Regeneration of rice double haploids using a onestep culture procedure

Abstract

Summary Double haploid plants of rice (Oryza sativa L.) were regenerated using two protocols. In protocol 1, anthers from the variety Colonia Mascias 5 MAG were cultured on basal N6 and B5 medium. Both media were supplemented with 5% sucrose and different combinations and concentrations of NAA, 2,4-D, KIN and BAR. All media tested induced calli after 15 d in culture, which appeared only from pollen grains. The best response was obtained with N6 + 2.0 mg·L−1 NAA + 0.5 mg·L−1 KIN. Two types of callus arose from the explants: embryogenic and non-embryogenic. The first type was firm, white, and had a nodular surface. Scutella with coleoptile-like leaf structures at different stages of development were observed. After 30 d in culture, calli were transferred to the light. Within 3 d, shoots turned green from more than 85% of the explants that had produced white shoots. These shoots continued growing and produced a good root system. In protocol 2, pieces of callus, obtained in the same medium used in protocol 1, were subcultured on basal medium MS (3% sucrose) with different concentrations and combinations of NAA, BAP, and KIN. Maximum shoot regeneration was achieved with MS + 0.1 mg·L−1 NAA + 1 mg·L−1 BAP, but root development was poor. Shoots without roots were then transferred to basal medium without hormones plus 8% sucrose; roots then formed and tillering occurred within 15 d. Protocol 1 was successful with seven additional rice varieties. Plants from protocol 1 and 2 were successfully established in soil.