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Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l–1 hygromycin in liquid MS medium that contained 10 mg l–1N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA.

An efficient system for clonal mass propagation in liquid culture was established for the propagation of ornamental gentian. In a test of the requirements for three cytokinins [6-benzylaminopurine, N-(2-chloro-4-pyridyl)-N′-phenylurea and N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ)] in combination with 1-naphthaleneacetic acid (NAA), we found that effective propagation of shoots occurred with 0.01 mg l–1 TDZ in a 300 ml conical flask that contained 100 ml of medium. The propagation of shoots was also affected by the concentrations of macronutrients (KNO3, NH4NO3 and CaCl2) and sucrose in Murashige and Skoog's (MS) medium, and it was influenced to some extent by the speed of agitation on an orbital shaker. The most efficient propagation of shoots was achieved in full-strength MS medium supplemented with 0.01 mg l–1 TDZ and 20 g l–1 sucrose with agitation at 150 rpm. The propagation of shoots was maximal after 6 weeks of culture (140 shoots from five nodal segments in one flask). Large-scale propagation in a 5-l fermenter was attempted using 3 l of MS medium that contained 0.01 mg l–1 TDZ and 20 g l–1 sucrose. More than 2,000 shoots were obtained in the fermenter in 5 weeks following the initial cultivation of five nodal segments for 6 weeks in one 300-ml flask. The shoots that had propagated in the fermenter were transferred directly to soil without prior rooting in vitro and were easily acclimatized within 1 month.

An antifungal protein fraction against Botrytis cinerea was purified from the leaves of Gentiana triflora. Polypeptides with molecular weight ca. 8.0 K (8.0 KP) and 7.0 K (7.0 KP) were detected by Tricine-SDS PAGE under reducing conditions. The deduced amino acid sequence of 8.0 KP cDNA showed amino acid identities with lipid transfer proteins from Apium graveolens (32.4%), Lycopersicon pennellii (31.5%), L. esculentum (31.5%), Nicotiana tabacum (30.7%), non-specific lipid transfer protein from L. esculentum (28.0%), and an unknown protein from Prosopis juliflora (30.7%) (GtLTP1). The deduced amino acid sequence of 7.0 KP showed amino acid identities with putative lipid transfer proteins from Arabidopsis thaliana (26.3% and 22.9%), Gossypium hirsutum (21.0%), Tamarix hispida (19.5%), Populus trichocarpa (15.9%) and an unknown protein from Vitis vinifera (17.2%) (GtLTP2). Both GtLTP1 and GtLTP2 were present as a multi-gene family in the genome and were strongly expressed in the roots and stems. Overexpression of GtLTP1 in tobacco plants improved tolerance against B. cinerea, demonstrating that GtLTP1 is a useful molecular tool for genetic engineering of disease-resistant plants.

Genetic relationships among 50 Gentiana accessions, comprising 36 wild species and 14 cultivars, were determined based on analysis of sequence data for the chloroplast trnL(UAA) intron, the rpl16 coding region and the rpl16-rpl14 intergenic spacer (IGS), together with nuclear DNA content as determined by flow cytometric analysis. The combined chloroplast DNA (cpDNA) data set was analyzed using both neighbor-joining (NJ) and maximum parsimony (MP) methods. The NJ and the strict consensus trees were generally congruent with previous phylogenetic and taxonomic studies, whereas G. cachemirica and G. yakushimensis were classified in different sectional affinities from their prevailing classifications. Three major cpDNA haplotypes (designated A, B and C), comprising 30 accessions in the sections Pneumonanthe, Cruciata, and Kudoa (ser. Monanthae), were each distinguished by two single-nucleotide polymorphisms in the rpl16-rpl14 IGS and the rpl16 coding region. Nuclear DNA content varied from 6.47 to 11.75 pg among the taxa possessing cpDNA haplotype A. These results provide genetic information that will assist in the development of future Gentiana breeding strategies.

Blue-flowered gentian (Gentiana triflora) is known to accumulate gentiodelphin, a unique polyacylated delphinidin-type anthocyanin, in the petals. Almost all of the structural genes involved in gentiodelphin biosynthesis have been isolated, but an important gene encoding UDP-glucose:anthocyanin 5-O-glucosyltransferase (5GT) remained to be identified. In this study, an attempt was made to isolate and characterize gentian 5GT, which is responsible for glucosylation of anthocyanidin 3-glucoside. A PCR-based cloning strategy identified seven 5GT candidates from gentian flowers. Among them, the deduced amino acid sequence of the 5GT gene from gentian petal cDNA, designated Gt5GT7, exhibited 36.0-41.7% identities with those of 5GTs from other plant species, and phylogenic analysis also suggested that Gt5GT7 belongs to the 5GT subfamily. The expression analysis showed that Gt5GT7 transcripts were detected predominantly in petals and weakly in filaments but not in leaves, stems, and other floral organs. In addition, increased levels of Gt5GT7 transcripts in petals coincided with flower development, a pattern identical to that of 5GT enzymatic activity as determined by in vitro assay using petal crude proteins. The substrate specificity of Gt5GT7 was analysed in vitro using the recombinant enzyme produced by Escherichia coli. Gt5GT7 could transfer a glucosyl moiety to anthocyanidin 3-glycosides but not to other flavonoid compounds. Delphinidin 3-glucoside, the precursor of gentiodelphin, was the best substrate among several anthocyanidin 3-glycosides tested. Heterologous expression of Gt5GT7 in tobacco plants led to additional accumulation of cyanidin 3-rutinoside-5-glucoside, confirming that Gt5GT7 has a valid enzymatic activity in planta.

Manipulation of horticultural plants' traits using genetic engineering has been a challenge because of gene redundancy and limited information concerning genome or other factors necessary for successful engineering. Recently we have developed a powerful tool with potential to overcome these difficulties, a novel gene silencing technology targeting transcription factor, which is designated Chimeric REpressor gene-Silencing Technology (CRES-T). Using this system, we are now analyzing biological functions of transcription factors in Arabidopsis and trying to manipulate morphological traits of various floricultural plants. To provide these information for genetic engineering of horticultural plants, we have developed the ‘FioreDB’ database in a web-based interface (http://www.cres-t.org/fiore/public_db/), which stores phenotypic information induced by various chimeric repressors in Arabidopsis and six floricultural plants, namely torenia, chrysanthemum, gentian, cyclamen, eustoma, morning glory. Users can find gene constructs that induce their preferred phenotype in Arabidopsis using simple searches, and can browse induced phenotypes in floricultural plants. Most phenotypic information has photo data. FioreDB is continually updated by addition of new data derived from the CRES-T analyses. FioreDB will help to improve traits of horticultural plants using the CRES-T system.

RNA interference (RNAi) is an efficient and powerful technique for gene silencing compared with antisense and sense suppression. Here we report adaptation of RNAi technology to modify flower colors in gentian, targeted for suppression of three anthocyanin biosynthetic genes; chalcone synthase (CHS), anthocyanidin synthase (ANS) and flavonoid 3′,5′-hydroxylase (F3′5′H). The petals of transgenic gentian plants with a suppressed CHS gene exhibited pure white to pale-blue color, while those with a suppressed ANS gene showed only pale-blue. The suppression of the F3′5′H gene decreased delphinidin derivatives and increased cyanidin derivatives, and led to magenta flower colors. Northern blot analyses confirmed that all transgenic gentian plants showing typical phenotypes had strongly suppressed transcriptions of the targeted genes, corresponding with a change in anthocyanin accumulation and composition in their petals. Some rolCpro-CHSir transgenic gentians exhibited bicolor phenotypes with reduced anthocyanin accumulation along the vascular bundles. These data demonstrated that the suppression of anthocyanin biosynthetic genes by RNAi was successfully applied to gentian plants to change flower color, and this could be useful for designing novel flower color and patterns. Transgenic gentian plants produced in this study might be utilized as elite materials in the breeding of gentian plants in the near future.

We attempted to evaluate the effects of promoters on flower color modification by RNA interference (RNAi). An inverted repeat of a tobacco chalcone synthase (CHS) fragment was used as a trigger under control of three different promoters and transformed into tobacco plants. The flowers of CaMV35S:CHSir displayed a completely white color, whereas those of Agrobacterium rhizogenes rolC:CHSir kept slight pigmentation. On the other hand, gentian GtCHS:CHSir showed decreased pigmentation in the tip only but not at the base of the petal limbs, which resulted in the appearance of bicolor-like flowers. The observed suppression of flower pigmentation corresponded with tissue-specificity of each promoter activity determined by histochemical GUS assay using transgenic tobacco plants with each promoter–GUS construct. These results demonstrate for the first time that RNAi-mediated silencing can be used to produce novel-colored flowers with high horticultural value.

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3'-hydroxylase [F3'H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully.

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding beta-carotene ketolase (4,4'-beta-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.

An antimicrobial protein was purified from leaves of Wasabia japonica L., and designated as WjAMP-2. WjAMP-2 strongly inhibited growth of four phytopathogenic fungi. The deduced amino acid sequence from WjAMP-2 cDNA showed a homology with pathogenesis-related protein 1 (PR-1) from several higher plants. Expression of WjAMP-2 was induced by inoculation with fungal pathogens and with treatments by salicylic acid, but not by methyl jasmonate. Overexpression of WjAMP-2 in tobacco plants conferred resistance against Botrytis cinerea. These results suggest that WjAMP-2 (PR-1) is an antifungal protein with a defensive role against phytopathogenic fungi in wasabi plants.

A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

Two different heterologous expression systems, microsomal fractions of Saccharomyces cerevisiae and transgenic tobacco plants, were used to investigate the enzymatic activities of flavonoid 3′-hydroxylase (GtF3′H) and flavone synthase II (GtFSII) homologues isolated from gentian petals. Recombinant GtF3′H expressed in yeast showed hydroxylation activities in the 3′ position with several flavonoid substrates, while recombinant GtFSII was able to produce flavone from flavanone. GtF3′ H-expressing transgenic tobacco plants showed a slight increase in anthocyanin content and flower color intensity, and conversion of the flavonol quercetin from kaempferol. On the other hand, GtFSII-expressing plants showed a remarkable reduction in anthocyanin content and flower color intensity, and additional accumulation of flavone, especially luteolin derivatives. We demonstrated that two cytochrome P450s from gentian petals have F3′H and FSII enzymatic activities both in vitro and in vivo, and might therefore be useful in modification of flower color using genetic engineering.

Transgenic plants over-expressing wasabi defensin gene were successfully produced in Phalaenopsis orchid by Agrobacterium-mediated transformation method. Embryogenic cell suspension culture of Phalaenopsis Wataboushi '#6.13' was infected with A. tumefaciens strain EHA101 carrying a plasmid containing wasabi defensin gene and selectable marker nptII, hpt genes. Plantlets were regenerated through somatic embryogenesis from the calli selected on hygromycin- containing medium. Transformation of plantlets with wasabi defensin gene was confirmed by PCR analysis. Southern blot analysis confirmed successful integration of 1-4 copies of the gene. Production of the 5 kDa wasabi defensin protein with varying levels was confirmed in the leaf extracts of different transgenic clones using Western blot analysis. Most of the transgenic plants showed strong resistance to Erwinia carotovora, which causes soft rot disease in the control plant. These results suggest the usefulness of this gene for conferring the resistance to various diseases of Phalaenopsis and possibly other orchids.

Leguminous plants have a unique pathway for production of 5-deoxy-type flavonoids and isoflavonoids that is distinct from the general flavonoid pathways. 5-Deoxy(iso)flavonoids are believed to play important eco-physiological roles as antimicrobial compounds and symbiotic signals toward rhizobia. The branching point of the 5-deoxyflavonoid pathway is the formation of the deoxy-type chalcone (isoliquiritigenin), which is catalyzed by the co-action of chalcone synthase and polyketide reductase (PKR). In the course of the comparative genomics of legume-specific genes, we cloned a putative cDNA for PKR (cPKR1) of a model legume Lotus japonicus (Regel) K. Larsen. Genomic Southern analysis showed that L. japonicus has a gene family composed of two to four paralogous PKR genes. The overexpression of cPKR1 in a red- flowered cultivar of petunia, "Polo Red Target", reduced anthocyanin accumulation and caused the formation of isoliquiritigenin and its putative derivatives. These results suggested that PKR1 encodes a PKR that functions in planta.

We successfully produced dwarf potted gentian using wild-type Agrobacterium rhizogenes A4 strain (ATCC43057) harboring an agropine type plasmid (pRiA4). A number of hairy roots were induced by direct inoculation with A. rhizogenes to the stem and leaf tissues in vitro. Adventitious shoots were regenerated from the hairy roots on Murashige and Skoog medium containing 10 mg l � 1 N-phenyl-N� -(1,2,3-thiadiazol-5-yl) urea, 0.1 or 1.0 mg l � 1 1-naphthaleneacetic acid, and 3 g l � 1 gellan gum. Regenerated plants were then cultured in vitro for at least six months and tested for the absence of viable A. rhizogenes within their tissues. After receiving authorization from the Ministry of Agriculture, Forestry and Fisheries, Japan, the plants were acclimated and grown in a greenhouse and/or field. Opine synthesis and rolC gene expression were analyzed to demonstrate successful introduction and expression of the transferred DNA. In total, 122 lines of variant types of dwarf plants with blue, white, and pink flowers were obtained, which might be useful for molecular breeding of a series of dwarf potted gentian cultivars.

To produce white-flowered gentian plants, we attempted to suppress the chalcone synthase (chs) gene by Agrobacterium-mediated transformation. A binary vector, pSMABcCHS, harboring antisense cDNA of chs isolated from the gentian Gentiana triflora cv. Maciry under control of the CaMV35S promoter was transformed into an interspecific hybrid gentian (cv. Albireo; G. scabraG. triflora). The vector also contained the bar gene as a selectable marker. Three out of seventeen transgenic plants showed completely white flowers with 10 to 25% reduced anthocyanin content compared with the wild-type. Molecular analyses confirmed integration of the foreign genes and suppression of chs mRNA accumulation in their petals. Application of commercial herbicide including bialaphos showed that the transformants were strongly resistant. Segregation of the white-flowered and herbicide-resistant traits was tested using a T1 progeny obtained from crossing with a blue-flowered parental line. The results clearly showed that these two traits are inherited dominantly as linked traits in the T1 progeny, suggesting that these transgenic plants are useful resources for production of white-flowered gentians. These results also demonstrated for the first time the inheritance of foreign genes and genetic modification of flower color in transgenic gentian plants.

Japanese cultivated gentian plants have naturally blue flowers, but some white-flowered cultivars are being bred through the utilization of spontaneous mutants. To determine the molecular basis of white coloration in gentian flowers, we compared two white-flowered cultivars Homoi and Polano White to a blue-flowered cultivar Maciry using biochemical and molecular approaches. High performance liquid chromatography (HPLC) analyses showed that flavone levels in cv. Polano White were only about one-half the amounts measured in the other two cultivars, while anthocyanins were absent in the two white-flowered cultivars compared to cv. Maciry in which high levels accumulated. Northern blot analysis of 10 flavonoid biosynthetic structural genes, previously reported to be temporally regulated in cv. Maciry [1] showed that cv. Homoi lacked transcripts for the anthocyanidin synthase (ANS) gene while cv. Polano White had decreased expressions for ANS as well as for chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3′,5′-hydroxylase (F3′,5′H), dihydroflavonol 4-reductase (DFR), UDP-glucose:flavonoid 3-glucosyltransferase (3GT) and anthocyanin 5-aromatic acyltransferase (5AT). Southern blot analysis confirmed the deficiency of one of two ANS loci in cv. Homoi. Transient expression of ANS in flower petals also strongly suggested that white flowers of cv. Homoi were derived from ANS mutation. Furthermore, analysis of stress-induced flower pigmentation suggested that rather than mutations in multiple structural genes being the cause, a defect in one or more regulatory factors controlling the later steps of flavonoid biosynthesis is responsible for white coloration in cv. Polano White.

Summary In this study, no transgenic gentian (Gentiana triflora × Gentiana scabra) plants produced via Agrobacterium-mediated transformation exhibited transgene (GtMADS, gentian-derived MADS-box genes or sGFP, green fluorescent protein) expression in their leaf tissues, despite the use of constitutive Cauliflower mosaic virus (CaMV) 35S promoter. Strikingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed. To investigate the possible cause of this drastic transgene silencing, methylation-specific sequences were analysed by bisulfite genomic sequencing using tobacco transformants as a control. Highly methylated cytosine residues of CpG and CpWpG (W contains A or T) sites were distinctively detected in the promoter and 5′ coding regions of the transgenes 35S-bar and 35S-GtMADS in all gentian lines analysed. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. The methylation frequencies in the other transgene, nopaline synthase (NOS) promoter-driven nptII, and the endogenous GtMADS gene coding region, were much lower and were variable compared with those in the 35S promoter regions. Transgene methylation was observed in the bialaphos-selected transgenic calluses expressing the transgenes, and methylation sequences were distributed preferentially around the as-1 element in the 35S promoter. Calluses derived from leaf tissues of silenced transgenic gentian also exhibited transgene suppression, but expression was recovered by treatment with the methylation inhibitor 5-aza-2′-deoxycytidine (aza-dC). These results indicated that cytosine methylation occurs exclusively in the 35S promoter regions of the expressed transgenes during selection of gentian transformants, causing transcriptional gene silencing.

Arabidopsis (Arabidopsis thaliana) ethylene-responsive element binding protein (AtEBP) gene was isolated as a suppressor of Bax-induced cell death by functional screening in yeast (Saccharomyces cerevisiae). To further examine the cell death suppressive action of AtEBP in plant cells, we established transgenic tobacco (Nicotiana tabacum) plants overexpressing AtEBP as well as transgenic tobacco plants ectopically expressing mouse Bax protein under a dexamethasone-inducible promoter. We prepared the crosses of the selective lines of each transgenic plant, which were evaluated in terms of cell death suppression activity. Results indicate that AtEBP suppressed Bax-induced cell death in tobacco plants, an action also associated with a lowered level of ion leakage. Furthermore, tobacco Bright Yellow-2 cells overexpressing AtEBP conferred resistance to hydrogen peroxide (H2O2) and heat treatments. AtEBP protein localized in the nucleus and functioned as an in vivo transcription activator as confirmed in transient assays and experiments using stable transgenic system. Up-regulation of defense genes was observed in transgenic Arabidopsis plants overexpressing AtEBP. Based on the analysis of mRNA accumulation in ethylene-related mutants, the position of AtEBP in signaling pathway is presented.

The cDNA clones encoding homologues of flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), flavonoid 3′-hydroxylase (F3′H) and flavone synthase II (FSII) genes were isolated from petals of Gentiana triflora. Deduced amino acid sequences exhibited 60–80% identities with the corresponding sequences from other dicotyledonous species. Southern blot analysis showed that they were present as multiple copies in the gentian genome, and Northern blot analysis showed that the flavonoid biosynthesis-related genes could be classified into three groups by their temporal expression patterns during gentian flower development. The first included chalcone synthase (CHS) and chalcone isomerase (CHI) expressing during all flower development stages; the second included F3′H and FSII expressing at flower early developmental stages; and the third included F3H, flavonoid 3′,5′-hydroxylase (F3′,5′H), dihydroflavonol 4-reductase (DFR), ANS, UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT) and anthocyanin 5-aromatic acyltransferase (5AT) expressing at flower late developmental stages. In general, low or undetectable levels of expression were observed in both the leaves and stems. High performance liquid chromatography (HPLC) analysis revealed that flavone accumulates from the early flower bud stage, but anthocyanin accumulation peaked at the later flower anthesis stage. A significant correlation between gene expression and pigment accumulation has been found, indicating that flavonoid biosynthesis during gentian flower development is regulated by temporal expression of these genes.

Little is known concerning the mechanisms by which auxin and cytokinin exert their effects on proliferation and differentiation. Cyclin-dependent kinases (CDKs) are major regulators of the eukaryotic cell cycle, thus they are assumed to control cell differentiation as well as proliferation in response to phytohormone signals. Here, we overexpressed rice R2 cDNA, which encodes a CDK-activating kinase, in tobacco leaf explants by using the glucocorticoid-mediated transcriptional induction system. Transient expression of R2 during the first 7 days of culture triggered callus formation in the absence of cytokinin. This phenotype was enhanced by higher expression of R2 or coexpression of cyclin H, and suppressed by treatment with roscovitine, a CDK inhibitor. R2 expression at a later stage did not prevent cells from differentiation into roots, suggesting a restricted period for sensing CDK activities that control differentiation fate of cells during organogenesis.

An antimicrobial protein, designated WjAMP-1, was purified from leaves of Wasabia japonica L. WjAMP-1 showed antimicrobial activity against both fungi and bacteria. The deduced amino acid sequence of cDNA of WjAMP-1 showed 60% and 70% identity with a hevein from Hevea brasiliansis and a hevein-like protein from Arabidopsis thaliana, respectively. However, matured WjAMP-1 lacked the hevein domain and may correspond to the C-terminal domain of hevein. Southern blot analysis showed that one or two copies of the WjAMP-1 gene were presented in the genome of wasabi. Expression of WjAMP-1 was detected in all organs tested, and was especially strong in petioles. Expression of WjAMP-1 was induced by the inoculation with fungal pathogens and treatment with methyl jasmonate. Recombinant WjAMP-1 expressed in Nicotiana benthamiana using potato virus X vector also inhibited not only growth of fungi but also bacteria. These results suggest that WjAMP-1 may be the C-terminal domain of hevein and one of the defense gene in W. japonica. WjAMP-1 gene may be useful genes to generate resistant plants against fungal and bacterial pathogens.

Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.

All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a ζ-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and ζ-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.

For analysis of the promoter of a gene for chalcone synthase ( GTCHS1 ) from Gentiana triflora , a cDNA was cloned for chalcone synthase (CHS) from a cDNA library of petals of Gentiana . Using the sequence of GTCHS1 , the promoter region of GTCHS1 was cloned by the inverse polymerase chain reaction. The promoter was fused to a gene for β -glucuronidase (GUS) and the construct was introduced into Petunia hybrida . Measurements of the GUS activities of transformants indicated that the GTCHS1 promoter strongly directed the expression of GUS in flower limbs, while the 35S promoter of cauliflower mosaic virus (CaMV) directed expression of the reporter gene in all tissues. Histochemical staining of GUS activity revealed that the GTCHS1 promoter strongly directed the expression of GUS in the inner epidermis, at sites where most of the anthocyanin accumulated. The sequence of the GTCHS1 promoter included a consensus sequence of the MYB protein-binding site, five consensus sequences of the MYC protein-binding site, one core sequence of a G-box and three P-box-like sequences.

Three novel anthocyanins, delphinidin 3,3′- di -O-β- d -glucoside -5-O-(6-O- caffeoyl -β- d -glucoside ) , delphinidin 3,3′- di -O-β- d -glucoside -5-O-(6-O-trans-p- coumaroyl -β- d -glucoside ) and delphinidin 3-O-β- d -glucoside -5-O-(6-O-trans-p- coumaroyl -β- d -glucoside ) , named albireodelphin A, B and C, respectively, were isolated from the blue flowers of Gentiana cv. Albireo, along with two known anthocyanins, gentiodelphin and gentiocyanin A. The complete structure of each anthocyanin was unambiguously determined by one- and two-dimensional NMR and other spectral methods. Two other novel anthocyanins, delphinidin 3-O-β- d -glucoside -5-O-(6-O- caffeoyl -β- d - glucoside )-3′-O-(6-O-trans-p- coumaroyl -β- d -glucoside ) and dephinidin 3-O-β- d -glucoside -5-O-(6-O-trans-p- coumaroyl -β- d -glucoside )-3′-O-(6-O- caffeoyl -β- d -glucoside ) , named albireodelphin D and E, respectively, were obtained as a mixture and the structures estimated by one- and two-dimensional NMR and other spectral methods.

Agrobacterium rhizogenes A4 harboring the agropine type root-inducing plasmid (pRiA4) induced hairy roots on stem sections of gentian plants. Transgenic gential plants were regenerated from the hairy roots at a frequency of 19% adventitious shoot regeneration on Murashige and Skoog medium containing 30 g l-1 sucrose, 10 mg l-1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg l-1 1-naphthaleneacetic acid and 2 g l-1 gellan gum. The shoots were grown to maturity in a growth chamber after acclimatization. The mature transformed plants showed distinct variations in their phenotypes, such as dwarfness. Polymerase chain reaction and Southern blot analysis revealed stable integration of transferred DNA.

Three novel anthocyanins, cyanidin 3-O-β- d -glucoside -5-O-(6-O- caffeoyl -β- d -glucoside ) , cyanidin 3-O-β- d -glucoside -5,3′- bis -O-(6-O- caffeoyl -β- d -glucoside ) and cyanidin 3-O-β- d -glucoside -5-O-(6-O-trans-p- coumaroyl -β- d -glucoside ) , gentiocyanin A, B and C, respectively, were isolated from pink flowers of gentian, along with a known anthocyanin, cyanidin 3-O-β- d -glucoside . The complete structure of each anthocyanin was unambiguously determined by 1D- and 2D-NMR and other spectral methods.

A protoplast-to-plant system was developed in Gentiana using a gellan gum-embedding culture. Viable protoplasts could be routinely isolated from in vitro-grown plantlets, and they were embedded in 0.2% gellan gum-solidified B5 medium containing 2 mg l-1 NAA, 0.1 mg l-1 TDZ, 0.1 M sucrose and 0.4 M mannitol. Weekly addition of fresh liquid medium was essential for preventing cell browning. Colony growth was promoted by lowering mannitol concentration of the culture media after one month, and visible colonies were produced after 2 months of culture. Shoot regeneration from protoplast-derived calluses was stimulated by 1 to 10 mg l-1 TDZ in combination with 0.1 mg l-1 NAA. Protoplast-derived plants were recovered following rooting of the shoots in plant growth regulator-free medium and they were successfully transferred to soil.

Although there have been lots studies about vehicular maneuvers on land or sea, maneuvers within a small area that require direction changes have rarely been discussed. Many reports on the arrival or departure of ships from ports have revealed more about what are safe and effective turning maneuvers within a narrow area. Convenient navigation systems for ship maneuvering should allow quick avoidance of obstacle to find the shortest distance in the whole seaway is important. This study proposes a review model that will satisfy optimal turning maneuvering anywhere and be applicable to any type of ship. However, the water-based environment is more complex and there are many factors that will affect the formula. By using a nonlinear unified state-space model to discuss another model we can divide and conquer the problem. In recent studies some categories have been evaluated to determine where the main attention should be directed. In this review study we look at how to construct the optimal turning maneuver within a limited sea area.

Although there have been a variety of studies about vehicular maneuvers on land or sea, maneuvers within a small area that require direction changes have rarely been discussed. The arrival or departure of ships to or from ports requires safe and effective turning maneuvers within a narrow area. In this study, we decompose the maneuver procedure into several operations, then establish an operational model, simplifying the behaviors of rudders and thrusters that operate the turning maneuver, ignoring the environmental factors. This proposed model is a unified model that can be applied to all types of ships, regardless of individual characteristics. Each operation is defined as an isolated procedure that has its own effect on the maneuver behavior and the whole maneuver procedure can be treated as a combination of these operations. A case study using this model to construct the optimal turning maneuver within a limited sea area is also provided.

Ornamental gentian plants have vivid-blue flowers. The main factor contributing to the flower colour is the accumulation of a polyacylated delphinidin 'gentiodelphin' in their petals. Although in vitro studies proposed that acylation plays an important role in the stability and development of gentian blue colour, the in vivo stability of the polyacylated anthocyanin was not clearly demonstrated. Thus, to reveal the importance of anthocyanin modification, especially acylation, and to engineer new colours of gentian flowers, we used chimeric RNAi technology to produce transgenic gentian plants with downregulated anthocyanin 5,3'-aromatic acyltransferase (5/3'AT) and flavonoid 3',5'-hydroxylase (F3'5'H) activities, which are both essential enzymes for gentiodelphin biosynthesis. Two lines of flower colour-modified plants were obtained from fifteen transgenic gentian plants. Clone no. 1 exhibited a lilac flower colour and clone no. 15 exhibited pale-blue flowers. RNA gel blot analysis confirmed that both transgenic lines had markedly suppressed 5/3'AT transcripts, whereas clone no. 15 had fewer F3'5'H transcripts than clone no. 1 and untransformed control plants. HPLC analysis of anthocyanin compositions showed that downregulation of the 5/3'AT gene led to increased accumulation of non-acylated anthocyanins, as expected. These results demonstrated that genetic engineering to reduce the accumulation of polyacylated anthocyanins could cause modulations of flower colour.

Gentian plants have vivid blue-colored flowers, caused by accumulation of a polyacylated anthocyanin 'gentiodelphin'. We previously performed expression analysis of gentiodelphin biosynthetic genes, and hypothesized that the white-flowered gentian cultivar 'Polarno White' might have resulted from the mutation of certain regulatory factors responsible for anthocyanin biosynthesis in flower petals. In this study, we isolated 26 R2R3-MYB gene fragments including four full-length cDNAs (GtMYB2a, GtMYB2b, GtMYB3 and GtMYB4) and one basic helix-loop-helix (bHLH) gene (GtbHLH1) from blue-flowered gentian by degenerate PCR and rapid amplification of cDNA ends (RACE). Phylogenetic tree analysis showed that GtMYB3 was categorized into a clade involved in anthocyanin biosynthesis including petunia AN2 and Arabidopsis PAP1. On the other hand, GtbHLH1 exhibited high identity with petunia AN1 based on both phylogenetic and genomic structural analyses. Temporal profiles of GtMYB3 and GtbHLH1 transcript levels corresponded well with those of gentiodelphin accumulation and their biosynthetic genes in petals. Yeast two-hybrid analysis showed that GtbHLH1 interacted with GtMYB3. Moreover, transient expression analysis indicated that the co-expression of GtMYB3 and GtbHLH1 could enhance the promoter activities of late anthocyanin biosynthetic genes in tobacco BY2 cells. We also revealed that in cv. 'Polarno White' the GtMYB3 genes were mutated by insertions of transposable elements or uncharacterized sequences, indicating that the white coloration was caused by GtMYB3 mutation. These results strongly suggested that GtMYB3 and GtbHLH1 are involved in the regulation of gentiodelphin biosynthesis in gentian flowers.

Morphogenetic alterations of rice spikelets grown under the influence of applied chemical hybridizing agent HGR-626 was studied using SEM and stereo microscope. Either a 10 ppm or 50 ppm solution of HGR 626 was sprayed at the spikelet primordium differentiation stage (Stage I), and in the middle of the meiosis stage (Stage II), respectively. Spikelets treated at Stage I showed such changes as: (1) spikelet and its component organs became smaller than control, (2) anthers of which bended toward axis, (3) no pollen mother cell differentiated in them, or (4) microspores with anther tissues ceased to develop, and, (5) exine of microspores developed irregularly to interconnect each other. Almost no content accumulated in them. On the contrary, 56.4% of pistils investigated in this study proliferated their ovaries and/or stigmata. Change of sex initiation namely pistil hyperplasia and stamen hypoplasia in rice spikelet as shown in this study indicated a similar tendency with the pistil hyperplasia and stamen hypoplasia induced by stressful environments studied previously. Skikelets treated at stage II showed no morphogenetic change as spikelets treated at stage I. This difference was considered to have occurred from the difference in developmental stage of apical meristem in spikelet primordia when the chemical was applied.

To establish a simple and an efficient system to minimize the environmental risk of genetically modified plants, we tested the applicability of the barnase/barstar system in conferring bisexual sterility; that is, in preventing plants setting seeds by self-fertilization and out-crossing. Transgenic tobacco plants were generated to express barnase, a cell death inducing ribonuclease, under the control of the gamete-specific AtDMC1 promoter, and barstar, a specific inhibitor of barnase, under the control of the ACT2 promoter, which is constitutively active in almost all tissues except gametes. In contrast to control plants harboring the barstar expression unit only, which set seeds normally with self-pollination, all transformants harboring both barnase and barstar were bisexually sterile. They produced aberrant anthers containing no detectable pollen and failed to set seeds even after pollination with wild-type tobacco pollen.

In this study, no transgenic gentian (Gentiana triflora x Gentiana scabra) plants produced via Agrobacterium-mediated transformation exhibited transgene (GtMADS, gentian-derived MADS-box genes or sGFP, green fluorescent protein) expression in their leaf tissues, despite the use of constitutive Cauliflower mosaic virus (CaMV) 35S promoter. Strikingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed. To investigate the possible cause of this drastic transgene silencing, methylation-specific sequences were analysed by bisulfite genomic sequencing using tobacco transformants as a control. Highly methylated cytosine residues of CpG and CpWpG (W contains A or T) sites were distinctively detected in the promoter and 5' coding regions of the transgenes 35S-bar and 35S-GtMADS in all gentian lines analysed. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. The methylation frequencies in the other transgene, nopaline synthase (NOS) promoter-driven nptII, and the endogenous GtMADS gene coding region, were much lower and were variable compared with those in the 35S promoter regions. Transgene methylation was observed in the bialaphos-selected transgenic calluses expressing the transgenes, and methylation sequences were distributed preferentially around the as-1 element in the 35S promoter. Calluses derived from leaf tissues of silenced transgenic gentian also exhibited transgene suppression, but expression was recovered by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (aza-dC). These results indicated that cytosine methylation occurs exclusively in the 35S promoter regions of the expressed transgenes during selection of gentian transformants, causing transcriptional gene silencing.

Pink-flowered gentian plants (Gentiana scabra) have been bred from spontaneous mutations of blue-flowered gentian plants, but the formation mechanism(s) is unknown so far. To investigate the process, two independent pink-flowered gentian plant lines were analyzed by a molecular biological approach. HPLC analysis showed that petals of the blue-flowered cultivar contained a small amount of cyanidin derivatives and major delphinidin derivatives, whereas pink petals had only a small amount of cyanidin derivatives. To find the causal factor(s) of this change, we focused on flavonoid 3',5'-hydroxylase (F3',5'H), which is a key enzyme for delphinidin biosynthesis in the flavonoid biosynthetic pathway. Molecular analyses confirmed that the loss of delphinidin synthesis could be attributed to the insertions of different transposable elements in the F3',5'H gene in each independent pink-flowered gentian plant. Sequence analysis showed that these transposable elements were classified into an hAT superfamily and terminal-repeat retrotransposon in miniature (TRIM), by which normal F3',5'H transcripts were interrupted. Southern blot analysis indicated that they belong to high copy number elements and are also found in a related gentian species (G. triflora). These results suggest that the transposable elements inserted in F3',5'H are the source of the mutations and may also play a substantial role in the genomic evolution of the genus Gentiana.

A cDNA encoding chalcone isomerase (CHI) was isolated from the petals of Nicotiana tabacum and the effect of its suppression on flavonoid biosynthesis was analyzed in transgenic tobacco plants. CHI-suppression by RNA interference (RNAi) showed reduced pigmentation and change of flavonoid components in flower petals. The plants also accumulated high levels of chalcone in pollen, showing a yellow coloration. Our results first demonstrated that suppression of CHI by genetic transformation is possible in higher plants. This suggests that CHI plays a major part in the cyclization reaction from chalcone to flavanone, and that spontaneous reactions are few, if any, in tobacco plants.