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  • Author or Editor: Ryutaro Tao x
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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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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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Correct assignment of self-incompatibility alleles (S-alleles) in sweet cherry (Prunus avium L.) is important to assure fruit set in field plantings and breeding crosses. Until recently, only six S-alleles had been assigned. With the determination that the stylar product of the S-locus is a ribonuclease (RNase) and subsequent cloning of the S-RNases, it has been possible to use isoenzyme and DNA analysis to genotype S-alleles. As a result, numerous additional S-alleles have been identified; however, since different groups used different strategies for genotype analysis and different cultivars, the nomenclature contained inconsistencies and redundancies. In this study restriction fragment-length polymorphism (RFLP) profiles are presented using HindIII, EcoRI, DraI, or XbaI restriction digests of the S-alleles present in 22 sweet cherry cultivars which were chosen based upon their unique S-allele designations and/or their importance to the United States sweet cherry breeding community. Twelve previously published alleles (S1, S2, S3, S4, S5, S6, S7, S9, S10, S11, S12, and S13 ) could be differentiated by their RFLP profiles for each of the four restriction enzymes. Two new putative S-alleles, both found in `NY1625', are reported, bringing the total to 14 differentiable alleles. We propose the adoption of a standard nomenclature in which the sweet cherry cultivars `Hedelfingen' and `Burlat' are S3S5 and S3S9 , respectively. Fragment sizes for each S-allele/restriction enzyme combination are presented for reference in future S-allele discovery projects.

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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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To understand the molecular basis of the endodormancy of buds of perennial plants, we searched for the genes that are expressed preferentially in endodormant lateral buds of the deciduous fruit tree japanese apricot (Prunus mume Sieb. et Zucc.) using suppression subtractive hybridization with mirror orientation selection (SSH/MOS). We generated two SSH/MOS libraries containing gene pools that are expressed preferentially in endodormant buds in comparison with paradormant or ecodormant buds to search for the genes that are upregulated by endodormancy induction or down-regulated by endodormancy release, respectively. Differential screening and sequencing indicated that genes involved in gibberellin metabolism, stress resistance, cell wall modification, and signal transduction, such as transcription factors, are upregulated in endodormant buds. After a further expression survey and full-length cDNA cloning, we found that a gene similar to the SVP/AGL24-type MADS-box transcription factor showed endodormancy-associated expression. Seasonal expression analysis suggested that the SVP/AGL24 homolog in japanese apricot might be involved in endodormancy regulation of its lateral buds.

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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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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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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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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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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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