Evaluating methods for storability assessment and determination of vitality status of container grown Norway spruce transplants after frozen storage

ABSTRACT

Autumn sown small seedlings for later transplanting into large containers have been introduced in Swedish forest tree nurseries. Containerized transplants of Norway spruce (Picea abies (L.) Karst.) from three Swedish nurseries were frozen stored during the autumn of 2014 to find out storability and post-storage vitality. Seedling storability was determined by measuring electrolyte leakage after freezing shoots to ?25°C (SE L _diff?25), by measurements of dry matter content (DMC) of seedling shoots and by the commercial molecular test ColdNSure?. Vitality of seedlings after storage was determined by measuring the leakage of electrolytes from shoots (SEL), and seedlings were also tested in regrowth tests. All three methods for storability assessment gave similar predictions, except in one case where DMC showed “not storable” for successfully stored seedlings. Our results indicated that young transplants can be successfully short term stored before reaching the target levels for safe long-term storage of conventional seedlings. Early storage of young transplants resulted in low post-storage survival and vitality expressed as root growth capacity and shoot electrolyte leakage (SEL). A prolonged duration in storage generally resulted in lower survival as well as lower root growth capacity and higher levels of SEL, especially for seedlings stored at earlier dates.     

Storability measures of Norway spruce and Scots pine seedlings and assessment of post-storage vitality by measuring shoot electrolyte leakage

As indoor frozen storage is increasing in forest tree nurseries it is important to have accurate methods for assessing seedling storability in autumn and methods to determine post-storage vitality. Storability of spruce (Picea abies (L.) Karst.) and pine (Pinus sylvestris L.) seedlings can be based on determination of dry matter content (DMC) of seedling shoots or by freezing shoots at –25°C and thereafter measure electrolyte leakage (SEL_diff–25). To compare these two methods we stored 1-year-old spruce and pine seedlings at different occasions during the autumn. To test if leakage of electrolytes from shoots (SEL) could indicate deteriorated vitality, we measured SEL at the end of storage. After storage seedling viability was determined in a three-week growth test, measuring shoot and root growth capacity (RGC). Determination of freezing tolerance (SEL_diff–25) before storage had a better ability to predict the outcome of storage compared to the DMC test. Measuring SEL at the end of the frozen storage period accurately indicated seedling vitality. Seedlings with SEL of 0–5% had a high survival rate whereas SEL over 10% indicated low survival and growth capacity after storage. The SEL method has a potential to become a screening test for identifying batches of seedlings that have been damaged during storage in the nursery.     

Overwinter Storability of Conifer Planting Stock: Operational Testing of Fall Frost Hardiness

Operational stock-testing facilities that estimate overwinter storability of seedlings (ability to survive and grow after storage) need a reliable method that provides fast results to forest nurseries. We compared three methods using container-grown seedlings of Douglas-fir, interior spruce, lodgepole pine, and western larch from forest nurseries in British Columbia. On three to nine dates in autumn, frost hardiness at −18°C was estimated using visible injury of foliage or stems (VI), electrolyte leakage from needles or stems (EL), and chlorophyll fluorescence of shoots (CF). Seedlings were placed into overwinter cold storage (−2°C). In the spring, stored seedlings were planted in nursery beds; survival and growth were assessed after one growing season. There were close correlations (r ≥ 0.93) between the assessment methods. Seedlings lifted after they reached thresholds of 69% or higher for CF and 25% or lower for EL and VI had over 90% survival at harvest and doubled shoot dry weight compared with seedlings lifted earlier. Measuring CF was the fastest and most easily replicated method to estimate successful storability, and reduced testing time by 6 days relative to VI tests.     

Physiological Characteristics and Carbohydrate Contents of Spring-lifted Picea glauca Bareroot Seedlings Following Low-temperature Storage

This study examined the effects of low-temperature storage of white spruce [ Picea glauca (Moench) Voss] bareroot seedlings to determine whether the time between lifting and planting of spring-lifted seedlings could be extended. Seedlings were lifted from the nursery beds on May 4, 1994, and stored at- 2C (frozen storage) and 4C (cold storage) for 3, 5 and 7 weeks. Frozen storage of spring-lifted seedlings resulted in an increase in sugar levels in roots and needles that progressed with storage duration. Seedlings stored in cold storage maintained a relatively constant sugar content. Needle starch content decreased with storage duration in both frozen and cold storage. In the roots of cold-stored seedlings, starch content remained relatively constant during storage; however, in the frozen-stored seedlings, root starch levels sharply declined during the initial 3 weeks of storage. The levels of total non-structural carbohydrates (starch and sugars) decreased in both types of storage in needles, but not in roots. However, the decrease was more pronounced in the cold-stored than in the frozen-stored seedlings. Gas exchange, root growth potential and number of days to bud break were similar in frozen- and cold-stored seedlings planted in the greenhouse. However, following planting in the forest, cold-stored seedlings flushed buds earlier than did frozen-stored seedlings. The results indicate that tree nurseries could consider frozen storage of spring-lifted white spruce seedlings to facilitate lifting and planting schedules.     

Root preparation technique and storage affect results of seedling quality evaluation in Norway spruce

Evaluation of tree seedling quality is necessary for improving technology for forest nursery production and seedling handling. Nutrient status can be measured to determine seedling quality, but it can be affected by seedling handling techniques. In this study effects of root preparation technique and storage regime on content of macro nutrients (N, P, K, S, Ca, Mg) in fine roots of Norway spruce (Picea abies (L.) Karst.) plants were investigated. The root preparation techniques were: (a) rinse in tap or (b) deionised water, and (c) dry preparation. These techniques were tested on seedlings subjected to four storage regimes: (1) no storage, (2) deep freezing below −20°C, and long-term (3) cold (+1 to +3°C) and (4) frozen (−3 to −4°C) storage. From the results it was concluded that the nutrient status in needles is not sufficient to describe the whole plant nutrient status in stored dormant plants, fine roots should also be included. The results also showed that deep freezing of fine roots before nutrient analyses should be avoided. Losses of K, P, S, and Mg were substantial with this method. Deionised water or dry preparation is preferred since tap water contains substantial amounts of ions that may affect the analyses.     

Assessment of seedling storability of Quercus robur and Pinus sylvestris

Pedunculate oak (Quercus robur L.) and Scots pine (Pinus sylvestris L.) seedlings were lifted on several occasions during autumn 1997 to determine the relationships between storability and frost hardiness. On each lifting date their physiological status was determined by assessment of shoot and root electrolyte leakage and frost hardiness, assessed as freeze-induced electrolyte leakage. Additional seedlings were simultaneously cold-stored for field planting and assessment of preplanting root growth potential in April 1998. First year field performance was determined the following winter. Storability and cold acclimation patterns differed between the two species. Both were negatively affected by early lifting, but oak was less sensitive with respect to survival, and pine attained tolerance to cold storage more rapidly and earlier with respect to growth increment. The correlations between shoot frost hardiness and performance suggest that freeze-induced shoot electrolyte leakage (SEL_diff?20) below a threshold of 5% is a good storability predictor for Scots pine in Denmark. A completely reliable criterion for pedunculate oak could not be established.     

Cryopreservation of dormant orthodox seeds of forest trees: mazzard cherry (Prunus avium L.)

? The sensitivity of dormant seeds of mazzard cherry (Prunus avium L.) of Polish provenances to extreme desiccation and/or the ultra-low temperature of liquid nitrogen, LN (?196 °C), was investigated to identify the optimum seed water content (w.c.) at this temperature.

? Germination and seedling emergence tests in this study showed that there is no critical seed w.c. for deeply desiccated seeds (stone w.c. 1.6%), although such desiccated seeds were sensitive to the temperature of LN in the seedling emergence test. For seeds frozen in LN, the highest germinability was observed at w.c. of 9.0–16.9%, but seedling emergence was then significantly lower than in nonfrozen seeds. The 2-year of storage in LN of seeds desiccated to 7.8% w.c. did not decrease germinability after thawing, in comparison with 2-year of storage at ?3 °C. Storage in LN showed that if seeds were stored after breaking of their dormancy, germinability after storage was lower because of the necessity of seed desiccation to the lower level (～ 8%) of w.c. after stratification (before storage). Secondary dormancy was induced in seeds desiccated after stratification.

? The results of this study demonstrate the potential for long-term cryopreservation of mazzard cherry seeds in forest gene banks.

     

Influence of nutrient supply on spring frost hardiness and time of bud break in Norway spruce (Picea abies (L.) Karst.) seedlings

When spring frosts occur on recently planted forest sites, severe damage may occur to the seedlings. The aim of the present study was to test how different low levels of nutrient concentrations in Norway spruce (Picea abies (L.) Karst.) seedlings affected spring frost hardiness and time of bud break. Seedlings were grown in a greenhouse for one season and supplied with fertiliser containing 22, 43 and 72 mg N l^–1, respectively. The treatments resulted in needle nitrogen concentrations ranging from 0.9 to 1.8% in autumn. After winter storage at 0 °C, bud break was recorded on seedlings growing in the greenhouse, outdoors and in growth chambers at 12 °C and at 17 °C. Freezing tests were performed on seedlings directly removed from winter storage and following one week growth in the greenhouse. Seedlings receiving fertiliser with 43 mg N l^–1 had less freezing injury than the two other fertilisation treatments in the present study. The earliest bud break occurred in seedlings receiving 72 mg N l^–1.     

Cold hardiness and transplant response of Juglans nigra seedlings subjected to alternative storage regimes

? Effects of overwinter storage regimes on seedling cold hardiness and physiological vigor are relatively unexplored, particularly for temperate deciduous forest tree species.

? We evaluated influence of storage duration (0, 66, 119, or 175 d) on electrolyte leakage of stem and root collar tissues following exposure to a series of freeze-test temperatures in black walnut (Juglans nigra L.) seedlings sampled from cold (3 °C) or freezer (?2 °C) storage. Seedlings were subsequently transplanted into a controlled growth chamber environment for two months.

? Regardless of storage temperature, mean LT₅₀ was lowest for seedlings stored for 66 d (≤ ?34 °C) and increased dramatically after 119 d (≥ ?13 °C).

? Root collar tissue had lower LT₅₀ than stem tissue after 119 d for cold-stored seedlings, reflecting importance of evaluative tissue type. Days to bud break shortened with increasing storage duration up to 119 d and stabilized thereafter for both storage regimes. Root growth potential was maximized after 119 d of storage, and subsequently declined for cold-stored seedlings. Height growth increased following storage, regardless of duration.

? To promote stress resistance and transplant growth response, we recommend that black walnut seedlings from this genetic source be outplanted after approximately 66–119 d of storage.

     

Studies on Electrolyte Conductivity and Activity of Dehydrogenase of Chinese Fir and Masson Pine Bare—Root Seedling under Water and Cold Stress

The electrolyte conductivity and activity of dehydrogenase of bare-root seedlings of both Chinese fir (Cunninghamia lanceolata (Lamb.)Hook.)and Masson pine (Pinus massoniana Lamb.) under freezing and desiccation treatments were studied.The results showed that needle electrolyte conductivity of both speices increase significantly after freezing treatment and there are no significant differences in needle electrolyte conductivity between the two species.The dehydrogenase activity(ARD) of fine roots of both Chinese fir and Masson pine was negatively correlated with increasing freezing and desiccation.The results suggest that both electrolyte conductivity and dehydrogenase activity could be used as quick indicators of Chinese fir and Masson pine bare-root seedling quality.     

Effects of pulp freezing and frozen pulp storage on fibre characteristics

Summary A requirement of long-term research on pulp fibres in that the material for study be stored for prolonged periods without deterioration and without changes in properties. In this paper effects of pulp freezing and thawing and of frozen pulp storage on fibre, wet web, and handsheet properties are discussed. A variety of radiata pine kraft pulps, a radiata pine sodium bisulphite pulp, and silver beech and hard beech (Nothofagus species) kraft pulps are examined.The expanded walls and diameters of beaten fibres were contracted by pulp freezing. This behaviour made fibres less flexible and less able to collapse during papermaking operations. The freezing treatment also caused fibre kinks and other fibre configurations which existed in a pulp before freezing to be fixed into position and made somewhat resistant to straightening when in strained wet webs. It was found that extents of fibre kink can be varied depending on the degree to which fibre configurations are forced into a pulp network before freezing. Increasing periods of frozen storage caused the intensity and distribution of bonds redeveloped by the freezing treatment to be progressively modified. Fibre walls were, however, not contracted further by increasing periods of frozen storage.The technical assistance of Miss D. Brookes is gratefully acknowledged     

Influence of ectomycorrhizal fungal inoculation on growth and root IAA concentrations of transplanted conifers

We determined whether in vitro plant growth regulator production by mycorrhizal fungi is correlated with conifer seedling growth and root IAA concentrations. Container-grown seedlings of interior Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), lodgepole pine (Pinus contorta Dougl.) and ponderosa pine (Pinus ponderosa Dougl.) were inoculated at seeding with ectomycorrhizal fungi having a high, moderate or low capacity to produce either IAA or ethylene in vitro. Inoculated seedlings were grown for one season in the nursery, harvested in December, cold stored over winter and then transplanted to either a nursery field or a forest site in the spring. Seedling morphology and endogenous IAA in roots were measured immediately after cold storage and again six and 12 months after transplanting. Morphological responses to inoculation varied among different mycorrhizal fungi. Free IAA concentration of roots was increased in some inoculation treatments for all conifer species. In seedlings transplanted to a nursery field, in vitro ethylene-producing capacity of the ectomycorrhizal fungi was highly correlated with more morphological features than in vitro IAA-producing capacity. Both IAA- and ethylene-producing capacity were significantly correlated with more morphological features in seedlings transplanted to a forest site than in seedings transplanted to a nursery field. One year after transplanting, only in vitro IAA-producing capacity was correlated with endogenous IAA concentration of roots of the inoculated seedlings. We conclude that growth responses of conifer seedlings can be partially influenced by IAA and ethylene produced by ectomycorrhizal fungal symbionts.     

Short-day photoperiods affect expression of genes related to dormancy and freezing tolerance in Norway spruce seedlings

Key Message

Gene expression analysis showed that prolonged short day (SD) treatment deepened dormancy and stimulated development of freezing tolerance of Picea abies seedlings. Prolonged SD treatment also caused later appearance of visible buds in autumn, reduced risks for reflushing, and promoted earlier spring bud break.

Context

Short day (SD) treatment of seedlings is a common practice in boreal forest tree nurseries to regulate shoot growth and prepare the seedlings for autumn planting or frozen storage.

Aims

The aim of this study was to examine responses of Norway spruce (Picea abies (L.) Karst.) to a range of SD treatments of different length and evaluate gene expression related to dormancy induction and development of freezing tolerance.

Methods

The seedlings were SD treated for 11 h a day during 7, 14, 21, or 28 days. Molecular tests were performed, and the expression profiles of dormancy and freezing tolerance-related genes were analyzed as well as determination of shoot growth, bud set, bud size, reflushing, dry matter content, and timing of spring bud break.

Results

The 7-day SD treatment was as effective as longer SD treatments in terminating apical shoot growth. However, short (7 days) SD treatment resulted in later activation of dormancy-related genes and of genes related to freezing tolerance compared to the longer treatments which had an impact on seedling phenology.

Conclusion

Gene expression analysis indicated an effective stimulus of dormancy-related genes when the SD treatment is prolonged for at least 1–2 weeks after shoot elongation has terminated and that seedlings thereafter are exposed to ambient outdoor climate conditions.

     

Use of short-day treatment in the production of Norway spruce mini-plug seedlings under plant factory conditions

Mini-plug transplant seedlings of Norway spruce have been cultivated in closed growth systems, so-called plant factories, for few years. The aim of the experiment was to define a short-day treatment (SD) that harden seedlings to sustain 3 months of cold storage, but does not have adverse effects on growth, morphology, and vitality. The seedlings were subjected to one of the following treatments: (1) 12?h photoperiod + 3 weeks duration; (2) 8?h photoperiod + 3?wk duration; (3) 12?h photoperiod + 5?wk duration; and (4) 8?h photoperiod + 5?wk SD. All the SD treatments yielded healthy seedlings that grew well after the cold storage. The frost hardiness of the seedlings improved when the photoperiod was reduced from 12 to 8?h, and when the SD duration was increased from 3 weeks to 5 weeks, but reducing the photoperiod from 12?h to 8?h caused growth reductions. The root and shoot regrowth after cold storage was highest in seedlings that had received 12?h photoperiod and 5?wk duration. However, 12?h photoperiod and 3?wk duration may be an adequate practice for nurseries that treat multiple crops in their SD facilities.     

Effect of nursery storage and site preparation techniques on field performance of high-elevation Pinus contorta seedlings

After five years of growth at high-elevations (∼3000 m) in Utah, container lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) seedlings survived well (80–95%) and grew to similar heights regardless of nursery storage method and site preparation technique. Seedlings received one of three storage treatments: (1) spring-sown in the nursery, overwintered in cooler storage and outplanted in July; (2) spring-sown, overwintered in freezer storage, and outplanted in July; or (3) winter-sown, no storage, and hot-planted in late August. We outplanted seedlings at two locations that were clearcut and had received two treatments of surface organic matter (coarse wood, logging slash, and forest floor) removal: surface organic matter (OM) piled with a bulldozer and burned or surface OM remaining in situ. Compared to adjacent uncut stands, both site preparation treatments increased total soil bulk density, but retaining surface OM in situ maintained soil OM, carbon, and nitrogen levels. After one growing season, seedlings planted where surface OM had been bulldozed were taller and had more biomass, although survival was similar (≥96%) across site preparation treatments. The height growth advantage disappeared after five growing seasons and although overall survival was good, survival was highest where site preparation involved removal of surface OM and freezer-stored seedlings were planted. Total non-structural carbohydrates tended to be higher in roots than in shoots and were also higher in hot-planted seedlings than in stored seedlings. Our results indicate that nursery and forest managers have several options for successful nursery production and outplanting of container lodgepole pine seedlings in the central Rocky Mountains. Using hot-planted seedlings allows for a faster turnaround time (from seed to plantable seedling) and maintaining surface OM may be a cost-effective alternative to dozer piling and burning.     

楸树、灰楸花粉离体萌发特性及其保存方法

以灰楸、楸树花粉为试材,在固体培养基上离体培养,研究花粉萌发的最适温度、适温下的最佳培养时间及外源钙离子浓度对花粉萌发的影响。结果表明,30～35℃是灰楸花粉发芽的最适温度,25～35℃是楸树花粉发芽的最适温度;2种楸树花粉的最佳培养时间均为3h。外源钙离子能促进楸树、灰楸花粉的萌发,灰楸花粉以1.36～2.04 mmol／L CaCl2·2H2 O作用最为显著,楸树花粉以3.4 mmol／L CaCl2·2H2 O作用最为显著。贮藏条件对花粉的贮藏时间起决定作用。其中在常温常湿条件下,灰楸、楸树的花粉贮藏10天时完全失活;在－20℃、－80℃冷冻条件下灰楸、楸树花粉的活力分别可保持120天、90天以上。表明灰楸、楸树花粉适宜的贮藏条件依次为－20℃、－80℃冷冻。此研究可为楸树、灰楸生产上的花粉贮藏和人工授粉提供参考依据。     

Using morphological characteristics for assessing seedling vitality in small-scale tree nurseries in Kenya

Small-scale tree nurseries are important in fulfilling the goals of reforestation and agroforestry implementation schemes in Kenya and other developing countries. The focus in seedling production has been on quantity, instead of quality, but a change can be seen in recent tree nursery manuals. These manuals are emphasising morphological characteristics as tools for assessing potential field performance of seedlings. However, morphological criteria are debatable and their value is questioned. A survey was carried out among tree nursery operators in the Meru area, in the Eastern province of Kenya, to determine how operators perceived seedling vitality, and how they separated acceptable seedlings from those of poor vitality. Based on the survey, 3 pairs of criteria were chosen, size (tall versus small), colour (green versus yellowish), and sturdiness quotient (sturdy versus lanky). These criteria were tested on survival and growth in a field trial, a controlled bench trial, and in a root growth potential test. The results showed that the nursery operators were aware of quality differences in seedlings, but they did not cull accordingly. The results from the field trial showed that mango (Mangifera indica L.) performed poorly compared to grevillea (Grevillea robusta A. Cunn. ex. R. Br.), probably due to the high altitude. The altitudinal range for mango and grevillea are 0-1,200 m and 0-2,300 m, respectively, and the trial site was located on an altitude of 1,725 m. In grevillea, small seedlings grew better than tall in the field trial, but no differences could be found in the other trials. In mango, sturdy seedlings grew better than lanky ones in the field trial, while in the controlled trials tall seedlings grew better than small ones. The results showed that morphological characteristics as seedling quality assessment criteria could be unreliable as the effect differs with species and planting site.     

Survival of southern pine seedlings after inoculations with Pythium and cold storage in the presence of peat moss

Cold storing bareroot pine (Pinus spp.) seedlings grown in the southern U.S. for as little as 1 week in a cooler (just above freezing) in the fall (November to mid‐December) has been shown to reduce seedling survival after outplanting. In contrast, survival of container‐grown seedling is typically not affected when stored for 4 weeks in coolers in November and December. Wounds sustained by seedlings as they are lifted from nursery beds may allow Pythium spp. to infect bareroot seedling roots. Once in the cool, moist storage environment, Pythium multiplies and may result in seedling mortality after outplanting. Bareroot loblolly pine (Pinus taeda) and container‐grown loblolly, longleaf (Pinus palustris), slash (Pinus elliottii) and shortleaf pine (Pinus echinata) seedlings were inoculated with either Pythium dimorphum or Pythium irregulare, cold stored with or without peat moss and monitored for survival after outplanting. Peat moss did not increase bareroot loblolly pine survival or reduce Pythium populations when seedlings were inoculated with Pythium prior to storage. Pythium irregulare reduced survival of longleaf and shortleaf pine grown in peat moss and perlite, respectively. Pythium did not affect loblolly or slash pine, but wounding their roots did reduce seedling survival when grown in containers.     

寒害天气对永州市赤桉组培苗造林的影响

2004年冬季永州市经历了严重的寒害天气。灾害过后,调查了东安县种植的赤桉组培苗受冻和冻后恢复情况,分析比较了赤桉组培苗种植后的耐寒性和冻后恢复能力。调查结果表明：2004年冬季是永州市罕见的寒害天气,所有引种的桉树幼林均受到不同程度的冻害,但赤桉组培苗幼林受冻程度较轻,冻后恢复较快,类似2004年冬季的寒害天气,对永州市赤桉组培苗造林没有影响。根据永州南桉北移造林的现状,提出了发展桉树产业的建议。     

Effects of Previous High N Addition on Nutrient Conditions in Above-ground Biomass of a Picea abies Stand in Sweden

The effect of different thawing procedures on seedling quality in Norway spruce (Picea abies) was evaluated. Freezer-stored seedlings were thawed rapidly for 20 h by immersion in water with an initial water temperature of 8°C or thawed more slowly by gradually increasing the temperature in storage over a period of 8 weeks. Seedlings from these treatments were also compared with seedlings thawed rapidly overnight at 15°C. Frost tolerance, carbohydrate content and the time of bud break were used as indicators of the vitality of the seedlings. Seedlings thawed rapidly in water or air showed significantly better frost tolerance after storage than slowly thawed seedlings. The contents of sucrose and raffinose in seedlings thawed rapidly were also significantly higher than in seedlings thawed more slowly. Bud break occurred later in seedlings thawed rapidly than in slowly thawed seedlings. Rapid thawing in water may be a good method to ensure the delivery of high-quality thawed seedlings throughout the planting season.