Imaging sweetness: Rapid permeation of sucrose into rice callus cells as a component of cryoprotectant solutions

Sucrose was first included in plant vitrification solutions (PVS) in 1989 and has since remained a primary component of most formulations. Although sucrose is known to stabilize cell membranes, prevent protein denaturation, and promote cellular dehydration, its specific role in successful plant cryopreservation remains unclear. With the growing need to preserve plant germplasm, less toxic yet effective PVS are being developed. However, empirical knowledge on the permeation of sucrose is insufficient for the optimization of PVS and cryopreservation methods. The study reported here evaluates sucrose permeation into rice callus cells for each step of a model cryopreservation cycle. By deuterating sucrose, its C-D bond can be tracked using coherent anti-Stokes Raman scattering microscopy, a highly sensitive, non-invasive vibrational microscopy technique. Results show that sucrose permeates cells within 6 s of initial exposure and takes only 20-30 s to reach steady-state concentration. Sucrose is a permeating cryoprotecting agent regardless of pre-existing intracellular sucrose from preculture and loading steps. Beyond the permeation of sucrose as a CPA component, the results presented here suggest that the method we used could measure permeation by other molecules interacting with live plant material, such as the permeation of herbicides, showing kinetics and sequestration into plant organelles.