The term direct or vector less transfer of DNA is used when the foreign DNA is directly introduced into the plant genome. Direct DNA transfer methods rely on the delivery of naked DNA into the plant cells. This is in contrast to the Agrobacterium or vector-mediated DNA transfer which may be regarded as indirect methods. Majority of the direct DNA transfer methods are simple and effective. And in fact, several transgenic plants have been developed by this approach.
1. Liposome-Mediated Transformation: Liposomes are artificially created lipid vesicles containing a phospholipid membrane. They are successfully used in mammalian cells for the delivery of proteins, drugs etc. Liposomes carrying genes can be employed to fuse with protoplasts and transfer the genes.The efficiency of transformation increases when the process is carried out in conjunction with polyethylene glycol (PEG). Liposome-mediated transformation involves adhesion of liposomes to the protoplast surface, its fusion at the site of attachment and release of plasmids inside the cell.
2. Electroporation: Electroporation basically involves the use of high field strength electrical impulses to reversibly permeabilize the cell membranes for the uptake of DNA. This technique can be used for the delivery of DNA into intact plant cells and protoplasts. The plant material is incubated in a buffer solution containing the desired foreign/target DNA, and subjected to high voltage electrical impulses. This results in the formation of pores in the plasma membrane through which DNA enters and gets integrated into the host cell genome.
3. Transfection: Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production of proteins using the cell’s own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. Therefore, the DNA can be transiently expressed for a short period of time, or become incorporated into the genomic DNA, where the change is passed on from cell to cell as it divides.
4. Transformation: Transformation is the method of introducing foreign DNA into bacterial cells (e.g. E.coli). The uptake of plasmid DNA by E.coli is carried out in ice-cold CaCl2 (0-5°C), and a subsequent heat shock (37-45°C for about 90 sec). By this technique, the transformation frequency, which refers to the fraction of cell population that can be transferred, is reasonably good e.g. approximately one cell for 1000 (10-3) cells.The mechanism of the transformation process is not fully understood. It is believed that the CaCl2 affects the cell wall, breaks at localized regions, and is also responsible for binding of DNA to cell surface. A brief heat shock (i.e. the sudden increase in temperature from 5°C to 40°C) stimulates DNA uptake. In general, large-sized DNAs are less efficient in transforming.
