Recalcitrant unlike orthodox seeds do not show a sharp border between maturation and germination and remain highly hydrated and desiccation-sensitive at all developmental and post-harvest stages. In contrast with recalcitrant seeds, orthodox types retain viability for predictably long periods in the dry state and hence can be stored under low relative humidity and temperature conditions. Storage of recalcitrant seeds under conditions allowing little to no water loss, at moderate temperatures, allows for short- to medium-term storage but only facilitates viability retention for a matter of a few weeks to months, at best, because the seeds are metabolically active and initiate germination while stored. Cryopreservation, i.e. storage at ultra-low temperatures (usually in liquid nitrogen [LN] at -196°C), is a promising option for the long-term germplasm conservation of recalcitrant-seeded species but their seeds present some unavoidable difficulties in terms of the amenability of their germplasm to cryopreservation. Pre-conditioning treatments can reduce the amount of ‘free’ water available for freezing and may increase the chances of cells or tissues surviving exposure to cryogenic temperatures. Such conditioning may be imposed by physical dehydration or cryoprotection, i.e. exposure to compounds that depress the kinetic freezing point of water and so reduce the likelihood of lethal ice-crystal formation during cooling (i.e. exposure to LN at -196°C or sub-cooled LN at -210°C) and subsequent thawing. Partial dehydration is presently a standard pre-treatment for the cryopreservation of recalcitrant zygotic germplasm and explant cryoprotection has been shown to improve postthaw survival in some recalcitrant-seeded species. However, there is a paucity of information on the physiological and biochemical basis of post-thaw survival or death in recalcitrant seeds, and this is the major focus of the current contribution. Additionally, in light of the lack of understanding on how cryo-related stresses imposed at the embryonic stage are translated or manifested during subsequent seedling growth, this study also investigated the effects of partial dehydration and the combination of partial dehydration and cooling of recalcitrant zygotic embryos on subsequent in and ex vitro seedling vigour. All studies were undertaken on the zygotic embryos of two recalcitrant-seeded members of the Amaryllidaceae, viz. Amaryllis belladonna (L.) and Haemanthus montanus (Baker); both of which are indigenous to South Africa. Studies described in Chapter 2 aimed to interpret the interactive effects of partial dehydration (rapidly to water contents > and <0.4 g g-1), cryoprotection (with sucrose [Suc; nonpenetrative] or glycerol [Gly; penetrative]) and cooling rate (rapid and slow) on subsequent zygotic embryo vigour and viability, using three stress markers: electrolyte leakage (an indicator of membrane integrity); spectrophotometric assessment of tetrazolium chloride-reduction (an indicator of respiratory competence); and rate of protein synthesis (an indicator of biochemical competence). These studies showed that in recalcitrant A. belladonna and H. montanus zygotic embryos, stresses and lesions, metabolic and physical, induced at each stage of the cryopreservation protocol appear to be compounded, thus pre-disposing the tissues to further damage and/or viability loss with the progression of each step. Maximum post-thaw viability retention in both species appeared to be based on the balance between desiccation damage and freezing stress, and the mitigation of both of these via Gly cryoprotection. Post-thaw viabilities in both species were best when Gly cryoprotected + partially dried zygotic embryos were rapidly, as opposed to slowly, cooled. However, the rate at which water could be removed during rapid drying was higher in A. belladonna and this may explain why the optimum water content range for post-thaw survival was <0.40 g g-¹ for A. belladonna and >0.40 g g-¹ for H. montanus. These results suggest that to optimise cryopreservation protocols for recalcitrant zygotic germplasm, attention must be paid to pre-cooling dehydration stress, which appears to be the product of both the ‘intensity’ and ‘duration’ of the stress. Cryoprotection and dehydration increased the chances of post-thaw survival in A. belladonna and H. montanus zygotic embryos. However, transmission electron microscopy studies on the root meristematic cells from the radicals of these embryos (described in Chapter 3) suggest that their practical benefits appear to have been realised only when damage to the sub-cellular matrix was minimised: when (a) pre-conditioning involved the combination of cryoprotection and partial dehydration; (b) the cryoprotectant was penetrating (Gly) as opposed to non-penetrating (Suc); and (c) embryos were rapidly cooled at water contents that minimised both dehydration and freezing damage. The ability of A. belladonna and H. montanus embryos to tolerate the various components of cryopreservation in relation to changes in extracellular superoxide (.O2 -) production and lipid peroxidation (a popular ‘marker’ for oxidative stress) was investigated in studies featured in Chapter 4. Pre-conditioning and freeze-thawing led to an increase in oxidative stress and the accompanying decline in viability suggests that oxidative stress was a major component of cryoinjury in the embryos presently investigated. Post-thaw viability retention in Gly cryoprotected + partially dried embryos was significantly higher than noncryoprotected + partially dried embryos, possibly due to the relatively lower post-drying lipid peroxidation levels and relatively higher post-drying and post-thawing enzymic antioxidant activities in the former. Exposure of certain plant tissues to low levels of oxidative or osmotic stress can improve their tolerance to a wide range of stresses. In contrast, exposure of H. montanus zygotic embryos to low levels of oxidative stress provoked by exogenously applied hydrogen peroxide (H2O2) or exposure of A. belladonna embryos to low levels of osmotic stress provoked by low water potential mannitol and polyethylene glycol solutions (in studies featured in Chapter 5) increased their sensitivity to subsequent dehydration and freeze-thaw stresses. Exposure of Gly cryoprotected and non-cryoprotected amaryllid embryos to such stress acclimation treatments may pre-dispose A. belladonna and H. montanus embryos to greater post-drying and post-thaw total antioxidant and viability loss than untreated embryos. To assess the vigour of seedlings recovered from partially dried H. montanus embryos, seedlings recovered from fresh (F) and partially dried (D) embryos in vitro were hardened-off ex vitro, and subsequently subjected to either 42 days of watering or 42 days of water deficit (in studies described in Chapter 6). In a subsequent study (described in Chapter 7), seedlings recovered from fresh (F), partially dried (D) and cryopreserved (C) A. belladonna embryos were regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8 days of water stress (S) followed by 3 days of re-watering. Results of these studies suggest that the metabolic and ultrastructural lesions inflicted on A. belladonna and H. montanus zygotic embryos during cryopreservation may compromise the vigour (e.g. development of persistent low leaf water and pressure potentials and reduced photosynthetic rates) and drought tolerance of recovered seedlings, compared with seedlings recovered from fresh embryos. While the adverse effects of freeze-thawing were carried through to the early ex vitro stage, certain adverse effects of partial drying were reversed during ex vitro growth (e.g. the increased relative growth rate of seedlings from partially dried embryos). The reduced vigour and drought tolerance of seedlings recovered from partially dried and cryopreserved embryos in the present work may therefore disappear with an extension in the period afforded to them for hardening-off under green-house conditions, and in the field. The results presented in this thesis reinforce the notion that each successive manipulation involved in the cryopreservation of recalcitrant zygotic germplasm has the potential to inflict damage on tissues and post-thaw survival in such germplasm relies on the minimisation of structural and metabolic damage at each of the procedural steps involved in their cryopreservation. The results also highlight the need to design research programmes aimed not only at developing protocols for cryopreservation of plant genetic resources, but also at elucidating and understanding the fundamental basis of both successes and failures. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2010.

The conservation of germplasm of indigenous plant species is vital not only to preserve valuable genotypes, but also the diversity represented by the gene pool. A complicating factor, however, is that a considerable number of species of tropical and sub-tropical origin produce recalcitrant or otherwise non-orthodox seeds. Such seeds are hydrated and metabolically active when shed and cannot be stored under conventional conditions of low temperature and low relative humidity. This poses major problems for the longterm conservation of the genetic resources of such species. Presently, the only strategy available for the long-term conservation of species that produce recalcitrant seeds is cryopreservation. Ekebergia capensis is one such indigenous species that produces recalcitrant seeds. The aim of the present study was to develop methods for the cryopreservation of germplasm of this species. Different explant types were investigated for this purpose, viz. embryonic axes (with attached cotyledonary segments) excised from seeds, and two in vitro-derived explants, i.e. ‘broken’ buds excised from in vitro-germinated seedlings and adventitious shoots generated from intact in vitro-germinated roots. Suitable micropropagation protocols were developed for all explant types prior to any other experimentation. Before explants could be cryopreserved it was necessary to reduce their water content in order to limit damaging ice crystallisation upon cooling. All explants tolerated dehydration (by flash drying) to 0.46 – 0.39 g gˉ¹ water content (dry mass basis) with survival ranging from 100 – 80%, depending on the explant. In addition, penetrating and non-penetrating cryoprotectants were used to improve cryo-tolerance of explants. The cryoprotectants tested were sucrose, glycerol, DMSO and a combination of sucrose and glycerol. Explant survival following cryoprotection and dehydration ranged from 100 – 20%. Cryoprotected and dehydrated explants were exposed to cryogenic temperatures by cooling at different rates, since this factor is also known to affect the success of a cryopreservation protocol. The results showed that ‘broken’ buds could not tolerate cryogen exposure. This was likely to have been a consequence of the large size of explants and their originally highly hydrated condition. Adventitious shoots tolerated cryogenic exposure slightly better with 7 – 20% survival after cooling in sub-cooled nitrogen. Limited shoot production (up to 10%) was obtained when axes with attached cotyledonary segments were exposed to cryogenic temperatures. In contrast, root production from axes cooled in sub-cooled nitrogen remained high (67 – 87%). Adventitious shoots were subsequently induced on roots generated from cryopreserved axes by applying a protocol developed to generate adventitious shoots on in vitrogerminated roots. In this manner, the goal of seedling establishment from cryopreserved axes was attained. Each stage of a cryopreservation protocol imposes stresses that may limit success. To gain a better understanding of these processes the basis of damage was investigated by assessing the extracellular production of the reactive oxygen species (superoxide) at each stage of the protocol, as current thinking is that this is a primary stress or injury response. The results suggested that superoxide could not be identified as the ROS responsible for lack of onwards development during the cryopreparative stages or following cryogen exposure. The stresses imposed by the various stages of a cryopreservation protocol may affect the integrity of germplasm. Since the aim of a conservation programme is to maintain genetic (and epigenetic) integrity of stored germplasm, it is essential to ascertain whether this has been achieved. Thus, explants (axes with cotyledonary segments and adventitious shoots) were subjected to each stage of the cryopreservation protocol and the epigenetic integrity was assessed by coupled restriction enzyme digestion and random amplification of DNA. The results revealed little, if any, DNA methylation changes in response to the cryopreparative stages or following cryogen exposure. Overall, the results of this study provided a better understanding of the responses of germplasm of E. capensis to the stresses of a cryopreservation protocol and two explant types were successfully cryopreserved. Future work can be directed towards elucidating the basis of damage incurred so that more effective protocols can be developed. Assessment of the integrity of DNA will give an indication as to the suitability of developed protocols, or where changes should be made to preserve the genetic (and epigenetic) integrity of germplasm. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Embryogenic tissue of Pinus patula Scheide et Deppe was initiated from immature green female cones during the months of November 1996 to February 1997 and December 1997 to January 1998. Tissue was maintained on MSG3 medium (BECWAR, NAGMANI and WANN 1990) supplemented with maltose. A comparison of various sugars as a carbohydrate source for maintaining embryogenic tissue showed that maltose was far superior to sucrose and the other sugars tested. Embryogenic tissue was successfully cryopreserved for up to 8 weeks using 0.3 M sorbitol and 5 % DMSO. Recovered tissue initially underwent a lag phase in tissue regrowth, but by the end of 5 weeks post-thaw, tissue proliferation was as vigorous as the unfrozen, untreated control. Fluoresceine diacetate (FDA) staining revealed that the embryonal head survived cryopreservation, but the highly vacuolated suspensor tissue had ruptured and died. Embryogenic tissue from two different families and four genotypes were successfully cryopreserved using this protocol. A comparison of commonly used cryopreservation techniques was conducted. It was found that the slow addition of the cryoprotectants over two days slowed the recovery rate of the tissue and increased the chances of contamination. Embryogenic tissue did not respond well to cryopreservation using a combination of the cryoprotectants PEG, glucose and DMSO (10-8-10%). Only a small proportion of the tissue survived, and initial tissue regrowth took up to 5 weeks. Embryogenic tissue was also set in gel and immersed directly in liquid nitrogen in an effort to cryopreserve tissue using the process of vitrification. However, none of the tissue survived, possibly due to insufficient dehydration prior to immersion in liquid nitrogen. Tissue recovery was highest when the tissue was precooled to -70°C in a container filled with isopropyl alcohol placed in a static freezer prior to immersion in liquid nitrogen. Recovery of tissue was improved by suspending the tissue on polyester grids and removing the liquid medium prior to placing onto MSG3 medium. Recovered tissue was bulked up using suspension cultures, and then paced onto MSG5 (BECWAR, NAGMANI and WANN 1990) or 240 medium (PULLMAN and WEBB 1994) to mature. Mature embryos were isolated from both media and germinated. Somatic plantlets were successfully hardened-off under greenhouse conditions. The successful cryopreservation of a number of genotypes and lines, and the maturation of recovered tissue has been achieved. This technique is now being actively incorporated into P. patula somatic embryo research, enabling the long-term storage of juvenile reference tissue while field trials are carried out and evaluated. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1999.

No description available.

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen.

Thesis (Ph.D.)--University of Melbourne, School of Forest and Ecosystem Science, 2006. / Typescript. Includes bibliographical references (leaves 184-216).

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 122-124). Also available on the Internet.

Thèse--Paris.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 122-124). Also available on the Internet.

Thesis (Ph. D.)--Illinois State University, 1969. / Title from title page screen, viewed Aug. 24, 2004. Dissertation Committee: Robert M. Chasson, M.J. Nadakavukaren (co-chairs), Arthur D. Bond, John L. Frehn, David F. Weber. Includes bibliographical references (leaves 106-114). Also available in print.