Gene transfer is one of the most recent areas of plant cell and tissue culture. It has brought a revolution in agriculture for the improvement of several genetic traits. Even in India voice of cotton sown in Andhra Pradesh and Gujarat was raised in Parliament.
Indian plant biotechnologists have made impressive progress in transformation of crops like mustard, rice, potato, tomato, and brinjal. Efforts are being made to transform the other crops too.
1. Direct Gene Transfer Methods :
There are five different methods that transfer foreign DNA of desired function into plant cells. These methods are briefly discussed herewith.
(a) Stimulation by Chemical Helpers:
In 1977, Lurquin and Kado demonstrated the stimulation by poly-L-ornithine in uptake of plasmid DNA by cowpea protoplasts. In the presence of calcium ions, polyethylene glycol (PEG) also enhances DNA uptake by the protoplasts in the range 10"2 and 10"5.
(b) Liposome-entrapped Plasmid Transfer:
Liposomes are microscopic lipid molecules. These are produced when phospholipids are dispersed in aqueous phase. Charges can be developed on liposomes as desired. Liposomes entrap DNA and interact with many cells in such a way that plasmid will be transferred into plant genome.
For example, unilamellar liposomes have been used for transformation and expression of a cloned cDNA copy of potato spindle tuber viroid (PSTV) and a chimeric DNA of E. coli plasmid into protoplasts.
Introduction of foreign DNA into the living cells (as protoplasts) by exposure of brief electric pulse (250-350 V) is known as electroporation. Minimum manipulation of protoplasts ensures their high survival rates. Using electroporation, transient expression of foreign DNA has been demonstrated in maize, wheat and rice.
In 1986, Crossway and co-workers used microinjection technique for direct gene transfer into plant cells. Animal cells can be transformed at high frequency by microinjection than the plant cells.
In this technique protoplasts are surface attached on a slide by embedding in agarose using a holding pipette. DNA is transferred by injecting a microneedle or a glass micropipette having fine tip CO.5 - 1µ diameter). The microinjected protoplasts are cultured as described earlier.
(e) Particle (Microprojectile) Bombardment Gun:
It is also called as particle gun, shot gun, microprojectile gun and biolistic missile. In this method foreign DNA containing desired genes are coated onto the surface of tungusten or gold particles (4 micrometer diameter).
These particles are bombarded to a high velocity (by particle gun) onto plant cells or tissues. The particles pierce cell walls and membrane.
2. Vector-mediated Gene Transfer in Plant Cells:
Agrobacterium consists of Ti-plasmid which is directly responsible for tumour induction. Transfer of small DNA segment (T-DNA) from Ti-plasmid and its integration into host genome induces tumour formation in plants. T-DNA region of Ti-plasmid is essential for Agrobacterium-mediated transformation of plants.
The Ti-plasmid is taken out from A. tumifaciens and a foreign DNA of desired function is inserted into T- DNA region by using restriction enzyme. The recombinant T-plasmid is incorporated into A. tumifaciens cells. The steps of Agrobacterium-mediated gene transfer in a plant.
(a) Co-cultivation with Protoplasts:
Freshly isolated protoplasts are co-cultivated with transformed A. tumifaciens for a few days. Then the protoplasts are washed and cultured in medium containing antibiotic. The regenerated protoplasts are transferred onto culture medium that enables the development of transformed callus and plants.
(b) Leaf-disc Infection Method:
In this approach leaves (of tomato, tobacco, Petunia, etc.) are surface sterilised and inoculated with a genetically modified A. tumifaciens cells. Then the leaves are cultured for two days. During this time infection takes place.
The leaf discs containing infection are transferred to a selection cum regeneration medium containing kanamycin. Kanamycin selectively eliminates the non-transformed cell/tissues. Within 2-4 weeks, these cultures regenerate into transformed plantlets.
Then, the genetically modified plantlets are transferred into soil after hardening of regenerated plant so that they can be acclimatised with the new environment. Presence and function of foreign gene in transgenic plant are found out by molecular techniques as given in transgene analysis.
3. Transgene Analysis :
In plant transformation experiments selection of transformed cells from the mixed populations of both transformed and untransformed cells is an important event. A marker gene (e.g. antibiotic resistance or herbicide resistance) is also introduced along with foreign DNA into the host cells.
A selection medium is prepared into which antibiotics or herbicide (for which marker gene has been introduced) is mixed. The transformed cells/tissues are cultured on selection medium.
Only those cells will grow on such medium which contains marker gene such cells are said to be transformed. The untransformed cells will not grow because they lack marker gene.
After selection subsequent maintenance of marker in transgenic plant is not necessary. Since the herbicide-resistance gene may be transformed to bacterium or herbicide-resistance gene may be transferred to weeds, the marker genes must be eliminated. The removal of marker enables the transgene to function and express properly.
Integration and expression of transgenes are further studied following molecular techniques like PCR, Southern blot, Northern blot, Western blot, etc.