Fiziol. rast. genet. 2020, vol. 52, no. 6, 528-537, doi:

Influence of picloram on the morphogenesis of calli cultures of selection-value genotypes of winter wheat under Agrobacterium-mediated transformation

Dubrovna O.V., Slivka L.V.

  • Iнститут фізіології рослин і генетики Національної академії наук України 03022 Київ, вул. Васильківська, 31/17

The influence of synthetic auxin — picloram on the frequency of morphogenic callus formation and regeneration of shoots after Agrobacterium-mediated transformation of selection-valuable genotypes of winter wheat was studied. It was shown that the presence of picloram in the nutrient medium contributed to the formation of a callus of greater mass, which is capable to longer cultivation in vitro compared to callus formed on a medium with 2,4-D, while maintaining regenerative capacity. In calluses obtained on medium with 2,4-D, a relatively higher number of necrosis was observed and the transition of tissues to the morphogenic state occurred 3—5 days later compared to calluses obtained on media with picloram. The formation of the maximum amount of morphogenic callus for all studied genotypes was observed on medium with the addition of picloram at a concentration of 2 mg/l. The viability of the morphogenic callus obtained on the medium with picloram exceeds the duration of cultivation of the callus obtained on the medium with 2,4-D, on average by 30 days. It is established that the use of picloram in regeneration media allows to increase the frequency of shoot formation during genetic transformation of wheat. On the medium with the addition of this auxin at a concentration of 2 mg/l it was able to obtain the largest number of regenerants. At this concentration of picloram it revealed a significant increase in the number of plants compared to the medium containing IOC. The regeneration potential of callus obtained on medium with 2,4-D after genetic transformation was maintained for a maximum of 2 passages, while regenerating plants from callus formed on medium with picloram were obtained for 3—4 passages.

Keywords: Triticum aestivum, picloram, morphogenesis, Agrobacterium-mediated transformation

Fiziol. rast. genet.
2020, vol. 52, no. 6, 528-537

Full text and supplemented materials

Free full text: PDF  


1. Abdul, R., Ma, Z. & Wang, H. (2010). Genetic Transformation of Wheat (Triticum aestivum L.): A Review. Triticeae Genomics and Genetics, 1, No. 2, pp. 1-7.

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

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

4. Bhalla, P., Ottenhof, H. & Singh, M. (2006). Wheat transformation an update of recent progress. Euphytica, 149, pp. 353-366.

5. Ding, L. (2009). Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Mol. Biol. Rep., 36, pp. 29-36.

6. Hayta, S., Smedley, M.A., Demir, S.U., Blundell, R., Hinchliffe, A., Atkinson, N. & Harwood, W.A. (2019). An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.). Plant Methods, 15, 121.

7. Abid, N., Maqbool, A. & Malik, K. (2014). Screening commercial wheat (Triticum aestivum L.) varieties for Agrobacterium mediated transformation ability. Pakistan J. Agricult. Sci., 51, No. 1, pp. 83-89.

8. 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 (Triticum turgidum L. var. durum cv Stewart) with improved efficiency. J. Exp. Bot., 61, pp. 1567-1581.

9. Khurana, J., Chugh, A. & Khurana, P. (2002). Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum diccocum Schuble). Indian J. Exp. Biol., 40, pp. 1295-1303.

10. Mahalakshmi, A. & Khurana, P. (1995). Agrobacterium-mediated gene delivery in various tissues and genotypes of wheat (Triticum aestivum L.). J. Biochem. and Biotechnol., 4, No. 2, pp. 55-59.

11. Tao, L., Du, L., Xu, H. & Ye, X. (2011). Improvement of plant regeneration from immature embryos of wheat infected by Agrobacterium tumefaciens. Agricult. Sci. in China, 10, pp. 317-326.

12. Wang, Y., Xiao, X. & Zhang, A. (2002). Factors affecting Agrobacterium tumefaciens-mediated transformation of wheat (Triticum aestivum L.). Acta Genetica Sinica, 29, No. 3, pp. 260-265.

13. Tamas, C., Szucs, P., Rakszegi, M., Tamas, L. & Bedo, Z. (2004). Effect of combined changes in culture medium and incubation conditions on the regeneration from immature embryos of elite varieties of winter wheat. Plant Cell, Tissue and Organ Culture, 79, pp. 39-44.

14. Ishida, Y., Tsunashima, M., Hiei, Y. & Komari, T. (2015). Wheat (Triticum aestivum L.) transformation using immature embryos. Methods Mol Biol., 1223, pp. 189-198.

15. Sarker, R.H. & Biswas, A. (2002). In vitro plantlet regeneration and Agrobacterium-mediated genetic transformation of wheat (Triticum aestivum L.). Plant Tissue Culture, 12, No. 2, pp. 155-165.

16. Medvecka, E. & Harwood, W. (2015). Wheat (Triticum aestivum L.) transformation using mature embryos. Methods Mol. Biol., 1223, pp. 199-209.

17. Tang, Z., Ren, Z., Wu, F., Fu, S., Wang, X. & Zhang, H. (2006). The selection of transgenic recipients from new elite wheat cultivars and study on its plant regeneration system. Agricultural Sciences in China, 5, pp. 417-424.

18. Tran, T.N. & Sanan-Mishra, N. (2015). Effect of antibiotics on callus regeneration during transformation of IR 64 rice. Biotechnol. Rep., No. 7, pp. 143-149.

19. Goncharuk, O.M., Bavol, A.V. & Dubrovna, O.V. (2014). Increase in frequency of wheat callus cultures regeneration for Agrobacterium-mediated transformation. Bul. Ukr. Soc. Genet. Breeders, 12, No. 2, pp. 159-165 [in Ukrainian].

20. Yu, Y. & Wei, Z. (2008). Influences of cefatoxime and carbenicillin on plant regeneration from wheat mature embryos. Biologia Plantarum, 52, pp. 553-556.

21. Dubrovna, O.V., Bavol, A.V., Zinchenko, M.O., Goncharuk, O.M. & Lyalko, I.I. (2012). Influence of cefotaxime on morphogenesis in the culture of apical meristems and mature embryos of wheat. Physiology and biochemistry of cult. plants, 44, No. 3, pp. 218-224 [in Ukrainian].

22. Sharma, V., H¬nsch, R. & Mendel, R. (2005). Influence of picloram and thidiazuron on high frequency plant regeneration in elite cultivars of wheat with long-term retention of morphogenecity using meristematic shoot segments. Plant Breeding, 124, No. 3, pp. 242-246.

23. Ren, J., Wang, X. & Yin, J. (2010). Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat. Agricultural Sciences in China, 9, No. 1, pp. 31-37.

24. Bavol, A.V., Dubrovna, O.V., Lyalko, I.I. & Zinchenko, M.O. (2011). The influence of thidiazuron on processes of morphogenesis in culture in vitro of bread wheat. Physiology and biochemistry of cult. plants, 43, No. 5, pp. 412-418 [in Ukrainian].

25. Mendoza, M. & Kaeppler, H. (2002). Auxin and sugar effects on callus induction and plant regeneration frequencies from mature embryos of wheat (Triticum aestivum L.) In Vitro Cellular and Development Biology. Plant, 38, No. 1, pp. 39-45.

26. Barro, F., Martin, A., Lazzeri, P. & Barcel, P. (1999). Medium optimization for efficient somatic embryogenesis and plant regeneration from immature inflorescences and immature scutella of elite cultivars of wheat, barley and tritordeum. Euphytica, 108, No. 3, pp. 161-167.

27. Bavol, A.V., Dubrovna, O.V. & Lyalko, I.I. (2007). Regeneration of plants from the explants of the top of wheat seedlings shoots. Bul. Ukr. Soc. Genet. Breeders, 5, No. 1-2, pp. 3-10 [in Ukrainian].

28. Bavol, A.V., Dubrovna, O.V. & Lyalko, I.I. (2008). Regeneration of plants from different types of bread wheat explants. Physiology and biochemistry of cultivated plants, 40, No. 2, pp. 150-156 [in Ukrainian].

29. Kumar, R., Mamrutha, H., Kaur, A., Venkatesh, K., Grewal, A., Kumar, R. & Tiwari, V. (2017). Development of an efficient and reproducible regeneration system in wheat (Triticum aestivum L.). Physiol. Mol. Biol. Plants, 23, No. 4, pp. 945-954.

30. Gorbatyuk, I.R., Bavol, A.V., Holubenko, A.V. & Morgun, B.V. (2015). Effect of synthetic auxin like growth regulators on callus regenerative ability of common wheat. Biotechnologia acta, 8, No. 1, pp. 56-62.

31. Collins, G.B., Vian, W.E. & Phillips, G.C. (1978). Use of 4-amino-3,5,6-trichloropicolinic acid as an auxin source in plant tissue cultures. Crop Sci., 18, No. 2, pp. 286-288.

32. Ozgen, M., Turet, M., Ozcan, S. & Sancak C. (1996). Callus induction and plant regeneration from immature and mature embryos of winter durum wheat genotypes. Plant Breed., 115, pp. 455-458.

33. Bavol, A.V., Dubrovna, O.V., Goncharuk, O.M. & Voronova, S.S. (2014). Agro­bacterium-mediated transformation of bread wheat using callus cultures. Factors of experimental evolution of organisms, 15, pp. 16-19 [in Ukrainian].

34. Vishnudasan, D., Patnaik, D. & Khurana, P. (2006). Agrobacterium-mediated transformation of mature embryos of Triticum aestivum and Triticum durum. Current science, 91, pp. 307-317.

35. Ishida, Y., Tsunashima, M., Hiei, Y. & Komari, T. (2015). Wheat (Triticum aestivum L.) transformation using immature embryos. Methods Mol. Biol., 1223, pp. 189-198.

36. Wu, H., Doherty, A. & Jones, H. (2009). Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos. In: Transgenic wheat, barley and oats (pp. 93-103), New York: Humana Press.

37. Wu, H., Sparks, C., Amoah, B. & Jones, H. (2003). Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Reports, 21, pp. 659-668.

38. Ahmad, A., Zhong, H., Wang, W. & Sticklen, M. (2002). Shoot apical meristem: in vitro regeneration and morphogenesis in wheat (Triticum aestivum L.). In vitro Cellular & Developm. Biology - Plant, 38, No. 2, pp. 163-167.

39. Karp, A. (1994). Origins, causes and uses of variation in plant tissue cultures. In: Vasil, I.K., Thorpe, T.A. [et al.] Plant cell and tissue culture. (pp. 139-151), Kluwer Publ., Dordrecht.

40. Ying-Hua, S., Yu-Bo, L. & Xian-Sheng, Z. (2011). Auxin-cytokinin interaction regulates meristem development. Mol. Plant, 4, No. 4, pp. 616-625.