Biopolym. Cell. 2019; 35(2):118-128.
http://dx.doi.org/10.7124/bc.00099D
Molecular and Cell Biotechnologies
Crambe aspera plants in vitro propagation and its effect on fatty acids and phenolic compounds content and genome stability
1Pushkarova N. O., 2Lakhneko O. R., 2Morgun B. V., 2Kuchuk M. V., 1Blume Ya. B., 1Yemets A. I.
  1. Institute of Food Biotechnology and Genomics NAS of Ukraine
    2A, Osipovskogo Str., Kyiv, Ukraine, 04123
  2. Institute of Cell Biology and Genetic Engineering, NAS of Ukraine
    148, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680

Abstract

In vitro culture can be used for endangered species preservation but its effect on biochemical properties and genetic stability of plants requires further research. Aim. The study on fatty acids content, phenolic compounds and phylogenetic relationships of Crambe aspera plants upon aseptic cultivation in vitro. Methods. Morphogenic potential of Crambe aspera leaf, root and petiole explants was studied in the Murashige and Skoog (MS) medium with different concentrations of growth factors. Fatty acids (FA) content was determined by gas chromatography-mass spectrophotometry of FA ethers, phenolic compounds were determined by spectrophotometry. Polymerase chain reactions were used to study phylogenetic relationships. Results. The efficient protocols of seed surface sterilization as well as methods of fast microclonal multiplication and obtention of C. aspera callus tissue for were developed. Conclusions. Leaves of both in vitro and in vivo cultured plants had a high content of α-linolenic acid whereas erucic acid was absent. At the same time, the difference in biochemical composition between the plants grown in aseptic and non-aseptic conditions was shown. In vivo populations of C. aspera showed a high level of polymorphism but its genome did not undergo changes after the in vitro establishment.
Keywords: Crambe aspera, in vitro regeneration, fatty acids, phenolic compounds, SSR and ISSR markers.
Full text: (PDF, in English)

References

[1] Didukh YP. Crambe aspera. Red data book of Ukraine. Plant kingdom. K.: Globalconsulting, 2009:358.
[2] Belokurova V. Methods of Biotechnology in system of efforts aimed at plant biodiversity preservation. Cytol Genet. 2010;44(3):174–85.
[3] Nayak S, Kaur T, Mohanty S, Ghosh G, Choudhury R, Acharya L, Subudhi E. In vitro and ex vitro evaluation of long-term micropropagated turmeric as analyzed through cytophotometry, phytoconstituents, biochemical and molecular markers. Plant Growth Regul. 2011;64(1):91–8.
[4] Nehra N, Kartha K, Stushnott C, Giles K. The influence of plant growth regulator concentrations and callus age on somaclonal variation in callus culture regenerants of strawberry. Plant Cell Tissue Organ Cult. 1992;29(3):257–68.
[5] Sun X-Q, Pang H, Guo J-l, Peng B, Bai M-m, Hang Y-y. Fatty acid analysis of the seed oil in a germplasm collection of 94 species in 58 genera of brassicaceae. Chem Ind Forest Prod. 2011;31(6):46–54.
[6] Haslam TM, Kunst L. Extending the story of very-long-chain fatty acid elongation. Plant Sci. 2013;210:93-107.
[7] Taylor D, Guo Y, Katavic V, Mietkiewska E, Francis T, Bettger W. New seed oils for improved human and animal health and as industrial feedstocks: Genetic manipulation of the Brassicaceae to produce oils enriched in nervonic acid, in: Krishnan AB. (Ed.), Modification of Seed Composition to Promote Health and Nutrition, ASA-CSSA-SSSA Publishing, Madison, Wisconsin, USA, 2009; 219–33.
[8] Meot-Duros L, Magné C. Antioxidant activity and phenol content of Crithmum maritimum L. leaves. Plant Physiol Biochem. 2009;47(1):37-41.
[9] Ghasemi Pirbalout A, Rahnama GH, Malekpoor F, Roohi Broujenl H. Variation in antibacterial activity and phenolic content of Hypericum scabrum L. populations. J Med Plants Res. 2011;5(17):4119–25.
[10] Haq M, Sani W, Hossain ABMS, Taha RM, Monneruzzaman KM. Total phenolic contents, antioxidant and antimicrobial activities of Bruguiera gymnorrhiza. J Med Plants Res. 2011;5(17):4112–8.
[11] Pushkarova N, Belokurova V, Kuchuk M. Seed surface sterilization efficiency as an important prerequisite in formation of endangered plant species in vitro collections. Fact Exp Evol Organis. 2015;17:241–4.
[12] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473–97.
[13] Konvalyuk II, Kravets NB, Drobyk NM, Mel’nyk VM, Kunakh VA. Direct organogenesis in vitro of Gentiana lutea L. Biotechnology. 2010;3(5):66-73.
[14] Hlushchenko AV. Quantitative determination of polyphenols in extracts of Salsola collina l. Sb. nauk. sprats' sotrud. NMAPO imeni P. L. Shupika. 2014;23(4):240–5.
[15] State Pharmacopoeia of Ukraine. 1st ed. Appendix 3. Kharkiv: SE «Scientific Expert Pharmacopoeia Center», 2009:280 p.
[16] Yevdokimova OV. Development and validation of the method of quantitative determination of the amount of flavonoids in the grass of the tortoiseshell. VSU Herald, series: Chemistry. Biology Pharmacy. 2007;2:155–60.
[17] Garcés R, Mancha M. One-step lipid extraction and fatty acid methyl esters preparation from fresh plant tissues. Anal Biochem. 1993;211(1):139-43.
[18] Brody JR, Kern SE. History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 2004;333(1):1-13. Review.
[19] Qi W, Lin F, Liu Y, Huang B, Cheng J, Zhang W, Zhao H. High-throughput development of simple sequence repeat markers for genetic diversity research in Crambe abyssinica. BMC Plant Biol. 2016;16(1):139.
[20] Lowe AJ, Jones AE, Raybould AF, Trick M, Moule CL, Edwards KJ. Transferability and genome specificity of a new set of microsatellite primers among Brassica species of the U triangle. Mol Ecol Notes. 2002;2(1):7–11.
[21] Werner ET, Soares TC, Gontijo AB, Souza Neto JD, do Amaral JA. Genetic stability of micropropagated plants of Crambe abyssinica Hochst using ISSR markers. Genet Mol Res. 2015;14(4):16450-60.
[22] Khalin RMA, Soliman KhA, Rashed NAK, Ibrahim SA. Genetic polymorphism of some medicinal plants belonging to Brassicaceae using molecular markers. Egypt J Genet Cytol. 2010;39(1):41–55.
[23] Singhal H, Ren YR, Kern SE. Improved DNA electrophoresis in conditions favoring polyborates and lewis acid complexation. PLoS One. 2010;5(6):e11318. PubMed Central
[24] Sambrook J, Russell D. Molecular cloning: a laboratory manual. New York: CSHL Press, 2001; 2344 p.
[25] Perrier X, Flori A, Bonnot F. Data analysis methods. In: Hamon P, Seguin M, Perrier X, Glaszmann JC Ed. Genetic diversity of cultivated tropical plants. Enfield, Science Publishers. Montpellier. 2003:43–76.
[26] Bowes B. In vitro Morphogenesis of Crambe maritima L. Protoplasma. 1976;89(1-2):185–8.
[27] Li X, Ahlman A, Lindgren H, Zhu L-H. Highly efficient in vitro regeneration of the industrial oilseed crop Crambe abyssinica. Ind Crops Prod. 2011;33(1):170–5.
[28] Piovan A, Cassina G, Filippini R. Crambe tataria: actions for ex situ conservation. Biodivers Conserv. 2011;20(2):359–71.
[29] Kunakh VA, Mozhylevska LP, Bublyk OM, Kolonina IV, Muzyka VI. Microclonal propagation of Ungernia victoris Vved. ex. Artjuschenko. Biotechnology. 2008;1(4):57-63.
[30] The Science of flavonoids. Grotewold E. (Ed.). New York: Springer Science. 2006; 273 p.
[31] Comlekcioglu N, Karaman S, Ilcim A. Oil composition and some morphological characters of Crambe orientalis var. orientalis and Crambe tataria var. tataria from Turkey. Nat Prod Res. 2008;22(6):525-32.
[32] Sakhno LA. Transgene cruciferous plants as producents of long chain unsaturated fatty acids. Biotechnology. 2010;3(2):9–18.
[33] Sakhno L, Ostapchuk A, Klochko V, Kuchuk M. Fatty acid oil composition of Canola carrying cytochrome P450 SCC CYP11A1 transgene. Biotechnology. 2012;5(5):27–33.
[34] Lakhneko O, Pushkarova N, Stepanenko A, Kuchuk M, Morgun B. Phylogenetic study of Crambe species with ISSR markers. “VESTNIK” of the South-Kazakhstan state pharmaceutical academy Republican Scientific Journal. 2016;4(77):7–10.
[35] Twardovska M, Strashniuk N, Mel’nyk V, Konvalyuk I, Kunakh V. RAPD-analysis of the genome polymorphism for some Gentiana L. species from the Ukrainian flora. Dopv Nat Acad Sci Ukraine. 2009;5:200–4.
[36] Gaudeul M, Taberlet P, Till-Bottraud I. Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers. Mol Ecol. 2000;9(10):1625-37.