An efficient method for in vitro clonal propagation of Stevia rebaudiana Bertoni accession MS007

Abstract

Stevia rebaudiana, commonly known as Stevia, is an economically important sweetening medicinal herb species from the family Asteraceae. Although this plant is native to Paraguay, many countries including Malaysia have shown interest to cultivate this plant in large commercial-scale. The accession MS007 produced more axillary branching, high leaf number with big sizes which implies good quality sweetening compounds. To overcome conventional propagation method, in vitro clonal propagation is a potential alternative to ensure regular supply of planting material. Therefore, this study aims to explore the possibility of mass producing S. rebaudiana accession MS007 using in vitro clonal propagation technique with two major purposes i.e., to establish an effective surface sterilization protocol for shoot tips and nodal explants of Stevia and to develop multiple shoot induction and plant regeneration. Shoot tips and nodal explants collected from the field were sterilized with 5% NaOCl for 10 minutes with 0.2% carbendazim (fungicide) pre-treatment demonstrated least contamination 16.67% (shoot tips) and 18.89% (nodes). After sterilization, explants were inoculated on MS medium with different concentrations of cytokinins, BAP (0.5-3 mg/L) and Kn (0.5-3 mg/L) either in singly or in combination, and combination with auxin NAA (0.5 mg/L) for shoot induction. The highest frequency (85.19% for shoot tips and 86.67% for nodes) of multiple shoot regeneration with maximum number of shoots (14.30 shoots/explant for shoot tips and 12.77 shoots/explant for nodes) was noticed on MS medium supplemented with 1.0 mg/L BAP. The proliferated healthy shoots (>2 cm) were dissected out from in vitro shoot cluster and cultured on MS medium for root induction either on half strength or full strength MS medium fortified with different concentrations of auxins IBA, NAA and IAA (0.5, 1.0, and 1.5 mg/L). Highest frequency of rooting (92.22% and 91.11%) with highest number of roots (14.92 and 13.82 roots/explant) and length (4.29 and 3.09 cm) from shoot tips and nodes, respectively was noticed on half-strength MS medium augmented with 0.5 mg/L IBA. Finally, the rooted plantlets were successfully transferred into plastic cups containing peat moss and subsequently established in the field and it was noticed that 86.67% of plantlets survived which is phenotypically similar to the parental mother plant. This present research offers a reliable method for clonal propagation of Stevia (MS007) for large scale production.