Juntaro Kato

Different genomic combinations in inter-section hybrids obtained from the crosses between Primula sieboldii (section cortusoides) and P. Obconica (section obconicolisteri) by the embryo rescue technique

Abstract Inter-section hybrids were successfully obtained by rescuing the immature embryos produced in the cross between Primulasieboldii of Sect. Cortusoides and P. obconica of Sect. Obconicolisteri. In these hybrid plants two types of triploids with different genome combinations were found in addition to the normal diploid hybrids which each had one genome of the parents. Among the five triploids obtained, four had two genomes of P. sieboldii and one genome of P. obconica, whereas the remaining one had one genome of P. sieboldii and two genomes of P. obconica. The possibilities of diploid female gamete formation in P. sieboldii and diploid pollen formation in P. obconica as the causal factors for these triploid formations were discussed.

Variation of ploidy level in inter-section hybrids obtained by reciprocal crosses between tetraploid Primula denticulata (2n = 4x = 44) and diploid P. rosea (2n = 2x = 22)

Summary Inter-section hybrids were successfully obtained from reciprocal crosses between tetraploid Primula denticulata (section Denticulata) and diploid P. rosea (section Oreophlomis) by applying ovary culture 4 weeks after pollination. Differences in ploidy levels were found in these hybrids by flow cytometric analysis and by counting chromosome numbers. When P. denticulata was used as maternal parent, all the hybrids were triploid, with two genomes of P. denticulata and one genome of P. rosea. In contrast, when P. rosea was used as maternal parent, two genome-type hybrids were obtained: one was a triploid hybrid, with the same genome combinations as those obtained when P. denticulata was used as maternal parent, and the other type was a tetraploid hybrid with two genomes of each species. These results suggest that unreduced (2x) gametes were formed only in the female side of P. rosea.

Interspecific hybrid production by reciprocal crosses between Dianthus caryophyllus L. and Dianthus × isensis Hirahata et Kitamura

Summary To introduce the characters of extremely early flowering and unique flower shape of Dianthus isensis Hirahata et Kitamura into Dianthus caryophyllus L., reciprocal interspecific crosses were carried out between seven lines of D. caryophyllus and one line of D. isensis. When D. caryophyllus was used as the maternal parent, 79 progeny were obtained from only four lines of D. caryophyllus. Flow cytometric analysis revealed that they consisted of 30 diploid hybrid plants and 49 diploid maternal-type progeny. When D. isensis was used as the maternal parent, five progeny including three diploid hybrids, one triploid hybrid and one maternal-type were obtained by pollination with only two lines of D. caryophyllus. The ploidy level of these hybrids was confirmed by counting chromosome numbers in root-tip cells. Flow cytometric analysis also revealed that the triploid hybrid had two genomes of D. isensis and one genome of D. caryophyllus, suggesting that D. isensis had produced an unreduced female gamete. Since the diploid interspecific hybrids were fertile and have some of the profitable characters of D. isensis such as early flowering and a unique flower shape, they are expected to be used for future breeding in carnation.

Sporogenesis in haploid sporophytes of Osmunda japonica (Osmundaceae)

Three out of four haploid sporophytes of Osmunda japonica (2n=x=22), produced artificially by induction of apogamy, developed fertile fronds and spores. The spores that germinated (up to 25% of the total green spores) were monoploid, determined by the nuclear DNA content and the somatic chromosome numbers of the gametophytes. The haploid 2 sporophyte produced more fertile spores than the two other haploids, providing an opportunity to observe the details of fertile spore development. In some spore mother cells of this plant, the random separation of 22 univalent chromosomes did not occur at the first meiotic division, but restitution nuclei were formed. The observation of chromosome behavior in the haploid 2 sporophyte showed that sporogenesis in haploid O. japonica resembles the Braithwaite scheme in apogamous ferns. Gametophytes, developed from spores of the three haploid sporophytes, produced a few sporophytes with diploid chromosome numbers. One of the new sporophytes was trisomic.

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.

Amphidiploids produced by natural chromosome-doubling in inter-specific hybrids between Dianthus × isensis Hirahata et Kitam. and D. japonicus Thunb.

Summary Inter-specific crosses were carried out between two lines of Dianthus isensis Hirahata et Kitam., and one line of D. japonicus Thunb. Germinating seeds were obtained from only one line of D. x isensis used as the seed parent, and a total of 46 progeny from this cross were all confirmed to be hybrids by RAPD analysis and flow cytometry. The hybrids had intermediate characters of both parents with respect to leaf-width and flower-size, but flowered at the same time as each other with a uniform flower colour, purplish-pink. All 46 hybrids initially had sterile pollen and seeds, but two later produced some flowers of larger size with fertile pollen. Flow cytometric analysis revealed that flowers with restored pollen fertility originated from a chimaeric tetraploid branch induced by natural chromosome-doubling. Amphidiploid progeny were obtained from seeds produced by self-pollination of flowers with restored pollen fertility. Since these amphidiploids had some desirable characters, such as the early flowering of D. isensis, and the vigorous Summer growth, robust upright stems and broad leaves of D. japonicus, they can be used in carnation breeding programmes to incorporate useful traits from these two species such as disease resistance, heat tolerance and unique flower morphologies.

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.