Artificial induction of tetraploidy in Humulus lupulus L. using oryzalin

Abstract

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 <em>Humulus lupulus</em> (2<em>n</em> = 20), using antimicrotubule agent oryzalin. For this purpose, the polyploidization was carried out for <em>H. lupulus</em> 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 <em>H. lupulus</em>. 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.

Polyploidy in Industrial Crops: Applications and Perspectives in Plant Breeding

“…As reported by Trojak-Goluch and Skomra [73] as well as Švécarová et al [78], hop tetraploids showed poorer acclimatisation to field conditions and poorer rooting. Similar traits were displayed by tetraploids of T. vulgaris [79] and C. sativa [74].…”

“…On the other hand, shorter and narrower leaves, especially in the central part of the main and lateral shoots, were obtained in hop tetraploids [73] (Figure 1). Contrasting results were obtained by Švécarová et al, where the leaves of tetraploids of H. lupulus were larger than in diploids, and their shape changed [78]. For example, in certain species, C. roseus, polyploidisation did not significantly change the leaf size, thickness or colour, but increased the total number of leaves, resulting in increased biomass [107].…”

“…Also, Švécarová et al demonstrated that among the four tested genotypes of H. lupulus, one (maple No. 72) was distinguished by a significantly higher frequency of polyploid induction, regardless of oryzalin concentration and application method [78]. Differences in genome polyploidisation efficiency depending on the genotype of the donor plant were also confirmed by studies on S. viminalis [75].…”

“…The number of polyploids obtained this way represented 10.3 to 66.7% of the regenerants [79]. The study by Švécarová et al showed that for H. lupulus, it is possible to obtain a greater number of polyploids (70% of all tested) using this method [78]. An important issue related to plant regeneration from somatic tissues is the emerging somaclonal variability, resulting from the genetic variation of the primary explant (occurrence of cells with different ploidy), mutations in the genome or epigenetic changes.…”

“…In the following years, Heinz and Mee [85] treated cell suspension with colchicine and produced polyploids of S. officinarum. Nowadays, the most commonly used plant material for in vitro polyploid induction are shoot tips, axillary buds, petiole or leaf explants, nodal segments, roots and callus [74,77,78,81]. A technique for the induction of tetraploid Humulus lupulus L. from apical meristems incubated in colchicine solution and shaken at 100 rpm has been developed [73].…”