Plant breeding has several other serious limitations for use as a tool to increase disease resistance. There are only a limited number of plant species which are able to cross-fertilize, thus restricting the transfer of potentially useful traits. Moreover, having found useful traits, it is impossible to prevent the co-transfer of undesirable ones, which can take many years to breed out again by back-crossing.
How can genetic engineering be of use in the quest for new answers to the problems of providing plants with the ability to resist disease? The aim of crop plant genetic engineering is to insert a gene (or genes) which improve an existing plant variety whilst retaining the desirable genetic make up of the original plant.
The main tool at the disposal of the scientist is the use of nature’s own genetic engineer, Agro-bacterium tumefaciens. The manipulation of this bacterium’s natural functions has allowed the biologist to transfer many foreign genes into plants. The bacterium is soil-borne and infects plants at the crown, usually through a wound site, causing cancerous growths of proliferating plant cells known as crown gall tumors. This disease in itself is ergonomically important and effects most dicotyledonous plants causing millions of dollars’ worth of damage to plants. In the 1940s, from experimental observations, it was concluded that a factor is transmitted from the invading bacteria to the host plant cell. Further studies demonstrated that the disease is actually the direct result of the transfer of a particular DNA fragment (genes) from the bacterium to the plant cell. In addition to its chromosomal DNA, Agro-bacterium contains a much smaller circular DNA molecule called a Ti (tumor-inducing) plasmodia, of which a small piece, called the T-DNA (Transferred-DNA), is the factor transferred into plant cells (see Figure 1). The T-DNA becomes stably integrated into the plant’s chromosomes, from where it is able to perturb the natural functions of the plant. The T-DNA encodes genes, which, when expressed, bring about the production of new enzymes that are able to alter the hormone balance within the infected cell. This brings about de-differentiation and cell division, leading to proliferation of cells and the formation of tumors. This appears to be of little benefit to the bacterium. However, other genes are also present in the T-DNA which, when expressed, are able to synthesize novel compounds from naturally occurring plant precursors. These novel compounds cannot be metabolized by the plant but are a good source of nutrients for the bacterium.
Mechanism of gene (T-DNA) transfer from Agrobacterium to the plant cell
Mutation analysis of the T-DNA revealed two regions, the left and right borders, which were essential for integration into the chromosome. It was also found that any piece of DNA inserted between these borders was stably inserted into the host chromosome on transformation. Deletion of the genes for tumor formation (Disarmed Ti-Plasmid) were found to have no effect on the transfer efficiency from bacterium to plants. Availability of disarmed Ti-plasmids, tissue culture methods for the regeneration of whole fertile plants from single cells, and marker genes (such as antibiotic resistance) for the selection of transformed cells, have allowed for the production of a whole new range of plants containing foreign genes. Several genes responsible for pathogen and herbicide resistance proteins have been isolated from the bacteria and viruses. These genes have then been inserted into the T-DNA region of Agro-bacterium and introduced into plants, giving rise to insect, virus or herbicide resistant plants.
By using Agro-bacterium as a plant genetic engineer, many crop plants such as the tomato, potato and cucumber have now been engineered for virus resistance. Field tests showed that these genetically modified plants appeared to be highly resistant to viral infections. Similarly genetically-engineered cotton plants have proved to be resistant to insect attack and many herbicides (weed killers).
Another powerful new genetic engineering technique is ‘antigens’ technology, whereby specific gene transcripts are prevented from being translated into proteins. By using antigens technology, it has been possible to produce genetically engineered tomato plants that have a much increased shelf-life. Although many aspects of gene transfer from Agro-bacterium to plants are not fully understood, the use of Agro-bacterium for gene transfer will continue to increase; and it is likely that genetically engineered crops carrying traits for resistance to herbicides, insects and viral diseases will soon reach the market-place.