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Rice (Oryza sativa L.) is a staple crop in Korea, which is cultivated over 700,000 ha and produces a half million tons of rice every year. Rice research and varietal improvement have been dynamic, changing in line with public demands and needs. Since the 1970s, plant architecture has been improved to increase yield components. In the 2000s, a new emphasis was placed on grain quality and biotic and abiotic stress resistance, and the rice varieties became more diversified according to reduced consumption and increased demands of processing industries. As a result of extensive research, 232 (71.4%) were categorized as good quality for eating in rice bowls, 27 (8.3%) were high yielding, and 21 varieties (6.5%) were for waxy rice among 325 varieties. The culm length was continuously reduced for lodging resistance, and panicle length and number of seeds per panicle increased. Finally, milled rice yields increased by 13% from 491 kg/10a in the 1980s to 555 kg/10a in the 2010s. One of the potential risks in Korean rice breeding is the narrow pool of genetic diversity. The parental origins of 325 Korean varieties show that 66% of the varieties originate from two Korean-bred stocks and 26% of the varieties have at least one parent from Japanese stocks. The limited genetic diversity has a negative influence on hybrid vigor and may lead to potential problems for Korean rice breeding in the future.

Currently, approximately 900,000 tons of rice is produced annually in Turkey from a rice cultivation area of around 120,000 ha, which is equivalent to 28% of the rice growing area in the European Union. Since 1990, 63 rice varieties have been bred in Turkey and national rice production has increased 3.5-fold. Therefore, we reviewed the shifts in agronomic traits and the genetic resources used in rice varietal improvements in Turkey from 1990 to the present. We also compared the agronomic traits of Turkish-bred and Italian rice varieties. The shifts in agronomic traits in this period showed that the number of days to flowering decreased by about one day from S1 (1990-2003) and S2 (2004-2015) to S3 (2016-present). Additionally, culm length decreased by 6 cm from S1 to S2 and S3, and 1000 grain weight decreased continuously from S1 to S3 to the current average value of 24.1 g. In contrast, the head rice ratio increased continuously from 59.8% in S1 to 64.9% in S3. The rice sector in Turkey must overcome new challenges of market competition with Italian rice varieties; we found that three Italian rice varieties (Cameo, Ronaldo, and Luna CL) shared 25% of the 2016 rice market in Turkey. In addition, Turkish rice had longer maturation periods, shorter caryopsis, and lower head rice recovery relative to Italian varieties. Pedigree analysis of Turkish varieties also revealed that 62% had at least one Italian variety as a hybridization parent. We suggest that extensive use of Italian varieties in Turkish rice breeding programs may result in reduced genetic diversity; hence, low genetic diversity could be a critical constraint in future rice breeding in Turkey.

This paper investigates the effects of vertical integration on screening decisions using data from the Korean movie industry. We find that integrated theaters show their affiliated movies more than nonaffiliated movies, and more than nonintegrated theaters do. However, this gap reduces during the peak times of the day and after the opening week. Our evidence suggests that the integrated theaters’ screening decisions favorable to their own movies are better understood as the result of dealing with the incentive misalignment than as the result of foreclosing other movies.

Background Understanding late pollen development, including the maturation and pollination process, is a key component in maintaining crop yields. Transcriptome data obtained through microarray or RNA-seq technologies can provide useful insight into those developmental processes. Six series of microarray data from a public transcriptome database, the Gene Expression Omnibus of the National Center for Biotechnology Information, are related to anther and pollen development. Results We performed a systematic and functional study across the rice genome of genes that are preferentially expressed in the late stages of pollen development, including maturation and germination. By comparing the transcriptomes of sporophytes and male gametes over time, we identified 627 late pollen-preferred genes that are conserved among japonica and indica rice cultivars. Functional classification analysis with a MapMan tool kit revealed a significant association between cell wall organization/metabolism and mature pollen grains. Comparative analysis of rice and Arabidopsis demonstrated that genes involved in cell wall modifications and the metabolism of major carbohydrates are unique to rice. We used the GUS reporter system to monitor the expression of eight of those genes. In addition, we evaluated the significance of our candidate genes, using T-DNA insertional mutant population and the CRISPR/Cas9 system. Mutants from T-DNA insertion and CRISPR/Cas9 systems of a rice gene encoding glycerophosphoryl diester phosphodiesterase are defective in their male gamete transfer. Conclusion Through the global analyses of the late pollen-preferred genes from rice, we found several biological features of these genes. First, biological process related to cell wall organization and modification is over-represented in these genes to support rapid tube growth. Second, comparative analysis of late pollen preferred genes between rice and Arabidopsis provide a significant insight on the evolutional disparateness in cell wall biogenesis and storage reserves of pollen. In addition, these candidates might be useful targets for future examinations of late pollen development, and will be a valuable resource for accelerating the understanding of molecular mechanisms for pollen maturation and germination processes in rice. Electronic supplementary material The online version of this article (10.1186/s12284-018-0219-0) contains supplementary material, which is available to authorized users.

NH4+ is an important nitrogen resource for rice plants in paddy soil. Therefore, it is likely that NH4+-triggered plant growth interacts with phytohormone-mediated developmental mechanisms. Our previous transcriptomic analysis revealed that many genes involved in auxin signaling and efflux are sensitive to NH4+. In the current study, we found that NH4+ treatment causes a delayed gravity response in rice roots. To further elucidate the interlocking relationship between NH4+ and auxin signaling during root development, we utilized mutants and overexpressors of a key NH4+ signaling transcription factor INDETERMINATE DOMAIN 10 (IDD10), encoding a transcription factor that regulates the expression of NH4+ uptake and N-assimilation genes. We obtained several lines of evidence that auxin affects NH4+-mediated gene expression and root development in rice plants via IDD10. First, the gravity response was delayed in idd10 roots and accelerated in IDD10 overexpressor (IDD10 OX) roots in the absence and (especially) presence of NH4+. Second, idd10 plants showed strong root coiling only in the presence of NH4+. However, treatment of 1-N-naphthylphthalamic acid (NPA), a polar auxin transport inhibitor suppressed the NH4+-specific root phenotype of idd10. Third, the expression of NH4+-responsive auxin-related genes was affected in idd10 and IDD10 overexpressors. Finally, IDD10 expression was induced by IAA and suppressed by NPA. These findings suggest that the gene expression patterns and phenotypes triggered by NH4+ are influenced by the actions of auxin during root development, pointing to a regulatory circuit between NH4+ and auxin signaling that functions in root development in rice.

Figure S1. a. Order of developmental stages for anatomical samples. ACF, formation of archesporial cells; BG, bi-cellular gametophyte; Fl, flowering; Me, meiosis; Me1, meiotic leptotene; Me2, meiotic zygotene-pachytene; Me3, meiotic diplotene-tetrad; MP, mature pollen; PMe, pre-meiosis; GP, germinating pollen; TG, tri-cellular pollen; UG, uni-cellular gametophyte. Red bar, sample containing late pollen. b. Expression graph of 36 clusters after KMC analysis with 57,382 probes. Clusters 2 and 35 exhibited mature pollen-preferential patterns of expression and are marked with red boxes. Figure S2. Schematic representation of 3 promoter trap lines for T-DNA insertions. a. T-DNA was inserted into 17th intron of SacI homology domain-containing protein (LOC_Os11g20384) in Line 1A-13,819 (mtd1â 1). b. Line 3A-05916 has T-DNA insertion in B12D protein (LOC_Os07g17310). BL, left T-DNA border; RB, right T-DNA border; Gray boxes, exons; lines, introns. Figure S3. Expression graph after KMC analysis of meta-expression data from Arabidopsis. Clusters marked with red box showed late pollen-preferred patterns. Figure S4. Heatmap for expression profiles of late pollen-preferred genes in Arabidopsis. Figure S5. Heatmap of genes involved in GA biosynthesis and signaling. CPS, GA3ox1, KAO, GA20ox3, and KO2 showed late pollen-preferred expression patterns and are outlined with red boxes. (DOCX 227 kb)

Table S1. Six series of microarray data comprising 64 slides (GPL2025) associated with anthers/pollen in rice. Table S2. Locus IDs and putative functions of late pollen-preferred genes from rice. Table S3. Locus IDs and promoter regions of genes used for promoter analysis with GUS reporter. Table S4. Classification of GO terms for biological processes associated with late pollen-preferred genes. Table S5. MapMan classification of late pollen-preferred genes. Table S6. Genes related to hormone metabolism term in MapMan. Table S7. Late pollen-preferred genes in Arabidopsis. Table S8. Assignment of rice orthologs to Arabidopsis late pollen-preferred genes. Locus numbers are shown in by red. Table S9. Assignment of Arabidopsis orthologs to rice late pollen-preferred genes. Locus numbers are shown in red. Table S10. MapMan terms related to cell wall organization and modifications in rice and Arabidopsis. Table S11. Primer sequences used in genotyping and real-time PCR. (DOCX 1980 kb)

This study was carried out to evaluate the expression of yellow endosperm color in the progeny of Psy-2A-CrtI (β-carotene gene, PAC) transgenic plants crossed with IR36. The selected 25 lines of F2 progeny exhibited stable expression of yellow endosperm color and high agronomic characteristics. IR36 and PAC transgenic plant of the grain length, width and ratio showed the same tendency of normal distribution. For progeny selection, colorimeters were as employed to distinguish differing visible colors. Correlation analysis was conducted to investigate the relationship between b* value and the expression of specific genes. b* value was associated with the relative expression of the PAC-Rev and MAR product by real-time PCR and t-test revealed significant difference. Based on this study, correlation between colorimetric values and real-time PCR was effective to detect gene expression. Yellow endosperm progenies represent a new genetic pool that might be useful to increase the genetic diversity of yellow endosperm rice.

During rice cultivation, brown planthopper (BPH, Nilaparvata lugens Stal.) and white-backed planthopper (WBPH, Sogatella furcifera) infestations cause decreased yield and rice quality. In this study, we attempted to develop multi-resistance plants and increase the marker-assisted selection (MAS) efficiency. The frequency of callus induction in 95 F1 doubled haploid lines (DHLs) derived from JSNDH13 (BPH resistance)/CNDH32 (WBPH resistance) ranged from 3.8 to 15.2 %. The plant regeneration rates varied from 2.9 to 39.3 %. Selected 75 DH plants were used to compare the results of WBPH bioassay and DNA marker reaction. The DHL bioassay showed continuous gene distribution and resistance score ranged from 0.5 to 9.0 (mean, 4.2). The broad-sense heritability for WBPH resistance was 51.7 %. The DHLs from the cross of JSNDH13/CNDH32 were segregated into 39 resistant (R):36 susceptible (S) to WBPH, i.e., 1 RR:SS. For the bioassay and MAS, RM11669 with WBPH resistance was selected. Genetically, the use of resistant cultivars, MAS, and DH breeding has proven to be environment friendly, efficient, and economical. This study confirmed the genetic basis for MAS to improve WBPH resistance in DHLs of WBPH/BPH resistant rice plants.

The concentration of nutrients in brown rice is mainly associated with embryo size. Various beneficial components have been purified from rice bran. Recently developed black waxy rice with a giant embryo (‘Milyang 263’, BGE), which is the ge t mutant of the GE gene, was selected and analyzed to produce high quality nutritional components. γ-Aminobutyric acid (GABA) content in BGE rice bran, one of the most important nutritional compounds in rice, showed a 10.6-fold increase (2.66±0.48 mg/g) compared to that of BGE brown rice (0.25±0.01 mg/g). In addition, brown rice and BGE rice bran showed enriched amylopectin (94.5±0.5 and 97.0±0.0%) and bioactive anthocyanin [Cy-3-G: 75.15±4.18 (brown rice), 82.97±0.81 (rice bran) and Pn-3-G: 1.52±0.50 (brown rice), 4.33±0.20 (rice bran); mg/100 g] contents. These results suggest that BGE rice bran can be used as an excellent raw material to efficiently produce high quality essential amino acids, cyanidin-3 glycoside-enriched anthocyanins, and GABA.

Recently, improving eating quality and processing properties has become one of the most important objectives in japonica rice breeding programs in Korea. This study was carried out to determine the agronomy and physicochemical characteristics of the opaque endosperm rice "S-21-3-8" regenerated from seed-derived callus culture of a rice cultivar, "Ilpum". S3 generation of opaque endosperm mutants selected from pedigree breeding was used for analysis of agronomic and physicochemical traits. Genetic segregation was observed at the highest frequency among opaque endosperm mutants, being present in 85.7% (12/14 lines) of the entire opaque lines. However, the major agricultural characteristics and grain traits of "S-21-3-8" were similar to those of a donor cultivar, "Ilpum". "S-21-3-8" showed significantly lower (10.6%) amylose than those (17.7%) of "Ilpum" in brown rice, while the protein levels were similar to those of the donor plant. The grain hardness of "S-21-3-8" (1.67 kgf/mm2) was lower than that of "Ilpum" (1.97 kgf/mm2), resulting in a high flour-milling percentage. The loosely packed starch granules of "S-21-3-8" in the opaque endosperm were demonstrated by SEM analysis of cross-sectioned rice grains. The opaque endosperm mutants that were of somaclonal variations in the tissue culture will lead to improved eating quality and processing properties of rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious threats to rice production. In this study, screening of rice for resistance to BLB was carried out at two different times and locations; that is, in a greenhouse during winter and in an open field during summer. The pathogenicity of Xoo race K1 was tested on 32 Korean rice cultivars. Inoculation was conducted at the maximum tillering stage, and the lesion length was measured after 14 days of inoculation. Five cultivars, Hanareum, Namcheon, Samgdeok, Samgang, and Yangjo, were found to be resistant in both the greenhouse and open-field screenings. Expression of the plant defense-related genes JAmyb, OsNPR1, OsPR1a, OsWRKY45, and OsPR10b was observed in resistant and susceptible cultivars by qRT-PCR. Among the five genes tested, only OsPR10b showed coherent expression with the phenotypes. Screening of resistance to Xoo in rice was more accurate when conducted in open fields in the summer cultivation period than in greenhouses in winter. The expression of plant defenserelated genes after bacterial inoculation could give another perspective in elucidating defense mechanisms by using both resistant and susceptible individuals.

The importance of genetic stability and bio-safety in the environment has recently been recognized for many (genetically modified) GM plants. This study evaluated the GM safety of transgenic rice and its environmental variance. Data on agronomic characters and principal component were collected for vitamin A-enriched GM rice and four check cultivars in a large GM field trial during 2009-2011. The cultivation environment was a large GM field and a greenhouse. In this experiment, there was no significant difference between the agronomic characters of the GM rice and those of a donor plant, ‘Nagdong’. In terms of grain characteristics, the appearance and physicochemical characteristics of the GM rice and those of the donor plant were similar. However, the grain of the GM rice developed a white core and a white belly when planted in the greenhouse. The type and distribution of dominant weed species were not different in the GM rice and the ‘Nagdong’. In addition, gene flow was not detected in the dominant weed species based on PCR analysis.

*ABSTRACT This study was carried out to investigate the agronomic traits, comparison of weed characteristics and possibility of gene flow in ‘vitamin A enforced GM rice' and the donor plant, ‘Nagdong'. The GM rice was not significantly different agronomic traits compared to the donor plant, Nagdong. Weed population changes were investigated in the cultivation of the GM rice and the donor plant, Nagdong. Dominant weed species and their dry matter did not show the difference between GM rice and the donor plant, Nagdong in macro-GM crop field. Dominant weed species with the GM rice and the donor plant, Nagdong were Monochoria vaginalis, followed by Eleocharis kuroguwai, Echinochloa crus-galli and Lindernia procumbens. The detection of gene from the GM rice was done using PCR, gene flow can't be detected by weed species. Results of this study on the agronomic traits, weed characteristics and possibility of gene flow has elucidated that GM rice might not be different from the donor plant, Nagdong.Key words: agronomic trait; gene flow; GM rice; weed-mediated.

Nutritional and physicochemical properties of new giant embryo mutant rice (YR23517Acp79, YR) were analyzed. YR exhibited increased total protein (9.3±0.3%), lipid (3.7±1.1%), amino acid (663.28±1.9 mg/g), and mineral contents (Ca=284.0±6.2, Mg=1417.5±13.6 mg/kg). In YR brown rice four major physicochemicals, including gamma-aminobutyric acid (brown rice= 0.46±0.014 mg/g), γ-oryzanol (0.43±0.021 mg/g), vitamin B1 (6.42±0.3 mg/kg), and tocopherols (alpha= 2.68±0.1, beta=0.11±0.01, gamma=0.05±0.001 mg/100 g) increased in comparison to reported giant embryo (Keunnunbyeo, KB) and normal embryo rice (Ilmibyeo, IB). YR showed higher scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (0.2 g/mL=57.1±2.25) and 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (0.2 g/mL=50.2±1.45) radicals but also inhibited lipopolysaccharide-induced nitric oxide production without cytotoxicity. These results indicate YR is a high quality functional rice due to its high nutrition content and antioxidant effects of physicochemicals.

Transposon tagging and insertional mutagenesis provide one of the most powerful tools in gene function studies. Here, we report a comparison between two novel drooping leaf (DL) mutants from transposon and T-DNA insertion lines of rice. DL is distinct from well-known ABC genes and a member of the YABBY gene family, and it is closely related to the CRABS CLAW (CRC) gene of Arabidopsis thaliana. Based on phenotypic analysis, DL regulated midrib formation by promoting cell proliferation in the central region of rice leaf and was necessary for the specification of carpel identity. We identified two DL mutants by screening the Ac/Ds and T-DNA insertional mutant pool of rice. Flanking sequence tag analysis indicated that both Ds and T-DNA segments were inserted in the promoter region at 3.4 kbs and 5.4 kb upstream, respectively, of the previously known OsYABBY domain. Interestingly, the progenies of DL lines of two different pools showed various degrees of leaf drooping and abnormal carpel formation. Flower structures revealed that there were more than two stigmas with normal stamens and pistils per panicle in the Ds-induced mutants. However, T-DNA induced mutant had extra stamens with staminoid carpels. These results indicate that the promoter region of DL plays an important function in regulating anther and carpel formation.

The purposes of this study were to analyze the weediness potential associated with 'Vitamin A enforced rice' and to evaluate the relationships between growth characteristics and environmental adaption related to survival ability in the natural environment. 'Vitamin A enforced rice', 'Nagdong', donor variety of 'Vitamin A enforced rice' and three other varieties were used for this study. The 'Vitamin A enforced rice' was experimented according to ratio of ripened grain, shattering of grain, germination traits, germination ratio of low temperature, emergence ratio after wintering, ratio of viviparity, and growth ratio after ratooning ability. 'Vitamin A enforced rice' was not significant between 'Nagdong' and other varieties in seven characters. Germination ratio was similar of Vitamin A enforced rice' and 'Nagdong', but average germination ability of was different at low temperature. Also, shattering of grain and germination ratio of alternating temperature were significant between 'Nagdong' and other varieties. These results suggested that the relationships between growth characteristics and environmental adaption in 'Vitamin A enforced rice' could be applied to the stability of weediness p otential and biosafety guide in GM rice.

Importin β1 interacts with nuclear transport factors and mediates the import of nuclear proteins. We isolated a pollen-expressed gene, rice Importin β1 (OsImpβ1), from a T-DNA insertional population that was trapped by a promoterless β-glucuronidase (GUS) gene. The GUS reporter was expressed in the anthers and ovaries from early through mature developmental stages. Its expression was also observed in all floral organs. However, these patterns changed as the spikelet developed. T-DNA was inserted into the OsImpβ1 gene at 339 bp downstream from the translation initiation site. We obtained another T-DNA insertional allele by searching the flanking sequence tag database. In both lines, the wild-type and T-DNA-carrying progeny segregated at a ratio close to 1:1. The latter genotype was heterozygous (OsImpβ1/osimpβ1). Reciprocal crosses between WT and heterozygous plants demonstrated that the mutant alleles could not be transmitted through the male gametophyte. Close examination of the heterozygous anthers revealed that the mutant pollen matured normally. However, in vitro assays showed that tube elongation was hampered in the mutant grains. These results indicate that OsImpβ1 is specifically required for pollen tube elongation.

This study was carried out to facilitate the functional analysis of rice genes. Some 297 insertion plants (1.7%) of the entire lines with the endogenous retrotransposon Tos17 were produced. Phenotypes of these plants in the S2 genera-tion were observed in the field according to different leaf types. Rolling leaf mutants showed thinner sclerenchyma-tous cells, defective arrangement of vascular bundles, and well-formed bulliform cells as compared to the parental cultivar. Two new copies of Tos17 were detected in the rolling leaf type. In the new leaf type, the copy number and activation of Tos12, 15 did not appear as ‘Ilpum’. Flanking sequence tag (FST) analysis of Tos17 in the rolling leaf mutant indicated that new copies of Tos17 were transposed on chromosomes 11 and 12. Annotated homologues of the tagging genes on chromosome 11 were arabinoxylan rabinofuranohydrolase isoenzyme AXAH-I and II. The tagging gene in chromosome 12 was highly correlated with 6 kinds of genes including a transcript regulated factor and a rough sheath 2-like protein. This rolling leaf and flanking sequence data will stimulate the functional analysis of rice genes.

Rice is the staple food of more than 50% of the worlds population. Cultivated rice has the AA genome (diploid, 2n=24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized transposable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos (Hirochika. 1997) have been used to generate gene-tagged populations. To date T-DNA and maize transposable element systems has been utilized as main insertional mutagens in rice. A main drawback of a T-DNA scheme is that Agrobacteria- mediated transformation in rice requires extensive facilities, time, and labor. In contrast, the Ac/Ds system offers the advantage of generating new mutants by secondary transposition from a single tagged gene. Revertants can be utilized to correlate phenotype with genotype. To enhance the efficiency of gene detection, advanced gene-tagging systems i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of insertionally mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been launched by collaborative works from 2001 in Korea.

A phosphate starvation-induced, purple, acid phosphatase cDNA was cloned from rice, Oryza sativa. The cDNA encoding the phosphatase (OsPAP2) has 1,893 bp with an open reading frame of 630 amino acid residues. The deduced amino acid sequence of OsPAP2 shows identities of 60–63% with other plant purple acid phosphatases and appears to have five conserved motifs containing the residues involved in metal binding. OsPAP2 expression is up-regulated in the rice plant and in cell cultures in the absence of phosphate (P i ). The induced expression of OsPAP2 is a specific response to P i starvation, and is not affected by the deprivation of other nutrients. OsPAP2 expression was responsive to the level of P i -supply, and transcripts of OsPAP2 were abundant in P i -deprived roots. The OsPAP2 cDNA was expressed as a 69 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsPAP2 gene was introduced into Arabidopsis via an Agrobacterium-mediated transformation. Functional expression of the OsPAP2 gene in the transgenic Arabidopsis line was confirmed by northern and western blot analyses, as well as by phosphatase activity assays. These results suggest that the OsPAP2 gene can be used to develop new transgenic dicotyledonous plants that are able to adapt to P i -deficient conditions.

The rice embryo is rich in lipid and protein bodies, bioactive chemicals such as dietary fiber, phytic acids, vitamin B and E, and gamma aminobutyric acid (GABA) than the endosperm. In this paper, we report a new giant embryo mutant,get, induced from somaclonal variation derived by anther culture in rice. Sequence analysis of Cytochrome P450 of the get mutant revealed thatget is a new allele of theGE gene with a single point mutation with substitution of amino acid, W395 to L395. The weight of theget mutant embryo was 3.7 times higher than normal embryo. Tocopherol and mineral content were also higher than the previously reported giant embryo rice variety, Keunnun. These results indicated that this new giant embryo rice (get) offers a promising source of genetic material in improving nutritional quality of rice especially tocopherol, essential minerals, and GABA.

A gene detection strategy using two-component Ac/Ds construct, with the mobile Ds transposon, has been developed to better understand gene functions in crops. Currently, 115,000 Ds insertion lines have been generated through the Ac/Ds gene trap system in Korea using japonica rice Dongjin as donor. Four hundred and thirty-seven mutants from 12,162 Ds-tagged lines were catalogued, including physiological and agronomic traits. Different traits were identified with distinct characteristics in terms of tillers, panicles, leaves, flowers, seed, chlorophyll content, and height. Culm and panicle length, number of panicles, and days to flowering of the Dongjin Ds population revealed high standard deviations compared with the donor cultivar. An evaluation of the Ds distribution on the chromosome revealed that 74.5% of the Ds were reinserted into gene-rich regions, making this Ac/Ds-mediated gene trap system useful in helping to gain an understanding of the function of genes and thus improve the gene-tagging system in rice.

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice. To understand the molecular basis of Pi5-mediated resistance to M. oryzae, we cloned the resistance (R) gene at this locus using a map-based cloning strategy. Genetic and phenotypic analyses of 2014 F2 progeny from a mapping population derived from a cross between IR50, a susceptible rice cultivar, and the RIL260 line carrying Pi5 enabled us to narrow down the Pi5 locus to a 130-kb interval. Sequence analysis of this genomic region identified two candidate genes, Pi5-1 and Pi5-2, which encode proteins carrying three motifs characteristic of R genes: an N-terminal coiled-coil (CC) motif, a nucleotide-binding (NB) domain, and a leucine-rich repeat (LRR) motif. In genetic transformation experiments of a susceptible rice cultivar, neither the Pi5-1 nor the Pi5-2 gene was found to confer resistance to M. oryzae. In contrast, transgenic rice plants expressing both of these genes, generated by crossing transgenic lines carrying each gene individually, conferred Pi5-mediated resistance to M. oryzae. Gene expression analysis revealed that Pi5-1 transcripts accumulate after pathogen challenge, whereas the Pi5-2 gene is constitutively expressed. These results indicate that the presence of these two genes is required for rice Pi5-mediated resistance to M. oryzae.

Most high-affinity phosphate transporter genes (OsPTs) in rice were highly induced in roots when phosphate was depleted. OsPT1, however, was highly expressed in primary roots and leaves regardless of external phosphate concentrations. This finding was confirmed histochemically using transgenic rice plants that express the GUS reporter gene under the control of the OsPT1 promoter, which exhibited high GUS activity even in the phosphate sufficient condition. Furthermore, transgenic rice plants overexpressing the OsPT1 gene accumulated almost twice as much phosphate in the shoots as did wild-type plants. As a result, transgenic plants had more tillers than did wild-type plants, which is a typical physiological indicator for phosphate status in rice.

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.

The development of rice varieties (Oryza sativa L.) that are resistant to the brown planthopper (BPH; Nilaparvata lugens Stål) is an important objective in current breeding programs. In this study, we generated 132 BC(5)F(5) near-isogenic rice lines (NILs) by five backcrosses of Samgangbyeo, a BPH resistant indica variety carrying the Bph1 locus, with Nagdongbyeo, a BPH susceptible japonica variety. To identify genes that confer BPH resistance, we employed representational difference analysis (RDA) to detect transcripts that were exclusively expressed in one of our BPH resistant NIL, SNBC61, during insect feeding. The chromosomal mapping of the RDA clones that we subsequently isolated revealed that they are located in close proximity either to known quantitative trait loci or to an introgressed SSR marker from the BPH resistant donor parent Samgangbyeo. Genomic DNA gel-blot analysis further revealed that loci of all RDA clones in SNBC61 correspond to the alleles of Samgangbyeo. Most of the RDA clones were found to be exclusively expressed in SNBC61 and could be assigned to functional groups involved in plant defense. These RDA clones therefore represent candidate defense genes for BPH resistance.

A phosphate starvation-induced acid phosphatase cDNA was cloned from the rice, Oryza sativa. The cDNA encoding O. sativa acid phosphatase (OsACP1) has 1100 bp with an open reading frame of 274 amino acid residues. The deduced amino acid sequence of OsACP1 cDNA showed 53% identity to tomato acid phosphatase and 46-50% identity to several other plant phosphatases. OsACP1 expression was up-regulated in the rice plant and in cell culture in the absence of phosphate (Pi). The induced expression of OsACP1 was a specific response to Pi starvation, and was not affected by the deprivation of other nutrients. OsACP1 expression was responsive to the level of Pi supply, with transcripts of OsACP1 being abundant in Pi-deprived root. The OsACP1 cDNA was expressed as a 30 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsACP1 gene was introduced into Arabidopsis via Agrobacterium-mediated transformation. Functional expression of the OsACP1 gene in the transgenic Arabidopsis lines was confirmed by Northern blot and Western blot analyses, as well as phosphatase activity assays. These results suggest that the OsACP1 gene can be used to develop new transgenic dicotyledonous plants able to adapt to Pi-deficient conditions.

The spikelet identity gene "fzp" (frizzy panicle) is required for transformation of the floral meristems to inflorescent shoots. In fzp mutants, spikelets are replaced by branches and spikelet meristems produce massive numbers of branch meristems. We have isolated and characterized a new fzp mutant derived from anther culture lines in rice and designated as fzp-9(t). The fzp-9(t) mutant showed retarded growth habit and developed fewer tillers than those of the wild-type plant. The primary and secondary rachis branches of fzp-9(t) appeared to be normal, but higher-order branches formed continuous bract-like structures without developing spikelets. The genetic segregation of fzp-9(t) showed a good fit to the expected ratio of 3: 1. The sequence analysis of fzp-9(t) revealed that there is a single nucleotide base change upstream of the ERF (ethylene-responsive element-binding factor) domain compare to wild-type plant. The mutation point of fzp-9(t) (W66G) was one of the six amino acids of the ERF domain that contributed to GCC box-specific binding. The premature formation of a stop codon at the beginning of the ERF domain might cause a non-functional product.

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.

This study was carried out to establish a new breeding scheme which is connected with conventional breeding method and anther culture method. To develop a herbicide resistant and direct seeding rice, plants were subjected to anther culture and regenerated plants from 5 crosses were studied to confirm the introduction of bar gene. After PCR analysis, we selected 227 plants which were carrying herbicide resistance gene (bar) out of 1,508 regenerated plants from anther culture. Among 169 lines carrying herbicide resistant gene from 5 crosses including YR23235 (Dongjin Ds3(Ba )/ Milyang165), 42 lines that had superior agronomic characters were selected for further research. Among them, YR23235Acp79 which showed herbicide resistance, direct seeding adaptability and superiority in major agronomic characters was named Milyang 204. This breeding scheme proved that the anther culture of plants crossed between transformant and cultivar or transform ant alone could be utilized in breeding programs for a rapid progeny fixation and development of a variety.y.

Two genes, SinIFS1 and SinIFS2 from Korean soybean cultivar, Sinpaldalkong known as one of isoflavonerich cultivars, were cloned with PCR and degenerate primers. The sequences of two genes were analyzed with previously reported IFS genes of leguminous plants and their expression pattern in various environmental conditions was surveyed. The genomic clone of SinIFS1 contained 1,828bp nucleotides and encoded a polypeptide of 521 amino acids, and 1912bp nucleotides and a polypeptide of 521 amino acids for SinIFS2. Both genes included several conserved motifs, oxygen binding and activation (A/G-G-X-E/D-T-T/S), ERR triad (E...R....R), and heme binding (F-X-X-G-X-R-X-C-X-G) domain, which are typical in any member of cytochrome P45O superfamily. Very high sequence homology (>98%) was observed in the comparison with other IFSs of legumes. In the northern blot analysis to check the expression and increase of SinIFS1 to various environmental renditions (low temperature, light, dark, UV, and fungal elicitor), the most significant induction, more than 6 times of transcript level compared to the dark treatment as a control, was observed from the fungal elicitor treatment. The next up-regulated expression was from UV treatment (4), low temperature and light conditions.

Variations of agronomic traits were evaluated on the progenies of regenerated rice plants of different callus origin and subculture period. From eighty-eight percent to ninety percents of ovary culture-derived plants (OCP) and anther culture-derived plants (ACP) were not segregated within the lines in major agronomic characters. Compare to ACP and OCP lines, the seed culture derived lines (SCP) showed more segregation (17％) in major agronomic traits among the lines. The most frequent segregating traits were grain fertility (6.1％) in ACP, leaf color (4.2％) in OCP and grain fertility (4.9％) and leaf color (4.9％) in SCP lines. The SCP line showed more variation in culm length, panicle length and heading date than those of OCP and ACP lines. The variation of agronomic traits in SCP lines was tended to increase with prolonged subculture. Culm and panicle length were shorter than those of original cultivar in all three types of tissue culture-derived lines (OCP, ACP and SCP).

In spite of potential benefits of anther culture, low productivity of plant regeneration in some genotypes; e.g. tonsil and indica rice, is one of the major obstacles for practical use of anther culture. This study was conducted to improve cold shock method and carbohydrate source for increasing the efficiency of anther culture in rice. The most common carbon source, sucrose was replaced to maltose, which has two molecules of glucose. Maltose increased callus induction 1.4-to 1.8-fold higher in japonica rice, 3.2-to 11.6-fold in tongil types and 2.7-fold in indica rice IR50. Callus induction was increased from 0.2％ to 12.5％ in maltose medium compared to the medium supplemented with sucrose plus glucose in indica rice "Tetep". A simple procedure of vacuum packaging of panicles during cold shock treatment prolonged not only anther viability more than 15 days but also increased callus induction more than 2-fold compared to open-air storage (conventional method). Combining of above two methods, callus induction was increased 28 to 56％ in japonica, 13 to 33％ in tonsil type and 12 to 31％ in indica rice. Plant regeneration was increased 14 to 35％ in japonica, 10 to 20％ in tonsil and 4 to 15％ in indica rice, respectively.

Plant architecture is an important factor that controls several characteristics, such as light interception, harvest index, and lodging. Plant architecture is controlled by a group of regulatory genes. It was previously found that ectopic expression of some MADS-box genes resulted in early flowering and a dwarf habit in Arabidopsis and tobacco (Nicotiana tabacum) plants. To explore ways to alter heading time and heading height, we expressed ectopically several OsMADS (Oryza sativa MADS-box) genes in rice plants. The OsMADS cDNA clones were connected to the maize ubiquitin promoter in the sense orientation, and the constructs were introduced into rice plants by the Agrobacterium-mediated transformation. Ectopic expression of the rice MADS-box genes hastened flowering to varying extents. RNA-blot analyses revealed that the phenotypes of early flowering and dwarfism were correlated with the transcript level of the transgene. In addition, weak expression of OsMADS1 using the nopaline synthase (nos) promoter, shortened the heading time by a few days and resulted in a mild reduction of heading height without any pleiotropic effects. The present study suggests that the rice MADS box genes could be used as sources of early-flowering and dwarfing traits in monocot species.

An anther-specific cDNA clone of rice, RA8, was isolated from an anther cDNA library by differential screening. RNA blot analysis indicated that the RA8 transcript is present specifically in anthers and the transcript level increased as flowers matured, reaching the highest level in mature flowers. The RA8 clone contains an open reading frame of 264 amino acid residues with a hydrophobic N-terminal region. The deduced amino acid sequences did not show significant homology to any known sequences. Genomic DNA blot analysis showed that RA8 is a single-copy gene. A genomic clone corresponding to the RA8 cDNA was isolated and its promoter region was fused to the β-glucuronidase (GUS) gene. Transgenic rice plants exhibited anther-specific expression of the GUS reporter gene. Histochemical GUS analysis showed that the RA8 promoter was active in the tapetum, endothecium, and connective tissues of anthers. Experiments showed that expression of the gene starts when microspores are released from tetrads, and it reaches to the maximum level at the late vacuolated-pollen stage. The RA8 promoter may be useful for controlling gene expression in anthers of cereal plants and for generating male-sterile plants.