# Structural Biochemistry/Nucleic Acid/DNA/Transgenic Plants

Transgenic plants are genetically engineered to have genes from other organisms inserted into their genome. Transgenic plants are identified as a class of genetic modified organisms (GMO). The introduced genes do not have to be from the plant kingdom, but can come from animals, viruses, or bacteria as well. The uses of exogenous gene introduction include virus immunity, a replacement for pesticides, the ability to grow in acidic soil, and greater nutritional content.

## Making Transgenic Plants

Transgenic plants are constructed by inserting genes from other organisms into the host plant's DNA sequence. For this to happen a desired gene must be isolated and cloned. A few changes must be made to the gene so that it can effectively be inserted into the plant. First, a promoter sequence must be added to the gene. The promoter sequence is an on/off switch that controls where and under what cues the gene is expressed. The gene must also sometimes be modified (e.g. The Bt gene for insect resistance has a greater amount of A-T nucleotide pairs than plants, which tend to have more C-T pairs. The A-T nucleotides can be substituted for with C-T pairs in a manner that does not significantly change the amino acid sequence, leading to greater protection of the inserted gene in plant cells.). A terminal sequence must also be added to signal when the end of the gene sequence has been reached. Finally, a selective marker gene must be inserted to identify plant cells which have successfully integrated the transgene.

### Agrobacterium System

A method that is used to transform plants is the Agrobacterium method and the "Gene Gun" method. The Agrobacterium method uses Agrobacterium tumefaciens, a soil-dwelling bacterium that has the ability to infect plant cells by introducing transfer DNA, or T-DNA of a tumor-inducing (Ti) plasmid (i.e. a DNA sequence that can replicate independently of chromosomal DNA and is often circular) to the host's nuclear DNA. The bacteria is part of the rhizobiaceae family which is responsible for many tumors found in plants. The Ti plasmid contains the T-DNA as well as a series of vir (virulence) genes that direct the infection process. Agrobacterium tumefaciens can be used as a vector for gene transfer into plants. First, a hybrid plasmid that carries only the T-DNA from a Ti plasmid is cut open with a restriction enzyme and a foreign gene is inserted, creating a recombinant plasmid. The recombinant plasmid is then transferred into an Agrobacterium tumefaciens cell that contains a Ti plasmid that has had its T-DNA removed. The Agrobacterium with the engineered plasmid is then used to infect a plant and integrates the T-DNA with the foreign gene into the plant genome. For the Agrobacterium to be used the DNA must be able to penetrate into the plant cells. This is often done with electroporation, where brief high-voltage electrical pulses are administered to naked protoplasts (i.e. plant tissues and DNA). The electrical pulses open the pores in the plasma membrane allowing the DNA to enter the protoplast (which can then be grown into a mature plant by treating it with hormones). In the "Gene Gun" method, gold or tungsten microspheres (about 1 micrometer in diameter) are coated with the DNA or RNA from the specific gene of interest. The microspheres are then accelerated into undifferentiated target cells in a petri dish. Once inside the cells, the gene from the DNA coating the microsphere is released and can be incorporated into the host plant genome. The advantage of this method is that a high percentage of a single copy of T-DNA can used to transform the plant. In addition, they are an abundant of vector system available to carry out this method.

### Biolistic Method

This method delivers microprojectiles that are coated with DNA by accelerating it into the cell of interest. The microprojectiles are usually made up of tungsten or gold. To carry out the acceleration, an explosion is made with gunpowder under high pressure of helium. Plants that are made using the boilistic method have multiple copies of a gene that is still able to segregate in a Mendelian pattern. This method helps increase the diversity seen in plants. There are some advantages to the biolistic method compared to the Agrobacterium method. The plants that undergo the bombardment of genes in this method are still fertile. Other advantages includes this is the only reliable method to transform the chloroplast and this method does not need any transformation vector.

## Importance of Transgenic Plants

The new methods developed to transform plants have opened a new field of interest. Transgenic plants are used to solve a lot of problems in the agriculture sector. In addition, transgenic plants can be used in the medical field

### Nutrients of Transgenic Plants

When people go to the supermarket, they often buy fruits that are not soft or overly ripened. The major problem in the agriculture field with fruits is that the fruits often become soft during processing and transporting because they are being ripened. Using one of the methods for creating transgenic plants, scientists are able to slow down the process of ripening. Three companies have been able to apply this technology to slow down the ripening of tomatoes. And now other companies are hoping to be able to do the same for other fruits such as mangos or papayas. Cereal grains and legume seeds are a big source of protein for many people. However, the cereal grains and legumes seed often lack certain amino acids such lysine in cereal grains and methionine in legume seeds. Many efforts have been put into creating seeds that are higher in nutritional values. Currently, transgenic tobacco and canola seeds have a 33% increase in methionine due to the transgenic technology. In addition, the nutritional values have potatoes have increased by transforming it with AmA1, a gene from amaranth.
Increasing the nutritional values in plants and fruits can address many malnutrition problems and diseases. Vitamin A deficiency is a huge problem in Asia that affects around 124 million children and causes blindness. The main staple in Asia is rice, but rice does not contain any vitamin A. Researches are being performed in hope of developing rice that is rich in vitamin A. Currently, scientist have found the genes that encode for B-carotene (pro-vitamin A) enzymes in the endosperm of transgenic rice seed and they hope to use this information to engineer rice in a way that vitamin A can be produce through the rice.

## Uses of Transgenic Crops

The use of transgenic plants for pathogen resistance has received the most attention from popular media. The use of GMOs has been a topic of debate since their introduction in the mid-1990s. The two best known cases were virus-immunity in papayas and insect immunity in crops such as corn through a gene from Bacillus thuringiensis (BT). The papaya ringspot virus (PRSV) that severely damages papaya trees was causing a major toll on the papaya industry in Hawaii. Genes for the protein coat of the virus were inserted into papaya tissue by using the gene gun. Some of the papaya cells incorporated the viral genes into their DNA, giving the plant immunity to PRSV. This saved the Hawaiian papaya industry. The introduction and use of BT crops is even more publicized. The BT gene codes for the Cry proteins which are toxic to and that specifically target and kill the larvae of butterflies and moths. By introducing this into plants, crops such as corn, rice, and potatoes were able to exhibit the Cry proteins, and have proved to be very effective at stopping insect pests such as the European corn borer caterpillar. The protein is very selective and does not harm other insects (e.g. beetles, flies, bees, wasps) and is also considered safe for human consumption. The use of the BT endotoxic has greatly reduced the use of pesticides on crops. However, issues concerning immunity of the pests to the BT corn are a problem, and refuge crops that do not contain the toxin are planted to reduce the evolution of the caterpillar immunity to the Cry proteins.
GMOs have also been bred to improve food nutritional quality, to induce a longer shelf-life by delaying senescence, to allow corn to grow in acidic soil, to protect strawberries from cold temperatures, and a variety of other uses.

## References

Bessin, Ric. Bt-Corn: "What It Is And How It Works". University of Kentucky College of Agriculture. January 2004.
Transgenic Crops: An Introduction and Resource Guide. Colorado State University Soil and Crop Sciences. March 2006.
Lipps G (editor). (2008). Plasmids: Current Research and Future Trends. Caister Academic Press. Raven, Peter. "Biology of Plants". W.H. Freeman and Company. New York. 2005. "Harvest of Fear" (Film) - Nova. PBS. 2004
Peña, Leandro. Transgenic Plants: Methods and Protocols. Totowa, NJ: Humana, 2005. Print.