Agrobacterial transformation of spring rape without phase of regeneration in vitro
Bohulska C. V., Postgraduate, , Uman National University of Horticulture, Ukraine
Transformation of spring rape with the help of soil bacterium Agrobacterium tuimfaciens by inplanta method is conducted. It’s generally known that Agrobacterium is capable to infect dicotyledons and some of monocotyledonous plants and to cause formations of specific tumours —crowns galls. In connection with these features, agrobacterium strated to use in the genetic engineering as a vector for the transfer of heterogenous DNA. To create such vector with the help of genic-engineering methods, was cut out from it the areas encoding formations of tumours and with the help of Ecoli embedded given genes. Becoming numb important is the origin of promoters to provide the expression of built-in transgene.
For transformation of spring rape used Agrobacterium tuimfaciens with plasmids that contains gene-bar (bialaphos resistance), that determines stability to herbicide of "Basta" the operating substance of which is phosphinothricin. Also plasmids carries the marker genes of stability to the antibiotics and put under promoters 35S CaMV of virus of cauliflower mosaic.
As a plant recipient the varieties of a spring rape were taken: Aydar, VNIS 100 and Dobrobut.
Upbuilding of Agrobacterium and subsequent treatment of flowering plants of rape was produced in the field terms. Received seeds with bacterial treatment plants sowed in soil in spring and the selection of steady forms was made by sprinkling herbicides with the operating substance of phosphinothricin. Resistent form received: 5 plants of sort Aydar, 7 plants of sort VNIS 100, 9 plants of sort Dobrobut.These plants was covered with insulators for self-pollination and receiving of seed which is sown in the soil in spring. After appearance of sprouts, the treatment of a spring rape plants with herbicide was made. The received forms saved stability to herbicide. Almost all plants of these varieties are resistant to phosphinothricin.
Substantial advantage of this method is the absence of the stage of regeneration in vitrо. And also its easiness in use and small expenses.
agrobacterial transformation, spring rape, T-DNA, inplanta