Chromosome Doubling of Japanese Native Lychnis spp . by in vitro Oryzalin Treatment

The genus Lychnis, a member of Caryophyllaceae, consists of about 30 species, which are distributed throughout the temperate regions of the Northern Hemisphere (Magnus et al., 2008). Among them, 6 species, L. fulgens, L. gracillima, L. kiusiana, L. miqueliana, L. sieboldii and L. wilfordii, are native to Japan. Most Lychnis spp.,including Japanese native ones, are cultivated as pot and garden plants for their beautiful flowers. However, all the species have few variations in horticultural traits, such as flower color, flower form and plant form. Previously we examined effect of in vitro spindle toxin treatment on chromosome doubling of a triploid genotype of L. senno in order to widen its variability in horticultural traits (Nonaka et al., 2011). Efficient chromosome doubling was achieved by treating nodal segments with 10 mg L oryzalin (ORY), and this method was successfully applied to diploid genotypes of L. fulgens and L. sieboldii (Nonaka et al., 2011). In the present study, we examined chromosome doubling of 3 Japanese native Lychnis spp., L. gracillima, L. kiusiana and L. miqueliana, by using the in vitro oryzalin treatment method developed by Nonaka et al. (2011). Chromosome doubling of plantlets derived from ORY-treated nodal segments was confirmed by both flow cytometry (FCM) analysis and chromosome observation. MATERIALS AND METHODS Plant materials and shoot culture L. gracillima (2n=2x=24), L. kiusiana (2n=2x=24) and L. miqueliana (2n=2x=24) were used in the present study. Potted plants were cultivated in the greenhouse without heating at the Faculty of Agriculture, Niigata University. Shoot cultures of 3 Lychnis spp. were established according to Nonaka et al. (2011). Briefly, nodal explants were prepared from vigorously growing stems of potted plants and placed on a shoot culture medium [Murashige and Skoog (1962) (MS) medium supplemented with 10 mg L benzyl adenine, 30 g L sucrose and 2 g L gellan gum, pH 5.7]. Axillary bud-derived shoots were subcultured every 2 months by transferring nodal segments to fresh medium of the same composition. Cultures were maintained at 25°C under continuous illumination with fluorescent light (35 μmol m s).