The trees belonging to the genus Podocarpus, of which only four species are native to South Africa, are renowned for their superior quality timber. Prior to 1880, Podocarpus henkelii, together with P. falcatus and P. latifolius, played a significant role in the development of the country as they were heavily utilised as timber trees for the building of dwellings, furniture and other necessary items. Due to this over-exploitation in the timber trade, all the Podocarpus species in South Africa have been afforded a ‘Protected’ status on the IUCN red data list of species that are either threatened or in danger of extinction. However, despite the obvious need to conserve the threatened genetic diversity of these species, few attempts (aside from in vitro micropropagation) have been made to explore ex situ Podocarpus germplasm conservation in the long-term. Consequently, the primary aim of this study was to establish a protocol for the long-term conservation of germplasm of Podocarpus henkelii Stapf ex Dallim. Jacks. The seeds of Podocarpus henkelii exhibit recalcitrant behaviour and can therefore not be stored in conventional seed banks. This has necessitated the investigation of alternative methods of germplasm conservation with a focus on cryopreservation which is presently considered the most reliable, efficient and cost-effective means of storing the genetic resources of recalcitrant-seeded species for prolonged periods. The first objective of this study was to investigate the effect of slow (two-step) and ultra-rapid cooling on the post-thaw survival of variously treated P. henkelii embryos. The results of this investigation revealed that the rate of cooling employed had a significant effect on explant viability as none of the precultured, cryoprotected embryos that were slowly cooled survived cryostorage while some of the preconditioned embryos responded to ultra-rapid cooling (i.e. 36% shoot production and 88% callus formation). For ultra-rapid cooling, it was found that flash-drying prior to cooling was a prerequisite for survival as osmotic dehydration alone did not effectively prepare the tissues for the stresses imposed during cryostorage. Furthermore, for those flash drying intervals that yielded positive results, preconditioning explants with 10% glycerol proved the most effective pre-cooling treatment. However, due to the low recovery numbers after ultra-rapid cooling, a third cryopreservation technique i.e. cryogenic vitrification, was investigated. For cooling by vitrification, data obtained from preliminary experiments showed that precultured explants needed to be initially loaded with 18% sucrose (w/v) + 14% glycerol (v/v) for 20 min and subsequently immersed in Plant Vitrification Solution 3 (PVS3) at 0°C for 10 min prior to cooling. However, relatively low success was achieved for P. henkelii embryos cooled by vitrification as the highest post-cooling survival obtained was only 20% germination, 27% shoot formation and 37% callus formation. Due to the low post-thaw survival obtained despite the rigorous manipulations employed in the development of the slow cooling, ultra-rapid cooling and vitrification protocols, it was decided that an alternative explant should be investigated for the conservation of P. henkelii germplasm. The explant of choice was adventitious buds induced to form on, and subsequently excised from, mature P. henkelii embryos. The first objective was to develop a suitable protocol for the induction of adventitious buds on P. henkelii embryos. The medium that induced in the highest percentage of embryos (85%) to form adventitious buds consisted of Douglas-fir cotyledon revised (DCR) basal medium supplemented with 30 g L-1 sucrose, 0.05 mg L-1 NAA, 0.5 mg L-1 BA and 6 g L-1 agar. This medium also resulted in the highest average number of buds formed per embryo (i.e. 35 ± 3 buds per embryo). Once the adventitious bud induction medium was developed, it was necessary to optimise the size of adventitious bud clumps to be used as explants for cryopreservation. Three bud clump sizes were investigated: ca 3, 5 and 10 buds per clump. However, none of the bud clumps survived excision from the mother-tissue despite the investigation of three different types of bud-break media. The resultant tissue mortality is suggested to have occurred because the adventitious bud clumps were excised prior to bud break and shoot development which could have exacerbated excision-related cellular and sub-cellular damage. It was therefore decided that attempts should be made to induce adventitious buds directly on P. henkelii embryos post-cooling, thereby eliminating the possibility of potentially lethal excision-related damage. The protocols that yielded the best results after ultra-rapid cooling and cooling by vitrification were used in this experiment. For ultra-rapid cooling, embryos were first cryoprotected with 5% followed by 10% glycerol for 1 h in each and subsequently flash dried for 30 min prior to immersion in nitrogen slush. For cooling by vitrification, embryos that were first precultured on 0.3 M sucrose for 1 d were loaded with 10% glycerol + 14% sucrose (LS4). The loaded explants were then immersed in ice-cold PVS3 and maintained on ice for 10 min prior to cryostorage. The effect of each pretreatment (either independently or in combination) on adventitious bud production pre-cooling was also investigated. For both protocols the various pretreatments decreased not only the capacity of the embryos to form buds but also the average number of buds formed per embryo (i.e. 7 ± 2 buds per embryo and 14 ± 2 buds per embryo were formed on treated embryos prior to ultra-rapid cooling and cooling by vitrification, respectively). Thus, it was predicted that even if the percentage of cryopreserved embryos forming buds was minimal, the number of possible plantlets that could be regenerated from adventitious buds per cryopreserved explant would compensate for the low recovery of embryos post-cooling. However, none of the embryos that were cryopreserved by either ultra-rapid cooling or by vitrification formed adventitious buds after eight weeks in culture. The very restricted success achieved in this study despite the investigation of three cryopreservation techniques and two different explants only serves to reinforce the difficulties associated with the conservation of recalcitrant germplasm. The large size and structural complexity of P. henkelii embryos, coupled with their high water content post-shedding, are just some of the characteristics to which their intractability to the manipulations involved in the development of a successful cryopreservation protocol could be attributed. For future investigations, development of adventitious buds produced on cryopreserved root segments (as opposed to entire roots), and/or use of seedling meristems as explants which might be amenable to cryopreservation are suggested as possible avenues for the long-term conservation of P. henkelii genetic diversity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Ctenocephalides felis is a major pest of companion animals worldwide. This project aimed to generate novel genetic resources for C. felis and develop tools to aid drug-target identification and validation. Sample handling methods were assessed and candidate reference genes validated, to ensure quality of RNA samples and reliable gene expression normalisation. Piercing C. felis samples prior to storage in RNAlater ensured RNA integrity was maintained over time. Glyceraldehyde 3-phosphate dehydrogenase , 60S ribosomal protein L19 and elongation factor-1α were demonstrated as stable reference genes across all comparisons tested. A C. felis transcriptome encompassing multiple developmental stages, sexes and tissues was sequenced and de novo assemblies produced with two assemblers, Trinity and Oases. Each assembly contained >100000 contigs. Annotation of the assemblies generated functional insight, such as top BLAST hits, GO annotations and signal peptide predictions. The Trinity assembly was deemed the highest quality and searched for genes of interest, involved in development. Expression analysis of selected transcripts across stadia gave insight into developmental processes, and demonstrated the utility of the transcriptome. This study was the first to demonstrate that C. felis can mount an RNAi response upon exposure to dsRNA. Knockdown of glutathione S-transferase σ (GSTσ), was demonstrated in adult C. felis: ≈80 % knockdown following microinjection of dsGSTσ; ≈64 % knockdown after soaking in dsGSTσ; ≈96 % knockdown after continuous feeding on dsGSTσ. RNAi machinery was identified in C. felis. siRNAi pathway components, Dicer 2 and Argonaute 2, were upregulated following dsRNA exposure. Dicer 2 was knocked-down by soaking in dsDicer2, although results of an “RNAi of RNAi” experiment were inconclusive. Transcripts encoding machinery putatively involved in dsRNA uptake and breakdown were also identified. Through these studies, this project has generated novel insights into C. felis biology and opened up new avenues for research.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-08-22T19:15:30Z No. of bitstreams: 1 2013 - Evandro Silva Pereira Costa.pdf: 2748177 bytes, checksum: 3632d387d4cb90bea88d42726fae79ca (MD5) / Made available in DSpace on 2018-08-22T19:15:30Z (GMT). No. of bitstreams: 1 2013 - Evandro Silva Pereira Costa.pdf: 2748177 bytes, checksum: 3632d387d4cb90bea88d42726fae79ca (MD5) Previous issue date: 2013-12-18 / CAPES / The present work had the objective to characterize 59 accessions of tomato of the cherry group regarding its agronomic characteristics and resistance to the late blight and to select those more adapted to the organic agriculture. As standards were used: Carolina, Perinha ?gua Branca, Pending Yashi, Joanna and the hybrids Super Sweet, Sweet Million and Mascot F1. The experiments were conducted under field conditions in the Horticulture Sector of the Federal Rural University of Rio de Janeiro (UFRRJ) from June 2010 to November 2013 during which eight trials were carried out. In the different trials, productivity, number of total fruits, disease progression, morphological characteristics (coloration, shape, number of locules), physical (longitudinal and equatorial diameter), and physicochemical characteristics of fruits (total soluble solids - TSS , titratable total acidity - TTA, pH, TSS / TTA ratio). It was also evaluated the accumulation of dry mass of the stem, leaves and fruits, content and content of nitrogen (N), potassium (K) and phosphorus (P) in the respective organs. It was observed a great genetic variability between the accessions as to the physical and physical-chemical morphological attributes. Promising accesses were selected for use in breeding programs and with great productive potential and potential for cultivation in organic systems. Among these, we highlight the ENAS 1040, ENAS 1037, ENAS 1031 and ENAS 1026 accessions for cultivation in the spring / summer period and the accesses ENAS 1228, ENAS 1214, ENAS 1227 and ENAS 1220 by the highest total soluble solids (?Brix). The accessions ENAS 1121, EAS 1013, ENAS 1143 and ENAS 1029 were distinguished by the production of fruits with differentiated formats and the accesses ENAS 1007, ENAS 1008, ENAS 1037, ENAS 1033, ENAS 1017, ENAS 1036, ENAS 1062 and ENAS 1029 by coloring of the fruits. The accessions ENAS 1227 and 1026 stood out by the partial resistance to the late blight, equivalent to the standards 'Carolina' and 'Perinha ?gua Branca'. The accessions ENAS 1227, ENAS 1216, ENAS 1153 and ENAS 1060 were the ones that stood out the most regarding the resistance to the late blight and productivity. / O presente trabalho teve como objetivo caracterizar 59 acessos de tomateiro do grupo cereja quanto ?s suas caracter?sticas agron?micas e resist?ncia ? requeima e selecionar aqueles mais adaptados ? agricultura org?nica. Como padr?es foram utilizadas: Carolina, Perinha ?gua Branca, Pendente Yashi, Joanna e os h?bridos Super Sweet, Sweet Million e Mascot F1. Os experimentos foram conduzidos em condi??es de campo, no Setor de Horticultura da Universidade Federal Rural do Rio de Janeiro (UFRRJ) no per?odo de junho de 2010 a novembro de 2013 durante o qual realizaram-se oito ensaios. Nos diferentes ensaios, avaliaram-se produtividade, n?mero de frutos totais, progresso da requeima, caracter?sticas morfol?gicas (colora??o, formato, n?mero de l?culos), f?sicas (di?metro longitudinal e equatorial), e f?sico-qu?mica dos frutos (s?lidos sol?veis totais - SST, acidez total titulav?l - ATT, pH, rela??o SST/ATT). Avaliou-se, ainda, ac?mulo de massa seca do caule, folhas e frutos, teor e conte?do de nitrog?nio (N), pot?ssio (K) e f?sforo (P) nos respectivos ?rg?os. Observou-se grande variabilidade gen?tica entre os acessos quanto aos atributos morfol?gicos f?sicos e f?sico-qu?micos. Selecionaram-se acessos promissores para uso em programas de melhoramento e com grande potencial produtivo e com potencial para cultivo em sistemas org?nicos. Dentre estes, destacam-se os acessos ENAS 1040, ENAS 1037, ENAS 1031 e ENAS 1026 para cultivo no per?odo de primavera/ver?o e os acessos ENAS 1228, ENAS 1214, ENAS 1227 e ENAS 1220 pelos maiores teores de s?lidos sol?veis totais (?Brix). Os acessos ENAS 1121, EANAS 1013, ENAS 1143 e ENAS 1029 destacaram-se pela produ??o de frutos com formatos diferenciados e os acessos ENAS 1007, ENAS 1008, ENAS 1037, ENAS 1033, ENAS 1017, ENAS 1036, ENAS 1062 e ENAS 1029 pela colora??o dos frutos. Os acessos ENAS 1227 e 1026 destacaram-se pela resist?ncia parcial ? requeima, equivalente ? dos padr?es ?Carolina? e ?Perinha ?gua Branca?. Os acessos ENAS 1227, ENAS 1216, ENAS 1153 e ENAS 1060 foram os que mais se destacaram quanto a resist?ncia ? requeima e produtividade.

Exotic germplasm has potential to provide new alleles for disease and insect resistance. US maize (Zea mays L.) currently lacks genetic resistance to Aspergillus flavus, a fungal pathogen that produces aflatoxin in maize kernels. Aflatoxin is one of the main limitations to maize production in hot, dry regions like the Southern US because of the harmful effects on humans and animals and subsequent marketing regulations. Two experiments were conducted to evaluate different exotic maize collections for response to aflatoxin. Exotic adapted maize lines, known as LAMA lines, were found to accumulate less aflatoxin than US hybrids in tests across Southern Texas. Exotic introgression lines developed by The International Center for Maize and Wheat Improvement (CIMMYT) including inbred lines, yellow hybrids, and white hybrids, were more resistant to aflatoxin than US inbred lines and hybrids in field trials in Texas, Georgia, and Mississippi. Another experiment evaluated the grain composition of hybrids with exotic adapted LAMA maize lines and a collection of US hybrids, quality protein maize (QPM) hybrids, and advanced breeding lines using near-infrared spectroscopy. Individual LAMA lines and advanced breeding lines have higher starch content than US hybrid checks. Starch content was the primary grain composition trait of interest as an enhanced-value market has emerged for high starch maize hybrids. Limited germplasm has been analyzed for grain composition because wet chemistry analysis methods required large sample sizes and were time and labor intensive. The near infrared spectroscopy (NIR) method requires a relatively small sample and is a non-destructive analysis method. In this study, NIR was effective at ranking genotypes based on starch, oil, and protein content of the grain.

Exotic germplasm has potential to provide new alleles for disease and insect resistance. US maize (Zea mays L.) currently lacks genetic resistance to Aspergillus flavus, a fungal pathogen that produces aflatoxin in maize kernels. Aflatoxin is one of the main limitations to maize production in hot, dry regions like the Southern US because of the harmful effects on humans and animals and subsequent marketing regulations. Two experiments were conducted to evaluate different exotic maize collections for response to aflatoxin. Exotic adapted maize lines, known as LAMA lines, were found to accumulate less aflatoxin than US hybrids in tests across Southern Texas. Exotic introgression lines developed by The International Center for Maize and Wheat Improvement (CIMMYT) including inbred lines, yellow hybrids, and white hybrids, were more resistant to aflatoxin than US inbred lines and hybrids in field trials in Texas, Georgia, and Mississippi. Another experiment evaluated the grain composition of hybrids with exotic adapted LAMA maize lines and a collection of US hybrids, quality protein maize (QPM) hybrids, and advanced breeding lines using near-infrared spectroscopy. Individual LAMA lines and advanced breeding lines have higher starch content than US hybrid checks. Starch content was the primary grain composition trait of interest as an enhanced-value market has emerged for high starch maize hybrids. Limited germplasm has been analyzed for grain composition because wet chemistry analysis methods required large sample sizes and were time and labor intensive. The near infrared spectroscopy (NIR) method requires a relatively small sample and is a non-destructive analysis method. In this study, NIR was effective at ranking genotypes based on starch, oil, and protein content of the grain.

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 36-41). / vi, 41 leaves, bound col. ill. 29 cm

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.

Genome resource banking (collection, storage and use of germ plasm) has emerged as a potentially powerful tool to assist in managing and conserving endangered species. The development of genome resource banks is multi-faceted. This thesis focused on aspects of population genetics and the phenomenon of sperm capacitation, a fundamental biological process crucial to the use of germ plasm for assisted breeding (i.e., artificial insemination, in vitro fertilization). Computerized simulation modeling, using two endangered species models, the Eld's deer (Cervus eldi thamin ) and Pr-zewalski's horse (Equus przewalskii) clearly demonstrated the value of certain sperm storage and use strategies for maintaining genetic diversity in captive populations. Laboratory studies with Eld's deer sperm examining a host of conventional strategies (developed in livestock) demonstrated clear challenges in provoking capacitation and the acrosome reaction in the Eld's deer. This finding reaffirmed the importance of species-specific studies in wildlife research.

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