In vitro plant regeneration of Stevia rebaudiana through indirect organogenesis

Stevia rebaudiana plants produce non-caloric sweeteners that can be used as an alternative to sugar and also synthetic sweetening agents. Under natural conditions, seed germination is poor, and propagation through stem cuttings has limitations due to poor rooting. In the present study, a protocol was developed to obtain healthy plants of S. rebaudiana through organogenesis via callus cultures. Freshly collected seeds were surface sterilized using 0.2% Carbendazim with 5 minutes exposure time and 10% Clorox with 10 minutes exposure time. Seeds were then cultured on MS medium supplemented with 3.0 mgL-1 GA3 to obtain to In-Vitro germinated seedlings. Leaf discs (1cm2) and nodal explants (1 cm) were taken from 2 weeks old seedlings and cultured on MS medium supplemented with different concentrations and combinations of BAP and NAA. MS medium supplemented with 2.0 mgL-1 BAP and 1.0 mgL-1NAA was found to be the best medium for callus induction. To determine the best explant source, leaf discs, intermodal and nodal segments from seedlings were cultured on best callus induction medium, and leaf disc was found to be the best explants type for callus initiation. Leaf derived calli were transferred into shoot induction media and MS medium supplemented with 0.5 mgL-1 NAA and 2.0 mgL-1BAP was found to be the best medium for shoot induction. Shoots more than 5.0 cm in length were transferred into root induction media and half-strength MS medium supplemented with 0.5 mgL-1 IBA was found to be the best medium for root induction.