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DOI： 10.1016/j.scienta.2019.05.003

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Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

Ornamental plants with red, purple and/or yellow leaves are generally called colored-leaf plants. Colored leaves are resulted from biosynthesis and accumulation of pigments in leaf cells. Among such pigments, anthocyanins are the most major ones, and they are synthesized through the flavonoid biosynthetic pathway. R2R3-MYB, a member of the MYB transcription factor family, is known to activate the flavonoid biosynthetic pathway. In the present study, we examined genetic transformation of Torenia concolor with R2R3-MYB genes from Tricyrtis sp. (TrMYB1) or Arabidopsis thaliana (PAP1) for producing novel colored-leaf plants. Totally ten and eight independent transgenic plants have so far been obtained by transformation with TrMYB1 and PAP1, respectively. Transgenic plants could be classified into three types according to the leaf color phenotype: green leaves as the wild-type plants (Type I), light red-purple leaves (Type II), and deep red-purple leaves (Type III). Three Type I, two Type II and five Type III transgenic plants were obtained for TrMYB1, whereas two Type I, three Type II and three Type III transgenic plants were obtained for PAP1. Total anthocyanin contents in leaves of Type III transgenic plants were significantly higher than the vector control. Real-time RT-PCR analysis showed that TrMYB1 and PAP1 expression levels generally correlated with the degree of leaf color alteration. These results indicate that ectopic expression of the heterogeneous R2R3-MYB genes in T. concolor may activate the flavonoid biosynthetic pathway in leaves leading to anthocyanin accumulation and leaf color alteration.

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Other Link： http://hdl.handle.net/10191/50915

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DOI： 10.1007/s13562-019-00487-2

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2503/hortj.UTD-036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

R2R3-MYB transcription factors are known to activate the flavonoid biosynthetic pathway. In the present study, R2R3-MYB gene isolated from the liliaceous ornamental plant Tricyrtis sp. (TrMYB1) under the control of the cauliflower mosaic virus 35S promoter was introduced into Pelargonium crispum via Agrobacterium-mediated transformation in order to alter leaf color phenotype. Ten independent transgenic plants have been obtained, and they could be classified into three types according to the leaf color phenotype: six transgenic plants had deep yellowish-green leaves as non-transgenic plants (Type I)
two had deep red-purple leaves (Type II)
and two had deep red leaves (Type III). Spectrophotometric analysis showed that the amount of total anthocyanins significantly increased in leaves of Type II and Type III transgenic plants compared with non-transgenic and Type I transgenic plants. In addition, several anthocyanins were newly produced in leaves of Type II and Type III transgenic plants as revealed by high performance liquid chromatography analysis. Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that TrMYB1 expression level correlated with the degree of leaf color alteration. Our results indicate the validity of genetic transformation with TrMYB1 for producing colored foliage in heterologous ornamental plants.

DOI： 10.1016/j.scienta.2018.06.029

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

Gibberellins (GAs) are diterpenoid hormones, control various physiological developments in plants. The role of gibberellins on morphology and secondary metabolite production was examined in Artemisia annua, a medicinal plant that has been acknowledged as a source of artemisinin, an antimalarial compound. Subsequently, the GA20ox gene from Torenia fournieri (TfGA20ox2) was transferred to A. annua by Agrobacterium-mediated transformation. Compared with wild type plants, all nine transgenic plants showed significantly higher plant heights and artemisinin contents. The highest artemisinin content and yield in TfGA20ox2-overexpressing plants was around two-fold higher than wild type. Moreover, transgenic plants had higher numbers of branches (52.4%) and greater branch lengths (60–203%), but smaller leaf size (77.6%). Interestingly, relative to wild type the number and size of glandular trichomes in transgenic leaves was about 30 and 35% higher, respectively. From GC–MS analysis, the proportion of diterpenes in transgenic plant extracts was 1.5-fold lower than those noticed in wild type, while the proportion of sesquiterpenes was increased about 1.6 times when compared to wild type. However, the content proportion of monoterpenes showed a slightly increase, whereas the level of triterpenes showed no variation. In addition, two monoterpenes (eucalyptol and borneol), four sesquiterpenes (α-caryophyllene, β-guaiene, δ-cadinene and β-cubebene) and one triterpenes (isomultiflorenone) were detected only in transgenic extract, whereas d-α-tocopherol, a diterpenoid compound was found only in wild type but not transgenic plant. These results suggested that gibberellins play a significant role in regards to morphology, trichome formation and terpenoid metabolite production in A. annua.

DOI： 10.1007/s11240-017-1171-1

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Language：English   Publisher：SPRINGER  

DOI： 10.1007/s11240-017-1269-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

B class MADS-box genes play important roles in petal and stamen development. Some monocotyledonous species, including liliaceous ones, produce flowers with petaloid tepals in whorls 1 and 2. A modified ABCE model has been proposed to explain the molecular mechanism of development of two-layered petaloid tepals. However, direct evidence for this modified ABCE model has not been reported to date. To clarify the molecular mechanism determining the organ identity of two-layered petaloid tepals, we used chimeric repressor gene-silencing technology (CRES-T) to examine the suppression of B function in the liliaceous ornamental Tricyrtis sp. Transgenic plants with suppressed B class genes produced sepaloid tepals in whorls 1 and 2 instead of the petaloid tepals as expected. In addition, the stamens of transgenic plants converted into pistil-like organs with ovule-and stigma-like structures. This report is the first to describe the successful suppression of B function in monocotyledonous species with two-layered petaloid tepals, and the results strongly support the modified ABCE model.

DOI： 10.1038/srep24549

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Other Link： http://orcid.org/0000-0002-9762-4842

Language：English   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/srep42322

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Language：English   Publisher：新潟大学農学部  

The family Colchicaceae contains some important ornamental plants, such as Gloriosa spp. and Sandersonia aurantiaca. We have produced various intergeneric hybrid plants in this family in order to obtain wide variability in horticultural traits and to develop novel cultivars. In the present study, we examined chromosome doubling of a diploid intergeneric hybrid between L. modesta and G. superba 'Lutea' (Lit×Gsu-1; 2n=2x=22) for fertility restoration and for further widening the variability. Shoot apical segments harvested from in vitro shoot cultures were treated with various concentrations of amiprophos-methyl (APM; 10, 20 or 40 mg L^<-1>), colchicine (COL; 100, 500 or 1000 mg L^<-1>) or oryzalin (ORY; 5, 10 or 20 mg L^<-1>) for 24 h. Two months after spindle toxin treatment, the highest percentage apical segments with shoot elongation (80.0%) was obtained in 10 mg L^<-1> APM treatment. Flow cytometry analysis of elongated shoots showed that a solid tetraploid (4x) was obtained from 40 mg L^<-1> APM treatment and ploidy chimeras (2x+4x) were obtained from treatments with 10 or 20 mg L^<-1> APM and 5 or 10 mg L^<-1> ORY. Tetraploid and polyploidy chimera shoots showed compact forms

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Language：Japanese   Publisher：Japanese Society of Breeding  

DOI： 10.1270/jsbbr.18.34

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Language：Japanese   Publisher：一般社団法人 園芸学会  

<p>Ornamental values of cut tulip (<i>Tulipa gesneriana</i> L.) flowers are reduced by elongation of the flower stalks and leaf yellowing. The effect of pretreatments with 2-chloroethylphosphonic acid (CEPA), 6-benzylaminopurine (BA), and gibberellic acid (GA<sub>3</sub>) at various concentrations on the vase life of cut 'Christmas Dream' flowers was investigated. Pretreatments with CEPA at 25, 50, and 100 mg・L<sup>–1</sup> significantly suppressed flower stalk elongation, but shortened the flower longevity by 2 days. Pretreatments with BA at 5, 25, and 125 mg・L<sup>–1</sup> suppressed leaf yellowing, but pretreatment with GA<sub>3</sub> suppressed leaf yellowing only slightly. Flower longevity was not affected by pretreatments with BA or GA<sub>3</sub>. A combined pretreatment with 50 mg・L<sup>–1</sup> CEPA and 25 mg・L<sup>–1</sup> BA suppressed flower stalk elongation and leaf yellowing in cut 'Christmas Dream' flowers. Similar results were obtained from 7 cultivars of cut flowers. These results indicate that combined pretreatment with CEPA and BA is effective for suppressing flower stalk elongation and leaf yellowing of cut tulip flowers.</p>

DOI： 10.2503/hrj.15.445

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Complex flowers containing large numbers of petals are especially prized as ornamental plants. Double-flowered Tricyrtis macranthopsis, in which stamens and carpels are converted to petaloid organs, is related to AGAMOUS (AG) gene expression. So we isolated AGAMOUS gene from T. macranthopsis, named as TrimAG. Since TrimAG expression was limited to whorls 3 and 4 and ectopic expression of TrimAG in Arabidopsis thaliana caused the same phenotype as overexpression of other AG orthologs in transgenic Arabidopsis, this gene has similar function as its AG orthologs. In the double-flowered T. macranthopsis cultivar, the level of TrimAG expression was reduced in the two inner reproductive whorls (stamens and carpels) and caused reproductive organ replacement with petaloid tepals and floral indeterminacy. Southern blot analysis of genomic DNA indicated that there were no significant differences between the double-flowered cultivar and wild-type plants. Analysis of promoter and AG intron II sequences did not identify any critically mutated sequences in the doubled-flowered cultivar. These results suggest that reduced TrimAG expression in whorls 3 and 4 might be caused by mutation in one of the genes in regulatory network which controls AGAMOUS expression or transcriptional silencing by methylation of TrimAG promoterfintron sequences. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scienta.2015.06.050

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC BREEDING  

'Niigata S3' is a new strawberry (Fragaria x ananassa Duch.) cultivar that is early flowering and possesses high soluble solid content and good coloration. It was selected from a cross between Kei812 (seed parent) and 'Astika-R.uby' (pollen parent). The first harvest date of 'Niigata S3' was December 27, 34 days earlier than 'Echigohime' and 9 days earlier than 'Asuka-Ruby' (means of 2007 and 2008). The marketable yield of 'Niigata S3' was 85% of 'Echigohime', 107% of 'Astika-Ruby', while the early yield was 145% of 'Echigohime', 85% of 'Asuka-Ruby' (based on 2007 and 2008 means). The shape of the fruit is long conical, and its skin color medium-red. The fruit skin hardness of 'Niigata S3' was 31.5 g/mm(2), which was harder than 'Echigohime', and its average soluble solid content was 11.4%, which was higher than the values for 'Echigohime' and 'Asuka-Ruby' (2008). Furthermore, 'Niigata S3' did not bear apical overripe fruit. This new cultivar is adaptable to the climatic conditions of Niigata, as well as other regions that experience low winter temperatures and insolation.

DOI： 10.1270/jsbbs.64.427

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Diploid and triploid intergeneric hybrids obtained by crosses among Gloriosa superba 'Lutea' (2n = 2x = 22), G. 'Marron Gold' (2n = 4x = 44), Littonia modesta (2n = 2x = 22), and Sandersonia aurantiaca (2n = 2x = 24) were analyzed for their meiotic chromosome pairing in pollen mother cells by genomic in situ hybridization (GISH) with digoxigenin-labeled total DNA of one parent as probe. Chromosomes from each parent could be clearly distinguished in pollen mother cells of all the five intergeneric hybrids by GISH. For three diploid hybrids, L. modesta x G. superba 'Lutea' (2n = 2x = 22), L. modesta x S. aurantiaca (2n = 2x = 23) and S. aurantiaca x G. superba 'Lutea' (2n = 2x = 23), 0.04-0.27 autosyndetic bivalents (intragenomic pairing of non-homologous chromosomes) and 0.13-0.36 allosyndetic bivalents (intergenomic chromosome pairing) were observed per pollen mother cell, indicating that there are some homologous chromosomal regions within each genome and among the genomes of Gloriosa, Littonia and Sandersonia. Differences in the average number of allosyndetic bivalents per pollen mother cell among different genome combinations may reflect the evolutionary distances among the three genera, and Gloriosa and Littonia may be closely related to each other, while Sandersonia may have relatively distant relationships with Gloriosa and Littonia. For two triploid hybrids, L. modesta x G. 'Marron Gold' (2n = 3x = 33) and S. aurantiaca x G. 'Marron Gold' (2n = 3x = 34), no allosyndetic bivalents were observed. Based on the results obtained in the present study, possible utilization of the diploid and triploid intergeneric hybrids for further breeding of colchicaceous ornamentals is discussed.

DOI： 10.1007/s10681-014-1152-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Artemisia annua L. is a commercial source of artemisinin. Nevertheless, artemisinin content within the plant is relatively low and varies depending on genotype and environment. To broaden the genetic variability, the mutation effect of C-12-ion beam irradiation on A. annua was examined. Irradiation at 2.5 Gy had a slight lethal effect to nodal segments while a noticeable lethal effect was observed at 5 and 10 Gy. Furthermore, at higher doses (20 and 50 Gy), a severe lethal effect was observed. Mutations at the DNA level of axillary bud-derived shoots were performed by RAPD. The mutation frequency at 10 Gy was about 1.7 and 2.1 times higher than that at 2.5 and 5 Gy, respectively. After growth and artemisinin production observation of 72 irradiated mutants, around 14 and 7 % of them showed higher artemisinin content and artemisinin yield compared to the controls, respectively. The highest artemisinin content in a mutant was 1.43 % DW, which was 3.2-fold higher than the original wild type. Additionally, the highest artemisinin yield in mutants was 3.68 mg/plant, which was around 1.4-fold higher than in the wild type. Moreover, irradiated mutants exhibited antibacterial activity against S. aureus, but the wild types did not. This study presents an effective application of heavy ion beam irradiation to create variations and improve artemisinin production in A. annua.

DOI： 10.1007/s11240-014-0519-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The CYCLOIDEA (CYC) gene controls the development of zygomorphic flowers and the determination of adaxial identity of floral organs in the model developmental system of Antirrhinum majus. However, whether CYC homologue genes also control floral zygomorphy in monocotyledon Alstroemeria plants is yet unknown. In this study, we investigated CYC-like genes in the monocotyledons Alstroemeria aurea, Alstroemeria magenta, and Alstroemeria pelegrina var. rosea, all of which have zygomorphic flowers. Since the CYC gene belongs to the T-complex protein (TCP) gene family of transcription factors, cloning of CYC-like sequences was performed using rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR) by using degenerate primers designed for the TCP domain. We cloned 1 CYC-like sequence each from A. aurea (AaTCP1, accession number AB714967 in the GenBank/EMBL/DDBJ databases) and A. magenta (AmTCP1, AB714970), and two CYC-like sequences from A. pelegrina var. rosea (ApTCP1, AB714968; and ApTCP2, AB714969). The deduced amino acid sequences of AaTCP1, AmTCP1, ApTCP1, and ApTCP2 shared 67.7%, 67.7%, 71.0%, and 64.5% identities, respectively, with the TCP domain in CYC. Molecular phylogenetic analysis indicated that three CYC-like genes from Alstroemeria belonged to the ZinTBL1b clade in the CYC-/tbl -like subfamily. Reverse transcription (RT)-PCR and in situ hybridization analyses showed that AaTCP1 transcripts were specifically detected in flower buds and localized in the base of adaxial inner perianth of A. aurea. These results suggest that CYC-like genes are also involved in the development of floral asymmetry and the determination of adaxial identity of floral organs in the monocotyledon Alstroemeria. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scienta.2014.01.046

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：International Society for Horticultural Science  

Gibberellins (GAs) are plant hormones controlling many aspects of plant growth and development including stem elongation, germination and transition from vegetative growth to flowering. GA 20-oxidase (GA 20ox) and GA 3-oxidase (GA 3ox), classes of 2-oxoglutarate-dependent dioxygenases, catalyze the conversion of precursor GAs to their bioactive forms, and therefore play a direct role in determining the levels of bioactive GAs in plants. Transgenic plants of the liliaceous ornamental Tricyrtis sp. 'Shinonome' overexpressing the GA 20ox or GA 3ox gene from Torenia fournieri (TfGA20ox2 and TfGA3oxl) were produced. After 3 years of cultivation, 4 and 2 independent transgenic plants containing TfGA20ox2 and TfGA3oxl, respectively, were subjected to morphological characterization at the flowering stage. Because GA 20ox and GA 3ox catalyze the last step in the formation of bioactive GAs, overexpression of TfGA20ox2 or TfGA3oxl was initially expected to induce a GA-overproduction phenotype in transgenic plants, such as internode elongation. However, on the contrary, all the transgenic plants exhibited reduced plant height, reduced internode length and reduced stem diameter compared with the control, non-transgenic plants, irrespective of the kind of transgene. In addition, all the transgenic plants had slender leaves and narrow flower tepals. Exogenous treatment of transgenic plants with gibberellic acid and a GA biosynthesis inhibitor, uniconazol, resulted in increased and decreased plant height, respectively. Possible factors leading to morphological alterations, observed in the present study, of transgenic Tricyrtis sp. were proposed.

DOI： 10.17660/ActaHortic.2014.1025.2

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：International Society for Horticultural Science  

The class B genes, which belong to the MADS-box gene family, play important roles in regulating petal and stamen development in flowering plants. These genes exist in two different types termed DEFLCLENS (DEF)- and GLOBOSA (GLO)-like genes, and the B-function is provided by heterodimers of DEF- and GLO-like gene products. In order to understand the molecular mechanism of floral development in the agapanthaceous ornamental Agapanthus praecox ssp. orientalis, we produced and characterized transgenic A. praecox ssp. orientalis plants ectopically expressing the DEF- or GLCMike gene of the same plant, ApDEF or ApGLO. No visible morphological alterations were observed both in vegetative and floral organs of all the 7 independent transgenic plants containing ApDEF. On the other hand, in 4 out of 6 independent transgenic plants containing ApGLO, organs developed in whorl 4 showed noticeable morphological alteration: they were thick compared with carpels of non-transgenic plants, and had a branch tip. No apparent morphological alterations were observed in floral organs of the other 3 whorls. Scanning electron microscopic observations showed that the tip surface of whorl 4 organs of non-transgenic plants was covered with papilla cells, while there were few papilla cells on the tip surface of the morphologically altered whorl 4 organs of transgenic plants. In addition, epidermal cells of the morphologically altered whorl 4 organs of transgenic plants showed an intermediate morphology between those of ovaries and filaments of non-transgenic plants. Since it has been reported that ApDEF is expressed in all the 4 whorls of non-transgenic A. praecox ssp. orientalis, endogenous ApDEF products and transgenic ApGLO products may form heterodimers causing homeotic conversion in whorl 4 of transgenic plants.

DOI： 10.17660/ActaHortic.2014.1025.14

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Agapanthus praecox ssp. orientalis (Leighton) Leighton, a monocotyledonous ornamental plant belonging to Agapanthaceae, has recently become popular as,a potted plant for landscaping and as a cut flower. As a first step toward molecular breeding for flower color alteration in this plant, we isolated and characterized a gene encoding dihydroflavonol 4-reductase (DFR), a pivotal enzyme of the flavonoid biosynthetic pathway. A full-length cDNA clone for DFR was isolated from flower tepals of a cultivar with deep-blue flowers, and its genomic clone, designated ApDFR1 (accession number AB099529 in the GenBank/EMBL/DDBJ databases) was isolated from leaves by a polymerase chain reaction (PCR)-based strategy. Nucleotide sequence analysis revealed that ApDFR1 contains four introns and an open reading frame encoding a polypeptide of 378 amino acid residues. Deduced amino acid sequence shows 59-75% identities with those of previously reported DFR genes. Southern blot analysis showed that there are one or two copies of the DFR gene in the genome of A. praecox ssp. orientalis. ApDFR1 transcripts were detected in young flower tepals, stamens, pistils and bracts, but not in pedicles, scapes and leaves as revealed by reverse transcription-PCR analysis. When ApDFR1 was expressed under the control of the cauliflower mosaic virus 35S promoter in transgenic Petunia hybrida 'W85', a dfr-recessive line, some transgenic plants showed drastic flower color alteration froth white to red purple. High-performance liquid chromatography analysis showed that colored flower limbs of transgenic plants accumulate anthocyanidins, mainly cyanidin and petunidin. These results indicate that ApDFR1 encodes DFR and is active in a heterologous plant species. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scienta.2013.12.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY  

The gibberellin 2-oxidase catalyzes the bioactive gibberellins or their immediate precursors to inactive forms. We have previously produced transgenic plants of the liliaceous plant Tricyrtis sp. containing the GA2ox gene from the linderniaceous plant Torenia fournieri. These transgenic plants showed dwarf phenotypes as expected but unfortunately had no flowers or only small, unopened flowers. Recently, one newly produced transgenic line (G2-55) formed fully opened flowers. G2-55 showed a moderately dwarf phenotype and the shoot length decreased to 63.4% of that of the control, non-transgenic plants. No significant differences in the number of flowers per shoot and in the flower size were observed between G2-55 and the control. Flow cytometry analysis and chromosome observation showed that G2-55 was tetraploid (2n=4x=52), whereas the other transgenic lines producing no or only small flowers were diploid (2n=2x=26) as the mother plant. Pollen fertility of G2-55 was 81.2% as determined by acetocarmine staining. The tetraploidy in G2-55 might be resulted from somaclonal variation of embryogenic calluses used as a target material for Agrobacterium-mediated transformation. The tetraploid transgenic plant G2-55 may be useable not only directly as a potted plant, but also as a material for further breeding of Tricyrtis spp.

DOI： 10.5511/plantbiotechnology.14.0916a

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：INT SOC HORTICULTURAL SCIENCE  

Both Gloriosa spp. and Sandersonia aurantiaca are tuberous plants belonging to the family Colchicaceae. They have recently become popular worldwide as cut flowers or potted plants because of their beautiful, unique flowers and good vase life. Since no large variations in horticultural traits are found within each genus, we have examined intergeneric hybridization among colchicaceous ornamental plants in order to obtain wide variability in horticultural traits and to develop novel cultivars. To date, hybrid plants have been produced via ovule culture in various intergeneric cross-combinations. In the present study, horticultural characterization of an intergeneric hybrid of G. superba 'Verschild' (Gve; 2n=7x=77). S. aurantiaca (Sau; 2n=2x=24) was carried out at the flowering stage. This hybrid (Gve. Sau-1) was shown to have 44 chromosomes from Gve and 12 chromosomes from Sau by genomic in situ hybridization analysis. Gve. Sau-1 produced flowers in late June, whereas Gve and Sau did in early July and early June, respectively. Gve. Sau-1 showed a climbing habit as Gve did, and had vegetative shoots below the first flower. Plant height of Gve. Sau-1 was much higher than both parents. Gve. Sau-1 had sessile leaves with leaf tip tendrils as Gve did. Flowers of Gve. Sau-1 were pendulous and had undulate and reflexed petals like those of Gve, but the degree of petal reflection in Gve. Sau-1 was lower than in Gve. Flower color of Gve. Sau-1 was different from both parents: the petal center was bright red (JHS 0106) and the petal rim was light yellow (JHS 2504). Gve. Sau-1 had dehiscent anthers, and its pollen viability was 2.0% as assessed with acetocarmine staining. Thus Gve. Sau-1 was clearly distinguishable from the parents and had novel horticultural characteristics. Fertility restoration of Gve. Sau-1 through in vitro chromosome doubling is now in progress.

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Gibberellins (GAs) are the plant hormones that control many aspects of plant growth and development, including stem elongation. Genes encoding enzymes related to the GA biosynthetic and metabolic pathway have been isolated and characterized in many plant species. Gibberellin 2-oxidase (GA2ox) catalyzes bioactive GAs or their immediate precursors to inactive forms
therefore, playing a direct role in determining the levels of bioactive GAs. In the present study, we produced transgenic plants of the liliaceous monocotyledon Tricyrtis sp. overexpressing the GA2ox gene from the linderniaceous dicotyledon Torenia fournieri (TfGA2ox2). All six transgenic plants exhibited dwarf phenotypes, and they could be classified into two classes according to the degree of dwarfism: three plants were moderately dwarf and three were severely dwarf. All of the transgenic plants had small or no flowers, and smaller, rounder and darker green leaves. Quantitative real-time reverse transcription-polymerase chain reaction (PCR) analysis showed that the TfGA2ox2 expression level generally correlated with the degree of dwarfism. The endogenous levels of bioactive GAs, GA1 and GA4, largely decreased in transgenic plants as shown by liquid chromatography-mass spectrometry (LC-MS) analysis, and the level also correlated with the degree of dwarfism. Exogenous treatment of transgenic plants with gibberellic acid (GA3) resulted in an increased shoot length, indicating that the GA signaling pathway might normally function in transgenic plants. Thus, morphological changes in transgenic plants may result from a decrease in the endogenous levels of bioactive GAs. Finally, a possibility of molecular breeding for plant form alteration in liliaceous ornamental plants by genetically engineering the GA metabolic pathway is discussed. © 2013 Elsevier GmbH.

DOI： 10.1016/j.jplph.2013.05.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HORTICULTURAL SCI  

Lychnis belongs to Caryophyllaceae and contains a number of horticulturally attractive species. In order to widen their variations in horticultural traits, interspecific cross-pollination and subsequent immature seed culture were carried out using 8 Lychnis species, L. chalcedonica, L. coronata, L. fulgens, L. gracillima, L. kiusiana, L. miqueliana, L. sieboldii, and L. wilfordii. Enlarged fruits containing immature seeds were obtained in the 26 cross-combinations 4 weeks after pollination. Immature seeds were isolated from the fruits and cultured on half-strength Murashige and Skoog medium without plant growth regulators, on which germination occurred in 11 cross-combinations and seedlings were produced in 8 cross-combinations. However, green seedlings were obtained only in L. fulgens x L. sieboldii, and seedlings obtained from the other 7 cross-combinations, L. coronata x L. gracillima, L. gracillima x L. coronata, L. gracillima x L. fulgens, L. kiusiana x L. sieboldii, L. kiusiana x L. wilfordii, L. wilfordii x L. kiusiana, and L. wilfordii x L. sieboldii, were albino and died during acclimatization. The hybridity of all 55 green seedlings obtained from L. fulgens x L. sieboldii was confirmed by random amplified polymorphic DNA analysis. Although some morphological variations were observed among hybrids, hybrid plants generally showed intermediate morphologies between the parents. Hybrids showed high pollen fertility and their self-pollination yielded viable seeds. The present study shows the possibility of interspecific cross-breeding in Lychnis. Further studies are necessary to improve the culture conditions of immature seeds and to examine interspecific cross-compatibility using a wide range of Lychnis species.

DOI： 10.2503/jjshs1.82.57

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HORTICULTURAL SCI  

Lychnis belongs to Caryophyllaceae and contains a number of horticulturally attractive species. In order to widen their variations in horticultural traits, interspecific cross-pollination and subsequent immature seed culture were carried out using 8 Lychnis species, L. chalcedonica, L. coronata, L. fulgens, L. gracillima, L. kiusiana, L. miqueliana, L. sieboldii, and L. wilfordii. Enlarged fruits containing immature seeds were obtained in the 26 cross-combinations 4 weeks after pollination. Immature seeds were isolated from the fruits and cultured on half-strength Murashige and Skoog medium without plant growth regulators, on which germination occurred in 11 cross-combinations and seedlings were produced in 8 cross-combinations. However, green seedlings were obtained only in L. fulgens x L. sieboldii, and seedlings obtained from the other 7 cross-combinations, L. coronata x L. gracillima, L. gracillima x L. coronata, L. gracillima x L. fulgens, L. kiusiana x L. sieboldii, L. kiusiana x L. wilfordii, L. wilfordii x L. kiusiana, and L. wilfordii x L. sieboldii, were albino and died during acclimatization. The hybridity of all 55 green seedlings obtained from L. fulgens x L. sieboldii was confirmed by random amplified polymorphic DNA analysis. Although some morphological variations were observed among hybrids, hybrid plants generally showed intermediate morphologies between the parents. Hybrids showed high pollen fertility and their self-pollination yielded viable seeds. The present study shows the possibility of interspecific cross-breeding in Lychnis. Further studies are necessary to improve the culture conditions of immature seeds and to examine interspecific cross-compatibility using a wide range of Lychnis species.

DOI： 10.2503/jjshs1.82.57

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Language：Japanese   Publisher：園芸学会  

On the assumption that consumers treat cut flowers continuously with sugar, the effects of continuous treatments with glucose, sucrose, fructose, and trehalose at different concentrations were investigated on the vase life of cut tulip flowers. Continuous treatments with all 4 sugars extended the vase life of cut flowers of ‘Ile de France’. Continuous treatments with glucose, sucrose, and fructose were more effective than that with trehalose. However, all continuous treatments except for that with 1% glucose caused damage to leaves to various extents. Continuous treatment with 1% glucose increased the relative fresh weight of cut flowers in all 12 cultivars examined, and significantly extended the vase life of cut flowers in 9 cultivars. On the other hand, the effects of pulse treatments with glucose were also investigated based on the assumption that producers treat cut flowers for a short period with sugars before shipping. However, such pulse treatments had no effects on the vase life of cut flowers of ‘Ile de France’. Thus, continuous treatment with 1% glucose is effective for extending the vase life of tulip cut flowers.<br>

DOI： 10.2503/hrj.12.201

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PONTIFICIA UNIV CATOLICA CHILE, FAC AGRONOMIA INGENIERIA FORESTAL  

A. K. Vidal, D.-S. Han, M. Nakano and Y. Niimi. 2012. Decreased time from seed to flowering corm size in Zephyra elegans via in vitro cultivation. Cien. Inv. Agr. 39(3): 577-584. Experiments were performed to establish a method to reduce the time from seed to flowering in Zephyra elegans. Seedlings took at least four years to produce a flowering corm. Although germination was highest in a water-agar medium, plant necrosis occurred when plants were later transferred; therefore, MS (Murashige and Skoog) medium was selected as the medium for germination. An increase in the light intensity to 9500 lux and in the pH to 6.7, significantly increased the germination rate. Eight weeks after seed germination, seedlings were transferred to MS media with sucrose concentrations of 45, 60, 75, 90 or 105 g L-1 and a pH 5.7 or 6.7, with the aim of achieving the greatest corm weight gain. After 16 weeks, the best weight gain was obtained in the MS medium with 75 g L-1 of sucrose and a pH of 6.7. The resulting corms were transferred to pots and grown under greenhouse conditions. Corms weighing under 0.12 g did not sprout, whereas all corms over 0.3 g sprouted and all those over 0.4 g at the end of the in vitro culture stage bloomed in the second year of greenhouse cultivation. The time required for development from seedling to flowering corm was reduced to two years.

DOI： 10.4067/S0718-16202012000300017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Chalcone synthase (CHS) is the key enzyme in an early stage of the flavonoid biosynthetic pathway. In the present study, a full-length cDNA clone for CHS was isolated from flower tepals of the liliaceous ornamental Tricyrtis sp., in which tepals have many reddish-purple spots resulting from accumulation of cyanidin derivatives. The deduced amino acid sequence of the isolated cDNA clone, designated TrCHS1 (accession number AB478624 in the GenBank/EMBL/DDBJ databases), shows 79.4-91.4% identity with those of previously reported CHS genes. An RNA interference (RNAi) construct targeting TrCHS1 was introduced by Agrobacterium-mediated transformation in order to alter the flower color of Tricyrtis sp. Seven transgenic plants that produced flowers could be classified into three types according to flower color phenotype: one transgenic plant had tepals with as many reddish-purple spots as non-transgenic plants (Type I); one had tepals with reduced numbers of reddish-purple spots (Type II); and five had completely white tepals without any spots (Type III). High-performance liquid chromatography analysis showed that tepals of Type III transgenic plants did not accumulate detectable amounts of anthocyanidins. In addition, TrCHS1 mRNA levels in tepals of Type II and Type III transgenic plants decreased substantially compared with non-transgenic plants, as determined by quantitative real-time reverse transcription-polymerase chain reaction analysis. Our results indicate the validity of RNAi suppression of the flavonoid biosynthetic pathway genes for flower color alteration in Tricyrtis sp. To the best of our knowledge, this is the first report on flower color alteration by genetic transformation in monocotyledonous ornamentals.

DOI： 10.1007/s11032-011-9653-z

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Language：English   Publisher：新潟大学農学部  

センノウ属(Lychnis spp. ;ナデシコ科)は約30種で構成され、そのうちの6種は日本に自生している。多くのセンノウ属植物は観賞的価値が高く、そのため鉢植え植物や庭植え植物として栽培されている。本研究では、園芸形質の拡大を目的として、日本原産の3種のセンノウ属植物、センジュガンピ(L. gracillima;2n=2x=24)、オグラセンノウ(L. kiusiana;2n=2x=24)およびフシグロセンノウ(L. miqueliana;2n=2x=24)について、培養物のオリザリン(ORY)処理による染色体倍加を試みた。これは、三倍体のセンノウ(L. senno)において最初に確立された方法である(Nonakaら、2011)。培養小植物体から調製した節切片を10mg L-1 ORYで24時間処理したところ、2ヵ月後にはいずれのセンノウ属植物においても70%以上の節切片が生存していた。また、ORY処理した節切片由来の小植物体についてフローサイトメトリー分析を行ったところ、センジュガンピ、オグラセンノウおよびフシグロセンノウの小植物体のそれぞれ15.7、15.1および7.8%が四倍

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Some intergeneric hybrids produced by the crosses among several colchicaceous ornamentals, Gloriosa superba 'Lutea' (2n = 2x = 22), G. 'Marron Gold' (2n = 4x = 44), G. 'Verschild' (2n = 7x = 77), Littonia modesta Hook. (2n = 2x = 22), and Sandersonia aurantiaca Hook. (2n = 2x = 24), were subjected to genomic in situ hybridization (GISH) analysis in order to clarify their genome constitutions. Chromosome preparation was made from root tip cells of L. modesta x G. superba 'Lutea', L. modesta x S. aurantiaca, S. aurantiaca x G. superba 'Lutea', L. modesta x G. 'Marron Gold', S. aurantiaca x G. 'Marron Gold' and G. 'Verschild' x S. aurantiaca. Total DNA of one parent was labeled with digoxigenin or biotin and used as probe, and chromosomes were counterstained with 4'-6-diamidono-2-phenylindole (DAPI). For all the nine intergeneric hybrids, chromosomes from each parent could be clearly distinguished by GISH analysis. Thus GISH analysis is a powerful tool for identifying the genome constitution of intergeneric hybrids in colchicaceous ornamentals. The results obtained by GISH analysis study may be important for further progress in breeding of colchicaceous ornamentals.

DOI： 10.1007/s10681-011-0393-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Lychnis (Caryophyllaceae) consists of about 30 species distributed throughout the temperate regions of the Northern Hemisphere, from East Asia to Europe. Many Lychnis spp. have high ornamental value and cultivated as pot or garden plants. In the present study, in vitro chromosome doubling of several Lychnis spp. was examined in order to widen their variability in horticultural traits. Initially effect of various spindle toxin treatments [100, 500 or 1000 mg l(-1) colchicine (COL), 10,20 or 50 mg l(-1) oryzalin (ORY), or 1, 5, 10 mg l(-1) amiprophos-methyl (APM)] of nodal segments of a triploid genotype of L. senno (3x) was investigated on survival of nodal segments and chromosome doubling in nodal segment-derived plantlets. Significantly higher percentage (75.0%) of surviving segments after spindle toxin treatment was obtained in 10 mg l(-1) ORY treatment. Flow cytometry (FCM) analysis of leaf tissues showed that 9.4-13.8% of plantlets, which were derived from 10 to 20 mg l(-1) ORY, or 5 mg l(-1) APM treatments, were hexaploid (6x) or ploidy chimera (3x + 6x, 4x + 6x, 5x + 6x, 3x + 4x + 6x). The results obtained by FCM analysis were confirmed by chromosome observation in root tip cells. Thus 10 mg l(-1) ORY treatment of nodal segments is suitable for in vitro chromosome doubling of triploid L. senno. Efficient chromosome doubling was also achieved in diploid L. fulgens (2x) and L sieboldii (2x) by treating nodal segments with 10 mg l(-1) ORY: 68.9-88.7% of nodal segments survived after ORY treatment, and 24.7-26.5% of plantlets derived from ORY-treated nodal segments were tetraploid (4x) or ploidy chimera (2x + 4x) in both species. These results indicate that the in vitro chromosome doubling method established for triploid L. senno may be applicable to a wide range of Lychnis spp. Tetraploid L. fulgens and L sieboldii showed a compact plant form, and had thick stems and deep green leaves compared with the diploid mother plants. On the other hand, hexaploid L. senno showed very poor growth and died before flowering. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scienta.2011.05.004

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The gesneriaceous perennial plant Titanotrichum oldhamii has beautiful foliage and attractive bright yellow flowers. However, breeding of T. oldhamii by conventional sexual hybridization may be difficult because sexual reproduction of this species is very rare. In the present study, plant regeneration systems via both direct and indirect formation of adventitious shoots from leaf explants were established as the first step toward breeding T. oldhamii by using biotechnological techniques. Adventitious shoots were formed efficiently on medium containing 0.1 mg l(-1) benzyladenine. Histological observation showed that shoot formation on this medium occurred directly from leaf epidermal cells without callus formation. On the other hand, leaf explants formed calluses on medium containing 0.1 mg l(-1) 2,4-dichlorophenoxyacetic acid. The calluses could be maintained by monthly subculturing to fresh medium of the same composition. When the calluses were transferred to plant growth regulator-free medium, they formed adventitious shoots. Directly and indirectly formed shoots rooted well on medium containing 0.1 mg l(-1) indole-3-butyric acid. Plantlets thus obtained were successfully acclimatized and grew vigorously in the greenhouse. Flow cytometry analysis indicated that no variation in the ploidy level was observed in plants regenerated via direct shoot formation, whereas chromosome doubling occurred in several plants regenerated via indirect shoot formation. Regenerated plants with the same ploidy level as the mother plants showed almost the same phenotype as the mother plants, whereas chromosome-doubled plants showed apparent morphological alterations: they had small and crispate flowers, and round and deep green leaves.

DOI： 10.1007/s11816-011-0172-5

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Effect of heavy-ion beam irradiation on the growth and development of embryogenic calluses was examined in the liliaceous monocotyledon Tricyrtis hirta and the umbelliferous dicotyledon Daucus carota. Embryogenic calluses of T. hirta were irradiated with 5, 10, 20 or 50 Gy of (12)C(6+), (14)N(7+) or (20)Ne(10+) ions, and those of D. carota were irradiated with 5, 10, 20 or 50 Gy of (14)N(7+) ions. In both species, irradiation at 10-50 Gy inhibited growth of embryogenic calluses, and callus growth rate decreased as irradiation dose increased. Interestingly irradiation at low doses greatly promoted somatic embryo production from embryogenic calluses in both species. In T. hirta, calluses irradiated with 5 and 10 Gy (12)C(6+) ions, 10 Gy (14)N(7+) ions, and 5 Gy (20)Ne(10+) ions produced more than twice as many embryos as the control, non-irradiated calluses. In D. carota, embryogenic calluses irradiated with 5 Gy (14)N(7+) ions produced more than one and a half times as many embryos as the control. Somatic embryo production in both species was inhibited by irradiation at 20 and 50 Gy.

DOI： 10.1007/s10725-009-9438-0

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Language：Japanese  

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J-GLOBAL

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Language：English   Publisher：富山県中央植物園  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

In order to induce horticulturally valuable mutants in the Liliaceous ornamental Tricyrtis hirta, embryogenic calluses of this species were irradiated with (12)C(+6) ion beams. Morphological characterization was performed on 35, 37 and 15 plants regenerated from calluses irradiated with 5, 10 and 20 Gy ion beams, respectively, and on 10 plants from non-irradiated calluses after 3 years of cultivation in pots. No plants were regenerated from calluses irradiated with 50 Gy ion beams. There were no large differences in the mean values of leaf length, leaf width, soil and plant analyzer development (SPAD) value of leaves, flower length and flower diameter between the control (division-derived plants from the mother plant of the embryogenic calluses) and the irradiation treatments at different doses. On the other hand, the mean number of shoots per plant increased, and the mean shoot length and the mean number of nodes per shoot decreased in the irradiation treatments. The mean number of flowers per plant was increased in the 20 Gy irradiation treatment. For most morphological characteristics investigated, the variation spectrum widened with increase in the irradiation dose. Several horticulturally attractive variations such as dwarfism, slender and deep green leaves, and large flowers were observed in regenerants from the irradiation treatments, and these variations were stable after additional 2 years of cultivation in pots or garden. Thus mutation induction by heavy ion beam irradiation of embryogenic calluses is a valuable tool for improving horticultural value of T. hirta.

DOI： 10.5511/plantbiotechnology.27.155

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SEJANI PUBL  

Chirita flavimaculata, C. eburnea, and C. speciosa, which belong to Gesneriaceae, have high ornamental values such as beautiful foliages and showy flowers. An efficient plant regeneration system via adventitious shoot formation was established for micropropagation of these species. For all species, adventitious shoots elongating over 5 mm in length were efficiently regenerated from leaf explants on half-strength MS medium containing 0.1 mg l(-1) of both NAA and BA (8.1-15.6 shoots per explant). Histological observation indicated that adventitious shoot regeneration on a medium containing 0.1 mg l(-1) of both NAA and BA occurred directly from leaf epidermal cells without callus formation. Plantlets were obtained by rooting the shoots on half-strength MS medium containing 0.1 mg l(-1) IBA. No variations in the ploidy level were observed in the regenerated plants of three Chirita species as indicated by flow cytometry analysis.

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Language：Japanese   Publisher：日本植物園協会  

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Language：English   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

Colchicaceous plants, Gloriosa spp., Littonia modesta and Sandersonia aurantiaca, are cultivated as ornamentals. However, unfortunately no large variations in horticultural traits are found within each genus. We examined intergeneric hybridization using 6 genotypes of Gloriosa spp., 1 genotype of L. modesta and 2 genotypes of S. aurantiaca to obtain wider variability and to develop novel cultivars in those groups. Following intergeneric cross-pollination, putative hybrid plantlets were obtained via ovule culture in various combinations. Early confirmation of the hybridity of ovule culture-derived plantlets was accomplished by flow cytometry and random amplified polymorphic DNA analyses. Several intergeneric hybrids have so far been produced flowers and subjected to morphological characterization. All the hybrids examined, i.e. L. modesta x S. aurantiaca, L. modesta x S. aurantiaca `Phoenix', L. modesta x G. superba `Lutea', L. modesta x G. `Marron Gold', S. aurantiaca x G. superba `Lutea', S. aurantiaca x G. `Marron Gold' and S. aurantiaca 'Phoenix' x G. `Marron Gold', had novel morphological characteristics compared with their parents, some of which were horticulturally attractive. The results obtained in our series of studies indicate the validity of intergeneric hybridization in the improvement programs of colchicaceous ornamentals. We are now examining to develop a rapid and efficient micropropagation system and to restore fertility by artificial chromosome doubling of intergeneric hybrids that had been produced in our series of experiments.

DOI： 10.5511/plantbiotechnology.26.535

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY  

Senno (Lychnis senno, Caryophyllaceae), a traditional ornamental plant in Japan. was introduced from China and all the strains of Senno found in Japan are triploid with 2n=3x=36. Hand-pollination was conducted between triploid Senno and allied Lychnis taxa, L. kiusiana (2n=24), L. mniqueliana (2n=24), L. miqueliana f. albescens (2n=24), L. chalcedonica (2n=24), L. wilfordii (2n=24), L. sieboldii (2n=24), and L. coronata (2n=24). Immature seeds 17-42 days after pollination were cultured on half-strength Murashige and Skoog media with or without 6-benzyladenine. Although immature seeds were obtained in most cross combinations, seed germination was observed only in three cross combinations, L. senno x L. kiusiana, L. kiusiana X L. senno, and L. senno X L. sieboldii. Seedlings derived from reciprocal crosses between L. senno and L. kiusiana showed an abnormal morphology or a chlorophyll-deficiency, xantha. Seedlings derived from L. senno X L. sieboldii also showed an abnormal morphology or chlorophyll-deficiency, but one seedling grew into a green plantlet. The hybridity of these seedlings was confirmed by random amplified polymorphic DNA analysis. The chromosome number of the interspecific hybrid plantlet between L. senno and L. sieboldii was determined to be 2n=32. The present study shows the possibility of using triploid Senno for breeding by interspecific hybridization, and the effectiveness of immature seed culture for producing interspecific hybrids in the genus Lychnis.

DOI： 10.5511/plantbiotechnology.26.301

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Language：English   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY  

DOI： 10.5511/plantbiotechnology.25.509

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Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1-2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation. © 2008 Springer Science+Business Media B.V.

DOI： 10.1007/s10535-008-0099-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC HORTICULTURAL SCIENCE  

Three intergeneric hybrids among colchicaceous ornamentals, Gloriosa superba 'Lutea' (2n = 2x =: 22), Littonia modesta (2n = 2x = 22), and Sandersonia aurantiaca (2n = 2x = 24), were subjected to morphological characterization and chromosome observation. Hybrid plants produced flowers 2 to 3 years after transplantation of ovule culture-derived plantlets to the greenhouse. All the hybrid plants, L. modesta x S. aurantiaca, L. modesta x G. superba 'Lutea', and S. aurantiaca x G. superba 'Lutea', showed a climbing habit like those of L. modesta and G. superba 'Lutea'. Plants of L. modesta x S. aurantiaca and L. modesta x G. superba 'Lutea' were taller and shorter than their respective parents, whereas plant height of S. aurantiaca X G. superba 'Lutea' was nearly intermediate between the parents. Leaves of all the hybrids had a tendril at the tip like those of L. modesta and G. superba 'Lutea'. Flower morphologies of all the hybrids were nearly intermediate between the parents. Flower colors of all the hybrids were similar to the seed or pollen parent. Although hybrids of L. modesta X G. superba 'Lutea' showed low pollen fertility, as assessed with acetocarmine staining, the other two kinds of hybrids had nondehiscent anthers or no fertile pollen grains. Chromosome observation in root tip cells revealed that all the hybrids had a diploid number of chromosomes: L. modesta x S. aurantiaca (2n = 2x = 23), L. modesta x G. superba 'Lutea' (2n = 2x = 22), and S. aurantiaca x G. superba 'Lutea' (2n = 2x = 23). Novel morphological characteristics of the hybrids may be valuable for future breeding of colchicaceous ornamentals.

DOI： 10.21273/HORTSCI.43.1.115

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The class B genes, which belong to the MADS-box gene family, play important roles in regulating petal and stamen development in flowering plants. These genes exist in two different types termed DEF- and GLO-like genes, and the B-function is provided by heterodimers of a DEF- and a GLO-like gene product. In the present study, dicot (tobacco and lettuce) and monocot (Tricyrtis hirta) plants were transformed with the GLO-like gene of Agapanthus praecox ssp. orientalis ApGLO alone or in combination with the DEF-like gene of the same plant ApDEF. In two out of 10 transgenic tobacco plants containing ApGLO, sepals partially converted into petaloid organs. For lettuce, ray florets of four out of nine transgenic plants containing ApGLO also developed additional petaloid organs. In two out of five transgenic T. hirta plants containing both ApGLO and ApDEF, organs developed in whorl 4 showed noticeable morphological alteration: they were much longer compared with carpels of non-transgenic plants, and had purple spots overall on the surface as filaments of non-transgenic plants. No morphological alterations were observed in vegetative organs between transgenic and non-transgenic plants for all the three species. The results obtained in the present study indicate a possibility of molecular breeding for flower form alteration by genetic transformation with the class B MADS-box gene(s) of heterologous plant species.

DOI： 10.1007/s11032-007-9103-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HORTICULTURAL SCI  

Plantlets derived from ovule culture following intra- or intergeneric crosses among Colchicaceous ornamentals, Gloriosa spp., Littonia modesta Hook. and Sandersonia aurantiaca Hook., were subjected to flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses in order to verify their hybridity. For crosses between plants with apparently distinct relative fluorescence intensity (RFI) of nuclei, i.e., intrageneric crosses between Gloriosa genotypes with different ploidy levels, and most intergeneric crosses, detection of hybrid plantlets could be readily accomplished by FCM analysis. The results of hybrid identification by RAPD analysis supported those of FCM analysis. In addition, RAPD analysis allowed the verification of the hybridity of intra- or intergeneric cross-derived plantlets, which could not be identified as hybrids by FCM analysis due to the similarity of RFI of the parents or appearance of the RFI peak in an unexpected position. Totally, 110 independent hybrid plantlets (60 intrageneric and 50 intergeneric hybrids) have so far been identified by FCM and/or RAPD analyses. Thus, FCM in combination with RAPD analyses offer simple and rapid means for the early detection of intrageneric and intergeneric hybrids in Colchicaceous ornamentals.

DOI： 10.2503/jjshs.76.73

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Agapanthus (Agapanthus praecox ssp. orientalis), a liliaceous perennial, is cultivated as an ornamental plant because of its beautiful, blue-violet to white flowers. We have previously produced transgenic plants containing the beta-glucuronidase (GUS) reporter gene under the control of cauliflower mosaic virus (CaMV) 35S promoter in an experimental strain of Agapanthus via Agrobacterium-mediated genetic transformation. These transgenic plants have been cultivated for 5 yr after transplantation to pots. In the present study, we carried out morphological characterization, and examination of the ploidy level, pollen fertility and stability of GUS gene expression in 12 transgenic plants derived from five independent lines at the flowering stage. Flow cytometry (FCM) analysis indicated that transgenic plants of four lines kept the diploid level, but those of the remaining one line were tetraploid. For all of the 12 transgenic plants, some morphological variations were observed both in vegetative and floral organs such as decreased number of leaves per inflorescence, smaller leaves, shorter inflorescence stalks, decreased number of florets per inflorescence and smaller florets. Pollen fertility of all the transgenic plants was below 5% as determined by acetocarmine staining. All the 12 transgenic plants showed stable expression of the GUS gene in leaves, roots, tepals, pistils, and stamens, as indicated by histochemical GUS assay, fluorometric GUS assay, and/or real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. No apparent GUS gene silencing was observed in transgenic agapanthus plants even after 5 yr of cultivation.

DOI： 10.1007/s11627-006-9012-7

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Language：Japanese   Publisher：一般社団法人 園芸学会  

Plantlets derived from ovule culture following intra- or intergeneric crosses among Colchicaceous ornamentals, <i>Gloriosa</i> spp., <i>Littonia modesta</i> Hook. and <i>Sandersonia aurantiaca</i> Hook., were subjected to flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses in order to verify their hybridity. For crosses between plants with apparently distinct relative fluorescence intensity (RFI) of nuclei, i.e., intrageneric crosses between <i>Gloriosa</i> genotypes with different ploidy levels, and most intergeneric crosses, detection of hybrid plantlets could be readily accomplished by FCM analysis. The results of hybrid identification by RAPD analysis supported those of FCM analysis. In addition, RAPD analysis allowed the verification of the hybridity of intra- or intergeneric cross-derived plantlets, which could not be identified as hybrids by FCM analysis due to the similarity of RFI of the parents or appearance of the RFI peak in an unexpected position. Totally, 110 independent hybrid plantlets (60 intrageneric and 50 intergeneric hybrids) have so far been identified by FCM and/or RAPD analyses. Thus, FCM in combination with RAPD analyses offer simple a

DOI： 10.2503/jjshs.76.73

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The Liliaceous perennial Tricyrtis hirta, sometimes called 'Japanese toad lily', has recently become popular as an ornamental for pot and garden uses. Highly embryogenic callus cultures of this plant predominately consisted of diploid cells but also contained tetraploid cells after 1 year of establishment. In the present study, plans regenerated from the 1-year-old embryogenic callus cultures were subjected to ploidy level analysis and morphological characterization following 3 years of cultivation. Among 37 plants examined, 28 kept the diploid level (2n = 2x = 26) but nine were tetraploid (2n = 4x = 52) as indicated by FCM analysis and chromosome observation. Although no morphological alterations were detected in 26 out of 28 diploid regenerants, the remaining two showed noticeable variations: both were severely dwarf and had crimped leaves and many malformed flowers. The tetraploid regenerants had several horticulturally attractive characteristics compared with the diploid controls, such as longer shoots, thicker stems, and larger flowers. Thus regeneration of tetraploid plants from 1-year-old embryogenic callus cultures offers a possibility to improve the horticultural value of T hirta, although regeneration of trueness-to-type plants is essential for utilizing the cultures for micropropagation and genetic transformation. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scienta.2006.07.026

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Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis 'Fanfare' (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l(-1) sucrose, 2.5 g l(-1) gellan gum, 0.1 mg l(-1) a-naphthaleneacetic acid (NAA), 0.1 mg l(-1) 6-benzyladenine (BA) and 50 mg l(-1) gibberellic acid (GA(3)). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l(-1) sucrose and 3 g l(-1) gellan gum, without plant growth regulators (PGRs) or with I mg l(-1) zeatin and 0.1 mg l(-1) NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used alpha as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scienta.2006.06.027

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In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM, 4-amino-3,5,6-trichloropicolinic acid (picloram
PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium. © 2005 Society for In Vitro Biology.

DOI： 10.1079/IVP2005707

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The class B genes, which belong to the MADS-box gene family, play important roles in regulating the development of petals and stamens in flowering plants. To understand the molecular mechanisms of floral development in Agapanthus praecox ssp. orientalis (Agapanthaceae), we isolated and characterized the homologs of the Antirrhinum majus genes GLOBOSA and DEFICIENS in this plant. These were designated as ApGLO and ApDEF, respectively. ApGLO and ApDEF contain open reading frames that encode deduced protein with 210 and 214 amino acid residues, respectively. Phylogenetic analysis indicated that ApGLO and ApDEF belong to the monocot class B gene family. In situ hybridization experiments revealed that hybridization signals of ApGLO and ApDEF were observed in whorl 1 as well as in whorls 2 and 3. Moreover, the flowers of transgenic Arabidopsis plants that ectopically expressed ApGLO formed petal-like organs in whorl 1. These observations indicate that the flower developmental mechanism of Agapanthus follows the modified ABC model.

DOI： 10.1007/s11103-005-5218-z

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Embryogenic calli of <i>Muscari armeniacum</i> cv. Blue Pearl were co-cultivated with <i>Agrobacterium tumefaciens</i> EHA101/pBH, which harbored a binary vector that carries the phosphinothricin acetyltransferase (<i>bar</i>) and hygromycin phosphotransferase (<i>hpt</i>) genes. The calli were then transferred onto the selection media containing either 4 mg · liter<sup>−1</sup> bialaphos or 75 mg · liter<sup>−1</sup> hygromycin. Four to five weeks after transfer to the selection media, both bialaphos-resistant (Bia<sup>r</sup>) and hygromycin-resistance (Hyg<sup>r</sup>) cell clusters were produced. Over 90% of callus lines selected on a bialaphos-containing medium were verified to be transgenic by PCR analysis; this selection efficiency is comparable to that based on the <i>hpt</i> gene. Leaf segments of plantlets regenerated from the transgenic Bia<sup>r</sup> callus lines showed resistance to 4 mg · liter<sup>−1</sup> of bialaphos, indicating that the <i>bar</i> gene is useful not only as a selectable marker but also as a producer of herbicide-resistant transgenic plants of <i>M</i>. <i>armeniacum</i>.

DOI： 10.2503/jjshs.74.60

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HORTICULTURAL SCI  

Embryogenic calli of Muscari armeniacum cv. Blue Pearl were co-cultivated with Agrobacterium tumefaciens EHA101/pBH, which harbored a binary vector that carries the phosphinothricin acetyltransferase (bar) and hygromycin phosphotransferase (hpt) genes. The calli were then transferred onto the selection media containing either 4 mg (.) liter(-1) bialaphos or 75 mg (.) liter(-1) hygromycin. Four to five weeks after transfer to the selection media, both bialaphos-resistant (Bia(r)) and hygromycin-resistance (Hyg(r)) cell clusters were produced. Over 90% of callus lines selected on a bialaphos-containing medium were verified to be transgenic by PCR analysis; this selection efficiency is comparable to that based on the hpt gene. Leaf segments of plantlets regenerated from the transgenic Bia(r) callus lines showed resistance to 4 mg (.) liter(-1) of bialaphos, indicating that the bar gene is useful not only as a selectable marker but also as a producer of herbicide-resistant transgenic plants of M. armeniacum.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：International Society for Horticultural Science  

Flavonoid 3'5'-hydroxylase (F3'5'H), a member of the cytochrome P450 family, is the key enzyme for the expression of blue or purple flower color. By a polymerase chain reaction (PCR)-based strategy using a degenerate primer designed from the conservative region of the F3'5'H genes, a full-length cDNA (accession number AB127340) was cloned from blue flower tepals of Muscari armeniacum 'Blue Pearl', and its genomic clone designated MaP450 (accession number AB127341), was isolated from leaves. Nucleotide sequence analysis revealed that MaP450 contains an open reading frame (ORF) of 1512 bp, which consists of two exons (888 and 624 bp) encoding a polypeptide of 503 amino acid residues. The deduced amino acid sequence of MaP450 has several conserved regions of the cytochrome P450 proteins, but shows only 34-37% identities with those of previously reported F3'5'H genes. Southern blot analysis showed that there are 5-6 copies of MaP450 in the genome of M. armeniacum 'Blue Pearl'. High-performance liquid chromatography (HPLC) and reverse transcription- polymerase chain reaction (RT-PCR) analyses revealed that MaP450 is expressed developmentally paralleling anthocyanidin accumulation in the tepal. The expression pattern of MaP450 is similar to those of the majority of genes involved in the flavonoid biosynthetic pathway. Transcripts of MaP450 were markedly detected in tepals of Muscari genotypes with colored flowers, but only slightly detected in those of genotypes with white flowers. These results indicate that MaP450 may be a P450 gene involved in the flavonoid biosynthetic pathway in Muscari species.

DOI： 10.17660/ActaHortic.2005.673.54

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：International Society for Horticultural Science  

We have previously developed a system for Agrobacterium-mediated transformation of the Oriental hybrid lily, Lilium 'Acapulco' (Hoshi et al., 2004). Since no transformed plants of L. longiflorum could be obtained with this system, we made some modifications in this system. Following a scratching-treatment of filament-derived calluses of L. longiflorum 'Georgia' with sandpaper, they were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm, which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt), and intron-containing ?-glucuronidase (gus-intron) genes in the T-DNA region. By increasing the concentration of gellan gum in co-cultivation medium from 3 to 10 g L-1, about 3-fold increase was obtained in the number of blue spots resulting from transient expression of the gus gene. The concentration of hygromycin for selecting putative transformants was decreased from 50 to 35 mg L-1. By using an improved system, 5 hygromycin-resistant (Hyg r) culture lines have so far been obtained from 280 co-cultivated calluses of 'Georgia'. Plantlets were regenerated from all of these Hyg r lines, and they were confirmed to be transgenic by inverse PCR analysis and GUS histochemical assay.

DOI： 10.17660/ActaHortic.2005.673.73

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

Due to indiscriminate collection, the natural habitat of Hylotelephium sieboldii and H. sieboldii var. ettyuense have been significantly reduced. For ex situ conservation and efficient vegetative propagation, a micropropagation system based on adventitious shoot regeneration was developed for these two endangered species. Leaves, stems and roots of in vitro-grown plantlets, and flower buds of greenhouse-grown plants were used as explants. For H. sieboldii, adventitious shoots were most efficiently regenerated from flower bud explants on a medium containing 1 mg l-1 each of NAA and BA. Adventitious shoot regeneration from flower bud explants under this condition was also obtained in H. sieboldii var. ettyuense, but with lower efficiencies. Adventitious shoots of both species rooted and developed into plantlets on a medium containing 0.1 mg l-1 IBA. Almost all of these plantlets were successfully transplanted to the greenhouse. At least at early stage of growth, they showed no apparent morphological alterations. Copyright © 2005 The Japanese Society for Plant Cell and Molecular Biology.

DOI： 10.5511/plantbiotechnology.22.221

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：International Society for Horticultural Science  

A system for producing transgenic plants has been developed for the Liliaceous geophyte Tricyrtis hirta via Agrobacterium-mediated transformation. Tepal-derived embryogenic calluses were co-cultivated with A. tumefaciens EHA101/pIG121Hm, which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing ?-glucuronidase (GUS-intron) genes in the T-DNA region. The duration of co-cultivation and acetosyringone (AS) treatment during co-cultivation affected the frequency of transient expression of the GUS gene: the best result was obtained when embryogenic calluses were co-cultivated for 7 days in the presence of 50 mg l-1 AS. Following co-cultivation, the calluses were transferred to a medium lacking plant growth regulators (PGRs) but containing 40 mg l-1 hygromycin and 300 mg l-1 cefotaxime, on which several hygromycin-resistant (Hygr) somatic embryos were produced 8 weeks after transfer. These embryos were transferred to the same medium but containing lower concentrations of antibiotics (20 mg l -1 hygromycin and 100 mg l-1 cefotaxime) to promote germination. Finally, Hygr embryo-derived plantlets were established on a medium without both PGRs and antibiotics. Most of them were verified to be stable transformants by GUS histochemical assay and PCR analysis.

DOI： 10.17660/ActaHortic.2005.673.52

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The liliaceous perennial plants, Tricyrtis spp., are cultivated as ornamental plants in Japan. Natural populations of several Japanese Tricyrtis spp. are severely threatened by indiscriminate collection and habitat destruction. In this study, a plant regeneration system based on somatic embryogenesis has been developed for efficient clonal propagation of T. hirta, T. hirta var. albescens, T. formosana, T. formosana cv. Fujimusume, T. flava ssp. ohsumiensis, and T. macrantha ssp. macranthopsis. Flower tepal explants of these genotypes were cultured on media containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) alone or in combination with N-(1,2,3-thiadiazol-5-yl)-N′-phenylurea (thidiazuron, TDZ). Calluses induced on media containing 2,4-D produced somatic embryos following their transfer to a plant growth regulator-free medium, indicating that these calluses were embryogenic. A combination of 4.5 μM 2,4-D and 0.45 μM TDZ was most effective for inducing embryogenic calluses from tepal explants. Among various explant sources, filaments were most suitable for inducing embryogenic calluses on a medium containing 4.5 μM 2,4-D and 0.45 μM TDZ. Embryogenic calluses were only obtained from filament explants for T. macrantha ssp. macranthopsis. Embryogenic calluses could be maintained by subculturing monthly onto the same medium, and a 1.5-3.5-fold increase in fresh weight was obtained after 1 mo. of subculture. Depending on the plant genotype, 50-500 somatic embryos per 0.5 g fresh weight of embryogenic callus was obtained 1 mo. after transfer to a plant growth regulator-free medium. Most of the embryos developed into plantlets, and they were successfully acclimatized to greenhouse conditions. Regenerated plants showed no alteration in the ploidy level as indicated by chromosome observation and flow cytometric analysis.

DOI： 10.1079/IVP2003506

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Flavonoid 3′,5′-hydroxylase (F3′5′H) is the key enzyme for the expression of blue or purple flower color. A full-length cDNA for the F3′5′H gene was cloned from petals of Vinca major, and its genomic clone, designated VmFH1 (accession number AB078781 in the GenBank/ EMBL/DDBJ databases), was isolated from leaves by a PCR-based strategy. Nucleotide sequence analysis revealed that VmFH1 contains one intron and an open reading frame encoding a polypeptide of 506 amino acid residues. The deduced amino acid sequence shows between 51% and 83% identity with those of previously reported F3′5′H genes. Southern blot analysis showed that there are 3-4 copies of the F3′5′H gene in the genome of V. major. Transcripts of the F3′5′H gene were detected in young flower petals but not in leaves as revealed by RT-PCR analysis. When VmFH1 was expressed in transgenic Petunia hybrida under the control of the cauliflower mosaic virus 35S promoter, some transgenic plants showed drastic flower color alteration from red to deep red with deep purple sectors. These transgenic plants accumulated 3′,5′-hydroxylated anthocyanins in their petals, which were never detected in non-transgenic plants by high-performance liquid chromatography analysis. These results indicate that VmFH1 isolated from V. major encodes F3′5′H and is active in a heterologous plant species.

DOI： 10.1007/s00299-003-0709-3

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A system for the production of transgenic plants was developed for the Oriental hybrid lily, Lilium cv. Acapulco, by Agrobacterium-mediated genetic transformation. Filament-derived calli were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm, which harbored a binary vector carrying the neomycin phosphotransferase II, hygromycin phosphotransferase, and intron-containing β-glucuronidase genes in the T-DNA region. Six hygromycin-resistant (Hygr) culture lines were obtained from 200 calli by scratching them with sandpaper prior to inoculation and using NH4NO3-free medium for co-cultivation and a hygromycin-containing regeneration medium for selection. Hygr culture lines regenerated shoots, which developed into plantlets following transfer to a plant growth regulator-free medium. All of these plantlets were verified to be transgenic by GUS histochemical assay and inverse PCR analysis.

DOI： 10.1007/s00299-003-0700-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.

DOI： 10.1007/s00299-003-0608-7

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Language：Japanese   Publisher：新潟大学農学部  

サイトカイニン(CKs)の定量において、精度と感度の向上のために、安定同位体標識とコールド(非標識の天然型)標品のHPLCでの保持と MSでの質量スペクトルの特性を試験Lた。ODSとPhタイプのカラムの両方で、ゼアフチン(Z)とゼアチンリボシド(ZR)の両者とも、重水素ラベルの標品がコールドよりも早く溶出した。negative modeにおいて、ZRの m/z133/134と218の消長のパターンはZ と同様で、2H5-ZRでは分子イオンの m/z355巴断片イオンの133/134と223が見られた。 positive modeにおいて、 Z では、 20Vから60V で分子イオンの m/z220と断片の136が見られた。2H5-Z では、 Z の質量スペクトルの変化と同様に、分子イオンの m/z225と断片の137/136が見られた。 ZR では、 60V までは分子イオンのm/z352が見られ、 m/z136/137と220の消長のパターンはZ と同様であった。しかし、negative modeと同様に、 ZR の方が高いドリフト電圧が必要であった。2H5-ZRでは、分子イオンの m/z357と断片の m/z136/137と225が見られ、これらはZR および2H5-Z のビ

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

A system for plant regeneration from protoplasts was developed for the Liliaceous ornamental plant, Agapanthus praecox ssp. orientalis (Leighton) Leighton 'Royal Purple Select' (2n=2x=32). Viable protoplasts were routinely isolated from leaf-derived embryogenic calluses with yields of 0.8 to 1.5x10(6) protoplasts per g FW of calluses. Protoplasts started to divide 5 to 7 days after isolation, and protoplast-derived colonies consisting of 50 to 100 cells were obtained after 1 month. A plating efficiency of 0.8% was obtained after 2 months of culture using a gellan gum-solidified medium containing 1 mg l(-1) each of PIC and BA under continuous illumination. Protoplast-derived calluses produced somatic embryos at a frequency of 46.7% on PGR-free medium, whereas 68.3% of the calluses regenerated adventitious shoots on a medium containing 1 mgl(-1) BA. Somatic embryos and adventitious shoots developed into plantlets, which were successfully transplanted to pots. Flow cytometric analysis and chromosome observation revealed that both diploid and tetraploid plants were regenerated from protoplasts.

DOI： 10.1023/A:1021290106221

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Ploidy level was estimated for nine species and 94 cultivars in the genus Hemerocallis by flow cytometry (FCM) analysis. By using parsley (Petroselinum crispum (Mill.) Nyman ex A.W. Hill) as an internal control, the ratio of the relative fluorescence intensity (RFI) of nuclei from each Hemerocallis genotype and parsley was calculated. The values (Hemerocallis/parsley ratio) for 103 Hemerocallis genotypes were clearly classified into three groups: 1.84-2.13 (group 1), 2.94-3.10 (group 2), and 3.74-4.26 (group 3). Since the diploid H.fulva var. littorea (2n = 22) and the triploid H. fulva var. kwanso (2n = 33) belonged to groups 1 and 2, respectively, the other Hemerocallis genotypes belonging to groups 1, 2 and 3 were considered to be di-, tri- and tetraploid, respectively. Chromosome counting in root tip cells revealed that Hemerocallis genotypes belonging to groups 1, 2 and 3 had 22, 33 and 44 chromosomes, respectively. No cytochimeras and polysomaty were observed. Thus, FCM analysis has proven useful for simple and rapid estimation of the ploidy level of Hemerocallis species and cultivars. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4238(02)00150-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

We have established a system for isolating gametic microprotoplasts from developing microspores of Lilium longiflorum 'Hinomoto' (2n = 2x = 24) as a first step toward the production of intergeneric hybrid plants with one or a few alien chromosomes via microprotoplast fusion in the Liliaceous ornamentals. Anthers of this cultivar containing microsporocytes at mainly diakinesis to metaphase I were cultured for 3-4 days in a medium containing half-strength MS salts, double-strength MS vitamins, 1 g l(-1) casamino acids, 100 g l(-1) sucrose, and one of four spindle toxins: amiprophosmethyl, isopropyl N-(3-chlorophenyl)carbamate (CIPC), colchicine or propyzamide. Of the four spindle toxins examined, CIPC at concentrations of 5 or 10 muM efficiently induced micronucleation with the mean number of nuclei per meiocyte being 7.5. CIPC treatment also efficiently induced micronucleation in the other five Lilium genotypes evaluated (L. regale, L. longiflorum 'Georgia', L. speciosum 'Uchida', the Asiatic hybrid lily 'Connecticut King' and the Aurelian hybrid lily 'Golden Splendor') with the mean number of nuclei per meiocyte falling within the range 5.4-11.7. In 'Hinomoto', each meiocyte nucleus formed a microcell 4-5 days after initiation of CIPC treatment. Following isolation of such meiocytes from the anthers and their incubation in a cell-wall-digesting enzyme solution, gametic (micro)protoplasts of less than 10 mum, 10-20 mum and 20-50 mum in diameter were obtained with yields of 5.5x10(4), 6.6x10(4) and 4.9x10(4) per anther, respectively. Smaller microprotoplasts with DNA content below the 2C level, as indicated by flow cytometric analysis, were enriched by sequential filtration through nylon sieves of decreasing pore size (50, 20 and 10 mum). The relative fluorescence intensity in some of their nuclei corresponded to that of one or only a few chromosomes.

DOI： 10.1007/s00497-002-0153-5

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Systems for establishing suspension cultures and for inducing plant regeneration from these cultures for the Liliaceous ornamental plant, Hosta sieboldiana (Lodd.) Engl. have been developed. Pale-yellow and nodular calluses were induced from more than 20% of scape segments on MS medium containing 1 mg l(-1) picloram (PIC), 30 g l(-1) sucrose, and 2 g l(-1) gellan gum. Upon transfer of calluses to the same medium lacking gellan gum, stably-growing suspension cultures were established after 1 month. Suspension cell clusters regenerated a large number of adventitious shoots following transfer to MS media containing 0.1 mg l(-1) NAA in combination with either BA or TDZ. Over 20 shoots per 0.3 g FW of cell clusters were obtained on media containing 0.1 mg l(-1) NAA and either 1 or 5 mg l(-1) TDZ. Shoots rooted easily on plant growth regulator (PGR)-free MS medium, and plantlets were successfully transferred to soil. Plants showed no visible morphological alterations and maintained the diploid level as indicated by flow cytometric analysis.

DOI： 10.1023/A:1016574513545

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Highly embryogenic callus cultures were established from leaf explants in the Liliaceous ornamental plant, Agapanthus praecox ssp. orientalis (Leighton) Leighton, as the first step toward the development of an efficient transformation system. Embryogenic calli were induced and then maintained by monthly subculturing onto a medium containing 1 mg l(-1) picloram. Upon transfer to a plant growth regulator-free medium, the calli produced numerous somatic embryos, most of which could develop into plantlets. Histological observations revealed that, following the transfer of the embryogenic calli to a plant growth regulator-free medium, 2- to 6-cell proembryos, probably of unicellular origin, were produced, which passed through the globular and oval stages, and developed into club-shaped embryos with cotyledon, shoot apex and radicle. For establishing an efficient selection system in future transformation, the effects of selective agents (kanamycin, 6418, hygromycin and bialaphos) as well as antibiotics for eliminating Agrobacterium (carbenicillin and cefotaxime) were examined on the growth and development of the embryogenic calli. Callus growth was completely inhibited by 50 mg l(-1) hygromycin or 4 mg l(-1) bialaphos, and somatic embryo formation was completely inhibited by 50 mg l(-1) hygromycin, 75 mg l(-1) G418 or 3 mg l(-1) bialaphos. On the other hand, carbenicillin and cefotaxime rather promoted both growth and development of the embryogenic calli. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4238(02)00033-X

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Language：Japanese   Publisher：日本育種学会  

マイクロプロトプラストを用いた非対称融合により,ユリ科花卉園芸植物において属間の染色体添加系統を作出することを目的として,微小核を形成したヘメロカリス`ステラ・デ・オロ'(Hemerocallis hybrida cv. Stella d'Oro)の懸濁培養細胞からマイクロプロトプラストを単離する方法を確立した.培養細胞を2mMハイドロキシウレア(HU)で24時間,その後8μMプロピザミド(PRO)で60時間処理し,さらにPRO処理を開始して20時間後に20μMサイトカラシン-B(CB)を添加することにより,微小核の形成が効率的に誘導され,そのときの微小核形成率は19.1%であった.微小核を形成した細胞を細胞壁消化酵素溶液で処理し,単離されたプロトプラストをパーコール連続密度勾配を用いて超遠心分離を行うことにより,わずかな細胞質に囲まれた小核をもつマイクロプロトプラストが単離された.それらを孔径50,20および10μmのナイロンメッシュに連続して通すことにより,直径10μm以下のマイクロプロトプラストが1ml圧縮細胞量の培養細

DOI： 10.1270/jsbbs.52.51

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan International Research Center for Agricultural Sciences  

Studies on Agrobacterium-mediated transformation in 3 Liliaceous ornamental plants, Lilium formosanum, Agapanthus praecox ssp. orientalis and Muscari armeniacum, were described. Three different strains of A. tumefaciens were used, all of which harbored the binary vector carrying the nptII, hpt and gus-intron genes in the T-DNA region. For L. formosanum, no transgenic tissues nor plants were obtained after co-cultivation of organogenic calli with A. tumefaciens, although transient expression of the gus gene could be detected in the calli during co-cultivation. On the other hand, several hygromycin-resistant (Hygr) cell clusters were obtained for both A. praecox ssp. orientalis and M. armeniacum following the transfer of co-cultivated embryogenic calli onto hygromycin (Hyg)-containing media. Hygr calli developed into complete plants via somatic embryogenesis, and most of them were confirmed to be transgenic plants based on GUS histochemical assay and PCR analysis. Southern blot analysis revealed the integration of 1 to 5 copies of the transgene into the genome of the transgenic plants of both 2 species, but most of them had 1 or 2 copies. Agrobacterium-mediated transformation systems developed for A. praecox ssp. orientalis and M. armeniacum may be useful as a tool for their genetic improvement as well as molecular biology studies.

DOI： 10.6090/jarq.36.119

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To produce intergeneric hybrid plants with one or a few chromosomes via microprotoplast fusion in the Liliaceous ornamentals, we developed a system for isolating microprotoplasts from micronucleated cells in suspension cultures of Hemerocallis hybrida cv. Stella d'Oro (2n = 2x = 22). Micronucleation was efficiently induced with a micronucleus index up to 19.1% by the following sequential treatment of suspension cultures: initially with 2 mM hydroxyurea for 24 h and then with 8 μM propyzamide for 60 h with the addition of 20 μM cytochalasin-B to the cultures 20 h after the initiation of the propyzamide treatment. Following the enzyme treatment of the micronucleated cells and ultra-centrifugation with a continuous iso-osmotic gradient of Percoll solution, microprotoplasts were isolated, each of which had a small nucleus surrounded by a thin rim of cytoplasm. Microprotoplasts less than 10 μm in diameter were obtained with yield of 2.9 × 104 cells per 1 ml packed cell volume of suspension cells through sequential filtration with nylon sieves with decreasing pore sizes (50, 20 and 10 μm). The DNA content of the microprotoplasts was less than the 2C level as indicated by flow cytometric analysis, and the relative fluorescence intensity in some of their nuclei corresponded to one or a few chromosomes.

DOI： 10.1270/jsbbs.52.51

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan International Research Center for Agricultural Sciences  

We aimed to produce intergeneric hybrid plants with only one or a few alien chromosomes via microprotoplast fusion for genetic improvement and chromosome studies in Liliaceous ornamental plants. In order to apply this technique, it is essential to establish an efficient system for mass-preparation of microprotoplasts. We have established 2 different systems for isolating microprotoplasts, one from partially synchronized cell suspension cultures of Hemerocallis hybrida and the other from developing microspores of Lilium longiflorum. Here, the induction of micronucleated cells, isolation of microprotoplasts, and enrichment of smaller microprotoplasts containing one or a few chromosomes are described for both systems.

DOI： 10.6090/jarq.36.129

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Language：English   Publishing type：Research paper (scientific journal)  

A system for the production of transgenic plants has been developed for the Liliaceous ornamental plant Muscari armeniacum Leichtl. ex Bak via Agrobacterium-mediated transformation of embryogenic cultures. Leaf-derived embryogenic cultures were co-cultivated with each of three A. tumefaciens strains, all of which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt) and intron-containing β-glucuronidase (gus-intron) genes in the T-DNA region. Following co-cultivation, the embryogenic cultures were cultured on a medium containing 500 mg l-1 cefotaxime for 1 week followed by a medium containing 75 mg l-1 hygromycin in addition to cefotaxime. After 4-5 weeks, several hygromycin-resisrant (Hygr) cell clusters were produced from the co-cultivated embryogenic cultures. The highest efficiency of production of Hygr cell clusters was obtained when embryogenic cultures were inoculated with A. tumefaciens EHA101/pIG121Hm in the presence of 100 μM acetosyringone (AS) and 0.1% (v/v) of a surfactant (Tween20) followed by co-cultivation in the presence of 100 μM AS. Hygr embryogenic cultures developed into complete plants via somatic embryogenesis, and most of them were verified to be transgenic by GUS histochemical assay and polymerase chain reaction analysis. Southern blot analysis revealed the integration of one to five copies of the transgene into the genome of transgenic plants, but most of them had one or two copies.

DOI： 10.1007/s00299-001-0405-0

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Language：Japanese   Publisher：日本育種学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC BREEDING  

For the production of intergeneric hybrid plants with partial alien genome via microprotoplast fusion in the Lillaceous ornamentals, the effects of the DNA synthesis inhibitor, hydroxyurea (HU), and the spindle toxins, colchicine (COL), oryzalin (ORY), amiprophos-methyl (APM), butamiphos (BUT), isopropyl N-(3-chlorophenyl)carbamate (CIPC) and propyzamide (PRO) on the metaphase index (MI) and the percentage of micronucleated cells (micronucleus index; MNI) were examined in cell suspension cultures of Hemerocallis hybrida cv. Stella d'Oro. Suspension cells were subcultured every three days in MS medium containing 10 mg l(-1) picloram. Although MI was only 2-3% in the asynchronous control cultures, it increased up to 8.9% and 9.7% by treatment of the cultures with HU and COL, respectively. In addition, MI was further increased by using the sequential treatments of the cultures with HU and each spindle toxin: the highest MI of 30.5% was obtained by treatment with 2 mM HU for 24 h followed by that with 250 muM COL for 20 h. COL and ORY were more effective for synchronizing cell division than the other four spindle toxins. The effects of various spindle toxin treatments on the micronucleation of suspension cultures were also examined by combining the HU pre-treatment for 24 h. Among the six spindle toxins, COL and ORY induced few micronuclei, whereas APM, BUT, CIPC and PRO induced micronucleation in cells to various extents. The most effective treatment for micronucleation was that with 8 muM PRO for 66 h, where MNI was 14.7% and the number of micronuclei per cell ranged from 1-7 were obtained.

DOI： 10.1270/jsbbs.51.285

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for In Vitro Biology  

Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44-44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses were maintained for over 2 yr.

DOI： 10.1007/s11627-001-0067-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

Cell division of suspension cultures of Hemerocallis hybrida was partially synchronized by treating with hydroxyurea (HU), a DNA synthesis inhibitor, followed by treatment with colchicine (COL), a spindle toxin, as the first step toward an efficient preparation of microprotoplasts. During COL treatment, suspension cells were analyzed for relative DNA contents using flow cytometry (FCM) as well as mitotic index (MI) under microscopy. MI reached a peak after 20 h of COL treatment. In FCM analyses, histogram of asynchronous cultures showed three peaks (2C, 4C and 8C nuclei), and the numbers of both 4C and 8C nuclei gradually increased until 18 h after the initiation of COL treatment. After 20 h of COL treatment, the numbers of 4C and 8C nuclei transiently decreased, indicating that a large number of cells were at the prometaphase to metaphase stages. These results indicate that FCM analyses offer a simple and rapid means of preliminary examination for arresting cell-cycle at metaphase in partially synchronized suspension cultures of H. hybrida.

DOI： 10.5511/plantbiotechnology.18.229

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A system for producing transgenic plants was developed for the Liliaceous ornamental Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacterium-mediated genetic transformation. Leaf-derived embryogenic calli were inoculated with A. tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing β-glucuronidase (GUS-intron) genes in the T-DNA region. Following co-cultivation, the calli were transferred to a medium containing 1 mg 1-1 picloram (PIC), 50 mg 1-1 hygromycin and 500 mg 1-1 cefotaxime, on which several hygromycin-resistant (Hygr) cell clusters were obtained 5-6 weeks after transfer. Agrobacterium strain, co-cultivation period and acetosyringone (AS) treatment during co-cultivation affected the number of Hygr callus lines produced: the best result was obtained when embryogenic calli were co-cultivated with LBA4404/pTOK233 for 7 days in the presence of 20 mg 1-1 AS. Hygr calli were transferred to the same medium, but lacking PIC, for inducing somatic embryos. Somatic embryos thus obtained developed into complete plantlets following their transfer to a medium without PIC and antibiotics. All of them were verified to be stable transformants by GUS histochemical assay, PCR and Southern blot analyses. © 2001 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0168-9452(01)00393-4

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Language：Japanese   Publisher：新潟大学  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

Cell suspension cultures of Lilium formosanum Wallace were initiated from bulb scale-derived calli and subcultured every 2 weeks using a medium containing 5 mu M 4-amino-3,5,6-trichloropicolinic acid (picloram). Almost all cell clumps from the suspension cultures developed numerous somatic embryos following their transfer onto a plant growth regulator-free medium, while they vigorously produced shoot buds on media containing 0.5 or 5 mu M 6-benzyladenine (BA). The high regeneration potential on a plant growth regulator-free medium was maintained for up to 54 months, but it gradually decreased thereafter, and only a few adventitious shoots and embryos were obtained from 75-month-old cultures. For restoring the regeneration potential of these cultures, various treatments with plant growth regulators were applied, among which about 10-fold increases in the number of regenerated shoot buds were obtained with 0.5 or 5 mu M 2,3,5-triiodobenzoic acid (TIBA) in combination with 0.5 or 5 mu M BA or N-(1,2,3-thiadiazol-5-yl)-N'-phenylurea (thidiazuron). Only shoot buds were produced from the cell clumps cultured on TIBA-containing media, and these shoot buds developed into complete plantlets after they were excised from the calli and transferred to a plant growth regulator-free medium. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0168-9452(00)00313-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Reciprocal pollination was made between Lilium nobilissimum and L. regale. Pollen tubes reached the base of style within 144 h after pollination, but no mature seeds were obtained in either cross combination. Explants, or ovules-with-placental-tissue excised from each carpel 30 and 40 days after pollination (DAP), were cultured on a medium composed of major salts of B-5 macronutrient (Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Exp. Cell Res. 50, 151-158), micronutrient, Fe-EDTA and vitamins of MS (Murashige, T., Skoog, F., 1962. Physiol. Plant. 15, 473-497), 5% sucrose and 0.2% gellan gum. In L. regale x Lilium nobilissimum, 3% of ovules excised at 30 DAP and 9% of those excised at 40 DAP developed into seedlings. In Lilium nobilissimum x L. regale, only 3.6% of ovules excised 40 DAP developed into seedlings, and none of the ovules excised 30 DAP produced any seedlings. Bulbs of L;. regale and hybrids transplanted to soil showed some resistance to bulb-rot, leaf-top scorch, browning spots and/or streaking on leaves, but those of Lilium nobilissimum were sensitive to these diseases. Flowering individuals were nearly intermediate between parents in their morphological characteristics. All flowering individuals (2n = 24 chromosomes) were identified as hybrids based on karyotype, isozyme and random amplified polymorphic DNA analyses. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4238(99)00115-6

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Language：English   Publisher：園芸学会  

1. Bulblets of Lilium rubellum were cultured for 16 weeks in liquid MS medium containing 100, 150 and 250 mmol·liter<SUP>-1</SUP> (mM) sucrose (referred to as 100, 150 and 250mM sucrose medium, respectively). The 250mM sucrose medium enhanced bulblet growth the most. The greatest gain in fresh weight occurred between 4 and 8 weeks after culture. The ratio of dry to fresh weights (DW/FW) of bulblets cultured in the 250mM sucrose medium was always highest. 2. Three sugars (sucrose, glucose, and fructose) were detected in the autoclaved MS medium. When bulblets were cultured in the liquid sucrose medium, the concentration of glucose and fructose in the medium increased until week 4 as the sucrose concentration decreased. Sugars in the 150mM sucrose medium were nearly depleted by week 8, whereas those in the 250mM sucrose medium persisted for 12 weeks. The growth rate of the bulblets declined as sugar concentration in the medium decreased. 3. The sucrose content in bulblets, cultured in each medium, increased until week 4, then decreased. However, in bulblets, cultured in the 250mM sucrose medium, the sugar content increased again after 12 weeks of culture, while starch content

DOI： 10.2503/jjshs.69.161

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HORTICULTURAL SCI  

1. Bulblets of Lilium rubellum were cultured for 16 weeks in liquid MS medium containing 100, 150 and 250 mmol . liter(-1) (mM) sucrose (referred to as 100, 150 and 250 mM sucrose medium, respectively). The 250 mM sucrose medium enhanced bulblet growth the most. The greatest gain in fresh weight occurred between 4 and 8 weeks after culture. The ratio of dry to fresh weights (DW/FW) of bulblets cultured in the 250 mM sucrose medium was always highest.
2. Three sugars (sucrose, glucose, and fructose) were detected in the autoclaved MS medium. When bulblets were cultured in the liquid sucrose medium, the concentration of glucose and fructose in the medium increased until week 4 as the sucrose concentration decreased. Sugars in the 150 mM sucrose medium were nearly depleted by week 8, whereas those in the 250 mM sucrose medium persisted for 12 weeks. The growth rate of the bulblets declined as sugar concentration in the medium decreased.
3. The sucrose content in bulblets, cultured in each medium, increased until week 4, then decreased. However, in bulblets, cultured in the 250 mM sucrose medium, the sugar content increased again after 12 weeks of culture, while starch content decreased. Hence, it appears that the starch accumulated in the bulblets was hydrolyzed to glucose.
These results indicate that the growth of bulblets of L. rubellum cultured in liquid medium can be promoted by subculturing the bulblets after 8 to 12 weeks of culture.

DOI： 10.2503/jjshs.69.161

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HEADLEY BROTHERS LTD  

Histological examinations of callogenesis and adventitious embryogenesis in immature ovary culture of grapevine (Vitis vinifera L. 'Neo Mat') were carried out during different phases of ontogenetic development. Adventitious embryos and/or embryogenic calli were obtained when immature ovary explants were cultured on a callus induction medium (C medium) for two months followed by transfer of callus-forming explants onto an embryogenesis induction medium (E medium). Microscopic observations of serial sections of the explants revealed that the calli formed on C medium were initiated preferentially from receptacle parenchyma cells. No cell division in the embryo sac was observed and most of them degenerated four weeks after the onset of culture. Two to four weeks after transfer of the explants onto E medium, calli characterized by dense cytoplasm, conspicuous nuclei and thick cell walls were newly formed in the initially-formed, receptacle-derived ones. Proembryos simultaneously developed in the newly formed calli, indicating that they were embryogenic calli. Cell division of embryo sacs was never observed even on E medium, and adventitious embryos and embryogenic calli were hence of somatic origin. Adventitious embryos developed asynchronously and passed through globular, heart, torpedo and cotyledonary stages. These adventitious embryos germinated and developed into plantlets following their transfer onto a plant growth regulator-free medium.

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Language：English   Publisher：園芸学会  

Calli from microspores were induced for the first time in Lilium species. The uninucleate microspores of the Asiatic hybrid lily 'Connecticut King' were isolated and cultured in liquid medium, containing half strength of MS macronutrients, full strength of micronutrients, Fe-EDTA and vitamins of MS, 100 mg·liter<SUP>-1</SUP> cosamino acids, 500 mg·liter<SUP>-1</SUP> glutamine, 1 mg·liter<SUP>-1</SUP> picloram and 0.25 M sucrose or maltose. Microspore viability and development of cultured microspores were influenced by the carbohydrate sources in culture medium. A relatively high viability rate of microspores was observed in the maltose medium, compared with that of sucrose. Cell divisions of microspores and callus formation were found in the maltose medium, whereas in the sucrose medium, a large number of swollen microspores containing many starch grains remained undivided. Hence, maltose is the preferred carbohydrate source for microspore culture of 'Connecticut King'.

DOI： 10.2503/jjshs.69.52

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HORTICULTURAL SCI  

Calli from microspores were induced for the first time in Lilium species. The uninucleate microspores of the Asiatic hybrid lily 'Connecticut King' were isolated and cultured in liquid medium, containing half strength of MS macronutrients, full strength of micronutrients, Fe-EDTA and vitamins of MS, 100 mg . liter(-1) cosamino acids, 500 mg . liter(-1) glutamine, 1 mg . liter(-1) picloram and 0.25 M sucrose or maltose. Microspore viability and development of cultured microspores were influenced by the carbohydrate sources in culture medium. A relatively high viability rate of microspores was observed in the maltose medium, compared with that of sucrose. Cell divisions of microspores and callus formation were found in the maltose medium, whereas in the sucrose medium, a large number of swollen microspores containing many starch grains remained undivided. Hence, maltose is the preferred carbohydrate source for microspore culture of 'Connecticut King'.

DOI： 10.2503/jjshs.69.52

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Headley Brothers Ltd  

Histological examinations of callogenesis and adventitious embryogenesis in immature ovary culture of grapevine (Vitis vinifera L. 'Neo Mat') were carried out during different phases of ontogenetic development. Adventitious embryos and/or embryogenic calli were obtained when immature ovary explants were cultured on a callus induction medium (C medium) for two months followed by transfer of callus-forming explants onto an embryogenesis induction medium (E medium). Microscopic observations of serial sections of the explants revealed that the calli formed on C medium were initiated preferentially from receptacle parenchyma cells. No cell division in the embryo sac was observed and most of them degenerated four weeks after the onset of culture. Two to four weeks after transfer of the explants onto E medium, calli characterized by dense cytoplasm, conspicuous nuclei and thick cell walls were newly formed in the initially-formed, receptacle-derived ones. Proembryos simultaneously developed in the newly formed calli, indicating that they were embryogenic calli. Cell division of embryo sacs was never observed even on E medium, and adventitious embryos and embryogenic calli were hence of somatic origin. Adventitious embryos developed asynchronously and passed through globular, heart, torpedo and cotyledonary stages. These adventitious embryos germinated and developed into plantlets following their transfer onto a plant growth regulator-free medium.

DOI： 10.1080/14620316.2000.11511215

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

A haploid callus line from anther cultures of the Asiatic hybrid lily 'Connecticut King' was maintained for a long term. The survival and growth of the haploid calluses were affected by auxins of picloram, alpha-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) and temperatures of 25, 15 and 7 degrees C during culture. Picloram was more suitable for maintenance of the haploid calluses, whereas NAA and 2,4-D led to root and shoot formation from the haploid calluses. The best temperature for maintenance was 25 degrees C. About 90% of cells in calluses were maintained in haploid level during 60 weeks of subculture, and about 80% of cells were haploid in the calluses maintained over 2 years with the MS medium containing 4 mu M picloram in the dark at 25 degrees C.

DOI： 10.1023/A:1006425307530

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Language：English   Publisher：園芸学会  

An effective method for producing doubled haploid plants of the Asiatic hybrid lily 'Connecticut King' was established with an in vitro colchicine treatment of haploid calli. With an increase in the concentration and duration of colchicine treatment for the haploid calli, both the survival rate and the shoot regeneration from the calli decreased, but the frequency of diploid cells in the callus increased. Treatments of 0.25 and 0.5 mM colchicine for 48 or 72 hrs induced doubled haploid cells and shoot differentiation from calli. Haploid and diploid plantlets were regenerated from colchicine-treated calli, and only haploid plantlets were formed in colchicine-free treatments. This result suggests that these diploid plantlets originated from doubled haploid cells. Double haploids developed bulblets with more scaly leaves with longer stomatal guard cells than did the haploid plantlets.

DOI： 10.2503/jjshs.68.979

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HORTICULTURAL SCI  

An effective method for producing doubled haploid plants of the Asiatic hybrid lily 'Connecticut King' was established with an in vitro colchicine treatment of haploid calli. With an increase in the concentration and duration of colchicine treatment for the haploid calli, both the survival rate and the shoot regeneration from the calli decreased, but the frequency of diploid cells in the callus increased. Treatments of 0.25 and 0.5 mM colchicine for 48 or 72 hrs induced doubled haploid cells and shoot differentiation from calli. Haploid and diploid plantlets were regenerated from colchicine- treated calli, and only haploid plantlets were formed in colchicine-free treatments. This result suggests that these diploid plantlets originated from doubled haploid cells. Double haploids developed bulblets with more scaly leaves with longer stomatal guard cells than did the haploid plantlets.

DOI： 10.2503/jjshs.68.979

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Language：Japanese   Publisher：新潟大学  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Adventitious shoots elongating over 10 mm in length were efficiently obtained (8-9 elongated shoots per explant) from both leaf and petiole segments of the 'rose-formed' strain of hybrid tuberous begonia (Begonia x tubeuhybrida Voss) on MS media containing 0.54 mu M NAA and 0.44 mu M BA. Shoots were efficiently multiplied by shaking shoot-forming leaf segments in a liquid medium containing 0.54 mu M NAA and 0.44 mu M BA, while shoot growth was stimulated by shaking them in a liquid medium without plant growth regulators. Plantlets were obtained by rooting the elongated shoots on half-strength MS media containing 0.54 mu M NAA and solidified with 8 g l(-1) agar or 2 g l(-1) gellan gum, and successfully transferred to the greenhouse. Regenerated plants grew into the flowering stage and showed no apparent morphological alterations. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4238(98)00198-8

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Language：English   Publisher：園芸学会  

Excised scales of lily bulbs were kept at 15°C and 25°C, and under continuous light or dark to study the regeneration and development of bulblets. The seven species were examined : Lilium rubellum Baker, L. speciosum Thunb. 'Uchida', L. nobilissimum Makino, L. formosanum Wallace, L. longiflorum Thunb. 'Hinomoto', L. maculatum Thunb., and the Asiatic hybrid L. X 'Benisugata'. 1. Generally, more bulblets were regenerated at 25°C than at 15°C in all Lilium spp : bulblets of L. rubellum and L. X 'Benisugata' were formed equally well at 15°C and 25°C. Regardless of temperatures, more bulblets of L. formosanum, L. longiflorum 'Hinomoto', and L. X 'Benisugata' regenerated in the light more than they did in the dark. 2. Regenerated bulblets grew better under light at 25°C than at 15°C and the light stimulated the formation of scaly leaves from bulblets of all species and cultivars, except in L. nobilissimum. In L. nobilissimum, bulblets failed to form scaly leaves under any cultural conditions. Growth of bulblets of L. formosanum, L. longiflorum 'Hinomoto', and L. maculatum was promoted in darkness, whereas the bulblets of L. rubellum and L. nobilissimum gre

DOI： 10.2503/jjshs.68.28

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HORTICULTURAL SCI  

Excised scales of lily bulbs were kept at 15 degrees C and 25 degrees C,and under continuous light or dark to study the regeneration and development of bulblets. The seven species were examined: Lilium rubellum Baker, L. speciosum Thunb. 'Uchida', L. nobilissimum Makino, L. formosanum Wallace. L. longiflorum Thunb. 'Hinomoto', L maculatum Thunb., and the Asiatic hybrid L. X 'Benisugata'.
1. Generally, more bulblets were regenerated at 25 degrees C than at. 15 degrees C in all Lilium spp: bulblets of L. rubellum and L. X 'Benisugata' were formed equally well at 15 degrees C and 25 degrees C. Regardless of temperatures, more bulblets of L. formosanum, L. longiflorum 'Hinomoto', and L. X 'Benisugata' regenerated in the light more than they did in the dark.
2. Regenerated bulblets grew better under light at 25 degrees C than at 15 degrees C and the light stimulated the formation of scaly leaves from bulblets of all species and cultivars, except in L, nobilissimum. In L. nobilissimum, bulblets failed to form scaly leaves under any cultural conditions. Growth of bulblets of L, formosanum, L. longiflorum' Hinomoto', and L. maculatum was promoted in darkness? whereas the bulblets of L. rubellum and L. nobilissimum grew best under light at 25 degrees C.
3. Bulblets regenerated at 15 degrees C tended to rot during cold treatments compared with those regenerated at 25 degrees C, and the latter bulblets sprouted more frequently than the former ones after transplantation in a greenhouse.

DOI： 10.2503/jjshs.68.28

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

Embryogenic calli induced from leaf segments of grapevine (Vitis vinifera L. cv. Koshusanjaku) were cocultivated for 5 days with Agrobacterium tumefaciens strains EHA101 (pIG121Hm) or LBA4404 (pTOK233), both of which contained the plasmid carrying the neomycin phosphotransferase II (NPT II), hygromycin phosphotransferase (HPT) and the β-glucuronidase (GUS) genes. Putative transgenic calli were selected on 2g/l gellan gum-solidified Nitsch's medium (1969) containing 50 mg/l kanamycin and 20 g/l sucrose after co-cultivation with A. tumefaciens. Transformation frequency of the embryogenic calli evaluated by GUS histochemical assay was increased by the addition of acetosyringone to co-culture medium. Complete transgenic plants were selected among secondary embryos formed on the surface of embryos in the presence of kanamycin. Finally, kanamycin-resistant plants expressing GUS gene were obtained. PCR analysis confirmed their transgenic nature by detecting GUS and NPT II genes.

DOI： 10.5511/plantbiotechnology.15.29

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DOI： 10.5511/plantbiotechnology.15.213

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Bulblets of Lilium rubellum Baker were cultured for 16 weeks in a 20-, 30- or 40-ml liquid medium renewed 0, 1, or 3 times. The bulblets were assessed for growth and rot infection. 1. Bulblets with fresh weight of more than 600 mg were obtained by using a 20- or 30-ml medium with 3 renewals, or 40 ml with one renewal. 2. Bulblets frequently turned brown when they were cultured in a 30- or 40-ml medium with 3 renewals: they frequently rotted both during a cold treatment and after transplantation. 3. Bulblets cultured in a 20-ml medium did not rot after a cold treatment irrespective of the number of medium renewals; they grew well after transplantation to the greenhouse. 4. Bulblets cultured in a 20-ml medium with 3 renewals most frequently developed into plantlets with elongated axes 15 weeks after transplantation. These results indicate that the 20-ml medium with 3 renewals appears to be most suitable for bulblet culture of L. rubellum in a liquid medium.

DOI： 10.2503/jjshs.66.113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BUNDESANSTALT ZUCHTUNGS FORSCHUNG KULTURPFLANZEN  

Establishment of embryoenic cultures was examined for different tissue explants in 23 cultivars of grapevine (Vitis spp.). The explants were initially cultured on callus induction media (C media) for 2 months; and those producing calli were then transferred to an embryogenesis induction medium (E medium) containing 1 mu M 2,4-dichlorophenoxyacetic acid (2,4-D), on which adventitious embryos or embryogenic calli were induced 4 to 6 months after transfer. C media containing 10 mu M 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in combination with 10 mu M N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) or N-(1,2,3-thiadiazol-5-yl)-N'-phenylurea (TDZ) were suitable for inducing subsequent adventitious embryogenesis from leaf explants of V. vinifera Koshusanjaku. Adventitious embryogenesis was more efficiently induced from immature ovary explants than leaf and anther ones. Among 23 cultivars examined, embryogenic cultures, such as embryogenic calli or adventitious embryos proliferating via secondary embryogenesis, were established in 10 cultivars including V. vinifera Sekirei, Rosario Bianco, Semillon and Merlot, and V. x labruscana Delaware. These embryogenic cultures could be maintained without loosing a high regeneration capacity for over 20 months by subculturing onto fresh E medium. They could be useful as a target material for Agrobacterium- or particle gun-mediated genetic transformation.

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Bulblets of <I>Lilium rubellum</I> Baker were cultured for 16 weeks in a 20-, 30- or 40-ml liquid medium renewed 0, 1, or 3 times. The bulblets were assessed for growth and rot infection.<BR>1. Bulblets with fresh weight of more than 600 mg were obtained by using a 20- or 30-ml medium with 3 renewals, or 40 ml with one renewal.<BR>2. Bulblets frequently turned brown when they were cultured in a 30- or 40-ml medium with 3 renewals; they frequently rotted both during a cold treatment and after transplantation.<BR>3. Bulblets cultured in a 20-ml medium did not rot after a cold treatment irrespective of the number of medium renewals; they grew well after transplantation to the greenhouse.<BR>4. Bulblets cultured in a 20-ml medium with 3 renewals most frequently developed into plantlets with elongated axes 15 weeks after transplantation.<BR>These results indicate that the 20-ml medium with 3 renewals appears to be most suitable for bulblet culture of <I>L. rubellum</I> in a liquid medium.

DOI： 10.2503/jjshs.66.113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

A simple protocol is described for high frequency plant regeneration from protoplasts isolated from leaf-derived embryogenic calli of grapevine (Vitis vinifera L. cv. Koshusanjaku). The protoplasts successfully divided to form somatic embryos by culturing in gellan gum disc-method in which protoplasts were embedded in 2 g/l gellan gum-solidified Nitsch's medium containing 2.0 mg/l NAA, 0.5 mg/l BA, 0.09 M sucrose and 0.3 M glucose at a density of 1 x 10(5) protoplasts/ml. For the continuous growth of the colonies without browning, it was essential to add 0.3% (w/v) AC in the liquid reservoir medium from the beginning of the culture. In this culture condition, protoplasts started to divide after IO days of culture and grew into torpedo embryos 4 months after initiation of culture. The torpedo embryos thus obtained germinated normally by transferring onto 2 g/l gellan gum-solidified PGR-free Nitsch's medium containing 30 g/l sucrose. The regenerated plants were successfully transferred to the greenhouse and showed normal morphology. (C) 1997 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0168-9452(96)04557-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A system for producing haploid plants from anther cultures was developed for the Asiatic hybrid lily 'Connecticut King'. Anthers containing microspores at the mid- to late-uninucleate stages were cultured on MS media supplemented with various plant growth regulators. Microspores containing 3 or 4 vegetative-like nuclei were observed 2 to 3 weeks later, and yellowish nodular calluses appeared within dehisced anthers 2 to 3 months after culture. Picloram was superior to 2,4-D for inducing nodular calluses. Anthers from greenhouse-grown plants required higher concentrations of both picloram and cytokinins than those from field-grown plants and most frequently produced nodular calluses (17.6%) on MS medium containing 2 mg l(-1) picloram and 2 mg l(-1) zeatin. The nodular calluses regenerated many bulblets following transfer to MS medium supplemented with 0.1 or 0.5 mg l(-1) picloram and 0.01 mg l(-1) BA, and the bulblets developed into plantlets (bulblets with scaly leaves and roots) after transfer to MS medium containing 0.1 mg l(-1) NAA. Chromosome counts of root-tip cells of 11 plantlets revealed that five were haploids (2n = 12), two diploids (2n = 24), and four mixoploid. This result suggests that at least some plantlets were of gametophytic origin.

DOI： 10.1007/BF02318951

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Language：Japanese   Publisher：園芸学会  

The present study was conducted to examine whether pre-pollination is effective in improving growth of pollen tubes and seed-sets of <I>Lilium longiflorum</I> 'Georgia' by comparing cut-style pollination (CSP) and stigmatic pollination (SP).<BR>1. Pollen tubes reached the basal end of the style by 48 hr in self-pollination of 'Georgia' and cross-pollination of 'Georgia' × 'Hinomoto' by CSP. Using the pre-pollination technique in CSP enhanced the growth of pollen tubes both in self-and cross-pollination.<BR>2. Generative cell division of pollen tubes in the style was observed 24 hr after pollination. In cross-pollination by SP, 78% of the pollen tubes had 2 sperm nuclei and 1 vegetative nucleus ('2 S + V'), while only 15% of these appeared in the self-combination. Using CSP, pollen tubes with '2 S+ V' were 32%in cross-and 9%in self-pollination. The percentages increased to 54% and 35%, respectively, when pollen of 'Hinomoto' was used in the pre-pollination treatment.<BR>3. In SP, numerous pollen tubes which penetrated the style grew in a bunch between two rows of ovules in an ovary ; they reached the ovules near the mid-region of the loculi. In CSP, pollen tubes elongated poor

DOI： 10.2503/jjshs.65.135

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<I>Lilium regale</I> and <I>L. rubellum</I> were crossed reciprocally to introduce economically and horticulturally desirable traits of <I>L. regale</I>, such as vigorous growth and disease resistance, into <I>L. rubellum</I>.<BR>1. In <I>L. regale</I> ×<I>L. rubellum</I>, seeds containing an embryo were obtained with a low frequency (3.3 %) by stigmatic pollination at anthesis, but they did not germinate in soil or in vitro. Seedlings were successfully obtained with frequencies of 5.3 to 6.7 % by applying ovule culture 30 to 60 days after pollination.<BR>2. In <I>L. rubellum</I> ×<I>L. regale</I>, the growth of pollen tubes was inhibited in the style after stigmatic pollination at anthesis; hence, no ovule with an embryo was obtained. Growth of pollen tubes was promoted by stigmatic pollination when the pollination was done 2 to 5 days after anthesis. Embryos definitely formed if stigmatic pollination was carried out 5 days after anthesis, but they could not be rescued even when ovule culture was utilized. Cut-style pollination had no effect on production of ovules with an embryo.<BR>3. Hybridity of seedlings resulted from <I>L. regale</I> × <I>L. rubellum</I> wa

DOI： 10.2503/jjshs.64.919

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Verlag  

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5-10 mg/l and 0.1 mg/1 for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30-100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.

DOI： 10.1007/BF00232456

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Verlag  

Hypocotyl-derived protoplasts of Dianthus barbatas that had been pretreated with iodoacetamide were fused electrically with cell suspension culture-derived protoplasts of Gypsophila paniculata that could divide to form callus but could not regenerate shoots under the culture conditions used in this study. Electrofusion-derived calli which produced shoots were selected as putative somatic hybrids, and plantlets were subsequently regenerated from 2 of these selected calli. These plantlets, which in vitro produced flowers precociously, were identified as intergeneric somatic hybrids by nuclear ribosomal DNA analysis. Normal plants have not been established up to the present.

DOI： 10.1007/BF00223372

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Language：English   Publisher：日本植物細胞分子生物学会  

タカサゴユリ (<i>Lilium formosanum</i>) において, 幼胚からの実生獲得方法を確立するために, 4通りの胚救出方法 (胚培養, 胚珠培養, 胎座付き胚珠培養, 子房輪切り培養) を比較したところ, 胚珠培養, 胎座付き胚珠培養および子房輪切り培養を行った場合には, 自家受粉後10日目の幼胚からも実生を獲得することができた. 特に, 胎座付き胚珠培養は比較的簡便に行え, また外植片の汚染も少ないといった点から, 最も実用的であると考えられた.

DOI： 10.5511/plantbiotechnology1984.12.317

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

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 1(-1) TDZ in combination with 0.1 mg 1(-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.

DOI： 10.1007/BF00045085

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Language：English   Publisher：日本植物細胞分子生物学会  

Protoplast isolation, culture and shoot regeneration from protoplast-derived calli were compared among different organs of <i>Dianthus</i> species under the same conditions. Leaves, petals and seedling hypocotyls of <i>D. caryophyllus</i> cv. Chabaud (a seed-propagated cultivar) and <i>D. barbatus</i> were used as protoplast sources. Protoplasts isolated from petals of these two species showed only low yield and low division frequency. On the other hand, division frequency of hypocotyl protoplasts was higher than that of leaf protoplasts, although, in <i>D. caryophyllus</i>, protoplast yield in hypocotyls was slightly lower than in leaves. In both species, relatively high frequency of shoot regeneration was obtained in hypocotyl- and petal-derived protoplasts, while only low or no shoot regeneration occurred in leaf-derived protoplasts.

DOI： 10.5511/plantbiotechnology1984.12.62

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l(-1) 2,4-D and 2 g l(-1) casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1-3 x 10(7)/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l(-1) 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1 x 10(5)/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l(-1) each of NAA and BA for 2 months followed by 0.01 mg l(-1) NAA and 5 mg l(-1) BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.

DOI： 10.1007/BF00238593

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Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and alpha-naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 muM BA with or without 5 muM NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N'-phenylurea and N-1,2,3-thiadiazol-5-yl-N'-N'-phenyl-urea were more effective than BA, kinetin, N6-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a1 significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral, Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.

DOI： 10.1007/BF00048310

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Protoplasts isolated from leaf mesophyll cells of Dianthus chinensis and D. barbatus were fused by polyethylene glycol (PEG). Calli exhibiting vigorous growth were selected from the PEG-treated protoplasts and shoots were regenerated from one of these calli after 5 months of culture. These shoots readily rooted and continuously produced flowers in the in-vitro condition. The data on flower color, chromosome number, and esterase isozyme patterns indicated that this plantlet was an interspecific somatic hybrid. The hybridity of the plantlet was also confirmed by nuclear rDNA analysis. This report provides the possibility of applying the somatic hybridization technique for the genetic improvement of the genus Dianthus.

DOI： 10.1007/BF00223802

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Protoplasts isolated from cell suspension cultures of two caryophyllaceous species, Dianthus caryophyllus L. and Gypsophila paniculata L., were fused using polyethylene glycol and cultured in Murashige and Skoog (1 962) (MS) medium containing 1 mg l-1 picloram and 0.7 M sorbitol. Among the calli produced, one showing green coloration and vigorous growth was identified as an intergeneric somatic hybrid based on isozyme analyses for esterase and glutamate dehydrogenase. Hybridity of this callus was also confirmed by Southern hybridization analysis using a rice rDNA probe. This somatic hybrid callus regenerated roots after 2 months of culture on MS medium containing 1 mg l-1 picloram, but shoot regeneration was not observed on any of the media tested.

DOI： 10.1016/0304-4238(93)90133-B

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A range of antibiotics were screened for their ability to induce somatic embryogenesis from leaf explants in several Dianthus cultivars. Among the antibiotics tested, cefotaxime was most efficient for inducing somatic embryogenesis. Penicillin G and carbenicillin also induced somatic embryogenesis, but with lower frequencies. Auxins such as NAA, 2,4-D, and picloram had no effect on somatic embryogenesis. Somatic embryos transferred to a medium without both antibiotics and plant growth regulators developed into plantlets that could be grown to maturity following transfer to the green house. © 1993, Gustav Fischer Verlag, Stuttgart. All rights reserved.

DOI： 10.1016/S0176-1617(11)81581-6

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Language：English   Publisher：日本植物細胞分子生物学会  

DOI： 10.5511/plantbiotechnology1984.10.115

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Protoplasts isolated from mesophyll cells of Dianthus chinensis L. and Dianthus caryophyllus L. were fused by polyethylene glycol. Prior to fusion treatment, protoplasts of D. chinensis were inactivated with iodoacetamide to inhibit cell division. Protoplasts of D. caryophyllus divided to form callus under the culture conditions used in this study but no shoot regeneration was observed. Therefore, fusion-derived calli which regenerated shoots could be tentatively selected as somatic hybrid cell lines. Plants were regenerated from four cell lines, and three of them exhibited intermediate characteristics of both parents in plant height and flower morphology. Esterase isozyme analyses and chromosome counts indicated that they were interspecific somatic hybrid plants. The hybrid nature of these plants was also confirmed by random amplified polymorphic DNA analysis.

DOI： 10.1016/0168-9452(93)90092-E

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Seventeen cultivars belonging to the genus Dianthus were examined for protoplast isolation, culture and shoot regeneration under the same conditions. These included D. caryophyllus, D. chinensis, D. barbatus, D. plumarius, D. superbus and D. japonicus as well as interspecific hybrid cultivars (D. caryophyllus x D. chinensis and D. chinensis x D. barbatus). In all cultivars, viable protoplasts were isolated at high yields from leaves of axenic shoot cultures and some of these protoplasts divided and formed colonies. However, shoot regeneration frequencies were markedly different among the species. High frequency shoot regeneration was obtained from D. chinensis and interspecific hybrid cultivars, while only low frequency or no shoot regeneration was obtained from other species.

DOI： 10.1007/BF00235070

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Factors affecting shoot regeneration from hypocotyl segments of spinach (Spinacia oleracea L.) were investigated. When explants were cultured on medium containing 10 mg/l IAA for 7 weeks, 3 out of 9 cultivars showed relatively high shoot regeneration response (15 - 35%). The other PGRs tested had no effect on shoot regeneration. However, the transfer of explants from auxin-containing medium to auxin-free medium 20 d after culture induced shoot formation from explants cultured on media containing each of the auxin sources tested individually. By applying this short term auxin treatment, more than 80% shoot regeneration was obtained on medium containing 5 - 20 mg/l 5,6-Cl2-IAA, compared to less than 30% with 10 - 20 mg/l IAA treatment.

DOI： 10.1007/BF00235253

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Effects of 4 potentially selective agents for transformed cells, 3 antibiotics [kanamycin, geneticin (G418) and hygromycin] and bialaphos, as well as 2 antibiotics for eliminating Agrobacterium, carbenicillin and cefotaxime on growth and somatic embryogenesis of embryogenic calli of Muscari armeniacum cv. Blue Pearl were evaluated. Callus growth was completely inhibited by 75 mg dm-3 hygromycin or 4 mg dm-3 bialaphos, and somatic embryos were never produced on media containing 25 mg dm-3 hygromycin or 3 mg dm-3 bialaphos. Kanamycin and G418 less inhibited growth and somatic embryogenesis of the calli. On the contrary, carbenicillin and cefotaxime promoted both callus growth and somatic embryogenesis at all concentrations tested.

DOI： 10.1023/B:BIOP.0000023887.16716.f7

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Many monocots have two whorls of petaloid organs. To explain this floral morphology, modified ABC model was hypothesized that class B genes express in whorl 1 (W1), besides W2 and W3. This model was supported by the expression analysis of class B genes in tulip and agapanthus, but not in asparagus. Thus, applicability of this model in monocots remains unclear.<br>To inspect this model, we analyzed the tepal morphology and class B genes expression in alstroemeria, a monocot which has distinguishable outer and inner petaloid perianth. Outer and inner tepals differ in shapes but resemble in vascular bundles and epidermal cells. We isolated three class B genes (<I>AlsDEFa</I>, <I>AlsDEFb</I> and <I>AlsGLO</I>). <I>AlsDEFb</I> and <I>AlsGLO</I> were expressed in W1, W2 and W3, whereas <I>AlsDEFa</I> was detected only in W2 and W3. This supports modified ABC model and suggests that <I>AlsDEFa</I> expression may correlate with morphological difference of outer and inner tepals.

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap

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researchmap