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A. Yamada, R. Tao and A. Sugiura

The efficacy of ploidy breeding using unreduced pollen in japanese persimmon (Diospyros kaki Thunb.) is not high because of the low frequency of unreduced pollen in most cultivars. This study was conducted in 2002 and 2003 to determine if the exposure to a low temperature before flowering could enhance the unreduced pollen formation in five cultivars of japanese persimmon including two cultivars that barely produce unreduced pollen under the field condition. The results showed that low-temperature treatment (4 °C for 48 hours) increased the occurrence of unreduced pollen at 15 to 17 and 17 to 18 days after the end of the low-temperature treatment in 2002 and 2003, respectively, in all five cultivars tested. Naturally occurring temperatures below 5 °C in the field also appeared to enhance the unreduced pollen formation in the cultivars that naturally produce unreduced pollen in the field.

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A. Sugiura, G.H. Zheng and K. Yonemori

Fruit temperature of persimmons (Diospyros kaki L. f. cv. Hiratanenashi) was regulated at a constant 14, 22, or 30C during growth stage III. Fruit growth and ripening were greatly accelerated at 22C compared with control fruit grown at ambient air temperature (range 9.3 to 28.5C). At harvest (30 days after treatment), fruit kept at 22C was much heavier, more deeply colored, and softer than the controls. In contrast, 30C delayed the onset of rapid fruit expansion and ripening. At harvest, however, the fruit, though still green, were about the same size as tbe controls. With the exception of rapid chlorophyll degradation, 14C had little effect on fruit growth and ripening. Little difference in sugar content and composition was found between temperature treatments and controls.

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K. Yonemori, M. Oshida and A. Sugiura

In order to study the nature of tannins in vivo, we developed a method for collecting the vacuolar contents from intact tannin cells in persimmon fruit. We used a micropipette controlled with a MMS-77 micromanipulator system (Shimadzu Co., Kyoto, Japan) under an inverted microscope. Fruit flesh of mature persimmon fruit (cv. Miyazakimukaku) was cut into 300-μm-thick sections with a DSK-100 miaoslicer (Dosaka EM, Kyoto, Japan). The sections were then put on a glass slide, and a micropipette was inserted into a tannin cell to withdraw its contents. After determination of the sap volume collected, the sample was injected into a 25-μl drop of water on a glass slide. Then, the water-drop containing the tannin sample was transferred to a small microfuge tube and stored in a freezer until analysis. Based on calculations, we could collect approximately 7 to 12 nl of vacuolar contents per tannin cell. When tannin and sugar contents per tannin cell were determined, we found that tannin cells contain tannins at 10% to 15% as catechin equivalents (w/v) and 8% to 10% total sugars (w/v), while a whole fruit contains tannins at 1% to 1.5% as catechin equivalents and 10% to 13% total sugars on fresh weight basis. We are currently continuing more detailed analysis of tannin cell constituents.

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H. Yamane, R. Tao, A. Sugiura, N. Hauck and A. Iezzoni

Most fruit tree species of Prunus exhibit gametophytic self-incompatibility, which is controlled by a single locus with multiple alleles (S-alleles). One interesting aspect of gametophytic self-incompatibility is that it commonly “breaks down” as a result of polyploidy, resulting in self-compatible individuals. This phenomenon is exhibited in the diploid sweet cherry (P. avium) and the tetraploid sour cherry (P. cerasus), in which most cultivars are self-compatible. Recently, S-gene products in pistil of Prunus species were shown to be S-RNases. As sour cherry is one Prunus species, it is likely to possess S-alleles encoding pistil S-RNases. To confirm this, we surveyed stylar extracts of 11 sour cherry cultivars, including six self-compatible and five self-incompatible cultivars, by 2D-PAGE. As expected, all 11 cultivars tested yielded glycoprotein spots similar to S-RNases of other Prunus species in terms of Mr, immunological characteristics, and N-terminal sequences. A cDNA clone encoding one of these glycoproteins was cloned from the cDNA library constructed from styles with stigmas of a self-compatible cultivar, `Erdi Botermo'. Deduced amino acid sequence from the cDNA clone contained two active sites of T2/S type RNases and five conserved regions of rosaceous S-RNases. In order to determine the inheritance of self-incompatibility and S-allele diversity in sour cherry, we conducted genomic DNA blot analysis for sour cherry germplasm collections and mapping populations in MSU using the cDNA as a probe. To date, it appears as if self-compatibility in sour cherry is not simply controlled by a self-fertile allele as demonstrated in other Prunus species.

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Young-A Choi, R. Tao, K. Yonemori and A. Sugiura

Multi-color genomic in situ hybridization (MCGISH) was performed for mitotic cells of the somatic hybrids of Diospyros kaki (2n = 6x = 90) and D. glandulosa (2n = 2x = 30). Total DNA of D. kaki and D. glandulosa were isolated and labeled with biotin-16-UTP and digoxigenin (DIG)-11-UTP, respectively. The labeled DNAs were used as probes to differentiate parental chromosomes. The biotin-labeled probe was detected with avidin-rhodamine, and the DIG-labeled probe was detected with anti-DIG-FITC (fluorescein isothiocyanate). Ninety chromosomes from D. kaki that showed reddish-orange and 30 chromosomes from D. glandulosa that showed greenish-yellow were observed under a fluorescence microscope. Some chromosomes showed cross-hybridization with both probes at their terminal or other chromosome regions. These results indicated that MCGISH could be used to analyze genomes of Diospyros species whose chromosomes are small and numerous.

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K. Yonemori, A. Itai, R. Nakano and A. Sugiura

The role of calyx lobes in CO2 exchange in persimmon (Diospyros kaki Thunb.) fruit was investigated during fruit growth and development. Carbon dioxide exchange rates of attached and detached fruit were measured in light and dark conditions in the field after calyx lobes were removed. Calyx lobes were removed at fruit growth stages I and III, which were defined as the period when fruit diameter increases >0.3 mm·d-1 (Zheng et al., 1990). Removing calyx lobes at stage I significantly inhibited fruit growth, while removing them at stage III had no effect on growth. Two weeks after calyx lobes were removed at stage I, CO2 exchange decreased 80% in light and dark conditions compared with the control fruit. The rapid decreases of CO2 exchange rate by calyx lobe removal at stage I were obvious if expressed per fruit or on a fresh weight basis. In contrast, treatment at stage III had no effect on CO2 exchange rate of fruit and no effect on fruit growth. However, when the calyx lobe scars were sealed with Vaseline soon after calyx lobe removal at stage III, an immediate decline in CO2 exchange rate in the dark occurred with simultaneous inhibition of the final swell in fruit growth. A possible relationship between fruit growth potential and gas-exchange capacity is discussed.

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R. Tao, T. Ohkuma, M. Tamura and A. Sugiura

Distribution of pollen diameter of Japanese persimmon cv. Zenjimaru (2n = 6x, × = 15) was determined using pollen grains hydrated with CPW solution supplemented with 0.9 M mannitol. Mean diameter of giant pollen grains (65 μm) was 1.3 times longer than that of normal pollen grains (50 μm). The occurrence of giant pollen was estimated to be about 5% of the pollen population. The hydrated giant pollen grains could be sorted out from normal pollen grains by filtering through a layer of nylon mesh (62 μm). Flow cytometric analysis of nuclear DNA content confirmed that giant pollen was unreduced 2n pollen. 2n giant pollen grains were pollinated to cn. Jiro (2n = 6x) callie and plantlets could be obtained from immature embyros excised from seeds 70 days after pollination.

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S.K. Kang, H. Motosugi, K. Yonemori and A. Sugiura

Microcomputer-based thermal analysis (TA) was conducted on dormant mixed buds of Japanese persimmon (`Hiratanenashi'). The exotherms of buds were detected by thermoelectric modules. Flower buds of peach (Prunus persica Batsch cv. Shimizuhakuto) were also analyzed. When TA was used on a whole excised bud, including bud scales, the persimmon buds had only one exotherm at –14.3 °C, while the peach buds had high and low exotherms at –8.4 °C and –14.1 °C, respectively. However, when the exotherm was measured for the primordium, with the bud scales and transitional leaves removed, each primordium showed only one exotherm at –20.7 °C in persimmon and –11 °C in peach. Determination of killing temperature by visual observation, electrolyte leakage method, and triphenyltetrazolium chloride test revealed that the primordium of the persimmon bud was killed at about –14 °C as the excised whole bud or as the whole bud attached to the branch segment. Using the same method, the naked primordium was killed between –22 °C and –25 °C as the primordium was cooled. The peach primordium was killed at –14 °C when examined as a whole bud and at –11 °C as a naked primordium. Furthermore, the exotherm temperatures of persimmon buds and stem segments were measured at appropriate intervals during the two winter seasons 1993–95. Exotherm temperatures of persimmon buds were always higher than the low-temperature exotherm (LTE) temperatures of the stem segments and lower than the high-temperature exotherm (HTE) temperatures of the stem segments. LT50 of persimmon buds almost coincided with the exotherm temperatures of buds. A postulated role of bud scales in supercooling is discussed.

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Young A Choi, Ryutaro Tao, Keizo Yonemori and Akira Sugiura

5S ribosomal DNA (rDNA) was visualized on the somatic metaphase chromosome of persimmon (Diospyros kaki) and ten wild Diospyros species by fluorescent in situ hybridization (FISH). The digoxigenin (DIG)-labeled 5S rDNA probe was hybridized onto the chromosomes and visualized by incubation with anti-DIG-fluorescein isothiocyanate (FITC). Strong signals of 5S rDNA probe were observed on several chromosomes of Diospyros species tested. Furthermore, multicolor FISH using 5S and 45S rDNA probes differently labeled with DIG and biotin, revealed separate localization of the two rDNA genes on different chromosomes of Diospyros species tested, suggesting that 5S and 45S rDNA sites can be used as chromosome markers in Diospyros. The number of 5S rDNA sites varied with the Diospyros species. More 5S rDNA sites were observed in four diploid species native to Southern Africa than in three Asian diploid species. The former had four or six 5S rDNA sites while the latter had two. Three Asian polyploidy species had four to eight 5S rDNA sites. Among the Asian species, the number of 5S rDNA sites seemed to increase according to ploidy level of species. These features of 5S rDNA sites were very similar to those of 45S rDNA sites in Diospyros. Phylogenetic relationship between D. kaki and wild species tested are discussed based on the number and chromosomal distribution of 5S and 45S rDNA.

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K. Yonemori, A. Sugiura, K. Tanaka and K. Kameda

Patterns of floral differentiation were studied in two monoecious-type Japanese persimmon (Diospyros kaki L.) cultivars Hana-gosho and Kakiyama-gaki. In both cultivars, the pistillate and staminate floral primordium started to differentiate in early June, and differentiation progressed until August, when the sepal primordia in pistillate flowers and petal primordia of staminate flowers had become evident. The buds then entered a quiescent, overwintering state. Thus, flower sex of monoecious-type persimmons was determined at a relatively early stage of floral development. Moreover, in both cultivars, sex differentiation was associated with previous history of the current season's shoots. Current season's shoots that bore pistillate flowers differentiated pistillate buds (mixed buds from which pistillate flowers emerge) at significantly higher rates than for shoots that bore staminate flowers. Similarly, shoots that bore staminate flowers produced staminate buds (mixed buds from which staminate flowers emerge) at a higher percentage than shoots that had borne pistillate flowers. With `Hana-gosho', the flower type was also predictable with fair accuracy by bud position on the current season's shoot, i.e., pistillate flowers emerged from distal mixed buds, whereas staminate flowers arose predominantly from basal buds.