ABSTRACT
In order to develop a high-efficiency and reproducible regeneration protocol for Stevia protoplasts, various factors such as type and concentration of enzymes, osmoticum, incubation time, plant material type and age were studied. Protoplasts were successfully isolated from leaves of fourweek- old in vitro grown plants using an enzyme mixture comprising of 2% (w/v) Cellulase Onozuka R-10, 1.5% (w/v) Macerozyme Onozuka R-10, 0.2% (w/v) Driselase and 0.1%(w/v) Pectolyase Y- 23 in 0.5 M mannitol, 2.5 mM CaCl2.2H2O and 5 mM 2 (N-morpholino)-ethanesulfonic acid (MES ) at pH of 5.8. Approximately 8.4±0.40x106 protoplasts g-1fresh weight with 98.8±1.39% viability was obtained after incubating in enzyme solution for 4 hours in dark. Viable protoplasts were collected by centrifugation in the presence of 16% sucrose solution. Protoplasts at density of 5x105 mL-1were cultured on modified KM8P medium supplemented with 0.2 mg L-1 2,4-dicholorophenoxyacetic acid (2,4-D), 1 mg L-1 α-naphthalene acetic acid (NAA), 0.5 mg L-1 zeatin, 0.15 M sucrose and 0.3 M mannitol by agarose-bead or thin layer liquid culture technique. The protoplasts regenerated cell walls within 24 hours. First cell division was observed after culturing for 2-3 days and microcolonies were formed within 4 weeks. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Compared to liquid culture, agarose bead culture improved division frequency almost 1.5 times effectively and showing a plating efficiency of 13% and 9.1% respectively with survival rate of 23.5% to 14.8%. Upon transfer to Murashige and Skoog’s medium (MS) with 1 mg L-1BA, alone or in combination with NAA or 2, 4-D at 0.1 mg L-1, protoplast-derived calli produced complete plantlets through somatic embryogenesis in 8-weeks. The regenerated plants survived in soil and all were normal with respect to morphology and growth characters. This protocol might lead to the improvement of the Stevia through somatic hybridization, somaclonal variation and genetic engineering by using protoplast based regeneration System.