Physiological and biochemical analysis of transgenic wheat plants of seed generation T2 with double-stranded RNA suppressor of the proline dehydrogenase gene

  • S. S. Kulesh
  • O. V. Dubrovna
  • L. V. Slivka

Abstract

Aim. To carry out physiological and biochemical analysis of genetically modified plants of bread wheat of seed generation T2 with the double-stranded RNA suppressor of the proline dehydrogenase gene. Methods. Biochemical determination of free L-proline content and activity of the enzyme proline dehydrogenase; physiological examination of plant growth in vitro and in vivo. Results. It has been shown that transgenic plants, in contrast to control groups, grow more intensively in a selective medium with mannitol, maintaining a green color. It has been established that under normal conditions and under conditions of water deficit, plants of the T2 have an elevated level of free proline in the leaves, compared with the control genotypes. It was found that transformants are characterized by reduced activity of the enzyme of proline dehydrogenase, which manifests itself when the norm-stress-norm conditions change. Transgenic T2 plants had a higher resistance to aqueous deficiency compared to baseline, which was reflected in the nature of their growth. In conditions of soil moisture shortage, the yield of the most of the transformed lines was significantly higher than non-transformed plants. Conclusions. The obtained results allow us to conclude that the use of the pBi2E vector construct with the double-stranded RNA suppressor of the pdh gene is effective for the production of transgenic bread wheat plants with a high level of resistance to water deficiency.

Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, RNA suppressor of the proline dehydrogenase gene, T2 plants, physiological-biochemical analysis.

References

Bhalla P.L., Ottenhof H.H., Singh M.B. Wheat transformation – an update of recent progress. Euphytica. 2006. Vol. 149 (3). P. 353–366. doi: 10.1007/s10681-006-9087-6.

Sparks C., Doherty A., Jones H. Genetic transformation of wheat via Agrobacterium-mediated DNA delivery. Methods Mol Biol. 2014. Vol. 1099. P. 235–250. doi: 10.1007/978-1-62703-715-0_19.

Kolodyazhna Ya.S., Kutsokon N.K., Levenko B.A., Syutikova O.S., Rakhmetov D.B., Kochetov A.V. Transgenic plants toler-ant to abiotic stresses. Cytology and Genetics. 2009. Vol. 43 (2). P. 72–93. [in Russian]

Lehmann S., Funck D., Szabados L., Rentsch D. Proline metabolism and transport in plant development. Amino Acids. 2010. Vol. 39 (4). P. 949–962. doi: 10.1007/s00726-010-0525-3.

Verbruggen N., Hermans C. Proline accumulation in plants: A review. Amino Acids. 2008. Vol. 35 (4). Р. 753–759. doi: 10.1007/s00726-008-0061-6.

Carvalho K., Campos M.K., Domingues D.S., Pereira L.F., Vieira L.G. The accumulation of endogenous proline induces changes in gene expression of several antioxidant enzymes in leaves of transgenic Swingle citrumelo. Mol. Biol. Rep. 2013. Vol. 40 (4). P. 3269–3279. doi: 10.1007/s11033-012-2402-5.

Titov S.E. Poluchenie geneticheski modificirovannykh rasteniI tabaka (Nicotiana tabacum L.), ekspressiruiushchikh antismyslovoI supresor gena prolindegidrogenazy: avtoref. dis. … kand. biol. nauk. Novosibirsk, 2008. 18 р. [in Russian]

Nanjo T., Kobayashi M., Yoshiba Y., Kakubari Y., Yamaguchi-Shinozaki K., Shinozaki K. Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Letters. 1999. Vol. 461 (3). P. 205–210.

Manavalan L.P., Chen X., Clarke J., Salmeron J., Nguyen H.T. RNAi-mediated disruption squalene synthase improves drought tolerance and yield in rise. Journal of Experimental Botany. 2012. Vol. 63 (1). P. 163–175. doi: 10.1093/jxb/err258.

Mykhalska S.I., Sergeeva L.E., Matveyeva A.Yu., Kobernik N.I., Kochetov A.V., Tishchenko O.M., Morgun V.V. The eleva-tion of free proline content in osmotolerant transgenic corn plants with dsrna suppressor of proline dehydrogenase gene. Plant Physiology and Genetics. 2014. Vol. 46 (6). P. 482–489. [in Russian]

Komisarenko A.G., Mykhalska S.I., Kurchii V.M, Tishchenko O.M. The characterization transgenic sunflower (Helianthus annuus L.) Plants with suppressor of proline dehydrogenase gene. Factors in Experimental Evolution of Organisms. 2016. Vol. 19. P. 143–147. [in Ukrainian]

Bavol A.V., Dubrovna O.V., Goncharuk О.М., Voronova S.S. Agrobacterium-mediated transformation of wheat using calli culture. Factors in Experimental Evolution of Organisms. 2014. Vol. 15. P. 16–19. [in Ukrainian]

Andriushchenko V.K., Saianova V.V., Zhuchenko A.A., D'iachenko N.I., Chilikina L.A., Drozdov V.V., Korochkina S.K., Cherep G.I., Medvedev V.V., Niutin Iu.I. Modification of the method for determining proline to identify drought-resistant forms of the genus Lycopersicon Tourn. Izvestiia Akademii Nauk Moldavskoi SSR. 1981. Vol. 4. P. 55–60. [in Russian]

Mattioni C., Lacerenza N.G., Troccoli A.D., De Leonardis A.M., Di Fonzo N. Water and salt stress-induced alterations in proline metabolism of Triticum durum seedlings. Physiol Plant. 1997. Vol. 101. P. 787–792. doi: 10.1111/j.1399-3054.1997.tb01064.x.