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
Ryutaro Tao and Akira Sugiura
Leaf extracts of 163 Japanese persimmon cultivars (Diospyros kaki L.) and six other Diospyros species were analyzed for isozyme variation of glucose phosphate isomerase (GPI, E C 188.8.131.52) and malate dehydrogenase (MDH, E C 184.108.40.206). With both systems, the bands were sharp and well-resolved, and intracultivar polymorphism was absent. Isozyme phenotypes were more varied with GPI than with MDH. With Japanese persimmons, GPI yielded 24 different banding patterns, and six cultivars were uniquely discriminated by this enzyme alone. MDH produced only three different banding patterns, with none of the cultivars discriminated. When both enzyme systems were taken together, 18 cultivars were uniquely discriminated and the rest could be classified into 22 groups of 2 to 18 cultivars each.
Akira Sugiura and Takashi Tomana
Ethanol production in the seeds and its accumulation in the flesh were compared among 47 Japanese persimmon cultivars (Diospyros kaki L.) in relation to their degree of astringency. Those which produce relatively high amounts of ethanol and accumulate it in the flesh, i.e. pollination-variant/nonastringent (PVNA) cultivars, lose astringency on the tree, while those producing less ethanol, i.e. pollination-variant/astringent (PVA) ones, remain astringent. Pollination-constant/nonastringent (PCNA) and pollination-constant/astringent (PCA) cultivars generally produced little ethanol in the seeds and accumulated small amounts or none in the flesh. Thus, 2 different mechanisms exist that are involved in the loss of astringency. One is associated with PVNA, PVA, and PCA types and is dependent upon the production and accumulation of ethanol and presumably acetaldehyde. The second is associated with PCNA types which apparently do not produce these volatile substances.
Mihoko Tamura, Ryutaro Tao, and Akira Sugiura
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.
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.
Hirotoshi Kitagawa, Akira Sugiura, and Misako Sugiyama
Gibberellic acid at 50, 100, and 200 ppm sprayed on leaves of bearing Kaki shoots 3 clays before harvest greatly increased the storage life of fruit. In Hiratanenashi cultivar, GA sprayed fruits had more than double the storage life of unsprayed fruits. Sprayed fruits also remained firm on the tree, so period of harvest could be extended. Sprayed trees showed delay in defoliation. No injurious effects of the spray were observed.
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).
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.
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.