Optimising methods for embryonic axis fixation and micropropagation of Syzygium cordatum Hochst.
Syzygium cordatum Hochst. (family - Myrtaceae), commonly called the umDoni (Zulu) tree, is found throughout South Africa. The tree is utilised for its fruit, bark and wood by many villagers, and this demand has placed potential pressure on existing populations. It is necessary to conserve this widely used tree before it becomes threatened by over-utilisation. Seeds of S. cordatum are recalcitrant and storable only in the short-term at 16oC over moist paper towel (hydrated storage). The study was initiated to follow deterioration of the embryonic axes, in relation to dehydrated versus and hydrated storage. However, for electron microscopic investigations, it was crucial that material was properly fixed to obtain samples that accurately represented the in vivo conditions. This proved to be challenging, as explained below, and changed the original aim of the project. The high phenolic content of S. cordatum seeds and axes makes fixation, using an aldehyde-based fixative, such as glutaraldehyde, difficult, as the aldehyde groups bind to phenolic compounds, forming large oligomers that tear out during sectioning. This causes sections to become fragmented, making viewing with the transmission electron microscope (TEM) impossible. The quest to visualise the ultrastructure, consequently became an additional focus of the project. Substituting glutaraldehyde with alternate primary fixatives including potassium permanganate (KMnO4) and 1% osmium tetroxide (OsO4) did not improve the situation. Cryo-fixation followed by freeze substitution was then attempted. Three substitution media, comprising glutaraldehyde, tannic acid, osmium tetroxide and acetone were used, all providing similar, unsatisfactory results showing ice crystal damage. Eventually, glutaraldehyde fixation was modified where samples were fixed in glutaraldehyde while being exposed to microwave energy. Results from this method of fixation were far better, with fine structure adequately preserved. A second facet of the project was aimed at producing explants alternative to seed-derived zygotic axes. Cotyledonary explants used in an attempt to produce somatic embryos, were cultured onto media which incorporated various concentrations of 2,4-D, BAP and NAA. The callus produced was sub-cultured onto regeneration media, which included NAA and BAP or PGR-free media, did not develop further. Zygotic axes cultured onto shoot multiplication medium containing BAP and NAA produced adventitious shoots which produced roots when sub-cultured onto media containing GA3.