Induced mutagenesis in varietal development of stevia

Abstract

Stevia rebaudiana or Stevia is a highly recommended alternative natural sweetener plant. Being a photoperiod sensitive plant, commercialisation of Stevia in Malaysia may cause higher cost production. Responding to increased interest by Malaysian, many agencies have shown their interest in research and development of Stevia, especially in the development of photoperiod insensitive variety. To develop this new variety, determination of concentration or dose of mutagens used in mutation induction was done, together with the assessment on effects of each mutagens to morphology and germination. Any genetic variabilities were evaluated to complete the objectives of the study and lastly, any mutants with photoperiod insensitivity and other useful traits were selected. For mutation breeding purposes, Stevia genetic variability was induced with methyl nitrosourea (MNU), acute gamma ray and chronic gamma ray in the production of better varieties. Novel approach to induce mutation in Stevia using Methyl nitrosourea (MNU) showed the presence of seedlings with tricots and chlorophyll mutation. Seeds soaked in MNU showed lower percentage of germination and germination rate as compared to control. From this study, for treatment on seeds, the suggested LC50 was 0.75 mM, 0.58 mM, 0.32 mM and 0.35 mM for 15, 30, 45, and 60 minutes, respectively. Meanwhile, the suggested LC50 for treatment on flower buds of Stevia was 0.15 mM. Acute gamma irradiation on the stems of Stevia was done for the first time with recommended LD50 for this treatment was 88.75 Gy. Meanwhile treatment on seeds, the suggested LD50 was 53.95 Gy. In general, the percentage of germination and germination rate decreased when seeds were irradiated with acute gamma rays. Mutation induction on Stevia using chronic gamma irradiation was another approach initiated in this study. Two mutants were developed, the first mutant M1F1V1 (obtained from plant irradiated with 0.01 Gy/h) and the second mutant M1V2 (from plants irradiated with 0.03 Gy/h). M1F1V1 (0.01 Gy/h) had the longest day before flowering (approximately 6 months) compared to control (approximately 43 days). The second mutant, M1V2 (0.03 Gy/h), possessed the largest leaves surface areas (15 cm2) compared to control plants (4 cm2). There was stimulation effect observed in plants irradiated with chronic gamma rays. There were increases in height, number of buds, number of flowers and number of branches, which might happen because of hormesis. Suggested ED50 which was based on the height of chronically irradiated plants was 0.013 Gy/h. Meanwhile, suggested LD50 which was based on the percentage of germination was 0.018 Gy/h. ISSR was used as molecular marker to study the genetic variation between nonmutant and mutant. Among all mutagens, treatment with 0.25mM MNU for 60 minutes was the most effective, and the most efficient mutagenesis treatment was acute gamma irradiation with dose 65 Gy.