Gene transfer METHODS in PLANTS
For the genetic transformation in plants, we must build a vector (a genetic vehicle), which transports the genes of interest, on the sides of the necessary control sequences that promoter and terminator, and delivery of genes in the host plant. Two types of data transfer channels in the genes of plants:
Vector-mediated and indirect gene flow
Among the various vectors used in plant transformation, Agrobacterium tumefaciens plasmid Ti is widely used. These bacteria, known as "natural" genetic engineering of plants, because these bacteria have natural ability to transfer t-plasmid DNA into the genome of a plant infection cells at the wound and cause growth of unorganized mass of cells, known as Crown gall, Ti plasmids as gene vectors for delivery of alien genes into plant cells and tissue targets. Alien gene clone of t-DNA Ti-plasmid in place unwanted sequences.
For the transformation of plants, leaves of wheels (in the case of dicots) or embryogenic calluses (in the case of the Monocots) are infected with Agrobacterium carrying a recombinant plasmid vector-Ti disarmed. infected tissue then cultivate (joint cultivation) on the regeneration of shoots environment within 2-3 days, during which time the transfer of t-DNA, together with alien genes occurs. After that, when transformed tissue (leaf disks/calluses) is sent to the regeneration of the plants sampled honors environment augmented usually lethal concentrations of the antibiotic to selectively exclude non-transformed tissues. Through 3-5 weeks, recovered shoots (from leaf discs) are passed to the root cause, and after another 3-4 weeks complete plants transfer in soil after hardening (acclimatization) regenerated plants. Molecular methods such as POLYMERASE CHAIN REACTION and Southern hybridization-used to detect the presence of alien genes in transgenic plants.
Vectorless gene transfer or direct
In direct methods of gene transfer, the interest of foreign gene comes in plant host cell, without the aid of a vector. The methods used for the direct transfer of the gene in plants are:
Chemical mediated gene transfer, for example, chemicals such as polyethylene glycol (peg) and dextran sulfate cause DNA uptake in plant protoplasts.Calcium phosphate is also used to transfer DNA into the cultivated cells.
Microinjection of DNA is injected directly into protoplasts and plants or cells (specifically in the nucleus or the cytoplasm), using a thin tip (0.5-1.0 micrometerdiameter) Glass needle or mikropipetku. This method of gene transfer is used to introduce DNA into large cells, such as the oocytes, egg and early embryo cells.
Electroporation includes high voltage pulse applied to the protoplast or cell/tissue to the interim (temporary) pores in the plasma membrane, which facilitates the absorption of alien DNA.
The cells are placed in a solution containing DNA and subjected to electric shocks to cause holes in the membranes. Foreign DNA fragments to enter through the holes in the cytoplasm and the nucleus.
Shotguns for particle bombardment of particles/-in this way it is DNA containing genes to be turned over to put on a surface a minute gold or tungsten particles (1-3 micrometers) and bombard the target tissue or cells using shotguns for particles (also called the gene gun/shot gun/pistol microparticles). Hotel microparticles method bombing was originally named after its inventor of Ballistic Sanford (1988). Two types of plant tissue, usually used for particle bombardment-primary explants and proliferating embryonic tissues.
Conversion-this method is used for the introduction of foreign DNA into bacterial cells such as e. Coli. Conversion Rate (fraction of cellular population that can be) very good in this method. Naprimerpogloŝenie e. coli plasmid DNA, CaCl2 gelid conduct (0-50 c), followed by heat shock treatment on 37-450 c for about 90 seconds. Conversion efficiency refers to the number of transformants per microgram of added DNA.CaCl2 breaks the cell wall in certain regions of DNA and bind to the cell surface.
A bundle is a natural process of microbial recombination and is used as a method of gene transfer. In conjunction with two live bacteria come together and single-stranded DNA is transferred through cytoplasmic bridges from the donor to the recipient bacteria bacteria.
Liposome mediated gene transfer or Lipofection-Liposomes constitute a lipid molecule with water round the Interior that can transfer of nucleic acids. Liposome encapsulation of DNA fragments and ADHER cell membrane and fuse with them, to pass DNA fragments.