Field Evaluation and Genetic Stability Assessment of Regenerated Plants Produced Via Direct Shoot Organogenesis from Leaf Explant of an Endangered 'Asthma Plant' (Tylophora indica) Along with Their In Vitro Conservation

Adventitious shoot buds were induced directly without intervening callus phase from the cut edge of the 'aged leaves' explants as well as via nodular meristemoids from the cut edge of the 'young leaves' of Tylophora indica an endangered medicinal climber of the family Asclepiadaceae. Shoot primordia developed directly from the cut edge of the 'aged leaf' explant without any callus phase within 26-30 days and maximum 18.6 +/- A 0.36 shoot per explant were induced on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 6-benzyl-aminopurine (BAP). Whereas maximum 25.2 +/- A 0.57 shoots per explant were produced from 'young leaf' explant via nodular meristemoids on 2.0 mg/l BAP and 0.2 mg/l indole-3-acetic acid fortified medium within 40-45 days. Multiplied shoots were successfully rooted with maximum 8.3 +/- A 0.36 root per shoot with 15.5 +/- A 0.44 cm length on half strength MS medium supplemented with 0.2 mg/l indole-3-butyric acid. Finally, the rooted plants were hardened and transferred to soil with a 93.3 % success rate. After 18 months of field transfer, a 69.0 % of regenerated plants produce flowers and 28.5 % produce fruits. Seeds of these fruits show 78.3 % viability more or less similar with in vivo grown plants with 71.7 % viability on in vitro germination, which ensures the fertility of the regenerants. These regenerated plants were conserved in vitro for up to 21 months without sub-culturing in presence of osmoticums (Mannitol and Sorbitol) and best results found on MS medium supplemented with 3 % sucrose and 2 % mannitol, where 53.3 +/- A 3.33 % plants survive up to 21 months and 86.7 +/- A 3.33 % of these survived plants could turn to normal plants after a re-growth for 2 month which showed no obvious difference in morphology. Further, clonal fidelity assessment of the micropropagated plants was carried out using cytological studies as well as using randomly amplified polymorphic DNA (RAPD) markers analysis. All the micropropagated plants have shown normal diploid 2n = 22 chromosomes, same as that of the mother plant. A result of RAPD markers analysis indicates all the regenerants are true-to-type and there were no somaclonal variations among these regenerants.