Optimization of Hairy Root Induction and Biomass Production of Chicory (Cichorium intybus L.)

علوم باغبانی

Article 3, Volume 32, Issue 3, November 2018, Pages 393-405   

Document Type: Research Article

DOI: 10.22067/jhorts4.v32i3.63287

Authors

Roghayeh Fathi; Mehdi Mohebodini^* ; Esmaeil Chamani

University of Mohaghegh Ardabili

Abstract

Introduction: Transformed hairy roots obtained by infection of plants with Agrobacterium rhizogenes strains are used as a tissue culture tool for secondary metabolite production since hairy roots are genetically and biologically stable and they are able to produce metabolite within a short time. Chicory (Cichorium intybus L.) is a medicinal plant from Asteraceae. This plant contains many important metabolites include chicoric asid, inulin, scoline, coumarin and flavonoids.
Materials and Methods: The seeds were surface-sterilized by rinsing with 2% benomil for 30 min and later sterilized with 5% sodium hypochlorite for 20 min and subsequently with 70% ethanol for 90 s. the wounded leaf of 20 and 28-day-old seedlings inoculated with Agrobacterium rhizogenes 11325. Bacterial colonies were cultured on liquid LB medium for 1 day. The explants were submerged in the solution for 15 min and were shaken gently. After that, the explants were drained on sterile filter paper and then transferred to MS solid medium. The explants were incubated at (25 ± 2) ◦C under dark condition for three different co-cultivation period (24, 48 and 72 hours). In the next step, the explants were transferred to MS solid medium supplemented with 500 mg/L cefotaxime. The explants were incubated at (25 ± 2) ◦C under a 16-h photoperiod condition for four weeks. At the end of incubation time, the percentage of hairy root induction, root number and root length were investigated. Genomic DNA of Cichorium intybus L. hairy root was extracted following the method of CTAB and subjected to PCR analysis. Non-transformed root DNA was used as a negative control and pRi11325 plasmid DNA was used as positive control. The pair of primers specific to the rolB fragment sequences was: 5-ATGGATCCCAAATTGCTATTCCCCACGA -3 and 5-TAGGCTTCTTTCATTCGGTTTACTGCAGC-3. The amplification protocol for the rolB was a 5 min melting at 94 ◦C followed by 35 cycles of a 5 min melting at 94 ◦C, a 45 s annealing at 55 ◦C and a 1 min elongation at 72 ◦C, and final elongation at 72 ◦C for 7 min. PCR products were electrophoretically separated on 0.8 % agarose gels (w/v) and stained with gel red. To determine the best media composition for growth of hairy roots, approximately 100 mg FW roots were cultured in basal liquid MS, solid MS and liquid 1/2MS medium supplemented with 500 mg/L cefotaxime on a rotary shaker (90 rpm) at 25 ◦C in dark for 5 weeks, and increase in fresh weight and dry weight was recorded at 5 week later. Three replicates were made for each experimental set. Non-transformed roots were cultured on same mediums.
Results and Discussion: Each of the two different explants – leaf and petiole showed different levels of hairy root induction in different co-culture time. Hairy root induction was observed 7 days after co-cultivation. There were low adventitious roots formed from control explants. 72 hours co-cultivation period was found to be more efficient in inducing hairy root phenotype in both explant types. The infection of explants by A. rhizogenes is a process that implies a succession of events, including the transfer and integration of T-DNA, into the host plant genome. Therefore, the time of contact between A. rhizogenes and the explants determines the transformation efficiency. A maximum of transformation frequency (53.33%) was observed in leaf explants in 72 hours co-cultivation followed by 33.33% in petiole explants in 72 hours co-cultivation. In leaf explants maximum root number (8.5 roots per explant) and maximum length of root (9.16 cm) induced from 20-day-old seedling in 72 hours co-culture. In petiole explants maximum root number (3.66 roots per explant) and maximum root length (11.46 cm) induced from 28-day-old seedling in 72 hours co-culture. The “hairy root” phenotype characterized by high branching and fast growth was previously reported in “reactive species” like Datura innoxia but not for “difficult species”. The high phenolic content of woody plant species could be responsible for the reduced hairy root phenotype as reported for gingko or pine. Three different culture media (solid MS, liquid MS and 1/2 MS media) were tested to determine the best suitable media for the maximum Biomass of hairy roots line.
Conclusions: Genetically transformed hairy roots obtained by infection of plants with Agrobacterium rhizogenes are suitable source for production of bioactive molecules due to their genetic stability and generally show fast growth in culture media free of growth hormones. This study describes the protocol for hairy root induction which could further be useful for the production of secondary metabolites and biomass.

Keywords

Agrobacterium Rhizogenes; Co- culture; Hairy Roots; Rol gene; Secondary metabolites

References

1- Ayadi R., and Tremouillaux-Guiller J. 2003. Root formation from transgenic calli of Ginkgo biloba. Tree Physiology, 23: 713–718.

2- Azarmehr B., Karimi F., Taghizade M., and Mousavi Gargari S.L. 2012. Comparative study of growth and secondary metabolite production ability in transformed hairy roots from Cichorium intybus L. Journal of Plant Research, 26: 476-485. (In Persian with English ََabstract(

3- Azarmehr B., Karimi F., taghizadeh M., and Mousavi Gargari S.L. 2013. Secondary metabolite contents and antioxidant enzyme activities of Cichorium intybus hairy roots in response to z inc. Journal of Medicinal Plants and By-Products, 2: 131-138.

4- Bertani G. 1952. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. Journal of Bacteriology, 62: 293-300.

5- Brijwal A., and Tamta S. 2015. Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC, SpringerPlus, 4: 443-453.

6- Cho H.J., Widholm J.M., Tanaka N., Nakanishi Y., and Murooka Y. 1998. Agrobacterium rhizogenes mediated transformation and regeneration of the legume Astragalus Sinicus (Chinese milk). Plant Science, 138: 53-65.

7- Chu C.C., Wang C.S., Sun C.C., Hsu C., Yin K.C., and Chu C.Y. 1975. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Scientia Sinica, 18: 659-668.

8- Georgiev M.I., Agostini E., Ludwig-Müller J., and Xu J. 2012. Genetically transformedroots: from plant disease to biotechnological resource. Journal of Trends in Biotechnology, 30: 528–537.

9- Grzegorczyk-Karolak I., Kuzma L., Skała E., and Kiss A. 2018. Hairy root cultures of Salvia viridis L. for production of polyphenolic compounds. Industrial Crops and Products, 117: 235-242.

10- Hasanlu T., Rezazadeh S., and Rahnama H. 2008. Hairy roots sources for the production of valuable pharmaceutical compounds. Journal of Medicinal Plants, 29: 1-17.

11- Huang S.H., Vishwakarma R.K., Lee T.T., Chan H.S., and Tsay H.S. 2014. Establishment of hairy root lines and analysis of iridoids and secoiridoids in the medicinal plant Gentiana scabra. Botanical Studies, 55: 1- 17.

12- Jaiswal R., Kiprotich J., and Kuhnert N. 2011. Determination of the hydroxycinnamate profile of 12 members of the Asteraceae family. Journal of Phytochemistry, 72: 781–790.

13- Kabirnataj S., Nematzadeh G., Zolala J., and Talebi A. 2016. High-efficient transgenic hairy roots induction in chicory: redawn of a traditional herb. Acta Agriculturae Slovenica, 107(2): 321-334

14- Kabirnetaj S., Zolala J., Nematzadeh G.A., and Shokri, E. 2012. Optimization of hairy root cultue establishment in chicory plants (Cichorium intybus L.) through inoculation by Agrobacterium rhizogenes. Iranian Journal of Agricultural Biotechnology, 4: 61–75. (In Persian with English abstract(

15- Karthikeyan A., Palanivel S., Parvathy S., and Bhakya R.R. 2007. Hairy root induction from hypocotyl segment of groundnut (Arachis hypogaea L.). African Journal Biotechnology, 15: 1817-1820.

16- Kedari P., and Malpatak N. 2014. Hairy root culture of Chonemorpha fragrans (moon) Alston plant for compothecin production. Indian Journal of Biotechnology, 13: 231-235.

17- Khan S., Irfan Q. M., Kamaluddin A., and Abdin M. 2007. Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. African Journal of Biotechnology, 6: 175-178.

18- Kodjo D., Atsou V.A., Melin C., Bland N., Oudin A., Courdavault V., Creche J., and Lanoue A. 2013. Optimized genetic transformation of Zanthoxylum zanthoxyloides by Agrobacterium rhizogenes and the production of chelerythrine and skimmiamine in hairy root cultures. Engineering in Life Sciences, 14: 95–99.

19- Lanoue A., Shakourzadeh K., Marison I., and Laberche J. C. 2004. Occurrence of circadian rhythms in hairy root cultures grown under controlled conditions. Journal of Biotechnology and Bioengineering, 88: 722–729.

20- Lourenco P.M.L., Castro S.D., Martins T.M., and Domingos A.C. 2002. Growth and proteolytic activity of hairy roots from Centaurea calcitrapa: effect of nitrogen and sucrose. Enzyme and Microbial Technology, 31: 242–249.

21- Mano Y., Ohkawa H., and Yamada Y. 1989. Production of tropane alkaloids by hairy root cultures of Duboisia leichhardtii transformed by Agrobacterium rhizogenes. Plant Science, 59: 191-201.

22- Manuhara Y. S., Kristanti A.N., Utami E.S., and Yachya A. 2015. Effect of sucrose and potassium nitrate on biomass and saponin content of Talinum paniculatum Gaertn. Hairy root in balloon-type bubble bioreactor. Asian Pacific Journal of Tropical Biomedicine, 5(12): 1027-1032.

23- Murashige T., and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiolgia Plantarum, 15: 473-476.

24- Pakdin Parizi A., Farsi M., Nematzadeh G.A., and Mirshamsi A. 2014. Impact of different culture media on hairy roots growth of Valeriana officinalis L.. Acta Agriculturae Slovenica, 103: 299-305

25- Park S.U., Li X., Eom S.H., Lee C.Y., and Lee S.Y. 2010. E-P-Methoxycinnamic acid production in hairy root cultures of Scrophulari buergeriana miquel. Archives of Biological Sciences Belgrade, 62(3): 649-652.

26- Rischer H., Hakkinen S.T., Ritala A., Seppanen-Laakso T., Miralpeix B., Capell T., Christou P., and Oksman-Caldentey K.M. 2013. Plant cells as pharmaceutical factories. Current Pharmaceutical Design, 19: 5640–5660.

27- Ritala A., Dong L., Imseng N., Seppänen-Laakso T., Vasilev N., and Krol S. 2014. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committedstep in terpenoid indole alkaloid pathway. Journal of Biotechnology, 176: 20-28.

28- Samadi A., Carapetian J., Heidary R., Jafari M., and Hssanzadeh A. 2012. Hairy root induction in Linum mucronatum sp. an anti-tumor lignans production plant. Nothlae Botanicae Hortiagrobatanici Cluj- Napaca, 40(1): 125-131.

29- Sang U., Xiaohua L., Seok H., Chung Y., and Sook Y. 2010. E-P-Methoxycinamic acid production in hairy root culture of Scrophularia buergeriana miquel. Archives of Biological Sciences, 62(3): 649-652.

30- Sevonand N., and Oksman-Caldentey K.M. 2002. Agrobacterium rhizogenes-mediated transformation: root cultures as source of alkaloids. Planta Medica, 68: 859–868.

31- Shinde A., Malpathak N., and Fulzele D. 2010. Impact of nutrient components onproduction of the phytoestrogens daidzein and genistein by hairy roots of Psoralea corylifolia. Journal of Natural Medicines, 64: 346-353.

32- Singh R., Kamal S., Rani D., Mukhopadhyay K., and Banerjee M. 2014. Development of hairy root culture system of Phlogacanthus thyrsiflorus Nees. Journal of Applied Research on Medicinal and Aromatic Plants, 1(3): 107-112.

33- Sivakumar G., Yu K.W., and Paek K.Y. 2005. Production of biomass and ginsenoides from adventitious roots of Panax ginseng in bioreactor cultures. Engineering in Life Sciences, 5: 333-342.

34- Srivastava V., Kaur R., Chattopadhyay S.K., and Banerjee S. 2013. Production of industrially important cosmaceutical and pharmaceutical derivatives of betuligenolby Atropa belladonna hairy root mediated biotransformation. Industrial Crops and Products, 44: 171–175.

35- Sujatha G., Zdravkovic-Korac S., Calic D., Flamini G., and Ranjitha Kumari B.D. 2013. High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia vulgaris: Hairy root production and essential oil analysis. Industrial Crops and Products, 44: 643–652.

36- Thiruvengadam M., Rekha K., and Chung I. 2016. Induction of hairy roots by Agrobacterium rhizogenes-mediatedtransformation of spine gourd (Momordica dioica Roxb. ex. willd) forthe assessment of phenolic compounds and biological activities. Scientia Horticulturae, 198: 132-141.

37- Young-Am C., Yu H.S., Song J.S., Chun H.K., and Park S.U. 2000. Indigo production in hairy root cultures of Polygonum tinctoium Lour. Biotechnology Letters, 22: 1527-1530.