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Characterisation and role of sugarcane invertase with special reference to neutral invertase.Vorster, Darren James. January 2000 (has links)
The relationship between extractable invertase activities and sucrose
accumulation in the sugarcane (Saccharum spp. hybrids) culm and in vivo
invertase mediated sucrose hydrolysis was investigated to determine the
significance of invertases in sucrose utilisation and turnover. In vitro activities
were determined by assaying the soluble acid invertase (SAI), cell wall bound
acid invertase (CWA) and neutral invertase (NI) from internodes three to ten in
mature sugarcane plants of cultivar NCo376. Extractable activities were verified
by immunoblotting. In vivo invertase mediated sucrose hydrolysis was
investigated in tissue discs prepared from mature culm tissue of the same
cultivar. Sugarcane NI had a higher specific activity than SAI (apoplastic and
vacuolar) in the sucrose accumulating region of the sugarcane culm. CWA was
also present in significant quantities in both immature and mature tissue.
Sugarcane NI was partially purified from mature sugarcane culm tissue to remove
any potential competing activity. The enzyme is non-glycosylated and exhibits
catalytic activity as a monomer, dimer and tetramer. Most of the activity elutes as
a monomer of native Mr ca 60 kDa. The enzyme displays typical hyperbolic
saturation kinetics for sucrose hydrolysis. It has a Km of 9.8 mM for sucrose and a
pH optimum of 7.2. An Arrhenius plot shows the energy of activation of the
enzyme for sucrose to be 62.5 kJ.mol-1 below 30°C and -11.6 kJ.mol-1 above
30°C. Sugarcane NI is inhibited by its products, with fructose being a more
effective inhibitor than glucose. Sugarcane NI is significantly inhibited by HgCI2,
AgNO-3, ZnCI2, CuSO4 and CoCI2 but not by CaCI2, MgCI2 or MnCI2. Sugarcane NI
showed no significant hydrolysis of cellobiose or trehalose.
When radiolabelled fructose was fed to sugarcane internodal tissue, label
appeared in glucose which demonstrates that invertase mediated hydrolysis of
sucrose occurs. A combination of continuous feeding and pulse chase
experiments was used to investigate the in vivo contribution of the invertases and
the compartmentation of sugars.
Sucrose is synthesised at a rate greater than the rate of breakdown at all stages
of maturity in sugarcane culm tissue. The turnover time of the total cytosolic label
pool is longer for internode three than internode six. A higher vacuolar:cytosolic
sugar molar ratio than previously assumed is indicated. Developmentally, the
greatest change in carbon allocation occurs from internodes three to six. The
main competing pools are the insoluble and neutral fractions. As the tissue
matures, less carbon is allocated to the insoluble and more to the neutral fraction.
The neutral fraction consists mainly of sucrose, glucose and fructose.
The compartmented nature of sugarcane storage parenchyma carbohydrate
metabolism results in a system that is complex and difficult to investigate. A
computer based metabolic flux model was developed to aid in the interpretation of
timecourse labelling studies. A significant obstacle was the global optimization of
the model, while maintaining physiologically meaningful flux parameters. Once
the vacuolar:cytosolic molar ratio was increased, the model was able to describe
the internode three and six labelling profiles. The model results were in
agreement with experimental observation. An increase in the rate of sucrose
accumulation was observed with tissue maturation.
Only the internode three glucokinase activity was greater than the experimentally
determined limit. The rate was however physiologically feasible and may reflect
the underestimation of the in vivo rate. SAI and NI contributed to sucrose
hydrolysis in internode three but not in internode six. The rates in internode six
were set to fixed low values to enable the model to fit the experimental data. This
does not however preclude low levels of in vivo SAI and NI activity, which would
prove significant over a longer time period. The flow of label through the individual
pools, which comprise the experimentally measured composite pools could be
observed. This provides insight into the sucrose moiety label ratio, SPS:SuSy
sucrose synthesis ratio, and the rate of 14CO2 release. The model provides a
framework for the investigation and interpretation of timecourse labelling studies
of sugarcane storage parenchyma. / Thesis (Ph.D.)-University of Natal, Durban, 2000.
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Desiccation tolerance and sensitivity of vegetative plant tissue.Sherwin, Heather Wendy. January 1995 (has links)
There is a great deal of work currently being done in the field of desiccation tolerance. Generally workers studying desiccation-tolerant plant tissues have concentrated on the
mechanisms of desiccation tolerance without concomitant studies on why most plants cannot survive desiccation. The present study considers both a desiccation-tolerant plant
as well as a range of desiccation-sensitive plants. The work incorporates physiological, biophysical, biochemical and ultrastructural studies in an attempt to get a holistic picture
of vegetative material as it dries and then rehydrates.
The plant species used in this study are: Craterostigma nanum, a so-called resurrection plant; Garcinia livingstonei, a drought-tolerant small tree; Isoglossa woodii, an
understorey shrub which shows a remarkable ability to recover from wilting; Pisum sativum seedlings, which have a very high water content at full turgor; and finally, Adiantum raddianum, the maiden hair fern, which wilts very quickly and does not recover from wilting. The desiccation-tolerant plant, C. nanum, had an unusual pressure-volume (PV) curve which indicated that while large volume changes were taking place there was little concomitant change in pressure or water potential. The unusual nature of this PV curve
made it difficult to assess the relative water content (RWC) at which turgor was lost. The desiccation-sensitive plants exhibited standard curvi-linear PV curves. The amount of nonfreezable water in the five species was studied and found to show no correlation with the ability to withstand dehydration or with the lethal water content. There were no differences in the melting enthalpy of tissue water between the tolerant and most of the sensitive plants. Isoglossa woodii had a lower melting enthalpy than the tolerant and the other sensitive species. Survival studies showed that the desiccation-sensitive plants all had similar lethal RWCs.
The tolerant plant survived dehydration to as low as 1% RWC, recovering on rehydration within 24 hours. Membrane leakage studies showed that the sensitive plants all exhibited membrane damage at different absolute water contents, but very similar RWCs and water potentials. The increase in leakage corresponded to the lethal RWC for all the sensitive species. The desiccation-tolerant plant recovered from dehydration to very low water contents and did not show an increase in membrane leakage if prior rehydration had taken place. Without prior rehydration this tolerant plant exhibited an increase in leakage at similar RWCs and water potentials to that of the sensitive species. There did not appear to be much difference in the RWC at which damage to membranes occurred whether the material was dried rapidly or slowly. Respiration and chlorophyll fluorescence were studied to determine what effect drying and rehydration have on the electron transport· processes of the leaf. The chlorophyll fluorescence studies gave an indication of damage to the photosynthetic apparatus. Both
qualitative changes as well as quantitative changes in fluorescence parameters were assessed. Characteristics like quantum efficiency (Fv/Fm)remained fairly constant for a
wide range of RWCs until a critical RWC was reached where there was a sharp decline in Fv/Fm. Upon rehydration, C. nanum recovered to pre-stress levels, I. woodii showed no recovery and no further damage on rehydration, whilst the other species exhibited even
more damage on rehydration than they had on dehydration.
Respiration remained fairly constant or increased slightly during drying until a critical RWC was reached at which it suddenly declined. The RWC at which this decline occurred
ranged from 15% and 20% in P. sativum and C. nanum respectively, to 50% for G. livingstonei. On rehydration respiration exceeded the levels measured in dehydrated
material for the sensitive species. Unsuccessful attempts were made to fix material anhydrously for ultrastructural studies so standard fIxation was used. The ultrastructural studies revealed that changes had occurred in the ultrastructure of leaves of the sensitive species dried to 30% RWC particularly in A. raddianum and P. sativum. Drying to 5% RWC revealed extensive ultrastructural
degradation which was worsened on rehydration in the sensitive species. The tolerant species showed ultrastructural changes on drying but these were not as severe as occurred in the sensitive species. The cell walls of the tolerant species folded in on drying. This folding was possibly responsible for the unusual PV curves found in this species. At 5% RWC the cells were closely packed and very irregular in shape. The cell contents were clearly resolved and evenly spread throughout the cell. The large central vacuole appeared to have subdivided into a number of smaller vacuoles. On rehydration the cells regained their shape and the cell contents had moved towards the periphery as the large central vacuole was reformed. Beading of membranes, which was common in the sensitive
species, was not found in the tolerant species suggesting that membrane damage was not as severe in the tolerant species. Western Blot analysis of the proteins present during drying was performed to determine whether a class of desiccation-induced proteins, called dehydrins, were present. These proteins have been suggested to play a protective role in desiccation-tolerant tissue. It was found that C. nanum did, in fact, possess dehydrins, but so did P. sativum. The other three sensitive species did not show any appreciable levels of dehydrin proteins. The presence of dehydrins alone is, therefore, not sufficient to confer desiccation tolerance. While physiologically the damage occurring in the sensitive plants was similar to that of the tolerant plant, at an ultrastructural level the damage appeared less in the tolerant plant. On rehydration from low RWCs damage appeared to become exacerbated in the sensitive plants. This was in contrast to the tolerant plant where damage was apparently repaired. There appears, therefore, to be a combination of protection and repair mechanisms responsible for the ability of C. nanum to tolerate desiccation. The lethal RWC of the sensitive species was higher than that at which protective mechanisms, such as water replacement, might come into play. So it is not just the possible ability to replace tightly
bound water that set the tolerant plant aside. It must also have mechanisms to tolerate damage at the higher RWCs which were damaging and lethal to the sensitive plants. The
lethal damage to sensitive species appeared to be related to a critical volume, thus it is concluded that the tolerant plant had the ability to tolerate or avoid this mechanical damage during drying as well as the ability to remain viable in the dry state. It is hypothesised that the ability of the walls to fold in and the unusual nature of the PV curve may provide some answers to the enigma of desiccation tolerance. / Thesis (Ph.D.)-University of Natal, 1995.
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Investigations into the responses of axes of recalcitrant seeds to dehydration and cryopreservation.Wesley-Smith, James. January 2002 (has links)
Achieving long-term storage of germplasm is critical for the conservation of plant biodiversity. Seed storage practices require that degradative reactions causing ageing be limited. By reducing the water content, cytoplasmic viscosity is increased to levels that minimise deteriorative reactions. Reducing the storage temperature additionally increases the storage lifespan by further reducing the rate at which such deleterious processes occur. Two broad categories of seeds can be distinguished based on their storage behaviour. Orthodox seeds are desiccation-tolerant; generally shed in the dry state and are metabolically quiescent. Such seeds are usually stored at low water contents (e.g. 5%), and their high cytoplasmic viscosity prevents freezing damage during cooling to subzero temperatures. On the other hand, desiccation-sensitive (recalcitrant) seeds do not undergo a maturation-drying phase, they are metabolically active at shedding, and sensitive to extreme or prolonged drying. Accordingly, recalcitrant seeds cannot be stored under conventional conditions because they do not survive drying to low water contents and are damaged by sub-zero temperatures, even when dried to the lowest water content tolerated. Therefore, procedures that facilitate harmless drying and cooling to low temperatures are required to achieve long-term storage of recalcitrant germplasm. Recalcitrant seeds that are dried rapidly can attain relatively lower water contents without injury. However, these seeds are usually large and this limits the drying rates that can be achieved even under favourable conditions. Isolating embryonic axes from the rest of the seed facilitates faster drying, and a consequent reduction in the water content at which damage occurs. In axes of many species, the level of drying attained before lethal desiccation damage occurs is sufficient to limit freeZing damage during cryogenic exposure and facilitate survival in vitro. However, many others are damaged when dried to water contents that preclude freezing, and also are killed if cooled to sub-zero temperatures at higher water contents. In such instances, the window of permissible water contents leading to survival may be small or nonexistent. A basic premise explored in this thesis is that by restricting the growth of intracellular ice crystals using increasingly rapid cooling rates, the range of permissible water contents can be widened, facilitating survival of axes at higher water contents. An overview of the problems associated with the long-term storage of recalcitrant germplasm, and the rationale behind such rapid cooling approach are presented in Chapter 1 of the present thesis. Subsequent chapters report investigations on the effects of variables required to dry and cryopreserve embryonic axes with minimum damage, in keeping with this approach. Collectively, those studies aimed at establishing a robust cryopreservation procedure for the conservation of recalcitrant germplasm with broad applicability across species. The approach presently adopted entailed manipulating the water content of excised axes using rapid drying to discrete water content ranges, and also using different methods to cool axes to cryogenic temperatures at various rates. The calorimetric properties of water in axes were investigated for Camellia sinensis (L.) O. Kuntze using differential scanning calorimetry (DSC). For all species, the effect of any drying or cooling treatment tested was determined by assessing the survival of axes in vitro, which provided the most reliable indicator of cellular damage. Additionally, the effects of different treatments upon the structural and functional integrity of axes were assessed using light and electron microscopy as well as measurement of electrolyte leakage. The studies undertaken are presented in a similar sequence to that in which they took place during the course of the experimental phase of this work. These are summarised below. Partial drying plays a pivotal role in the approach developed, and microscopy has contributed towards increasing present understanding of desiccation damage. Microscopy was used to determine the effects of drying rate upon the ultrastructure of recalcitrant axes. It was necessary to find reliable protocols to prepare specimens for light and electron microscopy that did not alter the architecture of the cells in the dry state. Freeze-substitution and conventional aqueous fixation were compared in Chapter 2 using variously dried material from three species. The results obtained revealed that an unacceptably high extent of artefactual rehydration occurs during aqueous fixation, and highlight the need for anhydrous processing of dehydrated samples. Significantly, that study also revealed that many cellular events commonly associated with desiccation damage (e.g. withdrawal, tearing and/or vesiculation of the plasmalemma) are not seen in freeze-substituted preparations, and are likely artefacts of aqueous fixation. Freeze-substitution was used subsequently (Chapter 3) to assess the effects of slow drying (2 - 3 days) or rapid drying (min) upon the survival of embryonic axes of jackfruit (Artocarpus heterophyllus Lamk.) Results confirmed the beneficial effects of rapid drying, and also provided insights into ultrastructural changes and probable causes underlying cellular damage that occur during a drying/rehydration cycle. Efforts subsequently focused on determining the effect of cooling rate upon survival of recalcitrant axes at various water contents. The study on embryonic axes of recalcitrant camellia sinensis (tea; Chapter 4) tested the hypothesis that rapid cooling facilitates survival of axes at higher water content by restricting the growth of ice crystals to within harmless dimensions. The presence of sharp peaks in DSC melting thermograms was indicative of decreased survival in vitro. These peaks were attributed to the melting of ice crystals sufficiently large to be detected by DSC as well as to cause lethal damage to axes. Increasing the cooling rate from 10°C min-1 to that attained by forcibly plunging naked axes into sub-cooled nitrogen increased the upper limit of water content facilitating survival in vitro from c. 0.4 to 1.1 - 1.6 g H20 g-1 (dry mass [dmb]). Subsequent studies tested whether a similar trend occurred in other recalcitrant species cooled under similar conditions. In order to investigate further the relationship between water content, cooling rate and survival it was necessary to achieve cooling rates reproducibly, and to quantify these reliably. The plunging device required to achieve rapid cooling, and instruments required to measure the cooling rates attained, are described in Chapter 5. That study investigated the effects of cryogen type, depth of plunge and plunging velocity on the cooling rates measured by thermocouples either bare or within tissues of similar size and water content as encountered in cryopreservation experiments. This plunger was used in subsequent studies to achieve the fastest cooling conditions tested. Favourable cooling conditions were selected, and the efficacy of this procedure to cryopreserve relatively large axes was tested (Chapter 6) using embryonic axes of horse chestnut (Aesculus hippocastanum L.) Axes at water contents above c. 0.75 g g-1 could not be cooled faster than c. 60°C S-1, but cooling rates of axes below this water content increased markedly with isopentane, and to a lesser extent with subcooled nitrogen. Axes were killed when cooled at water contents above 1.0 g g-1 but survived fully (albeit abnormally) when cooled in isopentane between 1.0 and 0.75 g g-1. Complete survival and increasingly normal development was attained at water contents below 0.75 g g-1, especially if isopentane was used. The study on horse chestnut axes emphasised that water content and cooling rate are co-dependent during non-equilibrium cooling. Accordingly, that study could not determine whether survival at lower water contents increased because of the corresponding increase in cooling rates measured, or because of the higher cytoplasmic viscosity that resulted from drying. That uncertainty was addressed by the study discussed in Chapter 7, using axes of the trifoliate orange (Poncirus trifoliata [L.] RAF.) That study investigated the effect of cytoplasmic viscosity upon survival of axes cooled and warmed at different rates. Survival and normal development was high at lower water contents, and seemingly independent of cooling rate at about 0.26 g g-1. At higher water contents the range of cooling rates facilitating survival became narrower and displaced towards higher cooling rates. This study revealed two conspicuous inconsistencies that questioned the beneficial effect of rapid cooling. Firstly, the fastest cooling rates did not necessarily facilitate the highest survival. Secondly, survival of fully hydrated axes was higher when cooled under conditions that encouraged - rather than restricted - the growth of intracellular ice crystals. These inconsistencies were explored further using embryonic axes of silver maple (Acer saccharinum L.). Freeze-fracture replicas and freeze-substitution techniques provided valuable insights into the way in which ice crystals were distributed in cells cooled using different methods at rates ranging between 3.3 and 97°C S-1. Extensive intracellular freezing was common to all treatments. Unexpectedly, fully hydrated axes not only survived cryogenic exposure, but many axes developed normally when cooled using the relatively slower methods (77 and 3.3°C S-1) if warming was rapid. The most conspicuous ultrastructural difference between plunge cooling and the relatively slower methods was the exclusion of ice from many intracellular compartments in the latter. It is possible that even the fastest warming cannot prevent serious cellular damage if ice crystals form within such 'critical' compartments. It is proposed that the intracellular location of ice is a stronger determinant of survival that the size attained by ice crystals. The study of A. saccharinum also investigated the recovery of axes cooled fully hydrated either rapidly (97°C S-1) or slowly (3.3°C S-1). This facet of the study showed that cell lysis became apparent immediately after warming only where damage was most extensive. In other cells damage became apparent only after 2.5 to 6 h had elapsed, thus cautioning against inferring survival from the ultrastructural appearance of cells immediately after warming. Microscopy enabled cell repair as well as the pattern of growth of cryopreserved tissues to be appraised at the cellular, tissue and organ levels. Similar studies are required to understand further the nature of freezing damage, and how those events affect cell function. The salient trends observed in previous chapters are brought together in Chapter 9. / Thesis (Ph.D.)-University of Natal, Durban, 2002.
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Studies on factors influencing viability after cryopreservation of excised zygotic embryos from recalcitrant seeds of two amaryllid species.Naidoo, Sershen. January 2010 (has links)
Recalcitrant unlike orthodox seeds do not show a sharp border between maturation and germination
and remain highly hydrated and desiccation-sensitive at all developmental and post-harvest
stages. In contrast with recalcitrant seeds, orthodox types retain viability for predictably long
periods in the dry state and hence can be stored under low relative humidity and temperature
conditions. Storage of recalcitrant seeds under conditions allowing little to no water loss, at
moderate temperatures, allows for short- to medium-term storage but only facilitates viability
retention for a matter of a few weeks to months, at best, because the seeds are metabolically
active and initiate germination while stored. Cryopreservation, i.e. storage at ultra-low
temperatures (usually in liquid nitrogen [LN] at -196°C), is a promising option for the long-term
germplasm conservation of recalcitrant-seeded species but their seeds present some unavoidable
difficulties in terms of the amenability of their germplasm to cryopreservation. Pre-conditioning
treatments can reduce the amount of ‘free’ water available for freezing and may increase the
chances of cells or tissues surviving exposure to cryogenic temperatures. Such conditioning may
be imposed by physical dehydration or cryoprotection, i.e. exposure to compounds that depress
the kinetic freezing point of water and so reduce the likelihood of lethal ice-crystal formation
during cooling (i.e. exposure to LN at -196°C or sub-cooled LN at -210°C) and subsequent
thawing. Partial dehydration is presently a standard pre-treatment for the cryopreservation of
recalcitrant zygotic germplasm and explant cryoprotection has been shown to improve postthaw
survival in some recalcitrant-seeded species. However, there is a paucity of information on
the physiological and biochemical basis of post-thaw survival or death in recalcitrant seeds, and
this is the major focus of the current contribution. Additionally, in light of the lack of
understanding on how cryo-related stresses imposed at the embryonic stage are translated or
manifested during subsequent seedling growth, this study also investigated the effects of partial
dehydration and the combination of partial dehydration and cooling of recalcitrant zygotic
embryos on subsequent in and ex vitro seedling vigour. All studies were undertaken on the
zygotic embryos of two recalcitrant-seeded members of the Amaryllidaceae, viz. Amaryllis
belladonna (L.) and Haemanthus montanus (Baker); both of which are indigenous to South
Africa.
Studies described in Chapter 2 aimed to interpret the interactive effects of partial
dehydration (rapidly to water contents > and <0.4 g g-1), cryoprotection (with sucrose [Suc; nonpenetrative]
or glycerol [Gly; penetrative]) and cooling rate (rapid and slow) on subsequent
zygotic embryo vigour and viability, using three stress markers: electrolyte leakage (an indicator
of membrane integrity); spectrophotometric assessment of tetrazolium chloride-reduction (an
indicator of respiratory competence); and rate of protein synthesis (an indicator of biochemical
competence). These studies showed that in recalcitrant A. belladonna and H. montanus zygotic
embryos, stresses and lesions, metabolic and physical, induced at each stage of the
cryopreservation protocol appear to be compounded, thus pre-disposing the tissues to further
damage and/or viability loss with the progression of each step. Maximum post-thaw viability
retention in both species appeared to be based on the balance between desiccation damage and
freezing stress, and the mitigation of both of these via Gly cryoprotection. Post-thaw viabilities
in both species were best when Gly cryoprotected + partially dried zygotic embryos were
rapidly, as opposed to slowly, cooled. However, the rate at which water could be removed
during rapid drying was higher in A. belladonna and this may explain why the optimum water
content range for post-thaw survival was <0.40 g g-¹ for A. belladonna and >0.40 g g-¹ for H.
montanus. These results suggest that to optimise cryopreservation protocols for recalcitrant
zygotic germplasm, attention must be paid to pre-cooling dehydration stress, which appears to
be the product of both the ‘intensity’ and ‘duration’ of the stress.
Cryoprotection and dehydration increased the chances of post-thaw survival in A.
belladonna and H. montanus zygotic embryos. However, transmission electron microscopy
studies on the root meristematic cells from the radicals of these embryos (described in Chapter
3) suggest that their practical benefits appear to have been realised only when damage to the
sub-cellular matrix was minimised: when (a) pre-conditioning involved the combination of
cryoprotection and partial dehydration; (b) the cryoprotectant was penetrating (Gly) as opposed
to non-penetrating (Suc); and (c) embryos were rapidly cooled at water contents that minimised
both dehydration and freezing damage.
The ability of A. belladonna and H. montanus embryos to tolerate the various
components of cryopreservation in relation to changes in extracellular superoxide (.O2
-)
production and lipid peroxidation (a popular ‘marker’ for oxidative stress) was investigated in
studies featured in Chapter 4. Pre-conditioning and freeze-thawing led to an increase in
oxidative stress and the accompanying decline in viability suggests that oxidative stress was a
major component of cryoinjury in the embryos presently investigated. Post-thaw viability
retention in Gly cryoprotected + partially dried embryos was significantly higher than noncryoprotected
+ partially dried embryos, possibly due to the relatively lower post-drying lipid
peroxidation levels and relatively higher post-drying and post-thawing enzymic antioxidant
activities in the former.
Exposure of certain plant tissues to low levels of oxidative or osmotic stress can improve
their tolerance to a wide range of stresses. In contrast, exposure of H. montanus zygotic
embryos to low levels of oxidative stress provoked by exogenously applied hydrogen peroxide
(H2O2) or exposure of A. belladonna embryos to low levels of osmotic stress provoked by low
water potential mannitol and polyethylene glycol solutions (in studies featured in Chapter 5)
increased their sensitivity to subsequent dehydration and freeze-thaw stresses. Exposure of Gly
cryoprotected and non-cryoprotected amaryllid embryos to such stress acclimation treatments
may pre-dispose A. belladonna and H. montanus embryos to greater post-drying and post-thaw
total antioxidant and viability loss than untreated embryos.
To assess the vigour of seedlings recovered from partially dried H. montanus embryos,
seedlings recovered from fresh (F) and partially dried (D) embryos in vitro were hardened-off ex
vitro, and subsequently subjected to either 42 days of watering or 42 days of water deficit (in
studies described in Chapter 6). In a subsequent study (described in Chapter 7), seedlings
recovered from fresh (F), partially dried (D) and cryopreserved (C) A. belladonna embryos were
regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8
days of water stress (S) followed by 3 days of re-watering. Results of these studies suggest that
the metabolic and ultrastructural lesions inflicted on A. belladonna and H. montanus zygotic
embryos during cryopreservation may compromise the vigour (e.g. development of persistent
low leaf water and pressure potentials and reduced photosynthetic rates) and drought tolerance
of recovered seedlings, compared with seedlings recovered from fresh embryos. While the
adverse effects of freeze-thawing were carried through to the early ex vitro stage, certain
adverse effects of partial drying were reversed during ex vitro growth (e.g. the increased relative
growth rate of seedlings from partially dried embryos). The reduced vigour and drought
tolerance of seedlings recovered from partially dried and cryopreserved embryos in the present
work may therefore disappear with an extension in the period afforded to them for hardening-off
under green-house conditions, and in the field.
The results presented in this thesis reinforce the notion that each successive manipulation
involved in the cryopreservation of recalcitrant zygotic germplasm has the potential to inflict
damage on tissues and post-thaw survival in such germplasm relies on the minimisation of
structural and metabolic damage at each of the procedural steps involved in their
cryopreservation. The results also highlight the need to design research programmes aimed not
only at developing protocols for cryopreservation of plant genetic resources, but also at
elucidating and understanding the fundamental basis of both successes and failures. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2010.
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Conservation of select South African Disa Berg. species (Orchidaceae) through in vitro seed germination.Thompson, David Ian. January 2003 (has links)
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.
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Growth and nutrient cycling in cultivated protea neriifolia R.Br.Heinsohn, Rolf-Dieter. January 1991 (has links)
The family Proteaceae is distributed predominantly in the south-western Cape Province of
South Africa and south-western and south-eastern Australia, areas which fall within the
climate term mediterranean ecosystems. A major characteristic of these areas is the low level
of total nutrients in the soil, particularly nitrogen and phosphorus. In their natural habitat,
therefore, Proteaceae occur on well-drained and highly leached soils of low nutrient status.
Efficient nutrient cycling processes, combined with morphological adaptations designed to
facilitate maximum absorption of available nutrients (for example, proteoid roots in the
Proteaceae), conserve the limited nutrients available, allowing for the continued growth of
these shrubs under conditions of low nutrient availability. In recent years, flowers of certain species of Proteaceae have become popular as cut-flowers. As a result, many species of Proteaceae are currently cultivated worldwide, under conditions that match as closely as possible those found in mediterranean ecosystems. Traditionally, the
shrubs are cultivated on nutrient poor soils and of concern is the loss of nutrients through the removal of flowers for commercial sale. Therefore, the aim of the present study was to evaluate growth and mineral cycling in the proteaceaous shrub, Protea neriifolia R.Br., cultivated in a summer rainfall area in South Africa. Nutrient loss through flower removal and its effect on nutrient cycling was quantified. Optimum levels of ammonium nitrogen, phosphorus and potassium for the growth of P. neriifolia seedlings was determined and this formed the basis for the fertilization of mature P. neriijolia shrubs. The effects of inorganic fertilizers on growth and mineral cycling in mature P. neriijolia shrubs was monitored and the effectiveness of inorganic fertilizers, applied to redress nutrient loss, assessed.
The primary response of seedlings of P. neriifolia to applied ammonium nitrogen,
phosphorus and potassium was to ammonium nitrogen, with increased growth with increasing levels of applied ammonium nitrogen, to a maximum of 7 mM applied as 60 ml per week. Seedling response to applied phosphorus and potassium became noticeable only at higher levels of ammonium nitrogen supply, and at these levels seedlings were observed to respond favourably to relatively high phosphorus (0.65 mM) and potassium (1.25 mM) levels, also applied as 60 ml per week. Since nitrate nitrogen has been shown to be toxic to certain
Proteaceae it was not tested in this investigation. However, results from the nursery trial
suggested favourable P. neriijolia growth with a non-nitrate inorganic fertilizer with an NPK
ratio of approximately 5: 1:3 (mass basis) and this was used as a basis for testing the effects of inorganic fertilizers on growth and nutrient cycling in mature P. neriijolia shrubs.
Growth and nutrient cycling was monitored in mature P. neriijolia shrubs for four years: two
years prior to the application of inorganic fertilizers and two years with the application of
inorganic fertilizers, including unfertilized control shrubs. Two inorganic fertilizer
preparations were tested. Both had as their base the commercial slow-release urea based
fertilizer, Plantosan, which has an NPK ratio of 5: 1:3. This was supplemented with either
ammonium sulphate or urea at a rate of 80 g per running metre every three months.
Whole shrub dimensions showed similar growth of P. neriijolia shrubs cultivated in a
summer rainfall area to the growth of the species in its natural habitat. Applied fertilizers did
appear to increase growth, although these results became apparent only after 18 months. As recorded in other Proteaceae, the stem length of all shrubs decreased with increasing age of the shrubs although this decrease was less in shrubs receiving inorganic fertilizers. Branching did not appear to be affected by shrub age or the application of inorganic fertilizers. However, shrub reproductive productivity did increase with age, with greater increases in fertilized shrubs. Furthermore, flowers from fertilized shrubs were larger than those from unfertilized shrubs, although this phenomenon also only became apparent after 18 months. There was also a change in nutrient allocation patterns with those shrubs growing on soils of lowest nutrient availability directing more resources to root growth. This appeared to occur at the expense of stem material which, in each case, accounted for more than 50% of the total shrub biomass.Although shrub age and the application of fertilizers did influence total shrub growth, the timing of growth events were not affected. They were, however, not synchronous to growth events in P. neriijolia growing in its natural habitat. Vegetative growth showed a peak in early spring (September) and the peak reproductive period was in autumn (March, April and May). Maximum litter production (comprising more than 90% leaf litter) occurred in late autumn to early summer (May to December) and this, too, was not affected by shrub age or the application of inorganic fertilizers. Proteoid root occurrence was greatest in late winter/spring (August to November), co-incident with peak above-ground vegetative growth. There are two models that have been developed to describe the growth of overstorey shrubs in mediterranean ecosystems. In the first, the availability of nutrients is described as being of over-riding importance in determining growth events, while in the second model, soil moisture and temperature are regarded as primary growth detenninants. Neither model could adequately explain the shift in phenophase observed in P. neriifolia cultivated in a summer rainfall area. However, soil moisture and temperature do appear more important in determining phenophase events, particularly since the application of inorganic fertilizers did not appear to alter the timing of these growth events. Nevertheless, the importance of nutrients cannot be ignored as growth can occur only provided sufficient nutrients are available. Seasonal variations in nutrient concentrations of leaf, stem, floret and bract material were
observed both prior to and after the application of inorganic fertilizers. However, of
importance is that only shrubs receiving Plantosan plus ammonium sulphate retained
comparable nutrient levels in tissue types compared with nutrient levels in the corresponding
tissue types prior to the application of inorganic fertilizers. Shrubs receiving Plantosan plus
urea and unfertilized shrubs had lower nutrient levels suggesting growth at the expense of
previously absorbed nutrients. This was supported by a change in nutrient allocation patterns, particularly more nutrients in below-ground biomass in shrubs of the latter two treatments. The cycling of nitrogen, phosphorus and potassium was measured prior to and after the
application of inorganic fertilizers. During nutrient cycling, the amount of nitrogen circulated
was larger than the amount of potassium which, in turn, was larger than the amount of
phosphorus. Three nutrient pools, in the above- and below-ground biomass and the soil, were measured and these comprise the plant/soil system. Inputs into the plant/soil system measured were from rainfall and inorganic fertilizers. Nutrient flows within the plant/soil system measured were leaching, due to rainfall, from the shrub onto the soil, litter production and decomposition, and nutrient uptake by the shrubs, into above- and below-ground nutrient pools. Losses from the plant/soil system recorded in this study were losses from the soil through stream-water, and the largest loss, loss through the removal of flowers for commercial sale.
In the absence of flower harvesting the flow of nutrients in the plant/soil system, combined
with inputs from rainfall, appeared adequate for the continued growth of the shrubs.
However, in the presence of flower harvesting there appeared to be a nutrient budget deficit.
This deficit appeared to worsen with increasing shrub age and increasing reproductive
productivity in the absence of inorganic fertilizer applications. This was confirmed by
nutrient depletion from the soil available nutrient pool. Although inorganic fertilizers did not
dramatically alter soil total nutrient pools, soil levels of soluble nitrogen and available
phosphorus did show slight increases but not in accordance with the levels of fertilizers
applied. It is likely that a high proportion of the applied fertilizers was lost to the plant/soil
system before becoming available to the system. This could have occurred through leaching
through the soil profile although this was not measured in this study. Nevertheless, inorganic
fertilizers did appear to compensate for nutrient loss through the removal of flowers for
commercial sale, and the ammonium sulphate supplemented preparation resulted in the most
favourable response.
Due to a number of cultivation practices which affected the growth of the P. neriifolia
shrubs, results are not always strictly comparable with natural systems. However, a number
of results obtained in this invegstigation do have horticultural implications and these are
briefly discussed with regard to the cultivation of the Proteaceae, particularly P. neriifolia
cultivation. / Thesis (Ph.D.)-University of Natal, Durban, 1991.
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Systematic studies in Gnidia L. (Thymelaeaceae)Beaumont, Angela Jane. January 2010 (has links)
Gnidia L., variously estimated to contain 100–160 species, is the largest genus in
the sub-cosmopolitan family Thymelaeaceae. Most species are shrubby, and
occur in tropical and southern Africa, with one species reaching southern India and
Sri Lanka, and 14 species endemic to Madagascar. Assorted segregate genera
have been established using characters considered by some as too few, too trivial
or unreliable. Generic limits have been contentious with authors following either a
narrower concept of Gnidia or a broader circumscription within which segregate
genera are placed in synonymy under Gnidia. Regional treatments for African and
Madagascan floras have been published over the last century until very recently,
but the genus was last revised in its entirety 153 years ago. Today, a broad-based
concept of Gnidia is generally recognised, but there is no modern infrageneric
classification, and species relationships are poorly understood.
Homogenous groups of species are identified by their similarities of leaf length and
width or bract length and width ratios. Species comprising the homogenous groups
for leaf ratios differ to those comprising the homogenous groups based on bract
ratios, and there is no correlation between leaf and bract length and width ratios.
This suggests that the factors influencing leaf diversity differ from those influencing
bract diversity. Bracts differ most from leaves in species with capitate
inflorescences, and involucres of several layers of bracts likely protect
reproductive organs (flowers) in heads. Previously overlooked morphological and
micromorphological details, and morphometric analyses of leaf, bract and floral
dimension data help define individual species, and clades of species derived from
phylogenetic analyses of molecular data. Evidence from a phylogenetic analysis of
nuclear ribosomal and plastid DNA sequence data confirms the polyphyly of
Gnidia. Three lineages contain Gnidia species and species of genera from
southern Africa, southern South America or Australia, while another lineage
corresponds largely to the previously recognized genus Lasiosiphon. The genus
Lasiosiphon is reinstated characterised by flowers mostly in heads, bracts different
from the leaves, and the presence of smooth hairs; it now includes species with
tetramerous flowers as well as ones with pentamerous flowers. Gynodioecy is
recorded for the first time in a single species and represents the first documented
example of sexual polymorphism involving unisexual flowers in Gnidia and sub-
Saharan Thymelaeaceae.
The findings of this thesis are discussed in terms of their phylogenetic value and
contribution to our better understanding of the generic limits of Gnidia and its
relationships with other southern hemisphere Thymelaeoideae. The
circumscription and generic affinities of Gnidia as suggested by results presented
in this thesis are compared to previous classification systems for congruence and
dissimilarity. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.
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Pharmacological properties of members of the Sterculiaceae.Reid, Kirsty Ann. January 2002 (has links)
There is a resurgence of interest in many countries in medicinal plants and their
curative properties (HARBORNE & BAXTER, 1993). Little work has previously
been conducted on the Sterculiaceae species, especially those located within
South Africa. This was a perfect opportunity to broaden the available
information on the medicinal properties and chemical constituents of this family,
within KwaZulu-Natal. Of the 50 genera of the Sterculiaceae family, six are located in South Africa:
Cola, Oombeya, Hermannia, Melhania, Sterculia and Waltheria .
Seven Sterculiaceae species were chosen for investigation. They varied in
growth type and use in traditional medicine. These species included: Oombeya
rotundifolia, D. burgessiae, D. cymosa, Cola natalensis, C. greenwayi,
Hermannia depressa and Sterculia murex. Plant material used in the study was
collected from a variety of areas, all within KwaZulu-Natal or the Northern
Province. There were two collection sites for D. rotundifolia, from differing
habitats, and results were compared. The material was screened pharmacologically for anti-bacterial activity using the disc-diffusion assay and Minimal Inhibitory Assay (MIC), and for antiinflammatory
activity using the COX-1 assay. Only D. rotundifolia and C. natalensis were tested for anti-bacterial activity using the disc-diffusion assay as the disc-diffusion asay was found to show
inconsistencies in the results obtained. Bacteria used included: Escherichia coli
and Klebsiella pneumoniae being Gram-negative, and Micrococcus luteus,
Staphylococcus aureus and Staphylococcus epidermidis being Gram-positive.
D. rotundifolia exhibited activity, both anti-bacterial and bacteriostatic, in the
leaf, twig and bark extracts from both collection sites. Only the water extract
obtained from the leaf material of C.natalensis exhibited slight anti-bacterial
activity against S. epidermidis. Minimal inhibitory concentration (MIC) values
were determined using a microdilution assay (25 mg ml-1 serially diluted 50 %
to 0.195 mg ml-1). Bacteria used in the screening were: B. subtilis, E. coli, K.
pneumoniae and S. aureus. None of the water extracts showed any antibacterial
activity. Good MIC values were exhibited by D. cymosa ethanolic leaf
extracts, C. greenwayi leaf ethyl acetate extracts especially against K.
pneumoniae (0.78 mg ml-1) and S. aureus (0.39 mg rnl-1) and H. depressa
ethanol and ethyl acetate leaf, stem and root extracts. D. burgessiae and S.
murex showed low activity, with insignificant MIC values.
D. rotundifolia plant material yielded the highest anti-inflammatory activity of all
the plant species, with the extracts from the Umgeni Valley Nature
Reserve(UVNR) showing the best results. The lowest activity was recorded in
the aqueous bark extracts (5% inhibition)and the highest from the ethanolic leaf
extract (97% inhibition). D. cymosa extracts showed high activity in ethanolic
leaf and twig extracts with low activity in all the other extracts. D. burgessiae,
C. greenwayi and S. murex extracts showed high activity in both ethanolic and
dichloromethane extracts from leaf and twig material. Activity occurred in the
dichloromethane extracts of H. depressa obtained from the stem (78%) and
root (81%) extracts. C. natalensis extracts showed insignificant activity.
Plant material was phytochemically screened for alkaloids, saponins, tannins,
cardiac glycosides and cyanogenic glycosides. No alkaloids were detected
using pH-partitioning and no cyanogenic glycosides were observed (TLC
sandwich method) in any of the extracts of the seven species screened. Using
the gelatin salt-block test, tannins were found to be present in the leaf and twig
material of D. rotundifolia, the leaf material of C. greenwayi and the leaf, stem
and root material of H depressa. The froth test indicated that saponins were
present in the leaf and twig material of D. rotundifolia and leaf, root and stem
material of H. depressa. The haemolysis test indicated the presence of
saponins in the D. rotundifolia bark material. Screening for cardiac glycosides
detected cardienolides in the leaf, twig and bark material of D. rotundifolia, and
bufadienolides were detected in D. rotundifolia , D. cymosa, D. burgessiae and
S. murex.
Five species screened were selected for isolation of active anti-bacterial
compounds: D. rotundifolia, D. burgessiae, D. cymosa, C. greenwayi and H.
depressa. Material was extracted by Soxhlet and isolation techniques employed
were VLC, TLC separation, Sephadex LH-20 column chromatography and
HPLC techniques. The isolated compounds were analysed by NMR and GCMS.
All isolated compounds were fatty acids: Palmitic acid, Myristic acid, Lauric
acid, Stearic acid, Acetic acid as welll as stearyl alcohol, eicosane and
octadecane.
The aqueous eaf extract of H. Depressa exuded a thick mucilage. The
production of this mucilage from the H. depressa aqueous extract may have
medicinal or commercial value. A technique to separate the mucilaginous
extract from the leaf material was devised. After extraction, the extract was
screened to determine its sugar content through gas chromatography. It was
screened for its pharmacological properties: antibacterial and anti-inflammatory
activity. The hydrolysing effect of -amylase and HCI on the extract was
determined to find its potential use as a bulking agent for use as an appetite
suppressant, laxative or against the effects of diarrhoea. It was concluded that
the extract is not likely to break down easily in the human digestive system and
may be effective against the three listed ailments . / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.
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Development of strategies towards the cryopreservation of germplasm of Ekebergia capensis Sparrm. : an indigenous species that produces recalcitrant seeds.Hajari, Elliosha. January 2011 (has links)
The conservation of germplasm of indigenous plant species is vital not only to preserve
valuable genotypes, but also the diversity represented by the gene pool. A complicating
factor, however, is that a considerable number of species of tropical and sub-tropical
origin produce recalcitrant or otherwise non-orthodox seeds. Such seeds are hydrated
and metabolically active when shed and cannot be stored under conventional conditions
of low temperature and low relative humidity. This poses major problems for the longterm
conservation of the genetic resources of such species. Presently, the only strategy
available for the long-term conservation of species that produce recalcitrant seeds is
cryopreservation.
Ekebergia capensis is one such indigenous species that produces recalcitrant seeds. The
aim of the present study was to develop methods for the cryopreservation of germplasm
of this species. Different explant types were investigated for this purpose, viz.
embryonic axes (with attached cotyledonary segments) excised from seeds, and two in
vitro-derived explants, i.e. ‘broken’ buds excised from in vitro-germinated seedlings
and adventitious shoots generated from intact in vitro-germinated roots. Suitable
micropropagation protocols were developed for all explant types prior to any other
experimentation.
Before explants could be cryopreserved it was necessary to reduce their water content in
order to limit damaging ice crystallisation upon cooling. All explants tolerated
dehydration (by flash drying) to 0.46 – 0.39 g gˉ¹ water content (dry mass basis) with
survival ranging from 100 – 80%, depending on the explant. In addition, penetrating
and non-penetrating cryoprotectants were used to improve cryo-tolerance of explants.
The cryoprotectants tested were sucrose, glycerol, DMSO and a combination of sucrose
and glycerol. Explant survival following cryoprotection and dehydration ranged from
100 – 20%. Cryoprotected and dehydrated explants were exposed to cryogenic
temperatures by cooling at different rates, since this factor is also known to affect the
success of a cryopreservation protocol. The results showed that ‘broken’ buds could not
tolerate cryogen exposure. This was likely to have been a consequence of the large size
of explants and their originally highly hydrated condition. Adventitious shoots tolerated
cryogenic exposure slightly better with 7 – 20% survival after cooling in sub-cooled
nitrogen. Limited shoot production (up to 10%) was obtained when axes with attached
cotyledonary segments were exposed to cryogenic temperatures. In contrast, root
production from axes cooled in sub-cooled nitrogen remained high (67 – 87%).
Adventitious shoots were subsequently induced on roots generated from cryopreserved
axes by applying a protocol developed to generate adventitious shoots on in vitrogerminated
roots. In this manner, the goal of seedling establishment from cryopreserved
axes was attained.
Each stage of a cryopreservation protocol imposes stresses that may limit success. To
gain a better understanding of these processes the basis of damage was investigated by
assessing the extracellular production of the reactive oxygen species (superoxide) at
each stage of the protocol, as current thinking is that this is a primary stress or injury
response. The results suggested that superoxide could not be identified as the ROS
responsible for lack of onwards development during the cryopreparative stages or
following cryogen exposure.
The stresses imposed by the various stages of a cryopreservation protocol may affect the
integrity of germplasm. Since the aim of a conservation programme is to maintain
genetic (and epigenetic) integrity of stored germplasm, it is essential to ascertain
whether this has been achieved. Thus, explants (axes with cotyledonary segments and
adventitious shoots) were subjected to each stage of the cryopreservation protocol and
the epigenetic integrity was assessed by coupled restriction enzyme digestion and
random amplification of DNA. The results revealed little, if any, DNA methylation
changes in response to the cryopreparative stages or following cryogen exposure.
Overall, the results of this study provided a better understanding of the responses of
germplasm of E. capensis to the stresses of a cryopreservation protocol and two explant
types were successfully cryopreserved. Future work can be directed towards elucidating
the basis of damage incurred so that more effective protocols can be developed.
Assessment of the integrity of DNA will give an indication as to the suitability of
developed protocols, or where changes should be made to preserve the genetic (and
epigenetic) integrity of germplasm. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.
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Medicinal properties and in vitro responses of Mayenus senegalensis (Lam.) exell.Matu, Esther Ng'endo. 21 November 2013 (has links)
No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.
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