A PROMISING DEBUT FOR BT HYBRID RICE

A distinguished scientist, Swapan Datta has pioneered the genetic engineering of improved varieties of tropical rice and contributed to the creation of rice varieties resistant to bacterial blight, sheath blight, and stem borer. Currently he is working to develop genetically enhanced, vitamin A-enriched rice suitable to tropical climates.

Two technological advances have been combined to greatly improve rice productivity—hybrid rice and a fusion Bt endotoxin gene. Hybrid rice, developed and commercialized in China in 1976, became popular because it has a 20% yield advantage over inbred varieties. Bt (Bacillus thuringiensis) has been used for more than 50 years as a biological insecticide. Cloning the insecticidal delta-endotoxin Bt gene into plants further enhanced its effectiveness. Ten years ago Monsanto showed that the fusion of two Bt genes could further improve plant protection against insects¹. In 1999, we developed a promising tool for the use of Bt technology to improve hybrid rice². Subsequently, we developed commercial hybrid rice with a fusion hybrid Bt gene. This hybrid rice showed a 28% yield advantage in field conditions³. The advancement of this product will considerably help to reduce pesticide use.

Rice is the major staple food for about 2.5 billion people, almost all of whom live in developing countries. To maintain an adequate supply of rice for the tremendous annual increase in population between now and 2020 and beyond is a formidable challenge to the scientific community. This increase must be achieved in the face of declining arable land and water supplies, and in a manner that protects the environment (soil, water, and biotic resource base) from which all food must come. The combination of genetic engineering with improved plant breeding offers a solution to the demand for food security. The total global area under cultivation with transgenic crops as of 1999 was 8.9 million hectares. The commercialization of other Bt crops such as canola, cotton, and maize is in progress in several countries including Asian countries such as India and China.

Hybrid rice lines have been observed to produce about 20% higher yields than inbred semidwarf varieties. Rice hybrids are now cultivated on about 55% of the rice-growing areas in China and contribute 66% of China's total rice production. During the past four years, India, Vietnam, Bangladesh, and the Philippines have been using this technology successfully. The production of hybrid rice involves a three-line system: cytoplasmic male sterile (CMS), maintainer, and restorer lines. Incorporation of resistance gene(s) in a CMS line makes this technology widely applicable to the development of resistant rice hybrids. Since a CMS line is maintained by backcrossing to its isogenic maintainer line, the presence of resistance gene(s) in the latter will lead to the development of a CMS line possessing these genes. Alternatively, the gene(s) can be incorporated into a restorer line, and a homozygous restorer line carrying the gene, when hybridized with the CMS line, will produce a desirable hybrid plant with a gene of interest.

Stem borer is a serious problem in rice, causing estimated losses of 5-30% of the total yield. Recovery of 5% of this yield loss could provide food for one year for the approximately 140 million people of Bangladesh. Yellow stem borer (Scirpophaga incertulas) and striped stem borer (Chilo suppressalis) are the major stem borer pests and are widely distributed in Asia. Stem borer larvae start their attack by boring through the inner portion of the leaf sheath. The subsequent boring through the stem by caterpillars causes considerable damage, resulting in "deadheart" symptoms, and the affected tillers do not bear panicles. Panicles often appear with empty grains, a condition called "whitehead."

Bt, a common soil bacterium, produces crystals containing insecticidal proteins. These toxins kill insects by binding to and creating pores in the midgut membranes. Bt toxins are highly specific and therefore are not toxic to beneficial insects, birds, and mammals, including humans⁴. The question arises whether we can express this Bt toxin in tissues other than seeds. Tissue-specific promoters, particularly the green-tissue-specific promoter (PEPcP) or pith tissue-specific promoter-driven Bt genes, were introduced in rice and showed preferential tissue-specific expression and significantly reduced expression in grain⁵.

It took 10 years to develop the first transgenic plant with the Bt gene. However, the expression of the Bt endotoxin protein was too low (<0.001% of leaf soluble protein) to provide adequate protection against insect pests. The situation has now changed dramatically as truncated and/or synthetic genes have been developed by removal of potential mRNA-processing (improper splicing sites) and polyadenylation signals during resynthesis of the Bt gene⁶.

What are the prospects for current Bt rice and acceptance by farmers? A restorer line, Minghui 63, was transformed with a fusion of two genes, cryIA(b) and cryIA(c), driven by the actin1 promoter. A selected homozygous MH63 Bt line was hybridized with CMS line Zhenshan 97A to produce a hybrid Shanyou 63 Bt rice. Shanyou 63 has been the most widely used popular hybrid in rice production in China for the past 15 years. Transgenic cultivars were selected based on co-transformation using hph (selectable antibiotic marker gene, hygromycin phosphotransferase). However, during the subsequent progeny selection, careful molecular analysis helped us select a MH63-CMS-Bt line without the marker gene. Finally, our hybrid rice was selected and used in the field carrying only hybrid Bt genes but without any selectable marker gene. This was possible because of the integration of the transgenes (Bt and hph) in two different loci.

Hybrid Bt rice (Shanyou 63) was field-tested in Wuhan, China, in 1999 and 2000. Transgenic plants were field-tested in natural and repeated heavy manual infestations of two lepidopteran insects, leaffolder and yellow stem borer. The transgenic hybrid plants showed high protection against both insect pests. The yield of the hybrid Bt rice was 28.9% more than that of the non-Bt hybrid. Considering that the field trial was conducted without the use of chemicals after transplanting, these results demonstrate that expression of the Bt fusion protein in the genome of the transgenic hybrid rice provided season-long protection against the natural outbreak of heavy manual infestation of the two lepidopteran insects.

Where do we go from here?

Rice grows in different ecosystems. Adaptive cultivars already developed by plant breeders should be used for incorporation of the Bt gene. For example, we have developed deepwater rice (DWR) with the Bt gene. DWR is grown in areas usually flooded deeper that 50 cm (sometimes up to 400 cm) for one month or longer during the growing season. Consequently, any traditional tall and elongating rice cultivar can be grown in these areas. Yield is generally low (1-2 t/ha) and very often reduced further by insect attack. The application of insecticides to DWR causes many problems. Ordinary ground applications are limited to the pre-flood period and spraying is not possible when the water is deeper than 50 cm. Moreover, pesticides could affect beneficial natural predators and cause fish mortality. Fish harvested from DWR are a major source of income and protein for the people living in these areas. The development of DWR varieties with resistance to yellow stem borer will help farmers in flood-prone ecosystems significantly. We have successfully introduced a cryIA(b) gene (provided by Novartis) into an elite DWR Vaidehi variety⁷ and homozygous material is now ready to transfer to India for field testing and further use.

Bt will play an important role in meeting the increasing global need for food, but its use is subject to social, economic, and ecological pressures. Several adaptive varieties must be used for each crop. A system well adapted and optimized in United States farms for a larger land area may not be applicable for a country such as India having a large number of small farms. Suitable systems need to be developed for various crops and countries, which can only be achieved by increased field testing, evaluation, and data collection. Bt toxins are insecticidal and, as with conventional chemical insecticides, insects may quickly adapt to them unless Bt plants are carefully designed and deployed. A greater assurance of durable resistance can be achieved if a Bt toxin is combined with a second type of toxin¹.

It is critical that Bt remains a viable option for agriculture. After 30 years of successful use, Bt is considered one of the safest pesticides available. It is biodegradable and has no adverse effects on beneficial insects, other wildlife, and farm workers. Successful Bt crops including the first commercial Bt hybrid rice, have now been thoroughly evaluated in fields. In the future, the use of Bt crops within an adaptable Integrated Pest Management strategy may lead to durable and environmentally friendly plant protection. The successful expression of the Bt fusion gene in hybrid rice provides a good resource for management of rice pests in tropical Asian countries.

Sources

1. Perlak FJ, et al. 1990. Insect-resistant cotton plants. Bio/Technology 8: 939-943.

2. Alam MF, et al. 1999. Transgenic insect-resistant maintainer line (IR68899B) for improvement of hybrid rice. Plant Cell Reports 18: 572-575.

3. Tu J, et al. 2000. Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis delta-endotoxin. Nature Biotechnology 18: 1101-1104.

4. Koziel MG, et al. 1993. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11:194-200.

5. Datta K, et al. 1998. Constitutive and tissue-specific differential expression of cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest. Theoretical and Applied Genetics 97: 20-30.

6. Frutos R, et al. 1999. Managing insect resistance to plants producing Bacillus thuringiensis toxins. Critical Reviews in Biotechnology 19: 227-276.

7. Alam MF, et al. 1998. Production of transgenic deepwater indica rice plants expressing a synthetic Bacillus thuringiensis cryIA(b) gene with enhanced resistance to yellow stem borer. Plant Science 135: 25-30.

GREEN DRUG FACTORY NOT FAR AFIELD

The application of biotechnology to agriculture is creating a broad spectrum of new high-value traits for traditional crop species. Whole industries are arising out of these new discoveries and low-value commodity crops are being turned into specialty products. Research conducted during the past 10 years clearly demonstrates that plants can be engineered to produce a wide range of pharmaceutical proteins in a broad array of crop species and tissues¹.

Evidence that plants are a strong base from which to approach the prevention of autoimmune disease was presented with the expression of murine GAD67 in plants and its application to the prevention of diabetes in the NOD mouse ². Autoimmune diseases are debilitating to individuals and represent a major challenge for society³. As a group, they represent some of the most expensive diseases to treat and clearly require effective low-cost therapies. Our interest is to use interleukin-10 for the oral treatment of inflammatory bowel disease and to augment oral immune tolerance in the prevention of autoimmune disease. Knowing that oral treatment will require large amounts of recombinant protein, we felt it was important to evaluate low-cost modes of interleukin-10 production.

Plants offer several distinct advantages over conventional expression systems for the production of recombinant proteins. Since plants are higher eukaryotes, they can properly fold and assemble proteins in a manner similar to mammalian cells and therefore plant recombinant proteins will not require expensive in vitro refolding. The upstream cost of producing the raw plant material will be significantly lower than that of fermentation¹. In addition, plant systems produce recombinant proteins that are free of pathogens of mammalian origin. Unlike fermentation-based bacterial and mammalian cell systems, protein production in plants is not restricted by physical facilities. Agricultural scale production ensures the availability of recombinant proteins in theoretically unlimited amounts that are certainly sufficient for extensive clinical studies and therapeutic use.

Though many crops are being evaluated for use in the production of recombinant proteins, it is not yet clear which crop platform is best. Research is currently focused on technical issues, such as intracellular targeting and optimization of recombinant protein yield and purification. However, as the production of plant recombinant proteins is scaled-up, practical issues such as safety and containment also need consideration. The recombinant therapeutic proteins produced in crops are biologically active, and the effects of chronic exposure or escape into the environment are not known. Field scale systems for containment are therefore essential, but plants are only competitive if production costs are not inflated by costly restrictions. Only a non-food crop platform used in conjunction with "good agricultural practices" will limit safety issues and the potential for environmental impact so that both regulatory and economic concerns can be satisfied.

Tobacco is a non-food crop that has been the subject of many years of breeding and agronomic research and can be used as a strong base for a field production system for molecular farming. We have developed a production system based on hybrids between male-sterile low-nicotine females and homozygous transgenic lines. The low-nicotine background genotype begins to address issues associated with unwanted secondary metabolites. Minimizing the presence of nicotine may also allow for in vivo human use. The background genotype is also optimized for agricultural production. The resultant hybrids express the transgene uniformly and recombinant protein production is based on leaves, not seeds or tubers, which further limits the potential for escape. The plants are grown at a high density to maximize biomass yield and are harvested after 30-40 days, eliminating flower production and allowing the system to be adapted to a broad range of production environments. Male-sterility further reinforces containment. Multiple staggered plantings ensure the availability of plant material for processing throughout the growing season.

We have tested the system by producing biologically active human interleukin-10 and then scaled production up to the level of confined field trials. Interleukin-10 is a contra-inflammatory cytokine that has multiple roles in the regulation of immune responses. Recombinant IL-10 can be produced by insect cells or by bacteria when coupled with in vitro refolding. We have successfully completed experiments aimed at producing biologically active human interleukin-10 in plants. We have gone through the process of pureline selection and identified material that shows maximum levels of human IL-10 accumulation. Two cycles of field trials aimed at the evaluation of different types of targeting and to assess the value of hybrid male-sterility to the overall system have been completed. We are in the process of publishing a detailed description of those results, and in the early stages of testing the material in a mouse model of chronic enterocolitis.

Sources

1. Kusnadi AR, Nikolov ZL and Howard JA. 1997. Production of recombinant proteins in transgenic plants: Practical considerations. Biotechnology and Bioengineering 56: 473-485.

2. Ma SW et al. 1997. Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. Nature Medicine 3: 793796.

3. Autoimmune disease. 2000. Nature Biotechnology, Supplement 18: IT7-IT9.

GENE TECHNOLOGY IN THE LAND DOWN UNDER

The following is a report of an agricultural tour and the World Congress of International Federation of Agricultural Journalists attended by Tracy Sayler in Australia in September 2000.

With about 90% of its population urban dwellers who live on the coasts, and a dependence on exports to move about 80% of its agricultural production, it is not surprising that biotechnology is a sensitive issue in Australia. Government and industry leaders realize that Australia could risk domestic and export markets if genetically modified products are commercialized. They also realize they could risk their market share if Australia does not commercialize GM products.

BIOTECHNOLOGY POLICY IN IRELAND—AN EXAMPLE TO EUROPE?

"On the basis of the best knowledge available to us, therefore, the development of world-class competence in biotechnology on a basis compatible with the protection of human health and the environment is essential, not optional, for Ireland and Europe¹."

Recently, a major clarification in modern biotechnology policy took place in Ireland with the publication of a new 235 page Government report. The report is likely to have ripple effects across the European Union in regards to its faith in GM technology. Modern applications of biotechnology have been subject to biopolitical influences² in Ireland where "Widely different points of view are expressed and challenged daily"³ on the subject. In the course of this ongoing biopolitical debate, biotechnology itself has been successively defined and redefined, negotiated and renegotiated, as professional and political interests have sought to shape the technology according to differing priorities.

Current Irish Situation
In Ireland, the implementation of experimental field trials of GM crops has been a focus point for all the actors involved in the debate surrounding the application of modern biotechnology— pressure groups, competent authorities, the political establishment, industry, media, academic scientists, etc. In December 1996, the multinational life science/chemical company Monsanto applied to the Environmental Protection Agency (EPA) in Ireland to carry out experimental field trials of the glyphosate tolerant GM sugar beet (Beta vulgaris). This application was made in accordance with the EU Directive 92/220, which has been embodied into Irish law within the 1994 GMO Regulations made by the Minister for the Environment pursuant to powers contained in the 1992 Environmental Protection Act. The EPA granted conditional permission to Monsanto to test the GM sugar beet for a period of four years—three for growing purposes and one for subsequent test site monitoring. On September 28^th, 1997, the first test plot was destroyed by a group called the Gaelic Earth Liberation Front. Clare Watson, a leading member of Genetic Concern, a pressure group set up in April 1997, was granted leave to seek a judicial review of the EPA's procedure in granting the license to Monsanto. This hearing concluded in October 1998 with the High Court ruling against Clare Watson on all the twelve main areas of contention. Since then, the GM sugar beet field trials have continued, and a total of six attacks on the test plots have occurred at several different locations.

Up until last week, the current Irish Government had not officially stated its GM food policy. During the final days of the 1997 election, the then current Minister of the Environment and the Minister of Agriculture issued a joint statement describing GM food as a `mass experiment', and vowed to end the experimental trials of GM crops in Ireland (Fianna Fail, Press Release, April 1997). Since gaining office, the new Government has instituted a unique two-stage public consultation process to allow input into the formulation of its policy on `GMOs and the Environment' under the specific auspices of the Department of Environment and Local Government (DOE). The first stage called for written submissions from interested members of the public. By the submission deadline, September 30^th, 1998, over 200 people and organizations had made submissions.

The second stage of the consultation process allowed for a two-day debate. A panel of stakeholder representatives participated in the debate sessions, chaired by an independent panel. The stakeholder panels consisted of two representatives from each of three groups: industry; the academic science community; and Non-Government Organizations (NGO)/pressure groups. These representatives where chosen from those who had responded to the advertised Government call for submissions on `GMOs and the Environment'. The debate process encountered several severe problems, which resulted in a boycott of the final day of a two-day debate by the vast majority of the anti-GM NGO/pressure groups. The panel issued a report of the debate, which was accepted by the Minister. On receipt of the report in October 1999, the Government referred a number of specific issues to the Inter-Departmental Group on Modern Biotechnology, which had been established in March 1999 under political pressure and direct suggestion from the main opposition party, Fine Gael. These issues included the dissemination and coordination of information on genetic engineering, the case for a biotechnology ethics committee, and future policy and administrative coordination on genetic engineering.

The Inter-Departmental Group issued a report on Monday, November 20^th, 2000 in which they made recommendations on the coordinated inter-departmental government positions on a wide range of issues related to the development of modern biotechnology. The Group's main recommendations include the following:

• Ireland should take a positive but precautionary approach to GM issues, at both EU and in international forums, which acknowledges the potential benefits of modern biotechnology while maintaining a fundamental commitment to human safety and environmental sustainability;

• Irish trials of GM crops should continue, subject to compliance with EU legislation and with the conditions laid down by the EPA;

• the Department of Agriculture, Food, and Rural Development should, in consultation with the Environmental Protection Agency, draw up detailed protocols governing the management of GM crops in field trials;

• the Department of Agriculture, Food, and Rural Development should, in cooperation with other bodies, devise a program for the managed development of GM crops that would provide for a phased, monitored progression to full commercial cultivation;

• the Irish State Laboratory should be designated as the national reference laboratory for the detection of GM materials in foods and other products;

• in the interests of transparency and public awareness, regulatory bodies should, as a matter of standard practice, make available the fullest possible information on the applications for release and marketing approvals of GMOs;

• a national biotechnology ethics committee should be established under the auspices of the Royal Irish Academy to consider the ethical issues raised by biotechnology in an informed and dispassionate way;

• independent genetic research should be conducted in Ireland into all aspects of GMOs, giving consideration to distinctive Irish climatic and geological conditions;

• new ways of informing the public about biotechnology, its existing and potential benefits, and the possible risks to health and the environment should be devised and deployed: A central Government Web site should be established that provides a broad range of relevant, up-to-date information in a manner readily accessible to the public;

• new means of promoting public consultation and involvement in debates about biotechnology should be developed and piloted; and

• the Inter-Departmental Group should be permanently supported to ensure that the Government has an integrated view of the full range of relevant issues, and the Group should be expanded to include representatives of the Environmental Protection Agency; the Food Safety Promotion Board; Teagasc; and the Department of Arts, Heritage, Gaeltacht, and the Islands.

Several noteworthy points can be made regarding this new inter-departmental report. First, the report identifies several factors deemed to have aggravated public disquiet about genetic engineering (pg. 94), and it also comments that "the introduction of GM crops and foods was insensitively managed by industry and effectively opposed by environment groups" (ch. 3, pg 105)¹. However, the report fails to mention the lack of political leadership in Ireland on the issue and the current government's own 1997 negative pre-election statement that stimulated public concern. The section outlining the public perceptions toward GM technology in Ireland is dated and omits the 1999 Eurobarometer results⁴. It is also worrisome that the report completely ignores previous public communication recommendations compiled largely by the Irish government^5,6.

The report also acknowledges two important aspects regarding the public disquiet over GM food. First, it points out that the media's "desire for strong, stand-alone stories is not always easy to reconcile with the incremental, provisional nature of scientific finding" (pg. 98)¹. The report also states that "in Britain in particular, there have been no shortage of instances in which newspapers—some of which have a sizable readership in [Ireland]—have misled rather than informed the public about genetic modification. Distorted presentations of the facts, lurid accounts of `Frankenstein foods', have been commonplace in the treatment of the issue in the mass-circulation newspapers" (pg. 97)¹.

The second interesting acknowledgment is that the Irish government has officially underlined the role the organic movement has played in the campaign against GM crops and foods. The report also points out that the organic movement has much to gain in this opposition, which is reflected in the report's comments that "Representatives of the organic farming and food sector have also been prominent in the campaign against GM crops and foods…. We appreciate also that the debate about genetic modification has given organic producers an opportunity to draw attention to the merits of their own produce" (pg. 103)¹. The Irish Government concludes that, even if they accept the concerns of organic farmers regarding possible gene transfer, they "see no reason why [approved GM crops] cannot form part, with organic farming, of a broad mix of crop types and farming practices" (pg. 103-104)¹.

On the basis of this Government report, it seems that Ireland is set to move the debate regarding GM technology to a more mature level at home and abroad.

Sources

COLOMBIA PUTS FIRST LEGAL TRANSGENIC CROP IN GROUND

POSITION ANNOUNCEMENT

Information Systems for Biotechnology is seeking a project coordinator; this is a half-time position with potential for expansion to full-time.

Qualifications include a Ph.D. in biological science; working knowledge of current agricultural biotechnology research, products, and regulations; and familiarity with environmental issues associated with transgenic crops, including safety assessment and risk management. Excellent speaking and writing skills are essential; proficiency in MS-Word and use of the Internet is required.

Responsibilities include the following: