ANNEX III

Applications of Biotechnology Relevant to the CGIAR Mission
References

Applications of Biotechnology Relevant to the CGIAR Mission

1. Introduction

Some donors suggested that the Panel's report should not only analyze the issues for decision-makers, but should also be informative for the layman. This Annex has been prepared with the second of these requests in mind but makes no attempt to cover the whole spectrum of possible applications. It elaborates some of the statements made elsewhere in the report and provides a few specific examples of relevant biotechnological applications.

Some of those who responded to the Panel's questions on present and future contributions of biotechnology (see Annex II), did so primarily in relation to improved products (such as drought tolerant plants), rather than to new techniques that can be used in their development (such as marker assisted selection). The Panel has not attempted to forecast in detail what improved products are likely to emerge as a result of greater CGIAR investment in biotechnology. Rather, it has focused on the technology that will increasingly become available for accelerating the development of improved products, and on the steps that will be needed for such technology to become readily available for use by the Centres and the developing countries they serve.

2. Genomics, Synteny and Bioinformatics

Molecular biology is providing new insights into the structure of the genome at an exponential rate of growth. Such studies and the knowledge they generate are broadly referred to as "genomics".

The term "synteny" was originally applied to genes presumed, from the results of marker analysis, to occur on the same chromosome. More recently, however, the term has been used in the same sense as "collinearity of genetic maps" to mean the similarity in the order of genes that occurs along chromosome segments over a wide range of organisms.

The insights provided by genomics have been likened to the understanding of hieroglyphics provided by studies of the Rosetta stone. Thus, knowledge of the mouse genome, for example, provides valuable information on the human genome, while more closely related organisms, such as rice and wheat, show remarkable similarities in gene organisation, sequence and function. It follows that, because of genomic synteny, there is enormous potential for sharing and exchanging information across a wide range of organisations and individuals working on different organisms. The computerised accumulation and interpretation of this knowledge has acquired the new name of "bioinformatics".

3. The Relevance of Genomics

Just as classical genetics provides indirect knowledge of whether genes are linked or inherited independently, so genomics provides direct information on the sequence, function, activity, position and grouping of genes on the chromosome. The techniques of molecular biology and genetics that have evolved since the dawn of the recombinant DNA era have given rise to systematic, high throughput, whole-genome sequencing and associated activities, such as the development of molecular markers for detecting the presence of genes or groups of genes, directly or by genetic linkage.

The future will see substantial reductions in cost and increased speed of large-scale sequencing through automation, robotics and, eventually, microarray chip-based techniques. These offer the prospect of capturing vast amounts of genomic information on a single chip and using it to determine how the activity of each and every gene varies with the environment and genetic background.

Once the complete catalogue of all genes in a plant has been determined (year 2000) then it will be relatively simple to find the equivalent gene in any plant. Similarly, the sequence of the human genome will lead to relatively rapid identification of genes in the principal farm animals. Once a gene has been shown to play a role in controlling a trait in one species, it will be straightforward to investigate the equivalent trait in another species. The development of such activities is already accelerating and the Centres need to remain aware of the new opportunities that are constantly emerging.

4. Gene Transfer

Improvements in the technology required to design genes, insert them into plant cells and regenerate transformed, fertile plants, continue to be made, even for the more difficult crops such as wheat and maize. However, many of the orphan crops of critical importance to the CGIAR remain to be transformed efficiently and some general problems associated with the expression of inserted genes remain to be solved. Nevertheless, the recent commercial introduction of transgenic crops gives grounds for supposing that such problems will eventually be solved.

Although arguments have been raised against the release of transgenic crops, in many cases there is no reason why, ultimately, they should present a greater hazard to the environment than varieties produced as a result of classical hybridisation techniques. Perceived dangers, such as the use of antibiotic markers to select transformed cells, will be overcome by improved methods already in the pipeline.

The extent to which transgenic crops are now entering agriculture is illustrated by the following table extracted from ISAAA Briefs (1997), and by Annex IV.

Traits already Commercialised in Field Trials, and under Development for Selected Crops, 1997

Crop	Traits already commercialised	Traits in Field Trials/Development
Canola	1. Herbicide Tolerance	1. Improved disease resistance
	2. Hybrid technology	2. Other oil modifications
	3. Hybrid technology and herbicide tolerance
	4. High lauric acid
Corn	1. Control of Corn-Borer	1. Control of Asian Corn-Borer
	2. Herbicide tolerance	2. Control of Corn Rootwonn
	3. Insect protected/herbicide tolerance	3. Disease resistance
	4. Hybrid technology	4. Higher starch content
	5. Hybrid/herbicide tolerance	5. Modified starch content
		6. High lysine
		7. Improved protein
		8. Resistance to storage grain pests
		9. Apomixis
Cotton	1. Bollworm control with single genes	1. Bollworm control with multiple genes
	2. Herbicide resistance	2. Control of Boll Weevil
	3. Insect protected/herbicide tolerance	3. Improved fibre/staple quality
		4. Disease resistance
Potato	1. Resistance to Colorado Beetle	1. Resistance to Colorado Beetle + virus
		2. Multiple Virus resistance (PVX, PVY, PLRV)
		3. Fungal disease resistance
		4. Higher starch/solids
		5. Resistance to potato weevil/storage pests
Rice		1. Resistance to bacterial blight
		2. Resistance to rice-borers
		3. Fungal disease resistance
		4. Improved hybrid technology
		5. Resistance to storage pests
		6. Herbicide tolerance
Soybean	1. Herbicide tolerance	1. Modified oil
	2. High oleic acid	2. Insect resistance
		3. Virus resistance
Tomato	1. Delayed/Improved ripening	1. Virus resistance
		2. Insect resistance
		3. Disease resistance
		4. Quality/high solids
Vegetables & Fruits	1. Virus resistance	1. Insect resistance
		2. Delayed ripening

Source: Clive James, 1997, Global Status of Transgenic Crops, ISAAA Briefs 5

A few transgenic farm animals have been produced but it will be some time before this technology is suitable for widespread use.

5. Applications to Germplasm Improvement

Knowledge of the genome enables molecular markers to be developed. Breeders make progress through selection among varying genotypes often based, during the early stages of selection, primarily on phenotypic differences. At best, they can only estimate the genotypic variation, using the results of replicated progeny tests. However, molecular markers permit direct assessment of genotypic variation and they can be used to detect the presence of both single genes and polygenic complexes. This applies whether the genes are dominant or recessive. Moreover they can be used at the juvenile stage, such as seedlings, to detect genes that are not expressed until maturity. Provided the marker techniques are reliable and can be applied on a large enough scale, they provide valuable tools for the plant breeder and accelerate selection processes. They are also extremely helpful in characterising germplasm collections (see below).

Numerous examples of how the tools of biotechnology are being used to improve crop varieties, have been given above and in Annex IV. In addition the following have been selected simply to give a few examples of the types of approach that are now possible across a wide range of crops and problems.

(a) Recognition of Valuable Genes and Marker Assisted Selection

The rapid break-down of single gene resistance through changes in the pathogen is well known. Its consequences are loss of yield, which can be avoided only through the use fungicides that may not be accessible to the poor. In the past, some progress has been made towards achieving "durable" resistance by breeding two or more resistance genes into the same variety, through prolonged schemes of crossing and selection. However, biotechnology gives new impetus to such approaches. Insights into the genomics of both host and pathogen are providing greater understanding of host-pathogen interactions which, together with techniques such as marker assisted selection, are providing new strategies for building multigenic resistance into crop plants.

For example, recent work on rice blast disease by CIAT scientists and their collaborators has focused on genes resistant to "lineages" of pathotypes rather than on those resistant to specific isolates, as revealed by traditional pathotype analysis. Biotechnology has provided ways of defining the genetic organization and distribution of diversity in the pathogen and has facilitated the incorporation of appropriate resistance genes into breeding lines. Such a strategy for attacking a formerly intractable problem could have wide application to other crops and diseases.

(b) Production of Transgenics

Although rice is the world's most important food crop, it is not a good source of vitamins or iron. Vitamin A deficiency is a serious problem in many rice dependent poor populations, with children who are weaned on rice gruel at particularly high risk. The carotenoid biosynthetic pathway is now well understood and genes for all steps have been cloned from plants and microbes. It is now possible to manipulate the pathway in rice through genetic engineering with the objective of increasing the beta carotene content of the grain. Similar approaches could be applied to other crops in which the harvested product is deficient in vitamin A.

Another example of nutritional improvement relates to the iron. Several different strategies are being pursued for increasing the bio-available iron in rice. The most advanced involves adding the gene for a protein such as ferritin which accumulates iron and makes it available. It has been shown that rice seeds transformed with a ferritin gene from soybean store up to three times more iron than normal seeds. Other strategies focus on phytate, a derivative of phytic acid, which binds iron in a non bio-available form. It may well be possible to reduce the phytate content in the grain or to introduce the gene for phytase which breaks down the phytate and releases iron in an available form.

(c) Rhizomania-resistant Sugarbeet

Although sugar beet does not feature strongly in the agriculture of developing countries, recent work on Rhizomania provides a good example of the application of molecular techniques to breeding for resistance to a disease in which expression is strongly influenced by the environment.

Rhizomania (root madness or bearded root) is a destructive disease of sugar beet (Beta vulgaris L.) caused by the beet necrotic yellow vein virus whose vector is a soil borne fungus. Rhizomania is widespread in the temperate regions of Europe and Asia and occurs in USA. The disease causes losses as high as 80% in some parts of Europe and reductions of 20-50% in sugar yield and juice purity are common in infested fields. Viruliferous resting spores of the vector have been reported to survive in uncultivated fields for as long as 15 years. No chemical control of the virus is possible.

Breeding for resistance to Rhizomania began in the eighties when a single source of tolerance to the virus was discovered. Tolerance was weakly expressed and the trait was found to be inherited through many genes. The level of tolerance was difficult to maintain when introducing it into the then elite lines. Early recognition of the environmental hazards of using fungicides and the then intractable nature of breeding for high levels of tolerance led Kleinwanzlebener Saatzucht (KWS) to mount a two-pronged effort, one through conventional breeding methods and the other through molecular approaches, to search for a means to introduce Rhizomania resistance into sugarbeet. Both methods have since yielded results.

New sources with higher levels of tolerance found later were used successfully to introduce, through backcrossing, Rhizomania tolerance into elite varieties. These varieties suffer no significant damage when the crop is attacked by the virus. However, they do not inhibit virus replication. With the development of molecular techniques, investigations showed that plants expressing viral coat protein genes interfere with the multiplication of this virus. A viral gene that synthesizes the beet necrotic yellow vein virus coat protein and inhibits its replication has been successfully incorporated into sugarbeet. Transgenic sugarbeet now offers cross protection against Rhizomania. These transformants compare well with the results of classically bred sugarbeet hybrids for plant growth and yield. Early tests have shown that hybrids that combine transgenic resistance and classical tolerance perform better than current classically bred Rhizomania-resistant varieties under heavy infection.

The basis for Rhizomania-resistance breeding at KWS was and continues to be the identification of genetic sources of high level resistance that can be inherited easily, an efficient selection for resistance which includes both screening methods and marker-aided selection, and short breeding cycles. Through the use of various kinds of molecular markers closely linked to the Rhizomania resistance genes, screening for the presence of the gene and fingerprinting parental genotypes is achieved. Conventional breeding techniques combined with biotechnological ones offer an efficient means to produce Rhizomania-resistant varieties that have high yield and sugar content.

Biosafety aspects related to the release of transgenic sugarbeet have been investigated by independent agencies. No negative effects have been reported.

6. Applications to Germplasm Banks

Genomic knowledge is fundamental to the classification and utilisation of germplasm collections. Without such knowledge, germplasm banks have been likened to "stamp collections" which spend most of their time in unopened albums. Over the next few years, there will be major technical advances that will facilitate the automated screening of chromosomal segments in large numbers of samples.

Molecular genetics also opens up entirely new avenues for the transfer of desirable genes from germplasm accessions to advanced cultivars, through transformation and marker assisted selection. Recent work has shown that, as well as providing genes for resistance to pests and diseases, primitive ancestors of crop plants also contain genes, not present in advanced cultivars, that can enhance yield and quality. These can be recognised through molecular assays.

In this general context, IPGRI comments on the rapid technical advances in gene transfer, in-vitro conservation and diversity analysis. Key technologies include cryopreservation and artificial seed development, as well as a range of molecular genetic methods. Further advances in sequencing technologies and in chip-based analyses are likely to be of considerable significance in the analysis of diversity and in determining useful traits. Other important applications relate to the rationalisation of germplasm management, such as the identification of duplicate samples within and between collections, as well as the development of core collections.

7. Applications to Agroforestry

The following outline is based on information supplied by ICRAF.

An important aspect of agroforestry is the domestication of tree species, in contrast to the unsustainable exploitation that has typified their use in the past. Some 2,500 tree species are known to be used in agroforestry systems in one way or another, making it essential to focus on a few carefully chosen species. The large number of species involved is only one of several ways in which the problems of agroforestry differ from those of crop productivity.

ICRAF does not see biotechnology as being amongst its top priorities in the immediate future but recognises the potential for biotechnology to contribute to certain specific problems.

For example, most trees are outbreeding and heterozygous and also have a prolonged generation time. The most useful techniques in the foreseeable future are likely to be molecular markers for selection at the juvenile stage, methods for the large-scale multiplication of selected trees, such as by cloning or apomixis, and techniques for inducing flowering to shorten the generation cycle.

8. Applications to Livestock Improvement

ILRI points out that, in considering the future exploitation of biotechnology for livestock research and development, it is important to note the major differences between livestock and plants. Many aspects of biotechnology that are relevant to crop improvement also apply to livestock improvement. These include genome analysis, molecular genetics and marker assisted selection. These techniques are even more beneficial in livestock improvement, however, because the generation time for livestock is generally longer than in crops, the number of offspring per generation is relatively small and the consequent cost of breeding programmes is high.

Other important applications to livestock improvement include a wide range of diagnostic techniques, vaccines and new drug technologies, as well as transfection/transgenics for livestock, forages and rumen micro-organisms.

9. Applications to Aquatic Organisms

The following outline of applications of biotechnology to aquatic organisms has been condensed from information provided by ICLARM.

ICLARM draws attention to the growing importance of molecular markers for biodiversity research, genome mapping and trait selection in fish and other aquatic organisms. International groups are already collaborating on genetic maps of tilapia and common carp (of most direct relevance to ICLARM) as well as maps for salmonids, cattish, zebrafish and pufferfish. Maps for commercially important invertebrate species including shrimp and oysters are being initiated.

ICLARM is not currently working on transgenesis, but several other institutes and companies in the fisheries and aquaculture sectors are conducting such research on various species including tilapia. It is anticipated that there will be an increase in the number of species and strains into which genes are introgressed, and the number of gene constructs available for transgenesis (governing biological functions in addition to growth) will also be increased. Transgenesis may become a cost effective means of enhancing indigenous species important to one or a few countries and not covered by international breeding efforts.

Sex manipulation (e.g. the production of all male populations of fish, especially tilapia) is also an active area of research, designed to avoid the detrimental production effects of early maturation and cessation of growth. In carp species, however, all-female populations are required and are being requested by developing countries. It is also anticipated that sex reversal will be used more widely in breeding programmes to increase the speed of production of inbred lines. Haploid fish will be important for similar reasons.

A wide range of diagnostic techniques is being developed for applications such as disease diagnosis, sexing of juvenile fish and for assessing progeny relationships in large populations of fish raised together to reduce environment-specific variations in production. Other techniques include tissue culture, or other manipulations of embryos or embryonic cells, for the isolation of viruses, bacteria and fungi pathogenic to fish.

10. Additional Information

To supplement the information in this Annex, the reader might wish to refer to some of the earlier reports and publications that cover relevant biotechnology in the context of developing countries. Among these, a paper by Gary Toenniessen (1995) and two reports edited by Gabrielle Persley (1990) are particularly informative in the context of this report. Other reports are mentioned in the introduction to the main text.

References

Persley, G. J. (ed.) (1990a). Agricultural Biotechnology: Opportunities for International Development. CAB International, Wallingford, UK.

Persley, G. J. (1990b). Beyond Mendel's Garden: Biotechnology in the Service of World Agriculture. CAB International, Wallingford, UK.

Toenniessen, G. H. (1995). Plant Biotechnology and Developing Countries. TIBTECH 13, James, C. (1997). Global Status of Transgenic Crops in 1997. ISAAA Briefs 5.