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

1. 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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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%

9. Experiment 1: Days of prechilling and temperature SpeciesPrechillTemp(oC) • Abies amabilis 21 days 15-25 • Abies concolor 21days20-30 • Abies x shastensis 21 days20-30 • Calocedrus decurrens 30 days 20-30 • Picea engelmannii 21 days 20-30 • Pinus contorta 21 days20-30 • Pinus jeffreyi 21 days 20-30 • Pinus monticola 90 days 20-30 • Pseudotsuga menziesii 21 days 20-30

10. 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

11. 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®

12. 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

13. Experiment 1: Germination PercentagesControl Liquid Nitrogen

14. Experiment 2: Species List

15. 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)

16. Experiment 2:Average Germination

17. 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

18. 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

19. 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

20. 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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