OPTIMIZATION OF CELL LINES GROWTH PARAMETERS OF SOME DICOTYLEDONOUS PLANTS CULTURED IN SUSPENSION CULTURE

Seven species of dicotyledonous plants selected for their ability to accumulate high levels of total soluble protein in vegetative tissues were introduced into in vitro culture. To obtain an intensively growing loose callus, suitable for the preparation of a suspension culture, nutrient media compositions were optimized and the morphogenetic reactions of various types of explants were evaluated. After screening for the ability of callus formation of seven species of dicotyledonous plants, it was established that fast-growing suspension lines can be obtained for the cells of three plant species - Nicotiana tabacum, Daucus сarota, Amaranthus hypochondriacus, using the noted protocols of media and explant types. Based on the evaluation of the growth characteristics of the suspension cultures obtained from the calli of the three isolated plant species, it was established that all three cell lines analyzed were characterized by high viability and intensive biomass increment. The results of a comparative analysis of the growth of biomass in the three analyzed cell cultures revealed that the cell density increased most in the suspension culture of A. hypochondriacus. Using the reporter gfp-gene, the selected cell lines were evaluated for their ability to accumulate a recombinant protein. A direct correlation was observed between the accumulation of total soluble and recombinant proteins in the cells of the suspension culture. Cell cultures obtained on the basis of these plant species are of interest for biotechnology and will be used to create production lines for recombinant proteins, including medicinal products.

Plant cell culture, biotechnology, transgenesis, agrobacterial transformation, recombinant protein, GFP, культуры растительных клеток, биотехнология, трансгенез, агробактериальная трансформация, рекомбинантный белок, GFP

1. Пермякова Н. В., Уварова Е. А., Дейнеко Е. В. Состояние исследований в области создания растительных вакцин ветеринарного назначения// Физиология растений. -2015. - Т. 62, № 1. - С. 28-44.

2. Large-scale production of pharmaceutical proteins in plant cell culture - the protalix experience / Y. Tekoah, A. Shulman, T. Kizhner [et al.] [Electronic resources] // Plant Biotechnology Journal. - 2015. - Vol. 13. - P. 1199-1208. - URL: http://onlinelibrary.wiley.com/doi/10.1111/pbi.12428/epdf.

3. Persistence Market Research. Global Market Study on Biopharmaceuticals: Asia to Witness Highest Growth by 2020. - NY, 2015 [Electronic resources]. - URL: http://www.persistencemarketresearch.com/market-research/biopharmaceutical-market.asp.

4. Nagata T., Nemoto Y., Hasezawa S. Tobacco BY-2 cell line as the «HeLa» cell in the cell biology of higher plants// International Review of Cytology 132. - 1992. - P. 1-30.

5. Yang S.-T. Bioprocessing for Value-Added Products from Renewable Resources: New Technologies and Applications. - Elsevier, 2011. - 684 p.

6. Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures / N. S. Kim, H. Y. Yu, N. D. Chung [et al.] [Electronic resources] // Protein Expr Purif. - 2011. - Vol. 76. - P. 121-126. - URL: http://www.sciencedirect.com/science/article/pii/S1046592810002846.

7. Identification and сharacterization of a heat-inducible ftsH gene from tomato (Lycopersicon esculentum Mill.) / A. Q. Sun, S.Y Yi., Yang J. Y. [et al.] // Plant Sci. - 2006. - Vol. 170. - P. 551-562.

8. Physical characteristics and nutritional composition of some new soybean (Glycine max (L.) Merrill) genotypes / S. Sharma, M. Kaur, R. Goyal [et al.] // [Electronic resources] // J. Food Sci. Technol. - 2014. - Vol. 51, N 3. - P. 551-557. - URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931876/.

9. Горковенко Л. Г., Потехин С. А., Кондратьева Л. Ф. Рациональное использование протеина люцерны // Зоотехния. - 2010. - № 3. - С. 12-15.

10. Красовская А. В., Веремей Т. М. Сравнительное изучение зернобобовых культур в Западной Сибири// Изв. Оренбург. гос. аграр. ун-та. - 2010. - № 1 (25). - С.14-17.

11. Antioxidant activity and nutritional assessment of under-utilized medicinal plants / A. A. Shad, J. Bakht, H. U. Shah [et al.] // Pak. J. Pharm. Sci. - 2016. - Vol.29, N 6. - P. 2039-2045.

12. Quality of carrots as affected by pre- and postharvest factors and processing / R. Seljasen, H. L. Kristensen, C. Lauridsen [et al.] // Sci Food Agric. - 2013. - Vol.93, N 11. - P. 2611-2626.

13. Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals. / R. Colgan, C. J. Atkinson [et al.] // Transgenic Res. - 2010. - Vol. 19, N 2. - P. 241-256.

14. Murashige T., Skoog F. A revised medium for rapid growth and bioassay with tobacco tissue cultures// Physiol Plantarum. -1962. - Vol. 15. - P. 473-497.

15. Gamborg O. L., Miller R. A., Ojima K. Nutrient requirements of suspension cultures of soybean root cells// Exp. Cell Res. -1968. - Vol. 50. - P. 151-158.

16. Паушева З. П. Практикум по цитологии растений. - М.: Агропромиздат, 1988. - 271 c.

17. Use of Transgenic Carrot Plants Producing Human Interleukin-18 for Modulation of Mouse Immune Reactions / U. V. Yakushenko, Yu.A. Hrapov, E. V. Deineko [et al.] // New research on Biotechnology and Medicine/ Eds.: A. M. Egorov, G. E. Zaikov. - Nova Science Publishers, NY, 2006. - 762 p.

18. Bradford M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding// Anal. Biochem. - 1976. - Vol. 72. - P. 248-254.

19. Мокроносов А. Т., Березенкова Р. А. Методика количественной оценки структуры и функциональной активности фотосинтезирующих тканей и органов// Тр. по приклад. ботанике, генетике и селекции. - 1978. - Т. 61, вып. 3. - С. 119-133.