Biotechnological methods of in vitro propagation in willows (Salix spp.)

Abstract

Many populations of high-mountainous relic dioecious willows in Central Europe only consist of female individuals and are thus limited in their reproductive potential. We completed micropropagation experiments with shoot apexes and nodal segments of common and endangered willow (Salix) species, which can help to reintroduce autochthonous genotypes to their natural sites. Until recently, cultivation of green young shoot apexes of S. alba and S. lapponum showed the highest percentage of regeneration. We successfully applied the two-times-sterilisation due to high contamination of natural explants. The OK medium was the most efficient culture medium. In vitro propagation of willows with unisexual catkins, anther and ovule cultures were tested and optimised. Isolated anthers were cultivated on selected media and then microcallus and calluses of S. caprea and calluses of S. viminalis were formed on the A medium. Among various tested and optimised media for the ovule culture, the CP medium was the most efficient one. In this case, only the microcalluses of S. viminalis were observed. We developed biotechnological procedures that can be useful in conserving fragmented populations of high-mountainous willows.

Efficient method for rapid multiplication of clean and healthy willow clones via in vitro propagation with broad genotype applicability

“…In previous reports, depending on the genotypes studied, it proved necessary to use cytokines to obtain the regeneration of new shoots from axillary buds. The most frequently used cytokine was 6-Benzylaminopurine (Bergman et al 1985;Mashkina et al 2010;Park et al 2008;Skálová et al 2012); however, thidiazuron was also used for shoot induction with S. tetrasperma (Khan and Anis 2012). For propagation of S. caprea, the most suitable medium was reported to be hormone free with the addition of activate charcoal (Liesebach and Naujoks 2004;Naujoks 2007).…”

“…We also observed good shoot formation in the absence of cytokinin, but we found it necessary to use the auxin IBA to facilitate root development. Although spontaneous in vitro rooting of some willow cultivars has been described (Khan et al 2011;Lyyra et al 2006;Park et al 2008), rooting has been more commonly induced by the inclusion of some auxin such as indol-3-acetic acid (IAA) (Mashkina et al 2010), naphthaleneacetic acid (NAA) (Bergman et al 1985), or, in most cases, IBA (Khan et al 2011;Khan and Anis 2012;Naujoks 2007;Park et al 2008;Skálová et al 2012). In this paper, we reported the use of a simple, single medium, one-half MS with 0.1 mg·L −1 IBA, which was successful in a wide range of genotypes for every culture stage, i.e., shoot induction, elongation, multiplication, and rooting of in vitro willow plants.…”

“…In this paper, we propose a complementary approach in which the phenotype selection and multiplication functions of the "preliminary" and "observation" trials are partly or completely substituted by tissue culture to multiply clones that could be incorporated into willow breeding schemes to save 4 or 5 years. Explants used previously to initiate direct organogenesis for regeneration in tissue culture include buds, adventitious shoots, and shoot apexes (reviewed in Skálová et al (2012)). Explants are commonly cultured on woody plant medium (WPM) (Lloyd and McCown 1980) or Murashige and Skoog medium (MS) (Murashige and Skoog 1962) supplemented with hormones, sugars, and other compounds.…”

Genetic Stability of the Endangered Species Salix lapponum L. Regenerated In Vitro during the Reintroduction Process

“…Willows grow in river valleys and wetlands, forming riparian forests or dense shrubs worldwide, from the Arctic to the tropical zone [1]. The genus Salix is also of biogeographic importance, because most high mountain willow species in Central Europe are relic taxa with fragmented distribution [2][3][4]. Populations of various species of this genus are spatially limited, and many are present only as female-biased sex ratio populations [5].…”

“…Tissue cultures have been widely used for propagation of various rare and endangered plant species [12][13][14]. The method has been also applied to propagate various Salix species [15][16][17]; however, only one study has been undertaken on micropropagation of S. lapponum, and it concerned specimens growing in a mountainous area [4].…”