Tomato Agrobacterium – Mediated Genetic Transformation with Mouse Metallothionein Gene
Nora Gorinova, Velichka Rodeva, Stanislava Grozeva, Milka Nedkovska and Elena Yordanova
Induction of changes by genetic trans formation with mouse metallothionein gene mMTF1 for tolerance to heavy metals, inherited in tomatoes, was studied on leaf explants of two Bulgarian tomato lines – XXIV A and N 120. It was established that a pre-cultivation of the explants on initial MS basal medium supplemented with 10.74 µM a-naphtalene acetic acid (NAA) and 0.89 µM 6-benzyladenine (BA) for 24 hours was an important step for obtaining a higher percentage of regenerated explants after treatment with Agrobacterium tumefaciens -49.2% for line XXIV A and 6.7% for line N 120. It was found that the optimal bacterium dilution was 1:15 (vol/vol), at 10 min optimal duration of the treatment. The period of co-cultivation with A. tumefaciens for 48 hours was adapted. Later on the inoculated leaf explants were transferred to selective medium, containing MS macro- and microelements, vitamins B5, 8.90 µM BA, 1.14 µM indole-3-acetic acid (IAA), kanamycin (100 mg l -1) and claforan (500 mg l -1 ), for induction of organogenesis. Only in the studied tomato genotype XXIV A was achieved 7.7% transformation efficiency. The presence of mouse metallothionein gene (mMTF1), responsible for immobilization of heavy metals, was proved by PCR analysis.