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  • Author or Editor: Haruki Komatsu x
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With crosses between colchicine-induced tetraploid shashanbo (Vaccinium bracteatum section Bracteata) and tetraploid highbush blueberry ‘Spartan’ (Vaccinium corymbosum section Cyanococcus), intersectional hybrids were produced. The hybridity of these plants was confirmed based on DNA markers and morphological characteristics. The morphological characteristics, blooming date, and ripening period of the hybrids were intermediate between those of the parents. Ploidy analyses by flow cytometry and chromosome counting revealed that these hybrids were tetraploid. Four hybrids set fruit in the field and these two hybrids showed high pollen stainability. It was noteworthy that fruit of two hybrids had high soluble solids concentration compared with ‘Spartan’ and the fruit pulp of the hybrids was tinged with red as shashanbo. These hybrids could be useful in breeding new cultivars with high sugar content, abundant phytochemicals, extensive environmental adaptability as well as late flowering and fruit maturity.

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We tested efficient in vitro methods for screening the genotypes with higher pH tolerance using multiple shoots of intersectional hybrids between Vaccinium corymbosum ‘Spartan’ and V. bracteatum. The response of the four hybrid clones tested to different pH levels was clone-dependent in vitro. An apparent difference was found in the rooting rate among the hybrid clones even at higher pH levels; the rooting rates of JM4 (91%) at pH 8.0 indicated a significantly high value compared with other clones (JM1: 24%, JM2: 9%, JM3: 8%, ‘Spartan’: 0%). Furthermore, JM4 showed constantly high rooting rates (91% to 100%) at all pH levels with no significant differences. Similar differences in the root characters of the hybrids were also confirmed by checking the viability of roots using fluorescein diacetate (FDA)/propidium iodide (PI) staining after dipping the roots of in vitro-produced shoots in liquid medium at different pH levels for 6 hours. These results suggest that an in vitro screening method using the rooting rate of multiple shoots and the viability test of roots by FDA/PI staining as a marker could become a very useful tool for the selection of germplasm with tolerance to higher pH within a short time using small planting spaces. In addition, JM4, which showed a high rooting rate at pH 8.0, could be useful in breeding new cultivars with higher pH tolerance.

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The morphological characteristics and reproductive potential of a haploid plant obtained from the cross between `Banpeiyu' pummelo (Citrus grandis) and `Ruby Red' grapefruit (C. paradisi Macf.) were investigated. The haploid was confirmed to be derived from female gamete of `Banpeiyu' pummelo by isozyme and random amplified polymorphic DNA (RAPD) analysis. Flow cytometry analysis revealed that the haploidy was maintained in several tissues and organs of this plant. It also had the typical morphology of a haploid, such as small leaves and flowers, and had slightly fertile pollen grains. Furthermore, diploid progenies were obtained from the cross between `Kiyomi' tangor and the haploid. The hybridity of these seedlings was confirmed by assessment of the leaf characteristics and RAPD analysis. These results suggest that this haploid produced fertile unreduced pollen grains (n = 9).

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To produce the homozygous strain of a haploid plant derived from small seed-derived seedlings of `Banpeiyu' pummelo (Citrus grandis Osbeck), we carried out colchicine treatment to axillary shoot buds of the haploid. Many shoots with cytochimeras (X+2X and 2X+4X) arose from the colchicine-treated axillary buds. When cytochimeric buds of 2X+4X were top-grafted onto trifoliate orange [Poncirus trifoliata (L.) Raf.], a complete diploid shoot with 18 chromosomes was obtained from the cytochimera. This diploid strain showed vigorous growth compared with the original haploid. The leaf weight per unit area and the stomata size in this diploid were significantly larger than those of the original haploid plant, and were almost equal to those of `Banpeiyu' pummelo. The diploid strain was confirmed to be a doubled haploid of a haploid from `Banpeiyu' pummelo, based on random amplified polymorphic DNA (RAPD) analysis and chromosome composition analysis by chromomycin A3 (CMA) staining.

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The reciprocal crosses between two citrus cultivars and Citropsis schweinfurthii (Engl.) Swing. & M. Kell. were conducted. The cross between `Nanpu' tangor {`Kiyomi' tangor (Citrus unshiu Marc. × C. sinensis Osbeck) × `Fairchild' tangerine-tangelo [clementine (C. clementina hort. ex Tanaka) × `Orlando' tangelo (C. paradisi Macf. × C. reticulata Blanco)]} and C. schweinfurthii produced some developed seeds with an average weight approximately 1/10 of that of the seeds obtained from open pollination in `Nanpu' tangor. These seeds germinated on Murashige and Tucker medium, and three and 28 seedlings were obtained from crosses using C. schweinfurthii as the female and the male parent, respectively. The absolute nuclear genome size of these seedlings [∼0.84 pg of DNA content per somatic nucleus (2C)] was intermediate of that of the `Nanpu' tangor (0.78 pg/2C) and C. schweinfurthii (0.90 pg/2C) seedlings. The chromosome counts of the young leaves revealed that they were diploids (2n = 2X = 18). Furthermore, the hybridity of the seedlings obtained from the reciprocal crosses between `Nanpu' tangor and C. schweinfurthii was confirmed by randomly amplified polymorphic DNA (RAPD) analysis and cleaved amplified polymorphic sequence (CAPS) analysis. These hybrids will be utilized as important materials for investigating the phylogenic relationships between these genera in the subfamily Aurantioideae.

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