Ph.D. (Zoology) / This project involves a detailed study of three South African antelope species, springbok, impala and blesbok. The study investigates the origins of sperm in terms of testicular histology and subsequently the major storage organ, the cauda epididymis. Sperm of these species were characterized in terms of their quality (morphology, motility, vitality characteristics among others and their physiology: when exposed to different media and cryopreservation protocols. Finally sperm fertilization biology of the three species and evaluation of fertilization and developmental success when using homologous and heterologous oocytes (relative comparison) were assessed. Cauda epididymal spermatozoa was recovered post-mortem from the testes of culled springbok (n =38); impala (n =26) and blesbok (n =42) during winter months in South Africa and cryopreserved in a Tris-fructose-citric acid extender (Biladyl) supplemented with 20% egg yolk and 7% glycerol under field conditions. Longevity of sperm was assessed in Tris and Citrate extenders and modified Tyrode lactate in vitro fertilization (IVF) media. Oocytes were collected from the ovaries of domestic cows (n =165), springbok (n = 72) and blesbok (n = 42) and matured in domestic cattle M199 maturation media supplemented with 10% FCS, 10IJg/mi LH, 10IJg/mi FSH and antibiotics. Heterologous (zona intact and zona free) and homologous fertilization was carried using a domestic cattle IVF protocol. Results were analysed using SPSS version 18.0 (Statcon, South Africa). Interspecies comparisons were made using parametric tests: paired t-test for the freezing effect, one-way analysis of variance (ANOVA), Mixed between-within subjects ANOVA for longevity, Non Parametric test for motility characteristics and least squares ANOVA for...

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.

Doctor of Philosophy / Department of Plant Pathology / Bernd R. Friebe / Hexaploid wheat (Triticum aestivum L., 2n=6x=42, AABBDD) is a recent polyploid and originates from a limited number of founder genotypes. Domestication bottlenecks further reduced genetic diversity. The wheat gene pool, which consists of landraces and wild relatives such as rye (Secale cereale L.), Leymus racemosus Tien and Elymus tsukushiensis Honda, is a rich source of genetic diversity. Agronomically important traits can be transferred from these gene pools to wheat through chromosome engineering. This dissertation describes chromosome engineering and pre-breeding efforts for resistance to Hessian fly and Fusarium head blight (FHB) in wheat. The germplasm with a whole-arm rye translocation, T2BS.2R#2L, contains the highly effective Hessian fly resistance gene, H21, and an unnamed powdery mildew resistance gene. Directed chromosome engineering was used to shorten the whole-arm rye segment. The recovered wheat-rye recombinant chromosome, T2BS.2BL-2R#2L, had a shorter rye segment but still contained the H21 gene and was transferred through backcrosses to adapted winter and spring wheat cultivars. This study released the germplasm KS09WGGRC51, which is used in wheat breeding programs in the U.S.A. Two novel sources of FHB resistance were identified in L. racemosus and E. tsukushiensis. Fhb3 present in the wheat-L. racemosus T7AL.7Lr#1S Robertsonian translocation was transferred into the adapted Kansas winter wheat cultivar Fuller. The wheat-E. tsukushiensis disomic addition translocation line confers FHB resistance. Ph1b-induced homoeologous recombination was used to produce wheat-E. tsukushiensis recombinants. The distal and interstitial recombinants were identified using molecular markers and genomic in situ hybridization (GISH). A combination of molecular cytogenetic analyses determined that the distal recombinant involved wheat chromosome 1A and a small distal segment originating from the E. tsukushiensis chromosome arm, 1E[superscript]ts#1S, resulting in the recombinant chromosome T1AL.1AS-1E[superscript]ts#1S. The interstitial recombinant involves an unidentified wheat chromosome and appears to be highly rearranged. Both recombinants confer high levels of type II FHB resistance (resistance to spread within the head) based on point inoculations in the greenhouse. To date, these two recombinants are the smallest alien introgression with FHB resistance in common wheat. This germplasm material has been released as KS14WGRC61. The distal recombinant can be used directly for breeding of FHB-resistant cultivars worldwide.

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

Cheng, Chun Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 164-183). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / General Abbreviations --- p.viii / Abbreviations of Chemicals --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / Table of Contents --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Salt stress in plants --- p.1 / Chapter 1.2 --- Overview of the molecular basis of salt tolerance in plants --- p.2 / Chapter 1.2.1 --- Stress perception --- p.3 / Chapter 1.2.2 --- Signal transduction --- p.3 / Chapter 1.2.2.1 --- Protein phosphatases --- p.4 / Chapter 1.2.2.2 --- The SOS pathway for ion homeostasis --- p.4 / Chapter 1.2.3 --- DNA and RNA helicases in post-transcriptional control --- p.6 / Chapter 1.2.4 --- ROS scavengers --- p.7 / Chapter 1.2.5 --- Proteases and proteinase inhibitors --- p.8 / Chapter 1.2.6 --- Heat shock proteins (Hsps) --- p.9 / Chapter 1.2.7 --- Highlights on DnaJ/Hsp40 --- p.9 / Chapter 1.3 --- Review on functional genomics of salt stress responses in plants --- p.11 / Chapter 1.3.1 --- Genomics on model organisms --- p.12 / Chapter 1.3.2 --- Transcriptomics for identifying salt stress responsive genes --- p.12 / Chapter 1.3.2.1 --- Multiple stress transcriptome analysis --- p.13 / Chapter 1.3.2.2 --- Genome-wide transcriptome analysis on molecular crosstalk --- p.14 / Chapter 1.3.2.3 --- Tissue specific transcriptome analysis --- p.16 / Chapter 1.3.2.4 --- Comparative transcriptome analysis --- p.17 / Chapter 1.3.2.5 --- Transcriptome analysis of soybean --- p.24 / Chapter 1.3.3 --- Proteomics in plant salt stress studies --- p.26 / Chapter 1.3.4 --- Beyond the transcriptome and proteome --- p.27 / Chapter 1.4 --- Significance of using soybean germplasms for identifying salt stress responsive genes --- p.28 / Chapter 1.5 --- Objectives --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Plants, bacterial strains，and vectors" --- p.30 / Chapter 2.1.2 --- Enzymes and major chemicals --- p.33 / Chapter 2.1.3 --- Primers --- p.34 / Chapter 2.1.4 --- Commercial kits --- p.34 / Chapter 2.1.5 --- Equipment and facilities --- p.34 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- cDNA microarray analysis --- p.35 / Chapter 2.2.1.1 --- Construction of cDNA subtraction libraries --- p.35 / Chapter 2.2.1.2 --- Assembly of cDNA microarray --- p.36 / Chapter 2.2.1.3 --- External control RNA synthesis --- p.39 / Chapter 2.2.1.4 --- Probe labelling and hybridization --- p.40 / Chapter 2.2.1.5 --- Hybridization signal collection --- p.41 / Chapter 2.2.1.6 --- Image analysis --- p.41 / Chapter 2.2.1.7 --- Data analysis --- p.42 / Chapter 2.2.1.8 --- Selection of salt responsive genes using fold difference in expression --- p.45 / Chapter 2.2.1.9 --- DNA sequencing --- p.46 / Chapter 2.2.1.10 --- Real-time PCR analysis --- p.47 / Chapter 2.2.2 --- Growth conditions and treatments of plants --- p.48 / Chapter 2.2.2.1 --- Soybean for microarray hybridization and real-time PCR --- p.48 / Chapter 2.2.2.2 --- Soybean for the study of GmDNJ1 expression under ABA treatment --- p.48 / Chapter 2.2.2.3 --- Wild-type and transgenic Arabidopsis for functional analysis --- p.49 / Chapter 2.2.2.4 --- Wild-type and transgenic rice for functional analysis --- p.49 / Chapter 2.2.3 --- "DNA, RNA, and protein extraction" --- p.50 / Chapter 2.2.3.1 --- Plasmid DNA extraction from E. coli cells --- p.50 / Chapter 2.2.3.2 --- RNA extraction from plant tissues --- p.51 / Chapter 2.2.3.3 --- Soluble protein extraction from plant tissues --- p.51 / Chapter 2.2.4 --- Blot analysis --- p.51 / Chapter 2.2.4.1 --- Northern blot analysis --- p.52 / Chapter 2.2.4.2 --- Western blot analysis --- p.53 / Chapter 2.2.5 --- Subcloning of GmDNJ1 into pGEX-4T-1 --- p.53 / Chapter 2.2.5.1 --- "Restriction digestion, DNA purification and ligation" --- p.53 / Chapter 2.2.5.2 --- Transformation of competent Escherichia coli (DH5a and BL21) --- p.54 / Chapter 2.2.6 --- Luciferase refolding assay --- p.54 / Chapter 2.2.6.1 --- Culture of E. coli strain BL21 (DE3) --- p.54 / Chapter 2.2.6.2 --- Cell lysis --- p.55 / Chapter 2.2.6.3 --- Purification of the GST-GmDNJ1 fusion protein --- p.55 / Chapter 2.2.6.4 --- Quantitation of protein --- p.55 / Chapter 2.2.6.5 --- Luciferase refolding assay --- p.56 / Chapter Chapter 3 --- Results --- p.57 / Chapter 3.1 --- Overview of cDNA microarray analysis --- p.57 / Chapter 3.2 --- Identification of salt responsive genes in subtraction libraries concerning two contrasting soybean germplasms --- p.61 / Chapter 3.3 --- Data processing before selection of salt stress responsive genes --- p.75 / Chapter 3.3.1 --- M-A plots --- p.75 / Chapter 3.3.2 --- Boxplots --- p.76 / Chapter 3.3.3 --- Scatterplots --- p.76 / Chapter 3.4 --- Selection of salt responsive genes using fold difference in expression --- p.77 / Chapter 3.4.1 --- Selection of genes with differential expression between tolerant and sensitive germplasms --- p.77 / Chapter 3.4.2 --- Selection of genes with differential expression between cultivated and wild germplasms --- p.89 / Chapter 3.4.3 --- Data validation by real-time PCR analysis --- p.91 / Chapter 3.5 --- Selection of salt responsive genes using statistical tools --- p.95 / Chapter 3.5.1 --- Quantitative trait analysis for salt responsive genes --- p.95 / Chapter 3.5.2 --- Identification of salt stress correlation genes --- p.100 / Chapter 3.5.3 --- Cluster analyses --- p.104 / Chapter 3.5.3.1 --- Clustering genes --- p.104 / Chapter 3.5.3.2 --- Clustering samples --- p.108 / Chapter 3.5.4 --- Data validation by real-time PCR analysis --- p.111 / Chapter 3.6 --- Summary of cDNA microarray analysis --- p.112 / Chapter 3.7 --- Studies on GmDNJ1 --- p.120 / Chapter 3.7.1 --- Sequence analysis of GmDNJ1 --- p.120 / Chapter 3.7.2 --- GmDNJ1 was induced by salt stress and ABA treatment in soybean (Glycine max) --- p.127 / Chapter 3.7.3 --- Expressing GmDNJ1 in transgenic Arabidopsis (Arabidopsis thaliana) enhances the tolerance to salt stress and dehydration stress --- p.129 / Chapter 3.7.4 --- Expressing GmDNJ1 in transgenic rice (Oryza sativa) enhances the tolerance to salt stress and dehydration stress --- p.135 / Chapter 3.7.5 --- The GmDNJ1 protein can replace DnaJ in the in vitro luciferase refolding assay --- p.141 / Chapter Chapter 4 --- Discussion --- p.145 / Chapter 4.1 --- Overview of expression profiling of the 20 soybean germplasms --- p.145 / Chapter 4.2 --- Identification of salt responsive genes from subtraction libraries --- p.146 / Chapter 4.3 --- Normalization of data from microarray experiments --- p.148 / Chapter 4.4 --- The fold difference analysis --- p.149 / Chapter 4.4.1 --- Response to stress --- p.149 / Chapter 4.4.2 --- Gene expression --- p.150 / Chapter 4.4.3 --- Molecular function --- p.150 / Chapter 4.4.4 --- Metabolic activity --- p.151 / Chapter 4.4.5 --- Cellular component --- p.152 / Chapter 4.4.6 --- Genes with 2.5-fold difference in expression between cultivated and wild germplasms --- p.153 / Chapter 4.5 --- Selection of salt responsive genes using statistical tools --- p.153 / Chapter 4.5.1 --- Quantitative trait analysis --- p.153 / Chapter 4.5.2 --- Cluster analyses --- p.154 / Chapter 4.6 --- Studies on GmDNJ1 --- p.157 / Chapter 4.6.1 --- GmDNJ1 is a good candidate for gene studies --- p.157 / Chapter 4.6.2 --- Sequence analysis of GmDNJ1 suggested it to be a DnaJ/Hsp40 homologue in soybean --- p.158 / Chapter 4.6.3 --- GmDNJ1 was induced by salt stress and ABA treatment --- p.158 / Chapter 4.6.4 --- GmDNJ1 has a higher expression in salt tolerant soybean germplasms over sensitive ones --- p.159 / Chapter 4.6.5 --- Ectopic expression of GmDNJ1 enhanced the tolerance to salt stress and dehydration stress in transgenic Arabidopsis --- p.159 / Chapter 4.6.6 --- Ectopic expression of GmDNJ1 enhanced the tolerance to salt stress and dehydration stress in transgenic rice --- p.160 / Chapter 4.6.7 --- Luciferase activity assay showed that GmDNJ 1 functioned as a DnaJ/Hsp40 in vitro --- p.161 / Chapter Chapter 5 --- Conclusion --- p.162 / References --- p.164 / Appendix I - Enzymes and major chemicals --- p.184 / Appendix II - Primers --- p.188 / Appendix III - Major commercial kits --- p.192 / Appendix IV - Major equipment and facilities --- p.193 / "Appendix V - Formulation of buffer, solution, gel, and medium" --- p.194 / Appendix VI - Plots in microarray experiments --- p.198 / Appendix VII - Clones with differential expression (>2.5-fold or >1.8-fold) between germplasms --- p.208 / Appendix VIII - Salt responsive genes revealed by quantitative trait analysis --- p.216 / Appendix IX - Supplementary data in real-time PCR analysis --- p.221 / Appendix X - Supplementary data in functional analyses --- p.233

Cryopreservation of gametes is a valuable tool for the fast-growing aquaculture industry in New Zealand. In the present study, research was aimed to improve the cryopreservation of Pacific oyster (Crassostrea gigas) oocytes. For this, two main approaches were used: the modification of an existing published (standard) cryopreservation protocol for oyster oocytes and the modification of the oocytes themselves prior to cryopreservation. The objectives in the chapters of this thesis were: (a) determination of the cryobiological characteristics of oyster oocytes; (b) assessment and reduction of intracellular ice formation (IIF) in oocytes; and (c) modification of the lipid fraction (cholesterol and fatty acids) of oocytes prior to cryopreservation. Knowledge of the membrane permeability parameters in response to concentrations of water and ethylene glycol (EG), the influence of temperature upon these parameters, and the osmotic tolerance limits of oyster oocytes were used to develop computer models that simulated the cellular volume changes that oocytes underwent during EG addition and removal. The models predicted that when one part of EG was added in one step to one part of oocyte suspension and equilibrated for 20 min at 20 �C, similar volume changes in oocytes would be obtained, compared to a more complicated multi-step addition method. This method of addition resulted in similar post-thaw fertilization rates to those obtained by using the multi-step addition method, thus reducing oocyte handling. Cryomicroscopy was used to assess the effect of cooling rates and EG concentration on the temperature at which oocytes underwent IIF. It was found that IIF occurred at higher subzero temperatures when fast cooling rates were used (30 and 5 �C min⁻�) and at EG concentrations ranged between 0 and 15%. At a relatively slower cooling rate of 0.3 �C min⁻� and with 10% EG, which are the conditions employed in the standard cryopreservation protocol, no IIF occurred. The steps of the standard protocol that were more likely to cause oocyte damage were identified by evaluating the fertilization rate of oocytes at each step. Results showed that oocytes were most damaged by cooling them to -35 �C and followed by plunging them in liquid nitrogen. Contrary to what had been observed under the cryomicroscope, transmission electron microscopy (TEM) analysis revealed that all oocytes cryopreserved by the standard protocol contained cytoplasmic ice. In addition, it was also observed that oocytes were at two developmental stages when frozen (prophase and metaphase I). These observations prompted the development of alternative cooling programmes aimed to reduce intracellular ice. The effect of cooling rate, plunge temperature and time held at the plunge temperature were thus evaluated, based on post-thaw fertilization rate of oocytes. Overall, neither the cooling rate nor the holding time had an effect on oocyte fertility. However, the plunge temperature had an effect, where oocytes plunged at -60 �C had lower post-thaw fertilization rates than oocytes plunged at -35 �C. Through the slowing of the cooling rate, lengthening of the holding time and lowering of the plunge temperature, it was possible to reduce the amount of ice in the cytoplasm. However, the reduction of intracellular ice did not improve the post-thaw fertilization rate of the oocytes; on the contrary, post-thaw fertilization decreased notoriously. From these results, it can be suggested that oyster oocytes are more likely to be damaged by exposure to high intra and extracellular solute concentration than IIF during cryopreservation. In an effort to modify the lipid content of oyster oocytes prior to cryopreservation and thus, making them more resistant during cryopreservation, oocytes were incubated in solutions that would add or remove cholesterol or in solutions rich in long chain fatty acids (EPA or DHA). Oocytes incubated in cholesterol-rich solutions showed a positive uptake of fluorescently labelled cholesterol and this effect was dose dependent. Nevertheless, this uptake did not improve the post-thaw fertilization rate nor did it increase the total cholesterol content of the oocytes. When oocytes were incubated in non-conjugated or conjugated EPA or DHA, no increase in the proportion of these fatty acids was identified in the fatty acid profiles of whole oocytes and no improvement of the post-thaw fertilization rate was recorded. Given that there was no uptake of fatty acids from the incubation media by the oocytes, a different approach was taken. This involved the supplementation of lipid-rich diets to the oyster broodstock during gametogenesis (cold-conditioning) and vitellogenesis (warm-conditioning). Despite results showing that lipid content and, indeed, fatty acid profile was altered through the diet, the results also showed that fresh oocytes from broodstock fed during cold-conditioning did not show any improvement in their fertilization rates, nor did they benefit from a lipid-rich diet during warm-conditioning. On the other hand, cryopreserved oocytes did have higher post-thaw fertilisation rates when broodstock were fed during cold-conditioning and, although no effect was found from feeding broodstock with either of the lipid-rich diets during warm-conditioning, trends indicated that a diet consisting of fresh microalgae or the commercial supplement Algamac would yield the highest post-thaw fertilization rates. This thesis has furthered the understanding of some of the factors that determine cryosurvival in oyster oocytes and has demonstrated that both physical and biological issues must be taken into consideration for cryopreservation. Specifically, the results in this thesis helped to modify an empirically developed cryopreservation protocol for Pacific oyster oocytes. In addition, the results also showed strong evidence of the survival of oyster oocytes to intracellular ice and highlighted the importance of supplying the broodstock with lipid-rich food during the periods of gamete formation and maturation in order to obtain oocytes that are more amenable to cryopreservation. These benefits could be of significant practical importance and may be extended for the development or refinement of cryopreservation protocols for other shellfish species of commercial importance to the aquaculture industry of New Zealand.

Thesis (doctoral)--Universität, Gfiessen, 2007. / Includes bibliographical references (p. [287]-303) and index.

Graduation date: 1999

The freedom to operate (FTO) and the costs of acquiring and protecting intellectual property (IP) has become a major concern among both private and public plant breeders, especially in the IP intensive transgenic crops. Despite the developments in biotechnology, crop breeding still remains a sequential process where the best new varieties build on the successful varieties of the past. Given this breeding process, if FTO reduces the ability of breeders to access the best germplasm this could slow the rate of global crop improvement in both transgenic and non-transgenic crops. This potential problem has led many agricultural science leaders to raise concern about the possibility of an anti-commons developing because of growing freedom to operate issues.<p>One of the solutions that is proposed for the growing FTO issue in plant breeding is the development of open source research platforms similar to those that led to the development of the Linux computer operating system. With an open source research process anyone is able to use the research platform to develop commercial products but any improvements made to the research platform become part of the platform for future users. The proponents of this approach, such as CAMBIA, argue that it will maintain access to critical intellectual property and allow optimal sharing of knowledge to take place.<p>In this paper we examine the intellectual property rights associated with crop germplasm and varieties in Canada. We show that the breeder rights that are built into many current systems of breeder rights systems create a de facto open source system. This system allows breeders to use previously released varieties as breeding material for their own breeding programs allowing them to improve their own germplasm base. Once this is done, and a new variety is released from the program, it then becomes available for other breeders to use in the same manner. Few would argue that this system has not had a long history of success.<p>As a counterfactual we consider the case where provisions of the UPOV 1991 act are used to give plant breeders the rights to not only protect their current varieties from being illegally copied but would also give them claim over any future varieties developed that use their variety as breeding material. In the factual, a three stage model has two public sector breeders seeking to maximize the benefits of their varieties over a heterogeneous group of iii farmers. In the first stage of the model the breeders decide the optimum amount of germplasm to share between each other. The second stage of the model requires the breeders to decide the optimal level of yield it should set as a plant breeding target. In the final stage farmers make an adoption choice basing their decision on the variety that best suits their farm. Backward induction is then used to solve both of the models. Applying the results of this simulation to the wheat plant breeding system in western Canada, shows that such a revised breeders rights system would quickly lead to a large number of potential owners for each variety released, which would then increase transactions costs and eventually lead to an anti-commons or FTO issue. In the case where there are no intellectual property rights on varieties breeders are able to produce a variety that more farmers will adopt because breeders costs will be lower due to germplasm sharing. Once intellectual property rights are introduced into the system, breeders choose to reduce the amount of variety sharing, which then reduces the number of farmers who would adopt the new variety, thus decreasing the benefits for farmers. Given this outcome, jurisdictions that implement the provisions of UPOV 1991 which may hinder FTO, may find benefits from developing other legal measures to maintain an open source type access to germplasm.

The freedom to operate (FTO) and the costs of acquiring and protecting intellectual property (IP) has become a major concern among both private and public plant breeders, especially in the IP intensive transgenic crops. Despite the developments in biotechnology, crop breeding still remains a sequential process where the best new varieties build on the successful varieties of the past. Given this breeding process, if FTO reduces the ability of breeders to access the best germplasm this could slow the rate of global crop improvement in both transgenic and non-transgenic crops. This potential problem has led many agricultural science leaders to raise concern about the possibility of an anti-commons developing because of growing freedom to operate issues.<p>One of the solutions that is proposed for the growing FTO issue in plant breeding is the development of open source research platforms similar to those that led to the development of the Linux computer operating system. With an open source research process anyone is able to use the research platform to develop commercial products but any improvements made to the research platform become part of the platform for future users. The proponents of this approach, such as CAMBIA, argue that it will maintain access to critical intellectual property and allow optimal sharing of knowledge to take place.<p>In this paper we examine the intellectual property rights associated with crop germplasm and varieties in Canada. We show that the breeder rights that are built into many current systems of breeder rights systems create a de facto open source system. This system allows breeders to use previously released varieties as breeding material for their own breeding programs allowing them to improve their own germplasm base. Once this is done, and a new variety is released from the program, it then becomes available for other breeders to use in the same manner. Few would argue that this system has not had a long history of success.<p>As a counterfactual we consider the case where provisions of the UPOV 1991 act are used to give plant breeders the rights to not only protect their current varieties from being illegally copied but would also give them claim over any future varieties developed that use their variety as breeding material. In the factual, a three stage model has two public sector breeders seeking to maximize the benefits of their varieties over a heterogeneous group of iii farmers. In the first stage of the model the breeders decide the optimum amount of germplasm to share between each other. The second stage of the model requires the breeders to decide the optimal level of yield it should set as a plant breeding target. In the final stage farmers make an adoption choice basing their decision on the variety that best suits their farm. Backward induction is then used to solve both of the models. Applying the results of this simulation to the wheat plant breeding system in western Canada, shows that such a revised breeders rights system would quickly lead to a large number of potential owners for each variety released, which would then increase transactions costs and eventually lead to an anti-commons or FTO issue. In the case where there are no intellectual property rights on varieties breeders are able to produce a variety that more farmers will adopt because breeders costs will be lower due to germplasm sharing. Once intellectual property rights are introduced into the system, breeders choose to reduce the amount of variety sharing, which then reduces the number of farmers who would adopt the new variety, thus decreasing the benefits for farmers. Given this outcome, jurisdictions that implement the provisions of UPOV 1991 which may hinder FTO, may find benefits from developing other legal measures to maintain an open source type access to germplasm.