3. Analysis of the FAO-BioDeC data on non-GM biotechnologies

A number of non-GM biotechnologies are currently being used in developing countries in the agricultural sector. These techniques have been grouped in four main clusters: microbial, cell biology, molecular marker and diagnostic, each of them divided into subgroupings for specific techniques. Subgroupings under “other” have been included to cover unspecified activities under each cluster. The information gathered is analysed by region, species and technique, and summary tables have been included to facilitate the analysis. In the tables, research on one trait or technique in one crop in one country is counted as one initiative.

3.1 MICROBIAL PRODUCTS FOR AGRICULTURE

Microbiological techniques are central to the production, harvesting and use of many agricultural products. The use of microbial techniques includes the design or delivery of microorganisms for the control of pests (biocontrol agents or biopesticides), as fertilizers (biofertilizers) and for fermentation and food processing techniques. From data in the FAO-BioDeC (Table 1), it seems that research towards the scientific development of biocontrol agents (biopesticides), biofertilizers and fermentation and food processing microbial techniques, is at the early stages in Africa and Asia, (with the exception of China and India), while Latin American countries are already using advanced techniques on a more routine basis and some of the results obtained are already being tested. It should be borne in mind that many production and post-production processes already in use by farmers have a microbiological basis and that the extent of use of microbiological techniques in agriculture is grossly under reported by focusing only on the use of microbiological techniques which emanated from `modern' laboratories, rather than also on the use of microbiological techniques developed as indigenous or local knowledge of farmers and consumers.

3.1.1 Microbial agents for the biocontrol of pests

Although considerable use of classical pesticides persists, in some countries there is a trend towards using newer pesticides that are more selective, less toxic to humans and the environment, and require less application per hectare to be effective. A small but growing percentage of these are biopesticides, including microbial pesticides like Bacillus thuringiensis (Bt), and biocontrol agents such as pheromones, growth regulators and hormones. In addition, there is growing acceptance of use of such alternative pest control agents in various forms of Integrated Pest Management (IPM). Biocontrol agents, or biopesticides, range from the classic Bacillus thuringiensis (Bt) to Trichoderma, Verticillium, Bauveria, Bacillus subtilis to plant extracts, entomophagic nematodes or entomopathogenic viruses, such as nucleopolyhedrosis virus (NPV).

TABLE 1
Number of initiatives to develop and use microbial techniques

C: technology used on a routine basis and products available on the market; T: results being tested; E: number of activities at experimental level (including laboratory or glasshouse activities); U: activities in unknown phase; N: number of countries involved. (Totals of this column have been calculated taking into account that sometimes more than one activity is being carried out by the same country, although that country will only be counted once).

Table 1 shows that reported research on biological control agents is only at the laboratory phase in Africa, with emphasis on application to cowpea in Ghana and Kenya, sorghum in Ethiopia, Kenya and Zimbabwe, banana in South Africa, Uganda and Zimbabwe, cassava in Malawi and sugar cane in South Africa. In Eastern Europe, both Georgia and the Republic of Moldova have trials underway on use of Trichoderma spp as a biocontrol agent. Research on Metarhizium anisopliae, Entomophora spp. is undertaken in Georgia and on pathogenic bacteria, fungi and viruses in the Republic of Moldova . In Asia, use of Steinernema thailandensis is established and laboratory research is underway on Bt and NPV for use as biopesticides in Thailand. In Latin America, Guatemala is testing botanical extracts and Bacillus subtilis on melons and tomato, B. subtilis on pea, entomopathogenic fungi on sugar cane and pasture, and Bt/NPV on ornamental, horticultural and other crops. Chile is researching biocontrol of scab in apple. In the Near East, Egypt and Morocco are researching biocontrol of Fusarium in date palm.

3.1.2 Biofertilizers

Biological nitrogen fixation (BNF) refers to the process of microorganisms fixing atmospheric nitrogen, mostly within subsoil plant nodules, and making it available for assimilation by plants. Nitrogen availability is a key limiting factor in crop production. Rhizobia are the most studied and important genera of nitrogen fixing bacteria, but also a number of endophytic bacteria are now known as nitrogen fixing organisms. Use of biofertilizers, such as Rhizobium or other symbiotic and non-symbiotic species for atmospheric nitrogen fixation represents a more environmentally-friendly alternative to chemically generated fertilizers. Other microorganisms, such as Mycorrhiza, are active in establishing symbiosis with cultivated plants and forest trees, and facilitate phosphorus uptake. Inoculation with these fungi has proven to be an efficient way to substitute or complement phosphorus-based chemical fertilization.

The only reported research on biofertilizers in the Near East is research into Rhizobium in food legumes in Morocco. In Latin America, techniques for increasing nitrogen fixation in soybean, bean and cowpea are being tested in Venezuela, and researched in rice in Brazil, where arbuscular mycorrhiza are also being tested in native trees. Argentina has started nitrogen-fixation research on unspecified microorganisms. In Asia, India is researching Rhizobium with high capacity for nitrogen fixation, and China is studying Rhizobium in rice and maize. Indonesia is investigating vascular arbuscular mycorrhiza, the Philippines mycorrhiza and Rhizobium in forest species, and Viet Nam is experimenting with Rhizobium strains for the Mekong delta soils.

In Africa, research into biofertilizers for sorghum is ongoing in Ethiopia, Kenya and Zimbabwe, for cowpea in Cameroon, for groundnut and bambara groundnut in Madagascar, and for rice in Rwanda, with unspecified work in Burkina Faso, Cote d'Ivoire, the Democratic Republic of the Congo, Kenya, Rwanda and Senegal. The UNESCO Microbiological Resources Centre (MIRCEN) project at the University of Nairobi in Kenya has, since 1981, developed a Rhizobium inoculant known as BIOFIX, currently the main inoculant available on the local market. In Eastern Europe, Armenia and the Republic of Moldova are testing Azotobacter and Rhizobium.

3.1.3 Fermentation technology and food processing

Biotechnology in the food processing sector targets the selection and improvement of microorganisms with the objectives of improving process control, yields and efficiency as well as the quality, safety and consistency of bioprocessed products (FAO, 2004a). These microorganisms are used in fermentation; the process of bioconversion of organic substances by microorganisms and/or enzymes (complex proteins) of microbial, plant or animal origin. Fermentation is one of the oldest forms of food processing which is applied globally. In developing countries, fermented foods are produced primarily at the household and village level, where they find wide consumer acceptance. Food fermentations contribute substantially to food safety and food security, particularly in the rural areas of many developing countries.

The extent of application of biotechnologies such as fermentation technology and various food processing techniques is immense and difficult to monitor using a database such as the FAO-BioDeC. Nevertheless, it indicates that fermentation technology and food processing is quite commercialized in Latin America for sorghum in Brazil, where food enzymes are also being tested in several species of grains and vegetables, and researched in sugar cane. In Eastern Europe, Lactobacillus, Acetobacter and Saccharomyces are reported as being used commercially in Armenia, Georgia and the Republic of Moldova , where Bacillus subtilis, is also being used. Actinomycetes are being tested in Azerbaijan, and Bacillus vulgaris researched in Armenia. In Africa, research phase initiatives are underway in the fermentation of banana in Kenya and Zimbabwe, cowpea in Cameroon, cassava in Nigeria, with unspecified research in Burkina Faso. In Asia, Indonesia is researching lactic acid bacteria, dextranase, xanthan gum production and soybean fermentation. China and Indonesia are reported to be working on the use of the phytase enzyme in improving animal feed.

3.1.4 Environmental biotechnology

Environmental biotechnology can be defined as the development, use and regulation of biological systems for remediation of contaminated environments (land, air, water), and for environment-friendly processes (green manufacturing technologies and sustainable development). The FAO-BioDeC reveals few environmental biotechnology initiatives of relevance to food and agriculture underway in the countries surveyed. However, it is reported that microbiologial techniques for environmental management are being investigated in Indonesia, for biological waste and water treatment and bioconversion of solasodine, and in Thailand for biodegradable plastics and waste treatments. In Eastern Europe, Azerbaijan is testing Basidiomycetes, Deyteromycetes, and Fungi imperfecti and Georgia is researching Mycobacteria, Halobacterium halovium, and Aspergillus terreus. In Latin America, Chile is testing Thermomyces lanuginosus for cellulase production, and bacterial biofilm for bioremediation.

3.2 APPLIED PLANT CELL BIOLOGY TECHNIQUES

3.2.1 Micropropagation

Micropropagation is the use of tissue culture methods to propagate plants. Using micropropagation, millions of new (clonal) plants can potentially be derived from a single plant. Micropropagation encompasses a range of tissue culture techniques for propagation of plant species. In essence, tissue from a plant (explant) is isolated to create a sterile culture of that species in vitro. Once a culture is stabilized and growing well in vitro, multiplication of the tissue or regeneration of entire plants can be carried out. Shoots (tips, nodes or internodes) and leaf pieces are commonly used but cultures can be generated from many different tissues. Juvenile tissues generally respond best. This method of cultivation of plant material is used for:

•  rapid, large-scale, year round production of desired horticultural varieties (e.g. many of the popular miniature rose varieties are produced this way);
• propagation of plant species that are difficult to grow from seed;
• production of genetically uniform plant material (“clones”);
• development of plant culture systems that can be used for genetic transformation, e.g. to introduce disease resistance; and
• production of disease-free plant material (e.g. potato microtubers).

The benefits of plant tissue culture propagation include potentially unlimited multiplication of superior plant lines or elite individuals, avoidance of contamination with pathogens, production of true-to-type multiplication material of desirable plant lines suited for indefinite storage through cryopreservation, or for long-term maintenance of propagule inventories. In comparison to other plant propagation techniques, the major limitation of the application of this technology is the need for technically skilled labourers and some essential equipment. It is therefore commercially applied to high value added crops, which are worth the necessary investment.

Micropropagation is now a `mature' plant biotechnology and is among the most widely used plant biotechnologies, reportedly being applied in 21 countries in Africa, ten in Asia, nine in Eastern Europe, nine in Latin America and eight in the Near East (Tables 2 and 3).

The success of micropropagation may be explained by its relatively low costs and generally positive effects on productivity (especially of clonally propagated root and tuber crops). Micropropagation has become an irreplaceable tool for many clonally propagated species for the production of pathogen-free plantlets (among such clonally propagated crops are 10 of the 30 most cultivated crops worldwide). Emphasis on the development of cost-effective micropropagation techniques is expected to increase even further.

TABLE 2
Number of tissue culture initiatives

C: technology used on a routine basis and products available on the market; T: results being tested; E: number of activities at experimental level (including laboratory or glasshouse activities); U: activities in unknown phase; N: number of countries involved. (Totals of this column have been calculated taking into account that sometimes more than one activity is being carried out by the same country, although that country will only be counted once).

The FAO-BioDeC indicated that micropropagation is routinely being used, with many commercial products available on the market. In Latin America, micropropagation is used for blueberry, raspberry, sugar cane and potato in Argentina, and sugar cane, blackberry, strawberry, apple, potato, banana, cassava, pineapple, violet, fruits, cereals and ornamentals in Brazil. In Asia, Bangladesh has micropropagated plants of banana, orchid, chrysanthemum, potato, jackfruit, pineapple and unspecified timber trees; Nepal has micropropagated potato, orchids and Miscanthus spp. In Eastern Europe, Yugoslavia has micropropagated potato, while in the Near East, date palm is being commercially propagated in the Islamic Republic of Iran, Kuwait, Morocco, Tunisia and the United Arab Emirates. Tunisia also has available micropropagated Prunus rootstocks, almond, citrus, grape, olive and pistachio. The Islamic Republic of Iran, Jordan and the Syrian Arab Republic have potatoes, and in addition banana is available in the Islamic Republic of Iran. In Africa, micropropagated banana is promoted in Cameroon, Gabon, Kenya and Uganda. In addition, plantain and cassava are promoted in Gabon, and yam, potato and sweet potato in Uganda.

A wide range of micropropagated ornamentals, trees and food crops, with emphasis on root and fruit crops, are being tested for commercial production, or are in the preliminary research phase in all regions.

3.2.2 Anther and pollen culture

Anther culture involves the aseptic culture of immature anthers to generate fertile haploid plants from microspores. The production of haploid plants through anther culture is widely used for breeding purposes, as an alternative to the numerous cycles of inbreeding or backcrossing usually needed to obtain pure lines in conventional breeding. The success achieved with anther culture has led to the development of microspore culture. This involves the isolation of the microspores from the anthers, culturing them in specialized media and subsequent regeneration of fertile homozygous plants. Furthermore, isolated microspores are very attractive for protoplast isolation and applications aiming at transformation as they are unicellular and transgenic homozygous plants could be provided in a comparatively short time.

In vitro anther culture is now routinely used for improving some vegetable crops such as asparagus, sweet pepper, eggplant, watermelon and Brassica vegetables. In addition, anther culture is being increasingly used in cereal crop improvement both as a source of haploids and new genetic variation. Isolated microspore culture has been successfully carried out with some Brassica vegetables such as cabbage, broccoli and Chinese cabbage-petsai and pakchoi. Most genotypes respond better to isolated microspore culture and embryo yield is generally higher than with anther culture. Therefore, isolated microspore culture has been preferred as a breeding tool and as an experimental system for various genetic manipulations. A Chinese cabbage breeding programme using this technique is underway in the Beijing Vegetable Research Center.

In Asia, three countries are reportedly using anther culture (Table 2). In China, for example anther culture is being applied in different species such as wheat, rice, maize, rubber, hot pepper, poplar and sugar beet. Bangladesh is conducting trials on anther culture derived rice with salt tolerance and anther culture research on mulberry, maize, jute barley and tea, and Nepal has research on rice and wheat. In the Near East, anther culture research emphasis is on cereals. An anther culture generated durum wheat variety has reached the commercial phase in Tunisia, and anther culture techniques are being researched in Morocco, where an anther cultured bread wheat is in commercial use. Anther culture techniques are being tested in the Sudan, and applied to barley in the Islamic Republic of Iran, Iraq, Morocco and the Syrian Arab Republic. Wheat is also being researched regarding anther culture in Eastern Europe in the former Yugoslav Republic of Macedonia and Serbia and Montenegro.

TABLE 3 
Some examples of countries using plant propagation techniques for different crops at the commercial level 

In Latin America, varieties obtained through anther culture of unspecified fruits, cereals and ornamentals have reached the commercial stage in Brazil. Advanced testing of anther culture derived varieties of `many plants' is reported in Costa Rica, and in Venezuela cocoa, rice, sugar cane, papaya, mango and bamboo are also being tested. Anther culture derived rice varieties are under advanced testing also in Argentina, and in Brazil, where this technique is specifically used in breeding for blast resistance. In Chile, wheat, barley and rice, are in the research phase. Other anther culture research is carried out on maize and onion in Argentina, wheat in Ecuador and asparagus in Peru. In Africa, anther culture research has started on banana in Cameroon and on sorghum and rice in Mali.

3.2.3 Embryo rescue

In embryo rescue/culture, the embryo is removed before seed abortion occurs and is grown outside the parent plant to produce a new plant to enable crosses to be made between species which would not normally be sexually compatible. In vitro embryo rescue techniques are therefore often used to rescue plant embryos from aborting progeny seeds that result when two distantly related plants (e.g. two species) are crossed together. Such `wide crosses' are often desirable to transfer genetic traits from the secondary and tertiary genepools (i.e. crop wild relatives) to the cultivated primary genepools of crop plants. This technique is used in breeding of many crop species and allowed synthesis of triticale, a new hybrid species resulting from the cross between rye and wheat.

In the Near East, embryo rescue techniques are being developed in Algeria, Morocco and the Sudan. In Eastern Europe, embryo culture research is underway on sweet and sour cherry in the former Yugoslav Republic of Macedonia, sunflower in Yugoslavia, and maize in the Republic of Moldova. Only in Latin America has extensive use been made of the embryo culture technique, where it has given rise to commercially viable new varieties in banana, citrus, papaya, fruits, cereals and ornamentals in Brazil. New embryo rescue-derived varieties are tested in maize, Celtis tala, Erthytrina crista galli, Acacia caven and Elymus spp. in Argentina, in grape in Chile, in orchids in Costa Rica and in rice, sugar cane, papaya, Solanum nigrum, Yantén, Spathiphyllum sp., Aster sp., and gloxinia in Venezuela. Research is at an early stage in Africa, on yam in Nigeria and unspecified work is being carried out in Cameroon, and also in Asia, where the only embryo culture report is of work on rice and orchid in Bangladesh.

3.2.4 In vitro regeneration and somaclonal variation

Plant regeneration from cell cultures is central to the application of gene transfer techniques such as biolistics and Agrobacterium-mediated transformation. Not all plants are readily amenable to in vitro regeneration and there is thus a need to continuously develop regeneration protocols for the recalcitrant species if they are to benefit from genetic modification technologies. In vitro regeneration usually results in high genetic and phenotypic variability in individuals derived from cultures, which is called somatic variation. Somatic variation can be beneficial in crop improvement especially on traits for which somaclonal mutants can be enriched during in vitro culture, including resistance to disease pathotoxins, herbicides and tolerance to environmental or chemical stress, as well as for increased production of secondary metabolites.

Advanced in vitro regeneration work (protocol testing) is reported in Acacia caven, Erythrina crista galli, potato, sweet potato, cassava and garlic in Argentina, in blueberry in Chile, in cassava, aroids, Musa sp., Coffea sp., banana and plantains in Costa Rica, in Ullucus tuberosus, Oxalis tuberosa, Tropaeolum tuberosum, Arracacia xanthorrhiza, Mirabilis expansa, Canna edulis, and manihot in Ecuador and in tropical fruits, roots and tubers, cassava, potato, Musa, Solanum nigrum, Yantén, Spathiphyllum sp., Aster sp., and gloxinia in Venezuela. Research is in the laboratory stage (protocol development) on Parkinsonia aculeate, Elymus sp, and Ilex paraguariensis in Argentina, red clover and garlic in Chile, and on Mycosphaerella fijiensis, Rosellinia spp. and selected plant species in Costa Rica. In Asia, the only reported initiative is research on somaclonal variation in mulberry in Bangladesh.

3.2.5 In vitro germplasm conservation and cryopreservation

Biotechnology can play an important role in the conservation of germplasm of many crop species, whether the germplasm is maintained as vegetative or seed propagules. In vitro conservation protects germplasm from possible contamination with pathogenic agents and preserves the genetic identity of the stored material. Germplasm regeneration techniques (in vitro or in vivo) coupled to cryopreservation protocols ensure the long-term, safe storage of much of the world's germplasm, even if this technique is well established only for a number of plant species. The FAO-BioDeC include information on the use of in vitro conservation and cryopreservation. In this regard, the only work reported in Asia is experiments in Viet Nam on sugar cane and in Bangladesh on orchid, bamboo and hybrid Acacia. Similarly in Africa, reported work is only in the experimental phase, concentrating on root crops, cassava in Cameroon, Ghana and Nigeria, yams in Cameroon, Malawi and Nigeria, sweet potato and potato in Kenya, also on banana in Ghana, Malawi, Nigeria and Uganda, with unspecified activities in Cote d'Ivoire. In Latin America, use in cassava and violet is reported in Brazil, where experiments are also underway on sugar cane. Chile has developed cryopreservation of Fragaria chiloensis at the laboratory stage. In the Near East, only Egypt has any activity, in the laboratory, on potato conservation. In Eastern Europe, the former Yugoslav Republic of Macedonia is conducting trials on techniques for in vitro conservation of grape, apple, strawberry and cherry, and Yugoslavia for sunflower and potato. Experimental work is ongoing for potato in Albania and Bosnia and Herzegovina, also on Populus, Genthiana sp., and 30 wild species in Bosnia and Herzegovina and on Salvia hydrangea, Bryonia alba, Atropa belladonna, Rubia tinctorum, Hypericum perforatum, and Melissa officinalis in Armenia and grapevine in Azerbaijan.

3.3 MOLECULAR MARKER TECHNIQUES

Molecular marker techniques (employing RFLP, RAPD, microsatellites, AFLP, SNP and other kinds of marker systems) represent a rapidly evolving suite of powerful research tools for the characterization and management of genetic polymorphism (variation and diversity) in plant breeding and germplasm characterization programmes. Where they are available and cost-effective, molecular markers can have a wide number of applications in plant breeding, the most commonly considered being marker-assisted selection (MAS). The FAO-BioDeC suggests that the earliest generation forms of DNA-based molecular markers (RAPD, RFLP) are more widely used than the more recently developed types of molecular markers (microsatellite and AFLP markers).

MAS is based on the identification and use of markers which are linked to the gene(s) controlling the trait of interest (FAO, 2003). By virtue of that linkage, selection may be applied to the marker itself. The advantage consists in the opportunity of speeding up the application of the selection procedure. For instance, a character which is expressed only at the mature-plant stage, may be selected at the plantlet stage if selection is applied to a molecular marker. Also, selection may be applied simultaneously to more than one character, and selection for a resistance gene can be carried out without needing to expose the plant to the pest, pathogen or deleterious agent. Finally, when there is linkage between a molecular marker and a quantitative trait locus (QTL), selection may become more efficient and rapid. The construction of detailed genetic and molecular maps of the genome of the species of interest is a prerequisite for most forms of MAS. However, the current cost of the application of these techniques is significant, and the choice of one technique rather than others may be dictated by cost factors. There are still very few examples of crop varieties in farmers' fields which have been developed based on MAS, largely because of the currently prohibitive cost of incorporating large-scale MAS into the budgets of most plant breeding programmes.

In addition to MAS, molecular markers can be used in germplasm characterization. Compared to morphological and protein markers, DNA-based genetic markers are often considered to be the most useful for genetic diversity studies because they are highly polymorphic and heritable (their expression is not affected by environmental variability). The FAO-BioDeC indicates that molecular markers are being extensively used in Latin America (Table 4) with 93 trials and 165 molecular marker projects at the research phase in nine countries. The assessment of the genetic diversity of Andean local roots and tubers in Latin America using molecular markers is an interesting development. Species reported to be included in molecular marker programmes in these countries are sugar cane, rice, cocoa, banana, bean and maize. The survey also indicates that most countries in Asia are undertaking a wide spectrum of crop research using molecular markers. Molecular marker-related research activities in Africa are reported to be underway in only a few of the countries, such as Ethiopia, Nigeria, South Africa and Zimbabwe; the range of Africa crops under study with molecular markers, however, is very wide: from traditional commodities to tropical fruits.

3.4 DNA AND IMMUNO-DIAGNOSTIC TECHNIQUES

Many crop diseases are difficult to diagnose, especially at the earliest stages of infection by the pathogen. In particular, many diseases caused by viruses can exhibit similar symptoms and therefore it is difficult to identify the causative virus. Knowledge of the nature of the pathogen can be used to develop and apply proper management practices. For instance, some viruses are seed transmitted

TABLE 4
Number of research initiatives to develop and use molecular markers

C: technology used on a routine basis and products available on the market; T: results being tested; E: number of activities at experimental level (including laboratory or glasshouse activities); U: activities in unknown phase; N: number of countries involved. (Totals of this column have been calculated taking into account that sometimes more than one activity is being carried out by the same country, although that country will only be counted once). whereas others are not, or some bacterial pathogens can be managed by changing the growing environment.

TABLE 5
Number of initiatives to develop and use diagnostic techniques

C: technology used on a routine basis and products available on the market; T: results being tested; E: number of activities at experimental level (including laboratory or glasshouse activities); U: activities in unknown phase; N: number of countries involved. (Totals of this column have been calculated taking into account that sometimes more than one activity is being carried out by the same country, although that country will only be counted once).

The development of cheap diagnostic techniques could assist decision-making in relation to pest and disease management. The development of diagnostic kits, such as enzyme linked immunosorbent assay (ELISA), and other molecular assays, can enable the precise identification of viruses, bacteria and other disease-causing agents, and is now an established tool in disease management in many farming systems. Diagnostic assays have also been developed to identify a wide range of other organisms, chemicals (such as undesirable by-products, e.g. aflatoxin), or impurities involved with food quality.

The FAO-BioDeC (Table 5) suggests that ELISA is only in commercial use in Eastern Europe, although this is unlikely to be unique to Eastern Europe as ELISA is undoubtedly used in plantation farming in many other regions. Most of the use of ELISA reported in Eastern Europe is to detect plant viruses (e.g. PLRV, PVX, PVA, PVM, PVY) in crops such as potato, fruits and strawberries. In the Near East, all ELISA-related work is reported to be in the experimental phase, with Iraq, Morocco and the Syrian Arab Republic working on potato, Morocco also on sugar cane, Tunisia on grapevine and Egypt on detection of ZYMV in cucurbits. In Africa, use of ELISA for cassava diagnostics is under study in Malawi, Nigeria, Uganda and Zimbabwe, for cowpea in Cameroon and Nigeria, and maize in South Africa and Zimbabwe. In Latin America, Brazil is testing ELISA in sugar cane, potato, plum, unspecified Solanaceae and soybean, and researching cucurbits, Solanaceae, and Rhizobium in association with beans and forage and tree legumes, Chile is researching potato, tomato, tobacco and grapevine, Paraguay is testing ELISA in citrus, and Peru is testing potato.

DNA diagnostics are also a powerful technique for identification of pathogens and other organisms in agriculture. Most DNA diagnostics are now based on the use of the polymerase chain reaction (PCR), a common research tool used in most molecular biology laboratories worldwide, which can be used to specifically amplify segments of DNA. Most PCR techniques require the use of the enzyme Taq polymerase which until recently was protected by patents requiring that any commercial use of the PCR technique would have to pay royalties to the holders of the Taq polymerase patent. The enzyme Taq polymerase is now in the process of becoming a `generic' biochemical reagent which will substantially reduce the cost of PCR applications in research and commerce.

At present, most of the reported work on PCR diagnostics is in the experimental phase. In the Near East, Egypt is working on PCR analysis of ZYMV in cucurbits and TYLCV in tomato. In Europe, Yugoslavia is researching PCR for PPV in plum, ASPV in apple and PVYV in pear. Armenia is working on diagnostic techniques for disease of tobacco, potato, linen, wheat and other crops, Azerbaijan on chickpea, tobacco and other crops, and the Republic of Moldova on maize, tobacco and other crops. In Africa, maize disease diagnostics is the subject of PCR studies in South Africa, Uganda and Zimbabwe, while diagnosis tools are studied for diseases of cowpea in Cameroon and Nigeria, cassava in South Africa and unspecified work is underway in Burkina Faso and Ghana. In Latin America, Brazil is researching on PCR for unspecified uses in sugar cane, bean, rice, tomato, carrot, unspecified Solanaceae and banana and is conducting experiments on sweet potato, garlic, apple, genome sequencing of Herbaspirillum in soybean, Rhizobium in association with beans and forage and tree legumes, peanut and wild related species, studies on fungi, eucalyptus, Brazilian forest trees, soybean, coffee, sugar cane, Xylella fastidiosa, and heart of palm for food quality. Chile is researching molecular diagnostics for pathogens of potato, tomato, tobacco and grapevine, and Peru of potato.