Otimização de protocolos para transformação genética de arroz (Oryza sativa L.), mediada por Agrobacterium tumefaciens

Abstract

Rice (Oryza sativa L.) is one of the world's most important food crops, playing a fundamental role in global food security and serving as a staple food for billions of people. The development of efficient tissue culture and genetic transformation protocols is essential to accelerate breeding programs and the development of cultivars with superior agronomic traits. This study aimed to optimize in vitro protocols for rice (Oryza sativa L.), encompassing seed asepsis procedures, morphogenic responses in tissue culture, and bacterial growth parameters of Agrobacterium tumefaciens. In the first experiment, the effects of physical pre-treatments (washing with running water and neutral detergent), sodium hypochlorite (NaClO) exposure time, and post-asepsis drying on contamination and callus induction were evaluated in different rice genotypes. The results showed that the physical washing and drying steps were decisive in reducing contamination, whereas increasing the exposure time to the chemical disinfectant had no consistent effect. Among the evaluated genotypes, CMG 1590 and Nipponbare showed the best performance, with lower contamination rates and greater stability in callogenesis. In the second experiment, the in vitro growth of the Agrobacterium tumefaciens strains EHA105 and LBA4404 was evaluated, and no significant differences in bacterial density were observed, indicating equivalent performance under controlled conditions. Additionally, the response of rice genotypes to callus induction and regeneration was evaluated on N6 medium supplemented with different concentrations (0.0, 1.0, 1.5, 2.0, 2.5, and 3.0 mg L⁻¹) of 2,4- dichlorophenoxyacetic acid (2,4-D). The results demonstrated a concentration-dependent response, with the highest efficiency observed between 2.0 and 2.5 mg L⁻¹, whereas the absence of the growth regulator prevented callus formation, and higher concentrations (above 2.5 mg L⁻¹) reduced the morphogenic response. Statistical analysis confirmed a significant interaction between genotypes and 2,4-D concentrations. It is concluded that the efficiency of in vitro rice protocols depends on the integration of physical asepsis procedures, appropriate hormonal adjustments, and genotype selection, all of which are essential for improving regeneration systems and supporting future applications in genetic improvement.