X-ray phase contrast imaging of Vitis spp. buds shows freezing pattern and correlation between volume and cold hardiness

Grapevine (Vitis spp.) buds must survive winter temperatures in order to resume growth when suitable conditions return in spring. They do so by developing cold hardiness through deep supercooling, but the mechanistic process of supercooling in buds remains largely unknown. Here we use synchrotron X-ray phase contrast imaging to study cold hardiness-related characteristics of V. amurensis, V. riparia, and V. vinifera buds: time-resolved 2D imaging was used to visualize freezing; and microtomography was used to evaluate morphological changes during deacclimation. Bud cold hardiness was determined (low temperature exotherms; LTEs) using needle thermocouples during 2D imaging as buds were cooled with a N-2 gas cryostream. Resolution in 2D imaging did not allow for ice crystal identification, but freezing was assessed by movement of tissues coinciding with LTE values. Freezing was observed to propagate from the center of the bud toward the outer bud scales. The freezing events observed lasted several minutes. Additionally, loss of supercooling ability appears to be correlated with increases in bud tissue volume during the process of deacclimation, but major increases in volume occur after most of the supercooling ability is lost, suggesting growth resumption processes are limited by deacclimation state.