en   ru   uk  
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

Full text and suplimented materials

Free full text: PDF  


1. Hiei, Y., Ishida, Y. & Komari, T. (2014). Progress of cereal transformation technology mediated by Agrobacterium tumefaciens. Frontiers in Plant Sci., 5, pp. 1-11. https://doi.org/10.3389/fpls.2014.00628

2. Sparks, C., Doherty, A. & Jones, H. (2014). Genetic transformation of wheat via Agrobacterium-mediated DNA delivery. Methods Mol. Biol., 1099, pp. 235-250. https://doi.org/10.1007/978-1-62703-715-0_19

3. Sramkova, Z., Gregova, E. & Sturdik, E. (2009). Genetic improvement of wheat - a review. Nova Biotechnologica, 9, No. 1, pp. 27-51.

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

5. Zale, J., Agarwal, S., Loar, S. & Steber, C. (2009). Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens. Plant Cell Rep., 28, pp. 903-913. https://doi.org/10.1007/s00299-009-0696-0

6. Zhao, T., Zhao, S., Chen, H., Zhao, Q., Hu, Z., Hou, B. & Xia, G. (2006). Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling. Plant Cell Rep., 25, pp. 1199-1204. https://doi.org/10.1007/s00299-006-0184-8

7. Supartana, P., Shimizu, T., Nogawa, M., Shioiri, H., Nakajima, T., Haramoto, N., Nozue, M. & Kojima, M. (2006). Development of simple and efficient in planta transformation method for wheat (Triticum aestivum L.) using Agrobacterium tumefaciens. Journal of Bioscience and Bioengineering, 102, No. 3, pp. 162-170. https://doi.org/10.1263/jbb.102.162

8. Puhalsky, V.A., Smirnov, S.P., Korostyleva, T.V., Bilinskaya, E.N. & Eliseeva, A.A. (1996). Genetic transformation of wheat (Triticum aestivum L.) with Agrobacterium tumefaciens. Genetika, 32, No. 11, pp. 1596-1600 [in Russian].

9. Sawahel, W. & Hassan, A. (2002). Generation of transgenic wheat plants producing high levels of the osmoprotectant proline. Biotechnol. Letters, 24, pp. 721-725. https://doi.org/10.1023/A:1015294319114

10. Agarwal, S., Loar, S., Steber, C. & Zale, J. (2009). Floral transformation of wheat. Methods in Mol. Biol., 478, pp. 105-113. https://doi.org/10.1007/978-1-59745-379-0_6

11. Hussain, J. Manan, S., Ahmad, S., Ahmed, T. & Shah, M. (2013). Biotechnologies used in genetic transformation of Triticum aestivum: A mini overview. Fuuast J. Biol., 3, pp. 105-109.

12. Jasdeep, C. & Avijit, T. (2015). Genetic transformation and transgenic wheat development: an overview. Clon Transgen., 5, No. 1, pp. 147-148. https://doi.org/10.4172/2168-9849

13. Yang, B., Ding, L., Yao, L., He, G. & Wang, Y. (2008). Effect of seedling ages and inoculation durations with Agrobacterium tumefaciens on transformation frequency of the wheat wounded apical meristem. Mol. Plant Breed., 6, pp. 358-362.

14. He, D., Li, Z. & Wang, H. (2003). The efficient transformation of wheat in planta by Agrobacterium tumefaciens. Scientia Agricultura Sinica, 36, pp. 1437-1441.

15. Langridge, P., Brettschneider, R., Lazzeri, P. & Lorz, H. (2002). Transformation of cereals via Agrobacterium and the pollen pathway: a critical assessment. Plant J., 2, pp. 631-638. https://doi.org/10.1111/j.1365-313X.1992.00631.x

16. Voronova, S.S., Bavol, A.V. & Dubrovna, O.V. (2015). In planta genetic transformation of bread wheat, using AGL0 strain, containing pBi2E with dsRNA suppressor of ProDH gene. Faktory eksperymental'noi evoliucii orhanizmiv, 17, pp. 126-130 [in Ukrainian].

17. Goncharuk, O.M., Bavol, A.V. & Dubrovna, O.V. (2015). Agrobacterium-mediated transformation of wheat with ornithine-aminotransferase gene by an in planta method. Faktory eksperymental'noi evoliucii orhanizmiv, 17, pp. 131-135 [in Ukrainian].

18. Gorbatiuk, I.R., Bavol, A.V., Bannikova, M.O. & Morgun, B.V. (2015). Agrobacterium-mediated in planta transformation of bread winter wheat cv. Podolianka. Visnyk Kharkivskoho nacionalnoho universytetu im. V.N. Karazina, Ser. Biol., Iss. 24 (1153), pp. 47-53 [in Ukrainian].

19. Cheng, M. (2004). Invited Review: Factors Influencing Agrobacterium-mediated Transformation of Monocotyledonous Species. In Vitro Cellular Develop. Biology Plant, 40, No. 1, pp. 31-45. https://doi.org/10.1079/IVP2003501

20. Chumakov, M. I. & Moiseeva, E. M. (2012). Agrobacterial transformation technology of plants in planta. Biotekhnologiya, 1, pp. 8-20.

21. Fullner, K. J. & Nester, E. W. (1996). Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J. Bacteriol., 178, pp. 1498-1504. https://doi.org/10.1128/jb.178.6.1498-1504.1996

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

23. Sidorov, V. & Duncan, D. (2009). Agrobacterium-mediated maize transformation: immature embryos versus callus. Methods Mol. Biol., 526, pp. 47-58. https://doi.org/10.1007/978-1-59745-494-0_4

24. Chumakov, M. I., Rozhok, N.A., Velikov, V.A., Tyrnov, V.S. & Volokhina, I.V. (2006). Agrobacterium-mediated in planta transformation of maize via pistil filaments. Russian J. Genet., 42, No. 8, pp. 893-897. https://doi.org/10.1134/S1022795406080072

25. Salas, M., Park, S., Srivatanakul, M. & Smith, R. (2001). Temperature influence on stable T-DNA integration in plant cells. Plant Cell Rep., 20, pp. 701-705. https://doi.org/10.1007/s002990100374

26. Dale, P., Marks, M., Brown, M., Woolston, C., Gunn, H., Mullineaux, P., Lewis, D., Kemp, J., Chen, D., Gilmour, D. & Flavell, R. (1989). Agroinfection of wheat: inoculation of in vitro grown seedlings and embryos. Plant Sci., 63, pp. 237-245. https://doi.org/10.1016/0168-9452(89)90249-5

27. Arencibia, A., Carmona, E., Tellez, P., Chan, M., Yu, S., Trujillo, L. & Oramas, P. (1998). An efficient protocol for sugarcane (Saccharum spp. L.) transformation diated by Agrobacterium tumefaciens. Transgenic Res., 7, pp. 213-222. https://doi.org/10.1023/A:1008845114531

28. Hashizume, F., Tsuchiya, T., Ugaki, M., Niwa, Y., Tachibana, N. & Kowyama, Y. (1999). Efficient Agrobacterium-mediated transformation and the usefulness of a synthetic GFP reporter gene in leading varieties of japonical rice. Plant Biotechnol., 16, pp. 397-401. https://doi.org/10.5511/plantbiotechnology.16.397

29. Moiseeva, Y.M., Velikhov, V.A., Volokhina, I.V., Gusev, Yu.S., Yakovleva, O.S. & Chumakov, M.I. (2014). Agrobacterium-mediated transformation of maize with antisense suppression of the proline dehydrogenase gene by an in planta method. British Biotechnology Journal, 4, No. 2, pp. 116-125. https://doi.org/10.9734/BBJ/2014/6504

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

31. Gorbatyuk, I.P. (2016). Optimization of Agrobacterium-mediated biotechnology of transformation of Triticum aestivum in culture in vitro and method in planta (Extended abstract of Doctor thesis). Institute of Cells Biology and Gene Engineering of NAS of Ukraine. Kyiv, Ukraine [in Ukrainian].