4 Seedling Quality Testing at the Gene Level By Thomas D. Landis and Monique F. van Wordragen Nursery managers are all too familiar with the critical importance of determining the proper lifting window for nursery stock. Plants that are harvested too early are damaged during the lift-and-pack process and also store poorly. Currently, the best physiological test for determining the lifting window is to measure cold hardiness by the whole plant freezing or electrolyte leakage tests. For example, conifer nurseries in British Columbia use 0 o F (-18 o C) as the hardiness level when it is safe to begin harvesting. While these cold hardiness tests are useful, they typically take one to several weeks to produce results and a series of tests must be done during the fall to track cold hardiness development. Wouldn’t it be great if there were a quick and accurate test to determine exactly when the cold hardiness process started? Genomic Testing— Genomics, or gene-expression analysis, is a relatively new discipline that allows us to look inside plant tissues at the chemical signals that trigger specific physiological events such as the development of cold hardiness (Figure 1). In living organisms, each developmental step and every interaction with the environment is orchestrated by DNA encoded genes. Therefore, the physiological condition of a plant can be determined by analyzing the activity profile of its genes. Sounds great, but the trick is to identify which gene or genes are involved in the cold hardiness process. Gene expression analysis uses microarrays or biochips to simultaneously examine thousands of genes from a sample of plant tissue and determine their level of activity. In this way, plant response to environmental cues can be closely examined and this information used to identify the genes that are involved in hardening. Once these indicator genes have been identified, then a chemical assay can be developed to measure their activity. Changes in the expression of specific genes are thus an accurate and early indicator for the development of cold tolerance. And, because it can identify the start of the hardening process, genomic testing is much more useful that traditional cold hardiness tests that only provide information several weeks after hardiness has already developed (Figure 1). The Research— I know that this sounds like Star Wars technology but researchers in Europe have already identified the genes involved in the cold hardiness of Scots pine (Pinus sylvestris) and European beech (Fagus sylvatica) seedlings. The study was performed in 4 countries (Denmark, the Netherlands, Scotland, and Sweden) and involved both research institutes and operational forest nurseries. The main objective was to monitor shoot cold tolerance and bud dormancy of pine and beech seedlings before, during, and after refrigerated storage with the shoot electrolyte leakage (SEL) test. Because pine and beech represent broadleaved and gymnosperm trees, they differ in the morphological and perhaps physiological development of cold hardiness. These cold hardiness test results were correlated with gene expression using genomics technology, which led to the development of a rapid, predictive molecular diagnostic test. Seedlings were grown in climate-controlled environments for the initial identification of the relevant hardiness genes, followed by outdoor nursery trials to monitor the actual development of cold hardiness. A standard provenance of each species was tested at each research location along with seedlings from a local seed source. This testing procedure allowed comparisons of most parameters that are known to influence dormancy and cold hardiness such as geographic origin, genetic background, and nursery cultural history Dehydrins are one of several proteins that were already known to be specifically associated with the onset of cold hardiness in red-osier dogwood, rhododendron, and blueberry. The European research trials identified the specific dehydrins and other proteins that are linked to cold hardiness in Scots pine and European beech seedlings. Once the specific genes were identified, the researchers used genomics technology to identify when they were activated. These genetic response data were analyzed with sophisticated statistical techniques, which revealed 3 different gene groups that were correlated to the cold hardening process. In samples from different provenances, genes from each group displayed a characteristic gene expression profile during the acquisition of frost hardiness. Figure 1 – Genomics tests of physiological and morphological processes such as cold hardening will give nursery managers an early warning, compared to traditional seedling quality testing.