Thermal History Between Field Collection and Laboratory Analysis Critically Impacts Cold Hardiness Assessment in Grapevine Bud and Phloem Tissues During Mid-Winter Dormancy

Abstract Although considerable progress has been made in understanding cold hardiness mechanisms in grapevine, the extent to which post-sampling handling conditions influence the reliability of freezing tolerance measurements has received comparatively little attention. In this study, we compared bud and phloem tissue survival, water content, electrical conductivity, and carbohydrate status in three grapevine cultivars (‘Narince’, ‘Alphonse Lavallée’, and ‘Trakya İlkeren’) subjected to controlled freezing tests (-16 to -24°C) following two handling protocols: immediate controlled cold storage at + 4°C for one week, and direct field handling without controlled storage. Viability was assessed in primary, secondary, and tertiary buds as well as phloem tissue by cross-sectioning and electrolyte leakage. Both handling protocol and freezing temperature significantly affected all viability parameters. Controlled-storage samples consistently outperformed field-handled samples, with approximately 60% versus 40% survival across all tissue types. Critical damage thresholds occurred between − 20 and − 22°C, and mortality peaked at -24°C regardless of protocol. Field-handled samples retained higher tissue moisture and showed elevated electrical conductivity, suggesting that warming-cooling cycles during transport triggered partial deacclimation and altered the physiological baseline prior to testing. Carbohydrate analyses revealed cultivar-specific patterns; Narince accumulated the highest total carbohydrates and exhibited superior cold tolerance across tissue types. These findings demonstrate that thermal history between field collection and laboratory analysis introduces substantial and systematic bias into cold hardiness measurements. Immediate temperature stabilization after sampling is therefore essential for obtaining accurate and reproducible freezing tolerance data. The consistent magnitude of this bias across cultivars, tissue types, and years underscores the need for standardized sample handling protocols in both cold hardiness research and practical frost risk assessment for vineyard management.