Efficient plant regeneration from protoplasts of Kalanchoe blossfeldiana via organogenesis

A simple and efficient protocol for plant regeneration from protoplasts of the potted plant Kalanchoe blossfeldiana Poelln. is reported. Mesophyll protoplasts were isolated from axenic leaves after a preculture. The enzymatic digestion of the tissue with a solution containing 0.4% Cellulase Onozuka R-10 and 0.2% Driselase yielded 6.0 x 10(5) protoplasts per gram fresh weight after density gradient purification. Protoplasts were cultured in the dark at an initial density of 1 x 10(5) protoplasts per milliliter in a liquid medium with 320 mM mannitol, 130 mM sucrose, 2.3 mu M 2,4-dichlorophenoxy acetic acid (2,4-D), 5.4 mu M 1-naphthaleneacetic acid (NAA) and 2.2 mu M 6-benzyladenine (BA). Cell wall regeneration was observed within 4 days of culture and cell division began after 5-7 days. When cultured in a liquid medium with 5.4 mu M NAA and 8.9 mu M BA, protoplast-derived colonies proliferated until small visible calli, and adventitious buds appeared after transfer to photoperiod conditions. Developed shoots were rooted on a solid medium supplemented with 0.6 mu M indole-3-acetic acid (IAA) and successfully established under greenhouse conditions. The process required 4 months from isolation to rooted plants and the best conditions found gave a plant regeneration efficiency of 6.4 plants per 1 x 10(5) protoplasts. This is the first protocol reported for plant regeneration from protoplasts for a Crassulaceae family species.