Physical and chemical mutagenesis in Stevia rebaudiana: variant generation with higher UGT expression and glycosidic profile but with low photosynthetic capabilities
2016
Khan, Shamshad Ahmad | Rahman, Laiq ur | Verma, Rajesh | Shanker, Karuna
The leaf explants of Stevia rebaudiana were exposed to a range of EMS (chemical mutagen) and gamma radiation (physical mutagen) doses. Among all the treatments, doses of 0.4 % v/v EMS and 0.95 KR gamma radiation were found to be most effective treatment for selection of variants via direct shoot bud induction. The field-established plants designated as EMS (E), gamma (G), and control (C) plants were subjected to photosynthetic parameter studies where E and G plants showed lower chlorophyll content, transpiration rate, CO₂ exchange rate, and stomatal conductance. The implanted field soil at the time of harvesting of plants showed reduction in organic carbon (OC), electrical conductivity (EC), phosphorus (P)/potassium (K), and total Kjeldahl nitrogen (TKN). The ISSR profiling of E, G, and C scored total of 107 bands out of which 63 % were polymorphic bands, and plant E was found to be more distant from the C plant phylogenetically. On phytochemical analysis, the G plant registered twofold enhanced rebaudioside A with lower stevioside (3.2 ± 0.22 % dry wt.) content, whereas E plant showed more than 1.5- and 2.0-fold increase in stevioside and rebaudioside A as compared to control plants. Both the E (3.1 ± 0.15 % dry wt.) and G (2.3 ± 0.21 % dry wt.) plants registered lower steviol. The enhanced steviol glycoside profile was supported by the RT-PCR analysis of UGT74G1 and UGT76G1 that corresponds to stevioside and rebaudioside A biosynthesis, respectively. While the E plant showed 5–6-fold increase in the UGT74G1 (RQ = 5.51 ± 0.5) and UGT76G1 (RQ = 6.61 ± 0.5) gene expression, the G plant showed 5-fold increase in UGT76G1 (RQ = 5.29 ± 0.2) gene expression.
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