Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (<i>Gentiana</i> spp.)

Abstract

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.

Haploid and Doubled Haploid Plant Production in Gentian (Gentiana spp.)

“…These authors demonstrated that G. scabra and the hybrid G. triflora × G. scabra, which is a recalcitrant species in anther culture, produced many ELSs via gynogenesis. Recently, similar results were obtained when this method was applied to many other genotypes (Doi et al 2013). From these results, Doi et al (2011Doi et al ( , 2013 indicated that ELS production from the culture of unfertilized ovules in gentian was not affected by genotype when compared with androgenesis.…”

“…Recently, similar results were obtained when this method was applied to many other genotypes (Doi et al 2013). From these results, Doi et al (2011Doi et al ( , 2013 indicated that ELS production from the culture of unfertilized ovules in gentian was not affected by genotype when compared with androgenesis. The developmental stage of ovules has an intense influence on gynogenesis, but the responsive stage depends on the species (Mukhambetzhanov 1997;Chen et al 2011).…”

“…Pathirana et al (2011) treated regenerants with 15.5 mg/l oryzalin for 4 weeks, but did not report frequency of diploidization. When G. triflora haploids were treated with 50 μM oryzalin for 1-4 weeks, 37.5-23.9 % of diploids and 50.0-37.5 % of tetraploids were obtained (Doi et al 2013). …”

Haploid induction via unpollinated ovule culture in Gerbera hybrida

“…Cold pretreatment (4 °C) of flower buds for 1-2 weeks also promotes effective induction of embryo-like structures in plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) 23 . Cold pretreatment also has a positive effect on direct embryo induction in pepper (Capsicum annuum L.) 24 .…”

“…These results are consistent with a previous analysis of unfertilized ovule/ovary culture in gentians (Gentiana spp. ), with 57.9% haploid and 34.3% diploid among the total of 1,515 regenerated plantlets 23 . Miyoshi and Asakura cultured unpollinated ovules of several genotypes of pot gerbera (G. jamesonii) and found that 80.4% were haploid, 15.2% were diploid, and 4.3% were mixoploid 11 .…”