The effect of dispersion on plant embryo development
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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.