Improved Agrobacterium-mediated transformation of Carica papaya cultivar ‘Kapoho’ from embryogenic cell suspension cultures

Abstract

The adoption of Agrobacterium-mediated transformation to agronomically important Carica papaya cultivars and genotypes, such as “Kapoho’, has been challenging. To address this problem, an alternative transformation protocol was developed for papaya using embryogenic suspension-derived cultures. The ability of the tissues (cultivar ‘Kapoho’) to regenerate plantlets from these cultures was demonstrated for both transgenic lines and untransformed controls. Suspension-derived cultures at the young globular calli stage contained embryos highly receptive to Agrobacterium infection and did not require the problematic wounding treatments inherent in other protocols. The optimum parameters selected to obtain transgenic calli were as follows: 1-d co-cultivation with Agrobacterium, the cell density used for transformation was A₆₀₀ of 0.005, and elimination of Agrobacterium post-transformation was done using 250 mg/L carbenicillin and cefotaxime. Putative transgenic calli were confirmed positive for the presence of the eGFP transgene, Cp45 promoter sequence, and hygromycin resistance gene (hptII) using PCR. The presence of eGFP messenger RNA (mRNA) and protein were detected using reverse transcription PCR (RT-PCR) and Western blot analysis, respectively. Visualization of qualitative eGFP fluorescence in roots, stems, and leaves further confirmed the expression of Cp45:eGFP fusion in the transformed papaya plantlets. This technique serves as an alternative and efficient method to generate transgenic plants in a simple laboratory setup that facilitates Agrobacterium-mediated transformation of previously difficult papaya cultivars and genotypes.