In this article we will discuss about the role of Ti plasmids in genetic engineering of plants.
Genetic engineering of plants is carried out by introducing DNA into a cell in culture that can grow into a mature plant. An efficient vector for introduction of recombinant DNA into plant cells has been developed from plant viruses. Furthermore, the Ti plasmid isolated from the bacterium Agrobacterium tumefaciens also serves as a vector for inserting foreign DNA.
Agrobacterium tumefaciens is a gram negative, soil bacterium and a plant pathogen that induces tumour-like growths on plants called crown gall tumours. Gene transfer from the bacterium to the plant occurs naturally, resulting in tumours. Tumours can also be induced in gymnosperms and dicotyledonous angiosperms by inoculation of wound sites with A. tumefaciens.
Evidence suggests that crown gall tissue represents true oncogenic transformation because the undifferentiated cell mass of the tumour (callus) can be cultured in vitro even if bacteria are killed by antibiotics, still retaining its tumour-like properties. These properties include unlimited growth as a callus, and synthesis of opines, such as octopine and nopaline which are unusual amino acid derivatives not present in normal plant tissue.
The metabolism of opines is a central feature of crown gall disease. Plant cells acquire the property of opine synthesis when they are colonised by A. tumefaciens. The bacterium utilizes opine as its sole source of nitrogen and carbon.
The virulent strains of A. tumefaciens contain a Ti plasmid that confers tumour-inducing properties on the bacterium. Earlier investigators on crown gall tumours had observed that continued presence of Agrobacterium is not required to maintain plant cells in their transformed state.
In 1974 Zaenen and colleagues demonstrated presence of large-sized plasmids, of 140 to 235 kilo base pairs length in virulent strains of A. tumefaciens. Further experiments revealed that virulence and ability to use as well as induce synthesis of opines are plasmid-borne traits.
These plasmids were known as tumour-inducing plasmids (Ti plasmids). Ti plasmids also contain information about the specific type of opine that is synthesised in the transformed tissue and utilised by the bacterium.
Furthermore, the complete Ti plasmid is not found in the plant tumour cells. A specific segment of the plasmid, about 23 base pair in size is found integrated in plant nuclear DNA at a random site. This segment of DNA transferred from the plasmid is called T-DNA (transferred DNA).
T-DNA carries genes responsible for conferring unlimited growth and ability to synthesise opines upon the transformed plant tissue. The genes responsible for T-DNA transfer are located in a separate part of the Ti plasmid called vir (virulence) segment.
Two of these genes (virA and virG) are expressed constitutively at a low level of control. The vir gene expresses a protein that forms a conjugative plasmid through which T-DNA is transferred to the nucleus. Once inside the nucleus, T-DNA is incorporated randomly into nuclear DNA by a process of illegitimate recombination. In addition to plants, Agrobacterium can transfer DNA to other bacteria, yeasts and filamentous fungi.
Thus, Ti plasmid serves as a natural vector in genetic engineering of plant cells because it can transfer its T-DNA from the bacterium to the plant genome. The wild type Ti plasmids, however, are not suitable as vectors because of the presence of oncogenes in T-DNA which result in dis-organised growth in recipient plant cells.
To accomplish efficient regeneration in plants, attempts have been made to delete all of the oncogenes from the plasmid. Indeed, when Agrobacterium carrying non-oncogenic plasmids were allowed to transfer the modified T-DNA to plant cells, no tumours were produced.
Transgenic Plants from Transformed Protoplasts:
Somewhat parallel to transfection in animal cells, protoplast transformation has been achieved in plants. Plant protoplasts from which the rigid cell wall has been removed, are induced to take up DNA. A number of chemicals, in particular polyethylene glycol promote gene transfer across the protoplast membrane. Alternatively, DNA uptake can be induced by electroporation.
In a small proportion of the protoplasts, the DNA is incorporated stably into the genome (transformed protoplasts). The first successful experiments on plant regeneration using protoplasts were carried out on tobacco and petunia, subsequently in monocots such as Lolium.
Gene Transfer in Plants by Particle Bombardment:
In contrast with animal cells that are bounded by a unit membrane, plant cells are distinguished by presence of a thick, often multilayered cell wall. A technique involving particle bombardment was developed in 1987 for gene transfer in a wide range of plant species. A modified shotgun is used to accelerate small metal particles (1 to 4 µm) into plant cells at a velocity sufficient to penetrate the cell wall.
In the first experiments, onion skin cells were bombarded with tungsten particles coated in tobacco mosaic virus (TMV) RNA. About 40% of onion cells were found to contain particles three days after bombardment. These cells also showed evidence of TMV replication. Subsequently, explants from several plant species have been stably transformed by this technique.