plant molecular farming in australia and...

Plant molecular farming in Australia and overseas

Osman Mewett, Hilary Johnson and Ruth Holtzapffel

© Commonwealth of Australia 2007

This work is copyright. Apart from any use as permitted under the Copyright Act 1968, no part may be reproduced by any process without prior written permission from the Commonwealth. Requests and inquiries concerning reproduction and rights should be addressed to the Commonwealth Copyright Administration, Attorney General’s Department, Robert Garran Offices, National Circuit, Barton ACT 2600 or posted at http://www.ag.gov.au/cca.

ISBN 1 921192 05 4

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http://www.ag.gov.au/cca

Foreword Plant molecular farming is the cultivation of genetically modified plants as ‘biofactories’ to produce novel pharmaceutical and industrial products. Applications in plant molecular farming are being developed around the world and adoption (whether overseas or domestically) could have implications for Australia.

Plant molecular farming offers many opportunities for Australian agriculture, including: the diversification of existing industries; the development of new industries for new products; a greater return on rotational break crops such as canola and lupins; and the opportunity to focus on regional animal and human disease priorities.

This report provides information on the potential benefits and limitations of using genetically modified plants as biofactories, discusses which plants have the most potential for use as biofactories, discusses issues surrounding the use of food crops as biofactories, identifies current research projects in Australia and overseas and highlights those with the most potential to affect Australian agriculture. It is based on reviews of published literature and structured discussions with experts involved in the development of plant molecular farming applications in Australia.

Dr Colin J. Grant Executive Director Bureau of Rural Sciences

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Executive summary This report explores what third generation genetically modified (GM) plants are, why they are being developed, which traits are being developed, when they can be expected to be commercialised and how they may be relevant to Australian agriculture.

Third generation genetically modified (GM) plants are plants designed to act as ‘biofactories’, producing pharmaceutical and industrial products. In this way, third generation GM plants are distinct from first generation plants, designed to reduce the inputs required for their growth and second generation plants, designed to have altered outputs.

The cultivation of third generation GM plants has been termed ‘plant molecular farming’. Pharmaceutical-producing plants are often called ‘pharma’ crops and the cultivation of these plants is commonly referred to as ‘pharming’ or ‘biopharming’.

Plants have been a source of pharmaceutical and industrial compounds for centuries…

The use of plants to produce pharmaceutical and industrial products has a long history. For example: willow bark has been used to reduce fever since ancient Roman times – the active ingredient was later isolated and called aspirin; opiates have been extracted from poppies for centuries; and rubber was originally made from the sap of the rubber tree but is now made mainly from petrochemicals. Vegetable oils are used for the production of industrial compounds such as lubricants, flow enhancers and hydraulic oils.

..and this is not the first time that gene technology has been used to produce pharmaceutical and industrial compounds.

The use of gene technology to produce pharmaceutical and industrial compounds is not new. Medical proteins such as insulin have been produced from GM bacteria since the early 1980s. Some industrial enzymes, such as those in detergents, are also produced from GM bacteria.

A wide variety of traits are being expressed in third generation GM plants, including antibodies for diagnosis and treatment of medical conditions, human and animal vaccines…

Plants are able to produce many different compounds and researchers have taken advantage of this to make GM plants that produce quite complex proteins including antibodies. These antibodies may be used to diagnose pregnancy or cancer, to prevent tooth decay or as a treatment for cancer.

Plants are also being used to produce simpler proteins that can be used to vaccinate people and animals against diseases. It is hoped that oral vaccines can be developed from these GM plants to decrease the costs and risks associated with injectable vaccines.

Human and animal disease priorities are often regional, giving Australia a potential local market for plant-made vaccines. Avian Influenza in South-East Asia is an example of a disease that is being targeted by Australian researchers working on third generation GM applications.

…other proteins for use in laboratory research and medical treatments…

Globally a variety of other proteins are also being expressed commercially in plants. These have a number of different applications for use in laboratory research and diagnostics (e.g. β-glucuronidase, used in plant research).

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…bioplastics… 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. Australian researchers are currently working to produce bioplastic from sugarcane. Researchers overseas are using maize and oilseeds as factories for producing bioplastic.

…and biofuels. 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.

The main aims of developing third generation GM plants are to decrease the costs of production of these compounds, increase their availability, reduce our reliance on petrochemical resources and provide alternative agricultural products for Australian farmers.

Many pharmaceutical and industrial products are now made: by GM or non-GM bacteria, yeast or mammalian cell cultures; purified from animal tissue; or using non-renewable petrochemical resources. It is widely believed that third generation GM plants will:

• provide cheaper sources of drugs and food additives • improve access to both human and livestock vaccines • provide more environmentally friendly industrial

chemicals • provide farmers with alternative agricultural products

and higher farm-gate returns and • provide improved renewable resources for producing

biofuels.

Plant-made pharmaceuticals should also be safer by decreasing the risk of pharmaceuticals being contaminated with human or animal pathogens.

These aims may well be achieved through the use of GM technology, however, most third generation GM plants are at an early stage of research and the proposed benefits are yet to be proven.

Developments in third generation GM plants may provide opportunities for Australian agriculture, particularly where large production areas are required.

Possible opportunities for Australian agriculture include:

• diversification from traditional food and feed markets into new markets that may have higher profit margins

• the development of new industries, based on new crop plants

• a greater return on break crops used in rotational cropping systems, as these break crops may be modified to produce higher value products and

• the opportunity to focus on animal and human disease priorities in our region.

Products requiring a high volume of production, such as biofuels or bioplastics, are likely to provide the greatest opportunities for Australian agriculture. The adoption of these technologies could help to maintain or increase the value of Australian broadacre cropping systems. This in turn should increase the value of the agricultural industry as a whole.

However, some traits will require low levels of production and will be less applicable to Australian agriculture.

Many other applications, in particular those relating to pharmaceuticals, will require smaller scale production and are unlikely to provide extensive opportunities for agricultural cropping systems. They may be grown by a few farmers on small areas of land, or kept contained within glasshouses or laboratories. Some of these applications may not even occur in whole plants but make use of plant cell culture systems analogous to bacterial or yeast fermentation systems.

Although commercial production of many of these traits in Australia is a long way off, there are some projects that can be expected to be commercialised within the next five years.

There are many research groups in Australia and overseas developing plant molecular farming applications. Much of this research is at an early stage of the product development pipeline and is at least 10 years from commercialisation, but some products have already been commercialised overseas. These include a number of proteins that are used in laboratory research or diagnostics.

In Australia, a few applications have developed further and could be commercialised within the next five years. For example, an Australian company, Farmacule BioIndustries, has developed GM tobacco plants that express the high-value human protein, vitronectin that is widely used in medical research. Currently vitronectin is isolated from blood serum, but vitronectin is also being expressed in GM tobacco plants. One gram of vitronectin per month could be produced in a single biosecure glasshouse. This level of production would meet the total world demand for vitronectin, which currently retails for $2.3–6.8 million per gram.

Overseas, Syngenta is close to commercialising a GM maize plant that can be used to produce biofuel ethanol more cheaply. The plants produce a heat stable enzyme that converts starch to sugar and will simplify the processing required for ethanol production.

A number of different plant species are being considered for expression of third generation GM traits.

The choice of plant species for expressing third generation GM traits will affect every stage of product development, from the initial design of the modified DNA to purification of the final product. Using easily identifiable, non-food plants for these applications would reduce the potential for admixture with the food or feed supply chains. However, for many reasons, existing food crops may provide the best opportunities for the efficient production of pharmaceuticals.

There are a number of issues that need to be addressed when considering the adoption of this technology, including regulation…

In Australia, third generation GM plants will be subject to the same regulatory control by the Gene Technology Regulator as are all other GM plants and will be assessed for risks to human health and safety and the environment on a case-by-case basis. The Gene Technology Regulator has the capacity to issue a licence that contains specific conditions to manage risks.

Third generation GM plants or products from these plants may also be subject to regulation by the Therapeutic Goods Administration, Food Standards Australia New Zealand, the Australian Pesticides and Veterinary Medicines Authority or the National Industrial Chemicals Notification and Assessment

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Scheme, depending on the trait, the plant and the intended use.

…segregation from other crops…

The development of separate supply chains, even closed loop supply chains, for each pharmaceutical and industrial product is likely to be important. Each third generation GM application will have particular and valuable properties that need to be protected in the growing and post-harvest phases of production. Mixing lower-value, non-GM crops with higher-value, third generation GM crops could jeopardise this higher value.

Some third generation products may not be approved for use in the food chain and will need to be kept segregated for safety reasons. Also, third generation GM crops will be subject to the same marketing issues as all GM crops (such as tolerance levels for the presence of GM products) and will need to be managed in such a way as to meet these requirements.

If a third generation GM crop required strict segregation from conventional varieties of that crop or from other crops, there could be significant costs involved. Whether this affected the economic viability of the crop would need to be evaluated by the developer.

…and the potential for a specific product to be commercially successful.

It is important that developers of third generation GM products consider the product’s potential for commercial success. Products must meet market specifications, have legal access to all required enabling technologies (e.g. intellectual property) and receive the necessary regulatory and industry approvals. The product also needs to provide value throughout the supply chain. If a product is missing the ability, freedom, permission or incentive to operate, it is unlikely to succeed in the marketplace.

If the value of rotation break crops could be increased significantly, there could be opportunities to develop niche export markets for Australian farmers, for example a potential niche market could be developed for high quality industrial oils, such as those used in the automotive industry.

This report concludes that third generation GM plants producing industrial compounds will have the greatest potential to benefit broadacre Australian agriculture in the future.

Although plant molecular farming is still developing in Australia, it should provide opportunities for Australia to add value to its broadacre cropping systems. In particular, growing GM plants that make industrial products may help farmers to maintain their profitable, competitive and sustainable cropping systems.

There are still issues to be resolved, but the world’s human population will continue to increase, along with its demands for pharmaceuticals, energy and industrial products. These demands are likely to exceed global production capacity in the future. Adopting third generation GM plants may, therefore, become important in the near future, making it necessary for the scientific, regulatory and policy issues that surround this technology to be discussed sooner rather than later.

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Contents Foreword ................................................................................................................................. iii

Executive summary.......................................................................................................................... v

Contents ................................................................................................................................. ix

List of abbreviations ..................................................................................................................... xiii

Glossary ................................................................................................................................ xv

Chapter 1 Introduction............................................................................................................. 1

1.1 What are third generation GM plants?...................................................................... 1

1.2 Why are third generation GM plants being developed? ........................................... 2

1.3 When will third generation GM crops be commercialised? ...................................... 2

1.4 How was information collected for this report?......................................................... 4

Chapter 2 Potential benefits and opportunities from developing third generation GM plants ....................................................................................................................... 7

2.1 Potential benefits ...................................................................................................... 7

2.1.1. Potential for decreased costs and increased safety................................................. 7

2.1.2. Increased functionality .............................................................................................. 8

2.2 Relevance to Australian agriculture.......................................................................... 8

2.2.1 Which plant species will be used?............................................................................ 8

2.2.2 Where should the protein be expressed within the plant?...................................... 10

2.2.3 Adding value to rotation cropping systems............................................................. 11

2.2.4 Using plants to make industrial products................................................................ 12

2.2.5 Biofuels ................................................................................................................... 12

2.2.6 Using plants to make pharmaceutical products...................................................... 13

2.3 Technical constraints .............................................................................................. 13

2.3.1 Difficulty in obtaining high yields and consistent expression levels ....................... 14

2.3.2 Plant specific glycosylation and potential for allergic reactions.............................. 14

2.3.3 Potential for gene flow and admixture with food and/or feed crops ....................... 15

Chapter 3 Recent developments in plant molecular farming ............................................ 17

3.1 Introduction ............................................................................................................. 17

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3.2 Pharmaceutical applications of plant molecular farming ........................................ 17

3.2.1 Antibodies ............................................................................................................... 17

3.2.2 Vaccines ................................................................................................................. 17

3.2.3 Other therapeutic and research/analytical proteins................................................ 18

3.3 Industrial applications of plant molecular farming .................................................. 18

3.3.1 Biofuels ................................................................................................................... 18

3.3.2 Sweeteners............................................................................................................. 18

3.3.3 Bioplastics............................................................................................................... 18

3.3.4 Spider silk ............................................................................................................... 19

3.3.5 Resilin ..................................................................................................................... 19

3.4 Plant molecular farming in Australia ....................................................................... 19

3.4.1 Broadacre grain crops and oilseeds ....................................................................... 20

3.4.2 Tobacco .................................................................................................................. 20

3.4.3 Sugarcane .............................................................................................................. 20

3.4.4 Horticultural crops................................................................................................... 21

3.4.5 Other applications................................................................................................... 22

3.4.6 Summary table........................................................................................................ 22

3.5 Plant molecular farming internationally................................................................... 22

3.5.1 Broadacre grain crops and oilseeds ....................................................................... 23

3.5.2 Tobacco .................................................................................................................. 25

3.5.3 Horticultural crops................................................................................................... 26

3.5.4 Other or non-specified host .................................................................................... 27

3.5.5 Summary table........................................................................................................ 28

Chapter 4 Discussion and conclusions ............................................................................... 29

4.1 Choice of platform crops......................................................................................... 29

4.1.1 Sugarcane in Australia............................................................................................ 29

4.1.2. Maize in the United States...................................................................................... 30

4.2 Perceived constraints of the technology................................................................. 30

4.2.1 Regulation of third generation GM plants ............................................................... 30

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4.2.2. Segregation and identity preservation .................................................................... 31

4.2.3 Intellectual property ................................................................................................ 33

4.3 International initiatives ............................................................................................ 34

4.4 Conclusions ............................................................................................................ 35

Acknowledgements........................................................................................................................ 37

References ................................................................................................................................ 39

Appendix A Consultation questions........................................................................................ 47

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List of abbreviations

CaMV35S Cauliflower Mosaic Virus 35S promoter

CRC SIIB Cooperative Research Centre for Sugar Industry Innovation through Biotechnology

CSIRO Commonwealth Scientific and Industrial Research Organisation

DAFF Department of Agriculture, Fisheries and Forestry

FDA Food and Drug Administration

FSANZ Food Standards Australia New Zealand

GM Genetically modified

GMP Good Manufacturing Practice

GRDC Grains Research and Development Corporation

HEAR High Erucic Acid Rape

HSV-2 Herpes Simplex Virus type 2

mAb Monoclonal antibody

ML Mega litres

OGTR Office of the Gene Technology Regulator

PHA Polyhydroxyalkanoate

PHB Poly(3-hydroxybutyrate)

PMV Plant-made vaccine

sIgA Secretory Immunoglobulin A

TGA Therapeutic Goods Administration

UCS Union of Concerned Scientists

USDA United States Department of Agriculture

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Glossary Where these words appear in the main body of the report they are highlighted in bold.

Adjuvant A substance used in conjunction with a vaccine to enhance the immune response.

Antibody Proteins produced by the immune system in response to the presence of a specific antigen.

Antigen Any substance, for example a protein or glycoprotein, which is capable of causing an immune response.

Apomixis The production of fertile seeds without fertilisation. The resulting seeds are genetically identical to the parent plant.

Bagasse The biomass remaining after sugarcane stems are crushed to remove their juice.

Biofuel Any fuel which is derived from biomass.

Biomass Plant material that can be used as a fuel or energy source

Biomimetics The application of methods and systems found in nature to the study and design of engineering systems and modern technology.

Bioplastic Plastic derived from biological sources.

Biotechnology A broad term to describe the process of using living things to make products or perform tasks for people.

Break crop A crop grown as part of a crop rotation system that helps control weeds and plant diseases, while also providing income through sale of produce from the crop.

C3 photosynthesis The most common type of photosynthesis. It is more efficient than C4 photosynthesis under cool, moist conditions and normal light levels.

C4 photosynthesis A type of photosynthesis found, for example, in many tropical grasses in which photosynthesis occurs faster and more efficiently under high temperature and light and conserves more water in comparison to the more typical C3 photosynthesis.

Chloroplast Organelle present in green algae and plants that contains chlorophyll and carries out photosynthesis.

Ending stocks The volume of product remaining at the end of a given time frame (e.g. at the end of a year).

Extensible Capable of being extended or stretched.

Genetic Use Restriction Technologies (GURTs)

Applications of biotechnology that provide the means to turn genes ‘on’ or ‘off’. This includes technologies that prevent the seeds of some GM plants germinating or otherwise prevent gene flow occurring.

Glycoprotein Any protein with one or more sugar chains linked to it.

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Glycosylation The process of adding one or more sugars to a protein.

Human serum albumin A plasma protein that helps to maintain blood pressure in humans.

Immunogenic A term used to describe a substance which can induce an immune reaction.

Interferon A type of cytokine released by certain types of immune cells to enhance the response to infection.

Interleukin A type of cytokine that mainly mediates interactions between white blood cells during inflammation and immune responses.

Isomer A compound that contains exactly the same constituents as another compound except that the constituents are arranged differently.

Male sterility The result of altered pollen formation where no pollen or infertile pollen is produced by a plant.

Monoclonal antibodies Specific antibodies that have been produced in a laboratory, are identical, and recognise a single antigen.

Native In relation to genes or proteins: those that are naturally present in an organism.

Peptide A constituent molecule of proteins, formed by the linking of two or more amino acids.

PHA Polyhydroxyalkanoate

A compound used to produce a certain type of bioplastic.

PHB Poly(3-hydroxybutyrate)

The most extensively studied PHA.

Promoter The region of DNA to which RNA polymerase binds to begin transcription. This region of DNA helps to determine where each gene is expressed within an organism.

Vaccine An antigenic preparation — protein, peptide, attenuated (weakened) living organism or a dead organism — which is able to elicit an immune response that prevents the development of an infectious disease.

Vegetative tissue Plant tissue, such as a leaf and stem, which does not have a floral or reproduction function.

Volunteer plant An unwanted plant resulting from natural propagation, rather than deliberate planting. For example a plant that emerges after harvest.

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Plant molecular farming 1

Chapter 1 Introduction Many advances in plant biotechnology are occurring rapidly in Australia and internationally. A selection of broadacre genetically modified (GM) crops with the potential to be commercially released within the next 15 years were identified in the recent BRS report, ‘What’s in the pipeline?’ (Glover et al., 2005b). This report contained a brief discussion of the research being conducted in Australia and internationally into third generation GM plants, which are designed to act as biofactories, producing pharmaceutical or industrial compounds.

This current report is the result of a further study — funded by the Australian Government Department of Agriculture, Fisheries and Forestry (DAFF) under the National Biotechnology Strategy which aims to improve the understanding of and encourage development in, innovative applications of biotechnology in the agriculture and food sectors. This report provides information on Australian and international developments in third generation GM plants, current and future research trends and identifies issues that will need to be addressed. It is based on reviews of published literature and consultations with experts involved in the development of third generation GM crops in Australia (the methodology is described in more detail at the end of this chapter).

This chapter introduces the topic and outlines the main reasons for developing third generation GM plants. The remaining chapters look at particular issues, as follows:

• Advantages and disadvantages of developing third generation plants (Chapter 2)

• Examples of third generation developments relevant to Australian agriculture (Chapter 3)

• Discussion of the major findings (Chapter 4)

Appendix A shows the questions used as the basis for consultation with the experts.

1.1 What are third generation GM plants? Plants that have been modified to express pharmaceutical and industrial products are referred to as third generation GM plants, to distinguish them from GM plants containing first and second generation genetic modifications, which generally aim to reduce the inputs required for growing plants and to alter the output of the plants, respectively (e.g. Glover et al., 2005b and Table 1.1).

Table 1.1 Classification of GM plant traits used in this report

Category Specific traits Also called Environmental (abiotic) stress tolerances Improved pest and disease control

First generation

Improved nutrient use

Input traits

Enhanced nutrition Improved oil quality Longer post-harvest life

Second generation

Improved feed and pastures

Output traits

Pharmaceutical crops Third generation Industrial crops

Non-food industrial products or processes

Source: Modified from Glover et al. (2005b).

The cultivation of third generation GM plants has been termed ‘plant molecular farming’. Pharmaceutical-producing plants are often called ‘pharma’ plants and the cultivation of these plants is commonly referred to as ‘pharming’ or ‘biopharming’.

The classification of GM plants as second or third generation is not always clear cut, because both describe plants with altered output traits. Glover et al. (2005b) defined second generation modifications as those relating to enhancements of the food or feed quality or nutritional value and third generation as those relating to non-food, industrial or pharmaceutical products. For example,


in this report, modifications relating to taste (e.g. production of alternative sweeteners) are classified as third generation traits, because the modification is not designed to make a particular crop taste sweeter; the crop is being used as a biofactory for the production of the sweetener, which in most cases, will then be purified and added to processed foods. Another example where this classification is not clear cut is for ornamental plants, such as the GM carnations altered for flower colour.

It is worth noting that using living organisms to produce pharmaceutical and industrial products is not new. Plants and their extracts have been used to treat human disease since the earliest civilisations: for example, willow bark was used to relieve pain and fever in ancient Roman times and it was only in the late 19th century that the active ingredient, aspirin, was isolated. Other pharmaceutical examples include opiates, which have been extracted from poppies for centuries (Miller and Tran, 2000) and insulin, which has been produced in GM bacteria since 1982 (Swartz, 2001). Rubber is an example of a plant-produced industrial product. Although most rubber is now produced from petroleum products, about 25% of rubber still comes from the rubber tree, Hevea brasiliensis (ANU Forestry, 1999). Enzymes produced by GM bacteria are used in industrial applications, such as detergents, with the first one produced in 1988 (Sasson, 2005). Indeed Australia already has experience in producing a conventionally bred ‘pharma’ crop, the opium poppy. Alkaloids that are produced by commercial poppy crops are essential elements in analgesics and Tasmania grows over 40% of the world’s legal opiates.

1.2 Why are third generation GM plants being developed? Traditionally, the commercial production of pharmaceutical proteins has used mainly microbial or mammalian systems and many modern industrial commodities are produced using non-renewable petrochemical resources. The use of plants as replacement sources of similar products is widely expected to:

• provide cheaper sources of drugs and food additives, such as artificial sweeteners

• improve access to vaccines for people

• provide more environmentally friendly industrial chemicals

• provide farmers with alternative agricultural products and higher returns

• provide improved renewable resources for producing biofuels.

However, the ability of third generation GM plants to live up to these expectations has yet to be proven. Only a small number of plant-made proteins have made it through the ‘pipeline’ to commercialisation. These include a small number of proteins used in scientific and medical research, which are being commercially produced from GM plants and the first plant-made veterinary vaccine, which has recently achieved regulatory registration in the United States (Chapter 3).

1.3 When will third generation GM crops be commercialised? The ‘pipeline’ referred to above is the product development pipeline for GM plants, as defined by Glover et al (2005a; 2005b) (Figure 1.1). Where possible, predictions of the time to commercialisation have been made for the products discussed in this report (Chapter 3). Some of the third generation applications reported in Chapter 3 are at an advanced stage, with commercialisation expected in less than five years, while other applications are at a very early stage of development and commercialisation is not likely to happen for at least 10–15 years. However, as Glover et al (2005b) discussed, the time taken for a GM plant to make its way through the pipeline depends on many factors, including the plant itself, the complexity and type of introduced product, business negotiations regarding access to intellectual property and the regulatory regime.

Negotiations around the use of intellectual property and other enabling technologies aim to provide freedom to operate. A lack of freedom to operate and the necessary regulatory and industry permissions to operate, are often seen as the main impediments to commercialising GM products. However, there are other important considerations that may sometimes be over-looked: a company must make a product that meets market specifications, thus demonstrating its ability to operate and the product itself must provide an incentive to operate through increased profitability for both the producer and the whole supply chain. This set of requirements is summarised in Figure 1.2. These requirements are not unique to GM plants. Failure to meet any one of these requirements is likely to prevent commercial success for a new product.

For successful characteristics, regulatory approvals are gained and seeds/planting material produced and marketed

Genes of interest are tested in a crop under field conditions to determine the likely success of the crop

Genes of interest are tested, usually in non-crop plants, for those which show the most promise for application in crops

The research stage of the process, where an idea is explored

Figure 1.1 The four stages of the product development pipeline for GM plants Source: Glover et al. (2005a).

Figure 1.2 Requirements for making commercial products

Source: Allan Green (personal communication)

Therapeutic products that contain substances produced in third generation GM plants and are intended for import into, manufacture or supply in or export from Australia will be subject to the

+ Ability

+ +PermissionFreedom Incentive to

operate to operate to operate to operate

e.g. Product can be made to meet

market specification

e.g. No patent infringement

e.g. Regulatory approval

e.g. Profitable production

Industry approval

Creates value throughout

supply chain


same regulatory controls as apply to other therapeutic products, i.e. regulation by the Therapeutic Goods Administration (TGA) to ensure quality, safety and effectiveness.1

Pharmaceutical therapeutic products (e.g. medicines and medical devices) that contain substances produced in third generation GM plants will also need to satisfy a regulated clinical trial process to ensure their safety and efficacy effectiveness. Clinical trials are research studies using human volunteers to test new treatments, medicines or therapeutic products. They aim to answer specific health questions that cannot be answered using animal models. Clinical trials are commonly split into four phases, with each phase having a different purpose and allowing scientists to answer different questions (Figure 1.3).

Researchers test an experimental drug or treatment in a small group of people (20-80) for the first time to evaluate its safety, determine a safe dosage range and identify side effects.

Figure 1.3 The four phases of the clinical trial process

Source: Text adapted from http://www.clinicaltrials.gov and http://www.medicinesaustralia.com.au/pages/page39.asp.

There is also continuing debate regarding the feasibility of significantly reducing the world’s dependence on fossil fuels through increased use of vegetable oils, wood and other sources of biomass, particularly if these are produced on land or with water that would otherwise be used for food crops (Giampietro et al., 1997; Berndes, 2002; Bungay, 2004; Kim and Dale, 2005; Pearce, 2005).

1.4 How was information collected for this report? This report is based on a combination of literature review and expert advice from people involved in plant molecular farming in Australia. Appendix A presents the list of questions that were used as the basis for discussions with these experts. Although not all who were contacted agreed to speak with us, most were keen to share their knowledge, experience and views of this exciting and rapidly developing area of agricultural biotechnology. These consultations provided a valuable insight into

1 Information about the relevant regulatory requirements is available on the TGA website: www.tga.gov.au

Phase I

Phase II

Phase III

Phase IV

The experimental drug or treatment is given to a larger group of people (100-300) to see if it is effective and to evaluate its safety further.

The experimental drug or treatment is given to large groups of people (1000-3000) to confirm its effectiveness, monitor side effects, compare it to commonly used treatments and collect information that will allow it to be used safely.

Post-marketing studies provide additional information including the drug’s risks, benefits and optimal use.


the opportunities that this technology could bring to Australia, as well as raising the issues that should be addressed before this happens. In total 14 people from eight organisations were consulted for this report. Where information from these discussions is used in the text, it is referenced as ‘consultations with experts’.



Chapter 2 Potential benefits and opportunities from developing third generation GM plants

There are a number of factors driving the development of third generation GM plants, including the need to:

• produce large volumes of vaccines for livestock diseases, such as Avian Influenza;

• reduce the world’s dependence on petrochemical resources, while also reducing pollution; and

• deliver affordable drugs to large populations of people.

These factors are likely to become increasingly important in the future as the world’s human population continues to grow.

Advocates of third generation GM plants predict cost, safety and environmental benefits although these have yet to be proven.

It is likely that some third generation GM plants will be able to produce sufficient amounts of a specific pharmaceutical or research protein on a relatively small area of land or in a laboratory, and will not have a large impact on the agricultural sector. However, other developments will require production on an extensive scale, suitable for incorporation into broadacre production systems and have the potential to provide additional or alternative sources of income for the Australian agricultural sector. Developments in third generation GM plants are discussed in the following chapter with an emphasis on those with the greatest potential to provide benefits to Australian agriculture. This chapter discusses the benefits that may arise from developing third generation GM plants and the opportunities that exist to direct and support further developments in this area. A number of technical constraints that are currently limiting developments in this field are also identified and discussed.

2.1 Potential benefits

2.1.1. Potential for decreased costs and increased safety Many people predict that plant-based production systems for pharmaceutical and industrial products will be cheaper and easier to scale up or down than the traditional production methods such as using animals, mammalian cell cultures, or bacterial/yeast fermentation systems to make the products. Although it is relatively inexpensive to grow bacteria and yeast, the demands of Good Manufacturing Practice (GMP; discussed in Section 2.3.1) at downstream processing stages increase the required technology costs (Kirk and Webb, 2005).

If sufficiently high yields can be obtained, proteins could be produced in plants at 2–10% of the cost of bacterial or yeast fermentation systems and at 0.1% of the cost of mammalian cell cultures (Twyman et al., 2003). However, despite the high cost of mammalian cell culture production systems, it is also worth noting that the protein yield obtained is 10–100 fold larger than current plant-based production systems (consultations with experts).

If vaccines can be produced in plants in an economically viable way, it could reduce the cost of vaccine production, purification, storage and administration — crucial cost reductions for developing countries and the livestock industry. However, it is important to remember that GMP requires that a regulated vaccine must be delivered in a standardised dose (for efficacy and lack of toxic effects), so some level of processing of the plant tissue is still likely to be required (Kirk and Webb, 2005; Ma et al., 2005a).

Expressing proteins in the storage tissues of plants (e.g. seeds or tubers) has been suggested as a way to remove the need for cold storage of products such as vaccines; however, current data indicates that drying is still required for long term stability of proteins. The expression of oral vaccines in plant tissues will also remove the need for needles. Both of these factors will result in a decreased cost of delivery.

Local production has been cited as another way that plant-made vaccines (PMVs) will contribute to decreased costs through reduced transport requirements (Goldstein and Thomas, 2004; Dalal et al., 2006), but processing will still be required and it is likely to be more cost effective to build a few dedicated processing facilities rather than attempt local processing.

Pharmaceuticals produced from plants should be inherently safer for the recipient than those produced from animals or cell culture, as they are much less likely to be contaminated by human or animal pathogens (Goldstein and Thomas, 2004; Peterson and Arntzen, 2004; Howard and Hood, 2005). Oral PMVs should also increase safety by replacing needles with capsules or powders, thus reducing the risk of injury for health professionals.

2.1.2. Increased functionality Plants have an advantage over some other organisms used for protein expression, such as bacteria, because they are able to make, fold and correctly assemble proteins consisting of multiple subunits. This allows them to be used to make antibodies and other complex proteins. As an example, yeast, bacterial and mammalian cell culture systems are unable to make the immune protein molecule, Secretory Immunoglobulin A (sIgA)—which consists of four linked proteins—while GM tobacco plants have been used to demonstrate that sIgA can be successfully made by plants (Goldstein and Thomas, 2004). It may also be possible to create multi-component vaccines by inserting a number of different antigens into the same GM plant or by crossing GM plant lines expressing different antigens (Goldstein and Thomas 2004).

Another advantage of using plants for the production of vaccines and other therapeutics is the higher stability of the protein as compared to other protein expression systems, under a wide range of conditions either within the plant cell or following extraction (consultations with experts).

Administration to people of antibodies produced by animals can lead to adverse immune reactions because the immune system recognises the proteins as non-human in origin. By producing human antibodies in plants, the likelihood of these reactions can be reduced. However, differences in the way plants and humans glycosylate proteins can still produce adverse immune responses (see Section 2.3.2).

2.2 Relevance to Australian agriculture

2.2.1 Which plant species will be used? Many different plant species have been investigated as platforms for the production of pharmaceutical and industrial compounds. These range from simple plants (e.g. moss) and model plants (such as Arabidopsis thaliana or tobacco) that are easy to grow in laboratories or glasshouses, to domesticated food crops that are grown on a broadacre scale (such as maize and wheat). Pharmaceutical or research and diagnostic proteins will often be of high value and require relatively low production volumes. As a result, they are likely to be produced in laboratory or glasshouse containment and utilise model plants (for example see section 3.4.2 on the production of the high-value human protein vitronectin in tobacco).

However, economies of scale may be necessary to provide cheap drugs and oral PMVs for large populations and these, like many industrial compounds (for example, applications of GM technologies to biofuel and bioplastic production discussed in Chapter 3), may be grown by the


agricultural sector once approved by the Australian regulatory system. These crops could provide an alternative source of income for Australian farmers.

Simple plants Algae, moss and aquatic plants, such as duckweed, have been used as model laboratory species for many years. Some research companies are developing systems using these organisms to produce pharmaceutical proteins such as antibodies. The potential benefits of these production systems include high protein expression levels, rapid growth, lack of pollen and seed production, secretion of the proteins into the growth medium and low cost2. Production of functional antibodies has been demonstrated in the green algae Chlamydomonas reinhardtii (Mayfield et al., 2003). However, such projects will not produce GM plants that are relevant to Australian agriculture.

Wild species The use of wild species not usually grown as crops may have the advantage of the plants being clearly distinguishable from food or feed crops. This decreases the chance of their products being mistakenly incorporated into the food or feed chain. However, disadvantages to this approach are that wild species have not been adapted to production in agricultural systems and may possess traits such as seed shatter, dormancy or low yields, making them difficult for farmers to produce and contain. Also, little may be known about the safety of such crops; for example, they may produce substances that are allergenic or toxic for people or animals. These are some of the issues that the Crop Biofactories Initiative (see Box 2.1) will be addressing.

Domesticated food and feed crops Food and feed crops often have advantages such as fast growth, high yield and ease of transformation and many have been used for experimental expression of vaccines, including potatoes, tomatoes, bananas, carrots, lettuce, maize, alfalfa and white clover (Fischer et al., 2004; Goldstein and Thomas, 2004). If plant-made pharmaceutical products were to be consumed either uncooked, unprocessed or partially processed, cereal and vegetable crops might prove to be the most attractive future option for PMVs (Twyman et al., 2003). However, as discussed in Section 2.3.1, pharmaceutical products are unlikely to be delivered in an unprocessed form, due to the need to provide standardised and effective doses.

Domesticated non-food crops Tobacco has been the non-food plant of choice when expression in leaf tissue is desired, with advantages including established technology for gene transfer and protein expression, high biomass and seed production and existing infrastructure for large scale processing (Twyman et al., 2003). Since tobacco is a non-food and non-feed crop, there is a reduced risk of its products entering food or feed chains. A disadvantage of using tobacco is its high content of toxic alkaloids, the removal of which adds to the downstream processing requirements (Howard and Hood, 2005). However, low-alkaloid tobacco cultivars have been developed (Rymerson et al., 2003) and may alleviate this problem.

Choice of an appropriate plant platform for third generation GM traits in Australia The choice of plant platform will vary, depending on a number of factors such as volumes required, nature of the substance being produced and end use of the product. It is widely accepted that no one plant will be suitable for all third generation applications. A suitable crop platform may need to be developed from a plant species that is not currently grown commercially in Australia or perhaps anywhere in the world. Breeding and agronomy programs would need to be set up if a new crop species were to be successfully domesticated in Australia. It has been estimated that it could take

2 http://www.biolex.com/technologies.html;


10 years or more before cultivars suited to growth in Australia could be released from such breeding programs if new crops are chosen as platforms. To this end, the Grains Research and Development Corporation (GRDC) has recently commissioned a study to identify a number of possible plants that could be targeted for development as third generation GM crop platforms. This study forms part of the Crop Biofactories Initiative (Box 2.1).

Box 2.1 Crop Biofactories Initiative The GRDC is investigating ways for its stakeholders to benefit from plant molecular farming of industrial products with the end goal of sustainable higher farm-gate prices. The Commonwealth Scientific and Industrial Research Organisation (CSIRO) and the GRDC are initially investing $13 million over four years in the Crop Biofactories Initiative. This 12-year initiative (2005–2016) aims to demonstrate that plants can be used as cheap, reliable, renewable and environmentally friendly sources of raw industrial materials; it aims to identify a range of polymers and other industrial compounds currently derived from petrochemical sources that could be produced in plants. It is predicted that the world-wide value of this technology to the chemical industry alone could reach $160 billion by 2010 (CSIRO Plant Industry, 2005).

Initiatives such as these have the potential to identify ways through which Australian farmers can increase the value of their broadacre crops by diversifying from traditional food and feed markets into new markets with higher margins for producers, which in turn will lead to an increase in the value of the grains industry as a whole.

For any non-food or feed crop platform that is selected, additional acute and chronic toxicity testing may be required to develop it for use in producing human therapeutic applications and this in turn will add significant time and cost to the project (consultations with experts).

2.2.2 Where should the protein be expressed within the plant? A number of tissue-specific promoters are available for targeting expression of an inserted gene to either vegetative or reproductive tissues. There are advantages and disadvantages to each of these approaches, as outlined below.

Expression in leaves

Expression in the leaves can result in a high level of protein production and leafy crops produce more biomass than seed crops. Leafy crops can often be planted or have leaves harvested several times per year, as they do not need to reach maturity, flower and produce seeds (Abranches et al., 2005). This would allow increased production volumes per area of land and also avoid the dissemination of pollen to other nearby, sexually compatible crops.

However, leaves are highly physiologically active, meaning that proteins may quickly be broken down unless tissue is dried soon after harvest or some other method is used to inactivate the enzymes that degrade proteins (Twyman et al., 2003; Fischer et al., 2004; Goldstein and Thomas, 2004). Hence, expression in vegetative tissue may result in poor recovery of proteins if immediate processing is not possible. There is also greater potential for co-extraction of secondary compounds such as phenolics and alkaloids, which can be difficult to remove and are likely to increase processing costs. Therefore, the use of these systems may be limited to high value proteins (Giddings et al., 2000; Howard and Hood, 2005).

Expression in seeds

Conversely, seeds have evolved to be stable, long-term protein storage units (Stoger et al., 2005). Compared to vegetative tissue, they generally contain fewer protein-degrading enzymes during seed dormancy and a less complex mixture of proteins, thus simplifying extraction and purification (Fischer et al., 2004; Goldstein and Thomas, 2004).



If expression in seeds is chosen, a plant that has been optimised to produce large quantities of seeds is the most desirable option. Many crop plants have been bred for food or feed production and already produce large quantities of high-protein seeds (Goldstein and Thomas 2004). Disadvantages of seed production include a lower overall protein yield in comparison to species that produce a large amount of leaf tissue (such as tobacco) and the necessity for the GM plants to go through a flowering cycle to produce seeds (Twyman et al., 2003; Abranches et al., 2005). Crop plants that have been investigated for seed-based production of pharmaceuticals include cereals (rice, wheat and maize) and legumes (soybean and pea) (Delaney, 2002; Twyman et al., 2003).

Australian scientists are performing research to identify seed specific promoters in various cereal crops (Furtado and Henry, 2005). They are focusing on the achieving high and stable expression levels of introduced proteins in the seed and are particularly interested in those promoters which show both species and tissue specificity. This research is still at the technology discovery phase of the pipeline and has not yet been applied to any specific third generation project.

2.2.3 Adding value to rotation cropping systems Modification of current rotational break crop plants to produce pharmaceutical or industrial products could add value to Australia’s agricultural industries. The decreasing price currently received for many break crops in Australia means that some farmers find that it is no longer economically viable to grow them, despite the agronomic and soil productivity benefits of established cereal/oilseed rotations. Benefits of break crops to Australian agriculture are discussed in more detail in the BRS report ‘GM oilseed crops and the Australian oilseeds industry’ (Holtzapffel et al., In Preparation), but can be summarised as follows:

• the average yield of wheat grown after a canola rotation can be almost 20% higher than for wheat grown after wheat

• a grain-legume (e.g. lupin, field pea, chickpea) rotation can increase the yield of the following wheat crop by 40–50%, with the benefit of both returning nitrogen to the soil and providing a cereal disease break (Rice, 2005).

Adding value to break crops in rotation cropping systems could encourage Australian broadacre farmers to continue growing them and perhaps increase the area on which they are grown with resulting benefits for the whole agricultural industry (consultations with experts).

To be of advantage to wheat, Australia’s most widely grown broadacre crop, break crops need to be grown on millions of hectaresP

3P; therefore, low- to mid-value, high-volume industrial products

are most likely to be suitable. As Australian farmers already have the agronomic knowledge to grow lupins, oilseed Brassica species and other legumes, such as field or chick peas, these could be good rotation crops in which to introduce third generation industrial traits. Another rotational crop, linseed, has the advantage of being produced for both industrial and food purposes, with segregated supply chains already in existence (consultations with experts). The Crops as Biofactories Initiative is also investigating a series of undomesticated plants that could be developed as new rotational broadacre crops for Australia (consultations with experts).

One important consideration, however, is whether volunteer plants of the rotation crops will re-grow in the following wheat or barley crop and what impact this would have on the safety and marketability of these crops.

The key gain for most Australian farmers will not necessarily be the money they receive from the third generation GM plant itself, but rather the value that the break crop will add to the following wheat or barley rotation. Many of the people consulted for both this report and by Holtzapffel et al (In Preparation), stated that ceasing to grow break crops would have negative effects on

TP

3PT In 2005/06 the total area sown to wheat in Australia was 12.6 million hectares:

http://www.abareconomics.com/publications/2006/e_report/AustGrains_06_1_[0352].pdf

Australian agriculture. If the value of the break crops can be increased significantly, this may open up new niche export markets for Australian farmers. For example, a potential niche market could be developed for high-quality industrial oils, such as those used in the automotive industry (consultations with experts).

2.2.4 Using plants to make industrial products Consultations with experts suggested that, to maximise potential benefits from third generation GM applications, Australia must develop an independent position from which to market these products and not simply follow international competitors. Because of factors of scale, opportunities for Australian agriculture are likely to exist in industrial rather than pharmaceutical applications of plant molecular farming. Some of the experts consulted for this report suggested that Australian agriculture should focus on low- to mid-value, mid- to high-volume plant molecular farming applications.

Australian research into the expression of industrial third generation traits, including bioplastics, in GM sugarcane is discussed in Chapter 3. A challenge for sugarcane researchers is to decide where to express the proteins. If they are expressed in the leaves, it may be possible to develop bioplastics as a co-product, allowing sugar to continue to be extracted from the stem by conventional means. In this way, value could be added to the existing sugarcane industry.

The aim of expressing industrial products in sugarcane is to add increased value to an industry already based on bulk (i.e. low-value, high-volume products) and for this reason, sugarcane may be more suited to the expression of industrial rather than pharmaceutical products.

In Queensland, the sugar industry is tightly self-regulated within milling regions and, with the exception of a small number of independent mills, the majority of production is administered by Queensland Sugar. It was suggested during the consultation process that individual milling areas could be segregated along GM or non-GM lines, although this would require the cooperation and support of all growers in each milling area.

There are a number of other research projects that also aim to produce industrial substances in GM plants. These include spider silk and artificial sweeteners. Most of these projects are still at an early stage of research (described in Chapter 3), but if they were successfully developed to commercialisation, large volumes of raw material could be required. GM plants producing these substances could be grown on agricultural land, providing alternative cropping options for Australian farmers.

2.2.5 Biofuels The two biofuels relevant to this report are biodiesel and ethanol. Biodiesel can be made from either vegetable oils or animal fat. Ethanol is produced from the fermentation of sugar. This sugar can be extracted directly from plants (e.g. sugarcane), derived from plant starch (e.g. maize or wheat) or made from cellulose (e.g. wood, leaves or stems).

Biofuels currently cost more to produce than petroleum fuels and are likely to require continuing government support and subsidies (Biofuels Taskforce, 2005; Hill et al., 2006). In addition to processing costs, biofuel crops require planting, fertilising, pesticide and herbicide applications, harvesting and transportation. First generation GM plants that decrease production costs and inputs may, therefore, contribute to the future viability of biofuel production from crop plants. Efforts are also being made to optimise the processing steps to increase the efficiency of turning biomass into biofuel. For instance, some third generation GM plants that are being developed specifically for more efficient ethanol production are described in Chapter 3. These plants would need to be grown on a large area of land and could provide alternative cropping options for farmers.

As part of its 2001 election commitment, Biofuels for Cleaner Transport, the Australian Government committed to a target of producing 350 million litres of biofuels from renewable


sources by 2010 as the first step in diversifying Australia’s fuel supply. The Biofuels Taskforce (2005) considered the costs and benefits to Australia of meeting the 350 ML target. The costs were estimated to be approximately $72 million per year in the long term, with higher costs initially. Benefits were thought to include possible increases in regional employment, reduced greenhouse gas emissions and increases in air quality. Biofuels currently account for about 0.1% of fuel use in Australia and the government target of 350 ML by 2010 will increase this 10-fold (Biofuels Taskforce, 2005).

Other countries are also encouraging greater use of biofuels. For example, the Biofuels Research Advisory Council of the European Union has recently published a draft report discussing the feasibility of replacing 25% of the European Union’s transport fuel with biofuels by 2030. The report estimates that between 4 and 13% of total agricultural land in the European Union would be needed to produce this amount of fuel (Biofuels Research Advisory Council, 2006).

Biofuels are viewed by many people as renewable, alternative sources of energy that will deliver environmental benefits, such as improved air quality and reduced greenhouse gas emissions, although the size of their potential to reduce greenhouse emissions has recently been questioned (Cannell, 2006). There are also practical considerations: large areas of land and water will need to be used for growing biofuel crops and it is not certain that these resources will be available, considering the expected increases in the human population in the future (Giampietro et al., 1997; Berndes, 2002; Bungay, 2004; Kim and Dale, 2005; Pearce, 2005).

Concern has been raised recently about the increasing demand for vegetable oils in the European Union to supply the biofuel market. This demand has led to increased use of imported palm and soy oil to supplement locally produced rapeseed oil. Some people believe that this increased demand is encouraging destruction of forests to supply land for palm oil plantations and soybean crops (Pearce, 2005).

Questions have also been raised about the ability of biofuel production to keep pace with increasing global energy demands, which are forecast to increase by 1–3% each year (Bungay, 2004). In addition, the economic feasibility of biodiesel production often depends on cheap inputs (e.g. waste wood or used cooking oil) and/or saleable by-products (e.g. glycerol or protein meal). It is worth considering that waste materials are likely to increase in value if demand for them develops and by-products are likely to decrease in price if an oversupply is produced. Bungay (2004) describes a methane-producing factory in Florida that became unviable when its by-products stopped being used by the animal feed industry.

2.2.6 Using plants to make pharmaceutical products PMVs are more likely to be used in developing countries where production, storage and delivery costs need to be minimised. PMVs with the most potential to have an impact on Australian agriculture are livestock vaccines such as the Avian Influenza vaccine for poultry that is being developed through collaboration between Dow AgroSciences, the University of Melbourne, the Macfarlane Burnet Institute for Medical Research and Public Health and Monash University (Chapter 3). Livestock vaccines such as this will need to be produced on a large scale at low cost to ensure that they can be used by the livestock industry.

Human and animal disease priorities in our region are different to those in North America, giving Australia a potential regional market for vaccine production. Avian Influenza in South-East Asia is an example of a disease that is being targeted by Australian researchers working on third generation GM applications.

2.3 Technical constraints Most developments of third generation GM traits are still at a relatively early stage and there are a number of technical problems that need to be resolved to ensure that expression of these traits in



GM plants is effective and that further uses of the products are economically viable. Some of the technical problems are discussed in this section.

2.3.1 Difficulty in obtaining high yields and consistent expression levels Currently, the greatest barrier to the economic viability of third generation GM plants is the technical complexity of producing sufficient levels of protein in the plant. Traditionally, the strongest promoters available (for example ‘CaMV35S’) have been used to drive expression of inserted genes in GM plants. This results in relatively non-specific expression throughout the plant. For pharmaceutical and industrial applications, targeted, tissue-specific promoters are likely to be preferred. Tissue-specific promoters will allow the protein to be targeted to a desired tissue within the plant (meaning that processing will be easier) with potential for non-GM co-products to be extracted from other tissues. For example, expression of bioplastic molecules in sugarcane could be targeted to the leaves, allowing sugar to continue to be extracted from the stem of the plant (consultations with experts). The utility of seed-specific promoters is also being investigated (Fischer et al., 2004 and consultations with experts).

Natural variations in plant growth and environmental conditions affect the consistency of protein expression levels (Ma et al., 2005b). The uncertainty of final protein levels makes it difficult to accept the concept of vaccine delivery by direct consumption of GM plant tissue, because of concerns about efficacy of the vaccine if the dose is uncontrolled and the ability to meet the requirements of GMP (Kirk et al., 2005; Ma et al., 2005b and consultations with experts).

In traditional pharmaceutical production terms, GMP is a component of quality assurance used to ensure that a pharmaceutical product is made to an appropriate and consistent quality (see Box 2.2). The same standards of GMP that are applied to traditional bacterial or mammalian cell culture systems must also be applied to new pharmaceutical products derived from plants (Goldstein and Thomas, 2004). Although it may be difficult to establish an equivalent level of control for plant-made pharmaceuticals (Ma et al., 2005b), implementation of GMP will help to address and manage a number of the risks associated with PMVs (Kirk et al., 2005 and Box 4.1).

Box 2.2 Good manufacturing practice The Australian Code of Good Manufacturing Practice for Medicinal Products defines GMP as “…that part of Quality Assurance which ensures that products are consistently produced and controlled to the quality standards appropriate to their intended use and as required by the marketing authorisation or product specification” (TGA, 2002).

When manufacturing pharmaceuticals, GMP requires the process to be defined from start to finish, in terms of both materials and procedures. Production should take place in certified facilities using approved equipment, validated processes and analytical methods and trained staff. Importantly, there must be an unbroken information chain that allows a final product to be traced back to source, together with a consistent protocol for manufacturing every batch of product (Ma et al., 2005b).

2.3.2 Plant specific glycosylation and potential for allergic reactions Proteins are glycosylated differently in plants and humans; plant-specific glycosylation of introduced proteins could alter or negate the function of such proteins. Also, there are concerns that these changes in glycosylation could lead to an adverse immune response if proteins produced by expression in GM plants were to be administered to susceptible people.

Altered glycosylation is not limited to human proteins expressed in plants. Experiments in Australia expressed a protein (the α-amylase inhibitor) from the common bean in GM field peas in order to protect the GM peas from weevil attack during storage. During this research, it was shown that the GM peas, when fed to mice, induced an immune response (Prescott et al., 2005). Analysis


of the protein indicated altered post-translational modifications to the protein, including glycosylation. In view of the results of these experiments, the research project was discontinued. This incident reinforces the need for case-by-case assessments of GM applications, the strategy followed by Australian regulatory authorities.

This problem with plant-specific glycosylation is being addressed by a number of researchers who are modifying various plants to enable them to perform human-like glycosylation of proteins (Huether et al., 2005; Bakker et al., 2006).

2.3.3 Potential for gene flow and admixture with food and/or feed crops An issue to be considered in making pharmaceutical and industrial products in plants is the potential for the product to enter the human food or animal feed chain through gene flow or admixture, particularly where existing crop plants are modified. Unintended presence in the food or feed supply chain could occur through inadvertent mixture of seeds, opportunistic growth of volunteer GM plants or cross-pollination of nearby crops (Goldstein and Thomas, 2004). In assessing any application to make pharmaceutical or industrial products in plants the relevant Australian regulators, as part of their case-by-case assessment procedure, would take into account risks that might arise via gene flow or admixture. If necessary licence conditions would be imposed with the appropriate level of containment of the GM plant, its pollen and seeds (See Section 4.2.1 for further details of the regulatory environment in Australia).

For the long-term production of plant-made pharmaceutical and industrial products to be effective, Ma et al (2005b) suggest that adequate segregation measures would need to be enforced, both on-farm and post-harvest, including transport and processing.

There are a number of possible measures that could be adopted to prevent gene flow between third generation GM plants and food or feed crops. An interesting recent development is the growth of GM plants underground, in disused mines. A North American company, Controlled Pharming Ventures, has designed and built a plant-growth facility inside a 60-acre former limestone mine and has shown that crop plants can be successfully grown underground in contained facilities. They have created a series of different facilities in which temperature, humidity, light, airflow and other plant-growth requirements are tightly regulated (Cutraro, 2005). The major advantage of such a facility is complete plant containment, thus preventing the release of any GM material into the environment. While facilities such as these are likely to increase the costs associated with production and could only be suitable for high-value products, they may have advantages where above-ground containment measures are considered unsuitable. They will be most useful for applications requiring small scale production.

Other possible measures may include the following (drawn from Goldstein and Thomas, 2004; Mascia and Flavell, 2004 and consultations with experts):

• production in geographical areas where the crop plant is not normally grown and in isolation from any breeding material, to avoid the prospect of genetic or mechanical mixing of material

• the use of temporal isolation to prevent cross-pollination (i.e. planting GM crops at different times to nearby crops)

• the use of dedicated planting and harvesting equipment, together with dedicated transport, grain-handling, drying and storage systems

• the use of genetic traits to control gene transfer, including

o male sterility

o apomixis

o transformation of the chloroplast genome


o other Genetic Use Restriction Technologies

• ready availability of cheap and simple test kits for all commercialised traits, to allow monitoring of any admixture early in the supply chain.

Some of these techniques are not yet available for some of the species being considered for production of third generation GM traits and not all techniques may be appropriate for all traits.

Chapter 3 Recent developments in plant molecular farming

3.1 Introduction Plant molecular farming using GM techniques is, for the most part, still developing, with only a small number of plant-made research and analytical-grade proteins having reached the commercialisation stage of the product development pipeline. This chapter explains some of the different types of pharmaceutical and industrial applications of plant molecular farming before highlighting a selection of recent developments. For the main part, the applications selected are those that may have relevance to agriculture. However, some other well advanced applications have also been included.

3.2 Pharmaceutical applications of plant molecular farming Pharmaceutical therapeutics are products which have medical applications. Three broad categories of pharmaceutical therapeutics can be produced in plants — antibodies, vaccines and ‘other therapeutics’.

3.2.1 Antibodies Antibodies are proteins produced by the immune system in response to the presence of a specific antigen. An antigen is any substance, for example, a bacterial or viral protein, which is capable of causing an immune response. Monoclonal antibodies (mAbs) are specific antibodies that have been produced in a laboratory, purified and recognise single antigens.

Certain mAbs can be used for human therapeutic purposes. Traditionally, such mAbs were derived from mice and cell cultures, but there are several disadvantages to this system, including the high set-up and running costs, the limited opportunities to scale up production and the potential for contamination with human and animal pathogens (Fischer et al., 2003). Furthermore, mAbs derived from mice may result in an immunogenic reaction in humans; a reaction which may be avoided by using mAbs derived from plants (Fischer et al., 2003).

Plants have the potential to produce large amounts of mAbs, with low production costs, the ability to be rapidly scaled up to meet market demand and reduced risk of contamination with human and animal pathogens (Fischer et al., 2003; Teli and Timko, 2004). Systems for expressing mAbs have been extensively trialled in tobacco, but cereal grains are now being used because protein accumulation in dry seeds allows long-term storage at ambient temperatures with little degradation or loss of activity (Ma et al., 2003). Other crop plants used to produce antibodies include potatoes, alfalfa and rice (Goldstein and Thomas, 2004).

3.2.2 Vaccines A vaccine is an antigenic substance — either a protein, peptide, attenuated (weakened) living organism or a dead organism — which is able to elicit an immune response that prevents the development of an infectious disease. Vaccines are designed to ‘educate’ the immune system, so that upon exposure to the infectious agent it will already have the antibodies that will enable it to combat the infection.

There has been considerable research into the use of transgenic plants as an alternative to conventional vaccine production systems. PMV production appears to offer advantages in protein production and storage; and potential advantages in the distribution of vaccines to developing countries. However, as discussed in Chapter 2, the commercial feasibility of PMVs has yet to be



demonstrated. PMVs could either be processed to a powder for oral administration or purified into a liquid form to allow administration orally or by injection.

3.2.3 Other therapeutic and research/analytical proteins In addition to antibodies and PMVs, scientists have also examined the potential for using plants to produce a wide assortment of other therapeutic agents, including hormones, enzymes, interleukins, interferons and human serum albumin (Goldstein and Thomas, 2004).

Proteins found in breast milk, such as lactoferrin, lysozyme and β-casein, have been expressed in plants. Addition of these therapeutic proteins to fortify other food products could aid in improving infant health.

The production of research or analytical proteins in plants has been quite successful, with a number having been commercialised in the United States (see Table 3.2).

3.3 Industrial applications of plant molecular farming Plant molecular farming can also be used to make products with industrial applications including biofuels (biodiesel and ethanol), industrial oils, natural sweeteners, taste-modifying proteins, bioplastics, spider silk and elastic proteins.

3.3.1 Biofuels As discussed in Chapter 2, biofuels currently cost more to produce than petroleum fuels, a situation which has generated considerable interest in developing new technologies to reduce the production costs of biofuels. Plant molecular farming is seen by many researchers to be one way of reducing these costs. Australian Government policy initiatives and related concerns about biofuel production were discussed in Chapter 2.

3.3.2 Sweeteners Artificial sweeteners with a low calorific value (e.g. saccharin) have been developed as a replacement for sucrose and other natural sugars, especially for individuals who suffer from diseases linked to the consumption of sugar, such as diabetes, hyperlipemia (presence of excess lipids in the blood), dental caries and obesity (Dalal et al., 2006; Sun et al., 2006). However, some artificial sweeteners have undesirable baking properties and aftertaste (Alonso and Setser, 1994; Zubare-Samuelov et al., 2005), which has led to research into alternatives. Potential alternatives to current artificial sweeteners that can be produced in plants include palatinose, brazzein, miraculin, isomaltulose and sorbitol.

3.3.3 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 (Metabolix, 2005b). 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 (Moire et al., 2003).

Biopol, a PHA made by TAlcaligenes eutrophus bacteria fermenting carbohydrates from wheat, was marketed by ICI, Zeneca and then Monsanto in the 1990s in biodegradable products such as bottles and cycle helmetsT (Immel, 2002)T, however, low yields resulted in a high price (£6-9/kg in 2002)

and production ceased (BenBrahim, 2002). Another company, Metabolix, has since purchased the Biopol assets from Monsanto and intends to commercialise PHA-made products with much reduced costs (Metabolix, 2005a). Metabolix research into bioplastic-producing GM plants is described in Section 3.5.1.

Synthesis of PHAs in GM or non-GM bacteria is expensive, with bacterial PHA currently priced five to ten times higher than petroleum-derived plastics. This limits the use of bacterial PHAs to high-value products such as medical applications. Attempts have been made to synthesise PHA in other organisms, including agricultural crop plants, to develop large scale production of biodegradable plastics at a low cost (Moire et al., 2003; Scheller and Conrad, 2005). It has been calculated that bioplastic concentrations in plants would have to reach 15% dry weight for commercial production to be economically feasible (Moire et al., 2003; Michael, 2004; Wrobel et al., 2004; Scheller and Conrad, 2005).

The polymer poly(3-hydroxybutyrate) (PHB) is the most extensively studied PHA; it is naturally synthesised by the bacterium Ralstonia metallidurans from glucose. PHB has similar properties to polypropylene, although it is more brittle, making it less stress resistant for industrial applications (Scheller and Conrad, 2005).

PHB is naturally present at low levels in the cell walls of some plants (for example carrots and a species of willow) (Hartley and Harris, 1981). 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 (where possible, the percent PHB expressed in terms of dry weight is shown); including oilseed rape (up to 7.7% dry weight), maize (up to 5.7%), sugarcane (1.5–7.3%), flax (up to 0.5%), cotton (0.34% fibre weight), soybean, palm oil, tobacco, switch grass, sugar beet (up to 5%), potato and alfalfa (Moire et al., 2003; Michael, 2004; Wrobel et al., 2004; McQualter et al., 2005; Scheller and Conrad, 2005)

3.3.4 Spider silk Spiders produce silks with varied properties for constructing webs, cocoons and draglines. For example, ‘dragline’ silk, used for the frames of a spider’s web and for safety lines, is stronger than high-tensile steel, but weighs less than one-tenth as much. Its tensile strength compares to that of the synthetic fibre, Kevlar, but it is more extensible. ‘Capture spiral’ silk has a lower tensile strength but can be stretched to more than twice its length before breaking. These exceptional material properties have prompted research into developing spider silks for industrial applications (Moire et al., 2003; Scheller and Conrad, 2005). Creating GM plants that can synthesise spider silk may allow large-scale production of these fibres.

3.3.5 Resilin Resilin is an elastic protein that is found in specialised regions of the cuticle (the outer exoskeleton) of most insects. With low stiffness, high strain and efficient energy storage, resilin facilitates flight and jumping in insects. The resilience and durability of resilin could have both industrial and medical applications; for example, in spinal disc implants, vascular prostheses and high-efficiency rubber (Elvin et al., 2005). CSIRO is currently investigating whether it will be possible to use GM plants as a production system for resilin.

3.4 Plant molecular farming in Australia This section discusses specific applications of plant molecular farming in relation to Australia’s agricultural industries. It is split into five broad parts, based on the predicted plant platform, namely: broadacre grain crops and oilseeds; tobacco; sugarcane; horticultural crops; and other types of plants.


Sub-sections within each part describe the relevant plant molecular farming applications. Non-agricultural applications of plant molecular farming that are being developed in Australia are also discussed. These research activities are summarised at the end of the section in Table 3.1.

3.4.1 Broadacre grain crops and oilseeds Antibodies

In Australia, researchers have investigated the feasibility of using wheat to produce high value antibodies for use in unspecified medical applications (GBA, n.d.). This research is currently in the technology discovery and proof of concept stage of the development pipeline and is likely to be some years from commercialisation.

Other applications

The Crop Biofactories Initiative (Box 2.1) aims to identify a number of potential crops to be developed as platforms for third generation GM applications. These will be broadacre crops and may be able to be used as break crops in rotation cropping systems.

3.4.2 Tobacco Research-grade analytical protein

An Australian company, Farmacule BioIndustries, has produced GM tobacco plants expressing a high-value human protein (vitronectin) used in medical research. Vitronectin is involved in cell adhesion in vivo and when isolated from cells, can adhere to glass, plastic, organic molecules, such as collagen, and cells (Hogasen et al., 1992). It is widely used to coat cell culture plates in laboratory research and its function in the body is of interest to researchers working in thrombosis, inflammation and wound repair, cancer, infection and biomimetics. Currently derived from blood serum, the production of the protein in tobacco is close to commercialisation. Farmacule could produce 1 gram of vitronectin per month from its biosecure glasshouse that contains several dozen tobacco plants. This would meet the total world demand for this protein, which currently retails for approximately $2.3-6.8 million per gram (O'Neill, 2006 and consultations with experts). Since production of this protein is on a small scale, it is unlikely to have an impact on Australian agricultural systems. Rather it can be classified as a small-scale industrial production system.

3.4.3 Sugarcane Production of precursors of bioplastics

In Australia, the Cooperative Research Centre for Sugar Industry Innovation through Biotechnology (CRC SIIB) is currently investigating the production of PHAs in sugarcane. The group has successfully produced PHAs in both the leaves and stems of sugarcane. In deciding the future direction of this research, a decision has to be made whether to express the PHA in the sugarcane’s leaves or the stem. If expressed solely in the leaves, it could potentially be treated as a co-product; with sugar being extracted from the cane as it would normally be with no requirement for GM labelling. If expressed in the stem, then the whole of the sugarcane would be used for bioplastic production (consultations with experts). Expressing PHA as a co-product with conventional sugar may not be ideal due to possible problems with public perception of the sugar derived from such cane being GM. In this scenario, dedicated bioplastic crop production would be the preferred option.

Production of alternative sugars

Isomaltulose is an isomer of sucrose that is produced naturally by some microbes. In Australia, field trials are currently being conducted with sugarcane that produces isomaltulose (OGTR, 2005a). Isomaltulose does not encourage tooth decay; it is also digested more slowly than sucrose, with resultant health benefits for both diabetics and non-diabetics. The cost of producing


isomaltulose in sugarcane is expected to be much lower than for the current industrial processes (OGTR, 2005a). These GM sugarcanes are particularly interesting because they accumulate more total sugars (i.e. sucrose and isomaltulose) than conventional canes (consultations with experts).

Sorbitol is a commercially available sweetener with fewer calories per gram than sucrose that can be made from cornstarch. Australian researchers recently developed GM sugarcane that expresses sorbitol (CRC SIIB, 2005). However, as accumulation levels were not high enough in the cane and the economics were not favourable, this research is not being pursued (consultations with experts).

Cost-effective biofuels

A need to reduce the cost of biofuel production to make it competitive with traditional fuels was identified through consultations with experts (Chapter 2). Genetic modifications being investigated to reduce costs of ethanol production include those that lead to increased sugar accumulation in plants. Farmacule BioIndustries and its research partner, the Queensland University of Technology, have developed sugarcane plants that may be able to produce ethanol more cost-effectively from leaf material, without compromising the plant’s commercial sugar products located in the cane. The modification includes insertion of cellulases, enzymes which operate after harvest to convert cellulose in leaf material into fermentable sugars in a highly efficient manner. Fermentable sugars are a key input to ethanol production and mean that sugarcane waste can be partially transformed into the main component of ethanol. This process, known as ‘cellulosic bioethanol production’, increases ethanol yield without affecting the output of commercial sugar products from the cane and is expected to be available to Australian farmers within the next five years (Farmacule, 2006).

3.4.4 Horticultural crops Avian influenza vaccine for poultry

In Australia, Dow AgroSciences have recently entered into collaboration with the University of Melbourne, the Macfarlane Burnett Institute and Monash University under a Linkage Grant from the Australian Research Council to develop a PMV for the Avian Influenza Virus in poultry. The project aims to provide proof of concept for the rapid production of PMVs for the Australian poultry industry. The production platform for this technology is yet to be decided, but it is likely to be tomato, tobacco or a plant cell culture system (consultations with experts). Until the production system is confirmed, it remains uncertain whether this product will be produced at an agricultural or laboratory scale.

Norwalk Virus vaccine for humans

Australian researchers are attempting to develop a PMV against the Norwalk Virus, a common cause of gastroenteritis and ‘traveller’s diarrhoea’. The production platform for this technology has yet to be decided, but it is likely to be a choice between tomato, tobacco or a plant cell culture system (consultations with experts). This research is still at the proof-of-concept stage of the product development pipeline and its potential application to Australian agricultural systems is uncertain.

Measles vaccine for humans

Australian scientists are also working towards a plant-made measles vaccine4. The problem with existing measles vaccines is that they need to be administered by injection and are inactivated by both heat and maternal antibodies (consultations with experts). GM lettuce plants have been developed that express a measles virus protein which has elicited an immune response in mice. The next step for these researchers is to secure the funding to test their vaccine in primates.

It is unlikely that the production of these vaccines in plants will be on a broadacre scale; they are more likely to be produced as a high-value, low volume product in contained glasshouses.

4 http://www.burnet.edu.au/researchandprograms/vaccine/plantderived/171


3.4.5 Other applications Resilin

Scientists at CSIRO Livestock Industries have successfully cloned and expressed the resilin gene from the fruit fly Drosophila melanogaster as a soluble protein in Escherichia coli. CSIRO researchers have also been able to cast the resulting resilin protein into a rubber-like material. Resilin has a resilience (recovery after deformation) of 97%, greater than that of either high-resilience rubber (80%) or elastin (90%) (Elvin et al., 2005). CSIRO is currently investigating whether resilin could be produced in plants, but the project is still at least 10 years from any possible commercial release (consultations with experts).

Opium poppies

Using genetic modification to alter alkaloids produced by the opium poppy plant has also been researched in Australia as reported previously (Glover et al, 2005b). This research is at proof of concept stage with no commitment to commercialisation.

3.4.6 Summary table The third generation GM applications under development in Australia which have been highlighted in this report are summarised in Table 3.1 below.

Table 3.1 Some of the third generation GM applications under development in Australia

Crop Application Stage of development Lettuce Production of a human measles vaccine in plants Proof-of-concept; animal

feeding trials Production of precursors of biodegradable plastics Technology discovery;

proof-of-concept Cellulosic bioethanol production Proof-of-concept

Sugarcane

Alternative sugars for food ingredient and industrial applications — isomaltulose

Proof-of-concept; field trial

Tobacco Production of vitronectin — high value human protein used in medical research

Proof-of-concept; Glasshouse production

Undecided Production of the elastic protein resilin in plants Technology discovery Avian Influenza Virus vaccine for poultry Proof-of-concept Undecided — tomato,

tobacco or plant cell culture Production of a human Norwalk Virus vaccine in plants Proof-of-concept Wheat Expression of antibodies for use in topical medical

applications Technology discovery; proof-of-concept

Poppy Alkaloid production Proof of concept

3.5 Plant molecular farming internationally This section discusses specific international applications of plant molecular farming. It is split into four broad sections, based on the predicted plant platform, namely: broadacre grain crops and oilseeds; tobacco; horticultural crops; and other types of plants. Where a number of plant platforms are being investigated, the research appears under one heading only.

Sub-sections within each section describe relevant plant molecular farming applications. Non-agricultural applications of plant molecular farming that are being developed overseas are also discussed. These research activities are summarised at the end of the section in Table 3.2.



3.5.1 Broadacre grain crops and oilseeds Antibodies

The antibody ‘T84.66’ recognises a marker for colorectal, lung, breast and pancreatic cancers. It has been tested in both cancer imaging and therapy and has been produced in several different plant systems, including wheat, rice, tobacco and pea in Germany (Fischer et al., 2003).

An anti-Herpes Simplex Virus-2 (HSV-2) antibody has been developed and expressed in soybeans in the United States. This antibody has been shown to prevent vaginal HSV-2 transmission in mice. If a similar efficacy is shown in human clinical trials, then this antibody could represent an inexpensive protective measure for this disease (Zeitlin et al., 1998; Fischer et al., 2003).

Vaccines

A vaccine for the E. coli Heat-Labile Toxin protein has been expressed in maize and potato in the United States to combat diarrhoea caused by this toxin. The safety of the vaccine from each source is currently being tested in two independent phase I clinical trials (Tacket et al., 1998; Ma et al., 2005a).

Treatment of allergic reactions

Two proteins isolated from Japanese cedar pollen (Cryj1 and Cryj2) have been expressed at a high level in GM rice in Japan. Initial testing in mice has indicated that the consumption of this rice successfully treated allergic reactions to Japanese cedar pollen by causing immune system tolerance to develop (Takagi et al., 2005). If this concept proves to work in people, treatments for a range of allergic reactions could be devised.

Research-grade proteins for analytical, diagnostic or manufacturing uses

It is unlikely that the applications described under this heading will be suitable for broadacre agricultural production in Australia as they are more likely to be cultivated in secure contained glasshouses. However, it is important to be aware of them as they have been approved in the United States where they are being produced on a commercial scale.

Aprotinin is an intravenously administered protein that helps prevent bleeding following cardiac surgery. It works by blocking the action of certain enzymes in the bloodstream that dissolve blood clotsTP

5PT. It also has some other related research and manufacturing applications through maintaining

protein stability. Traditionally, aprotinin is extracted from bovine lung tissue, however, the aprotinin gene derived from cows has been successfully expressed in maize and tobacco plants LSBC commercialised a research-grade version of aprotinin in 2004 and announced that it would be conducting field production trials to prove that year-round supply was possible. Rather than the tobacco itself being genetically modified, the aprotinin gene was first inserted into a GM plant virus. Expression of the aprotinin protein was achieved by infecting the tobacco with the GM virus. Aprotinin derived from GM maize and tobacco infected with a GM virus has been commercialised in the United States (LSBC, 2005). As LSBC has recently gone into voluntary receivership the current status of this protein on the market is unclear.

Avidin is a protein naturally found in egg whites. It has applications in research and diagnostics. The chicken avidin gene has been successfully expressed in maize. Avidin from GM maize was commercialised in the United States in 1997 by ProdiGene and Sigma Chemical Co. Despite ProdiGene recently going into voluntary receivership, it is still possible to purchase avidin derived from corn from the Sigma-Aldrich chemical catalogueTP

6PT.

β-glucuronidase is an easily visualised reporter gene that is used in plant research and diagnostics. The β-glucuronidase gene is derived from bacteria and has been expressed in many plant species,

TP

5PT http://tchin.org/resource_room/c_art_15d.htm

TP

6PT http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/A8706


including maize. Since β-glucuronidase derived from maize was commercialised in the United States by ProdiGene (Witcher et al., 1998), the current status of its availability is unclear.

Lactoferrin is an iron-binding protein found in milk and some white blood cells. It can be used to treat gastrointestinal infections in addition to topical infections and inflammations. The lactoferrin gene from humans has been successfully expressed in rice and maize. Lactoferrin derived from rice for research purposes has been commercialised in the United States by Ventria Bioscience (Ventria Bioscience, 2002a).

Lysozymes are enzymes that can destroy the cell walls of certain bacteria and have practical application as antimicrobial agents. A human version of lysozyme has been successfully expressed in GM rice plants. Lysozyme isolated from this GM rice has been commercialised in the United States by Ventria Bioscience (Ventria Bioscience, 2002b).

Trypsin is an enzyme that cleaves peptides and proteins at specific places. The trypsin gene from cows has been successfully expressed in GM maize plants. Trypsin isolated from this GM maize is manufactured by Sigma-Aldrich using a protein expression system developed by ProdiGeneTP

7PT.

Treatment of gastrointestinal infections

Lactoferrin derived from maize to treat gastrointestinal infections has been developed by Meristem Therapeutics in France (Meristem Therapeutics, 2005a). Phase I clinical trial studies of this product were completed in 2002. It is unclear how far this research has since advanced in the development pipeline.

Treatment for cystic fibrosis

Gastric lipase may be of use as a treatment for cystic fibrosis and pancreatitis. Researchers from the French company Meristem Therapeutics have successfully expressed gastric lipase in GM maize plants (Meristem Therapeutics, 2005b). The resulting drug has been called MerispaseP

®P and the

company estimates the value of the market for similar drugs (made from porcine pancreatic extracts) to be €262 million in 2003 with a projected value of €300–500 million in 2010. Their research is at the phase II clinical trial stage and the drug achieved orphan (small market) drug status from the European Agency for the Evaluation of Medicinal Products in 2003. In 2005, the company received permission to plant 20 ha of field trials of MerispaseP

®P-producing maize plants in

France and is also conducting field trials in the United States and South America. The drug is currently undergoing ‘formulation optimisation’ to optimise the delivery and activity of Merispase P

®P.

Cost-effective biofuels

Syngenta has produced GM maize seeds with increased sugar production. The modified seeds contain an enzyme that turns maize starch into sugar for ethanol production. This heat-stable enzyme should make ethanol easier to produce and reduce costs by eliminating the need for mills to add liquid enzymes Syngenta initiated a consultation with the US Food and Drug Administration (FDA) and filed a Pre-market Biotechnology Notification (PBN) in September 2005. In addition, a Petition for the Determination of Nonregulated Status for Corn Line 3272 was submitted to the US Department of Agriculture (USDA) in October 2005. Food Standards Australia New Zealand (FSANZ) is also currently assessing an application from Syngenta to amend the Australia New Zealand Food Standards Code to approve food derived from this GM corn line for use in food. During 2006, dossiers for food and/or feed approvals will be submitted to the relevant authorities in the European Union, Canada, China, Japan, Korea, Philippines, Taiwan, Russia, South Africa and Switzerland (FSANZ, 2006). It would appear that Syngenta is preparing to release Line 3272 commercially in the future — possibly by 2007 (Checkbiotech, 2006b) — although it is unclear at this stage in which countries it will be grown.

TP

7PT http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Analytical_Enzymes/TrypZean.html

Alternative sweeteners

Brazzein is a protein that has an intrinsic sweetness 500–2000 times that of sucrose. It can be isolated from the fruit of the African plant Pentadiplandra brazzeana, but it is uneconomical to produce on a commercial scale due to the limited availability of the fruit and complications associated with large-scale production of the native plant. Widespread commercial production of brazzein is, therefore, only likely to occur if the protein is expressed in an existing crop plant. GM maize plants expressing high levels of brazzein were developed in the United States by ProdiGene, who reported brazzein accumulation of up to 4% of total soluble protein in maize seed. Tests indicated that maize germ flour containing brazzein could be directly used in food sweetening applications (Lamphear et al., 2005). However, ProdiGene officially shut down all its operations in early 2006 and its product development pipeline has ceased with no further research being performed. The associated intellectual property portfolio will be retained by the new owner, Stine Seed Company.

Production of bioplastics

It has been calculated that PHB concentrations in plants would have to reach 15% dry weight for commercial production to be economically feasible (Scheller and Conrad, 2005). Nevertheless, moderate accumulation of PHB in commercial crop plants has been reported, with concentrations of 5.7% dry weight and 7.7% mature seed dry weight achieved for maize and rapeseed, respectively. Leaf chlorosis developed at higher levels of PHB accumulation in maize. No changes in size, appearance and germination frequency of GM rapeseed seeds were observed (Moire et al., 2003; Scheller and Conrad, 2005).

The US company, Metabolix, is developing switchgrass as a platform crop for PHA production and claims to be making significant progress towards a high yielding plant that can be used for commercial production of bioplastics with the residue biomass used for biofuel production (Metabolix, 2005b).

3.5.2 Tobacco Tobacco produces abundant biomass, making it a useful platform crop for many plant molecular farming applications.

Antibodies

The antibody ‘CaroRx™’ binds specifically to Streptococcus mutans (the bacterial cause of tooth decay) and prevents the bacteria from adhering to the teeth. CaroRx™ produced in tobacco has reached phase II clinical trials in the United States (Fischer et al., 2003; Ma et al., 2005a). CaroRX™ is designed to be applied by dentists to the teeth several times over a two week period following the use of a normal antiseptic to eradicate oral bacteria. No further treatments are then required for up to one year. It is intended that the antiseptic treatment kills existing S. mutans along with other bacteria. After antiseptic treatment and during CaroRx™ application, adhesion of S. mutans to the teeth is blocked, while colonisation of the teeth by other oral bacteria can proceed unimpeded. This prevents S. mutans recolonising after the treatment is stopped (Planet Biotechnology, 2004).

A mAb recognising human chorionic gonadotropin is being expressed in tobacco in Germany (Fischer et al., 2003). This antibody could potentially be used for pregnancy detection, emergency contraception and diagnosis or therapy of tumours that produce the pregnancy hormone human chorionic gonadotropin.

Antibodies have been developed that could be used to treat non-Hodgkin’s Lymphoma. These have been expressed in a GM tobacco virus, which is used to infect tobacco plants, which then express the antibodies in the infected plant tissue. There were at least 12 antibodies submitted by the US company LSBC for phase I clinical trials (Ma et al., 2005a). However, since LSBC recently went into voluntary receivership, the current position of this research is unclear.


Researchers in Cuba have developed GM tobacco plants that make a mAb that is used for purifying a component of a vaccine for Hepatitis B. These GM tobacco plants have been registered with Cuban authorities and a licence has been issued for their use (Checkbiotech, 2006a).

Vaccines

An oral PMV for hepatitis B in humans has been successfully produced in tobacco plants, potato and lettuce. Preliminary clinical trials with this vaccine were performed in a joint US-Polish research project and were reported to have produced promising results (Kapusta et al., 1999; Ma et al., 2003). However, this research does not appear to have progressed to the phase II clinical trial stage (Robert and Kirk, In Press).

A PMV to combat the infectious agent of bubonic plague has been developed in the United States. Researchers have modified tobacco plants to produce high levels of different antigens, from the plague bacterium Yersinia pestis. Early animal trials have shown that the vaccine elicits a protective immune response (Alvarez et al., 2006; Checkbiotech, 2006d). Genetically modified tomato plants have also been used for plague vaccine proof-of-concept studies (Alvarez et al., 2006).


Palatinose is a low calorie alternative to sucrose. Palatinose has been produced industrially by using bacteria such as Erwinia rhapontici to convert sucrose into palatinose. However, this method is costly and the scale is limited, preventing the widespread use of palatinose as an alternative sweetener. The enzyme that converts sucrose to palatinose has been successfully cloned from E. rhapontici and expressed in both tobacco and potatoes in Germany. A considerable amount of palatinose accumulated in the GM tobacco, but the plants suffered severe growth retardation as a result, suggesting that tobacco is not the ideal plant for this application. In contrast, GM potatoes converted almost all their sucrose into palatinose, without affecting growth or development (Bornke et al., 2002).

Spider silk

Creating GM plants that can synthesise spider silk may allow large-scale production of these fibres. Successful expression of spider silk proteins has been achieved in tobacco and potato in Germany, with an accumulation of up to 2% of total soluble proteins in leaves (Scheller et al., 2001; Scheller et al., 2004). Spider silk proteins have also been synthesised in potatoes and A. thaliana. However, silk proteins isolated from these GM plants have not yet been successfully spun into a fibre. Given that silk properties are dependent on the structure of the fibre, which is influenced by the way the proteins are spun, advances in artificial spinning technologies are required before spider silk fibres will be commercially available (Ma et al., 2003; Scheller and Conrad, 2005).

3.5.3 Horticultural crops Vaccines

Oral PMVs for bacterial and Norwalk Virus-induced diarrhoea have been produced in GM potatoes. Preliminary human phase I clinical trials of these vaccines in the United States have demonstrated an immune response (Tacket et al., 2000; Teli and Timko, 2004; Ma et al., 2005a), however it was not yet at the appropriate level for the next stage of product development to continue (Robert and Kirk, In Press).

A vaccine against a glycoprotein from rabies has been expressed in spinach. The spinach itself has not been genetically modified; rather a GM plant virus expressing the rabies vaccine has been used to infect spinach plants. The vaccine is then expressed in the infected plant tissue. This vaccine has undergone phase I clinical trials in the United States (Yusibov et al., 2002; Teli and Timko, 2004; Ma et al., 2005a), but it is uncertain whether or not it will progress to phase II (Robert and Kirk, In Press).



Miraculin is a taste-modifying protein that does not elicit a sweet taste by itself, but can modify a sour taste into a sweet taste. The maximum sweetness was estimated to be approximately 400 000 times that of sucrose on a molar basis; which means the same sweetening effect can be achieved with only minute amounts of miraculin. As with brazzein, commercial production of miraculin is currently not economically viable, because the protein comes from a tropical plant that is difficult to cultivate outside its natural environment. The expression of miraculin in GM plants could provide an attractive alternative production method. To date, the protein has been successfully expressed in GM lettuce in Japan and displayed strong sweetness-inducing activity (Sun et al., 2006).

3.5.4 Other or non-specified host Livestock vaccine

Livestock vaccines produced in plants are closer than human vaccines to achieving regulatory approval and commercialisation. In January 2006, the first PMV achieved regulatory registration from the USDA’s Centre for Veterinary Biologics. The vaccine was developed by Dow AgroSciences to protect poultry against Newcastle disease. The scientists chose to produce this vaccine in plant cell culture, rather than a whole plant. This strategy helped them to mitigate some of the concerns associated with pharmaceutical production in whole plants, particularly food crops. Currently the company does not intend to commercialise this vaccine as there are a number of other vaccines available for Newcastle disease. The company used the vaccine as a test case for the US regulatory system. The company has several other plant-made animal vaccines in development, but the first product is not expected to reach the market before 2009–10 (Checkbiotech, 2006c).

Human intrinsic factor

Human Intrinsic Factor (HIF) is used to treat vitamin B12 deficiency in humans. However, native HIF is no longer available in many countries. Researchers in Denmark have expressed HIF in the model plant Arabidopsis thaliana, which is grown in glasshouses. Phase II clinical trials have been completed8. The results of the trial suggested that plant-made HIF is able to promote the uptake of vitamin B12 in patients with evident vitamin B12 deficiency (Hvas et al., 2006).

Production of bioplastics

There has been limited success in achieving high concentrations of PHB without adversely affecting plant growth. For example, scientists in the United States achieved a PHB accumulation of 14% dry leaf weight in A. thaliana, with little indication of reduced growth, although some yellowing of the leaves (chlorosis) was observed (Nawrath et al., 1994). However, researchers in Germany have observed that PHB accumulations as little as 3% of dry leaf weight in A. thaliana affect plant growth rates (Bohmert et al., 2000). These authors managed to increase PHB accumulation in A. thaliana to 40% of dry weight, but reported significantly retarded growth and loss of fertility in the resulting plants.

8 http://www.clinicaltrials.gov/ct/show/NCT00279552



3.5.5 Summary table Table 3.2 summarises the third generation GM applications under development internationally which have been highlighted in this report. The stage of development refers to either the GM crop product development pipeline (Figure 1.1), or the ‘phase’ system for clinical trials (Figure 1.3).

Table 3.2 A selection of third generation GM applications under development internationally

Crop or plant species Application Stage of Development Treatment for vitamin B12 deficiency in humans

Phase II clinical trials A. thaliana

Production of bioplastics Technology discovery; proof of concept Lettuce Miraculin, an alternative sweetener Proof of concept

Avidin protein for use in research and diagnostics

Commercialised

β-glucuronidase protein for use in research and diagnostics

Commercialised

Lactoferrin protein for treatment of gastrointestinal infections

Phase I clinical trials

Trypsin protein for use in research and manufacturing

Commercialised

Brazzein, an alternative sweetener Proof of concept

Maize

Treatment for cystic fibrosis and pancreatitis

Phase II clinical trials

Maize and oilseed rape Production of bioplastics Technology discovery; proof of concept Maize and potato Vaccine to combat diarrhoea Two independent phase I clinical trials Maize P

bP and tobacco Aprotinin protein used in research and

manufacturing Commercialised

Plant cell culture Vaccine to protect poultry against Newcastle disease

Regulatory approval given for commercialisation

Potato Vaccine for bacterial and Norwalk Virus induced diarrhoea (gastroenteritis)

Clinical trials — phase I

Treatment for pollen allergy Proof of concept; animal immunological trials

Lactoferrin protein used in research Commercialised

Rice

Lysozyme for use in research Commercialised Soybean Antibody to prevent vaginal transmission

of Herpes Simplex Virus-2 Clinical trials — phase not specified

Spinach P

bP Vaccine for rabies Phase I clinical trials (have not

progressed to phase II) Antibody to prevent tooth decay Phase II clinical trials Antibody to detect a pregnancy hormone Proof of concept

Tobacco

Antibodies to treat non-Hodgkin’s Lymphoma

At least 12 different antibodies submitted for phase I clinical trials P

aP

Tobacco and potato Palatinose, an alternative sweetener Proof of concept Tobacco, tomato Vaccine for the plague Proof of concept; animal immunological

trials Tobacco, potato and A. thaliana Spider silk for industrial purposes Technology discovery; proof of concept Tobacco, potato and lettuce Vaccine for hepatitis B Virus Clinical trials — phase I (have not

progressed to phase II) Wheat, rice, tobacco and pea Antibody that recognises a marker for

various cancers Clinical trials — phase uncertain

P

aP The company developing this product, LSBC, has recently gone into voluntary receivership, so the current status of this

research is unclear. P

b PInfection of the plant tissue with a modified plant virus results in expression of the introduced proteins.

Chapter 4 Discussion and conclusions

4.1 Choice of platform crops Despite the early stage of development of third generation crops in Australia, there is a significant amount of high quality research being undertaken. Research into third generation traits in broadacre crops has focussed mainly on sugarcane. Other applications are likely to be in either plants that can be grown in contained glasshouses or plant cell culture systems grown in laboratories.

While sugarcane is the most common platform crop of third generation GM traits in Australia (Table 3.1), maize is the most common overseas (Table 3.2). These two crops have similar characteristics that may explain their current popularity as crop platforms. For example, both sugarcane and maize are very efficient producers of biomass because they, along with many other tropical grasses, perform C4 photosynthesis, which fixes carbon dioxide into carbohydrates more efficiently in hot conditions than the more typical C3 photosynthesis process. Sugarcane is the most efficient plant at converting of light into biomass, converting around 1% of energy over a year, compared to other plants that range from 0.1–0.4% (Whitmarsh and Govindjee, 1999).

4.1.1 Sugarcane in Australia In terms of crop production volume, sugarcane is currently ranked third behind wheat and barley in Australia. Australian sugarcane production is the sixth highest in the world (USDA FAS, 2006). However, world sugar prices are volatile and the Australian sugar industry needs to identify innovative ways to remain internationally competitive. The world demand and price for wheat and barley are more stable and there is less incentive to develop new uses for these commodities. As a result of the need to stabilise farmers’ income from sugarcane, there is a drive from within the industry to develop higher value products from sugarcane. As a result, the CRC SIIB was established in 2003. Two goals of the CRC SIIB are to increase sucrose production by 10% within the seven years of its operation and to double the value of the sugarcane plant within 10–15 years (CRC SIIB, 2004). Increased ethanol use in Australia in the future could increase the demand for sugarcane as a feedstock and increases in the efficiency of converting sugarcane to ethanol will increase the economic benefits from this use of sugarcane.

The majority of Australia’s sugarcane is grown in Queensland, the only State that does not currently have a moratorium in place on the cultivation of GM food crops. The absence of a moratorium may be encouraging investment in this research in Queensland, as it removes one of the obstacles to bringing a GM product to market that are present in other Australian states. However, it should be noted that GM sugarcane would only be feasible with the support of the sugarcane industry.

There are scientific, economic and social reasons to select sugarcane as a platform crop for third generation traits. As mentioned above, sugarcane is highly efficient at producing carbohydrates through photosynthesis; it is vegetatively propagated in the field (i.e. grown from cuttings rather than from seed) and generally harvested before flowering occurs, reducing the potential for gene flow to non-GM sugarcane crops; and efficient transformation and regeneration processes have been developed for sugarcane that have decreased the lead-time for creating GM lines (Kortschak et al., 1965; McQualter et al., 2005).

The sugar industry currently only produces four products: sugar, molasses, ethanol and bagasse (CRCA, 2004). Volatility of global sugar prices makes relying on a few products risky. Diversification should provide a broader base and greater stability for the industry (CRC SIIB, 2006). One example of potential diversification is the production of the liquid ‘furfural’ from bagasse. Recently, as part of the Australian Government’s sugar industry reform package, up to $12 million was awarded to the Proserpine Sugar Co-op to produce furfural at the Proserpine Mill.


This will allow the Mill to produce up to 5,000 tonnes of furfural per year, worth approximately $3 million. Furfural is not currently produced in Australia and can be used in casting resins, brake linings, fibre glass and pharmaceuticals. Local production of furfural would mean more money for both farmers and the local community (Kelly, 2006).

From a social perspective, people may be more accepting of GM technologies if industrial products are not expressed in a seed crop. Creating stability within the sugarcane sector should also have social benefits for sugarcane farmers, their families and their communities.

4.1.2. Maize in the United States In the United States, maize, soybean and wheat have the highest production volumes. The drive to find alternative uses for maize in the United States is likely to be based on production issues: production of maize exceeds demand with average ending stocks for the three years since 2002 equalling 35 million metric tonnes (USDA Foreign Agriculture Service, 2006). This was more than double the average ending stocks for wheat and seven times the average ending stocks for soybean. This over-supply creates an incentive for the development of new uses for maize. For example, maize is already widely used for biofuel ethanol production. The photosynthetic efficiency of maize, its wide-geographic range and production of seeds that stay attached to the cobs during harvest are reasons for choosing this species as a platform for third generation GM traits.

4.2 Perceived constraints of the technology

At the time of writing, no plant-made human vaccine had completed the product development and clinical trial pipelines to reach commercialisation. There are a number of perceived constraints to commercialising this technology and these are outlined in this section.

4.2.1 Regulation of third generation GM plants In Australia, the regulatory system put in place by the Gene Technology Act 2000 (Commonwealth) is applied on a case-by-case basis to all GM plants, including third generation GM plants. GM plants, or products from these plants, may also be subject to regulation by the TGA, FSANZ and APVMA and NICNAS, depending on the plant, the trait and the intended use. For example, GM food ingredients and processing aids would be regulated by FSANZ; human vaccines would be regulated by the TGA; animal vaccines would be regulated by the APVMA, and industrial chemicals such as enzymes would be regulated by the NICNAS. More information on which Commonwealth Government agencies would regulate particular GM products and compliance steps involved is available online9.

Early discussions about the potential for production of vaccines in plants suggested that the plants could be grown locally, harvested and eaten without further processing. However, this is not realistic as it will be important for the vaccine to be extracted from the plant and processed to allow the dose to be standardised to control the level of exposure and ensure efficacy of the vaccine. It is now accepted that vaccines and other drugs produced in GM plants will have to meet the standard requirements for pharmaceuticals before they can be approved by regulators (Kirk et al., 2005; Kirk and Webb, 2005). These regulatory requirements will add to the cost originally envisaged for developing these pharmaceutical products and may alter some optimistic assumptions that making pharmaceutical products in plants will be substantially cheaper than by other means.

9 http://www.bioregs.gov.au


Uncertainty about how third generation GM plants will be regulated may affect investment in research and development of such plants. Between 2003 and 2004, Australian states and territories (with the exception of Queensland the Northern Territory) introduced moratorium legislation preventing the commercial planting of GM canola crops or more broadly, GM crops. International biotechnology companies may be reluctant to conduct business in Australia because of these moratoria. This is illustrated by Monsanto Australia’s decision to close its canola business in Australia.

Kirk et al (2005) identify some potential risks that may need to be assessed in the production of one example of third generation GM plants, plant made vaccines. The European Medicines Agency has also released draft guidelines for assessing the quality of biologically active substances produced in GM plants (European Medicines Agency, 2006). Both of these papers acknowledge that there are certain plant-specific considerations that need to be taken into account, such as the protein-processing in plants that may change the allergenicity of the produced vaccine. In Australia these risks would be assessed on a case-by-case basis by the relevant regulatory authorities.

The potential production of third generation GM crop plants on a broadacre scale raises the issue of whether there is a need to segregate their products from the food supply chain. GM plants and GM products that have not been assessed for use as human food may pose risks to human health and safety if they are not adequately segregated. The StarLink case in the United States is a particular example where a GM maize line was approved for use in animal feed but not human food (Box 4.1). In Australia the Gene Technology Regulator has developed a policy to manage this risk and prevent such split approvals (OGTR, 2005b). For commercial release applications, the Regulator will impose a licence condition that neither GMOs nor their products can be used in animal feeds unless the same GMO’s/products have been approved for human food use by FSANZ.

No third generation GM plants or products have yet been commercialised in Australia, although FSANZ is currently assessing an application by Syngenta for use in food of a GM maize line modified for biofuel production (FSANZ, 2006). Depending on the type of protein or chemical being produced by a third generation GM plant, the regulatory pathway may require approval from several different agencies.

4.2.2. Segregation and identity preservation As identified by Glover et al. (2005b), marketing uncertainties continue to be a major barrier to the progression of GM crop plants through the development pipeline in Australia. Some perceive that the commercialisation of GM grains in Australia could result in market access problems for our agricultural products; particularly if some markets, both domestic and export, require non-GM products and if there are no segregation or identity preservation systems in place. To meet any non-GM market demands, segregation and identity preservation systems will be important and could result in increased costs. Markets may or may not evolve to require segregated products and this may or may not be associated with price premiums.

Where there is a need to do so, containment and segregation of the third generation GM plants should be possible. Potential measures that may be needed are considered in Section 2.3.3.

Segregation practices are already in operation for the production of some non-GM crop plants that are used for production of non-food industrial products. Ma et al. (2005a) give the example of the production of high erucic acid rape (HEAR), used to make industrial oils in Europe, the United States and Canada. Due to the effects of erucic acid as an antinutrient, production is tightly managed and controlled to prevent mixing of HEAR seeds and edible rapeseed (e.g. canola) intended for food and feed. For example, in Canada farmers are required to grow HEAR varieties


under contract registration. This means that during the growing season, regulators from the Canadian Food Inspection Agency have the right to inspect HEAR fields to ensure farmers are complying with the segregation requirements (Smyth and Phillips, 2002).

Box 4.1 discusses two cases in the United States where segregation systems did not work effectively. Both cases highlight the importance of ensuring that pharmaceutical and industrial crops are adequately segregated from food and feed crops and that inspections occur to enforce appropriate measures.

Box 4.1 ProdiGene and StarLink cases In 2002, volunteer maize expressing a pharmaceutical protein was found by the USDA to be growing in some soybean fields in Nebraska. The previous year, those fields had been used by ProdiGene Inc to field trial and harvest maize that expressed a vaccine for viral diseases in pigs. The same year, in field trials of the same pharmaceutical maize line in Iowa, cross-pollination with conventional maize that surrounded a field-trial site occurred. Although there was no evidence that such contamination posed a health risk, the FDA considered the presence of any unapproved substance, such as a pharmaceutical crop, to be an illegal adulterant that must be removed from the food chain. As a result, ProdiGene paid a fine of US$250 000, as well as paying approximately US$3 million for destruction of the 500 000 bushels of soybeans and 155 acres of maize that had been contaminated (Hileman, 2003; Graff and Moschini, 2004). As of early 2006, ProdiGene had officially shut down all of its research operations and development pipeline. Its intellectual property portfolio will be retained by its owner, Stine Seed Company, but no further research and development will be carried out.

Another example of the unintended presence of a GM trait is StarLink, a variety of maize produced by Aventis CropScience that was genetically modified to produce the insecticidal protein, Cry9C. Due to concerns about the potential human allergenicity of the Cry9C protein, StarLink was approved by the US Environmental Protection Authority for use only in animal feed and other non-food uses (Taylor and Tick, 2001). However, StarLink maize was detected in the human food supply in September 2000, leading to an extensive recall of more than 300 maize products, at a cost of tens of millions of dollars (Taylor and Tick, 2001; Hileman, 2003).

The ProdiGene and StarLink cases described in Box 4.1 raised questions about the efficacy of regulations in the United States for preventing admixture of pharmaceutical crops with food crops and prompted calls from the Union of Concerned Scientists (UCS) for a halt to all field trials of pharmaceutical crops. A senior scientist for the UCS Food and Environment Program stated that

“[the ProdiGene incident] illustrates the vulnerability of the US food system as pharm[aceutical]-crop technology has outpaced the government’s ability to control risks … the government must establish a strong, transparent regulatory system … [and] should impose a moratorium of at least one year on field tests and commercial production of pharmaceutical and industrial crops.”10

However, supporters of pharmaceutical crops argued the opposite, with the Executive Director of Food and Agriculture at the Biotechnology Industry Organization (BIO), stating that

“[the] ProdiGene incident demonstrates that the regulatory framework governing plant-made pharmaceutical crops works. USDA/APHIS [Animal and Plant Health Inspection Service] identified violations of the federal permitting system and has been effective in ensuring isolation of pharmaceutical-producing plant material from grain intended for food and feed production, thus ensuring the integrity of the food supply.”11

10 See http://www.ucsusa.org/news/press_release/pharmaceutical-maize-contaminates-soybean-harvest-in-nebraska.html 11 See http://www.bio.org/news/newsitem.asp?id=2002_1115_01


Both these cases emphasise that stringent control of pharmaceutical and industrial GM crop plants will probably be required (depending on the case) in Australia, particularly with regards to containment and segregation. The Gene Technology Regulator and other relevant agencies are responsible for regulating GM plants and their products in Australia and have an important role in assessing and managing risks as specified under the Gene Technology Act 2000 (Commonwealth) and other relevant legislation.

At the BIO2006 conference in Chicago, a farmer described the methods that he uses to maintain segregation and identity preservation of GM pharmaceutical crop trials that he grows for a variety of companies on his farms. A summary of his seminar is presented in Box 4.2.

Box 4.2 Case Study: Horan Bros Farms, Iowa, USA (Horan, 2006) Bill Horan is a fourth generation Iowa farmer who has grown field crops expressing pharmaceutical products for different biotechnology companies over a number of years. He believes that the techniques used for identity preservation of a field crop are neither disruptive nor unusual for farmers.

There are four layers of redundancy built into his standard operating procedures for growing pharma crops.

1. Geographic separation – pharmaceutical-expressing maize plants are surrounded by an area of fallow land and grown a minimum distance away from non-pharma commercially grown maize as specified by the US regulatory authorities.

2. Temporal separation – the pharma maize cannot be sown until at least 28 days after the last non-pharma commercially grown maize has been sown in the surrounding area. This ensures that the flowering times for the pharma and non-pharma maize do not overlap.

3. Use of male-sterile lines.

4. All tassels are hand removed to make certain that no pollen is released.

Bill uses dedicated equipment for each protein and each plant platform and stated that this is not an onerous measure for farmers. The equipment required can be purchased for approximately US$20,000 and can be shared among different farmers who are growing the same product. All equipment used for the cultivation and harvest of the pharma crop is locked in sheds when not in use. Bill has modified some of the equipment so that the pharma maize can be harvested, dried and stored in the one wagon. The wagon is weighed on electronic scales so he knows exactly how much material has been harvested. Waste material is incinerated in a two foot deep trench and covered with soil as required by the regulatory authorities. By following these procedures, Bill can keep track and account for everything harvested from the plot.

4.2.3 Intellectual property One of the barriers to commercialisation of GM plants in Australia that was identified by Glover et al. (2005b) was lack of freedom to operate - in other words, a lack of intellectual property ownership by Australian biotechnology providers, which prevented them accessing the technology required for innovation in this field. Industry members recognise that consideration of intellectual property issues is essential for any biotechnology provider seeking to develop third generation GM applications in Australia.

The biotechnology industry is heavily dependent on patents. Although patents act as an incentive for innovation, patents can also act as a barrier to further research and may discourage commercialisation as many companies hold patents with broad claims that may block further research in an area. Most of the gene patents granted in Australia relate to overseas inventions (ALRC, 2004). A recent Australian Law Reform Commission report (ALRC, 2004) identified a lack of commercialisation experience in Australia, with only a small pool of people having the skills to allow new companies to manage intellectual property issues effectively in the



biotechnology industry. In order to begin to address this problem, the Australian Law Reform Commission recommended that:

“Biotechnology Australia, in conjunction with its member departments, and in consultation with state and territory governments and other stakeholders, should:

a) develop further programs to assist biotechnology companies in commercialising inventions involving genetic materials and technologies; and

b) develop strategies to ensure widespread participation of biotechnology companies in these programs.” (Recommendation 18-1, ALRC, 2004).

Green and Salisbury (2001) comment that ownership of particular gene technologies is not always clear cut, particularly when different groups have independently developed and patented parts of a technology in parallel. This may also be called a ‘patent thicket’ where there are multiple overlapping patents that must be negotiated through in order to commercialise a product (ALRC, 2004). Green and Salisbury (2001) also comment on the uncertainty of the scope of many patents, particularly those that make broad, wide-ranging claims. This uncertainty can lead to legal challenges of patents, which can take many years and considerable cost to resolve. Freedom to operate will not always be achievable if the technology owner chooses not to license its use or parties are unable to reach a licence agreement because of pricing issues (Green and Salisbury, 2001; ALRC, 2004).

4.3 International initiatives TAustralia has not pursued third generation GM crop technologies as actively as other countries have, particularly the United States. Although Australia has the research ability, there is been a relative lack of funding (both private and public sector), particularly a lack of venture capital in comparison to the United States (ALRC, 2004). International agricultural biotechnology companies may have been reluctant to invest research money in these technologies in Australia, because the state and territory government moratoria mean that thereT is no clear path to market (see Section 4.2.1).

TA recent US initiative (the Specialty Crops Regulatory Initiative) aims to establish an organisation that will help developers of biotechnology-derived, specialty crop plants (another term for third generation GM plants) navigate through the regulatory approval process. Similar programmes exist in the FDA and USDA for small market (orphan) drugs and pesticides (McHugen, 2005).

The United States and European Commission initiated a joint Task Force on Biotechnology Research in 1990 to promote information exchange and coordination of biotechnology research programmes funded by the European Commission and the US governmentTP

12PT. There are three

current flagship programmes (Working Group on Bio-based Products, 2005 and John Dyer, personal communication):

• The Plant Cell Wall Flagship — The main target of research in this flagship programme will be the development of easily digested plant cell walls in order to improve the processing of new products to replace petroleum-based products; the creation of new value from agricultural residues; and the maximisation of value and resource utilisation per acre of land.

• Oilseed Crop Flagship — This research programme aims to develop oilseed crop platforms for the production of industrial oils. Non-food oilseed plants often have high levels of conjugated, epoxy, hydroxy and waxy oils suited for industrial uses, however these plants often have poor agronomic traits including small seeds, low yields and limited geographical growing areas. Developments may focus on domesticating new oilseed plants or transferring genes from these plants into domesticated oilseed crops.

TP

12PT http://ec.europa.eu/research/biotechnology/ec-us/tf_mission_en.html


• Biopolymer Flagship — Research conducted in this programme will study the various challenges and benefits associated with the use of plants for the production of biopolymers (starch, protein and/or novel polymers). This will include the development of polymers with plastic and elastomeric properties and adhesives, films and resins based on proteins.

4.4 Conclusions This project identified both opportunities that may be provided by adopting third generation GM applications in Australia and issues that should be discussed further.

Third generation GM plants are now being developed around the world and their adoption overseas or domestically could have implications for Australian agriculture. Possible opportunities that may be provided by adopting third generation GM plants in Australia include:

• the diversification of existing industries (for example the production of alternative sugars in sugarcane),

• the development of new industries producing new products (for example the production of biofuels or industrial proteins such as bioplastics),

• a greater return on break crops used in crop rotations, such as canola and lupins, which can be modified to produce industrial materials,

• the opportunity to focus on animal or human disease priorities in our region (for example the PMV to combat Avian Influenza in poultry currently being developed in Australia).

Ensuring that industrial and pharmaceutical crops that have not been approved for food or feed use in Australia are segregated from the food and feed supply chains is a significant issue commonly raised for third generation GM crops. Containment of these types of crops may be required and suitable supply chains will need to be developed.

Other issues that must also be discussed are:

• How can this technology be used to add value to Australian cropping systems? This will include identifying how regulatory, on-farm and post-farm gate issues will be addressed to enable broad-scale adoption of this technology in Australia.

• How should Australia utilise available farming land and water resources to balance production of food with high volume industrial products such as biofuels?

• How to ensure that Australian researchers can access the enabling technologies (such as intellectual property) related to plant molecular farming? Lack of access could limit the opportunities for Australia to develop its own plant molecular farming technologies in the future.

In Australia, plant molecular farming is at an early stage of development in comparison to the United States, which has commercialised a number of applications (Table 3.2). In the short term there are likely to be opportunities for small scale operations (for example glasshouse production) of high-value, specialised third generation GM crops. In the longer term, third generation GM crops could provide opportunities for Australia to add value to its broadacre cropping systems. In particular, growing GM plants that make industrial products such as those used in the biofuels or bioplastics industries may help farmers to maintain and improve the profitability, competitiveness and sustainability of their cropping systems.

The world’s demand for therapeutics and diagnostics is likely to exceed its capacity to produce these compounds in the future and the world’s population and energy demands will also continue to increase. The use of plants as biofactories for pharmaceutical and industrial products may therefore become important in the near future, making it necessary for the scientific, regulatory and policy issues that surround this technology to be discussed sooner rather than later.

Acknowledgements This study was financially supported by the Australian Government Department of Agriculture, Fisheries and Forestry, Rural Policy and Innovation Division using funds provided under the National Biotechnology Strategy.

The authors wish to acknowledge the people who contributed to this report through taking part in our consultations. They were generous with their time and provided valuable input into this report.

Thanks also to the reviewers of this report: Dwayne Kirk, David Cunningham, Julie Glover, the DAFF Biotechnology Policy Section, and officers from the Office of the Gene Technology Regulator and the Therapeutic Goods Administration who all provided valuable feedback on the report.


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Appendix A Consultation questions

1. Can you give examples of current Australian research into using GM crops as factories to produce pharmaceutical or industrial compounds?

2. With reference to the product development pipeline shown below (Figure 1), into what stage does the research you referred to in Q1 fit?

If you feel that this pipeline does not adequately reflect the product development process for pharmaceutical and industrial crops, how do you feel it could be better described?

3. What do you see as the advantages and disadvantages of using GM plants to produce pharmaceutical and industrial compounds?

4. Do you think they are likely to offer broad benefits to agricultural communities by offering higher value crop options? Or, is it possible that gains would be offset by the higher cost of containment and meeting regulatory requirements?

5. Which crops have the most potential as platforms for the application of third generation traits, and why?

6. What do you see as the main issues surrounding the use of food plants as platforms for third generation GM traits?

7. What might the impacts on Australia be of either adopting or not adopting these technologies, particularly if they are adopted overseas?

8. What do you see as options for Australia in this area, particularly in regard to niche markets?

Figure A1 The product development ‘pipeline’ for GM crops

Genes of interest are tested, usually in non-crop plants, for those which show the most promise for application in crops

The research stage of the process, where an idea is explored

For successful characteristics, regulatory approvals are gained and seeds/planting material produced and marketed

Genes of interest are tested in a crop under field conditions to determine the likely success of the crop


plant molecular farming in australia and...

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