Molecular farming

Molecular farming
[New face of plant biotechnology]

Submitted by:
LalithyaKantamneni 110605012

Supervised by:
Mr. Saleemulla Khan Dr. Aswatha Ram

Manipal College of Pharmaceutical Sciences

Molecular farming

M.Pharm Part-I Department of Pharmacognosy MCOPS

Manipal College of Pharmaceutical Sciences

Molecular farming

CONTENTS
Introduction to molecular farming
Biofarming

History of molecular farming Plants as expression systems Medical molecular farming Plant made pharmaceuticals Transgenic plants Edible vaccines Plantigens Plantibodies Non medical molecular farming Bioplastics Bioremediation Choice of plant species Concerns regarding molecular farming Current areas of research Conclusion Abstract

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WHAT IS MOLECULAR FARMING?

Biotechnology in agriculture has two categories: (1)"improvements" to existing livestock and crops, and (2) Development of entirely new uses for both animals and plants (Biopharming) So called "improvements", include input traits such as crops with extra resistance to insect attack and improved weed control. These "GM" or "GMO" crops, are modified food crops made more commercially viable. e.g. "Roundup ready" soya, "Starlite" corn, or "Frost-tolerant" tomatoes (7).
MOLECULAR FARMING is the term for new use plants only [not animals] and is different in that this does not affect and has nothing to do with Food. OTHER RELATED FIELDS

TRANSGENICS 'biofarming animals' or 'pharming' is the related Animal field. This is the direct application of biotechnology to develop new uses from Animals and has already produced potential treatments for conditions as diverse as Diabetes and CysticFibrosis (7). HISTORY OF MOLECULAR FARMING For the first time Hiatt et al. (1989) produced antibodies in plants which could produce positive immunization. But the first report on production of edible vaccine appeared in 1990 in the form of a patent application. In 1992, C.J. Arntzen and co-workers expressed hepatitis B surface antigen in tobacco to produce immunologically active ingredients via genetic engineering of plants. During 1980s, great effort has been made to transform plant by foreign genes. Various foreign proteins including serum albumin, human a-interferon, human erythropoietin, and murine IgG and IgA immunoglobulins have been successfully expressed in plants. In 1999, the Indian scientists at ICGEB, New Delhi have successfully produced transgenic maize, tobacco, rice, etc. capable of producing interferon gamma (INF-) (Barros et al., 2005).

ADVANTAGES OF PLANTS AS EXPRESSION SYSTEMS

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The organism or material into which the new genetic information is inserted is often referred to as the expression system, since it serves as the system for expressing the new product. Several expression systems have been explored, and each has advantages and disadvantages. The ideal expression system must: produce the desired, functional product; be costeffective; allow for convenient storage and distribution of the desired product; come with little or no risk (perceived or real); and not be time-consuming. Expression systems that have been studied in addition to bacteria include plant viruses, yeast, animal cell cultures, transgenic plants and transgenic animals. Plants offer advantages over live animals and animal cell cultures in terms of safety, cost, time involved, and storage and distribution issues; plant expression systems are also believed to be better than microbes in terms of cost, protein complexity, and storage and distribution issues. Global demand for pharmaceuticals is at unprecedented levels, and current production capacity will soon be overwhelmed. Expanding the existing microbial systems, although feasible for some therapeutic products, is not a satisfactory option on several grounds. First, it would be very expensive for the pharmaceutical companies. Second, other proteins of interest are too complex to be made by microbial systems. These proteins are currently being produced in animal cell cultures, but the resulting product is often prohibitively expensive for many patients. Finally, although it is theoretically possible to synthesize protein molecules by machine, this works only for very small molecules, less than 30 amino acid residue in length. Virtually all proteins of therapeutic value are larger than this and require live cells to produce them. For these reasons, science has been exploring other options for producing proteins of therapeutic value. The use of plants offers a number of advantages over other expression systems. Plants can be used in two ways. One way is to insert the desired gene into a virus that is normally found in plants, such as the tobacco mosaic virus in the tobacco plant. The other way is to insert the desired gene directly into the plant DNA to produce a transgenic plant (Twyman et al., 2003).

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COMPARISION OF DIFFERENT EXPRESSION SYSTEMS (Twyman et al., 2003)

Animal Transgeni Transgenic Cell c Plants Animals Cultures Cost of inexpensiv inexpensiv inexpensiv inexpensiv expensive expensive maintaining e e e e Type of -2.0C -2.0C -2.0C RT* N2** N/A storage Gene size (protein) Unknown Unknown Limited Not limited Limited Limited restriction Production Medium Medium Low Low High High cost Medium Protein yield High Medium Very high High High to high Therapeutic Unknown yes Unknown Unknown yes yes risk Expressions Yeast System Bacteria Plant viruses *RTroom temperature. ** N2 culture must be maintained under nitrogen gas. MEDICAL MOLECULAR FARMING Medical Molecular Farming is the growing and harvesting of genetically engineered crops of transgenic plants, to produce biopharmaceuticals or 'farmaceuticals'. The idea is to use these molecular crops as biological factories to generate drugs difficult or expensive to produce in any other way. Combining plant genetics, molecular biology and gene delivery, scientists take genes from other sources, such as microorganisms, and splice them into the plant's genome. During normal growth these genetically engineered plants synthesize 'recombinant' proteins which can be Therapeutics, Vaccines, Blood substitutes, Enzymes or Diagnostics which are then extracted from the crop. These transgenic techniques are already being used to produce vaccines for some animal diseases, such as mink enteritis virus. Many others are at advanced stages, such as measles vaccine in Australian potatoes and drugs to fight cancer, heart disease, infant diabetes and Crohn's disease. Therapeutic Proteins, edible vaccines, 'plantigens' and 'plantibodies' are already in Clinical trials. Tobacco plants are especially used for this purpose. Other transgenic plants to date include Alfalfa, Safflower, Canola, Flax, Potatoes, Corn, Barley, Soybeans, Peas, Beans, Bananas, Tomatoes, Wheat, Carrot, Lettuce, Moss and Rice (7).
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NONMEDICAL MOLECULAR FARMING Nonmedical molecular faming includes Industrial Enzymes and Polymers. Potentially the biggest development in this field could be the development of plants growing biodegradable plastics. Other uses could be Industrial oils such as hydraulic oil or high yielding biodiesels, new solid Biofuels, new Fibres and Papers, and agents such as Bioremediation and Phytoremediation, environmentally cleaning up contamination (7). PRODUCTION OF THERAPEUTIC PROTEINS USING PLANTS Transgenic plant expression systems were developed as alternative sources for the production of biologics, known as plant-made pharmaceuticals or PMPs. Instead of a large capital investment in cell culture facilities, plant production systems can be expanded simply by growing and harvesting additional plants. However, about 50% of the cost of production is in extraction and purification of proteins, which is required in both systems. This reduces the potential cost advantage for PMPs. Nonetheless, plant expression systems can potentially produce hundreds of kilograms per year of a purified protein whereas the cost of a similar production capacity using mammalian cells may be prohibitive. Like mammalian cells, plant production systems have the advantage over microbial systems of being able to produce active forms of complex proteins with appropriate posttranslational modifications (e.g. glycosylation). However, production of proteins using plant expression systems possess some unique challenges, such as containment to prevent gene transfer to conventional crops during plant growth and possibly higher costs to extract the desired protein due to the presence of interfering compounds in plants (Thomas et al., 2002).

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Potential application or Plant host human protein Anti coagulant Thrombin inhibitor Neutropenia Growth hormone Anemia Hepatitis C and B Liver cirrhosis, surgery Blood constitute Collagen Antimicrobial Tobacco Canola (Brassica napus) Tobacco Tobacco Tobacco Rice, turnip, tobacco burns, Tobacco Tobacco Tobacco Potato

Protein

Protein C Hirudin Granulocyte-macrophage colony stimulating factor Somatropin, chloroplast Erythropoietin Interferon-, Serum albumin Haemoglobin , Homotrimeric collagen Lactoferin

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Molecular farming

Non-human proteins

Hypertension Gauchers disease

Tobacco, tomato Tobacco

Angiotensin enzyme

converting

Glucocerebrosidase

TRANSGENIC PLANTS Genetically modified plants whose DNA is modified using genetic engineering techniques are called transgenic plants. In most cases the aim is to introduce a new trait to the plant which does not occur naturally in this species. Examples include resistance to certain pests, diseases or environmental conditions, or the production of a certain nutrient or pharmaceutical agent. Transgenic plants have genes inserted into them that are derived from another species. The inserted genes can come from species within the same kingdom (plant to plant) or between kingdoms (bacteria to plant). In many cases the inserted DNA has to be modified slightly in order to correctly and efficiently express in the host organism. Transgenic plants are used to express proteins like the cry toxins from Bacillus thuringiensis, herbicide resistant genes and antigens for vaccinations. Cisgenic plants are made using genes found within the same species or a closely related one, where conventional plant breeding can occur. Some breeders and scientists argue that cisgenic modification is useful for plants that are difficult to crossbreed by conventional means (such as potatoes), and that plants in the cisgenic category should not require the same level of legal regulation as other genetically modified organisms. In research plants are engineered to help discover the functions of certain genes. One way to do this is to knock out the gene of interest and see what phenotype develops. Another strategy is to attach the gene to a strong promoter and see what happens when it is over expressed. A common technique used to find out where the gene is expressed is to attach it to GUS or a similar reporter gene that allows visualization of the location. The first commercialized genetically modified plants (Flavr Savr tomatoes) used RNAi technology, where the inserted DNA matched an endogenous gene already in the plant. When the inserted gene is expressed it can repress the translation of the endogenous protein. Host delivered RNAi systems are being developed, where the plant will express RNA that will interfere with insects, nematodes and other

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Molecular farming

parasites protein synthesis. This may provide a novel way of protecting plants from pests. Ex: BT cotton
What are edible vaccines? Molecular farming involves the use of plants, and potentially also animals, as the means to produce compounds of therapeutic value. Charles Arntzen was the pioneer of edible vaccines. A "subunit vaccine" refers to a pathogen-derived protein (or even just an immunogenic domain of a protein, i.e., "an epitope") that cannot cause disease but can elicit a protective immune response against the pathogen. Very often the subunit vaccine candidate is a recombinant protein made in transgenic production-hosts (such as cultured yeast cells), then purified, and injected into vaccines to immunize against a specific disease. Subunit vaccines are generally considered safer to produce (eliminating the need to culture pathogenic organisms) and more importantly, to use (7). MOLECULAR TOMORROW FARMINGHOW PLANTS PRODUCE VACCINES OF

It relies on the same method used to produce genetically modified (GM) crops the artificial introduction of genes into plants. A number of vaccines, antibodies and other therapeutic substances made in plants such as tobacco, maize, potato and carrot are already commercially available or in advanced clinical trials. Producing pharmaceuticals in plants is easy and efficient compared to conventional production methods. Typically, animal or microbial cell cultures are used to produce vaccines but costs associated with maintenance, safety, storage and transport are 80% higher compared to plant-derived vaccines. (Moret al 2002)

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Molecular Farming: A DNA molecule carrying the genetic information for a pharmaceutical substance is introduced into the plant genome. This process (1) is called transformation. The genes can be incorporated permanently (stable transformation) or for a short period of time (transient transformation). The transformed plant acts as a bioreactor producing large quantities of the pharmaceutical using its protein making machinery (2). Through industrial processing, the pharmaceutically active substance is extracted from the plant (3) and made into in a formulated product (4), for example a pill. Step 1: The genetic information necessary to make the therapeutic substance is carried on a DNA molecule. During a process called transformation, this DNA molecule is introduced into the plant where it becomes part of the plant genome. Step 2: The genetic information carried on the incorporated DNA molecule is read by the plant protein-making machinery and used to produce the pharmaceutical along with other plant proteins. In this way the plant acts somewhat as a bioreactor, producing large quantities of pharmaceutically active substances (Mor and Arntzen 2002). "SECOND GENERATION" EDIBLE VACCINES Multicomponent vaccines that provide protection against several pathogens are very desirable. An elegant approach to achieve this goal, based on epitope fusion to both subunits of the cholera toxin (CT), was recently demonstrated by Yu and Langridge (2001). CT provides a scaffold for presentation of protective epitopes of rotavirus and ETEC, acts as a vaccine candidate by its own right and as a mucosal adjuvant devoid of
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Molecular farming

toxicity. The trivalent edible vaccine elicited significant humoral responses, as well as immune memory B cells and T-helper cell responses (Mor and Arntzen 2002).

PLANTIGENS Plantigens [Plant Antigens] are those Plant substances which cause Human/Animal production of Antibodies (6). Antigens (e.g., of pathogenic bacteria) produced in plants which are genetically engineered to produce those (specific) antigens. That process (i.e., genetically engineering plants to cause them to produce specific antigens) can be utilized to produce edible vaccines for the pathogenic bacteria possessing those antigens. Then, people could be "vaccinated" against disease merely by eating the genetically engineered plant (e.g., banana) (7). PLANTIBODIES Plantibodies [Plant Antibodies] are the Human/Animal Antibodies made by and in Transgenic Plants (6). BIOPLASTICS Due to their biodegradability and potential to decrease our reliance on petrochemical resources, bioplastics are considered an environmentally beneficial alternative to synthetic plastics. Current research into biodegradable plastics has largely focused on polyhydroxyalkanoates (PHAs) polyesters of hydroxy acids that are produced by more than 100 different genera of bacteria. The monomers found in PHAs are highly diverse, meaning that these plastics have a wide spectrum of physical properties and could be used as replacements for polyethylene, polystyrene, polypropylene and PET. PHAs are also biocompatible, breaking down into molecules that are naturally found in animals, so they may have medical applications, for example, as implants, gauzes and suture filaments. The polymer poly(3-hydroxybutyrate) (PHB) is the most extensively studied PHA; it is naturally synthesised by the bacterium Ralstoniametalliduransfrom glucose. PHB has similar properties to polypropylene, although it is more brittle, making it less stress resistant for industrial applications. PHB is naturally present at low levels in the cell walls of some plants (for example carrots and a species of willow). Higher levels of PHB were first produced in plants in 1992 in the model plant Arabidopsis thaliana (Moire et al., 2003; Scheller and Conrad, 2005). Since this initial achievement, researchers have attempted to produce PHB in a number of different plants with varying results including oilseed rape (up to 7.7% dry weight), maize (up to 5.7%), sugarcane (1.57.3%), flax (up to 0.5%), cotton (0.34%

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Molecular farming

fibre weight), soybean, palm oil, tobacco, switch grass, sugar beet (up to 5%), potato and alfalfa. Plants can be used to express precursors of bioplastics. These bioplastics are biodegradable and unlike conventional plastics, do not use petrochemical resources as the starting material. Some plants are being modified to express enzymes that convert starch or cellulose into sugars for ethanol production, thus allowing biofuels to be produced more efficiently from these plants (Barros et al. 2005) BIOREMEDIATION/ PHYTOREMEDIATION The use of biological agents such as bacteria, fungi, or green plants, to remove or neutralize contaminants, as in polluted soil or water. Bacteria and fungi generally work by breaking down contaminants such as petroleum into less harmful substances. Plants can be used to aerate polluted soil and stimulate microbial action. They can also absorb contaminants such as salts and metals into their tissues, which are then harvested and disposed of. The use of green plants to decontaminate polluted soil or water is called phytoremediation (6). CHOICE OF PLANT SPECIES Tobacco has a long history as a successful crop system for molecular farming and is therefore one of the strongest candidates for the commercial production of recombinant proteins. The major advantages of tobacco include the well-established technology for gene transfer and expression, high biomass yield, prolific seed production and the existence of a large-scale processing infrastructure. Because tobacco is neither a food nor a feed crop, there is little risk that tobacco material will contaminate either the food or feed chains. Although many tobacco cultivars produce high levels of toxic alkaloids, there are low-alkaloid varieties that can be used for the production of pharmaceutical proteins, and these metabolites are absent from tobacco cell suspensions, which can also be used to produce recombinant proteins. Alternative leafy crops that are being investigated for molecular farming include alfalfa, soybean and lettuce. Alfalfa and soybean have the major advantage of using atmospheric nitrogen through nitrogen fixation, therefore reducing the need for chemical fertilizers. Alfalfa is particularly useful because it has a large dry biomass yield per hectare and can be harvested up to nine times a year. Both of these legumes have been used to produce recombinant antibodies. Soybean has been used to produce Aspergillus phytase.

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Lettuce is also being investigated as a production host for edible recombinant vaccines and has been used in one series of clinical trials for a vaccine against the hepatitis B virus. One of the greatest disadvantages of leafy crops is that recombinant proteins are synthesized in an aqueous environment and are often unstable, resulting in low yields. The leaves must be frozen or dried for transport, or processed soon after harvest to extract useful amounts of the product. Tobacco leaves contain phenolic substances that are released during grinding and protein extraction, which can interfere with downstream processing. The expression of recombinant proteins in vegetative organs such as leaves could potentially interfere with plant growth and development. Biosafety concerns include the potential exposure of herbivores to pharmaceutical products expressed in leaves, and the leaching of recombinant proteins into the environment. In contrast to leafy crops, in Cereal and legume seeds, the expression of proteins in seeds enables long-term storage, even at room temperature, because seeds have the appropriate biochemical environment to promote stable protein accumulation. It has been demonstrated that antibodies expressed in seeds remain stable for at least three years at ambient temperatures with no detectable loss of activity. Cereal seeds also lack the phenolic compounds present in tobacco leaves, thus improving the efficiency of downstream processing. However, the overall yields of recombinant proteins in seed crops are much lower than in tobacco, and the most appropriate expression system must be determined on a case-by-case basis. The specific expression of recombinant proteins in seeds has biosafety advantages in that it reduces exposure to herbivores and other nontarget organisms. However, it is necessary for the transgenic plants to go through a flowering cycle to produce seeds, whereas proteins produced in vegetative organs can be harvested before flowering, therefore preventing the release of pollen and eliminating gene flow by pollen transfer (Twyman et al. 2003) CONCERNS REGARDING MOLECULAR FARMING While molecular farming is one application of genetic engineering, there are concerns that are unique to it. In the case of genetically modified (GM) foods, concerns focus on the safety of the food for human consumption. In contrast, molecular farming is not intended for crops destined for the food chain. It produces plants that contain physiologically active compounds that accumulate in the plants tissues. Considerable attention is focussed, therefore, on the restraint and caution necessary to protect both consumer health and environmental biodiversity. A. Use of Food Crops In 2004, the National Academy of Sciences in the United States published a report entitled Biological Confinement of Genetically Engineered Organisms. The report noted

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that absolute containment of crops is virtually impossible, and made two significant observations:

the need for bio-confinement, or crop containment, must be explored early in the development of the plant; and non food crops should be considered for those products that need to be kept out of the food supply.

Ensuring that transgenic crops do not contaminate crops destined for the food supply requires that there be no cross-pollination between the crops, that no transgenic seeds be left behind in fields where food crops are to be planted, and that there be no contamination of a harvested food crop with a harvested transgenic crop. There have already been examples of these containment breaches. Cross-pollination of GM corn with traditional crops occurred in Iowa in 2002, despite the observance of the required distance between crops. Recent interest in edible vaccines has also yielded to caution as to the prudence of this idea. While the concept is intriguing, in practice it could prove quite unsafe. Even the creator of the edible vaccine, Charles Arntzen, now concedes that the idea should be abandoned. Although the ease of administering a vaccine as a food is very appealing, the risk of the vaccine producing food becoming integrated into the food supply is too great. Research in this area now focuses on non-food crops where the vaccine is subsequently purified and packaged as a pill or capsule. The development of an orally administered vaccine, packaged as a pill or capsule, is in itself novel. It permits wider distribution and easier storage of the product. (Mewettet al. 2005) B. Biodiversity Whether molecular farming ultimately uses food or non-food crops, certain environmental concerns need to be addressed. All field crops are subject to ingestion by wildlife. It would even be difficult to guarantee that all wildlife could be kept away from greenhouse crops. The products that accumulate in these plants could be toxic to an animal or could lead to more subtle physiological or behavioural effects. Another aspect that has not yet been adequately researched is whether the altered plants produce a change in their surrounding soil due to alterations in the exudates from the root systems. Such changes could require longer-term studies than those that have been conducted so far. In addition, if a change in the soil composition is established, further investigations would be required as to whether and how such a change would affect biodiversity. Molecular farming could potentially occupy a significant portion of currently farmed land. Despite its many advantages, molecular farming requires much more space than any of the other expression systems. Its critics warn that the practice could lead to: more intensive or aggressive agricultural practices, such as reducing crop rotations or increased herbicide use; encroachment of cropland onto field margins; and

Conversion of natural habitats to cropland.

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These criticisms are also used to condemn research into biofuels, such as ethanol from corn, and it is unlikely that pharma-crops would be grown on nearly the same scale as biofuel crops. (Mewett et al. 2005) C. Allergies Concern over allergies has been raised for PMPs, as it has for GM crops, but in a reverse sense. Many critics of GM crops argue that allergies could be triggered by inserting a gene from an allergen such as peanuts into another plant. Despite scientists assertion that the inserted gene does not code for the allergen, critics fear that expression of the transgenic peanut gene in the new host plant could provoke an allergic response. With respect to PMP, the concern is not with the transgene, but with the host plant itself. Critics point out that allergies exist to a wide variety of plants, and that purification of the PMP from the plant could include contaminants that might induce an allergic response. For example, many vaccines in use now are produced in eggs, and individuals with an allergy to eggs are advised not to get such vaccines. Therefore, regulations may include a requirement to disclose the source of the PMP. (Mewett et al. 2005)
CURRENT AREAS OF RESEARCH

Plant molecular farming is currently being pursued to address either the increased demand for proteins that cannot be produced in sufficient quantities in either microbial or animal cell cultures, or as a means to produce proteins that cannot be expressed in microbial or animal cell cultures. There are several human diseases for which the underlying cause has been determined to be an ineffective, deficient or absent enzyme. Fabry disease, for example, is a rare disorder in which the affected enzyme is alpha galactosidase A and which affects metabolism and storage of fats and carbohydrates. Although recombinant forms of this enzyme are currently available to sufferers of Fabry disease, such forms are prohibitively expensive and production capacity is limited. Large Scale Biology Corporation has developed ENZAGAL, which the company claims can be biomanufactured more efficiently and in greater abundance than competing products currently produced in animal cell cultures. With regard to products that are too complex to lend themselves to microbial transgenics, there are several examples of efforts being focused on new and innovative therapeutic products. The worlds first plantibody, a plant-produced antibody, has been developed by Planet Biotechnology Inc. to help prevent tooth decay. The product, called CaroRx, is an antibody that specifically binds to S. mutans, the bacteria that cause tooth decay, which prevents the bacteria from adhering to teeth. CaroRx is in clinical trials in the United States. Vaccines are another area of research in molecular farming. Early-stage clinical trials have been completed on customized, patient-specific vaccines for Non-Hodgkins Lymphoma. These plant-produced vaccines can be generated in 6 to 10 weeks, a much shorter time frame than conventional methods. As mentioned previously, edible vaccines, although enthusiastically discussed in recent years, have virtually been abandoned. Despite promising results from early clinical trials of an edible vaccine in potato against

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Hepatitis B, fears that the engineered crops could become mixed in with food crops have prompted researchers to turn to non-food crops instead, primarily tobacco. The issue of the use of food crops in plant molecular farming was discussed earlier in this paper (8). CONCLUSION Increased pharmaceutical demands, as well as advances in gene identification following the completion of the Human Genome Project, have led to an interest in plants as expression systems for therapeutic products. Despite numerous benefits to this approach, however, the concerns that have been raised must be adequately addressed. Although molecular farming offers an exciting alternative for pharmaceutical production, industry and government must proceed cautiously in this area in order to gain public acceptance.

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Pollen-mediated gene flow from transgenic safflower (Carthamus tinctorius L.) intended for plant molecular farming to conventional safflower McPherson, Marc A, Good, Allen G, Topinka, A. Keith C, Yang, Rong-Cai, McKenzie, Ross H, Cathcart, R. Jason, Christianson, Jed A, Strobeck, Curtis and Hall, Linda M. Published by: Environmental Biosafety Research, January 2009 8 : pp 19-32

Abstract Field experiments were conducted in Chile and western Canada to measure shortdistance (0 to 100 m) outcrossing from transgenic safflower (Carthamus tinctorius L.) intended for plant molecular farming to non-transgenic commodity safflower of the same variety. The transgenic safflower used as the pollen source was transformed with a construct for seed-specific expression of a high-value protein and constitutive expression of a gene conferring resistance to the broad-spectrum herbicide glufosinate. Progeny of non-transgenic plants grown in plots adjacent to the transgenic pollen source were screened for glufosinate resistance to measure outcrossing frequency. Outcrossing frequency differed among locations: values closest to the transgenic pollen source (0 to 3 m) ranged from 0.48 to 1.67% and rapidly declined to between 0.0024 to 0.03% at distances of 50 to 100 m. At each location, outcrossing frequency was spatially heterogeneous, indicating insects or wind moved pollen asymmetrically. A power analysis assuming a binomial distribution and a range of alpha values (type 1 error) was conducted to estimate an upper and lower confidence interval for the probable transgenic seed frequency in each sample. This facilitated interpretation when large numbers of seeds were screened from the outcrossing experiments and no transgenic seeds were found. This study should aid regulators and the plant molecular farming industry in developing confinement strategies to mitigate pollen mediated gene flow from transgenic to non-transgenic safflower.

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REFERENCES
1) Barros E, Bock R, Christou P, et al. 2005, Current perspectives on the production

of pharmaceuticals in transgenic plants, Published by: The European Union Framework 6 PharmaPlanta Consortium.
2) Mewett O, Johnson H and Holtzapffel R 2007, Plant molecular farming in Australia and overseas, Australian Government: Bureau of rural sciences.
3) Mor T S and Arntzen C J 2002, Plants and human health: delivery of vaccines via

transgenic plants; Arizona Biomedical Institute and the Plant Biology Department.
4) Thomas B R, Van Deynze A, Bradford K J 2002,Production of therapeutic

proteins in plants, Published by: Agricultural Biotechnology in California Series, Publication: 8078.

5) Twyman R M, Stoger E, Schillberg S, et al. 2003,Molecular farming in plants:

hostsystems and expression technology,Trends in Biotechnology, Vol. 21, Issue 12, pp. 570-578. 6) www.biotechterms.org 7) www.molecularfarming.com 8) www.parl.gc.ca 9) www.the-gist.org

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