Isolated microspore culture of maize: effects of isolation technique, reduced temperature, and sucrose level

Improvements in ab initio microspore culture of maize are presented using a modified isolation technique, reduced temperature during early stages of culture, and an elevated sucrose level in the culture medium. Blending-isolation, using excised anthers, was less stressful on microspores than pressing anthers against a stainless steel sieve and resulted in a 3-fold increase in the yield of embryo-like structures (ELS). Exposure to reduced temperature (15°C) during the first 4 days of culture improved microspore viability and increased by 2-fold the number of ELS produced. Higher levels of sucrose (8.0-9.5%) also resulted in improved response. Maximum yield in the present study was 92 ELS per 100 anther equivalents, exceeding previously reported values of 15 ELS per 100 anther equivalents for ab initio microspore culture of maize. The increase in the total number ELS produced had no observable effect on their quality as evidenced by the frequency of formation of callus capable of regenerating plants.