A fast and effective protocol for obtaining genetically diverse stevia (Stevia rebaudiana Bertoni) regenerants through indirect organogenesis

Abstract

Plant regeneration through indirect organogenesis allows obtaining genetic variability that can be used in the creation of new cultivars. The study presents a fast and effective protocol of one-step preparation of stevia (Stevia rebaudiana Bertoni) regenerants. To obtain callus tissue and shoot regeneration, leaves and nodal segments were used as primary explants, which were placed on MS (Murashige and Skoog) medium supplemented with plant growth regulators (PGRs): NAA (1-naphthaleneacetic acid – 2.0 mg·dm–3, BA (6-benzylaminopurine – 4.0 mg·dm–3), 2,4‑D (2,4-dichlorophenoxyacetic – 2.0 mg·dm–3). Callus tissue was formed on both types of explants, however, only those derived from nodal segments were proliferating. An average of 3.92 shoots per explant were obtained from leaf explants on the applied medium after 6 weeks of culture. The analysis of the morphogenetic capacity of the obtained regenerants was carried out on MS medium supplemented with PGRs – kinetin (0.25 mg·dm–3), BA (0.5 mg·dm–3). The evaluation of the mean number of shoots, mean shoot length (cm), and the mean number of nodes per shoot indicated phenotypic variability of regenerants. The use of RAPD (randomly amplified polymorphic DNA) markers confirmed the differences also at the DNA level. The proposed one-step indirect organogenesis regeneration protocol induced somaclonal variation of Stevia rebaudiana Bertoni and the obtained regenerants, after selection, could be used in the breeding of this species.

Keywords

• micropropagation
• molecular markers
• RAPD
• shoot regeneration
• somaclonal variation