A Freezing Protocol for Diversity Assessments in St. Augustinegrass: Freezing, Acclimation, and Evaluation Methods.

While field evaluations provide the best assessment of winter survivability, reliable lab-based freezing methods offer breeders an expedited, economical alternative for cold tolerance selection in St. Augustinegrass (Stenotaphrum secundatum [Walt.] Kuntze). Therefore, the development of a freezing protocol that illustrates the range of the freezing tolerances found in St. Augustinegrass would be advantageous for breeders. The objectives of this study were to i) identify a freezing temperature suitable to quantify differences among nine St. Augustinegrass genotypes varying in their response to freezing, ii) assess the differences between non-acclimated (NCA) and cold-acclimated (CA) plants, and iii) identify a reliable way to evaluate and document the survivability and recovery of St. Augustinegrass plants post-freeze. Nine genotypes were evaluated at different cold-acclimation and freezing temperatures for effects of freezing injury and recovery. The experimental design was a 9x4x2 factorial (9 genotypes x 4 freezing temperatures x 2 acclimation treatments) with 2 runs/temperature and three replications/run. Digital imaging and visual ratings were used to evaluate recovery of genotypes for six weeks after freezing. Temperature had a significant impact on freezing injury and recovery; both -3°C and -4°C showed large response variances between cultivars in comparison to -5°C and -6°C. Recovery varied between cultivars in response to acclimation treatments and was higher for CA plants, except for cultivars ‘Floratam’ and ‘Sapphire’. Evaluation of freezing injury and recovery responses over time indicated that data collection at 2 weeks post-freeze provided adequate separation of cold-tolerant vs cold-susceptible genotypes, while collecting data up to 6 weeks post-freeze only increases genotype separation. Pearson correlation coefficients indicated that digital imaging and visual ratings were highly correlated at 2, 4, and 6 weeks. Based on these results, the methodology used in this study appears to be adequate and reliable to test genotypes for freezing tolerance and is currently being validated using a mapping population developed specifically for cold tolerance evaluation.