1. DNA changes in naturally and artificially aged longleaf pine (Pinus palustris Mill) seeds E.L. Tolentino, Jr 1., W.W. Elam 2 and F.T. Bonner 3 1 University of the Philippines Los Baños, 2 Mississippi State University, 3 US Forest Service

2. Outline of Presentation Objectives Objectives Germination changes Germination changes DNA changes DNA changes Natural vs artificial aging Natural vs artificial aging Summary and Conclusions Summary and Conclusions

3. Objectives Determine if AAT conditions simulate natural seed aging process Determine if AAT conditions simulate natural seed aging process Compare DNA changes in naturally and artificially-aged longleaf pine seeds Compare DNA changes in naturally and artificially-aged longleaf pine seeds

4. Germination of artificially aged seeds

5. Naturally-aged seeds (Alabama Lot)

6. Naturally-aged seeds (Florida Lot)

7. Naturally-aged seeds (Louisiana Lot)

8. Naturally-aged seeds (Mississippi Lot)

9. DNA content of artificially aged seeds DNA content ( g g –1 seed) AGING TIME (hr) ALABAMAFLORIDALOUISIANAMISSISSIPPI 0 b 272.75 bcb 332.50 ba 567.67 ba 498.83 b 48 a 375.00 ba 556.25 aa 422.33 da 633.83 ab 96 a 697.00 aa 470.75 aba 443.00 da 629.50 ab 144 a 632.33 ab 422.50 abb 463.50 cda 660.17 ab 192 a 654.67 ab 401.50 aba 750.50 aa 801.67 a 240 a 307.17 bca 426.00 bcb 235.83 ca 433.50 bc 288 c 207.83 cb 448.75 aba 549.00 bcc 213.83 c

10. DNA content of naturally-aged seeds LOTSTORAGE CONDITION DNA content ( g g -1 seed) 0 days90 days180 days270 days360 days ALForest conditions a 272.75 ba 582.17 a nd 4oC4oC a 272.75 bb 227.00 ba 542.75 aa 541.00 aa 561.50 a 30 o C a 272.75 bb 379.75 ab 373.25 ab 323.50 ab 354.25 a FLForest conditions a 332.50 bb 578.00 a nd 4oC4oC a 332.50 cb 531.33 abcb 552.33 abb 457.00 b c a 739.75 a 30 o C a 332.50 ba 748.75 aa 690.75 aa 727.17 aa 632.00 a

11. DNA content of naturally-aged seeds LOTSTORAGE CONDITION DNA content ( g g -1 seed) 0 days90 days180 days270 days360 days LAForest conditions a 567.67 ba 675.00 a nd 4oC4oC a 567.67 ab 341.00 bb 421.25 bb 525.75 aa 521.67 a 30 o C a 567.67 aa 725.83 aa 696.67 aa 679.17 aa 583.17 a MSForest conditions a 498.83 ab 340.25 b nd 4oC4oC a 498.83 aa 618.33 ab 570.25 aa 494.00 ab 502.00 a 30 o C a 498.83 aa 707.17 aa 694.67 aa 542.00 aa 715.25 a

12. Natural vs Artificial Aging In AAT-treated seeds: In AAT-treated seeds: Very low and insignificant correlations between DNA and % G Very low and insignificant correlations between DNA and % G In Naturally-aged seeds In Naturally-aged seeds No clear pattern of relationship No clear pattern of relationship Naturally aged and artificially aged seeds similar % G when projected DNA value is 0. Naturally aged and artificially aged seeds similar % G when projected DNA value is 0. Rate of change between aging regimes not the same Rate of change between aging regimes not the same

13. 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).

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

15. 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).

16. Summary and Conclusions For AAT-treated seeds For AAT-treated seeds Low-high-low pattern of DNA change coinciding with viable-non-viable cycle. Low-high-low pattern of DNA change 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 hr Sudden drop in DNA after 240 and 288 hr Fragmented DNA after 144 hr Fragmented DNA after 144 hr High MW fragments in seeds after 240 and 288 hr (crosslink between DNA and lipid peroxidation products) High MW fragments in seeds after 240 and 288 hr (crosslink between DNA and lipid peroxidation products)

17. Summary and Conclusions Storage under forest conditions Increase in DNA after 90 days (dead seeds) Fragmented DNA Storage in 4oC and 30oC No distinct changes in DNA Fragmentation began at 270 days of storage at 30oC No fragmentation for seeds stored at 4oC

18. Summary and Conclusions AAT may not be useful for examine aging process in seeds AAT may not be useful for examine aging process in seeds AAT still valid as vigor test for longleaf pine and even other species. AAT still valid as vigor test for longleaf pine and even other species.