In the first of a fortnightly series that demystifies genetic engineering and its impact on the environment and health, Suman Sahai explains the cut-and-paste processes that go into making a transgenic plant
A genetically-engineered (GE) plant is a plant into whose DNA a gene from an unrelated species has been incorporated. Such plants are also called transgenics. Transgenic plants currently in use have a limited number of genes in them because only a few genes have so far been identified. The Bt gene is the most frequently used gene in transgenic crops today. In fact, identifying and locating genes for agriculturally important traits is the most limiting step in the transgenic process. We still know relatively little about the specific genes required to enhance yields, improve stress tolerance, improve nutrient uptake, or otherwise affect plant character. Even after a gene is identified, it need not necessarily be available for use. For that to happen, scientists must understand how the gene is regulated, what other effects it might have on the plant, and how it interacts with other genes active in the same biochemical pathway.
Public and private research programmes invest heavily in plant genomics to rapidly sequence and determine the function of genes in the most important crop species.
Making a transgenic plant is essentially a cut-and-paste job. The gene of interest is cut out from one source and pasted into the genome of the host plant to make a transgenic plant. Special enzymes mediate this process. After the gene is identified, it has to be isolated and then cloned. Cloning is done by multiplying the gene in a bacterial vector. Once several copies of the gene are available, a 'gene construct' has to be made using other genes, before the gene of interest can be introduced into the host plant. This is done to ensure that the gene will express the protein it is supposed to in the new organism.
A gene construct typically consists of:
- A promoter sequence. This is attached to the gene for it to be correctly expressed, ie translated, into a protein. The promoter is the on/off switch that controls when and where in the plant the gene will be expressed.
- The terminating sequence. This is also referred to as 'the terminator', but is not to be confused with the other use of the term 'terminator', which refers to sterile seed technology. The termination sequence signals to the cellular machinery that the end of the gene sequence has been reached.
- Marker gene. A selectable marker gene is attached to the gene 'construct' in order to identify plant cells or tissue in which the transgene has been successfully incorporated. Identifying the incorporation event is a critical step, since it is a rare event. Of several thousand cells that are transformed, only a small percentage actually integrate with the new gene. The most common markers are for antibiotic resistance.
- Reporter gene. A reporter gene is often used to do a quick assessment to see if the introduced gene has been expressed. Usually, the introduced gene and reporter gene are attached to a common promoter.
1 The Agrobacterium method
The plant pathogen Agrobacterium tumefaciens that causes 'crown gall' disease in plants is able to colonise its host plant because it can transfer some of its own genes directly and permanently into the host plant genome. The ability of Agrobacterium tumefaciens to transfer its DNA into plant cells is exploited to deliver alien DNA to make transgenic plants.
The Agrobacterium gene transfer system has some limitations. It does not infect all species of plants. Commercially important plants, notably cereal grain, are not hosts for Agrobacterium and the gene transfer system does not work well in these plants.
2 The 'gene gun' method
A second method for gene transfer in plant cells is the 'gene gun', which was developed at Cornell University, USA. This technique has been especially useful in transforming species like corn and rice. In this case, the gene construct is coated onto tiny gold or tungsten beads that are blasted into the living plant cells at high velocities. The DNA coat leaches off the micro-projectile surface once it is inside the recipient cell, and a small fraction of the DNA becomes incorporated into the cell's genome through a process that is still not very clear. The 'gene gun' method has a much lower efficiency than Agrobacterium-mediated gene transfer, and the incorporated DNA sequence has often been re-organised by the time it is stably inserted into the plant genome.
Nevertheless, the 'gene gun' method has one advantage -- it will, in principle, allow any plant species to be transformed, including those that are not suitable hosts for Agrobacterium.
Making a transformed plant from transformed cells
Both the Agrobacterium and 'gene gun' methods are capable only of transforming a very small percentage of all the cells in the plant tissue being treated. To create a transformed plant in which all cells are transformed and carry the new gene, two further steps must be completed:
- A full plant must be regenerated only from one or more of the original transformed cells.
- All non-transformed cells must be eliminated
Generating a full-size fertile plant from a single cell is usually more difficult than the actual gene transfer process itself. While gene transfer technologies are now well established, the process of plant regeneration is still trial and error. Different varieties of the same species often differ drastically in their ability to be regenerated from small starting tissue pieces. Procedures need to be customised for each new genotype of interest. In fact genetically-engineered plants are, by definition, custom-made.
The insertion of single genes into plant genomes using either the Agrobacterium or 'gene gun' method is now a routine laboratory procedure, but the initial population of transformed plants created in the laboratory is far from homogeneous. Both the genetic-engineering techniques lead to random insertion events, that is, the location of the new gene within the host genome cannot be predicted. Therefore, each transformed individual will carry the transgene at a different location. In many cases, they will carry multiple copies of the transgene, some of which will be functional while others will not.
Sorting through the transgenic population and identifying those individuals that express the new gene construct requires considerable time, effort and expertise. Eventually, a limited number of lines that display the desired trait in the laboratory or greenhouse trials in a genetically stable manner will be chosen for more extensive testing and analysis, including field trials for several years, at multiple locations.
It is important to note that genetic engineering will only lead to a product when it is integrated into conventional breeding programmes where it can provide a source of genetic variation. The value of a transgenic variety depends on the quality of the parent plant into which the gene construct is integrated. The transgene cannot convert a poor variety into a good variety. It will only add a particular property, for instance, the Bt gene will add toxin to fight bollworm. If the cotton variety that has received the Bt gene is a poor-to-medium-yielder, the addition of the Bt gene will not change it into a high-yielder.
(Suman Sahai has a PhD in genetics. She is the Director of Gene Campaign, a leading research and advocacy organisation working on farmer and community rights, bioresources, IPR, indigenous knowledge and GE food and crops)