Preliminary Study on Polyploidy Induction and Associated Physiological Stress Responses in Stevia Rebaudiana Seed and Callus Cultures

Ang, Wan Hua (2026) Preliminary Study on Polyploidy Induction and Associated Physiological Stress Responses in Stevia Rebaudiana Seed and Callus Cultures. Final Year Project (Bachelor), Tunku Abdul Rahman University of Management and Technology.

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Abstract

Stevia rebaudiana (Bertoni) is a perennial herb of the Asteraceae family highly valued for its non-caloric steviol glycosides, such as stevioside and rebaudioside A, which are significantly sweeter than sucrose. Despite its high pharmaceutical and commercial potential as a natural sweetener, cultivation is often challenged by low seed viability and slow germination rates. This study aimed to optimize callus induction from internode explants using Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), to evaluate the survival and germination performance of Stevia seeds and calli following colchicine-induced polyploidy and determine the best materials of Stevia plant for polyploidy induction. The methodology involved culturing sterilized, slightly matured internodes on optimized MS media under controlled environmental conditions with a 16/8-hour photoperiod. Polyploidy was induced in seeds using colchicine concentrations of 0.1% and 0.5% for durations of 6 and 24 hours. Concurrently, healthy calli were treated with 0.05% and 0.1% colchicine for 2 and 6 hours. To mitigate post-induction oxidative stress and browning, treated calli underwent a recovery process using an antioxidant solution containing citric and ascorbic acids, followed by transfer to media supplemented with 500 mg/L activated charcoal. Results demonstrated that matured internodes successfully initiated callus formation. For seed germination, the control group reached 32% germination rate, while colchicine-treated seeds showed reductions to as low as 2% at highest concentration with longest exposure time. In contrast, colchicine–treated calli maintained recovery rates between 30 – 60% following antioxidant treatment. In conclusion, the research successfully established an optimized protocol for callus induction and identified the physiological impacts of colchicine–mediated polyploidy induction in Stevia. These findings provide a framework for future efforts to enhance the production of high-value secondary metabolites through polyploid breeding.