) GUS( و بتا- گلوكورنيداز SN روش تراريزش سريع با كارآيي بالا در يونجه هاي ديپلوييد و تتراپلوييد براي تظاهر ژن هاي

Rapid Transformation Method of Diploid and Tetraploid Medicago with High Efficiency for Expression of Sn and Gus Genes

A simple and efficient method for regeneration --transformation of two Medicago: the perennial M.sativa var.Rgsy27 (2n=4x=32)and the annual M. truncatula var.R108-1(c4) (2n=2x=16) with Sn and gus genes has been hereby described. Here embryo regeneration of 8108-1 to complete plants was further improved by four successive in vitro regeneration cycles resulting in the line R108-1(c4). According to the employed protocol transgenic plants were produced on all leaf explants within 1.5-2 months with the same ploidy levels as before, with their all being fertile. This protocol appears to be the most efficient and fastest reported so far for medicago genus plants. Agrohacterium tumefaciens -mediated transformation of leaf explants was carried out with the plasmid 121.1(Binary vector containing gus gene) and the plasmid 121. Sn was derived from pBI 121.1 where the gus gene was replaced with the full length cDNA of Sn, The Sn gene, a maize bH1.H gene, modulates anthocyanin and condensed tannin pathways. The functions of CT are due to their ability to form reversible complexes with proteins, the formation of these protein-tannin complexes making protein unavailable for microbial enzyme, activating other mechanisms like deprivation of metal ions and inhibition of oxidative phosphorylation which also result in the prevention of foam formation and bloating. For confirmation of transgenic plants, southern blot, RT-PCR, Enzyme assay and hystochemical assay (DMACA) were used. The efficiency of transformation in Rgsy27 plants was dependent on Agrobacterium strains, in contrast the efficiency of transformation in R108-1 (c4) plants did not depend on Agrobacterium strains. The expression of Sn gene on CT levels in leaves and roots of transgenic plants is either suppressed or unsuppressed.