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Takuya Tetsumura and Kensuke Yamashita

Japanese chestnut (Castanea crenata Sieb. et Zucc.) was micropropagated from nodal explants of 2-month-old seedlings, and the regenerated plantlets were acclimatized after potting. For in vitro establishment, 5 μm zeatin was more effective than 5 μm BA or TDZ, and Sato's (BW) medium was as effective as MS(½NO3) medium. Driver-Kuniyuki walnut medium produced more shoots showing hyperhydricity symptoms. In the multiplication culture, the higher the concentration of zeatin added to BW medium, the greater the number of shoots showing hyperhydricity, although the longest shoot was obtained when 6.9 μm zeatin was added. After planting in ½BW medium with 15 μm IBA for 5 d to induce rooting, shoots planted in ½BW medium plus vermiculite gelled with Gellan Gum (SV substrate) rooted better than those in either the gelled medium without vermiculite (S substrate) or vermiculite plus liquid medium (V substrate). One third of the shoots planted in the V substrate died, although 62% of the surviving shoots rooted well. Of the shoots planted in the S substrate, 83% survived, but only 35% of these survived rooting. Shoots rooted in the SV and V substrates survived well after the completion of acclimatization, and shoots rooted in the SV substrate grew more vigorously after potting. Chemical names used: 6-benzyladenine (BA); 6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin); indole-3-butyric acid (IBA); 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ).

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Masaki Yahata, Hisato Kunitake, Tsutomu Yabuya, Kensuke Yamashita, Yukiko Kashihara and Haruki Komatsu

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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Masaki Yahata, Seiichi Harusaki, Haruki Komatsu, Kayo Takami, Hisato Kunitake, Tsutomu Yabuya, Kensuke Yamashita and Pichit Toolapong

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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Masaki Yahata, Hisato Kunitake, Kiichi Yasuda, Kensuke Yamashita, Haruki Komatsu and Ryoji Matsumoto

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.