ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
Biotechnologia Acta V. 15, No. 3, 2022
P. 13-22. Bibliography 26, Engl.
UDC: 604.6:577.13
https://doi.org/10.15407/biotech15.03.013
PECULIARITIES OF THE GROWTH OF Artemisia tilesii Ledeb. “HAIRY” ROOTS WITH DIFFERENT FOREIGN GENES
Т. А. Bohdanovych 1, B. V. Morgun 1, 2, О. R. Lakhneko 1, 2, А. М. Shakhovsky 1, N. А. Matvieieva 1
1Institute of Cell Biology and Genetic Engineering of the National Academy of Sciences of Ukraine, Kyiv
2 National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”
Aim. To compare Artemisia tilesii “hairy” root lines with different transferred genes in terms of the relationship between the total content of flavonoids, the levels of antioxidant activity (AOA) and reducing power (RP), as well as the activity of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), rolB and rolC genes.
Methods. We compared the root lines Nos. 10 and 16, obtained by transformation with the wild Agrobacterium rhizogenes strain A4, lines Nos. 2 and 4, obtained using A. rhizogenes carrying pSV124 vector with ifn-α2b and nptII genes, as well as the roots of non-transformed plants that were cultivated in vitro. The presence and activity of rolB, rolC, PAL, and CHS genes were determined by PCR and real-time PCR, respectively. The content of flavonoids, AOA and RP were determined by standard tests with AlCl3, DPPH (2,2-diphenyl-1-picrylhydrazyl) and K3[Fe(CN)6] accordingly.
Results. The content of flavonoids in most of the lines was higher than in the control, and correlated with AOA and RP. Roots No. 10 were characterized by the fastest growth, which coincided with higher activity of rolB and rolC genes. The activities of PAL and CHS in “hairy” roots were lower than those in non-transformed ones.
Conclusions. Root lines carrying only rolB and rolC and lines with additional ifn-α2b and nptII genes had similar ranges of flavonoids concentration, AOA and RP levels that exceeded those in the control. The dependence of the root growth rate, and lack of the dependence of the flavonoids content with the activity of the rol genes were demonstrated. PAL activity inversely correlated with flavonoids content in all experimental lines, which may be the result of overproduction of compounds in transgenic roots.
Key words: Artemisia tilesii Ledeb., Agrobacterium rhizogenes, “hairy” roots, flavonoids, rol genes, PAL and CHS genes, real-time PCR, antioxidant activity.
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2022
References
1. Wani K. I., Choudhary S., Zehra A., Naeem M., Weathers P., Aftab T. Enhancing artemisinin content in and delivery from Artemisia annua: a review of alternative, classical, and transgenic approaches. Planta. 2021, 254(2), 29. https://doi.org/10.1007/s00425-021-03676-3
2. Slezakova S., Ruda-Kucerova J. Anticancer Activity of Artemisinin and its Derivatives. Anticancer Res. 2017, 37(11), 5995–6003. https://doi.org/10.21873/anticanres.12046
3. Nair M. S., Huang Y., Fidock D. A., Polyak S. J., Wagoner J., Towler M. J., Weathers P. J. Artemisia annuaArtemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants. J. Ethnopharmacol. 2021, 274, 114016. https://doi.org/10.1016/j.jep.2021.114016
4. Saarela J. M., Sokoloff P. C., Gillespie L. J., Bull R. D., Bennett B. A., Ponomarenko S. Vascular plants of Victoria Island (Northwest Territories and Nunavut, Canada): a specimen-based study of an Arctic flora. PhytoKeys. 2020, 141, 1–330. https://doi.org/10.3897/phytokeys.141.48810
5. Stiles A .R., Liu C. Z. Hairy root culture: bioreactor design and process intensification. Adv. Biochem. Eng. Biotechnol. 2013, 134, 91–114. https://doi.org/10.1007/10_2013_181
6. El-Esawi M. A., Elkelish A., Elansary H. O., Ali H. M., Elshikh M., Witczak J., Ahmad M. Genetic Transformation and Hairy Root Induction Enhance the Antioxidant Potential of Lactuca serriola L. Oxid. Med. Cell. Longev. 2017, V. 2017, P. 5604746. https://doi.org/10.1155/2017/5604746
7. Zafari S., Sharifi M., Chashmi N.A. A comparative study of biotechnological approaches for producing valuable flavonoids in Prosopis farcta. Cytotechnology. 2018, 70(2), 603–614. https://doi.org/10.1007/s10616-017-0143-y
8. Tavassoli P., Safipour Afshar A. Influence of different Agrobacterium rhizogenes strains on hairy root induction and analysis of phenolic and flavonoid compounds in marshmallow (Althaea officinalis L.). 3 Biotech. 2018, 8(8), 351. https://doi.org/10.1007/s13205-018-1375-z
9. Li C., Wang M. Application of Hairy Root Culture for Bioactive Compounds Production in Medicinal Plants. Curr. Pharm. Biotechnol. 2021, 22(5), 592–608. https://doi.org/10.2174/1389201021666200516155146
10. Roy A. Hairy Root Culture an Alternative for Bioactive Compound Production from Medicinal Plants. Curr. Pharm. Biotechnol. 2021, 22(1), 136–149. https://doi.org/10.2174/1389201021666201229110625
11. Casanova E., Trillas M.I., Moysset L., Vainstein A. Influence of rol genes in floriculture. Biotechnol. Adv. 2005, 23(1), 3–39. https://doi.org/10.1016/j.biotechadv.2004.06.002
12. Dilshad E., Ismail H., Haq I.U., Cusido R.M., Palazon J., Ramirez-Estrada K., Mirza B. Rol genes enhance the biosynthesis of antioxidants in Artemisia carvifolia Buch. BMC Plant. Biol. 2016, 16(1), 125. https://doi.org/10.1186/s12870-016-0811-7
13. Dilshad E., Zafar S., Ismail H., Waheed M.T., Cusido R.M., Palazon J., Mirza B. Effect of Rol Genes on Polyphenols Biosynthesis in Artemisia annua and Their Effect on Antioxidant and Cytotoxic Potential of the Plant. Appl. Biochem. Biotechnol. 2016, 179(8), 1456–68. https://doi.org/10.1007/s12010-016-2077-9
14. Shkryl Y. N., Veremeichik G. N., Bulgakov V. P., Tchernoded G. K., Mischenko N. P., Fedoreyev S. A., Zhuravle Yu. N. Individual and combined effects of the rolA, B and C genes on anthraquinone production in Rubia cordifolia transformed calli. Biotechnol. Bioeng. 2007 (published online). https://doi.org/10.1002/bit.21727
15. Dilshad E., Cusido R. M., Palazon J., Estrada K. R., Bonfill M., Mirza B. Malar J. Enhanced artemisinin yield by expression of rol genes in Artemisia annua. 2015, 14, 424. https://doi.org/10.1186/s12936-015-0951-5
16. Matvieieva N. A., Morgun B. V., Lakhneko O. R., Duplij V. P., Shakhovsky A .M., Ratushnyak Y. I., Sidorenko M., Mickevicius S., Yevtushenko D. P. Agrobacterium rhizogenes-mediated transformation enhances the antioxidant potential of Artemisia tilesii Ledeb. Plant. Physiol. Biochem. 2020, 152, 177–183. https://doi.org/10.1016/j.plaphy.2020.04.020.
17. Matvieieva N. A., Shakhovsky A. M., Belokurova V. B., Drobot K. O. Artemisia tilesii Ledeb. hairy roots establishment using Agrobacterium rhizogenes-mediated transformation. Prep. Biochem. Biotechnol. 2016, 46(4), 342–345. https://doi.org/10.1080/10826068.2015.1031393
18. Aboul-Maaty N. A. F., Oraby H. A. S. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bull. Nat. Res. Centre. 2019, 43(25). https://doi.org/10.1186/s42269-019-0066-1
19. Box M. S., Coustham V., Dean C., Mylne J. S. Protocol: a simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis. Plant Methods 2011, 7, 7. https://doi.org/10.1186/1746-4811-7-7
20. Pfaffl M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29, e45. https://doi.org/10.1093/nar/29.9.e45
21. P?kal A., Pyrzynska K. Evaluation of Aluminium Complexation Reaction for Flavonoid Content Assay. Food Anal. Methods. 2014, 7, 1776–1782. https://doi.org/10.1007/s12161-014-9814-x
22. Brand-Williams W., Cuvelier M. E., Berset C. Use of a free radical method to evaluate antioxidant activity. LWT – Food Science and Technology. 1995, 28(1), 25–30. https://doi.org/10.1016/S0023-6438(95)80008-5
23. Zhao H., Fan W., Dong J., Lu J., Chen J., Shan L., Lin Y., Kong W. Evaluation of antioxidant activities and total phenolic contents of typical malting barley varieties. Food Chem. 2008, 107(1), 296–304. https://doi.org/10.1016/j.foodchem.2007.08.018
24. Ahmad P., Jaleel C. A., Salem M. A., Nabi G., Sharma S. Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress. Crit. Rev. Biotechnol. 2010, 30(3), 161–175. https://doi.org/10.3109/07388550903524243
25. He L., He T., Farrar S., Ji L., Liu T., Ma X. Antioxidants Maintain Cellular Redox Homeostasis by Elimination of Reactive Oxygen Species. Cell Physiol. Biochem. 2017, 44(2), 532–553. https://doi.org/10.1159/000485089
26. Srivastava S., Srivastava A. K. Hairy root culture for mass-production of high-value secondary metabolites. Crit. Rev Biotechnol. 2007, 27(1), 29–43. https://doi.org/10.1080/07388550601173918