An efficient Agrobacterium-mediated genetic transformation method for foxtail millet (Setaria italica L.)

A simple and robust Agrobacterium-mediated gene expression system in the C4 panicoid model crop, foxtail millet has been developed with up to 27 % transformation efficiency. Foxtail millet (Setaria italica L.) is a model crop to study C₄ photosynthesis, abiotic stress tolerance, and bioenergy traits. Advances in molecular genetics and genomics had identified several potential genes in this crop that would serve as candidates for imparting climate-resilient traits in related millets, cereals, and biofuel crops. However, the lack of an efficient genetic transformation system has been impeding the functional characterization of these genes in foxtail millet per se. Given this, an easy and efficient regeneration and transformation protocol was optimized using mature seeds as a choicest explant. The suitability of secondary embryogenic calli over primary calli is underlined due to their high competence. The use of perfect combinations of plant growth regulators together with the ionic strength of organic and inorganics salts was found to influence regeneration and genetic transformation. We studied and optimized various crucial factors that affect the genetic transformation of foxtail millet calli using Agrobacterium tumefaciens-mediated approach. Secondary embryogenic calli and LBA44404 strain were found to be the best targets for transformation. The use of high sucrose and glucose, together with freshly prepared tobacco leaves extract, Silwet L-77 and acetosyringone, improved the efficiency of the genetic transformation of foxtail millet. Moreover, the use of an in vitro regeneration system with 84% callusing efficiency and 70-74% regeneration frequency led to a high recovery of transformants. Altogether, the present study reports a highly efficient (~ 27%) transformation system in foxtail millet that will expedite forward and reverse genetic studies in this important crop.