Investigation of in vitro somatic embryogenesis and haploidization methods in naturally distributed and endemic Helleborus vesicarius species in Turkey

The Helleborus genus is distributed in the world with 22 species. Helleborus vesicarius is endemic to Turkey. Plant genetic resources naturally distributed in the flora of Turkey are very important materials for the ornamental plants sector. Pure line breeding is one of the most commonly used methods to develop new varieties from wild species. Dihaploidization is an effective method to obtain 100% homozygous pure lines. In order to obtain haploid plants of this species, which grow naturally in Turkey, in vitro ovule, ovary and anther culture experiments were established. Morphological characterization of species collected from nature was carried out. Appropriate bud sizes were determined for anther, ovule and ovary cultures in H. vesicarius. In anther culture experiments, different concentrations of NAA (0, 0.5, 1.5, 2 mgL-1) and BA (0, 0.1, 0.3, 0.5 mgL-1) were combined with 30-60-90-120 gL-1 sucrose. MS and B5 medium supplemented with +0.4% gelrite were used. Development in anther culture remained limited. In ovule culture experiments, the effects of MS medium supplemented with NAA, BA and TDZ (0, 0.5, 1, 2 mgL-1) were examined. In H. vesicarius, callus formation occurred at a rate of 38% on MS medium containing 2 mgL-1NAA + 0.5 mgL-1 TDZ, and haploid embryo development was achieved. The effects of MS medium supplemented with NAA and BA (0, 0.5, 1, 2 mgL-1) were examined in ovary culture experiments. Somatic embryogenesis method was used to propagate the endemic species. In somatic embryogenesis experiments, different concentrations of NAA and BA (0, 0.5, 1, 2 mgL-1) were tested in MS medium. Callus and embryo structures were obtained from leaf expanses, and the best development was observed at a concentration of 2 mgL-1 NAA + 1 mgL-1 BA. In the ovule culture and SE methods applied to H. vesicarius species, explants remained in the embryo stage and transformation into plants could not be achieved .Key words: Anther culture, Haploidization, Helleborus vesicarius, Ovule culture, Somatic Embryogenesis