Micropropagation of Stevia rebaudiana Betroni and assessment of genetic stability of in vitro regenerated plants using inter simple sequence repeat (ISSR) marker

摘要

Stevia rebaudiana Bertoni has become a new sweetener crop in different regions in the world. Stevia produces few seeds, therefore, micropropagation is a prevalent method to obtain sufficient amount of uniform plants. In the present study, in vitro propagation of Stevia was attempted through multiple shoot regeneration from nodal segments cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), α-napthyalene acetic acid (NAA), indole acetic acids (IAA) and thidiazuran (TDZ). The maximum of number axillary shoots per explant (3.24) and highest shoot length (3.12?cm) were observed with MS medium supplemented with 1.0?mg?l?1 BA+0.05?mg?l?1 NAA and 2.0?mg?l?1 BA+0.05?mg?l?1 NAA, respectively. Roots were produced within two weeks and the highest percentage (98.72%) of root induction, the maximum number of roots (9.46 roots/shoot) and root length (9.87?cm) were recorded on MS medium fortified with 0.5?mg?l?1 IAA and 1.5?mg?l?1 IAA, respectively. The ex-vitro plantlets were successfully acclimatized with a survival rate of 70% at the hardening phase. Inter simple sequence repeat (ISSR) markers were used to analyze the genetic stability of micro-propagated and mother plants of Stevia. Four ISSR primers generated clear, distinct and reproducible bands. All ISSR profiles from micro-propagated plants were monomorphic and similar to mother plants, while low variation was induced in the next sub-culture. The results indicated that Stevia plantlets regenerated using micropropagation techniques standardized at our lab were genetically stable especially in the early sub-cultures.