Synergetic effect of TDZ and BA on minimizing the post-exposure effects on axillary shoot proliferation and assessment of genetic fidelity in Rauvolfia tetraphylla (L.)

The stimulatory effect of different concentrations of thidiazuron (TDZ) on in vitro bud induction and proliferation of a potent medicinal woody shrub R. tetraphylla were investigated. Multiple shoots were induced from nodal explants on Murashige and Skoog medium augmented with different concentrations (0.2-1.0 mu M) of TDZ. Continuous exposure beyond 5 weeks of singly supplemented TDZ resulted in hyperhydricity, fasciation and distortion of cultures. To avoid these deleterious effects, benzyl adenine (BA) was supplemented to the media. It not only mitigated the negative effects of prolonged TDZ exposure, but also enhanced shoot multiplication rate. About 30 shoots per explant with an average shoot length of 5.8 cm were obtained after 20 weeks of culture on MS medium fortified with 0.4 mu M TDZ and 2.0 mu M BA, regeneration frequency was also enhanced by 24% on this media as compared to singly supplemented TDZ. Microshoots (similar to 5 cm) were successfully rooted on filter paper bridge in MS liquid medium augmented with different concentrations of indole-3-butyric acid (IBA) after 4 weeks of incubation. Best rooting response (80%) was achieved on MS medium supplemented with 0.3 mu M IBA with (9.3 +/- 1.45) mean number of roots and (3.3 +/- 0.33 cm) root length per shootlet. The in vitro raised healthy plantlets with well developed root and shoot were successfully hardened off and acclimatized in thermocol cups containing sterilized planting substrate (R) for 8 weeks under culture room conditions prior to field transfer and successfully established in earthen pots with 90% survival rate. The genetic fidelity of in vitro regenerated plantlets was evaluated using random amplified polymorphic DNA markers. No polymorphism was observed in banding pattern among micropropagated and the donor plant, thus, exhibiting their genetic uniformity and clonal fidelity.