The introduction of pre-culture, washing and pre-selection stages enhances rice transformation efficiency by reducing Agrobacterium overgrowth

Abstract

Agrobacterium tumefaciens-mediated rice transformation efficiency depends on several factors such as genotype, type of explant, media composition and growth regulators, co-cultivation conditions, antibiotics, temperature, A. tumefaciens strain and their cell density, gene construct, explant competence after wounding, and control of A. tumefaciens overgrowth. Most of these issues have been addressed earlier, but only for a few elite indica rice cultivars like IR64, IR72, Pusa Basmati 1, Samba Mahsuri, etc. Besides these, there are some local cultivars having considerable consumers' preference and farmers' choice. Considering these aspects, the objective of the study was to standardize the callus transformation technique. In our earlier study, a popular rice variety, Khitish (IET 4094) from West Bengal, was found to be the most amenable genotype for indirect regeneration. Hence, this variety was selected for transformation experiments with A. tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA2301. Here, the co-cultivation-based transformation protocol was optimized by introducing several steps such as pre-culture in co-cultivation media before callus infection, washing with antibiotics dissolved in MS instead of water after co-cultivation, and pre-selection with only Agrobacterium elimination antibiotics after washing–drying step. To control the Agrobacterium, a combination of cefotaxime and timentin was used during these steps. This resulted in lowest Agrobacterium overgrowth and highest transformation frequency (28.2% in Khitish). The transient expression and stable integration of foreign gene were confirmed by GUS assay and PCR amplification of nptII selection marker gene in T₀ plant genomic DNA, respectively.