Darryl R. J. Macer, Ph.D. Eubios Ethics Institute 1990
Microbial Production of Biochemicals
There is no major ethical debate about the use of microorganisms to produce products ranging from industrial chemicals to alcohol. Microorganisms, because of their size, life habits and versatility have long been used for the production of both simple chemicals and complex brews. In the last decade the long history of human use of microorganisms has been extended as genetically engineered bacteria and yeasts have become commonly used. Bacteria and fungi are being used for the production of an increasing variety of commercially important products.
Organisms can be made to produce new products, and/or made to grow under different and sometimes extreme conditions. Bacteria that can grow in a 70% solution of toluene, and in a high concentration of other organic solvents, could be useful for industrial reactions requiring those conditions. Thermophilic bacteria, that can grow at 100C, and the enzymes that they produce, are useful for industry. Reactions proceed faster at higher temperatures and thus genes being isolated from thermophilic bacteria and transferred to other organisms will improve production.
There are a great variety of naturally occurring proteins to be exploited. The genes can also be altered, which further expands the potential of the new technology. Protein engineering allows specific alterations to be made using a technique called site-directed mutagenesis, where specific DNA sequences in genes can be changed. Modified proteins can be made, which can alter the catalytic properties of natural enzymes, or the stability, or the antigenicity of proteins.
Scientists have made a new enzyme completely synthetically, opening up the possibility of designer enzymes. Stewart, Kahn and Klis of the University of Colorado Medical School have designed an enzyme on a computer screen, then produced the 73 amino acid enzyme, chymohelizyme-1, in the laboratory and studied the biochemical properties. The enzyme displayed the expected biochemical properties, and it is based on the naturally occuring enzyme chymotrypsin (Amato 1990). It opens the way for many future possible constructions of novel enzymes.
Production of Chemicals
Microorganisms have been, and will continue to be, used for the production of many chemicals. They have also been made to produce enzymes for industrial use (Imanaka 1986). Enzymes are the catalysts that carry out all the synthetic and degradative reactions of living organisms. The enzymes produced from microorganisms are particularly suitable for commercial applications (Neidleman 1989). The world market for enzymes was worth more than US$ 500 million in 1985, and is expanding as only a fraction of the enzymes existing in nature have been characterised. One everyday use of a genetically engineered product is of the enzyme lipase (there are many different types), which breaks down fat, and is added to washing powders, so that the amount of washing powder needed is greatly reduced.
Genetic engineering is being used for the production of compounds for cosmetics, especially in Japan where the industry is already promoting "bBio-cosmetics" (Scheidegger 1989).
Any organism that can be grown in cultivation can be used as a source of materials for human use. The range of materials can be diversified by genetic engineering. Within ethical limits, the best production system will be one that is cheapest to produce good quality product. There should be use of organisms that are primary producers, that is plants, because the energy conversion is most efficient. An example of an alternative system that could be explored is the use of algae that can grow in saline water, such as Dunaliella. It is currently used for production of carotene and glycerol, and may be grown cheaply in shallow pools of salty water with a few nutrients, open to the sun (Ben-Amotz & Avron 1990). This culture system presents a cheap way of producing proteins, and since it is a eucaryote it will be able to produce eucaroytic proteins that are processed properly. The feature of genetic engineering is that it allows any organism to be used, and researchers should target attention on organisms that will be the most cost efficient, and ones which can be used with little technological complication.
Medical Drugs, Proteins and Vaccines
Microorganisms have been used for the production of medically important drugs such as antibiotics. With the advent of recombinant DNA techniques this role has greatly expanded. The genes that direct the synthesis of mammalian proteins have been inserted into bacteria, which are then grown in large cultures, to produce large quantities of medically important proteins at low cost. The availability of these products makes therapies for many previously untreated or uncured diseases possible (Clark & Kamen 1987). It would not be an overstatement to say that they have and are revolutionising the treatment of disease. Many human proteins are now being commercially manufactured using this technology. These include blood clotting factor VIII, interferons, interleukins, growth hormone, erythropoietin, insulin, tissue plasminogen activator (TPA) and various growth factors, which have medical uses (Gilbert & Villa-Komaroff 1980, Anderson & Diacumakos 1981, PMA 1988). They can be produced in yields of several grams per litre of broth, in bacteria such as Escherichia coli (Davies 1988).
Recombinant DNA techniques are being used to produce human vaccines. A vaccine against Hepatitis B has already been approved for worldwide use (Zuckerman 1988), and it costs about US$ 1 per dose. There has been much research on preparing vaccines against malaria, AIDS, and other major diseases, and there are vaccines under trial for some. Many pharmaceutical products can potentially be made (Vane & Cuatrecasas 1984). Unfortunately there is relatively little investment in research by the pharmaceutical industry, as vaccines account for less than 1% of their profit, but a greater percentage of their liability (Bloom 1989).
Veterinary drugs and vaccines have been made (Van Brunt 1987b, Bishop 1988). They have been produced even for diseases that were impossible to vaccinate against previously, such as tapeworms in sheep (Anderson 1989). Rabies vaccine is in widespread use in France, and Belgium, though regulatory delays in the USA have meant it only began field trials in August 1990.
Use of Fungi
Fungi are commercially important in producing compounds such as penicillin and other antibiotics. Fungi are also used as food, or for food additives, such as citric acid, and beverages such as beer or wine. As in bacteria, individual genes and even base sequences can be altered, allowing precise genetic control. Like bacteria, they have potential for producing many substances (Timberlake & Marshall 1989).
There are many possible improvements to be made in processes with a long history of microbial use, such as malting and brewing (Wettstein 1989, Chater 1990). This could be done by improving secretion efficiency, or relieving rate limiting steps in metabolic pathways.
Using Plants and Animals
Mammalian Cell Culture
There are new developments which may allow the use of mammalian tissue culture cells in commercial ways. Recently mammalian cells have been used to produce useful protein products (Ramabhadran 1987). Some eucaryotic proteins made by bacteria are not biologically active because bacteria lack the mechanisms to modify newly synthesised proteins in order to activate them. Fungi such as yeast are being used, as the current best production obtainable from mammalian cells is still an order of magnitude (one tenth) below that in the best bacterial systems (Friedman et al. 1989), though this may be sufficient for commercial production in some cases.
Plant Cell Culture
Plant cell tissue culture is being used to produce plant-derived products. The first tissue culture product commercialised was Shikonin, a dye and pharmaceutical manufactured by Mitsui Ltd. in Japan, in 1983. Ginseng, one of the most valuable plant products, can be harvested only after 5-6 years of plant growth, and the plant is destroyed. Large yields have been produced by culture of chlorophyll-accumulating aggregates of ginseng cells (Odnevall & Bjork 1989). These two examples did not use genetic engineering techniques, which will expand the range enormously. The technology is still at an early stage, and many pharmaceutical products can be produced (approximately a quarter of all pharmaceuticals are plant-derived) (Fowler et al. 1988). The advantage of using plant tissue culture is that factory control makes the supply independent of seasonal plant yields or imports.
Use of Whole Plants
Transgenic plants are also being used to produce industrial products. Very recently two American biotechnology companies have begun to use plants to produce melanin, the natural pigment that darkens skin. This will be used in new sunscreen lotions (Buck 1989). There have also been pharmaceutical peptides produced in oilseed rape plants. Some of these proteins could be economically produced in the seeds of plants (Gasser & Fraley 1989). There is much research interest in the production of thaumuatin, one of the sweetest known substances. It is normally extracted from a West African plant, however, the gene has been isolated. For example, in New Zealand the gene has been transferred to potatoes, with the idea of using potato as a source of supply. Thaumatin is already used as a food additive to increase weight gain in pigs by making them eat more of the nicer tasting sweet food (Witty 1990). If thaumatin is included in the crops directly it will be simpler. Also, the worldwide market for human low calorie sweeteners is over US$ 3 billion annually, so there is much possibility for entering this market with a lower cost product.
Human serum albumin has been produced in potato and tobacco plants (Sijmons et al. 1990). Animal antibodies have been produced in transgenic plants (Hiatt et al. 1989), and this could bring the cost of producing monoclonal antibodies down by a factor of 10,000 (Hiatt 1990). Animal antibodies could also be used to protect plants against viral diseases, or the antibodies could be used to scavenge small organic pollutants such as toxins, from the environment. Obviously, the potential range of proteins is enormous.
Bacteria can be used to produce polymers that can be processed into polypropylene-like plastic. Biopolymers can be made using the precise enzymatic control that is not possible to use with synthetic polymers, with the advantage of biodegradibility to avoid pollution problems. New types of products, like synthetic rubbers, are also objectives of this research. Bacteria can also be made to produce the raw material for biodegradable plastic bags. The genes for polymer production may be put into foodcrops, such as potato tubers. This would also avoid using nonrenewable and energy intensive production techniques. This research area is attracting much commercial research, and it is already feasible to produce industrially one type of polymer, based on polyhydroxybutyrate, as a speciality plastic. It will take further work before bioplastics can compete financially for the commodity plastic market (Pool 1989).
There is also research into developing a process for converting food wastes, such as potato peels, into a source for biodegradable plastics. The food waste is converted to glucose, then into lactic acid, which is used as a feedstock for biopolymers.
Animals as Bioreactors
There are experiments underway to use animals to produce desired proteins in their milk, as protein factories or "bioreactors". Currently there has only been reasonable success using sheep which make human blood-clotting factor IX or human alpha-1 antitrypsin. The protein alpha-1 antitrypsin can be used to treat emphysema, a lung disorder caused by a deficiency of this protein. There are advantages to use of bioreactors over bacteria for producing proteins, as many proteins require processing by mammalian enzymes after initial protein synthesis. The mammary gland is very useful, as in sheep about 400 litres can be collected per lactation cycle (in cattle the figure is 8000 litres) (Clark et al. 1989). The production of transgenic sheep and the subsequent breeding of a flock of sheep probably costs about US$ 1 million, which is much cheaper than establishment of an industrial genetic engineering plant.
However, sheep take a relatively long time to grow in order to test expression, whereas mice produce only small quantities of milk, and rabbits have a useful ratio of size versus development time. Transgenic rabbits have been made that produce biologically active human interleukin-2 in their milk (Buhler et al. 1990). The milk yield from rabbits is about 100g per day, with a three fold greater protein level than cow's milk. Also rabbits are cheap, and can be housed in an enclosed and controlled environment.
Amgen, a Californian company, is designing chickens that will lay eggs in which the normal protein, albumin, is replaced by precious drugs. From 10 chickens it may be possible to produce a gram of Interferon daily, a very large quantity (Hill 1989). Silkworm caterpillars have been modified to produce human insulin, but this is still to be developed commercially.
Production of Biomass
Lignocellulose is the most available renewable resource for the production of food, fuel and chemicals. Approximately 2 X 1011 tons of carbon are fixed every year as plant biomass through photosynthesis. Biomass consists primarily of cellulose, hemicellulose and lignin, and there is about a 100 fold excess of it over current uses as animal feeds. There has been much research on the microbial conversion of these polymers, and there has been conversion of this material to protein. Genetic manipulation has been used to transfer enzymes into microorganisms, to enhance their ability to degrade polymers to usable products (Srinivisan & Cary 1988). There is much potential for additional uses of this resource for many purposes, and it is important in view of the desire to move to renewable resources and environmentally friendly technology.
Expanding Plant Breeding
For millenium plants and animals have been selectively bred to develop varieties that are more productive, or suitable for human use. The welfare of humanity is inextricably bound up with efficient agriculture. Plant breeding as a science began in the 19th century after the discoveries of how plant traits are inherited (Simmonds 1979). Our modern varieties originated from gene transfers within crop species, by selective breeding. There are, however, some major exceptions. For example, about 5,000 years ago wheat was created, when the three genomes of Triticum monococcum, Triticum tauschii, and a species of Aegilops came to be combined. The definition of a species rests on the concept of genetic isolation but sexual exchange of genes between species can and does occur in nature without human intervention.
Often, the crop species does not contain sufficient genetic diversity to allow the desired improvements, hence the search for diversity has led plant breeders to use new genetic technology (Goodman et al. 1987). The same concern has led animal breeders to use the same technology, as well as a desire to increase the desirable traits beyond those obtainable by normal breeding. In both cases, the aim is to arrive at a breeding population consistently expressing the desired trait(s). One of the main weaknesses of conventional plant breeding is its dependency upon sexual crosses and thus to genes that exist only in one species.
There are dozens of examples of agriculturally important genes and traits transferred to crop plants by interspecific or intergenic hybridisation and they have been reviewed by several authors (Goodman et al. 1987, Gasser & Fraley 1989). Recombinant DNA technology allows the transcendence of inter-species barriers and makes very novel genetic combinations possible. The first transgenic plants were created in 1983. One of the most popular methods of gene transfer is the use of the soil bacterium Agrobacterium tumefaciens, which can transfer genes to many plants at wound sites. However, it works mainly on the dicotyledonous plants which excludes many crop plants, such as cereals. Direct DNA transfer can be used to transfer genes to protoplasts (cells which lack a cell wall) from which plants can be regenerated. About 150 species of plants have already been regenerated from protoplasts, so the potential application of the technique is already very large.
Among the techniques for gene transfer another common one is "biolistics", the use of particle guns to shoot DNA into cells. Some techniques use tungsten particles, or gold beads. There may also be advantages of up to a 40% reduction in time for crop production via some biolistic based approaches over using Agrobacterium (Christou et al. 1990). This approach has recently been used to transform corn, and for the first time to produce genetically-modified fertile corn. The gene for resistance to the herbicide bialaphos has been inserted and the corn are resistant (Gordon-Kamm et al. 1990). Rice and corn are the only major crops that have been genetically modified to produce fertile transgenic offspring. Further improvements should be expected and developed, and the final proof of success must wait until field studies are completed. Microinjection has also got potential (Potrykus 1989).
Gene transfer technology has advanced at a far faster pace than our understanding of plant biotechnology and the factors which are important within the plant in determining other useful agronomic traits. Because of this, attention has been focused largely on characters which might be determined by single genes (Walden 1989).
The main focus of most biotechnology programmes is to produce new cultivars with improved pest and disease resistance to promote more environmentally acceptable alternatives for food production. Yield is no longer the only goal, improving the quality and marketing appeal, using genetically engineered pest and disease resistance to produce healthier products, are goals. Traditional methods of cross-breeding are not only limited, but slow and costly (Sansavini 1989). Transgenic plants are also being used to study gene expression and control, which will be useful for further practical applications (Benfey & Chua 1989).
Plant Disease Resistance
Natural disease resistance is complicated. Plant breeders have long sought to increase the disease resistance of crops through selection of resistant varieties and by hybridising crops with wild relatives. About one third of total crop losses are directly attributable to plant disease. Molecular techniques, such as insertion of antiviral or antibacterial genes from other species into plants, and cellular methods to allow rapid screening for the desired phenotype, have led to more rapid progress. Plant viruses are not well understood, but opportunities for improved disease resistance exist as the plant-pathogen relationship is easier to modify (Wilson 1989).
Viruses cause serious diseases in many crops. The genetic basis of viral resistance in plants is narrow, so resistance breaking strains of virus frequently appear. Isolating the plant's own resistance genes to combat disease is not practical until they have been isolated. The function of such genes depends on complex factors, such as the right genomic background. However, they could be used as good starting materials for protein engineering. Good viral disease control has been obtained using three different approaches:
* Cross protection occurs when plants are deliberately inoculated with a mild strain of virus. The plants are then resistant to infection with normal virulent strains of the same virus. Cross protection is probably due to the presence of the coat protein of the protecting strain. Coat protein genes of several viruses have been inserted into transgenic plants to provide protection (Fraser 1989). The expression of a single coat protein gene can protect a plant against several different viruses. This heterologous protection is important, as it reduces the number of genes required for multiple viral resistance. This has protected crops from infections of alfalfa mosaic virus, cucumber mosaic virus, potato viruses X and Y, soybean mosaic virus, tobacco mosaic virus, tobacco rattle virus (Conner et al. 1990), and others. Importantly, it has been found that transgenic plants that express the coat protein genes of tobacco mosaic virus or alfalfa mosaic virus also have some protection against other viruses. The mechanism of this protection is unknown (Anderson et al. 1989).
*Insertion of Satellite viruses (which are unable to replicate themselves) into the plants' genome to provide protection has been used for cucumber mosaic virus and tobacco ringspot virus (Conner et al. 1990).
*Antisense RNA the translation of a specific mRNA can be inhibited if the plant contains a complementary antisense RNA, which will form a double-stranded RNA molecule with part of the messenger mRNA, preventing translation of the protein, and thus protecting the plant (Day 1989).
Tobacco plants that have been made resistant to tobacco mosaic virus (TMV) infection (Abel et al. 1986), are of great commercial importance. Cotton plants have successfully expressed genetically transferred marker enzymes, and commercially important genes are being tested. Douglas fir has also expressed marker genes, and the seedlings were micropropagated from shoots. The importance of forestry trees is very high, and disease resistance genes are perhaps the most immediate targets. There have also been genes inserted into tobacco, cotton, corn and soybeans to make them resistant to crown gall disease (OTA 1988b).
Potato is one of the most important stable food crops worldwide. Because it is a tetraploid it is laborious to use traditional breeding to improve varieties. Viral diseases are of major importance: potato virus X can cause yield depressions of over 10%, and potato virus Y can decrease yields by up to 80%. Thus virus resistance holds a major key to potato crop improvement. Potato plants containing resistance genes to these viruses, have been tested in many countries for several years. The inserted genes may not affect other cultivar characteristics. Current work is aimed at obtaining simultaneous resistance to more of the major viruses (Elzen et al. 1989). One team in Monsanto has generated resistance to both virus X and Y in a commercial potato variety, Russet Burbank (Lawson et al. 1990).
Bacteria can be used for disease resistance. In 1988 a bacterial fungicide called "Dagger G" was introduced for the control of cotton diseases caused by Rhizoctonia and Pythium. It was given approval in the USA by the EPA after only five months. This is in contrast to the common ten year period required for approval of chemicals. Dagger G should have fewer negative side effects than chemicals and will probably be improved by genetic modification.
The current pesticide market in the United States is worth US$ 3 billion annually. There are many problems associated with pesticide use, including pest resistance to chemicals and negative environmental effects. There are also insecticides being developed that are of plant origin (Arnason et al. 1989).
Biological control has a long history and is becoming more important (Campbell 1989). In 1889 Vedalia beetles were introduced to California to control cottony cushion scale in citrus orchards. They are still being used. In recent years imported parasitic wasps have been used to control alfalfa weevils. Another example is the introduction of musk thistle weevil which is a long term alternative to chemical herbicide for the control of musk thistle. It has been well used in North America (Cramer 1989). Another is the Ecogen product "Collego", for controlling northern gointretch, a costly weed for rice farmers. It is a dry formulation of a naturally occurring fungus. There are other similar products on the market, and more are being developed.
Crop rotation and tillage that disrupt pest life cycles are at the first level of control. At the second level is the introduction of self-sustaining control agents. Expansion of the marketing of biocontrol agents by major companies has been slow, as they will lose much profit from the sale of chemicals. There has been more introduction of repeated application of biological pest control, such as bacteria or fungi-based pesticides (Cramer 1989). Pests take longer to develop resistance to biocontrols, and although the results may take longer to see then with chemicals, the management can be long term. They have the additional advantage of specificity, not seen with insecticidal chemicals.
Plants expressing the insecticidal protein of a bacterium, Bacillus thuringiensis are pest resistant. Insect pests will die if they eat the plants. Larvae of moths and butterflies can be selectively killed by different insecticidal proteins. Many companies have put this gene into crop plants including corn, cotton, soybean, tobacco and tomato and this protected the plants from insect larvae (Buck 1989, Cramer 1989). Some crops such as cotton may normally have over ten applications of insecticide over the growing season. The Belgium company Plant Genetic Systems which has been testing plants expressing insecticidal proteins since 1986 has found the resistance to be very good without affecting yield. Like several US companies, including Monsanto, they will be producing several commercial varieties soon.
The control of caterpillar pests with plants expressing this insecticidal gene offers several advantages. Control is independent of the weather, and in conditions which would be unsuitable for spraying chemicals or bacteria, the crop is still protected. All parts of the plant are protected, such as the roots, or new growth previously susceptible between sprayings (Meeusen & Warren 1989). The pests are affected as soon as they begin to feed. Broad spectrum insecticides kill all insects, which includes spiders and beetles which are useful predators. The B. thuringiensis endotoxin kills only killing leaf-eating species. Different insecticidal proteins have been expressed to kill larvae of Lepidoptera (moths) and Coleoptera (beetles). There are different proteins produced by different strains with varying specificity. Being proteins, they are biodegradable (Gould 1988), and can be much cheaper to develop, and to obtain environmental release approval for use. To develop and register a new chemical insecticide costs about US$ 25 million, but to develop a new plant variety costs about US$ 1 million. However, the Environmental Protection Agency (EPA) regulations may require that a new crop variety needs to be registered as a pesticide in the USA, which would add about US$ 10 million to the cost (Meeusen & Warren 1989). The situation remains uncertain, and test crops are under review.
An alternative way to control herbivorous insect pests is by introducing the gene for digestive protease inhibitors into the plants, so the animals cannot digest food. The expression of these plant genes, which are thought to be a defensive response to insect attack, can be enhanced. Wounded plants produce a factor which induces the synthesis of protease inhibitors specific against insect and microbial proteases. They have an effect on a wide range of insects and are known not to be harmful to humans (Waldman 1989). The big environmental advantage is that only insects that eat the plants are affected. Commercial seed are soon expected to be available. The first field tests in Belgium were in 1985. In New Zealand, researchers are trying to identify the best plant protease inhibitors for insertion into clover or pasture crops, there is also work using insecticidal proteins to improve pest resistance in clover.
There has also been work on the development of insecticidal microorganisms to be sprayed onto plants. The current application costs of spraying microorganisms containing a toxin gene are similar to the costs of applying chemicals, but with the significant environmental advantages. These need continual application, but may not require additional regulatory approval for human consumption, as they will need to be if they contain novel genes. Losses to crops also occur during storage after harvest. It is possible that increased levels of antifeedant could be added to plants to reduce such losses.
Herbicide Tolerance and Weed Control
The use of agrochemicals is still expanding. Weeds reduce crop productivity by at least 12%. Overall worldwide sales of herbicides are worth over $US 5 billion annually, and are double the market for insecticides, and fungicides. Even in developing countries where labour for weed control is very cheap, large quantities of herbicides are used. The effects of this large-scale chemical use are still unknown, and many effects are indirect. The function of soil ecosystems is poorly understood, and the major biological cycles of organic matter breakdown and nitrification are the most sensitive to these chemicals (Edwards 1989).
Genes that give plants tolerance to herbicides have been isolated and incorporated into some plants (Shields 1985, Shah et al. 1986). There has been a variety of herbicide resistant plants developed, and there are some recent reviews on this (Mazur & Falco 1989, Schulz et al. 1990). Work has concentrated on herbicides that are more environmentally friendly than those commonly used. The gene from bacteria that confers resistance to the herbicide glyphosate (Roundup) has been expressed in higher plant chloroplasts (Cioppa et al. 1987). Since 1987 tomato plants expressing the gene, have been made (Fillatti et al. 1987). The gene is under patent by an American company Calgene, and is called "GlyphoTol". It has also been tested in cotton (OTA 1988b) and has been transferred to many other crops.
Resistance to sulfonyl-urea compounds, the active ingredients in Glean and Oust herbicides, has been obtained by the introduction of a mutant acetolactate synthase gene. Many researchers have transferred the gene to a wide range of plants. Plants resistant to phosphinothricin (Basta), have also been made by Plant Genetic Systems. Potato and tomato plants that can grow with concentrations of herbicide ten times higher than normal herbicide application level have been grown (Newark 1987). The field can be sprayed to kill weeds without affecting the crops. The results indicate there is no yield penalty. Many field trials have been conducted. Resistance to other herbicides such as atrazine (AAtrex) is also being developed.
There are different ways to alter herbicide tolerance and a recent comprehensive review by Schulz et al. (1990) summarises these. Sensitive enzymes can be over-expressed, by increasing the copy number and/or the expression of the gene. It therefore requires more herbicide to kill the plant. Site-directed mutagenesis can be used to alter the herbicide binding to enzymes. The herbicide uptake can be reduced by altering transport systems or the morphological changes such as increasing the number of cuticular wax layers obstructing penetration of the herbicide. Enzymes that degrade the herbicide can be expressed, and this approach has been effective against bromoxynil, phosphinothricin and 2,4-D (Conner et al. 1990, Schulz et al. 1990).
Research has mainly been conducted on those herbicides with properties such as high unit activity, low toxicity, low soil mobility, and rapid biodegradation and with broad spectrum activity against various weeds. The development of crop plants that are more tolerant to such herbicides should prove more effective, less costly and more environmentally attractive weed control. The commercial strategy for chemical companies is to gain increasing market share through a shift in herbicide use, not to increase overall use (Gasser & Fraley 1989). Herbicide tolerant plants will have the positive impact of reducing the overall herbicide use and will also lead to substitution by more effective and environmentally acceptable products. The first herbicide resistant crop to be released for use was atrazine-resistant Canola (Mazur & Falco 1989). Imidazolinone resistant corn and sulfonylurea resistant soybeans and potatoes are under evaluation. The technology is widely applicable, so once the food is judged to be safe, a large range of crops may be available.
It is possible to use phenotypic selection to attempt to develop herbicide-tolerant crop cultivars. Cellular selection has often been used, but the whole plant response can be different to the cellular response. Phenotypic recurrent selection has been used to select for 2,4-D tolerance in red clover. The levels of 2,4-D tolerance were increased by 35% over four cycles (Taylor et al 1989). This is an alternative to gene insertion. The advantage is that the gene is found "naturally" in that species. The Soybean variety, Tracy M, was bred by classical means for resistance to the herbicide Metribuzin (Schulz et al. 1990). However, this sort of selection method is not necessarily intrinsically safer for animal or human consumption. We need to look at genetic engineering as an extension of traditional crop breeding goals. All alternatives are potentially useful.
There are several advantages of herbicide tolerant plants (Conner et al. 1990). The obvious use is in removing weeds from crops. It also allows the maintenance of genetic purity during seed multiplication of new cultivars. It could allow chemical thinning of crops after the mixing of parent and resistant seeds. It can also be linked to other characters, as a selection method. There is no need to apply herbicides until weed infestation reaches an intolerable level, therefore less herbicide is used. This reduces soil erosion. A single safe herbicide can be used instead of a mixture of herbicides, reducing the chance of weeds becoming resistant to several herbicides.
Better Crop Varieties
The most obvious improvement accomplished by traditional breeding is increased yield. In the USA from 1930-1975 the average yield per unit land increased by the following percentages: soybeans 70%, wheat 115%, corn 320%, and grain sorghum 358%. This increase was accomplished by small increments, but genetic engineering techniques have the potential to rapidly increase yield, as they complement the traditional technology. In the USA the average annual increase in corn yield is 1%, however, in parts of southeast Asia where more people live the rice yield is no longer increasing. This presents problems that will require new approaches to improving yield. During the Green Revolution the average rice yield increased 4% annually, but principally by converting stalk to seed. There is much potential to increase yield, for example by improving the efficiency of photosynthesis so that more carbon is fixed into plant material. There are efforts to alter the cell membranes of plants, as well as basic enzymes involved in photosynthesis, to increase the efficiency of plant growth. Any approach must also involve the associated good farming practise.
Tolerance of Environmental Extremes
Plants may be able to be more resistant to drought, flooding, salinity or sensitivity to heavy metals, so that they can be grown in areas of the earth currently beyond the tolerance range of species, or even those areas unable to be used for agriculture at all. About 30% of the world's land area has major plant stress conditions, including insufficient soil nutrients or water, or toxic excesses of minerals and salts. To exploit other environments, tolerance to low temperature is also important. The antifreeze gene from an arctic fish has been transferred to soybean, with the goal of creating plants tolerant to low temperature. There is research by a number of groups on the development of aluminium resistance in plants. Aluminium toxicity is a problem in low pH soils, where it may reduce plant growth. By making plants tolerant, they will grow better in such soils.
Pine trees are being made more drought resistant and suited to warmer weather, because of the expected climatic changes due to global warming expected in North America in 30 or 40 years when the trees mature. Due to the long reproductive cycle, and the need to wait 20-30 years before mature traits can be evaluated, we are now using only the second and third generations of genetically improved trees. The long juvenile periods, large size and high natural heterozygosity limit the application of conventional breeding techniques, so genetic engineering is more applicable to tree improvement than to herbaceous agronomic crops. The traits that will be targeted include climatic adaptation, fusiform rust resistance (losses exceed US$ 100 million a year), and herbicide resistance to allow better plantation establishment. There are other long term targets such as nitrogen fixation, lignin biosynthesis, cellulose biosynthesis, photosynthetic efficiency, cytoplasmic male sterility, and apical dominance (Olsen 1988).
A major long term project for crop improvement is to characterise, then transfer, the genes for nitrogen fixation into plants to enable them to fix atmospheric nitrogen to save using nitrogen fertilisers. However, the nitrogen fixing pathway involves 17 different genes and regulation is important. The transfer of the unit of 17 linked genes has been done from Rhizobium to Aztobacter strains. The strains could then fix atmospheric nitrogen. To fix nitrogen for plants, there is also the requirement to have the genes for a symbiotic relationship between the legume and the symbiont (Paul & Clark 1989). The importance of this technology is highlighted by the growing pollution of ground water by nitrogenous fertilisers. Expensive biological and mechanical filtering to remove nitrates from drinking water is the current "solution". The only success has been in improving the nitrogen fixing ability of some Rhizobium strains. It is a much more distant goal to insert the genes into plants in a way in which they could be used (Postgate 1990). In terms of economics, the market for a nitrogen-fixing crop plant would probably be small in the developed world. The advantages for the third world would be greater, saving the costs of importing chemical fertilisers.
Improved Nutritional Qualities
The food content of seeds, and plant products can be altered to improve their nutritional qualities. New techniques are continually being developed. One such technology involves using antisense RNA sequences to bind to the mRNAs of undesired proteins. One application was the reduction of the concentration of an enzyme (polygalacturonase) which is produced by ripening tomatos causing softening of the tomato. The concentration of this enzyme was reduced by 99%, so the fruit stay firm (Day 1989). These tomatoes have been developed to improve shelf life (about 300% longer) and taste since growers can leave the tomatoes on the plant longer (Kramer et al. 1989). These tomatos are being patented by Calgene, and are currently under consideration for FDA approval for human consumption. There is work in New Zealand on the control of ripening and softening in apples. The goals are to improve storage life, as well as quality (texture, colour, and sugar content).
Entirely synthetic proteins have been designed to supplement the overall production of essential amino acids in potatoes. The appropriate DNA sequence can be designed and synthesised in the laboratory, then inserted into the crop (Jaynes et al. 1986). Insertion of an artificial gene coding for a protein rich in essential amino acids can reduce from 1.8 to 0.8 kg the amount of potato one must eat each day to get all the daily protein intake (Dodds 1988). In CSIRO, Australia and DSIR, New Zealand, pea albumin (which has many sulphur amino acids) is being inserted into white clover, so that it will lead to better wool growth in the sheep that feed on the improved clover. Improving the amount of methionine-rich protein in plants is important for other animal based systems also.
Widening Consumer Choice
There is work at the DSIR, in New Zealand, on introducing new characteristics to increase the number of varieties of kiwifruit. These include altering internal colour, texture, seed content and shape, and making smooth skin. There are also goals involving nutritional characteristics such as altering sweetness and increasing vitamin C. The aim is to introduce new varieties for consumer choice. There is also work on improving the flavour of onions (Conner et al. 1990, Macer 1990b). As discussed later, this type of food product requires examination before being used for human consumption.
There have also been advances in the breeding of ornamental plants. Exports of ornamentals from the Netherlands alone were worth US$ 200 million in 1987. Ornamental plants were one of the first concerns of early plant hybridisers. However, they have used less sophisticated methods than those for breeding field crops. With the development of genetic engineering, and given the economic importance, there is renewed interest. The choice of flower colour can be extended, as novelty is added, such as rare blues or purples (Mol et al. 1989). Flower colour manipulation was first reported in Germany in 1987 with petunias. There have been some changes made using antisense methods. More long term objectives will be altering flower morphology, and improving vase life. Productivity will also be improved, as with other plants, by disease and pest resistance. A Netherlands company, Florigene, is producing genetically engineered roses, carnations, chrysanthemums and gerberas, with new colours (Day 1989). Unlike foodstuffs, there will not need to be proof that they are safe for human consumption.
Other Plant Properties
Other desirable properties in crops include the development of plants that require minimum attention, a low number of cultivation operations, and developing crops that can be mechanically harvested. Orchard trees need to be bred that conform to a given space, so control of branching is another goal. The crops should all be ripe for harvesting at the same time. The characteristics of crops sent to further food processing steps after harvesting can also be altered to suit the machinery, e.g. the milling and baking qualities of wheat can be improved. A basic improvement is to increase the ratio of edible to nonedible parts in a plant, as was the basis of the changes to rice during the green revolution two decades ago.
Animal Genetic Engineering
Farm animals will continue to be bred using existing methods of gene transfer and artificial insemination or embryo transfer, with help from bioengineering to improve fertility and reduce disease. However, recent developments may make gene transfer techniques important in the near future, and field testing of transgenic pigs and sheep is taking place. Genetic alteration can be used to improve weight gain, disease resistance and fertility. Historically, animal breeders have used new biotechnologies soon after their development, and there has been active research in this area. Genetic techniques are being increasingly used to alter animals used in both medical and agricultural research (Evans et al. 1986). In the past, animal breeders have had to rely on the opportune use of stud animals which show the qualities, using selected mating, by natural or artificial insemination or in vitro fertilisation (IVF) and embryo transfer. The new techniques for the isolation of genes, their manipulation, and transfer into other organisms of different species has meant that individual characteristics can be altered, introduced or removed from any organism. These techniques were developed both for research and for foreseen applications to technological use (Jaenich 1988).
Technology for manipulating embryos of farm animals has developed at the same time as genetic manipulation experiments in laboratory animals. The term "transgenic" was first applied to a mice strain that had foreign genes integrated into its genome (Gordon & Ruddle 1981). Much excitement was generated by the production of "supermice": large mice expressing a genetically transferred human growth hormone gene (Palmiter et al. 1982). The transfer of DNA into the male pronucleus of fertilised mammalian eggs by microinjection is one method for introducing novel DNA into the germ line, but this method results in multiple copies of DNA inserted at random sites. It has been found that regulatory sequences do aid controlled expression.
The enhanced growth of mice after transfer of a human growth hormone gene is an effect that is being repeated in other animals, most effectively in fish (Gill et al. 1985). One of the problems in injecting embryos of pig, sheep and cattle is that the cytoplasm is opaque, making the pronuclei invisible under light microscopy (unlike mice or rabbits). However, methods have been developed to overcome this, and fusion genes (mouse metallothionein promoter with human growth hormone gene) have been injected into pronuclei of rabbit, pig and sheep ova. The DNA was integrated and expressed in rabbits and pigs (Hammer et al. 1987) in several laboratories which have published similar results (Jaenisch 1988). Pigs that are being tested, were found to grow more rapidly, but have a high morbidity. The ability to produce pigs exhibiting only the beneficial side of growth hormone gene expression, increased weight gain and less fat, requires better control of gene expression, and alternatives are being pursued (Pursel et al. 1989). A deeper understanding of genetic regulation will come as more alternatives are tested. In British experiments the side-effects were absent. They have inserted the gene so that it can be turned on or off. The idea is to put a chemical trigger into the feed to control the amount of fat on these transgenic animals. There are transgenic pigs in Adelaide, and sheep in Sydney. Researchers in Texas, USA, have produced transgenic cows with inserted human oestrogen receptor gene, insulin-like growth factor gene, and bovine growth hormone genes. The aim of all these insertions is to grow leaner, faster growing cows, and cows that produce more milk (MacKenzie 1990).
It has recently been found that it is possible to make mini-mice by genetic engineering of the growth hormone gene. Researchers, Kopchick and Chen, inserted an altered growth hormone gene into mice expecting larger mice, but found that they obtained half sized mice (Weiss 1990). The altered growth hormone may bind to growth hormone receptors without stimulating growth. It may be useful for treating people who have giantism, or disorders leading to above normal growth. It will further help elucidate the mechanism
In addition to improving growth rate a major target of genetic engineering in sheep is to improve wool production. Selective breeding has been used to improve fleece densities and wool growth. One limiting factor is nutrient and energy supply to the wool follicles (Rogers 1990). An increase in wool growth rate has been observed in genetically engineered sheep with higher levels of growth hormone. Another approach is to improve the balance of amino acids, particularly increasing sulphur amino acids, in the forage (Altenbach & Simpson 1990).
The discovery of a fertility gene factor in merino sheep is of potential use for animal breeding(Radke & Lagaris 1986). The genotype confers increased ovulation rates and large litter sizes on merino sheep, without detriment to body size or wool production. This type of gene would be of particular value in rapidly increasing any new breeding populations' size, but is also of general application to improve fertility. Other genes which may be introduced into livestock include disease-resistance genes which may have simpler physiological repercussions, but still need careful assessment on economic and environmental grounds.
The way that animals respond to new genes will only been known after experiments. Over-expression of an exogenous gene such as that for growth hormone may affect the complex processes regulating growth rate, body composition and reproductive characters in a number of ways.
There have been attempts to make chicken resistant to common viruses, by transforming developing chick embryos with genes to increase egg production, and growth rate. Salmonella resistance would help to avoid use of antibiotics, which cause problems as they are passed on to human consumers.
Fish are more easily genetically manipulated using current techniques as natural fertilisation of eggs is external, and there are numerous large eggs which makes microinjection relatively easy (Maclean et al. 1987). Development is rapid, and does not require reintroduction into the reproductive tract of a receptive female. The best results have been obtained by transferring the regulatory sequences surrounding the genes in addition to the actual genes. There are many laboratories actively working on fish genetic engineering. The genes being transferred are both mammalian and piscine. The genes are usually spliced to a strong promoter from another gene, such as the promoter of the mouse metallothionein I gene, or the promoter of the animal virus SV40. Usually about a million copies of the gene are injected per cell, as most of the DNA is degraded in the cytoplasm before reaching the small nucleus. Some genes are correctly integrated, and expressed appropriately, and some are transmittable in the germ line. Genes of immediate usefulness that are already available are the growth hormone genes, globin genes, "antifreeze" genes, disease resistance genes and digestive enzyme genes (Maclean et al. 1987). So far many fish species have been genetically altered using this technique. About 5% of rainbow trout hatching from microinjected eggs have integrated genes. The initial projects are aimed at improving the growth rate in commercially important fish species. It will still be some time before stable, regulatable and heritable alterations of genes in other animals will be made.
Of obvious commercial value is the ability to control the sex ratio of offspring in breeding populations of livestock. It has not yet been found possible to separate semen into X- and Y-bearing sperm. However, it appears that there are male-specific antigens expressed in 8-cell embryos of mice, cattle, pigs and sheep that can be identified. A high degree of success has been claimed for sex separation in bovine embryos, and it has been reported to work also for pig and sheep embryos. There is a commercial embryo sexing and splitting kit sold in Australia. Seven days after fertilisation the embryos are flushed from the cow, then a cell sample is taken from each embryo to screen for the presence of the Y-chromosome (a male). After 2-3 hours the result is known, and the desired embryo can be split (to produce twins) and reimplanted (Glasgow 1989). It should be possible to select for the presence of other genes also.
The method used in making clones of salmon by Onosato did not involve direct gene transfer (Johnstone 1983). The sperm were eradiated before fertilisation, so that the sperm's genes were destroyed, but the sperm still stimulated the eggs to complete division so allow fertilisation to occur. The fertilised eggs were treated by high pressure to prevent the formation of polar bodies, then the eggs were incubated producing a 60% hatch rate, and 90% were clones. The purpose is to increase the number of females in the farm, as a single male produces enough sperm for the fertilisation of many more females' eggs. Males can be produced by treating the hatched fry with a male hormone, so while remaining chromosomally female about 80% could function as males.
Parthenogenesis occurs when a female egg develops without incorporating any of the male chromosomes into the offspring, as is seen in many birds. This was reported to be inducible in rabbits in 1939 (Pincus 1939), but is not widely seen as a source of clones. It appears that for proper development of mammalian embryos, genes from both parents are needed, as genes are differentially used from paternal or maternal chromosomes (Monk 1988, Thomson & Solter 1988).
The most successful type of cloning that has been reported for mammals involves the splitting of preembryos into more preembryos which can then develop into several clones. Much early work was done by Beatrice Mintz who began to manipulate mouse embryos over twenty years ago (Mintz 1967). She used an enzyme to break up the gelatinous coat of blastocysts, then mixed the cells, blastomeres, from several sources and found that the cells would aggregate. The cells of 32-cell embryos were found to be totipotent, and she reported that in the first thousand born mice there were no "monsters". The aim of these experiments was to trace cell lineages from embryo to adult tissues. These experiments showed that embryos could be split and reformed. Blastomeres of early cleavage-stage embryos of cattle, horses, pigs, sheep and mice have resulted in normal development from as little as half or even a quarter of the normal complement of cells (Solter 1987). There have been live births of 2-4 clones. Development and the ease of manipulation may be species dependent.
A related alternative that has been used for sheep is to combine whole blastomeres (cells of a blastocyst) from 8- and 16-cell embryos with enucleated or nucleated halves of unfertilised eggs. Single egg cells were bisected, then the egg halves were fused with single blastomeres resulting in live, normal, sheep (Willadsen 1986). This type of technique is useful for agricultural breeders to rapidly increase the number of a breeding stock. Single blastomeres from 8-cell embryos can be combined with single blastomeres from 4-cell embryos, and there is a tendency for the nucleus of the 8-cell blastomere to become the genotype of the sheep. This does not make many clones, but rather groups of 5-10 clones (Willadsen 1985).
The use of embryo transfer is becoming as common as artificial insemination in agriculture, and is similar in price and success rate. Artificial insemination has been standard practice in agriculture for 30 years, and embryo transfer for several years (Womack 1987). It is possible to recover a dozen or more embryos from individual superovulated cows. There are thousands of pregnancies a year produced by embryos that have been transferred.
One major development in embryonic manipulation is the use of embryonic stem (ES) cell lines. These ES cell lines are established in culture from preimplantation blastocysts and can colonise both the somatic and germ cell lineages of chimeric animals following their injection into host blastocysts. In order to characterise the mutations in new mice a cultured totipotent cell line was needed. Cells from teratocarcinomas, tumours that arise from undifferentiated embryonic cells (Stewart & Mintz 1981, Marx 1982) can be grown as tumours in mice, but not in culture. By mixing in these cells to make chimeric mouse embryos (Hogan et al. 1986), some of the mice had these cells established as the germ line (these tumour cells did not lead to tumours in the mouse). The breakthrough was in 1981 when Evans produced the first ES cell line (Evans & Kaufman 1981). These ES cell lines can be genetically-manipulated, the desired transformed somatically growing ES cells selected, and used to make chimeric embryos which when born give rise to new strains of mice.
There have been, and are many future possible uses of microorganisms in the environment, and this range has been greatly expanded by genetic engineering. The novelty of biotechnology is its ability to exploit the universality of the genetic code to combine, in a single organism, major adaptive traits developed by very different organisms. More details will be given in chapter 8 on the release of GMOs into the environment. Some of the current uses are summarised below.
* Bacteria can be used as pesticides, for example to carry the Bacillus thuringiensis insecticidal proteins.
* Bacteria (Pseudomonas syringae) that have reduced ice-nucleating ability have been sprayed onto plants to reduce frost damage. Conversely, iIce-nucleating bacteria and the ice-nucleating protein from P. syringae have been successfully tested for use in artificial snow-making.
* Viruses (Baculovirus) can also be used as pesticides.
* There are many possible uses of viral vaccines, against human diseases such as Hepatitis A, B, and C polio, rabies, malaria and against animal diseases such as foot and mouth disease, sheep foot rot, or tapeworms.
* Plant symbionts can be introduced, such as nitrogen fixing bacteria, which reduce the need for fertilisers. Mycorrhizal fungi can be used to increase plant growth rates by improving the efficiency of root uptake of nutrients.
* Bacteria can be used to degrade toxic compounds, such as heavy metals, organic compounds, phosphorus, ammonia or other pollutants. The first major use of bacterial degradation of an oil spill followed the Exon oil spill in Prince William Sound, Alaska in 1988. In this case, genetically modified bacteria were not added, but fertiliser to allow the low number of naturally occuring bacteria present in any soil that can degrade hydrocarbons, to multiply. The fertiliser is called Inipol. The technique is only applicable to beach areas, as the fertiliser will not cling to rock surfaces. The treated areas were dramatically improved within fifteen days, and even the underlying soil at a depth of one foot was degraded within 40-50 days. This was particularly important in the low temperature arctic environment where oil degrades very slowly. There was further areas treated in the 1990 summer, as there are large areas. The technique is not easy, but effective. It has stimulated research into this area. There has also been use of oil-degrading bacteria in the open sea in an oil tanker accident in the Gulf of Mexico in June 1990. It would be an advantage to destroy the oil before it reaches land, and also before it kills marine life.
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