Apogamous fern species are often difficult to distinguish from related species because of their continuous morphological variations. To clarify the genetic relationships among the members of the Dryopteris varia complex, we analyzed the nucleotide sequences of the plastid gene rbcL and the nuclear gene PgiC. We also analyzed the diploid sexual species D. caudipinna and D. chinensis, which have not been included in the complex, but were recently shown to be closely related to the complex in a molecular phylogenetic study. The PgiC sequences of the diploid sexual species, D. varia, D. saxifraga, D. sp. 'protobissetiana' (undescribed diploid sexual species), D. caudipinna, and D. chinensis, were well differentiated and hence designated A, B, C, D, and E, respectively. Thus, the PgiC constitution of apogamous species in the complex was as follows: D. bissetiana, B + C; D. kobayashii, B + C + E); D. pacifica, A + C, A + B + C, or A + C + D; D. sacrosancta, A + C + E; and D. saxifragivaria, B + C. These results suggest that these apogamous species are formed by hybridizations of species including not only the three diploid sexual species of the D. varia complex (A, B, and C) but also the two diploid sexual species D. caudipinna (D) and D. chinensis (E), which do not belong to the complex.

Reciprocal interspecific crosses were carried out between six lines of Dianthus caryophyllus L. and one line of Dianthus japonicus Thunb. Although no seed was set when D. japonicus was used as the seed parent, six seedlings were successfully obtained from 2,380 immature ovules by applying the embryo-rescue technique. However, they showed seed parent-like morphology and no evidence for the hybridity by flow cytometry and RAPD analyses. When six lines of D. caryophyllus were used as seed parents, a total of 192 seedlings were successfully obtained without using the embryo-rescue technique. Among these seedlings, 12 out of 25 progenies obtained from the carnation line '98sp1651' were confirmed to be the hybrids. The remaining 13 progenies of this line, and the total 167 progenies obtained from the other carnation lines, had carnation-like morphology without any evidence of hybridity by flow cytometry and RAPD analyses. The progenies confirmed as hybrids had intermediate characters of the parents with respect to leaf width and flower size, but they had a uniform flower color, reddish purple, which was different from that of either parent. Since the hybrids obtained in the present study have some profitable characters such as vigorous growth in summer time, upright robust stem, broad leaves and early flowering, they are expected to be used for the breeding of carnation which is suitable for growing under the Japanese climate.

Amphidiploid induction by chromosome-doubling is the most efficient method of utilizing a sterile interspecific hybrid for breeding. Previously, although we successfully obtained an interspecific hybrid between carnation (Dianthus caryophyllus L.) and Dianthus japonicus Thunb., the hybrid was sterile. In the present study, therefore, attempts were mode to induce amphidiploids to restore the fertility by artificial chromosomedoubling, i.e., by applying drops of a colchicine solution on to the shoot tips of greenhouse-grown plants or by treating nodal segments excised from in vitro-grown plants by incubation in a colchicine or amiprophosmethyl (APM) solution. Among 156 hybrid plants regenerated after these treatments, nine were revealed to be tetraploids, in addition to one octoploid and 88 mixoploids. Colchicine dropping treatment at a concentration of 2,000 mg⋅l −1 for one day led to the highest rate (14%) of tetraploid formation, whereas 10% tetraploid formation was obtained by in vitro shaking culture with 5 and 10 mg⋅l −1 APM for 24 hrs. The amphidiploids exhibited a larger flower size and later flowering time than the original diploid hybrid, and the fertility of both pollen and seed was restored. These amphidiploids are expected to be used for the breeding of carnation cultivars, which are suitable for growing under the Japanese climatic conditions.

Four bread wheat (Triticum aestivum L.) cultivars, 'Aobakomugi', 'Chinese Spring', 'Norin 61' and 'Shinchunaga', were pollinated with five barley lines/cultivars consisting of three cultivated barley (Hordeum vulgare L.) lines, 'Betzes', 'Kinai 5' and OHL089, and two wild barley (Hordeum spontaneum C. Koch) lines, OUH602 and OUH324. Crossability, expressed as the percentage of embryo formation, varied from 0 to 55.4% among the cross combinations. The two wild barley lines generally had a higher crossability than the previously reported best pollinator, 'Betzes', and some Japanese wheat cultivars were better as the female parent than 'Chinese Spring'. Ninety four hybrid plants were obtained from 250 embryos cultured, and their somatic chromosome numbers ranged from 21 to 36. Eighteen plants were mosaic in chromosome number. Twenty one-chromosome plants appeared most frequently (45.7%) followed by 28-chromosome plants (14.9%). C-banding analysis revealed that elimination of barley chromosomes was mainly responsible for the occurrence of aneuploid plants. In hypoploids derived from 'Betzes'-crosses, chromosome 5 was preferentially eliminated as previously reported, while in hypoploids derived from OUH602-crosses, chromosome 4 was preferentially eliminated. The wild barley line OUH602 may be a useful parent for producing a new wheat-barley addition set because of its high crossability with wheat and a different pattern of chromosome elimination.