Reticulate evolution in the apogamous Dryopteris varia complex (Dryopteridaceae, subg. Erythrovariae, sect. Variae) and its related sexual species in Japan.

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.

Production of interspecific hybrids in ornamental plants.

In breeding of ornamental plants, interspecific hybridization and polyploidization have successfully been used to produce novel cultivars with blended traits of both parents and to introgress useful traits of one species to another. Embryo rescue techniques and molecular cytogenetic methods have successfully been used to produce and characterize interspecific hybrids in various genera. In this paper, recent advances in interspecific hybridization are described based on the results obtained in Primula, Cosmos, and Kalanchoe with special references to the use of embryo culture techniques for rescuing the abortive hybrid embryos. The methods for production and characterization of interspecific hybrids are categorized into three steps, i.e., (1) pollination, (2) rescue culture of immature embryo, and (3) confirmation of hybridity and ploidy level of the plants obtained. For interspecific crosses, emasculation step is usually needed to avoid self-pollination even in the genera with self-incompatibility system, such as Primula and Cosmos, since self-incompatibility is not always complete. Since interspecific crosses are usually hindered by various cross-incompatibility mechanisms, successful production of interspecific hybrids could be achieved only from limited crosses among those using many cultivars/strains of both parents, suggesting the importance of the selection of the compatible genotypes. Unilateral cross incompatibility is commonly observed in interspecific cross combinations, so reciprocal crosses should be conducted as an indispensable step. At the rescue culture step, addition of plant hormones, e.g., auxin cytokinin and gibberellin, to the culture medium at the appropriate concentrations is proved to be effective and necessary. The hybridity of the plants is efficiently confirmed at the seedling stage by DNA analysis in addition to the observation of morphological characters. The analysis of relative DNA contents by flow cytometry is an easy and rapid means to confirm hybridity and to estimate ploidy level and genomic combination.

Ploidy chimeras induced in haploid sporophytes of Osmunda claytoniana and Osmunda japonica.

Haploid sporophytes of Osmunda claytoniana (2n = x = 22) were apogamously produced from calli when cultivated on a hormone-free medium. Flow cytometric analysis showed that ploidy chimeras were spontaneously produced in a haploid sporophyte of O. claytoniana and those of O. japonica that were obtained in the previous study. In the haploid sporophyte of O. claytoniana, a diploid pinnule and a partially diploid terminal segment were produced in a haploid pinna. In O. japonica, a haploid sporophyte yielded a diploid pinna in a haploid frond, and another haploid sporophyte yielded a diploid pinnule in a haploid pinna. Diploid chimeras were large in size and could be readily distinguished from other haploid parts of the fronds. It is likely that the chimeras were produced clonally from a single diploid cell that established chromosome doubling.

Production of interspecific hybrid plants in Primula.

The methods of production of inter-specific hybrids in Primula are categorized into four steps: (1) emasculation, (2) pollination, (3) rescue culture of immature embryo, and (4) confiration of hybridity and ploidy level of the regenerated plants. Although most of the Primula pecies have a heteromorphic self-incompatibility system, an emasculation step is usually needed to avoid self-pollination since self-incompatibility is not always complete. At the resue culture step, addition of plant hormones (e.g., auxin, cytokinin, and gibberellin) to the culture medium is proved to be effective. The hybridity of the plants is efficiently confirmed at seedling stage by DNA analysis in addition to the comparison of morphological characters. The analysis of relative DNA contents by flow cytometry is easy and rapid technique to confirm hybridity and to estimate ploidy level and genomic combination.