Agrobacterium-mediated transformation of rice flowering locus T1 gene in MR219 rice shoot apex

Abstract

Rice is one of the staple foods that contributes significantly to global food security. In Malaysia, rice occupies the commanding height of staple food basket. However, local rice production fails to satisfy domestic demand as domestic expansion of rice production is hampered by the low yield of Malaysian rice, its long reproductive phase and poor amenability to genetic transformation. Biotechnology methods employed to improve the Malaysia rice proved unsatisfactory due to its recalcitrance to transformation. This study sought to address these challenges by identifying the most efficient and rapid methods for indica rice transformation up to transgenic recovery. For this, RFT1 gene was isolated from Malaysia upland rice (cultivar Wai) and constructed into pCAMBIA1305.2 expression vector. The construct was further mobilized into Agrobacterium tumefaciens LBA4404. MR219 Malaysian wetland rice was used for shoot apex induction. Five-day-old shoot explants were used for transformation with A. tumefaciens LBA4404 harbouring pCAMB::RFT1 construct. The transformants were regenerated and analyzed for transient expression of RFT1, hpt and GUS. Stable integration of transgene and GUS were validated by PCR amplification and histochemical analysis. The RT-PCR and bioinformatics analyses showed that full-length RFT1 gene was isolated and shared 99% nucleotide and 80% amino acid identity with other rice cultivars. pCAMB::RFT1 construct was successfully developed and transformed into MR219 wetland rice. Optimum shoot apices regeneration frequency of 71.64±0.74% was recorded in MS medium supplemented with 3 mg/L TDZ and genetically transformed. The molecular analysis of the transgenic rice confirmed the integration of the RFT1 transgene, hpt and GUS gene into the T0 plant. Histochemical and PCR analyses of established transgenic MR219 also confirmed the presence of the transgenes. Total transformation efficiency was recorded in the range of 12.5±5.37% to 17.5±4.91%. This is the first report of full-length RFT1 gene isolation and transformation into MR219 shoot apex. Findings from this study could serve as a new procedure for genetic manipulation and a fundamental stage for producing transgenic rice.