Optimization of an Enhanced Micropropagation Protocol of Stevia rebaudiana for Mass Production in an Indian Sub-humid Region

Abstract

The present study was an attempt to optimize the regeneration protocols of Stevia rebaudiana (Bert.). Leaf and nodal explants were inoculated in MS medium supplemented with various plant growth regulators in different combinations and concentrations in order to attain direct and indirect regeneration. Among the treatments used, 2 mg/L of indole-3-acetic acid (IAA) + 1 mg/L of 6-Benzylaminopurine (BAP) displayed better results in stevia for callus induction when leaf and nodal explants were used, but in the case of nodal explants, it took more days for callus initiation compared to leaf segment. Among the treatments, 2.0 mg/L of IAA + 1.0 mg/L of BAP were found to be better for callus induction and subsequent plantlets regeneration (66.15%) from leaf explant of stevia. MS medium fortified with 0.5 mg/L of kinetin produced a high regeneration response (46.92%), the highest number of plantlets (40.67%) per explant, and the maximum number of roots (5.00) per plantlet in case of direct in vitro regeneration. While the MS medium fortified with 1.0 mg/L of BAP + 2.0 mg/L of IAA was found to be the best indirect in vitro regeneration of stevia plantlets from nodal explants concerning the highest regeneration response (68.10%), number of plantlets per explant and shoot length. MS medium added with 1.0 mg/L of BAP + 2.0 mg/L of IAA was reported to have the highest root regeneration ability (67.41%) and considerable length of roots for rooting of in vitro derived plantlets of stevia. The Soilrite was the best for acclimatizing in vitro regenerated plantlets of stevia. This is the first successful report on micropropagation in Stevia rebaudiana from sub-Himalayan region of West Bengal, India. A combination of IAA and BAP proved better for direct and indirect organogenesis. The Soilrite media was the best for acclimatization of in vitro regenerated plantlets with the highest (68.57%) survival rate in field conditions. Results of the present study will be useful for mass multiplication of Stevia rebaudiana using direct and indirect in vitro approaches. Moreover, direct in vitro shoot formation from leaves of Stevia rarely reported may open up possibilities of more successful large-scale cultivation of Stevia in the region and elsewhere.