Transgenic Plants Abiri et al.

A Critical Review of the Concept of Transgenic Plants:

Insights into Pharmaceutical Biotechnology and
Molecular Farming

Rambod Abiri1,2, Alireza Valdiani1*, Mahmood Introduction

Maziah1,3,4*, Noor Azmi Shaharuddin1*, Mahbod Currently, critical challenges in soil and water resources, as
Sahebi4, Zetty Norhana Balia Yusof1, Narges Atabaki5, well as climate change, have resulted in the human
Daryush Talei6 population outpacing the available agricultural products.
Thus, scientists are attempting to find various ways to
1Department of Biochemistry, Faculty of Biotechnology and increase the quality and quantity of the food,
Biomolecular Sciences, Universiti Putra Malaysia, 43400 pharmaceutical and the industrial products of plants within
UPM Serdang, Selangor DE, Malaysia. limited arable spaces (De La Fuente et al., 2013).
2Young Researchers and Elite Club of IAU, Kermanshah, Phenotypic selection was the first action taken by humans
Iran. to establish plant breeding as a reliable classical approach
3Institute of Bioscience, 5Institute of Tropical Agriculture, to this problem. Indeed, the history of plant breeding can
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor be traced to the dawn of civilization,10,000 years ago,
DE, Malaysia. when early societies began to shift from being hunter-
4Institute of Tropical Agriculture, Universiti Putra Malaysia, gatherers to being members of agrarian communities
43400 UPM Serdang, Selangor DE, Malaysia (Tilman et al., 2002).
5IAU of Tehran Science and Research Branch, Tehran, Iran Gregor Mendel reached the first milestone in modern
6Medicinal Plant Research Center, Shahed University, plant breeding. The results of his plant-breeding
Tehran, 3319118651, Iran. investigations led to the discovery of genetic functions
through focusing on DNA as the basis of trait heredity
*Corresponding authors: (Acquaah, 2009). Although, conventional plant breeding
Alireza Valdiani [email protected] has been used to overcome the lack of sufficient food or
Mahmood Maziah [email protected] feed (Lopes et al., 2012), the success of this method is
Noor Azmi Shaharuddin [email protected] largely dependent on the breeder's experience and on
phenotypic selection; consequently, inaccurate predictions
http://dx.doi.org/10.21775/cimb.018.021 can be made and low-efficiency breeding can occur
(Mewett et al., 2007). Moreover, other limiting factors, such
Abstract as the method being time consuming, the difficulty in
Using transgenic plants for the production of high-value finding the most related parents of a new generation of
recombinant proteins for industrial and clinical applications plants, selecting the best crossing method according to the
has become a promising alternative to using conventional plant traits desired, the high number of back-crosses
bioproduction systems, such as bacteria, yeast, and required and the difficulties in increasing the expression of
cultured insect and animal cells. This novel system offers the favoured traits are the main concerns in plant breeding
several advantages over conventional systems in terms of (De La Fuente et al., 2013).
safety, scale, cost-effectiveness, and the ease of Genetic modification (GM) is a new method that
distribution and storage. Currently, plant systems are being researchers have been using to increase the yield of plant
utilised as recombinant bio-factories for the expression of products by improving certain traits, including the
various proteins, including potential vaccines and responses of herbs to abiotic and biotic stresses (Tait,
pharmaceuticals, through employing several adaptations of 1999; Ashraf et al., 2008). Background genetic
recombinant processes and utilizing the most suitable tools transformation is the most significant application of GM; in
and strategies. The level of protein expression is a critical this process various methods are utilized to introduce
factor in plant molecular farming, and this level fluctuates desirable traits into the host genome while concentrating
according to the plant species and the organs involved. on preserving the individual characteristics of the plant
The production of recombinant native and engineered (Ziemienowicz, 2013). The most important plants that have
proteins is a complicated procedure that requires an inter- been successfully subjected to gene transformation
and multi-disciplinary effort involving a wide variety of programs are industrial plants, cereal crops, legumes, root
scientific and technological disciplines, ranging from basic plants, vegetables, turf grasses, tropic plants, woody
biotechnology, biochemistry, and cell biology to advanced species, medicinal and ornamental plants, as well as fruit
production systems. This review considers important plant plants (Wang, 2006a; Wang, 2006b). These plants and
resources, affecting factors, and the recombinant-protein their related species have been presented in Table 1. In
expression techniques relevant to the plant molecular gene transformation processes, the gene(s) of interest of
farming process. donor plants, bacteria or viruses are transferred to host

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Transgenic Plants Abiri et al.

Table 1. Examples of genetically transformed plant species using various transformation methods.
Model Plants References Model Plants References
Plants Plants
Root Carrot (Daucus (Zhang and Huang, Ornamental Carnation (Dianthus caryophylus) (Burana et al., 2014)
plants carota) 2010) plants
Cassava (Manihot (Xu et al., 2012) Orchids (Cymbidium spp., (Shu-Hong et al.,
esclenta) Oncidium, Phalaenopsis) 2015)
Potato (Solanum (Park and Cheong., Rose (Rosa hybrida) (Condliffe et al.,
tuberosum) 2002) 2003)
Sweet potato (Goo et al., 2015) Petunia (Petunia hybrida) (Kamenarova et al.,
(Ipomoea batatas) 2005)
Turf Perennial ryegrass (Fischer et al., 2012) Industrial Sunflower (Helianthus annuus) (Sankararao and
grasses (Lolium perenne) plants Rohini., 1999)
Bermudagrass (Fischer et al., 2012) Indian Mustard (Brassica juncea) (Fischer et al., 2012)
(Cynodon spp.)
Switchgrass (Wang, 2006a) Canola (Brasicca napus) (Wang, 2006a)
(Panicum virgatum)
Tropic Banana (Musa spp.) (Wang, 2006a) Cotton (Gossypium hirsutum) (Ashraf et al., 2008)
plants
Pineapple (Ananas (Wang, 2006a) Legume Alfalfa (Medicago sativa) (Wang, 2006a)
comosus) plants
Sugarcane (Ziemienowicz, 2013) Beans (Phaseolus spp.) (Wang, 2006a)
(Saccharum spp.)
Citrus spp., coffee (Ziemienowicz, 2013) Soybean (Glycine max) (Ziemienowicz, 2013)
(Coffea spp.)
Papaya (Carica (Ziemienowicz, 2013) Pigeonpea (Cajanus cajan) (Ziemienowicz, 2013)
papaya)
Woody Eucalyptus, Pine (Ziemienowicz, 2013) Peanut (Arachis hypogaea) (Wang, 2006a)
species (Pinus radiate)
Cork oak (Quercus (Dunwell, 2009) Peas (Pisum sativum) (Ziemienowicz, 2013)
suber)
Poplar (Populus (Wang, 2006b)
spp.)
Rubber tree (Hevea (Wang, 2006b) Chickpea (Cicer arietinum) (Ziemienowicz, 2013)
brasiliensis)
Medicinal Ginseng (Panax (Wang et al., 2005) Cereal crop Rice (Oryza sativa) (Dunwell, 2009)
plants ginseng
Hemp (Cannabis (Wang et al., 2005) Maize (Zea mays) (Ziemienowicz, 2013)
sativa)
Nuts and Blueberry (Dunwell, 2009) Rye (Secale cereal) (Dunwell, 2009)
fruits (Vaccinium
corymbosum)
Walnut (Juglans (Fischer et al., 2012) Sorghum (Sorghum bicolor) (Dunwell, 2009)
spp.)
Strawberry (Dunwell, 2009) Wheat (Triticum aestivum) (Dunwell, 2009)
(Fragaria x
ananassa)
Apple (Malus x (Ziemienowicz, 2013) Barley (Hordeum vulgare) (Ziemienowicz, 2013)
domestica)
Vegetable Tomato (Wang, 2006a) Model plants Arabidopsis (Arabidopsis (Wang et al., 2005)
plants (Lycopersicum thaliana)
esculentum)
Lettuce (Lectuca (Ziemienowicz, 2013) Tobacco (Nicotiana tobaccum, N. (Fischer et al., 2012)
sativa) benthamiana)

34

plants using various methods, such as Agrobacterium improve their nutrient uptake (Sahebi et al., 2014), to
mediation, particle bombardment (biolistics), the reduce the effects of harmful agrochemicals and increase
microprojectile gun method, electroporation, a silicon- crop yields (Ziemienowicz, 2013), to allow phosphorus
carbide fibre-based technique, polyethylene glycol (PEG)- absorption and reduce the use of fertilizer (Hirsch and
mediated protoplast fusion, and liposome-mediated gene Sussman, 1999), to enhance grain production and the
transfer, allowing the investigation of their effects (Rivera et growth rate under the condition of low-iron availability
al., 2012). (Takahashi et al., 2001) and to increase the aluminium
To date, transgenic plants have been engineered for tolerance of papaya and tobacco (De la Fuente et al.,
the following purposes: to increase their tolerance of abiotic 1997). Some of the most significant genes transformed in
and biotic stresses (Lau et al., 2014; Wang et al., 2005), to the four plants of tobacco, rice, potato and Arabidopsis

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Transgenic Plants Abiri et al.

have been listed in Figure 1. By introducing Bt (Bacillus molecular farming (PMF) researchers do not focus on
thuringiensis) toxin genes into rice, tomato, maize, tobacco, conventional products, such as food, feed or fibre
potato, cotton and other crop species, their insect applications, but on producing industrial and
resistance has been significantly increased (Singh et al., pharmaceutical products (Sourrouille et al., 2009; Torrent et
2010). al., 2009). The first plant-based pharmaceutical protein was
There is evidence showing that the first application of produced by Barta et al., 1986 during their investigations of
herbs for medicinal purposes occurred during the period producing human growth hormone in tobacco (Nicotiana
when Neanderthals lived (approximately 130,000 years tabacum). Since then, several other plant species, such as
ago), whereas more reliable evidence indicated that plant banana and potato, have been tested for the production of
products have been used for therapeutic purposes as early medicinal proteins (Schillberg et al., 2003). Although the
as when the major human civilizations began, when the notion of producing human proteins and antibodies in
Egyptians collected at least 700 different medicinal plants plants was initially met with great scepticism, nevertheless,
in approximately 1600 BC. Consistent with this trend, the crops offer a unique combination of advantages as well as
Romans used willow bark (Salix alba L.) to treat fevers interesting features compared with those of the traditional
(Ahmad, 2014; Kleiner, 1995). The functions of the production systems. Generally, during the last several
ingredients of medicinal plants have been recently decades, plants have been widely investigated as
identified, allowing their use as prescription drugs (Mewett unconventional systems for the production of
et al., 2007). Modern biotechnology is expanding the pharmaceutical proteins.
application of herbs in medicine well beyond the usual
borders. Because plants can be the sources of medicinal Plant molecular farming compared with conventional
proteins, including a blood-component substitute bioproduction systems
(Magnuson et al., 1998), mammalian antibodies (Fischer et Conventional molecular farming began in 1920 with the
al., 1999), and vaccine subunits (Walmsley and Arntzen, extraction of insulin from animal tissues by Fredrick
2000), plant molecular farming can play a constructive role Banting, as described by Dynkevich et al., 2013, but the
in the development of suitable transgenic plants. Using this drawbacks of this approach most likely led to the
technology, scientists have succeeded in introducing genes establishment new molecular farming methods through
into host plants that induced them to produce considering novel sources. New methods arose according
pharmaceuticals (Torrent et al., 2009). In contrast to those to the molecular sources (e.g., plant cells, transgenic plant
who focus on other aspects of transgenic plants, plant cells, virus-infected plants, animal cells, transgenic animals

Figure 1. Groups various genes were transformed to model plants.

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Transgenic Plants Abiri et al.

and microbial cells). Accordingly, plant molecular farming is suitable for molecular farming than are dry-tissue plants,
one of the outcomes of this process, and the major source such as cereals. This phenomenon could be related to the
in this field is the transgenic plant cell (da Cunha et al., ease of extraction of squashy tissues (Horn et al., 2004).
2014). Despite all of the advantages of using higher plants, there
Compared with other transgenic products obtained are limits to the products than can be obtained using plant
from transgenic bacteria, fungi, and animals, which are the molecular farming, such as the unknown mechanisms that
most common models for recombinant-protein production, cause certain post-translational disorders in plant cells. The
plant-based therapeutics are produced with the lowest challenging issue of how to fine-tune the systems that are
cost, leading to an economic justification for their use essential for the preservation of the structural integrity of
(Häkkinen et al., 2014). The availability of personnel with the nascent recombinant proteins and their activities in
experience and expertise in planting, harvesting, and their new cellular environments are still debated within the
processing plant material are some of the other benefits of field of plant molecular farming (Walsh and Jefferis, 2006).
recombinant-protein production using molecular farming of The advantages and disadvantages of plant molecular
higher plants. In addition, the dissimilarity of the pathogenic farming compared with using other molecular farming
factors of plants and humans decreases the risk of systems are presented in Figure 2.
microbial interactions that could negatively affect the quality
of the final products. Technically, the stability of Factors affecting the expression of recombinant
recombinant proteins within plants stressed by proteins in different hosts
environmental factors is greater than that of recombinant According to the classification of final plant molecular
proteins produced in other hosts. Furthermore, higher farming products by Horn et al. (2004), proteins are
plants typically produce recombinant proteins with the clustered as parental pharmaceuticals, therapeutical
correct folding, activity and glycosylation (Schillberg et al., intermediates, monoclonal antibodies (MAbs), industrial
2005; Yano et al., 2010). Another beneficial characteristic proteins, and antigens for use as edible vaccines (Figure
of these systems is that recombinant plants can be stored 3). As green bioreactors, plants have various advantages,
at room temperature. In contrast, the storage temperature such as the capacity to accomplish eukaryotic post-
for plant viruses, bacteria and yeasts is -20 ºC. The storage translational modifications that are necessary for biological
condition for cultured mammalian cells is even more activities, such as disulphide bridging and glycosylation, in
stringent because they must be maintained in liquid many different mammalian proteins (Ma et al., 2003). The
nitrogen (Faye et al., 2005). Among plant species, plants genetic transformation procedures employed are governed
with watery tissues, such as tomato plants, are more by various vital factors, such as the plant genotype, the

Figure 2. Comparison between various hosts in molecular farming.

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Transgenic Plants Abiri et al.

type of explant, the plasmid vector, the agrobacterial strain, Types of PGRs and plant regeneration
and the composition of the culture medium (Kavitah et al., The complicated procedure of plant tissue culture
2010; Sood et al., 2011). These factors play important roles prompted the development of somatic embryogenesis (SE)
in the transformation process that depends upon the plant methods, in which PGRs play an essential role. Two
species involved. Therefore, efficient plant molecular mainstream PGRs that have been used for plant tissue
farming researchers tend to develop new transformation culturing are auxins and cytokinins. Members of the sub-
methods and novel construct designs, to incorporate well- groups of each family are involved in different metabolic
defined transgenes and to search for more effective pathways that affect plant physiology. For examples, auxins
methods of introducing multiple genes into plants (Bregitzer cause the production of undifferentiated cells called callus
and Brown, 2013; Karimi et al., 2013). cells. Apparently, PGRs induced methylation of DNA,
whereas cytokinins affect root formation in tissue cultures.
Types of explants The auxins generally used for the induction of different
As mentioned above, the type of explant involved is one of callus tissues are 2,4-D (2,4-Dichlorophenoxyacetic acid),
the most vital factors for genetic transformation. For IAA (Indole-3-acetic acid), NAA (1-Naphthaleneacetic acid),
instance, the use of different sources of plant cells or Dicamba, Picloram and 2,4,5-T (2,4,5-trichlorophenoxy-
tissues (e.g., seed, root, shoot, leaf, and shoot and root acetic acid) (Yokoya et al., 2014). In contrast, cytokinins
apical meristems) leads to variable results for callus are the best candidates for inducing the production of roots
induction, plant regeneration, and the efficacy of genetic and of plant regeneration via calli. The main function of
transformation. In this regard, to produce a mass of calli, cytokinins appears to involve the synthesis of proteins
numerous protocols have been established for different necessary for the formation and functionality of the mitotic
plant-tissue culturing systems. Although these protocols spindle apparatus (George et al., 2008). BAP (6-
are well-established, difficulties in callus induction in some Benzylaminopurine), Kinetin, Zeatin and TDZ (Thidiazuron)
plants, such as Indica rice varieties, are still experienced are the most important cytokinins that display regenerative
(Ikeuchi et al., 2013). activity in cultured plant tissues (Alam et al., 2010). Despite
the undeniable effects of PGRs on cultured plant tissues,
other essential factors, such as sugars, vitamins, salt
concentrations, the type of medium and certain chemicals,

Figure 3. Examples of plant molecular farming products in different types of plants.

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Transgenic Plants Abiri et al.

have been found to be a key factors in somatic embryo- nuts and fruits, such as apple (Malus × domestica),
genesis (Enríquez-Obregón et al., 1999; Kavitah et al., American chestnut (Castanea dentata), strawberry
2010; Murashige and Skoog, 1962). ( F r a g a r i a × a n a n a s s a ) , b l u e b e r r y ( Va c c i n i u m
Achieving the effective gene transformation systems in corymbosum), walnut (Juglans spp.) and grapevine (Vitis
molecular farming largely depends on the efficiency of vinifera); and final, medicinal plants, such as hemp
tissue culture methods. These methods are mainly affected (Cannabis sativa), ginseng (Panax ginseng) and the opium
by the plant source, such as calli, root and shoot. In several poppy (Papaver somniferum) (Dunwell, 2009;
plants, the lack of effective regeneration system is the most Ziemienowicz, 2013).
important limiting factor preventing the successful gene The major concerns that have arisen regarding
transfer technologies. transgenic plants include the low efficacy of plant
regeneration during tissue culture, as well as production
Plant vectors bottlenecks, such as in the spatial and temporal aspects of
Scientists have many devices that allow them to achieve transgenic expression, target production, and the high-level
their goals of producing transgenic plants. In addition to yield of recombinant products (Stoger et al., 2002a;
various methods, such as the gene gun and biolistic Streatfield, 2007). Considering that Agrobacterium is a
techniques, natural bacterial vectors of plants, such as pathogen of dicotyledonous species, the efficacy of the
Agrobacterium tumefaciens, have been introduced as Agrobacterium-based genetic transformation of
efficient tools, particularly in the case of cereals. monocotyledonous plants is still limited due to the low
Agrobacterium tumefaciens has the ability to transfer a integration rate. The above-mentioned difficulties prompted
portion of its DNA or so-called transferred DNA (T-DNA) to researchers to create new approaches to increase
the genome of a host plant (Sheng and Citovsky, 1996). transformation efficiency. An Agrobacterium T-DNA derived
Agrobacterium-mediated gene transfer to plant cells nano-complex has been introduced as a promising method
includes five essential steps, as follows: a) induction of the f o r i n c r e a s i n g t h e t r a n s f o r m a t i o n e ff i c i e n c y o f
bacterial virulence system, b) generation of a T-DNA monocotyledonous plants (Ziemienowicz et al., 2012). This
complex, c) transfer of the T-DNA from Agrobacterium to nano-complex was first transferred into triticale (Triticum ×
the nucleus of the host cell, d) integration of the T-DNA into Secale) microspores. The most important nano-complexes
the plant genome, and e) the expression of T-DNA genes involve Agrobacterium T-DNA, virulence protein VirD2 and
(Gelvin, 2012; Ziemienowicz et al., 2012). To date, single stranded DNA- and double stranded DNA-binding
Agrobacterium-based transformation methods have proven RecA protein (Chugh et al., 2010).
to useful approaches to genetically modify plants of various
levels, including model plants such as the barrel clover Promoters
(Medicago truncatula) Arabidopsis (Arabidopsis thaliana) As the upstream elements of genes, promoters play an
and tobacco (Nicotiana tobaccum, N. benthamiana); eminent role in gene expression. Promoters contain sites
cereals, such as maize (Zea mays), rye (Secale cereale), with core sequences to which RNA polymerase and
barley (Hordeum vulgare), wheat (Triticum aestivum), rice transcription factors bind. Although constitutive promoters,
(Oryza sativa) and sorghum (Sorghum bicolor); legume such as the cauliflower mosaic virus 35S (CaMV35S) and
plants, such as chickpea (Cicer arietinum); bean the maize ubiquitin-1promoter, have been utilized in dicots
(Phaseolus spp.); pea (Pisum sativum); peanut (Arachis and monocots, respectively, tissue-specific and inducible
hypogaea), pigeon pea (Cajanus cajan); alfalfa (Medicago promoters provide better control of gene expression (Ko et
sativa); soybean (Glycine max); clover (Trifolium spp.); al., 2005; Nguyen et al., 2004). Inducible promoters can be
industrial plants, such as cotton (Gossypium hirsutum), prompted to induce gene expression within selected
canola (Brasicca napus), sunflower (Helianthus annuus), spaces and at chosen times through the addition of the
Indian mustard (Brassica juncea), Camelina sativa and appropriate chemicals. Reportedly, applying these specific
Brassica oleracea; vegetable, such as tomato chemicals can inhibit the occurrence of the undesirable
(Lycopersicum esculentum), eggplant (Solanum effects of gene expression on different stages of plant
melongena), cucumber (Cucumis sativus), lettuce (Lectuca growth and development (Stöger et al., 2000). Another
sativa), root plants; cassava (Manihot esclenta), carrot great advancement in the field of plant genetic
(Daucus carota), sweet potato (Ipomoea batatas) and transformation was the identification of tissue-specific
potato (Solanum tuberosum); ornamental plants, such as promoters. These promoters control gene expression by
carnation (Dianthus caryophylus), orchids (Cymbidium restricting it to specific tissues, which decreases the extent
spp., Oncidium, Phalaenopsis), chrysanthemum of the diverse effects of transformation on the growth and
(Dendranthema × glandiflora hybrid), rose (Rosa hybrida) development on the host plant (Stahl et al., 2002).
and petunia (Petunia hybrida); tropical plants, such as However, the efficacy of gene expression in specific tissues
Citrus spp., banana (Musa spp.), sugarcane (Saccharum depends on the availability of a promoter that induces the
spp.), coffee (Coffea spp.), pineapple (Ananas comosus), production of a sufficient level of protein (Ko et al., 2005;
papaya (Carica papaya); grasses, such as turf grasses, Ko et al., 2003). The level of gene expression obtained
perennial ryegrass (Lolium perenne), bentgrasses (Argostis using a tissue-specific promoter is not always adequate for
spp.), bermudagrass (Cynodon spp.), tall fescue (Festuca a molecular farming process designed to generate
arundinacea), switchgrass (Panicum virgatum); woody biotherapeutic proteins. This results could be due to the
species, such as cork oak (Quercus suber), American elm limited number of existing promoters, their activity during
(Ulmus americana), poplar (Populus spp.), eucalyptus, the regeneration process, and the unpredictability and
rubber tree (Hevea brasiliensis) and pine (Pinus radiate); complexity of the effect of environmental factors, all of

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Transgenic Plants Abiri et al.

which can cause unwanted and unexpected pleiotropic living organisms by increasing the translational efficacy of
effects (Du et al., 2003). Indeed, expressing the series of the transgene (Mechold et al., 2005).
genes necessary for the production of a full-size antibody Additionally, extensive modification of the coding
requires the application of specific promoters that control region and of certain molecular factors, such as
the activity of each of these genes. In addition to the accommodating codon bias, altering the GC content,
problems regarding plant protein expression mentioned eliminating cryptic splicing sites, putative polyadenylation
above, the use of adverse promoters may cause signals and mRNA-instability sequences and incorporating
homology-based gene silencing (De Neve et al., 1999; Ko 5′ and 3′ regulatory sequences, are required to achieve the
et al., 2005). Applying both a constitutive promoter and an high-level expression of foreign genes in plants (Streatfield
inducible promoter is a feasible solution to the and Howard, 2003).
aforementioned problems. For example, the combination of Many candidate plant species for the production of
the 35S promoter and the potato proteinase inhibitor II recombinant proteins have been evaluated and developed
(pin2) promoter has been used to control the expression of during the past 25 years based on their particular
the heavy- and light-chain antibody genes, respectively (Ko advantages and disadvantages. These candidates from the
et al., 2003). plant Kingdom includes those that can be applied in cell
cultures (carrot, tobacco, and rice), leafy crops (alfalfa,
Composition of RNA and DNA tobacco, and lettuce), aquatic plants (Lemna minor) and
The process for the production of a recombinant protein in seed crops (canola, soybeans, corn, and rice) (Franconi et
transgenic plants is similar to that of normal plants, in al., 2010; Karg and Kallio, 2009).
which DNA is transcribed to produce mRNA and then,
through the harmonized activities of mRNA (messenger Tobacco
RNA), tRNA (transfer RNAs) and rRNA (ribosomal One of the most suitable platforms typically used as a
ribonucleic acid) mRNA is translated to yield a recombinant green producer is tobacco (Nicotiana tabacum). Despite
protein (Gebauer and Hentze, 2004). The untranslated the application of tobacco in smoking, it has many unique
regions of mRNA (the 5′ and 3′ untranslated regions) have advantages over other plant species for the production of
direct effects on the transcriptional and translational pharmaceutically relevant proteins. Scientists believe that
processes via the activity of regulator- protein complexes the role of tobacco in recombinant-protein investigations is
(Amrolia et al., 1995; Costanzo and Fox, 1995). The core similar to that of the white mouse in mammalian studies
sequence of an untranslated region (UTR) of a gene may over the last 20 years. As a leafy species, tobacco has
function as a gene-expression repressor when it binds numerous advantages over other plants, which have
translation–initiation factors. As a consequence of this encouraged researchers to focus on this plant as an
activity, the translation of the gene product may be down- undeniable alternative for recombinant protein expression.
regulated (Curie and McCormick, 1997). A motif within the This herb is capable of producing a biomass of up to 100
5′ UTR sequence of a gene can cause the specific ton/ha. Moreover, a well-established system for
induction of the transcription of its mRNA (Dickey et al., transforming tobacco that results in a high level of soluble
1992). In parallel, the 5′-untranslated region of tobacco protein exists. The potential of utilizing various strategies
mosaic virus (Mitsuhara et al., 1996) and that of alfalfa for the expression of proteins in a stable or transient
mosaic virus (AMV) RNA3 have been shown to enhance manner using this species, as well the possibility of using
the translation efficiency of a β-glucuronidase (GUS) chloroplast genome-based methods mediated via
transgene and stabilize its mRNA (Datla et al., 1993), Agrobacterium or viral induction are its other advantages
which was also reported for the luciferase (Gallie and (Figure 2) (Karg and Kallio, 2009). Nevertheless, the high
Kobayashi, 1994) and β-phaseolin (Oliver et al., 1993) concentrations of alkaloids and nicotine in some tobacco
genes. The cytoplasmic polyadenylation element (CPE) varieties are disadvantageous to utilizing this plant for
motif at the 3′ end of transcripts reportedly exhibited a molecular farming. These drawbacks have been
regulatory function in mRNA translational events. In this compensated for by breeding new cultivars such as
regard, the above-described motif can stimulate protein “81V” (Ma et al., 2003). The instability of the products of
translation by hastening the occurrence of polyadenylation tobacco is one of the disadvantages of this plant in
(Kervestin and Amrani, 2004). Additionally, transgenic molecular farming. Alternatively, its products can be
plants containing the untranslated leader sequence of AMV immediately processed on farms; alternatively, the leaves
had higher levels of attacin and a recombinant lytic protein can be dried or frozen before transport to processing plants
than did the plants that did not contain this sequence (Ko et (Kamenarova et al., 2005). Tobacco is not a feed or food
al., 2000). crop, which decreases the probability of its contamination
Thus, modifying the structural stability of mRNA and within the feed or food chains. In addition, tobacco can
controlling RNA polyadenylation via 5′ and 3′ untranslated produce a wide variety of therapeutic immune-modulatory
sequences of mRNAs, respectively, are useful molecules, such as cytokines, vaccines and antibodies
translational-based approaches to increasing the (Tremblay et al., 2010). A full-length monoclonal antibody
production of recombinant proteins in transgenic plants. (mAb) directed against a mouse catalytic IgG1(6D4) was
Differences in the codon usage of the transgene and the the first antibody produced in tobacco plants (Hiatt and
expression host will also affect the level of recombinant- Pauly, 2006). Other antibodies that were generated using
protein expression. Codon optimization has been used to tobacco plants include anti-PA (protective antigen) mAb,
improve the level of recombinant-protein expression in anti-BoNT/A scFv (botulinum neurotoxin/antibody single-
chain variable fragment) idiotype specific antibodies, anti-

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Transgenic Plants Abiri et al.

Lewis Y mAb, H10 mAb, CO17-1A mAb, TheraCIM®, B294 against several human diseases, including Type-1 diabetes
mAB, B303 mAb, 2F5 mAb, R12 mAb, CaroRX™, and anti- mellitus (T1DM), transgenic tobacco can be used as a
LPS scFv, as well as antibodies targeted against different reliable system for the generation of high levels of human
disease entities, such as anthrax (Hull et al., 2005), IL-13 (Thompson and Debinski, 1999; Wang et al., 2008).
botulism (Almquist et al., 2006), cancer (B-cell lymphoma) Transplastomic plants are novel alternatives to nuclear
(Young et al., 2009), (breast and colon) (Brodzik et al., transgenic plants, which are created by introducing the
2006), (broad spectrum) (Svanes et al., 2010), (colon) (Ko recombinant DNA into the genome of chloroplasts rather
et al., 2005), (skin) (Rodriguez-Oroz et al., 2005), as well than the nuclear genome using gene-gun bombardment.
as hepatitis (Gleba et al., 2005), HIV (Human Some of the most important accessible products that have
immunodeficiency virus) (Ward et al., 2007), rabies (Girard been produced in tobacco chloroplasts are a tetanus-toxin
et al., 2006), S. mutans colonization (Ma et al., 1998), and fragment, serum albumin, and human growth hormone.
Salmonella (Makvandi-Nejad et al., 2005). Vaccines are However, plastids do not have the capacity to perform
antigenic materials that are administered to produce glycosylation; thus, chloroplast cannot be used to produce
immunity to diseases. To date, various types of these human glycoproteins (Ma et al., 2003).
vaccines have been generated in tobacco and some of
them have been tested in mammals. The cysteine protease The expression of tobacco systems
Der p 1 (Dermatophagoides pteronyssinus allergen) The availability of various tobacco expression systems with
(Johnston et al., 2009; Lienard et al., 2007), Der p 2 their particular strengths is another advantage of this model
(Lienard et al., 2007), protective antigen (Koya et al., plant over other plant species. Nuclear transformation is
2005), L1 major capsid protein (Lenzi et al., 2008), CTB the appropriate method to achieve the long-term production
(cholera toxin B) (Jani et al., 2004), multi-epitope vaccine of glycoproteins, such as antibodies. Alternatively,
(Soria-Guerra et al., 2009), VCA (viral capsid antigen) (Lee chloroplast-based expression systems allow the production
et al., 2006), VP1 (viral protein-1) (Wu et al., 2007), VP21 of large amounts of proteins that require certain types of
(Huang et al., 2009), HSP-A (heat shock protein) (Brodzik post-translational processing. Tobacco is amenable to
et al., 2006), UreB (urease subunit beta) (Gu and Glatz, several different simultaneous applications and has the
2007), HBsAg (Hepatitis B antigen) (Thanavala et al., potential to excel at each of them. When a product that
1995), Hep C (Hepatitis C) core protein (Nanou and requires rapid modification, as in the case of the above-
Azzouz, 2009), HIV p24 capsid protein (Zhang et al., mentioned idiotypic anti-cancer vaccine, transient
2002), HIV-Nef (human immunodeficiency virus- Negative expression can be anticipated to yield the proper quantities
regulatory factor) (Marusic et al., 2007), F1-V, SARS-CoV- of the desired protein within a short period (Tremblay et al.,
S1protein (severe acute respiratory syndrome coronavirus) 2010). Using transient protein-expression systems,
(Pogrebnyak et al., 2005), Tet-C (tetanus toxin Fragment C) researchers can produce considerable amounts of
(Tregoning et al., 2005), GAD65 (Mr 65,000 isoform of recombinant protein during a short period, which is crucial
glutamic acid decarboxylase) (Ma et al., 2004), GAD67 for the rapid response to a disease outbreak, as is the case
(glutamic acid decarboxylase) (Ma et al., 1997), HSP-60 when a new influenza appears, e.g., during the A/H1N1
(Heat shock protein 60) (Tremblay et al., 2010), insulin pandemic, as well as for patient-specific cancer treatments.
(Lee et al., 2006) and GLP-1 (Glucagon-like peptide-1)
(Brandsma et al., 2009) are some of the proteins that have Nuclear transformation
been expressed in tobacco, which target diseases/disease Stable nuclear transformation involves the incorporation of
entities such as allergy-dust mites, anthrax, cancer an exogenous gene into the nuclear genome of a plant,
(cervical), cholera, DPT (diphtheria), Epstein–Barr virus, which leads to the expression of the new heritable traits by
foot and mouth disease, Helicobacter pylori, hepatitis B/C, the next generation of transgenic plants. This
HIV, plague, SARS, tetanus, Type 1 diabetes and Type 2 transformation method is commonly utilized for the
diabetes, respectively. production and accumulation of a recombinant protein in
Cytokines, some of which are glycoproteins, are certain explants, such as the dry seeds of cereals (Horn et
members of a family of strong immunoregulators that are al., 2004).
produced by different types of cells. The function of these Agrobacterial infection and biolistic delivery are the two
small proteins is regulating the period and intensity of the mainstream methods used to transform an exogenous
immune response by inhibiting or stimulating the activation, gene into the nuclear genome of plants, including tobacco.
proliferation, and differentiation of a variety of cells. This Agrobacterium transfers genes to dicots with great efficacy
procedure is accomplished through regulating the secretion and the Agrobacterial-infection method has been improved
of Abs or other cytokines by target cells (Parkin and Cohen, to increase the efficacy of the genetic transformation of
2001). Cytokines bind to cytokine receptors expressed on certain monocotyledonous plants. The best example of the
the membrane of the responsive target cells (Thomson and success of the latter involves rice (Chan et al., 1993; Chen,
Lotze, 2003). Despite the availability of various commercial 2008; Hiei et al., 1997).
recombinant cytokinins, considering the limitations of some Furthermore, decreased costs and simplification of the
conventional bioproduction systems, such as low levels of production process are the results of gene delivery leading
expression and the lack of glycosylation of E. coli-derived to stable genetic integration, which frequently leads to the
recombinant cytokines, scientists are interested in production of recombinant proteins with the least external
producing such recombinant proteins using tobacco. Due input. The exogenous proteins produced due to nuclear
to the low efficiency of E. coli production of a pleiotropic transformation can be directed to various secretory
regulatory cytokine, such as human IL-13,which protects

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Transgenic Plants Abiri et al.

organelles or other organelles for standard eukaryotic post- accidentally spreading the infection to wild species. In
translational modifications. addition, this method is limited to controlled conditions,
Although nuclear transformation systems are such as those of a greenhouse or laboratory. Although, the
commonly employed, the low level of production of the latter drawback is less of a concern because the controlled
resultant recombinant protein remains a matter of concern. environments eliminate the risks of cross-fertilization that
Among the many strategies to address this issue, the most could potentially occur when transgenic plants are grown in
important are the use of 5′ enhancer sequences to increase open fields. The agro-infiltration method that was
translational productivity, the use of promoters with strong developed by (Dillen et al., 1997) is another technique for
tissue-specific constitutive activity, the use of a sub-cellular transient expression. This method involves the infiltration of
localization signal (Benchabane et al., 2008), the use of 3′ a suspension of recombinant Agrobacterium tumefaciens
untranslated regions that were modified to increase into tobacco leaf tissue, which in turn facilitates the transfer
transcript stability and the optimization of the coding of T-DNA to a very high percentage of the cells. The
sequence of the transgene using the typical tobacco method also positively affects the intensity of transient
codons (Streatfield, 2007; Streatfield and Howard, 2003), expression in transgenic crops. A method allowing transient
the addition of fusion partners that increase protein expression in tobacco has been specifically developed as a
stability/proteolysis resistance (Floss et al., 2007), and very rapid and high-yield strategy for the production of
targeting proteins to the endoplasmic reticulum (ER) via the clinical-grade bio-pharmaceuticals (Pogue et al., 2010;
attachment of a C-terminal (K/H) DEL signal (Denecke et Vézina et al., 2009).
al., 1992). The most important factors that directly affect
the performance of stable transformation are the variety Plastid transformation
and the physiology of the plant. Plastid transformation is an efficient alternative to nuclear
transformation because has several advantages that latter
Transient transformation method lacks. For example, despite the enormous
Transient transformation is the proper method to achieve importance of the delivery of a normal bio-containment of
the rapid production of vital proteins in tobacco. In contrast transgene flow by out-crossing, the transgene cannot be
to the case of stable genomic integration, a large amount of transferred due to the lack of chloroplasts in pollen, thereby
recombinant protein can be produced in less than five days allaying public concerns regarding genetically modified
post-infection using the transient transformation method. plants (Meyer et al., 2010; Pantaleoni et al., 2014).
Nevertheless, nuclear and transient transformation Transgenic plants subjected to homoplastomic chloroplast
methods have similar advantages. For the production of transformation are selected after several generations of
pharmaceutical materials using viral coding sequences via plants have been regenerated from the gene-gun
Agrobacterium tumefaciens, transient expression is the bombarded leaf explants, meaning that the plant
proper method because it offers a high level of expression chloroplast genome has had opportunity to incorporate the
with the lowest input. Different strategies have been transgene. The selection of the above-mentioned
developed to increase the efficiency of transient bombarded leaf explants is conducted on a medium
expression. One of the successes achieved by scientists containing a either spectinomycin or streptomycin.
as a promising approach to directly transforming cells with Researchers have achieved a noticeable yield of
a virus using Agrobacterium tumefaciens has decreased therapeutic human and bacterial proteins, ranging from
the energy required for genetically transforming tobacco 3-6% of the total soluble proteins, using the tobacco
(Gleba et al., 2005). The principle of this method to transfer chloroplast-transformation technique (Reddy et al., 2002;
specific components of the viral expression platform by Oey et al., 2009) achieved very high-level expression of a
mixing a dissimilar Agrobacterium strain harbouring proteinaceous antibiotic, which comprised approximately
portions of the viral machinery, with recombination 70% of the soluble proteins, through employing a
occurring intracellularly once infection has occurred. By chloroplast transformation system, which is the most
manipulating the viral codons and applying classic significant yield of recombinant protein attained using
eukaryotic introns, the efficiency of gene delivery has been transgenic plants to date. Although plastid transformation
significantly increased. Thus, the required amount of the has an enormous potential, its application remains
essential infectious Agrobacterium was reduced. For restricted. In that regard, even though plastid
example, 1 litre of an overnight culture could be used to transformation has been attained in plant species, such as
infect nearly 1000 kg of tobacco leaf tissue, yielding up to 4 lettuce, eggplant, soybeans and tomatoes (Bock, 2007),
kg of recombinant protein at 40% TSP (Marillonnet et al., the plant most commonly modified using a chloroplast
2005). This example demonstrates represents the scale transformation system is tobacco, which is highly regulated
that are presently being explored to maximize protein yield and is inedible due to its high level of toxic alkaloids.
while reducing the input costs, resulting in an estimated $1/ Finally, whether the protein stability will change over time,
kg of raw protein or $50/kg of purified protein. Another even with refrigeration, is a matter of concern (Horn et al.,
alternative is to insert the viral machinery required to 2004).
prevent tobacco-gene silencing via stable nuclear
transformation. Applying this method will decrease the Plant-cell suspension cultures
delivery requirements of the deconstructed viral vectors One of the best plant-based alternatives to mammalian
(Azhakanandam et al., 2007). In general, transient cells for the production of biopharmaceuticals is a plant-cell
expression has two main problems, including the high suspension, which is a robust system involving a simple
technical requirements for induction and high risk of purification procedure and easy downstream processing

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Transgenic Plants Abiri et al.

(Kim et al., 2008). Although this system requires a high organelles are preferable to systems involving the flowering
level of sterility to control contamination, pharmaceuticals cycle (Twyman et al., 2003).
with a high level of purity can be produced using it Cereals have particular characteristics that have
(Franconi et al., 2010). Additionally, in contrast to plant-cell encouraged scientists to use members of this family as
culture systems, plant-cell suspension systems eliminate model plants in molecular farming. Cereal endosperms are
the regeneration process, and accordingly, is a rapid the most important components for researchers due to their
procedure (Shaaltiel et al., 2007). Despite having soluble-protein content. Soluble proteins can be recognized
numerous advantages over other systems, plant-cell and separated easily in cereals by displaying antibody role
suspension systems have been established only for a small in molecular farming (Han et al., 2006). Among the cereals,
number of plants, such as tobacco, carrot, Arabidopsis and wheat is not as an appropriate model plant because of the
rice. Moreover, due to the certain limiting factors, such as low efficiency of its transformation (Stevens et al., 2000).
increasing proteolytic activity, which leads to a low Some of the recombinant proteins that have been extracted
concentration of the recombinant protein during the late from seed bioreactors are the most efficient vaccine
stationary phase, this system is not the best method of antigens (Wu et al., 2007), cell-culture proteins (Nandi et
protein expression (Obembe et al., 2011). al., 2002), industrial enzymes (Hood et al., 2007),
therapeutic antibodies (Stöger et al., 2000) and cytokines
Arabidopsis (Zhu et al., 1994). Among the seed-based bioreactors,
Plants with appropriate traits have been discovered in maize is the major viable plant that produces recombinant
genetic-engineering investigations. Of all the known plants proteins in large amounts. Some of the remarkable
in the Kingdom, Arabidopsis has been the main plant used features regarding this model plant compared with those of
for genetic transformation. Among the traits of Arabidopsis, other plant systems used in molecular farming are the
the short generation period, small genome size, presence existence of well-established techniques for its tissue
of a self-pollination mating system, ease of in vitro culture and transformation, its production of high levels of
culturing, easy regeneration and in vivo transformation, as biomass and the ease of scaling up its use. Moreover, this
well as its lack of food and feed applications, are plant has the ability to produce recombinant antibodies as
interesting characteristics that have led scientists to exploit well as protease inhibitors and enzymes for
this model plant (Koornneef and Meinke, 2010). Due to its pharmaceutical/technical applications, such as aprotinin,
prolific seed production and rapid propagation rate, laccase, and trypsin (Hood, 2002).
Arabidopsis appears to be capable of generating a range of One of the initial plant molecular-farming investigations
various products. Like other seed-producing plant was conducted in 2003 by Xue, who used barley as a
bioreactors, for the production of recombinant proteins, bioreactor to produce a highly active and thermo-tolerant
Arabidopsis should be transformed during the early stages hybrid cellulase (1,4-ß-glucanase) (Xue, 2003). Other types
of growth, because its development toward the flowering of recombinant proteins and materials have been produced
stage may increase the risk of environmental in barley, of which serum albumin, lactoferrin, lysozyme,
contamination (Ruebelt et al., 2006). High levels of α1-antitrypsin and human antithrombin III are the most
recombinant protein accumulation in Arabidopsis seeds important examples (Stahl et al., 2002). Using rice as the
have been achieved using a seed-specific expression host plant, Anzai and colleagues have successfully
cassette (Van Droogenbroeck et al., 2007). expressed human lactoferrin (Anzai et al., 2000). The
recombinant single-chain Fv antibody directed against
Cereals and legumes carcinoembryonic antigen that was produced in wheat and
As protein synthesis and protein storage organelles, seeds rice could be preserved for up to 4 or 5 months at room
have a critical role in plant molecular farming. The most temperature without any loss in activity or of the product
important reasons for a seed-based system being an ideal (Stöger et al., 2000). Although soybean and alfalfa plants
platform for molecular farming are that seeds allow the produce a relatively smaller amount of green biomass
long-term storage of proteins due to having an appropriate compared with that of tobacco and maize, their ability to
biochemical environment, a low water content and low directly utilize atmospheric nitrogen through nitrogen
protease activities and that they possess biosafety and are fixation make them ideal plants for the production of
easy to transport (Stoger et al., 2002b). The lack of recombinant antibodies and other proteins (Ma et al.,
phenolic components is another advantage of cereal seeds 2003).
compared to other plant bioreactors, such as tobacco, that
contain phenolic components in their leaves. Generally, the Vegetables and Fruits
downstream process of removing phenol from the products Notably, fruits, vegetables and leafy crops are consumed in
is time consuming and expensive (Ma et al., 2003). As is the form of moderately processed or entirely raw foods. In
the case for other plant bioreactors used in molecular addition, these groups of plant products are normally free
farming, seed-based systems have some drawbacks. The of toxicants and are rich in nutrients, which make them
seed-based transgenic plants must reach the flowering particularly appropriate for the production of recombinant
stage before the recombinant proteins can be extracted, vaccine subunits, antibodies, and food additives for active
which significantly increases the possibility of immunotherapy (Ma et al., 2003). Potatoes have been
environmental contamination by the pollen of the used extensively for the production of plant-derived
transgenic plants. To reduce the risk of this type of vaccines, which have been administered to humans in
environmental contamination, plant bioreactors that many clinical trials.
accumulate recombinant proteins in their vegetative

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Transgenic Plants Abiri et al.

Tomato (Lycopersicon esculentum), potato (Solanum proteins must be detached so that they can be extracted,
tuberosum), banana (Musa acuminata colla) and carrot which can be achieved using a simple procedure, namely,
(Daucus carota spp. sativus) plants have been successfully endoprotease digestion (Boothe et al., 1997). Safflower-
utilized for the expression of vaccine subunits. Interestingly, derived insulin and hirudin are new-generation
among these plants, tomato plants have been utilized as pharmaceutical proteins that have produced by oilseed
model genetically transformed producers of the first plant- plants (Spök et al., 2008).
derived rabies-vaccine component, HIV-gag and HBsAg
proteins (Sala et al., 2003). The accumulation of beta- Aquatic plants
amyloid (Aβ) in the brain initiates the development of Green-cell factories are also options for plant molecular
Alzheimer's disease (AD), which is a neurodegenerative farming. Aquatic plants are promising green-cell platforms
disease. The accumulation of this toxic protein in the brain for the introduction of genes and the production of novel
can lead to neuronal destruction and intensification of the recombinant proteins. To this end, the chloroplast and
disease process. Administering an antigen directed against nuclear genomes of five different microalgal species,
this toxic protein might be the most useful strategy to treat namely, Cylindrotheca fusifornzis, Symbiodinium
AD or at least to arrest its progress. To this end, scientists microadriaticum, Amphidinium carterae, Pheaeodactylum
have successfully expressed Aβ in tomatoes (Youm et al., tricornutu and Chlamydomonas reinhardtii, have been
2008). Moreover, thymosin α1 has been expressed in successfully transformed. Among the above-mentioned
tomato fruits through Agrobacterium-mediated algal species, Chlamydomonas reinhardtii is the most
transformation, and this recombinant protein also been suitable host for the production of edible vaccine subunits
used to treat cancer and viral infections (Cheng et al., (Potvin and Zhang, 2010; Rasala et al., 2010), antibodies
2003). The potato plant, which is another member of the (Mayfield and Franklin, 2005), blood proteins (Manuell et
Solanaceae family, has attracted the attention of al., 2007), industrial phytases (Yoon et al., 2011), and a
researchers wishing to produce plant-based recombinant growth factor within chloroplasts (Rasala et al., 2010). Their
proteins. Ma et al. (2003) demonstrated that the potato lack of toxicity, low expression levels, low cost, short
tuber is a suitable host for the production of diagnostic growth period, high yield and capability for fresh use are
antibody-fusion proteins, human milk proteins, and other the most significant advantages of employing microalgae to
antibodies. Banana plants have been considered as green overcome the current obstacles in plant molecular farming
bioreactors for the production of recombinant antibodies (Pryer et al., 2002; Zaslavskaia et al., 2000).
and vaccines. The transgenic fruits of different varieties of Another reliable plant bioreactor for the production of
banana commonly grown in countries in which vaccines pharmaceutical products is duckweed. Duckweed is a
are most desperately needed can be consumed as raw monocotyledonous plant belonging to the Lemnaceae
materials by both children and adults or the vaccine family, which includes four major subspecies, namely,
subunits within them can be purified (Ma et al., 2003). The Wolffiella, Spirodela, Lemna and Wolffia (Zhang et al.,
specific potential of carrots for plant engineering have led 2010). Like other plant bioreactors, duckweed has many
to this plant becoming an ideal candidate for tissue culture advantages for the production of recombinant proteins
and genetic transformation, as well as for plant molecular (Stomp, 2005). Some of specific features of this plant are
farming (Muller et al., 2003). that it is fast-growing, easy to harvest, safe, accumulates
Celery cabbage (Brassica rapa var. pekinensis), high levels of protein, and can produce complex proteins
lettuce (Lactuca sativa), and cauliflower (Brassica oleracea (Popov et al., 2006). Agrobacterium tumefaciens and
var. botrytis) are other vegetables that have been biolistics are the main methods used to transfer genes to
considered for plant molecular farming application, duckweed, such in the case for other plants. Interferon α2
although their low regeneration rates and recombinant (De Leede et al., 2008), avian influenza H5N1
expression levels remain challenging (Tacket and Mason, hemagglutinin (Gu and Glatz, 2007), aprotinin (Rival et al.,
1999). 2008), anti-CD20 mAb (BLX-301), human plasmin
(BLX-155) (Paul and Ma, 2011), a monoclonal antibody
Oilseeds (Cox et al., 2006) and plasminogen (Spencer et al., 2011)
Plants bearing seeds that are rich in oil are known as are some of the recombinant proteins that have been
oilseed plants. Safflower, coconut, peanut, sunflower, palm, produced at various levels by duckweed (Stomp, 2005;
sesame, olive, rapeseed and rice (bran) are examples of Vunsh et al., 2007).
oilseed plants (Moloney et al., 2003). Safflower and
rapeseed have been employed as useful sources of Glycosylation as a modification mechanism in
recombinant proteins due to their protein-production transgenic plants
capability and the simplicity of purifying the proteins they Modifying recombinant proteins through post-translational
produce. In addition to the low cost and low acreage processes plays an important role in their functions. Post-
associated with their use, the high protein yield obtained translational modifications, such as glycosylation,
and their being self-pollinating plants are other advantages phosphorylation, sulphation and methylation, are vital in
of these oilseed organisms. To produce recombinant transgenic higher eukaryotic organisms (Gomord et al.,
proteins in such organisms, scientists exploited oleosin 2004). One of the most important post-translational
proteins, which are structural components of safflower modifications is glycosylation. Certain aspects of this
cells. Oleosins are small structural proteins that are enzymatic process, in which glycans are attached to
attached to the surface of oil bodies and subcellular organic molecules such as proteins and lipids, are
organelles that store oils. Oleosin-recombinant fusion significantly different in plants and mammalian cells. The

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Transgenic Plants Abiri et al.

basic N-acetyl glucosamine (GlcNAc)-mannose precursor expression of highly demanded anti-cancer antibodies (Cox
structures added to the glycosylation sites of proteins et al., 2006).
within the endoplasmic reticulum of plant and mammalian
cells are identical, but evolution of the Golgi apparatus has The effect of environmental factors on transgenic
caused considerable variation. Glycosylation affects the plants
basic biological functions of proteins, such as their ligand- The effects of environmental factors on the health, biomass
receptor interactions, specific activity and immunogenicity. production and activity of plants are matters of concern
A chain of oligosaccharides can be formed via either O- or even under normal conditions; however, these factors
N-linked glycosylation. N-linked glycosylation occurs within become more serious concerns during the post-
the endoplasmic reticulum (ER), and the primary transformational period. Indeed, genetically transformed
oligosaccharide chain is further processed during its exit plants are extremely susceptible to the effects of
from the ER and passage through the Golgi apparatus environmental factors immediately after being transferring
(GA) (Balen et al., 2006). to natural soil. Environmental condition directly affects the
Recent reports concerning glycosylation within quality and quantity of recombinant proteins produced. The
transgenic plants suggested that the pattern of factors that may negatively affect transgenic plants are
glycosylation of an Fc antibody did not have a large effect divided into biotic and abiotic stresses. In this regards,
on antibody-dependent cellular cytotoxicity (ADCC) or the light, drought, salinity, nutritional deficits and cold have
stimulation of the complement cascade. However, four negative effects on plant products. These factors are
aspects are worth mentioning in relation to the potential abiotic stresses (Jamal et al., 2009). Light plays a crucial
effects of adding carbohydrate moieties and replacing them role in regulating photosynthesis and the phenological
are the following: a) the immunogenicity of plant N-glycans events that drive the growth and development of plants
per se, b) the immunogenicity of plant N-glycans in the toward the flowering stage (Dahl et al., 1995). Generally,
presence of a given glycoprotein, c) probable allergies to plants use light of 400- and 740-nm wavelengths to
plant N-glycans, and d) the impact of these carbohydrate conduct photosynthesis. Light of these wavelengths is
moieties on pharmacokinetics (Pujol et al., 2007). A general called photosynthetically active radiation. Photons of
problem is that some antibodies are not produced against wavelengths lower and higher than this range are either
compounds consisting of a large number of impractical or destructive for photosynthesis due to their
monosaccharides “linked glycosidically”, when inoculated insufficient or extremely high energy levels (Zhu et al.,
with plant-based enzymes. The best example of this 2008). To protect recombinant proteins against stresses,
phenomenon is the antagonistic reaction of N-glycans with scientists try to optimize the light condition, such as the
horseradish peroxidase. In addition, these antibodies length of the day and the light intensity. Other stresses,
interact with glycoproteins from plants, insects and snails such as drought, restrict the establishment of crops. This
that contain the same α(1,3)-fucose and β(1,2)-xylose stress decreases the productivity and quality of plants and
glycoepitopes (Chargelegue et al., 2000). Unfortunately, leads to morphological changes. Consequently, the
the products of plant glycosylation can occasionally lead to expression of recombinant proteins may be down-
side effects, such as allergic reactions. Because persons regulated. Similar to the effects of other stresses, drought
who are prone to pollen allergies have IgE (Ig: insoluble leads to the accumulation of reactive oxygen species
glycoproteins) and IgG4 reactivity to glycoepitopes, it is (ROS) in plants, causing oxidative stress (Pastori and
rational to attribute the allergenicity of plant-based Foyer, 2002). Moreover, physiological traits, such as the
glycosylated antibodies to the existence of allergenic carbon-assimilation and stomatal-conductance rates are
factors, such as glycoproteins containing α(1,3)-fucose and prominent determinants of fitness under drought conditions
β(1,2)-xylose. An appropriate example is the allergenic due to their relevance to the efficiency of water-use and
factors of olive-tree pollens, which contain a glycoprotein photosynthesis (Heschel and Riginos, 2005). Another
with α(1,3)-fucose and β(1,2)-xylose groups that are restricting factor of plant molecular farming is salinity.
capable of inducing histamine release by basophils Approximately one-third of the world's irrigated farms are
(Cárdaba et al., 2000). Therefore, it may be impossible to ineffective due to the excess salt content of the soil
predict the side effects of plant-based glycosylated (Munns, 2005). The adverse effects of salt on plants are
antibodies in humans to whom they are administered. manifested in two ways. Firstly, a high concentration of salt
Eventually, engineering the N-glycosylation processes of in the soil directly hampers water absorption by the roots
plants will enhance the efficacy of plant-made by affecting root-soil osmotic regulation. Secondly, salt
pharmaceuticals (PMPs) not only regarding the accumulation in various organs poisons plants (Munns and
immunogenicity of N-glycans in humans but also through Tester, 2008). The two toxic ions derived from NaCl, Na+
generating glycovariants of therapeutic proteins with a and Cl-, can damage plant cells through both osmotic and
higher bioactivity levels than those produced in cultured ionic mechanisms (Chinnusamy et al., 2005). Quantitative
mammalian cells (Saint-Jore-Dupas et al., 2007). In this and qualitative changes in metabolite synthesis, as well as
regard, priority should be given to antibodies containing a the occurrence of enhanced metabolic toxicity are a few of
single major N-glycan species and lacking detectable the most usual indicators of stressed plants (Karimi et al.,
levels of plant-specific N-glycans (Cárdaba et al., 2000). 2005). Furthermore, salt stress alters the expression of
Such antibodies have been shown to induce better cell-cycle progression genes through affecting mitotic cell
a n t i b o d y d e p e n d e n t c e l l - m e d i a t e d c y t o t o x i c i t y, division (Burssens et al., 2000). All of these cellular
demonstrating the potential of plants as systems for the processes may be affected by altered hormonal
homeostasis occurring under salt-stress conditions (Lee et

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Transgenic Plants Abiri et al.

al., 2001). It has been well documented that the abscisic providing balanced nutrition would enhance the production
acid (ABA) content of plants increases under salt-stress of pharmaceutical proteins by transgenic plants.
conditions (Bray, 2002). Adaptation to saline stress is
accompanied by alterations in the level of numerous Large-scale transient gene transfection, climate risk-
metabolites, proteins, and mRNAs (García et al., 1997). A free production systems, and biosafety considerations
variety of genes, the expression of which is activated in Plant biotechnology typically relies on two strategies for
response to salt stress, have been identified and have delivery and expression of heterologous genes in plants,
been transferred to plants (Rensink et al., 2005). Because including a) stable genetic transformation, and b) transient
high salinity conditions promote plant-cell dehydration (Liu expression using viral vectors (Marillonnet et al., 2005). In
et al., 2011), many of the genes that are activated by saline recent years, the technological progression in virus-based
stress are also activated by drought. The expression of the vectors has allowed plants to become a feasible platform
majority of these genes is regulated by abscisic acid (ABA), for recombinant proteins (RPs) production, while RPs were
a plant hormone that is generated in response to saline only able to be produced from cultures of mammalian,
stress (Wilkinson and Davies, 2002). insect, and bacteria cells, previously. The plant-based RPs
Another important environmental factor that affects are more preferable in terms of versatility, speed, cost,
plant growth and productivity is temperature. High- scalability, and safety over the current production
temperature stress induces certain physiological, paradigms (Chen and Lai, 2014). In spite of being a faster
biochemical, and genetic changes in plants, including method, the transient approach is hampered by low
protein denaturation, lipid liquefaction, and perturbation of contagiosity of viral vectors carrying average- or large-
membrane integrity (Levitt, 1980). Among the suppositions sized genes. Fortunately, these drawbacks have been
regarding high-temperature acclimation are that several subject to troubleshooting by developing constructs for the
mechanisms of adaptation to high temperature involving efficient delivery of RNA viral vectors as DNA precursors.
the induction of protein synthesis or altered protein The mentioned efforts have tended to expanding systemic
functions may exist (Turner et al., 2001). High Agrobacterium tumefaciens-mediated transfection of viral
temperatures decrease the rate of synthesis of normal replicons for efficient transient expression in plants. As
cellular proteins and induce the synthesis of heat-shock such, Agrobacterium-mediated delivery of the target
proteins (Parsell and Lindquist, 1993). High temperatures constructs using results in gene amplification in all
are harmful to plant cells, leading to a loss of viability. In developed leaves of a plant simultaneously. This process is
Medicago sativa transgenic plants, heat can inactivate the also referred to as "magnifection" that can be performed on
gene that encodes the phosphinothricin/N- a large scale and with different plant species. The
acetyltransferase recombinant protein (Walter et al., 1992). mentioned technique incorporates advantages of three
Sometimes, transferring plants to an environment with a biological systems consisting of: a) the transfection
stressful heat level initiates the expression of members of efficiency of A. tumefaciens, b) the high expression yield
heat-shock protein/chaperone cascades that prevent the obtained with viral vectors, and c) the post-translational
misfolding, denaturation and aberrant aggregation of capabilities of a plant. This procedure does not entail
cellular proteins (Wang et al., 2004). Plants lacking genetic modification of plants and is faster than other
temperature adaptation may be incapable of inducing current methods (Marillonnet et al., 2005). Transient
structural or functional changes in their proteins. According expression systems have been established to eliminate the
to Stevens et al. (Stevens et al., 2000), exposure to 25 °C long-time frame of generating transgenic plants, so that the
and high light conditions can increase the biomass and transgene is not integrated into the plant genome but rather
total soluble protein content of plants, whereas exposure to quickly directs the production of the RP while residing
high light conditions and 15 °C favoured the production of a transiently within the plant cell. In addition to the significant
recombinant monoclonal antibody by transgenic tobacco acceleration of production timeline, this approach improves
plants. the recombinant proteins accumulation level by excluding
The temperature also affects the glycosylation of the “position effect” of variable expression instigated by the
recombinant proteins (antibodies) in plants. Sulphur (S) is a random integration of transgene within the genome
major component of any protein molecule; hence, its (Komarova et al., 2010). In another word, the climate risk-
uptake and assimilation can affect the production of free molecular farming systems have become more
recombinant proteins in transgenic plants. Of course, S achievable by conducting the transient gene transfection.
uptake is dependent upon a constant supply of the Beside all these advances achieved by the transient
precursor of cysteine, O-acetylserine, which in turn, is expression technology, some complementary strategies
dependent on the presence of adequate nitrogen and have been taken into consideration to limit the potential
carbon sources (Kopriva and Rennenberg, 2004). As a environmental and human health impacts linked to PMF.
good example, the combined application of S and N affects Specifically, cell cultures of transgenic plants, physical
the accumulation of lipids in rapeseed (Brassica napus L.) containment, dedicated land, plastid transformation,
via the induced increase in the rate of protein synthesis biological confinement, male sterility, gene use restriction
(Fazli et al., 2005). The significantly positive correlation of technologies (GURTs), expression from or in roots,
the antibody and total protein contents of transgenic plants expression in edible parts and seeds, post-harvest
allows the prediction of the fluctuating trend of antibody inducible expression, and temporal confinement have been
accumulation through monitoring changes in the amounts suggested as additional solutions to minimize the risks of
of total protein. The above-mentioned facts suggest that PMF (Breyer et al., 2009).

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Transgenic Plants Abiri et al.

Industrialization, current status and perspectives References

As plant molecular farming has come of age, there have Acquaah, G. (2009). Principles of plant genetics and
been technological progresses on many aspects, including breeding, 1th edn. (John Wiley & Sons).
transformation methods, regulating gene expression, Ahmad, K. (2014). Molecular farming: Strategies,
protein targeting and accumulation, as well as the use of expression systems and bio-safety considerations.
different crops as production platforms (Twyman et al., Czech J. Genet. Plant Breed. 50, 1-10.
2003). Recently, plant molecular farming has been Alam, I., Sharmin, S. A., Naher, K., Alam, J., Anisuzzaman,
proposed as an example of a green development scheme M., and Alam, M. F. (2010). Effect of growth regulators on
in convergence with sustainable agricultural industries. meristem culture and plantlet establishment in sweet
Despite, the yield improvement remains as one of the most potato [Ipomoea batatas (L.) Lam.]. Plant Omics. 3, 35.
challenging issues, because the product yield has a Alam, I., Sharmin, S.A., Naher, K., Alam, J., Anisuzzaman,
significant impact on economic feasibility of any related M., and Alam, M.F. (2010). effect of growth regulators on
project. meristem culture and plantlet establishment in sweet
The advantages of transgenic plants over other potato [Ipomoea Batatas (L.) Lam.]. Plant Omics 3, 35.
expression systems make them become industrialized as Almquist, K.C., McLean, M.D., Niu, Y., Byrne, G., Olea-
economic alternatives to the conventional pharmaceutics. Popelka, F.C., Murrant, C., Barclay, J., and Hall, J.C.
Several plant-made pharmaceuticals, including the enzyme (2006). Expression of an anti-botulinum toxin A
glucocerebrosidase (GCase), insulin and Interferon alfa 2b neutralizing single-chain Fv recombinant antibody in
[IFN-alpha (2b)], have approached commercialization with transgenic tobacco. Vaccine 24, 2079-2086.
low costs and large-scale production. Interestingly, these Amrolia, P.J., Cunningham, J.M., Ney, P., Nienhuis, A.W.,
achievements have been attached to substantial patenting and Jane, S.M. (1995). Identification of two novel
activities as well. Reportedly, there was a tangible regulatory elements within the 5′-untranslated region of
downward trend in the number of patents filed from 2002 to the human-globin gene. J. Biol. Chem. 270,
2008, and a greater number of patents were filed by public 12892-12898.
sector institutions or inventors than by the private sector An, C. H., Lee, K. W., Lee, S. H., Jeong, Y. J., Woo, S. G.,
(Drake and Thangaraj, 2010). The USA dominated Chun, H., and Kim, C. Y. (2015). Heterologous
patenting activity providing nearly 30% of inventors. Most expression of IbMYB1a by different promoters exhibits
of the patents were related to vaccine candidates (55%), different patterns of anthocyanin accumulation in
followed by therapeutics and antibodies with 38 and 7%, tobacco. Plant Physiol. Biochem. 89, 1-10.
respectively (Drake and Thangaraj, 2010). Anzai, H., Takaiwa, F., Katsumata, K., Shimazaki, K.,
Tsuda, H., Tomita, M., Kuwata, T., and Perraudin, J.
Conclusions (2000). Production of human lactoferrin in transgenic
Plant molecular farming has been shown to be a promising plants. Paper presented at: Lactoferrin: structure,
biotechnological approach; however, because this function and applications. Proceedings of the 4th
approach is novel, its efficacy may be disputed. Methods International Conference on Lactoferrin: Structure,
that facilitate plant cultivation under extremely controlled Function and Applications. Sapporo, Japan 18-22 May
conditions should be developed for the subsequent stages 1999.
of this process, as we move away from aseptic plant-cell Ashraf, M., Athar, H., Harris, P., and Kwon, T. (2008). Some
cultures to non-aseptic conditions in which plants are prospective strategies for improving crop salt tolerance.
grown traditionally or are grown hydroponically using Adv Agron. 97, 45-110.
compost. Plant molecular farming has significant potential Azhakanandam, K., Weissinger, S.M., Nicholson, J.S., Qu,
for the development of medicinal products. With regard to R., and Weissinger, A.K. (2007). Amplicon-plus targeting
the history of plant molecular farming, the current major technology (APTT) for rapid production of a highly
focus is to accelerate the improvement of plant unstable vaccine protein in tobacco plants. Plant Mol.
biotechnological procedures for the generation of new Biol. 63, 393-404.
products, as well as conventional products. The most Balen, B., Krsnik-Rasol, M., Zamfir, A.D., Miloševic, J.,
important challenges in this field are identifying new plant Vakhrushev, S.Y., and Peter-Katalinic, J. (2006).
resources and optimizing protocols for producing high Glycoproteomic survey of Mammillaria gracillis tissues
levels of recombinant proteins. The cryptic medicinal plant grown in vitro. J. Proteome Res. 5, 1658-1666.
such as Andrographis paniculata (Valdiani et al., 2013) can Barta, A., Sommergruber, K., Thompson, D., Hartmuth, K.,
be introduced as an impending candidates (Valdiani et al., Matzke, M.A., and Matzke, A.J. (1986). The expression
2012), while the genetic (Valdiani et al., 2014), and of a nopaline synthase-human growth hormone
proteomic (Talei et al., 2014) analyses of the herb have chimaeric gene in transformed tobacco and sunflower
both performed promising horizons for being subjected to callus tissue. Plant Mol. Biol. 6, 347-357.
plant molecular farming. Benchabane, M., Goulet, C., Rivard, D., Faye, L., Gomord,
V., and Michaud, D. (2008). Preventing unintended
Acknowledgements proteolysis in plant protein biofactories. Plant Biotechnol.
The authors would like to appreciate the Long-term J. 6, 633-648.
Research Grants Scheme (LRGS), Food Security Rice Bock, R. (2007). Plastid biotechnology: prospects for
Research Program of the Ministry of Higher Education, herbicide and insect resistance, metabolic engineering
Malaysia, for creating an opportunity to conduct the present and molecular farming. Curr. Opin. Biotechnol. 18,
review article. 100-106.

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !34

Transgenic Plants Abiri et al.

Boothe, J.G., Saponja, J.A., and Parmenter, D.L. (1997). Cheng, M., Hu, T., Layton, J., Liu, C.-N., and Fry, J.E.
Molecular farming in plants: oilseeds as vehicles for the (2003). Desiccation of plant tissues post-Agrobacterium
production of pharmaceutical proteins. Drug Dev. Res. infection enhances T-DNA delivery and increases stable
42, 172-181. transformation efficiency in wheat. In Vitro Cell. Dev. Biol.
Brandsma, M., Wang, X., Diao, H., Kohalmi, S., Jevnikar, -Plant 39, 595-604.
A., and Ma, S. (2009). A proficient approach to the Chinnusamy, V., Jagendorf, A., and Zhu, J.-K. (2005).
production of therapeutic glucagon-like peptide-1 Understanding and improving salt tolerance in plants.
(GLP-1) in transgenic plants. Open Biotechnol J 3, Crop Sci. 45, 437-448.
57-66. Chugh, A., Eudes, F., and Shim, Y.-S. (2010). Cell-
Bray, E. (2002). Abscisic acid regulation of gene expression penetrating peptides: Nanocarrier for macromolecule
during water-deficit stress in the era of the Arabidopsis delivery in living cells. IUBMB Life 62, 183-193.
genome. Plant, Cell Environ. 25, 153-161. Condliffe, P. C., Davey, M. R., Brian Power, J., Koehorst-
Bregitzer, P., and Brown, R.H. (2013). Long-term van Putten, H., and Visser, P. B. (2003). An optimized
assessment of transgene behavior in barley: Ds- protocol for rose transformation applicable to different
mediated delivery of bar results in robust, stable, and cultivars. In: XXI International Eucarpia Symposium on
heritable expression. In Vitro Cell. Dev. Biol. -Plant 49, Classical versus Molecular Breeding of Ornamentals-
231-239. Part I. 612, 115-120.
Breyer, D., Goossens, M., Herman, P., and Sneyers, M. Costanzo, M.C., and Fox, T.D. (1995). A point mutation in
(2009). Biosafety considerations associated with the 5′-untranslated leader that affects translational
molecular farming in genetically modified plants. J. Med. activation of the mitochondrial COX 3 mRNA. Curr.
Plants Res. 3, 825-838. Genet. 28, 60-66.
Brodzik, R., Glogowska, M., Bandurska, K., Okulicz, M., Cox, K.M., Sterling, J.D., Regan, J.T., Gasdaska, J.R.,
Deka, D., Ko, K., van der Linden, J., Leusen, J.H., Frantz, K.K., Peele, C.G., Black, A., Passmore, D.,
Pogrebnyak, N., and Golovkin, M. (2006). Plant-derived Moldovan-Loomis, C., and Srinivasan, M. (2006). Glycan
anti-Lewis Y mAb exhibits biological activities for efficient optimization of a human monoclonal antibody in the
immunotherapy against human cancer cells. Proc. Natl. aquatic plant Lemna minor. Nat. Biotechnol. 24,
Acad. Sci. U. S. A. 103, 8804-8809. 1591-1597.
Burana, C., Kurokura, T., Yamaki, Y., and Yamane, K. Curie, C., and McCormick, S. (1997). A strong inhibitor of
(2014). Modified atmosphere (MA) and 1- gene expression in the 5'untranslated region of the
methylcyclopropene (1-MCP) combination treatment pollen-specific LAT59 gene to tomato. Plant Cell 9,
extends the postharvest life of carnations. Environ. 2025-2036.
Control Biol. 52, 131-136. da Cunha, N.B., Vianna, G.R., da Almeida Lima, T., and
Burssens, S., Himanen, K., Van De Cotte, B., Beeckman, Rech, E. (2014). Molecular farming of human cytokines
T., Van Montagu, M., Inzé, D., and Verbruggen, N. and blood products from plants: Challenges in
(2000). Expression of cell cycle regulatory genes and biosynthesis and detection of plant produced
morphological alterations in response to salt stress in recombinant proteins. Biotechnol. J. 9, 39-50.
Arabidopsis thaliana. Planta 211, 632-640. Dahl, M.-L., Johansson, I., Bertilsson, L., Ingelman-
Cai, S., Jiang, G., Ye, N., Chu, Z., Xu, X., Zhang, J., and Sundberg, M., and Sjöqvist, F. (1995). Ultrarapid
Zhu, G. (2015). A key ABA catabolic gene, OsABA8ox3, hydroxylation of debrisoquine in a Swedish population.
is involved in drought stress resistance in rice. Plos One Analysis of the molecular genetic basis. J. Pharmacol.
10, e0116646. Exp. Ther. 274, 516-520.
Cárdaba, B., Cortegano, I., Florido, F., Arrieta, I., Aceituno, Datla, R.S., Bekkaoui, F., Hammerlindl, J.K., Pilate, G.,
E., Del Pozo, V., Gallardo, S., Rojo, M., Palomino, P., Dunstan, D.I., and Crosby, W.L. (1993). Improved high-
and Lahoz, C. (2000). Genetic restrictions in olive pollen level constitutive foreign gene expression in plants using
allergy. J. Allergy Clin. Immunol. 105, 292-298. an AMV RNA4 untranslated leader sequence. Plant Sci.
Chan, M.-T., Chang, H.-H., Ho, S.-L., Tong, W.-F., and Yu, 94, 139-149.
S.-M. (1993). Agrobacterium-mediated production of De La Fuente, G.N., Frei, U.K., and Lübberstedt, T. (2013).
transgenic rice plants expressing a chimeric α-amylase Accelerating plant breeding. Trends Plant Sci. 18,
promoter/β-glucuronidase gene. Plant Mol. Biol. 22, 667-672.
491-506. De la Fuente, J.M., Ramıŕ ez-Rodrıg ́ uez, V., Cabrera-
Chargelegue, D., Vine, N.D., van Dolleweerd, C.J., Drake, Ponce, J.L., and Herrera-Estrella, L. (1997). Aluminum
P.M., and Ma, J.K. (2000). A murine monoclonal antibody tolerance in transgenic plants by alteration of citrate
produced in transgenic plants with plant-specific glycans synthesis. Science 276, 1566-1568.
is not immunogenic in mice. Transgenic Res. 9, 187-194. De Leede, L.G., Humphries, J.E., Bechet, A.C., Van
Chen, Q. (2008). Expression and purification of Hoogdalem, E.J., Verrijk, R., and Spencer, D.G. (2008).
pharmaceutical proteins in plants. J. Biol. Eng. 1, Novel controlled-release lemna-derived IFN-α 2b
291-321. (locteron): pharmacokinetics, pharmacodynamics, and
Chen, Q., and Lai, H. (2014). Gene delivery into plant cells tolerability in a phase I clinical trial. J. Interferon Cytokine
for recombinant protein production. BioMed Res. Int. doi: Res. 28, 113-122.
10.1155/2014/932161 De Neve, M., De Buck, S., De Wilde, C., Van Houdt, H.,
Strobbe, I., Jacobs, A., Van Montagu, M., and Depicker,
A. (1999). Gene silencing results in instability of antibody

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !35

Transgenic Plants Abiri et al.

production in transgenic plants. Mol. Gen. Genet. 260, Franconi, R., Demurtas, O.C., and Massa, S. (2010). Plant-
582-592. derived vaccines and other therapeutics produced in
Denecke, J., De Rycke, R., and Botterman, J. (1992). Plant contained systems. Expert Rev. Vaccines 9, 877-892.
and mammalian sorting signals for protein retention in Gallie, D.R., and Kobayashi, M. (1994). The role of the 3'-
the endoplasmic reticulum contain a conserved epitope. untranslated region of non-polyadenylated plant viral
EMBO 11, 2345. mRNAs in regulating translational efficiency. Gene 142,
Dickey, L.F., Gallo-Meagher, M., and Thompson, W. (1992). 159-165.
Light regulatory sequences are located within the García, M.J., Ríos, G., Ali, R., Bellés, J.M., and Serrano, R.
5'portion of the Fed-1 message sequence. EMBO 11, (1997). Comparative physiology of salt tolerance in
2311. Candida tropicalis and Saccharomyces cerevisiae.
Dillen, W., Clercq, J., Kapila, J., Zambre, M., Montagu, M., Microbiology 143, 1125-1131.
and Angenon, G. (1997). The effect of temperature on Gebauer, F., and Hentze, M.W. (2004). Molecular
Agrobacterium tumefaciens-mediated gene transfer to mechanisms of translational control. Nat. Rev. Mol. Cell
plants. Plant J. 12, 1459-1463. Biol. 5, 827-835.
Drake, P.M., and Thangaraj, H. (2010). Molecular farming, Gelvin, S.B. (2012). Traversing the cell: Agrobacterium T-
patents and access to medicines. Expert Rev. Vaccines DNA’s journey to the host genome. Front. Plant Sci. 3,
9, 811-819. doi: 10.1586/erv.10.72. 52.
Du, Y., Wei, X., Dodel, R., Sommer, N., Hampel, H., Gao, George, E.F., Hall, M.A., and De Klerk, G.-J. (2008).
F., Ma, Z., Zhao, L., Oertel, W.H., and Farlow, M. (2003). Micropropagation: uses and methods, Vol 1, 3rd edn
Human anti β-amyloid antibodies block β-amyloid fibril (Dordrecht, The Netherlands: Springer).
formation and prevent β-amyloid induced neurotoxicity. Girard, L.S., Fabis, M.J., Bastin, M., Courtois, D., Pétiard,
Brain 126, 1935-1939. V., and Koprowski, H. (2006). Expression of a human
Dunwell, J.M. (2009). Transgenic wheat, barley and oats: anti-rabies virus monoclonal antibody in tobacco cell
future prospects. In Methods in Molecular Biology, H.D. culture. Biochem. Biophys. Res. Commun. 345, 602-607.
Jones, and P.R. Shewry, eds. (Humana Press, Springer), Gleba, Y., Klimyuk, V., and Marillonnet, S. (2005).
pp. 333-345. Magnifection-a new platform for expressing recombinant
Dynkevich, Y., Rother, K.I., Whitford, I., Qureshi, S., vaccines in plants. Vaccine 23, 2042-2048.
Galiveeti, S., Szulc, A.L., Danoff, A., Breen, T.L., Kaviani, Gomord, V., Sourrouille, C., Fitchette, A.C., Bardor, M.,
N., and Shanik, M.H. (2013). Tumors, IGF-2, and Pagny, S., Lerouge, P., and Faye, L. (2004). Production
Hypoglycemia: Insights From the Clinic, the Laboratory, and glycosylation of plant made pharmaceuticals: the
and the Historical Archive. Endocr. Rev. 34, 798-826. antibodies as a challenge. Plant Biotechnol. J. 2, 83-100.
Enríquez-Obregón, G.A., Prieto-Samsónov, D.L., Gustavo, Goo, Y. M., Han, E. H., Jeong, J. C., Kwak, S. S., Yu, J.,
A., Pérez, M., Selman-Housein, G., and Vázquez- Kim, Y. H., and Lee, S. W. (2015). Overexpression of the
Padrón, R.I. (1999). Agrobacterium-mediated Japonica sweet potato IbOr gene results in the increased
rice transformation: a procedure assisted by an accumulation of carotenoid and confers tolerance to
antinecrotic treatment. Plant Cell Tissue Organ Cult. 59, environmental stresses in transgenic potato. C. R.
159-168. Biol. 338, 12-20.
Faye, L., Boulaflous, A., Benchabane, M., Gomord, V., and Gu, Z., and Glatz, C.E. (2007). A method for three-
Michaud, D. (2005). Protein modifications in the plant dimensional protein characterization and its application
secretory pathway: current status and practical to a complex plant (corn) extract. Biotechnol. Bioeng. 97,
implications in molecular pharming. Vaccine 23, 1158-1169.
1770-1778. Guan, C., Li, X., Jin, C., Ji, J., and Wang, G. (2015). LcBiP,
Fazli, I., Abdin, M., Jamal, A., and Ahmad, S. (2005). an endoplasmic reticulum chaperone binding protein
Interactive effect of sulphur and nitrogen on lipid gene from Lycium chinense, confers cadmium tolerance
accumulation, acetyl-CoA concentration and acetyl-CoA in transgenic tobacco. Biotechnol. Prog. doi: 10.1002/
carboxylase activity in developing seeds of oilseed crops btpr.2046
(Brassica campestris L. and Eruca sativa Mill.). Plant Sci. Häkkinen, S.T., Raven, N., Henquet, M., Laukkanen, M.L.,
168, 29-36. Anderlei, T., Pitkänen, J.P., Twyman, R.M., Bosch, D.,
Fischer, R., Drossard, J., Commandeur, U., Schillberg, S., Oksman-Caldentey, K.M., and Schillberg, S. (2014).
and Emans, N. (1999). Towards molecular farming in the Molecular farming in tobacco hairy roots by triggering the
future: moving from diagnostic protein and antibody secretion of a pharmaceutical antibody. Biotechnol.
production in microbes to plants. Biotechnol. Appl. Bioeng. 111, 336-346.
Biochem. 30, 101-108. Han, M., SU, T., ZU, Y.-G., and AN, Z.-G. (2006). Research
Fischer, R., Schillberg, S., Hellwig, S., Twyman, R. M., & advances on transgenic plant vaccines. Acta Genet. Sin.
Drossard, J. (2012). GMP issues for recombinant plant- 33, 285-293.
derived pharmaceutical proteins. Biotechnol. Adv. 30, Heschel, M.S., and Riginos, C. (2005). Mechanisms of
434-439. selection for drought stress tolerance and avoidance in
Floss, D.M., Falkenburg, D., and Conrad, U. (2007). Impatiens capensis (Balsaminaceae). Am. J. Bot. 92,
Production of vaccines and therapeutic antibodies for 37-44.
veterinary applications in transgenic plants: an overview.
Transgenic Res. 16, 315-332.

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !36

Transgenic Plants Abiri et al.

Hiatt, A., and Pauly, M. (2006). Monoclonal antibodies from Kavitah, G., Taghipour, F., and Huyop, F. (2010).
plants: a new speed record. Proc. Natl. Acad. Sci. U. S. Investigation of factors in optimizing Agrobacterium-
A. 103, 14645-14646. mediated gene transfer in Citrullus lanatus cv. Round
Hiei, Y., Komari, T., and Kubo, T. (1997). Transformation of Dragon. J. Biol. Sci. 10, 209-216.
rice mediated by Agrobacterium tumefaciens. Plant Mol. Kervestin, S., and Amrani, N. (2004). Translational
Biol. 35, 205-218. regulation of gene expression. Genome Biol. 5, 359.
Hirsch, R.E., and Sussman, M.R. (1999). Improving Kidokoro, S., Watanabe, K., Ohori, T., Moriwaki, T.,
nutrient capture from soil by the genetic manipulation of Maruyama, K., Mizoi, J., Myint Nang, P. S. H., Sachiko
crop plants. Trends Biotechnol. 17, 356-361. S., Kazuo, S., and Yamaguchi-Shinozaki, K. (2015).
Hood, E.E. (2002). From green plants to industrial Soybean DREB1/CBF-type transcription factors function
enzymes. Enzyme Microb. Technol. 30, 279-283.
in heat and drought as well as cold stress-responsive
Hood, E.E., Love, R., Lane, J., Bray, J., Clough, R., Pappu,
gene expression. Plant J. 81, 505-518.
K., Drees, C., Hood, K.R., Yoon, S., and Ahmad, A.
Kim, H., Lee, K., Hwang, H., Bhatnagar, N., Kim, D. Y.,
(2007). Subcellular targeting is a key condition for high-
Yoon, I. S., Byun, M. O., Kim, S. T., Jung, K. H., and Kim,
level accumulation of cellulase protein in transgenic
B. G. (2014). Overexpression of PYL5 in rice enhances
maize seed. Plant Biotechnol. J. 5, 709-719.
drought tolerance, inhibits growth, and modulates gene
Horn, M., Woodard, S., and Howard, J. (2004). Plant
expression. J. Exp. Bot. 65, 453-464.
molecular farming: systems and products. Plant Cell
Kim, T.-G., Baek, M.-Y., Lee, E.-K., Kwon, T.-H., and Yang,
Rep. 22, 711-720.
M.-S. (2008). Expression of human growth hormone in
Huang, T.K., Plesha, M.A., Falk, B.W., Dandekar, A.M., and
transgenic rice cell suspension culture. Plant Cell Rep.
McDonald, K.A. (2009). Bioreactor strategies for
27, 885-891.
improving production yield and functionality of a Kleiner, S. (1995). The true nature of herbs. Physician
recombinant human protein in transgenic tobacco cell Sportsmed. 23, 13-14.
cultures. Biotechnol. Bioeng. 102, 508-520. Ko, K., Norelli, J.L., Reynoird, J.-P., Boresjza-Wysocka, E.,
Hull, A.K., Criscuolo, C.J., Mett, V., Groen, H., Steeman, Brown, S.K., and Aldwinckle, H.S. (2000). Effect of
W., Westra, H., Chapman, G., Legutki, B., Baillie, L., and untranslated leader sequence of AMV RNA 4 and signal
Yusibov, V. (2005). Human-derived, plant-produced peptide of pathogenesis-related protein 1b on attacin
monoclonal antibody for the treatment of anthrax. gene expression, and resistance to fire blight in
Vaccine 23, 2082-2086. transgenic apple. Biotechnol. Lett. 22, 373-381.
Ikeuchi, M., Sugimoto, K., and Iwase, A. (2013). Plant Ko, K., Steplewski, Z., Glogowska, M., and Koprowski, H.
callus: mechanisms of induction and repression. Plant (2005). Inhibition of tumor growth by plant-derived mAb.
Cell 25, 3159-3173. Proc. Natl. Acad. Sci. U. S. A. 102, 7026-7030.
Jamal, A., Ko, K., Kim, H.-S., Choo, Y.-K., Joung, H., and Ko, K., Tekoah, Y., Rudd, P.M., Harvey, D.J., Dwek, R.A.,
Ko, K. (2009). Role of genetic factors and environmental Spitsin, S., Hanlon, C.A., Rupprecht, C., Dietzschold, B.,
conditions in recombinant protein production for and Golovkin, M. (2003). Function and glycosylation of
molecular farming. Biotechnol. Adv. 27, 914-923. plant-derived antiviral monoclonal antibody. Proc. Natl.
Jani, D., Singh, N., Bhattacharya, S., Meena, L., Singh, Y., Acad. Sci. U. S. A. 100, 8013-8018.
Upadhyay, S., Sharma, A., and Tyagi, A. (2004). Studies Koornneef, M., and Meinke, D. (2010). The development of
on the immunogenic potential of plant-expressed cholera Arabidopsis as a model plant. Plant J. 61, 909-921.
toxin B subunit. Plant Cell Rep. 22, 471-477. Komarova, T. V., Baschieri, S., Donini, M., Marusic, C.,
Johnston, R.J., Poholek, A.C., DiToro, D., Yusuf, I., Eto, D., Benvenuto, E., and Dorokhov, Y. L. (2010). Transient
Barnett, B., Dent, A.L., Craft, J., and Crotty, S. (2009). expression systems for plant-derived
Bcl6 and Blimp-1 are reciprocal and antagonistic biopharmaceuticals. Expert Rev. Vaccines 9, 859-876.
regulators of T follicular helper cell differentiation. Kopriva, S., and Rennenberg, H. (2004). Control of
Science 325, 1006-1010.
sulphate assimilation and glutathione synthesis:
Kamenarova, K., Abumhadi, N., Gecheff, K., & Atanassov,
interaction with N and C metabolism. J. Exp. Bot. 55,
A. (2005). Molecular farming in plants: an approach of
1831-1842.
agricultural biotechnology. J. Cell Mol. Biol. 4, 77-86.
Koya, V., Moayeri, M., Leppla, S.H., and Daniell, H. (2005).
Kamenarova, K., Abumhadi, N., Gecheff, K., and
Plant-based vaccine: mice immunized with chloroplast-
Atanassov, A. (2005). Molecular farming in plants: an
derived anthrax protective antigen survive anthrax lethal
approach of agricultural biotechnology. J. Cell Mol. Biol.
toxin challenge. Infect. Immun. 73, 8266-8274.
4, 77-86.
Krivosheeva, A. B., Varlamova, T. V., Yurieva, N. O.,
Karg, S.R., and Kallio, P.T. (2009). The production of
Sobol’kova, G. I., Kholodova, V. P., and Belyaev, D. V.
biopharmaceuticals in plant systems. Biotechnol. Adv. 27,
(2014). Potato transformation with the HvNHX3 gene and
879-894.
the improvement of transformant salt tolerance. Russ. J.
Karimi, G., Ghorbanli, M., Heidari, H., Nejad, R.K., and Plant Physiol. 61, 792-800.
Assareh, M. (2005). The effects of NaCl on growth, water
Kumar, D., and Kirti, P. B. (2015). Pathogen-induced SGT1
relations, osmolytes and ion content in Kochia prostrata.
of Arachis diogoi induces cell death and enhanced
Biol. Plant. 49, 301-304.
disease resistance in tobacco and peanut. Plant
Karimi, M., Inzé, D., Van Lijsebettens, M., and Hilson, P.
Biotechnol. J. 13, 73-84.
(2013). Gateway vectors for transformation of cereals.
Trends Plant Sci. 18, 1-4.

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !37

Transgenic Plants Abiri et al.

Lau, W., Fischbach, M.A., Osbourn, A., and Sattely, E.S. Magnuson, N.S., Linzmaier, P.M., Reeves, R., An, G.,
(2014). Key applications of plant metabolic engineering. HayGlass, K., and Lee, J.M. (1998). Secretion of
Plos Biol. 12, e1001879. biologically active human interleukin-2 and interleukin-4
Lee, H., Xiong, L., Gong, Z., Ishitani, M., Stevenson, B., from genetically modified tobacco cells in suspension
and Zhu, J.-K. (2001). The Arabidopsis HOS1 gene culture. Protein Expr. Purif. 13, 45-52.
negatively regulates cold signal transduction and Makvandi-Nejad, S., McLean, M.D., Hirama, T., Almquist,
encodes a RING finger protein that displays cold- K.C., MacKenzie, C.R., and Hall, J.C. (2005). Transgenic
regulated nucleo–cytoplasmic partitioning. Genes Dev. tobacco plants expressing a dimeric single-chain variable
15, 912-924. fragment (scfv) antibody against Salmonella enterica
Lee, M.Y., Zhou, Y., Lung, R.W., Chye, M.L., Yip, W.K., serotype Paratyphi B. Transgenic Res. 14, 785-792.
Zee, S.Y., and Lam, E. (2006). Expression of viral capsid Manuell, A.L., Beligni, M.V., Elder, J.H., Siefker, D.T., Tran,
protein antigen against Epstein-Barr virus in plastids of M., Weber, A., McDonald, T.L., and Mayfield, S.P. (2007).
Nicotiana tabacum cv. SR1. Biotechnol. Bioeng. 94, Robust expression of a bioactive mammalian protein in
1129-1137. Chlamydomonas chloroplast. Plant Biotechnol. J. 5,
Lee, S. H., Li, C. W., Liau, C. H., Chang, P. Y., Liao, L. J., 402-412.
Lin, C. S., and Chan, M. T. (2015). Establishment of an Marillonnet, S., Thoeringer, C., Kandzia, R., Klimyuk, V.,
Agrobacterium-mediated genetic transformation and Gleba, Y. (2005). Systemic Agrobacterium
procedure for the experimental model orchid Erycina tumefaciens–mediated transfection of viral replicons for
pusilla. Plant Cell Tissue Organ Cult. 120, 211-220. efficient transient expression in plants. Nat. Biotechnol.
Lenzi, P., Scotti, N., Alagna, F., Tornesello, M.L., Pompa, 23, 718-723.
A., Vitale, A., De Stradis, A., Monti, L., Grillo, S., and Marusic, C., Nuttall, J., Buriani, G., Lico, C., Lombardi, R.,
Buonaguro, F.M. (2008). Translational fusion of Baschieri, S., Benvenuto, E., and Frigerio, L. (2007).
chloroplast-expressed human papillomavirus type 16 L1 Expression, intracellular targeting and purification of HIV
capsid protein enhances antigen accumulation in Nef variants in tobacco cells. BMC Biotechnol. 7, 12.
transplastomic tobacco. Transgenic Res. 17, 1091-1102. Mayfield, S.P., and Franklin, S.E. (2005). Expression of
Levitt, J. (1980). Responses of plants to environmental human antibodies in eukaryotic micro-algae. Vaccine 23,
stresses. Volume II. Water, radiation, salt, and other 1828-1832.
stresses (Academic Press.). Mechold, U., Gilbert, C., and Ogryzko, V. (2005). Codon
Lienard, D., Dinh, O.T., Van Oort, E., Van Overtvelt, L., optimization of the BirA enzyme gene leads to higher
Bonneau, C., Wambre, E., Bardor, M., Cosette, P., expression and an improved efficiency of biotinylation of
Didier-Laurent, A., and Borne, D. (2007). Suspension- target proteins in mammalian cells. J. Biotechnol. 116,
cultured BY 2 tobacco cells produce and mature 245-249.
immunologically active house dust mite allergens. Plant Mewett, O., Johnson, H., and Holtzapffel, R. (2007). Plant
Biotechnol. J. 5, 93-108. molecular farming in Australia and overseas (Bureau of
Liu, X., Hong, L., Li, X.-Y., Yao, Y., Hu, B., and Li, L. (2011). Rural Sciences).
Improved drought and salt tolerance in transgenic Meyer, M., de Angelis, M.H., Wurst, W., and Kühn, R.
Arabidopsis overexpressing a NAC transcriptional factor (2010). Gene targeting by homologous recombination in
from Arachis hypogaea. Biosci. Biotechnol. Biochem. 75, mouse zygotes mediated by zinc-finger nucleases. Proc.
443-450. Natl. Acad. Sci. U. S. A. 107, 15022-15026.
Lopes, M., Reynolds, M., Manes, Y., Singh, R., Crossa, J., Mitsuhara, I., Ugaki, M., Hirochika, H., Ohshima, M.,
and Braun, H. (2012). Genetic yield gains and changes Murakami, T., Gotoh, Y., Katayose, Y., Nakamura, S.,
in associated traits of CIMMYT spring bread wheat in a Honkura, R., and Nishimiya, S. (1996). Efficient promoter
“historic” set representing 30 years of breeding. Crop Sci. cassettes for enhanced expression of foregin genes in
52, 1123-1131. Dicotyledonous and Monocotyledonous plants. Plant Cell
Ma, J., KC Drake, P.M., and Christou, P. (2003). The Physiol. 37, 49-59.
production of recombinant pharmaceutical proteins in Moloney, M., Boothe, J., and Van Rooijen, G. (2003). Oil
plants. Nat. Rev. Genet. 4, 794-805. bodies and associated proteins as affinity matrices (USA:
Ma, J.K.-C., Hikmat, B.Y., Wycoff, K., Vine, N.D., Google Patents).
Chargelegue, D., Yu, L., Hein, M.B., and Lehner, T. Muller, C.P., Fack, F., Damien, B., and Bouche, F.B. (2003).
(1998). Characterization of a recombinant plant Immunogenic measles antigens expressed in plants: role
monoclonal secretory antibody and preventive as an edible vaccine for adults. Vaccine 21, 816-819.
immunotherapy in humans. Nat. Med. 4, 601-606. Munns, R. (2005). Genes and salt tolerance: bringing them
Ma, S., Huang, Y., Yin, Z., Menassa, R., Brandle, J.E., and together. New Phytol. 167, 645-663.
Jevnikar, A.M. (2004). Induction of oral tolerance to Munns, R., and Tester, M. (2008). Mechanisms of salinity
prevent diabetes with transgenic plants requires glutamic tolerance. Annu. Rev. Plant Biol. 59, 651-681.
acid decarboxylase (GAD) and IL-4. Proc. Natl. Acad. Murashige, T., and Skoog, F. (1962). A revised medium for
Sci. U. S. A. 101, 5680-5685. rapid growth and bio assays with tobacco tissue cultures.
Ma, S.-W., Zhao, D.-L., Yin, Z.-Q., Mukherjee, R., Singh, Physiol. Plant 15, 473-497.
B., Qin, H.-Y., Stiller, C., and Jevnikar, A. (1997). Nandi, S., Suzuki, Y.A., Huang, J., Yalda, D., Pham, P., Wu,
Transgenic plants expressing autoantigens fed to mice to L., Bartley, G., Huang, N., and Lönnerdal, B. (2002).
induce oral immune tolerance. Nat. Med. 3, 793-796. Expression of human lactoferrin in transgenic rice grains

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !38

Transgenic Plants Abiri et al.

for the application in infant formula. Plant Sci. 163, (2010). Production of pharmaceutical-grade recombinant
713-722. aprotinin and a monoclonal antibody product using plant-
Nanou, A., and Azzouz, M. (2009). Gene therapy for based transient expression systems. Plant Biotechnol. J.
neurodegenerative diseases based on lentiviral vectors. 8, 638-654.
Prog. Brain Res. 175, 187-200. Popov, S.V., Golovchenko, V.V., Ovodova, R.G., Smirnov,
Nguyen, H.T., Leelavathi, S., and Reddy, V.S. (2004). V.V., Khramova, D.S., Popova, G.Y., and Ovodov, Y.S.
Bacteriophage T7 RNA polymerase-directed, inducible (2006). Characterisation of the oral adjuvant effect of
and tissue-specific over-expression of foreign genes in lemnan, a pectic polysaccharide of Lemna minor L.
transgenic plants. Plant Biotechnol. J. 2, 301-310. Vaccine 24, 5413-5419.
Obembe, O.O., Popoola, J.O., Leelavathi, S., and Reddy, Potvin, G., and Zhang, Z. (2010). Strategies for high-level
S.V. (2011). Advances in plant molecular farming. recombinant protein expression in transgenic microalgae:
Biotechnol. Adv. 29, 210-222. a review. Biotechnol. Adv. 28, 910-918.
Oey, M., Lohse, M., Kreikemeyer, B., and Bock, R. (2009). Pryer, K.M., Schneider, H., Zimmer, E.A., and Ann Banks,
Exhaustion of the chloroplast protein synthesis capacity J. (2002). Deciding among green plants for whole
by massive expression of a highly stable protein genome studies. Trends Plant Sci. 7, 550-554.
antibiotic. Plant J. 57, 436-445. Pujol, M., Gavilondo, J., Ayala, M., Rodríguez, M.,
Oliver, M.J., Armstrong, J., and Bewley, J. (1993). González, E.M., and Pérez, L. (2007). Fighting cancer
Desiccation and the control of expression of β-phaseolin with plant-expressed pharmaceuticals. Trends
in transgenic tobacco seeds. J. Exp. Bot. 44, 1239-1244. Biotechnol. 25, 455-459.
Pantaleoni, L., Longoni, P., Ferroni, L., Baldisserotto, C., Rasala, B.A., Muto, M., Lee, P.A., Jager, M., Cardoso,
Leelavathi, S., Reddy, V.S., Pancaldi, S., and Cella, R. R.M., Behnke, C.A., Kirk, P., Hokanson, C.A., Crea, R.,
(2014). Chloroplast molecular farming: efficient and Mendez, M. (2010). Production of therapeutic
production of a thermostable xylanase by Nicotiana proteins in algae, analysis of expression of seven human
tabacum plants and long-term conservation of the proteins in the chloroplast of Chlamydomonas reinhardtii.
recombinant enzyme. Protoplasma 251, 639-648. Plant Biotechnol. J. 8, 719-733.
Park, Y., and Cheong, H. (2002). Expression and Reddy, V.S., Leelavathi, S., Selvapandiyan, A., Raman, R.,
production of recombinant human interleukin-2 in potato Giovanni, F., Shukla, V., and Bhatnagar, R.K. (2002).
plants. Protein Expr. Purif. 25, 160-165. Analysis of chloroplast transformed tobacco plants with
Parkin, J., and Cohen, B. (2001). An overview of the cry1Ia5 under rice psbA transcriptional elements reveal
immune system. Lancet 357, 1777-1789. high level expression of Bt toxin without imposing yield
Parsell, D., and Lindquist, S. (1993). The function of heat- penalty and stable inheritance of transplastome. Mol.
shock proteins in stress tolerance: degradation and Breed. 9, 259-269.
reactivation of damaged proteins. Annu. Rev. Genet. 27, Rensink, W.A., Iobst, S., Hart, A., Stegalkina, S., Liu, J.,
437-496. and Buell, C.R. (2005). Gene expression profiling of
Pastori, G.M., and Foyer, C.H. (2002). Common potato responses to cold, heat, and salt stress. Funct.
components, networks, and pathways of cross-tolerance Integr. Genomics 5, 201-207.
to stress. The central role of “redox” and abscisic acid- Rival, S., Wisniewski, J.-P., Langlais, A., Kaplan, H.,
mediated controls. Plant Physiol. 129, 460-468. Freyssinet, G., Vancanneyt, G., Vunsh, R., Perl, A., and
Paul, M., and Ma, J.K.-C. (2011). Plant-made Edelman, M. (2008). Spirodela (duckweed) as an
pharmaceuticals: Leading products and production alternative production system for pharmaceuticals: a
platforms. Biotechnol. Appl. Biochem. 58, 58-67. case study, aprotinin. Transgenic Res. 17, 503-513.
Peng, X. C., Qiu, D. W., Zeng, H. M., Guo, L. H., Yang, X. Rivera, A.L., Gómez-Lim, M., Fernández, F., and Loske,
F., and Liu, Z. (2015). Inducible and constitutive A.M. (2012). Physical methods for genetic plant
expression of an elicitor gene Hrip1 from Alternaria transformation. Phys. Life Rev. 9, 308-345.
tenuissima enhances stress tolerance in Rodriguez-Oroz, M., Obeso, J., Lang, A., Houeto, J.-L.,
Arabidopsis. Transgenic Res. 24, 135-145. Pollak, P., Rehncrona, S., Kulisevsky, J., Albanese, A.,
Pérez-Díaz, J., Wu, T. M., Pérez-Díaz, R., Ruíz-Lara, S., Volkmann, J., and Hariz, M. (2005). Bilateral deep brain
Hong, C. Y., and Casaretto, J. A. (2014). Organ-and stimulation in Parkinson's disease: a multicentre study
stress-specific expression of the ASR genes in rice. Plant with 4 years follow-up. Brain 128, 2240-2249.
Cell Rep. 33, 61-73. Ruebelt, M.C., Lipp, M., Reynolds, T.L., Astwood, J.D.,
Pilsyk, S., Natorff, R., Gawińska-Urbanowicz, H., and Engel, K.-H., and Jany, K.-D. (2006). Application of two-
Kruszewska, J. S. (2015). Fusarium sambucinum astA dimensional gel electrophoresis to interrogate alterations
gene expressed during potato infection is a functional in the proteome of genetically modified crops. 2.
orthologue of Aspergillus nidulans astA. Fungal Biol. doi: Assessing natural variability. J. Agric. Food Chem. 54,
10.1016/j.funbio.2015.02.002 2162-2168.
Pogrebnyak, N., Golovkin, M., Andrianov, V., Spitsin, S., Sahebi, M., Hanafi, M.M., Akmar, A.S.N., Rafii, M.Y., Azizi,
Smirnov, Y., Egolf, R., and Koprowski, H. (2005). Severe P., and Idris, A. (2014). Serine rich protein is a novel
acute respiratory syndrome (SARS) S protein production positive regulator for silicon accumulation in mangrove.
in plants: development of recombinant vaccine. Proc. Gene 556, 170-81.
Natl. Acad. Sci. U. S. A. 102, 9062-9067. Saint-Jore-Dupas, C., Faye, L., and Gomord, V. (2007).
Pogue, G.P., Vojdani, F., Palmer, K.E., Hiatt, E., Hume, S., From planta to pharma with glycosylation in the toolbox.
Phelps, J., Long, L., Bohorova, N., Kim, D., and Pauly, M. Trends Biotechnol. 25, 317-323.

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !39

Transgenic Plants Abiri et al.

Sala, F., Manuela Rigano, M., Barbante, A., Basso, B., expressed in transgenic tobacco. Plant Physiol. 124,
Walmsley, A.M., and Castiglione, S. (2003). Vaccine 173-182.
antigen production in transgenic plants: strategies, gene Stoger, E., Sack, M., Fischer, R., and Christou, P. (2002a).
constructs and perspectives. Vaccine 21, 803-808. Plantibodies: applications, advantages and bottlenecks.
Sankararao, K., and Rohini, V. K. (1999). Gene transfer Curr. Opin. Biotechnol. 13, 161-166.
into Indian cultivars of safflower (Carthamus tinctorius L.) Stoger, E., Sack, M., Perrin, Y., Vaquero, C., Torres, E.,
using Agrobacterium tumefaciens. Plant Biotechnol. 16, Twyman, R.M., Christou, P., and Fischer, R. (2002b).
201-206. Practical considerations for pharmaceutical antibody
Schillberg, S., Fischer, R., and Emans, N. (2003). production in different crop systems. Mol. Breed. 9,
'Molecular farming'of antibodies in plants. 149-158.
Naturwissenschaften 90, 145-155. Stöger, E., Vaquero, C., Torres, E., Sack, M., Nicholson, L.,
Schillberg, S., Twyman, R.M., and Fischer, R. (2005). Drossard, J., Williams, S., Keen, D., Perrin, Y., and
Opportunities for recombinant antigen and antibody Christou, P. (2000). Cereal crops as viable production
expression in transgenic plants-technology assessment. and storage systems for pharmaceutical scFv antibodies.
Vaccine 23, 1764-1769. Plant Mol. Biol. 42, 583-590.
Shaaltiel, Y., Bartfeld, D., Hashmueli, S., Baum, G., Brill- Stomp, A.-M. (2005). The duckweeds: a valuable plant for
Almon, E., Galili, G., Dym, O., Boldin-Adamsky, S.A., biomanufacturing. Biotechnol. Annu. Rev. 11, 69-99.
Silman, I., and Sussman, J.L. (2007). Production of Streatfield, S.J. (2007). Approaches to achieve high-level
glucocerebrosidase with terminal mannose glycans for heterologous protein production in plants. Plant
enzyme replacement therapy of Gaucher's disease using Biotechnol. J. 5, 2-15.
a plant cell system. Plant Biotechnol. J. 5, 579-590. Streatfield, S.J., and Howard, J.A. (2003). Plant-based
Sheng, J., and Citovsky, V. (1996). Agrobacterium-plant vaccines. Int. J. Parasitol. 33, 479-493.
cell DNA transport: have virulence proteins, will travel. Sun, N., Liu, M., Zhang, W., Yang, W., Bei, X., Ma, H., and
Plant Cell 8, 1699. Qi, X. (2015). Bean metal-responsive element-binding
Singh, A., Verma, S., and Bansal, K. (2010). Plastid transcription factor confers cadmium resistance in
transformation in eggplant (Solanum melongena L.). tobacco. Plant Physiol. 167, 1136-1148.
Transgenic Res. 19, 113-119. Svanes, C., Sunyer, J., Plana, E., Dharmage, S., Heinrich,
Sood, P., Bhattacharya, A., and Sood, A. (2011). Problems J., Jarvis, D., de Marco, R., Norbäck, D., Raherison, C.,
and possibilities of monocot transformation. Biol. Plant and Villani, S. (2010). Early life origins of chronic
55, 1-15. obstructive pulmonary disease. Thorax 65, 14-20.
Soria-Guerra, R.E., Alpuche-Solís, A.G., Rosales- Tacket, C.O., and Mason, H.S. (1999). A review of oral
Mendoza, S., Moreno-Fierros, L., Bendik, E.M., vaccination with transgenic vegetables. Microbes Infect.
Martínez-González, L., and Korban, S.S. (2009). 1, 777-783.
Expression of a multi-epitope DPT fusion protein in Tait, R.C. (1999). The Application of Molecular Biology.
transplastomic tobacco plants retains both antigenicity Curr. Issues Mol. Biol. 1, 1-12.
and immunogenicity of all three components of the Takahashi, M., Nakanishi, H., Kawasaki, S., Nishizawa,
functional oligomer. Planta 229, 1293-1302. N.K., and Mori, S. (2001). Enhanced tolerance of rice to
Sourrouille, C., Marshall, B., Liénard, D., and Faye, L. low iron availability in alkaline soils using barley
(2009). From Neanderthal to nanobiotech: from plant nicotianamine aminotransferase genes. Nat. Biotechnol.
potions to pharming with plant factories. In Recombinant 19, 466-469.
Proteins From Plants (Springer), pp. 1-23. Talei, D., Valdiani, A., Rafii, M.Y., Maziah, M. (2014)
Spencer, D., Dickey, L.F., Gasdaska, J.R., Wang, X., Cox, Proteomic analysis of the salt-responsive leaf and root
K.M., and Peele, C.G. (2011). Expression of plasminogen proteins in the anticancer plant Andrographis paniculata
and microplasminogen in duckweed (United States Nees. Plos One. 9, e112907. doi:10.1371/journal.pone.
Google Patents). 0112907
Spök, A., Twyman, R.M., Fischer, R., Ma, J.K., and Tarte, V. N., Seok, H. Y., Woo, D. H., Le, D. H., Tran, H. T.,
Sparrow, P.A. (2008). Evolution of a regulatory Baik, J. W., and Moon, Y. H. (2015). Arabidopsis Qc-
framework for pharmaceuticals derived from genetically SNARE gene AtSFT12 is involved in salt and osmotic
modified plants. Trends Biotechnol. 26, 506-517. stress responses and Na+ accumulation in
Srinivasan, A., Yamini, K. N., Reddy, S. S., and Kumar, V. vacuoles. Plant Cell Rep. doi: 10.1007/
D. (2015). Tapetum specific expression of unedited nad3 s00299-015-1771-3
gene from safflower and targeting the protein into Thanavala, Y., Yang, Y., Lyons, P., Mason, H., and Arntzen,
mitochondria induces male sterility in transgenic tobacco C. (1995). Immunogenicity of transgenic plant-derived
plants. Plant Cell Tissue Organ Cult. 120, 387-398. hepatitis B surface antigen. Proc. Natl. Acad. Sci. U. S.
Stahl, R., Horvath, H., Van Fleet, J., Voetz, M., von A. 92, 3358-3361.
Wettstein, D., and Wolf, N. (2002). T-DNA integration into Thompson, J.P., and Debinski, W. (1999). Mutants of
the barley genome from single and double cassette interleukin 13 with altered reactivity toward interleukin 13
vectors. Proc. Natl. Acad. Sci. U. S. A. 99, 2146-2151. receptors. J. Biol. Chem. 274, 29944-29950.
Stevens, L.H., Stoopen, G.M., Elbers, I.J., Molthoff, J.W., Thomson, A.W., and Lotze, M.T. (2003). The Cytokine
Bakker, H.A., Lommen, A., Bosch, D., and Jordi, W. Handbook, Two-Volume Set (Gulf Professional
(2000). Effect of climate conditions and plant Publishing).
developmental stage on the stability of antibodies

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !40

Transgenic Plants Abiri et al.

Tilman, D., Cassman, K.G., Matson, P.A., Naylor, R., and Walter, C., Broer, I., Hillemann, D., and Pühler, A. (1992).
Polasky, S. (2002). Agricultural sustainability and High frequency, heat treatment-induced inactivation of
intensive production practices. Nature 418, 671-677. the phosphinothricin resistance gene in transgenic single
Torrent, M., Llompart, B., Lasserre-Ramassamy, S., Llop- cell suspension cultures of Medicago sativa. Mol. Gen.
Tous, I., Bastida, M., Marzabal, P., Westerholm-Parvinen, Genet. 235, 189-196.
A., Saloheimo, M., Heifetz, P.B., and Ludevid, M.D. Wang, K. (2006a). Methods in Molecular Biology, Vol 1, 2nd
(2009). Eukaryotic protein production in designed edn (New Jersey, USA: Humana press Totowa).
storage organelles. BMC Biol. 7, 5. Wang, K. (2006b). Methods in Molecular Biology, Vol 2, 2nd
Tregoning, J.S., Clare, S., Bowe, F., Edwards, L., edn (New Jersey, USA: Humana press Totowa).
Fairweather, N., Qazi, O., Nixon, P.J., Maliga, P., Wang, L. C., Wu, J. R., Hsu, Y. J., & Wu, S. J. (2015).
Dougan, G., and Hussell, T. (2005). Protection against Arabidopsis HIT4, a regulator involved in heat-triggered
tetanus toxin using a plant based vaccine. Eur. J. reorganization of chromatin and release of transcriptional
Immunol. 35, 1320-1326. gene silencing, relocates from chromocenters to the
Tremblay, R., Wang, D., Jevnikar, A.M., and Ma, S. (2010). nucleolus in response to heat stress. New
Tobacco, a highly efficient green bioreactor for production Phytologist 205, 544-554.
of therapeutic proteins. Biotechnol. Adv. 28, 214-221. Wang, T, E., Sandberg, R., Luo, S., Khrebtukova, I., Zhang,
Turner, N.C., Wright, G.C., and Siddique, K. (2001). L., Mayr, C., Kingsmore, S.F., Schroth, G.P., and Burge,
Adaptation of grain legumes (pulses) to water-limited C.B. (2008). Alternative isoform regulation in human
environments. Adv. Agron. 71, 194-233. tissue transcriptomes. Nature 456, 470-476.
Twyman, R.M., Stoger, E., Schillberg, S., Christou, P., and Wang, Y., Xue, Y., and Li, J. (2005). Towards molecular
Fischer, R. (2003). Molecular farming in plants: host breeding and improvement of rice in China. Trends Plant
systems and expression technology. Trends Biotechnol. Sci. 10, 610-614.
21, 570-578. Ward, J., Bonaparte, M., Sacks, J., Guterman, J., Fogli, M.,
Valdiani, A., Javanmard, A., Talei, D., Tan, S.G., Nikzad, S., Mavilio, D., and Barker, E. (2007). HIV modulates the
Kadir, M.A., Abdullah, S.N.A. (2013) Microsatellite-based expression of ligands important in triggering natural killer
evidences of genetic bottlenecks in the cryptic species cell cytotoxic responses on infected primary T-cell blasts.
“Andrographis paniculata Nees”: A potential anticancer Blood 110, 1207-1214.
agent. Mol. Biol. Rep. 40, 1775-1784. Wilkinson, S., and Davies, W.J. (2002). ABA-based
Valdiani, A., Kadir, M.A., Tan, S.G., Talei, D., Abdullah, chemical signalling: the co-ordination of responses to
M.P., Nikzad, S. (2012) Nain-e Havandi “Andrographis stress in plants. Plant, Cell Environ. 25, 195-210.
paniculata” present yesterday, absent today: a plenary Woo, H. J., Sohn, S. I., Shin, K. S., Kim, J. K., Kim, B. G.,
review on underutilized herb of Iran’s pharmaceutical and Lim, M. H. (2014). Expression of tobacco tocopherol
plants. Mol. Biol. Rep. 39, 5409-5424. cyclase in rice regulates antioxidative defense and
Valdiani, A., Talei, D., Tan, S.G., Kadir, M.A., Maziah, M., drought tolerance. Plant Cell Tissue Organ Cult. 119,
Rafii, M.Y., Sagineedu, S.R. (2014) A classical genetic 257-267.
solution to enhance the biosynthesis of anticancer Wu, J., Yu, L., Li, L., Hu, J., Zhou, J., and Zhou, X. (2007).
phytochemicals in Andrographis paniculata Nees. Plos Oral immunization with transgenic rice seeds expressing
One. 9, e87034. doi:10.1371/journal.pone.0087034 VP2 protein of infectious bursal disease virus induces
Van Droogenbroeck, B., Cao, J., Stadlmann, J., Altmann, protective immune responses in chickens. Plant
F., Colanesi, S., Hillmer, S., Robinson, D.G., Van Biotechnol. J. 5, 570-578.
Lerberge, E., Terryn, N., and Van Montagu, M. (2007). Xiong, L., and You, J. (2014). Use of OsPP18 Gene in
Aberrant localization and underglycosylation of highly Controlling Rice Drought Resistance. US Patent
accumulating single-chain Fv-Fc antibodies in transgenic 20,140,373,193.
Arabidopsis seeds. Proceedings of the National Xu, J., Dolan, M. C., Medrano, G., Cramer, C. L., and
Academy of Sciences 104, 1430-1435. Weathers, P. J. (2012). Green factory: plants as
Vézina, L.-P., Faye, L., Lerouge, P., D’Aoust, M.-A., bioproduction platforms for recombinant proteins.
Marquet-Blouin, E., Burel, C., Lavoie, P.-O., Bardor, M., Biotechnol. Adv. 30, 1171-1184.
and Gomord, V. (2009). Transient co-expression for fast Xue, G.P. (2003). The DNA binding activity of an AP2
and high-yield production of antibodies with human-like transcriptional activator HvCBF2 involved in regulation of
N-glycans in plants. Plant Biotechnol. J. 7, 442-455. low temperature responsive genes in barley is modulated
Vunsh, R., Li, J., Hanania, U., Edelman, M., Flaishman, M., by temperature. Plant J. 33, 373-383.
Perl, A., Wisniewski, J.-P., and Freyssinet, G. (2007). Yang, G., Li, J., Liu, W., Yu, Z., Shi, Y., Lv, B., and Han, D.
High expression of transgene protein in Spirodela. Plant (2015). Molecular cloning and characterization of
Cell Rep. 26, 1511-1519. MxNAS2, a gene encoding nicotianamine synthase in
Walmsley, A.M., and Arntzen, C.J. (2000). Plants for Malus xiaojinensis, with functions in tolerance to iron
delivery of edible vaccines. Curr. Opin. Biotechnol. 11, stress and misshapen flower in transgenic tobacco. Sci.
126-129. Hortic. 183, 77-86.
Walsh, G., and Jefferis, R. (2006). Post-translational Yang, X., Wang, X., Ji, L., Yi, Z., Fu, C., Ran, J., and Zhou,
modifications in the context of therapeutic proteins. Nat. G. (2015). Overexpression of a Miscanthus lutarioriparius
Biotechnol. 24, 1241-1252. NAC gene MlNAC5 confers enhanced drought and cold

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !41

Transgenic Plants Abiri et al.

tolerance in Arabidopsis. Plant Cell Reports. doi: Zhang, G.G., Rodrigues, L., Rovinski, B., and White, K.A.
10.1007/s00299-015-1756-2 (2002). Production of HIV-1 p24 protein in transgenic
Yano, M., Hirai, T., Kato, K., Hiwasa-Tanase, K., Fukuda, tobacco plants. Mol. Biotechnol. 20, 131-136.
N., and Ezura, H. (2010). Tomato is a suitable material Zhang, H., Liu, J., Hou, J., Yao, Y., Lin, Y., Ou, Y., Song,
for producing recombinant miraculin protein in genetically Botao., and Xie, C. (2014). The potato amylase inhibitor
stable manner. Plant Sci. 178, 469-473. gene SbAI regulates cold-induced sweetening in potato
Yokoo, T., Matsumoto, K. I., Ooba, T., Morimoto, K., and tubers by modulating amylase activity. Plant Biotechnol.
Taguchi, S. (2015). Enhanced poly (3-hydroxybutyrate) J. 12, 984-993.
production in transgenic tobacco BY-2 cells using Zhang, Q., Walawage, S. L., Tricoli, D. M., Dandekar, A. M.,
engineered acetoacetyl-CoA reductase. Biosci. and Leslie, C. A. (2015). A red fluorescent protein
Biotechnol. Biochem. doi: (DsRED) from Discosoma sp. as a reporter for gene
10.1080/09168451.2014.1002448 expression in walnut somatic embryos. Plant Cell Rep.
Yokoya, N.S., Ávila, M., Piel, M.I., Villanueva, F., and doi: 10.1007/s00299-015-1749-1
Alcapan, A. (2014). Effects of plant growth regulators on Zhang, Z., Huang, R. (2010). Enhanced tolerance to
growth and morphogenesis in tissue culture of freezing in tobacco and tomato overexpressing
Chondracanthus chamissoi (Gigartinales, Rhodophyta). transcription factor TERF2/LeERF2 is modulated by
J. Appl. Phycol. 26, 819-823. ethylene biosynthesis. Plant Mol. Biol. 73, 241–9.
Yoon, S.-M., Kim, S.Y., Li, K.F., Yoon, B.H., Choe, S., and Zhao, Y., Ma, Q., Jin, X., Peng, X., Liu, J., Deng, L., Yan,
Kuo, M.M.-C. (2011). Transgenic microalgae expressing H., Sheng, L., Jiang, H., and Cheng, B. (2014). A novel
Escherichia coli AppA phytase as feed additive to reduce maize homeodomain–leucine zipper (HD-Zip) I gene,
phytate excretion in the manure of young broiler chicks. Zmhdz10, positively regulates drought and salt tolerance
Appl. Microbiol. Biotechnol. 91, 553-563. in both rice and arabidopsis. Plant Cell Physiol. 55,
Yoshida, T., Fujita, Y., Maruyama, K., Mogami, J., Todaka, 1142-1156.
D., Shinozaki, K., and Yoshida T, Fujita Y, Maruyama K, Zhong, L., Chen, D., Min, D., Li, W., Xu, Z., Zhou, Y., and
Mogami J, Todaka D, Shinozaki K, Yamaguchi-Shinozaki Ma, Y. (2015). AtTGA4, a bZIP transcription factor,
K. (2015). Four Arabidopsis AREB/ABF transcription confers drought resistance by enhancing nitrate transport
factors function predominantly in gene expression and assimilation in Arabidopsis thaliana. Biochem.
downstream of SnRK2 kinases in abscisic acid signaling Biophys. Res. Commun. 457, 433-439.
in response to osmotic stress. Plant Cell Environ. 38, Zhu, S., Li, Y., Vossen, J. H., Visser, R. G., and Jacobsen,
35-49. E. (2012). Functional stacking of three resistance genes
Youm, J.W., Jeon, J.H., Kim, H., Kim, Y.H., Ko, K., Joung, against Phytophthora infestans in potato.Transgenic
H., and Kim, H. (2008). Transgenic tomatoes expressing Res. 21, 89-99.
human beta-amyloid for use as a vaccine against Zhu, X.-G., Long, S.P., and Ort, D.R. (2008). What is the
Alzheimer’s disease. Biotechnol. Lett. 30, 1839-1845. maximum efficiency with which photosynthesis can
Young, A., Lyons, J., Miller, A.L., Phan, V.T., Alarcón, I.R., convert solar energy into biomass? Curr. Opin.
and McCormick, F. (2009). Ras signaling and therapies. Biotechnol. 19, 153-159.
Adv. Cancer Res. 102, 1-17. Zhu, Z., Hughes, K.W., Huang, L., Sun, B., Liu, C., and Li,
Zaslavskaia, L.A., Lippmeier, J.C., Kroth, P.G., Grossman, Y. (1994). Expression of human α-interferon cDNA in
A.R., and Apt, K.E. (2000). Transformation of the diatom transgenic rice plants. Plant Cell Tiss. Org. Cult. 36,
Phaeodactylum tricornutum (Bacillariophyceae) with a 197-204.
variety of selectable marker and reporter genes. J. Ziemienowicz, A. (2013). Agrobacterium-mediated plant
Phycol. 36, 379-386. transformation: Factors, applications and recent
Zhang, D., Nandi, S., Bryan, P., Pettit, S., Nguyen, D., advances. Biocatal. Agric. Biotechnol. 3, 95-102.
Santos, M.A., and Huang, N. (2010). Expression, Ziemienowicz, A., Shim, Y.-S., Matsuoka, A., Eudes, F., and
purification, and characterization of recombinant human Kovalchuk, I. (2012). A novel method of transgene
transferrin from rice (Oryza sativa L.). Protein Expr. Purif. delivery into triticale plants using the Agrobacterium
74, 69-79. transferred DNA-derived nano-complex. Plant Physiol.
158, 1503-1513.

Curr. Issues Mol. Biol. (2016) 18: 21-42. horizonpress.com/cimb !42

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