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Hirotoshi Tsuda, Hisato Kunitake, Mai Yamasaki, Haruki Komatsu, and Katsunori Yoshioka

With crosses between colchicine-induced tetraploid shashanbo (Vaccinium bracteatum section 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 hybrids were 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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Jing-Tian Ling, Nobumasa Nito, Masao Iwamasa, and Hisato Kunitake

Embryos were obtained from unfertilized and undeveloped seeds of satsuma (Citrus unshiu Marc.) cultured on a modified Murashige and Tucker (MT) medium. Embryogenic callus was induced from the hypocotyl region of the embryos. The callus was successfully maintained through subculturing on MT medium with 185 μm ade-nine, 2.8 μm GA3 and 400 mg malt extract/liter, solidified with Gelrite. Somatic embryogenesis occurred from callus subculture on medium containing 50 g lactose/liter and in the absence of plant growth regulators. Somatic embryos developed into plants on medium with sucrose and GA3. Protoplasts isolated from this callus produced somatic embryos through colony formation: subsequently, normal, entire plants were regenerated.

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Ena Akamatsu, Takanori Kai, Hideaki Hirabaru, Chizuko Yukizaki, Miho Sakai, Hirofumi Uto, Hirohito Tsubouchi, and Hisato Kunitake

Blueberry (Vaccinium sp.) fruits contain high concentrations of polyphenols such as anthocyanin. It is well known that polyphenols have antioxidant activity, so it is likely that the fruit has a possible preventative effect against several diseases like cancer. However, only a few reports so far have studied the human health benefits of the leaves. In this study, the antioxidant activity and antiviral effects of blueberry leaves were investigated. The leaves of three groups of blueberry, northern highbush blueberry (NHB), southern highbush blueberry (SHB), and rabbiteye blueberry (REB), were examined. These leaves were harvested in July and extracted with 80% ethanol. Samples were analyzed for antioxidant activity (DPPH radical scavenging activity) and antiviral activity against hepatitis C virus using the replicon cell assay (Lomann et al., 1999). The antioxidant activity showed significant variability between cultivars and species, with REB having about two times the activity of NHB and SHB. Antiviral activity was observed in the extracts of the leaves and the fruit, and the activity of the leaves was higher than that of the fruit. Among the cultivars and species evaluated, the antiviral activity of REB was higher than that of NHB and SHB. In addition, we discovered a positive correlation (r=0.68) between the antioxidant activity and the antiviral activity, using the leaves of hybrid seedlings of REB. Therefore, it is possible to speculate that the antiviral activity bears some relation to the antioxidant activity.

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Yiran Li, Asuka Uchida, Akiha Abe, Akihiro Yamamoto, Tomonari Hirano, and Hisato Kunitake

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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Masaki Yahata, Hisato Kunitake, Tsutomu Yabuya, Kensuke Yamashita, Yukiko Kashihara, and Haruki Komatsu

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 A3 (CMA) staining.

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Masaki Yahata, Hisato Kunitake, Kiichi Yasuda, Kensuke Yamashita, Haruki Komatsu, and Ryoji Matsumoto

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 [clementine (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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Yiran Li, Akiha Abe, Takuichi Fuse, Tomonari Hirano, Yoichiro Hoshino, and Hisato Kunitake

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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Masaki Yahata, Seiichi Harusaki, Haruki Komatsu, Kayo Takami, Hisato Kunitake, Tsutomu Yabuya, Kensuke Yamashita, and Pichit Toolapong

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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Hirotoshi Tsuda, Hisato Kunitake, Yo Aoki, Akiko Oyama, Takuya Tetsumura, Haruki Komatsu, and Katsunori Yoshioka

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.