Disa comprises 163 species, 131 of which occur in South Africa (SA). The
genus is distributed across winter- and summer-rainfall areas, but few species
transverse both climatic regions. Species are therefore regarded as winter-rainfall
or summer-rainfall endemics - yet release their seeds in autumn, irrespective of
provenance. Disa contributes 40 % of threatened Orchidaceae in SA, with half of
the local species requiring conservation initiatives. In vitro seed germination is a
potential conservation tool for producing large numbers of genetically diverse
plants in relatively short periods. However, only 11 winter-rainfall Disa species are
easily germinated ex situ. Studies were therefore undertaken on summer-rainfall
taxa, which are ungerminated in vitro, in an effort to define their germination
parameters. This thesis describes mechanisms that control germination in Disa
and establishes practical propagation methods for seed culture.
Two seed types occur in Disa; i) comparatively large, pale and pyriform
seeds in members of the D. uniflora sub-c1ade, which populate streamside habitats
under conditions of winter-rainfall maxima, and ii) smaller, variously brown and
fusiform seeds in the remainder of the genus. Seed morphometrics distinguished
seed types, although embryo dimensions were similar. Testa continuity, which is
disrupted in the large seeds, also supported separation. Typically, small seeds
are ungerminated in vitro, whilst large seeds germinated readily. Increased seed
size did not necessarily impart increased germ inability, as several germinable,
small-seeded species exist - being winter-rainfall species
Attempts to establish in vitro germinability revealed that increased water
availability and charcoal supplementation promoted germination in intractable
species. The control of germination was therefore proposed as a trade-off
between water availability and the presence of phyto-inhibitors - two features
typical of seeds exhibiting water-impermeable dormancy. Three germinability
categories were recognized; i) easily germinable species, ii) poorly germinable
species through media manipulation, and iii) ungerminated species. Germination
of immature seed in the absence of media modification was comparable to mature
seed germination under modified conditions, providing evidence of the role of an
impermeable seed testa in regulating germination.
Testa impermeability in mature, small-seeded species was demonstrated
using aqueous EVANS' blue dye and was linked to i) testa integrity and ii) increased
levels of leachable phenolics (LPC) - which are hydrophobic and phytotoxic. In
addition, this research revealed an impervious and elaborate embryo carapace in
small seeds. Large-seeded species were highly permeable at dehiscence, with
perforated testae and negligible LPC. Germinability was ultimately defined by a
significant regression with LPC. Phenolic deposition increased exponentially with
increasing seed maturity and reflected decreased permeability and the
development of testa colouration. The testa precludes the use of viability stains
such as nc and FDA, unless rendered permeable through scarification. This
was achieved using NaOCI. Viability and germinability percentages did not
correlate well for the small-seeded Disa species, indicating that i) the methods
used to break dormancy are inadequate, ii) additional factors may be acting in
concert with the testa to regulate germination and iii) that the determination of
mature Disa seed viability is ineffective. As an alternative, the germination
potential of immature seed was estimated as the ratio between the proportion of
embryos stained with TTC and the proportion of seeds permeable to EVANS' blue.
Attempts to relieve water-impermeable dormancy in Disa resulted in the
formulation of a dual-phase protocol - with the specific aim of increasing water
availability to the embryo. Dual-phase cultures comprised a solid, charcoal-rich
medium overlaid with a reduced strength, liquid medium fraction of the same type.
The solid fraction negated the influence of leached phenols and allowed
protocorms to establish polarity, whilst the fluid fraction increased water
availability. The dual-phase protocol allowed germination of nine summer-rainfall
Disa species, usually in percentages that approximated their estimated
germination potential.
For the remaining species, germination is controlled by more complex
factors. Large seeds are atypical in containing starch, the hydrolysis of which
facilitated their rapid, autonomous germination. Small-seeded Disa species stored
lipids and proteins and germinable species accumulated starch post-germination.
The embryo protoplasts of all species contained appreciable amounts of soluble
sugars, irrespective of germinability. However, decreased sucrose and increased
fructose correlated significantly with decreased seed germinability. This study
provides evidence of the nutritional value of mycotrophy, with endophytes
liberating soluble carbohydrate and non-carbohydrate compounds upon lysis.
However, few species were germinated symbiotically, suggesting that endophytes
isolated from adult roots do not necessarily support germination in the same
species. Similar endophytic fungi occur in Australian and Holarctic orchids. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/5579 |
| Date | January 2003 |
| Creators | Thompson, David Ian. |
| Contributors | Edwards, Trevor J., Van Staden, Johannes. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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