Studies of Somatic Embryogenesis of Stevia Rebaudiana (Bertoni) Bertoni and Clonal Fidelity of Regenerated Plants

Abstract

Stevia rebaudiana (family Asteraceae) is an important medicinal plant mainly known for its non-caloric natural sweetness. It is propagated through seeds and/or cuttings. Seed raised populations are showing variations and cutting raised plants are available in limited numbers. Therefore, a rapid cloning of elite plants is required by the growers. The present study was focused to study the effect of plant growth regulators on somatic embryo induction and shoot organogenesis from leaf explants taken from microshoots of S. rebaudiana for its mass multiplication. Microshoots of the herb growing on basal MS medium have very small rudimentary leaves which may be due to ethylene accumulation in culture vessels. Thus to get better explants for experimentation, microshoots were cultured on medium supplemented with silver nitrate (AgNO₃; 10.0µM). Addition of silver nitrate resulted in expansion of leaves in these explants, which were subsequently used for experimentation. To evaluate the effect of PGRs on shoot organogenesis and somatic embryogenesis, MS medium supplemented with different concentrations of 2,4-D, NAA and BA either singly or in combination with each other. Explants cultured on MS medium supplemented 1µM 2,4-D and 5µM NAA showed maximum (94.44%) explant differentiating into somatic embryos. It was interesting to note that although combination of BA and NAA resulted in shoot organogenesis but no somatic embryo induction was observed on this medium. The developed embryo like structure were further studied through histological and Scanning Electron microscopic (SEM) studies. Histology studies revealed the epidermis layer with actively dividing meristematic pockets and SEM studies revealed different development stages of embryos like globular, heart, torpedo shape of embryos as well. Regenerated plantlets from somatic embryos were checked for any somaclonal variations using PCR based molecular marker such as RAPD and ISSR and these were found true to type.