Wheat protoplast culture: embryogenic colony formation from protoplasts

Wheat (Triticum aestivum L. cv Chinese Spring) protoplasts were isolated from immature embryos or embryogenic calli (3-4 weeks of culture on MS medium with 32 mg/1 dicamba) and cultured in R2 medium containing 2 mg/1 2,4-D by the nurse culture methods originally developed for rice protoplasts (Kyozuka et al. 1987). Protoplasts isolated from embryogenic calli started to divide within 3-5 days and formed colonies at frequencies up to 2% after 3-4 weeks of culture, while protoplasts isolated from immature embryos formed colonies at much lower frequency (less than 0.1%). Some of these colonies were "embryogenic", and they appeared at a frequency of approximately 0.5% of colonies formed when callus-derived protoplasts were used. From two of those "embryogenic" colonies, calli were regenerated and albino shoots and roots were obtained.