Actions for Ex Situ Conservation of Gloriosa superba L. - Springer Link

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Actions for Ex Situ Conservation of Gloriosa superba L. - an. Endangered Ornamental Cum Medicinal Plant. Kuldeep Yadav, Ashok Aggarwal, Narender Singh*.
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J. Crop Sci. Biotech. 2012 (December) 15 (4) : 297 ~ 303 DOI No. 10.1007/s12892-012-0045-7 RESEARCH ARTICLE

Actions for Ex Situ Conservation of Gloriosa superba L. - an Endangered Ornamental Cum Medicinal Plant Kuldeep Yadav, Ashok Aggarwal, Narender Singh* Department of Botany, Kurukshetra University, Haryana, India Received: May 3, 2012 / Revised: August 14, 2012 / Accepted: September 15, 2012 Ⓒ Korean Society of Crop Science and Springer 2012

Abstract Factors affecting in vitro propagation and microtuberization were evaluated for Gloriosa superba L., an endangered ornamental cum medicinal plant having limited reproductive capacity. Surface sterilization of tuber explants with 0.1% mercuric chloride (HgCl2) for 5 min eliminated the contamination effectively with highest survival rate. Among the various combinations used, Murashige and Skoog (MS) medium with 2.0 mg L-1 6-benzylaminopurine (BAP) + 0.5 mg L-1 α-naphthalene acetic acid (NAA) containing 3% sucrose with 16-h photoperiod exhibited the greatest in vitro tuberization (3.2) with the highest shoot regeneration frequency (90%). The longest tuber regeneration occurred on MS media containing 4% sucrose. Transfer of in vitro-regenerated shoots to half-strength MS medium with 1.0 mg L -1 indole-3-butyric acid (IBA) + 0.5 mg L -1 NAA showed maximum root induction (66.6%). The in vitro-grown plantlets were successfully acclimatized and transplanted to sterilized soil and sand mixture (3:1) in the glasshouse with 70% survival. The colchicine content was determined in the tubers of ex vitro plants by HPLC using the same retention time (1.5 min) as that of the standard colchicine. This revealed that the micropropagation protocol developed by us for rapid mass production could be used as raw material for colchicine extraction and provides a basis for germplasm conservation and genetic improvement of G. superba. Key words : conservation, endangered, Gloriosa superba, HPLC, microtuber induction

Introduction Gloriosa superba L. (family Colchicaceae) commonly known as Kalihari and Malabar glory lily is a herbaceous climber with brilliant wavy-edged yellow and red flowers (Anonymous 1956; Satyavati et al. 1976). It is an inhabitant of tropical Africa and now found growing in many countries of tropical Asia including India. In India, it occurs commonly in tropical forests of Bengal and Karanataka (Sivakumar and Krishnamurthy 2002). The advantage of using this species for colchicine extraction is that both its tubers and seeds contain colchicine. Colchicine alkaloid is high in its demand due to its diverse uses (Amoroso 1935). It is well-documented for its uses for treating gout, rheumatism and cancer control (Jana and Shekhawat 2011a; Kala et al. 2004). It is also capable of inducing polyploidy in plants (Ade and Rai 2009). Narender Singh ( ) E-mail:[email protected] Tel: +91 1744 238410 / Fax: +91 1744 238277 The Korean Society of Crop Science

The conventional method of propagation of G. superba through tubers is a commonly followed practice but is considered slow with a poor multiplication ratio of 1: 1 every year (Krause 1986). Its production is seasonal having susceptibility toward many pests (Maiti et al. 2007). In nature, less seed germination with poor viability is responsible for its diminishing population size. The poor propagation coupled with over-exploitation by the local population as well as pharmaceutical companies has put this plant into acutely threatened species. So, it has been affirmed as an endangered plant by IUCN Red Data Book (Sivakumar and Krishnamurthy 2000). Tuberization in many plant species represents the most important physiological process for reproduction and survival (Ovono et al. 2007). Root tubers are typical vegetative plant propagules, which can survive in dry or cold conditions as dormant organs (Sarkar 2008). Storage and transport of

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tubers are also easier, facilitating germplasm exchange across national borders (Mantell and Hugo 1989). In vitro methods of propagation provide an alternate and effective means for rapid multiplication of species by the continuous production to meet the demand for commercial exploitation (Yadav and Singh 2012). In vitro tubers can be planted directly in the soil without acclimatization. The success of in vitro regeneration depends on a series of stages, each with a specific set of requirements (Fernie and Willmitzer 2001). Some of these include day-length (Vaillant et al. 2005), temperature (Grison 1991), presence or absence of growth regulators either singly or in combination (Ovono et al. 2009) and presence of sucrose concentration (Forsyth and Van Staden 1984). Tissue culture-derived material provides an industrial source of different necessary metabolic compounds which are of medicinal value (Kumar et al. 2011). Although attempts have been made by several workers for in vitro studies and biosynthesis of secondary metabolites from this highly valuable endangered medicinal plant species (Ghosh et al. 2002, 2006; Hassan and Roy 2005; Sivakumar and Krishnamurthy 2000, 2002, 2004), but considerable efforts are still required to make it more economical and practical. In the light of the above-referred importance and demand, the present investigation was undertaken on G. superba to evaluate the various other factors such as sterilization treatments, growth regulators, sucrose concentration and photoperiod influencing the different stages of rapid in vitro multiplication and to analyze the content of colchicine in ex vitrogrown plants.

Materials and Methods Explant source and sterilization study Mature, non-dormant tubers of G. superba plants procured from Ch. Devi Lal Herbal Garden, Chuharpur, YamunaNagar, Haryana (India) were dissected into small portions (1 - 1.5 cm) and surface sterilized with Tween-20 (2 drops per 100 mL water) for 7 min followed by washing under running tap water for 10 min. In order to solve the serious contamination problem encountered in tuber culture, different concentrations of HgCl2 (0.05 - 0.15% w/v) were tested at three selected exposure timings (2, 5, and 8 min). The tubers were washed thoroughly with sterilized distilled water thrice.

basal media consisted of MS macro and micro salts, 3% sucrose and 0.8% agar (w/v) (Himedia, India). The pH of the medium was adjusted to 5.8 prior to adding agar. The medium was autoclaved for 20 min at 1.5 kg cm-2 at 121°C. All aseptic manipulations were carried out under a laminar airflow chamber. All the cultures were maintained by sub-culturing at 4 week-intervals on the same medium and culture conditions. All cultures were incubated at 24 ± 1°C under 16 h photoperiod with a photosynthetic photon flux density (PPFD) of 40 µmol m-2 s-1 with 60 - 70% humidity.

Influence of different sucrose levels and photoperiod duration on in vitro tuber induction In order to investigate the effect of different sucrose levels on microtuberization development, 2, 3, and 4% (w/v) of sucrose was incorporated into the MS media containing 2.0 mg L-1 BAP and 0.5 mg L-1 NAA keeping all other parameters (hormones, pH, etc.) constant. Different durations of photoperiod, 3 light/dark cycles, i.e. 12/12, 16/8 and 20/4 h were also tested to find out their optimum level for in vitro tuberization. The percentage of explants producing shoots, number of shoots per explants, average shoot length, number of microtubers and length of microtubers produced per explants were recorded after 9 weeks of culture.

In vitro rooting and acclimatization For in vitro root induction, individual shoots (2 - 3 cm long) were excised from the shoot clump and transferred to half-strength MS medium (3% sucrose and 0.7% agar) with different concentrations and combinations of NAA and IBA (0.5 - 2.0 mg L-1). The cultures were maintained under the same conditions as for shoot induction. When adequate rooted shoots were obtained, the plantlets were thoroughly washed to remove the adhering agar particles and transferred to thermocol cups containing sterilized soil and sand mixture (3:1). The cups were covered with polyethylene membranes to ensure about 80% relative humidity. The potted plants were irrigated with ¼ MS basal salt solution devoid of sucrose and myo-inositol every 3 days. After about 4 weeks, these plants were transferred to larger pots and were maintained under glasshouse conditions. Data were recorded in percentage of rooting, mean number of days required for root initiation and mean length of roots after 5 weeks of transferring onto rooting medium.

Culture conditions

Extraction and quantification of colchicines from tubers

The explants were then trimmed gently with a sterilized blade to remove the sterilizing agent affected brown parts and inoculated in modified MS (Murashige and Skoog 1962) medium supplemented with different concentrations (0.5 - 2 mg L-1) of 6-benzylaminopurine (BAP) alone and in combination with 0.5 mg L-1 α-naphthalene acetic acid (NAA). The

Extraction and HPLC analysis of colchicine from the tubers of acclimatized (ex vitro) and wild plants were carried out, according to the protocol described by Alali et al. (2004). All chemicals and reagents used were of HPLC grade. Identification of colchicine was done by comparing the retention time of the sample with that of the standard colchicine.

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For HPLC analysis, an Agilent Technologies, 1200 series liquid chromatogram system comprising a quaternary pump with a XDB-C18 column (150 mm X 4.6 mm X 5 µm) was used. A mixture of acetonitrile and 3% acetic acid (60:40) at a 1 mL min-1 flow-rate was used as the mobile phase. All the samples were filtered through 0.22 µm filter, 20 µL of samples were injected and the chromatograms were recorded at 245 nm.

Statistical analysis Each experiment consisted of 10 replicates with one explant per culture tube and was repeated thrice. Data were analyzed for significance using one-way analysis of variance (ANOVA) and the differences contrasted using a Duncan’s multiple range test (DMRT) at P ≤ 0.05. All statistical analyses were performed using the SPSS (version 11.5).

Results and Discussion The first and vital condition for the success of a culture is surface sterilization of explants, minute error can lead to loss of whole culture with wastage of time and labor (Chawla 2003). Tubers are difficult explants to disinfect. The sterilants are also toxic to the plant tissues, hence proper concentration of sterilants, duration of exposing the explant to the various sterilants and the sequences of using these sterilants has to be standardized to minimize explant injury and achieve better survival (Yadav and Singh 2011). In the present study, various concentrations of mercuric chloride (0.5, 1.0, and 1.5%) for different durations (3, 5, and 8 min) were used to standardize the best sterilization protocol for in vitro culture of G. superba. Of the various pretreatments tried, 0.1% HgCl2 for 5 min gave the maximum survival percent (Fig. 1). Lower concentration of HgCl2 (0.05%) was found less effective with maximum percentage of contamination. Increasing exposure time and concentration significantly reduced the contamination, but was found deleterious with very less viability (Table 1). HgCl2 has been very effectively used for surface sterilization for different explants of several important medicinal plant species (Goyal and Bhadauria 2006). Ang and Chan (2003) used 0.08% (w/v) HgCl2 to obtain aseptic Spilanthes acmella nodal segments efficiently and 0.1% (w/v) HgCl2 was used by Tiwari et al. (2000) for Centella asiatica axilary buds sterilization. Cytokinins are the most important phytohormones involved in the regulation of storage organ formation by promoting cell division in the growing tuber (Sarkar 2008). BAP has been one of the most successfully used cytokinin for in vitro tuberization in several species (Medina et al. 2009; Omokolo et al. 1995, 2003; Poornima and Rai 2007). Initially, all treated cultures showed no significant difference with an apical bud formation on the surface of the tuber explants, which slowly elongates into aerial shoot (Fig. 1A).

Fig. 1. In vitro propagation of Gloriosa superba. A: Apical bud initiation on MS medium + BAP (2.0 mg L-1), B: Formation of aerial shoot from apical bud, C: Microtubers and shoots developed on MS medium with BAP (2.0 mg L-1), BAP (1.0 mg L-1) + NAA (0.5 mg L-1) and BAP (0.5 mg L-1), respectively, after 5 weeks, D: Microtuber formation on MS medium with BAP (2.0 mg L-1) + NAA (0.5 mg L-1) under 3, 4 and 2% (w/v) sucrose concentration, respectively, after 4 weeks, E: In vitro-derived plantlet with shootlets, tubers, and rootlets, F: Regenerated plantlets transplanted to greenhouse. Bar = 1.0 cm and it represents the length of the plant.

Table 1. Effect of different concentrations with varying time of exposure of HgCl2 on contamination, aseptic culture and survival percentage on tuber explants of Gloriosa superba recorded after 4 weeks of culture on MS medium containing 2.0 mg L-1 BAP + 0.5 mg L-1 NAA Concentration of mercuric chloride (HgCl2 w/v) 0.05 %

0.1 %

0.15 %

Exposure time (min)

Survival (%)

2 5 8 2 5 8 2 5 8

0e 6.66de 46.66b 20.0cde 90.0a 80.0a 26.66bcd 76.66a 40

Contamination Aseptic (%) culture (%) 93.33e 80.0de 43.33b 66.66cd 10.0a 6.66a 56.66bc 3.33a 0a

6.66e 20de 56.66b 33.33cd 90.0a 93.33a 43.33bc 96.66a 100a

Values represent mean ± standard error, n = 30. Means were compared by using the least significant difference (LSD) test (P ≤ 0.05). Data within each column followed by dissimilar letters differ significantly at P ≤ 0.05.

But the elongation of the aerial shoots from the tuber explants differed with different treatments. Later, when the aerial shoot attained maximum height, one globular bulb-like structure developed at its base near the junction of shoot and the explants which ultimately turned into tubers. The buds showed elongation at varying rates with varying concentrations of BAP (Fig. 1B). The MS medium supplemented with BAP (2.0 mg L-1) + NAA (0.5 mg L-1) took the shortest time (6.33 days) for bud break and favored maximum bud break (90%). The medium supplemented with BAP (0.5 mg L-1) showed the lowest percent of bud break (40%) with the maximum days (14.66) to sprout. The rate of shoot induction and

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Table 2. Effect of various concentrations of BAP alone and in combination with NAA on shoot formation and microtuber induction per explant in Gloriosa superba after 9 weeks of culture Plant growth regulators concentration (mg L-1) BAP

NAA

0.5 1.0 2.0 1.0 2.0

0.5 0.5

Bud break (%)

Number of days required for bud break

Number of shoots per explant

Shoot length (cm)

40 53.33 70 76.66 90

14.66 ± 3.11d 10.75 ± 2.08c 9.66 ± 2.33bc 8.39 ± 1.97b 6.33 ± 1.68a

1.16 ± 0.38c 1.31 ± 0.47c 1.85 ± 0.57b 2.13 ± 0.86b 2.81 ± 0.83a

6.05 ± 0.35e 7.82 ± 0.59d 10.29 ± 0.74a 8.67 ± 0.40c 9.14 ± 0.33b

No. of in vitro tubers Length of each per explant tuber (cm) 0.66 ± 0.49d 1.43 ± 0.72c 2.04 ± 0.58b 2.39 ± 0.78b 3.2 ± 0.80a

0.65 ± 0.07e 1.47 ± 0.08d 2.30 ± 0.07a 1.50 ± 0.14c 1.73 ± 0.07b

Values represent mean ± standard error, n = 30. Means were compared by using the least significant difference (LSD) test (P ≤ 0.05). Data within each column followed by dissimilar letters differ significantly at P ≤ 0.05.

shoot length increased with an increase in BAP concentration. The globular structures that developed from the mother explant tissue were also able to induce in vitro tubers directly (Fig. 1C). The NAA concentration tended to favor microtuber production (Table 2). BAP at low concentration (0.5 mg L-1) could result in formation of only one single shorter shoot with the smallest-sized tuber or no tuber at all, whereas the medium with BAP (1.0 mg L-1) showed medium-sized shoots (7.82 cm) and yielded medium-sized tubers. The largestsized microtubers were obtained on media with hormonal supplements of 2.0 mg L-1 BAP. The microtubers produced were initially light green in color but later turned yellowish brown. The inner part of the microtuber was white. The globular bulb-like structures further elongated to form V-shaped structures after 7 weeks in culture. The data presented in Table 2 indicate that the highest shoot induction frequency with maximum number of tubers was obtained on media with 2.0 mg L-1 BAP and 0.5 mg L-1 NAA. However, BAP and NAA combination also promoted vigorous rooting in more than 70% of the regenerated shoots along with small greenish-white tuberous growth at the origin of the roots. The results obtained are in accordance with the findings that the interaction of auxin and cytokinin is necessary for plant in vitro organogenesis and cytokinins of high concentrations and auxins of low concentrations are prerequisites for differentiation of adventitious buds (Chen et al. 2007). Similarly, Behera et al. (2009) reported that the combination and interaction of BAP and NAA are effective inducers of microtuberization. The auxin NAA regulates not only vegetative growth but also organ growth, whereas the cytokinin BAP facilitates cell division and sprouting (Pan 2001). Sucrose is the most widely used carbohydrate source in plant tissue culture media and seems to be the most critical stimulus for in vitro tuber formation (Lawrence and Barker 1963). Our earlier finding (Table 2) revealed that MS media supplemented with 2.0 mg L -1 BAP + 0.5 mg L -1 NAA showed the best result on tuber production. Hence, only this concentration was considered for the present study. The earliest sprouting was observed after 4 days of inoculation on MS medium having 4.0% sucrose. More than 60% of the tubers sprouted after 1 week and after 9 days the maximum (90%) number of tubers had sprouted. The reduction of the sucrose level in media not only reduced the percent sprouting but

also reduced the tuber size. On the media with 3.0% sucrose content, the first sprouting was also observed after 4 days and the sprouting percentage after 4 weeks was limited to 90%. Only 70% of the explants showed tuber formation after 4 weeks in the presence of 2.0% sucrose. Accordingly, in the presence of 2.0% sucrose levels in the culture medium, a decrease of the tuber length (1.2 cm) and the tuber number (1.8) was observed in comparison to in vitro tuberization on 4.0% sucrose medium (Table 3; Fig. 1D). On the other hand, same MS medium supplemented with 3% sucrose stimulated profuse microtuber initiation. As suggested by Jo et al. (2009), sucrose may be essential as an osmoticum, as an energy source and at higher concentrations it may have a role as a signal for microtuber formation. Aksenova et al. (2000) reported that cytokinins and sucrose at high concentrations stimulated tuber initiation. A positive effect of concentration of sucrose on size and weight of microtubers has also been observed in Dioscorea nipponica (Chen et al. 2007), Dioscorea cayenensis (Ovono et al. 2009) and Habenaria bractescens (Medina et al. 2009). Photoperiod plays an important role in the tuberization process and should be optimized to enhance tuber size (Bernier and Perilleux 2005). Photoperiod affects plant growth due to photosynthesis and photomorphogenesis both in vivo and in vitro (Pierik 1967). In vitro tuberization potentiality of G. superba was also influenced by photoperiod. Due to the best response of MS medium supplemented with BAP (2.0 mg L-1) + NAA (0.5 mg L-1) at 3.0% sucrose, it was used to study the effect of different photoperiods on in vitro microtuberization (Table 4). The number of days required for bud break was the lowest Table 3. Effect of different levels of sucrose on in vitro tuberization on MS medium containing 2.0 mg L-1 BAP + 0.5 mg L-1 NAA after 9 weeks Sucrose concentration

Bud break (%)

Number of days required for bud break

No. of in vitro tubers per explant

Length of each tuber (cm)

2% 3% 4%

70.0 90.0 90.0

12.25 ± 2.51b 6.33 ± 1.68a 7.44 ± 1.91a

1.81 ± 0.65c 3.2 ± 0.80a 2.48 ± 0.65b

1.20 ± 0.07c 1.73 ± 0.07b 2.72 ± 0.08a

Values represent mean ± standard error, n = 30. Means were compared by using the least significant difference (LSD) test (P ≤ 0.05). Data within each column followed by dissimilar letters differ significantly at P ≤ 0.05.

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Table 4. Effect of different photoperiods on in vitro tuberization on MS medium containing 2.0 mg L-1 BAP + 0.5 mg L-1 NAA at 3% sucrose after 9 weeks Photoperiod (light/dark)

Bud break (%)

Number of days required for bud break

No. of in vitro tubers per explant

Length of each tuber (cm)

12/12 16/8 20/4

70.0 90.0 90.0

9.38 ± 1.71b 6.33 ± 1.68a 6.14 ± 1.68a

1.9 ± 0.66b 3.2 ± 0.80a 2.6 ± 0.67a

1.61 ± 0.15c 1.73 ± 0.07b 2.23 ± 0.10a

Values represent mean ± standard error, n = 30. Means were compared by using the least significant difference (LSD) test (P ≤ 0.05). Data within each column followed by dissimilar letters differ significantly at P ≤ 0.05.

Table 5. Root formation on different concentrations of IBA alone and in combination with NAA (0.5 mg L-1) in Gloriosa superba after 6 weeks Media composition NAA MS IBA strength (mg L-1) (mg L-1) Full Half Half Half Half Half Half Half

0 0 0.5 1.0 2.0 0.5 1.0 2.0

0 0 0 0 0 0.5 0.5 0.5

Rooting (%)

Days required for root induction

Extent of rooting

26.66 33.33 66.66 56.66

26.0 ± 1.06d 24.9 ± 0.56c 20.2 ± 1.19a 21.6 ± 1.00b

+ ++ +++ +++

- : No Response The number of ‘+’ sign donates extent of rooting +: fair rooting; ++: good rooting; +++: extensive rooting Values represent mean ± standard error, n = 30. Means were compared by using the least significant difference (LSD) test (P ≤ 0.05). Data within each column followed by dissimilar letters differ significantly at P ≤ 0.05.

Table 6. Comparison of colchicine content (% dw) in the tubers obtained from in vivo and micropropagated plantlets of Gloriosa superba Treatments

Cochicine contents (% dw)

Tubers derived from wild plants Tubers derived from micropropagated-raised plants

0.08b 0.10a

Values represent mean ± standard error, n = 3. Means were compared by using the least significant difference (LSD) test (P ≤ 0.05). Data within each column followed by dissimilar letters differ significantly at P ≤ 0.05.

(6.14 days) under 20 h photoperiod and it was the highest (9.38 days) under 12 h photoperiod. The highest number of tubers per explant (3.2) was under 16 h photoperiod while the average length of microtuber (2.23 cm) was higher under 20 h photoperiod. Seabrook et al. (1993) observed that the longer the photoperiod was, the better the tuberization was. There are contradictory reports on photoperiod requirements for in vitro microtuberization formation. Several authors studied the effects of light on in vitro tuberization in the presence of PGRs in the medium and found that environmental factors had permitting effects rather than regulating ones during tuberization and the PGRs applied in the medium had a regulating role (Charles et al. 1992; Pelacho and MingoCastle 1991; Perl et al. 1991). The requirement of different photoperiods may be potentially attributed to the different genetic makeup of the species.

Production of plantlets with profuse rooting under in vitro is an important step for successful establishment of regenerated plants in soil (Ohyama 1970). Roots were visible after 19 days; however, the results were recorded after 4 weeks. Out of all combinations tried for root induction, the best response (66.6%) was obtained with ½ MS with 1.0 mg L-1 IBA and 0.5 mg L-1 NAA (Fig. 1E; Table 5). A lower percentage with fair extent was also observed on ½ MS with medium 2.0 mg L-1 IBA alone. Use of auxin/s singly or in combination for in vitro rooting has also been reported (Rai 2002; Sahoo and Chand 1998). Similar responses were observed in different plant species like Gymnema sylvestra (Komalavalli and Rao 2000) and Anethum graveolens (Jana and Shekhawat 2011b). After successful hardening, plantlets were transferred to greenhouse with 70% establishment (Fig. 1F). The applied HPLC analysis of methanolic extracts of tubers of micropropagated plants along with wild plants showed the presence of colchicine at the same retention time (1.5 min) as that of the standard reference colchicine. Concentration of colchicine varied among the different tested samples. Sharp symmetrical peaks were recorded in all the treatments. The colchicine content in the tubers of G. superba micropropagated plants was found to be significantly higher than those from the wild field plants (Table 6). Accumulation of most of the secondary metabolites appears to be developmentally regulated (Bhojwani and Razdan 2005). Cytokinins have been reported to stimulate alkaloid synthesis in cultures from different plants also (Hara et al. 1994). The concentration of the carbon source also affects the cell growth and yield of secondary metabolites in many cases (Robins et al. 1990). Thus, this protocol produced 2 - 4 tubers with healthy green shoots in the course of 4 - 5 months from a small portion of tuber, while each tuber produces only 2 tubers per year in vivo. In conclusion, the present study on the optimization of cultural conditions for micropropagation of G. superba may be highly useful for raising quality planting material for commercial and off-season cultivation which will not only help in ex-situ conservation but also in the restoration of the genetic stock of the species.

Acknowledgements The authors are grateful to Kurukshetra University, Kurukshetra for providing laboratory facilities and other institutional support. Funding for this work came from the University Grant Commission (UGC), New Delhi, India in the form of a Major Research Project. Thanks are owed to Prof. O.P. Arora (Institute of Pharmaceuticals Studies), Prof. R.K. Sharma and Satender Yadav (Department of Zoology) Kurukshetra University, Kurukshetra for assistance with HPLC analysis.

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In vitro Tuberization in Gloriosa superba L.

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