Introduction

Given that the term "biotechnology" is generally applied quite loosely, it may be useful to commence this discussion of biotechnology with a definition of the term. Semantically, the word is formed from the union of bios (life) with technology (techniques), and it encompasses all technologies and processes involving living beings. The Convention on Biological Diversity (UNEP/CBD/94/1, 1998) defines biotechnology as "any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use." Interpreted in this broad sense, the definition covers many of the tools and techniques that are commonplace in agriculture and food production.

While the "official" definition of biotechnology is quite broad, biotechnology is generally considered by the public to be applicable on a much narrower sense, one which restricts itself to applications of DNA technology, genomics, modern reproductive techniques, most of which were developed over the last 30 years, whereas their application to agriculture dates to the last 10 or 15 years or so.1 Because of the novelty of this set of techniques, and the controversies over its potential impacts and applications, the subject can stir passionate debates. Controversies over the uses of biotechnology aside, this set of tools has potentially important applications, especially when used in conjunction with other technologies, for the production of food, agricultural products and services, applications that may be of significance in meeting the food supply, and nutritional needs of an expanding and increasingly urbanized world population, not to mention other applications in areas that go beyond agriculture.

The goal of this chapter is to provide a basic overview of the key agronomic and biologic concepts and applications of biotechnology that can supplement the economic issues and perspectives addressed in the rest of the book. This chapter will begin with a discussion of the evolution of agriculture. Next it will present and examine some of the key concepts related to the applications of modern biotechnologies to agriculture, agricultural production, and for biosafety. Finally, the current state of agricultural biotechnology will be assessed, implications drawn for its application to developing countries, and future prospects of its development and application will be addressed.

2. DOMESTICATION, AGRICULTURE, AND PLANT AND ANIMAL BREEDING

About 130,000 years ago, while people were still hunters and gatherers, they began developing the knowledge of plants and animals that set the stage for the beginnings of agriculture (Harlan, 1975). They began to recognize which plants and animals could be eaten and which could not due to toxicities or unpalatabilities. People began to learn which plants needed to be cooked to be edible, and those that could be eaten raw. Later, they also discovered how to use some plants for treating illnesses. Even so, at this stage, humans were heavily dependent on the available food in the

1 DNA (deoxyribonucleic acid) is the long chain of molecules in most cells that carries the genetic message and controls all cellular functions in most forms of life. It is the information-carrying genetic material that comprises the genes. RNA (Ribonucleic acid) is molecule derived from DNA that may carry information (messenger RNA (mRNA)), provide subcellular structure, transport amino acids, or facilitates the biochemical modification of itself or other RNA molecules.

surroundings of their dwellings, and their average life expectancy was relatively short.

Considering the natural division of tasks at the time, the male was the hunter and forager and the female likely gathered fruits and grains, close to the setting, to feed both herself and the children. At some stage, Man began selecting plants for their ease of use (e.g., cereals with larger kernels whose ears did not shed the grains easily and plants with less toxic compounds) and began to cultivate them. People began the act of planting simply as the process of throwing seeds of fruits around their dwellings, noticing that those seeds would develop into plants that were easier to find at the time of harvest. The step from understanding that seeds were the reproductive part of a plant to promoting plant growth near their dwellings was a short one. The selection of the most suitable types for further growing was the logical next step that marked the beginnings of agriculture. This fact happened around 10,000 years ago at several different locations around the globe (Harlan, 1975).

As humans started nurturing plants and animals, they protected and modified the growth environment and, as a consequence, these organisms were gradually modified to become ever more adapted to the new conditions. Domestication is the process that resulted in inherited modifications in plants and animals that made them more suited to human needs (Harlan, 1975; Futuyma, 1979).

Wild plants have many traits that make them different from their domesticated counterparts. Wild wheat collected by the original aboriginal societies in South West Asian countries were very different from cultivated wheat, in which a single ear has many grains which mature simultaneously and are not shed. The loss of wild traits was not fortuitous nor was it due to deliberate selection pressure. When a plant with the convenient combination of characteristics appeared and was recognized, it allowed man to transform a species into a cultigen (a category of plants found only in cultivation). Probably the next discovery was that seed germination could be improved if seeds were thrown in a soil that was not too hard or if seeds were not left uncovered under the hot sun. It was also noticed that seeds needed water to grow and early growers began nurturing plants in order to increase the probability of adequately feeding the family. Plants were additionally selected for better traits or to give a better response to the care provided by the humans.

Even though there are about 250,000 to 300,000 plant species on Earth, only a small number of them sustain humans (The Crucible Group, 1994). The bulk of calories (80%) in the human diet are furnished by only 30 crop species. Wild types of these species are so different from domesticated types that some may think that they are not related, and many have compounds that are toxic to humans. The process of domestication not only reduced or eliminated such compounds, but it also reduced to a much lower level the amount of genetic variation present in those plants. Crop plants have been highly modified to the point that they are unique and dependent upon humans for their survival.

A similar pattern can be found in the domestication of animals. Initially, animals were simply hunted. At some point in time, they began to live around human settlements, where they discovered that they could get leftovers to eat or could steal food. The proximity of animals meant more abundant and easier hunting and the process of encouraging this proximity became deliberate. For both plants and animals, such practices modified the genetic structure of the populations of wild plants and animals that were targeted, restricting their variability to the types more adequate to the human needs and making some of those species dependant on the humans for survival. Domestication was an important factor of plant and animal evolution (Futuyma, 1979).

2.1 Plant and animal breeding

According to Vavilov (1951), breeding is the human-controlled "improvement" or modification of plants and animals to suit human needs (Allard, 1960). While the origins of breeding of plants and animals goes back over 100,000 years (more or less coinciding with the origin of the domestication process), scientific breeding gained momentum in the 20th Century with the rediscovery of the genetic laws that had been first described by Mendel at the end of the 19th Century. All scientific breeding procedures pass through two steps: (1) the creation and or release of variability; and (2) the identification and selection of the useful genotypes (the genetic constitution, or gene makeup, of an organism). In this section we will first give a brief overview of both steps.

2.1.1 Creation and release of variability

The creation and release of variability can be made by induced mutations or by the production of new recombinations among existing genotypes.2 The induction of mutations creates a new form (allele) of an element (gene) that was not formerly present in a population. Natural mutations created most of the existing variations and forms. Spontaneous mutations take place at low rates, and in most cases mutants are disadvantageous for the genotype survival and or for reproduction. Hence, mutant individuals tend to be naturally eliminated from the population or are

2 In classical genetics, recombination (crossing-over) is done through traditional breeding techniques, e.g., Parents: AB/ab and ab/ab produce recombinant offspring: Ab/ab. In molecular genetics, it refer to the process that yields a molecule containing DNA from different sources. The word is typically used as an adjective, e.g., recombinant DNA.

present in very low frequencies. The very rare ones that are advantageous or have become advantageous in some environments are the few that tend to predominate in the populations and, because of their numbers, have a larger probability of surviving (Falconer, 1960; Futuyma, 1979).

Mutagenic agents are chemical or physical means that increase the mutation rate. A number of them have been identified and are exploited by scientists for inducing mutations with greater or lower efficiency in plants, animals, and microorganisms (Allard, 1960). Unfortunately, mutations occur at random in the genome, and there are no ways of directing the induction. Molecular genetics allows a better understanding of the action of some mutagenic agents; however, mutation breeding is a time-consuming, expensive, and largely random procedure that most breeders avoid in their programs.

Polyploidy is the natural state of some species or individuals who have more than two complete sets of chromosomes in their somatic cells. Induction of polyploidy is a random process (Allard, 1960), and not all genotypes in a species will survive the treatments that have that effect (i.e., colchicine, heat treatment), although the reason why this happens is not yet clear. Polyploidy, although being of great importance for the evolution of many species of plants and to some extent of animals, is not used very much for breeding purposes.

Formation of new recombinants is usually obtained by sexually crossing two compatible genotypes belonging either to the same biological species or to closely related species. In general, recombination can only be obtained among individuals with similar genetic background due to close evolutionary histories and thus similar traits. Most, if not all, breeding progress has so far been based on the production of new recombinations by crossing followed by selection. Breeding was, and will continue to be highly successful based on these procedures, but it is well known that some characteristics cannot be improved, due to the lack of adequate variability among sexually compatible genotypes. Interspecific (i.e., interspecies) crossing is usually very difficult and only in a few cases within each genus can it be performed successfully and/or produce fertile offspring. Inter-generic crosses are usually impossible, as the isolating mechanisms are very strong between two different genus.

Another way of producing distant hybrids through bypassing sexual barriers is by protoplast (plant cells whose cell wall have been removed) fusion, and plant regeneration, e.g., "in vitro" procedures. Cells are submitted to a treatment that degrades cell walls, making them amenable to fusion, and plants from the fused protoplasts are regenerated. The process takes place in the "in vitro" cell culture medium. After being submitted to selection for some generations, they may end up by being regenerated as hybrid genotypes. Although theoretically all plants could be submitted to this procedure, protocols have been developed only for a few species

(Nakano and Mii, 1990; Ohgawara et al., 1989). Furthermore, irregularities of cell division during the subsequent growth (and mitotic divisions) make the process much less useful and reliable than it may sound. Protocols have recently been developed that improve the process involved, and this process will likely have greater use in the future. Almost certainly it will be a useful tool in combination with other techniques to help in genetic engineering procedures, for instance, to obtain transient gene expression within the hybrid protoplasts or cells.3

The existence of useful variation for a given trait has always been the main issue in plant breeding, and all possible steps have been taken by breeders to increase its amount. Induction of mutations and interspecific crosses are two methods, but these require large expenditure in terms of time and money. Hence, use of these approaches is often avoided. Furthermore, the potential rate of success in terms of the number of commercial genotypes is extremely low, the results in terms of the gene affected are largely random, and several generations of careful selection have to follow in order to obtain a useful product. Frequently, the desired mutation is obtained together with many other undesirable traits, which have to be selected against and eliminated in a number of generations of selection.

It is also known that wild relatives of a crop species have much higher variability than domesticated types. When a genotype possessing a desirable trait is identified in a related species, it is never known a priori if that genotype can be crossed. Once the cross is made, the hybrid usually has viability problems and is fully or partially sterile. Many backcrosses are needed before the offspring is fully viable, fertile, and possesses the desirable trait introduced in the genetic background of the recurrent parent. There are a few really successful cases that have resulted in excellent commercial varieties that have contributed to solving some pressing agricultural problems in specific areas and, as such, they are a good justification for breeding programs to continue investing in these interspecies crossing techniques. On the other hand, intra-specific crosses have always been the main way by which breeders produce new variability, but unfortunately, the variability for agronomic traits, and especially for disease and pest resistance, is many times limited.

Most breeders, when they cannot identify the desired trait in the available genetic material, usually abandon their breeding objective because it is considered an impossible task. In some cases the problem may be bypassed by cultural practices, such as the use of chemical inputs. In other

3 Genetic engineering produces changes in the genetic constitution of cells (apart from selective breeding) resulting from the introduction or elimination of specific genes through modern molecular biology techniques. This technology is based on the use of a vector for transferring useful genetic information from a donor organism into a cell or organism that does not possess it.

cases, the problem may be so serious that the only solution for farmers is to change the crop. That has been the case in some areas of Brazil, for common bean (Phaseolus vulgaris), in spite of the fact that it represents a staple crop in Brazil and is the main protein source for the poorer population strata. However, due to the golden mosaic virus affecting beans, it cannot be cultivated anymore in some areas of the country (Farias et al., 1996).

In most such cases where the desired trait is not identified among the available genetic material, biotechnology, or more precisely, genetic engineering techniques, can help breeders to incorporate specific desired traits into the otherwise good genotypes. Namely, if the suitable gene or genes can be found in any other organism (plant, animal, or microorganism), that gene can be isolated using the proper laboratory procedures, the gene can be cloned and inserted into a bacterium or virus that will act as a vector to transfer it to the organism of interest (plant or animal). The isolated gene can also be introduced into the plant, animal or microorganism cell (depending on the case), using "biolistic" procedures (techniques which shoot DNA- coated micro-particles into cells).

Besides allowing the transfer of genes that would otherwise be impossible due to sexual barriers, the other advantage of genetic engineering over the other mentioned procedures is that it is specifically targeted. One single gene (or a few genes) is introduced into a genotype that is usually composed of thousands of other genes; therefore, it is a very small change and the genotype will continue to have the same genetic background, except for the additional characteristic. The total re-shuffling of the genome that happens when any type of crossing is made (intraspecific, with wild types or interspecific) will not occur, and the general behavior of that genotype is expected to be very similar to that of the same genotype before the introduction of the alien gene.

2.1.2 Identification and selection of useful genotypes

All known breeding strategies are based on selection (Allard, 1960). Selection procedures are only efficient when genetic variability is present in the material to be selected. Obviously, the variability caused by environmental factors cannot be fixed by selection. One difficulty breeders have always been confronted with is that selection acts on the phenotypic expression. However, a phenotype (the visible appearance or set of traits of an organism resulting from the combined action of genotype and environment) is the outcome of the genotype as well as of the environment action. Consequently, selection is often a complicated and lengthy process that limits the rate of success of breeding programs (Falconer, 1960; Ramalho, Dos Santos, and Zimmermann, 1993).

A number of characteristics are difficult to measure, and the recognition of the amount and type of genetic variations may require complicated manipulations that limit the number of individuals who can be evaluated and, by consequence, the rate of progress that can be realized. That is the case for many physiological traits, e.g., rate of plant photosynthesis, root growth, disease resistance, cold and/or drought tolerance, physiological efficiency, rate and efficiency of nutrient uptake, and many others. In general, plant and animal improvements have relied on indirect measures and on evaluations of some final products. Also, traditional breeding procedures are often expensive, as they require a somewhat large number of plants, with replications and grown at different locations, before a few superior individuals are recognized.

Researchers in quantitative and population genetics have developed theories and procedures for evaluating progenies and understanding breeding value of genotypes, but many of these are difficult to apply as they refer to one or a few genes, and extension to several genes becomes cumbersome and unrealistic. The net result is that breeding is largely "a numbers' game" in which the most successful programs are those that have more resources and are capable of evaluating larger populations from many crosses at many different locations. Many potentially very useful populations are discarded after a few generations simply because the breeder was not capable of identifying interesting genotypes at an early stage. Therefore, in every breeding program there is a large waste of potentially good genetic material.

The application of molecular marker technology to breeding provides new opportunities to improve selection procedures. Researchers can use molecular markers to detect variations either at the level of DNA sequences or of polypeptides (storage proteins, enzymes), which are direct products of genes. Those markers can also be part of, or closely linked to, a gene of interest. When the isolation of these molecules is an easy, nondestructive, and not very expensive process, large populations can be screened in a short period and individual genotypes can be unambiguously identified. The application of such technology can accelerate the breeding programs, improve their precision, and reduce the number and size of populations that must be planted at each generation. Selection, based on molecular markers, is named marker-assisted selection (McCouch et al., 1988).

The efficiency of selection is another important issue to breeding programs and is necessary in identifying genotypes to be crossed and later selected in the offspring. No cross can be superior if the parents are not well selected for true genetic superiority and complementarity. This selection procedure can be difficult to implement. It requires tests to be performed in a wide range of environments and using the proper experimental designs.

It is also very important to utilize all possible procedures that allow the correct identification of a sought characteristic, such as inoculating a pathogen for obtaining disease resistance, inducing drought or cold, etc. Some of the procedures may be very sophisticated, but they are necessary given that nature does not assure the occurrence of the selective atmospheric phenomena.

Breeders can use molecular markers to produce linkage maps to which agronomic traits should be added, and linkage relationships among the markers and the traits established. Based on the knowledge of the linkage relationships, selection can be applied over molecular markers in order to change some linked characteristics that are difficult to measure or to visualize in individuals. This is the case, for example, of root traits in plants. As an example of the use of molecular markers, take the case of rice. Markers were identified in rice that relate to root diameter and to drought resistance (Champoux et al., 1995). Using prior techniques, crosses were made using selected parents, and the segregating population had to be selected for root diameter in a nondestructive manner, and before harvest time, when roots were already degenerating. Using molecular markers, it was possible to take one or a few leaves per plant, extract its DNA, and identify the presence or absence of the marker. The selection was done without any influence of the environment in which plants were growing.

There are several reasons why these marker procedures are not widely incorporated in breeding programs, some of which are expected to change in the near future. One of them is that molecular marker linkage maps are yet not available for many species and, even when the maps exist, the location of useful genes has still to be determined. Other limitations are due to the fact that most breeders do not have access to adequate laboratory facilities, molecular markers are still rather expensive, and the pleiotropic effects of single genes are entirely unknown. Pleiotropy is the property of many genes by which a particular gene has a recognizable effect on several different traits. An area of knowledge that is developing and will help significantly in the application of molecular markers to breeding is bio-informatics.4

These difficulties do not change the fact that molecular markers offer the only hope for breeders to be able to begin purposely selecting for characteristics that so far have been almost impossible, such as those linked to some physiological traits. The potential results of molecular marker applications are new genotypes similar to those that could be obtained by traditional procedures. However, they will be obtained at a much faster rate, and more precisely even for characteristics that formerly were considered impossible to be individually recognized and selected.

As with traditional breeding techniques, molecular biotechnology can be used to improve the performance of plants and animals in different

4 Bio-informatics is the use and organization of information of biological interest. In particular, it is concerned with organizing bio-molecular databases, in getting useful information out of such databases, in utilizing powerful computers for analyzing such information, and in integrating information from disparate biological sources.

agricultural conditions and even for harsh environments. With the latest techniques, plants can also be bred to recover degraded environments (bio-remediation) utilizing the genes of plants and microorganisms that are able to live on soils or water that contains heavy metals or other toxic compounds. The resulting products could be used to detoxify some areas or water and to restore them to normal uses. Besides plant and animal breeding, biotechnology can also help in improving microorganisms for industrial purposes. Microorganisms are already being used to help clean up oil spills and to cleanse water from sugarcane processing residues. Genetically modified yeast is used for cheese and wine making procedures and for other fermentation products.

The breeding of interspecific hybrids predates molecular biotechnology as a method of bringing together desirable traits from different species. Although interspecific hybrids are difficult to produce, often plants of a species receive pollen from plants of other species and in rare cases fertilization can take place. The resulting hybrids are usually not fully viable or sterile, but they may survive and backcross with one or both of their parents, producing an offspring which will be more viable than the hybrid itself. In some cases the hybrid plants are able to explore new specialized niches or to adapt to a changed environment. The phenomenon of an occasional interspecific or even intergeneric cross, followed by hybrid survival and subsequent backcross to one or both parental species for several generations, results in the introduction in one species of some traits of the other and is called introgression. Hybrids that exploit new niches may also end up by evolving into new species (Futuyma, 1979). Genetic engineering is the method for the precise transfer of known genes from an organism to another beyond the limits imposed by the reproductive isolation of the species and can be considered a method of producing targeted introgression.

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