CONSTRUCTION OF A NEW VECTOR CONFERRING METHOTREXATE RESISTANCE IN NICOTIANA-TABACUM PLANTS

A new binary vector encoding for Candida albicans dihydrofolate reduct ase (DFR1) has been constructed and used as a dominant selectable mark er for plant transformation. Transgenic tobacco plants with an increas ed resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotian a tabacum were directly selected for and rooted on medium containing b oth kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assa yed for capacity of regeneration at different Mtx concentrations. Anal ysis of transcripts was performed on total RNA extracted from two Mtx- resistant plants. The transgenic plants increased resistance to Mtx ca n be explained by the exceptionally low capacity of Mtx to bind C. alb icans dihydrofolate reductase, accountable by the presence of two amin o acid residues strategically important in Mtx binding.