In this text, we shall see how selection data have been used to assist the design of experiments on the modification of plants using recombinant DNA (genetic engineering). It is evident that in some situations the potential of in vitro selection is limited by a number of difficulties. It is partly because of these difficulties that genetic engineering methods are assuming an increasingly important role in the modification of plants. We now consider some of these methods.The plant pathogen Agrobacterium tumefaciens causes crown gall disease in higher plants. Infection by the bacterium commonly results in the formation of timorous outgrowths, the morphology of which depends on the plant species. The bacterium is now known to possess a large (95-160 Md) tumour-inducing (Ti) plasmid, part of which (the T-DNA) is transferred and incorporated into the host genome during infection.
[...] Protoplasts are incubated with the DNA at 22-24 C for about 30 min in the presence of PEG. Alternatively, protoplasts are subjected to electroporation in the presence of the DNA to be inserted. During these treatments the recombinant DNA construct is taken up into the protoplasts which are then washed and cultured. In this way it has been possible to transfer genes directly into protoplasts of graminaceous plants such as Trilicum monococcum and Lolium multiflorum. The presence of the genes in the transformed cells can be determined by assessing levels of antibiotic resistance. [...]
[...] Clearly therefore, CaMV has potential for the transfer of agriculturally desirable traits into crops. Conclusion Attempts are being made to circumvent this problem through the use of binary vectors, i.e. one viral genome carrying foreign genes, and the second carrying viral genes essential for replication, etc. However, there are also general problems of disease symptoms which may be produced after expression of viral genes in the host plant. Clearly, much more research will be required before viruses acquire a major role in plant genetic engineering. SUMMARY It is [...]
[...] Genetic engineering Genetic engineering is also now being used to develop new plant varieties with reduced symptom expression during viral infection. Some cucumber mosaic viruses contain a small satellite RNA molecule which is dependent on the virus for replication. The satellite RNA molecule is, therefore, rather like a parasite of the virus. The consequence of this is that symptoms produced by the virus containing the satellite RNA are much reduced compared with the effects of satellite-free virus on plants. Presence of the satellite RNA may also lead to a reduced number of viruses in the infected plant. [...]
[...] Early work on Agrobacterium- mediated transfer of foreign genes into plant cells used selectable antibiotic-resistance markers inserted into the T-DNA. In order to ensure expression of the integrated resistance marker in the host cell it was necessary to include transcription signals which were recognized by the plant transcription mechanisms. Typically this was done by inserting the new gene into the coding region for nopaline synthase which is expressed constitutively during tumour induction. This placed the antibiotic- resistance gene under the transcriptional control of the mopaline synthase promoter in the T-DNA causing its expression in the growing tumour. [...]
[...] Specially designed Ti plasmid ‘transformation vectors' has now been constructed which, typically, have the following characteristics: - Absence of genes which interfere with normal plant differentiation - A genetic marker such as the GUS gene and a selectable marker, for example conferring antibiotic resistance, to indicate the presence of the incorporated T-DNA - Presence of the T-DNA ‘border regions', i.e. the DNA regions which flank the left and right ends of the T-DNA and are required for the insertion of T-DNA into the plant genome. [...]
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