Establishment of glyphosate selective concentrations for winter rapeseed (Brassica napus L.) transgenic tissues efficient selection in vitro
Abstract
Aim. To establish the optimal concentration of glyphosate for efficient selection of transgenic tissue culture in biotechnology of rapeseed by in vitro selection of glyphosate-resistant and non-resistant winter rape. Methods. As an explants 7–10 mm fragments of 6-day rapeseed hypocotyls were used to cultivate on the medium, supplemented with 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) for 12 days at 24°C under dark conditions for the initiation of callusogenesis. For regeneration of plants, the MS nutrient medium was supplemented with 3 mg/L of 6-benzylaminopurine (BAP), 2 mg/L of zeatin, 0.1, 0.5 and 1 mM of glyphosate, respectively. As a control, glyphosate-free regenerative nutrient medium was used. The results were statistically processed using Microsoft Excel. Results. It has been shown that glyphosate-susceptible rape explants form leaves and shoots with a frequency up to 8.3 % ± 0.8 % on the medium supplemented with 0.1 mM glyphosate compared to 57.2 % ± 0.6 % in the control. However, the morphogenic structures did not pass selection after transferring shoots to the elongation medium supplemented with 0.1 mM glyphosate. Conclusions. For the in vitro selection of rapeseed 0.1 mM glyphosate can be used in a nutrient medium at the stage of adventitious buds formation. But at the regeneration stage, the amount of herbicide should be increased to avoid false-positive results.
Keywords: winter rapeseed, Brassica napus L., in vitro culture, selective marker, herbicide, glyphosate.
References
Nandula V.K. Glyphosate resistance in crops and weeds: history, development, and management. Wiley: Hoboken, 2010. 344 p.
Green J.M. Current state of herbicides in herbicide-resistant crops. Pest Manag Sci. 2014. Vol. 70, № 9. P. 1351–1357. doi: 10.1002/ps.3727.
USDA (2015) Determinations of non-regulated status. www.aphis.usda.gov/biotechnology/petitions_table_pending.shtml. Accessed 21 Sept 2015.
OECD (1999) Consensus document on general information concerning the genes and their enzymes that confer tolerance to glyphosate herbicide. Series on harmonization of regulatory oversight in biotechnology. Report No. 10. URL: https://www.oecd.org/env/ehs/biotrack/46815618.pdf. Accessed 24 Sept 2015 (Last accessed: 1.04.2019).
OECD (1999) Consensus document on general information concerning the genes and their enzymes that confer tolerance to Phosphinothricin herbicide. Series on harmonization of regulatory oversight in biotechnology. Report No. 11. URL: https://www.oecd.org/env/ehs/biotrack/46815628.pdf (Last accessed: 1.04.2019).
Schütte G., Eckerstorfer M., Rastelli V., Reichenbecher W., Restrepo-Vassalli S., Ruohonen-Lehto M., Wuest Saucy A.-G., Mertens M. Herbicide resistance and biodiversity: agronomic and environmental aspects of genetically modified herbicide-resistant plants. Environ Sci Eur. 2017. Vol. 29, № 5. doi: 10.1186/s12302-016-0100-y.
Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum. 1962. Vol. 15. P. 473–497.
Rahnama H., Sheykhhasan M. Transformation and light inducible expression of cry1Ab gene in oilseed rape (Brassica napus L.). J. Sci. I. R. Iran. 2016. Vol. 27, № 4. P. 313–319.
Xing X.-J., Tian Y.-S., Peng R.-H., Xu J., Zhao W., Yao Q.-H., Sun S. Functional characterization of 5-enopyruvylshikimate-3-phosphate synthase from Alkaliphilus metalliredigens in transgenic Arabidopsis. J. Microbiol. Biotechnol. 2014. Vol. 24, № 10. P. 1421–1426. URL: http://dx.doi.org/10.4014/jmb.1404.04023 (Last accessed: 1.04.2019).
Zhao Q., Liu M., Zhang X., Lin C., Zhang Q., Shen Z. Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene. J Zhejiang Univ-Sci B (Biomed & Biotechnol). 2015. Vol. 16, № 10. P. 824–831. doi: 10.1631/jzus.B1500056.
Hu T., Metz S., Chay C., Zhou H. P., Biest N., Chen G., Cheng M., Feng X., Radionenko M., Lu F., Fry J.. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep. 2003. Vol. 21. P. 1010–1019. doi: 10.1007/s00299-003-0617-6.
Howe A.R., Gasser C.S., Brown S.M., Padgette S.R., Hart J., Parker G.B., Eromm M.E., Armstrong C.L. Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants. Molecular breeding. 2002. Vol. 10. P. 153–164.
