Research review paper Transgenic hairy roots: recent ... · PDF fileHyderabad 500028, India...

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)

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 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


, 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


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].


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

4


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


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]


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


Nicotiana tabacum


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]

6 A. Giri, M.L. Narasu / Biotechnology Advances 18 (2000) 1–22

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

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/


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


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]


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-


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-


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


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].


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.


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.

References

[1] Hamill JD, Parr AJ, Rhodes MJC, Robins RJ, Walton NJ. New routes to plant secondary products. Bio/Technol 1987;5:800–4.

[2] Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempe J. Agrobacterium rhizogenes insertsT-DNA into the genome of the host plant root cells. Nature 1982;295:432–4.

[3] Ooms G, Twell D, Bossen ME, Hoge JHC, Burrell MM. Development regulation of Ri T DNA gene ex-pression in roots, shoots and tubers of transformed potato (Solanum tuberosum cv. Desiree). Plant MolBiol 1986;6:321–30.

[4] Shen WH, Petit A, Guern J, Tempe J. Hairy roots are more sensitive to auxin than normal roots. Proc NatlAcad Sci USA 1988;35:3417–21.

[5] Stachel SE, Messens E, Van Montagu M, Zambryski P. Identification of signal molecules produced bywounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 1985;318:624–9.

[6] Hu ZB, Alfermann AW. Diterpenoid production in hairy root cultures of Salvia miltiorrhiza. Phytochemis-try 1993;32:699–703.

[7] Kumar V, Jones B, Davey MR. Transformation by Agrobacterium rhizogenes and regeneration of trans-genic shoots of the wild soybean Glycine argyrea. Plant Cell Reports 1991;10:135–8.

[8] Giri A, Banerjee S, Ahuja PS, Giri CC. Production of hairy roots in Aconitum heterophyllum wall. UsingAgrobacterium rhizogenes. In Vitro Cell Dev Biol-Plant 1997;33:280–4.

[9] Nguyen C, Bourgaud F, Forlot P, Guckert A. Establishment of hairy root cultures of Psoralea species.Plant Cell Reports 1992;11:424–7.

[10] Linsmaier EM, Skoog F. Organic growth factor requirements of tobacco tissue cultures. Physiol Plant1965;18:100–27.

[11] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol Plant 1962;15:473–97.

[12] Gamborg OL, Murashige T, Thorpe TA, Vasil IK. Plant tissue culture media. In Vitro 1976;7:473–8.[13] Hamill JD, Parr AJ, Robins RJ, Rhodes MJC. Secondary product formation by cultures of Beta vulgaris

and Nicotiana rustica transformed with Agrobacterium rhizogenes. Plant Cell Reports 1986;5:111–4.


[14] Mano Y, Nabeshima S, Matsui C, Ohkawa H. Production of tropane alkaloids by hairy-root cultures ofScopolia japonica. Agric Biol Chem 1987;50:2715–22.

[15] Signs MW, Flores HE. The biosynthetic potential of plant roots. Bio-Essays 1990;12:282–5.[16] Benjamin BD, Roja G, Heble MR. Alkaloid synthesis by root cultures of Rauwolfia serpentina trans-

formed by Agrobacterium rhizogenes. Phytochemistry 1994;35:381–3.[17] Mukundan U, Rai A, Dawda H, Ratnaparkhi S, Bhinde V. Secondary metabolites in Agrobacterium rhizo-

genes mediated transformed root cultures. In: Srivastava PS, editor. Plant Tissue Culture and Molecular Bi-ology Applications and Prospects. New Delhi: Narosa Publishing House, 1998. pp. 302–31.

[18] Sevon N, Drager B, Hiltunen R, Oksman-Caldentey KM. Characterization of transgenic plants derivedfrom hairy roots of Hyoscyamus muticus. Plant Cell Reports 1997;16:605–11.

[19] Nussbbaumer P, Kapetanidis I, Christen P. Hairy roots of Datura candida X D. aurea: Effect of culturemedium composition on growth and alkaloid biosynthesis. Plant Cell Reports 1998;17:405–9.

[20] Rhodes MJC, Parr AJ, Ceielutt A, Aird ELH. Influence of exogenous hormone on the growth and secondarymetabolite formation in transformed root cultures. Plant Cell Tissue and Organ Culture 1994;38:143–51.

[21] Srinivasan V, Pestchanker L, Moser S, Hirasuna TJ, Taticek RA, Shuler ML. Taxol production in bioreac-tors: Kinetics of biomass accumulation, nutrient uptake and taxol production by cell suspension of Taxusbaccata. Biotechnol Bioeng 1995;47:666–76.

[22] Boitel CM, Gontier E, Laberche JC, Ducrocq C, Sangvan-Norreel BS. Inducer effect of Tween 20 permea-belization treatment used for release of stored alkaloids in Datura innoxia Mill. Hairy root cultures. PlantCell Reports 1996;16:241–4.

[23] Shimomura K, Sudo H, Saga H, Kamada H. Shikonin production and secretion by hairy root cultures ofLithospermum erythrorhizon. Plant Cell Reports 1991;10:282–5.

[24] Lee KT, Yamakawa T, Kodama T, Shimomura K. Effects of chemicals on alkaloid production by trans-formed roots of Belladonna. Phytochemistry 1998;49:2343–7.

[25] Subroto AM, Kwok KH, Hamill JD, Doran PM. Co-culture of genetically transformed roots and shoots forsynthesis, translocation and biotransformation of secondary metabolites. Biotech Bioeng 1996;49:481–94.

[26] Mahagamasekera MGP, Doran PM. Intergeneric co-culture of genetically transformed organs for the pro-duction of scopolamine. Phytochemistry 1998;47:17–25.

[27] Sauerwein M, Wink M, Shimomura K. Influence of light and phytohormones on alkaloid production intransformed root cultures of Hyoscyamus albus. J Plant Physiol 1992;140:147–52.

[28] Saito K, Yamazaki M, Anzai H, Yoneyama K, Murakoshi I. Transgenic herbicide-resistant Atropa bella-donna using an Ri plasmid vector and inheritance of the transgenic trait. Plant Cell Reports 1992;11:219–24.

[29] Mazarei M, Ying Z, Houtz RL. Functional analysis of the Rubisco large subunit AN-methyltransferasepromoter from tobacco and its regulation by light in soybean hairy roots. Plant Cell Reports 1998;17:907–12.

[30] Kwok KH, Doran PM. Kinetic and stoichiometric analysis of hairy roots in a segmented bubble column re-actor. Biotechnol Prog 1995;11:429–35.

[31] Taya M, Yoyama A, Kondo O, Kobayashi T. Growth characteristics of plant hairy roots and their culturesin bioreactors. J Chem Eng Japan 1989;22:84–9.

[32] Hilton MG, Rhodes MJC. Growth and hyoscyamine production of “hairy root” cultures of Datura stramo-nium in a modified stirred tank reactor. Appl Microbiol Biotechnol 1990;33:132–8.

[33] Tescione LD, Ramakrishan D, Curtis WR. The role of liquid mixing and gas-phase dispersion in a sub-merged, sparged root reactor. Enzyme Microbiol Tech 1997;20:207–13.

[34] Buitelaar RN, Langenhoff AAN, Heidstra R, Tramper J. Growth and thiophene production by hairy rootcultures of Tagetus patula in various two liquid phase bioreactors. Enzyme Microbiol Technol 1991;13:487–94.

[35] Kondo O, Honda H, Taya M, Kobayashi T. Comparison of growth properties of carrot hairy root in variousbioreactors. Appl Microbiol Biotechnol 1989;32:291–4.

[36] Dilorio AA, Cheetham RD, Weathers PJ. Growth of transformed roots in a nutrient mist bioreactor: Reac-tor performance and evaluation. Appl Microbiol Biotechnol 1992;37:457–62.

[37] Whitney PJ. Novel bioreactors for the growth of roots transformed by Agrobacterium rhizogenes. EnzymeMicrobiol Technol 1992;14:13–7.


[38] Yoshikawa T, Furuya T. Saponin production by cultures of Panax ginseng transformed with Agrobacte-rium rhizogenes. Plant Cell Reports 1987;6:449–53.

[39] Sauerwein M, Yamazaki T, Shimomura K. Hernandulcin in hairy root cultures of Lippia dulcis. Plant CellReports 1991;9:579–81.

[40] Yu S, Kwok KH, Doran PM. Effect of sucrose, exogenous product concentration and other culture condi-tions on growth and steroidal alkaloid production by Solanum aviculare hairy roots. Enzyme MicrobiolTechnol 1996;18:238–43.

[41] Kim S, Hoppa E, Hjortso M. Hairy root growth models: Effect of different branching patterns. BiotechnolProg 1995;11:178–86.

[42] Albiol J, Campmajo C, Casas C, Poch M. Biomass estimation in plant cell cultures: A neural network ap-proach. Biotechnol Prog 1995;11:88–92.

[43] Wyslouzyl BE, Whipple M, Chatterjee C, Walcerz DB, Weathers PJ, Hart DP. Mist deposition onto hairyroot cultures: Aerosol modeling and experiments. Biotechnol Prog 1997;13:185–94.

[44] Cardarelli M, Mariotti D, Pomponi M, Spano L, Capone I, Constantino P. Agrobacterium rhizogenesT-DNA genes capable of inducing hairy root phenotype. Mol Gen Genet 1987;209:475–80.

[45] Van Altvorst AC, Bino RJ, Van Dijk AJ, Lamers AMJ, Lindhout WH, Mark FV, Dons JJM. Effects of theintroduction of Agrobacterium rhizogenes rol genes on tomato plant and flower development. Plant Sci-ence 1992;83:77–85.

[46] Palazon J, Cusido RM, Roig C, Pinol MT. Expression of the rol gene and nicotine production in transgenichairy roots and their regenerated plants. Plant Cell Reports 1998;17:384–90.

[47] Perez-Molphe E, Ochoa-Alejo N. Regeneration of transgenic plants of Mexican lime from Agrobacteriumrhizogenes transformed tissues. Plant Cell Reports 1998;17:591–6.

[48] Hoshino Y, Mii M. Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinummajus L.) transformed by Agrobacterium rhizogenes. Plant Cell Reports 1998;17:256–61.

[49] Gutierrez-Pesce P, Taylor K, Muleo R, Rugini E. Somatic embryogenesis and shoot regeneration fromtransgenic roots of cherry rootstock Colt (Prunus avium X P.pseudocerasus) mediated by pRi 1855 T-DNAof Agrobacterium rhizogenes. Plant Cell Reports 1998;17:581–5.

[50] Giovanni A, Pecchioni N, Rabaglio M, Allavena A. Characterization of ornamental Datura plants trans-formed by Agrobacterium rhizogenes. In Vitro Cell Dev Biol–Plant 1997;33:101–6.

[51] Pellegrineschi A, Damon JP, Valtorta N, Paillard N, Tepfer D. Improvement of ornamental characters andfragrance production in lemon scented geranium through genetic transformation by Agrobacterium rhizo-genes. Bio/Technol 1994;12:64–8.

[52] Handa T, Sujimura T, Kato E, Kamada H, Takayanagi K. Genetic transformation of Eustoma grandiflorumwith rol genes. Acta Hort 1995;392:209–18.

[53] Damiani F, Aricioni S. Transformation of Medicago arborea L. with Agrobacterium rhizogenes binaryvector carrying the hygromycin resistance genes. Plant Cell Reports 1991;10:300–3.

[54] Huang Y, Diner A, Karnosky F. Agrobacterium rhizogenes mediated genetic transformation of a conifer:Larix decidua. In Vitro Cell Dev Biol–Plant 1991;27:201–7.

[55] Zhan XC, Jones DD, Kerr A. Regeneration of flax plants transformed by Agrobacterium rhizogenes. PlantMol Biol 1988;11:551–60.

[56] McGranahan GH, Leslie CA, Uratsu SL. Agrobacterium-mediated transformation of walnut somatic em-bryos and regeneration of transgenic plants. Bio/Technol 1988;6:800–4.

[57] McGranahan GH, Leslie CA, Dandekar AM. Transformation of pecan and regeneration of transgenicplants. Plant Cell Reports 1993;12:634–8.

[58] Cabrera-Ponce JC, Vegas-Garcia A, Herrera-Estrella L. Regeneration of transgenic papaya plants via so-matic embryogenesis induced by Agrobacterium rhizogenes. In Vitro Cell Dev Biol–Plant 1996;32:86–90.

[59] Roy MC. Plant growth response to Agrobacterium rhizogenes M Appl Sc Thesis, Lincoln College, Univer-sity of Canterbury, New Zealand, 1989.

[60] McAfee BJ, White EE, Pelcher LE, Lapp MS. Root induction in pine (Pinus) and larch (Larix) Spp. usingAgrobacterium rhizogenes. Plant Cell Tiss Organ Cult 1993;34:53–62.

[61] Rugini E, Mariotti D. Agrobacterium rhizogenes T-DNA genes and rooting in woody species. Acta Hort1991;300:301–8.


[62] Stougaard J, Abildsten D, Marcher KA. The Agrobacterium rhizogenes pRi TL-DNA segment as a genevector system for transformation of plants. Mol Gen Genet 1987;207:251–5.

[63] Zambryski P, Tempe J, Schell J. Transfer and function of T-DNA genes from Agrobacterium Ti and Riplasmids in plants. Cell 1989;56:193–201.

[64] Hashimoto T, Yun DJ, Yamada Y. Production of tropane alkaloids in genetically engineered root cultures.Phytochemistry 1993;32(3):713–8.

[65] Hamill JD, Robins RJ, Parr AJ, Evans PM, Furze JD, Rhodes MJC. Over expressing a yeast ornithine de-carboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.Plant Mol Biol 1990;15:27–38.

[66] Berlin J, Ruegenhagen C, Dietze P, Fecker LF, Goddijn OJM, Hoge JHC. Increased production of serato-nin by suspension and root cultures of Peganum harmala transformed with a tryptophan decarboxylasecDNA clone from Cathranthus roseus. Transgenic Res 1993;2:336–44.

[67] Christey MC, Sinclair BK, Braun RH, Wyke L. Regeneration of transgenic vegetable Brassicas (Brassicaoleracea and B. campestris) via Ri-mediated transformation. Plant Cell Reports 1997;16:587–93.

[68] Hatamoto H, Boulter ME, Shirsat AH, Croy EJ, Ellis JR. Recovery of morphologically normal transgenictobacco from hairy roots co-transformed with Agrobacterium rhizogenes and a binary vector plasmid.Plant Cell Reports 1990;9:88–92.

[69] Boulter ME, Croy E, Simpson P, Shields R, Croy RRD, Shirsat AH. Transformation of Brassica napus L.(oilseed rape) using Agrobacterium tumefaciens and Agrobacterium rhizogenes: A comparison. Plant Sci1990;70:91–9.

[70] Downs CG, Christey MC, Davies KM, King GA, Seelye JF, Sinclair BK, Stevenson DG. Hairy roots ofBrassica napus: II glutamine synthase over expression alters ammonia assimilation and the response tophosphinothricin. Plant Cell Reports 1994;14:41–6.

[71] Lodhi AH, Bongaerts RJM, Verpoorte R, Coomber SA, Charlwood BV. Expression of bacterial isochoris-mate synthase (E C 5.4.99.6) in transgenic root cultures of Rubia peregrina. Plant Cell Reports 1996;16:54–7.

[72] Sommer S, Siebert M, Bechthold A, Heide L. Specific induction of secondary product formation in trans-genic plant cell cultures using an inducible promoter. Plant Cell Reports 1998;17:891–6.

[73] Damiani F, Paoloci F, Consonni G, Crea F, Tonelli C, Aricioni S. A maize anthocyanin transactivator in-duces pigmentation in hairy roots of dicotyledonous species. Plant Cell Reports 1998;17:339–44.

[74] Wongsamuth R, Doran PM. Hairy roots as an expression system for production of antibodies. In: DoranPM, editor. Hairy roots culture and application. Amsterdam: Harwood Academic Publishers, 1997. pp.89–97.

[75] McLauchlan WR, Mc Kee RA, Evans DA. The purification and immunocharacterization of N-methyl pu-trescine oxidase from transformed root cultures of Nicotiana tabcum L. cvSc58. Planta 1993;191:440–5.

[76] Walton NJ, Peerless ACJ, Robins RJ, Rhodes MJC, Boswell HD, Robins DJ. Purification and properties ofPutrescine N-methyltransferase from transformed roots of Datura stramonium L. Planta 1994;193:9–15.

[77] Nakashimada Y, Uozemi N, Kobayashi T. Production of plantlets for use as artificial seeds from horserad-ish hairy roots fragmented in a blender. J Ferment Bioeng 1995;79:458–64.

[78] Yoshimatsu K, Yamaguchi H, Shimomura K. Traits of Panax ginseng hairy roots after cold storage andcryopreservation. Plant Cell Reports 1996;15:555–60.

[79] Phunchindawan M, Hirata K, Sakai A, Miyamoto K. Cryopreservation of encapsulated shoot primordia in-duced in horseradish (Armoracia rusticana) hairy root cultures. Plant Cell Reports 1997;16:469–73.

[80] Uozumi N, Ohtake Y, Nakashimada Y, Morikawa Y, Tanaka N, Kobayashi T. Efficient regeneration froma GUS transformed ajuga hairy roots. J Ferment Bioeng 1996;81:374–8.

[81] Matsumoto T, Tanaka N. Production of phytoecdysteroides by hairy root cultures of Ajuga reptans var at-ropurpurea. Agric Biol Chem 1991;55:10–25.

[82] Flores HE, Pickard JJ, Hoy MW. Production of polyacetylenes and thiophenes in heterotrophic and photo-synthetic root cultures of Asteraceae. In: Lam J, Breheler H, Arnason T, Hansen L, editors. Chemistry andBiology of Naturally Occurring Acetylenes and Related Compounds (NOARC) Bioactive Molecules 7.1988. pp. 233–54.

[83] Jobanovic V, Grubisic D, Giba Z, Menkovid N, Ristic M. Alkaloids from hairy root cultures of Anisodusluridus (Scopolia lurida Dunal Solanaceae Tropane alkaloids). Planta Medica 1991;2:102.


[84] Taya M, Yoyama A, Nomura R, Kondo O, Matsui C, Kobayashi T. Production of peroxidase with horse-radish hairy root cells in a two step culture system. J Ferment Bioeng 1989;67:31–4.

[85] Babakov AV, Bartova LM, Dridze IL, Maisuryan AN, Margulis GU, Oganian RR, Voblikova VD,Muromtsev GS. Culture of transformed horseradish roots as source of Fusicoccin-like ligands. J PlantGrowth Reg 1995;14:163–7.

[86] Nin S, Bennici A, Roselli G, Mariotti D, Schiff S, Magherini R. Agrobacterium mediated transformation ofArtemisia absinthum L. (worm wood) and production of secondary metabolites. Plant Cell Reports 1997;16:725–30.

[87] Jaziri M, Shimomura K, Yoshimatsu K, Fauconnier ML, Marlier M, Homes J. Establishment of normal andtransformed root cultures of Artemisia annua L. for artemisinin production. J Plant Physiol 1995;145:175–7.

[88] Teoh KH, Weathers PJ, Cheetham RD, Walcerz DB. Cryopreservation of transformed (hairy) roots of Arte-misia annua. Cryobiology 1996;33:106–17.

[89] Weathers PJ, Cheetham RD, Follanskie E, Teoh K. Artemisinin production by transformed roots of Artemi-sia annua. Biotechnol Lett 1994;16:1281–6.

[90] Rao KV, Venkanna N, Lakshmi Narasu M. Agrobacterium rhizogenes mediated transformation of Artemi-sia annua. J Sci Ind Res 1998;57:773–6.

[91] Ionkava I, Kartnig T, Alfermann W. Cycloartane saponin production in hairy root cultures of Astragalusmongholicus. Phytochemistry 1997;45(8):1597–600.

[92] Christen P. Centranthus species: In vitro culture and the production of valepotriates and other secondarymetabolites. In: Bajaj YPS, editor. Biotechnology in Agriculture and Forestry, Medicinal and AromaticPlants XI, Vol. 43. Berlin: Spinger-Verlag, 1999. pp. 42–56.

[93] Allan EJ, Stuchbury T, Mordue (Luntz) AJ. Azadirachta indica A. Juss. (Neem Tree): In vitro culture, mi-cropropagation and the production of Azadiractine and other secondary metabolites. In: Bajaj YPS, editor.Biotechnology in Agriculture and Forestry, Medicinal and Aromatic Plants XI, Vol. 43. Berlin: Spinger-Verlag, 1999. pp. 11–41.

[94] Taya M, Mine K, Kinoka M, Tone S, Ichi T. Production and release of pigments by cultures of transformedhairy roots of red beet. J Ferment Bioeng 1992;73:31–6.

[95] Giulietti AM, Parr AJ, Rhodes MJC. Tropane alkaloids production in transformed root cultures of Brug-mansia candida. Planta Medica 1993;59:428–31.

[96] Jung G, Tepfer D. Use of genetic transformation by the Ri T-DNA of Agrobacterium rhizogenes to stimu-late biomass and tropane alkaloid production in Atropa belladonna and Calystegia sepium roots grown invitro. Plant Sci 1987;50:145–51.

[97] Bhadra R, Vani S, Shanks JV. Production of indole alkaloids by selected hairy root lines of Catharanthusroseus. Biotechnol Bioeng 1993;41:581–92.

[98] Tada H, Nakashima T, Kuntake H, Mori K, Tanaka M, Ishimaru K. Polyacetylenes in hairy root cultures ofCampanula medium L. J Plant Physiol 1996;147:617–9.

[99] Asamizu T, Abiyam K, Yasuda I. Anthraquinones production by hairy root culture in Cassia obtusifolia.Yakagaku Zasshi 1988;108:1215–8.

[100] Ko KS, Ebizuka Y, Noguchi H, Sankawa U. Production of polypeptide pigments in hairy root cultures ofCassia plants. Chem Pharm Bull (Tokyo) 1995;43:274–8.

[101] Parr AJ, Peerless ACJ, Hamill JD, Walton NJ, Robins RJ, Rhodes MJC. Alkaloid production by trans-formed root cultures of Catharanthus roseus. Plant Cell Reports 1988;7:309–12.

[102] Toivonen L, Ojala M, Kauppinen V. Indole alkaloid production by hairy root cultures of Catharanthusroseous: Growth kinetics and fermentation. Biotechnol Lett 1990;12:519.

[103] Toivonen L. Utilization of hairy root cultures for production of secondary metabolites. Biotechnol Progr1993;9:12–20.

[104] Davioud E, Kan C, Hamon J, Tempe J, Husson HP. Production of indole alkaloids by in vitro root culturesfrom Catharanthus trichophyllus. Phytochemistry 1989;28:2675–80.

[105] Granicher F, Cristen P, Kaptanidis I. Production of valepotriates by hairy root cultures of Centranthus ru-ber DC. Plant Cell Reports 1995;14:294–8.

[106] Constabel CP, Towers GHN. Thiarubrine accumulation in hairy root cultures of Chaenactis douglasei. JPlant Physiol 1988;133:67–72.


[107] Hamill JD, Robins RJ, Rhodes MJC. Alkaloid production by transformed root cultures of Cinchona ledge-riana. Planta Medica 1989;55:354–7.

[108] Sasaki K, Udagava A, Ishimaru H, Hayashi T, Alfermann AW, Nakanishi F, Shimomura K. High forskolinproduction in hairy roots of Coleus forskohlii. Plant Cell Reports 1998;17:457–9.

[109] Marchant YY. Agrobacterium rhizogenes-transformed root cultures for the study of polyacetylene metabo-lism and biosynthesis. In: Lam J, Breheler H, Arnason T, Hansen L, editors. Chemistry and Biology of Natu-rally Occurring Acetylenes and Related Compounds (NOARC), Bioactive Molecules 7. 1988. pp. 217–31.

[110] Christen P, Roberts MF, Phillipson JD, Evans WC. High yield production of tropane alkaloids by hairyroot cultures of a Datura candida hybrid. Plant Cell Reports 1989;8:75–7.

[111] Payne J, Hamill JD, Robins RJ, Rhodes MJC. Production of hyoscyamine by hairy root cultures of Daturastramonium. Planta Medica 1987;29:2545–50.

[112] Furze JM, Rhodes MJC, Parr AJ, Robins RJ, Whitehead IM, Threlfall DR. Abiotic factors elicit sesquiter-penoid phytoalexin production but not alkaloid production in transformed root cultures of Datura stramo-nium. Plant Cell Reports 1991;10:111–4.

[113] Hilton MG, Rhodes MJC. Factors affecting the growth and hyosyamine production during batch culture oftransformed roots of Datura stramonium. Planta Medica 1993;59:340–4.

[114] Bel-Rhlid R, Chabot S, Piche Y, Chenevert T. Isolation and identification of flavanoids from Ri T-DNAtransformed roots (Daucus carota) and their significance in vesicular–arbuscular Mycorrhiza. Phytochem-istry 1993;35:381–3.

[115] Kim CH, Lee SW, Chung IS. Hairy root cultures of Dacus carota for anthocyanin production in fluidized-bed bioreactor. Agric Chem Biotechnol 1994;37:237–42.

[116] Saito K, Yoshimatsu K, Murakoshi T. Genetic transformation of foxglove (Digitalis purpurea) by chimericforeign genes and production of cardioactive glycosides. Plant Cell Reports 1990;9:121–4.

[117] Deno H, Yamagata T, Emoto T, Yoshioka T, Yamada Y, Fujita Y. Scopolamine production by root cul-tures of Duboisia myoporoides II. Establishment of a hairy root culture by infection with Agrobacteriumrhizogenes. J Plant Physiol 1987;131:315–23.

[118] Muranaka T, Ohakawa H, Yamada Y. Scopolamine release into media by Duboisia leichhardtti hairy rootclones. Appl Microbiol Biotechnol 1992;37:554–9.

[119] Mukundan U, Hjortso MA. Growth and thiophene accumulation by hairy root cultures of Tagetus patula inmedia of varying initial pH. Plant Cell Reports 1990;9:627–30.

[120] Trypsteen M, Van-Lijsekettens M, Van Severen R, Van Montagu M. Agrobacterium rhizogenes mediatedtransformation of Echinacea purpurea. Plant Cell Reports 1991;10:85–9.

[121] Couilterot E, Caron C, Trentesaux C, Chenieux JC, Audran JC. Fagara zanthoxyloides Lam. (Rutaceae):In vitro culture and the production of benzophenanthridine and furoquinoline alanine. In: Bajaj YPS, edi-tor. Biotechnology in Agriculture and Forestry, Medicinal and Aromatic Plants XI, Vol. 43. Berlin: Sp-inger-Verlag, 1999. pp. 136–56.

[122] Trotin F, Moumou Y, Vasseur J. Flavonol production by Fagopyrum esculentum hairy roots and normalroot cultures. Phytochemistry 1993;33:929–31.

[123] Motomari Y, Shimomura K, Mori K, Kunitake H, Nakashima T, Tanaka M, Miyazaki S, Ishimaru K.Polyphenol production in hairy root cultures of Fragaria X ananassa. Phytochemistry 1995;40:1425–8.

[124] Ishimaru K, Shimomura K. Tannin production in hairy root cultures of Geranium thunmbergii. Phy-tochemistry 1991;30:825–8.

[125] Asada Y, Li W, Yoshikawa T. Isoprenylated flavonoids from hairy root cultures of Glycyrrhiza glabra.Phytochemistry 1998;47:389–92.

[126] Fei HM, Mei KF, Shen X, Ye YM, Lin ZP, Peng LH. Transformation of Gynostemma pentaphyllum byAgrobacterium rhizogenes. Saponin production in hairy root cultures. Acta Bot Sincia 1993;35:626–31.

[127] Kuroyanagi M, Arakava T, Mikami Y, Yoshida K, Kawahar N, Hayashi T, Ishimaru H. Phytoalexins fromhairy roots cultures of Hyoscyamus albus treated with methyl jasmonate. J Nat Prod 1998;61:1516–9.

[128] Vanhala L, Hiltunen R, Oksman-Caldentey KM. Virulence of different Agrobacterium strains on hairy rootformation of Hyoscyamus muticus. Plant Cell Reports 1995;14:236–40.

[129] Halperin SJ, Flores HE. Hyoscyamine and proline accumulation in water stressed Hyoscyamus muticushairy root cultures. In Vitro Cell Dev Biol–Plant 1997;33:240–4.


[130] Jaziri M, Yoshimatsu K, Homes J, Vanhaelen M. Tropane alkaloid production by hairy root cultures ofDatura stamonium and Hyoscyamus niger. Phytochemistry 1988;27:419–20.

[131] Kisiel W, Stojakowska A, Malarz J, Kohlmunzer S. Sesquiterpene lactones in Agrobacterium rhizogenestransformed hairy root cultures of Lactuca virosa. Phytochemistry 1995;40:1139–40.

[132] Hook I. Secondary metabolites in hairy root cultures of Leontopodium alpinum cass (edelweiss). Plant CellTissue and Organ Culture 1994;38:321–6.

[133] Oostdam A, Mol JNM, Vanderplas LHW. Establishment of hairy root cultures of Linum flavum producingthe lignan 5-methoxy podophyllotoxin. Plant Cell Reports 1993;12:474–7.

[134] Fukui H, Feroj Hasan AFM, Ueoka T, Kyo M. Formation and secretion of a new brown benzoquinone byhairy root cultures of Lithospermum erythrorhizon. Phytochemistry 1998;47:1037–9.

[135] Yamanaka M, Ishibashi K, Shimomura K, Ishimaru K. Polyacetylene glucosides in hairy root cultures ofLobelia cardinalis. Phytochemistry 1996;41(1):183–5.

[136] Yonemitsu H, Shimomura K, Satake M, Mochida S, Tanaka M, Endo T, Kaji A. Lobeline production byhairy root cultures of Lobelia inflata L. Plant Cell Reports 1990;9:307–10.

[137] Carron TR, Robbins MP, Morris P. Genetic modification of condensed tannin biosynthesis in Lotus cornic-ulatus: 1. Heterologous and antisense dihydroflavanol reductase down-regulate tannin accumulation in“hairy root” cultures. Theor Appl Genet 1994;87:1006–15.

[138] Parr AJ, Hamill JD. Relationship between Agrobacterium rhizogenes transformed hairy roots and intactuninfected Nicotiana plants. Phytochemistry 1987;26:3241–5.

[139] Flores H, Filner P. Metabolic relationships of putriscine GABA and alkaloids in cell and root cultures ofSolanaceae. In: Neumann K, Barz W, Reinhard E, editors. Primary and Secondary Metabolism of Plant CellCultures. Berlin: Springer-Verlag, 1985. pp. 174–85.

[140] Washida D, Shimomura K, Nakajima Y, Takido M, Kitanaka S. Ginsenosides in hairy roots of a Panax hy-brid. Phytochemistry 1998;49:2331–5.

[141] Yoshimatsu K, Shimomura K. Transformation of opium poppy (Papaver somniferum L.) with Agrobacte-rium rhizogenes MAFF 03-01724. Plant Cell Reports 1992;11:132–6.

[142] Williams RD, Ellis BE. Alkaloids from Agrobacterium rhizogenes transformed Papaver somniferum cul-tures. Phytochemistry 1993;32:719–23.

[143] Arellano J, Vazquez F, Villegas T, Hernandez G. Establishment of transformed root cultures of Perezia cu-ernavacana producing the sesquiterpene quinone perezone. Plant Cell Reports 1996;15:455–8.

[144] Santos PM, Figueiredo AC, Olivera MM, Barroso JG, Pedro LG, Deans SG, Younus AKM, Scheffer JJC. Essen-tial oils from hairy root cultures and from fruits and roots of Pimpinella anisum. Phytochemistry 1998;48:455–60.

[145] Tada H, Shimomura K, Ishimaru K. Polyacetylenes in Platycodon grandiflorum hairy roots and campanu-laceous plants. J Plant Physiol 1995;145:7–10.

[146] Ahn JC, Hwang B, Tada H, Ishimaru K, Sasaki K, Shimomura K. Polyacetylenes in hairy roots of Platy-codon grandiflorum. Phytochemistry 1996;42(1):69–72.

[147] Sato K, Yamazaki T, Okuyama E, Yoshihira K, Shimomura K. Anthraquinone production by transformedroot cultures of Rubia tictorum: Influence of phytohormones and sucrose concentration. Phytochemistry1991;30:2977–8.

[148] Zhou Y, Hirotani M, Yoshikava T, Furuya T. Flavonoids and phenylethanoids from hairy root cultures ofScutellaria baicalensis. Phytochemistry 1997;44(1):83–7.

[149] Delbeque JP, Beydon P, Chapuis L. In vitro incorporation of radio labelled cholesterol and mevalonic acidinto ecdysteron by hairy root cultures of a plant Serratula tinctoria. Eur J Entomol 1995;92:301–7.

[150] Ogasawara T, Cheba K, Tada M. Production in high-yield of a naphthoquinone by a hairy root culture ofSesamum indicum. Phytochemistry 1993;33:1095–8.

[151] Ikenaga T, Oyama T, Muranaka T. Growth and steroidal saponin production in hairy root cultures ofSolanum aculeatissi. Plant Cell Reports 1995;14:413–7.

[152] Ermayanti TM, McComb JA, O’Brien PA. Stimulation of synthesis and release of swainsonine from trans-formed roots of Swainsona galegifolia. Phytochemistry 1994;36:313–7.

[153] Ishimaru K, Sudo H, Salake M, Malsugama Y, Hasagewa Y, Takamoto S, Shimomura K. Amarogenetinand amaroswertin and four xanthones from hairy root cultures of Swertia japonica. Phyotchemistry1990;29:1563–5.


[154] Arroo RRJ, Develi A, Meijers H, Van de Westerlo E, Kemp AK, Croes AF, Williems GJ. Effects of exog-enous auxin on root morphology and secondary metabolism in Tagetes patula hairy root cultures. PhysiolPlant 1995;93:233–40.

[155] Kisiel W, Stojakowska A. A sesquiterpene coumarin ether from transformed roots of Tanacetum parthe-nium. Phytochemistry 1997;46(3):5515–6.

[156] Savary BJ, Flores HE. Biosynthesis of defense related proteins in transformed root cultures of Tricosantheskirilowii Maxim. Var. japonicum (Kitam). Plant Physiol 1994;106:1195–204.

[157] Merkli A, Christen P, Kapetanidis I. Production of diosgenin by hairy root cultures of Trigonella foenum-graecum L. Plant Cell Reports 1997;16:632–6.

[158] Tanaka N, Takao M, Matsumoto T. Agrobacterium rhizogenes mediated transformation and regenerationof Vinca minor L. Plant Tiss Cult Lett 1994;11:191–8.

[159] Banerjee S, Naqui AA, Mandal S, Ahuja PS. Transformation of Withania somnifera (L.) Dunal by Agro-bacterium rhizogenes: Infectivity and phytochemical studies. Phytotherapy Res 1994;8:452–5.

[160] Rhodes MJC, Robins RJ, Hamill JD, Parr AJ, Walton NJ. Secondary product formation using Agrobacte-rium rhizogenes transformed hairy root cultures. TCA Newsletter 1987;53:2–15.

[161] Rodriguez-Mendiola MA, Stafford A, Cresswell R, Aria-Castro C. Bioreactors for growth of plant roots.Enzyme Microb Technol 1991;13:697–702.

[162] Sharp JM, Doran PM. Characteristics of growth and tropane alkaloid synthesis in Atropa belladonna rootstransformed by Agrobacterium rhizogenes. J Biotechnol 1990;16:171–86.

[163] Flores HE, Curtis WR. Approaches to understanding and manipulating the biosynthetic potential of plantroots. Ann NY Acad Sci 1992;665:188.

[164] Stiller J, Nasinec V, Svoboda S, Nemcova B, Machackova T. Effects of agrobacterial oncogenes in kidneyvetch (Anthyllis vulneraria L.). Plant Cell Reports 1992;11:363–7.

[165] Christey MC, Sinclair BK. Regeneration of trasgenic kale (Brassica oleracea var. acephal), rap (B. napus)and turnip (B. campestris var rapifera) plants via Agrobacterium rhizogenes mediated transformation.Plant Sci 1992;87:161–9.

[166] Trulson AJ, Simpson RB, Shahin EA. Transformation of cucumber (Cucumis sativus L.) plants with Agro-bacterium rhizogenes. Theor Appl Gene 1986;73:11–5.

[167] Rech EL, Golds TJ, Husnain T, Vainstein MH, Jones B, Hammat N, Mulligan BJ, Davey MR. Expressionof a chimaeric kanamycin resistance gene introduced into the wild soybean Glycine canescens using a co-integrate Ri plasmid vector. Plant Cell Reports 1989;8:33–6.

[168] Otani M, Mu M, Handa T, Kamada H, Shimada T. Transformation of sweet potato (Ipomoea batatus (L.)Lam.) plants by Agrobacterium rhizogenes. Plant Sci 1993;94:151–9.

[169] Morgan AJ, Cox PN, Turner DA, Peel E, Davey MR, Gartland KMA, Mulligan BJ. Transformation of to-mato using an Ri plasmid vector. Plant Sci 1987;49:37–49.

[170] Shin DI, Podila GK, Huang Y, Karnosky DF. Transgenic larch expressing genes for herbicide and insectresistance. Can J For Res 1994;4:2059–67.

[171] Forde BG, Day HM, Turton JF, Shen WJ, Cullimore V, Oliver JE. Two glutamine synthase genes fromPhaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenicLotus corniculatus plants. Plant Cell 1989;1:391–401.

[172] Shahin EA, Sukhainda K, Simpson RB, Spivey R. Transformation of cultivated tomato by a binary vectorin Agrobacterium rhizogenes: Transgenic plants with normal phenotypes harbor binary vector T DNA, butno Ri-plasmid T-DNA. Theor Appl Genet 1986;72:770–7.

[173] Thomas MR, Rose RJ, Nolan KE. Genetic transformation of Medicago truncatula using Agrobacteriumwith genetically modified Ri and disarmed Ti plasmid. Plant Cell Reports 1992;11:113–7.

[174] Davey MR, Mulligan BJ, Gartland KMA, Peel E, Sargent AW, Morgan AJ. Transformation of Solanumand Nicotiana species using an Ri plasmid vector. J Exp Bot 1987;38:1507–16.

[175] Pythoud F, Sinbar VP, Nester EW, Gordon MP. Increased virulence of Agrobacterium rhizogenes conferred bythe vir region of ptiB0542: Application to genetic engineering of Poplar. Bio/Technol 1987;5:1323–7.

[176] Han KH, Keathley DE, Davis JM, Gordon MP. Regeneration of a transgenic woody legume Robiniapseudoacacia L., (Black locust) and morphological alterations induced by Agrobacterium rhizogenes-mediated transformation. Plant Sci 1993;88:149–57.


[177] Visser RGF, Hesseling-Meinders A, Jacobsen E, Nijdam H, Witholt B, Feenstra WJ. Expression and inher-itance of inserted markers in binary vectors carrying Agrobacterium rhizogenes transformed potato(Solanum tuberosum L.). Theor Appl Genet 1989;78:705–14.

[178] Visser RGF, Jacobsen E, Witholt B, Feenstra WJ. Efficient transformation of potato (Solanum tuberosumL.) using a binary vector in Agrobacterium rhizogenes. Theor Appl Genet 1989;78:594–600.

[179] McInnes E, Morgan AJ, Mulligan BJ, Davey MR. Phenotypic effects of isolated pRiA4 TL-DNA rol genesin the presence of intact TR-DNA in transgenic plants of Solanum dulcamara L. J Exp Bot 1991;42:1279–86.

[180] Manners JM, Way H. Efficient transformation with regeneration of the tropical pasture legume Stylosan-thes humilis using Agrobacterium rhizogenes and a Ti plasmid–binary vector system. Plant Cell Reports1989;8:341–5.

[181] Stummer BE, Smith SE, Langridge P. Genetic transformation of Verticordia grandis (Myrtaceae) usingwild-type Agrobacterium rhizogenes and binary Agrobacterium vectors. Plant Sci 1995;111:51–62.

[182] Nakano M, Hoshino Y, Mii M. Regeneration of transgenic plants of grape vine (Vitis vinifera L.) via Agro-bacterium rhizogenes mediated transformation of embryogenic calli. J Exp Bot 1994;45(274):649–56.

[183] Padmanabhan K, Cantliffe DJ, Harrell RC, Harrison J. Computer vision analysis of somatic embryos ofsweet potato [Ipomoea batatas (L.) Lam.] for assessing their ability to convert to plants. Plant Cell Reports1998;17:681–4.

[184] Granicher F, Christen P, Vuagnat P. Rapid high performance liquid chromatographic quantification ofValeriana officinalis L. var. sambutifolia hairy roots. Phytochemistry 1994;38:103–5.

Research review paper Transgenic hairy roots: recent ... · PDF fileHyderabad 500028, India...

Documents

Transcript of Research review paper Transgenic hairy roots: recent ... · PDF fileHyderabad 500028, India...