Preliminary Study on Seed Germination and Callus Induction Optimization in Stevia Rebaudiana Leaf and Internode Explants Using 2,4-Dichlorophenoxyacetic Acid (2,4-D)

Yap, Wen Nie (2026) Preliminary Study on Seed Germination and Callus Induction Optimization in Stevia Rebaudiana Leaf and Internode Explants Using 2,4-Dichlorophenoxyacetic Acid (2,4-D). Final Year Project (Bachelor), Tunku Abdul Rahman University of Management and Technology.

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Abstract

Stevia rebaudiana is a commercially important plant known for producing steviol glycosides, which are widely used as natural sweeteners. The initial aim of this study was to induce polyploidy at the seedling stage using colchicine treatment as a strategy to potentially increase steviol glycoside content. However, seed germination trials conducted on half-strength Murashige and Skoog (0.5 MS) medium and on wetted blotting paper resulted in 0% germination, further reinforcing the well-documented limitation of poor seed germination in Stevia and rendering the original colchicine approach unfeasible. Consequently, the focus shifted toward in vitro callus induction from leaf explants as an alternative platform. Leaf explants were cultured on MS medium supplemented with 2.0, 2.5, and 3.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). The overall induction rate was low, with less than 6% of explants forming callus. Most cultures were compromised by fungal contamination, and although the fungicide pentachloronitrobenzene (PCNB, 25 μg/L) was incorporated into the medium, contamination persisted. Among uncontaminated explants, progressive tissue browning suggested oxidative stress and loss of regenerative capacity. Callus formation was observed only in three explants cultured on 2.0 mg/L 2,4-D and in a single plate of explants at 2.5 mg/L, while no induction occurred at 3.0 mg/L. The calli produced were small and limited in proliferation. These findings indicate that Stevia rebaudiana is highly recalcitrant to callus induction under the tested conditions, with fungal contamination and tissue browning representing major challenges. The results underscore the need for improved sterilization protocols, alternative fungicides or antioxidants, and optimization of hormonal combinations to enhance callus induction. Although induction success was limited, the few calli obtained confirm that Stevia rebaudiana leaves can respond to auxin treatment, providing a preliminary foundation for further refinement of tissue culture protocols aimed at improving propagation and genetic studies of this species.