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Ryutaro Tao and Akira Sugiura

Callus cultures were initiated in the dark from leaf primordia, stem internodes, and young leaves of adult Japanese persimmon (Diospyros kaki L.) to induce adventitious buds. A high frequency of regeneration occurred on Murashige and Skoog medium (MS) with half the normal NH4NO3 and KNO3 concentration (1/2N) and containing 10 μm zeatin or 1 μm 4PU-30 in combination with 0.1 μm IAA, or MS(1/2N) medium containing 0.03 to 0.1 μ m IAA or 0.01 to 0.03 μm NAA combined with 10 μm zeatin. No significant differences in the capacity of regeneration were observed among the calli from different explant sources. Only eight of 16 cultivars formed adventitious buds on MS(1/2N) medium containing 10 μm zeatin and 0.1 μm IAA, with the percentage of explants forming adventitious buds ranging from 2% to 72%. Chemical names used: indole3-acetic acid (IAA); 1-naphthaleneacetic acid (NAA); N-phenyl-N'-(2-chloro-4-pyridyl)urea (4PU-30).

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Takuya Tetsumura, Ryutaro Tao and Akira Sugiura

A potentially dwarfing rootstock for japanese persimmon (Diospyros kaki L.) was propagated by single-node stem cuttings taken from root suckers. When a mature tree was cut down at ground level and part of the roots was exposed to the air, numerous suckers formed on the exposed parts of the roots. Single-node stem cuttings 3 to 4 cm (1.2 to 1.6 inches) long survived and rooted better than 10-cm (3.9-inch) and 25-cm (9.8-inch) leafy stem cuttings with several buds. Dipping cuttings in 3000 mg·L-1 (ppm) IBA for 5 s or in 25 mg·L-1 IBA for 24 h resulted in similar rooting. Most of the single-node stem cuttings taken in late-June and July survived and rooted well, whereas those prepared in late August rooted poorly and few survived. The survival and rooting percentages were unaffected by the position on the suckers (top vs. base) from which cuttings were taken. High relativehumidity in the propagation frame appeared to enhance survival and rooting. This clonal propagation method will make a rapid multiplication of japanese persimmon, a difficult-to-root species, possible. Chemical name used: indole-3-butyric acid (IBA).

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Mihoko Tamura, Ryutaro Tao and Akira Sugiura

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Mihoko Tamura, Ryutaro Tao and Akira Sugiura

Interspecific hybrids between Diospyros glandulosa (2n = 2x = 30) and D. kaki cv. Jiro (2n = 6x = 90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using modified KM8p medium. Relative nuclear DNA contents of calli derived from fusion-treated protoplasts were determined by flow cytometry. One-hundred-forty-nine of 166 calli obtained had the nuclear DNA content of the sum of those of D. glandulosa and D. kaki cv. Jiro. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and they appeared to be interspecific somatic hybrid calli. Shoots were regenerated from 63 of the 149 interspecific hybrid calli. PCR-RFLP of chloroplast DNA analysis, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained nuclear genome from both the parents but only chloroplast genome from D. glandulosa. Microscopic observation of root tip cells confirmed that somatic chromosome numbers of the interspecific hybrids were 2n = 8x = 120.

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Yong-Ping Gao, Hino Motosugi and Akira Sugiura

Ungrafted trees of seven apple rootstock cultivars, M.4, M.7, M.11, M.26, M.27, MM.106, and Maru. bakaidou (Malus prunifolia Borkh. var. ringo Asami; weeping type), and `Fuji' (Malus domestics Borkh.) trees grafted on these seven plus M.9 and M. 16 rootstock were grown in sand. They were regularly supplied with nutrient solutions of N as ammonium alone (A), nitrate alone (T), and both (AT). With both ungrafted and grafted trees, the shoot growth of six rootstock (M.11, M.4, M.7, MM.106, M.26, and M.27) was significantly less with A than with T. With `Fuji' trees grafted on the above six rootstock, the number of flowering buds and the ratios of flowering buds to total emerged buds were significantly enhanced by treatments A and AT, especially in the formation of axillary flowering buds. Flowering and shoot growth of `Fuji' trees grafted on M. prunifolia and M.16 were slightly affected by the form of supplied N. In the xylem sap, cytokinin-like activity was detected in a single zone in paper chromatography in all rootstock and `Fuji' trees. The activity in six ungrafted rootstock (M.4, M.7, M.11, M.26, M.27, and MM.106) and `Fuji' trees grafted on these plus M.9 rootstock were higher with A than with T. Gibberellin-like activity in the same sap was detected in two zones, Rfs 0.3 to 0.4 and Rfs 0.7 to 0.8 in paper chromatography. In the six ungrafted rootstock and in `Fuji' trees grafted on these plus M.9, A led to higher activity at Rfs 0.7 to 0.S, but T led to higher activity at Rfs 0.3 to 0.4. Cytokinin-like and gibberellin-like activities in ungrafted M. prunfolia and `Fuji' trees grafted on M. prunifolia or M.16 were not affected by the form of N.

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Ayako Ikegami, Keizo Yonemori, Akira Sugiura, Akihiko Sato and Masahiko Yamada

Japanese persimmon (Diospyros kaki Thunb.) cultivars are classified into four types depending on the nature of astringency loss of the fruit. The pollination-constant, non-astringent (PCNA) persimmons lose their astringency on the tree as the fruits develop. This PCNA trait is qualitatively inherited and recessive to the other three types, pollination-constant, astringent (PCA), pollination-variant, nonastringent (PVNA), and pollination-variant, astringent (PVA). In fact, crosses among Japanese PCNA cultivars yield only PCNA type in F1 generation as shown in recent breeding programs at the National Institute of Fruit Tree Science. Despite these previous results, we demonstrated here that non-PCNA (PVNA, PVA, and PCA) type offspring were derived at relatively high rates in the F1 generation from a cross between `Luo Tian Tian Shi', a PCNA accession from China, and the Japanese PCNA cultivar, `Taishu', despite the fact that `Luo Tian Tian Shi' was confirmed to be a true PCNA type by measuring tannin cell size, a principal morphological characteristic to distinguish PCNA cultivars from non-PCNA ones. When segregations of tannin cell size and tannin content in three progenies of the breeding populations derived from Chinese PCNA `Luo Tian Tian Shi' × Japanese PCNA `Taishu', Japanese PCNA `Shinshu' × Japanese PCNA `Taishu', and Japanese PVNA (non-PCNA) `Kurokuma' × Japanese PCNA `Taishu' were investigated, all offspring between Japanese PCNA cultivars contained only small tannin cells and were PCNA types, and those between Japanese PVNA × PCNA cultivars contained only large tannin cells and were non-PCNA types. However, hybrids between `Luo Tian Tian Shi' and `Taishu' segregated into populations of small and large tannin cells, indicating that `Luo Tian Tian Shi' is likely heterozygous for astringency. Therefore, Chinese PCNA `Luo Tian Tian Shi' should be different from Japanese PCNA cultivars in genetic makeup.

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Keizo Yonemori, Masayoshi Oshida, Fumio Fukuda and Akira Sugiura

A method for collecting the vacuolar contents of intact tannin and parenchyma cells of persimmon (Diospyros kaki Thunb.) fruit using a micropipette was developed. Thin sections of the mesocarp tissue from mature persimmon fruit, `Miyazaki-mukaku' and `Hiratanenashi', were placed on a glass slide. Using a micromanipulator and an inverted microscope, a micropipette was inserted into a vacuole and its contents were withdrawn. A 5-nL sample of vacuole sap was collected per tannin cell from `Hiratanenashi' and 7 nL from `Miyazaki-mukaku', whereas only 2 nL was withdrawn from adjacent parenchyma cells. Analyses of the vacuolar sap revealed that the tannin cells of both cultivars contained 10% to 12% (m/v) of tannin as (+)-catechin equivalents and 10% to 13% (m/v) of soluble sugars, whereas the parenchyma cells contained trace amounts of tannins and ≈20% of soluble sugars. Tannin cells contain only a slight amount of sucrose, in contrast to a relatively large amount in parenchyma cells.

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Ryutaro Tao, Tsuyoshi Habu, Hisayo Yamane, Akira Sugiura and Kazuya Iwamoto

Self-compatible cultivars of Japanese apricot (Prunus mume Sieb. et Zucc.) have a horticultural advantage over self-incompatible ones because no pollinizer is required. Self-incompatibility is gametophytic, as in other Prunus species. We searched for molecular markers to identify self-compatible cultivars based on the information about S-ribonucleases (S-RNases) of other Prunus species. Total DNA isolated from five self-incompatible and six self-compatible cultivars were PCR-amplified by oligonucleotide primers designed from conserved regions of Prunus S-RNases. Self-compatible cultivars exhibited a common band of ≈1.5 kbp. Self-compatible cultivars also showed a common band of ≈12.1 kbp when genomic DNA digested with HindIII was probed with the cDNA encoding S 2-RNase of sweet cherry (Prunus avium L.). These results suggest that self-compatible cultivars of Japanese apricot have a common S-RNase allele that can be used as a molecular marker for self-compatibility.

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Yasutaka Kano, Hiroshi Nakagawa, Masami Sekine, Hideyuki Goto and Akira Sugiura

Head and leaf weight of cabbage plants grown using half the nitrogen fertilizer applied to control plants (hereafter referred to as the half treatment) were markedly less than those obtained for control plants to which the standard amount of nitrogen fertilizer was applied. Sugar content 33 d after sowing (DAS) did not differ between treatments, but glucose and fructose content in the half treatment 82 DAS was higher than that of the control. Although the number of cell layers in cross-section for the leaves from both treatments was ≈20, cells from the half treatment appeared smaller than those of the control. Therefore, it is suggested that the higher sugar content in leaves of cabbage plants grown on media containing less nitrogen fertilizer occurs in response to the smaller cells in the leaves.

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Shinya Kanzaki, Keizo Yonemori, Akira Sugiura, Akihiko Sato and Masahiko Yamada

Japanese persimmon (Diospyros kaki Thunb.) cultivars are classified into four types depending upon the nature of astringency loss of the fruit. Among them, the pollination-constant and nonastringent (PCNA) type is the most desirable for fresh fruit consumption due to the trait of stable loss of astringency on the tree with fruit development. Lack of tannin accumulation is the main cause of natural astringency loss in PCNA-type fruit, and is qualitatively inherited. The PCNA trait is recessive to the non-PCNA trait. In this study, we investigated amplified fragment length polymorphism (AFLP) markers for the trait of natural astringency loss of PCNA-type fruit using bulked segregant analysis (BSA) for efficient selection of PCNA type plants in a breeding population. A total of 128 primer combinations were tested and one AFLP marker was found to be linked to the dominant allele controlling the trait for astringency. This marker, EACC/MCTA-400, was absent in all of the PCNA-type plants tested, whereas it was present in about half of the non-PCNA-type plants tested. However, RFLP analysis using this marker enabled the detection of the other dominant allele, and all PCNA-type plants could be distinguished from the non-PCNA-type plants. Application of this marker system will be useful for the selection of PCNA-type plants in persimmon breeding.