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REVIEW ARTICLE

Hairy Root Culture: An Experimental System for Secondary Metabolite Production

Shailju G. Gurunani*, Manjusha P. Yeole, Yogesh N. Gholse, Chaple D.R.

Priyadarshini J. L. College of Pharmacy (Degree), Electronic Zone Bldg., MIDC, Hingna Road, Nagpur-16

*Corresponding Author E-mail: shailju_g07@yahoo.com

ABSTRACT:

Mankind is totally dependent on plants for pharmaceuticals , agrochemicals, food additives, flavor and fragrance etc. Plants contain secondary metabolites having medicinal values. Secondary metabolites are biosynthetically derived from primary metabolites but are distributed in the limited amount and are valued at several thousand dollars per pound. Plant tissue culture technology is an important tool for production of the plant natural products via. micropropagation, cell culture, scaled up bioreactors and many more. One such tool is transformed root cultures also called as hairy root culture (HRC). It have productive capacity similar to the plants from which they have been initiated and they maintain the stability of growth and secondary metabolite production in successive generation. HRC is beneficial for root derived compounds. The revolutionized products obtained using HRC are Atropine, Diosgenin, Emetine, Quinine, Nicotine, Vincristine, Vinblastine, Glycerrhizin, Anthraquinones Shikonin and many more medicinally active ingredients which are in demand in the pharmaceutical industries. The production of HRC involves 3 steps viz., root induction, decontamination of roots and establishing liquid medium. The induction of hairy roots are done by genetic inseration of T- DNA of R-Plasmid, lodge in Agrobacterium rhizogenes into wounded tissue and proliferates, causing the growth of very fine adventitious roots. Hairy roots are genetically stable and have features similar to that of the normal roots. Thus, HRC provides promising approach to the biotechnological exploitation of plant cell culture for fine chemical production commercially.

KEYWORDS:

1. INTRODUCTION:

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of the secondary metabolites are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots also called as Hairy Root Culture (HRC) ¹.

Received on 25.02.2015 Modified on 15.03.2015

Accepted on 19.03.2015 © RJPT All right reserved

Research J. Pharm. and Tech. 8(6): June, 2015; Page 728-730

DOI: 10.5958/0974-360X.2015.00115.8

Hairy root cultures are hormone independent and can grow as fast as unorganized plant cell suspensions while maintaining a stable differentiated phenotype; furthermore secondary metabolites synthesis is not strictly limited to those that are normally produced in roots of differentiated plants. ²

It is a type of plant tissue culture. Hairy roots are formed by genetic transformation of plant cells using . Integration into the plant genome of T-DNA from the bacterial root-inducing (Ri) plasmid results in differentiation and growth of hairy roots at the infection site. Hairy roots can be excised, cleared of excess bacteria using antibiotics, and grown indefinitely in vitro by subculture of root tips in liquid medium. Practical techniques for initiation, culture, genetic manipulation, and molecular analysis of hairy roots are well explored ³.

2. Establishment of Axenic HRC

The protocol for successful establishment of axenic HRC involves three steps viz. root induction, decontamination of roots and establishment in liquid medium. Seedling grown under sterile conditions is inoculated with an appropriate strain of Agrobacterium rhizogene. A commonly used technique is to wound the axenic seedling or suitable plant material with an overnight culture of A. rhizogene . Hairy root emerge from the site of infection within 2-4 weeks. Putative hairy roots are established on MS medium. The hairy roots are decontaminated by subculturing on MS solid medium containing 250 mg / l antibiotic. The bacteria free hairy roots can be successfully grown in a phytoharmone free MS liquid medium. The cultures are incubated on a rotary shaker at 100 RPM , with 25⁰C temperature ^4,5. Fig. no. 1 shows the overview for the production of hairy root culture. ⁶

Fig. 1. An overview of establishment of Hairy Root Culture

3. Properties of Hairy Roots

There are several general features of hairy roots that confer significant technical advantages to them compared with untransformed roots or dedifferentiated plant cells. The attention given to hairy roots and their increasing adoption in scientific studies are due largely to properties such as, genotype and phenotype stability , biochemical stability, autotrophy in plant hormones, fast growth by addition of elicitors, high levels of secondary metabolites are produced. It is important to realize, however, that not every hairy root culture displays these characteristics. In addition, as well as advantages, many researchers have experienced problems with hairy root initiation and maintenance. ⁷

4. Applications

For 15-20 years, hairy roots have been applied in a wide range of fundamental studies of plant biochemistry, molecular biology, and physiology, as well as for agricultural, horticultural, and large-scale tissue culture purposes. Several recent reviews describe current and potential uses of hairy root cultures in research and industry.

These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. Hundreds of plant species have been successfully transformed to hairy roots, lists of amenable species are provided in table 1. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel.

Metabolic studies: Hairy root cultures can be used for phytoremediation, and are particularly valuable for studies of the metabolic processes involved in phytoremediation. Further applications include detailed studies of fundamental molecular, genetic and biochemical aspects of genetic transformation and of hairy root induction.

Genetically transformed cultures: The Ri plasmids can be engineered to also contain T-DNA, used for genetic transformation (biotransformation) of the plant cells. The resulting genetically transformed root cultures can produce high levels of secondary metabolites, comparable or even higher than those of intact plants.

Use in plant propagation: Hairy root culture can also be used for regeneration of whole plants and for production of artificial seeds. The only limitation to the commercialization of hairy root cultures of this species is the complexity of the secondary metabolic profile 8,9.

5. CONCLUSION:

Hairy roots (HRs) are differentiated cultures of transformed roots generated by the infection of wounded higher plants with Agrobacterium rhizogenes. This pathogen causes the HR disease leading to the neoplastic growth of roots that are characterized by high growth rate in hormone free media and genetic stability. HRs produce the same phytochemicals pattern of the corresponding wild type organ. High stability and productivity features allow the exploitation of HRs as valuable biotechnological tool for the production of plant secondary metabolites. In addition, several elicitation methods can be used to further enhance their accumulation in both small and large scale production. However, in the latter case, cultivation in bioreactors should be still optimized. HRs can be also utilised as biological farm for the production of recombinant proteins, hence holding additional potential for industrial use. HR technology has been strongly improved by increased knowledge of molecular mechanisms underlying their development. The present review summarizes aspects of the hairy root induction, genetics and metabolite production.

Currently the main constraint for commercial utilization of hairy root culture is the development and up-scaling of appropriate (bioreactors) vessels for the delicate and sensitive hairy roots. Japan is the leader in adopting hairy root culture technology for production of Shikonin and Ginseng extracts. However so far, the company based on Hairy Root Cultures as an industrial level is a Swiss company called ROOTec bioactives AG in Witterswil (SO) ¹⁰.

Table no. 1 Plant species successfully transformed to hairy roots

Sr.No.	Plants	Secondary metabolites produced	Use
1.	Atropa belladonna, Datura innoxia, Hyoscymus niger	Atropine, Scopolamine, Hyoscyamine	Cholinergic blockers
2.	Nicotiana tabaccum	Nicotine	Ganglion blockers
3.	Catharanthus roseus	Indole alkaloids	Anti cancer
4.	Cinchona ledgeriana	Quinine	Antimalarial
5.	Lithospermum erythrorhizon	Shikonin	Biolipstick Antibacterial
6.	Beta vulgaris	Betalains	Colorants
7.	Panax ginseng, Solanum species	Saponins and Steroidal alkaloids	Steroids
8.	Lippia dulcis Glycyrrhiza uralensis	Hernandukin Glycyrrhizin	Sweetener
9.	Dioscorea deltoidea	Diosgenin	Contraceptive
10.	Trichosanthes krilowii	Trichosanthin	Anti-HIV

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