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Chapter: Basic Concept of Biotechnology - Tools and Techniques in Biotechnology

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Transformation Techniques - Techniques of Biotechnology and Innovations

Agrobacterium mediated transformation - 1) Co-culturing 2) In plant transformation

TRANSFORMATION TECHNIQUES

The uptake of foreign DNA by cell is called transformation. This technique incorporates a new DNA fragment into a host cell. Therefore, incorporated cell will get extra trait/ improved its one of the trait. It can be achieved by two methods 1) Agrobacterium medium transformation 2) Direct gene transfer. Both the techniques aim at stable integration of foreign DNA molecule into the plant genome. Hence, the incorporated gene will inherent from generation to generation.


Agrobacterium mediated transformation

The binary / co-integrated vector carrying the GOI transferred into the plants by Agrobacterium in two ways 1) co-culturing 2) In plant transformation.

 

1) Co-culturing

The Agrobacterium carrying GOI carrying T-DNA and vir plasmid is cultured with plant cell/s (explants). The explants are the part from which has potentiality to regenerate into whole plant. Explants may be cotyledon leaves, thin cell layers, protoplast, tissue slice, leaf disc and section of roots, shoots or floral tissues. Before the co-culturing with Agrobacterium the explants are surface sterilized with 0.1% mercurychloride for 1 min (varies with explants and type of crop). The traces of mercury chloride washed off by sterilized water for 3-4 times. The dried explants incubated with log phase grown desired gene carrying Agrobacterium culture. The culture incubated for 2 days under darkcondition during the acetosyringone released from tissue (dicot) or artificially added (monocot) induce the vir genes expression. The series of vir protein act on T-DNA containing GOI and kanamycin resistant gene (antibiotic resistant gene) to transfer the gene/s into cultured explants genome. Later, cultured explants are washed in sterile water for couple of time to remove the Agrobacterium from explants. The washed explants are dried in sterile blotting sheet and later kept in regeneration medium containing antibiotic selection pressure such as carbenicillin to restrict the Agrobacterium growth and kanamycin to screen the transformed cell. In about 3-4 weeks the shoot will regenerate and it is transferred to rooting medium. In 15 days roots are produced and these plants are subjected for hardening. The two level of hardening carried primary hardening and secondary hardening. In primary hardening the tissue culture planting are transplanted into peat and kept under humidcondition with temperature of 25±1 after 2 weeks these plants are transferred to soil / green house.

 

2) In plant transformation

This technique circumvents the tissue culture practice and direction the sterilized plant tissue will be soaked Agrobacterium culture. Tissue may be seed, flower and leaf, tissue is immersed in a fresh culture of Agrobacterium, for an efficient transformation the vacuum is created, and it will facilitate the Agrobacterium to enter the cell. The plants are grown from these seeds and progeny obtained from this is believed to be containing the GOI in its genome.

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