Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal

• Published in: Plant cell, tissue and organ culture Vol. 105; no. 1; pp. 135 - 140
• Main Authors: Jain, Rohit, Sinha, Arunima, Jain, Devendra, Kachhwaha, Sumita, Kothari, S. L
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.04.2011; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Benzyladenine; Biological and medical sciences; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Callus; Choline; Deoxyribonucleic acid; DNA; Explants; Fundamental and applied biological sciences. Psychology; Genetic transformation; High performance liquid chromatography; Kinetin; Lactones; Leaves; Life Sciences; Liquid chromatography; Metabolic engineering; Micropropagation; Phloroglucinol; Plant Genetics and Genomics; Plant Pathology; Plant Physiology; Plant Sciences; Random amplified polymorphic DNA; Regeneration; Research Note; Rooting; Shoots; Thin layer chromatography; Withania coagulans; Micropropagation; TLC; Withanolides; RAPD; HPLC; Vegetals; Adventitious bud; HPLC chromatography; Withania coagulans; Lactone; Biosynthesis; In vitro; Random amplified polymorphic DNA marker; Regeneration; Dicotyledones; Angiospermae; Spermatophyta; Solanaceae
• ISSN: 0167-6857; 1573-5044
• DOI: 10.1007/s11240-010-9840-3

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.