Overview

This lesson is a general description of the overall process of genetic engineering. A basic explanation of the five steps for genetically engineering a crop is provided. Details for each step are given in later lessons of this course. The five steps are:

1. Locating an organism with a specific trait and extracting its DNA.
2. Cloning a gene that controls the trait.
3. Designing a gene to express in a specific way.
4. Transformation, inserting the gene into the cells of a crop plant.
5. Plant breeding to get the transgene into an elite background.

Authors

• Patricia Hain, Department of Agronomy and Horticulture, University of Nebraska-Lincoln
• Don Lee, Department of Agronomy and Horticulture, University of Nebraska-Lincoln

Published 2000

Introduction

Genetic engineering is the directed addition of a foreign gene or genes to the genome of an organism. A gene holds information that will give the organism a trait (Fig. 1).

Genetic engineering is one type of genetic modification. Traditional plant breeding also modifies the genetic composition of plants. Plant breeding, which involves crossing and selection of new superior genotype combinations, has been going on for hundreds of years. Every time someone crosses two plants in order to improve their traits, they are genetically modifying the plants.

Plant breeding is an important tool, but has limitations. First, breeding can only be done between two plants that can sexually mate with each other. This limits the new traits that can be added to those that already exist in that species. Second, when plants are mated, (crossed), many traits are transferred along with the trait of interest including traits with undesirable effects on yield potential.

Genetic engineering, on the other hand, is not bound by these limitations. It physically removes the DNA from one organism and transfers the gene(s) for one or a few traits into another. Since crossing is not necessary, the ‘sexual’ barrier between species is overcome. Therefore, traits from any living organism can be transferred into a plant. This method is more specific in that a single trait can be added to a plant.

Try out the genetic engineering steps yourself through Who Wants to be a Genetic Engineer – CinnaApples or Bt Corn.

Step 1: DNA Extraction

The process of genetic engineering requires the successful completion of a series of five steps and discoveries. To better understand each of these, the development of Bt corn will be used as an example.

Before the genetic engineering process can begin, a living organism that exhibits the desired trait must be discovered. The trait for Bt corn (resistance to European corn borer) was actually discovered around 100 years ago. Silk worm farmers in the Orient had noticed that populations of silk worms were dying. Scientists discovered that a naturally occurring soil bacteria was causing the silk worm deaths. These soil bacteria, called Bacillus thuringiensis, or Bt for short, produced a protein that was toxic to silk worms, the Bt protein.

Although the scientists didn’t know it, they had made one of the first discoveries necessary in the process of making Bt corn. The same Bt protein found to be toxic to silk worms is also toxic to European corn borer because both insects belong to the Lepidoptera order. The production of the Bt protein in the Bt soil bacteria is controlled by the soil bacteria’s DNA.

In order to be able to work with the DNA, scientists have to extract it out of the soil bacteria (Fig. 2). DNA extraction is the first step in the genetic engineering process. This is accomplished by taking a sample of bacteria containing the gene of interest and taking it through a series of steps that separate the DNA from the other parts of a cell.

Step 2: Gene Cloning

The second step of the genetic engineering process is gene cloning (Fig. 3). During DNA extractionDNA extraction, all of the DNA from the organism is extracted at once. This means the sample of DNA extracted from the Bacillus thuringiensis bacteria will contain the gene for the Bt protein, but also all of the other bacterium’s genes. Scientists use gene cloning to separate the single gene of interest from the rest of the genes extracted.

The next stages of genetic engineering will involve further study and experimentation with this gene. In order to do that, a scientist needs to have thousands of exact copies of it. This copying is also done during the gene cloning step.

Step 3: Gene Design

Gene design relies upon another major discovery. This was the ‘One gene One enzyme‘ Theory first proposed by George W. Beadle and Edward L. Tatum in the 1940’s. Discoveries made during their research laid the groundwork for the theory that a single gene stores the information that directs the cell in how to produce a single enzyme (protein). Therefore, there is a single gene that controls the production of the Bt protein. It is called the Bt gene.

Once a gene has been cloned, genetic engineers begin the third step, designing the gene to work once inside a different organism. This is done in a test tube by cutting the gene apart with enzymes and replacing certain regions (Fig. 4).

The first Bt gene released was designed to produce a level of Bt protein lethal to European corn borer and to only produce the Bt protein in green tissues of the corn plant, (stems, leaves, etc.). Later, Bt genes were designed to produce the lethal level of protein in all tissues of a corn plant, (leaves, stems, tassel, ear, roots, etc.).

Step 4: Transformation

The modified gene is now ready for the fourth step in the process, transformation or gene insertion.

Since plants have millions of cells, it would be impossible to insert a copy of the transgene into every cell. Therefore, tissue culture is used to propagate masses of undifferentiated plant cells called callus. These are the cells to which the new transgene will be added.

The new gene is inserted into some of the cells using various techniques. Some of the more common methods include the gene gun, agrobacterium, microfibers, and electroporation. The main goal of each of these methods is to transport the new gene(s) and deliver them into the nucleus of a cell without killing it. (For example, the gene gun method of transformation shoots gene-coated, microscopic gold particles at plant cells to deliver the DNA into the nucleus. Transformed plant cells are then regenerated into transgenic plants. (Fig. 5))

From there, the new DNA may or may not be successfully inserted into a chromosome. The cells that do receive the new gene are called transgenic and are selected from those that are not transgenic. Many types of plant cells are totipotent meaning a single plant cell can develop into an entire plant. Therefore, each transgenic cell can then develop into an entire plant which has the transgene in every cell. The transgenic plants are grown to maturity in greenhouses and the seed they produce, which has inherited the transgene, is collected. The genetic engineer’s job is now complete. He/she will hand the transgenic seeds over to a plant breeder who is responsible for the final step.

Step 5: Backcross Breeding

The fifth and final part of producing a genetically engineered crop is backcross breeding (Fig. 6). Transgenic plants are crossed with elite breeding lines using traditional plant breeding methods to combine the desired traits of elite parents and the transgene into a single line. The offspring are repeatedly crossed back to the elite line to obtain a high yielding transgenic line. The result will be a plant with a yield potential close to current hybrids that expresses the trait encoded by the new transgene.

The Process of Plant Genetic Engineering

The entire genetic engineering process is basically the same for any plant (Fig. 7). The length of time required to complete all five steps from start to finish varies depending upon the gene, crop species, and available resources. It can take anywhere from 6-15+ years before a new transgenic hybrid is ready for release to be grown in production fields.

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Summary

• Genetic engineering is the directed addition of foreign DNA (genes) into an organism. Five basic steps in crop genetic engineering:
  • DNA extraction – DNA is extracted from an organism known to have the desired trait.
  • Gene cloning – The gene of interest is located and copied.
  • Gene modification – The gene is modified to express in a desired way by altering and replacing gene regions.
  • Transformation – The gene(s) are delivered into tissue culture cells, using one of several methods, where hopefully they will land in the nucleus and insert into a chromosome.
  • Backcross breeding – Transgenic lines are crossed with elite lines to make high yielding transgenic lines.
• Differences between genetically engineered crops and non-genetically engineered crops:
  • Genetically engineering crops is done by manually adding totally new gene(s) to an organism. Therefore there is no need for sexual mating to pass on genetic material. As a result, a gene from any organisms can be introduced into another organism regardless of which species they belong to. This allows for many more possibilities of genetically engineered crops especially for crops that contain beneficial traits that their species never had before. Genetic engineering is very precise allowing scientists to move a single gene or just a few genes.
  • Plant breeding is done by mating two plants to obtain improved offspring. Sexual mating is required in plant breeding. Therefore, plant breeding can only be done between two organisms/plants that can sexually mate with each other, and genetic improvement is limited to selecting for traits that already exist within a species. When mating occurs, the offspring get half of their genes from each parent. As a result, many genes are transferred to the offspring, even those with undesirable effects on yield potential.

Acknowledgements

Development of this lesson was supported in part by Cooperative State Research, Education, & Extension Service, U.S. Dept of Agriculture under Agreement Number 98-EATP-1-0403 administered by Cornell University and the American Distance Education Consortium (ADEC). Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.