open, the width of a crack reflects closely the wood temperature. Cracks fully close when
the mean daily air temperature is above freezing for 24 to 48 hr.
Many explanations of freeze cracks continue to be based on the theory of thermal
contraction: cracks result from the rapid cooling and contraction of the bark and outer
wood while the inner wood remains warm with less contraction (Kramer and Kozlowski
1960). If this theory were valid, radial, vertical cracks would occur only in the exterior
rings of the bole with extensive ring shakes beneath. Although the trees studied here could
not be sectioned to examine the exact penetration depth of cracks, they appeared to have
a form and depth consistent with the definition of freeze cracks given by Kubler (1983)
(i.e., complete arc segments reaching to the center of the tree).
The freeze-dry hypothesis that is proposed by Kubler (1983) to explain freeze crack
formation in trees in winter is largely based on experimentation on wood samples in the
laboratory. Our measurements of mature trees confirm that at temperatures below 0°C
there is substantial trunk shrinkage. The wood shrinkage in trees with freeze cracks is
greater than in trees without freeze cracks. Our temperature measurements at varying depths
in the trunk establish that there is relative uniformity of wood temperatures. The temper­
ature data do not indicate that a temperature differential between exterior wood and interior
wood is a likely cause for freeze cracks. Our shrinkage and temperature measurements
wholly support the freeze-crack mechanism given by Kubler (1983).
LITERATURE CITED
HIMEUCK, E. B. 1970. Frost cracks on London plane tree: An important winter thermometer. Arbor.
News 35(1):5-6.
KRAMER, P. ]., and T. T. KoZLOWSKI. 1960. Physiology of trees. McGraw-Hill, New York. 642 p.
KUBLER, H. 1983. Mechanism of frost crack formation in trees-a review and synthesis. For. Sci.
29:559-568.
KUBLER, H., L. LIANG, and L. S. CHANG. 1973. Thermal expansion .of moist wood. Wood and Fiber
5(3):257-267.
KUBLER, H., and H. TRABER. 1964. Temperatur- und Dimensionsanderungen von Baumstammen
im Winter. Forstwiss Centralbl. 83:88-96.
PANSHIN, A. ]., and C. DEZEEUW.
1980.
Textbook of wood technology. Ed. 4. McGraw-Hill, New
York. 722 p.
Forest Sci., Vol. 33, No. 1, 1987, pp. 244-246
Copyright 1987, by the Society of American Foresters
Long-Term Storage of Douglas-fir Pollens
Donald L. Copes
ABsTRAcr. A test of pollen fertility after 3 years of cold storage is reported. Douglas-fir (Pseu­
dotsuga menziesiz) pollen was as fertile after 1, 2, or 3 years of storage in liquid nitrogen as was
fresh pollen. Fertility of the same pollen lots decreased significantly after I or 2 years of storage
at OOC. FoR. Sci. 33(1):244-246.
ADDITIONAL KEY
woRDS.
Pseudotsuga menziesii, cryogenic, pollination, breeding.
DouGLAS-FIR (Pseudotsuga menziesii [Mirb.] Franco) pollen retains high fertility after I
year of storage in liquid nitrogen ( -196°C) (Copes 1985). The liquid nitrogen procedure
for storing pollen uses inexpensive equipment and simple techniques. The possibility that
the same procedure might be used to extend viability of Douglas-fir pollen for many more
years was suggested because most biological activity ceases at -1960C (Mazur 1966). Pollen
that remains viable after short-term storage in liquid nitrogen should theoretically remain
viable indefinitely in liquid nitrogen (Sakai 1978).
The author is Principal Plant Geneticist, Forestry Sciences Laboratory, Pacific Northwest Research
Station, USDA Forest Service, Corvallis, OR 97331. Manuscript received July II, 1986.
244 / FOREST SCIENCE
TABLE 1. Percent fertility of fresh and 1- to 3-year-old Douglas-fir pollen stored
at O"C and -196"C.a
Year of
collection
1982
1983
Percent fertility by year of pollination
Type of storage
-196"C
Fresh
1983
1984
1985
Number of
cones
79.2b
81.4
80.9
93
8l.9b
-196"C
O"C
1984
Fresh
X
X
71
86.2
72.5
63
34.9
46.6
58
80.7
X
10
83.4
33
58.9
23
68.6
20
-196"C
O"C
1985
•
Fresh
Fertility was determined by dividing the number of filled seeds per cone by the number of round
seeds per cone and multiplying the quotient by 100.
b Data from Copes (1985).
MATERIALS AND METHODS
The data from a continuation of the 1-yr storage test on Douglas-fir pollen are reported
here (Copes 1985). The same pollen collection, extraction, storage, and pollination pro­
cedures described in the 1985 report were also used in the present study. Pollen fertility
after storage periods of up to 3 yr at -196°C and up to 2 yr at ooc are described. Ramets
of the same two and three clones used as females and males, respectively, in the 1983
pollinations were retested in 1984 and 1985. The 1982 pollen samples were stored at
-196°C for 2 or 3 yr; pollen collected in 1983 was evaluated after 1 and 2 yr of storage
at either ooc or -196°C. Additional pollen from the same clones was gathered in 1984
and 1985.
Moisture content of fully mature pollen ranged from 4 to 7% after air drying when
samples were placed in 1.2- or 5-cc screw-top plastic vials. The filled vials were placed
directly in a 29-liter liquid nitrogen refrigerator without being prechilled. The pollen was
kept in the liquid nitrogen until the day of pollination. At that time the tubes were placed
in a conventional freezer for approximately 30 min at -20°C in order to prevent implosion
and then gradually warmed to ambient air temperature prior to pollination.
Pollen fertility was measured as the capacity to promote the formation of filled seeds.
The percentage of filled seeds was calculated from the ratio of the number of filled seeds
to round seeds per cone; the value for seeds resulting from pollinations with the fresh,
unstored pollen was the fertility standard against which stored pollen was compared in
following years. Seed processing, x-ray techniques, and analysis of variance methods used
were nearly identical to those described in the 1985 report, except no germination of filled
seed was done because the previous report showed all filled seeds to be viable (Copes 1985).
Data from the three males were combined for the statistical analysis because of small
sample size.
RESULTS AND DISCUSSION
Pollen lots retained their original fertility values even after 3 yr in liquid nitrogen (Table
1). Analysis of variance tests of filled seed data indicated that differences in fertility between
pollen stored for 1, 2, or 3 yr in liquid nitrogen were not significantly different at the 5%
level. Pollen stored at -196°C was not significantly different at the 5% level from fresh
pollen in promoting fertilization after pollination. In both 1984 and 1985, fertilization
rates with both fresh pollen and pollen stored in liquid nitrogen were significantly higher
at the 5% level than those for pollen stored at ooc for 1 and 2 yr. Fertility of pollen stored
at OOC dropped 22 to 65% below rates for fresh pollen or for pollen stored in liquid nitrogen
(all pollens were from the same lots).
Effective long-term storage of Douglas-fir pollen in liquid nitrogen was demonstrated.
Little or no change in fertility occurred, even after 3 yr, and it is unlikely that additional
MARCH 1987 I 245
change will occur even after many more years of storage (Mazur 1966). The ability to
collect pollen whenever it is produced and to store it without loss of viability enables forest
geneticists and tree improvement specialists to work more efficiently and to complete crosses
that might not otherwise be possible.
LITERATURE CITED
CoPES, D. L. 1985. Fertility of Douglas-fir pollen after one year of storage in liquid nitrogen. For.
Sci. 31(3):569-574.
MAzuR, P. 1966. Physical and chemical basis of injury in single-celled and micro-organisms subjected
to freezing and thawing. P. 213-315 in Cryobiology, H. Meryman (ed.). Academic Press, New
York. 775 p.
SAKAI, A.
1978. Survival of plant germplasm in liquid nitrogen. P. 345-359 in Plant cold hardiness
and freezing stress: Mechanisms and crop implications, P. H. Li (ed.). Nat. Sci. Found. Academic
Press, New York. 416 p.
246 / FOREST SCIENCE