ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
"Biotechnologia Acta" V. 8, No 2, 2015;
https://doi.org/10.15407/biotech8.02.052
Р. 52-57, bibliography 15, English
Universal Decimal classification: 577.21+604.6:582.983.2+631.348
A. M. Taranenko, L. O. Sakhno, B. V. Morgun, M. V. Kuchuk
Institute of Cell Biology and Genetic Engineering of the National Academyof Sciences of Ukraine, Kyiv
The purpose of research was to analyze 10 developed at the Institute of Cell Biology and Genetic Engineering lines of rapeseed to confirm the presence and functionality of the transferred transgene CP4 epsps, as well as the differences among those lines from registered transformation events GT73 and GT200 (Monsanto).
During the study extraction of total rapeseed DNA, PCR analysis, electrophoretic separation and visualization of amplicons in agarose gel were conducted, as well as testing of green plants for resistance to glyphosate in greenhouse. The structural difference among 7 transgenic lines from registered transformation events GT73 and GT200 was revealed. Plants showing the presence of synthetic CP4 epsps sequence were resistant to the herbicide in a closed soil. The uniqueness of the obtained transformation events was confirmed, as well as the prospect of using them in breeding.
Key words: rapeseed, glyphosate, genetic transformation, CP4 epsps gene.
© Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, 2015
References
1. Pests and diseases of rape. Available at ttp://www.agrocounsel.ru/vyraschivanie-rapsa.
2. Rape supply and demand balances, February 2012. Available at http://www.ukragroconsult.com/data/analytics/balansy-sprosa-i-predlozheniya-rapsa-fevral-2012g
3. Opinion of the Scientific Panel on Genetically Modified Organisms on a request from the Commission related to the Notification (Reference C/NL/98/11) for the placing on the market of herbicide-tolerant oilseed rape GT73, for import and processing, under Part C of Directive 2001/18/EC from Monsanto. EFSA J., 2004, V. 29, P. 1–19.
4. GMO Detection method Database (GMDD) – Event Information GT73. Available at http://www.gmdd.shgmo.org/event/view/109
5. GMO Detection method Database (GMDD) – Event Information GT200. Available at http://www.gmdd.shgmo.org/event/view/108
6. Sakhno L. A., Gocheva E. A., Komarnitsky I. K., Kuchuk N. V. Stable expression of promoterless bar gene in transgenic rape plants. Tsitol. Genet. 2008, 42 (1), 21–28. (In Russian).
7. Sakhno L. O., Gerasymenko I. M., Komarnitsskii I. K., Sheludko Y. V., Goldenkova-Pavlova I. V. Creation of glyphosate-resistant Brassica napus L. plants expressing DesC desaturase of cyanobacterium Synechococcus vulcanus. Biopolymers and Cell. 2012, 28 (6), 449–455. (In Russian).
http://dx.doi.org/10.7124/bc.000135
8. Sakhno L. O., Komarnitsskii I. K., Maystrov P. D., Kuchuk M. V. Creation of rapeseed plants resistant to glyphosate-based herbicides by introduction a synthetic gene epsps. Factors in Experimental Evolution of Organisms. Collected Works, edited by Kunah V. A. Kyiv: Lohos, 2011, V. 11, P. 388–393. (In Ukrainian).
9. Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962, V. 15, P. 473–497.
http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x
10. Sakhno L. O., Morgun B. V., Kvasko E. J., Kuchuk N. V. Creation of transformed rapeseed plants expressing animal gene cyp11A1 of P450SCC cytochrome. Biotekhnolohiia. 2010, 3 (5), 74–82. (In Russian).
11. Morgun B. V., Stepanenko O. V., Stepanenko A. I. Method for detecting the rapeseed transformation event GT73 by multiplexed PCR. Ukraine Patent 2013 09296, July 24, 2013.
12. Somma M. Extraction and Purification of DNA. Session 4. In: Training Course on the Analysis of Food Samples for the Presence of Genetically Modified Organisms - User Manual. Edited by M. Querci, M. Jermini, G. Van den Eede. European Commission, DG Joint Research Centre, Institute for Health and Consumer Protection. Luxembourg, 2006, 229 p.
13. Sambrook J., Fritsch E. F., Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 1989, 1626 p.
14. NCBI database. Brassica napus cruA gene for cruciferin. Available at http://www.ncbi.nlm.nih.gov/nuccore/X14555.
15. NCBI database. Synthetic construct CP4 EPSPS glyphosate tolerance protein gene. Available at http://www.ncbi.nlm.nih.gov/nuccore/JF445290.