Plant genetic engineering can be used for many different purposes
The method of genetic engineering via the Agrobacterium system has pro-duced revolutionary results in basic as well as in applied plant science. In basic science, it has led in a very short time to the identification and char-acterization of very many new proteins, such as enzymes and translocators. When investigating the function of a protein in a plant, it is now common to increase or decrease the expression of the protein by molecular genetic transformation. From the effects of these changes on the phenotype or others, conclusions can be drawn about the role of the corresponding protein in metabolism.
`In agriculture, plant genetic engineering has been utilized in many ways to augment protection against pests, to increase the qualitative and quan-titative yield of crop plants, and to produce sustainable raw materials for industrial purposes. By now many genetically altered (transgenic) culti-vars are grown worldwide, especially in North and South America and in China. The exception is Europe, where until now only one single transgenic species of a cultivated plant (a BT maize, see below) is grown, due to lack of public acceptance. Of the worldwide cultivated transgenic plants by far the largest part was developed with the aim of improving pest and weed management, by generating resistance to insects, viruses and herbicides. In order to increase the harvest yield, genetically engineered male-sterile plants (e.g., of rape seed (canola)) have been generated for producing hybrids. Transformants were also produced to improve the quality of the harvest (e.g., to improve storage properties of tomatoes) or the production of customized fats in rape .
Field crops are in great danger of being attacked by insects. Some examples may illustrate this:
1. The Colorado beetle, originating in North America, can cause complete defoliation of potato fields.
2. The larvae of the corn borer, penetrating maize shoots, causes large crop damage by feeding inside the shoots.
3. In a similar way, the cotton borer prevents the formation of cotton flow-ers by feeding inside shoots.
It has been estimated that about one-sixth of the global plant food pro-duction is lost due to insect pests. In order to avoid serious crop losses, the farmer very often has no option but to use chemical pest protection. In former times, chlorinated hydrocarbons such as DDT or Aldrin were used as a very potent means of protection against insects. Since these com-pounds degrade only very slowly and therefore accumulate in the food chain, they cause damage to the environment and are now restricted in their use or even forbidden by law in many countries. Nowadays, mostly organophosphorous compounds are used as insecticides, which, as phos-phoesterase inhibitors, impair the nerve function at the site of the synapses. These compounds are readily degraded, but unfortunately destroy not only pests, but also useful insects such as bees, and are poisonous for humans. The threat to humans lies not so much in the pesticide residues in con-sumed plant material, but primarily to the people applying the insecticides.
For more than 30 years, preparations from Bacillus thuringensis have been used as alternative biological insecticides. These bacteria form toxic peptides (BT proteins) that bind to receptors in the intestine of certain insects, thus impairing the uptake of food. This inactivation of the intes-tinal function causes the insect to starve to death. More than 100 bacte-rial strains are known, which form different BT proteins with a relatively specific toxicity towards certain insects. Toxicological investigations have shown that BT proteins are not harmful to humans. For many years now, bacterial suspensions containing the BT protein have been used as a biolog-ical spray to protect crops from insects. They are admitted for the produc-tion of so-called “organic” products. Unfortunately, these preparations are relatively expensive and are easily washed off the leaves by rain. Spraying also has the disadvantage that it does not reach larvae which are already inside plant shoots (e.g., the corn borer and cotton borer).
The genes for various BT proteins have been cloned and used to trans-form a number of plants. Although transgenic plants produce only very low amounts of the toxic BT protein (0.1% of total protein), it is more than enough to deter insects from eating the plant. The BT protein is decom-posed in soil and is degraded in the human digestive tract just like all other proteins. On this basis, insect-resistant transformed varieties of, e.g., maize and cotton are grown worldwide on a large scale. In certain countries more genetically modified cotton is grown than traditional varieties. Traditional cotton varieties, due to very high pest infestation, have to be sprayed with pesticides between 2 and12 times, and in single cases up to 30 times, which is a great hazard for the entire insect population. The cultivation of insect-resistant transformed varieties has resulted in a substantial reduction in the use of pesticides, easing threats to the entire fauna spectrum. The use of such transgenic plants thus may contribute to preserving the environment.
The insertion by genetic engineering of foreign genes encoding proteinase inhibitors is an alternative way to protect plants from insect pests (see sec-tion 14.4). After wounding (e.g., by insect attack or by fungal infection), the formation of proteinase inhibitors, which inhibit specific proteinases of animals and microorganisms, is induced in many plants. Insects feeding on these plants consume the inhibitor, whereby their digestive processes are disrupted with the result that the insect pest starves to death. The synthesis of the inhibitors is not restricted to the wound site, but often occurs in large parts of the plant and thereby protects them from further attacks. The introduction of suitable foreign genes in transgenic potato, lucerne (alfalfa), and tobacco plants enabled a high expression of proteinase inhibitors in these plants, protecting them efficiently from being eaten by insects. This strategy has the advantage that the proteinase inhibitors are not specific to certain insect groups. These proteinase inhibitors are contained naturally in many of our foods, sometimes in relatively high concentrations, but they are destroyed by cooking.
The expression of an amylase inhibitor in pea seeds, which prevents stor-age losses caused by the larvae of the pea beetle, is another example of how genetic engineering of plants offers protection from insect damage.
Virus diseases can result in catastrophic harvest losses. Many crop plants are threatened by viruses. Infection with thecucumber mosaic virus can lead to the total destruction of pumpkin, cucumber, melon, and courgette crops. In sugar beet, losses of up to 60% are caused by the viral disease rhizomania. In contrast to fungal or animal pests, viruses cannot be directly combated by the use of chemicals. Traditional procedures, such as decreasing the propa-gation of the viruses by crop rotation, are not always successful. Another way to control virus infections has been to attack the virus-transferring insects, especially aphids, with pesticides.
It has long been known that after infection with a weak pathogenic strain of a certain virus, a plant may be protected against infection by a more aggressive strain. This phenomenon has been applied successfully in the biological plant protection of squash plants. It was presumed that a sin-gle molecular constituent of the viruses caused this protective function, and this has been verified by molecular biology: the introduction of the coat protein gene of the tobacco mosaic virus into the genome of tobacco plants makes them resistant to this virus. This has been confirmed for many other viruses: if a gene for a coat protein of a particular plant virus is expressed sufficiently in a plant, the plant usually becomes resistant to infection by this pathogen. This principle has already been used several times with suc-cess to generate virus-resistant plants by genetic engineering. In the United States, a virus-resistant squash variety generated in this way has been licensed for cultivation. In Hawaii, where the cultivation of papayas had broken down completely due to virus infection, the utilization of virus-resistant papaya varieties made this valuable crop possible again.
The use of gene technology to generate resistance to fungal infections in plants is still at an early stage. An attempt is being made to utilize the natu-ral protective mechanisms of plants. Some plants protect themselves against fungi by attacking the cell wall of the fungi. The cell walls of most fungi con-tain chitin, an N-acetyl-D-glucosamine polymer, which does not occur in plants. Some plants express chitinases in their seeds, which lyse the cell wall of fungi. This protective function has been transferred to other plants. Plant cultivars have been generated by transformation which contains a chitinase gene from beans, thereby gaining an increased resistance against certain fungi. Another strategy lies in the expression of enzymes for the synthesis of fungicide phytoalexins (e.g., stilbenes). However, it may still take some time until fungus-resistant plants are ready for cultivation.
The most economically successful her-bicide is glyphosate (trade name Round Up, Monsanto) (Fig. 10.18), which inhibits specifically the synthesis of aromatic amino acids at the EPSP syn-thase of the shikimate pathway (Fig. 10.19). Animals are not affected by glyphosate since they lack the shikimate pathway. Glyphosate is from its structure a simple compound and therefore degraded rapidly by soil bacte-ria. Therefore it can be applied only by spraying the leaves. As a nonselec-tive herbicide, glyphosate even destroys many of the very persistent weeds. For example, it is widely used to clear vegetation from railway tracks, to control the weeds on the grounds of fruit and wine plantations, and to kill weeds before crops are planted. In order to apply this powerful herbicide as a selective post-emergence herbicide, glyphosate-resistant transformants have been generated for a number of crop plants by means of genetic engi-neering. To generate glyphosate resistance in plants, the bacterial EPSP synthase was isolated, which is less sensitive to glyphosate than the plant enzyme. Transgenic plants that express the bacterial EPSP synthase activity therefore acquired protection against the herbicide. Glyphosate-resistant cotton, rape seed, and soybean are now available to the farmer. In a simi-lar way, crop plants have been made resistant to the herbicide glufosinate (trade name Basta, Bayer, Crop Science) (Fig. 10.7) by the expression of bacterial detoxifying enzymes in transgenic plants. Herbicide-resistant cultivars of soybean, maize, rape and cotton, also in combination with insect resistance by Bt protein, represent the large majority of the trans-genic crop plants grown worldwide today.
The application of genetic engineering for generating resistance against pests or herbicides requires usually that only one additional gene is transferred into the plant. To alter the quality or the yield of harvest products, however, it is often necessary to transfer several genes which is more difficult.
A promising way to increase crop yields is the generation of hybrids from genetically engineered male-sterile plants. Another strategy for the improvement of crop yield is to alter the partition-ing of biomass between the harvestable and nonharvestable organs of the plants. An improvement of the tuber yield has been observed in transgenic potato plants , but these results have yet to be confirmed by field trials.
Genetic engineering is now being utilized in multiple ways to improve the quality and in particular the health value of food and fodder. Examples of this are the formation of highly unsaturated fatty acids in rape seed oil, the generation of rice containing provitamin A (golden rice), or the increase of the methionine content in soy beans, as discussed earlier.
Genetic engineering is a method with a great promise for the production of plants as renewable resources for industry. Transgenic plants which pro-duce customized fats, with short chain fatty acids for the detergent and cosmetic industries, and with high erucic acid content for the production of synthetic materials. Transgenic pota-toes which contain only the branched starch amylopectine (AMFLORA, BASF) are grown as raw material for industry, e.g., for the production of glues. Transgenic potatoes which produce starch consisting only of long-chain -amylose would be an interesting supplier of raw mate-rial for the production of plastics. Amylose ethers have polymer properties similar to those of polyethylenes, but with the advantage that they are bio-degradable (i.e., can be degraded by microorganisms).
Transgenic plants, especially plastidic transformants (transplastomes), are well suited for the production of peptides and proteins, such as human serum albumin or interferon. Progress is being made in the attempts to use plants for the production of human monoclonal antibodies (e.g., for curing intestinal cancer). Antibodies against bacteria causing caries have been pro-duced in plants, and it is feasible that they could be added to toothpaste. To make such a project economically viable, it would be necessary to produce very large amounts of antibody proteins at low cost. Plants would be suit-able for this purpose. There also has been success in using transgenic plants for the production of oral vaccines. The fodder plant lucerne (Medicago sativa) was transformed to produce an oral vaccine against foot and mouth disease. Oral vaccines for hepatitis B virus have been produced in pota-toes and lupines, and a vaccine for rabies has been produced in tomatoes. Although these experiments are still at an initial experimental phase, they open up the possibility of vaccination by ingesting plant material (e.g., fod-der for animals or fruit for humans).
Genetic engi-neering opens up the prospect of increasing the resistance of cultivated plants to these stresses by overexpression of enzymes involved in the stress responses. Thus, an increase of the number of double bonds in the fatty acids of membrane lipids through genetic engineering has improved the cold tolerance of tobacco. The generation of plants accumu-lating heavy metals, such as mercury and cadmium, in order to detoxify polluted soils, may have a great future.
With the growth of the world’s population, the availability of sufficient arable land becomes an increasing problem. Large areas of the world can no longer be utilized for agriculture, because of the high salt content of the soil, often caused by inadequate irrigation management. In 1990, 20% of the total area used worldwide for agriculture (including 50% of the artificially irrigated land) has been classified as salt stressed. Investigations are in progress to develop salt-tolerant plants by increasing the synthesis of osmotically com-patible compounds, such as mannitol, betaine, or proline , but also by increasing the expression of enzymes, which eliminate reactive oxy-gen species (ROS). ROS cause serious damages dur-ing drought and salt stress. Results obtained so far are promising, although it may still take considerable time until these efforts can be put into general practice. So far one drought-resistant wheat variety is in the licensing phase. If plant genetic engineering were to succeed in generating salt-resistant crop plants, this would be a very important contribution in securing the world’s food supply.
At present, plant genetic engineering, on the one hand, raises sometimes exaggerated expectations and, on the other hand, induces fear in parts of the population. Responsible application of plant genetic engineering requires that for each plant licensed for cultivation, a risk analysis be made according to strict scientific criteria as to whether the corresponding plant represents a hazard to the environment. Among other criteria, it has to be examined whether crossing between the released transgenic and wild plants is possible, and what are the potential consequences for the environment. For example, crossing can occur between transgenic rape seed and other Brassicaceae such as wild mustard. This could be prevented by generating chloroplast transformants . Moreover, transgenic plants them-selves could grow in the wild. In this way herbicide-resistant weeds may develop from herbicide-resistant cultivars. However, it also should be noted that in the conventional application of herbicides, herbicide-resistant weeds have evolved, namely, by natural selection. Experiments in the laboratory as well as controlled field tests are required for such risk analyses. It is beyond the scope of this book to deal with the criteria set by legislation to evaluate the risk entailed by the release of a transgenic culti-var. It is expected that, when used responsibly, plant genetic engineering may contribute to the improvement of crop production, an increase of the health benefit of foods, and the provision of sustainable raw materials for industry. If plant protection based on plant genetic engineering were simply to have the result that “fewer chemicals are put on the field,” then this would be an improvement for the environment.