Search

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.

Most Bt transgenic rice events have an underlying grain cost depending on the insect pressure in the field. This study was conducted (1) to determine if the cry1Ac/bar genetically modified (GM) rice (designated Agb0101) based on a Korean japonica rice cultivar has any underlying grain cost and (2) to develop cry1Ac/bar GM rice lines with yield performance similar to those of Korean elite cultivars via crossbreeding. A three-year field experiment showed that Agb0101 had a high underlying grain cost equivalent to 18% grain yield loss, and this cost was mainly due to the grain number per panicle. Moreover, it was found that the cry1Ac/bar transgene was inherited as a single dominant gene. BC1F4―F5 crossbred progeny derived from an anther culture of Agb0101―showed phenotypic resemblance to non-transgenic Korean elite cultivars, resulting in a decrease in the grain yield cost of Agb0101 from 27% to 10%. These results suggest that Bt-GM rice-oriented cross-fertilization with a non-GM elite cultivar followed by two- or threefold selection is a feasible method to minimize or remove the underlying grain cost of the Bt transgenic insect-resistant rice line.

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 less simple approach developed for generation of marker-free transgenic plants is to select transformants without the use of selective marker genes. Some results about development of marker-free transgenic plants were obtained using a non-selective approach in several crops such as rice, potato and tobacco. However, the study did not provide evidence on detailed characterization of introduced gene on genome, a critical step for confirming the stable integration and transmission of a foreign gene. In this study, we evaluated structure and integration sites of transgene (m Cry1Ac ) in the transgenic Bt rice plants which were made via conventional Agrobacterium -mediated transformation by non-selective method. Structure and integration sites of transgene in these transgenic plants had similar fashion as those recovered under selection. Keywords Non-selective, Marker-free transgenic rice, Agrobacterium -mediated transformation, Transgene, integration site 서 론 선발마커유전자(Selective marker gene)는 목표유전자와 연결하여, 도입유전자가 형질전환 된 식물체를 선택적으로 선발하는데 사용된다. 통상적으로

Composition analysis of genetically modified crops is an important consideration in the assessment of food safety. Agb0101 (Oryza sativa L. cv. Nakdongbyeo) developed in Korea is a form of insect-resistant rice that contains a synthetic truncated cry1Ac gene isolated from Bacillus thuringiensis (Bt), and demonstrates high resistance to rice leaf folder under field conditions. Nutrients, anti-nutritive components, and secondary metabolites of Agb0101 were analyzed and compared with those of its non-transgenic counterpart. The amounts of proximates, amino acids, fatty acids, minerals, vitamins, trypsin inhibitors, phytic acid, and ferulic acid in brown rice from Agb0101 were comparable to those of its non-transgenic counterpart. Statistical comparisons to test for equivalence showed that all the analyzed components in the insect-resistant rice plants were substantially equivalent to those of its non-transgenic counterpart. Furthermore, most of the measured values from Agb0101 were within the range of values reported for other commercial rice varieties.

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is one of the most commonly used in the development of genetically modified (GM) crops. In this research, we analyzed Bt rice showing lepidopteran pest-resistance. The Bt gene is a synthetic Cry1Ac composed of optimal codons for plants, and the Bt protein is targeted to the chloroplast by a transit peptide. Three Cry1Ac rice events (C103-3, C127-1, and C7-1) were analyzed for molecular characterization. C103-3 contains two copies of T-DNA where the left border (LB) region is truncated. Both C7-1 and C127-1 have a single copy of T-DNA, but a part of the vector backbone DNA is inserted into the genome of C127-1; thus, only C7-1 had intact T-DNA. Progenies of C7-1 crossed with the original cultivar, Nakdong, and double-haploid lines from anther culture of lines crossed with the elite cultivar, Dongjin, were analyzed for T-DNA flanking genomic DNA and genotyping. Results showed that an intact T-DNA region without the vector backbone was inserted into the genome and was stably inherited through generations. The C7-1 homozygous event could be used as breeding material to develop GM rice with pest resistance.

Genetic components introduced into approved GM crops are a key subject for safety assessment and provide a basis for the development of detection methods for GM crops. In order to understand the genetic components in approved GM crops comprehensively, we screened the genetic vector maps of GM crops that had been approved for commercialization around the world. A total of 64 varieties from 5 major GM crop species (maize, canola, cotton, soybean, and tomato) were subjected to analysis. The genetic components included genes, promoters, terminators, and selection marker. This survey may be useful for researchers who develop GM crops and methods for detecting GM crops.

In recent years, several genetically modified (GM) crops have been developed worldwide through the recombinant DNA technology and commercialized by various agricultural biotechnological companies. Commercialization of GM crops will be required the assesment of risks associated with the release of GM crops. In advance of the commercial release of GM crops, developer should submit the several information on GM crops for approval. In this study, we carried out to provide the molecular data for the risk assessment of GM rice containing insect-resistant gene, modified Cry1Ac (CryIAc1). Through the molecular analysis with CryIAc1 induced GM rice, we confirmed the steady integration and expression of transgene, the transgene copy number, the adjacent region sequences of inserted gene into rice genome, and the transgene stability in progenies. For the qualitative PCR detection methods, specific primer pairs were designed on the basis of integration sequences, and construct- and event-specific detection markers were developed for leaf folder-resistant rice, Cr7-1 line. From these results, we demonstrated that the molecular data and the PCR detection methods of leaf folder- resistant GM rice could be acceptable to conduct the biosafety and environment risk assessment.

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.

Tocopherols are essential lipophilic antioxidant in human cells, while little is known about its function in plant tissues. To study the impact of composition and content of tocopherols on stress tolerance, tobacco (Nicotiana tabacum) was transformed with a construct containing a cDNA insert encoding -tocopherol methyltransferase (-TMT/VTE4) from perilla under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic tobacco was confirmed by PCR and RT-PCR. The total content and composition of tocopherols in the transgenic lines were similar with wild type controls. However, chlorophyll-a and carotenoid content in the transgenic lines were increased by up to 45% (P-TMT increased the salt stress tolerance in tobacco plants. These results demonstrate that over-expression of -TMT gene in tocopherol bio-synthetic pathway can increase salt stress tolerance and contents of chlorophyll-a and carotenoid in transgenic tobacco plants.

The Agrobacterium-mediated transformation has been successfully used method to introduce foreign genes into some monocotyledonous as well as a large number of dicotyledonous plants genome, We developed transgenic Chinese cabbage plants with insect-resistance gene, modified CryIAc, by Agrobacterium-transformation and confirmed transgene copy number by Southern blot analysis. We confirmed that twenty-nine out of 46 transgenic Chinese cabbage plants have single copy of CryIAc. To obtain the sequences information on the transferred DNA (T-DNA) integration into plant genome, we analyzed left border (LB) flanking sequences by genome walking (GW) PCR method. Out of 46 transgenic Chinese cabbage plants examined, 37 carried the vector backbone sequences. This result indicates that the transfer of the vector backbone from the binary vectors resulted mainly from inefficient termination of LB site. Analysis of T-DNA LB flanking region of 9 transgenic Chinese cabbage plants without vector backbone revealed that all LB ends were not conserved and nucleotides up to 36bp from the LB cleavage site were deleted.

The influence of light quality on growth and development of in vitro grown Doritaenopsis hort. (Orchidaceae) plants was investigated. Growth parameters like leaf and root fresh/dry mass and leaf area were highest with plants grown under red plus blue light emitting diodes (LEDs). Leaf length was greater with the plants grown under red LED. Carbohydrate (starch, sucrose, glucose and fructose) and leaf pigment (chlorophylls and carotenoids) biosynthesis of the plants was significantly increased in plants grown under red plus blue LEDs compared to red or blue LED and fluorescent light treatments. This study suggests that the production of quality Doritaenopsis plants is possible by culturing the plants in vitro under a mixture of blue plus red light sources.

We investigated the effects of various qualities of light-emitting diode (LED) light sources on the growth and carbohydrate accumulation of grape rootstock ‘Teleki 5BB’ culturedin vitro. Shoot fresh and dry weights and net photosynthetic rates were increased when plants were exposed to fluorescent lighting (control), red light, or a mixture of blue plus red, but were unaffected by blueonly radiation. Shoot elongation was significantly stimulated by red light whereas the combination of blue and red light was associated with the shortest shoots. However, the number of nodes did not differ among these treatments. Under monochromic blue or red light, sugar content and starch accumulation increased under the mixed-radiation treatment.

An efficient protocol has been developed for rapid mass propagation of sweetpotato from shoot-tips derived embryogenic callus. Optimal embryogenic callus was induced from shoot apical meristem explants on Murashige and Skoog (MS) medium supplemented with 1mg/L 2,4-D. The addition of casein hydrolysate in the media increased the embryogenesis efficiency of sweetpotato. Somatic embryos were easily induced from the embryogenic callus on MS basal medium containing 300-500mg/L casein hydrolysate without phytohormon. Treatment of casein hydrolysate (100∼300mg/L) with 1mg/L 2,4-D also improved the secondary embryonic efficiency from somatic embryos below 2mm in length. Plant regeneration was achieved via somatic embryogenesis and direct organogenesis. Regenerated planlets with well developed shoots and roots on MS basal medium were successfully transferred to soil.

For the identification of a novel insecticidal protein, a two-dimensional liquid chromatography (PF-2D) system was used in a quantitative proteomic analysis of Xenorhabdus nematophila CBNU strain isolated from entomophagous nematode Steinernema carpocapsae . Protein patterns obtained from minimum and maximum insecticidal activities during cultivation were contrasted, and a novel toxin protein (Txp40) was identified by MALDI-TOF/MS. The DNA sequence of the cloned toxin gene (1089 bp) has an open reading frame encoding 363 amino acids with a predicted molecular mass of 41162 Da. The txp40 identified in this study is most closely related to the known txp40 cloned from X. nematophila EB (ADQ92844) with 94.4% identical sequence residues. Following the expression of the newly identified toxin gene in Escherichia coli , the insecticidal activity of the recombinant toxin protein was determined against Plutella xylostella larvae; a 56.7% mortality rate was observed within 24 h.

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.

To increase insect resistance in transgenic rice plants, a synthetic truncated cry1Ac gene was linked to the rice rbcS promoter and its transit peptide sequence (tp) for chloroplast-targeted expression. Several transgenic lines were generated by the Agrobacterium-mediated transformation method and the expression levels of the transgene were compared with untargeted expression. Use of the rbcS-tp sequence increased the cry1Ac transcript and protein levels by 25- and 100-fold, respectively, with the accumulated protein in chloroplasts comprising up to 2% of the total soluble proteins. The high level of cry1Ac expression resulted in high levels of plant resistance to three common rice pests, rice leaf folder, rice green caterpillar, and rice skipper, as evidenced by insect feeding assays. Transgenic plants were also evaluated for resistance to natural infestations by rice leaf folder under field conditions. Throughout the entire period of plant growth, the transgenic plants showed no symptoms of damage, whereas nontransgenic control plants were severely damaged by rice leaf folders. Our results demonstrate that the targeting of cry1Ac protein to the chloroplast using the rbcS:tp system confers a high level of plant protection to insects, thus providing an alternative strategy for crop insect management.

Food crisis is one of the most important issues around the world. With a population explosion, Harvesting crops in a classical manner no longer keep up with population explosion. In addition, agricultural pesticide and insect pest give rise to environmental problems. Recent advances in biotechnology ameliorates both issues by inserting productivity amplifying gene and pesticide neutralizing protein encoded gene, maximizing crop productivity and producing pesticide resistant crop, so-called genetically modified (GM) crops. Due to the bioethical issues and potential risk to human, people are concerned about the GM crops, thus many countries make regulations for the GM crops. In order to regulate GM crops, we need fast, accurate and simple methods to the GM crops. In this study we have devised a biosensor to detect phosphinothricin acetyltransferase (PAT) protein that makes GM crops resistant to herbicides. In order to detect PAT protein, anti-PAT antibody was conjugated to polydiacetylene (PDA) vesicles. PDA vesicles have unique colorimetric characteristics, changing their color from blue to red upon external stimuli. Anti-PAT antibody conjugated PDA vesicles are subsequently encapsulated within hydrogel matrix to maximize the intensity of color change, resulting in the sensitivity enhancement of the sensor. Our biosensor could detect as low as the 20nM of PAT protein. Furthermore we developed a microfluidic device to automate the production of uniform sized immunohydrogel beads. Our simple biosensor can be identified by the naked eyes without the aid of special instrument or expertise, widening the potential applications in the area of point-of-care testing (POCT).