallus induction from nodal explants of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e using MS basal medium supplemented with different concentrations of BAP and NAA is mentioned in Table 1 and Figure 2A. Brownish callus was produced after 6 days of incubation on MS media supplemented with BAP (4ppm). Brownish, White and Green callus was produced in the nodal explants inoculated in MS media supplemented with NAA (1ppm to 5pmm) after 6-7 days of inoculation (Figure 2B). Callus induction from nodal explants of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e using MS basal medium supplemented with different concentrations of 2,4-D is mentioned in Table 1. Brownish callus was produced in the nodal explants inoculated in MS media supplemented with 2,4-D (0.2 mg/lit.), 2,4-D (1.5 mg/lit.) and 2,4-D (5 mg/lit.) after 15 and 6 days of inoculation respectively. Callus induction in the nodal explants was observed in the different combinations of NAA and BAP (Table 1). Brownish callus was produced in the nodal explants inoculated in MS media supplemented with different combinations of hormones BAP (0.5mg/lit) + NAA (5mg/lit), BAP (1mg/lit) + NAA (4.5mg/lit) and BAP (3.5mg/lit) + NAA (2mg/lit) after 6-8 days of inoculation (Figure 2B). Mehul and Kalpesh 2015 reported that a combination of BAP and NAA was more suitable combination for callus induction of\u0026nbsp;\u003cem\u003eM. dioica\u0026nbsp;\u003c/em\u003e\u003cu\u003e\u003csup\u003e22\u003c/sup\u003e\u003c/u\u003e. Hoque et al., (1995) found that a combination of 1.5mg/l BAP and 0.1 mg/l NAA was more suitable combination for adventitious multiple shoots formation of\u0026nbsp;\u003cem\u003eM. dioica\u003c/em\u003e \u003cu\u003e\u003csup\u003e23\u003c/sup\u003e\u003c/u\u003e.\u0026nbsp;Where present investigated\u0026nbsp;BAP + NAA combination is most suitable for callus induction. Devendra et al. (2012) reported that the callus initiation started from nodal base on 10th day 2.0 mg/l NAA, 2.0 mg/l BAP auxin or cytokinin either alone or in combinations were efficient for the induction of callus depending on the varied concentrations of the growth regulators \u003cu\u003e\u003csup\u003e24\u003c/sup\u003e\u003c/u\u003e.\u0026nbsp;The different combinations of KN and 2,4-D shows callus induction in the nodal explants (Table 1). Off white and brownish callus was produced in the nodal explants inoculated in MS media supplemented with different combinations of hormones KN(1mg/lit) + 2,4-D(1mg/lit), KN(0.1mg/lit) + 2,4-D(1mg/lit), KN(2mg/lit) + 2,4-D(0.2mg/lit) and KN(0.2mg/lit) + 2,4-D(2mg/lit) after 4-5 days of inoculation (Figure 2B). Salvador et al., (2009) reported the \u003cem\u003eAlternanthera tenella\u003c/em\u003e, an amaranthaceae family, MS basal medium was supplemented with 1.0 mg/lit 2, 4-D and 1.0 mg/lit KN and 2.5 mg/lit NAA and 1.0 mg/lit BA for callus induction \u003cu\u003e\u003csup\u003e25\u003c/sup\u003e\u003c/u\u003e.\u0026nbsp;The different combinations of NAA and BA show callus induction in the nodal explants (Table 1). Green callus was produced in the nodal explants inoculated in MS media supplemented with different combinations of hormones BA (2mg/lit) + NAA (1mg/lit), BA (1mg/lit) + NAA (1mg/lit), BA (0.5mg/lit) + NAA (1mg/lit) after 4-5 days of inoculation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Callus induction from tuber explants of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e using MS basal medium supplemented with different concentrations of BAP in combination with NAA is mentioned in Table 1 and Figure 2F. The different combinations of NAA and BAP show callus induction in the tuber explants. Brownish callus was produced in the tuber explants inoculated in MS media supplemented with different combinations of hormones BAP (0.5mg/lit) + NAA (5mg/lit), BAP (1mg/lit) + NAA (4.5mg/lit) and BAP (1.5mg/lit) + NAA (4.0mg/lit) after 7 days of inoculation (Figure 2I, 2J, 2K, 2L). \u0026nbsp;Castillo \u003cem\u003eet al\u003c/em\u003e., (1998) reported that auxin 2,4-D by itself or in combination with cytokinin has been widely used to enhance callus induction and maintenance \u003cu\u003e\u003csup\u003e26\u003c/sup\u003e\u003c/u\u003e. Moreover, many researchers observed 2,4-D as the best auxin for callus induction as common as in monocot and even in dicot \u003cu\u003e\u003csup\u003e27, 28, 29, 30, 31\u003c/sup\u003e\u003c/u\u003e. This combination is more suitable for more production of the biomass for secondary metabolites. Callus induction from tuber explants of \u003cem\u003eC. epigaeus\u003c/em\u003e using MS basal medium supplemented with different concentrations of BAP is mentioned in Table 1. Brownish, green and yellow callus was produced after 6 days of incubation on MS media supplemented with BAP (1 to 5 mg/lit). Callus induction from tuber explants of \u003cem\u003eC. epigaeus\u003c/em\u003e using MS basal medium supplemented with different concentrations of NAA is mentioned in Table 1. Brownish, yellow and green callus was produced in the tuber explants inoculated in MS media supplemented with NAA (3 and 5 mg/lit) after 9 days of inoculation (Figure 2H). Callus induction from tuber explants of \u003cem\u003eC. epigaeus\u003c/em\u003e using MS basal medium supplemented with different concentrations of 2,4-D is mentioned in Table 1. Brownish callus was produced in the tuber explants inoculated in MS media supplemented with 2,4-D (0.1mg/lit to 5mg/lit) after 9 days of inoculation (Figure 2H). Callus induction from tuber explants of \u003cem\u003eC. epigaeus\u003c/em\u003e using MS basal medium supplemented with different concentrations of BAP in combination of NAA is mentioned in Table 1. The different combinations of NAA and BAP show callus induction in the tuber explants. Cream white callus was produced in the nodal explants inoculated in MS media supplemented with different combinations of hormone KN (2mg/lit) + 2,4-D (0.2mg/lit) after 4-5 days of inoculation (Figure 2G). No callus was produced in the tuber explants inoculated in MS media supplemented with different combinations of hormones BA in combination with NAA after 40 days of inoculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Micropropagation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;MS media supplemented with higher concentration of cytokinin along with lower concentration of auxins were used for the shoot multiplication. The nodal explants were selected for shoot multiplication and inoculated on MS media supplemented with higher concentration of BAP and lower concentration of IBA. The combination (BAP 1ppm : IBA 0.01ppm) of PGR shows the positive results of shoot multiplication and maximum shoot (3 shoot) obtained after 15 days of inoculation (Table 2 and Figure 2C). The \u003cem\u003ein vitro\u003c/em\u003e raised shoots were inoculated on to MS supplemented with various combinations of auxin and cytokinin for root formation. Root formed in all combination and concentration of hormone but highest frequencies of rooting were observed on MS fortified with 1.0 mg/lit of IAA and 0.5 mg/lit NAA after 15 days of inoculation (Figure 2D). But prior to the root initiation callus was taking place (Figure 2B). The reduced rooting may be due to the imbalance between the endogenous auxin and exogenous auxin, IAA. In the present study, initially the rooting percentage was very low, these results are in accordance with those on \u003cem\u003eWithania somnifera\u003c/em\u003e \u003cu\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/u\u003e, when IAA was tested for rooting, there was not only decrease in the rooting response but also enhanced basal callusing from the \u003cem\u003ein vitro\u003c/em\u003e raised shoots were observed. These results conform to those of Anand et al., 1997 in \u003cem\u003eKaempfera rotunda\u0026nbsp;\u003c/em\u003e\u003cu\u003e\u003csup\u003e33\u003c/sup\u003e\u003c/u\u003eand Ankita and Kalpesh, (2015) in \u003cem\u003eCoccinia grandis\u003c/em\u003e (L.) Voigt. \u003cu\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/u\u003e. After shoot and root production transfer to primary and secondary hardening of the plants. After 3 week plants transfer to natural condition (Figure 2E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Phytochemical analysis of callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1 Qualitative analysis of callus from tuber\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The callus obtained in different combination of growth hormones MS + BAP (0.5mg/lit) + NAA (5mg/lit), MS + BAP (0.5mg/lit) + NAA (5mg/lit) and MS + BAP (0.5mg/lit) + NAA (5mg/lit) was extracted by methanol. Methanolic extract of various calluses was subjected to qualitative analysis. The analysis shows the presence of alkaloids, flavonoids, saponins, phenols and steroids (Table 3). Kattamanchi et al., (2013) reported the ethanolic extract of \u003cem\u003eCorallocarpus epigaeus\u0026nbsp;\u003c/em\u003erhizomes was found the presence of phenolic compounds, Flavonoids, terpenoids and phytosterols \u003cu\u003e\u003csup\u003e15\u003c/sup\u003e\u003c/u\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2 Quantitative analysis of callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Estimation of alkaloids \u003cem\u003ein vitro\u003c/em\u003e callus obtained in the medium supplemented different combinations of PGRs (BAP-0.5mg/lit + NAA-5mg/lit; BAP-1mg/lit + NAA-4.5mg/lit; BAP-1.5mg/lit + NAA-4mg/lit) was done by Harborne method. The evaluation alkaloids of callus were done after 25 days of inoculation. Then remaining callus was subjected to sub culture on same medium and again analysis of alkaloids was done after sub culture. The percent yield of alkaloids in callus was calculated by back calculation and mentioned in Table 4. After 25 days maximum percentage yield present in BAP-1mg/lit + NAA-4.5mg/lit and after sub culture percentage yield of alkaloids was obtained in same concentration.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Estimation of phenols \u003cem\u003ein vitro\u003c/em\u003e callus obtained in the medium supplemented different combination of PGRs (BAP-0.5mg/lit + NAA-5mg/lit; BAP-1mg/lit + NAA-4.5mg/lit; BAP-1.5mg/lit + NAA-4mg/lit) was done by Folin’s method. The evaluation alkaloids of callus were done after 25 days of inoculation. Then remaining callus was subjected to sub culture on same medium and again analysis of alkaloids was done after sub culture. The percent yield of phenols in callus was calculated by back calculation and mentioned in Table 4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEstimation of flavonoids \u003cem\u003ein vitro\u003c/em\u003e callus obtained in the medium supplemented different combination of PGRs (BAP-0.5mg/lit + NAA-5mg/lit; BAP-1mg/lit + NAA-4.5mg/lit; BAP-1.5mg/lit + NAA-4mg/lit) was done by aluminum chloride method. The evaluation flavonoids of callus were done after 25 days of inoculation. Then remaining callus was subjected to sub culture on same medium and again analysis of flavonoids was done after sub culture. The percent yield of flavonoids in callus was calculated by back calculation and mentioned in Table 4.\u0026nbsp;\u0026nbsp;Kattamanchi et al., (2013) reported the\u0026nbsp;ethanolic extract of \u003cem\u003eCorallocarpus epigaeus\u0026nbsp;\u003c/em\u003erhizomes possesses the antidiabetic action which is comparable with that of the standard Glibenclamide drug employed \u003cu\u003e\u003csup\u003e15\u003c/sup\u003e\u003c/u\u003e. This work supports the traditional claim of the rhizomes for their use in diabetes. This ethanolic extract of \u003cem\u003eCorallocarpus epigaeus\u0026nbsp;\u003c/em\u003epresents the flavonoids. In our study show the in vitro callus induction more production of theflavonoids compares to the normal is less produce. This work supports the traditional claim of the rhizomes for secondary metabolites production through callus induction increases the biomass.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCallus initiation of nodal explants took place after 4-6 days of inoculation while the tuber explants shows callus initiation after 7-9 days of inoculation. The best degree for callus formation of tuber explants was obtained on MS medium supplemented with BAP:NAA (0.5mg/lit:5.0mg/lit, 1.0mg/lit:4.5mg/lit and 1.5mg/lit:4mg/lit) while the nodal explants shows highest degree for callus formation on MS medium supplemented with BA (2mg/lit) in combination with NAA (1mg/lit). The nodal explants were selected for shoot multiplication and inoculated on MS media supplemented with higher concentration of BAP and lower concentration of IBA. The combination (BAP 1ppm: IBA 0.01ppm) of PGR shows the positive results of shoot multiplication and maximum shoot (3 shoot) obtained after 15 days of inoculation. \u0026nbsp;NAA (3ppm) of PGR shows the positive results of root production and maximum root (1 root) and length (4cm) obtained after 25 days of inoculation. The present study showed successfully micropropagation of \u003cem\u003eCorallocarpus epigaeus.\u0026nbsp;\u003c/em\u003ePhytochemical investigation the tuber callus indicated the presence of alkaloids, flavonoids, phenols, steroids and saponins. The yield of alkaloids, flavonoids and phenols are 4.71%, 0.23% and 0.013% respectively in 25-day old callus of tuber explants. Further study required for the large-scale production of secondary metabolites through callus production and also large-scale production of propagules required the standardization.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) received the financial support from its institutional research grant of P. G. Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India for the research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data include in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest about this manuscript and research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are thankful to P. G. Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India for providing necessary support for research and laboratory facility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKaushik Nakum and Vipul Vaja design the methodology and perform the experiments. Dr. Kalpesh Ishnava interpretation of experiment results and preparing the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStafford, A., Morris, P. \u0026amp; Fowler, M. W. Fowler plant cell biotechnology: A perspective. \u003cem\u003eEnzym. Microb. Technol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 578\u0026ndash;587 (1986).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoumya, P., Rout, K. A., Choudary, D. M., Kar, L., Das, A. \u0026amp; Jain Plants in traditional medicinal system - Future source of new drugs. \u003cem\u003eInt. J. Pharm. Pharm. Sci.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e (1), 1\u0026ndash;23 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFarnsworth, N. R. Ethnopharmacology and drug development. Ciba Foundation Symposium 185:42\u0026ndash;59. (1994).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCowan, M. M. Plant products as antimicrobial agents. \u003cem\u003eClin. Microbiol. Rev.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (4), 564\u0026ndash;582 (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh, P. et al. 2009 Somatic embryogenesis and \u003cem\u003ein vitro\u003c/em\u003e regeneration of an endangered medicinal plant sarpgandha (\u003cem\u003eRauvolfia serpentina\u003c/em\u003e L). \u003cem\u003eLife Sci. J.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e(3):57\u0026ndash;62 .\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhani, A. \u003cem\u003eMedicinal plants of Bangladesh - Chemical constituents and uses\u003c/em\u003e 2nd edn pp 337\u0026ndash;338 (Asiatic Society of Bangladesh, 1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrishnan, P. N. \u0026amp; Seeni, S. Rapid micropropagation of \u003cem\u003eWoodfordia fruticosa\u003c/em\u003e (L.) Kurz (Lythraceae), A rare medicinal plant. \u003cem\u003ePlant Cell Rep.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 55\u0026ndash;58 (1994).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrown, D. C. W. \u0026amp; Thorpe, T. A. Crop improvement through tissue culture. \u003cem\u003eWorld J. Microbiol. Biotechnol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 409\u0026ndash;415 (1995).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkin-Idowu, P. E., Ibitoye, D. O. \u0026amp; Ademoyegun, O. T. Tissue culture as a plant production technique for horticultural crops. \u003cem\u003eAfr. J. Biotechnol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (16), 3782\u0026ndash;3788 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYaadwinder \u0026amp; Sidhu \u003cem\u003eIn vitro\u003c/em\u003e micropropagation of medicinal plants by tissue culture. \u003cem\u003ePlymouth Student Sci.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e (1), 432\u0026ndash;449 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar, V., Cotran, R. S., Robbins, S. L. \u0026amp; Saunders, W. B. \u003cem\u003eBasic pathology\u003c/em\u003e 5th edn pp 25\u0026ndash;40 (Harcourt, Brace \u0026amp; Company, 1992).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShah, G. L. \u003cem\u003eFlora of Gujarat State\u003c/em\u003e (University press, Sardar Patel University, Vallabh Vidyanagar, 1978).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKirtikar, K. R. \u0026amp; Basu, B. D. \u003cem\u003eIndian Medicinal Plants\u003c/em\u003eVol. IIpp 1166\u0026ndash;1167 (International Book Distributors, 1918).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePandey, C. N., Raval, B. R., Mali, S. \u0026amp; Salvi, H. \u003cem\u003eMedicinal plants of Gujarat\u003c/em\u003epp 1\u0026ndash;5 (Gujarat Ecological Education and Research (GEER) Foundation, 2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKattamanchi Gnananath, K. R., Reddy, G. P., Kumar, B. \u0026amp; Krishna Karka Srinivas Reddy, Avvari Sanjeeva Kumar, 2013 Evaluation of antidiabetic activity in \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e Rhizomes. \u003cem\u003eInt. Curr. Pharm. J.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e(3):53\u0026ndash;56 .\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKapesh Ishnava, R., Kotadia, S. \u0026amp; Patel Nutritional properties and chemical composition of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e (Arn.) Cl: As remedy to control diabetes mellitus. \u003cem\u003eChiang Mai J. Sci.\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e (4), 806\u0026ndash;815 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTejas, P. Genetic Transformation of Medicinal Plant, \u003cem\u003eWithania somnifera\u003c/em\u003e (L.) Dunnal by \u003cem\u003eAgrobecterium tumefaciens\u003c/em\u003e (MTCC-431). Dissertation, Sardar Patel University. (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMurashige, T. \u0026amp; Skoog, F. Revised medium for rapid growth and bioassays with Tobacco tissue cultures. \u003cem\u003ePhysiol. Plant.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 473\u0026ndash;499 (1962).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeghana \u003cem\u003eIn vitro\u003c/em\u003e study of \u003cem\u003eTinospora cordifolia\u003c/em\u003e (Wild.) Miers (Menispermaceae) - \u0026lsquo;A multipurpose plant\u0026rsquo; by using different plant bark extracts for secondary metabolite production. Dissertation, Sardar Patel University. (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarborne, J. B. \u003cem\u003ePhytochemical methods - A guide to modern techniques of plant analysis\u003c/em\u003e (Chapman and Hall, 1973).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHossain, M. A., AL-Raqmi, K. A., AL-Mijizy, Z. H., Weli, A. M. \u0026amp; Al-Riyami, Q. Study of total phenol, flavonoids contents and phytochemical screening of various leaves crude extracts of locally grown \u003cem\u003eThymus vulgaris.\u003c/em\u003e Asian Pacific. \u003cem\u003eJ. Trop. Biomed.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (9), 705\u0026ndash;710 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel, M. G. \u0026amp; Ishnava, K. B. \u003cem\u003eMomordica dioica\u003c/em\u003e Roxb. (Spine Gourd): Multiple shoot induction from nodal cultures and its antidiabetic activity. Journal of Medicinal Plants Studies 3(6):82\u0026ndash;88. (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoque, A., Islam, R. \u0026amp; Joarder, O. I. \u003cem\u003evitro\u003c/em\u003e plantlets differentiation in kakrol (\u003cem\u003eMomordica dioica\u003c/em\u003e Roxb). \u003cem\u003ePlant. Tissue Cult.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 119\u0026ndash;124 (1995 In).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDevendra, N. K., Everaldo, G. A., Raghunandan, D. \u0026amp; Seetharam, Y. N. \u003cem\u003evitro\u003c/em\u003e production of Cucurbitacins from \u003cem\u003eTrichosanthes cucumerina\u003c/em\u003e L. var. \u003cem\u003ecucumerina Adv. Life Sci.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e (4), 108\u0026ndash;111 (2012 In).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSalvador, M. J. et al. Bioactive chemical constituents and comparative antimicrobial activity of callus culture and adult plant extracts from \u003cem\u003eAlternanthera tenella\u003c/em\u003e. \u003cem\u003eZ. f\u0026uuml;r Naturforschung C\u003c/em\u003e. \u003cb\u003e64\u003c/b\u003e (5\u0026ndash;6), 373\u0026ndash;381 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCastillo, A. M., Egana, B., Sanz, J. M. \u0026amp; Cistue, L. Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain. \u003cem\u003ePlant. Cell. Rep.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 902\u0026ndash;906 (1998).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEvans, D. A., Sharp, W. R. \u0026amp; Bravo, J. E. Cell culture methods for crop improvement. Hand Book of Plant Cell Culture. Vol.2. Macmillan Publishing Company, New York. (1984).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHo, W. J. \u0026amp; Vasil, I. K. Somatic embryogenesis in sugarcane (\u003cem\u003eSaccharum officinarum\u003c/em\u003e L.) I. the morphology and physiology of callus formation and the ontogeny of somatic embryos. \u003cem\u003eProtoplasma\u003c/em\u003e \u003cb\u003e118\u003c/b\u003e, 169\u0026ndash;180 (1983).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJaiswal, V. S. \u0026amp; Naryan, P. Regeneration of plantlets from the callus of stem segment of adult plants of \u003cem\u003eFucus religiosa\u003c/em\u003e L. \u003cem\u003ePlant Cell Rep.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 256\u0026ndash;258 (1985).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChee, P. P. High frequency of somatic embryogenesis and recover of fertile cucumber plants. \u003cem\u003eHortScience\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (7), 792\u0026ndash;793 (1990).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMamun, A. N., Islam, R., Reza, M. A. \u0026amp; Joadar, O. I. \u003cem\u003evitro\u003c/em\u003e differentiation of plantlet of tissue culture of \u003cem\u003eSamonea saman\u003c/em\u003e. \u003cem\u003ePlant. Tissue Cult.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 1\u0026ndash;5 (1996 In).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eManickam, V. S., Mathavan, R. E. \u0026amp; Antonisamy, R. Regeneration of Indian ginseng plantlets from stem callus. \u003cem\u003ePlant. Cell. Tissue Organ. Cult.\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e, 181\u0026ndash;185 (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnand, P. H. M., Harikrishnan, K. N., Martin, K. P. \u0026amp; Hariharan, M. \u003cem\u003evitro\u003c/em\u003e propagation of \u003cem\u003eKaempferia rotunda\u003c/em\u003e Linn. Indian crocus - a medicinal plant. \u003cem\u003ePhytomorphology\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e (3), 281\u0026ndash;286 (1997 In).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel, A. \u0026amp; Ishnava, K. \u003cem\u003evitro\u003c/em\u003e shoot multiplication from nodal explants of \u003cem\u003eCoccinia grandis\u003c/em\u003e (L.) Voigt. and it\u0026rsquo;s antidiabetic and antioxidant activity. \u003cem\u003eAsian J. Biol. Sci.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (2), 57\u0026ndash;71 (2015 In).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Callus induction from tuber and nodal explants of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"692\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNutrient media\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 281px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTuber Explants\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 276px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNodal explants\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS- media\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGrowth hormones\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% of explants showing callus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDuration of callus production\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResponses\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% of explants showing callus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDuration of callus production\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResponses\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e11 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e6-7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eGreen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrown green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAA (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6-7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAA (2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6-7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAA (3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6-7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAA (4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e5-6 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eWhite\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAA (5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrown green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6-7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eGreen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (0.1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (0.2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (0.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (0.7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e22 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (1.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e22 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e22 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2,4-D (5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (0.5) + NAA (5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight brown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6-7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eCream brown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (1) + NAA (4.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight brown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6-7 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003ebrown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (1.5) + NAA (4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e7days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight brown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (2) + NAA (3.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight brown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (2.5) + NAA (3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight brown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (3) + NAA (2.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e11 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight brown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (3.5) + NAA (2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e8 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrownish\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (4) + NAA (1.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (4.5) + NAA (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (5) + NAA (0.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (3) + NAA (3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKN (1) + 2,4-D (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4-5 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKN (0.1) + 2,4-D (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4-5 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKN (2) + 2,4-D (0.2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eCream white\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4-5 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eOff white\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKN (2) + 2,4-D (2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKN (0.2) + 2,4-D (2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4-5 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrown\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBA (1) + NAA (0.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4-5 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eGreen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBA (1) + NAA (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBA (0.5) + NAA (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4-5 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eBrown green\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBA (2) + NAA (1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4-5 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eLight Green\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBA (1) + NAA (0.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4-5 days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eGreen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Effect of hormones on shoot multiplication and root production\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"654\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSr. No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS medium\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBAP : IBA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of shoots\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in Days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength of shoot (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of Roots\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(in Days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength of Root (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003csup\u003eth\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e1 ppm : 0.1 ppm\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e1 ppm : 0.01 ppm\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e1\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e4\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e6\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e2 ppm : 0.2 ppm\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e2 ppm : 0.02 ppm\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e3 ppm NAA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e1\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e3\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42px;\"\u003e\n \u003cp\u003e5\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Qualitative analysis of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e callus from tuber\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"565\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecondary\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMetabolite\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS + BAP (0.5mg/l) + NAA (5mg/l)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS + BAP (1mg/l) + NAA (4.5mg/l)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS + BAP (1.5mg/l) + NAA (4mg/l)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlkaloids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlavonoids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSaponins\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTerpenoids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhenols\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTannins\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGlycosides\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSteroids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Quantity analysis of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e callus from tuber\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"680\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCallus inoculated on MS-media\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e+ PGRs\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlkaloids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhenols\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlavonoids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWt. of callus (gm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWt. of alkaloids (mg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% yield\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWt. of callus (gm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWt. of\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePhenols (mg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% yield\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWt. of callus (gm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWt. of flavonoids (mg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% yield\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (0.5) + NAA (5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e199.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.088\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (1) + NAA (4.5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e201.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.225\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBAP (1.5) + NAA (4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e3.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1.541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e219.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1.541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.097\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Corallocarpus epigaeus, Antidiabetic, MS medium, Callus, Micropropagation, Phytochemical analysis","lastPublishedDoi":"10.21203/rs.3.rs-6739131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6739131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study deals with the \u003cem\u003ein vitro\u003c/em\u003e callus induction, micropropagation and phytochemical analysis of \u003cem\u003eCorallocarpus epigaeus\u003c/em\u003e (Arn.) Cl. The tuber, node and leaf were used as explants and cultured on MS medium with NAA, 2,4-D, BAP and KN alone and different combinations for callus induction and micropropagation. The best callus formation of tuber explants was obtained on BAP: NAA after 20 days of inoculation while the nodal explants show best callus formation on BA: NAA after 10 days. Leaf explants not show callus induction in any combination. The nodal explants were selected for shoot multiplication and inoculated on MS media supplemented with the combination (BAP and IBA) of PGR shows the positive results of shoot multiplication and maximum shoot obtained after 15 days of inoculation. NAA of PGR shows the positive results of root production and maximum root and length obtained after 25 days of inoculation. After 25-day callus of tuber explants was collected and subjected to phytochemical investigation which indicates the presence of alkaloids, flavonoids, Phenols, steroids and saponins. The yield of alkaloids, flavonoids and Phenols are 4.71%, 0.23% and 0.013% respectively. Further study required for the large-scale production of secondary metabolites through callus production and also large-scale production of propagules required the standardization.\u003c/p\u003e","manuscriptTitle":"Callus production and Micropropagation and Phytochemical Investigation of Corallocarpus epigaeus (Arn.) Cl. – A Potential Antidiabetic Medicinal Plant","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 18:51:33","doi":"10.21203/rs.3.rs-6739131/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"750ef3e4-7715-4feb-bb74-92f46fb0d381","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52682916,"name":"Biological sciences/Biotechnology"},{"id":52682917,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-04-11T13:25:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-06 18:51:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6739131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6739131","identity":"rs-6739131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}