Plant Transformation With Osmotine And Defensines Genes

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Shadenkov A.A., Lunin V.G., Kovaleva M.V., Kolobova O.S., Michajlyuk T.A., Kolesnikova S.V., Varochobin S.N., Sharova A.P., Davydova Yu.V., Avetisov V.A.

All-Russian Institute of Agricultural Biotechnology,
Moscow, Thimirjasevskaja str. 42, Russia

The transformation of the plants are conducted in plant genetic engineering laboratory using different methods. Genes transfer in tomato is carrying out using pollen as 'supervector'. In these experiments exogenous plasmid pK22Ap24 DNA or Agrobacterium tumefaciens strain LBA4404, containing this plasmid were applied to flower pistils after artificial pollination. Plasmid pK22Ap24 (size about 9,5 t.p.n.) contains Ap24 gene of fungicidal taumatin-osmotin-like protein of the tobacco under the control of 35S promoter and terminator of VI CaMV gene in a structure of vector pH22Kneo. The pH22Kneo vector was constructed on the basis of the broad-host-range plasmid RSF1010. The sites of pRSF1010, carrying genes of resistance to streptomycin, in pH22Kneo were replaced on plant marker gene nptll conferring resistance to aminoglycoside antibiotics. Thus, pH22Kneo vector is capable of providing the transfer of gene Ap24 in plant genome and selection of transgenic plants. Development of the fruits on experimental plants is being observed at present. The peptide antibiotics ?defensines are also characterized by wide fungicidal activity. Potato root, leaf and stem explants were transformed with defensine gene rs of Raphanus sativus with the purpose of creation plants more resistant to phytopathogenic fungi. This gene was under the control of 35S- promoter in strains LBA4404 and LGV3850 A.  tumefaciens and A4 A.  rhizogenes. nptll was used as a marker gene. Kanamycin resistant regenerants were obtained and now are checked for presence of rs-gene in genome.