Genetic Manipulation In Plants
Over the past decade there has been a revolution in the ability of geneticists to manipulate the genomes of plants, as before the 1980's there was no sustained expression of foreign genes in a genetically engineered plant. However, now we are able to transfer any gene into a plant by routine procedures, as was previously possible with prokaryotic cells. This is mainly based on the Agrobacterium tumefacien vector system as well as other mechanisms, for the transfection of plant protoplasts (lack cell walls), including transfection via biolistic devices. Plant biology has therefore advanced rapidly, providing important biotechnological implications for creating plants with both novel and advantageous, engineered characteristics such as the agriculturally significance of crop herbicide resistance.
There are problems associated with transfection that limit the range of species available for routine transgenic plant production, mainly due to the fact that
Agrobacterium related gene transfer is generally only efficient in the dicotyledons (eg. Brassicae). Also, transgenic plant production by either Agrobacterium related systems or protoplast transfection requires the ability to regenerate plants from a single, or few isolated cells. These techniques have been extensively studied in a small collection of experimental species, focusing upon those of agricultural importance (ie. economically significant). For example, dicotyledonous species such as the tomato, tobacco, B. napus and petunia have well developed developmental systems associated, although surprisingly legumes which are classed as an essential world wide food crop don't have such developed procedures.
Mechanism For Agrobacterium Gene Transfer
Agrobacterium tumefaciens is a bacterium that has, and still, plays an essential role in the history in plant engineering. As early as 1907, it was found that inoculation of wound sites in some dicotyledons with Agrobacterium tumefaciens induced tumour formation (Crown gall disease), thus representing an oncogenic transformation. It was found that the tumour cells synthesise opines (amino acid derivatives) when transformed, and the specific opine produced is determined by the bacterial strain. Later it was discovered that a genetic element must be transferred from bacterium, to plant, at the wound site for tumour formation because the cancerous properties were hereditary. Tumour-forming A. tumefacien strains all contain large plasmids, found to be essential for virulence and are referred to as Ti plasmids (Tumour inducing).
However, analysis of plant tumour cells showed no sign of the plasmid, but specific segments (T-DNA) had been found to undergo stable integration into the genome.
Integration Of Foreign Genes Into Plant Genome By The Ti Plasmid
Agrobacterium was simply a natural tumour inducing system that genetic manipulation has now utilised, based on Ti plasmid mechanism (ie. similar to pBR322 plasmid in prokaryotes) as a means of inserting specific fragments of foreign DNA into the genome of plant cells (integration at random sites). For engineering usage the Ti plasmids have been modified to prevent the associated abnormal growth (ie. oncogenic growth), whilst maintaining their high levels of integration and expression of the foreign genes.
