A limiting factor for native species restoration at high elevation sites in Hawai‘i is exposure of planted seedlings to winter frost. Frost damage reduces survival (Scowcroft et al. 1999, 2000) until seedlings grow tall enough to escape the frost zone concentrated at the soil surface. Artificial frost protection devices, consisting of a single layer of vertically oriented shade cloth placed on the east side of seedlings, may reduce frost damage associated with less radiative cooling (Scowcroft et al. 1999, 2000). But these devices are expensive and time-consuming to install. Selecting genetic sources of Acacia koa (koa) that exhibit high cold tolerance during the establishment phase of regeneration may help to improve the efficiency of restoration. Previous research in Hawai‘i (Krauss 2014) did not detect differences among koa families in frost tolerance, but used 9-year-old trees (rather than seedlings) and included a limited number of populations. Significant seasonal variation was observed in frost tolerance, however, indicating that koa does adapt according to phenological cues.
The objective of this project is to determine 1) the degree to which koa develops cold hardiness as a mechanism to tolerate frost, 2) whether populations of koa from higher elevations show greater cold tolerance than koa populations from lower elevations, and 3) the extent to which exposure to environmental conditions to induce hardening may affect cold tolerance.
In November 2018, 4-month-old seedlings (grown in a greenhouse at Purdue University) from 13 Acacia koa populations along an elevational gradient (603-2050 m) on the island of Hawai‘i were divided into two groups that were either cold acclimated (i.e., exposed to reduced photoperiod and lower temperature) in growth chambers or remained in the greenhouse. Electrolyte leakage, which quantifies the amount of tissue damage as a measure of the proportion of cell solutes lost due to freezing damage at a range of temperatures (Hasse 2011), was used to assess frost tolerance. Five test temperatures, consisting of one control temperature + four below-freezing temperatures (˗5, ˗10, ˗15 and ˗ 20°C), were selected based on the expected hardiness of the samples. Upon reaching each freeze test temperature, samples of leaflets were maintained in the freezer for 60 minutes, removed and placed in a refrigerator for thawing for 24 hours, and then placed at room temperature for 18 hours for complete thawing. Initial electroconductivity was then measured and the samples were placed in a refrigerator at ~4°C overnight. The following day, samples were autoclaved (which achieves 100% electrolyte leakage) in samples at 120°C for 20 minutes and then allowed to cool at room temperature. Once the vials were cooled, electroconductivity was measured again to determine total electrolytes. Electrolyte leakage of samples from each test temperature was expressed as a percentage of total electrolytes. This test provides a measurement of the LT50, which is the interpolated temperature at which 50% of the tissue is killed (Burr, 1990). Statistical analyses will be carried out to determine whether seedlings of populations from higher elevations show higher cold tolerance than populations from lower elevations, and how this may interact with cold acclimation.
We will repeat this study with a whole plant freeze test, which corresponds more closely than electrolyte leakage to field conditions. Additionally, we will use RNA sequence data to help develop probes for characterizing the expression of genes related to cold tolerance and the development of cold hardiness. Differences in gene expression may then be related to differences in genome sequence, so that the genetic basis for observed physiological differences can be determined. A comprehensive characterization of the allelic differences among koa populations at genes of interest will enable rapid assessment of the cold tolerance of any population and, possibly, the identification of rare genes that can be used to improve the survival of koa at high altitudes where it formerly thrived.