Online

Grasso, Matthew

Plant Biology

2023

Ph. D.

Plant morphogenesis requires the coordinated growth between cells and their neighbors. This coordination is regulated by internal and external signals that are both physical and chemical. It is understood that both mechanical and molecular signals are involved in the regulation of plant morphogenesis however, how mechanical signals induce molecular signals and vice versa is not. This is due to a lack of research tools for manipulating and measuring the mechanical environment of growing plant cells. Such tools would ideally provide individual plant cells with mechanically tunable physical microenvironments. This may allow changes in the cells physical environment to be linked to molecular changes such as gene expression or cellular architecture. The field of microfluidics has provided biology with a suite of tools for handling cells and tissues precisely at small scales. In this work, droplet microfluidics was used to encapsulate individual plant protoplasts in congealed agarose microspheres. Encapsulated protoplasts were viable and able to regenerate their cell wall and resume growth eventually breaking out of the microsphere. To manipulate the growth of the encapsulated protoplasts agarose microspheres were modified with the addition of pectin or polyelectrolyte coatings. Agarose microspheres were also modified by encapsulating them in a layer of alginate methacrylate creating a two-layer hydrogel microsphere. Alginate methacrylate is capable of dual crosslinking by either ionic or covalent bonding of the polymer chains. Viable protoplasts were successfully encapsulated in each of the described hydrogel microsphere environments. Cell expansion and the orientation of cortical microtubules was compared between cells growing in the agarose, agarose-pectin blend, and agarose-polyelectrolyte coated microspheres. Agarose-polyelectrolyte microspheres decreased anisotropic growth and disrupted the cortical microtubule orientation of encapsulated protoplasts. Alginate methacrylate was shown to be a suitable material for plant cell culture as well as hydrogel microsphere fabrication. Protoplast viability was confirmed within photo crosslinked alginate methacrylate blocks and viable protoplasts were encapsulated within agarosealginate methacrylate two-layer hydrogel microspheres. This work shows that agarose microsphere encapsulation of living plant protoplasts is feasible with high yield and viability using a small microfluidic system. Agarose microspheres provide delicate protoplasts with physical support and a protective layer that is suitable for cell culture. They also may serve as a template that can be modified to create cellular microenvironments tuned for specific applications. This methodology may contribute to studies in plant cell reprogramming and cell differentiation.