Some aspects of megagametophyte development and post-shedding seed behaviour of Encephalartos natalensis (Zamiaceae)

Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2009.

Very little is known about the post-shedding seed behaviour and megagametophyte development of the cycads, the most primitive extant seed-bearing plants, which pre-date the dinosaurs. In the present investigation, seeds of Encephalartos natalensis Dyer and Verdoorn were shed with relatively high mean embryo (3.33 g g-1) and megagametophyte (1.25 } 0.16 g g-1) WCs, when the developing embryo consisted primarily of the coiled, elongated suspensor bearing a rudimentary sporophyte at its tip. It was not surprising that these seeds were revealed as desiccation sensitive in the present investigation, as the embryos continued to develop after seed-shed, reaching a germinable size (.15 mm) only 4 . 6 months after seed abscission from the strobilus. Maintenance of the seeds in hydrated storage conditions was precluded by the proliferation of fungi, despite the application of the fungicide: BenlateR. Some seeds were also found to germinate in hydrated storage, despite the hard physical barrier to germination imposed by the enclosing sclerotesta. Seeds dusted with BenlateR and placed in eopen f storage in loosely closed paper bags had a longer life-span than those placed in hydrated storage; however, seeds stored in open storage were also overcome by fungi, but only around 18 months after seed-shed. Therefore, while the vigour and viability of the seeds appeared to decline slowly in the months after the embryos reached a germinable size, the life-span of stored E. natalensis seeds devoid of fungi is yet to be determined and will be the subject of further research. The current investigation also combined ultrastructural and viability retention studies to observe the post-shedding behaviour of the storage tissue, the megagametophyte. The cells of the megagametophyte became progressively packed with starch and protein as the two main storage reserves, a limited number of discrete lipid bodies, and occasional mitochondria all of which appeared to be embedded in an homogeneous matrix. When the development of the megagametophyte cells was analysed ultrastructurally, it was found that the unusual matrix was present from the inception of megagametophyte cellularisation, and contained microtubules and numerous very faintly-visible vesicles. Newly-formed megagametophyte cells were thus not highly vacuolated as previously thought, but dominated by an homogeneous matrix. Enzyme-gold localisation was employed in an attempt to determine the organelles responsible for the deposition of cell wall components during cellularisation of the megagametophyte. It appeared that ER-derived vesicles (and not Golgi-derived vesicles) were the principal contributors of the primary cell wall components, pectin and xylan. While cellularisation took place over approximately 1 - 2 weeks, subsequent development of the megagametophyte cells involved the accumulation of storage reserves, this phase lasting approximately 8 months -when the seeds were shed whether pollination/fertilisation had recently occurred, or not. At seed-shed, the cells of the megagametophyte were nucleated and contained a few mitochondria of a metabolically-active appearance. The occurrence of aerobic metabolism in these cells was confirmed by the tetrazolium (TTZ) test. Judging from the TTZ reactivity, the viability of the megagametophyte cells of fertilised seed appeared to decline slowly in the months after seed-shed, in parallel with extension growth of the embryo. The cell layer comprising the external surface of the megagametophyte showed marked ultrastructural differences from the inner cells, and may emerge as having an ‘aleurone-like’ function. It is, however, possible that the cells of the body of the gametophyte participate actively – at least in the earlier stages of post-shedding seed development – in mobilisation of stored reserves, which must support the development of the embryonic sporophyte.