Improved experimental protocols to evaluate cold tolerance thresholds in Miscanthus and switchgrass rhizomes

Abstract

AbstractThis study investigates protocols to evaluate cold tolerance thresholds for overwintering rhizomes of perennial bioenergy grasses. Protocols examined include the temperature at which ice formation occurs, cooling rate, incubation time at the treatment temperature, and the electrolyte leakage (EL) method to assess mortality thresholds. Using these protocols, we assessed low temperature injury in two genotypes of Miscanthus and two genotypes of lowland switchgrass (Panicum virgatum). Ice formed near −1 C in the rhizomes cooled at 1 C h−1, but at variable temperatures at cooling rates of 3 and 5 C h−1. Rhizome temperature followed chamber temperature at a cooling rate of 1 C h−1, whereas at faster cooling rates, there was a lag in rhizome temperature that affected treatment exposure time. A 1 C h−1 cooling rate is thus suitable. In rhizomes incubated for <4 h at the treatment temperature, EL values were variable, while there was no change in EL when samples were incubated 4–20 h. A continuous, steady rate of cooling at 1 C h−1 demonstrated the Miscanthus and lowland switchgrass varieties exhibited lethal levels of electrolyte leakage below −6 C. Continuous cooling does not allow for subzero acclimation and reflects thermal tolerances of sampled tissue in situ. To allow for maximum acclimation at subzero temperatures, a prolonged, staged‐cooling procedure was adopted. This procedure showed diploid Miscanthus rhizomes could acclimate and adjust their tolerance limit to −12 C, while a triploid Illinois line showed little acclimation and was still killed below −6 C.