Introduction
Introduction
One of the most studied group of secondary metabolites is alkaloids. Today more than
16.000 alkaloids have been identified from plants [1]. Alkaloids also represent an important pharmaceutical and economical value, and
many of them are currently isolated from plant material since no alternative production
method exists which is economically feasible. For the past few decades considerable
interest has been shown in the production of the secondary products by plant cell
cultures [2]. The success, however, has been moderate mainly because the biosynthesis pathways
of the secondary metabolites, including the enzymes and the regulatory mechanism governing
expression and function of the pathways, are poorly understood [3], [4]. Secondary product formation is often low and unstable in undifferentiated callus
and suspension cultures; the metabolism of secondary products seems to correlate with
the degree of organisation of cell structures. Therefore, the roots are capable of
accumulating a large range of secondary metabolites reflecting biosynthetic capacity
[5]. As an example several alkaloids that are scarcely synthesised in undifferentiated
cells are produced at relatively high levels in cultured roots. This suggests that
alkaloid production is associated with the root organogenesis [6]. The major problem associated with the in vitro culture of conventional roots is usually the slow growth rate. In contrast, Agrobacterium-mediated hairy roots are fast-growing and genetically stable which can also be successfully
cultured in large-scale bioreactors (e. g., [7]). Transformed roots are able to produce secondary metabolites at levels that are
often comparable to, or greater than that of the intact plants [2], [8], [9].
In this review we describe Agrobacterium rhizogenes-mediated transformation of plants to obtain hairy root cultures, recent achievements
and improvements on alkaloid production in transformed roots, and finally the possibilities
for metabolic engineering using hairy root system.
Hairy Root Disease
Hairy Root Disease
Hairy root and the crown gall tumor are two plant diseases caused by two Gram-negative
soil bacteria, Agrobacterium rhizogenes and A. tumefaciens, respectively. Depending on the strains of Agrobacterium involved, one or both of the two pieces (TL and TR) of the Ti-(tumor inducing) or
Ri- (root inducing) plasmid are transferred from the bacteria into the nuclear genome
of the host plant. The transferred T-DNA derived from the Ti-plasmid causes the plant
cells to proliferate, to form crown gall tumors, and in the case of the Ri-plasmid,
the extensive formation of adventitious roots at or near the site of infection. The
transformed plant tissues are also directed by T-DNA genes to produce unusual metabolites
called opines, that serve as specific nutrients for the bacteria [10], [11].
The molecular mechanism of T-DNA transfer to the plant is most likely the same for
both Agrobacterium species, but the physiological basis of tumorigenesis is totally different. Crown
gall tumors result from the overproduction of the phytohormones auxin and cytokinin
specified by A. tumefaciens T-DNA genes [12]. The physiological basis of the hairy root disease is not totally understood. Alteration
of auxin metabolism in transformed cells has been supposed to play an important role
in expression of the hairy root phenotype [12], [13]. TL-DNA plays the major role in hairy root induction, and the genes encoding auxin
synthesis have a somewhat accessory role [14], [15]. Auxin is necessary for hairy root induction, but it seems likely that auxin does
not play a role in T-DNA expression in transformed plant cells [14]. Physiological studies have indeed shown that the transformed cells are more sensitive
to extracellularly supplied auxins than the normal roots [16]. Spanó and co-workers [17] have suggested that the genes responsible for increased sensitivity of hairy root
cells to auxin are located on the TL-DNA. On the other hand, hairy roots of Hyoscyamus muticus L. have been demonstrated to tolerate high auxin levels. The sensitivity is most
probably restricted to certain plant species [18].
Characterization of Agrobacterium plasmids
Several classes of both Ri- and Ti-plasmids have been characterised. The plasmids
are large (200 to greater than 800 kb) and contain one or two regions of T-DNA and
a vir (virulence) region, all of which are necessary for tumorgenesis [12], [19]. The classification of plasmids depends to a large extent on the type of opines
that the plasmids direct the infected plants to synthesise. The Ri-plasmids are grouped
into two main classes according to the opines synthesised by hairy roots. Agropine-type
strains (e. g., A4, 15 834, LBA9402, 1855) induce roots to synthesise agropine, mannopine
and the related acids, and mannopine,-type strains (e. g., 8196, TR7, TR101) induce
roots to produce mannopine and the corresponding acids [20]. Other types of opines (e. g., cucumopine, mikimopine) have also been described
[21], ]22].
The agropine-type Ri-plasmids are very similar as a group, and a quite distinct group
from the mannopine-type plasmids [23]. Perhaps the most studied Ri-plasmids are agropine-type strains, which are considered
to be the most virulent and therefore more often used in the establishment of hairy
root cultures [24].
The plasmids of A. tumefaciens were used as vectors for many years before the interaction between Agrobacterium rhizogenes and the plant cell was discovered [10], [19], [25]. The use of Agrobacterium as a vector is based on its unique capacity to transfer a piece of its own DNA (T-DNA)
into the nuclear genome of plant cells. Any DNA placed between the borders will be
transferred to a plant cell. This property has been extremely useful for the introduction
of new genes into plants, either for research or for practical applications [26].
The genes responsible for hairy root formation
The T-DNA of the agropine-type Ri-plasmid consists of two separate T-DNA regions designed
the TL-DNA and TR-DNA [23]. Each of the T-DNA fragments spans a 15 - 20 kb region, and they are separated from
each other by at least 15 kb of non-integrated plasmid DNA. These two fragments can
be transferred independently during the infection process [27]. White and co-workers [23] made a comparison between the T-DNA region of the agropine and mannopine-type Ri-plasmids
and the octopine and nopaline-type Ti-plasmids. The agropine-type Ri T-DNA has limited
sequence homology to either the nopaline- or octopin-type Ti-DNA sequences, and share
homology only in the region corresponding to two loci encoding auxin synthesis and
the agropine synthesis loci [23]. No homology has been found between Ri T-DNA and the tmr locus of the Ti T-DNA. The genes encoding auxin synthesis (tms1 and tms2) and agropine synthesis (ags) have been localised on the TR-DNA of the agropine type Ri-plasmid [23], [28]. The mannopine type Ri-plasmids contain only one T-DNA that shares considerable
DNA sequence homology with TL of the agropine-type plasmids [12].
Mutation analysis of the TL-DNA has led to identification of four genetic loci, designed
locus rolA, rolB, rolC, and rolD, which affect hairy root induction [15], [23]. The complete nucleotide sequence of the TL-region revealed the presence of 18 open-reading
frames (OFRs), 4 of which, ORFs 10, 11, 12 and 15, respectively, correspond to the
rolA, rolB, rolC, and rolD loci. It was also shown that rolA, rolB, and rolC play the most important role in hairy root induction. In particular, rolB seems to be the most crucial in the differentiation process of transformed cells,
while rolA and rolC provide with accessory functions [15].
Although the TR-DNA is not essential for hairy root formation it has been shown that
the aux1 gene harboured in this segment provides to the transformed cells with an additional
source of auxin. Recently, Moyano and co-workers [29] found that aux genes play a significant role in the morphology and alkaloid production of transformed
roots of Datura metel and Duboisia hybrid. The studies with Panax ginseng c. v. Meyer hairy roots also support this finding (Mallol et al., unpublished results).
Mechanism of Agrobacterium-plant cell interaction
One of the earliest stages in the interaction between Agrobacterium and a plant is the attachment of the bacterium to the surface of the plant cell.
A plant cell becomes susceptible to Agrobacterium when it is wounded. The wounded cells release phenolic compounds, such as acetosyringone,
that activate the vir-region of the bacterial plasmid [30]. It has been shown that the Agrobacterium plasmid carries three genetic components that are required for plant cell transformation
[13]. The first component, the T-DNA that is integrated into the plant cells, is a mobile
DNA element. The second one is the virulence area (vir), which contains several vir genes. These genes do not enter the plant cell but, together with the chromosomal
DNA (two loci), cause the transfer of T-DNA. The third component, the so-called border
sequences (25 bp), resides in the Agrobacterium chromosome. The mobility of T-DNA is largely determined by these sequences, and they
are the only cis elements necessary for direct T-DNA processing.
Zupan and Zambryski [31] have described in details the mechanism for the transfer of T-DNA into the plant
cell. The early steps of the transfer are relatively well studied but the mechanics
of integration are not completely understood. According to Zambryski [32], it is a multistep process involving recombination, replication and repair activities,
most likely mediated by host cell enzymes. The overall process of integration is probably
very similar to any illegitimate recombination of foreign DNA into eukaryotic genomes
[26].
Comprehensive reviews on Agrobacterium transformation can be found in Zhu and co-workers [19] and Zupan and co-workers [25].
Establishment of Hairy Root Cultures
Establishment of Hairy Root Cultures
The transformation is induced on aseptic, wounded plants or plant parts by inoculating
them with a thick, viable A. rhizogenes suspension. After 1 - 4 weeks, when roots emerge at the site of inoculation, they
are individually cut off and transferred into a hormone-free growth medium e. g.,
MS [33] or B5 [34], containing antibiotics to kill the bacteria. The protocol of the establishment
of hairy root cultures has been described in detail by [8], [35], [36].
The susceptibility of plant species to Agrobacterium strains varies greatly. However, plant species, which were shown to be insusceptible
to A. rhizogenes, e. g., strain A4, have been successfully transformed with other strains [37], [38]. Significant differences were observed between the transformation ability of different
strains of Agrobacterium [36], [39]. The age and differentiation status of plant tissue can also affect the chances
of successful transformation. The level of tissue differentiation also determines
the ability to give rise to transformed roots after A. rhizogenes inoculation [40]. Successful infection of some species can be achieved by the addition of acetosyringone
[41].
The genetic transformation can be confirmed by assaying the opines. Opine production
can, however, be unstable in hairy roots and may disappear after a few passages [42]. For this reason, detection of T-DNA by Southern blot hybridization is often necessary
to confirm the genetic transformation [43]. The polymerase chain reaction (PCR) simplifies the detection of transformation
[44], [45].
Characteristics of the Hairy Roots Cultures
Characteristics of the Hairy Roots Cultures
Hairy roots are fast growing and laterally highly branched, and are able to grow in
hormone-free medium. Moreover, these organs are not susceptible to geotropism anymore.
They are genetically stable and produce high contents of secondary metabolites characteristic
to the host plant. The secondary metabolite production of hairy roots is stable compared
to other types of plant cell culture. The alkaloid production of hairy roots cultures
has been reported to remain stable for years [6]. The secondary metabolite production of hairy roots is highly linked to cell differentiation.
Alkaloid production decreased clearly when roots were induced to form callus, and
reappeared when the roots were allowed to redifferentiate [6], [42]. An interesting characteristic of some hairy roots is their ability to occasionally
excrete the secondary metabolites into the growth medium [46]. However, the extent of secondary product release in hairy root cultures varies
between species [47], [48], [49].
The average growth rate of hairy roots varies from 0.1 to 2.0 g dry weight/litre/day.
This growth rate exceeds that of virtually all-conventional roots and is comparable
with that of suspension cultures. However, the greatest advantage of hairy roots compared
to conventional roots is their ability to form several new growing points and, consequently,
lateral branches [2]. The growth rate of hairy roots may vary greatly between species, but differences
are also observed between different root clones of the same species [6], [50], [51], [52]. The pattern of growth and secondary metabolite production of hairy root cultures
can also vary. Secondary production of the hairy roots of Nicotiana rustica L. was strictly related to the growth, whereas hairy roots of Beta vulgaris L. exhibited non-growth-related product accumulation [47]. In the case of the hairy roots of Scopolia japonica Jacq. and H. muticus, the secondary products only started to accumulate after growth had ceased [36], [46], [53]. Secondary metabolite synthesis dissociated from growth would be desirable for commercial
production, as it would allow the use of continuous systems.
Improvement of the Production in Transformed Root
Cultures
Improvement of the Production in Transformed Root
Cultures
Hundreds of plant species have been successfully transformed to hairy roots (see the
reviews by Tepfer [54] and Giri and Narasu [9]). For the past ten years hairy roots have also been investigated as a potential
source of pharmaceuticals [2], [9]. Table [1] summarises some of the most important alkaloids produced by the hairy root cultures
of medicinal plants. The comparison of hairy roots is not always possible, since the
product yield is calculated in many different ways (e. g., mg/g f. w. or d. w., mg/flask,
% of d. w., mg/l), and all the parameters are not always given. Additionally, high
contents of the product could be associated with poor growth, and thus the real productivity
(mg/l) remains low.
The secondary metabolites of hairy roots are strictly limited to those that are normally
produced in the roots. On the other hand, if the biosynthesis of secondary metabolites
normally takes place in the green parts of plants, it is necessary to utilise modified
hairy roots, e. g., ”green hairy roots” or, alternatively, transformed shoot teratomas
[42], [55]. Conversely, the shoot teratomas of Nicotiana tabacum L., A. belladonna L. and Solanum tuberosum L. failed to produce alkaloids, indicating that the biosynthesis site of these alkaloids
is in the roots [56].
Selection of high-producing cell lines
Somaclonal variation has been used widely as a breeding tool in the search for agriculturally
interesting traits. Cultured plant cells are heterogeneous and it is therefore possible
to select the cells with respect to a particular desired property [57]. The selection of highly productive cell lines has for long been a well-known strategy
for the production of secondary metabolites by cell cultures. Considerable somaclonal
variation was found in cell cultures derived from protoplasts of H. muticus [58], [59]. Thus high alkaloid-producing plants can be also obtained by selection [60]. However, there is no complete agreement on the reasons for the diversity in alkaloid
production among clones. Somaclonal variation is caused by genetic changes, which
may alter the gene expression of the cells and the synthesis of secondary metabolites.
Genetic changes certainly cause the occasional variability in secondary metabolites,
but the expression of many secondary pathways is easily altered by external factors
and, furthermore, the responses of the cells to external factors depend on their physiological
stage. However, a different level of a particular metabolite is the result of differential
and reversible gene expression [61].
Mano and co-workers [50] derived forty-five hairy root clones of D. leichhardii F.v. M. from individual root meristems and found that there was considerable variation
in growth rate, alkaloid content and productivity between the clones. Generally hairy
roots are considered to be stable and not easily manipulated. However, hairy roots
also possess a certain amount of heterogeneity even though derived from a single root
tip, because repeated selection has shown to be applicable to hairy root cultures
in order to obtain high scopolamine-producing hairy root lines [62]. Nicotinic acid can be used as a selective agent in order to isolate high nicotine-producing
root lines of N. rustica hairy root cultures. However, the selected root clones also had a higher ability
to detoxify nicotinic acid to nicotine and anatabine [63]. Amino acid analogues have also been used for establishing hairy root lines with
a high yield of l-hyoscyamine [64].
Protoplasts also offer a possibility to isolate high-producing variants at the single
cell level. There are some reports of protoplasts having been isolated from root material
[65]. Selected plants with high contents of the desired products should be used as the
starting material for protoplast isolation [58]. Statistically high-producing plants give rise to high producing cell lines. The
hairy root clones of N. rustica regenerated from protoplasts showed variation in morphology, alkaloid formation and
T-DNA structure. Some clones also showed increased alkaloid production [66]. Clear differences were also observed in the growth rate, morphology and in the
l-hyoscyamine content between the protoplast-derived hairy root clones of H. muticus. Most of the protoplast-derived hairy root clones showed increased alkaloid synthesis
characteristics compared to the parent line. The l-hyoscyamine content ranged from 0.04 % to 1.5 %. The mean content of the clones (0.49
%) was, however, almost the same as in that the parent clone (0.57 %) that was used
as the starting material for the protoplast isolation [65]. The most comprehensive study on somaclonal variation in transformed roots and protoplast
derived hairy root clones has been performed by Sevón and co-workers [6]. They could show that the clones were stable over long-term cultivation and the
large variation between the clones remained several years unchanged.
Optimizing the growth conditions and the medium
Several physical and chemical factors have been found that could influence the growth
and productivity of hairy root cultures. However, hairy roots are not so easily modified
by changing the culture conditions as cell suspension cultures [67]. Several studies have been made on the effect of medium composition on growth and
the production of secondary metabolites. Most of the investigations have been carried
out with hairy roots of Catharanthus roseus L. [68] and Solanaceous species [52], [69].
Factors such as the carbon source and its concentration, ionic concentration of the
medium [70], pH of the medium [71], light [70], phytohormones [18], [72], [73], temperature [74] and inoculum [8], [50], [75], are known to influence the growth and alkaloid production of hairy roots. A. rhizogenes strain could also have effect on biomass and alkaloid productivity of hairy roots.
The atropine yield of root lines of Hyoscyamus albus L. induced by A. rhizogenes strain A4 were significantly higher than the root lines of H. albus induced by A. rhizogenes strain LBA9402. Such relationship between the bacterial strain and alkaloid productivity
could not be found in case of root lines of H. muticus [76].
Gamborg’s B5 medium is the most widely used medium for the hairy roots of many species
[69]. Supplementation of heavy metal ions, such as Cu2+, has been shown to stimulate alkaloid production [46], [70]. Concentrations of inorganic phosphate above or below that present in Gamborg B5
medium (1.0 mM) has reduced the cell yield of hairy root cultures of Datura stramonium L., but low levels of phosphate stimulated l-hyoscyamine production. Nitrate also reduced cell yield and l-hyoscyamine production at concentrations above that present in Gamborg B5 medium (30
mM) [77].
Toivonen and co-workers [68] studied the effect of varying concentrations of sucrose, phosphate, nitrate and
ammonium on growth and indole alkaloid production in hairy root cultures of C. roseus. They found that low nutrient levels enhanced alkaloid production, but biomass yields
were maximal in media containing high concentrations of sucrose and ammonia. Similar
results have been obtained by Payne and co-workers [77], who reported that the optimum concentrations of phosphate and nitrate for product
formation were lower than that for growth.
Hairy root cultures of H. muticus also produced the highest l-hyoscyamine content at a sucrose concentration of 30 g/l, but higher than this stimulated
the growth of the hairy roots. The root clones of H. muticus could not utilise ammonium as the sole nitrogen source. Maximum growth and l-hyoscyamine production was achieved when the content of ammonium was not more than
2 mM [52]. Ammonium had a strong influence on the growth of hairy roots of A. belladonna while nitrate had clear effect on the alkaloid production and the scopolamine and
hyoscyamine ration [78]. Modifying the culture conditions can increase the growth rates and biomass yields
of the hairy roots of D. stramonium. However, the specific extracellular productivity (mg alkaloid /g biomass) cannot
be significantly increased by varying either the temperature or the relative nutrient
levels of sucrose and minerals in the medium [70], [74].
The hairy roots of different species behave differently in the same culture conditions.
Hilton and Wilson [69] investigated the growth and uptake of sucrose and minerals ions by six tropane alkaloid-producing
transformed root cultures and found that their requirements for certain mineral ions
varied when grown in batch cultures on Gamborg’s B5 medium. Individual hairy root
clones can also have different optimum concentrations of sucrose or mineral ions [52]. The different requirements make optimisation work difficult, because the culture
conditions have to be optimised separately for each species and for individual clones.
Effect of elicitors
Elicitation is one of the methods that have been used to enhance secondary metabolites
of cell cultures [79]. Not many publications have appeared on the elicitation of root or hairy root cultures,
most of the results being from experiments with cell suspension cultures [67[, [80]. Table [2] lists the reports where elicitors have been applied to hairy root cultures of medicinal
plants. There are only a limited number of alkaloids whose production can be induced
by elicitors. Those compounds, which defend the plants against micro-organisms, namely,
phytoalexins, are often easily formed in response to the elicitors, but the accumulation
of the alkaloids of interest has not usually been induced.
Although the use of elicitors does not directly increase the alkaloid content of hairy
roots, cell permeability increases and this often has a positive effect on the formation
of secondary metabolites [81]. The fungal elicitors and agents that increase the excretion of desired compounds
have on occasions been combined successfully in the treatment of hairy roots of C. roseus [82].
Enhancement of cell permeability may increase the formation of secondary products,
because feedback inhibition and intracellular degradation of the products decrease.
The economical benefit of the production process also depends on the capacity of the
producing cells to secrete the desired metabolite into the surrounding medium. Permeability
of plant membranes for the release of secondary metabolites has often been connected
with the loss of viability of the plant cells treated with permeabilizing agents and
methods [81]. Some attempts have been made to increase the permeability of the hairy roots. Biotic
and abiotic elicitors including solvents and detergents have been reported to release
the products from hairy roots into the medium without any loss of viability and production
capacity of the hairy roots [82], [83], [84]. Cusidó and co-workers [85] reported that tween 20 treatment encouraged both growth and alkaloid productivity
of hairy roots of Datura metel L. Additionally tween 20 treatment clearly increased the extracellular content of
scopolamine.
Chitosan has been used as an effective elicitor, but it also enhances the permeability
of the cells [46], [86], [87]. Permeabilization studies with chitosan have mainly been performed with cell suspension
cultures which, however, are not directly connected with hairy roots. This polycationic
polysaccharide induces pore formation in the plasmalemma of the cell cultures of Chenopodium rubrum. It has been suggested that pore formation is related to the degree of the deacetylation
(positive charges) of the chitosan. Consequently, highly charged chitosan polymers
induce a higher degree of pore formation and cause faster secondary product release
than the less charged ones. This means that there is a critical charge density, which
leads to loss of cell viability. Unfortunately, most of the permeabilization agents
are not, like chitosan, membrane-specific [87].
Table 1 Alkaloid production of the hairy root cultures of some medicinal plants
| Plant |
Alkaloid |
Content (mg/d d. w.) |
Reference |
|
Aconitum heterophyllum
|
aconites |
29.6 |
[102]
|
|
Atropa belladonna
|
atropine
cuscohygrine
l-hyoscyamine
scopolamine
atropine
scopolamine
littorine |
3.7
2.8
9.5
3.0
7.6
0.3
0.9 |
[103]
[37]
[37]
[98]
[104]
[104]
[105]
|
|
Brugmansia candida
|
scopolamine
hyoscyamine
scopolamine
hyoscyamine |
2.5 mg/g f. w. *
1.0 mg/g f. w.*
0.26*
0.86* |
[83]
[83]
[106]
[106]
|
|
Catharanthus roseus
|
ajmalicine
catharanthine
serpentine
vindoline
vinblastine |
4.0
2.0
2.0
4.0
0.003 μg/g f. w. |
[51]
[51]
[51]
[51]
[107]
|
|
Catharanthus tricophyllus
|
crude alkaloids |
9.2 |
[88]
|
|
Calystegia sepium
|
cuscohygrine |
3.0 |
[37]
|
|
Cinchona ledgerina
|
cinchonine
cinchonidine
quinidine
quinine |
1.6 μg/g f. w.
18.0 μg/g f. w.
15.9 μg/g f. w.
24.3 μg/g f. w. |
[38]
[38]
[38]
[38]
|
|
Cinchona officinalis
|
cinchonine +
cinchonidine
quinidine
quinine
strictosidine |
0.4
1.0
0.5
1.9 |
[107], [108]
[107], [108]
[107], [108]
[107], [108]
|
|
Datura candida
|
scopolamine
l-hyoscyamine |
5.7
1.1 |
[109]
[109]
|
|
Datura innoxia
|
l-hyoscyamine |
1.7 |
[110]
|
|
Datura metel
|
scopolamine
hyoscyamine |
4.1*
1.0* |
[85]
[85]
|
|
Datura stramonium
|
l-hyoscyamine
scopolamine
hyoscyamine
scopolamine |
5.6
5.6
6.4
1.9 |
[111]
[112]
[113]
[113]
|
|
Duboisia hybrid
|
l-hyoscyamine
scopolamine |
2.1
2.5 |
[110]
[110]
|
|
Duboisia leichhardtii
|
scopolamine |
18.0 |
[50]
|
|
Duboisia myoporoides
|
l-hyoscyamine
scopolamine
scopolamine |
8.0
2.4
32.0 |
[35]
[35]
[62]
|
|
Hyoscyamus albus
|
l-hyoscyamine
scopolamine
hyoscyamine
scopolamine |
8.0
4.6
15.1
5.4 |
[114]
[110]
[76]
[76]
|
|
Hyoscyamus niger
|
l-hyoscyamine |
12.5 |
[110]
|
|
Hyoscyamus muticus
|
l-hyoscyamine
atropine
scopolamine |
12.2
1.8
1.0 |
[6]
[76]
[99]
|
|
Nicotiana tabacum
|
nicotine
nicotine |
1.1 mg/g f. w.
0.1 mg/g f. w. |
[48]
[115]
|
|
Nicotiana rustica
|
nicotine
anatabine
nicotine |
0.3 mg/g f. w.
0.4 mg/g f. w.
0.9 mg/g f. w.* |
[63]
[63]
[116]
|
|
Peganum harmala
|
β-carbolines |
17.0 |
[117]
|
|
Scopolia carniolica
|
l-hyoscyamine |
2.0 |
[111]
|
|
Scopolia japonica
|
l-hyoscyamine
scopolamine |
13.0
5.0 |
[118]
[118]
|
|
Scopolia tangutica
|
l-hyoscyamine
scopolamine |
0.5
0.2 |
[110]
[110]
|
|
Solanum tuberosum
|
steroidal alkaloids
such as solanine |
0.1 mg/g f. w. |
[56]
|
|
Weigelia ”Styrica” |
ajmalicine
serpentine |
1.4 μg/g
0.2 μg/g |
[119]
[119]
|
| * calculated from the figure |
Table 2 Some elicitation studies carried out with the hairy root cultures of the medicinal
plants
| Hairy roots |
Elicitor |
Effect |
Reference |
|
Brugmansia candida
|
Hemisellulase
Theophylline
CaCl2
|
Stimulation of
hyoscyamine and
scopolamine |
[83]
|
|
Brugmansia candida
|
Salicylic acid
Yeast extract
CaCl2
AgNO3
CdCl2
|
Stimulation of
hyoscyamine and
scopolamine |
[84]
|
|
Catharanthus roseus
|
Methyl jasmonate |
Stimulation of catharanthine and ajmalicine |
[120]
|
|
Catharanthus roseus
|
Penicillium sp
homogenate |
Stimulation of catharanthine and ajmalicine |
[121]
|
|
Datura stramonium
|
Wide range of abiotic elicitors
(metal ions) |
Accumulation of sesquiterpene phytoalexins
(lubimin, 3-hydroxylubimin, rishitine) |
[122]
|
|
Hyoscyamus muticus
|
Rhizoctonia solani
|
Accumulation of
sesquiterpene phytoalexin |
[123]
|
|
Hyoscyamus muticus
|
Rhizoctonia solani
|
Accumulation of
solavetivone |
[124], [125]
|
|
Hyoscyamus muticus
|
Rhizoctonia solani
|
Accumulation of
solavetivone and lubimin |
[126]
|
|
Hyoscyamus muticus
|
Inonotus obliquus
|
Stimulation of hyoscyamine |
[8]
|
|
Hyoscyamus muticus
|
CuSO4
|
Stimulation of hyoscyamine |
[8]
|
|
Hyoscyamus muticus
|
Purifield chitosan |
Stimulation of
l-hyoscyamine |
[46]
|
|
Hyoscyamus muticus
|
Jasmonic acid
Methyl jasmonate |
Slight stimulation of hyoscyamine. Strong
stimulation of polyamines |
[127]
|
|
Hyoscyamus muticus
|
Methyl jasmonate +
wounding +
Rhizoctonia solani
|
Solavetivone and lubimin accumulation |
[128]
|
|
Nicotiana tabacum
|
Yeast extract, Botrytis fabae extract |
Accumulation of
sesquiterpene phytoalexins (capsidiol and debneyol) |
[129]
|
Large-Scale Cultivation of Transformed Roots
Large-Scale Cultivation of Transformed Roots
Much work has been carried out with bioreactors and process development during the
last decades. Design of the mixing system for bioreactors has been the most problematic.
Mechanical agitation is seldom suitable for hairy roots because they are susceptible
to shear stress that causes disorganisation and callus formation, with consequently
lowered productivity.
Conventional stirred-tank reactors have been successfully applied to hairy roots even
though the mixing system of such biorectors has been reported to cause shear damage
[88]. However, this is the only reported study in the literature, where hairy roots were
successfully cultivated in a simple stirred reactor. It seems to be clear that standard
reactors are not suitable for hairy root cultures.
However, the best growth characteristics were obtained with bioreactors without mechanical
stirring. The use of airlift reactors makes it possible to avoid shear stress completely,
and up to 13-litre vessels have been used for the growth of hairy roots [89]. Wilson and co-workers [7], [90] have described so called droplet reactors in which the medium is sprayed over the
roots and periodically sucked out, the roots being in contact with the air for most
of the time. The most promising bioreactors for the cultivation of hairy roots seem
to be so-called wave reactors. This reactor system has three components: a rocker
unit, the disposable bioreactor chamber, and the measuring and control units. The
wave reactor is a mechanically driven reactor system. The energy input is caused by
rocking the chamber forth and back putting the cell culture and the medium in a wave
movement. This reactor has been demonstrated to increase the growth of hairy root
cultures producing tropane alkaloids and ginsenosides significantly more than optimised
stirred reactors, rotating drum reactors and droplet phase reactors [91]. Pilot-scale studies with wave reactors are currently running up to 100 litre working
volume (R. Eibl, personal communication). More comprehensive review on large-scale
cultivation of hairy roots is presented by Eibl and Eibl [92].
Genetic Engineering as a Tool to Increase Alkaloid Production
Genetic Engineering as a Tool to Increase Alkaloid Production
Metabolic engineering has been successful in micro-organisms for the increased production
of pharmaceuticals and for the production of new compounds, for instance antibiotics.
Although efficient methods for gene cloning, including organ-specific promoters, transformation
(e. g., particle bombardment and Agrobacterium-mediated gene transfer), and regeneration of transgenic plants, are available, the
progress to improve medicinal plants has so far been relatively slow. This is due
to the fact that still very little is known about the biosynthesis of secondary metabolites.
Often the biosynthetic routes are very long and complicated requiring several steps
and key enzymes before the desired end product is formed [4]. The majority of plant genes involved in primary metabolism have now been identified
due to large-scale DNA sequencing projects whereas only very limited numbers of genes
involved in secondary metabolism are available.
Most enzymes in a given pathway are co-ordinately regulated, and it is speculated
that there are, in the case of secondary metabolites, no clear rate-limiting enzymes
as is the case for primary metabolism. Catalytic activities of individual enzymes
in a pathway often vary considerably, which may result in accumulation of some intermediates
unless metabolic channelling or compartmentation occurs. Pathways are controlled by
the cellular development but also partly induced by exogenous and endogenous signals.
One major limitation to modifying an existing biosynthetic pathway by introducing
a foreign enzyme is the substrate specificity because the enzyme must act on an intermediate
in that specific pathway. The regulation of enzyme levels and activity is the most
important factor in the control of the production of pharmaceutical compounds [2], [93].
Different approaches can be applied for obtaining higher yields of a desired compound
in transgenic plants or cell cultures: engineering single biosynthetic steps, mapping
regulatory genes, reducing flux towards competitive pathways and/or catabolism, and
increasing the number of producing cells [93]. Several reporter genes, such as neomycin phosphotransferase II (kan) and β-glucuronidase (gus), have been used as models in investigating gene expression in several medicinal
plants of interest [94], [95]. Although reporter genes were not expected to have an effect on secondary metabolite
production, there are a few examples where the formation of secondary metabolites
has either been stimulated or inhibited by the reporter gene [94], [96].
In recent years characterisation of the enzymes involved in the biosynthesis of alkaloids
has increased exponentially [3]. From a pharmaceutical point of view, the first successful example of engineering
a medicinal plant was performed by Yun and co-workers [97]. They cloned the hyoscyamine 6β-hydroxylase gene (h6h) of H. niger and introduced it to A. belladonna, which produces hyoscyamine as the main alkaloid and very little scopolamine. The
engineered A. belladonna hairy roots exhibited increased hydroxylase activity and produced five-fold higher
concentrations of scopolamine than the wild-type hairy roots [98]. The transgenic A. belladonna plants almost exclusively produced scopolamine [97]. The same gene alone or in combination with other regulatory genes has been further
transferred to different tropane alkaloid-producing plant species, and the effects
on secondary metabolism vary considerably. Transgenic root cultures of H. muticus carrying the h6h transgene were able to produce over 100 times more scopolamine than the control ones
[99]. Interestingly in this study also the hyoscyamine levels remained high contrary
to the findings in A. belladonna.
Besides the effective research carried out on the tropane alkaloid pathway [100], a lot of effort has been put in the understanding of regulation of indole alkaloid
(particularly vincristine and vinblastine, two high-value anticancer drugs) production
in C. roseus. These investigations, however, have so far had only limited success [101]. It might be due to the fact that the introduced enzymes do not catalyse critical
rate-limiting steps in the target biosynthetic pathways [98].
Conclusions
Conclusions
Despite the promising features and developments, the production of plant-derived pharmaceuticals
by hairy roots has not yet been commercially exploited. The main reasons for this
reluctance shown by industry to produce pharmaceuticals by means of hairy roots compared
to the conventional extraction of whole plant material are mainly economical ones
based on the too low contents. Furthermore, the compound produced by this novel production
system has to be re-evaluated by authorities for quality, efficacy and safety reasons
which might inhibit the industry for using this technology. However, we are convinced
of the rapid development of genomics, proteomics and metabolomics tools will create
great opportunities to engineer the often complex pathways of plant secondary metabolites
and thus increase the contents of high-value pharmaceuticals in plant cell or organ
cultures.
