Influence of Different Agrobacterium rhizogenes Strains on Hairy Roots Induction and Secondary Metabolites Production in Ocimum basilicum L.

علوم باغبانی

Article 7, Volume 34, Issue 2 - Serial Number 46, September 2020, Pages 273-284   

Document Type: Research Article

DOI: 10.22067/jhorts4.v34i2.82167

Authors

Maryam Zare Hasan Abadi¹; Ali Ganjeali^{* 1}; Mehrdad Lahouti²; Nasrin Moshtaghi²

¹Ferdowsi University of Mashhad

²Ferdowsi university of Mashhad

Abstract

Introduction: Agrobacterium rhizogenes hairy roots induction is used for secondary metabolite production in plants. A. rhizogenes is a genus of gram-negative soil bacteria belonging to the Rhizobiaceae family that causes hairy roots at the site of infection. Hairy roots have various advantages, including high growth rate, more genetic stability than the callus and suspension cultures, growing well on hormone-free media that have been reported effective for producing high levels of secondary metabolites. Basil (Ocimum basilicum) is a popular herb with important economical applications in food, cosmetic, and pharmaceutical industry. It is a digestive stimulant with anticarcinogenic, antibacterial, and anticonvulsant properties. The main phenolics reported in basil plants are in the classes of phenolic acids and flavonoids, some of which have human health benefits. This study was designed to develop hairy root culture from O. basilicum using different of A. rhizogenes strains for the production of total phenols and introduce the best strain of A. rhizogenes to induce hairy root and growth and production of total phenol.
Materials and Methods: Different A. rhizogenes strains (ATCC-15834, A4, MSU, and R1000) were studied to investigate their effects for the transformation and production of secondary metabolites in O. basilicum. Therefore, shoot and leaf explants and nodes of the seedlings were used for Agrobacterium-mediated transformation. These explants were inoculated with four A. rhizogenes strains and transferred to ½ MS medium. About four weeks after cultivation with A. rhizogenes, hairy roots were excised from the seedlings and subcultured to fresh medium MS liquid culture containing 500 mg/l cefotaxime. After 60 days of inoculation, various parameters, including dry weight, infection percentage, number of hairy roots per explant, and total phenol contents were measured. The growth rate and phenolic contents of the transformed hairy roots were compared with normal ones. Total genomic DNA was isolated from non-transgenic and transgenic hairy root lines using the Cetyl Trimethyl Ammonium Bromide (CTAB) method. Isolated genomic DNA was used to detect the rolC gene through polymerase chain reaction (PCR) analysis. The PCR using specific primers for rolC of T-DNA and virD2 was used to confirm the nature of resulted transgenic hairy roots.
Results and Discussion: Selecting efficient A. rhizogenes strains, as well as the type of explants, are crucial factors for hairy root induction. All used A. rhizogenes strains were able to produce hairy roots. Hairy roots appeared on the nodes at the point of injection, but were not forming on the shoot and leaf explants. So, the choice of the plant material is crucial for successful transformation with A. rhizogenes and usually, transformation of young tissues gives the best results. The transgenic status of the hairy roots was confirmed using PCR with rolC and virD specific forward and reverse primers. All lines showed the presence of 612 bp rolC amplified products, indicating the integration of T-DNA of A. rhizogenes and O. basilicum. Hairy roots could synthesize phenolic compounds, which was significantly higher in hairy roots than non-transformed control. Four hairy root lines were independently evaluated for their content and these lines showed variation in total phenolic contents, with the highest amount (312 mgGAE/ g DW) in hairy roots induced by ATCC-15834 strain and the lowest amount (113.2 mgGAE/ g DW) in hairy roots induced by R1000 strain. The results showed that the strain ATCC-15834 caused the highest infection percentage (68.1%) along with the highest number of hairy roots (4.8) per explant and root length (1.8 cm). The growth rate and phenolics production were investigated in each hairy root of O. basilicum from infection by four different A. rhizogenes strains. The highest growth rate (103.2 mg DW) and production of total phenol (312 mg/g DW) were found in ATCC-15834. The growth rate of transformed hairy roots was more than that of normal ones.Total phenol contents in all hairy roots were also increased significantly compared with non-transformed control plants (4.6 times in hairy roots induced by A. rhizogenes strain ATCC-15834). ATCC-15834 has been reported as the most widely used A. rhizogenes strain owing to its strong induction ability, and the variation in hairy root induction could be due to disparity in the virulence of different A. rhizogenes strains.
Conclusion: The hairy roots of O. basilicum had shown promising results in terms of significant yield of phenolic contents and had the potential for being scaled-up further for phenol production. It could be concluded that A. rhizogenes strains had different abilities in hairy roots induction. Therefore, the selection of an effective A. rhizogenes strain for the production of transformed root cultures is important, highly dependent on the plant species, and must be determined in future experiments.

Keywords

Agrobacterium rhizogenes hairy root Ocimum basilicum total phenol

References

1. Ahmadi Moghadam Y., Piri K., Bahramnejad, B., and Ghiasvand, T. 2014. Dopamine production in hairy root cultures of Portulaca oleracea (Purslane) using Agrobacterium rhizogenes. Journal of Agricultural Science and Technology, 16:409-420.

2. Bais H.P., Walker T.S., Schweizer H.B., and Vivanco J.M. 2002. Root specific elicitation and antimicrobial activity of rosmarinic acid in hairy root cultures of Ocimum basilicum. Plant Physiology Biochemistry, 40:983 95.

3. Chen H., and Chen F. 2000. Induction of phytoalexin formation in crown gall and hairy root culture of Salvia miltiorrhiza by methyl viologen. Biotechnology Letters, 22(8):715–720.

4. Chung I.M., Rekha K., Rajakumar G., and Thiruvengadam, M. 2016. Production of glucosinolates, phenolic compounds and associated gene expression profiles of hairy root cultures in turnip (Brassica rapa ssp. rapa). 3 Biotech, 6:175.

5. Danial M., Keng C.L., Alwee S.S., and Subramaniam S. 2011. Seed histology of recalcitrant Eurycoma longifolia plants during germination and its beneficial attribute for hairy roots production. Journal of Medicinal Plants Research, 5(1):93-98.

6. Farsi M., Moshtaghi N., Shahriari F.A., and Raeisi M. 2005. Investigation on growth stability and alkaloid content of transformed hairy roots in Datura stramonium. Agricultural sciences and technology, 19(2):47-56.

7. Gabr A.M., Sytar O., Ahmed A.R., and Smetanska I. 2012. Production of phenolic acid and antioxidant activity in transformed hairy root cultures of common buckwheat (Fagopyrum esculentum M.). Australian Journal of Basic and Applied Sciences, 6(7): 577-586.

8. Gangopadhyay M., Chakraborty D., Bhattacharyya S., and Bhattacharya S., 2010. Regeneration of transformed plants from hairy roots of Plumbago Indica. Plant cell tissue and organ culture, 102(1):109-114.

9. Gupta S.K., Liu R.B., Liaw S.Y., Chan H.S., and Tsay H.S. 2011. Enhanced tanshinone production in hairy roots of ‘Salvia miltiorrhiza Bunge’ under the influence of plant growth regulators in liquid culture. Botanical Studies, 52:435-443.

10. Ionkova I., Karting T., and Alfermann W. 1997. Cycloartanesaponin production in hairy root cultures of Astragalus mongholicus. Phytochemistry, 45:1597-1600.

11. Karam N.S., Jawad F.M., Arikat N.A., and Shibli R.A., 2003. Growth and rosmarinic acid accumulation in callus, cell suspension, and root cultures of wild Salvia fruticosa. Plant cell tissue and organ culture, 73:117-121.

12. Khezerluo M., Hosseini B., and Amiri J. 2018. Sodium nitroprusside stimulated production of tropane alkaloids and antioxidant enzymes activity in hairy root culture of Hyoscyamus reticulatus L. Acta Biologica Hungarica, 69(4):437-448.

13. Kim Y., Wyslouzil B.E., and Weathers P.J. 2002. Secondary metabolism of hairy root cultures in bioreactors. In Vitro Cellular & Developmental Biology - Plant 38(1):1–10.

14. Kumar V., Desai D., and Shriram V. 2014. Hairy root induction in Helicteres isora L. and production of Diosgenin in hairy roots. Naural Products and Bioprospecting, 4:107–112.

15. Kwee E.M., and Niemeyer E.D. 2011. Variations in phenolic composition and antioxidant properties among 15 basil (Ocimum basilicum L.) cultivars. Food Chemistry, 12:1044-1050.

16. Lee S.Y., Cho S.I., Park M.H., Kim Y.K., Choi J.E., and Park S.U. 2007. Growth and rutin production in hairy root cultures of buckwheat (Fagopyrum esculentum M.). Preparative Biochemistry and Biotechnology, 37(3):239-246.

17. Lee S.Y., Xu H., Kim Y.K., and Park S.U. 2008. Rosmarinic acid production in hairy root cultures of Agastache rugosa Kuntze. World Journal of Microbiology and Biotechnology, 24:969-972.

18. Lee S.U., Kimi S.U. , Song W.S., Kim Y.K., Park N.I., and Park S.U. 2010, Influence of different strains of Agrobacterium rhizogenes on hairy root induction and production of alizarin and purpurin in Rubia akane Nakai. Romanian Biotechnological Letters, 15(4):5405-5409.

19. Majumdar S., Garai S., and Jha S. 2011. Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants. Plant Cell Reports, 30(5):941-954.

20. Mateus L., Cherkaout S., Christen P., and Oksman-Caldentey K.M. 2000. Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography. Phytochemistry, 54:517-523.

21. Modnicki D., and Balcerek M. 2009. Estimation of total polyphenols contents in Ocimum basilicum L., Origanum vulgare L. and Thymus vulgaris L. commercial samples. Herba Polonica, 55(1):35-42.

22. Mohiuddin A.M., Cabdullah Z., Chowdhury K., Harikrishna K., and Napis S. 2011. Enhanced virulence gene activity of Agrobacterium in Muskmelon (Cucumis melo L.) cv. `Birdie´. Notulae Scientia Biologicae, 3:71-79.

23. Murashige T., and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiology, 15:473-497.

24. Park S.U., Kim Y.K., and Lee S.Y. 2009. Establishment of hairy root culture of Rubia akane Nakai for alizarin and purpurin production. Scientific Research and Essay, 4(2):094-097.

25. Park N.I., Xiaohua L., Uddin R.M., and Park S.U. 2011. Phenolic compound production by different morphological phenotypes in hairy root cultures of Fagopyrum tataricum Gaertn. Archives of Biological Sciences, Belgrade, 63(1):193-198.

26. Rahnama H. 2007. Silimarin production using hairy root culture of Silybum marianum. In: Proceedings of Symposium of Medicinal Plants. Iran, Shahed University, 554-573. (In Farsi)

27. Scagel C.F., and Lee J. 2012. Phenolic composition of basil plants is differentially altered by plant nutrient status and inoculation with mycorrhizal fungi. Horticultural Science, 47:660–671.

28. Sharma M., Ahujab A., Guptac R., and Mallubhotla S. 2014. Enhanced bacoside production in shoot cultures of Bacopa monnieri under the influence of abiotic elicitors. Natural Product Research, 29(8):745-749.

29. Sharp P.I., Kries M., Shewry P.R., and Gale M.D. 1988. Location of β-amylase sequences in wheat and its relatives. Theoretical and applied genetics, 75 (2):286-290.

30. Shen B., Hohmann S., Jensen R.G., and Bohnert, H.J. 1999. Roles of sugar alcohols in osmotic stress adaptation. Replacement of glycerol by mannitol and sorbitol in yeast. Plant Physiology, 121(1):45-52.

31. Singleton V.L., and Rossi J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American journal of enology and viticulture, 16:144-158.

32. Srivastava S., Cahill D.M., Conlan X.A., and Adholeya A. 2014. A novel in vitro whole plant system for analysis of polyphenolics and their antioxidant potential in cultivars of Ocimum basilicum. Journal of Agricultural and Food Chemistry, 62(41):10064–10075. Srivastava S., Conlan X.A., Adholeya A., and Cahill D.M. 2016. Elite hairy roots of Ocimum basilicum as a new source of rosmarinic acid and antioxidants. Plant Cell Tissue Organ Culture, 126:19–32.

33. Stojakowska A., Malarz J., Szewczyk A., and Kisiel W. 2012. Caffeic acid derivatives from a hairy root culture of Lactuca virosa. Acta Physiologiae Plantarum, 34(1):291-298.

34. Thilip C., Raju C.S., Varutharaju K., Aslam A., and Shajahan A. 2015. Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera L.Dunal using sonication and heat treatment. 3 Biotech, 5(6):949-956.

35. Tiwari R.K., Trivedi M., Guang Z.C., Guo G.Q., and Zheng G.C. 2007. Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures. Plant Cell Reports, 26:199-210.

36. Winans SC. 1992. Two-way chemical signaling in Agrobacterium-plant interactions. Microbiology and Molecular Biology Reviews 56(1):12-31.

37. Zhao J., Lou J., Mou Y., Li P., Wu J., and Zhou L. 2011. Diterpenoidtanshinones and phenolic acids from cultured hairy roots of Salvia miltiorrhiza Bunge and their antimicrobial activities. Molecules, 16(3):2259-2267.