Development of a molecular test to determine the vitality status of Norway spruce (Picea abies) seedlings during frozen storage

New Forests, Sep 2012

In boreal forest regions, a great portion of forest tree seedlings are stored indoors in late autumn to prevent seedlings from outdoor winter damage. For seedlings to be able to survive in storage it is crucial that they store well and can cope with the dark and cold storage environment. The aim of this study was to search for genes that can determine the vitality status of Norway spruce (Picea abies (L.) Karst.) seedlings during frozen storage. Furthermore, the sensitivity of the ColdNSure™ test, a gene activity test that predicts storability was assessed. The storability of seedlings was tested biweekly by evaluating damage with the gene activity test and the electrolyte leakage test after freezing seedlings to −25 °C (the SELdiff-25 method). In parallel, seedlings were frozen stored at −3 °C. According to both methods, seedlings were considered storable from week 41. This also corresponded to the post storage results determined at the end of the storage period. In order to identify vitality indicators, Next Generation Sequencing (NGS) was performed on bud samples collected during storage. Comparing physiological post storage data to gene analysis data revealed numerous vitality related genes. To validate the results, a second trial was performed. In this trial, gene activity was better in predicting seedling storability than the conventional freezing test; this indicates a high sensitivity level of this molecular assay. For multiple indicators a clear switch between damaged and vital seedlings was observed. A collection of indicators will be used in the future development of a commercial vitality test.

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Development of a molecular test to determine the vitality status of Norway spruce (Picea abies) seedlings during frozen storage

Eva Stattin 0 1 Nathalie Verhoef 0 1 Peter Balk 0 1 Monique van Wordragen 0 1 Anders Lindstro m 0 1 0 N. Verhoef P. Balk M. van Wordragen NSure, Binnenhaven 5, 6700 AA Wageningen, The Netherlands 1 E. Stattin (&) A. Lindstrom Dalarna University , 791 88 Falun, Sweden In boreal forest regions, a great portion of forest tree seedlings are stored indoors in late autumn to prevent seedlings from outdoor winter damage. For seedlings to be able to survive in storage it is crucial that they store well and can cope with the dark and cold storage environment. The aim of this study was to search for genes that can determine the vitality status of Norway spruce (Picea abies (L.) Karst.) seedlings during frozen storage. Furthermore, the sensitivity of the ColdNSureTM test, a gene activity test that predicts storability was assessed. The storability of seedlings was tested biweekly by evaluating damage with the gene activity test and the electrolyte leakage test after freezing seedlings to -25 C (the SELdiff-25 method). In parallel, seedlings were frozen stored at -3 C. According to both methods, seedlings were considered storable from week 41. This also corresponded to the post storage results determined at the end of the storage period. In order to identify vitality indicators, Next Generation Sequencing (NGS) was performed on bud samples collected during storage. Comparing physiological post storage data to gene analysis data revealed numerous vitality related genes. To validate the results, a second trial was performed. In this trial, gene activity was better in predicting seedling storability than the conventional freezing test; this indicates a high sensitivity level of this molecular assay. For multiple indicators a clear switch between damaged and vital seedlings was observed. A collection of indicators will be used in the future development of a commercial vitality test. - The commercially managed forests require a supply of billions of seedlings annually. Historically, many areas have also been left inadequately reforested after cutting and these areas need restoration and therefore are also in the need of a supply of vital seedlings. The coniferous planting stock for reforestation or restoration, annually more than 2 billion seedlings in Northern Europe, USA, and Canada alone, is mainly produced by large scaled forest tree nurseries. This requires a tight scheduling of operations to be able to deliver vital seedlings to the planting site. In late autumn, a great portion of cultured seedlings in Scandinavia are transferred to frozen storage to prevent seedlings from damage caused by low and fluctuating winter temperatures, fungae, or feeding by rodents. In many northern regions there is a substantial risk that roots of container seedlings will be exposed to detrimental low temperatures due to lack of insulating snow cover. Root damage is probably one of the main reasons for deteriorated vitality of outdoor stored container seedlings (Lindstro m 1986). Besides protection, another advantage with the artificial storage environment is that the resting seedlings become available for planting for an extended period. To survive storage it is crucial that seedlings are in such a state that they can cope with the dark and cold environment (reviewed by Colombo et al. 2001). Seedling storability can be assessed by measurement of dry matter content or water content (Rosvall-A hnebrink 1985; Calme et al. 1993), bud dormancy status (Holby et al. 1981), tolerance to freezing by determining electrolyte leakage (Lindstro m and Hakansson 1996; Colombo 1997; Bro nnum 2005), chlorophyll fluorescence and visible damage (Perks et al. 2004; LHirondelle et al. 2006) or by measuring gene activity of genes involved in frost hardiness development (Joosen et al. 2006; Balk et al. 2007; Balk et al. 2008). Nevertheless, seedling vitality at the time of delivery may be low even though seedlings were considered storable at the time of storage. Because most storability measurement methods are usually performed on above ground tissues, pre-storage damages to seedling roots may not be recognized. Lindstro m and Stattin (1994) showed deteriorated vitality of Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.), particularly in frozen storage, when roots had been exposed to freezing before storage. Also, inappropriate storage conditions can negatively affect the vitality in different ways (Aldhous 1964; Webb and von Althen 1980; Ritchie 1982) and fungae infections is often a major problem (Landis 1989; Capieau 2004). Therefore, methods are needed to estimate seedling vitality during frozen storage. To establish a reliable general measure of seedling quality before seedlings are delivered to the customer is desirable, but hard to obtain. Besides morphological parameters, the vitality of the seedling determines the field performance of the seedling. Numerous morphological (e.g. height, weight, root/shoot ratios) and phys (...truncated)


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Eva Stattin, Nathalie Verhoef, Peter Balk, Monique van Wordragen, Anders Lindström. Development of a molecular test to determine the vitality status of Norway spruce (Picea abies) seedlings during frozen storage, New Forests, 2012, pp. 665-678, Volume 43, Issue 5-6, DOI: 10.1007/s11056-012-9320-1