RITCHIE, G. A., and T. M. HINCKLEY. 1975. The pressure chamber as an instrument for ecological
research. Adv Ecol Res 9:165-254.
RITCHIE, G. A., and R. G. SHULA. 1984.
Seasonal changes of tissue-water relations in shoots and
root systems of Douglas-fir seedlings. Forest Sci 30:538-548.
RocHow, J. J.
1972. A vegetational description of a mid-Missouri forest using gradient analysis
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RUNDEL, P. W., J. EHLERINGER, H. A. MOONEY, and S. L. GULMON.
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TESKEY, R. 0., J. L.CHAMBERS, G. S. Cox , T. M. HINCKLEY, and J. E. ROBERTS.
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Forest Sci Vol.31, No.3, 1985, pp. 569-574
Copyright 1985, by the Society of American Foresters
.•
Fertility of Douglas-fir Pollen After One Year of Storage in
Liquid Nitrogen
Donald L. Copes
ABSTRACT.
Results indicated that Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) pollen
stored in liquid nitrogen ( - I96"C) for I year was nearly as fertile as fresh pollen when used for
controlled pollinations. Percent of filled seed was 79.9 for liquid nitrogen-stored pollen and 81.9
percent for fresh pollen of the current year. Filled seeds per cone averaged 46.3 and 47.9 for liquid
nitrogen-stored pollen and fresh pollen, respectively. Aowers pollinated with fresh or stored pollen
experienced significantly greater levels of flower abortion than flowers that were isolated but not
pollinated; survival of flowers was 45.3, 64.5, and 93.8 percent, respectively.
Methods used in cryogenic storage with liquid nitrogen are simple and enable tree improvement
workers and forest geneticists to effectively store Douglas-fir pollen for at least I year without
significant loss of fertility. FoREST Sci. 31:569-574.
ADDITIONAL KEY WORDS.
Pseudotsuga menziesii, pollination, viability, cold storage, cryogenics,
Douglas-fir.
THE ABILITY to effectively store Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) pollen
for a year or more is desirable because it is not unusual for a small proportion of the trees
in a study or breeding program to fail to develop adequate numbers of male flowers in the
same year as the desired female flowers. Some clones also produce pollen too late in the
growing season to be used on earlier flowering clones. Breeding programs can thus suffer
long delays or be adversely affected by lack of fertile pollen. A storage method that uses
simple procedures and equipment, yet consistently yields highly fertile pollen after a year
or more in storage, would be extremely useful.
Current pollen storage technology used by Douglas-fir tree breeders consists of air-drying
The author is Principal Plant Geneticist, U.S. Department of Agriculture, Forest Service, Pacific
Northwest Forest and Range Experiment Station, Forestry Sciences Laboratory,Corvalis, OR 97331.
Manuscript received 17 May 1984.
VOLUME 31, NUMBER 3, 1985/569
pollen and storing it at near ooc in a refrigerator or at approximately -200C in a freezer.
A moisture content from 5 to 8 percent is considered best (Livingston and Ching 196 7,
Jensen 1970). Generally, the resistance of pollen cells to freezing decreases with increasing
water content (Ching and Ching 1964, Ichikawa and Shidei 19 71). These methods work
for 1 year of storage for most clones, but fertility drops severely in succeeding years, and
some clones do not maintain high pollen fertility for even 1 year.
Livingston and Ching (1967) evaluated freeze-drying of Douglas-fir pollen. They report
that pollen could be effectively stored for 1 year if air-dried to 8.5-percent moisture content
and then subjected to a 36-day chill at ooc prior to freeze-drying. Unfortunately, filled seed
counts were much reduced when 2-year-old freeze-dried pollen was used in pollinations.
Attempts by researchers and tree improvement organizations to utilize the freeze-dried
technique in their own programs have been unsuccessful because of low fertility after
storage, and erratic or variable results from year to year or even within the same pollen
lots of the same year. Much of the irregularity may be the result of variable pollen mat­
uration, extraction conditions, and pollen handling techniques.
Storage technology for Douglas-fir pollen is not at the stage· where a person can be
confident that specific lots will remain fertile following a year or more of conventional
storage. Workers who have had similar problems with other species have, however, reported
excellent success with cryogenic storage. Pollen storage in liquid nitrogen (-196°C} has
been used successfully for maize (Barnabas and Rajki 19 76), date palm (Tisserat and others
1983), hop (Haunold and Stanwood 1980), and potato (Towill 1981). These pollens were
stored for a year or more and showed no decrease in viability or fertility when compared
to fresh pollen.
If a pollen survives freezing and thawing, little decline in fertility or viability should
occur even after years of storage at -196°C (Mazur 1966). Douglas-fir pollen has remained
viable after a few minutes of immersion in liquid nitrogen (Livingston and Ching 1967).
This report describes the effects of cryogenic storage for 1 year on fertility of Douglas­
fir pollen. Comparisons of conelet survival, percent of filled seeds, and number of filled
seeds per cone were made for cones pollinated with liquid nitrogen-stored pollen, fresh
pollen, and no pollen.
MATERIALS AND METHODS
Viability was tested using two clones as females and three clones as pollen parents. Three
pollen treatments were evaluated: 1982 pollen stored for 1 year in liquid nitrogen, fresh
1983 pollen with no long-term storage or freezing, and a nonpollinated control.
Pollen catkins were gathered in April 1982 and 1983 from grafted ramets of the same
three Douglas-fir clones (16-13-1, 16-15-1, and 65) in a breeding orchard near Monmouth,
Oregon. Pollen sacs of the microstrobili were almost fully expanded at that time, but pollen
was usually not being shed into the air. Collection was accomplished by cutting off the
branch tips containing the catkins and placing them in paper bags. The collected catkins
were dried in the bags for 2 days in a closed room at 32°C. Low relative humidity of the
room was maintained by a portable dehumidifier. After 2 days, bags were shaken to extract
the pollen, which was cleaned further by putting it through a 60-mesh wire screen. The
pollen was then spread in a thin layer on sheets of paper in a room free of air turbulence.
Room temperature was approximately 20°C.
After air-drying for 2 to 3 days, the moisture content of each pollen lot was determined
prior to storage. The moisture content was calculated from the difference in weight between
air-dried and oven-dried pollen divided by the oven-dried weight. Oven-dry weight was
obtained from 1 to 2 g of pollen dried at 80°C for 2 days. Moisture content of air-dried
pollen averaged 6.5 percent prior to freezing or refrigerator storage.
In April 1982 air-dried pollen was poured into 1.2-cc screw-top plastic vials. Each vial
containing approximately 0. 75 g of pollen was placed directly in a 29-liter liquid nitrogen
refrigerator without being prechilled. The pollen was kept in liquid nitrogen until April
1983. At that time the vials of pollen were placed in a conventional freezer for 30 minutes
at 20°C in order to prevent implosion and then were carried to the field in an insulated
ice chest along with other containers of fresh 1983 pollen. The pollen gradually warmed
to ambient air temperature when removed from the ice chest 5 to 10 minutes prior to
pollination.
570 I FOREST SCIENCE
Fresh 1983 pollen was extracted and processed in the same manner as 1982 pollen except
it was stored in glass containers and refrigerated near 3°C until needed. Fresh pollen storage
was generally less than 7 days.
Pollination in April l 983 was done using grafted ramets of clones 202-37 and 92-18 as
female parents. All three pollen treatments (1982 pollen stored in liquid nitrogen, fresh
1983 pollen, and nonpollinated control) from each of the three male clones were tested on
each mother tree. Branch tips with female flowers were emasculated and covered with
protective kraft pollination bags several weeks before the flowers became receptive. Pol­
lination was done within the first 1 or 2 days after the bud scales opened by gently removing
the pollination bags, one at a time, and carefully pouring a small amount of pollen over
the top of each flower. Syringe or brush applications of pollen were not done because both
methods result in greater variation in seed set than occurs with pouring pollen over the
flowers. Each flower was pollinated once. One 0.75-g vial had sufficient pollen for 9 to 24
female flowers. The amount needed to adequately pollinate each flower varied according
to flower size and velocity of the wind. At least two bags were pollinated of each treatment
combination. Pollinations were done the same day the pollen was removed from the liquid
nitrogen. Each isolation bag was placed over the branch immediately after pollination.
Each bag was off the branch for only 30 to 60 seconds. The number of flowers pollinated
in each bag was recorded at this time. The same procedures were used for the treatment
using the fresh 1983 pollen. The nonpollinated control involved carefully removing each
pollination bag from the branch tip for 30 to 60 seconds and then replacing each bag. All
pollination bags remained over the flowers until cone collection. Douglas-fir flowers grow
into normal cones even when they do not receive pollen, but their seeds are empty if no
pollen is present for fertilization.
Cones were collected at maturity in late August 1983. The number of surviving cones
in each bag was recorded. Seed was extracted by hand from open cones by tearing off one
scale at a time. All round seeds (nonshriveled) were counted and x-rayed to nondestructively
identify seeds containing an embryo. The percentage of filled seeds and numbers of filled
seeds per cone were calculated from the total number of nonshriveled seeds. Two hundred
filled seeds of each parentage-pollen treatment were given cold stratification and sown in
outdoor nursery beds in May 1984. Seed germination was recorded in July 1984.
Seed and flower data were evaluated by analysis of variance in which female clones were
used as blocked replication. Percentage data were subjected to arcsin transformation prior
to analysis. Pollen treatments and male clones were assigned randomly to bags within each
female tree. Data from the two pollen storage treatments and the no-pollen treatment were
analyzed for conelet survival, but because seed set was extremely low or absent in the
nonpollinated controls, only liquid nitrogen and fresh pollen treatments were analyzed for
seed information.
REsULTS
The percentage and number of filled seeds per cone did not vary significantly for cones
grown from flowers pollinated with 1982 liquid nitrogen-stored pollen or with 1983 fresh
pollen (Tables 1 and 2). The values were extremely similar (79.9 vs. 81.99 percent filled
seeds and 46.3 vs. 47.9 filled seeds per cone) for liquid nitrogen-stored vs. fresh pollen,
respectively. Nonsignificant differences for the two pollen treatments were found for the
average number of nonshriveled seeds per cone (potentially viable) and the proportion of
the nonshriveled seeds which were filled (viable). Nursery bed tests of filled seeds averaged
97.3 percent germination.
Only 0.3 percent filled seeds were produced by the nonpollinated controls (Table 1).
Slightly more filled seeds occurred in cones of female tree 202-37 than for tree 92-18. The
female parent 202-37 had 0.6 percent filled seeds and 92-18 had only 0.1 percent filled
seeds. Significance was at the 12-percent level (Table 2). The low seed set for the control
indicated that practically all filled seeds in controlled pollinations resulted from pollens of
the three male clones and not from open pollination.
A greater percentage of female flowers developed into mature cones when they did not
receive any pollen (Table 1). Aborted flowers never completed normal development, as
evidenced by turning down from the receptive upright flower position to a normal pendant
cone position and failing to change from succulent to woody in the weeks following pol-
VOLUME 31, NUMBER 3, 1985 /571
TABLE 1.
Influence ofpollen type on conelet survival and numbers of seed.
Parents
Pollen type
1982 pollen
Male
Female
202-37
X
stored in liquid
nitrogen
Female
flowers
pollinated
Conelet
survival
Nonshriveled
seed
Filled
seed
Filled
seeds
per cone
Number
Percent
Number
Percent
Number
16-13-J
13
38.5
309
75.4
46.6
16-15-1
9
33.3
176
74.4
43.7
52.9
585
76.2
49.6
75.7
47.6
65
17
43.6
Mean
--
92-18
X
J6-J3-J
24
54.2
752
84.2
48.7
16-15-1
22
40.9
506
79.4
44.7
65
21
42.9
453
46.3
Mean
83.0
41.8
82.5
45.5
79.9
46.3
48.8
--
45.3
Mean
Fresh,
202-37
X
1983 pollen
!6-13-J
II
72.2
450
86.7
16-15-1
8
100.0
425
71.3
37.9
65
17
88.2
907
80.9
48.9
!6-13-J
22
27.3
381
81.1
51.5
16-15-1
15
40.0
369
75.1
46.2
65
37
75.7
1,626
85.7
49.8
86.1
Mean
--
92-18
X
54.1
Mean
80.1
46.0
--
--
83.3
--
64.5
Mean
No pollen
49.5 --
81.9
47.9 202-37
x
None
20
90.0
1,162
0.6
92-18
x
None
28
96.4
1,755
.1
.04
.3
.2
Mean
93.8
0.4
lination. Instead they died and dried up. The 93.6-percent survival by the nonpollinated
control treatment exceeded the survival of the fresh pollen treatment by 29.1 percent and
the liquid nitrogen treatment by 48 percent (Table 1). The difference was significant at the
5-percent level (Table 3). The difference in conelet survival results for fresh and liquified
nitrogen-stored pollen treatments was not significant.
DISCUSSION AND CONCLUSIONS
The most important finding was that Douglas-fir pollen stored for 1 year under cryogenic
conditions remained highly viable. The average seed set of 46 fertile Douglas-fir seeds per
cone from controlled pollinations was very good because syringe or brush controlled pol­
linations in Douglas-fir averaged only from 10 to 30 filled seeds per cone (Carlson and
Hsin 1976). The high percentage of filled seeds demonstrated that the liquid nitrogen­
stored pollen remained extremely fertile after 1 year. High pollen fertility should be main­
tained, theoretically, even after a number of years of storage because little deterioration
from respiration or other metabolic processes can occur at -196°C (Mazur 1966). Trials
over a longer period of time will be made with stored Douglas-fir samples from the same
three pollen lots described in this report.
The pollen handling and cryogenic procedures used for this study were simple and
required only that mature, freshly collected pollen be air-dried to an average of 6.5-percent
moisture content prior to liquid nitrogen storage. No prechilling or prefreezing treatments
were required and pollen did not have to be rehydrated prior to use. The simplicity of the
572 I FOREST SCIENCE
TABLE 2.
Analysis ofvariance of seed data.
Degrees
freedom
Source of variation
Mean squares
Fvalues
Significance
of Fvalues
Proportion filled seeds
Female clones
I
0.0310
3.50
0.12
Male clones
2
.0151
1.70
.27
Pollen storage (fresh vs. LN2)
I
.0114
1.29
.31
Male clones
2
.0102
1.16
.39
5
.0089
x
pollen storage
Experimental error
II
Total
-
Number filled seeds per cone
Female clones
I
5.0722
.10
0.76
Male clones
2
124.4852
2.50
.18
Pollen storage (fresh vs. LN2)
I
42.6704
.86
.40
Male clones
2
19.0397
.38
.70
5
49.6645
x
pollen storage
Experimental error
II
Total
-
Number of nonshriveled seeds per cone
Female clones
I
19.6010
.23
.65
Male clones
2
85.5492
1.013
.42
Pollen storage (fresh vs. LN2)
I
9.9979
.12
.74
pollen storage
2
2.3924
.28
.97
5
84.6734
Male clones
x
Experimental error
II
Total
procedure probably contributed t o obtaining relatively uniform results both within and
among pollen lots. The procedures also posed little danger for significant pollen damage.
Pollen removed from liquid nitrogen storage was nearly as fertile as fresh pollen.
One drawback of storing Douglas-fir pollen in liquid nitrogen is the increase in the
number of aborted conelets. Why previously frozen pollen should cause more flowers to
abort than fresh pollen of the same parentage, or why that same fresh pollen also causes
more conelet abortion than is observed in the bagged nonpollinated flowers on the same
tree are unknown. One hypothesis is that the pollen is involved in some phase of the
abortion process. It is possible, but unlikely, that too much pollen was applied to each
flower in the 1983 pollinations because similar pollination procedures have been used for
many years and extensive flower abortion is rare. Excess pollen did not promote conelet
abortion in slash pine (Bramlett 1977).
TABLE 3.
Source of variation
Pollen treatments
Male clones (M)
Fresh
M
x
Female
x
x
LN2
x
LN2
0.1632
Fvalues
Significance
of Fvalues
1.2892
0.30
.64
.0612
.4837
1.3445
10.6236
.02
I
.5874
4.6411
.08
2
.0799
.6314
.56
6
.1266
2
pollen treatments
(experimental error)
Total
Mean squares
(6)
no-pollen
Fresh
x
Degrees
freedom
I
Female clones
Pollen
Analysis ofvariance of conelet survival data.
13
VOLUME 31, NUMBER 3, 1985/573
Some contamination from extraneous pollen was possible whenever pollination bags
were removed to pour pollen over each female flower. The number of filled seeds, obtained
from flowers of the nonpollinated controls when the bags were temporarily removed,
showed that about 0.3 percent of the ovules were open-pollinated from undesired sources.
The actual amount of pollen contamination experienced in the two controlled pollination
treatments was probably even lower than 0.3 percent because of the great dilution of
contaminating pollen by the desired pollen when it was poured over each flower. Based
on Ho and Owens' (1974) value of 3 million Douglas-fir pollen grains per gram, the
pollinations with liquid nitrogen-stored and fresh pollen were done with about 92,000 to
240,000 pollen grains for each flower. The time of exposure of flowers from both the
nonpollinated controls and the pollinated treatments to the external environment was equal.
Undesired pollen could have come from surrounding trees, adjacent branches, or catkins
on the same branch as the female flowers that were missed during emasculation. Past field
experience suggests that pollen contamination results primarily from catkins within the
bag that were missed during emasculation. Contamination from that source would be
present equally in syringe, brush, or pouring procedures. Self-fertility in Douglas-fir varies
from 0.1 to 4.5 percent (Sorensen 1971). In any case, the 0.3 percent of undesired male
contribution is not thought to be a serious problem when weighed against the greatly
increased filled seed yields obtained by pouring pollen over the receptive conelets.
Tree improvement workers and researchers should be able to collect Douglas-fir pollen
whenever it is available and store it in liquid nitrogen without having to worry about short­
term fertility loss. One year of storage caused little decrease in fertility and, theoretically,
the Douglas-fir pollen may remain fertile for more years than a worker is likely to need it.
Cryogenic refrigerators have no moving parts and are not dependent upon an external
energy source. Trouble-free storage of pollen should enable tree breeders and others to
undertake desired crosses more easily than if pollen availability and fertility were problems.
LITERATURE CITED
BARNABAS, B., and E. RA.IKI.
1976.
Storage of maize (Zea mays L.) pollen at -1960C (Celsius) in
liquid nitrogen. Euphytica 25(3):747-752.
BRAMLETT, D. L.
1977. Pollen quantity affects cone and seed yields in controlled slash pine polli­
nations. Proc South Forest Tree lmprov Conf (Gainesville, Fla., June 1977) 14, p 28-34.
CARLSON, M., and L. Y. HsiN. 1976. Comparative effectiveness of two control pollination techniques.
Weyerhaeuser Co., Centralia, WA, For Res Tech Rep 50-3201-76-19, 7 p.
CHING, T. M., and K. K. CHING. 1964. Freeze-drying pine pollen. Plant Physiol 39:705-709.
HAUNOLD, A., and P. C. STANWOOD. 1980. Effect of short-term liquid nitrogen storage on hop pollen
fertility (Abstr). Am Soc Agron, Madison, Wisconsin, p 57. (Submitted to Euphytica.)
Ho, R.,and J. OwENS. 1974. Microstrobili of Douglas-fir. Can J Forest Res 4:561-562.
IcHIKAWA, S., and T. SHIDEI.
1971.
Fundamental studies on deep-freezing storage of tree pollen.
Kyoto Univ For Bull 42:51-82.
JENSEN, C. J. 1970. Some factors influencing survival of pollen on storage conditions. Proc IUFRO
Sect 22. Working group on sexual reproduction of forest trees, Varparanta, Finland, 28.5-5.6.
Pap No. 38, 18 p.
LIVINGSTON, G. K., and K. K. CHING. 1967. The longevity and fertility of freeze-dried Douglas-fir
pollen. Silvae Genet 16(3):98-101.
MAZUR, P. 1966. Physical and chemical basis of injury in single-celled and micro-organisms subjected
to freezing and thawing. In Cryobiology
(H. Meryman, ed), p 213-315. Academic Press, New
York. 775 p.
SoRENSEN, F.
1971.
Estimate of self-fertility in coastal Douglas-fir from inbreeding studies. Silvae
Genet 20(4):115-120.
TrssERAT, B., J. M. ULRICH, and B. J. FINKLE. 1983. Survival of Phoenix pollen grains under cryogenic
co1}ditions. Crop Sci 23(2):254-256.
TowiLL, L. E.
1981.
Liquid nitrogen preservation of pollen from tuber-bearing Solanum species.
Hort Sci 16(2):177-179.
574 I FOREST SCIENCE