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Insect-resistant Agb0101 rice was recently developed by modifying the cry1ac1 gene by changing codon usage changes relative to the native truncated cry1ac1 gene. To assess the toxicity of genetically modified Agb0101 rice, we conducted bioinfomational comparisons of the amino acid sequences that are not similar to known toxic proteins. Sufficient quantities of mCry1Ac1 protein were produced in Escherichia coli for in vitro evaluation and animal study. We compared the amino acid sequences and molecular mass. There have the same amino acid sequences and molecular masses after purifying the modified Cry1Ac1 (mCry1Ac1) protein from highly expressed bacteria and genetically modified rice were identical. We also investigated the acute and 90-days oral toxicities. No adverse effects were observed in mice following acute oral exposure to 2,000 mg/ kg body weight mCry1Ac1 protein of body weight and 90 days oral exposure to Agb0101. These results indicate that mCry1Ac1 proteins and Agb0101 rice demonstrate no adverse effects in these tests when applied via gavage and feed, respectively. Key words: Modified Cry1Ac1, food safety assessment, toxicity, insect- resistant rice Agb0101.

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.

Four transgenic rice lines harboring insect-resistant gene cry3A showed ideal field performances characterized by high considerable resistance to rice water weevil ( Lis-sorhoptrus oryzophilus Kuschel). In this study, we estimated the insertion number of foreign genes, and analyzed the flanking sequences of T-DNAs in rice genome. As a result, T-DNA of BT12R1 line was inserted in exon region of rice chromosome 10. Two copies of T-DNAs were inserted in line BT12R2. BT12R3 line was analyzed at only left border flanking sequence. BT12R4 line was confirmed one copy of foreign gene insertion at the position 24,516,607 ~ 24,516,636 of rice chromosome 5, accompanied by a deletion of 30 bp known genomic sequences. This intergenic position was confirmed none of expressed gene and any deletion/addition of T-DNA sequence. In conclusion, these molecular data of rice water weevil resistant Bt rice would be used to conduct the biosafety and environment risk assessment for GM crop commercialization.

본 연구는 종자로부터 유도된 캘러스를 아그로박테리움을 이용해 감염하여 고체배지에서 배양하는 기존의 방법과 달리, 형질전환에 소모되는 노동력과 시간, 비용을 단축하고자 감염과 공동배양, 균제거와 캘러스 선발까지 액체배양을 시도하였다. 성숙 종자로부터 유도된 캘러스를 바로 아그로박테리움으로 감염함으로써 조직배양으로 인한 체세포 변이의 발생을 최소화하고 감염부터 그 후 캘러스선발까지 총 4단계의 시간과 비용을 최소화하였으며, 감염된 캘러스로부터 재분화 시킴으로써 형질전환 식물체를 얻는 방법을 새롭게 수립하였다. 배양과정 중 감염과 공동배양 기간을 3일로 단축시킴으로써 캘러스의 스트레스를 최소화 하였고, PCR 분석을 통해 원하는 목표 유전자가 형질전환체에 안정적으로 도입이 되는 것도 확인할 수 있었다. 이러한 결과를 종합해 볼 때, 본 실험을 통해 얻어진 새로운 액체배양 방법은 우수한 농업적 형질을 가진 벼 품종 개발시 효율적으로 이용할 수 있을 것으로 생각된다. 【Rice is one of the most important cereal crops as a model plant for functional genomics of monocotyledons and usually transformed using Agrobacterium tumefaciens. However, the transformation's process using previous method is still time consuming and uneconomical, low efficiency. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the infection and co-cultivation, Agrobacterium elimination, infected calli's selection steps using liquid media. We directly inoculated Agrobacterium containing a ZjLsL gene under the control of constitutive promoter into the 1- to 3-week-old rice calli derived from mature seeds. After 3 days of co-cultivation, the infected calli were transferred onto liquid media of Agrobacterium elimination and calli's selection for 3 days. The calli were transferred to calli's growth solid media for 14 days and then the calli transferred to shoot induction and root induction media. Putative transformants were initially selected on the medium containing phosphinothricin, and the PAT protein verified by PAT strip test. This method in this study would lead to reduction of substantial labor and time to generate transgenic plants.】

Insect-resistant transgenic rice was developed by inserting the mCry1Ac1 a modified gene fromthe soil bacterium Bacillus thuringiensis (Bt). For biosafety assessment, we studied the effects onsurvival of cantor Daphnia magna, a commonly used as a model organism in ecotoxicologicalstudies. D. magna fed on Bt rice and its near non-genetically modified (GM) counterparts(Nakdong) grown in the same environment (100% ground rice suspension). The Bt rice wascomfirmed to have the insertion of T-DNA and protein expression by the polymerase chainreaction and ELISA analysis. Feeding study showed similar cumulative immobility and abnormalresponse of D. magna between Bt rice and non-GM counterparts. 48 h-EC

Lactoferrin is an 80-kDa iron-binding glycoprotein that is found in high concentrations in human milk. Human lactoferrin (hLF) has several beneficial biological activities including immune system modulation and antimicrobial activity. In the present study, we devolved a method of hLF expression through introducing the hLF gene construct into Oriza sativa cv. Nakdong using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the rice glutelin promoter was detected in the seeds of transgenic rice plants. Transformed rice plants were selected on media containing herbicide(DL-phosphinothricin) and the integration of hLF cDNA was confirmed by Southern blot analysis. The expression of the full length hLF protein from the grains of transgenic rice plants was verified by Western blot analysis. The lactoferrin expression levels in the transformed rice grains determined by enzyme-linked immunosorbant assay accounted for approximately 1.5% of total soluble protein. Taken together, these data indicate that rice grains expressing hLF can be directly incorporated into infant formula and baby food.

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.

Selectable marker gene systems are vital for the development of transgenic plants, but the presence of selectable marker genes encoding antibiotic or herbicide resistance in genetically modified plants poses a number of problems. A lot of research results and various techniques have been developed to produce marker-free GM plants. The aim of this review is to describe the principal methods used for eliminating selectable marker genes to generate marker-free GM plants, concentrating on the three significant methods(co-transformation, site-specific recombinase-mediated ex-cision, non-selected transformation) in several marker-free techniques. 서 론 1996년 처음 상업화된 유전자변형 (Genetically Modified, GM) 작물의 재배면적은 매년 급속한 성장을 기록하여 2009년에는 25개국 1억 3천 4백만 헥타르에 이르고 있다(James 2009). 이와 같은 GM작물의 성장세는 개발도상국과 선진국의 재배농민 모두에게 GM작물이 경제적으로 도움이 되며 식물형질전환기술의 급속한 발전으로 주요작물에서 높은 효율로 형질전환 (transformation)이 가능해졌기 때문이다. GM작물 개발을 위한 식물형질전환기술의 급속한 발전은 새로운 배지나 형질전환법의 개발이 빠르게 이루어졌기 때문이기도 하지만, 항생제 내성 유전자와 같은 선발표지 시스템을 식물형질전환에 적용했기 때문이다 (Miki and McHung 2004). 일반적으로 식물체 내에 목적유전자를 형질전환하기 위해서는 운반체 (vector)라는 DNA 구성물을 사용하여 목적유전자와 함께 항생제 내성 유전자와 같은 선발표지 유전자를 토양미생물인 아그로박테리움 (

Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h, from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F(2) plants and five F(3) lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3' splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 (OsCPL1). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development.

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.

For the development of qualitative PCR detection method of genetically modified (CM) rice, rice species-specific gene, OsCc-1 (rice cytochrome c gene), was selected as suitable far use as an endogenous gene in rice. The primer pair OsCytC-5`/3`with 111 bp amplicon was used for PCR amplification of the rice endogenous gene, OsCc-1 and no amplified product was observed from 8 different crops as templates. Qualitative PCR method was carried out with stack traits of L528CryIAc1 GM rice developed in Korea. For the qualitative PCRs, some primer pairs were designed with a construct-specific and event-specific type based on T-DNA and junction sequences of T-DNA in GM rice. Actck-5`/3` amplifying between actin promoter and OsCK1 gene introduced in LS28 gave rise to an amplicon 306 bp; also, CrLB-5`/3` from CryIAcl and CKRB-5`/3`amplifying the junction region of T-DNA and genome sequence from LS28 as event-specific primers gave rise to an amplicon 142 bp and 91 bp, respectively. These primer pairs for the detection of event-specific targets not produced PCR amplicons on non-CM rice and various crops in contrast to event lines. Therefore, in this study we verified that event-specific primers were effective to specifically detect stack trait lines and demonstrated that this method presented could be provided with the detection-method data for risk assessment analysis of GM rice to be commercialized.

Transposon-mediated insertional mutagenesis is one of powerful strategy for assessing functions of genes in higher plants. In this report, we have selected highly susceptible and tolerance plant by screening about high salt (3% NaCl) and cold stresses () from F2 seeds of 30,000 Ac/Ds insertional mutagenesis lines in rice (Oryza sativa L. cv. Dongjin). In order to identify the gene tagging, insertion of Ds element was analyzed by Southern blot and these results revealed that 19 lines were matched genotype of selected lines with phenotype from the first selected 212 lines, and 13 lines have one copy of Ds elements. The Franking Sequence Tags (FSTs) of selected mutant lines showed high similarities with the following known function genes: signal transduction and regulation of gene expression (transpoter, protease family protein and apical meristem family protein), osmotic stress response (heat shock protein, O-methyltransferase, glyceraldehyde-3-phosphate dehydrogenase and drought stress induce protein), vesicle trafficking (SYP 5 family protein) and senescence associated protein. The expression pattern of 19 genes were analyzed using RT-PCR under the abiotic stresses of 9 class; 250mM NaCl, osmotic, drought, 3% , ABA, IAA, 0.1 ppm 2,4-D, cold and high temperature. Isolated knock-out genes showed the positive response about 250 mM NaCl, drought, , PEG, IAA, 2,4-D, ABA treatment and low () and high temperature (). The results from this study indicate that function of selected knock-out genes could be useful in improving of tolerance to abiotic stresses as an important transcriptional activators in rice.

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.

본 연구는 동진벼 유래의 Ac/Ds 삽입변이집단의 GUS 분석을 통하여 뿌리 및 미숙종자에서 강하게 GUS가 발현한 개체를 선발하여 FST(flanking sequence tag) 분석 한 결과 Ds 전이 인자가 3번 염색체 zinc finger RING-H2 관련 Oszinc626 유전자의 첫 번째 exon 부위에 single copy로 삽입되어 있었으며, 선발변이체는 뿌리 및 종자 발달이 정상인 동진벼에 비해 매우 낮은 것으로 나타났다. Oszinc626 유전자는 RING-H2 type(C3-H2-C3)으로 $Cys-X_2-Cys-X_{28}-Cys-X-His-X_2-His-X_2-Cys-X_{14}-Cys-X_2-Cys$ 배열이 C-terminus 가장 말단에 위치하며, 49 kDa의 분자량을 가지고 있다. 또한 Southern blot 분석에서 Oszinc626 유전자는 벼 게놈상에 single copy로 존재하였다. RT-PCR을 통한 돌연변이 유전자의 발현분석 결과 250 mM의 염과, $4^{\circ}C$ 저온등과 같은abiotic stress에 의해 발현이 증가함을 보였고, 호르몬처리에 있어서 ABA와 IAA의 식물호르몬을 처리했을 경우 24시간까지 계속해서 발현양이 증가하는 것을 보이는 반면, 2,4-D 처리의 경우 30분 후에 발현이 일시적으로 증가되었으나 이후 발현이 급속히 감소한 것을 보였다. 벼의 조직 별 발현 검정에서 미성숙한 종자, 뿌리 분열조직 및 신초 등 주로 생장점 부위에서 강하게 발현되는 것을 보임에 따라 Oszinc626 유전자의 경우 식물의 생장에 관여하는 주동 유전자의 하나로 판단된다. 【Ac/Ds mutant lines of this study were transgenic rice plants, each of which harbored the maize transposable element Ds together with a GUS coding sequence under the control of a promoterless(Ds-GUS). We selected the mutants that were GUS expressed lines, because the GUS positive lines will be useful for identifying gene function in rice. One of these mutants was identified knock-out at Oszinc626(NP_001049991) gene, encoding a RING-H2 zinc-finger protein, by Ds insertion. In this mutant, while primary root development is normal, secondary root development from lateral root was very poor and seed development was incomplete compare with normal plant. RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, a few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. The consensus of the RING-H2(C3-H2-C3 type) domain for this group of protein is $Cys-X_2-Cys-X_{28}-Cys-X-His-X_2-His-X_2-Cys-X_{14}-Cys-X_2-Cys$ . Oszinc626 encodes a predicted protein product of 445 amino acids residues with a molecular mass of 49 kDa, with a RING-zinc-finger motif located at the extreme end of the C-terminus. RT-PCR analysis indicated that the expression of Oszinc626 gene was induced by IAA, cold, dehydration, high-salinity and abscisic acid, but not by 2,4-D, and the transcription of Oszinc626 gene accumulated primarily in rice immature seeds, root meristem and shoots. The gene accumulation patterns were corresponded with GUS expression.】

The Korean Rice Ds-tagging lines Database (KRDD) is designed to provide information about Ac/Ds insertion lines and activation tagging lines using japonica rice. This database has provided information on 18,158 Ds lines, which includes the ID, description, photo image, sequence information, and gene characteristics. The KRDD is visualized using a web-based graphical view, and anonymous users can query and browse the data using the search function. It has four major menus of web pages: (i) a Blast Search menu of a mutant line; Blast from rice Ds-tagging mutant lines; (ii) a primer de-sign tool to identify genotypes of Ds insertion lines; (iii) a Phenotype menu for Ds lines, searching by identi-fication name and phenotype characteristics; and (iv) a Management menu for Ds lines. Availability: The KRDD can be accessed at the web site of the National Institute of Agricultural Biotechnology (http://www. niab.go.kr/RDS/) Keywords: Ac/Ds database, Ds phenotype, Ds-tagging line, insertion line, KRDD

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.

A nonpathogenic rhizobacterium, Pseudomonas putida LSW17S, elicited systemic protection against Fusarium wilt and pith necrosis caused by Fusarium oxysporum f. sp. lycopersici and P. corrugata in tomato (Lycopersicon esculentum L.). LSW17S also confers disease resistance against P. syringae pv. tomato DC3000 (DC3000) on Arabidopsis ecotype Col-0. To investigate mechanisms underlying disease protection, expression patterns of defense-related genes PR1, PR2, PR5, and PDF1.2 and cellular defense responses such as hydrogen peroxide accumulation and callose deposition were investigated. LSW17S treatment exhibited the typical phenomena of priming. Strong and faster transcription of defense-related genes was induced and hydrogen peroxide or callose were accumulated in Arabidopsis treated with LSW17S and infected with DC3000. In contrast, individual actions of LSW17S and DC3000 did not elicit rapid molecular and cellular defense responses. Priming by LSW17S was translocated systemically and retained for more than 10 days. Treatment with LSW17S reduced pathogen proliferation in Arabidopsis ecotype Col-0 expressing bacterial NahG; however, npr1, etr1, and jar1 mutations impaired inhibition of pathogen growth. Cellular and molecular priming responses support these results. In sum, LSW17S primes Arabidopsis for NPR1-, ethylene-, and jasmonic acid-dependent disease resistance, and efficient molecular and cellular defense responses.

Ahn, I.-P., and Suh, S.-C. 2007. Calcium restores prepenetration morphogenesis abolished by methylglyoxal-bis-guanyl hydrazone in Cochliobolus miyabeanus infecting rice. Phytopathology 97:331-337. Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect its host rice plants. Curtailment of prepenetration development by spermidine and spermine was more evident in appressorium development and germination remained unaffected, whereas putrescine and methylglyoxal-bis-guanyl hydrazone (MGBG) impaired both morphogenetic events. Exogenous calcium nullified the inhibitory effect of MGBG on the prepenetration development in vitro and in vivo and the disease progression. High levels of polyamines were detected in freshly collected conidia, but the amounts were reduced during germination and appressorium formation. MGBG fortified the decrease of polyamines within conidia under development and calcium amendment did not affect the reduction. Hard-surface contact augmented messenger RNA synthesis of calmodulin gene (CmCaM) and protein kinase C (PKC) activity in germinating or appressorium-forming conidia. Calcium restored transcription of CmCaM and upregulation of PKC activity suppressed by MGBG. Taken together, fine-tuning of intracellular polyamine transition is indispensable for the conidial germination and appressorium formation in C. miyabeanus. Biochemical and molecular analyses revealed that the MGBG-acting site or sites are upstream of Ca2+-dependent signaling pathways regulating prepenetration morphogenesis of C. miyabeanus causing rice brown leaf spot. Additional keywords: Bipolaris oryzae, Oryza sativa.

Thiamine confers systemic acquired resistance (SAR) on susceptible plants through priming, leading to rapid counterattack against pathogen invasion and perturbation of disease progress. Priming reduces the metabolic cost required for constitutive expression of acquired resistance. To investigate the effects of priming by thiamine on defense-related responses, Arabidopsis (Arabidopsis thaliana) was treated with thiamine and effects of pathogen challenge on the production of active oxygen species, callose deposition, hypersensitive cell death, and pathogenesis-related 1 (PR1)/Phe ammonia-lyase 1 (PAL1) gene expression was analyzed. Thiamine did not induce cellular and molecular defense responses except for transient expression of PR1 per se; however, subsequent Pseudomonas syringae pv tomato challenge triggered pronounced cellular defense responses and advanced activation of PR1/PAL1 gene transcription. Thiamine treatment and subsequent pathogen invasion triggered hydrogen peroxide accumulation, callose induction, and PR1/PAL1 transcription activation in Arabidopsis mutants insensitive to jasmonic acid (jar1), ethylene (etr1), or abscisic acid (abi3-3), but not in plants expressing bacterial NahG and lacking regulation of SAR (npr1 [nonexpressor of PR genes 1]). Moreover, removal of hydrogen peroxide by catalase almost completely nullified cellular and molecular defense responses as well as SAR abolishing bacterial propagation within plants. Our results indicated that priming is an important cellular mechanism in SAR by thiamine and requires hydrogen peroxide and intact NPR1.

We isolated HvRAF (Hordeum vulgare root abundant factor), a cDNA encoding a novel ethylene response factor (ERF)-type transcription factor, from young seedlings of barley. In addition to the most highly conserved APETALA2/ERF DNA-binding domain, the encoded protein contained an N-terminal MCGGAIL signature sequence, a putative nuclear localization sequence, and a C-terminal acidic transcription activation domain containing a novel mammalian hemopexin domain signature-like sequence. Their homologous sequences were found in AAK92635 from rice and RAP2.2 from Arabidopsis; the ERF proteins most closely related to HvRAF, reflecting their functional importance. RNA blot analyses revealed that HvRAF transcripts were more abundant in roots than in leaves. HvRAF expression was induced in barley seedlings by various treatment regimes such as salicylic acid, ethephon, methyl jasmonate, cellulase, and methyl viologen. In a subcellular localization assay, the HvRAF-GFP fusion protein was targeted to the nucleus. The fusion protein of HvRAF with the GAL4 DNA-binding domain strongly activated transcription in yeast. Various deletion mutants of HvRAF indicated that the transactivating activity was localized to the acidic domain of the C-terminal region, and that the hemopexin domain signature-like sequence was important for the activity. Overexpression of the HvRAF gene in Arabidopsis plants induced the activation of various stress-responsive genes, including PDF1.2, JR3, PR1, PR5, KIN2, and GSH1. Furthermore, the transgenic Arabidopsis plants showed enhanced resistance to Ralstonia solanacearum strain GMI1000, as well as seed germination and root growth tolerance to high salinity. These results collectively indicate that HvRAF is a transcription factor that plays dual regulatory roles in response to biotic and abiotic stresses in plants.

Responses of rice to Magnaporthe grisea and Cochliobolus miyabeanus were compared. In Tetep, a rice cultivar resistant to both fungi, pathogen inoculation rapidly triggered the hypersensitive reaction (HR), resulting in microscopic cell death. In rice cv. Nakdong, susceptible to both pathogens, M. grisea did not cause HR, whereas C. miyabeanus caused rapid cell death similar to that associated with HR, which appeared similar to that observed in cv. Tetep, yet failed to block fungal ramification. Treatment with conidial germination fluid (CGF) from C. miyabeanus induced rapid cell death in both cultivars, suggesting the presence of phytotoxins in CGF. Pretreatment of cv. Nakdong with CGF significantly increased resistance to M. grisea, while the same treatment was ineffective against C. miyabeanus. Similarly, in cv. Nakdong, benzothiadiazole (BTH) significantly increased resistance to M. grisea, but was ineffective against C. miyabeanus. Methyl jasmonate (MeJA) treatment appeared to be ineffective against either fungus. Increased resistance of cv. Nakdong to M. grisea by BTH or CCF treatment was correlated with more rapid induction of three monitored PR genes. Application of MeJA resulted in the expression of JAmyb in cv. Nakdong being induced faster than in untreated plants in response to M. grisea infection. In contrast, the expression pattern of the PR and JAmyb genes in response to C. miyabeanus was nearly identical between cvs. Nakdong and Tetep, and neither BTH nor MeJA treatment significantly modified their expression patterns in response to C. miyabeanus infection. Our results suggest that rice employs distinct mechanisms for its defense against M. grisea versus C. miyabeanus.

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.

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.

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.