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Review

Bioreactor technology: A novel industrial tool for high-tech

production of bioactive molecules and biopharmaceuticals from
plant roots
Ganapathy Sivakumar
Biotech Genomics, ENEA, Casaccia, Rome, Italy

Plants are the richest source for different bioactive molecules. Because of the vast number of side
effects associated with synthetic pharmaceuticals, medical biotechnologists turned to nature to
provide new promising therapeutic molecules from plant biofactories. The large-scale availability
of the disease- and pesticide-free raw material is, however, restricted in vivo. Many bioactive plant
secondary metabolites are accumulated in roots. Engineered plants can also produce human therapeutic proteins. Vaccines and diagnostic monoclonal antibodies can be won from their roots, so
that engineered plants hold immense potential for the biopharmaceutical industry. To obtain sufficient amounts of the plant bioactive molecules for application in human therapy, adventitious
and hairy roots have to be cultured in in vitro systems. High-tech pilot-scale bioreactor technology for the establishment of a long-term adventitious root culture from biopharmaceutical plants
has recently been established. In this review, I briefly discuss a technology for cultivating bioactive
molecule-rich adventitious and hairy roots from plants using a high-tech bioreactor system, as well
as the principles and application of genome-restructuring mechanisms for plant-based biopharmaceutical production from roots. High-tech bioreactor-derived bioactive phytomolecules and
biopharmaceuticals hold the prospect of providing permanent remedies for improving human
well-being.

Received 7 July 2006

Revised 5 October 2006
Accepted 6 October 2006

Keywords: Adventitious roots Bioactive molecules Biopharmaceuticals Bioreactor Plantibodies

Introduction

Plant molecules have a higher bioactivity, and are safer

and better for human health than synthetic drugs. This is
so because human beings have co-evolved with plants
over the past few million years. For 5.1 billion people
worldwide, natural plant-based remedies are used for
both acute and chronic health problems, from treating
common colds to controlling blood pressure and cholesterol. Today, plants are the raw source materials for as
many as 40% of the pharmaceuticals in use in the United

Correspondence: Dr. Ganapathy Sivakumar, Biotech Genomics, ENEA

Centro Ricerche, Casaccia, Via Anguillarese 301, 00060 Rome, Italy
E-mail: sivakumar@libero.it
Abbreviations: OUR, oxygen uptake rate; CER, carbon dioxide evolution rate

2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

States and Europe. Some commercial medicines, such as

the cancer drugs taxol from Taxus brevifolia, colchicine
from Gloriosa superba, camptothecin from Camptotheca
acuminate, ginsenosides from Panax ginseng, vinblastine
from Catharanthus roseus, the anti-malarial drugs quinine from Cinchona pubescens, and artemisin from
Artemisia annua, are manufactured from plants. Synthetic drugs often act in the body as irritants and toxins, upsetting the balance of whole systems, and producing side
effects that can be lethal. The relative potency of
stereoisomers of natural and synthetic drugs differs for
each effect, which may result in differences in therapeutic and adverse effects and in the benefit to risk ratio.
Thus, pharmacologists consider drug products that consist of different stereoisomers as different drugs rather
than different formulations of the same drug. It is impossible to obtain equally high bioactive stereoisomers
through chemical synthesis. For these reasons, plant

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drugs can provide better natural bioactive medicine than

synthetic drugs without harmful side effect.
Most of the bioactive molecules, such as camptothecin, vinblastine, and ginsenosides, are of root origin.
Harvesting roots is destructive for the plants and hence
there has been increasing interest in developing in vitro
cultures of adventitious and hairy roots from several medicinal plant species. The gap between discovery and
commercialization can be bridged when biotechnologists
and bioengineers join forces and integrate their research
disciplines to develop bioreactor technology for the production of natural therapeutic molecules. Recently, the
adventitious and hairy root culture technology for the production of pharmaceuticals has reached from small-scale
laboratory to large-scale industrial production: the German company ROOTec has described commercial-scale
cultures of hairy roots, producing camptothecin and
podophyllotoxin for medicine. Normally, adventitious root
cultures need an exogenous phytohormone supply for
growth [14]. However, the transgenic hairy roots can
grow actively without phytohormone [5]. The ability to
manipulate roots at a cellular and molecular level shows
great potential for commercial production of bioactive
molecules. In the future, demand for existing biopharmaceuticals (e.g., erythropoietin to treat anemia and insulin
to treat diabetes), as well as new therapeutic proteins discovered through genomics efforts, is expected to rise considerably. It is prudent, therefore, to evaluate alternative
plant biopharmaceutical production systems and determine how the future availability of safe recombinant biopharmaceuticals can be ensured in a cost-effective manner. Producing human therapeutic proteins in plants has
many economic and qualitative benefits, including reduced health risks from pathogen contamination, comparatively high yields, and production in roots or other
storage organs [6]. In addition, plants have a big advantage over mammalian cell systems, in that plant viruses
are not pathogenic to humans unlike mammalian viruses.
In just 25 years, the biopharmaceutical industry has
boomed, from a few early companies to todays $ 50-billion market [7]. Currently, more than 300 plant-made biopharmaceuticals are under various stages of discovery,
development and production at various academic and
company sites around the world. Several plant-derived
biopharmaceutical proteins are reaching the late stages
of commercial development. These products include antibodies, vaccines, human blood products, hormones and
growth regulators [8]. Basically, the genetic engineering
process involves incorporating the desired foreign gene
into the genome of the plant to create a biopharmaceutical-producing plant, or introducing the gene through external means into the plant and just transforming that
plant to make the desired product. The biopharmaceutical hairy root system offers tremendous potential for introducing additional genes, along with the Ri T-DNA
genes, for alteration of metabolic pathways and produc-

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tion of useful human therapeutic molecules. The hairy

roots continuously secrete recombinant proteins into a
liquid medium or accumulated them in the intercellular
spaces, thus simplifying the purification process.
Globally, the field cultivation of biopharmaceuticals
plants requires the application of pesticides, which results in the serious problem of pesticide residues. Furthermore, major problems in biopharmaceutical plant
cultivation are that geographical constraints and higher
production costs. To overcome these problems, we established high-tech bioreactor technology for commercialscale production of plant biopharmaceuticals, creating an
alternative way to produce the corresponding bioactive
secondary metabolites and vaccines through a root culture system. High-tech bioreactors are essential in adventitious and hairy root engineering, not only because
they provide an aseptic environment mimicking controlled conditions for the growth of roots, but also because
they enable high-tech systematic development of the
responses of higher root biomass and biopharmaceutical
production.

Bioreactor technology

Caring for a better world, the major challenge is how to

translate the laboratory-scale product designs into largescale production of high-tech biopharmaceuticals from
plant roots that are reproducible, safe, and economically
competitive. Bioreactor culture is the key step towards
commercial production of bioactive therapeutics by plant
biotechnology. The engineering of adventitious and hairy
roots represents one of todays fastest-growing biotechnology areas of world pharmaceutical and nutraceutical
economy. The in vitro cultivation of adventitious and hairy
roots in the bioreactor that supports efficient nutrition of
cells, possibly combined with the application of mechanical stimulation to direct cellular activity, differentiation
and function, is an important step towards the development of high-tech products.
At the heart of bioreactor control is the ability to monitor important process variables such as pH, temperature,
dissolved oxygen, oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), temperature and specific gravity. Mixing, oxygen transfer and shear stress remain the
biggest challenges as far as the scale-up to industrial-size
high-tech bioreactors is concerned. These parameters are
generally linked, and compromises need to be made, for
instance, in aeration to avoid excessive shear stress. The
development of bubble-free bioreactor systems without
conventional agitation technologies is needed to address
this problem. At initial stages in a bioreactor oxygen
transfer is not difficult as the medium contains enough
dissolved oxygen to support the growth of the plant root
inoculum. Mixing is a very important factor because it
serves the dual purpose of supplying dissolved oxygen

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and driving away the carbon dioxide. The OUR by a unit

of root biomass in a set time is known as the oxygen transfer coefficient. Other dissolved gaseous metabolites, i.e.,
carbon dioxide and ethylene, also affect the overall productivity of the roots. Although cells at the core of the
roots would be exposed to adequate oxygen tensions,
high oxygen levels near the surfaces of the roots may become toxic to peripheral cells and cause localized oxidative stress [9]. High-tech bioreactor systems typically employ mathematical models that describe the process being controlled via a software sensor [10]. To enable the kinetic analysis of bioreactors, Wu et al. [11] developed a
black-box transfer function model, and were able to identify the system parameters from input-output data. They
obtained excellent agreement between the measured and
estimated values of the OUR and CER within seconds following a system perturbation.
A stirred tank bioreactor is not suitable for mass production of adventitious and hairy roots because of shear
stress and high electric charge. Among the various kinds
of bioreactors, airlift and bubble column bioreactor are
commercially successful for adventitious and hairy root
culture because of low shear stress and the simplicity of
their design and construction. This reactor comprises a
main body, air bubbling device, steam generator for sterilization, air inlet, air vent system and various control system for checking temperature, oxygen, and pH, and
pipeline systems for transferring steam, air, medium and
root masses [1].
Bioreactors with computer control systems theoretically offer various advantages over conventional culture
procedures due to possibilities of automation, saving labor, reduction of production costs and high-tech. The
bioreactor culture system is more advanced than the traditional tissue culture system because the culture conditions in a bioreactor can be optimized by on-line manipulation of temperature, pH, concentrations of oxygen, carbon dioxide and nutrients in the medium. Nutrient uptake
of roots can also be enhanced by continuous medium circulation. Furthermore, cell proliferation and regeneration
rates can be increased. Thus, production cost and time
can be substantially reduced, product quality can be controlled and standardized, products can be free of pesticide
contamination, and production can be conducted all year
round without geographical constraints [1]. In this regard,
high-tech bioreactor technology is useful for the largescale cultivation of biopharmaceuticals from plant roots.

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genes under sterile conditions. Indeed, compared to cell

cultures, adventitious roots have a higher stability in the
physical chemical conditions and accumulate large quantities of secondary metabolites on intercellular spaces,
which can be more easily isolated. Adventitious roots,
once established, can be grown in a medium with low inoculum with a high growth rate. The change in carbon assimilation mode has dramatic effects on the patterns of
phytochemicals produced by adventitious root cultures,
thus providing a system to study the coordination between primary and secondary metabolism.
Adventitious roots can also express their photosynthetic abilities in vitro. We have induced green adventitious roots under phototropic condition. Chloroplast-dependent reactions are a vital part of certain metabolic
pathways and could result in a novel pattern of compounds produced by adventitious roots. Green adventitious roots are known to produce certain metabolites that
are normally synthesized in green parts of the plant. For
example, in plants tocopherol biosynthesis takes place in
the plastid, and the enzymes are associated with the plastidial envelop [12]. Homogentisic acid derived from aromatic amino acid metabolism, and phytyldiphosphate derived from the plastidic methylerythritol phosphate pathway [13], serve as precursors for tocopherol biosynthesis
[14]. -Tocopherol is the major vitamin E compound found
in leaf chloroplast, where it is located in the chloroplast
envelope, thylakoid membranes and plastoglobuli [15].
Natural -tocopherol contains three stereogenic centers
(RRR), and thus exists as a mixture of eight stereoisomers
in all racemic forms that are provided in most commercially available vitamin preparations. The arrangement of
atoms influences the properties and the biological activity of a substance. The natural -tocopherol isomer (RRR)
disappears more quickly from the liver after supplementation than the enantiomer (SSS) [16]. This implies that
the stereoisomers of -tocopherol are discriminated from

Adventitious root culture

Several secondary metabolites of pharmaceutical interest

are accumulated in adventitious roots. According to our
experience, the adventitious root cultures (Fig. 1) constitute a good biological material for stable commercial production of higher secondary metabolites without foreign

2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. High-Tech bioreactor cultivation of hazelnut adventitious roots

for the production of human therapeutic molecules.

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each other and suggests that there is an interaction with

another chiral entity, likely a macromolecule. It is impossible to obtain equally highly bioactive -tocopherol
through chemical synthesis [17]. Thus, we produced RRR-tocopherol through a green adventitious root culture
system. Furthermore, glucosinolates can be derived from
amino acids and contain a sulfate and thioglucose
residue. Upon tissue damage, glucosinolates are brought
into contact with myrosinases and hydrolyze into unstable aglycons. These are further converted into a range of
biologically active compounds like isothiocyanates and
nitriles that possess antimicrobial, cancer-preventing,
goitrogenic or inflammatory activities, depending on the
nature of the glucosinolate precursor amino acid and the
type of degradation product formed. Sulforaphane is one
of a class of bioactive molecule called isothiocyanates.
Sulforaphane predominantly found in broccoli, may help
to reduced risk of developing several types of cancers [18].
It is an indirect antioxidant; it does not neutralize free radicals directly, but rather boosts Phase 2 enzymes that trigger ongoing and long-lasting antioxidant activity [19].
Sulforaphane is a very expensive molecule, costing approximately 72 Euro/5 mg. Sulforaphane from chemical
synthesis requires several highly toxic substances, and final products from these reactions still contain toxic
residues and require further purification. This disadvantage limits the use of synthesized sulforaphane as food additives. Thus, natural sulforaphane from plants is more favorable for the consumer [20]. We therefore produced sulforaphane through an adventitious root culture system.
Many traditional strategies can be used to increase the
production of bioactive secondary metabolites, but jasmonic acid elicitation is usually one of the most commercially successful.

Elicitation

An elicitor can be defined as any compound or mixture of

compounds that induces a plant defense reaction, which
may enhance secondary metabolism in plant cell and root
cultures. Besides, biogenetic precursors, salicylic acid,
chitosan and heavy metals, the jasmonic acid elicitation
has been commercially successful. Jasmonic acid and its
volatile derivative methyl jasmonate (MeJA) are collectively called jasmonates. Jasmonic acid is an important
plant stress signaling molecule. It induces the biosynthesis of defense proteins and protective secondary metabolites. Jasmonates are fatty acid derivatives with a 12-carbon backbone that are synthesized from 18-carbon intermediates via the so-called octadecanoid pathway [21].
The induction of secondary metabolite accumulation is
an important stress response that depends on jasmonates
as a regulatory signal [22, 23]. The effect of jasmonates on
secondary metabolism have been studied in detail for alkaloid biosynthesis. In alkaloid metabolism, jasmonate

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acts by coordinate activation of the expression of multiple

biosynthesis genes. However, how the jasmonic acid signal is transduced to affect gene expression is largely unknown. Two regions were identified that dictated elicitor
and jasmonate responsive reporter gene activation: the
so-called BA region and a region close to the TATAbox
called jasmonate and elicitor responsive element (JERE).
The JERE interacts with two jasmonic acid responsive
transcription factors called octadecanoid responsive
Catharanthus AP2-domain proteins (ORCAs). In terpenoid indole alkaloid metabolism and primary precursor
pathways, jasmonate induces gene expression and metabolism via ORCAs, which are members of the AP2/ERFdomain family of plant transcription factors. Other jasmonate-regulated secondary metabolic pathways might
also be controlled by ORCA-like AP2/ERF-domain transcription factors. [24]. We assume that it could be a mechanism in adventitious root cultures, as MeJA treatment
increased accumulation of bioactive secondary metabolites. This treatment consists in applying chemical stresses to the adventitious cultures that triggers the higher
production of secondary metabolites. This offers the possibility of obtaining adventitious roots with improved nutritional value and anti-carcinogenic properties through
natural molecules without foreign genes [25].

Hairy root culture

In the past few decades, a lot of attention has been paid

to hairy root research for the production of important secondary metabolites. Hairy roots also offer a valuable
source of root-derived phytochemicals that are useful as
pharmaceuticals and cosmetics [5]. Hairy roots develop as
the consequence of the interaction between Agrobacterium rhizogenes, a gram-negative soil bacterium, and the
host plant. To generate hairy roots, wounded plant tissues
are inoculated with A. rhizogenes, which transfers the TDNA comprising the loci between the TR and TL regions
of the Ri plasmid into the plant genome. The rhizogenic
strains contain a single copy of a large Ri plasmid [26]. The
T-DNA carries the rol and aux genes. The rol genes are responsible for the phenotype of hairy roots and the aux
genes direct auxin synthesis, involved in root induction
[27, 28]. A. rhizogenes A4 type (A4, ATCC, 15834, 1855,
TR105, etc.) can synthesis both agropine and mannopine.
A. rhizogenes 8196 type (TR7, TR101, etc) synthesize the
mannopine only. The vir region is about 35 kb in the Ri
plasmid, and encodes six transcriptional loci, vir AG,
which have important functions in gene transfer. Genes
involved in agropine and auxin synthesis are located in
the TR DNA region. Genes of Ri TL-DNA such as rolA, rolB,
rolC and rolD direct the synthesis of a substance that
stimulates hairy root differentiations under the influence
of endogenous auxin synthesis. These four genes correspond to ORF10, ORF11, ORF12 and ORF15 of the

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eighteen ORFs of TL-DNA, respectively. ORFs of these sequences were named c-rol genes. Ngrol (NgrolB, NgrolC,
NgORF13 and NgORF14) and trol (trolC, torf 13 -1 and torf
13 -2 ) genes are c-rol genes contained in the genome of
Nicotiana glauca and N. tabacum, respectively. Introduction of a DNA fragment that contained RirolC, RiORF13
and RiORF14 resulted in more intense and earlier root formation than that of RirolB. Ngrol genes (NgrolB, NgrolC,
NgORF13, and NgORF14) in the genome of Nicotiana
glauca are similar in sequence to Rirol genes [29]. The culture requirements and applications of hairy roots have
been extensively investigated in recent years [5]. Hairy
roots induced by rol genes are usually characterized by
extensive secondary branching and an increase in the
number of root hairs with a higher ratio of metabolically
active meristem tissues. In addition, hairy root is an excellent starting material for the production of bioactive
secondary metabolites and plant-based biopharmaceuticals. In contrast to ordinary root cultures, which often require a balanced supplement of auxin and cytokinin to
maintain growth and phenotype, transformed roots are
auxoautotrophic and so vigorous, stable cultures can be
maintained on hormone-free media. Also, fast growing
hairy roots can be used as a continuous source for the production of valuable human therapeutic molecules.

Bioengineering

Metabolic engineering now offers new possibilities to improve the metabolic pathway of specific molecules in
hairy root for the production of human therapeutic molecules. Metabolic engineering is based on integrating
genes that encode enzymes of a given plant biosynthetic
pathway between the T-borders of the Ri plasmid, then
transferring this construct into plant genome. For example, the Hyoscyamus niger gene for hyoscyamine 6-hydroxylase has been placed under the control of the 35S
CaMV promoter and introduced into hyoscyamine-rich
Atropa belladonna plants by a binary vector system using
A. rhizogenes. The resulting hairy roots showed increased
levels of hydroxylase activity and contained up to fivefold
higher concentrations of scopolamine than control [30].
Putrescine:SAM N-methyltransferase (PMT) catalyzes
the N-methylation of the diamine putrescine to form Nmethylputrescine, the first specific precursor of both
tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of
Nicotiana tabacum was placed under the regulation of the
CaMV 35S promoter and introduced into the genome of a
scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed A. tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The N-methylputrescine
levels of the resulting engineered hairy roots increased
fourfold [31]. The cDNA encoding farnesyl diphosphate
synthase (FDS) placed under a CaMV35S promoter was

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transferred into Artemisia annua using A. rhizogenes

strain ATCC15834. As a result a fourfold higher accumulation of artemisinin was found compared to control [32].
Hairy root cultures of C. acuminata were transformed with
A. rhizogenes strains ATCC 15834 and R-1000. The hairy
roots produced and secreted camptothecin as well as the
more potent and less toxic natural derivative, 10-hydroxycamptothecin (HCPT) into the medium. Remarkably, the
hairy roots were able to synthesize higher alkaloids than
in vivo roots, i.e., 1.0 and 0.15 mg/g dry weight for camptothecin and the 10-hydroxycamptothecin, respectively
[33].

Biopharmaceuticals

Besides bioactive secondary metabolites, hairy roots can

also produce human therapeutic proteins, vaccines and
diagnostic monoclonal antibodies from engineered roots,
and thereby hold immense potential for the biopharmaceutical. Plants are capable of carrying out acetylation,
phosphorylation, and glycosylation as well as other posttranslational protein modifications required for the biological activity of many eukaryotic proteins. Plants have
now been modified, on the basis of peptide epitopes of
pathogens, to produce vaccines against a variety of human and animal diseases [8]. Indeed, numerous heterologous (recombinant) proteins have been produced in plant
leaves, fruits, roots, tubers, and seeds, and targeted to different subcellular compartments, such as cytoplasm, endoplasmic reticulum (ER), or apoplastic space [34]. Several biotechnology companies are now actively developing,
and patenting hairy root expression systems, while clinical trials are proceeding on the first biopharmaceuticals
derived from them. Producing therapeutic proteins in
hairy roots has many economical and practical benefits,
including reduced health risks from pathogen contamination and comparatively high yields. The bioreactor cultivation, harvesting, storage, and processing of hairy roots
can also use an existing high tech infrastructure. Hairy
roots are potentially a cheap source of recombinant products, and it has been estimated that the cost of producing
recombinant proteins in roots could be 10- to 15-fold lower than producing the same protein by E. coli fermentation. Biopharmaceuticals produced in animal cell culture
systems have to be purified from the culture supernatant,
an expensive process. Hairy roots can be made to store
proteins in intercellular or extracellular spaces, from
where they can be easily extracted [35]. Borisjuk et al. [34]
demonstrated that root secretion can be successfully exploited for the continuous production of recombinant proteins in a process called rhizosecretion. Recombinant
proteins targeted to the root intercellular space through
the secretion pathway would also be secreted continuously by the roots [36]. Root-derived recombinant biopharmaceutical proteins, whether purified or not, are less

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likely to be contaminated with human pathogenic microorganisms than those derived from animal cells, because hairy roots do not act as hosts for human infectious
agents.
Using recombinant approaches, biotechnologist began turning DNA sequences into protein drugs or biopharmaceuticals. The design and construct used to express the recombinant protein is a key factor in determining the yield. Promoter choice affects the yield by determining the rate of transcription [37]. Regulated promoters
can be used in preference to constitutive promoters because these often have both practical and biosafety advantages. The rate of translation can be optimized by ensuring that any mRNA instability sequences are eliminated from the transgene construct, and that the translational start-site matches the Kozak consensus for plants. It
might be necessary to modify codon usage in some transgenes, not only to maximize the rate of protein synthesis,
but also to eliminate cryptic introns and instability sequences [38]. Plant viral proteins offer new opportunities
to activate immunity against linked T cell epitopes to attack cancer [39]. Genes were expressed from either the
strong constitutive cauliflower mosaic virus (CaMV) 35S
promoter, or the strong modified mannopine synthase
(mas2) promoter from Agrobacterium, preferentially active in plant roots [40]. The two host-virus systems most
frequently used are tobacco with tobacco mosaic virus
(TMV) or cowpeas with cowpea mosaic virus (CPMV).
Many forms of recombinant antibodies (plantibodies) can
be produced in biopharmaceutical plants and the variety
ranges from single chain antibody fragments to full-size
antibodies and antibody complexes, as well as antibody
fusion proteins. Vectors derived from TMV have been
used to produce oral anti-hypertensive peptide (angiotensin-1-converting enzyme inhibitor) in tomato and
tobacco [41] and an inhibitor of HIV replication (a-trichosanthin) in N. bethamiana [42]. Chimeric CPMV particles, displaying human rhinovirus-14 and HIV-1, have
been purified from infected cowpea plants [43]. They were
found to elicit antibody production and, in the case of the
HIV chimera, to neutralize the infection of T cells by HIV1 in vivo. Recombinant plantibodies include fully assembled whole immunoglobulins [44], antigen-binding fragments of immunoglobulins, and synthetic single-chain
variable fragment gene fusions [45]. Single-chain Fv plantibodies are encoded by an artificial gene made by joining
together light- and heavy-chain variable sequences [46].
A novel Tobravirus-based system [47] for root expression
was reported, and new vector technologies continue to be
developed. For example, internal ribosome entry sites
(IRES) have been used to direct the expression of bicistronic mRNA [48]. Hairy root cultures have been shown
to produce recombinant proteins [49]. Recently, hairy root
cultures of potato were transformed with A. rhizogenes
strains ATCC 15834 with a 681-bp BamHI fragment from
pBSHER containing the hepatitis B surface antigen (HB-

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sAg) s gene. These hairy roots expressed higher levels of

HBsAg [50]. In the case of hairy root cultures expressing
monoclonal antibodies, it was shown that antibodies
were not only associated with the root tissue, but were released extracellularly [51, 52]. Sharp and Doran [53] reported that expression of a murine immunoglobulin (Ig)
G1 in hairy root cultures led to secretion of the recombinant antibodies into the medium. Assembly and posttranslational modifications in the ER are important for the
synthesis of full-size antibodies and Fab fragments, and
these molecular forms must be directed to the secretory
pathway. Peeters et al. [54] demonstrated that an Fab
fragment was efficiently secreted to the apoplast of roots.
Secretion of recombinant proteins into tobacco root exudates was developed for the production of human secreted alkaline phosphatase and has recently been used for
the secretion of recombinant antibodies [55]. Green fluorescent protein (GFP) was genetically fused to ricinB and
expressed in tobacco hairy root cultures. Tobacco-synthesized ricinB:GFP hairy root media was used for nasal
immunization and oral gavage of mice and systemic and
mucosal immune responses against GFP [56].
Plantibodies could be of particular benefit for topical
immunotherapy. Plantibodies derived from roots have a
multitude of applications, including binding to pathogenic organisms, binding to serum or body fluid effector proteins, binding to tumor antigens to deliver imaging or
anti-tumor agents, or binding to a cellular receptor site to
up- or down regulate receptor function. The ability to assemble immunoglobulins is a major advantage that plants
have over bacterial expression systems [6]. More remarkably, plants produce functional secretory plantibodies,
which are dimers of the typical serum immunoglobulins
and have two additional polypeptide components, making ten separate polypeptides in total [57]. Two different
cell types are required to assemble such antibodies in
mammals [58]. The biggest component of the cost involved in production of plantibodies will be for purification. However, expression roots opens up the possibility
of oral administration of some therapeutic antibodies
without the need for expensive purification. The expression of antigens in hairy roots has opened up a new avenue for the development of oral vaccines. Oral delivery of
vaccines is an attractive alternative to injection, largely
for reasons of low cost and easy administration. Therefore,
oral vaccines are particularly applicable to combating diseases that affect very large populations. Furthermore, oral
root-based vaccines are stable during storage at ambient
temperatures. However, silencing of introduced transgenes has frequently been observed in roots, constituting
a major commercial problem. Post-transcriptional gene silencing is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region
of the silenced gene and with the accumulation of small
RNA molecules homologous to the silenced gene. Efforts

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to understand and eliminate factors responsible for transgene silencing are therefore useful. The greatest advantages of biopharmaceuticals in roots are that recombinant
proteins are synthesized in an aseptic non-toxic aqueous
environment and are stable, resulting in high yields.

Conclusion

Plant biotechnology can lead to the commercial production of pharmacologically important proteins which are, in
many cases, fully functional and nearly identical to their
mammalian counterparts. Biopharmaceutical production
in roots necessitates a series of careful decisions regarding two critical areas: the gene expression system to be
used and the type of plant to be used. The use of roots as
factories for the production of novel plant-based bioactive
molecules, vaccines, antibodies and other therapeutic
proteins will undoubtedly continue to develop. With a
growing number of antibodies moving into clinical evaluation, the utility of biopharmaceutical production will be
evaluated alongside plant root culture for the production
of therapeutic antibodies. Biopharmaceuticals derived
from engineered roots will have to meet the standards of
performance and safety of biopharmaceuticals originating from other systems. Many companies, such as Phytomedics and Photosynthetic in the US, are now producing commercial-scale plant based on bioactive molecules
and vaccines through root system. Biopharmaceutical in
roots from high-tech pilot-scale bioreactor system hold
great potential for the safe production of relatively inexpensive plant-based pharmaceutical bioactive human
therapeutic molecule and vaccines that would open
prospects for improving world health. Easy scale-up of
production is a major advantage of biopharmaceutical
root system, thus in terms of cost-effectiveness the full potential of roots may be realized best at higher production
requirements. Long-term targets for high-tech bioreactors may therefore encompass high-volume, low-cost antibodies, which do not require extensive purification [59].
Roots have many advantages over established production
technologies for the large-scale expression of recombinant proteins, but several challenges remain to be addressed in terms of improving yields and product quality.
A small number of root-derived bioactive molecules are
approaching commercialization, but these are the minority that have met the technological challenges, cleared
the regulatory hurdles and overcome inertia in the
biotechnology industry. Furthermore, high-tech bioreactor technology would, of course, block certain safety
locks to prevent the release of biopharmaceutical plant
materials into the open environment and to protect the
operating personnel. Thus, we are at an important platform in global health where rapid advances are possible.
In the future plant root bioactive molecules and vaccines
from high-tech bioreactor system may be the premier ex-

2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.biotechnology-journal.com

pression system for diagnostic and therapeutic safe drugs

for human well-being.

The authors thank Dr. Loretta Bacchetta for helpful

suggestions during the course of this project and for bioreactor facilities. This work was supported in part by
research grants from ENEA, Rome, Italy.

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Biotechnol. J. 2006, 1, 14191427

Dr. G. Sivakumar is a scientist in plant

molecular biotechnology, Department
of Biotech Genomics, ENEA, Rome,
Italy, since 2004. He did his Ph.D. in
Plant Biotechnology at Bharathidasan
University, Tamilnadu, India, in 2001.
He first performed postdoctoral research in natural products molecular
chemistry and biotechnology, at the
Calabria University, Italy, from
20012003. His area of research interests includes advanced bioreactor technology, bioengineering, biopharmaceuticals (plant vaccines
and functional proteins), bioactive plant secondary metabolites, natural products molecular chemistry and pilot-scale plant tissue culture.
His research was mainly focussed on laboratory to high-tech industrial-scale production of biosafe plant based biopharmaceuticals, therapeutic, nutraceutical and cosmetic molecules from plant roots using
pilot-scale bioreactor technology.

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