DNA manipulation is changing agriculture

The cultivation of plants and the husbandry of animals provide the world’s oldest examples of biotechnology, dating back more than 10,000 years. Over the centuries, people have adapted crops and farm animals to their needs, producing organisms with desirable characteristics such as large seeds, high fat content in milk, or resistance to disease.

Until recently, the most common way to improve crop plants and farm animals was to identify individuals with desirable phenotypes that existed as a result of natural variation. Through many deliberate *crosses—a process called selective breeding—the genes responsible for the desirable trait could be introduced into a widely used variety or breed of that organism.

*connect the concepts In genetics experiments, typically only a few genes and alleles are involved. But crop plants have phenotypes determined by many genes and alleles. This makes selecting genetically stable, desirable offspring from crosses between two varieties challenging. See Key Concepts 12.2 and 12.3.

Despite some spectacular successes—among them the breeding of high-yielding varieties of wheat, rice, and hybrid corn—such deliberate crossing can be a hit-or-miss affair. Many desirable traits are controlled by multiple genes, and it is hard to predict the results of a cross or to maintain a prized combination as a true-breeding variety year after year, especially because in sexual reproduction combinations of desirable genes are quickly separated by meiosis. Furthermore, traditional breeding takes a long time: many plants and animals take years to reach maturity and then can reproduce only once or twice a year—a far cry from the rapid reproduction of bacteria.

Here are just some of the advantages that modern recombinant DNA technology offers over traditional methods of breeding:

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investigating life

Producing TPA

experiment

For a protein such as TPA to become useful in the clinic, the expression of recombinant DNA (in this case, the TPA gene coupled to an active promoter) in cells must result in a large amount of the protein. The protein is then purified and used to treat patients with a stroke, by catalyzing the dissolution of a blood clot in an artery leading to the brain.

Original Paper: Collen, D. et al. 1984. Biological properties of human tissue-type plasminogen activator obtained by expression of recombinant DNA in mammalian cells. Journal of Pharmacology and Experimental Therapeutics 231: 146–152.

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work with the data

It is essential that a purified molecule made by biotechnology have the desired biological properties. In the case of TPA, the goal was to mimic the properties of the natural clot-dissolving molecule. Désiré Collen, a physician-scientist in Belgium, led an international team that compared laboratory-made TPA with the natural molecule made by human cells. They performed two types of experiments, one on clots suspended in human blood in the lab, and the other on clots that had developed in rabbits. The team also investigated the time course of clot dissolution and the dose of TPA needed for the effect.

QUESTIONS

Question 1

Human blood clots were prepared in the lab using a radioactively labeled clotting molecule. Dissolution of the clot resulted in the radioactive molecules becoming hydrolysis products (monomers), so comparing soluble to insoluble radioactivity was a measure of clot dissolution. Table A shows the results of clot dissolution over time after injection with natural TPA, TPA made by biotechnology, and a control injection without TPA. Plot the data in a graph of percent clot dissolution over time. What can you conclude about the experiment?

With no TPA there was no clot dissolution, but with TPA there was dissolution. Lab-made TPA was somewhat superior to natural TPA both in terms of the rate of clot dissolution and the final percentage of dissolution.

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Question 2

To determine whether lab-made TPA could dissolve clots in the mammalian blood system, clots were induced in a large vein in rabbits. After 20 h, either natural or lab-made TPA was injected at the clot region and the dissolution of the clot measured 4 h later. The results are shown in Table B. What can you conclude about this experiment? The results show the SEM (standard error of the mean) for the data. How would you use a statistical test to examine the significance, if any, between the two treatment groups and compare them with the control?

Again, lab-made TPA was somewhat superior to natural TPA in dissolving clots in the rabbits. A comparison could be made between the final percent of dissolution using a t-test.

Table A
Percent clot dissolution
Time (h) No TPA (Control) Natural TPA Lab-made TPA
1 0 4 5
2 0 10 20
3 1 20 35
4 1 35 55
5 2 50 65
Table B
Percent clot dissolution (SEM)
Dose of TPA (units) Natural TPA Lab-made TPA
0 (control) 14.3 (1.4) 14.3 (1.4)
12,000 19.8 (5.4) 22.3 (6.0)
24,000 24.5 (7.9) 30.6 (0.8)
48,000 38.9 (4.8) 49.3 (9.7)
96,000 66.0 (6.3) 75.4 (3.9)

A similar work with the data exercise may be assigned in LaunchPad.

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Consequently, recombinant DNA technology has found many applications in agriculture (Table 18.2). We will describe a few examples to demonstrate the approaches that plant scientists have used to improve crop plants.

table 18.2 Agricultural Applications of Biotechnology under Development
Goal Technology/genes
Improving the environmental adaptations of plants Genes for drought tolerance, salt tolerance
Improving nutritional traits High-lysine seeds; β-carotene in rice
Improving crops after harvest Delay of fruit ripening; sweeter vegetables
Using plants as bioreactors Plastics, oils, and drugs produced in plants

PLANTS THAT MAKE THEIR OWN INSECTICIDES Plants are subject to infections by viruses, bacteria, and fungi, but probably the most important crop pests are herbivorous insects. From the locusts of biblical (and modern) times to the cotton boll weevil, insects have continually eaten the crops people grow.

The development of insecticides has improved the situation, but insecticides have their own problems. Many, including the organophosphates, are relatively nonspecific and kill beneficial insects in the broader ecosystem as well as crop pests. Some pesticides even have toxic effects on other groups of organisms, including people. What’s more, many insecticides persist in the environment for a long time.

Some bacteria protect themselves by producing proteins that can kill insects. For example, the bacterium Bacillus thuringiensis produces a protein that is toxic to the insect larvae that prey on it.

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The toxicity of this protein to insects is 80,000 times greater than that of a typical commercial insecticide. When a hapless larva eats the bacteria, the toxin becomes activated and binds specifically to the insect’s gut, producing holes and killing the insect. Dried preparations of B. thuringiensis have been sold for decades as safe insecticides that break down rapidly in the environment. But the biodegradation of these preparations is their limitation, because it means that the dried bacteria must be applied repeatedly during the growing season.

A longer-acting approach is to have the crop plants themselves make the toxin, and this is exactly what plant scientists have done. The toxin gene from B. thuringiensis has been isolated, cloned, and extensively modified by the addition of a plant promoter and other regulatory sequences. Transgenic corn, cotton, soybeans, tomatoes, and other crops are now being grown successfully with this added gene. Farmers growing these transgenic crops use less of other pesticides.

CROPS THAT ARE RESISTANT TO HERBICIDES Herbivorous insects are not the only threat to agriculture. Weeds may grow in fields and compete with crop plants for water and soil nutrients. Glyphosate is a widely used and effective herbicide, or weed killer, that works only on plants. It inhibits an enzyme system in the chloroplast that is involved in the synthesis of amino acids. Glyphosate is a broad-spectrum herbicide that kills most weeds, but unfortunately it also kills crop plants. One solution to this problem is to use it to rid a field of weeds before the crop plants start to grow. But as any gardener knows, when the crop begins to grow, the weeds reappear. If the crop were not affected by the herbicide, the herbicide could be applied to the field at any time without harming the crop.

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Scientists have used expression vectors to make plants that synthesize a different form of the target enzyme for glyphosate that is unaffected by the herbicide. The gene for this enzyme has been inserted into corn, cotton, and soybean plants, making them resistant to glyphosate. This technology has expanded rapidly, and a large proportion of cotton and soybean plants now carry this gene.

GRAINS WITH IMPROVED NUTRITIONAL CHARACTERISTICS To remain healthy, humans must consume adequate amounts of β-carotene, which the body converts into vitamin A. About 400 million people worldwide suffer from vitamin A deficiency, which makes them susceptible to infections and blindness. One reason is that rice grains, which do not contain β-carotene, make up a large part of their diets. Rice grains lack the two-enzyme biochemical pathway that synthesizes β-carotene.

Plant biologists Ingo Potrykus and Peter Beyer isolated one of the genes for the β-carotene pathway from the bacterium Erwinia uredovora and the other from daffodil plants. They added a promoter and other signals for expression in the developing rice grain and then transformed rice plants with the two genes. The resulting rice plants produce grains that look yellow because of their high β-carotene content. A newer variety with a corn gene replacing the one from daffodils makes even more β-carotene and is golden in color (Figure 18.11). A daily intake of about 150 g of this cooked rice can supply all the β-carotene a person needs. This new transgenic strain has been crossed with strains adapted for various local environments, in the hope of improving the diets of millions of people.

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Figure 18.11 Transgenic Rice Rich in β-Carotene Middle and right: The grains from these transgenic rice strains are colored because they make the pigment β-carotene, which is converted to vitamin A in the human body. Left: Wild-type rice grains do not contain β-carotene.

CROPS THAT ADAPT TO THE ENVIRONMENT Agriculture depends on ecological management—tailoring the environment to the needs of crop plants and animals. A farm field is an unnatural, human-designed system that must be carefully managed to maintain optimal conditions for crop growth. For example, excessive irrigation can cause increases in *soil salinity. The Fertile Crescent, the region between the Tigris and Euphrates rivers in the Middle East where agriculture probably originated 10,000 years ago, is no longer fertile. It is now a desert, largely because the soil has a high salt concentration. Few plants can grow on salty soils, partly because of osmotic effects that result in wilting, and partly because excess salt ions are toxic to plant cells.

*connect the concepts Learn about the effects of salt and heavy metals on plants in Key Concept 38.3.

Some plants can tolerate salty soils because they have a protein that transports Na+ ions out of the cytoplasm and into the vacuole, where the ions can accumulate without harming plant growth (see Key Concept 5.3 for a description of the plant vacuole). Scientists developed a highly active version of the gene for this transporter protein and used it to transform crop plants that are less tolerant to salt, including rapeseed, wheat, and tomatoes. When this gene was added to tomato plants, they grew in water that was four times as salty as the typical lethal level (Figure 18.12). This finding raises the prospect of growing crops on what were previously unproductive soils.

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Figure 18.12 Salt-Tolerant Tomato Plants Transgenic plants containing a gene for salt tolerance thrive in salty water (A), whereas plants without the transgene die (B). This technology may allow crops to be grown on salty soils.

Perhaps the most spectacular transgenic crop developed so far is a strain of rice that is resistant to both salt stress (using the gene above) and drought stress (using a gene from a bacterium) and has the ability to grow on nitrogen-deficient soils (using a gene from barley). This triply-tolerant rice is undergoing field tests.

These examples illustrate what could become a fundamental shift in the relationship between crop plants and the environment. Instead of manipulating the environment to suit the plant, biotechnology may allow us to adapt the plant to the environment. As a result, some of the negative effects of agriculture, such as water pollution, could be lessened.