In-planta Agrobacterium-mediated Stable hva1 and EPSPS Integration into Potato var. Agria Genome

Abstract

BackgroundAfter wheat, maize, and rice, potato is not only an important food crop but also a substantial source of income throughout the world. Developing a practical and effective transformation method for cultivars that are recalcitrant in tissue culture is vital. Hva1 encodes the protein of the LEA III superfamily that involves in reactions to abiotic stresses, which holds considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.ResultsHere, a protocol has been designed for an Agrobacterium-mediated transient transformation in tissue culture-independent conditions in-planta. The protocol establishes for hva1 and EPSPS transformations by direct injection of the bacterial suspension into the potato tuber sprout to encode resistance to cold and against glyphosate herbicide. A two-stage selection was involved using 1% and 2% Glyphosate to eliminate the chimeric and non-transformed plants. Ultimately, the protocol enabled confirmation of gene integration into the plant, transgene expression of the gene and transgene expression, which was made possible by competitive PCR reaction, RT-PCR, and ELISA, respectively. In this research, the transformation efficiencies acquired in potatoes (up to 46%) were higher than those reported using conventional Agrobacterium-mediated approaches in previous studies. ConclusionsThe constitutive expression of the integrated T-DNA neither slowed down the growth rate nor affected potato tuberization significantly. The hva1 gene was expressed successfully leading to the accumulation of the hva1 protein in transgene-generated tubers. This study is the first report on a successful transformation of potato in-planta whereby Agrobacterium can be directed at potato seed sprouts through injection.