Electroporation
Electroporation (using electric field) is now used to transfer the foreign DNA into the fragile cells. Brief pulses of high voltage electricity (about 350 V) is applied to protoplasts suspension containing naked or recombinant plasmids. The electric pulses induce the formation of large pores in the cell membrane. These pores give a passage through which the foreign DNA can enter into the protolasts and thus, increase the transformation frequency. The transformed protoplasts are cultured for about one month, These develop microcalli which are plated on solid medium containing selective marker (e.g. kanamycin). After 37-45 days, the calli are analyzed for the presence of differences in transformed cells.
By using electroporation method, genes of choice have been successfully transferred in protoplasts of wheat, rice, petunia, sorghum, maize and tobacco. Moreover, transformation frequency can be improved by using linear DNA instead of circular one, by giving heat sock to protoplast at 45°C for 5 minutes, by adding PEG and using 1.25 kV/cm voltage. |
