Tanasiienko I. Genetic transformation of barley (Hordeum vulgare L.) by human lactoferrin gene

The results of callusogenesis and the study of the regenerative potential of commercial spring barley cultivars are representative in this thesis. Variants with the best characteristics of above-mention positions was selected for the further work on genetic transformation. It is well known fact that Agrobacterium tumefaciens is not a natural tool for the monocots transformation. Thus, a range of additional physical factors such as vacuum-infiltration, sonication and their combination were tested for Agrobacterium-mediated transformation of barley calli. The 1,35-fold increase of transient GUS expression after sonication of callus tissue during 1-3 s was reveled. In turn, vacuum-infiltration induced almost two-fold increase of GUS expression. Moreover, the combination of sonication and vacuum infiltration led to 2,6-fold increase in transient transformation efficiency as compared to control. Thus, optimal conditions for transient expression of reporter gus (uid A) were developed. Moreover, barley mature embryos were selected for Agrobacterium-mediated transformation and F1 generation of transgenic plants was obtained. Detection of appropriate mRNA confirmed the expression of human lactoferrin gene in the transgenic plants. In addition barley embryogenic callus culture was used for the biolistic transformation. The present study demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation. Biolistic plant transformation and selection system have been developed utilizing a mutant ?-tubulin gene as a selective marker. The pHLFTuBA vector carrying hLF and mutant ?-tubulin gene from goosegrass (Eleusine indica) conferring resistance to dinitroaniline herbicides were created for barley transformation. Trifluralin, the main representative of dinitroaniline herbicides, was used foe selection of the transgenic tissues in concentration 10 mM. mRNA analyses of F1 generation of the transformed barley lines with a specific probe to hLF gene were performed to confirm the transgenic nature of the obtained barley regenerants. Band specific for hLF gene was identified in all transformed plant lines. Therefore, the stable integration of the hLF gene into the plant genomes was confirmed. Moreover, it was clearly demonstrates that using of plant mutant alpha-tubulin as a selective gene for plant transformation was compatible to utilize of antibiotic resistance genes.