1.6. The pollen-tube pathway method
The transformation method via pollen-tube pathway has great function in agriculture molecular breeding. Foreign DNA can be applied to cut styles shortly after pollination. The DNA reaches the ovule by flowing down the pollen-tube. This procedure, the so-called pollen-tube pathway (PTP), was applied first time for the transformation of rice. The authors obtained transgenic plants at remarkably high frequency. Afterward PTP was used for other species e.g. wheat, soybean, Petunia hybrida and watermelon. A bacterial inoculum or plasmid DNA can also be injected into inflorescence with pollen mother cells in the pre- meiotic stage without removing the stigma. In that case, it is expected that foreign DNA will be integrated with the gamete genome. Such an approach has been employed for rye. Pollen collected from inflorescences injected with a suspension of genetically engineered A. tumefaciens strain was predestined for the pollination of the emasculated spikes of the maternal plant. But the transformation efficiency was about 10-fold lower than that approximately reached for this species via microprojectile bombardment. Shou et al. (2002) also reported they were unable to reproduce the pollen-tube pathway transformation for delivering plasmid DNA into soybean. They concluded that the pollen-tube pathway transformation in cotton and soybean was not reproducible. This might have been because of the manipulation of transformation, the growth stage of plants, the effects of environment and weather
2. Chemical gene transfer method
This involves plasma membrane destabilizing and/or precipitating agents. Protoplasts are mainly used which are incubated with DNA in buffers containing PEG, poly L-ornithine, polyvinyl alcohol or divalent ions. The chemical transformation techniques work for a broadspectrum of plants.
2.1. Polybrene–Spermidine Treatment
The combination polybrene–spermidine treatment greatly enhanced the uptake and expression of DNA and, hence, the recovery of nonchimeric germline transgenic cotton plants. The major advantages of using the polybrene–spermidine treatment for plant genetic transformation are that polybrene is less toxic than the other polycations; spermidine protects DNA from shearing because of its condensation effect; and because no carrier DNA is used, and the integration of plasmid DNA into the host genome should enable direct analysis of the sequences surrounding the site of integration. To deliver plasmid DNA into cotton suspension culture obtained from cotyledon-induced callus, polybrene and/or spermidine treatments were used. The transforming plasmid (pBI221.23) contained the selectable hpt gene for hygromycin resistance and the screenable gus gene. Primary transformant cotton plants were regenerated and analyzed by DNA hybridization and b-glucuronidase assay.
2.2. PEG mediated gene transfer
In this method protoplasts are isolated and a particular concentration of protoplast suspension is taken in a tube followed by addition of plasmid DNA (donor or carrier). To this 40% PEG4000(w/v) dissolved in mannitol and calcium nitrate solution is slowly added because of high viscosity, and this mixture is incubated for few minutes (ca 5 min.). As per the requirements of the experiments, transient or stable transformation studies are conducted. Among the most important parameters that affect the efficiency of PEG-mediated gene transfer are the concentration of calcium and magnesium ions in the incubation mixture, and the presence of carrier DNA. The linearized dsDNA are more efficiently expressed and integrated in the genome than the supercoiled forms. The advantage of the method is that the form of DNA applied to the protoplast is controlled entirely by the experimenter and not by intermediate biological vector. Main disadvantage is that the system requires a protoplast.
2.3. Calcium-Phosphate co-precipitation
DNA when mixed with calcium chloride solution isotonic phosphate buffer DNA-CaPO 4 precipitate. The precipitate is allowed to react with actively dividing cells for several hours, washed and then incubated in the fresh medium. Giving them a physiological shock with DMSO can increase the efficiency of transformation to a certain extent. Relative success depends on high DNA concentration and its apparent protection in the precipitate.
2.4. DEAE dextran procedure
Transformation of cells with DNA complexed to the high molecular weight diethyl amino ethyl (DEAE) dextran is used to obtain efficient transient expression. The efficiency increasewhen 80% DMSO shock is given. But this technique does not produce stable transformants.
2.5. The polycation DMSO technique
It involves use of a polycation, polybrene, to increase the absorption of DNA to the surface followed by a brief treatment by 25-30% DMSO to increase the membrane permeability and enhance the uptake. The major advantage of polybrene is that it is less toxic than other polycations and a high transformation efficiency requires very small quantities of plasmid DNA to be used.
3. Direct gene transformation through imbibition
During imbibition the uptake of exogenous DNA of dehydrated plant tissues is a direct gene transfer method which has been studied since the 1960s and for which the literature contains a number of both claims and refutations. The physical and biochemical changes which are already known occur in plant tissues during dehydration (e.g. a large water potential between the dry tissue and external solution, rapid cell expansion, cell wall rupture, cell membrane structural changes and leakiness; suggest that under these conditions DNA uptake might be possible. DNA uptake and expression was observed under simple dehydration conditions, but was stimulated by the presence of 20% DMSO, suggesting that membrane permeabity was an important factor in the process. A number of lines of evidence supported the conclusion that reporter gene expression was the result DNA uptake into cells and plants were recovered from treated embryos, but no evidence of stable transformation was presented. Subsequent research on the imbibition transformation has extended its application to dessicated somatic embryos of alfalfa, which showed transient GUS expression at frequencies upto 70%. The stable transformation of rice by embryo imbibition was also reported. The frequency of transient expression of gusA and hpt genes using the CaMV35S promoter was about 30 to 50%. The main sites of gusA gene expression were meristems of roots and vascular bundles of leaves. Also, DNA uptake, integration and expression of the hpt gene in selected rice were investigated by various PCR methods and Southern blot analysis of genomic DNA. It was shown that the hygromycin phosphotransferase (HPT) DNA was present in the rice genome in an integrated form and not as a plasmid form. These methods are technically the most simple of DGT methods, as they require no specialist equipment and the preparation of target plant tissues are generally simple. This simplicity constitutes the advantage of these techniques, while their limitations are i) they can be applied only to very specific organs or tissues (i.e. newly pollinated flowers or hydrating embryos) and ii) it is still not clear that they lead to stable, and heritable transformation. While they add support to the observation that many different plant cells may be amenable to DNA uptake and expression, at present these techniques are subjects to further analysis and development rather than usable gene transfer methods.