Improvement of rice transformation using bombardment of scutellum-derived calli

Although several reports on rice transformation have been published, producing transgenic plants of Indica rice varieties is still problematic. We report an improved protocol for transforming Indica rice genotypes. An important agronomic Mexican Indica rice variety, Morelos A-92, was used. Calli derived from scutellum seeds were produced by using auxins and bombarded with 2 vectors, one harboring the reporter UidA gene and the other with the hptII gene conferring hygromycin resistance. The influence of the molar relation of these vectors (uidA-hptII) in generating callus and plants expressing the uidA reporter gene was analyzed. Selection of bombarded calli was performed under 2 conditions: 50 mg/L of hygromycin with 3 subcultures and 80 mg/L of hygromycin with no subcultures. The best conditions were a 20:1 uidA-hptII molar vector relationship and selection at 80 mg/L of hygromycin, producing 14% of calli expressing GUS. The minimal callus size in regenerating plants was 3 mm. Transformed rice plants were generated with 4.6% efficiency, considering the initial number of bombarded calli. Heredity of the uidA gene behaved as a single locus in transformed rice plants. Homozygous plants were identified in the T, generation by means of pollen staining.