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Gymnosperm seedling sensitivity to gamma radiation: Its relation to seed radiosensitivity and nuclear variables
Radiation Botany, 1971, Vol. 11, pp. 45 to 51. Pergamon Press. Printed in Great Britain.
GYMNOSPERM SEEDLING SENSITIVITY TO GAMMA RADIATION: ITS RELATION TO SEED RADIOSENSITIVITY AND NUCLEAR VARIABLES T. D. R U D O L P H
North Central Forest Experimental Station, U.S.D.A. Forest Service, Institute of Forest Genetics, Rhinelander, Wisconsin, U.S.A. (Received 17 July 1970)
RUDOLPH,T. D., Gymnospermseedling sensitivity to gamma radiation: Its relation to seed radiosensitivity and nuclear variables. RADIATIONBOTANY 11~ 45--51, 1971.--Newly germinated seedlings of Larix laricina, Picea glauca, Picea mariana, Pinus banksiana, Pinus contorta, Pinus resinosa, and Thuja occidentalis were gamma irradiated at exposures of 150, 300, 450, 600, 900, 1200, 1800, 2400 and 3600 R. Number of leaves produced and shoot dry weight were determined 50 days after irradiation and survival and shoot dry weight after 130 days. Of the 4 endpoints scored, shoot dry weight at 50 days showed greatest radiosensitivity with a Ds0 ranging from 195 R for Pinus resinosa to 380 R for Picea mariana. Except for shoot dry weight at 50 days, all endpoints showed a sharp response over a relatively narrow range ofexposures, generally between 600 and 900 R. The 7 species differed in seedling radiosensitivity by a factor of less than 2 for all endpoints. Comparison with seed radiosensitivity of the same populations showed that seedlings are from 7 to 15 times more sensitive than seeds. Correlation analyses showed that the only relationship among the endpoints studied was between Ds0 for number of leaves at 50 days and shoot dry weight at 130 days: the seedlings with the greatest number of leaves at 50 days had the greatest dry weight at 130 days. No relation was found between the Ds0 following seedling irradiation and the Ds0 for irradiated seed previously reported for the same populations of the 7 species. The observed seedling radiosensitivity was not correlated with predicted mature plant sensitivity, thus adding further evidence on the variation in relative radiosensitivity patterns between stages of the life cycle among gymnosperm species suggested previously. Seedling radinsensitivity as determined from 3 of the 4 endpoints studied was related to D N A content per cell determined cytophotometrically in Feulgen absorption units, but unrelated to D N A content determined chemically or to nuclear volume. INTRODUCTION
PHYSIOLOGICALLY active plants are more sensitive to ionizing radiation than the same species in d o r m a n t or lower metabolic states of development. (2°) A relative radiosensitivity factor of 4, i.e. c o m p a r i n g the most sensitive with least sensitive, has been reported for d o r m a n t or inactive seed o f 9 representative N o r t h T e m perate gymnosperms and a factor o f more than 3 for the 7 species in the present study.Oe) T h e radiosensitivity o f seedlings and trees of certain g y m n o s p e r m species also has been studied by 45
numerous investigators and found to vary a m o n g species as well as experiments, p r o b a b l y due to different experimental conditions.O,a,s,~,1014,17-~5) However, information on the relative radiosensitivity of y o u n g seedlings of gymnosperm species obtained u n d e r comparable experimental conditions is lacking. N o r has the relative radiosensitivity of seedlings of g y m n o sperm species been adequately studied in relation to seed sensitivity of the same populations. This study was initiated to investigate the relative radiosensitivity of seedlings from the
46
T. D. RUDOLPH
same populations of most of the gymnosperm species for which relative seed sensitivity had been determined at this laboratory/x6) and to compare the seed and seedling responses. Seed radiosensitivity of this group of gymnosperms showed no relation to nuclear volume and DNA quantity per cell. (9) The present study also investigated the relation between gymnosperm seedling radiosensitivity and these nuclear variables. MATERIAL A N D M E T H O D S
The species in which seedling radiosensitivity was studied were Larix laricina (DuRoi) K. Koch, Picea glauca (Moench) Voss, Picea mariana (Mill.), Pinus banksiana Lamb., Pinus contorta Dougl., Pinus resinosa Ait., and Thuja occidentalis L. The seed origin of each species was the same as that used in the study of seed relative radiosensitivityA16) Unfilled seeds were removed with a commercial seed blower before use. Prior to sowing, the seed was cold-soaked in water to full imbibition.O.le) The seeds were sown in a greenhouse in 4-in. cylindrical paperboard containers coated with paraffin. A germination substrate of sand, potting soil and peat (1 : 1 : 1) was used. Each container was sown with 75 seeds with 4 containers or replications per exposure. The seeds were sown in a uniformly spaced arrangement in the containers into small holes prepunched into the soil mixture with a pegged template. During the course of the experiment, greenhouse temperatures oceasionally varied from 68 to 84°F. but normally were at 72°F. A 20-hr photoperiod was provided throughout the experiment using fluorescent and incandescent lamps. The seedlings were fertilized with water-soluble commercial fertilizer when necessary to maintain plant vigor as manifested by the controls. Irradiation of all species was done at that point in seedling development when one-haft of the population had shed their seedcoat from the expanding cotyledons. At this stage, the hypocotyl appeared to be fully extended and the epicotyl was beginning to elongate. Seedlings reached this stage of development as early as 15 days after sowing in Pinus banksiana and as late as 25 days in Thuja occidentalis. Total exposures of 150, 300, 450, 600, 900, 1200, 1800, 2400
and 3600 R were applied to all species. The irradiation was done in a 260-Ci x3VCs selfcontained irradiator. The containers with the seedlings were rotated on a turntable within an average radiation field of 6,000 R/hr. Detailed lithium fluoride dosimetry showed that the exposure of the roots was reduced by an average of 20 per cent due to attenuation by the soil mixture. The irradiated seedlings together with controls were returned to the greenhouse into a randomized block design immediately after exposure. Fifty days after irradiation, 10 randomly chosen seedlings per replication in each treatment were harvested for determination of number of leaves produced and shoot dry weight. Leaf number was determined by examination of the seedlings under a dissecting microscope. Because the hypocotyl and the cotyledons were already well developed at the time of ir?adiation, they were not included in the dry weight determinations. At 130 days after irradiation, survival was scored and shoot dry weight was obtained for all surviving seedlings.
R E S U L T S A N D DISCUSSIONS
The Dso (dose required to reduce performance to 50 per cent of control), determined graphically (Fig. 1), for each of the 4 endpoints (characteristics measured) in each species is given in Table 1. The endpoint showing greatest sensitivity to gamma radiation was shoot dry weight at 50 days with a D60 ranging from 195 R for Pinus resinosa to 380 R for Picea rnariana. The other 3 endpoints had consistently higher D60 exposures ranging from approximately 400 to 750 R. Species differed in seedling radiosensitivity by a factor of less than 2 for all endpoints. This is a much smaller range of variation than was found for seed sensitivity of the same populations of these 7 species where the species differed by a factor of more than 3.~tO As expected the radiosensitivity of the actively growing seedlings was much greater than that reported for inactive soaked seed of the same species.tie) Comparison of the mean Ds0 for all endpoints for the seeds and seedlings shows that seedlings are from about 7 to more than 15 times more sensitive than seeds.
GYMNOSPERM SEEDLING SENSITIVITY
47
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Fro. l. The response to gamma irradiation in seedlings of 7 gymnosperm species. Young seedlings were irradiated when half of the seedcoats had been shed. Response scored 50 and 130 days after irradiation.
T. D. RUDOLPH
48
Table 1. Radiosensitivi~ ofyoung gymnosperm seedlings determinedfrorn four endpoints
Species Larix laricina Picea glauca Picea mariana Pinus banksiana Pinus contorta Pinus resinosa Thuja occidentalis
Range of variation factor
No. leaves at 50 days
Dso in R and S.E. for Shoot dry wt. Survival at 50 days at 130 days
Shoot dry wt. at 130 days
Mean Dso
550+ 12 366 -+ 9 480_+25 562 -+ 19 560 _+17 417-+ 4 708 _+47
261 +42 224-1- 39 380+28 280 -+ 18 336 + 26 195-+ 4 339 + 75
750_+26 550 -+32 640+_44 655 _+29 702 -+20 519-+ 2 650 -+38
534+ 69 460-+ 65 560_+ 20 660 _+ 45 651 +_120 505-+ 33 690 -+ 125
524 400 515 539 562 409 597
1-94
1.95
1.45
1.50
1.49
The pattern of response to increasing exposure of g a m m a radiation for each of the endpoints is illustrated in Fig. 1. Leaf n u m b e r and shoot dry weight at 50 days had consistently lower Ds0 exposures than survival and shoot dry weight at 130 days, suggesting that repair and recovery from radiation injury did occur in some of the seedlings during the period 50-130 days postirradiation (Fig. 1 and Table 1). This increase in Dn0 with time post-irradiation contrasts with a decrease in D60 with seedling age following seed irradiation,~,15) and older seedling irradiation.{X) Figure 1 also shows that the response curves for leaf number and shoot dry weight at 50 days do not change significantly at exposures higher than 600 R for some species and 900 K for others, nor do they reach Da00. This simply indicates the number of leaf primordia present and the dry weight of the shoot at the time of irradiation or at the stage of seedling development when one-half of the seedcoats are shed. The pines had a larger n u m b e r of leaf primordia at this stage than Picea or Larix; Thuja was intermediate. The presence of leaf primordia also indicates that not only was growth occurring by cell enlargement, but mitosis was already underway in the seedlings at the time of irradiation. All of the endpoints showed a sharp response over a relatively narrow range of exposures, generally between 600 and 900 R (Fig. 1). This is especially evident for 130-day survival in all species except Pinus contorta in which a significant percentage of seedlings survived exposures
higher than 900 R. The inflection and flattening of the survival curve over a relatively wide range of near-total lethal exposures ~uggests that variation in radiosensitivity m a y exist within this population ofPinus contorta, or that variation in germination rate and seedling growth rate m a y have resulted in increased variability in the stage of development at the time of irradiation in this species. In Pinus contorta and Picea glauca some unexpected increases in shoot dry weight occur in the upper radiation exposures (Fig. 1). These are for a few surviving seedlings that apparently suffered little radiation injury, probably as a result of delayed germination. Since the values for this endpoint are based on the mean shoot dry weight per seedling, they fluctuate more widely with decrease in n u m b e r of seedlings. Correlation analyses showed that the only relationship among the endpoints studied was between Ds0 for number of leaves at 50 days and shoot dry weight at 130 days (Table 2). Thus, plants with the greatest number of leaves at 50 days had the greatest dry weight at 130 days. Survival Ds0 at 130 days was not related to any of the other endpoints. Similarly, n u m b e r of leaves at 50 days was not related to shoot dry weight at the same age, nor was shoot dry weight at 50 days related to that at 130 days. Hence, survival Ds0 at 130 days cannot be predicted from leaf production or dry weight Ds0 at 50 days, nor does shoot dry weight Ds0 at 50 days indicate the radiation response at 130 days. These results are not in agreement with those of
GYMNOSPERM SEEDLING SENSITIVITY
49
Table 2. Correlation coefficients between seedling Ds0 endpoints
Dso
for
Survival at 130 days
Survival at 130 days No. of leaves at 50 days Shoot dry wt. at 50 days Shoot dry wt. at 130 days
No. of leaves at 50 days
Ds0 for Shoot dry wt. at 50 days
0.64 --
Shoot dry wt. at 130 days
0.54 0"58 --
0-51 0"89t 0.64
tSignificant at I per cent level. CAP~'.LLAand CONOERtl) who reported that early measurement of damage to growth was a fair predictor for later survival in the gymnosperms. Their results are based on 1- and 2-year-old seedlings, however. Comparison of the relative radiosensitivity rank of the species for the different endpoints indicates that survival at 130 days results in a different ranking of species radiosensitivity than do the other 3 endpoints (Table 3). N u m b e r of
No relation was found between the Ds0 following seedling irradiation and the Ds0 for irradiated seed previously reported(16) for the same populati6ns of the 7 species (Table 4). In the same report it was noted that seed radiosensitivity for these species was not related to mature plant sensitivity predicted by SPARROW et al.(10) from nuclear variables. The seedling radiosensitivity determined in the present study also is not correlated with the predicted mature
Table 3. Correlation coefficients between species relative radiosensitivity rank for seedling Ds0 endpoints (Rank Correlation Test, Dixon and M~LssEY(4))
Ds0 for
~)50 for Survival at No. of leaves Shoot dry wt. 130 days at 50 days at 50 days
Survival at 130 days No. of leaves at 50 days Shoot dry wt. at 50 days Shoot dry wt. at 130 days
0-61 --
0.36 0.73* --
Shoot drywt. at 130 days 0.46 0"96t 0.82*
*Significant at 5 per cent level. tSignificant at I per cent level. leaves at 50 days, shoot dry weight at 50 days and shoot dry weight at 130 days, on the other hand, result in species relative sensitivity ranks that are significantly correlated. Both these comparisons and the correlation coefficients between Ds0 endpoints indicate that early measures of radiation induced growth inhibition are later modified by species differences in recovery ability, resulting in dissimilar species relative radiosensitivity patterns between early and later endpoints.
plant sensitivity. While the number of gymnosperm species studied in these comparisons is small, the extremely small correlation coefficients given in Table 4 suggest that increasing the number of species would not prove a significant relation. Thus, the results of the present study add further evidence on the variation in relative radiosensitivity patterns between stages of the life cycle among gymnosperm species suggested previously. {e, 9,t 6) Seedling radiosensitivity for each of the 4
50
T.D. RUDOLPH
Table 4. Correlation coeffcients between seed and seedling Dsofor various endpoints Ds0 for seedling endpoints
Dso for seed endpoints*
Survival at 130 days
No. of leaves at 50 days
Shoot dry wt. at 50 days
Shoot drywt. at 130 days
Survival at 36 days Survival at 75 days Shoot dry wt. at 75 days
0.27 0"09 0.20
0" 16 0.08 0.03
0-17 0.01 0"07
0.60 0.26 0.51
*Seed sensitivity data used in these analyses are from RUDOLPH and MmSCHF..(16) endpoints in r e l a t i o n to n u c l e a r variables previously r e p o r t e d for the same p o p u l a t i o n s o f the 7 speciesC9) is presented in T a b l e 5. Seedling radiosensitivity as d e t e r m i n e d from 3 o f the 4 endpoints studied was r e l a t e d to D N A c o n t e n t p e r cell d e t e r m i n e d c y t o p h o t o m e t r i c a l l y in
b r y o roots a n d shoots a n d in seedling roots were not significantly r e l a t e d to seedling r a d i o sensitivity ( T a b l e 5). A similar lack of relationship b e t w e e n these n u c l e a r variables a n d seed radiosensitivity of the same p o p u l a t i o n s from these species has also been d e m o n s t r a t e d . (9) Thus,
Table 5. Correlation coefficients between nuclear variables and seedling Dso for various endp~ints
Dso for Nuclear~ variable
Nuclear vol. of embryo root Nuclear voh of embryo shoot Nuclear voh of seedling root DNA/cell in Feulgen absorption units (root) DNA/cell in pieograms (root)
Survival at 130 days -
0.56 0.56 0.24 0-83* 0.56
No. of leaves at 50 days -
0.54 0.68 0.44 0.83* 0.55
Shoot dry wt. at 50 days -
0.42 0-40 0.44 0.72" 0.56
Shoot d r y w t . at 130 days -
0"22 0.42 0.16 0"59 0-40
*Significant at 5 per cent level. J'The nuclear variable data used in these analyses are from MIKSCHE and X~UDOLPH(9) and MIKSOHE.(s) F e u l g e n a b s o r p t i o n units. D N A p e r cell in picog r a m s d e t e r m i n e d c h e m i c a l l y was not signific a n t l y related to seedling radiosensitivity, alt h o u g h the correlation coefficients a p p r o a c h significance a n d w o u l d possibly prove significant with a l a r g e r n u m b e r of species. A n o t h e r consideration is t h a t D N A p e r cell d e t e r m i n e d c h e m i c a l l y includes 2C, S, 4C, a n d possibly some e n d o p o l y p l o i d nuclei, thus r e d u c i n g a c c u r a c y b y i n c l u d i n g some higher D N A values. Thus, D N A p e r cell m e a s u r e d c y t o p h o t o m e t r i c a l l y using the F e u l g e n t e c h n i q u e on cells in the 2C condition a p p e a r s to be a b e t t e r p r e d i c t o r o f g y m n o s p e r m seedling sensitivity t h a n D N A content d e t e r m i n e d chemically, even t h o u g h these 2 m e t h o d s o f D N A d e t e r m i n a t i o n a r e strongly correlated.(s) N u c l e a r volumes in em-
insofar as these p o p u l a t i o n s r e p r e s e n t the specie3 studied, n u c l e a r variables, w i t h the possible exception o f D N A content p e r cell d e t e r m i n e d c y t o p h o t o m e t r i c a l l y , a p p e a r to be u n r e l i a b l e predictors o f radiosensitivity n o t o n l y o f g y m n o s p e r m seeds, b u t seedlings as well. W h e t h e r o t h e r p o p u l a t i o n s or p r o v e n a n c e s w i t h i n these species will r e s p o n d to g a m m a r a d i a t i o n in the s a m e m a n n e r is b e i n g investigated.
Acknowledgements--The author wishes to express his appreciation to EDMUND O. BA~R, ROBERT A. CECICH and THOMhS F. KING for their technical assistance. These studies were supported in part by the Atomic Energy Commission under contract AT(49-7)3016.
GYMNOSPERM SEEDLING SENSITIVITY REFERENCES
I. CAPELLAJ. A. and CONGER A. D. (1967) Radioscnsitivity and intcrphasc chromosome volumc in the gymnospcrms. RadiationBotany 7, 137-149. 2. CLARK G. M., SWEANEY W. P., B U N T m O W. R. and BAKER D. G. (1968) Germination and survival of conifers following chronic gamma irradiation of seed. Radiation Botany 8, 59-66. 3. DAvxs T. S. (1962) Effect of cobalt-60 gamma radiation on pine seed and one-year-old seedlings. Forest Sci. 8, 411-412. 4. DIXON W. J. and MA$SEY F. J. JR. (1957) hztroduction to statistical analysis, 2nd ed. McGraw-Hill, New York, 488 p. 5. DoNim B. (1967) Effects of chronic gammairradiation on Pinus pinea and Pinus halepensis. Radiation Botany 7, 183-192. 6. MCCORMICKJ. F. and MCJUNKINR. E. (1965) Interactions of gamma radiation and other environmental stresses upon pine seeds and seedlings. Health Phys. U , 1643-1652. 7. M.e'RGENF. and THmLOES B. A. (1966) Effect of chronic exposures to Co e° radiation on Pinus rigida seedlings. Radiation Botany 6, 203-210. 8. MIKSCHEJ. P. (1967) Variation in DNA content of several gymnosperms. Can. 07. Genet. Cytol. 9, 717-722. 9. MIKSCHEJ. P. and RUDOLPHT. D. (1968) Use of nuclear variables to investigate radiosensitivity of gymnosperm seed. Radiation Botany 8, 187-192. 10. MILLER L. N. (1965) Changes in radiosensitivity of pine seedlings subjected to water stress during chronic gamma irradiation. Health Phys. 11, 1653-1662. 11. MURAl M. and OHBA K. (1966) Studies on radiosensitivity of forest trees--III. Effects of chronic gamma irradiation on the growth and foliar cell division of Japanese larch, Larix leptolepis in a gamma field; J. Japan. Forestry Soc. 48, 62-68. 12. OHBA K. (1964) Studies on radiosensitivity and induction of somatic mutations in forest trees, pp. 111-141. In, Gammafield symposia No. 3, Inst. Radiation Breeding, Ibaraki-ken, Japan. 13. OKUNEWaCKJ. (1966) Effects of acute doses of x-radiation on pine trees. Rand Corporation, Santa Monica, California, RM-4904-TAB, 30 p.
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14. PEDIGO R. A. (1960) The effects of inonizing radiation on the ecology of Pinus taeda. Bull. Ecol. Soc. Am. 41, 94--95. 15. RUDOLPH T. D. (1967) Effects of x-irradiation of seed on X t and X2 generations in Pinus banksiana Lambert. Radiation Botany 7, 303-312. 16. RUDOLPH, T. D. and MXmSCHEJ. P. (1970) The relative sensitivity of the soaked seed of nine gymnosperm species to gamma radiation. Radiation Botany 10, 401-409. 17. SPARROWA. H., CUANYR. L., MIKSCHEJ. P. and SCHAIRERL. A. (1961) Some factors affecting the responses of plants to acute and chronic radiation exposures. Radiation Botany 1, 10-34. 18. SPARROWA. H. and GUNCKELK. E. (1956) The effects on plants of chronic exposure to gamma radiation from radiocobalt. Proc. Intern. Conf. Peaceful Uses At. Energy, 1955, 12, 52-59. 19. SPARROWA. H., ROGERSA. F. and SCI-rWEMMER SUSAN S. (1968) Radiosensitivity studies with woody plants--I. Acute gamma irradiation survival data for 28 species and predictions for 190 species. Radiation Botany 8, 149-186. 20. SPARROW A. H., SCHAIRER L. A., SPARROWR. C. and CAMPBELLW. F. (1963) The radiosensitivityof gymnosperms--I. The effect of dormancy on the response of Pinus strobus seedlings to acute gamma irradiation. Radiation Botany 3, 169-173. 21. SPARROWA. H., SCHAIRERL. A. and WOODWELL G. M. (1965) Tolerance ofPinus rigida trees to a ten-year exposure to chronic gamma irradiation from cobalt-60. Radiation Botany 5, 7-22. 22. SPARROW R. C. and SPARROW A. H. (1965) Relative radiosensitivities of woody and herbaceous spermatophytes. Science 147, 1449-1451. 23. WOODWELL G. M. (1962) Effects of ionizing radiation on terrestrial ecosystems. Science 138, 572-577. 24. WOODWELL G. M. and SPARROW A. H. (1963) Predicted and observed effects of chronic radiation on a near-climax forest ecosystem. Radiation Botany 3, 231-238. 25. WOODWELL G. M. and SPARROW A. H. (1965) Effects of ionizing radiation on ecological systems, pp. 20-38. In G. M. Woodwell (Ed.), Ecological effects of nuclear war. Brookhaven Natl. Lab., Upton, New York BNL 917 (C-43).
GYMNOSPERM SEEDLING SENSITIVITY TO GAMMA RADIATION: ITS RELATION TO SEED RADIOSENSITIVITY AND NUCLEAR VARIABLES T. D. R U D O L P H
North Central Forest Experimental Station, U.S.D.A. Forest Service, Institute of Forest Genetics, Rhinelander, Wisconsin, U.S.A. (Received 17 July 1970)
RUDOLPH,T. D., Gymnospermseedling sensitivity to gamma radiation: Its relation to seed radiosensitivity and nuclear variables. RADIATIONBOTANY 11~ 45--51, 1971.--Newly germinated seedlings of Larix laricina, Picea glauca, Picea mariana, Pinus banksiana, Pinus contorta, Pinus resinosa, and Thuja occidentalis were gamma irradiated at exposures of 150, 300, 450, 600, 900, 1200, 1800, 2400 and 3600 R. Number of leaves produced and shoot dry weight were determined 50 days after irradiation and survival and shoot dry weight after 130 days. Of the 4 endpoints scored, shoot dry weight at 50 days showed greatest radiosensitivity with a Ds0 ranging from 195 R for Pinus resinosa to 380 R for Picea mariana. Except for shoot dry weight at 50 days, all endpoints showed a sharp response over a relatively narrow range ofexposures, generally between 600 and 900 R. The 7 species differed in seedling radiosensitivity by a factor of less than 2 for all endpoints. Comparison with seed radiosensitivity of the same populations showed that seedlings are from 7 to 15 times more sensitive than seeds. Correlation analyses showed that the only relationship among the endpoints studied was between Ds0 for number of leaves at 50 days and shoot dry weight at 130 days: the seedlings with the greatest number of leaves at 50 days had the greatest dry weight at 130 days. No relation was found between the Ds0 following seedling irradiation and the Ds0 for irradiated seed previously reported for the same populations of the 7 species. The observed seedling radiosensitivity was not correlated with predicted mature plant sensitivity, thus adding further evidence on the variation in relative radiosensitivity patterns between stages of the life cycle among gymnosperm species suggested previously. Seedling radinsensitivity as determined from 3 of the 4 endpoints studied was related to D N A content per cell determined cytophotometrically in Feulgen absorption units, but unrelated to D N A content determined chemically or to nuclear volume. INTRODUCTION
PHYSIOLOGICALLY active plants are more sensitive to ionizing radiation than the same species in d o r m a n t or lower metabolic states of development. (2°) A relative radiosensitivity factor of 4, i.e. c o m p a r i n g the most sensitive with least sensitive, has been reported for d o r m a n t or inactive seed o f 9 representative N o r t h T e m perate gymnosperms and a factor o f more than 3 for the 7 species in the present study.Oe) T h e radiosensitivity o f seedlings and trees of certain g y m n o s p e r m species also has been studied by 45
numerous investigators and found to vary a m o n g species as well as experiments, p r o b a b l y due to different experimental conditions.O,a,s,~,1014,17-~5) However, information on the relative radiosensitivity of y o u n g seedlings of gymnosperm species obtained u n d e r comparable experimental conditions is lacking. N o r has the relative radiosensitivity of seedlings of g y m n o sperm species been adequately studied in relation to seed sensitivity of the same populations. This study was initiated to investigate the relative radiosensitivity of seedlings from the
46
T. D. RUDOLPH
same populations of most of the gymnosperm species for which relative seed sensitivity had been determined at this laboratory/x6) and to compare the seed and seedling responses. Seed radiosensitivity of this group of gymnosperms showed no relation to nuclear volume and DNA quantity per cell. (9) The present study also investigated the relation between gymnosperm seedling radiosensitivity and these nuclear variables. MATERIAL A N D M E T H O D S
The species in which seedling radiosensitivity was studied were Larix laricina (DuRoi) K. Koch, Picea glauca (Moench) Voss, Picea mariana (Mill.), Pinus banksiana Lamb., Pinus contorta Dougl., Pinus resinosa Ait., and Thuja occidentalis L. The seed origin of each species was the same as that used in the study of seed relative radiosensitivityA16) Unfilled seeds were removed with a commercial seed blower before use. Prior to sowing, the seed was cold-soaked in water to full imbibition.O.le) The seeds were sown in a greenhouse in 4-in. cylindrical paperboard containers coated with paraffin. A germination substrate of sand, potting soil and peat (1 : 1 : 1) was used. Each container was sown with 75 seeds with 4 containers or replications per exposure. The seeds were sown in a uniformly spaced arrangement in the containers into small holes prepunched into the soil mixture with a pegged template. During the course of the experiment, greenhouse temperatures oceasionally varied from 68 to 84°F. but normally were at 72°F. A 20-hr photoperiod was provided throughout the experiment using fluorescent and incandescent lamps. The seedlings were fertilized with water-soluble commercial fertilizer when necessary to maintain plant vigor as manifested by the controls. Irradiation of all species was done at that point in seedling development when one-haft of the population had shed their seedcoat from the expanding cotyledons. At this stage, the hypocotyl appeared to be fully extended and the epicotyl was beginning to elongate. Seedlings reached this stage of development as early as 15 days after sowing in Pinus banksiana and as late as 25 days in Thuja occidentalis. Total exposures of 150, 300, 450, 600, 900, 1200, 1800, 2400
and 3600 R were applied to all species. The irradiation was done in a 260-Ci x3VCs selfcontained irradiator. The containers with the seedlings were rotated on a turntable within an average radiation field of 6,000 R/hr. Detailed lithium fluoride dosimetry showed that the exposure of the roots was reduced by an average of 20 per cent due to attenuation by the soil mixture. The irradiated seedlings together with controls were returned to the greenhouse into a randomized block design immediately after exposure. Fifty days after irradiation, 10 randomly chosen seedlings per replication in each treatment were harvested for determination of number of leaves produced and shoot dry weight. Leaf number was determined by examination of the seedlings under a dissecting microscope. Because the hypocotyl and the cotyledons were already well developed at the time of ir?adiation, they were not included in the dry weight determinations. At 130 days after irradiation, survival was scored and shoot dry weight was obtained for all surviving seedlings.
R E S U L T S A N D DISCUSSIONS
The Dso (dose required to reduce performance to 50 per cent of control), determined graphically (Fig. 1), for each of the 4 endpoints (characteristics measured) in each species is given in Table 1. The endpoint showing greatest sensitivity to gamma radiation was shoot dry weight at 50 days with a D60 ranging from 195 R for Pinus resinosa to 380 R for Picea rnariana. The other 3 endpoints had consistently higher D60 exposures ranging from approximately 400 to 750 R. Species differed in seedling radiosensitivity by a factor of less than 2 for all endpoints. This is a much smaller range of variation than was found for seed sensitivity of the same populations of these 7 species where the species differed by a factor of more than 3.~tO As expected the radiosensitivity of the actively growing seedlings was much greater than that reported for inactive soaked seed of the same species.tie) Comparison of the mean Ds0 for all endpoints for the seeds and seedlings shows that seedlings are from about 7 to more than 15 times more sensitive than seeds.
GYMNOSPERM SEEDLING SENSITIVITY
47
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Fro. l. The response to gamma irradiation in seedlings of 7 gymnosperm species. Young seedlings were irradiated when half of the seedcoats had been shed. Response scored 50 and 130 days after irradiation.
T. D. RUDOLPH
48
Table 1. Radiosensitivi~ ofyoung gymnosperm seedlings determinedfrorn four endpoints
Species Larix laricina Picea glauca Picea mariana Pinus banksiana Pinus contorta Pinus resinosa Thuja occidentalis
Range of variation factor
No. leaves at 50 days
Dso in R and S.E. for Shoot dry wt. Survival at 50 days at 130 days
Shoot dry wt. at 130 days
Mean Dso
550+ 12 366 -+ 9 480_+25 562 -+ 19 560 _+17 417-+ 4 708 _+47
261 +42 224-1- 39 380+28 280 -+ 18 336 + 26 195-+ 4 339 + 75
750_+26 550 -+32 640+_44 655 _+29 702 -+20 519-+ 2 650 -+38
534+ 69 460-+ 65 560_+ 20 660 _+ 45 651 +_120 505-+ 33 690 -+ 125
524 400 515 539 562 409 597
1-94
1.95
1.45
1.50
1.49
The pattern of response to increasing exposure of g a m m a radiation for each of the endpoints is illustrated in Fig. 1. Leaf n u m b e r and shoot dry weight at 50 days had consistently lower Ds0 exposures than survival and shoot dry weight at 130 days, suggesting that repair and recovery from radiation injury did occur in some of the seedlings during the period 50-130 days postirradiation (Fig. 1 and Table 1). This increase in Dn0 with time post-irradiation contrasts with a decrease in D60 with seedling age following seed irradiation,~,15) and older seedling irradiation.{X) Figure 1 also shows that the response curves for leaf number and shoot dry weight at 50 days do not change significantly at exposures higher than 600 R for some species and 900 K for others, nor do they reach Da00. This simply indicates the number of leaf primordia present and the dry weight of the shoot at the time of irradiation or at the stage of seedling development when one-half of the seedcoats are shed. The pines had a larger n u m b e r of leaf primordia at this stage than Picea or Larix; Thuja was intermediate. The presence of leaf primordia also indicates that not only was growth occurring by cell enlargement, but mitosis was already underway in the seedlings at the time of irradiation. All of the endpoints showed a sharp response over a relatively narrow range of exposures, generally between 600 and 900 R (Fig. 1). This is especially evident for 130-day survival in all species except Pinus contorta in which a significant percentage of seedlings survived exposures
higher than 900 R. The inflection and flattening of the survival curve over a relatively wide range of near-total lethal exposures ~uggests that variation in radiosensitivity m a y exist within this population ofPinus contorta, or that variation in germination rate and seedling growth rate m a y have resulted in increased variability in the stage of development at the time of irradiation in this species. In Pinus contorta and Picea glauca some unexpected increases in shoot dry weight occur in the upper radiation exposures (Fig. 1). These are for a few surviving seedlings that apparently suffered little radiation injury, probably as a result of delayed germination. Since the values for this endpoint are based on the mean shoot dry weight per seedling, they fluctuate more widely with decrease in n u m b e r of seedlings. Correlation analyses showed that the only relationship among the endpoints studied was between Ds0 for number of leaves at 50 days and shoot dry weight at 130 days (Table 2). Thus, plants with the greatest number of leaves at 50 days had the greatest dry weight at 130 days. Survival Ds0 at 130 days was not related to any of the other endpoints. Similarly, n u m b e r of leaves at 50 days was not related to shoot dry weight at the same age, nor was shoot dry weight at 50 days related to that at 130 days. Hence, survival Ds0 at 130 days cannot be predicted from leaf production or dry weight Ds0 at 50 days, nor does shoot dry weight Ds0 at 50 days indicate the radiation response at 130 days. These results are not in agreement with those of
GYMNOSPERM SEEDLING SENSITIVITY
49
Table 2. Correlation coefficients between seedling Ds0 endpoints
Dso
for
Survival at 130 days
Survival at 130 days No. of leaves at 50 days Shoot dry wt. at 50 days Shoot dry wt. at 130 days
No. of leaves at 50 days
Ds0 for Shoot dry wt. at 50 days
0.64 --
Shoot dry wt. at 130 days
0.54 0"58 --
0-51 0"89t 0.64
tSignificant at I per cent level. CAP~'.LLAand CONOERtl) who reported that early measurement of damage to growth was a fair predictor for later survival in the gymnosperms. Their results are based on 1- and 2-year-old seedlings, however. Comparison of the relative radiosensitivity rank of the species for the different endpoints indicates that survival at 130 days results in a different ranking of species radiosensitivity than do the other 3 endpoints (Table 3). N u m b e r of
No relation was found between the Ds0 following seedling irradiation and the Ds0 for irradiated seed previously reported(16) for the same populati6ns of the 7 species (Table 4). In the same report it was noted that seed radiosensitivity for these species was not related to mature plant sensitivity predicted by SPARROW et al.(10) from nuclear variables. The seedling radiosensitivity determined in the present study also is not correlated with the predicted mature
Table 3. Correlation coefficients between species relative radiosensitivity rank for seedling Ds0 endpoints (Rank Correlation Test, Dixon and M~LssEY(4))
Ds0 for
~)50 for Survival at No. of leaves Shoot dry wt. 130 days at 50 days at 50 days
Survival at 130 days No. of leaves at 50 days Shoot dry wt. at 50 days Shoot dry wt. at 130 days
0-61 --
0.36 0.73* --
Shoot drywt. at 130 days 0.46 0"96t 0.82*
*Significant at 5 per cent level. tSignificant at I per cent level. leaves at 50 days, shoot dry weight at 50 days and shoot dry weight at 130 days, on the other hand, result in species relative sensitivity ranks that are significantly correlated. Both these comparisons and the correlation coefficients between Ds0 endpoints indicate that early measures of radiation induced growth inhibition are later modified by species differences in recovery ability, resulting in dissimilar species relative radiosensitivity patterns between early and later endpoints.
plant sensitivity. While the number of gymnosperm species studied in these comparisons is small, the extremely small correlation coefficients given in Table 4 suggest that increasing the number of species would not prove a significant relation. Thus, the results of the present study add further evidence on the variation in relative radiosensitivity patterns between stages of the life cycle among gymnosperm species suggested previously. {e, 9,t 6) Seedling radiosensitivity for each of the 4
50
T.D. RUDOLPH
Table 4. Correlation coeffcients between seed and seedling Dsofor various endpoints Ds0 for seedling endpoints
Dso for seed endpoints*
Survival at 130 days
No. of leaves at 50 days
Shoot dry wt. at 50 days
Shoot drywt. at 130 days
Survival at 36 days Survival at 75 days Shoot dry wt. at 75 days
0.27 0"09 0.20
0" 16 0.08 0.03
0-17 0.01 0"07
0.60 0.26 0.51
*Seed sensitivity data used in these analyses are from RUDOLPH and MmSCHF..(16) endpoints in r e l a t i o n to n u c l e a r variables previously r e p o r t e d for the same p o p u l a t i o n s o f the 7 speciesC9) is presented in T a b l e 5. Seedling radiosensitivity as d e t e r m i n e d from 3 o f the 4 endpoints studied was r e l a t e d to D N A c o n t e n t p e r cell d e t e r m i n e d c y t o p h o t o m e t r i c a l l y in
b r y o roots a n d shoots a n d in seedling roots were not significantly r e l a t e d to seedling r a d i o sensitivity ( T a b l e 5). A similar lack of relationship b e t w e e n these n u c l e a r variables a n d seed radiosensitivity of the same p o p u l a t i o n s from these species has also been d e m o n s t r a t e d . (9) Thus,
Table 5. Correlation coefficients between nuclear variables and seedling Dso for various endp~ints
Dso for Nuclear~ variable
Nuclear vol. of embryo root Nuclear voh of embryo shoot Nuclear voh of seedling root DNA/cell in Feulgen absorption units (root) DNA/cell in pieograms (root)
Survival at 130 days -
0.56 0.56 0.24 0-83* 0.56
No. of leaves at 50 days -
0.54 0.68 0.44 0.83* 0.55
Shoot dry wt. at 50 days -
0.42 0-40 0.44 0.72" 0.56
Shoot d r y w t . at 130 days -
0"22 0.42 0.16 0"59 0-40
*Significant at 5 per cent level. J'The nuclear variable data used in these analyses are from MIKSCHE and X~UDOLPH(9) and MIKSOHE.(s) F e u l g e n a b s o r p t i o n units. D N A p e r cell in picog r a m s d e t e r m i n e d c h e m i c a l l y was not signific a n t l y related to seedling radiosensitivity, alt h o u g h the correlation coefficients a p p r o a c h significance a n d w o u l d possibly prove significant with a l a r g e r n u m b e r of species. A n o t h e r consideration is t h a t D N A p e r cell d e t e r m i n e d c h e m i c a l l y includes 2C, S, 4C, a n d possibly some e n d o p o l y p l o i d nuclei, thus r e d u c i n g a c c u r a c y b y i n c l u d i n g some higher D N A values. Thus, D N A p e r cell m e a s u r e d c y t o p h o t o m e t r i c a l l y using the F e u l g e n t e c h n i q u e on cells in the 2C condition a p p e a r s to be a b e t t e r p r e d i c t o r o f g y m n o s p e r m seedling sensitivity t h a n D N A content d e t e r m i n e d chemically, even t h o u g h these 2 m e t h o d s o f D N A d e t e r m i n a t i o n a r e strongly correlated.(s) N u c l e a r volumes in em-
insofar as these p o p u l a t i o n s r e p r e s e n t the specie3 studied, n u c l e a r variables, w i t h the possible exception o f D N A content p e r cell d e t e r m i n e d c y t o p h o t o m e t r i c a l l y , a p p e a r to be u n r e l i a b l e predictors o f radiosensitivity n o t o n l y o f g y m n o s p e r m seeds, b u t seedlings as well. W h e t h e r o t h e r p o p u l a t i o n s or p r o v e n a n c e s w i t h i n these species will r e s p o n d to g a m m a r a d i a t i o n in the s a m e m a n n e r is b e i n g investigated.
Acknowledgements--The author wishes to express his appreciation to EDMUND O. BA~R, ROBERT A. CECICH and THOMhS F. KING for their technical assistance. These studies were supported in part by the Atomic Energy Commission under contract AT(49-7)3016.
GYMNOSPERM SEEDLING SENSITIVITY REFERENCES
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51
14. PEDIGO R. A. (1960) The effects of inonizing radiation on the ecology of Pinus taeda. Bull. Ecol. Soc. Am. 41, 94--95. 15. RUDOLPH T. D. (1967) Effects of x-irradiation of seed on X t and X2 generations in Pinus banksiana Lambert. Radiation Botany 7, 303-312. 16. RUDOLPH, T. D. and MXmSCHEJ. P. (1970) The relative sensitivity of the soaked seed of nine gymnosperm species to gamma radiation. Radiation Botany 10, 401-409. 17. SPARROWA. H., CUANYR. L., MIKSCHEJ. P. and SCHAIRERL. A. (1961) Some factors affecting the responses of plants to acute and chronic radiation exposures. Radiation Botany 1, 10-34. 18. SPARROWA. H. and GUNCKELK. E. (1956) The effects on plants of chronic exposure to gamma radiation from radiocobalt. Proc. Intern. Conf. Peaceful Uses At. Energy, 1955, 12, 52-59. 19. SPARROWA. H., ROGERSA. F. and SCI-rWEMMER SUSAN S. (1968) Radiosensitivity studies with woody plants--I. Acute gamma irradiation survival data for 28 species and predictions for 190 species. Radiation Botany 8, 149-186. 20. SPARROW A. H., SCHAIRER L. A., SPARROWR. C. and CAMPBELLW. F. (1963) The radiosensitivityof gymnosperms--I. The effect of dormancy on the response of Pinus strobus seedlings to acute gamma irradiation. Radiation Botany 3, 169-173. 21. SPARROWA. H., SCHAIRERL. A. and WOODWELL G. M. (1965) Tolerance ofPinus rigida trees to a ten-year exposure to chronic gamma irradiation from cobalt-60. Radiation Botany 5, 7-22. 22. SPARROW R. C. and SPARROW A. H. (1965) Relative radiosensitivities of woody and herbaceous spermatophytes. Science 147, 1449-1451. 23. WOODWELL G. M. (1962) Effects of ionizing radiation on terrestrial ecosystems. Science 138, 572-577. 24. WOODWELL G. M. and SPARROW A. H. (1963) Predicted and observed effects of chronic radiation on a near-climax forest ecosystem. Radiation Botany 3, 231-238. 25. WOODWELL G. M. and SPARROW A. H. (1965) Effects of ionizing radiation on ecological systems, pp. 20-38. In G. M. Woodwell (Ed.), Ecological effects of nuclear war. Brookhaven Natl. Lab., Upton, New York BNL 917 (C-43).