Получение трансгенных линий сахарной свеклы, содержащих синтетический ген cry1C
Анотація
Aim. Insect pest’s impact makes a significant limitation of the sugar beet crop yield. Integration of cry-genes of Bacillus thuringiensis into the plant genome is one of the promising strategies to ensure of plant resistance. The aim of this work was the production of sugar beet lines (based on the MM1/2 line) expressing cry1C genes. Methods. Genetic transformation of sugar beet was performed using the method of co-cultivation of leaf explants with Agrobacterium tumefaciens. Results. Sugar beet line MM1/2 was transformed by Agrobacterium-mediated method of transformation using binary vector pRD400-cry1CST, containing synthetic cry1C gene and selectable marker gene neomycin phosphotransferase II (nptII) conferring resistance to kanamycin. After the optimization protocol of genetic transformation and direct regeneration from leaf discs a transgenic sugarbeet lines were obtained. Conclusions. PCR analysis revealed integration of cry1C into the genome of transgenic lines of B. vulgaris.
Keywords: genetic transformation, Agrobacterium tumefaciens, Beta vulgaris, cry-genes.
Посилання
Rankovic J., Dodic J., Dodic S., Popov S. Bioethanol production from intermediate products of sugar beet processing with different types of Saccharomyces cerevisiae. Chem. Ind. & Chem. Engineer. Quarter. 2009. V. 15. P. 13−16. doi: 10.2298/CICEQ0901013R
Dodic S., Popov S., Dodic J., Rankovic J., Zavargo Z., Mucibabic R.J. Bioethanol production from thick juice as intermediate of sugar beet processing. Biomass and Bioenergy. 2009. V. 33. P. 822-827. doi: 10.1016/j.biombioe.2009.01.002
Menzel G., Harloff H.J., Jung C. Expression of bacterial poly(3-hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.). Appl. Microbiol. Biotechnol. 2003. V. 60. P. 571-576. doi: 10.1007/s00253-002-1152-z
Margara J. Néoformation de bourgeons in vitro chez la Betterave sucrière (Beta vulgaris L.). C.R. Hebd. Seances Acad. Sci. 1970. V. 270. P. 698-701.
Gurel E., Wren M. J. In vitro development from leaf explants of sugar beet (Beta vulgaris L.). rhizogenesis and the effect of sequential exposure to auxin and cytokinin. Annals of Botany. 1995. V. 75. P. 31-38. doi: 10.1016/S0305-7364(05)80006-X
Snyder G.W., Ingersoll J.C., Smigocki A.C., Owens L.D. Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugar beet (Beta vulgaris L.) by Agrobacterium or particle bombardment. Plant Cell Rep. 1999. V. 18. P. 829-834. doi: 10.1007/s002990050669
Bannikova M.A., Golovko A.E., Khvedynich O.A., Kuchuk N.V., Gleba Y.Y. Regeneration of sugar beet (Beta vulgaris L.) plants in in vitro culture. Histological analysis of regeneration. Cytology and Genetics. 1995. V. 29. P. 14-21.
Norouzi P., Malboobi M.A., Zamani K., Yazdi-Samadi B. Using a competent tissue for efficient transformation of sugarbeet (Beta vulgaris L.). In Vitro Cell. Dev. Biol. 2005. V. 41. P. 11-16. doi: 10.1079/IVP2004589
Kishchenko E.M., Komarnitskii I.K., Gleba Yu.Yu., Kuchuk N.V. Production of transgenic sugarbeet (Beta vulgaris L.) plants of O-type by Agrobacterium tumefaciens, Tsitol Genet. 2004. V. 38(5). P. 3-8.
Jafari M., Norouzi P., Malboobi M.A. et al. Enhanced resistance to a lepidopteran pest in transgenic sugar beet plants expressing synthetic cry1Ab gene. Euphytica. 2009. V. 165. P. 333-344. doi: 10.1007/s10681-008-9792-4
Ingersoll J.C., Huette T.M., Owens L.D. Effect of promoter-leader sequences on transient expression of reporter gene chimeras biolistically transferred into sugarbeet (Beta vulgaris) suspension cells. Plant Cell. Rep. 1996. V. 15. P. 836-840. doi: 10.1007/s002990050131
Golovko A. E., Dovzhenko A.A., Gleba Yu.Yu. Genetycheskaya transformaciya sakharnoi svekly: evolyuciya vzglyadov i metodicheskikh podkhodov. Cytologiya i genetika. 2005. V. 39(3). P. 30-36.
De Marchis F., Wang Y., Stevanato P., Arcioni S., Bellucci M. Genetic transformation of the sugar beet plastome. Italian National Research Council. Transgenic Res. 2009. V. 18. P. 17-30. doi: 10.1007/s11248-008-9193-4
Fromm M.E., Taylor L.P., Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Nat. Acad. Sci. U.S.A, 1985. V. 82. P. 5824-5825. doi: 10.1073/pnas.82.17.5824
Lindsey K., Jones M.G.K. Stable transformation of sugarbeet protoplasts by electroporation. Plant Cell Reports. 1989. V. 8. P. 71-74. doi: 10.1007/BF00716841
Lytvyn D. I., Syvura V. V., Kurylo V. V., Olenieva V. D., Yemets A. I., Blume Ya. B. Creation of transgenic sugar beet lines expressing insect pest resistance genes cry1C and cry2A, Cytol Genet. 2014. V. 48(2). P. 69-75. doi: 10.3103/S0095452714020078
Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 1962. V. 15. P. 473-476. doi: 10.1111/j.1399-3054.1962.tb08052.x
Mishutkina Ya.V., Gaponenko A.K. Sugar beet (Beta vulgaris L.) Morphogenesis in Vitro: Effects of Phytohormone Type and Concentration in the Culture Medium, Type of Explants, and Plant Genotype on Shoot Regeneration Frequency. Russian Journal of Genetics. 2006. V. 42. P. 150-157. doi: 10.1134/S1022795406020086
Bekheet S.A., Taha H.S., Maharik N.T. Response of Sugarbeet Tissue Cultures to Salinity and Identification of Tolerants Using RAPD Analysis. Journal of Applied Sciences Research. 2009. V. 5. P. 2515-2519.
Bekheet S.A., Taha H.S., Matter M.A. In vitro regeneration of sugarbeet propagules and molecular analysis of the regenerants. Arab J. Biotech. 2007. V. 10. P. 321-332.
Davidson M.M., Butler R.C., Wratten S.D., Conner A.J. Field evaluation of potato plants transgenic for a cry1Ac gene conferring resistance to potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae). Crop Protection. 2006. V. 25 - P. 216-224. doi: 10.1016/j.cropro.2005.04.010
Sedighi L., Rezapanah M., Aghdam H.R. Efficacy of Bt transgenic sugar beet lines expressing cry1Ab gene against Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae). J. Entomol. Res. Soc. 2011. V. 13. P. 61-69.
Coumans-Gilles M.F., Kevers C.I., Coumans M., Ceulemans E., Gaspar Th. Vegetative multiplication of sugarbeet through a vitro culture of inflorescence pieces. Plant Cell. Tiss. Org. Cult. 1981. V. 1. P. 93-101. doi: 10.1007/BF0231890824.
Kurylo V.V., Yemets A.I. Genetychna transformaciya czukrovogo buryaku syntetychnym genom cry1C. Faktory eksperymental'noi evoliutsii orhanizmiv. 2015. V. 17. P. 193–196.
Ahmad M.S., Shakoori A.R. Isolation, molecular characterization and toxicity of cry1C gene harboring Bacillus thuringiensis from different habitats and localities of Pakistan. Pakistan J. Zool. 2013. V. 45. P. 261–271.
