More food, cleaner food – gene technology and plants
Box 1 | Adding a gene to an organism
The characteristics of all organisms are determined by genes. Genes determine characteristics by providing information to make proteins, basic biochemical units that control biological processes. One gene provides the information to make one protein. It is now possible to identify a gene that specifies a desirable characteristic, clone the gene (ie, clone the DNA) in a test tube and deliver the gene into the DNA of a cell from a different species. For example, scientists might add a gene for resistance to a particular disease to a plant of economic importance, such as wheat. When a new gene has been added, the cell is said to be transformed. The transformed cell can be grown into a wheat plant that also has the gene. The plant grown from the transformed cell is described as a transgenic wheat plant, as are any offspring carrying the gene.
Enzymes that cut and splice DNA are important tools for a genetic engineer
There are two basic techniques in manipulating DNA: cutting and splicing (ligating). Enzymes are used in both techniques. Restriction enzymes make breaks in the DNA, at specific sites. These enzymes are found in bacterial cells where they break down the DNA of invading viruses; scientists use restriction enzymes to cut DNA into manageable fragments. Another enzyme present in cells, DNA ligase, joins fragments of DNA together and can join DNA fragments from different organisms as easily as DNA fragments from the same organism. If DNA fragments from two different sources are mixed in a test tube with DNA ligase, sometimes the re-joining will put together the DNA from the two sources. DNA molecules composed of sequences derived from different sources are described as recombinant.
How scientists add a gene to an organism
Identifying a gene. To identify the gene for a particular characteristic from the huge amount of DNA within an organism is a daunting task. Before you begin, you need to know something about the gene - for example, what protein it contains instructions to make, or its base sequence.
Initially, scientists used information about the protein, such as its amino acid sequence, to eventually isolate the DNA molecule that contained the instructions for that protein. More recently, scientists have determined the entire sequence of bases that make up the entire genome of single-celled and more complex organisms. In 2000, a small plant called Arabidopsis, a relative of commercially important plants such as oil-seed rape, was the first plant to have its entire genome sequenced. The map of the human genome was announced in 2001.
Proteins with similar functions often have similar structures, and the genes coding for these proteins will have a related base sequence. So, as more and more genes are identified, from more and more organisms, the task of identifying a new gene for a particular characteristic becomes easier.
Cloning DNA using bacteria. Scientists use a restriction enzyme to cut all the DNA of a donor organism into manageable fragments of a few thousand bases in length. They then splice each fragment into a bacterial plasmid, a small circular DNA molecule, to create a recombinant plasmid. Scientists reintroduce each recombinant plasmid into a separate bacterium, creating a bacterial 'library' of the donor DNA. To clone the DNA the bacteria are spread thinly on a nutrient agar plate so that each bacterium is well separated from the others. Each bacterium grows into a colony of millions of cells, each of which contains an identical recombinant plasmid with its DNA fragment from the donor organism. Since there are millions of cells, there are now millions of copies of each DNA fragment.
Finding the fragment that you want. Fragments are recognised by their sequence of bases. A gene probe is like a template that will recognise only the bacterial colony containing the DNA of the matching fragment. Once the desired colony is identified, the number can be increased (cloned) to produce more copies of the DNA fragment.
Getting DNA into the cell. Getting the cloned and purified fragment of DNA into a living cell is the next step. This step is more difficult in plants than animals, because plant cells have a cell wall in addition to a cell membrane. The following methods are those most commonly used to introduce DNA into a plant cell:
- Transporting DNA into a cell via a bacterium. The bacterium Agrobacterium tumefaciens infects many plants, causing tumours to form. The tumour-inducing DNA resides in a plasmid of the bacterium. When the bacterium infects a plant, part of this plasmid is transferred to the plant cell nucleus. Scientists have capitalised on this ability and now use Agrobacterium as a vehicle for introducing new DNA into plant cells. They ligate the DNA of a desirable gene into the bacterium's plasmid, and the new DNA is delivered into the nucleus of the plant cell. The transformed plant cell doesn't produce a tumour because scientists removed the tumour-inducing genes from the plasmid.
The first version of Agrobacterium tumefaciens that did not initiate tumours was made in 1983 and has proved very successful. Using Agrobacterium to deliver a desired gene to a plant is now the most widely used method of gene delivery in plant genetic engineering.
- Removing the barrier of the plant cell wall. Not all plants are natural hosts of Agrobacterium tumefaciens. For example, wheat, rice and corn cannot be readily infected. So, for these plants, scientists remove the plant cell wall, producing a 'naked' plant cell. Without the barrier of the cell wall, DNA can more easily be delivered into the cell. One method used to introduce DNA into a 'naked' plant cell is to inject cloned DNA fragments using a very fine needle. Another is to produce pores in the cell membrane using short bursts of electric current. The DNA molecules can move through the pores which later mend.
Removing the cell wall makes the plant cell fragile, so the success rate of delivering DNA into these 'naked' cells is low.
- Shooting the DNA through the cell wall using a 'gun'. The DNA gun (or particle gun) is the most recent technique used to modify plants. This gun shoots the DNA in through the cell wall, so the problems with naked cells do not arise. It is crucial that scientists fire the DNA-coated bullets – pieces of metal about 2 micrometres across – with just the right amount of force. If there is too much force, the cells will be destroyed by the blast. If there is too little, the bullet will not pass through the thick wall and into the cell. The holes in the cell wall and membrane mend quickly and the bullets, although they remain in the cell, appear to do no lasting damage. The DNA diffuses away from the bullet and, with luck, is incorporated into the DNA of the plant. CSIRO scientists have recently transformed wheat using this method.
Low rate of success
With any one of these three methods, however, the chances are still very small of getting the DNA to be accepted within one of the cell's chromosomes where it can function as a gene. To ensure that at least one cell receives the DNA at an acceptable spot on its chromosomes, scientists expose tens of thousands of plant cells to cloned DNA fragments. Easily identified genes, called marker genes, are sent in as part of the cloned DNA so that scientists can tell whether the inserted DNA has been accepted by the plant cell. A successfully transformed cell is then carefully nurtured into a fully developed plant. If this plant produces seed, some of the resulting seedlings will contain the transformed gene.
Boxes
Box 2. Some examples of Australian gene technology research
Box 3. Concerns about gene technology
Related sites
Tissue culture for gene transfer
(CSIRO Plant Industry, Australia)
Transforming genes into a bacterial ‘syringe’
(CSIRO Plant Industry, Australia)
Molecular scissors slice DNA to isolate genes
(CSIRO Plant Industry, Australia)
Understanding the ABC of DNA technology
(CSIRO Plant Industry, Australia)
Plant transformation (Partnership for Plant Genomics Education, University of California, Davis, USA) Transforming plants – basic genetic engineering techniques (Access Excellence, USA)
Page updated December 2007.