Microbead encapsulation of living plant protoplasts: A new tool for the handling of single plant cells

Abstract

Although the likely significance of cell and tissue mechanics in plant development has been appreciated for many years, the study of cellular micromechanics at the level of the individual cell has proven to be problematic. The universal presence of the cellulosic cell wall and the apoplastic continuity that it provides endows plant tissues with a unique level of mechanical coupling. In principle, this makes it possible for plant tissues to transmit stress-mechanical information precisely and instantaneously over multicellular distances. However, the same apoplastic continuity that makes stress-mechanical signaling attractive as a possible developmental effector also makes it difficult to interpret responses and isolate mechanical variables at the level of the individual cell. With the advent of droplet microfluidics, it is now possible to manipulate individual cells in novel ways, potentially revealing levels of developmental control that have previously been experimentally inaccessible. In this study, we present a reliable procedure for capturing large numbers of individual plant protoplasts in mechanically isotropic hydrogel microbeads, thereby isolating them from the physical influence of neighboring cells and allowing them to regenerate their walls and proceed through cell division in a precisely controlled physical environment. A detailed protocol is provided as Appendix 1. Microfluidic devices designed to facilitate the handling and analysis of individual cells are now becoming available and have already been used to encapsulate animal cells (Kumachev et al., 2011), but in studies with plant cells they have found only limited use (Agudelo et al., 2012; Ghanbari et al., 2014). Here we show that droplet microfluidic systems are capable of rapidly and efficiently capturing large numbers of individual plant protoplasts in precisely sized spherical hydrogel beads, providing plant scientists with new ways of dissecting the biophysical background of plant development.\r