Direct gene transfer methods for producing transgenic plants, DNA mediated transformation of protoplasts, electroporation, balistic methods used for gene transfer. herbicide, insect résistance plants
Direct Gene Transfer:- When stable transformation is achieved by incorporating the desired gene into the genome of plant cells without the help of any biological factor, it is called direct gene transfer.
1. Gene gun method:-
> It is a physical method of gene transfer.
> It is also called Biolistic method.
> In this, tungsten or gold particles of 1-2µm diameter are coated with DNA and fired them into the cell by a particle gun.
> Gold is expensive, but it is less harmful to cells.
> The cost of a particle gun is almost 15 lakhs.
> The particles are accelerated by compressed helium gas in the particle gun.
> DNA reaches inside the nucleus and becomes incorporated into the plant's genome.
> By this method, the gene is transferred to the meristem and embryo.
2. PEG mediated Gene Transfer:-
> It is a chemical method of gene transfer.
> In this method gene transfer is induced by PEG. (PEG = Poly Ethylene Glycol)
> Transformation medium is added in a beaker that has a high concentration of Mg2 + ions.
> Now plant protoplasts are put into this nutritive medium.
> Now put the desired gene in the beaker.
> Now take the pH to 8.
> Now add 20% PEG.
> Now decrease the concentration of PEG and increase the concentration of Ca2+ ions.
> Now incubate for some time.
> The transformation frequency increases 10 to 1000 times when plant protoplasts are transferred to ice after heat-shock at 45°C for 5 minutes just before adding DNA.
3. Electroporation:- Introduction of DNA into plant cells by making minute pores in the plant cell membrane is called as electroporation.
> This method involves suspension of plant protoplasts in a suitable ionic solution containing linearized recombinant plasmid DNA.
> This mixture is then exposed to low voltage-long pulses or high voltage short pulses for the desired number of cycles.
> The electrical pulses are thought to induce transient pores in the plasma lemma through which the DNA molecules are incorporated.
> Treated protoplasts are then cultured to obtain cell colonies and plants.
4. Microinjection:-
> It involving the mechanical insertion of the desirable DNA into a target cell.
> The target cell may be the one identified from intact cells, protoplasts, callus, embryos, meristems, etc.
> Microinjection is used for the transfer of cellular organelles and for the manipulation of chromosomes. > The technique of microinjection involves the transfer of the gene through a micropipette (0.5-10.0 pm tip) into the cytoplasm/nucleus of a plant cell or protoplast.
> While the gene transfer is done, the recipient cells are kept immobilized in agarose embedding, and held by a suction holding pipette. > As the process of microinjection is complete, the transformed cell is cultured and grown to develop into a transgenic plant.
> In fact, transgenic tobacco and Brassica napus have been developed by this approach.
> The major limitations of microinjection are that it is slow, expensive, and has to be performed by trained and skilled personnel.
Herbicide resistance plants:-
Insect resistance plants:-
Insect Resistance:-
Cry gene transfer:- The cry gene is found in the plasmid of Bacillus thuringiensis (Bt) bacteria. This cry gene makes Crystal protein which is insecticidal.
Ø The cry gene was isolated from the plasmid of Bt and integrated into the cotton genome to form a transgenic plant called Bt-cotton.
Ø This Bt - cotton is resistant to boll worm.
Ø Crystal protein perforates the larva's alimentary canal leading to its death.
