Agrobacterium-mediated transformation of potato with human lactoferrin gene

  • A. Yu. Buziashvili
  • A. I. Yemets
DOI: https://doi.org/10.7124/FEEO.v25.1162

Abstract

Aim. Obtaining of the new Ukrainian potato cultivars with complex resistance to phytopathogens. Methods. Agrobacterium-mediated transformation of potato cultivars Vernisage and Zarevo with human lactoferrin gene possessing antimicrobial activity was carried out. For transformation, supervirulent A. tumefaciens strain EHA 105 with hLf gene and gene for the resistance to kanamycine was used. Selection was carried out for 3 months on MSK-S1 and MSK-S2 media supplemented with 100 mg/l kanamycine. Integration of lactoferrin gene in transgenic plant lines was confirmed by polymerase chain reaction with specific for hLf gene primers. Results. After selection, potato lines with the resistance to kanamycine were obtained. The transgenic potato lines with integrated into their genomes hLf gene were obtained. Integration of lactoferrin gene was confirmed for 1 line of Zarevo cultivar and 3 lines of Vernisage cultivar. Transformation frequency was of 6.8% for cv. Vernisage and 6.25% for cv. Zarevo. Conclusions. Selected potato lines would be used in further studies on their resistance to bacterial and fungal pathogens.

Keywords: human lactoferrin gene hLf, Solanum tuberosum, Agrobacterium-mediated transformation, transgenic plants.

References

Nemerytska L.V. The causals of the mixed potato rots and limitation measures of their spreading under the Ukrainian Polissya conditions: avtoref. dys. of cand. of biol. nauk. Kyiv, 2005. 23 p. [in Ukrainian]

Klyachenko О.L., Koltunov V.А., Boroday V.V., Kozhemyakina L.М., Voyceshina N.І. The features of in vitro growth and development of different potato cultivars depending on the composition of culture medium. Potato Gr. 2012. Vol. 41. P. 103–111. [in Ukrainian]

Zaviriuha P.D., Tymoshenko І.І. Potato selection in Lviv NAU: theoretical and applied aspects. Mater. of Іnternat. scientif. and pract. forum. Lviv, 2009. Vol. 1. P. 122–127. [in Ukrainian]

Yemets A., Tanasienko I., Krasylenko Yu., Blume Ya. Plant-based biopharming of recombinant human lactoferrin. Cell Biol. Int. 2014. Vol. 38. P. 989–1002. doi: 10.1002/cbin.10304.

Lakshman D.K., Natarajan S., Mandal S., Mitra A. Lactoferrin derived resistance against plant pathogens in transgenic plants. J. Agric. Food Chem. 2013. Vol. 61, № 48. P. 11730–11735. doi: 10.1021/ jf400756t.

Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962. Vol. 15. P. 473–497.

Buziashvili А., Yemets А. Obtaining of tomato and potato plants with human lactoferrin gene hLf . Rep. of the Nat. Acad. Of Sci. of Ukraine. 2018. Vol. 10. P. 88–94. doi: https://doi.org/10.15407/dopovidi2018.10.088 [in Ukrainian]

Rogers S., Bendich A. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Molec. Biol. 1985. Vol. 5. P. 69–76. doi: 10.1007/BF00020088.

Zhuk V.P., Oliynyk Т.М., Yemets А.І., Blume Ya.B. Regeneration and genetic transformation of Ukrainian potato cultivars with synthetic CRY-genes. Collent. of scientif. papers of Nikit. bot. garden. 2009. Vol. 131. P. 197–201. [in Ukrainian]

Veale M.A., Slabbert M.M., van Emmenes L. Agrobacterium-mediated transformation of potato cv. Mnandi for resistance to the potato tuber moth (Phthorimaea operculella). South Afr. Jour. of Bot. 2012. Vol. 80. P. 67–74. doi: 10.1016/j.sajb.2012.02.007

El-Shawaf I., Hasan A., Bekhit M., Hasanein E., Salim T., Hagemann M. Agrobacterium-mediated transformation of potato. Arab J. Biotech. 2013. Vol. 16, № 1. P. 91–104.

Han E.H., Goo Y.M., Lee M.K., Lee S.W. An efficient transformation method for a potato (Solanum tuberosum L. var. Atlantic). J. Plant Biotechnol. 2015. Vol. 42. P. 77–82. doi: 10.5010/JPB.2015.42.2.77.