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Stevia rebaudiana is a herbaceous perennial plant with great global demand due to its beneficial steviol glycosides(SGs) content. Current conventional breeding technique is unable to cater the need for more S. rebaudiana planting materials. Therefore, an improved in vitro shoot regeneration protocol was developed for S. rebaudiana by using chitosan. The highest fresh weight of plant (0.586 ± 0.176 g/explant), dry weight of plant (0.056 ± 0.02 g/explant) and plant height (4.94 ± 1.17 cm/explant) with maximum number of leaves (25.33 ± 6.95 /explant) were observed on explants grown in optimun treatment of MS basal medium supplemented with 1.0 mg/L of 6-benzyaminopurine (BAP) and 60 mg/L of low molecular weight (MW) chitosan after 4 weeks of culture. Scanning electron microscopy (SEM) analysis showed that new shoot primordia and shoot bud formation can be seen as early as day 3 and 5 of cultures on optimun treatment. Further biochemical assays showed that total phenolic acid content, total protein content and total hydrolyzed sugar content were recorded higher in explants cultured in optimun treatment as compared to control media. In contrast, total chlorophyll content and total flavonoids content were reduced in optimum treatment. Meanwhile, no significant difference in antioxidant activity was observed. All cultures from the optimal treatment were successfully regenerated, acclimatized and grew well with 100% survival rate. Taken together, an enhanced and efficient shoot regeneration protocol of S. rebaudiana was successfully developed which will be useful for rapid and large-scale micropropagation in future.

Stevia rebaudiana Bertoni from Asteraceae family is commercially valuable for its steviol glycosides (SGs) contents, which is 300 times sweeter than commercial sugar. The bottleneck in Malaysia is the lack of suitable stevia varieties that are able to thrive well under her climatic conditions and still produce high SGs. Mutation induction including gamma irradiation is effective in generating genetic variations and developing new plant varieties with desired traits. This study was aimed to determine the effects of acute gamma irradiation on phenotypic changes and enhancement of SGs contents of Stevia rebaudiana Bertoni variety AKH L1 (herein after will be designated as AKH L1). In vitro shoot tip explants of AKH L1 were subjected to a gamma doses regime of 10Gy to 50Gy, following which phenotypic changes of the irradiated explants and subsequent regenerated plantlets were observed. All irradiated explants exhibited different survival rates, with the lowest at 9.33±8.33% when subjected to 50Gy, while all the control (non-irradiated explants) survived. The LD50 was found to be at 23Gy. Subsequent irradiation of 900 shoot tip explants at 23Gy, produced 468 surviving shoot tips, which were all capable to develop and successfully sub-cultured until the fourth generation, M4. These M4 in vitro mutant plantlets exhibited significant increase in the numbers of leaf (16.07±5.19) and average leaf size (1.12±0.26cm x 0.54±0.15cm). HPLC analysis performed in parallel further revealed the mutant plants contained higher concentrations of stevioside (387.04ppm), rebaudioside A (670.18ppm) and rebaudioside D (106.26ppm) compared to the non-irradiated plantlets, which exhibited 96.87, 194.42 and 28.25ppm, respectively.