Improvement of Plant Regeneration and Agrobacterium-mediated Genetic Transformation Efficiency in Red Clover (Trifolium pratense L.)

Red clover (Trifolium pretense L.) is one of the important forage crops in most temperate agricultural regions. Red clover germplasm has limited genetic variation for disease resistance. Application of an efficient gene transformation protocol to introduce genes against diseases is considered the only approach for developing new resistant cultivars. In this study, in order to establish an efficient regeneration system in red clover, highly regenerative genotypes were utilized for evaluation of different media sequences for their suitability to utilize potentiality of regeneration capacity. Media sequence KBC-SPL-SPL significantly increased the efficiency of somatic embryo genesis of red clover. There was no relationship between regeneration ability and fresh callus weight. Also, no interaction was observed between genotypes and media sequences for fresh callus weight, number of embryos and number of developed shoots. To develop an efficient gene transfer system, some parameters were investigated, including evaluation of suitable concentration of selection agent, examination of the effect of pre-culturing of explants on efficiency of transformation and comparison of different Agrobacterium strains for efficiency of gene transformation. The effect of pre-culturing explants was increased slightly by the frequency of gene transferring. Highly infective Agrobacterium strain C58C1 can increase the transformation efficiency for red clover to 83%. PCR and histochemical GUS assay of transformed plants confirmed successful integration of the T-DNA into the red clover genome. Our established system will be a platform for genetic manipulation of red clover to broaden its genetic variation and for germplasm innovation.