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 developed molecular markers for discrimination of white and blue flower color in Japanese gentian plants. White-flowered gentians can be classified into two types, based on genetic and physiological features. One type includes four allelic variations (gtmyb3-1, gtmyb3-2, gtmyb3-3, and gtmyb3-4) of an anthocyanin biosynthetic regulator gene (GtMYB3), distinguished by three PCR-based molecular markers. The other type contains a newly identified inactive allele (ans1) of the anthocyanidin synthase (ANS) gene with a premature stop codon generated from a 4-bp deletion in the second exon. The ans1 allele was distinguished from the active ANS allele by a cleaved amplified polymorphism sequence (CAPS) marker. The genotypes of 12 white-flowered gentian cultivars/lines could be identified and classified as either ans1 or gtmyb3 using these four molecular markers. No white-flowered gentians contained ans1 and gtmyb3 alleles simultaneously. The mutated ANS gene co-segregated with white flower color in an F 2 population, demonstrating that the CAPS marker is useful to discriminate between white and blue flowers in gentian. Markers to discriminate flower color in Japanese gentian will be useful for early selection of progeny and for breeding management.
The development of new varieties of perennial plants generally requires lengthy and laborious procedures. In this study, we used ion beam irradiation mutagenesis in an attempt to accelerate the breeding process for perennial plants. We evaluated the biological effects of five ion beam sources (carbon, neon, argon, silicon, and iron) and neutron irradiation on Japanese gentian and apple. These treatments were applied at the National Institute of Radiological Sciences (NIRS) using the Heavy Ion Medical Accelerator in Chiba (HIMAC) and the Neutron-exposure Accelerator System for Biological Effect Experiments (NASBEE). Biological effects were observed in in vitro gentian plants after irradiation with ion beams at <10 Gy, whereas apple trees were less sensitive to ion beam irradiation. The growth of gentians in vitro was repressed by 3 Gy neutron irradiation, while that of grafted apple trees was not affected by 4 Gy neutron irradiation. During in vitro proliferation, seven pink-flowered lines were obtained from originally blue-flowered gentian after C and Ne ion beam irradiation treatments. Genomic and reverse transcription-PCR analyses of these lines suggested that the mutations occurred in the genomic region containing F3′5′H (encoding flavonoid 3′,5′-hydroxylase). These results provide useful information for the mutagenesis and breeding of gentian, apple, and other perennial plants.
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