Biopolym. Cell. 2004; 20(5):421-428.
http://dx.doi.org/10.7124/bc.0006C5
Molecular and Cell Biotechnologies
Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts
1Kovalenko P. G.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680

Abstract

The bacterial chloramphenicol acetyltransferase (CAT) gene was expressed in licorice G. glabra L. suspension protoplasts by electroporation after introduction of the chimeric plasmid pDNt23-CaMV35S-nos-cat (pDNt23-root-specific and CaMV 35S promoters, nopaline synthase nos-terminator, and selectable NPT-II gene). Maximum of CAT activity in the licorice protoplasts was observed in 50 h after electroporation. Together with recent advances in the cell isolation and electroporation procedures, this system allows to study expression of the root-specific promoter introduced into the licorice cells. The level of total flavonoids production in 4 weeks culture was tested in the electroporated cell lines. Additionally, these electroporated cells were treated by elicitors (yeast extract and fungal bioextract). The total flavonoids production increased by 2–5 times in comparison with the non-electroporated licorice cells.
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References

[1] Aoki T., Akashi T., Ayabe S.-I. Flavonoids of leguminous plants: Structure, biological activity, and biosynthesis. Journal of Plant Research. 2000; 113 (1112):475-488.
[2] Kovalenko P.G., Antonjuk V.P., Maliuta S.S. Secondary metabolites production from transformed cells of Glycyrrhiza glabra and Potentilla alba as a producents of radioprotective compounds. Ukr. bioorg. acta., 2002;1 (1):21-31.
[3] Li W, Asada Y, Yoshikawa T. Antimicrobial flavonoids from Glycyrrhiza glabra hairy root cultures. Planta Med. 1998;64(8):746-7.
[4] Christie S, Walker AF, Lewith GT. Flavonoids--a new direction for the treatment of fluid retention? Phytother Res. 2001;15(6):467-75.
[5] Hamil J.D., Parr A.J., Rhodes M.J., Robins S.R., Walton N.J. New routes to plant secondary productst. Bio/Technology. 1987; 5:800-804.
[6] Shillito R.D., Saul M.W., Paszkowski J., Muller M., Potrykus I. High efficiency direct gene transfer to plants. Bio/Technology., 1985; 3:1099-1103.
[7] Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473–97.
[8] Widholm JM. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Biotech Histochem. 1972;47(4):189-94.
[9] Domanskii NN, Gening LV, Kovalenko PG, Medvedeva TV, Galkin AP, Gazarian KG. Cloning of tobacco DNA fragment with promoter properties in a transgenic plant. Mol Biol (Mosk). 1989;23(5):1391-9.
[10] Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982;2(9):1044-51.
[11] Jones H, Ooms G, Jones MG. Transient gene expression in electroporated Solanum protoplasts. Plant Mol Biol. 1989;13(5):503-11.
[12] Bandyukova VA. Photocolorimetric methods for the determination of flavonoids. Proc. of the 1st All-Russia Congr. of Pharmacists. Moscow, Medicine, 1964:227-30.
[13] Georgiyevskiy VP, Komisssarenko NF, Dmitruk SYe. Biologically active substances of medicinal plants. Novosibirsk, Nauka, 1990: 328 p.
[14] Gallois P., Lindsey K., Malone R. Electroporation of tobacco leaf protoplasts using plasmid DNA or total genomic DNA. Methods in molecular biology (Clifton, N.J.), 1995; 55:89-107.
[15] Kovalenko P.G., Schuman N.V., Ponomarenko S.P. Biotechnological advances of electroporation of grapevine and sugar beet cells. Bioelectrochem Bioenerg. 1997 43 (1):165-168.
[16] Rathus C., Birch R.G. Optimization of conditions for electroporation and transient expression of foreign genes in sugarcane protoplasts. Plant Science. 1992; 81 (1):65-74.
[17] Bekkaoui F, Datla RS, Pilon M, Tautorus TE, Crosby WL, Dunstan DI. The effects of promoter on transient expression in conifer cell lines. Theor Appl Genet. 1990;79(3):353-9.
[18] Fromm ME, Taylor LP, Walbot V. Stable transformation of maize after gene transfer by electroporation. Nature. 1986 Feb 27-Mar 5;319(6056):791-3.
[19] Dillen W, Engler G, Van Montagu M, Angenon G. Electroporation-mediated DNA delivery to seedling tissues ofPhaseolus vulgaris L. (common bean). Plant Cell Rep. 1995;15(1-2):119-24.
[20] Zhou H, Stiff CM, Konzak CF. Stably transformed callus of wheat by electroporation-induced direct gene transfer. Plant Cell Rep. 1993;12(11):612-6.
[21] Laursen CM, Krzyzek RA, Flick CE, Anderson PC, Spencer TM. Production of fertile transgenic maize by electroporation of suspension culture cells. Plant Mol Biol. 1994;24(1):51-61.
[22] Ou-Lee TM, Turgeon R, Wu R. Expression of a foreign gene linked to either a plant-virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat, and sorghum. Proc Natl Acad Sci U S A. 1986;83(18):6815-9.
[23] Simonenko YuV, Kuchuk HV. Genetic transformation of pea (Pisum sativum L.) by electroporation of protoplasts. Fiziologiia i biokhimiia kul'turnykh rasteniy. 1999; 31(3):203-7.
[24] Srinivasan V, Ciddi V, Bringi V, Shuler ML. Metabolic inhibitors, elicitors, and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures. Biotechnol Prog. 1996;12(4):457-65.
[25] Dornenburg H., Knorr D. Strategies for the improvement of secondary metabolite production in plant cell cultures. Enzyme and Microbial Technology. 1995; 17 (8):674-684.
[26] Flores-Sanchez IJ, Ortega-Lopez J, del Carmen Montes-Horcasitas M, Ramos-Valdivia AC. Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa. Plant Cell Physiol. 2002;43(12):1502-9.
[27] Yoon H.J., Kim H.K., Ma C.-J., Huh H. Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor. Biotechnology Letters, 2000; 22 (13):1071-1075.
[28] Ignatov A, Clark WG, Cline SD, Psenak M, Krueger J, Coscia CJ. Elicitation of dihydrobenzophenanthridine oxidase in Sanguinaria canadensis cell cultures. Phytochemistry. 1996;43(6):1141-4.