Optimization of Agrobacterium-mediated transformation conditions of prospective genotypes of winter bread wheat by in planta method

  • O. V. Dubrovna Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine
  • L. V. Slivka Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine


Aim. Optimization of conditions for genetic transformation of new promising genotypes of winter bread wheat (T. aestivum L.) by in planta method. Methods. Agrobacterium-mediated transformation by in planta method using the strain AGL0 and vector construct pBi2E. Results. The influence of air temperature, optical density of cells of agrobacterial suspension, inoculation day and composition of inoculation medium on the frequency of obtaining transgenic plants of new winter wheat genotypes was studied. The dependence of the frequency obtaining of transgenic plants from environmental conditions, in particular temperature, has been established. It was found that the temperature regime of 20-22°C provided the largest number (4.8%) of wheat transformants, and when the temperature is reduced to 16-18°C there is a decrease in the efficiency of T-DNA transfer into the plant genome and the lowest frequency of transformation (0.7%). Conclusions. The largest number of transformants was obtained using a inoculation medium without sucrose, the optical density of cells of the agrobacterial suspension of 0.6 op.od. and inoculation on the third day after castration of ears.

Keywords: T. aestivum, Agrobacterium-mediated transformation in planta, optimization of conditions.


Hiei Y., Ishida Y., Komari T. Progress of cereal transformation technology mediated by Agrobacterium tumefaciens. Fron-tiers in Plant Sci. 2014. Vol. 5. P. 1–11. doi: 10.3389/fpls.2014.00628.

Sparks C., Doherty A., Jones H. Genetic transformation of wheat via Agrobacterium-mediated DNA delivery. Methods Mol. Biol. 2014. Vol. 1099. P. 235–250. doi: 10.1007/978-1-62703-715-0_19.

Risacher T., Craze M., Bowden S., Paul W., Barsby T. Highly efficient Agrobacterium-mediated transformation of wheat via in planta inoculation. Methods Mol. Biol. 2009. Vol. 478. P. 115–124. doi: 10.1007/978-1-59745-379-0_7.

Zale J., Agarwal S., Loar S., Steber C. Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefa-ciens. Plant Cell Reports. 2009. Vol. 28. P. 903–913. doi: 10.1007/s00299-009-0696-0.

Borisjuk N., Kishchenko O., Eliby S., Schramm C., Anderson P., Jatayev S., Kurishbayev A., Shavrukov Y. Genetic mod-ification for wheat improvement: from transgenesis to genome editing. BioMed Research International. 2019. Article ID 6216304. 18 p. doi: 10.1155/2019/6216304.

Agarwal S., Loar S., Steber C., Zale J. Floral transformation of wheat. Methods in Mol. Biol. 2009. 478. P. 105–113. doi: 10.1007/978-1-59745-379-0_6.

Razzaq A., Hafiz I., Mahmood I., Hussain A. Development of in planta transformation protocol for wheat. African Journal of Biotechnology. 2011. Vol. 10 (5). P. 740–750.

Supartana P., Shimizu T., Nogawa M., Shioiri H., Nakajima T., Haramoto N., Nozue M., Kojima M. Development of simple and efficient in planta transformation method for wheat (Triticum aestivum L.) using Agrobacterium tumefaciens. Journal of Bioscience and Bioengineering. 2006. Vol. 102 (3). P. 162–170. doi: 10.1263/jbb.102.162.

Chumakov M.I., Moiseeva E.M. Agrobacterial transformation technology of plants in planta. Biotekhnologiya, 2012. No. 1. P. 8–20.

Moiseeva Y.M., Velikhov V.A., Volokhina I.V., Gusev Yu.S., Yakovleva O.S., Chumakov M.I. Agrobacterium-mediated transfоrmation of maize with antisense suppression of the proline dehydrogenase gene by an in planta method. British Biotechnology Journal. 2014. Vol. 4 (2). P. 116–125. doi: 10.9734/BBJ/2014/6504.

Sidorov V., Duncan D. Agrobacterium-mediated maize transformation: immature embryos versus callus. Methods Mol. Biol. 2009. Vol. 526. P. 47–58. doi: 10.1007/978-1-59745-494-0_4.

Voronova, S.S., Bavol, A.V., Dubrovna, O.V. In planta genetic transformation of bread wheat, using AGLO strain, containing pBi2E with dsRNA suppressor of ProDH gene. Factors of experimental evolution of organisms. 2015. Vol. 17. P. 126–130 [in Ukrainian].

Dillen W., De Clereq J., Kapila J., Zamnbre M., Van Montagu M., Angenon G. The effect of temperature on Agrobacterium tumefaciens method of gene transfer to plants. Plant J. 1997. Vol. 12. P. 1459–1462. doi: 10.1046/j.1365-313x.1997.12061459.x.

Salas M., Park S., Srivatanakul M., Smith R. Temperature influence on stable T-DNA integration in plant cells. Plant Cell Rep. 2001. Vol. 20. P. 701–705. doi: 10.1007/s002990100374.

Frame B., McMurray J., Fonger T., Main M., Taylor K., Torney F., Paz M., Wang K. Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts. Plant Cell Rep. 2006. Vol. 25. P. 1024–1034. doi: 10.1007/s00299-006-0145-2.