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Rakesh Verma, Dr. Dilip Gena and Dr. Bhanwar Lal Jat*


Stevia rebaudiana is a self-incompatible plant. So it cannot pollinate regularly and not produced viable seed. The seed of Stevia are very small and have very little endosperm. By these reasons the germinated seedling plant not survives. The objective of present investigation has been to derive the method of mass propagation by developing efficient for Stevia rebaudiana. The germination rate of seed can be low except half the seed slouch not to germinate. It is propagated vegitatively. Through stem cutting are used for vegetative propagation. Tissue culture plants have proven to be best planting material for stercia. Tissue culture plants of Stevia are genetically pure, free from pathogen and have exillent vigour. Systematic approaches was made in order to achieve multiple shoot induction from node of mature and juvenile stem explants of Stevia rebaudiana NAA (0.1 mg-1) with BAP 2.0mgl-1) in MS medium was found optimum for induction of multiple shoot buds (14-18) from explant. The incorporation of NAA (0.1 mgl-1) in medium not caused multiplication of the shoot but also affected the subsequent rooting of the In vitro raised shoots of Stevia rebaudiana. The shoot buds excised from explant after 4 weeks were subcultured on MS medium containing NAA (0.1mgl-1) with various concentrations of BAP. Amongst all the concentration of BAP tried, 0.5 - 2.0 mgl-1 was found the best for further multiplication of shoots. On this medium each shoot bud formed 25-28 shoots within a week. In a period of 4 weeks newly produced shoots attained a length of 2-3 cms and could be transformed for rooting. In Stevia rebaudiana the original mature explant produced 14-18 shoot-buds on MS + 2.0 mgl-1BAP + 0.1 mgl-1 NAA. Each of the In vitro regenerated shoot buds subcultured on fresh medium proliferated into 25-28 shoots in one week. The same explants could be subcultured on fresh medium for five times and after each subcultured it produced 20-28. In the nodal explant produced 10-12 shoots and the same explant after excision of regenerated shoots was subcultured to fresh MS medium containing 1.0 mgl-1BAP + 0.1 mgl-1 IAA where it again produced 15-20 shoots with in a period of 10 days. This explant could be sub-cultured six times after every 25-30 days, on fresh medium. The formation of new shoots from subcultured explant and development of older shoots took place simultaneously. In Stevia rebaudiana the callus was raised from leaf and juvenile segments. This was achieved on MS medium containing 2.4-D (1.0-2.0 mgl-1) or NAA (1.0- 5.0 mgl-1). These calluses multiplied rapidly in the same medium on surface and margins of callus. Before the formation of these structures the callus became hard. For the callus culture on MS medium containing 1.0 – 2.0 mg/lit concentration of 2,4-D was found to be best for induction of callus.The rooting of In vitro shoots was achieved on MS ½ strength salt medium. Various media tried for rooting of shoots of these plants were white’s, B5, WP, and MS dilution of ¾. ½, ¼ salts. Although rooting was observed in all the media with or without growth regulators in Stevia rebaudiana rooting was obtained only on MS medium. In Stevia rebaudiana 6-7 sturdy fast growing roots per shoot with lateral roots were produced on medium containing 0.5 mgl-1IBA+0.1 mgl-1 NAA. These roots grow up to a length of 5-10 cms in 5-6 weeks. This medium is optimum for ideal rooting in the aseptically grown shoots of Stevia rebaudiana. The MS full strength medium containing 1.0 mgl-1IBA was found the best for regeneration if roots with 8-10 cms length in 6 weeks in Stevia rebaudiana. In Stevia rebaudiana strong 7-8 roots withfast growth (8-10 cms in 6 weeks) were formed from each shoot MS ½ strength salt +0.5 mgl-1IBA. Lowering of agar-agar from 0.8-0.6 percent improved rooting of shoots and subsequent growth of roots. Higher concentration of agar may be inhibitory to some growth factors of cultures. The better growth of roots can be explained by easy penetration of the growing roots in the medium due to less solidification.

Keywords: In vitro, NAA, BAP, 2,4-D, IBA,Relative Humidity (RH).

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