Title: Unit 10 Learning Module
Course: SAT 170 Introductory Biotechnology > Section: SAT 170 Introductory Biotechnology

Unit 10 Learning Module

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Biotechnology in the Field of Agriculture-Green Biotechnology

You may recall from unit 1 that agriculture is a field which focuses on the cultivation of organisms such as plants and animals, often times using large plots of land. These agricultural products are primarily intended for generating a supply of food to support the human population, but are also used as a source of fiber, biofuels and other raw materials. This task of supplying humans with the food that they need is daunting and one that has its fair share of obstacles. One of these obstacles is the large and rapidly growing human population. There are currently more than 7 billion people alive on the planet, and that number continues to grow exponentially with an estimated population of nearly 8 billion people by the year 2025. Another major obstacle is the distribution of food to all of these people. Food security is a measurement of how available and how accessible food is to an individual. Research indicates that there is currently enough food being produced to provide everyone on the planet with the nutrition that they need. However, because of uneven distribution of the food supply, as well as problems with economic and social access to that food, there are still many people on the planet not receiving proper nutrition. Additionally, there is growing concern over whether or not the food supply is going to be able to keep up with the rapidly growing human population. The field of biotechnology is believed by many to be essential in improving the food security world-wide. For this reason, many organizations have been erected to help evaluate the safety and efficacy of biotechnology practices in agriculture. An example of one such organization is the Advisory Committee on Biotechnology and 21^st Century Agriculture (AC21) of the US Department of Agriculture (USDA). The objectives of this committee include assessing the applications of biotechnology in agriculture and the potential long-term effects of such applications. Agricultural biotechnology is a field which focuses on using biotechnology techniques for specific agricultural purposes. For many people, when they hear about agricultural biotechnology, they think of the use of genetically modified organisms for food crops.

Transgenic Plants

Figure 1

Since the first domestication of plants, crop alteration has been an important technique in optimizing the yield from a crop. Such alterations can include selective breeding and, more recently, things such as cloning and the creation of transgenic plants. You may recall from unit 1, that selective breeding is the process by which organisms are bred to have certain characteristics. For plants this often relies on creating hybrids which result when two different plants are mated to get unique offspring. This is in contrast to self-fertilization where a single parent has both male and female gametes which it uses to create offspring identical to that original parent plant. Another form of creating hybrid plants is known as protoplast fusion. During this technique, the rigid cell wall is removed from two different plant cells creating cells that are known as protoplasts. These protoplasts can subsequently be fused to create a hybrid plant. Often the cells used for protoplast fusion are from different species of plants.

Figure 1 - Notice how the plant cells in this image have a circular shape, this shape results once these cells become protoplasts after the removal of their cell walls. The absence of the cell wall allows these cells to readily fuse in a technique known as protoplast fusion as shown here.

In unit 8, we also discussed various methods of creating clones of plants. These methods include cuttings, grafting and micropropagation. See learning module from unit 8 for a complete description of these methods. For this unit, we will focus on transgenic plants. Transgenic organisms are those which have received a foreign gene or segment of DNA from another, unrelated organism. Another form of genetic engineering among plants that is less commonly discussed is known as cisgenesis. This is the process of incorporating a foreign gene or segment of DNA into an organism from a closely related organism with which it could naturally breed. The traits which are the primary focus for creating genetically engineered crops include resistance to insects and pathogens as well as tolerance to herbicides. Other genetic modifications focus on increasing the nutritional value or even the hardiness of a particular crop. In addition to improving crop yield, the introduction of transgenic plants is aimed at reducing the use of agrochemicals such as pesticides and fertilizers. As we will discuss further in our next two units, some crops are also used for the production of pharmaceutical and industrial compounds.

Figure 2 - This diagram compares conventional selective breeding methods with that of cisgenesis and transgenesis. Notice the key difference between these last two methods is whether or not the organism supplying the transgene is a close relative to the organism being transformed.
Figure 2
These are the cuvettes used for the process of electroporation.

As we discussed in unit 7, the main steps for generating a genetically modified organism are as follows: isolate a gene of interest from one organism, insert that gene of interest into a vector, and finally, use that vector to introduce the gene of interest into a new organism. One of the primary vectors that is used to genetically modify plants is known as tumor inducing (TI) plasmid. The TI plasmid was originally isolated from a bacterium known as Agrobacterium tumefaciens (which has recently been reclassified as Rhizobium radiobacter). The TI plasmid originally isolated from this bacterium is one that is capable of causing the formation of tumors in plant cells as it enters the genome of those plant cells causing uncontrollable growth of those cells. These tumors are known as crown galls. Because of its ability to enter plant cells and incorporate its DNA into the genome of those cells, it is an ideal vector for creating transgenic plants. Researchers need only modify the plasmid to carry their gene of interest rather than the tumor causing genes to introduce new genes in a plant cell. The major obstacle of this vector is that it is only capable of transforming cells of dicotyledonous plants. Such dicotyledons are those which grow from two seed halves and include crops such as tomatoes, potatoes and soybeans. Crops such as corn and wheat are unaffected by this plasmid as they are known as monocotyledons which means that they grow from a single seed embryo. In some cases, an electrical current can be applied to cells in order to increase the permeability of their cell membranes. This increased permeability can then be taken advantage of as a way to introduce foreign DNA into these cells. This is often performed on plant protoplasts as they are most susceptible when their cell walls have already been removed. This process is known as electroporation.

Figure 3 - The process of electroporation involves the application of an electrical current to a suspension of cells in an attempt to increase the permeability of the membranes of those cells. This method is used for the creation of transgenic plants and animals.

Gene guns are another method used to carry genes into plant cells. For this method, small metal particles are coated with the DNA of interest and are then loaded into a special type of device known as the gene gun. The gene gun is used to fire the DNA coated particles into the cells. The efficacy of this technique is relatively low as the DNA makes it into the nucleus mostly by chance. Thus, antibiotic resistance genes are often included along with the gene of interest as a way to select the plant cells that have been transformed. You may recall from our last unit that antibiotic resistance genes can be used as markers for the identification of genetically modified cells by placing the cells on a media which contains that particular antibiotic.

Figure 4 - This particle delivery system is similar to the hand held gene gun, both of which are used to introduce DNA coated metal particles into cells.

The chloroplasts, the target of chloroplast engineering, are shown as green intracellular structures. This form of transgenic plant carries the transgene in the chloroplast rather than the nucleus.

The tomatoes shown here contain a gene for an enzyme that is shown to slow the ripening process.

In some cases, the chloroplast, rather than the nucleus becomes the target for introducing new genes into a plant cell. You may recall from unit 2, that the chloroplast is the cellular organelle that is found in plants and algae which serves as the site of photosynthesis. Chloroplasts are known to contain DNA, and they are also known to readily accept new DNA. Such chloroplast engineering, as it is known, is advantageous over nuclear engineering as the chloroplasts of a transformed plant are not present in the plants pollen. Thus, there is little risk for the trait being carried unintentionally to another plant.

As mentioned in unit 1, the first genetically modified organism which was introduced to the public for human consumption occurred in 1994 and was known as the FlavrSavr tomato. It had been genetically modified to carry an anti-ripening gene which would prolong its shelf-life. This modification was based on what is known as antisense technology. Such technology relies on antisense molecules, which are molecules that are complementary to a particular gene. Recall from unit 4 that a gene is transcribed into a complementary mRNA molecule which is then translated into a protein. An antisense gene can be created which will be copied into an mRNA molecule that is complementary to the mRNA of the original gene. When introduced into the cell, this antisense RNA will prevent the expression of the original gene. In the context of the FlavrSavr tomato, the gene that was silenced in this way was the polygalactonurase (PG) gene which is involved in the process of ripening. Thus, by creating a genetically modified plant which had this gene deactivated, the shelf-life of the tomatoes was greatly extended. Although the FlavrSavr tomato is no longer commercially available, several other transgenic plants have been created for the same purpose.
Figure 3

Figure 4
Increasing crop yield is one reason why transgenic plants are desirable. This is primarily accomplished by introducing resistance genes to particular insects and plant pathogens. A gene known as the Cry gene is most often used for the production of insect resistant crops. This Cry gene codes for a crystalline protein, known as Bt, which is a toxin to many insects. The bacterium from which the Bt protein was originally isolated is known as Bacillus thuringiensis. Once ingested by an insect, the Bt toxin begins to destroy the intestinal lining of the insect, thus resulting in the death of that insect. After the widespread use of Bt in crops including corn, soybean, tomatoes, cotton and soybean, there was suspicions that the toxin may have unintended effects on non-pest insects, including the monarch butterfly. After significant amounts of research, the USDA has decided that the environmental exposure that these butterflies would receive would not have negative impacts on their populations.

Herbicide tolerance genes can also be used to generate crops with higher yields. The most famous example of this was the round-up ready strain of soybeans introduced by the company Monsanto, and brought to the United States in 1996. Round-up is an example of a glyphosphate herbicide which is commonly used by farmers to treat weeds. The mechanism of action of this herbicide is to block an enzyme known as ESPS which is vital for plant growth and survival. Because this enzyme is ubiquitous among plants, the use of glyophosphate can also affect the crop plants. The introduction of transgenic glyphosphate resistant crop plants has overcome this problem. Unfortunately, the widespread use of these transgenic glyphoshpate resistant plants has led to the evolution of glyphosphate resistant weeds. New strategies are being examined and introduced in attempt to address this problem.

Figure 5 - Although there are many different types of genetically modified plants that are used worldwide, the Round Up ready plants such as the soybean first introduced by Monsanto have received a great deal of attention. Part of the controversy over the introduction of such transgenic plants by Monsanto, is that they are also the ones that sell Round Up.

Crops of golden rice are thought to be important in the fight against diseases associated with vitamin A deficiency because they carry a gene for beta carotene, a precursor to this essential vitamin.

Improving flavor, texture and nutritional value of food can also be accomplished through the use of transgenic plants. Golden rice is a great example of this. Golden rice is that which has been modified to contain the genes necessary for the production of beta-carotene. Beta-carotene is known to be a precursor to vitamin A, thus, the consumption of such rice can be used to help alleviate some of the symptoms of vitamin A deficiency. Dispute over whether or not the amount of beta carotene available in Golden rice can effectively impact the global problem of vitamin A deficiency is currently underway.
Figure 5

Transgenic plants are also being used as a source of nutritional supplements and pharmaceuticals including protein treatments and vaccines. We will explore these applications further in unit 11.

Outcrossing is used to describe the movement of unrelated genetic material into a breeding line of organisms. In the context of transgenic plants that are used as crops in fields that may lie adjacent to wild-type crops, the term outcrossing is used to describe the unintentional movement of a transgene into another species. The potential for outcrossing is one of the major concerns with regards to the introduction of transgenic plants as crops. To try to control for this outcrossing, as well as address many other concerns associated with the introduction of transgenic plants, strict governmental regulations are placed on these genetically modified organisms.