Study of transgene expression in Triticum aestivum L. after Agrobacterium-mediated in planta transformation
Aim. Detection of sequences of target transgenes nptII and bar in the genome of probable transformants of bread winter wheat Triticum aestivum L. cultivars Zymoiarka and Podolianka obtained as a result of Agrobacterium-mediated transformation in planta and determination of their expression level. Methods. Polymerase chain reaction (PCR) method was used independently and in combination with reverse transcription (RT-PCR), electrophoresis of DNA in agarose gel. Tolerance to the herbicide was evaluated in the physiological test. Results Through PCR analysis, the sequence of nptII transgene was detected in 30 samples of 145 analyzed, the frequency of transformation was 20.7 %. The sequence of the gene bar was observed in 85 experimental plants, and the frequency of transformation was 15.6 %. mRNAs of both transgenes were detected, indicating their transcriptional activity and stable expression. Conclusions PCR analysis allowed to detect nptII transgenic signal in 20.7 % of plants, while the presence of the bar gene was detected in 15.6 % of cases, indicating a higher efficiency of this genetic construct. The transcription is shown in all the specimens studied for both transgene. According to the results of the physiological test, 25 % of plants containing the gene bar showed resistance to the Basta® herbicide.
Keywords: genetically modified organisms, transgenic plants, biotechnological cultures, bread winter wheat, genetic engineering.
Method of creation of transgenic plants with high level of expression of transgenic proteins: pat. 2507736 Russian Federation. No2507736; app. 11.01.2011; pub. 27.02.2014, Bull. No 6.
Altpeter F., Vasil V., Srivastava V., Stöger E., Vasil I. K. Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Reports. 1996. Vol. 16. P. 12-17. doi: 10.1007/BF01275440
Dai S. H., Zheng P., Marmey P., Zhang S. P., Tian W. Z., Chen S. Y., Beachy R. N., Fauquet C. Comparative analysis of transgenic plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed. 2001. Vol. 7. P. 25–33. doi: 10.1023/A:1009687511633
Ding L., Li S., Gao J., Wang Y., Yang G., He G. Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Mol Biol Rep. 2009. Vol. 36. P. 29–36. doi: 10.1007/s11033-007-9148-5
Hamada H., Linghu Q., Nagira Y., Miki R., Taoka N., Imai R. An in planta biolistic method for stable wheat transformation. Scientific Reports. 2017. Vol. 7:11443. doi: 10.1038/s41598-017-17188-2
Ishida Y., Tsunashima M., Hiei Y., Komari T. Wheat (Triticum aestivum L.) transformation using immature embryos. In Agrobacterium Protocols Vol. 1, Methods in Molecular Biology; Wang, K., Ed. Springer Science + Business Media: New York, NY, USA. 2014. Vol. 1223. P. 189–198. doi: 10.1007/978-1-4939-1695-5_15
IshidaY., Yukoh H., Toshihiko K. High efficiency wheat transformation mediated by Agrobacterium tumefaciens. Advances in Wheat Genetics: From Genome to Field. 2015. Vol. 5. P. 167-175. doi: 10.1007/978-4-431-55675-6_18
Lazzeri P. A., Jones H. D. Transgenic wheat, barley and oats: production and characterization. Methods in Molecular Biology. 2009. Vol. 478. P. 3–22. doi: 10.1007/978-1-59745-379-0_1
Qin J B, Wang Y, Zhu C Q. Biolistic transformation of wheat using the HMW-GS 1Dx5 gene without selectable markers. Genetics and Molecular Research. 2014. Vol. 13. P. 4361–4371. doi: 10.4238/2014.June.10.3
Richardson T., Thistleton J., Higgins T., Howitt C., Ayliffe M. Efficient Agrobacterium transformation of elite wheat germplasm without selection. Plant Cell Tiss. Org. 2014. Vol. 119. P. 647–659. doi: 10.1007/s11240-014-0564-7
Sestili F., Janni M., Doherty A., Botticella E., D'Ovidio R., Masci S., Jones H. D., Lafiandra D. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes. BMC Plant Biology. 2010. Vol. 10. P. 1–12. doi: 10.1186/1471-2229-10-144
Vasil V., Castillo A.M, Fromm M.E, Vasil I.K. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Biotechnol. 1992. Vol. 10. P. 667–674. doi: 10.1038/nbt0692-667
Weeks J. T., Anderson O. D., Blechl A. E. Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum L.). Plant Physiology. 1993. Vol. 102. P. 1077–1084. doi: 10.1104/pp.102.4.1077
Zhang Z., Liu X., Wang X., Zhou M., Zhou X., Ye X. et al. An R2R3 MYB transcription factor in wheat, TaPIMP1, mediates host resistance to Bipolaris sorokiniana and drought stresses through regulation of defense- and stress-related genes. New Phytol. 2012. Vol. 196. P. 1155–1170. doi: 10.1111/j.1469-8137.2012.04353.x
Zhou H., Arrowsmith J. W, Fromm M. E., Hironaka C. M., Taylor M. L., Rodriguez D., Pajeau M. E., Brown S. M., Santino C. G., Fry J. E. Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation. Plant Cell Reports. 1995. Vol. 15. P. 159–163. doi: 10.1007/BF00193711