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154 Biotechnological Production of Lignans

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©CAB International 2010. Medicinal Plant Biotechnology 156

(ed. Rajesh Arora)

Chapter 10

Hairy Root Culture: Copying Nature in New

Bioprocesses

Milen I. Georgiev, Jutta Ludwig-Müller and Thomas Bley

Copying Nature: Transformation with Agrobacterium rhizogenes

Conventional cell cultures need to be sustained using plant growth hormones, but this is not

necessary after transformation of plants with Agrobacterium rhizogenes, which leads to the

production of ‘hairy roots’ that do not normally need exogenous hormones and can grow

indefinitely. In addition, such cultures may produce different metabolites from cell cultures,

and they have much greater resemblance to the root system of a normal plant, i.e.

morphologically A. rhizogenes-induced hairy roots are very similar to wild-type roots,

except that they are more branched, agravitropic and their root hairs are longer and more

numerous (Veena and Taylor, 2007). In addition, since there is still concern about

transgenic plants, if the Agrobacteria used for the transformation are not transformed with

foreign DNA (see the section on metabolic engineering), these cultures may be more

readily accepted than possible transgenic alternatives. Furthermore, production of hairy

roots is less time consuming than most transformation procedures for whole plants.

A wide range of plants can be transformed with Agrobacterium, including numerous

dicot and monocot angiosperm species (Gelvin, 2003; Li and Leung, 2003) and

gymnosperms (Li and Leung, 2003; McAfee et al., 1993; Yibrah et al., 1996). However,

the outcome of transformation/infection in nature varies between Agrobacterium species.

While A. tumefaciens causes the widely distributed crown gall disease, A. rhizogenes

infection leads (as mentioned above) to hairy root formation. In the laboratory, so-called

disarmed vectors without the tumour-inducing genes, but containing the genes of interest to

be introduced into the target plant, are used for A. tumefaciens transformation (Tzfira and

Citovsky, 2006). In contrast, when plants are transformed with A. rhizogenes, the rol genes

(see below) are still present, which facilitates the generation of the root cultures (either with

or without foreign DNA, see section on metabolic engineering).

The molecular mechanism of hairy root induction is not completely understood (Veena

and Taylor, 2007; Georgiev et al., 2008). However, the transferred DNA (T-DNA) is

known to be a segment of the Agrobacterium Ri-plasmid. Hairy root formation in plants

Hairy Root Culture 157

infected with A. rhizogenes is not thought to be dependent on changes in the

phytohormonal balance in plant cells, probably because the cells become more sensitive to

auxins (Nilsson and Olsson, 1997; Altamura, 2004). It should be noted that Park and

Facchini (2000) found that although hairy root cultures should, in theory, be able to grow

without exogenous plant hormones, the growth rates of transformed roots of two species,

opium poppy and Californian poppy, was improved by the addition of auxin to the liquid

culture medium. Exogenous application of auxin can also reportedly stimulate growth in

hairy root cultures of Lippia dulcis Trev. (Sauerwein et al., 1991). The minimum doubling

times, of approximately 2 weeks for California poppy and 3–4 weeks for opium poppy hairy

root cultures are comparable to those of other species (Loyola-Vargas and Miranda-Ham,

1995).

The products of the four rol (A, B, C and D) genes play key roles in hairy root

formation, but the rolB gene appears to be the most important in hairy root induction, since

loss-of-function mutation at this locus renders the plasmid avirulent (Nilsson and Olsson,

1997; Altamura, 2004). Further, when rolB is introduced into the host plant genome as a

single gene, it is capable of hairy root induction (Altamura, 2004). Auxin has been

proposed to be synthesized from tryptophan (Trp) by a two-step reaction in hairy roots,

because Trp can be converted by the aux1 gene product tryptophan monooxygenase to

indole-3-acetamide (IAM), which is then converted by indoleacetamide hydrolase, the aux2

gene product, to auxin (indole-3-acetic acid; IAA) (Thomashow et al., 1984; Offringa et al.,

1986). However, very recently it was shown that introduction of the rolABCD genes alone

does not affect the auxin requirement of tobacco BY-2 cells. Instead, the aux1 and aux2

genes, rather than the rolABCD genes, appear to be responsible for the auxin autotrophy of

these transgenic lines (Nemoto et al., 2009). These findings, together with older reports

(see above), imply that although the rolABCD genes may be sufficient to generate hairy-

root tumours, they are irrelevant to auxin autotrophy. A further interesting observation was

made by Nemoto et al. (2009), that the BY-2 cells were able to grow without

supplementary auxin even when only the aux1 gene was present, suggesting that there

might be plant enzymes capable of catalyzing the amidase reaction. Accordingly, Pollmann

et al. (2003) found an amidase in Arabidopsis and Ishikawa et al. (2007) reported that

amidase genes are present in Brassica sp., so such genes may be quite ubiquitous, and

could contribute to the increased auxin levels in hairy root cultures. Understanding the

mechanisms that lead to rhizogenesis may also be useful for establishing faster growing

cultures in sub-optimal culture conditions, e.g. in bioreactors (see the section on

bioprocessing). However, auxin production can also have effects on secondary metabolite

production, which need to be taken into account. For example, Rischer et al. (2006) found

that addition of auxin to cell cultures of Catharanthus roseus inhibited accumulation of the

terpenoid indole alkaloid ajmalicine, but stimulated the accumulation of other alkaloid

metabolites, such as tabersonine and catharanthine, and when methyl jasmonate (MeJA)

was added to auxin-treated cells it restored their ability to produce all alkaloids. Such

responses may also occur in hairy roots, which already produce auxin via the aux genes,

and possibly change the desired metabolic pattern. Since the rol genes alone are sufficient

to induce hairy roots, the aux genes may warrant further consideration before planned

transformations. However, the rol genes themselves can also influence secondary

metabolite production. For instance, Palazon et al.

(1997) observed striking differences in

nicotine production between transgenic root lines of tobacco transformed with the rolA,

rolB and rolC genes singly and in combination. While the rolC gene strongly stimulated

alkaloid production indirectly, by increasing growth, the rolA gene stimulated it mainly by

a direct effect. Further, the presence of rolA, rolB and rolC together dramatically enhanced

158 Hairy Root Culture

both the growth rate and nicotine production, showing that the effects of these three rol

genes were synergistic.

Besides transformation via A. rhizogenes, it should be noted that there are alternative

approaches for producing hairy root cultures, i.e. establishment of root cultures from

mutants with a hairy root phenotype. For example, Suzuki et al. (2008) developed an

alternative root culture system using an allelic mutant of Arabidopsis thaliana superroot 1

(sur1). When the mutant was cultured in liquid medium developed for hairy root cultures, it

grew rapidly, developing a globular, rooty phenotype and was easily subcultured in the

medium. This mutant is advantageous because the rooty phenotype is recessive, hence the

desired characteristics can likely be maintained via seeds. Crossing heterozygous sur1

plants with fatty acid desaturase mutants in the cited study by Suzuki et al. (2008) resulted

in the accumulation of high amounts of oleic acid and linolenic acid, indicating that the

sur1 root culture system is an alternative tool for the production of useful compounds in

roots.

The long-term maintenance of induced hairy root strains is of both practical and

financial significance. Usually, transformed root cultures, grown on solid media are

subcultured every 3–4 weeks. However this is expensive in terms of time, labour and

resources, with consequently high risks of contamination and eventual loss of the original

strains. Conservation of the original strains by cryopreservation could avoid these

problems. However, despite the progress made in the last decade on cryopreservation of

plant material, there have been few reports on the cryopreservation of hairy root cultures

(Lambert et al., 2009). Generally the main approaches used for cryopreservation of plant

tissues and organs include slow cooling, vitrification, encapsulation-dehydration and

encapsulation-vitrification, the last two of these appear to be most effective (Xue et al.,

2008). Lambert et al. (2009) studied the potential applicability of encapsulation in calcium-

alginate beads (containing 0.1 M sucrose), followed by dehydration (in air streams, to 35%

of the initial mass) for hairy root cultures of Maesa lanceolata and Medicago truncatula

and established survival rates of 90% and 53%, respectively, which was concluded to be

sufficient for large-scale purposes.

Applications of Transformed Root Cultures

Currently, the main applications of transformed root cultures include production of high-

value metabolites and biotransformation, phytoremediation, regeneration of whole plants

and production of artificial seeds (Giri et al., 2001; Guillon et al., 2006). Plant regeneration

and artificial seed production are beyond the scope of this chapter and, thus, will not be

further considered here. Further applications include their use in detailed studies of

fundamental molecular, genetic and biochemical aspects of genetic transformation and

hairy root induction.

Production of metabolites

The enormous biosynthetic potential of hairy root cultures was largely neglected for years,

in which investigations mainly focused on the mechanisms underlying the hairy root

syndrome. Nowadays, transformed root cultures receive more attention as technological

objects for production of valuable metabolites, for which they have several attractive

features, including high genetic and biochemical stability, and relatively fast growth rates in

Hairy Root Culture 159

hormone-free media (Georgiev et al., 2007). Figure 10.1 shows the structures of some high-

value metabolites that can be produced by transformed root cultures, including anticancer

(paclitaxel, podophyllotoxin, camptothecin, justicidine B and berberine), antimalarial

(artemisinin), and anti-inflammatory (harpagoside) substances.

To illustrate the economic potential of substances from natural sources, Potterat and

Hamburger (2008) noted that over 60% of all anti-cancer drugs are of natural origin, and

that annual sales of such drugs exceed several billion US dollars. In 1971 Wall and Wani

reported the isolation of paclitaxel from the stem bark of the pacific yew Taxus brevifolia.

Paclitaxel (a complex diterpenoid that is the active ingredient of Taxol

) has received

considerable attention because of its activity against various kinds of cancer, inter alia

breast cancer, ovarian cancer, AIDS-related Kaposi’s sarcoma, and non-small cell lung

cancer. The annual sales of paclitaxel and related taxanes in 2004 exceeded 2 billion US

dollars (Tabata, 2004). Obtaining commercial supplies of paclitaxel from natural sources is

not economically feasible because the source Taxus plants grow very slowly, and their

paclitaxel contents are very low (~0.01% of the dry weight of the bark, where levels are

highest). Tabata (2004) calculated that a 100-year-old yew tree would yield just 300 mg of

the compound. The total synthesis of paclitaxel has been achieved, but this too is not

economically viable. Therefore, paclitaxel is now produced commercially in large-scale

plant cell suspension cultures (by Phyton and Samyang Genex Corporation), in one of the

most remarkable examples of the potential value of plant in vitro culture. Hairy root

cultures of Taxus x media var. Hicksii have also been found to produce paclitaxel at

amounts of 69 g/g dry weight. Further, Furmanowa and Syklowska-Baranek (2000) found

that the content of paclitaxel in transformed roots can be enhanced through elicitation with

MeJA; a week after addition of 100 M MeJA the paclitaxel content increased to 210 g/g

dry weight. Another compound that is produced by transformed root cultures (of

Ophiorrhiza mungos) is camptothecin, an alkaloid with anticancer activities, in amounts

(~0.3% per unit dry weight) significantly exceeding those obtained from cell suspension

cultures (~0.01% per unit dry weight) of the same species (Wink et al., 2005).

Podophyllotoxin, a lignan with antimitotic activity, and the closely related 6-

methoxypodophyllotoxin are also accumulated in hairy root cultures, of Linum album and

L. persicum (Wink et al., 2005). Therapeutic application of podophyllotoxins is hampered

due to severe side effects. However, they serve as useful bases for production of the

semisynthetic etoposide and teniposide, which have found clinical use for the treatment of

various cancers (Potterat and Hamburger, 2008).

In addition, Vasilev et al. (2006) recently reported the production of justicidine B (an

arylnaphthalene lignan with cytotoxic, antiviral, fungicidal, antiprotozoal and antiplatelet

properties) by transformed root cultures of L. leonii. After two weeks of submerged

cultivation the justicidine B content reached 10.8 mg/g dry; 5-fold higher than yields from a

callus culture of the same species. Berberine (an alkaloid with anti-inflammatory and

antimicrobial activities, which was recently also found to possess anticancer properties)

was also found to accumulate at higher amounts in transformed root cultures of

Phellodendron chinense than in the callus cultures of the same species. All of these studies

support the hypothesis that the biosynthesis of some metabolites (e.g. alkaloids) is

associated with organogenesis.

160 Hairy Root Culture

Fig. 10.1. Chemical structures of valuable metabolites produced by transformed root

cultures.

Finally, for this section, artemisinin (a sesquiterpene lactone produced by Artemisia

annua; Fig. 10.1) is highly active against the parasitic protozoa of the genus Plasmodium

that cause malaria, but it has low toxicity to humans. Malaria is a very common and

virulent tropical disease, causing ~2 million deaths (mainly of infants and young children)

amongst 300–500 million cases per year (Potterat and Hamburger, 2008). When extracted

from the plant yields of artemisinin are very low (0.01–0.8%), making large-scale

production costly (Potterat and Hamburger, 2008). However, Kim et al. (2001) examined

artemisinin production by transformed root cultures of A. annua, and obtained yields up to

2.64 g/g, and ca. ~3-fold higher levels from cultivation in mist reactors than in bubble

column reactors.

Hairy Root Culture 161

Biotransformation

Biotransformation refers to the chemical modification of the molecular structure of organic

compounds, by microorganisms, plant in vitro systems or enzymes. Plant in vitro systems

can perform a wide range of reactions, such as glucosylation, hydroxylation, reduction of

carbon double bonds ( = ), hydrolysis, epoxidation and many others (Giri et al., 2001).

Glucosylation reactions are of particular interest in this respect because they facilitate the

conversion of water-insoluble compounds (e.g. phenols, phenylpropanoid acid or their

analogues) to water-soluble compounds (Giri et al., 2001). More specifically, in an analysis

by Faria et al. (2009) of the capacity of Anethum graveolens hairy roots to transform

menthol and geraniol, neither of these monoterpenes adversely affected growth of the roots,

menthyl acetate was formed following menthol addition and ten new biotransformation

products appeared following geraniol addition (including the alcohol linalool and the

aldehyde geranial). In another example of the biotransformation capacity of hairy root

cultures, Chen et al. (2008) observed regiospecific glucosylation of three isomers of

hydroxybenzoic acid (p-, m- and o-hydroxybenzoic acid) in hairy root cultures of Panax

ginseng. p-Hydroxybenzoic acid and m-hydroxybenzoic acid were converted into their

corresponding glycosides and glycosyl esters, but no metabolite of o-hydroxybenzoic acid

was detected.

Phytoremediation

In phytoremediation plants and/or plant in vitro systems (e.g. hairy root cultures) are used

to remove, or reduce the concentrations of, toxic organic pollutants or to absorb and

accumulate heavy metals (Oller et al., 2005; Guillon et al., 2006). Use of plants or plant

cultures has considerable advantages for these purposes, including the scope to use them in

situ or ex situ and their environmental acceptability (Oller et al., 2005). Most successful

applications of hairy root cultures for phytoremediation have been for removing phenol

(Table 10.1), since the process is mainly catalyzed by peroxidases, which are produced

abundantly by hairy roots. Furthermore, Alderete et al. (2009) recently reported the

production of transgenic Nicotiana tabacum hairy roots, expressing heterologous basic

peroxidases, with removal efficiencies that were significantly higher than those of control

‘wild type’ hairy roots.

Another major application of phytoremediation with hairy roots is for heavy metal

detoxication. The exploitation of plants and plant in vitro systems to remove toxic heavy

metals from the environment is currently of considerable commercial interest. Of particular

importance for such uses are hyperaccumulator plants/in vitro systems, which are capable

of taking up and storing elevated concentrations of heavy metals (Cd, Hg, Ag, Pb, Cu, Zn

etc.), without displaying symptoms of severe metal toxicity or cell damage (Boominathan

and Doran, 2003). Hairy roots of Daucus carota have been found to be sensitive to uranium

exposure by Straczek et al. (2009), but their study provides further insights for application

of hairy root cultures for phytomining purposes.

In confirmation, Boominathan and Doran (2003) observed that hairy roots of Thlaspi

caerulescens are hyperaccumulators of Cd ions, since exposure to 178 M Cd neither

suppressed their growth, nor significantly enhanced their H

levels. In addition, 300-fold

greater constitutive catalase activity was detected in the hairy roots of T. caerulescens (than

in N. tabacum), illustrating the further scope for overexpressing catalase and/or superoxide

dismutase in order to improve Cd tolerance (Boominathan and Doran, 2003).

162 Hairy Root Culture

Table 10.1. Selected examples of phytoremediation and metal accumulation by hairy root

cultures.

Hairy root culture Organic or

inorganic

pollutant

Enzyme(s) involved Reference

Lycopersicon

esculentum

Phenol Peroxidase Gonzales et al.,

2006

Brassica napus

Phenol Peroxidase Coniglio et al.,

2008

Daucus carota,

Ipomoea batatas,

Solanum aviculare

Phenol and

chlorophenols

Peroxidase Santos de

Araujo et al.,

2006

Armoracia

rusticana

Acetaminophen

(paracetamol)

P450 monooxygenase;

Glutathione S-transferases

Huber et al.,

2009

Brassica napus

Prochiral

diketones

Dehydrogenases Orden et al.,

2006

Solanum nigrum

Polychlorinated

bisphenyls

- Rezek et al.,

2007

Helianthus annuus

Tetracycline,

Oxytetracycline

- Gujarathi et al.,

2005

Daucus carota

Uranium - Straczek et al.,

2009

Thlaspi

caerulescens

Cadmium Catalase Boominathan

and Doran, 2003

Metabolic Engineering

While production of naturally occurring metabolites in tissue cultures can be elicited by

various signalling molecules, or simply by the presence of A. rhizogenes genes from the

TL-DNA (see the section on transformation with A. rhizogenes), it may also be desirable to

modify the genetic features of hairy root cultures so that a targeted metabolite can be

produced in higher amounts if there is a rate-limiting step in its production pathway.

However, such a strategy requires thorough knowledge of the pathway(s) leading to the

desired metabolite, and other pathways that may compete for precursors or intermediates to

avoid potentially adverse secondary effects if a necessary precursor such as an amino acid

is depleted (see, for instance, Verpoorte et al., 2000). Other potential factors to be

considered include: (i) feedback inhibition of enzymes in the pathway; (ii) the availability

of cofactors; and (iii) differences in the compartmentation of enzymes and substrates, and

hence the possible need to transport an intermediate to another compartment (see Fig.

10.2A). However, it is also possible to block pathways competing for intermediates. In the

schematic diagram presented in Fig. 10.2A, for instance, the precursor is converted to

intermediate A which is not rate-limiting. However, if the next step (conversion of A to B)

would be rate limiting, then increasing the levels of enzymes that catalyze the conversion of

A to B would increase the amount of intermediate B. It might also be helpful to increase

levels of intermediate A by adding the compound. Intermediate B is then converted to C

(not rate-limiting), but B is also a precursor for metabolite K. Hence, it would be desirable

to reduce production of K, by either blocking the reaction chemically or using antisense

Hairy Root Culture 163

approaches. Alternatively, the expression (or dosage) of the gene encoding the protein

required for conversion of B to C could be increased. Metabolite C might have to be

transported to a different compartment, which could lead to a drop in the concentration of

C. A solution could be to start engineering the pathway after the transport step. In addition,

high rates of synthesis of the end product D could require an additional cofactor. It is also

possible that the end product (or an intermediate) could reduce production of one of the

important rate-limiting intermediates (e.g. B) by feedback inhibition, if so steps should be

taken to prevent this happening.

Strategies for genetic engineering of more complex pathways could also include: (i)

heterologous gene expression to produce compounds that are not normally produced from a

precursor molecule present in the given species (see below); (ii) antisense inhibition or co-

suppression (RNAi) of alternative pathways; (iii) expression of multiple genes in a

pathway; and (iv) expression of genes encoding transcription factors that regulate multiple

steps in a pathway (Fig. 10.2B).

The potential utility of the last of these approaches has been demonstrated in a study

where the efficiency of the maize Sn gene, which transactivates the anthocyanin pathway in

various tissues, for regulating anthocyanin biosynthesis in several dicotyledonous species

was tested using transformed root cultures (Damiani et al., 1998). The Sn gene was found to

be capable of inducing anthocyanin biosynthesis in some heterologous roots, such as

lucerne (Medicago sativa L.) and lotus (Lotus angustissimus L.), but not in others, such as

petunia (Petunia hybrida L.).

Knowledge of control factors that could be easily transferred to hairy root cultures has

been obtained in several studies, including the following. Conserved regulatory elements in

the strictosidine synthase promoter have been used to isolate various regulatory genes that

control JA-inducible expression of monoterpene indole alkaloid (MIA) biosynthesis in C.

roseus cell cultures (Memelink and Gantet, 2007). In another study, T-DNA activation

tagging led to the identification of the cDNA encoding the transcription factor ORCA3 (van

der Fits and Memelink, 2000), which regulates several early steps in monoterpene indole

alkaloid biosynthesis, and over-expression of ORCA3 in transformed C. roseus cell

cultures increased the expression of several MIA pathway genes. Interestingly, treating C.

roseus cell cultures with JA resulted in a post-translational modification of ORCA3,

possibly via phosphorylation (Memelink and Gantet, 2007).

Genetically engineered root cultures have also been used as model systems to study

various aspects of the metabolic and molecular regulation of several natural product

pathways. For example, expression of a cDNA encoding Antirrhinum majus L.

dihydroflavonol reductase in hairy roots of Lotus corniculatus L. caused an increase in the

content, and alteration in the structure, of condensed tannins in a manner consistent with the

substrate specificity of the transgene product (Bavage et al., 1997).

In addition, the same precursor can sometimes be used for the production of several

different metabolites, e.g. tryptophan can be converted to either indole glucosinolates or

tryptamine in canola (De Luca, 2000), the latter leading to the desired terpenoid indole

alkaloids, while coumaryl-CoA and malonyl-CoA can be substrates for either stilbene

synthase or chalcone synthase, leading to resveratrol and flavonol/anthocyanidin

production, respectively (Hain and Grimmig, 2000). Sometimes it is sufficient to

overexpress genes encoding a single key enzyme in the pathway (see above), notably genes

encoding hyoscyamine 6-hydroxylase (Table 10.2), which catalyzes the conversion of

hyoscyamine to the medicinally important scopolamine (Yun et al., 1992).

An engineered A. rhizogenes strain, harbouring the 35S-H6H gene construct, was

introduced into Hyoscyamus muticus by Jouhikainen et al. (1999), and into hyoscyamine-