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Hisayo Yamane, Megumi Ichiki, Ryutaro Tao, Tomoya Esumi, Keizo Yonemori, Takeshi Niikawa and Hino Motosugi

Fruit size is one of the most important traits that affect the economic value of fruit. In persimmon (Diospyros kaki Thunb.), somatic and bud-sport mutations that affect the fruit traits are frequently observed. Recently, a small-fruit mutant, ‘Totsutanenashi’ (TTN), was discovered in Japan as a bud-sport mutant of the leading cultivar Hiratanenashi (HTN). In this study, we investigated the morphological and physiological characteristics of TTN and HTN focusing on the tree architecture, fruit size, and the fruit flesh chemical composition. The objectives of the study were to evaluate the potential horticultural use of TTN and to characterize the differences between HTN and TTN. Both TTN and HTN are nonaploid plants, indicating that a difference in ploidy is not the cause of the small-fruit mutation. The vegetative growth of trees and tissue-cultured shoots of TTN was more compact than that of HTN. The floral organs of TTN appeared similar to those of HTN before flowering, but the TTN flowers opened earlier, resulting in smaller ovaries than in HTN flowers. The fruit size of TTN was consistently lower than that of HTN at all fruit developmental stages. TTN fruit had a higher sugar content and a higher proportion of sucrose to total sugars than HTN fruit. TTN fruits contained lower levels of secondary metabolites such as soluble tannins and ascorbate than HTN fruits. These results suggest that the fruit size mutation also affects the fruit biochemistry, leading to alterations in the fruit flesh composition. TTN may be a valuable genetic resource because compact trees require less labor and maintenance, and small, sweeter fruits may meet the various needs of consumers. The use of TTN in studies of the genetic control of fruit size is also discussed.

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Akira Sugiura, Yoshiko Matsuda-Habu, Mei Gao, Tomoya Esumi and Ryutaro Tao

In persimmon, plant regeneration from cultured cells usually takes place through adventitious bud formation. If somatic embryogenesis were possible, the efficiency of mass propagation and genetic engineering would be greatly improved. We attempted to induce somatic embryogenesis from immature embryos and plant regeneration from the induced embryos. Hypocotyls and cotyledons from immature ‘Fuyu’ and ‘Jiro’ seeds were cultured in the dark in Murashige and Skoog medium solidified with gellan gum and supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) at various concentrations. Callus formation started at ≈2 weeks of culture, and the callus formation rate was highest at 3 or 10 μm combinations of 2,4-D and BA. The initially formed calli gradually became brown or black from which white embryogenic calli (EC) appeared secondarily. After ≈8 weeks of culture, globular embryos were formed from these EC, and the formation proceeded until 20 weeks of culture. Formation of globular embryos was higher with ‘Fuyu’ than ‘Jiro’, especially with hypocotyls. When EC with globular embryos were transferred to fresh medium with no plant growth regulators, ≈70% developed to the torpedo-type embryo stage in 6 weeks. The torpedo-type embryos thus formed were germinated and rooted in agar medium with or without zeatin in several weeks without entering dormancy. After germination and rooting, the plantlets were transferred to the same medium and acclimatized for another 4 weeks. As the embryos germinated and rooted simultaneously, the plantlets were easy to grow in pots without transplanting shock. This is the first report on plant regeneration through somatic embryogenesis of persimmon.

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Takanori Takeuchi, Miwako Cecile Matsushita, Soichiro Nishiyama, Hisayo Yamane, Kiyoshi Banno and Ryutaro Tao

Endodormancy release and the fulfillment of the chilling requirement (CR) are critical physiological processes that enable uniform blooming in fruit tree species, including apple (Malus ×domestica). However, the molecular mechanisms underlying these traits have not been fully characterized. The objective of this study was to identify potential master regulators of endodormancy release and the CR in apple. We conducted RNA-Sequencing (RNA-seq) analyses and narrowed down the number of candidates among the differentially expressed genes (DEGs) based on the following two strict screening criteria: 1) the gene must be differentially expressed between endodormant and ecodormant buds under different environmental conditions and 2) the gene must exhibit chill unit (CU)–correlated expression. The results of our cluster analysis suggested that global expression patterns varied between field-grown buds and continuously chilled buds, even though they were exposed to similar amounts of chilling and were expected to have a similar dormancy status. Consequently, our strict selection strategy resulted in narrowing down the number of possible candidates and identified the DEGs strongly associated with the transition between dormancy stages. The genes included four transcription factor genes, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), FLOWERING LOCUS C (FLC)-LIKE, APETALLA2 (AP2)/ETHYLENE-RESPONSIVE 113 (ERF113), and MYC2. Their expressions were upregulated during endodormancy release, and were correlated with the CU, suggesting that these transcription factors are closely associated with chilling-mediated endodormancy release in apple.

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Ryutaro Tao, Hisayo Yamane, Akira Sugiura, Hideki Murayama, Hidenori Sassa and Hitoshi Mori

This report identifies S-RNases of sweet cherry (Prunus avium L.) and presents information about cDNA sequences encoding the S-RNases, which leads to the development of a molecular typing system for S-alleles in this fruit tree species. Stylar proteins of sweet cherry were surveyed by two dimensional polyaclylamide gel electrophoresis (2D-PAGE) to identify S-proteins associated with gametophytic self-incompatibility. Glycoprotein spots linked to S-alleles were found in a group of proteins which had Mr and pI similar to those of other rosaceous S-RNases. These glycoproteins were present at highest concentration in the upper segment of the mature style and shared immunological characteristics and N-terminal sequences with those of S-RNases of other plant species. cDNAs encoding these glycoproteins were cloned based on the N-terminal sequences. Genomic DNA and RNA blot analyses and deduced amino acid sequences indicated that the cDNAs encode S-RNases; thus the S-proteins identified by 2D-PAGE are S-RNases. Although S1 to S6-alleles of sweet cherry cultivars could be distinguished from each other with the genomic DNA blot analysis, a much simpler method of PCR-based typing system was developed for the six S-alleles based on the DNA sequence data obtained from the cDNAs encoding S-RNases.

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Yuto Kitamura, Hisayo Yamane, Akira Yukimori, Hiroyoshi Shimo, Koji Numaguchi and Ryutaro Tao

Flower bud development and the timing of blooming are mainly affected by genotype-dependent chilling requirements (CRs) during endodormancy and subsequent heat requirements (HRs) during ecodormancy. However, little information is available regarding the responses of flower buds to temperatures during endodormancy and ecodormancy in japanese apricot. We exposed japanese apricot ‘Nanko’ trees to various temperatures to estimate the CRs and HRs using development index (DVI) models specific for the endodormant (DVIendo) and ecodormant (DVIeco) stages. These models were based on the experimentally determined development rate (DVR). The DVRendo value was calculated as the reciprocal of the chilling time required to break endodormancy. The relationship between the DVRendo value and temperature was estimated using a three-dimensional curve. Our results indicated that 5–6 °C was the most effective temperature for breaking endodormancy in ‘Nanko’ flower buds. Additionally, exposure to −3 °C negatively affected endodormancy release, whereas 15 °C had no effect. We also determined that the DVReco values for temperatures between 5 and 20 °C were the reciprocal values of the time required for blooming after endodormancy release. The values outside this range were estimated using linear functions. The DVI was defined as the sum of the DVR values ranging from 0 to 1. Models for predicting the blooming date were constructed using the functions of sequentially combined DVIendo and DVIeco models. The accuracy of each model was assessed by comparing the predicted and actual blooming dates. The prediction of the model in which DVIeco = 1 corresponded to a 40% blooming level and DVIeco = 0 was set to DVIendo = 0.5 had the lowest root mean square error (RMSE) value (i.e., 3.11) for trees in commercial orchards exposed to different climates. Our results suggest that the developed model may have practical applications.

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Tsuyoshi Habu, Fumio Kishida, Miki Morikita, Akira Kitajima, Toshiaki Yamada and Ryutaro Tao

Japanese apricot (Prunus mume Sieb. et Zucc.) exhibits S-RNase-based gametophytic self-incompatibility as do other Prunus species. Both self-incompatible and self-compatible Japanese apricot cultivars are grown commercially in Japan. These self-compatible cultivars are shown to have a common S-haplotype called S f that contains S f-RNase and SFB f (S-haplotype-specific F-box protein). This study describes a simple and rapid detection of SFB f, in Japanese apricot, based on loop-mediated isothermal amplification (LAMP) method. A set of 4 primers, F3, B3, FIP, and BIP primer, were designed from the exon and the putative inserted sequence of SFB f. Optimal reaction time at 63 C was determined to be 90 minutes. It appeared that the LAMP method combined with the ultrasimple DNA extraction efficiently detected SFB f. The advantage of the marker-assisted selection of self-compatibility based on the LAMP method was discussed.

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Hisayo Yamane, Ryutaro Tao, Akira Sugiura, Nathanael R. Hauck and Amy F. Iezzoni

This report demonstrates the presence of S-ribonucleases (S-RNases), which are associated with gametophytic self-incompatibility (SI) in Prunus L., in styles of self-incompatible and self-compatible (SC) selections of tetraploid sour cherry (Prunus cerasus L.). Based on self-pollen tube growth in the styles of 13 sour cherry selections, seven selections were SC, while six selections were SI. In the SI selections, the swelling of pollen tube tips, which is typical of SI pollen tube growth in gametophytic SI, was observed. Stylar extracts of these selections were evaluated by two-dimensional polyacrylamide gel electrophoresis. Glycoproteins which had molecular weights and isoelectric points similar to those of S-RNases in other Prunus sp. were detected in all selections tested. These proteins had immunological characteristics and N-terminal amino acid sequences consistent with the S-RNases in other Prunus sp. Two cDNAs encoding glycoproteins from `Erdi Botermo' were cloned. One of them had the same nucleotide sequence as that of S4-RNase of sweet cherry (Prunus avium L.), while the amino acid sequence from the other cDNA encoded a novel S-RNase (named Sa-RNase in this study). This novel RNase contained two active sites of T2/S type RNases and five regions conserved among other Prunus S-RNases. Genomic DNA blot analysis using cDNAs encoding S-RNases of sweet cherry as probes indicated that three or four S-RNase alleles are present in the genome of each selection regardless of SI. All of the selections tested seemed to have at least one S-allele that is also found in sweet cherry. Genetic control of SI/SC in tetraploid sour cherry is discussed based on the results obtained from restriction fragment length polymorphism analysis.

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Akira Sugiura, Takeshi Ohkuma, Young A Choi, Ryutaro Tao and Mihoko Tamura

To produce nonaploid Japanese persimmon (Diospyros kaki L.f.) by artificial hybridization, we surveyed the natural occurrence of unreduced (2n) pollen among hexaploid cultivars and sorted them from normal reduced (n) pollen. The sorted 2n pollen was crossed with a hexaploid female cultivar and the resultant embryos were rescued by in vitro culture techniques to obtain plantlets. Three out of six male-flower-bearing cultivars (2n = 6x = 90) produced 2n pollen at rates of 4.8% to 15.5% varying with the cultivar, which was estimated by both pollen size and flow cytometry. After sorting giant (2n) from normal pollen grains by using nylon mesh, they were crossed with a hexaploid female cultivar. The seeds obtained from pollination with normal pollen were perfect, but those obtained from pollination with giant pollen were mostly imperfect, with embryo growth being suspended at the globular stage. Although the rate of survival was very low, some embryos at the globular stage were rescued successfully and grown in vitro. Both flow cytometric analysis and chromosome counting proved that the plantlets obtained were nonaploid.

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Ryutaro Tao, Abhaya M. Dandekar, Sandra L. Uratsu, Patrick V. Vail and J. Steven Tebbets

Japanese persimmon (Diospyros kaki L. `Jiro') was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA101, carrying the binary plasmid vector, pDU92.710. The T-DNA region of pDU92.710 contained the kanamycin resistance gene (nptII), the β-glucuronidase gene (uidA), and a synthetic reconstruct of cryIA(c) encoding the insecticidal crystal protein fragment of Bacillus thuringiensis subsp. kurstaki HD-73. Leaf discs made from leaves of shoot cultures were cocultivated with Agrobacterium and cultured on a callus-induction medium containing kanamycin and cefotaxime. Among 720 infected leaf discs, 17 putative transformed callus lines showing kanamycin resistance were obtained after 8 weeks of culture. When these were cultured on a regeneration medium containing kanamycin, 15 formed adventitious buds. Of the 15 shoot lines, 11 grew well on a shoot-proliferation medium containing kanamycin, while 4 lines did not grow well. Of the 11 shoot lines, 10 showed GUS activities by fluorometric assay and were subjected to polymerase chain reaction (PCR) and Southern analyses. Except for two lines, all results were consistent with a stable integration of T-DNA into the persimmon genome. The production of CryIA(c) protein in transformed shoot lines was confirmed with Western analysis using anti-CryIA(c) serum. Insect bioassays were conducted with 10 lines showing GUS activity. Many of these lines showed high significant mortality of the test insects, Plodia interpunctella Hüber and Monema flavescens Walker, when compared to nontransformed controls.

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Akiko Watari, Toshio Hanada, Hisayo Yamane, Tomoya Esumi, Ryutaro Tao, Hideaki Yaegaki, Masami Yamaguchi, Kenji Beppu and Ikuo Kataoka

Most commercial cultivars of japanese plum (Prunus salicina Lindl.) exhibit S-RNase-based gametophytic self-incompatibility (GSI), although some self-compatible (SC) cultivars exist. In this study, we characterized S-RNase and SFB, the pistil and pollen S determinants of the specificity of the GSI reaction, respectively, from four S-haplotypes, including a SC (Se) and three SI (Sa, Sb, and Sc) S-haplotypes of japanese plum. The genomic organization and structure of the SC Se-haplotype appear intact, because the relative transcriptional orientation of its S-RNase and SFB and their intergenetic distance are similar to those of the other three SI S-haplotypes of japanese plum and other Prunus L. species. Furthermore, there is no apparent defect in the DNA sequences of Se-RNase and SFBe. However, a series of transcriptional analyses, including real-time reverse transcriptase–polymerase chain reaction, showed that the Se-RNase transcript levels in the pistil are significantly lower than those of the Sa-, Sb-, and Sc-RNases, although transcripts of SFBa, SFBb, SFBc, and SFBe are present at similar levels in pollen. Furthermore, no Se-RNase spot was detected in two-dimensional polyacrylamide gel electrophoresis profiles of stylar extracts of the cultivars with the Se-haplotype. We discuss the possible molecular basis of SC observed with the Se-haplotype with special reference to the insufficient Se-RNase accumulation incited by the very low transcriptional level of Se-RNase in pistils.