To obtain basic information about doubled haploid plants in Citrus, in the present study, we investigated the morphological characteristics in doubled haploid induced by colchicine-treated axillary shoot buds of a haploid plant from 'Banpeiyu' pummelo [C. maxima (Burm.) Merr.]. We also evaluated the reproductive potential of the doubled haploid as a male or a female parent by crossing with some diploids. In term of the results, this doubled haploid had significantly large leaf, flower and fruit compared with those of the original haploid plant. Moreover, the doubled haploid showed higher pollen fertility (84.1% stainability and 32.9% pollen germination rate) and a larger number of seeds (47.2 developed seeds per open-pollinated fruit) than the haploid. In the reciprocal crosses between the doubled haploid and some diploids, many developed seeds were obtained. These seeds germinated normally and developed into diploid seedlings. These results show that the doubled haploid will be valuable for genetic analysis and possibly for planned breeding.

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A ploidy chimera of the Meiwa kumquat (Fortunella crassifolia Swingle), which had been induced by treating the nucellar embryos with colchicine, and had diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36) cells, was examined for its ploidy level, morphological characteristics, and sizes of its cells in its leaves, flowers, and fruits to reveal the ploidy level of each histogenic layer. Furthermore, the chimera was crossed with the diploid kumquat to evaluate the ploidy level of its reproductive organs. The morphological characteristics and the sizes of the cells in the leaves, flowers, and fruits of the chimera were similar to those of the tetraploid Meiwa kumquat and the ploidy periclinal chimera known as “Yubeni,” with diploids in the histogenic layer I (L1) and tetraploids in the histogenic layer II (L2) and III (L3). However, the epidermis derived from the L1 of the chimera showed the same result as the diploid Meiwa kumquat in all organs and cells. The sexual organs derived from the L2 of the chimera were significantly larger than those of the diploid. Moreover, the ploidy level of the seedlings obtained from the chimera was mostly tetraploid. In the midrib derived from the L3, the chimera displayed the fluorescence intensity of a tetraploid by flow cytometric analysis and had the same size of the cells as the tetraploid and the Yubeni. According to these results, the chimera is thought to be a ploidy periclinal chimera with diploid cells in the outermost layer (L1) and tetraploid cells in the inner layers (L2 and L3) of the shoot apical meristem. The chimera had desirable fruit traits for a kumquat such as a thick pericarp, a high sugar content, and a small number of developed seeds. Furthermore, triploid progenies were obtained from reciprocal crosses between the chimera and diploid kumquat.

The morphological characteristics and fruit quality of an autotetraploid plant selected from nucellar seedlings of Satsuma mandarin (Citrus unshiu Marcow.) were investigated. Additionally, in order to evaluate the reproductive potential of male and female gametes of the tetraploid Satsuma mandarin, reciprocal crosses with diploid cultivars were also carried out. The tetraploid had significantly thick and round leaves, as compared to those of the diploid Satsuma mandarin. The sizes of the flowers and pollen grains of the tetraploid were significantly larger than those of the diploid. Pollen fertility of tetraploid was high compared with that of the diploid. The tetraploid produced seedless fruits. The fruit weight of the tetraploid was equal to that of the diploid. Compared to the diploid fruits, the tetraploid fruit had less sugar contents and more organic acid contents. Although the tetraploid fruits showed similar traits to other Citrus tetraploids such as thick and hard peels, the tetraploid had a higher content of carotenoids in the flavedo than the diploid, and the rind color of the tetraploid was much better. In the reciprocal crosses between the tetraploid Satsuma mandarin and diploid cultivars, some seeds were obtained, and triploid progenies were obtained in all cross combinations.

We previously obtained two intergeneric hybrids with different ploidies, i.e., aneuploid (2n = 28) and eutriploid, from diploid−diploid crosses between ‘Kiyomi’ tangor (Citrus unshiu Marcow. × C. sinensis (L.) Osbeck) and Meiwa kumquat (Fortunella crassifolia Swingle) as novel breeding materials for a seedless kumquat. In this study, we attempted to clarify the construction of the parental genomes of these hybrids by SSR genotyping and genomic in situ hybridization (GISH)−chromomycin A3 (CMA) analysis. SSR genotyping in NSX43 (LG5) and CiBE2227 (LG8) loci revealed that both hybrids inherited one allele from ‘Kiyomi’ tangor and two heterozygous alleles from Meiwa kumquat. The GISH analysis failed due to the high genomic homology between Citrus and Fortunella. At the same time, the CMA karyotype compositions of the two intergeneric hybrids (H15-701: 2A + 1B + 3C + 13D + 7E + 1F + 1Dst; H15-702: 3A + 1B + 2C + 15D + 4E +1F + 1Dst) and both parents (‘Kiyomi’ tangor: 1A + 2B + 2C + 6D + 7E; Meiwa kumquat: 2A + 2C + 12D + 1F + 1Dst) were completely revealed. We identified the parental genome construction and polyploidization processes in both intergeneric hybrids on the basis of these SSR genotypes and CMA karyotype compositions according to the following theory: the SSR genotypes and chromosome compositions were the same as those of the somatic chromosome and two-fold after the first division (even number) in unreduced gametes caused by first-division restitution (FDR) and second-division restitution (SDR), respectively. Consequently, we determined that both intergeneric hybrids may have had two genomes derived from the 2n male unreduced gamete as a result of the FDR of the Meiwa kumquat. In addition, most horticultural traits of the leaves, flowers, and fruits of both hybrids showed intermediate traits of the parents, but the fruit sizes and flowering habits were more like those of the two inherited genomes of Meiwa kumquat.