Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components period of co ld pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore divis ion and the yields of microspore-derived calli. The best results were obtai ned by pretreating genotype G459 newer buds at 4 degrees C for 7-9 days, co -culturing anthers with shed microspores for 14 days and including 6% sucro se, 2 mg l(-1) alpha-naphthaleneacetic acid and 1 mg l(-1) N-6-benzylaminop urine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6-21% and 3.9-8.0% of mic rospore-derived calli produced shoots and embryos, respectively, and ultima tely plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid.