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Nishihara M.,Iwate Biotechnology Research Center | Hikage T.,Hachimantai City Floricultural Research and Development Center | Yamada E.,Iwate Biotechnology Research Center | Nakatsuka T.,Iwate Biotechnology Research Center
Molecular Genetics and Genomics | Year: 2011

We investigated the genetic basis for the derivation of pink coloration in petals from blue flowers in cultivated gentians. Using a revertant blue-flower phenotype that arose spontaneously from a pink-flowered cultivar, we sought to elucidate the molecular mechanism of flower color restoration caused by a suppressor mutation. Detailed sequencing analysis identified three novel deficient flavonoid 3',5'-hydroxylase (F3'5'H) alleles in pinkflowered gentians in addition to two mutations identified previously (Nakatsuka et al. in Mol Genet Genomics 275:231-241, 2006). Among the deficient alleles, one allele that contained a novel miniature inverted-repeat transposable element (GtMITE1) insertion in an intron of F3'5'H was shown to cause missplicing, resulting in abnormal F3'5'H transcripts and the pink-flower phenotype. The other two mutations were identified as a singlenucleotide insertion and gypsy-Ty3 retrotransposon (Tgt1) insertion within exon 1 and exon 2 of the F3'5'H gene, respectively. The blue-flowered revertant mutant contained a single-nucleotide spontaneous mutation immediately 3' of the TAA target site duplication and the GtMITE1 insertion, which caused restoration of normal splicing of F3'5'H and the normal blue-flower phenotype. Transient expression assays in gentian flowers in vivo demonstrated that normal F3'5'H splicing pattern was recovered from missplicing induced by the GtMITE1 insertion by the single- nucleotide substitution. These findings extend our knowledge of genomic evolution by transposable elements and spontaneous mutations in Gentiana species of economic and medical importance. © Springer-Verlag 2011. Source

Doi H.,Iwate University | Hoshi N.,Iwate Agricultural Research Center | Yamada E.,Iwate Biotechnology Research Center | Yokoi S.,Iwate University | And 3 more authors.
Breeding Science | Year: 2013

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes. ©2013 by JAPANESE SOCIETY OF BREEDING. Source

Doi H.,Iwate University | Takahashi R.,Hachimantai City Floricultural Research and Development Center | Hikage T.,Hachimantai City Floricultural Research and Development Center | Takahata Y.,Iwate University
Plant Cell, Tissue and Organ Culture | Year: 2010

The overall goal of this study is to develop an anther culture system to produce doubled haploid (DH) lines of gentian (Gentiana triflora), an ornamental flowering plant, for use in an F1 hybrid breeding program. Embryogenesis was induced from anther cultures incubated on half-strength modified Lichter (NLN) medium containing a high concentration of sucrose (130 g/l) and subjected to heat shock treatment. Among the various parameters investigated, anthers collected from buds 9-12 mm in length induced the highest frequency of androgenesis. Moreover, among three genotypes tested, cvs. Ashiro-no-Aki and Ashiro-no-Natsu produced 21.3 and 3.7 embryos per 100 anthers, respectively, whereas, cv. Lovely-Ashiro failed to produce embryos. Among a total of 427 embryos transferred to a regeneration medium consisting of Murashige and Skoog (MS) medium, 138 plants were regenerated. The ploidy levels of regenerants were determined by flow cytometry and chromosome counts, revealing the presence of 5% haploids, 25% diploids, and 70% triploids. Inter simple sequence repeat (ISSR) analysis using the 6PS line obtained following self-pollination of the diploid plant obtained from anther culture confirmed that the diploid plant was indeed a DH. © 2010 Springer Science+Business Media B.V. Source

Doi H.,Iwate University | Yokoi S.,Iwate University | Hikage T.,Hachimantai City Floricultural Research and Development Center | Nishihara M.,Iwate Biotechnology Center | And 2 more authors.
Plant Cell Reports | Year: 2011

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0. 93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding. © 2011 Springer-Verlag. Source

Hikage T.,Iwate University | Hikage T.,Hachimantai City Floricultural Research and Development Center | Yamagishi N.,Iwate University | Takahashi Y.,Iwate University | And 3 more authors.
Molecular Genetics and Genomics | Year: 2015

Overwinter survival has to be under critical regulation in the lifecycle of herbaceous perennial plants. Gentians (Gentiana L.) maintain their perennial life style through producing dormant and freezing-tolerant overwinter buds (OWBs) to overcome cold winter. However, the mechanism acting on such an overwinter survival and the genes/proteins contributing to it have been poorly understood. Previously, we identified an OWB-enriched protein W14/15, a member of a group of α/β hydrolase fold superfamily that is implicated in regulation of hormonal action in plants. The W14/15 gene has more than ten variant types in Gentiana species. However, roles of the W14/15 gene in OWB survival and functional difference among those variants have been unclear. In the present study, we examined whether the W14/15 gene variants are involved in the mechanism acting on overwinter survival, by crossing experiments using cultivars carrying different W14/15 variant alleles and virus-induced gene silencing experiments. We found that particular types of the W14/15 variants (W15a types) contributed toward obtaining high ability of overwinter survival, while other types (W14b types) did not, or even interfered with the former type gene. This study demonstrates two findings; first, contribution of esterase genes to winter hardiness, and second, paired set or paired partner among the allelic variants determines the ability of overwinter survival. © 2015 Springer-Verlag Berlin Heidelberg Source

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