Ph.D (Ag) Molecular and Subject: ADVANCES IN MBB 602: (3+0)

Topic :- General overview of transgenic plants; Case studies:; Teacher:- Dr. R.S. Sharma Biotechnology Centre, JNKVV, Jabalpur Introduction

Genetically modified crops (GM crops) are plants used in agriculture, the DNA of which has been modified using genetic engineering methods. In most cases, the aim is to introduce a new trait to the plant which does not occur naturally in the species. Examples in food crops include resistance to certain pests, diseases, environmental conditions, reduction of spoilage, resistance to chemical treatments (e.g. resistance to a herbicide), or improving the nutrient profile of the crop. Examples in non-food crops include production of pharmaceutical agents, biofuels, and other industrially useful goods, as well as for bioremediation.

ADOPTION OF BIOTECH CROPS-2018

 High adoption of biotech crops continued in 2018 with 191.7 million hectares worldwide.

On the 23rd year of commercialization of biotech/GM crops in 2018, 26 countries grew 191.7 million hectares of biotech crops – an increase of 1.9 million hectares (4.7 million acres) or 1% from 189.8 million hectares in 2017. Except for the 2015 adoption, this is the 22nd series of increases every single year; and notably 12 of the 18 years with double-digit growth rates.

 The adoption rates of the top five biotech crop-growing countries reached close to saturation. The average biotech crop adoption rate in the top five biotech crop-growing countries increased in 2018 to reach close to saturation, with USA at 93.3% (average for soybeans, maize, and canola adoption), Brazil (93%), Argentina (~100%), Canada (92.5%), and India (95%). Expansion of biotech crop areas in these countries would be through immediate approval and commercialization of new biotech crops and traits to target problems related to climate change and the emergence of new pests and diseases.

 Biotech crops increased ~113-fold from 1996 with accumulated biotech area at 2.5 billion hectares; thus, biotechnology is the fastest adopted crop technology in the world.

Global area of biotech crops has increased ~113-fold from 1.7 million hectares in 1996 to 191.7 million hectares in 2018 – this makes biotech crops the fastest adopted crop technology in recent times. An accumulated 2.5 billion hectares or 6.3 billion acres were achieved in 23 years (1996-2018) of biotech crop commercialization.

 A total of 70 countries adopted biotech crops – 26 countries planted and 44 additional countries imported.

The 191.7 million hectares of biotech crops were grown by 26 countries – 21 developing and 5 industrial countries. Developing countries grew 54% of the global biotech crop area compared to 46% for industrial countries. An additional 44 countries (18 plus 26 EU countries) imported biotech crops for food, feed, and processing. Thus, a total of 70 countries in total have adopted biotech crops.

 Biotech crops provided more diverse offerings to consumers in 2018. Biotech crops have expanded beyond the big four (maize, soybeans, , and canola) to give more choices for many of the world's consumers and food producers. These biotech crops include alfalfa, beets, , squash, eggplant, potatoes, and apples, all of which are already in the market. Two generations of Innate® potatoes with non-bruising, non-browning, reduced acrylamide, and late blight resistant traits as well as non-browning Arctic® apples were already planted in the USA. Brazil planted the first insect resistant (IR) sugarcane; Indonesia, the first drought tolerant sugarcane; and Australia planted the first high oleic acid safflower for R&D and seed propagation. Various trait combinations were also approved including high oleic acid canola, isoxaflutole herbicide tolerant (HT) cotton, stacked herbicide tolerant and high oleic acid soybean, HT and salt tolerant soybean, IR sugarcane, and biotech maize with various IR/HT combinations in stack. Additionally, biotech crop research conducted by public sector institutions include rice, banana, potatoes, wheat, chickpea, pigeon pea, and mustard with various economically-important and nutritional quality traits beneficial to food producers and consumers in developing countries.

 Biotech soybeans covered 50% of global biotech crop area.

The four major biotech crops -- soybeans, maize, cotton, and canola -- in decreasing area, were the most adopted biotech crops by 26 countries. Soybeans lead at 95.9 million hectares at 50% of the global biotech crop adoption, a 2% increase from 2017. This is followed by maize (58.9 million hectares), cotton (24.9 million hectares), and canola (10.1 million hectares). Based on the 2017 FAO global crop area for individual crops, 78% of soybeans, 76% of cotton, 30% of maize, and 29% of canola were biotech crops in 2018.  The area planted to biotech crops with stacked traits increased by 4% and occupied 42% of the global biotech crop area.

Stacked traits with insect resistance and herbicide tolerance increased by 4% and covered 42% of the global area, a testimony to farmers' adherence to smart agriculture with no till and reduced insecticide use. Herbicide tolerance in soybeans, canola, maize, alfalfa, and cotton has consistently been the dominant trait, which in 2018 covered 46% of the global area – a decrease of 1% compared to 2017.

 The top five countries (USA, Brazil, Argentina, Canada, and India) planted 91% of the global biotech crop area of 191.7 million hectares.

The USA led the biotech crop planting in 2018 at 75 million hectares, followed by Brazil (51.3 million hectares), Argentina (23.9 million hectare), Canada (12.7 million hectares), and India (11.6 million hectares) for a total of 174.5 million hectares, representing 91% of the global area. Thus, biotechnology benefitted more than 1.95 billion people in the five countries or 26% of the current world population of 7.7 billion.

India: IR (Bt) cotton adoption increased to 95%

In 2017-18, the adoption of officially approved IR cotton represents 95% of 12.24 million hectares of cotton planted in India. Due to the successful control of the spread of unapproved IR(Bt)/HT cotton, India achieved higher planting of officially approved IR cotton to 11.6 million hectares in 2018-19, an increase of 200,000 hectares over 2017-18 and planted by over 6 million farmers. Adoption rate went down to 93% in 2017 after an all-time high of 96% in 2016 when unapproved IR/HT cotton estimated at 3.5 million packets were planted over approximately 760,000 hectares. Thus, attaining 95% adoption rate again and 6% biotech crop area indicate the restoration of farmers’ confidence on the technology and a sign of demand for the approval of next generation biotech cotton technology including stacked IR/HT cotton. The nationwide management of pink bollworm campaign, which was implemented in cotton growing States, focused on dryland farmers in Maharashtra in 2018. The campaign included farmers educational programs, workshops, and awareness and training programs involving key stakeholders. This contributed to increasing farmer awareness resulting in significant control of pink bollworm in the 2018 Kharif season. However, reports from different maize growing States of India indicated the devastating infestation of fall armyworm on maize, causing heavy damages in both Kharif and Rabi seasons. Activities to raise awareness on the control of fall armyworm is in full swing which could push IR maize planting in India.

STATUS OF APPROVED EVENTS FOR BIOTECH CROPS USED IN FOOD, FEED, PROCESSING, AND CULTIVATION

A total of 70 countries (42 + EU 28, counted as one) have issued regulatory approvals to genetically modified or biotech crops for consumption either as human food, animal feed, as well as for commercial cultivation. Since 1992, there have been 4,349 approvals granted by regulatory authorities of these 70 countries. These were granted to 387 biotech events from 27 biotech crops, excluding carnation, rose, and petunia.

Of these approvals, 2,063 were food, either for direct use or for processing, 1,461 were feed use, for direct use or processing, while 825 were for environmental release or cultivation. United States had the most number of GM events approved, followed by Mexico, Japan, Canada, South Korea, Taiwan, Australia, New Zealand, Philippines, EU, Colombia, and Brazil. Maize still had the most number of approved events (137 in 35 countries), followed by cotton (63 events in 27 countries), potatoes (49 events in 13 countries), soybeans (38 events in 31 countries), and canola (37 events in 15 countries).

The HT maize event NK603 (61 approvals in 28 countries + EU 28) still had the most number of approvals. It was followed by HT soybeans GTS 40-3-2 (57 approvals in 28 countries + EU 28), IR maize MON810 (55 approvals in 26 countries + EU 28), HT/IR maize Bt11 (54 approvals in 25 countries + EU 28), HT/IR maize TC1507 (53 approvals in 25 countries + EU 28), IR maize MON89034 (51 approvals in 24 countries + EU 28), HT maize GA21 (50 approvals in 23 countries + EU 28), HT soybeans MON89788 (45 approvals in 25 countries + EU 28), HT soybeans A2704-12 (45 approvals in 24 countries + EU 28), HT/IR maize MON88017 (45 approvals in 23 countries + EU-28), IR cotton MON531 (44 approvals in 20 countries + EU 28), IR maize MIR162 (43 approvals in 23 countries + EU 28), and HT maize T25 (43 approvals in 20 countries + EU 28).

Ph.D (Ag) Molecular Biology and Biotechnology Subject: ADVANCES IN GENETIC ENGINEERING MBB 602: (3+0)

Topic :- Genetic engineering of herbicide resistance, Teacher:- Dr. R.S. Sharma Biotechnology Centre, JNKVV, Jabalpur Introduction Genetic engineering (GE) refers to techniques used to manipulate the genetic composition of an organism by adding specific genes. The enhancement of desired traits has traditionally been undertaken through conventional plant breeding. GE crops are often broken down into two categories, herbicide tolerant and Plant- incorporated protectants (PIPs).Crops are also engineered or “stacked” to express multiple traits like crops that are resistant to multiple herbicides or are resistant to herbicides and incorporates insecticides. Herbicide tolerant crops are designed to tolerate specific broad-spectrum herbicides, which kill the surrounding weeds, but leave the cultivated crop intact. Currently, the only varieties Cultivated in the U.S. are engineered to be tolerant to glyphosate. However, the U.S. Department of Agriculture (USDA) is currently in the process of deregulating other new varieties of crops that are resistant to 2,4- D and other herbicides.

Strategies for engineering herbicide resistance The engineering of herbicide resistance demonstrates the way in which quite different strategies can be used to achieve the same objective. Mullineaux (1992) identifies four distinct strategies for engineering herbicide resistance. These are: 1. Overexpression of the target protein. This strategy effectively involves titrating the herbicide out by overproduction of the target protein. For example, if the herbicide is a specific inhibitor of one particular enzyme, production of sufficient excess enzyme will partially overcome the inhibition. Overexpression can be achieved by the integration of multiple copies of the gene and/or the use of a strong promoter plus translational enhancer to drive expression of the gene. 2. Mutation of the target protein. The logic behind this approach is to find a modified target protein that substitutes functionally for the native protein and which is resistant to inhibition by the herbicide, and to incorporate the resistant target protein gene into the plant genome. Several sources of resistant proteins can be exploited. Note that both the overexpression and mutated target protein strategies require knowledge of the mode of action of the herbicide. 3. Detoxification of the herbicide, using a single gene from a foreign source. Detoxification is a means of converting the herbicide to a less toxic form and/or removing it from the system. This strategy can be contrasted with the previous two because it does not require a detailed knowledge of the site of action. Table 5.4 shows several examples of specific detoxification reactions for common herbicides. 4. Enhanced plant detoxification. The aim here is to improve the natural plant defences against toxic compounds. This requires detailed information about endogenous plant detoxification pathways and the mechanisms by which compounds are recognised and targeted for detoxification by the plant.

Table-Classification of herbicides according to their mode of action HRAC Mode of action Chemical family Example group ♦ Aryloxyphenoxypropionates Inhibition of acetyl-CoA A ‘FOPs’ Cyclohexanediones carboxylase (ACCase) ‘DIMs’ B Inhibition of acetolactate Sulfonylureas Chlorsulfuron

Imidazolinones synthase (AFS) Triazolopyrimidines (acetohydroxyacid synthase Pyrimidinyl(thio)benzoate Imazapyr

AH AS) Sulfonylaminocarbonyl triazolinones

Triazines Triazinones Inhibition of photosynthesis Triazolinone Cl Atrazine at photosystem II Uracils Pyridazinones Phenylcarbamates

Inhibition of photosynthesis Ureas C2 at photosystem II Amides Nitriles Inhibition of photosynthesis C3 Benzothiadiazinone Bromoxynil at photosystem II Phenylpyridazines

Photosystem-I-electron D Bipyridyliums Paraquat diversion

Inhibition of Diphenylethers E protoporphyrinogen Phenylpyrazoles

oxidase (PPO) N-phenylphthalimides Thiadiazoles Oxadiazoles Triazolinones Oxazolidinediones Pyrimidindiones Others

Bleaching: Inhibition of Pyridazinones carotenoid biosynthesis at F Pyridinecarboxamides the phytoene desaturase Others step (PDS)

Bleaching: Inhibition of 4- Triketones hydroxyphenyl- Isoxazoles F2 pyruvatedioxygenase (4- Pyrazoles HPPD) Others Table captionTable 5.2 Continued HRAC Mode of action Chemical family Example group

Triazoles Bleaching: Inhibition of Isoxazolidinones F3 carotenoid biosynthesis Ureas (unknown target) Diphenylethers G Inhibition of EPSP synthase Glycines Glyphosate

Glufosinate Inhibition of glutamine H Phosphinic acids ammonium synthetase Bialaphos Inhibition of DHP I Carbamates Asulam (dihydropteroate) synthase

Dinitroanilines Phosphoroamidates Microtubule assembly K1 Pyridines Oryzalin inhibition Benzamides Benzenedicarboxylic acids

Inhibition of mitosis/ K2 Carbamates microtubule organisation

Chloroacetamides Acetamides K3 Inhibition of cell division Oxyacetamides

Tetrazolinones Others

Nitriles Inhibition of cell wall L Benzamides (cellulose) synthesis Triazolocarboxamides Uncoupling (membrane M Dinitrophenols disruption)

Thiocarbamates Inhibition of lipid Phosphorodithioates N synthesis—not ACCase TCA Benzofuranes Chlorocarbonic inhibition acids

O Action like indole-acetic Phenoxycarboxylic acids 2,4-D acid (synthetic auxins) Benzoic acids Dicamba

Pyridine carboxylic acids Picloram Quinoline carboxylic acids Others

Phthalamates P Inhibition of auxin transport Semicarbazones

Arylaminopropionic acids z Unknown Pyrazolium

Organoarsenicals

CASE STUDY 1 Glyphosate resistance Glyphosate is a broad-spectrum herbicide that is reputedly effective against 76 of the world’s worst-78-weeds, and is marketed as ‘Roundup’ by the American chemical company . It is a simple glycine derivative (see Table) that acts as a competitive inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase. (EPSPS). Glyphosate binds more tightly to the EPSPS-shikimate- 3phosphate complex than does PEP—its dissociation rate from the complex is 2300 times slower than PEP. Consequently, EPSPS is effectively inactivated once glyphosate binds to the enzyme-substrate complex. EPSPS is a key enzyme in the biosynthetic pathways of the aromatic amino acids phenylalanine, tyrosine and tryptophan. Thus, the herbicidal activity of glyphosate results from its inhibition of the biosynthesis of aromatic amino acids and other products of the shikimate pathway. From this knowledge of the mode of action of glyphosate, is it possible to predict what effects the herbicide is likely to have on the growth of a treated plant? The first deduction is that protein synthesis will be blocked due to the insufficient supply of aromatic amino acids. The most immediate effects of this inhibition would be expected in regions of the plant involved in rapid growth and division, including the meristems. Certain specialised organs, such as the developing endosperm, also vigorously accumulate proteins. In addition, other pathways will be affected by the depletion of aromatic amino acids. The shikimate pathway The biosynthesis of the aromatic amino acids and related compounds shares a common biochemical pathway up to the formation of chorismate. The pathway to chorismate starts with the condensation of phosphoenolpyruvate (PEP) (from glycolysis) and erythrose 4-phosphate (from the pentose phosphate pathway) to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). The next step involves a complex redox/cyclisation reaction producing 3-dehydroquinate. The next two reactions (to form 3-dehydro-shikimate and then shikimate) are catalysed by a bifunctional enzyme in plants. Shikimate kinase then phosphorylates shikimate to produce shikimate 3-phosphate, one of the substrates of 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS adds the enolpyruvyl side chain to shikimate 3-phosphate to form EPSP. Finally, chorismate is formed by the elimination of phosphate from EPSP. Chorismate is the precursor of the phenolic and indole rings of the aromatic amino acids, and of other aromatic compounds. Chorismate mutase is the committing enzyme for phenylalanine and tyrosine synthesis, forming prephenate. Prephenate aminotransferase utilises glutamate or aspartate as amino donors to form arogenate, the immediate precursor of phenylalanine (via arogenate dehydratase) and tyrosine (via arogenate dehydrogenase). These amino acids are themselves the precursors of a wide range of secondary products, including lignins, flavonoids, hydroxycinnamic acids and alkaloids. Alternatively, chorismate may be converted to anthranilate by anthranilate synthase, an enzyme complex with two catalytic subunits—the a-subunit catalysing the amination of chorismate and the removal of the enolpyruvyl side chain, whilst the (3-subunit has glutamine aminotransferase activity. Four subsequent steps are involved in the biosynthesis of tryptophan. Tryptophan itself is the precursor of several important secondary products, including LAA, indole alkaloids, indole glucosinolates, phytoalexins and acridone alkaloids supplies aromatic precursors for a range of phenolic compounds, including lignins, alkaloids and flavonoids. Indeed, 20% of the carbon fixed by plants flows through this pathway, primarily for lignin biosynthesis. Indole compounds other than tryptophan are produced by the same pathway, so auxin (indole-acetic acid, IA A) biosynthesis will also be affected by this herbicide. It is therefore not surprising that glyphosate has such a profound effect on plants. Strategy 1 for glyphosate resistance: overexpression of a plant EPSPS gene Of the four strategies described above, three have actually been tested in the laboratory, two of which form the basis of the current commercial plantings of glyphosate-resistant crops. It is therefore instructive to compare the three strategies. One of the earliest approaches to engine ering glyphosate resistance involved the overexpressiory of a plant EPSPS gene. This was facilitated by the isolation of petunia cDNA from glyphosate-resistant tissue cultures. The stepwise selection of petunia cells capable of growing in the presence of increasing amounts of glyphosate led to the isolation of cultures in which the levels of EPSPS enzyme were much higher than normal. This was found not to be due to increased expression of the EPSPS gene, but was rather the result of gene amplification, such that there were multiple (up to 20) copies of the EPSPS gene in an otherwise normal petunia genome. The EPSPS enzyme was not itself mutated—i.e. the resistance was simply due to the increased amount of enzyme. However, the high levels of EPSPS mRNA made it simpler to isolate the cDNA for this gene and use it for re-introduction into plants. In prokaryotes, the isolation of a strain resistant to a particular toxic compound would usually result from the selective advantage derived from mutations in the gene coding for the affected protein itself, or in related genes involved in transport, detoxification or gene regulation. In contrast, the selection of eukaryotic cells in culture resistant to a particular toxic compound often results from the amplification of the gene encoding the target enzyme, rather than from mutations in that gene. This process is particularly observed during a step-wise selection procedure, in which the level of is increased gradually in each round of selection. One of the best characterised examples is that of methotrexate resistance in cultured mammalian cells. The anticancer drug methotrexate is an inhibitor of the enzyme dihydrofolate reductase (DHFR), which supplies single carbon units for, amongst other things, thymidine synthesis. It is therefore particularly important in cycling cells for the replication of DNA. A step-wise selection of methotrexateresistant cells led to the predominance of cells in which the DHFR gene had been specifically amplified in tandem 40-400-fold. This indicates that gene duplication occurs more frequently than mutation in eukaryotes. The step-wise selection applies a quantitative selection pressure for repeated gene duplications, resulting in a process of ‘accelerated evolution’. The dhfr gene can be amplified to give unstable copies that are extrachromosomal (double minutes) or stable (chromosomal). Extrachromosomal copies arise at early times. The genetic manipulation of herbicide resistance The observation that the endogenous mechanism of resistance that had been selected for in the petunia cells was one of excess normal EPSPS (as a result of gene amplification) indicates that Strategy 1—overexpression of the target protein—should be feasible. The effect of overexpressing the EPSPS gene in the .transgenic petunia was tested in one of the earliest experiments on the engineering of herbicide resistance. T he EPSPS cDNA was fuse d to the CaMV 35S promoter and a nos terminator sequence in the vector pMON 546 and transformed into petunia using . The use of thepianlgene enabled the researchers to avoid one ofthe major obstacles to expression—protein targeting. As noted above, many of the potential target pathways of herbicides are located in the plastids. The targeting of proteins to the plastid. Note that no further manipulation ofthe cDNA was required in order to target this protein to the plastid site of activity, since the plant EPSPS cDNA sequence contains its own transit peptide. Strategy 2 for glyphosate resistance: mutant EPSPS genes Mutated EPSPS genes have been isolated from a number of glyphosate- resistant bacteria. It is instructive to compare two of the early experiments using glyphosate-resistant genes from bacteria. In one, a mutated aroA gene from typhimurium was inserted between the promoter and terminator sequences of the ocs gene of the Ag robacterium tumefaciens Ti plasmid. Only a moderate increase in herbicide tolerance was obtained. The requirement for a functional plastid transit peptide was demonstrated by the construction of a hybrid EPSPS gene by fusion of the C-terminal end of a mutated aroA gene from E. coli to the N-terminal end of the petunia EPSPS cDNA sequence containing the transit peptide sequence. Expression of the chimeric enzyme increased glyphosate tolerance in transgenic from 0.01 mmol I' 1 to 1.2 mmol I -1 glyphosate. This experiment was an important validation of the strategy, and demonstrated the feasibility of expressing prokaryotic genes incorporated into the plant nuclear genome, given appropriate promoter and termination signals. Flowever, note that the expression of prokaryotic genes is often not optimal in transgenic plants and extensive modifications may be required to obtain high levels of expression. These early experiments provided useful evidence for the feasibility of Strategy 2 (the resistant target protein strategy), but also revealed problems associated with the strategy. One is that a mutant enzyme with reduced affinity for a competitive inhibitor may also have a lower affinity (increased K M ) for the substrate. This proved to be the case with the E. coli and S. typhimurium genes. For this reason, glyphosate-resistant genes from other sources have been tested for their effectiveness in different plants. A gene from the herbicide-resistant A. tumefaciens strain CP4 encodes an EPSPS that is resistant to glyphosate, but retains a low K M for phosphoenolpyruvate. This gene, in conjunction with an enhanced CaMV 35S promoter and a chloroplast transit peptide sequence from Arabidopsis or petunia, is incorporated into the current range of Monsanto’s major dicotyledonous Roundup Ready crops (soybean, cotton and oilseed rape). On the other hand, Roundup Ready maize contains a construct optimised for monocotyledonous crops, with a resistant EPSPS gene from maize (isolated after mutagenesis and selection in tissue culture), fused to a rice promoter and maize chloroplast transit peptide sequence. Chloroplast transit peptides and protein targeting Some chloroplast proteins are encoded by the plastid genome and translated on 70S ribosomes in the organelle. However, most chloroplast proteins are encoded in the nuclear genome and translated on 80S ribosomes in the cytoplasm. There must therefore be mechanisms for the recognition and transport of proteins destined for the chloroplast. Note that the chloroplast envelope comprises an outer and inner membrane, and that the thylakoids comprise an additional internal membrane system. Thus there are three distinct compartments in the chloroplast: the intermembrane space of the envelope; the stroma; and the thylakoid lumen. Depending upon the precise destination, a chloroplast protein might be transported across three membrane systems. The means by which all plastid proteins are transported into the chloroplast is by recognition of a sequence of about 40-50 amino acids at the N-terminal end of the protein (the transit peptide). This peptide directs translocation into the stroma, where a specific peptidase removes the transit peptide. Note that the process is ‘post-translational’, i.e. the protein is transported after synthesis, unlike the co-translational transfer of membranebound and secreted proteins into the ER during synthesis on rough ER-bound ribosomes. The transport process into the stroma appears to involve a complex protein import apparatus that spans the inner and outer membranes. Targeting into the thylakoids requires a bipartite transit peptide. Removal of the stromal targeting sequence exposes a second transit peptide that acts as a lumenal targeting peptide. This directs the protein across the thylakoid membrane into the thylakoid lumen, where it is also removed by a specific protease. Strategy 3 for glyphosate resistance: detoxification by heterologous genes An alternative strategy has been developed for engineering glyphosate tolerance based upon a specific detoxification mechanism. In soil , glyphosate can be degraded by cleavage of the C-N bond, catalysed by an oxidoreductase, to form aminomethylphosphonic acid (AMPA) and glyoxylate. A gene encoding the enzyme glyphosate oxidase (GOX) has been isolated from a soil organism, Ochrobactrum anthropi strain LBAA, and modified by addition of a transit peptide. Transgenic crops such as oilseed rape transformed with this gene show very good glyphosate resistance in the field. However, this strategy is not generally used in isolation. Monsanto now employ a dual strategy for oilseed rape (canola), in which both the resistant Agrobacterium CP4 EPSP5 gene and the GOX gene are expressed. In addition to enhanced glyphosate resistance, this approach avoids the accumulation of the herbicide in the resistant plant, because the glyphosate is broken down into relatively harmless products (glyoxylate is a normal plant metabolite, and AMPA can be converted to glycine). This highlights one significant difference between Strategy 2 and Strategy 3. Strategy 2 enables the plant to function in the presence of the herbicide and therefore the herbicide may accumulate to higher levels than those normally found in that crop. In contrast, the detoxification strategy should result either in the destruction of the herbicide, or in the accumulation of a conjugate less harmful than the original compound. Pleiotropic effects of It should be noted at this stage that the insertion of a transgene into a plant may result in unforeseen and perhaps undesirable effects. Roundup Ready crops have not been without problems of this type. One phenomenon encountered by Roundup Ready soybeans during hot weather has been splitting of the stems. It has been suggested that this occurs due to the 20% higher lignin content of these plants. A look back to the mode of action of glyphosate should indicate why the introduction of an additional EPSPS gene could result in increased lignin biosynthesis. It should be remembered that the plant EPSPS enzyme will still be functional under normal growth conditions in the absence of the herbicide, and that the expression of additional enzyme from the transgene may affect the balance of the relevant metabolic pathways. This lesson will be returned to in subsequent chapters, particularly Chapters 10 and 11 dealing with the manipulation of plant metabolic pathways. CASE STUDY 2 Phosphinothricin Glyphosate resistance is one of the most widespread commercial GM traits. The closest rival to glyphosate in terms of the number and acreage of resistant crops is the herbicide phosphinothricin (PPT) or . Whilst both are broad-spectrum herbicides, glyphosate is particularly effective against grasses, whilst phosphinothricin is more effective against broad-leafed weeds and least effective against perennials and volunteer cereals. (In this chapter, phosphinothricin will be used in preference to glufosinate to avoid confusion between glufosinate and glyphosate. Note also that glufosinate is usually applied as the ammonium salt, and is commonly called glufosinate ammonium.) Phosphinothricin is unusual amongst herbicides in being derived from a natural product. Bialaphos is a tripeptide of the form PPT-Ala-Ala produced by certain Streptomyces species. It can be applied directly as a herbicide and has been marketed as such under various trade names, for example 'Herbiace' (Meiji Seika). Bialaphos is converted to the active form lphosphinothricin by proteolytic removal of the alanine residues. Phosphinothricin was marketed by the German company Hoechst, under the trade name ‘Basta’. One point to be aware of in tracking the progress of a particular transgenic crop is the dynamic nature of the agrochemical and biotechnology sector—there have been a number of take-overs and mergers resulting in company name changes. Thus, Hoechst has since undergone a series of mergers such that the Basta brand name has been owned successively by AgrEvo and now Aventis. A different formulation of phosphinothricin is also marketed by Aventis under the brand name Liberty, and complements the LibertyLink lines of transgenic phosphinothricin-resistant crops produced by the same company, which are described below. The herbicidal action of phosphinothricin is a result of its competitive inhibition of glutamine synthetase (GS). The immediate effect of inhibiting GS is the accumulation of ammonia to toxic levels, which rapidly kills the plant cells. The disruption of glutamine synthesis also inhibits photosynthesis, and it is the combined effects of ammonium toxicity and inhibition of photosynthesis that account for the herbicidal activity of phosphinothricin. Uptake of phosphinothricin is through the leaf, the speed of which is dependent on many factors, including plant species, stage of growth, air humidity, temperature and rate of application. Translocation within the plant is limited (unlike glyphosate), and varies according to species. Some limited systemic activity may occur as a result of movement around the leaf, from leaf to leaf and from leaf to roots. This may be sufficient to suppress the regrowth of perennial weeds that are not killed outright by contact activity. However, it will often not provide the ‘roots and all’ kill seen by glyphosate for many perennial grass weeds. Strategy for Basta resistance The natural occurrence of bialaphos provides a lead to follow in devising a strategy for engineering resistance. Toxic compounds such as PPT, with a simple structural homology to a common substrate such as glutamate, are likely to be toxic to the host organism. The fact that the compound is synthesised as an inactive precursor is indicative of this fact. It is therefore not surprising that Streptomyces spp. also contain a detoxification gene that protects the organism from the toxic effects of PPT. The bar gene of Streptomyces hygroscopicus and the closely related pat gene of Streptomyces viridochromogenes code for the enzyme phosphinothricin acetyltransferase (PAT). The addition of an acetyl group to the amino group of phosphinothricin inactivates the compound. Thus, transferring this gene to a plant should, in theory, provide resistance against phosphinothricin. This approach to the engineering of phosphinothricin resistance was developed by Plant Genetic Systems under contract from Hoechst. The conversion of bialaphos to phosphinothricin involves removal of the two alanine residues by a peptidase. The compound acts as a competitive inhibitor of glutamine synthase (GS), and the figure highlights the structural similarity between phosphinothricin and the substrate L-glutamate. The detoxification reaction catalysed by phosphinothricin acetyltransferase (PAT) is also shown the 35S promoter. The current major LibertyLink crop lines supplied by Aventis are oilseed rape, maize and chicory. The bar gene has also proved to be useful as a selectable marker for the transformation and regeneration of transgenic plants. It provides an alternative to selection with antibiotics such as kanamycin, to which different species have a highly varied response. Transgenic plants can be selected directly on PPT medium, but caution is required. Inhibition of GS by PPT causes NH 3 accumulation in non- transgenic material and hence death of the plant tissue, but accumulation of NH 3 in the non-transformed tissue can also cause problems of toxicity to neighbouring transformed cells. Prospects for plant detoxification systems One other strategy that has yet to be fully exploited is the possibility of enhancing endogenous plant detoxification mechanisms. Many xenobiotic (foreign) compounds are detoxified in plants but the pathways may involve more than one step, such as hydroxylation, conjugation and transport stages, so it may prove difficult to identify single-gene mechanisms to engineer resistance. The hydroxylation of compounds involves enzymes such as the cytochrome P450 monooxygenases, which form a large gene family. For example, the analysis of weeds resistant to the herbicide bromoxynil revealed that the bromoxynil was being detoxified by an endogenous cytochrome P450 monooxygenase. This offers the opportunity to use endogenous plant genes to enhance resistance against a range of herbicides. However, more research is required to identify which members of the cytochrome P450 gene family are specific for particular classes of xenobiotics, and which have roles in normal metabolic pathways. Plant detoxification pathways often involve conjugation to glutathione by glutathione 5-transferase (GST) activity, and specific transport of the conjugate into the vacuole. GSTs also comprise a large gene family, some members of which are known to be involved in endogenous metabolic reactions. In some cases, the hydroxylation and conjugation pathways operate in concert. Hence, the resistance of maize to atrazine is ascribed to a twostep pathway involving both 2- hydroxylation and conjugation to glutathione. Thus, there is the potential here to enhance endogenous systems, or transfer systems between plant species, once more information about the functions of these large gene families is available. The exploitation of functional genomics techniques will accelerate the acquisition of this knowledge. The detoxification of atrazine is a two stage process involving a 2- hydroxylation step (removing the chlorine residue) prior to the addition of glutathione. Glutathione is a tripeptide in which the key residue is the middle cysteine. The SH group is involved in a number of redox and conjugation reactions.

Ph. D (Ag) Molecular Biology and Biotechnology Subject: ADVANCES IN GENETIC ENGINEERING MBB 602: (3+0)

Topic:- Transgenic plants resistant to insects/pests, Teacher:- Dr. R.S. Sharma Biotechnology Centre, JNKVV, Jabalpur Introduction The large scale of the problem of world crops lost to weeds, pests and diseases was discussed. It has been estimated that 13% of the potential world crop yield is lost to pests (see Figure 5.1). Plant pests range from nematodes to birds and mammals, but insect pests cause a major proportion of the total pest damage to crops. This chapter will therefore focus upon biotechnological solutions to the problem of insect pest damage to crops. Far from reducing pest damage, modern agricultural practices have, if anything, exacerbated the problem. One of the contributory factors to this exacerbation has been the breeding out of endogenous pesticidal traits. Some defensive characters may have been lost accidentally during the selection process for other properties, but pesticidal traits may also have been removed ‘deliberately’ because they affect other characteristics like yield and quality. For whatever reason, the fact remains that many current elite cultivars have less natural resistance to pests than did their predecessors. This problem is compounded by the practice of growing monocultures, since the growing of a single crop over a wide area in repeated years encourages the build-up of pests. These negative effects of modern agriculture on pest damage have encouraged an increasing reliance on chemical , but this has tended to drive a cycle in which each new just keeps pace with the appearance of resistance to the previous pesticide in the insect population. Against this background, the possibility of developing insect-resistant crops that could help to reduce insect damage, whilst reducing the reliance on chemical insecticides, has a number of attractions. This chapter will consider the scientific strategies for achieving pest resistance before returning to discuss the potential benefits and drawbacks of GM approaches. The nature and scale of insect pest damage to crops Before examining GM strategies for developing insect resistance, it is useful to consider some of the characteristics of the insects causing the damage. The Table Common insect pests of major crops first point to make is that, whilst some adult insects feed off plants and can damage crops (think of a plague of locusts), most of the problems are caused by insect larvae. The major classes of insect that cause crop damage are the orders Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes), Orthoptera (grasshoppers, crickets), Homoptera (aphids) and Coleoptera (beetles).

Table-lists a few of the common pests that cause extensive damage to some of the major crops of the world. Insect species Common name of pest Order Crops affected

Ostrinia nubilalis European corn borer Lepidoptera Maize

Heliothis virescens Tobacco budworm Lepidoptera Tobacco, cotton

Old world bollworm tomato Heliothis armigera Lepidoptera Cotton, tomato fruitworm

Helicoverpa zea Cotton bollworm Lepidoptera Cotton

Manduca sexta Tobacco hornworm Lepidoptera Tobacco, tomato, potato

Spodoptera Maize, rice, cotton, Cotton leafworm Lepidoptera littoralis tobacco

Leptinotarsa Colorado beetle Coleoptera Potato decemlineata Callosobruchus Cowpea seed beetle Coleoptera Cowpea, soybean maculatus

Tribolium Confused flour beetle Coleoptera Cereal flours confusum

Locusta migratoria Locust Orthoptera Grasses

Nilaparvata lugens Brown plant hopper Homoptera Rice

GM strategies for insect resistance: The approach

Here, two approaches will be compared: first, the use of bacterial insecticidal genes to provide protection from pest damage; and, second, the potential for using endogenous plant protection mechanisms (a ‘Copy Nature’ approach). By far the most widespread example of the first approach is the use of the cry endotoxin genes from Bacillus thuringiensis.

B. thuringiensis was discovered by Ishiwaki in 1901 in diseased silkworms, and was subsequently classified and named after its isolation from the gut of diseased flour moth larvae in Thuringberg, by Ernst Berliner. The adverse effect of the bacteria on the insect larvae was subsequently identified as arising from a number of produced by the bacteria. The bacterium produces an insecticidal crystal protein (ICP) which forms inclusion bodies of regular bipyramidal or cuboidal crystals during sporulation. ICPs are one of several classes of endotoxins produced by the sporulating bacteria, hence they were originally classified as 5- endotoxins, to distinguish them from other classes of a-, p- and y-endotoxins.

Table-Classification of the insecticidal crystal protein genes of Bacillus thuringiensis Protein cry gene B. thuringiensis subspecies/strain of size Susceptible insect class families holotype (kDa) crylAa(l-14) 133 kurstaki Lepidoptera crylAb(l-16) 130 berliner Lepidoptera crylAc(l-15) 133 kurstaki Lepidoptera crylAd-g 133 aizawai Lepidoptera crylBa(l-4) 140 kurstaki Lepidoptera crylBb-g 1340 EG5847 Lepidoptera cryl Ca(l-8) 134 entomocidus Lepidoptera crylCb(l-2) 133 galleriae Lepidoptera crylDa(l-2) 132 aizawai Lepidoptera crylDb(l-2) 131 BTS00349A crylEa(l-6) 133 kenyae Lepidoptera crylEbl 134 aizawai Lepidoptera crylFa(l-2) 134 aizawai Lepidoptera crylFb(l-5) 132 morrisoni crylGa(l-2) 132 BTS00349A crylGb(l-2) 133 wuhanensis Lepidoptera crylHa-b 133 BTS02069AA crylIa(l-9) 81 kurstaki Lepidoptera Lepidoptera & cryllb-e 81 entomocidus Coleoptera crylJa-d 133 EG5847 Lepidoptera crylKal 137 morrisoni Lepidoptera cry2Aa(l-10) 71 kurstaki Lepidoptera & Diptera cry2Ab(l-5) 71 kurstaki Lepidoptera cry2Ac(l-2) 70 shanghai Lepidoptera cry3Aa(l-7) 73 tenebrionis Coleoptera cry3Ba(l-2) 75 tolworthi Coleoptera cry3Bb(l-3) 74 EG4961 Coleoptera cry 3 Cal 73 kurstaki Coleoptera cry4Aa(l-3) 135 israelensis Diptera cry4Ba(l-5) 128 israelensis Diptera cry5Aal 152 darmstadiensis Nematodes crySAbl 142 darmstadiensis Nematodes cry5Acl 135 PS86Q3 Hymenoptera cry5Bal 140 PS86Q3 Hymenoptera cry6Aa(l-2) PS52A1 Nematodes cry6Bal PS69D1 Nematodes cry7Aal 129 galleriae Coleoptera cry7Ab (1-2) 130 dakota Coleoptera cry8A-D 131 kumamotoensis Coleoptera cry9Aa(l-2) 130 galleriae Lepidoptera cry9Bal galleriae Lepidoptera cry 9 Cal 130 tolworthi Lepidoptera cry9Da(l-2) 132 japonensis crylOAal 78 israelensis Diptera cryl lAa(l-2) 72 israelensis Diptera cryllBa-b 81 jegathesan Diptera cry 12 - 40 various various various

The structure of the cry genes and their 5-endotoxin products have been well characterised. The cry genes are carried on plasmids and belong to a superfamily of related genes. Table shows the classification of the cry gene superfamily according to size and sequence similarities. This classification was introduced in 1998 and supersedes previous nomenclature that may still appear in the literature. The sequence comparison indicates a large number of distinct families (cryl-cry40 at the time of publication) and within each family there may be three further levels or ranks of subfamily. Hence, crylAal is very closely related to the other crylAa genes, which are all closely related to the other crylA genes, which all form part of the cryl family. Table shows that the strains of B. tburingiensis produce a wide range of different crystal proteins. Thus, it is not just a case of each strain containing one specific cry gene encoding one particular crystal.

Apart from sequence similarity, it is apparent that there is a large difference in size between different Cry proteins, though they tend to cluster as either large (~130kDa) or small (~70kDa) proteins. Although there is a considerable difference in size between the different subfamilies, they share a common active core comprising three domains. Figure 6.1 shows a simple alignment of different classes of Cry protein, showing the common core domains. It can be seen that the N- terminal end of each gene has a similar organisation, despite the great difference in overall length. In fact, the larger proteins such as the Cryl group are inactive, and are activated by proteolytic removal of the Cterminal sequence.

Figure shows the structure of the active core of the CrylA protein, exemplifying the three discrete domains, each with a quite different folding structure. Domain I at the N-terminal end comprises a series of a-helices arranged in a cylindrical formation. The open cylindrical structure of this domain is thought to be responsible for creating a pore through the membrane of the insect midgut. Domain II comprises a triple p-sheet and is involved in receptor recognition. Domain III is a P-sandwich, with a number of putative roles including protection from degradation, toxin/bilayer interactions and receptor binding.

The mode of action of 5-endotoxins involves a specific interaction between the protein and the insect larva midgut. After ingestion by an insect larva, the protein crystals are solubilised in its midgut. The larger proteins such as the 130- kDa Cryl group are proteolytically cleaved at this stage to release the active 55-70- kDa active fragment of the protein. This interacts with highaffinity receptors in the midgut brush-border membrane. The result of this binding is to open cation- selective pores in the membrane. The flow of cations into the cells results in osmotic lysis of the midgut epithelium cells, causing their destruction. Thus, the 8- endotoxins are extremely toxic and can be lethal to susceptible insect larvae at relatively low concentrations. On the other hand, their toxicity to other animals (e.g. mammals) is extremely low. Table The range of insecticidal crystal proteins in individual Bacillus thuringiensis strains

B.t. subpecies and Crystal protein strains CrylAa, CrylAb, CrylAd, CrylCa, CrylDa, CrylEb, CrylFa, Cry9Ea, aizawai Cry39Aa, Cry40Aa entomocidus CrylAa, CrylBa, CrylCa, Cryllb galleriae CrylAb, CrylAc, CrylDa, CrylCb, Cry7Aa, Cry8Da, Cry9Aa, Cry9Ba israelensis CrylOAa, CryllAa japonensis Cry8Ca, Cry9Da jegathesan CryllBa, Cryl9Aa, Cry24Aa, Cry25Aa kenyae Cry2Aa, CrylEa, CrylAc kumamotoensis Cry7Ab, Cry8Aa, Cry8Ba kurstaki HD-1 CrylAa, CrylAb, CrylAc, Crylla, Cry2Aa, Cry2Ab kurstaki HD-73 CrylAc kurstaki NRD-12 CrylAa, CrylAb, CrylAc morrisoni CrylBc, CrylFb, CrylHb, CrylKa, Cry3Aa tenebrionis Cry3Aa tolwortbi Cry3Ba, Cry9Ca wuhanensis CrylBd, CrylGa, CrylGb

Figure- Ribbon model of CrylAa toxin molecule. The structure of the activated toxin is shown, demonstrating the three distinct domains. The a-helical cylinder that forms domain I is involved in membrane insertion and pore formation.

The conditions in the insect larva midgut vary according to insect class. The midgut of Lepidoptera and Diptera is mildly alkaline, whilst the coleopteran gut is generally either more alkaline or acidic. These different conditions favour the solubilisation and activation of different Cry subfamilies. Furthermore, the specificity of the interaction between the endotoxin and the midgut receptor means that individual Cry proteins are active against particular insect larvae.

The use of Bt’ as a biopesticide

Preparations of B. thuringiensis spores or isolated crystals have now been used as an ‘organic’ pesticide for half a century. The isolated crystals have a limited persistence on foliage of a few days, whilst the spore preparations are effective for about 40 days on foliage and up to 2 years in soil. Neither method of application has a particularly effective penetration, with regard to concealed surfaces and organs of the plant, or against sap-sucking insects—i.e. they are not systemic. However, the agronomic experience of using B. thuringiensis spores or isolated crystals as a biopesticide, coupled to the associated safety data and regulatory approvals, has been useful for the rapid development of the ‘ Bt’ strategy to genetic modification.

Note that Bt is generally used as a shorthand for a crop transformed with a cry gene (hence Bt cotton, etc.), and also for the Cry proteins (hence Bt protein). This can be confusing, bearing in mind that we have now met four different terms for effectively the same group of proteins—ICP, 8-endotoxin, Cry and now Bt.

Bt-based genetic modification of plants Although the GM approach to using the cry genes to obtain pest resistance in plants is conceptually simple, it does provide an object lesson in the detailed molecular biology that may be required to achieve high levels of expression of a bacterial gene in a transgenic plant. This goes beyond the obvious requirements for plant promoter and terminator sequences to regulate transcription. The first attempts t o express CrylA and Cry3A proteins under the control of the CaMV 35S or Agrobacterium T-DNA promoters resulted in very low levels of expression in tobacco, tomato and potato plants. It was realised that the prokaryotic gene sequence itself would need to be expensively modified in order to obtain high levels of stable expression. The eventual result was that expression was enhanced by 100-fold to give much better levels of expression, of the order of 100 ng Bt protein per mg total protein. Subsequent laboratory tests showed that the effect of producing a specific Cry protein at this level was to provide a considerable degree of protection against damage by susceptible insect larvae. Further confirmation of the effectiveness of these plants in providing protection against insect damage in small scale field trials resulted in the first Bt crops gaining approval for commercial planting in the USA in the mid-1990s.

The success of the Bt approach led to the development of Bt crops by several of the major biotechnology companies involved in crop protection (Table). This table demonstrates the point that the specificity of Cry proteins permits the targeting of specific pests by particular transgenes, and that different crops may have different cry genes inserted. We will see later that the nature of the cry gene construct can be important for the success or failure of a particular transgenic crop, and that some of the lines shown in Table. Indeed, maize and cotton are the only Bt crops that are currently commercially grown in the USA, since the New-Leaf Bt potato has now been discontinued. Table-Commercialisation of Bt technology

Company Trade name Bt protein Crops Insect pests Monsanto New-Leaf Cry3A Potato Colorado beetle Monsanto Bollgard Cry 1 Ac Cotton Tobacco budworm, cotton bollworm, pink bollworm Monsanto YieldGard CrylAb Maize European corn borer YieldGard, Novartis Knockout Mycogen NaturGard

DeKalb Bt-Xtra Cry 1 Ac Maize European corn borer Aventis StarLink Cry9C Maize European corn borer Mycogen Herculex 1 Cry IF Maize European corn borer Pioneer Monsanto pending Cry3Bb Maize Corn rootworm larvae

CASE STUDY 1 Resistance of Bt-maize to the European corn borer and other pests

The European corn borer (Ostrinio nubilalis or ECB) is a major pest of maize. As the name suggests, the larvae damage maize crops by tunnelling into the central pith of the stalks and ears. However, despite its name, it is not confined to Europe, and causes considerable damage to the maize crop worldwide. Developing an ECB-resistant maize has therefore become one of the main targets of the agricultural biotechnology industry. Table shows that several different companies have each produced a Bt maize line which is resistant against ECB. The rate of adoption of Bt-corn has been rapid in the USA, growing from <5% of the crop acreage in 1996 to ~25% in 2000.

The development of commercial lines involves testing a large number of transformed plants to find the optimal line in terms of the quality of the desired trait, the copy number and stability of integration of the transgene, as well as the stability, level and pattern of transgene expression. Regulatory approval is normally given only for the varieties ultimately descended from a single, fully characterised transformation event.

Three different transformation events with the crylAb gene (176, Btll and Mon810) have been developed by different companies and subsequently licensed to, and marketed by, a number of others. In all three cases, a truncated crylAb gene with modified codon usage optimised for maize expression was used. The transgene in the Btl 1 (YieldGard, Novartis) and Mon810 (YieldGard, Monsanto) events is regulated by a constitutive promoter. On the other hand, the Btl 76 event (Knockout, Novartis; and NaturGard, Mycogen) was produced by bombardment with two separate crylAb constructs: one controlled by a maize PEP carboxylase promoter (specific for green tissue), and the other under the control of a promoter from a pollen-specific protein kinase gene. Varieties derived from the Btl 76 event proved to be less effective against the second brood of ECB than the other two, and have now been phased out. Not surprisingly, the Btl 76 event was also shown to produce the Cry 1 Ab toxin in pollen at much higher levels than the other two, with consequences for the potential toxicity of the pollen to non-pest lepidopterans (see Box 6.3). Other cry genes have also been tried, including cry Me (Bt-Xtra, DeKalb), cry9C (StarLink, Aventis) and crylF (Herculex, Mycogen). The particular problems experienced by the StarLink lines.

The current Bt maize hybrids have generally provided better control against ECB than could be achieved with a single, well-timed insecticide treatment. Btl 1 and Mon810 also provide some control against corn earworm. On the other hand, Cry 1F provides additional protection against the fall armyworm and western bean cutworm. However, these Cry 1 proteins are not effective against other maize pests such as corn rootworm beetles, corn. The Aventis Bt cry9C maize event, marketed as StarLink, has been the topic of considerable controversy in the USA and Europe. The problem arose when AgrEvo (subsequently Aventis) sought approval for registration of StarLink in the USA. There were doubts raised by the US Environmental Protection Agency about the suitability of the Cry9C protein for human consumption, because it is more stable in acid than other approved Cry proteins (hence more slowly digested in the stomach), and because of a lack of data about allergenicity. AgrEvo accepted a limited and conditional approval for StarLink in order to get the product launched into what was a very competitive marketplace for GM maize. Under the terms of this limited registration, StarLink products could only be used for animal feed, and had to be prevented from contaminating food for human consumption. AgrEvo proceeded with the commercial planting, given that the bulk of US maize is used for animal feed and industrial purposes, whilst carrying out further allergenicity tests on the protein. Unfortunately, in 2000, before StarLink products had been approved for human consumption, traces of StarLink corn were identified in taco shells manufactured by Kraft Foods. There was an immediate recall of these products, followed by costly recalls of other maize products from other suppliers.

The case raises a number of issues around the decision to give conditional approval, and the mechanisms required for ensuring that the growers and processors of GM products complied with the conditions placed upon the biotechnology company that registered the transgenic crop. The case has also demonstrated the requirement to investigate the allergenicity of GM products, and highlighted the need for test procedures to detect the presence of GM materials in non-GM products. rootworm larvae or spider mites. Maize hybrids containing the cry3Bb gene are being tested by Monsanto and may soon appear for the control of corn rootworm larvae. Ph. D (Ag) Molecular Biology and Biotechnology

Subject: ADVANCES IN GENETIC ENGINEERING MBB 602: (3+0)

Topic:- Genetic engineering of abiotic stress tolerance, Teacher:- Dr. R.S. Sharma Biotechnology Centre, JNKVV, Jabalpur

Introduction

Crop plants are subject to a range of external factors that adversely affect their growth and development. In the previous four chapters, we have considered the effects of various biological factors (weeds, pests and diseases) on crop yields, and biotechnological strategies to resist them. In this chapter, we will look at the effects of environmental conditions such as temperature, water availability and salinity on crop plants. Figure shows a number of physical factors that may impose an abiotic stress on plants and adversely affect their growth and development. These include a number that can be grouped together as temperature stresses (heat, chilling and freezing), which in turn belong to a larger subgroup that can be categorised as stresses that result in water-deficit. The figure also emphasises the point that most abiotic and biotic stresses directly or indirectly lead to the production of free radicals and reactive oxygen species (ROS), creating oxidative stress.

The impact of abiotic stresses on crop yield compared to biotic stresses (weed, pest and disease effects) is shown in Table. One of the first things to notice is the large difference between the average yields of crops and the record yields (an indicator of the maximum yields possible under ideal conditions). It is clear from this data that the major difference between record yield and average yield is accounted for by abiotic stress. Thus, the variation in environmental conditions from one year to the next produces such a variation in yield for wheat that the average yield is only 13% of the maximum. The control of biotic stresses in industrialised farming is such that they tend to reduce the annual yield by a fairly stable proportion, which is generally less than the most adverse abiotic stresses. Improving the tolerance of crops to abiotic stresses could therefore enable them to maintain growth and development during the normal fluctuations of adverse conditions, and consequently buffer crops against the large swings in yield experienced from one year to the next.

In the longer term, the predicted depletion of the ozone layer and climate changes associated with global warming are likely to add to the burden of environmental stresses on crop plants, and increase the imperative to develop stress-tolerant varieties. Furthermore, there is increasing pressure to extend the area of crop cultivation to environments which are not optimal for the growth of major crops (e.g. desert or high-salt conditions). The development of stress-tolerant plants is therefore a major goal of crop biotechnology, and one that is likely to become increasingly important.

Figure-The different types of external stresses that affect plant growth and development. The figure shows some of the external stresses impacting upon plant growth and development. The stresses are grouped according to common characteristics. Thus, abiotic and biotic stresses are defined, and the range of abiotic stresses resulting in water deficit are shown. The figure emphasises the point that virtually all stresses result in the production of reactive oxygen species (ROS) and this creates oxidative stress.

The nature of abiotic stress

When discussing the subject of stress tolerance, it is necessary first to try to define stress in relation to plant physiology. Plants are subject to many types of fluctuation in the physical environment. Many of the strategies used by animals to avoid the effects of these fluctuations are not available to plants, because of the sessile nature of their growth habit. Plants therefore depend largely upon internal mechanisms for tolerating variations in the external environment. Not all such fluctuations present a stress to plants, since they are able to cope with normal variation by virtue of their plasticity (the ability to alter metabolic profiles and developmental trajectories in response to external conditions. Thus, plants are adapted to function in a fluctuating environment, and normal external changes are countered by internal changes without detriment to growth and development. It is only acute or chronic extremes of environmental condition that lead to environmental stress which has the potential to cause physical damage to the plant. Note also that the ‘extremes of environmental condition’ are defined by the normal environment of the particular plant species in question—desert plants do not prosper in the ‘mild’ climate of the temperate regions.

Table-Average and record yields of some major crops

Record Average Average yield (% of Average loss (% of record Average loss (% of record Crop yield yield record yield) yield) Biotic yield) Abiotic (kg/ha) (kg/ha)

Wheat 14500 1880 13.0 5.0 82.1

Barley 11400 2050 18.0 6.7 75.4

Soybean 7390 1610 21.8 9.0 69.3

Corn 19300 4600 23.8 10.1 65.8

Potato 94100 28 300 30.1 18.9 54.1

Sugar 121000 42600 35.2 14.1 50.7 beet

Given the range of abiotic stresses to which plants are exposed, it might be thought that a wide range of different strategies would be required to engineer particular types of stress tolerance. However, many different stresses cause similar types of damage to plants. This chapter will concentrate on the two major types of damage that result from a variety of different stresses. One of these is the damage that results from water deficit caused by a number of different environmental conditions, including drought, salinity, heat and cold. The other type of damage results from the production of reactive oxygen species (ROS), causing chemical damage to the cellular constituents of plants. Most abiotic and biotic stresses cause the production of reactive oxygen species either directly, or indirectly.

The nature of water-deficit stress Several of the physical changes result in a water deficit, i.e. a situation in which the demand exceeds the supply of water. The supply is determined by the availability of water in the soil to the roots. The demand for water is determined mainly by the plant transpiration rate. Transpiration, the loss of water from the aerial parts of the plant via the leaf stomata, is required to dissipate the energy received from solar radiation and the ambient air temperature. Thus, transpiration cause leaves to cool relative to the ambient temperature when the environmental energy load on the plant is high. The rate of transpiration is also affected by the relative humidity and wind speed.

Two parameters are used to describe the water status of plant cells and tissues: water potential and relative water content. Water potential is measured in units of pressure (M pascal or MPa) and is similar in concept to electrical potential—current flows from a compartment of high potential to one of low potential if a connection is made between them. Thus, water is driven through the plant from the soil to the atmosphere by the difference in water potential between the atmosphere (very low potential) and the soil (relatively high potential when wet). As water transpires from the leaf, leaf water potential is reduced; but if water is available in the soil, water will flow into the leaf to replenish the loss. However, if the soil water potential starts to reduce (because the soil is drying out, for example), a deficit between the supply from the root and demand from the leaf is created. Clearly, a continued water deficit would lead to dehydration and death, but plants can respond to water deficit in a number of different ways. One way is to reduce the water potential in the leaf in order to create the necessary gradient to maintain the flow of water from soil.

Two major factors determine water potential: the concentration of solutes that creates the solute- or osmotic potential; and the physical pressure of the cell or tissue boundaries that forms the pressure- or turgor potential. The osmotic potential is generally lower (more negative) than the water potential, and the turgor potential is the difference between them. Leaf turgor is lost (turgor potential = 0) when the leaf water potential drops to a value equal to the osmotic potential. As leaf turgor falls, the stomata close to reduce transpiration. When turgor is lost, the leaf cells collapse and the leaf wilts. These effects have the result of reducing water loss from the leaf, by closure of the stomata, and because wilting or rolling up of leaves reduces their exposure to sunlight.

However, the reduction in stomatal conductance also causes a reduction in photosynthetic assimilation, thus reducing growth. There may also be an increase in leaf temperature to a level that could cause heat damage to the leaf. The maintenance of turgor and transpiration are therefore important in order to maintain the growth of plants under drought stress. Turgor could be sustained either by keeping the leaf water potential high (by maintaining water uptake from the drying soil) or by reducing the osmotic potential. The latter could be achieved by solute accumulation (remember that an increase in solute concentration creates a more negative osmotic potential, i.e. a steeper gradient for the movement of water)—a process called osmotic adjustment.

Different abiotic stresses create a water deficit

Drought is, by definition, a water-deficit stress, because the environmental conditions either reduce the soil water potential and/or increase the leaf water potential due to hot, dry or windy conditions. High-salt conditions can result in water deficit because the soil water potential is decreased (because the osmotic potential of a salt solution is lower than that of water), making it more difficult for roots to extract water from the environment. As stated above, high ambient temperatures cause increased water loss by evaporation, so heat stress creates a water deficit. However, freezing temperatures also cause osmotic stress because the formation of ice crystals in the extracellular space reduces the water potential and results in the efflux of intracellular water. In general, these various causes of water deficit result in the efflux of cellular water, leading to plasmolysis and eventually cell death. Water deficit is a particular problem to plant cells because it inhibits photosynthesis via its effect on the thylakoid membranes. Water deficit is also potentially damaging to all cells because of the increase in the concentration of toxic ions and the loss of the protective hydration ‘shell’ around vulnerable molecules.

As described above, cells are able to accommodate some fluctuation in the water potential by osmotic adjustment—increasing the solute concentration and hence reducing the osmotic potential. However, as shown in Box 9.1, an increase in ions such as Na + and CF would be counterproductive, given their effects on vulnerable molecules. Instead, plant cells respond to osmotic stress by producing non-toxic compounds called ‘compatible solutes’ (or ‘osmolytes’, or ‘osmoprotectants’) to reduce the osmotic potential. It has been proposed that one reason for the non-toxicity of these compounds is that they can accumulate to high intracellular concentrations without disrupting the hydration shell around proteins and membranes.

Figure shows a range of compatible solutes produced by plants. These fall into two broad classes: and sugar alcohols; and zwitterionic compounds (i.e. carrying a positive and negative charge). The former class includes sugar alcohols such as mannitol, , pinitol and D-ononitol, and oligosaccharides such as trehalose and fructans. The latter class includes amino acids such as proline, and quaternary ammonium compounds such as glycine betaine. Different plant species tend to produce different compatible solutes. For example, mannitol is produced in large amounts by celery in response to salt stress, whereas spinach accumulates glycine betaine. In fact, spinach cells grown in a high-salt medium accumulate glycine betaine to a concentration of 300 mmol F 1 in the cytosol. This maintains the osmotic potential of the cytoplasm relative to the apoplast (the connected extracellular space outside the cell; the symplast is the continuous intracellular compartment within protoplasts, connected by plasmodesmata) whilst keeping the cytoplasmic concentration of Na + and CF ions low (<50mmoll _1 ) and storing high concentrations of Na + (200 mmol F 1 ) and CF (150mmolF a ) ions in the vacuole. It is particularly significant that certain crop plants (e.g. rice and tobacco) lack significant amounts of any of these major classes of osmoprotectants. Basic strategies for engineering resistance to water-deficit stress have therefore focused on the production of

The water shell around macromolecules

The precise folding of soluble proteins is determined not only by intramolecular interactions between residues in the molecule, but by its interactions with the surrounding solution. For example, hydrophobic side chains are normally coiled within the interior of the molecule in order to minimise the interaction with the surrounding water, whereas polar and charged groups are often positioned around the external surface of the molecule. The water molecules around the protein form a highly ordered (and therefore low entropy) structure called a ‘hydration’ or ‘water shell’, which stabilises the protein and serves to buffer it from interactions with polar solutes. The water shell around a protein is disrupted when the concentration of ions is increased, and the protein is denatured. An equivalent concentration of a compatible solute does not disrupt the water shell and the protein is not denatured.

Increases in the ionic concentration of the medium surrounding the protein will disrupt the structure of the protein, because Na + and Cl" ions can effectively penetrate the hydration shell, and interfere with the non-covalent bonding that contributes to the stability of the protein.

CASE STUDY 1 Glycine betaine production

Glycine betaine is a quaternary ammonium compound found in at least 10 flowering plant families and also in marine algae. The charged groups mean it is extremely soluble in water, and is electrically neutral over a wide range of physiological pH values. The three methyl groups that comprise the quaternary ammonium group enable it to interact with hydrophobic as well as hydrophilic molecules. Thus, glycine betaine appears to stabilise proteins and membranes via these interactions, in addition to acting as a cellular osmolyte.

The synthesis of glycine betaine in plants has been studied mainly in species of Chenopodacieae (spinach, beet, etc.). In these species, glycine betaine is formed by a two-stage oxidation of choline, with the first step catalysed by choline monooxygenase (CMO). CMO encodes an unusual ferredoxin-dependent enzyme with an iron-sulphur cluster similar to that found in the Rieske protein of the cytochrome bjf complex. This enzyme is localised in the chloroplast stroma, where its activity is strongly light-dependent due to its requirement for reduced ferredoxin generated by photosynthetic electron transport. The second step in glycine betaine synthesis is catalysed by betaine aldehyde dehydrogenase (BADH), which is also stromal in Chenopodacieae. CMO and BADH are both induced by osmotic stress and have both been cloned from several plants.

The biosynthetic pathway of glycine betaine is different in Escherichia coli compared to plants, in that the production of betaine aldehyde from choline is catalysed by choline dehydrogenase (CDH), but the subsequent step still involves a betaine aldehyde dehydrogenase enzyme. In contrast, the synthesis of glycine betaine in Arthrobacter globiformis involves a single enzyme, choline oxidase (encoded by the codA gene), which catalyses the reaction directly from choline to glycine betaine , producing hydrogen peroxide.

Attempts have been made to manipulate glycine betaine production by transformation with the relevant plant, E. coli or A. globiformis genes. Transformation of plants with the betaine aldehyde dehydrogenase genes from plants or E. coli permitted the accumulation of glycine betaine when plants were supplied with betaine aldehyde, and conferred resistance to this toxic compound. However, plants did not accumulate glycine betaine in the absence of betaine aldehyde, presumably because the conversion of choline to betaine aldehyde does not occur in plants that do not normally accumulate glycine betaine.

Transformation of tobacco with the plant CMO gene targeted to the chloroplast resulted in very low levels of glycine betaine accumulation. It has been suggested thatthis is a result of the very low pool of choline present in tobacco cells, or it could be attributable to the lack of betaine aldehyde dehydrogenase activity in the tobacco chloroplasts. Tobacco plants transformed with the E. coli choline dehydrogenase gene, betA, also failed to accumulate glycine betaine, even though this enzyme can also catalyse the conversion of betaine aldehyde to betaine. However, in this case, the gene was not targeted to the chloroplast, which might explain this failure. But there was an observed increase in the salt tolerance of these transgenic plants, that can not be accounted for by the accumulation of glycine betaine to high concentrations. Higher levels of glycine betaine in transgenic plants have been obtained using the choline oxidase gene from certain Arthrobacter species particularly when transformed into plant Arthrobacter gtobiformis

The different pathways from choline to glycine betaine in plants, E. coli and Arthrobacter globiformis are compared species (e.g. Arabidopsis and rice) that have significantly higher choline pools than does tobacco. In a number of cases where glycine betaine accumulation has been achieved, the tolerance to various water-deficit stresses has been determined, including salt, chilling, freezing heat and drought. It is apparent that production of glycine betaine in transgenic plants can improve tolerance to a range of osmotic stresses under the test conditions applied, validating the initial concept that osmoprotectants can enhance tolerance to a variety of stresses that cause water deficit. However, it is.not entirely clear what the protective effect of the glycine betaine is. The levels of glycine betaine produced in most of these transgenic plants is relatively low (about 10% of the levels that accumulate in plants such as spinach) and is not sufficient to account for the improved osmotic stress tolerance by osmotic adjustment. It may be that the glycine betaine exerts a protective effect on vulnerable macromolecular structures at a lower concentration than its effect as an osmolyte.

In the same way that production of glycine betaine increases tolerance to a range of stresses that cause water deficit, other osmoprotectants have been produced in transgenic plants to test the feasibility of this approach. Table shows the range of genes and products that have been used to test this strategy in a range of crop plants. Table-Glycine betaine production in transgenic plants

Stress Host plant Accumulation of Transgene tolerance glycine betaine tested

Barley badh (betaine Tobacco Not tested Not tested aldehyde dehydrogenase) peroxisome

20 pmol g' 1 FW (in Tobacco Spinach badh 5 mmol 1 _1 betaine Not tested chloroplast aldehyde)

Spinach cmo (choline Tobacco <0.05 (imol g" 1 Not tested monooxygenase) chloroplast FW

Tobacco E. coli betB (betaine chloroplast or Not tested Not tested aldehyde dehydrogenase) cytosol

E. coli betA Tobacco

(choline cytosol Not detected Salt dehydrogenase) (membranes)

Chilling betA/betB Tobacco 0.035 (imol g' 1 FW Salt Drought betA Rice 5.0 pmol g" 1 FW Salt

A. globiformis Arabidopsis 1.2 (imol g -1 FW Salt

Chilling codA Freezing chloroplast (choline oxidase) Heat

Strong light

Salt codA Rice 5.3 (imol g- 1 FW Chilling

A. pascens cox (choline Freezing Arabidopsis 19 (imol g _1 DW oxidase) Salt

Drought cox Brassica napus 13 pmol g- 1 DW Salt cox Tobacco 13 (imol g- 1 DW Salt

Table FW, fresh weight DW, dry weight.

What is clear from this table is the existence of a range of effective transgenes for the production of osmoprotectants in plants, and that these generally have a measurable effect on the tolerance of the plant to various stresses that cause water deficit. In some cases, tolerance to more than one stress has been demonstrated for the same transgenic plant, confirming the view that generic strategies to combat water-deficit stress could provide tolerance against a range of different stresses. However, several aspects of osmolyte accumulation still require further investigation and explanation. One point to note from Tables is that the level of accumulation of most of the compatible solutes, apart from proline and D- ononitol, is considerably lower than could account for their protective effects by osmotic adjustment.

The reasons for stress tolerance may be different in each case, with some compounds like glycine betaine acting primarily to stabilise macromolecular structures, whilst other compatible solutes such as mannitol, sorbitol and proline are effective scavengers of reactive oxygen species (see below). Another point to make is that the tests of stress tolerance of these transgenic plants are often performed under highly controlled laboratory conditions that present an acute osmotic shock rather than the chronic water-deficit stresses more generally experienced in the field. It should be noted that few of these transgenic plants have been tested for stress tolerance in the field. Another consideration for developing these strategies further is that the accumulation of trehalose and sorbitol was found to inhibit plant growth.

A final note of caution relates to the assumption that increasing osmolyte accumulation in transgenic plants to levels that can maintain cell and tissue turgor pressure under water-deficit conditions will improve crop yield under drought conditions. Under conditions of reduced soil water, the maintenance of leaf turgor and hence transpiration in order to sustain growth will reduce soil water further and hasten the dehydration of the leaves. What needs to be understood is that under conditions where the water deficit is severe enough to threaten plant survival, crop yields are so low that even large increases in fractional yield are of little practical benefit to the grower.

Targeted approaches towards the manipulation of tolerance to specific water- deficit stresses

The production of osmoprotectants is not the only possible strategy to engineer resistance to the water-deficit stresses shown in Figure. Individual stresses may be susceptible to specific mechanisms to improve tolerance. Some of these will be described in the following section.

Alternative approaches to salt stress

Salt tolerance is increasingly becoming a major target for crop improvement as substantial areas of irrigated land are damaged by the accumulation of salt. Furthermore, the pressure for land has made it necessary to consider the possibility of growing crops in more saline conditions, with poorer quality water. As described above, saline conditions lead to osmotic stress by preventing water uptake by the roots and water efflux from the cells. However, the accumulation of Na + and CF ions in the cytoplasm may also have direct toxic effects by inhibiting protein synthesis, photosynthesis and susceptible enzymes. Thus, strategies for engineering water stress tolerance via the production of compatible solutes may provide protection against the osmotic effects of saline conditions, but not against ion toxicity. Additional approaches to minimise the toxic effects of specific ions may also be required.

CASE STUDY 2 Vacuolar Na/H antiport in transgenic plants improves salt tolerance One approach to tackling this problem is to look at the response of plants to salt stress. Some halophytes actually excrete salt via specialised glands on their leaf surfaces. However, it is more common for plants to avoid sodium ion accumulation in the cytoplasm by transporting them into the vacuole. Thus, one strategy would be to improve the transport of ions out of the cytoplasm and into the vacuole, particularly using older leaves as a sink and avoiding salt accumulation in sensitive tissues such as meristems.

In order to put this into practice, it is necessary to consider the mechanism of ion transport into the vacuole. Since this transport is working against a concentration gradient, it requires the input of energy. This is achieved by coupling the transport protein to a proton pump, transporting H + ions in the opposite direction (hence it is called an antiport protein). The vacuolar Na/H antiport protein AtNHXI of Arabidopsis. has been extensively studied and is known to be coupled to proton pumps such as AVP1, a vacuolar H + translocating pyrophosphatase. An analogy that has been used is to compare AtNHXI with a revolving door, and AVP1 as providing the energy for the door to spin. To increase the traffic through the membrane, one could therefore either increase the number of doors, or provide more energy for the existing doors to spin faster.

The first approach has been successfully used to engineer salt tolerance in tomato and canola plants by transformation with the Arabidopsis AtNHXI antiport protein gene. These normally susceptible plants were able to grow in a high-salt environment to fertile maturity. Tomato plants grown in 200 mmol/I NaCl accumulated salt in the leaves to a level 20-fold higher than normal, but, significantly, the fruits had a low sodium and chloride content. The transgenic canola plants accumulated up to 6% sodium, but the oil quality and yield of the seeds were normal. The other approach has been to overexpress the encoding gene AVP1, initially in Arabidopsis, in order to increase the proton-pumping potential of the vacuole, and hence its ability to transport sodium. This has improved not only the salt tolerance of these experimental plants, but also the drought tolerance, since the altered ion balance has enabled the plants to retain more water.

Alternative approaches to cold stress

Different plants vary enormously in their ability to withstand cold and freezing temperatures. Most tropical plants have virtually no capacity to survive freezing conditions. On the other hand, many temperate plants can survive a range of freezing temperatures from -5 to -30 °C depending upon the species. Plants from colder regions routinely withstand temperatures even lower than this. It is known that plants are better able to withstand cold or freezing stress if they first undergo a period of cold acclimation, at a low but non-freezing temperature. For example, wheat plants grown at normal warm temperatures are killed by freezing at -5 °C, but after a period of cold acclimation when the plant grows at temperatures below 10°C, they can survive freezing temperatures down to -20 °C.

Plants differ in their ability to withstand cold or freezing conditions and cold tolerance is one of the traits that plant breeders have selected for over many centuries. However, there has been little improvement in the cold tolerance of major crop species over the past two decades by conventional breeding, prompting the search for molecular solutions to this problem.

CASE STUDY 3 The COR regulon

One approach has been to study the mechanisms of freezing resistance that exist in some plant species. During the period of acclimation, plants produce a number of cold-induced proteins that are assumed to play a role in the subsequent cold-resistance. About 50 coldinduced proteins have been identified in different plant species. These fall into a small number of groups, but they all share the property of being extremely hydrophilic. Many of them also have relatively simple amino acid compositions, with repeated motifs. Some of these groups had previously been identified as ‘late embryogenesis abundant’ (LEA) proteins. Other groups of proteins are encoded by a class of genes designated as COR (coldresponsive) genes according to their patterns of expression. The precise function of these cold-induced genes is not yet known, but it has been speculated they might contribute directly to freezing tolerance by mitigating the potentially damaging effects of dehydration associated with freezing. Overexpression or ectopic expression of these cold-induced proteins could therefore be a possible route to the specific engineering of cold or freezing stress tolerance.

There are some examples of the expression of cold-induced proteins in transgenic plants. For example, constitutive expression of the small, hydrophilic, chloroplast-targeted COR protein COR15a in Arobidopsis improved the freezing tolerance of chloroplasts frozen in situ and of protoplasts frozen in vitro. However, COR15a expression has no discernible effect on the survival of frozen plants. One explanation for this observation is that the coldinduced proteins may be targeted to different vulnerable cell components, and that they are all required to provide complete protection to the cell. By implication, many COR genes would need to be transformed into a transgenic crop in order to obtain an appreciable improvement in cold tolerance. As discussed in Chapter 4, insertion of several genes into a transgenic crop on the same vector is technically difficult, and the process of ‘pyramiding’ or ‘stacking’ genes by crossing transgenic lines is time-consuming. A different strategy is therefore needed. One solution to the problem of engineering a multigene trait has emerged following the recognition that several different cold tolerance-related genes contain a similar regulatory element in their promoters—the CRT/DRE element. Furthermore, it has been found thatthe transcription factor CBF1 binds to the CRT/DRE element and activates expression of this group of genes, which comprise the COR regulon. Therefore, the strategy is to overexpress the CBF7 gene, leading to the induction of this entire group of COR coldtolerance genes. Transgenic Arabidopsis plants carrying a 35S promot er::CBFl gene construct have been produced. These plants express a number of COR genes without cold acclimation and have been shown to be freezing-tolerant without prior cold acclimation. As a control, transgenic plants overexpressing a single COR protein, C0R15a, were found to be less freezing-tolerant than the CBF1 plants. The interrelated nature of different stress responses was demonstrated in a similar experiment. The expression of a CBF1 homologue, DREB1A (dehydration responsive-element binding protein) under the control of a stressinduced promoter in transgenic Arabidopsis resulted in plants that had improved drought, salt and freezing tolerance.

Tolerance to heat stress

For many years it has been known that heat stress applied to a wide range of organisms induces a specific set of ‘heat shock’ proteins (HSPs); they fall into five classes, four of which are highly conserved in prokaryotes and eukaryotes. These four are categorised according to size as the HSP100, HSP90, HSP70 and HSP60 classes whose members appear to function as molecular chaperones. Some of them are expressed constitutively and are involved in normal protein synthesis and folding. Those induced by heat appear to be involved in countering the effects of heat stress by protecting or refolding denatured proteins. Their expression is induced by heat treatment and, in some cases, can be correlated with the acquisition of thermotolerance. The fifth group of several classes of small heat- shock proteins are particularly abundant in plants, but their function is not yet clear.

In a way analogous to the cold/freezing tolerance strategies, individual heatshock proteins have been transformed into plants in order to enhance heat tolerance. However, it is also known that the rapid heat-shock response is co- ordinated by a heat-shock transcription factor (HSF). This protein is expressed constitutively, but in normal conditions exists as a monomer bound to one of the HSP70 heat-shock proteins. Upon heat stress, the HSP70 dissociates and the HSF assembles into a trimer which binds to a heat-shock element (HSE) common to the promoters of heat-shock protein genes. The HSE is made up of 5-bp repeats in alternating orientations with the consensus NGAAN ; five to seven of these repeats occur in the promoter close to the TATA box.

When the AtHSFl gene was overexpressed in Arabidopsis, the transcription factor was not active, and there was no effect on thermotolerance. However, fusion of AtHSFl to the N- or C- terminus of the gusA reporter gene produced a fusion protein that was able to trimerise in the absence of heat. Transformation of this fusion protein into Arabidopsis produced transgenic

COR and heat-shock regulons

A number of the cold-induced, COR, genes have been characterised, and the sequences of their promoters compared. One of the features of several different COR genes is that their promoters share a common regulatory element termed the ‘Crepeat’ (CRT) or ‘low temperature-response element’ (LTRE) which is 5 nucleotides long and has a consensus sequence of CCGAC. This element had already been linked to drought resistance and termed the ‘dehydration responsive element’ (DRE). The CRT/LTRE/DRE is bound by a transcription factor termed ‘CBF1’. The structure of the CBF1 transcription factor is shown, indicating the nuclear localisation sequence, DNA-binding domain and an acidic region that may be involved in interactions with other proteins. CBF1 expression is induced by cold acclimation, and leads to the expression of the COR genes. This group of genes, sharing a common regulatory mechanism, has been termed the ‘COR regulon’.

A number of cold-induced/drought responsive genes contain the DRE sequence element in their promoters, which is bound by transcription factors of the CBF family, activating transcription. Overexpression of a single CBF gene therefore induces the expression of several cold-induced/drought responsive genes. Heat shock-induced genes also contain a heat shock element (HSE) in their promoters. Heat shock factor (HSF) proteins bind the HSE as a trimer. Overexpression of HSF encoding gene induces the expression of several heat shock proteins. CBF1 is a member of a small gene family; CBF1-3. CBF2 and CBF3 are also transcription factors, and constitutive expression improves freezing tolerance. Expression of all three CBF genes is induced rapidly by low temperatures (but not by dehydration, ABA or high salt concentrations). In addition, CBF3 overexpression results in several biochemical changes associated with cold acclimation, such as elevated levels of compatible osmolytes, proline and soluble sugars.

Although low temperature-induced gene expression, mediated by the CRT element, appears to be well conserved in plants, not all cold-induced genes (including the CBF genes) have the CCGAC element in their promoters. Other pathways of low temperature-induced gene expression, not mediated via CRT/CBF, appear then to be present in plants. Another sequence element, CCGAAA, has been identified as conferring low temperature inducibility in some genes.

The heat-shock genes also comprise a regulon. They all contain the heat- shock element (HSE) consensus sequence in their promoter regions. The transcription factor HSF binds to the HSE element as a trimer. Plants that expressed heat-shock proteins constitutively and demonstrated enhanced thermotolerance without requiring prior heat treatment.

Secondary effects of abiotic stress—the production of reactive oxygen species

Many, if not all, forms of environmental stress (abiotic and biotic) result in oxidative stress. This occurs most directly as a result of ozone or ionising radiation. However, oxidative stress is a secondary effect of many type of stress, from pathogen attack to water-deficit stress. The oxidative stress arises from the production of free radicals and the subsequent ensuing cascade of reactions. Much of the oxidative stress-induced damage to cells occurs as a result of the formation of reactive oxygen species (ROS). Figure shows the cascade of successive reactions that form superoxide, hydrogen peroxide and hydroxyl-radical species. The ROS cause damage as a result of their reactions with cellular macromolecules. Damage to cellular membranes may result from a low concentration of hydroxyl radicals triggering a chain reaction of lipid peroxidation. Oxidative damage to proteins may involve specific amino acid modifications, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electrical charge and increased susceptibility to proteolysis. In DNA, both the sugar and bases are susceptible to oxidation by reactive oxygen species, causing base degradation, single-strand breakage, and cross-linking to protein. These lesions in DNA can result in deletions, mutations and other damaging genetic effects. Table-Transgenes used to manipulate heat tolerance

Gene Protein Transgenic plant

AtHSFl Heat-shock transcription factor HSF1::GUS fusion Arabidopsis

HsplOl HSP100 class heat-shock protein Arabidopsis

Hsp70 HSP70 class heat-shock protein Arabidopsis

Hspl7.7 SmHSP small heat-shock protein family Carrot

TLHS1 Class I smHSP Tobacco

Plants contain a number of enzymes that catalyse the cascade of reactive oxygen species and convert them to less reactive products (Figure). One key point to notice is the key role of certain enzymes in this cascade, notably superoxide dismutase, catalase and peroxidases in neutralising these reactive species. Also shown in the figure are a number of ‘antioxidant’ compounds that react with the activated oxygen compounds to produce harmless, regenerable products. These include three different vitamin compounds: |3-carotene (provitamin A), ascorbic acid (vitamin C) and a-tocopherol (vitamin E). Other important antioxidants include glutathione and zeaxanthin. Central to this antioxidant system is the ascorbate-glutathione cycle (Figure). The coupling of ascorbate and glutathione redox-cycling has been most extensively characterised in the chloroplast. Chloroplasts produce superoxide and hydrogen peroxide in the light, particularly from photosystem I. The superoxide generated is itself converted into hydrogen peroxide by either spontaneous dismutation or by the activity of superoxide dismutase. The hydrogen peroxide produced is scavenged by ascorbate and the enzyme ascorbate peroxidase. The monodehydroascorbate produced from this reaction may be regenerated in two ways. One is by reduction of NAD(P)H catalysed by monodehydroascorbate reductase. The other occurs via the dismutation of two monodehydroascorbate molecules to form ascorbate and dehydroascorbate, followed by the reduction of the dehydroascorbate by glutathione, catalysed by dehydroascorbate reductase.

From the outline of plant antioxidants and oxygen-scavenging activities given above, two general strategies for engineering tolerance to oxidative stresses are apparent—either to increase the level of enzymes that remove ROS, or to increase the level of antioxidant compounds that react with ROS.

Strategy 1: Expression of enzymes involved in scavenging ROS

Superoxide dismutases (SODs) catalyse the reaction shown in Figure, in which the superoxide radical combines with two hydrogen ions to form Reactive oxygen species.

In order to understand the significance of oxidative stress and the production of reactive oxygen species, it is necessary to review some of the basic chemistry of oxygen. Atmospheric oxygen in its ground-state has two unpaired electrons. These two unpaired electrons have parallel spins and, in consequence, oxygen is usually non-reactive to most organic molecules, which have paired electrons with opposite spins. (Oxygen cannot react with a divalent reductant unless it has two unpaired electrons with a parallel spin opposite to the oxygen. This spin restriction means that the most common mechanisms of oxygen reduction in biochemical reactions are those involving transfer of only a single electron). This means that oxygen has to be ‘activated’ before it can participate in reactions with organic molecules. The activation of oxygen may occur by two different mechanisms: 1. Absorption of sufficient energy to reverse the spin on one of the unpaired electrons to form the singlet state, in which the two electrons have opposite spins (see Figure). Singlet oxygen can participate in reactions involving the simultaneous transfer of two electrons, and since paired electrons are common in organic molecules, singlet oxygen is much more reactive towards organic molecules than its triplet counterpart. In photosynthetic plants, singlet oxygen is often formed by the photosystems and plays a role in metabolic reactions such as the oxidation of xenobiotics and the polymerisation of lignin. However, it may also be formed by the dysfunctioning of enzymes or electron transport systems, as a result of perturbations in metabolism caused by chemical or environmental stress.

2. The stepwise reduction of oxygen to form reactive oxygen species— superoxide (02), hydrogen peroxide (H 2 02 ) and the hydroxyl radical (OH ).

Superoxide is a powerful oxidant or a reductant. It can oxidise sulphur, ascorbic acid or NADPH, but it can also reduce cytochrome c and metal ions. The reaction leading to the formation of hydrogen peroxide and oxygen is catalysed by the enzyme superoxide dismutase, but it can also occur spontaneously (see Figure).

Hydrogen peroxide is a substrate in a number of oxidation reactions catalysed by peroxidases involving the synthesis of complex organic molecules. It readily permeates membranes and is therefore not compartmentalised in plant cells. The reactivity of hydrogen peroxide is the result of its reduction by metal ions to form the highly reactive hydroxyl radical.

The hydroxyl radical oxidises organic molecules by two different reaction mechanisms: addition of the hydroxyl radical to form a hydroxylated compound; or the formation of an organic radical plus water. The organic radical can react with triplet oxygen to form a peroxyl radical, which can produce another organic radical. This can proceed to generate a chain reaction in which further organic radicals are produced.

The activity of this enzyme therefore determines the concentration of the two substrates (superoxide and hydrogen peroxide) that react to form the hydroxyl radical. There is a broad family of SOD enzymes, requiring different metal ions for activity, and predominating in different cellular compartments. Thus, Mn", Fe" and Cu7Zn" forms are found, specific to different organelles.

Transgenic tobacco plants containing each class of enzyme have been produced and tested for their ability to withstand oxidative stress. In each case, there has been some measurable increase in the ability to withstand oxidative stresses such as exposure to ozone. The Mn-SOD has also been transformed into alfalfa and targeted to the chloroplast or mitochondria. In this case, the results of a 3-year field trial indicated significant improvements in the yield and survival of the transgenic plants. However, it is not clear whether the protective effect is due to the removal of superoxide by

Antioxidants in plants

L-Ascorbicacid

Ascorbate (vitamin C) has an important role in plant metabolism, growth and development. Amongst other functions, it acts a reductant for many free radicals to minimise the damage caused by oxidative stress. Ascorbate can directly scavenge reactive oxygen species with and without enzyme catalysts, and can indirectly scavenge them by recycling a-tocopherol (see below) to the reduced form. Indeed, it reacts with ROS more readily than any other aqueous component. For example, ascorbate reacts with superoxide to form hydrogen peroxide and dehydroascorbate. It also reacts with hydrogen peroxide to from monodehydroascorbate and water, in a reaction catalysed by ascorbate peroxidase. It also regenerates membrane-bound antioxidants such as a-tocopherol that scavenge peroxyl radicals and singlet oxygen. The tocopheroxyl radical is converted to a-tocopherol via the oxidation of ascorbate to monodehydroascorbate.

Note that there are two different products of ascorbate oxidation— monodehydroascorbate and dehydroascorbate—which represent one- and two- electron transfers, respectively. Two molecules of monodehydroascorbate may spontaneously dismutate to form ascorbate and dehydroascorbate, or can be reduced to ascorbate by NAD(P)H monodehydroascorbate reductase. Dehydroascorbate is unstable at pH > 6, forming tartrate and oxalate. To prevent this, dehydroascorbate is rapidly reduced to ascorbate by dehydroascorbate reductase using reducing equivalents from glutathione (GSH) (see below).

Glutathione

Glutathione (GSH) is a tripeptide (Glu-Cys-Gly) which was met to herbicide detoxification. The antioxidant function of GSH involves the sulphydryl group of the cysteine residue, which forms a thiyl radical on oxidation, and then reacts with a second oxidised glutathione to form a disulphide bond (GSSG). The redox potential of GSH enables it to reduce dehydroascorbate to ascorbate or to reduce the disulphide bonds of proteins.

GSH functions directly as a free-radical scavenger by reacting chemically with singlet oxygen, superoxide and hydroxyl radicals. It also stabilises membranes by removing acyl peroxides formed by lipid peroxidation reactions. It also functions to regenerate reduced ascorbic acid from dehydroascorbate.

The reduction of GSSG to GSH is catalysed by the enzyme glutathione reductase (GR), which exists in plants in multiple forms associated with different subcellular compartments. Glutathione peroxidase provides an alternative means of detoxifying activated oxygen, by using GSH to reduce hydrogen peroxide, producing GSSG.

a-Tocopherol

a-Tocopherol (vitamin E), is a membrane-bound scavenger of oxygen free radicals, lipid peroxyl radicals and singlet oxygen. It also serves as a membrane stabilising agent. The reaction with peroxyl radicals formed in the lipid bilayer appears to be particularly important. The tocopheroxyl radical produced is stable and therefore does not propagate further free radicals. a-Tocopherol can be regenerated by ascorbate, as described above.

Carotenoids

Carotenoids are C 40 isoprenoids and tetraterpenes, and include the carotenes and xanthophylls (see Chapter 10). They are located in the plastids of both photosynthetic and non-photosynthetic plant tissues. In chloroplasts, they protect the photosystems by detoxifying ROS and triplet chlorophyll produced as a result of excitation of the photosynthetic complexes by light. Thus, they can perform a similar role to a-tocopherol in scavenging the products of lipid peroxidation. They can react with singlet oxygen and dissipate the energy as heat. They also dissipate excess excitation energy through the xanthophyll cycle, and they can quench triplet or excited chlorophyll molecules to prevent the formation of singlet oxygen.

SOD enzyme family

Superoxide dismutase (SOD) catalyses the dismutation of superoxide to hydrogen peroxide and oxygen. SOD is present in most (if not all) subcellular compartments of the plant cell that generate activated oxygen, and is assumed to play a central role in the defence against oxidative stress. There are three distinct types of SOD classified on the basis of the metal cofactor: the copper/zinc (Cu/Zn- SOD), the manganese (Mn-SOD) and the iron (Fe-SOD) isozymes. The Mn-SOD is found in the mitochondria; whilst Cu/Zn-SOD isozymes are found in the cytosol, or in the chloroplasts. The Fe-SOD isozymes, though not always detected in plants, are usually found in the chloroplast. All of the plant SOD genes are in the nuclear genome and are targeted to their respective subcellular compartments by an amino- terminal targeting sequence. Flowever, they are not regulated co-ordinately, but independently according to the degree of oxidative stress experienced in the respective subcellular compartments.

Strategy 2: Production of antioxidants

Table shows the alternative strategy for oxidative stress tolerance in action. Thus, three of the enzymes shown in Figure—ascorbate peroxidase, glutathione peroxidase and glutathione reductase—have been transformed into Arabidopsis and tobacco plants and shown to have some effect on tolerance to various abiotic stresses such as heat, cold and salinity. Glutathione reductase also provided resistance to the oxidative stress resulting from paraquat treatment. On the other hand, simply increasing the cellular concentration of glutathione by expressing glutathione synthase had no effect on stress tolerance.

Table Transgenes used to engineer tolerance to oxidative stress

Gene Host Stress tolerance

Mitochondrial Mn-SOD Alfalfa 2x increase in SOD Increased Tobacco chloroplast field drought tolerance

Increased freezing tolerance

Mitochondrial Mn-SOD Tobacco 2-4x increase in SOD Tobacco chloroplast Increased ozone tolerance

8x increase in SOD Mitochondrial Mn-SOD Tobacco No effect on ozone Tobacco mitochondria tolerance

Increased aluminium Mn SOD Canola tolerance

Chloroplast Cu/Zn- 3-15x increase in SOD Tobacco SOD Increased tolerance to high light chloroplast Pea and chilling

1.5-6x increase in SOD Tobacco Cytosolic Cu/Zn-SOD Reduced damage from acute cytosol ozone exposure

Protected plants from Fe-SOD Arabidopsis Tobacco ozone damage

Apx3 Increased protection Tobacco (ascorbate peroxidase) against oxidative stress Apxl Arabidops Heat tolerance (ascorbate peroxidase) is

GST/GPX

(glutathione S- Tobacco Increased stress tolerance transferase with glutathione peroxidase)

Ntl07 Sustained growth under (glutathione S- Tobacco cold and salinity stress transferase)

ParB Arabidops Protects against aluminium (glutathione S- is toxicity and oxidative stress transferase)

NtPox Arabidops Protects against aluminium (glutathione is toxicity and oxidative stress peroxidase)

3-6x increase in foliar GR Glutathione reductase Tobacco Increased tolerance to S0 2 E. coli chloroplast and paraquat

Tobacco l-35x increase in GR Glutathione reductase cytosol Increased tolerance to paraquat E. coli

lOOx increase in GS

Glutathione synthetase Poplar GSH not increased cytosol E. coli No effect on paraquat tolerance

Increased tolerance to MsFer Tobacco oxidative damage caused by Alfalfa ferritin excess iron

Table captionSOD, superoxide dismutase; GST, glutathione S-transferase; GPX, glutathione peroxidase; GR, glutathione reductase; GS, glutathione synthetase; GSH, glutathione.

Ph. D (Ag) Molecular Biology and Biotechnology

Subject: ADVANCES IN GENETIC ENGINEERING MBB 602: (3+0)

Topic:- Engineering food crops for quality, Teacher:- Dr. R.S. Sharma Biotechnology Centre, JNKVV, Jabalpur Introduction Earlier we have considered a variety of ways in which the productivity of crop plants can be improved by enhancing their ability to resist or tolerate biotic and abiotic stresses. These strategies can all contribute to an improvement in crop yield by allowing the plants to better withstand external factors that reduce the amount and quality of harvestable plant material. However, the performance of crop plants is also determined by endogenous factors that affect the yield and quality of the harvestable material produced. This chapter will look at a number of examples of how crop productivity can be genetically enhanced by increasing the amount, or improving the quality, of material produced by the crop. The yield of a crop is ultimately determined by the amount of solar radiation intercepted by the crop canopy, the photosynthetic efficiency (i.e. the efficiency of conversion of radiant to chemical potential energy) and the harvest index (the fraction of dry matter allocated to the harvested part of the crop). Thus, manipulation of crop yield requires some understanding of the fundamental processes that determine photosynthetic efficiency and dry-matter partitioning. On the other hand, the quality of a crop is determined by a wide range of desirable characteristics such as nutritional quality, flavour, processing quality and shelf-life. The nature of yield and quality are therefore much more varied and wide-ranging than the other traits described to this point, and the structure of this chapter is therefore somewhat different to reflect this. Rather than try to cover the entire range of targets for different crops, a small number of yield and quality traits have been selected to exemplify the types of approaches that have been adopted in this area. The chapter therefore starts with an extended discussion of one system— tomato ripening—because it is possible to draw several general lessons from the techniques and concepts that were deployed in this project.

The genetic manipulation of fruit ripening

The predominance of herbicide and pest resistance amongst the commercially developed GM traits has been discussed. However, amongst the first GM products to reach the market, around 1994, were Calgene’s FlavrSavr fresh tomatoes, and processed tomato products containing delayed-softening tomato fruit developed in the UK by Zeneca and the University of Nottingham. This may seem rather puzzling, because the tomato crop is relatively small compared to the major GM crops of Northern America (maize, soybean, oilseed rape and cotton). Furthermore, it is not immediately clear why a large biotechnology company like Zeneca would be involved, when they had no direct interest in tomato seeds or processed products when the project was started (when the company was still ICI). Part of the answer lies in the early recognition by ICI and Calgene of the value of using tomato ripening as a model system on which to develop expertise and test the potential of plant biotechnology to modify crop quality. In order to understand why tomato ripening was seen as such a good model system, it is necessary to look at some of the basic biology of fruit ripening.

Fruit ripening is an active process that, in climacteric fruit such as tomatoes, is characterised by a burst of respiration (the respiratory climacteric), ethylene production, softening and changes to colour and flavour. The transient peaks in respiration and ethylene production at the start of tomato ripening, which are accompanied by softening, a change in colour from green to red and enhanced flavour. The peak of ethylene production is significant, because ethylene is known to be the phytohormone that triggers ripening in climacteric fruit. The colour change results from the degradation of chlorophyll and the production of the red pigment, lycopene. Flavour changes occur as starch is broken down and sugars accumulate. A large number of secondary products that improve the smell and taste of the fruit are also produced. The softening of the fruit is largely the result of the cell wall degrading activity of the enzymes polygalacturonase (PG) and pectin methylesterase (PME). The PG enzyme is synthesised de novo during ripening and acts to break down the polygalacturonic acid chains that form the pectin ‘glue’ of the middle lamella, which ‘sticks’ neighbouring cells together. The realisation that the ripening process involved the activation of specific ripening-related genes, such as the one encoding polygalacturonase, enabled these genes to be cloned. cDNA of ripening-related genes

The three ripening genes that will be discussed in this chapter were isolated from the same cDNA library made from ripe tomato fruit. Ripening-related clones were identified by differential screening of the cDNA library with cDNA sequences prepared from mRNA populations from mature green fruit and ripe fruit. Clones that hybridised to the ripe cDNA but not to the mature green cDNA were classed as ripening-related. Over 150 clones were isolated, grouped and characterised. Three groups of clones, represented by pTOM5, pTOM6 and pTOM13, were confirmed as ripening-related and were also shown to be induced by ethylene. Their identities and roles in the ripening process were subsequently determined, as shown in the table below. Clone Gene product Function Role in ripening

pTOM5 Phytoene synthase Lycopene synthesis Red coloration

pTOM6 Polygalacturonase Cell wall degradation Fruit softening pTOM13 ACC oxidase Ethylene formation Ripening trigger

CASE STUDY 1 The genetic manipulation of fruit softening

Sequencing of the TOM 6 clone showed that the sequence matched the N- terminal sequence of the polygalacturonase (PG) protein. In other words, one of the genes implicated in a particular part of the ripening process—softening—had been cloned. Northern blot analysis confirmed that expression of the PG gene was ripening-specific, and was induced by ethylene. The availability of the PG gene led to two types of question: (1) was it possible to use the cloned gene to investigate the precise role of PG in ripening; and (2) was it possible to modify the expression of endogenous PG in order to manipulate the ripening process. This led to one of the first examples of using antisense techniques to alter plant gene expression.

The initial antisense PG construct comprised a partial cDNA fragment of pTOM6 inserted in reverse orientation downstream of the 35S promoter (Figure 10.2). Note that expression of the antisense gene will therefore be ‘constitutive’, whilst expression of the endogenous gene is, of course, ripening-specific.

The effect of transforming this construct into tomato plants was quite instructive. Table- summarises the results of Northern blots of RNA from mature green and ripe fruit from both transgenic and non-transgenic tomatoes, hybridised with probes to sense and antisense mRNA. There are a number of things to note from this blot. PG mRNA is not present in mature green wild-type fruit, but is induced during ripening. In ripe transgenic fruit, the level of PG mRNA is much reduced. The antisense RNA probe reveals that the antisense RNA accumulates to high levels in mature green fruit, but its level is also reduced in ripe fruit. The conclusion is that the interaction of sense and antisense mRNA leads to a reduction in both transcripts.

In these transgenic fruit, it could be shown that polygalacturonase activity was much reduced during ripening. Furthermore, this caused a specific delay in softening, but not PG coding sequence.

Non Nontransgenic Mature Transgenic Mature Transgenic Fruit transgenic green green Ripe Ripe

Probe Sense AS Sense AS Sense AS Sense AS

ND Signal ND ND Intense ND Faint Faint Intense

band bands band bands

Size PG

Co-suppression of PG gene expression by sense PG constructs

In addition to pioneering the use of antisense techniques, the tomato ripening system was also one of the first to reveal the phenomenon of co-suppression. This was observed in fruit in which sense rather than antisense PG constructs were expressed. In several cases, the overall level of polygalacturonase (PG) activity was less than in non-transgenic controls. This implied some mechanism whereby the plant cell was able to detect the presence of extra gene copies and/or increased levels of specific gene expression of the endogenous gene, and to suppress both endogenous and transgene expression. The term ‘co-suppression’ is used specifically to describe this mutual suppression of endogenous gene and transgene expression. It is one example of a more general phenomenon called ‘transgene silencing’, in which the expression of a transgene, even though intact and stably integrated, is suppressed.

Viscosity in low-PG fruit (PG activity reduced by 99%) was enhanced by >80% as a result of the increase in size of the cell wall pectins, indicating a significant increase in the yield of paste from these tomatoes. This characteristic enabled Zeneca to exploit these transgenic tomatoes for the production of paste for processed foods such as sauces and pizzas.

Calgene in the USA exploited the delayed softening characteristics of low- PG fruit in quite a different way. Their low-PG varieties were found to remain firmer on the vine and during storage than unmodified fruit. Fresh tomatoes are generally harvested before they are fully ripe, to ensure they are firm enough to withstand handling during harvest, processing and storage. Ripening may then be induced by ethylene prior to sale. Whilst this ensures the fruit are sold intact, at a uniform size, colour and stage of ripeness, it is a common perception of consumers that tomatoes processed in this way have lost much of the flavour and aroma of those eaten straight from the vine. In contrast, the Calgene 'FlavrSavr’ tomatoes were allowed to ripen and develop their full flavour on the vine, but the ripe fruit were still firm enough to withstand damage from handling and also from postharvest fungal infections. CASE STUDY 2 The genetic modification of ethylene biosynthesis

The use of pT0M6 indicated that, although the ripening process is co- ordinated, specific elements of the process could be manipulated without affecting others. Thus, antisense PG fruit still change colour and accumulate sugars and flavour compounds. However, the cloning of several ripening-related sequences led to the proposal that some of these might be involved in the transient burst of ethylene production, and that the study of these clones could provide some insight into the regulation of the entire ripening process. Investigation of the expression of ripening-related clones in wounded leaves (which also produce ethylene) showed that the expression of pTOM 13 was related to ethylene production rather than to ripening perse. Sequence analysis led to the preliminary identification of pTOMI 3 as the second ethylene-forming enzyme (EFE) in the two-step pathway from S- adenosylmethionine to ethylene. The protein sequence, plus the availability of the clone for protein expression studies, also allowed the classification of the previously uncharacterised ethylene forming enzyme as ACC oxidase.

The antisense strategy was initially used to confirm the role of pTOM13 in ethylene synthesis. Transgenic tomato plants carrying the antisense pTOMI 3 construct produced much less ethylene, either in wounded leaves, or during ripening than did control plants.

CASE STUDY 3 Modification of colour

The third, ripening-related clone, pT0M5, highlighted at the beginning of this chapter (Box 10.1) encodes phytoene synthase, a key enzyme in the biosynthesis of the red pigment, lycopene. The position of this step in the isoprenoid pathways of plants is shown. Many classes of compound are synthesised by building up molecules from the 5carbon isoprenoid unit. In particular, this route forms the basis of the biosynthetic pathways of three classical plant hormones. Cytokinins such as zeatin are produced by the addition of a single isoprenoid unitto adenosine. The biosynthesis of gibberellins (GA) starts from the 20-carbon unit geranyl geranyl diphosphate (GGDP). Abscisic acid (ABA) is formed from (3-carotene, as are a wide range of carotenoids and xanthophylls.

As might be expected, antisense pTOM5 tomato plants produce yellow fruit during ripening. On the other hand, plants overexpressing pTOM5 under the control of the 355 promoter were found not only to produce lycopene ectopically, but also to have a shorter height. Analysis of gibberellin synthesis in these plants showed the ectopic phytoene synthase activity was channelling 20-C units into the lycopene pathway, leading to a reduction in GA synthesis. Gibberellins play a role in stem elongation, so the reduction in GA synthesis caused the dwarf phenotype.

The manipulation of lycopene synthesis, and its effect on gibberellin synthesis, leads on to three different examples of the manipulation of yield and quality. The first general area to consider is the modification of colour. There are many examples of this, particularly amongst the ornamental flowers.

Flower colour

Flower colours are due mainly to flavonoids, carotenoids and betalains. The most important flavonoids in this respect are the anthocyanins, which contribute to red and blue flowers. Part of the anthocyanin pathway is shown below.

The first enzyme indicated is chalcone synthase, in the context of gene silencing. The anthocyanin synthesis pathway is quite complex, at various stages the compounds can be modified by enzymes such as dihydroflavonol-4-reductase (DFR) to produce different coloured pigments. However, not all plants have all the enzymes of the pathway, so it is impossible to get some colours of flowers in some species. A combination of mutation and genetic manipulation of the anthocyanin pathway has the potential to generate colours in plants that previously were not possible. For example, petunias are unable to produce the pelargonidin-related pigments (brick red/orange) as their DFR is unable to use dihydrokaempferol. However, a line of petunias has been isolated with mutations in the flavonoid-3'- hydroxylase (F3'H) and the flavonoid-3',5'hydroxylase (F3'5'H) genes. The plants are unable to use dihydrokaempferol in the other parts of the pathway, so there is a build-up of the substrate. This plant line has been transformed with a maize DFR gene. The expression of the maize protein allows the dihydrokaempferol to be utilised to produce the brick-red pigment pelargonidin-3-glucoside, so producing brick-red petunia flowers.

THE‘GREEN REVOLUTION’

The development of dwarf, high-yielding varieties of wheat and rice by conventional breeding during the 1960s enabled several developing countries, most notably in the Indian subcontinent, to move from a position of food scarcity to become net exporters of these cereals. This step-change in crop yield was termed the ‘Green Revolution’, and the key wheat breeder involved, Norman Borlaug, was awarded the Nobel Peace Prize for his contribution to global food security. The key to his success lay in the development of semi-dwarf, high-yielding, winter wheat varieties adapted to heavy rain conditions in the USA after the Second World War. This wheat was unsuitable for growing in the tropics or semi-tropical climates because, as a winter wheat, it required a period of cold during early growth. It was also found to be susceptible to rust fungus. Using the semi-dwarf wheat lines as a starting point, a breeding programme was established in Mexico to develop wheat strains that were adapted to the growing conditions found in poorer countries. This was done by crossing a number of desirable traits, from wheat varieties collected from around the world, into the semi-dwarf lines. The result was the production of spring wheat varieties that matured quickly and were insensitive to photoperiod, allowing them to be grown more than once per year. They were also rust-resistant and adapted to a variety of warm climates. These characteristics meant that yields could be doubled compared to those of traditional varieties, given adequate fertiliser.

At about the same time, similar breeding programmes were implemented for rice at the International Rice Research Institute (IRRI) in The Philippines. Some of the most successful crosses were between semi-dwarf japonica and relatively highyielding indica lines, and rice varieties that were semi-dwarf, high yielding, early maturing, photoperiod-independent and blast fungus-resistant were produced. At IRRI, the new varieties could yield 3-4-fold more than traditional varieties under appropriate experimental conditions.

The Green Revolution required that farmers not only adopted the new cereal seeds, but also signed up to a high-input method of agriculture—including the use of nitrogen fertilisers, herbicides and pesticides as well as equipment for tilling and irrigation—to get the maximum yields from these varieties. There were significant cereal yield improvements, though often not as much as predicted from the experimental studies. Many small farmers were unable to afford to get the best out of the new seeds, whilst the richer farmers were able to produce more crops for less input of labour and thus started to acquire more land. The result was that smaller farmers were displaced from the land. Thus, the impressive increase in global food production was achieved at some cost in terms of higher agrochemical inputs, the loss of locally adapted crop varieties and increased poverty for some. Analyses of current trends in population growth indicate the need for another step-change in crop yields comparable to the Green Revolution. It is clear that the genetic modification of crops could make an important contribution to raising the ‘yield ceiling’ reached by conventional breeding efforts. However, it is also important that the implementation of this ‘Second Green Revolution’ takes account of the negative aspects of the first Green Revolution.

genes encode transcription factor-like proteins that contain domains indicative of phosphotyrosine signalling. The mutant gai genes encode proteins altered in a conserved gibberellin signalling domain. Transgenic rice and wheat plants into which a mutant gai gene was introduced were shown to have a reduced response to gibberellin, and to have a dwarf phenotype. This result is particularly significant because, whilst the dwarf character has been found in rice, attempts to breed a dwarf strain of basmati rice have failed to date because the resulting short plants have lost their characteristic flavour. The success of the gai gene experiments suggests that the dwarf character could be introduced into a wide range of crop species to improve crop yield, without having to shuffle all the other genes that contribute to the desirable characteristics of the current elite lines. An important lesson here is that transgenic approaches to crop improvement, particularly those that use well-characterised genes, can be much more precise and predictable than conventional breeding. However, as we look at effects of modifying phytochromes in a later section, it will become clear that this is not always the case.

The final example of crop quality enhancement that flows from the study of carotenoid biosynthesis is that of provitamin A production in rice grains—so called ‘’. CASE STUDY 4 Golden Rice

Rice is the most important food crop in the world, and is eaten by some 3.8 billion people. In some regions of the world where rice forms a staple component of the diet, vitamin A deficiency is a major nutritional problem. Deficiency of this vitamin can cause symptoms ranging from night blindness to total blindness as a result of xerophthalmia and keratomalacia. It has been estimated that around 124 million children are vitamin-A deficient, causing about 500 000 children to go blind each year. Vitamin A deficiency also exacerbates other health problems, for instance diarrhoea, respiratory diseases and childhood diseases such as measles. In consequence, it is estimated that improved vitamin A nutrition could prevent 1-2 million childhood deaths per year. One of the causes of vitamin A deficiency in regions where the majority of calories consumed comes from rice, is that milled rice contains no 13carotene (provitamin A). One of the solutions proposed for this problem is to engineer rice to produce provitamin A in the rice endosperm. Since the successful manipulation of 13carotene synthesis in the rice grains gives them a characteristic yellow/orange colour, rice which is genetically enriched in provitamin A has been described as 'Golden Rice’.

The biosynthetic pathway of provitamin A is a continuation of the lycopene pathway already discussed in the previous section on tomato ripening. Immature rice endosperm is capable of synthesising geranyl geranyl diphosphate, but subsequent stages of the pathway are not expressed in this tissue. Early transformation experiments with a phytoene synthase (psy) gene from daffodil fused to a rice endosperm-specific promoter indicated that phytoene could be synthesised from GGDP in the rice grain. However, three subsequent steps are required to convert phytoene to (3-carotene; phytoene desaturase and ^-carotene desaturase to introduce the double bonds to form lycopene, and lycopene (3- cyclase to form the rings in p-carotene. Fortunately, a bacterial carotene desaturase gene capable of introducing all four double bonds can be substituted for the phytoene desaturase and ^-carotene desaturase. Nevertheless, the manipulation of Golden Rice requires the introduction of three genes: phytoene synthase, carotene desaturase and lycopene p-cyclase.

The constructs used to target expression of the appropriate genes to the rice endosperm are shown in Figure 10.10. The daffodil psy gene::rice glutelin promoter construct was inserted into the vector pZPsC, along with the bacterial carotene desaturase gene {Ctrl) from Erwinia uredovora controlled by the 35S promoter. Both enzymes were targeted to the plastid (the site of GGDP synthesis): the psy gene by its own transit peptide, and the Ctrl gene by fusion to a pea rbcS (ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit) transit peptide sequence. The lycopene p-cyclase gene from daffodil with a functional transit peptide was inserted into the vector pZLcyH under the control of the rice endosperm-specific glutelin promoter, along with a hygromycin-resistance selectable marker gene.

Rice immature embryos were inoculated with a mixture of Agrobacte rium L BA4404 containing each of the two plasmids (see Chapter 3). A total of 60 hygromycin-resistant regenerated lines were selected at random, all of which were shown to contain the pZCyH construct. Of these, 12 were also found to contain the pZPsC cassette. Most of the seeds from these transgenic lines containing both constructs were found to be yellow, indicating carotenoid synthesis. A range of carotenoids was found in some of these lines, whereas pcarotene was the only carotenoid in others. The highest producing line of this type was found to contain 1.6 fig p-carotene g~’ endosperm from a mixed population of segregating grains. It is therefore calculated that the homozygous grains of the same line should produce at least 2 |ig g~' provitamin A, which corresponds to 100 fig retinol equivalents daily intake, assuming 300 g rice is consumed per day. This is sufficient to make a significant contribution to the daily intake of vitamin A, though it is probably not enough to provide the complete dietary requirement of the vitamin (see Box 10.6)

Figure 10.10 Constructs for the production of Golden Rice. The most successful strategy for the production of Golden Rice involved transformation with two independent constructs. The first one contains a daffodil phytoene synthase {psy) gene fused to a rice glutelin promoter in tandem with a bacterial carotene desaturase gene {ctrl) driven by the 35S promoter. The second construct contains the hygromycin-resistance apblV selectable marker gene in tandem with the lycopene P-cyclase gene (Icy) of daffodil also fused to an endosperm-specific rice glutelin promoter.

Before carrying on to discuss the exploitation of Golden Rice, it is important to emphasise the scientific significance of these results. As shown in Figure 10.9, four enzymes are involved in catalysing five reactions from GGDP to (3-carotene. The genetic manipulation of this multistep pathway, via the insertion of three genes into rice, required several years of intensive work and represents a considerable achievement. However, obtaining the laboratory-scale result is only the first of a series of hurdles that have to be negotiated before provitamin A-enriched rice reaches those who could benefit from it. Subsequent challenges included the clarification of intellectual property rights (IPR) and the mechanisms for technology transfer.

Engineering plant protein composition for improved nutrition

The example of Golden Rice has dealt with one aspect of nutritional quality. Another that has become a target for genetic engineering is the amino acid content of plant foods. Humans are only capable of synthesising 10 of the 20 naturally occurring amino acids. The other, ‘essential’, amino acids are obtained from the diet. Obtaining balanced quantities of the amino acids can be problematic if certain foods predominate within a diet. This is the case with cereal grains, which are commonly used as the principal energy source, and with legume seeds, which are important sources of proteins in the diet of humans and livestock. Cereal grains are often limiting for lysine, while the legume seeds have an adequate level of lysine but are limiting for the sulphur-containing amino acids, methionine and cysteine. It is important to note that animals can convert methionine into cysteine, but not the reverse, so it is possible to make up any short fall in the S-amino acids by increasing the level of the methionine. Transgenic routes have been used to increase the content of several different amino acids and some of the approaches will be discussed here.

One approach used has been to isolate the gene for a sulphur-poor protein and modify and enrich its nucleic acid sequence for the S-amino acid. This has been attempted for proteins such as (-phaseolin from Phaseolus vulgaris and vicilin from Vida faba, but the modified proteins were either unstable or contained too little methionine to make them useful. A more successful approach has been to construct totally artificial genes that code for proteins containing a high S-amino acid content. One such totally synthetic protein, containing 13% methionine residues, has been successfully expressed in sweet potato (Ipomoea batatas). Several methionine-rich proteins have been identified in maize (21-kDa zein— 28% methionine, 10-kDa zein—23% methionine); rice (10-kDa prolamin—20% methionine); sunflower (2S sunflower seed albumin—16% methionine, 8% cysteine); Brazil nut (Brazil nut albumin—18% methionine, 8% cysteine). The genes for these proteins have been introduced into a number of crops (maize, soybean, lupin, canola) to increase the level of S-amino acids in blended stock feeds containing cereal and legume grains. One major problem associated with moving proteins between species has become apparent with the Brazil nut albumin (BNA), i.e. the protein responsible for the potent allergenicity of Brazil nuts. This property is maintained in the seeds of BNA-containing transgenic plants, which makes them unacceptable for human consumption and has been a timely warning that safety issues should be paramount when dealing with food crops.

The genetic manipulation of crop yield by enhancement of photosynthesis

Crop yield is totally dependent upon light and its conversion (light harvesting and electron transport) into the usable energy (ATP, NADP) that drives the dark reactions of photosynthesis (carbon dioxide conversion into carbohy drates). These reactions usually take place in chloroplasts within the leaves, the source, but the efficiency of the process is also dependent upon the capacity of sink tissues and organs to assimilate this fixed carbon. These are complex processes and outside the scope of this book, but some studies have been carried out to investigate the potential for enhancing photosynthesis by biotechnological means. Although these have been very preliminary in nature, several examples will be briefly discussed to describe the potential for manipulating complex physiological interactions. Direct manipulation of light harvesting, electron transfer or the processes of photoprotection and photoacclimation will not be considered here.

Manipulation of light harvesting and the assimilate distribution— phytochromes

When grown in the field or in their natural environments, plants are not always under optimum light conditions, even during daylight hours. They are faced with changes in the amount, direction, duration and quality of incident light radiation. Plants have evolved mechanisms that optimise the acquisition of available light energy for photosynthesis. In dense populations or in shady conditions, plants display the shade response. The plants use available resources to increase stem and petiole elongation to outgrow any shading plant; there is also a reduction in chlorophyll synthesis, leaf thickening and an increase in apical dominance. From an agricultural point of view, this shade response has important implications for yield, as assimilates are used to re-establish optimal light conditions for photosynthesis rather than being stored. The shade response is regulated through a series of photoreceptors. The phytochrome family of proteins are the best characterised of these proteins (there are at least five Phy proteins in dicots). They are able to detect the level and quality of the incident light and then control growth and development via a series of signal transduction pathways that regulate gene expression. Phy proteins are photochromic proteins, they have a protein moiety connected to a tetrapyrrole chromophore, and they can exist in two forms: physiologically inactive (P R , red-light absorbing) and active (P FR , far- red light absorbing). These forms are interconvertible by red or far-red light, respectively. The photosynthetic pigments in leaves absorb most visible radiation (400-700 nm) but reflect light beyond 700 nm (far red, 700-800 nm). This leads to a high proportion of FR radiation being found in dense plant stands. Plants are able to perceive this radiation and the proximity of other plants because the equilibrium of the phytochromes is shifted towards the inactive form. Two of the major phytochromes involved in this response are PhyA and PhyB. PhyA accumulates in the dark and is rapidly degraded upon conversion, by the absorption of red light, into the labile P FR form. Despite this, it is responsible for the detection of continuous FR light and dampens the shade avoidance response. PhyB is responsible for the detection of red light, so under high light conditions (high relative proportions of red light) it is converted to the active form, in which form it suppresses the shade-avoidance response.

Genetic engineering for male sterility in plants

Navaneetha Krishnan J L-2016-A-18-D What is male sterility? • Male sterility is a situation where the male reproductive parts of a plant are either absent, aborted, or nonfunctional, and hence they fail to participate in the process of natural sexual reproduction. • This situation can arise due to any developmental defect at any stage of microsporogenesis or release of pollen grains. • First observed by Koelrouter in 1763-anther abortion within intraspecific hybrids of tobacco. • Darwin (1877) recognized the importance of this phenomenon and hypothesized that the loss of reproducing ability of plant helps evolutionary processes in enhancing adaptation through gene transfer from various related and unrelated individuals through cross- pollination.

Sterile Fertile What about female sterility?

• Occurs rarely in nature. • Not useful in Plant Breeding. • Very difficult to detect when compared to male sterility (large no: of pollens produced). • Does not have the ability of self propagation(seed set). • Cannot be stained for preparing assays as that of male sterility. Need for male sterility ??? Classification of male sterility

1) Based on the type of malfunctioning of the androecium, (i) Structural (absence or deformity of anthers) (ii) Sporogenous (defective microsporogenesis) and (iii) Functional (failure of mature pollen to germinate) male sterility 2) On the basis of genetic control mechanisms, (i) Genetic Male Sterility (GMS) (a) Environment insensitive GMS (b) Environment sensitive GMS (ii) Cytoplasmic Male Sterility (CMS) and (iii) Cytoplasmic Genetic Male Sterility (CGMS) 3) Artificiallly induced male sterility (i) Chemically induced male sterility and (ii) Genetically engineered male sterility Generalized hereditary constitution of the nucleus and cytoplasm of the three male sterility systems (B. Bahadur et al. 2015) Environment sensitive Genetic male Sterility(EGMS)

• Induction of male sterility in response to fluctuations in environmental conditions. • Particularly Photoperiod(PGMS) and Temperature(TGMS). • Eg:Rice • Controlled by nuclear genes. • No need to use maintainer line. • Two-line hybrid system-Only male sterile line(female parent) and Pollinator(male parent) present. Contd… • PGMS lines(Rice) : Nong-Ken 58 S,Zennong s,X-88. • TGMS lines(Rice):Annong S,Hennong S,Novin PL 12,IR 68945. • Any genotype can be used as a Pollinator parent. INHERITANCE OF EGMS Origin of male sterility systems

• Selection from natural variation • Integration of cultivated genome in to alien cytoplasm (using wild sp. as female parent) • Intergeneric and interspecific hybridisation • Selection from recombinant populations • Induced mutations (physical and chemical mutagens) Chemical induced Male Sterility

• The chemical which induces male sterility artificially called as male gametocides are used. • These chemicals are also known as Chemical hybridizing agents. • It is rapid method but the sterility is non-heritable. • In this system A, B and R lines are not maintained. • Some of the male gametocides used are Gibberellins (Rice, Maize), Sodium Methyl Arsenate (Rice) and Maleic hydrazide (Wheat, Onion). • Could be used in the large scale commercial production of hybrid seed (Hybrid wheat in UK, Germany).

Induced GMS (Transgenic male sterility)

regeneration

Agrobacterium- mediated transformation male-sterile plant

Promoter which Gene which disrupts induces transcription normal function of cell in male reproductive specifically First successful experiment on GE male sterility… Abstract

• The first transgene designed to confer GMS was reported and were used to transform Tobacco and oilseed rape plants. • Tapetal-specific transcriptional activity of the tobacco TA29 gene. • Upstream regulatory elements of TA29 gene used to drive the expression of transgenes (extracellular RNAses from bacteria). Two genes were used:  barnase from Bacillus amyloliquefaciens  RNAse-T1 from Aspergillus oryzae • RNase genes selectively destroyed the tapetal cells during anther development and prevented pollen formation • Herbicide (bialophos) resistant gene (bar) used as selectable marker Robert B. Goldberg et.al. 1993 Pollen grain differentiation and release Gene expression is temporally and spatially regulated

Koltunow et al. (1990) 17 Results… Fig 1. RNA gel blot analysis of organ-specific gene expression Fig 2. Floral morphology of male sterile (transgenic) and untransformed plants Fig 3. Tissue abnormalities in male sterile tobacco Fig 4. SEM micrographs of pollen grains produced by and oilseed rape anthers (Bright field microscopy) male sterile tobacco and oilseed rape anthers

E- epidermis; C- connective tissue; F- filament; PS- pollen sac and T- tapetum Another Breakthrough Abstract

• Barstar gene codes for an intracellular bacterial protein which is an inhibitor of barnase. • Oilseed rape plants were transformed with a binary vector containing barstar gene along with bar gene as selectable marker using agrobacterium mediated transformation. • When the transgenic plants containing barstar were crossed with the male sterile transgenic plants (barnase), the F1 progenies segregated in the ratio of 2:1 (male fertile : sterile) (viable progenies) after selection using herbicide spray at the seedling stage. • Around 25% male sterile plants produced in the F1 will be lacking bar gene and they will die. • The plant transformed with the barstar gene serves as the restorer of fertility. Fig 1. Restoration of male fertility by crossing oilseed rape plants containing the TA29-barstar and TA29-barnase genes

Note: ms, rf and hr refer to the hemizygous chromosomal loci that lack the TA29- barnase, TA29-barstar and bar genes respectively Results… Fig 2. Oilseed rape flowers and anther cross sections

Male fertile plants containing the TA29-barstar gene

Male sterile plants containing the TA29-barnase gene

Male fertile plants restored to fertility containing both the TA29-barstar and TA29-barnase gene

E-epidermis; En-endothecium; PG- pollen grain; PS- pollen sac and T-tapetum Fig 3. Scanning electron micrographs of oilseed rape pollen grains and dehiscing anthers

Dehiscing anther from an untransformed plant Pollen grains – untransformed anthers

Dehiscing male sterile anther from a plant containing TA29-barnase gene Pollen grains – anthers containing both the TA29- Dehiscing anther from a barstar and TA-29 barnase plant restored to male genes – restored to fertility fertility containing both the TA29-barstar and TA29-barnase gene Fig 4. Presence of barnase and barstar mRNAs in anthers of oilseed rape plants restored to male fertility Fig 5. Presence of barstar and barnase proteins in oilseed rape anthers restored to male sterility

MS plants (TA29- Anther proteins from barnase gene) wild type plants

Barstar and barnase proteins circled in the MS/RF plants (TA29- immunoblot barnase+TA29-barstar) Purified barstar-barnase complexes (denatured, fractionated by 2D gel electrophoresis) in a nutshell … Whats going on now?? Abstract

• L-ornithinase (argE) gene of E.coli was fused to the OSIPA promoter sequence which is known to function specifically in the pollen grains. • OSIPA – Oryza sativa indica pollen allergen • argE gene – involved in arginine biosynthesis in E.coli and also can deacetylate N-acetyl Phosphinothricin (N-ac-PPT) to yield phosphinothricin (PPT). • N-ac-PPT, a non toxic compound, but when converted in to PPT becomes cytotoxic. • Phosphinothricin is the active ingredient of the herbicides Basta (bialophos) and Glufosinate.

• Homozygous transgenic rice (variety BPT 5204) plants (T2) were obtained with argE gene by selection with hygromycin (hyg) following Agrobacterium mediated transformation. Contd…

• Transgenic rice plants expressing argE gene became completely male sterile after application of N-ac-PPT (inducer) due to the pollen specific expression of argE. • The argE transgenic plants produced fertile seeds in the absence of N- ac-PPT treatment. • Normal fertile seeds were obtained when male sterile argE transgenics were cross pollinated with untransformed control plants. • Female fertility male of sterile argE transgenics is not affected by the N-ac-PPT treatment. • First report of induction of complete male sterility in Rice. • This system does not require the use of restorer line. Results… Table I. Effect of N-ac-PPT on pollen fertility of argE rice transformants after topical application (0.075 mg/ml @ 100-110 days, 25 ml/plant)

Table 2. Effect of N-ac-PPT on pollen fertility of argE rice transformants after treatment with irrigation (0.2 mg/ml @ 80-90 days, 50ml/plant) Fig 2. Induction of pollen sterility in argE transgenic rice plants by N-acetyl-PPT treatment. (Alexander staining was carried out before and after N-ac-PPT treatment)

Control

Transformed

Untreated anther and gynoecium Treatment with N-ac-PPT by Treatment with N-ac-PPT spraying through irrigation Fig 3. Pollen grains germination ability of UC and argE-transgenic rice plants before and after N-ac-PPT treatment

Control

Transformed

Untreated pollen Treated with N-ac- Treated with N-ac- grains PPT by spraying PPT by irrigation Fig 4. Effect of N-acetyl-PPT on male fertility of argE transgenic rice plants

(A) Plants treated with N-acetyl-PPT (B) Plants treated with N-acetyl- by spraying topically PPT through irrigation Fig 5. Seed setting ability of argE transgenic rice plants after treating with N-acetyl- PPT

(A) Treated with N-ac-PPT by (B) Treated with N-ac-PPT through spraying irrigation Later, they were allowed to self pollinate. UC: Panicle of untransformed control showing normal seed setting. 9-6 & 25-3: Panicles of two argE transformants showing the failure of seed setting. Fig 6. Seed setting ability of argE male-sterile rice lines after pollination with UC plants.

(A) Treated with N-ac-PPT by (B) Treated with N-ac-PPT spraying through irrigation The list continues…

Commercial exploitation of GE male sterility

GM Canola – barnase/barstar system • Aventis has successfully introduced a GE canola hybrid, using the barnase/barstar gene system in 1996. • Regulatory agencies in such countries as Canada , USA , Mexico , Europe, Australia and Japan have approved consumption of this GE canola Issues in developing Genetically engineered hybrid crops – the Indian case

• One of the DMH-11 genes, called the bar gene, made the plant resistant to a herbicide (or weed killer) brand-named Basta, a product sold by multinational company Cropscience. • Two other genes — that weren’t patented in India — called barnase and barstar — were used to make mustard varieties more amenable to becoming hybrids. • DMH-11 developed by crossing Indian and East European mustard varieties. 25-30% more yield. • Hybrid varieties have had the greatest impact on increasing the world’s feed or food resources. • Usable male sterility systems have not yet been generated through conventional breeding methodologies for several important crop plants. • In such a scenario, genetic engineering becomes an efficient and rapid approach for developing male sterile lines in these crops. • The creation of new and efficient means of pollination control to produce better hybrids will soon be the plant biotechnologist’s contribution to this effort. Queries? Thank You… Plant Breeding, 136, 287–299 (2017) doi:10.1111/pbr.12473 © 2017 Blackwell Verlag GmbH

Review Article The use of genetic, manual and chemical methods to control pollination in vegetable hybrid seed production: a review

1 2,3,4 N OEMI C OLOMBO and C LAUDIO R OMULO G ALMARINI 1Instituto de Genetica, CICVYA, CNIA, INTA, CC 25, B1712WAA, Castelar, Buenos Aires, Argentina; 2Estacion Experimental Agropecuaria La Consulta, Instituto Nacional de Tecnologıa Agropecuaria (INTA), CC8 (5567), La Consulta, Mendoza, Argentina; 3Consejo Nacional de Investigaciones Cientıficas y Tecnicas (CONICET), Buernos Aires, Argentina; 4Facultad de Ciencia Agrarias, Universidad Nacional de Cuyo, Almirante Brown 500, M5528 AHB, Chacras de Coria, Lujan, Mendoza, Argentina; Corresponding author, E-mail: [email protected] With 2 figures and 2 tables Received July 26, 2016 / Accepted February 4, 2017 Communicated by M. Havey

Abstract Types of Pollination Control Production of hybrid varieties of vegetable crops is currently a desired breeding goal, due to their remarkable agronomic performance and to Taking into account the mechanism affecting pollen production, the possibility of intellectual property protection. However, efficient pollination control systems can be classified as follows: hybrid production requires a careful pollination control to guarantee the hybrid nature of F1 seed. Several technologies ranging from manual emasculation to genetic transformation are used to inhibit pollen pro- Systems determined by genetic control duction in mother plants. In this review, we examine the principles Male sterility underlying strategies like genetically determined systems (genic male Male sterility has been defined as the failure of plants to produce sterility, cytoplasmic–genic male sterility, self-incompatibility) and other functional anthers, pollen or male gametes (Kaul 1988). Anther methods (manual emasculation, chemical-hybridizing agents) in different and pollen development can be considered as a pathway with species, considering the benefits and drawbacks of their adoption. several stages: anther cell specification – comprising stamen pri- Finally, we present the current state of the art for vegetable hybrid seed mordial initiation and archaesporial initiation –, mature pollen production. formation – in which pollen mother cell meiosis and microspore Key words: Hybrid seed production — pollination control — maturation take place – and anther maturation and pollen release, male sterility — vegetables — chemical-hybridizing agents when dehiscence occurs. Many of the key genes involved in anther and pollen formation have been identified (Wilson et al. 2011). In this process, the tapetum plays a central role as sup- Hybrid varieties are major components of vegetable production plier of nutrients, proteins, lipids and polysaccharides which are systems due to their vigour, uniformity, horticultural quality, used in microspore release and pollen-wall formation (Parish and biotic and abiotic stress resistances and high yields. Another Li 2010). Male sterility can be conditioned by nuclear or cyto- important reason is the inherent biological intellectual property plasmic factors affecting any of the stages of microsporogenesis protection offered by hybrids. In some cases, hybrid parents or gametogenesis, resulting in genic male sterility (GMS) and are owned by a company. The inbred parents may be trade cytoplasmic male sterility (CMS), respectively. secrets, and thus, the hybrid has a built in form of protection Genic male sterility (GMS) for seed companies. Worldwide the share of hybrid seed is increasing at a fast pace of 8–10% annually in most of the Spontaneous and induced mutants vegetables crops (da Silva Dias 2014). However, their adoption Nuclear genes affecting normal pollen development have been by vegetable growers is limited by the high cost of hybrid reported in over 175 species. Most of the male sterile mutants seed. One of the bottlenecks in hybrid seed production is pol- have arisen spontaneously with a high frequency and some have lination control required to eliminate pollen from the mother been induced using physical or chemical mutagens, singly or in line in order to avoid undesirable selfing, thus obtaining true combination. In all cases, the pattern of inheritance and expres- F1 seed. Different technologies – ranging from manual emas- sion of GMS is Mendelian (Kaul 1988). The majority of the ms culation to transformation – provide alternative pathways to genes are recessive, in accordance with a loss-of-function muta- avoid pollen production in female plants (Fu et al. 2014, tion. However, some dominant genes have also been reported Kempe and Gils 2011). The choice among them is made (Kaul 1988, Fang et al. 1997, Shu et al. 2012). More than 55 according to the species of interest, the economic value of the recessive ms genes have been identified in tomato, over a dozen final product and the hybrid seed industry characteristics. This in pepper and six in broccoli (Kumar 2014). In common bean, review deals with the principles and applications of current faba bean, cauliflower, cabbage and turnip both recessive and available technologies of pollination control for vegetable dominant ms genes have been reported. In other species, like hybrid seed production. In the first section, different systems Brussels sprouts, radish, beetroot, Swiss chard, onion and carrot, are presented and their advantages and restrictions are dis- recessive ms genes are available, but they are not used to pro- cussed. The second part describes the current state of the art duce hybrids. Similarly, recessive ms genes in spinach, cucum- in the field of hybrid production of vegetable crops. ber, pumpkin, zucchini and summer squash have little practical 288 N. COLOMBO AND C. R. GALMARINI value due to the use of gynoecious lines or sex regulating chemi- is achieved by expressing the barstar gene, encoding an intracel- cals (Kaul 1988, Delourme and Budar 1999, Kumar 2014). lular inhibitor of barnase (Mariani et al. 1992, Reynaerts et al. When recessive ms genes are used in hybrid seed production, 1993), thus allowing hybrid seed production. This strategy has they are maintained by crossing msms plants with a heterozygous been successfully developed in cauliflower and chicory (Rey- fertile maintainer line producing 1 : 1 male sterile and male fertile naerts et al. 1993), tomato (Zhang et al. 1998, Bai et al. 2002), plants. Selection to discard male fertile genotypes before flower- Chinese cabbage (Yu et al. 2000), cabbage (Shen et al. 2001), ing must be carried out, using morphological or molecular mark- flowering Chinese cabbage (Cao et al. 2008) and eggplant (Cao ers tightly linked to the ms gene (Fig. 1). In tomato, some et al. 2010). Male sterility has been successfully engineered in markers linked to ms genes are as follows: absence of anthocyanin different vegetable crops using different approaches, like down- in the seedling stem (Gardner 2000), woolly phenotype (Durand regulation or over expression of essential genes (Sinha and 1981), potato leaf shape (Kaul 1988) and enzyme markers (Tanks- Rajam 2013, Nandy et al. 2013), transcription factor gene silenc- ley et al. 1984). A lack of suitable markers, as well as laborious ing (Toppino et al. 2011), callose degradation (Curtis et al. maintenance processes and poor free outcrossing in certain highly 1996) and metabolic engineering (Goetz et al. 2001, Cheng et al. self-pollinated species limit the use of GMS (Dhall 2010). 2015). Factors affecting the wide adoption of genetically engi- Some male sterility genes change their expression under dif- neered male sterility are as follows: availability of efficient gene ferent environmental conditions like temperature and photoperiod constructs, possible dispersion of transgene to other related spe- inducing thermosensitive genic male sterility (TGMS) and pho- cies, availability of efficient transformation techniques, very high toperiod-sensitive genic male sterility (PGMS), respectively. This initial investment and biosafety and regulatory matters (Singh means that msms plants will be male sterile at a particular tem- et al. 2012, Ananthi et al. 2013). At present, only the barnase- perature or photoperiod at a sensitive stage, whereas they will be barstar system has been used at commercial level in vegetable male fertile at another condition (Virmani and Ilyas-Ahmed crops to produce chicory hybrids (Singh et al. 2012). However, 2001). When grown under restrictive environments, TGMS and the licence to produce F1 hybrids using this system is no longer PGMS lines serve as the male sterile female parent; the same valid, and the marketing of these transgenic plants is not allowed lines grown in permissive conditions are fertile and allow the in the European Union (Klocke et al. 2010). propagation of the sterile line. This reversible system eliminates Cytoplasmic and cytoplasmic–genic male sterility (CMS-CGMS) the requirement of crossing to propagate the male sterile line and allows for the efficient development of the ‘two-line’ hybrids Cytoplasmic male sterility (CMS) is determined by mitochon- (Chen and Liu 2014). TGMS and PGMS lines have been drial genes resulting from mitochondrial DNA rearrangements reported in several horticultural crops (Dhall 2010). Production which disturb the normal development of pollen. This maternally of hybrids using GMS has been shown in pepper, chilli, cab- inherited character has been described in more than 140 species bage, cauliflower and tomato (Dhall 2010, Radkova et al. 2009). of higher plants. Although the mechanism involved in CMS has not been elucidated, evidence supports roles for energy defi- ciency, programmed cell death (PCD) and reactive oxygen spe- Genetically engineered male sterility cies (ROS) (Chen and Liu 2014, Horn et al. 2014, Hu et al. In the last 25 years, genetic engineering has become a new 2014, Touzet and Meyer 2014). Cytoplasmic–genic male sterility source of dominant genic male sterility, especially in those veg- (CGMS) results from the interaction of mitochondrial genes etable crops where other efficient sources of male sterility are causing male sterility and nuclear genes, which specifically not available. The objective of this technology is to disrupt any restore male fertility – Rf genes –, thus representing an example step during microsporogenesis or microgametogenesis by insert- of the crosstalk between mitochondrial and nuclear genomes. ing cloned gene sequences through genetic transformation. The Besides spontaneously arising CMS, the character has been first report was based on the tapetum specific expression of the obtained through wide crosses as a result of intra/interspecific or toxic enzyme barnase, a chimeric ribonuclease gene from Bacil- intergeneric nuclear/cytoplasmic incompatibilities (Hanson and lus amyloliquefaciens, which leads to the precocious degenera- Bentolila 2004, Kubo et al. 2011, Kaminski et al. 2015). tion of the tapetum cells, the arrest of microspore development Another source of new CMS lines is protoplast fusion, where and male sterility (Mariani et al. 1990). Male fertility restoration recombination between the two parental mitochondrial genomes occurs prior to cytoplasmic segregation in the somatic hybrids (Rambaud et al. 1993, Carlsson et al. 2007). Recently, CMS was achieved by genetic engineering of the chloroplast genome using the b-ketothiolase gene, a component of the polydroxybu- tyrate pathway (Ruiz and Daniell 2005). Additionally, repro- ducible transgenic induction of mitochondrial rearrangements leading to CMS was accomplished by disrupting the expression of Msh1, a nuclear gene involved in the suppression of mito- chondrial DNA rearrangements in tomato and tobacco (Sandhu et al. 2007). So far, no mitochondria transformation technology has been developed in higher plants. Although these biotechno- logical approaches offer a vast range of possibilities, they have not been adopted in commercial production of hybrids yet. CMS was first described in onion by Jones and Emsweller (1936), and its use for hybrid seed production was established by Jones and Clarke (1943) who reported that male sterility is originated by the interaction of the male sterile (S)cytoplasm Fig. 1: Hybrid seed production using recessive GMS Pollination control in hybrid seed production 289 with the homozygous recessive genotype at the male fertility specificity is expressed by both pollen and pistil. Current knowl- restoration locus (Ms) in the nucleus. The dominant allele Ms edge indicates that S-locus consists of at least two linked genes, restores male fertility in plants with S cytoplasm, while plants each of them coding for the male and female determinants possessing the normal (N) cytoplasm are male fertile irrespective expressed in the pollen grain and pistil, respectively. The vari- of the genotype of the restorer gene. For hybrid production, a ants of the gene complex are called S-haplotypes and the SI male sterile inbred line – A line – (S msms) and a maintainer response occurs when both determinants are issued by the same line to propagate the A line – B line – (N msms) are needed. S-haplotypes (Takayama and Isogai 2005). SI systems have been The A line is pollinated by the pollinator inbred line for hybrid classified as gametophytic (GSI) and sporophytic (SSI). In GSI, production (Fig. 2). The pollinator line will carry Ms alleles if the most widespread system, the incompatibility type of the pol- seeds are the product of economic value; on the other hand, len is controlled by its own haploid genotype, whereas in SSI, nuclear restoration will be not required for vegetable, fruit or for- the pollen incompatibility type is controlled by the diploid age crops (Havey 2004). Although CGMS is widely applied in (sporophyte) genotype of the parental anther in which it was pro- hybrid production, it presents some limitations which may duced (Hiscock and Tabah 2003, Franklin-Tong and Franklin restrict its adoption (Dhall 2010). 2003). The identities of female and male determinants have been Some of these constraints are related to adverse effects of the determined and molecular models for different types of SI have sterile cytoplasm, like (i) pleiotropic undesirable horticultural been developed recently (Kaothien-Nakayama et al. 2010, Ser- traits (Pelletier et al. 1983, Kaminski et al. 2012), (ii) poor rano et al. 2015). A major advantage of using SI for hybrid seed cross-pollination of male sterile flowers due to changes in flower production is that only two self-incompatible lines carrying dif- morphology and chemical composition of nectar (Soto et al. ferent S alleles are necessary (Kucera et al. 2006). Besides, in 2013) and (iii) instability of male sterility in different environ- this mechanism, pollen and nectar production are unaltered ments, which causes contamination of true hybrid seed with (Singh et al. 2013). Hybrid seed production requires the mainte- selfed seed (Weider et al. 2009). These topics will be discussed nance of self-incompatible lines. Several methods have been on family basis in the second part of this review. Besides, hybrid effectively used to break self-incompatibility like bud pollination seed production using CGMS is technically complex. A, B and (Dixon 2007), manipulation of plant age (Horisaki and Niikura pollinator lines must be developed and produced in isolated 2008), tissue culture (Razdan 2003), exposure to high tempera- areas, special distribution of rows in the field is needed, optimal ture (Parkash et al. 2015), carbon dioxide gas treatment (Palloix pollination must be ensured, and careful handling of hybrid seed et al. 1985, Lao et al. 2014) and stigmatic treat- is essential. Finally, male sterile cytoplasms and restorer genes ment (Monteiro et al. 1988, Kucera et al. 2006). Main difficul- are not always available. Successful application of CGMS to ties in SI adoption for hybrid seed production are the cost of vegetable hybrid production has been achieved in onion, chive, maintenance of SI lines, depression in SI lines due to continuous Japanese bunching onion, carrot, pepper, cabbage, cauliflower, inbreeding, effects of environmental factors on the expression of broccoli, Swiss chard, beetroot and sweet corn (Dhall 2010, self-incompatibility, pseudo compatibility, lack of synchroniza- Havey 2004). However, little attention has been paid to diversifi- tion of flowering (Parkash et al. 2015). SI systems have been cation of CMS sources. Sustainable breeding should emphasize extensively studied in the Brassicaceae, a family carrying SSI, the search, characterization and introgression of several cytoplas- and hybrid seed has been successfully obtained in cabbage, cau- mic types in order to prevent harmful effects associated to uni- liflower and broccoli (Singh 2000, Singh et al. 2013, Parkash formity (Kubo et al. 2011, Kumar 2014, Saxena and Hingame et al. 2015). However, self-incompatibility also exhibits some 2015). Information about different sources of cytoplasmic male limitations in this family. Some cauliflower genotypes, for sterility is given on family basis in sections 3.1, 3.2, 3.3, 3.7 instance, present a weak SI at high temperature, resulting into and 3.9 of this review. selfing and sibling among the plants of the female parent. This severe limitation can be overcome by the use of parental lines Self-incompatibility with synchronized flowering and similar morphology or by polli- Self-incompatibility (SI) is a genetically determined prezygotic nation by stored pollen (Kumar and Singh 2005). mate-recognition system preventing self-pollination and is very common in Angiosperms (Kao and McCubbin 1996, Ferrer and Systems not determined by genetic control Good 2012). SI response is comprised of a self- and non-self- recognition process between pollen and pistil that is followed by Manual emasculation selective inhibition of the self-pollen development. In most spe- Manual emasculation consists on manual removal of the stamens cies, SI is controlled by a single multi-allelic locus, the S-locus, from hermaphrodite flowers or the elimination of complete male which determines pollen inhibition when the same ‘S-allele’ flowers when they are separated from the female ones. This method is labour intensive and requires highly skilful human resources to ensure complete emasculation without affecting female organs (Kumar and Singh 2005, Adhikari 2012, Ozores- Hampton 2014). It is not suitable in species with small hermaph- rodite flowers, like carrot and onion, where other methods of pollination control are required. Subsequent pollination after emasculation can be carried out either by hand or by pollinator insects. To be cost-effective, it is appropriate for species that will produce many seeds from each pollination, like solanaceous veg- etables and cucurbits as compared to legumes. Nevertheless, availability of a genetic based system of pollination control in Fig. 2: Hybrid seed production and multiplication of lines using CMS. tomato or pepper would effectively reduce the cost of hybrid (a) F1 seed production and multiplication of R line; (b) multiplication of A and B lines seed production. Hand-pollinated hybrid seed production occurs 290 N. COLOMBO AND C. R. GALMARINI mainly in East Asia (China and Taiwan) and South-East Asian Table 1: Chemical-hybridizing agents probed for hybrid seed highlands (northern Thailand and northern Philippines), India production in vegetable crops (Karnataka and Andhra Pradesh), Mexico, Chile and, in recent Chemicals Crops References years, Argentina, where local conditions fulfil the requirements of vegetable seed industry (Tay 2006, Gallardo 2012). This sys- TIBA Tomato Rehm (1952) tem is successfully used in hybrid seed production of tomato, (Triiodobenzoic Squash Wittwer and Hillyer (1954) eggplant, pepper, cucurbits and sweet corn (Tay 2006, Kumar Acid) MH (Maleic Pepper Chauhan (1980) and Singh 2005). In Argentina, it is widely used for hybrid hydrazide) Onion Chopra et al. (1960) squash seed production (Della Gaspera 2013). Tomato Rehm (1952) Squash Wittwer and Hillyer (1954) Coriander Kalidasu et al. (2009) Chemical-hybridizing agents (CHAs) NAA (Naphthalene Tomato Mc Rae (1985) Since the middle of the 20th century, many attempts have been acetic acid) Squash, Wittwer and Hillyer (1954) made to find substances effective to selectively disrupt pollen cucumber development without affecting the female functionality. These Dalapon Pea, tomato Brauer (1959) compounds, known as gametocides, pollen suppressors or chemi- (Dichloropropionic acid) cal-hybridizing agents (CHAs) belong to different chemical Dalapon and a- Pepper Hirose and Fujime (1973) groups: auxins and auxin inhibitors (NAA, IBA, 2,4-D, TIBA, chloropropionate MH), gibberellins, ethylene (ethephon–ethrel), halogenated ali- FW-450 (sodium 2,3- Tomato Moore (1959) phatic acids (FW450; Dalapon), arsenicals and brassinosteroids. dichloroisobutyrate) Gibberellic acid Lettuce Eenick (1977) Not surprisingly, most of the evaluated products are plant growth Pepper Sawhney (1981) regulators. Cytokinins, auxin, gibberellins, ethylene, jasmonic Tomato Chandra Sekhar and Sawhney acid and brassinosteroids play a role in anther development, and (1990) male sterility has been associated with changes in many plant Onion van der Meer and van Bennekom growth regulators, suggesting that normal male development is (1976) Brussels van der Meer and van Dam (1979) controlled in concert by multiple hormones (Mc Rae 1985, sprouts, Huang et al. 2003, Hirano et al. 2008, Ye et al. 2010). The main cabbage, advantages of this technology are as follows: ease of making and cauliflower evaluating hybrid combinations, labour efficient seed production and kale CCC ((2-chloroethyl) Tomato Rastogi and Swahney (1988) and no need for developing male sterile and restorer lines. The trimethylammonium major drawbacks are as follows: toxicity effects on the female or chloride) – F1 seed, difficulties in field applications due to precise stage of Etephon ethrel (2- Lettuce Han and Lee (1972) plant development and environmental factors and less effective- Chloroethyl Eggplant Helal and Zaki (1981) ness due to interaction with genotypes and environment (Cross phosphonic acid) Squash Della Gaspera (2013) ABA (Abscisic acid) Tomato Rastogi and Swahney (1988) and Ladyman 1991, Adhikari 2012, Fu et al. 2014). In Table 1, products tested in different vegetable crops are presented. Although CHAs efficiency has been verified experimentally, their cytoplasm has been exhaustively studied and is used worldwide use at commercial scale in vegetable crops has not been demon- in F1 breeding of Brassicaceae. Discovered by Ogura (1968) in strated (Kumar and Singh 2005). Ethephon is regularly used for a Japanese radish (R. sativus L.), Ogura cytoplasm was intro- squash seed production, mainly for interspecific hybrids between gressed in B. oleracea by repeated backcrosses to cabbage and Cucurbita maxima Duchesne 9 Cucurbita moschata Duchesne, broccoli (Bannerot et al. 1974, Mc Collum 1981) and later from but the male sterility is never permanent, and several applications broccoli to cauliflower (Dickson 1975, Hoser-Krauze 1987). of the hormone are required (Della Gaspera 2013). However, these first alloplasmic male sterile lines carrying Ogura cytoplasm presented chlorophyll deficiency at low temper- atures, underdeveloped nectaries and malformed ovaries and Pollination Control Methods in Different Vegetable pods which reduced the seed set. These defects were assumed to Crops be caused by negative interactions between the Brassica nucleus Brassicaceae and the Raphanus chloroplasts and were overcome by somatic The main vegetable crops belonging to this family are cabbage, hybridization. Protoplast fusion between a normal B. oleracea cauliflower and broccoli (Brassica oleracea var. capitata L., var. line and a CMS (Ogura-radish) B. oleracea line allowed the botrytis L. and var. italica Plenck, respectively), turnip (Brassica selection of cybrids carrying only Brassica chloroplasts that rapa L. spp rapa) and radish (Raphanus sativus L.). F1 hybrid grew normally (Pelletier et al. 1983). These improved CMS lines production is a goal for breeders due to the high heterosis are known as Ogu-INRA and are widely used to produce hybrids observed in these species as well as greater uniformity. As men- in Brassicaceae (Pelletier and Budar 2015, Kaminski et al. tioned earlier, several pollination control systems are available in 2015). Extensive research on Ogura cytoplasm has revealed the this family: GMS, CGMS and SI. Hybrids have historically been molecular basis of male sterility and fertility restoration (Bon- produced using sporophytic SI, a natural method lacking adverse homme et al. 1992, Tanaka et al. 2012, Brown et al. 2003, Des- side effects. This system, however, has been replaced by an effi- loire et al. 2003, Koizuka et al. 2003, Uyttewaal et al. 2008). As cient CGMS method due to the occurrence of some self-pollina- a result, genomic data are available for development of molecu- tion (Singh et al. 2013, Kucera et al. 2006, Havey 2004). lar markers to assist the selection of CMS and restorer lines Several male sterile cytoplasms exist in this family, arising from (Kim et al. 2007, Yu et al. 2016). intraspecific variation, interspecific or intergeneric hybridizations In China, GMS-based pollination control is an alternative to and cell fusion (Yamagishi and Bhat 2014). Among them, Ogura CGMS used to produce hybrids in the Brassica family (Fang Pollination control in hybrid seed production 291 et al. 1997). Hybrid cabbage seed is produced at commercial et al. 2000, Leite et al. 1999). On the other hand, T cytoplasm is scale using a dominant male sterile gene, Ms-cd1 (Fang et al. commercially used in Europe and Japan (Havey 2000) and is 1997), for which SCAR and SSR are available to assist breeding present in Brazilian onion populations (Fernandes Santos et al. programmes (Zhang et al. 2011). 2010). To diversify the male sterile cytoplasms used in hybrid onion production, Havey (1999) introduced the cytoplasm of Allium galanthum Kar. et Kir in onion populations. In galan- Apiaceae thum CMS, complete absence of anthers is observed and nuclear Carrot (Daucus carota L.): Commercial production of hybrid restorer genes appear to be rare or non-existent (Havey 1999). carrot was feasible only after CGMS systems were available. Determination of cytoplasm types by test crossing demands 4– The main types of CMS are ‘brown anther’ (Sa), characterized 8 years in onion due to its biennial cycle. Several molecular by shrivelled, yellow-to-brown anthers with no pollen (Welch markers have been developed to identify the different cytoplasm and Grimball 1947) and ‘petaloid’ (Sp), in which anthers are types, thus reducing time and labour (Havey 1995, Sato 1998, replaced by a whorl of petals (Thompson 1961, Eisa and Wal- Engelke et al. 2003, Kim et al. 2009, Cho et al. 2006, Kohn lace 1969, Peterson and Simon 1986, Morelock et al. 1996). et al. 2013). Likewise, many attempts have been made to Both systems show instability due to high temperatures, dry con- develop molecular markers tightly linked to the Ms locus (Gokc€ ße ditions, growing time or long-day conditions. As carrot is par- et al. 2002, Bang et al. 2011, Huo et al. 2012, Yang et al. 2013, tially andromonoecious, the development of CMS lines requires Havey 2013, Huo et al. 2015, Kim et al. 2015), but their valida- stringent selection. Hybrid seed production is largely based on tion is needed before applying them in MAS of maintainer lines the use of petaloid CMS type because of less frequent reversion (Saini et al. 2015, Khar and Saini 2016). to male fertility; however, seed yields on the brown-anther CMS Hybrid chive (Allium schoenoprasum L.) and Japanese bunch- are generally higher (Havey 2004, Dhall 2010). Eleven molecu- ing onion (Allium fistulosum L.) are also produced using CGMS lar markers developed by Bach et al. (2002) distinguished all Sp systems (Havey 2004). In leek (Allium ampeloprasum L.), from N cytoplasms and are being used to identify the type of hybrids were initially produced using genic male sterility and cytoplasm and test seed purity in breeding programmes, to select ‘in vitro’ propagation of male sterile lines (Smith and Crowther cybrids after protoplast fusion and to study basic diversity in the 1995). genus Daucus. Besides these main CMS types, other CMS sources with potential use in hybrid production have been described (Linke et al. 1999, Nothnagel et al. 2000). Regarding Asteraceae fertility restoration, multiple nuclear genes with complex interac- In chicory (Cichorium intybus L.), hybrid seed has been obtained tions have been involved in different studies (Thompson 1961, on the basis of a male sterile nuclear mutation known as ‘Edith’ Hansche and Gabelman 1963, Borner€ et al. 1995, Wolyn and (Desprez 1993, Desprez et al. 1994, Gonthier et al. 2013). Chahal 1998). Recently, Alessandro et al. (2013) found that Recently, Barcaccia et al.(2011) and Barcaccia and Tiozzo(2014) restoration of petaloid cytoplasmic male sterility was due to a reported four new male sterile mutants of red chicory and devel- single dominant gene, Rf1, and developed a linkage map using oped molecular markers to assist hybrid breeding. An attempt to molecular markers, some of which can be used to develop PCR- develop an alternative source of male sterility was made by based markers for marker-assisted selection (MAS) in hybrid somatic hybridization through protoplast fusion between chicory breeding programmes. and the CMS line Pet 1 of sunflower (Rambaud et al. 1993, Celery (Apium graveolens L.): A recessive single locus 1997, Dubreucq et al. 1999, Varotto et al. 2001, Delesalle et al. involved in genic male sterility was found in an accession from 2004, Habarugira et al. 2015), but no reliable production of Iran, and efforts to use it for hybrid seed production have been hybrid chicory was achieved. Another approach, which reached carried out (Quiros et al. 1986, Quiros 1993, Tay 2006). Look- commercial application, was based on genetic transformation ing for an alternative system, cytoplasmic male sterility has been using the Barnase system (Reynaerts et al. 1993, Kempken experimentally induced by protoplast fusion between celery and 2010). CMS carrot (Tan et al. 2009). Recently, the use of CMS to obtain celery hybrids has been patented in China (Zhu et al. 2011, Gao et al. 2015). Amaranthaceae subfamily Chenopodioideae The former Chenopodiaceae family includes important vegetable crops like beetroot (Beta vulgaris varrubra L.), Swiss chard Amaryllidaceae (Beta vulgaris var cicla L.) and spinach (Spinacia oleracea L.). Onion (Allium cepa L.) hybrid seed has been extensively pro- In the case of beetroot and Swiss chard, hybrid production relies duced all over the world using CGMS-based systems. There are on CGMS systems. The most commonly used cytoplasm is two main sources of CMS, identified as S and T, which have Owen type originally described in sugar beet as the result of the been genetically characterized. S type (Jones and Emsweller interaction between a sterilizing cytoplasm and at least two 1936) results from the interaction of a cytoplasmic factor S and nuclear restorer genes and environmental factors (Owen 1942, a single nuclear restorer gene Ms (Jones and Clarke 1943). T 1945). Restorer gene Rf1 has been cloned and sequenced (Hagi- type is controlled by the interaction of the cytoplasmic factor T hara et al. 2005, Matsuhira et al. 2012), and molecular markers and three independent restorer genes (Berninger 1965, Sch- are available for MAS (Moritani et al. 2013). Recently, Honma weisguth 1973). Both cytoplasm types have been independently et al. (2014) have reported the molecular mapping of Rf2. Owen isolated from different sources (Havey 2000). S type is the most cytoplasm was introduced in beetroot between the 1950s and the widely used due to the relatively common occurrence of the 1960s, and hybrids have been produced since then. The associ- recessive allele at Ms, the stability of male sterility over environ- ated use of the annual gene B (conditioning annual flowering ments and no reduction of female fertility. Besides, it was the habit) has helped to efficiently develop sterile inbred lines and first source of CMS available in different germplasms (Goldman the introduction of the SF allele permitted self-pollination thus 292 N. COLOMBO AND C. R. GALMARINI allowing the development of inbred maintainer lines (Bliss and pollen abortive type (conferred by the ms-series) and functional Gabelman 1965, Goldman and Navazio 2008). Three other CMS sterility (conditioned by ps-2 gene) are often used in hybrid pro- types – E, G and H – have been characterized in Beta vulgaris duction, generating reductions in the costs of hybrid seed pro- (Cuguen et al. 1994, Satoh et al. 2004, Darracq et al. 2011), but duction as compared to manual procedures (Yordanov 1983, there is no information about their use in hybrid production. Georgiev 1991, Dhall 2010, Atanassova and Georgiev 2002). In Spinach (S. oleracea L.) is a dioecious species with an even particular, the ms1035 gene (Rick 1948, Jeong et al. 2014) linked ratio of female-to-male individuals. However, occasional monoe- to the marker gene anthocyanin-less aa has been widely used cious plants are observed in some populations, among which the due to its stability and lack of growth defects. proportion of female-to-male (or hermaphrodite) flowers per Attempts to establish a system based on CMS in tomato have plant varies widely (Janick and Stevenson 1955, Onodera et al. been made by exploring interspecific crosses (Andersen 1963, 2008, 2011, Yamamoto et al. 2014). Hybrids were initially pro- 1964, Valkova-Achkova 1980) and protoplast fusion (Melchers duced alternating female and male rows of two promising dioe- et al. 1992, Petrova et al. 1999); however, no CMS system is cious lines and removing the staminate flowers from the female currently available for this crop (Stoeva-Popova et al. 2007). rows as soon as differentiation was possible (Webb and Thomas Eggplant (Solanum melongena L.) is another example of the 1976). At present, it is preferred to use a highly female monoe- use of manual emasculation and hand pollination to commer- cious inbred as female parent and a highly male monoecious cially produce hybrid seed (Kumar and Singh 2005). A recessive inbred as male parent (Thompson 1955, Janick 1998, van der gene conferring functional male sterility – fms – (Phatak et al. Vossen 2004) . 1991) was tested, but the occasional presence of pollen in the indehiscent anthers due to environmental factors inhibited its application to hybrid seed production (Daunay 2008). Well-char- Solanaceae acterized genetic resources related to CMS systems and molecu- Pepper (Capsicum annuum L.) hybrid cultivars are commercially lar tools designed to assist selection will provide new obtained using manual emasculation with hand pollination or possibilities for eggplant hybrid breeding in the future (Mizanur methods based on genetic control of pollen, like GMS and et al. 2016, Krommydas et al. 2016). CGMS, with hand pollination or natural pollination (Kumar and In potato (Solanum tuberosum L.), self-incompatibility and Singh 2005, Tay 2006). genic and cytoplasmic male sterility have been described (Jansky Nearly 20 genes for GMS have been found or induced. All of 2009). However, hybrid cultivars have received a strong boost them are highly stable, and a few of them are linked with mark- with the recent adoption of Sli gene originating from Solanum ers which allow early identification of the sterile plants (Shifriss chacoense Bitter which makes diploid potato self-compatible 1997, Wang and Bosland 2006, Dhaliwal and Jindal 2014). (Hosaka and Hanneman 1998a,b, Phumichai et al. 2005). Using Commercial production of hybrids in India and Hungary are this strategy, Lindhout et al. (2011) obtained inbred lines that based on the use of the line ‘MS-12’ (ms10/ms10) and ms3 gene, combined self-compatibility with good agronomic performance respectively (Kumar 2014). Molecular markers have been devel- as well as hybrids which were uniform and showed good tuber oped and will help the introgression of ms genes in different quality; nevertheless, agamic propagation is mainly used world- backgrounds and selection of sterile plants in hybrid seed pro- wide. duction (Bartoszewski et al. 2012, Aulakh et al. 2016). The only source of CMS in this species is S cytoplasm from the ‘PI 164835’ line introduced from India (Peterson 1958, Liu Fabaceae et al. 2013). Kim and Kim (2005) developed two CMS-specific In common bean (Phaseolus vulgaris L.), hand emasculation and SCAR markers to distinguish N cytoplasm from S cytoplasm by pollination have been experimentally used. However, the low PCR. Restoration of fertility for S cytoplasm has been attributed outcrossing rate in field conditions yields insufficient number of to a single dominant nuclear gene (Peterson 1958, Gulyas et al. seeds for large agronomic evaluations, thus limiting the advance (2006) or to one major and four minor quantitative trait loci of hybrid breeding (Palmer et al. 2011). A male sterile cyto- which were mapped in the pepper genome by Wang et al. plasm found in several accessions has been characterized at the (2004). Molecular markers tightly linked to the major restorer molecular level (Bannerot 1989, Mackenzie 1991), and its single gene Rf were obtained (Zhang et al. 2000, Kim et al. 2006, Min dominant restorer gene has been identified (Mackenzie and Bas- et al. 2009). CGMS is commercially used to produce hot (chilli) set 1987). pepper hybrid seed, but it has not been successful for sweet pep- Faba bean (Vicia faba L.) presents considerable levels of allo- per due to the lack of restorer genes or instability of restorer gamy and high heterosis for productive traits (Le Guen et al. lines in most of sweet pepper genotypes. Recently, Lin et al. 1991). Genetic resources related to male sterility include reces- (2015) introgressed the Rf allele from hot pepper into several sive and dominant male sterility genes and several sterile cyto- sweet pepper lines opening the way to an efficient CMS applica- plasms (CMS 447, CMS 350, CMS 199 and CMS 297), tion for sweet pepper hybrid seed production. together with their restorer of fertility genes (reviewed in Palmer In tomato (Solanum lycopersicum L.), commercial hybrid seed et al. 2011). However, spontaneous reversion to fertility production using manual emasculation and hand pollination is observed in these male sterile cytoplasms has precluded their use economically viable and predominates in the seed industry at in commercial hybrid seed production. present. However, the availability of alternative suitable methods to avoid selfing and optimize crossing will considerably reduce Cucurbitaceae the cost of F1 seed (Kumar and Singh 2005, Sharma et al. 2015). More than 55 male sterile genes are known, and sterility Most of the species in this family are monoecious, with big they confer can be grouped in four groups: pollen sterile (pollen flowers which allow commercial hybrid production by pinching abortive), stamenless (stamens absent), positional sterility (stigma staminate flowers followed by hand or natural pollination usually exerted) and functional sterility (anthers do not dehisce). Both helped by pollinators like honeybees and bumblebees (Kumar Pollination control in hybrid seed production 293

Table 2: Available ( ) and commercially applied ( ) systems for pollination controlling F1 hybrid seed production of vegetable crops Systems not determined by Systems determined by genetic control genetic control Pollination control Genic male steril- ity Cytoplasmic Species S/I GE Male Sterility Self-incompatibility Manual Chemical

Cabbage Cauliflower Broccoli Turnip Radish Carrot Celery Onion Chive Bunching onion Leek Chicory Beetroot Swiss chard Spinach Pepper Tomato Eggplant Common bean Faba bean Potato Cucumber Squash Pumpkin Zucchini Sweet corn and Singh 2005, Gajc-Wolska et al. 2011, Petersen et al. 2013). over genotypes and environments (Weider et al. 2009). How- In cucumber (Cucumis sativus L.), gynoecious lines are available ever, the relatively short life of sweet corn hybrids makes it very and their widespread use has hampered the applicability of GMS difficult to develop new inbred CMS lines soon enough, thus based on recessive ms genes (Kumar 2014, Call and Wehner favouring hand emasculation (Havey 2004). Recent incorporation 2010). Plant growth regulators are frequently employed in spe- of transgenic traits like herbicide resistance and insect control in cies of this family to increase the number of female flowers. commercial sweet corn cultivars has reduced the use of CMS Application of ethylene or ethylene-releasing compounds because of the time necessary to transfer CMS into transgenic increases female flower production in zucchini (Cucurbita pepo lines (Havey 2004, Williams et al. 2015). Nevertheless, CMS L.), pumpkin (Cucurbita maxima Duchesne) and monoecious has recently been proposed as a tool to prevent transgene disper- cucumber (Robinson et al. 1969, Rudich et al. 1969, Hume and sal through pollen, thus enabling the coexistence of GM and Lovell 1981, Manzano et al. 2011). Similarly, auxins and brassi- non-GM crops (Buckmann€ et al. 2013). nosteroids promote femaleness in cucumber probably stimulating Table 2 presents a summary of the methods for pollination ethylene production (Shannon and De La Guardia 1969, Papado- control cited in this review. poulou and Grumet 2005). Hand pollination and applications of ethephon are regularly used for squash seed production, mainly for interspecific hybrids between Cucurbita maxima Duch- Perspectives esne 9 Cucurbita moschata Duchesne (Della Gaspera 2013). The global market of vegetable seeds is expected to expand in future years, due to the increase in world population and con- sumption. There is a clear association between human health and Poaceae vegetable consumption that is increasing vegetable demand Sweet corn (Zea mays L.) hybrid breeding has assimilated meth- worldwide. Moreover, markets are getting more refined in terms ods and genetic resources developed in corn hybrid seed produc- of quality and yield and there is a clear demand for excellent tion. Hybrid seed is produced by hand emasculation hybrid vegetable cultivars (da Silva Dias 2014). The exploitation (detasseling) and wind pollination or using CGMS systems. of heterosis is one of the leading causes for that trend, but it is Three male sterile cytoplasms are available in maize, T, C and even more important the protection of breeder rights. Although S, each of them being restored by specific Rf genes (Chen and OP cultivars can perform in many cases as well as F1 hybrids, Liu 2014). Male sterile and normal cytoplasms can be easily dis- the latter are preferred by the industry. Any contribution to criminated by multiplex PCR (Liu et al. 2002). T cytoplasm has increase the efficiency of hybrid seed industry will help to been banned from hybrid seed production because of its suscep- reduce seed price and alleviate grower’s costs. In this context, tibility to Bipolaris maydis (Nisikado and Miyake) Shoemaker; the availability of a safe and cost-effective method to control race T; C and S cytoplasms need to be checked for their stability pollination is of major relevance during hybrid seed production. 294 N. COLOMBO AND C. R. GALMARINI

In the last decades, a trend favouring the incorporation of Ogura-specific fragment correlated with cytoplasmic male-sterility in genetic control of male fertility can be observed. Advanced Brassica cybrids. Mol. Gen. Genet. 235, 340—348. knowledge in and genomics, microsporogenesis and Borner,€ T., B. Linke, T. Nothnagel, R. Scheike, B. Schulz, R. Steinborn, gametogenesis, plant cell biology and plant biotechnology have A. Brennicke, M. Stein, and G. Wricke, 1995: Inheritance of nuclear allowed the characterization of genetic resources and the devel- and cytoplasmic factors affecting male sterility in Daucus carota. In: U. Kuck,€ and G. Wricke (eds), Genetic Mechanisms for Hybrid Breeding opment of new technologies for different vegetable crops thus Advances in Plant Breeding 18, 111—122. Blackwell Science, Berlin. broadening the range of choices. Molecular breeding has also Brauer, H. O., 1959: Male sterility obtained with dalapon. Agric. Tec. facilitated the introgression of desired characters by the use of Mex. 9,6—8. MAS and MABC (marker-assisted backcross). On these bases, Brown, G. G., N. Formanova, H. Jin, R. Wargachuk, C. Dendy, P. Patil, it is reasonable to predict continuous progress in the adoption M. Laforest, J. Zhang, W. Y. Cheung, and B. S. Landry, 2003: The of these technologies by breeders and seed producers in the radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a future. protein with multiple pentatricopeptide repeats. Plant J. 35, 262—272. Buckmann,€ H., A. Husken,€ and J. Schiemann, 2013: Applicability of cytoplasmic male sterility (CMS) as a reliable biological confinement method for the cultivation of genetically modified maize in Germany. References J. Agric. Sci. Technol. A 3, 385—403. Adhikari, N. R., 2012: Chemical emasculation in plant breeding. http:// Call, A. D., and T. C. Wehner, 2010: Gene list 2010 for cucumber. Rep. highmettle.blogspot.com/2013/11/v-behaviorurldefaultvmlo_4.html. Cucurbit. Genet. Coop. 33–34,69—103. Alessandro, M. S., C. R. Galmarini, M. Iorizzo, and P. W. Simon, 2013: Cao, B. H., C. M. Meng, J. J. Lei, and G. J. Chen, 2008: The pTA29- Molecular mapping of vernalization requirement and fertility restora- barnase chimeric gene transformation of Brassica campestris L. subsp. tion genes in carrot. Theor. Appl. Genet. 126, 415—423. chinensis makino var. parachinensis mediated by Agrobacterium. Chin. Ananthi, M., P. Selvaraju, and P. Srimathi, 2013: Transgenic male steril- J. Biotechnol. 24, 881—886. ity for hybrid seed production in vegetables -A review. Weekly Sci. Cao, B. H., Z. Y. Huang, G. J. Chen, and J. J. Lei, 2010: Restoring pol- Res. J. 1,1—10. len fertility in transgenic male-sterile eggplant by Cre/loxP-mediated Andersen, W. R., 1963: Cytoplasmic sterility in hybrids of Lycopersicon site-specific recombination system. Genet. Mol. Biol. 33, 298—307. esculentum and Solanum pennellii. Rep. Tomato Genet. Coop. 13, Carlsson, J., M. Leino, and K. Glimelius, 2007: Mitochondrial genotypes 7—8. with variable parts of DNA affect development Andersen, W. R., 1964: Evidence for plasmon differentiation in Lycoper- in Brassica napus lines. Theor. Appl. Genet. 115, 627—641. sicon. Rep. Tomato Genet. Coop. 14,4—6. Chandra Sekhar, K. N., and V. K. Sawhney, 1990: Regulation of the Atanassova, B. B., and H. Georgiev, 2002: Using genic male sterility in fusion of floral organs by temperature and gibberellic acid in the nor- improving hybrid seed production in tomato (Lycopersicon esculen- mal and solanifolia mutant of tomato (Lycopersicon esculentum). Can. tum). Acta Hortic. 579, 185—188. J. Bot. 68, 713—718. Aulakh, P. S., M. S. Dhaliwal, S. K. Jindal, R. Schafleitner, and K. Chauhan, S. V. S., 1980: Effect of maleic hydrazide, FW 450 and dala- Singh, 2016: Mapping of male sterility gene ms10 in chilli pepper pon on anther development in Capsicum annuum. J. Indian Bot. Soc. (Capsicum annuum L.). Plant Breed. 18,1—5. 59, 133—136. Bach, I. C., A. Olesen, and P. W. Simon, 2002: PCR-based markers to Chen, L., and Y. G. Liu, 2014: Male sterility and fertility restoration in differentiate the mitochondrial genomes of petaloid and male fertile crops. Annu. Rev. Plant Biol. 65, 5.1—5.28. carrot (Daucus carota L.). Euphytica 127, 353—365. Cheng, J., Z. Wang, F. Yao, L. Gao, S. Ma, X. Sui, and Z. Zhang, Bai, L., F. Liu, S. P. Li, and M. Q. Cao, 2002: Agrobacterium mediated 2015: Down-regulating CsHT1, a cucumber pollen-specific hexose barnase gene transfer of tomato (Lycopersicon esculentum). J. Henan transporter, inhibits pollen germination, tube growth, and seed devel- Univ. 32,16—19. opment. Plant Physiol. 168, 635—647. Bang, H., D. Y. Cho, K. S. Yoo, M. K. Yoon, B. S. Patil, and S. Kim, Cho, K. S., T. J. Yang, S. Y. Hong, Y. S. Kwon, J. G. Woo, and H. G. 2011: Development of simple PCR-based markers linked to the Ms Park, 2006: Determination of cytoplasmic male sterile factors in onion locus, a restorer-of-fertility gene in onion (Allium cepa L.). Euphytica plants (Allium cepa L.) using PCR RFLP and SNP markers. Mol. 179, 439—449. Cells 21, 411—417. Bannerot, H., 1989: The potential of hybrid bean. In: S. Beeke (ed.), Chopra, V. L., S. K. Jain, and M. S. Swaminathan, 1960: Studies on the Current Topics in Breeding of Common Bean, 111—134. CIAT, Cali. chemical induction of pollen sterility in some crop plants. Indian J. Bannerot, H. O., L. Boulidard, Y. Cauderon, and T. Tempe, 1974: Cyto- Genet. Plant Breed. 20, 188—199. plasmic male sterility transfer from Raphanus to Brassica. In: Proc. Cross, J. W., and J. A. R. Ladyman, 1991: Chemical agents that inhibit Eucarpia Meet. Cruciferae 52—54. pollen development: tools for research. Sex. Plant Reprod. 4, Barcaccia, G., and S. C. Tiozzo, 2014: Cichorium spp. male sterile 235—243. mutants. US Patent 20140157448 A1. Cuguen, J., R. Wattier, P. Saumitou-Laprade, D. Forcioli, M. Morchen,€ Barcaccia, G., S. Collani, G. Galla, A. Ghedina, S. Tiozzo, and R. H. Van Dijk, and P. Vernet, 1994: Gynodioecy and mitochondrial Tiozzo, 2011: Discovery of nuclear male-sterility in red chicory: DNA polymorphism in natural populations of Beta vulgaris ssp mar- genetic analysis and methods for the marker-assisted breeding of F1 itima. Genet. Sel. Evol. 26,87—101. hybrid varieties. Proc. Joint Meeting AGI-SIBV-SIGA, Oral Commu- Curtis, I. S., C. He, R. Scott, J. Brian Power, and M. R. Davey, 1996: nication Abstract – 6A.04. Genomic male sterility in lettuce, a baseline for the production of F1 Bartoszewski, G., C. Waszczak, P. Gawronski, I. Stepien, H. Bolibok- hybrids. Plant Sci. 113, 113—119. Bragoszewska, A. Palloix, V. Lefebvre, A. Korzeniewska, and K. Darracq, A., J. S. Varre, L. Marechal-Drouard, A. Courseaux, V. Castric, Niemirowicz-Szczytt, 2012: Mapping of the ms8 male sterility gene in P. Saumitou-Laprade, S. Oztas, P. Lenoble, B. Vacherie, V. Barbe, sweet pepper the chromosome P4 using PCR-based markers useful for and P. Touzet, 2011: Structural and content diversity of mitochondrial breeding programmes. Euphytica 186, 453—461. genome in beet: a comparative genomic analysis. Genome Biol. Evol. Berninger, E., 1965: Contribution al’etude de la sterilitem^ale de 3, 723—736. l’oignon (Allium cepa L.). Ann. Amelior. Plant. 15, 183—199. Daunay, M. C., 2008: Eggplant. In: J. Prohens, and F. Nuez (eds), Bliss, F. A., and W. H. Gabelman, 1965: Inheritance of male sterility in Vegetables II, 163—220. Springer, New York. beets Beta vulgaris L. Crop Sci. 5, 403—406. Delesalle, L., C. Dhellemmes, and M. Desprez, 2004: Methods of mak- Bonhomme, S., F. Budar, D. Lancelin, I. Small, M. C. Defrance, and ing cytoplasmic male sterile chicory plants comprising the ORF 522 of G. Pelletier, 1992: Sequence and transcript analysis of the Nco2.5 Helianthus annuus. US Patent 6803497 B1. Pollination control in hybrid seed production 295

Della Gaspera, P., 2013: Manual del cultivo del zapallo anquito (Cucur- Georgiev, H., 1991: Heterosis in tomato breeding. In: G. Kalloo (ed.), bita moschata Duch.), 1a ed. Ediciones INTA, San Carlos, Mendoza. Genetic Improvement of tomato, 83—98. Springer Verlag, Berlin, Hei- Delourme, R., and F. Budar, 1999: Male sterility. In: C. Gomez-Campo delberg. (ed.), Biology of Brassica Coenospecies, 185—216. Elsevier Science Goetz, M., D. E. Godt, A. Guivarch, U. Kahmann, D. Chriqui, and B.V., Amsterdam. T. Roitsch, 2001: Induction of male sterility in plants by metabolic Desloire, S., H. Gherbi, W. Laloui, S. Marhadour, V. Clouet, L. Cat- engineering of the carbohydrate supply. Proc. Natl Acad. Sci. USA 98, tolico, C. Falentin, S. Giancola, M. Renard, F. Budar, I. Small, M. 6522—6527. Caboch, R. Delourme, and A. Bendahmane, 2003: Identification of the Gokc€ ße, A. F., J. McCallum, Y. Sato, and M. J. Havey, 2002: Molecular fertility restoration locus, Rfo, in radish, as a member of the pentatri- tagging of the Ms locus in onion. J. Am. Soc. Hortic. Sci. 127, copeptide-repeat protein family. EMBO Rep. 4, 588—594. 576—582. Desprez, B., 1993: Recherche de methodes d’obtention de plantes hap- Goldman, I. L., and J. P. Navazio, 2008: Table beet. In: J. Prohens, and lo€ıdes chez la chicoree (Cichorium intybus L.). Ph.D. thesis, University F. Nuez (eds), Vegetables I, 219—238. Springer, New York. of Paris, Paris, France. Goldman, I. L., G. Schroeck, and M. J. Havey, 2000: History of public Desprez, B. F., L. Delesalle, C. Dhellemmes, and M. F. Desprez, 1994: onion breeding programs and pedigree of public onion germplasm Genetique et amelioration de la chicoree industrielle. CR Acad. Agric. releases in the United States. Plant Breed. Rev. 20,67—103. 80,47—62. Gonthier, L., C. Blassiau, M. Morchen,€ T. Cadalen, M. Poiret, T. Hen- Dhaliwal, M. S., and S. K. Jindal, 2014: Induction and exploitation of driks, and M. C. Quillet, 2013: High-density genetic maps for loci nuclear and cytoplasmic male sterility in pepper (Capsicum spp.): a involved in nuclear male sterility (NMS1) and sporophytic self-incom- review. J. Hortic. Sci. Biotechnol. 89, 471—479. patibility (S-locus) in chicory (Cichorium intybus L., Asteraceae). The- Dhall, R. K., 2010: Status of male sterility in vegetables for hybrid or. Appl. Genet. 126, 2103—2121. development. A review. Adv. Hortic. Sci. 24, 263—279. Gulyas, G., K. Pakozdi, J. S. Lee, and Y. Hirata, 2006: Analysis of Dickson, M. H., 1975: G1117A, G1102A and G1106A cytosterile broc- fertility restoration by using cytoplasmic male-sterile red pepper coli inbreds. HortScience 10, 535. (Capsicum annuum L.) lines. Breed. Sci. 56,31—334. Dixon, G. R., 2007: Vegetable Brassicas and related crucifers. In: J. Habarugira, I., T. Hendriks, M. C. Quillet, J. L. Hilbert, and C. Ram- Atherton, and A. Rees (eds), Crop Production Science in Horticulture baud, 2015: Effects of nuclear genomes on anther development in Series No 14, ISBN 9780851993959, CABI, Oxfordshire, UK. cytoplasmic male sterile chicories (Cichorium intybus L.): morphologi- Dubreucq, A., B. Berthe, J. F. Asset, L. Boulidard, F. Budar, J. Vasseur, cal analysis. Sci. World J. 20, 13. doi:10.1155/2015/529521 and C. Rambaud, 1999: Analyses of mitochondrial DNA structure and Hagihara, E., N. Itchoda, Y. Habu, S. Iida, T. Mikami, and T. Kubo, 2005: expression in three cytoplasmic male-sterile chicories originating from Molecular mapping of a fertility restorer gene for Owen cytoplasmic somatic hybridization between fertile chicory and CMS sunflower pro- male sterility in sugar beet. Theor. Appl. Genet. 111, 250—255. toplasts. Theor. Appl. Genet. 99, 1094—1105. Han, D. W., and J. J. Lee, 1972: Effect of ethrel, RH 531 and gibberellin Durand, V., 1981: Relationships between the marker genes aa and wo on the sterile pollen induction in lettuce. PhD. Thesis. Seoul Municipal and the male sterility gene ms 35. In: J. Philouze (ed.), Genetics and College of Agriculture, Plant Breeding Abstracts, 4774, 1974. Breeding of Tomato, Proc. Meet. Eucarpia Tomato Working Group, Hansche, P. E., and W. H. Gabelman, 1963: Digenic control of male 225—228. Avignon, France. sterility in carrots Daucus carota L.. Crop Sci. 3, 383—386. Eenick, A. H., 1977: Induction of male sterility in lettuce (Lactuca sativa Hanson, M. R., and S. Bentolila, 2004: Interactions of mitochondrial and L.) by application of gibberellic acid: a technical note. Euphytica 26, nuclear genes that affect male gametophyte development. Plant Cell 31—32. 16, S154—S169. Eisa, H. M., and D. H. Wallace, 1969: Morphological and anatomical Havey, M. J., 1995: Identification of cytoplasms using the polymerase aspects of petaloidy in the carrot, Daucus carota L. J. Am. Soc. Hor- chain reaction to aid in the extraction of maintainer lines from tic. Sci. 94, 545—548. open-pollinated populations of onion. Theor. Appl. Genet. 90, Engelke, T., D. Terefe, and T. Tatlioglu, 2003: A PCR-based marker 263—268. system monitoring CMS-(S), CMS-(T) and (N)-cytoplasm in the onion Havey, M. J., 1999: Seed yield, floral morphology and lack of male-ferti- (Allium cepa L.). Theor. Appl. Genet. 107, 162—167. lity restoration of male-sterile onion (Allium cepa) populations possess- Fang, Z. Y., P. T. Sun, Y. M. Liu, L. M. Yang, X. W. Wang, A. F. ing the cytoplasm of Allium galanthum. J. Am. Soc. Hortic. Sci. 124, Hou, and C. S. Bian, 1997: A male sterile line with dominant gene 626—629. (Ms) in cabbage (Brassica oleracea var. capitata) and its utilization Havey, M. J., 2000: Diversity among male-sterility-inducing and male- for hybrid seed production. Euphytica 97, 265—268. fertile cytoplasms of onion. Theor. Appl. Genet. 101, 778—782. Fernandes Santos, C. A., D. Lopes Leite, V. Rodrigues Oliveira, and M. Havey, M. J., 2004: The use of cytoplasmic male sterility for hybrid seed Amorim Rodrigues, 2010: Marker-assisted selection of maintainer lines production. In: H. Daniell, and C. Chase (eds), Molecular Biology and within and onion tropical population. Sci. Agric. 67, 223—227. Biotechnology of Plant Organelles, 623—634. Springer, Dordrecht. Ferrer, M. M., and S. V. Good, 2012: Self-sterility in flowering plants: Havey, M. J., 2013: Single nucleotide polymorphisms in linkage disequi- preventing self-fertilization increases family diversification rates. Ann. librium with the male-fertility restoration (Ms) locus of onion. J. Am. Bot. 110, 535—553. Soc. Hortic. Sci. 138, 306—309. Franklin-Tong, N. V., and F. C. Franklin, 2003: Gametophytic self- Helal, R. M., and M. E. Zaki, 1981: Effect of 2, 4-D and Ethephon foliar incompatibility inhibits pollen tube growth using different mechanisms. sprays on induction of pollen sterility in eggplant. Egypt. J. Hortic. 8, Trends Plant Sci. 8, 598—605. 101—108. Fu, D., M. Xiao, A. Hayward, Y. Fu, G. Liu, G. Jiang, and H. Zhang, Hirano, K., K. Aya, T. Hobo, H. Sakakibara, M. Kojima, R. A. Shim, 2014: Utilization of crop heterosis: a review. Euphytica 197, 161—173. Y. Hasegawa, M. U. Tanaka, and M. Matsuoka, 2008: Comprehensive Gajc-Wolska, J., K. Kowalczyk, J. Mikas, and R. Drajski, 2011: Effi- transcriptome analysis of phytohormone biosynthesis and signaling ciency of cucumber (Cucumis sativus L.) pollination by bumblebees genes in microspore/pollen and tapetum of rice. Plant Cell Physiol. 49, (Bombus terrestris). Acta Sci. Pol. 10, 159—169. 1429—1450. Gallardo, G. S., 2012: Desarrollo institucional y polıtica cientıfica: el Hirose, T., and Y. Fujime, 1973: Studies of chemical emasculation in caso de la produccion nacional de semilla hortıcola. Tesis de Maestrıa pepper. III. Effect of repeated application of 2, 2-dichloropropionate en Gestion de la Ciencia, Tecnologıa e Innovacion. UNGS, 171 pp. (dalapon) and the gametocidal action of dalapon and related com- Gao, G., W. Wang, F. Wu, and H. Liu, 2015: Hybrid pollination method pounds. J. Japan. Soc. Hortic. Sci. 42, 235—240. of celery and application of method. Patent CN 104396730. Hiscock, S. J., and D. A. Tabah, 2003: The different mechanisms of Gardner, R. G., 2000: A male-sterile cherry tomato breeding line, NC 2C sporophytic self-incompatibility. Philos. Trans. R. Soc. Lond. B 358, ms-10, aa. HortScience 35, 964—965. 1037—1045. 296 N. COLOMBO AND C. R. GALMARINI

Honma, Y., K. Taguchi, H. Hiyama, R. Yui-Kurino, T. Mikami, and T. to obtain male-sterile Chinese cabbage inbred lines. Euphytica 208, Kubo, 2014: Molecular mapping of restorer of fertility 2 gene identi- 519—534. fied from a sugar beet (Beta vulgaris L. ssp. vulgaris) homozygous for Kao, T. H., and A. G. McCubbin, 1996: How flowering plants discrimi- the non-restoring restorer of fertility 1 allele. Theor. Appl. Genet. 127, nate between self and non-self pollen to prevent inbreeding. Proc. Natl 2567—2574. Acad. Sci. USA 93, 12059—12065. Horisaki, A., and S. Niikura, 2008: Developmental and environmental Kaothien-Nakayama, P. K., A. Isogai, and S. Takayama, 2010: Self- factors affecting level of self-incompatibility response in Brassica rapa incompatibility systems in flowering plants. In: E. C. Pua, and M. R. L. Sex. Plant Reprod. 21, 123—132. Davey (eds), Plant Developmental Biology-Biotechnological Perspec- Horn, R., K. J. Gupta, and N. Colombo, 2014: Mitochondrion role in tives 1, 459—486. Springer Verlag, Berlin, Heidelberg. molecular basis of cytoplasmic male sterility. Mitochondrion 19, 198 Kaul, M. L. H., 1988: Male Sterility in Higher Plants. Springer-Verlag, —205. Berlin, Heidelberg. Hosaka, K., and R. E. Hanneman, 1998a: b: Genetics of self-compatibil- Kempe, K., and M. Gils, 2011: Pollination control technologies for ity in a self-incompatible wild diploid potato species Solanum cha- hybrid breeding. Plant Breed. 27, 417—437. coense. 2. Localization of an S locus inhibitor (Sli) gene on the potato Kempken, F., 2010: Engineered male sterility. In: F. Kempken, and genome using DNA markers. Euphytica 103, 265—271. C. Jung (eds), Genetic Modification of Plants, Agriculture, Horticulture Hosaka, K., and R. E. Hanneman, 1998b: Genetics of self-compatibility in and Forestry, 253—268. Springer Verlag, Berlin, Heidelberg. a self-incompatible wild diploid potato species Solanum chacoense.1. Khar, A., and N. Saini, 2016: Limitations of PCR-based molecular mark- Detection of an S locus inhibitor (Sli) gene. Euphytica 99, 191—197. ers to identify male-sterile and maintainer plants from Indian onion Hoser-Krauze, J., 1987: Influence of cytoplasmic male sterility source on (Allium cepa L.) populations. Plant Breed. 135, 4:519—524. some characters of cauliflower (Brassica oleracea var. botrytis L.). Kim, D. H., and B. D. Kim, 2005: Development of SCAR markers for Genet. Pol. 28, 101. early identification of cytoplasmic male sterility genotype in chili pep- Hu, J., W. Huang, Q. Huang, X. Qin, C. Yu, L. Wang, S. Li, R. Zhu, per (Capsicum annuum L.). Mol. Cells 20, 416—422. and Y. Zhu, 2014: Mitochondria and cytoplasmic male sterility in Kim, D. S., D. H. Kim, J. H. Yoo, and B. D. Kim, 2006: Cleaved ampli- plants. Mitochondrion 19, 282—288. fied polymorphic sequence and amplified fragment length polymor- Huang, S., R. E. Cerny, Y. Qi, D. Bhat, C. M. Aydt, D. D. Hanson, K. phism markers linked to the fertility restorer gene in chili pepper P. Malloy, and L. A. Ness, 2003: Transgenic studies on the involve- (Capsicum annuum L.). Mol. Cells 21, 135—140. ment of cytokinin and gibberellin in male development. Plant Physiol. Kim, S., H. Lim, S. Park, K. H. Cho, S. K. Sung, D. G. Oh, and K. T. 131, 1270—1282. Kim, 2007: Identification of a novel mitochondrial genome type and Hume, R. J., and P. H. Lovell, 1981: Reduction of the cost involved in development of molecular markers for cytoplasm classification in rad- hybrid seed production of pumpkin (Cucurbita maxima Duchesne). N. ish (Raphanus sativus L.). Theor. Appl. Genet. 115, 1137—1145. Z. J. Exp. Agr. 9, 209—210. Kim, S., E. T. Lee, D. Y. Cho, T. Han, H. Bang, B. S. Patil, Y. K. Ahn, Huo, Y., J. Miao, B. Liu, Y. Yang, Y. Zhang, Y. Tahara, Q. Meng, Q. and M. K. Yoon, 2009: Identification of a novel chimeric gene, He, H. Kitano, and X. Wu, 2012: The expression of pectin methyles- orf725, and its use in development of a molecular marker for distin- terase in onion flower buds is associated with the dominant male-ferti- guishing among three cytoplasm types in onion (Allium cepa L.). The- lity restoration allele. Plant Breed. 131, 211—216. or. Appl. Genet. 118, 433—441. Huo, Y. M., B. J. Liu, Y. Y. Yang, J. Miao, L. M. Gao, S. P. Kong, Z. Kim, S., C. W. Kim, M. Park, and D. Choi, 2015: Identification of can- B. Wang, H. Kitano, and X. Wu, 2015: AcSKP1, a multiplex PCR didate genes associated with fertility restoration of cytoplasmic male based co-dominant marker in complete linkage disequilibrium with the sterility in onion (Allium cepa L.) using a combination of bulked seg- male-fertility restoration (Ms) locus, and its application in open-polli- regant analysis and RNA-seq. Theor. Appl. Genet. 128, 2289—2299. nated populations of onion. Euphytica 204, 711—722. Klocke, E., T. Nothnagel, and G. Schumann, 2010: Vegetables. In: F. Janick, J., 1998: Hybrids in horticultural crops. In: K. R. Lamkey, and Kempken, and C. Jung (eds), Genetic Modification of Plants, Agricul- J. E. Staub (eds.), Concepts and Breeding of Heterosis in Crop Plants, ture, Horticulture and Forestry, 499—552. Springer Verlag, Berlin, 45—56. Crop Science Society of America Special Publication number Heidelberg. 25, Madison, WI, United States of America. Kohn, C., A. Kiełkowska, and M. J. Havey, 2013: Sequencing and anno- Janick, J., and E. C. Stevenson, 1955: Genetics of the monoecious char- tation of the chloroplast DNAs and identification of polymorphisms acter in spinach. Genetics 40, 429—437. distinguishing normal male-fertile and male-sterile cytoplasms of Jansky, S. H., 2009: Breeding, genetics, and cultivar development. In: onion. Genome 56, 737—742. J. Singh, and L. Kaur (eds), Advances in Potato Chemistry and Tech- Koizuka, N., R. Imai, H. Fujimoto, T. Hayakawa, Y. Kimura, J. Kohno- nology, 27—61. Elsevier, San Diego. Murase, T. Sakai, S. Kawasaki, and J. Imamura, 2003: Genetic charac- Jeong, H. J., J. H. Kang, M. Zhao, J. K. Kwon, H. S. Choi, J. H. Bae, terization of a pentatricopeptide repeat protein gene, orf687 that H. Lee, Y. H. Joung, D. Choi, and B. C. Kang, 2014: Tomato Male restores fertility in the cytoplasmic male-sterile Kosena radish. Plant J. sterile 1035 is essential for pollen development and meiosis in anthers. 34, 407—415. J. Exp. Bot. 65, 6693—6709. Krommydas, K. S., Z. Tzikalios, P. Madesis, F. A. Bletsos, A. Mavro- Jones, H., and A. Clarke, 1943: Inheritance of male sterility in the onion matis, and D. G. Roupakias, 2016: Development and fertility restora- and the production of hybrid seed. Proc. Am. Soc. Hortic. Sci. 43, tion of CMS eggplant lines carrying the cytoplasm of Solanum 189—194. violaceum. J. Agric. Sci. 8,10—26. Jones, H., and S. Emsweller, 1936: A male sterile onion. Proc. Am. Soc. Kubo, T., K. Kitazaki, M. Matsunaga, H. Kagami, and T. Mikami, 2011: Hortic. Sci. 34, 582—585. Male sterility-inducing mitochondrial genomes: how do they differ? Kalidasu, G., C. Sarada, P. Venkata Reddy, and T. Yellamanda Reddy, Crit. Rev. Plant Sci. 30, 378—400. 2009: Use of male gametocide: an alternative to cumbersome emascu- Kucera, V., V. Chytilova, M. Vyvadilova, and M. Klıma, 2006: Hybrid lation in coriander (Coriandrum sativum L.). J. Hortic. Forest. 1, breeding of cauliflower using self-incompatibility and cytoplasmic 126—132. male sterility. Hortic. Sci. (Prague) 33, 148—152. Kaminski, P., B. Dyki, and A. A. Stezpowska, 2012: Improvement of cau- Kumar, S., 2014: Male sterility in vegetables. In: K. P. Singh, and liflower male sterile lines with Brassicanigra cytoplasm, phenotypic A. Bahadur (eds), Olericulture-Fundamental of Vegetable Production, expression and possibility of practical application. J. Agric. Sci. 4, I, 431—439. Kalyani, Ludhiana. 190—200. Kumar, S., and P. K. Singh, 2005: Mechanisms for hybrid development Kaminski, P., M. Podwyszynska, M. Starzycki, and E. Starzycka-Korbas, in vegetables. J. New Seeds 6, 381—407. 2015: Interspecific hybridisation of cytoplasmic male-sterile rapeseed Lao, X., K. Suwabe, S. Niikura, M. Kakita, M. Iwano, and

with Ogura cytoplasm and Brassica rapa var. pekinensisas a method S. Takayama, 2014: Physiological and genetic analysis of CO2- Pollination control in hybrid seed production 297

induced breakdown of self-incompatibility in Brassica rapa. J. Exp. Monteiro, A. A., W. A. Gabelman, and P. H. Williams, 1988: Use of Bot. 65, 939—951. sodium chloride solution to overcome self-incompatibility in Brassica Le Guen, J., P. Berthelem, and G. Duc, 1991: Breeding for heterosis and campestris. HortScience 23, 876—877. male sterility in faba bean. In: J. I. Cubero, and M. C. Saxena (eds), Moore, R. H., 1959: Male sterility induced in tomato by sodium 2, 3- Present Status and Future Prospects of Faba Bean Production and dichloroisobutyrate. Science 129, 1738—1740. Improvement in the Mediterranean Countries, 41—49. CIHEAM, Morelock, T. E., P. W. Simon, and C. E. Peterson, 1996: Wisconsin Zaragoza. wild: another petaloid male-sterile cytoplasm for carrot. HortScience Leite, D. L., C. R. Galmarini, and M. J. Havey, 1999: Cytoplasms of 31, 887—888. elite open-pollinated onions from Argentina and Brazil. Allium Moritani, M., K. Taguchi, K. Kitazaki, H. Matsuhira, T. Katsuyama, T. Improv. Newsl. 9,1—4. Mikami, and T. Kubo, 2013: Identification of the predominant non Lin, S. W., H. C. Shieh, Y. W. Wang, C. W. Tan, R. Schafleitner, W. J. restoring allele for Owen-type cytoplasmic male sterility in sugar beet Yang, and S. Kumar, 2015: Restorer breeding in sweet pepper: intro- (Beta vulgaris L.): development of molecular markers for the main- gressing Rf allele from hot pepper through marker-assisted backcross- tainer genotype. Mol. Breed. 32,91—100. ing. Sci. Hortic. 197, 170—175. Nandy, S., R. Sinha, and M. V. Rajam, 2013: Over-expression of argi- Lindhout, P., D. Meijer, T. Schotte, R. C. B. Hutten, R. C. F. Visser, nine decarboxylase gene in tapetal tissue results in male sterility in and G. J. van Eck, 2011: Towards F1 hybrid seed potato breeding. tomato plants. Cell Dev. Biol. 2, 117. Potato Res. 5, 301—312. Nothnagel, T., P. Straka, and B. Linke, 2000: Male sterility in popula- Linke, B., T. Nothnagel, and T. Borner,€ 1999: Morphological characteri- tions of Daucus and the development of alloplasmic male-sterile carrot zation of modified flower morphology of three novel alloplasmic male lines. Plant Breed. 119, 145—152. sterile carrot sources. Plant Breed. 118, 543—548. Ogura, H., 1968: Studies on the new male-sterility in Japanese radish Liu, Z., A. O. Peter, M. Long, U. Weingartner, P. Stamp, and O. Kaeser, with special reference to the utilization of this sterility toward the 2002: A PCR assay for rapid discrimination of sterile cytoplasm types practical raising of hybrid seeds. Mem. Fac. Agric. Kagoshima Univ. in maize. Crop Sci. 42, 566—569. 6,39—78. Liu, C., N. Ma, P. Y. Wang, N. Fu, and H. L. Shen, 2013: Transcrip- Onodera, Y., I. Yonaha, S. Niikura, S. Yamazaki, and T. Mikami, 2008: tome sequencing and de novo analysis of a cytoplasmic male sterile Monoecy and gynomonoecy in Spinacia oleracea L.: morphological line and its near-isogenic restorer line in chili pepper (Capsicum and genetic analyses. Sci. Hortic. 118, 266—269. annuum L.). PLoS ONE 8, e65209. Onodera, Y., I. Yonaha, H. Masumo, A. Tanaka, S. Niikura, S. Yamaza- Mackenzie, S. A., 1991: Identification of a sterility-inducing cytoplasm ki, and T. Mikami, 2011: Mapping of the genes for dioecism and in a fertile accession line of Phaseolus vulgaris L. Genetics 127, monoecism in Spinacia oleracea L.: evidence that both genes are clo- 411—416. sely linked. Plant Cell Rep. 30, 965—971. Reference mentioned in Mackenzie, S. A., and M. Basset, 1987: Genetics of restoration in cyto- page 24. plasmic male sterile Phaseolus vulgaris L. Theor. Appl. Genet. 74, Owen, F. V., 1942: Male sterility in sugar beets produced by comple- 642—645. mentary effects of cytoplasmic and Mendelian inheritance. Am. J. Bot. Manzano, S., C. Martınez, Z. Megıas, P. Gomez, D. Garrido, and M. 29, 692. Jamilena, 2011: The role of ethylene and brassinosteroids in the con- Owen, F. V., 1945: Cytoplasmically inherited male-sterility in sugar trol of sex expression and flower development in Cucurbita pepo. beets. J. Agric. Res. 71, 423—440. Plant Growth Regul. 65, 213—221. Ozores-Hampton, M., 2014: Hand pollination of tomato for breeding and Mariani, C., M. D. Beuckeleer, J. Truettner, J. Leemans, and R. B. Gold- seed production. Univ. Florida Inst. Food Agric. Sci. HS1248. berg, 1990: Induction of male sterility in plants by a chimaeric ribonu- Palloix, A., Y. Herve, R. B. Knox, and C. Dumas, 1985: Effect of car- clease gene. Nature 347, 737—741. bon dioxide and relative humidity on self-incompatibility in cauli- Mariani, C., V. Gossele, M. De Beuckeleer, M. De Block, R. B. Goldberg, flower, Brassica oleracea. Theor. Appl. Genet. 70, 628—633. W. De Greef, and J. Leemans, 1992: A chimaeric ribonuclease inhibitor Palmer, R. G., J. Gai, V. A. Dalvi, and M. J. Suso, 2011: Male sterility and gene restores fertility to male sterile plants. Nature 357, 384—387. hybrid production technology. In: A. Pratap, and J. Kumar (eds), Biol- Matsuhira, H., H. Kagami, M. Kurata, K. Kitazaki, M. Matsunaga, Y. ogy and Breeding of Food Legumes, 193—207. CABI, Oxfordshire. Hamaguchi, E. Hagihara, M. Ueda, M. Harada, A. Muramatsu, R. Papadopoulou, E., and R. Grumet, 2005: Brassinosteriod-induced female- Yui-Kurino, K. Taguchi, H. Tamagake, T. Mikami, and T. Kubo, ness in cucumber and relationship to ethylene production. HortScience 2012: Unusual and typical features of a novel restorer-of-fertility gene 40, 1763—1767. of sugar beet (Beta vulgaris L.). Genetics 192, 1347—1358. Parish, R. W., and S. F. Li, 2010: Death of a tapetum: a programme of Mc Collum, G. D., 1981: Induction of an alloplasmic male sterile Bras- developmental altruism. J. Plant Sci. 178,73—89. sica oleracea by substituting cytoplasm from “Early Scarlet globe” Parkash, C., R. B. Dey, S. S. Dey, and M. Dhiman, 2015: Use of self- radish (Raphanus sativus). Euphytica 30, 855. incompatibility and cytoplasmic male sterility in F1 hybrid seed pro- Mc Rae, D. H., 1985: Advances in chemical hybridization. Plant Breed. duction of temperate vegetable crops. In: R. Kumar, C. Parkash, V. Rev. 3, 169—191. Kumar, R. S. Suman, M. R. Dhiman, S. S. Dey, R. B. Dey, and S. S. van der Meer, Q. P., and J. L. Van Bennekom, 1976: Gibberellic acid as Kumar (eds), Emerging Trends in Hybrid Vegetable Seed Production a gametocide for the common onion (Allium cepa L.). II. The effect of for Temperate Regions, 21—30. ICAR, Katrain. GA 4/7. Euphytica 25, 293—296. Pelletier, G., and F. Budar, 2015: Brassica Ogu-INRA cytoplasmic male van der Meer, Q. P., and R. Van Dam, 1979: Gibberellic acid as gameto- sterility: an example of successful plant somatic fusion for hybrid seed cide for cole crops. Euphytica 28, 717—722. production. In: X. Q. Li, D. J. Donnelly, and T. G. Jensen (eds), Melchers, G., Y. Mohri, K. Watanabe, S. Wakabayashi, and K. Harada, Somatic Genome Manipulation, 199—216. Springer, New-York, Hei- 1992: One-step generation of cytoplasmic male sterility by fusion of delberg, Dordrecht, London. mitochondrial-inactivated tomato protoplasts with nuclear inactivated Pelletier, G., C. Primard, F. Vedel, P. Chetrit, R. Remy, P. Rousselle, Solanum protoplasts. Proc. Natl Acad. Sci. USA 89, 6832—6836. and M. Renard, 1983: Intergeneric cytoplasmic hybridization in Cru- Min, W. K., S. Kim, S. K. Sung, B. D. Kim, and S. Lee, 2009: Allelic ciferae by protoplast fusion. Mol. Gen. Genet. 191, 244—250. Refer- discrimination of the restorer-of-fertility gene and its inheritance in ence mentioned in page 17. peppers (Capsicum annuum L.). Theor. Appl. Genet. 119, Petersen, J. D., S. Reiners, and B. A. Nault, 2013: Pollination services 1289—1299. provided by bees in pumpkin fields supplemented with either Apis Mizanur, M., R. Khan, and S. Isshiki, 2016: Cytoplasmic male sterility mellifera or Bombus impatiens or not supplemented. PLoS ONE 8, in eggplant. Hortic. J. 85,1—7. e69819. 298 N. COLOMBO AND C. R. GALMARINI

Peterson, P. A., 1958: Cytoplasmically inherited male sterility in Cap- Saxena, K. B., and A. J. Hingame, 2015: Male sterility systems in major sicum. Am. Nat. 92, 111—119. field crops and their potential role in crop improvement. In: B. Baha- Peterson, C. E., and P. W. Simon, 1986: Carrot breeding. In: M. J. Bas- dur, M. V. Rajam, L. Sahijram, and K. V. Krishnamurthy (eds), Plant sett (ed.), Breeding Vegetable Crops, 321—356. AVI, Westport. Biology and Biotechnology, I, 639—656. Springer, India. Petrova, M., Z. Yulkova, N. Gorinova, S. Izhar, N. Firon, J. M. Jac- Schweisguth, B., 1973: Etude d’un nouveau type de sterilitem^ale chez quemin, A. Atanassov, and P. Stoeva, 1999: Characterization of a l’oignon, Allium cepa L. Ann. Amelior. Plant. 23, 221—233. cytoplasmic male sterile hybrid between Lycopersicon peruvianum Serrano, I., M. C. Romero-Puertas, L. M. Sandalio, and A. Olmedilla, Mill. x Lycopersicon pennellii Corr. and its crosses with tomato. The- 2015: The role of reactive oxygen species and nitric oxide in pro- or. Appl. Genet. 98, 825—830. grammed cell death associated with self-incompatibility. J. Exp. Bot. Phatak, S. C., J. Liu, C. A. Jaworski, and A. F. Sultanbawa, 1991: Func- 66, 2869—2876. tional male sterility in eggplant: inheritance and linkage to the purple Shannon, S., and M. D. De La Guardia, 1969: Sex expression and the fruit color gene. J. Hered. 82,81—83. production of ethylene induced by auxin in the cucumber (Cucumis Phumichai, C., M. Mori, A. Kobayashi, O. Kamijima, and K. Hosaka, sativum L.). Nature 223, 186. 2005: Towards the development of highly homozygous diploid potato Sharma, M., M. N. Adarsh, P. Kumari, M. Thakur, R. Kumar, R. lines using the self-compatibility controlling Sli gene. Genome 48, 977 Sharma, and N. Gautam, 2015: Hybrid breeding in tomato. Int. J. —984. Farm Sci. 5, 233—250. Quiros, C. F., 1993: Celery breeding program at the department of veg- Shen, G. Z., X. Q. Wang, Y. Y. Zhu, H. J. Yang, G. H. Lu, J. Wang, X. etable crops, University of California, Davis. HortScience 28, 250. Wan, and J. Zhang, 2001: Male sterile transgenic cabbage plants with Quiros, C. F., A. Rugama, Y. Y. Dong, and T. J. Orton, 1986: Cytologi- TA 29-barnase gene. Acta Phytophysiol. Sin. 27,43—48. cal and genetical studies of a male sterile celery. Euphytica 35, 867— Shifriss, C., 1997: Male sterility in pepper (Capsicum annuum L.). 875. Euphytica 93,83—88. Radkova, M., E. Balacheva, B. Atanassova, A. Iantcheva, and A. Atanas- Shu, Z., Z. Wang, X. Mu, Z. Liang, and H. Guo, 2012: A dominant gene sov, 2009: Study on the potential of genic male sterility in tomato as a for male Sterility in Salvia miltiorrhiza Bunge. PLoS ONE 7, e50903. tool for pollen flow restriction. Biotechnol. Biotechnol. Equip. 23, da Silva Dias, J. C., 2014: Guiding strategies for breeding vegetable cul- 1303—1308. tivars. Agric. Sci. 5,9—32. Rambaud, C., J. Dubois, and J. Vasseur, 1993: Male-sterile chicory Singh, P. K., 2000: Utilization and seed production of vegetable varieties cybrids obtained by intergeneric protoplast fusion. Theor. Appl. Genet. in India. J. New Seeds 2,37—42. 87, 347—352. Singh, S. P., J. K. Roy, D. Kumar, and S. V. Sawant, 2012: Tools for Rambaud, C., A. Bellamy, A. Dubreucq, J. C. Bourquin, and J. Vasseur, generating male sterile plants. In: A. Goyal, and P. Maheshwari (eds), 1997: Molecular analysis of the fourth progeny of plants derived from Frontiers of Recent Developments in Plant Science, I, 67—85. Ben- cytoplasmic male sterile chicory cybrid. Plant Breed. 116, 481—486. tham Science, Sharjah. Rastogi, R., and V. K. Swahney, 1988: Suppression of stamen develop- Singh, P. K., V. Pandey, M. Singh, and S. R. Sharma, 2013: Genetic ment by CCC and ABA in tomato floral bud cultured in vitro. J. Plant improvement of cauliflower. Veg. Sci. 40, 121—136. Physiol. 9, 529—537. Sinha, R., and M. V. Rajam, 2013: RNAi silencing of three homologues Razdan, M. K., 2003: Introduction to , 2nd edn. of S-adenosylmethionine decarboxylase gene in tapetal tissue of Science Publishers Inc, Enfield. tomato results in male sterility. Plant Mol. Biol. 82, 169—180. Rehm, S., 1952: Male sterile plants by chemical treatment. Nature 179, Smith, B., and T. Crowther, 1995: Inbreeding depression and single cross 38—39. hybrids in leeks (Allium ampeloprasum ssp. porrum). Euphytica 86, Reynaerts, A., H. Van de Wiele, G. de Sutter, and J. Janssens, 1993: 87—94. Engineered genes for fertility control and their application in hybrid Soto, V. C., I. B. Maldonado, R. A. Gil, I. E. Peralta, M. F. Silva, and seed production. Sci. Hortic. 55, 125—139. C. R. Galmarini, 2013: Nectar and flower traits of different onion male Rick, C. M., 1948: Genetics and development of nine male sterile tomato sterile lines related to pollination efficiency and seed yield of F1 mutants. Hilgardia 18, 599—633. hybrids. J. Econ. Entomol. 106, 1386—1394. Robinson, R. W., S. Shanno, and M. D. La Guardia, 1969: Regulation of Stoeva-Popova, P. K., D. Dimaculangan, M. Radkova, and Z. Vulkova, sex expression in the cucumber. Bioscience 19, 141—142. 2007: Towards cytoplasmic male sterility in cultivated tomato. Rudich, J., A. Halevy, and N. Kedar, 1969: Increase in femaleness of J. Agric. Food Environ. Sci. 1,1. three cucurbits by treatment with ethrel, an ethylene releasing com- Takayama, S., and A. Isogai, 2005: Self-incompatibility in plants. Annu. pound. Planta 86,69—76. Rev. Plant Biol. 56, 467—489. Ruiz, O. N., and H. Daniell, 2005: Engineering cytoplasmic male sterility Tan, F., H. Shen, and S. Wang, 2009: Preliminary study of asymmetric via the chloroplast genome by expression of b-ketothiolase. Plant protoplast fusion between celery (Apium graveolens L.) and CMS car- Physiol. 138, 1232—1246. rot (Daucus carota L.). Acta Hortic. Sin. 36, 1169—1176. Saini, N., N. K. Hedau, A. Khar, S. Yadav, J. C. Bhatt, and P. K. Agra- Tanaka, Y., M. Tsuda, K. Yasumoto, H. Yamagishi, and T. Terachi, wal, 2015: Successful deployment of marker assisted selection (MAS) 2012: A complete mitochondrial genome sequence of Ogura-type for inbred and hybrid development in long-day onion (Allium cepa male-sterile cytoplasm and its comparative analysis with that of nor- L.). Indian J. Genet. Plant Breed. 75,93—98. mal cytoplasm in radish (Raphanus sativus L.). BMC Genom. 13, Sandhu, A. P. S., R. V. Abdel Noor, and S. A. Mackenzie, 2007: 352. Transgenic induction of mitochondrial rearrangements for cytoplasmic Tanksley, S. D., C. M. Rick, and C. E. Vallejos, 1984: Tight linkage male sterility in crop plants. Proc. Natl Acad. Sci. USA 104, 1766— between a nuclear male-sterile locus and an enzyme marker in tomato. 1770. Theor. Appl. Genet. 68, 109—113. Reference corrected in page 6. Sato, Y., 1998: PCR amplification of CMS-specific mitochondrial Tay, D., 2006: Vegetable hybrid seed production in the world. In: A. S. nucleotide sequences to identify cytoplasmic genotypes of onion Basra (ed.), Handbook of Seed Science and Technology, 703—718. (Allium cepa L.). Theor. Appl. Genet. 96, 367—370. The Haworth Press, Binghamton. Satoh, M., T. Kubo, S. Nishizawa, A. Estiati, N. Itchoda, and T. Mikami, Thompson, A. E., 1955: Methods of producing first-generation hybrid 2004: The cytoplasmic male-sterile type and normal type mitochon- seed in spinach. Cornell Univ. Agr. Exp. St. Mem. 336, 48. drial genomes of sugar beet share the same complement of genes of Thompson, D. J., 1961: Studies on the inheritance of male sterility in the known function but differ in the content of expressed ORFs. Mol. carrot (Daucus carota L. var. sativa). Proc. Am. Soc. Hortic. Sci. 78, Genet. Genomics 272, 247—256. 332—338. Sawhney, V. K., 1981: Abnormalities in pepper (Capsicum annuum) Toppino, L., M. Kooiker, M. Lindner, L. Dreni, G. L. Rotino, and M. flowers induced by gibberellic acid. Can. J. Bot. 59,8—16. M. Kater, 2011: Reversible male sterility in eggplant (Solanum Pollination control in hybrid seed production 299

melongena L.) by artificial microRNA mediated silencing of general Wolyn, D. J., and A. Chahal, 1998: Nuclear and cytoplasmic interactions transcription factor genes. Plant Biotechol. J. 9, 684—692. for petaloid male-sterile accessions of wild carrot (Daucus carota L.). Touzet, P., and E. H. Meyer, 2014: Cytoplasmic male sterility and mito- J. Am. Soc. Hortic. Sci. 123, 849—853. chondrial metabolism in plants. Mitochondrion 19, 166—171. Yamagishi, H., and S. R. Bhat, 2014: Cytoplasmic male sterility in Bras- Uyttewaal, M., N. Arnal, M. Quadrado, A. Martin-Canadell, N. Vrie- sicaceae crops. Breed. Sci. 64,38—47. lynck, S. Hiard, H. Gherbi, A. Bendahmane, F. Budar, and H. Mireau, Yamamoto, K., Y. Oda, A. Haseda, S. Fujito, T. Mikami, and Y. Ono- 2008: Characterization of Raphanus sativus pentatricopeptide repeat dera, 2014: Molecular evidence that the genes for dioecism and proteins encoded by the fertility restorer locus for Ogura cytoplasmic monoecism in Spinacia oleracea L. are located at different loci in a male sterility. Plant Cell 20, 3331—3345. chromosomal region. Heredity 112, 317—324. Valkova-Achkova, Z., 1980: L. peruvianum a source of CMS. Rep. Yang, Y., Y. Huo, J. Miao, B. Liu, S. Kong, L. Gao, C. Liu, Z. Wang, Tomato Genet. Coop. 30, 36. Y. Tahara, H. Kitano, and X. Wu, 2013: Identification of two SCAR Varotto, S., E. Nenz, M. Lucchin, and P. Parrini, 2001: Production of markers co-segregated with the dominant Ms and recessive ms alleles asymmetric somatic hybrid plants between Cichorium intybus L. and in onion (Allium cepa L.). Euphytica 190, 267—277. Helianthus annuus L. Theor. Appl. Genet. 102, 950—956. Ye, Q., W. Zhu, L. Li, S. Zhang, Y. Yin, H. Ma, and H. Wang, 2010: Virmani, S. S., and M. Ilyas-Ahmed, 2001: Environment-sensitive genic Brassinosteroids control male fertility by regulating the expression of male sterility (EGMS) in crops. Adv. Agron. 72, 139—195. key genes involved in Arabidopsis anther and pollen development. van der Vossen, H. A. M., 2004: Spinacia oleracea L. In: G. J. H. Grub- Proc. Natl Acad. Sci. USA 107, 6100—6105. ben, and O. A. Denton (eds), Internet Record from PROTA4U. Yordanov, M., 1983: Heterosis in tomato. In: Heterosis, R. Frankel (ed.), Wageningen, PROTA. Monograph on Theoretical and Applied Genetics 6, 189—219. Wang, D., and P. W. Bosland, 2006: The genes of Capsicum. HortS- Springer Verlag, Berlin. cience 41, 1169—1187. Yu, P. T., W. Wang, Y. K. He, and R. J. Shen, 2000: Transformation of Wang, L. H., B. X. Zhang, V. Lefebvre, S. W. Huang, A. M. Daubeze, barnase gene in Chinese cabbage. Acta Agric. Shanghai 16,17—19. and A. Palloix, 2004: QTL analysis of fertility restoration in cytoplas- Yu, H., Z. Fang, Y. Liu, L. Yang, M. Zhuang, H. Lv, Z. Li, F. Han, X. mic male sterile pepper. Theor. Appl. Genet. 109, 1058—1063. Liu, and Y. Zhang, 2016: Development of a novel allele-specific Rfo Webb, R. E., and C. E. Thomas, 1976: Development of F1 spinach marker and creation of Ogura CMS fertility-restored interspecific hybrids. HortScience 11, 546. hybrids in Brassica oleracea. Theor. Appl. Genet. 129, 1625—1637. Weider, C., P. Stamp, N. Christov, A. Husken,€ X. Foueillassar, K. H. Zhang, H., B. Wang, A. Xue, J. Cao, B. Li, Z. Tan, and W. Huang, Camp, and M. Munsch, 2009: Stability of cytoplasmic male sterility in 1998: The construction and tomato transformation of male sterile chi- maize under different environmental conditions. Crop Sci. 49,77—84. meric gene. Hereditas 20,5—7. Welch, J. E., and E. L. Grimball, 1947: Male sterility in carrot. Science Zhang, B. X., S. W. Huang, G. M. Yang, and J. Z. Guo, 2000: Two 106, 594. RAPD markers linked to a major fertility restorer gene in pepper. Williams II, M. M., C. A. Bradley, S. O. Duke, J. E. Maul, and K. N. Euphytica 113, 155—161. Reddy, 2015: Goss’s wilt incidence in sweet corn is independent of Zhang, X., J. Wu, H. Zhang, Y. Ma, A. Guo, and X. Wang, 2011: Fine transgenic traits and glyphosate. HortScience 50, 1791—1794. mapping of a male sterility gene MS-cd1 in Brassica oleracea. Theor. Wilson, Z. A., J. Song, B. Taylor, and C. Yang, 2011: The final split: Appl. Genet. 123, 231—238. the regulation of anther dehiscence. J. Exp. Bot. 62, 1633—1649. Zhu, X., L. Jin, and G. Gao, 2011: Celery cytoplasmic male sterile line Wittwer, S. H., and I. G. Hillyer, 1954: Chemical induction of male breeding method and cross-breeding method using celery cytoplasmic sterility in cucurbits. Science 120, 893—894. male sterile line. Patent CN 101999311 A. Ph. D (Ag) Molecular Biology and Biotechnology Subject: ADVANCES IN GENETIC ENGINEERING MBB 602: (3+0) Topic:- Molecular farming of plants for applications in veterinary and human medicine systems, Teacher:- Dr. R.S. Sharma Biotechnology Centre, JNKVV, Jabalpur Introduction ‘Molecular farming’ is a term coined to describe the application of molecular biological techniques to the synthesis of commercial products in plants. The term can be applied to a broad spectrum of activities, from the enhanced production of products that are already extracted from plants through to the manufacture of compounds that are completely novel to plants. A wide range of products have already been identified as likely targets for molecular farming, these include a variety of carbohydrates, fats and proteins, as well as secondary products. This chapter will consider examples of some of the major classes of molecules produced in transgenic plants. Many of the proteins being ‘farmed’ in plants are antibodies, vaccines or biopharmaceuticals aimed at improving human and animal health, and it is in the area of molecular farming that many of the most exciting and potentially beneficial developments in plant biotechnology are taking place. At the end of the chapter, we will also review some of the economic reasons why molecular farming might, in some cases, be an attractive alternative to current forms of manufacture of these compounds.

Carbohydrates and lipids

We will look at some examples of the production of novel or modified carbohydrates and oils (and their derivatives) in plants. Many of these examples have multiple applications (both as modified food- or feedstuffs and industrially) that will be highlighted here. Carbohydrate production Plants produce a range of commercially valuable carbohydrates. The two most abundant carbohydrates are cellulose (used for fibres from cotton and flax, for paper-making from trees and for a range of industrial products such as paints and polymers) and starch (used for food, feed and industrial purposes). Some biotechnological effort is directed towards improving the yield and quality of these bulk carbohydrates. However, there are a number of other carbohydrates that it could be attractive to produce in transgenic plants, including oligofructans, cyclodextrins and trehalose.

CASE STUDY Starch

The major starch-producing crops—cereals and potatoes—are already widely grown to produce starch as a chemical feedstock. Approximately 70% of the starch produced in Europe and the USA is used for a variety of industrial purposes, with only 30% used for human consumption and animal feed. In Figure 11.2, the pathway for starch synthesis in chloroplasts is shown, starting with triose- phosphate, which is generated in the Calvin cycle. This is subsequently converted to hexose phosphate and then ADP-glucose, which is the substrate for starch synthesis. In the case of amyloplasts, which are plastids specialised for starch accumulation, the hexose molecules are transported directly into the amyloplast. In the cereal endosperm, ADP-glucose may be synthesised in the cytosol and transported directly into the plastids. Starch synthesis involves two classes of enzymes. Starch synthase (SS) catalyses the addition of glucose residues from ADP-glucose to the non-reducing end of the growing chain, forming a(1 ->4) links. One form of starch molecule, amylose, is composed entirely of unbranched chains of glucose of about 1000 residues long. There are several SS isoenzymes, some of which are soluble in the plastid stroma, and others which are bound to the insoluble starch granules (granule-bound starch synthase, or GBSS).

The other class of starch biosynthetic enzymes is the starch branching enzymes (SBE) which create a(1-^6) branches in the starch molecule. These branched starch molecules are called amylopectin and typically contain 104 -105 glucose residues.

The simple diagram of starch biosynthesis shown in Figure illustrates some important principles in the genetic engineering of metabolic pathways. There is a single step from a pool of common primary metabolites (hexose phosphate sugars) to the immediate precursor of starch, ADP-glucose, catalysed by the enzyme ADP- glucose pyrophosphorylase. This precursor molecule is specific to the starch pathway, so this step commits hexose sugars to starch biosynthesis. Starch comprises amylose and amylopectin, so effectively there is a branch in the biosynthetic pathway leading to one or the other form of starch. Thus, this pathway could be manipulated at three points, in a number of different ways. The production of ADP-glucose could be altered, which would affect the overall level of starch biosynthesis. Thus, increasing the amount or activity of the enzyme ADP- glucose pyrophosphorylase should increase the amount of starch produced. On the other hand, manipulation of one of the branches in the pathway would affect the ratio of amylose to amylopectin.

Figure-Metabolic pathways for the biosynthesis of carbohydrate products for molecular farming. The biosynthetic pathways for the production of the compounds shown in Figure 11.1 are shown schematically in this stylised cell. Starting from the pool of hexose-phosphate sugars in the cytoplasm, sucrose is synthesised via UDP-glucose and transported into the vacuole, where it may be stored as fructans. UDP-glucose and glucose 6-phosphate are used to form trehalose in the cytoplasm, and glucose 6-phosphate is also the precursor of the sugar alcohols. In leaf cells, triose-phosphates are transported in and out of the chloroplast, where they can be converted to hexose-phosphate and then ADP- glucose for starch synthesis. Cyclodextrins are not found in plants, but starch is a substrate for some bacterial biosynthetic enzymes. The proportion of amylose:amylopectin is normally about 20-30% amylose to 70- 80% amylopectin, and it is this ratio that has the greatest influence on the physicochemical properties of the starch. For some applications it would be advantageous to increase the proportion of amylopectin. For example, many of the uses of starch in food production involve the formation of a gel after heating the starch in water and cooling. Amylose molecules tend to aggregate and crystallise on cooling, whereas amylopectin gels are more stable and generably more desirable for food processing. On the other hand, a high- amylose starch with limited branching would be a valuable feedstock for industrial purposes. In potatoes, starch synthesis involves the activities of three isoforms of soluble starch synthase (SS), one granule-bound starch synthase (GBSS1) and two isoforms of the starch branching enzyme SBE (SBE A and B). The GBSS is responsible for the synthesis of amylose chains; the successful antisense inhibition of GBSS1 in potato produced an amylose-free starch. On the other hand, the engineering of a high-amylose starch required the antisense inhibition of both SBE A and SBE B. The resulting high-amylose starch also had much higher phosphate levels and could prove to be useful for food and industrial applications.

Cyclodextrins from starch

Whilst starch itself is a bulk feedstock for industry, the potential exists for carrying out further biotransformations on the starch in the plant, rather than by chemical or fermentation processes after extraction. One type of high-value product that could be made from starch is the cyclodextrins. These compounds are typically 6-, 7- or 8-membered rings comprising glucopyranose subunits attached in a(l—>4) linkages. These compounds are normally produced by bacterial fermentation of maize starch, particularly for pharmaceutical applications. The structure of the cyclodextrins is such that they form a cone-shaped ring, with the hydrophilic residues on the exterior and a hydrophobic pocket in the centre of the ring. The 7-membered ring has the ideal dimensions to form a pocket for small hydrophobic compounds. Thus, in a concentrated suspension, the cyclodextrins will effectively solubilise hydrophobic pharmaceuticals such as steroids. At lower concentrations, for example after injection into the bloodstream, the therapeutic agent is released.

Having been identified as a suitable target for molecular farming, it is striking that only one report, in 1991, of an attempt to produce cyclodextrins has been published. A bacterial cyclodextrin glycosyltransferase gene from Klebsiella pneumoniae was fused to a plastid-targeting sequence and placed under the control of the promoter from the patatin gene. Patatin is a protein that accumulates in potato tubers, and the promoter directs high levels of expression in the tubers of transgenic potatoes. However, transformation of potatoes with this construct resulted in very little conversion (0.001-0.01%) of starch to cyclodextrins. It was concluded that the insoluble starch granules may have been inaccessible to the bacterial enzyme, or that the enzyme became trapped in the growing granule. Whatever the reason, no subsequent attempts to produce this commodity have appeared in the literature.

CASE STUDY 2 Polyfructans

Another example of a carbohydrate targeted for production in transgenic plants is polyfructans. These compounds are soluble polymers of fructose that are synthesised and 1-kestose type neokestose type stored in the vacuole. They have a typical structure of glucose-fructose-(fructose) n (G—F—F n ) as shown in Figure 11.3. The use of fructans as a carbohydrate reserve is widespread throughout the plant kingdom. As with glucose polymers, there are different glycosidic linkages possible between the fructose residues, giving different straight and branched polymers. The inulins are the major storage carbohydrate found in bulbs such as onion, and storage roots such as chicory and Jerusalem artichoke, and are formed by (1 —>2(3) linkages. Levans are widespread in the leaves and stems of grasses, including major cereal crops such as wheat, and comprise (6->2[3) linkages. Graminae-type fructans found, for example, in grasses, are a mixed type and have both (1 ->2(3) and (6->2|3) linkages.

The biosynthetic pathway of fructans in plants is a two-stage process. The first step involves the transfer of fructose from a donor sucrose molecule to an acceptor sucrose molecule to form kestose by the enzyme sucrose-sucrose fructosyltransferase (SST).

G-F + G-F—> G-F-F + G, where G = glucose and F = fructose.

In the second step, the kestose (GFF or GF 2 ) acts as the fructose donor to the growing fructan chain, via fructan-fructan fructosyltransferase (FFT) activity, and a sucrose molecule is recycled.

G-F-Fn + G-F-F G-F-F n+1 + G-F.

In certain bacteria, such as Bacillus subtilis, very high molecular weight levans are produced by a single reaction in which sucrose acts directly as the fructose donor to the growing chain (sucrose-fructan fructosyltransferase, SFT). Note that in all cases, sucrose is the initial acceptor molecule of the chain, so the first sugar in the fructan chain is always glucose. However, for each remaining fructose residue added to the chain a glucose residue is released, which in plants is transported back out of the vacuole into the cytosol.

A number of transgenic plants producing polyfructans have now been developed. In some of the first experiments with tobacco, two different bacterial genes were compared. The sacB gene of 8. subtilis encodes a levansucrase catalysing a 6->2(3 linkage, whilst the ftf gene from Streptomyces spp. encodes a fructosyltransferase that forms 1 ->2p linkages. The construct used to express sacB demonstrating how the sacB gene was modified with a vacuolar targeting sequence from the yeast carboxypeptidase gene (cpy). The features of vacuolar signal sequences. The targeting sequence directs the enzyme to the vacuole, where sucrose is stored and polyfructan synthesis normally occurs, in those plants that store this carbohydrate reserve.

In tobacco, both transgenes were responsible for the production of significant amounts of fructans, with the sacB gene proving more effective than ftf. The transgenic tobacco plants carrying the sacB gene were found to be more tolerant to drought stress induced by growth in medium containing polyethylene glycol to reduce the water concentration. In Chapter 9, the production of osmoprotectants to provide stress tolerance against the effects of water deficit was described, and this provides another example of this principle in action. More recently, expression of the sacB gene in sugar beet has also been shown to improve tolerance to polyethylene glycol-mediated drought stress.

Polyfructans have also been produced in potatoes using the same vacuolar-targeted sacB gene, but under the control of the patatin gene promoter (see the earlier example of cyclodextrins). In potato, the formation of a new sink, by diversion of sucrose away from starch accumulation in the tuber and towards fructans in the vacuole, has provided a useful model system for studying the regulation of sucrose metabolism and the partitioning of

Metabolic engineering of lipids The next class of compounds that we will explore as targets for molecular farming is the lipids and their derivatives. As with the carbohydrates, lipids are already produced in large quantities from major crops such as oilseed rape (canola), soybean and maize, for industrial as well as food purposes. Molecular farming will therefore have a role in the same broad areas as shown for carbohydrates, that is: • Improvement of existing lipid products; • Engineering of novel lipid products. Improvement of plant oils In daily life, our closest association with plant oils comes by way of their rapid displacement of animal fats as cooking oils. This association alone should indicate that oils are already extracted from a number of different types of crop. Oilseed rape or canola, and soybean are major sources of oil that are often unspecified, but sunflower oil, corn oil and olive oil are often identified because of the improved characteristics of these oils for particular purposes. Plant oils are also used in a wide range of processed foods, and for animal feeds. They also have an increasing number of applications in industry for the manufacture of soaps and detergents, lubricants and biofuel. However, the non-food applications currently comprise only about 10% of the total vegetable oils produced. One of the reasons for this relatively low level of industrial use is the complex and variable nature of plant oils, which generally makes them less suitable for oleochemical applications than cheaper alternatives from mineral oils. A major long-term goal of this sector is therefore the improvement of plant oils for industrial applications, since they must eventually replace non-renewable, petroleum-based products. Storage oils that are used as carbon/energy reserves in plant seeds and some fruits (such as olives and avocados) are generally glycerol esters of fatty acids, called triacylglycerols (TAGs). The differences between oils from different sources are largely due to the particular proportions of different fatty acids comprising TAGs. A few of the common fatty acids. These differ in their length, and the number and positions of double bonds. Saturated fats contain no double bonds— the more double bonds there are, the greater the degree of unsaturation. The site of de novo fatty acid biosynthesis is exclusively in the stroma of the plastid, whereas most modification of the fatty acids occurs in the cytoplasm and on the endoplasmic reticulum (ER). Assembly of TAGs takes place in the membrane of the ER. The TAGs are stored in oil bodies, which are essentially oil droplets surrounded by a lipid monolayer formed from one half of the ER membrane lipid bilayer. The oil bodies of seeds (and other tissues that undergo extreme desiccation) also contain proteins, called ‘oleosins’, in the lipid monolayer. The first committed step in fatty acid biosynthesis in the plastid is the formation of malonyl-coenzyme A (CoA) from acetyl-CoA, in an ATP-dependent reaction catalysed by acetyl-CoA carboxylase (ACCase). The CoA residue is then exchanged for the acyl carrier protein (ACP). Acetyl-CoA is then condensed to the malonyl-ACP, forming acetoacetate-ACP, and liberating C02. Three subsequent steps (reduction of the carbonyl group, removal of water to form a double bond and reduction of the double bond) produce an acyl-ACP, which is two carbons longer than the original. The entire sequence of elongation reactions from the initial binding to ACP is catalysed by a fatty acid synthase multienzyme complex. The elongated fatty acid chain is then transferred to another ACP protein and this is then condensed to a new malonyl-ACP. Thus, the formation of fatty acids occurs by the stepwise addition of 2-carbon units at the carboxyl end, hence the even numbers of carbon atoms in the fatty acids. In many of the oil producing crops, this process stops at the 16-carbon stage, and the palmitoyl-ACP (16:0) is elongated to stearoyl-ACP (18:0) by a specific synthase. (The standard nomenclature of fatty acids indicates the number of carbon atoms and the number of double bonds.) At this stage, desaturation by a soluble A 9 C-stearoyl desaturase in the plastid stroma converts most of the stearoyl-ACP to oleoyl-ACP (18:1 A 9 ). (The A 9 indicates that the double bond starts at the 9th carbon counting from the carboxylic acid ‘front end’ of the molecule.)

After termination of synthesis in the plastid, the fatty acids (mainly palmitic, stearic and oleic acids) are released from the ACP and exported to the cytoplasm, where they are converted to acyl-CoA esters. TAGs are formed by stepwise acylation of glycerol-3-phosphate in the ER membrane. Further modifications to the fatty acids normally occur after they have been attached to various glycerophosphatides. Additional double bonds may be added (typically to A 12 , A 15 and A 6 as well as A 9 ). Other modificatons may include hydroxylation by the addition of water across a double bond.

Production of shorter chain fatty acids The oils produced by the major oil crops of the world consist mainly of palmitic, stearic, oleic, linoleic and linolenic acids, which are all Cl6 or Cl 8 in length. Coconut and palm kernel oils are largely C8-C14, and lauric acid (12:0), in particular, is an important raw material for the production of soaps, cosmetics and detergents (think of the widespread use of sodium lauryl sulphate, or SDS, in these products). The synthesis of fatty acids at a particular length is terminated by the hydrolysis of the acyl-ACP by a thioesterase. The California bay tree contains a very high proportion of lauric acid in its seeds, and an acyl-ACP thioesterase that specifically hydrolyses lauroyl-ACP has been cloned from this source. The introduction of this gene into oilseed rape causes fatty acid synthesis to terminate at the 12:0 stage, and a high proportion of lauric acid to accumulate in the seed oil. Most importantly, field tests show that these plants grow normally and produce normal yields. Production of longer chain fatty acids One of the important targets is to produce fatty acids longer than Cl 8 for use as industrial oils. In oilseed rape and other Brassica species, there is a two-step elongation pathway from oleoyl-CoA (18:1 A 9 ) to erucoyl-CoA ( 22:1A 13 ), such that erucic acid is one of the constituents of brassica oils. However, whilst erucic acid is valuable as an industrial oleochemical, it is nutritionally unsuitable for human consumption. Conventional breeding has led to the development of two distinct oilseed rape crops—high-erucic acid rape (HEAR) for industrial purposes, and low-erucic acid rape (LEAR) with virtually no erucic acid, for food products. (NB: the existence of two distinct crops that need to be kept apart means that systems are in place in the seed production, agriculture and processing industries to ensure minimal cross-contamination, a process called ‘identity preservation’. Thus, requirements to keep GM and non-GM seeds and products separate do not create entirely new demands on this sector.) However, the highest erucic acid content of HEAR is about 50% of the total fatty acids, which makes the cost of separating out and disposing of the other fatty acids uncompetitive with mineral oil sources. Attempts are being made to overexpress the genes that encode the elongases, and also to transfer enzyme activities that preferentially incorporate erucic acid into TAGs. Modification of the degree of saturation As the erucic acid example shows, the ideal products for industrial purposes are as uniform as possible. This requires uniformity of desaturation as well as length. (The same does not necessarily apply to food constituents, where complex mixtures may produce the ideal product. In this case it is consistency, rather than uniformity, that is required.) One of the first attempts in this area was to increase the level of stearic acid (18:0) at the expense of oleic acid (18:1 A 9 ) by inserting an antisense construct of the A 9 desaturase gene into oilseed rape. The antisense gene was under the control of the napin (a seedspecific protein of brassica) gene promoter. There was a marked decrease in the amount of the desaturase enzyme, resulting in a decreased formation of oleic acid, and a rise in the proportion of stearic acid from 1-2% up to 40% of total fatty acids. This high stearic acid oil has potential as a cocoa butter substitute. On the other hand, there is considerable potential in the production of a very high (>90%) oleic acid oil for food purposes and as a uniform oleochemical feed- stock. Conventional breeding has produced mutant oilseed rape lines with an oleic acid content close to 80%, but attempts to increase this level have produced plants with poor cold tolerance, presumably as a result of the lack of unsaturated fatty acids in the cellular membranes. Antisense repression and co-suppression of the A 12 desaturase in the seeds of oilseed rape have, however, facilitated the raising of oleic acid levels to 87-88%, without affecting cold tolerance. Similar experiments in soybean increased the oleic acid level from 22% to 79%. Production of rare fatty acids There are 210 known types of fatty acids produced in plants, but most of these are not found in the major crop plants and would be difficult to produce commercially in their host plants. However, it is possible to transfer genes from these plants in order to manipulate the profile of fatty acids produced in the major oil crop plants. One such fatty acid is petroselenic acid (18:1 A 6 ), which is found in certain Umbelliferae, such as coriander, and has potential as a raw material for industry. Oxidation of petroselenic acid by ozone produces lauric acid (12:0) (for soaps and detergents) and adipic acid (6:0), which can be used for nylon production. Transformation of tobacco with a coriander acyl-ACP desaturase cDNA led to the production of petroselenic acid in calli to a level of 5% of total fatty acids. Certain polyunsaturated fatty acids have pharmaceutical or nutraceutical value. These include y-linolenic acid (18:3A 6,9,12 ) and arachidonic acid (20:4A 5 ’ 8,11 ’ 14 ), which are essential fatty acids for humans and precursors of eicosanoids (including prostaglandins, leukotrienes and thromboxanes). Recently, it has become clear that the synthesis of these compounds involves a specific subclass of microsomal desaturases called ‘front-end’ desaturases. These enzymes insert additional double bonds between existing bonds and the carboxyl ‘front-end’ of the fatty acid, whereas most desaturases in plants add sequentially towards the methyl end. The transformation of a front-end desaturase from borage (which is one of a few plant species to produce ylinolenic acid) into tobacco resulted in the accumulation of high levels of A 6 unsaturated fatty acids. A A 5 desaturase has been cloned from the filamentous fungus Mortierella alpina (which accumulates arachidonic acid) and expression in oilseed rape produced significant levels of polyunsaturated fatty acids. Ricolenic acid (A 12 -hydroxyoleic acid) is produced in castor beans to a level of 90% of the total fatty acids. However, the castor oil crop has a number of problems, including the presence of toxic compounds such as ricin in the residual meal. The synthesis of ricolenic acid involves the direct hydroxylation of oleic acid bound to phosphatidylcholine on the ER membrane. The cDNA for the 12- hydroxylase has been cloned from castor bean and could be used to produce ‘castor oil’ in major oil crops. Another fatty acid modifying enzyme has been cloned from Crepis acetylenics. This non-haem, di-iron protein catalyses triple bond and epoxy group formation in fatty acids, and would be a valuable way of inserting chemically reactive sites into oils. CASE STUDY 3 Bioplastics One of the most imaginative examples of molecular farming has been the attempt to produce biodegradable plastics (‘bioplastics’) in plants. These compounds are currently produced by microbial fermentation, but a number of experimental studies have been carried out to determine the feasibility of producing them in bulk in plants. The structure of the polyhydroxyalkanoates (PHAs) which form bioplastics is shown in Figure. The length of the R side chain alters the properties of the plastics, and can vary from 0 carbon (3hydroxypropionate), 1 carbon (3- hydroxybutyrate) or 2 carbons (3-hydroxyvalerate) up to long carbon chains. Polyhydroxybutyrate (PHB) is the best-characterised PHA and is found as intracellular inclusions in a wide variety of bacteria. In Alcaligenes eutrophus, PHB accumulates as a high molecular weight polymer up to 80% of the bacterial dry weight.

R O

generic polyhydroxyalkanoate

polyhydroxybutyrate Figure-Chemical structure of the major polyhydroxyalkanoates. The repeating subunit of polyhdroxyalkanoates is shown, along with a short section of a polyhydroxybutyrate chain (where R = methyl group). The R group can be anything from 0 carbons (i.e. H) in hydroxypropionate, to >10. The length of the R-group side chain affects the physical properties of the plastic formed by these polymers. The pathway for polyhydroxybutyrate synthesis. It can be seen that this is a relatively simple three-stage pathway starting from acetyl-CoA. The genes for the three enzymes involved in the pathway (successively the phaA, phaB and phaC genes) have been cloned from Alcaligenes eutrophus. In the initial experiments, it was recognised that the first step of the pathway, production of acetoacetyl-CoA, occurs in the cytoplasm of plants, at the start of the pathway to isoprenoids (see Chapter 10). Thus, only phaB and phaC, encoding acetocetyl-CoA reductase and PHB synthase, respectively, were transformed into Arobidopsis, without targeting sequences (Figure 11.8(A)). Microscopic observation of the Arobidopsis leaves indicated the formation of microbodies of bioplastics accumulating in the cytosol, nucleus and vacuole. However, the amount of bioplastic was relatively low (20- 100 pg g _1 fresh weight) and the plants were severely stunted in growth. Subsequently, all three genes were transformed into Arobidopsis, and targeted to the chloroplast. In the first generation of experiments, each gene was separately fused to a sequence encoding the transit peptide plus N-terminal fragment of the Rubisco small subunit protein, and expression of each construct was directed by the CaMV 35S promoter. Each construct was transformed into separate Arobidopsis plants, and brought together by a series of sexual crosses between the individual transformants. In this case, the bioplastics accumulated as 0.2-0.7-pm granules in the plastids, to levels of up to 14% of plant dry weight, and there was no observable effect on growth or fertility. Increased PHB production in Arobidopsis has been achieved using a triple construct, so that all three genes are transferred into the plant in a single transformation event. A rapid gas chromatography—mass spectrometry (GC-MS) procedure was used to screen a large number of transgenic plants, permitting the selection of plants accumulating PHB in the leaf chloroplasts to more than 4% of their fresh weight (40% dry weight). However, the highproducing lines showed stunted growth and loss of fertility. Interestingly, the production of PHB did not affect fatty acid accumulation or composition, but there was a significant impact on the levels of various organic acids, amino acids, sugars and sugar alcohols. Whilst Arabidopsis is a valuable model species for these analyses, some research has been focused on the potential for the commercial production of plastics in oil crops, effectively diverting the pool of acetyl-CoA away from fatty acid biosynthesis (for oil body production—see above) towards bioplastic production. A team from Monsanto used the three genes for the PHB biosynthetic pathway from the bacterium Ralstonio eutropha and fused each one to a seed-specific promoter (from Lesquereila fendleri oleate 12-hydroxylase). These were transferred into a single, multigene vector, which was used to transform oilseed rape. PHB was found to accumulate in mature oilseed leucoplasts to levels up to 7.7% of fresh seed weight. However, apart from in Arabidopsis, it has proved difficult to obtain very high levels of PHB in transgenic plants. Recent work indicates that constitutive expression of the (3-ketothiolase gene is detrimental to the efficient production of PHB in some plant species, and the use of inducible or developmental^ regulated promoters to drive the phbA gene permitted some production of PHBs in tobacco and potato.

A

B Figure-Expression of the PHB biosynthesis pathway in transgenic Arabidopsis. In scheme A, the presence of 3-ketothiolase activity in the plant cytoplasm removes the necessity to transfer three genes to the plant. The phaB and phaC genes from Alcaligenes eutrophus were transformed into Arabidoposis without protein targeting sequences. PHB granules were produced in the cytoplasm, but they also accumulated in the nucleus and vacuole. In scheme B, all three genes of the pathway are targeted to the chloroplast using Rubisco small-subunit transit peptide sequences. In this case, PHB accumulates in the plastids. Another interesting direction taken by this research has been the production of bioplastics in cotton fibres. At the start of this work it was recognised that cotton fibres contain |3ketothiolase activity (the first enzyme in the PHB pathway. Therefore, the A. eutrophus phaB and phaC genes were transformed into cotton by particle bombardment of seed axis meristems. The phaB gene was driven by a cotton fibre-specific gene promoter, whilst phaC was fused to the 35S promoter. Clusters of small PHB granules were found in the cytoplasm of the fibre cells (i.e. in the fibre lumen). The thermal properties of these fibres were found to be altered, indicative of enhanced insulation characteristics. One of the limitations of this approach to bioplastic production is that polyhydroxybutyrate is a highly crystalline polymer, which produces rather stiff and brittle plastics. Polyhydroxyalkanoate co-polymers made from longer monomers have better physical properties in terms of being less crystalline and more flexible. Pseudomonads accumulate medium chain-length PHAs when grown on fatty acid substrates. These PHAs are synthesised from 3-hydroxy-acyl-CoA intermediates generated by the (3-oxidation of the fatty acids, hence the PHA monomer size is related to the length of the fatty acid substrate. Medium chain- length PHAs have been produced in Arabidopsis using the phaCI gene from Pseudomonas aeruginosa with a peroxisome targeting sequence from an oilseed rape isocitrate lyase. The enzyme was targeted to leaf-type peroxisomes in light- grown plants and glyoxisomes in dark-grown plants. These plants accumulated PHAs in the glyoxisomes and peroxisomes, and in the vacuole, to a level of 4 mg g _1 dry weight. The PHAs contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from 6 to 16 carbons, with 41% being a mixture of the 8-carbon saturated and mono-unsaturated monomer. Molecular farming of proteins Throughout this book we have presented to you examples where transgene expression is used as a tool to, for example, modify a biosynthetic pathway, manipulate growth and development or alter some other property of the plant such as herbicide resistance. However, in some cases it is the transgenic protein itself that is important. We saw how attempts to improve the amino acid content of grain are being addressed by expression of proteins with a high content of lysine. In this section of the chapter, we will consider the production of bulk enzymes and potentially high-value proteins such as antibodies and vaccines. The production of functional proteins in plants on an industrial scale does not, at first sight, suggest too many difficulties. However, the production of a complex protein that is folded, processed, easily purified and can be shown to be safe for pharmaceutical use, all at low cost, is a real challenge. The economics of protein production in plants is complicated. The actual cost will depend on many factors, amongst them being the cost of growing the plant, transport costs and processing and protein purification costs. The costs of proteins produced in plants may undercut the costs of producing proteins by existing methods, but if too much (in economic terms) is produced, then the market price will fall. This has already proved to be the case for some vaccines produced by existing methods. In this remaining section, the protein products that will be considered fall into two main types. First, enzymes for industrial and agricultural uses will be looked at. Second, we will consider medically related proteins such as: • antibodies—full immunoglobulins and engineered types such as scfvs (single chain antibodies); • subunit vaccines—vaccines based upon short peptide sequences that act as antigens rather than whole organism-based vaccines; and • protein antibiotics. Production systems The interest in producing such proteins in plants comes in part from the problems associated with existing fermentation or bioreactor systems. Mammalian cell systems are expensive and cannot easily be scaled up (input costs can approach $US1 million per kg of crude product). Bacterial systems can be scaled up, but often the recombinant proteins are not properly processed (they are not properly folded and disulphide bridges are not formed), so intracellular precipitation of non- functional proteins can occur. Plant systems can be scaled up so that large amounts of material can be harvested and processed, allowing industrial-scale amounts of protein to be purified. In some cases it may be possible to omit purification, as is the case with vaccines from plant sources, and where plant material containing recombinant enzymes can be added directly to animal feed or industrial processes. There are some differences between plant and animal post-translational modification systems, and the difference in glycosylation patterns between plants and animals has caused some concerns, particularly when expressing antibodies in plants. However, plants are able to fold, cross-link, and post-translationally modify (by, for example, glycosylation) non-plant proteins sufficiently well to ensure that, in most cases, functional proteins are obtained. The production of recombinant proteins in plants also benefits from the ability to target the proteins to cellular compartments where processing occurs, or where the proteins may be more stable. Another advantage of using plant systems is that the potential for contamination of the protein products with toxins or pathogens of animals and humans is very much reduced. The plants initially chosen for expression studies related to the ability of scientists to transform the plant material. A lot of work was first done with tobacco, but this was not a suitable plant for feeding to animals. For work in which edible products are being developed, plants such as potatoes, tomato, maize and lettuce have been used. For bulk production, the leaves of plants such as tobacco and alfalfa remain a favourite, because they can be harvested several times a year. Flowever, plants such as rice, wheat, maize and soybean have also been used, although their biomass yields are lower (Table).

Table-Potential biomass yields from various plants considered as potential vehicles for protein production Crop Potential annual biomass yield (tonne per hectare)

Tobacco >100

Alfalfa 25

Rice 6

Wheat ~3

Strategies for protein production Two major strategies have been developed for the production of proteins in plants: the stable integration approach, which has been used for many systems; and the use of plant viruses as transient vectors. Stable expression The stable transgene expression approach, in which the transgene is regulated by a strong, constitutive promoter (such as the 35S promoter), is perhaps the most suitable for the bulk production of soluble proteins in leaves, although yields can be low using this approach. A more sophisticated approach has been to target gene expression and protein production to specific tissues. Restricting gene expression to particular organs or tissues often leads to higher yields of recombinant proteins. Targeting expression has a number of advantages, some of which are related to the increased yield. The resources of the plant are effectively wasted if proteins are produced in parts of the plant that are not harvested. So, for vaccines that are to be ingested, after the minimum amount of processing, it makes sense to target the protein only to the parts of the plant that are normally eaten. Another important point is that storage organs, such as tubers and seeds, are designed to maintain their biological integrity over long periods. Targeting proteins to these structures has been shown to increase the stability of recombinant proteins from weeks to years. Seeds are also of great interest because they can offer simple strategies for protein purification. In tobacco, seeds contain a simpler mixture of proteins and lipids, with fewer phenolic compounds, than do green leaves.

GM Crops & Food Biotechnology in Agriculture and the Food Chain

ISSN: 2164-5698 (Print) 2164-5701 (Online) Journal homepage: https://www.tandfonline.com/loi/kgmc20

Heterologous protein production in plant systems

Siddhesh B. Ghag, Vinayak S. Adki, Thumballi R. Ganapathi & Vishwas A. Bapat

To cite this article: Siddhesh B. Ghag, Vinayak S. Adki, Thumballi R. Ganapathi & Vishwas A. Bapat (2016): Heterologous protein production in plant systems, GM Crops & Food, DOI: 10.1080/21645698.2016.1244599 To link to this article: https://doi.org/10.1080/21645698.2016.1244599

Accepted author version posted online: 27 Oct 2016.

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Heterologous protein production in plant systems Siddhesh B. Ghag1, Vinayak S. Adki2, Thumballi R. Ganapathi3,*, Vishwas A. Bapat4 1UM-DAE Centre for Excellence in Basic Sciences, Kalina, Mumbai, India 2Lokmangal Biotechnology College, Solapur, Maharashtra, India 3Plant Cell Culture Technology Section, Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India 4Department of Biotechnology, Shivaji University, Vidyanagar, Kolhapur, Maharashtra, India *Corresponding author Email: [email protected] Abstract Production of recombinant proteins is primarily established in cultures of mammalian, insect, and bacterial cells. Concurrently, concept of using plants to produce high-value pharmaceuticals such as vaccines, antibodies and dietary proteins have received worldwide attention. Newer technologies for plant transformation such as plastid engineering, agroinfiltration, magnifection and deconstructed viral vectors have been used to enhance protein production in plants along with the inherent advantage of speed, scale and cost of production in plant systems. Production of therapeutic proteins in plants has now a more pragmatic approach when several plant-produced vaccines and antibodies successfully completed Phase I clinical trials in humans and were further scheduled for regulatory approvals to manufacture clinical grade products on large scale safely, efficacious and meeting the quality standards. The main thrust of this review is to summarize the data accumulated over the last two decades and recent development and achievements of the plant derived therapeutics. It also attempts to discuss different strategies employed to increase the production so as to make plants more competitive with the established production systems in this industry. Keywords Protein, plantibodies, vaccines, magnifection, therapeutics, biopharmaceuticals, clinical trials

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Introduction Protein synthesis is a very complex process, which involves translation of mRNA on ribosomes and post-translational modifications of protein (glycosylation, phosphorylation and proper folding), required for its stability and precise expression of biological activity. Being a tightly regulated process, protein synthesis involves sequential activities of enzymes and co-factors at various steps that is also linked to other physiological and biochemical factors. Production of a protein outside its natural host system is called heterologous protein production (Mahmoud, 2007; Rai and Padh, 2001). Heterologous proteins are divided into three major groups: therapeutic proteins (for pharmaceutical use), reagent proteins (used for research and study purposes) and industrial proteins (used for various industrial applications). Among these, proteins used as biopharmaceuticals and therapeutic purposes form an extraordinary class with the stringent quality standards demanding high value. Today, the share of biopharmaceuticals is 28.7% of the total global pharmaceutical industry. The opportunities in biopharmaceuticals are growing very rapidly with an expected increase from US$200 billion in 2013 to US$500 billion in the year 2020. There are at present approx. 2500 biotech drugs in discovery phase and 1500 biopharmaceuticals undergoing clinical trials (Walsh 2014; Ritala et al. 2014; http://www.marketresearchstore.com/report/global-biopharmaceuticals-industry-2015- market-growth-trends-and-45928). Bacterial (Itakura et al. 1977), fungal (Su et al. 2012; Nevalainen and Peterson 2014), plant (Sijmons et al. 1990) and animal systems (Dyck et al. 2003; Wurm 2004) have been used since 1970s to produce a variety of recombinant proteins and later explored to produce biopharmaceuticals in addition to industrially relevant proteins. E. coli was the pioneer expression system in which first therapeutic protein, somatostatin, was successfully expressed (Itakura et al. 1977). Insulin was the first FDA approved therapeutic protein produced from E. coli expression system (Pillai and Panchagnula 2001). ATryn was first approved as a therapeutic protein produced from transgenic animal (http: //www. transgenics.com/products/atryn.html). The first examples, reported in 1990, of heterologous protein production using transgenic plant cell suspension cultures, is a recombinant human serum albumin (Sijmons et al. 1990) and chloramphenicol acetyltransferase (Hogue et al. 1990).

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However, it is challenging to meet the high-volume demand of heterologous proteins by all those systems because of limitations of cost, cumbersome procedures and problems in upscale. Production costs are the key issue for recombinant protein production in non-plant sources, and several factors contribute for higher costs. Nevertheless, plants offer remarkable platform for large scale production of eukaryotic proteins and are found to be safe, reasonably cheap and efficacious. The upfront cost of generating recombinant plant material includes standardization of the experimental protocols, cost of growing and harvesting plant material, and the downstream processing and purification cost. Improvement in expression of heterologous protein at cellular and molecular level is one of the practical ways to alleviate these limitations. An ideal expression system shoud possess (i) capability to produce the required protein with correct conformation, (ii) good productivity, (iii) easy handling and maintenance, (iv) safe and economic (v) affordable downstream processing and (vi) easy amenability for experimental manipulations. Plants as expression systems: advantages and disadvantages Microbial fermentations, animal cultures or plant systems have been employed for recombinant protein production based on the type of recombinant proteins, demand and production costs. Each of these production systems are accompanied by their advantages and disadvantages. Animal cell cultures and transgenic animals carrying harmful pathogens or prions increase the downstream processing cost for their separations. Microbial systems even though considered as best systems to obtain large quantities of proteins, they are unable to carry out post-translational modifications involving folding and glycosylation essential for eukaryotic protein activity and efficacy. Further, large scale production of heterologous proteins in microbial system is confined to inclusion bodies and extraction poses hurdles which enhances the downstream cost. Plant cells on the other hand offer advantages over mammalian and microbial systems by producing different types of eukaryotic proteins having precise folding and modification required for its activity. The dual options of using plant platform are, proteins can be produced in field grown plants or in cell cultures to achieve the required yield. Additionally, use of plants ensures enhanced production and inexpensive scale up cost, easy scaling up, high protein quantity, homogeneity and relative safety. Albeit, there are few shortcomings in

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use of plants as production platforms such as slow growth cycle, variation in the yield in each cycle and downstream processing cost. However, some of these limitations can be overcome in some exceptional cases whereby therapeutic proteins are secreted out into the liquid broth or expressed in edible tissues which can be consumed raw. These will ensure in lowering the downstream costs and decrease the yield losses. Plants are gaining increasing importance as production platforms because of their ability to glycosylate therapeutic proteins which eventually increases the immunogenic potential and receptor binding in addition to resistance to thermal denaturation and protection from proteolytic degradation. Plants are amenable for glyco-engineering wherein efforts are directed mainly on silencing plant-specific N-glycan-processing genes, and/or the introduction of the enzymatic machinery required for synthesis, transport and transfer of human type sugars onto the recombinant proteins (Séveno et al. 2004; Strasser et al. 2004; Daskalova et al. 2010; Nagels et al. 2011; Castilho et al. 2012; Strasser et al. 2014). Moreover, Cox et al. (2006) demsonstrated the production of monoclonal antibody without plant specific glycosylation by knocking down the Lemna minor endogenous α-1,3-fucosyltransferase and β-1,2-xylosyltransferase genes which resulted in the production of a single major N-glycan species without any noticeable plant-specific N-glycans. Further, this monoclonal antibody had better antibody- dependent cell-mediated cytotoxicity and effector cell receptor binding activities than the ones expressed in cultured Chinese hamster ovary (CHO) cells. Various strategies used for high-level expression of heterologous protein in plant system (Fig. 1.) Plants form the main source of food and medicine ever since ancient times and are generally considered to be safe for human use providing a safer alternative host system for production of desired proteins. Plants have been the major intruders on this earth suggesting the production of heterologous protein at larger quantity and at wider host range. In the most recent decade, tremendous progress has been made in heterologous protein production in plants and plant systems have become competitive alternative to the established production technologies that use bacteria, yeast, fungi, insect and or cultured mammalian cells.

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Stable expression from DNA integrated into the plant genome Expression of heterologous protein from plant genome involves stable nuclear transformation and integration wherein whole plants can be regenerated, ultimately producing seeds or vegetative tissues which can be maintained under tissue culture conditions or in experimental farms. This involves screening of large number of transgenic lines to select a best line for protein production (Ling et al. 2010). However, nuclear transformation ensures posttranslational modifications and subsequent storage or secretion of the proteins depending on the fused signal peptide. The accumulation of protein in cellular compartments, such as ER, chloroplast, mitochondria, and vacuole ensure appropriate folding and assembly results in protein stability and bioactivity. Nuclear transformation and expression of proteins have certain merits, but incurs high cost and consumes more time with generally low levels of expression of proteins (Yap and Smith 2010). The expression levels in nuclear transformation can be increased by utilizing strong promoters, enhancer elements and augmenting other molecular strategies like codon usage or addition of strong signal sequences. But long production cycles and ability to cross with native plant species has limited its scope for commercialization.

Traditionally, interferon gamma (IFN-γ) which is decisive for immunity against viral, some bacterial and protozoal infections has been produced using a variety of transgenic systems including bacteria, cultured animal cells, and viruses (Sekellic et al. 1994; Digby and Lowenthal 1995; Schultz et al. 1995; Arora et al. 1996; Song et al. 1997; Argyle et al. 1998; Yashiro et al. 2001; Takehara et al. 2002; Wu et al. 2002, 2008; Leelavathi and Reddy 2003; Chen et al. 2004; Sun et al. 2005; Jiménez-Bremont et al. 2008; Rupa et al. 2008). Recombinant IFN-γ expressed in plant and animal systems exhibit different glycosylation profiles, and therefore behaves differently with respect to susceptibility to proteolysis, shorter survival times in blood (Sareneva et al. 1993), and high production costs.The heat-labile enterotoxin B (LTB) subunit from enterotoxigenic Escherichia coli and the cholera toxin B (CTB) subunit from Vibrio cholerae was produced in transgenic rice with a view to produce vaccines against these strains causing diarrhea. LTB and CTB genes were inserted between globulin promoter and potato protease inhibitor II terminator for expression in transgenic rice plants (Soh et al. 2015).

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Stable expression from the plastid genome (chloroplast transformation/ transplastomic technology) Apart from expression of recombinant protein from plant genome, alternately, plastid is also explored as a useful target for genetic manipulation and expression of heterologous proteins. The advantages of chloroplast transformation over the nuclear transformation are: thousands of plastids present in photosynthetic cells of higher plants result in higher level expression (46-70% of the total soluble leaf protein) of transgene with proper folding and formation of disulfide bonds (Staub et al. 2000; Daniell et al. 2001; Daniell and Dhingra 2002; Fernandez-San Millan et al. 2003; Lee et al. 2003; Koya et al. 2005; Oye et al. 2009; Gao et al. 2012). Transgene integrated into chloroplast DNA do not appear to undergo silencing or suffer from position effects due to their site-specific transgene integration into the chloroplast genome; scope for multi-gene engineering in a single transformation event (De Cosa et al. 2001; Jeong et al. 2004); and chloroplast genes are inherited in a strictly maternal fashion providing a natural containment method for transgenic plants, since transgene cannot be transmitted through pollen in majority of the plants (Maliga 1993; Daniell et al. 2002). Integration of the transgene in the transcriptionally active spacer region between the trnI and trnA genes within the ribosomal operon resulted in the highest levels of transgene expression (Ruhlman et al. 2010). Although, chloroplast transformation has been achieved in a few important crop plants such as carrot, potato, tomato, soybean and eggplant, expressing therapeutic proteins like subunit vaccines in the non-green edible portion is a challenging task. Edible leafy vegetables (lettuce and brassica) could be the suitable options for chloroplast transformation and these could serve as the best source of edible vaccines (Bock and Khan 2005; Daniell 2005; Singh and Verma 2010; Verma and Daniell 2007). The chief capsid protein L1 of human papillomavirus HPV-16 was expressed in tobacco chloroplast genome to a level as high as 240 mg per mature plant. The chloroplast-derived L1 protein exhibited proper conformation and assembled into virus- like particles (Fernández-San Millán et al. 2008). Further, intraperitoneal injection of the leaf extract in mice showed presence of neutralizing antibodies (Lenzi et al. 2008). The hepatitis C viral core gene and a codon-optimized gene encoding a C-terminal truncated

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16 kDa core polypeptide were expressed in tobacco chloroplasts. Anti-core antibodies in HCV-infected human sera were detected by the 16 kDa core polypeptide in total leaf protein (Madesis et al. 2010). Youm et al. (2010) demonstrated successful production of the human b-site APP cleaving enzyme (BACE) in tobacco plants by transplastomic technology. Molecular analysis revealed the integration of tobacco aadA and BACE genes between trnI and trnA site in the plastome and transcribed as dicistron. The transgenic tobacco lines accumulated BACE protein at a level of 2% of total soluble proteins and were found to be immunogenic in mice. Transient expression The transient production system is the highest and the most convenient platform for the production of heterologous proteins in plants. The methods employed for transient expression in plants includes Agrobacterium-mediated transformation or Agroinfiltration (Chen et al. 2013; Leuzinger et al. 2013), use of virus based expression system (Wagner et al. 2004; Lico et al. 2008) and Magnifection technology (Gleba et al. 2005). Magnifection technology utilizes viral vectors delivered by Agrobacterium for high level expression of several polypeptides. Among plant-based approaches, viral expression systems have shown great promise and flexibility to produce recombinant proteins on a large scale in short time (Pogue et al. 2002). Plant virus expression vectors have always been under development as one of the efficient systems for recombinant protein production in plants. Plant RNA virus (Tobacco mosaic virus, Potato virus X, and Cowpea mosaic virus) expression vector systems have been broadly classified as the ones which are engineered for the production of immunogenic peptides and proteins in plants (short epitopes fused to the CP that are displayed on the surface of assembled virus particles) and the other as polypeptide expression systems (expressing the entire recombinant protein) (Yusibov et al. 2011). Viruses are the chosen system for transient expression and transformation as they can be engineered as deconstructed viruses to prevent movement from cell-to-cell, be transmitted by insects to other plants or undergo unnecessary and unexpected recombination events (Gleba et al. 2004). Fujiki et al. (2008) developed a Cucumber mosaic virus (CMV)-based expression vector for the production of heterologous proteins in plants. The CMV-based expression

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vector utilized truncated 3a protein, which expresses the target genes from the strong coat protein (CP) sub genomic promoter and without the need for providing CP in trans for cell-to-cell spread. A maximum expression level of ~450 mg/kg and ~170 mg/kg of leaf tissue of green fluorescent protein (GFP) and human growth hormone (hGH) respectively was obtained in Nicotiana benthamiana plants transformed using agroinfiltration. Recombinant virus-like particles (VLPs) represent one of the effective vaccine strategy. A stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines have been described previously. On the other hand, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major obstructions in the practical development of plant-based VLP production. Geminivirus- derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs has been reported by Huang et al. (2009). Co-delivery of bean yellow dwarf virus (BeYDV)- derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and protein production up to 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. Concurrently, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. This method has advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. Chemical induction using a chemically inducible viral amplicon expression system to increase expression of a heterologous protein, α-1-antitrypsin (AAT), in plants was optimized by Plesha et al. (2008). A cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce a recombinant human blood protein (AAT), by co-infiltrating intact and detached Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Application of induction solution every 2 days via topical application resulted in AAT improvement of a 1.8-fold of the total soluble protein.

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Recent developments in transient expression system involves the combination of plant viral sequences (based on TMV) and Agrobacterium binary plasmid (based on pBI121) termed as hybrid Launch vector (pBID4). The launch vector contains the CaMV 35S promoter transcribing the viral sequences. After agroinfiltration multiple single stranded DNA copies present between the left and right borders of the T-DNA of Agrobacterium plasmid are produced in the plant cells thereby producing multiple copies of the gene of interest and high expression level. This system has been successfully demonstrated for the expression of influenza H5N1 antigens (Musiychuk et al. 2007; Shoji et al. 2009). Magnifection is the new strategy employed at the industrial level to assure the requirements like high-yield, rapid scale-up, and rapid production in bulk quantity. MagnICON® technology (ICON Genetics GmbH, Halle, Germany) is based on in planta assembly of functional viral vectors from two separate 5’ and 3’ pro-vector modules. Agrobacterium-mediated transformation helps in delivering these viral sequences into the plant cells which are then assembled in the presence of a site-specific recombinase (Marillonnet et al. 2004; Gleba et al. 2005). It was proven highly effective in the production of plague antigens (Santi et al. 2006), hepatitis B virus core antigen (HBcAg) VLPs (Huang et al. 2006), Norwalk virus VLPs (Santi et al. 2008), and anti-ebola monoclonal antibodies (Qiu et al. 2014). Organ Specific expression (Fig. 2) Heterologous protein production in leaves The idea of producing heterologous protein using plant system gained importance due to its ability to produce large biomass (leaves) under natural conditions. Several tones of biomass can be collected from one to few acres of plantations. Tobacco species namely Nicotaina tabacum and Nicotiana benthamania have been the appropriate plant platforms and commonly employed to produce antibodies in leaves. The protocol for stable and transient transformation of tobacco using Agrobacterium is very well established. Nevertheless, tobacco plants produce large amount of leaf biomass in a single harvest cycle and also multiple harvest cycles can be performed per year to increase the yield per hectare. Transplastomic technology has provided maximum usage potential of plant leaves for production for therapeutic proteins and vaccines as plastids are capable of

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producing multimeric proteins with appropriate folds. High yield of active human growth hormone i.e. somatotropin (Gils et al. 2005) and serum albumin protein (Fernandez-San Millan et al. 2003) have been produced in tobacco chloroplasts. A 47 kDa tetanus toxin fragment (TetC) was successfully expressed in tobacco chloroplasts, and was capable for inducing protective levels of TetC antibodies in mice (Tregoning et al. 2003). Similarly cholera toxin B fragment fused with human proinsulin CTB-pins (Ruhlman et al. 2007) and exendin 4 (Nitynandam, 2011) was expressed in high quantities in tobacco leaves and tested for their therapeutic role in treatment of diabetes. E. coli enterotoxin B (Rosales- Mendoza et al. 2008) and plague F1-V (Arlen et al. 2008) expressed in tobacco leaves were successful in inducing an immune response and protected mice against these diseases. Nevertheless, several viral antigens were expressed in tobacco leaves in high yield and were effective in eliciting an immune response in mice. virus HEV E2 (Zhou et al. 2006), Swine fever virus CFSV E2 (Shao et al. 2008), and human papillomavirus L1 (Lenzi et al. 2008) were produced in tobacco chloroplasts. Protozoan antigens such as LecA from Entamoeba (Chebolu et al. 2007) and CTB-ama1 and CTB- msp1 from plasmodium parasite (Davoodi-Semiromi et al. 2010) were produced in tobacco leaves and its potential as vaccine candidates have been evaluated. Simultaneously, besides tobacco, lettuce and alfalfa are the most preferred choice of plants for production of heterologous protein in leaves as they both produce large quantity of leaf biomass and lack harmful phenolic compounds like nicotine. Cholera toxin B subunit fused with the known Mycobacterial antigens; secretory antigens (ESAT6) and cell wall based lipase (LipY) expressed in lettuce chloroplasts were used for oral delivery of TB vaccine antigens (Lakshmi et al. 2013). Malarial vaccines namely, Pfama1 and Pfmsp1 were produced in lettuce leaves (Davoodi-Semiromi et al. 2010) and were highly immunogenic in mice. Further, the antibodies generated in this response prevented the invasion of Plasmodium into red blood cells. A chimeric peptide (MLC) consisting of the merozoite surface protein-1 (MSP-1) and the circumsporozoite protein (CSP) separated by a poly-Gly linker motif expressed in Brassica napus was identified as a potential oral vaccine against vivax malaria (Lee et al. 2011). Transgenic lettuce expressing the hepatitis B surface antigen (HBsAg) was administered as oral vaccines in mice and humans. This antigen induced HBsAg antibodies in both populations to a

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protective level (Kapusta et al. 1999). Geminiviral replicon delivery system was used for high-level expression of virus like particle (VLP) derived from the Norwalk virus capsid protein (NVCP) and therapeutic humanized mAbs against Ebola (EBV) (6D8) or West Nile (WNV) (hE16) viruses in lettuce (Lai et al. 2012). Alfalfa has a strong regenerative capacity in addition to low quantities of phenolic compounds and high content of proteins which makes it suitable for expressing proteins in large quantities. The sVP6 protein of human group A expressed in alfalfa elicited sera antibodies in mice and provided passive immunization when subjected to a challenge with simian rotavirus SA-11 (Dong et al. 2005). The most successful achievement is the production of animal vaccines in alfalfa. Protective levels of serum antibody were generated to foot and mouth disease virus in mice after oral administration or parenteral immunization with transgenic alfalfa plants expressing the Foot and Mouth disease viral structural protein VP1 (Wigdorovitz, et al. 1999). .A truncated version of the structural protein E2 from Bovine Viral Diarrhea Virus (BVDV) was expressed as a fused protein in transgenic alfalfa plants (Aguirreburualde et al. 2013). Mice and bovine studies demonstrated production of BVDV specific antibodies that were able to protect the animals completely form infection. Heterologous protein production in seeds Ease of collection, processing and concentration of protein in seed tissues has made plant seeds as natural bioreactors for successful heterologous protein production. Transgenic plants are engineered to express and accumulate recombinant proteins in seeds by using seed specific regulatory sequences such as tissue-specific soybean seed storage β- conglycinin promoter (Vianna et al. 2011). Soybeans (Glycine max) are the chosen plants for seed specific expression, as these seeds are richest source of proteins and contain nearly 40% protein by dry mass (Hudson et al. 2011). Transgenic soybean seed derived vaccine was used to protect cattle from infection by enterotoxigenic E. coli (ETEC). The ETEC fimbriae subunit protein FanC antigen was over expressed in soybean and was found to elicit immune response in mice even in the absence of cold chain transportation and storage (Oakes et al. 2009). Human proinsulin gene was expressed in transgenic soybean seeds driven by monocot tissue-specific promoter from sorghum γ-kafirin seed storage protein gene and the α-coixin cotyledonary vacuolar signal peptide from Coix

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lacryma-jobi. Proinsulin accumulated in soybean seeds and was found to be stable even after 7 years under room temperature conditions (Cunha et al. 2010). Heat-labile enterotoxin B (LTB) subunit from enterotoxigenic E. coli was expressed in soybean seeds (Moravec et al. 2007) and rice seeds (Soh et al. 2015) and used as oral vaccines to immunize mice against ETEC. The toxin-neutralizing activity of LTB in serum of orally immunized mice was due to the action both IgG and IgA responses. Murine single-chain variable fragment (scFv) was accumulated in transgenic Arabidopsis seeds, as high as 12.5 % and 36.5% of total soluble proteins using the 5' and 3' regulatory sequences of the seed storage protein gene arcelin 5-I and beta-phaseolin seed storage protein promoter from Phaseolus vulgaris, respectively (De Jaeger et al. 2002; De Jaeger et al. 2003). Additionally, single-chain variable fragment-Fc antibodies neutralizing virus (Van Droogenbroeck et al. 2007), porcine reproductive and respiratory syndrome virus glycoproteins (Piron et al. 2014) and human lysosomal acid β-glucosidase protein (He et al. 2012) was expressed in transgenic Arabidopsis seeds and accessed for their immunogenicity. Numerous antigens have been expressed in maize/corn seeds owing to high biomass yield, bigger endosperm, absence of active proteases in dry seeds and presence of a rich mix of molecular chaperones and disulfide isomerases for proper folding of proteins and the very well-established processing technology (Moeller et al. 2010; Naqvi et al. 2011). Heat labile B subunit from enterotoxigenic E. coli, cholera toxin B subunit from Vibrio cholerae, spike protein of swine transmissible gastroenteritis, hepatitis B oral vaccine and human recombinant proinsulin was efficiently expressed in maize plants (Streatfield et al. 2002; Chickwamba et al. 2002; Lamphear et al. 2004; Streatfield, 2005; Farinas et al. 2007; Karaman et al. 2012). Rice is another apt candidate next to maize to express heterologous proteins with the additional advantage of self-pollination and cultivated in most parts of the world. Rice seeds were engineered to produce therapeutic proteins like human serum albumin (He et al. 2011) single chain variable fragment against a tumor-associated marker antigen- carcinoembryonic antigen (Stoger 2000), and interleukin 10 (Fugiwara et al. 2010). The major T-cell epitopes from Japanese cedar pollen allergens Cryj1 and Cryj2 was expressed in the endosperm of rice seeds (Takaiwa et al. 2006; Wakasa et al. 2015).

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Mice fed with these transgenic rice seeds daily for three weeks and then challenged with cedar pollen allergen showed significant suppression of allergen-specific CD4+ T-cell proliferation, IgE and IgG levels. Such studies can be further used for developing potential human vaccines for oral administration as tolerogen formulations (Wakasa et al. 2013). Few reports in recent past also demonstrated production of therapeutic proteins in seed tissues such as hepatitis B surface antigen in tobacco seeds (Hernández et al. 2013), hepatitis C core antigen in Brassica napus seeds (Mohammadzadeh et al. 2015) and anti- hypertensive peptide in rice seeds (Yang et al. 2006). Other plants have also been investigated for production of recombinant proteins in seeds but cereals and legumes have been the favored choice. However, there is a strong opposition for producing therapeutic proteins in such food crops because of the contamination issues with non- transgenic plants under non-stringent conditions. Heterologous protein production in fruits and vegetables Fruit can serve as one of the best plant organs for production of therapeutic proteins and oral delivery of vaccines because they are consumed raw and hence the expressed proteins will retain their natural conformation imparting more immunogenicity. Tomatoes have been used for expression of heterologous proteins owing to the easy availability of transformation protocol, capability to increase biomass with low cost under greenhouse conditions and the short life cycle. Additionally, the amount of protein content in raw tomatoes is more as compared to the ripened tomatoes. Yersinia pestis F1-V antigen fusion protein expressed in tomato fruits and when administered orally developed immunogenicity in mice after they were challenged subcutaneously with bacterially produced F1-V (Alvarez et al. 2006). An edible diphtheria-pertussis-tetanus (DPT) multicomponent vaccine was synthesized by combining the exotoxin epitopes from Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani and expressed satisfactorily in transgenic tomato plants (Soria-Guerra et al. 2007). Proteins expressed in fruit tissue undergo post-translation modification and form biological active oligomers. E. coli heat-labile enterotoxin B subunit when expressed in plant was able to form active pentamers and specifically bind to GM1 ganglioside (Loc et al. 2014). A range of viral antigenic proteins, such as rabies virus glycoprotein G (McGarvey et al. 1995), respiratory syncytial virus F glycoprotein (Sandhu et al. 2000), a

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hepatitis E virus surface protein (Ma et al. 2003), rotavirus capsid protein VP2 and VP6 (Saldana et al. 2006), a synthetic HBV/HIV antigen (Shchelkunov et al. 2006), Norwalk virus capsid antigen (Zhang et al. 2006), hepatitis B virus surface antigen (HBsAg) (Lou et al. 2007) chimeric human papilloma virus HPV-16 L1 proteins were expressed in tomatoes (Paz de la Rosa et al. 2009). Other industrially important product engineered in tomato fruits includes a taste-modifying protein, miraculin which functions to change the perception of a sour taste to a sweet one (Hirai et al. 2011). Transgenic banana plants have been used to produce HBsAg in fruits with a view to use it for vaccination through banana fruits (Kumar et al. 2005). But the lower yield of this antigenic protein in banana fruits raised a problematic technical issue for its use as a vaccine. The difficulty of accumulating heterologous proteins in ripened fruits such as banana has always hindered the system to be used for vaccine delivery. Another study claimed production of cholera toxin B subunit in transgenic banana callus (Renuga et al. 2010). Strawberries have been used as candidate for production of recombinant proteins as fruits are eaten raw, store good amount of protein and strawberry plants are propagated vegetatively thus lessening the risk of gene contamination. A Japanese firm had setup a plant under the METI project for production of canine interferon (IFNα) from transgenic strawberries (Rubicki 2009). Later these transgenic strawberries expressing dog interferon-α were powdered and sold as an oral drug from 2014 and was found to be effective in the treatment of periodontal disease (Tabayashi and Matsumura 2014; Hiwasa-Tanase and Ezura 2016). Heterologous protein production in roots/tubers Transgenic potato tubers are used for the last few years as a source for heterologous proteins due to key properties such as long-term storage of accumulated protein in stable form, plentiful of biomass, short growth cycle and certainly the ability to induce mucosal immunity on oral administration (Thanavala et al. 2005 ; Kim et al. 2009). Transgenic potato plants constitutively expressing the synthetic E. coli heat-labile enterotoxin subunit B (LT-B) gene which has increased accumulation and pentamers assemblage of LT-B in leaves and tubers. Raw tubers fed to mice in three doses were able to generate higher levels of serum and mucosal anti-LT-B (Mason et al. 1998). Cholera toxin B

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subunit protein was expressed in potato tubers and was able to induce serum and intestinal anti-CTB antibodies (Arakawa et al. 1998). Transgenic potato tubers have also been used to produce viral antigens such as hepatitis B surface antigen (Richter et al. 2000; Kong et al. 2001; Shulga et al. 2004), Norwalk virus coat protein (Mason et al. 1996; Tacket et al. 2000), epitope of porcine epidemic diarrhea virus (Kim et al. 2005), GP5 protein of Porcine reproductive and respiratory syndrome virus (Chen and Liu 2011), Rotavirus VP7 (Wu et al. 2003; Li et al. 2006; ), Rabbit hemmorhagic disease virus VP60 (Castanon et al. 1999), infectious bronchitis virus (IBV) S1 glycoprotein (Zhou et al. 2003) and human papilloma virus major capsid protein L1 (Warzecha et al. 2003; Biemelt et al. 2003) or oncogene E7 (Bříza et al. 2007) as oral vaccine candidates. Yu and Langridge (2001), constructed a combination of Cholera toxin B and A2 subunit, rotavirus enterotoxin and enterotoxigenic E. coli fimbrial antigen genes were expressed in transgenic potatoes. This fusion protein was assembled into cholera holotoxin-like structures having enterocyte-binding affinity and elicited immune response against diarrheal symptoms. Transgenic potato tubers have also been used to produce other therapeutic proteins and nutraceuticals. Soybean agglutinin (SBA), an N-acetylgalactosamine- binding plant lectin finds application in screening and treatment of breast cancer, fetal cell screening, purification of tagged proteins and carrier for drug delivery were accumulated in transgenic potato tubers which were highly resistant to degradation of gastric secretions and also retained their specific binding activity (Tremblay et al. 2011). Human beta-casein protein was expressed in potato plants with a view to replace bovine milk in baby foods which is responsible for gastric and intestinal diseases in children (Chong et al. 1997). Further, Chong and Langridge (2000) also expressed human lactoferrin gene in potato tubers to a level approx. 0.1% of total soluble proteins. A cholera toxin B subunit-Insulin fusion protein was produced in transgenic potato tubers at a concentration of 0.1% of the total soluble proteins. Non-obese diabetic mice fed with these potato tuber tissues showed reduction of inflammation of pancreatic cells and delay in diabetes disease progression (Arakawa et al. 1998). In another study, ricin subunit B from castor bean was used as an immunomodulatory molecule capable of enhancing immunosuppression associated with Type I diabetes (Carter et al. 2010). Subtypes of

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human and salmon interferon alpha was produced in transgenic potatoes and effectively tested for their bioactivity against viral strains (Ohya et al. 2001; Sawahel et al. 2002; Fukuzawa et al. 2010). De Wilde and team demonstrated bulk production of full length IgGs and Fab fragments in transgenic potato tubers (De Wilde et al. 2002). To increase the nutritional quality of potato tubers, AmA1 gene from Amaranthus hypochondriacus was transformed into potato plants in a tuber specific manner. This protein is rich in all essential amino acids for optimal human nutrition (Chakraborty et al. 2000). Transgenic potato tubers have also been transformed for production of recombinant spider (Nephila clavipes) dragline proteins spidroin useful for medical and industrial purposes (Scheller et al. 2001). Other plant tubers used for heterologous protein expression includes carrot, sweet potatoes and turnips. The fusion protein CFP10-ESAT6-dIFN derived from Mycobacterium tuberculosis genes was synthesized and used to transform carrot tissue. The protein was produced in transgenic carrot root tissues and was able to induce both humoral and cell-mediated immune responses in mice (Permyakova et al. 2015). Yersinia pestis F1 and V antigens were produced in transgenic carrot roots and were able to elicit protective levels of antibodies (Rosales-Mendoza et al. 2011). Likewise, studies also demonstrated production of interferon alpha (Daniell et al. 2001) in turnip and human lactoferrin in sweet potatoes (Min et al. 2009). Hairy root cultures have been standardized for the commercial-scale production of secondary metabolites. These root cultures have also been exploited to produce heterologous proteins in larger amounts in continuous bioreactor cultures. Hairy roots offer unique advantages because of their genetic and biosynthetic stability, fast doubling time, require simple hormone free medium and easy scalable protein production. Transgenic tobacco hairy root cultures were engineered to produce human acetylcholinestarase (Woods et al. 2008) and active antimicrobial peptide, ranalexin (Aleinein et al. 2015). Tobacco hairy root cultures were also used for the production of murine interleukin 12 (Liu et al. 2008). A study demonstrated use of Brassica rapa (turnip) hairy root cultures over tobacco cultures for better production capability and stability of heterologous proteins (Huet et al. 2014). An isoform of human growth hormone was produced in Brassica oleracea hairy root cultures (Lopez et al. 2014).

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Although the recombinant proteins are accumulated or secreted by hairy root cultures in large quantities, proteins are degraded by the proteolytic activity of peptidases (Lallemand et al. 2015). Nevertheless, studies are underway to inactivate these peptidases genetically or biochemically. Boosting plants as heterologous protein production systems Research in heterologous protein production in plant is now oriented for increasing the recombinant protein production to cut down overall production and downstream cost. Several strategies have been implemented to enhance protein production in plants by modulating regulatory sequences like promoters, 3’ UTRs, increasing transcription rate and transcript stability, efficient translation and compartmentalization for higher accumulation (Fig. 3). A number of constitutive and tissue specific promoters have been used for high level protein production. The most widely used constitutive promoter is the Cauliflower mosaic virus 35S promoter (CaMV35S) which has shown to drive strong expression of the downstream gene of interest in most of the plant species (Dutt et al. 2014). Alternatively, other constitutive promoters used for plant transformation include ubiquitin, actin, histone, tobacco cryptic promoter and nopaline synthase promoter to list a few (Stefanov et al. 1991; Herman et al. 2001; Menassa et al. 2004). Chimeric promoters or combination of viral promoters have been created to enhance the expression level. Elements from the Commelina Yellow Mosaic Virus (CoYMV), the Cassava Vein Mosaic Virus (CsVMV) and activating sequences from the CaMV 35S promoter were combined together to drive strong expression of the downstream reporter gene (Rancé et al. 2002). Selected tissue specific promoters have also been utilized to accumulate and compartmentalize recombinant proteins which affect protein stability and downstream processing. Several root, fruit, tuber and seed specific promoters have been isolated (Hernández et al. 2013; Piron et al. 2014; Dutt et al. 2014; Opabode and Akinyemiju 2015; Streatfield et al. 2016). Furthermore, to achieve high level of expression of the heterologous protein multiple enhancer elements from strong promoters can be stacked upstream of gene of interest which will increase the transcription rate. Another way to increase the transcriptional activity is to insert scaffold or matrix attachment regions next to the promoter sequence which helps in the recruitment of transcription factors and polymerases (Li et al. 2002).

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Inducible promoter systems offer the opportunity to regulate gene expression levels at particular stages of plant growth and development and in particular tissues of interest. Thus the overexpression of heterologous protein will not hinder normal growth and development and can be accumulated with higher yields at the right stage for easy downstream processing. The expression can be triggered by external elicitors like ethanol, tetracycline, dexamethasone, copper, salicyclic acid, salts, and sugars and by environmental factors like temperature and oxidative stress or by pathogens (Borghi 2010; Kinkema et al. 2014; Gatz 2014). Dugdale and his team (2013) developed a technology termed as INPACT (In Planta Activation) used for inducible high level expression of protein in tobacco plants. The INPACT cassette is assembled in such a way that on infection by tobacco yellow dwarf virus, the recombinant gene is split and expressed from the extra chromosomal, replicating episomes which are set free from the host chromosome in the presence of the virus-encoded replication associated proteins, Rep/RepA transcriptionally controlled by the AlcA:AlcR switch responding to ethanol application. Using INPACT technology bovine trypsinogen and human vitronectin was expressed in tobacco leaves to a level of up to 196 mg/kg (dry weight) and ~100 mg/kg (fresh weight) respectively. Transcription levels mostly cannot be correlated to the amount of protein accumulated as it depends on several factors such as efficient 5’capping, mRNA splicing, polyadenylation, nuclear export and mRNA stability in the cytosol. However, all these factors can be manipulated to enhance protein production in plant systems. Insertion of a strong 3’ UTR downstream to the gene of interest in the expression vector takes care of the polyadenylation and mRNA stability. Commonly used terminators are nopaline synthase, cauliflower mosaic virus 35S, heat shock protein and potato proteinase inhibitor II 3’UTRs (Hood 2007; Nagaya et al. 2009; Limkul et al. 2015). Position and the sequence of an intron have significant effects on expression levels of the proteins in plant cells. Thus, intron sequences are modulated in the synthetic gene constructs to achieve maximum expression levels (Bourdon et al. 2001; Morella et al. 2008; Dugdale et al. 2013). Translational efficiency of the protein sequences can be increased by inserting a leader sequence or manipulating native 5’UTR sequence with the objective to increase

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ribosome binding and thereby translation. In addition to augment protein production, codon optimization can be carried out during designing the gene of interest. During this procedure some rarely utilized codons can be removed and replaced by the codons commonly recognized from the available pool of t-RNAs in plants. Precise engineering is warranted depending upon the type of transformation as codon usage for nuclear and plastid genes are varied. To obtain proteins in their biological active form, they undergo post translational modifications such as glycosylation, phosphorylation, methylation and ribosylation. Post translational degradation of proteins can be effectively reduced by subcellular compartmentalization. Proteins can be sequestered to organelles like endoplasmic reticulum, vacuoles, mitochondria and plastids. Addition of ER retention signal (KDEL/ HDEL) or mitochondrial/plastid signal sequences can direct the transport of nascent peptide to the respective organelle. ER targeting helps in safeguarding the proteins and ensures proper folding and assembly because of the resident chaperone machinery (Vitale and Denecke 1999; De Mayer and Depicker 2014). C-terminal fusion of the KDEL peptide, an ER retention signal to single-chain antibody variable-region fragments has been found to increase antibody levels by a factor of up to 10-100 as compared to either extracellular secretion to apoplast or expression in the cytosol (Fischer et al. 1999). Proteins which can accumulate and are stable under acidic conditions can be sequestered to the vacuoles. Few N-terminal and C-terminal propeptide sequences have been identified that routes the protein to vacuoles. Plastids in plants are the next important organelles to accumulate proteins up to 70% of the total soluble proteins. Proteins can be diverted to plastids by adding unique signal sequences or can be inherently produced in plastids by transplastomic technology described before. Most proteins produced and channelized through endomembrane system find their way to the apoplast. The proteins secreted into the apoplastic region avert intracellular and vacuolar proteinases. If these proteins are large enough, they accumulate between the cell membrane and cell wall and if small then are secreted into the medium. Secretion of proteins into the medium reduces the downstream cost and helps in easy recovery of the proteins. However, these secreted proteins are constantly exposed to harsh conditions or some extracellular proteinases which degrade the proteins and reduce the production yield. Co-expression of proteinase inhibitors or protease-resistant fusion constructs have

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increased the production of recombinant proteins in plant systems (Outchkourov et al. 2003; Benchabane et al. 2008; Kim et al. 2008; Sainsbury et al. 2012; Pillay et al. 2014) Bioreactors for recombinant protein production in plant cells Plant cell suspension cultures offer best platform for stable accumulation and secretion of recombinant protein in large quantities. Generally, plant nutrient media are relatively simple salt solutions with no added proteins and if a heterologous protein is produced in tissue culture and secreted into the medium, product recovery and purification could be easily and efficiently worked out due to absence of large intracellular contaminating proteins. Several studies have demonstrated secretion of proteins into medium which are less than 30 KDa whereas higher molecular weight ones are retained inside the cell due to plant cell wall. This problem is overcomed by incorporating a signal sequence before the gene of interest so as to secrete the protein into the culture medium. Use of bioreactors have facilitated up scaling and increase the production of recombinant proteins to a level equivalent to bacterial or mammalian system (Hellwig et al. 2004; Huang and McDonald 2012). Although, the technology of producing recombinant proteins in cell suspension cultures was proven more than 25 years before, the cost and potential to scale to industrial level was not a very successful venture (Sijmons et al. 1990). During this time, different bioreactors were designed to overcome this hurdle that included the stirred tank and wave bioreactor. Both stirred tank and airlift bioreactor were used for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in transgenic Nicotiana tabacum cells (Lee et al. 2001). A new type of bioreactor designed by Protalix Biotherapeutics (ProCellEx production system) consisted of large polyethylene bags serially arranged and filled with medium and supplied with sterile air to produce ELELYSO™ (taliglucerase alfa), which was approved by the U.S. Food and Drug Administration in May 2012. Carrot cells and tobacco BY-2 cells are commonly used cell cultures for production of therapeutic, pharmaceutical and vaccine proteins (Rosales-Mendoza and Tello-Olea 2015; Tekoah et al. 2015). A number of therapeutic proteins have also been expressed in rice cell cultures. Following are few proofs of principle studies demonstrating the production of foreign protein in rice cell suspension cultures. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g) driven by rice alpha-

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amylase 3D (RAmy3D) promoter was expressed in transgenic rice cell suspension culture. Purified hCTLA4IgP was found to be biologically active and suppressed T-cell proliferation (Lee et al. 2007). mGM-CSF fused with the αAmy3 signal peptide was produced in rice cell suspension cultures scaled up in a 2 liter bioreactor and yielded highest concentration of 24.6 mg/L protein in the culture filtrate (Liu et al. 2012). A Der p2-FIF-Fve fusion protein was expressed in rice cells under the control of sucrose starvation induced α-amylase gene (αAmy8) The fusion protein elicited IgE immunogenicity and histamine release (Su et al. 2012). Similarly, recombinant alpha-1- antitrypsin (McDonald et al. 2005), human growth hormone (Kim et al. 2008) and Interferon-gamma (IFN-γ) (Chen et al. 2004) was also expressed in rice cells. Currently mosses such as Physcomitrella patens, have been employed as platforms for production of therapeutic proteins in bioreactors (Decker et al. 2014; Reski et al. 2015). Owing to their photosynthetic property and glycosylation capability, Physcomitrella patens has become a best alternative to produce high value humanized proteins with reduced production costs. Simple designed bioreactors are employed to grow moss for production of therapeutic proteins such as vascular endothelial growth factor (Baur et al., 2005), erythropoietin (Weise et al. 2007), the complement-regulatory protein factor H (Büttner-Mainik et al., 2011), HIV epitopes from gp120 and gp41 (Orellana-Escobedo et al. 2015), glyco-optimized antibody IgG1 IGN314 (Kircheis et al. 2012) and enzymes such as human alpha-galactosidase and glucocerebrosidase (Niederkrüger et al. 2014). With increasing knowledge and technical know-how plant cell cultures can be efficiently utilized to produce large quantities of therapeutic proteins at par with bacterial and mammalian platforms with reduced cost. Plant derived protein in clinical trials Antibodies To date, several antibodies have been synthesized in a variety of plants having applications in medical, industrial and research fields. Production of antibodies in plants has gained importance owing to its reduced cost. Effective protection against some serious diseases like AIDS requires continuous application of HIV neutralizing antibodies (Strasser et al. 2009; Arcalis et al. 2013) which will dramatically increase the cost per application. In such cases, scaling up production becomes more important than speeding

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the reaction; and therefore transgenic plant production platform become more pertinent because of their rapid scaling up capacity. In one prominent study antibody cocktails (ZMapp) produced in tobacco plants (Nicotiana benthamiana) using MagnICON technology was used for treating Ebola infection in non-human primates (Qui et al. 2014). A few number of antibodies expressed in plants reached the clinical trails and soon to be launched in the market. A chimeric secretory antibody CaroRx (IgG-IgA) that binds to the bacteria Streptococcus mutans, a causative agent of tooth decay, was expressed in tobacco plants. This antibody is effective as it protects against dental caries and prevents the recolonization of the bacteria up to 2 years after 3 weeks of application (Ma et al. 1998). An anti-CD20 optimized antibody BLX-301 was produced in aquatic plant Lemna minor (duckweed) by Biolex Inc. which entered the phase I trials for the treatment of non-Hodgkin's B cell lymphoma and rheumatoid arthritis (Cox et al. 2006). MAPP66 is an antibody cocktail produced in Nicotiana benthamiana by Icon Genetics (Bayer’s) MagnIcon technology and used as a HSV/ HIV microbiocide which entered first phase clinical trial (Mapp Biopharmaceutical, Inc.). Four anti-HIV neutralizing monoclonal antibodies namely b12, 2G12, 2F5, 4E10 were found to be effective in controlling the transmission of virus (Rosenberg et al. 2013). Out of these four antibodies, 2G12 antibody produced in transgenic tobacco by Pharma-Planta has been approved for the first-in human phase I clinical trial in UK (Ma et al. 2015). The success of these trials will mark a significant achievement in the field of plant derived pharmaceuticals and will further boost in the transfer of proof of principle studies to commercialization. Few plant derived antibodies have also been used in manufacturing other biopharmaceuticals. CIGB, a Cuban company, has developed and produced ScFv monoclonal antibody (CB-Hep1) in tobacco plants which has been used for several years for the purification of recombinant Hepatitis B subunit vaccine (Pujol et al. 2005; Triguero et al. 2011). Antigen/ subunit vaccines Plant based recombinant vaccines can be categorized as one used for veterinary and other as human vaccines. Plant-based human vaccines are not yet commercialized, although,

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proofs of principle studies of viral and bacterial subunit vaccines have been successfully demonstrated in transgenic plants such as tomato, potato, banana, maize, alfalfa and soybean (Kristina and Luthar 2016). Developing countries are most of the time inflicted with diseases that results in mortality. Plants serve as the best alternative for production of vaccine candidates with reduced cost and thereby decrease the overall cost burden involved in conventional methods for procuring recombinant vaccines. The first plant derived vaccines approved for clinical trial was for the veterinary use which protect against Newcastle disease (Rybicki 2010). Dow AgroSciences (USA) produced USDA approved hemagglutinin and neuraminidase of Newcastle disease virus in tobacco cells for subcutaneous application, but is yet to be marketed (Yusibov et al. 2011). Only few of the plant derived human vaccine subunits reached the clinical trials. Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic E. coli (ETEC) produced either in potato or maize was the first to enter clinical trial to protect against diarrhea. Raw mashed transgenic potato or corn meal suspended in water and administered orally to healthy volunteers was observed for its safety and immunogenic potential. The study showed that volunteers fed with transgenic tissues had increased levels of LTB-specific serum IgG and IgA as compared to the placebo controls who were fed with non-transgenic potato/corn (Tacket et al. 1998; Tacket et al. 2004). In another study a major capsid protein of Norwalk virus was expressed in transgenic potato and used to feed 24 healthy volunteers (Tacket et al. 2000). Each volunteer was administered 2-3 doses as raw diced potatoes containing 215–751 mg of NVCP. 95% of the volunteers showed significant levels of serum IgG and stool IgA. This vaccine is currently being optimized for commercialization under the trade name NoroVAXX (Fischer et al. 2012). Hepatitis B virus disease persists even after vaccines were developed more than three decades ago. Despite of several attempts to develop oral vaccines for Hepatitis B virus disease in plants like potato and banana, no plant derived vaccine could be commercialized so far but expected to yield promising results in the near future. However, purified antigens derived from plants were immunogenic, but the inherent levels of these antigens in the tissues were found to be considerably low for using it as edible vaccines. Nevertheless, HBsAg expressed in transgenic potato and lettuce plant was used as oral vaccines for phase I trials. More than 50% of the volunteers fed with

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transgenic potato showed increased levels of anti-HBsAg in the serum and developed systemic resistance response to Hepatitis B infection (Richter et al. 2000; Kong et al. 2001; Thanavala et al. 2005). Likewise, HBsAg expressed in transgenic lettuce leaves (0.1–0.5 µg of HBsAg per 100 g of fresh tissue) were given to adult volunteers with reducing doses in two consecutive months (Kapusta et al. 1999). Two of three vaccinated volunteers showed presence of HBsAg-specific IgG 2 weeks after the second vaccination. Further, no IgA specific for HBsAg was detected and there were no noticeable side effects observed after ingestion of transgenic lettuce. Endemic rabies is the common cause of mortality in some parts of the world. Therefore, there is a need for constant supply of rabies vaccines in these regions. Two rabies virus epitopes, glycoprotein (GP) and nucleoprotein (NP) were fused together and expressed in transgenic spinach under the driving control of recombinant Alfalafa mosaic virus machinery (Dietzschold et al. 1990; Modelska et al. 1998; Yusibov et al. 2002). Raw spinach leaves were fed to two groups of volunteer, one group who were previously vaccinated for rabies virus and the other non-vaccinated group. In all more than 50 % of the volunteers from both the groups displayed elevated levels of sera IgG able to neutralize rabies virus particles. Influenza virus is a frequently mutating strain that results in the antigenic shift which quite often obliterates cross-protective immunity of the host. In such a scenario, strain specific vaccines produced on a large scale in short period of time are the prime requirement to prevent disease pandemics. D’Aoust and team (2008) expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. The virus like particles assembled and accumulated in the apoplastic region of tobacco cells and was able to elicit immune response in mice. Phase I / II clinical trial of the VLP composed of HA protein of H5N1 influenza virus (A/Indonesia/5/05) (H5-VLP) has been completed. Both H1 and H5 VLP vaccines elicited significantly greater CD4+ T cell responses than placebo and persisted even after 6 months of vaccination (Landry et al. 2010; Landry et al. 2014). Additionally, some volunteers developed antibody response to plant glycans which subsided within 6 months in most volunteers (Ward et al. 2014). Medicago Inc. have

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taken up this project and completed phase I trial with the H1N1 epitope and further the test for trivalent synthetic vaccine for other strains of influenza is underway. Few years back the rapid spread of H1N1 strain in the developing countries urged the need to produce cheap vaccines in a large quantity. Plant based VLPs were produced to develop subunit vaccines which are immunogenic. Recombinant hemagglutinin proteins from A/California/04/09 (H1N1) and A/Indonesia/05/05 (H5N1) strains of influenza virus were produced in Nicotiana benthamiana plant on a large scale. The production of serum hemagglutination inhibition and virus neutralizing antibodies was studied in laboratory animals (Shoji et al. 2011). To further enhance the overall potency of these antigens as vaccine candidates, Shoji et al. (2015) constructed H1 HA VLPs (HAC-VLPs) using ectodomain of HA from A/California/04/09 strain (Shoji et al. 2015). The recombinant hemagglutinin protein (HAI-05) from the A/Indonesia/05/2005 (H5N1) strain of influenza virus was produced transiently in Nicotiana benthamiana using 'launch vector. In the phase I clinical trial, the immune response elicited in volunteers by the HAI-05 vaccine was variable with respect to both hemagglutination-inhibition and virus neutralization antibody response (Chichester et al. 2012). Correspondingly, a first-in- human, Phase 1 dose dependent study was conducted to investigate safety, reactogenicity and immunogenicity of an HAC1 formulation at three dosages with and without Alhydrogel(®), in healthy adults 18-50 years of age (Cummings et al. 2014). In both the phase I trials, the vaccine was generally safe and was well tolerated, with no reported serious adverse events. Fraunhofer Center for Molecular Biotechnology, Plymouth, MI, USA completed phase I trials for recombinant protective antigen (rPA) against anthrax disease in the year 2014. Plasmodium falciparum surface protein Pfs25 expressed in Nicotiana benthamiana plants by tobacco mosaic virus-based launch vector was able to induce serum antibodies with complete transmission blocking activity (Jones et al. 2013). The Pfs25-VLP is in phase I trial and currently undergoing optimization (Fraunhofer Center for Molecular Biotechnology, USA). All these studies demonstrate the safety and immunogenicity of a plant-produced subunit vaccine in healthy adults and propose testing of novel candidate vaccines in human volunteers and further commercialization of these plant derived vaccines to combat severe diseases.

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Therapeutic proteins/ nutraceuticals Several recent studies have validated the potential of nutraceuticals to improve health and prevent chronic illnesses like cardiovascular, cancer, diabetes, obesity and multiple immune dysfunctions. Because of its nutritional benefits, safety and therapeutic role, the market for nutraceuticals is expanding every year. Plant derived products such as flavonoids, sterols, tannins and glucosinolates are the important source of such nutraceuticals and increasing the amount of these metabolites is the primary goal for commercialization. Recombinant glucocerebrosidase called as Elelyso (taliglucerase alfa) was produced in carrot cells for the treatment of Gaucher disease (Shaaltiel et al. 2007). Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD) resulting in the deposition of lipids in spleen, liver and other organs. Taliglucerase alfa produced by Protalix Biotherapeutics (Israel) and licensed by Pfizer (USA) was administered orally into human patients. Phase I clinical trials displayed presence of the enzyme in the blood stream of the patients with no side effects. Phase 2a, 2b and 3 trials are under way along with other pharmacokinetics studies. SemBioSys Genetics Inc., a Canadian biotechnology company produced an insulin molecule in safflower (Carthamus tinctorius) at commercially viable levels (Molony et al. 2009). The Phase I/II clinical trial conducted in Europe has demonstrated clinically significant results and safety profile comparable to pharmaceutical grade human insulin. The Company submitted the Investigational New Drug application to the US Food and Drug Administration and prepared for first clinical trial in 2008 and planned phase 3 trials in 2009-10. Recombinant gastric lipase for the treatment of pancreatic and cystic fibrosis was produced in maize seeds and entered phase II clinical trials in Europe in the year 2004 and currently marketed under the brand name Meripase (http://www.meristem- therapeutics.com). Maize modified with human lactoferrin (LacrominTM) was field tested by Biochem SA Company and by Meristem Therapeutics Company in France for the treatment of gastrointestinal infections (Samyn-Petit et al. 2001). Ventria Biosciences obtained approvals to carry out field trials (later disapproved by US Food and Drug Administration) of transgenic rice expressing lactoferrin and lysozyme intended for

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production of iron supplements and antimicrobial activity (Humprey et al. 2003; Hennegan et al. 2005; Mulvaney et al. 2011). Recombinant Human Intrinsic Factor useful for vitamin B12 absorption was engineered in Arabidopsis seeds and is marketed by Cobento Biotech AS (Fedosov et al. 2003). Field trials of low-nicotine tobacco varieties expressing human interleukin-10 (HIL-10) was carried out in Canada (Southern Crop Protection and Food Research Centre, Canada). HIL-10 which is used for the treatment of inflammatory bowel syndrome and Crohn's disease was produced in tobacco. Oral administrations of transgenic tobacco expressing HIL-10 reduce the severity of colitis by down-regulating TNF-alpha expression in IBD-susceptible IL-10(-/-) mice (Menassa et al. 2007). Biolex (USA) used duckweed plants to produce fibrinolytic drug for blood clots and lactoferrin for Hepatitis B & C virus disease which are under phase I and phase II trials respectively (Biolex Therapeutics Inc.). Planet Biotechnology’s (http://www.planetbiotechnology.com) α-galactosidase (for Fabry disease) produced in tobacco and SemBioSys’s (http://www.sembiosys.com) Apolipoprotein (for cardiovascular problems) produced in safflower were also approved for phase I trials and expected to reach market soon. Few of the therapeutic proteins such as virtonectin (Farmacule Bioindustries Pty Ltd) and thyroid stimulating hormone receptor (NEXGEN , Inc.) are used mostly for research purposes and available from the company. Epidermal growth factor produced in tobacco plants are supplied by Plantderma (http://plantaderma.es/es/), used in cosmetology for improving skin properties. Concluding remarks Production of antibodies , vaccines and other therapeutic proteins in plants shows great promise as recombinant proteins can be rapidly produced on large scale with low cost compared to other production systems. Most of the plant derived pharmaceuticals are in clinical trials and many are under investigation. Governing bodies in developed countries are involved in addressing and conceptualizing the manufacturing and application guidelines to ensure safety, efficacy and consistency of these plant derived pharmaceuticals. Over the last few decades biofarming in plants has made significant progress to retort several shortcomings in the production system and regulatory issues

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(biosafety and risk assessment) and reached a stage where it can challenge the established production technologies that use bacteria, yeast and mammalian cells. With the recent successful application of plant derived ebola vaccine ZMapp by Mapp Biopharmaceutical Inc., to combat the 2014 Ebola virus outbreak in Africa has once again evoked the interest of plant derived pharmaceuticals. Current scenarios have changed the mindset from using plants as edible vaccines to utilizing them as production platforms. Additionally, proteins produced in plants are properly folded and post-translationally modified which can further be formulated as vaccines and therapeutics. Bioreactor is the most promising approach to effectively use plant cells to produce heterologous proteins which involves low capital investment with enhanced product yield. Multinational companies like Protalix, Bayer, Icon Genetics, Meristem therapeutics are testimony to this. The studies carried out at several institutes and industries all over the world with a view to commercialize these products explain the tremendous potential of protein production in plants. Although the scientific community is aware of this fact, still an extensive work is warranted to establish plant production platform as unanimously accepted approach for vaccine and therapeutic protein production. Remarkable progress in molecular biology currently underway across the globe will definitely open new feasible options for the production of several bio pharmaceuticals. In this review, we highlighted the past attempts and recent progress in heterologous protein production in plants and its potential for commercial drug development and production. In conclusion, although plant systems as production platform face problems for public acceptance, undoubtedly it will definitely find its way into future as the best production platforms with constant support from the government, medical field, companies and scientific community. Conflict of Interest Authors declare that no conflict of interest exists. Acknowledgements SBG would like to thank UM-DAE Centre for Excellence in Basic Sciences, Mumbai for constant support and encouragement and Department of Science and Technology (Govt. of India) for financial support. VAB thanks Indian National Science Academy, New Delhi for Senior Scientist fellowship.

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References Aguirreburualde MSP, Gómez MC, Ostachuk A, Wolman F, Albanesi G, Pecora A, Odeon A, Ardila F, Escribano JM, Santos MJD, Wigdorovitz A (2013) Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants. Vet. Immunol. Immunopathol. 151:315–324. Aleinein RA, Schafer H, Wink M (2015) Rhizosecretion of the recombinant antimicrobial peptide ranalexin from transgenic tobacco hairy roots. Research & Reviews: Journal of Botanical Sciences: Phytopathology/ Genes & Diseases- S1:45- 55. Alvarez ML, Pinyerd HL, Crisantes JD, Rigano MM, Pinkhasov J, Walmsley AM, Mason HS, Cardineau GA (2006) Plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice. Vaccine. 24:2477-2490. Arakawa T, Chong DKX, Langridge WHR (1998a) Efficacy of food plant based oral cholera toxin B subunit vaccine. Nat. Biotechnol. 16:292-297. Arakawa T, Yu J, Chong DK, Hough J, Engen PC, Langridge WH (1998b) A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes.Nat. Biotechnol. 16:934-938. Arcalis E, Stadlmann J, Rademacher T, Marcel S, Sack M, Altmann F, Stoger E (2013) Plant species and organ influence the structure and subcellular localization of recombinant glycoproteins. Plant Mol. Biol. 83:105-117. Argyle DJ, Harris M, Lawrence C, McBride K, Barron R, McGillivray C, Onions DE (1998) Expression of feline recombinant interferon-γ in baculovirus and demonstration of biological activity. Vet. Immunol. Immunopathol. 64:97-105. Arlen PA, Singleton M, Adamovicz JJ, Ding Y, Davoodi-Semiromi A, Daniell H (2008) Effective plague vaccination via oral delivery of plant cells expressing F1-V antigens in chloroplasts. Infect. Immun. 76:3640-3650. Arora D, Khanna N (1996) Method for increasing the yield of properly folded recombinant human gamma interferon from inclusion bodies. J. Biotechnol. 52:127– 133. Baur A, Reski R, Gorr G (2005) Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens. Plant Biotechnol. J. 3:331–340. Benchabane M, Goulet C, Rivard D, Faye L, Gomord V, Michaud D (2008) Preventing unintended proteolysis in plant protein biofactories. Plant Biotechnol J. 6:633-648 Biemelt S, Sonnewald U, Galmbacher P, Willmitzer L, Muller M (2003) Production of human papillomavirus type 16 virus-like particles in transgenic plants. J. Virol. 77:9211-9220. Bock R, Khan MS (2004) Taming plastids for a green future. Trends in Biotechnol. 22:311-318. Borghi L (2010) Inducible gene expression systems for plants in plant developmental biology. Hennig L and Kohler C (eds.) Volume 655 of the series Methods in Molecular Biology pp. 65-75. Doi : 10.1007/978-1-60761-765-5_5. Bourdon V, Harvey A, Lonsdale DM (2001) Introns and their positions affect the translational activity of mRNA in plant cells. EMBO Rep. 2:394-398.

29

Bříza J, Pavingerova D, Vlasak J, Ludikova V, Niedermeierova H (2007) Production of human papillomavirus type 16 E7 oncoprotein fused with β-glucuronidase in transgenic tomato and potato plants. Biologia Plantarum. 51:268. Büttner-Mainik A, Parsons J, Jérôme H, Hartmann A, Lamer S, Schaaf A, Schlosser A, Zipfel PF, Reski R, Decker EL (2011) Production of biologically active recombinant human factor H in Physcomitrella. Plant Biotechnol. J. 9:373-383. Carter JE, Odumosu O, Langridge WHR (2010) Expression of a ricin toxin B subunit: insulin fusion protein in edible plant tissues. Molecular Biotechnology. 44:90-100. Castanon S, Marin MS, Martin-Alonso JM, Boga JA, Casais R, Humara JM, Ordas RJ, Parra F (1999) Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. J. Virol. 73:4452-4455. Castilho A, Neumann L, Daskalova S, Mason HS, Steinkellner H, Altmann F, Strasser R (2012) Engineering of sialylated mucin-type O-glycosylation in plants. J. Biol. Chem. 287:36518-36526. Chakraborty S, Chakraborty N, Datta A (2000) Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proc Natl Acad Sci USA 97:3724-3729. Chebolu S, Daniell H (2007) Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis. Plant Biotechnol J. 5:230-239. Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M (2013) Agroinfiltration as an effective and scalable strategy of for production of pharmaceutical proteins. Adv. Tech. Biol. Med. 1:103. Chen TL, Lin YL, Lee YL, Yang NS, Chan MT (2004) Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures. Transgenic Res. 13:499- 510. Chen X, Liu J (2011) Generation and immunogenicity of transgenic potato expressing the GP5 protein of porcine reproductive and respiratory syndrome virus. J. Virol. Methods. 173:153-158. Chichester JA, Jones RM, Green BJ, Stow M, Miao F, Moonsammy G, Streatfield SJ, Yusibov V (2012) Safety and immunogenicity of a plant-produced recombinant hemagglutinin-based influenza vaccine (HAI-05) derived from A/Indonesia/05/2005 (H5N1) influenza virus: a phase 1 randomized, double-blind, placebo-controlled, dose- escalation study in healthy adults. Viruses. 4:3227-3244. Chikwamba R, Cunnick J, Hathaway D, McMurray J, Mason H, Wang K (2002) A functional antigen in a practical crop: LT-B producing maize protects mice against Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT). Transgenic Res. 11:479-493. Chong DK, Langridge WH (2000) Expression of full-length bioactive antimicrobial human lactoferrin in potato plants.Transgenic Res. 9:71-78. Chong DK, Roberts W, Arakawa T, Illes K, Bagi G, Slattery CW, Langridge WH (1997) Expression of the human milk protein beta-casein in transgenic potato plants. Trangenic Res. 6:289-296. Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM, Dickey LF (2006)

30

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat. Biotechnol. 24:1591-1597. Cummings JF, Guerrero ML, Moon JE, Waterman P, Nielsen RK, Jefferson S, Gross FL, Hancock K, Katz JM, Yusibov V (2014) Safety and immunogenicity of a plant- produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza A (H1N1) pdm09 virus: a Phase 1 dose-escalation study in healthy adults. Fraunhofer USA Center for Molecular Biotechnology Study Group Vaccine. 32:2251- 2259. Cunha NB, Araujo ACG, Leite A, Murad AM, Vianna GR, Rech EL (2010) Correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds. Genet Mol Res 9:1163-1170. Daniell H, Chebolu S, Kumar S, Singleton M, Falconer R (2005) Chloroplast derived vaccine antigens and other therapeutic proteins. Vaccine. 2:1779-1783. Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant. Sci. 7:84–91. Daniell H, Lee SB, Panchal T, Wiebe P (2001) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Bio. 311:1001-1009. Daniell H, Streatfiled SJ, Wycoff K (2001) Medical molecular farming: Production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant, Sci. 6:219-226. Daskalova SM, Radder JE, Cichacz ZA, Olsen SH, Tsaprailis G, Mason H, Lopez LC (2010) Engineering of N. benthamiana L. plants for production of N- acetylgalactosamine-glycosylated proteins-towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation. BMC Biotechnology 10:62. Davoodi-Semiromi A, Schreiber M, Nalapalli S, Verma D, Singh ND, Banks RK, Chakrabarti D, Daniell H (2010) Dual chloroplast derived vaccine for cholera and malaria confers immunity in vaccinated mice. Plant Biotechnol. J. 8:223-242. Decker EL, Parsons J, Reski R (2014) Glyco-engineering for biopharmaceutical production in moss bioreactors 2. De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 19:71-74. De Jaeger G, Angenon G, Depicker A (2003) Exceptionally high heterologous protein levels in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Commun. Agric. Appl. Biol. Sci. 68:359-366. De Jaeger G, Scheffer S, Jacobs A, Zambre M, Zobell O, Goossens A, Depicker A, Angenn G (2002) Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nat. Biotechnol. 20:1265-1268. De Meyer T, Depicker A (2014) Trafficking of endoplasmic reticulum-retained recombinant proteins is unpredictable in Arabidopsis thaliana. Front. Plant Sci. 5:473. De Wilde C, Peeters K, Jacobs A, Peck I, Depicker A (2002) Expression of antibodies and Fab fragments in transgenic potato plants: a case study for bulk production in crop plants. Mol. Breed. 9:271-282

31

Dietzschold B, Gore M, Marchadier D, Niu HS, Bunschoten HM, Otvos L Jr, Wunner WH, Ertl HC, Osterhaus AD, Koprowski H (1990) Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine. J. Virol. 64:3804-3809. Digby MR, Lowenthal JW (1995) Cloning and expression of the chicken interferon- gamma gene. J. Interferon Res. 15:939-945. Dong J-L, Liang B-G, Jin Y-S, Zhang W-J, Wang T (2005) Oral immunization with pBsVP6-transgenic alfalfa protects mice against rotavirus infection. Virology. 339:153-163. Dugdale B, Mortimer CL, Kato M, James TA, Harding RM, Dale JL (2013) In Plant Activation: An inducible, hyperexpression platform for recombinant protein production in plants. Plant Cell. 25:2429-2443. Dutt M, Dhekney SA, Soriano L, Kandel R, Grosser JW (2014) Temporal and spatial control of gene expression in horticultural crops. Horticulture Res. 1:14047. Dyck MK, Lacroix D, Pothier F, Sirard M-A (2003) Making recombinant proteins in animals- different systems, different applications. Trends Biotechnol. 21:394-399. Farinas CS, Leite A, Miranda EA (2007) Recombinant human proinsulin from transgenic corn endosperm: solvent screening and extraction studies. Brazilian Journal of Chemical Engineering. 24:315-323. Fernandez-San Millan A, Mingo-Castel A, Miller M, Daniell H (2003) A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol J. 1:71-79 Fernández-San Millán A, Ortigosa SM, Hervás-Stubbs S, Corral-Martínez P, Seguí- Simarro JM, Gaétan J, Coursaget P, Veramendi J (2008) Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic. Plant Biotechnol. J. 6:427-441. Fischer R, Schumann D, Zimmermann S, Drossard J, Sack M, Schillberg S (1999) Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants. Eur J Biochem 262:810-816. Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J (2012) GMP issues for plant-derived recombinant proteins. Biotechnol Adv. 30:434-439. Fujiki M, Kaczmarczyk JF, Yusibov V, Rabindran S (2008) Development of a new cucumber mosaic virus-based plant expression vector with truncated 3a movement protein. Virology. 381:136-142. Fujiwara Y, Aiki Y, Yang L, Takaiwa F, Kosaka A, Tsuji NM, Shiraki K, Sekikawa K (2010) Extraction and purification of human interleukin-10 from transgenic rice seeds. Protein Expr. Purif. 72:125-130. Fukuzawa N, Tabayashi N, Okinaka Y, Furusawa R, Furuta K, Kagaya U, Matsumura T (2010) Production of biologically active Atlantic salmon interferon in transgenic potato and rice plants. J. Biosci Bioeng. 110:201-207. Gao M, Li Y, Xue X, Wang X, Long J (2012) Stable plastid transformation for high level recombinant protein expression: promises and challenges. J. Biomed. Biotechnol. 158232, doi:10.1155/2012/158232. Gatz C (1996) Chemically inducible promoters in transgenic plants. Curr. Opin. Biotechnol. 7:168-172.

32

Gils M, Kandzia R, Marillonnet S, Klimyuk V, Gleba Y (2005) High-yield production of authentic human growth hormone using a plant virus-based expression system. Plant Biotechnol. J. 3:613-620. Gleba Y, Klimyuk V, Marillonnet S (2005) Magnifection- a new platform for expressing recombinant vaccines in plants. Vaccine. 23:2042-2048. Gleba Y, Marillonnet S, Klimyuk V (2004) Engineering viral expression vectors for plants: the 'full virus' and the 'deconstructed virus' strategies. Curr. Opin. Plant Biol. 7:182-188. He X, Galpin JD, Tropak MB, Mahuran D, Haselhorst T, von Itzstein M, Kolarich D, Packer NH, Miao Y, Jiang L, Grabowski GA, Clarke LA, Kermode AR (2012) Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants. Glycobiology. 22:492-503. Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for production of recombinant proteins. Nat. Biotechnol. 22:1415-1422. Hennegan K, Yang D, Nguyen D, Wu L, Goding J, Huang J, Guo F, Huang N, Watkins SC (2005) Improvement of human lysozyme expression in transgenic rice grain by combining wheat (Triticum aestivum) puroindoline b and rice (Oryza sativa) Gt1 promoters and signal peptides.Transgenic Res. 14:583-592. Herman SR, Harding RM, Dale JL (2001) The banana actin 1 promoter drives near constitutive transgene expression in vegetative tissue of banana (Musa spp.). Plant Cell Rep. 20:525-530. Hernández A, López A, Ceballo Y, Rosabal L, Rosabal Y, Tiel K, Pérez M, González EM, Ramos O, Enríquez G (2013) High-level production and aggregation of hepatitis B surface antigen in transgenic tobacco seeds. Biotecnología Aplicada. 30:97-100. Hirai T, Kim Y-W, Kato K, Hiwasa-Tanase K, Ezura H (2011) Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter. Transgenic Res. 20:1285-1292. Hiwasa-Tanase K, Ezura H (2016) Molecular breeding to create optimized crops: from genetic manipulation to potential applications in plant factories. Front. Plant Sci. 7:539. Hogue RS, Lee JM, An GH (1990) Production of a foreign protein product with genetically modified plant-cells, Enzyme Microb. Technol. 12:533-538. Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, Flynn P, Register J, Marshall L, Bond D, Kulisek E, Kusnadi A, Evangelista R, Nikolov Z, Wooge C, Mehigh RJ, Hernan R, Kappel WK, Ritland D, Li C-P, Howard JA (1997) Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol. Breed. 3:291- 306. Huang TK, McDonald KA (2012) Bioreactor systems for in vitro production of foreign proteins using plant cell cultures. Biotechnol Adv. 30:398-409. Huang Z, Chen Q, Hjelm B, Arntzen C, Mason H (2009) A DNA replicon system for rapid high-level production of virus-like particles in plants. Biotechnol. Bioeng. 103:706-714 Huang Z, Santi L, LePore K, Kilbourne J, Arntzen CJ, Mason HS (2006) Rapid, high- level production of hepatitis B core antigen in plant leaf and its immunogenicity in mice. Vaccine. 24:2506-2513.

33

Hudson LC, Bost KL, Piller KJ (2011) Optimizing recombinant protein expression in soybean. In: Sudaric A, (ed.) Soybean - molecular aspects of breeding. InTech Open: InTech pp. 19-42. Huet Y, Ekouna JP, Caron A, Mezreb K, Boitel-Conti M, Guerineau F (2014) Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots. Biotechnol Lett. 36:181-190. Humphrey B, Haung N, Klasing K (2002) Rice expressing lactoferrin and lysozyme has antibiotic like properties when fed to chicks. J. Nutr. 132:1214-1218. Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar F, Boyer HW (1977) Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198:1056-1063. Jeong SW, Jeong WJ, Woo JW, Choi DW, Park YI, Liu JR (2004) Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for selection and tracking of plastid-transformed cells in tobacco. Plant Cell Rep. 22:747- 751. Jiménez-Bremont JF, Ordonez Acevedo LG, De León Rodríguez A (2008) Periplasmic expression and recovery of human interferon gamma in Escherichia coli. Protein Expr. Purif. 59:169-174. Kapila J, DeRycke R, VanMontagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci.122:101-108. Kapusta J, Modelska A, Figlerowicz M, Pniewski T, Letellier M, Lisowa O, Yusibov V, Koprowski H, Plucienniczak A, Legocki AB (1999) A plant-derived edible vaccine against hepatitis B virus. FASEB J. 13:1796-1799. Karaman S, Unnick J, Wang K (2012) Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea.Plant Cell Rep. 31:527-537. Kim TG, Baek MY, Lee EK, Kwon TH, Yang MS (2008) Expression of human growth hormone in transgenic rice cell suspension culture. Plant Cell Rep. 27:885-891. Kim TG, Lee HJ, Jang YS, Shin YJ, Kwon TH, Yang MS (2008) Co-expression of proteinase inhibitor enhances recombinant human granulocyte-macrophage colony stimulating factor production in transgenic rice cell suspension culture. Protein Expr. Purif. 61:117-121. Kim TW, Goo YM, Lee CH, Lee BH, Bae JM, Lee SW (2009) The sweet potato ADP- glucose pyrophosphorylase gene (ibAGP1) promoter confers high-level expression of the GUS reporter gene in the potato tuber. C. R. Biol. 332:876-885. Kim Y-S, Kang T-J, Jang Y-S, Yang M-S (2005) Expression of neutralizing epitope of porcine epidemic diarrhea virus in potato plants. Plant Cell Tiss Org Cult. 82:125-130. Kinkema M, Geijskes RJ, Shand K, Coleman HD, De Lucca PC, Palupe A, Harrison MD, Jepson HI, Dale JL, Sainz MB (2014) An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Mol. Biol. 84:443-454. Kircheis R, Halanek N, Koller I, Jost W, Schuster M, Gorr G, Hajszan K, Nechansky A (2012) Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314. MAbs 4:532–541.

34

Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS, Thanavala Y (2001) Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc. Natl. Acad. Sci. USA. 98:11539-11544. Koya V, Moayeri M, Leppla SH, Daniell H (2005) Plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect. Immun. 73:8266-8274. Kristina LEDL, Luthar Z (2016) Production of vaccines for treatment of infectious diseases by transgenic plants. Acta Agriculturae Slovenica 107:191-217. Kumar GB, Ganapathi TR, Revathi CJ, Srinivas L, Bapat VA (2005) Expression of hepatitis B surface antigen in transgenic banana plants. Planta. 222:484-493. Lai H, He J, Engle M, Diamond MS, Chen Q (2012) Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce. Plant biotechnol. J. 10:95-104. Lakshmi PS, Verma D, Yang X, Lloyd B, Daniell H (2013) Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro. PLoS ONE 8:e54708. Lallemand J, Bouche F, Desiron C, Stautemas J, de Lemos Esteves F, Perilleux C, Tocquin P (2015) Extracellular peptidase hunting for improvement of protein production in plant cells and roots. Front. Plant Sci. 6:37. doi: 10.3389/fpls.2015.00037. Lamphear BJ, Jilka JM, Kesl L, Welter M, Howard JA, Streatfield SJ (2004) A corn- based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine. Vaccine. 22:2420-2424. Landry N, Pillet S, Favre D, Poulin JF, Trépanier S, Yassine-Diab B, Ward BJ (2014) Influenza virus-like particle vaccines made in Nicotiana benthamiana elicit durable, poly-functional and cross-reactive T cell responses to influenza HA antigens. Clin. Immunol. 154:164-177. Landry N, Ward BJ, Trépanier S, Montomoli E, Dargis M, Lapini G, Vézina LP (2010) Preclinical and clinical development of plant-made virus-like particle vaccine against avian H5N1 influenza. PLoS ONE. 5:e15559. Lee C, Kim HH, Choi KM, Chung KW, Choi YK, Jang MJ, Kim TS, Chung NJ, Rhie HG, Lee HS, Sohn Y, Kim H, Lee SJ, Lee HW (2011) Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus. Malar. J. 10:106. Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun MO, Daniell H (2003) Accumulation of a trehalose within transgenic chloroplasts confers drought tolerance. Mol. Breed. 11:1-13 Lee SJ, Park CI, Park MY, Jung HS, Ryu WS, Lim SM, Tan HK, Kwon TH, Yang MS, Kim DI (2007) Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures. Protein Expr. Purif. 51:293-302. Lee S-Y, Hur W, Cho GH, Kim D-I (2001) Cultivation of transgenic Nicotiana tabacum suspension cells in bioreactors for the production of mGM-CSF. Biotechnol. Bioprocess Eng. 6:72-74. Leelavathi S, Reddy VS (2003) Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol. Breed. 11:49-58.

35

Lenzi P, Scotti N, Alagna F, Tornesello ML, Pompa A, Vitale A, De Stradis A, Monti L, Grillo S, Buonaguro FM, Maliga P, Cardi T (2008) Translational fusion of chloroplast- expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco. Transgenic Res. 17:1091-1102. Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q (2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J. Vis. Exp. 77:e50521. doi:10.3791/50521. Li JT, Fei L, Mou ZR, Wei J, Tang Y, He HY, Wang L, Wu YZ (2006) Immunogenicity of a plant-derived edible rotavirus subunit vaccine transformed over fifty generations. Virology. 356:171-178. Li X-G, Zeng Q-C, Chen S-B, Xu J-W, Chang T-J, Zhu Z (2002) Influence of matrix attachment regions from maize on transgene expression level in tobacco. Acta Bot. Sinica, 44:804-808. Lico C, Chen Q, Santi L (2008) Viral vectors for production of recombinant proteins in plants. J. Cell. Physiol., 216:366-377. Limkul J, Misaki R, Kato K, Fujiyama K (2015) The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants. Plant Sci. 240:41-49. Ling HY, Pelosi A, Walmsley AM (2010) Current status of plant made vaccines for veterinary purposes. Expert Rev. Vaccines 9:971-982 Liu C, Towler MJ, Medrano G, Cramer CL, Weathers PJ (2009) Production of mouse interleukin-12 is greater in tobacco hairy roots grown in a mist reactor than in an airlift reactor. Biotechnol Bioeng 102:1074-1086. Liu YK, Huang LF, Ho SL, Liao CY, Liu HY, Lai YH, Yu SM, Lu CA (2012) Production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture. Biotechnol Bioeng 109:1239-1247. Loc NH, Long DT, Kim T-G, Yang M-S (2014) Expression of Escherichia coli heat- labile enterotoxin B subunit in transgenic tomato (Solanum lycopersicum L.) fruit. Czech J. Genet. Plant Breed., 50:26-31. López EG, Ramírez EG, Gúzman OG, Calva GC, Ariza-Castolo A, Pérez-Vargas J, Rodríguez HG (2014) MALDI-TOF characterization of hGH1 produced by hairy root cultures of Brassica oleracea var. italica grown in an airlift with mesh bioreactor. Biotechnol Prog. 30:161-171. Lou XM, Yao QH, Zhang Z, Peng RH, Xiong AS, Wang HK (2007) Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants. Clin. Vaccine Immunol. 14:464-469. Ma JK, Drossard J, Lewis D, Altmann F, Boyle J, Christou P, Cole T, Dale P, van Dolleweerd CJ, Isitt V, Katinger D, Lobedan M, Mertens H, Paul MJ, Rademacher T, Sack M, Hundleby PA, Stiegler G, Stoger E, Twyman RM, Vcelar B, Fischer R (2015) Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants. Plant Biotechnol J. 13:1106-1120. Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, Hein MB, Lehner T (1998) Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat. Med. 4:601-606.

36

Ma Y, Lin SQ, Gao Y, Li M, Luo WX, Zhang J, Xia NS (2003) Expression of ORF2 partial gene of hepatitis E virus in tomatoes and immunoactivity of expression products. World J. Gastroenterol. 9:2211-2215. Madesis P, Osathanunkul M, Georgopoulou U, Gisby MF, Mudd EA, Nianiou I, Tsitoura P, Mavromara P, Tsaftaris A, Day A (2010) A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera. J. Biotechnol.145:377-386. Mahmoud K (2007) Recombinant protein production: strategic technology and a vital research tool. Res. J. Cell. Mol. Biol. 1:9-22. Maliga P (1993) Towards plastid transformation in flowering plants. Trends Biotechnol. 11:101-107. Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc. Natl. Acad. Sci. USA. 101:6852–6857. Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK, Arntzen CJ (1996) Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc. Nat. Acad. Sci. USA. 93:5335-5340. Mason HS, Haq TA, Clements JD, Arntzen CJ (1998) Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine. 16:1336-1343. McDonald KA, Hong LM, Trombly DM, Xie Q, Jackman AP (2005) Production of human alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor. Biotechnol. Prog. 21:728-734. McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Dietzschold B, Koprowski H, Michaels FH (1995) Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnology. 13:1484-1487. Menassa R, Du C, Yin ZQ, Ma S, Poussier P, Brandle J, Jevnikar AM (2007) Therapeutic effectiveness of orally administered transgenic low-alkaloid tobacco expressing human interleukin-10 in a mouse model of colitis. Plant Biotechnol J. 5:50-59. Menassa R, Zhu H, Karatzs CN, Lazaris A, Richman A, Brandle J (2004) Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production. Plant Biotechnol J. 2:431-438. Michael PA, Huang T-K, Dandekar AM, Falk BW, McDonald KA (2008) High-level transient production of a heterologous protein in plants by optimizing induction of a chemically inducible viral amplicon expression system. Biotechnology Prog. 23:1277- 1285. Min SR, Kim JW, Jeong WJ, Lee YB, Liu JR (2009) Development of transgenic sweet potato producing human lactoferrin. http://agris.fao.org/agris- search/search.do?recordID=KR2010002888 (Accessed on 28th January 2016) Modelska A, Dietzschold B, Sleysh N, Fu ZF, Steplewski K, Hooper DC, Koprowski H, Yusibov V (1998) Immunization against rabies with plant-derived antigen. Proc. Natl. Acad. Sci. USA. 95:2481-2485. Moeller L, Taylor-Vokes R, Fox S, Gan Q, Johnson L, Wang K (2010) Wet-milling transgenic maize seed for fraction enrichment of recombinant subunit vaccine. Biotechnol Prog. 26:458-465.

37

Mohammadzadeh S, Roohvand F, Ajdary S, Ehsani P, Salmanian AH (2015) Heterologous expression of hepatitis C virus core protein in oil seeds of Brassica napus L. Jundishapur J. Microbiol. 8:e25462. Molony MM, Boothe J, Keone R, Nykiforuk C, Van Rooijen G (2009) Method for production of insulin in plants. US Patent US7547821B2. Moravec T, Schmidt MA, Herman EM, Woodford-Thomas T (2007) Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine. Vaccine. 25:1647-1657. Morello L, Breviario D (2008) Plant spliceosomal introns: not only cut and paste. Curr. Genomics. 9:227-238. Mulvaney D, Krupnik T, Koffler K (2011) Transgenic rice evaluated for risks to marketability. Cal. Ag. 65:161-167. doi: 10.3733/ca.E.v065n03p161. Musiychuk K, Stephenson N, Bi H, Farrance CE, Orozovic G, Brodelius M, Brodelius P, Horsey A, Ugulava N, Shamloul AM, Mett V, Rabindran S, Streatfield SJ, Yusibov V (2007) A launch vector for the production of vaccine antigens in plants. Influenza Other Respir. Viruses. 1:19-25. Nagaya S, Kawamura K, Shinmyo A, Kato K (2010) The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol. 51:328-332. Nagels B, Van Damme EJ, Pabst M, Callewaert N, Weterings K (2011) Production of complex multiantennary N-glycans in Nicotiana benthamiana plants. Plant Physiol. 155:1103-1112. Naqvi S, Ramessar K, Farré G, Sabalza M, Miralpeix B, Twyman RM, Capell T, Zhu C, Christou P (2011) High-value products from transgenic maize. Biotechnol. Adv. 29:40-53. Nevalainen H, Peterson R (2014) Making recombinant proteins in filamentous fungi- are we expecting too much? Front. Microbiol. 5:75. doi:10.3389/fmicb.2014.00075. Niederkrüger H, Dabrowska-Schlepp P, Schaaf A (2014) Suspension culture of plant cells under phototrophic conditions. In Industrial Scale Suspension Culture of Living Cells Meyer H-P and Schmidhalter DR (eds), pp. 259-292. Oakes JL, Bost KL, Piller KJ (2009) Stability of a soybean seed-derived vaccine antigen following long-term storage, processing and transport in the absence of a cold chain. J. Sci. Food Agric. 89:2191-2199. Oey M, Lohse M, Kreikemeyer B, Bock R (2009) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J. 57:436-445. Ohya K, Matsumura T, Ohashi K, Onuma M, Sugimoto C (2001) Expression of two subtypes of human IFN-alpha in transgenic potato plants. J. Interferon Cytokine Res. 21:595-602. Opabode JT, Akinyemiju OA (2015) Tissue- and organ-specific promoters for expression of heterologous genes in transgenic cassava (Manihot Esculenta Crantz) plants. Gene Technol. 4:125. doi:10.4172/2329-6682.1000125. Orellana-Escobedo L, Rosales-Mendoza S, Romero-Maldonado A, Parsons J, Decker EL Monreal-Escalante E, Moreno-Fierros L, Reski R (2015) An Env-derived multi- epitope HIV chimeric protein produced in the moss Physcomitrella patens is immunogenic in mice. Plant Cell Rep. 34:425-433.

38

Outchkourov NS, Rogelj B, Strukelj B, Jongsma MA (2003) Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production. Plant Physiol. 133:379-390. Paz de la Rosa G, Monroy-Garcia A, Mora-Garcia ML, Pena CG, Hernandez-Montes J, Weiss-Steider B, Gomez-Lim MA (2009) An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice. Virol. J. 6:2. Permyakova NV, Zagorskaya AA, Belavin PA, Uvarova EA, Nosareva OV, Nesterov AE, Novikovskaya AA, Zav’yalov EL, Moshkin MP, Deineko EV (2015) Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice. BioMed Res. Int. 2015:417565. doi:10.1155/2015/417565. Pillai O, Panchagnula R (2001) Insulin therapies-past, present and future. Drug Discov Today. 6:1056–1061. Pillay P, Schlüter U, van Wyk S, Kunert KJ, Vorster BJ (2014) Proteolysis of recombinant proteins in bioengineered plant cells. Bioengineered 5:15-20. Piron R, De Koker S, De Paepe A, Goossens J, Grooten J, Nauwynck H, Depicker A (2014) Boosting in planta production of antigens derived from the porcine reproductive and respiratory syndrome virus (PRRSV) and subsequent evaluation of their immunogenicity. PLoS ONE 9:e91386. Pogue GP, Lindbo JA, Garger SJ, Fitzmaurice WP (2002) Making an ally from an enemy: plant virology and the new agriculture. Annu. Rev. Phytopathol. 40:45-74. Potera C (1999) EPIcyte produces antibodies in plants- Plantibodies step in to fulfill the promise of mabs. Genetic Engineering News. 19:22-23. Pujol M, Ramírez NI, Ayala M, Gavilondo JV, Valdés R, Rodríguez M, Brito J, Padilla S, Gómez L, Reyes B, Peral R, Pérez M, Marcelo JL, Milá L, Sánchez RF, Páez R, Cremata JA, Enríquez G, Mendoza O, Ortega M, Borroto C (2005) An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification. Vaccine. 23:1833-1837. Qiu X, Wong G, Audet J, Bello A, Fernando L, Alimonti JB, Fausther-Bovendo H, Wei H, Aviles J, Hiatt E, Johnson A, Morton J, Swope K, Bohorov O, Bohorova N, Goodman C, Kim D, Pauly MH, Velasco J, Pettitt J, Olinger GG, Whaley K, Xu B, Strong JE, Zeitlin L, Kobinger GP (2014) Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. 514:47-53. Rai M, Padh H (2001) Expression systems for production of heterologous proteins. Curr. Sci. 80:1121–1128. Rancé I, Norre F, Gruber V, Theisen M (2002) Combination of viral promoter sequences to generate highly active promoters for heterologous therapeutic protein over- expression in plants. Plant Sci. 162:833-842. Renuga G, Saravanan R, Babu Thandpani A, Arumugam KR (2010) Expression of Cholera toxin B subunit in banana callus culture. J.Pharm. Sci. & Res. 2:26-33. Reski R, Parsons J, Decker EL (2015) Moss made pharmaceuticals: from bench to bedside. Plant Biotechnol. J. 13:1191-1198. Richter LJ, Thanavala Y, Arntzen CJ, Mason HS‐ (2000) Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotechnol. 18:1167-1171. Ritala A, Häkkinen ST, Schillberg S (2014) Molecular in plants and plant cell cultures: a great future ahead? Pharm. Bioprocess. 2:223-226.

39

Rosales-Mendoza S, Soria-Guerra RE, Lopez-Revilla R, Moreno-Fierros L, Alpuche- Solis AG (2008) Ingestion of transgenic carrots expressing the Escherichia coli heat- labile enterotoxin B subunit protects mice against cholera toxin challenge. Plant Cell Rep. 27:79-84. Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, Han Y, Alpuche-Solís AG, Korban SS (2011) Transgenic carrot tap roots expressing an immunogenic F1-V fusion protein from Yersinia pestis are immunogenic in mice.J Plant Physiol. 168:174-180. Rosales-Mendoza S, Tello-Olea MA (2015) Carrot cells: a pioneering platform for biopharmaceuticals production. Mol. Biotechnol. 57:219-232. Rosenberg Y, Sack M, Montefiori D, Forthal D, Mao L, Hernandez -Abanto S, Urban L, Landucci G, Fischer R, Jiang X (2013) Rapid high-level production of functional HIV broadly neutralizing monoclonal antibodies in transient plant expression systems. PLoS ONE 8:e58724. Ruhlman T, Verma D, Samson N, Daniell H (2010) The role of heterologous chloroplast sequence elements in transgene integration and expression. Plant Physiol. 152:2088- 2104. Rupa P, Monedero V, Wilkie BN (2008) Expression of bioactive porcine interferon gamma by recombinant Lactococcus lactis. Vet. Microbiol. 129:197-202. Rybicki EP (2009) Third International Conference on PlantBased Vaccines and Antibodies. Expert. Rev. Vaccines. 8:1151-1155. Rybicki EP (2010) Plant-made vaccines for humans and animals. Plant Biotechnol J. 8: 620-637. Sainsbury F, Benchabane M, Goulet M-C, Michaud D (2012) Multimodal protein constructs for herbivore insect control.Toxins 4:455-475. Saldaña S, Esquivel Guadarrama F, Olivera Flores TdeJ, Arias N, López S, Arias C (2006) Production of rotavirus-like particles in tomato (Lycopersicon esculentum L.) fruit by expression of capsid proteins VP2 and VP6 and immunological studies. Viral Immunol. 19:42-53. Samyn-Petit B, Gruber V, Flahaut C, Wajda-Dubos JP, Farrer S, Pons A, Desmaizieres G, Slomianny MC, Theisen M, Delannoy P (2001) N-glycosylation potential of maize: The human lactoferrin used as a model. Glycoconj J. 18:519-527. Sandhu JS, Krasnyanski SF, Domier LL, Korban SS, Osadjan MD, Buetow DE (2000) Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response. Transgenic Res. 9:127- 135. Santi L, Batchelor L, Huang Z, Hjelm B, Kilbourne J, Arntzen CJ, Chen Q, Mason HS (2008) An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles. Vaccine. 26:1846-1854. Santi L, Giritch A, Roy CJ, Marillonnet S, Klimyuk V, Gleba Y, Webb R, Arntzen CJ, Mason HS (2006) Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system. Proc. Natl. Acad. Sci. USA. 103:861-866. Sawahel WA (2002) The production of transgenic potato plants expressing human alpha- interferon using lipofectin-mediated transformation. Cell. Mol. Biol. Lett. 7:19-29. Scheller J, Guhrs K-H, Grosse F, Conrad U (2001) Production of spider silk proteins in tobacco and potato. Nat. Biotechnol. 19:573-577.

40

Schultz U, Rinderle C, Sekellick MJ, Marcus PI, Staeheli P (1995) Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active. Eur. J. Biochem. 229:73-76. Sekellick MJ, Ferrandino AF, Hopkins DA, Marcus PI (1994) Chicken interferon gene: cloning, expression, and analysis. J. Interferon Res. 14:71-79. Séveno M, Bardor M, Paccalet T, Gomord V, Lerouge P, Faye L (2004) Glycoprotein sialylation in plants?. Nature Biotech. 22:1351-1352. Shaaltiel Y, Bartfeld D, Hashmueli S, Baum G, Brill-Almon E, Galili G, Dym O, Boldin- Adamsky SA, Silman I, Sussman JL, Futerman AH, Aviezer D (2007) Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. Plant Biotechnol J. 5:579-590. Shao HB, He DM, Qian KX, Shen GF, Su ZL (2008) The expression of classical swine fever virus structural protein E2 gene in tobacco chloroplasts for applying chloroplasts as bioreactors. C. R. Biol. 331:179-184. Shchelkunov SN, Salyaev RK, Pozdnyakov SG, Rekoslavskaya NI, Nesterov AE, Ryzhova TS, Sumtsova VM, Pakova NV, Mishutina UO, Kopytina TV, Hammond RW (2006) Immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis B and human immunodeficiency viruses. Biotechnol. Lett. 28:959-967. Shulga NY, Rukavtsova EB, Krymsky MA, Borisova VN, Melnikov VA, Bykov VA, Buryanov YI (2004) Expression and characterization of hepatitis B surface antigen in transgenic potato plants. Biochemistry(Mosc) 69:1158-1164. Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ, Hoekema A (1990) Production of correctly processed human serum albumin in transgenic plants. Biotechnology. 8:217-221. Sijmons PC, Dekker BMM, Schrammeijer B, Verwoerd TC, Vandenelzen PJM, Hoekema A (1990) Production of correctly processed human serumalbumin in transgenic plants, BioTechnology 8:217-221. Singh AK, Verma SS (2010) Plastid transformation in eggplant (Solanum melongena L.). Transgenic Res. 19:113-119. Soh HS, Chung HY, Lee HH, Ajjappala H, Jang K, Park J-H, Sim J-S, Lee GY, Lee HJ, Han YH, Lim KW, Choi I, Chung IS, Hahn B-S (2015) Expression and functional validation of heat-labile enterotoxin B (LTB) and cholera toxin B (CTB) subunits in transgenic rice (Oryza sativa). SpringerPlus 4:148. Song KD, Lillehoj HS, Choi KD, Zarlenga DJ, Han Y (1997) Expression and functional characterization of recombinant chicken interferon-gamma. Vet. Immunol. Immunopathol. 58:321-333. Soria-Guerra RE, Rosales-Mendoza S, Marquez-Mercado C, Lopez-Revilla R, Castillo- Collazo R, Puche-Solis AG (2007) Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene. Plant Cell Rep. 26:961-968. Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18:333-338.

41

Stefanov I, Illubaev S, Feher A, Margoczi K, Dudits D (1991) Promoter and genotype dependent transient expression of a reporter gene in plant protoplasts. Acta Biologica Hungarica. 42:323-330. Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y, Christou P, Fischer R (2000) Cereal crops as viable production and storage systems for pharmaceutical ScFv antibodies. Plant. Mol. Biol. 42:583-590. Strasser R, Altmann F, Mach L, Glössl J, Steinkellner H (2004) Generation of Arabidopsis thaliana plants with complex N-glycans lacking β1,2-linked xylose and core α1,3-linked fucose. FEBS Lett. 561:132–136. Strasser R, Castilho A, Stadlmann J, Kunert R, Quendler H, Gattinger P, Jez J, Rademacher T, Altmann F, Mach L, Steinkellner H (2009) Improved virus neutralization by plant-produced anti-HIV antibodies with a homogeneous β1,4- galactosylated N-glycan profile. J. Biol. Chem. 284:20479-20485. Strasser R, Altmann F, Steinkellner H (2014) Controlled glycosylation of plant-produced recombinant proteins. Curr. Opin. Biotechnol. 30:95-100. Streatfield SJ (2005) Oral candidates produced and delivered in plant material. Immunol. Cell Biol. 83:257-262. Streatfield SJ, Bray J, Love RT, Horn ME, Lane JR, Drees CF, Egelkrout EM, Howard JA (2010) Identification of maize embryo preferred promoters suitable for high-level heterologous protein production, GM Crops. 1:162-172. Streatfield SJ, Mayor JM, Barker DK, Brooks C, Lamphear BJ, Woodard SL, Beifuss KK, Vicuna DV, Massey LA, Horn ME, Delaney DE, Nikolov ZL, Hood EE, Jilka JM, Howard JA (2002) Development of an edible subunit vaccine in corn against enterotoxigenic strains of Escherichia coli. In Vitro Cell Dev. Biol. Plant. 38:11-17. Su CF, Kuo IC, Chen PW, Huang CH, Seow SV, Chua KY, Yu SM (2012) Characterization of an immunomodulatory Derp2-FIP-fve fusion protein produced in transformed rice suspension cell culture. Transgenic Res. 21:177-192. Su X, Schmitz G, Zhang M, Mackie RI, Cann IK (2012) Heterologous gene expression in filamentous fungi. Adv. Appl. Microbiol. 81:1-61. Sun YK, Wang YF, Zhi HD, Liu SW, Wang M, Tong GZ (2005) Construction and characterization of a recombinant Fowl pox virus expressing chicken type II interferon. Chin. J. Agr. Biotechnol. 2:143-148. Tabayashi N, Matsumura T (2014) Forefront study of plant biotechnology for practical use: development of oral drug for animal derived from transgenic strawberry. Soc. Biotechnol. J. Japn. 92:537-539. Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen CJ (1998) Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat Med. 4:607-609. Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM, Arntzen CJ (2000) Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. J. Infect Dis. 182:302-305. Tacket CO, Pasetti MF, Edelman R, Howard JA, Streatfield S (2004) Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn. Vaccine. 22:4385- 4389.

42

Takehara K, Kamikawa M, Ohnuki N, Nagata T, Nakano A, Amaguchi D, Yokomizo Y, Nakamura M (2002) High level expression of C-terminal truncated recombinant chicken interferon-γ in baculovirus vector system. J.Vet. Med. Sci. 2:95-100. Tekoah Y, Shulman A, Kizhner T, Ruderfer I, Fux L, Nataf Y, Bartfeld D, Ariel T, Gingis-Velitski S, Hanania U, Shaaltiel Y (2015) Large-scale production of pharmaceutical proteins in plant cell culture- the Protalix experience. Plant Biotech J. 13:1199-1208. Thanavala Y, Mahoney M, Pal S, Scott A, Richter L, Natarajan N, Goodwin P, Arntzen CJ, Mason HS (2005) Immunogenicity in humans of an edible vaccine for hepatitis B. Proc. Natl. Acad. Sci. USA. 102:3378-3382. Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N, Ytterberg J, Van Wijk KJ, Dougan G, Maliga P (2003) Expression of tetanus toxin fragment C in tobacco chloroplasts. Nucleic Acids Res. 31:1174-1179. Tremblay R, Feng M, Menassa R, Huner NP, Jevnikar AM, Ma S (2011) High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems. Transgenic Res. 20:345-356. Triguero A, Cabrera G, Rodríguez M, Soto J, Zamora Y, Pérez M, Wormald MR, Cremata JA (2011) Differential N glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice.‐ Plant Biotechnol. J. 9:1120-1130. Van Droogenbroeck B, Cao J, Stadlmann J, Altmann F, Colanesi S, Hillmer S, Robinson DG, Van Lerberge E, Terryn N, Van Montagu M, Liang M, Depicker A, De Jaeger G (2007) Aberrant localization and underglycosylation of highly accumulating single- chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proc. Natl. Acad. Sci. USA. 104:1430-1435. Verma D, Daniell H (2007) Chloroplast vector systems for biotechnology applications. Plant Physiol. 145:1129–1143. Vianna G, da Cunha N, Rech E (2011) Expression and accumulation of heterologous molecules in the protein storage vacuoles of soybean seeds. Protocol Exchange. doi:10.1038/protex.2011.206 Vitale A, Denecke J (1999) The endoplasmic reticulum- gateway of the secretory pathway. Plant Cell. 11:615-628. Wagner B, Fuchs H, Adhami F, Ma Y, Scheiner O, Breiteneder H (2004) Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana. Methods. 32:227-234. Wakasa Y, Takagi H, Hirose S, Yang L, Saeki M, Nishimura T, Kaminuma O, Hiroi T, Takaiwa F (2013) Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens, Cry j 1 and Cry j 2, against Japanese cedar pollinosis. Plant Biotechnol J. 11:66-76. Wakasa Y, Takagi H, Watanabe N, Kitamura N, Fujiwara Y, Ogo Y, Hayashi S, Yang L, Ohta M, Thet Tin WW, Sekikawa K, Takano M, Ozawa K, Hiroi T, Takaiwa F (2015) Concentrated protein body product derived from rice endosperm as an oral tolerogen for allergen-specific immunotherapy- A new mucosal vaccine formulation against japanese cedar pollen allergy. PLoS ONE 10:e0120209. Walsh G (2014) Biopharmaceutical benchmarks. Nat. Biotechnol. 32:992-1000.

43

Ward BJ, Landry N, Trépanier S, Mercier G, Dargis M, Couture M, D'Aoust MA, Vézina LP (2014) Human antibody response to N-glycans present on plant-made influenza virus-like particle (VLP) vaccines. Vaccine. 32:6098-6106. Warzecha H, Mason HS, Lane C, Tryggvesson A, Rybicki E, Williamson A-L, Clements JD, Rose RC (2003) Oral immunogenicity of human papillomavirus-like particles expressed in potato. J. Virol. 77:8702-8711. Wigdorovitz A, Carrillo C, Dus Santos MJ, Trono K, Peralta A, Gomez MC, Rios RD, Franzone PM, Sadir AM, Escribano JM, Borca MV (1999) Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology. 255:347-353. Woods RR, Geyer BC, Mor TS (2008) Hairy-root organ cultures for the production of human acetylcholinesterase. BMC Biotechnology. 20088:95. Wu D, Murakami K, Liu N, Inoshima Y, Yokoyama T, Kokuho T, Inumaru S, Matsumura T, Kondo T, Nakano K, Sentsui H (2002) Expression of biologically active recombinant equine interferon-γ by two different baculovirus gene expression systems using insect cells and silkworm larvae. Cytokine. 2:63-69. Wu Y, Zhao D, Song L, Xu W (2009) Heterologous expression of synthetic chicken IFN- γ in transgenic tobacco plants. Biologia 64:1115–1122. Wu YJ, Zhao DG, Song L, Xu WZ (2008) Construction of plant expression vector consisting of ChIFN-γ gene and its transient expression. J Yunnan Univ (Nat Sci Ed) 6:630–635. Wu YZ, Li JT, Mou ZR, Fei L, Ni B, Geng M, Jia ZC, Zhou W, Zou LY, Tang Y (2003) Oral immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA. Virology. 313:337-342. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22:1393-1398. Yang L, Tada Y, Yamamoto MP, Zhao H, Yoshikawa M, Takaiwa F (2006) A transgenic rice seed accumulating an anti-hypertensive peptide reduces the blood pressure of spontaneously hypertensive rats. FEBS Lett. 580:3315-3320. Yap YK, Smith DR (2010) Strategies for the plant-based expression of dengue subunit vaccines. Biotechnol Appl Biochem. 57:47-53. Yashiro K, Lowenthal JW, O’Neil TE, Ebisu S, Takagi H, Moore RJ (2001) High-level production of recombinant chicken interferon-γ by Brevibacillus choshinensis. Protein Expr. Purif. 23:113-120. Youm JW, Jeon JH, Kim H, Min SR, Kim MS, Joung H, Jeong WJ, Kim HS (2010) High-level expression of a human β-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice. Transgenic Res. 19:1099-1108. Yu J, Langridge WH (2001) A plant-based multicomponent vaccine protects mice from enteric diseases. Nat. Biotechnol. 19:548-552. Yusibov V, Hooper DC, Spitsin SV, Fleysh N, Kean RB, Mikheeva T, Deka D, Karasev A, Cox S, Randall J, Koprowski H (2002) Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. Vaccine. 20:3155-3164. Yusibov V, Streatfield SJ, Kushnir N (2011) Clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond. Hum Vaccin. 7:313- 321.

44

Zhang X, Buehner NA, Hutson AM, Estes MK, Mason HS (2006) Tomato is a highly effective vehicle for expression and oral immunization with Norwalk virus capsid protein. Plant Biotechnol. J. 4:419-432. Zhou JY, Wu JX, Cheng LQ, Zheng XJ, Gong H, Shang SB, Zhou EM (2003) Expression of immunogenic S1 glycoprotein of infectious bronchitis virus in transgenic potatoes. J. Virol. 77:9090-9093. Zhou YX, Lee MY, Ng JM, Chye ML, Yip WK, Zee SY, Lam E (2006) A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice. World J. Gastroenterol. 12:306-312.

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Fig 1.Approaches for heterologous protein production in plants.

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Fig 2.Schematic representation of heterologous proteins expression in different plant parts.

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Fig 3.Schematic representation of stages involved in heterologous protein production in plants along with factors affecting final product output.

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Transgenic plants as green factories for vaccine production

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Vol. 12(43), pp. 6147-6158, 23 October, 2013 DOI: 10.5897/AJB2012.2925 ISSN 1684-5315 ©2013 Academic Journals African Journal of Biotechnology http://www.academicjournals.org/AJB

Review

Transgenic plants as green factories for vaccine production

B. Vinod Kumar1, T. K. Raja2, M. R. Wani3, S. A. Sheikh3, M. A. Lone3, Gowher Nabi4, M. M. Azooz5, Muhammad Younis6, Maryam Sarwat7 and Parvaiz Ahmad8*

1Department of Microbiology, Faculty of Applied Medical Sciences, Jazan University, Jazan, Saudi Arabia. 2 KMR College of Pharmacy, Perundurai – 638052, Tamilnadu, India. 3Department of Botany, GDC Anantnag 192102, Jammu and Kashmir, India. 4Molecular Biology and Genetics Laboratory, Faculty of Applied Medical Sciences, Jazan University, Jazan, Saudi Arabia. 5Department of Botany, Faculty of Science, South Valley University, 83523 Qena, Egypt. 6Department of Biochemical Engineering and Biotechnology, IIT, Delhi, Hauz Khas, New Delhi 110016, India. 7Pharmaceutical Biotechnology, Amity Institute of Pharmacy, Amity University, NOIDA, Uttar Pradesh 201303. 8Department of Botany, S.P. College, Srinagar 190001, Jammu and Kashmir, India.

Accepted 23 July, 2013

Edible vaccine technology represents an alternative to fermentation based vaccine production system. Transgenic plants are used for the production of plant derived specific vaccines with native immunogenic properties stimulating both humoral and mucosal immune responses. Keeping in view the practical need of new technology for production and delivery of inexpensive vaccines, especially in developing world, plant derived edible vaccines is the best option in hand to combat infectious diseases. Plant derived vaccine is easy to administer, cost effective, readily acceptable, have increased safety, stability, versatility and efficacy. Several plant derived vaccines are under research, some are under clinical trials for commercial use. Like most biotechnology products, the IP situation for edible vaccines is complex as IP rights influence every stage of vaccine development.

Keywords: Transgenic plants, edible vaccines, chimeric viruses, bacterial diseases, viral diseases.

INTRODUCTION

Transgenic plants are the plants in which foreign genes 2012a; b; Sarwat et al., 2012). Apart from the above, of desired characters have to be inserted. Transgenic transgenic plants have been employed for the production plant have been found to have many advantages like, of vaccines for the treatment of various infectious development of high yielding varieties of crop plants and diseases (Kant et al., 2011; Vianna et al., 2011; Yoshida disease resistant, and are plants with improved tolerance et al., 2011; Sharma and Sood, 2011; Twyman et al., to biotic and abiotic stress (Ahmad et al., 2008; 2010a; b; 2012). Infectious diseases are major cause of mortality 2011; Ahmad and Umar, 2011; Ahmad and Prasad, and morbidity worldwide (Goldblatt and Ramsay, 2003)

*Corresponding author. E-mail: [email protected].

Abbreviations: IP, Intellectual Property; HBsAg, hepatitis B surface antigen;HIVgag, HIV Gag protein ; LT-B, heat labile enterotoxin B subunit; CT-B, cholera toxin B subunit; ETEC, entero toxigenic Escherichia coli; M cells, microfold cells; CaMV, Cauliflower mosaic virus; CpMV, Cow pea mosaic virus; TMV, Tobacco mosaic virus; CTB, Eholera toxin B subunit; PA, protective antigen; LF, pethal factor; HBV, hepatitis B virus; JEV, Japanese encephalitis virus 6148 Afr. J. Biotechnol.

and one-third of the deaths are caused by the infectious The immunogenicity and safety of plant derived vaccines agents. Vaccine is an immuno-biological substance, used was declared in phase I clinical studies (Tacket, 2009). for specific protection against both infectious and non- During the last decade, different types of efficient plant- infectious diseases (reviewed by Ahmad et al., 2012; based expression systems have been studied and more Twyman et al., 2012). Vaccine is responsible for the stimu- than 100 different types of recombinant proteins including lation of protective antibody and other immune mecha- plant-derived vaccine antigens have been successfully nisms. The vaccines can be made from live or killed expressed in different types of plant tissues (Tiwari et al., inactivated organisms, extracted cellular fractions, toxoid 2009; Rybicki, 2010; reviewed by Ahmad et al., 2012). or combination of these. Recent preparations are sub-unit Positive effects of edible vaccines include decrease in vaccines and recombinant vaccines. The main limitation potential hazards such as toxic compounds, responses to with vaccines is their dependence on cold chain system, allergy and risk of attenuated strains reverting to pathogenic which is used to store and transport the vaccine under strains associated with established production technologies strict controlled conditions (Park, 2005). Other limitations that use bacteria, yeast and mammalian cells (Pelosi et are risk of adverse reactions such as reactions inherent al., 2012). to inoculation, reactions due to faulty techniques etc (Goldblatt and Ramsay, 2003). Thus, for the implemen- tation of a successful global vaccination strategy, a well TRANSGENIC PLANTS FOR THE PRODUCTION OF designed subunit oral vaccine system should satisfy the PLANT DERIVED VACCINES following criteria (Chargelegue et al., 2005; Levine et al., 2006; Nochi et al., 2007): (a) Produce sufficient quantities Through recombinant DNA technology, different level of of desired antigen; (b) preserve the expressed antigen for antigen expression for each independent line has been a long time at room temperature; (c) induce protective observed in plants (Karg and Kallio, 2009; Shih and immunity; (d) be protected from enzymatic digestion in Doran, 2009; Wilken and Nikolov, 2012). In 1990 first the gastrointestinal tract. edible vaccine, surface protein A from Streptococcus Therefore, in the 1990s, an International campaign was mutans was expressed in tobacco (Curtis and Cardineray, initiated to immunize all the world's children against six 1990). Plant derived vaccine in the form of seed or fruit devastating diseases. The target was to reach 80% of can be easily stored and transported from one place to infants and reduce the annual death toll from these another without the worry of its degradation or damage. A infections by roughly three million. Still, 20% of infants large amount of plant derived vaccine can be easily are un-immunized by six vaccines against polio, measles, produced by cultivation in fields with relatively few inputs. diphtheria, pertusis, tetanus and tuberculosis. In many Autoimmune disorders like Type I diabetes, multiple developing countries, millions of children still die from sclerosis, rheumatoid arthritis etc., can also be suppressed infectious diseases due to immunizations being non- by using plant derived vaccines (Prakash, 1996). existent, unreliable or too costly (Ramsay et al., 1999). Plants are selected which expresses highest level of None will be entirely safe until every child has routine antigen and least number of adverse effects. Till date access to vaccines. Hence, there is an urgent need to various types of antigens are successfully expressed in search for vaccines which are easy to administer, easy to different plants (Mason and Arntzen, 1995; reviewed by store, cost effective, easy to transport and possess Ahmad et al., 2012). With the development of plant readily acceptable delivery system. Hence, there is a lot genetic engineering, the expression system for transgenic of scope in developing plant derived vaccine (Streatfield plants are no longer limited to model plants, but extended et al., 2001; Ahmad et al., 2012). Now the question arises to some orally or high protein content plants. Various what is plant derived vaccine? Advances in transgenic plant plateforms have been demonstrated for production research have made use of crop plants to serve as of recombinant proteins in plants, including leafy crops, bioreactor for the production of recombinant molecules cereals and legume seeds, oil seeds, fruits, vegetables, (Raskin et al., 2002; Kant et al., 2011; Vianna et al., higher plant tissue and cell cultures, hydroponic systems, 2011; Yoshida et al., 2011; Sharma and Sood, 2011). algae and halobios (reviewed by Mei et al., 2006). Co- This means that transgenic plants are used to express expression of adjuvant along with antigen has also been antigen proteins induced by plant transgenic vectors and done in the same plant (Lal et al., 2007). The use of rice to produce certain special vaccines with high anti-disease storage protein gene promoters to express transgenes in ability (reviewed by Mei et al., 2006; Malabadi et al., rice grain is well documented (Nicholson et al., 2005). 2012) (Figure 1). Plant derived vaccines significantly Furtado et al. (2008) compared use of storage protein increase availability of vaccines in places where mainte- gene promoter and non-storage gene promoter with nance of cold chain system is difficult (Webster et al., regard to spatial and temporal control of expression from 2002; Kant et al., 2011; Vianna et al., 2011; Yoshida et barely, wheat and rice. Storage protein promoter from al., 2011; Sharma and Sood, 2011; Twyman et al., 2012). barley and wheat directed the expression in endosperm Important examples on the development of plant bio- but not in embryo; expression was leaky, as it was reactors are shown in Table 1. observed in seed maternal tissues, leaf and root tissues; Kumar et al. 6149

Figure 1. Strategy for the production of candidate vaccine antigen in plants

Table 1. Representative plant-based vaccines: under clinical development or in market.

Expression Route of Product stage S/N Product Plant Host Indication Reference system administration development Potato Transgenic Diarrhea Oral Phase 1 Tacket et al. (2007) Tacket et al. (2009) 1 E. coli LT-B Maize kernels Transgenic Phase 1 Chikwamba et al. (2003) Potato , Tobacco Tacket et al. (2000) Tobacco 2 Norwalk virus Transgenic Diarrhea Oral N A Santi et al. (2008) (VLP’s) Zhong et al. (2005) Tomato fruit (Capsid protein) Kong et al. (2001) Potato HBsAg Kumar et al. (2005) Banana Hepatitis Kostrzak et al. (2009) 3 Tobacco Transgenic B Oral Phase 1 IgG (hepatitis Gao et al. (2003); Cherry, tomato B virus) Tobacco Valdes et al. (2003) Spinach Rabies virus Transient (viral Modelska et al. (1998) 4 Rabies Oral Phase 1 GP/NP vector) Roy et al. (2010) Tobacco Tobacco Cell Newcastle Newcastle USDA Yusibov et al. (2011) 5 disease virus Suspension Transgenic disease Subcutaneous approved (not Gómez et al. (2008) HN Potato marketed) Personalized Non- Nicotiana Transient (viral 6 anti-idiotype Hodgkin’s Subcutaneous Phase 1 Yusibov et al. (2011) benthamiana vectors) scFVs lymphoma 6150 Afr. J. Biotechnol.

Table 1. Contd.

Personalized Transient Non- Nicotiana Phase 1 7 anti-idiotype (magnICON Hodgkin’s Subcutaneous Yusibov et al. (2011) benthamiana (ongoing) dcFVs vectors) lymphoma Phase 1 (ongoing) H5N1 Transient H5N1 Nicotiana Phase 2 8 influenza HA (agrobacterial “avian” Intramuscular Yusibov et al. (2011) benthamiana (Health VLP binary vector) influenza Canada approved) H5N1 H5N1 Nicotiana Transient (launch Phase 1 9 influenza Intramuscular Yusibov et al. (2011) benthamiana vector) “avian” HAI1 influenza H1N1 H1N1 Nicotiana Transient (launch Phase 1 10 influenza Intramuscular Yusibov et al. (2011) benthamiana vector) “swine” (ongoing) HAC1 influenza Potato Transient Subcutaneous NA Biemelt et al. (2003) Transient Human 11 L1 capsid Oral Warzecha et al. (2003) (biolistic delivery Papilloma protein Tobacco Subcutaneous NA Kohl et al. (2006) system) virus Intramuscular Transient Tobacco Transient Anthrax Subcutaneous NA Aziz et al. (2002) Protective Koya et al. (2005) 12 Transgenic antigen (PA) Anthrax Subcutaneous NA (Microprojectiles) Tobacco Watson et al. (2004) Tomato Transient Oral Pogrebnyak et al. 13 S protein SARS NA Tobacco Transient Oral (2005) Transient Oral NA Webster et al. (2002) Tobacco Measles 14 MV-H protein NA Intraperitoneal NA Webster et al. (2005) Lettuce Virus NA intranasal NA Webster et al. (2005) Swine transmissi 15 Spike protein Maize NA ble Oral NA Lamphear et al. (2004) gastroente ritis virus D2 peptide of fibronectin- Staphylo- Intranasal 16 binding Cowpea Transient coccus NA Brennan et al. (1999) protein aureus oral (FnBP) E. coli 17 Intimin protein Tobacco Transient Oral NA Judge et al. (2004) 0157:H7 Entero- FaeG of K88 toxigenic 18 fimbrial Tobacco Transient E. coli Intraperitoneal NA Huang et al. (2003) antigen (Strain K88) Japanese Cedar 19 Cry jI, Cry jII Rice NA Oral NA Takagi et al. (2005) pollen allergens Alfalfa Foot and Wigdorovitz et al. Mouth Parenterally (1999) 20 VP1 NA NA Tobacco Disease oral chloroplasts Virus Li et al. (2006) Kumar et al. 6151

Table 1. Contd.

Respirator 21 F protein Tomato NA y Syncytial Oral NA Sandhu et al. (2000) Virus Sunflower Narrow Leaf 22 SSA NA seed oral NA Smart et al. (2003) Lupin albumin Influenza 23 B5 Tobacco NA NA NA Shoji et al. (2008) Virus F1-V fusion 24 Tomato Transient Plague Oral NA Alvarez et al. (2006) protein Parenteral route Tobacco Canine 25 2L2I peptide NA with Oral NA Molina et al. (2005) Chloroplast Parvovirus booster Japanese Appaiahgari et al. Envelope Tobacco 26 encephaliti (2009) protein (E) Rice s virus Wang et al. (2009) ESAT-6 Tuberculo 27 Arabidopsis NA Oral NA Rigano et al. (2005) antigen sis VP6 Alfalfa Rotavirus Yuan and Saif (2002) 28 NA Oral NA HRV-VP7 Potato Yu-Zhang et al. (2003)

whereas, rice promoters directed the endosperm-specific are: Chlamydomonas reinhardii (Sun et al., 2003), expression in transgenic rice (Furtado et al., 2008). Phaeodactylum tricornutum (Zaslavskaia et al., 2000), Alfalfa (Medicago sativa) is considered as a good Amphidinium carterae, Symbiodinium microadriaticum bioreactor for production of recombinant proteins as it (ten Lohuis and Miller, 1998) and Cylindrotheca fusiformis contains high levels of protein content and low levels of (Fischer et al., 1999). Exciting progress has been made secondary metabolites (Dus Santos et al., 2002). Cereal with the chloroplast based production of two particularly crops can be the most suitable candidate and can be important classes of pharmaceuticals, vaccines and anti- used to enhance the antigen concentration and help to bodies (Bock and Warzecha, 2010; Scotti et al., 2012). reduce oral dose as they have ample amount of soluble Extraordinarily high expression levels and the prospects protein in endosperm (Ahmad et al., 2012). Potato, of developing edible pharmaceuticals make transgenic tomato and carrot have been successfully reported to chloroplasts a promising platform for the production of express vaccine candidates (Walmsley and Arntzen, next-generation vaccines and antimicrobials (Waheed et 2000). Antigen genes encoding HBsAg, HIVgag and al., 2012). During the past few years, several vaccine Rabies Capsid Proteins have been successfully trans- candidates have been produced successfully via plastid formed to tomato (Sala et al., 2003). High levels of transformation, which emphasizes that transplastomic recombinant protein expression were observed in plants, as a second generation expression system, have proplastids of cultured carrot cells (Daniell et al., 2005). great potential to fill gaps in conventional production Oral delivery of the therapeutic proteins via edible carrot platforms. A salient feature of plastids is that they combine preserved the structural integrity of their target proteins characteristics of prokaryotic and eukaryotic expression as no cooking is needed (Muller et al., 2003). Other systems, which is exemplified by the production of virus vegetable crops like lettuce (Lactuca sativa), celery like particles and of bacterial antigens (reviewed by Bock cabbage (Brassica rapa var. pekinensis), cauliflower and Warzecha, 2010). Successful expression of antigens (Brassica oleracea var. botrytis) are under study for the in plants was carried out for Escherichia coli, heat labile production of vaccines. The only problem in these enterotoxin B subunit (LT-B) in tobacco and potato (Hirst vegetables is low expression levels (Koprowski, 2005; and Holmgren, 1987), Rabies virus G protein in tomato Tacket and Mason, 1999). The earliest fruit used for the (Mc Garvery et al., 1995), Hepatitis B virus surface plant transgenic programme is banana (Musa acuminate) antigen in tobacco and potato (Thanavala et al., 1995), (Mason et al., 2002). Norwalk virus capsid protein in tobacco and potato According to Trivedi and Nath (2004) papaya (Carica (Mason et al., 1996) and cholera toxin B subunit (CT-B) papaya) is another ideal plant species for vaccine in potato (Arakawa, 1997). production. Apart from fruit, vegetable and cereal crops Antigen expressed in plant or plant products can be scientists have used algae to produce metabolites and administered orally or by intramuscular or by intravenous heterologous proteins for pharmaceuticals applications injection. Homogenized leaves, fruits or vegetables are (Mayfield and Franklin, 2005). The species under study used through oral route. Purified antigen containing plant 6152 Afr. J. Biotechnol.

tissue can be delivered in a capsule or powder (pill) form. responses (Streatfield, 2006). Capsule may be suitable because capsule coating can be Edible vaccines have multi-component ability that is modified in such a way that coating material dissolves in possible due to the crossing of 2 plant lines (Lal et al., particular area of stomach, and vaccine can be released 2007). These vaccines with multi-component abilities are in a specific area of the body. Purified component can known as second generation edible vaccines as they also be used by intramuscular and intravenous admini- allow for several antigens to approach M cells (microfold stration. Oral administration of plant derived vaccine cells) simultaneously (Lal et al., 2007). The multi- induces both mucosal and systemic immunity. When component edible vaccines can prevent multiple diseases antigen is administered orally, it induces more mucosal for example ETEC, chlorea and ratovirus (Lal et al., response than intramuscular or intravenous injections. 2007). Injected vaccines do not have this property, so So, more importance has been given to those antigens, there are less effective than edible vaccines (Ramessar which induce mucosal immune response to produce et al., 2008a; b; Naqvi et al., 2011). secretory Ig A at mucosal surfaces. Mucosal immunity is very effective in diarrhoeal diseases caused by rotavirus, Norwalk virus, Vibrio cholerae, entero toxigenic E. coli Chimeric viruses (ETEC) and also in respiratory diseases such as pneumonia. Over-coat and epi-coat technology is used to produce Second generation plant derived vaccines are known chimeric viruses. Over-coat technology provides as multi component vaccines, provides protection against expression of entire protein, whereas epi-coat technology several pathogens. Both Enterotoxigenic Escherichia coli permits the plant to produce only the foreign proteins (ETEC) heat-labile enterotoxin (LT-B) and the capsid (http://www.geocities.com/plantvaccines/transgenicplants protein of Norwalk virus were successfully expressed in .html). Plant viruses redesigned to carry the desired plants and induced immune response against both E. coli genes and used to infect differently in different parts of and Norwalk virus in mice (Huang et al., 2001). the plant. Alfalfa mosaic virus, CaMV (Cauliflower mosaic virus), CpMV (Cow pea mosaic virus), TMV (Tobacco mosaic virus), Tomato bushy stunt virus and Potato virus ADVANTAGES OF EDIBLE VACCINES OVER are redesigned to express fragments of antigens on their INJECTED VACCINES surface. There are reports that they produce plant based chimeric virus such as foot and mouth disease virus; mint Edible vaccines have many advantages over the injected enteritis virus. Fragment of gp41 surface protein of HIV vaccines like: virus put into CpMV could evoke a strong neutralizing antibody response in mice (Moffat, 1995; Wang et al., 1. Edible vaccines are cost effective, have low risk of 2012). contamination and no cost for transportation. Pharmaceutical companies spend million dollars for the production of vaccines and to preserve vaccines. APPROACHES TO PRODUCE PLANT DERIVED Transgenic plants does not need cold chain storages. VACCINES 2. Pharmaceutical companies need the hitech machines for the production of vaccines. In the case of edible Plants serve as an important source to produce cost- vaccines production we need soil rich land instead of effective vaccine derivatives. Plant based production of machines. vaccine candidates can help to reduce the economic 3. Long distance transportation is not required in the case burden on the developing countries and can be made of edible vaccines. easily available to every individual. Various models to 4. The cost of materials needed for field grown plants is produce vaccine candidates are described below. lower compared to cell culture grown in bioreactors (Xu et al., 2011). 5. Edible vaccines have a low cost for medical equipment Bacterial as well, because needles and syringes are not needed for delivery (Streatfield, 2006; Xu et al., 2011). Enterotoxigenic Escherichia coli (ETEC) 6. Medical professionals are not needed for oral delivery (Streatfield, 2006). Enterotoxigenic Escherichia coli strains are a major 7. Transgenic plants have low contamination risks as cause of enteric diseases in live stock and humans. compared to injected vaccines ETEC is attached to specific receptors on the surface of 8. Needles and syringes are responsible for spreading of enterocytes in the intestinal lumen by fimbriae. ETEC second hand diseases (Nochi et al., 2007). produces a heat-stable enterotoxin (ST) which consists of 9. Oral delivery has efficiency to provoke a mucosal five B subunits and one A subunit. B subunit binds to immune response, which produces cell mediated sugar residues of ganglioside Gm1 on the cells lining the Kumar et al. 6153

villi and crypts of the small intestine. Insertion of the B which after endocytosis, blocks the adenyl cyclase pathway subunit into the host cell membrane forms a hydrophilic within the cell. The main effect of this toxin complex is to transmembrane channel through which the toxic A increase vascular permeability, which leads to a shock. subunit can pass into the cytoplasm (Roy et al., 2010). Protective antigen was expressed in transgenic tobacco Raw transgenic potato expressing LT-B were fed to 11 chloroplasts by inserting the pag A gene into the chloroplast volunteers, out of which 10(91%) developed neutralizing genome. Cytotoxicity measurements in macrophage lysis antibodies and 6(55%) of individuals also showed assays showed that chloroplast-derived PA was equal in mucosal response (Tacket et al., 1998). Different reports potency to PA produced in B. anthracis. Chloroplast- are there on synthetic heat-labile enterotoxin (LT-B) gene derived protective antigen provides cleaner and safer and their expression in plants such as potato, banana, anthrax plant-derived-vaccine at a lower production cost tobacco and tomato; and all were tested in mice (Mason (Koya et al., 2005). Koya et al. (2005) published for the et al., 1998). Expression of E. coli fimbrial subunit protein first time the PA expression in plants from stable nuclear- in transgenic plants can be used to vaccinate against transgenic tobacco. Aziz et al. (2002) also reported the these diseases. Joensuu et al. (2006) evaluated expression of PA in leaves of stable nuclear-transgenic transgenic plants to produce Fae G protein and adhesion tomato plants. Expression of PA in tobacco or tomato of F4 fimbriae. Oakes et al. (2007) reported the edible was enhanced in combination with a second B. anthracis transgenic soyabean plant producing E. coli fimbrial protein, lethal factor (LF), and showing cytolytic activity subunit proteins. Tacket (2009) discussed early human when applied to macrophage-like cell lines. Also, when studies of oral transgenic plant-derived vaccines against tomato leaf material was injected into mice, antisera enterotoxigenic Escherichia coli. Genetic combination of could be recovered with neutralizing activity to anthrax gene coding for an LTB:ST protein in tobacco by lethal toxin (LT), which is a combination of PA and LF. Agrobacterium mediated transformation displays antigenic determinants from both LTB and ST. Presence of mucosal and systemic humoral responses in mice Porphyromonas gingivalis when dosed orally with transgenic tobacco leaves also confirmed that plant-derived LTB:ST can lead to Periodontal diseases are caused by oral anaerobic immunogenicity development via oral route (Rosales- bacterium Porphyromanas gingivalis. It is thought to be Mendoza et al., 2011). initiated by the binding of P. gingivalis fimbrial protein to saliva coated oral surfaces. Shin et al. (2009) has success- fully transferred FIM A protein producing gene into potato Vibrio cholera tuber tissues and produced native FIM A protein in edible plant cells. Cholera is due to contaminated food or water which triggers an acute intestinal infection by V. cholera (López- Gigosos et al., 2011). Enterotoxin such as cholera toxin Viral (CT) was expressed in tobacco plant (Arakawa et al., 1998). Nochi et al. (2007), showed oral immunization with Norwalk virus transgenic rice encoding the cholera toxin B subunit (CTB) which stimulates secretory Ig A, shows resistant to Calci viruses are a major cause of food and water gastrointestinal digestion. Karaman et al. (2012) associated outbreaks of diarrhoea and vomiting, affecting introduced synthetic gene encoding for CT-B by the individuals of all age groups. A capsid protein of Norwalk control of a γ-zein promoter in maize seeds. CT-B levels virus was expressed in transgenic tobacco and potato were checked via ganglioside dependent ELISA. Anti- plants. Potato tubers expressing Norwalk virus antigen CTB IgG and anti-CTB IgA were found in the sera and were fed to mice, it developed serum IgG specific for fecal samples of the orally immunized mice protected Norwalk virus (Mason et al., 1996). According to Tacket against holotoxin challenge with CT. et al. (2000) volunteers fed with transgenic potato expressing Norwalk virus antigen showed seroconversion.

Anthrax Hepatitis B virus Anthrax is a disease most commonly occur by inoculation of B. anthracis through the skin of infected animals, their It is estimated that 3 to 6% of the world population has products and inhalation of spores in dust or wool fibers. been infected with Hepatitis B virus (HBV) and there are Virulence factors is a toxin complex, which consists of 300 to 400 million carriers in the world. India alone has three proteins. The protective antigen (PA) binds the over 40 million carriers. In the acute stage there are signs complex receptors on the macrophage surface. After of inflammation in the portal triads: the infiltrate is mainly proteolysis, oedema factor and lethal factor are released lymphocytic. In the liver parenchyma, single cells show 6154 Afr. J. Biotechnol.

ballooning and form acidophilic (councilman) bodies as derived MV-H showed MV-specific IgG. they die. In chronic hepatitis, damage extends out from the portal tracts, giving the piecemeal necrosis appearance. Some lobular inflammation is also seen. As the disease Japanese encephalitis progresses fibrosis develops and eventually, cirrhosis. Hepatitis B virus replicates in the hepatocytes, reflected JE virus is a single stranded positive sense RNA virus in the detection of viral DNA and HBs Ag in the nucleus belonging to family flaviviridae transmitted through a and HBs Ag in the cytoplasm and at the hepatocyto- zoonotic cycle between mosquitoes, pigs and water birds. membrane (Simmonds and Peutherer, 2003). Hepatitis B It causes encephalitis all over the world especially in virus is carried in the blood and blood derived bodily Eastern and South-eastern Asia. JE affects some primary fluids of infected persons and can be transferred through organs like thalamus, corpus striatum, brainstem and contact with a carrier’s blood caused by unsafe injections spinal cord. With the absence of specific antiviral therapy, or transfusions, sexual contact and tattooing. Long term it is managed mainly by its symptom and by supportive protection against Hepatitis B virus is possible with therapies along with preventive measurements (Misra vaccine. HBs Ag was expressed in transgenic potato and Kalita, 2010). Transgenic rice expressing the envelope plant and tested in mice for production of antibodies protein of Japanese encephalitis virus (JEV), under control (Richter et al., 2000). of a dual cauliflower mosaic virus (CaMV 35s) promoter, Pniewski et al. (2011) has shown the production of was generated. JEV specific neutralizing antibody was small surface antigen for HBV (S-HBsAg) in genetically detected in mice after immunization of mice with protein modified glufosinate-resistant lettuce. They orally immu- extracts from transgenic rice plant by intraperitoneal or nised mice by using lyophilised form of plant material and oral immunization (Wang et al., 2009). Appaiahgari et al. showed the presence of secretory IgA (S-IgA) and total (2009) showed the expression of Japanese encephalitis serum antibodies. Li et al. (2011) also demonstrated the viral envelope protein (E) in transgenic tobacco can transformation of HBsAg (hepatitis B surface antigen) produce immunogenic response in mammalian system. gene in to tomato mediated by Agrobacterium tumifaciens. Lou (2007) has experimentally expressed hepatitis B virus large surface antigen in transgenic tomato plant. Influenza virus H5N1 Transgenic lettuce plant carrying recombinant hepatitis B virus antigen HBs Ag was demonstrated in Brazil Shoji et al. (2009) described the production of hema- (Marcondes and Hansen, 2008). Tacket (2009) has gglutinin from A/Indonesia/05/05 strain of H5N1 influenza discussed early human studies of oral transgenic plant- virus by transient expression in plants. The results indicate derived vaccines against hepatitis B virus. A phase I that immunization of ferrets with plant-derived hema- clinical trial with plant derived hepatitis B vaccine has gglutinin elicited serum hemagglutinin-inhibiting antibodies boosted antigen-specific serum antibodies titer (Tacket, and protected the ferrets against challenge infection with 2009). a homologous virus. Plant derived vaccine may be the solution in the rapid, large scale production of influenza vaccine in the face of pandemic. Measles Kalthoff et al. (2010) showed the expression of full- length recombinant hemagglutinin (rHA0) of H5N1 in Millions of people live in areas where measles are Nicotiana benthamiana with optimize expression levels. endemic and resources are scare. Measles are Their results showed to provide an immunogenic protection transmitted from person to person by respiratory droplets. protect chicken against lethal challenge infection with Measles in an acute febrile illness, the onset is flu-like heterologous HPAIV H5N1 of 96% homology to rHA0 by with high fever, cough and conjunctivitis, red spots with a plant-expressed hemagglutinin. Jul-Larsen et al. (2012) bluish-white centre on the buccal mucosa called Koplik’s demonstrated recombinant influenza haemagglutinin spots. Measles antigens expressed in plants have been antigen (HAC1) that was derived from the 2009 pandemic shown to be antigenic and immunogenic both after H1N1 virus and expressed in tobacco plants. They showed invasive and oral vaccination (Marcondes and Hansen, that the tobacco derived recombinant HAC1 antigen is a 2008). Crude Quillaja saponin extracts stimulates measles’ promising vaccine candidate recognized by both B- and T virus specific immune responses in mice, following oral cells. immunization with plant based measles virus haema- Shoji et al. (2011) showed the advantages provided by gglutinin protein (Pickering et al., 2006). the plant system for influenza vaccine antigen production Webster et al. (2002) confirmed that the transgenic is their independence from pathogenic viruses, and cost tobacco plants-derived MV-H protein vaccine, which when, and time efficiency. They produced large-scale of modified to MV-H DNA vaccine, to prime-boost vaccination recombinant hemagglutinin proteins from strategy demonstrated the MV hemagglutinin protein A/California/04/09 (H1N1) and A/Indonesia/05/05 (H5N1) (MV-H) expression. Orally immunized mice with plant- strains of influenza virus in N. benthamiana plants, and Kumar et al. 6155

tested their immunogenicity (serum hemagglutination ization of plant derived vaccine technology and to prevent inhibition and virus neutralizing antibodies), and safety in misuse of technology because it possesses great risk on animal models. environment and human health. Development of vaccine Madhun et al. (2011) produced influenza subunit antigen into a stable seed form or production in leaf is mostly in transient plant expression systems as an alternative. A favoured but its to spoilage to prevent loss/leaking out of needle-free intranasal influenza vaccine is an attractive antigen into environment is to be checked. The amount of approach to be followed. Plant-derived influenza H5N1 plant which can be taken up as raw food is to be strictly (A/Anhui/1/05) antigen, alone or formulated with bis-(3', monitored as over dose may cause toxic/allergic 5')-cyclic dimeric guanosine monophosphate (c-di-GMP) reactions. Most of the edible crops are destroyed by as adjuvant induces strong mucosal and systemic attack of insects and hence their effect on vaccine humoral immune responses. Search for safe and producing plant has to be evaluated. Even though plant effective adjuvant to enhance H5N1 intranasal vaccine derived vaccines have shown promising results but with extracts of mushroom mycelia was found to be good evaluation of their tolerance needs in-depth study (Ichinolhe et al., 2010). (Ahmad et al., 2012).

LIMITATIONS CONCLUSION AND FUTURE PERSPECTIVE

Before the commercial production of plant derived Edible plant derived vaccine may lead to a future of safer vaccines, there is urgent need to consider the following and more effective immunization. They would overcome points; some of the difficulties associated with traditional vaccines like production, distribution and delivery and 1. Searching for suitable plant which will give ideal they can be incorporated in to the immunization plans. antigen expression. Edible vaccines have lot of advantages over injected 2. Identification of proper dosage (whether plant parts, vaccines like, well established cultivation, low cost of plant products, pill, intramuscular or intravenous injection production, no need for “cold chain” delivery, rapid scale- of purified antigen) can produce proper dose. up, simple distribution by seeds, ease of genetic 3. Verification of allergens in the plant and plant products. manipulation, oral delivery and low health risks from 4. Study the impact of plant derived vaccines on the human pathogen and toxin contamination, etc. Significant environment and human health. progress has been achieved in employing plants as 5. Genetically altered crops producing plant derived vaccine expression system, for example vegetables, vaccine could get mixed with human food supply or fruits, cereal crops, etc. Tobacco, tomato, maize, rice are animal feed, causing potential threat to public health. leading production plateforms for recombinant protein 6. Cross pollination and their problems. production. The basic advantage of using plants as 7. Effects on insects and soil microbes. vaccine production system is that plants being higher 8. Regulation of plant derived vaccines in the form of eukaryotes provide opportunities for unlimited production, food, drug or agricultural product. the range and diversity of recombinant molecules namely 9. Cultivation of plant derived vaccines and their delivery peptides, polypeptides and complex multimeric proteins in capsule or pill form. that cannot be made in microbial systems. Plant production system provides a wider flexibility in designing of new pharmaceutical proteins. Days are not too far Risks of plant derived vaccines when we eat delicious vegetables, fruits etc, to prevent ourselves from infectious diseases. Developing and Plant derived vaccines pose serious risks to the public if under-developed countries will be benefited more by this they are not handled with care. Safety of transgenic edible vaccine production system because the methods plants includes many aspects like ecology, agronomy and in production are reasonably affordable and the vaccine molecular biology which focus on food and environmental products would be more openly accessible to the safety (Ahmad et al., 2012). Environmental issues and population. biodiversity concern are raised because of the transgenic One of the most important bottlenecks in edible vaccine seeds or plants that escape into the wild. Moreover, plant technology is yield improvement, as this factor has a derived vaccines cannot be distinguished from non-plant major impact on economic feasibility. Different strategies derived vaccines of the same plant. Plant derived vaccine in hand which can lead to improved production of edible tomato plant looks like a traditional tomato. There is vaccines include the development of novel promoters, always a risk of mis-administration. improvement in protein stability by protein engineering Although, plant derived vaccine technology can save approach, targeted expression of protein of interest and many lives in developing countries. At the same time, last but not least the improvement in downstream process- there is an urgent need to address proper commercia- sing. The potential concern of edible vaccine technology 6156 Afr. J. Biotechnol.

is differential glycosylation of proteins in in vitro systems maize kernels. PNAS 100: 11127–11132. or in non-native species. Strategies should be devised to Curtis RI, Cardineray CA (1990). World patent App WO 90/02484. Daniell H, Kumar S, Dufourmantel N (2005). Breakthrough in humanize the plant glycosylation machinery by inhibiting chloroplast genetic engineering of agronomically important crops. glycosylation enzymes. The use of plastids as vaccine Trend. Biotechnol. 23:238–245. production platform is quite promising to prevent Dus-Santos MJ,Wigdorovitz A, Trono K, Rios RD, Franzone PM, Gil F, transgene escape through pollens or seed dispersal and Moreno J, Carrillo C, Escribano JM, Borca MV (2002). A novel methodology to develop a foot and mouth disease virus (FMDV) it needs an extensive research to improve expression peptide-based vaccine in transgenic plants. Vaccine 20:1141–1147. levels and prevention of proteolysis in plastids. Fischer R, Liao YC, Hoffmann K, Schillberg S, Emans N (1999). Molecular farming of recombinant antibodies in plants. Biol. Chem. 380:825–839. REFERENCES Furtado A, Henry RJ, Takaiwa F (2008). Comparison of promoters in transgenic rice. Plant Biotechnol. J. 6 (7): 679-693. Ahmad P, Jaleel CA, Salem MA, Nabi G, Sharma S (2010a). Roles of Gao Y, Ma Y, Li M, Cheng T, Li SW, Zhang J, Xia NS (2003). Oral Enzymatic and non-enzymatic antioxidants in plants during abiotic immunization of animals with transgenic cherry tomatillo expressing stress Crit. Rev. Biotechnol. 30(3): 161–175. HBsAg. World J. Gastroenterol. 9(5): 996-1002. Ahmad P, Nabi G, Jeleel CA, Umar S (2011). Free radical production, Goldblatt D, Ramsay M (2003). Immunization, In: D.A. Warrel, T.M. oxidative damage and antioxidant defense mechanisms in plants Cox, J.D. Firth, Jr. E.J. Benz (ed.) Oxford text book of medicine fourth under abiotic stress, In: P. Ahmad, S. Umar (ed.) Oxidative stress: edition. Oxford University Press. Role of antioxidats in Plants. Studium Press Pvt. Ltd. New Delhi, Gómez E, Zoth SC, Asurmendi S, Rovere CV, Berinstein A (2009). India. Expression of hemagglutinin-neuraminidase glycoprotein of Ahmad P, Prasad MNV (2012a). Environmental adaptations and stress newcastle disease virus in agroinfiltrated Nicotiana tolerance in plants in the era of climate change. benthamiana plants. J. Biotechnol. 144(4): 337-340. http://f6mail.rediff.com/prism/@mail/http:/help.yahoo.com/l/in/yahoo/ Hirst RT, Holmgren J (1987). Conformation of protein secreted across mail/classic/context/context-07.htmlSpringer Science+Business bacterial outer Membranes: A study of enterotoxin translocation from Media, LLC, New York Vibrio cholerae. Proc. Nat. Acad. Sci. USA 84: 7418-7422. Ahmad P, Prasad MNV (2012b). Abiotic stress responses in Plants: Huang Y, Liang W, Pan A, Zhou Z, Huang C, Chen J, Zhang D (2003). metabolism,productivity and sustainability. Production of FaeG, the major subunit of K88 fimbriae, in transgenic http://f6mail.rediff.com/prism/@mail/http:/help.yahoo.com/l/in/yahoo/ tobacco plants and its immunogenicity in mice. Infect. Immun. 71(9): mail/classic/context/context-07.htmlSpringer Science+Business 5436–5439. Media, LLC, New York. Huang Z, Dry I, Webster D, Strugnell R, Wesselingh S (2001). Plant- Ahmad P, Sarwat M, Sharma S (2008). Reactive oxygen species, derived measles virus hemagglutinin protein induces neutralizing antioxidants and signaling in plants. J. Plant Biol. 51(3): 167-173. antibodies in mice. Vaccine 19: 2163-2171. Ahmad P, Umar S (2011). Oxidative stress: Role of antioxidants in Ichinolhe T, Ainai A, Nakamura T, Akiyama Y, Maeyama J, Odagiri T, plants. Studium Press Pvt. Ltd. New Delhi, India Tashiro M, Takahashi H, Sawa H, Tamura S, Chiba J, Kurata T, Sata Ahmad P, Umar S, Sharma S (2010b). Mechanism of free radical T, Hasegawa H (2010). Induction of cross-protective immunity scavenging and role of phytohormones during abiotic stress in plants, against influenza A virus H5N1 by an intranasal vaccine with extracts In: M Ashraf, M Ozturk, M.S.A. Ahmad (ed.) Plant adaptation and of mushroom mycelia. J. Med. Virol. 82 (1):128-137. phytoremediation. Springer Dordrecht Heidelberg, London, New Joensuu JJ, Kotiaho M, Teeri TH, Valmu L, Nuutila AM, Oksman- York. Caldentey KM, Niklander-Teeri V (2006). Glycosylated F4 (K88) Ahmad P, Ashraf M, Younis M, Hu X, Kumar A, Akram N A, Al-Qurainy fimbrial adhesin FaeG expressed in barley endosperm induces F (2012). Role of transgenic plants in agriculture and biopharming. ETEC-neutralizing antibodies in mice. Transgenic Res. 15(4):359– Biotechnol. Adv. 30(3): 524-540. 373. Alvarez ML, Pinyerd HL, Crisantes JD, Rigano MM, Pinkhasov J, Judge NA, Mason HS, O'Brien AD (2004). Plant cell-based intimin Walmsley AM, Mason HS, Cardineau GA (2005). Plant-made subunit vaccine given orally to mice primed with intimin reduces time vaccine against pneumonic and bubonic plague is orally of Escherichia coli O157:H7 shedding in Feces. Infect. Immun. immunogenic in mice. Vaccine 24: 2477-2490. 72:168-175. Appaiahgari MB, Abdin MZ, Bansal KC, Vrati S (2009). Expression of Jul-Larsen A, Madhun A, Brokstad K, Montomoli E, Yusibov V, Cox R Japanese encephalitis virus envelope protein in transgenic tobacco (2012). The human potential of a recombinant pandemic influenza plants.J. Virol. Meth. 162 (1-2): 22-29. vaccine produced in tobacco plants. Hum. Vaccin. Immunother. 8(5): Arakawa T, Chong DKX, Langridge WHR (1998). Efficacy of a food 653-661. plant-based oral cholera toxin B subunit vaccine. Nat. Biotechnol. 16: Kalthoff D, Giritch A, Geisler K, Bettmann U, Klimyuk V, Hehnen HR, 292-297. Gleba Y, Beer M (2010). Immunization with plant-expressed Arakawa T, Chong DKX, Merritt JL, Langridge WHR (1997). hemagglutinin protects chickens from lethal highly pathogenic avian Expression of cholera toxin B subunit oligomers in transgenic potato influenza virus H5N1 challenge infection. J. Virol. 84(22): 12002– plants. Transgenic Res. 6(6): 403-413. 12010. Aziz MAS, Singh P, Kumar A, Bhatnagar R (2002). Expression of Kant A, Reddy S, MM Shankraiah, Venkatesh JS, Nagesh C (2011). protective antigen in transgenic plants: a step towards edible vaccine Plant made pharmaceuticals (PMP’s)-A protein factory: A Overview. against anthrax. Biochem. Biophys. Res. Commun. 299: 345–351. Pharmacology online 1: 196-209. Bock R, Warzecha H (2010). Solar-powered factories for new vaccines Karaman S, Cunnick J, Wang K (2012). Expression of the cholera toxin and antibiotics. Trend. Biotechnol. 28: 246-252. B subunit (CT-B) in maize seeds and a combined mucosal treatment Brennan FR, Bellaby T, Helliwell SM, Jones TD, Kamstrup S, against cholera and traveler’s diarrhea. Plant Cell Rep. 31: 527–537. Dalsgaard K, Flock J, Hamilton WDO (1999). Chimeric plant virus Karg SR, Kallio PT (2009). The production of biopharmaceuticals in particles administered nasally or orally induce systemic and mucosal plant systems. Biotechnol. Adv. 27:879–894. immune responses in mice. J. Virol. 73(2): 930-938. Kohl T, Hitzeroth II, Stewart D, Varsani A, Govan VA, Christensen ND, Chargelegue D, Drake PMW, Obregon P, Prada A, Fairweather N, Williamson AL, Rybicki P (2006). Plant-produced cottontail rabbit Ma JKC (2005). Highly immunogenic and protective recombinant Papilloma virus L1 protein protects against tumor challenge: a proof- vaccine candidate expressed in transgenic plants. Infect. Immun. 73: of-concept study. Clin. Vaccin. Immunol. 13(8): 845-853. 5915-5922. Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS, Thanavala Y Chikwamba RK, Scott MP, Mejia LB, Mason HS, Wang K (2003). (2001). Oral immunization with hepatitis B surface antigen expressed Localization of a bacterial protein in starch granules of transgenic in transgenic plants. Proc. Natl. Acad. Sci. USA. 98: 11539–11544. Kumar et al. 6157

Koprowski H (2005). Vaccines and sera through plant biotechnology. neutralizing antibodies by a tobacco chloroplast-derived vaccine Vaccine 23:1757–1763. based on a B cell epitope from canine parvovirus. Virol. 342(2): 266- Kostrzak A, Gonzalez MC, Guetard D, Nagaraju DB, Hobson SW, 275. Tepfer D, Pniewski T, Sala M (2009). Oral administration of low Muller CP, Fack F, Damien B, Bouche FB (2003). Immunogenic doses of plant based HBsAg induced antigen-specific IgAs and IgGs measles antigens expressed in plants: role as an edible vaccine for in mice, without increasing levels of regulatory T cells. Vaccine 27: adults. Vaccine 23:816–819. 4798–4807. Naqvi S, Ramessar K, Farré G, Sabalza M, Miralpeix B, Twyman RM, Koya V, Moayeri M, Leppla SH, Daniell H (2005). Plant-based vaccine: Capell T, Zhu C, Christou P (2011) High-value products from mice immunized with chloroplast-derived anthrax protective antigen transgenic maize. Biotechnol. Adv. 29: 40–53. survive anthrax lethal toxin challenge. Infect. Immun. 73:8266–8274. Nicholson L, Gonzales-Menlendi P, van Dolleweerd C, Tuck H, Perrin Kumar GB, Ganapathi TR, Revathi CJ, Srinivas L, Bapat VA (2005). Y, Ma JKC, Fischer R, Christou P, Stoger E (2005). A recombinant Expression of hepatitis B surface antigen in transgenic banana multimeric immunoglobulin expressed in rice shows assembly- plants. Planta 222(3): 484–493. dependent subcellular localization in endosperm cells. Plant Lal P, Ramachandran VG, Goyal R, Sharma R (2007). Edible vaccines: Biotechnol. J. 3: 115–127. Current status and future. Ind. J. Med. Microbiol. 25: 93-102. Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, Nakanishi Lamphear BJ, Jilka JM, Kesl L, Welter M, Howard JA, Streatfield SJ U, Matsumura A, Uozumi A, Hiroi T et al. (2007). Rice-based (2004). A corn-based delivery system for animal vaccines: an oral mucosal vaccine as a global strategy for cold-chain and needle-free transmissible gastroenteritis virus vaccine boosts lactogenic immunity vaccination. Proc. Natl. Acad. Sci. 104: 10986-10991. in swine. Vaccine 22(19): 2420-2424. Oakes JL, Garg R, Bost KL, Piller KJ (2007). Expression and Levine MM (2006). Enteric infections and the vaccines to counter them: subcellular targeting of a model subunit vaccine in transgenic Future directions. Vaccine 24:3865–3873. soyabean. J. immunol. (Meeting Abstract Supplement) S32 Li T, Sun JK, Lu ZH, Liu Q (2011). Transformation of HBsAg (Hepatitis 178:41.13. B Surface Antigen) gene into tomato mediated by Agrobacterium Park K (2005). Park’s Preventive Social Medicine. M/S Banarsidas tumefaciens. Czech J. Genet. Plant Breed. 47(2): 69–77. Bhanot pub. 95-100. Li YU, Sun M, Liu J, Yang Z, Zhang Z, Shen G (2006). High expression Pelosi A, Shepherd R, Walmsley AM (2012). Delivery of plant-made of foot-and-mouth disease virus structural protein VP1 in tobacco vaccines and therapeutics. Biotechnol. Adv. 30(2):440-4488. chloroplasts. Plant Cell Rep. 25(4): 329–333. Pickering RJ, Smith SD, Strugnell RA, Wesselingh SL, Webster DE López-Gigosos RM, Plaza E, Díez-Díaz RM, Calvo MJ (2011). (2006). Crude saponins improve the immune response to an oral Vaccination strategies to combat an infectious globe: Oral cholera plant-made measles vaccine. Vaccine 24(2): 144-150. vaccines. J. Glob. Infect. Dis. 3(1): 56–62. Pniewski T, Kapusta J, Bociąg P, Wojciechowicz J, Kostrzak A, Gdula Lou XM, Yao QH, Zhang Z, Peng RH, Xiong AS, Wang HK (2007). M, Fedorowicz-Strońska O, Wójcik P, Otta H, Samardakiewicz S, Expression of the human hepaptitis B virus large surface antigen Wolko B, Płucienniczak A (2011). Low-dose oral immunization with gene in transgenic tomato plant. Clin. Vaccin. Immunol. 14(4): 464- lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B 469. surface antigen for prototype plant-derived vaccine tablet formulation. Madhun AS, Haaheim LR, Nøstbakken JK, Ebensen T, Chichester J, J. Appl. Genet. 52:125–136. Yusibov V, Guzman CA, Cox RJ (2011). Intranasal c-di-GMP- Pogrebnyak N, Golovkin M, Andrianov V, Spitsin S, Smirnov Y, Egolf adjuvanted plant-derived H5 influenza vaccine induces R, Koprowski H (2005). Severe acute respiratory syndrome (SARS) S multifunctional Th1 CD4 (+) cells and strong mucosal and systemic protein production in plants: Development of recombinant vaccine. antibody responses in mice. Vaccine 29(31): 4973-82. Proc. Natl. Acad. Sci. USA. 102(25): 9062–9067. Malabadi RB, Meti NT, Mulgund GS, Nataraja K, Kumar SV (2012). Prakash CS (1996) Edible vaccines and antibody producing plants. Recent advances in plant derived vaccine antigens against human Biotechnol. Dev. Monitor. 27: 11-13. infectious diseases. Res. Pharm. 2(2): 8-19. Ramessar K, Rademacher T, Sack M, Stadlmann J, Platis D, Stiegler Marcondes J, Hansen E (2008). Transgenic lettuce seedlings carrying G, et al. (2008b). Cost-effective production of a vaginal protein hepatitis B virus antigen HBs Ag. Braz. J. Infect. Dis. 12(6): 469-471. microbicide to prevent HIV transmission. Proc. Natl. Acad. Sci. USA. Mason HS, Arntzen CZ (1995). Transgenic plants as vaccine production 105:3727–3732. systems. Trend Biotechnol. 13: 388-392. Ramessar K, Sabalza M, Capell T, Christou P (2008a). Maize plants: an Mason HS, Ball JM, Shi JJ, Estes MK, Arntzen CJ (1996). Expression of ideal production platform for effective and safe molecular pharming. Norwalk virus capsid protein in transgenic tobacco and potato and its Plant Sci. 174:409–419. oral immunogenicity in mice. Immunology 93(11): 5335-5340. Ramsay AJ, Kent SJ, Strugnell RA, Suhrbier A, Thomson SA, Mason HS, Haq TA, Clements JD, Arntzen CJ (1998). Edible vaccine Ramshaw IA (1999). Genetic vaccination strategies for enhanced protects mice against Escherichia coli heat-labile enterotoxin (LT): cellular, humoral and mucosal immunity. Immunol. Rev. 171: 27-44. potatoes expressing a synthetic LT-B gene. Vaccine 16(13): 1336- Raskin I, Ribnicky DM, Komarnytsky S, Ilic N, Poulev A, Borisjuk N, 1343. Brinker A, Moreno DA, Ripoll C, Yakoby N, O’Neal JM, Cornwell T, Mason HS, Warzecha H, Mor T, Arntzen CJ (2002). Edible plant Pastor I, Fridlender B (2002). Plants and human health in the twenty- vaccines: applications for prophylactic and therapeutic molecular fi rst century. Trend. Biotechnol. 20:522–531. medicine. Trends Mol. Med. 8:324–329. Richter LJ, Thanavala Y, Arntzen CJ, Mason HS (2000). Production of Mayfield SP, Franklin SE (2005). Expression of human antibodies in hepatitis B surface antigen in transgenic plants for oral immunization. eukaryotic micro algae. Vaccine 23(15):1828-1832. Nat. Biotechnol. 18: 1167-1171. McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Rigano MM, Dreitz S, Kipnis AP, Izzo AA, Walmsley AM (2005). Oral Dietzschold B, Koprowski H, Michaels FH (1995). Expression of the immunogenicity of a plant-made subunit tuberculosis vaccine. Rabies Virus lycoprotein in transgenic tomatoes. Bio/Technol. 13: Vaccine 24(5): 691-695. 1484-1487. Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, Govea- Mei H, Tao S, Zu YG, An ZG (2006). Research advances on plant Alonso DO, Herrera-Díaz A, Korban SS, Alpuche-Solís AG (2011). vaccine. Acta Genet. Sin. 33(4): 285-293. Immunogenicity of nuclear-encoded LTB:ST fusion protein Misra UK, Kalita J (2010). Overview: Japanese encephalitis. Progress from Escherichia coli expressed in tobacco plants. Plant Cell Neurobiol. 91(2):108-120. Rep. 30(6): 1145-1152. Modelska A, Dietzschold B, Sleysh N, Fu ZF, Steplewski K, Hooper Roy K, Bartels S, Qadri F, Fleckenstein JM (2010). Enterotoxigenic DC, Koprowski H, Yusibov V (1998). Immunization against rabies Escherichia coli elicit immune responses to multiple surface proteins. with plant-derived antigen. PNAS 2481-2485. Infect. Immun. 78: 3027–3035. Moffat AS (1995). Exploring transgenic plants as a new vaccine source. Rybicki EP (2010) Plant-made vaccines for humans and animals. Plant Science 268: 658-660. Biotechnol. J. 8(5): 620-637. Molina A, Veramendi J, Hervas-Stubbs S (2005). Induction of Sala F, Rigano MM, Barbante A, Basso B, Walmsley AM, Castiglione S 6158 Afr. J. Biotechnol.

(2003). Vaccine antigen production in transgenic plants: strategies, in microalgae using heterologous promoter constructs. Plant J. gene constructs and perspectives. Vaccine 21:803–808. 13:427–435. Sandhu JS, Krasnyanski SF, Domier LL, Korban SS, Osadjan MD, Thanavala Y, Yang Y, Lyons P, Mason HS, Arntzen C (1995). Buetow DE (2000). Oral immunization of mice with transgenic tomato Immunogenicity of transgenic plant-derived hepatitis B surface fruit expressing respiratory syncytial virus-F protein induces a antigen. Proc. Nat. Acad. Sci. USA. 92: 3358-3361. systemic immune response. Transgenic Res. 9: 127–35. Tiwari S, Verma PC, Singh PK, Tuli R (2009). Plants as bioreactors for Santi L, Batchelor L, Huang Z, Hjelm B, Kilbourne J, Arntzen CJ, the production of vaccine antigens. Biotechnol. Adv. 27(4): 449-467. Qiang Chen, Mason HS (2008). An efficient plant viral expression Trivedi PK, Nath P (2004). MaExpl, an ethylene-induced expansin from system generating orally immunogenic Norwalk virus-like particles. ripening banana fruit. Plant Sci. 167: 1351–1358. Vaccine 26(15): 1846-1854. Twyman RM, Schillberg S, Fischer R (2012). The production of vaccines Sarwat M, Ahmad P, Nabi G, Hu X (2013). Ca2+ signals: the versatile and therapeutic antibodies in plants, In: A. Wang, S. Ma (ed.) decoders of environmental cues. Crit. Rev. Biotechnol. 33(1):97-109 Molecular farming in plants: Recent advances and future prospects. Scotti N, Rigano MM, Cardi T (2012). Production of foreign proteins Springer Science+Business Media, New York. pp. 145-159. using plastid transformation. Biotechnol. Adv. 30(2): 387-397. Valdes R, Gómez L, Padilla S, Brito J, Reyes B, Álvarez T, Mendoza Sharma M, Sood B (2011). A banana or a syringe: journey to edible O, Herrera O, Ferro W, Pujol M, Leal V, Linares M, Hevia Y, Garc a vaccines. World J. Microbiol. Biotechnol. 27(3): 471-477. C, Milá L, Garcı a O, Sánchez R, Acosta A, Geada D, Paez R, Vega J Shih SMH, Doran PM (2009). Foreign protein production using plant cell L, Borroto C (2003). Large-scale purification of an antibody directed and organ cultures: Advantages and limitations. Biotechnol. Adv. 27: against hepatitis B surface antigen from transgenic tobacco plants. 1036–1042. Biochem. Biophy. Res. Commu. 308(1): 94-100. Shin EA, Park YK, Lee KO, Langridge WH, Lee JY (2009). Synthesis Vianna GR, Cunha NB, Murad AM, Rech EL (2011). Soybeans as and assembly of Porphyromonas gingivalis fimbrial protein in potato bioreactors for biopharmaceuticals and industrial proteins. Gene. Mol. tissues. Mol. Biotechnol. 43(2): 138-147. Res. 10: 1733-1752. Shoji Y, Bi H, Musiychuk K, Rhee A, Horsey A et al. (2009). Plant- Waheed MT, Gottschamel J, Hassan SW, Lössl AG (2012) Plant- derived hemagglutinin protects ferrets against challenge infection derived vaccines: An approach for affordable vaccines against with the A/Indonesia/05/05 strain of avian influenza. Vaccine 27(7): cervical cancer. Hum. Vaccin. Immunother. 8(3): 403-406. 1087-1092. Walmsley AM, Arntzen CJ (2000). Plants for delivery of edible vaccines. Shoji Y, Chichester J A, Jones M, Manceva S D, Damon E, Mett V, Curr. Opin. Biotechnol. 11: 126–129. Musiychuk K, Bi H, Farrance C, Shamloul M, Kushnir N, Sharma S, Wang Y, Deng H, Zhang X, Xiao H, Jiang Y, Song Y, Fang L, Xiao S, Yusibov V (2011). Plant-based rapid production of recombinant Zhen Y, Chen H (2009). Generation and immunogenicity of Japanese subunit hemagglutinin vaccines targeting H1N1 and H5N1 influenza. encephalitis virus envelope protein expressed in transgenic rice. Hum. Vaccin. 7 Suppl: 41–50. Biochem. Biophys. Res. Commun. 380(2): 292-297. Shoji Y, Chichester JA, Bi H, Musiychuk K, de la Rosa P, Goldschmidt Wang Y, Shen Q, Jiang Y, Song Y, Fang L, Xiao S, Chen H (2012). L, Horsey A, Ugulava N, Palmer GA, Mett V, Yusibov V (2008). Plant- Immunogenicity of foot-and-mouth disease virus structural expressed HA as a seasonal influenza vaccine candidate. Vaccine polyprotein P1 expressed in transgenic rice. J. Virol. Meth. 181(1): 26(23): 2930- 2934. 12-17. Simmonds P, Peutherer JF (2003). Hepadnaviruses, In: D. Greenwood, Warzecha H, Mason HS, Lane C, Tryggvesson A, Rybicki R, Williamson R.C.B. Slack, J.F. Peutherer (ed.) Medical microbiology sixteenth A, Clements JD, Rose RC (2003). Oral immunogenicity of human edition. Churchill Living stone Elsevier Science Limited. pp. 438-447. Papillomavirus-like particles expressed in potato. J. Virol. 77(16): Smart V, Foster PS, Rothenberg ME, Higgins TJV, Hogan SP (2003). A 8702-8711. plant-based allergy vaccine suppresses experimental asthma via an Watson J, Koya V, Leppla SH, Daniell H (2004). Expression of Bacillus + low IFN-γ and CD4 CD45RB T Cell-dependent mechanism. J. anthracis protective antigen in transgenic chloroplasts of tobacco, a Immunol. 171: 2116-2126. non-food/feed crop. Vaccine 22: 4374–4384. Streatfield SJ (2006). Mucosal immunization using recombinant plant- Webster DE, Cooney ML, Huang Z, Drew DR, Ramshaw IA, Dry IB, based oral vaccines. Methods 38: 150–157. Strugnell RA, Martin JL, Wesselingh SL (2002). Successful boosting Streatfield SJ, Jilka JM, Hood EE, Turner DD, Baily MR, Mayor JM, of a DNA measles immunization with an oral plant derived measles Woodard SL, Beifuss KK, Horn ME, Delaney DE, Tizard IR, Howard virus vaccine. J. Virol. 76(15): 7910–7912. JA (2001). Plant-based vaccines: unique advantages. Vaccine 19: Webster DE, Thomas MC, Huang Z, Wesselingh SL (2005). The 2742-2748. development of a plant based vaccine for measles. Vaccine 23: Sun M, Qian K, Su N, Chang H, Liu J, Shen G (2003). Foot-and- 1859–1865. mouth disease virus VP1 protein fused with cholera toxin B subunit Wigdorovitz A, Carrillo C, Dus-Santos MJ, Sadir AM, Rios R, Franzione expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol. P, Escribano JM, Borca MV (1999). Induction of a protective antibody Lett. 25 (13): 1087-1092. response to foot and mouth disease virus in mice following oral or Tacket CO (2007). Plant-based vaccines against diarrheal diseases. parenteral immunization with alfalfa transgenic plants expressing the Trans. Am. Clin. Climatol. Assoc. 118: 79–87. viral structural protein VP1. Virology 255: 347–353. Tacket CO (2009). Plant-based oral vaccines: Results of human trials. Wilken LR, Nikolov ZL (2012). Recovery and purification of plant-made Current Topics Microbiol. Immunol. 332: 103-117. recombinant proteins. Biotechnol. Adv. 30(2): 419-433. Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen Xu J, Ge X, Dolan MC (2011). Towards high-yield production of CJ (1998). Immunogenicity in humans of a recombinant bacterial- pharmaceutical proteins with plantcell suspension cultures. antigen delivered in transgenic potato. Nat. Med. 4: 607-609. Biotechnol. Adv. 29(3): 278–299. Tacket CO, Mason HS, Losonsky G, Estes M K, Levine M M, Arntzen Yoshida T, Kimura E, Koike S, Nojima J, Futai E, Sasagawa N, CJ (2000). Human immune responses to a novel Norwalk Virus Watanabe Y, Ishiura S (2011). Transgenic rice expressing amyloid β- vaccine delivered in transgenic potatoes. J. Infect. Disease. 182: 302- peptide for oral immunization. Int. J. Biol. Sci. 7(3): 301-307. 305. Yuan L, Saif LJ (2002). Induction of mucosal immune responses and Tacket CO, Mason HS (1999). A review of oral vaccination with protection against enteric viruses: rotavirus infection of gnotobiotic transgenic vegetables. Microb. Infect. 1: 1-7. pigs as a model. Vet. Immunol. Immunopathol. 87: 147-160. Takagi H, Hiroi T, Yang L, Tada Y, Yuki Y , Takamura K, Ishimitsu R, Yusibov V, Streatfield S J, Kushnir N (2011). Clinical development of Kawauchi H, Kiyono H, Takaiwa F (2005) A rice-based edible plant-produced recombinant pharmaceuticals: Vaccines, antibodies vaccine expressing multiple T cell epitopes induces oral tolerance for and beyond. Hum. Vaccin. 7(3):313-321. inhibition of Th2-mediated IgE responses. Proc. Natl. Acad. Sci. USA Zaslavskaia LA, Lippmeier JC, Kroth PG, Grossman AR, Apt KE (2000) 102(48): 17525–17530. Transformation of the diatom Phaeodactylum tricornutum ten Lohuis MR, Miller DJ (1998). Genetic transformation of (Bacillariophyceae) with a variety of selectable marker and reporter dinoflagellates (Amphidinium and Symbiodinium): expression of GUS genes. J. Phycol. 36(2):379-386.

View publication stats 613272 TAV0010.1177/2051013615613272Therapeutic Advances in VaccinesN. Takeyama et al. research-article2015

Therapeutic Advances in Vaccines Review

Ther Adv Vaccines Plant-based vaccines for animals and 2015, Vol. 3(5-6) 139 –154 DOI: 10.1177/ humans: recent advances in technology 2051013615613272 © The Author(s), 2015. Reprints and permissions: and clinical trials http://www.sagepub.co.uk/ journalsPermissions.nav Natsumi Takeyama, Hiroshi Kiyono and Yoshikazu Yuki

Abstract: It has been about 30 years since the first plant engineering technology was established. Although the concept of plant-based pharmaceuticals or vaccines motivates us to develop practicable commercial products using plant engineering, there are some difficulties in reaching the final goal: to manufacture an approved product. At present, the only plant- made vaccine approved by the United States Department of Agriculture is a Newcastle disease vaccine for poultry that is produced in suspension-cultured tobacco cells. The progress toward commercialization of plant-based vaccines takes much effort and time, but several candidate vaccines for use in humans and animals are in clinical trials. This review discusses plant engineering technologies and regulations relevant to the development of plant-based vaccines and provides an overview of human and animal vaccines currently under clinical trials.

Keywords: GMP-compliant, human vaccine, plant-based vaccine, plant transformation, veterinary vaccine

Introduction 2002. TrypZean is particularly useful in animal Correspondence to: Yoshikazu Yuki, PhD In the past quarter century, plant genetic engineer- cell cultures because it has no contaminants of ani- Division of Mucosal ing technologies have progressed dramatically. mal origin. Rice has been used to manufacture Immunology, Department of Microbiology and Barta and colleagues were the first to transcribe a human lysozyme and lactoferrin [Hennegan et al. Immunology, The Institute chimeric gene of nopaline synthase and human 2005; Yang et al. 2002]. Protalix, an Israeli com- of Medical Science, The University of Tokyo, 4-6-1 growth hormone in sunflower and tobacco plants pany, has developed a method to produce plant- Shirokanedai, Minato-ku, using the Ti plasmid [Barta et al.1986]. Shortly based biopharmaceuticals in cultured transgenic Tokyo 108-8639, Japan [email protected] thereafter, mouse monoclonal antibody was pro- carrot or tobacco cells [van Dussen et al. 2013; Natsumi Takeyama, PhD, duced and functionally assembled in tobacco leaf Zimran et al. 2011]. In 2012, Protalix and its part- DDS Division of Mucosal segments [Hiatt et al. 1989]. As bioreactors, plants ner Pfizer received approval from the United States Immunology, The Institute may yield high amounts of recombinant proteins; Food and Drug Administration (FDA) of the of Medical Science, The University of Tokyo, Tokyo, these proteins are not contaminated with patho- United States for taliglucerase alfa for Gaucher’s Japan gens of animals or humans and can be stored with- Disease. Research Department, Nippon Institute for out refrigeration at low cost. A number of Biological Science, Ome, recombinant proteins have been produced in On the other hand, plant-based human vaccines Tokyo, Japan plants, and the production of protein-based phar- are not yet commercialized, although production Hiroshi Kiyono, PhD Division of Mucosal maceuticals has partially shifted from bacterial, of dozens of viral and bacterial subunit vaccines is Immunology, The fungal, and mammalian cell cultures to plants and attempted in transgenic plants. Recombinant Institute of Medical Science, The University plant cell cultures [Lico et al. 2012; Merlin et al. subunit vaccines are safer than traditional vac- of Tokyo, Tokyo, Japan 2014; Twyman et al. 2005]. Commercialized cines, because they contain no live pathogens. International Research and Development Center enzymes and reagents produced in plants are avail- Various plants such as tobacco, rice, maize, for Mucosal Vaccines, able. For instance, human type I collagen, which potato, alfalfa, lettuce, tomato, carrot, peanut, The Institute of Medical Science, The University of can self-assemble into fine homogenous fibrils, is and soybean are used as hosts for gene introduc- Tokyo, Tokyo, Japan manufactured in tobacco plants [Shoseyov et al. tion, which is achieved in vitro by using protoplast 2014]. Bovine trypsin produced in maize, TrypZean or cell culture, or hairy root culture. Nuclear or (Sigma-Aldrich), has been on the market since chloroplast genome recombination is routinely

http://tav.sagepub.com 139 Therapeutic Advances in Vaccines 3(5-6)

used to obtain transgenic plants. The choice of Agrobacterium-based nucleus transformation the plant species and technology determines the Plant recombinant technologies are being con- vaccine administration route because some plants stantly improved and diversified [Hefferon, can be consumed only when processed, whereas 2014] (Figure 1). Genes can be introduced into heat or pressure treatments may destroy the anti- plants either directly or by using the gram-nega- gen. Cereal crops are attractive for subunit vac- tive bacterium Agrobacterium tumefaciens. Plant cine production because vaccines produced in transformation with polyethyleneglycol or by seeds are stable over long storage periods usually includes protoplast prep- [Hefferon, 2013]. aration by removing the cell wall, which requires time and skill. Almost half of plant transforma- There are two options for vaccine administration: tion technologies use A. tumefaciens, which injection (intramuscular or subcutaneous) and infects plants naturally. The T-DNA region mucosal (oral or nasal) administration. Injection- between the left and right borders of the A. type vaccines elicit strong protective immunity by tumefaciens Ti plasmid is introduced into plant preferentially inducing IgG production. They are genome and transcribed in the plant cell; this most suitable against pathogens that infect via a process induces abnormal plant hormone pro- systemic or respiratory route; however, the anti- duction, resulting in crown gall disease. The gens have to be purified before administration. T-DNA region can be replaced with a gene of These vaccines are often produced in tobacco interest, and the Ti plasmid has been modified plants using transient expression. into a binary vector that can be manipulated in Escherichia coli [Bevan, 1984]. Selection pressure Oral- or nasal-type vaccines induce mucosal and is used to establish stable integration of the gene systemic immunity [Azegami et al. 2014; of interest in the nuclear genome. Once the trans- Lamichhane et al. 2014]. In a conceptual sense, oral genic line stably producing the target protein is plant-based vaccines are ideal because the manufac- established, it can be used as a permanent source turing process is simple; no additional medical of vaccine and established as a master seed bank. devices are needed for injection; and the antigen A stable and characterized line can be adopted as immunogenicity and biological activities are pre- Good Manufacturing Practice (GMP)-compliant served in the gastrointestinal tract due to their natu- production. Yet, the development of such lines is ral bioencapsulation in a plant cell organelle. Oral time-consuming and can be complicated by gene plant-based vaccines have been developed in edible silencing, host genome damage, or hybridization plants, including rice, maize, potato, lettuce, and with non-transgenic crops cultivated without carrot. Once these vaccines pass through the gastric strict regulations. environment and reach the small intestine, antigens are incorporated into M cells in the follicle-associ- ated epithelium (FAE) for the induction of mucosal Plastid transformation and systemic immune responses [Azegami et al. A new technology, which targets the chloroplast 2014; Holmgren and Czerkinsky, 2005]. genome instead of the nuclear genome, is now available. Chloroplasts originate from cyanobac- This review discusses technologies and regula- teria that were incorporated into algae. tions in the development of plant-based vaccines Chloroplast and nuclear genomes coevolved, and and recent achievements in the production of vac- now chloroplast genomes possess only 100–250 cines that are already or expected to be under genes, which is smaller than nuclear genomes. clinical trials and are intended for worldwide dis- The chloroplast genome is maternally inherited, tribution in the near future. and plants can stably produce protein without transgene outcrossing via pollen. Multiple chloro- plasts with a high transgene copy number can Recombinant technologies accumulate large amounts of recombinant pro- To use plants as bioreactors for commercial vac- tein (as much as 70% of total leaf protein [Oey cines, one needs to (a) attain a high expression level et al. 2009]). Foreign genes are usually trans- of recombinant genes, (b) be able to quickly and formed into chloroplast DNA by biolistic process easily design and produce new antigens in response or polyethylene glycol treatment of protoplasts to new pathogen subtypes, and (c) identify the [Cardi et al. 2010]. For plastid transformation, a genes to be transfected and ensure the safety of pro- target gene and selectable marker genes are placed duced proteins for use in humans or animals. between the two flanking sequences originated

140 http://tav.sagepub.com N Takeyama, K Hiroshi et al.

Figure 1. Plant transgenic technologies and their advantages and disadvantages. VLP, virus-like particle.

from the chloroplast genome to induce homolo- virus, and plum pox virus [Salazar-González et al. gous recombination between the vector and plas- 2015]. First-generation plant viral expression tid genome [Verma and Daniell, 2007; Scotti vectors encode almost all viral complements; the et al. 2012]. The chloroplasts of tobacco and gene encoding the protein or peptide of interest is other leafy plants such as carrot, petunia, and let- placed downstream of viral polymerase, move- tuce have mainly been used; nonphotosynthetic ment protein, and coat protein genes. This tech- plant organs are less efficient in producing target nology has been employed to produce various proteins [Rigano and Walsley, 2005; Verma and plant-made vaccines, such as those against human Daniell, 2007]. papilloma virus [Cerovska et al. 2012; Noris et al. 2011] and influenza virus [Ravin et al. 2012; Shoji et al. 2011], by modifying PVX or TMV and a Transient expression with plant virus vaccine against [Lai and Chen, 2012] expression vectors by modifying TMV. These recombinant viruses The benefit of transient expression using plant retain infectivity to plants and then shed the viral vectors is their high replication ability in the transgene, which may spread to other plants, thus target plant, resulting in high vaccine yield. prompting safety concerns. Second-generation Several plant viruses are used for this purpose: viral vectors, which are safe in natural environ- tobacco mosaic virus (TMV), cowpea mosaic ments, have been developed. These vectors rely virus (CPMV), potato virus (PVX), alfalfa mosaic on an integrated system that has the minimal

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Table 1. GMP-compliant plant factories for biopharmaceuticals.

Company Location Plant Bioproduct Kentucky BioProcessing Owensboro, KY, USA Tobacco, potato Norovirus VP1 Ebola LLC (KBP) virus antibody (ZMapp) Sigma-Aldrich Fine St. Louis, MO, USA Maize Trypsin Chemicals Medicago Inc. Quebec, Canada Nicotiana benthamiana Influenza HA-VLP Protalix Carmiel, Israel Carrot cells, tobacco Alphataliglicerase cells Caliber Biotherapeutics Byran, TX, USA Tobacco Influenza HA LLC Fraunhofer CMB USA Newark, DE, USA Nicotiana benthamiana Influenza HA Fraunhofer IME Aachen, Germany Tobacco Antibody (for HIV) National Institute of Hokkaido, Japan Strawberry Canine interferon alpha Advanced Industrial Science and Technology Institute of Medical Science, Tokyo, Japan Rice Cholera toxin B subunit The University of Tokyo

number of viral elements required for vector rep- 2013]. Another advantage of this protocol is that lication, whereas other functions, such as DNA a variety of T-DNA vectors can be used. delivery, are provided by nonviral elements. Agroinfiltration has also been applied to other These ‘deconstructed’ expression vectors usually leafy plants, such as lettuce [Chen et al. 2013]. provide higher yields than those attained with full-virus vectors [Peyret and Lomonossoff, 2013; ‘Magnifection’ has been developed to address vari- Salazar-González et al. 2015]. ous safety concerns, namely the use of intact viral expression vectors and possible transgene loss dur- ing systemic spreading. It combines the agroinfil- Infiltration technologies for transient tration method with the delivery of cDNA encoding expression a ‘deconstructed’ TMV-based vector [Gleba et al. New time-saving technologies to introduce 2004, 2005, 2014; Marillonnet et al. 2004]. The recombinant genes into plants have also been magnifection system is restricted to N. benthamiana. developed. Protocols called ‘agroinfiltration’ and Icon Genetics, a German plant biotechnology ‘magnifection’ use vacuum or syringe to infiltrate company, has developed and adapted this technol- leaves of 6-week-old plants such as Nicotiana ogy as MagnICONTM for the manufacturing of benthamiana or Arabidopsis with Agrobacterium various plant-based vaccines, including high containing either binary vectors or deconstructed amounts of hepatitis B virus (HBV) surface anti- viral vectors [Leuzinger et al. 2013]. The use of gen (HBsAg; up to 300 mg/kg N. benthamiana agroinfiltration in vaccine production was pio- fresh leaves) in the form of VLP [Huang et al. neered by the Canadian biotechnology company 2006], norovirus capsid proteins [Scotti and Medicago, which developed virus-like particle Rybicki, 2013; Rybicki, 2014], and non-Hodgkin (VLP) vaccines for influenza HA antigens, and lymphoma vaccines, which proceeded to a phase I these vaccines were used in a clinical trial [Landry clinical trial (Table 1) [McCormick et al. 2008]. et al. 2010]. Antigenicity of human and animal viruses is often determined by the conformation A group from Fraunhofer USA Center for of their surface proteins, and to acquire protective Molecular Biotechnology (CMB) has developed immunity, it is desirable to express antigens in a ‘launch vector’, an advanced gene infiltration sys- VLP form [Kushnir et al. 2012; Vacher et al. tem that combines the elements of TMV vector 2013]. Agroinfiltration uses suspensions of A. and A. tumefaciens binary plasmids [Musiychuk tumefaciens; this method allows the production of et al. 2007]. The hybrid launch vector pBID4 large amounts of vaccine proteins within a few contains the 35S promoter from cauliflower days to a couple of weeks, which is much faster mosaic virus (35S CaMV) that drives transcrip- than in stable expression systems [Leuzinger et al. tion of the viral genome, the nopaline synthase

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(nos) terminator, genes for virus replication and (RNAi) using the same T-DNA vector enhanced cell-to-cell movement proteins, and the target the production of botulinum neurotoxin A or gene cloned under the transcriptional control of cholera toxin B subunit in rice seeds [Kuroda the coat protein subgenomic mRNA promoter. et al. 2010; Kurokawa et al. 2013; Yuki et al. Following infiltration, primary transcripts pro- 2012]. Other options to increase protein produc- duced in the nucleus are transported into the tion in plants include modification of codon usage cytoplasm, resulting in robust protein production from that of the host to that of the plant [Hiwasa- [Musiychuk et al. 2007]. Tanase et al. 2011; Jackson et al. 2014], intron introduction in TMV before the target gene The pEAQ system is based on full-length or trun- [Chakravarthi et al. 2015], and co-expression of a cated versions of CPMV RNA-2 and permits effi- suppressor of gene silencing [Garabagi et al. 2012; cient and rapid protein production without viral de Ronde et al. 2014]. replication [Peyret and Lomonossoff, 2013; Sainsbury et al. 2009]. In pEAQ system, a series N-glycosylation is relatively well-conserved in eukar- of small binary vectors, which may be used for yotes; however, there are several sugar-modification production of a wide variety of proteins in both enzymes specific to plants. Knockout of genes for transient and stable expression systems, was engi- plant-specific N-glycan-processing enzymes and the neered. These vectors contain the 35S CaMV introduction of the enzymatic machinery catalyzing promoter, nos terminator, the P19 sequence synthesis, transport, and addition of mammalian encoding a suppressor of silencing, and 5′- and sugars have been reported [Gomord et al. 2010]. 3′-UTRs from CPMV RNA-2. The gene of inter- Whereas this strategy is effective for producing native est is inserted between the UTRs. A new-genera- forms of viral antigens, unexpected N-glycosylation tion vector, pCPMV-HT, provides extremely of vaccine antigens of bacterial origin produced in high translational efficiency and, consequently, a plants may result in molecular heterogeneity and dif- high level of the recombinant protein [Peyret and ficulties in recognition by immune cells. Because Lomonossoff, 2013]. N-glycosylation occurs at asparagine residues, the substitution of asparagine with aspartic acid or another amino acid can solve this problem [Yuki Other methods with improved performance et al. 2013]. of Engineering of genetically modified plants with- out the use of antibiotic resistance genes as selec- Risk analysis and regulations tion markers eliminates the potential risk of transfer of these genes to gut microbes when the Potential risks vaccine is orally administered or to the environ- Several considerable risks are associated with ment. Using Agrobacterium-mediated nucleus plant-based pharmaceuticals [Kirk et al. 2005]. transformation, Mejima and colleagues cotrans- Plant-made vaccines, particularly the oral ones, formed the selection marker hygromycin phos- might induce either allergenicity or oral tolerance, photransferase gene and cholera toxin B subunit which are two conflicting phenomena. Post- gene encoded by separate T-DNA vectors and translational modifications such as N-glycosylation selected marker-free candidate plants by segrega- might induce allergic responses, whereas co- tion in the seed progeny [Mejima et al. 2015]. administration with oral adjuvants to broadly Daniell and colleagues used betaine aldehyde stimulate the mucosal immune system might dehydrogenase (BADH) gene from spinach as a induce hypersensitive responses to other proteins selectable marker for plastid transformation in daily food [Guan et al. 2013]. Frequent admin- [Daniell et al. 2001]. This enzyme is naturally istration of plant-made vaccines via the oral route produced in the chloroplast and converts toxic can enhance regulatory T-cell activation against betaine aldehyde into non-toxic glycine betaine. vaccine antigen [Fujihashi et al. 1999], as seen in BADH transgenic tobacco plant showed higher successful hyposensitization therapy by oral anti- BADH activity than did nontransgenic plants, gen intake in cases of pollen allergy or food allergy indicating that transgenic plants could be selected [Cox et al. 2012; Sato et al. 2014]. by the level of enzyme activity. Another potential risk of the use of plant-made vac- Knocking down mRNAs for rice storage proteins, cines is their influence on the environment. Small- glutelin and 13K prolamin, by RNA interference scale production of genetically modified plants

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(including their production for research purposes) The European Medicines Agency (EMA) pub- must be managed. Even manufacturing in regulated lished draft guidance notes on ‘the quality of bio- facilities to control the quality and safety of the logical active substances produced by stable products may pose difficulties in the use of geneti- transgene expression in higher plants’ [EMA, cally modified plants. Another aspect is that open- 2008], which complement the FDA and USDA field production of stably transfected plants increases regulations. However, these notes cover only the possibility of contamination of nontransgenic plants with stable expression of transgenes and crops intended for human or animal consumption exclude transiently transfected plants and plant [Bawa and Anilakumar, 2013; Fischer et al. 2012]. cell cultures. The use of several transient expres- Recombinant genes can spread to field crops via sion technologies, which rely on plant viral vectors pollen and accidentally result in genetic modifica- or agroinfiltration (or both), requires advanced tions in nontarget plants. Genetically modified regulation applicable to all plant transgenic tech- plants might be eaten by wild animals or acciden- nologies. An interesting point in the EMA guide- tally harvested by humans. Contact with insects and lines is a statement of the importance of the the release of contaminated water to the environ- establishment of master and working seed banks ment are also possible mechanisms for DNA or from the final transformant. Both banks should be antigen escape. The probability and severity of each well-characterized in respect to the transgene risk will depend on the plant species and the antigen (sequence, integrity, site of insertion, copy num- and has to be determined on a case-by-case basis for ber, and the fate of the marker sequence), recom- each plant-based vaccine. binant protein expression (tissue and organ specificity, regulation, and expression level), and unintended changes in the levels of endogenous Government regulations plant proteins. The storage properties (conditions, In the United States, the FDA ensures the safety shelf-life, and closure criterion) of the master for both manufacturing and clinical use of plant- transgenic bank should be defined also. The based biopharmaceuticals and vaccines; it also authorities of European Authority approves and licenses them. Similar to other (EFSA) and EMA partially overlap. The former biopharmaceuticals, plant-based vaccines should cares for the cultivation of transgenic plants, and be free of impurities, including other transgenes the latter cares for biopharmaceutical products and resistance marker products, which must all from transgenic plants. Importantly, EMA regula- be evaluated under the same criteria. The FDA tory guidelines [EMA, 2008] indicate that all also approves GMP facilities for plant manufac- biopharmaceutical products intended for phase I turing. In 2005, the World Health Organization trials should be manufactured according to GMP. (WHO) conducted an ‘informal consultation on Thus, GMP compliance appears to be a key point scientific basis for regulatory evaluation of can- in developing plant-based vaccines for clinical use. didate human vaccines from plants’ [van der Laan et al. 2006]. In its report, the WHO recom- mended that the guidelines on Good Agricultural GMP facilities for plant bioreactors and Collection Practices (GACP), which are Production of recombinant proteins used in phar- typically applied to herbal plants, should also be maceutical applications requires certain quality applied to plants producing biopharmaceuticals. standards. The GMP grade is compulsory for A report of quality-control methods for medici- clinical applications. At least two large biomanu- nal plant materials recommended tests to assess facturing facilities are capable of producing plant- the identity, purity, and content of biopharma- derived HA proteins under GMP conditions, ceutical plant materials. The United States Fraunhofer CMB [Shoji et al. 2011, 2015] in the Department of Agriculture (USDA) also plays a United States and Medicago Inc. in Canada key role in the introduction of plant-made phar- [Yusibov et al. 2014]. Fraunhofer CMB is situated maceuticals. The USDA approves veterinary in Delaware. It possesses a GMP pilot plant that is biologics such as vaccines. The USDA considers capable of dealing with regulatory and clinical the nature of the plant, the probability of cross- affairs and technology transfer. The key process- contamination, and the genetic background of ing areas in the GMP plant are equipped with stably transfected plants. The USDA also con- complete processing cycles for transient expres- siders the risk-management strategies, taking sion in plants, such as plant and bacterium culti- into account physical and geographical aspects vation, infiltration, plant harvest, and protein and plant reproduction. purification. The major product in this facility is

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Table 2. Plant-based human vaccines in clinical trials.

Pathogen or Antigen Plant Expression Administration Clinical Reference disease system route trial Enterotoxigenic LTB Potato Transgenic Oral Phase I Tacket et al. [1998] E. coli Enterotoxigenic LTB Maize Transgenic Oral Phase I Tacket et al. [2004] E. coli Norovirus Capsid protein Potato Transgenic Oral Phase I Tacket et al. [2000] Hepatitis B virus Viral major surface Lettuce Transgenic Oral Phase I Kapusta et al. [1999] protein Hepatitis B virus Viral major surface Potato Transgenic Oral Phase I Thanavala et al. protein [2005] Rabies virus Glycoprotein and Spinach Viral vector Oral Phase I Yusibov et al. [2002] nucleoprotein (fusion) (transient) Influenza virus HA Nicotiana Launch vector Intramuscular Phase I Chichester et al. (H5N1) benthamiana (transient) [2012] Influenza virus HA Nicotiana Launch vector Intramuscular Phase I Cummings et al. (H1N1; 2009 benthamiana (transient) [2014] pandemic) Influenza virus HA (H5; VLP) Nicotiana Agrobacterial Intramuscular Phase I D’Aoust et al. [2008] (H5N1) benthamiana binary vector Phase II Landry et al. [2010] (transient) Influenza virus HA (H7; VLP) Nicotiana Agrobacterial Intramuscular Phase I Medicago Inc. (http:// (H7N9) benthamiana binary vector www.medicago.com) (transient) Influenza virus HA (VLP) (seasonal; Nicotiana Agrobacterial Intramuscular Phase I Medicago Inc. (http:// quadrivalent) benthamiana binary vector www.medicago.com) (transient) Cholera CTB Rice Transgenic Oral Phase I Nochi et al. [2009] Yuki et al. [2013]

influenza HA antigen, which is produced by using Human vaccines in clinical trials plant-based Proficia™ technology, VLPs, and the Only a few plant-based human vaccines have VLPExpress™ platform for transient expression reached clinical trials (Table 2). Non-toxic B sub- in N. benthamiana. Medicago Inc., located in unit of heat-labile enterotoxin (LTB) of entero- Québec, is now proceeding to phase II clinical toxigenic E. coli (ETEC) produced either in potato trial with influenza VLP (H5) produced in N. or maize was administered orally to healthy volun- benthamiana by using the agroinfiltration method teers to examine its safety and immunogenicity. (see http://www.medicago.com). Rabies and rota- Raw, diced, transgenic potato tubers containing virus antigens produced by Medicago Inc. are at a LTB (0.4 or 1.1 mg) were given to volunteers on preclinical stage. Some university-launched plant- days 0, 7, and 21 [Tacket et al. 1998, 2007; made vaccines that reached phase I clinical trials Yusibov et al. 2011]. For maize-derived LTB, the have been produced in collaboration with GMP clinical study was placebo-controlled, and each facilities. Norovirus capsid protein subunit vac- group received 2.1 g of either transgenic or wild- cine produced in potato tubers was developed in type maize germ meal suspended in water on days Arizona State University and manufactured at 0, 7, and 21 [Tacket et al. 2004, 2007]. No adverse Kentucky BioProcessing GMP plant [Tacket et al. effects of vaccination were noticed in comparison 2000]. In Japan, rice-based cholera vaccine has with the placebo control. LTB-specific IgA- been developed at the Institute of Medical Science, secreting cells were detected in peripheral blood 1 The University of Tokyo (IMSUT); test vaccine week after the first vaccination. Serological survey produced at the GMP facility at IMSUT is now in revealed that vaccinated volunteers had increased a phase I clinical trial [Yuki et al. 2013; Mejima levels of LTB-specific serum IgG (91% of the vol- et al. 2015] (Table 1). unteers) and IgA (55%) on day 59.

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Norovirus capsid protein VP1 was produced in rabies vaccine in endemic areas is needed. potato tubers in the same way as LTB. As norovi- Antigenic determinants of rabies virus G and N rus is a nonenveloped virus, VP1 is the only pro- proteins have been mapped, and a synthetic chi- tein of the capsid. Vaccination with approximately meric peptide (G5–24-N31D) containing a linear 500 µg of recombinant VP1 was done on days 0, epitope of G protein and an epitope of N protein 7, and 21 (three doses) or on days 0 and 21 (two was found to be immunogenic in mice [Dietzschold doses). Some volunteers had VP1-specific anti- et al. 1990]. Yusibov et al. [2002] fused fragments bodies before the study because norovirus is encoding a chimeric protein of G protein (amino highly infectious, and repeated epidemics are fre- acids 253–275) and N protein (amino acids 414– quent, especially in winter [Campos and Lees, 418) with that of alfalfa mosaic virus coat protein 2014; Teunis et al. 2008]. However, 20% of vac- and introduced this fusion construct into TMV cinated volunteers developed VP1-specific serum lacking native coat protein. Spinach was infected IgG titers. The geometric mean of IgG titers (four with recombinant virus to obtain transient expres- responders) was 1:67 before vaccination and sion of the chimeric rabies peptide. Three of 5 vol- 1:757 after vaccination [Tacket et al. 2000]. unteers previously vaccinated with a commercial injection-type vaccine had elevated rabies-specific According to the WHO, almost 780 000 people IgG after having ingested 3 doses of spinach (20 g; die every year from HBV infections worldwide. In 84 µg of chimeric rabies peptide each) at 2-week different regions, 1–10% of the adult population intervals. Another protocol involved volunteers are chronically infected with HBV, whereas with no history of rabies vaccination; five of nine 80–90% of infants infected under the age of 6 participants responded to the rabies antigen. An months develop chronic infections that lead to additional single dose of commercial vaccine cirrhosis or liver cancer [MacLachlan and Cowie, enhanced rabies virus-neutralizing antibody pro- 2015; Norkrans, 1990]. HBV surface antigen duction (three out of nine participants). Regardless (HBsAg) was produced in plants. HBsAg trans- of the vaccination order, spinach oral rabies vac- genic lettuce leaves (0.1–0.5 µg of HBsAg per cine in combination with currently available vac- 100 g of fresh tissue) were given to adult volun- cines might enhance immunity against rabies virus teers (initially 200 g, then 150 g within 2 months). [Yusibov et al. 2002]. Two of three vaccinated volunteers showed tran- sient protective levels of HBsAg-specific IgG Influenza virus has 16 hemagglutinin (HA) sub- (above 10 IU/l) 2 weeks after the second vaccina- types, and even in the strains with the same sub- tion, but no HBsAg-specific serum IgA was type, antigenic shift often occurs to abolish detected [Kapusta et al. 1999]. cross-protective immunity of the host. For exam- ple, in 2009, H1N1-type influenza virus became Another clinical study was conducted with oral pandemic [Itoh et al. 2009; Neumann et al. HBsAg produced in transgenic potato [Thanavala 2009]. To control its spread by vaccination, fast et al. 2005]. All volunteers enrolled in this study production of a new HA antigen was required. As had received three doses of HBV injection-type mentioned above, Medicago Inc. developed the vaccine within 15 years. The placebo group was technology to produce VLPs of influenza virus given nontransgenic potato, the two-dose group HA in N. benthamiana with the A. tumefaciens- was vaccinated at 0 and 28 days with 100 g of based transient expression system [D’Aoust et al. transgenic potato (850 ± 210 µg of antigen), and 2008; Yusibov et al. 2011; US patent application the three-dose group was vaccinated with the number 20130183341]. VLPs of the expected same doses at 0, 14, and 28 days. The authors size were found between the plasma membrane found that 52.9% of participants in the two-dose and the cell wall of N. benthamiana cells [D’Aoust group and 62.5% of participants in the three-dose et al. 2008]. Phase I and II clinical trial of the group had elevated serum HBsAg antibody titers VLP composed of HA protein of H5N1 influenza over the 70-day follow-up period after the first virus (A/Indonesia/5/05) (H5-VLP) has been immunization. completed. In a phase I clinical trial, 5, 10, or 20 µg of H5-VLP was subcutaneously injected Current human rabies vaccines are efficacious twice with alum adjuvant [Landry et al. 2010]. both pre- and post-exposure to rabies virus The vaccine-induced hemagglutinin inhibition [Toovey, 2007]. Endemic rabies is spreading all titer at all tested doses. In addition, a phase II over the world except in a few countries. To reduce clinical trial of H5-VLP was conducted as a ran- mortality caused by rabies, a regular stock of safe domized, placebo-controlled, dose-ranging study

146 http://tav.sagepub.com N Takeyama, K Hiroshi et al. that used 20, 30, or 45 µg of H5-VLP [Landry titers and were observed in the group immunized et al. 2014]. After 6 months of vaccination with with the highest dose (90 μg) without adjuvant. H5-VLP, the volunteer group showed cross-pro- tective CD4+ T-cell responses, which were not observed in the placebo group, indicating strong Development of veterinary vaccines induction of long-term cell-mediated immunity Antigens of farm-animal pathogens have been by plant-made H5-VLP. The immunogenicity of expressed in plants, and a few of them have been plant-specific glycans has also been studied in tested in host species. Plant-made vaccines for this clinical trial; some vaccine recipients devel- veterinary use are listed in Table 3. The first oped plant N-glycan-specific allergic or hyper- USDA-approved plant-made vaccine was for sensitivity symptoms. Some volunteers (34%) veterinary use: Newcastle disease vaccine for developed transient IgG and, in some cases, IgE poultry from the USDA Center for Veterinary to plant glyco-epitopes, but no IgE responses to Biologics was approved in 2006 [reviewed in mannose residues (MMXF motifs) were Floss et al. 2007; Kolotilin et al. 2014; Rybicki observed. The levels of antibodies returned to 2010]. Dow AgroSciences LCC (Indianapolis, baseline by 6 months in most participants [Ward IN, USA) produces hemagglutinin and neurami- et al. 2014]. Medicago Inc. also completed phase nidase of Newcastle disease virus in suspension- I clinical trial using 5, 13, or 28 µg of H1N1 cultured tobacco cells [reviewed in Yusibov et al. influenza (A/California/7/09) VLP (H1-VLP) 2011]. Two subcutaneous doses of this vaccine vaccine [Landry et al. 2010]. All doses tested at a 2-week interval administered to neonatal were safe and well-tolerated and induced immune chicks ensure 90% protection against Newcastle response to the virus, including cell-mediated disease virus challenge. For farm animals, the immunity. The company intends to proceed cost of immunization tends to limit profit from phase IIa trial of its seasonal trivalent vaccine selling products such as meat, milk, and eggs. with antigens from the recommended pandemic Therefore, plant-based vaccines are an asset for H1N1, H3N2, and B influenza strains the animal use if they can be manufactured at [Redkiewicz et al. 2014]. low cost. Moreover, edible vaccines require little effort for administration. In poultry, in addition Another plant-based influenza vaccine that has to the approved vaccine mentioned above, glyco- completed phase I clinical trial is in production at protein of Newcastle disease virus has been Fraunhofer CMB USA. HA from A/California/ expressed in potato, tobacco, maize, and rice 04/2009 H1N1 (HAC1) and A/Indonesia/05/ [Guerrero-Andrade et al. 2006; Kolotilin et al. 05 H5N1 (HAI-05) has been produced in 2014; Zhou et al. 2004]. S1 glycoprotein gene of N. benthamiana by using an infiltration method of chicken infectious bronchitis virus (IBV) has A. tumefaciens in which genes are regulated by the been introduced into potato [Zhou et al. 2004]. ‘launch vector’ [Shoji et al. 2011, 2015]. Pre- Oral immunization with transgenic potato tubers clinical studies using mice and rabbits were con- (5 g; 12.45 µg of S protein) three times or intra- ducted by the injection of HAC1 and HAI-05 muscular immunization with transgenic potato twice with 3-week-intervals. Seropositive rate of extracts two or three times elicited high neutral- serum HA antibody responses were 100% in mice izing antibody titers against IBV in chicken with the dose of 5 µg for HAC1, and 45 µg for serum. These levels were similar to those in HAI-05 in a dose-dependence test with alum- chickens immunized with live attenuated intra- adjuvant. Rabbits also showed seropositive for nasal vaccine and conferred 60–80% protection HA with the dose of 90 µg in both HAC1 and HAI- from a virulent IBV strain. Lymphocyte prolifer- 05 [Shoji et al. 2011]. A phase I clinical trial for ation and IL-2 production by spleen cells were HAC1 and HAI-05 was conducted as a rand- confirmed in vitro, indicating that the potato- omized, double-blind, placebo-controlled study based vaccine induced protective immunity. To with healthy 18–49-year-old volunteers [Chichester protect chicken against infectious bursal disease et al. 2012; Cummings et al. 2014]. In both cases, virus (IBDV), the protective antigen VP2 was three doses (15, 45, and 90 μg) of purified antigen expressed in rice and tobacco [Gómez et al., with or without Alhydrogel® (as adjuvant) were 2013; Wu et al. 2007], and efficiently protected administered twice intramuscularly. Nearly all chickens from a highly virulent IBDV strain. adverse events were mild to moderate; the highest responses were detected by hemagglutining inhibi- Plant-based vaccines for pig protection from tion (HI) and viral-neutralizing (VN) antibody ETEC and foot and mouth disease virus (FMDV)

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Table 3. Plant-based vaccines for veterinary use.

Host Pathogen Antigen Plant Administration Treated animal Reference route Chicken Newcastle Hemagglutinin- Tobacco Subcutaneous Chicken Vermij et al. [2006] disease neuraminidase suspension cells Approved by USDA Chicken Newcastle F protein Maize Oral Chicken Guerrero-Andrade disease et al. [2006] Chicken Newcastle F protein Rice Oral Mice Yang et al. [2007] disease Chicken IBV S1 glycoprotein Potato Oral Chicken Zhou et al. [2004] Chicken IBDV VP2 Rice Oral Chicken Wu et al. [2007] Pig ETEC Fimbriae (F4) Tobacco N/D Pig (in vitro Kolotilin et al. [2012] (chloroplast) assay in intestines) Pig ETEC Fimbriae (F4) Alfalfa Oral Piglet Joensuu et al. [2006] Pig ETEC Cholera toxin B Rice Oral Pig Takeyama et.al. [2015] subunit Pig ETEC Fimbriae (F4) Barley Subcutaneous Mice Joensuu et al. [2006] Pig Foot and VP1 Nicotiana Intramuscular Pig Yang et al. [2007] mouth bentamiana disease virus Pig TGEV S protein Tobacco Intramuscular Pig Tuboly et al. [2000] Cattle Bovine gD protein Tobacco Intramuscular Cattle Pérez Filgueira et al. Herpesvirus and [2003] subcutaneous Cattle Bovine Viral E2 protein Alfalfa Intramuscular Cattle Peréz Aguirreburualde Diarrhea et al. [2013] Virus Cattle Rinderpest Hemagglutinin Peanut Oral Cattle Khandelwal et al. virus [2003]

have been well characterized. ETEC fimbriae (of amino acid sequence and conformation of this type F4) were produced in alfalfa chloroplasts toxin are similar to those of cholera toxin. The use and remained stable for 2 years when alfalfa was of CTB-producing rice, named MucoRice-CTB, dried and stored at room temperature [Joensuu ensured high antigen stability in the gastrointesti- et al. 2006]. Recombinant proteins in combina- nal tract and mucosal immune induction in a tion with cholera toxin as an adjuvant were intro- mouse model [Nochi et al. 2007; Tokuhara et al. duced intragastrically into piglets, resulting in a 2010]. Pregnant sows and weaned minipigs pro- reduction in ETEC excretion in their feces. duced antigen-specific IgG and IgA in their sera Kolotilin and colleagues stably expressed F4 fim- upon MucoRice-CTB immunization. MucoRice- brial adhesin FaeG in tobacco chloroplasts (1% CTB also induced maternal CTB-specific IgG of dry leaf weight or 11.3% of total soluble leaf and IgA in the colostrum and milk of sows after protein) [Kolotilin et al. 2012]. Although the farrowing. These antigen-specific maternal anti- authors did not challenge vaccinated animals, bodies offer newborn piglets passive immuniza- they showed that recombinant F4 competitively tion with ingested milk. CTB-specific antibodies inhibited the attachment of F4-positive ETEC to also were secreted into the gut lumen of weaned pig small-intestinal villi in vitro [Kolotilin et al. piglets and reduced intestinal loop fluid accumu- 2012]. We have produced rice-based cholera lation upon ETEC challenge, indicating a protec- toxin B subunit (CTB) vaccine, which is efficient tive effect of MucoRice-CTB against ETEC against pig ETEC heat-labile toxin because the diarrhea. Therefore, MucoRice-CTB could be a

148 http://tav.sagepub.com N Takeyama, K Hiroshi et al. candidate oral vaccine for inducing both passive vaccines in humans and animals. First, we need to and active immunity to protect both suckling and determine the eligible combinations of target plants weaned piglets from ETEC diarrhea [Takeyama and transgenic protocols. Unlike open-air farming, et al. 2015]. the production of transgenic plants for biothera- peutic use is strictly regulated. Plant selection FMDV infects pigs and cows but not humans and would affect the whole procedure throughout com- causes a major animal disease, the prevention of mercialization. Transient expression systems ena- which requires international cooperation. Yang ble the rapid production of high amounts of target and colleagues expressed VP1 of FMDV on the proteins, but their implementation is complex surface of bamboo mosaic virus in N. benthamiana because it requires infiltration and the large-scale and Chenopodium quinoa [Yang et al. 2007]. use of A. tumefaciens or viral vectors. Second, we Purified VP1-expressing bamboo mosaic virus need to determine the most suitable cultivation sys- was intramuscularly injected with an oil adjuvant tem for transgenic plants (open-field or in-house into 2-month-old piglets twice with a 6-week cultivation). Open-field cultivation is less expense interval. The neutralization titer was elevated in than greenhouse or in-house cultivation, but plant all piglets that received 0.5, 1, or 5 mg of purified factories offer controllable, reproducible cultivation antigen even after single vaccination. FMDV VP1 conditions suitable for GMP manufacturing. and the structural polyprotein P1 produced in Finally, we need to define the procedures for man- tobacco, potato, alfalfa, or tomato protected mice ufacturing and processing of plant-based pharma- and guinea pigs from FMDV challenge [Carrillo ceuticals. The challenge is to facilitate the et al. 2001; Dus Santos et al. 2005]. procedures without compromising quality, which is a prerequisite for manufacturing plant-based Rabies virus causes a zoonotic disease transmitted human and animal vaccines. from wild animals such as bats, raccoons, and foxes to pet animals and humans. To control Funding rabies in humans, vaccination of wildlife and pets The author(s) received no financial support for is needed [Fooks et al. 2014]. Capturing large the research, authorship, and/or publication of numbers of wild animals for vaccine injection is this article. almost impossible, and distribution of bait mixed with oral vaccine in the risk regions could be a Declaration of Conflicting Interests major solution for rabies control [Yang et al. The author(s) declared no potential conflicts of 2013]. Rabies G protein has been expressed in interest with respect to the research, authorship, several species, including tobacco, tomato, spin- and/or publication of this article. ach, carrot, and maize [Loza-Rubio et al. 2012; McGarvey et al. 1995; Rojas-Anaya et al. 2009]. Oral immunization with rabies G protein pro- duced in maize (0.5–2 mg) protected sheep from References rabies strain CASS-88; the mortality rate from Azegami, T., Yuki, Y. and Kiyono, H. (2014) virus challenge 120 days after vaccination was Challenges in mucosal vaccines for the control of reduced in a dose-dependent manner [Loza- infectious diseases. Int Immunol 26: 517–528. Rubio et al. 2012]. Plant-made rabies antigens may be used at different doses both in humans Barta, A., Sommergruber, K., Thompson, D., Hartmuth, K., Matzke, M. and Matzke, A. (1986) and animals. The expression of a nopaline synthase–human growth hormone chimaeric gene in transformed tobacco and Aquafarming relies on ocean water, and excessive sunflower callus tissue. Plant Mol Biol 6: 347–357. use of antibiotics contaminates the environment. The use of oral vaccines for disease prevention in Bawa, A. and Anilakumar, K. (2013) Genetically fisheries and aquaculture may ameliorate this modified foods: safety, risks and public concerns – a review. J Food Sci Technol 50: 1035–1046. problem [Clarke et al. 2013]. Bevan, M. (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12: 8711– Conclusions 8721. Several key points are essential for the development Campos, C. and Lees, D. (2014) Environmental of a broadly effective GMP-compliant regulatory transmission of human in shellfish waters. framework for clinical application of plant-based Appl Environ Microbiol 80: 3552–3561.

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Cardi, T., Lenzi, P. and Maliga, P. (2010) D’Aoust, M., Lavoie, P., Couture, M., Trépanier, S., Chloroplasts as expression platforms for plant- Guay, J., Dargis, M. et al. (2008) Influenza virus- produced vaccines. Expert Rev Vaccines 9: 893–911. like particles produced by transient expression in Nicotiana benthamiana induce a protective immune Carrillo, C., Wigdorovitz, A., Trono, K., Dus Santos, response against a lethal viral challenge in mice. Plant M., Castañón, S., Sadir, A. et al. (2001) Induction Biotechnol J 6: 930–940. of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 de Ronde, D., Pasquier, A., Ying, S., Butterbach, expressed in transgenic potato plants. Viral Immunol P., Lohuis, D. and Kormelink, R. (2014) Analysis of 14: 49–57. Tomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence Cerovska, N., Hoffmeisterova, H., Moravec, T., and RNA silencing suppression. Mol Plant Pathol 15: Plchova, H., Folwarczna, J., Synkova, H. et al. (2012) 185–195. Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of Dietzschold, B., Gore, M., Marchadier, D., Niu, H., coat protein of potato virus X in plants. J Biosci 37: Bunschoten, H., Otvos, L. Jr. et al. (1990) Structural 125–133. and immunological characterization of a linear virus- neutralizing epitope of the rabies virus glycoprotein Chakravarthi, M., Philip, A. and Subramonian, N. and its possible use in a synthetic vaccine. J Virol 64: (2015) Truncated ubiquitin 5’ regulatory region from 3804–3809. Erianthus arundinaceus drives enhanced transgene expression in heterologous systems. Mol Biotechnol 19: Dus Santos, M., Carrillo, C., Ardila, F., Ríos, R., 820–835 Franzone, P., Piccone, M. et al. (2005) Development of transgenic alfalfa plants containing the foot and Chen, Q., Lai, H., Hurtado, J., Stahnke, J., mouth disease virus structural polyprotein gene P1 Leuzinger, K. and Dent, M. (2013) Agroinfiltration as and its utilization as an experimental immunogen. an effective and scalable strategy of gene delivery for Vaccine 23: 1838–1843. production of pharmaceutical proteins. Adv Tech Biol Med 1: 103. European Medicines Agency (EMA) (2008) Guideline on the Quality of Biological Active Substances Produced Chichester, J., Jones, R., Green, B., Stow, M., by Stable Transgene Expression in Higher Plants (EMEA/ Miao, F., Moonsammy, G. et al. (2012) Safety and CHMP/BWP/48316/2006). London, UK: European immunogenicity of a plant-produced recombinant Medicines Agency. hemagglutinin-based influenza vaccine (HAI-05) derived from A/Indonesia/05/2005 (H5N1) influenza Fischer, R., Schillberg, S., Hellwig, S., Twyman, M. virus: a phase 1 randomized, double-blind, placebo- and Drossard, J. (2012) GMP issues for recombinant controlled, dose-escalation study in healthy adults. plant-derived pharmaceutical proteins. Biotech Adv Viruses 4: 3227–3244. 30: 434–439. Clarke, J., Waheed, M., Lössl, A., Martinussen, I. and Floss, D., Falkenburg, D. and Conrad, U. (2007) Daniell, H. (2013) How can plant genetic engineering Production of vaccines and therapeutic antibodies contribute to cost-effective fish vaccine development for veterinary applications in transgenic plants: an for promoting sustainable aquaculture? Plant Mol Biol overview. Transgenic Res 16: 315–332. 83: 33–40. Fooks, A., Banyard, A., Horton, D., Johnson, N., Cox, L., Casale, T., Nayak, A., Bernstein, D., McElhinney, L. and Jackson, A. (2014) Current Creticos, P., Ambroisine, L. et al. (2012) Clinical status of rabies and prospects for elimination. Lancet efficacy of 300IR 5-grass pollen sublingual tablet in a 384: 1389–1399. US study: the importance of allergen-specific serum Fujihashi, K., Dohi, T., Kweon, M., McGhee, J., IgE. J Allergy Clin Immunol 130: 1327–1334. Koga, T., Cooper, M. et al. (1999) Gammadelta T Cummings, J., Guerrero, M., Moon, J., Waterman, cells regulate mucosally induced tolerance in a dose- P., Nielsen, R., Jefferson, S. et al. (2014) Safety and dependent fashion. Int Immunol 11: 1907–1916. immunogenicity of a plant-produced recombinant Garabagi, F., Gilbert, E., Loos, A., McLean, M. monomer hemagglutinin-based influenza vaccine and Hall, J. (2012) Utility of the P19 suppressor of derived from influenza A (H1N1)pdm09 virus: a gene-silencing protein for production of therapeutic phase 1 dose-escalation study in healthy adults. antibodies in Nicotiana expression hosts. Plant Vaccine 32: 2251–2259. Biotechnol J 10: 1118–1128. Daniell, H., Muthukumar, B. and Lee, S. (2001) Gleba, Y., Klimyuk, V. and Marillonnet, S. (2005) Marker free transgenic plants: engineering the Magnifection – a new platform for expressing chloroplast genome without the use of antibiotic recombinant vaccines in plants. Vaccine 23: 2042– selection. Curr Genet 39: 109–116. 2048.

150 http://tav.sagepub.com N Takeyama, K Hiroshi et al.

Gleba, Y., Marillonnet, S. and Klimyuk, V. (2004) production of hepatitis B core antigen in plant leaf and Engineering viral expression vectors for plants: the its immunogenicity in mice. Vaccine 24: 2506–2513. ‘full virus’ and the ‘deconstructed virus’ strategies. Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Curr Opin Plant Biol 7: 182–188. Y., Hatta, M. et al. (2009) In vitro and in vivo Gleba, Y., Tusé, D. and Giritch, A. (2014) Plant characterization of new swine-origin H1N1 influenza viral vectors for delivery by Agrobacterium. Curr Top viruses. Nature 460: 1021–1025. Microbiol Immunol 375: 155–192. Jackson, M., Sternes, P., Mudge, S., Graham, M. and Gómez, E., Lucero, M., Chimeno Zoth, S., Birch, R. (2014) Design rules for efficient transgene Carballeda, J., Gravisaco, M. and Berinstein, A. expression in plants. Plant Biotechnol J 12: 925–933. (2013) Transient expression of VP2 in Nicotiana Joensuu, J., Verdonck, F., Ehrström, A., Peltola, M., benthamiana and its use as a plant-based vaccine Siljander-Rasi, H., Nuutila, A. et al. (2006) F4 (K88) against infectious bursal disease virus. Vaccine 31: fimbrial adhesin FaeG expressed in alfalfa reduces 2623–2637. F4+ enterotoxigenic Escherichia coli excretion in Gomord, V., Fitchette, A., Menu-Bouaouiche, L., weaned piglets. Vaccine 24: 2387–2394. Saint-Jore-Dupas, C., Michaud, D. and Faye, L. Kapusta, J., Modelska, A., Figlerowicz, M., Pniewski, (2010) Plant-specific glycoprotein patterns in the T., Letellier, M., Lisowa, O. et al. (1999) A plant- context of therapeutic protein production. Plant derived edible vaccine against hepatitis B virus. Biotechnol J 8: 564–587. FASEB J 13: 1796–1799. Guan, Z., Guo, B., Huo, Y., Guan, Z., Dai, J. and Khandelwal, A., Lakshmi, S. and Shaila, M. (2003) Wei, Y. (2013) Recent advances and safety issues Oral immunization of cattle with hemagglutinin of transgenic plant-derived vaccines. Appl Microbiol protein of rinderpest virus expressed in transgenic Biotechnol 97: 2817–2840. peanut induces specific immune responses. Vaccine Guerrero-Andrade, O., Loza-Rubio, E., Olivera- 21: 3282–3289. Flores, T., Fehérvári-Bone, T. and Gómez-Lim, M. Kirk, D., McIntosh, K., Walmsley, A. and Peterson, (2006) Expression of the Newcastle disease virus R. (2005) Risk analysis for plant-made vaccines. fusion protein in transgenic maize and immunological Transgenic Res 14: 449–462. studies. Transgenic Res 15: 455–463. Kolotilin, I., Kaldis, A., Devriendt, B., Joensuu, Hefferon, K. (2013) Plant-derived pharmaceuticals J., Cox, E. and Menassa, R. (2012) Production of for the developing world. Biotechnol J 8: 1193–1202. a subunit vaccine candidate against porcine post- Hefferon, K. (2014) Plant virus expression vector weaning diarrhea in high-biomass transplastomic development: new perspectives. BioMed Res Int 2014: tobacco. PLoS One 7: e42405. 1–6. Kolotilin, I., Topp, E., Cox, E., Devriendt, B., Hennegan, K., Yang, D., Nguyen, D., Wu, L., Conrad, U. and Joensuu, J. (2014) Plant-based Goding, J., Huang, J. et al. (2005) Improvement solutions for veterinary immunotherapeutics and of human lysozyme expression in transgenic rice prophylactics. Vet Res 45: 117. grain by combining wheat (Triticum aestivum) Kuroda, M., Kimizu, M. and Mikami, C. (2010) A puroindoline b and rice (Oryza sativa) Gt1 simple set of plasmids for the production of transgenic promoters and signal peptides. Transgenic Res 14: plants. Biosci Biotechnol Biochem 74: 2348–2351. 583–592. Kurokawa, S., Nakamura, R., Mejima, M., Kozuka- Hiatt, A., Cafferkey, R. and Bowdish, K. (1989) Hata, H., Kuroda, M., Takeyama, N. et al. (2013) Production of antibodies in transgenic plants. Nature MucoRice-cholera toxin B-subunit, a rice-based 342: 76–78. oral cholera vaccine, down-regulates the expression Hiwasa-Tanase, K., Nyarubona, M., Hirai, T., Kato, of a-amylase/trypsin inhibitor-like protein family as K., Ichikawa, T. and Ezura, H. (2011) High-level major rice allergens. J Proteome Res 12: 3372–3382. accumulation of recombinant miraculin protein in Kushnir, N., Streatfield, S. and Yusibov, V. (2012) transgenic tomatoes expressing a synthetic miraculin Virus-like particles as a highly efficient vaccine gene with optimized codon usage terminated by platform: diversity of targets and production systems the native miraculin terminator. Plant Cell Rep 30: and advances in clinical development. Vaccine 31: 113–24. 58–83. Holmgren, J. and Czerkinsky, C. (2005) Mucosal Lai, H. and Chen, Q. (2012) Bioprocessing of plant- immunity and vaccines. Nat Med 11: S45–S53. derived virus-like particles of Norwalk virus capsid Huang, Z., Santi, L., LePore, K., Kilbourne, J., protein under current Good Manufacture Practice Arntzen, C. and Mason, H. (2006) Rapid, high-level regulations. Plant Cell Rep 31: 573–584.

http://tav.sagepub.com 151 Therapeutic Advances in Vaccines 3(5-6)

Lamichhane, A., Azegami, T. and Kiyono, H. Merlin, M., Gecchele, E., Capaldi, S., Pezzotti, M. (2014) The mucosal immune system for vaccine and Avesani, L. (2014) Comparative evaluation development. Vaccine 32: 6711–6723. of recombinant protein production in different biofactories: the green perspective. Biomed Res Int Landry, N., Pillet, S., Favre, D., Poulin, J., Trépanier, 2014: 1–14. S., Yassine-Diab, B. et al. (2014) Influenza virus-like particle vaccines made in Nicotiana benthamiana Musiychuk, K., Stephenson, N., Bi, H., Farrance, elicit durable, poly-functional and cross-reactive T C., Orozovic, G., Brodelius, M. et al. (2007) cell responses to influenza HA antigens. Clin Immunol A launch vector for the production of vaccine 154: 164–177. antigens in plants. Influenza Other Respir Viruses 1: 19–25. Landry, N., Ward, B., Trépanier, S., Montomoli, E., Dargis, M., Lapini, G. et al. (2010) Preclinical and Neumann, G., Noda, T. and Kawaoka, Y. (2009) clinical development of plant-made virus-like particle Emergence and pandemic potential of swine-origin vaccine against avian H5N1 influenza. PLoS One 5: H1N1 influenza virus. Nature 459: 931–939. e15559. Nochi, T., Takagi, H., Yuki, Y., Yang, L., Leuzinger, K., Dent, M., Hurtado, J., Stahnke, J., Lai, Masumura, T., Mejima, M. et al. (2007) Rice-based H., Zhou, X. et al. (2013) Efficient agroinfiltration mucosal vaccine as a global strategy for cold-chain- of plants for high-level transient expression of and needle-free vaccination. Proc Natl Acad Sci USA recombinant proteins. J Vis Exp 77: e50521. 104: 10986–10991. Lico, C., Santi, L., Twyman, R., Pezzotti, M. and Nochi, T., Yuki, Y., Katakai, Y., Shibata, H., Avesani Tywa, L. (2012) The use of plants for the Tokuhara, D., Mejima, M. et al. (2009) A rice- production of therapeutic human peptides. Plant Cell based oral cholera vaccine induces macaque-specific Rep 31: 439–451. systemic neutralizing antibodies but does not Loza-Rubio, E., Rojas-Anaya, E., López, J., Olivera- influence pre-existing intestinal immunity. J Immunol Flores, M., Gómez-Lim, M. and Tapia-Pérez, G. 183: 6538–6544. (2012) Induction of a protective immune response to rabies virus in sheep after oral immunization Noris, E., Poli, A., Cojoca, R., Rittà, M., Cavallo, F., with transgenic maize, expressing the rabies virus Vaglio, S. et al. (2011) A human papillomavirus 8 E7 glycoprotein. Vaccine 30: 5551–5556. protein produced in plants is able to trigger the mouse immune system and delay the development of skin MacLachlan, J. and Cowie, B. (2015) Hepatitis B lesions. Arch Virol 156: 587–595. virus epidemiology. Cold Spring Harb Perspect Med 5: a021410. Norkrans, G. (1990) Epidemiology of hepatitis B virus (HBV) infections with particular regard to Marillonnet, S., Giritch, A., Gils, M., Kandzia, current routes of transmission and development of R., Klimyuk, V. and Gleba, Y. (2004) In planta cirrhosis and malignancy. Scand J Infect Dis Suppl 69: engineering of viral RNA replicons: efficient assembly 43–47. by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci USA 101: Oey, M., Lohse, M., Kreikemeyer, B. and Bock, R. 6852–6857. (2009) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable McCormick, A., Reddy, S., Reinl, S., Cameron, T., protein antibiotic. Plant J 7: 436–445. Czerwinkski, D., Vojdani, F. et al. (2008) Plant- produced idiotype vaccines for the treatment of non- Peréz Aguirreburualde, M., Gómez, M., Ostachuk, Hodgkin’s lymphoma: safety and immunogenicity in A., Wolman, F., Albanesi, G., Pecora, A. et al. (2013) a phase I clinical study. Proc Natl Acad Sci USA 105: Efficacy of a BVDV subunit vaccine produced in 10131–10136. alfalfa transgenic plants. Vet Immunol Immunopathol 151: 315–324. McGarvey, P., Hammond, J., Dienelt, M., Hooper, D., Fu, Z., Dietzschold, B. et al. (1995) Expression of Pérez Filgueira, D., Zamorano, P., Domínguez, M., the rabies virus glycoprotein in transgenic tomatoes. Taboga, O., Del Médico Zajac, M., Puntel, M. et al. Biotechnology 13: 1484–1487. (2003) Bovine herpes virus gD protein produced in plants using a recombinant tobacco mosaic virus Mejima, M., Kashima, K., Kuroda, M., (TMV) vector possesses authentic antigenicity. Takeyama, N., Kurokawa, S., Fukuyama, Y. et al. Vaccine 21: 4201–4209. (2015) Determination of genomic location and structure of the transgenes in marker-free rice- Peyret, H. and Lomonossoff, G. (2013) The pEAQ based cholera vaccine by using whole genome vector series: the easy and quick way to produce resequencing approach. Plant Cell, Tissue Organ recombinant proteins in plants. Plant Mol Biol 83: Cult 120: 35–48 51–58.

152 http://tav.sagepub.com N Takeyama, K Hiroshi et al.

Ravin, N., Kotlyarov, R., Mardanova, E., Kuprianov, Tacket, C., Mason, H., Losonsky, G., Clements, J., V., Migunov, A., Stepanova, L. et al. (2012) Plant- Levine, M. and Arntzen, C. (1998) Immunogenicity produced recombinant influenza vaccine based on in humans of a recombinant bacterial antigen virus-like HBc particles carrying an extracellular delivered in a transgenic potato. Nat Med 4: 607–609. domain of M2 protein. Biochemistry 77: 33–40. Tacket, C., Mason, H., Losonsky, G., Estes, M., Redkiewicz, P., Sirko, A., Kamel, K. and Góra- Levine, M. and Arntzen, C. (2000) Human immune Sochacka, A. (2014) Plant expression systems for responses to a novel norwalk virus vaccine delivered in production of hemagglutinin as a vaccine against transgenic potatoes. J Infect Dis 182: 302–305. influenza virus. Acta Biochim Pol 61: 551–560. Tacket, C., Pasetti, M., Edelman, R., Howard, J. and Rigano, M. and Walsley, A. (2005) Expression Streatfield, S. (2004) Immunogenicity of recombinant systems and developments in plant-made vaccines. LT-B delivered orally to humans in transgenic corn. Immunol Cell Biol 83: 271–277. Vaccine 22: 4385–4389. Rojas-Anaya, E., Loza-Rubio, E., Olivera-Flores, M. Takeyama, N., Yuki, Y., Tokuhara, D., Oroku, K., and Gomez-Lim, M. (2009) Expression of rabies virus Mejima, M., Kurokawa, S. et al. (2015) Oral rice- G protein in carrots (Daucus carota). Transgenic Res based vaccine induces passive and active immunity 18: 911–919. against enterotoxigenic E. coli-mediated diarrhea in pigs. Vaccine 33: 5204–5211. Rybicki, E. (2010) Plant-made vaccines for humans and animals. Plant Biotechnol J 8: 620–637. Teunis, P., Moe, C., Liu, P., Miller, S., Lindesmith, L., Baric, R. et al. (2008) Norwalk virus: how Rybicki, E. (2014) Plant-based vaccines against infectious is it? J Med Virol 80: 1468–1476. viruses. Virol J 11: 205. Thanavala, Y., Mahoney, M., Pal, S., Scott, Sainsbury, F., Thuenemann, E. and Lomonossoff, A., Richter, L., Natarajan, N. et al. (2005) G. (2009) pEAQ: versatile expression vectors for Immunogenicity in humans of an edible vaccine for easy and quick transient expression of heterologous hepatitis B. Proc Natl Acad Sci USA 102: 3378– proteins in plants. Plant Biotechnol J 7: 682–693. 3382. Salazar-González, J., Bañuelos-Hernández, B. and Tokuhara, D., Yuki, Y., Nochi, T., Kodama, T., Rosales-Mendoza, S. (2015) Current status of viral Mejima, M., Kurokawa, S. et al. (2010) Secretory expression systems in plants and perspectives for oral IgA-mediated protection against V. cholerae and vaccines development. Plant Mol Biol 87: 203–217. heat-labile enterotoxin-producing enterotoxigenic Sato, S., Yanagida, N., Ogura, K., Asaumi, T., Escherichia coli by rice-based vaccine. Proc Natl Acad Okada, Y., Koike, Y. et al. (2014) Immunotherapy Sci USA 107: 8794–8799. in food allergy: towards new strategies. Asian Pac J Toovey, S. (2007) Preventing rabies with the Allergy Immunol 32: 195–202. Verorab vaccine: 1985–2005 twenty years of clinical Scotti, N., Rigano, M. and Cardi, T. (2012) experience. Travel Med Infect Dis 5: 327–348. Production of foreign proteins using plastid Tuboly, T., Yu, W., Bailey, A., Degrandis, S., Du, S., transformation. Biotech Adv 30: 387–397. Erickson, L. et al. (2000) Immunogenicity of porcine Scotti, N. and Rybicki, E. (2013) Virus-like particles transmissible gastroenteritis virus spike protein produced in plants as potential vaccines. Expert Rev expressed in plants. Vaccine 18: 2023–2028. Vaccines 12: 211–224. Twyman, R., Schillberg, S. and Fischer, R. (2005) Shoji, Y., Chichester, J., Jones, M., Manceva, S., Transgenic plants in the biopharmaceutical market. Damon, E., Mett, V. et al. (2011) Plant-based rapid Expert Opin Emerg Drugs 10: 185–218. production of recombinant subunit hemagglutinin Vacher, G., Kaeser, M., Moser, C., Gurny, R. and vaccines targeting H1N1 and H5N1 influenza. Hum Borchard, G. (2013) Recent advances in mucosal Vaccin 7: 41–50. immunization using virus-like particles. Mol Pharm Shoji, Y., Prokhnevsky, A., Leffet, B., Vetter, N., 10: 1596–1609. Tottey, S., Satinover, S. et al. (2015) Immunogenicity van der Laan, J., Minor, P., Mahoney, R., Arntzen, of H1N1 influenza virus-like particles produced in C., Shin, J., Wood, D. et al. (2006) WHO informal Nicotiana benthamiana. Hum Vaccin Immunother 11: consultation on scientific basis for regulatory 118–123. evaluation of candidate human vaccines from plants, Shoseyov, O., Posen, Y. and Grynspan, F. (2014) Geneva, Switzerland, 24–25 January 2005. Vaccine Human collagen produced in plants, More than just 24: 4271–4278. another molecule. Bioengineered 5: 49–52. van Dussen, L., Zimran, A., Akkerman, E., Aerts, J., Tacket, C. (2007) Plant-based vaccines against diarrheal Petakov, M., Elstein, D. et al. (2013) Taliglucerase diseases. Trans Am Clin Climatol Assoc 118: 79–87. alfa leads to favorable bone marrow responses in http://tav.sagepub.com 153 Therapeutic Advances in Vaccines 3(5-6)

patients with type I Gaucher disease. Blood Cells Mol of rice endogenous storage proteins enhances the Dis 50: 206–211. production of rice-based Botulinum neutrotoxin type A vaccine. Vaccine 30: 4160–4166. Verma, D. and Daniell, H. (2007) Chloroplast vector systems for biotechnology applications. Plant Physiol Yuki, Y., Mejima, M., Kurokawa, S., Hiroiwa, T., 145: 1129–1143. Takahashi, Y., Tokuhara, D. et al. (2013) Induction Vermij, P. (2006) USDA approves the first plant- of toxin-specific neutralizing immunity by molecularly based vaccine (News In Brief). Nature Biotechnol 24: uniform rice-based oral cholera toxin B subunit 233–234. vaccine without plant-associated sugar modification. Plant Biotechnol J 11: 799–808. Ward, B., Landry, N., Trépanier, S., Mercier, G., Dargis, M., Couture, M. et al. (2014) Human Yusibov, V., Hooper, D., Spitsin, S., Fleysh, N., antibody response to N-glycans present on plant-made Kean, R., Mikheeva, T. et al. (2002) Expression influenza virus-like particle (VLP) vaccines. Vaccine in plants and immunogenicity of plant virus-based 32: 6098–6106. experimental rabies vaccine. Vaccine 20: 3155– 3164. Wu, J., Yu, L., Li, L., Hu, J., Zhou, J. and Zhou, X. (2007) Oral immunization with transgenic rice Yusibov, V., Kushnir, N. and Streatfield, S. (2014) seeds expressing VP2 protein of infectious bursal Advances and challenges in the development and disease virus induces protective immune responses in production of effective plant-based influenza vaccines. chickens. Plant Biotechnol J 5: 570–8. Expert Rev Vaccines 9: 1–17.

Yang, D., Guo, F., Liu, B., Huang, N. and Watkins, Yusibov, V., Stephen, J., Streatfield, S. and Kushnir, S. (2002) Expression and localization of human N. (2011) Clinical development of plant-produced lysozyme in the endosperm of transgenic rice. Planta recombinant pharmaceuticals: vaccines, antibodies 216: 597–603. and beyond. Human Vaccin 7: 313–321. Yang, D., Kim, H., Lee, K. and Song, J. (2013) The Zhou, J., Cheng, L., Zheng, X., Wu, J., Shang, S., present and future of rabies vaccine in animals. Clin Wang, J. et al. (2004) Generation of the transgenic Exp Vaccine Res 2: 19–25. potato expressing full-length spike protein of Yang, C., Liao, J., Lai, C., Jong, M., Liang, C., Lin, infectious bronchitis virus. J Biotechnol 111: 121–130. Y. et al. (2007) Induction of protective immunity Zimran, A., Brill-Almon, E., Chertkoff, R., Petakov, in swine by recombinant bamboo mosaic virus M., Blanco-Favela, F., Muñoz, E. et al. (2011) expressing foot-and-mouth disease virus epitopes. Pivotal trial with plant cell-expressed recombinant BMC Biotechnol 7: 62. Visit SAGE journals online glucocerebrosidase, taliglucerase alfa, a novel enzyme http://tav.sagepub.com Yuki, Y., Mejima, M., Kurokawa, S., Hiroiwa, T., replacement therapy for Gaucher disease. Blood 118: SAGE journals Kong, I., Kuroda, M. et al. (2012) RNAi suppression 5767–5773.

154 http://tav.sagepub.com High-yield production of therapeutic proteins in chloroplast

Shrikant Yankanchi Research Scholar IGKV, Raipur Top 10 causes of deaths . Global life expectancy for children born in 2015 was 71.4 years (India – 68.33)

U.S. Food and Drug Administration (FDA)-approved therapeutic proteins (2011–2016)

drug class

Therapeutic area Lagassé et al. 2017 Therapeutic Proteins?

• Therapeutic Proteins - engineered in the laboratory for pharmaceutical use • Cancer, Infectious Diseases, Haemophilia, Anaemia, Multiple Sclerosis, Hepatitis B/C,

Eg: Monoclonal antibodies, Protective antigen of Bacillus anthracis Plant-made therapeutics - protein products with pharmaceutical applications produced in recombinant plant systems Landmarks of therapeutic prteins

 Insulin was the first therapeutic protein to be introduced to treat diabetes in the 1920s  The first pharmaceutically relevant protein made in plants was human growth hormone, which was expressed in transgenic tobacco in 1986  First IgM expressed in plants and protein targeted to chloroplast for accumulation (Tobacco)  cholera toxin subunit B (CTB), the first vaccine antigen expressed in chloroplasts by (Kang et al. 2003) Ma et al., 2003 Different Production systems - therapeutic proteins

Production systems - Bacteria, Yeast, Animal or human cell, Transgenic animals

The majority of therapeutic proteins produced today are made in bacteria (Escherichia coli), yeast (Saccharomyces cerevisiae) or mammalian cell culture (Chinese hamster ovary cells, CHO)

Traditional recombinant production systems such as bacterial and mammalian cell cultures are limited in their scalability and production cost, due to requirement for complicated fermentation Equipment and expensive downstream processing

Rasala et al., 2010 Comparison of different Production systems

v

v v

Lack of Glycosylation •Bacterial products require expensive post-processing posttranslational Phosphorylatio procedures to form usable proteins modifications n in E. coli Disulfide bridge formation it is necessary to explore alternate production methods that should facilitate cost reduction at different levels Plants offer several advantages

 Refereed as “Green bioreactors”  Transgenesis is relatively easy in plants  Ability to perform eukaryotic post translational modification  Plant systems are robust and inert, allowing for symplified handling/ purification and the ability in case of pharmaceutical relevant proteins to be administered orally with minimal processing  plant-derived therapeutic proteins are free of human pathogens and mammalian viral vectors The tobacco plant

 Is often chosen because of its large biomass, yielding ~170 MT of biomass per hectare it is easy to engineer tobacco chloroplast and nuclear genome and regenerate transgenic lines within a few months Each transgenic plant is capable of producing up to a million seeds and, therefore, it is possible to scale up from a single transgenic plant to 100 acres within 1 year. Non-food crop and is self pollinated, minimizing transgene escape By using this technology, large quantities of vaccine antigen can be produced. Ways of expressing protein of interest……

 Nuclear genome based expression

 Chloroplast genome based expression - Transplastomic expression Advantages of chloroplast genome transformation over Nuclear genome

Daniell et al., 2012 Production of therapeutic proteins in chloroplasts

Eliminates

 Expensive fermentation technology

 Oral delivery of chloroplast-derived therapeutic proteins

 expensive purification steps

 cold storage

 cold transportation, and delivery via sterile needles,

 thereby further decreasing their cost Steps in Chloroplast genome based expression

 Delivery of DNA by : Biolistic process or occasionally by polyethylene glycol (PEG) treatment of protoplasts

 This is followed by transgene integration into the chloroplast genome via homologous recombination facilitated by a RecA-type system between the chloroplast-targeting sequences of the transformation vector and the targeted region of the chloroplast genome

 LTR and RTR 400 bp homologous to chosen target site Mode of transgene integration components used in chloroplast vectors Jin and Daniell., 2015 Biolistic method of chloroplast transformation

Examples of therapeutic protein folding and modifications reported in plant chloroplasts zhang et al., 2017 Sourrouille et al. 2010 Chloroplast derived biopharmaceutical therapeutic proteins/vaccine/antigens Chloroplast derived biopharmaceutical therapeutic proteins. Chloroplast-derived vaccine antigens. Limitations

 Homoplasmic transplastomic plants have not yet been created after two decades of research  There is a great need to transform edible leafy crops, especially for oral drug delivery or enhanced nutrition but except lettuce chloroplast, no other edible system has yielded reproducible results so far  Edible non-green parts of plants (e.g., tomato fruits) have very low levels of foreign protein accumulation compared to transplastomic leaves  A serious limitation in plastid biotechnology has been the low expression levels of plastid genes in non-green tissues, such as fruits, tubers and seeds Future prospects...

Marker free transplastomic plants must be developed in edible crops to advance the chloroplast made biopharmaceuticals for clinical use More importantly, further studies are required to understand the post-translational modifications of foreign proteins expressed in plant chloroplasts Drug dosage determination is currently feasible only in purified biopharmaceuticals - drug dosage determination in-planta (targeted proteomic quantitation) Conclusion

 The chloroplast genetic engineering approach is ideal for economical production of vaccine antigens and biopharmaceuticals in an environmentally friendly manner

 High expression levels of vaccine antigens and therapeutic proteins in transgenic chloroplasts hold the promise for unlimited quantities of production, but the cost of purification is still prohibitive

 This can be overcome by the oral delivery of vaccine antigens and therapeutic proteins or by using the novel purification strategies

References...

• Zhang et al., 2017, Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts. Current Opinion in Chemical Biology. 38: 17-23. • Jin, S and Daniell, H (2015), The engineered chloroplast genome just got smarter. Trends Pl Sci, 20(10): 620-640 • Rasala et al., 2010, Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Pl ant Biotechnol J, 8:719–733. • Ma et al., 2003, The production of recombinant pharmaceutical proteins in plants, Nature Gen (4): 794-805.