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Doctoral Degrees (Plant Pathology)

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Browsing Doctoral Degrees (Plant Pathology) by Author "Berjak, Patricia."

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    Aspects of post-harvest seed physiology and cryopreservation of the germplasm of three medicinal plants indigenous to Kenya and South Africa.
    (2002) Kioko, Joseph Ivala.; Berjak, Patricia.; Pammenter, Norman W.
    The current state of global biodiversity is one of sustained and increasing decline especially in developing countries such as South Africa, where, medicinal plants face a particular threat due the herbal medicine trade, and because in situ conservation measures have not stemmed the exploitation of these plants (Chapter 1). Furthermore, seed storage, which offers an efficient ex situ conservation technique, cannot presently be applied to many medicinal plants, either because these species produce short-lived, recalcitrant seeds, or the post-shedding behaviour of the seeds is altogether unknown. This study investigated three medicinal plant species indigenous to Kenya and South Africa: Trichilia dregeana and T. emetica, of which no population inventories exist and no wild populations were encountered locally during the course of this study; and Warburgia salutaris, one of the most highly-utilised medicinal plants in Africa, and which is currently endangered and virtually extinct in the wild in some countries such as South Africa. Aspects of post-shedding seed physiology (Chapter 2) and the responses of the germplasm of the three species to cryopreservation (Chapter 3) were studied using viability and ultrastructural assessment, with the aim of establishing methods for both short-term and the long-term preservation, via appropriate seed storage and cryopreservation, respectively. The effect of cryopreservation on genetic fidelity, a crucial aspect of germplasm conservation, was assessed by polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPDs), using W. salutaris as a case-study (Chapter 4). The seeds of all three species were found to exhibit non-orthodox behaviour. On relatively slow-drying, seeds of T. dregeana and T. emetica lost viability and ultrastructural integrity at axis water contents of 0.55 g g-l (achieved over 6 d) and 0.42 g g-l (after 3 d) respectively, while flash-drying of embryonic axes facilitated their tolerance of water contents as low as 0.16 g g-l (T. dregeana, flash-dried for 4 h) and 0.26 (T. emetica, flash-dried for 90 min). Seeds of W. salutaris were relatively more tolerant to desiccation, remaining viable at axis water contents below 0.1 g g-l when desiccated for 6 d in activated silica gel. However, excised embryonic axes flash-dried to similar water contents over 90 min lost viability and were ultrastructurally damaged beyond functionality. In terms of storability of the seeds, those of T. dregeana could be stored in the fully hydrated state for at least 5 months, provided that the quality was high and microbial contamination was curtailed at onset of storage, while those T. emetica remained in hydrated storage for about 60 d, before all seeds germinated in storage. Seeds of W salutaris, even though relatively tolerant to desiccation, were not practically storable at reduced water content, losing viability within 49 d when stored at an axis water content of 0.1 g g-l. The seeds of all three species were sensitive to chilling, suffering extensive subcellular derangement, accompanied by loss of viability, when stored at 6 °C. Thus, T. dregeana and T. emetica are typically recalcitrant, while those of W. salutaris are suggested to fit within the intermediate category of seed behaviour. For either recalcitrant or intermediate seeds, the only feasible method of long-term germpalsm preservation may be cryopreservation. Subsequent studies established that whole seeds of W. salutaris could be successfully cryopreserved following dehydration in activated silica gel. However, whole seeds of T. dregeana and T. emetica were unsuitable for cryopreservation, and excised embryonic axes were utilised. For these, in vitro germination methods, as well as cryoprotection, dehydration, freezing and thawing protocols were established. Post-thaw survival of the axes of both species was shown to depend on cryoprotection, rapid dehydration and cooling (freezing) in cryovials. Embryonic axes of T. dregeana regenerated only as callus after cryopreservation, while those of T. emetica generated into apparently normal plantlets. Thawing/rehydration in a 1:1 solution of 1 µM CaC12.2H2O and 1 mM MgC12.6H2O increased the percentage of axes surviving freezing, and that of T. emetica axes developing shoots. The effect of the extent of seed/axis development on onward growth after cryopreservation was apparent for seeds of W. salutaris and excised axes of T. emetica. The seeds of W. salutaris surviving after cryopreservation germinated into seedlings which appeared similar to those from non-cryopreserved seeds, both morphologically and in terms of growth rate. Analysis using PCR-RAPDs revealed that there were no differences in both nucleotide diversity or divergence, among populations of seedlings from seeds which had been sown fresh, or those which had either been dehydrated only, or dehydrated and cryopreserved. Thus, neither dehydration alone, nor dehydration followed by cryopreservation, was associated with any discernible genomic change. The above results are reported and discussed in detail in Chapters 2 to 4, and recommendations and future prospects outlined in Chapter 5.
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    Some effects of drying rate and wet storage on aspects of the physiology and biochemistry of embryonic axes from diesiccation- sensitive seeds.
    (2004) Ntuli, Tobias M.; Berjak, Patricia.; Pammenter, Norman William.; Smith, Michael Trevor.
    Desiccation-sensitive seeds show differential viability characteristics during drying at different rates. A number of studies have demonstrated that rapid dehydration permits survival to lower water contents than does slower desiccation. The aim and objective of the present study was to test the hypothesis which states that rapid drying of desiccation-sensitive seeds removes water sufficiently fast to reduce the accumulation of metabolic damage. In addition, the hypothesis that wet storage subjects desiccation-sensitive seeds to mild, but increasingly severe, water stress causing oxidative damage if additional water is not supplied, was tested. In the present study, axes of germinating orthodox seeds of Pisum sativum and newlyshed recalcitrant counterparts of Quercus robur, Strychnos madagascariensis, Trichilia emetica, Trichilia dregeana and Avicennia marina were subjected to rapid or slow drying or wet storage. For those species where more than one harvest was investigated, differences were observed in water contents at shedding. For all the species studied, the dehydration rate could be described by an exponential and a modified inverse function for both desiccation regimes, and the water content remained constant with wet storage. The level of tetrazolium staining and germination percentage of axes decreased sharply drying and hydrated storage such that the marked decline took place at lower water contents upon rapid than slow dehydration. The conductivity of electrolyte leachate increased progressively during desiccation and moist storage of axes of all species investigated. Greater membrane leakage occurred upon slow, than rapid dehydration in axes of all species studied. Activities of respiratory enzymes which have a potentially regulatory role in glycolysis, phosphofructokinase (PFK), or the tricarboxylic acid cycle, malate dehydrogenase (MDH), and levels of the oxidized form of the coenzyme, nicotinamide adenine dinucleotide (NAD), of the enzymes of the electron transport chain, NADH dehydrogenases ofNADH-ubiquinone (coenzyme Q) reductase (complex I) and NADHcytochrome c reductase (complex IV), were monitored in the present investigation. v In addition, the role of free radical activity in the form of lipid peroxidation, which has been implicated in loss of viability in seeds, was examined by assaying the levels of hydroperoxides. The involvement of the free radical processing enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), and the antioxidant, ascorbic acid (AsA), was also ascertained. The activity of PFK in axes of P. sativum remained constant during drying and wet storage. However, PFK activity increased as rapid dehydration and hydrated storage of Q. robur axes proceeded. In contrast, the activity of PFK in axes of Q. robur decreased during slow desiccation. Similarly, PFK activity was reduced upon drying, and moist storage, of T. dregeana axes such that higher activity of PFK was seen during rapid than slow dehydration. The activity ofPFK inA. marina axes also declined upon desiccation. The activity ofMDH in axes of P. sativum was also unchanged during drying and wet storage. However, an increase in MDH activity was recorded in Q. robur axes during dehydration and hydrated storage such that the activity of MDH was higher upon slow than rapid desiccation. In contrast, MDH activity in axes of T. dregeana decreased as drying proceeded. Similarly, the activity of J\.1DH declined during dehydration and moist storage of A. marina axes. An increase in the level of NAD occurred in axes of P. sativum during drying. In contrast, a decrease in NAD levels was seen upon dehydration and wet storage of Q. robur axes such that the level of NAD was higher upon rapid than slow desiccation. There was an enhancement of the level of NAD in axes of T. dregeana during hydrated storage. Conversely, NAD levels declined during drying ofA. marina axes. A decrease in the level of hydroperoxides in axes of P. sativum was seen as rapid drying proceeded. In contrast, hydroperoxide levels increased during wet storage of P. sativum axes. Similarly, the levels of hydroperoxides were enhanced upon dehydration and hydrated storage of Q. robur axes such that they were higher in axes during slow desiccation compared to those dried rapidly. Conversely, the hydroperoxide level in axes of T. dregeana was reduced upon rapid dehydration. In contrast, an elevation of the level of hydroperoxides was observed during moist storage. The levels of hydroperoxides remained constant as desiccation and wet storage ofA. marina axes proceeded. vi The activity of SOD in axes of P. sativum decreased during rapid drying. In contrast, SOD activity increased upon slow dehydration and wet storage ofP. sativum axes. There was a decline in the activity of SOD in Q. robur axes during slow desiccation. Similarly, SOD activity was diminished upon drying of axes of T. dregeana. The activity ofSOD in T. dregeana axes was enhanced during hydrated storage. An elevation in SOD activity also took place during rapid dehydration and moist storage of axes ofA. marina. The activity of CAT did not change during drying of axes of P. sativum. However, a decrease in CAT activity in Q. robur axes was seen upon slow dehydration and wet storage. Similarly, the activity of CAT declined as desiccation of axes of T. dregeana proceeded. In contrast, CAT activity inA. marina axes increased during slow drying. Whereas the activity of GR in axes of P. sativum increased during drying and wet storage, GR activity decreased in A. marina axes upon all treatments such that the activity ofGR was higher during rapid than slow dehydration. GR activity also declined upon slow desiccation and hydrated storage ofaxes of Q. robur. Similarly, the activity of GR in T. dregeana axes was reduced during moist storage. Finally, a decrease in the level of AsA in axes of P. sativum took place during drying. Nonetheless, dehydration and wet storage of Q. robur axes were associated with no siginificant change in AsA levels. There was also a decline in the level of AsA in axes of T. dregeana as rapid desiccation proceeded. Similarly, a reduction in AsA level occurred upon slow drying ofaxes ofA. marina. The results presented here are consistent with the observation that drying and wet storage adversely affected the respiratory enzymes, PFK, MDH and NADH dehydrogenase. It is suggested that the resultant metabolic imbalance led to more leakage of electrons from the mitochondrial electron transport chain than normal, and through lipid peroxidation increased levels of hydroperoxides. In addition, dehydration and hydrated storage may depress the activities of free radical processing enzymes, SOD, CAT and GR and levels of antioxidant, AsA. This phenomenon was less pronounced during rapid, in comparison to slow, desiccation and moist storage. However, it appears that the above biochemical events are overtaken by physical damage at higher water contents in the highly recalcitrant seeds. It was concluded that the differential effects of VII the drying rate and wet storage on responses of desiccation-sensitive seeds varies with tissue, harvest, species and the degree of desiccation sensitivity.