Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal
- 567 Downloads
A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3 μM kinetin (Kn) and lower levels of 6–benzyladenine (BA) (13.3 μM) while multiple adventitious shoot bud differentiation occurred on medium fortified with 2.3 μM kinetin (Kn) and higher levels of BA (22.2 μM). Shoot buds were transferred to proliferation medium containing 2.2 μM BA, 2.3 μM Kn, and 3.9 μM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7 days in a medium containing 71.6 μM choline chloride (CC) and 3.9 μM PG and then transferred to rooting medium containing ½ MS, 1.2 μM IBA, 3.6 μM PAA, and 14.3 μM CC for 3 weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplification of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby confirming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantification through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the field-grown mother plant. Accumulation of withaferin A and withanolide A increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides.
KeywordsMicropropagation HPLC TLC RAPD Withania coagulans Withanolides
Diode array detector
Murashige and Skoog
Random amplification of polymorphic DNA
Thin layer chromatography
Financial support from Council of Scientific and Industrial Research (CSIR) in the form of R&D project: CSIR–38(1178) EMR–II/2007 is gratefully acknowledged. Rohit Jain, Arunima Sinha and Devendra Jain thank CSIR for the award of Senior Research Fellowships.
- Bhandari MM (1995) Flora of the Indian desert. MPS Repros, JodhpurGoogle Scholar
- Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
- Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research. Wiley, New York, p 680Google Scholar
- Jain R, Sinha A, Kachhwaha S, Kothari SL (2009b) Micropropagation of Withania coagulans (Stocks) Dunal: a critically endangered medicinal herb. J Plant Biochem Biotechnol 18:249–252Google Scholar
- Kachhwaha S, Kothari SL (1996) Plant regeneration from immature embryo explants of Hordeum spontaneum and Hordeum vulgare. Cer Res Comm 24:27–32Google Scholar
- Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN, Tuli R (2004) Phytochemical variability in commericial herbal products and preparations of Withania somnifera (ashwagandha). Curr Sci 86:446–461Google Scholar
- Sinha A, Jain R, Kachhwaha S, Kothari SL (2010) Optimization of the level of micronutrient copper in the culture medium improves shoot bud regeneration in Indian ginseng [Withania somnifera (L.) Dunal]. Natl Acad Sci Lett 33:11–16Google Scholar