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Two types of vectors are used for the introduction of foreign genes into plant cells. Among them, the Ti-plasmid of Agrobacterium tumefaciens and a few plant viruses such as caulimoviruses and geminiviruses are the most important ones.
Vector # 1. Agrobacterium Tumefaciens Ti Plasmids:
Plants cells do not contain natural plasmids that can be utilised as cloning vectors. However, the bacterium Agrobacterium tumefaciens which primarily infects dicotyledonous plants (e.g., tomato tobacco, peas, etc.), but has been shown recently to infect the monocots (e.g., rice), contains the 200 kb Ti plasmid (i.e., Tumour inducing plasmid).
On infection, part of the Ti plasmid, the T-DNA, is integrated into the plant chromosomal DNA resulting in uncontrolled growth of the plant cells directed by genes in the T-DNA, and the development of a crown gall.
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Recombinant Ti plasmids with a target gene inserted in the T-DNA region can integrate that gene into the plant DNA, where it may be expressed. In practice, however, several refinements are made to this simple scheme.
The size of the Ti plasmid makes it difficult to manipulate, but it has been discovered that if T-DNA and the remainder of the Ti-plasmid are on separate molecules within the same bacterial cell, integration will still take place.
The recombinant T-DNA can be constructed in a standard E. coli plasmid, and then transformed into the A. tumefaciens cell carrying a modified Ti plasmid without T-DNA. A further improvement is made by deleting the genes for crown gall formation from the T- DNA.
So-called disarmed T-DNA shuttle vectors can integrate cloned genes benignly, and, if the host cells are growing in culture, complete recombinant plants can be reconstituted from the transformed cells.
Vector # 2. Caulimoviruses:
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Caulimoviruses are a group of plant viruses which have double-stranded DNA. These viruses have a very narrow host range. One such virus is Cauliflower Mosaic Virus (CaMV). This virus infects plants of the family Cruciferae.
The caulimoviruses are isometric particles and are 50 nm in diameter. The cells of the infected plants accumulate virus-containing proteinaceous inclusion bodies (i.e., viroplasma). These infected regions are regarded as the sites of virus assembly.
The DNA of the CaMV particles is a circular, double- stranded molecule which is 824 bp long. It has three discontinuities (gaps) at specific sites on each strand. Two gaps are present on one strand whereas other strand has only one gap.
These gaps have short regions of overlapping DNA produced by strand displacement. The free genome of CaMV is infectious and it has no tissue restriction. It has six genes, Gene I produces the factors needed for cell to cell spread of the virus.
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Gene II is responsible for the production of the aphid transmission factor. Gene III encodes a virion structural protein Gene IV produces the coat of protein. Gene V produces the enzyme reverse transcriptase. Gene VI produces the inclusion body protein.
Multiplication cycle of CaMV:
Cauliflower mosaic virus enters the host cell. Immediately after its entry, the coat proteins are shed and the DNA molecule enters the nucleus of the host cell. Inside the nucleus the single- stranded overlapping at the gaps are digested and the gaps are thus sealed.
This circular DNA molecule becomes supercoiled and associates with the host proteins to form a mini-chromosome. This mini-chromosome acts as a template and produces mRNA under the direction of the host RNA polymerase II.
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Two major RNA transcripts, 19S RNA and 35S RNA, are produced. The 19S RNA is the mRNA for the gene VI which encodes the subcellular inclusion bodies. The 35S RNA is genome length and this RNA comes out of the nucleus.
The 35S RNA also functions as a template for the synthesis of the minus-strand DNA which will act as template for the plus-strand DNA synthesis. Thus, a new complete viral DNA is synthesized.
The circular DNAs are finally assembled into viral particles, which can move from cell to cell within the host plant. During the life cycle of CaMV, its DNA does not integrate with the host DNA. This virus also does not cause cell lysis.
CaMV as a Gene Vector:
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The naked DNA of CaMV is capable of infecting host cells. Infections can be initiated by inoculating the host plant with DNA. DNA of CaMV has a single Sal I site. The DNA is linearised by Sal I site. The DNA linearised by Sal I site is infectious in vitro even in the absence of re- ligation.
Re-circularization occurs in the plant cell. The viral particles assembled in the host cells have the ability to spread from cell to cell as it is a systemic virus. Plant cell infected by the CaMV can be cultured in vitro to produce clones.
Limitations of CaMV as the Vector:
1. The envelope (=capsid) of CaMV does not accommodate larger DNA than the normal size.
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2. Genes of CaMV are closely packaged. CaMV lacks long non-essential sequences that could be replaced by foreign genes.
3. If DNA longer than few hundred nucleotides is inserted, the infectivity of vector is destroyed. However, a small foreign DNA having the bacterial dhfr gene (dihydrofolate reductase) replacing gene II, has been successfully expressed in plants.
The promoter sequences of the DNA of CaMV are very efficient. These promoters are isolated and are used to construct vector systems in which foreign genes are efficiently expressed.
