Effect of Liquid Nitrogen Effect of Liquid Nitrogen Storage on Seed Storage on Seed

Germination of 51 Tree Germination of 51 Tree SpeciesSpecies

Jill Barbour, Victor Vankus, Jill Barbour, Victor Vankus, Gary Johnson - Gary Johnson - USDA Forest USDA Forest

Service, National Tree Seed Laboratory;Service, National Tree Seed Laboratory;

Bernard Parresol - Bernard Parresol - USDA Forest USDA Forest

Service, Southern Research StationService, Southern Research Station

Purpose of studyPurpose of study

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

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

• Large storage facilities are costlyLarge storage facilities are costly• Viability loss may change original Viability loss may change original

genetic makeup of populationgenetic makeup of population

Why cryo-storage?Why cryo-storage?

• Reduces metabolic seed Reduces metabolic seed deteriorationdeterioration

• Long-term maintenance of plant Long-term maintenance of plant genetic resourcesgenetic resources

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

• Used by USDA National Center for Used by USDA National Center for Genetic Resources Preservation Genetic Resources Preservation

Study PlanStudy 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 Liquid Nitrogen TankTank

• 30 liter tank, no vapor phase 30 liter tank, no vapor phase • Seed housed in 1.8 and 2.0 ml Seed housed in 1.8 and 2.0 ml

cryogenic vials; vials snapped cryogenic vials; vials snapped into aluminum canesinto aluminum canes

• Nylon stockings held seed in Nylon stockings held seed in experiment 2experiment 2

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

Experiment 1Experiment 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 Species in Experiment 1Experiment 1

• Abies amabilis Pacific silver fir Abies amabilis Pacific silver fir • Abies concolorAbies concolor White fir White fir• Abies x shastenis Shasta red firAbies x shastenis Shasta red fir• Calocedrus decurrens Incense cedarCalocedrus decurrens Incense cedar• Picea engelmannii Engelmann Picea engelmannii Engelmann

spruce spruce • Pinus contorta Lodgepole pinePinus contorta Lodgepole pine• Pinus jeffreyi Jeffrey pinePinus jeffreyi Jeffrey pine• Pinus monticola Western white Pinus monticola Western white

pinepine• Pseudotsuga menziesii Douglas-firPseudotsuga menziesii Douglas-fir

Experiment 1: Moisture Content Experiment 1: Moisture Content (wet weight)(wet weight)

• Abies amabilis Abies amabilis Pacific silver fir 6.85%Pacific silver fir 6.85%• Abies concolorAbies concolor White fir White fir 6.88%6.88%• Abies x shastensis Abies x shastensis Shasta red fir 6.51%Shasta red fir 6.51%• Calocedrus decurrens Calocedrus decurrens Incense cedar 4.44%Incense cedar 4.44%• Picea engelmannii Picea engelmannii Engelmann spruceEngelmann spruce 5.44%5.44%• Pinus contorta Pinus contorta Lodgepole pine 6.85%Lodgepole pine 6.85%• Pinus jeffreyi Pinus jeffreyi Jeffrey pine 5.60%Jeffrey pine 5.60%• Pinus monticola Pinus monticola Western white pine 5.60%Western white pine 5.60%• Pseudotsuga menziesii Pseudotsuga menziesii Douglas-fir 10.22%Douglas-fir 10.22%

Experiment 1: Days of prechilling and Experiment 1: Days of prechilling and temperature temperature SpeciesSpecies PrechillPrechill Temp(Temp(ooC)C)

• Abies amabilisAbies amabilis 21 days21 days 15-2515-25• Abies concolorAbies concolor 2121 daysdays 20-3020-30• Abies x shastensis Abies x shastensis 21 days21 days 20-3020-30• Calocedrus decurrens Calocedrus decurrens 30 days30 days 20-3020-30• Picea engelmanniiPicea engelmannii 21 days21 days 20-3020-30• Pinus contorta Pinus contorta 21 days21 days 20-3020-30• Pinus jeffreyiPinus jeffreyi 21 days21 days 20-3020-30• Pinus monticolaPinus monticola 90 days90 days 20-3020-30• Pseudotsuga menziesii Pseudotsuga menziesii 21 days21 days 20-3020-30

Experiment 2:Experiment 2:• 42 species examined42 species examined• Treatment was overnight exposure to Treatment was overnight exposure to

liquid nitrogenliquid nitrogen• Control sample with each treatment per Control sample with each treatment per

speciesspecies• Control – 4 dishes of 100 seeds; Control – 4 dishes of 100 seeds;

treatment – 2 dishes of 100 seeds treatment – 2 dishes of 100 seeds • KimpakKimpak®® used as media unless AOSA used as media unless AOSA

rules states other media requiredrules states other media required• AOSA rules followed for each speciesAOSA rules followed for each species• Germination - radicle, hypocotyl, & Germination - radicle, hypocotyl, &

cotyledons present cotyledons present

Statistical Analysis: SAS® Statistical Analysis: SAS®

Experiment 1:Experiment 1:• Controls contrasted Controls contrasted

with treatments in with treatments in Proc glmProc glm

• Controls contrasted Controls contrasted to each otherto each other

• Treatments not Treatments not contrasted with each contrasted with each otherother

• No significant No significant correlation between correlation between moisture content and moisture content and treatmentstreatments

Experiment 2:Experiment 2:• Proc TTest Proc TTest • Folded F test Folded F test

calculated to test calculated to test for equality of for equality of variancesvariances

• Transformed data Transformed data analysis same as analysis same as percentage data percentage data analysisanalysis

• Moisture correlated Moisture correlated with germination on with germination on 8 species8 species

Experiment 1: Experiment 1: ResultsResults• 24 hour liquid nitrogen exposure had no 24 hour liquid nitrogen exposure had no

effect effect • 2 species exhibited a negative response 2 species exhibited a negative response

to 4 weeks liquid nitrogen exposure to 4 weeks liquid nitrogen exposure • 4 species exhibited a positive response 4 species exhibited a positive response

to 222 day liquid nitrogen exposure to 222 day liquid nitrogen exposure when compared with control Dwhen compared with control D

• Germination declined for 5 species in Germination declined for 5 species in Control D Control D

• Seed may have degraded in cold storageSeed may have degraded in cold storage

Experiment 1: Germination PercentagesExperiment 1: Germination Percentages

Control Liquid Nitrogen

Species A B D 24 hrs 4 wks 222 days

Abies amabilisAbies amabilis 4141 4040 1717 4242 4040 34**34**

Abies concolorAbies concolor 4545 3838 1313 3737 3434 39**39**

Abies x Abies x shastensisshastensis

4848 5858 4343 4444 46*46* 3333

Calocedrus Calocedrus decurrensdecurrens

1111 1010 00 1414 88 33

Picea Picea engelmanniiengelmannii

9696 8585 8989 9797 70*70* 8585

Pinus contortaPinus contorta 4242 4848 4444 5151 4848 4343

Pinus jeffreyiPinus jeffreyi 9494 9393 9090 9393 8787 8989

Pinus Pinus monticolamonticola

6969 7070 3030 7373 7272 70**70**

Pseudotsuga Pseudotsuga menziesiimenziesii

7171 6161 2323 6767 5555 38*38*

Experiment 2: Species ListExperiment 2: Species ListAbies balsamea Liriodendron

tuliperaPinus contorta Pinus strobis

Abies basamea var. canaan

Lonicera tartarica

Pinus echinata Pinus sylvestris

Abies fraseri Maclura pomifera

Pinus elliottii Pinus taeda

Acer rubra Malus prunifolia Pinus michoacana

Pinus virginiana

Celtis occidentalis

Morus rubra Pinus nigra Platanus occidentalis

Cersis canadensis

Physiocarpus opulifolius

Pinus oocarpa Pseudotsuga menziesii

Fraxinus pennsylvanica

Picea abies Pinus palustris Taxodium distichum var. distichum

Ilex glabra Picea glauca Pinus ponderosa Thuja occidentalis

Juniperus virginiana

Picea pungens Pinus resinosa Vitex-agnus-castus

Larix larcina Pinus banksiana Pinus rigida

Liquidambar styraciflua

Pinus clausa Pinus serotina

Experiment 2: Experiment 2: ResultsResults

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

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

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

Experiment 2:Experiment 2: Average Average GerminationGermination

SpeciesSpecies Average Average Control germ Control germ %%

Average Average

LN germ % LN germ %

Acer rubra Acer rubra 4848 21**21**

Celtis occidentalisCeltis occidentalis 8585 40**40**

Lonicera tartaricaLonicera tartarica 6262 44**44**

Malus prunifoliaMalus prunifolia 7474 49*49*

Physiocarpus Physiocarpus opulifoliusopulifolius

2424 10*10*

Pinus banksianaPinus banksiana 8989 83*83*

Pinus clausaPinus clausa 9292 84*84*

Pinus nigraPinus nigra 1111 25**25**

Pinus rigida Pinus rigida 8686 95*95*

Experiment 2: Correlation Experiment 2: Correlation between moisture & between moisture & germinationgermination

Negative Negative correlationscorrelations

• Abies fraseriAbies fraseri r = r = -0.79, n=36, P< -0.79, n=36, P< 0.0010.001

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

Positive correlationsPositive correlations • Pinus Pinus

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

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

Problems in experiments Problems in experiments • Germination not high enough for gene Germination not high enough for gene

conservation- upgrade seedlots before conservation- upgrade seedlots before testingtesting

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

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

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

Conclusions Conclusions

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

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

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

Conclusions continued Conclusions continued • 10 tree species tested at the USDA 10 tree species tested at the USDA

National Center for Genetic Resources National Center for Genetic Resources Preservation were not adversely Preservation were not adversely affected by 1 to 3 years liquid nitrogen affected by 1 to 3 years liquid nitrogen exposureexposure

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

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