Expression of Bacillus thuringiensis insecticidal protein genes in transgenic crop plants

ELSEVIER Agriculture, Ecosystems and Environment 49 (1994) 85-93

A g r i c u l t u r e E c o s y s t e m s & E n v i r o n m e n t

Expression of Bacillus thuringiensis insecticidal protein genes in transgenic crop plants

D a n n y Llewellyn*, Yvonne Cousins, Anne Mathews, Lynn Hartweck, Bruce Lyon 1

CSIRO Division of Plant Industry, GPO Box 1600, Canberra 2601, Australia

Abstract

The crystals and spores of Bacillus thuringiensis (Bt) have been used for many years as microbially produced insecticides with mixed success. Many of the problems of using Bt as a spray, such as environmental inactivation of the proteins or poor crop coverage, can be circumvented by modern genetic engineering techniques. These can now be used to transfer the genes for the toxic Bt crystal proteins from the bacteria into crop plants and so protect them from attack by economically important insect pests. For many years, the two major obstacles limiting the potential commercial use of transgenic plants expressing these insecticidal Bt proteins were the introduction of Bt genes into important agricultural species and having them expressed at sufficiently high levels to achieve insect control. Many of the technical limitations have now been overcome and the first commercial releases of transgenic insect resistant crops, like cotton are now, or soon will be, in the hands of regulatory bodies. Transgenic seed should hopefully come on the market over the next 4 or 5 years if general approval is given. One of the major considera- tions that might delay commercialisation is the possibility that insects may become resistant to the Bt proteins expressed in transgenic plants. Considerable research into the deployment of transgenic Bt plants on farms and/ or in the production of multiply resistant transgenic plants will still be needed to ensure the effective use of this valuable agricultural resource.

1. Introduction

Many people are by now familiar with the biological pesticide Bt, sold under the tradenames DiPel, Thuricide and Delfin as a wettable or flowable formulation of spores and protein crystals of the bacterium Bacillus thuringiensis var. kurstaki (see review by Aronson, 1986). Bt can be applied like a traditional chemical pesticide, but there is, however, an alternative packaging for Bt proteins and that is in the crop plant it

*Corresponding author. ~Present address: School of Biological Sciences, University of Sydney, Sydney, N.S.W. 2006, Australia.

would normally be used to protect. Conceptually this is relatively straightforward. The active agent of DiPel, for example, is an insecticidal protein produced by the bacterium during the transition into its resting or sporulation stage. Because the protein is encoded by a gene, modern recombi- nant DNA techniques can be used to isolate the gene from the bacterium and transfer it into the genome of a crop plant so that all of the cells of that plant now produce their own protein insecticide.

This has a number of economic and environmental advantages, including the potential for a substantial reduction in the use of chemical pesticides, considerable savings to the farmer be-

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cause there are no application costs, and high specificity, because only insects eating the crop are exposed to the biopesticide.

Although the idea of expressing Bt insecticidal proteins in plants is as old as the technology to manipulate and introduce genes into plants, its realisation has been slow. Two major factors have limited the implementation of the concept: ( 1 ) it has proved more difficult to introduce new genes into agronomically important plants than experimental plants such as tobacco; (2) the technical difficulties of making plants express sufficiently high levels of the Bt insecticidal protein have taken a long time to resolve.

become model systems for the testing of genetic engineering concepts because of the short time necessary to produce transgenics (as little as 6- 8 weeks) and the ease with which genetic anal- yses on the inheritance of the introduced genes can be performed. The first artificial virus resistant (Powell-Abel et al., 1986), herbicide tolerant (Comai et al., 1985) and insect resistant (Barton et al., 1987 ) plants were all produced in tobacco, but once the concepts had been proven, attention was turned to the major crop species and, in most cases until quite recently, were unsuccessful.

2. Gene transfer into crop plants

The first plant to contain a foreign introduced gene was produced in 1983 by Zambryski et al. by means of Agrobacterium tumefaciens, the causal agent for crown gall disease (see review by Gheysen et al., 1985 ). This bacterium carried out a natural gene transfer, passing some of its own genes into the chromosomes of the plant cells that it infected. Researchers were able to 'piggy- back' foreign genes into plant cells using modi- fied strains of Agrobacterium that were no longer pathogenic, and then regenerate those transgenic cells into whole plants. This first transgenic tobacco plant contained a harmless Agrobacterium gene in all of its cells (Zambryski et al., 1983). Subsequently, other tobacco plants with selectable marker genes, genes for resistance to phyto- toxic antibiotics, were produced. These ad- vances enabled the rapid selection of transgenic cells and the routine production of whole transgenic plants in tobacco and shortly after in petunia, tomato and potato (Herrera-Estrella et al., 1983 ). More recently, physical gene transfer systems, such as micro-injection (e.g. Reich et al., 1986 ) or bombardment of cells with tiny gold or tungsten pellets coated with DNA (e.g. Finer and McMullen, 1990), were developed. These new techniques depended on the availability of efficient tissue culture systems and reliable selectable marker genes.

Tobacco, and to some extent petunia, have now

3. Gene transfer into cotton

The problems of introducing new genes into important crop species will be illustrated for cotton but they are similar to those found for all the dicot crops such as soya bean, oilseed rape, sugar beet and sunflower. Monocot crops, and in particular the cereals, such as maize, rice and wheat, have proved to be recalcitrant to the rapidly ex- panding techniques of biotechnology, but over the past couple of years, they too have become accessible with the production of the first transgenic rice (Shimamoto et al., 1989) and maize plants (Rhodes et al., 1988).

Regardless of the method of gene transfer, either Agrobacterium mediated or physically in- duced, an essential criterion is that once a single cell has been transformed (received a foreign piece ef DNA), an efficient tissue culture system to nurture that cell back to a whole plant must be available. Unfortunately, with most of our highly bred crop species this is not a characteristic for which they have been selected, and the ability to regenerate plants from single ceils is highly gen- otype-dependent. Success in the genetic engineering of crop species has depended on identifying a particular cultivar(s) with good tissue culture characteristics. In cotton, Trolinder and Goodin (1987) identified a number of 'Coker'

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varieties (e.g. 'Coker 312' or 'Coker 315' ) with excellent tissue culture performance and this was shortly followed by reports of Agrobacterium- mediated transformation of 'Coker' cotton (Fi- roozabody et al., 1987; Umbeck et al. 1987). 'Coker' varieties are not high performing or high quality varieties, but if necessary, useful genes, such as insect tolerance, introduced into 'Coker' can be crossed into elite commercial cultivars followed by several years of traditional back- crossing to remove undesirable characteristics carried across with the new genes.

In Australia, the authors have persevered with identifying good quality commercial cotton cultivars with acceptable tissue culture performance. Thus at least one cultivar, 'Siokra 1-3', has been identified with tissue culture characteristics as good as those of 'Coker' varieties and efficient transformation with engineered genes for insect and herbicide resistance has been achieved (Cousins et al., 1991 ). 'Siokra 1-3' is an elite breeding line that has never been sold commer- cially, although it has quality and performance characteristics that would allow it to be a suc- cessful commercial variety. Its sister line, 'Siokra 1-4', is currently an important commercial variety for the Australian cotton industry, an industry worth over $900 million in export earnings. 'Siokra 1-4' is itself regenerable, but only at a low frequency and it would probably be difficult to transform this cultivar directly. The authors have, however, just recently transformed another Australian cultivar, 'Siokra $324', a short season variety released for commercial use only last year.

The process of introducing new genes into 'Siokra' and 'Coker' varieties is now routine, but not rapid (Cousins et al., 1991 ). Pieces of young seedling hypocotyls or cotyledons are incubated for 2 days with an engineered Agrobacterium strain carrying a Bt gene and a selectable marker conferring resistance in plant cells to the antibiotic kanamycin. During co-cultivation, a transfer of genetic material occurs between the Agrobacterium and some of the wounded cells along the cut edges of the cotton tissues. The tissue pieces are transferred to a solid tissue culture medium containing plant hormones that encour-

age cell division, and the antibiotics kanamycin and cefotaxime. The kanamycin selects for the proliferation of only those cotton cells that have received the Bt gene (and the antibiotic resistance gene ) and the cefotaxime kills any residual Agrobacterium once their gene transfer job is completed. Over the next couple of months the transformed cells multiply into a callus, a disor- ganised mass of cells, and are then transferred into liquid culture without plant hormones. This encourages a switch in growth of the plant cells to an organised or embryogenic growth state in which tiny embryos, similar in many ways to those that would be found at the centre of a normal seed, are produced out of the callus. This may take from 2 to 4 months. The embryogenic tissues are then increased and eventually individ- ual embryos are placed in a different medium and allowed to germinate into small plantlets that can eventually be transferred into soil and into a containment glasshouse. Each cell of these transgenic plants now contains the Bt gene, say, which is then passed from one generation to the next as would any natural gene for flower colour, seedling vigour or yield. The whole process, from a small explant to a small transgenic plant in soil, takes from 9 to 12 months.

Alternative methods to Agrobacterium-mediated transformation are being investigated by the authors and others (e.g. Finer and Mc- Mullen, 1990), but the proven success of the Agrobacterium method with amenable geno- types is likely to ensure that it remains the method of choice for introducing Bt crystal protein genes and other genes into cotton and many other dicot crop species. The authors have suc- cessfully used the method to introduce into 'Siokra 1-3' and 'Siokra $324', a Bt gene developed by Monsanto (described below), and have produced a number of plants that show high toxicity to lepidopteran insects in an in vitro bioas- say of callus or leaf segments.

4. Expressing Bt proteins in plants

As indicated previously, the technical difficulties of expressing Bt proteins in plants are not

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trivial. Over 12 years have elapsed from the in- ception of the idea to the fulfilment of commer- cially useful Bt genes for crop improvement and even more research will be required before transgenic Bt crops become available from seed wholesalers.

A number of factors had to be explored and difficulties overcome in reaching the current po- sition with Bt genes in transgenic plants, and these are considered below.

4. I. Bt strains--a perfect Bt for each pest?

The choice of the appropriate Bt strain to use in transgenic plants has been governed by the target pest and the availability of the appropriate Bt gene. The lepidopteran-active Bts of the kurstaki type, which were discovered in the early 1960s and their genes (cryIA types) isolated in the early 1980s, are by far the most thoroughly characterised (see review by H~ifte and White- ley, 1989 ). Because lepidopteran larvae are also the most important pests of many summer crops in Australia and overseas, it is not surprising that these Bts have formed the bulk of research into transgenic plants expressing Bt (Barton et al., 1987; Fischoff et al., 1987; Vaeck et al., 1987). Dipteran- and coleopteran-specific Bt proteins and genes (see H/Sfte and Whiteley, 1989) have now been isolated and are beginning to receive attention in transgenic research for the control of these other classes of economically important insect pests.

Word-wide, a great deal of research effort has been put into searching for new isolates of Bt that are more effective against specific pests. Al- though this has proved effective for pests such as beetle larvae for which there were initially very few effective isolates, as indicated below, it is becoming doubtful whether this is a useful strategy for the commonly occurring lepidopteran-active Bts.

In Australian cotton, the major lepidopteran pests are two species of Helicoverpa (formerly Heliothis), Helicoverpa armigera (the cotton boUworm) and Helicoverpa punctigera (the na- tive budworm) which cause extensive crop damage if left uncontrolled (see Thomson, 1987).

Helicoverpa armigera, in particular, is becoming a serious problem as it becomes resistant to all of the currently available chemical pesticides, such as synthetic pyrethroids and organophosphates, and has been targeted by the authors for control with transgenic cotton plants expressing Bt proteins. Both pests are controlled by the standard kurstaki type Bt strains. Of the dozen or so best new isolates obtained from a few hundred isolates generated by a screening programme (R.J. Milner and D.E. Evans, unpublished data, 1990), none were any more effective than DiPel and those tested appeared to be identical to the kurstaki standard crylA type at the DNA level (by Southern blotting). It is the authors' view that the known cryIA genes are therefore likely to be the most appropriate for use in transgenic cotton and the authors have abandoned any search for better strains. As a result there is now a research agreement with the Monsanto Company to use their proprietary cryIA (b) and cryIA (c) genes in Australian cotton cultivars.

5. Achieving adequate expression levels of Bt in plants

The initial approach to expressing Bt genes in plants was simply to place the bacterial coding region between a highly active promoter functional in plants and a region providing transcription termination and polyadenylation functions (Barton et al., 1987; Fischoff et al., 1987). In most cases, the promoter was the region of the cauliflower mosaic virus responsible for the transcription of the abundant 35S RNA (Odell et al., 1985) and is a promoter that has proven useful in the expression of a variety of engineered genes in plants. The 3' ends of the chimeric Bt genes have usually come from the T- DNA genes of A. tumefaciens. The early examples of transgenic tobacco or tomato gave very poor expression of Bt protein (often undetecta- ble by even the most sensitive biochemical techniques) and consequently very poor protection against insect predation. Protection was im- proved when only the toxic N-terminal half of the protein was expressed in plants, but protein

D. Llewellyn et al. / Agriculture, Ecosystems and Environment 49 (I 994) 85-93 89

levels were still often below detection limits (Fischoff et al., 1987). The data looked consid- erably better when highly susceptible insect pests such as Manduca sexta were used in the bioas- says. These early transgenic plants would never have held up to heavy pressure from the more hardy insects normally encountered in field situations.

The Belgian group at Plant Genetic Systems (PGS) achieved a substantial increase in Bt expression levels by exploiting a coupled selection system that linked the expression of Bt with that of an antibiotic resistance gene (Vaeck et al., 1987). In any particular gene transfer experi- ment, there is generally a normal distribution of expression levels of the introduced gene, probably because of local influences of chromosome structure on gene activity. A small number of transformants will have very high expression and the PGS scientists devised a strategy for select- ing out these relatively rare events. The coding region of a truncated Bt toxin (the N-terminal toxic fragment) was fused in frame with a bacterial neomycin phosphotransferase (NptlI) gene such that in the fusion protein generated from this chimeric gene, the phosphotransferase could still fold normally and confer resistance to ami- noglycoside antibiotics such as kanamycin, neomycin or G418. The transformed plants are selected for high levels of antibiotic resistance, and then co-selected for high levels of expression of the Bt gene. Because Bt normally produces a pro- toxin that is proteolytically processed in susceptible insect guts, the additional protein frag- ments fused to the C-terminus of Bt made little difference to the toxicity of the fusion protein. Protein levels were still relatively low (just de- tectable by enzyme-linked immunosorbent as- says), but reasonable control of Manduca sexta was achieved.

In 1990 researchers at Monsanto made a sig- nificant advance in the expression of Bt genes in plants (Perlak et al., 1990 ). They noticed that Bt genes were excessively AT-rich in comparison with normal plant genes. This bias in nucleotide composition of the DNA could have a number of deleterious consequences to gene expression since AT-rich regions in plants are often found

in introns or have a regulatory role in determin- ing polyadenylation. There are also cases in ani- mal systems where AT-rich regions can signal rapid degradation of specific mRNAs. In addi- tion, plants have a tendency to use G or C in the third base of redundant codons, A or T being rarer. Bt genes have the opposite tendency and as codon preference is thought to be linked to the abundance of the corresponding tRNAs, the overuse of rare codons would decrease the rate of synthesis of a Bt protein in plant cells. Mon- santo reconstructed the Bt gene using a DNA synthesiser, maintaining the encoded peptide se- quence, but altering AT-rich regions to a more balanced GC content (see Perlak et al., 1991 for details of the modifications). This synthetic gene when expressed from a 35S promoter in tobacco, tomato or cotton plants gave exceptionally high levels of expression of Bt protein (over 0.2% of total soluble protein) in transgenic tissues, with a corresponding increase in the effectiveness of insect control (Perlak et al., 1990).

Preliminary field tests on these Bt cotton plants indicate very effective performance without the use of chemical pesticides. The gene modifications required for the high level of expression of Bt toxins in plants are probably not economically practicable for more than one or two genes and many groups are looking for other ways of achieving the same result. The authors are ex- ploring the intracellular targeting of Bt proteins into stable cellular compartments where their levels can accumulate despite a low overall rate of synthesis, but as yet no promising data are available.

6. Target tissues for the expression of Bt proteins

The promoter most often used to express Bt toxins in plants is the CaMV 35S promoter or some derivative thereof. Although it is not ab- solutely constitutive, this promoter does result in gene expression in most tissues of the plant. The authors have linked the 35S promoter to an eas- ily assayed reporter gene, the bacterial fl-glucu- ronidase gene and assayed its expression in transgenic cotton tissues. The product of this gene

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can be detected histochemically in vascularised tissues such as leaves, roots, stems, floral bracts, filaments, ovaries and stigmas, but not in mature petals or pollen (Cousins et al., 1991 ). Bt toxins expressed from the same promoter would therefore be expressed in the same tissues and initial data from the authors' transgenic cotton con- firms this. There have, however, been some con- cerns raised that this general expression of Bt prr eins throughout the plant may encourage resis nce development by the target insect (Har- ris 991 ). Helicoverpa species, for example, have an gg laying and larval feeding behaviour that shows a strong preference for the young terminal leaves and floral buds (squares) of cotton. Be- cause the squares are the precursors to the cotton boll, any loss in square production from heliothis attack can have a drastic effect on produc- tivity and hence the current need for extensive pesticide use in the cotton industry. Bt expression could be targeted into these susceptible tissues alone by the use of tissue-specific pro- moters, thereby protecting only those tissues that are really vulnerable to the pest. The authors are currently isolating floral bud-specific genes from cotton to modify the expression of Bt in transgenic cotton.

7. Stability of the introduced transgene

New genes introduced into plants by genetic engineering are integrated into the chromosomes of the plant and are therefore inherited just as any other gene. There are examples, however, where the activity of the introduced gene is lost. The genetic material is not lost but the gene is switched off, probably by the chemical modifi- cation or methylation of the DNA itself (John and Amasino, 1989 ). All the cells in a plant contain the same DNA but not all the potentially active genes are functional. Methylation appears to be a natural way in which unwanted gene activity is suppressed in higher organisms, and for un- known reasons, some introduced genes become methylated and hence inactive. This is particu- larly true when the genes are maintained in the hemizygous state, i.e. when the transgene is pres-

ent on only one of the two homologous chromosomes. In the case of the Bt genes that the authors and Monsanto have introduced into cotton, the genes have been inherited and have re- mained active over at least a couple of genera- tions, but more research will need to be done on a longer time scale before there is full under- standing of the factors that affect the stability of transgenes in their new hosts.

8. Development of resistance by the target pest

It seems clear at present that transgenic crops expressing reasonable levels of Bt protein will be tolerant to attack by those specific insect pests targeted by specific Bt toxins. The overriding question is, however, just how long will these insect tolerances based on Bt last in the field, before the insects become completely resistant to the insecticidal action of Bt? That insects can overcome their susceptibility to Bt was learned in the mid-1980s with stored grain pests and more recently with horticultural pests in field situations where Bt has been used as a microbial spray (McGaughey, 1985; Dixon, 1991; Harris, 1991 ). No cases of resistance to transgenic plants expressing Bt have yet been reported, but these plants have still only been released on a relatively small scale.

Considerable controversy still reigns over the potential large-scale commercial release oftrans- genic crops containing only a single type of Bt- toxin gene, for example transgenic cotton containing only the cryIA (c) gene. Some argue that the development of resistance to transgenic plants will be very rapid and will negate the potential beneficial use of similar types of Bt as microbial pesticide sprays (Harris, 1991 ). Resistance development is a serious concern with Bt, but does not only apply to the use of Bt in transgenic plants. The first examples of field resistance development were to Bt insecticidal sprays, the use of which may increase rapidly in the Australian cotton industry over the next few years. In the short term, it is more worrying that excessive use of the microbial Bt pesticide in general agriculture might select for resistant heliothis before

D. Llewellyn et al. / Agriculture, Ecosystems and Environment 49 (1994) 85-93 91

transgenic cotton can be evaluated and commer- cialised (in Australia, commercialisation may be 4-5 years away). Although there are now better commercial formulations and application technologies, the environmental decay characteristics for the potency of microbial Bt and the ina- bility for the insecticide spray to penetrate all parts of the plant, mean that some insects will always survive sublethal exposure. This is the classic scenario for resistance development to Bt that has been observed in both the laboratory and the field (McGaughey, 1985). In overwintering insect species, like H. armigera, or isolated is- land populations, like Plutella in Hawaii, that have limited gene flow between populations, the potential for resistance development to both microbial Bts and transgenic Bt plants might even be as high as that for the hard chemical pesticides they are gradually replacing. Careful use of both forms of Bt is clearly required.

As yet there are insufficient field data available on the absolute potential for resistance development to transgenic Bt plants on their own, although Bt plants will probably not be used alone, but as part of existing pest management strategies that will include the use of some of the softer chemical pesticides. The authors intend to adopt a cautious approach to the use of Bt genes in transgenic crops and will explore a number of strategies that may extend the useful life of Bt transgenic plants. In the short term, the pattern of deployment within fields of the first generation of transgenic Bt plants that the authors and others have produced, is a possible strategy that needs to be assessed. The use of transgenic and non-transgenic plants in mixtures, mosaics or ro- tations may provide refuges for sensitive insects and dilute out any resistance that does develop. Some crop damage may result, but this should be relatively minor. Such strategies can be partially assessed using computer modelling, but effective field evaluation will only come once the transgenic crops are released on a reasonably large scale. It should also be emphasised that, in Aus- tralia at least, transgenic plants (probably cotton) expressing Bt are unlikely to be released on a large scale for another 4 or 5 years, pending preliminary field evaluation and seed increase.

The potential for resistance development in H. armigera will be examined at each stage in the stepwise seed increases that lead towards cultivar registration and commercial release. In the longer term, the authors hope to produce better transgenic cultivars with more durable insect resistance characteristics.

Durable host plant resistances to diseases have been achieved in the past by classical plant breeding techniques using the pyramiding of several major and minor disease resistance genes within the same cultivar, and the possibility of using similar pyramided insect resistances to ensure the extended usefulness of Bt genes in agriculture is being explored. Evidence is accumulat- ing from studies on those insects that have become resistant to microbially produced Bt that the resistance mechanism is not universal, but only affects the type of Bt used to elicit the resistance. Diamondback moth (Plutella xylostella), for example, which has become resistant to Bt kurstaki-type insecticides (the CryIA type) is resistant to other Bt isolates producing CryIA toxins, but is not cross-resistant to CryIB or CryIC Bt types (discussed in Dixon, 1991 ). These other Bt types might therefore be useful as a second tier in a resistance pyramid if combined into the same cotton variety already carrying the cryIA toxin gene. The authors have tested two Bt strains, HD2 and entomocidus 60.5, producing CryIB and CryIC toxins, respectively for toxicity to their target pest, H. armigera, and found that both are toxic (as purified protoxins), although only about a third as potent as the CryIA toxins. The authors are currently engineering the cloned genes from both of these strains for expression in transgenic plants and will test them in transgenic tobacco before introducing them into 'Siokra' cotton.

There are also possibilities for a third or even fourth tier to the resistance pyramid, using genes that plants already possess as deterrents for insect pests. Many crop plants have been bred for increased yield, palatability, etc., often to the detriment of their natural tolerance to insect pests. Other plants, however, produce high levels ofprotease inhibitors in their seeds to act as anti- nutritional compounds against the insects that eat

92 D. Llewellyn et al. / Agriculture, Ecosystems and Environment 49 (I 994) 85-93

their seeds. Some plants even have wound-indu- cible protease irthibitors that are produced at high levels around plant tissues that have been wounded during predation by phytophagous insects (Ryan, 1973). Genetic engineering techniques have recently been used to transfer some of these protease inhibitors from one plant to another with a dramatic increase in tolerance of the recipient plant to some insect pests (Hilder et al., 1987; Johnson et al., 1989). The authors have investigated the use of a number of protease inhibitors against H. armigera by adding purified inhibitors to synthetic diets, but so far have only identified one that has any appreciable effect on the growth o f neonate larvae. This inhibitor comes from the giant taro (Alocasia macror- rhiza), a tropical root crop that shows a natu- rally high resistance to attack by insects (Ham- mer et al., 1989). The gene for this inhibitor is currently being cloned and will be tested in transgenic tobacco before introduction into cotton. Other plant-produced insect inhibitors, such as lectins (Boulter et al., 1990) and enzyme inhibitors, are also being investigated and will hopefully add to the arsenal of genes that can be used to protect cotton and other crops from attack by economically important insect pests.

9. The future of transgenic Bt-expressing crops

Although the commercial use of transgenic plants expressing Bt is still several years away, it is clear that the technology has advanced to the stage where dramatic protection from insect attack can be demonstrated in a range of important crops including cotton, oilseed rape and maize. Two factors remain to be assessed and tested in the marketplace. The first is the dura- bility of insect tolerance based on Bt genes. The use of transgenic plants will have little value if the important insect pests become resistant to Bt after only a couple of years, and considerable research and thought will have to go into the deployment of transgenic crops in agricultural systems in the Short term, so that resistance is delayed or prevented. As indicated previously, this may be achieved by using combinations of

transgenic and non-transgenic crops or by pyramiding different sorts of insect resistance genes together in the same plant or in different plants in rotation. The second factor is achieving public acceptance for transgenic crops. This may not be too difficult for fibre crops like cotton, but will probably require considerable public education for food crops like tomato or potato, despite the good toxicological data already existing for Bt. As with many aspects of genetic engineering, politics can impact on the success of a project, irrespective of its apparent social, economic or environmental benefits. Public education will be essential to ensure the widespread adoption of genetic engineering technologies in agriculture, and scientists will have to play an active role in this process.

Bt toxins and their genes are a unique resource for agricultural systems and the authors consider that the most cost-effective and environmentally appropriate form of packaging for this biological insecticide is the seeds that the farmer buys and plants. The techniques exist for producing such seeds and this goal may be realised in the fore- seeable future.

Acknowledgements

The authors acknowledge the generous sup- port of this research by the Australian Cotton In- dustry through the Cotton Research and Devel- opment Corporation and Cotton Seed Distributors Pty. Ltd. Thanks are expressed to Jenny Thistleton and Merran Brown for excellent technical assistance.

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Expression of Bacillus thuringiensis insecticidal protein genes in transgenic crop plants