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Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing 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.

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.

Blueberry leaf is currently a popular dietary supplement. Effects of dietary blueberry leaf and its active components on body fat accumulation were examined. C57BL/6J mice were fed high-fat, high-sucrose diet with or without 3% blueberry leaf extract (BLEx) or 3% concentrated-polyphenolic BLEx (CP BLEx) for 8 weeks. Compared to mice fed a high-fat, high-sucrose diet without blueberry leaf, BLEx and CP BLEx significantly reduced body weight and adipose tissue weight gain. Adipocytes were also smaller and and liver lipid accumulatioin was significantly inhibited in mice fed either BLEx or CP BLEx. These effects tended to be more pronounced in mice fed CP BLEx compared to in mice fed BLEx. Together, results suggest that blueberry leaf inhibits body fat accumulation typically observed in mice fed a high-fat, high-sucrose diet, and that inhibition is attributable to polyphenolic components in leaf extracts.

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.

Metabolic profiling is often used to identify possible correlations between a compoundʼs 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.

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. Keywords: Blueberry leaf; blueberry stem; quercetin; hepatic lipid; rat; functional food

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.

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. Lev., 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.

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.

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.

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.

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.

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.

ニンポウキンカンの種子を用いた試験管内染色体倍加について検討した．有糸分裂阻害剤であるコルヒチンとオリザリンを様々な濃度や時間で種子に処理し，その後500 mg・liter−1麦芽抽出物を添加したMT培地で培養した．処理2か月後，発芽率を調査し，フローサイトメーターと根端の染色体観察により得られた実生の倍数性を解析した．四倍体実生率は有糸分裂阻害剤の種類，処理濃度および時間に依存しており，本処理条件内では，コルヒチンの方がオリザリンより染色体倍加の効果が高かった．0.05％，48時間でコルヒチン処理を行った時，約50％と最も高い頻度で四倍体を獲得することができた．これらの四倍体の初期成長は貧弱であったが，3年生のカラタチ台に接ぎ木すると二倍体と同様に旺盛な成長を示した．以上のように，胚培養を組合せた種子へのコルヒチン処理は，ニンポウキンカンの四倍体を効率的に誘導できることが明らかとなった．

Summary 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 ½MS 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.

Antioxidants are abundant in blueberries, and while there are many studies concerning the bioactive compound of fruit, it is only recently that the wild Vaccinium species has attracted attention for their diverse and abundant chemical components. The aim of this study was to investigate the bioactive compounds of blueberry cultivars and wild species found in Japan. Among the five extracts of the Vaccinium species, Natsuhaze (Vaccinium oldhamii Miq.) was found to be the most effective at inhibiting the growth of HL-60 human leukemia cells in vitro. Although all ethanol extracts showed a growth inhibitory effect on HL-60 cells, the degree of the effects differed among the species. The extract of Natsuhaze induced apoptotic bodies and nucleosomal DNA fragmentation in the HL-60 cells. Of the extracts tested, that of Natsuhaze contained the largest amount of total polyphenols and showed the greatest antioxidant activity, but the anthocyanin content of Natsuhaze was similar to that of rabbiteye blueberry (V. virgatum Ait.). The results showed that total polyphenols contributed to the high antioxidant activity and growth inhibitory effect on HL-60 human leukemia cells of Natsuhaze extract.

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.

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.

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.

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.

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 GA3 and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.