Radio-Sensitivity Assessment of In Vitro Tissues of Stevia (Stevia rebaudiana Bert.) for Induced Mutagenesis

In vitro mutagenesis approach using gamma irradiation has its proven advantages over conventional breeding methods, since the same exhibits more potential for mutation induction (with desirable traits) with minimal negative effects, within a short time-span, in various plant species. Present study reports an assessment of sensitivity of stevia to gamma irradiation in order to optimize the irradiation doses [median lethal dose (LD 50 ) and median growth reduction dose (GR 50 ) along with LD 25 , LD 75 , GR 25 , and GR 75 ] for induced mutagenesis. Nodal segments from in vitro-regenerated stevia shoots were exposed at six different doses of gamma irradiations (5, 10, 15, 20, 25, and 30 Gy). The irradiated nodal segments were then cultured on Murashige and Skoog basal medium supplemented with 1.5 mg/L meta -Topolin and 1 mg/L indole-3-butyric acid along with the control (non-treated) explants for 3 weeks to assess the effect of irradiation on multiple shoot–root formation. Upon exposure to different gamma ray doses from 5 to 30 Gy, a gradual and morphological trait-specific differential decline of in vitro growth was detected. The individual as well as the cumulative impact of gamma irradiation doses on the growth and development traits were assessed using hierarchical clustering heat map (based on ward distance matrix) and principal component analyses. In addition, based on the probit analysis on trends of gamma irradiation effect, the LD 25, 50, 75 values were calculated to be 9.1, 18.2, and > 30 Gy, respectively. On the other hand, GR 25, 50, 75 values were calculated to be in between 7.1–12.6 Gy, 15.8–21.3 Gy, and 25.5 to way beyond 30 Gy, respectively. On studying the response of all the in vitro growth traits, it was deduced that in order to induce desirable mutations and also to develop novel mutants with adequate survival rate, the optimum irradiation dose (from LD 25, 50, 75 or GR 25, 50, 75 values) should be calculated based on LD 50 /GR 50 , which was determined to be 15–20 Gy in stevia. Hence, this optimum dose can be utilized to produce a higher percentage of beneficial mutations, resulting in maximal desirable genetic diversity in M 1 V 2 and its subsequent generations.