Plant regeneration from suspension cultured-derived protoplasts of apomictic dallisgrass (Paspalum dilatatum Poir.)

Abstract

Protoplasts were isolated from embryogenic suspension cells of apomictic dallisgrass (Paspalum dilatatum Poir.). The respective suspension cultures were initiated from immature inflorescence-derived embryogenic callus. Previous to protoplast isolation, suspension cells were treated with Murashige and Skoog (MS) liquid medium without sucrose and hormones. Due to this pretreatment protoplast yield and viability were dramatically increased. A maximum protoplast yield of 4–6 × 106· −1 fresh weight was obtained. Cell division and colony formation from pretreated protoplasts were found to be best in an agarose solidified KM8p medium at a density of 5–8 × 105· ml−1. The plating efficiency, based on colony formation after 2 weeks of culture, was 0.5–0.8%. Protoplast-derived colonies were transferred to a solidified MS medium containing 1.0 mg · 1−1 2,4-dichlorophenoxyacetic acid (2,4-D) for callus proliferation. The calli formed embryonic structures which gave rise to green plants in 0.2% (w/v) Gellan Gum solidified MS medium with 1.0 mg·l−1 naphthaleneacetic acid (NAA) and 0.2 mg·l−1 benzylaminopurine (BAP). The regenerated plantlets were transferred to 12 MS hormone-free medium for further growth and root formation. Rooted plants could be transferred to soil.