Agrobacterium Rhizogenes-mediated Transformation of Peganum multisectum (Maxim) Bobrov and Harmine Production in Hairy Roots

Author

Department of Biotechnology, Payame Noor University, Tehran, Iran

Abstract

Using Agrobacterium rhizogenes due to create hairy roots is a useful method to product secondary metabolites in many medicinal plants. The transgenic hairy roots were induced from Peganum multisectum (Maxim) Bobrov a medicinally important species, by infecting leaf and stem explant with wild type Agrobacterium rhizogenes strain ATTCC 15834, which led to the induction of hairy roots from 19% of the explants. Polymerase chain reaction with primers for rol A confirmed the integration of T-DNA fragment of Ri plasmid of A. rhizogenesis into the genome of hairy roots obtained after transformation. Four transformed lines of hairy roots were established. Doubling time of the faster growing hairy root lines was about 11 days and these cultures showed about 12-fold increase in biomass at the end of 6 weeks as compared to non-transformed roots. The hairy roots showed an ability to synthesize harmine, a β-carboline alkaloid of medicinal value. The effect of the initial sucrose and ammonium nitrate concentration in biomass and harmine production of the liquid MS cultural medium cultures was studied. The highest values for harmine accumulation and fresh weight were obtained between 30-45 g l-1 of sucrose. The results also showed that the addition of extra ammonium nitrate up to optimum level (2475 mg l-1), as a source of nitrogen was significantly effective than standard ammonium nitrate in MS basal medium for growth of hairy roots and harmine production. This is the first report on the induction of hairy roots in P. multisectum (Maxim) Bobrov.

Keywords

References

1. Javzan S, Selenge D, Nedelcheva D, Christov V, Philipov S. Alkaloids from Mongolian species of Peganum multisectum (Maxim). Int J Com Mat. 2017;7:155-160.
2. Nida A, Aijaz AW, Irshad AN, Bhat MA. Distribution and medicinal importance of Peganum harmala A review. Int J Adv Res.2014;2:751-755.
3. Sobhani AM, Ebrahimi SA, Hoormand M, Rahbar N, Mahmoudian M. Cytotoxicity of Peganum harmala L. seeds extract and its relationship with contents of β-carboline alkaloid activity-guided isolation of P. nigellastrum Bunge. Arch Pharm Res. 2009;32:1245-1251.
4. Hemmateenejad B, Abbaspour A, Maghamia H, Miri R. Panjehshahin MR. Partial least squares-based multivariate spectral calibration method for simultaneous determination of beta-carboline derivatives in Peganum harmala seed extracts. Anal Chim Acta. 2006;575:290-299.
5. Fathiazada FYA, Khodaie L. Pharmacological effects of Peganum harmala seeds extract on isolated rat uterus. Iranian J Pharmaceut Sci. 2006;2:81-86.
6. Mazandarani M, Sineh Sepehr K, Baradaran B, Khuri V. Autecology, Phytochemical and Antioxidant Activity of Peganum harmala L. Seed Extract in North of Iran (Tash Mountains). JMPB. 2012;2:151-156.
7. Kirtikar KR, Basu BD. Indian Medicinal Plants, 2nd ed. Lalit Mohan Basu, Allahabad, 1935;1:457.
8. Chopra RN, Chopra IC, Handa KL and Kapur LD. Chopra’s Indigenous Drugs of India, 2nd ed. UN Dhur and Sons Pvt. Ltd., Calcuttta, India. 2002;370.
9. Jinao D, Ronghan  Z, Shouxun Z, Mingshi W, Chuntao C. Studies on the chemical constituents of Peganum multisectum Maxim. The alkaloids from seeds and antitumor activity. J China Pharm Uni.  1998;29:21-23.
10. Lamchouri F, Settaf A, Cherrah A, Zemzami Y, Lyoussi M, Zaid B, Attif N, Hassar M.Antitumor principles from Peganum harmala. Therapie. 1999;54:753-758.
11. Bruinvels J, Sourkes TL. Influence of drugs on the temperature lowering effect of harmaline. Euro J Pharm. 1998;4:31-39.
12. Aarons DH, Rossi GV, Orzechowski RF. Cardiovascular actions of three harmala alkaloids: harmine, harmaline and harmalol. J Pharm Sci. 1997;66:1244-1248.
13. O’Hearn E, Molliver ME. Degeneration of Purkinje cells in parasagittal zones of the cerebellar vermis after treatment with ibogaine or harmaline. Neuroscience. 1993;55:303-310.
14. Nelson DL, Herbet A, Petillot Y, Pichat L, Glowinski J, Hamon M. [3H] Harmaline as a specific ligand of MAOA-I. Properties of the active site of MAOA from rat and bovine brains. J Neurochem. 1979;32:1817-1827.
15. McCormick SJ, Tunnicliff G. Inhibitors of synaptosomal gammahydroxybutyrate transport. Pharmacology. 1998;57:124- 31.
16. Saeed SA, Farnaz S, Simjee RU, Malik A. Triterpenes and Bsitosterol from piper betel: isolation, antiplatelet and anti-inflammatory effects. Biochem Soc T. 1993;21:462-426.
17. Li WK. Extraction of alkaloids from Peganum harmala L. and study on their antihydatid chemical composition. J Lanzhou Med Coll. 1996;22:8-16.
18. Wink M, Schmeller T, Latz-Bruning B. Modes of action of allelochemical alkaloids: interaction with neuroreceptors, DNA and other molecular targets. J Chem Ecol. 1998;11:1881-937.
19. Wakabayashi K, Totsuka Y, Fukutome K, Oguri A, Ushiyama H, Sugimura T. Human exposure to mutagenic/carcinogenic heterocyclic amines and comutagenic beta-carbolines. Mutat Res. 1997;376:253-9.
20. Picada N, da Silva V, Erdtmann B, Henriques T, Henriques A. Genotoxic effects of structurally related beta-carboline alkaloids. Mutat. Res. 1997;379:135–149.
21. Bergstrom M, Westerberg G, Langstrom B. 11C-harmine as a tracer for monoamine oxidase A (MAO-A): in vitro and in vivo studies. Nucl Med Biol. 1997;24:287-93.
22. Rivas P, Cassels K, Morello A, Repetto Y. Effects of some beta-carboline alkaloids on intact Trypanosoma cruzi epimastigotes. Comp. Biochem. Physiol. C Pharmacol Toxicol Endocrinol. 1999;122:27-31.
23. EI-Gengaihi E, Dimetry Z, Mohamed M. Chemical and biological investigation of harmala plant. 2. Alkaloidal investigation. J Appl Ent. 1997;121:165-167.
24. Albores R, Neafsey J, Drucker G, Fields Z, Collins A. Mitochondrial respiratory inhibition by N-methylated b-carboline derivatives structurally resembling N-methyl-4-phenylpyridine. Proc Natl Acad Sci. 1990;87:9368–9372.
25. Réus GZ, Stringari RB, de Souza B, Petronilho F, Dal-Pizzol F, Hallak JE, Zuardi AW, Crippa JA, Quevedo J. Harmine and imipramine promote antioxidant activities in prefrontal cortex and hippocampus. Oxid Med Cell Longev. 2010;3:325-31.
26. Berlin J, Rügenhagen C, Greidziak N, Kuzovkina IN, Witte L, Wray V. Biosynthesis of serotonin and β-carboline alkaloids in hairy root cultures of Peganum harmala. Phytochem. 1993;33: 593-597.
27. Zayed R. Efficient in vitro elicitation of b-carboline alkaloids in transformed root cultures of Peganum harmala. B. Fac. Pharmacy, 2011;49:7–11
28. Jahaniani F, Ebrahimi SA, Rahbar-Roshandel N, Mahmoudian M. Xanthomicrol is the main cytotoxic component of Dracocephalum kotschyii and a potential anticancer agent. Phytochem., 2005;66:1581–1592.
29. Verdian R, Mohammad R, Hajiakhoondi A. Cytotoxicity and antimicrobial activity of harmane alkaloids. J Pharmacol Toxicol. 2007;2:677-680.
30. Di Giorgio C, Delmas F, Ollivier E, Elias R, Balansard G, Timon-David P. In vitro activity of the beta-carboline alkaloids harmane, harmine, and harmaline toward parasites of the species Leishmani infantum. Exp. Parasitol. 2004;106: 67-74.
31. Giri A, Narasu ML. Transgenic hairy roots: recent trends and applications, Biotechnol. Adv. 2000;18:1-22.
32. Christey MC. Use of Ri mediated transformation for production of transgenic plants. In vitro Cell Dev Biol. 2001;37:687–700.
33. Allan EJ, Eeswara JP, Jarvis AP, Mordue AJ, Morgan ED. Stuchbury, Induction of hairy root cultures of Azadirachta indica A. Juss. and their production of azadirachtin and other important insect bioactive metabolites, Plant Cell Rep. 2002;21:374-379.
34. Rao SR, Ravishankar GA. Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv. 2002;20:1-53.
35. Sevon N, Oksman-Caldentey KM. Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids, Planta Med. 2002;68:859-868.
36. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 1962;15:473–497.
37. Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small amount of fresh leaf tissue, Phytochem. Bull. 1987;5:547–555.
38. Kumar V, Kotamballi N, Murthi G, Hamidi S, Sudha CG, Ravishankar GA. Genetically modified hairy root of Withania somuifera Dunal. A potent source of rejuvenating principles. Rejuvenation Res. 2005;8:37-45.
39. Kittipongpatana N, Hock RS, Porter JR. Production of solasodine by hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. Plant Cell Tiss Org Cult. 1998;52:133-143.
40. Wodnicka M, Guarino RD, Hemperly JJ, Timmins MR, Stitt D, Pitner JB. Novel Fluorescent technology platform for high throughput cytotoxicity and proliferation assays, J Biomol Screen. 2000;5:141-152.
41. Mani S, Purwar S, Singh BR, Garg GK, Kumar A. Effect of jasmanic acid on growth characteristic of derided from mature embryos of Wheat cultures showing differential resistance to Tilletia indica. Plant Cell Biotechnol Mol Biol. 2006;1:97-108.
42. Kartal M, Altun ML and Kurucu S. HPLC method for the analysis of harmol, harmalol, harmine and harmaline in the seeds of Peganum harmala L. J Pharmaceut. BiomedAna. 2003;31:263-269.
43. Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soyabean root cells. Exp. Cell Res. 1968;50:151–158.
44. Zayed R, Winka M. β-Carboline and Quinoline Alkaloids in Root Cultures and Intact Plants of Peganum harmala. Z. Naturforsch. 2005;60:451-458.
45. Willims RR. The chemical microenvironment. In: Automation and Environmental Control in Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht. 1995;405-439
46. Lin HW, Kwok KH, Doran PM. Development of Linum flavum hairy root cultures for production of coniferin, Biotechnol Lett. 2003;25:521-525.
47. 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;199–210.
48. Bonhomme V, Mattar DL, Lacoux J, Fliniaux MAL, Dubreuil AJ. Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only. J Biotechnol. 2000;81:151-157.
49. Jacob A, Malpathak N. Manipulation of MS and B5 components for enhancement of growth and solasodine production in hairy root cultures of Solanum khasianum Clarke. Plant Cell, Tiss Org Cult. 2005:80:247-257.
50. Kuzovkina N, Gohar A, Alterman E. Production of b-carboline alkaloids in transformed root cultures of Peganum hamala L. Z Naturforsch. 1990;45c:727-728.
51. SrinivasanV, Pestchanker L, Moser S, Hirasuna TJ, Taticek RA, Shuler ML. Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake and taxol production by cell suspension of Taxus baccata. Biotechnol Bioeng. 1995;47:666-676.
52. Yu S, Kwok KH, Doran PM. Effect of sucrose, exogenous product concentration, and other culture conditions on growth and steroidal alkaloid production by Solanum avidare hairy roots. Enzyme Microb Tech. 1996;18:238-243.
53. Lourenço PML, Castro SD, Martins TM, Clemente A, Domingos A. Growth and proteolytic activity of hairy roots from Centaurea calcitrapa: effect of nitrogen and sucrose. Enzy Microbial Technol. 2002;31:242-249.
54. Sturm A, Tang GQ. The sucrose-cleaving enzymes of plants are crucial for development, growth and carbon partitioning. Trends Plant Sci. 2000;4:401-407.
55. Bensaddek L, Gillet F, Nava Saucedo JE, Fliniaux MA. The effect of nitrate and ammonium concentration on growth and alkaloid accumulation of Atropa belladonna hairy roots. J Biotechnol. 2001;85:35-40.
56. Payne J, Hamill JD, Robins JR, Rhodes MJC. Production of hyoscyamine by hairy root cultures of Datura stramonium. Planta Med. 1987;53: 474-478.
57. SugimotoY, Sugimura Y, Yamada Y. Effects of culture conditions on bisbenzylisoquinoline alkaloid production in cultured roots of Stephania cepharantha. Agric Biol Chem. 1988;52:1495-1498.
58. Oksman-Caldentey KM, Sevón N, Vanhala L, Hiltunen R. Effect of nitrogen and sucrose on the primary and secondary metabolism of transformed root cultures of Hyoscyamus muticus.Plant Cell Tiss Org Cult. 2002;38:263-272.
59. Wang WJ, Tan RX. Artemisinin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium. Biotechnol Lett.2002;24:1153-1156
60. Sawada H, Leki H, Matsuda L. PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains. Appl. Environ Microbiol. 1995;61:828-31.
61. Waller GR, Nowacki EK. Alkaloid biology and metabolism in plants. Plenum Press, New York and London. 1987.
62. Xu T, Zhang L, Sun X, Zhang H, Tang K. Production and analysis of organic acids in hairy-root cultures of Isatis indigotica Fort. (Indigo woad). Biotechnol Appl Biochem. 2004;39:123-128.
63. Jacob A, Malpathak N. Plantlet regeneration enhances solasodine productivity in hairy root cultures of Solanumkhasianum Clarke. In Vitro Cell Dev-Pl. 2005;41:291-295. 
64. Jacob A, Malpathak N. Manipulation of MS and B5 components for enhancement of growth and solasodine production in hairy root cultures of Solanum khasianum Clarke. Plant Cell Tiss Org. 2005;80:247-257.
65. Lourenço PM, Castro S, Martine TM, Clemente A, Domingos A. Growth and proteolytic activity of hairy roots from Centaurea calcitrapa: effect of nitrogen and sucrose. Enzyme Microb Tech. 2002;31:242-249.
Journal of Medicinal plants and By-product
Volume 6, Issue 2
September 2017
Pages 201-212

Files

Share

How to cite

Statistics