Fiziol. rast. genet. 2016, vol. 48, no. 1, 65-74, doi:

Establishing transgenic wheat plants of cv. Zymoyarka resistant to the herbicide phosphinothricin in vitro

Gorbatyuk I.R.1, Shcherbak N.L.1, Bannikova M.O.1, Velykozhon L.H.1,2, Kuchuk M.V.1, Morgun B.V.1,2

  1. Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine 148 Akademika Zabolotnoho St., Kyiv, 03143, Ukraine
  2. Institute of Plant Physiology and Genetics, National Academy of Sciences of Ukraine 31/17 Vasylkivska St., Kyiv, 03022, Ukraine

Phosphinothricin resistant wheat plants of native breeding cv. Zymoyarka carrying bar gene of bacterium Streptomyces hygroscopicus have been obtained by two methods of genetic transformation. The biolistic transformation was performed with vector pAHC25 while the Agrobacterium-mediated one with pCB203 in strain GV3101. Both vectors in addition to the selective gene (bar) of phosphinothricin acetyl transferase contained the reporter gene (uidA) of b-glucuronidase from Escherichia coli. The immature embryos were used as primary explants. The integration of transgene bar into regenerants’ genome and the lack of agrobacterium infection were proved by means of PCR. The expression of gene uidA was confirmed by histochemical analysis. The transformation efficiency amounting to 0.5 % for biolistic and to 1.25 % for Agrobacterium-mediated allowed us to select 3 and 12 transgenic lines, respectively. This is the first report on the successful establishment of transgenic wheat resistant to the herbicide phosphinothricin in in vitro culture in Ukraine.

Keywords: Triticum aestivum L., bread wheat, genetic transformation of plants, plant biotechnology, herbicides

Fiziol. rast. genet.
2016, vol. 48, no. 1, 65-74

Full text and supplemented materials

Free full text: PDF  


1. Gorbatyuk, I.R., Gnatyuk, I.S. & Bannikova, M.O.(2015). Effect of growth regulators on the regenerative capacity of bread wheat varieties Zimoyarka. Fiziol. rast. genet., 47, No. 6, pp. 514-525 [in Ukrainian].

2. Dubrovna, O.V., Morgun, B.V. & Bavol, A.V. (2014). Wheat biotechnology: cell selection and genetic engineering. Kyiv: Logos [in Ukrainian].

3. Altpeter, F., Vasil, V., Srivastava, V., Stoger, E. & Vasil, I.K. (1996). Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Reports, 16, pp. 12-17.

4. Bertani, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. Journal of Bacteriology, 62, pp. 293-300.

5. Binka, A., Orczyk, W. & Nadolska-Orczyk, A. (2012). The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (xTritocosecale Wittmack): role of the binary vector system and selection cassettes. Journal of Applied Genetics, 53, pp. 1-8.

6. Birch, R.G. (1997). Plant transformation problems and strategies for practical application. Annal Review of Plant Physiology and Plant Molecular Biology, 48, pp. 297-326.

7. Christensen, A.H. & Quail, P.H. (1996). Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Research, 5, pp. 213-218.

8. Dai, S.H. (2001). Comparative analysis of transgenic plants obtained by Agrobacterium-mediated transformation and particle bombardment. Molecular Breeding, 7, pp. 25-33.

9. Ding, L. (2009). Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Molecular Biology Reports, 36, pp. 29-36.

10. Gamborg, O.L. & Eveleigh, D. (1968). Culture methods and detection of glucanases in cultures of wheat and barley. Canadian Journal Biochemistry, 46, No. 5, pp. 417-421.

11. He, Y., Jones, H.D., Chen, S., Chen, X.M., Wang, D.W., Li, K.X., Wang, D.S. & Xia, L.Q. (2010). Agrobacterium-mediated transformation of durum wheat (Agrobacterium-mediated transformation (Triticum turgidum L. var. durum cv. Stewart) with improved efficiency. Journal of Experemental Botany, 61, pp. 1567-1581.

12. Hiei, Y., Ishida, Y. & Komari, T. (2014). Progress of cereal transformation technology mediated by Agrobacterium tumefaciens. Frontiers in Plant Sciences, 5, pp. 1-11.

13. Hu, T., Metz, S., Chay, C., Zhou, H.P., Biest, N., Chen, G., Chenq, M., Fenq, X., Radionenko, M., Lu, F. & Fry, J. (2003). Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Reports, 21, pp. 1010-1019.

14. Khanna, H.K. & Daggard, G. (2003). Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Reports, 21, pp. 429-436.

15. Lazzeri, P.A. & Jones, H.D. (2009).Transgenic wheat, barley and oats: production and characterization. Methods in Molecular Biology, 478, pp. 3-22.

16. Murashige, T., Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15, pp. 473-497.

17. Nadolska-Orczyk, A., Orczyk, W. & Przetakiewicz, A. (2000). Agrobacterium-mediated transformation of cereals - from technique development to its application. Acta Physiologiae Plantarum, 22, pp. 77-88.

18. Ombori, O., Vincent, J., Muoma, O. & Machuka, J. (2013). Agrobacterium-mediated genetic transformation of selected tropical inbred and hybrid maize (Zea mays L.) lines. Plant Cell, Tissue and Organ Culture, 113, pp. 11-23.

19. Rashid, H., Afzal, A. & Khan, M.H. (2010). Effect of bacterial culture density and acetosyringone concentration on Agrobacterium-mediated transformation in wheat. Pakistan Journal of Botany, 42, pp. 4183-4189.

20. Sawada, H., Leki, H. & Matsuda, I. (1995). PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains. Applied and environmental microbiology, 61, No. 2, pp. 828-831.

21. Sestili, F., Janni, M., Doherty, A., Botticella, E., D Ovidio, R., Masci, S., Jones, H.D. & Lafiandra, D. (2010). Increasing the amylose content of durum wheat through silencing of the SBEIIa genes. BMC Plant Biology, 10, pp. 1-12.

22. Sidorov, V. & Duncan, D. (2009). Agrobacterium-mediated maize transformation: immature embryos versus callus. Methods in Molecular Biology, 526, pp. 47-58.

23. Sparks, C.A., Doherty, A. & Jones, H.D. (2014). Genetic transformation of wheat via Agrobacterium-mediated DNA delivery. Methods in Molecular Biology, 1099, pp. 235-250.

24. Vasil, V., Castillo, A.M., Fromm, M.E. & Vasil, I.K. (1992). Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Biotechnology, 10, pp. 667-674.

25. Weeks, J.T., Anderson, O.D. & Blechl, A.E. (1993). Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum L.). Plant Physiology, 102, pp. 1077-1084.

26. Zhou, H., Arrowsmith, J.W., Fromm, M.E., Hironaka, C.M., Taylor, M.L., Rodriquez, D., Paieau, M.E., Brown, S.M., Santino, C.G. & Fry, J.E. (1995). Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation. Plant Cell Reports, 15, pp. 159-163.

27. Ziemienowicz, A. (2013). Agrobacterium-mediated plant transformation: Factors, applications and recent advances. Biocatalysis and Agricultural Biotechnology, pp. 1-8.

28. Ziemienowicz, A. (2014). Agrobacterium-mediated plant transformation: Factors, applications and recent advances. Biocatalysis and Agricultural Biotechnology, 3, pp. 95-102.