Example 1: Crop Plants

Crop plants are grown in an artificial environment, the so-called agricultural environment, but escape into the 'natural' environment may be a concern.

Historically crop plants have not been subjected to formalised risk/safety analysis or risk management as there is a mass of knowledge, understanding and experience of the procedures for managing the introductions of crop plants developed by a wide range of breeding methods. In traditional breeding, crops are improved by cross pollination between plants with desirable characters, followed by selection of progeny with new gene combinations. Improvement by plant breeding methods is possible when the genes controlling the characters of interest are found within the crop species itself or within species that are sexually compatible with it. Various techniques have been used over the history of traditional plant breeding to increase the choice of genes available through traditional breeding and now include embryo culture, ovary culture and protoplast fusion. Even when a novel hybrid plant can be obtained there may be failure of chromosome pairing, or the genetic recombination necessary to introduce the foreign genes into the crop species.

One of the principal hurdles that have prevented the routine modification of crop plants, is the difficulty of introducing foreign DNA into a plant cell. The process of DNA introduction into plants (called transformation) was first achieved in tobacco, and has been relatively easy to achieve in other solanaceous species (potato, petunia, various Nicotiana species). In certain other crop species, especially the cereals, it has been less easy. Many approaches to transformation have been attempted and the successful methods used at present fall into 4 groups. It is a digression to briefly discuss the principle methods for transformation of plants, but they do provide some information useful in risk-assessment:

The Agrobacterium method of transformation is used widely to transform dicotyledonous species. There are two principal species. Agrobacterium tumefaciens and A. rhizogenes which in their wild type form are pathogens causing crown gall disease and hairy root disease, respectively. Many dicotyledonous species are susceptible to infection by Agrobacterium species brought about by the incorporation of genes from an independently replicating plasmid within the Agrobacterium cell which then become incorporated into the host plant. The introduced DNA modifies the phytohormonal levels within infect cells and either causes a disorganised proliferation of cells and the formation of a gall or the production of a mass of roots covered with root-hairs.

The disease causing genes are carried between specialised T-DNA (transforming DNA) border sequences on the independently replicating circular plasmid DNA molecule. By recombinant DNA methods, it has been possible to remove the disease causing genes, so the Agrobacterium organism is no longer pathogenic. New vector plasmids have also been constructed which enable foreign genes to be inserted between the T-DNA borders. Agrobacterium cells carrying the gene(s) of interest are then incubated with cultured cells of the recipient crop plant and transgenic plants regenerated from them. Only a small proportion of the treated plant cells eventually become transformed, so it is usually necessary to incorporate selectable marker genes (usually conferring resistance to a particular antibiotic) between the T-DNA border sequences. To select the transgenic plants, the corresponding antibiotic is incorporated into a plant regeneration medium on which only transgenic plants are able to grow normally.

The major restriction to the use of this technique is that many plants, particularly cereals, are extremely difficult to transform because of the lack of a wound response.

Protoplasts are plant cells that have had their cell walls removed by enzymatic treatment. They can be produced from various parts of the plant (often from leaves or hypocotyls) and are bounded by the plasma membrane. This membrane is delicate and its integrity can be affected by polyethylene glycol treatment or by passing an electrical current through a protoplast suspension. The DNA to be introduced is added to the medium surrounding the suspended protoplasts and the chemical or electrical treatment allows DNA to enter. In a small proportion of protoplasts the foreign DNA becomes incorporated into the cell genome.

As with the Agrobacterium method, it is usual to use an antibiotic resistance selectable marker gene in order to select the transformed protoplasts and the cell colonies that develop from them. Plant tissue culture procedures are subsequently used to regenerate whole transgenic plants.

The Agrobacterium and protoplast methods have often proved inadequate for the transformation of recalcitrant species. Cereals are not normally hosts to Agrobacterium, and routine and reliable regeneration of plant from cereal protoplasts is difficult and often genotype dependant. The technique of particle bombardment was developed in an attempt to overcome some of these problems and involves coating metal particles (usually tungsten or gold particles, 1 m in diameter) with DNA and shooting them into plant cells capable of subsequent plant regeneration. Particles can be propelled by various means, including 0.22" (inch) blank cartridges, compressed gasses or by the instantaneous evaporation of a water droplet caused by electrical discharge. The small metal particles, with their DNA covering, enter the plant cells and become lodged there. In a tiny proportion of the recipient cells the DNA becomes incorporated into the genome, and transgenic plants can be regenerated from them. As with the other methods, it is usual to incorporate a selectable marker gene.

Partial digestion of cells in multicellular structures: A method which now looks promising for the transformation of cereals, is the partial digestion of immature embryos with enzymes, followed by stimulation of DNA uptake by exposure to an electrical current (electroporation). Eventually plants are regenerated from the transformed cells of the embryo. This method has the potential advantage of using immature embryos which often have a high capacity for plant regeneration. More experience with this approach will be required before it can be established how widely applicable it may be for the transformation of a range of gramineous species.

What are the hazards posed by modified plants? -- Unwanted attributes of crop plants may include the tendency of a self-pollinated line to outcross because of self-sterility or other factors. There may also be a tendency to become a weed. The modified plants may produce toxic substances in the product or the target range of the toxin deliberately inserted into the modified plant may differ from that of the donor organism. The response of the modified organism to other organisms in the environment and the reaction of other organisms to the modified plant may change. Any of these may pose a risk to either humans working with or consuming the products, or to the environment. Where pesticides are introduced into the modified crops, the actual response may be much wider than that expected, with consequences for the ecosystem. In addition, the modified plant may display unwanted changes in appearance, susceptibility to environmental stress or end-use characteristics. "In many cases, the effects have been scale dependent and, therefore, became apparent during the scale-up process"

It has proved virtually impossible to formulate a 'quantitative', structured method for the assessment of risk to the environment resulting from the deliberate release of a modified plant. For example, it is not thought possible to estimate the probability of a plant becoming a weed when released, as the characteristics of 'weediness' are not easily defined . The complexities of the natural environment and ecological interactions mean that risk estimation is, in most cases, more a question of qualitative evaluation rather than of quantitative analysis. Risk assessment procedures for the release of transgenic plants therefore require a detailed comparison of the transgenic plant with the plant genotype it was derived from. The procedures also require consideration of the interaction of the modified organism with the particular environment into which it is to be introduced. Although the methodologies in various countries differ, most ask similar detailed questions about the modified organisms and the environment in attempting to perform a risk assessment. The system used by most countries requires a response to a very large number of questions relating to the organism, release site and wider environment. The information required is essentially the same in all countries. It involves a number of steps which identify the hazards associated with an introduction into the environment .

An example of the information required for risk assessment is given in Table 1. The method of presenting the data varies from one authority to another, but the information required is essentially the same. In 1995 the Organisation for Economic Co-operation and Development assessed the processes within member countries for the "commercialisation of agricultural products derived for modern biotechnology". 23 countries responded to their survey:

• 12 countries had a regulatory oversight system in place specifically for agricultural crop plants derived from modern biotechnology
• 6 countries had a modified form of an existing regulatory system
• 3 countries were in the process of developing a new system of regulatory oversight
• National expert committees had been established in nearly all countries to deal with products of agricultural biotechnology
• There was wide agreement as to the type of data needed in order to perform a risk assessment

The categories outlined in Table 1 will need some explanation. The process involves the identification of possible hazards associated with the modified organism, the environment into which it is to be released, and the interaction of organism and environment. Hazard having been identified, it is possible to assess both the risk of that hazard occurring and the magnitude of harm if the hazard is realised. If the hazard is small and the probability of it happening is high, the risk to the environment remains small.

Table 1:

A summary of the information required to submit a proposal for the field release of transgenic plants in the European Community

It is considered important that the staff and the institution responsible for carrying out the release have a sufficiently high level of expertise and experience to carry out the proposed release of transgenic plants and to be responsible for any field containment and monitoring that is considered necessary.

The DNA donor organism, the recipient plant species and the transgenic plant:

It is essential to have information on the donor organism and the recipient plant species. Information on the recipient species will establish a baseline against which to compare the transgenic plants. Knowledge of the donor species will highlight the kind of information required from the transgenic plant. If the donor is a plant pathogen, for example, this will raise questions in the risk assessment exercise about the possibility of recombination between the integrated DNA from the pathogen and pathogens that may infect the transgenic plant subsequently. The possibility of transcapsidation may also need to be considered where the sequences inserted code for a viral coat protein to confer resistance to certain viral diseases. When the modified cell is infected by another virus, transcapsidation of the virus with the coat protein of original donor virus might be possible and this may, in turn, affect viral host range.

Transformation Vector:

Information is required on the DNA vector used during the transformation process employed to introduce the transgenes. Antibiotic resistance genes are generally used to facilitate the screening of transformed cells. Other DNA sequences may act as linking sequences with the vector or may provide other functions associated with the use of recombinant DNA methods. With certain transformation systems carrier DNA is sometimes used to aid the transformation process. It is therefore necessary to know the nature of this DNA so that any consequences can be considered during the assessment process.

Transgenic Plant:

It is important to give a description of the transgenic plant including molecular data on the inserted transgenes, the stability of expression, whether there is any change in allergenicity, toxicity and the capacity of the transgenic plant to persist in agricultural habitats or invade natural habitats. It is essential here that the corresponding unmodified plant genotype is used as a control so that changes in plant phenotype caused by the transgenes can be measured.

The conditions of the release and the receiving environment: Although the scientific or commercial purpose of the release may not have any consequences for risk, it is important that the biosafety groups charged with the assessment of risk have perspectives against which to assess the release. The risk to the environment requires qualitative judgements and, therefore, an essential part of the risk assessment philosophy is case by case analysis and that based on the accumulated experience there is a progression towards streamlined and simplified procedures where appropriate (see conclusions). Providing information on the objectives of the release, its size and design and the agronomic treatments to be used are important both for risk assessment of the particular release and for the longer term national and international learning process.

Ecological information on the release site environment is also important. This should include a survey of plant species that might be growing in the vicinity of the release and information on what is known about the nature of pollen dissemination and the distances over which pollen can give successful pollination.

The location and type of the anticipated target organisms must be specified. The target organisms are those which the transgenes are targeted to affect. For transgenes containing the insecticidal Bt protein it may be a particular class of insect pest, for a viral coat gene it would be a particular viral pathogen. Non-target organisms are those which are not the primary target of the modification and include those that are affected inadvertently. If the transgene has an effect on an insect that is not considered to be a pest, this should be noted, There should also be a consideration of whether the transgenic plant becomes a better or worse host and/or harmful to organisms that might be associated with the crop. The risk of harm to the environment includes harming non-target organisms.

The interaction between the transgenic plants and their environment: In order to determine the impact of the transgenic plant on its environment it is important to describe changes in the transgenic plant that may change its invasiveness in wild habitats, its persistence in agricultural habitats or changes in its ability to propagate itself sexually or asexually. It is also important to take a note of earlier studies with similar transgenic plants. It is also necessary to determine the possibility of the transfer of transgene to the same or related plant species (wild and cultivated) or to micro-organisms and if this is possible what the consequences of that gene transfer might be.

Monitoring, control, waste treatment and the emergency response plans:

Once plants are released from containment and particularly if allowed to flower and set seeds there is the possibility of plants, seeds, pollen carrying the transgenes being transferred out of the immediate release environment. An important part of risk assessment is to determine the extent to which it is possible to monitor transgenes after the release and the efficiency with which it is possible to destroy plant material if it becomes necessary. Efficient methods of identifying transgenic plants or transgenes in species they may have transferred to may be necessary. This may be by a visual marker (e.g. beta-glucuronidase), a selectable marker (e.g. antibiotic resistance) or by molecular analysis e.g. PCR and Southern hybridisation.

There are ways of minimising genetic exchange which might be considered (see later). It may also be appropriate to describe ways in which plant material can be destroyed at the end of the release experiment or if considered to be necessary during the course of the experiment.

Carrying out the risk assessment: The first releases of modified plants have been experimental. Commercial releases are now happening as the results of the experimental release have been analysed. It is at this experimental stage that the major risk assessment will have to have been done, and evaluated. The aim of the risk analysis is to identify either changes to the experimental protocol or methods by which the GMO may be confined in order to minimise risk to the environment or human health.

Assessing risk is not an exact science. It is difficult to put a value on the degree of risk. It is never possible to establish that releasing a transgenic plant will have no risk. All of the activities people are involved in pose a degree of risk, no matter what kind of precautions are taken. The essential feature of risk assessment is to determine how the transgenes might alter risk compared with the non-transgenic crop, hence the starting point must be the use of the unmodified crop plant as a baseline against which to compare the effect of the inserted transgenes. There will undoubtedly be questions that cannot be answered because the relevant data are not available. For instance it is not known for certain whether plant genes can be transferred to micro-organisms, and there is much we do not know about the nature and consequences of gene flow from our conventionally bred crops to related weed species. Biosafety committees have to take into account both knowledge and ignorance to arrive at a decision that on the acceptability of a release and where this is not the case, what additional information or precautions may be necessary.

In cases where detailed scientific knowledge is not available, it is important to use the experience of conventional plant breeding to aid the risk assessment process. Plant breeding has been carried out (first the result of serendipity and later of intention) for thousands of years and many of the genes being inserted by recombinant transformation fall into classes very similar to those manipulated by the conventional plant breeder.

One of the major concerns associated with the release of modified organisms is that the inserted information may be transferred to wild populations. The process of introgression is of concern to many authors as a mechanism which may lead to undesirable traits being transferred from modified organisms. Inter-specific hybridisation is a ubiquitous process, notwithstanding the barriers that exist to cross-breeding, but most hybrids are rare and the majority are sterile. 'Gene Flow' is believed to be highly restricted , but there is some evidence that this may be misleading. Modified plants could, in theory, become weeds difficult to control, possibly in contexts other than their normal agricultural environment.

A transgene may escape from a crop if the transgene is transferred to another crop or to another, wild related crop (possibly by introgression) and the plant containing it persists after the crop on the agricultural land, in verges, ditches or waste-tips or if the plant invades semi-natural habitats. It is likely that the spread of transgenes can be used using similar methodology to any other single gene trait.

What then, are the risks associated with the release of modified organisms? Is it possible to confine the modified organism within the released site, and if it were to 'escape' would it pose a problem for the environment? Could it, for example, survive or persist outside the managed 'site' within which it had been released? There are differences in the potential of crop plants to transfer from the environment in which they are placed, and in their ability to establish feral populations. If this happened, would it matter? Could the inserted genes be transferred to other plants of the same type or to wild relatives?

The risk assessment must take the host or parental plant as its starting point. Is the host plant capable of surviving outside the normal agricultural environment? Does it have relatives in the external or agricultural environment with which gene transfer is possible? The modification must then be considered, both in terms of making any resulting transgenic plant more likely to survive and the safety of the gene product in the environment.

Some areas that need to be considered are outlined below as examples.

• Selectable marker genes: Most transgenic plants contain a selectable marker gene that is used during the transformation process. The characteristics of a marker gene are that
• the marker should enable stringent selection with a minimum of non-transformed plants escaping the selection;
• the selection should result in a large number of independent transformation events and not significantly interfere with regeneration;
• the marker should work well in a large number of species; and
• there should be an assay which allows confirmation of the presence of the marker.

Antibiotic resistance genes are used as they meet all of the above criteria. The nptII gene which confers resistance to the antibiotic kanamycin is most commonly used. It is an antibiotic which has been superseded for clinical use in the USA. The resistance marker also gives resistance to the antibiotic neomycin, which is still prescribed in some countries for clinical and veterinary use. There are also other aminoglycoside antibiotics that are also used as markers. Although several extensive studies have concluded that the likelihood of the transfer of this gene from transgenic plants to micro-organisms is negligible and probably of little consequence if it did happen, there is a continuing debate about whether it is acceptable for this kind of selectable marker gene should be present in commercial transgenic varieties.

Alternatives to antibiotic resistance markers have been used. These include herbicide resistance markers. There have been a number of suggestions for replacing antibiotic marker genes in plant systems. The Cre-lox system involves the production of two sister lines of modified plants, one containing a gene coding for a bacterial recombination enzyme, the other containing the antibiotic resistance gene flanked by sites for the action of the enzyme. When the two lines are crossed, the marker gene should be excise. It is believed that this system would be difficult to apply in vegetatively propagated food crops, such as potatoes.

Gene-fusion markers are used to confirm the expression of proteins within the inserted gene, rather than for selection of the modified organism. One of the most commonly used markers is the ß-glucuronidase gene from Escherichia coli

A range of herbicide tolerance genes have been introduced into various crop plants. Herbicide resistance was one of the first traits subject to genetic modification as the mechanisms of resistance had been characterised. In general the resistance is a dominant single gene trait. One of the principal attractions for this application is that it provides a means of providing selectivity for herbicides that are quickly degraded in the environment. An environmental risk that needs to be considered is whether the transgenic crop plant that is herbicide tolerant may become a weed that is then difficult to control. Another factor that needs to be considered is the likelihood of the herbicide resistance genes becoming established in weed populations by hybridisation between crop and weeds. If the hybrids are fertile, they may be difficult to control in an agricultural system which depends for weed control on the same herbicide, or in adjacent crops that depend on the use of the herbicide for weed control. If there are other adjacent populations of the same crop which have been modified to be tolerant to different herbicides, would a crop plant resistant to multiple herbicides pose extra problems within either the agricultural or natural environment?

Were the herbicide tolerance to be transferred to non-managed, non-agricultural species within the 'wild' environment, would there be cause for concern? Such environments are not normally subject to herbicide treatment and the presence of wild relatives of crop species displaying resistance may be of little significance. The risk assessment should attempt to identify the possible consequences of such a transfer.

Transgenes conferring pest or disease resistance could potentially confer a selective advantage on a crop plant and make it more persistent on agricultural land and more invasive in wild habitats. The transgenes could similarly confer a selective advantage if transferred to related wild plant species. Another aspect that needs to be considered is the effect of the resistance genes on pest and pathogen populations. If the transgene provides a very efficient defence it is possible that the pest or pathogen will rapidly become resistant. This is a phenomenon that is well known in conventional plant breeding and is arguably more about devising a sound agricultural strategy than assessing risk, but the possibility of using the same resistance gene in a range of different crops by transformation means that this prospect has to be taken seriously.

One of the most important uses of this technology for the insertion of genes leading to pest-resistance has been the use of Bt toxins. A problem that may be associated with the use of Bt toxins is the evolution of resistance in the target pests. This resistance is due to reduced affinity for the toxin to a mutant membrane receptor. Transgenic crops containing proteinase inhibitors may pose similar problems, and their use should be carefully planned to avoid the evolution of resistant pests.

Viral coat protein genes are frequently used to give protection against particular viruses. There is currently a debate about whether this strategy might modify the host range of plant viruses by a process of transcapsidation. Transcapsidation events have been detected in mixed infections of luteoviruses in the field. This process does not in itself create a new virus, as the coat-protein genes are unaltered, but may temporarily alter the specificity. There are also concerns over the recombination between the viral sequences expressed by the plant and those of the infecting virus. The concerns will relate to the presence of other viruses capable of transcapsidation within the receiving environment.

Resistance to stress conditions such as drought, saline soils, heavy metals, cold, high temperature are complex characters so the isolation of genes conferring enhanced stress resistance will take time. Plant genes induced by stress have been identified (e.g. heat, cold, salinity, heavy metals) so it is likely that plants with enhanced tolerance will become available in the next few years. Transgenic plants of this type will present a particular challenge for risk assessment because this change may enable plants to grow in habitats where they were unable to grow before and may confer a selective advantage on plants to which the transgenes may transfer by cross pollination.

The exotic species model where a plant is transferred to a different country and become a dominant species is sometimes used to illustrate what might result from the release of transgenic plants. While this is not a good general model for assessing the consequences of inserting one or a few genes into a crop to modify it in very specific ways, it may be relevant to instances where plants are modified to make them grow in new kinds of habitats. The results of a risk assessment in these cases may be that more information is required on the nature of any competitive advantage conferred by the transgene under the specific conditions of the release.

The gene product may be toxic in the plant or parts of the plant or it may modify the allergenic properties of the crop for food or products in the environment (e.g. pollen). Toxicity may apply to organisms other than the target organism, and harm may be to the ecosystem.

The risk assessment required when considering the widespread use of a transgenic crop variety are essentially the same as for small scale releases, but there are some important differences. Field containment measures including those to prevent flower production and to destroy all plants material on the release site (field plot) are no longer possible, the risk assessment must therefore take account of the possibility of cross pollination between transgenic crop and adjacent non-transgenic crops and weed species. Transgenic plants will have the opportunity to become established in a range of natural habitats and there will be the opportunity for recurrent migration of pollen containing the transgenes into wild populations. In habitats where the crop plants vastly outnumber the sexually compatible wild species, the transgenes may become established and those wild populations even if they confer a selective disadvantage compared with the wild species counterparts not carrying the transgene.

There will be the opportunity for transgenic plants to be taken intentionally or inadvertently to other countries, including to those geographical areas that have a different spectrum of sexually compatible weed species. A small scale risk assessment considers the distribution of sexually compatible species in the location of the release site large scale releases raise the question as to what geographical limits should be placed on the environment considered in the assessment scheme.

Rare events which might not be evident in small scale release may be significant on a large scale. For example transgene instability on a small scale be unacceptable in crop varieties used on a large scale.

There are also questions of agricultural strategy, for example, how far is it prudent to progress towards the introduction of several different herbicide resistance genes into a crop species? Will this give the possibility of multiply resistant weeds? Also, what will be the consequences of the same or similar pest or disease resistance genes being present in the many different crops. The experience of conventional plant breeding may make it more likely for resistant pests and pathogens to emerge.

Other scale dependent questions regard the toxicity and allergenicity of the crop and the nature of the breakdown products of the transgenes when the plant decays.

The opportunities for managing risk in small scale releases are principally concerned with achieving an acceptable level of field containment and of monitoring the site during the release and afterwards. Increasing containment can be achieved by the inclusion of buffer crops, cages or bagging flowers in the field. Permission for the first release experiments in the United Kingdom required the de-flowering of all plants in the experimental plot. While ensuring that there the probability of transferring the introduced gene to other plants was low, this procedure could only be applied to small scale field trials.

The options for containment following widespread release of a transgenic crop variety are limited and a risk assessment before the commercial release of a transgenic variety must take this into consideration. It is important that the risk assessment leading to commercial release is thorough and open to scrutiny and considers all the evidence available from the small scale releases carried out with the same and similar transgenic plant material. As mentioned in the previous section, there may be difficulties associated with large scale releases. Some mitigating action might be considered for large scale releases.

There is now a wide range of plant promoters available for giving transgene expression in specific tissues in the plant. It may be desirable to restrict their expression of a particular protein to the parts of the plant where it is required. A pest that attacks leaves, for instance, may need an insecticide protein expressing only in leaves. There may also be opportunities in the future to use a constitutive promoter to give expression in most parts of the plant, but to use a strategy to switch off transgene action in very specific tissues, for example in pollen where there may be the possibility of an allergic response.

The use of male sterile transgenic plants may be considered desirable to prevent the dispersal of transgenes in pollen.

There may also be instances where it is possible or desirable to grow transgenic crops in areas where no sexually compatible wild relatives grow naturally.

In order to prevent genetic contamination, it may be occasionally desirable to grow a particular type of transgenic crop (e.g. oilseed crops with a particular fatty acid composition) in areas free from non-modified crops.

When considering crop plants, it is not only their impact on the environment which needs to be considered, but also that they may be intended to be used for animal feed or human food. In addition, when the crop is out in the field, the farm-workers may come into contact with the plants or pollen at high concentrations. The possible toxic, allergenic, teratogenic or carcinogenic properties of the modified plants has to be considered as part of the risk assessment. In order to determine if the gene product new to the modified plant is allergenic sequence epitope homology with known allergens, heat stability, sensitivity to pH, digestibility by gastrointestinal proteases, detectable amounts in plasma and molecular weight may be considered. The allergenic potential of the donor and of the recipient organisms should be considered.(30)