The present investigation can be divided into two main sections: the first dealing with the
post-shedding embryogenesis of Encephalartos natalensis and the second concerned with the
cell wall properties of immature and mature embryos of this species.
Development of the embryo of E. natalensis from a rudimentary meristematic structure
approximately 700 μm in length, extends over six months after the seed is shed from the
strobilus. Throughout its development the embryo remains attached to a long suspensor.
Differentiation of the shoot meristem flanked by two cotyledonary protuberances occurs over
the first two months, during which peripheral tannin channels become apparent. Tannins,
apparently elaborated by the endoplasmic reticulum, first accumulate in the large central
vacuole and ultimately fill the channel. By the fourth month of development the root
meristem is apparent and procambial tissue forming discrete vascular bundles can be
discerned in the elongating cotyledons. Between four and six months, mucilage ducts
differentiate, and, after six months when the seed becomes germinable, the embryo is
characterised by cotyledons far longer than the axis. Shoot and root meristem cells remain
ultrastructurally similar throughout embryo ontogeny, containing small vacuoles, many welldifferentiated
mitochondria and ER profiles, abundant polysomes, plastids containing small
starch deposits and Golgi bodies. Unusually however, Golgi bodies are infrequent in other
cells including those elaborating mucilage which is accumulated in distended ER and
apparently secreted into the duct lumen directly by ER-derived vesicles. The nonmeristematic
cells accumulate massive starch deposits to the exclusion of any protein bodies,
and only very sparse lipid, features which are considered in terms of the prolonged period of
embryo development and the high atmospheric oxygen content of the Carboniferous Period,
when cycads are suggested to have originated.
With regard to plant cell walls, the present investigation employed immunofluorescence
microscopy and immunocytochemistry to characterise the cell walls of immature and mature
embryos of the recalcitrant-seeded E. natalensis to determine wall composition and potential
changes with development. These techniques, together with cryo-scanning- and transmissionelectron
microscopy (TEM) were used to analyse potential changes in the cell walls of mature
embryos upon desiccation. Immature cell walls appeared to be composed of low- and high
methyl esterified epitopes of pectin, rhamnogalacturonan-associated arabinan, and the
hemicellulose xyloglucan, while partially-esterified epitopes of pectin appear to have a punctuate distribution in the wall. Arabinogalactan protein recognised by the LM2 antibody,
along with rhamnogalacturonan-associated galactan and the hemicellulose xylan, were not
positively localised using immunological probes, suggesting that the embryo of the current
species does not possess these epitopes. Interestingly, mature embryos appeared to be
identical to immature ones with respect to the cell wall components investigated, implying
that these may not change during the protracted post-shedding embryogeny of this species.
Analysis of the monosaccharide composition of the walls by gas liquid chromatography
complemented the immuno-labelling work. However, there appeared to be abnormally high
levels of glucose (Glc), which may indicate the presence of Glc-rich polymers not accounted
for by the antibodies used in the current study. Preliminary Glc-normalised data revealed that
there may be considerable quantities of arabinose polymers in the wall comparable to that
found in desiccation tolerant plants. Drying appeared to induce some degree of cell wall
folding in mature embryos, correlating with their possession of wall plasticisers such as
arabinose polymers, but this was limited, due to the abundance of amyloplasts, which filled
the cytoplasmic space. From the results of this study, it is proposed that the embryo cell walls
of E. natalensis are constitutively prepared for the flexibility required during cell growth and
expansion, which may facilitate the observed moderate cell wall folding in mature embryos
upon drying. This, together with an abundant supply of amyloplasts in the cytomatrix may
provide sufficient mechanical stabilisation during desiccation even though the seeds of this
species are highly desiccation sensitive.
Overall, this study has been a relatively comprehensive coverage of histological and
ultrastructural aspects of embryogenesis in E. natalensis. This work will form a pivotal basis
for future studies, which may ultimately lead to the successful germplasm cryopreservation
and in vitro production on a commercial scale of these, and other, endangered cycad species.
Furthermore, the work on cell walls in this investigation has provided improved
comprehension of the responses of seed cell walls to dehydration. / Ph.D. University of KwaZulu-Natal, Durban 2013.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/11176 |
| Date | 11 September 2014 |
| Creators | Woodenberg, Wynston. |
| Contributors | Berjak, Pat., Pammenter, Norman., Farrant, Jill. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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