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Fiziol. rast. genet. 2019, vol. 51, no. 4, 283-294, doi: https://doi.org/10.15407/frg2019.04.283

Optimization of conditions of Agrobacterium-mediated transformation of bread wheat by the in planta

Dubrovna O.V., Kulesh S.S., Slivka L.V.

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

The conditions of Agrobacterium-mediated transformation of bread wheat of Zymoyarka variety by the in planta method with the use of strains LBA4404 and AGL0 have been optimized. The dependence of the seed setting efficiency and the frequency of obtaining transgenic T. aestivum plants on the environmental conditions, in particular, temperature and air humidity, have been established. It was shown that using the AGL0 strain, the largest percentage of seed set (38.7 %) was obtained at a temperature of 20 °C and humidity of 45 %, and the smallest amount of grains (16.1 %) was obtained at a temperature of 16 °C and high air humidity 75 %. At a temperature of 27 °C, the setting of seeds was twice higher compared to 16 °C, that is, lowering the temperature in combination with high humidity has a more negative effect on pollination than it increasing. When comparing temperatures of 22 °C and 27 °C at practically the same air humidity between the experimental variants with using one strain of Agrobacterium, there was no significant difference in the seed setting index. Also, there were no significant differences in the frequency of seeds setting at the same temperature when using different strains. The temperature of 22 °С was more effective, as with its increase to 27 °С the number of grains formed somewhat decreased. It was established that the temperature regime of 20 °С and air humidity of 45 % ensured the greatest number (4,3 %) of Zimoyarka wheat transformants, and when the temperature decreased to 16 °С, the efficiency of T-DNA transfer to the plant genome decreased and the lowest transformation frequency was observed (0.5 %). The negative effect of sucrose in the inoculation medium on the process of seed setting during the Agrobacterium-mediated transformation of bread wheat by the in planta method was revealed. The largest number of seeds was obtained using an inoculation medium without sucrose, the optical density of cells in the agrobacterial suspension was 0.4—0.6 o.u. and under inoculation on the third day after castration of the ears.

Keywords: T. aestivum, Agrobacterium-mediated transformation in planta, temperature, humidity, inoculation medium

Fiziol. rast. genet.
2019, vol. 51, no. 4, 283-294

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