The overall goal of this study is to develop an anther culture system to produce doubled haploid (DH) lines of gentian (Gentiana triflora), an ornamental flowering plant, for use in an F1 hybrid breeding program. Embryogenesis was induced from anther cultures incubated on half-strength modified Lichter (NLN) medium containing a high concentration of sucrose (130 g/l) and subjected to heat shock treatment. Among the various parameters investigated, anthers collected from buds 9-12 mm in length induced the highest frequency of androgenesis. Moreover, among three genotypes tested, cvs. Ashiro-no-Aki and Ashiro-no-Natsu produced 21.3 and 3.7 embryos per 100 anthers, respectively, whereas, cv. Lovely-Ashiro failed to produce embryos. Among a total of 427 embryos transferred to a regeneration medium consisting of Murashige and Skoog (MS) medium, 138 plants were regenerated. The ploidy levels of regenerants were determined by flow cytometry and chromosome counts, revealing the presence of 5% haploids, 25% diploids, and 70% triploids. Inter simple sequence repeat (ISSR) analysis using the 6PS line obtained following self-pollination of the diploid plant obtained from anther culture confirmed that the diploid plant was indeed a DH.

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.

We have identified multiple alleles for a single gene termed W14/15. This gene encodes closely related but not identical proteins W14 and W15 that accumulate in overwinter buds of Gentiana triflora (Takahashi et al. in Breed Sci 56:39-46, 2006; Hikage et al. in Mol Genet Genomics 278:95-104, 2007). In this study, structural analysis of the W14/15 gene was carried out for 21 different gentian lines/cultivars consisting of 5 different species, to survey species- or line/cultivar-specific haplotypes. Within the samples examined, multiple variant forms were found. Those were categorized into seven major types (type I-VII) and ten subtypes based on the presence of three short insertion/deletion sites, three RFLP sites, and several SNP sites. Each line/cultivar had a distinct set of W14/15 gene variants for an allelic pair. Phylogenetic analysis showed that the W14/15 alleles cluster into groups that are characteristic of gentian species, i.e., G. triflora, G. scabra, G. pneumonanthe, G. septemfida and an unknown species other than the former four. In addition, within the same gentian species, different sets of haplotypes were found. Thus, the W14/15 alleles provide useful landmarks to resolve phylogenies of the genus or section Gentiana, as well as to analyze pedigree and breeding history of the cultivars derived from those Gentiana sp.

Successful production of haploid and doubled haploid (DH) plants from male and female gametophytic cells by in vitro culture has been established in some gentians (Gentiana triflora, G. scabra, and their hybrid), which are used as ornamental plants. Plant regeneration is obtained by both anther culture (androgenesis) and unfertilized ovule/ovary culture (gynogenesis). Attention is given to culture techniques, factors influencing androgenesis and gynogenesis, the ploidy of regenerants, and utilization of the molecular genetic marker for identification of DH. Although plant regeneration is obtained by both anther culture and unfertilized ovule/ovary culture, the latter has more advantages than the former. In gentian, the establishment of the haploid and DH method has application to F1 breeding and also to basic genetic studies.