DNA changes in naturally and DNA changes in naturally and artificially aged longleaf pine artificially aged longleaf pine ((Pinus palustrisPinus palustris Mill) seeds Mill) seeds

E.L. Tolentino, JrE.L. Tolentino, Jr11., W.W. Elam., W.W. Elam22 and F.T. and F.T. BonnerBonner33

11University of the Philippines Los Baños, University of the Philippines Los Baños, 22Mississippi State University, Mississippi State University, 33US Forest US Forest

ServiceService

Outline of PresentationOutline of Presentation

ObjectivesObjectives Germination changesGermination changes DNA changesDNA changes Natural vs artificial agingNatural vs artificial aging Summary and ConclusionsSummary and Conclusions

ObjectivesObjectives

Determine if AAT conditions simulate Determine if AAT conditions simulate natural seed aging processnatural seed aging process

Compare DNA changes in naturally Compare DNA changes in naturally and artificially-aged longleaf pine and artificially-aged longleaf pine seedsseeds

Germination of artificially aged Germination of artificially aged seedsseeds

0102030405060708090

0 48 96 144 192 240 288

Aging period (h)

Germ

inati

on (

%) Alabama

FloridaLouisianaMississippi

Naturally-aged seeds Naturally-aged seeds (Alabama Lot)(Alabama Lot)

0

10

20

30

40

50

60

70

80

0 90 180 270 360

Storage period (days)

Ger

min

atio

n (

%)

Forest conditions

4 degrees C30 degrees C

Naturally-aged seeds Naturally-aged seeds (Florida Lot)(Florida Lot)

0102030405060708090

0 90 180 270 360

Storage period (days)

Ger

min

atio

n (

%)

Forest Conditions4 degrees C30 degrees C

Naturally-aged seeds Naturally-aged seeds (Louisiana Lot)(Louisiana Lot)

0102030405060708090

0 90 180 270 360

Storage period (days)

Ger

min

atio

n (

%)

Forest conditions4 degrees C30 degrees C

Naturally-aged seeds Naturally-aged seeds (Mississippi Lot)(Mississippi Lot)

0102030405060708090

0 90 180 270 360

Storage period (days)

Ger

min

atio

n (

%)

Forest conditions4 degrees C30 degrees C

DNA content of artificially aged DNA content of artificially aged seedsseeds

DNA content (g g –1 seed)

AGING TIME (hr)

ALABAMA FLORIDA LOUISIANA MISSISSIPPI

0 b272.75bc b332.50b a567.67b a498.83b

48 a375.00b a556.25a a422.33d a633.83ab

96 a697.00a a470.75ab a443.00d a629.50ab

144 a632.33a b422.50ab b463.50cd a660.17ab

192 a654.67a b401.50ab a750.50a a801.67a

240 a307.17bc a426.00bc b235.83c a433.50bc

288 c207.83c b448.75ab a549.00bc c213.83c

DNA content of naturally-aged DNA content of naturally-aged seedsseeds

LOT STORAGE CONDITION

DNA content (g g-1 seed)

0 days 90 days 180 days 270 days 360 days

AL Forest conditions

a272.75b a582.17a nd nd nd

4oC a272.75b b227.00b a542.75a a541.00a a561.50a

30oC a272.75b b379.75a b373.25a b323.50a b354.25a

FL Forest conditions

a332.50b b578.00a nd nd nd

4oC a332.50c b531.33abc b552.33ab b457.00bc a739.75a

30oC a332.50b a748.75a a690.75a a727.17a a632.00a

DNA content of naturally-aged DNA content of naturally-aged seedsseeds

LOT STORAGECONDITION

DNA content (g g-1 seed)

0 days 90 days 180 days 270 days 360 days

LA Forestconditions

a567.67b a675.00a nd nd nd

4oC a567.67a b341.00b b421.25b b525.75a a521.67a

30oC a567.67a a725.83a a696.67a a679.17a a583.17a

MS Forestconditions

a498.83a b340.25b nd nd nd

4oC a498.83a a618.33a b570.25a a494.00a b502.00a

30oC a498.83a a707.17a a694.67a a542.00a a715.25a

Natural vs Artificial AgingNatural vs Artificial Aging

In AAT-treated seeds:In AAT-treated seeds: Very low and insignificant correlations between Very low and insignificant correlations between

DNA and % GDNA and % G In Naturally-aged seedsIn Naturally-aged seeds

No clear pattern of relationshipNo clear pattern of relationship Naturally aged and artificially aged seeds Naturally aged and artificially aged seeds

similar % G when projected DNA value is similar % G when projected DNA value is 0.0.

Rate of change between aging regimes not Rate of change between aging regimes not the samethe same

Gel electrophoresis of artificially aged long leaf pine seeds. DNA was treated with 1 l RNAase (Legend: Lane 1 – 1 kb DNA ladder; Lane 2 – control/unaged seeds; Lane 3 – 48 hr AAT; Lane 4 – 96 hr AAT; Lane 5 – 144 hr AAT; Lane 6 – 192 hr AAT; Lane 7 – 240 hr AAT; Lane 8 – 288 hr AAT).

Gel electrophoresis of naturally aged long leaf pine seeds. DNA was treated with 1 l RNAase (Legend: Lane 1 – 4oC, 90 days; Lane 2 – 4oC, 180 days; Lane 3 – 4oC, 270 days; Lane 4 – 4oC, 360 days; Lane 5 –30oC, 90 days; Lane 6 – 30oC, 180 days; Lane 7 – 30oC, 270 days; Lane 8 – 30oC, 360 days).

Gel electrophoresis of naturally aged long leaf pine seeds. DNA was treated with 1 l RNAase (Legend: Lane 1 - 1 kb DNA ladder; Lane 2 – forest condition, 90 days, without RNAase; Land 3 – forest conditions, 90 days with RNAase).

Summary and ConclusionsSummary and Conclusions

For AAT-treated seedsFor AAT-treated seeds Low-high-low pattern of DNA change Low-high-low pattern of DNA change

coinciding with viable-non-viable cycle.coinciding with viable-non-viable cycle. After 192 hr, abrupt increase in DNA After 192 hr, abrupt increase in DNA Sudden drop in DNA after 240 and 288 hrSudden drop in DNA after 240 and 288 hr Fragmented DNA after 144 hrFragmented DNA after 144 hr High MW fragments in seeds after 240 High MW fragments in seeds after 240

and 288 hr (crosslink between DNA and and 288 hr (crosslink between DNA and lipid peroxidation products)lipid peroxidation products)

Summary and ConclusionsSummary and Conclusions

Storage under forest conditionsStorage under forest conditions Increase in DNA after 90 days (dead seeds)Increase in DNA after 90 days (dead seeds) Fragmented DNAFragmented DNA

Storage in 4Storage in 4ooC and 30C and 30ooCC No distinct changes in DNANo distinct changes in DNA Fragmentation began at 270 days of Fragmentation began at 270 days of

storage at 30storage at 30ooCC No fragmentation for seeds stored at 4No fragmentation for seeds stored at 4ooCC

Summary and ConclusionsSummary and Conclusions

AAT may not be useful for examine AAT may not be useful for examine aging process in seedsaging process in seeds

AAT still valid as vigor test for AAT still valid as vigor test for longleaf pine and even other species.longleaf pine and even other species.