| Nanotechnology is a high technology which is born in nineteen nineties.The application of nanotechnology in life sciences progressed very rapidly in recent years. Currently this new tool has been widely used on the animal and/or medical researches, for instance, bioimaging, DNA and drug delivery, biosensors and so on. Nanoparticles as gene carrier become popular in the mammalian cultured cells whereas its application in plant cells is still very limited. Compared to mammalian systems, gene delivery to plant cells is much more challenging due to the presence of cell walls. Nanoparticle gene carriers have significant advantages than traditional carriers. Firstly, it is applicable to both animal and plant cells, and any types of organs; secondly, it can effectively overcome transgenic silencing by controlling the DNA copies combined to nanoparticles; thirdly, nanoparticles can easily be functionalized and further enhance transformation efficiency; finally, nanoparticles-mediated multigene transformation can be achieved without involving traditional building method of complex carrier. The key properties of nanoparticles as gene carrier suggest the potential of its application in plant transformation. In order to overcome the limitations of cell walls, Nanoparticle gene carriers have to combine with the traditional transgenic methods.This study we constructed3-5nm ZnS nanoparticles and500nm silica nanoparticles, the two kinds of nanoparticles were modified with positively charged poly-1-lysine (PLL) successfully deliver GUS-encoding plasmid DNA into tobacco cells assisted by ultrasonic and gene gun methods. The stable genetic modified plants have been obtained. Our research also reveals the size of nanoparticles, quantity of gene, types of receptor materials, transgenic methods and experiment conditions can affect the expression efficiency. The detail results are as follows.Section Ⅰ:ZnS nanoparticles deliver DNA into plant cell. ZnS nanoparticles modified with positively charged poly-1-lysine (PLL) successfully deliver GUS-encoding plasmid DNA into tobacco cells using ultrasound-assisted methods. PCR detection, southern blotting analysis and GUS histochemical staining were carried out for the regenerated plants. The results indicate that ZnS-PLL can not only effectively combine with DNA, but also can prevent DNA from ultrasonic damage. Moreover, when the receptor material was tabacco leaves, the PCR-positive transformation efficiencies of43.42%,52.67%and48.57%were obtained under different experiment conditions (60W,15min;60W,20min;60W,25min); when the receptor material was callus, the PCR-positive transformation efficiencies of47.56%,55.47%and39.73%were obtained under different experiment conditions (30W,5min;30W,10min;30W,15min). The transformation efficienciy of callus is higher than that of leaves. In addition, the transformation efficiency of nanoparticle-mediated ultrasound-assisted is higher than that of the conventional ultrasound-mediated plant genetic transformation. This article demonstrates the great potential of nanoparticles as gene carrier in plant transformation and proves a novel approach for plant genetic decoration.Section II:Silica nanoparticles-mediated stable gene transformation in plant.500nm silica nanoparticles (SiNPs) modified with positively charged poly-1-lysine (PLL) was used to be gene carrier to combine with GUS-encoding plasmid DNA for tobacco transformation. The results indicate that SiNP-PLL can not only effectively combine with DNA, but also can control DNA copies combined to SiNP-PLL by adjusting the quantity of DNA and prevent DNA from DNaseI cleavage. The stable genetic modified plants have been obtained based on the silica nanoparticle-mediated gene gun-assisted method. For callus receptor material, the average transformation efficiencies of40.99±2.27%,47.11±0.4%and42.95±2.37%were found in the regenerated plantlets from the three different experimental conditions with DNA/SiNP mass ratios of1:9,1:10and1:11, respectively. For leaves receptor material, the transformation efficiencies of regenerated plantlets transformed by DNA/SiNP (1:9,1:10and1:11) are34.15±6.92%,39.53±3.4%,37.26±1.26%. For the three different mass ratios of DNA/SiNP, the transformation efficiency of the regenerated plantlets with MSN/DNA (1:10) is higher than that of1:9and1:11.For the two different transformed receptor materials, callus and leaves, the transformation efficiencies of callus is higher than that of leaves. Moreover, all the transformation efficiencies of the regenerated plantlets mediated by SiNPs are higher than that of the regenerated plantlets mediated by conventional gold particles. The silica nanoparticles have the great potential to replace the gold particles for the gene gun method.In general, all both kinds of nanoparticles (3-5nm ZnS nanoparticles and500nm silica nanoparticles) papared here can successfully deliver GUS-encoding plasmid DNA into tobacco cells by ultrasonic and gene gun transform methods. And all the transformation efficiencies of the regenerated plantlets mediated by nanoparticles are higher than that of the regenerated plantlets mediated by conventional ultrasonic and gene gun transform methods. For the two different transformed receptor materials, callus and leaves, the transformation efficiencies of callus is higher than that of leaves. When using the ultrasonic transform method, for the same receptor material, the size of nanoparticle is smaller, the transformation efficiency is higher. The DNA copies combinition to nanopartices is larger, the transformation efficiency is higher. For the two different methods, the transformation efficiency from ultrasonic is higher from gene gun. In this article, a novel approach for delivering gene into plant cells was established. More importantly, this is first time that stable genetic modified plants mediated by nanoparticles were obtained. Our research also reveals the size of nanoparticles, quantity of gene, types of receptor materials, transgenic methods and experiment conditions can affect the expression efficiency. And the optional method of nanoparticles as gene carrier is also been constructed for the first time. The use of the delivery system developed here with tobacco as a target virtually can be further extended to all plant species having successful regeneration systems. |
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