SUMMARY

Somatic embryogenesis is a clonal propagation method that encompasses many advantages for large-scale propagation of plants. Application of somatic embryogenesis is however limited due to unresolved problems in culture techniques. One major obstacle is the inhibition from culture of embryos in liquid culture medium, i.e. in a bioreactor environment, on subsequent plant development. To overcome this obstacle, it is important to gain insight into the effects of flow on somatic embryo development. Specifically, preliminary data points to the importance of the effect from hydrodynamically-induced mechanical stress, on the development of a polarized somatic embryo in the proliferation phase. This doctoral research program intends to establish the correlation between shear stress in liquid culture conditions and somatic embryo development. The results from this research program will support development of a culture technique that allows the embryo to continue develop into a plant after a multiplication stage in liquid culture medium. The effect of steady shear stress on somatic embryos were investigated in a flow chamber and evaluated at different time intervals using microscopy technique. The development of meristematic cell clusters, i.e. the immature embryos, into a polarized somatic embryo, and the effect on the localization of the suspensor cells that form during development of the immature embryos, were studied as a function of shear stresses. With the distribution and growth rate of the meristematic and suspensor cells, the effect of stress on the embryo development was established. Furthermore, the effect of shear stress on the cells at molecular level, the reaction of integrin-like proteins, the production of reactive oxygen species and the pore size of the cell walls involved in the shear stress responses, were investigated with molecular techniques. In general, shear stress inhibits meristematic cells growth. Meristematic cells grow fastest at shear rate of 86 s-1 among all the tested shear stress conditions. By combining the results of meristematic cells growth and suspensor cells formation, it suggests that there is a critical shear rate between 86 and 140 s-1, at which no suspensor cells form. The reactive oxygen species and integrin-like protein are detected in the stressed cells as responses to shear stresses. By monitoring the pore size change on cell walls with solute-exclusive experiments, it confirms that the stressed cells decrease the pore size on the cell wall as one of the means of defense.