-
Biotechnology Advances 18 (2000) 1–22
0734-9750/00/$–see front matter © 2000 Elsevier Science Inc. All
rights reserved.
PII: S0734-9750(99)00016-6
Research review paper
Transgenic hairy roots: recent trends and applications
Archana Giri, M. Lakshmi Narasu*
School of Biotechnology, Jawaharlal Nehru Technological
University,Hyderabad 500028, India
Abstract
Agrobacterium rhizogenes
causes hairy root disease in plants. The neoplastic roots
produced by
A. rhizogenes
infection is characterized by high growth rate and genetic
stability. These geneticallytransformed root cultures can produce
higher levels of secondary metabolites or amounts comparableto that
of intact plants. Hairy root cultures offer promise for production
of valuable secondary metabo-lites in many plants. The main
constraint for commercial exploitation of hairy root cultures is
theirscaling up, as there is a need for developing a specially
designed bioreactor that permits the growth ofinterconnected
tissues unevenly distributed throughout the vessel. Rheological
characteristics of heter-ogeneous system should also be taken into
consideration during mass scale culturing of hairy
roots.Development of bioreactor models for hairy root cultures is
still a recent phenomenon. It is also neces-sary to develop
computer-aided models for different parameters such as oxygen
consumption and ex-cretion of product to the medium. Further,
transformed roots are able to regenerate genetically stableplants
as transgenics or clones. This property of rapid growth and high
plantlet regeneration frequencyallows clonal propagation of elite
plants. In addition, the altered phenotype of hairy root
regenerants(hairy root syndrome) is useful in plant breeding
programs with plants of ornamental interest. In vitrotransformation
and regeneration from hairy roots facilitates application of
biotechnology to tree spe-cies. The ability to manipulate trees at
a cellular and molecular level shows great potential for
clonalpropagation and genetic improvement. Transgenic root system
offers tremendous potential for intro-ducing additional genes along
with the Ri T-DNA genes for alteration of metabolic pathways and
pro-duction of useful metabolites or compounds of interest. This
article discusses various applications andperspectives of hairy
root cultures and the recent progress achieved with respect to
transformation ofplants using
A. rhizogenes.
© 2000 Elsevier Science Inc. All rights reserved.
Keywords: Agrobacterium rhizogenes
; Hairy roots; Secondary metabolites; Bioreactor; Genetic
manipulation;
Transgenics
* Corresponding author. Fax:
1
91-339-7648
E-mail address:
[email protected] (M.L. Narasu)
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A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22
1. Introduction
Plants remain a major source of pharmaceuticals and fine
chemicals. Despite considerableefforts, only a few commercial
processes have been achieved using cell cultures (e.g. shiko-nin,
berberine). The major constraint with cell cultures is that they
are genetically unstableand cultured cells tend to produce low
yields of secondary metabolites. A new route for en-hancing
secondary metabolite production is by transformation using the
natural vector system
Agrobacterium rhizogenes
, the causative agent of hairy root disease in plants.
Geneticallytransformed hairy roots obtained by infection of plants
with
A. rhizogenes
, a gram-negativesoil bacterium, offers a promising system for
secondary metabolite production [1]. The fastgrowing hairy roots
are unique in their genetic and biosynthetic stability and their
fast growthoffers an additional advantage. These fast growing hairy
roots can be used as a continuoussource for the production of
valuable secondary metabolites. Moreover, transformed rootsare able
to regenerate whole viable plants and maintain their genetic
stability during furthersubculturing and plant regeneration.
2.
Agrobacterium
and Ri T-DNA genes
Agrobacterium
recognizes some signal molecules exuded by susceptible wounded
plantcells and becomes attached to it (chemotactic response).
Infection of plants with
A. rhizo-genes
causes development of hairy roots at the site of infection. The
rhizogenic strains con-tain a single copy of a large Ri plasmid. In
the Agropine Ri plasmid T-DNA is referred to asleft T-DNA (T
L
-DNA) and right T-DNA (T
R
-DNA). T
R
T-DNA contains genes homologousto Ti plasmid tumor inducing
genes. Genes involved in agropine synthesis are also located inthe
T
R
DNA region. T-DNA is transferred to wounded plant cells and it
gets stably integratedinto the host genome [2]. Genes encoded in
T-DNA are of bacterial origin but have eukary-otic regulatory
sequences enabling their expression in infected plant cells.
Synthesis of aux-ins can be ascribed to the T
R
-DNA. However, even in the absence of T
R
-DNA directed auxinsynthesis as in the mannopine type which
lacks
tms
loci, root induction occurs. Genes of RiT
L
-DNA direct the synthesis of a substance that recruits the cells
to differentiate into rootsunder the influence of endogenous auxin
synthesis [3,4].
With the exception of border sequences, none of the other T-DNA
sequences are requiredfor the transfer. Virulence genes that form
the
vir
region of the Ri plasmid, and
chv
genes foundon bacterial chromosomes mediate transfer of T-DNA.
Transcription of the
vir
region is in-duced by various phenolic compounds released by
wounded plant cells such as acetosyringoneand
a
-hydroxy-actosyringone. Recalcitrant plant species for
transformation can be transformedby inducing the
vir
genes of the bacteria by signal molecules or it can be achieved
in vitro byco-cultivating
Agrobacterium
with wounded tissues or in media that contains signal
molecules[5]. Acetosyringone or related compounds have been
reported to increase
Agrobacterium
medi-ated transformation frequencies in a number of plant
species [6]. Various sugars also act syner-gistically with
acetosyringone to induce high level of
vir
gene expression. Different strains of
Agrobacterium rhizogenes
vary in their transforming ability [7,8]. Hairy roots obtained
by in-fection with different bacterial strains exhibit different
morphologies. The differences in viru-lence and morphology can be
explained by the different plasmids harbored by the strains
[9].
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A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22
3
The growth medium has a significant effect on hairy root
induction. High salt media suchas LS [10] or MS [11] favors hairy
root formation in some plants. Low salt media such as B
5
[12] favor excessive bacterial multiplication in the medium and
therefore the explant needsto be transferred several times to fresh
antibiotic containing medium before incubation. Thebacterial
concentration also plays an important role for the production of
transformed roots,suboptimal concentrations may result in lower
availability of bacteria for transforming theplant cells while high
concentrations may decrease it by competitive inhibition [7].
Hairyroots are fast growing and plagiotropic and require no
external supply of growth hormones;the plagiotropic characteristic
is advantageous as it increases the aeration in liquid mediumand
roots grown in air have an elevated accumulation of biomass.
2.1. Secondary metabolite production
Hairy root cultures are characterized by a high growth rate and
are able to synthesize root de-rived secondary metabolites.
Normally, root cultures need an exogenous phytohormone supplyand
grow very slowly, resulting in poor or negligible secondary
metabolite synthesis. However,the use of hairy root cultures has
revolutionized the role of plant tissue culture for
secondarymetabolite synthesis. These hairy roots are unique in
their genetic and biosynthetic stability.Their fast growth, low
doubling time, ease of maintenance, and ability to synthesize a
range ofchemical compounds offers an additional advantage as a
continuous source for the productionof valuable secondary
metabolites. To obtain a high-density culture of roots, the culture
condi-tions should be maintained at the optimum level. Hairy root
cultures follow a definite growthpattern, however, the metabolite
production may not be growth related. Hairy roots also offer
avaluable source of root derived phytochemicals that are useful as
pharmaceuticals, cosmetics,and food additives. These roots can also
synthesize more than a single metabolite and thereforeprove
economical for commercial production purposes. Transformed roots of
many plant spe-cies have been widely studied for the in vitro
production of secondary metabolites [13–17] (Ta-ble 1). Transformed
root lines can be a promising source for the constant and
standardized pro-duction of secondary metabolites. Hairy root
cultures produce secondary metabolites oversuccessive generations
without losing genetic or biosynthetic stability. This property can
be uti-lized by genetic manipulations to increase biosynthetic
capacity. Sevon et al. [18] characterizedtransgenic plants derived
from hairy root cultures of
Hyoscyamus muticus
and concluded that asingle hairy root that arises from the
explant tissue is a clone.
Secondary metabolite biosynthesis in transformed roots is
genetically controlled but it isinfluenced by nutritional and
environmental factors. The composition of the culture mediumaffects
growth and secondary metabolite production [8,19]. The sucrose
level, exogenousgrowth hormone, the nature of the nitrogen source
and their relative amounts, light, tempera-ture and the presence of
chemicals can all affect growth, total biomass yield, and
secondarymetabolite production [20]. Sucrose is the best source of
carbon and is hydrolyzed into glu-cose and fructose by plant cells
during assimilation; its rate of uptake varies in different
plantcells [21]. In hairy roots the source of new cells are in the
tips so proliferation occurs only atthe apical meristem and
laterals form behind the elongation zone. Such a defined growth
pat-tern leads to steady accumulation of biomass in root cultures.
To obtain a high density rootculture, the culture conditions should
be maintained at the optimum level. Hairy root cultures
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Table 1Secondary metabolite production from hairy root
cultures
Plant Secondary metabolite References
Aconitum heterophyllum
Aconites [8]
Ajuga replans var. atropurpurea
Phytoecdysteroids [81]
Ambrosia sps.
Polyacetylenes and thiophenes [82]
Amsonia elliptica
Indole alkaloids [39]
Anisodus luridus
Tropane alkaloids [83]
Armoracia laphthifolia
Peroxidase, Isoperoxidase, Fusicoccin [84,85]
Artemisia absinthum
Essential oils [86]
Artemisia annua
Artemisinin [87–90]
Astragalus mongholicus
Cycloartane saponin [91]
Atropa belladonna
Atropine [24,92]
Azadirachta indica
A. Juss. Azadirachtin [93]
Beta vulgaris
Betalain pigments [13,94]
Bidens sps.
Polyacetylenes and thiophenes [82]
Brugmansia candida
Tropane alkaloids [95]Calystegia sepium Cuscohygrine [96,97]
Campanula medium
Polyacetylenes [98]
Carthamus
Thiophenes [82]
Cassia obtusifolia
Anthraquinone [99,100]Polypeptide pigments
Catharanthus roseus
Indole alkaloids, Ajmalicine [101–103]
Catharanthus tricophyllus
Indole alkaloids [104]
Centranthus ruber
Valepotriates [92,105]
Chaenatis douglasis
Thiarubrins [106]
Cinchona ledgeriana
Quinine [107]
Coleus forskohlii
Forskolin [108]
Coreopsis
Polyacetylene [109]
Datura candida
Scopolamine, Hyoscyamine [110]
Datura stramonium
Hyoscyamine, Sesquiterpene [111–113]
Daucus carota
Flavonoids, Anthocyanin [114,115]
Digitalis purpurea
Cardioactive glycosides [116]
Duboisia myoporoides
Scopolamine [117]
Duboisia leichhardtii
Scopolamine [118]
Echinacea purpurea
Alkamides [119,120]
Fagra zanthoxyloids
Lam. Benzophenanthridine [121]Furoquinoline alanine
Fagopyrum
Flavanol [122]
Fragaria
Polyphenol [123]
Geranium thubergee
Tannins [124]
Glycyrrhiza glabra
Flavonoids [125]
Gynostemma pentaphyllum
Saponin [126]Hyoscyamus albus Tropane alkaloids, Phytoalexins
[27,127]
Hyoscyamus muticus
Tropane alkaloids [18,128]Hyoscyamine, Proline [129]
Hyoscyamus niger
Hyoscyamine [130]
Lactuca virosa
Sesquiterpene lactones [131]
Leontopodium alpinum
Anthocyanins & Essential oil [132]
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5
are able to synthesize stable amounts of phytochemicals but the
desired compounds arepoorly released into the medium and their
accumulation in the roots can be limited by feed-back inhibition.
Media manipulations have been reported to aid in the release of
metabolites.Betacyanin release from hairy roots of
Beta vulgaris
was achieved by oxygen starvation. Per-meabilization treatment
using Tween-20 (Polyoxy ethylene sorbilane monolaurate)
releasedhigh yield of hyoscyamine from roots of
Datura innoxia
without any detrimental effects[22]. Addition of XAD-2, liquid
paraffin stimulated production of shikonin [23]. Lee et al.[24]
reported that treatment with 5 mM H
2
O
2
induced a transient release of tropane alkaloidsfrom transformed
roots without affecting its viability.
Table 1(Continued)
Plant Secondary metabolite References
Linum flavum
Lignans (5-methoxy podophyllotoxins) [133]Lippia dulcis
Sesquiterpenes, (hernandulcin) [39]
Lithospermum erythrorhizon
Shikonin, Benzoquinone [23,134]
Lobelia cardinalis
Polyacetylene glucosides [135]
Lobelia inflata
Lobeline, Polyacetylene [136]
Lotus corniculatus
Condensed tannins [137]
Nicotiana hesperis
Nicotine, Anatabine [138]
Nicotiana rustica
Nicotine, Anatabine [13]
Nicotiana tabacum
Nicotine, Anatabine [139]
Panax ginseng
Saponins [38,78]
Panax
Hybrid
(P. ginseng X P. quinqifolium)
Ginsenosides [140]Papaver somniferum Codeine [141,142]
Perezia cuernavcana
Sesquiterpene quinone [143]
Pimpinella anisum
Essential oils [144]
Platycodon grandiflorum
Polyacetylene glkucosides [145,146]
Rauwolfia serpentina
Reserpine [16,147]Rubia peregrina Anthraquinones [71]Rubia
tinctorum Anthroquinone [147]Rudbeckia sps. Polyacetylenes and
thiophenes [82]Salvia miltiorhiza Diterpenoid [6]Scopolia japanica
Hyoscyamine [14]Scutellaria baicalensis Flavonoids and
phenylethnoids [148]Serratula tinctoria Ecdysteroid [149]Sesamum
indicum Naphthoquinone [150]Solanum aculeatissi Steroidal saponins
[151]Solanum lacinialum Steroidal alkaloids [1]Solanum aviculare
Steroidal alkaloids [40]Swainsona galegifolia Swainsonine
[152]Swertia japonica Xanthons [153]Tagetus patula Thiophenes
[82,154]Tanacetum parthenium Sesquiterpene coumarin ether
[155]Tricosanthes kirilowii maxim var japonicum Defense related
proteins [156]Trigonella foenum graecum Diosgenin [157]Valeriana
officinalis L. Valepotriates [184]Vinca minor Indole alkaloids
(vincamine) [158]Withania somnifera Withanoloides [159]
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Production of certain secondary metabolites requires
participation of roots and leaves.Metabolic precursors produced by
organ-specific enzymes in roots are presumed to be trans-located to
aerial parts of the plant for conversion to another product by the
leaves. If the ex-pression and activity of enzymes retain the organ
specificity in vitro then the end productsynthesis will be
difficult. A solution to this problem is the root-shoot co-culture
using hairyroots and their genetically transformed shoot
counterparts shooty teratomas [25,26].
Intergeneric co-culture of genetically transformed hairy roots
and shooty teratomas is effec-tive for improving tissue specific
secondary metabolites. It resembles the whole plant in local-ized
metabolite synthesis and translocation of compounds between organs
for further biocon-version. Developments in transgenic organs make
co-culture feasible by sharing commonmedium requirement without any
hormone supplement. Besides this, transformed green rootshave been
obtained in a few species belonging to Asteraceae, Solanaceae, and
Cucurbitaceae[27]. Green hairy roots are known to produce certain
metabolites that are normally synthesizedin green parts of the
plant [28]. Chloroplast-dependent reactions are a vital part of
certain meta-bolic pathways and could result in a novel pattern of
compounds produced by roots. This aspecthas been studied recently
using soybean hairy roots by functional analysis of the
tobaccoRubisco large subunit AN-methyltransferase promoter and its
light controlled regulation [29].
2.2. Scaling up of hairy roots and bioreactors
Hairy roots once established can be grown in a medium with low
inoculum with a highgrowth rate. The main constraint for commercial
exploitation of hairy root cultures is thescaling up at industrial
level. Hairy roots are complicated biocatalysts when it comes to
scal-ing up and pose unique challenges. Mechanical agitation causes
wounding of hairy roots andleads to callus formation. With a
product of sufficiently high value it is feasible to use
batchfermentation, harvest the roots, and extract the product. For
less valuable products it may bedesirable to establish a packed bed
of roots to operate the reactor in a continuous process forextended
periods collecting the product from the effluent stream. Scale up
becomes difficultin providing nutrients from both liquid and gas
phases simultaneously. Meristem dependentgrowth of root cultures in
liquid medium results in a root ball with young growing roots onthe
periphery and a core of older tissue inside. Restriction of
nutrient oxygen delivery to thecentral mass of tissue gives rise to
a pocket of senescent tissues. Due to branching, the rootsform an
interlocked matrix that exhibits a resistance to flow. The main
problem with hairyroots is supply of oxygen. The ability to exploit
hairy root culture as a source of bioactivechemicals depends on
development of suitable bioreactor system where several physical
andchemical parameters must be taken into consideration.
2.3. Chemical parameters
Nutrient availability is the major chemical factor involved in
scaling up. For large-scalecultivation in a bioreactor several
aspects play an important role. Periodic estimations of spe-cific
nutrients at different periods provide information regarding
nutrient uptake, biomass,and metabolite production in bioreactors.
Carbon, nitrogen, oxygen, and hydrogen depletionin the medium along
with the biomass increase and alkaloid production has been studied
inAtropa belladonna by Kwok and Doran [30]. These types of studies
can be extended to other
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A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22
7
plant species, where product leaches into the medium and can be
recovered by adsorbents.The medium can be rejuvenated to maintain
the supply of nutrients. By leaching of second-ary metabolite
synthesized by hairy roots, the uptake of nutrients gets altered so
leachateneeds to be removed regularly. Leaching of phenolics by
hairy roots and their oxidation leadsto inhibition of uptake of
other nutrients which can be avoided by passing the spent
mediumthrough adsorbents or metabolite traps.
Mass transfer is also an important factor that influences the
uptake of nutrients by hairyroot cultures. The availability of
water and nutrients to any region of a hairy root network ina
bioreactor at different periods is known as mass transfer. Hairy
root bioreactor chambersbecome more heterogeneous owing to
continuous growth of culture. Oxygen is the most im-portant
chemical that needs to be supplied continuously to a bioreactor,
judicious mixingleads to efficient oxygen transfer. At initial
stages in a bioreactor oxygen transfer is not diffi-cult as the
medium contains enough dissolved oxygen to support the growth of
the inoculum.Mixing is a very important factor because it serves
the dual purpose of supplying dissolvedoxygen and driving away the
carbon dioxide. The rate of uptake of oxygen by a unit of bio-mass
in a unit of time is known as the oxygen transfer coefficient.
Other dissolved gaseousmetabolites namely carbon dioxide and
ethylene also affect the overall productivity. A highbiomass
transfer resistance by hairy roots will result in development of
stagnant zones andnon-uniform gaseous metabolite concentrations.
The sampling of the inlet and exit gases bypassing through
rotameter and then to a mass spectrophotometer interphased to a
computer isan important factor in analysis of bioreactor
functioning. Few attempts have been made forscaling up hairy root
cultures for secondary metabolite production. Several bioreactor
de-signs have been reported for hairy roots taking into
consideration their complicated morphol-ogy and shear sensitivity.
These features call for a specially designed bioreactor that
permitsthe growth of interconnected tissue unevenly distributed
throughout the culture vessel. Thedesign of bioreactors for hairy
root cultures should take into consideration factors such as
therequirement for a support matrix and the possibility of flow
restriction by the root mass incertain parts of the bioreactor.
Moreover, for optimal biomass yields, an even distribution ofroots
is needed within the bioreactor. For a continuous mode of operation
in a bioreactor, theproduct must be in part released from the
roots, and it should be possible to maintain a highdensity of
packed root cultures without loss of viability. Several bioreactor
designs havebeen formulated for hairy root cultures (Table 2).
2.3.1. Stirred tank reactor (STR)This type of bioreactor
includes impeller or turbine blades which facilitate mass
transfer,
and is not usually suitable for hairy root cultures because of
the wound response and callusformation that results from the shear
stress caused by the impeller rotation [31,32]. However,recently
some modified stirred tank bioreactors have been developed. These
modified STRshave large impellers and baffles that are agitated at
a very low speed; alternatively, hairyroots can be grown in a steel
cage inside the STR.
2.3.2. Airlift or submerged bioreactorsThese are similar to STRs
but lack an impeller. Plants cells have large vacuoles and slow
growth so hairy roots require comparatively low oxygen supply of
about 0.05–0.4 vol of air/
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vol of liquid/min. Humidified air is passed through glass grid
that functions as aerators.These have been found to be successful
for hairy roots [31,33].
2.3.3. Bubble column reactorLike an airlift bioreactor, in a
bubble column the bubbles create less shear stress, so that it
is useful for organized structures such as hairy roots. In this
case, the bubbling rate needs to
Table 2Bioreactor types used for the growth and secondary
metabolite production from hairy roots
Bioreactor Volume Plant species Secondary metabolite
References
Air-sparged vessel 880 mL Nicotiana rustica Nicotine
[160]Stirred tank 330 mL Armoracia rusticana [84]
1.0 L Atropa belladonna Tropane alkaloids [96]1.0 L Calystegia
sepium Tropane alkaloids [96]
Stirred tank with impeller isolated
1.0 L Atropa belladonna The impeller isseparated by a meshfrom
the roots
Tropane alkaloids [96]
1.0 L Calystegia sepium Tropane alkaloids [96]12.0 L Datura
stramonium Tropane alkaloids [32]1.0 L Duboisia
leichhardtiiScopolamine [118]
Fermenter with mechanical stirring
Catharanthustricophyllus
Indole alkaloids [104]
Air lift 300 mL Armoracia rusticana Roots immobilized
inreticulatedpolyurethane foam
[84]
9.0 L Trigonella foenum-graceum
Draft tube Diosgenin [161]
9.0 L Trigonella foenum-graceum
Nylon mesh replacing draft tube
Diosgenin [161]
Panax ginseng Saponins [38]Lippia dulcis Hernandulcin [39]
Concentrically arranged three sparged set-up used to provide air
bubbles
2.0 L Lithospermum erythrorhizon
Reactor connected tocolumn containingpolymeric adsorbentfor
continuousproduction of shikonin
[23]
15 L Solanum tuberosum [33]Bubble column 2.5 L Atropa belladonna
Tropane alkaloids [162]
1.0 L Catharanthus roseus Indole alkaloids [102]6.0 L Tagetes
patula Thiophene [34]2.5 L Atropa belladonna Atropine [30]
Trickle bed nutrient mist rotating drum
2.0 L Hyoscyamus muticus Tropane alkaloids [163]
1.4 L Beta vulgaris Betacyanins [36]1.0 L Daucus carota
Anthocyanins [35]
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be gradually increased with the growth of hairy roots. Moreover,
the division of a bubble col-umn into segments, and installation of
multiple spargers increases the mass transfer [34].
2.3.4. Gas sparged bioreactorHere humidified air is introduced
from the bottom of the reactor through a sintered glass
sparger. This is useful for mixing and oxygenation.
2.3.5. Turbine blade reactorThis is a combination of
airlift/stirred tank reactor. Here cultivation space is
separated
from agitation space by stainless steel mesh, so that hairy
roots do not come in contact withimpeller and the air is introduced
from the bottom and dispersed by an eight-blade impellerthat stirs
the medium. This is efficient for hairy roots [35].
2.3.6. Mist bioreactor (trickle bed reactor)Here the medium
trickles over a Whatman filter paper containing the biomass, then
spent
medium is drained from the bottom of the bioreactor to a
reservoir and is recirculated at aspecific rate. The degree of
distribution of liquid varies according to the mechanism of
liquiddelivery at the top of the reactor chamber. For better
dispersion spraying is done by mixinghumidified air with medium
that creates the mist [36,37].
2.3.7. Rotating drum bioreactorThis consists of a drum-shaped
container mounted on rollers for support and rotation. The
drum is rotated at only 2–6 rpm to minimize the shear pressure
on the hairy roots. Kondo etal. [35] used this system for hairy
roots from carrot. Hairy roots adhere to the walls of the re-actor
and as the drum rotates the roots tend to break up. To overcome
this problem, a poly-urethane foam sheet was fixed on to the
surface of the drum, to which the hairy roots get at-tached. This
resulted in higher growth without any detachment.
In a gas sparged reactor the oxygen is delivered by local
transfer from gas bubbles that risethrough the reactor and the
inoculum gets distributed evenly in the vessel and circulates.
Be-sides the cultivation of free roots in a stirred tank reactor
and an airlift column, the growth ofhairy roots was also tested
after immobilization in polyurethane foam. Buitelaar et al.
[34]tested growth and thiophene production by Tagetes patula hairy
roots in three different typesof fermenters and found the best
productivity with a bubble column bioreactor. Shimomuraet al. [23]
used an airlift reactor connected to a column containing a
polymorphic adsorbentfor continuous production of shikonin by hairy
root cultures of Lithospermum erythrorhizon.Yoshikawa and Furuya
[38] successfully used an airlift reactor with Panax ginseng
hairyroots and for hernandulin production from Lippia dulcis
[39].
2.3.8. Spin filter bioreactorIn this bioreactor the rotating
filter mixes the cultures and simultaneously allows for spent
medium removal and fresh medium addition.
2.4. Parameters that affect productivity
Although roots do not require additional illumination, certain
hairy roots produce higherlevels of metabolites in the presence of
light. Bioreactors can be illuminated externally or in-
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ternally. Temperature also plays an important role. Yu et al.
[40] studied the effects of tem-perature on Solanum aviculare hairy
roots and found 258C to be optimal. Root morphology isan important
parameter for scale-up. The shear sensitivity of hairy root systems
is of specialinterest because their rheology changes continuously
because of their indefinite proliferation.Their cell walls are
relatively weak and rupture easily which makes them more sensitive
to-ward shear stress. Asepsis is another parameter that plays an
important role; it can beachieved through effective system design,
operating procedures, scheduled checks, andmaintenance [36].
All the above-mentioned parameters and variables result in
highly complicated opera-tional procedures for successfully running
a bioreactor. Computer-aided models can help inplanning for
efficient product formation and recovery. Kim et al. [41] developed
hairy rootmodels based on a branching pattern that helps to monitor
shear stress and stoichiometry. Al-biol et al. [42] used an
artificial neural network model for plant cell cultures and adapted
itfor hairy roots. Wyslouzyl et al. [43] found good agreement
between experimental modelsand predicted values. Padmanabhan et al.
[183] have done computer vision analysis of so-matic embryos for
assessing their ability to be converted to plants; the same type of
analysisfor hairy roots may be beneficial for assessing their
growth, genetic and biosynthetic stabil-ity. For complex hairy root
cultures modeling involves multiple factors as rheology,
oxygenconsumption, and product excretion.
3. Plant regeneration
Transformed roots are able to regenerate whole viable plants;
hairy roots as well as theplants regenerated from hairy roots are
genetically stable. However, in some instances trans-genic plants
have shown an altered phenotype compared to controls. Plants
regenerated fromRi transformed roots display ‘hairy root syndrome,’
combined expression of the rolABC lociof the Ri plasmid is
responsible for this expression. Each locus is responsible for a
typicalphenotypic alteration; that is, rolA is associated with
internode shortening and leaf wrinkling;rolB is responsible for
protruding stigmas and reduced length of stamens; rolC causes
intern-ode shortening and reduced apical dominance [44–46].
Plants can be regenerated from hairy root cultures either
spontaneously (directly fromroots) or by transferring roots to
hormone-containing medium. The advantage of Ri plasmid-based gene
transfer is that spontaneous shoot regeneration is obtained
avoiding the callusphase and somaclonal variations. Ri
plasmid-based gene transfer also has a higher rate oftransformation
and regeneration of transgenic plants; transgenic plants can be
obtained with-out a selection agent thereby avoiding the use of
chemicals that inhibit shoot regeneration;high rate of co-transfer
of genes on binary vector can occur without selection. Further,
Agro-bacterium tumefaciens mediated transformation results in high
a frequency of escapeswhereas Agrobacterium rhizogenes mediated
transformation consistently yields only trans-formed cells that can
be obtained after several cycles of root tip cultures. These hairy
rootscan be maintained as organ cultures for a long time and
subsequent shoot regeneration can beobtained without any
cytological abnormality.
Rapid growth of hairy roots on hormone-free medium and high
plantlet regeneration fre-quency allows clonal propagation of elite
plants. In in vitro cultures, the hairy root regener-
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A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22
11
ants show rapid growth, increased lateral bud formation, and
rapid leaf development, theseregenerants are useful for
micropropagation of plants that are difficult to multiply
[47–49].Altered phenotypes are produced from hairy root regenerants
and some of these have provento be useful in plant breeding
programs [50]. Morphological traits with ornamental value
areabundant adventitious root formation, reduced apical dominance,
and altered leaf and flowermorphology. Dwarfing, altered flowering,
wrinkled leaves, or increased branching may alsobe useful for
ornamentals. Dwarf phenotype is an important characteristic for
flower cropssuch as Eustoma grandiflorum and Dianthus [50]. Higher
levels of some target metaboliteshave been found in the leaves of
plants regenerated from hairy roots so plant regeneration isan
important aspect for production of these chemicals. Pellegrineschi
et al. [51] improved theornamental quality of scented Pelargonium
spp. This plant has pleasant odor but its long in-ternodes and
ungainly growth makes it unattractive, and hairy root regenerants
are of shorterstature. In snapdragon, the flower number was
increased upon transformation [52]. Some pe-rennial forage legumes
turned annual after transformation [53].
3.1. Tree improvement
A major limitation of tree improvement programs is their long
generation cycle. Classicalbreeding programs in trees are slow and
tedious and it is difficult to introduce specific genesfor genetic
manipulation by crossing parental lines. Agrobacterium rhizogenes
mediatedtransformation can be a useful alternative, as a rapid and
direct route for introduction and ex-pression of specific traits
[54]. Transformation of trees and subsequent regeneration of
trans-genic plants has been reported for only a few genera [55–58].
The ability to manipulate treespecies at cellular and molecular
level shows great potential and in vitro transformation
andregeneration from hairy roots facilitates application of
biotechnology to tree species. Thissignificantly reduces the time
necessary for tree improvement and gives rise to new
genecombinations that cannot be obtained using traditional breeding
methods. In some tree spe-cies root initiation limits vegetative
propagation; by using A. rhizogenes rooting of cuttingsfrom
recalcitrant woody species have been improved. Roy [59]
demonstrated this for somefruit trees such as peach, apple, cherry,
and olive. McAffe et al. [60] reported it for Pinus andLarix spp.
Rugini and Mariotti [61] demonstrated successful rooting of some
tree species.These methods have the potential to increase the
efficiency of plant propagation in cropswhere propagation is
difficult. A. rhizogenes mediated transformation has the potential
to in-troduce foreign genes specifically into root systems (e.g.
resistance to pathogens or pests andresistance to heavy
metals).
3.2. Genetic manipulation
Transformed roots provide a promising alternative for the
biotechnological exploitation ofplant cells. A. rhizogenes mediated
transformation of plants may be used in a manner analo-gous to the
well-known procedures employing A. tumefaciens. A. rhizogenes
mediated trans-formation has also been used to produce transgenic
hairy root cultures and plantlets havebeen regenerated. With the
exception of the border sequences, none of the other T-DNA
se-quences are required for the transfer. The rest of the T-DNA can
be replaced with the foreignDNA and introduced into cells from
which whole plants can be regenerated. These foreign
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12 A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000)
1–22
DNA sequences are stably inherited in a Mendelian manner
[62,63]. The A. rhizogenes me-diated transformation has the
advantage that any foreign gene of interest placed in binaryvector
can be transferred to the transformed hairy root clone.
It is also possible to selectively alter some plant secondary
metabolites or to cause them tobe secreted by introducing genes
encoding enzymes that catalyze certain hydroxylation, meth-ylation,
and glycosylation reactions. An example of a gene of interest with
regard to second-ary metabolism that was introduced into hairy
roots is the 6-b-hydroxylase gene of Hyoscy-mus muticus which was
introduced to hyoscymin rich Atropa belladona by a binary
vectorsystem using Agrobacterium rhizogenes. In another instance,
engineered roots showed an in-creased amount of enzyme activity and
a five-fold higher concentration of scopolamine [64].Hairy root
cultures of Nicotiana rustica with ornithine decarboxylase gene
from yeast [65]and Peganum harmala with tryptophan decarboxylase
gene from Catharanthus roseus [66]have been shown to produce
increased amounts of the secondary metabolites nicotine and
se-ratonin when expressing transgenes from yeast. Transgenic plants
produced either by binaryor co-integrate vectors are summarized in
Table 3.
In 12 Brassica cultivars transgenic plants with genes from
binary vectors have been ob-tained and the plant showed hairy root
phenotype to varying degrees and were fertile. Segre-gation
analysis confirmed the transmission of traits to the progeny [67].
Due to independentinsertion of the Ri T-DNA and binary vector T-DNA
in subsequent generations, phenotypi-cally normal transgenic plants
were produced in tobacco [68] and in Brassica napus [69].Downs et
al. [70] reported transgenic hairy roots in Brassica napus
containing a glutaminesynthase gene from soybean showed a
three-fold increase in enzyme activity. When a bacte-rial
isochorismate synthase gene was cloned in a binary vector and then
mobilized into A.rhizogenes, the transgenic hairy root Rubia
peregrina cultures containing this gene expressedtwice as much
isochorismate synthase activity as the roots of control plants and
accumulated20% higher levels of total anthraquinones [71].
Recently, there has been considerable atten-tion given to the
specific induction of secondary metabolite in transgenic plant cell
culturesusing inducible promoters [72]. This approach can be
extrapolated to hairy root cultures foryield enhancement. In
addition new secondary metabolites can be induced in
transgenichairy roots by introducing anthocyanin transactivators
[73]. In the near future, this approachmay be a reality for the
commercial production of pharmaceutically important compoundsusing
transgenic hairy root culture system. Recently a number of genes
including tryptophandecarboxylase, strictosidine synthase,
tropinone reductase, berberine bridge enzyme, andberbamunine
synthase have been isolated and used for the metabolic engineering
of second-ary metabolic pathways
Recently, Wongsamuth and Doran [74] reported production of
monoclonal antibodies byhairy roots. They initiated hairy roots
from transgenic tobacco plants expressing a full-lengthIgG
monoclonal antibody and also tested the long-term stability of
antibody expression inhairy roots, variation between clones, the
time course of antibody accumulation in batch cul-ture, and the
effect of different factors on antibody accumulation and
secretion.
An additional advantage of hairy root cultures is for
enzymological studies. Abundantquantities of sterile, rapidly
growing tissue can be generated. In hairy roots the proportion
ofmeristematic tissue is high and phenolic contents are lower than
in normal plant roots, lead-ing to an increased level of enzyme
activity [75,76].
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A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22
13
Table 3Transgenic plants obtained by Agrobacterium rhizogenes
mediated transformation
Plant Gene introduced References
Ajuga sps. GUS [80]Anthyllis vulneraria NPT II, ipt [164]Atropa
belladonna Bar, 6 bH [28,64]Brassica napus GUS, NPT II, ALS [165]B.
napus NPT II [69]B. campestris GUS, NPT II, ALS [165]B. oleracea
NPT II, GUS [67]B. oleracea GUS, NPT II, ALS [165]B. campestris NPT
II [67]B. napus GS [70]Brassica sps. NPT, Bt, GUS, 35 S-EFE5979
gene [67]Cucumis satives NPT II [166]G. canescens NPT II
[167]Glycine argyrea NPT II [7]Ipomoea batatus NPT II, GUS [168]L.
peruvianum NPT II [169]Larix decidua NPT II, aro A, BT [170]Lotus
corniculatus GS from Phaseolus vulgaris [171]Lycopersicon
esculentum NPT II [172]Medicago truncatula NPT II [173]M. arborea
HPT [53]Nicotiana debneyi NPT II [174]Nicotiana plumaginifolia, N.
tabacum NPT II [68]N. rustica ODS [65]Nicotiana sps. Rol
[46]Peganum harmala* TDS [66]Populus tricocarpa X P. deltoides NPT
II [175]Robinia pseudoacasia NPT II [176]Rubia peregrina* ICS
[71]S. nigrum NPT II [174]S. tuberosum NPT II, GUS [177,178]Solanum
dulcamara NPT II, rol [179]Stylosanthes humilis NPT II
[180]Verticordia grandis NPT II, GUS [181]Vinca minor NPT II, GUS
[158]Vitis vinifera NPT II, GUS [182]
Abbreviations: ALS, Acetolactate synthase; aro A,
5-enolpyruvylshikimate-3-phosphate synthase; bar,Phosephinothicin
acetyltransferase; 6 bH, 6-b-hydroxylase from Hyoscyamus muticus;
BT, Bacillus thuringene-sis protein; GS, Glutamine synthase from
Soyabean; GUS, b-glucuronidase; HPT, Hygromycin
phosphotrans-ferase; ipt, Isopentinyl transferase; NPT II, Neomycin
phsphotransferase; ODS, Ornithine decarboxylase fromYeast; 35
S-EFE5979, Coding region of ethylene forming enzymes from tomato in
antisense orientation; rol, Rootloci genes.
*Only up to the hairy root stage.
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14 A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000)
1–22
Artificial seeds have been developed by encapsulating root
segments and shoot primordia[77]. Root tips of hairy roots of Panax
ginseng [78] and shoot tips of hairy roots regenerantshave been
cryopreserved in horseradish [79]. These can be regenerated and
cultured whenneeded. Hairy roots in the form of transformed plant
organs provide a promising means forthe biotechnological
exploitation of plant cells. Artificial seeds are a reliable
delivery systemfor clonal propagation of elite plants with genetic
uniformity, high yield, and low cost of pro-duction. Plant cells
used for artificial seed production must have a good ability to
regenerate.Micropropagation can be done from hairy roots using
artificial seeds. In Ajuga reptans GUS-transformed hairy roots have
been used for producing artificial seeds [80]. Artificial
seedsusing hairy roots has further potential for mass propagation,
and modifications in bioreactordesign, image analysis with
computers and robotics can improve the process.
Acknowledgments
The financial support to A.G. by Council of Scientific and
Industrial Research (CSIR)Govt. of India is duly acknowledged. The
authors thank Dr C.C. Giri, Centre for Plant Mo-lecular Biology,
Osmania University, Hyderabad, India, for his critical suggestions
duringthe preparation of the manuscript.
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