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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.

Policosanols (PCs) are a group of long-chain alcohols that have been reported to have many beneficial physiological activities. In this study, the total content and composition of PCs in 15 rice (Oryza sativa L.) cultivars from Korea were characterized by gas chromatography–time-of-flight mass spectrometry. This method proved to be sufficiently precise and accurate with respect to the degree of endogenous biological variability found in the rice samples. Octacosanol (C28) and triacontanol (C30) were the major components of PCs in all cultivars. In addition, there were positive correlations among the determined PC contents. Given its high PC content, the Heughyangbyeo cultivar may appear to be a good candidate for future breeding programs.

An accurate and sensitive method for determining 14 lipophilic compounds, including tocopherols, tocotrienols, sterols and policosanols from rice (Oryza sativa L.) grain was developed using gas chromatography (GC) coupled to time-of-flight mass spectrometry (TOFMS). The method is fast, requiring only 13 min for the GC–TOFMS run. During the validation procedure, the method proved to be sufficiently precise and accurate with respect to the degree of endogenous biological variability found in rice samples. The profiles of the 14 lipophilic compounds of pigmented rice, as well as genetically modified (GM) rice to improve β-carotene, were subjected to principal component analysis (PCA) to evaluate the differences among cultivars. PCA could fully distinguish pigmented rice from non-pigmented rice. However, GM rice could not be separated from its non-transgenic counterpart (Nakdongbyeo). This protocol provides a rapid and feasible method for monitoring the bioactive compounds of rice.

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.

Coordination of multiple transgenes is essential for metabolic engineering of biosynthetic pathways. Here, we report the utilization of two bicistronic systems involving the 2A sequence from the foot-and-mouth disease virus and the internal ribosome entry site (IRES) sequence from the crucifer-infecting tobamovirus to the biosynthesis of carotenoids in rice endosperm. Two carotenoid biosynthetic genes, phytoene synthase (Psy) from Capsicum and carotene desaturase (CrtI) from Pantoea, were linked via either the synthetic 2A sequence that was optimized for rice codons or the IRES sequence under control of the rice globulin promoter, generating PAC (Psy-2A-CrtI) and PIC (Psy-IRES-CrtI) constructs, respectively. The transgenic endosperm of PAC rice had a more intense golden color than did PIC rice, demonstrating that 2A was more efficient than IRES in coordinating gene expression. The 2A and IRES constructs were equally effective in driving transgene transcription. However, immunoblot analysis of CRTI, a protein encoded by the downstream open reading frame of the bicistronic constructs, revealed that 2A was ninefold more effective than IRES in driving translation. The PAC endosperms accumulated an average of 1.3 μg/g of total carotenoids, which was ninefold higher than was observed for PIC endosperms. In particular, accumulation of β-carotene was much higher in PAC endosperms than in PIC endosperms. Collectively, these results demonstrate that both 2A and IRES systems can coordinate the expression of two biosynthetic genes, with the 2A system exhibiting greater efficiency. Thus, the 2A expression system described herein is an effective new tool for multigene stacking in crop biotechnology.

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.

A gene encoding a putative guanosine 5′-diphosphate (or 5′-triphosphate) 3′-diphosphate ((p)ppGpp) synthetase, designated PepRSH ( Pep per RelA / SpoT homologue), was isolated from hot peppers. PepRSH was found to contain five introns and six exons and a 2166-bp open reading frame encoding a protein of 721 amino acids; this protein displayed significant homology with other plant (p)ppGpp synthetases. A genomic DNA gel blot analysis revealed that the pepper genome has at least a single copy of PepRSH . PepRSH transcripts were highly accumulated in non-host resistance response-induced leaves and in leaves following induction with salicylic acid, methyl jasmonate, wounding, hydrogen peroxide, and ultraviolet-B. The expression of PepRSH was also influenced by abiotic stresses, such as flooding and high salinity. The deduced PepRSH protein has a putative chloroplast-targeting transit peptide at its N-terminus, and immunolocalization studies verified the translocation of PepRSH to the chloroplast. The predicted PepRSH protein is markedly similar to known plant and bacterial RSH proteins. Expression of a putative (p)ppGpp synthetase domain in an Escherichia coli single mutant ( RelA − SpoT + ) complemented growth of the mutant but not of an E. coli double mutant ( RelA − SpoT − ), demonstrating that PepRSH has (p)ppGpp synthetase activity only in the (p)ppGpp synthetase domain. Site-directed mutagenesis of the conserved histidine and aspartic acid (HD) site in the putative HD domain of PepRSH revealed that the histidine and aspartic acid dual sites were critical residues for the (p)ppGpp synthetase activity of PepRSH protein. Mutation of the HD site limited the tolerance of bacteria to both salt and osmotic stress. Our results indicate that pepper plants have a (p)ppGpp regulatory system that is similar to that of bacteria and which may transduce stress-related signals through the regulation of (p)ppGpp by PepRSH localized in chloroplasts.

Using an F 2:3 population with 200 lines derived from a cross between the japonica rice ( Oryza sativa L.) varieties Gaochan 106 and Changbai 9, quantitative trait loci (QTLs) for root number, root length, seedling height, and their relative alkaline damage rates at early seedling stage were identified under 0.15% Na 2 CO 3 alkaline stress. A total of 26 QTLs associated with these traits were detected on chromosomes 1, 5, 6, 7, 8, 9, and 11, consisting of 4 QTLs for root number, 5 QTLs for relative alkaline damage rate of root number, 6 QTLs for root length, 2 QTLs for relative alkaline damage rate of root length, 5 QTLs for seedling height, and 4 QTLs for relative alkaline damage rate of seedling height. QTLs qRN6-1 , qRN11 , qRRN11-2 , qRRL11-2 , qSH1 , qSH11-2 , qRSH5 , and qRSH6-2 accounted for 29.91, 13.42, 23.86, 21.06, 15.81, 16.53, 29.89, and 34.63% of the phenotypic variations, respectively. These QTLs, except for qRN6-1 with the marker flanking distance of 19.0 cM, were located with relatively large marker flanking distances over 26.3 cM. The positive alleles at 13 loci originated from the tolerant parent Changbai 9, and the positive alleles at other loci were from the sensitive parent Gaochan 106. Partial dominance and over dominance were the main effects at these loci.

OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a "blade to sheath transformation" phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::DeltaOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles.

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes.

Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect rice. Contacting a hard surface induces appressorium formation in C. miyabeanus, while the hydrophobicity of the substratum does not affect this morphogenic infection event. To determine whether the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. miyabeanus, the effects of a calcium chelator (ethylene glycol tetraacetic acid; EGTA), phospholipase C inhibitor (neomycin), intracellular calcium channel blocker (TMB-8), calmodulin antagonists (chlorpromazine, phenoxybenzamine, and W-7), and calcineurin inhibitor (cyclosporin A) on morphogenesis and infection were examined. Addition of Ca2+ and the calcium ionophore A23187 did not affect conidial germination, while the number of appressoria decreased with higher concentrations. EGTA inhibited conidial germination and appressorium formation. The calcium channel blocker did not affect appressorium formation at any concentration; however, calmodulin antagonists and the calcineurin inhibitor specifically reduced appressorium formation at the micromolar level. One of the calmodulin antagonists, W-7, also inhibited accumulation of mRNA of the calmodulin gene within germinating conidia and/or appressorium-forming germ tubes. Thus, biochemical processes controlled by the calcium/calmodulin signaling system seem to be involved in the induction of prepenetration morphogenesis on rice.

Coordination of multiple transgenes is essential for metabolic engineering of biosynthetic pathways. Here, we report the utilization of two bicistronic systems involving the 2A sequence from the foot-and-mouth disease virus and the internal ribosome entry site (IRES) sequence from the crucifer-infecting tobamovirus to the biosynthesis of carotenoids in rice endosperm. Two carotenoid biosynthetic genes, phytoene synthase (Psy) from Capsicum and carotene desaturase (CrtI) from Pantoea, were linked via either the synthetic 2A sequence that was optimized for rice codons or the IRES sequence under control of the rice globulin promoter, generating PAC (Psy-2A-CrtI) and PIC (Psy-IRES-CrtI) constructs, respectively. The transgenic endosperm of PAC rice had a more intense golden color than did PIC rice, demonstrating that 2A was more efficient than IRES in coordinating gene expression. The 2A and IRES constructs were equally effective in driving transgene transcription. However, immunoblot analysis of CRTI, a protein encoded by the downstream open reading frame of the bicistronic constructs, revealed that 2A was ninefold more effective than IRES in driving translation. The PAC endosperms accumulated an average of 1.3 μg/g of total carotenoids, which was ninefold higher than was observed for PIC endosperms. In particular, accumulation of β-carotene was much higher in PAC endosperms than in PIC endosperms. Collectively, these results demonstrate that both 2A and IRES systems can coordinate the expression of two biosynthetic genes, with the 2A system exhibiting greater efficiency. Thus, the 2A expression system described herein is an effective new tool for multigene stacking in crop biotechnology.

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.