Alginate Encapsulation of Shoot Tips and Their Regeneration for Enhanced Mass Propagation and Germplasm Exchange of Genetically Stable Stevia rebaudiana Bert.

An accelerated, unique and efficient alginate-encapsulation (synthetic seed)-based genetically true-to-type mass propagation system in stevia (Stevia rebaudiana Bert) was successfully developed. Multiple shoot cultures in stevia were initially established from shoot tip explants cultured on Murashige and Skoog (MS) semi-solid medium supplemented with 0.5 mg/l N-6-benzyladenine. Synthetic seeds were developed from individual shoot tips (obtained from in vitro multiple shoots) using 3% (w/v) sodium alginate and 75 mM calcium chloride solutions. In comparison with non-encapsulated in vitro shoot tips, alginate-encapsulated ones (i.e. synthetic seeds) exhibited accelerated germination (in the form of fresh shoot emergence) (similar to 2 days), higher shoot proliferation rate and elongation (9.47 shoots with 10.94 mm length, and 64.33 leaves) as well as quicker root initiation ( similar to 9 days), multiplication and elongation ( similar to 13 roots with 38 mm length) after 30 days of inoculation in filter paper (M-bridge)-immersed half-strength MS liquid medium without plant growth regulator. Non-encapsulated in vitro shoot tips failed to initiate roots and showed reduced shoot growth. Synthetic seed-regenerated complete plantlets were successfully acclimatized in cocopeat. Clonal fidelity analysis of synthetic seed-regenerated plantlets was performed using ten inter-simple sequence repeats primers that exhibited monomorphic banding pattern, ensuring no genetic variations among the plantlets as well as with their mother plant. The present protocol will be beneficial not only for the smooth exchange of germplasms but also as an alternative approach for accelerated-cum-enhanced in vitro mass propagation of stevia.

Automated summary

An accelerated, unique and efficient alginate-encapsulation-based mass propagation system was developed for stevia, which exhibited accelerated germination, higher shoot proliferation rate and elongation, and quicker root initiation and multiplication. Synthetic seed-regenerated plantlets showed clonal fidelity with their mother plant. This protocol is important for germplasm exchange and production of stevia.