Nowadays, available phosphorus (P) deficiency in soil and weed resistance to herbicides have emerged as two severe limiting factors for sustainable agriculture. Therefore, it is of urgent needs to improve plant absorption/utilization ability of the soil P, seek phosphate (Pi)-alternative P fertilizers, and develop new forms of weed control systems. Phosphite (Phi), as a P resource of relatively high amount only less than Pi in Earth, can be converted to utilizable Pi uniquely in some bacterial species by oxidization via its specific dehydrogenase (PTDH), but inhibits plant growth and development. This implies that Phi might rather become a suitable P fertilizer for plants if introducing a PTDH detoxifier from bacteria. Herein, we created the transgenic tobaccos harboring a Pseudomonas PTDH gene (PsPtx) amplified from the soil metagenome previously. RT-PCR showed that the exotic PsPtx gene could express similarly in root, stem and leaf tissues of all transgenic lines. PsPtx transgenic tobaccos could utilize Phi by oxidization as the sole Pi supply, and also outperformed wild-type tobacco with a remarkably dominant growth under Phi stress conditions. Moreover, the PsPtx gene was preliminarily evaluated with a notable quality as a potential candidate of the selection marker in plant genetic transformation. Conclusively, PsPtx and its encoded phosphite dehydrogenase might be applicable for developing a dual system of plant phosphorus utilization and weed control using Phi as P fertilizer and herbicide, and provide an effectual solution to some obstacles in the current crop transgenic studies.
Oxidoreductases ; Phosphites ; Phosphorus ; Plants, Genetically Modified ; Weed Control

Genetically modified (GM) crops were first introduced to farmers in 1995 with the intent to provide better crop yield and meet the increasing demand for food and feed. GM crops have evolved to include a thorough safety evaluation for their use in human food and animal feed. Safety considerations begin at the level of DNA whereby the inserted GM DNA is evaluated for its content, position and stability once placed into the crop genome. The safety of the proteins coded by the inserted DNA and potential effects on the crop are considered, and the purpose is to ensure that the transgenic novel proteins are safe from a toxicity, allergy, and environmental perspective. In addition, the grain that provides the processed food or animal feed is also tested to evaluate its nutritional content and identify unintended effects to the plant composition when warranted. To provide a platform for the safety assessment, the GM crop is compared to non-GM comparators in what is typically referred to as composition equivalence testing. New technologies, such as mass spectrometry and well-designed antibody-based methods, allow better analytical measurements of crop composition, including endogenous allergens. Many of the analytical methods and their intended uses are based on regulatory guidance documents, some of which are outlined in globally recognized documents such as Codex Alimentarius. In certain cases, animal models are recommended by some regulatory agencies in specific countries, but there is typically no hypothesis or justification of their use in testing the safety of GM crops. The quality and standardization of testing methods can be supported, in some cases, by employing good laboratory practices (GLP) and is recognized in China as important to ensure quality data. Although the number of recommended, in some cases, required methods for safety testing are increasing in some regulatory agencies, it should be noted that GM crops registered to date have been shown to be comparable to their nontransgenic counterparts and safe . The crops upon which GM development are based are generally considered safe.
Agriculture ; Animal Feed ; Animals ; Biotechnology ; China ; Consumer Product Safety ; Food, Genetically Modified ; Humans ; Models, Animal ; Plants, Genetically Modified ; Safety

Agroinfiltration is a newly developed plant transient gene expression technique, which is simple, rapid and reproducible. It has been widely used in analyses of foreign gene expression, hypersensitive reaction, gene silencing, promoter activity and identification of new disease-resistance genes. In this paper, we describe the principle and the operation procedure of Agroinfiltration and its application in diverse aspects of plant molecular biology research. Our experiences in modification of the Agroinfiltration technique are also provided.
Agrobacterium tumefaciens ; genetics ; Genetic Vectors ; genetics ; Plants ; genetics ; Plants, Genetically Modified ; Research Design

Animals ; Genetic Engineering ; Humans ; Plants, Genetically Modified ; genetics ; Plasmids ; Vaccines, Synthetic ; biosynthesis ; genetics

Chloroplast genetic engineering, offers several advantages over nuclear transformation, including high level of gene expression, increased biosafety, remedying some limitations associated with nuclear genetic transformation, such as gene silencing and the stability of transformed genes. It is now regarded as an attractive new transgenic technique and further development of biotechnology in agriculture. In this article we reviewed the characteristics, applications of chloroplast genetic engineering and its promising prospects were discussed.
Biotechnology ; methods ; Chloroplasts ; genetics ; Genetic Engineering ; methods ; Plants, Genetically Modified ; genetics ; Transformation, Genetic

Resveratrol synthase (RS) plays a key role in resveratrol (Res) biosynthesis. RS gene has been formerly reported to be transformed into many plant species and microorganisms, and to play certain roles in metabolic and regulation processes. In this paper, the transformations of RS gene in plants, and the related changes of biological properties, such as metabolites, anti-pathogen activities, anti-radical properties, and developmental characters in transgenic plants, as well as the production of resveratrol in microbes by utilizing RS gene were summarized. Moreover, the application prospects of RS gene in bioengineering were also addressed.
Acyltransferases ; genetics ; Genetic Engineering ; Plants, Genetically Modified ; enzymology ; genetics ; Stilbenes ; metabolism

In this article primary studies of the application of hyperbranched rolling cycle amplification (HRCA) in exogenous genes detection of transgenic plants were done. Four padlock probes were designed according to the sequences of four genes/DNA fragments that are used widely in transgenic plants; part of the sequence of pKK233 was chosen as the linking part of padlock probes and a pair of HRCA primers was designed according to the sequence of linking part. Study of the specificity of ligation in HRCA with isotope labeled padlock probes indicated padlock probes could be ringed effectively only when corresponding target DNA exited in the same reaction system and could not be ringed when there was no corresponding target DNA exited. Ligation time is very different according to the characteristic of target DNA being used. 5 min to 10 min is enough if the target DNA is plasmid; 30 min to 60 min is needed if the target is genome DNA of plant because it's sequence is more complex than that of plasmid's. HRCA time was analyzed which indicated longer reaction time can obviously increase the amount of products. Quantity of enzyme in HRCA was also analyzed. Different amount of enzyme (from 0.5 unit to 4 units) can give similar result when other conditions are not changed. On the basis of the research, transgenic tobacco was detected with these four padlock probes and the results were just as prospective. In order to increase the efficiency of detection, multiplex HRCA (MHRCA)was used. In MHRCA more than one padlock probes are used at the same time in the same reaction system to detect more than one targets. Because the amplification products of MHRCA will be complex and it is almost impossible to analyze with electrophoresis, so reverse-blot is used. Detection results of transgenic tobacco with this method are the same with anticipation. Compare to MPCR method we established before MHRCA is more convenient to operate and more effective in detecting exogenous genes in transgenic plants.
Nucleic Acid Amplification Techniques ; methods ; Plants, Genetically Modified ; genetics ; Plasmids ; Tobacco ; genetics

Using plants to remove or inactivate heavy metal pollutants from soils and surface waters provide a cheap and sustainable approach of Phytoremediation. However, field trials suggested that the efficiency of contaminant removal using natural hyperaccumulators is insufficient, due to that many of these species are slow growing and produce little shoot biomass. These factors severely constrain their potential for large-scale decontamination of polluted soils. Moreover, both the micronutrient and toxic metal content accumulated in crops determine the quality and safety of our food-chain. By a transgenic approach, the introduction of novel genes responsible for hyperaccumulating phenotype into high biomass plants and/or stable crops uptaking minerals as food is a promising strategy for the development of effective techniques of phytoremediation and improvement of nutritional value of stable food through a viable commercialization. Recently, the progress at molecular level for heavy metal uptaking, detoxification and hyperaccumulation in plants, and also the clarification of some functional genes in bacteria, yeasts, plants and animals, have advanced the research on genetic engineering plants of heavy metal resistance and accumulation, and on the functional genes (e . g. gsh1, MerA and ArsC) and their genetic transformated plants. These studies demonstrated commercialization potentials of phytoremediation. In this paper, the molecular approach, effects and problems in gene transformation were discussed in details, and also the strategy and emphases were probed into the future research.
Biodegradation, Environmental ; Genetic Engineering ; methods ; Metals, Heavy ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Soil Pollutants ; metabolism

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.
Genetic Markers ; Green Fluorescent Proteins ; genetics ; Plants, Genetically Modified ; genetics ; Plasmids ; Recombination, Genetic ; Tobacco ; genetics

Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. The disease can seriously reduce melon yield and quality. However, little information is available on the genetics and functional genomics of the fungal pathogen. In this study, we developed an Agrobacterium-mediated transformation system for D. bryoniae by using a universal pathogenic isolate DB11 and the Agrobacterium tumefaciens strain C58C1 carrying plasmid pBIG2RHPH2 harboring the hygromycin B phosphotransferase gene (hph). Total 45 transformants could be obtained per 1 x 10(5) spores when 1 x 10(6) spores per milliliter of D. bryoniae spore suspension were cocultivated with Agrobacterium cells at OD600 = 0.15 for 48 h in the presence of induction medium (pH 5.2) containing acetosyringone at 200 microg/mL and selection medium contained 100 microg/mL of hygromycin B and 200 microg/mL of cefotaxime sodium, ampicillin and tetracycline, respectively. The transformants were stable when grown on PDA medium without hygromycin B for five times and were verified by PCR amplification with the hph primers and by Southern blot analysis with the hph probe. The transformation system will be useful for further studies of functional genes in D. bryoniae.
Agrobacterium tumefaciens ; genetics ; Ascomycota ; genetics ; Cucumis melo ; microbiology ; Plant Diseases ; microbiology ; Plants, Genetically Modified ; Transformation, Genetic