Wyoming Big Sagebrush
Germination After 41 Months
Storage in Sealed Containers
Storage of Sagebrush in Sealed
Containers –Why?
• Sagebrush seeds are relatively short lived
• Genetic conservation via long term
(decades long) seed storage
• Have seed sources on hand for
emergency rehabilitation
• Preserve seed viability in multiyear
collections needed for common gardens
and other genetics work
Materials
• 5 commercially produced seed lots were purchased
in fall of 2006 – co-investigator Nancy Shaw
• Lots split in half
– One half designated low purity
– One half cleaned to remove most of the trash (as
determined by simple inspection) and designated
high purity
• Cleaned by nursery personnel at FS Lucky
Peak nursery at Boise ID
• Cleaned on a Westrup LA-LSU seed cleaner
Methods
• Each lot was mixed in a riffle seed divider
to ensure uniformity among subsamples
– 4 fractions each, one for each of 4 ERH
• After moisture equilibration
– Each ERH Fraction again divided with the
riffle divider into 4 fractions , one for each
temperature
– 16 subsamples are now prepared
Methods
• Seed Moisture
– Equilibrium relative humidity (ERH)
– Adjusted seeds to 30, 40, 50, 70 ERH
• Found ambient conditions and placed the seeds in
that condition – some indoors some out of doors
• Repeat measures of ERH until measurements
indicated equilibration was achieved
• Storage Temperature: 20o, 3o, -8o, - 20oC
• Storage containers were 6mil poly bags with a subtreatment of foil laminate bags at -20o C
Humid air (70%)
Seeds take up water
in moist air
Moist Seed
The condition of a high equilibrium relative humidity. At
equilibrium this seed will measure an ERH of 70%.
Dry air (30% relative humidity)
Seeds lose
water in dry
air
Dry Seed
The condition of a low equilibrium relative humidity. At
equilibrium this seed will measure an ERH of 30%.
Hygrometer measuring ERH of seeds in a covered box
Probe
Sample holder
A water activity (Aw)
meter used to measure ERH.
Meter
Water
Activity
Meter
Comparison of Meters
• Both meters gave almost exact same
reading
– Variation among meters about .2 or so
– So close the readings were not recorded
Artemisia tridentata ssp wyomingensis
24.0
M
o
i
s
t
u
r
e
22.0
y = 7E-05x3 - 0.0071x2 + 0.2975x + 2.409
R² = 0.9899
20.0
18.0
16.0
14.0
12.0
Series1
Poly. (Series1
C 10.0
o
n 8.0
t 6.0
e
n 4.0
t 2.0
0.0
0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Equilibrium Relative Humidity
Artemisia tridentata ssp wyomingensis
24.0
M
o
i
s
t
u
r
e
22.0
y = 7E-05x3 - 0.0071x2 + 0.2975x + 2.409
R² = 0.9899
20.0
18.0
Water loss =
Membrane damage
16.0
14.0
12.0
Tightly bond
water
Hard to
remove
C 10.0
o
n 8.0
t 6.0
e
n 4.0
t 2.0
Biologically
active
water
Series1
Poly. (Series1
More water
= more
enzyme
activity
Water in this range
promotes microbial
activity and seed
deterioration
0.0
0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Equilibrium Relative Humidity
Artemisia tridentata ssp wyomingensis
24.0
22.0
y = 7E-05x3 - 0.0071x2 + 0.2975x + 2.409
R² = 0.9899
20.0
18.0
16.0
14.0
12.0
Series1
Poly. (Series1)
10.0
8.0
6.0
4.0
Storage ERH of this study
2.0
0.0
0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Storage Containers
• 16 fractions (4 ERH x 4 Temps) sealed in
6 mil poly bags for each of the 5 seed lots
– 80 bags of seed
• 2 fractions (30 and 40 ERH) for each of
the 5 seed lots was sealed in a foil
laminate bag
– 10 foil bags
– Done to see if this might be a better container
– This is the foil bag used at NCGRP, security
backup
Germination Methods
•
•
•
•
•
4 x 50 seeds (4 x 25 in 2010 tests)
Constant 15o C with light (AOSA rule)
Counts made at 7, 14, 21, 28 days
Final germ often at 14 days (rule is 10 dy)
Blue blotters in plastic jar with screw lid
– Jar petri dish diameter
– Inverted jar using lid as dish and jar as cover
Sagebrush Seed Storage Lot WP High Purity Seed
3 months
30
40
50
70
Blank
-20
-8
2
Storage Temperature C
20
%
70
60
50
40
30
20
10
0
ER
H
% Germination
Initial
germination
was 56%
Sagebrush Seed Storage Lot WP High Purity Seed
6 months
Initial
germination
was 56%
60
40
30
20
50
0
70
Blank
-20
-8
2
Storage Temperature C
20
%
40
ER
H
% Germination
80
Sagebrush Seed Storage Lot WP High Purity Seed
15 months
Initial
germination
was 56%
60
40
30
20
50
0
70
Blank
-20
-8
2
Storage Temperature C
20
%
40
ER
H
% Germination
80
Germination at 29 months, High
Purity Seeds, Average of 5 seed
lots
Germination at 41 months,
High Purity Seeds,
G
e
r
m
i
n
a
t
i
o
n
%
70
60
50
40
30
20
10
0
30
N/T
-20 foil
40
-20
50 ERH
-8
Storage Temperature
2
80
G
e
r
m
i
n
a
t
i
o
n
%
Comparing 29 month
and 41 month data
70
60
50
50-29
50-41
40-29
40-41
30-29
30-41
40
30
20
10
0
-20 foil
-20
-8
Storage Temperature
2
Number of Seedlings per Gram
Sagebrush Seed Storage Low Purity Lot CA2
6 months
Initial
germination
was 48
seedlings per
gram of
seeds
100
50
30
50
0
70
Blank
-20
-8
2
20
Storage Temperature C
Number of Seedlings per Gram
Sagebrush Seed Storage Low Purity Lot CA2
15 months
Initial
germination
was 48
seedlings per
gram.
100
50
30
50
0
70
Blank
-20
-8
2
20
Storage Temperature C
Number of Seedlings per Gram
Sagebrush Seed Storage Low Purity Lot CA2
29 months
Initial
germination
was 48
seedlings
per gram.
100
50
30
40
50
0
70
Blank
-20
-8
2
20
Storage Temperature C
ERH %
Findings
Low Purity Seeds
• Cleaning the seed appears to make it live
longer in seal container storage.
• If the seed is not cleaned it must be at
30% ERH and frozen when put in sealed
containers.
Findings
High Purity Seeds
• ERH was maintained well in both poly and foil
bag
• 30, 40, and 50% ERH were equivalent in
preserving germ at 41 months of storage
• -20oC was the best storage temperature
• Wyoming sagebrush seed will store well at
least 5 years in sealed containers
Artemisia tridentata ssp wyomingensis
24.0
22.0
y = 7E-05x3 - 0.0071x2 + 0.2975x + 2.409
R² = 0.9899
20.0
18.0
16.0
14.0
12.0
Series1
Poly. (Series1)
10.0
8.0
6.0
4.0
Storage ERH of this study
2.0
0.0
0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
A FEW OTHER THINGS
Variation in Equilibration Time
by Species
• Artimesia tridentata ssp wyomingensis
– 4 to 5 million seeds per kilo
– Equilibrates very quickly
• Pinus palustris
– 8 to 10 thousand seeds per kilo
– Can take16 to 24 hours to equilibrate in test chamber
if not at a stabile moisture condition.
Key points of ERH
 Can be used on any seed lot regardless of
 Viability
 Purity
 Stage of extraction
 Species
 No conversion charts needed
 Traceable standards available for calibration
 Non destructive
 Fast – Results obtained in a few minutes
 Very easy to use
 Low cost equipment ($70 to $2700)
Germination Protocols
• Indian ricegrass and penstemons
• Have tried moist, 3o C stratification
– 30, 60, 90 days little or no germnation
– Longer strat periods are now being tested
• Indian ricegrass did give uniform germ at
about 120 days at 3o C
– Will strat for 60, 90, 120 days
– Germ at 5-10 and other cool temps