Different operational areas of plant biotechnology are: 1. Plant Transformation 2. Gene Expression 3. Methods of Gene Discovery 4. Gene Stability 5. Insect Resistance 6. Weed Control 7. Disease Resistance 8. Stress Resistance 9. Food Processing 10. Specialty Chemical Products 11. Pharmaceuticals.
1. Plant Transformation:
Remarkable progress has been made in the development and application of gene transfer systems, to crops since the first demonstration of transgenic plant production just over ten years ago.
Today, over 80 species of crop plants can be genetically manipulated using available Agrobacterium tumefaciens or a variety of free DNA delivery transformation systems (Table 1). This list includes nearly all major dicotyledonous crops and a rapidly increasing number of monocotyledonous crops, including wheat, rice and maize.
It is highly likely that routine gene transfer systems will exist for nearly all crops within the next two or three years. While technical improvements will lead to further increases in transformation efficiency, extend transformation to elite commercial germplasm and lower transgenic plant production cost, there is no significant barrier, even today, to the application of plant transformation to crop improvement.
2. Gene Expression:
Plant genetic engineers currently have in hand a large battery of regulatory sequences that provide for both constitutive expression as well as highly accurate targeting of gene expression to specific tissues within transgenic plants. Moreover, established differential screening methods allow for ready isolation of regulatory sequences that may be required for even more sophisticated expression requirements.
The ability to decrease endogenous gene expression in plants represents a remarkably powerful tool, and striking phenotypic alterations have been observed by selective inactivation of genes using antisense technology.
Table 1: Species in which Transgenic Plants have been produced:
Achieving even higher levels of gene expression in selected plant organs would increase opportunities for more economic specialty chemical or pharmaceutical production in plants, and site-specific insertion could minimize the variability of gene expression among transform-ants. However, current expression systems appear sufficient for meeting immediate crop improvement needs.
3. Methods of Gene Discovery:
Advances in methods for the identification and isolation of new gene coding sequences are of great importance to the engineering of improved plants. The interspecies- specific use of transposons and T-DNA insertion has permitted the tagging and isolation of novel genes from several plant sources.
The availability of high resolution physical maps in tomato and Arabidopsis has already led to mapping of several novel loci and new methods will allow direct testing of the isolated DNA for its ability to complement the mutation of interest at each step of the walking process.
Advances in the redesign of coding sequences for plant expression allow for predictable, high-level expression of a variety of non-plant genes in crop plants. Again, while ongoing research efforts will predictably and dramatically increase the probability and efficiency of gene discovery and isolation, it would appear that even with today’s methods most genes can be identified and isolated. Even assuming only modest advances in gene discovery methods and progress on the sequence analysis of the Arabidopsis genome in the next ten years, one could infer that gene discovery will not be a limiting element for very long.
4. Gene Stability:
By the end of 1991, nearly five hundred field test experiments evaluating the performance of genetically engineered plants had been carried out in the USA and Europe alone. The overwhelming conclusions from these extensive studies are that newly introduced genes are stable, inherited and are expressed like any other plant gene. This includes a variety of new genes which provide for control of insects, weeds, and plant diseases as well as for quality improvement.
Such traits have already been successfully introduced into several important crop species and genetically engineered soybean, cotton, rice, rapeseed, sugar beet, tomato, alfalfa, potato and maize crops are expected to enter the marketplace between the years 1995 and 2000. Broad germplasm access will likely require extensive backcrossing or micro-propagation efforts. These established methods, although cumbersome, appear adequate for ensuring large germplasm access in most annual and perennial crops.
Plant biology is entering a unique period where both basic research and commercial applications will be limited only by the creativity of the researcher and by funding levels. While there is an obvious need for substantial expansion of our understanding of basic plant biochemistry and physiology in order to fully exploit scientific advances, there are no significant technical hurdles remaining.
The initial wave of research in plant biotechnology has been driven by the seed and agrochemical industries and has appropriately concentrated on the engineering of “agronomic traits” that relate directly to the traditional roles of these industries in farming, such as the control of insects, weeds, and plant diseases. Progress in this area has been exceedingly rapid, and genes conferring these new traits have already been successfully introduced into several important crop species. The status of some of these product candidates is discussed below.
5. Insect Resistance:
The production of plants that naturally control insects has obvious, important implications for crop improvement, and for both the seed and agrochemical industries. Progress in developing insect control in transgenic plants has been initially achieved through the expression in plants of the insect control protein genes of Bacillus thuringiensis (B.t.). B. t. is a naturally-occurring soil bacterium that produces an insect control protein which is lethal to selected insect pests. Most strains of B.t. are toxic to lepidopteran (caterpillar) larvae, although some strains with toxicity to coleopteran (beetle) or dipteran (fly) larvae have also been described.
The insect toxicity of B.t. resides in a large protein; this protein has no toxicity to beneficial insects, other animals or humans. The mode-of-action of the B.t. insect control protein involves disruption of K+ ion transport across brush border membranes of susceptible insects.
Transgenic tomato, tobacco, cotton and maize plants containing the B.t. gene have exhibited tolerance to caterpillar pests in laboratory tests. A novel approach for increasing expression of B.t. genes in plants, which involves restructuring of the DNA coding sequence without altering the encoded amino acid sequence, has led to substantial enhancement in insect control. Cotton plants with a high level of resistance to boll damage by caterpillars have been developed, and commercial levels of control have been achieved with both potato and cotton. Both products are being reviewed by the EPA.
Field tests have confirmed excellent protection (equivalent to weekly insecticide spraying) from bollworm, budworm and pink bollworm. Excellent protection from defoliation by Colorado potato beetle has also been observed in greenhouse and field experiments with potato plants containing the novel coleopteran-active B. t. tenebrionis gene. The insect resistant plants sustained no damage from Colorado beetles throughout the growing season under conditions of high insect pressure.
Other types of insecticidal molecules are clearly necessary to extend biotechnology approaches for controlling additional insect pests in these and other target crops. Extensive efforts are under way to identify other microbial and plant insecticidal proteins. It has been demonstrated that plants genetically engineered to express a proteinase inhibitor gene demonstrate enhanced resistance to a range of insect pests; in vitro studies indicate the alpha-amylase inhibitor protein has broad- spectrum insecticidal activity.
It is highly likely that a large percentage of insect control in annual crops such as cotton, maize and vegetables will be provided by introduced genes in the next 10-20 years. These developments will provide new solutions for insect control and allow for significant reduction in insecticide usage. An important focus of seed companies introducing these new crops will be ensuring that appropriate agronomic and farm management practices are utilised to minimise any possibility of insects developing resistance to the plants.
6. Weed Control:
Engineering tolerance to a specific herbicide into a crop plant represents a new alternative for conferring selectivity and enhancing the crop safety of herbicides. While laboratory experiments have shown that it is possible to achieve resistance to nearly a dozen different herbicides, R&D efforts by private companies have concentrated only on those herbicides with minimal environmental impact, with emphasis on properties such as high unit activity, low toxicity and rapid biodegradation. Care has also been taken to ensure that herbicide-tolerant genes will not be introduced into crops which could become “volunteer” weeds in subsequent crop rotations or which outcross readily with weed species.
The development of crop plants which are tolerant to such herbicides would provide for more effective, less costly and more environmentally attractive weed control options than exist today. The commercial strategy behind engineering herbicide to clearance is to gain market share through a shift in herbicide use, not to increase the overall use of herbicides as is popularly held by critics.
Two general approaches have been pursued in engineering herbicide tolerance:
1) Altering the level and sensitivity of the target enzyme for the herbicide; and
2) Incorporating a gene encoding an enzyme which can inactivate the herbicide.
As an example of the first approach, Roundup herbicide acts by specifically inhibiting the enzyme 5- enolpyruvylshikimate-3-phosphate syntheses. Roundup herbicide is active against annual and perennial broad leaf and grassy weeds, it has very low animal toxicity, and it is rapidly inactivated and degraded in all soils.
Tolerance to Roundup herbicide has been engineered into canola, soybean, cotton and maize by introducing genetic constructions for the overproduction of herbicide resistant EPSPS enzymes. Similarly, resistance to sulfonylurea compounds, the active ingredients in Glean, and Oust herbicides, has been produced by the introduction of mutant acetolactate synthase (ALS) genes into canola and cotton.
Sulfonylureas are broad spectrum herbicides that are effective at very low application rates. Resistance to gluphosinate, the active ingredient in Basta, and bromoxynil has been achieved by the alternative approach of introducing bacterial genes encoding enzymes that inactivate the herbicides by acetylation or nitrile hydrolysis respectively. In field tests, gluphosinate-tolerant canola, soybean and maize have shown excellent tolerance to the herbicide. Similarly, bromoxynil tolerant cotton has been extensively evaluated and shown to provide excellent control of broadleaf weeds.
The current crop targets for engineered herbicide tolerance include soybean, cotton, maize, rapeseed and sugar beet. For the farmer, many factors such as weed spectrum, herbicide performance, environmental impact, seed and chemical cost, application timing and flexibility have to be considered when choosing a particular weed control system. The availability of herbicide tolerance in annual crops over the next decade will give farmers more flexibility in choosing effective and less costly options for weed control.
Herbicide-tolerant plants will have the positive impact of shifting overall herbicide usage through substitution of more effective and environmentally acceptable products. Such improvements in chemical weed control will also allow for higher adoption of minimum tillage practices, and encourage crop rotations which will further reduce soil erosion.
7. Disease Resistance:
Significant resistance to a variety of plant viral diseases has been achieved by coat protein-mediated protection, which involves expressing the coat protein gene of a particular virus in transgenic plants. The mechanism for coat protein-mediated cross protection is likely to involve interference with the un-coating of virus particles in cells prior to translation and replication.
Using this approach, results have been obtained for transgenic tomato, alfalfa, tobacco, potato, melon and rice against a broad spectrum of plant viruses, including alfalfa mosaic virus, cucumber mosaic virus, potato virus X (PVX), potato virus Y (PVY) and potato leaf roll virus.
Excellent tolerance has been observed in field tests of Russet Burbank potatoes containing coat protein genes to both PVY and PVX. Recently, very significant resistance to tobacco mosaic virus in tobacco plants has also been obtained by an alternative method which involves expression of a sub-genomic viral replicase component.
Rapid progress is also being made in engineering resistance to bacterial and fungal pathogens by several groups around the world. Resistance to the bacterial pathogen Pseudomonas syringae, which causes wildfire in tobacco, has been introduced in transgenic tobacco by expressing a tabtoxin resistance gene that codes for an acetyltransferase. This result demonstrates a successful approach to engineering disease resistance in plants by detoxification of pathogenic toxins.
Some success in engineering resistance to fungal diseases has also been reported. A chitinase gene from the soil bacterium Serratia marcescens was stably expressed in transgenic tobacco, the preliminary results indicated that the expression of the bacterial chitinase in transgenic ‘ tobacco leaves resulted in significantly reduced severity of disease caused by a brown-spot pathogen, Alternaria longipes. The plants were reported to have significantly reduced fungal lesions as well as delayed susceptibility to the pathogen.
A bean chitinase gene driven by a high level, constitutive-promoter has been expressed in tobacco plants. These plants exhibit increased resistance to the pathogenic fungus Rhizoctonia solani, resulting in significantly reduced root damage and enhanced ability to survive in infested soil. Genes conferring fungal resistance based on the plant’s own defense response are being cloned by a number of research groups; one of these proteins, termed osmotin, has been shown to have potent in vitro activity against Phytophthora infestans, the causal agent of late blight disease in potato. Manipulating existing defense mechanisms may also prove to be useful.
8. Stress Resistance:
A variety of abiotic stresses including water, temperature and soil composition are known to impact crop productivity. Although the complexity of plant stress responses has eluded early demonstration of improved phenotypes using plant biotechnology methods, these tools are being applied to dissect and understand the molecular basis for plant response.
A number of plant genes induced by exposure to heat, cold, salt, heavy metals, phytohormones, nitrogen etc., have been identified. Additionally, rapid progress is being made in identifying ion transport pumps and proteins which regulate transport of molecules through channels and plasmodesmata.
Metabolites such as proline and betaines have been implicated in stress tolerance in both bacteria and plants-experiments are in progress in a number of laboratories to evaluate the potential of these metabolites to alleviate stress in engineered plants and understand their mode of action. As these advances accelerate, it is highly likely that there will be demonstrations of heat, cold, drought and salt tolerance in the near future.
The world-wide seed and agrochemical industries have been the leading commercial sponsors of agricultural biotechnology research. However, the technical advances and the success of the first products have increased interest by the food processing, specialty chemical and pharmaceutical industries (Figure 1).
9. Food Processing:
Plant biotechnology offers exciting opportunities for the food processing industry to develop new and more nutritious food products and cost effective processes. Examples of such applications include production of higher quantities of sugars, starch or specialised starches with various degrees of branching and chain length to improve texture, storage and cooking properties.
Starch levels have been increased by 20-40% in potato by expression in tubers of a bacterial ADP glucose pyrophospho-rylase gene (ADPGPPase), which has been shown to be the rate-limiting step in starch production in plants. Expression of the gene encoding sucrose phosphate syntheses in transgenic tomato plants has been shown to elevate sucrose levels and reduce starch. The opportunity to specifically create specialty oils or to eliminate particular fatty acids in seed crops is quickly becoming a reality.
The identification and cloning of several key enzymes in fatty acid biosynthesis has allowed for antisense inhibition of key steps in the pathway; by expressing antisense constructs to stearyl ACP desaturase in canola, significant increases in stearic acid levels have been observed. The production of proteins with nutritionally balanced amino acid compositions has been shown to result in significant changes in seed amino acid composition. In tomato, it has been possible to increase bruise resistance by expression 6f antisense RNA to polygalacturonase and to delay fruit, ripenirig by expression of antisense RNA to enzymes involved in ethylene production.
Recently, the expression of ACC deaminase, an enzyme that degrades the immediate precursor to ethylene, has also been shown to confer controlled ripening. The enzymes and genes involved in biosynthesis of coloring materials and flavors are also important to the food industry and to the consumer; it has been possible to manipulate color of flowers by sense and antisense expression of the flavonoid biosynthetic genes.
10. Specialty Chemical Products:
Enormous opportunity lies in the successful exploitation of crops for both commodity arid specialty chemical products. Plants have traditionally been a source of a wide range of monomeric and polymeric materials. These range from sugars and fatty acids to polymers such as starch and celluloses, which are carbohydrate based, and to polyhydrocarbons such as rubber and waxes. Many of these polymers have been replaced in the last two to three decades by synthetic materials derived from petroleum- based products.
However, the cost, supply and waste-stream problems often associated with petroleum-based products are issues which have focused new attention on the use of renewable, biological materials. Genetic engineering will significantly enlarge the spectrum and composition of available plant monomers and polymers. Expression of an Escherichia coli mannitol dehydrogenase gene in tobacco has allowed for increased levels of mannitol in plants.
Cyclodextrins are interesting specialty starches which have application in catalysis, formulations, and food processing; introduction of a bacterial cyclodextrin glucosyl transferase has been reported to result in low but detectable levels of cyclodextrins in potato tubers.
Recently, researchers have reported the expression of acetoacetyl CoA synthetase and the acetoacetyl CoA reductase in transgenic plants-these two enzymes constitute the two steps leading to the production of poly-hydroxybutyrate, an interesting thermoplastic polymer. It has been proposed that expression of a novel fatty esterase could result in production of C-12 fatty acids in temperate crops like soybean and canola.
Plants also offer the potential for production of foreign proteins with a variety of health-care applications. Proteins such as neuropeptides, blood factors and growth hormones could be produced in plant seeds and this may ultimately prove to be an attractive economical means of production. Several mammalian proteins have already been produced in genetically engineered plants, including pharmaceutically-active peptides, enkephalins, in rapeseed and human serum albumin in potato.
Plant virus vectors containing desired genes may represent a particularly interesting route for producing large quantities of proteins in plants, due to their high copy number. In the longer term, there is little doubt that biotechnology will be used to improve the nature of both the micro- and macro-ingredients in plant-derived foods. The relationship between diet and disease is slowly emerging, and the consumer demand for more healthy foods is expected to grow.
From the above examples, it is clear that plant biotechnology will have an enormous impact on the seed, agrochemical, food processing, specialty chemical and pharmaceutical industries of the industrialised world. However, the majority of the five billion additional people that will live on our planet in 2030 will be in the less developed or developing countries.
Most experts agree that biotechnology will, and in fact must, have a positive impact on agriculture in the developing world. It will not be a “quick fix” to the problems of food production and world hunger, and it will not substitute for conventional agricultural applications, economic and political reform, education, solutions to rural landlessness, international debt relief or population control, among others.
But biotechnology can make big contributions in helping to ensure a sustainable supply of adequate food. One of the key reasons plant biotechnology will have this impact is because of its inherently low capital cost for delivery and implementation.
Biotechnology enables delivery of the latest technology to a rural farmer in the package he is most familiar with-the seed. In fact, “plant biotechnology” has been identified by the World Bank, the United Nations Educational, Scientific and Cultural Organisation (UNESCO), the French Institute for Scientific Research for Development and Cooperation (ORSTOM), the United States Agency for International Development (USAID), the Rockefeller Foundation, the International Service for the Acquisition of Agri-Biotech Applications (ISAAA) and numerous other international aid and scientific groups as a high priority in order to meet the future agricultural production needs of developing countries.
Because of the unique challenges associated with regional population density, food distribution, and dietary preference, agricultural biotechnology must be developed locally in developing countries.
Some of the issues which currently must be addressed to facilitate biotechnology in developing countries are:
1. Identification of near-term crop/trait targets; mobilisation of resources and funding;
2. Establishment of technology transfer mechanisms with private/public organisations;
3. Development of a regulatory oversight framework; and
4. Addressing of property rights and trade issues.
Success will clearly require a coordinated effort between international agencies and governments as well as private companies and public institutions. Several private companies have taken a leadership role in technology transfer to the developing world by establishing relationships which bring these important constituent groups together.
Monsanto Company, for example, has projects with Mexico and Africa, which are sponsored by the Rockefeller Foundation and USAID respectively, to develop virus resistant potatoes and other root crops. The benefits of such interactions are both short and long term. Obvious and most important, in the short term, is the opportunity to help alleviate suffering and poverty; in the long term, it is hoped these countries will become important trading partners.