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).
Ryutaro Tao and Akira Sugiura
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.
Mihoko Tamura, Ryutaro Tao, and Akira Sugiura
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).
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.
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.
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.
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.
Chitose Honsho, Keizo Yonemori, Akira Sugiura, Songpol Somsri, and Suranant Subhadrabandhu
Flower bud differentiation and the flowering habit of durian (Durio zibethinus Murray) `Mon Thong' from budbreak to anthesis were investigated at the Chantaburi Horticultural Research Center in Thailand. Clusters of flower buds appeared at the end of November on primary or secondary scaffold branches near where a flower cluster occurred the previous year. Anatomical observations revealed that the development of floral organs was acropetal; the five fused epicalyx forming a large, elongated envelope enclosing the sepals, petals, stamen and fused multi-carpellate pistil. Floral organ development was completed in early January. The mature flower bud more than doubled in size one day before anthesis, with anthesis starting around 1600 hr and ending ≈1900 hr. The anthers did not dehisce until the completion of flowering. This change induced heterostyly in this cultivar, which promoted out-crossing by reducing the possibility of self-pollination. Aromatic nectar that attracted insects to the flower was secreted during anthesis. This is the first report to have clarified the overall flowering process in durian and provides the basic information for elucidating reproductive biology of durian in future research.
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.