Protocols for Micropropagation of Woody Trees and Fruits

214 G. NAUJOKS
somatic embryos developed after transfer to hormone-free medium. Bergman et al.
(1985) tested the influence of BA on the micropropagation potential of five
different willow clones, including one of S. caprea. Neuner and Beiderbeck (1993)
analysed nine clones of S. caprea for their tissue culture ability. Both publications
emphasised the difficulties in cutting and microcutting propagation of sallow and the
strong influence of genotypes on rooting success in this species. Callus proliferation
in Salix exigua with 2,4-D was induced by Stoehr et al. (1989), followed by shoot
regeneration with BA and rooting without hormones. A recent publication by Santos
et al. (2005) also described possibilities for callus induction in Salix humboldtiana
Willd with BA, 2,4-D and 1-naphthaleneacetic acid (NAA). Chung and Carrasco
(2000) presented experiments with 13 different Salix species (e.g. Salix stipularis,
S. triandra, S. purpurea, S. dasyclados, S. burjatica, S. calodendron, S. sericans, S.
eriocephala and others). They tested the influence of basic nutrient elements and the
concentration and effect of BA and gibberellic acid (GA3) on micropropagation
behaviour. As a result they reported that the responses obtained were dependent on
the type and concentration of growth-regulators used and were strongly dependent
on species and genotype.
Gebhardt (1992) reported that cytokinin-containing media resulted in shoot tip
browning of Salix viminalis, S. fragilis, S. x lispoclados, S. x rubens and S. petandra.
He recommended a shoot-division-method on medium without cytokinins. The suitability
of this micropropagation mode could be partly confirmed by Liesebach and
Naujoks (2004) during their investigations on vegetative propagation of Salix caprea.
Furthermore, there are reports on application of tissue culture methods for special
purposes. Crossing products from artificial pollination of Salix fragilis x S. lispoclados
are unable to survive under conventional conditions. Agrawal and Gebhardt (1994)
established and propagated these hybrid seedlings via embryo rescue in vitro on BA
containing medium. In a project dealing with alpine willow species as pioneer wood
for revegetation of areas endangered by erosion, Büttner et al. (2006) presented their
first results on tissue culture establishment for 11 difficult-to-propagate Salix species
selected in alpine regions. On media with cytokinin/auxin combinations, the first
step of establishment was successful for the majority of these clones.
In the following, a protocol which was developed for micropropagation of Salix
caprea clones is presented. This method may also be suitable for many other willow
species.
2. EXPERIMENTAL PROTOCOL
2.1. Explant Preparation
2.1.1. Growing Conditions of Mother Plants.
Accelerated sprouting of twigs in the greenhouse. One-year-old twigs, 40–50 cm long,
were harvested from adult donor trees at the end of January, put into vessels filled with
tap water and placed on tables in the greenhouse. To reduce surface contamination,
the twigs were sprayed with 0.2% Euparen (fungicide by BAYER, 50% dichlorfluanide,
w/v). The temperature was in the range from 18 to 26°C. Additional artificial
lighting was installed during winter and early spring for prolonging the illumination
period to about 12 hours a day aiming to force sprouting.