PLANT HAIRY ROOT CULTURE: A PROMISING SYSTEM TO PRODUCE

SECONDARY METABOLITE
Yosephine Sri Wulan Manuhara
Department of Biology,Faculty of Science and Technology
Airlangga University, Surabaya, Indonesia

ABSTRACT
Many plant secondary metabolites of interest are accumulated in roots.
Harvesting roots is destructive for the plants and hence there has been increasing
interest in developing hairy root cultures from several medicinal plant species. Hairy
roots can be produced by transformation with the soil bacterium Agrobacterium
rhizogenes, resulting in the so-called hairy roots disease. Hairy roots are induced
when a plant is infected by an A. rhizogenes, by a part of a root inducing (Ri) plasmid
in bacteria, called transfer DNA (T-DNA), which is transferred into the plant cell and
expressed therein. The interest in hairy roots is mainly due to their ability to grow fast
without needing an external supply of auxins. Many times, they do not need
incubation under light. They are fairly well stable in metabolite yield due to their
genetic stability. Several hairy roots have been put to scale-up studies in bioreactors.
The use of bioreactor in micropropagation revealed its commercial applicability, and
recently gained attention to commercial microprapagation process and also to
produced valuable metabolite from plant. So, the hairy root cultures in bioreactor
have opportunity to developing in the future, because many experiments on hairy
root culture that produce valuable secondary metabolite have already established.
Keywords: hairy root, secondary metabolite, Agrobacterium rhizogenes
Introduction
Plants synthesize a wide range of secondary metabolites such as alkaloids,
anthocyanins, flavonoids, quinins, lignans, steroids, and terpenoids, which play a
major role in the adaptation of plants to their environment. The secondary
metabolites have been used as food additives, drugs, dyes, flavours, fragrances, and
insecticides. Such chemicals are extracted and purified from naturally grown plants.
However, production of secondary metabolites from plants is not always satisfactory.
It is often restricted to a limited species or genus, and geographically to a specific
region. Many important medicinal plants were endangered by overexploitation. Some
plants are difficult to cultivate and grow very slowly or are endangered in their natural
habitats. The biotechnological approach by utilizing plant cell and organ culture
system can offer an opportunity to produce the secondary metabolites. However,
there are many problems in the production of metabolites by plant cell and organ
culture technology due to the high cost to natural counterparts, and the low yield of
metabolites in cultured plant cells. Although there are many efforts for establishing
the cell and organ culture systems.
The secondary metabolites can be produced by developed organ and
plantlets. An alternative method for the production of plant materials for secondary
metabolite production is the culture of shoots, roots, or whole plants. However, the
organ culture tends to grow slowly and renders the difficulty of the large-scale
cultivation compared to cell culture. Agrobacterium rhizogenes-transformed hairy
roots synthesize the same component as does the roots of the intact plants and have
a fast growth property in hormone-free medium.

What is a Hairy Root?
Hairy roots can be produced by transformation with the soil bacterium
Agrobacterium rhizogenes, resulting in the so-called hairy roots disease. Hairy roots
are induced when a plant is infected by an A. rhizogenes, by a part of a root inducing
(Ri) plasmid in bacteria, called transfer DNA (T-DNA), which is transferred into the
plant cell and expressed therein. The T-DNA between the TR and TL regions of the
Ri-plasmid in the bacterium is transferred and integrated into the nuclear genome of
the host plant. The transformation process produces a valuable by-product, hairy
root, which will form at or near the site of infection. In addition opines are produced
and serve as spesific food for the bacteria (Chilton at al., 1982). The T-DNA of the
A.rhizogenes strain consists of two non-contiguous stretches of DNA, the TL- and
TR-DNA, which are transferred separately into plant chromosomes [Huffman et al,
1984 and Jouain, 1984]. The integration of the TL-DNA, especially rol genes (rolA,
rolB, rolC and rolD), into the plant genome is sufficient to induce hairy root formation.
These rol genes on TL-DNA are primarily responsible for the hairy root syndrome
[Nilsson et al, 19975, Spena et al, 1987 White et al, 1985]. The TR-DNA contains
aux genes (aux1 and aux2), which may play a conditional but nonessential role in
hairy root formation [White et al. 1985 and Cardarelli, 1985].
In nature, the soil bacterium Agrobacterium rhizogenes genetically engineers
dicotyledonous plant species into chemical producers of an Agrobacterium food
source (opines). This transformation process delivers a valuable by-product, hairy
roots, tissues that are capable of unlimited propagation in culture media. Hairy root
cultures have several propertiesfast growth, genetic and biochemical stability, and
growth in hormone-free media that have promoted their use in plant
biotechnology. First, hairy root cultures serve as model systems for plant metabolism
and physiology, and second, as a technical alternative to plant cell suspension
cultures for the production of therapeutics and specialty chemicals [Shanks and
Morgan, 1999].
To succeed in establishing a hairy root culture system for a certain plant
species, several essential conditions should be taken into consideration. These
condition include the bacterial strain of A. rhizogenes, an appropriate explant, a
proper antibiotic to eliminate redundant bacteria after cocultivation, an suitable
culture medium [Hu and Du, 2006]. Most plants materials, such as hypocotyl, leaf,
stem, stal, petiole, shoot tip, cotyledon, protoplast, storage root, or tuber, can be
used to induce hairy roots [Mugnier, 1988; Han et al. 1993; Drewes et al. 1995; Giri
et al. 2001; Krolicka et al. 2001; Azlan et al. 2002]. However, for different species,
the proper explant material may vary and the age of material is most critical, with
juvenile material being optimal. To induce hairy root, explants are separately
wounded and cocultivation or inoculated with A. rhizogenes.

Prospect of Hairy Root Culture

Many plant secondary metabolites of interest are accumulated in roots. Harvesting
roots is destructive for the plants and hence there has been increasing interest in
developing hairy root cultures from several medicinal plant species. A survey of
literature on hairy root cultures published during the past eight years provides
evidence of continuous progress in this area (Table 1).

Table 1. Recent report on valuable metabolites produced by hairy root culture
___________________________________________________________________
Plant species Metabolite References
_________________________________________________________________________
Pueraria phaseoloides Puerarin Shi and Kintzlos (2003)
Rauvolfia micrantha Ajmalicine, ajmaline Shuda et al. (2003)
Solidago altissima Polyacetylene Inoguchi et al. (2003)
Linum flavum Coniferin Lin et al. (2003)
Panax ginseng Ginsenoside Palazon et al. (2003)
Yu et al. (2005)
Saussurea medusa Jaceosidin Zhao et al. (2004)
Camptotheca acuminate Camptothecin Lorence et al. (2004)
Papaver somniferum Morphine, sanguinarine, Le Flem-Bonhomme,
codeine (2004)
Gmelina arborea Verbascoside Dhakulkar et al. (2005)
Gynostemma pentaphyllum Gypenoside Chang CK et al. (2005)
Azadirachta indica Azadirachtin Satdive et al. (2007)
Plumbago zeylanica L. Plumbagin Sivanesan & Jeong(2009)
Plumbago rosea Plumbagin Yogananth & Basu(2009)
_________________________________________________________________________

The interest in hairy roots is mainly due to their ability to grow fast without
needing an external supply of auxins. Many times, they do not need incubation under
light. They are fairly well stable in metabolite yield due to their genetic stability.
Because of these advantages, many of the root-derived plant products once not
considered feasible for production by cell culture are being reinvestigated for
production using the hairy root culture technology [Rao and Ravishankar, 2002].
Several factors influence the yield of secondary metabolites of pharmaceutical
interest in hairy root cultures. Optimizing of the composition of nutriens for hairy root
cultures is crtical to gain a high production of secondary metabolites. Factor such as
the carbon source and its concentration, the ionic concentration of the medium, the
pH of the medium, light, temperature, and inoculum are known to influence growth
and secondary metabolism [Hu and Du, 2006]. Beside that, elicitation and
biotranformation are known to improved secondary metabolites. Satdive et al. 2007
got the increase production of azadirachtin by hairy root culture of Azadirachta indica
A. Juss by elicitation and media optimization. Elicitation and biotransformation
method can also improved ginsenoside production of Panax ginseng transformed
root [Palazon et al. 2003; Chen et al. 2008]. Temperature and light quality influence
the production of ginsenoside of hairy root cultures of Panax ginseng [Yu et al.
2005]. Shiao and Doran, 2000 described that the fluid flow and oxygen transfer
influence of the biomass of hairy root of Arabidobsis thaliana.
Based on the progress of the development of the hairy root cultures at above,
the hairy root culture system is potential approach for the production of secondary
metabolites, especially pharmaceuticals, because it has many good traits, such as
rapid growth rate, easy culture and genetic manipulation, and most importantly, an
increase ability to synthesize useful metabolites that cannot be produced by
unorganized cell even higher than plant roots. Therefore, in the recent year the hairy
root culture are developed in the large scale in bioreactor.

Scale-up of Hairy Root Culture
Several hairy roots have been put to scale-up studies in bioreactors. The term
bioreactor is generally used to describe a vessel carrying out a biological reaction,
and to refer a reactor vessel for the culture of aerobic cells, or to columns of packed
beds of immobilized cells or enzymes. The bioreactors are widely used for industrial
production of microbial, animal and plant metabolites. The bioreactor technique
applied to plant propagation was first reported by the present author in 1981 on
Begonia propagation using a bubble column bioreactor. Since then, bioreactor
technology for plant propagation has developed and aerobic bioreactor culture
techniques have been applied for large-scale production of plant propagules
[Takayama and Akita, 2008].
The use of bioreactor in micropropagation revealed its commercial
applicability, and recently gained attention to commercial microprapagation process
and also to produced valuable metabolite from plant. The advantages of the use of
bioreactor compare with agar culture are (1) large number of planlets can easily be
produced in one batch in the bioreactor and scaling up of bioreactor, (2) since
handling of cultures such as inoculation or harvest is easy, reducing the number of
culture vessels, and the area of culture space results in the reduction of cost, (3)
whole surface of culture are always in contact with medium, uptake of nutrients are
stimulated and growth rate is also increased, (4) forced aeration (oxygen supply) is
performed which improves the growth rate and final biomass, and (5) culture are
moving in the bioreactor, which results in the disappearance of apical dominance
and stimulates the growth of numerous shoots buds into planlets [Takayama and
Akita, 2008]. Nevertheless, it must be noted that a chance contamination will quickly
develop and disperse in liquid medium and is likely to lead to total loss of culture
[Preil, 2005].
Since production of secondary metabolites is generally higher in differentiated
tissues, there are attempts to cultivate shoot culture and root culture for the
production of medicinally important compounds, because these are relatively more
stable. The interest in scale-up of hairy root cultures is mainly due to their ability to
grow fast without needing an external supply of auxins. Many times, they do not
need incubation under light. They are fairly well stable in metabolite yield due to their
genetic stability. Because of these advantages, many of the root-derived plant
products once not considered feasible for production by cell cultured are being
reinvestigated for production using the hairy root culture technology, for instance in
culture of Panax ginseng. A survey of literatures on many type of in vitro culture that
are developed in bioreactor are given in Table 2.

Table 2. Many type of in vitro culture that are developed in bioreactor
___________________________________________________________________
Plant species Type of culture Reference
_________________________________________________________________________
Solanum tuberosum Hairy root Tescione et al.(1997)
Panax notoginseng Cell suspension Zhong et al. (1999)
Panax notoginseng Cell suspension Woragidbumrung et al.(2001)
Panax notoginseng Cell suspension Hu & Zhong (2001)
Elaeis guineensis Cell suspension Gorret et al. (2004)
Panax ginseng Cell suspension Ali et al. (2005)
Panax ginseng Adventitious roots Jeong et al. (2006)
Panax ginseng Cell suspension Thanh et al. (2006)
Azadirachta indica Cell suspension Prakash & Srivastava (2008)
Hypericum perforatum L. Adventitious roots Cui et al. (2010)
Oncidium Sugar Sweet Protocorm-like bodies Yang et al. (2010)
___________________________________________________________________

Based on the literature study above, the hairy root cultures in bioreactor have
opportunity to developing in the future, because many experiments on hairy root
culture that produce valuable secondary metabolite have done.

Conclusions
The hairy root culture system is potential approach for the production of secondary
metabolites, because it has many good traits, such as rapid growth rate, easy culture
and genetic manipulation, and, most importantly, an increase ability to synthesize
useful metabolites that cannot be produced by unorganized cells even higher than
plants roots. Research of hairy root culture in bioreactor in the future have an
opportunity to developing in a commercial bioprocess, so hairy root can play a
significant role in developing design principles for plant metabolic engineering.


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