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From small-scale to large-scale releases of transgenic crops: are there unresolved issues regarding impacts on the ecosystem?

Philip J Dale
John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom

Introduction

During the history of crop improvement, plant breeders have become familiar with the nature and extent of the genetic variation released by sexual recombination. Where potentially undesirable or harmful plant products are obtained, procedures have been adopted to eliminate them from the breeding programme. Within the past decade there have been significant advances in the way we can genetically modify crop plants. The essential feature of these advances for biosafety, is that we can now incorporate genes from microbes, unrelated plants, animals, humans, and even those synthesised in the laboratory. It is likely, therefore, that plant phenotypes will be produced that are outside the experience of conventional plant breeding. Some of the genes introduced, and recent examples include the ability to produce pharmaceutical substances (Goddijn and Pen, 1995; Mason and Arntzen, 1995), will provide particular challenges for biosafety assessment. Other modifications may produce phenotypes similar to those in conventional breeding, such as virus resistance or pest resistance, but use novel genetic mechanisms (Shah et al., 1995).

Many thousands of transgenic plants have been obtained in a wide range of crop species. There have also been many hundreds of field releases worldwide. In a recent review (Dale, 1995), with data up to the end of 1993, 38 crop plant species had been tested in field releases in 31 different countries. During the past 2 years, several transgenic crop lines have been approved for widespread unrestricted use, and several other crop/transgene combinations are being considered for similar approval. Even though there is growing experience in assessing transgenic crops under field conditions, it is sometimes argued that because the majority of releases to date have been small scale and confined, we have learned little that is relevant to assessing the potential impact when those transgenic plants are grown extensively in agriculture.

The objective in this paper is to summarise what I believe we have learned about transgenic plants over the past decade, from laboratory, glasshouse and field studies. I will then discuss some of the questions and uncertainties I believe need to be considered in assessing the possible impact of transgenic plants when used widely in agriculture.

What have we learned about transgenic plants that is relevant to biosafety assessment?

Variation in transgene expression and stability. There is often substantial variation between independently transformed plants in transgene expression levels and tissue specificity. Transgene expression can become switched off during plant development and over sexual generations. Transgenes can be unstable and become structurally modified over time. Some of this variation is influenced by the number of transgene constructs inserted, and the position of insertion of the transgene in the plant genome (Meyer, 1995).

Environmental effects. Environment is known to influence transgene expression and stability (Meyer, 1995).

Pleiotropic effects. Transgenes can influence the expression of genes that would not be expected to be associated with the action of a specific transgene (Dale and McPartlan, 1992).

Background genotype effects. The background genotype of a plant affects transgene expression and stability. Transgenes moved to different background genotypes by sexual hybridization have displayed variation in expression and stability (Irwin et al., 1995).

Somaclonal effects. Independently transformed plants can display variation originating from the plant tissue culture process used during plant transformation (Dale and McPartlan, 1992).

Co-suppression and transgene interaction. Transgenes have been observed to interfere with the expression of endogenous genes, and transgenes can sometimes modify the expression of genes present on other transgene constructs in the same plant (Meyer, 1995).

Characteristics of specific genes. Companies have carried out extensive studies on the characteristics of the transgenes they are incorporating into plant varieties for commercialisation. This has included assessments on allergenicity, toxicity, non-target organisms, and on the general growth and physiology of the plant (Fuchs et al., 1992).

Characteristics of transgene dissemination by pollen. There have been various studies to assess the distance transgenes are transported by pollen, and the likelihood of cross pollination with related sexually compatible species. This information is being used by Regulatory Authorities to specify isolation distances for the field evaluation of novel transgenic plants, and in assessing the likelihood and possible impact of gene transfer to related plants in wild or weedy plant populations (Scheffler et al., 1993; McPartlan and Dale, 1994; Scheffler and Dale, 1994).

Many of the characteristics of transgenes and transgenic plants could have been predicted from our knowledge of conventional genetics. What is perhaps surprising is the extent of the variation in expression and transgene instability between different plants transformed with the same gene construct. There are also very specific interactions between different transgenic constructs introduced into the same plant, and interactions between introduced genes and endogenous genes. It is reasonable to assume that these phenomena reflect genetic principles that are normal features of interactions between endogenous genes, but the use of transgenes enables them to be studied with a degree of precision that was difficult before the advent of transformation.

Information obtained on transgenic plants over the past 13 years makes biosafety assessment more informed, and helps with determining whether the introduced transgene changes the crop plant in ways that affect its biosafety and its possible impact on the ecosystem.

Are there unresolved issues relevant to assessing the impact of the widespread use of transgenic crops in agriculture?

What I have included here as unresolved issues will rarely have a significant impact on the biosafety of transgenic crop plants. However, I raise them because I believe they are questions that need to be considered and debated as we attempt to assess the possible impacts of specific transgenic plants in agricultural production. The use of transgenic plants as food was the subject of the 1994 Basel Forum on Biosafety, and is outside the scope of this presentation.

Subtle or accumulative effects. Companies developing transgenic plant varieties usually carry out extensive tests, on non-target organisms, on toxicity and on allergenicity. These are essentially short term and small scale tests. With some of the characters like Bt (Bacillus thuringiensis) insect resistance, there has already been several decades of applying the Bt insecticidal protein in sprays of the Bacillus thuringiensis bacterial suspension. However it is important that long term impact assessments consider subtle accumulative or less obvious effects on the environment from plants able to produce the transgene protein, throughout most of their lifetime, and in the majority of their cells.

Herbicide tolerance. There are now many different herbicide tolerance genes available for incorporation by transformation, and there have been field trials with transgenic plants with at least 9 different herbicide tolerance genes (Dale, 1995). There is now the potential for plant breeders to produce different varieties of a crop carrying a range of different individual herbicide tolerance genes. This could, potentially, make it difficult to control ground keeper weeds of that crop in set-aside land, or in subsequent cropping, or lead to the production of multiply resistant weed species that receive the transgenes by cross pollination. Although there has been some debate on this subject, the results have generally been inconclusive. The options appear to be:

1. to leave the future use of herbicide tolerance genes to market forces,
2. to develop integrated weed management strategies for different crops and
3. to restrict the use of herbicide resistance genes.

It is reasonable to assume that it would not be in the interest of plant biotechnology companies to introduce a herbicide tolerance transgene that would be unsuccessful in a new plant variety or would jeopardise the long term usefulness or commercial future of a particular herbicide. This is a complex issue, however, which many regulatory authorities find challenging. Part of the problem is that the use of herbicide tolerance may well impact more on agricultural strategies, rather than biosafety or the ecosystem, and that orchestrating agricultural strategies for the future are not usually the responsibility of authorities regulating the release and commercialisation of transgenic crops. However, if the use of herbicide tolerance genes results in difficulties with weed control or the requirement to use less environmentally friendly herbicides, then this could well impact on the agricultural, and possibly the wider ecosystem.

Disease and pest resistance. One of the attractive features of modifying plants by transformation is that many novel pest and disease strategies become available. There is the opportunity to be much more imaginative in designing resistance mechanisms, than have hitherto been possible from within the sexual gene-pool available to the conventional plant breeder. A possible disadvantage is that the same, or very similar resistance mechanisms, can potentially be used in a wide range of crops plants. For example Bt insecticidal proteins can be used in many important crop plant species (Shah et al., 1995). It is important to develop intelligent strategies for the use of particular resistance mechanisms, because their widespread use is likely to result in very intense selection pressure on pests and pathogens to overcome the resistance mechanism. There are already indications of insects becoming multiply resistant to Bt proteins (Gould, 1991), and the Bt working group in the USA is considering long term strategies for the use of Bt in agriculture (Kidd, 1994).

Virus resistance. The incorporation of part of the viral genome to confer resistance to certain viral diseases is the subject of some biosafety debate, especially with regard to transcapsidation and template switching. Virus resistance is the subject of another presentation at this meeting, but there is a growing belief that these phenomena probably do not present a new biosafety hazard. Mixed infections with different viruses are believed to give opportunities for these two phenomena to occur in conventionally bred plants. There is still uncertainty, however, about whether mixed infections with different viral strains provide the same opportunity for these phenomena as do transgenic plants containing viral DNA sequences in every cell of the plant (Dale, 1995).

Transgene instability. Transgene instability and loss of expression is often regarded as of little significance for biosafety assessment because the silencing of a transgene will make the plant like the non-transgenic crop genotype it was derived from. There are some instances, however, where gene silencing may need to be considered carefully in biosafety assessments. Transgenes are sometimes used to down-regulate undesirable proteins eg allergenic proteins in rice (Tada et al., 1995). Silencing of the transgene in this case, even if only a rare event, could result in expression of the undesirable allergenic protein .

Transgene interactions. Different transgene constructs present in the same plant are found to interact with one another. When there are homologous sequences in the two constructs, transgene expression can be modified. This modification appears to be influenced by the position of the transgene within the plant genome (Delannay et al., 1989; Matzke et al., 1993). This phenomena may have consequences for biosafety in two respects:

1. when plant breeders begin to combine different transgenes into the same variety and
2. different transgenes may be transferred to wild species by cross pollination.

International transfer of transgenic plant material. Most regulatory authorities concentrate their biosafety assessment on the release of a transgenic crop plant within their own country. When transgenic plants are in widespread agricultural production, seeds will be transported deliberately or unintentionally from one continent to another. National regulatory authorities should take this possibility into account in their biosafety assessments, and consider wider ecosystem impacts, including the possibility that the modified plant may be transferred to the centre of origin or diversity where it may hybridize freely with plants in natural populations.

Monitoring after commercialisation

Although detailed monitoring of transgenic crops is often required for small-scale releases, there is frequently no statutory requirement to monitor crops once they have been approved for widespread commercial use. To bridge the gap between small-scale releases and the large scale commercial use of transgenic crop plants, it makes sense to search for answers to some of the questions raised above, by a monitoring programme. There are a number of difficulties with monitoring, eg. who should do it, who should fund it, what should be measured, is the data meaningful for assessing biosafety? It is important that any monitoring has a strong scientific base, but because of the nature of the work, it is often not very satisfying scientifically. The question of monitoring needs to be considered further to determine what is possible, sensible and relevant to long term assessments of the impact of transgenic crops on biosafety and the ecosystem.

Acknowledgements

thank the Biotechnology and Biological Sciences Research Council, the Ministry of Agriculture Fisheries and Food and the European Union, for financial support.

References

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