Plant germplasm conservation :|bdevelopment of field collection and transport techniques for eucalyptus species and trichilia dregeana

Abstract

Lack of suitable techniques for field collection of the germplasm of different species, and spoilage of samples in transit, hinder efforts to collect, conserve, distribute and regenerate most plant germplasm in vitro. The aims of this investigation, therefore, were to address problems encountered in collection of field germplasm from species and hybrids of Eucalyptus (TAG5, TAGI4, ZG14, GC550 and GU2IO) that are propagated vegetatively and Trichilia dregeana, which has recalcitrant seeds. Simple in vitro culture-based protocols were developed to minimise contamination and maintain viability of plant material for sufficient time for it to be transported from the field to the tissue culture laboratory. From the two simulations of 48 h 'transportation' conditions for explants of Eucalyptus species investigated, those in bottles containing sterile vermiculite exhibited no contamination and greater than 50% bud break, regardless of whether or not field surface sterilization with alcohol had been done. In contrast, when explants were enclosed in cling wrap, contamination was high and bud break levels low. For selection of the more suitable Eucalyptus explant, two types were investigated: nodal explants each with one half leaf (type 1) and stem segments with three nodes (type 2). As type 2 showed considerably better shoot yields (up to 1624 shoots per 100 explant), and were more practical to use with respect to space, such trinodal stem segments were deemed best for collection. Of the sterilization procedures investigated, treatment with 70% (v/v) alcohol prior to storage was found to be most suitable in almost all cases. For plant material with high endogenous microbial contamination, the bud break medium was supplemented with Benomyl and calcium hypochlorite, each at 0.5 and 1.0 g.r). Alcoholtreated, stored explants cultured on bud break medium with 1.0 g.r) calcium hypochlorite exhibited low levels of contamination and an increased final yield (up to a maximum of 930 shoots per 100 explants). Thus, this protocol was employed for field material of E. grandis clones TAG5, TAGI4 and ZGI4. For these clones, stored type 2 explants