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

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

Bernard Parresol - USDA Forest Service, Southern Research Station


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?

  • 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

  • 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

  • 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

  • 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

  • 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)

  • 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 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:

  • 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


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

  • 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 PercentagesControl Liquid Nitrogen


Experiment 2: Species List


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


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

  • 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

  • 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

  • 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


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