Survey on and common protocols for the assessment of frost hardiness of young trees

Heino Wolf and Tore Skrøppa

Partner N° 8, Staatsbetrieb Sachsenforst (SBS) and Partner N° 17, Norwegian Forest and Landscape Institute (NFLI)

1  Introduction

In forestry, frost may affect significantly the success of plantation and cultivation of trees, since the hardiness to this factor is depending on the origin of the basic material to be reproduced. Therefore, there is a common interest in the response of tree species and their provenances to frost occurrences.

Despite these general aspects, the effects of global warming and the subsequent climate change will also create a specific interest in the response of forest reproductive material to frost. Most climate models especially for Central Europe expect an increase of temperature on the annual as well as on the seasonal level (e. g. IPCC 2007, MPI-M 2006, Spekat et al. 2007). The number of days with ice and frost will decrease, but the possibility of heavy frost periods will still remain. The consequences will be effects on the length of the winter season and the vegetation period, on the stability of temperature over time as well as on the beginning of dormancy, the continuity of dormancy, the dehardening and the beginning of growth. Therefore, the risk for trees to be damaged by frost may be higher in future than today since the processes of frost hardiness may not run parallel to the course of temperature within the year.

Therefore, the assessment of the response of species, provenances, progenies, clones to frost will play a more important role in future for the evaluation of suitability of forest reproductive material in question for the utilization in forestry as well as in land scape management. Since frost has an important impact on the survival of plantations, the results of the assessment can be used as a tool for early selection of suitable material. The following methods can also be used as a base for the development of non-destructive estimation methods, e.g. root electrolyte leakage as described by Schüte & Sarvas (1999) or for the assessment of quality deterioration in coldhouse storage of plants or parts of plants (e. g. O´Reilly et al. 1999, 2000).

The purpose of this paper is to describe the state of art and to present different common protocols for the assessment of the response of tree species and their provenances, progenies or clones to frost. The state of art as well as the common protocols are based on the results of two questionnaires done among the participants of the Treebreedex consortium. After the evaluation of the questionnaires the partners were asked to send relevant literature which was completed by additional publications (see list of literature).

2  Frost hardiness as a dynamic process

Frost resistance is the ability of trees to survive the effects of temperatures below the freezing point. The several aspects of frost resistance such as avoidance, tolerance, processes during freezing or development of primary and secondary damages were described for example by Levitt (1980) and Libbert (1987). In the following, the term frost hardiness will be used as synonym for the ability of trees to tolerate frost of different levels.

The development of frost hardiness within trees is influenced by several exogenous factors e. g. course of temperature during the year, day length or light. It is also affected by endogenous factors such as the development rhythm of the tree, the accumulation of plant contents or the speed of metabolism. Therefore, frost hardiness can not be described as a fixed condition lasting constantly for a long period. It should be understood as a process which is object of permanent change.

Different phases of frost hardiness should be considered if frost hardiness should be assessed (Scheumann 1964, 1968):

-  The ability of plants for hardening (early frost hardiness)

-  The degree of hardening (winter frost hardiness)

-  The response on significant changes of temperature during winter period (stability of frost hardiness)

-  The beginning of dehardening (late frost hardiness)

-  The ability to regenerate damages caused by frost

Since the natural conditions may chance in a not predictable way, frost hardiness should be assessed under controlled conditions in climate-/frost-chambers along the course of the year. Therefore, the material in question should be tested immediately after the vegetation period in front of the first frosts, during the winter period and finally at the end of the winter season. In addition, assessments of the timing of annual growth rhythm events in field trials, such as bud flushing in spring and growth cessation in the autumn, may provide valuable information for ranking provenances, families or clones for their frost hardiness in different periods of the growth season.

3  State of art

3.1. Experience and species of interest

Out of the 27 partner institutions participating in the questionnaire, 11 institutes (39 %) have already experience with the assessment of frost hardiness. The priority species of interest was Douglas fir, which was included in frost experiments in 36 % of the institutions. Apart from Douglas fir, Scots pine, oak species and walnut gained interest in 18 % (2) of the institutes each. Four institutions have experience with Med. pines, Noble fir, Norway spruce, Sitka spruce as well as with Common ash (Tab. 1).

Tab. 1: Experience with the assessment of frost hardiness and species of interest

Partner N° / Institute / Assessment on the response to frost done with
Douglas fir / Med. pines / Noble fir / Norway spruce / Scots pine / Sitka spruce / Common ash / Engl. Walnut / Oaks
8 / SBS / X
9 / UoC / X / X / X
12 / CRA / X / X / X
13 / Collite / X
17 / NFLI / X
18 / IDPAN / X
21 / SKOGFORSK / X
23 / INIA / X
25 / SLU / X
26 / FCBO / X
27 / CITA-Aragon / X

3.2. Material and methods used for the assessment of frost hardiness

The review on material and methods used is based on 22 publications and different protocols delivered by partners with experience of the assessment of frost hardiness. Only experiments with plant material under controlled conditions are mentioned which lead finally to the destruction of the material in question. Assessments of frost damages in the field or indirect methods like the assessment of cell sap concentration or the measurement of the osmotic freezing point are not included.

3.2.1.  Plant material used and sample size

The genetic background of the material investigated varied from clones on the one hand to full-sib-families and open pollinated progenies on the other hand. In most investigations, generatively reproduced plant material such as seedlings was used in the experiments. Clonal material was considered only in exceptional cases. The age of plants used reached mostly from one to 10 years, in one case up to 18 years. The sample size varied from 3 up to 30 individuals per genetic unit. In one case the sample size was not specified at all. The plant material used for the freezing experiments depended from the category of trees: In the case of conifers mainly lignified twigs and needles of the current year were used, sometimes together, sometimes separately. In the case of deciduous tree species whole plants and parts of plants (twigs) were collected (Tab. 2).

Tab. 2: Kind of plant material, age of plants, sample size and method of sampling for the assessment of frost hardiness

Species / Genetic units (GU) / Age of plants / Sample-Size / Material / Reference
Douglas fir / Full sibs, half sibs / 4 years / 5 trees/GU / Lignified twigs / Rehfeldt 1977
Douglas fir / Half sibs / 10 years / 30 trees/GU randomly / Lignified twigs / Nowatzki 1998
Scots pine / Full sibs, open polls / 1 – 2 years / 15 x 9 seedlings /GU / Seedlings / Andersson 1994
Scots pine / Clones / 15 – 18 years / Not specified / Current-year needles / Nilsson & Walfridsson 1995
Scots pine / Full sibs, half sibs / 8 years / 8 trees/GU, randomly / Current-year needles / Nilsson 2001
Norway spruce / Full sibs, half sibs / 8 years / 15 trees/GU / Rooted cuttings / Johnson 1989a
Norway spruce / Clones / 2 years / 3 plants/GU / 2-year old twigs / Wagner 1991
Sitka spruce / Open polls / 3 years / 10 seedlings/treatment / Current-year shoots / Black et al. in press
Silver fir / Open polls / 3 years / 10 seedlings/GU and treatment / 6 one-year-old needles/tree / Larsen et al. 1990
Beech / Open polls / 9 years / 7 trees/GU / One-year-old twigs / Visnjic & Dohrenbusch 2004
Oak / Open polls / 1 year / 10 - 30 trees/GU / Plants / Liepe 1993
Oak / Open polls / 2 years / 17 plants/GU / Twigs / Jensen & Deans 2004

The number of samples to be included in the experiments depends also on the way, the data are evaluated statistically (Johnsen 1989b).

3.2.2.  Freezing protocols and test levels

In most experiments, the test material was taken for a certain period under a temperature from +2°C up to +20°C for a uniform pre-treatment before starting the freezing experiments.

After this procedure two different approaches were used to produce frost damages in plant tissues:

Firstly, the test material is cooled down straight after the pre-treatment to the test temperature. After freezing the material for a certain period, the material in question is thawed back straight to temperatures between +2 and +10 °C for post freezing treatment. Again, after keeping the material under these temperatures up to 10 hours, the tissues are transferred to glasshouse, laboratory or nursery facilities for cultivation (Tab. 3). The instant freezing method was used for plant material e.g. from Douglas fir, Noble fir, Norway spruce, Scots pine or Sessile and Pedunculate oak (Braun & Scheumann 1989; Jensen & Deans 2004; Nielsen & Rasmussen 2009; Nilsson & Walfridsson 1995; Scheumann 1964, 1968; Schüte & Sarvas 1999; Skrøppa 1991).

Tab. 3: Freezing protocols used for the assessment of frost hardiness

Species / Method / Pre-treat-ment / Cooling / Dura-tion test level / Thawing / Post-treatment / Cultivation / References
Noble fir / Instant freezing / 4h: +2°C / 4h to test level / 2h / 4h to 2°C / 4h: +2°C / Not specified / Nielsen & Rasmussen 2009
Sessile & Peduncu-late oak / Instant freezing / 3h: 5°C / 4h to test level / 4h / 5h to 3°C / 3h to 5°C / Sand, 20°C, 25d / Jensen & Deans 2004
Scots pine / Instant freezing / +5°C / 1.5h to test level / 1h / 1.5h to 10°C / 10h: 10°C / GH, 10°C, 14h light, 21d / Nilsson & Walfridsson 1995
Douglas fir, Grand fir / Constant freezing / 3h: +2°C / -6K/h / 4h / +6K/h / 3h: +2°C / Sand, 20°C / Larsen 1978a, b
Sessile oak / Constant freezing / +6°C / -3K/h / 4h / +3K/h / 4h: +6°C / Outdoor; 8 weeks / Liepe 1993
Sitka spruce / Constant freezing / +20°C / -6K/h / 3h / +10K/h / +10°C / Water, 20°C, 16h light, 14d / Black et al. in press

Secondly, the temperature in the climate chamber is constantly decreased to the test temperature with a fixed rate from 3 to 6 K per hour. After a certain period under the constant test temperature, the temperature is increased again with a fixed rate from 3 to 10 K per hour until a temperature from +2 to +10 °C was reached. After keeping the material under these temperatures up to 4 hours, the tissues are transferred to glasshouse, laboratory or nursery facilities for cultivation (Tab. 3). However, for this procedure a climate chamber with flexible temperature processing is a precondition. This constant freezing method was used for plant material e.g. from Douglas fir, Grand fir, Norway spruce, Sitka spruce, Silver fir, Common beech or Sessile oak (Black et al. in press; Johnson et al. 2005a, b; Kohmann & Johnson 2007; Larsen 1978a, b, 1986; Larsen & Ruetz 1980; Liepe 1993; Nowatzki 1998; O´Reilly et al. 1999, 2000; Perks et al. 2004; Rehfeldt 1977; Søgaard et al. 2009; Visnjic & Dohrenbusch 2004).

The differences between these two approaches seem to be little. Groß et al. (1991) demonstrated at the example of Norway spruce, that cooling and thawing rates between 4 K per hour and 8 K per hour produce no differences in the damages independent which approach is used. However, the increase of the cooling and thawing rates from 5 K per hour up to 20 K per hour leads to an increase of frost damages from 6 % to 100 % in the case of a high-altitude spruce. This increase of frost damage corresponds to a decrease of the frost hardiness (LT50) of about 3 K (Groß et al. 1991).

For causing frost damages and injuries, 4 to 5 different test levels of freezing temperatures were used commonly. Depending on the type of frost damage to be observed, the test levels varied from -4 °C to –25.5 °C in the case of early frosts, from -5 °C to -40 °C in the case of winter frost and from -3 °C to -22 °C in the case of late frost. The differences between the different test levels were quite different and ranged from 3.5 K to 12 K (Tab. 4).

For the evaluation of the storage capacity of Sitka spruce seedlings 5 different test levels from -4 °C to -30 °C were applied (Tab. 4).

Tab. 4: Freezing test levels for the assessment of frost hardiness

Purpose / Species / I / II / III / IV / V / References
Early frost hardiness / Norway spruce / -11.0°C / -14.5°C / -18.0°C / -21.5°C / -25.5°C / Wagner 1991
Early frost hardiness / Oak / -4.0°C / -8.0°C / -11.0°C / -14.0°C / -18.0°C / Jensen & Deans 2004
Winter frost hardiness / Oak / -8.0°C / -20.0°C / -25.0°C / -28.0°C / -32.0°C / Jensen & Deans 2004
Winter frost hardiness / Beech / -5.0°C / -10.0°C / -15.0°C / -20.0°C / -25.0°C
-30.0°C / Visnjic & Dohren-busch 2004
Winter frost hardiness / Noble fir / -20.0°C / -27.0°C / -34.0°C / -40.0°C / ---- / Nielsen & Rasmussen 2008
Late frost hardiness / Noble fir / -3.0°C / -8.0°C / -13.0°C / -22°C / Nielsen & Rasmussen 2008
Storage capacity / Sitka spruce / -4.0°C / -8.0°C / -12.0°C / -20.0°C / -30.0°C / Black et al. in press

3.2.3.  Assessment of frost damages and injuries