Companion Publication Biotechnology and Sustainable Development

Chapter 2  

Emerging Scientific Trends

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The contributions of gene technology to today’s agriculture are already substantial. Discoveries in gene technology have led to:
·    Better Understanding of how plants function, and how they respond to the environment.
·    More targeted selection objectives in breeding programs to improve the performance and productivity of crops, trees, livestock and fish, and post harvest quality of food.
·    Use of molecular (DNA) markers for smarter breeding, by enabling early generation selection for key traits, thus reducing the need for extensive field selection.
·   Molecular tools for the characterization, conservation and use of genetic resources.
·   Powerful molecular diagnostics, to assist in the improved diagnosis and management of parasites, pests and pathogens.
·   Vaccines to protect livestock and fish against lethal diseases.

In crop agriculture, applications of gene technology are making major contributions to present day agriculture by the development of new (conventional) crop varieties, through the use of marker assisted selection. The other important application of gene technology in agriculture is in the development of novel, transgenic plant varieties. Here new genetic instructions are introduced into the crop by laboratory-based molecular methods, leading to new plant varieties that have been genetically modified for a specific trait.

It seems likely that in most countries the applications of gene technology to agriculture will be a two-stage process. Firstly, there are many applications of gene technology that can be used to improve the management and efficiency of present agricultural practices. These include the use of molecular markers in smarter breeding, new diagnostics and vaccines. Secondly, there are options for the targeted introduction of transgenic strains, genetically modified for one or more specific traits. Although transgenic strains of various species of crops, trees, livestock and fish have been developed experimentally, only transgenic crop varieties are in widespread commercial use in agriculture. 

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Commercial cultivation of transgenic crops

Broadly, the first wave of genetically modified crops, which are in commercial use, address production traits; the second wave, which are mainly under development, address quality and nutritional traits; and the third wave for the future address complex stress response traits and novel products able to be produced in plants. The scientific basis of dealing with each of these three groups of traits is increasingly complex.

The production traits targeted in the first wave of transgenic plant varieties specifically addressed the economic and environmental costs of chemical management in large-scale agriculture. An important factor in the initial choice of production traits was the fact that the major early private investors in plant biotechnology were several multinational chemical companies. The long-term viability of chemically based agriculture was being questioned as potentially:  

·   damaging to human health;
·   damaging to the environment, due to chemical damage to living organisms, and excess chemical run off into water courses;
·   less effective, due to the build-up of pesticide tolerance in target pests and diseases, thus shortening the economic life of agri-chemicals;  
·   less feasible due to the difficulty in discovering new agri-chemical compounds.

Biologically based management strategies were been sought, not only  to reduce chemical use in agriculture, but .also to find more powerful ways to increase sustainable productivity and improve quality. Strategies for integrated pest management and later integrated crop management were developed in some areas. These strategies aimed to reduce inputs of chemical pesticides, herbicides and fertilisers, maximise the effectiveness of natural enemies for pest control and make judicious use of host-plant resistance for disease and pest control.

In regard to the development of novel genetic approaches for specific production traits, a combination of new scientific possibilities, business opportunities, and decreasing viability of chemically-based agriculture led to the targeting of particular production traits (insect resistance and herbicide tolerance) and their subsequent commercial development into new transgenic crop varieties.

The first transgenic plant was produced experimentally in 1983. The first commercial cultivation was in 1995. By 2001, there were almost 53 million hectares of genetically modified crops growing in 13 countries (James, 2001). The most commercially important of these crops are soybean, corn, cotton and canola (oil seed rape). The traits these new plant varieties contain are mainly insect resistance (corn, cotton), herbicide resistance (corn, soybean), delayed fruit ripening (tomato) and virus resistance (papaya) (Figure 1.1, Tables 1.2, 1.3). Many other crops and traits are under investigation but most have yet to be taken through to practical use. 

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Trait Selection

Single gene production traits

The developers of the first generation of genetically modified (transgenic) crops faced a number of technical limitations that constrained the choice of crops and traits that have been taken through to full product development. These constraints included:

(1)   The availability of genes controlling traits that could be manipulated. Initially, only traits controlled by single genes could be manipulated; and single genes control only a limited number of traits.
(2)   The efficiency of the methods to produce genetically modified plants that express the desired trait consistently under field conditions;
(3)   The need to meet evolving regulatory requirements for new crop varieties (and other genetically modified organisms) containing genes from outside their normal range of hybridization.

Choice of single gene traits

Initially, only certain traits that were controlled by a single gene could be genetically manipulated for the development of transgenic crops. Thus, single genes that conveyed resistance to certain species of insects (Lepidoptora) by producing a toxin, were derived from a soil-borne bacterium (Bacillus thuringiensis) and transferred to several plant species. This discovery led ultimately to the commercial production of Bt cotton and Bt corn. Similarly, various single genes that conferred tolerance to selected herbicides were transferred to soybean, canola and corn. In some instances, these two traits have been combined, to produce insect and herbicide tolerant corn and cotton. These applications have led to reductions in the amount of pesticide being used for insect control in cotton and corn (Carpenter et al 2002; James 2001).

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Management of single gene traits

The careful targeting and correct management of single gene traits is critical for their successful use in agriculture. The management of single gene traits is important so as to avoid the boom/bust cycles typical of single gene resistance when used previously in agriculture.

For example, when single gene traits are being manipulated to enhance pest or disease resistance in a plant, there is a risk that the pest or pathogen will evolve so as to overcome this host resistance. This occurs with conventionally bred plant varieties, which are usually replaced after a number of years as the pest or pathogen evolves (eg wheat varieties with rust resistance). Yet, there are also instances where conventionally bred plant varieties with single-gene based disease resistance has been stable over many years, especially for bacterial and virus diseases.

In the deployment of new transgenic varieties of Bt-crops, such as cotton and corn, in broad scale agriculture, much effort has gone into devising and implementing specific crop management arrangements that mitigate against the evolution of resistance in the target pest. These crop cultivation regimes include leaving some of the field as non-transgenic, susceptible crops (refugia for the insects), which reduces the evolutionary pressure on the pest to evolve to overcome the pest resistance in the plant (Gould, 1996; Rousch, 1996).

Another strategy is to include in the plant two or more different genes for pest resistance (eg two different Bt genes). This gene stacking strategy makes it more difficult for the pest to evolve, as it has to overcome two resistance genes with different modes of action.

Dealing with complex traits

Most characteristics of food are controlled by more than one gene. Thus taste, aroma, color, nutritional composition and other aspects of food quality are the result of complex biochemical reactions within the plant before and after harvest. Crop yield is also a complex characteristic, with many genes involved in plant development, flowering, and yield components.

Crop responses to stress during cultivation are often controlled by many genes, which stimulate complex biochemical pathways within individual plant cells. For example, many genes control plant responses to fungal infections. Similarly, some plants have developed means to respond to environmental stresses such as drought by changing their metabolism to accommodate having less water available (eg sorghum).

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New targets

Emerging scientific developments are enabling complex traits to be addressed, with the intention of developing new products of potential value for agriculture, human health and the environment (Table 2.1). These include traits in the following categories:

Increasing sustainable agricultural production, by the cultivation of crops that are better able to tolerate biotic stresses (pests, diseases and weeds) and abiotic stresses (drought, salinity, and temperature stress).

Delivering health benefits through more nutritionally beneficial foods, with higher content of essential vitamins and minerals, especially in staple crops such as rice. Reducing allergenic, carcinogenic and/or toxic compounds in certain plants may also be possible, so that they are safer sources of food. (eg reduced cyanide content in cassava; removing allergenic content of nuts; modifying oil content of certain plants to produce more long chain, poly-unsaturated fatty acids).

Using plants for pharmaceutical production: Certain plants may provide a platform (bioreactor) for the more economical and efficient production of specific proteins, such as vaccines against human diseases, and other pharmaceuticals. This approach is being used successfully in microbes and transgenic animals that have been genetically engineered to produce certain high value pharmaceuticals (eg insulin in microbes; blood clotting factor in sheep). (Larrick and Thomas, 2001).

Using plants for production of products for industrial purposes: These compounds may include novel compounds such as biodegradable plastics and industrial strength fibers, as well as the more efficient production of common plant products such as starch and alcohol used for industrial purposes.

Environmental benefits: Using plants (and microbes) to mitigate the effects of industrial pollution (bioremediation), by increasing their ability to remove and/or break down toxic compounds in the soil. Recent demonstrations include transgenic plants changing mercury into a less toxic form (Bizily et al., 2000); plants with improved abilities of Cd accumulation (Cobbett, 2000); and plants degrading industrial waste and harmful substances (Hannink et al., 2001).

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From promises to reality

The attractiveness of the new targets amongst complex traits is tempered by the fact that they are technically difficult, requiring the expression and control of multiple genes, often involved in different biochemical pathways.

Recent scientific developments confer the ability to study the structure and function of all the genes within an organism simultaneously (through genomics), as well as the protein products they code for (through proteomics). It is also possible to study the role of all the chemical compounds in the metabolism of the cell (through metabolomics). These emerging scientific developments are being greatly assisted by powerful computing and statistical techniques that enable the assembly, interrogation and interpretation of large databases (through bioinformatics). New terms are being coined to describe these rapidly evolving branches of science and the techniques on which they are based (Boxes 2.1-2.3).

These new fields of science are using a series of sophisticated techniques to locate and characterize genes, proteins and other compounds, understand their functions and the means to manipulate them for new purposes. Most importantly, several methodologies have been developed that permit the study of genes, proteins and other metabolites collectively rather than individually (Box 2.4).

The emerging scientific possibilities also pose new challenges in the assessments of the risks and benefits of potential new products to human health and the environment. Some of these potential products are meant for food or feed use, while others are intended for use as pharmaceuticals, and others as compounds for industrial uses. Some will require inter-specific transfer and control of multiple genes. Others will rely on switching on (or off) and better regulating genes that are already present in the organism but not usually expressed.

New scientific developments also offer potential means to overcome some of the  risks involved in the cultivation of genetically modified crops. These include limiting the unintentional movement of genes out of the target crop (through gene containment), where such movement may pose a risk to biodiveristy or to the environment.  Better food safety assessments of any unintended changes in the composition of foods may be undertaken  by assessments of the content of whole foods (through metabolomics).

The challenge is how emerging scientific discoveries, such as those in the rapidly evolving fields of genomics, proteomics and metabolomics, amongst others, can be translated into safe applications of biotechnology that will lead to new varieties of crops, novel foods and new products that deliver benefits for society. These new applications and their risks and benefits will differ in different parts of the world. Careful thought needs to be given to identifying the most suitable targets and desirable traits for future research and development efforts, in different countries and environments.

Understanding Plant and Animal Genes

The past decade has seen dramatic advances in our understanding of how biological organisms function at the molecular level, as well as in our ability to analyze, understand, and manipulate DNA molecules, the biological material from which the genes in all organisms are made. The entire process has been accelerated by the Human Genome Project, which has invested substantial public and private resources into the development of new technologies, skills and equipment to work with human genes. The same technologies are directly applicable to all other organisms, including plants, animals, insects, and microbes. Thus, the new scientific discipline of genomics has arisen, which has contributed to powerful new approaches to identify the functions of genes and their applications in agriculture, medicine, and industry.

Genomics refers to means of determining the DNA sequence and identifying the location and function of all the genes contained in the genome of an organism. The advent of large-scale sequencing of entire genomes of organisms as diverse as bacteria, fungi, plants, and animals, is leading to the identification of the complete complement of genes found in many different organisms. This is dramatically enhancing the rate at which an understanding of the function of different genes is being achieved. This new knowledge is changing the ways of developing future improved strains of crops, livestock, fish, and tree species.

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Platform technologies

Rapid technical advances are occurring in three major areas: (1) DNA sequencing; (2) genome analysis, and (3) computational biology (bioinformatics). Firstly, developments in DNA sequencing have made the acquisition of whole genome sequences possible. These data, when interpreted with the assistance of bioinformatics, can provide a complete listing of all the genes present in an organism, (its genetic blueprint). The first genome sequence of a higher organism was published in 1996. More than 23 genome sequences are available, and some 60 or more genome sequencing projects of a wide variety of organisms, including plants, animals, parasites, and microbes, are under way (more details on structural genomic projects are available on The Institute for Genomic Research-TIGR- web site, http://www.tigr.org/).

Secondly, different types of technologies have been developed for genome analysis, which speed up the process. What puts this type of genome analysis into a different league is that, with the immense increase in the amount of DNA sequence data available, it is possible to scan whole genomes rapidly and to develop a systems approach for mapping genetic traits.

There are continuing improvements in molecular techniques so as to reduce their costs and increase their speed and efficiency in dealing with large numbers of genes. Particularly important are cDNA microarray techniques. These are used in functional genomics to identify how each gene responds to a specific environmental stress. Similar techniques are being developed for the study of proteins, through proteins arrays.

For example, in microarray analysis, when all the genes in a plant (about 25,000 in Arabidopsis thaliana) are placed on a glass slide and subjected to a sequence of environmental stresses, it is possible to determine the several genes that are most important in the organism’s overall response to stress. These genes will be those that react to all the stresses. This small number of genes can then be studied in more detail for their specific functions and potential use in the development of stress-tolerant plant varieties.

New developments in the molecular tools for gene, protein and metabolite studies have been reviewed recently by van Montagu and Burssens (2002). The molecular tools presently available and their uses are summarized in Box 2.4.

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Bioinformatics

It is possible to use the developments in bioinformatics to understand the complex genetic interactions involved in growth, development, and environmental responses. Developments in bioinformatics are allowing the prediction of gene function from gene sequence. Thus from genome sequences of DNA it is possible to build a theoretical framework of the biology of an organism (Flavell, 1999). This forms a powerful base for further experimentation. In addition, as the numbers of physical and genetic maps of different species increase, it becomes possible to compare these across different organisms ( through comparative genomics), be they microbes, plants or animals, and to significantly reduce the time required identifying important genes. Some of these genes are conserved (shared) between organisms. These technologies allow novel approaches to addressing biological problems. 

The results of the early genome mapping projects have shown that many genes are conserved (shared) amongst organisms as diverse as humans, animals, plants, fish and microbes.

Plant genome mapping (structural genomics)

Much research in plant sciences is done on model plants such as Arabidopsis thaliana, a small crucifer plant belonging to the Brassicaceae. Plant genomes vary greatly in size, ploidy, and chromosome number. Arabidopsis thaliana was chosen as the preferred model plant for genomic and related biological studies because of its small genome and short generation time. The knowledge acquired on model plants has spill over benefits to economically important crops in the development of novel traits.

The first plant genome that has been completely sequenced is Arabidopsis thaliana, as a result of the international Arabidopsis Genome Initiative, which commenced in 1996. The Arabidopsis genome sequencing shows both structural features of the genome and gives information about the function of several genes. The total genome holds about 25,500 genes. About half of these genes appear to be specific to plants.

The genomic sequencing of economically important crops is also being undertaken. The most advanced are the several sequencing projects on rice, to map the indica (Oryza sativa var indica) and japonica (Oryza sativa var japonica) rice types. Some of these projects are being undertaken by public consortia, led by scientists from China, Japan and the international rice genome sequencing project, for indica rice (Yu et al; 2002). Others are being undertaken by private companies, for japonica rice (Goff et al, 2002). Almost half the genes so far identified in rice are similar to genes that occur in Arabidopsis thaliana. A maize genome-sequencing project is also in progress. It appears likely that rice, maize and other cereals share a large number of common genes.

There are also many other genome sequencing projects, for over 100 plant species, based on the use of expressed sequence tags (ESTs). These species include soybean, oilseed rape, sugarcane, amongst others (see http://www.nature.com/genomics).

Functional genomics for trait discovery

The purpose of functional genomics is to understand the role that a particular gene plays in the life of a plant. Several techniques have been developed to assist in the identification of gene function. These include knock-out techniques (whereby an individual genes is disrupted and the resulting mutant phenotype compared with the wild type to identify any changes in phenotype). This technique is being used in rice to identify the function of all the genes in rice.

A completely sequenced plant genome such as rice, for example, will provide a pool of genetic markers and genes for rice improvement through marker-assisted selection or genetic transformation. To fully exploit the wealth of molecular data it is necessary to understand the specific biological functions encoded by DNA sequence through detailed genetic and phenotypic analyses. Thus unlike genome sequencing (structural genomics), functional genomics requires diversity of scientific expertise as well as genetic resources for evaluation. In many important food crops, national and international public sector research has a large investment in genetic resources and breeding materials, and a long history of understanding biological function and genotype x environment interactions. These scientific and biological resources will become increasingly important in gaining knowledge about the function of genes and in developing molecular markers to assist the breeding process (Fischer et al 2000).

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Proteomics

Most cellular functions are carried out by multi-protein complexes. New techniques are enabling these complexes to be unravelled, and the functions of individual proteins understood. Techniques for large-scale protein separation, combined with precise approaches that analyze, identify, and characterize the separated proteins, are enabling researchers to investigate cellular function at the protein level.

Proteomics has been enabled by the accumulation of both DNA and protein sequence databases, improvements in mass spectrometry, and the development of computer algorithms for database searching (van Montagu and Burssens 2002).

New techniques allow the identification and quantification of proteins expressed in a particular tissue or in a specific developmental or environmental condition, such as in response to stress. These techniques include 2-Dimensional -protein gel electrophoresis combined with mass spectrometry (Box 2.4). Recent improvements are making the techniques more sensitive so that they can detect small amounts of proteins (Mann 2001).

The recent development of protein arrays will be a powerful method to link genomics with proteomics. In this system, fully active proteins are spotted onto membranes to study protein interactions, protein-nucleic acid interactions or protein-ligand interactions (Ge, 2000).

Metabolomics

Besides the integration of data on protein function and activity, information on metabolite levels in the cell is critical to obtaining a holistic view on a biological process and its functional biocomplexity. Examining changes in metabolic profiles (through metabolomics), can be an important part of an integrative approach for assessing gene function and relationships of phenotypes (van Montagu and Burssens, 2002).

Modern high-resolution techniques allow the establishment of a profile of all the metabolites present in a specific plant tissue. By use of improved tools for analytical chemistry a variety of previously unidentified biochemical pathways can now be understood.

Metabolomics can provide information on metabolic network regulation in response to genetic and environmental perturbations, leading to a better understanding of plant responses to stress. Genes encoding the biosynthetic enzymes can also be more easily identified and consequently the production of secondary and intermediary metabolites in crop plants can be envisaged. The study of secondary metabolites is of particular interest to the pharmaceutical industry, since most drugs are based on plant derived products.

By exploring the three levels of genomic analysis (transcriptome, proteome and metabolome), extensive databases of quantitative information are being developed about the degree to which each gene responds to environmental stimuli. These stimuli may come from biotic and abiotic stresses such as pathogens, pests, drought, salt; from chemicals such as phytohormones, growth regulators, herbicides and pesticides; or from changes in developmental processes such as germination and flowering. These databases will provide insights into the set of genes that control complex responses and will create powerful opportunities to assign functional information to genes of otherwise unknown function (van Montagu and Burssens, 2002).

 

Improving Enabling Technologies for Gene Manipulation

The essential tools of gene technology are the techniques that enable one or more genes that control a particular trait to be transferred within or between species, switched on, and made to express themselves in the right place, at the right time and in the right amount. Thus several enabling technologies are required to facilitate the detection, transfer, and expression of genes (Box 2.5).

Initially, only a limited number of plant species and sometimes only a small number of strains within a species could be transformed and regenerated. Much current research is directed at improving the enabling techniques for gene technology, so that the techniques become faster, more precise, less expensive and more widely applicable to all plant species. For example, improved techniques are being developed to enable the introduction and simultaneous expression of multiple genes that control a particular trait. Other techniques being developed are concerned with new ways to manipulate the plant’s own genes, by switching them on and off, perhaps in response to particular environmental stimuli. 

Potential Applications from Emerging Scientific Developments

New approaches for disease resistance and stress tolerance  
Examples of the current approaches that are being used to develop new crop varieties with tolerance to plant diseases are described in Box 2.6.  New approaches to dealing with the complex traits associated with abiotic stress tolerance in the environment are summarised in Box 2.7  

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Metabolic engineering for producing specific compounds in plants

Metabolic engineering is the in vivo manipulation of biochemistry to produce non-protein products or to alter cellular properties (Elborough and Hanley, 2002). The products may be native to the plant or novel (expressed after the introduction of genes from another source). The non-protein products able to be produced by plants include:

·   alkaloids such as quinine,
 ·   lipids such as long-chain polyunsaturated fatty acids,
·   polyterpenes such as rubber,
·   structural components such as lignin,
·   osmoprotectants such as glycine betaine,
·   aroma compounds such as S-linalool in tomatoes,
·   pigments such as blue delphinidin in flowers,
·   vitamins such as folic acid,
·   biodegradable plastics such as polyhydroxyalkanoates.

Recent research shows that the following applications of metabolic engineering are technically possible in plants at the experimental level:  Increasing vitamin A content (Ye et al 2000); increasing essential oil production (Mahmoud and Croteau, 2001); decreasing lignin deposition (Abbott et al 2002); stimulating the bioconversion of secondary metabolites to medicinally important alkaloids (Van der Fits and Memelink, 2000); improving tomato flavor (Wang et al 2001) and producing biodegradable plastics in plants (Bohmert et al, 2000). Several of these products are now in development phase and are likely to be coming forward for regulatory approval over the next several years.

Meeting Evolving Regulatory Requirements

The revolutionary nature of the discoveries in gene technology has also raised concerns as to the safety of genetically modified foods for human consumption and the potential impact of genetically modified crops and other living modified organisms (LMOs) on the environment. These concerns arise largely because the first generation of genetically modified crops have been produced by the introduction into plants of genes from other phyla, with whom the plants would not normally cross in nature.

Regulatory/risk issues that are being addressed through new research developments include:
(1)   Limiting gene flow to related and wild species, by gene containment;
(2)   Control of trait expression and movement, to minimise impact on non-target species; for example, by limiting transgene expression to specific plant parts and times in the life cycle;
(3)   Removal of DNA from selectable markers and promoters, especially when these are derived from bacteria or viruses.

Selectable markers are used to demonstrate that a transformation event has occurred and to select these transformed individual plants in the laboratory. These selectable markers have often been genes for antibiotic resistance derived from bacteria. The use of antibiotic resistance markers especially has raised some concerns that these may contribute to the further development of antibiotic resistance in humans, by horizontal gene transfer of DNA from genetically modified foods and crops to humans and animal pathogens. The likelihood of this happening is remote. Nevertheless, new selectable markers such as those conveying a green fluorescent pigment are replacing these antibiotic resistant selectable makers. Another approach is to remove the selectable marker after the transformed plants have been selected in the laboratory but before they move into field testing and product development (Hare and Chau, 2002).

Emerging regulatory issues from new applications and novel products

There are also some additional regulatory issues emerging from the new applications of plant biotechnology. These issues include:
·   Ensuring that when plants are used to produce products for industrial uses, these products do not inadvertently enter the food chain; 
·   Inter-action with pharmaceutical regulations, where plants are used to produce vaccines and other medicinal products;
·   Identifying the extent of regulation required for nutritionally modified foods (nutriceuticals), to ensure that these foods meet their makers’ claims for improving nutritional quality, for example by the expression of nutritionally significant levels of essential vitamins and minerals;
·   Identifying where crops with modifications to genes normally present in the crop species may need to be regulated differently from those containing transgenes from other phyla;
·   Identifying where crops with complex traits regulated by several genes may have a reduced liklihood  of transferring these complex traits to related species and wild relatives, than transgenic crops carrying single gene traits, and any differences in regulation and safety assessments that may be required. 
·   Regulatory systems need to be sufficiently flexible to be able to respond to emerging scientific developments, both in terms of encouraging their use to address risks associated with current applications of gene technology, and in recognizing any new issues likely to arise when new scientific opportunities are applied to agriculture.

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Conclusion

Achieving any of the new applications of plant science will require substantial private and public investments, and a wide range of scientific and communication skills. The required scientific skills lie not only in gene technology, but also in the related fields of plant breeding, agronomy and physiology, food and nutrition and in natural resources management. There also needs to be greatly improved linkages amongst the social, scientific, industrial and environmental communities, so as to better define the ways in which science can benefit society and to design new technologies in ways that are socially and environmentally acceptable and beneficial in different countries and communities.

Further information and references

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