Optimization of Agrobacterium tumefaciens- Mediated Transformation Conditions in Coffea arabica cv. Catimor CIFC 7963
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Abstract
Agrobacterium-mediated plant genetic transformation is one of the established methods for plant breeding. In this study, conditions for Agrobacterium-mediated delivery of the GUS reporter gene into Coffea arabica cv. Catimor CIFC 7963 calli were optimized. The calli were cocultivated with Agrobacterium tumefaciens and cultured in a liquid medium for 20 and 24 hours. The Agrobacterium suspensions consisted of three treatments: undiluted (control), 1:5 dilution, and 1:10 dilution. The calli were transferred to MS/2 medium containing 500 mg/L cefotaxime for 1 month. Subsequently, they were moved to MS/4 medium with 17.76 μM BA, 50 mg/L hygromycin, and 250 mg/L cefotaxime for another month. Finally, they were subcultured on MS/4 medium with 17.76 μM BA, 50 mg/L hygromycin, and 125 mg/L cefotaxime for 1 month. The results showed that the survival rate of calli co-cultivated with Agrobacterium cultured for 20 hours was better than that of the 24-hour condition. The friable callus was then selected to co-cultivate with Agrobacterium cultured for 20 hours. The callus survival rates for the undiluted, 1:5 dilution, and 1:10 dilution treatments were 0%, 62%, and 64%, respectively. Notably, somatic embryos developed from the friable callus after the 1:5 dilution treatment. Additionally, GUS expression varied between compact and friable calli. Therefore, co-cultivation of friable calli with a 1:5 dilution of Agrobacterium cultured for 20 hours was considered the optimal treatment, as it enhanced the frequency of putative transformation. This transformation system offers an advantage for improving the development of desired traits in coffee.
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