Studies on the N_2-laser microbeam system for efficient gene delivery and the utilization of this system for plant protection

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An N_2-laser with a 337.1 nm wavelength and 3 ns pulse width burned localized hole ca.0.5 mum in diameter on the cell wall and cell membrane of cultured cells of melon or tobacco under selected irradiation conditions (1.5-6 muj/spot). No visible damage due to the irradiation was detected under these conditions. Fluotescent-isothyocyanate (FITC) was used to determine the osmotic value of the medium surrounding cultured cells and 0.3 Molarity was the best to get 66.3 % of cells which showed fluorescence. Efficiency of gene delivery into plant cells was tested using this N_2 laser treatment. Mature pollen of lmpatiens balsamina, I.walleriana and Lycoris albiflora was irradiated with a laser at 3muj/spot in medium containing 0.3 M mannitol and plasmid PBI221 harboring beta-glucuronidase. The maximum frequency of transient gene expression was 24% in these treatments. It is concluded that the osmotic value of medium surrounding the pollen grains is extremely important for the GUS gene epression to use N_2-laser microbeam and it is able to introduce any gene into pollen exactly one by one. When the melon cotyledons were treated with a laser in the presence of cucumber mosaic virus RNA,virus multiplication was observed in the epidermal and around the cells 24 hr after irradiation. These results suggest that the present laser method is useful for the transformation of plant cells. Resistance in the African wild plant, Cucumis figarei Des.et.Noude to CMV was found to related to restricted virus movement, and the rates of cell-to-cell and long distance movement in C.figarei is regulated by sequences in CMV RNA 3. A cDNA clone encoding the 3a and coat protein gene of CMV (pepo strain), linked to the cauliflower mosaic virus 35S promotor, was introdeced into tobacco by N_2-laser microbeam and the transgenic tobacco plants expressing the CP gene were protected from infection with CMV.