The focus of this research is to gain insight into the role of dispersion for synchronized development and yield of mature embryos on solid and in liquid culture medium. It is hypothesized that dispersion and synchronous development of embryos will result in high yield of plants. The increase in yield would be helpful in practical implementation of somatic embryogenesis for large-scale clonal propagation of plants and agricultural goods. This doctoral research investigates the yield and the synchronized development of mature embryos, if immature embryos in aggregates of proembryogenic masses (PEMs) have access to same nutritional environment. In order to explore this, a dispersion mechanism was automated and used to provide the same nutritional environment to all PEMs. The effectiveness of the dispersion system was investigated by culturing the PEMs of Norway spruce (Picea abies) on solid medium and in a well-functioning liquid culture system, i.e., bioreactor that could be used for large-scale clonal propagation of plants. Distribution of nutrient concentrations at different locations in an aggregate of PEMs during the culture period was studied using a mathematical model. Results have indicated that dispersion of aggregates of PEMs of Norway spruce has a favorable effect on the rate of proliferation of PEMs and subsequent development of mature somatic embryos. Compared to non-dispersed aggregates of PEMs with dispersed aggregates of PEMs, embryo development increased two folds on solid medium and three to five folds in liquid medium in bioreactors in this study. Bioreactor culture has shown significantly higher yield of mature embryos compared to that on solid culture. The effect of dispersion on synchronized development of mature embryos appears to be cell line dependent. Dispersion has improved synchronization of embryo development in one of two cell lines used in liquid medium experiments and two of four cell lines examined on solid medium. Cell line 11:12:02 has shown more synchronized development of embryos in dispersed PEMs in both medium. To further investigate the details to understand the association between development of mature embryos and nutrient uptake by cells and tissues, a nutrient diffusion model was developed using the volume averaging technique. It estimated the concentration distributions of nutrients, e.g. sugars, in a PEMs cluster on solid medium over a period of culture. The Michaelis-Menten enzyme kinetics was used in the model for uptake of nutrients by cells and tissues. Enzymatic assaying of soluble sugars was performed to determine concentrations of sugars (glucose, fructose, and sucrose) at different locations in tissue clusters. In both experiments and model simulation, sharp decline in concentrations of sucrose and fructose was observed in the first 12 hours after inoculation in glucose-containing ½ LP gel medium. A significant match between predicted and experimental outputs was observed over the culture period. Both experiments and simulation of the model showed a rapid uptake of glucose from the medium and saturation of PEMs cluster within 12 hours. Hence in a PEMs cluster there was no scarcity of nutrients that would inhibit growth and development of somatic embryos. Though dispersion resulted in a significant increase in development of mature somatic embryos, it might not play the decisive role in synchronized development of embryos. It seems that the major factor in synchronization is the initial developmental stage of immature embryos in a culture and genetic characteristics.

No description available.

No description available.

No description available.

No description available.

No description available.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Initially, we aimed to identify novel genes regulating vertebrate neurogenesis and somitogenesis by screening cDNAs derived from gastrulation/neurulation stage zebrafish embryos for clones revealing corresponding genes with expression patterns suggestive of roles in these processes. The lack of suitable cDNA libraries prompted us to devise a simplified method for producing randomly-primed, directionally cloned cDNA libraries from small amounts of embryonic tissue. To achieve this, several techniques were combined, including cDNA synthesis on a solid carrier, random priming of 1st cDNA strand synthesis, non-specific priming of 2nd cDNA strand synthesis and amplification of initially small amounts of cDNAs by suppression-PCR. A pilot-scale in situ screen using a cDNA library produced by the above method identified a gene, spadetail, that is expressed in presomitic mesoderm and in unidentified, apparently irregularly distributed cells of the spinal cord. spt functions in mesodermal development, yet its role in neural tissue remains unknown. Analysis of the spadetail-expressing neural cells' gene co-expression profile and dorsoventral location implied that they are Dorsal Longitudinal Ascending interneurons. Quantitative analysis of these cells' rostrocaudal distribution showed that there is a tendency to higher cell numbers in rostral spinal segments. The observation that spadetail-expressing neurons are frequently juxtaposed to somitic cells expressing spadetail at low levels suggests that the distribution of spadetail-expressing neurons may be 'inefficiently' patterned by spadetail-expressing somitic cells or that the expression of spadetail in both tissues is induced by a common positional cue. The strategy for non-specific was then extended to develop a simple technique for cloning unknown DNA sequences flanking known DNA. An initial nonspecific PCR amplification was performed with a single primer that binds specifically within known sequence and non-specifically in the unknown DNA region. In a second reaction, the sequences of interest were amplified from the primary reaction mixture (that also contains undesired sequences) with nested PCR using a primer that had been extended further downstream from the primer used in the initial PCR. This enabled isolation of a 0.5 kb region of amphioxus Notch cDNA, that, in turn, contributed to the subsequent analysis of the evolution of vertebrate Notch genes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1108027 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2004

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Initially, we aimed to identify novel genes regulating vertebrate neurogenesis and somitogenesis by screening cDNAs derived from gastrulation/neurulation stage zebrafish embryos for clones revealing corresponding genes with expression patterns suggestive of roles in these processes. The lack of suitable cDNA libraries prompted us to devise a simplified method for producing randomly-primed, directionally cloned cDNA libraries from small amounts of embryonic tissue. To achieve this, several techniques were combined, including cDNA synthesis on a solid carrier, random priming of 1st cDNA strand synthesis, non-specific priming of 2nd cDNA strand synthesis and amplification of initially small amounts of cDNAs by suppression-PCR. A pilot-scale in situ screen using a cDNA library produced by the above method identified a gene, spadetail, that is expressed in presomitic mesoderm and in unidentified, apparently irregularly distributed cells of the spinal cord. spt functions in mesodermal development, yet its role in neural tissue remains unknown. Analysis of the spadetail-expressing neural cells' gene co-expression profile and dorsoventral location implied that they are Dorsal Longitudinal Ascending interneurons. Quantitative analysis of these cells' rostrocaudal distribution showed that there is a tendency to higher cell numbers in rostral spinal segments. The observation that spadetail-expressing neurons are frequently juxtaposed to somitic cells expressing spadetail at low levels suggests that the distribution of spadetail-expressing neurons may be 'inefficiently' patterned by spadetail-expressing somitic cells or that the expression of spadetail in both tissues is induced by a common positional cue. The strategy for non-specific was then extended to develop a simple technique for cloning unknown DNA sequences flanking known DNA. An initial nonspecific PCR amplification was performed with a single primer that binds specifically within known sequence and non-specifically in the unknown DNA region. In a second reaction, the sequences of interest were amplified from the primary reaction mixture (that also contains undesired sequences) with nested PCR using a primer that had been extended further downstream from the primer used in the initial PCR. This enabled isolation of a 0.5 kb region of amphioxus Notch cDNA, that, in turn, contributed to the subsequent analysis of the evolution of vertebrate Notch genes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1108027 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2004

The consequence of a folate deficiency during organogenesis has been investigated in the rat embryo. In vitro culture of 9.5 day embryos in serum from dietary induced folate deficient rats frequently resulted in abnormal embryos. Many were growth retarded and exhibited a defect in the turning mechanism which inverts the embryo from ventrally to dorsally convex. Affected embryos displayed abnormal twisting or kinking of the neural tube. Gross anaemia was also frequently observed and the protein content of the embryos was markedly less than that of embryos grown in normal rat serum. Supplementation of the deficient serum with folic acid improved growth and greatly reduced the occurrence of deformities. It virtually eliminated the incidence of gross anaemia but only partially restored the protein content to the level observed in embryos cultured in normal rat serum. The effects of the folate deficiency could not be reversed by supplementation with multivitamins or by increasing the volume of culture serum. They were, however, eliminated by supplementing the deficient culture serum with normal rat serum. The effects could also be overcome by in vivo folate supplementation; rats which had previously been so folate deficient that culture in their serum would have resulted in malformed embryos, after restoration to a folate supplemented diet produced serum which supported completely normal embryonic growth. The results indicate that embryos undergoing organogenesis require adequate folate in order for normal growth and differentiation to take place. They also suggest that some of the embryopathic effects of maternal folate deficiency are mediated by secondary effects. These may involve complex growth or metabolic factors which can be corrected in vivo but are not readily reversed in vitro.

No description available.