Fiziol. rast. genet. 2022, vol. 54, no. 4, 311-327, doi:

Agrobacterium-mediated transformation of promising genotypes of winter wheat using the ornithine-d-aminotransferase gene

Dubrovna O.V., Slivka L.V.

  • Institute of Plant Physiology and Genetics, National Academy of Sciences of Ukraine 31/17 Vasylkivska St., Kyiv, 03022, Ukraine

Ornithine-d-aminotransferase (OAT) is an important regulator of cellular metabolism because the reaction catalyzed with this enzyme binds several biochemical systems: the urea cycle, the proline accumulation and degradation cycle, and the polyamine biosynthesis pathway. The introduction of the exogenous oat gene into the plant genome is one of the promising methods for creating abiotic stress-resistant wheat genotypes. The aim of our work was to optimize the conditions of Agrobacterium-mediated transformation of morphogenic calluses of new promising genotypes of winter bread wheat, and to obtain genetically modified plants with the heterologous gene of ornithine-d-aminotransferase. The main parameters of the transformation protocol were studied, in particular the influence of optical density of agrobacterial cells suspension, concentration of the antibiotic cefotaxime, the effect of duration of cocultivation on the frequency of kanamycin-resistant regenerants from callus cultures of apical origin. The regeneration environment has been optimized, which allows to accelerate the process of obtaining genetically modified wheat regenerants, and increase their number. This reduces the biotechnological process, and the material costs for its implementation. Through Agrobacterium-mediated transformation of morphogenic calluses of new promising genotypes of winter bread wheat, regenerants were obtained in the genome of which the complete incorporation of the genetic construct containing oat and nptII transgenes was revealed. The transgenic nature of all plants obtained was confirmed by PCR with primers specific for the oat and nptII genes. The frequency of transformation for the studied genotypes was 0.75—2.5 %.

Keywords: Triticum aestivum, Agrobacterium-mediated transformation, callus cultures, ornithine-d-aminotransferase gene

Fiziol. rast. genet.
2022, vol. 54, no. 4, 311-327

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