Arizona State University College of Liberal Arts and Sciences

Bio343

Plant Genetic Engineering: Methodology

(Chapter 17)


Plant transformation

Genetic engineering of plants is much easier than that of animals. There are several reasons for this: (1) there is a natural transformation system for plants (the bacterium Agrobacterium tumefaciens), (2) plant tissue can redifferentiate (a transformed piece of leaf may be regenerated to a whole plant), and (3) plant transformation and regeneration are relatively easy for a variety of plants.

The soil bacterium Agrobacterium tumefaciens ("tumefaciens" meaning tumor-making) can infect wounded plant tissue, transferring a large plasmid, the Ti plasmid, to the plant cell. Part of the Ti (tumor-inducing) plasmid apparently randomly integrates into the chromosome of the plant. The integrated part of the plasmid contains genes for the synthesis of (1) food for the bacterium, and (2) plant hormones. Genes from the Ti plasmid that are integrated in the plant chromosome are expressed at high levels in the plant. Overproduction of the plant hormones leads to continuous growth of the transformed cells, causing plant tumors. Rapid, cancerous growth of the transformed plant tissue obviously is advantageous to the bacterium: more food gets produced.

The Ti plasmid has been genetically modified ("disarmed") by deleting the genes involved in the production of bacterial food and of plant hormones, and inserting a gene that can be used as a selectable marker. Selectable marker genes generally are coding for proteins involved in breakdown of antibiotics, such as kanamycin. Any gene of interest can be inserted into the Ti plasmid as well. In principle, one can thus transform any plant tissue, and select transformants by screening for antibiotic resistance. However, unfortunately, there are some complications: (1) it has proven difficult to transform some monocots (grasses, etc.) by Agrobacterium, and (2) regeneration of plants from tissue culture or leaf discs is not always possible.

A number of genetically engineered plant varieties have been developed. Traits that have been introduced by transformation include herbicide resistance, increased virus tolerance, or decreased sensitivity to insect or pathogen attack. Traditionally, most of such genetically engineered plants were tobacco, petunia, or similar species with a relatively limited agricultural application. However, during the past decade it now has become possible to transform major staples such as corn and rice and to regenerate them to a fertile plant. Increasingly, the transformation procedures used do not depend on Agrobacterium tumefaciens. Instead, DNA can be delivered into the cells by small, Ám-sized tungsten or gold bullets coated with the DNA. The bullets are fired from a device that works similar to a shotgun. The modernized device uses a sudden change in pressure of He gas to propel the particles, but the principle of "shooting" the DNA into the cell remains the same. This DNA-delivery device is nicknamed "gene gun", and has been shown to work for DNA delivery into chloroplasts as well. Over the last several years, use of the "gene gun" has become a very common method to transform plants, and has been shown to be applicable to virtually all species investigated. For example, transformation of rice by this method is now routine. This is a very important development as rice is the most important crop in the world in terms of the number of people critically dependent on it for a major part of their diet.

Another method to get foreign genes into cereals is by electroporation: a jolt of electricity is used to puncture self-repairing holes in protoplasts (i.e., the cell without the cell wall), and DNA can get in through these holes. However, it is often very difficult to regenerate fertile plants from protoplasts of cereals. Nonetheless, significant advances in overcoming these practical difficulties have been made over the years. Now even transgenic trees have been created: for example, the gene for a coat protein of the plum pox virus has been introduced into apricot. The plum pox virus leads to the feared Sharka disease, for which there is no cure. The resulting transgenic tree shows a markedly decreased sensitivity to this virus. The reason why continuous exposure of the tree to the viral coat protein leads to tolerance against viral infection is not yet understood, however.

Thus, now there are a number of different techniques to introduce foreign genes into plants. Essentially all major crop plants can be (and have been or are being) genetically engineered, the procedures are now routine and the frequency of success is very high. Even though genetically engineered crops are more costly than the usual ones, they have been rather readily accepted by US farmers provided that tangible benefits can be demonstrated. However, it is questionable whether the farmer in poorer countries can come up with the funds to "try out" and use the new crops. Another issue in this respect is how genetically engineered crops are perceived by the consumer. Even though in the US there is little resistance to such crops as long as the products can be shown to be safe and advantageous, in other countries (for example in sections of Europe) genetically modified foods are received poorly by the consumer. It is unlikely that there is a rationally sound basis for this rather hostile reaction of the consumer, as most of the crops are the result of human manipulation (such as centuries of breeding) and may have been treated with harmful herbicides and pesticides. Time and education will need to be invested to provide consumers and consumer advocates with a balanced opinion on the acceptability of the origin of their foods. A website that is critical of plant genetic engineering and genetically engineered foods is http://www.biointegrity.org/. One area of particular concern for some people is the lack of labeling of genetically engineered foods, and legislation may be introduced to address this issue (for example, see http://www.thecampaign.org/ and http://www.safe-food.org/). On the other hand, as so many plants (soybean, corn, etc.) are genetically modified and the nature of the genetic modification is not necessarily easy to explain, it may be simpler to label those foods that are guaranteed free of "genetically modified organisms" or their products. However, keep in mind that essentially all agricultural products have been genetically modified by traditional breeding, so it may be difficult to define what is actually free of genetically modified organisms.

Several plant biotechnology companies have increased their efforts to provide information regarding the full, global scope of impacts of plant biotechnology. For example, Monsanto (http:/www.biotechknowledge.monsanto.com/) and Du Pont (http://www.dupont.com/biotech/) have useful web sites. These company sites probably are just as subjective as some of the sites listed above that are very critical of genetically modified organisms, and the best solution is to read information provided by both sides and to see what is reasonable.


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Instructors | Aims
Lecture Part: Schedule | Expected Background & Textbook Info | Historical Perspective
Intro to Biotechnology | DNA, RNA and Protein Synthesis | Chemical Synthesis, Sequencing, and Amplification of DNA |
Directed Mutagenesis and Protein Engineering | Vaccines | Antibiotics & Proteins | Bioremediation |
Microbial Insecticides | Plant Genetic Engineering: Methodology | Plant Genetic Engineering: Applications | Transgenic Animals
Human Molecular Genetics | Regulatory & Ethical Aspects | Biotech Inventions | Additional Materials
Lab Part: Aims and Expectations | Schedule

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