Regeneration of rice double haploids using a one step culture procedure

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 BAP. 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 multiplied by L super(-1) NAA + 0.5 mg multiplied by L super(-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 multiplied by L super(-1) NAA + 1 mg multiplied by L super(-1) BAP, but root development was poor.