Frost hardening in first-year eastern larch (Larix laricina) container seedlings

Eastern larch (Larix laricina [du Roi] K. Koch) container seedlings were tested to determine shoot frost hardiness development under short or long days and warm (15 to 25 °C) or cool (10/5 °C, day/night) temperatures, to aid in the development of greenhouse hardening strategies. Seedlings were sampled sequentially over time (25 seedlings per week) from a population of 1000 trees. Frost hardiness increased significantly after one week of fluctuated over the next 6 weeks, and increased thereafter through week 14. Seven weeks of warm, intermittent short days, followed by 6 weeks of cool, continuous short days, resulted in greater frost hardiness than 13 weeks of warm, intermittent short days. In contrast, seedlings exposed to 7 weeks of warm, intermittent short days, followed by six weeks of warm, long days were significantly less frost hardy. Stems with needles attached had lower Index of Injury than stems without needles.     

Frost hardiness gradients in shoots and roots of picea mariana seedlings

Frost hardiness of tissues along the length of the stem and the root was investigated in first‐year black spruce (Picea mariana (Mill.) B.S.P.) seedlings. Frost hardiness of 1 cm long stem and root segments was evaluated based on Index of Injury, calculated from post‐freezing electrolyte leakage. Frost hardiness was tested approximately weekly beginning seven weeks after seedlings were transferred from an 18 to a 10 h photoperiod, both at day/night temperatures of 26°C/16°C. Trees were transferred to temperatures of 10°C day and 5°C night at a 10 h photoperiod after a further 18 days. Frost hardiness was greater at the terminal bud and least at the root tips. Although shoots were generally more frost hardy than roots, differences in hardiness along the stem and root axes were gradual, rather than abruptly differing at the shoot‐root interface. All tissues, including root tips, increased in frost hardiness after conditioning for 18 days under short photoperiods (10 h) and warm temperatures (26?C/16°C, day/night). Under cold temperatures (10°C/5°C, day/night) all tissues, excepting the root tips, tolerated — 16°C with little subsequent electrolyte leakage.     

The effect of photosynthetic photon flux density on development of frost hardiness in top and roots of Larix leptolepis seedlings

Seedlings of Larix leptolepis were grown in a growth chamber under short day conditions (8 h day) at 4 different photosynthetic photon flux densities (PPFD's). After a period of 8 weeks frost hardiness of the shoot tips and roots, dry matter content, dry weight (dw), content of glucose and starch were determined. The frost hardiness in the shoot tips increased from ‐15°C at a PPFD of 55μmol m^‐2s^‐1 to about ‐35°C at 440 μmol m^‐2s^‐1. No effect of PPFD was found on frost hardiness of the roots. A high PPFD results in a high dry matter content. The effect on dry matter content was most pronounced for the shoot tips and less pronounced for the roots. The total dry weight increased for both root and top with increasing PPFD. The height of the plants increased when the PPFD increased up to 220 μmol m^‐2s^‐1.     

Frost hardening of Pinus radiata seedlings: effects of temperature on relative growth rate,carbon balance and carbohydrate concentration

Pinus radiata (D. Don) seedlings were grown for 100 days at day/night temperatures of 10/1, 15/1, 20/1 and 25/1 degrees C, to determine whether temperatures above a threshold of 5 degrees C influence frost hardiness development. Relationships between hardening and relative growth rate, carbohydrate concentration and net carbon balance were also investigated. Seedlings hardened at a nearly constant rate in each treatment, although the rate of hardening was strongly temperature dependent. It increased as the temperature declined, but in a curvilinear fashion. Temperatures below 9.5 degrees C were effective in hardening the seedlings. During the daily temperature cycle, dehardening occurred at temperatures above the threshold, whereas hardening occurred at temperatures below the threshold. The net difference between the two processes determined the development of frost hardiness. The development of frost hardiness was negatively correlated with relative growth rate and positively correlated with the accumulation of starch and sugars. We conclude that frost hardening is a complex process that is causally linked to carbohydrate concentrations.     

Temperature control of the development of frost hardiness in two populations of Leptospermum scoparium

Seedlings of Leptospermum scoparium J.R. et G. Forst (manuka) originating from seed from a low altitude coastal site (Auckland) and from a high altitude inland site (Desert Road) were grown for 96 days in four controlled environments to compare the relationship between growth temperature and frost hardening. Day/night temperature treatments were 12/6, 12/3, 12/0 and 12/-3 degrees C. Frost hardiness was determined at 14-day intervals by exposing whole seedlings to temperatures ranging from -2 to -8 degrees C. Frost damage differed significantly between the two populations: Desert Road seedlings were less affected than Auckland seedlings. At all growth temperatures, the time courses of frost hardiness of both populations followed curvilinear relationships reaching a maximum hardiness at about Day 50, after which the seedlings spontaneously dehardened. The rate of frost hardening increased linearly with decreasing temperature from 6 to 0 degrees C, but thereafter, no further increase occurred with decreasing temperature to -3 degrees C. The frost hardening process was more sensitive to temperature in the Desert Road seedlings than in the Auckland seedlings, and this difference may account for the intraspecific variation in frost hardening capacity of this species. Comparisons with Pinus radiata D. Don and Lolium perenne L. indicated that interspecific variation in frost hardening capacity can also be accounted for by differences in the sensitivity of the hardening process to temperature.     

Morphological and physiological differences in Scots pine seedlings of six seed origins

One-year-old seedlings of Scots pine (Pinus sylvestris L.) offour native seed origins (Loch Maree Islands, Glengarry/GlenMorriston, Glen Affric and Abernethy), a commercial Britishseedlot, and a seedlot from Hedesunda, in middle Sweden, werecompared at monthly intervals from October 1993 to April 1994.Seedling morphology, root condition, root frost hardiness andbud dry matter were determined at each date. There were clear morphological differences among seed origins.Seedlings raised from the commercial seedlot (A70) were largerbut had a poorer root:shoot ratio than the other seed origins.Of the native pines tested, the Loch Maree Islands origin allocateda larger proportion of its photosynthate to fine roots and needlesand smaller proportion to woody structures. Seedlings raised from the commercial British seedlot tendedto have poorer bud lignification than the other origins andalso, in autumn, higher electrolyte leakage rates from its fineroots. During winter, the Swedish origin had the lowest fineroot electrolyte leakage. Seedlings of all origins showed aprogressive increase in fine root hardiness towards mid-winterwith maximum hardiness (–7°C) in January. Dehardeningoccurred over subsequent months reaching –3°C in April.Differences among origins were evident. The Swedish seedlotdeveloped greater frost resistance than the other origins, hardeningbegan earlier in autumn and dehardening began later in spring.The commercial seedlot hardened later than the other originsbut reached a similar level of frost hardiness by January. Ofthe native pines, seedlings of the Loch Maree Islands originwere slowest to develop root hardiness.     

Frost hardening spruce container stock for overwintering in Ontario

Difficulties overwintering container stock in northern Ontario led to the development of the extended greenhouse culture hardening regime for spruce seedlings. Laboratories to measure shoot frost hardiness and evaluate terminal bud development were established to monitor nursery crops being hardened using this regime. Information on frost hardiness and bud development provided by these laboratories has been used by nursery managers to determine readiness of container seedlings for overwintering. Since 1982, over 200 stock lots have been monitored by these operational laboratories. This database can be used to determine the importance of nursery cultural factors and seed source on frost hardening. The database shows large differences between nurseries in approach to hardening seedlings which were reflected in levels of freezing damage, winter desiccation and overwintering success. Rates of frost hardening (i.e., the interval between terminal bud initiation and attainment of a --15°C level of shoot frost hardiness) of crops produced in north central Ontario failed to show significant seed source effects. The rate of frost hardening was faster in crops producing fewer needle primordia in terminal buds.     

Seasonal Changes in the Frost Hardiness of Provenances of Picea sitchensis in Scotland

Changes in the natural level of frost hardiness of shoots offour provenances of Picea sitchensis were monitored over twogrowing seasons by detaching shoots from 7 to 10-year-old treesgrowing in a nursery in Scotland, and subjecting them to freezingtemperatures under conditions which simulated night frosts. Six seasonal phases of frost hardiness were identified (Fig.3).

1. During each autumn, killing temperatures (the level of hardiness)decreased from –5°C to below –20°C, beginningseveral weeks after shoot elongation ceased. Alaskan provenanceshardened in September, apparently in response to shorteningday lengths alone, whereas an Oregon provenance did not hardenuntil November, after repeated frosts. Queen Charlotte Islandsprovenances were intermediate.
2. From November to March allprovenances were hardy to below –20°C,which is adequateto prevent direct freezing injury at mostplantation sites.
3. In March-April, several weeks before bud-burst, old shootsdehardenedto killing temperatures of about –10°Cin responseto warm temperatures, and southerly provenancesdid so beforenortherly ones.
4. During bud-burst the newly-emergingshoots were hardy to only–3°C to –5°C untilthey were about 3.5 cmlong. All provenances burst bud at thesame time and were equallyfrost susceptible at this time.
5. DuringMay-July the elongating shoots fluctuated in hardinessbetween–5°C and –10°C apparently in responsetofluctuating ambient temperatures.
6. In August 1980 there wasa period of late summer dehardeningto killing temperaturesof about –3°C.

Seasonal changes in hardiness are discussed in relation to changesin shoot growth and environmental factors. The main opportunitiesfor selecting frost hardy genotypes seem to be in the rate ofautumn hardening, the time of pre-bud burst dehardening, andthe time of bud-burst.     

Aftereffects of maternal environment on autumn frost hardiness in Pinus sylvestris seedlings in relation to cultivation techniques

Full-sib families of Pinus sylvestris (L.) from genetically identical parent clones, grown at three geographic locations (64, 59.5, and 56 degrees N), were tested for autumn frost hardiness. At one of the locations (64 degrees N), maternal clones were grown in plots subjected to different site treatments. Progenies raised in a heated greenhouse with additional artificial light were induced to undergo two shoot elongation periods, separated by a period with long nights and non-hardening temperatures, followed by a hardening period. Frost hardiness was tested by exposing seedlings to -17, -19, or -21 degrees C during the hardening period. Progenies produced at the northernmost locality (64 degrees N) were the most hardy. The hardiness ranking among localities and the magnitude of differences in hardiness were in accordance with earlier results obtained from the same crosses that had undergone only one shoot elongation period. Effects of maternal site treatments were significant but small compared with family differences. The growth regimes used in the present study did not eliminate maternal effects induced by geographic location, but maternal effects related to site treatment decreased substantially in relation to family variation when compared with seedlings that had undergone only a single shoot elongation period. The results are discussed in relation to other studies of the same crosses raised under different growth conditions.     

Frost hardiness of nutrient-loaded two-year-old <Emphasis Type="Italic">Picea abies</Emphasis> seedlings in autumn and at the end of freezer storage

The effects of nutrient loading (NLOAD) on the frost hardening and dehardening of Picea abies (L.) Karst. seedlings were investigated under nursery conditions. Before NLOAD, second-year container seedlings were either short-day (SD) treated for 3 weeks in July or left for the natural photoperiod (CO). By mid-September, after 5 weeks of NLOAD, the fertilization of three foliar nutrient concentration levels (low = L-SD, medium = M-SD, and high = H-SD) for the SD-treated seedlings and one (medium = M-CO) for the CO-seedlings was completed. The NLOAD resulted in foliar nitrogen concentration 10.6, 16.1, 22.3, and 17.5 g kg⁻¹ for L-SD, M-SD, H-SD and M-CO seedlings, respectively. The NLOAD had no effects on the morphology or dry mass variables of the seedlings, while SD-treatment reduced the dry mass of shoots, but not that of roots. The frost hardiness (FH) of different batches of the seedlings was assessed by the visual scoring of damage in their needles, stems and buds after their controlled exposure to freezing during frost hardening and dehardening. The low nutrient concentration in the SD-treated seedlings (L-SD seedlings) resulted in poor FH, to an even lower extent than that of the M-CO seedlings. The NLOAD did not affect the dehardening of the seedlings at the end of the freezer storage in the following spring.     

Frost tolerance and bud dormancy of container-grown yellow birch, red oak and sugar maple seedlings

Container-grown seedlings of red oak (Quercus rubra L.), sugar maple (Acer saccharum Marsh.) and yellow birch (Betula alleghaniensis Britton) in their first year of growth were overwintered outdoors. Tolerance of roots and stems to freezing was compared from late summer to the following spring. Mitotic activity in the apical bud was related more closely to air temperature than to bud dormancy as defined by days to bud break. In all species, stem hardening was observed before days to bud break reached a maximum. Dormancy release (days to bud break equal to zero) of yellow birch coincided with loss of stem hardening in the spring. Roots hardened more slowly, had a lower frost tolerance than stems in fall and winter, and dehardened earlier than stems in the spring. There were differences in stem and root hardiness among the species, with yellow birch being the most tolerant, followed by sugar maple and red oak. Primarily because of root sensitivity to frost, winter was a critical period for all three species, but particularly for red oak.     

The effect of late summer fertilization on the frost hardening of second-year Scots pine seedlings

In this study the effect of summer fertilization on the initiation of frost hardening of containerized second-year Scots pine (Pinus sylvestris L.) seedlings is studied. During the second growing season three different fertilization programs (water soluble NPK with micronutrients) determined by electrical conductivity of peat water extract (0.2, 0.5 and 1.2 mS cm^-1) were initiated. The growth and nutrient concentrations of needles were monitored during the fertilization period. The frost hardiness of seedlings was assessed on four separate occasions at two week intervals from August 7 to September 18. This assessment was based on artificial freezing tests and visual damage scoring of tissue browning on current-year needles. Clear differences in foliar N, P and K concentrations were observed between the fertilization treatments. Fertilization prolonged the growing period of needles and increased root collar diameter. In all the tests, the highest fertilization level resulted in the highest level of frost hardiness. The difference between the fertilization treatments ranged from 1 °C to 2.2 °C. Frost hardiness increased with an increase in foliar nitrogen concentration and slightly less consistently with increases in foliar phosphorus and potassium concentrations.     

Effects of nitrogen fertilization and temperature on frost hardiness of Aleppo pine (Pinus halepensis Mill.) seedlings assessed by chlorophyll fluorescence

In a 14-week study, 1-year-old Aleppo pine seedlings were grownin two growth chambers. Seedlings were artificially hardenedby decreasing photoperiod and temperature. In each chamber halfof the seedlings were fertilized with nitrogen (8.4 mg seedling^–1).In order to determine the relative importance of the hardeningenvironment versus fertilization, each chamber was programmedto decrease night temperatures down to a low of 8 or 4°C.Chlorophyll fluorescence and frost hardiness was measured fivetimes during the experiment. A sample of seedlings from eachtreatment was exposed to an artificial frost at –5°Cand the freezing effects were assessed by measurements of chlorophyllfluorescence and visual evaluation of needle damage. Seedlingsincreased their frost hardiness during the experiment in allthe treatments but the ratio of variable to maximal chlorophyllfluorescence (F_v/F_m) measured before freezing did not vary duringthe experiment. This indicates that Aleppo pine maintains itsphotosynthetic ability during hardening in contrast to otherconiferous species from colder climates. The effect of nitrogenfertilization on frost hardiness was small in comparison withchamber effect. Nitrogen fertilization slightly delayed theacquisition of hardening in the coldest chamber. Seedlings inthe warmest chamber did not become fully resistant to –5°C,but in the coldest chamber, where night temperature reached4°C, all the seedlings were resistant to the frost. Severedamage caused by frost could be related to a rapid rise of minimalfluorescence (F₀) but the best index of damage was the dropof F_v/F_m after freezing.     

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.     

Effects of nutritional factors on frost hardening in Larix leptolepis (Sieb & Zucc.) Gord.

A method for estimating frost hardiness in seedlings of Larix leptolepis in their first stage of acclimation is demonstrated. Cuttings of the shoot tip were frozen linearly from +2°C to ‐36°C. The viability was determined by TTC‐assay. Data were fitted to a logistic regression model, and hardiness was calculated from the fitting parameters. It was shown that hardiness is influenced by the duration of the fertilizer application period. No effect of the nutrient status of N, P or K in the shoots was found.     

The Influence of Desiccation, Rough Handling and Cold Storage on the Quality and Establishment of Sitka Spruce Planting Stock

In January 3-year-old Sitka spruce were lifted and treated eithercarefully or roughly. Plants in each treatment were either transferredto the laboratory for testing, or transferred to cold storageat –1°C. In the laboratory, half of the plants ineach treatment were desiccated to shoot water potentials ofless than –2.0 MPa. Plants were then tested for root growthpotential (RGP), bud dormancy, frost hardiness and carbohydratecontent before transplanting at a field site, Additional plantswere removed from the nursery and cold store at approximatelymonthly intervals until late April, and then treated as above.In April, plants in each treatment were transferred to on-sitefield storage (sheughs), for 2 or 4 weeks prior to testing asabove. Carefully handled plants maintained large RGP which was positivelycorrelated with plant water potential, root water content, shootrelative growth rate and field survival. Overall RGP was reduced: 59 per cent by desiccation; 85 percent by rough handling; and 98 per cent by desiccation and roughhandling in combination. On site storage for 4 weeks in April/May reduced RGP. Whereplants produced more than 30 new roots > 1 cm long in RGPtests, field survival exceeded 90 per cent. Cold storage at–1°C maintained RGP, (of carefully handled plants),bud dormancy and shoot frost hardiness at approximately pre-storagevalues. In contrast, total nonstructural carbohydrate contentdeclined in cold store but increased in March/April for plantsin the nursery.     

Carbohydrate reserve accumulation and depletion in Engelmann spruce (Picea engelmannii Parry): effects of cold storage and pre-storage CO(2) enrichment

The effects of pre-storage CO(2) enrichment on growth, non-structural carbohydrates and post-storage root growth potential of Engelmann spruce (Picea engelmannii Parry) seedlings were studied. Seedlings were grown from seed for 202 days in growth chambers with ambient (340 micro l l(-1)) or CO(2) enriched (1000 micro l l(-1)) air. Some seedlings were transferred between CO(2) treatments at 60 and 120 days. Photoperiod was reduced at 100 days to induce bud set and temperature was reduced at 180 days to promote frost hardiness development for storage at -5 degrees C for 2 or 4 months. Stored seedlings were planted in a growth chamber after thawing for one week at +5 degrees C. At 80, 120, 140 and 202 days, and at each planting time after storage, seedlings were harvested for growth measurements and analysis of starch and soluble sugar concentrations. Planted seedlings were assessed for bud break every two days and new roots > 5 mm long were counted after four weeks. Carbon dioxide enrichment increased root collar diameter and almost doubled seedling biomass, with the most obvious effects occurring after bud set. Stem height was affected only slightly and shoot/root ratios were not affected at all. Carbon dioxide enrichment increased the rate of reserve carbohydrate accumulation, but did not influence the final concentration attained before storage (accounting for 32% of seedling dry weight). Needles were the major storage organ for soluble sugars, whereas roots were the major storage organ for starch. Soluble sugars were not strongly affected by two or four months of storage, but starch was reduced by more than 50% in all plant parts. None of the CO(2) treatments had an impact on bud break or root growth potential.     

Frost tolerance of two-year-old Picea glauca seedlings grown under different irrigation regimes in a forest nursery

Abstract

This study examined the impact of increased irrigation efficiency on the hardening and frost tolerance of 2-year-old containerized white spruce seedlings in the context of groundwater protection, irrigation management and the maintenance of seedling quality in northern climates. The seedlings were grown under three different irrigation regimes (IR =30%, 40% and 55% v/v; cm³ H₂O/cm³ substrate) and were hardened under conditions of natural photoperiod and temperature. After being subjected to artificial frost tests on four sampling dates during autumn, the seedlings were compared for bud development and frost tolerance. IR had no influence on frost tolerance as determined by measurements of physiological (electrolyte leakage, root water loss) and morphological (shoot damage, root initiation) variables. At the end of the second growing season, there was no significant difference between IRs in seedling height, root collar diameter, shoot dry mass and root dry mass. The results indicate that the amount of water applied to large-dimension 2-year-old white spruce seedlings during the growing season can be significantly decreased without prematurely impeding their growth or hindering their acquisition of frost tolerance.     

Freezing temperatures in the root zone—effects on growth of containerized Pinus sylvestris and Picea abies seedlings

Roots of 1‐year‐old containerized seedlings of Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) were experimentally frozen in December. The seedlings were then grown for 3 weeks in a growth chamber and evaluated with regard to root growth capacity (RGC) and shoot elongation. The subsequent RGC of Scots pine declined as root zone temperatures were lowered from ‐6°C to ‐11°C and from ‐11°C to ‐16°C. Almost no root growth was observed after exposure to ‐20°C. Shoot growth was also negatively affected by low root temperatures but less than root growth. Low root temperatures did not affect Norway spruce as much as Scots pine, although root and shoot growth of Norway spruce were reduced after exposure to the lowest test temperatures (‐16°C and ‐20°C). The length of exposure, ranging between 1 and 8 hours had no effect on subsequent growth.     

Temperature step response of dehardening in Pinus sylvestris seedlings

The time constant of the dehardening of one‐year‐old Scots pine (Pinus sylvestris L.) seedlings was studied in three experiments started at different times of the year. The seedlings were transferred from outside to the different controlled conditions in January (Exp. 1), March (Exp. 2) and April (Exp. 3). Changes in the frost resistance of the seedlings were followed on the basis of the temperature response of the specific impedance difference in the shoot. The time constant for dehardening at a temperature of 7.5°C was found to be about 12 days in Exp. 1, about 8 days in Exp. 2, and about 5 days in Exp. 3. the terminal bud burst when the frost resistance of the shoot had risen to about ‐10°C.