APPLICATION OF MOLECULAR BIOLOGY TO AGRICULTURE

1. APPLICATION OF MOLECULAR BIOLOGY TO AGRICULTURE • WHAT ARE TRANSGENIC CROPS? • STEPS IN THE DEVELOPMENT OF TRANSGENIC CROPS • EXAMPLES OF TRANSGENIC CROPS • MERITS AND DEMERITS OF TRANSGENIC CROPS

2. INTRODUCTION TO BIOTECHNOLOGY LECTURE NOTES BY: PROF. E.H. KWON-NDUNG DEPARTMENT OF BOTANY FEDERAL UNIVERSITY LAFIA

3. WHAT ARE TRANSGENIC CROPS? Transgenic crops are simply crops that contain a gene that has been artifically inserted into that crop, instead of being inserted through pollenation. The gene that is inserted into the crop is called a transgene. For example the transgene inserted into corn is called Bacillus Thuringiensis. This transgene is a soil bacterium that produces a protein that is poisonous to insects.

4. STEPS IN THE DEVELOPMENT OF TRANSGENIC CROPSStep 1: Extracting the desired D.N.A. Step 2: Cloning the gene of interest.Step 3: Designing the gene so it can easily be inserted into the plant.Step 4: Transformation.Step 5: Plant breeding

5. Step 1: Extracting the desired D.N.A First before we are able to extract the desired D.N.A gene we must be able to identify the particular gene. Unfortunately in this day and age we do not know very much about which genes are responsible for what traits and therefore the hardest part of extracting the desired D.N.A gene is the identifying part. Most of the time, identifying one gene involved with the trait is insufficient because scientists must understand how the gene is regulated, what other effects it might have on the plant, and how it interacts with other genes. Scientists today still know very little on which genes are responsible for enhancing yield potential, improving stress tolerance, modifying chemical processes of the harvested crop or any other plant characteristics. Most of the research in transgenic is focused on identifying and sequencing these certain genes. Isolating a specific gene is not that hard to understand.

6. Steps in creating transgenic crops:

7. The two main tools involved with isolating a gene are the restriction enzymes and the D.N.A ligase. It will be easier to think of the restriction enzymes as “scissors” and the D.N.A ligase as “glue scissors”. The restriction enzymes recognize and cut the D.N.A at a specific region of the D.N.A, much like scissors. Note that there are different restriction enzymes for different regions of D.N.A that is required to be cut. The ligase then attaches the two ends of D.N.A fragments together, much like glue. These two enzymes along with many more allow for manipulation and amplification of DN.A which are essential components in joining the D.N.A of two unrelated organisms. Before the specific D.N.A region can be inserted into another organism, we must obtain the D.N.A in a significant amount. This brings us to the next step, cloning the gene.

8. Step 2: Cloning the gene of interest The first step in cloning is to extract the D.N.A gene that is required using the restriction enzymes and the D.N.A ligase. After the D.N.A is extracted from the cells it is placed in a bacterial plasmid. A plasmid is molecular biological tool that allows any segment of D.N.A to be put into a carrier cell (usually a bacterial cell such as E. coli) and replicated to produce more of the D.N.A. Along with the desired D.N.A, an antibiotic- resistance gene is also inserted into a bacterial plasmid, which in turn is inserted into a carrier cell. This allows for the carrier cell to be successfully amplified through a process called transformation. Transformation will amplify the carrier cells but at the same time only amplifying the carrier cells with the desired D.N.A. Transformation involves the carrier cell being placed into two mediums; one medium would have a specific antibiotic while the other medium would not have the antibiotic. The carrier cells are then placed onto both mediums. The medium that did not have the antibiotic would grow substantially. While the medium with the antibiotic would only grow slightly. This is because the carrier cells that did not have the desired D.N.A and antibiotic- resistance gene will not grow on the medium with the antibiotic on it. This ensures that the carrier cells are all going to have the desired D.N.A in it. So as the carrier cells grow the D.N.A inside the cell will grow with it, and therefore be amplified or cloned to a considerable amount. Once the D.N.A has been amplified it is now almost ready to be inserted into the desired crop.

9. Steps in creating transgenic crops:

10. Step 3: Designing a gene so it can be easily inserted into a crop. Before a gene can be successfully inserted into a crop, it must be slightly modified. First a promoter sequence must be added to the gene so that it can be correctly expressed (ex. So that it can be successfully translated into a protein product). This is considered an on/off switch which controls when and where the specific gene will be expressed. A common promoter is CaMV35S, which is from the cauliflower mosaic virus. This promoter generally results in a high degree of expression in plants. Sometimes a gene must also be modified so that it can achieve a greater expression in plants. For example, the BT gene was modified by replacing A-T nucleotides with G-C nucleotides (which are preferred in plants) without significantly changing the sequence of the gene. This resulted in more production of the gene product in plant cells. Another thing that must be added to a gene is a terminator sequence, which sends a signal to the cellular machinery that the end of a gene has been reached. The last thing that must be added to the gene is a selectable marker gene. This marker gene is added in order to identify plant cells or tissues that have successfully been inserted with the desired D.N.A gene. The marker gene can also encode proteins that provide resistance to toxins, such as herbicides and antibiotics. After the gene has been successfully modified it is now ready to be inserted into the plant.

11. Steps in creating transgenic crops:

12. Step 4: Transformation Transformation is a change in a cell or organism brought on by the introduction of new D.N.A. There are two main methods of accomplishing this 1: The gene gun method, and 2: The Agrobacterium method. • 1: The Gene Gun method. This is also known as the micro-projectile bombardment method. This method is mainly used in corn and rice. This involves high velocity micro-projectiles that deliver the desired D.N.A into living cells using a “gun”. The desired D.N.A is attached to the micro-projectiles and fired into the cell. This method is much like a universal delivery system and it can eliminate problems such as the gene being rearranged when it enters the cell. • 2: The Agrobacterium method. This method involves the use of soil-dwelling bacteria known as Agrobacteriumtumefaciens. This bacterium has the ability to infect plant cells with a piece of its D.N.A. The piece of D.N.A that is integrated into the plants chromosomes is a tumor inducing plasmid. This plasmid will take control of the plants cellular machinery and uses it to make copies of its own bacterial D.N.A. On this plasmid there is also a region where the scientist can insert the desired D.N.A, which will be transferred to the plant cell. This plasmid is also activated when the plant has been wounded because when the plant is wounded it sends off chemical signals, and these signals activate the plasmid. When the plasmid is activated it enters the plant cell through the wound. It is still unknown how the D.N.A moves from the cytoplasm to the nucleus of the plant cell or how it is integrated into the plant chromosome. To be able to use Agrobacteriumtumefaciens successfully as a vector, the tumor inducing part of the plasmid has been removed so that it will not harm the plant as it is inserted. This method is useful because it can allow for large fragments of D.N.A to be transferred very effectively but the limitations are that not all crops can be infected by this bacterium.

13. Steps in creating transgenic crops:

14. Step 5: Plant Breeding After the D.N.A has been successfully inserted the plant tissues are then transferred to a selective medium which contains an antibiotic or herbicide that matches the marker gene. As in the cloning process only plants expressing the selective trait will survive and it is assumed that these posses the desired gene. To obtain whole plants from these tissues, a process known as tissue culture is used. This process is when the plant tissues are grown under controlled environments in a series of mediums that contain nutrients and hormones. To be sure that these plants have the desired gene in them, they undergo a series of test. These tests pay specific attention to the activity of the gene, inheritance of the gene, and unintended effects on plant growth, yield, and quality.

15. Steps in creating transgenic crops:

16. EXAMPLES OF TRANSGENIC CROPS Genetically Engineered Corn - BT Corn What is Bt? Bt stands for Bacillus thuringiensis, which is a spore forming soil bacterium that produces protein crystals that are toxic to many types of insects. Bt can be found about almost anywhere. It is distributed in the soil sparsely but frequently, and that is why it can be found almost anywhere. Bt has been found in all type of environments from beaches to the desert to tundra type habitats. • There are also over a thousand types of Bt that produce over 200 types of protein crystals which are toxic against a wide variety of insects and some other invertebrates. Bt belongs to the bacteria family, Bacillus cerus, which cause food-poisoning in humans. Bt does not cause food poising to humans because it contains a plasmid that produces the certain protein crystals that are toxic to insects. • Proteins crystals bind specifically to certain receptors in the insect’s intestine. Not all insects have these certain receptors, which allow for high species specificity. Humans and other vertebrates also do not have these receptors and therefore the toxin does not affect us.

17. History of Bt Bt was first discovered in 1901 by a Japanese biologist, ShigetaneIshiwatari. He was investigating the cause of the sotto disease (sudden collapse disease) that was killing large populations of silkworms. Bt was then rediscovered in 1911 by Ernst Berliner when he had isolated a bacteria that had killed a Mediterranean flour moth. Berliner had mentioned the existence of protein crystals but the activity of the crystals was not discovered until much later. By 1920 farmers were using Bt as a pesticide to kill moth larvae, since that was the only strain of Bt that was known at the time. But in 1956 Fitz-James Hannay and Angus Hannay had discovered that the reason Bt killed the moths was due to the protein crystals. Research had begun on Bt and the Bt crystals. So by 1977 there were 13 different strains of Bt, all still only effective against moths. But also in 1977 the first strain was found that was toxic to flies. The next strain was found in 1983 that was toxic to beetles. Today there are thousands of strains and many encode for crystals that are toxic to a wide variety of insects. Also because of Bt’s ability to be effective and not harm the environment the government and private industries have funded research on Bt.

18. How does Bt work? • The only way that Bt can be poisonous is that if it is eaten. The toxin becomes active when it is dissolved in the high pH insect gut. These toxins then attack the gut cells of the insect by creating holes in the lining. Bt spores and bacteria then spill out into the gut which cause the insect to stop eating and die in a couple of days. Even though the toxin does not kill the insect immediately, parts of the plants that have been treated with Bt will not be affected because the insect stops eating within hours. Note that Bt does not spread to other insects and it does not cause disease outbreaks on its own. The Bt toxin is very specific though because of the many different strains of Bt. Each Bt strain is specific to different receptors inside the insect gut. So the toxicity of the Bt depends on the receptors involved, and damage to the gut upon binding of the toxins to the receptors. Each species of insects has certain receptors inside their gut that will match only certain toxins, much like a lock and a key.

19. How Bt toxins work : • Insect eats the Bt toxin (crystals and spores) • The toxin binds to certain receptors in the gut and the insect stops eating • The crystals cause the gut wall to break down which allow the spores and normal gut bacteria to enter body • The insect dies as spores and gut bacteria proliferate into body.

20. Merits of transgenic crops • As of 2003 U.S grew 63% of the world’s transgenic crops, while Argentina grew 21%, and Canada grew 6%. Other countries that grew transgenic crops include China and Brazil that both grew 4% and South Africa that grew 1%. The future seems that we will see an exponential growth in transgenic crops with researches gaining more information about the process and the traits involved. As the new technologies are being produced there are many merits and demerits surrounding the issue of transgenic crops. • Transgenic cropscan protect itself against predators that feed off of the crops • Transgenic crops allows for less tillage, especially if the herbicide resistance gets added into crop. The herbicide resistance enables the plants to be sprayed with herbicides and they will not be killed. This will enable the farmers to spray herbicides and remove only the unwanted plants such as weeds. This will also conserve fertility through minimizing soil damage through compressions • No insecticidal sprays will be needed on Transgenic crops, because the insecticide is already engineered into it. Transgenic crops are also likely to give rise to lower levels of mycotoxins in the final food product. Also less damage from organisms mean less opportunity to develop fungi to infect the plant and bring toxic substances. • Transgenic crops only targets the pests that attack the crop, because the pest is only effective if it is eaten. So it will only affect the pests that that eat the transgenic crops. For example, it has been proven that the Bt gene in corn is expressed in more leaves and stems than in Bt corn pollen, and therefore the risk to butterflies through pollen drift onto their plants is diminished.

21. Transgenic crops can also be produced more often and more quickly. More genes can be engineered into the crop. • Transgenic crops has also been tested for genetic stability, substantial equivalence, nutritive properties, toxicity and allergenicity. It is also known that conventional breeding can introduce increased levels of plant toxins into a new variety or can modify its digestibility or nutritiousness. It has also been proven that some organic crops have higher levels of toxic substances. • Transgenic crops can be engineered with the ability to resist pests and can also be engineered to have more vitamins, minerals and anti- cancer substances. • Transgenic crops can help solve hunger in the world because transgenes can have a higher growing yield and effective utilization of scarce land because of better pest resistance and nutrient utilization

22. Demerits of Bt Corn • The introduction of transgenic crops has raised a number of possible negative consequences. • The low tillage is also a prime spot for a monoculture in the fields. A monoculture is where all of the crops are the same, so with this it is a perfect situation for weeds. • Genetic pollution from transgenic crops is another concern. It is thought that the genes can spread into other organisms through pollen, seeds and microbial process. This is different from other types of pollution because once the genes are out they cannot be re-called. An example is canola seeds in Canada, when some canola seeds were contaminated with unapproved genetically modified rapeseed and accidentally shipped to the UK. The UK had already planted these seeds and the crops had to be destroyed. • The total herbicides used with the herbicide resistant crops will kill all weeds and reduce the biodiversity. This in turn will effect the crops because if one species is taken out completely, you cannot successfully predict what will happen to the other species

23. Transgenic crops can also kill other beneficial organisms and thus affect other forms of life in the ecosystem. • Inserting organisms into others will subject them to natural genetic resistance to the toxins. • Transgenic crops can produce unpredictable toxins and allergens into food plants and therefore into the final product. Thus, transgenic crops are considered unstable as the number of copies of an inserted gene changes through later generations.