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Methods of Gene Transfer in PlantsBY: sippy ISSAC | Category: Agriculture | Submitted: 2013-02-23 20:45:20
The totipotency of the plant cells and transformation with foreign DNA paved the way for genetic engineering of plants for desired traits. There are several methods had been tried over the years
VECTOR MEDIATED GENE TRANSFER
(I) Agrobacterium mediated (ii) viral vector III) Agroinfection
DIRECT GENE TRANSFER
(i) Pollen mediated,
(iv) Particle bombardments or biolistics,
(vi) ultrasound-induced DNA uptake
(vii) silicon carbide fibre -mediated DNA uptake,
(viii)laser-mediated DNA uptake,
(ix)liposome-mediated DNA uptake,
(x)PEG-mediated DNA uptake, and
Agrobacterium-mediated and biolistics have been predominately used in transformation work. While both of these methods are quite effective, each has distinct advantages and shortcomings and their deployment has been largerly influenced by the characteristics of the plant tissue to be transformed.
AGROBACTERIUM-MEDIATED GENE TRANSFER;
Agrobacterium tumefaciens is gram negative soil bacterium which causes tumor formation(called crown gall) on a large number of dicotyledonous as well as some monocots and gymnosperms. Crown gall induction is due to the transfer of a specific DNA fragment, the T-DNA (transferred DNA), from a large tumor-inducing (Ti) plasmid within the bacterium to the plant cell. There bacterial genetic elements are required for T-DNA transferred to plants: (i) T-DNA border sequences that consist of 25 bp direct repeats, flanking and defining the T-DNA. Usually all DNA sequences between the borders are transferred to the plant (II)The virulence (vir) gene-encoded by the Ti-plasmid in region outside of the T-DNA vir A and vir G form a 2 component regulatory system responsible for transcriptional activation of the vir operons. The other vir genes are involved in the processing of the T-DNA targeting to the nucleus and are probably involved in precise T-DNA integration into plant genome.(iii) The third bacterial element necessary for T-DNA transfer consists of a number of chromosomal gene--chvA, chvB, pscA, exocC and att--with functions like response to plant wounds and attachement of the bacterium to the plant cell.
The development of plant transformation vectors using A. tumefaciens is based on the fact beside the border repeats none of the T-DNA sequences is required for transfer and integration. This means that the T-DNA gene can be replaced by any other DNA of interest, which will be transferred to the plant genome. Also, length of the T-DNA is not critical. Small(a few Kb OR LESS) AS WELL AS LARGE t-dna'S (150KB0 can be transformed by the Agrobacterium into the plant cell.
Particle bombardment or biolistics: The invention of the direct Gene Transfer Technique of particle bombardment was a major development in plant genetic manipulation as it had enabled the transformation of many plants not amenable to Agrobacterium based gene transfer techniques.
The method involved particle gun accelerated microprojectiles, typically tungsten or gold particles (1.2 mm in diameter) coated with plasmid DNA of interest, to velocities at which they can penetrate the plant cell walls. The most widely used particle gun is PDS-1000/He marketed by BioRad, which uses helium gas for propulsion of DNA coated gold/tungsten particles. Gold is preferred over tungsten because gold is biologically inert whereas tungsten degrades DNA over time and can be toxic to some cell type.
Transgenics have been produced from bombardments of various explants like shoot apices immature zygotic embryos, inflorescences etc. in maize, wheat, tobacco, barley, Arabidopsis and some other plants.
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