An Efficient Micropropagation Protocol for Rauvolfia hookeri Srinivas and Chithra and Assessment of Clonal Fidelity by RAPD Analysis

An efficient in vitro propagation protocol via shoot multiplication was developed for Rauvolfia hookeri, a rare medicinal species endemic to the southern Western Ghats of India. Shoot tip explants cultured on half strength Murashige and Skoog medium supplemented with various cytokinins (6-benzylaminopurine, 6-furfurylaminopurine and Thidiazuron) either alone or in combinations produced multiple shoots. When either cytokinin was used alone, 6-benzylaminopurine was found nearly twice more successful than 6-furfurylaminopurine. The highest shoot proliferation was obtained when 6-benzylaminopurine and 6-furfurylaminopurine was used at 22.2 μM and 4.64 μM respectively. Thidiazuron gave the lowest response for shoot proliferation. The effect of indole-3-butyric acid and naphthalene acetic acid was evaluated for in vitro root induction. Rhizogenesis of excised shoots was of the shoots was readily achieved on half strength Murashige and Skoog medium containing various concentrations of indole-3-butyric acid and naphthale ne acetic acid. Indole-3butyric acid was found to be more effective than naphthalene acetic acid and resulted in the highest frequency of shoots that rooted (86.5%) and mean number of roots per shoot (3.66) when used at 7.38 μM concentration. The micropropagated plants were hardened and transferred to green house condition wherein 70% plants established and were morphologically similar to mother plant. Genetic stability of regenerated plants has been checked by Random Amplified Polymorphic DNA using ten selected decamer primers.

In vitro propagation of an endemic medicinal plant R. hookeri was attempted for the first time for conservation purpose

Shoot tip explants responded well in half strength MS medium augmented with BAP

BAP at 22.2μM concentration in combination with 4.64μM KIN induced maximum shoots per shoot tip explants (about 5 shoots)

Further shoot proliferation was achieved in the same hormonal combination (BAP and KIN) and yielded enough shoots for rooting

Rooting frequency and mean number of roots per shoot was high in ½ MS medium supplemented with 7.38 μM IBA

In vitro regenerated plants were successfully transferred to field condition and 70% of them were established

Genetic purity of the established plants were assessed by PCR based RAPD analysis and found that all are identical to the mother plant

The protocol developed was reproducible and can be utilized for the conservation of this endemic plant and can generate about 100 true-to-type plants within ten month culture period