Biopolym. Cell. 2020; 36(6):423-432.
http://dx.doi.org/10.7124/bc.000A40
Molecular and Cell Biotechnologies
Creation of winter rapeseed Brassica napus L. commercial line of biotechnological plants, resistant to the glyphosate action
1, 2Hnatiuk I. S., 1, 2Varchenko O. I., 1Kuchuk M. V., 2, 3Parii M. F., 1, 2Symonenko Yu. V.
  1. Institute of Cell Biology and Genetic Engineering, NAS of Ukraine
    148, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03143
  2. Ltd “Ukrainian Scientific Institute of Plant Breeding” (VNIS)
    30, Vasylkivska Str., Kyiv, Ukraine, 03022
  3. National University of Life and Environmental Sciences of Ukraine
    15, Heroiv Oborony Str., Kyiv, Ukraine, 03041

Abstract

Aim. To create biotechnological plants of a commercial line winter rapeseed, which carry the glyphosate and phosphinostricin resistance genes. Methods. In vitro tissue culture method, Agrobacterium-mediated genetic transformation, polymerase chain reaction and χ2 methods were used. Results. The PCR analysis results are presented for the biotechnological winter rapeseed plants obtained after genetic transformation to confirm the presence of CP4 epsps gene insert in 11 of 12 plants, as well as the presence of bar gene integration in 6 of 7 plants. All obtained regenerant plants were adapted to in vivo conditions, treated with glyphosate and vernalized. The obtained seeds were sterile germinated and the disjoining was determined based on the resistance to the selective agent. Conclusion. 11 biotechnological plants with the glyphosate (epsps) and kanamycin (nptII) resistance genes were obtained, as well as 6 biotechnological plants, which carry [the] phosphinothricin (bar) resistance genes in addition to the epsps gene. The presence of the CP4 EPSPS protein in transgenic rapeseed plants has been proven by the molecular analysis of protein expression using an immunoassay system. The expression of selective agents resistance genes was confirmed in T1 generation.
Keywords: Brassica napus, winter rapeseed, bar gene, epsps gene, nptII gene, genetic transformation
Full text: (PDF, in English)

References

[1] Gianessi LP. The increasing importance of herbicides in worldwide crop production. Pest Manag Sci. 2013;69(10):1099-105.
[2] Imran M, Asad S, Barboza AL, Galeano E, Carrer H, Mukhtar Z. Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide. Physiol Mol Biol Plants. 2017;23(2):453-460.
[3] Barry G, Kishore G, Padgette S, Taylor M, Kolacz K, Weldon M, Re D, Eichholtz D, Fincher K, Hallas L. Inhibitors of amino acid biosynthesis: strategies for imparting glyphosate tolerance to crop plants. In: Singh BK, Flores HE, Shannon JC (eds) Biosynthesis and molecular regulation of amino acids in plants. American Society of Plant Physiologists. 1992; 7:139-45.
[4] Kishore GM, Padgette SR, Fraley RT. History of herbicide-tolerant crops, methods of development and current state of the artemphasis on glyphosate tolerance. Weed Technology. 1992; 6(3):626-34.
[5] Zhao Z, Gu W, Cai T, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D. High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed. 2001; 8:323-33.
[6] Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, Beachy RN, Fauquet C. Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed. 2001; 7:25-33.
[7] Sakhno LO. Plant biomass increase: recent advances in genetic engineering. Biopolym Cell. 2013; 29(6):443-53.
[8] Sakhno LO, Lystvan KV, Kuchuk MV. Antioxidant activity of leaf of biotechnological rape (Brassica napus L.), resistant to herbicides based on glyphosate and glufosinate. Visnyk Kharkivskoho natsionalnoho ahrarnoho universytetu, Seriia: Biolohiia. 2015; 3(36):62-70.
[9] Sakhno LO, Komarnitsskii IK, Kuchuk MV. Inheritance of glyphosate and glufosinate resistance in T1-T2 generations of biotechnological canola (Brassica napus L.) plants. Visnyk Ukrainskoho tovarystva henetykiv i selektsioneriv. 2015; 13(1):3-10.
[10] Murashige T, Skoog FA. Revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962; 15:473-97.
[11] Hnatyuk IS, Varchenko OI, Kuchuk MV, Parii MF, Symonenko YuV. Development of an effective in vitro regeneration system for Ukrainian breeding winter rape Brassica napus L. Cytol Genet. 2020; 54(4):341-6.
[12] Hnatyuk IS, Varchenko OI, Bannikova MA, Kuchuk MV, Parii MF, Symonenko YuV. Development of an effective technique for in vitro Agrobacterium-mediated genetic transformation of winter rape Brassica napus L. AgroLife Scientific Journal. 2020; 9(1):149-55.
[13] Bertani G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951; 62(3):293-300.
[14] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989; 1626 p.
[15] Mashayekhi M, Shakib AM, Ahmad-Raji M, Ghasemi Bezdi K. Gene transformation potential of commercial canola (Brassica napus L.) cultivars using cotyledon and hypocotyl explants. Afr J Biotechnol. 2008; 7(24):4459-63.
[16] Rahnama H, Sheykhhasan M. Transformation and light inducible expression of cry1ab gene in oilseed rape (Brassica napus L.). J Sci Islamic Repub Iran. 2016; 27(4):313-9.
[17] Cardoza V, Stewart CN. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep. 2003;21(6):599-604. doi: 10.1007/s00299-002-0560-y.
[18] Savelieva EM, Tarakanov IG. Control of flowering in canola plants with various response to photoperiodic and low-temperature induction. Izvestiya of Timiryazev Agricultural Academy. 2014; 2:57-68.
[19] Filek M, Koscielniak J, Macháčková I, Krekule J. Generative development of winter rape (Brassica napus L.) - The role of vernalization. Int J Plant Develop Biol. 2007; 1(1):57-63.
[20] Waalen WM, Stavang JA, Olsen JE, Rognli OA. The relationship between vernalization saturation and the main-tenance of freezing tolerance in winter rapeseed. Environ Exp Bot. 2014; 106:164-73.