LT50

Evaluation location: Minnesota, United States

Seedlings, derived from wild collected seed which had germinated in Febraury 2018, were tested for cold hardiness in January 2019, with a controlled freezing test. These methods were based on those described previously for controlled hardiness screening (McNamara et al., 2002; McNamara and Hokanson, 2010). The container-grown seedlings were allowed to acclimate under ambient conditions in Chanhassen, MN until mid-November, when they were moved into a minimally heated greenhouse structure maintained at a minimum temperature of -9.5°C to avoid cold damage before sampling. The seedlings were sampled on four dates over the course of 2 weeks (6, 10, 13, and 17 Jan.) in a completely randomized experimental design. Each sample operation consisted of harvesting and preparing stem samples on the first day and freezing the stems the following day. On the first day of each sampling date, stems from seedlings of each population to be tested that day were taken to the laboratory and cut into 3.5-cm sections, color-coded, and then randomly assigned to one of six temperature treatments or a nonfrozen control. Stem sections from each population at each temperature treatment were placed into bags with moist paper towels (three replicate bags per temperature per sample date). The bags were placed in a ScienTemp freezer (ScienTemp Corporation, Adrian, MI) controlled by a Watlow series 942 temperature controller (Watlow, St. Louis, MO). Each bag contained stem sections from all populations, with a total of 8 to 12 stem samples per population per test temperature. A thermocouple was inserted into the pith of at least one stem section per temperature treatment to monitor the stem temperature throughout the experiment. Temperatures were slowly decreased to the first test temperature (-10°C) overnight to allow stem temperatures to equilibrate. The following day, temperatures were decreased at a rate of 3°C per hour, and three replicate bags for each treatment were removed from the freezer at increments of -5 to -35°C. Samples were removed from the freezer when each test temperature was reached. Stem sections were slowly thawed at 3°C overnight and incubated at room temperature for 1 week to allow damage symptoms to appear. Stem sections were evaluated for cold damage by slicing the stem longitudinally with a scalpel and rating stems as live or dead by observing the damage (oxidative browning of vascular tissue) under a dissecting microscope (12× magnification). Most stems were unambiguously dead or alive; however, some stems were intermediate. In that case, stems were classified according to having more than or less than 50% damage. Cold hardiness was determined as the mean lethal temperature (LT₅₀) and calculated for each population using a binomial logit model to interpolate the temperature at which 50% of the stems would have died (Suojala and Lindén, 1997). The binomial logit model was implemented in the R packages MASS (v7.3–50) and stats (v3.5.1).The experiment was repeated during late Fall 2019 through early Spring 2020, with a subset of populations that were tested in January 2019 plus a younger cohort of seedling populations (germinated in February 2019) as well as a selection of cultivars. During this experiment, plants were tested once per month from October to April to characterize the timing of fall acclimation and spring deacclimation in addition to midwinter hardiness using the methods described. The tested temperatures (and interval between test temperatures) were varied each month to exceed the expected range of LT₅₀ values. During each month, the sample from a population or cultivar comprised one random stem collected from multiple individual plants; therefore, each plant was resampled every month. Cultivars were tested with four to six stem samples per cultivar per temperature per month while the populations were tested with the same sample sizes as those during January 2019. See citations for additional details.