Hnatiuk I. Genetic engineering techniques application for biotechnological plants obtaining and genome editing of winter rape Brassica napus L.

The aim of thesis research was to develop an effective genetic transformation method of Ukrainian breeding winter rapeseed and to create a genome editing platform in order to improve valuable economic characteristics. The following research methods were used in work: analysis of scientific papers; laboratory methods (in vitro plant tissue culture, Agrobacterium-mediated genetic transformation, polymerase chain reaction, enzyme-linked immunosorbent assay, histochemical method for determining the β-glucuronidase activity, Golden Gate molecular cloning method); bioinformational (for in silico computer modeling of genetic vectors), physiological (for in vivo growing and analyzing of transgenic plants), statistical (for processing the results). The scientific novelty of the obtained results consist in the development of genome editing platform of Ukrainian breeding winter rapeseed, namely for the first time: - an effective regeneration system of B. napus L., which allows to obtain T0 seeds in one year after the beginning of experiments and does not lead to the of somaclonal variability in vitro, was developed; - the Bn1 line Ukrainian breeding winter rape Agrobacterium-mediated genetic transformation conditions were optimized and phosphinothricin resistant biotechnological plants were created; - glyphosate resistant transgenic plants of Bn1 line winter rapeseed were created as a result of genetic transformation by constructions pCB133, pCB135 and pSPG2254; - a genetic vector pSPE2053, which carries the bacterial endonuclease Cas9 gene was created; transgenic lines of winter oilseed rape were obtained using this vector, these lines will be used in breeding programs for genome editing; - a genetic construct pSPE2058, carrying the bacterial endonuclease Cas9 gene, Aa1 and Aa2 guide sequences for rapeseed genome editing in order to accumulate oleic acid, and the selective phosphinothricin-N-acetyltransferase bar gene was created; the construct will be used to create high oleic Ukrainian breeding winter rape lines. The practical significance of the results. Biotechnological plants of Ukrainian breeding Bn1 line winter rapeseed, resistant to the glyphosate, may be used in further experimental studies and breeding programs. In addition, a genetic construct based on CRISPR/Cas9 system was created and biotechnological plants carrying the bacterial endonuclease synthesis Cas9 gene were obtained as a platform for the rapeseed genome editing. The results of theoretical and practical research: - are applied in practical activity of Ltd "BETA NK" during creation of winter rape biotechnological plants with qualitatively new traits (the research results implementation act is presented in Appendix B); - may be of interest to research and educational institutions, in particular during the teaching of biotechnology and plant genetics.