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Beta-carotene-enhanced transgenic soybeans, harboring genes encoding phytoene synthase and carotene desaturase under the control of a seed-specific promoter, were developed to alleviate vitamin A deficiency in populations, the diet of which was deficient in this vitamin. However, metabolic engineering of carotenoid biosynthetic pathways often has unintended effects, leading to major metabolic changes in plants that harbor endogenous beta-carotene biosynthesis pathways. In the present study, we performed transcriptome profiling analysis using RNA-seq to investigate the changes in the transcriptome and some unintended pleiotropic effects on the leaves, stems, roots, and seeds of beta-carotene-enhanced transgenic soybean lines, and compared them to those of their non-transgenic counterpart donor variety Kwangan. We observed that transgenic soybeans showed significant changes in secondary metabolic biosynthesis in leaves and down-regulated galactose metabolism in roots. Differentially expressed genes in the transgenic group, which were significantly up-regulated, included those encoding glycine-aspartic acid-serine-leucine-motif esterase/lipase, known as cutin synthase and cutinase. These results suggested enhanced beta-carotene biosynthesis may affect related enzymes to carbohydrate metabolism and fatty acid metabolism. Hence, we speculated that upregulation of cutin polymerization resulted in thickened seed coat and delayed seed germination of transgenic soybeans. Furthermore, downregulation of raffinose family oligosaccharide biosynthesis may cause redundancy of myo-inositol, a substrate of phytin formation. This could lead to phytic globoids accumulation in transgenic soybean seeds. The present imformation would be important for transgenic plant development via carotenoid metabolic engineering, with focus on beta-carotene over-production. Gene expression changes related to secondary metabolite biosynthesis, cutin metabolism, and raffinose-family oligosaccharide biosynthesis in leaf, stem, and root, and phytin globoid accumulation in seeds, occurred in beta-carotene-enhanced transgenic soybeans.

Nutritional composition is important for assessing the safety of genetically modified (GM) crops for human consumption. Three beta-carotene-enhanced soybean lines were developed by introducing the β-conglycinin promoter::Phytoene synthase-2A-Carotene desaturase/t35S gene cassette into the genome of the commercial Kwangan (Glycine max L.) soybean variety. Transgenic soybeans were successfully detected on beta-carotene productions ranged from 170.47 to 213.58 µg/g. Comparative assessments of nutrition were conducted with 3 transgenic soybeans, their non-GM counterpart, and several commercial soybean varieties. Results indicated that most levels of proximate, fatty acids, amino acids, and vitamins showed non-significant differences between transgenic soybeans and their counterpart, and fit within the reference ranges established for other commercial soybeans and Organization for Economic Cooperation and Development Guidelines. However, significant differences on levels of crude fat, carbohydrate, δ-tocopherol, and oleic acid of transgenic soybeans comparing to those of non-transgenic counterpart Kwangan cannot eliminate the influences of transgene insertion. Alternations on compositions should be definite by further studies, such as transcriptome and metabolome profiling.

Public concern about the safety of genetically modified (GM) crops and potential pleiotropic effects of selectable marker gene insertion and expression can be avoided by developing a selectable marker-free transformation system. Various techniques have been developed to produce marker-free transgenic plants; however, an approach that does not require the use of selectable marker genes would not require post-transformation processes. We report such a method for generating of GM rice plants. To develop transgenic rice resistant to lepidopteran insects, a marker-free binary vector harboring the Bacillus thuringiensis mcyr1Ac gene was constructed and used for Agrobacterium tumefaciens-mediated transformation of rice scutellar calli. Initial screening of putative T0 transgenic rice plants was performed using polymerase chain reaction (PCR) on pools of DNA from 15 to 30 regenerated shoots, followed by genomic DNA PCR and Cry1Ac ImmunoStrip assays of individual plant extracts. Although the overall rice transformation efficiency of the non-selection approach is lower than that of traditional rice transformation by using marker genes, we derived six independent homozygous T3 events from Cry+ T0 shoots. Transgenes were detected and inheritance confirmed using DNA PCR, RT-PCR, and Southern blot analysis, and insertion sites were characterized by the sequencing of DNA flanking the transgenes. The transgene expression of cry1Ac was also stable and effective against rice leaf folder at levels comparable to those of a transgenic plant that had been derived by selection. This method could be applied to other plant species with similar transformation efficiencies to create selectable-marker-free transgenic plants for research and commercial uses.

Tocopherols (α-, β-, γ-, and δ-tocopherol) represent a group of lipophilic antioxidants that are synthesized only by photosynthetic organisms. It is widely believed that the main functions of tocopherols are protection of pigments and proteins of photosystem and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species. In the present study, we report on the cloning and characterization of NtTC, which is a tocopherol cyclase (TC) ortholog isolated from tobacco. To enhance tocopherol contents, we generated independent transgenic rice events in expressing NtTC or NtTC along with Perilla γ-tocopherol methyltransferase genes. The transgenic TC line significantly increased α-, total tocopherol, total glutathione, and total antioxidant status activity levels compared with the wild type. Furthermore, TC rice plants showed higher tolerance to drought than wild-type rice plants. On the basis of these studies, we concluded that overexpression of NtTC could increase the tolerance to drought stress and that the increase in tocopherol affects cellular signaling and antioxidant defense of plants in response to drought.

Stacked genetically modified (GM) crops are becoming popular for their enhanced production efficiency and improved functional properties. In this study, we developed an event-specific PCR method for simple qualitative detection of stacked events combining more than 2 transgenic traits. Ten primer sets were designed, including 9 that were event-specific and 1 that was specific for a maize endogenous gene. Five event-specific multiplex-PCR systems were built, based on the main type of stacked GM events approved in Korea. Multiplex PCR was performed with mixtures of template DNA extracted from certified reference materials. PCR amplicons (3 or 4 by type) of expected sizes and mutually similar intensities were detected. The limit of detection was approximately 0.1%(v/v) for stacked GM maize in all event-specific PCRs. This method may be useful for the specific detection and monitoring of stacked GM maize lines and individual parent GM maize lines, by effectively distinguishing genestacked events.

The rice water weevil (RWW), Lissorhoptrus oryzophilus Kuschel, is the most widely distributed and destructive early season insect pest of rice, Oryza sativa L. worldwide. The rice plants were transformed with cryIIIA insecticidal gene as well as with the bar gene coding phosphinothricin acetyltransferase. CryIIIA gene under the control of a modified RCg2 promoter drives the insect-toxic gene expression in roots and/or leaves. The cryIIIA gene was transferred into O. sativa L. cv. Nakdong by Agrobacterium-mediated transformation. Stable integration of the transgene was confirmed in putative transformed rice by Southern blot analysis. The expression of the cryIIIA toxin gene in the roots of transgenic rice plants was verified by RT-PCR and immunoblot analysis. Transgenic rice plants were also evaluated for resistance to natural infestations of the RWW under field conditions between 2007 and 2011. The transgenic Btt8R and Btt12R lines reduced the growth rate of RWW larvae and pupae populations compared with non-transgenic control plants by approximately 52 and 58 %, respectively. To further examine the efficacy of the RWW bioassay, we used pots and performed experiments in trays and under field conditions in 2012. The Btt12R line reduced the total populations of RWW larvae and pupae in trays and under field conditions by 56 and 45 %, respectively. The bioassay experiments conducted over 6 years, showed a significant reduction rate of RWW larvae and pupae populations demonstrating that the cryIIIA gene in transgenic rice confers resistance to RWW.

A novel genetically modified organism (GMO) detection method was developed using a simple and rapid procedure to identify a regulatory gene introduced into genetically modified (GM) maize. After DNA extraction from GM maize flours, fragments of the conserved sequence of the cauliflower masaic virus (CaMV) 35S promoter gene along with a competitive internal control gene were amplified using duplex polymerase chain reaction (PCR). Amplification products were then detected using a disposable detection device. The quantitative detection limit for GM maize 59122 was found to be 1.0% (w/w). Because the combination-method involving PCR technology coupled with a disposable detection device does not require expensive instrumentation or expertise, it will serve as a valuable alternative to immunoassays and traditional PCR-based tests in the detection of GMOs.

The development of marker-free transgenic plants has responded to public concerns over the safety of biotechnology crops. It seems that continued work in this area will soon remove the question of unwanted marker genes from the debate concerning the public acceptability of transgenic crop plants. Selectable marker genes are co-introduced with genes of interest to identify those cells that have integrated the DNA into their genome. Despite the large number of different selection systems, marker genes that confer resistance to the antibiotics, hygromycin (hpt) and kanamycin (nptII) or herbicide phosphinothricin (bar), have been used in most transgenic research and crop development techniques. The techniques that remove marker gene are under development and will eventually facilitate more precise and subtle engineering of the plant genome, with widespread applications in both fundamental research and biotechnology. In addition to allaying public concerns, the absence of resistance genes in transgenic plants could reduce the costs of developing biotechnology crops and lessen the need for time-consuming safety evaluations, thereby speeding up the commercial production of biotechnology crops. Many research results and various techniques have been developed to produce marker-free transgenic plants. This review describes the strategies for eliminating selectable marker genes to generate marker-free transgenic plants, focusing on the three significant marker-free technologies, co-transformation, site-specific recombinase-mediated excision, and non-selected transformation.

Antibiotic resistance marker genes are powerful selection tools for use in plant transformation processes. However, once transformation is accomplished, the presence of these resistance genes is no longer necessary and can even be undesirable. We herein describe the successful excision of antibiotic resistance genes from transgenic plants via the use of an oxidative stress-inducible FLP gene. FLP encodes a recombinase that can eliminate FLP and hpt selection genes flanked by two FRT sites. During a transformation procedure in tobacco, transformants were obtained by selection on hygromycin media. Regenerants of the initial transformants were screened for selective marker excision in hydrogen peroxide supplemented media and both the FLP and hpt genes were found to have been eliminated. About 13-41% of regenerated shoots on hydrogen peroxide media were marker-free. This auto-excision system, mediated by the oxidative stress-inducible FLP/FRT system to eliminate a selectable marker gene can be very readily adopted and used to efficiently generate marker-free transgenic plants.

Whole cells, cytoplasms and peptidoglycans of ten different lactic acid bacteria (LAB) were tested for in vitro cytotoxicity on diverse cancer cell lines using the 3H-thymidine incorporation assay. The peptidoglycans and cytoplasm fractions, as well as heat-killed whole cells of LAB, had significant antiproliferative activities against several cancer cell lines. In particular, the cytoplasm fractions exhibited marked direct antiproliferative activities against colon and gastric cancer cell lines, whereas the peptidoglycans retarded growth of colon and bladder cancer cell lines. The cytoplasm fractions of Bifidobacterium longum and Lactococcus lactis ssp. lactis inhibited proliferation of two cancer cell lines by 50% at 33 and 23 μg ml−1 for SNUC2A (a human colon adenocarcinoma cell line) and 17 and 11 μg ml−1 for SNU-1 (a human gastric cancer cell line), respectively.

Increasing vitamin E activity in economically important oil crops such as soybean will enhance the nutritional value of these crops. An improved soybean transformation system involving pre-culture of soybean seed on medium supplemented with 1 mg/L 6-benzylaminopurine under dark conditions was established. To improve the nutritional value of soybean by increasing the α-tocopherol content, soybeans were transformed with γ-tocopherol methyltransferase (γ-TMT) gene by Agrobacterium-mediated transformation. Frequency of soybean transformation was significantly increased from 0.5 to 4.3% by this modified system, and 13 lines of transgenic soybean plants containing γ-TMT were obtained. The molecular characterization using polymerase chain reaction (PCR), reverse transcriptase PCR, and Southern blot analysis confirmed insertion and inheritance of the transgene in the transgenic plants and their progeny. Morphologically normal and fertile transgenic plants were analyzed, and the majority of transgenic soybean transmitted herbicide resistance at 3:1 or 15:1 ratios to their progeny. Alpha-tocopherol content in transgenic soybean seeds was determined by high-performance liquid chromatography; over-expression of γ-TMT resulted in a 41-fold increase in α-tocopherol over wild-type soybean seeds.