Ryutaro Aida

National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

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Publications (44)93.76 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The class B genes DEFICIENS (DEF)/APETALA3 (AP3) and GLOBOSA (GLO)/PISTILLATA (PI), encoding MADS-box transcription factors, and their functions in petal and stamen development have been intensely studied in Arabidopsis and Antirrhinum. However, the functions of class B genes in other plants, including ornamental species exhibiting floral morphology different from these model plants, have not received nearly as much attention. Here, we examine the cooperative functions of TfDEF and TfGLO on floral organ development in the ornamental plant torenia (Torenia fournieri Lind.). Torenia plants co-overexpressing TfDEF and TfGLO showed a morphological alteration of sepals to petaloid organs. Phenotypically, these petaloid sepals were nearly identical to petals but had no stamens or yellow patches like those of wild-type petals. Furthermore, the inflorescence architecture in the co-overexpressing torenias showed a characteristic change in which, unlike the wild-types, their flowers developed without peduncles. Evaluation of the petaloid sepals showed that these attained a petal-like nature in terms of floral organ phenotype, cell shape, pigment composition, and the expression patterns of anthocyanin biosynthesis-related genes. In contrast, torenias in which TfDEF and TfGLO were co-suppressed exhibited sepaloid petals in the second whorl. The sepaloid petals also attained a sepal-like nature, in the same way as the petaloid sepals. The results clearly demonstrate that TfDEF and TfGLO play important cooperative roles in petal development in torenia. Furthermore, the unique transgenic phenotypes produced create a valuable new way through which characteristics of petal development and inflorescence architecture can be investigated in torenia.
    Plant Molecular Biology 08/2014; 86(3). DOI:10.1007/s11103-014-0231-8 · 4.07 Impact Factor
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    ABSTRACT: Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet-, or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3',5'-hydroxylase gene (F3'5'H), which encodes the key enzyme for delphinidin biosynthesis. In F3'5'H-transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3'5'H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3'5'H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5'-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from red-purple to purple/violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.
    Plant and Cell Physiology 08/2013; 54(10). DOI:10.1093/pcp/pct111 · 4.98 Impact Factor
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    ABSTRACT: Chrysanthemum is globally the second most important ornamental in terms of socioeconomic importance. Even though the vast range of flower colors, shapes and forms were initially created using conventional and mutation breeding, transgenic strategies are now more frequently used with Agrobacterium-mediated transformation being the most popular form of introducing foreign genes into chrysanthemums. Even so, transformation efficiency remains dependent on cultivar and regeneration procedure. Transgenic molecular breeding has seen the introduction of important traits such as novel flower color and form and plant architecture, prolonged cut-flower vase-life, resistance to biotic stresses such as viruses/viroids, pathogens and insects. However, chimerism and transgene silencing continue to be limiting factors. Transgenic strategies, despite opening up new avenues for creating new cultivars with improved agronomic and horticultural traits, may be limited due to the risk of transgenic pollen escaping into the wild.
    Critical Reviews in Plant Sciences 01/2013; 32(1):21-52. DOI:10.1080/07352689.2012.696461 · 5.29 Impact Factor
  • Tomoya Niki · Ryutaro Aida · Tomoko Niki · Takaaki Nishijima
    Journal- Japanese Society for Horticultural Science 01/2013; 82(4):328-336. DOI:10.2503/jjshs1.82.328 · 0.82 Impact Factor
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    ABSTRACT: We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY.
    The Plant Journal 05/2012; 71(6):1002-14. DOI:10.1111/j.1365-313X.2012.05047.x · 6.82 Impact Factor
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    ABSTRACT: It has long been proposed that white-flowered chrysanthemums (Chrysanthemum morifolium Ramat.) have a single dominant gene that inhibits carotenoid formation or accumulation in ray petals. However, the precise function of the proposed gene was unknown. We previously isolated a gene encoding carotenoid cleavage dioxygenase 4, designated CmCCD4a, which is specifically expressed in the ray petals of white-flowered chrysanthemums. Because CmCCD4a was a strong candidate for the single dominant gene, we analyzed the relationship between CmCCD4a expression and carotenoid content in two sets of petal color mutants. Here, we show that CmCCD4a represents a small gene family containing at least four members. Two of them, CmCCD4a-1 and CmCCD4a-2, were highly expressed in ray petals of two taxa with low carotenoid levels. In petal color mutants derived from these taxa, increases in carotenoid levels accompanied decreases in CmCCD4a expression levels in ray petals. Two different circumstances reduced the levels of CmCCD4a expression in the mutants: either a CmCCD4a gene was lost from the genome or the expression of a CmCCD4a gene was suppressed. In the latter case, suppression may be caused by the loss of a function that normally enhances CmCCD4a transcription. A stepwise decrease in the amount of CmCCD4a expression in either L1 or L2 resulted in a corresponding stepwise increase in the carotenoid content in ray petals. From these results, we propose that CmCCD4a expression is the key factor that controls the carotenoid content in ray petals of chrysanthemum.
    Euphytica 04/2012; 184(3). DOI:10.1007/s10681-011-0602-z · 1.69 Impact Factor
  • Ryutaro Aida
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    ABSTRACT: This chapter describes an Agrobacterium tumefaciens-mediated transformation protocol for torenia, a plant that has several useful characteristics and is primarily used for ornamental and experimental purposes. Leaf segments of torenia were co-cultured with A. tumefaciens containing a vector plasmid for 7 days at 22°C under dark conditions on Murashige and Skoog (MS) medium containing 1 mg/L benzyladenine, 1 mg/L indoleacetic acid, and 100 μM acetosyringone. Subsequent culturing at 25°C under a 16-h photoperiod with fluorescent light on MS medium containing 1 mg/L benzyladenine, 300 mg/L carbenicillin, and selection agent (300 mg/L kanamycin or 20 mg/L hygromycin) allowed for transformant selection. Transgenic shoots were obtained from green compact calli after 2-3 months of culture in the selection medium. This method can achieve a transformation rate of approximately 5% (transformants/explant).
    Methods in molecular biology (Clifton, N.J.) 01/2012; 847:267-74. DOI:10.1007/978-1-61779-558-9_23 · 1.29 Impact Factor
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    ABSTRACT: While heavy-ion beam irradiation is becoming popular technology for mutation breeding in Japan, the combination with genetic manipulation makes it more convenient to create greater variation in plant phenotypes. We have succeeded in producing over 200 varieties of transgenic torenia (Torenia fournieri Lind.) from over 2,400 regenerated plants by this procedure in only 2 years. Mutant phenotypes were observed mainly in flowers and showed wide variation in colour and shape. Higher mutation rates in the transgenics compared to those in wild type indicate the synergistic effect of genetic manipulation and heavy-ion beam irradiation, which might be advantageous to create greater variation in floral traits.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 847:275-89. DOI:10.1007/978-1-61779-558-9_24 · 1.29 Impact Factor
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    ABSTRACT: We give an overview of the advances of an Agrobacterium-mediated transformation system, clarifying its problems and their solutions, and then show the latest version of our transformation system and examples of the introduction of agronomically important traits into chrysanthemums. Typical problems with the Agrobacterium-mediated transformation in chrysanthemum include low transformation efficiency, high chimerism and cultivar specificity. Using a co-cultivation medium containing acetosyringone and casamino acids for high transformation efficiency and an antibiotic-selection step for transgenic calli before plant regeneration to eliminate the chimerism, we established an efficient and stable transformation system for chrysanthemum. In addition, this system was used to successfully introduce useful agronomical traits, such as insect resistance and new flower color, into chrysanthemums. These traits have been stably and highly expressed to confer the expected characteristics upon the transgenic chrysanthemums. Before applying a field trial of the genetically modified (GM) chrysanthemums, male and female sterility were introduced into the transformants to exclude the transgene flow from the GM plants to their wild relatives. So far, using RNAi technology, some of the transgenic chrysanthemums have displayed complete male sterility with very weak female fertility.
    Plant Biotechnology 01/2012; 29(4):323-337. DOI:10.5511/plantbiotechnology.12.0521a · 1.06 Impact Factor
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    ABSTRACT: Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most popular ornamental flowers in the world, and many agronomic traits have recently been introduced to chrysanthemum cultivars by gene transformation. Concerns have been raised, however, regarding transgene flow from transgenic plants to wild plants. In early studies, ethylene receptor genes have been used for genetic modification in plants, such as flower longevity and fruit ripening. Recently, overexpression of ethylene receptor genes from melon (CmETR1/H69A) caused delayed tapetum degradation of the anther sac and a reduction in pollen grains. We therefore introduced the ethylene receptor gene into chrysanthemums to induce male sterility and prevent transgene flow via pollen. The chrysanthemum cultivar Yamate shiro was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA105, carrying the binary vector pBIK102H69A, which contains the CmETR1/H69A gene. A total of 335 shoots were regenerated from 1,282 leaf discs on regeneration medium (26.1%). The presence of the Cm-ETR1/H69A gene was confirmed in all of the regenerated plantlets by Southern blot analysis. These genetically modified (GM) plants and their non-GM counterparts were grown in a closed greenhouse and flowered at temperatures between 10 and 35°C. In 15 of the 335 GM chrysanthemum lines, the number of mature pollen grains was significantly reduced, particularly in three of the lines (Nos. 91, 191 and 324). In these three lines, pollen grains were not observed at temperatures between 20 and 35°C but were observed at 10 and 15°C, and mature pollen grains were formed only at 15°C. In northern blot analyses, expression of the CmETR1/H69A gene was suppressed at low temperatures. This phenomenon was observed as a result of both the suppression of CmETR1/H69A expression at low temperatures and the optimal growth temperature of chrysanthemums (15–20°C). Furthermore, the female fertility of these three GM lines was significantly lower than that of the non-GM plants. Thus, the mutated ethylene receptor is able to reduce both male and female fertility significantly in transgenic chrysanthemums, although the stability of male and/or female sterility at varying growth temperatures is a matter of concern for its practical use.
    Molecular Breeding 02/2011; 29(2). DOI:10.1007/s11032-010-9546-6 · 2.28 Impact Factor
  • 01/2011; 80(1):113-120. DOI:10.2503/jjshs1.80.113
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    ABSTRACT: While torenia (Torenia fournieri Lind.) is a useful model flower for molecular biological studies of floral architecture, the maintenance of plant materials and resulting transgenic plants requires vegetative propagation due to its heterozygous nature. Reduction of labor and costs for maintaining thousands of in vitro torenia cultures is therefore a critical issue. We found that substituting trehalose for sucrose drastically extended the culture period to 70 days, which is more than twice as long as for the common, sucrose-based medium, without reduction in plant viability. Comparative measurement of the plant mass indicated that the increased survival benefit of the trehalose-based medium might be on account of improvement in the rhizosphere environment through reduction of root density in the culture, rather than by reduced plant growth. No harmful effects arising from the trehalose-based medium were observed in 1,800 laboratory lines during the bimonthly subculture for over 12 months, except for a wilting on the first transfer to the trehalose-based medium. In conjunction with the use of the commercial food additive, Okome-ni-TREHA (R) rather than reagent-grade trehalose, we have succeeded in reducing the costs and labor associated with the culture medium to less than one third of those for the sucrose-based system.
    Plant Biotechnology 01/2011; 28(2):263-266. DOI:10.5511/plantbiotechnology.11.0124c · 1.06 Impact Factor
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    ABSTRACT: Molecular breeding with genetic modification enables the production of novel floral traits in floricultural plants that could not be obtained by traditional breeding. To facilitate novel flower production, we collectively introduced 2 sets of 42 and 50 chimeric repressors of Arabidopsis transcription factors into Agrobacterium and then used these to co-transform torenia (Torenia fournieri). We generated 750 transgenic torenias, and identification of the transgenes revealed that more than 80% of the transgenic torenias had a single transgene. A total of 264 plants showed phenotypic modification, and 91.2% displayed modified flower colors and/or shapes, such as altered color patterns, curled petal margins, and wavy petals. These results indicated that the collective transformation system can be applied to molecular breeding of flowers. Detailed analysis of the phenotypes revealed that PETAL LOSS could control blotch sizes and that modification of cell shape could change the texture of petals. We found that the chimeric repressors of functionally unknown transcription factors also induced novel floral traits, and therefore, the transgenic torenias provide an understanding of the functions of transcription factors that could not be revealed by previous studies in Arabidopsis.
    Plant Biotechnology 01/2011; 28(2):189-199. DOI:10.5511/plantbiotechnology.10.1216a · 1.06 Impact Factor
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    ABSTRACT: Chimeric REpressor gene-Silencing Technology (CRES-T) is a powerful gene-silencing tool to analyze the function of Arabidopsis transcription factors. To investigate whether CRES-T is also applicable to horticultural plants inadequate for genetic engineering because of their limited molecular biological characterization and polyploidy, we applied CRES-T to torenia and the hexaploid chrysanthemum and produced their transgenic plants expressing the chimeric repressor derived from the Arabidopsis TEOSINTE BRANCHED1, CYCLOIDEA, and PCF family transcription factor 3 (TCP3) fused with a plant-specific transcriptional repression domain named SRDX, consisting of 12 amino acids originated from the EAR-motif (TCP3-SRDX). Transgenic torenia and chrysanthemum expressing TCP3-SRDX exhibited fringed leaves and short pistils, while those expressing TCP3 fused with either the mutated repression domain (TCP3-mSRDX) or the overexpressor of TCP3 (TCP3-ox) did not exhibit phenotypic changes. In addition to fringed leaves, TCP3-SRDX transgenic torenia plants exhibited petals with fringed margins, distinctive color patterns, and reduced anthocyanin accumulation. In TCP3-SRDX transgenic chrysanthemum plants, floral organ development was suppressed as compared with the wild type. These results indicate that the Arabidopsis-derived TCP3-SRDX induced morphological changes in transgenic torenia and chrysanthemum although the observed phenotypes partially differ from each other. CRES-T may function in various plant species including polyploid species and modify their biological characteristics.
    Plant Biotechnology 01/2011; 28(2):131-140. DOI:10.5511/plantbiotechnology.11.0121a · 1.06 Impact Factor
  • Koji Tanase · Ryutaro Aida · Akio Ohyama · Takashi Onozaki
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    ABSTRACT: Anther-specific promoters have been extensively studied in terms of the genetic engineering of male-sterile plants. Here, we fused the 5' upstream promoter regions of a sucrose synthase gene from tomato (TOMSSF) to the beta-glucuronidase (GUS) gene and used the construct to transform Chrysanthemum plants. Histochemical analysis of two transformants showed high GUS activity in ray florets and tubular florets. Analysis of the staining pattern in these florets reveled that staining was stamen-specific. GUS gene expression was highest in the stamen and remained at a steady-state level throughout the experiment. These results suggest that the TOMSSF promoter has high activity in the stamen of tubular florets and would be useful as a high stamen-specific promoter in Chrysanthemum.
    Plant Biotechnology 01/2011; 28(5):497-501. DOI:10.5511/plantbiotechnology.11.0823a · 1.06 Impact Factor
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    ABSTRACT: Homeotic class B genes GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) are involved in the development of petals and stamens in Arabidopsis. However, functions of these genes in the development of floral organs in torenia are less well known. Here, we demonstrate the unique floral phenotypes of transgenic torenia formed due to the modification of class B genes, TfGLO and TfDEF. TfGLO-overexpressing plants showed purple-stained sepals that accumulated anthocyanins in a manner similar to that of petals. TfGLO-suppressed plants showed serrated petals and TfDEF-suppressed plants showed partially decolorized petals. In TfGLO-overexpressing plants, cell shapes on the surfaces of sepals were altered to petal-like cell shapes. Furthermore, TfGLO- and TfDEF-suppressed plants partially had sepal-like cells on the surfaces of their petals. We isolated putative class B gene-regulated genes and examined their expression in transgenic plants. Three xyloglucan endo-1,4-beta-d-glucanase genes were up-regulated in TfGLO- and TfDEF-overexpressing plants and down-regulated in TfGLO- and TfDEF-suppressed plants. In addition, 10 anthocyanin biosynthesis-related genes, including anthocyanin synthase and chalcone isomerase, were up-regulated in TfGLO-overexpressing plants and down-regulated in TfGLO-suppressed plants. The expression patterns of these 10 genes in TfDEF transgenic plants were diverse and classified into several groups. HPLC analysis indicated that sepals of TfGLO-overexpressing plants accumulate the same type of anthocyanins and flavones as wild-type plants. The difference in phenotypes and expression patterns of the 10 anthocyanin biosynthesis-related genes between TfGLO and TfDEF transgenic plants indicated that TfGLO and TfDEF have partial functional divergence, while they basically work synergistically in torenia.
    MGG Molecular & General Genetics 11/2010; 284(5):399-414. DOI:10.1007/s00438-010-0574-z · 2.83 Impact Factor
  • Akemi Ohmiya · Katsuhiko Sumitomo · Ryutaro Aida
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    ABSTRACT: 'Jimba' is the most popular white-flowered chrysanthemum cultivar in Japan. A yellow-flowered cultivar with the same growth properties as 'Jimba' will benefit growers because both forms could be produced under the same conditions. Many breeders have therefore tried to produce a "Yellow Jimba" by mutation breeding but have not yet succeeded. Previously, we showed that color mutation from white to yellow in the petals of ray florets is caused by the loss of a carotenoid cleavage dioxygenase gene, CmCCD4a. Here we introduced two separate CmCCD4a RNAi constructs into 'Jimba' by Agrobacterium-medtated transformation. The double-transformation effectively suppressed CmCCD4a expression in petals, which became yellow. The yellowest transformants contained 102 μg·g-1 FW carotenoids in the petals, and the expression level of CmCCD4a was 0.4% of the wildtype level. Although the transformed plants were significantly smaller than the wild type, flower size was unchanged. We consider that CmCCD4a could become a powerful tool for petal color manipulation from white to yellow. JSHS
    10/2009; 78(4):450-455. DOI:10.2503/jjshs1.78.450
  • Ryutaro Aida
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    ABSTRACT: Torenia is an annual plant of the family Scrophulariaceae that is used as an ornamental summer bedding plant. Torenia is also an experimental plant with several useful characteristics, i.e., case of genetic transformation, ability to differentiate adventitious structures, protruding embryo sac, and capacity for in vitro flowering, Genetic transformation of torenia was first reported in 1995, and it has been used in various transgenic Studies. Torenia is a useful model plant for transgenic studies on ornamental plant characteristics Such as the color, shape, and longevity of flowers. In this paper, the characteristics of torenia as an experimental plant and the transgenic Studies performed with torenia are reviewed.
    Plant Biotechnology 12/2008; 25(6):541-545. DOI:10.5511/plantbiotechnology.25.541 · 1.06 Impact Factor
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    Plant Biotechnology 03/2008; 25:45-53. DOI:10.5511/plantbiotechnology.25.45 · 1.06 Impact Factor
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    ABSTRACT: 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.
    Plant Biotechnology 03/2008; 25(1):37-43. DOI:10.5511/plantbiotechnology.25.37 · 1.06 Impact Factor

Publication Stats

600 Citations
93.76 Total Impact Points


  • 2008–2013
    • National Agriculture and Food Research Organization
      Tsukuba, Ibaraki, Japan
  • 2011
    • The Graduate University for Advanced Studies
      Миура, Kanagawa, Japan
  • 2004
    • Nara Institute of Science and Technology
      Ikuma, Nara, Japan