Tissue culture and transformation for introducing genes useful for fungal diseases management in J-104 rice cultivar
2009
Pérez, M., Centro de Ingeniería Genética y Biotecnología, Sancti Spíritus (Cuba) | Hernández, C., Centro de Ingeniería Genética y Biotecnología, Sancti Spíritus (Cuba) | Barceló, M.T., Centro de Ingeniería Genética y Biotecnología, Sancti Spíritus (Cuba) | Armas, R., Centro de Ingeniería Genética y Biotecnología, Sancti Spíritus (Cuba) | Delgado, M., Centro de Ingeniería Genética y Biotecnología, Sancti Spíritus (Cuba)
J-104 is the main commercial rice cultivar in Cuba. Fungal diseases affect its crop and production. Breeders have developed some strategies to control fungal attack, but a higher level of resistance is needed. Pathogenesisrelated proteins ©¬-1.3-glucanase and chitinase are components of defence mechanisms for protecting plants against fungal pathogens. In this research it was established in vitro culture of J-104 rice cultivar and its transformation with chitinase-glucanase genes. It was proved different concentrations of 2.4-D, agar, proline and glutamine, in culture media for callus induction from seeds and plant regeneration. The highest frequency of callus induction was obtained with 2.5 mg/L, 2.4-D and 0.8 percent of agar. Addition of proline and glutamine promoted callus growth. The effect produced together suggested that amino acid interaction was involved in this instance. An efficient regeneration process was perceived using 1.3 percent of agar. An average of nine plants per callus in a month was obtained. Morphological characteristics of regenerated plants revealed the simultaneous occurrence of indirect somatic embryogenesis and organogenesis. For genetic transformation it was used Agrobacterium tumefaciens strain EHA 105 with pCAMBIA 1300 harbouring chitinase gene from bean, ©¬-1,3-glucanase gene from tobacco, and hpt gene as selectable marker. Calli were immersed in bacterial suspension for 10 min, followed by co-culture of three days on culture medium with 100 ¥ìM acetosyringone, at 20 deg C. Calli selection was conducted using 50 mg/L hygromycin during 15 days. Almost 40 percent of calli were hygromycinresistant, and they were transferred to regeneration media. PCR analysis was performed to confirm the presence of foreign genes in all regenerated plants.
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