Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 120-124). Also available via the Internet.

Thesis (doktorgrades)--Christian-Albrechts-Universität zu Kiel, 1998. / Includes bibliographical references (p. [225-239) and index.

Genetic diversity of cultivated species and their wild relatives, as well as of wild species encompasses plant genetic resources or germplasm, the ex situ preservation of which embodies a critical aspect of biological conservation. While seed storage affords an efficient ex situ conservation method, recalcitrant seeds are intolerant of desiccation and cannot be stored conventionally in seed banks. Seeds of the three indigenous tree species investigated in this study, viz. Trichilia emetica, T. dregeana and Protorhus longifolia are recalcitrant, with the species considered to be endangered. Cryopreservation, which involves storage at ultra-low temperatures of selected tissue(s) from which plants are subsequently able to be generated, is currently the only method available for long-term ex situ conservation of recalcitrant-seeded species and affords significant potential for the future. Many protocols that have been applied for the cryopreservation of the germplasm of recalcitrant zygotic embryonic axes excised from seeds of tropical/sub-tropical species have resulted in survival post-cryo which has been recorded only as root development or callus formation, with shoot formation seldom occurring. Successful cryostorage of genetic resources cannot be achieved until post-cryopreservation recovery facilitates normal seedling development, i.e. the formation of both a fully functional root and a shoot. Cryopreservation requires the utilisation of the smallest explant possible (greatest surface area to volume ratio), the most suitable for recalcitrant seeds in general being the zygotic embryonic axis. Based on preliminary studies it was demonstrated that shoot production by axes is inhibited in association with a burst of reactive oxygen species (ROS), produced in response to wounding upon excision of the axis from the cotyledons, when these are attached close to the shoot apical meristem. It was postulated that a combination of the oxidative burst at the site of excision coupled with inadequate antioxidant machinery within the recalcitrant axis tissue, precludes shoot production. It was further considered highly probable that each subsequent stressful manipulation throughout the cryopreservation process would be accompanied by a surge of uncontrolled oxidative activity within the tissue, in response to the stress. Therefore, the primary aim of the study was to investigate the underlying causes of failure of shoot production after procedures associated with cryopreservation and to focus on ways to ameliorate the consequences of unbalanced oxidative metabolism. Additionally, studies were carried out to optimise each step of the cryopreservation procedure, viz. cryoprotection, dehydration, rehydration and cooling, and subsequent recovery, in conjunction with assessment of oxidative responses, ultimately to achieve successful cryopreservation of the embryonic axes of these species. The experimental work conducted to achieve this aim assessed changes in various biomarkers of injury, those selected for this study being three ROS, viz. superoxide, the hydroxyl radical and hydrogen peroxide, after axes were exposed to various pre-treatments, cryopreservation and recovery. Concomitantly, the elicited responses of endogenous antioxidant systems accompanying these steps were assessed. Changes in the levels of ROS and antioxidant activity were determined using various biochemical assays, and these parametres, together with assessment of shoot development, were investigated after each step of the cryopreservation process. The effect of stress on oxidative metabolism was tested after exposure to pre-treatments with and without the provision of various antioxidants, viz. DMSO, ascorbic acid and cathodic water, so as to determine the efficacy of selected ROS scavengers and, in general, to develop the best protocol for cryopreservation of embryonic axes of the three species. Significant results, in terms of shoot development and regulated ROS generation, were obtained after three major processes of the cryopreservation procedure. The production of roots and shoots by excised axes of T. emetica, T. dregeana and P. longifolia after excision (75%, 80% and 75%, respectively), and by 40% of excised axes of T. dregeana after each of the two further stages, cryoprotection and desiccation, were major achievements towards cryopreservation of the recalcitrant germplasm. The modulation of ROS by ascorbic acid and cathodic protection significantly improved survival of axes of both Trichilia species. In its entirety, the present study made significant advancements towards cryopreservation of recalcitrant germplasm and also towards understanding oxidative events associated with cryogenic processing and exposure to cryogenic conditions. This study concludes that unregulated metabolism is one of the underlying causes of failure of recalcitrant germplasm represented by zygotic axes, to survive cryopreservation. The application of antioxidants and cathodic protection during cryopreservation facilitated survival that has been previously unattainable. The outcomes of this study provide an informative platform for further optimising cryopreservation procedures for the germplasm of the species investigated, and extending the work to other recalcitrant-seeded species, especially those of tropical/sub-tropical provenances. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology: Biotechnology, Technikon Natal, 2002. / Lack of suitable techniques for field collection of the germplasm of different species, and spoilage of samples in transit, hinder efforts to collect, conserve, distribute and regenerate most plant germplasm in vitro. The aims of this investigation, therefore, were to address problems encountered in collection of field germplasm from species and hybrids of Eucalyptus (TAG5, TAGI4, ZG14, GC550 and GU2IO) that are propagated vegetatively and Trichilia dregeana, which has recalcitrant seeds. Simple in vitro culture-based protocols were developed to minimise contamination and maintain viability of plant material for sufficient time for it to be transported from the field to the tissue culture laboratory. From the two simulations of 48 h 'transportation' conditions for explants of Eucalyptus species investigated, those in bottles containing sterile vermiculite exhibited no contamination and greater than 50% bud break, regardless of whether or not field surface sterilization with alcohol had been done. In contrast, when explants were enclosed in cling wrap, contamination was high and bud break levels low. For selection of the more suitable Eucalyptus explant, two types were investigated: nodal explants each with one half leaf (type 1) and stem segments with three nodes (type 2). As type 2 showed considerably better shoot yields (up to 1624 shoots per 100 explant), and were more practical to use with respect to space, such trinodal stem segments were deemed best for collection. Of the sterilization procedures investigated, treatment with 70% (v/v) alcohol prior to storage was found to be most suitable in almost all cases. For plant material with high endogenous microbial contamination, the bud break medium was supplemented with Benomyl and calcium hypochlorite, each at 0.5 and 1.0 g.r). Alcoholtreated, stored explants cultured on bud break medium with 1.0 g.r) calcium hypochlorite exhibited low levels of contamination and an increased final yield (up to a maximum of 930 shoots per 100 explants). Thus, this protocol was employed for field material of E. grandis clones TAG5, TAGI4 and ZGI4. For these clones, stored type 2 explants / M

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in the School of Molecular and Cell Biology. Johannesburg 2017 / With growing populations and climate change associated drought predicted for the future, cassava can provide one solution for food security and a source of starch for industrial use and biofuels in South Africa, and other countries in the SADC region. One of the severe constraints on cassava production is cassava mosaic disease (CMD) caused by cassava infecting begomoviruse species, including African cassava mosaic virus (ACMV), South African cassava mosaic virus (SACMV) and East African cassava mosaic virus (EACMV). Cassava begomoviruses (CBVs) are responsible for significant yield loss of the starchy tubers. Since no chemical control of virus diseases of plants is possible, one approach to develop virus resistance is via biotechnology, through genetic engineering (GE) of cassava with hairpin RNA (hpRNA) silencing constructs that express small interfering RNAs targeting CBVs and preventing severe disease development. The aim of this project was to subject previously transformed five CMM6 cassava lines (cv. 60444 transformed with a non-mismatched Africa cassava mosaic virus-[Nigeria:Ogorocco;1990] (ACMV-[NG:Ogo:90])-derived hpRNA construct, six AMM2 (cv. 60444 transformed with a mismatched ACMV-[NG:Ogo:90]-derived hpRNA construct), six CMM8 cassava lines (cv.60444 transformed with a non-mismatched SACMV BC1-derived hpRNA construct) and seven AMM4 cassava lines (cv.604444 transformed with a mismatched SACMV BC1-derived hpRNA construct) to reproducible trials, and evaluate for response to virus challenge. The ACMV-[NG:Ogo:90] hpRNAi constructs target 4 overlapping virus open reading frames (ORFs) (AC1 replication associated protein/AC4 and AC2 transcriptional/AC3 replication enhancer), while the SACMV hpRNAi constructs target the cell-to cell movement BC1 ORF. Non mismatched constructs consist of a transformation cassette that has an intron separating the sense and antisense arms of the viral transgene whilst mismatched constructs have the sense arm of the viral transgene treated with bisulfite to induce base mutation. This mutated sense arm is then separated from the non mutated antisense arm by a small spacer. Furthermore, a 229 bp inverted repeat hpRNA construct (DM-AES) was designed to target ACMV-[NG:Ogo:90] 117 nt putative promoter region (2714-49 nt), a 91 nt overlapping sequence (1530-1620 nt) between ACMV-[NG:Ogo:90] AC1 3’ end and AC2 5’ end (AC1 3’/AC2 5’-ter) as well as being efficient against SACMV and EACMV due to the inclusion of a 21 nt conserved sequence (1970-1990) of AC1/Rep shared between ACMV, EACMV and SACMV. Cassava landrace T200 friable embryogenic callus (FEC) were transformed with this construct. The selected transgenic lines were infected with either ACMV-[NG:Ogo:90] (CMM6 and AMM2 transgenic lines) or SACMV (CMM8 and AMM4 transgenic lines) by agro-inoculation and monitored at 14, 36 and 56, 180 and 365 days post infection (dpi) for symptom development, plant growth and viral load. From the ACMV trials 3 lines (CMM6-2, CMM6-6 and line AMM2-52) showed significantly lower symptom scores and lower viral load at 36, 56 and 365 dpi, compared with viral challenged untransgenic cv.60444. This phenotype is described as tolerance, not resistance, as despite ameleriorated symptoms virus replication persists at lower levels. From the SACMV infectivity trials even though all CMM8 and AMM4 transgenic lines had lower symptom severities and viral loads compared with infected untransformed cv.60444, the results were not highly significant (p˃ 0.05). From this study, tolerance or reduction of viral load and symptoms was attributed to the accumulation of transgene-derived siRNAs prior to infection. However there was no observable correlation between levels (semi-qauntitative northern blots) of siRNAs and tolerance or susceptible phenotypes. Tuber yield evaluation of the three tolerant lines (CMM6-2, CMM6-6 and line AMM2-52) showed that the tuber fresh and dry weight at 365 dpi was not affected by the viral presence. These are promising lines for larger greenhouse and field trials. A comparison between the two different constructs showed that the two tolerant CMM6 lines-2 and 6 appeared to perform better (viral load) compared with AMM2 tolerant line-52 with regards to levels of viral amplification. The mismatched construct in AMM4 lines and the nonmismatched construct in CMM8 lines induced the same viral and symptom severity score (sss) reduction. Transformation of T200 FECs with the DM-AES construct was unsuccessful due to the age (more than six months old) of the FECs. FECs are more likely to lose their regeneration and totipotent nature with age. We therefore propose the use of fresh T200 FECs in future transformation studies to test the DM-AES construct. / MT2017

Thesis (Ph. D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on Dec. 16, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science, Department of Agricultural, Food and Nutritional Sciences, University of Alberta." Includes bibliographical references.

Cryopreservation is the most promising method for the long-term conservation of germplasm of plants producing desiccation-sensitive seeds. While such seeds are generally termed recalcitrant in the context of conventional storage practices, the term ‘cryo-recalcitrant’ is used for germplasm which is not readily amenable to cryopreservation. Cryo procedures usually involve a sequential combination of steps which must be optimised to limit the stresses experienced by specimens, thus promoting their survival. The present contribution reports on the effects of some of the steps involved in cryopreservation on the survival of the embryos of the amaryllids, Ammocharis coranica, Brunsvigia grandiflora and Haemanthus albiflos, with the ultimate aim of developing a protocol(s) for the successful cryopreservation of the germplasm of these species. The main foci of the investigations were the effects of rapid (flash) drying, the use of the cryoprotectant additives, glycerol (5 & 10%) and DMSO (0.1 & 0.25%), and employment of different cooling rates on the zygotic embryos of the selected species, which are known to be recalcitrant as well as being cryo-recalcitrant. Furthermore, this study reports on attempts at improving the rapidity of dehydration during flash drying by applying a vacuum, and also of providing cathodic protection (via highly reducing cathodic water and/or direct exposure to a static {negatively-charged} cathodic field during flash drying) to the explants at various stages in the protocol. These techniques were employed in attempts to ameliorate the adverse effects of reactive oxygen species associated with stresses imposed by the procedures during the cryopreservation process. The embryos of Ammocharis coranica, Brunsvigia grandiflora and Haemanthus albiflos were initially at water contents (WCs, dry mass basis) of 3.28±0.52, 2.55±0.22, 4.48±0.92 g g-1, respectively, after harvest. These embryos proved to be tolerant to moderately rapid water loss in the short term, with >60% retaining germinability at water contents ≥0.5 g g-1. The results from this study confirmed that dehydration to water contents below 0.5 g g-1 (dry mass basis) compromised survival, and that this effect was exacerbated if the embryos were cryoprotected prior to drying. Interestingly, the rate of water loss in embryos of these species differed, with A. coranica and H. albiflos drying at a (comparably) much slower rate than those of B. grandiflora. Subsequent rapid cooling yielded promising results when compared with slow cooling, as 30% of glycerol cryoprotected, rapidly cooled A. coranica embryos that had been flash-dried to 0.36±0.10 g g-1 generated normal seedlings. It was clear, however, that the effects of these procedures were exacerbated when all the steps of the cryo procedure were applied sequentially. However, the work also showed that these adverse effects may be ameliorated if each step of the cryopreservation protocol is optimised on a species-specific basis, thus promoting the chances of survival after cryopreservation and facilitating subsequent seedling establishment. This was evident in the 30% germination obtained when embryos of A. coranica, which had been cryoprotected with glycerol prior to flash drying before exposure to rapid cooling, while those that had not been cryoprotected or were cryoprotected with DMSO before drying did not survive. The incorporation of cathodic protection during flash drying appeared promising as it promoted the survival of 10% of H. albiflos embryos dehydrated to WCs between 0.37 and 0.26 g g-1 (whereas no survival was achieved without the inclusion of this step), and 70% of A. coranica embryos that were dehydrated to 0.35±0.21. In addition, the reduction of the explant size, from a whole 6 mm embryo to a 3-4 mm excised axis, promoted survival by up to 30% for A. coranica and H. albiflos, even at higher WCs. However, survival in these cases was based on observations of abnormal development, i.e. the development of roots or shoots, or calli. No surviving embryos were obtained from B. grandiflora after cooling, regardless of the preconditioning treatment or rate of cooling, and this was accredited to the greater degree of sensitivity of these embryos to the cryo procedures than those of the other two species. The use of cathodic water to re-hydrate explants after dehydration and of applying a vacuum during flash drying did not result in any observable benefits, and require further investigation for optimisation. The very limited success towards establishing a cryopreservation protocol for the species investigated in this study reinforces the difficulties associated with the cryopreservation of recalcitrant germplasm, which informs the cryo-recalcitrance of some explants. However, the results obtained have helped to identify a number of intervention points that could be used to minimise the damage incurred during the various procedural steps involved in cryopreservation. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Thesis (Ph. D.)--Wageningen Universiteit, 2005. / Thesis propositions sheet inserted. Includes bibliographical references (p. [249]-262).

Gray leaf spot (GLS) of corn (Zea mays L.), caused by the fungus Cercospora zeaemaydis (CZM) (Tehon and Daniels) has increased in incidence and severity with increasing use of no-tillage and continuous corn practices. This disease can be yield limiting. Corn hybrids were evaluated under natural disease pressure for three years (1989, 90, and 91) at two locations (Montgomery and Wythe Co., VA). Yield losses ranged from 2127.4 kg/ha (Wythe Co., 1991) to 4242.2 kg/ha (Wythe Co., 1990). It was estimated that 77% of the variability in yield was due to GLS. Fungicides were evaluated for the control of GLS over three years on a susceptible hybrid, Pioneer Brand 3320. All fungicides, with the exception of mancozeb, provided significant control over nontreated check in all years. Benomyl, propiconazole and terbutrazole were the most effective fungicides. As much as 93% of the variablilty in yield was attributed to blighting. Reduction in blighting also increased the kernel weight. The toxin, cercosporin, produced by CZM was evaluated for its ability to elicit differential responses in corn germplasm by three methods, ie., vein inoculation, root, and shoot uptake. No consistant differential reponses were found with vein inoculation, but 31-day old plants were significantly more sensitive to the toxin than 21-day old plants, as measured by lesion width. Root and shoot uptake of the toxin by inbred germplasm produced lesions that resembled those produced by CZM in the field. Microscopic, yellow fluorescing crystals were found associated with necrotic tissue from toxin-treated inbreds. Significantly more injury occurred to toxin-treated inbreds exposed to light than to darkness. By chromatographic analysis, 407.1-1076.7 ng of toxin/g of tissue was recovered from leaf lesion extracts of plants exposed to light. Five inbreds (B73, H99, Va59, NC250a, and NC264) showed consistent and differential responses to the toxin. H99 and NC250a showed differential responses to the same concentration of toxin, thus suggesting that some germplasm are more sensitive to the toxin than others. Tests using the toxin as a means to identify resistant germplasm did not provide reliable predictions of germplasm response to CZM in the field. / Master of Science

Landolphia kirkii is scrambling shrub forming an integral part of the flora along the coastal areas of north-eastern South Africa. The non-sustainable harvesting of fruit as food source, by monkeys and rural communities and the highly recalcitrant nature of their seeds threatens the continuation of the species. In addition, the ability of the plants to produce high quality rubber makes its long-term conservation highly desirable. Previously, attempts have been made to cryopreserve germplasm of L. kirkii, but no survival had been recorded at cryogenic temperatures of below -140ºC. The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates, thawing and imbibition treatments on survival of embryonic axes excised with cotyledons completely removed, as well as with 3 mm portion of each cotyledon attached, from fresh, mature, recalcitrant seeds of L. kirkii. Survival was assessed by the ability for both root and shoot development in in vitro culture, the tetrazolium test and electrolyte leakage readings. At seed shedding, embryonic axes were at the high mean water content of 2.24 g gˉ¹ (dry mass basis). All axes (with and without attached cotyledonary segments) withstood rapid (flash) drying to a water content of c. 0.28 g gˉ¹; however, the use of chemical cryoprotectants, singly or in combination, before flash-drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g gˉ¹, with no explants showing shoot production after exposure to -196ºC and -210ºC. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology regardless of the thawing temperature employed. Furthermore, analysis of the lipid composition within embryos of L. kirkii revealed negligible amounts of capric and lauric acids, suggested to be the medium-chained saturated fatty acids responsible for triacylglycerol crystallisation when lipid-rich seeds are subjected to cryogenic temperatures. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent thawing and rehydration. Rapid rehydration of embryonic axes of L. kirkii by direct immersion in a calcium-magnesium solution at 25ºC for 30 min (as apposed to slow rehydration on moistened filter paper or with rehydration in water) was associated with highest survival post-dehydration. Cooling at 1ºC minˉ¹ and 2ºC minˉ¹ facilitated survival of 70 and 75% respectively of axes with attached cotyledonary segments at 0.28 g gˉ¹ after exposure to - 70ºC. Viability retention of 40 and 45% were recorded when embryonic axes with attached cotyledonary segments were cooled at 14 and 15ºC minˉ¹ to temperatures below -180ºC. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.