Culture and regeneration of protoplasts from shoots of asparagus cultures

Abstract

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 efficiency, colony formation, and plant regeneration. The optimized protocol 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 growing feeder cells were critical for high plating efficiency and colony formation. In the presence of growing asparagus feeder cells, asparagus protoplasts initiated cell divisions within 2 d of plating in agarose with plating efficiencies up to 20%. About 80% of the protoplasts initiating cell divisions developed into colonies of at least 25-30 cells, of which 25% initiated somatic embryos. Complete plants were regenerated from the embryos at a frequency over 20%. Overall, ca. 0.8% of the initially isolated protoplasts regenerated into complete plants with this protocol. Plants regenerated from protoplasts have been transferred 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.