Regeneration of stevia rebaudiana and analysis of somaclonal variation by rapd

The objectives of this experiment were to develop the optimal concentration of auxin and cytokinin for regeneration of Stevia rebaudiana and finally analysis somaclonal variation by RAPD. Various degrees of callus induced from the leaf segments cultured on MS medium supplemented with the different concentrations and combinations of NAA+BA and 2,4-D +BA. Among them. 1.5 mg L-1 NAA with 1.0 mg L-1 BA was the best for callus induction (91.67%) which also produced highest fresh weight (621.7 mg) and dry weight (79.00 mg) of callus. For shoot formation, calli were transferred on to MS medium supplemented with different concentrations and combinations of BA and NAA with control. The highest number of shoots (2.17) and the highest average length of the shoot (3.22 cm) per culture was observed at 1.8 mg L-1 of BA with 0.12 mg L-1 of NAA. The regenerated shoots were then transferred to MS liquid medium supplemented with same concentration of IBA and NAA. All the treatments produced roots and 1.50 mg L-1 IBA produced highest percentage of root (93.33%), but 1.00 mg L-1 NAA produced highest number (no. 7.66) of roots and highest length of roots (13.33 cm) per culture. The regenerated plantlets were successfully transferred into pots containing 75% soil and 25% sand and finally transferred into the field. Apparently somaclonal variations were examined among regenerated plants along with mother plant by RAPD. DNA samples from mother plant and 9 randomly selected regenerated plants were subjected to RAPD analysis. Bands generated through RAPD-PCR were scored according to whether they were present (1) or absent (0) to determine the extent of somaclonal variation. The estimation of genetic similarity coefficient based on RAPD band-sharing data analyzed indicated that some regenerated plants were 100% similar to the mother plants and some were 71, 57 or 14% similar may be due to variation in vitro condition. © 2009 Asian Network for Scientific Information.