Artificial induction of tetraploidy in Humulus lupulus L. using oryzalin

The aim of the research was to establish an efficient procedure for in vitro micropropagation in order to obtain numbers of identical plants for in vitro polyploidization of Humulus lupulus (2n = 20), using antimicrotubule agent oryzalin. For this purpose, the polyploidization was carried out for H. lupulus Osvald’s clones 31, 74, 114, and for ‘Sladek’ cultivar. The two experimental methods – the cultivation of nodal segments on medium with different concentrations of oryzalin (1, 5, and 10 µM) for 2 weeks and the irrigation of nodal segments with oryzalin (10 and 20 µM) for 24 and 48 h were chosen for inducing for polyploid plantlets of H. lupulus. This procedure provided tetraploids, which were identified by flow cytometry using internal standardization method and confirmed using chromosome counting of methaphasic cells from the root tips and morphological observations. The influence of chromosome doubling was also verified with stomata characterization. The polyploid plants were propagated for next evaluation, rooting and transfer to nonsterile conditions and into field. After chromosome doubling, using some different concentration of oryzalin, some plantlets became tetraploids, no mixoploids were detected. The highest efficiency of polyploidization was achieved for clone 72 after 2-week treatment of oryzalin supplemented medium. On the other hand, for method based on the irrigation of nodal segments with oryzalin, the most efficient conditions were treatment with 10 µM and 20 µM oryzalin for 24 and 48 h, respectively.