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 sub-

treatment of foil laminate bags at -20o C

Moist Seed

Humid air (70%)

The condition of a high equilibrium relative humidity. At

equilibrium this seed will measure an ERH of 70%.

Seeds take up water

in moist air

Dry Seed

Dry air (30% relative humidity)

The condition of a low equilibrium relative humidity. At

equilibrium this seed will measure an ERH of 30%.

Seeds lose

water in dry

air

Hygrometer measuring ERH of seeds in a covered box

Water Activity Meter

Probe

Meter

Sample holder

A water activity (Aw)

meter used to measure ERH.

Comparison of Meters

• Both meters gave almost exact same

reading

– Variation among meters about .2 or so

– So close the readings were not recorded

y = 7E-05x3 - 0.0071x2 + 0.2975x + 2.409R² = 0.9899

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

M

o

i

s

t

u

r

e

C

o

n

t

e

n

t

Equilibrium Relative Humidity

Series1

Poly. (Series1)

Artemisia tridentata ssp wyomingensis

y = 7E-05x3 - 0.0071x2 + 0.2975x + 2.409R² = 0.9899

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

M

o

i

s

t

u

r

e

C

o

n

t

e

n

t

Equilibrium Relative Humidity

Series1

Poly. (Series1)

Artemisia tridentata ssp wyomingensis

Biologically

active

waterTightly bond

water

Hard to

remove

Water loss =

Membrane damage

More water

= more

enzyme

activity

Water in this range

promotes microbial

activity and seed

deterioration

y = 7E-05x3 - 0.0071x2 + 0.2975x + 2.409R² = 0.9899

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Series1

Poly. (Series1)

Artemisia tridentata ssp wyomingensis

Storage ERH of this study

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

0

10

20

30

40

50

60

70

% G

erm

inati

on

-20 -8 2 20

70

50

40

30

Storage Temperature C

ERH %

Sagebrush Seed Storage Lot WP High Purity Seed

3 months

Initial

germination

was 56%

Blank

0

20

40

60

80

% G

erm

inati

on

-20 -8 2 20

70

50

40

30

Storage Temperature C

ERH %

Sagebrush Seed Storage Lot WP High Purity Seed

6 months

Initial

germination

was 56%

Blank

0

20

40

60

80

% G

erm

inati

on

-20 -8 2 20

70

50

40

30

Storage Temperature C

ERH %

Sagebrush Seed Storage Lot WP High Purity Seed

15 monthsInitial

germination

was 56%

Blank

Germination at 29 months, High

Purity Seeds, Average of 5 seed lots

50

40

30

0

10

20

30

40

50

60

70

-20 foil-20

-82

ERH

G

e

r

m

i

n

a

t

i

o

n

%

Storage Temperature

Germination at 41 months,

High Purity Seeds,

N/T

0

10

20

30

40

50

60

70

80

-20 foil-20

-82

G

e

r

m

i

n

a

t

i

o

n

%

Storage Temperature

50-29

50-41

40-29

40-41

30-29

30-41

Comparing 29 month

and 41 month data

70

50

30

0

50

100

-20 -8 2 20

Nu

mb

er

of

Seed

lin

gs p

er

Gra

m

Storage Temperature C

Sagebrush Seed Storage Low Purity Lot CA2

6 months

Initial

germination

was 48

seedlings per

gram of

seeds

Blank

70

50

30

0

50

100

-20 -8 2 20

Nu

mb

er

of

Seed

lin

gs p

er

Gra

m

Storage Temperature C

Sagebrush Seed Storage Low Purity Lot CA2

15 months

Initial

germination

was 48

seedlings per

gram.

Blank

70

50

40

30

0

50

100

-20 -8 2 20ERH %

Nu

mb

er

of

Seed

lin

gs p

er

Gra

m

Storage Temperature C

Sagebrush Seed Storage Low Purity Lot CA2

29 monthsInitial

germination

was 48

seedlings

per gram.

Blank

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

y = 7E-05x3 - 0.0071x2 + 0.2975x + 2.409R² = 0.9899

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Series1

Poly. (Series1)

Artemisia tridentata ssp wyomingensis

Storage ERH of this study

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