Papers - KUNITAKE Hisato
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Setoguchi Y., Fukutome H., Narasako Y., Hirano T., Otani M., Takeshita M., Kunitake H.
Journal of the American Society for Horticultural Science 150 ( 3 ) 159 - 167 2025.5
Authorship：Last author, Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of the American Society for Horticultural Science
Foot rot (Diaporthe destruens) of sweetpotato generally infects the stem of the plant and then spreads throughout the plant. In Japan, foot rot is prevalent in the main area of sweetpotato production, and there is a pressing need for the development of resistant cultivars. It is generally believed that polyphenols are involved in defense against stress. However, little is known about the relationship between polyphenols and the susceptibility to foot rot in sweetpotato. We thus analyzed the polyphenol of the disease-free healthy stems and investigated their relationship between susceptibility to the disease to establish basic techniques for selecting cultivars resistant to foot rot. Polyphenol was analyzed by the Folin–Ciocalteu method and high-performance liquid chromatography. Resistance tests were conducted using the direct inoculation method. We observed a correlation between the total polyphenol content and the degree of foot rot disease severity. Surprisingly, cultivars with a low stem polyphenol content (‘Tamaakane’) had lower susceptibility to foot rot, whereas the cultivars with high stem polyphenol content (‘Kokei No. 14’) had higher susceptibility to foot rot. In particular, high positive correlations were shown with several polyphenols, including 3,4-dicaffeoylquinic acid and chlorogenic acid. Furthermore, the results of in vitro experiments also suggested that foot rot may metabolize trace amounts of chlorogenic acid. This discovery has potential applications in the selection of resistant cultivars, contributing to the streamlining of disease resistance tests in breeding programs. It could ultimately shorten the breeding period and help overcome foot rot disease in sweetpotato.
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Seasonal Variation and Mean Degree of Polymerization of Proanthocyanidin in Leaves and Branches of Rabbiteye Blueberry (Vaccinium virgatum Aiton) Reviewed
Yasuko Koga, Yuno Setoguchi, Kazuhiro Sugamoto, Yo Goto, Tomonari Hirano and Hisato Kunitake
Plants 13 1864 2024.7
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
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Effects of fertilization of male gametes with heavy-ion beam irradiation on embryo and endosperm development in <i>Cyrtanthus mackenii</i> Reviewed
Shii Makiko, Kajiya Yuki, Murata Muneaki, Abe Tomoko, Kunitake Hisato, Hirano Tomonari
CYTOLOGIA 89 ( 2 ) 133 - 139 2024.6
Language：English Publishing type：Research paper (scientific journal) Publisher：公益財団法人 日本メンデル協会
Heavy-ion beams are widely used for mutagenesis. The type and size of the induced mutations vary depending on the ion species and velocity. DNA damage response of male gametes during pollen tube growth has been investigated using heavy-ion beam irradiation to the pollen of <i>Cyrtanths mackerii</i> (Amaryllidaceae), indicating that DNA damage induced by argon-ion beam is more difficult to repair than that by carbon-ion beam. In this study, we investigated the effects of DNA damage or mutations in the male gametes with argon-ion irradiation on double fertilization and subsequent embryo and endosperm development and compared the results with carbon-ion irradiation. In immature seeds after pollination with argon-ion-irradiated pollen grains at 10 Gy, there were two types of embryo sacs with embryo and endosperm and with egg cell/zygote and endosperm. The proportion of embryo sacs in the latter type was higher when using argon-ion-irradiated pollen than carbon-ion-irradiated pollen at 40 Gy, suggesting that qualitative differences between the two kinds of irradiation influenced embryo development. In many endosperms after pollination with the irradiated pollen, abnormal chromosome separation and enlarged endosperm nuclei were observed. This indicated that the chromosomal abnormalities in the irradiated male gametes were transmitted to the endosperm nuclei. The enlarged nucleus formation was higher in pollen irradiated with an argon-ion beam than in that with a carbon-ion beam. In conclusion, argon-ion beams, even at low doses, induce distinctive development of embryo and endosperm, making them valuable for investigating double fertilization.
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Changes in Carotenoids and Polyphenols during the Growth Stages of Orange-Fleshed Sweet Potato (Ipomoea batatas (L.) Lam.) Reviewed
Yuno Setoguchi 1, Yosuke Narasako, Tomonari Hirano, Motoyasu Otani and Hisato Kunitake 3
Horticulturae 10 629 2024.6
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
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Distinctive development of embryo and endosperm caused by male gametes irradiated with carbon-ion beam. Reviewed
Hirano T, Murata M, Watarikawa Y, Hoshino Y, Abe T, Kunitake H
Plant reproduction 2024.2
Authorship：Last author Language：English Publishing type：Research paper (scientific journal)
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Effect of Growth Stages on Anthocyanins and Polyphenols in the Root System of Sweet Potato. Reviewed
Setoguchi Y, Nakagawa S, Ohmura R, Toshima S, Park H, Narasako Y, Hirano T, Otani M, Kunitake H
Plants (Basel, Switzerland) 12 ( 9 ) 2023.5
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
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Blueberry Stem Extract Prevents Lacrimal Hyposecretion in Non-obese Diabetic Mice via Activation of AMPK. Reviewed
Ogawa K, Ohno Y, Tagashira A, Urata K, Satoh K, Fujimoto N, Sonoda H, Ikeda M, Matsuzaki T, Nishiyama K, Kunitake H, Goto YO, Yamasaki M
In vivo (Athens, Greece) 37 ( 3 ) 1003 - 1015 2023.5
Language：English Publishing type：Research paper (scientific journal)
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Characterization of a novel mutant with inhibition of storage root formation in sweet potato Reviewed International coauthorship
Park Hyungjun, Abe Tomoko, Kunitake Hisato, Hirano Tomonari
Ｂｒｅｅｄｉｎｇ Ｓｃｉｅｎｃｅ 22090 ( 0 ) 2023.4
Language：English Publishing type：Research paper (scientific journal) Publisher：日本育種学会
Sweet potato is a widely cultivated crop with storage roots. Although many studies have been conducted on the mechanism of its storage root formation, the details have not been fully elucidated. We screened mutant lines with inhibition of storage root formation to clarify parts of the mechanism. In this study, the process of storage root formation in one of the mutant lines, C20-8-1, was investigated. The inhibition of storage root formation was observed during the early stages of growth. The roots in C20-8-1 did not show histological differences compared to those in wild type. The transition from fibrous roots to pencil roots, which are the developmental stages prior to mature storage root formation, was delayed or inhibited in C20-8-1. The upregulation of starch biosynthesis-related genes and downregulation of lignin biosynthesis genes with storage root swelling were not confirmed in the root of C20-8-1 during the developmental transition stage, suggesting that most of the roots in C20-8-1 are in the pre-transition state toward the storage root swelling. C20-8-1 showed a mutant phenotype during the critical period of storage root swelling initiation, and further clarification of this mutation is expected to provide new insights into storage root formation.
DOI： 10.1270/jsbbs.22090
DOI： 10.1270/jsbbs.22090
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Toshima S., Katsumi I., Kai A., Yahata M., Hirano T., Kunitake H.
Scientia Horticulturae 312 2023.3
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
The fruit of the black raspberry (Rubus occidentalis L.) contains numerous bioactive components such as polyphenols, and the development of novel black raspberries with numerous functional components is eagerly desired. Rubus wild species in Asia, including Rubus parvifolius L., have been attracting a great deal of attention as sources of new traits in breeding raspberry and blackberry. In this study, we developed interspecific hybrids (BCP1 and BCP2) between black raspberry JP1 (Japanese accession 1) and the Asian Rubus parvifolius L. wild raspberry, and we confirmed interspecificity using simple-sequence repeat (SSR) markers. We then developed an amphidiploid using colchicine treatment and confirmed and evaluated it. Our results confirmed the interspecific hybridity of BCP1 and BCP2 using 4 SSR markers. In the colchicine treatment of BCP2, tetraploids were obtained after exposure to 0.1% colchicine for 2 days (7.7%) and 4 days (5.9%). The tetraploid of BCP2 showed vigorous growth, flowering, and fruiting in a warm region of Japan, and the flower diameters of BCP2 (4x) were significantly larger than those of BCP2 (2x). Also, the fruit color was confirmed to be black, the same as the fruit of JP1. We believe our research will support the development of black raspberry breeding and may contribute to ploidy breeding between black raspberries and wild Asian species as a new genetic resource for environmental adaptability.
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Comparison of Proanthocyanidin Content in Rabbiteye Blueberry (Vaccinium virgatum Aiton) Leaves and the Promotion of Apoptosis against HL-60 Promyelocytic Leukemia Cells Using 'Kunisato 35 Gou' Leaf Extract. Invited Reviewed
Toyama Y, Fujita Y, Toshima S, Hirano T, Yamasaki M, Kunitake H
Plants (Basel, Switzerland) 12 4 2023.2
Authorship：Last author, Corresponding author Language：English Publishing type：Research paper (scientific journal)
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Blueberry stem extract and stem active components prevent blue light-emitting diode light-induced retinal photoreceptor cell damage in vitro. Reviewed
Ogawa K, Urata K, Suzuki Y, Sugamoto K, Goto Y, Nakayama T, Nishiyama K, Kunitake H, Yamasaki M
Bioscience, biotechnology, and biochemistry 87 ( 378 ) 388 2023.1
Language：English Publishing type：Research paper (scientific journal)
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Ogawa K., Urata K., Maeda S., Ohno Y., Satoh K., Yamada Y., Suzuki Y., Koga Y., Sugamoto K., Kawaguchi M., Kunitake H., Nishiyama K., Goto Y., Nakayama T., Yamasaki M.
In vivo (Athens, Greece) 37 ( 1 ) 149 - 162 2023.1
Language：English Publishing type：Research paper (scientific journal) Publisher：In vivo (Athens, Greece)
BACKGROUND/AIM: This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren's syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice. MATERIALS AND METHODS: NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1β and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot. RESULTS: After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment. CONCLUSION: BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands.
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Blueberry Stem Extract Suppresses Blue Light-Emitting Diode Light-Induced Endoplasmic Reticulum Stress on Retinal Photoreceptor Cells
Ogawa Kenjirou, Karitani Takara, Otsu Wataru, Nishiyama Kazuo, Kunitake Hisato, Goto Yo, Nomiyama Shota, Hara Hideaki, Yamasaki Masao
BPB Reports 6 ( 3 ) 87 - 97 2023
Language：English Publishing type：Research paper (scientific journal) Publisher：公益社団法人 日本薬学会
<i>Background</i>: Blue light causes retinal photoreceptor damage via oxidative and endoplasmic reticulum (ER) stress. A previous study showed that blueberry stem extract (BStEx) and its active components have cytoprotective effects against blue-light-induced photoreceptor cell damage by suppressing oxidative stress. This study demonstrated the inhibitory effect of BStEx against blue light-induced ER stress in photoreceptor cells. <i>Methods</i>: The photoreceptor cells treated with BStEx or the antioxidant <i>N</i>-Acetyl-L-cysteine (NAC) as a positive control were used and then exposed to blue light. The cytoprotective effects of BStEx and NAC were evaluated using CCK-8. The ER stress-related protein expression changes over time, and its levels were measured after each exposure time to blue light in photoreceptor cells treated with BStEx or NAC. <i>Results</i>: BStEx and NAC showed protective effects against blue-light-induced photoreceptor morphological abnormalities and cell damage. Although blue light triggered ER stress factors such as BiP, PERK, ATF6, eIF2α, ATF4, and CHOP, which in turn stimulated cell cycle arrest factors p53 and p21 and upregulation of apoptosis-inducing factors caspase-3. However, BStEx suppressed the increase in expression of BiP, ATF4, ATF6, CHOP, p53, p21, and caspase-3, but not mitochondrial apoptotic factors Bax and cytochrome c. Furthermore, the antioxidant NAC showed similar suppressive effects on BStEx. <i>Conclusion</i>: Our findings suggest that blue light-induced ER stress is primarily caused by oxidative stress, and BStEx might suppress ER stress via an antioxidant effect. The antioxidant NAC contributes to the cell proliferative capacity and suppression of apoptosis in photoreceptor cells.
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Effects of In vitro Colchicine Treatment on Tetraploid Induction in Seeds of Polyembryonic Cultivars of the Genera Citrus, Fortunella, and Poncirus Invited Reviewed
YASUDA Kiichi, NUKAYA Tsunaki, SUDO Miki, YAHATA Masaki, TOMINAGA Akiyoshi, MUKAI Hiroo, KUNITAKE Hisato
Japanese journal of tropical agriculture 66 ( 2 ) 51 - 58 2022.9
Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：日本熱帯農業学会
To produce tetraploid plants effectively, we examined the effects of colchicine treatment on tetraploid induction from seeds of five polyembryonic genotypes in <i>Citrus</i>, <i>Fortunella</i>, and <i>Poncirus</i>. In the ‘Valencia’ sweet orange (<i>C. sinensis</i>), the germination rate decreased with increasing colchicine-treatment concentration and/or duration. The Meiwa kumquat (<i>F. classifolia</i>) and Hongkong kumquat (<i>F. hindsii</i>) showed high germination rates (>90%) in all treatments for 24–72 h, regardless of the colchicine concentration, although their germination rate decreased with any 96 h colchicine treatment. There was no regularity in the germination rates of the trifoliate orange variation ‘Flying-dragon’ trifoliate orange (<i>P. trifoliata</i>) or the ‘Swingle’ citrumelo [grapefruit (<i>C. paradis</i>) x trifoliate orange]. The effects of the treatment concentration and duration on the germination rate and the number of total seedlings differed among genera. Although the seedlings obtained after colchicine-treatment showed various ploidy levels such as tetraploids, octaploids, and chimeras (2x + 4x, 4x + 8x), the tetraploid induction rate varied greatly with the plant materials. The tetraploid induction rate per seed in ‘Valencia’ was highest (53.3%) with the 24 h treatment with 0.1% colchicine. The tetraploid induction rates in the Meiwa (50.0%) and Hongkong (30.0%) kumquats were greatest with 72 h treatment with 0.2%. The ‘Flying-dragon’ trifoliate orange and ‘Swingle’ showed the highest tetraploid induction rates with treatment of 0.1% for 24 h (10.0%) and 0.2% for 24 h (7.3%), respectively. Compared to <i>Citrus</i> and <i>Fortunella</i>, the tetraploid induction rate in <i>Poncirus</i> and its hybrid was low, and no regularity was observed among all of the conditions.
DOI： 10.11248/jsta.66.51
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Park Hyungjun, Narasako Yosuke, Abe Tomoko, Kunitake Hisato, Hirano Tomonari
Plant Biotechnology 39 ( 3 ) 311 - 316 2022.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Society for Plant Biotechnology
<p>Sweet potato is a major root crop with nutritious tuberous roots. The mechanism of tuberous root development has not yet been adequately elucidated. Genetic resources are required to develop the molecular understanding of sweet potato. Heavy-ion beams were applied to hexaploid sweet potato for an increase in genetic variation, after which the comprehensive effects of heavy-ion beam irradiation were investigated. In vitro cultured shoots with an axillary bud of ‘Beniharuka’ were irradiated with Ar-ions at a dose of 1–5 Gy and C-ions at a dose of 5–20 Gy, and three irradiated lines were separated from each irradiated shoot. The shoot regeneration was inhibited at high doses of each ion irradiation. Ar-ion irradiation had an especially high biological effect on shoot regeneration. A total of 335 lines were obtained, consisting of 104 and 231 lines derived from Ar- and C-ion irradiation, respectively. The change in the DNA content of the lines was analyzed by flow cytometry to evaluate the irradiation-induced damage to the DNA. The two lines demonstrated significant differences in the DNA content and changes at the chromosome level. The screening for the morphological mutants was conducted in the field. Some irradiated lines showed inhibited or no tuberous root phenotype as mutant candidates. Additionally, the high-yield mutant candidates were dominated by Ar-ion irradiation. It was indicated that heavy-ion beam mutagenesis is effective in broadening the range of the phenotypes corresponding to tuberous root formation in hexaploid sweet potato.</p>
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Kazuhiro Sugamoto, Yuri L. Tanaka, Akatsuki Saito, Yoh Goto, Takayuki Nakayama, Tamaki Okabayashi, Hisato Kunitake, Kazuhiro Morishita
Biochemical and Biophysical Research Communications 615 56 - 62 2022.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier {BV}
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Nakagawa S., Setoguchi Y., Ohmura R., Toshima S., Park H., Narasako Y., Hirano T., Otani M., Kunitake H.
Scientia Horticulturae 299 2022.6
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
Anthocyanins in sweetpotato (Ipomoea batatas L.) have attracted much attention in light of their multiple health benefits and excellent properties as processed foods. Although crossbreeding is an effective way to breed new cultivars of sweetpotato, there are few reports on how anthocyanin content and composition are passed on to F1 hybrid generations by crossbreeding because of a complex genome and self-incompatibility reactions. In this study, three purple-fleshed sweetpotao cultivars, ‘Akemurasaki’, ‘Kyushu No.137’, ‘Purple Sweet Lord’, and one yellow-fleshed cultivars, ‘HE306’ (Silk Sweet®) selected for their anthocyanin contents and compositions were crossed in six combinations. Flowering was induced by using a sweetpotato grafting technique to obtain a total of 143 F1 hybrid lines. Anthocyanin content, the percentage of acylated anthocyanins, and the peonidin/cyanidin ratio in each of these F1 hybrid lines were investigated. In the F1 hybrid generations derived from these six cross combinations, many lines tended to have anthocyanin contents similar to those of the parent cultivars. In particular, among the 36 hybrid lines derived from crosses between ‘Akemurasaki’ and ‘HE306’ (Silk Sweet®), 13 lines with high anthocyanin contents (≧200 mg·100 g FW−1) and 20 lines without anthocyanins were identified. The F1 hybrid lines of this combination had anthocyanin contents similar to those of the parent cultivars, with anthocyanin-free lines appearing more frequently than anthocyanin-containing lines. Interestingly, we also identified a line with an extremely low percentage of acylated anthocyanins (49.7%) from the F1 hybrid lines derived from a cross between ‘Purple Sweet Lord’ (seed parent) and ‘Kyushu No. 137’ (pollen parent). Moreover, the F1 hybrid lines derived from crosses between ‘Purple Sweet Lord’ and ‘Kyushu No.137’ tended to have high peonidin/cyanidin ratios. This study provides an example of the inheritance of anthocyanin content and its composition in sweetpotato, which supplies valuable information for selection of parents to breed purple-fleshed cultivars.
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Prediction of the Adult T-Cell Leukemia Inhibitory Activity of Blueberry Leaves/Stems Using Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics. Invited Reviewed International journal
Hisahiro Kai, Yoshihito Okada, Yo Goto, Takayuki Nakayama, Kazuhiro Sugamoto, Kenjirou Ogawa, Masao Yamasaki, Kazuhiro Morishita, Koji Matsuno, Hisato Kunitake
Plants (Basel, Switzerland) 11 ( 10 ) 2022.5
Language：English Publishing type：Research paper (scientific journal)
Although Vaccinium virgatum Aiton leaves and stems inhibit adult T-cell leukemia (ATL) cells, leaves and stems can differ between individual plants and by time and location. In this study, leaf and stem components were profiled in the same individual plant using direct-injection electron ionization-mass spectrometry (DI-EI-MS) metabolomics, with the aims of analyzing the anti-ATL activity, and quantifying proanthocyanidins (PACs). Leaves, stems, and leaf/stem mixtures showed distinct and characteristic spectra. Anti-ATL activity was stronger in stems than leaves, and the PAC content was higher in stems than leaves. These data were subjected to bivariate analysis to identify the factor (m/z) responsible for the inhibitory effect of ATL based on the highest coefficient of determination (R2). The results of this DI-EI-MS metabolomics analysis suggest that among PACs contained in V. virgatum stems and leaves, the fragment ion at m/z 149 contributes significantly to anti-ATL activity.
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ブルーベリーの流通・消費の実態と新品種の開発・普及の方向
宮下智人・玉木志穂・菊池 豊・大槻 優華・國武久登・大浦裕二
東京農総研研報 17 ( 1 ) 69 - 89 2022.4
Language：Japanese Publishing type：Research paper (scientific journal)
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キンカン属 （Fortunella） 植物の形態的特徴と果実特性 Reviewed
安田喜一・八幡昌紀・國武久登
東海大学紀要 農学部 41 17 - 27 2022.4
Authorship：Last author Language：Japanese Publishing type：Research paper (scientific journal)
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カンキツ属 4 種とキンカン属 5 種を用いた カンキツ類の遺伝解析に有用な SSR マーカーの選定 Reviewed
安田喜一・戸村大河・佐藤真衣・富永晃好・八幡昌紀・國武久登
東海大学紀要 農学部 41 29 - 37 2022.4
Authorship：Last author Language：Japanese Publishing type：Research paper (scientific journal)
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Mutagenic Effects of Heavy-Ion Beam Irradiation to Plant Genome Invited Reviewed
Hirano Tomonari, Kazama Yusuke, Kunitake Hisato, Abe Tomoko
ＣＹＴＯＬＯＧＩＡ 87 ( 1 ) 3 - 6 2022.3
Language：English Publishing type：Research paper (scientific journal) Publisher：公益財団法人 日本メンデル協会
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Effective Utilization of Vaccinium virgatum Aiton Stems as Functional Materials: Major Constituent Analysis and Bioactivity Evaluation Invited Reviewed
Hisahiro Kai 1, Kazuhiro Sugamoto , Saki Toshima, Yo Goto , Takayuki Nakayama , Kazuhiro Morishita and Hisato Kunitake
Plants 11 ( 568 ) 2022.2
Authorship：Last author Language：English Publishing type：Research paper (scientific journal)
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Inhibition of ATL cell proliferation by polymerized proanthocyanidin from blueberry leaves through JAK proteolysis. Reviewed
Ichikawa T, Sugamoto K, Matsuura Y, Kunitake H, Shimoda K, Morishita K
Cancer science 2022.1
Language：English Publishing type：Research paper (scientific journal)
DOI： 10.1111/cas.15277
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Induction and Characterization of Chromosome-doubled Nagabo-natsuhaze (Vaccinium sieboldii Miq.), a Wild Species Native to Japan, by Colchicine and Oryzalin Treatments of in vitro Shoots Invited Reviewed
Horticultural Research 21 ( 1 ) 1 - 10 2022.1
Authorship：Last author Language：Japanese Publishing type：Research paper (scientific journal)
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Sudo M., Yasuda K., Yahata M., Sato M., Tominaga A., Mukai H., Ma G., Kato M., Kunitake H.
Agronomy 11 ( 12 ) 2021.12
Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：Agronomy
The morphological characteristics and fruit quality of an autotetraploid plant selected from nucellar seedlings of Satsuma mandarin (Citrus unshiu Marcow.) were investigated. Additionally, in order to evaluate the reproductive potential of male and female gametes of the tetraploid Satsuma mandarin, reciprocal crosses with diploid cultivars were also carried out. The tetraploid had significantly thick and round leaves, as compared to those of the diploid Satsuma mandarin. The sizes of the flowers and pollen grains of the tetraploid were significantly larger than those of the diploid. Pollen fertility of tetraploid was high compared with that of the diploid. The tetraploid produced seedless fruits. The fruit weight of the tetraploid was equal to that of the diploid. Compared to the diploid fruits, the tetraploid fruit had less sugar contents and more organic acid contents. Although the tetraploid fruits showed similar traits to other Citrus tetraploids such as thick and hard peels, the tetraploid had a higher content of carotenoids in the flavedo than the diploid, and the rind color of the tetraploid was much better. In the reciprocal crosses between the tetraploid Satsuma mandarin and diploid cultivars, some seeds were obtained, and triploid progenies were obtained in all cross combinations.
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Yamasaki M., Kiue Y., Fujii K., Sushida M., Yamasaki Y., Sugamoto K., Suzuki Y., Koga Y., Kunitake H., Kai H., Ogawa K., Nishiyama K., Goto Y., Nakayama T.
Plants 10 ( 12 ) 2021.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plants
Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted in-creasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.
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Fruit Qualities of Interspecific Hybrid and First Backcross Generations between Red Raspberry and Rubus parvifolius Reviewed
Saki Toshima, Marika Fujii, Momoko Hidaka, Soya Nakagawa, Tomonari Hirano, Hisato Kunitake
J. Amer. Soc. Hort. Sci. 146 ( 6 ) 445 - 451 2021.10
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
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Yasuda K., Yahata M., Sato M., Sudo M., Tominaga A., Kunitake H.
Agronomy 11 ( 10 ) 2 - 16 2021.10
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Agronomy
We previously obtained two intergeneric hybrids with different ploidies, i.e., aneuploid (2n = 28) and eutriploid, from diploid−diploid crosses between ‘Kiyomi’ tangor (Citrus unshiu Marcow. × C. sinensis (L.) Osbeck) and Meiwa kumquat (Fortunella crassifolia Swingle) as novel breeding materials for a seedless kumquat. In this study, we attempted to clarify the construction of the parental genomes of these hybrids by SSR genotyping and genomic in situ hybridization (GISH)−chromomycin A3 (CMA) analysis. SSR genotyping in NSX43 (LG5) and CiBE2227 (LG8) loci revealed that both hybrids inherited one allele from ‘Kiyomi’ tangor and two heterozygous alleles from Meiwa kumquat. The GISH analysis failed due to the high genomic homology between Citrus and Fortunella. At the same time, the CMA karyotype compositions of the two intergeneric hybrids (H15-701: 2A + 1B + 3C + 13D + 7E + 1F + 1Dst; H15-702: 3A + 1B + 2C + 15D + 4E +1F + 1Dst) and both parents (‘Kiyomi’ tangor: 1A + 2B + 2C + 6D + 7E; Meiwa kumquat: 2A + 2C + 12D + 1F + 1Dst) were completely revealed. We identified the parental genome construction and polyploidiza-tion processes in both intergeneric hybrids on the basis of these SSR genotypes and CMA karyotype compositions according to the following theory: the SSR genotypes and chromosome compositions were the same as those of the somatic chromosome and two-fold after the first division (even num-ber) in unreduced gametes caused by first-division restitution (FDR) and second-division restitution (SDR), respectively. Consequently, we determined that both intergeneric hybrids may have had two genomes derived from the 2n male unreduced gamete as a result of the FDR of the Meiwa kumquat. In addition, most horticultural traits of the leaves, flowers, and fruits of both hybrids showed intermediate traits of the parents, but the fruit sizes and flowering habits were more like those of the two inherited genomes of Meiwa kumquat.
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宮崎大学医学部附属病院臨床研究支援センター食品臨床試験部門の取り組み Reviewed
有村保次、永濱清子、黒木光渉、國武久登、片岡寛章（他6名）
宮崎県医師会医学会誌 45 ( 2 ) 222 - 230 2021.9
Language：Japanese Publishing type：Research paper (scientific journal)
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Nhat V.Q., Kazama Y., Ishii K., Ohbu S., Kunitake H., Abe T., Hirano T.
Plants 10 ( 9 ) 2021.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Plants
Two growth processes, cell proliferation and expansion, determine plant species-specific organ sizes. A large flower mutant in Arabidopsis thaliana, ohbana1 (ohb1), was isolated from a mutant library. In the ohb1 flowers, post-mitotic cell expansion and endoreduplication of nuclear DNA were promoted. The whole-genome resequencing and genetic analysis results showed that the loss of function in MEDIATOR16 (MED16), a mediator complex subunit, was responsible for the large flower phenotypes exhibited by ohb1. A phenotypic analysis of the mutant alleles in MED16 and the double mutants created by crossing ohb1 with representative large flower mutants revealed that MED16 and MED25 share part of the negative petal size regulatory pathways. Furthermore, the double mutant analyses suggested that there were genetically independent pathways leading to cell size restrictions in the floral organs which were not related to the MED complex. Several double mutants also formed larger and heavier seeds than the wild type and single mutant plants, which indicated that MED16 was involved in seed size regulation. This study has revealed part of the size-regulatory network in flowers and seeds through analysis of the ohb1 mutant, and that the size-regulation pathways are partially different between floral organs and seeds.
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Saki Toshima, Tomonari Hirano, Hisato Kunitake
Scientia Horticulturae 285 2021.7
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
The breeding of raspberries (Rubus idaeus L.) and blackberries (R. fruticosus Agg.) has shifted in focus toward traits associated with the sensory qualities and potential health benefits of the fruit, and Rubus wild species have attracted a great deal of attention as new sources of genetic diversity. In this study, the anthocyanin and polyphenol contents and compositions, and three types of antioxidant capacities were compared in mature fruits including raspberries, blackberries, and Japanese wild Rubus species. ‘Blackcap’ black raspberry showed the significantly highest values of total anthocyanin content and total polyphenol content among 12 Rubus species or cultivars. Cyanidin-3-xylosyl-rutinoside was contained only in black raspberries, and was characteristic anthocyanin in Rubus species. R. croceacanthus H. Lév., a Japanese wild species that is adapted to warm regions, had a deep red fruit color, and had the highest total anthocyanin and total polyphenol contents among the four Japanese wild species examined. As for the antioxidant capacities, the highest values were observed in ‘Blackcap’ among 12 Rubus species. Furthermore, a high positive correlation was shown between total anthocyanin or total polyphenol content and antioxidant capacities. In particular, black raspberries and blackberries contained high concentrations of rutin. Thus, rutin might contribute to the high antioxidant capacities in these cultivars.
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Nakagawa S., Ohmura R., Toshima S., Park H., Narasako Y., Hirano T., Otani M., Kunitake H.
Scientia Horticulturae 284 2021.6
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
Anthocyanins and polyphenols in the sweetpotato (Ipomoea batatas L.) have attracted attention due to their multiple physiological functions. Therefore, sweetpotato breeders have focused on increasing the anthocyanin and polyphenol contents. In this study, we investigated the diversity of anthocyanins, polyphenols, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activities in sweetpotato genotypes. We also assessed how the anthocyanin and polyphenol contents and compositions were related to DPPH radical-scavenging activities during tuber growth in several cultivars of sweetpotatoes. In six purple-fleshed cultivars, the percentage of acylated anthocyanins that were evaluated was high (92.0–98.5 %), whereas in ‘Fukumurasaki’ this percentage was particularly low (63.6 %). Moreover, the percentages of anthocyanins that were acylated in the purple-fleshed cultivars ‘Tanegashimamurasaki’, ‘Purple Sweet Lord’, and ‘Akemurasaki’ had already reached high levels in the early stage of tuber development. On the other hand, these three cultivars with high anthocyanin contents had high DPPH radical-scavenging activities, and the activities during tuber growth showed a high correlation with total anthocyanin content. Elucidating the changes in acylated anthocyanin content and in antioxidant activities during tuber growth can help promote high-quality sweetpotato cultivation and breeding.
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Polyphenol contents, antioxidant activities, and anti-cancer cell proliferation properties at each stage of fruit development in intersectional hybrids between highbush blueberry and shashanbo (Vaccinium bracteatum Thunb.) Invited Reviewed
Toyama Yuki, | Toshima, Saki, Hirano, Tomonari, Yamasaki, Masao, Kunitake, Hisato
Journal of Berry Research 1 - 16 2021.6
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
DOI： 10.3233/JBR-210713
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Comparison of fruits quality and antioxidant activity between diploid and tetraploid in Satsuma mandarin Reviewed
Mai SATO, Kiichi YASUDA, Kohei KIKUCHI, Taiga TOMURA, Masaki YAHATA and Hisato KUNITAKE
Proceedings of the School of Agriculture, Tokai University 40 1 - 8 2021.3
Language：Japanese Publishing type：Research paper (bulletin of university, research institution)
To clarify the time-dependent change of fruit quality and health functional components in the tetraploid Satsuma mandarin
(Citrus unshiu Marcow.), the fruits were measured the coloring fruit skin, sugar and organic acid contents, total polyphenol
content and antioxidant activities by ABTS cation radical scavenging activity. As a result, the tetraploid fruit skin indicated the
deeper and redder color than that of the diploid at harvesting stage. There was no significant difference in the sugar contents
between the diploid and tetraploid. The organic acid content generally continued to be flat in the tetraploid pulp (juice sac),
whereas it was normally decreased in the diploid pulp with maturation of fruits. For the last period, this value of the tetraploid
was significantly higher than the diploid. Furthermore, the antioxidant activity the flavedo and/or albedo in the tetraploid was
significantly higher than the diploid during some investigation periods, although there was no significant difference between
the two in the total polyphenol content, expect for some investigation periods. Thus, it was considered that the tetraploid
Satsuma mandarin was difficult to use as a fresh fruit, but it was useful as a processed food such as marmalade, dried fruit and
juice whose raw material was pericarp. -
Relationship between morphology and weight of seeds and ploidy level of seedlings obtained by cross between diploid pummelo 'Banpeiyu' and triploid gragpefruit-type citrus 'Orobranco'. Reviewed
20 ( 1 ) 29 - 37 2021.3
Language：Japanese Publishing type：Research paper (scientific journal)
DOI： 10.2503/hrj.20.29
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Mik iKawano, Masaki Yahata, Tokurou Shimizu, Chitose Honsho, Tomonari Hirano, Hisato Kunitak
Scientia Horticulturae 277 2021.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
The genetic analysis of five haploid plants (BX1-BX5) of ‘Banpeiyu’ pummelo (BP), [Citrus maxima (Burm.) Merr.] using 32 simple sequence repeat (SSR) markers carried out for the purpose of confirming their gynogenetic origin and genetic differences. As the results, all the haploid plants possessed only either one of the BP alleles, confirming that they are gynogenetic origin. We also confirmed that BX1 and its doubled haploid (BX1-DH) have exactly the same SSR alleles. Interestingly, all the five haploids had common alleles on the linkage group (LG) 6 in the case of three markers. Then, we performed self-pollination of DH plants and reciprocal crosses between DH and BP, which has strong self-incompatibility. The seeds obtained by these crosses germinated in vitro or ex vitro normally and developed into diploid seedlings, except for one tetraploid seedling. Genetic analysis of these seedlings with 32 SSR markers revealed that segregation ratios of the 32 loci in the 30 BP inbred, 30 BP x DH and 30 DH x BP progenies mostly fitted to the expected ratios. However, significantly distorted segregation ratios were found in one, five and three SSR markers for BP inbred, BP x DH and DH x BP progenies, respectively. All of the 30 DH inbred progenies showed exactly the same SSR alleles as the original DH. This is the first example in which self and back cross progenies of DH in fruit crops were genetically analyzed using SSR markers.
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DNA Damage Response of Cyrtanthus mackenii Male Gametes Following Argon Ion Beam Irradiation Reviewed
Hirano T., Matsuyama Y., Hanada A., Hayashi Y., Abe T., Kunitake H.
Cytologia 86 ( 4 ) 311 - 315 2021
Language：English Publishing type：Research paper (scientific journal) Publisher：Cytologia
Heavy-ion beams have been applied as effective mutagens to various plant materials. Pollen has been used as material for mutant induction and genetic analysis. However, our knowledge of the DNA damage response of plant male gametes remains limited. In the present study, we irradiated Cyrtanthus mackenii pollen with an argon ion beam, which induced complex DNA damage, and investigated the DNA damage response of male gametes during pollen tube growth. Male gametes derived from the irradiated pollen grains were isolated from pollen tubes after 12 and 24 h of culture and subjected to cell cycle analysis. After 12 h of culture, the irradiated generative cells were frequently arrested at metaphase during pollen mitosis II (PMII), and the proportion of metaphase cells increased with increasing absorbed dose. These results suggest that the genomic lesions induced by the argon ion beam caused spindle assembly checkpoint (SAC)-dependent arrest. After 24 h of culture, the irradiated male gametes completed PMII, albeit forming sperm cells with abnormalities in chromosome separation, and chromosomal bridges were often formed between these cells. Moreover, phosphorylated H2AX foci, an indicator of DNA double-strand breaks, were detected in the irradiated male gametes after 24 h of culture, regardless of passing through the SAC. Taken together, these results indicate that male gametes activate functions to cope with radiation-induced complex DNA damage during pollen tube growth.
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Phylogeny and trait variation of Japanese Rubus subgenus Ideaobatus Reviewed
Okada A., Kikuchi S., Hoshino Y., Kunitake H., Mimura M.
Scientia Horticulturae 264 2020.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
© 2019 Elsevier B.V. Rubus L. subgenus Idaeobatus is one of the largest groups comprising mostly diploid species within the genus. Idaeobatus species is distributed throughout Japan, showing a wide range of trait variation and climatic distribution. We constructed phylogenetic trees of Idaeobatus that are naturally distributed in Japan. The Bayesian and ML phylogenetic tree found that this subgenus was polyphyletic with at least three genetic groups. The phylogenetic tree of Japanese Idaeobatus also reflects the trait variations for flower colors, fruit colors, and leaf shape (simple vs compound). The species that occurs in subtropical climates was found to be closely related to their sister temperate species in Japanese Idaeobatus. Crossability generally reflected the phylogenetic distances between species. Trait diversity and its phylogenetic relationship among Japanese Idaeobatus can provide additional insights for future breeding and ecological and evolutionary studies.
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Shoji K., Yamasaki M., Kunitake H.
Journal of Oleo Science 69 ( 2 ) 143 - 151 2020.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Oleo Science
©2020 by Japan Oil Chemists’ Society. Prevention of postprandial hypertriglyceridemia is an important consideration for reducing the risk of developing cardiovascular disease. While blueberry fruits have been reported to ameliorate lipid metabolism in humans, there are only few research reports on the effects of blueberry leaves (BL). Here, we investigated the efficacy of BL on postprandial hyperlipidemia in subjects with high fasting triacylglycerol (TG) concentrations. Randomized, double-blind, cross-over design study was conducted. The subjects consumed a BL containing beverage or a placebo beverage before a fat-enriched test meal. Blood samples were collected prior to and 1, 2, 3, 4, and 5 hours after consuming the test beverage. The postprandial serum TG and remnant-like particle cholesterol (RLP-C) concentrations were significantly lower in the BL beverage compared with those in the placebo beverage. Additionally, BL was more effective in subjects with high fasting ghrelin with gastric emptying function. In current study, fasting ghrelin correlated with the increase in postprandial serum TG, suggesting that BL ameliorates hypertriglyceridemia through delayed gastric emptying. In conclusion, this pilot study suggests that BL may be useful as an early dietary therapy for treating postprandial hyperlipidemia.
DOI： 10.5650/jos.ess19201
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Nakagawa Soya, Narasako Yosuke, Otani Motoyasu, Hirano Tomonari, Kunitake Hisato
Abstracts of Meeting of the CSSJ 249 ( 0 ) 91 - 91 2020
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：CROP SCIENCE SOCIETY OF JAPAN
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SAKAI Kentaro, IMAI Masato, IZUMI Nobuo, KOURIKI Haruna, KUNITAKE Hisato, MISAWA Naoaki, SUIKO Masahito
The Journal of Science Policy and Research Management 35 ( 1 ) 23 - 31 2020
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japan Society for Research Policy and Innovation Management
Recently, many universities have been working to promote the sharing of research facilities and equipment in Japan. University of Miyazaki is also developing a sharing service system of those through two projects supported by the Ministry of Education, Culture, Sports, Science and Technology. In addition, we have established the Miyazaki Facility Network, in which public research institutions in Miyazaki Prefecture participate, and are working to create an environment that can provide mutually advanced analysis technologies that contribute to regional revitalization.In this paper, we introduce a common facilities management system linked on-line to the existing financial accounting and the researcher database developed by University of Miyazaki, and describe the relationship with the administration policy of our university and examples of utilization in Miyazaki prefecture.
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Nukaya T., Sudo M., Yahata M., Ohta T., Tominaga A., Mukai H., Yasuda K., Kunitake H.
Agronomy 9 ( 9 ) 2019.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Agronomy
© 2019 by the authors. A ploidy chimera of the Meiwa kumquat (Fortunella crassifolia Swingle), which had been induced by treating the nucellar embryos with colchicine, and had diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36) cells, was examined for its ploidy level, morphological characteristics, and sizes of its cells in its leaves, flowers, and fruits to reveal the ploidy level of each histogenic layer. Furthermore, the chimera was crossed with the diploid kumquat to evaluate the ploidy level of its reproductive organs. The morphological characteristics and the sizes of the cells in the leaves, flowers, and fruits of the chimera were similar to those of the tetraploid Meiwa kumquat and the ploidy periclinal chimera known as "Yubeni," with diploids in the histogenic layer I (L1) and tetraploids in the histogenic layer II (L2) and III (L3). However, the epidermis derived from the L1 of the chimera showed the same result as the diploid Meiwa kumquat in all organs and cells. The sexual organs derived from the L2 of the chimera were significantly larger than those of the diploid. Moreover, the ploidy level of the seedlings obtained from the chimera was mostly tetraploid. In the midrib derived from the L3, the chimera displayed the fluorescence intensity of a tetraploid by flow cytometric analysis and had the same size of the cells as the tetraploid and the Yubeni. According to these results, the chimera is thought to be a ploidy periclinal chimera with diploid cells in the outermost layer (L1) and tetraploid cells in the inner layers (L2 and L3) of the shoot apical meristem. The chimera had desirable fruit traits for a kumquat such as a thick pericarp, a high sugar content, and a small number of developed seeds. Furthermore, triploid progenies were obtained from reciprocal crosses between the chimera and diploid kumquat.
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Blueberry Leaf Polyphenols Prevent Body Fat Accumulation in Mice Fed High-fat, High-sucrose Diet. Reviewed
Fujii K, Ota Y, Nishiyama K, Kunitake H, Yamasaki Y, Tari H, Araki K, Arakawa T, Yamasaki M
Journal of oleo science 2019.4
Language：English Publishing type：Research paper (scientific journal)
DOI： 10.5650/jos.ess18226
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Subchronic toxicity evaluation of leaves from rabbiteye blueberry (Vaccinium virgatum Aiton) in rats Reviewed
Tanaka , Daigo Yokoyama , Yasushi Matsuura , Masahiko Nozaki , Naoya Hirozawa ,Naoya Hirozawa ,Hisato Kunitake , Masanobu Sakono , Hiroyuki Sakakibara
Toxicology Reports 6 272 - 278 2019.3
Language：English Publishing type：Research paper (scientific journal)
Blueberry leaf may contain multiple compounds with beneficial effects. We conducted a 90-day toxicity study in rats to evaluate the safety of consuming the leaves of rabbiteye blueberry (Vaccinium virgatum Aiton; RB species). Powdered leaves were administered daily by oral gavage at doses of 500, 1000, and 2500 mg/kg body weight to male and female Sprague-Dawley rats for 90 days. Treatment did not result in death or changes in the behavior and external appearance of the animals. No alterations were observed in hematological and serum chemical parameters, urinalysis, food consumption, body weight gain, or absolute and relative organ weights at the end of the treatment period, with the exception of some leukocyte percentages in male rats treated with 500 and 1000 mg/kg blueberry leaf powder. The findings indicate that rabbiteye blueberry leaf is safe for consumption and should be investigated as a candidate functional food.
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Nukaya T., Sudo M., Yahata M., Nakajo Y., Ohta T., Yasuda K., Tominaga A., Mukai H., Kunitake H.
Scientia Horticulturae 245 ( 9 ) 210 - 217 2019.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
© 2018 Elsevier B.V. Three Fortunella (kumquat) species, the Meiwa kumquat (F. crassifolia Swingle), the Round kumquat [F. japonica (Thunb.) Swingle] and the Changshou kumquat (F. obovata hort. ex Tanaka), showing tetraploid, which had been induced by treating the seeds with colchicine, were examined for origin and horticultural characteristics (e. g. morphology of leaves, flowers, pollen and fruits). Additionally, these tetraploid kumquats were crossed to produce triploid kumquats. All of the tetraploids were confirmed to be derived from the nucellar embryo of each original kumquat by examining their chromosomal composition with chromomycin A3 staining. All of the tetraploid kumquats had the typical morphological characteristics of tetraploid Citrus plants, such as round and thick leaves, and large flowers and pollen grains. On the other hand, the fruits of these tetraploid plants showed desirable traits for kumquats such as thicker pericarp and higher soluble solids content. Furthermore, when these tetraploids were crossed with some diploid cultivars, triploid progenies were obtained from almost all of the cross combinations.
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Kai H., Uesawa Y., Kunitake H., Morishita K., Okada Y., Matsuno K.
Planta Medica 85 ( 1 ) 81 - 87 2019.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Planta Medica
© Georg Thieme Verlag KG Stuttgart. New York. Metabolic profiling is often used to identify possible correlations between a compound's metabolic profile and biological activity. Direct-injection electron ionization-mass spectrometry fingerprinting is useful for characterizing biological materials. We demonstrate the utility of direct-injection electron ionization-mass spectrometry for metabolic profiling using 100 different extracts of leaves from 20 blueberry cultivars collected at 5 time points from April to December 2008. A qualitative direct-injection electron ionization-mass spectrometry method was used to profile the major and/or minor constituents in the blueberry leaf extracts. Blueberry leaf extracts could be distinguished by principal component analysis based on the absolute intensity of characteristic fragment ions. Twenty cultivars were categorized into four species, and the most appropriate discriminative marker m/z value for identifying each cultivar was selected statistically. Correlated m/z values indicating the collection month were determined in the same analysis, and air temperature variance factors were extracted from score plots by principal component analysis. We previously reported that blueberry extracts inhibit the proliferation of adult T-cell leukemia cells. Leaves of Vaccinium virgatum collected in December of 2008 exhibited significantly greater inhibition of adult T-cell leukemia cell proliferation than other species. Highly bioactive cultivars or species were identified by direct-injection electron ionization-mass spectrometry metabolomics analysis of blueberry leaf extracts. The components extracted based on our direct-injection electron ionization-mass spectrometry analyses could be used to construct a model to predict anti-adult T-cell leukemia bioactivity. This is the first study to report a relationship between seasonal variation and bioactivity of natural products using a direct-injection electron ionization-mass spectrometry metabolomics method.
DOI： 10.1055/a-0725-8295
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Erratum: Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method for Analyzing Blueberry Leaf Metabolites That Inhibit Adult T-cell Leukemia Proliferation.
Kai H, Uesawa Y, Kunitake H, Morishita K, Okada Y, Matsuno K
Planta medica 85 ( 1 ) e3 2019.1
Language：English Publishing type：Research paper (scientific journal)
DOI： 10.1055/a-1238-9951
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Yasushi Matsuura, Hiroyuki Sakakibara, Maho Kawaguchi, Emi Murayama, Daigo Yokoyama, Chizuko Yukizaki, Hisato Kunitake, Masanobu Sakono
Functional Foods in Health and Disease 8 ( 9 ) 447 - 461 2018.9
Language：English Publishing type：Research paper (scientific journal)
Background: Blueberry stems, a by-product of blueberry leaf tea production, are typically discarded. We evaluated the effects of hot-water extracts of rabbiteye blueberry (Vaccinium virgatum Aiton; RB species) leaves and stems on hepatic lipid levels in rats consuming a high-sucrose diet.
Methods: Male Sprague-Dawley rats were divided into groups that received a control high-sucrose diet alone or supplementation with 2.0% blueberry leaf extract or 0.5% or 2.0% blueberry stem extract. Blood and hepatic lipid levels, hepatic lipogenic enzyme activity, and hepatic quercetin metabolites were evaluated after 28 days of ad libitum consumption.
Results: Supplementation with the extracts did not affect body weight gain, food intake, liver and white adipose tissue weights, or serum lipid levels. Hepatic triglyceride and total cholesterol levels were reduced in the groups that received 2.0% supplementation of either extract. Hepatic malic enzyme activity was also reduced in those groups. Quercetin and its glycosides, the major polyphenols identified in the extracts, accumulated in the liver as quercetin aglycone and quercetin metabolites.
Conclusion: We demonstrated how daily consumption of blueberry leaf and stem extracts can decrease hepatic lipid levels, potentially downregulating malic enzyme activity. These effects were intensive in leaf extracts. The active compounds existed in both extracts may be quercetin and its glycosides. Therefore, blueberry stems and leaves may be an attractive candidate novel functional food. -
YAHATA Masaki, KUNITAKE Hisato, KOMATSU Haruki
Japan Agricultural Research Quarterly: JARQ 51 ( 4 ) 293 - 298 2017.10
Language：English Publishing type：Research paper (scientific journal) Publisher：国立研究開発法人 国際農林水産業研究センター
To obtain basic information about haploid plants in <i>Citrus</i>, the morphological characteristics and reproductive potential of a haploid plant obtained from the 'Banpeiyu' pummelo [<i>C. maxima</i> (Burm.) Merr.] were examined. The leaves, flowers and fruits of the haploid were significantly smaller than those of the 'Banpeiyu' pummelo. Although the haploid showed remarkably reduced pollen fertility compared with the 'Banpeiyu' pummelo, it produced slightly fertile pollen grains. In crosses with diploid cultivars, when the haploid was the seed parent, there was no seed. But when diploid cultivars were pollinated with pollen of the haploid, diploid progenies were obtained. During female gamete formation, no embryo-sac mother cell was formed in the ovules, and consequently no normal embryo sac was observed at anthesis, owing to female sterility caused by the lack of embryo-sac mother cell (EMC) formation in the haploid. During male gamete formation, however, univalents were not distributed to opposite poles at anaphase I in some dividing cells; instead, they remained near the equatorial plate. The nine univalents then performed mitosis, and the univalents were equally distributed to opposite poles. And at the tetrad stage, dyads were also produced in the haploid at a ratio of 24.7%. These results suggest that fertile pollen grains in the haploid would be dyad derivation, produced by abnormalities in the first meiotic division such as first division restitution (FDR).
DOI： 10.6090/jarq.51.293
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Horticultural research 16 ( 3 ) 345 - 352 2017.8
Language：Japanese Publishing type：Research paper (scientific journal)
In order to develop new raspberry varieties adaptable to Japan with high quality and functionality, we aimed to produce interspecific
hybrids between Rubus parvifolius L., a wild species native to Japan, and the `Indian Summer' raspberry (R. idaeus L.).
The morphological characteristics, such as the leaf, flower, and fruit, and random amplified polymorphic DNA (RAPD) analysis
revealed that `07RUBIXP01' was an interspecific hybrid. Although `07RUBIXP01' had small thorns, it showed vigorous growth
in the warm environment and had a significantly larger fruit compared with the parents. Also, `07RUBIXP01' showed values
intermediate between those of the parents for total polyphenol, anthocyanin, and ellagitannin contents. In particular, it showed
an ellagitannin content that was about four times higher than that of `Indian Summer'. In sugar and organic acid analyses, a
higher sugar acid ratio was noted compared with the parents. `07RUBIXP01' was registered as a new variety in 2012 (variety
registration no. 21801), and it would be a useful raspberry for growing in warm regions of Japan. -
Dhungana Sabitri Adhikari, Kunitake Hisato, Niino Takao, Yamamoto Shin-ichi, Fukui Kuniaki, Tanaka Daisuke, Maki Shinya, Matsumoto Toshikazu
Ｐｌａｎｔ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ 34 ( 1 ) 1 - 5 2017.3
Language：English Publishing type：Research paper (scientific journal) Publisher：日本植物細胞分子生物学会
Cryopreservation is an important tool for long-term storage of plant germplasm that is currently used for plant germplasm storage at many institutes worldwide. Recently, novel cryogenic procedures (V and D cryo-plate methods) have been developed. In this study, the most suitable conditions for preserving blueberry shoot tips derived from in vitro and current shoots using the D cryo-plate method were investigated. The D cryo-plate method has advantages such as higher regrowth after cryopreservation and a more user-friendly process compared with conventional cryogenic methods. The optimum duration of desiccation for regrowth of shoot tips from each shoot type was 1 h. To induce dehydration tolerance for the shoot tips, the effects of two cryoprotection treatments (sucrose preculture and loading solution [LS] treatment) on shoot regrowth after cryopreservation were investigated. The combined effect of both treatments significantly increased percentage regrowth (approximately 90%). No regrowth of shoot tips was attained without the two treatments. Thus, preculture and LS treatment were effective to induce dehydration tolerance for cryopreservation of blueberry shoot tips. The optimized conditions for blueberry shoot tips using the D cryo-plate technique were: preculture with 0.3 M sucrose for 1 day, LS treatment (2 M glycerol +0.4–1.0 M sucrose) for 30 min, and air dehydration for 1 h. This optimized procedure was applied to additional blueberry cultivars shoot tips derived from in vitro shoots (regrowth 46.7–100%) and current shoots (regrowth 17.2–62.7%). Furthermore, in vitro shoot tips were suitable material for the D cryo-plate method in blueberry.
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Effect of temperature on self-incompatibility in Citrus pistil and mature pollen culture systems
Kawano S., Li Y., Yahata M., Kunitake H.
Acta Horticulturae 1135 117 - 122 2016.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Acta Horticulturae
© 2016, International Society for Horticultural Science. All rights reserved. Temperature has a clear effect on pollen germination and pollen tube growth. Recently, researchers have reported a relationship between self-incompatibility (SI) and temperature. However, the detailed behavior and mechanisms of this relationship have not been completely clarified in Citrus. In the present study, to reveal the relationship between SI and temperature in Citrus cultivars, we investigated pollen germination and pollen tube behavior using in vitro pistil and mature pollen systems under controlled environmental chamber conditions of 25, 30 and 35°C. We first evaluated pollen tube growth for one week in in vitro detached pistils of the SI cultivar 'Hyuganatsu' (Citrus tamurana hort. Ex Tanaka) after self- or cross-pollination. 'Hassaku' (C. hassaku) and 'Banpeiyu' pummelo [C. maxima (Burm.) Merr.] were used as compatible cultivars. Pollen tube growth was observed one week after pollination using aniline blue staining and a fluorescent microscope. Results indicated incompatible pollen tubes stopped in the upper part of styles at 25°C, whereas they reached the base of the styles at 30°C, similar to the behavior of compatible pollen tubes at 25°C. To investigate improvement of the SI-like responses of pollen tubes under different temperature conditions, we examined the inhibition of pollen tube growth and morphological changes of tube tips using the mature pollen culture of 'Hyuganatsu' (Abe et al., in press). The pollen was treated with stylar crude extract protein of 'Hyuganatsu' to simulate the SI-like response. Pollen tubes with the SI treatment showed tip inflation, content leakage and abnormal tip twisting at 25°C, and tube growth was inhibited. However, at 30°C, these SI-like responses were suppressed. These results suggested that temperature might be a trigger for avoiding SI reaction in Citrus.
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Li Y., Fuse T., Kawano S., Kunitake H.
Acta Horticulturae 1135 109 - 116 2016.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Acta Horticulturae
© 2016, International Society for Horticultural Science. All rights reserved. Self-incompatibility (SI) in Citrus strongly prevents self-pollination and promotes outcrossing. Even though it has been confirmed that the S-allele is a key gene in controlling the recognition between pollen and pistil in Rosaceae, Solanaceae and Papaveraceae, this has not been cloned in Citrus. A citrus mature pollen culture system was established in our lab in order to culture 'Banpeiyu' and 'Hyuganatsu' pollen in vitro. Stylar crude protein extracts were used to treat citrus pollen in order to induce the SI-like response in the pollen tube. We observed the alteration of the pollen tube shape, including tip twisting and content leakage. Programmed cell death (PCD), as a normal cellular process that eliminates unwanted cells, was reported in previous studies of incompatible pollen tubes, together with an increase in hydrogen peroxide (H 2 O 2 ), depolymerization of actin skeleton, the collapse of mitochondria, and leakage of cytochrome c. In our study, we investigated the cytosolic calcium ion concentration ([Ca 2+ ] cyt ) changes and identified alterations in the actin cytoskeleton in the pollen tube after exposure to the incompatible treatment. The [Ca 2+ ] cyt content increased after incompatible treatment, but there was no obvious difference between the compatible-treatment group and the control. Punctate foci of actin filaments were found in the incompatible-treated pollen tubes; the rates of punctate foci were significantly higher than in the compatible-treated pollen tubes in both 'Banpeiyu' and 'Hyuganatsu'. The results suggested that these alterations in the pollen tubes were directly induced by the SI-like response, and might cause pollen tube PCD.
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Phylogeny and Classification of Kumquats (Fortunella spp.) Inferred from CMA Karyotype Composition Reviewed
Kiichi Yasuda, Masaki Yahata and Hisato Kunitake
The Horticulture Journal 85 ( 2 ) 115 - 121 2016.3
Language：English Publishing type：Research paper (scientific journal)
DOI： 10.2503/hortj.MI-078
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Assessment of genetic relationships among cultivated and wild Rubus accessions using AFLP markers
Miyashita T., Kunitake H., Yotsukura N., Hoshino Y., Hoshino Y.
Scientia Horticulturae 193 165 - 173 2015.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
© 2015 Elsevier B.V.The genus Rubus comprises more than 740 species divided into 12 subgenera and contains the raspberries, blackberries, arctic fruits, and flowering raspberries, all of which have been utilized in breeding programs. The objective of this study was to evaluate the phylogenetic relationships among wild and cultivated Rubus species mainly collected in Japan. To evaluate genetic resources in Rubus, 81 accessions were analyzed with three amplified fragment length polymorphism (AFLP) primer pairs and data were analyzed with the neighbor-joining and unweighted pair group methods with arithmetic mean. Two of the generated phylogenetic trees grouped subgenera Anoplobatus, Eubatus, Idaeobatus, and Malachobatus into different clusters. Accessions of Rubus idaeus L. var. aculeatissimus collected from four regions in Hokkaido formed distinct clusters reflecting sampling sites. Four hybrid accessions between raspberry cultivars (R. idaeus L.) and R. idaeus L. var. aculeatissimus, and one hybrid accession between a raspberry cultivar and Rubus spectabilis Pursh were clearly distinguished from parental accessions. These results indicated that AFLP markers are a reliable technique for assessing genetic diversity and studying phylogenetic relationships in Rubus. Data from diversity and phylogenetic studies revealed valuable information on the availability of unique fragments in different accessions that would be useful for the development of improved genotypes through conventional breeding and marker-assisted selection.
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Li, Y., Abe, A., Fuse, T., Hirano, T., Hoshino, Y., Kunitake, H.
Journal of the American Society for Horticultural Science 140 ( 4 ) 339 - 345 2015.8
Language：English Publishing type：Research paper (scientific journal)
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Li Y., Abe A., Fuse T., Hirano T., Hoshino Y., Kunitake H.
Journal of the American Society for Horticultural Science 140 ( 4 ) 339 - 345 2015.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the American Society for Horticultural Science
© 2015, American Society for Horticultural Science. All rights reserved. Self-incompatibility (SI) is an important mechanism in higher plants that promotes outcrossing and prevents self-fertilization. ‘Banpeiyu’ (Citrus maxima) and ‘Hyuganatsu’ (Citrus tamurana), two of the Citrus cultivars distributed in Kyusyu, Japan, show gametophytic SI. In this study, we used the Citrus mature pollen culture system and stylar crude protein extracts to simulate compatible (C) and SI responses in ‘Banpeiyu’ pollen tubes. We analyzed the protein changes in pollen tubes with the C- and SI-like treatments by nano-liquid chromatography-mass spectrometry (nano-LC-MS); 14 and 27 proteins were identified in C- and SI-like treatments, respectively. We picked up some candidate genes that were particularly prevalent in SI-like treatment and analyzed their expression level changes during C- and SI-like treatments in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes. The expression levels of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese SOD (Mn SOD), catalase (CAT), and cysteine protease (CYP) increased after SI-like treatment. We used a fluorescent probe to visualize reactive oxygen species (ROS) level changes in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes after C- and SI-like treatments and found that 2-hour SI-like treatment induced ROS levels to increase in the pollen tubes of both cultivars. These results suggest that an ROS increase could be one of the key phenomena in the SI response of Citrus and that gene expression changes were responses to ROS generation.
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Li Yiran, Uchida Aska, Abe Akiha, Yamamoto Akihiro, Hirano Tomonari Kunitake Hisato
Journal of American Society of Horticultural Sciences 140 ( 2 ) 183 - 190 2015.2
Language：English Publishing type：Research paper (scientific journal)
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Analysis of expressed proteins in the pollen tube occurring self-incompatible response using mass or single-culture system of mature pollen in Citrus Reviewed
Abe A., Uchida A., Hoshino Y., Sakakibara Y., Suiko M., Kunitake H.
Acta Horticulturae 1267 - 1274 2015.2
Language：English Publishing type：Research paper (scientific journal)
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Yahata Masaki, Nukaya Tsunaki, Sudo Miki, Ohta Tomohiro, Yasuda Kiichi, Inagaki Hidehiro, Mukai Hiroo, Harada HIsashi, Takagi Toshihiro, Komatsu Haruki, Kunitake Hisato
The Horticulture Journal 84 ( 1 ) 30 - 36 2015.2
Language：English Publishing type：Research paper (scientific journal)
DOI： 10.2503/hortj.MI-005
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Functional genetic analysis of Arabidopsis thariana SYNC1 in Lotus corniculatus super-growing roots using the FOX gene-hunting system. Invited Reviewed
Yano Tsubasa, Tanaka Hidenori, Kurino Taiki, Yamamoto Akihiro, Kunitake Hisato, Saeki Yuichi, Akashi Ryo
Plant Root 9 6 - 14 2015.2
Language：English Publishing type：Research paper (scientific journal)
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Abe A., Uchida A., Hoshino Y., Sakakibara Y., Suiko M., Kunitake H.
Acta Horticulturae 1065 1267 - 1274 2015
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Acta Horticulturae
To analyze the protein expression of pollen grains and tubes having self-incompatible reactions in Hyuganatsu (Citrus tamurana Hort. ex Tanaka), we cultured mature pollen and treated it with crude extracts from the styles using a mass or single liquid culture system, and we collected pollen grains and tubes for proteome analysis. The relative expression of each identified protein was quantified by an imager and revealed as a value relative to the pollen grain. Eleven identified proteins were remarkably up-regulated (above 1.2-fold) or down-regulated (under 0.8-fold) in treatment with self-incompatible reactions. Eight of these proteins were predicted to be self-incompatible-related proteins with the reported functions. In this study, F-box protein was identified as showing minimum expression in the treatment with crude extracts from styles of Hyuganatsu (self-incompatible reactions). The S-locus gene product expressed in pollen has been identified as an F-box protein in Solanaceae and Rosaceae. It is not clear whether a mechanism similar to that of Solanaceae and Rosaceae exists in the self-incompatible reaction in Hyuganatsu; however, the reduced expression of F-box protein may induce a self-incompatible reaction.
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Yasuda Kiichi, Yahata Masaki, Kunitake Hisato
The Horticulture Journal 0 ( 0 ) 2015
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society for Horticultural Science
Kumquats (<i>Fortunella</i> spp.) is classified into the subfamily Aurantioideae (family Rutaceae). The taxonomy and phylogeny of this genus are complicated and controversial. Therefore, we carried out a estimation based on chromomycin A<sub>3</sub> (CMA) karyotype composition in order to understand the cytogenetics and evolution of the genus <i>Fortunella</i>. Among the 6 <i>Fortunella</i> species examined, <i>Fortunella hindsii</i> var. <i>chintou</i> Swing. (Hongkong kumquat) showed the simplest CMA karyotype composition. On the other hand, close relationships were found among the 3 species <i>F. margarita</i> (Lour.) Swing. (Oval kumquat), <i>F. japonica</i> (Thunb.) Swing. (Round kumquat), <i>F. crassifolia</i> Swing. (Meiwa kumquat). <i>Fortunella polyandra</i> (Ridl.) Tan. (Malayan kumquat) and <i>F. obovata</i> hort. ex Tan. (Changshou kumquat) had type E chromosomes, which is an elemental chromosome type for <i>Citrus</i>. We concluded that there are only two true species for the genus <i>Fortunella</i>, <i>F. hindsii</i> and <i>F. margarita</i> complex, which includes <i>F. margarita</i>, <i>F. japonica</i>, and <i>F. crassifolia</i>, and that <i>F. polyandra</i> and <i>F. obovata</i> should be classified as natural or horticultural hybrids.
DOI： 10.2503/hortj.MI-078
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Li Y., Uchida A., Abe A., Yamamoto A., Hirano T., Kunitake H.
Journal of the American Society for Horticultural Science 140 ( 2 ) 183 - 190 2015
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the American Society for Horticultural Science
© 2015, American Society for Horticultural Science. All rights reserved. Citrus is a genus with gametophytic self-incompatibility, which prevents fertilization by self-pollination. Polyamines (PAs), as a class of active small molecules, widely participate in various cellular activities. To investigate the effects of the changes in PA contents [putrescine, spermidine (SPD), and spermine] on pollen tube growth in self-incompatible (SI) plants, we cultured pollen of Citrus maxima ‘Banpeiyu’ and C. tamurana ‘Hyuganatsu’ in a Citrus mature pollen culture system and detected the PA content changes in pollen by high-performance liquid chromatography (HPLC) analysis during germination and after treatment with crude protein extracts of compatible or incompatible styles. We profiled the PA content changes during the germination course in pollen of ‘Banpeiyu’ and ‘Hyuganatsu’ by HPLC, and identified increases of free and perchloric-acid-insoluble SPD contents in pollen tubes in the incompatible treatment. Exogenous SPD treatment obviously increased the SPD contents in the pollen tube, inhibited pollen tube elongation, and resulted in some abnormal morphological alterations, such as tip inflation, tube content leakage and tip twist, which were observed in both ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes exposed to incompatible treatment. This finding implied that SPD might participate in the SI response in Citrus.
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クロマメノキとラビットアイブルーベリーT100との節間交雑から得られたF1系統の評価 Reviewed
執行 みさと、森田 恭代、西村 謙一、井上 論司、國武 久登、小松 春喜
園芸学研究 13 ( 4 ) 323 - 333 2014.10
Language：Japanese Publishing type：Research paper (scientific journal)
DOI： 10.2503/hrj.13.323
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The phenomenon of root elongation and high respiration actibity in the rolB-gene-enhanced FSL#35 variant of Lotus corniculatus FOX-SR line. Reviewed
Yano Tsubasa, Yamamoto Akihiro, Kunitake Hisato, Saeki Yuichi, Akashi Ryo
Plant Root 8 82 - 91 2014.8
Language：English Publishing type：Research paper (scientific journal)
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我が国自生スノキ属野生種クロマメノキとハイブッシュブルーベリー‘ブルークロップ’との節間交雑から得られたF1系統の評価 Reviewed
執行みさと、具志堅 文、桂川明広、臂 光昭、吉岡克則、鹿毛哲郎、國武久登、小松春喜
園芸学研究 13 ( 2 ) 97 - 106 2014.6
Language：Japanese Publishing type：Research paper (scientific journal)
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Comparison of Cultivars and Seasonal Variation in Blueberry (<i>Vaccinium</i> Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression.
Kai H, Fuse T, Kunitake H, Morishita K, Matsuno K
Medicines (Basel, Switzerland) 1 ( 1 ) 3 - 11 2014.6
Language：Japanese Publishing type：Research paper (scientific journal)
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我が国自生スノキ属野生種クロマメノキとハイブッシュブルーベリー ブルークロップとの節間交雑から得られたF1系統の評価 Reviewed
執行 みさと、具志堅 文、桂川 明広、臂 光昭、吉岡 克則、鹿毛 哲朗、國武 久登、小松 春喜
園芸学研究 13 ( 2 ) 97 - 106 2014.4
Language：Japanese Publishing type：Research paper (scientific journal)
DOI： 10.2503/hrj.13.97
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ブルーベリー近縁種ナツハゼとアラゲナツハゼにおける果実の成熟特性と品質評価 Reviewed
津田浩利, 小島祥子, 大坪早貴, 小松春喜, 國武久登
園芸学研究 13 ( 1 ) 1 - 9 2014.3
Language：Japanese Publishing type：Research paper (scientific journal)
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Efficient in vitro screening for higher soil pH adaptability of intersectional hybrids in blueberry
Tsuda H., Kunitake H., Aoki Y., Oyama A., Tetsumura T., Komatsu H., Yoshioka K.
HortScience 49 ( 2 ) 141 - 144 2014.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：HortScience
We tested efficient in vitro methods for screening the genotypes with higher pH tolerance using multiple shoots of intersectional hybrids between Vaccinium corymbosum 'Spartan' and V. bracteatum. The response of the four hybrid clones tested to different pH levels was clone-dependent in vitro. An apparent difference was found in the rooting rate among the hybrid clones even at higher pH levels; the rooting rates of JM4 (91%) at pH 8.0 indicated a significantly high value compared with other clones (JM1: 24%, JM2: 9%, JM3: 8%, 'Spartan': 0%). Furthermore, JM4 showed constantly high rooting rates (91% to 100%) at all pH levels with no significant differences. Similar differences in the root characters of the hybrids were also confirmed by checking the viability of roots using fluorescein diacetate (FDA)/propidium iodide (PI) staining after dipping the roots of in vitro-produced shoots in liquid medium at different pH levels for 6 hours. These results suggest that an in vitro screening method using the rooting rate of multiple shoots and the viability test of roots by FDA/PI staining as a marker could become a very useful tool for the selection of germplasm with tolerance to higher pH within a short time using small planting spaces. In addition, JM4, which showed a high rooting rate at pH 8.0, could be useful in breeding new cultivars with higher pH tolerance.
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Tsuda Hirotoshi, Kojima Shoko, Otsubo Saki, Komatsu Haruki, Kunitake Hisato
Hort. Res. (Japan) 13 ( 1 ) 1 - 9 2014
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE
To obtain fundamental data for using Natsuhaze and Aragenatsuhaze in the breeding and cultivation of blueberries, we examined the fruit growth characteristics and fruit components. Natsuhaze and Aragenatsuhaze had many fruits per fruit cluster compared with blueberry cultivars, and the fruit clusters were long and slender. Although the maturation period of the fruits within the same fruit cluster was different, the matured fruits did not fall for a long period. Therefore, this species may be harvested by cluster harvesting, as with grapes. The maturation periods of both Natsuhaze and Aragenatsuhaze were from September to November, one to three months later than the latest-maturing blueberry cultivars. Although the sugar-acid ratio in the fruits of Natsuhaze and Aragenatsuhaze was lower than in blueberry cultivars and the taste was inferior, the total anthocyanin content of fruits in Natsuhaze was about 6 times that of blueberries. The total polyphenol content, total proanthocyanidin content, and antioxidant activities showed high values in unripe fruits, and increased as maturity progressed in these species. Mature fruit of Aragenatsuhaze had 3 times more total polyphenols and 17 times more total proanthocyanidin than blueberry cultivars; it was suggested that total polyphenols including proanthocyanidin contribute to antioxidant activities. Natsuhaze and Aragenatsuhaze could be useful breeding materials for new cultivar production by expanding the crop season of blueberries, enabling cluster harvesting, and offering high biological functionality.
DOI： 10.2503/hrj.13.1
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Evaluation of Some Wild Species of Rubus Native to Japan as Breeding Materials. Reviewed
12 ( 4 ) 335 - 342 2013.12
Language：Japanese Publishing type：Research paper (scientific journal)
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Tsuda H., Kunitake H., Yamasaki M., Komatsu H., Yoshioka K.
Journal of the American Society for Horticultural Science 138 ( 4 ) 317 - 324 2013.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the American Society for Horticultural Science
With crosses between colchicine-induced tetraploid shashanbo (Vacciniumbracteatumsection Bracteata) and tetraploid highbush blueberry 'Spartan' (Vaccinium corymbosum section Cyanococcus), intersectional hybrids were produced. The hybridity of these plants was confirmed based on DNA markers and morphological characteristics. The morphological characteristics, blooming date, and ripening period of the hybridswere intermediate between those of the parents. Ploidy analyses by flow cytometry and chromosome counting revealed that these hybrids were tetraploid. Four hybrids set fruit in the field and these two hybrids showed high pollen stainability. It was noteworthy that fruit of two hybrids had high soluble solids concentration compared with 'Spartan' and the fruit pulp of the hybrids was tinged with red as shashanbo. These hybrids could be useful in breeding new cultivars with high sugar content, abundant phytochemicals, extensive environmental adaptability as well as late flowering and fruit maturity.
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Tsuda, H., Kunitake, H., Ryoko Kawasaki-Takaki, R., Nishiyama, K., Yamasaki, M., Komatsu, H., Yukizaki
Plants 2 ( 1 ) 57 - 71 2013.3
Language：English Publishing type：Research paper (scientific journal)
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Antioxidant Activities and Anti-Cancer Cell Proliferation Properties of Natsuhaze (Vaccinium oldhamii Miq.), Shashanbo (V. bracteatum Thunb.) and Blueberry Cultivars.
Tsuda H, Kunitake H, Kawasaki-Takaki R, Nishiyama K, Yamasaki M, Komatsu H, Yukizaki C
Plants (Basel, Switzerland) 2 ( 1 ) 57 - 71 2013.2
Language：Japanese Publishing type：Research paper (scientific journal)
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Evaluation of Some Wild Species of <i>Rubus</i> Native to Japan as Breeding Materials
Shigyo Misato, Chikuma Kouhei, Kunitake Hisato, Komatsu Haruki
Hort. Res. (Japan) 12 ( 4 ) 335 - 342 2013
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE
There is increasing interest in fruits of bramble crops (<i>Rubus</i> spp.) because of the possible benefical health effects associated with their phenolic content. Almost all cultivars are from Europe and the United States, although there are many wild <i>Rubus</i> species native to Japan. In order to obtain basic knowledge for the horticultural application of those wild species, we collected and evaluated some wild species native to Kyusyu Island as genetic resources. All raspberry cultivars were diploid, but almost all wild species were diploid with a different genome size, except for a few hexaploids. The aggregate fruit weights of wild species were not smaller than cultivars except for <i>Rubus lambertianus</i> Seringe. The ripening fruits of some wild species, <i>R. crataegifolius</i> Bubge., <i>R. trifidus</i> Thunb., and <i>R. palmatus</i> Thunb., were higher than the fruits of raspberry cultivars regarding total sugar contents and lower in total organic acid contents. The fruits of <i>R. crataegifolius</i> Bubge., <i>R. palmatus</i> Thunb., and <i>R. lambertianus</i> Seringe also showed a higher total polyphenol content and antioxidant activity than the cultivars. Some wild species native to Japan may be useful as breeding material to establish some new cultivars adaptable to Japan with high quality and functionality.<br>
DOI： 10.2503/hrj.12.335
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Effective micropropagation of rabbiteye blueberries for leaf tea production
Tetsumura T., Kajiwara Y., Honsho C., Sato-Yamauchi M., Sugimoto Y., Kunitake H.
Environmental Control in Biology 50 ( 3 ) 289 - 296 2012.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Environmental Control in Biology
Micropropagation of two clones of rabbiteye blueberry (Vaccinium virgatum Aiton) developed for leaf tea production was investigated for supply in high quantities to plantations and plant factories. Shoots of a new cultivar, 'Kunisato 35 Gou', were multiplied efficiently on MW, a mixture of equal parts of MS and WPM, supplemented with 20 μM zeatin by using 2-node segments. For No. 19, another promising clone, 5-node segments were the most productive for shoot multiplication. Among basal media tested for micropropagation, MW medium was found to be the most efficient. Although shoots of 'Kunisato 35 Gou' grew better in vitro than those of No. 19, both clones had equivalent rooting abilities. More than 90% of the shoots rooted and survived in vermiculite after culturing in Gellan gum-solidified rooting medium without plant growth regulators. The growth of shoots with roots in pots was significantly influenced by the potting soil. The plantlets in the soils mixed with peat moss, which made the soil acid, grew vigorously, while those in the soils without peat moss grew moderately or poorly, depending on soil type.
DOI： 10.2525/ecb.50.289
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Identification of self-incompatibility related proteins in the pistil of Japanese pear (Pyrus pyrifolia (Burm.f.)) by proteome analysis. Reviewed
1 Uchida, U., H. Sassa, S. Takenaka, Y. Sakakibara, M. Suiko, H. Kunitake
Plant Omics Journal ５ ( ４ ) 320 - 325 2012.8
Language：English Publishing type：Research paper (scientific journal)
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Uchida A., Sassa H., Takenaka S., Sakakibara Y., Suiko M., Kunitake H.
Plant OMICS 5 ( 4 ) 320 - 325 2012.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant OMICS
The differences between stylar protein of the Japanese pear (Pyrus pyrifolia (Burm.f.)) cultivars 'Kosui' (S 4 S 5 ) and 'Kikusui' (S 2 S 4 ) were compared by two-dimensional difference gel electrophoresis (2-D DIGE), and were labelled and visualized with different fluorescent dyes (IC3-OSu, IC 5 -OSu) on a single 2-D gel. The individual different expressed proteins spots were subjected to identification. The proteins were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) to identify proteins related to gametophytic self-incompatibility (GSI). S 4 -RNase and thaumatin-like protein 1 were successfully detected as expected in the pistils of 'Kosui' and 'Kikusui'. S 5 -RNase was also detected in the pistils of 'Kosui'. However, we could not detect S 2 -RNase in 'Kikusui' in this study, possibly because the level of expression of S 2 -RNase might be minuscule, or the estimated isoelectric point (pI) of S 2 -RNase (pI:9.26) was more basic than S 4 -RNase (pI: 9.17) and S 5 -RNase (pI: 9.01). These results indicate that proteomic studies are effective tools for detection of the expected proteins and might be helpful for finding the unknown key proteins related to the mechanism of self-incompatibility (SI) in many other SI plants.
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Uchida A., Takenaka S., Takenaka S., Sakakibara Y., Kurogi S., Honsho C., Sassa H., Suiko M., Kunitake H.
Journal of the Japanese Society for Horticultural Science 81 ( 2 ) 150 - 158 2012.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
In Citrus, self-incompatibility (SI) regulation is gametophytic, and this phenomenon is an economically critical problem for some Citrus cultivars without high parthenocarpic ability. Few molecular biological studies of SI in Citrus have been performed, and the molecular mechanism of SI has not been clarified. To investigate the effect of different stages of style development on pollen tube behavior, flower buds of self-incompatible 'Hyuganatsu' (Citrus tamurana hort. ex Tanaka) were histologically assayed. When the flower buds were observed 168 hours after pollination, pollen tubes in the self-pollinated flower buds 1 and 3 days before anthesis (DBA) were arrested in the upper part of the styles, while those in flower buds self-pollinated 5 DBA reached the base of the styles. These results revealed that SI in 'Hyuganatsu' has not yet occurred in the flower buds 5 DBA, but generated in the flower buds 3 DBA. To search SI-related pistil proteins in Citrus, we profiled a number of protein expressions in 'Hyuganatsu' styles of 1, 3, and 5 DBA by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. One-hundred thirty-eight protein spots were significantly different in abundance among the three stages, and 17 up-regulated and 26 down-regulated protein spots could be identified. Among the 17 up-regulated proteins, nine up-regulated proteins exhibited the expression pattern of 1 DBA ≥ 3 DBA > 5 DBA, a pattern which reflected the transmission from SC to SI, evaluated by pollen tube growth. BLASTP homology search against the peptide sequences of these proteins was carried out and predicted the proteins related to the maintenance of cell shape, the signaling of pollen tube growth, the flavonol biosynthesis, the responses to various stresses, photosynthesis, and the methionine metabolic process. Among the nine proteins, some may be the SI-related pistil proteins; however, it was not possible to determine the SI-related key protein of the style in Citrus. © 2012.
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Comprehensive Analysis of Expressed Proteins in the Different Stages of the Style Development of Self-incompatible ‘Hyuganatsu’ (Citrus tamurana hort. Ex Tanaka) Reviewed
Uchida, A., S. Takenaka, Y. Sakakibara, S. Kurogi,, C. Honsho, H. Sassa, M. Suiko and H. Kunitake
J Japan Soc Hort Sci. 81 ( 2 ) 150 - 158 2012.4
Language：English Publishing type：Research paper (scientific journal)
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オリザリンおよびコルヒチン処理によるスノキ属植物における倍数体の作出. Reviewed
津田浩利・小島祥子・鉄村琢哉・小松春喜・國武久登.
園芸学研究 11 ( 2 ) 205 - 212 2012.4
Language：Japanese Publishing type：Research paper (scientific journal)
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ヒュウガナツ（Citrus tamurana hort. ex Tanaka）における成熟花粉の液体培養系の確立. Reviewed
内田飛香・安部秋晴・星野洋一郎・國武久登.
園芸学研究 11 ( 2 ) 173 - 179 2012.4
Language：Japanese Publishing type：Research paper (scientific journal)
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我が国自生のスノキ属植物とブルーベリー栽培品種における植物組織培養と試験管外発根を利用したクローン増殖. Reviewed
山内（佐藤）真希子・津田浩利・荒木啓輔・内田飛香・安田喜一・鉄村琢哉・小松春喜・國武久登.
園芸学研究 11 ( 1 ) 13 - 19 2012.1
Language：Japanese Publishing type：Research paper (scientific journal)
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Unreduced 2n pollen production in 'Nishiuchi Konatsu' Hyuganatsu as inferred by pollen characteristics and progeny ploidy level. Reviewed
Honsho, C., E.Yamamura, K. Tsuruta, Y. Yoshimaru, K. Yasuda, A. Uchida, H. Kunitake and T. Tetsumura.
J Japan Soc Hort Sci. 81 ( 1 ) 19 - 26 2012.1
Language：English Publishing type：Research paper (scientific journal)
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Honsho C., Yamamura E., Tsuruta K., Yoshimaru Y., Yasuda K., Uchida A., Kunitake H., Tetsumura T.
Journal of the Japanese Society for Horticultural Science 81 ( 1 ) 19 - 26 2012.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
Hyuganatsu is a self-incompatible Citrus species with no parthenocarpic traits. Pollinizer requirements and seedy fruit production are therefore a limiting factor for Hyuganatsu growers. 'Nishiuchi Konatsu', a bud mutation of Hyuganatsu, can set fruit by self-pollination and aborts most seeds. To obtain a better understanding of the reproductive mechanisms of 'Nishiuchi Konatsu', its pollen was characterized by pollen observation and viability testing in vitro in addition to pollen tube observation of the pistil following various pollination combinations. Furthermore, the ploidy level of 'Nishiuchi Konatsu' and the progenies obtained after controlled pollination were analyzed using a flow cytometer. As a result, 'Nishiuchi Konatsu' pollen was shown to be functional in vitro, as indicated by both the pollen germination test and FDA staining. A curious feature of 'Nishiuchi Konatsu' pollen is that a giant pollen of about 42-44 μm in diameter, which is about 1.3-fold the average diameter of original Hyuganatsu pollen (33.4 μm), was partially produced. Observation of in vivo pollen germination and pollen tube elongation revealed that Hyuganatsu (or 'Nishiuchi Konatsu') × 'Nishiuchi Konatsu' showed imperfect incompatibility; that is, some pollen tubes were able to enter the style while others could not. Flow cytometry analysis indicated that 'Nishiuchi Konatsu' is diploid while all of its self-pollinated progenies, which were rarely obtained as normal seeds, were tetraploid. Progenies from other combinations were diploid. These results strongly suggest that unreduced 2n pollen production occurred in 'Nishiuchi Konatsu'. In addition to self-compatibilty and seed abortion of 'Nishiuchi Konatsu' self-pollination, it has been reported tha t the proportion of normal to aborted seeds differs according to the cultivar of the seed parent crossed with 'Nishiuchi Konatsu' pollen (Honsho et al., 2009). Taking into account the unreduced pollen production as well as the effect of polyploidization on self-incompatibility and embryo development, we finally discussed why such a phenomenon was induced by pollination with 'Nishiuchi Konatsu' pollen and why 'Nishiuchi Konatsu' became self-compatible. © 2012.
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本勝 千歳, 山村 恵梨, 鶴田 今日子, 芳丸 由佳子, 安田 喜一, 内田 飛香, 國武 久登, 鉄村 琢哉
Journal of the Japanese Society for Horticultural Science 81 ( 1 ) 19 - 26 2012
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：一般社団法人 園芸学会
ヒュウガナツは自家不和合性で，かつ単為結果性を持たない．そのため，受粉樹が必要であることと，果実中に多数の種子が含まれることが，生産者にとって問題であった．このヒュウガナツの枝変わり品種である'西内小夏'は自家受粉で結実し，また種子のほとんどがしいな化する性質を持つ．このメカニズムについての知見を得るために，本研究では花粉の観察，花粉稔性の調査，また花柱内における花粉管伸長の調査を行い，さらに'西内小夏'自身および'西内小夏'を交雑に用いて得られた後代の倍数性を，フローサイトメトリーによって分析した．その結果，まず'西内小夏'花粉は十分な稔性を持っていることが明らかとなった．そして興味深い結果として，'西内小夏'花粉には一般的なヒュウガナツの平均花粉径（33.4 μm）の約 1.3 倍の大きさである巨大花粉が一部生産されていることが確認された．次に花柱内の花粉管伸長を観察したところ，ヒュウガナツあるいは'西内小夏'の雌ずいに'西内小夏'花粉を受粉したとき，多くの花粉が柱頭と花柱の境界周辺で花粉管伸長が阻害されていた一方で，一部の花粉が花柱基部まで伸長していた．そしてフローサイトメトリーの結果，'西内小夏'自体は二倍体であったが，'西内小夏'を自家受粉してまれに得られた正常種子由来の実生は全て四倍体であった．これらの結果は，'西内小夏'が非還元花粉を形成していることを強く示唆している．これまでに'西内小夏'の自家和合化および種子のしいな化に加えて，'西内小夏'の花粉をいくつかの単胚性カンキツに受粉すると，種子親に応じてしいなの形成程度が異なることが報告されている（Honsho ら，2009）．これらの現象について，非還元花粉形成と倍数化が自家不和合性に及ぼす影響を考慮して，考察を行った．<br>
DOI： 10.2503/jjshs1.81.19
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産学官連携によるブルーベリー葉茶の開発(大学発!美味しいバイオ)
國武 久登, 甲斐 孝憲
生物工学会誌 : seibutsu-kogaku kaishi 90 ( 8 ) 520 - 522 2012
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本生物工学会
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自家不和合性の'ヒュウガナツ'（<i>Citrus tamurana</i> hort. ex Tanaka）花柱の異なる発達ステージにおける発現タンパク質の網羅的解析
内田 飛香, 竹中 聡, 榊原 陽一, 黒木 重文, 本勝 千歳, 佐々 英徳, 水光 正仁, 國武 久登
Journal of the Japanese Society for Horticultural Science 81 ( 2 ) 150 - 158 2012
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：一般社団法人 園芸学会
これまでにカンキツにおける自家不和合性の分子生物学的研究が行われてきたが，そのメカニズムについては未だ明らかになっていない．花柱の成育ステージの違いが引き起こす花粉管の挙動の経時的変化を調査するため，自家不和合性品種'ヒュウガナツ'の花蕾の組織学的調査を行った．自家受粉 168 時間後において，開花 1 日前と 3 日前に受粉した花蕾の花粉管は花柱上部に停滞していた．一方，開花 5 日前に受粉した花蕾では花粉管が花柱基部まで到達していた．これらの結果，'ヒュウガナツ'の開花 5 日前の花蕾においては自家不和合性反応は生じていないが，開花 3 日前の花蕾においては自家不和合性反応が生じていることが明らかとなった．そこで，カンキツにおける自家不和合性に関連する雌ずいタンパク質を探索するため，'ヒュウガナツ'の開花 1，3，5 日前の花蕾における発現タンパク質のプロファイリングを二次元電気泳動法と MALDI-TOF/MS 法を用いて行った．3 つの異なるステージにおいて，138 個のタンパク質の発現量に有意な差が観察され，そのうち 17 個の発現量が上昇しているタンパク質と，26 個の発現量が減少しているタンパク質が同定された．17 個の発現量が上昇していたタンパク質のうち，9 個のタンパク質が示す開花 1 日前 ≧ 開花 3 日前 ＞ 開花 5 日前の発現パターンが，花粉管の挙動に基づく自家和合性から自家不和合性への移行を反映していると思われた．BLASTP によるペプチド配列の相同性探索により，これらは細胞形成の維持や花粉管伸長におけるシグナル，フラボノイド生合成系，ストレス応答，光合成系やメチオニン代謝経路に関するタンパク質であると推定された．これら 9 個のタンパク質のうち，いずれかが自家不和合性と関連を持つ花柱タンパク質である可能性があると考えられた．しかしながら，これらの中からカンキツにおける自家不和合性反応の鍵となるタンパク質を明らかにすることはできなかった．<br>
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Nepal A., Sugimoto Y., Nishiwaki A., Kunitake H., Anugroho F., Kirimura M., Muranaka C.
European Journal of Scientific Research 52 ( 1 ) 123 - 131 2011.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：European Journal of Scientific Research
Microbial additive compost on microbial growth, activity and N mineralization of the soil amendments were examined, and their relationships were analyzed. Composts were prepared from commercial cattle manure with/without additive containing thermophilic bacteria. Soils unamended and amended compost with/without additive were incubated at 25oC for 125 days. The microbial additive increased microbial populations and activity during the first 13 days of incubation and accelerated N mineralization, which was enhanced throughout the rest of the incubation. Therefore, N mineralization can be increased by the increased microbial growth and activity caused by the microbial additive. © EuroJournals Publishing, Inc. 2011.
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Effects of microbial additive on composting process and on Swiss chard growth and nutrient uptake
Nepal A., Sugimoto Y., Nishiwaki A., Kunitake H., Anugroho F., Kirimura M., Muranaka C.
European Journal of Scientific Research 52 ( 1 ) 132 - 141 2011.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：European Journal of Scientific Research
We investigated the effect of a microbial additive on composting and on growth of Swiss chard (Beta vulgaris var. cicla). Composts were prepared from commercial cattle manure with/without a microbial additive containing thermophilic bacteria. Compost with microbial additive (AC) had greater temperature rise and microbial activity (measured by ATP levels) during first-phase composting than compost without additive (NAC), possibly because of increased thermophilic bacterial activity. Application of AC in Swiss chard cultivation gave higher SPAD values, photosynthetic activity, leaf yield, and nutrient contents than with NAC. The microbial additive including thermophilic bacteria produced good-quality compost that characterized by lower moisture and C:N ratio, and good growth of Swiss chard. © EuroJournals Publishing, Inc. 2011.
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Abnormality of gamete formation in a pummelo [Citrus maxima (Burm.) Merr.] haploid
Yahata M., Yahata M., Yahata M., Kunitake H., Yasuda K., Hirai T., Yabuya T., Yamashita K., Komatsu H.
Journal of the Japanese Society for Horticultural Science 80 ( 1 ) 14 - 18 2011.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
We investigated female and male gamete formation in a pummelo [Citrus maxima (Burm.) Merr.] haploid. During female gamete formation, no embryo-sac mother cell (EMC) was formed in ovules, and consequently no normal embryo sac was observed at anthesis, which was confirmed as the cause of female sterility in the haploid. During male gamete formation, on the other hand, meiosis in the pollen mother cells (PMCs) of the haploid occurred twice, but abnormalities were observed in most dividing cells. In some dividing cells, univalents did not distribute to the opposite poles at anaphase I; instead, they remained near the equatorial plate. The nine univalents then performed mitosis, which distributed equally forward opposite poles. Furthermore, at the tetrad stage, the tetrad comprised 99.3% of the 'Banpeiyu' pummelo, whereas sporads from monads to hexads with a relatively high frequency of dyads (24.7%) were produced in the haploid. These results suggest that fertile pollen grains observed in the haploid pummelo were produced by abnormalities in the first meiotic division such as first division restitution (FDR). JSHS © 2011.
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Relationship between Weight of Seeds and the Ploidy Level of the Seedlings Obtained from the Cross Between ‘Banpeiyu’ Pummelo and Somatic Hybrid ‘Citrus parental line No.4’.（共著） Reviewed
Yahata, M., T. Sekimoto, K. Yasuda, H. Komatsu and H. Kunitake
Bulletin of the Faculty of Agriculture, University of Miyazaki 57 63 - 70 2011.2
Language：English Publishing type：Research paper (bulletin of university, research institution)
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Analytical study on the relationships between microbial growth and N mineralization influenced by microbial additive compost. "jointly worked" Reviewed
Arati P. N., Y. Sugimoto, A. Nishiwaki, H. Kunitake, F. Anugroho, M. Kirimura and C. Muranaka
European Journal of Scientific Research 52 ( 1 ) 123 - 131 2011.2
Language：English Publishing type：Research paper (scientific journal)
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Effects of microbial additive on composting process and on Swiss chard growth and nutrient uptake. "jointly worked" Reviewed
Arati P. N., Y. Sugimoto, A. Nishiwaki, H. Kunitake, F. Anugroho, M. Kirimura and C. Muranaka
European Journal of Scientific Research 52 ( 1 ) 132 - 141 2011.2
Language：English Publishing type：Research paper (scientific journal)
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Morphological characteristics of fruit in a haploid pummelo."jointly worked" Reviewed
Yahata, M., H. Kunitake, K. Yasuda, T. Yabuya, K. Yamashita, H. Komatsu
宮崎大学農学部研究報告 57 57 - 61 2011.2
Language：English Publishing type：Research paper (bulletin of university, research institution)
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Abnormality of gamete formation in a pummelo [Citrus maxima (Burm.) Merr.] haploid. "jointly worked" Reviewed
Yahata M., H. Kunitake, K. Yasuda, T. Hirai, T. Yabuya, K. Yamashita and H. Komatsu.
Journal of Japanese Society for Horticultural Science 80 ( 1 ) 14 - 18 2011.1
Language：English Publishing type：Research paper (scientific journal)
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ニンポウキンカン珠心胚へのコルヒチン処理によって得た倍数体の特性とそれらの三倍体育種への利用（共著） Reviewed
糠谷綱希・太田知宏・安田喜一・八幡昌紀・國武久登・小松春喜・新居直祐・向井啓雄・原田久・高木敏彦
園芸学研究 10 ( 1 ) 1 - 8 2011.1
Language：Japanese Publishing type：Research paper (scientific journal)
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Abnormality of Gamete Formation in a Pummelo [Citrus maxima (Burm.) Merr.] Haploid
八幡 昌紀, 國武 久登, 安田 喜一, 平井 孝宜, 藪谷 勤, 山下 研介, 小松 春喜
Journal of the Japanese Society for Horticultural Science 80 ( 1 ) 14 - 18 2011
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：一般社団法人 園芸学会
半数体ブンタン［<i>C. maxima</i>（Burm.）Merr.］の雌性および雄性配偶子の形成過程を調査した．雌性配偶子形成では，胚のう母細胞が全く形成されないため，いずれの時期においても正常に発達した胚のうは全く観察されなかった．このことから，半数体ブンタンの雌性不稔は胚のう母細胞の無形成が原因であることが明らかとなった．一方，雄性配偶子形成では，半数体ブンタンは連続した 2 回の分裂を行っていたものの，ほとんどの分裂細胞で減数分裂の異常が認められた．一部の分裂細胞では，第一減数分裂後期においていくつかの一価染色体が両極に分配されず，赤道面付近に残るものや周りの細胞が第二減数分裂過程にもかかわらず，赤道面に 9 個の一価染色体が並列し，両極に分裂するものが観察された．さらに，花粉四分子期において，'晩白柚'では 99.3％が四分子であったのに対し，半数体ブンタンでは，一分子から六分子の小胞子型が観察され，二分子が 24.7％と，非常に高い頻度で出現していた．これらの結果は，半数体ブンタンの花粉が第一分裂復旧（FDR）のような異常によって稔性が回復していることを示唆した．<br>
DOI： 10.2503/jjshs1.80.14
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Nukaya Tsunaki, Ohta Tomohiro, Yasuda Kiichi, Yahata Masaki, Kunitake Hisato, Komatsu Haruki, Nii Naosuke, Mukai Hiroo, Harada Hisashi, Takagi Toshihiko
Hort. Res. (Japan) 10 ( 1 ) 1 - 8 2011
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE
Six lines of Meiwa kumquat (<i>Fortunella crassifolia</i> Swingle) showing tetraploidy (No.5, No.6, No.8, No.12, No.18 and No.19), which had been induced by treating the nucellar embryos with colchicines, were investigated for ploidy level, origin and horticultural characteristics. Furthermore, these tetraploid plants were crossed with diploid kumquats for triploid production. All tetraploid plants were confirmed to be derived from the nucellar embryo of Meiwa kumquat by investigation of chromosome composition with chromomycin A3 staining. Flow cytometric analysis and chromosome observation of young leaves demonstrated that No.8 was a ploidy chimera that was both diploid and tetraploid. Five lines showing tetraploidy had the typical morphological characteristics of tetraploid <i>Citrus</i> plants, such as large leaves, flowers and pollen grains with low pollen fertility. However, these tetraploid plants have desirable traits for kumquat such as thicker pericarp, high sugar content and smaller number of developed seeds. No.8 had horticultural characteristics that were similar to those of tetraploid plants excluding the dermal system. When their polyploids were crossed with some diploid cultivars, triploid progenies were obtained from almost all cross combinations.<br>
DOI： 10.2503/hrj.10.1
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Yahata, M., K. Yasuda, K. Nagasawa, S. Harusaki, H. Komatsu and H. Kunitake
Journal of the Japanese Society for Hortticultural Sciences 79 ( 3 ) 239 - 245 2010.9
Language：English Publishing type：Research paper (scientific journal)
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Investigation of sexual intergeneric haybrid Progenies between Citrus cultivar and Citropsis schweinfurthii
Yasuda, K., H. Kunitake, M. Yahata and R. Matsumoto
Proceedings of the International Society of Citriculture 1 121 - 125 2010.9
Language：English Publishing type：Research paper (international conference proceedings)
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Yahata M., Yasuda K., Nagasawa K., Harusaki S., Komatsu H., Kunitake H.
Journal of the Japanese Society for Horticultural Science 79 ( 3 ) 239 - 245 2010.8
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
To induce haploid plants in Citrus maxima (Burm.) Merr. 'Banpeiyu', we evaluated the effect of pollination with soft X-ray-irradiated pollen on fruit set and seed development, and carried out ovule culture. When 'Banpeiyu' pummelo pistils were pollinated with X-ray-irradiated pollen of 'Fukuhara' sweet orange [C. sinensis (L.) Osbeck], the exposure doses affected the fruit set. The number of seeds per fruit was also affected by the exposure dose, and tended to decrease as the dose increased; however, all developed seeds obtained from these crosses were diploid. In the ovule culture of 'Banpeiyu' pummelo, six embryoids shown haploidy were obtained in all treatments. One haploid plantlet with 9 chromosomes was regenerated from an embryoid in a culture of ovules established 40 days after pollination with 400 Gray (Gy)-irradiated pollen of 'Tosa-buntan' pummelo (C. maxima). This haploid was suggested to be derived from 'Banpeiyu' pummelo by random amplified polymorphic DNA (RAPD) and cleaved amplified polymorphic sequence (CAPS) analysis. JSHS © 2010.
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Yasuda K., Yahata M., Shigyo M., Matsumoto R., Yabuya T., Kunitake H.
Journal of the Japanese Society for Horticultural Science 79 ( 2 ) 129 - 134 2010.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
To obtain cytogenetic information on sexual intergeneric hybrid progenies between Citropsis schweinfurthii and 'Nanpu' tangor (Citrus spp.), we conducted Chromomycin A3 (CMA) chromosome banding and genomic in situ hybridization (GISH) analyses. The CMA karyotype composition of C. schweinfurthii was very simple (14D + 4E), while that of 'Nanpu' tangor showed more complicated chromosome types (1A + 4C + 5D + 8E). Two progenies with C. schweinfurthii as the maternal parent showed a karyotype of 1A + 1C + 11D + 5E, suggesting that each of the type A and C chromosomes was inherited from 'Nanpu' tangor. GISH-CMA analysis in one of these progenies showed a clear distinction between parental chromosomes, and revealed that the hybrid progeny arose due to fertilization between a female gamete with a CMA karyotype composition of 7D + 2E from C. schweinfurthii and a male 1A + 1C + 4D + 3E gamete from 'Nanpu' tangor. The result suggests that C. schweinfurthii had homomorphic homologous chromosomes as a wild species, while 'Nanpu' tangor had heteromorphic homologous chromosomes. © 2010 JSHS.
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Triploid and Aneuploid Hybrids from Diploid-Diploid Intergeneric Crosses between Citrus Cultivar ‘Kiyomi’ Tangor and Meiwa kumquat (Fortunella crassifolia Swingle) for Seedless Breeding of Kumquat.”jointly worked" Reviewed
「Yasuda K.」, 「M. Yahata」, 「H. Komatsu」,「Y. Kurogi」,「H. Kunitake」.
J Japan Soc Hort Sci 79 ( 2 ) 129 - 134 2010.4
Language：English Publishing type：Research paper (scientific journal)
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Yasuda K., Yahata M., Komatsu H., Kurogi Y., Kunitake H.
Journal of the Japanese Society for Horticultural Science 79 ( 1 ) 16 - 22 2010.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
In order to produce new seedless kumquat cultivars, we carried out an intergeneric cross between 'Kiyomi' tangor [Citrus unshiu Marcow. × C. sinensis (L.) Osbeck] and Meiwa kumquat (Fortunella crassifolia Swingle), obtaining 2 normal seeds and 7 undeveloped seeds. These seeds were cultivated on Murashige and Tucker medium, and the 2 normal seeds germinated and developed. The results of genome size analysis by flow cytometry revealed that both seedlings were triploids and that the difference in genome size corresponded to more than one chromosome in the 2 seedlings. Chromosome observation confirmed diploid (2n = 2x = 18) in both parents, aneuploid with 28 chromosomes (2n = 28) for one of the seedlings, and triploid (2n = 3x = 27) for the other seedling. Random amplified polymorphic DNA (RAPD) and cleaved amplified polymorphic sequence (CAPS) analyses proved that the seedlings were intergeneric hybrids between 'Kiyomi' tangor and Meiwa kumquat, with the maternal organelle genome. These hybrids have the potential to be released as a cultivar after further tree and fruit evaluations, and for use as cross-parents in seedless kumquat breeding. © 2010 JSHS.
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Phylogeny and classification of Fortunella (Aurantioideae) inferred from DNA polymorphism."jointly worked" Reviewed
「Yasuda K.」, 「M. Yahata」, 「H. Komatsu」, 「H. Kunitake」.
宮崎大学農学部研究報告 56 103 - 110 2010.2
Language：English Publishing type：Research paper (bulletin of university, research institution)
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Identification of Parental Chromosomes in Sexual Intergeneric Hybrid Progenies between Citrus Cultivar ‘Nanpu’ Tangor and Citropsis schweinfurthii in the Subfamily Aurantioideae. "jointly worked" Reviewed
「Yasuda, K」, 「M. Yahata」,「M. Shigyo」, 「R. Matsumoto」, 「T. Yabuya」, 「H. Kunitake」.
J Japan Soc Hort Sci 79 ( 1 ) 16 - 22 2010.1
Language：English Publishing type：Research paper (scientific journal)
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安田 喜一, 八幡 昌紀, 執行 正義, 松本 亮司, 藪谷 勤, 國武 久登
Journal of the Japanese Society for Horticultural Science 79 ( 2 ) 129 - 134 2010
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：一般社団法人 園芸学会
本研究では，カンキツ栽培品種'南風'タンゴールと <i>Citropsis schweinfurthii</i> 間の有性属間雑種における細胞遺伝学的知見を得るために，Chromomycin A3（CMA）核型分析と genomic <i>in situ</i> hybridization（GISH）を行った．その結果，<i>C. schweinfurthii</i> の CMA 核型構成が 14D + 4E と単純であったのに対し，'南風'タンゴールの CMA 核型構成は 1A + 4C + 5D + 8Eと複雑であった．また，<i>C. schweinfurthii</i> を種子親とする雑種 2 個体の CMA 核型構成はともに 1A + 1C + 11D + 5E であり，これらの A 型と C 型染色体は'南風'タンゴール由来のものと考えられた．さらに，その 1 個体について GISH-CMA 分析を行った結果，両親の染色体を明白に識別することができ，<i>C. schweinfurthii</i> 由来の雌性配偶子 7D + 2E と'南風'タンゴール由来の雄性配偶子 1A + 1C + 4D + 3Eとの受精によることが明らかになった．この結果から，野生種である <i>C. schweinfurthii</i> は同型の CMA 核型が相同染色体として対合するのに対し，'南風'タンゴールは，異型の CMA 核型染色体が対合することが示された．<br>
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安田 喜一, 八幡 昌紀, 小松 春喜, 黒木 義一, 國武 久登
Journal of the Japanese Society for Horticultural Science 79 ( 1 ) 16 - 22 2010
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：一般社団法人 園芸学会
キンカンの新しい無核性品種育成のために，'清見'タンゴール［<i>Citrus unshiu</i> Marcow. × <i>C. sinensis</i> (L.) Osbeck］とニンポウキンカン（<i>Fortunella crassifolia</i> Swingle）間の属間交雑を行った結果，2 個の完全種子と 7 個の不完全種子が得られた．交雑から得られた種子を MT 培地上で培養したところ，2 個の完全種子のみが発芽し，植物体へと成長した．まず，これらの実生の倍数性を確認するために，フローサイトメーターを用いたゲノムサイズ分析を行った．両実生は，ともに三倍性のピークを示したものの，それらの間には染色体 1 本以上に相当するゲノムサイズの差がみられた．このため，染色体の観察を行ったところ，一方の実生が 27 本の染色体を有する三倍体であったのに対し，他方は 28 本の染色体を有する異数体であることが明らかになった．次に，RAPD 分析を行った結果，両実生は両親に特異的なバンドを有しており，雑種であることが確認された．また，オルガネラ DNA 領域の CAPS 分析によって，両雑種が'清見'タンゴールのオルガネラゲノムを有することを確認した．これらの雑種は，すでに着花・結実に至っており，今後キンカンの新品種もしくは無核性品種育成のための中間母本となることが期待される．<br>
DOI： 10.2503/jjshs1.79.16
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八幡 昌紀, 安田 喜一, 長澤 幸治, 春崎 聖一, 小松 春喜, 國武 久登
Journal of the Japanese Society for Horticultural Science 79 ( 3 ) 239 - 245 2010
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：一般社団法人 園芸学会
ブンタン［<i>Citrus maxima</i> （Burm.）Merr.］'晩白柚'の半数体を誘導するために，軟 X 線照射花粉を受粉して着果と種子発育に及ぼす影響を調査するとともに，胚珠培養を行った．軟 X 線を照射した'福原'スイートオレンジ［<i>C. sinensis</i> （L.）Osbeck］の花粉を'晩白柚'に受粉すると，着果率はやや低下した．また，1 果実当たりの種子数は照射量の増加に伴い減少した．しかしながら，これらの交雑から得られた実生はすべて二倍体であった．一方，軟 X 線を照射した'福原'スイートオレンジと'土佐文旦'（<i>C. maxima</i>）花粉を受粉して胚珠培養を行うと，6 系統の半数性を示す胚様体が誘導された．その内，400 Gray 照射した'土佐文旦'花粉を受粉後 40 日に胚珠培養した胚様体から染色体数 9 本を有する半数体植物が 1 個体得られ，RAPD および CAPS 解析によりこの半数体は'晩白柚'由来であることが示された．<br>
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Investigation of sexual intergeneric hybrid progenies between Citrus cultivar and Citropsis schweinfurthii. Reviewed
Yasuda K., Kunitake, H., Yahata, M., Matsumoto, R.
Proceedings of the international society of citriculture 1 121 - 125 2009.9
Language：English Publishing type：Research paper (international conference proceedings)
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Fruit characteristics in autotetraploid meiwa kumquat induced by colchicine treatment to nucellar embryos. Reviewed
T. Nukaya, M. Yahata, K. Suzuki, K. Yasuda, H. Kunitake, H. Komatsu, H. Mukai, H. Harada, T. Takagi
Korean society for horticultural science 50 ( 3 ) 188 - 190 2009.9
Language：English Publishing type：Research paper (scientific journal)
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Evaluation of basal media for micropropagation of four highbush blueberry cultivars. Reviewed
Tetsumura, T., Y. Matsumoto, M. Sato, C. Honsho, K. Yamashita, H. Komatsu, Y. Sugimoto, H. Kunitake
Scientia Horticulturae 119 ( 1 ) 72 - 74 2008.12
Language：English Publishing type：Research paper (scientific journal)
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Evaluation of basal media for micropropagation of four highbush blueberry cultivars
Tetsumura T., Matsumoto Y., Sato M., Honsho C., Yamashita K., Komatsu H., Sugimoto Y., Kunitake H.
Scientia Horticulturae 119 ( 1 ) 72 - 74 2008.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
Murashige and Skoog medium (MS), woody plant medium (WPM), and a mixture of equal parts of MS and WPM (MW) were compared for in vitro shoot proliferation and rooting of four highbush blueberry (Vaccinium spp.) cultivars. During the multiplication stage, the shoots on WPM showed worse growth than the other shoots. The MW produced the best shoot growth. The shoots on MS grew well but tended toward hyperhydricity in the multiplication stage, especially in the case of 'Bluecrop' shoots. Rooting was worst in the shoots multiplied on WPM, while the best rooting percentage of 'Bluecrop' shoots was obtained on MS, and that of 'O'Neal' on MW. The rooted shoots developed a good root system and were easily acclimatized after potting. © 2008 Elsevier B.V. All rights reserved.
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The confirmation of ploidy periclinal chimera and its morphological characteristics in Meiwa kumquat `Yubeni.' Reviewed
Yasada K., H. Kunitake, S. Nakagawa, H. Kuroki, M. Yahata, R. Hirata, Y. Yoshikura, I. Kawakami, Y. Sugimoto
Horticultural Research 7 ( 2 ) 165 - 171 2008.4
Language：Japanese Publishing type：Research paper (scientific journal)
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Yasuda Kiichi, Kunitake Hisato, Nakagawa Syoko, Kurogi Hironori, Yahata Masaki, Hirata Rikiya, Yoshikura Yukihiro, Kawakami Ikuo, Sugimoto Yasuhiro
Hort. Res. (Japan) 7 ( 2 ) 165 - 171 2008
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE
The cultivar 'Yubeni' was discovered as a mutant of Meiwa kumquat (<i>Fortunella crassifolia</i> Swingle) in Kushima city, Miyazaki. According to the results of flow cytometry analysis and morphological investigation of several tissues and organs, 'Yubeni' was supposed to be a ploidy periclinal chimera with diploid (2n = 2X = 18) in the outermost layer (histogenic layer L-I) and tetraploid (2n = 4X = 36) inner layer (histogenic layer L-II and L-III) in the shoot apical meristem. Compared with the diploid line, it showed similar morphology to the tetraploid line such as larger leaves, flowers and pollen grains. The weight and size of the fruit were both greater than those of the diploid line. Furthermore, this cultivar has desirable traits for kumquat such as smaller number of developed seed and thicker pericarp.<br>
DOI： 10.2503/hrj.7.165
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Yasuda Kiichi, Kunitake Hisato, Nakagawa Syoko, Kurogi Hironori, Yahata Masaki, Hirata Rikiya, Yoshikura Yukihiro, Kawakami Ikuo, Sugimoto Yasuhiro
Horticultural research (Japan) 7 ( 2 ) 165 - 171 2008
Language：Japanese Publishing type：Research paper (scientific journal)
The cultivar 'Yubeni' was discovered as a mutant of Meiwa kumquat (Fortunella crassifolia Swingle) in Kushima city, Miyazaki. According to the results of flow cytometry analysis and morphological investigation of several tissues and organs, 'Yubeni' was supposed to be a ploidy periclinal chimera with diploid (2n = 2X = 18) in the outermost layer (histogenic layer L-I) and tetraploid (2n = 4X = 36) inner layer (histogenic layer L-II and L-III) in the shoot apical meristem. Compared with the diploid line, it showed similar morphology to the tetraploid line such as larger leaves, flowers and pollen grains. The weight and size of the fruit were both greater than those of the diploid line. Furthermore, this cultivar has desirable traits for kumquat such as smaller number of developed seed and thicker pericarp.
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Inoue K., Kato T., Nobukuni A., Kunitake H., Yabuya T.
Scientia Horticulturae 110 ( 4 ) 334 - 339 2006.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
Characteristics such as flower form, size and color of outer and inner perianths, anthocyanins in outer perianths, size, color and fertility of pollen and self-fertility of diploid and tetraploid lines regenerated via protoplast culture of Iris fulva were examined and compared with those of the diploid wild line. Among these characteristics, flower form, inner and outer perianth sizes of the tetraploid lines were noticeable, because these lines had upward flower forms and bigger flowers than diploid lines. Furthermore, reciprocal crosses between diploid or tetraploid lines of I. fulva and I. ensata and those of I. fulva and I. laevigata were performed. Three seedlings were obtained from the cross of tetraploid I. fulva × diploid I. laevigata through embryo rescue. One of them was identified as the interspecific hybrid between tetraploid I. fulva and I. laevigata by flow cytometoric (FCM), cytological and molecular (RAPD) analyses. This is the first report on production of hybrids from these lines. I. fulva has unique brown flowers, and this trait could be very useful for flower color breeding of I. laevigata which lacks this color. Therefore, the hybrid of I. fulva (4×) × I. laevigata may be the best available gene source for brown color breeding of this species. © 2006 Elsevier B.V. All rights reserved.
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Yahata, M., H. Kunitake, K. Yasuda, K. Yamashita, H. Komatsu, R. Matsumoto
Journal of American Society for Horticultural Science 131 ( 6 ) 764 - 769 2006.11
Language：English Publishing type：Research paper (scientific journal)
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Characterization of tetraploid plants regenerated via protoplast culture of Iris falva and their crossability with Japanese irises. （共著） Reviewed
K. Inoue, T. Kato, N. sami, H. Kunitake, T. Yabuya
Scientia Horticulturae 110 334 - 339 2006.11
Language：English Publishing type：Research paper (scientific journal)
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Yahata M., Kunitake H., Yasuda K., Yamashita K., Komatsu H., Matsumoto R.
Journal of the American Society for Horticultural Science 131 ( 6 ) 764 - 769 2006.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the American Society for Horticultural Science
The reciprocal crosses between two citrus cultivars and Citropsis schweinfurthii (Engl.) Swing. & M. Kell. were conducted. The cross between 'Nanpu' tangor {'Kiyomi' tangor (Citrus unshiu Marc. × C. sinensis Osbeck) × 'Fairchild' tangerine-tangelo [clementina (C. clementina hort. ex Tanaka) × 'Orlando' tangelo (C. paradisi Macf. × C. reticulata Blanco)]} and C. schweinfurthii produced some developed seeds with an average weight approximately 1/10 of that of the seeds obtained from open pollination in 'Nanpu' tangor. These seeds germinated on Murashige and Tucker medium, and three and 28 seedlings were obtained from crosses using C. schweinfurthii as the female and the male parent, respectively. The absolute nuclear genome size of these seedlings [∼0.84 pg of DNA content per somatic nucleus (2C)] was intermediate of that of the 'Nanpu' tangor (0.78 pg/2C) and C. schweinfurthii (0.90 pg/2C) seedlings. The chromosome counts of the young leaves revealed that they were diploids (2n = 2X = 18). Furthermore, the hybridity of the seedlings obtained from the reciprocal crosses between 'Nanpu' tangor and C. schweinfurthii was confirmed by randomly amplified polymorphic DNA (RAPD) analysis and cleaved amplified polymorphic sequence (CAPS) analysis. These hybrids will be utilized as important materials for investigating the phylogenic relationships between these genera in the subfamily Aurantioideae.
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Kunitake, H., H. Tsuda, R. Takaki, Y. Ohno, Y. Kuroki, K. Yoshioka, T. Kage, T. Ito, H. Komatsu
Horticultural Research 5 ( 2 ) 105 - 110 2006.6
Language：Japanese Publishing type：Research paper (scientific journal)
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Kunitake Hisato, Tsuda Hirotoshi, Takagi Ryoko, Ohno Yukinori, Kuroki Yoshikazu, Yoshioka Katsunori, Kage Tetsurou, Ito Toshiaki, Komatsu Haruki
Hort. Res. (Japan) 5 ( 2 ) 105 - 110 2006
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE
The possibility of using wild blueberry Shashanbo (Vaccinium bracteatum Thunb.) as a rootstock for cultivating northern highbush blueberry in a warm region was investigated. The survival ratios of three different cultivars ('Berkeley', 'Bluecrop', and 'Earliblue') grafted on 3-year-old seedlings of Shashanbo plants were 85.7 to 100%. Furthermore, 41 cultivars included in highbush blueberries and rabbiteye blueberries were successfully grafted on seedlings of Shashanbo rootstock. There were no signs of incompatibility even four years after grafting, and anatomical observations of graft unions supported this result. The shoot growth and fruit weight of 'Earliblue' grafted on seedlings of Shashanbo rootstock were significantly higher than those of plants on their own roots. However, the shoot growth of 'Earliblue' grafted on rooted cuttings of rabbiteye blueberries ('Homebell' and 'Tifblue') was significantly higher than that of those on seedlings of Shashanbo rootstock. In addition, there were no differences in concentrations or component ratio of sugars and organic acids on any rootstock. These findings suggest that Shashanbo has graft compatibility with blueberry and that seedlings could be used as a rootstock for cultivating northern highbush blueberries in southern Kyushu.
DOI： 10.2503/hrj.5.105
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共著者：Yahata, M., H. Kunitake, T. Yabuya, K. Yamashita, Y. Kashihara, H. Komatsu.
Journal of the American Society for Horticultural Science 130 ( 1 ) 34 - 40 2005.11
Language：English Publishing type：Research paper (scientific journal)
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Yahata M., Kunitake H., Yabuya T., Yamashita K., Kashihara Y., Komatsu H.
Journal of the American Society for Horticultural Science 130 ( 6 ) 899 - 903 2005.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the American Society for Horticultural Science
To produce the homozygous strain of a haploid plant derived from small seed-derived seedlings of 'Banpeiyu' pummelo (Citrus grandis Osbeck), we carried out colchicine treatment to axillary shoot buds of the haploid. Many shoots with cytochimeras (X+2X and 2X+4X) arose from the colchicine-treated axillary buds. When cytochimeric buds of 2X+4X were top-grafted onto trifoliate orange [Poncirus trifoliata (L.) Raf.], a complete diploid shoot with 18 chromosomes was obtained from the cytochimera. This diploid strain showed vigorous growth compared with the original haploid. The leaf weight per unit area and the stomata size in this diploid were significantly larger than those of the original haploid plant, and were almost equal to those of 'Banpeiyu' pummelo. The diploid strain was confirmed to be a doubled haploid of a haploid from 'Banpeiyu' pummelo, based on random amplified polymorphic DNA (RAPD) analysis and chromosome composition analysis by chromomycin A 3 (CMA) staining.
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共著者：Yahata, M., H. Kurogi, H. Kunitake, K. Nagano, T. Yabuya, K. Yamashita , H. Komatsu
Journal of the Japanese Society for Horticultural Science 74 ( 4 ) 281 - 288 2005.7
Language：English Publishing type：Research paper (scientific journal)
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Yahata M., Kurogi H., Kunitake H., Nagano K., Yabuya T., Yamashita K., Komatsu H.
Journal of the Japanese Society for Horticultural Science 74 ( 4 ) 281 - 288 2005.7
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
To evaluate the reproductive potential of female and male gametes in a haploid plant derived from seedlings of 'Banpeiyu' pummelo, we performed crosses between the haploid and several diploid citrus cultivars. In the crosses with the diploid cultivars as pollen parents, no fruit set on the haploid in all cross combinations. However, developed seeds were obtained in four cross combinations, when some monoembryonic diploid cultivars were pollinated with pollen of the haploid. These seeds germinated normally and developed into diploid, vigorous seedlings with large wing leaves, typical of 'Banpeiyu' pummelo. Furthermore, the hybridity of a seedling obtained from the cross between 'Kiyomi' tangor and the haploid was confirmed by random amplified polymorphic DNA (RAPD) analysis and chromosome composition by chromomycin A 3 (CMA) staining. These results suggest that the haploid produced fertile pollen grains (n=9).
DOI： 10.2503/jjshs.74.281
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K. Kawase, M. Yahata, S. Nakagawa, K. Haraguchi, H. Kunitake
Horticultural Research 4 ( 2 ) 141 - 146 2005.6
Language：Japanese Publishing type：Research paper (scientific journal)
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共著者：Takami, K., A. Matsumaru, M.Yahata, H. Kunitake, H. Komatsu.
Sexual Plant Reproduction 18 ( 1 ) 21 - 28 2005.6
Language：English Publishing type：Research paper (scientific journal)
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Takami K., Matsumaru A., Yahata M., Kunitake H., Komatsu H.
Sexual Plant Reproduction 18 ( 1 ) 21 - 28 2005.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Sexual Plant Reproduction
Embryogenic protoplasts of 'Shogun' mandarin (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from Citropsis gabunensis Swing. & M. Kell, and two green embryoids were regenerated after 3 months of culture. Two months after transfer to the regeneration medium, numerous plantlets were obtained from the embryoids. These plants grew vigorously, had well-developed root systems, and exhibited leaf characteristics intermediate to those of the parents. The absolute nuclear genome size of the regenerated plant SH2 (1.75 pg/4C) was the sum of those of the 'Shogun' mandarin (0.75 pg/2C) and C. gabunensis (0.97 pg/2C). The chromosome counts of the young leaves revealed that they were tetraploids (2n=4x=36). Random amplified polymorphic DNA (RAPD) analysis of the two lines (SH1 and SH2) verified their hybridity. Cytoplasmic genome analysis using universal primers reveal that their chloroplast (cp) DNA banding pattern is identical to that of C. gabunensis, while the banding pattern of the mitochondrial (mt) DNA is identical to that of the 'Shogun' mandarin. These somatic hybrids are important materials for investigating the phylogenetic relationships between these two genera in the subfamily Aurantioideae. © Springer-Verlag 2005.
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共著者：Yahata, M., S. Harusaki, K. Takami, P. Toolapong, H. Kunitake, T. Yabuya, K. Yamashita , H. Komatsu
Journal of the American Society for Horticultural Science 130 ( 1 ) 34 - 40 2005.1
Language：English Publishing type：Research paper (scientific journal)
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Yahata M., Harusaki S., Komatsu H., Takami K., Kunitake H., Yabuya T., Yamashita K., Toolapong P.
Journal of the American Society for Horticultural Science 130 ( 1 ) 34 - 40 2005.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the American Society for Horticultural Science
The morphological characteristics and reproductive potential of a haploid plant obtained from the cross between 'Banpeiyu' pummelo (Citrus grandis) and 'Ruby Red' grapefruit (C. paradisi Macf.) were investigated. The haploid was confirmed to be derived from female gamete of 'Banpeiyu' pummelo by isozyme and random amplified polymorphic DNA (RAPD) analysis. Flow cytometry analysis revealed that the haploidy was maintained in several tissues and organs of this plant. It also had the typical morphology of a haploid, such as small leaves and flowers, and had slightly fertile pollen grains. Furthermore, diploid progenies were obtained from the cross between 'Kiyomi' tangor and the haploid. The hybridity of these seedlings was confirmed by assessment of the leaf characteristics and RAPD analysis. These results suggest that this haploid produced fertile unreduced pollen grains (n = 9).
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Kunitake Ayumi, Iwasaki Tatsuma, Hidaka Nami, Nagamachi Shigeki, Katsuki Hiroshi, Uno Takeshi, Takasaki Mayumi
PAIN RESEARCH 20 ( 3 ) 117 - 125 2005
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本疼痛学会
The effects of spinal cord stimulation (SCS) on the neuronal activity of the brain were examined by single photon emission computed tomography (SPECT) in patients with chronic neuropathic pain. Regional cerebral blood flow (CBF) in each cortical area and the thalamus decreased in several patients without SCS. Patients with central pain due to thalamic hemorrhage showed a decrease in rCBF in the thalamus contralateral to the painful side. During the stimulation period in SCS, parietal rCBF decreased on the side contralateral to the pain. In contrast, rCBF increased in the bilateral frontal and anterior cingulate cortex and in the contralateral temporal lobe in half of the patients in whom SCS was effective in relieving pain. The decrease in thalamic rCBF in two patients with central pain was improved by the SCS therapy; however, pain was relieved in only one of them. In the majority of patients in whom SCS was not effective, there was no change in rCBF in various cortical areas, even after SCS. These results suggest that, in patients with chronic neuropathic pain, SCS modulates the neuronal activities of several brain areas that are believed to be associated with pain processing.
DOI： 10.11154/pain.20.117
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河瀬 憲次, 八幡 昌紀, 中川 匠子, 原口 加奈, 國武 久登
園芸学研究 4 ( 2 ) 141 - 146 2005
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE
実生の根の形態を指標としてニンポウキンカンの珠心胚実生から四倍体を選抜し, その形態調査を行った.<br>ニンポウキンカンの完全種子を播種して得られた457本の実生から, 太くて短い細根を有する1個体を見出した. この実生の倍数性を調査した結果, 36本の染色体を有しており, 四倍体であることが明らかとなった. また, RAPD分析により, この四倍体はニンポウキンカンの珠心胚由来であることが推察された. この四倍体の形態について調査したところ, 二倍体と比べ, 葉は丸くて厚く, 気孔, 花および花粉は大きくなっており, 四倍体特有の形態を示した. 四倍体の果実は, 果実の重さや大きさにおいて二倍体と同等の形質を示した. さらに, 可食部である果皮が厚くなり, 完全種子数が少なくなっていた. 将来, 本研究で得られたニンポウキンカンの四倍体は直接的な品種としての利用や三倍体育種の親として重要な素材となるであろう.
DOI： 10.2503/hrj.4.141
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二倍体カンキツ品種との交雑による半数体ブンタンの生殖機能の評価
八幡 昌紀, 黒木 宏憲, 國武 久登, 長野 克也, 藪谷 勤, 山下 研介, 小松 春喜
園藝學會雜誌 74 ( 4 ) 281 - 288 2005
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：園藝學會
'晩白柚'の実生から得られた半数体における雌性および雄性配偶子の生殖機能を評価するために, 半数体と種々の二倍体カンキツ品種との交雑を行った.半数体に二倍体の花粉を授粉した場合では全く着果しなかったが, 半数体を花粉親とした場合, 4つの交雑組合せにおいて完全種子が得られた.これらの完全種子は正常に発芽し, 多くの二倍体実生が得られた.これらの実生は旺盛に成長し, '晩白柚'の形態的特徴である翼葉を有していた.さらに, '清見'と半数体との交雑から得られた1個体の実生について, RAPD分析およびCMA染色による染色体構成を解析したところ, 雑種であることが確認された.これらの結果から, 本半数体では正常な花粉(n=9)が形成されていることが示唆された.
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Kawase Kenji, Yahata Masaki, Nakagawa Syoko, Haraguchi Kana, Kunitake Hisato
Horticultural research (Japan) 4 ( 2 ) 141 - 146 2005
Language：Japanese Publishing type：Research paper (scientific journal)
A tetraploid of Meiwa kumquat (Fortunella crassifolia Swingle) was selected from nucellar seedlings using root morphology, and its morphological characteristics were investigated. One seedling with thick short rootlets was obtained from 457 seedlings. Flow cytometry and chromosome observation demonstrated that the seedling was tetraploid (2n=4X=36). This tetraploid was presumed by Randam Amplified Polymorphic DNA (RAPD) analysis to have derived from nucellar embryos. The tetraploid showed similar morphology to other Citrus tetraploids such as thick, round leaves and larger stomata, flowers, and pollen grains compared with the original diploid. The tetraploid fruit was almost equal in weight and size to that of the diploid, but had a thicker peel and smaller number of developed seeds than the diploid fruit. In the future, the tetraploid obtained in the present study will be important material as a new cultivar and a parent of triploid breeding.
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Efficient Production of Polyploid Plants via Protoplast Culture of Iris fulva
INOUE Kouichi, KATO Tomomi, KUNITAKE Hisato, YABUYA Tsutomu
Cytologia : international journal of cytology 69 ( 3 ) 327 - 333 2004.11
Language：Japanese Publishing type：Research paper (scientific journal)
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Efficient production of polyploid plants via protoplast culture of Iris fulva. Reviewed
共著者：Inoue, K., T. Kato, H. Kunitake , T. Yabuya
Cytologia 69 ( 3 ) 327 - 333 2004.9
Language：English Publishing type：Research paper (scientific journal)
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Efficient production of polyploid plants via protoplast culture of Iris fulva
Inoue K., Kato T., Kunitake H., Yabuya T.
Cytologia 69 ( 3 ) 327 - 333 2004.9
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cytologia
In protoplast culture of the Louisiana iris species, Iris fulva (2n=42), N6 medium supplemented with 1 mg/l 2,4-D, 1 mg/l kinetin, 200 mg/l casein hydrolysate, 250 mg/l proline, 0.3 M glucose and 2.5 g/l gellan gum was suitable for cell division and colony formation. When colonies formed were transferred to plant growth regulator free MS medium, 61 plant lines were regenerated via somatic embryogenesis. Flow cytometric analysis revealed that leaf tissue of I. fulva consisted of 2C, 4C and 6C cells. Consequently, we identified this species as polysomatic in the genus Iris. Such polysomaty was also recognized in the suspension calli. Sixty one plant lines obtained by protoplast culture consisted of 25 diploid (41.0%), 1 triploid (1.6%), 29 tetraploid (47.5%) and 6 hexaploid lines (9.9%). In addition, chromosome analysis showed that somatic chromosome numbers of the diploid, hypertriploid and tetraploid lines were 2n=42, 67 and 84, respectively. On the other hand, all lines regenerated from suspension culture of I. fulva were diploid lines in contrast to the results from protoplast culture. Thus, it can be concluded that protoplast culture of this species is an efficient technique for production of polyploid plants.
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共著者：Takami, K., A. Matsumaru, M. Yahata, H. Kunitake , H. Komatsu
Plant Cell Reports 23 ( 1 ) 39 - 45 2004.8
Language：English Publishing type：Research paper (scientific journal)
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Takami K., Matsumara A., Yahata M., Imayama T., Kunitake H., Komatsu H.
Plant Cell Reports 23 ( 1-2 ) 39 - 45 2004.8
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant Cell Reports
Intergeneric somatic hybrids between embryogenic callus-derived protoplasts of round kumquat (Fortunella japonica Swingle) and 'Morita navel' orange (Citrus sinensis Osbeck) were produced by electrofusion. Among the eight different fusion strains obtained, six showed normal morphology, whereas the remaining two showed malformation. All the regenerated plants were intermediate in leaf morphology and had thick and round leaves, which are typical characteristics of polyploids. Ploidy analyses by flow cytometry and chromosome counting in root-tip cells revealed that these plants are amphidiploid (2n=4x=36). Hybridity of the fusion products was confirmed by random amplified polymorphic DNA and cleaved amplified polymorphic sequence (CAPS) analyses. Furthermore, analyses of chloroplast (cp) and mitochondrial (mt) DNA by CAPS showed that these somatic hybrids contained cp- and mt-DNA of round kumquat without recombination in the regions analyzed. © Springer-Verlag 2004.
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Yahata, M., Y. Kashihara, H. Kunitake, H. Komatsu
Horticultural Research 3 ( 1 ) 11 - 16 2004.4
Language：English Publishing type：Research paper (scientific journal)
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Yahata Masaki, Kashihara Yukiko, Kurogi Hironori, Kunitake Hisato, Komatsu Haruki
Hort. Res. (Japan) 3 ( 1 ) 11 - 16 2004
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE
In vitro chromosome doubling of nucellar embryos was investigated in Meiwa kumquat (Fortunella crassifolia Swingle). Nucellar embryos were aseptically treated with two antimitotic agents, colchicine and oryzalin at different concentrations and times. After these treatments, nucellar embryos were successively cultured on MT medium containing 500 mg/liter^<-1> malt extract. Ploidy levels of seedlings were evaluated by both flow cytometry and chromosome counting of root tips. Frequency of tetraploid production depended on the kind of antimitotic agents as well as on the concentration and time of treatment. Among the treatment conditions tested, colchicine gave better results than oryzalin on inducing chromosome doubling. Tetraploid plants were obtained from approximately 50% of the nucellar seedlings treated with colchicine at 0.05% for 48 hours. These tetraploids showed poor growth during the early stage after acclimatization, but grew as vigorously as the diploid plants when grafted onto 3-year-old seedlings of trifoliate orange. These results showed that colchicine treatment combined with embryo culture is particularly efficient to induce chromosome doubling of nucellar embryos in Meiwa kumquat.
DOI： 10.2503/hrj.3.11
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Yahata Masaki, Oka Nobutaka, Kunitake Hisato, Yamaguchi Seiji, Komatsu Haruki
Horticultural research (Japan) 2 ( 4 ) 247 - 252 2003.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society for Horticultural Science
Relationship between weight of seeds and ploidy level of the seed-derived plantlets obtained from reciprocal crosses between diploid 'Banpeiyu' pummelo and two tetraploid varieties were examined to efficiently select triploid progenies for further breeding of the pummelo. Percentages of fully developed seeds obtained from the cross of 'Banpeiyu' pummelo with 4X Yuzu or 4X Natsudaidai were decreased by 48.0% and 8.4% as compared with the control, respectively. Seedlings obtained from these two crosses included triploids, tetraploids and hexaploid. 'Banpeiyu' pummelo as seed parent yielded a high percentage of triploids (88.0%) when crossed with 4X Yuzu, but low percentage (30.2%) when crossed with 4X Natsudaidai. In both crosses, the average weight of triploid seeds was significantly lighter than that of tetraploid seeds. Furthermore, all seedlings derived from undeveloped seeds were triploids. Percentages of fully developed seeds obtained from 4X Yuzu or 4X Natsudaidai X 'Banpeiyu' pummelo were not different from those of seeds from open pollinated fruits, but weights of these seeds were extremely decreased compared to seeds from open pollinated fruits. Approximately 50% of in vitro cultured seedlings obtained from both crosses were triploid, while the others were tetraploids, which might be derived from nucellar embryos of seed parents. There was no difference in the average weights of seeds between have seeds yielding triploid seedlings and those yielding tetraploids that might have originated from nucellar tissue.
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M. Yahata, N. Oka, H. Kunitake, H. Komatsu
2 ( 4 ) 247 - 252 2003.12
Language：Japanese Publishing type：Research paper (scientific journal)
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Yahata Masaki, Oka Nobutaka, Kunitake Hisato, Yamaguchi Seiji, Komatsu Haruki
Hort. Res. (Japan) 2 ( 4 ) 247 - 252 2003
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE
Relationship between weight of seeds and ploidy level of the seed-derived plantlets obtained from reciprocal crosses between diploid 'Banpeiyu' pummelo and two tetraploid varieties were examined to efficiently select triploid progenies for further breeding of the pummelo. Percentages of fully developed seeds obtained from the cross of 'Banpeiyu' pummelo with 4X Yuzu or 4X Natsudaidai were decreased by 48.0% and 8.4% as compared with the control, respectively. Seedlings obtained from these two crosses included triploids, tetraploids and hexaploid. 'Banpeiyu' pummelo as seed parent yielded a high percentage of triploids (88.0%) when crossed with 4X Yuzu, but low percentage (30.2%) when crossed with 4X Natsudaidai. In both crosses, the average weight of triploid seeds was significantly lighter than that of tetraploid seeds. Furthermore, all seedlings derived from undeveloped seeds were triploids.<br> Percentages of fully developed seeds obtained from 4X Yuzu or 4X Natsudaidai × 'Banpeiyu' pummelo were not different from those of seeds from open pollinated fruits, but weights of these seeds were extremely decreased compared to seeds from open pollinated fruits. Approximately 50% of <i>in vitro</i> cultured seedlings obtained from both crosses were triploid, while the others were tetraploids, which might be derived from nucellar embryos of seed parents. There was no difference in the average weights of seeds between have seeds yielding triploid seedlings and those yielding tetraploids that might have originated from nucellar tissue.<br>
DOI： 10.2503/hrj.2.247
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共著者:Kunitake, H., K. Nagasawa, K. Takami, H. Komatsu
Plant Biotechnology 19 ( 5 ) 345 - 352 2002.12
Language：English Publishing type：Research paper (scientific journal)
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共著者:Rachanee C., H. Kunitake, N. Sattayasai, S. Nuchadomrong
Science Asia 28 ( 3 ) 221 - 225 2002.9
Language：English Publishing type：Research paper (scientific journal)
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Kunitake H., Nagasawa K., Komatsu H.
Plant Biotechnology 19 ( 5 ) 345 - 352 2002
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant Biotechnology
Somatic hybrids were produced between embryogenic callus protoplasts of 'Shogun' mandarin (Citrus reticulata Blanco, 2n=2x=18) and leaf protoplasts of grapefruit (C. paradisi Macf., 2n=2x=18) by electrofusion. Hybridity of the two regenerated plants was confirmed by leaf characteristics and nuclear genome analysis using RAPD and PCR RFLP. Flow cytometry and root-tip cell count of the two hybrids revealed a chromosome number of 27. These triploid somatic hybrids on transfer to field showed normal morphology and vigorous growth. Field performances as well as disease resistance are under investigation. © 2002, Japanese Society for Plant Cell and Molecular Biology. All rights reserved.
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Production of inter- specific hybrids between Lycoris incarnata and four other species through embryo culture. Reviewed
共著者:Biao Ma, I. Tarumoto, N. Nkakamura , H. Kunitake
Journal of the Japanese Society for Horticultural Science 70 ( 6 ) 697 - 703 2001.12
Language：English Publishing type：Research paper (scientific journal)
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Ma B., Tarumoto I., Nakamura N., Kunitake H.
Journal of the Japanese Society for Horticultural Science 70 ( 6 ) 697 - 703 2001.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the Japanese Society for Horticultural Science
Interspecific crosses were made between a sterile species (L. incarnata) and four fertile species (L. sanguinea, L. sprengeri, L. radiata var. pumila (L. pumila, hereafter), and L. aurea) to breed varieties with new characters. Embryos, excised from ovules 20-30 days after pollination, were cultured on a modified MS medium containing 500 mg· liter -1 casein hydrolysate (Ma et al., 2001). Interspecific crosses using L. incarnata as a female parent yielded 26 hybrid plants, whereas that of L. sanguinea X L. incarnata produced none. Cytological studies revealed that all hybrids were triploid, which would have originated from the fertilization of an unreduced 2n - egg of the female parent (L. incarnata) and a normal haploid gamete of the male parent. The hybridity was also confirmed by isozyme analysis in 26 hybrid plants and partly by character observation in hybrid plants of L. incarnata X L. pumila.
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Effects of temperature and CO2 concentration on the growth of Citrus plantlets. "jointly worked" Reviewed
K. Nagasawa, H. Kunitake, H. Komatsu
Proceedings of the school of Agriculture, Kyushu Tokai University 20 ( 1 ) 9 - 14 2001.4
Language：Japanese Publishing type：Research paper (bulletin of university, research institution)
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Production of triploid somatic hybrids between mandarin and grapefruit through electrofusion. Reviewed
H. Kunitake
Journal of Plant Biotechnology 2 ( 2 ) 109 - 113 2000.4
Language：English Publishing type：Research paper (scientific journal)
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Determination of ploidy levels of Citrus species using flow cytometry. "jointly worked" Reviewed
S. Harusaki, D. Kokuryo, H. Kunitake, H. Komatsu
Proceedings of the school of Agriculture, Kyushu Tokai University 19 ( 1 ) 45 - 52 2000.3
Language：Japanese Publishing type：Research paper (scientific journal)
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Survey and evaluation of old mango trees in Ishigaki city, Okinawa. "jointly worked" Reviewed
K. Sakihara, S. Ogawa, K. Kitajima, N. Nakazato, Y. Kawamitsu, H. Tsukayama, H. Kunitake, H. Komatsu
19 ( 1 ) 53 - 60 2000.3
Language：Japanese Publishing type：Research paper (scientific journal)
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Effect of in vitro environmental conditions on somatic embryogenesis and its germination from necellar calli of Ohta Ponkan(Citrus reticulata Blanco.)."jointly worked" Reviewed
Nagasawa, K., T. Kouichi, H. Kunitake, H. Komatsu
Proceedings of school of agriculture, Kyushu Tokai University 18 11 - 18 1999.3
Language：English Publishing type：Research paper (scientific journal)
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Polyphenols in Fararia x ananassa. "jointly worked" Reviewed
5 ( 2 ) 201 - 205 1998.12
Language：Japanese Publishing type：Research paper (scientific journal)
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Somaclonal and chromosomal effects of genotype, ploidy and culture duration in Asparagus officinalis L. Reviewed
共著者: Kunitake, H., T. Nakashima, K. Mori , M. Tanaka
Euphytica 102 ( 3 ) 309 - 316 1998.7
Language：English Publishing type：Research paper (scientific journal)
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Somatic embryogenesis and its application for breeding and micropropagation in asparagus (Asparagus officinalis L.). Reviewed
共著者: Kunitake, H. , M. Mii
Plant Biotechnology 15 ( 2 ) 51 - 61 1998.6
Language：English Publishing type：Research paper (scientific journal)
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Kunitake H., Nakashima T., Mori K., Tanaka M.
Euphytica 102 ( 3 ) 309 - 316 1998
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Euphytica
Chromosome and morphological variations of embryogenic calli-derived plants of gynogenic haploid, diploid, triploid and tetraploid asparagus (Asparagus officinalis L.) were investigated. Artificial tetraploids were produced using colchicine treatment of seeds of diploid cultivar, 'Poultom'. 'Haidel' (2X) was crossed with the artificial tetraploids, from which one gynogenic haploid, one diploid, 6 triploid, 3 mixoploids were obtained. Embryogenic calli were first obtained from crown buds, subsequently induced to form somatic embryos, and after 30 days, induced to germinate. Chromosome variation in embryogenic calli-derived plants increased with increasing duration of subculture, particularly when low ploidy levels of plants such as haploid and diploid were used as explants. Approximately 80% of haploid-derived plants showed morphological variations such as dwarfness and abnormal morphological characteristics, although no differences were observed in cladodes and stem characteristics between other polyploid-derived plants and their parents. The data presented here would supply important fundamental information for commercial mass-propagation using somatic embryogenesis.
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Kunitake H., Masahiro M.
Plant Biotechnology 15 ( 2 ) 51 - 61 1998
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant Biotechnology
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Normalization of asparagus somatic embryogenesis using a maltose-containing medium. Reviewed
共著者: Kunitake, H., T. Nakashima, K. Mori , M. Tanaka
Journal of Plant Physiology 150 ( 4 ) 458 - 461 1997.4
Language：English Publishing type：Research paper (scientific journal)
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Normalization of asparagus somatic embryogenesis using a maltose-containing medium
Kunitake H., Nakashima T., Mori K., Tanaka M.
Journal of Plant Physiology 150 ( 4 ) 458 - 461 1997
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Plant Physiology
Embryogenic calli derived from internal buds were cultured and maintained on Murashige and Skoog (MS) medium containing 2 mg/L 2,4-D, 3% sucrose and 0.2% gellan gum. Normal somatic embryos were induced from the embryogenic calli by pretreating with 1/4 MS liquid medium for 7 days, sieving through 600 μm stainless steel mesh and then partially desiccating on plant growth regulator-free MS medium containing a high concentration of gellan gum (1.0% w/v) for 1 week. Furthermore, use of maltose as a sugar and/or osmoticum in the embryo induction medium promoted development of normal somatic embryos. Using these methods, non-vitrified bipolor embryos were induced 30 days after transfer (approximately 1,500 embryos per 0.1 mL packed cell volume). The somatic embryos induced on maltose-containing medium exhibited a much higher germination rate more than 80%) with cold treatment (14 days at 4°C) than that induced on sucrose-containing medium.
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Polyacetylenes in hairly root cultures of Campanula medium L. Reviewed
共著者: Toda, H., T. Nakashima, H. Kunitake, K. Mori, M. Tanaka , K. Ishimaru
Journal of Plant Physiology 147 ( 5 ) 617 - 619 1996.12
Language：English Publishing type：Research paper (scientific journal)
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Production of interspecific somatic hybrid plants between Asparagus officina- lis and A. macowanii through electrofusion. Reviewed
共著者: Kunitake, H., T. Nakashima, K. Mori, M. Tanaka, A. Saito , M. Mii
Plant Science 116 ( 2 ) 213 - 222 1996.6
Language：English Publishing type：Research paper (scientific journal)
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Polyacetylenes in hairy root cultures of Campanula medium L.
Tada H., Nakashima T., Kunitake H., Mori K., Tanaka M., Ishimaru K.
Journal of Plant Physiology 147 ( 5 ) 617 - 619 1996.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Plant Physiology
Campanula medium L. hairy roots induced by infection with Agrobacterium rhizogenes A 13 grew well in three hormone-free liquid media, Murashige-Skoog (MS), Gamborg B5 (B5) and Woody Plant (WP). In these media, the hairy roots produced a high amount of the polyacetylene monoglucoside, lobetyolin (maximum content: 3.74% in MS medium, 2.69% in B5 medium and 3.13% in WP medium, dry weight) whose levels were over 130 times higher than that of the intact plant.
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Kunitake H., Nakashima T., Mori K., Tanaka M., Saito A., Mii M.
Plant Science 116 ( 2 ) 213 - 222 1996
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant Science
Interspecific somatic hybridization was performed between embryogenic callus protoplasts of Asparagus officinalis cv. Mary Washington and callus protoplasts of A. macowanii by electrofusion. By utilizing iodoacetamiae (IGA) inactivation treatment for A. officinalis protoplasts and the difference of cell division capacity between the two species, one callus line, which had an ability to produce somatic embryos, was selected as a putative hybrid. Normal germination of the somatic embryos was induced only when they were exposed to a period of cold treatment (14 days at 4°C). However, the regenerated plants showed intermediate but abnormal morphological characters in vitro as well as after transfer to soil. The hybrid nature of the plants derived from this callus was confirmed by isozyme, nuclear rDNA and random amplified polymorphic DNA (RAPD) analyses and counting the chromosome number. © 1996 Elsevier Science Ireland Ltd. All rights reserved.
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Plant regeneration from mesophyll protoplasts of lisianthus (Eustoma grandi- florum) by adding activated charcoal into protoplast culture medium. Reviewed
共著者: Kunitake, H., T. Nakashima, K. Mori, M. Tanaka , M. Mii
Plant Cell, Tissue and Organ Culture 43 ( 1 ) 59 - 65 1995.12
Language：English Publishing type：Research paper (scientific journal)
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Polyphenol production in hairly root cultures of Fragaria x ananassa. Reviewed
共著者: Motomori, Y., K. Shimomura, K. Mori, H. Kunitake, T. Nakashima, M. Tanaka, S. Miyazaki , K. Ishimaru
Phytochemistry 40 ( 5 ) 1425 - 1428 1995.12
Language：English Publishing type：Research paper (scientific journal)
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Polyphenol production in hairy root cultures of Fragaria x ananassa
Motomori Y., Shimomura K., Mori K., Kunitake H., Nakashima T., Tanaka M., Miyazaki S., Ishimaru K.
Phytochemistry 40 ( 5 ) 1425 - 1428 1995.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Phytochemistry
Hairy roots of Fragaria × ananassa cv. Reikou, induced with Agrobacterium rhizogenes ATCC 15834, grew well in hormone-free Murashige-Skoog (MS), root culture and Gamborg B5 liquid media. Particularly, in MS medium, hairy roots whowed maximum growth (539 mg per flask, dry wt at week 8) producing high contents of polyphenols (especially (+)-catechin (0.59% dry wt at week 8) and procyanidin B-3 (0.80% dry wt at week 7). Polyphenol contents in the intact plant (leaf blade, petiole, calyx, receptacle and root) were also investigated. © 1995.
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Kunitake H., Nakashima T., Mori K., Tanaka M., Mii M.
Plant Cell, Tissue and Organ Culture 43 ( 1 ) 59 - 65 1995.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant Cell, Tissue and Organ Culture
Plant regeneration from isolated protoplasts of 8 cultivars of lisianthus, Eustoma grandiflorum (Griseb.) Schinners, has been established by using activated charcoal. Protoplasts were isolated from lisianthus leaves grown in vitro and started to divide within 3-4 days of culture, but successful colony formation was only achieved by adding gellan gum blocks containing 1% (w/v) activated charcoal immediately after culture. Colonies consisting of as many as 50-100 cells formed after 30 days of culture and were transferred to fresh medium for callus proliferation and shoot regeneration, respectively. These shoots rooted on MS medium containing 0.5 mg l -1 indolebutyric acid(IBA) and the plantlets were finally transplanted to pots. Morphological characteristics, growth habit and pollen fertility of protoplast-derived plants of one cultivar were not different from those of seed-grown plants as control. © 1995 Kluwer Academic Publishers.
DOI： 10.1007/BF00042672
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Morphological and cytological characteristics of protoplast-derived plants of statice (Limonium perezii Hubbard).
共著者：Kunitake, H., K. Koreeda , M. Mii
Scientia Horticulturae 60 ( 3-4 ) 305 - 312 1995.3
Language：English Publishing type：Research paper (scientific journal)
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Kunitake H., Koreeda K., Mii M.
Scientia Horticulturae 60 ( 3-4 ) 305 - 312 1995.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
Morphological characteristics of 56 protoplast-derived plants (protoclones) of statice (Limonium perezii Hubbard) were compared with meristem-derived plants (controls). Some protoclones had smaller leaf shape index (length/width) and shorter length of flower stalk and calyx than the controls. However, no distinct somaclonal variations were observed in other characteristics such as color of calyx and number of petals and stamens, except for protoclone No. 1-7, which had flowers with abnormal petals and low pollen fertility (25%). Microscopic observation showed that all of the 17 protoclones examined, including clone No. 1-7, had the normal diploid chromosome number of 14. © 1995.
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Isolation and culture of asparagus microspore protoplasts. Reviewed
共著者：Kunitake, H., T. Godo , M. Mii
Japanese Journal of Breeding 43 ( 2 ) 231 - 238 1993.6
Language：English Publishing type：Research paper (scientific journal)
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Somatic embryogenesis and plant regeneration from immature seed-derived calli of rugosa rose (Rosa rugosa Thunb.). Reviewed
共著者：Kunitake, H., H. Imamizo , M. Mii
Plant Science 90 ( 2 ) 187 - 194 1993.4
Language：English Publishing type：Research paper (scientific journal)
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Kunitake H., Imamizo H., Mii M.
Plant Science 90 ( 2 ) 187 - 194 1993
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant Science
A procedure for plant regeneration from immature seed-derived calli of rugosa rose (Rosa rugosa Thunb.) via somatic embryogenesis is described. Embryogenic calli were initiated from immature seeds 2-3 weeks after anthesis on Murashige and Skoog (MS) medium without growth regulators. Induced calli had a white, friable and nodular appearance with several proembryos. These calli were subcultured at 20-day intervals on MS medium containing 0.1-0.2 M galactose on which they grew rapidly; but somatic embryogenesis was inhibited. Somatic embryos were again induced from the subcultured calli after transferring to MS medium containing 0.1 M M fructose or sucrose but lacking growth regulators. After transferring these embryos (1-2 mm) to MS medium containing 0.1 M sorbitol, 3% of them germinated and grew into plantlets which showed sustained growth on the MS medium containing only 0.1 M sorbitol as the sole carbon source. © 1993.
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Plant regeneration from protoplasts of ‘satsuma’mandarin (Citrus unshiu Marc.). Reviewed
共著者：Kunitake, H., H. Kagami , M. Mii
Japan Agricultural Research QuarterlyVol. 24, No. 4, 287-291 24 ( 4 ) 287 - 291 1991.7
Language：English Publishing type：Research paper (scientific journal)
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Kunitake H., Kagami H., Mii M.
Scientia Horticulturae 47 ( 1-2 ) 27 - 33 1991.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Scientia Horticulturae
Protoplasts were isolated from embryogenic calli of 'Satsuma' mandarin (Citrus unshiu Marc. cultivar 'Tokumori Wase') and cultured in Gellan gum-embedded Murashige and Tucker (MT) medium containing 40 mg l -1 adenine, 0.15 M sucrose and 0.45 M mannitol. The percentage of colony formation (plating efficiency) 40 days after culture was 46%, much higher than that in MT medium without adenine. Most of the colonies grew about 1 mm after 2 months of culture and were successfully transferred onto MT medium containing 5% lactose for embryoid formation. Five percent of the resulting embryoids germinated normally and grew into plantlets, 30-60 days after transplanting on MT medium containing 1 mg l -1 gibberellic acid, 3% sucrose and 0.2% Gellan gum. Plant regeneration from protoplasts was also obtained in other important selections, cultivars 'Miyagawa Wase' and 'Okitsu Wase', using the same methods. © 1991.
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Somatic embryogenesis and plant regeneration from protoplasts of ‘satsuma’mandarin (Citrus unshiu Marc.).
共著者：Kunitake, H., H. Kagami , M. Mii
Scientia HorticulturaeVol. 47, No. 1-2, 27-33 1991.2
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Plant regeneration from cell culture-derived protoplasts of statice (Limonium perezii). Reviewed
共著者：Kunitake, H. M. Mii
Plant Science 70 ( 1 ) 115 - 119 1990.11
Language：English Publishing type：Research paper (scientific journal)
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Plant regeneration from protoplasts isolated from embryogenic callus of satsuma(Citrus unshiu Marc.) Reviewed
共著者：Ling, J.T., N. Nito,M. Iwamasa, H. Kunitake
HortScience 25 ( 8 ) 970 - 972 1990.8
Language：English Publishing type：Research paper (scientific journal)
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Somatic embryogenesis and plant regeneration from protoplast of asparagus (Asparagus officinalis L.). Reviewed
共著者：Kunitake, H. M. Mii
Plant Cell Reports 8 ( 12 ) 706 - 710 1990.8
Language：English Publishing type：Research paper (scientific journal)
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Kunitake H., Mii M.
Plant Cell Reports 8 ( 12 ) 706 - 710 1990.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant Cell Reports
Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3-4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5-1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1-3 mm) in distilled water for a week, 30-40% of them germinated normally and grew into plantlets 20-30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA 3 and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes. © 1990 Springer-Verlag.
DOI： 10.1007/BF00272099
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Plant regeneration from cell culture-derived protoplasts of statice (Limonium perezii Hubbard)
Kunitake H., Mii M.
Plant Science 70 ( 1 ) 115 - 119 1990
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Plant Science
Protoplasts were isolated from leaf segment-derived suspension cultures of Limonium perezii Hubbard and cultured in Gellan Gum-solidified 1 2 Murashige and Skoog (MS) medium containing 1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 1 mg/l naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BA), 250 mg/l casein hydrolysate, 3% (w/v) sucrose and 0.5 M mannitol. The protoplasts started to divide within 2-3 days of culture and formed colonies (0.5-1 mm) after 2 months of culture. These colonies were then transferred to MS medium containing 1 mg/l 2,4-D, 3% sucrose and 0.2% (w/v) Gellan Gum for callus proliferation. The calli were then transferred for shoot regeneration to MS medium with or without 2 mg/l zeatin. These shoots were rooted on MS medium containing 0.5 mg/l indole-3-butyric acid (IBA), 3% sucrose and 0.2% Gellan Gum and transferred to pots. No abnormalities in leaf shape and growth habit were observed in these plants. © 1990.