The two species investigated in this study were Theobroma cacao and Barringtonia racemosa. Theobroma cacao has worldwide economic importance, as cocoa (the main ingredient in chocolate) is produced from the seeds of this tree; while B. racemosa has several applications in herbal medicine. The seeds of both T. cacao and B. racemosa are highly recalcitrant and therefore not amenable to storage for any significant periods. The long-term conservation of the germplasm of these species may only be feasible via cryopreservation. The aims of the present study were to: 1) optimize in vitro regeneration protocols for different types of explants that have the potential to be cryopreserved while maintaining the genetic integrity of these two species; and 2) develop cryopreservation protocols for selected explants. For T. cacao, protocols were established for bud-break and multiplication for both in vitro - and greenhouse-derived nodal explants, as well as a rooting medium for shoots derived from axillary buds. Parameters investigated towards the cryopreservation of axillary shoots, from greenhouse nodal segments, and nodal segments from in vitro plantlets, included the size of the explant and pre-treatments for cryopreservation. Nodal segments (6 - 7 mm) and axillary shoots (2 - 4 mm) needed to be soaked in 0.5% (w/v) ascorbic acid for 10 min to minimise phenolic production and subsequent tissue death, and surface-sterilized by soaking in 1% Ca(OCl)2 solution for 5 min to reduce microbial contamination. Subsequent cryopreservation attempts involved only in vitro nodal segments because of the lack of success in achieving elongation of excised axillary buds. Vitrification and slow freezing methods, with or without the application of cryoprotectants, did not achieve successful cryopreservation. Attempts to establish a protocol for producing somatic embryos, as an alternate to axillary shoots and in vitro nodal segments, resulted in the production of globular embryogenic callus for both leaf and cotyledon explants. Cryopreservation of these explants was not investigated in the scope of this study. The study on B. racemosa focused on the development of a somatic embryogenesis protocol. Segments of embryonic axes produced globular-stage embryos when placed on MS medium supplemented with 30 g 1-1 sucrose, 1.0 g 1-1 casein hydrolysate, 2.0 mg 1-1 2,4-D, 0.1 mg 1-1 BAP and 8.0 g 1-1 agar. Various strategies were employed to obtain embryo germination, which included 1) different time intervals on callus initiation medium; 2) the use of different auxins (IAA, NAA and 2,4-D) in combination with the cytokinins BAP and kinetin; 3) desiccation and 4) cold treatments. Although somatic embryo germination was not achieved, globular embryos proceeded with development to the cotyledonary stage when cold-treated for 8 h at 4°C. This study provides some fundamental bases for further investigation towards achieving long-term conservation for both T. cacao and B. racemosa. However, the use of meristems as explants for cryopreservation is suggested to be the way forward for the cryopreservation of both species. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2008.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 151-192) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 151-192) Also available online.

Thesis (M.S.)--University of North Texas, Dec., 2009. / Title from title page display. Includes bibliographical references.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Karlsson, Jens; Committee Co-Chair: Nerem, Robert; Committee Member: Meda, Paolo; Committee Member: Prausnitz, Mark; Committee Member: Sands, Jeff; Committee Member: Zhu, Cheng.

Thesis (MScMed)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

The aim of this study was to characterise and cryopreserve semen of the indigenous Namaqua Afrikaner breed, and to compare it to that of Dorper and Dohne Merino sheep, whose semen is commercially frozen on a large scale. The study was conducted between January and August 2015. September 2013-born Namaqua Afrikaner (12), Dohne Merino (12) and Dorper (9) rams were used in the study. The rams were kept under kraal conditions with adequate shade, and they received a high protein, high energy diet. Originally it was envisaged to collect semen samples using the artificial vagina (AV) method, which proved to be problematic with the Namaqua Afrikaner rams. Semen samples were subsequently collected twice a week by either AV (Dohne Merino and Dorper) or electro-ejaculation (EE; all three breeds). Macroscopic sperm traits were assessed and sperm concentration determined immediately after collection. Each semen sample was diluted with Triladyl® (1:3) and subsequently frozen in liquid nitrogen vapour in straws. Frozen straws were thawed and evaluated at 7, 30 and 90 days after cryopreservation. A droplet (0.5 ml) from each thawed sample was assessed microscopically for post-thaw motility and percentage live sperm..

No description available.

In an attempt to expand the knowledge of the diversity of algal taxa that are capable of successful recovery after freezing to and thawing from -196°C, 365 algal strains representing 13 algal classes were studied using various cryoprotectants at several concentrations. A cryopreservation protocol involving culturing the algae under low light (17-22 μE m⁻² s⁻¹) on semisolid (I % agar) media, controlled cooling at 1°C min⁻¹, rapid thawing, and the return of cultures to agar media was developed. This protocol produced successful recovery of >77% (283) of the algal strains tested. Of the 283 successfully cryopreserved strains comprising 240 species (68 genera), >220 species (34 genera) were cryopreserved for the first time. In addition, preliminary data suggest that some relationships exist among proposed systematic evolutionary lines, environmental preferences, cryoprotectants, and the organism's cryopreservability. / Master of Science

Submitted in fulfilment of the requirements for the degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2015. / In contrast to orthodox seeds, recalcitrant seeds are short-lived, shed at relatively high water contents (WCs), and are desiccation sensitive. Presently, the only option for long-term conservation of genetic resources of such plant species is by cryostorage in liquid nitrogen (LN; -196°C) or in the vapour phase over LN (at -150⁰C to -160⁰C). A number of cryopreservation protocols applied for recalcitrant zygotic embryos or embryonic axes of tropical/sub-tropical species have reported survival as either root or shoot development or callus formation, with no shoot or root production after cryopreservation. This is a consequence of the challenges encountered when optimising the WC for successful cryopreservation across species. Other shortcomings may also be the formation of ice or the sensitivity to desiccation resulting in lethal damage or poor re-growth. However, for successful cryopreservation, a normal plantlet with a shoot and a root needs to be obtained post-cryo. Specimens required for successful cryopreservation must be small; therefore embryonic axes excised from seeds have been often used as the explants of choice. However, in some cases, excised embryonic axes of mature recalcitrant seeds are too large to be cryopreserved, or, even if small, may be adversely affected by excision, dehydration and/or immersion in LN, thus failing to produce plantlets after cryopreservation. As a result, in such cases, there is a need to develop explants alternative to zygotic axes such as buds derived from in vitro shoots, shoot meristems, or shoot apices and somatic embryos. These alternative explants must have a high capacity for plantlet formation before and after cryopreservation. The present study aimed to successfully cryopreserve shoot apices of Trichilia emetica and T. dregeana, tropical recalcitrant-seeded tree species, and monitor the responses or effects of some of the procedural steps involved in cryopreservation on the survival and shoot production from these shoot apices. The main foci of the investigation were to produce vigorous plantlets after cryopreservation and ultimately develop a protocol for the successful cryopreservation of germplasm of these species. Furthermore, this study reports on a number of factors that may affect survival after cryopreservation, viz. WC of the explants, PVS2 treatment, production of reactive oxygen species (ROS) and levels of endogenous total aqueous antioxidants (TAA) during the various steps of cryopreservation. The effects of the various steps of cryopreservation on the ultra-structure of the shoot apices were also observed. Cathodic protection (by using highly reducing cathodic water; CW) of the explants was attempted to improve vigour and shoot production from the surviving shoot apices after cryopreservation as cathodic water has been reported to ameliorate the excessive burst of ROS, which often accompanies the stresses imposed by the procedural steps of cryopreservation. Experiments were also performed to optimise the medium for vigorous shoot formation from the shoot apices. Shoot apices of T. emetica in this study had an initial WC of ca. 2.2 g g -1 dry weight (DW) upon excision. Although the WC of the shoot apices decreased slightly after cryoprotection with PVS2, it did not result in sufficient dehydration before cooling. Upon retrieval from LN, 68% of the shoot apices survived and 40% of those produced shoots. Treatment of shoot apices with CW did not improve the survival or shoot production from the apices following cryo-retrieval. This could be a direct consequence of increase in WC of the shoot apices following CW treatment. Water content is not the only factor affecting successful cryopreservation; the production of ROS and the level of antioxidants may also have an impact on regrowth after cryogen exposure. Rapid changes in temperature when the samples are cryo-stored and then rewarmed result in an increase in ROS production, which could have affected the shoot production. More importantly the antioxidant activity showed a rapid decrease during recovery, especially in the CW treated shoot apices, which might have also led to the poor survival and shoot production from the shoot apices. Ultrastructural observations showed the injurious effects of PVS2 treatment typified by derangement of plastids, development of numerous small vesicles along the cell membrane and abnormalities in the structure of the nuclear envelope in the shoot apical cells both before and after cryogen exposure. Following cryo-retrieval, the meristem cells were extensively deteriorated – indicating non-survival, however, some shoot apices had areas of surviving cells which might have led to 40% shoot production after cryopreservation. Based on the studies on optimising medium composition for shoot formation from the apices, woody plant medium (WPM) with 1 mg L-1 BAP + 0.1 mg L-1 IBA was found to be the best medium which gave a higher shoot production of 67 – 70% before cryopreservation compared with only 18 – 20% shoot formation on media used previously. Therefore, this medium was used as the recovery medium. Encapsulation-dehydration of the shoot apices and the use of PVS3 instead of PVS2 for cryoprotection were also employed in an attempt to improve the survival and shoot production after post-cryo, but both methods did not result in any shoot production although 92% and 90% of the shoot apices survived cryogen immersion, respectively. While the shoot apices of T. emetica resulted in 40% shoot production following retrieval from LN and recovery on WPM with 1 mg L-1 BAP + 0.1 mg L-1 IBA, attempts to further improve the shoot production were not successful. The results of this study suggest that the shoot apices used were possibly not sufficiently developed, and with the commensurately high WC, proved to be unsuitable explants for germplasm conservation of T. emetica. The injurious effects of PVS2 treatment both before and after cryogen exposure as observed from the ultra-structural studies provide a clue to the repeated failure to cryopreserve embryonic axes of many tropical recalcitrant-seeded species after treatment with PVS2. Maintaining mother material in culture for longer durations before explant excision in order to allow better development of the axillary buds and render the cytosol more concentrated, and optimising the exposure duration to loading solution and concentration of sucrose in the loading solution might however, provide sufficient dehydration tolerance to PVS2 leading to successful vitrification up on cooling.