三吉 一光

<p>Flower color patterns are attractive traits of floricultural plants. However, the mechanisms underlying these traits remain mostly unknown. Carnation (Dianthus caryophyllus L.) and interspecific hybrids thereof exhibit many flower color patterns involving white margins on reddish petals, as observed in the cultivar 'Minerva'. Flowers with white margins also have white abaxial surfaces. We studied the factors regulating the formation of white margins and a white abaxial surface in flowers through analyses of pigments and related gene expression. HPLC analysis revealed an absence of anthocyanins in white margins, although the accumulation of other flavonoid pigments (flavonols) was almost identical between the dark-red central and white marginal regions of petals. RNA-seq analyses of the dark red central regions and white marginal regions of 'Minerva' petals resulted in the extraction of 18 genes related to anthocyanin biosynthesis and transportation, including some transcription factors, as candidate regulatory genes for the formation of white margins. Further analysis of the expression of these genes by real-time RT-PCR and a comparison of two white-margin-flowered cultivars and three red-unicolor-flowered cultivars indicated that the expressions of two bHLH transcription factor genes and seven structural genes were positively correlated with anthocyanin accumulation. Although DcbHLH1, which is a homolog of JAF13 in Petunia ×hybrida, was expressed in both flower color groups, DcbHLH2, a homolog of AN1, was expressed only in white-marginal-flowered cultivars. Moreover, in the petals of 'Minerva', the expression of those nine genes was repressed in the abaxial epidermal layer without red pigmentation conferred by anthocyanins. Therefore, we could postulate that the localized repression of both bHLH genes is involved in the formation of white margins in carnation petals by inducing the absence of anthocyanin synthesis and that the white abaxial surface of the petals may result from similar regulatory mechanisms. In particular, DcbHLH2 could act as a key gene because of its restricted expression only in cultivars with white-margined flowers.</p>

We examined the effects of five antimitotic agents using Antirrhinum majus L. ‘Maryland True Pink’ on the induction of adventitious shoots resulted in increase of frequencies of chromosome doubling without plant growth regulators. Seeds were treated in vitro with 0, 16.5, 32.9, 65.8, 131.6, or 263.2 µM oryzalin (ORY), amiprofos-methyl (APM), butamifos (BUT), or propham (IPC) or 800, 1,600, 3,200, 6,400, or 12,800 µM colchicine (COL) for 7 day. ORY, COL and APM promoted induction of adventitious shoots on the hypocotyls at maximum frequencies of 57.6% with 16.5 µM ORY, 5.6% with 800 µM COL and 88.8% with 131.6 µM APM. ORY and COL also induced adventitious shoots on the epicotyls adjacent to the cotyledons, particularly at high concentrations, with a maximum frequency of 26.0% at 12,800 µM COL. APM treatment increased frequencies of tetraploids from 0.0 to 93.1%, with a positive correlation between the frequency and concentration. By contrast, ORY and COL induced tetraploids at frequencies of 16.0 to 54.6% and 4.0 to 59.4%, respectively, with peaks at both low and high concentrations of each. Correlation analysis revealed that frequencies of adventitious shoot formation could be useful as an index for the induction of tetraploids. These results showed that three of the antimitotic agents tested induced both adventitious shoot and tetraploid without plant growth regulators, indicating that antimitotic action may play a common role in the induction of adventitious shoot.

Chromosome numbers of wild species in the genus Epidendrum have previously been reported in the range of 2n=24-240. However, the ploidy levels of cultivars in the given genus originating from interspecific hybridization among several wild species have never been reported. To elucidate their ploidy levels, we analyzed the chromosome number of cultivars and related wild species. Four wild species showed a new record of chromosome number in the present study, namely, E. radicans 2n=38, 80, E. secundum 2n=60, and E. cinnabarinum 2n=64. Six cultivars examined were revealed to have more chromosomes than the wild species, with a range of 2n=84-164, suggesting that the cultivars have high polyploid levels. A significant correlation between the nuclear DNA amount and the chromosome number in wild species as well as cultivars was observed. Those cultivars originated from hybridization. Thus, the results from the present study suggest that the polyploid cultivars in this genus could have resulted from pollination involving unreduced gametes during breeding.

Dendrobium Stardust ‘Fire Bird’ is an interspecific hybrid between D. unicum and D. Ukon ‘Arai’ and is known to be sterile and prevents further breeding. Chromosome doubling is an essential method to increase the size of plant organs, such as flowers, and restore fertility of interspecific hybrids, which enables further breeding for ornamental traits. To induce chromosome doubling, the protocorm-like bodies (PLBs) of a given variety were treated in vitro for 6 to 48 h with 10 to 160 mg L−1 amiprofos-methyl (APM). Higher APM concentrations resulted in lower rates of survival of treated PLBs. Furthermore, subsequent growth of PLBs was hampered with prolonged APM treatment, although 10 and 20 mg L−1 APM was optimal for doubling chromosomes. Plantlets with a swollen and shorter stem were observed after the APM treatment and these plants were also chromosome-doubled and became a distinctive morphological marker for the primary selection of chromosome-doubled plants. Chromosome-doubled plants of D. Stardust ‘Fire Bird’ will be utilized for the future breeding of unique Dendrobium cultivars.

Tetraploid plants of Antirrhinum majus L. were obtained from the seeds of the F1 variety ‘Maryland True Pink’ by amiprophos-methyl (APM) treatment and subsequent in vitro culture. Seeds were treated with 0, 10 and 20 mgl−1 APM for 1, 3, or 7 d (day) with or without osmopriming. Overall, the seedlings established from the seeds treated with 10 or 20 mgl−1 APM for 3–7 d exhibited malformation with rudimentary roots and swollen hypocotyls that eventually produced adventitious shoots. The highest adventitious shoot formation frequency reached 71.6% in non-primed seeds treated with 10 mgl−1 APM for 7 d; this decreased to 54.4% in osmoprimed seeds that received the same APM treatment. The plantlets from the adventitious shoots were identified as tetraploids at frequencies of 51.4% and 90.4%, respectively. Tetraploid frequency increased with higher concentration and/or longer duration of APM treatment and ranged from 0 to 65.9% in the non-primed seeds and 10.3% to 100% in the osmoprimed seeds. Osmopriming of seeds accelerated the induction of tetraploids by APM treatment at the same concentration and duration by 10.3–39.0%, except in the case of treatment with 20 mgl−1 APM for 3 d treatment. In the osmoprimed seeds treated with 20 mgl−1 APM for 7 d, all the plantlets from the adventitious shoots were revealed to be tetraploids. This study revealed that APM has dual effects, namely the inhibitory effect on the growth of seedlings and the stimulatory effect on the induction of adventitious shoots on the hypocotyl of seedlings.

We examined the effects of culture conditions, namely, illumination, temperature and the addition of 6-benzylaminopurine (BAP) to the germination medium, on the asymbiotic germination of mature seeds of Gastrodia pubilabiata Sawa, an achlorophyllous orchid. Mature seeds were harvested from plants at two sites, one in Toyama Prefecture and one in Chiba Prefecture, Japan. We used gellan gum-solidified New Dogashima medium as the basal medium. The maximum frequency of germination of the seeds in darkness was approximately 65%. BAP had no stimulatory effect on seed germination, but accelerated protocorm development. Necessary conditions for successful germination of seeds and subsequent development of seedlings from mature seeds of Gastrodia pubilabiata clarified in this study are prerequisites not only for conservation of biodiversity but also for the elucidation of the life cycle of this achlorophyllous orchid.

A cultivar of Catharanthus roseus, 'Jams 'N Jellies Blackberry', was investigated for its rare blackish flower color by comparing several factors affecting flower color with another ten C. roseus cultivars that had red to purple flower colors. Petals of 'Jams 'N Jellies Blackberry' accumulated only one novel anthocyanin, peonidin 3-robinobioside (Pn 3-rob), whereas those of all the other cultivars contained multiple anthocyanins. Moreover, although 'Jams 'N Jellies Blackberry' accumulated only one anthocyanin, Pn 3-rob, the total anthocyanin concentration was 2.5-fold higher in 'Jams 'N Jellies Blackberry' than in the cultivar with the second-highest anthocyanin concentration 'Equator Deep Apricot', which had orange red flowers. Microscopic observation of transverse sections of petals revealed that only 'Jams 'N Jellies Blackberry' exhibited aggregation of anthocyanins, namely anthocyanic vacuolar inclusions (AVIs), in the vacuoles of petal epidermal cells. Furthermore, the pH of petals of 'Jams 'N Jellies Blackberry' was approximately 6.0, which was significantly higher than that of all the other cultivars examined, which were in the range 5.0-5.6. Using purified Pn 3-rob and three other anthocyanins, 7-methylcyanidin 3-robinobioside, rosinidin 3-robinobioside, and hirstidin 3-robinobioside, which were the main anthocyanins accumulated in cultivars other than 'Jams 'N Jellies Blackberry', the color of their solutions was measured in vitro at pH values of 3.0, 5.0, and 6.0. Pn 3-rob did not exhibit the darkest color (lowest C-star and L-star) at all pH conditions investigated. However, at pH 5.0 and 6.0, Pn 3-rob recrystallized immediately after dissolution, and the solution retained the dark color even though slight discoloration occurred. The feature of aggregation of anthocyanins in the solution was almost identical to that in the AVIs observed in petal epidermal cells of 'Jams 'N Jellies Blackberry'. The color of the anthocyanin solution was darker at pH 6.0 than at pH 5.0 for all types of anthocyanin tested. Based on these results, we could postulate that multiple factors, such as the type and high concentration of anthocyanins, high pH of the petals, and formation of AVIs, could be involved in the development of the rare blackish flower color of 'Jams 'N Jellies Blackberry'. Further studies should be conducted to reveal the degree of contribution of each factor to blackish flower color using progenies of 'Jams 'N Jellies Blackberry' with various phenotypes and genotypes.

Among land plants, which generally exhibit autotrophy through photosynthesis, about 880 species are mycoheterotrophs, dependent on mycorrhizal fungi for their carbon supply. Shifts in nutritional mode from autotrophy to mycoheterotrophy are usually accompanied by evolution of various combinations of characters related to structure and physiology, e.g., loss of foliage leaves and roots, reduction in seed size, degradation of plastid genome, and changes in mycorrhizal association and pollination strategy. However, the patterns and processes involved in such alterations are generally unknown. Hybrids between autotrophic and mycoheterotrophic plants may provide a breakthrough in molecular studies on the evolution of mycoheterotrophy. We have produced the first hybrid between autotrophic and mycoheterotrophic plant species using the orchid group Cymbidium. The autotrophic Cymbidium ensifolium subsp. haematodes and mycoheterotrophic C. macrorhizon were artificially pollinated, and aseptic germination of the hybrid seeds obtained was promoted by sonication. In vitro flowering was observed five years after seed sowing. Development of foliage leaves, an important character for photosynthesis, segregated in the first generation; that is, some individuals only developed scale leaves on the rhizome and flowering stems. However, all of the flowering plants formed roots, which is identical to the maternal parent. © 2013 The Botanical Society of Japan and Springer Japan.

Three new acylated cyanidin 3,7-diglucosides (1-3) were isolated from the red-purple flowers of Sophronitis wittigiana as its main floral anthocyanins. These three pigments were based on cyanidin 3,7-diglucoside as the deacylanthocyanin, and their structures were determined to be cyanidin 3-O-[6-O-(malonyl)-β-glucopyranoside)]- 7-O-[6-O-(trans-caffeoyl)-β-glucopyranoside] as pigment 1, its demalonyl anthocyanin as pigment 2, and cyanidin 3-O-[6-O-(malonyl)-glucoside]-7-O-glucoside as pigment 3 by chemical and spectroscopic methods. On the other hand, five known acylated cyanidin 3,7,3'-triglucosides (4-8) were isolated from orange-red or red flowers of S. acuensis, S. brevipedunculata, S. cernua, S. coccinea var. xanthoglossa, and S. grandiflora as well as those of S. coccinea, and identified to be pigment 4 as Sophronitis coccinea anthocyanin 1 (SCA 1), pigment 5 as SCA 2, pigment 6 as SCA 3, pigment 7 as SCA 4, and pigment 8 as SCA 5 in comparison of the TLC, HPLC, and UVVis properties with standard samples of SCAs 1-5. These results showed that the three 3,7,3'-O-hydroxy groups of anthocyanins were all substituted with acylglucose and/or glucose residues in the orange-red or red flowers of six taxa of Sophronitis, whereas the 3'-O-hydroxy group was free from glucose in the red-purple flowers of S. wittigiana. Thus, the inactivation of 3'-O-glycosylation in cyanidin units might be involved in causing the blue color direction shift from orange-red or red to red-purple flower color of S. wittigiana. © 2014 The Japanese Society for Horticultural Science (JSHS), All right reserved.

Premise of the study: Mycoheterotrophic plants, which completely depend upon mycorrhizal fungi for their nutrient supply, have unusual associations with fungal partners. The processes involved in shifts in fungal associations during cladogenesis of plant partners from autotrophy to mycoheterotrophy have not been demonstrated using a robust phylogenetic framework.Methods: Consequences of a mycorrhizal shift were examined in Cymbidium (Orchidaceae) using achlorophyllous and sister chlorophyllous species. Fungal associates of the two achlorophyllous mycoheterotrophs (C. macrorhizon and C. aberrans), their close relatives, the chlorophyllous mixotrophs (C. goeringii and C. lancifolium) and an outgroup, the chlorophyllous autotroph C. dayanum, were identified by internal transcribed spacers of the nuclear ribosomal DNA sequences.Key results: Molecular identification of mycorrhizal fungi revealed: (1) the outgroup autotroph is predominantly dependent on saprobic Tulasnellaceae, (2) the mixotrophs associate with the Tulasnellaceae and ectomycorrhizal groups including the Sebacinales, Russulaceae, Thelephoraceae and Clavulinaceae, and (3) the two mycoheterotrophs are mostly specialized with ectomycorrhizal Sebacinales.Conclusion: Fungal partners in Cymbidium have shifted from saprobic to ectomycorrhizal fungi via a phase of coexistence of both nutritional types of fungi. These three phases correspond to the evolution from autotrophy to mycoheterotrophy via mixotrophy in Cymbidium We demonstrate that shifts in mycorrhizal fungi correlate with the evolution of nutritional modes in plants. Furthermore, gradual shifts in fungal partners through a phase of coexistence of different types of mycobionts may play a crucial role in the evolution of mycoheterotrophic plants. © 2012 Botanical Society of America.

The effect of ionic composition in medium on proliferation of shoot tip-derived PLBs of two Calanthe species, C. aristulifera and C. striata, was investigated by using the media with 12 different ionic compositions, which were systematically modified according to the method of Ichihashi (1992) based on the composition of modified Gamborg's B5 medium. These two species showed almost the same growth response to the ionic compositions and the growth of PLBs was inhibited on media with higher ratios of NH4+ and Ca2+, but promoted with higher ratios of both K+ and H2PO4-. Based on the results of PLB growth, optimum cationic compositions calculated for C. aristulifera and C. striata were NH4+ : K+ : Ca2+ : Mg2+ = 10.4 : 70.0 : 9.6 : 10.0 and 10.9 : 68.1 : 11.0 : 10.0, respectively, whereas optimum anionic compositions were NO3-: H2PO4-: SO42- = 42.1 : 43.7 : 14.2 and 43.0 : 42.9 : 14.1, respectively. The percentages of NO3- and H2PO4- in the optimum medium thus obtained were about half and 12-hold of the control B5 medium, respectively. Among the 100 micropropagated C. striata plants examined, no obvious variation in flower shape and color were observed except two plants which had larger and thicker flowers than original plants. Those two variants were confirmed to be chromosome doubled tetraploids by flow cytometric analysis.

Epiphytes begin their life history with seed germination on trees and live on the trees throughout the lives. In contrast, the seeds of terrestrial plants germinate on the ground. To clarify differences in early life history between epiphytes and terrestrial species, we compared the responses of in vitro asymbiotic seed germination and protocorm formation to various light intensities and temperatures between the epiphyte Liparis fujisanensis and two closely related terrestrial species, Liparis koreojaponica and Liparis kumokiri (Orchidaceae). Epiphytic germination was higher at low temperature and under high light intensity than that of the terrestrial species. Protocorms were formed only in the dark in the three species. Field measurements of temperature and photosynthetically active radiation in six natural habitats of the species from 2005 to 2006 showed that the habitats of the epiphyte were exposed to higher light intensities than those of the terrestrial species. The high germination capability of epiphyte seeds in wider ranges of light intensity and temperature may be beneficial to its seedling growth in the early growing season in exposed aboveground habitats.

The effects of different light wavelengths on in vitro asymbiotic germination of mature seeds and seedling growth of Bletilla ochracea Schltr. were examined using five wavelengths from light emitting diodes with peak wavelengths of 470 nm (blue), 525 nm (green), 590 nm (orange), 625 nm (red), and from white light emitting diodes at 40 mu mol m(-2) s(-1). Gellan gum solidified New Dogashima medium without plant growth regulators was used as germination medium. Cultures were maintained at 25 C under 24 h continuous lighting or darkness. The frequencies of seed germination three weeks after sowing reached more than 60% in all light conditions examined including continuous darkness. The highest frequencies of seed germination of 74% were achieved using lighting both with green and orange light emitting diodes, though strong inhibition of seed germination by the specific wavelength was not recognized. Seedling growth of this species was greatly inhibited by darkness. The most effective wavelength of light for rhizoid formation was revealed to be in the range of 590 nm (orange light) and 625 nm (red light), and almost no rhizoid was formed in the darkness. After 3 months of culture, leave width was expanded under white and blue light emitting diodes and was narrower under green, orange and red light. Seedlings grown under white and blue light emitting diodes resulted in thicker pseudobulbs.

The mature seeds of 7 Cymbidium species, including those from different climatic regions (temperate or tropical), with different life-forms (terrestrial or epiphytic), and different nutritional modes (autotrophic or mycoheterotrophic) were cryopreserved by vitrification to demonstrate the effectiveness of this method for germplasm preservation. The period of exposure to vitrification solution was initially optimized for seeds of 3 species, and the optimum periods were found to be 60 min for C. goeringii and C. macrorhizon seeds and 30 min for C. finlaysonianum seeds. Cryopreservation under optimum conditions did not have any deleterious effect on the seeds, and the germination rates of the cryopreserved seeds were as follows (these rates were almost identical to those of untreated control seeds): 32% for C. goeringii, 82% for C. macrorhizon, and 76% for C. finlaysonianum. The cryopreservation method was also successfully applied to the seeds of other members of the genus Cymbidium, although the period of exposure to the vitrification solution varied with life-form. The periods were optimized to 60 and 30 min for the terrestrial and epiphytic species, respectively. These results suggest that cryopreservation by vitrification has wide applicability for the seeds of several orchid species with varying ecological and physiological adaptations.

The effects of different light wavelengths on in vitro asymbiotic germination of mature seeds and seedling growth of Bletilla ochracea Schltr. were examined using five wavelengths from light emitting diodes with peak wavelengths of 470 nm (blue), 525 nm (green), 590 nm (orange), 625 nm (red), and from white light emitting diodes at 40μ μmolm -2 s -1. Gellan gum solidified New Dogashima medium without plant growth regulators was used as germination medium. Cultures were maintained at 25°C under 24 h continuous lighting or darkness. The frequencies of seed germination three weeks after sowing reached more than 60% in all light conditions examined including continuous darkness. The highest frequencies of seed germination of 74% were achieved using lighting both with green and orange light emitting diodes, though strong inhibition of seed germination by the specific wavelength was not recognized. Seedling growth of this species was greatly inhibited by darkness. The most effective wavelength of light for rhizoid formation was revealed to be in the range of 590 nm (orange light) and 625 nm (red light), and almost no rhizoid was formed in the darkness. After 3 months of culture, leave width was expanded under white and blue light emitting diodes and was narrower under green, orange and red light. Seedlings grown under white and blue light emitting diodes resulted in thicker pseudobulbs. © 2011 The Japanese Society for Plant Cell and Molecular Biology.

The effects of culture conditions on the asymbiotic germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial cool-climate orchid, were examined. Specifically, conditions such as illumination, temperature, and the addition of plant growth regulators to the medium were studied. Mature seeds were harvested from plants that had been collected in Toyama Prefecture, Japan, and maintained at the Botanic Gardens of Toyama. Solidified "New Dogashima" medium was used as the basal medium, and it was supplemented with 6-benzyladenopurine (BA) or alpha-naphthalene acetic acid (NAA). White light at 40 A mu mol m(-2) s(-1), with a 16-h photoperiod, inhibited the germination of seeds by 53-80%, as compared to dark controls in genotypes examined. The optimal temperature for the germination of seeds in darkness was 20A degrees C and the germination frequency reached 60%, whereas it was only 28% at 25A degrees C. While both NAA and BA stimulated germination, BA was more effective than NAA. After storage for 18 mo at 5A degrees C, seeds incubated on medium that contained 0.2 mg l(-1) BA germinated at a frequency of 36%, which was twice that of seeds grown without any plant growth regulators. The frequency of subsequent germination decreased during storage of seeds at 5A degrees C for approximately 2 yr, dropping from 61% to 13%. The protocorms obtained in this study were developed to plantlets readily after transferring to fresh 1/2 MS medium without any plant growth regulators. They were successfully acclimatized in green house after two to three subcultures in vitro. The significant role of a reproducible protocol for the germination of mature seeds is discussed in terms of the ex situ conservation of endangered orchid species.

Orchid seed capsules typically contain several thousand seeds, but obstacles to germination and development greatly constrain the establishment of new plants. This study compares the germination response of orchid seeds from the same capsule in situ and in vitro to assess the potential of soil conditions to influence recruitment. Mature capsules of the endangered Nervilia nipponica, a terrestrial orchid endemic to Japan, were harvested from two widely separated populations. On average, over 96% of seeds from all capsules contained an embryo. 2,3,5-Triphenyl tetrazolium chloride stainability was high and, when sown in vitro and cultured for 16 weeks asymbiotically, 76-99% of seeds from all but two capsules germinated and developed rapidly. However, when buried in seed packets for 7 months at the site of the source populations, germination was spatially variable and ranged from 0% to 12% per capsule. Embryo loss in buried seed was extensive, and germination was not significantly greater after a further 14 months of incubation. These results indicate that the window for successful germination under field conditions is short, and that recruitment is limited by microsite. Conservation efforts should focus on habitat management to promote conditions conducive to seed germination, coupled with ex situ propagation. © 2010 The Linnean Society of London.

We used a flow cytometer to investigate the variations in endopolyploidy (the frequencies of nuclei with DNA contents equivalent to 4C through 16C) during the short period of the early growing stage in vigorously growing young tissues of maize seedlings. We examined different portions of the root and leaves that had been growing for 7 (day 7) and 13 (day 13) days after germination. Endoreplication showed two opposing phenomena without aging. In one case, the endopolyploidy of the first leaf was higher on day 13 than on day 7. In the latter case, endopolyploidy decreased, as clearly revealed by a comparison of the endopolyploidy of the second leaves and the 160-170 mm portion of the seminal root on days 7 and 13. Endopolyploidy was also lower in the top of the leaf. In roots, endopolyploidy was increased by the exogenous application of abscisic acid for only 1 day. The levels of endopolyploidy increased without an increase in cell size in the roots. These results showed that endoreplication occurs in actively growing and young tissue and that the variation can be induced in the short period examined. © 2010 Korean Society for Plant Biotechnology and Springer.

A novel acylated anthocyanin was identified from the violet flowers of Bletilla striata 'Murasaki Shikibu' as amajor floral anthocyanin. This pigment was based on cyanidin 3,7-diglucoside as its deacylanthocyanin, and wasacylated with two molecules of caffeic acid. The structure was unambiguously elucidated by chemical andspectroscopic methods to be cyanidin 3-O-(β-glucopyranoside)-7-O-[6-O-(4-O-(6-O-(4-O-(β-glucopyranosyl)-trans-caffeoyl)-β-glucopyranosyl)-trans-caffeoyl)-β-glucopyranoside]. Based on the results of the present study,the bluing effect was discussed for the violet flowers of a given cultivar.

The effect of nitrous oxide gas (N2O) treatment on the induction of 2n male gametes and 4n embryo in Lilium × formolongi hort. was examined. Lilium × formolongi hort. is an artificial hybrid between Lilium longiflorum Thunb. and Lilium formosanum Wall. In general the members in the genus Lilium are propagated vegetatively. However, Lilium × formolongi hort. is exceptionally propagated by seeds and blooms 6-8 months after sowing. Seeds of Lilium × formolongi hort. cultivars 'Raizan' was sown in a plastic tray with 200 holes on 5 February 2008 and seedlings bearing 2-3 leaves were transplanted in 10.5 cm (in diameter) pots on 5 May, grown under ambient conditions in a green house. When the first flower bud of each plant were reached 15-30 mm in length, these buds were sampled consecutively and observed by the aceto-carmine squash method to determine the meiotic stage of pollen mother cells. The buds ranging from 19-23 mm in length contained pollen grains at prophase I to tetrad. Immediately after the staging of pollen grains, plants with the buds in the same length were treated with N2O gas for 48h at 6 atm at room temperature. The diameter of mature pollen grains in metaphase, obtained from the plants with buds of 22 mm in length at the time of exposure to N 2O gas, ranged from 76 to 169 μm, although those from untreated ones ranged from 62 to 95 μm. For the induction of 4n embryo, plants were treated with N2O gas for 72h at 6 atm 13 days after the pollination. Plants with pollinated ovaries grew visually normal after the treatment with N2O gas and these capsules were harvested 75-85 days after pollination. We obtained many plantlets from these capsules. Of 49 randomly selected seedlings, 22 were revealed to be tetraploid by flow cytometry. We could demonstrate that N2O gas treatment was useful for the manipulation of both male gamete as well as zygotic embryo for breeding Lilium × formolongi on polyploid level.

In this study we established reliable methods for conservation of seeds of Phaius tankervilleae as an orchid genetic resource. The seeds, which were dehydrated to 5% water content and preserved at 4°C, showed no decrease in viability and germinability after three months. After storage for six months, however, the seeds showed a drastic decrease in germinability, even though survival rate was high. For long-term preservation of seeds of P. tankervilleae, cryopreservation is applied to the freshly harvested seeds. When the seeds were cryopreserved by the vitrification method for up to 12 months there was no apparent deterioration effect of storage time. These results indicate that cryopreservation by the vitrification method is useful for long-term conservation of P. tankervilleae seeds, which are difficult to preserve for more than three months under dry and low-temperature conditions. © 2008 Korean Society for Plant Biotechnology and Springer.

A flexuous virus was isolated from cultivated Phaius flavus Lindl. plants in Japan with a latent infection. The virus was assigned to the genus Potexvirus based on morphology and analysis of its complete nucleotide sequence. The genome is 5,816 nucleotides in length, excluding the 3′-terminal poly (A) tail, and contains five open reading frames (ORFs), which is consistent with other members of the genus Potexvirus. The ORF nucleotide sequences differ from those of previously reported potexviruses, but the newly isolated virus is closely related to lily virus X and mint virus X. We propose that this virus should be designated as Phaius virus X (PhaVX). © 2007 Springer-Verlag.

The Asiatic hybrid lily cultivar 'Akita Petit White' (APW) is male-sterile with antherless stamens. This trait is advantageous for florists, who otherwise have to remove anthers carefully after the flowers have bloomed. However, unexpectedly, we observed one or two stamens with intact anthers in a few flowers, on fewer than 1% of a total of 400 plants in a field trial of this cultivar, in a horticultural setting. The trial was performed with bulbs that had been stored at -2°C for 9 months. The purpose of the present study was to identify those factors that regulate the expression and reversal of the antherless phenotype in APW lilies. We grew APW plants, from bulbs that had been stored frozen, under three temperature regimes in growth cabinets. Temperature conditions were: high 32°C/25°C (day/night), moderate 25°C/18°C, or low 18°C/H°C; all with a 14 h photoperiod. All stamens in plants which had been grown under the high temperature conditions had intact anthers. Under the moderate temperature conditions, the edges of all filaments, in all plants examined, were yellowish and swollen, resembling undeveloped anthers. Under the low temperature conditions, the antherless phenotype was stable, and no stamens had intact or undeveloped anthers. Pollen from intact anthers, from plants grown under the high temperature conditions, germinated in vitro and produced vegetative and generative nuclei in the normal manner. The yields of viable seeds produced after pollination with these pollen grains were almost equal to those of other Asiatic hybrid lilies (96 - 113 seeds per capsule were obtained with various hybrid combinations). Regulation of the antherless phenotype by temperature is discussed.

Although coevolution is acknowledged to occur in nature, coevolutionary patterns in symbioses not involving species-to-species relationships are poorly understood. Mycorrhizal plants are thought to be too generalist to coevolve with their symbiotic fungi; yet some plants, including some orchids, exhibit strikingly narrow mycorrhizal specificity. Here, we assess the evolutionary history of mycorrhizal specificity in the lady's slipper orchid genus, Cypripedium. We sampled 90 populations of 15 taxa across three continents, using DNA methods to identify fungal symbionts and quantify mycorrhizal specificity. We assessed phylogenetic relationships among sampled Cypripedium taxa, onto which we mapped mycorrhizal specificity. Cypripedium taxa associated almost exclusively with fungi within family Tulasnellaceae. Ancestral specificity appears to have been narrow, followed by a broadening after the divergence of C. debile. Specificity then narrowed, resulting in strikingly narrow specificity in most of the taxa in this study, with no taxon rewidening to the same extant as basal members of the genus. Sympatric taxa generally associated with different sets of fungi, and most clades of Cypripedium-mycorrhizal fungi were found throughout much of the northern hemisphere, suggesting that these evolutionary patterns in specificity are not the result of biogeographic lack of opportunity to associate with potential partners. Mycorrhizal specificity in genus Cypripedium appears to be an evolvable trait, and associations with particular fungi are phylogenetically conserved. © 2007 The Author(s).

Leaf samples of endangered endemic orchid Calanthe izu-insularis Ohwi & Satomi collected at the time of full-bloom from two natural habitats on Mikurajima Island on (33 degrees 50 ' N, 139 degrees 37 ' E), similar to 200 km south of Tokyo, Japan, were examined for nine species of virus. Cucumber mosaic virus (CMV) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and hybridisation in 4 of the 104 leaf samples from different plants of C. izu-insularis. Five plants were removed from their natural habitat and cultivated in a greenhouse. Concentrations of detectable CMV in these plants increased and CMV was detected in all five plants after 2 weeks of cultivation. However, after an additional 10 weeks of cultivation, CMV was not detected in any of the plants. Thus, it seems possible that almost all of the plants of this species in their natural habitat might harbour CMV at concentrations that are lower than the limit of detection by RT-PCR and hybridisation. The importance of these results for the conservation in situ is discussed.

Background and Aims Many Orchidaceous species are threatened globally by development and over-collection from their natural habitats for horticultural purposes. Artificial propagation from seeds is difficult in most terrestrial orchids native to temperate regions. Seed production is another limiting factor in the artificial propagation for these species because of the lessened probability of pollination and the destruction of fruit by insect larvae. Members of the genus Cephalanthera are distributed across Europe, Asia and North America. C. falcata is a temperate species of East Asia and an endangered species in Japan. As successful propagation from seeds of this species has never been reported, a reproducible method is described here for seed production in situ and propagation using immature seeds in asymbiotic culture in vitro.Methods Effects of hand-pollination and bagging treatment of ovaries were examined. Young capsules were collected every 10 d from 50 d after pollination until 120 d after pollination. Immature seeds obtained from these capsules were cultured asymbiotically on modified Kano medium and ND medium. Seed viability was examined within TTC (2,3,5-triphenyl tetrazolium chloride) test solution and histological observations were made on viable seeds by paraffin embedding at each collection stage.Key Results and Conclusions Hand-pollination followed by bagging treatment of ovaries with aluminium foil was effective for insect control during fruit development, and successfully yielded capsules. Of the capsules, 74.5 % survived to full maturity. The highest frequency (39.8 %) of seed germination was obtained with seeds harvested 70 d after pollination. The frequency declined with progress of seed maturity on the mother plant. Minimal germination was observed with seeds harvested 100 d or later after pollination. Histological observation suggests that accumulation of such substances as lignin in the inner integument surrounding the embryo during seed maturation plays an important role in induction of dormancy.

We examined the thermosensitivity of the restoration of stamens and genotypic differences in the formation of aberrant filaments and pistils in three male-sterile cultivars of Asiatic hybrid lily (Lilium sp.), namely, 'Akita Petit Lemon' (APL), 'Akita Petit Gold' (APG), and 'Akita Petit Cream' (APC). When cultivated in the traditional manner in early summer, APC had immature indehiscent anthers that contained no pollen grains, while the other two cultivars had antherless stamens. In a field trial of these cultivars, designed for harvesting of cut flowers in autumn, APC with intact anthers and APL and APG with immature anthers were unexpectedly observed in less than 1% of 400 plants in each case. Plants were then grown, from bulbs, under three temperature regimes in a set of growth cabinets, as follows: under high-temperature conditions (HT), at 32/25°C; under moderate-temperature conditions (MT), at 25/18°C; and under low-temperature conditions (LT), at 18/11°C. The restoration of stamens in APL, APG and APC was achieved under high-temperature conditions and the extent of restoration was dependent on temperature. The thermosensitivity of APL and APG with respect to the restoration of stamens was similar. Both cultivars had antherless stamens under LT and restored stamens with immature anthers under MT. Under HT, there was an obvious difference between these two cultivars. Both APL and APG developed intact anthers; however, those of APL contained viable pollen grains while those of APG did not. The thermosensitivity for restoration of stamens in APC was somewhat different from that in APL and APG APC had immature anthers even under LT but it had intact anthers with viable pollen grains under both MT and HT. Homeotic changes in floral components, such as transformation of filaments into tepals in APL and APC and split stigmas in APL were observed. The possible role of temperature in the restoration of stamens and genotypic differences in terms of the formation of aberrant filaments and pistils are discussed.

The effects of incubation temperature on germination of intact and dehusked japonica rice that had been harvested every 5 days from 10 days after anthesis (DAA) to 70 DAA were surveyed. At 30°C incubation, the germination percentage of intact seeds at 25 DAA exceeded that of dehusked seeds. This tendency persisted until 65 DAA, when around 95% of both intact and dehusked seeds germinated. Germination percentages of intact seeds incubated at 20°C were lower than seeds incubated at 30°C for all DAA. However, germination percentages of dehusked seeds incubated at 20°C were consistently higher than at 30°C. Germination percentages of intact and dehusked seeds were similar at 20°C incubation. Germination of intact seeds under 34/11°C (16/8 h) was higher than at 30°C. In contrast, germination of dehusked seeds under 34/11°C (16/8 h) was lower than at 30°C. Inhibitory effects of removing husk on germination were observed from 15 to 70 DAA. Almost all of the intact seeds at 45 DAA germinated when imbibed 35, 30 and 25°C, and germination percentages decreased with decreasing incubation temperatures from 20 to 15°C. By contrast, germination percentage of dehusked seeds was highest in 20°C incubation temperature; further increase or decrease of incubation temperature reduced germination percentage. The optimum germination temperature of intact and dehusked seeds of japonica rice differs during seed formation, such that the extent of germination inhibition by husk removal depends on the incubation temperature. © Springer 2005.

A new diacylated 8-C-glucosylanthocyanin was isolated from the purple flowers of Tricyrtis formosana 'Fujimusume' as one of the major anthocyanins along with four known pigments. The structure of this pigment was determined to be 8-C-(6-O-trans-sinapoyl)-β-glucopyranosylcyanidin 3-O-(6-O-malonyl- β-glucopyranoside) by chemical and spectroscopic methods. In addition, four known pigments, 8-C-glucosylcyanidin 3-malonylglucoside, cyanidin 3-glucoside, cyanidin 3-rutinoside and cyanidin 3-malonylglucoside, were identified as the major anthocyanins in the flowers. © 2004 Pharmaceutical Society of Japan.

A novel C-glycosylanthocyanin was isolated from the flowers of Tricyrtis formosana cultivar Fujimusume, and its structure was elucidated to be 8-C-β-D-glucopyranosylcyanidin 3-O-(6-O-malonyl)-β-D-glucopyranoside by chemical and spectroscopic methods. © 2003 Elsevier Ltd. All rights reserved.

We studied the effects of various treatments given before or at inoculation with Agrobacterium tumefaciens strain EHA105 (pIG121Hm) on the transient GUS gene expression in leaf segments of Saintpaulia ionantha Wendl. Sonication and vacuum infiltration treatments in the presence of Agrobacterium had no positive effect on GUS expression. In contrast, the explants cultured for 3 to 5 weeks on shoot induction medium containing 0. 5 mgr1 NAA and 0.5 mg1-1 BA, prior to Agrobacterium inoculation markedly increased transient GUS expression. The addition of acetosy-ringone to the co-cultivation medium enhanced the GUS expression. A similar enhancing effect was observed in all of the 4 cultivars examined, suggesting the wide applicability of the pre-culture treatment. © 2002, Japanese Society for Plant Cell and Molecular Biology. All rights reserved.