Biopolym. Cell. 2018; 34(6):461-476.
http://dx.doi.org/10.7124/bc.000991
Molecular and Cell Biotechnologies
Establishment and analysis of tissue and fast-growing normal root cultures of four Gentiana L. species, rare highland medicinal plants
1Drobyk N. M., 2Mel'nyk V. M., 1Hrytsak L. R., 1Kravets N. B., 2Konvalyuk I. I., 2Twardovska M. O., 2Kunakh V. A.
  1. Volodymyr Hnatiuk Ternopil National Pedagogical University
    2, M. Kryvonos Str., Ternopil', Ukraine, 46027
  2. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143

Abstract

Aim. To obtain tissue and isolated root cultures of four Gentiana L. species from Ukraine (G. lutea L., G. punctata L., G. acaulis L. and G. asclepiadea L.) and study peculiarities of their growth and content of flavonoids and xanthones. Methods. In vitro culture, chromatography, spectrophotometry and statistical methods. Results. The conditions were developed for callus induction, proliferation, and long-term maintenance of fast-growing root cultures from gentians. A comparative study of total flavonoid and xanthone content in the cultured tissues, isolated cultured roots and wild plants of gentians was carried out. The capacity was ascertained for synthesizing these biologically active substances in vitro. The content of compounds varied both in calli and isolated roots derived from the plants of different gentian species, and in tissue and organ cultures of these species. The morphogenic and non-morphogenic cultures showed much lower flavonoid and xanthone content than the shoots of intact plants, but similar to that of natural roots. The fast-growing normal root cultures displayed higher concentrations of these biologically active compounds than the callus tissues in most cases. Conclusions. A high yield of biomass from gentian cultures in vitro and their ability to synthesize and accumulate flavonoids and xanthones make them as a promising source of these biologically active compounds.
Keywords: Gentiana L. species, callus induction and proliferation, fast-growing normal root cultures, flavonoid and xanthone content, source of the biologically active compounds
Full text: (PDF, in English)

References

[1] Tutin TG. Flora Europea. vol 3. Cambridge: Cambridge University Press. 1972;374 p.
[2] Ho T-N, Liu S-W. The infrageneric classification of Gentiana (Gentianaceae). Bull British museum Natural History (Botany). 1990; 20(2):169–92.
[3] Strashniuk NM, Hrytsak LR, Les'kova OM, Mel'nyk VM. Gentiana L. species of Ukrainian flora in nature and in culture in vitro. Ukr Bot J. 2005; 62(3):337–48.
[4] Shiyan NM. A review of the taxonomy and distribution of the Gentianaceae in the Ukraine. In: Eds. Rybczyński JJ, Davey MR, Mikula A The Gentianaceae – Volume 1: Characterization and Ecology. Berlin, Heidelberg: Springer, 2014; 149–68.
[5] Harborne JB, Mabry TJ. The Flavonoids. Springer, 2014;760 p.
[6] Jensen SR, Schripsema J. Chemotaxonomy and pharmacology of Gentianaceae. In: Eds. Struwe L, Albert VA. Gentianaceae, Systematics and Natural History. Cambridge: Cambridge University Press, 2002;573–632.
[7] Krstić-Milošević D, Vinterhalter B, Janković T, Vinterhalter D. Biotechnology and phytochemistry of Gentianella species from the central regions of the Balkan peninsula. In: Eds Rybczyński JJ, Davey MR, Mikula A. The Gentianaceae – Volume 2: Biotechnology and Applications. Berlin, Heidelberg: Springer, 2015;93–112.
[8] Menković N, Šavikin-Fodulović K, Čebedžić R. Investigation of the activity of Gentiana lutea extracts against Mycobacterium bovis. Pharm Pharmacol Lett. 1999; 9(2): 74-5.
[9] Chericoni S, Testai L, Calderone V, Flamini G, Nieri P, Morelli I, Martinotti E. The xanthones gentiacaulein and gentiakochianin are responsible for the vasodilator action of the roots of Gentiana kochiana. Planta Med. 2003;69(8):770-2.
[10] Urbain A, Marston A, Queiroz EF, Ndjoko K, Hostettmann K. Xanthones from Gentiana campestris as new acetylcholinesterase inhibitors. Planta Med. 2004;70(10):1011-4.
[11] Savikin K, Menković N, Zdunić G, Stević T, Radanović D, Janković T. Antimicrobial activity of Gentiana lutea L. extracts. Z Naturforsch C. 2009;64(5-6):339-42.
[12] Vender C, Aiello N, Piovesana S. Survey of yellow gentian populations of the Central Alps and record of their main morphological and qualitative characteristics. Acta Hortic. 2010; 860:101–4.
[13] Kunakh VA. Biotechnology of medicinal plants. Genetic, physiological and biochemical basis. Kyiv: Logos, 2005; 730 p.
[14] Karuppusamy SA. A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. J Med Plants Res. 2009; 3(13): 1222-39.
[15] Srivastava S, Srivastava AK. Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotechnol. 2007;27(1):29-43. Review.
[16] Hosokawa K, Matsuki R, Oikawa Y, Yamamura S. Genetic transformation of gentian using wild-type Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult. 1997; 51:137–40.
[17] Momcilovic I, Grubisic D, Kojic M, Neskovic M. Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species. Plant Cell Tissue Organ Cult. 1997; 50(1):1–6.
[18] Vinterhalter B, Orbović V, Vinterhalter D. Transgenic root cultured of Gentiana punctata L. Acta Soc Bot Pol. 1999; 68(4):275–80.
[19] Rybczyński JJ, Davey MR, Tomiczak K, Niedziela A, Mikuła A. Systems of plant regeneration in Gentian in vitro cultures. In: Eds Rybczyński JJ, Davey MR, Mikula A. The Gentianaceae - Volume 2: Biotechnology and Applications. Berlin, Heidelberg: Springer, 2015:1–44.
[20] Menković N, Savikin-Fodulović K, Momcilović I, Grubisić D. Quantitative determination of secoiridoid and gamma-pyrone compounds in Gentiana lutea cultured in vitro. Planta Med. 2000;66(1):96-8.
[21] Menković N, Šavikin-Fodulović K, Vinterhalter B, Vinterhalter D, Janković T, Krstić D. Secoiridoid content in hairy roots of Gentiana punctata. Pharm Pharmacol Lett. 2000; 2:73–5.
[22] Tiwari RK, Trivedi M, Guang ZC, Guo GQ, Zheng GC. Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures. Plant Cell Rep. 2007;26(2):199-210.
[23] Zhang HL, Xue SH, Pu F, Tiwari RK, Wang XY. Establishment of hairy root lines and analysis of gentiopicroside in the medicinal plant Gentiana macrophylla. Russ J of Plant Physiol. 2010; 57(1):110–17.
[24] Krstić-Milošević D, Janković T, Uzelac B, Vinterhalter D, Vinterhalter B. Effect of elicitors on xanthone accumulation and biomass production in hairy root cultures of Gentiana dinarica. Plant Cell Tiss Organ Cult. 2017; 130(3): 631–40.
[25] Gamborg OL, Eveleigh DE. Culture methods and detection of glucanases in suspension cultures of wheat and barley. Can J Biochem. 1968;46(5):417-21.
[26] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15(3): 473-97.
[27] Drobyk NM, Grytsak LR, Mel'nyk VM, Kravets NB, Konvalyuk II, Twardovska MO, Kunakh VA. In vitro manipulation and propagation of Gentiana L. species from the Ukrainian flora. In: Eds Rybczyński JJ, Davey MR, Mikula A. The Gentianaceae – Volume 2: Biotechnology and Applications. Berlin, Heidelberg: Springer, 2015; 45–79.
[28] Drobyk NM, Mel'nyk VM, Twardovska MO, Konvalyuk II, Kunakh VA. Tissue and organ cultures of gentians as potential sources of xanthones and flavonoids. In: Eds Rybczyński JJ, Davey MR, Mikula A (). The Gentianaceae – Volume 2: Biotechnology and Applications. Berlin, Heidelberg: Springer, 2015:307–17.
[29] Li Y, Ma D, Sun D, Wang C, Zhang J, Xie Y, Guo T. Total phenolic, flavonoid content, and antioxidant activity of flour, noodles, and steamed bread made from different colored wheat grains by three milling methods. The Crop J. 2015; 3(4): 328–34.
[30] Skrzypczak L, Wesołowska M, Skrzypczak E. Gentiana species: in vitro culture, regeneration and production of secoiridoid glucosides. In: Ed Bajaj YPS. Biotechnology in agriculture and forestry, vol. 21. Medicinal and aromatic plants IV. Berlin, Heidelberg, New York: Springer, 1993:172–86.
[31] Jomori H, Takahata Y, Kaizuma N. Plant regeneration from leaf-derived calli of gentians and their protoplast culture. Acta Hort. 1995; 392:81–6.
[32] Vinterhalter B, Krstić-Milošević D, Janković T, Pljevljakušić D, Ninković S, Smigocki A, Vinterhalter D. Gentiana dinarica Beck. hairy root cultures and evaluation of factors affecting growth and xanthone production. Plant Cell Tissue Organ Cult. 2015; 121(3):667–79.
[33] Vinterhalter B, Janković T, Šavikin K, Nikolić R, Vinterhalter D. Propagation and xanthone content of Gentianella austriaca shoot cultures. Plant Cell Tissue Organ Cult. 2008; 94:329–35.
[34] Wadegaonkar PA, Bhagwat KA, Rai MK. Direct rhizogenesis and establishment of fast growing normal root organ culture of Withania somnifera Dunal. Plant Cell Tissue Organ Cult. 2006; 84(2): 223–5.