Effect of liquid nitrogen storage on seed germination of 51 tree species
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Effect of Liquid Nitrogen Storage on Seed Germination of 51 Tree Species. Jill Barbour, Victor Vankus, Gary Johnson - USDA Forest Service, National Tree Seed Laboratory; Bernard Parresol - USDA Forest Service, Southern Research Station. Purpose of study.

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Effect of Liquid Nitrogen Storage on Seed Germination of 51 Tree Species

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Effect of liquid nitrogen storage on seed germination of 51 tree species

Effect of Liquid Nitrogen Storage on Seed Germination of 51 Tree Species

Jill Barbour, Victor Vankus, Gary Johnson - USDA Forest Service, National Tree Seed Laboratory;

Bernard Parresol - USDA Forest Service, Southern Research Station


Purpose of study

Purpose of study

  • USDA Forest Service needs to store orthodox tree seed for gene conservation

  • Seed deterioration and loss of viability can occur with conventional seed storage

  • Large storage facilities are costly

  • Viability loss may change original genetic makeup of population


Why cryo storage

Why cryo-storage?

  • Reduces metabolic seed deterioration

  • Long-term maintenance of plant genetic resources

  • Liquid nitrogen in self contained tanks, not dependent on mechanized refrigeration

  • Used by USDA National Center for Genetic Resources Preservation


Study plan

Study Plan

  • 2 experiments

  • Experiment 1 – 9 western tree species

  • Experiment 2 – 42 tree species

  • Prechill time periods not used in analysis

  • AOSA rules followed for species


Liquid nitrogen tank

Liquid Nitrogen Tank

  • 30 liter tank, no vapor phase

  • Seed housed in 1.8 and 2.0 ml cryogenic vials; vials snapped into aluminum canes

  • Nylon stockings held seed in experiment 2

  • Both seed sets placed in aluminum tube, no contact with liquid


Experiment 1

Experiment 1

  • 9 western forest tree species

  • 3 time periods: 24 hours, 4 weeks, 222 days; each time period had a control (A, B, D)

  • Planted into 8 germination dishes of 25 seeds each – 200 seed samples

  • Kimpak® as media except with true firs where metromix® was used

  • 80 ml of water per germination dish; double for large dishes

  • Germination - Radicle, hypocotyl, &cotyledons present


Species in experiment 1

Species in Experiment 1

  • Abies amabilis Pacific silver fir

  • Abies concolor White fir

  • Abies x shastenis Shasta red fir

  • Calocedrus decurrens Incense cedar

  • Picea engelmannii Engelmann spruce

  • Pinus contorta Lodgepole pine

  • Pinus jeffreyi Jeffrey pine

  • Pinus monticola Western white pine

  • Pseudotsuga menziesii Douglas-fir


Experiment 1 moisture content wet weight

Experiment 1: Moisture Content (wet weight)

  • Abies amabilis Pacific silver fir 6.85%

  • Abies concolor White fir 6.88%

  • Abies x shastensis Shasta red fir 6.51%

  • Calocedrus decurrens Incense cedar 4.44%

  • Picea engelmannii Engelmann spruce5.44%

  • Pinus contorta Lodgepole pine 6.85%

  • Pinus jeffreyi Jeffrey pine 5.60%

  • Pinus monticola Western white pine 5.60%

  • Pseudotsuga menziesii Douglas-fir 10.22%


Experiment 1 days of prechilling and temperature species prechill temp o c

Experiment 1: Days of prechilling and temperature SpeciesPrechillTemp(oC)

  • Abies amabilis 21 days15-25

  • Abies concolor 21days20-30

  • Abies x shastensis 21 days20-30

  • Calocedrus decurrens 30 days20-30

  • Picea engelmannii 21 days20-30

  • Pinus contorta 21 days20-30

  • Pinus jeffreyi 21 days20-30

  • Pinus monticola 90 days20-30

  • Pseudotsuga menziesii 21 days20-30


Experiment 2

Experiment 2:

  • 42 species examined

  • Treatment was overnight exposure to liquid nitrogen

  • Control sample with each treatment per species

  • Control – 4 dishes of 100 seeds; treatment – 2 dishes of 100 seeds

  • Kimpak® used as media unless AOSA rules states other media required

  • AOSA rules followed for each species

  • Germination - radicle, hypocotyl, & cotyledons present


Statistical analysis sas

Experiment 1:

Controls contrasted with treatments in Proc glm

Controls contrasted to each other

Treatments not contrasted with each other

No significant correlation between moisture content and treatments

Experiment 2:

Proc TTest

Folded F test calculated to test for equality of variances

Transformed data analysis same as percentage data analysis

Moisture correlated with germination on 8 species

Statistical Analysis: SAS®


Experiment 1 results

Experiment 1: Results

  • 24 hour liquid nitrogen exposure had no effect

  • 2 species exhibited a negative response to 4 weeks liquid nitrogen exposure

  • 4 species exhibited a positive response to 222 day liquid nitrogen exposure when compared with control D

  • Germination declined for 5 species in Control D

  • Seed may have degraded in cold storage


Experiment 1 germination percentages control liquid nitrogen

Experiment 1: Germination PercentagesControl Liquid Nitrogen


Experiment 2 species list

Experiment 2: Species List


Experiment 2 results

Experiment 2: Results

  • 9 out of 42 species were significantly affected by liquid nitrogen

  • Exposure had a negative effect on 7 species (Acer rubra, Celtis occidentalis, Lonicera tartarica, Malus prunifolia, Physiocarpus opulifolius, Pinus banksiana, Pinus clausa)

  • Exposure had a positive effect on 2 species (Pinus nigra, Pinus rigida)


Experiment 2 average germination

Experiment 2:Average Germination


Experiment 2 correlation between moisture germination

Negative correlations

Abies fraseri r = -0.79, n=36, P< 0.001

Liriodendron tulipfera r = -0.996, n=12, P<0.001

Positive correlations

Pinus ponderosa r = 0.94, n=12, P<0.001

Pinus taeda r =0.23, n=96, P= 0.026

Experiment 2: Correlation between moisture & germination


Problems in experiments

Problems in experiments

  • Germination not high enough for gene conservation- upgrade seedlots before testing

  • Seed degradation, not liquid nitrogen was probable cause of viability loss in Control D and 222 day treatment

  • Seed coats in nylon stockings cracked when exposed to ambient conditions

  • Seed coats in cryovials did not crack when exposed to ambient conditions


Conclusions

Conclusions

  • Seed not adversely affected by liquid nitrogen over time periods tested

  • Extrapolation beyond scope of experiment is not valid- 24 hours not equal to 100 years

  • Longer exposure to liquid nitrogen may have adverse effect on germination


Conclusions continued

Conclusions continued

  • 10 tree species tested at the USDA National Center for Genetic Resources Preservation were not adversely affected by 1 to 3 years liquid nitrogen exposure

  • Abies concolor, Abies procera, Picea sp. Brewer, Pinus lambertiana, Pinus ponderosa, Pseudotsuga menziesii, Pyrus malus, Thuja plicata, Tsuga heterophylla, Ulmus americana


Effect of liquid nitrogen storage on seed germination of 51 tree species

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