A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables

Abstract

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.