Induction and growth of hairy roots for the production
of medicinal compounds
Lamine Bensaddek1, María Luisa Villarreal2,
and Marc-André Fliniaux1
1Laboratoire
de Phytotechnologie (EA 3900) Université de Picardie Jules
Verne, 80000 Amiens, France
2Centro
de Investigación en Biotecnología, Universidad Autónoma del
Estado de Morelos (UAEM), CP 62210 Cuernavaca, Morelos, México
*Corresponding author; email:
marc-andre.fliniaux@u-picardie.fr
Keywords:
hairy roots review, natural products, elicitation
ABSTRACT
The development of genetically transformed plant tissue cultures
and mainly of roots transformed by Agrobacterium rhizogenes
(hairy roots), is a key step in the use of in vitro
cultures for the production of secondary metabolites. Hairy
roots are able to grow fast without phytohormones, and to
produce the metabolites of the mother plant. The conditions of
transformation (nature and age of the explants, bacterial
strain, bacterial density, and the protocol of infection) deeply
influence the frequency of the transformation events as well as
the growth and productivity of the hairy roots. Then
optimization of the culture parameters (medium constituents,
elicitation by biotic or abiotic stress) may enhance the
capability of the hairy roots to grow fast and to produce
valuable compounds.
INTRODUCTION
The use of plants as medicines is a very ancient story and a
traditional medical practice in all the passed civilisations
(Samuelsson, 2004). Natural products and in particular plant
metabolites are still extensively used for therapeutic
applications, and it was evaluated that between 1981 and 2002,
28% of the 868 new chemical entities were natural products or
derived from natural products, with another 24% created around a
pharmacophore from a natural product (Raskin et al., 2002;
Newman et al., 2003; Balunas and Kinghorn, 2005). In the early
years of the twenty-first century, plants are economically
important pharmaceuticals and essential for human health.
Examples of important drugs obtained from plants are, morphine
and codeine from Papaver somniferum, vincristrine and
vinblastine from Catharanthus roseus, digoxin from
Digitalis lanata (Hollman, 1996), and quinine and quinidine
from Cinchona spp. Natural products have played a major
role in lead discovery, mainly in the following areas: oncology,
cardiovascular and metabolic diseases, and immuno suppression
(Butler, 2004). It has been estimated that between 1981 and
2002, 60% of anti-cancer drugs and 75% of anti-infectious drugs
already on the market or under clinical trial were of natural
origin (Cragg et al., 1997; Yue-Zhong Shu, 1998; Newman et al.,
2003; Lam, 2007). The antimalarial artemisinin, isolated from
Artemisia annua L. is effective against multidrug resistant
strains of Plasmodium, and a lead compound for the
discovery of new antimalarial drugs (van Agtmael et al., 1999).
Several clinically useful anti-cancer agents are plant products
or their close derivatives: vinblastine, irinotecan, topotecan,
etoposide, and paclitaxel (Cragg and Newman, 2005). Huperzine A
and galantamine (galanthamine) acting as acetylcholinesterase
inhibitors have been approved for the treatment of Alzheimer’s
disease and other neurodegenerative pathologies (Raves et al.,
1997; Scott and Goa, 2000).
The obtention of medicinal compounds from extraction of wild or
cultivated plants can be limited by various problems: plants
difficult to cultivate, risk of extinction for over exploited
plants, and geopolitical problems, among other causes (Verpoorte
et al., 2002). To try to overcome these problems, many attempts
were made during the last decades to evaluate the possibility of
producing medicinal compounds by in vitro plant cell and
organ cultures (Berlin, 1986; Alfermann and Petersen 1995).
However, in most cases, the compounds were undetectable or were
accumulated at low levels in the cultures. Several strategies
such as screening and selection of high producing cell lines,
cell immobilization, elicitation, and culture of differentiated
tissues were developed. In each case problems were encountered
and results did not allow the development of an economically
valuable commercialization of the biotechnologically produced
compounds (Verpoorte et al., 2002).
The in vitro transformation of plant material with
Agrobacterium rhizogenes strains allowed to overcome some of
the huge difficulties of in vitro plant organ cultures,
and led to the obtention of fast growing organs, exhibiting
extensive branching, and capable of producing the main
metabolites of the mother plant or even new metabolites
undetected in the mother plant nor in other kinds of in vitro
cultures (Nader et al., 2006). The so called “hairy roots”
offered a promising technology for secondary metabolite
production (Hamill et al., 1987) such as tropane alkaloids
(Flores and Filner 1985; Oksman-Caldentey and Arroo, 2000) and
many other metabolites (Giri and Narasu, 2000).
At the present time, the more precise knowledge about A.
rhizogenes transformation of plant material as well as about
hairy roots and their biotechnological use for the production of
pharmaceutical products offer new prospects (Guillon et al.,
2006a and b; Kuzovkina and Schneider, 2006; Georgiev et al.,
2007; Srivastava and Srivastava, 2007).
Agrobacterium rhizogenes
STRAINS AND THE INDUCTION OF HAIRY ROOTS
In the
hairy roots disease, the infectious process by A. rhizogenes
wild strains is characterized by the following four steps: 1)
chimiotactism induced movement of agrobacteria towards the plant
cells; 2) binding of the bacteria to the surface components of
the cell wall; 3) activation of the virulence (vir)
genes, and 4) transfer and integration of the transfer-DNA
(T-DNA) into the plant genome (Zupan and Zambryski, 1997). The
genetic information allowing this infection process is mainly
contained in the Ri plasmid (pRi) carried by the bacteria. In
the pRi, the vir region concentrates 6 to 8 genes
involved in the DNA transfer. The right and left T-DNA regions (TR-DNA
and TL -DNA) of the pRi, which are delimited by their
border sequences, are the regions that are transferred to the
plant.
Within
the TR section, loci involved in auxin biosynthesis
are transferred to the plant genome, thus increasing the auxin
level of the transformants. Other genes of the TR
section are responsible of the synthesis of opines which are
unusual amino acid sugar derivatives used by the bacteria for
their feeding (Gartland, 1995).
The wild A. rhizogenes strains, many of which have been
used to produce hairy roots from medicinal plants, can be
classified by their opine type. Agropine strains (A4, 15834,
1855, LBA 9402) induce agropine, mannopine and agropinic acid
production while the mannopine strains (8196) and the cucumopine
(Petit et al., 1983) strains induce the production of one single
opine. Agropine strains pRi transfer independently both the TL
-DNA and TR –DNA to the plant genome, while mannopine
strains only transfer the TL –DNA. This pRi region
contains the four rol genes A, B, C and D (Schmülling et
al., 1988; Petersen et al., 1989) which enhance the auxin and
cytokinin (Estruch et al., 1991) susceptibility of plant cells
and are responsible for the formation of roots by transformed
tissues (Bonhomme et al., 2000a; Hong et al., 2006). The hairy
root phenotype is mainly due to the rol genes (A, B, C
and D), and in particular the rolB gene (Nilsson and
Olsson, 1997), though hairy roots could also be obtained after
transformation of Atropa belladonna, with the rolC
gene alone (Bonhomme et al., 2000b). The choice of a bacterial
strain is very important since some plants are very resistant to
infection (monocots are for example harder to transform with
Agrobacterium than dicotyledonous plants). Moreover,
bacterial strains are more or less virulent according to the
plant species. The LBA 9402 strain is hypervirulent and has been
used to successfully transform Hyoscyamus muticus
(Vanhala et al., 1995), Centaurium erythraea (Piatczak et
al., 2006), Saponaria vaccaria (Schmidt et al., 2007),
Gentiana macrophylla (Tiwari et al., 2007) and P.
somniferum album (Le Flem et al., 2004).
Besides the use of wild strains, genetically engineered
bacterial strains with modified pRi or disarmed Agrobacterium
tumefaciens with a plasmid containing rol genes
together or separately have been also employed for the
transformation. Hairy roots may also be initiated, containing
constitutive expression constructs. This was the case for
Cinchona officinalis (Geerlings et al., 1999). These authors
developed a binary vector whose T-DNA contained
constitutive-expression versions (CaMV35S promoter with double
enhancer and nos terminator) of two genes encoding
rate-limiting enzymes: tryptophan decarboxylase (tdc) and
strictosidine synthase (str) from C. roseus,
together with an intron-possessing β-glucuronidase (gus-int)
reporter gene and a hygromycin phosphotransferase (hpt)
selection marker gene. This binary vector construct was used in
conjunction with A. rhizogenes strain LBA 9402 to obtain
tdc and str-gene-transformed hairy roots of C.
officinalis. This technique opened a wide field of
applications in the regulation of biosynthetic pathways and
bioconversions; however, plant transgenesis is still a discussed
subject which encounters a strong opposition of the public
opinion, more specifically in European countries.
INFECTION CONDITIONS OF THE PLANT MATERIAL
Several
protocols have been used for the infection of plant material by
A. rhizogenes. However, the success of the transformation
depends on various parameters such as the species and the age of
the plant tissue, with the younger ones being in general more
sensitive to bacterial infection (Sevon and Oksman-Caldentey,
2002). The bacterial strain used and the density of the
bacterial suspension are also influential (Park and Facchini,
2000). The explants most commonly used for infection are young
tissues of sterile plantlets, hypocotyl segments, cotyledons,
petioles and young leaves. The contact between bacteria and
plant cells can be induced by direct injection of the bacterial
suspension into the plantlet or by immersion of the plant
tissues in the bacterial suspension. This last procedure can be
enhanced with vacuum infiltration (Tomilov et al., 2007). In
these cases, the explants have to be wounded before they are
inoculated. The use of excised tissues, leaf disks (Wang et al.,
2002) or organ sections (Komaraiah et al., 2003) increases the
contact surface between the plant tissue and the bacteria.
With
hard to transform plants alternative procedures may be
implemented. Among these procedures, micro wounding through
electroporation (Matsuki et al., 1989) or sonication can be used
(in a process called sonication assisted Agrobacterium-mediated
transformation or SAAT) (Trick and Finer, 1997; Le Flem
et al., 2004). For the transformation of P. somniferum album,
several factors of the SAAT protocol were investigated for their
influence on transient gus expression: pre-culture
period, sonication and co-culture duration. The highest
number of GUS-positive hypocotyls (91%) was obtained after
60 seconds of sonication and 2 days of co-culture.
In
several experiments acetosyringone was used to activate the
virulence genes of Agrobacterium, and to enhance the
transfer of foreign genes into the plant genome (Stachel et al.,
1985; Gelvin, 2000; Tao and Li, 2006; Kumar et al., 2006). The
optimal concentration of acetosyringone varies from one
experiment to another. For the transformation of Torenia
fournieri, low concentrations (10-30 µM) enhanced the
transformation, but higher ones did not significantly increase
the transformation frequency. For Nicotiana tabacum or
P. somniferum the acetosyringone concentrations used varied
between 50 and 150 µM.
The
duration of the plant - bacterium contact during the inoculation
and the co-cultivation are parameters that can be optimized. The
average co-cultivation duration is about two or three days.
After that, the explants must be transferred to a solid medium
containing an antibiotic to eliminate the bacteria. Cefotaxime
(250-500 mg L-1) and Timentin (200-300 mg L-1)
are often used to eliminate the bacteria. The explants are then
transferred onto a solid hormone-free medium in the dark at
20-25°C, and the first roots appear after a few weeks (usually 1
to 4). The roots are then transferred to erlenmeyer flasks
containing liquid phytohormones-free medium. The typical
transformed root phenotype is a highly branched root covered
with a mass of tiny root hairs and these cultures do not require
phytohormones. Concerning the growth rate, the average doubling
time of hairy root lines is around 2-3 days. Species-related
anatomic, morphologic and cytologic changes have been reported
(Webb et al., 1990).
The
putatively transformed roots are usually analyzed to check for
T-DNA integration. Opine analysis using paper electrophoresis on
root extracts is one of the techniques used to confirm the
transgenic nature of the roots (Giri et al., 2000; Han et al.,
2006). Another alternative is the use of reporter genes. In this
area, the use of binary vectors has proven useful for assessing
the gene transfer to the plant genome, and following the long
term stability of this transfer using selection or marker genes.
Selection genes can be antibiotic –resistance genes such as
nptII or hpt coding for neomycin phosphotransferase
or hygromycin –phosphotransferase, respectively, expressed only
in the transformed tissues. A study on Astragalus sinicus
and Glycine max using feedback-insensitive anthranilate
synthase (ASA2) cDNA isolated from a 5-methyl tryptophan
(5MT)-resistant tobacco cell line showed that hairy roots
transformed with a 35s-asa2 construct could be directly
selected using 20–75 µM 5MT. GUS staining or fluorescence
microscopy following transformation with binary vectors
containing GUS, GFP, dsRED or EYFP are useful for the
identification of stable transformed roots (Tomilov et al.,
2007). PCR and Southern-blotting of rol genes is also
another way to confirm T-DNA integration into the plant genome (Lorence
et al., 2004; Tiwari et al., 2007). Additional PCR and
Southern-blotting of virC gene is sometimes performed to
check for total elimination of the agrobacteria (Shi et al.,
2006).
The
infection conditions are of capital importance and the choice of
the Agrobacterium strain is a first-rate parameter. The
strain virulence has strong repercussions on the transformed
material properties (morphology, growth rate, and metabolite
level). The A. rhizogenes strain LBA 9402 has showed
stronger infective ability on Rheum palmatum while A.
rhizogenes strain R1601 generated a faster growing clone. In
this paper, like often reported, the secondary metabolite
content and composition varied significantly between clones
(Yang et al., 2006). The T-DNA integration into the plant genome
which can be linked to the bacterial strain and the number of
transferred copies has consequences on the growth and secondary
metabolism of the transformed roots. In Whitania somnifera,
TL -DNA and TR –DNA integration frequency
was linked to the bacterial strain and had an effect on the
transformed material morphology. Typical transformed roots,
transformed rooty calluses and transformed calluses were
obtained where the whitasteroids level was more related to the
material morphology than to the inoculated bacterial strain (Bandyopadhyay
et al., 2007). In Gentiana macrophylla, transformed
roots, TL -DNA and TR –DNA integration had
an effect on the root specific secoiridoid glucoside
gentiopicroside accumulation (Tiwari et al., 2007).
EFFECT OF MEDIUM COMPONENTS ON GROWTH AND METABOLITE
ACCUMULATION
The hairy roots growth rate is generally high, but great
variations exist from one line to another. Mean doubling time
after inoculation ranges from 24 to 90 h (Payne et al., 1991),
but sometimes it is much longer. As an example, the doubling
time of Galphimia glauca hairy roots was 6 days (Nader et
al., 2006), and even 15 days in the case of Cinchona
hairy roots (Geerlings et al., 1999). Optimization of the medium
composition may sometimes increase the growth rate of the roots
and/or the yield of accumulated metabolites. The use of modified
culture media is generally required. These modifications involve
changes in sugars, nitrogen, and phosphorous sources. The effect
of nitrate and ammonium concentrations on growth and alkaloid
accumulation of A. belladonna hairy roots was studied (Bensaddek
et al., 2001). An increase of ammonium concentration in the
culture medium resulted in lowering the growth rate while an
increase of the nitrate concentration had a deleterious effect
on the alkaloid biosynthesis and accumulation. The highest
biomass and alkaloid yields were obtained with reduced levels of
both nitrogen sources. The results obtained by Sivakumar and
collaborators with ginseng hairy roots suggest that mineral
elements are an important regulatory factor of growth and
biomass (Sivakumar et al., 2005).
In
vitro
culture of plants cells usually requires the presence in the
medium of plant growth regulators, mainly auxins and cytokinins.
In the case of hairy roots, one characteristic of their
phenotype is the fast hormone-independent growth. The result is
that in media used for the culture of hairy roots hormones are
generally lacking. Even more, it has been demonstrated that in
transformed roots of Datura stramonium, treatment of the
cultures with 2.0 mg L-1 α-naphtalene acetic acid (NAA)
and 0.2 mg L-1 kinetin induced a de-differentiation
of the root tissue and a redirection of primary nitrogen
metabolism (Ford et al., 1996). In several experiments this
de-differentiation was accompanied by a significant decrease or
even a cessation of alkaloid production (Robins et al., 1991).
However, it was demonstrated more recently that when testing
systematically the effect of different types of phytohormones
upon root growth and secondary metabolite production, some of
them could enhance either growth or metabolites production. In
the case of A. annua hairy roots (Weathers et al., 2005),
the response of cultures to five types of hormones: auxins,
cytokinins, ethylene, gibberellins (GA) and abscissic acid (ABA)
was evaluated. The highest biomass was obtained when 1-5 mg L-1
ABA was supplied in the medium, while 0.5-1 mg L-1
2-isopentenyladenine inhibited root growth but stimulated the
production of artemisinin more than 2-fold. In other experiments
(Yu et al., 2006), Polygonum multiflorum hairy root
cultures were supplemented with 2,4-D, NAA and 6-BA at various
concentrations. The results showed that 0.1 mg L-1
2-4 D had a deleterious effect on the root cultures; in
contrast, NAA and 6-BA in certain conditions could stimulate the
growth (0.3-0.4 mg L-1 BA; or 0.4 mg L-1
NAA) and the production of anthraquinones (0.4 mg L-1
BA). With the combined treatment of P. ginseng hairy
roots with both 25 µM indole-3-butyric acid (IBA) and 100 µM
MeJA the productivity of ginsenoside went to 10 mg L-1
d-1, instead of 7.3 mg L-1 d-1
with MeJA alone (Kim et al., 2007).
ELICITATION
The use of biotic or abiotic stress on tissue cultures has been
shown to have an effect on the secondary metabolite
accumulation. The elicitation procedure consists in treating the
cultures with a physical or a chemical agent that will cause
phytoalexin production leading to defence mechanisms in the
plant cells. The eliciting agents are classified in two large
categories: abiotic elicitors (physical, mineral and chemical
factors), and biotic elicitors which are factors of plant or
pathogen origin (Yoshikawa, 1978). As the secondary metabolites
are generally produced in nature as a defence mechanism against
pathogenic and insect attack, elicitation is often used to
enhance their in vitro accumulation levels. Elicitation
is mainly used when the hairy root cultures have reached their
stationary phase, usually around 2-3 weeks after inoculation.
There are many recent examples combining hairy root culture and
elicitation treatments (Table I), some of which involving the
production of pharmacologically-active terpene-derived
compounds.
Abiotic elicitors such as NiSO4 (20 µM), selenium
(0.5 mM), and NaCl (0.1%) supplemented in transformed root
cultures of P. ginseng, increased the saponin content
1.15-1.33 times compared to controls (Jeong et al., 2006).
Sodium acetate (10.2 mM), added for 24 h to the culture medium
of Arachis hypogaea (peanut) hairy roots, lead to a
60-fold induction and secretion of trans-resveratrol into
the culture medium (Medina-Bolivar et al., 2007). Sorbitol added
as an osmoticum had a dramatic effect on tanshinone yield in
Salvia miltiorrhiza Bunge hairy roots: that yield was
increased 4.5-fold as compared to the control (Shi et al.,
2007).
The effect of biotic elicitors used at higher concentrations
(5-400 mg L-1) seems to be clearly efficient. In
transformed roots of P. ginseng, plant-derived
oligosaccharides from Paris polyphylla var. yunanensis
increased the saponin content by more than 3 times (Zhou et al.,
2007). Fungus-derived oligosacchrides (from the fungal endophyte
Colletotrichum gloeosporoides), yeast elicitor
(polysaccharide fraction of the yeast extract), and chitosan
increased artemisinin (anti-malarial sesquiterpene endoperoxide)
production in A. annua 1.5, 3 and 6-fold, respectively
(Wang et al., 2006; Putalun et al., 2007).
Table I:
Elicitation of hairy root cultures accumulating
pharmacologically-active compounds. a ratio as
compared to the content of not elicited hairy roots.
Type of elicitation |
Species |
Produced metabolites
and medicinal properties |
Elicitor |
Fold increase of the metabolite content
a |
Reference |
Abiotic |
Panax ginseng |
Total saponin content
tonic, stimulant, adaptogenic |
NiSO4 20 µM |
1.2 - 1.23 |
Jeong et al., 2006 |
Selenium 0.5 mM |
1.31 - 1.33 |
NaCl 1% |
1.13 - 1.15 |
Salvia miltiorrhiza
|
Tanshinone
antioxidant anti-inflammatory |
Sorbitol 50 g L-1 |
4.5 |
Shi et al., 2007 |
Arachis hypogaea |
trans-Resveratrol
antioxidant, atherosclerosis prevention |
Sodium acetate 10.2 mM |
60 |
Medina-Bolivar et al., 2007 |
Biotic |
Panax ginseng |
Total saponin content |
Oligosaccharides from Paris polyphylla
30 mg L-1, plant derived |
3 |
Zhou et al., 2007 |
Artemisia annua |
Artemisinin
antimalarial |
Oligosaccharides from Colletotricum
gloeosporoides 0.4 mg total sugar mL-1,
fungus derived. |
1.51 |
Wang et al., 2006
Putalun et al., 2007 |
Polysaccharide fraction of the yeast
extract 2 mg L-1 |
3 |
Chitosan 150 mg L-1 |
6 |
Azadirachta indica |
Azadirachtin
pesticidic |
Salicylic acid 100 mM |
6 |
Satvide et al., 2007 |
Jasmonic acid 100 mM |
9 |
Hyoscyamus niger,
PMT over-expression |
Polyamines and tropane alkaloids
mydriatic, parasympatholytic,
antiparkinsonian |
MeJA 50 µM |
2 |
Zhang et al., 2007 |
Centella asiatica |
Asiaticoside
anti-inflammatory |
MeJA 0.1 µM |
de novo
accumulation |
Kim et al., 2007 |
Signal compounds such as salicylic acid and
MeJA
can be used as elicitors to enhance the accumulation of secondary
metabolites already present in the cultures. In the case of
Azadirachta indica hairy roots, addition of 100 mM jasmonic acid
and salicylic acid showed a 6-9 fold enhancement of azadirachtin, a
tetranortriterpenoid with pesticidal activity, as compared to
control cultures (Satvide et al., 2007). These elicitors stimulate
biosynthetic pathways. In transgenic Hyoscyamus niger hairy
root cultures over-expressing putrescine N-methyltransferase, MeJA
treatment enhanced both polyamine and tropane alkaloid biosynthesis
(Zhang et al., 2007). Moreover, added to the medium of Centella
asiatica at a concentration of 0.1 µM, MeJA triggered de novo
accumulation of asiaticoside, an anti-inflammatory triterpene
saponin which was not initially accumulated in the hairy roots (Kim
et al., 2007). This accumulation followed a linear increase for 2
weeks and could be maintained at its top level (7.12 mg g-1)
for one additional week. The same authors also state that the
expression of CabAS, a putative beta-amyrin synthase, was
higher than controls 12 hours after MeJA addition and during the 2
following weeks.
CONCLUSION AND PERSPECTIVES
At the present time, a constantly increasing number of species have
been transformed for the establishment of hairy root cultures: 29
species in 1987, 116 in 1990, and 185 plants from 41 families in
2004 (Kuzovkina and Schneider, 2006). Hairy root cultures offer many
advantages among which we can highlight the high and continuous
yields of a wide range of metabolites and a high growth potential (Grzegorczyc
et al., 2006). The large number of initiated clones offers a
screening opportunity (Yu et al., 2006). Large scale culture
feasibility and long term stability make this biotechnological
approach not only a reliable source of secondary metabolites
(Peebles et al., 2007), but also an effective tool to study the
biosynthetic pathways of complex plant products (Robins, 1998).
However, scaling up hairy roots to industrial levels poses a great
challenge at the moment. The efficiency of the scaling up systems
still needs optimization before industrial exploitation becomes
valuable.
Considering their high efficiency at extremely low concentrations,
the use of MeJA and other signalling compounds for transformed root
cultures elicitation is opening new ways for a possibly profitable
in vitro secondary metabolite production. Genetic engineering
(Li et al., 2006), nutritional modelling (Cloutier et al., 2007),
and cross-species co-culture systems involving hairy roots, are
opening exciting future prospects in the field of enhanced
production and bioconversion (Lin et al., 2003).
ACKNOWLEDGMENTS
The authors would like to thank ECOS- Nord and ANUIES for supporting
the cooperation between the French and Mexican laboratories on the
biotechnological approach of promotion of Mexican medicinal plants.
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Accepted for
publication: 7 October 2008
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