The influence of in vitro and ex vitro conditions cultivation on free proline contents in plants of some Gentiana L. species

  • L. R. Hrytsak Ternopil Volodymyr Hnatiuk National Pedagogical University, Ukraine, 46027, Ternopil, M. Kryvonosa str., 2
  • N. V. Nuzhyna 2Educational and Scientific Centre “Institute of Biology and Medicine” of Taras Shevchenko National University of Kyiv, Ukraine, 03022, Kyiv, prospekt Akademika Hlushkova 2
  • N. M. Drobyk Ternopil Volodymyr Hnatiuk National Pedagogical University, Ukraine, 46027, Ternopil, M. Kryvonosa str., 2
Keywords: Gentiana L., plants in vitro, plants ex vitro, free proline, light conditions of cultivation


Aim. To establish the dependence of free proline concentration in plant tissues of in vitro and ex vitro alpine species of Gentiana lutea L., Gentiana punctata L., Gentiana acaulis L. on light conditions of their cultivation and the source of carbon in the nutrient medium; to analyze expediency of free proline amino acid use as a biological marker of physiological adaptation of biotechnological plants of these species to water deficit of in vitro and ex vitro conditions. Methods. Methods of in vitro and ex vitro cultivation of plants, the method of free proline detection with the use of ninhydrin. Results. It is shown that under in vitro conditions of free proline content in plants is dependent on light regime of their cultivation and the source of carbon in the composition of nutrient medium. The increased intensity of luminous flux within the range of photosynthetically active radiation (PAR) from 85 W/m2 to 100 W/m2 and 1.92 times raised share of red range waves in the light spectral composition in variant 2.1 (intensity of the luminous flux in the PAR range is 100  W/m2, the waves correlation of blue (Еb): green (Еg): red (Еr) ranges = 25 % : 27 % : 48 %) causes 11.5–37.1 %  increased amount of free proline in plants in vitro cultivated on nutrient media supplemented with sucrose. Substitution of sucrose in the medium for mannitol is accompanied by 1.64–1.84 times increased concentration of free proline in plants of the investigated species under the light conditions of 1.1 variant (85 W/m2, spectral composition Еb : Еg : Еr = 25 % : 27 % : 48 %= 33 % : 42 % : 25 %) and 1.3–2.57 times increased under light cultivation regime of 2.1 variant. The analysis of water balance of plants in vitro cultivated on mannitol under light conditions of 2.1 variant and the plants in conditions of natural growth doesn’t show any considerable distinctions in values of transpiration intensity, water deficit, general water content. The process of adaptation of plants in vitro to conditions ex vitro is accompanied by a change of proline content in leaves with dependence on water deficit in the substrate and light conditions of growing. Conclusions. The obtained results point to the expediency of using free proline content as a biochemical marker for assessing adaptive potential of plants in conditions in vitro and ex vitro.


Mathur A., Mathur A. K., Verma P. Biological hardening and genetic fidelity testing of microcloned progeny of Chlorophytum borivilianum. African Journal of Biotechnology. 2008. Vol. 7 (8). P. 1046–1053. URL:

Isah T. Adjustments to in vitro culture conditions and associated anomalies in plants. Acta biologica Cracoviensia. Series Botanica. 2015. Vol. 57/2. P. 9–28. doi: 10.1515/abcsb-2015-0026.

Sergieyeva L.Ye., Bronnikova L.I. Transformation proline in winter wheat shoots, obtained after genetic. Bìorìznomanìttâ, ekologìâ ta eksperimentalʹna bìologìâ. 2020. Vol. 22, No.1. P. 108–116 [in Ukrainian].

Hayat S., Hayat Q., Alyemeni M. N., Wani A. S., Pichtel J., Ahmad A. Role of proline under changing environments. Plant Signaling & Behavior. 2012. Vol. 7, No. 11. Р. 1456–1466. doі: 10.4161/psb.21949.

Phang J. M., Liu W., Zabirnyk O. Proline metabolism and microenvironmental stress. Annual Review of Nutrition. 2010. Vol. 30. P. 441–463. doi: 10.1146/annurev.nutr.012809.104638.

Kavi Kishor P. B., Hima Kumari P., Sunita M. S, Sreenivasulu N. Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny. Front Plant Sci. 2015. Vol. 6. 544. doi: 10.3389/fpls.2015.00544.

Hosseinifard M., Stefaniak S., Ghorbani J. M., Soltani E., Wojtyla Ł., Garnczarska M. Contribution of exogenous proline to abiotic stresses tolerance in plants: A Review. International Journal of Molecular Sciences. 2022. Vol. 23, No. 9. 5186. doi: 10.3390/ijms23095186

Dovgajuk-Semenuk M. V., Velychko O. І., Terek O. І. The content of free amino acids in the red clover plants under the influ-ence of oil polluted soil. Studia Biologica. 2016. Vol. 10, No.2. P. 115–122 [in Ukrainian].

Komisarenko A. G., Mykhalskaya S. I. The free proline levels in transgenic sunflower (Helianthus annuus L.) T3 plants with double-stranded proline dehydrogenase gene RNA-suppressor. Factors in Experimental Evolution of Organisms. 2017. Vol. 20. P. 211–214 [in Ukrainian]. doi: 10.7124/FEEO.v20.766.

Singh A., Sengar K., Sharma M. K., Sengar R.S., Garg S. K. Proline metabolism as sensors of abiotic stress in sugarcane // Biotechnology to Enhance Sugarcane Productivity and Stress Tolerance / ed by Sengar K. Boca Raton : CRC Press, 2018. doi: 10.1201/9781315152776.

Lédo A. S., Moura C. R. F., Machado C. A. et al. Mannitol for coconut ex situ conservation by minimum growth. Pesquisa Agropecuária Brasileira. 2014. Vol. 49, № 2. P. 148–151. doi: 10.1590/S0100-204X2014000200010.

Dragolova D., Stefanova M., Dimitrova M, Koleva D., Zhiponova M., Kapchina-Toteva V. In vitro cultivation and ex vitro adaptation of Nepeta nuda ssp. nuda – correlation between regeneration potential, leaf anatomy, and plastid pigments. Bulgarian Journal of Agricultural Science. 2015. Vol. 21, № 5. P. 1027–1032.