Inheritance of glyphosate and glufosinate resistance in T1-T2 generations of biotechnological canola (Brassica napus L.) plants
Aim. To obtain the biotechnological canola plants using the introduction of epsps and bar genes in the same cassette and investigate both inheritance of target genes and resistance to corresponding herbicides in Т1–Т2 generations. Меthods. Agrobacterium tumefaciens-mediated genetic transformation,molecular biological (PCR, RT-PCR),genetic (study of segregation for phosphinothricin resistance under aseptic conditions), physiological (evaluation of herbicide resistance, determination of biomass and total soluble proteins (TSP)). Results. The transgenic plants with epsps and bar genes of resistance to herbicides based on glyphosate and glufosinate have been obtained. The integration of target genes into nuclear genome and their activities on transcriptional level were shown. Resistance of biotechnological plants to herbicide treatments was proved in greenhouse. Stable and linked inheritance of transgenes was demonstrated. It was shown that transgenic plants grown in greenhouse and not treated with herbicide do not differ from the initial untransformed ones in biomass and TSP content in leaves, when they were. It was found that spraying of biotechnological plants with herbicides does not affect the growth rate, biomass, and TSP content. Conclusions. The biotechnological plants with genes of herbicide resistance to glyphosate (epsps gene) and glufosinate (bar gene) were obtained. Transgene expression was confirmed for Т1–Т2 generations. It did not influence biomass production and TSP content in transgenic plants under greenhouse conditions.
Кeywords: Brassica napus canola, epsps, bar, glyphosate, glufosinate.
Amrhein, N., B. Deus, P. Gehrke, H. C. Steinrücken. The site of the inhibition of the shikimate pathway by glyphosate, II: interference of glyphosate with chorismate formation in vivo and in vitro. Plant Physiol. 1980. V.66, N 5. – P.830–834.
James C. 2003. ISAAA Briefs: Preview, Global Status of Commercialized Transgenic Crops, In ISAAA Briefs 30, ISAAA, Ithaca, NY.
Pocket K No. 10: Herbicide Tolerance Technology: Glyphosate and Glufosinate. https://isaaa.org/resources/publications/pocketk/10/default.asp
Lea P.J., Joy K.W., Ramos J.L., Guerroro M.G. The action of 2-amino-4-(methylphosphiny)-butanoic acid (phosphinothricin) and its 2-oxoderivative on the metabolism of cyanobacteria and higher plants. Phytochem. 1984. V.23, N 1. P.1–6.
Kahrizi D., Salmanian A.H., Afshari A. et al. Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E. coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate. Plant Cell Rep. 2007. V.26, N 1. Р.95–104.
Chhapekar S., Raghavendrarao S., Pavan G. et al. Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosat. Plant Cell Rep. 2015. V.34, N 5. P.721–731.
Nicolia A., Ferradini N., Molla G. et al. Expression of an evolved engineered variant of a bacterial glycine oxidase leads to glyphosate resistance in alfalfa J. Biotechnol. 2014. V.184. P.201–208.
Vencill W.K., Nichols R.L., Webster T.M., et al. Herbicide resistance: toward an understanding of resistance development and the impact of herbicide-resistant crops. Weed Science. 2012. Special Issue. P.2–30.
Гочева Є.А., Сахно Л.О., Кучук М.В. Пат. 39205 UA 51 МПК А01Н1/00; А01Н4/00; А01Н5/00; С12N1/00; С12N5/00; С12N15/00. Спосіб отримання трансформованих рослин ріпаку методом агробактеріальної трансформації. Заявл. 03.10.2008. Публ. 10.02.2009, бюл. № 3.
Cheung W.Y., Hubert N., Landry B.S. A simple and rapid DNA microextraction method for plant, animal and insect suitable for RAPD and other PCR analyses. PCR Meths Applics. 1993. V.3, N 1. P. 69–70.
Logermann J., Schell J., Willmitzer L. Improved method for the isolation of RNA from plant tissues. Anal Biochem. 1987. V.163. P.16–20.
Bradford M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle to protein dye binding. Anal Biochem. 1976. V.72. P.248–254.
Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962. V.15, N 3. Р.473–497.
Лакин Г.Ф. Биометрия. М.: Высш.школа, 1990. 352 с.
Wang J. X., Zhao F. Y., Xu P. Use of aroA-M1 as a selectable marker for Brassica napus transformation. Crop Sci. 2006. V.46, N 2. Р.706–711.
Zang N., Zhai H., Gao S., et al. Efﬁcient production of transgenic plants using the bar gene for herbicide resistance in sweet potato. Scientia Horticulturae. 2009. V.122, N 4. P.649–653.
Song G.-Q., Sink K.C., Callow P.W., et al. Evaluation of a herbicide-resistant trait conferred by the bar gene driven by four distinct promoters in transgenic blueberry plants. JASHS. 2008. V.133, N. 4. P.605–611.
Jardak-Jamoussi R., Bouamama B., Mliki A., et al. The use of phosphinothricin resistance as selectable marker for genetic transformation of grapevine. Vitis. 2008. V.47, N 1. P.35–37.
Lohar D.P., Schuller K., Buzas D. M. еt al. Transformation of Lotus japonicus using the herbicide resistance bar gene as a selectable marker. J. Exp. Bot. 2001. V. 52, N 361. P.1697–1702.
De Block M., Botterman J., Vandewiele M. et al. Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J. 1987. V.6, N 9. P.2513–2518.
Spivak S. G. Berdichevets I. N., Yarmolinsky D. G., et al. Construction and characteristics of transgenic tobacco Nicotiana tabacum L. plants expressing CYP11A1 cDNA encoding cytochrome P450SCC. Rus. J. Genet. 2009. V. 45, N 9. P.1067–1073.
Vila-Aiub M.M., GohS.S., Gaines T.A., et al. No fitness cost of glyphosate resistance endowed by massive EPSPS gene amplification in Amaranthus palmeri. Planta. 2014. V.239, N 4. P.793–801.