- The term ‘direct gene transfer’ is used to discriminate between methods of plant transformation that rely on the use of Agrobacterium (indirect methods) and those that do not (direct method). Direct gene transfer methods rely on the delivery of large amounts of ‘naked’ DNA whilst the plant cell is transiently (rapidly) permeabilised. The main types of direct gene transfer methods will be considered in detail below. others, less reproducible methods are laser-mediated uptake of DNA, ultrasound and in plant exogenous application.
1. Particle bombardment / gene gun method/ biolistic method / micro projectile
- Particle bombardment or biolistic or gene gun is the most important and effective direct gene transfer method in regular use. In this technique, tungsten or gold particles are coated with the DNA that is to be used to transform the plant tissue. The particles are propelled at high speed into the target plant material, where the DNA is released within the cell and can integrate in to the genome.
- The delivery of the DNA using this technology has allowed transient gene expression (which does not depend on integration of the transgene into the plant genome) to be widely studied, but integration of the transgene occurs very rarely. In order to generate transgenic plants, the plant material, the tissue culture regime and the transformation conditions have to be optimized quite carefully. Developments to this technology led to the production of a number of systems, such as an electrostatic discharge device and others based on gas flow. Of the later type, a commercially produced, helium-driven, particle bombardment apparatus (PDS-1000 He) has become the most widely used.
- Plant tissues used for bombardment are generally of two types: primary explants that are bombarded and then induced to become embryogenic; or proliferating embryogenic cultures that are bombarded and then allowed to proliferate further and subsequently regenerate. This method is found to be suitable for cereals which are otherwise recalcitrant to Agrobacterium mediated transformation. However, the major complication is the vector DNA is often rearranged and transgene copy number can be very high. Despite the high copy number, the single locus may have benefits for subsequent breeding program.
Brief mechanism of biolistic method
- The recombinant plasmids are constructed in such a way that it carries the gene of interest, selectable marker gene (like hyg- confers resistance to hygromycin) and reporter gene (e.g. gusA- which can be easily assayed by histochemically). The plasmid is coated on to gold particles (which are termed as microcarrier) for bombardment into the plant cell. Gold or tungsten particles(beads or balls) are used as microcarrier and they are prepared by mixing plasmid DNA with beads along with spermidine (a polyamine).
- The microcarrier are then mixed, washed with ethanol and finally resuspended in ethanol. The microcarriers are then applied to the macrocarrier membrane as an ethanol suspension and are allowed to dry on to the macrocarrier. The plant material used for transformation is embryogenic callus derived from mature seeds on MS1 medium. The embryogenic callus is arranged in a petridish containing high concentration of maltose to generate a high osmoticum prior to particle bombardment of the plant tissue.
- Once the vacuum in the lower part of the apparatus is established, the helium pressure above the rupture disc is increased until at 1100 psi (or whatever pressure the rupture disc is designed to rupture at) the rupture disc bursts. This propels the fragments of the macrocarrier and the projectiles down the chamber.
- The macrocarrier is stopped at the stopping plate and allowing only the microcarrier to pass through and hit the plant material. Various parameters can be optimized in this system: the distance between the stopping plate and the plant material can be varied; the pressure at which the rupture disc bursts can also be selected. Varying these parameters allows the speed and pattern of the microcarriers to be adjusted to suit the needs of the plant material being transformed.
- One day after bombardment, the embryogenic callus is transferred to MS1 medium. After 1 week, the embryogenic callus is transferred to selection medium (MS1 medium+hygromycin) and incubated in dark for 2 weeks. This allows only the transformed cell to proliferate. Then the surviving callus is transferred to shoot and root regeneration medium.
- The presence (Southern blotting) and expression of the gene interest is verified by different means (Northern blotting and Western Blotting) and the positive plants are selected. Young plantlets are acclimatized in growth chambers and can then be transferred to soil and grown to maturity.
Polyethylene glycol (PEG) mediated transformation
- Plant protoplasts (plant cells without cell wall) can be transformed with naked DNA by treatment with PEG in the presence of divalent cations (usually calcium). The PEG and the divalent cations destabilize the plasma membrane of the plant protoplast and render it permeable to naked DNA. Once entered into the protoplast the DNA enters the nucleus and integrates into the genome.
- Plant protoplasts are not easy to work with, and the regeneration of fertile plants from protoplasts is problematical for some species, limiting the usefulness of the technique. The DNA used for transformation is also susceptible degradation and rearrangement. Despite these limitations, the technique does have the advantages that protoplasts can be isolated and transformed in large numbers from a wide range of plant species.
Electroporation
- The Electroporation of cells can be used to deliver DNA into plant cells and protoplasts. The vectors used can be simple plasmids. The genes of interest require plant regulatory sequences, but no specific sequences are required for integration.
- Material is incubated in a buffer solution containing DNA and subjected to high-voltage electrical pulses. The DNA migrates then through high voltage induced pores in the plasma membrane and integrates into the genome. Initially, protoplasts were used for transformation, but one of the advantages of the system is that both intact cells and tissues (such as callus cultures, immature embryos and inflorescence material) can be used.
- However, the plant material used for Electroporation may require specific treatments, such as pre- and post-electroporation incubations in high osmotic buffers. The efficiency of electroporation is very dependent on the condition of the plant material used and the electroporation and tissue treatment conditions chosen. The advantage of this system is all the electroporated cells are in the same physiological state. This method can be used both for plant and animal transformation.
Microinjection
- In this method, which is mostly used for animal cells, DNA solution is directly injected into the nucleus of the cell. Typically, a microinjection assembly consists of a low power stereoscopic dissecting microscope and two micromanipulators, one for a glass micropipette to hold the nucleus by partial suction and the other for a glass injection needle to introduce the DNA into the nucleus. Generally, 2 picolitre (2 X 10-12 litre) of DNA solution is injected into the nucleus. The transgene integration occurs at random sites in the genome.
- Other methods includes macroinjection, liposome mediated transformation, ultra sound mediated DNA transfer, DNA transfer via pollen etc is also used in plant transformation.
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