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; Jill Barbour, Victor Vankus, Gary Johnson - USDA Forest Service, National Tree Seed Laboratory; Bernard Parresol - USDA Forest Service, Southern Research Station Bernard Parresol - USDA Forest Service, Southern Research Station

2. Purpose of study Purpose of study USDA Forest Service needs to store orthodox tree seed for gene conservation USDA Forest Service needs to store orthodox tree seed for gene conservation Seed deterioration and loss of viability can occur with conventional seed storage Seed deterioration and loss of viability can occur with conventional seed storage Large storage facilities are costly Large storage facilities are costly Viability loss may change original genetic makeup of population Viability loss may change original genetic makeup of population

3. Why cryo-storage? Reduces metabolic seed deterioration Reduces metabolic seed deterioration Long-term maintenance of plant genetic resources Long-term maintenance of plant genetic resources Liquid nitrogen in self contained tanks, not dependent on mechanized refrigeration Liquid nitrogen in self contained tanks, not dependent on mechanized refrigeration Used by USDA National Center for Genetic Resources Preservation Used by USDA National Center for Genetic Resources Preservation

4. 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

5. Liquid Nitrogen Tank Liquid Nitrogen Tank 30 liter tank, no vapor phase 30 liter tank, no vapor phase Seed housed in 1.8 and 2.0 ml cryogenic vials; vials snapped into aluminum canes Seed housed in 1.8 and 2.0 ml cryogenic vials; vials snapped into aluminum canes Nylon stockings held seed in experiment 2 Nylon stockings held seed in experiment 2 Both seed sets placed in aluminum tube, no contact with liquid Both seed sets placed in aluminum tube, no contact with liquid

6. 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

7. Species in Experiment 1 Species in Experiment 1 Abies amabilis Pacific silver fir Abies amabilis Pacific silver fir Abies concolor White fir Abies concolor White fir Abies x shastenis Shasta red fir Abies x shastenis Shasta red fir Calocedrus decurrens Incense cedar Calocedrus decurrens Incense cedar Picea engelmannii Engelmann spruce Picea engelmannii Engelmann spruce Pinus contorta Lodgepole pine Pinus contorta Lodgepole pine Pinus jeffreyi Jeffrey pine Pinus jeffreyi Jeffrey pine Pinus monticola Western white pine Pinus monticola Western white pine Pseudotsuga menziesii Douglas-fir Pseudotsuga menziesii Douglas-fir

8. Experiment 1: Moisture Content (wet weight) Abies amabilis Pacific silver fir 6.85% Abies amabilis Pacific silver fir 6.85% Abies concolor White fir 6.88% Abies concolor White fir 6.88% Abies x shastensis Shasta red fir 6.51% Abies x shastensis Shasta red fir 6.51% Calocedrus decurrens Incense cedar 4.44% Calocedrus decurrens Incense cedar 4.44% Picea engelmannii Engelmann spruce 5.44% Picea engelmannii Engelmann spruce 5.44% Pinus contorta Lodgepole pine 6.85% Pinus contorta Lodgepole pine 6.85% Pinus jeffreyi Jeffrey pine 5.60% Pinus jeffreyi Jeffrey pine 5.60% Pinus monticola Western white pine 5.60% Pinus monticola Western white pine 5.60% Pseudotsuga menziesii Douglas-fir 10.22% Pseudotsuga menziesii Douglas-fir 10.22%

9. Experiment 1: Days of prechilling and temperature Species PrechillTemp( o C) Abies amabilis 21 days15-25 Abies amabilis 21 days15-25 Abies concolor 21 days 20-30 Abies concolor 21 days 20-30 Abies x shastensis 21 days 20-30 Abies x shastensis 21 days 20-30 Calocedrus decurrens 30 days20-30 Calocedrus decurrens 30 days20-30 Picea engelmannii 21 days20-30 Picea engelmannii 21 days20-30 Pinus contorta 21 days 20-30 Pinus contorta 21 days 20-30 Pinus jeffreyi 21 days20-30 Pinus jeffreyi 21 days20-30 Pinus monticola 90 days20-30 Pinus monticola 90 days20-30 Pseudotsuga menziesii 21 days20-30 Pseudotsuga menziesii 21 days20-30

10. Experiment 2: Experiment 2: 42 species examined 42 species examined Treatment was overnight exposure to liquid nitrogen Treatment was overnight exposure to liquid nitrogen Control sample with each treatment per species Control sample with each treatment per species Control – 4 dishes of 100 seeds; treatment – 2 dishes of 100 seeds Control – 4 dishes of 100 seeds; treatment – 2 dishes of 100 seeds Kimpak ® used as media unless AOSA rules states other media required Kimpak ® used as media unless AOSA rules states other media required AOSA rules followed for each species AOSA rules followed for each species Germination - radicle, hypocotyl, & cotyledons present Germination - radicle, hypocotyl, & cotyledons present

11. Statistical Analysis: SAS® Experiment 1: Controls contrasted with treatments in Proc glm Controls contrasted with treatments in Proc glm Controls contrasted to each other Controls contrasted to each other Treatments not contrasted with each other Treatments not contrasted with each other No significant correlation between moisture content and treatments No significant correlation between moisture content and treatments Experiment 2: Proc TTest Proc TTest Folded F test calculated to test for equality of variances Folded F test calculated to test for equality of variances Transformed data analysis same as percentage data analysis Transformed data analysis same as percentage data analysis Moisture correlated with germination on 8 species Moisture correlated with germination on 8 species

12. Experiment 1: Results Experiment 1: Results 24 hour liquid nitrogen exposure had no effect 24 hour liquid nitrogen exposure had no effect 2 species exhibited a negative response to 4 weeks liquid nitrogen exposure 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 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 Germination declined for 5 species in Control D Seed may have degraded in cold storage Seed may have degraded in cold storage

13. Experiment 1: Germination Percentages Experiment 1: Germination Percentages Control Liquid Nitrogen SpeciesABD24 hrs4 wks222 days Abies amabilis 414017424034** Abies concolor 453813373439** Abies x shastensis 4858434446*33 Calocedrus decurrens 111001483 Picea engelmannii 9685899770*85 Pinus contorta 424844514843 Pinus jeffreyi 949390938789 Pinus monticola 697030737270** Pseudotsuga menziesii 716123675538*

14. Experiment 2: Species List Abies balsameaLiriodendron tuliperaPinus contortaPinus strobis Abies basamea var. canaan Lonicera tartaricaPinus echinataPinus sylvestris Abies fraseriMaclura pomiferaPinus elliottiiPinus taeda Acer rubraMalus prunifoliaPinus michoacanaPinus virginiana Celtis occidentalisMorus rubraPinus nigraPlatanus occidentalis Cersis canadensisPhysiocarpus opulifolius Pinus oocarpaPseudotsuga menziesii Fraxinus pennsylvanica Picea abiesPinus palustrisTaxodium distichum var. distichum Ilex glabraPicea glaucaPinus ponderosaThuja occidentalis Juniperus virginianaPicea pungensPinus resinosaVitex-agnus-castus Larix larcinaPinus banksianaPinus rigida Liquidambar styraciflua Pinus clausaPinus serotina

15. Experiment 2: Results Experiment 2: Results 9 out of 42 species were significantly affected by liquid nitrogen 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 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 ) Exposure had a positive effect on 2 species ( Pinus nigra, Pinus rigida )

16. Experiment 2: Average Germination Species Average Control germ % Average LN germ % Acer rubra 4821** Celtis occidentalis 8540** Lonicera tartarica 6244** Malus prunifolia 7449* Physiocarpus opulifolius 2410* Pinus banksiana 8983* Pinus clausa 9284* Pinus nigra 1125** Pinus rigida 8695*

17. Experiment 2: Correlation between moisture & germination Negative correlations Abies fraseri r = - 0.79, n=36, P< 0.001 Abies fraseri r = - 0.79, n=36, P< 0.001 Liriodendron tulipfera r = -0.996, n=12, 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 ponderosa r = 0.94, n=12, P<0.001 Pinus taeda r =0.23, n=96, P= 0.026 Pinus taeda r =0.23, n=96, P= 0.026

18. Problems in experiments Germination not high enough for gene conservation- upgrade seedlots before testing 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 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 nylon stockings cracked when exposed to ambient conditions Seed coats in cryovials did not crack when exposed to ambient conditions Seed coats in cryovials did not crack when exposed to ambient conditions

19. Conclusions Conclusions Seed not adversely affected by liquid nitrogen over time periods tested 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 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 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 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 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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