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Green factory: Plants as bioproduction

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Green factory: Plants as bioproduction platforms for recombinant proteins

Jianfeng Xu a, Maureen C. Dolan a, Giuliana Medrano a, Carole L. Cramer a, Pamela J. Weathers b,
a
Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401, United States
b
Biology & Biotechnology Department, Worcester Polytechnic Institute, Worcester, MA 01609, United States

a r t i c l e i n f o a b s t r a c t

Available online 7 September 2011 Molecular farming, long considered a promising strategy to produce valuable recombinant proteins not only
for human and veterinary medicine, but also for agriculture and industry, now has some commercially avail-
Keywords: able products. Various plant-based production platforms including whole-plants, aquatic plants, plant cell
Molecular farming suspensions, and plant tissues (hairy roots) have been compared in terms of their advantages and limits.
Plant-made recombinant proteins Effective recombinant strategies are summarized along with descriptions of scalable culture systems and
Transgenic plants
examples of commercial progress and success.
Plant cell culture
Microalgae
2011 Elsevier Inc. All rights reserved.
Moss
Hairy roots
Duckweed

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172
2. Classes of recombinant proteins produced by plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172
2.1. Therapeutic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172
2.2. Industrial proteins/enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
2.3. Biopolymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
3. Plant expression platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
3.1. Whole plants stable expression systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
3.1.1. Leaf-based . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
3.1.2. Seed-based . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
3.2. Whole plants transient expression system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1176
3.3. In vitro culture systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1176
3.3.1. Plant cell suspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1177
3.3.2. Hairy roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1177
3.3.3. Moss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1177
3.4. Aquatic plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1178
3.4.1. Duckweed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1178
3.4.2. Microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1178
4. Culture scale-up and bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1178
5. Commercialization status and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179
5.1. Plant-made industrial proteins/enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179
5.2. Plant-made therapeutic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179
5.3. Plant-made biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
6. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1181
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1181

Corresponding author at: Dept. Biology & Biotechnology, Worcester Polytechnic Institute, 100 Institute Rd, Worcester, MA 01609, United States. Fax: + 1 508 831 5936.
E-mail address: weathers@wpi.edu (P.J. Weathers).

0734-9750/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.08.020
1172 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
1. Introduction
Plants possess exceptional biosynthetic capacity including the abil-
ity to use the sun (photosynthesis) and/or very simple media to sup-
port signicant biomass and protein accumulation. Their potential
for low-cost production of high quality and bioactive recombinant
protein is well documented (Obembe et al., 2011; Paul and Ma,
2011; Sharma and Sharma, 2009). Plants successfully perform the ma-
jority of post-translational modications important for many complex
eukaryotic proteins and provide tremendous exibility in bioproduc-
tion platforms that differentially address production scale, cost, safety,
and regulatory issues. Plant-based systems provide the spectrum of
production capacity ranging from plant/algal cell bioreactor systems
for lower volume, higher value product to eld-grown commodity
crops with potential for metric tons of recombinant protein at highly
competitive costs. Because plants cannot harbor the human and ani-
mal pathogens-of-issue for mammalian cell-based production sys-
tems, they bring signicant advantage in increased safety for
patients (Pogue et al., 2010; Xu et al., 2011a). These biosafety advan-
tages also impact commercial aspects; they reduce purication costs
and minimize risks associated with potential production shut-downs,
facility decontamination, and supply limitations leading to unmet
patient/customer demand. Although costs to purify proteins from
plant systems will be comparable to microbial or mammalian cell cul-
ture systems, lower up-front capitalization required to initiate com-
mercial production in plants and potential economies of scale
provide key advantages. Applications that directly use whole or mini-
mally-processed plants or plant parts (e.g., seeds) are also being ex-
plored for industrial/bioenergy applications as well as therapeutics
and vaccines to further reduce recombinant protein costs (Boothe
et al., 2010; Hood, 2002; Howard et al., 2011). In many systems, boost-
ing protein yields remains a challenge for economic feasibility (Paul
and Ma, 2011; Xu et al., 2011a) and key strategies used to boost prod-
uct recovery in plant expression system are summarized in Table 1
(see also Hood et al., 2011, in press; Rybicki, 2010). Plant-based pro-
duction systems involving eld-grown GM plants require regulatory
oversight and approvals that are unique to plants compared to other
bioproduction systems (Obembe et al., 2011); regulatory consider-
ations also impact production strategies and costs.
In contrast to other expression systems such as bacterial, mamma-
lian cell and yeast, plant expression systems encompass diverse forms
including whole-plants, suspension cells, hairy roots, moss, duck-
weed, microalgae, etc. (Fig. 1). Each of the platforms has its own
Table 1
Strategies used to boost recombinant protein yields in plants.
Production stages Strategies
Transcription Develop strong promoters, double enhanced
promoters, hybrid promoters
Use inducible promoters
Engineer better enhancers, activators or repressors
Translation Optimize 5- and 3-UTR (untranslated region);
Design preferred genetic codon.
Post-translation Target nascent proteins to sub-cellular compartments
such as ER
Co-express with protease inhibitor and protein
co-factor/subunit; co-express antibody with antigen
Express as fusion to a highly expressed and stable
peptide
In vitro cell/tissue culture Optimize medium composition
(for bioreactor system) Supplement protein-stabilizing agents
In situ remove expressed protein
Process development Select and/or improve bioreactor design
(for bioreactor system) Select culture strategy (e.g. batch vs. fed-batch vs.
continuous culture)
Evaluate secretion potential
strengths and weaknesses and is often best suited for certain classes
of recombinant proteins based on the market, scale, cost, and up-
stream and downstream processing constraints of the particular pro-
tein product. A multiplicity of plant species can serve as hosts for
plant-based bioproduction that comprise platforms ranging from in
vitro cell and plant tissue cultures to whole plants grown under
glass and in the eld. Access to such platform diversity makes it pos-
sible to respond rapidly for expressing novel recombinant proteins,
enables customizing and meeting scale-up needs, and provides op-
portunity for oral-based delivery of proteins. This review highlights
the advantages and challenges associated with each type of plant pro-
duction platform and production strategy, and where appropriate,
will comment on important issues related to regulation. It also high-
lights progress toward product commercialization.
2. Classes of recombinant proteins produced by plants
According to their functions and applications, plant-made recombi-
nant proteins can be generally categorized into three classes: therapeu-
tic proteins, industrial proteins/enzymes and biopolymers. Examples
of each of these classes are highlighted in Table 2 and discussed briey
in the following sections.
2.1. Therapeutic proteins
Many proteins of mammalian origin have been expressed in
plants, yielding a product with full function. These include monoclo-
nal antibodies (mAbs), vaccine antigens, therapeutic enzymes, blood
proteins, cytokines, growth factors and growth hormones (see ex-
amples in Table 2) (Davies, 2010; De Muynck et al., 2010; Xu et al.,
2011a). Bioactivity of these proteins often requires protein folding,
disulde bond formation, subunit assembly, proteolytic cleavage,
and/or glycosylation, highlighting the ability of plants to process
complex human/mammalian proteins. Plant-made antibodies (Plan-
tibodies) have received considerable interest as they are made at
much lower cost in plants than in mammalian cells without the asso-
ciated risks of potentially harboring animal pathogens (De Muynck
et al., 2010). Plants in fact are currently recognized as the only viable
production platform for the production of secretory antibodies
(sIgAs) (Paul and Ma, 2011). These recombinant therapeutic pro-
teins are produced using a multiplicity of plant-based platforms
that range from cultures cells to eld crops with the most common
being tobacco species with protein yields varying dramatically
from 0.01% of total soluble protein (TSP) (Mikschofsky et al., 2009)
and 0.1 g/L (Kwon et al., 2003), up to 25% of TSP (Vunsh et al.,
2007) and 247 mg/L (McDonald et al., 2005). Several recent reviews
of plant-made therapeutic proteins are available (Davies, 2010;
Obembe et al., 2011; Paul and Ma, 2011; Rybicki, 2010; Sharma and
Sharma, 2009; Tremblay et al., 2010; Weathers et al., 2010; Xu
et al., 2011a), so we will focus here on selective characteristics of
the plant-based systems permitting comparisons with bacterial, fun-
gal and mammalian cell systems.
Plants successfully glycosylate proteins at the signature recogni-
tion motif (N-X-S/T) for N-linked glycosylation. However, subsequent
processing in the Golgi to complex glycans differs from that found in
mammalian cells. Thus, a notable challenge in using plants as hosts
for production of glycosylated therapeutic proteins is that plant-
specic xylose and -1,3-fucose sugars may be added with a poten-
tial to alter bioactivity or immunogenicity in humans (Doran, 2000;
Gomord et al., 2010). Plants also do not naturally synthesize animal
or human-specic sugars, such as -1,4-galactose residues or sialic
acid (Gomord et al., 2005; Gomord et al., 2010; Paccalet et al.,
2007). Recent efforts involving RNAi strategies that knock down
fucosyl- and xylosyl-transferases in Nicotiana benthamiana, Arabidop-
sis and Physcomitrella patens and transgenic lines expressing a human
or chimeric -1,4-galactosyltransferase have been used to engineer
 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184 1173

Fig. 1. Various plant cell expression platforms for the production of recombinant proteins.

more human-like glycosylation machinery in plants (Bakker et al., chimaeric human -1,4-galactosyltransferase resulted in antibodies
2006; Schahs et al., 2007; Strasser et al., 2008). Furthermore, Agro-in- with human-like N-glycans (Vezina et al., 2009). Although signicant
ltrated N. benthamiana (plants inltrated with Agrobacterium tume- research has been focused on humanizing the glycan of plant-made
faciens) producing human antibodies, when co-expressed with a glycoproteins, the rst human-injected therapeutic with greatest

Table 2
Examples of the three classes of recombinant proteins being produced using plants.

Classication Recombinant proteins Plant expression platform References

Therapeutic proteins Antibody: anti-West Nile virus mAb Hu-E16a, Nicotiana benthamiana Lai et al. (2010), Ma et al. (1998)
Guy's 13 SIgA (CaroRx)b
Vaccine: H5N1, H1N1c N. benthamiana Shoji et al. (2008, 2011)
Therapeutic enzyme: glucocerebrosidased Carrot suspension cells Aviezer et al. (2009a, 2009b)
Blood protein: human serum albumine Potato Sijmons et al. (1990)
Cytokine: interlukin-12 Tobacco hairy roots Liu et al. (2009)
Growth factor: human epidermal growth factor Tobacco tissues Parsons et al. (2010)
Growth hormone: human growth hormonef Tobacco chloroplast Staub et al. (2000)
Oral therapeutic: human intrinsic factorg Arabidopsis thaliana Fedosov et al. (2003)
Industrial enzymes Cellulase Corn Hood et al. (2007, 2011)
-glucuronidaseh Corn Kusnadi et al. (1998)
Trypsinh Corn Woodard et al. (2003)
Avidinh Corn Hood et al. (1997), Murray et al. (2002)
-Amylasei Corn, tobacco Pen et al. (1992)
Laccase Corn Hood et al. (2003)
Biopolymers Spider silk proteins Tobacco, potato, Arabidopsis Menassa et al. (2004), Scheller et al. (2001), Yang et al. (2005)
Elastin-like polypeptides Tobacco Conley et al. (2009)
Collagensj Tobacco, corn Ruggiero et al. (2000), Xu et al. (2011b)
Plant gum Tobacco suspension cells Xu et al. (2005)
a
Recent on commercial route.
b
Phase II clinical trial completed by Planet Biotechnology; approved for use in the EU, but not marketed.
c
Phase II and phase I clinical trials for H5N1 and H1N1, respectively, completed by Medicago with positive results.
d
Phase III clinical trial completed by Protalix; the rst plant-made therapeutic protein entering commercial sector; FDA approval pending.
e
First complex human protein expressed in plant.
f
Early demonstration of high-yield protein expression in plant chloroplast.
g
Phase II clinical trial completed by Cobento Biotech AS; Marketed in the EU.
h
Sigma Aldrich products come as research enzyme or drug.
i
Recently (Feb, 2011) commercialized by Syngenta.
j
Under preclinical development by Medicago and Meristem Therapeutics.
1174 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
clinical experience (Protalix's taliglucerase; discussed further below)
did not trigger signicant antibody production in patients (Aviezer et
al., 2009a, 2009b).
Plant-specic post-translational modications (PTMs) may pre-
sent an opportunity for not only producing biosimilars, but also com-
plex recombinant proteins with enhanced function and efcacy (Faye
and Gomord, 2010). To this end, continued advances in understand-
ing and manipulating post-translational modications in plants will
be a key to the full adoption of plant-based therapeutic proteins. In
fact, the second generation of plant-made pharmaceutical proteins
is emerging at the research level whereby therapeutic targets are spe-
cically engineered to enhance the production or utility of plant-
based therapeutics. The goal in these systems is to exploit the biopro-
duction capacity of plants to produce new therapeutic proteins that
integrate novel motifs or fusion to facilitate protein assembly, deliv-
ery, trafcking, protein stability, serum longevity, or protein solubility
in either the production host or the target organism (e.g., Cramer
et al., 2010; Phoolcharoen et al., 2011; Xu et al., 2007, 2010).
2.2. Industrial proteins/enzymes
Industrial proteins are characterized by the fact that they are used
in very large quantities and must therefore be produced at very low
cost (Horn et al., 2004). Transgenic plants provide a viable technology
for producing industrial proteins, in particular enzymes, because of
low cost of agricultural production, stability of protein stored in spe-
cic organs such as seeds, ease and speed of scale-up as well as the
possibility of using crude plant materials directly in industrial pro-
cesses (Hood, 2002). Some plant-made industrial proteins/enzymes
that have received considerable interest are listed in Table 3; most of
them are hydrolases, including glycosidases (e.g., cellulase, -amylase
and -glucuronidase (GUS)) and proteases (e.g., trypsin). ProdiGene
Inc (formerly in College Station, TX) was the rst company to develop
and commercialize plant-based recombinant proteins/enzymes with
GUS and Avidin being ProdiGene's rst two commercialized products
(Basaran and Rodriguez-Cerezo, 2008; Hood, 2002; Sharma and
Sharma, 2009). ProdiGene, a little ahead of its time technically, is unfor-
tunately now out of business.
Corn seed is regarded as an ideal platform for the production of
industrial proteins/enzymes because corn has the largest annual
grain yield and relatively high seed protein content (10%), thus offer-
ing the highest potential recombinant protein yields per hectare
(Christou et al., 2005). Advantages of a seed-based production system
include easy protein storage and the ability to apply the product directly
to industrial processes, minimizing handling and enzyme manipulation
and preparation (Boothe et al., 2010; Hood, 2002). Corn-produced en-
zymes such as cellulases and hemicellulases involved in biomass
Table 3
Comparisons of different expression systems.
Expression system Commercially viable species
Whole plants
Stable transgenic plants Corn, soy, safower, rice
Transient plants Nicotiana sp., lettuce
In vitro cultured plant cells and species
Hairy roots Nicotiana sp.
Cell suspension culture Tobacco BY-2, carrot, rice
Moss Physcomitrella patens
Aquatic plants
Duckweed (closed system) Lemna sp., spirodela sp.
Microalgae Chlamydomonas reinhardtii
Open system
Photobioreactor
Conventional bioreactor
a
The time required to accumulate maximum amounts of recombinant proteins in a culture system after planting or bioreactor inoculation.
conversion to produce biofuels such as ethanol are currently interesting
candidates for commercialization (Obembe et al., 2011). A major hin-
drance to using plant-made industrial proteins/enzymes is regulatory;
very large acreage of transgenic plants will be planted. This will likely
require deregulation by federal regulatory agencies for eld growth
of those transgenic plants (Horn et al., 2004) and also wider acceptance
by the general public of open eld cultivation of transgenic plants for
protein production. Large scale production of GM biofuel corn,
which is a major commodity food/feed crop, remains controversial.
However, the regulatory route and public acceptance for crops expres-
sing this class of enzymes should be aided by the fact that these en-
zymes do not act on humans or animals (no substrate) and are
already widespread in the human diet (considered G.R.A.S, generally
recognized as safe). On the other hand, when as projected, global food
supplies become limited, then uses of a food/feed crop for non food/feed
products will eventually meet with resistance and new production solu-
tions will need to be developed.
2.3. Biopolymers
Differing from those abundant plant biopolymers such as cellulose
and starch, recombinant biopolymers discussed here specically refer
to those made of proteins such as spider silk proteins, elastin-like
polypeptides (ELPs), collagens and plant gums (Table 2). The proper-
ties and recombinant expression of these biopolymers in plants were
reviewed previously by Scheller and Conrad (2005). Of special inter-
est are the spider silk proteins (also called spidroins) that are modu-
lar brous proteins containing highly repetitive amino-acid
sequences consisting largely of glycine and alanine (Hinman et al.,
2000; Scheller et al., 2001). Spider silk bers spun from these spi-
droins are regarded as one of nature's most extraordinary materials
with exceptional exibility, elasticity, and toughnessthree times as
strong as Kevlar and ve times as strong as steel (Eisoldt et al.,
2011). Plants offer a more efcient and cheaper production platform
than bacteria for production of recombinant spidroins. The expres-
sion of Nephila clavipes dragline spidroins encoded by synthetic
genes in transgenic tobacco and potato plants has resulted in accu-
mulation of recombinant silk proteins up to a level of 2% of TSP in
the ER (Scheller et al., 2001). In another report, accumulation of drag-
line spidroins in Arabidopsis reached 18% of TSP in seeds (Yang et al.,
2005). Currently, the challenge to the application of such biomaterials
is the lack of appropriate techniques such as ber spinning to con-
vert the raw material into intermediate products (Scheller et al.,
2001). Another interesting biopolymer is the ELPs that are comprised
of the repetitive pentapeptide sequence (VGVPG), which mainly
serves as thermally responsive tags for the non-chromatographic pu-
rication of recombinant proteins (Conley et al., 2009). ELP tags were
Time for productiona Scalability Regulatory compliance
36 months Unlimited eld culture Difcult
27 days Greenhouse limited Moderate
1430 days 20,000 L Easy
720 days 100,000 L Easy
1430 days 200 L Easy
2040 days 10,000 L Moderate
2040 days Limited by water surface area Difcult
1430 days 10,000 L Moderate
720 days 200 L Easy
 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
found to signicantly enhance the production yield of a range of dif-
ferent recombinant proteins in plant leaves (Conley et al., 2011;
Patel et al., 2007). Compared with therapeutic proteins and industrial
enzymes that have shown high potential for commercialization,
plant-made biopolymers still have a long way to go before they are
cost effective to market.
3. Plant expression platforms
The rapid development of plant genetic engineering technologies
has expanded the diversity of well-established plant-based biopro-
duction platforms for recombinant proteins (Paul and Ma, 2011).
These platforms include transgenic plants (using both stable and
transient expression), duckweed, microalgae, and in vitro culture sys-
tems: cell suspensions, hairy roots and moss protonema (Fig. 1). Each
of the platforms has its own strengths and weaknesses, which are de-
scribed here. A generalized comparison of the time needed for pro-
duction, scalability and regulatory compliance of different platforms
is shown in Table 3.
3.1. Whole plants stable expression systems
To drive expression of foreign genes, many technologies for stable
integration of the transgene into the production plant have been de-
veloped and used over the years. The choices of 1) production host,
2) gene integration site (nuclear vs. plastid), 3) the cellular compart-
ment for recombinant protein accumulation, and 4) the plant tissue
targeted for product expression in establishing stable transgenic
plant lines will depend on a variety of considerations that include
the post-translational modications required for attaining functional
protein product, the stability of the expressed foreign protein in the
given plant host, desired expression levels of product and the down-
stream purication costs as well as the size and cost constraints of the
product market (see also Hood et al., 2011).
3.1.1. Leaf-based
Tobacco has emerged as the leading plant platform for leaf-based
recombinant protein expression (Tremblay et al., 2010). The rst
clinical trial of a plant-produced biopharmaceutical, secretory anti-
body variant of Guy's 13, was done with a product expressed in
eld grown tobacco leaves by Planet Biotechnology Inc. (Kaiser,
2008; Ma et al., 1998). Tobacco is genetically well studied and easily
manipulated, is classied as a non-food/non-feed crop, produces
high biomass (upwards of 300 tons per acre) and is one of the best
studied platforms to date for expressing recombinant biopharma-
ceuticals. The breeding of low nicotine and low alkaloid tobacco va-
rieties has been used to overcome potential toxicity carryover into
recombinant protein products (Menassa et al., 2001). In addition
to tobacco, many other leafy crops have been used for stable expres-
sion of proteins including lettuce, alfalfa and clover. The use of crops
like alfalfa has additional benets; it is a perennial that xes nitro-
gen and displays notable homogeneity of N-glycosylated recombi-
nant proteins (Fischer et al., 2004).
Both nuclear and plastid integration have been used when expressing
recombinant proteins in leaf tissue. The choice of gene integration within
the plant cell is generally dictated by the post-translational requirements
of the target protein. For glycosylated proteins, nuclear integration of the
transgene is needed to enable proper processing of the protein in the
endomembrane system (Gomord et al., 2010). This strategy has been
used in successfully expressing over 100 glycosylated proteins including
antibodies (e.g. Johansen et al., 1999; Ma et al., 1995; Fiedler et al.,
1997), vaccines (e.g. Pogrebnyak et al., 2005) cytokines (e.g. Wang et
al., 2008; Medrano et al., 2010), growth hormones and industrial en-
zymes in plant leaves. Benchabane et al. (2008) and Sharma and
Sharma (2009) have recently reviewed expression of endomembrane-
targeted proteins in plants.
1175
With an average of 100 chloroplasts per plant cell, the plastid pre-
sents an alternative integration site which, in practice, enables thou-
sands of copies of a given transgene to be expressed (Chebolu and
Daniell, 2007; Oey et al., 2009; Ruhlman et al., 2010). Chloroplastic
transformation and expression of chloroplast-targeted genes have
been reviewed in depth in a recent special issue on Chloroplast Bio-
technology in the Plant Biotechnology Journal (June 2011). To date,
over 100 recombinant proteins have been expressed through chloro-
plast transformation including industrial enzymes, antibodies, bio-
pharmaceutical proteins and vaccine antigens as noted in a recent
review by Clarke et al. (2011). While the chloroplast does support
many of the important post-translational modications desired for
expressing complex proteins, current plastid expression technology
is limited to production of non-glycosylated products.
Among the limitations of targeting recombinant protein expression
to the leaf is the nite shelf life of this tissue and the need to immediate-
ly process upon harvest to ensure product stability and quality. Al-
though plant leaves are advantageous in terms of biomass yield, this
tissue is generally more subject to damage than seeds. There are many
cases of recombinant proteins in leaves having associated stability and
accumulation issues. Product yields in eld-grown materials can be
highly variable due to environmental impacts (both biotic and abiotic
stresses) leading to increased consideration of growth in more con-
trolled conditions (e.g., under plastic or in greenhouses), especially for
pharmaceutical applications. In addition to associated instability of the
protein itself, the harvested material has a limited shelf life and there-
fore, must be processed immediately after harvest. It should also be
noted that at scale, regulatory-compliant disposal of transgenic bio-
mass waste may have volume and cost implications.
3.1.2. Seed-based
Recombinant protein expression targeted to plant seeds can over-
come limitations of protein stability and storage associated with leaf
tissue (Boothe et al., 2010; Lau and Sun, 2009; Ma et al., 2003). Several
cereal grains including rice, wheat, barley, soybean and maize are
commonly used as expression hosts (Ramessar et al., 2008a). Proteins
expressed in seeds are generally protected from proteolytic degrada-
tion, and storage upwards of three years at room temperature (longer
with cold storage) resulted in minimal loss of protein activity. The
high protein content of seeds, ranging from 7 to10%, has translated
into high expression for several seed-targeted proteins (e.g. Hood
et al., 1997; Horvath et al., 2000; Woodard et al., 2003; Xie et al.,
2008; Xue et al., 2003; Yang et al., 2006; Yang et al., 2008). Further-
more, maize seeds producing HIV neutralizing antibody 2 G12 (a po-
tential candidate for a topical microbicide), were not only shown to
have cost-effective production, but also simple downstream purica-
tion processes, which are considered the most signicant costs associ-
ated in the production of biopharmaceuticals (Ramessar et al., 2008c).
The potential to use agronomically important food/feed commod-
ity crops and other seed crops for non-food/feed GM-based applica-
tions such as biofuel enzymes or pharmaceuticals raises several
regulatory issues for both regulatory agencies and consumers. Regu-
latory compliance for eld growth (e.g., USDA APHIS/EPA) will en-
compass concepts of product segregation and stewardship. Crops
expressing therapeutics or vaccines will likely remain under regulato-
ry oversight for the life of the product and require containment pro-
tocols and documentation at each step in the process. In contrast,
for industrial enzymes which are/will be produced at large scale, pro-
ducers will likely seek deregulation. One of the regulatory consider-
ations for food-based grains is that while there is the need to
segregate the bioproduct source grain from the food/feed supply
chain, they do have a G.R.A.S. status from the US Food and Drug Ad-
ministration (FDA) which may reduce downstream hurdles linked
with pharmaceutical use of these plant-based products (Ramessar
et al., 2008b, 2009).
1176 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
While the rst plant-derived commercialized product was pro-
duced in maize (Hood et al., 1997; Witcher et al., 1998), oil-seeds
are emerging as a promising platform for recombinant protein pro-
duction due to their inherently low associated proteolytic activity
and simplied protein isolation via oil body separations. Among the
leading companies in this plant biotechnology sector is SemBioSys
Genetics (Calgary, Canada), which has developed the oleosin-fusion
platform, in which the target recombinant protein is produced as a fu-
sion with oleosin and accumulates in safower oilbodies (www.
sembiosys.com/). Application of this technology has highlighted the
development of an exceptionally low cost human insulin biosimilar
for improved accessibility and global distribution of insulin in coun-
tering the global diabetes epidemic. Over the past several years, a
number of other seed-based platforms have emerged, including Ven-
tria Biosciences and Meristem Therapeutics, which have invested in
eld-grown rice for the production of the human proteins lactoferrin
and lysozyme (www.ventria.com/). ORF Genetics Ltd., based in Ice-
land, has targeted barley grain as the expression site for a number
of human cytokines and growth factors (www.orfgenetics.com/).
3.2. Whole plants transient expression system
Transient expression systems, mediated by recombinant viral or
plant binary vectors, are being increasingly used for the expression of
biopharmaceuticals in plants due to speed, high protein yields, and reg-
ulatory considerations. During this process, foreign genes are typically
introduced into leaf tissue of intact plants (generally non-transgenic)
by vacuum inltration of engineered Agrobacterium (containing
gene(s) of interest within T-DNA with or without additional virus-der-
ived components). Recombinant protein production (based on extra-
chromosomal gene expression within the plant cell) is initiated in the
leaves within 24 h and continues for several days (Agro-mediated) to
several weeks (viral-mediated) depending on vector and protein.
While chromosomal integration is possible, it occurs at considerably
lower frequency in comparison to the number of cells transiently
expressing the desired gene (Circelli et al., 2010; Sheludko, 2008). In
contrast to stable transformation and when used as production systems,
these transgenic plants are grown only in contained environments, e.g.,
greenhouses. This system is widely used for assessing gene expression
constructs in plants and to validate activity of new recombinant pro-
teins (Circelli et al., 2010; Daniell et al., 2009; Dhillon et al., 2009;
Lombardi et al., 2009; Pogue et al., 2010; Tremblay et al., 2010,
2011; Voinnet et al., 2003; Whaley et al., 2011). Biomass production
for transient systems typically involves non-transgenic plants (gen-
erally Nicotiana benthamiana or other Nicotiana species) often
grown in contained environments, e.g., greenhouses, prior to inl-
tration, which is a highly automated process at production scales.
In addition to rapid transgene assessment and production runs and
high product yields, these systems also provide the ability to easily
co-express several genes at the same time (e.g., Huang et al., 2010;
Medrano et al., 2009). However, transiently expressed products re-
quire sizable greenhouse space and inltration infrastructure, imme-
diate processing of harvested leaf biomass, and, if using the product
in humans or animal, an additional purication step to remove endo-
toxins derived from the inltrated Agrobacterium.
Transient expression is typically carried out in leaves of N.
benthamiana, but also has been demonstrated to work in other plants
including potato, green pea, Arabidopsis, lettuce, and other Nicotiana
species such as N. debneyi, N. excelsior, N. exigua, N. maritime, and N.
simulans (Bhaskar et al., 2009; Green et al., 2009; Kim et al., 2009;
Negrouk et al., 2005; Sheludko et al., 2007; Sindarovska et al., 2008,
2010). There are two basic approaches for plant transient expression
based on the mechanism by which the transgene is transferred into
the plant cell: viral-mediated or plant binary vector-based (Fischer
et al., 1999; Matoba et al., 2011). In a transient expression system
using conventional plant expression vectors, the accumulation of
recombinant proteins in plants occurs within a shorter time, typically
24 days post-inltration, with recoveries typically ranging from ~0.1
to 180 g/g fresh leaf depending on the gene of interest (Condori
et al., 2009; Cramer et al., 1999, 2010; Liu et al., 2008; Medrano
et al., 2009, 2010). Recently, transient-expressed xylanase targeted
to the endomembrane system was reported with product recoveries
as high as 1 mg/g fresh leaf (Llop-Tous et al., 2011). A direct compar-
ison of stable versus transient expression of identical transgenes sug-
gests yields of 4 to 20-fold higher for the transient systems (using
de35S promoter) on a leaf fresh weight basis (Medrano et al., 2009).
Plant viral vectors also use the same Agro-inltration system to in-
troduce DNA copies into the plant cell. This transient expression process
generally produces higher levels of recombinant protein with yields
reported to be as high as 5 mg/g of fresh leaf for GFP (Marillonnet
et al., 2004). However, recombinant protein expression by this process
takes up to 14 days, which can present issues for proteins prone to pro-
tease degradation and instability. Geneware (Kentucky BioProcessing
LLC; originally developed by Large Scale Biology Corp.), MagnIcon
(Icon Genetics) and Geminiviral expression systems (Arizona State Uni-
versity) are some examples of effective viral vector expression systems
(Gleba et al., 2005, 2007; Huang et al., 2009a; Mason et al., 2002).
Alpha-trichosanthin and human lysosomal acid lipase have been
expressed using Geneware technology (Kumagai et al., 1993). MagnI-
con has been used to express hepatitis B core antigen (Huang et al.,
2006, 2008), Norwalk virus-like particles (Santi et al., 2008), and Yersi-
nia pestis antigens F1 and V (Santi et al., 2006). Geminivirus technology
was used to produce both the heavy and light chain subunits of IgG mAb
6D8 against Ebola virus GP1 (Wilson et al., 2000), hepatitis B core anti-
gen (HBc) and Norwalk virus capsid protein (Huang et al., 2009b). The
Launch vector system includes a combination of a plant viral vector ge-
nome and a binary plasmid of Agrobacterium in which several proteins
including inuenza targets have been expressed with recoveries of
60 g/g fresh leaf for recombinant H5HA-I (Musiychuk et al., 2007;
Shoji et al., 2009; Yusibov and Mamedov, 2010). Additional examples
and yield information of viral vector-mediated bioproduction are sum-
marized elsewhere (Daniell et al., 2009; Sheludko, 2008). Examples of
proteins in clinical development or commercialized were recently listed
by Yusibov et al. (2011).
Recombinant protein production using transient expression is now
being mobilized to large scale by industry. For example, the Fraunhofer
Center for Molecular Biotechnology (Newark, DE) (http://www.
fraunhofer.org) has developed a scalable, automated plant-based facto-
ry using plant viral vector technology to efciently produce large
amounts of pharmaceuticals within weeks. Medicago Inc. (Quebec, Can-
ada) (http://www.medicago.com) has a cGMP manufacturing facility
using a transient expression system to produce vaccines for Phase II
clinical trials. Icon Genetics (Bayer; Halle, Germany) (http://www.
icongenetics.com) also has a GMP manufacturing facility. Texas A&M
(College Station, TX) and Kentucky BioProcessing LLC (Owensboro,
KY) (http://www.kbpllc.com/) have constructed a GMP biomanufactur-
ing facility. Terrasphere (http://terraspheresystems.com/) has devel-
oped a high-density vertical hydroponic cultivation system that can
be used year round to produce large quantities of transient plants and
their products. The production of large quantities of recombinant pro-
tein offered by transient plant expression systems, coupled with use
of current technology to increase yields and many technical promising
solutions seem to favorably compare with mammalian or insect cell-
based systems in quality, cost, and scale.
3.3. In vitro culture systems
In vitro culture systems are characterized by the fact that plant
biomass is cultured in conned bioreactors under sterile conditions
for large-scale production of recombinant proteins. Plant suspension
cells, hairy roots and moss fall into this category.
 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
3.3.1. Plant cell suspensions
Like microorganisms, undifferentiated clusters of plant cells (callus)
can be dispersed and propagated in a liquid medium to generate stable
cell suspension cultures, which can retain the same production capacity
as whole plants. Increased concerns about regulatory compliance and
product safety of mammalian systems recently have renewed interest in
plant cell cultures as an alternative production platform for complex
pharmaceutical proteins (Huang and McDonald, 2009; Shih and Doran,
2009; Xu et al., 2011a). These systems provide cGMP-compatible produc-
tion environments more acceptable to the established pharmaceutical in-
dustry with added benets of complex protein processing compared to
bacteria and yeasts, and increase safety compared to mammalian cell sys-
tems which can harbor human pathogens. In fact, the rst plant-produced
commercial therapeutic protein for human infusion, glucocerebrosidase,
is being produced in carrot cells by Protalix Biotherapeutics, Inc. (Israel;
http://www.protalix.com), although proof-of-concept was originally
established previously using tobacco plants (Cramer et al., 1999; Radin
et al., 1999).
Plant cell suspension culture combines the advantages of whole-
plants with those of microbial cultures (reviewed in Hellwig et al.,
2004; Huang and McDonald, 2009; Xu et al., 2011a). Although plant
cell cultures do not share the perspectives of unlimited scalability of
eld-cultivated whole plants, culture of plant cells in a sterile environ-
ment allows for precise control over growth conditions, batch-to-batch
product consistency, and a production process aligned with current
good manufacturing practices (cGMP). The associated containment as-
pects of both production and product reduces the regulatory burden
compared to eld-grown plants and provides a platform more consistent
with established biopharmaceutical production systems potentially aid-
ing both regulatory and industry acceptance. The reduced regulatory
costs may partly outweigh the currently lower productivities of this pro-
duction platform along with its potentially higher capital costs. A wide
array of biologically active recombinant proteins have been successfully
expressed in plant cells, particularly in BY-2 (Bright Yellow-2) and NT-1
(Nicotiana tabacum-1). These strains are fast-growing, robust and readily
undergo Agrobacterium-mediated transformation and cell cycle synchro-
nization (Hellwig et al., 2004; Ozawa and Takaiwa, 2010; Su and Lee,
2007; Tremblay et al., 2010).
One bottleneck in exploiting plant cell suspension culture for com-
mercial purposes has been low productivity. Exceptions are expres-
sion of human proteins such as 1-antitrypsin, interleukin-12 and
hGM-CSF in rice cells using a sucrose-inducible RAmy3D promoter
(Huang et al., 2001; McDonald et al., 2005; Trexler et al., 2005; Shin
et al., 2003, 2010), where up to 247 mg/L was achieved (McDonald
et al., 2005). However, the growth rates and characteristics of rice
cell lines are inferior to those of tobacco BY-2 and NT-1 cell lines
(Hellwig et al., 2004) and the viability of rice cells is signicantly de-
creased when cultivated in a sucrose-starvation medium (Huang and
McDonald, 2009). To attain a reasonable prot margin, the productiv-
ity of plant cell cultures needs to increase 1050-fold, and this re-
quires a systematic strategy to maximize the efciency of all stages
of the production pipeline from gene expression to cell culture, pro-
cess development, and nally downstream protein purication
(Weathers et al., 2010; Xu et al., 2011a).
Although whole-plant produced pharmaceuticals do not yet account
for a signicant portion of the preclinical and clinical pipeline, plant
cells and tissues are emerging as a more compliant alternative factory
(Xu et al., 2011a). Currently, two biotechnology companies, Protalix
BioTherapeutics, Inc. (Israel; http://www.protalix.com) and Dow
AgroSciences (Indianapolis, IN; http://www.dowagro.com/) have fo-
cused on the development and commercialization of pharmaceutical
proteins expressed by proprietary plant cultures, named ProCellEx
and Concert Plant-Cell-Produced System, respectively. The commer-
cial successes of these companies will undeniably dene a new era in
the biopharmaceutical industry that promises to provide a growth op-
portunity for plant-produced products.
1177
3.3.2. Hairy roots
Hairy roots are generated by infection of plants with Agrobacterium
rhizogenes that harbors a large root-inducing (Ri) plasmid (Shanks
and Morgan, 1999). Integration of T-DNA carried on the Ri plasmid
into the plant genome results in differentiation and growth at the in-
fection site of neoplastic roots, called hairy roots (Guillon et al.,
2006b; Shanks and Morgan, 1999). These induced root tissues can be
grown indenitely in vitro and have become a viable alternative pro-
duction platform for heterologous proteins as well as other plant me-
tabolites (Giri and Narasu, 2000; Guillon et al., 2006a).
Similar to suspension cells, hairy roots can be axenically cultured
in a controlled environment suitable for the production of high-value
pharmaceutical proteins under cGMP requirements. In addition, the
possible extracellular secretion of expressed proteins from cultured
hairy roots, or rhizosecretion (Gaume et al., 2003), offers a simplied
method for the recovery of foreign proteins from an inexpensive and
well-dened medium. The advantages of rapidly growing hairy roots
over suspension cells include long-term genotype and phenotype sta-
bility, efcient productivity and the ability of hairy roots to grow on
hormone-free medium. Since the rst plant recombinant protein, a
full-length murine IgG1, was successfully produced in hairy root cul-
ture N10 years ago (Wongsamuth and Doran, 1997), nearly 20 recom-
binant proteins, including reporter proteins (e.g. GUS and GFP) (Lee
et al., 2007; Medina-Bolivar and Cramer, 2004), enzymes (Gaume et
al., 2003; Woods et al., 2008), antibodies (Martinez et al., 2005;
Sharp and Doran, 2001), antigens (Ko et al., 2006; Kumar et al.,
2006; Rukavtsova et al., 2007), growth factors (Komarnytsky et al.,
2006; Parsons et al., 2010), and immunomodulators such as ricin-B
(the non-toxic lectin subunit of ricin as antigen carrier) (Medina-
Bolivar et al., 2003) and interleukin-12 (Liu et al., 2008; Liu et al.,
2009) have been reported to be expressed by hairy roots with protein
yields up to 3.3% of TSP (Woods et al., 2008).
There are, however, challenges for large scale culture of hairy roots
because they have a tangled, lamentous morphology and often possess
unique physiological characteristics. Improved bioreactor designs, e.g.
the mist reactor, originally developed by Weathers and Giles (1988),
that offer low hydrodynamic stress, yet achieve high volumetric oxygen
transfer are needed for culture of hairy roots (Choi et al., 2006; Kim and
Yoo, 1993; Liu et al., 2009; Sivakumar et al., 2010). A Swiss biotechnol-
ogy company, ROOTec Bioactives Ltd (http://www.rootec.com/), is cur-
rently focusing on commercialization of plant-derived products with
hairy roots grown in a mist bioreactor. Hairy roots appear to be an at-
tractive in vitro expression system with several advantages compared
to eld-grown plants and suspension cultured cells. Future research
should be focused on establishing effective and economical bioreactors
for industrial production.
3.3.3. Moss
The moss, P. patens, with its fully sequenced genome (http://www.
cosmoss.org/) is also providing a promising platform for producing re-
combinant products. Knockout strains lacking xylosylation and fuco-
sylation activity have been produced so that recombinant proteins
can be humanized (Koprivova et al., 2004), and galactosylation has
been obtained by adding human glactosyltransferase to the xylosyl
and fucosyltranferase locus (Huether et al., 2005). Using the moss
platform, a variety of different engineered proteins have now been
produced including IgG1 and IgG4 antibodies, human VEGF, erythro-
poietin, and factor H (Buttner-Mainik et al., 2011; Decker and Reski,
2008). Secretion into the culture medium has also been achieved
and found to be most effective when P. patens endogenous signal se-
quences are used instead of human sequences (Schaaf et al., 2005).
The haploid juvenile gametophyte (protonema) stage of the plant
is morphologically similar to lamentous green algae and readily cul-
tured in liquid media in photobioreactors. By controlling pH or am-
monium tartrate levels in the medium, the moss can be maintained
indenitely in the protonema stage, thereby preventing development
1178 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
to the leafy mature gametophyte (Hohe et al., 2002). P. patens sus-
pensions also seem quite resistant to sheer stress; growth is not
inhibited even at 400500 rpm in stirred tank reactors (Hohe et al.,
2002). Greenovation GmbH has now developed both process technol-
ogy and proprietary strains of P. patens for contract production of de-
sired proteins up to 200 L using tubular or disposable rocker bag
bioreactors (http://www.greenovation.com/).
3.4. Aquatic plants
3.4.1. Duckweed
An aquatic higher plant, duckweed, is another promising biopro-
duction platform for recombinant proteins. Duckweed is the common
name for Lemnaceae, a monocot plant family consisting of four major
genera: Lemna, Spirodela, Wolfa and Wolfella. As the world's smal-
lest, fastest-growing and simplest owering plant (Zhang et al.,
2010), duckweed seems ideal for the production of recombinant pro-
teins (Stomp, 2005; Yamamoto et al., 2001). It is safe, fast-growing
(doubling time as short as 36 h), easy to grow and harvest, and has
a high protein content (up to 45% dry weight) (Stomp, 2005). Duck-
weed is capable of producing complex proteins that are not easily
made by bacteria and yeast, or made only at great cost over a long pe-
riod of time by mammalian cells. Moreover, duckweed is edible and
thus offers an attractive system for oral vaccines (Popov et al., 2006;
Rival et al., 2008).
Similar to other higher plants, duckweed can be transformed
using either biolistics or A. tumefaciens. Resulting transgenic lines
are stable for both gene insertion and expression. More than 20 ther-
apeutic proteins including plasminogen (Spencer et al., 2010), aproti-
nin (Rival et al., 2008), monoclonal antibody (Cox et al., 2006), avian
inuenza H5N1 hemagglutinin (Guo et al., 2009) and interferon 2
(De Leede et al., 2008) have been produced in duckweeds with ex-
pression levels up to 7% of TSP (Stomp, 2005). A reporter protein
gene, GFP, expressed in Spirodela oligorrhiza reached a protein yield
of more than 25% of TSP, making it among the highest expressing sys-
tems for nuclear transformation in a higher plant (Vunsh et al., 2007).
Biolex, Inc., based in Pittsboro, North Carolina (www.biolex.com),
is the major biotechnology company developing the duckweed (Lem-
na)-based expression (LEX) system for producing pharmaceutical
proteins. Currently, the leading product of Biolex is a controlled-re-
lease interferon 2b (Locteron) for the treatment of hepatitis C,
which offers tolerability and dosing advantages over PEGylated inter-
ferons currently on the market. Two other products that are in pre-
clinical development are a human plasmin (BLX-155) that is an
anticoagulant, and an anti-CD20 mAb (BLX-301) (Paul and Ma,
2011). For the duckweed platform to be economically viable for in-
dustrial production, several issues need to be addressed including im-
proved protein expression levels, human glycosylation, more
genomic information on duckweed and duckweed reproduction,
and methods for scale-up to commercial levels in both closed and
open systems (Stomp, 2005).
3.4.2. Microalgae
Microalgae also offer potential for large-scale and cost-effective
production of recombinant proteins. They integrate the merits of mi-
crobes including rapid growth and ease of culture with those of
higher plants in performing post-translational modication and pho-
tosynthesis (Franklin and Mayeld, 2005; Potvin and Zhang, 2010;
Specht et al., 2010). Use of transgenic microalgae for production of re-
combinant proteins has lagged behind other microorganisms, such as
bacteria and yeasts (Specht et al., 2010), but recently there has been a
surge of interest in microalgae as a viable bioproduction system
(Rasala et al., 2010). The majority of current work is performed
with the well-characterized green unicellular alga, Chlamydomonas
reinhardtii (Potvin and Zhang, 2010; Rosenberg et al., 2008; Rasala
et al., 2010). This alga has a rapid growth rate (doubling time of
10 h), supports easy nuclear and chloroplast transformation, and
can be grown either photoautotrophically or with acetate as a carbon
source. A variety of recombinant proteins have been successfully
expressed in microalgae as documented in a number of reviews
(Cadoret et al., 2008; Franklin and Mayeld, 2004, 2005; Leon-Ba-
nares et al., 2004; Mayeld et al., 2007; Potvin and Zhang, 2010;
Specht et al., 2010; Walker et al., 2005).
Both nuclear and chloroplast genomes of microalgae have been
successfully transformed for expressing recombinant proteins. How-
ever, nuclear transformants generally failed to accumulate recombi-
nant proteins to the levels observed in chloroplasts, most likely due
to nuclear silencing mechanisms (Specht et al., 2010). Thus, only
the chloroplast system is currently regarded as feasible for commer-
cial production (Potvin and Zhang, 2010). A variety of functional re-
combinant proteins including an antibody (Mayeld and Franklin,
2005), a vaccine (Streateld, 2006; Surzycki et al., 2009), a growth
factor (Rasala et al., 2010), a blood protein (Manuell et al., 2007),
and an industrial enzyme phytases (Yoon et al., 2011), have been suc-
cessfully expressed in the chloroplast of C. reinhardtii. The disadvan-
tage of chloroplast transformation is that the expressed proteins
lack posttranslational modications such as glycosylation (Franklin
and Mayeld, 2005). Although problematic for some products, in an-
tibody production, this can provide a benet because lack of glycosyl-
ation prevents antibodies from activating the immune system (Dance,
2010; Tran et al., 2009).
Recombinant protein production with microalgae is still in its in-
fancy. The development of economically viable microalgal expression
systems is currently hindered by a lack of effective and consistent
transformation methods for a wider variety of species, low (nuclear
expression) or inconsistent (chloroplast expression) recombinant
protein yields, as well as the lack of production systems optimized
for large-scale growth and harvesting under photoautotrophic condi-
tions (Specht et al., 2010). Systematic and concerted research efforts
that are both biological- and engineering-based such as optimization
of promoters, regulatory elements and codon usage as well as devel-
opment of improved photobioreactor culture systems will be critical
to the success of the microalgal production platform.
4. Culture scale-up and bioreactors
While the scale of eld-grown plants can be enlarged almost in-
denitely at low cost, scale-up of the elite genotype is the critical
rst step involving in vitro cultured plant cell and tissue systems.
Large scale culture of these in vitro cultures, especially to obtain
small plantlets (micropropagation) is still a major challenge. There
are two main categories of bioreactors typically used to culture plants
cells, tissues, and organs: liquid-phase and gas-phase reactors (Kim
et al., 2002). Liquid-phase reactors are the most commonly used,
but delivery of adequate oxygen to submerged cells or tissues re-
mains a challenge. Most plant cells and tissues are susceptible to
shear stress so, unlike microbial cells, aggressive agitation is not par-
ticularly benecial. As a result, gas-phase reactors were also devel-
oped to enhance O2 delivery, but with minimal shear. Since gasses
are very insoluble in liquids, gas-phase reactors expose the biomass
to air or a gas mixture, and nutrients are delivered as gas-infused
droplets. Hybrid versions of both types of reactor also exist wherein
the headspace gas spans a large area of relatively shallow liquid to en-
hance gas exchange to the submerged cells or tissues. Because there
are a number of recent reviews on this topic (Ducos et al., 2010;
Huang and McDonald, 2009; Weathers et al., 2010, 2011; Xu et al.,
2009), only a brief summary is provided here and the reader is direct-
ed to those for details.
Bioreactors typically used for larger scale culture of plant cells and
tissues include stirred tank, airlift, bubble column, temporary immer-
sion, balloon-type bubble, ebb and ow, mist reactor, and the rotating
drum. Typical micropropagation uses a very labor- and cost-intensive
 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
process involving small culture boxes with semi-solid medium. De-
sign and use of different reactor types and scale-up have been devel-
oped for micropropagation and other plant production systems
including cell suspensions, microalgae, hairy roots, moss protonema,
duckweed and other aquatic small plants, and even co-culture of mul-
tiple species. There are also many plant production systems being de-
veloped that instead use disposable (single use) bioreactors for
culture of plant cells and tissues. These usually employ plastic bags
of various designs including as a liner inside another vessel, a hanging
bag, the horizontal wave and wave and undertow bioreactors, the
slug bubble, the temporary immersion system (Box-In-Bag; RITA),
and a mist bag reactor. These newer disposable and issues regarding
their scale-up are described in greater detailed in recent reviews
(Eibl et al., 2010; Eibl and Eibl, 2008; Weathers et al., 2010, 2011).
5. Commercialization status and outlook
Plants and their cells or organs are essentially a production factory
for recombinant proteins. They provide both economic and safety ad-
vantages over conventional production systems. It was estimated that
with 1% (w/w) of protein expression in corn on the dry weight base
as well as 50% of protein recovery during purication, the cost of
plant-produced proteins is 10 to 50- fold lower than that of micro-
bial systems, and up to 1000-fold lower than that of mammalian
cell culture systems (Chen et al., 2005; Hood, 2002; Twyman et
al., 2003), implying the great potential of transgenic plants as an
economically competent production platform. The molecular farm-
ing industry has grown rapidly in the past 15 years. While some
plant-made industrial proteins/enzymes have been commercial-
ized, many plant-made pharmaceuticals are in various develop-
mental stages. Examples of some successful plant-produced
products, either on the market or in commercial development, are
listed in recent reviews (Obembe et al., 2011; Paul and Ma, 2011;
Sharma and Sharma, 2009), or on the Molecular Farming website
last updated in 2010 (http://www.molecularfarming.com/PMPs-
and-PMIPs.html). The major companies involved in research and
development of plant-produced recombinant proteins include: Biolex
Therapeutics Inc. (Pittsboro, NC; http://www.biolex.com), BioStrategies
LC (Jonesboro, AR; http://www.biostrategies-lc.com), Dow AgroSciences
(Indianapolis, IN; http://www.dowagro.com), Fraunhofer Center for Mo-
lecular Biotechnology (Newark, DE; http://www.fraunhofer.org), Ken-
tucky BioProcessing LLC (Owensboro, KY; http://www.kbpllc.com/),
Medicago Inc. (Qubec, Canada; http://www2.medicargo.com/en), Meri-
stem Therapeutics LLC (Cambridge, MA; http://www.meristem-
therapeutics.com), Protalix Biotherapeutics (Carmiel, Israel; http://
www.protalix.com), Planet Biotechnology (Hayward, CA; http://www.
planetbiotechnology.com), SemBioSys (Calgary, Canada; http://www.
sembiosys.com) and Ventria Bioscience (Fort Collins, CO; http://www.
ventria.com/).
5.1. Plant-made industrial proteins/enzymes
Plant-made industrial proteins/enzymes have spearheaded com-
mercialization over the other two classes of recombinant proteins;
this progress is mainly credited to ProdiGene's pioneering work on
the development and commercialization of hydrolases including
GUS, avidin and trypsin (TrypZean) and aprotonin (AproliZean)
in the late 1990s (Basaran and Rodriguez-Cerezo, 2008; Hood, 2002;
Hood et al., 1997; Sharma and Sharma, 2009; Witcher et al., 1998).
All of these products are produced in transgenic corn and sold by Sig-
ma-Aldrich (St Louis, MO) (Sharma and Sharma, 2009). The commercial
success of ProdiGene's products was followed by many efforts to de-
velop and commercialize other plant-made enzymes, in particular,
biomass-degrading enzymes such as cellulase, hemicellulase,
xylanase and ligninase (Hood et al., 2007, 2011; Sticklen, 2007),
-amylase (Pen et al., 1992), as well as an oxido-reductase fungal
1179
enzyme, laccase (Hirai et al., 2008; Hood et al., 2003). For example,
an Arkansas-based small company, Innite Enzymes (Jonesboro,
AR; http://www.inniteenzymes.com/index.htm) is engaged in de-
veloping a cost-effective corn seed production system for cellulases
for cellulosic ethanol production. However, few products entered
the marketplace until 2011 when a transgenic variety of corn expres-
sing -amylase was approved (deregulated for eld use) by the
USDA. This -amylase-producing crop was developed by Base-
l-based Syngenta and marketed as Enogen (http://www2.syngenta.
com/en/index.html). Unfortunately, it has sparked worries not only
from anti-GM organizations, but also from some biotechnology sup-
porters because of environmental and human health questions
(Waltz, 2011).
5.2. Plant-made therapeutic proteins
Because of the safety and economic concerns of using mammalian
cell cultures, plant-made therapeutic proteins are receiving renewed
interest by pharmaceutical companies. In fact, several plant-made
mammalian proteins such as human lactoferrin and lysozyme (Ven-
tria Bioscience), human aprotonin (ProdiGene), mammalian gastric
lipases (Meristem Therapeutics), and human intrinsic factor (Cobento
Biotechnology, Aarhus, Denmark) are now on the market (Obembe
et al., 2011; Paul and Ma, 2011; Sharma and Sharma, 2009). However,
they are regarded as either nutraceutical or diagnostic reagents rather
than pharmaceuticals. As of July 2011, there is no plant-made thera-
peutic protein approved for human systemic use by the US FDA.
Antibodies and vaccines are the two major classes of plant-made
therapeutic proteins that are under commercial development. Planet
Biotechnology took the lead in developing and commercializing Plan-
tibodies produced in tobacco; those being tested in clinical trials in-
clude CaroRX (for dental caries), DoxoRX (for side-effects of cancer
therapy), RhinoRX (for Rhinovirus prophylactic) and an IgG
(ICAM1) (for common cold) (Obembe et al., 2011). In addition, a
2G12 IgG used as prophylactic treatment for HIV is in Phase I clinical
trials by the Pharma-Planta Consortium (Paul and Ma, 2011), and a
plant-made scFv monoclonal antibody, used in downstream proces-
sing of a hepatitis B vaccine, has been commercialized in Cuba
(Pujol et al., 2005).
Subunit vaccine antigens produced in plants have received the most
attention in the molecular farming community. In 2006, Dow AgroS-
ciences received the world's rst regulatory approval by the USDA for a
tobacco cell-based vaccine against Newcastle disease virus, which served
as a landmark for production of therapeutic (veterinary) vaccines in
plants. One of the unique features of plant-produced vaccines is that
plants not only serve as the production bioreactor but also as the deliv-
ery vehicle for oral vaccines (Tiwari et al., 2009; Wang and Coppel, 2008).
Several oral vaccines have been subjected to Phase I clinical trials, e.g., a
potato tuber-derived HBsAg (Hefferon, 2010). The challenges of develop-
ing oral vaccines lie in the difculty in dose standardization, concerns
linked to potential contamination of the food chain, and the low efcacy
of the oral route for immunization. However, the lower production and
delivery costs of these technologies may be essential for bringing these
products to many areas of the world (Paul and Ma, 2011). The production
of vaccines using a transient plant expression system has been developed
rapidly, somewhat due to the recent outbreak of avian and swine u,
which led to the development of seasonal and pandemic inuenza vac-
cines. Such vaccines are currently in pre-clinical and clinical trials spon-
sored by Medicago Inc, The Texas Plant-Expressed Vaccine Consortium
and the Fraunhofer Center for Molecular Biotechnology. In addition, a re-
search group led by Charles J. Arntzen (a pioneer in plant-made edible
vaccines) at Arizona State University is engaged in developing several
plant-based vaccines including Hepatitis B surface antigen (Phase I in let-
tuce; Phase II in potato), Vibrio cholera vaccine, Heat-labile toxin B subunit
of Escherichia coli and the capsid protein of Norwalk virus (all are Phase I
in potato) (Obembe et al., 2011; Paul and Ma, 2011).
1180 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
Fig. 2. Comparison of benets and challenges associated with the three major types of plant production platforms. Because they are common to all plant platforms, the advantages
of plants over other production systems, e.g., eukaryotic protein processing, lack of human pathogens, scalability options, are not listed here.
In addition to antibodies and vaccines, there is special interest in
plant-produced therapeutic enzymes. The most encouraging example
is a plant (carrot) cell-produced recombinant glucocerebrosidase
(prGCD) used as enzyme replacement therapy for the treatment of
Gaucher disease (Shaaltiel et al., 2007). This prGCD was developed
by Protalix and will be marketed by Pzer with the commercial
name of taliglucerase alpha (Uplyso). It was awarded temporary
authorization for use in France in 2010, and thereby became the
rst plant cell-made human pharmaceutical to enter the commercial
market. Phases IIII clinical trials were accepted by the FDA and
nal approval is currently pending in the USA. Patient acceptance of
taliglucerase alpha was enhanced by supply and safety concerns
over Genzyme's CHO-based glucocerebrosidase, Cerezyme. Produc-
tion of Cerezyme was shut down several times in 2010 by the FDA
due to viral contamination and follow-on production issues leading
to drug shortages. Against this backdrop, the availability of Protalix's
taliglucerase alpha highlighted the key advantages in safety and
cost of the plant-derived enzyme. In addition, commercial partner-
ship with Pzer, the largest pharmaceutical company in the world,
suggests that plant-based bioproduction systems are being recog-
nized as competitive in this arena. Other plant cell-produced enzymes
in preclinical and clinical development by Protalix include a human
acetylcholinesterase (PRX-105) for several therapeutic and prophy-
lactic indications, -galactosidase (PRX-102) for the treatment of
Fabry disease, and a pr-antiTNF, a biosimilar version of etanercept
(Enbrel) for the treatment of rheumatoid arthritis.
5.3. Plant-made biopolymers
The commercial development of plant-made biopolymers lags far
behind the above two classes of recombinant proteins. Collagen, a
type of human protein that is brous and connects and supports
other bodily tissues, is under preclinical development by Medicago
Inc. (produced in alfalfa) and Meristem Therapeutics (produced in to-
bacco and corn) (Merle et al., 2002; Paul and Ma, 2011; Ruggiero
et al., 2000).
6. Conclusion
Plant-based bioproduction of proteins has evolved into a complex
arena supporting diverse production platforms that offer advantages
in scale, cost, safety, and regulatory acceptability. A comparison of
the advantages and remaining challenges of the main plant produc-
tion platforms is summarized in Fig. 2. As the eld matures, there is
greater differentiation between industrial and pharmaceutical appli-
cation with the industrial enzymes targeting eld-based production
primarily in seed/grain crops driven by scale and cost. In contrast,
therapeutic proteins are moving toward greater containment and
consistency through bioreactor-based systems or transient expres-
sion. It appears that plant-produced biologicals, long held as one of
the golden prospects of biotechnology, have nally come into their
own with commercialization of major plant-produced recombinant
proteins. With the exception of several plant-made enzymes that
were commercialized early, most plant-made pharmaceuticals only
recently entered the marketplace. Specically, the human therapeu-
tic, taliglucerase alpha by Pzer (Protalix technology), was the rst
to enter the commercial market. Others at various stages of clinical
trials are anticipated to appear in the market over the next few
years. Signicant progress has been made in the engineering of re-
combinant proteins for production by in vitro plant cell and tissue
cultures. Advances in bioreactor design and implementation have
further enhanced this potential, especially for growing more com-
plex 3-D cultures like hairy roots. Many early challenges related
to low protein yields and non-human glycosylation for therapeutic
 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
application have been overcome for select proteins and the eld
continues to develop innovative solutions to enhance the competi-
tiveness and economic impact of plant-based systems. The green
phyto-pharm is thus, emerging as an alternative and important
source of safe and cost effective therapeutics as well as industrial
enzymes.
Acknowledgments
This work was supported in part by the Worcester Polytechnic In-
stitute and the Plant Powered Production (P3) Center funds provided
through an NSF RII Arkansas ASSET Initiative (AR EPSCoR) grant and
the Arkansas Biosciences Institute, the major research component of
the Arkansas Tobacco Settlement Proceeds Act of 2000.
References
Aviezer D, Almon-Brill E, Shaaltiel Y, Galili G, Chertkoff R, Hashmueli S, et al. Novel en-
zyme replacement therapy for Gaucher disease: ongoing Phase III clinical trial with
recombinant human glucocerebrosidase expressed in plant cells. Mol Genet Metab
2009a;96:S134.
Aviezer D, Brill-Almon E, Shaaltiel Y, Hashmueli S, Bartfeld D, Mizrachi S, et al. A plant-
derived recombinant human glucocerebrosidase enzyme-a preclinical and phase I
investigation. PLoS One 2009b;4:e4792.
Bakker H, Rouwendal GJ, Karnoup AS, Florack DE, Stoopen GM, Helsper JP, et al. An an-
tibody produced in tobacco expressing a hybrid beta-1,4-galactosyltransferase is
essentially devoid of plant carbohydrate epitopes. Proc Natl Acad Sci U S A
2006;103:757782.
Basaran P, Rodriguez-Cerezo E. An assessment of emerging molecular farming activi-
ties based on patent analysis (2002 similar to 2006). Biotechnol Bioprocress Eng
2008;13:30412.
Benchabane M, Goulet C, Rivard D, Faye L, Gomord V, Michaud D. Preventing unin-
tended proteolysis in plant protein biofactories. Plant Biotechnol J 2008;6:63348.
Bhaskar PB, Venkateshwaran M, Wu L, Ane JM, Jiang J. Agrobacterium-mediated tran-
sient gene expression and silencing: a rapid tool for functional gene assay in pota-
to. PLoS One 2009;4:e5812.
Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, et al. Seed-based ex-
pression systems for plant molecular farming. Plant Biotechnol J 2010;8:588606.
Buttner-Mainik A, Parsons J, Jerome H, Hartmann A, Lamer S, Schaaf A, et al. Production
of biologically active recombinant human factor H in Physcomitrella. Plant Biotech-
nol J 2011;9:37383.
Cadoret JP, Bardor M, Lerouge P, Cabigliera M, Henriquez V, Carlier A. Microalgae as cell fac-
tories producing recombinant commercial proteins. Med Sci (Paris) 2008;24:37582.
Chebolu S, Daniell H. Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in
transgenic chloroplasts and immunogenicity in mice towards vaccine develop-
ment for amoebiasis. Plant Biotechnol J 2007;5:2309.
Chen M, Liu XW, Wang ZK, Song J, Qi QS, Wang PG. Modication of plant N-glycans
processing: the future of producing therapeutic protein by transgenic plants.
Med Res Rev 2005;25:34360.
Choi YE, Kim YS, Paek KY. Types and designs of bioreactors for hairy root culture. In:
Dutta Gupta S, Ibaraki Y, editors. Plant tissue culture engineering focus on bio-
technology. Dordrecht, The Netherlands: Springer; 2006. p. 16172.
Christou P, Stoger E, Twyman RM. Monocot expression system for molecular farming.
In: Fischer R, Schillbe S, editors. Molecular farming: plant-made pharmaceuticals
and technical proteins. Weinheim: John Wiley and Sons; 2005. p. 5567.
Circelli P, Donini M, Villani ME, Benvenuto E, Marusic C. Efcient Agrobacterium-based
transient expression system for the production of biopharmaceuticals in plants.
Bioeng Bugs 2010;1:2214.
Clarke JL, Daniell H, Nugent JM. Chloroplast biotechnology, genomics and evolution:
current status, challenges and future directions. Plant Mol Biol 2011;76:2079.
Condori J, Medrano G, Sivakumar G, Nair V, Cramer C, Medina-Bolivar F. Functional
characterization of a stilbene synthase gene using a transient expression system
in planta. Plant Cell Rep 2009;28:58999.
Conley AJ, Joensuu JJ, Jevnikar AM, Menassa R, Brandle JE. Optimization of elastin-like
polypeptide fusions for expression and purication of recombinant proteins in
plants. Biotechnol Bioeng 2009;103:56273.
Conley AJ, Joensuu JJ, Richman A, Menassa R. Protein body-inducing fusions for high-
level production and purication of recombinant proteins in plants. Plant Biotech-
nol J 2011;9:41933.
Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, et al. Glycan optimiza-
tion of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Bio-
technol 2006;24:15917.
Cramer CL, Boothe JG, Oishi KK. Transgenic plants for therapeutic proteins: linking up-
stream and downstream strategies. Curr Top Microbiol Immunol 1999;240:95-118.
Cramer CL, Dolan MC, Reidy MJ. 2010. Methods of delivery of molecules to cells using
ricin subunit and compositions relating to same. United State Patent Application,
No. 20100240597.
Dance A. From pond scum to pharmacy shelf. Nat Med 2010;16:1469.
Daniell H, Singh ND, Mason H, Streateld SJ. Plant-made vaccine antigens and biophar-
maceuticals. Trends Plant Sci 2009;14:66979.
1181
Davies HM. Commercialization of whole-plant systems for biomanufacturing of protein
products: evolution and prospects. Plant Biotechnol J 2010;8:84561.
De Leede LG, Humphries JE, Bechet AC, Van Hoogdalem EJ, Verrijk R, Spencer DG. Novel
controlled-release Lemna-derived IFN-alpha2b (Locteron): pharmacokinetics,
pharmacodynamics, and tolerability in a phase I clinical trial. J Interferon Cytokine
Res 2008;28:11322.
De Muynck B, Navarre C, Boutry M. Production of antibodies in plants: status after
twenty years. Plant Biotechnol J 2010;8:52963.
Decker EL, Reski R. Current achievements in the production of complex biopharmaceu-
ticals with moss bioreactors. Bioprocress Biosyst Eng 2008;31:39.
Dhillon T, Chiera J, Lindbo J, Finer J. Quantitative evaluation of six different viral sup-
pressors of silencing using image analysis of transient GFP expression. Plant Cell
Rep 2009;28:63947.
Doran PM. Foreign protein production in plant tissue cultures. Curr Opin Biotechnol
2000;11:199204.
Ducos JP, Terrier B, Courtois D. Disposable bioreactors for plant micropropagation and
mass plant cell culture. Adv Biochem Eng Biotechnol 2010;115:89-115.
Eibl R, Eibl D-. Design of bioreactors suitable for plant cell and tissue cultures. Phyto-
chem Rev 2008;7:5938.
Eibl R, Kaiser S, Lombriser R, Eibl D. Disposable bioreactors: the current state-of-the-art
and recommended applications in biotechnology. Appl Microbiol Biotechnol
2010;86:419.
Eisoldt L, Smith A, Scheibel T. Decoding the secrets of spider silk. Mater Today 2011;14:
806.
Faye L, Gomord V. Success stories in molecular farming-a brief overview. Plant Biotech-
nol J 2010;8:5258.
Fedosov SN, Laursen NB, Nexo E, Moestrup SK, Petersen TE, Jensen EO, et al. Human in-
trinsic factor expressed in the plant Arabidopsis thaliana. Eur J Biochem 2003;270:
33627.
Fiedler U, Philips J, Artsaenko O, Conrad U. Optimisation of scFv antibody production in
transgenic plants. Immunotechnol 1997;3:20516.
Fischer R, Vaquero-Martin C, Sack M, Drossard J, Emans N, Commandeur U. Towards
molecular farming in the future: transient protein expression in plants. Biotechnol
Appl Biochem 1999;30(Pt 2):1136.
Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM. Plant-based production of
biopharmaceuticals. Curr Opin Plant Biol 2004;7:1528.
Franklin SE, Mayeld SP. Prospects for molecular farming in the green alga Chlamydomonas
reinhardtii. Curr Opin Plant Biol 2004;7:15965.
Franklin SE, Mayeld SP. Recent developments in the production of human therapeutic
proteins in eukaryotic algae. Expert Opin Biol Ther 2005;5:22535.
Gaume A, Komarnytsky S, Borisjuk N, Raskin I. Rhizosecretion of recombinant proteins
from plant hairy roots. Plant Cell Rep 2003;21:118893.
Giri A, Narasu ML. Transgenic hairy roots: recent trends and applications. Biotechnol
Adv 2000;18:1-22.
Gleba Y, Klimyuk V, Marillonnet S. Magnifection a new platform for expressing re-
combinant vaccines in plants. Vaccine 2005;23:20428.
Gleba Y, Klimyuk V, Marillonnet S. Viral vectors for the expression of proteins in plants.
Curr Opin Biotechnol 2007;18:13441.
Gomord W, Chamberlain P, Jefferis R, Faye L. Biopharmaceutical production in plants:
problems, solutions and opportunities. Trends Biotechnol 2005;23:55965.
Gomord V, Fitchette AC, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson C, Michaud D,
et al. Plant-specic glycosylation patterns in the context of therapeutic protein
production. Plant Biotechnol J 2010;8:56487.
Green BJ, Fujiki M, Mett V, Kaczmarczyk J, Shamloul M, Musiychuk K, et al. Transient
protein expression in three Pisum sativum (green pea) varieties. Biotechnol J
2009;4:2307.
Guillon S, Tremouillaux-Guiller J, Pati PK, Rideau M, Gantet P. Hairy root research: re-
cent scenario and exciting prospects commentary. Curr Opin Plant Biol
2006a;9:3416.
Guillon S, Tremouillaux-Guiller J, Pati PK, Rideau M, Gantet P. Harnessing the potential
of hairy roots: dawn of a new era. Trends Biotechnol 2006b;24:4039.
Guo X, Bublot M, Pritchard N, Dickey L, Thomas C, Swayne DE. Lemna (duckweed)
expressed hemagglutinin from avian inuenza H5N1 protects chickens against
H5N1 high pathogenicity avian inuenza virus challenge. Abstracts of the 7th
International Symposium on Avian Inuenza, 2009; Athens, Georgia; 2009.
p. 62.
Hefferon K. Clinical trials fuel the promise of plant-derived vaccines. Am J Clin Med
2010;7:307.
Hellwig S, Drossard J, Twyman RM, Fischer R. Plant cell cultures for the production of
recombinant proteins. Nat Biotechnol 2004;22:141522.
Hinman MB, Jones JA, Lewis RV. Synthetic spider silk: a modular ber. Trends Biotech-
nol 2000;18:3749.
Hirai H, Kashima Y, Hayashi K, Sugiura T, Yamagishi K, Kawagishi H, et al. Efcient ex-
pression of laccase gene from white-rot fungus Schizophyllum commune in a trans-
genic tobacco plant. FEMS Microbiol Lett 2008;286:1305.
Hohe A, Decker EL, Gorr G, Schween G, Reski R. Tight control of growth and cell differ-
entiation in photoautotrophically growing moss (Physcomitrella patens) bioreactor
cultures. Plant Cell Rep 2002;20:113540.
Hood EE. From green plants to industrial enzymes. Enzyme Microb Technol 2002;30:
27983.
Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, et al. Commercial
production of avidin from transgenic maize: characterization of transformant, pro-
duction, processing, extraction and purication. Mol Breed 1997;3:291306.
Hood EE, Bailey MR, Beifuss K, Magallanes-Lundback M, Horn ME, Callaway E, et al. Cri-
teria for high-level expression of a fungal laccase gene in transgenic maize. Plant
Biotechnol J 2003;1:12940.
1182 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
Hood EE, Love R, Lane J, Bray J, Clough R, Pappu K, et al. Subcellular targeting is a key
condition for high-level accumulation of cellulase protein in transgenic maize
seed. Plant Biotechnol J 2007;5:70919.
Hood EE, Devaiah SP, Fake G, Egelkrout E, Teoh KT, Requesens DV, et al. Manipulating
corn germplasm to increase recombinant protein accumulation. Plant Biotechnol
J 2011. doi:10.1111/j.1467-7652.2011.00627.x.
Hood EE, Cramer CL, Medrano G, Xu J. Protein targeting: Strategic planning for optimiz-
ing protein products through plant biotechnology. In: Altman A, Hasagawa PM, ed-
itors. Plant Biotechnology and Agriculture: Prospects for the 21st Century. Elsevier;
in press. doi: 10.1016/B978-0-12-381466-1.00003-1.
Horn ME, Woodard SL, Howard JA. Plant molecular farming: systems and products.
Plant Cell Rep 2004;22:71120.
Horvath H, Huang J, Wong O, Kohl E, Okita T, Kannangara CG, et al. The production of recom-
binant proteins in transgenic barley grains. Proc Natl Acad Sci U S A 2000;97:19149.
Howard JA, Nikolov Z, Hood EE. Enzyme production systems for biomass conversion.
In: Hood EE, Nelson P, Powell R, editors. Plant Biomass Conversion. Ames, IA:
Wiley Press; 2011. p. 22753.
Huang TK, McDonald KA. Bioreactor engineering for recombinant protein production in
plant cell suspension cultures. Biochem Eng J 2009;45:16884.
Huang J, Sutliff TD, Wu L, Nandi S, Benge K, Terashima M, et al. Expression and puri-
cation of functional human alpha-1-Antitrypsin from cultured plant cells. Biotech-
nol Prog 2001;17:12633.
Huang Z, Santi L, LePore K, Kilbourne J, Arntzen CJ, Mason HS. Rapid, high-level produc-
tion of hepatitis B core antigen in plant leaf and its immunogenicity in mice. Vac-
cine 2006;24:250613.
Huang Z, LePore K, Elkin G, Thanavala Y, Mason HS. High yield rapid production of hep-
atitis B surface antigen in plant leaf by a viral expression system. Plant Biotechnol J
2008;6:2029.
Huang C, Xie Y, Zhou X. Efcient virus-induced gene silencing in plants using a modi-
ed geminivirus DNA1 component. Plant Biotechnol J 2009a;7:25465.
Huang Z, Chen Q, Hjelm B, Arntzen C, Mason H. A DNA replicon system for rapid high-level
production of virus-like particles in plants. Biotechnol Bioeng 2009b;103:70614.
Huang Z, Phoolcharoen W, Lai H, Piensook K, Cardineau G, et al. High-level rapid pro-
duction of full-size monoclonal antibodies in plants by a single-vector DNA repli-
con system. Biotechnol Bioeng 2010;106:9-17.
Huether CM, Lienhart O, Baur A, Stemmer C, Gorr G, Reski R, et al. Glyco-engineering of
moss lacking plant-specic sugar residues. Plant Biol 2005;7:2929.
Johansen FE, Natvig Norderhaug I, Roe M, Sandlie I, Brandtzaeg P. Recombinant expres-
sion of polymeric IgA: incorporation of J chain and secretory component of human
origin. Eur J Immunol 1999;;29:17018.
Kaiser J. Is the drought over for pharming? Science 2008;320:4735.
Kim YH, Yoo YJ. Development of a bioreactor for high-density culture of hairy roots.
Biotechnol Techniques 1993;7:85962.
Kim Y, Wyslouzil BE, Weathers PJ. Invited review: secondary metabolism of hairy root
cultures in bioreactors. In Vitro Cell Dev Biol Plant 2002;38:1-10.
Kim MJ, Baek K, Park CM. Optimization of conditions for transient Agrobacterium-
mediated gene expression assays in Arabidopsis. Plant Cell Rep 2009;28:115967.
Ko S, Liu JR, Yamakawa T, Matsumoto Y. Expression of the protective antigen (SpaA) in
transgenic hairy roots of tobacco. Plant Mol Biol Rep 2006;24:251.
Komarnytsky S, Borisjuk N, Yakoby N, Garvey A, Raskin I. Cosecretion of protease inhib-
itor stabilizes antibodies produced by plant roots. Plant Physiol 2006;141:118593.
Koprivova A, Stemmer C, Altmann F, Hoffmann A, Kopriva S, Gorr G, et al. Targeted
knockouts of Physcomitrella lacking plantspecic immunogenic N-glycans. Plant
Biotechnol J 2004;2:51723.
Kumagai MH, Turpen TH, Weinzettl N, della-Cioppa G, Turpen AM, Donson J, et al.
Rapid, high-level expression of biologically active alpha-trichosanthin in trans-
fected plants by an RNA viral vector. Proc Natl Acad Sci U S A 1993;90:42730.
Kumar GBS, Ganapathi TR, Srinivas L, Revathi CJ, Bapat VA. Expression of hepatitis B
surface antigen in potato hairy roots. Plant Sci 2006;170:91825.
Kusnadi AR, Evangelista RL, Hood EE, Howard JA, Nikolov ZL. Processing of transgenic
corn seed and its effect on the recovery of recombinant beta-glucuronidase. Bio-
technol Bioeng 1998;60:4452.
Kwon TH, Seo JE, Kim J, Lee JH, Jang YS, Yang MS. Expression and secretion of the het-
erodimeric protein interleukin-12 in plant cell suspension culture. Biotechnol
Bioeng 2003;81:8705.
Lai HF, Engle M, Fuchs A, Keller T, Johnson S, Gorlatov S, et al. Monoclonal antibody pro-
duced in plants efciently treats West Nile virus infection in mice. Proc Natl Acad
Sci U S A 2010;107:241924.
Lau OS, Sun SSM. Plant seeds as bioreactors for recombinant protein production. Bio-
technol Adv 2009;27:101522.
Lee KT, Chen SC, Chiang BL, Yamakawa T. Heat-inducible production of beta-glucuronidase
in tobacco hairy root cultures. Appl Microbiol Biotechnol 2007;73:104753.
Leon-Banares R, Gonzalez-Ballester D, Galvan A, Fernandez E. Transgenic microalgae as
green cell-factories. Trends Biotechnol 2004;22:4552.
Liu J, Dolan M, Reidy M, Cramer C. Expression of bioactive single-chain murine IL-12 in
transgenic plants. J Interferon Cytokine Res 2008;28:38192.
Liu CZ, Towler MJ, Medrano G, Cramer CL, Weathers PJ. Production of mouse interleu-
kin-12 is greater in tobacco hairy roots grown in a mist reactor than in an airlift re-
actor. Biotechnol Bioeng 2009;102:107486.
Llop-Tous I, Ortiz M, Torrent M, Ludevid MD. The expression of a xylanase targeted to
ER-protein bodies provides a simple strategy to produce active insoluble enzyme
polymers in tobacco plants. PLoS One 2011;6:e19474.
Lombardi R, Circelli P, Villani ME, Buriani G, Nardi L, Coppola V, et al. High-level HIV-1
Nef transient expression in Nicotiana benthamiana using the P19 gene silencing
suppressor protein of Artichoke Mottled Crinckle Virus. BMC Biotechnol 2009;9:
96.
Ma JK, Hiatt A, Hein MB, Vine ND, Wang F, Stabila P, et al. Generation and assembly of
secretory antibodies in plants. Science 1995;268:7159.
Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, et al. Characterization of a
recombinant plant monoclonal secretory antibody and preventive immunotherapy
in humans. Nat Med 1998;4:6016.
Ma JK, Drake PMW, Christou P. The production of recombinant pharmaceutical pro-
teins in plants. Nat Rev Genets 2003;4:794805.
Manuell AL, Beligni MV, Elder JH, Siefker DT, Tran M, Weber A, et al. Robust expression
of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant Biotechnol
J 2007;5:40212.
Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y. In planta engineering of
viral RNA replicons: efcient assembly by recombination of DNA modules deliv-
ered by Agrobacterium. Proc Natl Acad Sci U S A 2004;101:68527.
Martinez C, Petruccelli S, Giulietti AMA, Alvarez MA. Expression of the antibody 14D9
in Nicotiana tabacum hairy roots. Electron J Biotechnol 2005;8:1706.
Mason HS, Palmer KE, Hefferon KL, Mor TS, Arntzen C. 2002. Gemini virus vectors for
gene expression in plants. United State Patent, No. 6392121
Matoba N, Davis KR, Palmer KE. Recombinant protein expression in Nicotiana. Methods
Mol Biol 2011;701:199219.
Mayeld SP, Franklin SE. Expression of human antibodies in eukaryotic micro-algae.
Vaccine 2005;23:182832.
Mayeld SP, Manuell AL, Chen S, Wu J, Tran M, Siefker D, et al. Chlamydomonas rein-
hardtii chloroplasts as protein factories. Curr Opin Biotechnol 2007;18:12633.
McDonald KA, Hong LM, Trombly DM, Xie Q, Jackman AP. Production of human
alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor.
Biotechnol Prog 2005;21:72834.
Medina-Bolivar F, Cramer C. Production of recombinant proteins by hairy roots cul-
tured in plastic sleeve bioreactors. Methods Mol Biol 2004;267:35163.
Medina-Bolivar F, Wright R, Funk V, Sentz D, Barroso L, Wilkins TD, et al. A non-toxic lectin
for antigen delivery of plant-based mucosal vaccines. Vaccine 2003;21:997-1005.
Medrano G, Reidy MJ, Liu J, Ayala J, Dolan MC, Cramer CL. Rapid system for evaluating
bioproduction capacity of complex pharmaceutical proteins in plants. Methods
Mol Biol 2009;483:5167.
Medrano G, Dolan MC, Stephens NT, McMickle A, Erf G, Radin D, et al. Efcient plant-
based production of chicken interleukin-12 yields a strong immunostimulatory cy-
tokine. J Interferon Cytokine Res 2010;30:14354.
Menassa R, Hong Z, Karatzas CN, Lazaris A, Richman A, Brandle J. Spider dragline silk
proteins in transgenic tobacco leaves: accumulation and eld production. Plant
Biotechnol J 2004;2:4318.
Menassa R, Nguyen V, Jevnikar AN, Brandle JE. A self-contained system for the eld produc-
tion of plant recombinant human interleukin-10. Mol Breed 2001;8:17785.
Merle C, Perret S, Lacour T, Jonval V, Hudaverdian S, Garrone R, et al. Hydroxylated
human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient
expression and in transgenic tobacco plant. FEBS Lett 2002;515:1148.
Mikschofsky H, Schirrmeier H, Keil GM, Lange B, Polowick PL, Keller W, et al. Pea-der-
ived vaccines demonstrate high immunogenicity and protection in rabbits against
rabbit haemorrhagic disease virus. Plant Biotechnol J 2009;7:53749.
Murray C, Sutherland PW, Phung MM, Lester MT, Marshall RK, Christeller JT. Expres-
sion of biotin-binding proteins, avidin and streptavidin, in plant tissues using
plant vacuolar targeting sequences. Transgenic Res 2002;11:199214.
Musiychuk K, Stephenson N, Bi H, Farrance CE, Orozovic G, Brodelius M, et al. A launch
vector for the production of vaccine antigens in plants. Inuenza Other Respi Vi-
ruses 2007;1:1925.
Negrouk V, Eisner G, Lee H, Han K, Taylor D, Wong HC. Highly efcient transient expression
of functional recombinant antibodies in lettuce. Plant Sci 2005;169:4338.
Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming.
Biotechnol Adv 2011;29:21022.
Oey M, Lohse M, Kreikemeyer B, Bock R. Exhaustion of the chloroplast protein synthe-
sis capacity by massive expression of a highly stable protein antibiotic. Plant J
2009;57:43645.
Ozawa K, Takaiwa F. Highly efcient Agrobacterium-mediated transformation of sus-
pension-cultured cell clusters of rice (Oryza sativa L.). Plant Sci 2010;179:3337.
Paccalet T, Bardor M, Rihouey C, Delmas F, Chevalier C, D'Aoust MA, et al. Engineering
of a sialic acid synthesis pathway in transgenic plants by expression of bacterial
Neu5Ac-synthesizing enzymes. Plant Biotechnol J 2007;5:1625.
Parsons J, Wirth S, Dominguez M, Bravo-Almonacid F, Giulietti AM, Rodriguez Talou J.
Production of human epidermal growth factor (hEGF) by in vitro cultures of Nico-
tiana tabacum: effect of tissue differentiation and sodium nitroprusside addition.
Int J Biotechnol Biochem 2010;6:1318.
Patel J, Zhu H, Menassa R, Gyenis L, Richman A, Brandle J. Elastin-like polypeptide fu-
sions enhance the accumulation of recombinant proteins in tobacco leaves. Trans-
genic Res 2007;16:23949.
Paul M, Ma JKC. Plant-made pharmaceuticals: leading products and production plat-
forms. Biotechnol Appl Biochem 2011;58:5867.
Pen J, Molendijk L, Quax WJ, Sijmons PC, van Ooyen AJ, van den Elzen PJ, et al. Produc-
tion of active Bacillus licheniformis alpha-amylase in tobacco and its application in
starch liquefaction. Nat Biotechnol 1992;10:2926.
Phoolcharoen W, Bhoo SH, Lai H, Ma J, Arntzen CJ, Chen Q, et al. Expression of an im-
munogenic Ebola immune complex in Nicotiana benthamiana. Plant Biotechnol J
2011. doi:10.1111/j.1467-7652.2011.00593.x.
Pogrebnyak N, Golovkin M, Andrianov V, Spitsin S, Smirnov Y, Egolf R, et al. Severe
Acute respiratory syndrome (SARS) S protein production in plants: development
of recombinant vaccine. Proc Natl Acad Sci USA 2005;102:90627.
Pogue GP, Vojdani F, Palmer KE, Hiatt E, Hume S, Phelps J, et al. Production of pharma-
ceutical-grade recombinant aprotinin and a monoclonal antibody product using
plant-based transient expression systems. Plant Biotechnol J 2010;8:63854.
 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
Popov SV, Golovchenko VV, Ovodova RG, Smirnov VV, Khramova DS, Popova GY, et al.
Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of
Lemna minor L. Vaccine 2006;24:54139.
Potvin G, Zhang ZS. Strategies for high-level recombinant protein expression in trans-
genic microalgae: a review. Biotechnol Adv 2010;28:9108.
Pujol M, Ramirez NI, Ayala M, Gavilondo JV, Valdes R, Rodriguez M, et al. An integral
approach towards a practical application for a plant-made monoclonal antibody
in vaccine purication. Vaccine 2005;23:18337.
Radin DN, Cramer, CL, Oishi KK, Weissenborn DL. 1999. Production of Human Lysosom-
al Proteins in Plant-based Expression Systems. United State Patent. No. 5929304;
European Patent. No. 0865499.
Ramessar K, Capell T, Christou P. Molecular pharming in cereal crops. Phytochem Rev
2008a;7:57992.
Ramessar K, Sabalzaa M, Capell T, Christou P. Maize plants: an ideal production plat-
form for effective and safe molecular pharming. Plant Sci 2008b;174:40919.
Ramessar K, Rademacher T, Sack M, Stadlmann J, Platis D, Stiegler G, et al. Cost-effective
production of a vaginal protein microbicide to prevent HIV transmission. Proc Natl
Acad Sci U S A 2008c;105:372732.
Ramessar K, Capell T, Twyman RM, Quemada H, Christou P. Calling the tunes on trans-
genic crops: the case for regulatory harmony. Mol Breed 2009;23:99-112.
Rasala BA, Muto M, Lee PA, Jager M, Cardoso RMF, Behnke CA, et al. Production of ther-
apeutic proteins in algae, analysis of expression of seven human proteins in the
chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J 2010;8:71933.
Rival S, Wisniewski JP, Langlais A, Kaplan H, Freyssinet G, Vancanneyt G, et al. Spirodela
(duckweed) as an alternative production system for pharmaceuticals: a case study,
aprotinin. Transgenic Res 2008;17:50313.
Rosenberg JN, Oyler GA, Wilkinson L, Betenbaugh MJ. A green light for engineered
algae: redirecting metabolism to fuel a biotechnology revolution. Curr Opin Bio-
technol 2008;19:4306.
Ruggiero F, Exposito JY, Bournat P, Gruber V, Perret S, Comte J, et al. Triple helix assem-
bly and processing of human collagen produced in transgenic tobacco plants. FEBS
Lett 2000;469:1326.
Ruhlman T, Verma D, Samson N, Daniell H. The role of heterologous chloroplast se-
quence elements in transgene integration and expression. Plant Physiol
2010;152:2088104.
Rukavtsova EB, Abramikhina TV, Shulga NY, Bykov VA. Bur'yanov YI. Tissue specic ex-
pression of hepatitis B virus surface antigen in Transgenic plant cells and tissue cul-
ture. Russ. J Plant Physiol 2007;54:7705.
Rybicki EP. Plant-made vaccines for humans and animals. Plant Biotechnol J 2010;8:
62037.
Santi L, Giritch A, Roy CJ, Marillonnet S, Klimyuk V, Gleba Y, et al. Protection conferred
by recombinant Yersinia pestis antigens produced by a rapid and highly scalable
plant expression system. Proc Natl Acad Sci U S A 2006;103:8616.
Santi L, Batchelor L, Huang Z, Hjelm B, Kilbourne J, Arntzen CJ, et al. An efcient plant
viral expression system generating orally immunogenic Norwalk virus-like parti-
cles. Vaccine 2008;26:184654.
Schaaf A, Tintelnot S, Baur A, Reski R, Gorr G, Decker EL. Use of endogenous signal se-
quences for transient production and efcient secretion by moss (Physcomitrella
patens) cells. BMC Biotechnol 2005;5:30.
Schahs M, Strasser R, Stadlmann J, Kunert R, Rademacher T, Steinkellner H. Production
of a monoclonal antibody in plants with a humanized N-glycosylation pattern.
Plant Biotechnol J 2007;5:65763.
Scheller J, Conrad U. Plant-based material, protein and biodegradable plastic. Curr Opin
Plant Biol 2005;8:18896.
Scheller J, Guhrs KH, Grosse F, Conrad U. Production of spider silk proteins in tobacco
and potato. Nat Biotechnol 2001;19:5737.
Shaaltiel Y, Bartfeld D, Hashmueli S, Baum G, Brill-Almon E, Galili G, et al. Production of
glucocerebrosidase with terminal mannose glycans for enzyme replacement ther-
apy of Gaucher's disease using a plant cell system. Plant Biotechnol J 2007;5:
57990.
Shanks JV, Morgan J. Plant hairy root culture. Curr Opin Biotechnol 1999;10:1515. Wang DJ, Brandsma M, Yin ZQ, Wang AM, Jevnikar AM, Ma SW. A novel platform for
Sharma AK, Sharma MK. Plants as bioreactors: recent developments and emerging op-
portunities. Biotechnol Adv 2009;27:81132.
Sharp JM, Doran PM. Strategies for enhancing monoclonal antibody accumulation in
plant cell and organ cultures. Biotechnol Prog 2001;17:97992.
Sheludko YV. Agrobacterium-mediated transient expression as an approach to produc-
tion of recombinant proteins in plants. Recent Pat Biotechnol 2008;2:198208.
Sheludko YV, Sindarovska YR, Gerasymenko IM, Bannikova MA, Kuchuk NV. Compari-
son of several Nicotiana species as hosts for high-scale Agrobacterium-mediated
transient expression. Biotechnol Bioeng 2007;96:60814.
Shih SMH, Doran PM. Foreign protein production using plant cell and organ cultures:
advantages and limitations. Biotechnol Adv 2009;27:103642.
Shin YJ, Hong SY, Kwon TH, Jang YS, Yang MS. High level of expression of recombinant
human granulocytemacrophage colony stimulating factor in transgenic rice cell
suspension culture. Biotechnol Bioeng 2003;82:77883.
Shin YJ, Lee NJ, Kim J, An XH, Yang MS, Kwon TH. High-level production of bioactive
heterodimeric protein human interleukin-12 in rice. Enzyme MicrobTechnol
2010;46:34751.
Shoji Y, Chichester JA, Bi H, Musiychuk K, de la Rosa P, Goldschmidt L, et al. Plant-ex-
pressed HA as a seasonal inuenza vaccine candidate. Vaccine 2008;26:29304.
Shoji Y, Bi H, Musiychuk K, Rhee A, Horsey A, Roy G, et al. Plant-derived hemagglutinin
protects ferrets against challenge infection with the A/Indonesia/05/05 strain of
avian inuenza. Vaccine 2009;27:108792.
Shoji Y, Chichester JA, Jones M, Manceva SD, Damon E, Mett V, et al. Plant-based rapid
production of recombinant subunit hemagglutinin vaccines targeting H1N1 and
H5N1 inuenza. Hum Vaccin 2011;7:4150.
1183
Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ, Hoekema A.
Production of correctly processed human serum albumin in transgenic plants.
Nat Biotechnol 1990;8:21721.
Sindarovska YR, Sheludko YV, Gerasymenko IM, Bannikova MA, Kuchuk NV. Purica-
tion of recombinant GFP produced by Agrobacterium-mediated transient expres-
sion in Nicotiana excelsior. Tsitol Genet 2008;42:1620.
Sindarovska YR, Gerasymenko IM, Sheludko YV, Olevinskaya ZM, Spivak NY, Kuchuk
NV. Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agro-
bacterium-mediated transient expression. Tsitol Genet 2010;44:604.
Sivakumar G, Liu CZ, Towler MJ, Weathers PJ. Biomass production of hairy roots of Artemisia
annua and Arachis hypogaea in a scaled-up mist bioreactor. Biotechnol Bioeng 2010;107:
80213.
Specht E, Miyake-Stoner S, Mayeld S. Micro-algae come of age as a platform for re-
combinant protein production. Biotechnol Lett 2010;32:137383.
Spencer D, Dickey LF, Gasdaska JR, Wang X, Cox KM, Peele CG. 2010. Expression of plas-
minogen and microplasminogen in duck weed. United States Patent, No. 7659445
Staub JM, Garcia B, Graves J, Hajdukiewicz PTJ, Hunter P, Nehra N, et al. High-yield pro-
duction of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol
2000;18:3338.
Sticklen MB. 2007. Production of ss-glucosidase, hemicellulase and ligninase in E1 and
FLC-cellulase-transgenic plants. United States Patent Application, No. 20070192900
Stomp AM. The duckweeds: a valuable plant for biomanufacturing. Biotechnol Annu
Rev 2005;11:6999.
Strasser R, Stadlmann J, Schahs M, Stiegler G, Quendler H, Mach L, et al. Generation of
glyco-engineered Nicotiana benthamiana for the production of monoclonal anti-
bodies with a homogeneous human-like N-glycan structure. Plant Biotechnol J
2008;6:392402.
Streateld SJ. Engineered chloroplasts as vaccine factories to combat bioterrorism.
Trends Biotechnol 2006;24:33942.
Su WW, Lee KT. Plant cell and hairy-root cultures-process characteristics, products, and
application. In: Yang ST, editor. Bioprocessing for value-added products from renew-
able resources. Amsterdam, The Netherlands: Elsevier Science; 2007. p. 26392.
Surzycki R, Greenham K, Kitayama K, Dibal F, Wagner R, Rochaix JD, et al. Factors effect-
ing expression of vaccines in microalgae. Biologicals 2009;37:1338.
Tiwari S, Verma PC, Singh PK, Tuli R. Plants as bioreactors for the production of vaccine
antigens. Biotechnol Adv 2009;27:44967.
Tran M, Zhou B, Pettersson PL, Gonzalez MJ, Mayeld SP. Synthesis and assembly of a
full-length human monoclonal antibody in algal chloroplasts. Biotechnol Bioeng
2009;104:66373.
Tremblay R, Wang D, Jevnikar AM, Ma S. Tobacco, a highly efcient green bioreactor for
production of therapeutic proteins. Biotechnol Adv 2010;28:21421.
Tremblay R, Feng M, Menassa R, Huner NP, Jevnikar AM, Ma S. High-yield expression of
recombinant soybean agglutinin in plants using transient and stable systems.
Transgenic Res 2011;20:34556.
Trexler MM, McDonald KA, Jackman AP. A cyclical semicontinuous process for produc-
tion of human alpha(1)-antitrypsin using metabolically induced plant cell suspen-
sion cultures. Biotechnol Prog 2005;21:3218.
Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants:
host systems and expression technology. Trends Biotechnol 2003;21:5708.
Vezina LP, Faye L, Lerouge P, D'Aoust MA, Marquet-Blouin E, Burel C, et al. Transient
co-expression for fast and high-yield production of antibodies with human-like
N-glycans in plants. Plant Biotechnol J 2009;7:44255.
Voinnet O, Rivas S, Mestre P, Baulcombe D. An enhanced transient expression system in
plants based on suppression of gene silencing by the p19 protein of tomato bushy
stunt virus. Plant J 2003;33:94956.
Vunsh R, Li JH, Hanania U, Edelman M, Flaishman M, Perl A, et al. High expression of
transgene protein in Spirodela. Plant Cell Rep 2007;26:15119.
Walker TL, Purton S, Becker DK, Collet C. Microalgae as bioreactors. Plant Cell Rep
2005;24:62941.
Waltz E. Amylase corn sparks worries. Nat Biotechnol 2011;29:294.
biologically active recombinant human interleukin-13 production. Plant Biotech-
nol J 2008;6:50415.
Wang L, Coppel RL. Oral vaccine delivery: can it protect against non-mucosal patho-
gens? Expert Rev Vaccines 2008;7:72938.
Weathers PJ, Giles KL. Regeneration of plants using nutrient mist culture. In Vitro Cell
Dev Biol-Plant 1988;24:72432.
Weathers PJ, Towler MJ, Xu JF. Bench to batch: advances in plant cell culture for pro-
ducing useful products. Appl Microbiol Biotechnol 2010;85:133951.
Weathers PJ, Towler MJ, Wyslouzil BE. Beyond cells: culturing complex plant tissues.
In: El-Mansi EMT, Bryce CFA, Demain AL, Allman AR, editors. Fermentation Micro-
biology and Biotechnology. Boca Raton, FL: CRC Press 3rd edition; in press.
Whaley KJ, Hiatt A, Zeitlin L, Hum Vaccin. Emerging antibody products and Nicotiana
manufacturing. Hum Vaccin 2011:7.
Wilson JA, Hevey M, Bakken R, Guest S, Bray M, Schmaljohn AL, et al. Epitopes involved
in antibody-mediated protection from Ebola virus. Science 2000;287:16646.
Witcher DR, Hood EE, Peterson D, Bailey M, Bond D, Kusnadi A, et al. Commercial pro-
duction of -glucuronidase (GUS): a model system for the production of proteins
in plants. Mol Breed 1998;4:30112.
Wongsamuth R, Doran PM. Production of monoclonal antibodies by tobacco hairy
roots. Biotechnol Bioeng 1997;54:40115.
Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT, Lane JR, et al. Maize (Zea
mays)-derived bovine trypsin: characterization of the rst large-scale, commercial
protein product from transgenic plants. Biotechnol Appl Biochem 2003;38:12330.
Woods RR, Geyer BC, Mor TS. Hairy-root organ cultures for the production of human
acetylcholinesterase. BMC Biotechnol 2008;8:95.
1184 J. Xu et al. / Biotechnology Advances 30 (2012) 11711184
Xie T, Qiu Q, Zhang W, Ning T, Yang W, Zheng C, et al. A biologically active rhIGF-1 fusion
accumulated in transgenic rice seeds can reduce blood glucose in diabetic mice via
oral delivery. Peptides 2008;29:186270.
Xu JF, Shpak E, Gu TY, Moo-Young M, Kieliszewski M. Production of recombinant plant
gum with tobacco cell culture in bioreactor and gum characterization. Biotechnol
Bioeng 2005;90:57888.
Xu JF, Tan L, Goodrum KJ, Kieliszewski MJ. High-yields and extended serum half-life of
human interferon alpha 2b expressed in tobacco cells as Arabinogalactan-protein
fusions. Biotechnol Bioeng 2007;97:997-1008.
Xu JF, Okada S, Tan L, Goodrum KJ, Kopchick JJ, Kieliszewski MJ. Human growth hor-
mone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein
has a prolonged serum life. Transgenic Res 2010;19(5):84967.
Xu JF, Ge XM, Dolan MC. Towards high-yield production of pharmaceutical proteins
with plant cell suspension cultures. Biotechnol Adv 2011a;29:27899.
Xu L, Weathers PJ, Xiong XR, Liu CZ. Microalgal bioreactors: challenges and opportuni-
ties. Eng Life Sci 2009;9:17889.
Xu X, Gan Q, Clough RC, Pappu KM, Howard JA, Baez JA, et al. Hydroxylation of recom-
binant human collagen type I alpha 1 in transgenic maize co-expressed with a re-
combinant human prolyl 4-hydroxylase. BMC Biotechnol 2011b;11:69.
Xue GP, Patel M, Johnson JS, Smyth DJ, Vickers CE. Selectable marker-free transgenic
barley producing a high level of cellulase (1,4-beta-glucanase) in developing
grains. Plant Cell Rep 2003;21:108894.
Yamamoto YT, Rajbhandari N, Lin XH, Bergmann BA, Nishimura Y, Stomp AM. Genetic
transformation of duckweed Lemna gibba and Lemna minor. In Vitro Cell Dev Biol
Plant 2001;37:34953.
Yang JJ, Barr LA, Fahnestock SR, Liu ZB. High yield recombinant silk-like protein pro-
duction in transgenic plants through protein targeting. Transgenic Res 2005;14:
31324.
Yang L, Tada Y, Yamamoto MP, Zhao H, Yoshikawa M, Takaiwa F. A transgenic rice seed
accumulating an anti-hypertensive peptide reduces the blood pressure of sponta-
neously hypertensive rats. FEBS Lett 2006;580:331520.
Yang L, Wakasa Y, Takaiwa F. Biopharming to increase bioactive peptides in rice seed.
J AOAC Int 2008;91:95764.
Yoon SM, Kim SY, Li KF, Yoon BH, Choe S, Kuo MM. Transgenic microalgae expres-
sing Escherichia coli AppA phytase as feed additive to reduce phytate excretion
in the manure of young broiler chicks. Appl Microbiol Biotechnol 2011;91:
55363.
Yusibov VM, Mamedov TG. Plants as an alternative system for expression of vaccine
antigens. Proc ANAS (Biological Sciences) 2010;65:195200.
Yusibov V, Streateld SJ, Kushnir N. Clinical development of plant-produced recombi-
nant pharmaceuticals: vaccines, antibodies, and beyond. Hum Vaccin 2011;7:
31321.
Zhang Y, Hu Y, Yang B, Ma F, Lu P, Li L, et al. Duckweed (Lemna minor) as a model plant
system for the study of human microbial pathogenesis. PLoS One 2010;5:e13527.