Biotechnology Advances 18 (2000) 1–22
0734-9750/00/$–see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 4 - 9 7 5 0 ( 9 9 ) 0 0 0 1 6 - 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 genetically
transformed root cultures can produce higher levels of secondary metabolites or amounts comparable
to 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 their
scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of
interconnected 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 stable
plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency
allows 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 vitro
transformation 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 clonal
propagation 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 and
perspectives of hairy root cultures and the recent progress achieved with respect to transformation of
plants using
A. rhizogenes.
© 2000 Elsevier Science Inc. All rights reserved.
Keywords: Agrobacterium rhizogenes; Hairy roots; Secondary metabolites; Bioreactor; Genetic manipulation; Transgenics
2 A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22
1. Introduction
Plants remain a major source of pharmaceuticals and fine chemicals. Despite considerable
efforts, 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 unstable
and 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. Genetically
transformed hairy roots obtained by infection of plants with
A. rhizogenes
, a gram-negative
soil bacterium, offers a promising system for secondary metabolite production [1]. The fast
growing hairy roots are unique in their genetic and biosynthetic stability and their fast growth
offers an additional advantage. These fast growing hairy roots can be used as a continuous
source for the production of valuable secondary metabolites. Moreover, transformed roots
are able to regenerate whole viable plants and maintain their genetic stability during further
subculturing and plant regeneration.
2.
Agrobacterium
and Ri T-DNA genes
Agrobacterium
recognizes some signal molecules exuded by susceptible wounded plant
cells 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 as
left T-DNA (T
L-DNA) and right T-DNA (T
R-DNA). T
RT-DNA contains genes homologous
to Ti plasmid tumor inducing genes. Genes involved in agropine synthesis are also located in
the T
RDNA region. T-DNA is transferred to wounded plant cells and it gets stably integrated
into 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 auxin
synthesis as in the mannopine type which lacks
tms
loci, root induction occurs. Genes of Ri
T
L-DNA direct the synthesis of a substance that recruits the cells to differentiate into roots
under the influence of endogenous auxin synthesis [3,4].
With the exception of border sequences, none of the other T-DNA sequences are required
for the transfer. Virulence genes that form the
vir
region of the Ri plasmid, and
chv
genes found
on 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 acetosyringone
and
a
-hydroxy-actosyringone. Recalcitrant plant species for transformation can be transformed
by inducing the
vir
genes of the bacteria by signal molecules or it can be achieved in vitro by
co-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
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 such
as 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 needs
to be transferred several times to fresh antibiotic containing medium before incubation. The
bacterial concentration also plays an important role for the production of transformed roots,
suboptimal concentrations may result in lower availability of bacteria for transforming the
plant cells while high concentrations may decrease it by competitive inhibition [7]. Hairy
roots 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 medium
and 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 supply
and 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 secondary
metabolite 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 of
chemical compounds offers an additional advantage as a continuous source for the production
of 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 growth
pattern, however, the metabolite production may not be growth related. Hairy roots also offer a
valuable 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 therefore
prove 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 over
successive generations without losing genetic or biosynthetic stability. This property can be
uti-lized by genetic manipulations to increase biosynthetic capacity. Sevon et al. [18] characterized
transgenic plants derived from hairy root cultures of
Hyoscyamus muticus
and concluded that a
single hairy root that arises from the explant tissue is a clone.
4 A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22 Table 1
Secondary 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]
A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22 5
[image:5.486.41.443.113.533.2]are able to synthesize stable amounts of phytochemicals but the desired compounds are
poorly 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) released
high 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
2O
2induced a transient release of tropane alkaloids
from 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]
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 product
synthesis will be difficult. A solution to this problem is the root-shoot co-culture using hairy
roots 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 common
medium requirement without any hormone supplement. Besides this, transformed green roots
have 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 synthesized
in 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 aspect
has been studied recently using soybean hairy roots by functional analysis of the tobacco
Rubisco 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 high
growth rate. The main constraint for commercial exploitation of hairy root cultures is the
scaling up at industrial level. Hairy roots are complicated biocatalysts when it comes to
scal-ing up and pose unique challenges. Mechanical agitation causes woundscal-ing of hairy roots and
leads to callus formation. With a product of sufficiently high value it is feasible to use batch
fermentation, harvest the roots, and extract the product. For less valuable products it may be
desirable to establish a packed bed of roots to operate the reactor in a continuous process for
extended periods collecting the product from the effluent stream. Scale up becomes difficult
in providing nutrients from both liquid and gas phases simultaneously. Meristem dependent
growth of root cultures in liquid medium results in a root ball with young growing roots on
the periphery and a core of older tissue inside. Restriction of nutrient oxygen delivery to the
central mass of tissue gives rise to a pocket of senescent tissues. Due to branching, the roots
form an interlocked matrix that exhibits a resistance to flow. The main problem with hairy
roots is supply of oxygen. The ability to exploit hairy root culture as a source of bioactive
chemicals depends on development of suitable bioreactor system where several physical and
chemical parameters must be taken into consideration.
2.3. Chemical parameters
Nutrient availability is the major chemical factor involved in scaling up. For large-scale
cultivation 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 depletion
in the medium along with the biomass increase and alkaloid production has been studied in
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 leachate
needs to be removed regularly. Leaching of phenolics by hairy roots and their oxidation leads
to inhibition of uptake of other nutrients which can be avoided by passing the spent medium
through adsorbents or metabolite traps.
Mass transfer is also an important factor that influences the uptake of nutrients by hairy
root cultures. The availability of water and nutrients to any region of a hairy root network in
a bioreactor at different periods is known as mass transfer. Hairy root bioreactor chambers
become 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 mixing
leads 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 dissolved
oxygen 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 gaseous
metabolites namely carbon dioxide and ethylene also affect the overall productivity. A high
biomass transfer resistance by hairy roots will result in development of stagnant zones and
non-uniform gaseous metabolite concentrations. The sampling of the inlet and exit gases by
passing through rotameter and then to a mass spectrophotometer interphased to a computer is
an important factor in analysis of bioreactor functioning. Few attempts have been made for
scaling 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 permits
the growth of interconnected tissue unevenly distributed throughout the culture vessel. The
design of bioreactors for hairy root cultures should take into consideration factors such as the
requirement for a support matrix and the possibility of flow restriction by the root mass in
certain parts of the bioreactor. Moreover, for optimal biomass yields, an even distribution of
roots is needed within the bioreactor. For a continuous mode of operation in a bioreactor, the
product must be in part released from the roots, and it should be possible to maintain a high
density of packed root cultures without loss of viability. Several bioreactor designs have
been 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 callus
formation 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 STRs
have large impellers and baffles that are agitated at a very low speed; alternatively, hairy
roots can be grown in a steel cage inside the STR.
2.3.2. Airlift or submerged bioreactors
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 reactor
[image:8.486.36.448.114.536.2]Like 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 is separated by a mesh from 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 leichhardtii
Scopolamine [118]
Fermenter with mechanical stirring
Catharanthus tricophyllus
Indole alkaloids [104]
Air lift 300 mL Armoracia rusticana Roots immobilized in reticulated
polyurethane 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 to column containing polymeric adsorbent for continuous production 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]
A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22 9
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 bioreactor
Here 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 reactor
This 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 with
impeller and the air is introduced from the bottom and dispersed by an eight-blade impeller
that 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 a
specific rate. The degree of distribution of liquid varies according to the mechanism of liquid
delivery at the top of the reactor chamber. For better dispersion spraying is done by mixing
humidified air with medium that creates the mist [36,37].
2.3.7. Rotating drum bioreactor
This 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 et
al. [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 rise
through 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 of
hairy 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 types
of fermenters and found the best productivity with a bubble column bioreactor. Shimomura
et al. [23] used an airlift reactor connected to a column containing a polymorphic adsorbent
for continuous production of shikonin by hairy root cultures of
Lithospermum erythrorhizon.
Yoshikawa and Furuya [38] successfully used an airlift reactor with
Panax ginseng
hairy
roots and for hernandulin production from
Lippia dulcis
[39].
2.3.8. Spin filter bioreactor
In 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
in-ternally. Temperature also plays an important role. Yu et al. [40] studied the effects of
tem-perature on
Solanum aviculare
hairy roots and found 25
8
C to be optimal. Root morphology is
an important parameter for scale-up. The shear sensitivity of hairy root systems is of special
interest 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 be
achieved through effective system design, operating procedures, scheduled checks, and
maintenance [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 in
planning for efficient product formation and recovery. Kim et al. [41] developed hairy root
models 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 it
for hairy roots. Wyslouzyl et al. [43] found good agreement between experimental models
and 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 analysis
for 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, oxygen
consumption, and product excretion.
3. Plant regeneration
Transformed roots are able to regenerate whole viable plants; hairy roots as well as the
plants regenerated from hairy roots are genetically stable. However, in some instances
trans-genic plants have shown an altered phenotype compared to controls. Plants regenerated from
Ri transformed roots display ‘hairy root syndrome,’ combined expression of the
rolABC
loci
of the Ri plasmid is responsible for this expression. Each locus is responsible for a typical
phenotypic 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 from
roots) 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 callus
phase and somaclonal variations. Ri plasmid-based gene transfer also has a higher rate of
transformation 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 escapes
whereas
Agrobacterium rhizogenes
mediated transformation consistently yields only
trans-formed cells that can be obtained after several cycles of root tip cultures. These hairy roots
can be maintained as organ cultures for a long time and subsequent shoot regeneration can be
obtained without any cytological abnormality.
regener-A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22 11
ants show rapid growth, increased lateral bud formation, and rapid leaf development, these
regenerants 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 proven
to be useful in plant breeding programs [50]. Morphological traits with ornamental value are
abundant adventitious root formation, reduced apical dominance, and altered leaf and flower
morphology. Dwarfing, altered flowering, wrinkled leaves, or increased branching may also
be useful for ornamentals. Dwarf phenotype is an important characteristic for flower crops
such as
Eustoma grandiflorum
and
Dianthus
[50]. Higher levels of some target metabolites
have been found in the leaves of plants regenerated from hairy roots so plant regeneration is
an important aspect for production of these chemicals. Pellegrineschi et al. [51] improved the
ornamental 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 shorter
stature. 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. Classical
breeding programs in trees are slow and tedious and it is difficult to introduce specific genes
for genetic manipulation by crossing parental lines.
Agrobacterium rhizogenes
mediated
transformation 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 tree
species at cellular and molecular level shows great potential and in vitro transformation and
regeneration from hairy roots facilitates application of biotechnology to tree species. This
significantly reduces the time necessary for tree improvement and gives rise to new gene
combinations that cannot be obtained using traditional breeding methods. In some tree
spe-cies root initiation limits vegetative propagation; by using
A. rhizogenes
rooting of cuttings
from recalcitrant woody species have been improved. Roy [59] demonstrated this for some
fruit trees such as peach, apple, cherry, and olive. McAffe et al. [60] reported it for
Pinus
and
Larix
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 crops
where 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 and
resistance to heavy metals).
3.2. Genetic manipulation
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 binary
vector can be transferred to the transformed hairy root clone.
It is also possible to selectively alter some plant secondary metabolites or to cause them to
be 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 vector
system 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 binary
or 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 independent
insertion 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 glutamine
synthase 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 expressed
twice as much isochorismate synthase activity as the roots of control plants and accumulated
20% higher levels of total anthraquinones [71]. Recently, there has been considerable
atten-tion given to the specific inducatten-tion of secondary metabolite in transgenic plant cell cultures
using inducible promoters [72]. This approach can be extrapolated to hairy root cultures for
yield enhancement. In addition new secondary metabolites can be induced in transgenic
hairy roots by introducing anthocyanin transactivators [73]. In the near future, this approach
may be a reality for the commercial production of pharmaceutically important compounds
using transgenic hairy root culture system. Recently a number of genes including tryptophan
decarboxylase, strictosidine synthase, tropinone reductase, berberine bridge enzyme, and
berbamunine 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 by
hairy roots. They initiated hairy roots from transgenic tobacco plants expressing a full-length
IgG monoclonal antibody and also tested the long-term stability of antibody expression in
hairy 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.
A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22 13 Table 3
Transgenic 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 transphosphotrans-ferase; NPT II, Neomycin phsphotransphosphotrans-ferase; ODS, Ornithine decarboxylase from Yeast; 35 S-EFE5979, Coding region of ethylene forming enzymes from tomato in antisense orientation; rol, Root loci genes.
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 regenerants
have been cryopreserved in horseradish [79]. These can be regenerated and cultured when
needed. Hairy roots in the form of transformed plant organs provide a promising means for
the biotechnological exploitation of plant cells. Artificial seeds are a reliable delivery system
for 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 seeds
using hairy roots has further potential for mass propagation, and modifications in bioreactor
design, 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 during
the preparation of the manuscript.
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