CULTURE AND REGENERATION OF PROTOPLASTS FROM SHOOTS OF ASPARAGUS CULTURES

A reliable culture and regeneration protocol has been established for asparagus (Asparagus officinalis L.) protoplasts isolated from shoots of in vitro plants. Studies Focused on factors to maximize plating eff iciency, colony formation, and plant regeneration. The optimized proto col involved the culture of asparagus protoplasts embedded in agarose beads through a series of defined media. Etiolated shoot cultures were a superior source for protoplasts than green shoot cultures, and grow ing feeder cells were critical for high plating efficiency and colony formation. In the presence of growing asparagus feeder cells, asparagu s protoplasts initiated cell divisions within 2 d of plating in agaros e with plating efficiencies up to 20%. About 80% of the protoplasts in itiating cell divisions developed into colonies of at least 25-30 cell s, of which 25% initiated somatic embryos. Complete plants were regene rated from the embryos at a frequency over 20%. Overall, ca. 0.8% of t he initially isolated protoplasts regenerated into complete plants wit h this protocol. Plants regenerated from protoplasts have been transfe rred to soil and maintained phenotypically normal morphology for over 2 yr. Chromosome counts confirmed the diploid status (2n=20) of plants regenerated by somatic embryogenesis.