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Studies on the effects of ionizing radiation on some western coniferous species El-Lakany, Mohamed Hosny Hassan 1969

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5oe>7 .STUDIES ON THE EFFECTS OF IONIZING RADIATION ON SOME - WESTERN CONIFEROUS SPECIES by MOHAMED HOSNY HASSAN EL-LAKANY B . S c , U n i v e r s i t y of A l e x a n d r i a , 1962 M . S c , U n i v e r s i t y of A l e x a n d r i a , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Phi losophy i n the F a c u l t y of Fo re s t ry We accept t h i s t h e s i s as conforming to the r e q u i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA September, 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o lumbia, I agre e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f f:r<&/'&STt^Y The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date -Ce^?- 2 2 - / f£f i i ABSTRACT The primary objective of this study was to investigate the radiosensitivity of Pseudotsuga menziesli (Mirb.) Franco, Douglas-fir, from two different provenances representing the coastal and interior regions in British Columbia, Picea sitchensls (Bong.) Carr., Sitka spruce, and Tsuga heterophylla (Raf.) Sarg., Western hemlock. A secondary objective was to correlate the radiosensitivity with some cytogenetical and biochemical characteristics. Induc-tion of mutations and radiostimulation of seed germination and seedling growth were also sought. F i l l e d seeds of the above mentioned species were given five different dosages of gamma-irradiation from a cobalt-60-source control, 500, 2,000, 5,000, and 10,000 R. Stratification for 14 days at 0°-2°C as post-irradiation treatment was tested. Germination values were evaluated and germinants were transplanted. Survival and growth under controlled environmental conditions were recorded for 182 days. The species exhibited differential responses to seed irrad-iation. A l l showed drastic reduction in germination and survival at -the higher dosages, (5,000-10,000 R). There was some stimul-ation of Interior Douglas-fir seed germination and seedling survival at 500 and 2,000 R irradiation dosages over the control. The same exposures stimulated the height growth of Coastal Douglas-Fir. Stratification after irradiation reduced seed germination and seedling growth and survival in a l l the species. The tolerance to gamma-irradiation decreased in the following5; i i i order* Interior Douglas-fir, Coastal Douglas-fir, Western hemlock and Sitka spruce. Sitka spruce had a significantly larger nuclear volume than Western hemlock and Douglas-fir. No correlation was found between nuclear volume, or interphase chromosome volume, and L D 5 0 (germination). The amount of DNA per c e l l and per chromosome differed significantly among the three species with Sitka spruce having the highest DNA content followed by Western hemlock, Coastal Douglas-fir and Interior Douglas-fir. Signif-icant negative correlations were found between DNA content per c e l l and per chromosome, and LD^0 (germination). This indicated that DNA content plays a more important role than nuclear volume in determining the radiosensitivity of the species. The differences in radiosensitivity, nuclear volume and DNA content between the Coastal and Interior forms of Douglas-fir are discussed in relation to their ecogeographical distribution and taxonomy. Chromosome breaks, micro-nuclei and chromosome erosion were detected in irradiated seeds of Douglas-fir. Intraspecific hybridization was carried out in Douglas-fir using gamma-irradiated pollen grains. Pollen Irradiation up to 5,000 R increased the number of f i l l e d seeds/cone. Seedlings from pollen irradiated at 500 R, exhibited some increase in height growth. Similar effects were obtained for pollen germination in vitro. The possibilities of u t i l i z i n g seed and pollen irradiation in forest tree improvement are discussed' and recommendations are made for future mutation breeding work. i v ACKNOWLEDGMENTS The author wishes to express h i s most s ince re thanks to D r . 0 . S z i k l a i , F a c u l t y of F o r e s t r y , U . B . C . , f o r i n t r o d u c i n g him to the f i e l d of Fores t Gen e t i c s , and f o r h i s c o n s t r u c t i v e c r i t i c i s m and unders tanding guidance throughout the s tudy . The author would a l s o l i k e to acknowledge w i t h g r a t i t u d e h i s a p p r e c i a t i o n to D r . V . C. B r i n k , F a c u l t y of A g r i c u l t u r e , D r . K . Cole and D r . E . B . Tregunna, Department of Botany and B i o l o g y , U . B . C . , f o r t h e i r generous c o u n s e l , h e l p f u l a d v i c e , and f o r r ead ing the manuscr ip t . S p e c i a l thanks are extended to D r . A . Kozak, F a c u l t y of F o r e s t r y , f o r h i s a s s i s t ance i n s t a t i s t i c a l a n a l y s i s , and to Atomic Energy of Canada, L t d . , f o r s u p p l y i n g F igures 1-3* The f i n a n c i a l a s s i s t ance g iven by B r i t i s h Columbia Fores t P roduc t s , L t d . , (Vancouver) , i n the form of Fores t Genet ics S c h o l a r s h i p s , i s g r a t e f u l l y acknowledged. V TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGMENTS i v 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 4 21 P r o p e r t i e s of i o n i z i n g r a d i a t i o n ., 8 22 R a d i a t i o n u n i t s 10 23 A c t i o n of i o n i z i n g r a d i a t i o n 12 24 E f f e c t s of i o n i z i n g r a d i a t i o n on p l a n t s l 4 25 I o n i z i n g r a d i a t i o n i n f o r e s t r y . . l 6 251 R a d i o s e n s i t i v i t y of f o r e s t t r e e s . . . . . . 17 2511 E f f e c t s of i o n i z i n g r a d i a t i o n on seed germina t ion and subsequent s e e d l i n g growth and s u r v i v a l . . . . . . . . . 17 2512 Fac to r s modi fy ing seed r a d i o s e n s i t i v i t y 30 2513 E f f e c t s of i o n i z i n g r a d i a t i o n on young and mature t rees 37 2514 E f f e c t s of i o n i z i n g r a d i a t i o n on sexual r e p r o d u c t i o n of t r e e s . . . . . 45 2515 E f f e c t s of i o n i z i n g r a d i a t i o n of p o l l e n g ra in s and seeds produced by c o n t r o l l e d p o l l i n a t i o n . . . . . . . . 48 2516 C y t o g e n e t i c a l e f f e c t s of i o n i z i n g r a d i a t i o n on fores t , t r ees 52 2517 D i f f e r e n t i a l r a d i o s e n s i t i v i t y of f o r e s t t r e e s . . . . . . . . 55 2518 Nuc lea r c h a r a c t e r i s t i c s a f f e c t i n g r a d i o s e n s i t i v i t y . 58 252 I o n i z i n g r a d i a t i o n as a t o o l i n mutat ion breeding of f o r e s t t r e e s . . . . . . . . . . 66 3 MATERIALS AND METHODS 71 31 E f f e c t s of gamma- i r rad ia t ion on seed germina t ion and subsequent s e e d l i n g growth and s u r v i v a l . . . 74 32 C y t o l o g i c a l i n v e s t i g a t i o n s 84 321 De te rmina t ion of Nuclear volume and Interphase chromosome volume 84 322 Chromosome s tud i e s 86 323 De te rmina t ion of Deoxyr ibonuc le i c A c i d , DNA 87 33 P o l l i n a t i o n experiment 90 331 C o n t r o l l e d p o l l i n a t i o n 92 332 P o l l e n germina t ion i n v i t r o 99 4 RESULTS 101 4 l E f f e c t s of gamma- i r rad ia t ion on seed ge rmina t ion . 101 4 l l D i f f e rences w i t h i n each spec ies . . . . . . 101 v i Page 4111 D o u g l a s - f i r , c o a s t a l provenance . . . 101 4112 D o u g l a s - f i r , i n t e r i o r provenance. . . 104 4113 S i t k a spruce . 106 4 n 4 Western hemlock 108 412 Di f f e rences between spec ies . . 110 413 Germinat ion as a percentage of the c o n t r o l . 115 414 Rate of germina t ion 115 42 E f f e c t s o f gamma- i r rad ia t ion on s e e d l i n g s u r v i v a l and growth 129 43 Morpho log i ca l observa t ions 156 431 Germinat ing stage 156 432 Growing stage 157 44 C y t o g e n e t i c a l and b iochemica l o b se r v a t i o n s . . . . 158 441 Nuc lea r c h a r a c t e r i s t i c s . 158 4 4 l l R e l a t i o n s h i p between LDt-Q(germination) and N . V . and I . C . V . . / 158 442 Deoxyr ibonuc l e i c A c i d e s t ima t ions 16? 4421 R e l a t i o n s h i p between LD^ 0 (ge rmina t ion ) .and the amount of D N A / c e l l and DNA/ chromosome 167 443 Observat ions on the e f f e c t s of i r r a d i a t i o n on the chromosomes. . . . . . . 172 45 E f f e c t s of gamma- i r rad ia t ion of p o l l e n g ra ins on seed p roduc t ion and ge rmina t ion , s e e d l i n g growth and p o l l e n germina t ion i n v i t r o 174 5 DISCUSSION • 182 51 Seed r a d i o s e n s i t i v i t y and subsequent s e e d l i n g s u r v i v a l and growth . 182 511 D i f f e rences w i t h i n each spec ies 182 512 D i f f e rences between spec ies 190 52 Nuc lea r c h a r a c t e r i s t i c s as f a c t o r s i n r a d i o -s e n s i t i v i t y 197 521 V a r i a t i o n i n Nuclear and Interphase chromosome volumes. . . . 197 5211 The r e l a t i o n s h i p between nuc l ea r volume and in terphase chromosome volume and L D ^ 0 (germinat ion) . . . . 199 522 V a r i a t i o n i n the amount of DNA among the spec ies 201 53 E f f e c t s of i r r a d i a t i o n on the chromosomes . . . . 208 54 E f f e c t s of p o l l e n i r r a d i a t i o n on seed p roduc t ion and ge rmina t ion , s e e d l i n g s u r v i v a l and growth,and p o l l e n germina t ion i n v i t r o . 209 6 SUMMARY AND CONCLUSIONS 215 7 LITERATURE CITED 22l APPENDIX 1 L i s t of Some Common Names Used i n the Text . 249 v i i LIST OF TABLES Page 1. The age, h e i g h t , D . b . h . and crown r ad ius of Tree " E " and Tree "11" used i n c o n t r o l l e d p o l l i n a t i o n 9 2 2. A n a l y s i s of va r i ance f o r the germina t ion percent of Coas t a l D o u g l a s - f i r 102 3 . A n a l y s i s of va r i ance f o r the germina t ion percent of I n t e r i o r D o u g l a s - f i r 104 k. A n a l y s i s of va r i ance f o r the germina t ion percent of S i t k a spruce 1 0 6 5 . A n a l y s i s of va r i ance f o r the germina t ion percent of Western hemlock 108 6 . A n a l y s i s of va r i ance f o r the germina t ion percent of d i f f e r e n t spec ies I l l 7 . The combined r e s u l t s of germina t ion percentages of d i f f e r e n t s p e c i e s , t reatments and sub- t rea tments . . . 112 8. The R50 va lues i n days f o r d i f f e r e n t spec ies a f t e r d i f f e r e n t treatments 1 1 6 9 . A n a l y s i s of va r i ance f o r the s u r v i v a l of Coas t a l D o u g l a s - f i r s eed l ings from gamma-irradiated seeds . . 1 3 1 10. A n a l y s i s of va r i ance f o r the s u r v i v a l of I n t e r i o r D o u g l a s - f i r s eed l ings from gamma-irradiated seeds . . 1 3 2 1 1 . A n a l y s i s of va r i ance f o r the s u r v i v a l of S i t k a spruce seed l ings from gamma-irradiated seeds 1 3 3 12. A n a l y s i s of va r i ance f o r the s u r v i v a l of Western hemlock seed l ings from gamma-irradiated seeds . . . . 134 1 3 . A n a l y s i s of va r i ance f o r the s e e d l i n g he igh t s of C o a s t a l D o u g l a s - f i r l 4 l 14. A n a l y s i s of va r i ance f o r the s e e d l i n g he igh t s of - I n t e r i o r D o u g l a s - f i r l 42 1 5 . A n a l y s i s of va r i ance f o r the s e e d l i n g he igh t s of S i t k a spruce 143 1 1 6 . A n a l y s i s of va r i ance f o r the s e e d l i n g he igh ts of Western hemlock 144 17. A n a l y s i s of va r i ance f o r the he igh t of 182-days-o ld C o a s t a l D o u g l a s - f i r s eed l ings 1 5 0 v i i i Page 18. A n a l y s i s of va r i ance f o r the he igh t of 182-days-o ld I n t e r i o r D o u g l a s - f i r s e e d l i n g s . . . . 151 19. A n a l y s i s of va r i ance f o r the he igh t of 182-days -o ld S i t k a spruce seed l ings 152 20. A n a l y s i s of va r i ance f o r the he igh t of 182-days-o ld Western hemlock seed l ings . . . . 153 2 1 . Nuc lea r Diameter , Nuc lea r Volume and Interphase Chromosome Volume + Standard d e v i a t i o n i n f i v e embryos of Coas t a l D o u g l a s - f i r . . 159 22. Nuclear Diameter , Nuc lea r Volume and Interphase Chromosome Volume + Standard d e v i a t i o n i n f i v e embryos of I n t e r i o r D o u g l a s - f i r l 6 0 23 . Nuc lea r Diameter , Nuc lea r Volume and Interphase Chromosome. Volume + Standard d e v i a t i o n i n f i v e embryos of S i t k a spruce l 6 l 24. Nuc lea r Diameter , Nuclear Volume and Interphase Chromosome Volume + Standard d e v i a t i o n i n f i v e embryos of Western hemlock l 62 25. A n a l y s i s of va r i ance f o r the Nuc lea r Diameter of d i f f e r e n t spec ies l63. 26. * A n a l y s i s of va r i ance f o r the Nuc lea r Volume of d i f f e r e n t spec ies . ~l63 27. A n a l y s i s of va r i ance f o r the Interphase Chromosome Volume of d i f f e r e n t spec ies l64 28. The amount of DNA per c e l l and per chromosome + Standard d e v i a t i o n (gram X l O " 1 ^ ) i n d i f f e r e n t s p e c i e s . 168 29. A n a l y s i s of va r i ance f o r the amount of D N A / c e l l i n d i f f e r e n t spec ies 168 30. A n a l y s i s of va r i ance f o r the amount of DNA/chromosome i n d i f f e r e n t s p e c i e s . . . . . . . . . . . . . . . . . 169 31 . The number of p o l l i n a t e d cone le t s and c o l l e c t e d cones, and the f i l l e d , empty and t o t a l numbers of seeds u s i n g i r r a d i a t e d p o l l e n on t ree " E " 174 32. A n a l y s i s of va r i ance f o r the number of f i l l e d seeds/ cone from i r r a d i a t e d p o l l e n 175 33• A n a l y s i s of va r i ance f o r the germina t ion of seeds from c o n t r o l l e d p o l l i n a t i o n 177 ix Page 34. Analysis of variance for seedling height from a r t i f i c i a l p o l l i n a t i o n with i r r a d i a t e d pollen . . . . 178 35. Analysis of variance of pollen germination i n v i t r o a f t e r d i f f e r e n t i r r a d i a t i o n dosages . . 179 X • LIST OF FIGURES ! Page 1 . O v e r a l l view of gamma-cell 2 2 0 7 6 •22. Cross s e c t i o n of gamma-cell u n i t . . 7 8 3 . C o l l a r and sample chamber 7 9 4 . Tree " E " , used as a mother parent i n c o n t r o l l e d p o l l i n a t i o n 9 1 5 . Tree " 1 1 " , used as a p o l l e n source i n c o n t r o l l e d p o l l i n a t i o n . . 9 1 6 . Vege ta t ive ( V . ) , female ( F . ) , and male (M.) buds i n D o u g l a s - f i r •. . 9 3 7 . Female cone le t i n D o u g l a s - f i r 9 ^ 8 . Male cone le t i n D o u g l a s - f i r 9 ^ 9 . E f f e c t of d i f f e r e n t doses of / - i r r a d i a t i o n on the germina t ion of Coas t a l D o u g l a s - f i r 1 0 3 1 0 . E f f e c t of d i f f e r e n t doses of V - i r r a d i a t i o n on the germina t ion of I n t e r i o r D o u g l a s - f i r . . . . . . . . . 1 0 5 1 1 . E f f e c t of d i f f e r e n t doses of y - i r r a d i a t i o n on the germina t ion of S i t k a spruce 1 0 7 1 2 . E f f e c t of d i f f e r e n t doses of ^ " - i r r a d i a t i o n on the germina t ion of Western hemlock 1 0 9 1 3 . E f f e c t of d i f f e r e n t doses of / ^ - i r r a d i a t i o n on the germina t ion percentage of seeds of the three s p e c i e s . 114 14. E f f e c t of d i f f e r e n t doses of y ' ' - i r r a d i a t i o n on the germina t ion of Coas t a l D o u g l a s - f i r seeds, expressed as a percentage of the c o n t r o l . 117 1 5 . E f f e c t of d i f f e r e n t doses of / - i r r a d i a t i o n on the germina t ion of I n t e r i o r D o u g l a s - f i r seeds, expressed as a percentage of the c o n t r o l 1 1 8 1 6 . E f f e c t of d i f f e r e n t doses of Y-irradiation on the ge rmina t ion of S i t k a spruce seeds, expressed as a percentage of the c o n t r o l . 119 17. E f f e c t of d i f f e r e n t doses of i r r a d i a t i o n on the germina t ion of Western hemlock seeds, expressed as a percentage of the c o n t r o l . . . . . . . . . 1 2 0 18. E f f e c t of Y~irradiation on the germina t ion of s t r a t i f i e d seeds of Coas t a l D o u g l a s - f i r . . . . . . . 1 2 1 x i Page 1 9 . E f f e c t of f-irradiation on the germina t ion of u n s t r a t i f i e d seeds of Coas t a l D o u g l a s - f i r 1 2 2 2 0 . E f f e c t of " ^ i r r a d i a t i o n on the germina t ion of s t r a t i f i e d seeds of I n t e r i o r D o u g l a s - f i r 1 2 3 2 1 . E f f e c t of ) f - i r r a d i a t i o n on the germina t ion of u n s t r a t i f i e d seeds of I n t e r i o r D o u g l a s - f i r 1 2 4 2 2 . E f f e c t of / " - i r r a d i a t i o n on the germina t ion of s t r a t i f i e d seeds of S i t k a spruce 1 2 5 2 3 . E f f e c t of f-irradiation on the germina t ion of u n s t r a t i f i e d seeds of S i t k a spruce . . . . . 1 2 6 2 4 . E f f e c t of f-irradiation on the germina t ion of s t r a t i f i e d seeds of Western hemlock . 1 2 7 2 5 . E f f e c t of X~irradiation on the germina t ion of u n s t r a t i f i e d seeds of Western hemlock . 1 2 8 2 6 . The s u r v i v a l of C o a s t a l D o u g l a s - f i r s eed l ings f o l l o w i n g ^ " - i r r a d i a t i o n of seeds a t d i f f e r e n t dosages. 1 3 5 2 7 . The s u r v i v a l of I n t e r i o r D o u g l a s - f i r s eed l ings f o l l o w i n g ' { - i r r a d i a t i o n of seeds a t d i f f e r e n t dosages. 1 3 6 2 8 . The s u r v i v a l of S i t k a spruce seed l ings f o l l o w i n g j f - i r r a d i a t i o n of seeds a t d i f f e r e n t dosages 1 3 7 2 9 . The s u r v i v a l of Western hemlock seed l ings f o l l o w i n g ^ i r r a d i a t i o n of seeds a t d i f f e r e n t dosages • 1 3 8 3 0 . S u r v i v a l as a percentage of the c o n t r o l of 1 8 2 - d a y - o l d s eed l ings f o l l o w i n g ^ - i r r a d i a t i o n of seeds (weighted average of s t r a t i f i e d and u n s t r a t i f i e d seeds) . . . . 1 3 9 3 1 . The he igh t growth of Coas t a l D o u g l a s - f i r s eed l ings from seeds t r e a t ed w i t h d i f f e r e n t dosages of / " - i r r a d i a t i o n . . . l 4 5 • 3 2 . The he ight growth of I n t e r i o r D o u g l a s - f i r seed l ings from seeds t r e a t ed w i t h d i f f e r e n t dosages of ^ • i r r a d i a t i o n l 4 6 3 3 . The he igh t growth of S i t k a spruce seed l ings from seeds t r e a t ed w i t h d i f f e r e n t dosages of f - i r r a d i a t i o n . . . l 4 7 3 4 . The he igh t growth of Western hemlock seed l ings from seeds t r e a t ed w i t h d i f f e r e n t dosages of / - i r r a d i a t i o n . l 4 8 3 5 - 182 - d a y s o l d Coas t a l D o u g l a s - f i r s eed l ings from seeds t r e a t e d w i t h d i f f e r e n t dosages of gamma- i r rad ia t ion . 1 5 4 x i i Page 3 6 . 182 - d a y s o l d I n t e r i o r D o u g l a s - f i r s eed l ings from seeds t r e a t ed w i t h d i f f e r e n t dosages of gamma-i r r a d i a t i o n 1 5 4 3 7 . 182 - d a y s o l d S i t k a spruce seed l ings from seeds t r ea t ed w i t h d i f f e r e n t dosages of gamma- i r r ad ia t ion . . . . . 1 5 5 3 8 . 182 - d a y s o l d Western hemlock seed l ings from seeds t r e a t ed w i t h d i f f e r e n t dosages of gamma- i r r ad ia t ion . 1 5 5 3 9 . C o a s t a l D o u g l a s - f i r germinants r e p r e s e n t i n g 1 - c o n t r o l , 2 - 5 , 0 0 0 R, and 3 - 1 0 , 0 0 0 R-i r r a d i a t i o n treatments 1 5 6 4 0 . An a l b i n o s e e d l i n g from 5 . 0 0 0 R gamma-irradiated C o a s t a l D o u g l a s - f i r seed 1 5 7 4 1 . The c o r r e l a t i o n between nuc l ea r volume i n and L D ^ Q i n rads f o r three spec ies 1 6 5 4 2 . The c o r r e l a t i o n between in terphase chromosome volume i n and L D ^ Q i n rads f o r three spec ies 1 6 6 4 3 . The c o r r e l a t i o n between the amount of DNA per c e l l of the embryo and L D ^ Q f o r germina t ion 1 7 0 4 4 . The c o r r e l a t i o n between the amount of DNA per chromosome i n the embryo and L D ^ Q f o r ge rmina t ion . . 1 7 1 4 5 . C o a s t a l D o u g l a s - f i r chromosomes from u n i r r a d i a t e d seed 1 7 2 4 6 . Hftploid id iogram of Pseudotsuga m e n z i e s i i ( M i r b . ) Franco ( D o u g l a s - f i r ) 1 7 3 4 7 . E f f e c t of p o l l e n i r r a d i a t i o n on the number of f i l l e d seeds per cone i n D o u g l a s - f i r . . 1 7 6 4 8 . U n i r r a d i a t e d D o u g l a s - f i r p o l l e n g r a i n a f t e r 24 hours of ge rmina t ion i n v i t r o . 1 8 0 4 9 . 1 0 , 0 0 0 R- i r r a d i a t e d D o u g l a s - f i r p o l l e n g ra ins a f t e r 2 4 hours of germina t ion i n v i t r o 1 8 0 5 0 . E f f e c t of d i f f e r e n t dosages of " / ^ i r r a d i a t i o n on the germina t ion of D o u g l a s - f i r p o l l e n g ra in s i n v i t r o . . 1 8 1 1 INTRODUCTION A l l planned plant breeding i s based on the multitude of forms found in nature or released in experiments. Natural variation i s s t i l l the main source of genes and genotypes needed for the successive improvement of a cultivated species. It is a familiar fact nowadays that mutagenesis, such as ionizing radiations, as well as some chemical mutagens, increase genetic var i a b i l i t y , thus presenting the breeder with greater opportunities of controlling part of the evolution of his plants when subjecting them to breeding work. Although the classical work on radiation-induced mutations was done more than forty years ago by Muller (1927) on Drosophila, It was Gustafsson \-(195M and his colleagues (I956) in Sweden who, besides cereal research, have recognized the potential of radi-ation genetics in forestry. In gymnosperms each genus is remarkably stable cytologically. Spontaneous mutations arise very infrequently, something in the order of one per million, (Fraser et a l , 1966). Radiation increases this frequency many hundred-fold and might also favour a rearrangement of genes. Induced mutations as a method for forest tree improvement has been recommended by Gustafsson (i960), and mentioned by Duffield in 1962. The frequency of mutations i s proportional to the total dose, (Stadler, 1930), and the radiation dosage which maximate mutation frequency may k i l l about half of the exposed individuals. Study-ing the radiosensitivity of plant material i s , therefore, an essential step before starting a mutation breeding program. The 2. radiosensitivity of some woody speciesuhas been investigated and found to be varied depending on the species and several other factors. The fact that there has been no actual study of the radiosensitivity of some of the major Western coniferous species, except some predictions by Sparrow et a l , (1968) and Woodwell, (1966), may justify the present investigation. Considerable attention has been given to pollen irradiation especially when Osborne (1957) framed the hypothesis that the use of irradiated pollen causes an increase in the number of heteroz-ygotic plants and that the induction of heterosis i s possible. Induction of mutations by pollen irradiation and seeds i s partic-ularly attractive to forest tree breeder because, as Rudolph (19&5) pointed out, the increased variation in progenies can be tolerated since only 10-30$ of the i n i t i a l individuals comprise:the f i n a l harvest of the tree crop. In many agricultural crops, on the other hand, 90$ or more of the individuals i n i t i a l l y seeded may reach maturity. Consequently the trees may be subjected to both natural and a r t i f i c i a l selection during their development. This selection would favour the small proportion of heterotic individ-uals with the result that substantial improvement may be possible in the f i r s t generation. The objectives of this investigation may be restatedi (1) To obtain some information on the radiosensitivity of some selected Western coniferous species. (2) To correlate the differential radiosensitivity of the species with some cytogenetical and biochemical characteristics. 3. (3) To test the possibility of Induction of heterosis by intraspecific hybridization with irradiated pollen. 2 REVIEW OF LITERATURE Heritable individual variations are the basic materials of evolution. The forces acting on populations are the mechanisms which fashion these materials into an orderly, integrated pattern of variation. Stebbins (1950), stated that the variation seen between individuals of any population i s based on three factorst environmental modification, genetic recombination and mutation. Of these, environmental modification, although important to the breeder, i s the least important in evolution. Recombination is the main immediate source of variability for the action of selection and other external forces which direct the evolution of populations, however, the ultimate source of variation is mutation. The history and meaning of the term "mutation" have been given by Dobzhansky (19^1). For the purposes of this study Mayr's (19^2), definition of mutation as "A discontinuous chromosomal changes with genetic effect", is suitable. Plant breeding i s controlled evolution and as in evolution of natural population success in a breeding program depends upon the variations in the material available. New mutations are important chiefly as a means of replenishing the store of variab-i l i t y which i s continuously being depleted by selection. For a long time the raw material for breeding better types has been supplied by variation coming from the spontaneous mutation process or hybridization procedures. As has been emphas-ized by Dobzhansky (19^1) spontaneous rates of mutation are nearly always low and vary enormously from one race to another of the same species and from one gene locus to another of the same genom. More recently the use of induced mutations to increase variability in crop plants has received much attention. The idea of producing mutations a r t i f i c i a l l y and using them for breeding was clearly stated in, the years 1901-1903 when Hugo de Vries published "Die Mutationstheorie, I und II" in which he wrote t "The knowledge of the laws of mutation w i l l presumably lead to the induction of a r t i f i c i a l l y and intentional mutations and so produce entirely new characters in plants and animals. In the future i t may even be possible to create better species of cultivated plants and domestic animals by the control of mutation." At the beginning of this century, several investigators tried to induce mutations in many objects using physical and chemical agents of a varied nature. A historical survey i s given by Stubbe (1938) . In 1904, de Vries proposed using X-rays for a r t i f i c i a l production of mutations,(cited by Blakeslee 1936) . The conclusive proofs that ionizing radiations produce mutations were presented by Muller (192?) , in his paper " A r t i f i c i a l Transmutation of Genes", when he demonstrated that X-rays could induce mutations in Drosophila. Muller's findings were followed next year by those of Stadler (1928) , whose clear results pointed the way for producing: mutations in plants. Pioneer attempts to use a r t i f i c i a l l y produced mutations in breeding were those by Gustafsson (1944) in Sweden and Delaunay (1931) and Sapehin (1936) in Russia, where both produced mutations in wheat and published results indicating that the method has definite p o s s i b i l i t i e s . The Russian studies might have flowered into genuinely profitable contributions had they not been eclipsed by the chicanery of Lysenkoism. Opinions as to the practical possibilities of mutation breeding s t i l l d i f f e r widely, from pronouncedly sceptic via evasive to definitely positive and optimistic. Apart from the statement of de Vries (1901) quoted earlier in this review, Muller (1927) expressed the following cautious judgement "Similarly for the practical breeder i t i s hoped that the method w i l l prove useful." Stadler ( 1 9 2 8 ) , emphasized the negative side. "Something really new is hardly ever obtained; in addition to very few "favourable" mutations a great many "unfavourable" ones are found." It was not un t i l about 1950 that a sudden revival of interest was shown, Prakken (1959) * The usefulness of the methods of mutation breeding for developing useful strains has been repeatedly demonstrated during the past 20 years. Numerous characteristics, mostly for crop plants, have been observed to vary significantly under the influence of mutagens. A large number of papers have been pub-lished in this f i e l d and summarizing data were given by Gaul ( 1 9 6 1 ) , Gustafsson ( 1 9 5 9 ) , Gustafsson and Gadd ( 1 9 6 5 ) . Matsuo and Yamaguchi ( 1 9 6 2 ) , Nybom ( 1 9 6 1 ) , Prakken ( 1 9 5 9 ) . Sparrow, Binnington and Pond ( 1 9 5 8 ) , and Sparrow and Konzak ( 1 9 5 8 ) . Mackey (1956) concluded his paper on mutation breeding in Europe by saying that the evidence attests that "any" agronomic characteristlo^can be improved by induced mutations. -Gaul (1964) stated that there i s no doubt, in principle, of the usefulness of a r t i f i c a l l y produced mutations for breeding. Compared with breeding by crossing, this method may often be much more time-saving. In spite of the voluminous work done in many countries of the world, the number of new varieties obtained by mutation breed-ing i s relatively low. In the view of Gottechalk (1963) this may be chiefly due to two different facts. (l) Transition of a dominant gene to the recessive state does not only result in a specific anomaly as a consequence of the action of the mutant gene. In addition, a general inspecific reduction of v i a b i l i t y and f e r t i l i t y of the mutant organism can often be observed, being possibly due to a disturbance of the genetic balance within the genome by the presence of the mutant recessive gene. (2) In many cases a pleiotropic spectrum of gene action can be observed, and the useful action of the mutant gene is only one part of this spectrum while a different part causes an anomaly reducing the breeding value of the mutant type. Despite these d i f f i c u l t i e s many mutant strains have been developed which show useful properties of mainly agricultural value and the most comprehensive information about the character-i s t i c s which can be modified by Induced mutations has been obtained in barley and snapdragon. Gaul (I96l) l i s t s the more recent induced mutations in many other species as well. Unfort-unately, the induced useful mutations in forest trees are rare and have not received such attention. Many mutagenic agents are known and used by plant breeders. There are several reviews on the types and properties of different 8. mutagens, one of these reviews i s by Gunckel and Sparrow ( 1 9 6 1 ) . Apart from the earlier investigations on the treatment of plants with radium gamma-rays the classic papers by Muller (1927) and by Stadler ( 1 9 2 8 ) , rank f i r s t in a series of mutation experi-ments which have continued to the present day. Both used X-rays and in their experiments obtained a total mutation rate of a hundred times and more than that occurring spontaneously, thus really proving the existence of a r t i f i c i a l l y induced mutations and justifying the ut i l i z a t i o n of ionizing radiation by many plant breeders. A survey of mutagenic radiations known and used so far are given by Prakken (1959) and Sparrow ( 1 9 6 1 ) . 21 Properties of Ionizing Radiation Radiation may be considered as the movement of energy through space, in either corpuscular or electromagnetic form, (Gordon 1 9 5 7 ) . Corpuscular radiation consists of streams of atomic or subatomic particles which transfer their kinetic energy to any matter with which they collide-. . The particles may be negatively charged, such as the electrons in beta rays, positively charged, as the helium nuclei in alpha rays, or electrically neutral, as the neutrons. The energy of the particles is largely determined by i t s velocity. Its interaction with matter depends principally upon the atomic structure of the matter. Electromagnetic radiation, on the other hand, may be thought of a self-propogating stream of particles, called photons, that possess zero mass and no charge. It travels with constant velocity, the velocity of light (3 X 10l0cm/second in vacuum). In motion, however, i t behaves as i f i t were a series of waves 9. with both electrical and magnetic components; i t s energy depends on the frequency of vibration of the waves. Theoretical studies would indicate, (Bacq and Alexander 1 9 6 1 ) , that electromagnetic radiation consists of alternately oscillating electric and magnetic fields which are described by the basic wave equation C where C is the velocity of transmission, A. is the wavelength and V) is the frequency. The properties of light and a l l electromagnetic radiations are such that many phenomena and interactions, especially with matter, are inadequately explained by the classical concepts of wave phenomena. In 1901 Max Plank suggested a corpuscular concept of electromagnetic radiation,(Alexander 1959). This view proposes that electromagnetic radiation in many instances, in particular energy exchange, does not behave as a continuously distributed wave but rather like an energy package or quantum of discrete size. The energy of the quantum is related to the frequency of vibration by the ralatively simple relation E = h^ where "E" is the energy of the photon, "h" is Plank's constant (6.62 X 10~2^ ergs-sec.) and *\) Is*the frequency of vibration. Known electromagnetic radiation cover an enormous wavelength range and have strikingly different properties. Although methods of generation d i f f e r , a l l the various kinds are basically similar, differing only in the wavelength and hence photon energy. Ionizing radiation is that ^portion of the electromagnetic spectrum where the photon frequency is high enough so that photon absorption results in a charge separation in the absorbing entity, generally by electron ejection, (Bacq and Alexander, 1 9 6 l ) . In 10. addition to the process of ionization, energy transfer also occurs by a process known as excitation. The major effect of ionizing radiation is considered to result from their a b i l i t y to ionize and rupture chemical bonds. 22 Radiation Units There are several different units currently in use for the measurement of amounts of radiation produced in a i r or the amount of energy absorbed in tissue or similar materials. The oldest unit s t i l l in general use is the "roentgen" (abbreviation r ) . It i s defined as "that quantity of X- or gamma-radiation such that the associated corpuscular emission per 0.001293* gram of ai r produces, in a i r , ions carrying one electrostatic unit of elec t r i c i t y of either sign", (Lea 1962). A roentgen is equivalent to the absorption of approximately 98 ergs/gram in matter or tissue. One roentgen also corresponds to the liberation of 2.082X10^ ion-pairs per cm3 of ai r at standard temperature and pressure, 1.6X1012 ion pairs per gram of tissue, or about 1.8 ion pairs per u^. A second unit, less commonly used is known as the "roentgen equivalent physical" (abbreviation rep) and can be used for ion-izing radiation. A "rep" is defined as that quantity of corpus-cular radiation which produces, in tissue, per gram of tissue, an amount of ionization equivalent to that produced by one roentger of gamma radiation in a i r . It i s that dose of ionizing radiation which produces an energy absorption of 93 ergs per centimeter of tissue. Both of these units are units of exposure and have the * 0.001293 gram of ai r occupies 1 cm3 at 0 ° S and 760mm pressure. disadvantage that they do not measure the energy absorbed but depend on the amount of ionization produced in a i r , Sparrow ( 1 9 6 1 ) . In order to avoid this d i f f i c u l t y , a newer unit, the "rad" (R) (roentgen absorbed dose) has been adopted recently. One "rad" equals 100 ergs/gram. For X-rays, one rad equals the amount of energy released by 1.08 r in water. 1 kR = 1 ,000 R Other units that w i l l be mentioned throughout this investig-ation are: - 1 2 The electron-volt (eV) = 1 .602 X 10 ergs, is a unit of energy of suitable magnitude for dealing with energy changes in single atoms or molecules. It i s the energy which an electron acquires when accelerated by a potential of one volt. One eV per molecule = 2 3 . 0 5 kilogram calories per gram molecule (Lea 1 9 6 2 ) . 1 KeV = 1000 eV 1 MeV = 1 0 6 eV ergt energy required to move 1 gram through a distance of 1 Cm. rem1 (roentgen equivalent mammal)t that dose absorbed by a mammal when exposed to ionizing radiation biologically equivalent to the dose of one r of gamma radiation. rem = rep X RBE (Relative biological effectiveness). Curie 1 A radioactive isotope, or radio-nuclide w i l l spontaneously disintegrate into daughter products specified by i t s decay scheme with the release of energy. This decay occurs in an exponential fashion* = Ao E - ° « 6 9 3 / T 1 / 2 where* Ao = i n i t i a l amount of r a d i o a c t i v i t y , A = amount of nuclide at time t , X = decay constant, and Ti = physical h a l f - l i f e . The amount of radionuclide may be measured i n terms of the number of disintegrations per second. If the decay scheme fo r example involves the release of one gamma per d i s i n t e g r a t i o n , one can measure the number of quanta released from the material per second. If these are 3»7 X 1 0 1 0 disintegrations/second, there i s one curie of r a d i a t i o n present. mCl = VlOOO Ci 23 Action of Ionizing Radiation According to the p r i n c i p l e established by Gratthus (cited by Gordon, 1957). only r a d i a t i o n which i s absorbed can be chemic-a l l y a c t i v e . Ionizing r a d i a t i o n generally produces an e f f e c t proportional to the energy absorbed i n the tissue i n question. The f r a c t i o n of energy which passes r i g h t through a c e l l or tissue produces no e f f e c t nor does the energy absorbed i n the a i r or medium surrounding the object i n question (except for very small structures), Sparrow ( 1 9 6 1 ) . Thus, i n general terms, i t i s obvious that a r a d i a t i o n must have s u f f i c i e n t energy to penetrate to the p o s i t i o n where i t s e f f e c t i s sought and not so penetrating that most of i t passes through. The r e l a t i v e penetration i n various kinds of organic materials depends upon density of the tissue and i s given by Sun (1954). In addition to the penetration the pattern of d i s t r i b u t i o n 13. of absorbed energy i n the ir r a d i a t e d object i s important, Rossi ( i 9 6 0 ) . The dose at a p a r t i c u l a r position below the surface i s ca l l e d the depth dose and i s determined by a large number of factors such as» kind and energy of radiation, size of the area i r r a d i a t e d , chemical composition and density of the specimen or tissue, collimation of the beam, etc. These factors are gener-a l l y thoroughly discussed i n books on r a d i o l o g i c a l physics or radiotherapy such as Glasser ( i 9 6 0 ) . One other: important c h a r a c t e r i s t i c i s the r e l a t i v e density of ionizations along the path of the i o n i z i n g p a r t i c l e , t h i s varies widely f o r the d i f f e r e n t radiations, (Sparrow 1961) . Since each i o n i z a t i o n i s estimated to require 32 .5 eV, (Lea 1962), i t i s apparent that the denser the ionizations along the track, the higher the rate of energy transfer from the i o n i z i n g p a r t i c l e s to the atoms or molecules of the tissue. This concept, known as l i n e a r energy transfer (LET), i s defined as the energy released by the radiatio n per unit length of the track i n the absorbing material. I t i s usually expressed i n units of keV/ji and the LET varies d i r e c t l y with the square of the charge and inversely with the square of the v e l o c i t y (or energy) of the p a r t i c l e . Radiation i n the energy range of gamma-rays (up to several MeV, Sparrow 1961), interacts primarily by a process of cumpton scattering, i . e . photon rebound from c o l l i s i o n of electrons with scattered photons of lower energy; the energy l o s t i s transferred to electrons ejected by c o l l i s i o n r e c o i l . The ejected electrons often have s u f f i c i e n t energy themselves to expel electrons from other atoms, but as they lose energy thus are eventually captured. If the electron i s captured by a neutral atom a chemically active 14. ion is produced. A molecule capturing an electron is usually-unstable and decomposes to give reactive fragments termed "free radicals". These have different characteristics from the ions produced by the dissociation of salts:.: in that they contain an uneven number of electrons (Augenstein and Mason 1964) . The unpaired or odd electron makes the free radicals extremely reactive and short lived because of their reactivity (Bacq and Alexander 1 9 6 l ) . Complex organic radicals tend to have longer lives since resonance reduces the reactivity of the odd electron by distributing i t over the whole molecule, (Gordon 1957). 24 Effects of Ionizing Radiation on Plants Studies on the effects of ionizing radiations on plants have been carried out since the discovery of X-rays by Roentgen in 1895, of natural radioactivity by Bacquerel in 1896 and of induced radioactivity by Curie and F a l l a l in 1934, (Gunckel and Sparrow 1961). The amount of information accumulated on this subject is voluminous especially after 1950. Sparrow, Binnington and Pond (1958), l i s t e d the papers in plant radiobiology between 1896 and 1955 and showed an average of 200 papers a year between 1950 and 1955« This trend seems to be continuing. Another extensive bibliography has been compiled by Vasil'ev (1962) in U.S.S.R. The investigations in the general f i e l d of plant radiobiology f a l l into two main divisions, viz. genetic and non-genetic effects of ionizing radiation. The literature on radiation genetics is now enormous. 15. Several extensive reviews are available especially the followingJ Brookhaven symposia in Biology, number 14 (1961), Crow and Abrahamson (1965). Konzak (1957). Lea (1962), Muller (1959), and Wolff (1967). The genetic effects of small doses of ionizing radiation were discussed In a review by Shapiro (1959). Prakken (1959) l i s t e d 789 t i t l e s in a bibliography on induced mutations. The work on non-genetic effects of ionizing radiations such as biochemical, physiological, morphological...etc. effects, has been reviewed by many authors, e.g. Gordon (1957). Gunckel (1957, 196l), Gunckel and Sparrow (1961), Sparrow and Pond (1956), and others. Practically a l l of these reviews have indicated the drastic effects of high dosages of ionizing radiation on plants. Low doses may have some stimulatory effects, Breslavets (1946) has reviewed the early work, especially in U.S.S.H., on the stimul-ation of plant growth by ionizing radiation. Remarkable Increases in the growth and yield of rye were reported to be induced by giving the seed a dose of 250 r of X-rays and similar results were reported for peas and some other crops. In Europe, early work on the effects of ionizing radiations was carried out in Germany and France then followed by many other countries. This work, as well as the work in the United States, is well reviewed by Sax (1963). The experiments of Shull and Mitchell published in 1933 showed that low doses of X-rays given to corn seed resulted in a stimulation of early growth of the seedlings. Among the other investigators who reported stimul-ating effects of low doses of ionizing radiations are McCormik (1963), Stein and Steffensen (1959). 16. In an extensive series of experiments, Skok et aL (1965) investigated the stimulating effects of X-rays with seeds and plants of various genera. They found small but significant increase in growth in some cases but these results were not always reproducible. They questioned the value of ionizing radiation to stimulate plant growth of f i e l d crops. Negative results have also been reported by Osborne and Bacon (i960), and Vasil'ev et al. (1959). 25 Ionizing Radiation in Forestry As mentioned before, the research on the radiobiology of plants is extensive, however, relatively very few investigations were carried out on forest tree species. From the forestry point of view, the research on ionizing radiations has been mainly concerned with the presence of natural and fall-out radioactivity in the forest, and i t s distribution and movement through the plants and soils, the effects of radiation on various parts of the forest community (soil micro-organisms, tree seed, seedlings, trees ..etc.), and the application of radioactive isotopes in forestry research, e.g. tracer techniques and application of radio-active isotopes in wood science and techno-logy. A summary of the u t i l i z a t i o n of radioisotopes in Forestry research was given by Fraser and Gaertner in 1966. Since some of these subjects are beyond the scope of the work to be reported here, this review w i l l be mainly concerned with the radiosensitivity of forest trees and the ut i l i z a t i o n of ionizing radiation in tree breeding. 17. 251 Radiosensitivity of Forest Trees 2511 Effects of Ionizing Radiation on Seed Germination and  Subsequent Seedling Growth and Survival t Research on this subject is being carried out in a number of countries, notably in the U.S.A., Canada, Sweden, Japan, Korea, U.S.S.R., Yugoslavia, Rumania and a few others. Although the objectives are sometimes different, a wide variety of species has been studied, and i t is agreed that woody species are more susceptible to radiation than grasses, herbs...etc. and that conifers are more sensitive than deciduous trees. For years i t has been obvious to investigators working with seeds that they apparently represent a unique system and have special properties not possessed by the more actively metabol-izing tissues, such as vegetative buds or fresh pollen. One important characteristic of the seed in the radiobiological studies is i t s adaptability. It can be irradiated under a range of conditions that greatly alters the cellular environment. When dry i t Is resting, is almost biologically inert, and severe environmental treatments apparently cause l i t t l e or no biolog-i c a l damage. After such treatments and controlled rehydration, the irradiated seed can be measured for damage using several biological c r i t e r i a , Nilan et al. (196l). Besides the harmful effects of ionizing radiation on tree seed, some evidence of stimulation of germination and growth by treatment with small doses has been found. The results vary widely according to species, dosages, and the condition of the seed and there i s far more evidence for unfavourable effects. One of the f i r s t studies in this f i e l d was carried out by 18. Nlkltine In 193^ when he Investigated the influence of X-rays on seeds and pollen of some trees and shrubs. He found that exposure of seed of Picea ables and Plnus sylvestris to X-rays for periods of 10 minutes to 18 hours reduced germination. Baldwin (1936), in a t r i a l with European and American conifers, found that when seed was exposed to 100 kv of X-rays for 4 minutes germination of Plnus nigra, P. strobus, Plcea  rubrens and Picea abies was retarded. There was no stimulatory effect, and seedling growth was similar to that of the controls. He used one dosage only for a l l the species involved in the experiment, and no information was gained as to the behaviour of the species at other doses of radiation. The effects of X-rays on seed of Norway spruce (Plcea ables), Scots pine (Plnus sylvestris), and Alder (Alnus glutlnosa), were investigated by Simak and Gustafsson (1953)• In their experiments a weak effect was observedv/when using low dosages and an obviously depressive effect was observed at high dosages. The seeds of Scots pine were damaged by an X-ray dose of 900-1OOOr, 600-900r for Norway spruce and 5000r for alder. A dosage of 200r stimul-ated the germination of Alnus glutlnosa, but not of conifers. It appears that alder seed tolerates considerably higher dosages than seeds of conifers. In later experiments, (Gustafsson and Simak 1958), treatments with X- and gamma-rays at dosages higher than l50-300r reduced germinative capacity and germination rate of pine seed especially of the less ripe l o t s . Depressive effects on germin-a b i l i t y , rate of germination, number of cotyledons developed, hypocotyl length or dry weight of sound seedlings appeared at 19. dosages between 600 r and 900 r to dry Picea abies seeds. Germination was stimulated by 75 and 150 r and similar to controls at 300r. Miiller-Olsen and Simak (195^) and Muller-Olsen et a l . (1956) found that seeds of Scots pine and Norway spruce had an L D ^ Q with about 900r of X-radiation. Tralau (1957)» using Norway spruce seed, reported that irradiation with 300r increased the speed of germination but entailed some danger of physiological damage to the cells and that irradiation with 100 to 150 r slightly increased seedling growth, though results were not uniform. Seeds of Plnus sylvestrls. Pinus nigra, and Picea abies were exposed to 32 different dosages from 82 to 20,000 r from a cobalt-60 cource by Vidakovic (i960). Irradiation in small dosages was found to increase germination. The germination of Plnus sylvestris exposed to dosages of 82 to 2500r was greater than the controls, reaching 96.3$ vs. 84.8$. In Plnus nigra germination was greater than controls at 748 to 3500r, and in Picea abies at 500-5500r. Mean time taken for germination increased with increased dosages. The same author (Vidakovic, 1962), also reported that seed treatment with dosages up to 800 r had no significant effects on seedling growth of Pinus  sylvestrls. 784r gave temporary stimulus to growth in Plnus  nigra, but any difference had disappeared by the third year. 1-year plants of Plnus halepensls from seed given lOOr to 500r were t a l l e r and those from seed given 300, 800, 2000 and 5000r were smaller than controls. Plants of Larix decldua from seed given 332 and 748lr were nearly 50$ t a l l e r and had slightly larger diameter than controls. 20. Kamra and Simak (1965)t irradiated seeds of Plnus sylvestris and Plcea ables with the same X-ray apparatus normally used for seed testing by radiography but the tests included periods of irradiation longer than the usual. They found that even the longest period, (800 seconds), had no effect on germination percent after 30 days. With shorter irradiation periods, there was a weak tendency towards stimulation of germination in spruce seed, and with long exposures, a delay in pine. Seeds of Plnus sylvestris and Picea ables were irradiated with 300-3000r and those of Larlx s l b l r l c a with ?00 to 7000r by Karaban (1966). In the laboratory, germination percent was the same for a l l irradiation dosages. Field germination percent decreased sharply with dosages equal to or higher than l500r while with dosages of 3000r or more, no seedlings appeared at a l l . Heimburger (i960) obtained an L D 5 0 at 2000r for irradiated white pine seed, while Rhodes (cited by Snyder e_t al. 196l) found that germination of white pine was good after treatment with X-rays at 5000, f a i r after 2000r, and almost n i l after 8000r. Dudic (i960) stimulated growth of Plnus nigra seedlings by treating dry seed with 100-1000 rep from a cobalt-60 source. Following exposure of Plnus bankslana seeds to varying doses of cobalt-60 gamma radiation, La Croix (1964), recorded germination counts, seedling measurements and morphological effects. Percentage and rate of germination of seed were reduced following X-irradiation particularly with dosages exceeding 9,000r/hr. At the higher dosages, morphological effects included inhibited growth, thickening of the hypoeotyl and a swelling of the seed contents. 21. May and Poesy (1958), studied the effects of gamma-radiation on the germination of slash pine seeds. They con-cluded that irradiation at 500 r did not appear to affect total germination, but 5,000 r decreased germination significantly. LD^0 for germination was found to be approxim-ately 13,000 r. Irradiation doses of 500 and 5000 r delayed i n i t i a l germination of unstratified seeds, but after germination started, rates of germination paralleled that of non-irradiated seed. Germination of Slash and Longleaf pines, and seedling height growth decreased with increasing dosages in the range of 100-50,000 r, (McCormick 1965)« Seedlings from seed treated at 300 r were more resistant to experimental drought than those from untreated seeds, and a l l were s t i l l growing after 6 months. Although seedlings from seeds treated at 500 r survived for 40 days after the drought, this treatment Inhibited the formation of true leaves and a l l seedlings died on the completion of cotyledon stage. Also to identify environmental factors which modify species radiation sensitivity, another study was under-taken by McCormick and McGunkin (1965) to determine the effects of ionizing radiation upon the same above mentioned two species. They reported that percent germination, growth, physiological tolerance, and survival usually varied inversely with the radiation dose received. Slash pine seeds were more resistant than longleaf pine seeds but the order of seedling sensitivity was reversed. Results of these studies indicated that the condition of the organism and the nature of the environment prior 22. to and following irradiation must be rigorously defined in order to evaluate radiation effects. The Southern Forest Experiment station (1958), reported that the dosage of X-rays capable of k i l l i n g half of the strat-i f i e d loblolly pine (Plnus taeda) seed was approximately 1,000 r. Davis (1962), indicated that treatment of seeds with 10,000 r at 620 r/mlnute of gamma-radiation decreased the germination percent of Loblolly, Slash and Longleaf pines. None of the Longleaf pine survived after 4 months. Survival of Loblolly and Slash pines receiving 10,000 r was 28$ and 3 2 $ respectively after the same period, compared to 46 and 57$ for the controls. In another test, Loblolly pine seeds were exposed to different doses of gamma-radiation. Germination percentages were not recorded, but the surviving seedlings from each treatment were out—planted. Twelve months later, the average total growth showed no significant difference between treatments. In Korea, Yim (1964), studied the neutron and gamma-ray sensitivity of a i r dried seeds of three pine species (Pinus  densiflora. P. riglda and P. bankslana). The seeds of Plnus  banks!ana were most resistant to gamma-radiation with an L D ^ Q of 11.3 kr and the most sensitive was P. densiflora with an LD«jQ of 1 . 9 kr. Plnus riglda occupied an intermediate position with an LD50 of 7.2 kr. Clark et a l . ( 1 9 6 5 ) , irradiated dry seeds of jack pine, red pine, Scotch pine, white pine, black spruce and white spruce to simulate the gamma-component in an early fallout f i e l d over a period of 9 6 hours. They found that the 1 ^ 0 0 / 3 0 days f o r s e e d germination ranged from 3 2 0 0 R for white spruce to 8640 R for 2 3 . black spruce with exception of jack pine where germination occurred at a l l dosages employed. The L D i o o / i 2 0 days f ° r f i e l d grown seeds varied from 1720 R for white pine to 4730 R for jack pine. One-year-old seedlings survived higher dosages than did irradiated seeds scored at 4 months after irradiation. Under constant rate regime, the L D ^ Q Q / ^ O a a y s v a r i e d from 3260 R for white spruce to 8 6 4 0 R for jack pine. These findings were followed by another report from the same laboratory (Clark et a l . 1967), in which they indicated that a dose of 4 6 9 0 R to seeds of jack pine in combination with a 3 second thermal stress (425°C) resulted in a significant decrease in germination and survival (6 months), compared to other treatment groups. Seeds of Scotch pine exposed to the same thermal stress and 1020 R had an increased germination but a decreased survival. Dry seeds of the same species used by Clark e_t a l . (1965)» were exposed to chronic irradiation for a period of 1-250 days by Clark and. co-workers (1968) . An approximately 10-fold range in radiosensitivity was found between white pine, the most sensitive with an L D ^ Q / ^ Q days °^ R^ and jack pine, the most radioresistant with an ^ ~$Q/JQ ^ a y s of 6310 R. The highest exposures, producing no inhibition of germination (LD Q), varied from 4 0 0 to 3700 R. The lowest exposure preventing a l l germination (LD 1 0 0) varied from 1500 to 11,200 R. Survival of seedlings from irradiated seeds, 1-year post irrad-iation, showed white pine and white spruce to be most sensitive with an L D 5 0 of 575 R. and fack pine to be most radioresistant with an LD^0 of 2950 R. For a l l species combined the LD^Q ranged from approximately 8 0 to l l 6 0 R and the L D l 0 0 from 950 to 4300 R 24. for survival of 1-year old seedlings. Beers (1962) subjected seeds of Slash pine (Pinus e l l i o t t i i Englm. var. e l l i o t t i i ) to gamma-radiation dosages of 750 up to 12,000 r at a dose rate of 2868 r/minute from a cobalt-60 source. He found that lethal dose for germination after 30 days was 6300 r. Height measurements after nearly two months showed a small amount (1-3$) of radlostimulatiori at 750 r and height inhibitions were shown to occur by ca. 1000 r. Using Plnus sylvestrls seed, Ohba and Simak (1961) reported that X-ray dosages lower than 2400 r did not affect germinabllity of seed. No stimulative effect of X-rays on germination time was observed. Clearly an increase in time was required for germin-ation as dosages increased. Snyder, Grigsby and Hidalgo (1961), in studies of Southern pines, found that loblolly and shortleaf pine plants from seeds treated with X-rays at 1000 r were poorer in survival and growth than controls. For loblolly pine, in the f i r s t 7 years after out-planting, there were fewer female cones on plants from irradiated seeds than that on controls and 4000 r was lethal to the seed. 2000 r (not tested on loblolly and shortleaf pines) was lethal to seeds of Longleaf and Slash pines. Rudolph (1967), X-rayed Pinus banks1ana seeds at 1000 and 4000 R and the seeds were tested under commercial nursery and fi e l d planting conditions. At 4000 R, no seedling survived beyond 1 year of age. At the end of 2 years approximately 30$ of the seedlings from seeds given 1000 R survived as compared to the control. A decrease in LD^0 exposure was noted as the seedlings aged. Rudolph also found that the LD50 determined 25. from the survival response curves permitting prediction of dosage that would result in 50% plant survival at any point in the f i r s t two years. Seeds of Pinus riglda trees were exposed to It*-irradiation while they were s t i l l in the cones on the trees by Mergen and Johansen (1964). The results of a germination test showed that an exposure rate of 130 r/day and for a total exposure of 16,000 r, radiation did not affect germination, but 8 0 0 0 r at 295 r/day reduced i t . The L D ^ Q for seed irradiated at 84-0 r/day.was approximately 22,000 r. There was a temporary stimulation in root growth and an increase in fresh weight of the seedlings at exposures of 6,000 to 8 , 0 0 0 r and a temporary retardation in overall growth at exposures above 8 , 0 0 0 r, but these effects disappeared after the seedlings were one year old. Mergen and Stairs (1962) indicated that Plnus riglda was more sensitive than oaks to ^-irradiation at 3-15 r for 20 hours daily for several years, decrease in germination and seedling height being related to the total dosage accumulated by the tree. Decreased germination of Plnus densiflora. P. thunbergll. Cryptomeria .laponlca, and Chamaecyparls obtusa seeds treated with X-rays at 30 kv for 30-210 minutes was reported by Toyama (1954). He also indicated that germination percent decreased with increased duration of treatments. Seeds of Yucca brevlfolia were irradiated with 10-100 kr X-rays from cobalt-60 by Johnston and Klepinger (1967). In the resulting seedlings, the shoot-root ratio gradually Increased with increasing levels of irradiation. The authors interpreted the results as an indication of higher radiosensitivity of roots 26. than shoots. Survival percentages relative to controls varied with the time factor. Total mortality occurred at 60 kR and above. It appeared from their results that there was no significant difference between years of collection with respect to radiosensitivity. X-ray treatment of Ginkgobiloba seeds showed that the c r i t i c a l dose was very low, v i z . between 100 and 250 r, (Tacovic and Nikolic 1967). ' As indicated before, in spite of the extensive research on plant radiosensitivity, forest trees have received comparatively l i t t l e attention. Of those few studies most were carried out using gymnosperms and only a few used angiosperms. Practically a l l of these investigations have indicated the higher radio-sensitivity of gymnosperms. Privolov (1963) tabulated and discussed c r i t i c a l X- and gamma-rays doses determined for 31 f r u i t and forest tree species and varieties. He indicated that seed of conifers (5 species tested) were highly sensitive. Benea et a l . (1962) found that germination of Roblnla  pseudoaoacla was stimulated by 7400 and 14,700 rem dosages that prevented germination of Picea ables, and was scarcely affected by dosages of 5 and 2400 rem, which stimulated germination of spruce. They also found that germination of T l l l a tomentosa seed soaked for different periods in solution containing p^ 2, (0.25-1.00 u c / l i t e r activity), was ^highest for the shortest soaking period and was below that of controls after periods of 14 days or longer, (7 days for Plnus ponderosa). X-ray treated seeds of Betula verrucosa and Betula pubescens had a higher germination percent than the untreated seeds. 27. Irradiation for five minutes without a f i l t e r or for 30 minutes with a f i l t e r gave the best results, (Nikitin 1934). Scholz (1956) exposed air-dry Birch seeds in 1948 to X-rays at 130 kv at a distance of 30 cm. from the source. He reported that mortality in 1950-1955 of Betula verrucosa was 11.89$ for a 6-hr. and 14.93$ for 12-hr. exposures, and 11.17 and 17.80$ respectively for Betula pubescens. Germination of Betula  verrucosa seed exposed to X-rays in 1955 exceeded that of controls for a l l dosages except the-smallest (250 r ) , even exposures to 20,000 and 30,000 r gave germination somewhat above that of controls (Scholz, 1957). Jovanovic and Tucovic (1959)» exposed seeds of a natural cross between Populus x deltoldes and Populus x serotlna to d^-rays from a Co°^ source for doses of 500 to 50,000 r units. They found no significant difference in germination percent with difference in dose, but seedlings from seed treated with up to 10,000 r showed some increase'in growth over controls after 40 days, whereas stronger doses tended to reduce growth. After 2 months they noted that treatments with 500 and 1000 r caused most increase while doses higher than 10,000 r reduced the growth above the ground. At the end of the growing season, the stimul-ating effect was seen chiefly with the 500 r and 1000 r treatments, the latter causing greater increase in growth. Dose above 5000 r increasingly reduced growth both of parts above the ground and root system. Again in i960 the same authors, Jovanovic and Tucovic, described in detail the f i r s t year results of radiation treatments of seeds from crossing Populus x regenerata, P. x marllandlea and 28. P. x gelrlca with P. x serotina. They presented similar results to the previously recorded. At the end of the growing season, seedlings from the 1000 r and the 500 r - treatments were the ta l l e s t . An isolated stimulating effect was noted at 8000 r. The reduction in height growth with higher doses continued. Heaslip (1959), irradiated dormant seeds of 18 species native to the Eastern Deciduous Forests with accute doses of Cobalt-60 gamma rays. She indicated that seed irradiation aff-ected germination, seedling growth and survival of each species in an individual fashion. Survival of the seedlings from seeds irradiated with 10 kr was less than 30$ in relation to the control for one-half of the species in the laboratory and for ? out of 11 in the f i e l d . Llquldamber styraciflua and Fraxlnus  amerlcana were the only two species that germinated and had any survival following seed irradiation with 100 kR. Seed radio-sensitivity increased after dormancy had been broken. In the 6 0 meantime irradiation of 10,000 r from a Co source to Plnus  rlgida produced a germination of less than 20$ of the untreated seeds. Later (Heaslip 196?) , reported that combined doses of fast neutrons and "/-rays caused severe damage to seeds of Juglans nigra and Fraxlnus amerlcana at dosages that were harmless separately. Batches of 1000 winged stratified seeds of Fraxlnus  excelsior were exposed to radioactive cobalt for 1, 2, 3 , 24 or 48 hours, (dosages not stated), and then sown in 1956. By 1961 mortality was 80-90$ for a l l varieties and 84$ for untreated controls, but mean height and diameter were significantly greater for plants from treated seeds than for the controls. The 29. best treatments were 3 and 24 hours exposure, (Sapankovic" 1964). He also concluded that the possibility of selection for height was much greater in the irradiated seedlings. Stairs (1963). studied the effect of acute gamma-irradiation on the germination and seedling growth of Quereus alba, £. borealis. He found that the lethal exposures were the same for both but in the range 1-10 Kr there were inter-specific differences in germination, which for Quercus alba decreased linearly with increasing dosage, whereas for £. borealis the regression of germination on radiation was not significant although there was a significant difference between the 10 Kr dosage and the controls. Seedling height after one year did not give significant measure of damage. Many irradiated seedlings were as large as the controls, and at least one had outstanding total growth, but most of the growth alterations were generally harmful to total growth. Sinnes and Domingo (1967) presented tables and graphs showing the effects of 11 dose levels of gamma-irradiation (500-100,000 r) on Swletenla maorocarpa (Mahogany) seed, and on seedlings raised from irradiated seeds. A dose of 500 r slightly increased, but dose of 7500 r or more progressively decreased germination percent and there was less than 5% germination at 50,000 r or higher. Survival of 1-year seedlings was slightly reduced at the lower dose levels, but there was no survival at 50,000 r and up. At doses up to 10,000 r diameter at the collar, height, stem weight, and foliage weight a l l suggested a slight inverse relationship with increasing dose, except 500 r at which irradiation was apparently beneficial. 3 0 . 2512 Factors modifying seed radiosensitivity Radiosensitivity, as manifested by survival and development of biological material subsequent to irradiation exposure, is influenced by several factors. Lunden (1964) grouped such factors in two general categories for discussion of seed radio-sensitivityi (l) environmental conditions and (2) biological t r a i t s . Environmental conditions which affect Injury from exposure include temperature, atmosphere, moisture, chemicals, and pre-or post treatment variations. The second group of factors, biological t r a i t s , includes a l l physical and structural features of plants or, for seed radiosensitivity, embryos which have a bearing on radiation response of plant tissue. Low or high temperature and presence or absence of oxygen before, during or after irradiation can alter the injury and reviews on these points are given by Caldecott (1961), and Konzac e_t a l . (1961). Again most of the information on the factors affecting seed radiosensitivity were obtained from plants other than trees especially f i e l d crops, e.g. Biebb and Mostafa (1965)» Caldecott and North (I96l), Conger (1961), Konzak et a l . (1961), Mericle and Mericle (1961), Natarajan and Marie (1961), Osborne et a l . (1963), Poryadkova ( i 9 6 0 ) , Saric ( I 9 6 l ) , and Sax (1963). With respect to forest trees factors studied included seed moisture content, ripeness, dormancy, size, chemical factors, the position of seed on the crown of standing trees, provenance, and environment. Moist seed i s generally more resistant to radiation than dry one, but optimum moisture content and the effects of strat-i f i c a t i o n appear to vary between species. Gustafsson and Simak 31. (1958), reported that the germination of Picea abies dry seed (equilibrated at 0$ relative a i r humidity) was 8$ for treatment at 1,200 r of X-rays and 0$ at 2400 r, while the moist seed (equilibrated at 40$ a i r humidity) was 80$ at 2400 r. The differences in the mortality-of spruce seedlings grown from seeds exposed to various intensities of radiation were striking. Not only did exposed dry seed germinate poorly, but also the seedlings exhibited such low vigour that none of the seedlings emerging after 1200 r survived longer than 30 days. During the same length of time only two percent of the seedlings in the control series succumbed. They concluded that moisture content of seed at time of exposure is a factor of great importance in the effect of radiation on conifer seed. Experiments were conducted by McMahon and Gerhold (1965). using Scotch and white pine seeds to investigate how modific-ations in moisture content can influence the effects of gamma-Irradiation. Their data indicated that the highest dosages at which a i r dried seed germinated were 3.200 r for Scotch pine and 1,600 r for white pine. By adjusting the moisture content of the seeds of both species from the a i r dried condition of about 6$ to about 12$, i t was possible to obtain comparable germination results at four times the dosage. A decrease in moisture content depressed the germination only of white pine seed. Seed of Pinus densiflora with 6 different moisture contents in the range 2.2-16.3$ of dry weight were subjected to 7 different doses of X-irradiation (0-18,000 r) and the germination and survival percent, plant development and stage of development 3 2 . reached by k i l l e d plants were studied by Ohba (1961). Minimum sensitivity was found in seed with 12.7$ moisture content; irradiated seeds of higher, or especially lower, moisture content showed very low germination percent and high seedling mortality. Pinus rlglda seed samples were equilibrated by Mergen and Cummings (1965) for eight days at six moisture contents (2 .4-16.1$) and exposed for 16 hours to five gamma-ray dosages (4-34 kr). They reported that increased moisture content up to 8 .9$ decreased the radiosensitivity and between 10.9 and l 6 . 1 $ L D ^ 0 values for germination were similar, (120 kr). In this range there was no loss of germination capacity up to 12 kr, although seedling development was retarded. Recovery increased with increasing stratification period and moisture content. Snyder et a l . (1961) subjected seeds of long-leaf, slash and shortleaf pine to various moisture and X-ray treatments. Their results indicated severe irradiation damage at 33$ moisture content. Prestratification for 3 weeks in conjunction with dosages of 800 r and above caused significant reduction in germination percentages. L D ^ Q averaged 1600 r for the three species. Seed prestratifled for two weeks had the same moisture content as the 3-week seed, but was intermediate in sensitivity. Germination was also Intermediate for dry seed and seed exposed to 100$ relative humidity, while high germination was obtained from seed irradiated dry and then s t r a t i f i e d . X-lrradiation at 300-1800 r of Scotch pine seed, a i r dried or dried over P 2 0^, reduced the percent germination from 99$ to 90$, (Laura 1966a).. Then the author studied the effects of storage conditions on the extent to which this radiation damage 33. increased during storage for 24 hours. He found that the increase in damage, more marked in air-dried seed than over P 2 O^ , was greatest for storage in oxygen, less in a i r , and least in nitrogen atmosphere. With storage at 4l°C, damage Increased but only in seed dried over P 2 0^; at -186°C there was no increase in damage. Once more, Laura (1966b) indicated that in dried Pinus  sylvestrls seeds (4$ moisture content), and a i r dried seeds (ca. 6% moisture content), shortage up to few days after 1000 r X-lrradiation increased chromosome aberrations and decreased germination. The storage effect was not observed in moistened seeds. Yim, (1964) reported that the effect of the postirradiation storage of Plnus riglda seed irradiated with gamma- and X-rays were -that the radiation damage as determined by percent germin-ation, increased with lengthening the storage period after irradiation. Clark et a l . (1967) concluded that in dormant seeds high moisture content may increase resistance to irradiation while extreme dehydration resulted in increased sensitivity. Heaslip (1963)» found that the radiosensitivity of physiologically active Quereus velutina and Juglans nigra was inversely related to seed moisture content, however, moisture content, physiological activity, and radiosensitivity of seed increased as embryo dormancy was broken during stratification. Later she stated that the effect of increased water content on fast neutron and gamma-ray sensitivity varied between germination and survival in a given species and from one species to another. Increasing seed 34. water content preceding fast neutron-gamma-irradiation increased the sensitivity of physiologically active Quereus alba, decreased the sensitivity of dormant Fraxlnus amerlcana, and Platanus occidentalis and the effects were variable from level to level in Juglans nigra, (Heaslip, 1967)• Moist stratification of Plnus rlgida seeds for 4 weeks after irradiation of 0-48 kr caused an increase in germination that was paralleled by a decrease in cytological damage at a l l exposure levels, but there was no difference between 16 and 80 hour exposure periods within any post Irradiation treatment, (Mergen et a l . 1965)' Also stratification decreased the radiation effects, and the recovery period at lower levels of radiation was shorter than at higher levels. Dry stratification Increased germination at a l l radiation levels but not the rate of seedling development. Saric (1961) believes ? ithat much inter-sample variations in radiosensitivity i s caused by small differences in seed quality or maturity. The development of the embryos, i.e. the length of embryo in relation to the length of the embryo cavity, is a criterion of the ripeness of seed, the longer the embryo the more ripened,-is the seed, (Simak and Gustafsson, 1953)• The reaction of Pinus  sylvestris seeds of different ripeness to various intensities of radiation was studied by Gustafsson and Simak (1958). Their germination results showed clearly that unripened seed, (embryo class II), was much more sensitive to radiation than ripened seed, (embryo class IV). Ripened seed tolerated 2-3 times more X-irradiation than unripened seed. Simak, Ohba and Suszka (1961), working with Pinus sylvestris 35. also found that small seeds were more sensitive to X-rays than larger ones. Depression in germinability and seedling survival: increased simultaneously with and parallel to an increase in mitotic disturbance in the embryos. The ^ r a d i a t i o n sensitivity of Plnus riglda seeds varied with the seed size class. The smallest seeds were the most sensitive ones, (Yim 19640:. With regard to the genetical aspect of radio-sensitivity several interesting results have been reached. Simak, Ohba and Suszka (196l), X-irradiated Plnus sylvestrls seeds originated from 4 trees. They found difference in radiosensitivity between different trees and related this partly to the genetical constit-ution of the trees and partly to their environment. Seeds from four Plnus nigra trees were exposed to radiation from a cobalt-60 source at doses of 100-1,000 r by Dudic in i960. In nursery t r i a l s varying doses affected the progeny of each tree in different ways. Treatment of the seed after soaking for 24, 48, and 72 hours again showed different responses from each tree. Laboratory tests on germination and weekly seedling growth suggested that the effects of radiation was dependent on many factors including dose, length of soaking and parent character-i s t i c s . Ohba and Murai (1^67), studied the difference in radio-sensitivity to acute gamma-irradiation of self- and cross-pollinated seeds, and of seed with a marker gene in Crypotomeria  japonica. They reported that open pollinated seeds showed the highest resistance to radiation. Hybrid seeds between two trees showed intermediate germination percent than those from open 36. pollinated seed and selfed seeds of a r t i f i c i a l l y induced flowers (using Gibberellic acid), and a slightly higher value than those of selfed seeds of both of their parents, indicating a heterotic effect of the hybrid seeds on the radiosensitivity. The germin-ation percent of irradiated seed, and genetlcal and physiological shares of radiosensitivity were estimated. The genetlcal portion was dominant over the physiological in 12 and 15 kr exposures. Seeds of each of two Bulgarian provenances of Pinus  sylvestris, P. nigra and Picea ables, and one provenance of Plnus  mugo; were exposed to 200, 600 and 900 r of gamma radiation by Dobrinov and Atanasova (1967). A l l three dosages increased the germination percent of Plcea ables, Pinus mugo and 200 r also increased their germinative energy, but a l l other species/dosages combinations resulted in reduced germination percent and germin-ative energy. Ohba and Simak (1961) undertook experiments to obtain information about the radiation sensitivity of Pinus sylvestris seed from 10 different provenances in Sweden. The results showed great differences between the provenances with respect to the radiosensitivity of the seeds. Seeds from provenances, with short growing season northerly latitude and high altitude were more resistant to X-rays than seeds of southerly latitude and low altitude. Moreover, Tralau (1957) found that the generally rather greater resistance to gamma-rays of three southern provenances of Picea ables than that of a provenance from northern Sweden resulted from genetically controlled variation. Seedlings of Picea ables from high elevation sources were more resistant to gamma-rays than those of lowland origin, 37. (Laffers i960). Mergen and Johansen (1964) observed large differences in germination behaviour of seed from individual pitch pine trees subjected to ionizing radiation in the forest. In addition, Mergen and Commings (1965) stated that there was a highly-significant difference in response to radiation for the indiv-idual Plnus riglda trees and for the region within trees. With the exception of one tree out of 10, seed from the upper crown was more sensitive to gamma-irradiation than seed from the lower crown. That was not related to the seed size or weight. 2513 Effects of Ionizing Radiation on Young and Mature Trees The studies in this f i e l d have been conducted for several aims and objectives. The plants were exposed to natural or simulated fall-out radiation, radioactive wastes, or irradiated from experimental sources. Subjects covered include the effects of environmental conditions on damage, the sensitivity of young plants and trees, the effects of radioactivity on growth and development of seedlings and trees, the process of sexual reproduction, the rooting of cuttings, etc. Great attention has been given to the difference in radiosensitivity between species. This warrants special consideration. Davis (1962) found that dormant 1-year old seedlings of Plnus taeda exposed to 3.000-7,000 r of cobalt-60 gamma radiation were a l l dead after 8 months. Growth of the seedlings exposed to 500-2800 r at 2.7 or 6.95 r/minute was affected bybboth intensity of dose and total dosage. Survival was slightly less after treatment with 500 r than in controls, and decreased sharply with higher dosages. He also reported similar results for Plnus 38. e l l l o t t l l . Two-year-old seedlings of Plnus strobus were bundled and Irradiated with gamma-rays in different groups. The f i r s t group received only shoot exposure, the second only root exposure, and the third both shoot and root exposure. Needle moisture, length and mortality were measured for 6 months. Wltherspoon (1965)• found that needle moisture reached c r i t i c a l levels f i r s t in the second group and early needle mortality occurred in the third. In a young Plnus monophylla tree that received dosages from 2,000 r to 4 r in different parts, depending on the distance from the source, Brandenburg et a l . (1962), noticed that 1,300 r ki l l e d a l l l i v i n g tissues within 4 months, and 600 r k i l l e d actively growing tissue. Below 500 r, lateral buds replaced k i l l e d or inhibited terminal ones. Even l4 r sufficed to reduce stem elongation and radial xylem growth was reduced by 3-8$. Dwarfing, deformation, and reduction of needles occurred down to less than 50 r. Actively growing Giant sequoia seedlings (Sequoia glgantla) received acute y-radiation from a Co^° source at various dosages ranging from 42 to 1119 rad and Taylor (1968) found out that growth of primary stems was inhibited 28$ by 134 days post-irradiation. Microscopic examination of f o l i a r tissues revealed abnormal development of the vascular strand as the result of a reduction of elements per xylem row, extensive development of transfussion tissues, and increased c e l l wall thickness of some tracheids. Double or twin resin ducts also were observed in leaves that received 697 rad. 39. In a 12-year old f i e l d that had been exposed to portable Cs 1-^ source of 9200 Ci for 400 hr. Monk (1966) studied changes in f l o r i s t i c composition, root biomass...etc., including the effect on growth of 5-year-old planted Pinus palustrls trees. A l l dosages reduced terminal growth of the pines during and after exposure. One r/day reduced i t by 50$ vs. control and an accumulated dosage of 9.000 r was lethal. Sparrow, Schairer, Sparrow, and Campbell (1963). exposed seedlings of Plnus strobus for 16.5 hours to Cobalt-60 gamma radiation at dosages from 50 to 1000 r during a period of dormancy or a period of active growth. The effect on growth was determined by measurement of needle lengths. They showed dormant plants to be considerably more resistant%than the actively growing plants. After exposure of 400 r and above, the needle lengths of plants irradiated when actively growing were not appreciably greater than the time of treatment. They assumed that very l i t t l e or no c e l l division or c e l l elongation occurred in these plants after irradiation. While less sensitive than actively growing plants, needles of dormant plants exposed to 800 r or more exhibited no discernable growth changes. Young Plnus thunbergll trees, subjected to an acute dose of 715 to 440 r of gamma-radiation developed buds at the same time aswcontrols, a phenomenon not noted hitherto in trees subjected to chronic irradiation, Okunewick et a l . (1965). Piatt (1963) indicated thatterminal buds appeared to be more sensitive than lateral ones, and lateral buds near the apex more sensitive than those nearer to the main stem. He has attrib-uted the greater sensitivity of the terminal shoot than of the 40. lateral buds of Plnus taeda to auxin metabolism. Terminal growth was inhibited by 1000 and radial growth by 3000-4000 rad. In both Taxus media and Plnus strobus. after a 20-day exposure at dosages of 3*15 to 15 r/20 hr. day, cytohistological zonation became abnormal, after exposure for 50 to 60 days, some badly damaged shoot meristems appeared as broad disorganized structures, with adventitious merlstematic regions and fascia-tions, and others as completely "inoperative" sound tissue, (Miksche, Sparrow and Rogers 1962). They also found that dosages as low as 10 r/20-hr-day, given an accumulated dose of 600-800 r, produced nearly lethal effects. An experiment was designed by Mergen and Thielger (1966), to test the effects of differential chronic exposures to ionizing radiation at various stages of development and periods of growth on the survival, height growth, productivity and cytology of Plnus rlglda. Radiation had a marked effect on terminal shoot growth inhibition and in the stimulation of sprouting. Shoot growth decreased, and flushing was delayed with increasing exposure, while sprouting increased and was responsible for the survival of many seedlings. They also found that increased exposure to radiation resulted in a decrease in subterminal bud formation and, at the higher levels (15 and 20 r/day), axial primordia were delayed and the apical primordla were damaged. Needle length decreased with increasing exposure but c e l l damage in the needle meristems was slight. In deciduous trees, winter dormancy was prolonged by periods proportional to the dose received in the previous summer, delays ranging from 1-2 weeks for doses of several hundred rads to 7-8 41. weeks for 10,000-12,000 rad. The growing season of woody species was also shortened by the acceleration of summer and autumn growth processes, (Piatt 1963). Rebuck (1965)» concluded that bud mortality and crown condition in an Oak/Pine forest proved to be the best c r i t e r i a for assessing radiation damage, sprouting of oak species gave a resistance that was not predictable from the characteristics of the c e l l nucleus. Hamilton (1964) found that dosages of 9,800-35,000 rad over two years reduced the radial increment of Quercus alba, reduced the number of libriform fibers, and retarded c e l l elongation and c e l l wall development, however, Liquidamber styraclflua exposed to approximately 15,000 rad over the same period showed no pronounced tissue aberrations. Four-month-old plants of Swietenia humilis proved relatively resistant to chronic doses of gamma-radiation, (Moh 1963). Sax (1954), experimenting with young poplars showed that X-ray doses sufficient to suppress c e l l division can induce phloem blocks in the tree without k i l l i n g the stem tissue. 2,500 - 5,000 r caused phloem blocks and 6,000 r ki l l e d tissue above and below the irradiated areas. One-year-old seedlings of four Alnus and three Betula species and seven Populus varieties were outplanted and irrad-iated for 672 days by Ohba and Murai, (1966). The lethal dose rate was estimated at 60-160 r/day in the second year and 60-80 r/ day in the third year varying with species. Height growth was strongly inhibited at high doses. Blind buds and abnormal internodal branching increased rapidly above dose varying from 42. 50 to 200 r/day. The morphological and histological damage in Quereus alba and Quercus velutina. grown for 10 years in the vicinity of Co^° -gamma-source, were reported by Mericle, Mericle and Sparrow (1962). Duringtthat period plants were irradiated to a total of about 16 kR. Gross visible effects included sparseness of foliage due to death of many lateral or terminal buds or branches? internodes typically reduced in length and increased in thickness, leaves reduced in number, often enlarged, thickened and somewhat leathery in appearance, and abnormal inflorescences. Histological studies revealed many lateral and terminal buds outwardly appearing viable but lacking merlstematic tissue, deterioration of the central pith, reduced number of f l o r a l primordia, reduction of viable pollen grains, and other effects. Among other effects of ionizing radiation on forest trees, leaf abnormalities and abscission are well known. Dosages of 125-190 rad appeared to stimulate linear growth of the needles of Plnus taeda, Plnus echlnata. and Plnus virginlana. (Nelson et a l . 1963)f but chronic exposure to dosages as low as 1.5 r/ day reduced needle size of Pinus rlgida. (Sparrow, Schairer and Woodwell, 1965). Brandenburg et a l . (1962), indicated that a number and size of needles were reduced in Pinus monophylla by doses of 50 r or less. Trees of Plnus taeda within 750 feet of a reactor irradiated in June 1959» suffered an abnormal needle f a l l in the next month. Leaf malformation arising from irradiation have been observed in Lirlodendron tullplfera and Ginkgo:blloba, (Gunckel 43. and Sparrow, 1961), and in Quereus alba and £. velutlna. (Mircle, Mircle and Sparrow 1962). McGinnis (1964), reported that irradiation in an Oak/ Hickory forest in June, when leaves were ful l y expanded, had l i t t l e effect on leaf characters. 1500 rad caused leaf f a l l to begin 7 weeks early in the current year and almost completely inhibited leaf production in the following year, 4,000 rad induced leaf f a l l one week early and reduced total l i t t e r f a l l in the following year by 42$. Autumn coloration was normal, while the higher dosages caused leaves to turn brown before f a l l i n g . At 1200 rad differences in l i t t e r f a l l from controls were not significant. Irradiation in August, when buds were approaching dormancy, had less effect on the following year's l i t t e r production. Other effects of ionizing radiation included those on increment, stem form, natural regeneration and resistance to drought and insect attack. In Pinus monophylla, doses as low as 15 r reduced stem elongation, (Brandenburg et a l . 1962), and in Pinus riglda, exposed to 4 r/day increment declined after some years but cambial growth was resumed when environmental conditions improved, (Sparrow, Schairer and Woodwell, 1965). Experimental Plnus riglda trees that had been exposed to radiation over 9 years at rates of approximately 0.1 r/day in earlier years and higher rates, (maximum 5 r/day), later were felled by Woodwell and Miller (1963), and ring widths in each internode were measured and compared with non-irradiated controls. The irradiated trees showed reduced increment 44. throughout the stem, the effect being pronounced near i t s base. Trees with large crowns were less affected and the effect was lower in favourable climatic years. Murai and Ohba (1966) reported that the lethal dose for young Larix leptolepls grown in a gamma-radiation f i e l d in Japan from 1962 to 1965 w a s 135 r/day in the f i r s t year, with no cumulative effect on succeeding years. Height increment was affected by 2-5 to 4-5 r/day. Increasing the intensities of radiation caused a significant increase in abnormal c e l l division and developing leaves. Pedigo (1963a) indicated that the resistance of Pinus taeda to heat and tolerance of shade and drought were reduced by exposure to ionizing radiation. At the Lackhead reactor side, Dawsonville, Ga., Nelson e_t a l . (1963), correlated the significant increase in infestation of Pinus taeda and P. enchinata, but not P. vlrglnlana by the cecidomyild Itomlda pinirlgldae with exposure to a total of 2000-2599 rad two years before. A consideration of some of the major physiological lesions Induced by radiation may be helpful in expanding our under-standing of radiation syndrome in plants. Such physiological changes have an earlier onset for the most part than morphal-ogical damage (Giocomelll and Cervigi, 1964). The physiological processes of water exchange, nutrient uptake, Carbon dioxide exchange (Hadley and Woodwell, 1966), (Woodstock and Justice, 1967), food translocation, production of oxygen and nitrogen metabolism, have been considered in a wide range of plant speciest(Hasegawa et a l . i960). Most of the above mentioned physiological processes are quite radiosensitive at exposure proving lethal to the organism. Photosynthesis i s a sensitive process and reflects physiological imbalance related to radiation stress, ( Z i l l and Talbert, 1958), this process recovers quickly from radiation effects, (Hadley and Woodwell, 1966). Photosynthesis seems to respond to the effect of ionizing radiation by a rapid decrease in C02 fixation. This has been demonstrated in many plant species including pine seedlings, (Woodwell and Bourdeau, 1964). Hadley and Woodwell (1966), reported that at 8000 r/hr. even 1250 r permanently depressed net CO2 uptake and, after i n i t i a l stimulation, stem respiration of 2-year Pinus taeda and Pinus e l l i o t t i i seedlings. The effects Increased with dosage. Leaf respiration was stimulated by 50 r/hr. 2514 Effects of Ionizing Radiation on Sexual Reproduction of  Trees It has been realized by many investigators that ionizing radiation has harmful effects on sexual reproduction of a l l l i v i n g organisms. The reproductive organs of trees are particularly sensitive to radiation. Watanaba, and Ohba (1964), studied the effects of gamma irradiation at 13.4 r/day on flower setting of Pinus densiflora and P. thunbergii between August, 1962 and May, 1963* Male flowers were found to be more radiosensitive than female ones. Investigation of the pollen germination percent and length of the pollen tube showed no distinctive gradient because of the wide individual variation. Trees of Pinus taeda within 1500 feet of a reactor and 46. bearing cones with viable seeds in 1958 before irradiation began, produced only abortive cones in 1959-1960 after irradiation for a total accumulated dose of 1000 rad in the f i r s t year and 3400 rad in the second. Shielded trees and those beyond 2000 feet produced normal cones, (Pedigo, 1963a). Pinus riglda trees were exposed to radiation levels ranging from 960 r/day to a level of natural background radiation. Mergen and Johansen (1963). collected microsparangiate stroboli and evaluated them at regular Intervals to follow the effects of gamma-radiation on the cytology of microsporogenesis. They found that the trees receiving 82 r/day^and higher were unable to resume c e l l division and differentiation during the spring, while micro-spore mother cells were formed on trees receiving 75 r/day. A slight reduction in the length of the stroboli was observed at 0-4 r/day and at 3-7 r/day they were significantly shorter than the controls. The percentage of cells with visible chromosome aberrations increased with an increase in exposure. Pollen abortion increased with an Increase in radiation, and at 7 r/day approximately 50% of the pollen grains were aborted. As an addition to this work, the effect of radiation on megasporogenesis were reported by Mergen and Simpson, (1967). They made periodic collections of 1-year-old p i s t i l l a t e cones from Plnus rigida trees mentioned above. They concluded that f e r t i l i z a t i o n was delayed by chronic gamma exposure. In addition chromosome aberrations were also observed in several cells during mitosis. Chromosome bridging was observed at anaphase in the cells from nucellare:.1.; tissue of a cone exposed to 2-8 R/20 hr. day and micronuclei were present in an ovule which had received 47. 3.15 R/10 hr. day. Okunewick, Herrlck and Carlsen, (1965). reported that four-year- old trees of Plnus thunbergll exposed to acute gamma-Irradiation of 715 ± ^0 r showed reduction in terminal and lateral candle growth, and no pollen was released from the male stroboli, although they reached the same state of elongation as controls. Adventitious buds developed immediately below the candles, and buds also formed at the tips of candles; such bud formation has not been reported in conditions of chronic gamma-irradiation. A long term study by Sparrow, Shairer and Woodwell, (1965). showed that even 1.5 r/day affected the reproductive processes of Plnus riglda. Two microsporangiate stroboli were produced in 1 month on a sprout from a Pinus riglda seedling that had been exposed to a total of 6800 R at a rate of 20 R/day. This presented a consider-able shortening of normal phenological pattern, (Mergen and Thlelger, 1966). An investigation of Plnus riglda and Quercus spp. (Q. alba, £. cocclnea and £. l l l c l f o l l a ) , in the natural forest adjoining the gamma-field at Brookhaven showed that trees which had been severely damaged by chronic irradiation (4-12 r for 12 hr. day), were able to form flowers and to produce viable seeds. Flowering was retarded by 7 to l4 days In Plnus riglda and Quercus spp. Serious malformations and chromosome aberrations occurred and pollen abortion increased with increasing radiation. For Plnus  riglda there was a decrease in cone length that was associated with an Increase in the chronic gamma-irradiation accumulated 48. by the trees. Also pines showed decreased cone length and pollen germination, needle abnormality, lack of primordia on leading shoots and irregular apical growth patterns, in 1-year old progeny. Oaks were more resistant, (Mergen and Stairs, 1962). Stairs (1964), indicated that when branches of Quereus alba and £. coccinea bearing male buds were exposed to acute or semi-acute gamma-irradiation, the rate of chromosome-aberrations and of pollen abortion increased linearly with Increasing exposure, and a range of 1-4 kr is suggested for treating male flower buds in mutation breeding programs. Mericle et al.(l962)reported that the effect of ionizing radiation on Quercus alba and velutina exposed to ca. 16,000 r over 10 years included abnormal inflorescences and a reduction in flower primordia and viable pollen. 2515 Effects of Ionizing Radiation on Pollen Grains and Seeds  Produced by Controlled Pollination*. Considering the review of literature so far presented, and at the risk of generalization, i t may be noted that the majority of this work has been concerned with seed irradiation. Less work has been accomplished with mature tree irradiation or somatic irradiation of parts thereof; and much less with gametic irrad-iation. Despite the practical advantages of seed irradiation there is no a priori genetic reason to discriminate against gametic irradiation as a means of mutation induction. Furthermore, seeds and vegetative portions are multicellular, complex tissues presenting d i f f i c u l t i e s for induced mutation detection and selection. Osborne (I95?a)» related these d i f f i c u l t i e s to (a) the chemical nature of the ...mutated!!, sectors; 4 a . (b) a rapid loss of induced cytogenetic changes during subsequent mitosis and meiosis of M1 plants; and (c) over-growth of mutated cells by non mutated c e l l s . The pollen grains of flowering plants have proved to be tailored admirably to the demands of certain radiobiological queries. They offered a relatively simple, haploid radiation target and withstand an impressive variety of experimental abuses, (Brewbaker and Emery, 1 9 6 2 ) . Greater efficiency for induction of mutation might be obtained by irradiating a plant when half of i t s genes are located in an individual c e l l . This immediately suggests the application of mutagenesis to pollen or zygotes, (Nilan and Konzak, 1 9 6 1 ) . Pollen have been particularly suitable for cytogenetic inquiries involving most kinds of radiation. In addition the use of pollen or anthers for the induction and study of mutations has many commendable, although somewhat neglected features. Brewbaker and Emery ( 1962), presented a very lengthy account of the literature dealing with the effect of irradiation on the germin-ation of angiospermic pollen grains. They reported inhibition of germination, and pollen tube growth only after exposure to massive doses, with the L D ^ Q values ranging up to 550,000 r. By contrast, they reported that pollen tube divisions were restricted to an average dose of 250 r at the L D ^ Q level. Thus i t Is possible, they say, to irradiate pollen at very high levels which w i l l probably inactivate the nucleus but w i l l allow normalsor near normal germination and tube growth. In studies with pine and oak, Stairs and Mergen ( 1 9 6 4 ) , exposed pollen from Pinus riglda, Plnus strobus and Quercus alba 50. to '^-radiation. After exposure to 30,000 r up to 70$ of the pollen for a l l three species germinated and no significant reduction was found in pollen tube lengths. However, the germination results obtained were more variable after irrad-iation than the controls. Forking, balooning, and other growth phenomena were observed at a l l levels including the controls and there was no increase in forking as a result of radiation. Some of the irradiated pollen of Plnus rlgida was stored in a desiccator under refrigeration. After 10 months, the v i a b i l i t y was 6l$ for pollen exposed to 300,000 r. The pollen grains of Plnus rlgida were exposed to gamma-radiation by Mergen and Johansen (1963)* At 3 to 5 r/day germination in vitro was reduced to 50$ of the controls. Pollen tube length was not related to exposure and mature pollen of Plnus riglda and P. strobus produced normal pollen tubes in 60 vitro after 16 hr. exposure to 300 kr Co radiation. Gamma-irradiation of fresh pollen up to 75 kr had no effect on the germination of Plnus denslflora pollen, but pollen tube length of Plnus thunbergii decreased with increasing dosage, (Fujimata et a l . 1964). Erikson (1966), obtained pollen grains resembling the waxy pollen in maize and barley after 2.4 hour gamma-irradiation from 13? Cs J { of pollen mother cells in a clone of Larlx leptolepis using a dose range 45-500 rad. Doses of 90-500 rad gave an appreciably higher percent of giant, perhaps diploid, pollen grains (3-5-7$). The effects of acute^-irradiation on mature pollen of Quercus alba and Q. coccinea were studied by Stairs (1964). His 51. data showed that the pollen was highly resistant to radiation, no effect being found at 100 kr. Germination and tube growth were reduced at 300 kr. He found no significant difference between species for either chronic or acute irradiation. Stimulation of pollen germination and pollen tube growth by irradiation has been a subject of some interest to radio-biologists and one of which pollen research provides conflicting evidence. Nikitin (193^), indicated that the pollen of certain shrubs was stimulated by X-irradiation and that of others retarded in germination. Air dried pollen of Philadelphia caucaslca was injured by exposure to X-rays, but exposure of pollen of T l l l a spp. to X-rays for 30 seconds to 30 hours increased i t s germination percent and the length of the pollen tube. The total dose was not given, however. Pollen irradiation affects the characteristics of plants produced, (Rudolph 1965). Male-stroboli bearing branches were collected from white spruce (Picea glauca) trees and irradiated at the beginning of anthesis. After controlled pollination, the author reported that total seed yield per cone increased with increased gamma-ray dosage to the pollen, the 600 r and 800 r treatments resulting in 23 and 27% more seeds, respectively, that was significantly more than the controls. Seed germination as compared with the controls was 12% less at 400 r pollen dosage, 16% greater at 600 r and again 12$ poorer In the 800 r dosage. Seed weight showed an increasing trend with increases in pollen dosage. Vidakovic (1967), used two parent trees each of Austrian pine (Plnus nigra) and Scots pine (P. sylvestrls), to carry out 52. intraspecific hybridization with gamma-irradiated pollen (0.05-10 kR). Plants of Scots pine produced through controlled pollination, exhibited significantly greater heights and diameters than controls, i.e. parents whose male gametophyte of the male parent was not irradiated. In Austrian pine significant differences were obtained for the heights of 3-4-year old plants compared to their controls, while for the diameter no significant differences in 4-year old plants were obtained. In both species variability increased with increasing exposure and growth stimul-ation was evident after higher exposures. Exposures below 100 R did not produce stimulation. Jaarverslag Boskouwproeptation (1965), reported that when Elm pollen was treated with X-rays at 0 to 4000 r the lower doses failed to stimulate seed setting, but none of the dosages damaged the pollen. Also Bogdanov (1948), indicated that exposure of pollen of Populus spp. had no apparent effect on plants raised from such^material. 25l6 Cytogenetlcal Effects of Ionizing Radiation on Forest Trees It is of historical interest that even before the discovery of the radiation mutagenesis, i t was known that radiation would enhance, the frequency of chromosomal misbehaviour. Mavor showed as early as 1921 that Irradiation of Drosophila females caused them to produce eggs with too many or too few chromosomes. Sax (1957) stated that ionizing radiation produces three general classes of aberrations depending upon the stage of nuclear development at the time of irradiation* a) - irradiation at the resting stage results in chromosome aberrations, 53. b) - irradiation at prophase results in chromatid aberrations, and c) - irradiation in very late prophase or prometaphase produces half-chromatid or sub-chromatid aberrations. In a review of the research uses of the gamma f i e l d and related radiation f a c i l i t i e s at Brookhave National Laboratory, Sparrow (1966), indicated that considerable attention has been given to the types and frequency of chromosome aberrations produced in different species of chronically irradiated plants. It has been found that the aberration frequency gradually increases during the f i r s t few days of chronic exposure and plateaus except at the highest exposure rates. An example of such data from Vlcia faba is given by Evans and Sparrow (1961). This is in contrast to the well known sharp peak in the frequency of chromosome aberrations seen at the f i r s t day or so following acute irradiation, Evans (1962). It has been shown that daily exposure rates as low as 0.6 R produced a st a t i s t i c a l l y significant increase in micronuclei frequency, (chromosome fragments), (Sparrow and Pond 1956), and that the length of mitotic cycle is an important variable in determining total effect in chronically irradiated plants, (Sparrow, Cuany, Miksche and Schairer 1961), Vant Hof and Sparrow, (1963). Bevilaequa and Vidakovic (1963). studied the effects of gamma-irradiation on the chromosomes of somatic cells in Picea  abies. It was established that in cells produced from irradiated seeds there occurred changes of relative lengths of chromosomes In comparison to the controls. The changes occurred always in 54. larger and smaller chromosomes, while the medium sized ones have smaller changes. It was characteristic that the overall length of the chromosomes of the individual cells did not change in comparison with the controls at 100-1000 r dosages. At higher doses, i.e. from 3.000 to 10,000 r there occurred fragmentation of chromosomes as well as other abnormalities in the mitosis. Obtained were cells with a smaller number of chromosomes and cells with a double number of chromosomes arranged in pairs. At doses of 5,000, 7,000 and 10,000 the authors found that a large number of cells in anaphase showed bridges and other abnormalities. In a later paper, Bevilaequa (1965). stated that irradiation of Plnus nigra seeds induced changes in the daily rhythm of mitosis, the total number of mitotic stages in seedling root-tips;:-in 24 hours decreased with increasing dose in the range of 100-500 r. The time sequence of maxima was disturbed, and their number decreased in proportion to the intensity of radiation. In Picea ables and Pinus sylvestris, exposed to soft X-rays up to ca 800 seconds, Kamra and Simak (1965). reported that even short periods of irradiation caused some aberrations in mitotic c e l l division. Suszka, Ohba and Simak (i960), working with irradiated Plnus  sylvestris seed found that exposure to X-rays caused abnormal c e l l divisions, increased in number with increasing dosage, but some seedlings even after dosage of 4,800 r showed no abnormal c e l l division. In studies of the effects of irradiating standing trees of Plnus rlgida by Mergen and Johansen (1964), nuclear disturbances in seedlings from irradiated seeds increased with increasing 55. dosage, but normal mitotic divisions occurred up to 22,?00 r. Again, Mergen and Commings (1965)» reported that there was no divisions in root tips from Plnus riglda seeds exposed to levels greater than 8 kr gamma-radiation when the seed was stratified and no divisions in the levels greater than 4 kr when the seed received dry storage or sown immediately. Laura (1966b), indicated that chromosome aberrations increased to 26$ in Scots pine X-irradiated seeds compared to 1.5$ in the controls. A histological analysis has been made by Donini (1967), on shoot apices of Plnus plnea and P. halepensis collected from plants chronically irradiated in a gamma f i e l d at different exposure rates and doses. He evaluated the radiation response by mitotic inhibition of the apical and subapical meristem cells in shoot apices, necrosis of negative buds and plant death. The very high radiosensitivity of the two species has been ascertained even at low exposures. Delone (1968), studied the germination and number of telophase and chromosome reorganizations in seeds of 4 species that has spent ca. 22 days in space on board "Kosmos 110". In Plnus sylvestrls, germination percent was increased (85$ vs 65$ for control) and a st a t i s t i c a l l y significant increase occurred in the number of chromosome reorganizations in the primary rootlets of seedlings. 2517 Differential Radiosensitivity of Forest Trees Previous studies of the radiosensitivity of higher plants as determined by exposure to chronic irradiation have shown that radiosensitivity varies among species by as much as 500 folds. 56. If the algae are included, the difference is at least 5,000 folds, (Sparrow and Evans 196l , 5 Sparrow and Miksche 1961). A typical highly sensitive plant is Pinus strobus and highly resistant one is Gladiolus show severe growth inhibition at about 10 and 5000 r/day respectively, however, those values by no means represent either the upper or lower limits of tolerance of higher plants, (Sparrow and Woodwell 1962). It appears from the results presented by Sparrow, Cuany, Miksche and Schairer (1961), that there is a clear correlation between the tolerance as determined by acute compared with chronic exposure: that i s , .a plant which requires a high acute dose to stop growth also requires a relatively high daily chronic dose. Sparrow and Sparrow (1965)1 reported that lethal exposures of woody species have been found to range from 0.80 to 10 kr, those for conifers from 0.8 to 1.5 kr, and those for deciduous species (except Sambucus canadensis which is k i l l e d by 2 kr) from 8 to 10 kr. In the nursery, plants of l4 coniferous species were seriously injured or ki l l e d in a few weeks or months by 2,500 r, whereas growth of the angiosperms was stimulated by 2,500 to 5,000 r, and only dosages of 20,000 r retarded growth, (Flory and Singleton 1 9 5 9 ) • Miksche, Sparrow and Rogers (1962), indicated that Taxus  media cv. Hatfieldli is perhaps the most radiosensitive of the higher plants so far studied, and Sparrow et a l . (1961) found that chronic irradiation with 20 r/day k i l l e d a l l the plants within 2 months and 15 r/day within three. Sparrow and Gunckel (1956), reported that dosage rates of 57. 30-50 r/day produced severe effects in Taxus media compared to 101-200 r/day for Acer and Ilex spp., and 201-400 r/day for Llriodendron t u l i p l f e r a . The genus Pinus includes some of the most sensitive species known. Chronic exposures of 2 r/day for 8 months annually during 9 years produced detectable growth effects, and the lethal dose for Plnus strobus seedlings was 600 r, (Sparrow and Woodwell 1963). In Oak/Hickory forests containing 2-8$ pines, McGinnis, (1964), concluded that the Pine was almost completely eliminated by 5»000 rad, an exposure that reduced the numbers of leaves produced by Oaks and Hickories, but not by Dogwoodj 4,000 rad might have severe effects on pine and 10,000 r on deciduous species. Understory species were more resistant than those in the canopy. Working on Plnus tadea, Sparrow et a l . (1961), found that exposure to doses as low as 5 r/day k i l l e d a high percentage of trees in 7 years but had no effect in one season (8 months), and very much smaller doses k i l l e d young seedlings. In an undisturbed area irradiated for 8 months/year for 8 years, 75% of the trees of Plnus riglda receiving more than 5 r/ day were k i l l e d and Quercus alba receiving 10 r/day was severly damaged, while other woody species showed l i t t l e or no effect, (Sparrow, Schairer and Miksche 196l). In a later investigation of the radiosensitivity of Pinus  rigida, Sparrow, Schairer, and Woodwell (1965)» reported that after exposure to 3«1 r/day for 8 years (a cummulative dose of 5.8 kr), 50$ of the trees died, and none survived 13 kr. Differences in radiosensitivity of different parts of the 58. plant have been attributed to physiological effects. Pedigo (1963). found greater sensitivity of terminal shoots of Pinus  taeda compared with the lateral buds and suggested a relation-ship to auxin metabolism, the apical meristem being the region of most active auxin biosynthesis as well as that containing the most sensitive tissue. 2518 Nuclear Characteristics Affecting Radiosensitivity The various factors which influence radiosensitivity can interact in various ways and the unravelling of these inter-actions is often d i f f i c u l t and complex. Summaries of the relevant factors have been given by Gunckel and Sparrow (1961), Konzak (1957), Sparrow (1965a), and recently by Sparrow, Rogers and Schwemmer (1968). The primary site oof radiation:-damage to plants is the c e l l nucleus. The early work of Zirkle (1932), showed that two most important types of irradiation effects i.e. mitotic delay and ce l l death, were principally manifestations of radiation damage sustained by the nucleus. More recent investigations, particularly on the radiosensitivity of the nucleus versus the cytoplasm by Whiting (1955)• and many others have confirmed and amplified this conclusion. Therefore, considerable effort has been spent to explain the differences in radiosensitivity of different organisms on the basis of different nuclear characteristics. The radiosensitivity of a species, as indicated by degree of growth inhibition, is correlated with the average volume of interphase nuclei and with chromosome number, (Evans and Sparrow, 1961), Sparrow et a l . 196l). 59. If these variables are controlled one at a time, an Increase in nuclear size with chromosomes constant increases sensitivity and an increase in chr'omosome number with nuclear volume constant decreases sensitivity. Somatic mutation frequency per roentgen has also been found to be related to nuclear volume, (Sparrow et a l . 196l), and to chromosome volume, (Cuany et a l . 1958, Shaver and Sparrow, 1962). It has been reported, (Ostergren, Morris and Wakonig 1958, Sparrow and Christensen 1953» Sparrow and Gunckel 1956, and Sparrow and Schairer 1958), that the chromosome size is directly related to the amount of radiation damage produced by a given dose in some higher plants, but no qualitative measurements have been given partly because of the tedious nature of deter-mining chromosome size or volume accurately. Sparrow, Cuany, Miksche and Schairer (1961), assumed;that differences in chromosome size or volume in different species would be reflected in similar differences in size or volume of interphase nuclei. On this basis and because i t is relatively easy to determine nuclear volume, they have determined such volume for interphase nuclei. The value obtained by dividing the interphase nuclear volume by the chromosome number was referred to by Sparrow and Evans (1961), as "Interphase Chromosome Volume" (I.C.V.). They indicated that since no allowance has been made for nuclei on interchromosomal space and since in many species there is considerable variation in chromo-some size as seen at metaphase or anaphase (and presumably also in interphase), the average interphase chromosome volume is thus 6 0 . more of a concept than an actual biological entity. It is however highly useful s t a t i s t i c for calculation of energy absorption by genetic target. Sparrow and Woodwell ( 1962), believe that the relationship between N.V. and growth inhibiting daily dose probably has Its basis on chromosome volume rather than nuclear volume (N.V.) per se. Dry seed radiosensitivity to acute exposure can also be correlated with such variables as nuclear volume, c e l l volume, chromosome number and other embryonic characteristics, provided that moisture content is accurately controlled, (Sparrow and Woodwell, 1 9 6 2 ) . The relationship between interphase nuclear volume, and the size and number of chromosomes characteristic of a species has been discussed in detail by Sparrow, (1965&)• Extensive data on the relationship between nuclear volume and I.C.V. and radio-sensitivity of herbaceous plants, using lethality as an end point, have been published by Sparrow, Sparrow, Schairer and Thompson ( 1 9 6 5 ) , Yamakawa and Sparrow (1965, 1 9 6 6 ) , and by others. It has been demonstrated that woody species are more sensitive to radiation than herbaceous species of comparable interphase chromosome volume and the reason for that is not completely understood, (Sparrow and Sparrow, 1 9 6 5 ) ' When considering the chromosome complements of different plant species, i t appears that as the average metaphase chromosome volume increases, the average I.C.V. also increases (although these are not necessarily directly proportional). As the total metaphase volume increases, the total interphase nuclear volume w i l l also increase. Likewise, as the metaphase chromosome length 6 l . characteristic of a species increases, the interphase nuclear or chromosome volume w i l l also increase, (Sparrow, Sparrow, Thompson and Schairer, 1965)• Metaphase volume or metaphase length of a karyotype generally is closely related to the amount of Deoxyribonucleic acid (DNA) per nucleus, (Bhaskaran and Swaminathan, i960). As the total chromosome volume or nuclear volume goes up so does the DNA per nucleus provided that comparable stages are measured. At the plant population level, chronic irradiation can probably be expected to have i t s most severe effects on sexual reproduction because during and after meiosis nuclear volume is higher, chromosome number is reduced, the rate of nuclear division may be low, some species requiring two years between mieosis and f u l l maturation of seeds; and meiotic pairing and reduction tend to enhance the damage caused by aberrations, which may survive in diploid somatic c e l l s . The high radiosensitivity of sexual reproduction i s probably further enhanced by seed dormancy, during which damage accumulates. The relative dosage levels necessary to produce specific responses in growth rate, reproductive capacity or in degree of mortality vary greatly within a species, (Sparrow and Woodwell, 1963). It is generally accepted that diploid species are more radiosensitive and that polyploidy or increasing chromosome number in diploid increase tolerance to radiation. Data from plants (reviewed by Sparrow, Blnnington and Pond,,1958), and animals (Revesz and Norman, i960), indicate that the increasing degree of ploidy confer increasing degree of radioresistance. 62. Certain exceptions have been described, notably in yeast (Mortimer, 196l), and at certain stages of development in Habro-bracum (Clark, 1957). No adequate explanation for the difference between yeast and higher plants is yet available, (Sparrow and Woodwell 1962). For higher plants the average protective effect for eight species pairs, differing by factor of 2 in Chromosome number has been shown to be 1.67; Sparrow and Evans, (1961). Protection increased up to 22-ploid level in Chrysanthemum, (Sparrow and Schairer 1958). The protective effect of polyploidy in plants also has been shown in a tolerance study of 175 species of higher plants by Evans and Sparrow, (1961). Sparrow, Cuany, Miksche and Schairer (1961), have shown the general relationship between nuclear volume and DNA content/ nucleus in higher plant species. The relationship between estimated DNA content/chromosome and lethal exposure for acute and chronic irradiation of herbaceous plants was studied by Sparrow, (l965t>). He concluded that the radiosensitivity of species tends to increaseiias DNA content/chromosome increased for either acute or chronic exposure of higher plant species. The above correlations of nuclear characteristics with radiosensitivity make possible the prediction of radiosensitivity of vegetative growth of plants which have never been irradiated experimentally providing that nuclear volume,chromosome number and degree of polyploidy are known, (Sparrow and Woodwell 1963). However, extension of the technique to predict the effects on vegetation must take into consideration certain additional 63. factors, extrinsic to the c e l l , which may alter the extent of expected damage to the c e l l from a given dosage of radiation and hence radiosensitivity of the plant. Some of these factors include environmental factors modifying dosage, dose rate and dose fractionation, types of ionizing radiation, growth rate, rate of c e l l division and recovery...etc. Such predictions would be very useful for selecting in advance the appropriate exposures to deliver. Sparrow and Woodwell (1963)» indicated that both vegetative growth and the integrity of sexual reproduction process of pines appear to be highly susceptible to damage from ionizing radiation and that was explained by their large nuclear volumes, the long period between the production of meiocytes and seed maturation. It is unlikely that many gymnosperms w i l l be more than 2-3 times as resistant as pines, but the few polyploids should have significantly higher tolerance. Measurements of interphase nuclear volume in apical shoots of Pinus strobus meristems made at various times during the year showed a large volume during stages of active growth (970u^) and 3 much smaller values during dormancy (530u ) i.e. the ratio in average nuclear volume active to dormant was approximately 1.8. In the meantime Sparrow, Schairer, Sparrow and Campbell (1963)» found that the acute gamma-irradiation tolerance of the dormant plants was approximately 1.5 times that of the actively growing ones. Predictions of the seasonal radiosensitivity of some south-eastern tree species were made by Taylor, (1966). Vegetative shoot apices of 14 tree species at Oak Ridge, Tennessee were 6 4 . studied for 1 year, and the average interphase nuclear volume and estimated chromosome volume as well as the ratios of volumes in actively growing to dormant plants were determined. His results indicated that the predicted range of sensitivity between species for any month, approximately a 1 7 fold range, was constant even though the predicted sensitivity of individual species changed with respect to time. Bowen ( 1 9 6 2 ) measured c e l l fresh weight and DNA/cell in leaves of forty species of higher plants including Pinus  sylvestrls and Picea abies. The data were compared with the LD^Q for Co°^ f-irradiation of dry seeds of the same species. He found thatethe relation between L E ^ Q and c e l l weight was significant and the relation between LD^Q and amount of DNA/cell was highly significant. His conclusion was that "DNA/cell is a major factor determining plant radiosensitivity but other factors are also important". Yim (1964), failed to find a correlation between radiation-sensitivity and nuclear volume of Pinus densiflora, P. riglda, or P. banksiana. A relation between interphase nuclear volume of preblem, (cortical), i n i t i a l s of roots and radiosensitivity of several seed sources of Plnus roxburghli was reported by, Kedharnath, (1966). However, no relation was found when he used nuclear volumes from cells of the plerome, (stelar i n i t i a l s ) . Capella and Conger (1967) studied the radiosensitivity to acute Co gamma-irradiation of Pinus e l l i o t t i i , P. plastris, Junlperus conferta, Podocarpus macrophylla and Zamia florldona. The plants were irradiated in.the seedling stages and nuclear 65. and interphase chromosome volumes were calculated from measure-ments of shoot meristem nuclei for each species. They found that the LDJJQ for the species studied ranged from 500 to 760 R. The correlation between radiosensitivity and nuclear or interphase chromosome volumes agreed quite well with the values obtained from other acutely irradiated woody species. Using some nuclear variables to investigate the radio-sensitivity of some gymnospermic seeds Miksche and Rudolph (1968), determined the D N A content and nuclear volume of meristematic root cells of 9 gymnosperms. Basing their end point on root dry weight inhibition, they found no relation between L D ^ Q and either D N A quantity/cell or nuclear volume. However, when radiation responses of irradiated gymnosperm and some other angiosperm seeds were considered together, an inverse relation between nuclear parameters and sensitivity to ionizing radiation was found. The most extensive study on the radiosensitivity of woody species to date was carried out by Sparrow, Rogers and Schwemmer (1968). They determined survival curves for 12 species of woody angiosperms and 16 gymnosperms given 16 hour gamma-irradiation during the early period to spring growth. Second year survival data:iihave been used as an end point throughout. L D ^ Q in the gymnospermic species ranged from 460 to 1203 R with an average of 826 + 54 R and in angiosperms from 477 to 17,500 R with 8 of 12 species about 3»000 and 8,000 R. They confirmed the relationship previously found between interphase chromosome volume and radio-sensitivity for other end points i.e. the larger the I . C . V . the greater the sensitivity. Regression lines obtained from plotting L D . Q , L D K Q , L D Q O » L D I Q 0 against I . C . V . had similar slopewith an 66. average of -0.73. The authors used these lines to predict radiosensitivity of other 1 9 0 species with known I.C.V. About 50% of the 120 gymnosperms studied had predicted LD^Q of 1 kR or less and the predicted L D ^ Q for 95 angiosperms tended to be much higher, with over 8 0 $ between 2 and 8 kR. Differences in nuclear volume within a species were found between active vs. dormant plants and terminal vs. lateral buds. These differences are reflected in the expected differences in radiosensitivity. 252 Ionizing Radiation as a Tool in Mutation Breeding of  Forest Trees It has been known for a long time that the rate of spontaneous mutation is very low in woody species. In gymno-sperms each genus is remarkably constant cytologically, Occasionally some spontaneous polyploids arise, however, most of the spontaneous polyploids have been detected in the nurseries and only a few (e.g. in Larix and Pinus) are represented by full-grown trees. Among other mutations chlorophyll lethals constitute a characteristic mutation type that is frequent in higher plants. They are of various appearance including albina, xantha, v l r l d i l . . . e t c . (Gustaffson, 1 9 ^ 0 ) . Chlorophyll deviations of this kind have been registered for several genera of conifers and hardwoods. A detailed study with regard to population structure and provenance behaviour was published by Eiche in 1 9 5 5 » In some populations of Pinus sylvestrls such mutations are lethal and may diff e r from one place to another, some may survive however. Cases where they reach maturity are frequently reported in forestry literature, e.g. Langner (1953)• 67. Large accounts of spontaneous mutations in woody plants are given by Gustafsson (i960) and for Pinaceae in particular by Stairs and Mergen (1964). So far none of the spontaneous mutations in the family Pinaceae can be considered useful in a forest tree improvement program. Most of these are scientific curiosities and of interest in the study of abnormal cytological conditions, (Mergen 1963). The majority of spontaneous mutations in forest trees have been unfavourable, and they appear in seedlings that grow in nursery beds. These seedlings are not able to compete with normal ones and thus succumb to the unfavourable environment. Induced mutations have become an additional tool in plant breeding as a means of increasing variation. The usefulness of the methods of mutation breeding for developing useful strains of crop plants has been repeatedly demonstrated during the past two decades. For many horticultural crops with long reproductive cycles, Brock (1957), considers the method more promising than conventional ones. In forestry this method has been recommended by Gustafsson (I960). Snyder et a l . (1961) stated that with forest trees induced mutation may likewise have an advantage over time-consuming procedures such as backerossing. The use of irradiated pollen is of particular interest in forest tree breeding and improvement as i t has been suggested by Osborne (1957a, b). He advanced the hypothesis that induced structural heterozygosity in the chromosomes of forest tree progenies, from controlled pollination of non-irradiated females with irradiated pollen, may produce a heterotlc effect similar to 68. that observed In other organisms so treated. Confirmations of that hypothesis have been presented by Stairs and Mergen (1964), Rudolph (1965). and by Vidakovic (1967). Duffleld (1962) stated that the a r t i f i c i a l induction of mutation, or rather the a r t i f i c i a l acceleration of the natural mutation rate, should play an important role in forest tree improvement. However, he indicated that mutagenesis may have a place after the possibilities of hybridization are exhausted. Mutations form the basis for natural and a r t i f i c i a l selection. It i s the idea of Mergen (1963)» that we should look upon the induction of mutations as a supplementary tool that might contribute greatly to the improvement of trees, rather than a substitution for conventional approaches. Mutations bring about new genotypes, and their usefulness needs to be appraised objectively in each instance. Only a few works on the effects of ionizing radiation on forest trees aimed to Induce mutations and use them in mutation breeding programs. Some of such investigations were carried out by Beers (1962), Erlkson (1966), McMahon and Gerhold (1965), Rudolph (1967), Snyder et a l . ( l96l) , Stairs and Mergen (1964), and Yim (1964). When seeds of Pinus densiflora, P. thunbergll, Crypotomerla  japonlca and Chamaecypar1s obtusa were exposed to X-rays at 30 kV for 30-210 minutes, the number of mutants increased with increas-ing radiation time, but there was a considerable variation between species. Large number of mutants resulted from exposure of the pine seeds, but a l l quickly died or reverted to normal, and results for Chamaeoyparis obtusa were similar though less 69. pronounced. Se-ryyfew mutants were produced by Crypotemeria  .japonlca. but there was l i t t l e reversion. The only two mutants to survive of C. japonica from seed exposed for 150 or 180 minutes, differed considerably from normal; both showed dwarfing, increased size of "chlorophyll grains", and reduced size and number of resin canals in needles, (Toyama 1954). To induce somatic mutations, acute dosages of 500-1500 r were recommended by Gustafsson and Simak (1958), for grafted plants of Pinus sylvestris and 5,000 to 30,000 r for poplar cuttings. A study on poplars began in 1934 and assessed in 1948 showed that no hereditary mutations resulted from X-ray treatment of various types of material (seed, f r u i t , buds...etc.). Treatment of winter cuttings bearing dormant or flushing buds, green cuttings, and callus in process of forming adventitious buds sometimes produced chimaeras, differing chiefly in leaf shape and development of chlorophyll, in some the abnormality did not persist for more than a year, and more lasting effects were rare. A l l effects produced were detrimental, Bogdanov (1948). Ouellet and Pomerleau (19°5)» reported that exposure of seed of Ulmus americana to thermal neutrons or X-rays gave a few plants resistance to repeated innoculation with Dutch Elm disease; one showed no symptoms after 9 innoculations and i t s progeny from vegetative propagation, have resisted 2 innoculations. Scholz (1957), indicated that exposure of air-dry seed of Betula verrucosa and B. pubescens to X-rays at 130 kV gave 7 chlorophyll mutants in the F^ generation, with some aberrations in leaf size and shape and size of stomata. .70 . Studies on Quercus spp. by Stairs (1964), suggested to him that in breeding research exposures of 1-4 kr can be applied to buds and pollen and 1-10 kr to seed. Seven somatic mutations affecting the leaves were detected by Ohba and Murai (1966), under a dose of 50-70 r/day for 2 years for species of Alnus, Betula and Populus. They suggested that apparently these doses are the optimum radiation intensity for causing somatic mutations in broadleaved trees under chronic gamma-i rradi at i on. Some mutations that at f i r s t appear to be of l i t t l e or no value might be quite useful when crossed and combined with other genotypes and also be of importance as genetic markers. The importance of induced mutations w i l l steadily increase in the same degree as we know how best to apply the mutagenic agents and, which is no less important, how best to screen out the mutations desired. 71. 3 MATERIALS AND METHODS Three Western coniferous species were selected for this study: 1) Pseudotsuga menzlesli.(Mirb) Franco. (Douglas-fir), from two different provenances representing the coastal and interior forms or subspecies in British Columbia, 2) Plcea sitchensis (Bong.) Carr. - (Sitka spruce), and 3) Tsuga heterophylla (Raf.) Sarg. - (Western hemlock). Douglas-fir, one of the most important and valuable timber-trees, grows extensively through Western North America. It played an important part in the development of forest industries in the Pacific Northwest and, as an introduced exotic, in many parts of Europe, (Fowells, 1965). It extends in a north-south direction on the Pacific Coast from Kemano, British Columbia, to Salmon Creek in Santa Lucia mountains and Santa Barbara county in California. East of the Coast mountains the species reaches it s most northerly limit at Takla Lake, British Columbia, and extends to the Rocky Mountain range south into the Sierra Madre in Mexico, (Sziklai, 1967). Douglas-fir is the largest tree in Canada. On the Pacific coast i t commonly attains a height of 150 to 200 feet and a diameter of 3 to 6 feet. Occasional trees over 300 feet in height and 1 5 feet in diameter are found on the best sites. East of the coast mountains, i t frequently grows to about 100 feet, but seldom exceeds 1^ 0 feet. When rated with a l l other commercial conifers in the West, Douglas-fir isnsclassifled as intermediate in tolerance to shade. Over most of i t s range i t is a climax dominant on moist but well 7 2 . drained sites where a humid atmosphere prevails. In the wetter regions of the coastal forests, however, i t tends to be a pioneer or successional dominant and is ultimately replaced by such trees as Western hemlock, Western red cedar, or Amabilis f i r , as the forest approaches i t s climax stage. Douglas-fir comprises approximately sixty percent of the standing timber of the western forests. On the coast i t is noted for the large dimensions of the structural timber that makes i t a prime species in lumber production. It is also used extensively for plywood production since i t is strong and easy to peel and to glue. Sitka spruce is the most imposing of the four spruces native in Canada. It is a vigorous, fast growing tree that readily overtaps associated Western hemlock and Cedar to occupy a dominant position in the stand. Mature trees, often very impressive, are commonly 3 to 6 feet in diameter and 1 2 5 to 175 feet in height, but sometimes are found 8 to 1 2 feet in diameter and up to 2 5 0 feet in height, (Powells, 1 9 6 5 ) * In dense stands this tree produces a long, slightly tapering, clear trunk, with swollen and buttressed bases. Sitka spruce grows in a narrow strip along the Pacific coast from southern Alaska to northern California. In Canada i t is confined to the coastal region of British Columbia and the south-west corner of Yukon. It seldom extends inland more than 5 0 miles from the ocean or to an altitude exceeding 1 , 0 0 0 feet. It reaches maximum development on the Olympic Peninsula of Washington and the Queen Charlotte Islands of British Columbia. Here the rain forests exist, where Sitka spruce and Western hemlock grow under'?what may 73. be the best forest growth conditions in North America. This species is classified as shade tolerant placing i t second of five tolerance classes. Sitka spruce grows in pure stands or in mixtures of Douglas-f i r , Western red cedar, red alder, broadleaf maple and black cottonwood. It is one of the most important timber species in British Columbia. By virtue of i t s large size and high propor-tion of defect-free stem, i t was admirably suited for aircraft construction during the second world war. It is also an excellent pulp species. Western hemlock grows along the Pacific coast from Kenai Peninsula in Alaska to northern California. Its range extends also inland in a narrow, scattered pattern along the United States-Canadian borders, then spreads out fanlike through northern Montana and throughout the coastal and interior wet belts in British Columbia. It is a large graceful tree, frequently 120 to 160 feet in height and 3 to 4 feet in diameter. Western hemlock Is rated as very tolerant species, and in the coastal forests i t is more tolerant than Western red cedar and Sitka spruce at lower elevations, and mountain hemlock and Alpine f i r on high ground. It rarely forms pure stands. Western hemlock is a valuable pulpwood in British Columbia. It is also an important species in timber cut on the coast and used for lumber, general construction, boxes, crates, flooring and railway tie s . The investigation was divided into three main phases: (A) Study of the radiation sensitivity of each species to,gamma-4:rrad>latl0h;lusing seed germination, subsequent seedling 74. growth and seedling survival as c r i t e r i a . It was followed by a study of the differences among species with regard to radiation tolerance. An attempt was made to record morph-ol o g i c a l mutations. (B) Attempt to correlate the d i f f e r e n t r a d i o s e n s i t i v i t i e s of species with some cytogenetical and biochemical measure-ments, and to detect the effects of i r r a d i a t i o n on the chromosomal l e v e l . (G) I n t r a s p e c i f i c hybridization using i r r a d i a t e d Douglas-fir pollen. 31 E f f e c t s of Gamma-Irradiation on Seed Germination and  Subsequent Seedling Growth and Survival To study the effects of Y * -irradiation on seed germination, on subsequent seedling growth and on su r v i v a l , three experiments were conducted! F i r s t Experiment» It was a p i l o t experiment carried out between September 11, and October 25, 1967. A Cobalt-60 radiotherapy unit at " B r i t i s h Columbia Cancer I n s t i t u t e " was used and set to del i v e r gamma-radiation at approximately 40 Rads (R)/second. The following dosages were given to seed samples. 0 (control), 2,000 R, 5,000 R, 10,000 R, and 15,000 R. After i r r a d i a t i o n treatments the seeds were germinated as described i n the next experiment. Second Experiment: This experiment was conducted during the period between November 16 and December 28, 1967. Seeds from the following locations were usedi 1) Douglas-fir (a) Coastal provenance from Duncan, B.C. (48° ,47 ,N., 123°,43'W., 0-100 feet elevation), 1959 crop. 75. (b) I n t e r i o r provenance from R i c h t e r Pass , B . C . (49°,4 , N . , 119 ° , 3 5 , W . , 2200-2300 f ee t e l e v a t i o n ) , 1964 c r o p . 2) S i t k a spruces from Queen C h a r l o t t e I s l a n d , ( 5 3 ° . 3 ' N . , 1 3 2 ° , 2 ' W . , sea l e v e l ) , 1963 c rop , and 3) Western hemlocks from Marine D r i v e , U n i v e r s i t y of B r i t i s h Columbia Campus ( 4 9 ° , l 6 ' N . , 1 2 3 ° , 1 5 , W . , 100-200 fee t e l e v a t i o n ) , 1964 c r o p . The f i l l e d seeds were separated from the empty ones u s i n g X - r a y f l u o r o s c o p y . A "Softex" type EM(SEM-12) X - r a y u n i t , s u p p l i e d by "Nippon Softex Company L t d . , Japan" , was employed. I t has an X - r a y tube w i t h a (-kM-5-) Mica window and generates super so f t and l o n g wavelength X - r a y s . The equipment was operated a t . a p p r o x i m a t e l y 150 k i l l o v o l t s and 2.5 m i l l i a m p e r s . A "Softex Camera" f o r fluoroscopy was supp l i ed w i t h the u n i t . The seeds to be examined were put on the f l u o r e s c e n t p l a t e of the camera and the f i l l e d and the empty seeds d i s t i n g u i s h e d by l o o k i n g through the window. Only the f i l l e d seeds w i t h apparent l a c k of damage d u r i n g p roces s ing were used as the exper imenta l m a t e r i a l . Samples c o n t a i n i n g 150 seeds from each spec ies were sea led i n paper bags, ( 2 x 2 i n c h e s ) , and i r r a d i a t e d w i t h gamma-r a d i a t i o n from a cobalt-60 source . The r a d i o a c t i v e i s o t o p e , Cobal t -60, i s a source of pure gamma-rays of h i g h energy, 1.1 to 1.3 MeV. A "220 Gamma C e l l " was used . Th i s i s a Cobalt-60 i r r a d i a t i o n f a c i l i t y manufactured by "Atomic Energy of Canada L i m i t e d " . I t i s designed f o r use i n an unsh ie lded room. A p i c t u r e of the u n i t i n d i c a t i n g e x t e r n a l fea tures i s shown i n F igu re 1. B a s i c a l l y the u n i t c o n s i s t s of 76. FIG. CD O V E R A L L V I E W O F G A M M A C E L L 220 77. an annular shaped source, a lead shield around the source and long cylindrical drawer free to move vertically through the center of the source. The drawer carries seed samples from outside the shield to the source. A cross section of the unit from the front view is given in Figure 2. The sample chamber, (Figure 3 ) t is a hollow thin walled aluminium cylinder, 6 inch inside diameter by 8.125 inch inside height. A l i f t o u t type door is held in place by a register at the bottom and a snap-action locking ring at the top. Electrical interlocks are provided on both the door and lock ring to ensure that they are correctly located before the machine w i l l operate. The drawer is raised, for loading, and lowered, for irradiation, by a chain and sprocket system driven by an electric motor. This gamma c e l l is loaded with a source containing cobalt-60 as pellets in 6 pencils and as slugs in 18 pencils. The total activity is about 6200 curies. Radiation Dosimetry: Ferrous chemical dosimetry was used to measure the dose rate In the "220 Gamma Cel l " . It is a measurement system based on the effect of gamma radiation on a solution of ferrous sulfate and may be simply stated by the equation: Fe + Y Fe The number of ferric ions (Fe ) produced is a direct measure of the energy absorbed in the solution which is in turn proportional to the absorbed radiation dosage. The number of ferric ions can be accurately measured by spectrophotometry since they have an appreciable absorption in the ultraviolet region of the spectrum. Thus by measuring the amount of absorption, at a wave length of 78 . 7 9 . SHIELDING PLUG ACCESS HOLE MICRO SWITCH ASSEMBLY SHIELDING PLUG F I G .O) COLLAR AND SAMPLE CHAMBER 80. 305 millimicrons, in an irradiated sample and comparing this to absorption similarly measured for an unirradiated sample, the amount of dosage received can be calculated. The ferrous sulfate method is not an absolute one since the number of fer r i c ions produced is related to the radiation dosage received by a proportionality constant known as the "G" value of the system. This constant has been determined accurately by various means. The value used by Atomic Energy of Canada Ltd. is 15»5 ions per 100 eV of absorbed energy. Ferrous chemical dosimetry, properly performed has been shown to be independent of dose rate up to 10' Rad/second, independent of temperature between 0 and 50°C and independent of radiation energy between 0.1 and 2 Mev. The dose rate at the mid point of the sample chamber, that was measured and certified by Atomic Energy of Canada Ltd., was 5.01 X 10^ + 2% Rads per hour on June 7, 1967. The decay factor (1 month) = 0.01096 and T§ (half-life) of cobalt-60 is 5.25 years. Isodose curves of flux distribution in the sample chamber were also supplied. Seed were given the following dosages at approximately 135 rads/seconds 0 (control), 500 R, 2,000 R, 5,000 R, and 10,000 R. After exposure treatments each seed lot, (150 seeds), was divided into two parts: one part was sown immediately and the other s t r a t i f i e d . The seed to be stratified was presoaked in tap water for 24 hours at room temperature, reaching a moisture content, (oven-dry basis), of 60-70$. It was then surface-dried with paper towels u n t i l no damp spots showed on dry paper and put in small glass vials with loose-fitting cotton stoppers. The 8 1 . glass vials were placed in appropriate plastic containers, covered with moist paper towels, and stored in the cold storage room at 0° to 2°C for l 4 days, (Allen and Bientjes 1954). After the stratification period was completed the seeds were sown. In this experiment germination took place on "Perelite" in Petri-dishes. This material has many desirable properties, viz. i t holds moisture well, retains i t s porous structure long enough for the completion of the germination test and is not affected by microorganisms. Covered Petri dishes, 10 centimeters in diameter, were f i l l e d with "Peril i t e " to within about 3 to 5 mm from the tops. Water was added un t i l the medium was saturated and the free water was drained away. Gaseous interchange was facilated by placing small bent chain links over the edges of the lower dish, separating the upper and lower dishes. 25 seeds were placed, on their abaxial surface, on the "Perelite". "Arasan", a fungicide, was dusted on the seeds to prevent moulding during incubation. Tests with "Arasan" have shown that the fungicide has no detect-able effect upon germination, (Allen and Bientjes 1954). The o o Petri dishes were placed in incubators at 25 C + 1 C in the dark. Three replications (dishes) for each treatment were used in a factorial design. Germination counts were made every two days at 9 a.m. for a 42-day period for the nonstratifled seeds and for a 28-day period for the stratified ones. The seed was considered germinated when the radicle was normal in appearance and exceeded the length of the seed. Third Experiment! This experiment was carried out between June 22, and August 3, 1968. Seed samples were from the same lots used in 82. the second experiment except that of the interior Douglas-fir. In this experiment seed from Shuswap Lake, B.C. (50°,53'N, i i 9 ° . 2 3'E., 1140-1200 feet elevation), 1966 crop, was used. Moisture content was determined for a l l seed lots using representative samples. The moisture contents, based on oven-dry, were as follows: 1) Douglas-fir (a) from the coast = 10.43$ (b) from the interior = 9.37$ 2) Sitka spruce = 7.83$ 3) Western hemlock = 8.56$ The third experiment was on a larger scale than the second, since 5 replications, each containing 50 seeds, were assigned to every subtreatment. The same procedures were followed as described in the second experiment with regard,to seed prepar-ation, irradiation, and stratification. The dosage rate was c a . l 2 3 R/second and the following total exposures were given: 0, 500 R, 2,000 R, 5,000 R, and 10,000 R. Germination took place on "Jacobsen Germinator". Two units, supplied by ZEPHYR N.V. - ZOETERMEE - HOLLAND were employed. This is a Jacobsen seed germinator of the table type with an upper plate, (l60 x 71 cm.) constructed of stainless steel and has on regular distances 176 rectangular openings (35 x 5 mm). Under the upper plate a water basin is constructed in which the strips of blotting paper are placed. The temperature of the water is automatically controlled. 50 seeds were put on a germination pad "Number T300" supplied by (G. Schult & Zonen, Holland), that was placed on an 83. opening. Constant moisture regime was maintained by a strip of blotting paper that connects the water basin with the germination pad. After "Arasan" was sprayed on the seeds they were covered with a bell-shaped plastic cup to ensure high humidity. The cup has a. hole at the top to fa c i l i t a t e gas exchange. Temperature was kept at 25°C and light was provided for 12 hours a day from two fluorescent lamps. Germinating seeds were counted at 2-day intervals for 42-day periods and 28-day periods for unstratified and stratified seeds respectively. In this experiment the germinated seeds, when the radicle reached approximately 2 to 3 times the length of the seed, were transplanted to " J i f f y Pots". These are 8 cm x 8 cm square peat pots (No. 230, manufactured by J i f f y Pot, Denmark, Lyomgaard) 6 seedlings were planted in each pot. Sterilized "California Mix" solid was used. This s o i l is composed of sand and clay in 1tl ratio approximately. The pots were kept in a greenhouse where they were placed on wooden benches and distributed at random in a factorial design. The temperature in the greenhouse was 70 + 5°F and the relative humidity about 85$. Tap water was used for irrigation every 2-3 days or whenever i t was needed. Surviving seedlings were counted at 15 day intervals from the end of the germination period (August 3, 1968 to February 3, •1S>69). In order to obtain accurate information on the effect of Gamma-irradiation on the rate of growth of different species seedlings had to be grown under controlled environmental condit-ions. Therefore, one .pot, containing 6 average germinants, 84. representing each treatment was transferee! to a growth chamber. The treatments that gave no germination were not represented. The growth chambers used were two made by Percival as model PGC-78. J i f f y pots were placed in randomized design, on plastic trays and subirrigated with tap water daily. The two chambers were set for 9 hours continuous illumination (between 9 a.m. and 6 p.m<>). The light intensity was 3500 foot-candles, A temperature of 75 + 2°F and relative humidity of 75 •+ 5% were maintained during the illumination period and 60 + 2°F and 85 + 5% relative humidity were maintained during the dark period. Growth responses to seed irradiation treatments were determined by measuring the height of each seedling, from the so i l surface to the terminal bud to the nearest mm, every 15 days for 6 months. Each time, after height measurements, the trays were re-arranged randomly to further reduce biases that might have been caused by variations in the distance from the lights, observation windows or circulation fans. 32. Cytologlcal Investigations 321 Determination of Nuclear volume (N.V.) and Interphase chromosome volume (I.C.V.)i Since the c e l l nucleus is the principle target for radiation effect, (Lea, 1962), the nuclear volume and interphase chromosome volume as well as the amount of DNA per c e l l and per chromosome were measured for the three species and correlated with LD^0*s. It is well established that N.V. and consequently I.C.V. vary considerably in the different organs of the same plant and also in the several stages of growth development, (Sparrow and 85. Miksche 196l). Therefore, i t was necessary to estimate N.V. in the same stage that was subjected to irradiation treatment. The seeds were presoaked in tap water for 24 hours at room temper-ature, then embryos were excised carefully. Five embryos, representing each species were fixed in a k i l l i n g and fixing solution, glacial acetic acid» absolute ethanol in the ratio 1.3 for 2 hours and then hardened in 95$ ethyl alcohol overnight. The embryos were placed 10 minutes in each of 70$, 50$ and 10$ ethanol and then they were hydrolyzed in 1 N hydrochloric acid for 10 minutes and stained in leucobasic fuchsln for two hours, (Darlington and La Gour 1964). When the embryos stained properly they were washed thoroughly twice with tap water and l e f t for 10 minutes. The embryos were mounted on a glass slide, each species separately, dissected in a drop of 45$ acetic acid, covered with a cover s l i p and gentle pressure was applied, to flatten - but not to squash them. Measurements of 20 nuclei were taken for each embryo along i t s length. Two readings were taken for every nuclear diameter at right angles from each other and the diameter (D) was taken as the average of the two. The nuclear volume was calculated on the assumption that the nucleus is a sphere using the formula* N.V. = D^re . T The interphase chromosome volume was calculated by dividing nuclear volume by the number of somatic chromosomes for each speciesi (26 chromosomes for Douglas-fir, and 24 for both Sitka spruce and Western hemlock.) The average diameter, nuclear volume and interphase chromosome volume and their standard deviations were calculated. 86. The analysis of variance was carried out using an electronic computer. The relationship between N.V. and I.C.V. and LD,-~, was 50 s studied for each species. Correlation coefficients were estimated, (Snedecor and Cochran 1967). 322 Chromosome studies*. Squashes of root tips from irradiated and control germin-ating seeds were prepared as described by Darlington and La Cour, (1964) with some modifications as follows: a) Douglas-fir1 Root tips of germinating seeds were excised when they reached about 5-8 mm long and pretreated with 0.25$ colchicine solution for 4 hours. The material was then placed in a Carnoy k i l l i n g and fixing solution, (100 ml. absolute ethanol, 16 ml. glacial acetic acid: 50 ml. pure chloro-form), for 24 hours. It was hydrolyzed in 1N-H CI for 12 minutes at 60°C, then stained in Feulgen solution for 60 minutes. Treat-ment with pectinase enzyme, 10-15$ aqueous solution, at room temperature for one hour, was found to be very useful. The root tips were mounted and squashed in 45$ acetic acid. Chromosomes in different phases of cell division were examined in several slides prepared from both irradiated and un-irradiated seeds. Many micro-photographs were taken using an automatic camera attached to the microscope, (Leitz, Wetzlar). b) Sitka spruce and Western hemlock: The basic steps were followed as given above, but after several t r i a l s the following modifications were employed and gave better results: Pre-treatment with 1$ colchicine for 5 hours, fixation in acetic-alcohol solution under vacuum, hydrolysis in 1N-H CI for 15 minutes, 8?. treatment with pectinase for 30 minutes, staining with leucobaic fuchsin for 2 hours in the dark. 323 Determination of Deoxyribonucleic Acid, DNA: Embryos were also used for the DNA determination. 200 embryos were used from each of coastal and interior Douglas-fir lots, while 400 embryos were used from Sitka spruce and Western hemlock lots. Seeds were presoaked in water for 24 hours and the embryos were excised as stated above. The procedure used in determining DNA content could be divided into three steps: A) Preparation of finely disintegrated fat-free powder:-The embryos were disintegrated by means of a glass homogenizer, (Pyrex Number 7725, Fisher Scientific), in cold methanol. The suspension thus obtained was centrifuged at 0 to 2°C for 5 minutes at 8000 RPM, using a "Sorval superspeed automatic refrigerated centrifuge", and the clear supernatant fl u i d was discarded. The residue was washed twice with ether and dried "in vaccuo" using a flash-evaporator, at 50-60 cycles per minute and at 60°C. B) DNA extraction: The Ogur-Rosen and Schmidt-Thannhauser processes, as described by Kupila et a l . (1961), for extraction of fat-free tissue powders were followed without significant modifications. Extraction with hot salt solution was done in the following way: the tissue powder was dispersed in a minimal volume of boiling 10% sodium chloride solution (weight/volume) containing 0.05 M Tris (hydroxymethyl aminomethane, from Fisher Scientific Go.) at p of 7.0. The flask was placed in a boiling water bath. After 88. 30 minutes i t was removed from the bath, cooled and the suspension was centrifuged for 10 minutes at 10,000 RPM, the supernatant fl u i d being retained. That step was repeated again and the extracts were fi l t e r e d , combined and lyophilized to a dry powder. This powder was washed twice with cold 0.72 M trichloroacetic acid to remove excess sodium chloride. The extract was made 0 . 5 H with respect to perchloric acid and heated for 20 minutes at 70°C. C) DNA Analysis: The Dische reaction as modified by Burton (1956) was followed. Diphenylamine reagent: This was prepared by dissolving 1.5 gram recrystallized diphenylamine, (Reagent grade, Fisher Sci e n t i f i c ) , in 100 ml of concentrated sulfuric acid. The reagent was stored in the dark. On the day i t was used, 0.10 ml of aqueous acetaldehyde ( l 6 mg/ml.) was added for each 20 ml. of reagent required. Two volumes of reagent were added to one volume of appropri-ately diluted extract and the mixture was incubated for 18 hours at 25°C together with suitable standards and blanks. The optical density of the blue color formed on overnight incubation was determined at 595 (m |M millimicrons using a spectrophotometer, (Spectronic 20 manufactured by Bausch and Lomb). Four replications for each species were done at the same time and the average reading was calculated and used to determine the amount of DNA per embryo. Standard DNA solution: It was prepared also according to Burton (1956): The highly polymerized calf-thymus DNA, sodium salt preparation, (supplied by British Drug Houses), was used. A stock solution was prepared by dissolving DNA at 0.4 mg./ml. in 89. 5 millimolar-sodium hydroxide. From this working solution, standards were prepared by mixing a measured volume of 1N-perchloric acid and heating at 70°C for 15 minutes. Both stan-dards were stored in the refrigerator. A series of DNA concentrations were made, incubated with diphenylamine and read in the same instrument at the same wavelength (595 mji) • A standard curve for the relationship between DNA concentration and absorbance was drawn and used to determine the amount of DNA in the samples. Since i t was required to estimate the DNA content per c e l l , the number of cells per embryo was counted using the method of Brown and Rickless (1946). 10 embryos from each species were dispersed in 4 ml. 5% chromic acid, (chromium trioxide, as certified by the A.C.S.), for about 16 hours at room temperature. The whole mass in the tube was shaken vigorously by hand for about five minutes. This treatment yielded a turbid suspension in which normally there were no clumps visible to the naked eye. If occasional clumps remained they were dispersed by pressing them gently against the side of the vessel with a glass rod. When maceration was completed a sample of the suspension was with-drawn and a drop was introduced below the cover s l i p of a haemocytometer slide (Levy and Levy-Hausser Corpuscle Counting Chamber, made by CA. Hausser and Son, Phila., Pa.), an "Improved 1 1 Neubauer" with dimensions of /%00 square mm and /10 mm deep. The number of cells in a known volume was counted using the formula (supplied with the slide): Number of cells per cubic m.m. = No. of cells counted x dilution x 4000  No. of squares counted 90. 10 samples were counted for each species and their average was taken. If c e l l clusters were observed, or i f consecutive counts did not agree within + 10$ the chromic acid treatment was continued for 24 hours. That was the case with Douglas-fir. Once the amount of DNA per embryo was known and the number of cells per embryo was estimated, the DNA content per c e l l was calculated by dividing the former by the latter for each species. The amount of DNA per interphase chromosome was obtained from dividing the amount of DNA per c e l l by the somatic chromosome number. As with nuclear volume and interphase chromosome volume, the relationship between DNA content per c e l l and per chromosome was examined. 33 Pollination experiment: For the study of the effects of gamma-irradiation on the pollen grains and the resulting progeny from controlled pollin-ation two trees of Pseudotsuga menzlesii (Coastal form) were used. The two trees, designated E and 11, are located on the University of British Columbia Campus. These two, among others, were used previously in various experiments and voluminous data on the trees and their progenies are readily available, (Sziklai 1964). Tree E, (Figure 4), originated from natural regeneration and represents the local population of Douglas-fir, Tree 11, (Figure 5), was selected from a plantation established in 1934, and, although the origin of the seedlings is not known, i t is probably from a coastal provenance. Tree 11 was particularly used because i t showed a very high Figure 4-. Tree "E", used as a mother parent i n c o n t r o l l e d p o l l i n a t i o n , (0.005 X) F igure 5. Tree " 1 1 " , used as a p o l l e n source i n c o n t r o l l e d p o l l i n a t i o n , ( 0 . 0 0 5 X) 92. combining a b i l i t y , (Sziklai 1964). Both trees produce cones frequently and were readily accessible. The trees were measured for total height, diameter at breast height (ca. 135 cm), the crown radius and their ages were estimated, (Table 1). Table 1. The age, height, D.b.h. and crown radius of tree E and tree 11 used in controlled pollination, 1968. Tree Age "year" Height in meters D.b.h. in cm. (inside bark) Crown radius in meters E 46 24.4 57.2 6.2 11 39 22.5 34.4 4 .8 331 Controlled pollinationi The techniques of controlled pollination have been used and developed for a long time in forest tree breeding work. In Douglas-fir this technique was used by Duffield (1950), Orr-Owing (1956), and by Allen and Sziklai (1962). Detailed information on the methods of controlled pollination is given by Sziklai (1964). In this experiment the following steps were followeds Isolation of seed conelets: Tree "E" was used as the maternal parent. Douglas-fir is a monoecious species. The male and female flowers are born separately on different parts of the same branch. Morphologic-al l y , i t is known that f i r s t , second and third lateral buds, from the tip of the twig, are usually female while the laterals closer to the base are mostly male. The terminal bud is a vegetative one. The female buds are pointed and resemble the vegetative buds more than the male buds, yet they are consider-ably larger than the latter. The male buds are smaller than the 93. female ones, and more rounded. The c o l o r of the female f lowers ranges from deep red to green, (F igure 6 ) . Figure 6 . Vege ta t ive ( V . ) , female ( F . ) , and male (M.) buds i n D o u g l a s - f i r , ( 2 X ) Seed cone le t s are e rec t a t the time of p o l l i n a t i o n and composed of s e v e r a l s p i r a l l y arranged imbr i ca t ed th ree - lobed b r a c t s , each subtending a sma l l o v u l i f e r o u s s ca l e w i t h two b a s a l ovu les , (F igure 7 ) . I s o l a t i o n was c a r r i e d out on March 20, 1968, p r i o r to the opening of the female c o n e l e t s . A f t e r emascula t ion the female cone le t s were p ro tec t ed by i s o l a t i o n bags . 60 twigs were bagged on the no r th -eas t s ide of the middle p o r t i o n of the crown. Each i s o l a t e d b r anch l e t conta ined between two and n ine female f l o w e r s . The i s o l a t i o n bags, ( l 4 cm. w id th x 30 cm. l o n g ) , were made of " V i s c o s e " c a s i n g as recommended by D u f f i e l d (1950) and used by others v i z . A l l e n and S z i k l a i (1962) and S z i k l a i (1964). The 94. Figure 8. Male conelet in Douglas-fir, (3X) 95. viscose bag is permeable to water vapour, retains i t s inflated size, allows flower development, is transparent, and can be used several times. Sziklai (1964) mentioned that a d i f f i c u l t y encountered in using this type of bag was the fact that temper-ature increased within the bag and might have some undesirable effects on the conelets. Cotton was used for packing at the place of the t i e , that was made with "Twistem". Every branchlet was tagged for future reference. The sixty isolated branchlets were divided randomly into five groups each consisting of 12 bags and each group was assigned to a different treatment. Pollen extraction: Tree 11 was used as the male parent. The male conelets, (Figure 8), (microsporangiate stroboli), are easily distinguished from the female. They are usually close to the base of the twig, shorter than the vegetative bud and are oval in shape. The microsporangiate stroboli has approximately 80 microsporophyll spirally arranged on i t s axis. Each microsporophyll contains around 300 pollen grains. The pollen grain is unwinged, spherical in shape about 100 microns in diameter, and has a specific gravity greater than unity, (Sziklai 1964). Branchlets 40-50 cm. long, with pollen conelets were collected on March 25, 1968. The pollen grains were extracted by the method of Orr-Owing (1954) as modified by Sziklai, (1964). The branchlets were placed over "tracing paper" on clean trays and kept at room temperature in an isolated place in the laboratory to avoid contamination by other pollen grains. The branchlets were loosely covered with plastic sheets in order to maintain high and 96. constant level of relative humidity. The pollen grains as released were collected twice daily for three days and stored at 0°C. When a sufficient amount of pollen had been collected i t was cleaned by passing the grains through three sizes of wire screens, using 1.12, 0.62 and 0.11 mm openings. The cleaned pollen was then stored in plastic vi a l s , sealed tightly, put in a desiccator and kept in the cold storage room at 0°G until required for Irradiation treatments. Pollen Irradiation: The pollen moisture content was measured and found to be 11.54$. Five samples of pollen grains, approximately 0.5 gram each, were sealed in polyethylene bags separately. Each bag was assigned for a different treatment. The pollen was irrad-iated with gamma-radiation from the same source described above. The following dosages were given at l<2'5 R/second: 0 (control), 500 R, 2,000 R, 5,000 R and 10,000 R. After irradiation treatments the bags were kept separately in a desiccator at 0°C unti l pollination. A r t i f i c i a l Pollination: Seed conelets were checked frequently after isolation to find out the proper time for pollination. On April 6, 1968, when the seed conelet had completely emerged from the bud scale and had assumed erect position on the branch, a r t i f i c i a l pollin-ation took place. At this stage of development the bracts are greatly enlarged and nearly half the length of the ful l y developed cone bracts, while the scales are not more than one-sixth as long as the fully developed cone scales, -As a result of elongation of the central axis of the female strobolus the 9 7 . ovuliferous scale becomes separated allowing the pollen grains to r o l l along the channels formed by the upcurving of the lower edge of the bract to the unequal integuments. The nearly spherical tip of the integuments is well covered with un i c e l l -ular hairs, and the cleft between the two unequal lips of the integuments is facing upward, thus allowing easy access to the micropyle chamber (Allen, 1963 and Sziklai, 1964). The wet pollination technique, that developed by Allen and Sziklai, (1962) for Douglas-fir and practiced successfully by Sziklai, (1964) was used. Just before a r t i f i c i a l pollination, the pollen was diluted to about 100 times i t s volume with tap water and the suspension was applied to the seed conelets by means of a De Vilbiss atomizer. Five atomizers were used for the five groups of pollen i.e. for the five treatments. Protection of cones» The isolation bags were removed on June l4 , 1968 after the female conelets had closed and pointed downward. They were replaced by fiber-glass screens to protect the developing conelets against insects and animalsidamage, and to catch seed i f cones opened early. The screens were 16 -17 centimeters wide and 30-35 centimeters long with 1.5 mm openings. Due to bad weather conditions between the time of polli n -ation and screening many branches were broken. That was recorded as well as the number of cones which had not been f e r t i l i z e d or failed to develop. Collection of Cones and Extraction of Seedst As the cones reached maturity, the branchlets with tags, screens and cones were cut off with clippers on September 11, 1968. 5 branchlets containing open pollinated cones were also 98. collected. The screens were opened one by one and their contents were transferred to paper bags in the laboratory,in order to avoid the loss of the already shed seed within the screens. The bags were stored separately in a quiet warm greenhouse to fa c i l i t a t e cone opening. On September 23, 1968, when the cone opened a l l the seeds were extracted, dewlnged and cleaned by hand. The number of seeds per cone was counted then they were separated into f i l l e d and empty seeds, using the X-ray fluoroscopy as described before and the data was recorded. The seeds were packed in polyethylene bags and kept in the cold room at 0°C for further investigations. A germination test was carried out between December 1, 1968 and December 29, 1968 using only the f i l l e d seeds from controlled pollination. Since the number of f i l l e d seeds varied consider-ably between the different treatments, the number of replications was not the same for a l l irradiation treatments. A maximum of 100 seeds were used, in 10 replications, from the following groups: control, 2,000 R, 5,000 R, and open pollinated. 50 seeds, in 5 replications, were tested in the case of the 500 R treatment and only 1 replication, of 10 seeds, was tested in the case of the 10,000 R-treated pollen. Germination was commenced in the "Jacobsen Germinator" and the same procedure indicated before was followed. As the radicle of germinating seeds reached about twice the length of the seed, the germinants were transplanted into J i f f y pots and kept in the same greenhouse used in the other experiments. Seedling growth and survival were observed and recorded between December 29, 1968 and March 29, 1969. 99. 332 Pollen germination In vitro; As a corollary to the uti l i z a t i o n of Douglas-fir irradiated pollen in controlled pollination, pollen germination in vitro was investigated. The medium chosen to test pollen germinaMlity was described by Brewbaker and Kwack (1963) and the stock solutions were prepared as follows: Stock solution "A": BO^ 0.1 g Ca (N0 3) 2 4H20 0.3 g Mg S0^ 7H20 0.2 g K N 0 3 0.1 g in 100 ml. d i s t i l l e d water. Working medium: Stock solution "A" 1 ml. d i s t i l l e d water 9 ml. The |> of the medium was adjusted to about 7 and the procedure of testing pollen germinability was as follows: Petri-dishes were sterilized by autoclaving under 15 lb. pressure for 20 min. A f i l t e r paper was placed in a Petri-dish and moistened with a few drips of d i s t i l l e d water. Slides with two cavities were wiped with 70% ethyl alcohol and two slides were placed in each Petri-dish, then two to three drops of cultural medium were added to the slide cavities. Pollen samples from the irradiated lots used in a r t i f i c i a l pollination were dusted on the medium. Five replications were assigned for each pollen irradiation treatment. The Petri-dishes were kept in the transfer chamber at room temperature. After 24 hours pollen grains were picked up with a sterile 100. micro spatula, transferred to a slide, covered with a cover s l i p , and then examined under the microscope. Turgid pollen that began to elongate was considered viable while the ones with shrunken inclusions were considered non-viable. 20 random counts were made for each slide cavity and a total of approximately 50 pollen was counted each time. The number of germinating pollen grains was multiplied by 100 and divided by the total number of pollen counted each time to give the percentage pollen germin-ation. The average of 20 estimates was taken to represent one replication. 101. 4 RESULTS The f i r s t and second experiments were considered as pilot experiments and their data are reported elsewhere, (El-Lakany and Sziklai, 1969). The results obtained in the third experi-ment and to be presented here are based on larger number of replications and higher number of seeds in each replication. 4l Effects of Gamma-Irradiation on Seed Germination 4 l l Differences within each species 4 l l l Douglas f i r , coastal provenance Seed germination was markedly affected by irradiation. The data showed that as the dose of irradiation increased the germination percentage decreased. The analysis of variance, (Table 2), indicated that the differences among treatments were highly significant. The seeds treated with 500 R of gamma-radiation appeared to give the highest germination percent and those exposed to 10,000 R gave the lowest values, Figure 9. Duncan's Multiple range test showed that the differences among the values of the control, 500 or 2000 R treated seeds were not significant. Stratified seeds exhibited significantly lower percentage of germination than the unstratified ones. The interactions between treatments, (dosages), and subtreatments (stratified vs. unstratified), werewnot significant. 102. Table 2, Analysis of variance for the germination percent of Coastal Douglas-fir Source of variation d.f. M.S. F Irradiation treatments (I) 4 20944.80 5236.20 94.72** Stratification (ST) 1 278.48 278.48 5.04* I X ST 4 351.52 87.88 1.59 N - S Error 40 2211.20 55.28 Total 49 23786.00 ** Significant at 1% level * Significant at 5% level N.S. Not significant Duncan's multiple range test (5$ level of significance) 500 R Control 2,000 R 5,000 R 10,000 R 70.2 67.4 63.2 36.2 18.0 1 0 3 . Dose in kR Fig- 9- Effect of different closes of / - i rradiat ion on the germination of Coastal Douglas-fir seeds-104. 4112 Douglas-fir, Interior provenance The trend previously recognized in coastal Douglas-fir was also found in the interior source, (Figure 10), with l i t t l e differences i.e. as the dosage of radiation increased the germination percent decreased and the differences among treatment were highly significant, Table 3« Although the germination percent at 500 R irradiation level appeared to be higher than the control, i t was not significant. Results similar to those obtained in coastal seed source were found, with lower germination percent characterizing the stratifi e d seeds than the unstratified ones. The difference in this case was also significant. Table 3, Analysis of variance for the germination percent of Interior Douglas-fir Source of variation d.f. S.S. M.S. " F Irradiation treatments ( I) 4 26000.48 6500.12 124.24** Stratification (ST) 1 462.08 462.08 8.83** I X ST 4 157-92 39.48 o . 7 5 N , i Error 40 2092.80 52.32 Total 49 28713.28 ** Significant at 1% level N.S. Not significant Duncan's multiple range test (5% level of significance) 500 R Control 2, 000 R 5,000 R 10,000 R 79.6 79-0 67.8 61.6 17.6 s. 1 0 5 . Dose in kR Fig- lt> Effect of different doses of y - irradiation on the germination of Interior Douglas-fir seeds-106. 4113 Sitka Spruce As i t could be noticed in Figure 11, there was a sharp decrease in seed germination as the gamma-irradiation dosages increased. The differences among treatments were highly signif-icant, Table 4. No stimulation in germination was found, however, the seeds treated with 500 R did not give significantly lower germination percentage than the control. Also the difference between 5»000 R and 10,000 R treatments was not significant, i.e., both dosages reduced the germination with the same efficiency. The strati f i e d seeds gave significantly lower germination percent than the unstratified ones, except in the controls where the reverse occurred. There was a highly significant interaction between irradiation and stratification treatments. Table 4, Analysis of variance for the germination percent of Sitka Spruce Source of variation d.f. S.S. M.S. F Irradiation (I) 4 49678.88 12419.72 465.51** Stratification (ST) 1 2857.68 2857.68 107.11** I X ST 4 2415.52 603.88 22.63** Error 40 1067.20 26.68 Total 49 56019.28 ** Significant at 1% level Duncan's multiple range test (5$ level of significance) Control 500 R 2,000 R 5.000 R 10,000 R 75.6 69.4 30.6 4.8 0,2 1 0 7 . Unstrat i f ied seeds St ra t i f ied seeds Dose in kR F i g - II- E f f e c t of d i f f e r e n t d o s e s of y-irradiation on the g e r m i n a t i o n of S i t k a s p r u c e s eeds -108. 4ll4 Western Hemlock In this species also a marked decline in germination percentage was noted as the gamma-irradiation dose increased (Figure 12). The differences among the treatments were highly-significant, (Table 5 ) . Duncan's test indicated that the differences between the control and 500 R treatment on one hand, and between 5,000 and 10,000 R on the other were not significant. Like the other two species unstratified seed gave signif-icantly higher percentage of germination than the stratified seeds. There was a highly significant interaction between the treatments and subtreatments. Table 5» Analysis of variance for the germination percent of Western Hemlock Source of variation d.f. S.S. - M.S. F Irradiation treatments (I) 4 56545 .12 14136.28 581.26** Stratification (ST) 1 233.28 233.28 9 . 5 9 * * I x ST 4 2404.32 351.08 Error 40 972.80 24.32 Total 49 59155 .52 . . . . • . . ** Significant at 1% level Duncan's multiple range test (5$ level of significance) Control 500 R 2 , 000 R 5,000 R 10 ,000 R 7 8 . 0 7 5 . 8 4 8 . 0 4 . 0 0.4... 1 0 9 . Fig-12- E f f e c t of d i f f e r e n t d o s e s of y-irradiation on the g e r m i n a t i o n of W e s t e r n h e m l o c k seeds-110. 412 Differences between species The combined results of the effect of different dosages of gamma-irradiation on the germination of the species used were analysed s t a t i s t i c a l l y and the result is given in Table 6. Averages of species, treatments, subtreatments and their interactions are presented in Table 7. The data indicated highly significant differences among the species. On the average, Douglas-fir from the interior provenance gave the highest germination percentage of 6l.l2 followed by Douglas-fir from the coastal provenance with an average of 5l$? then Western hemlock, 4l . 3 6 $ and fi n a l l y Sitka Spruce with the lowest germination percent of 36.12$. Duncan's multiple range test indicated that the species were a l l significantly different from each other. With regard to the effects of treatments, the control seeds, i.e. those which received no irradiation exhibited the highest germination percentage. However, i t was not significantly different from that for the seeds irradiated with 500 R. As i t would be expected the seeds irradiated with 10,000 R had the lowest germination percent, (Figure 13)• Similar to the difference within individual species, the combined data also showed that the strati f i e d seeds had a signif-icantly lower germination percentage than the unstratified seeds. The interactions species X treatments, species X subtreat-ments were highly significant, while the interactions treatments X subtreatments and the three way interaction species X treatments X subtreatments were significant at 5$ level. I l l . Table 6 , Analysis of variance for the germination percent of different species Source of variation d.f. s.s. M.S. F Species (Sp) 3 16696 .00 5565 .33 95 -58** Irradiation treatments (I) 4 130730 .00 32682 .50 56l.28** Stratification (ST) 1 2394 .30 2394 .30 1 1 . 1 2 * * Sp x I 12 18136 .00 1511 .33 2 5 . 9 5 * * Sp x ST 4 2816 .50 704.12 1 2 . 0 9 * * I x ST 3 637 .83 212 .61 3 - 6 5 * Sp x I x ST 12 2690 .60 224.21 3 -85* Error 160 9316 .90 5 8 . 2 3 Total.. 199 183418.13 _ ** Significant at 1% level * Significant at 5% level Duncan's multiple range test (5$ level of significance) Species 1 (1) CDF IDF SS WH 51 6 1 . 1 2 3 6 . 1 2 41 .36 Treatments: Control 500 R 2 ,000 R 5 .000 R 1 0 , 0 0 0 R 7 5 . 1 5 7 3 . 7 5 5 2 . 5 5 26.90 1 0 . 5 5 Subtreatments t Stratified Unstratified 44 .32 51.24 (l) TheJ.following abbreviations w i l l be useds CDF = Coastal Douglas-fir IDF = Interior Douglas-fir SS •= Sitka spruce WH = Western hemlock 1 1 2 . Table 7 , The combined results of germination percentages of different species, treatments and sub-treatments (a) Species x Treatments Species Treatments Control 500 R 2 ,000 R 5,000 R 1 0 , 0 0 0 R (l)CDF 67.40 70 .20 63 .20 36 .20 18.00 SDF 79.00 79 .60 67.80 61 .60 17.60 ss 7 5 . 6 0 69.40 30 .60 4.80 0 . 2 0 WH 7 8 . 0 0 75.80 48 .60 4 . 0 0 ,0.40 (b) Species x subtreatments Species Subtreatments CDF IDF SS WH Stratified 48.64 58.08 3 0 . 5 4 3 9 . 2 0 Unstratified 53 .36 64.16 4 3 . 6 8 4 3 - 5 2 (a). Treatments x subtreatments Treatments Subtreatments Control 500 -R 2 ,000 R 5,000 R 1 0 , 0 0 0 R Strat- 72.40 i f i e d 69.80 42 .50 24.4 9 .00 Unstrat- 7 7 . 6 0 i f i e d 7 7 . 7 0 62.60 28 .9 9 .10 Table 7 (cont.) (d) Species x Treatments x Subtreatments (average values of five replications) Treatments Control 500 R 2 ,000 R 5 ,000 R 10 ,000 R Sp.- St. Unst; St. Unst. St. Unst. St. Unfit. St. Unst. CDF 66.00 68.80 64.80 75.60 58.40 68.00 34.00 38.40 20.00 16.00 IDF 74.00 84.00 74.40 84.80 65.20 70.20 60.80 62.40 16.00 19.20 SS 69 .20 82.00 62.40 76.40 10.00 51.20 1.20 8.40 0.00 0.40 WH 80.40 7 5 . 6 0 77.60 74.00 36.40 60.80 1 .60 6.40 0.00 0.80 Subtreatments t (1) St. = Stratified seeds (2) Unst. = Unstratified seeds 1 1 4 . Coastal Douglas-fir Interior Douglas-fir Western hemlock Sitka spruce 0 -5 Dose in kR F i g - I3- E f f e c t of d i f f e r e n t d o s e s of y-irradiation on t h e g e r m i n a t i o n p e r c e n t a g e of s e e d s of the t h r ee s p e c i e s -1 1 5 . 413 Germination as a percentage of the control In order to estimate the LD^Q (germination) the germination as a percentage of the control was calculated for each species and the results are presented in Figures 14, 1 5 , l6 and 17 for Coastal Douglas-fir, Interior Douglas-fir, Sitka spruce, and Western hemlock respectively. The LD^-, were found out by z>Q s reading the values on the horizontal axis in rad which corres-ponded to germination of 50$ of the control. The average of stratif i e d and unstratified seed readings was taken to represent each,species and the results showed that Interior Douglas-fir had the highest LD^Q (germination) of 7500 R, followed by Coastal Douglas-fir with 5525 R» Western hemlock with 2525 R and f i n a l l y Sitka spruce, the most sensitive with an LD^Q (germination) of 1915 R. 414 Rate of germination The rate of germination of different species as i t was affected by different irradiation dosages and post-irradiation 0 stratification or immediate sowing are presented in Figures 18 to 2 5 . These figures showed clearly the sigmoidal nature of the germination curve. The highest rate of germination was attained at different times depending on the treatment. Generally, in the case of unstratified seeds, the maximum rate of germination was reached between the eighth and the sixteenth day of the germin-ation period, while in the stratified seeds the same period was between the eighth and the twenty-fourth day. The rate of germination values (R^ Q), (number of days required to reach 50$ of total germination), were calculated and presented in Table 8. It could be observed that the 500 R-irradiated seeds of Douglas-fir, both from the coast and the interior provenances, required shorter time to reach 50$ of the total germination than the control. The R ^ Q values progressively increased as the total dosage of irradiation increased especially in Sitka spruce and Western hemlock. In a l l species the seed treated with 10,000 R required the longest R ^ Q values. Table 8, The R ^ Q values in days for different species after different treatments, (Average values of five replications) Treatments Subtreatments. Species Control 500 R 2,000 R 5,000 R 10,000 R St. Unst. St. Unst. St. Unst. St. Unst. St. CDF 9.0 13.4 8.6 12.6 12.0 19.4 16.2 25.6 16.0 IDF 7.0 16.0 7.4 18.2 8.8 21.4 10.4 23.0 14.4 SS 14.2 23.0 14.2 23.4 20.6 26.2 > 28 28.2 > 28 WH 8.2 13-6 9.4 14.0 17.4 22.0 25.2 28.4 > 28 1 1 7 . Unstratified seeds Stratified seeds Dose in kR F i g - I4- E f f ec t of d i f f e r e n t d o s e s of y-irradiation on the g e r m i n a t i o n of C o a s t a l D o u g l a s - f i r s e e d . e x p r e s s e d as a p e r c e n t a g e of the c o n t r o l -1 1 8 . 0-5 2 5 7-3 7-7 10 Dose in kR • I5- Effect of d i f fe ren t d o s e s of / - i r r a d i a t i o n cn the g e r m i n a t i o n of In te r io r D o u g l a s - f i r s e e d , e x p r e s s e d as a percentage of the c o n t r o l -1 1 9 . 0 -5 I25 2 2-6 5 10 Dose in kR Fig-16- Effect of different doses of y- irradiation on the germination of Sitka spruce seeds,expressed as a percentage of the control-1 2 0 . Unstratified seeds Stratified seeds L D 5 0 Dose in kR g- I7- Ef fec t of d i f fe ren t dose s of / - i r r a d i a t i o n on the g e r m i n a t i o n of W e s t e r n h e m l o c k s e e d s , e x p r e s s e d as a p e r c e n t a g e of the c o n t r o l -121 . F i g - I8- E f f e c t s of / - i r r o d i a t i o n on the g e r m i n a t i o n of s t r a t i f i e d s e e d s of C o a s t a l D o u g l a s - f i r -500 R Control ^ 2000R ^•5000R ,.• 10,000R 4 8 12 16 20 24 28 32 36 40 42 Days to Germination Fig-19- Effects of /-irradiation on the germination of unstratified seeds of Coastal Douglas-fir-500 R Control _ - - 2000 R 5000 R 10,000 R 8 12 16 20 24 Days to Germination F i g - 20- E f f e c t s of y-irradiation on the g e r m i n a t i o n of s t r a t i f i e d s e e d s of In te r io r D o u g l a s - f i r -1 2 4 . Days to Germination 1 2 5 . ,000 R Days to Germination Fig-22- E f f e c t s of / - i r r a d i a t i o n on the g e r m i n a t i o n of s t r a t i f i e d s e e d s of S i t k a s p r u c e -1 2 6 . Days to Germination Fig-23- Effects of y-irradiation on the germination of unstratified seeds of Sitka spruce-1 2 7 . 10,000 R 0 4 8 12 16 20 24 28 Days to Germination Fig- 24- Effects of / - i rradiat ion on the germination of stratified seeds of Western hemlock-80 1 2 8 . Control Days to Germination Fig* 25- Effects of / - irradiation on the gemination of unstratified seeds of Western hemlock-1 2 9 . 42 Effects of Gamma-irradiation on  Seedling Survival and Growth 421 Seedling survival As previously indicated, germinants were transplanted to J i f f y pots and kept in the greenhouse», Number of li v i n g seedlings was recorded every l4 days for 182 days and the results are presented in Figures 26, 2 7 , 28 and 29 for coastal Douglas-f i r , Sitka spruce, and Western hemlock respectively. Seedling mortality was greatest during the f i r s t 2 to 3 months followed by a lower rate. The response of each species differed from the others, from treatment to treatment, and between seedlings from stratif i e d or unstratified seeds. Tables 9 to 12 present the st a t i s t i c a l analysis of these data. In Coastal Douglas-fir the differences among irradiation treatments were highly significant with the control having the highest survival followed by 500 R then 2 ,000 R treatments. The difference between the control and 500 R was not significant. 5,000 and 1 0 , 0 0 0 R-irradiation treatments were eliminated from the analysis since they had no liv i n g seedlings-on the last day of counting. Post-irradiation stratification of seeds appeared to reduce seedling survival but not significantly lower than the unstratified seeds. For the effect of different irradiation doses on seedling survival of interior Douglas-fir, i t was found that seed irrad-iation at 500 and 2 ,000 R seemed to give higher seedling survival than the control however, the difference was insignificant, (Table 1 0 ) . The survival of the control seedlings was si g n i f i c -antly higher than those from seeds treated with 5 .000 R. Seed 1 3 0 . stratification reduced seedling survival to a significant degree. The interaction between irradiation and stratification was signif-icant . In Sitka spruce, seed irradiation with 500 R reduced seedling survival below the control and 2 ,000 R reduced i t more effectively. Only in this species seed stratification seemed to increase the seedling survival but i t was not significantly higher than non-stra t i f i c a t i o n . The interaction between treatments, (irradiation) and subtreatments (stratification) was significant. In Western hemlock 500 R dosage to the seeds had no significant effect on the survival of the seedlings compared to the control. In this species, as in Douglas-fir, post-irradiation stratification of the seeds reduced seedling survival. The interaction between irradiation and stratification was highly significant. The aforementioned data were transformed into survival as a percentage of the controls and shown In Figure 30 to.facilitate the estimations of L D ^ Q (survival) at age of 1 8 2 days. It was found that Interior Douglas-fir had the highest L D ^ 0 ( s u r v l v a D of 5950 R followed by Coastal Douglas-fir with 2500 R, Sitka spruce with 1400 R, and f i n a l l y Western hemlock'with 1290 R. ~ 1 3 1 . Table 9 . Analysis of variance for the survival of Coastal Douglas-fir seedlings from gamma-irradiated seeds Source of Variation d.f. s.s. M.S. F Irradiation (I) 2 5480.40 2740 .20 1 4 . 7 2 * * Stratification (ST) 1 2 6 3 . 0 9 2 6 3 . 0 9 1 . 4 l N.S. I x ST 2 •659 .93 329 .96 1.77 N.S. Error 24 4 4 6 9 . 1 0 186 .21 Total 29 10972 .52 ** Significant at 1% level N.S. Non significant Duncan's multiple range test (5$ level of significance) Treatments: Control 500 R 2 ,000 R 81.25 7 6 . 0 7 50 .34 1 3 2 . Table 1 0 , Analysis of variance for the survival of Interior Douglas-fir seedlings from gamma-irradiated seeds Source of Variation d.f. s.s. M.S. F Irradiation (I) 3 7270 .10 2423 .37 3 6 . 6 7 * * Stratification (ST) 1 613.42 613.42 9.28** I x ST 3 7 4 7 . 5 8 249.19 3 . 7 7 * Error 32 2115 .00 6 6 . 0 9 Total 39 10746 .10 ** Significant at 1% level * Significant at 5% level Duncan's multiple range test (5$ level of significance) Treatments: Control 500 R 2 , 000 R 5 ,000 R 79.41 84.62 83.47 5 1 . 6 9 133. Table 11, Analysis of variance for the survival of Sitka Spruce seedlings from gamma-irradiated seeds Source of variation d.f. S.S. M.S. F Irradiation (I) 2 14758.00 7379.00 48.4l** Stratification (ST) 1 108.23 108.23 0.71 N.S. I x ST 2 1358.08 679.04 4.45* Error 24 3658.20 152.43 Total 29 19882.51 Significant at 1% level * Significant at 5% level N.S. Not significant Duncan's multiple range test (5% level of significance) Treatments 1 Control 500 R 2 ,000 R 78.56 55.82 24.46 134. Table 12, Analysis of variance for the survival of Western Hemlock seedlings from gamma-irradiated seeds Source of Variation d.f. S.S. M.S. F I r r a d i a t i o n (I) 1 232.43 232 .43 2.70 N.S. S t r a t i f i c a t i o n (ST) 1 2107.40 2107.40 24.50** I x ST 1 937.08 937.08 10.89** Error 16 1376.40 86 .03 Total 19 4653 .31 ** S i g n i f i c a n t at 1% l e v e l N.S. Not s i g n i f i c a n t • Stratified o Unstratified 42 70 98 126 154 Age in Days after the Completion of Germination Fig-26- The survival of Coastal Douglas-fir seedlings following / - i rradiat ion of seeds at different dosages-• Stratified . o Unstratified 42 70 '98 126 Age in Days after the Completion of Germination Fig- 27- The survival of Interior Douglas-fir seedlings following /- irradiation of seeds at different dosages-Age in Days after the Completion of Germination g- 28- The survival of Sitka spruce seedlings following y-irradiation of seeds at different dosages 00 Age in Days after the Completion of Germination Fig- 29- The survival of Western hemlock seedlings following /-irradiation of seeds at different dosages- « v 1 3 9 . Coastal Douglas-fir 1400 0 500 2,000 5,000 10,000 Dose in Rads g- 30- Survival as a percentage of the control of 182 day-old seedlings following /-irradiation of seeds (weighted average of stratified and unstratified seeds)-140. 422 Height Growth of Seedlings The height measurements in millimeter for 30 seedlings representing each subtreatment in every species were combined and a s t a t i s t i c a l analysis was carried out for these data, (Tables 1 3 , l4, 15 and 16) for Coastal Douglas-fir, Interior Douglas-fir, Sitka spruce, and Western hemlock respectively. For a l l species studied, generally, there was a significant difference between age groups, (i.e. measurements at different ages), as i t might be expected. However, Duncan's multiple range test indicated that the differences in height for the same species were negligible after the third or the fourth measure-ments i.e. at age of 70 to 98 days. The differences among treatments were highly significant in a l l the species. Also, for a l l species, seed stratification after irradiation reduced seedling height to a significant extent. Only in the case of Coastal Douglas-fir, (Figure 31 ) , seed irradiation stimulated seedling growth, significantly at 500 R and not significantly at 2 ,000 R over the control. In the remaining species seed irradiation reduced seedling growth considerably however, 500 R seemed to be not considerably lower than the control, (Figures 32 to 3 4 ) . 141. Table 1 3 , Analysis of variance for the seedling heights of ... _ .. . Coastal Douglas-fir Source of Variation d.f. S.S. M.S. F Age (A) 6 1130600.00 188440.00 177.87** Irradiation (I) 2 33677.00 16838.00 15.89** Stratification (ST) 1 26772.00 26772.00 25.77** A x I 12 14680.00 1223.40 1 .15 N.S. A x ST 6 38207.00 6367.8O 6 . 0 1 * * I x ST 2 76552.00 38276.00 36 .13** A x I x ST 32 148650.00 4645.40 4.38** Error 1198 1269200.00 1059.40 Total - 1259 2738338.00 ** Significant at 1% level N.S. Not significant Averages and Duncan's multiple range test (5% level of significance) Age (days): 14 42 70 98 126 154 182 33.11 57.03 76.89 96.46 108.08 113.94 120.44 Irradiation: 500 R 2,000 R Control 93.86 83.25 82.57 Stratification: Stratified Unstratified 8 1 . 9 5 91 .17 l42. Table l4, Analysis of variance for the seedling heights of Interior Douglas-fir Source of Variation d.f. S.S. M-.-S-. F Age (A) 6 21347 .00 3557 .90 75.04** Irradiation (I) 3 47430 .00 15810 .00 3 3 3 . 4 7 * * Stratification (ST) 1 11274 .00 11274 .00 2 3 7 . 7 9 * * A x I 18 1027.80 57 .10 1.20 N.S. A x ST 6 447.80 7 4 . 6 3 1.57 N.S. I x ST 3 1472 .70 4 9 0 . 9 2 1 0 . 3 5 * * A x I x ST 45 3222.40 71.61 1 .51** Error 1597 75715.00 47.41 Total 1679 161936 .70 ** Significant at 1% level N.S. Not significant Averages and Duncan's multiple range test (5$ level of significance) Age (days): 14 42 70 98 126 154 182 2 5 . 9 6 30.40 3 3 . 1 3 3^.64 35.62 3 6 . 1 3 3 6 . 7 0 Irradiation: Control 500 R 2 ,000 R 5,000 R 3 8 . 3 2 37 .21 3 2 . 5 5 24.82 Stratification: Stratified Unstratified 30.64 35.82 143. Table 15» Analysis of variance for the seedling heights of Sitka Spruce Source of Variation d.f. S.S. M.S. F Age (A) 6 5307 .70 884.62 2 7 . 7 4 * * Irradiation (I) 2 46037 .00 23018 .50 7 2 1 . 9 4 * * Stratification (ST) 1 2 3 2 . 2 9 2 3 2 . 2 9 7 . 2 9 * * A x I 12 3 7 5 . 4 5 3 1 . 2 9 0 . 9 8 N.S. A x ST 6 7 7 2 . 1 3 128.69 4.04* I x ST 2 593 .75 296 .87 9 . 3 1 * * A x I x ST 32 2005.40 62 .66 1.97 N.S. Error 1198 38197 .00 3 1 . 8 8 Total 1259 93520 .72 ** Significant at 1% level * Significant at 5% level N.S. Not significant Averages and Duncan's multiple range test (5% level of significance) Age (days): 14 42 70 98 126 154 182 1 5 . 8 6 17 .99 19.31 2 0 . 1 2 20.71 21 .34 2 2 . 4 9 Irradiation: Control 500 R ; 2,000 R 24.50 23 .41 1 1 . 1 6 Stratification: Stratified 19.26 Unstratified 2 0 . 1 2 1 4 4 . Table .16 , Analysis of variance for the seedling heights of Western Hemlock Source of Variation d.f. S.S. M.S. F Age (A) 6 7635 .00 1272 .50 5 4 . 7 8 * * Irradiation (I) 1 1.38 1.38 0 .06 N.S. Stratification (ST) 1 1789-40 1789-40 7 7 . 0 3 * * A x I 6 3 3 4 . 1 5 5 5 . 6 9 2.40* A x ST 6 7 0 . 5 2 1 1 . 7 5 0 .51 N.S. I x ST 1 556.97 556 .97 2 3 . 9 8 * * A x I x ST 19 966 .67 5 0 . 8 8 2 . 1 9 * Error 799 18561 .00 2 3 . 2 3 Total 839 29915 .00 ** Significant at 1% level * Significant at 5% level ' N.S. Not significant Averages and Duncan's multiple range test {5% level of significance) Age (days): 14 42 70 98 126 154 182 13.26 14 .19 17.48 19.51 20.30 20.78 21.34 Irradiation: Control 500 R 18.08 18 .16 Stratification: Stratified Unstratified 1 6 . 6 6 19 .58 Control •5 K R 5 K R 2 K R • Seedlings from stratified seeds o Seedlings from unstratified seeds 14 42 70 98 126 Age in Days 154 182 T h e h e i g h t g r o w t h of i n t e r i o r D o u g l o s - f i r s e e d l i n g s f r o m s e e d s t r e a t e d w i t h d i f f e r e n t d o s a g e s of y-irradiation-L / U - l 1 L_ I I L L v 14 42 70 98 126 154 182 Age in Days F i g - 3 3 - T h e h e i g h t g r o w t h of S i t k a s p r u c e s e e d l i n g s f r o m s e e d s t r e a t e d wi th d i f f e r e n t d o s a g e s of / - i r r a d i a t i o n -25 to o> = 20 "O Q> V CO 15 -10 0L4-14 00 Control •5 KR • Seedlings from stratified seeds o Seedlings from unstratified seeds 1 42 70 1 126 154 182 98 Age in Days F i g - 34- T h e he igh t g r o w t h of W e s t e r n h e m l o c k s e e d l i n g s f r o m s e e d s t r e a t e d w i t h d i f f e r e n t d o s a g e s of y - i r r a d i a t i o n -149. In order to get better estimates of the differences between treatments within each species the analysis of variance was carried out for the measurements at the age of 182 days, (Tables 17, 18, 19 and 2 0 ) . In Coastal Douglas-fir, (Table 1 7 ) , i t was noticed that, as for a l l age groups, seed irradiation at 500 R and 2 , 000 R stimulated seedling growth over the control. The averages for the three treatments were as follows: 130.18 mm, 119.18 mm, and 1 1 1 . 9 5 mm respectively. However, such differences were not significant. Similarly there was no significant d i f f e r -ence between the height of seedlings from stratified seed and those from unstratified seed. In spite of the insignificant differences between treatment and between subtreatments the interaction between the two was significant. The highest mean seedling height obtained was 1 3 5 « 8 7 mm from 500 R irradiated strat i f i e d seeds and the lowest value was 1 0 1 . 4 3 mm from unirradiated unstratified seeds. For Interior Douglas-fir, (Table 18), and Sitka spruce, (Table 19), there was a significant difference between the irradiation treatments with a decrease in seedling height parallel to the increase in irradiation dosage to the seed. In Western hemlock, (Table 2 0 ) , no significant difference between the control and 500 R treatment was found. The rest of the irradiation treat-ments were eliminated because they had •,.insufficient survival. Stratification of ^ i r r a d i a t e d seeds of interior Douglas-fir and Western hemlock reduced seedling height at age of 182 days but in Sitka spruce the similar difference was insignificant. Photographs were taken for some seedlings representing a random replication from each irradiation exposure of unstratified seeds, (Figures 35 to 3 8 ) . 1 5 0 . Table 1 7 , Analysis of variance for the height of 182-days-old Coastal Douglas-f i r seedlings Source of Variation d.f. S.S. M.S. F Irradiation (I) 2 10115 .00 5057 .50 2 . 3 5 N.S. Stratification (ST) 1 565-34 565 .34 0.26 N.S. I x ST 2 15115 .00 7557 .50 3 . 5 2 * Error 174 373830 .00 2148.40 Total... 179 399265.34 * Significant at 5% level N.S. Not significant Averages and Duncan's multiple range test (5% level of significance) Irradiation dosages Control 500 R 2 ,000 R Average Stratified 1 2 2 . 4 7 135 .87 108.30 1 2 2 . 2 1 ( 1 ) Unstratified 1 0 1 . 4 3 124.50 130 .07 118.6 7 ( 1 ) (2) Average 1 1 1 . 9 5 1 3 0.19 ( 2 ) (2) 119.18V ; ( l ) , (2) Averages having the same number are not significantly different. 1 5 1 . Table 18, Analysis of variance for the height of 182-days -old Interior Douglas-fir seedlings Source of Variation d.f. S.S. M.S. - • -• F Irradiation (I) 3 8573 .30 2857 .77 5 0 . 4 4 * * Stratification (ST) 1 2653.40 2653.40 46 .84** I x ST 3 464.47 154.82 2 . 7 3 * Error 232 13143.17 Total 239 24834 .34 ** Significant at 1% level * Significant at 5% level Averages and Duncan's multiple range test (5% level of significance) Irradiation dosages Control 500 R 2 ,000 R 5 ,000 R Average Stratified Unstratified Average (l) Averages having the same number are not significantly different. 38.70 36 .23 32.20 26.37 33.38 47 .03 46.10 38.10 28.87 40.03 42.87 U ) 4 i . i 7 U ) 3 5 . 1 5 U ) 2 7 . 6 2 1 5 2 . Table 19, Analysis of variance for the height of 182-days-old -Sitka Spruce seedlings Source of Variation d.f. S.S. M.S. F Irradiation (I) 2 6815-50 3407-75 4 4 . 6 7 * * Stratification (ST) 1 0 . 0 5 0 . 0 5 0 .011 N.S. I x ST 2 80.13 40 .07 0 . 5 3 N.S. Error 174 13275-00 7 6 . 3 0 Total 179 20170 .68 ** Significant at \% level N.S. Not significant Averages and Duncan's multiple range test (5$ level of significance) Irradiation Dosages Control 500 R 2 ,000 R Average Stratified 27-73 25-13 1 4 . 6 7 (1) 22 .51 Unstratified 2 7 . 6 3 2 6 . 7 7 1 3 . 0 3 Average 2 7 . 6 9 (2) 25-95 1 3 . 8 5 ( l ) , (2) Averages having the same number are not significantly different. 1 5 3 . Table 2 0 , Analysis of variance for the height of 182-days-old Western Hemlock seedlings Source of Variation d.f. S.S. M.S. F Irradiation (I) 1 49.41 4 9 . 4 l 1.36 N.S, Stratification (ST) 1 185 .01 185 .01 5 . 1 0 * I x ST 1 7 8 . 4 i 7 8 . 4 i 2 . 1 6 N.S. Error 116 4208.20 36.28 Total 119 4 5 2 1 . 0 3 * .Significant at 5$ level N.S. Not significant Averages and Duncan's multiple range test (5$ level of significance) Irradiation dosages Control 500 R Average Stratified 20 .27 19 .93 2 0 . 1 0 Unstratified 2 1 . 1 3 24.03 2 2 . 5 8 (1) Average 2 0 . 7 0 2 1 . 9 8 (l) Averages having the same number are not significantly different. 1 5 4 . Figure 36. 182-days o ld I n t e r i o r D o u g l a s - f i r seed l ings from seeds t r ea ted w i t h d i f f e r e n t dosages of gamma- i r rad ia t ion . 155. Figure 38. 182-days o ld Western hemlock seed l ings from seeds t r ea ted w i t h d i f f e r e n t dosages of gamma-i r rad1at i on. 156. 4 3 Morpho log ica l Observat ions 4 3 1 Germinat ing stage The i r r a d i a t e d and u n i r r a d i a t e d germina t ing seeds e x h i b i t e d some morphologica l d i f f e r e n c e s and r e p r e s e n t a t i v e samples are shown i n F igure 39. In Coas t a l D o u g l a s - f i r , the c o n t r o l seeds, i . e . those which r e c e i v e d no i r r a d i a t i o n , were normal i n appear-ance w i t h hea l thy r a d i c l e s and showed no deformat ion . The seeds t r ea t ed w i t h 5,000 and 10,000 R had brown co loured r a d i c l e s which increased i n d e n s i t y as the dosage i n c r e a s e d . A l s o i n D o u g l a s - f i r , and the other spec ies as w e l l , the seeds i r r a d i a t e d a t h igh l e v e l s swe l l ed and abnormal germina t ion occur red , i . e . the co ty ledons ruptured the seed coat before the r a d i c l e emerged. In some seeds tha t underwent an i n i t i a l s p l i t t i n g , the e n t i r e "endosperm" s t i l l e n c l o s i n g the embryo emerged from the seed coat w i t h no f u r t h e r development o c c u r r i n g . Such seeds were s u s c e p t i b l e to fungus i n f e c t i o n . F igu re 3 9 . I r r a d i a t i o n t rea tments . ( 4 X ) 157. 432 Growing stage A search f o r v i s i b l e mutations was c a r r i e d on throughout the s tudy . One of the more pronounced probable e f f e c t s of i r r a d i a t i o n i s shown i n F igure 4 0 . Th i s a l b i n o s e e d l i n g i s from a 5,000 R i r r a d i a t e d Coas t a l D o u g l a s - f i r seed. I t d i f f e r e d from n a t u r a l l y o c c u r r i n g a l b i n o s i n tha t i t s cotyledons were normal ly green wh i l e the t rue leaves had no c h l o r o p h y l l . Th is may i n d i c a t e tha t the a p i c a l meristem was a f f ec t ed by i r r a d i a t i o n . Seedl ings from h e a v i l y i r r a d i a t e d seeds of ten produced t h i c k , sho r t , da rk green hypocoty ls and coty ledons which e x h i b i t e d pa le green spo t s . Other growth a b n o r m a l i t i e s were noted such as dwarf ism, i n h i b i t i o n of bud b u r s t i n g , death of the t e r m i n a l bud and i t s subsequent replacement w i t h a l a t e r a l one. In Douglas-f i r the seed l ings from seeds r e c e i v i n g h igh dosages of gamma-i r r a d i a t i o n (5 ,000 and 10,000 R ) , showed y e l l o w spot ted needles which then turned complete ly y e l l o w before they d i e d . In S i t k a spruce and Western hemlock the whole p l a n t turned pale y e l l o w before d r y i n g out . F igure 4 0 . An a l b i n o s e e d l i n g from 5 ,000 R gamma-irradiated Coas ta l D o u g l a s - f i r seed. (0.4 X) 1 5 8 . 44 Cytogenetical and Biochemical Observations  < 441 Nuclear characteristics The averages of 20 measurements of nuclear diameter (N.D.) in each of 5 embryos representing every species and the computed nuclear volumes (N.V.) and interphase chromosome volumes (I.C.V.) are given in Tables 2 l , 2 2 , 23 and 24 for Coastal Douglas-fir, Interior Douglas-fir, Sitka spruce and Western hemlock respectively. The differences among species were highly significant in nuclear diameter, (Table 2 5 ) , nuclear volume, (Table 2 6 ) , and in interphase chromosome volume, (Table 2 7 ) . Sitka spruce had the highest N.D., and consequently the highest N.V. and I.C.V., followed by Western hemlock, Coastal Douglas-fir, and f i n a l l y Interior Douglas-fir with the lowest N.D. and the smallest N.V. and I.C.V. The results indicated however, that there was no significant difference between Western hemlock, Coastal Douglas-f i r and Interior Douglas-fir in nuclear diameter nor nuclear volume. Also there was no significant difference between Western hemlock and Coastal Douglas-fir nor between Coastal Douglas-fir and Interior Douglas-fir when the interphase chromosome volume was considered. In a l l the above-mentioned estimations Sitka spruce exhibited significantly higher values than the other species. 4 4 l l Relationship between LD^Q (germination) and N.V. and I.C.V. The correlation between LD^Q for germination in rads and nuclear volume in jx is shown in Figure 4 l . The calculated correlation coefficient, r = - 0 . 7 4 9 4 , was not significant. 1 5 9 . The case was the same for the relationship between LD^Q (germination) and interphase chromosome volume, (Figure 4 2 ) . Although the correlation coefficient, r = - 0 . 7 9 4 6 , was slightly-higher than the f i r s t one, i t was s t i l l not significant at 5% level of significance. In the meantime, the general trend of the regression lines indicated an apparent inverse correlation between LD-J-Q (germination) and both nuclear volume and inter-phase chromosome volume. Table 2 1 . Nuclear Diameter, Nuclear Volume and Interphase Chromosome Volume + Standard deviation in five embryos of Coastal Douglas-fir, (Average values of 2 0 measurements). Embryo number 2 3 Average value of 100 5 measurements N.D. (P) 9 .68 9 . 2 6 1 0 . 4 3 1 0 . 3 0 9 .39 + 1 . 4 9 + 0 . 8 1 ± 1 . 2 7 + 1 . 0 8 + 1 . 3 9 3 N.V. (j1 ) 509 .00 4 2 5 . 4 5 619 .95 590 .06 4 6 2 . 2 0 +248.83 +112.90 +220.92 +169.46 +221.58 3 I.C.V. (f ) 1 9 . 5 7 1 6 . 3 6 23 .84 2 2 . 6 9 1 7 . 7 7 + 9 .57 + 4 . 3 4 + 8 .49 +,>6.24 + 8 .52 9 .815 + 1.248 521.331 +209.039 20 .051 + 8 . 0 4 0 1 6 0 . Table 2 2 , Nuclear Diameter, Nuclear Volume and Interphase Chromosome Volume ± Standard deviation in five embryos of Interior Douglas-fir, (Average values of 20 measurements). Embryo number 2 3 4 Average value of 100 5 measurements N.D. (P) 9 .33 9 .39 + 1 . 2 0 + 0 . 8 3 3 N.V. (f1 ) 4 4 5 . 4 9 4 4 3 . 4 7 ±177 .78 +109.61 I.C.V. (^3) 1 7 . 1 3 1 7 . 0 5 + 8 .38 + 4 . 2 1 9-78 9 .33 9 .13 9 .392 ± 1 .48 + 0 . 9 5 ± 0 . 6 5 ± 1.062 523 .22 4 3 7 . 9 8 404.83 4 5 0 . 9 9 8 +244.65 +141.77 ± 9 0 . 8 5 +163.816 2 0 . 1 2 16 .84 15 .57 17.346 ± 9.40 ± 5 .45 ± 3 . 4 9 + 6.301 1 6 1 . Table 2 3 . Nuclear Diameter, Nuclear Volume and Interphase Chromosome Volume ± Standard deviation in five embryos of Sitka Spruce, (Average values of 20 . measurements). Embryo number Average value of 100 1 2 3 ^ 5 measurements N.D. (/*) 1 3 . 1 0 11 .21 10 .01 1 1 . 9 6 11.24 II . 5 0 5 + 1.73 ± 1 .35 ± 1.06 ± 1.20 ± 1.45 ± 1.690 3 N.V. (J1 ) 1236 .54 7 6 9 . 2 2 542.30 922 .11 780.28 850.091 +503.79 ± 3 8 3 . 5 9 ± 1 7 2 . 3 5 ±281 .13 ±301.40 ± 3 9 3 - 8 3 9 3 I.C.V. (/* ) 5 1 . 5 2 3 2 . 0 5 22 .54 38.42 32 .51 35.42 + 2 0 . 9 9 ± 11 .81 ± 7.18 ± 11.71 ± 1 2 . 5 5 ± 1 6 . 4 i 162. Table 24. Nuclear Diameter, Nuclear Volume and Interphase Chromosome Volume + Standard deviation in five embryos of Western Hemlock, (Average values of 20 measurements). Embryo number 2 3 4 Average value of 100 5 measurements N.D. (P) 1 0 . 9 5 9.46 9 .07 9.91 9 .23 + 2.i4 + 1.4i +1.86 + 1.4i + 1 . 5 1 N.V. (P3) 767.18 4 7 2 . 1 0 +482.50 +228.13 3 i . c . v . ( f ) 3 1 . 9 6 1 9 . 6 7 + 2 0 . 1 0 + 9 .50 438.95 540.08 445.12 +284.51 +234.74 +243.24 18 .29 22.50 18.54 ± 11.85 ± 9.78 + 10.13 9 .724 ± 1.791 532 .687 +327.597 2 2 . 1 9 5 + 13 .650 163. Table 25. Analysis different of variance species. for the Nuclear Diameter of Source of variation d.f. S.S. M.S. F Species 3 269.75 89.92 40.51** Error 396 879.01 2.22 Total 399 1148.76 ** Significant at l; % level Duncan's multiple range test: Species S.S. CDF, wja- IDF. 11.505 9.815 9.724 9.393 Table 26. Analysis of variance for the Nuclear Volume of different species. Source of Variation d.f. S.S. M.S. Species Error Total 3 9496500.00 3165500.00 38.03** 396 32963000.00 83240.00 399 42459500.00 ** Significant at 1% level Duncan's multiple range test: S.S. W.H. 850.091 532.687 C . D . F . 521.331 I . D . F . 450.998 1 6 4 . Table 2 7 . Analysis of variance for the Interphase Chromosome Volume of different species. Source of variation d.f. S.S. M.S. F Species 3 19331 .00 6443 .6? 46 . 0 3 * * Error 396 55434.00 1 3 9 . 9 9 Total . 399 74765 .00 ** Significant at 1% level Duncan's multiple range tests Species (l) S.S. W.H. C.D.F. I.D.F. 35 .421 2 2 . 1 9 5 20 .051 17.346 (1) S.S. = Sitka Spruce C.D.F. = Coastal Douglas-fir W.H. = Western hemlock I.D.F. = Interior Douglas-fir D & Interphase Chromosome Volume in ft * 99 L 1 6 ? . 4 4 2 Deoxyribonucleic Acid Estimations The amount of DNA/cell was calculated by dividing the estimated amount of DNA/embryo by the number of cells/embryo. When each figure was divided by the somatic chromosome number of the species in question i t gave the amount of DNA content/ chromosome, (Table 2 8 ) . The analysis of variance was used to assess the differences among the species and the results, (Tables 29 and 30) indicated that such differences were highly significant for both the amount of DNA/cell and DNA/chromosome. Sitka spruce contained the highest amount of DNA/cell, and consequently per chromosome, followed by Western hemlock, Coastal Douglas-fir then Interior Douglas-fir which contained the least amounts of DNA/cell and per chromosome. A l l the averages were significantly different from each other as Duncan's multiple range test had indicated. 4 4 2 1 Relationship between L D ^ Q (germination) and the amount of DNA/cell and DNA/chromosome The correlation analysis revealed^highly significant negative relationship between the content of DNA per c e l l and per chromosome and LD^Q (germination), (Figures 43 and 4 4 ) . The computed correlation coefficients were - 0 . 9 9 4 8 with a standard error of estimate (S E E) of 65 .3758 for the amount of DNA/cell and - 0 . 9 9 7 3 with S E E of 0 .3514 for the amount of DNA/chromosome. It was noticed that the species characterized by larger nuclear volumes and interphase chromosome volumes contained higher amounts of DNA per c e l l and per chromosome. When such apparent positive correlations were tested s t a t i s t i c a l l y , they proved to be not significant. 1 6 8 . Table 28. The Amount of DNA per c e l l and per chromosome + -1 2 Standard deviation (gram x 10 ) in different species, (Average values of four replications). Coastal Interior Western Douglas-fir Douglas-fir Sitka spruce hemlock DNA/cell 6 9 . 3 3 + 1 . 9 3 42.28+2.71 1 1 1 . 3 3 +4.14 9 7 . 9 5 + 3 - 3 7 DNA/chro- 2 .67 + 0 .07 1.63 ± 0 .10 4 . 65 ± 0 . l 6 4.08 + 0 . l 4 mosome Table 2 9 . Analysis of variance for the amount of DNA/cell in different species. Source of variation d.f. S.S. M.S. F Species 3 11360 .88 37 86-.96 2 8 7 . 4 9 * * Error 12 1 5 8 . 1 2 13.18 Total 15 11519 .00 ** Significant at 1% level Duncan's multiple range test (5$ level of significance) Species S.S. W.H. C.D.F. I.D.F. 111 .33 9 7 . 9 5 6 9 . 3 3 42.28 (g x 10"" 1 2 ) 1 6 9 . Table 3 0 . Analysis of variance for the amount of DNA/chromosome in different species. Source of variation d.f. S.S. M.S. F Species 3 2 2 . 4 9 7 .50 375 -00** Error 12 0 . 2 5 0 .02 Total 15 22 .74 ** Significant at 1% level Duncan's multiple range test {$% level of significance) Species S.S. W.H. C.D.F. I.D.F. 4 . 6 5 4.08 2 .67 1-63 (g x 1 0 ~ 1 2 ) 1 7 0 . LD50 i n Rods Fig-43- The correlation between the amount of DNA per cell of the embryo and LD5Qfor germination-171 . 0 1,000 3,000 5,000 7,000 9,000 LD50 in Rads 44- T h e c o r r e l a t i o n be tween the a m o u n t of D N A per c h r o m o s o m e in the e m b r y o and L D 5 0 for g e r m i n a t i o n -1 7 2 . 4 4 3 Observat ion on the E f f e c t s of I r r a d i a t i o n on the Chromosomes A l a rge number of squaches have been prepared f o r roo t t i p s from both the c o n t r o l and i r r a d i a t e d germinants . F igure 4 5 i s a photomicrograph of the chromosome se t of Coas ta l D o u g l a s - f i r , ( 2 6 chromosomes), from n o n - i r r a d i a t e d seed. Chromosome fragmen-t a t i o n and e r o s i o n were f r equen t ly n o t i c e d i n p repara t ions from i r r a d i a t e d seeds. No d i f f e r ences were recognized between the c o a s t a l and i n t e r i o r forms of D o u g l a s - f i r a t the chromosomal l e v e l , and a r ep re sen t a t i ve id iogram i s shown i n F igure 4 6 . Observat ion on the chromosome prepara t ions from S i t k a spruce and Western hemlock d i d not r e v e a l any de tec tab le d i f f e r e n c e s between the c o n t r o l and the t r ea t ed seeds. F igure 4 5 . Coas ta l D o u g l a s - f i r chromosomes from u n i r r a d i a t e d seed, ( ca . 1 , 8 0 0 X ) . Figure 46- Haploid idiogram of Pseudotsuga menziesii (Mirb) Franco (Douglas - f i r )• 174. 45 Effects of Gamma-Irradiation of Pollen Grains on Seed Production and Germination, Seedling Growth and Pollen Germination in vitro 451 Production of f i l l e d seeds The number of pollinated conelets and collected cones are given in Table 31 for the five irradiation treatments of pollen grains. The total number of pollinated conelets was 2 9 5 , of these only 98 cones were screened and collected, i.e. approxim-ately 66$ of the cones were lost due to the unfavourable weather conditions. The loss occurred between the time of pollination and screening. Table 31• The numbers of pollinated conelets and collected cones and the f i l l e d , empty and total numbers of seeds using irradiated pollen on tree "E" Dosage to Number of Cones Number of seeds pollen conelets c o l l - F i l l e d Empty Total Total/ F i l l e d / grains pollinated ected cone cone Control 72 19 112 1075 1187 62 .47 5 . 8 9 500 R 65 14 52 737 789 56 .36 3.71 2 ,000 R 64 27 358 1065 1423 52 .70 13.26 5,000 R 46 23 167 956 1123 4 1 . 5 7 7 . 2 6 1 0 , 0 0 0 R 48 15 11 610 621 4 l .40 0 . 7 3 175. There was a highly significant difference between the treatments in the number of f i l l e d seeds/cones, (Table 3 2 ) . Pollen that received 2 ,000 R of gamma-irradiation before they were used in a r t i f i c i a l pollination appeared to give the highest number of f i l l e d seeds/cone(13.26) followed by 5 ,000 R with 7.26, control with 5 - 8 9 , 500 R with 3.71 and final l y 10,000 R which resulted in only-tan average of 0.73 f i l l e d seeds/ cone. Duncan's multiple range test indicated however, that only the 2 ,000 R irradiation exposure was significantly higher than 10,000 R. Figure (47) shows these finding in a diagramatic presentation. The lack of significant difference in the number of f i l l e d seeds/cone between the 10,000 R irradiation treatment and the control means that estimating LD^Q values is not jus t i f i e d . Table 32. Analysis of variance for the number of f i l l e d seeds/cone from irradiated pollen Source of variation d.f. S.S. M.S. F Irradiation 4 533.48 133-37 3 . 4 9 * Error 21 803.29 38.25 Total 25 1336.77 * Significant at $% level Duncan's multiple range test (5$ level of significance) Irradiation 2,000 R 5 ,000 R Control 500 R 10,000 R 13.26 7.26 5 . 8 9 3-71 0.73 176. a> c o u I— o. V ) •o a> <u CO TJ a> E z 0 500 2,000 5,000 Dose in Rads 10,000 Fig-47- Effect of pollen irradiation on the number of filled seeds per cone in Douglas-fir-177. 452 Germination of controlled pollinated seeds Due to the unequal number of seeds obtained from controlled pollination the number of replications in the germination exper-iment was unequal. The data was analysed s t a t i s t i c a l l y and given in Table 33• There was no significant difference between the treatments. When the germination percentage of seeds from a r t i f i c i a l pollination of tree "E" by unirradiated pollen from tree " 1 1 " , was compared with those from open pollination i t was found that the former gave higher germination percent than the latter, (98$ vs. 7^$). Table 33* Analysis of variance for the germination of seeds from controlled pollination Source of variation d.f. S.S. M.S. ' - - p - -Irradiation treatments 4 19 .52 4 . 8 8 2 . 5 9 N.S. Error 31 5 8 . 3 9 1.88 Total 35 77-91 N.S. = Not significant 453 Seedling survival and growth The survival of seedlings resulted from the above mentioned germination experiment was followed during the period between December 2 9 , 1968 and March 2 9 , 1 9 6 9 . It was found that the seedlings from 500 R irradiated pollen had the highest survival of 89 .58$ followed by the control with 86 .73$ with no pronounced difference from the 2 ,000 R dosage ( 8 6 . 3 6 $ ) . The seedling survival dropped considerably at 5 .000 and 10 ,000 R irradiation 1 7 8 . dosages to pollen which gave 63 .27 and 60 .00$ respectively. With regard to height growth, 10 randomly selected seedlings were measured from each treatment, except those seedlings from 1 0 , 0 0 0 R where only 6 survived. The differences among treatment were not significant, (Table 3*0, however, seedlings from 500 R irradiation treatment appeared to give the highest average height while those from 1 0 , 0 0 0 R treatment had the lowest value. The variation between the control, 2 , 000 or 5 ,000 R were not pronounced. The heights of seedlings from open pollination were taken for the sake of comparison. It could be realized that seedlings from the a r t i f i c i a l pollination (E x 11) showed a higher growth rate than those from wind pollination. Table 3 4 . Analysis of variance for seedling height from a r t i f i c i a l pollination with irradiated pollen Source of variation d.f. S.S. M.S. F Irradiation treatments 4 1579 .94 3 9 4 . 9 9 1.41 N Error 41 11503 .03 280.56 Total 45 13082 .97 N.S. Not significant Average heights in mm Irradiation treatments Control 500 R 2 ,000 R 5 ,000 R 10 ,000 R 6 2 . 7 7 3 . 5 6 2 . 0 6 3 . 8 5 3 . 8 179. 454 Pollen germination in vitro Irradiated pollen grains from Tree 11 were germinated in vitro to investigate the effects of different dosages of gamma-irradiation on pollen v i a b i l i t y . Pollen that received no irradiation treatment germinated and reached the two nuclei stage after 24 hours, (Figure 4 8 ) , while those irradiated with 1 0 , 0 0 0 R did not germinate and shrank after the same period, (Figure 4 9 ) . It xtfas found that irradiation had a significant effect on the germination of pollen, (Table 3 5 ) . The highest percentage of germination was attained when the pollen were given 2 , 000 R . 500 seemed to reduce the germination below the control, but that was not significant. Pollen irradiated with 1 0 , 0 0 0 R had the lowest v i a b i l i t y . The pollen germination as a percentage of the control was calculated for each treatment and plotted against the dosage, (Figure 5 0 ) . The L D ^ Q of pollen germination in vitro was found to be 9200 R . Table 3 5 . Analysis of variance for pollen germination in vitro after different Irradiation dosages Source of variation d.f. Irradiation 4 Error 20 Total 24 S.S. M.S. F 6002 .93 1 5 0 0 . 7 3 2 2 9 . 1 2 * * 1 3 0 . 9 8 6 . 5 5 6133.91 ** Significant at 1% level Duncan's multiple range test (5$ level of significance) Irradiation 2 , 000 R 5 , 000 R Control 500 R 10 ,000 R 64.33 48.98 42.54 4i.66 16.39 180. F igure 4 a . 10,000 R - l r r a d i a t e d D o u g l a s - f i r p o l l e n g ra ins a f t e r 24 hours of germina t ion in v i t r o . (Ca. 220 X) 181 . 50 E f f e c t of a d i f f e r e n t d o s a g e of / - i r r a d i a t i o n on the g e r m i n a t i o n o f D o u g l a s - f i r p o l l e n g r a i n s in vitro-182. 5 DISCUSSION 51 Seed Radiosensitivity and Subsequent Seedling Survival and Growth  511 Differences within each species A most necessary but sometimes overlooked pre-requisite to any discussion of radiobiological responses is a careful appraisal of the appropriateness of the c r i t e r i a being used to measure radiation effects. Failure to do so may often result in fallac-ious comparisons being made. Lethality based on germination studies and subsequent seedling survival are among the most objective of radiation effects and have been used for comparing the relative radiosensitivity of various species of plants. The harmful effects of ionizing radiation on seed germin-ation and seedling growth and survival have been known for a long time and were also demonstrated in this study. For a l l species investigated the germination percentage was reduced as the gamma-irradiation dosages were increased. Such a general trend has also been reported for various other coniferous species. Similarly the increase in seedling mortality and inhibition of growth are also in agreement with many other findings e.g. Beers (1962), Clark et a l . ( 1 9 6 8 ) , La Croix ( 1 9 6 4 ) , May and Posey ( 1 9 5 8 ) , Mc Cormick and Mc Junkin ( 1 9 6 5 ) , Mergen and Cummings ( 1 9 6 5 ) , Vidakovic ( i 9 6 0 ) , Yim ( 1 9 6 4 ) , and several others. The inhibition of seed germination and the retardation of seedling growth could be the result of inhibition of mitotic c e l l division, c e l l death, inhibition of the synthesis or inactivation of certain enzymes and other macromolecules, or a combination of these factors. Suppression of mitotic activity may result from 1 8 3 . radiation damage to the synthesis of the products that are necessary for c e l l division and/or damage to the mitotic apparatus with the blocking of mitosis even in the case where there is a sufficient quantity of products necessary for c e l l division, (Lebedinsky et a l . , 1 9 6 1 ) . In the present study the delay in germination observed in the irradiated seeds and the slow growth of seedlings from such seeds, while appearing to be dose-dependent, may have been a reflection of restitution of mitotic process. In other words, while the higher doses of irradiation may have inhibited c e l l division, the relatively moderate ones only delayed i t . Although the effects of irradiation on c e l l division may play an important role in inhibiting or delaying seed germination, the possibility of radiation damage to the metabolic processes during seed germination looks more attractive. The biochemical and physiological changes accompanying after ripening and seed germination have been reviewed by Hatano and Asakawa ( 1 9 6 4 ) . During the germination of coniferous seeds, generally, the metabolic processes include hydrolysis of carbo-hydrates, particularly oligasaccharides, to monosaccharides, the hydrolysis of fats by lipase, and changes in the amino and organic acids. Respiration is the evidence of energy-requiring processes and i t was reported that the rate of respiration changes during the germination of many seeds, (Hatano, 1 9 6 3 ) « As a result of c e l l irradiation polyphenol oxidase activity is released (Kuzin, I96l). This enzyme release could be related to an inhibition of oxidative phosphorylation. Woodstock and Justic ( 1 9 6 7 ) , reported that 80 kR caused marked inhibition of 1 8 4 . seedling growth and inhibited the rate of respiration in wheat, corn, sorghum and radish, and Sydoreuka (1962) found that, in corn 1-2 kR doses of gamma-radiation stimulated germination and increased activity of catalaze and peroxidase while germination inhibiting doses reduced the activity of these enzymes. Simil-arly, Grebinsky et a l . (1963) reported that small doses of radiation -500 r - applied to sprouting seeds, increased the activity of respiration and of glucose and i s o c i t r i c acid dehydrogenase, while the larger doses inhibited these a c t i v i t i e s . One of the most important factors in seed germination is the relative availability and activity' of growth substances. The breaking of seed dormancy may be interpreted as results of growth substances regulating the growth of the embryo. Radiation sensit-ivi t y of auxin per se, f i r s t reported by Skoog ( 1 9 3 5 ) , has been reaffirmed by Cervigni and B e l l i (1962) , and Weber and Gordon (1953) reported on the extreme radiosensitivity of auxin biosynthesis. Gordon (1956) traced the stunted growth of mug bean plants after X-ray exposure to the decrease of auxin formation from tryptophan. Again in,1957 he pointed out to the radiosensitivity of indolacetaldehyde conversion to IAA. Similar conclusions have been reached by King and Galston ( i 9 6 0 ) , and by Sax and Schairer ( 1 9 6 3 ) . Haber and Luippold ( 1959)1 demonstrated that the inhibition of lettuce seed germination by 300 kR of ^-v&ys could be reversed by Gibberellic acid, kinetin, thiourea, pricking or light. Other effects of radiation on lettuce seeds, such as reduced respiratory rates, chromosomal aberrations and reduction in seedling height, were not reduced by these materials or treatments. The authors thus concluded that dormancy may have been affected directly by radiation. 1 8 5 . Reversal of radiation effects by various plant growth promoters have been noted in several instances, e.g. Gaur and Notani, ( i 9 6 0 ) , Mathur ( l 9 6 l ) , and others. In trees, Krjnkova et a l . ( 1 9 6 8 ) , reported that Gibberellic acid not only stimulated the growth of non-irradiated poplar material, but helped to overcome the radiation damage. From the evidence presented above i t seems reasonable to assume that part, i f not most, of the inhibition or delayed seed germination and seedling growth results from the interaction of radiation with one or more of the aforementioned biochemical processes. A stimulation of seed germination and seedling survival was noticed in Douglas-fir particularly from the interior provenance and a more pronounced stimulation of height growth in Coastal Douglas-fir was recognized at 500 and 2 , 000 R irradiation doses. Accelerated germination of X-rayed seed was f i r s t reported by Maldiney and Thonvenin in 1898 (After Sax, 1 9 6 3 ) . In forest trees several other investigators have noticed such stimulation of seed germination and plant growth by exposing seeds or growing plants to low dosages of ionizing radiation. Some of those reports are mentioned in the "Review" section, e.g. Muller-Olsen and Simak ( 1 9 5 4 ) , Muller-Olsen et a l . ( 1 9 5 6 ) , Sato and Nishina (1951). Simak and Gustafsson ( 1 9 5 3 ) . Tralau ( 1 9 5 7 ) , Vldakovic ( i 9 6 0 ) , and Yim ( 1 9 6 4 ) . Although the early work on the stimulating effects of ionizing radiation was based upon inadequately controlled experiments, there is c r i t i c a l evidence that low levels of irradiation do have a stimulatory effect on certain stages of plant development. The 1 8 6 . older explanation of such effects, based upon the Arndt-Schultze Law that "Weak irradlants stimulate activity; medium irradiants depress i t ; strong ones halt i t " , has recently been replaced by a more: rational physiological interpretation, (Sax 1 9 6 3 ) . When Beers (1962) noticed 1-3$ stimulation of slash pine seed germination at 750 r of "^radiation, he concluded that stimulation of this type, possibly due to early activation of enzymes through premature breakdown of inhibitors, is usually transitory. Timofeev-Resovskii et a l . ( 1 9 5 7 ) , put forward the theory that the stimulatory effects of ionizing radiation is based on the slight intoxication of plant tissues by radiolysis products. Stimulation was mainly manifested'in a heightened rate of c e l l division brought about by small doses of Beta and Gamma-rays which cause only an insignificant increase in the percentage of abnormal mitosis. Low dosages of Ionizing radiation may stimulate seed germin-ation and/or seedling growth possibly through the increase in phytohormonal production. Several papers, primarily from works in Russia, suggest that irradiation of plants or plant parts may promote the production of physiologically active substances. Grebinskii et a l . (1963) attributed an abnormal growth of pea seedlings to increased auxin production resulting from seed irradiation. While the seed germination and seedling growth of Douglas-fir showed stimulation responses to the lower doses of irradiation, the seeds and seedlings of Sitka spruce and Western hemlock failed to exhibit such a response. Similar differential responses were 187* reported by Clark et a l . ( 1 9 6 5 ) , who indicated that low dosages of gamma-radiation produced a pronounced radiostimulatory effect on germination of Scotch pine, jack pine and black spruce, but no such effect was found in white spruce, white pine or red pine. Beatly and Beatly ( 1 9 5 6 ) , discovered that an X-ray dose that stimulated at 5 r/mlnute was inhibitory at 15-20 r/minute. No radiostimulation was observed however, by Baldwin ( 1 9 3 6 ) , La Croix ( 1 9 6 4 ) , Mergen and Johansen ( 1 9 6 4 ) , nor by Nikitin ( 1 9 3 4 ) , using some other coniferous species. It is interesting to notice that the species which tolerated higher doses of irradiation (Douglas-fir) is the one that showed stimulative effects of low doses, while Sitka spruce and Western hemlock were more sensitive and did not show any stimulation response. Similar findings were reported by Clark et_ a l . ( 1 9 6 8 ) . From the work reported here and the findings of some other investigators in a l l i e d fields the case of radio-stimulating action on certain plants seems to be established and the position is well summarized by Gunckel and Sparrow (1954). "It seems to us that results from one species or variety should not be applied to others and that different types of responses to ionizing radiation are to be expected in different plants. The fact that positive responses are not universal should not be used to invalidate those cases where a stimulation has been reported". It seems likely that in the same plant the responses of the seedling stages compared to older parts may be almost as variable as the responses of different plants to X-rays, (Sparrow and Christensen, 1953)-In a l l the three species studied here, unstratified seeds gave significantly higher germination percentages, had shorter R^0 1 8 8 . values, and, when developed, gave faster growing seedlings with lower rate of mortality, than the stratified seeds. This indic-ates that post-irradiation storage of seeds at 0-2°C for 14 days before germination had harmful effects. There have been indic-ations for a number of years that the effect of irradiation on dormant seeds may continue for some time after the irradiation ceases. Tasher (1919) was the f i r s t to demonstrate a post-irradiation effect in resting seeds. In 1938 Gustafsson found that storage of seed for a year after irradiation increased the X-ray damage and Sax (1941) reported increased chromosomal abnormalities after storing onion seeds for a week after irradiation. It has been also shown that under conditions where physiol-ogical activity was not detectable, the expression of damage to X-rayed seeds could progressively increase for weeks after i n i t i a l photon absorption, (Caldecott 1 9 5 8 ) . The effects of^temperature during or after irradiation with X-rays have been studied in a variety of biological material; e.g. in Tradescantia, (Faberge 1954), (Giles et a l . 1 9 5 1 ) , and in Drosophila, (Mickey, 1 9 3 8 ) . A l l these studies have demonstrated that low temperature (0 -5°C), increases the radiation-induced chromosome aberrations. In an attempt to explain such harmful effects, Van Huystee and Cherey ( 1 9 6 7 ) , found a decrease in nucleic acid synthesis following 2-4 weeks of storage after seed irradiation. They concluded that this points to a pattern of general c e l l damage, probably because of slow decay of c e l l components as an effect of incomplete recuperation from irradiation exposure. From 1 8 9 . electron microscopic observations von Wangenheim ( 1 9 6 5 ) , reported no significant disintegration of c e l l organelles occuring in wheat embryo un t i l 5 days after Irradiation. The decay process initiated by irradiation lasted for at least two weeks. The similarity between such storage effects and those reported for after-effects of aqueous solutions of proteins, enzymes^DNA, etc. (Mitchell and Holmes, 1 9 5 4 ) , is striking. This would lead to the conclusion that the in vivo effects reported here are essen-t i a l l y the same as in vitro effects. Another possibility is that the cytogenetic injury and biochemical lesions are d i f f i c u l t to heal at a low post-irradiation temperature. For example, chromosomes broken by irradiation may restitute rather easily i f c e l l division takes place soon after irradiation exposure. If seed is not germinated immediately and c e l l division is delayed, the broken chromosomes may d r i f t so far apart that restitution becomes very d i f f i c u l t . Studies complementary to those on the relation of time and temperature to the manifestation of post-irradiation injury have shown that the availability of oxygen to the seed, following X-irradiation also has a profound effect on the expression of injury. Thus seeds that are stored or hydrated in the presence of oxygen immediately after irradiation exposure, which was the case in this study, are more severely injured than seeds stored or hydrated anaerobically, Caldecott, ( 1 9 5 8 ) . Somewhat different results have been reported by some other workers, Yim ( 1 9 6 4 ) , found that post-irradiation stratification of dry pine seeds did not affect the germination. Furthermore, and contrary to the present findings, Mergen and Cummings ( 1 9 6 4 ) , 1 9 0 . working with Pinus rigida, pointed out that moist stratification caused an increase in germination that was paralleled by a decrease in cytological damage at a l l exposure levels they used. Cold-dry storage after irradiation seems to aggravate damage while stratification, (cold-moist storage),tends to enhance recovery, Tusujakova, (1959)^ Snyder et a l . (1961),.^demonstrated that an increase in the stratification period before irradiation from two weeks to three weeks further reduced the germination of pine seeds. They also found that post-irradiation stratification increased germination. The factors that alter the after-effect have been summarized by Conger ( 1 9 6 1 ) , as follows* the after-effect increases with storage time and is maximal when dry seeds that have 0^ present are stored at room temperature after irradiation. The presence of water, radical scavengers or hydrogen donors, the absence of 0^ and storage at low temperature diminish or prevent the after effect. 512 Differences between species The differences in seed germination among species are highly significant. Interior Douglas-fir gave the highest germination percentage followed by Coastal Douglas-fir, Western hemlock and Sitka spruce in a decreasing order. Also seedling survival and growth varied considerably among the species. Variation from species to species is most certainly to be expected since at least this muchvariation exists in the reported sensitivities for individual species in the literature. Pinus  sylvestris offers a good example to illustrate such variation since i t is one of the widely studied woody species. Simak and Gustafsson ( 1 9 5 3 ) , found that seeds were damaged by 900-1000 r of 1 9 1 . X-rays and Suszka et a l . (196O), reported that at 3000 and 4800 r less than 30$ of the irradiated seeds began to«germinate. Vidakovic (1966) noticed that germination was increased by 8 2 -2500 r and no germination beyond 14 ,000 r while Ohba and Simak ( I 9 6 l ) , found that 1200-1400 r reduced germinability, the rate of germination and subsequent seedling development. Kamra and Simak ( 1 9 6 5 ) , indicated that X-irradiation of the same species up to 8 0 0 seconds delayed the germination but did not affect the germination percent after 30 days. The L D ^ Q values for germination were found to be 7500 , 5525 . 2525 and 1915 R for Interior Douglas-fir, Coastal Douglas-fir, Sitka spruce and Western hemlock respectively. The L D ^ Q , ^ based on seedling survival were 5950 , 2500 , 1290 and 1400 R for the same species. To the best of our knowledge, there is no reported L D ^ Q values in the literature for the same species from actual experimentation. However, Woodwell ( 1 9 6 6 ) , gave some predicted figures based on nuclear measurements for slight growth Inhibition and 100$ mortality of 310 and 8 2 0 R for Douglas-fir and 377 and 990 R for Western hemlock. A comparison between these figures and the present findings would be on unequal basis. Based on nuclear measurements also, Sparrow e_t a l . ( 1 9 6 8 ) , predicted that the genus Pseudotsuga is the most sensitive of a l l gymnosperms, which is not the case in the present investigation. Variation in radiosensitivity between species under similar experimental conditions has been reported by many investigators. In this work, Interior Douglas-fir seed proved to be about four times more radioresistant than Western hemlock. Clark et,al. ( 1 9 6 8 ) , reported an approximate 10-fold range in sensitivity 1 9 2 . between whi te p i n e , the most s e n s i t i v e and jack pine the most r a d i o r e s i s t a n t . The p o s s i b i l i t y tha t seed moisture content p layed an important r o l e i n the d i f f e r e n t i a l s e n s i t i v i t y of the spec ies repor ted here i s not s t r o n g , f i r s t l y because the range of mois ture content between spec ies i s r e l a t i v e l y narrow, and second ly , even w i t h i n D o u g l a s - f i r , the i n t e r i o r form had lower seed moisture content than the seeds from the c o a s t a l provenance and the former i s more r a d i o r e s i s t a n t than the l a t t e r . However, t h i s does not exclude the p o s s i b i l i t y of some e f f e c t s on the r e s u l t s . The inve r se r e l a t i o n s h i p between moisture content and r a d i o s e n s i t i v i t y has been recogn ized f o r a long t ime, ' K l i n g m u l l e r , ( 1 9 6 1 ) , Natara jan and M a r i e , d 9 6 l ) , Osborne e t a l . ( 1 9 6 3 ) 1 and o the r s . Of cons ide rab le i n t e r e s t i s the d i f f e r e n c e i n r a d i o s e n s i t -i v i t y between the i n t e r i o r and c o a s t a l forms of D o u g l a s - f i r . The r e s u l t s showed tha t I n t e r i o r D o u g l a s - f i r t o l e r a t e d h igher gamma-i r r a d i a t i o n doses than Coas t a l D o u g l a s - f i r when both germina t ion and s u r v i v a l percentages were cons idered as end p o i n t s . L i t t l e ( 1 9 5 2 ) , c i t e d Murray i n I869 as the f i r s t to r e a l i z e the d i f f e r e n c e s between Coas t a l and I n t e r i o r D o u g l a s - f i r . Most of the d i f f e r e n t i a t i o n between the two forms i s based on morph-o l o g i c a l and ana tomica l c h a r a c t e r i s t i c s , (S t e rn , 1 9 6 6 ) . Tusko (1963) r ecogn ized two subspec ies : Pseudotsuga m e n z i e s i i ( M i r b . ) Franco s sp . m e n z i e s i i , Coas t a l or green D o u g l a s - f i r , and Pseudotsuga m e n z i e s i i ( M i r b . ) Franco s sp . glaucescens ( B a i l e y ) Schwer in , I n t e r i o r or Rocky Mountain D o u g l a s - f i r . A l l e n ( i 9 6 0 and 1961) separated c o a s t a l and i n t e r i o r provenances on the b a s i s of morphology of. seeds, and Dunlop (1964) confirmed h i s r e s u l t s . 1 9 3 . Important physiological differences between the two forms have been reported by many workers. Baldwin and Murphy (1956) reported that the forms of interior origin were more cold resistant than the coastal forms when tested in New Hampshire. Douglas-fir seedlings from three coastal sources were shown to be less drought resistant than those from five inland sources, (Ferrell and Woodard, 1966; and Pharis and Fer r e l l , 1 9 6 6 ) . The photoperiodic responses of Douglas-fir is different depending upon.the origin, (Irgens-Moller, 1962 ; and Nicholson, 1 9 6 3 ) . Also the photosynthetlc and respiratory responses are different, (Krueger and F e r r e l l , 1 9 6 5 ) . Differences in susceptibility to disease and insect infection and micorrhyzae formation were reported by Ching ( 1 9 6 2 ) . Haddock et a l . ( 1 9 6 7 ) , have presented the results of some studies of Douglas-fir provenances as well as their own findings. The genetic variability in Douglas-fir has been investigated by Campbell and Rediske ( 1 9 6 6 ) , Orr-Ewing ( 1 9 6 6 ) , and Sziklai ( 1 9 6 4 ) . From the above brief discussion i t could be concluded that morphological, anatomical and physiological, as well as genetlcal variations, do exist between coastal and interior forms of Douglas-f i r . The variation in radiosensitivity reported here adds to and substantiates these differences. The results also indicate that the seed source has a consid-erable influence on radiosensitivity and that the response to irradiation may be under genetic control. The influence of genotype on survival and growth reduction following acute irrad-iation with X or rays has been frequently demonstrated. In most cases many genes have been found to affect the radiosensit-1 9 4 . i v i t y , (Alper I 9 6 0 , Davies 1962 , and Davis 1 9 6 2 ) . In yeast, Resnick ( 1 9 6 8 ) , concluded that there are at least 13 genes that affect sensitivity to radiation. The influence of a single major gene has also been described, (Smith, 19^2) . Variation in radiosensitivity among strains within partic-ular species have been reported in some agricultural plants by Bianchi et a l . ( 1 9 6 3 ) , Nirula ( 1 9 6 3 ) 1 and Smalik et a l . ( i 9 6 0 ) , and in forest trees by Ohba and Murai, ( 1 9 6 7 ) . Intravarietal variations of radiation sensitivity have also been described by Notani ( 1 9 6 1 ) , and by Stailov and co-workers ( 1 9 6 6 ) . The differential radiosensitivity of Douglas-fir depending on the seed origin found here agrees quite well with the results of some other workers using different forest tree species. Tralau (1957)» indicated that the sensitivity of seeds to ionizing radiation may depend on the provenance. Varying doses of gamma-radiation affected the progeny of four Pinus nigra trees in different ways, (Dudic, i 9 6 0 ) . The differential effects of radiation on clones of Eastern cottonwood suggested to Witherspoon and Curling (1966) a genetic basis for radiosensitivity within this species and illustrated how ecotypes may exhibit differen-t i a l responses. Other supporting examples have been given by Mergen and Cummings ( 1 9 6 5 ) , Mergen and Johansen ( 1 9 6 4 ) , and by Simak, Ohba and Suszka ( 1 9 6 1 ) . Seedlings of Picea abies from high elevation sources were more resistant to gamma-radiation than those of lowland origin, (Loffers, i 9 6 0 ) . Ohba and Simak ( 1 9 6 1 ) , working with Pinus  sylvestris seeds found great differences between different provenances in Sweden. Since provenances studied were situated 1 9 5 . in different regions, (latitude, altitude, micro-climate, site conditions...etc.), they raised the question whether there is any relationship between radiation sensitivity of the seed lots and the ecological conditions prevailing at the original habitat. The authors used the length of the growing season, as the number of days per annum with a mean minimum temperature of 6°C, (Langlet, 1936), as a practical expression of the ecological conditions. They concluded that seeds from provenances with short growing season (northerly latitude and high altitude) were less sensitive to X-rays than seeds from provenances with long growing season (southerly latitude and low altitude). This relationship was significant for seedling mortality, hypocotyl length, and root length but insignificant for germinability. Using the same c r i t e r i a , the results reported here are in harmony with the findings of Ohba and Simak ( l 9 6 l ) . The provenance from which the Interior Douglas-fir seeds were obtained has a shorter growing season than the coastal one, (Canada Department of Transport, Metreological Branch, 1967), also the interior proven-ance originated from a higher altitude than the coastal one. When the results of the germination experiment were compared with those of survival, slight differences were recognized. In a l l species, seed germination was functional over a higher range of dosages than that over which survival occurred. This indicated multidamage to the seeds. The range of radiosensitivity between species was found to be about 3-fold in germination percentage compared to about a 5-fold range when survival at age of 182 days was employed as an end point. This is in contrast to the findings of Clark et a l . (1968), using different conifers. 1 9 6 . The fact that seed germination results are greater than seedling survival, as evident from the higher LD^Q (germination) than LD^ Q (survival), may be a reflection that physiological damage besides inhibition of mitosis accounts for death and reduced resistance to side stresses. Because of the more or less optimal conditions under which germination experiments were con-ducted in the laboratory one would naturally expect a greater number to germinate than subsequently survive in the greenhouse. Ohba and Simak (1961),'-likewise reported increased seedling mortality from irradiated Scotch pine seeds in the f i e l d . Germ-ination of irradiated seeds from a number of decidous species was reduced in the f i e l d compared to laboratory t r i a l s , (Heaslip 1 9 5 9 ) ' The higher rates of mortality occurred during the f i r s t 70 -98 days of the seedling l i f e indicate that most of the inhibitory mechanisms operate during this period. Once seedlings from irradiated seeds have survived several months, i t would seem that recovery is more or less complete. Some studies with irradiated pine seeds indicated much the same phenomenon. Davis (1962) reported that after one year out planted seedlings of Pinus  palustris, P. taeda and P. e l l i o t t i i could not be distinguished from the control. The same was reported for Pinus rigida by Mergen and Johansen ( 1 9 6 4 ) . Recovery could be thought of in terms of the replacement of damaged cells or recovery of factors involved in the essential metabolic pathways and the functional capacity of the cell's homeostatic mechanisms. Irreparable injury, such as at higher dosages of irradiation, may be thought of as cells which are not replaced or injury tolthe genetic mechanisms in which cases the 1 9 7 . damage (inherited functional derangement) is propagated in subsequent c e l l divisions resulting in a decreased capacity of the organism's homeostatic mechanisms. Another look at the differences between LD^Q (germination) and L D ^ Q (survival) indicates that the more radiosensitive the species, the less are the differences between the two values. This would imply that radiosensitivity may not be an index of sensitivity to environmental stresses as also was reported by Clark et a l . ( 1 9 6 8 ) . A further support to this view is the fact that Sitka spruce was the most radiosensitive species when germ-ination was used as an end point, while based on survival percentage, Western hemlock was more radiosensitive. 52 Nuclear Characteristics as Factors in Radiosensitivity 521 Variation in Nuclear and Interphase chromosome volumes The phenomenon of different radiosensitivity between species, as found by many students of radiobiology and demonstrated clearly in this investigation, is s t i l l far from satisfactorily understood or explained. Since the c e l l nucleus is a prime target and the major focus of radiation-induced injury, i t was obvious to seek such explanations through investigating the nuclear parameters. After extensive investigations Sparrow et a l . ( 1 9 6 l ) , and Sparrow and Woodwell ( 1 9 6 2 ) , arrived at the conclusion that* c e l l nuclear volume (measured in meristematic cells) polyploidy and chromosome number are the main factors determining radiation sensitivity. Larger nuclear volume means greater sensitivity; polyploidy and increasing chromosome number lead to lesser sensitivity. It must be kept in mind however, that these relations are complicated by the fact that numerous other biological factors are known which 1 9 8 . can be superimposed i n these "major f a c t o r s " i n a d e c i s i v e way, apar t from pure ly chemical and p h y s i c a l m o d i f i e r s . The present r e s u l t s showed h i g h l y s i g n i f i c a n t d i f f e r e n c e s i n nuc l ea r volume ( N . V . ) and in terphase chromosome volume, ( I . C . V . ) among s p e c i e s . The average nuc l ea r volumes were 8 5 0 . 0 9 1 , 5 3 2 . 6 8 7 , 521 .331 and 4 5 0 . 9 9 8 ja f o r S i t k a spruce , Western hemlock, Coas t a l D o u g l a s - f i r and I n t e r i o r D o u g l a s - f i r , r e s p e c t i v e l y . The i n t e r -phase chromosome volumes f o r the same spec ies were 35-421, 2 2 . 1 9 5 , 3 20 .051 and 17.346 u . These va lues are w i t h i n the range of e s t i m -3 ated I . C . V . ( 0 . 9 - 7 1 . 8 ji ) f o r a l l woody p l a n t s , Sparrow e_t a l . ( 1 9 6 8 ) . However, the f i g u r e s g iven i n the l i t e r a t u r e f o r the same spec ies used i n t h i s work are qu i t e v a r i a b l e and d i f f e r e n t from those i n d i c a t e d above. For D o u g l a s - f i r , Sparrow e t a l , ( 1 9 6 8 ) , 3 3 gave two v a l u e s : 2 2 . 9 + 1*5 Y f o r d o r m a n t P l a n t s and 28.5 ± 1»1 3 f o r a c t i v e ones, and f o r Western hemlock 2 3 . 7 + 0 . 9 u . S i m i l a r f i g u r e s were g iven by Woodwell ( 1 9 6 6 ) . The s l i g h t l y h igher va lues g iven by those two a u t h o r i t i e s than the present ones can be j u s t -i f i e d s ince they used shoot mer is temat ic t i s sues , and embryos were used w i t h cu r ren t study to measure N . V . The d i f f e r e n c e s i n N . V . and DNA content between d i f f e r e n t pa r t s of the same p l an t and i n the same par t depending on the season of sampling are w e l l r e cogn i zed , (Sparrow 1 9 6 2 ) . Mer i s t emat ic nuc l ea r volume g r a d u a l l y inc reases w i t h i n c r e a s i n g mer i s temat ic a c t i v i t y , and reaches a peak d u r i n g the p e r i o d of most r a p i d growth, and decreases as growth ra te decreases to a minimum d u r i n g dormancy, (Sparrow et a l . 1 9 6 3 , and T a y l o r , 1 9 6 6 ) . In genera l the e a r l y developmental stages of s eed l ings appear to have the l a r g e s t nuc l ea r volumes and mature p l a n t s the s m a l l e s t , (Sparrow et a l . 1 9 6 8 ) . A l s o the N . V . and DNA 1 9 9 . content increase during the early stages of germination, (Miksche, 1 9 6 5 ) . A high correlation between nuclear volume (and hence I.C.V.) of shoot and root nuclei of the same species were indic-ated by Baetcke et a l . ( 1 9 6 7 ) , and Baetcke et a l . ( 1 9 6 8 ) . This led Sparrow et a l . (1968) to consider i t valid to use data from either roots or shoots to predict radiosensitivity. For Sitka spruce, Burley (1965) gave a range of nuclear 3 volume as 2310-4445 u , (and interphase chromosome volume as 3 96.25-185*2 u consequently). These measurements are quite high, 3 since even the minimum volume ( 9 6 . 2 6 p ) is much higher than the 3 maximum I.C.V. of 71.8 u given by Sparrow et a l . (1968) for woody species. It is also about 3 times the value reported here 3 for the same species (35.421 u ). However, Burley obtained his measurements from root tip macerations which might have flattened the nuclei considerably and resulted in fallaciously higher diameter measurements. The same explanation could be also applied to the very high measurements given by Yim (1964). For instance 3 the N.V. in Pinus banksiana reported by Yim is 4 3 7 0 . 6 yr while 3 Miksche ( 1 9 6 7 ) , found i t to be 1356 + 90.8 . Miksche and Rudolph (1968) however, gave a different figure (1562 + 74) for the same species. 5211 The relationship between nuclear volume and interphase chromosome volume and LD^^ (germination) No significant correlation was found between either N.V. and LD^0 or I.C.V. and LD^Q. The correlation coefficient values were -0.7494 and -07946 respectively. Although those correlation coefficients are not significant they s t i l l indicate some kind of inverse relationship between N.V. and I.C.V. and LD^0 (germination). 200. In other words i t could be said that about 75% of the variation in L D ^ Q among species is due to difference in nuclear volume and consequently in interphase chromosome volume. Similar non signif-icant correlations were reported in the literature for other gym-nosperms by Miksche and Rudolph, (1968) and by Yim, ( 1 9 6 4 ) . But this is in disagreement with some other workers. A significant correlation was reported by Capella and Conger (196?) for four species of gymnosperms and for some woody species by Sparrow e_t a l . ( 1 9 6 5 ) » Sparrow et a l . (1968) pointed out that a potential p i t f a l l is to expect aihigh correlation between I.C.V. and radiosensitivity. Where the range of interphase chromosome volume among species is small the correlation with radiosensitivity may be poor because of s t a t i s t i c a l variation in determining I.C.V. and the inherent variab i l i t y in L D ^ Q , S routinely observed in a series of experi-ments with the same species. This no doubt accounts, in part, for the poor correlation obtained. Another possible reason for the nonsignificant correlation coefficients reported here and elsewhere is that they are based on relatively few number of species. Thus these findings should not eliminate N.V. and I.C.V. as useful estimates in predicting the radiosensitivity over a wide range of taxa. In any case, a large nuclear or chromosome volume results in high radiosensitivity because the amount of ionizing radiation/ roentgen delivered to such a structure is positively correlated with size. Hence a given dose measured in roentgens w i l l deliver more ionizations to a large nucleus than to a small one. The amount of damage produced per c e l l is consequently proportional to the average amount of radiation absorbed per nucleus, not 201 . merely to the Incident dose measured in a i r . It is pertinent i that both chromosome deletion (measured as micronuclei) and somatic mutations/roentgen also have been found to be highly correlated with nuclear volume, (Sparrow, Cuany, Miksche and Schairer 1 9 6 l , Sparrow and Evans 1 9 6 1 , Sparrow and Woodwell, 1 9 6 2 ) . 522 Variation in the amount of DNA among the species The differences in the estimated DNA content per c e l l and per chromosome were significant depending on the species. Sitka -12 spruce contained the highest amount of DNA/cell of 111 .33 g x 10 - 1 2 followed by Western hemlock with 9 7 . 9 5 x 10 , Coastal Douglas-fir -1 2 6 9 . 3 3 x 10 , and f i n a l l y Interior Douglas-fir with 42.28 g x 1 0 ~ 1 2 . The DNA contents/chromosome were 4 . 6 3 9 , 4 . 0 8 1 , 2 .667 and , , - 1 2 1.626 g x 10 for the order of species. Unfortunately there are no previously reported values for the same species to compare with, however, the values reported here are comparable to the amounts estimated for other species of gymnosperms. On the basis of chemical estimates for 10 gymnosperms used by Miksche ( 1 9 6 7 ) , Plnus resinosa had the highest amount of DNA/ -12 c e l l , 1 3 8 . 6 + 6 .2 g x 10 and Thuja occidentalis had the lowest -12 amount, 3 9 « 1 + 1.0 g x 10 . Bowen (1962) reported much lower values for Pinus sylvestrls and Picea abies than those of Miksche (1967) however, the former used leaves while the latter used root tips to estimate the amount of DNA/cell. The approximately three times variation in the amount of DNA/cell reported here is not particularly striking when compared to factors of 17 and 20 for diploid organisms indicated by McLeish and Sunderland, ( 1 9 6 1 ) , and by Sparrow and Miksche ( 1 9 6 1 ) . 2 0 2 . 5221 Relationship between DNA content and LD^Q (germination) A highly significant inverse correlation has been found between the amounts of DNA/cell and /chromosome and L D ^ Q (germination). The correlation coefficients were - 0 . 9 9 ^ 8 and - 0 . 9 9 7 3 respectively. This i s in agreement with the correspond-ing correlations reported by Bowen ( 1 9 6 2 ) , Miksche ( 1 9 6 5 ) » Sparrow and Miksche ( 1 9 6 l ) , for higher plants, and by Deering (1959) for Escherichia c o l l . On the other hand, Miksche and Rudolph (1968) found no relation between radiosensitivity and DNA quantity/cell in 10 gymnosperms. When they plotted their results in conjunction with Osborne and Lunden's (1965) data for some angiosperms a significant response was found. The high correlation coefficients for the amounts of DNA per chromosome and DNA per c e l l indicate that both are better parameters in predicting the radiosensitivity. From the known facts about the average nuclear size in diploids and polyploids and average amounts of DNA/nucleus, Sparrow, Cuany, Miksche and Schairer ( 1 9 6 1 ) , suggested that, at least in most cases, there is generally a direct correlation between nuclear size and DNA content/nucleus. Furthermore, Sparrow and Evans»(1961), actually showed that DNA content and nuclear volume are highly correlated in six species of higher plants. Similar positive correlations were also reported by Miksche ( 1 9 6 7 ) , Miksche and Rudolph ( 1 9 6 8 ) , Sparrow and Miksche ( 1 9 6 1 ) , and by van't Hoff and Sparrow ( 1 9 6 3 ) . A s t a t i s t i c a l analysis was carried out to test the relation-ship between DNA content/cell and N.V. and between DNA/chromosome and I.C.V. Although the correlation coefficients were found to be 2 0 3 . 0.7974 and 0 . 8 3 5 0 respectively, which are close to the value given by Miksche and Rudolph (1968), 0.864, they are s t i l l not significant probably because of the few number of species tested. Other nonsignificant correlations were reported in other plants by von Wagenheim and Walther ( 1 9 6 8 ) . It is a commonly held view in radiation biology that c e l l k i l l i n g by ionizing radiation is primarily a consequence of the deposition of energy in the c e l l nucleus. There are many reasons for such interpretations. The nucleus is the largest cellular organelle. It is the center of anabolic processes, i t undergoes more striking morphological changes during c e l l division than any other organelle, i t is of genetic interest, and, when studied by the usual techniques in an injured or abnormal -cell, i t s morphol-ogical deviations have been observed. If we accept the hypothesis that the nucleus is a l l important, then i t is logical to suspect that a large proportion of the lethal effects of radiations are simply consequences of the genetic loss which inevitably follows upon breakage and rearrangement of chromo-some structure. On the other hand, Deoxyribonucleic acid (DNA) has been always looked at as the major component of the c e l l nucleus on which radiation produces i t s major effects. When we make the assumption that damage to DNA is the primary radiation event, we can ask questions about what kind of damage is important. The complexity of the structure of the double helix and our ignorance of the metabolic processes which involve DNA make this a d i f f i c u l t question, even i f we were to ignore the protein part of the nucleoprotein complex in which DNA exists in the c e l l . 204. As far as DNA i t s e l f is concerned, Stacey (1963) listed seven kinds of damage: change of a base (especially deamination), loss of a base, single chain fracture, double chain fracture, cross linking within the helix, cross linking to other DNA molecules and cross linking to proteins. The DNA content of the nucleus is also affected by irradiation. Forssberg and Revesz (1957) found that the incorporation of C , labelled glycine, into DNA of the nucleus was very low compared to that in non-irradiated c e l l s . This indicates that the synthesis of DNA after irradiation may be suppressed. On quantitative grounds, DNA is a more likely candidate for the primary lesion caused by irradiation than enzymes, since i t is probable that every molecule is unique. It is possible there-fore that damage to only a few molecules per c e l l could affect survival. There are however a number of d i f f i c u l t i e s with this interpretation. A detailed investigation of the changes produced in DNA, when this is exposed to ionizing radiation under a variety of conditions, indicated that radiochemical damage to DNA is unlikely (according to Alexander and Bacq, 1961) to be the primary event that initiates the processes which eventually result in the death of irradiated c e l l s . Further support for this view is derived from the fact that the sensitivity of the nucleoproteins when irradiated within the cells is approximately the same for cells of widely different radiosensitivities. The fact that our data demonstrates a highly significant inverse correlation between the amount of DNA/cell and L D ^ Q (germination) and a nonsignificant correlation between nuclear volume and LD^ (germination) indicates that DNA plays a more 205. important role in determining the radiosensitivity of the species under consideration, and that predictions of radiosensitivity based on nuclear size measurements only are possible within certain limits. Although the nuclear volume in Douglas-fir, both from the coastal and interior provenances, does not differ significantly from that of Western hemlock, the f i r s t species is more radio-resistant than the second, (Table 30). This supports the evidence presented by Sparrow and Evans ( l Q 6 l ) , which suggests that in diploid species increased chromosome number without polyploidy and without a change in N.V. has a protective effect. In other words, increasing the chromosome number from 24 in Western hemlock to 26 in Douglas-fir without significant difference in nuclear volume could be a major factor in the relatively higher radio-resistance of Douglas-fir. In the meantime, this by no means eliminates the effect of other factors on radiosensitivity. The somewhat different but s t i l l related studies of Tsunewaki and Heyne (1959) seem pertinent here. They found that monosomies in Triticum aestivum were more sensitive than the normal diploids. The fact that Coastal Douglas-fir has a significantly higher DNA content/cell and /chromosome than Interior Douglas-fir is particularly interesting. The morphological, anatomical and physiological differences between these two forms have been discussed previously. The taxonomy of this species is not defined yet in spite of the considerable efforts spent by many workers. -Whether or not the differences in radiosensitivity, N.V., I.C.V. and DNA content would help in settling the problem needs further investigation. Comparable findings in the literature of forest 2 0 6 . genetics are relatively few. An ill u s t r a t i o n of change in nuclear volume with latitude was reported by Burley (1965)• He found that the nuclear volumes of Picea sitchensis increased significantly with increasing the latitude. The more southern spruce proven-ances have nuclear volumes one-half that of the northern counter-parts. He concluded that the variations in nuclear and karyotype constitution are associated with continuous variation in morphol-ogical and developmental characteristics of the species. Such continuous variation in Sitka spruce was also found by Daubenmire ( 1 9 6 8 ) . The relationship between N.V. and latitude described by Burley (1965) was again confirmed by Mergen and Thielges ( 1 9 6 7 ) , based on more reliable measurements. In addition to Picea  sitchensis, the latter authors reported a highly significant positive correlation between nuclear volume and latitude for Picea glauca, Pinus sylvestris and P. banksiana as well and they discussed the genetic and evolutionary significance of this relationship. The small sample of species studied by Miksche ( 1 9 6 7 ) , indicated that a relationship may exist between nuclear volume of meristematic root cells and the extent of species distribution, those with lower nuclear volumes tend to have a wider distribution. Our data confirm and support such observations. Douglas-fir has a significantly smaller N.V. than Sitka spruce and i t is well known that the latter species is confined to a relatively narrow strip along the Pacific coast and does not exceed 50 miles inland, while the range of Douglas-fir may extend up to 1000 miles inland, (Foxvells 5(1965) • Nuclear volume and DNA content of meristematic cells may not be related to evolutionary specialization, but to adaptive 207. characteristics of the species. In the Gramineae, as Avdulov (1931) (cited by Stebbins, 1 9 6 6 ) , pointed out variation in chromosome size is correlated not with phylogenetic primitiveness or advancement but with climatic adaption. The genera having large chromosomes are found extensively in temperate climates, whereas those having medium-sized or small chromosomes are predom-inantly tropical or subtropical. Stebbins ( 1 9 6 4 ) , confimed these findings and extended them for the variation in DNA content assuming DNA and nuclear volume to be closely related. These data as well as the data presented here, are s t i l l scanty, but they suggest that variation in DNA content, although not correlated with evolutionary advancement, is nevertheless not random and probably has adaptive significance. A clue to this significance is the positive correlation between DNA content and the length of mitotic cycle reported by van't Hoff and Sparrow ( 1 9 6 3 ) . Also the differences in chromosome size and nuclear volume may be closely related. Also the differences in chromosome size and nuclear volume are often associated with a different distribution of heteroch-romation in the metabolic or interphase nucleus, (Avdulov 1 9 3 1 ) . These evidences have been discussed by Stebbins ( 1 9 6 6 ) , and led him to the following hypothesis: "The.liarge chromosomes and nuclear DNA content which prevails in some plants and some animals indicates an increase in the number of genes but not the number of different gene-controlled metabolic processes. Instead, each of several different gene-controlled processes is controlled by a system consisting of many gene l o c i responsible for the same process". 208. 53 Effects of Irradiation on the Chromosomes Chromosome breakage and fragmentation were observed in the preparations from highly irradiated seeds. This is in general agreement with the well known effects of ionizing radiation on the chromosomes. Suszka et a l . ( i 9 6 0 ) , have reported for Scotch pine that the number of abnormalities in mitosis increased with an increase of X-ray doses. Sax and Burmfield (1943)» arrived at similar conclusions long before. The so-called chromosome erosion recognized in the 5,000 R irradiated Douglas-fir seeds were mentioned by Bevilaequa and Vidokovic (1963), and by Ehrenberg et a l . (1949). The production of chromosomal aberrations by ionizing radiation has been a classical study in the f i e l d of radiobiology. Chromosomal aberration results and dose curves furnished some of the most important data for developing and testing the target theory, (Lea 1962). The action of irradiation appears to be direct in the sense that breakage is caused by an ionizing particle passing through or in the immediate vi c i n i t y of the chromosome at the breakage point. It is known that a very high number of chromosomes-breaks rejoin again in a short time. Also in irradiated seeds the probability for detecting chromosome abnormalities increases as the resulting seedlings age. Thus, these two facts and others may account for the relatively few chromosomal aberrations observed in the root tips fixed 1 to 2 days after germination in this study. Further investigations are needed to study the effects of irradiation on the chromosomal level in these species. 209. 54 Effects of Pollen Irradiation on Seed Production and Germination, Seedling Survival and Growth and Pollen Germination in vitro When Douglas-fir pollen grains were irradiated at different dosages of gamma-radiation and used for controlled pollination, the number of developed f i l l e d seeds was significantly different depending upon the dosage. Increasing the irradiation dose up to 5,000 R caused an apparent increase in the number of f i l l e d seeds/ cone. However, dosage of 500 R gave nonsignificant lower number of f i l l e d seeds/cone than the control, i.e. seeds from unirradiated pollen. The amount of f i l l e d seeds obtained from pollen treated with 10,000 R was substantially lower than any other treatment. Although there are quite a few publications on similar studies, in none of them were actual counts made for empty and f i l l e d seeds resulting from controlled pollination with irradiated pollen. Some research workers have used other sound c r i t e r i a to assess the effect of pollen irradiation on seed production. The results reported here agree in a general way with those of Rudolph (1965) on White spruce. He indicated that the total seed yield per cone increased with increased gamma-ray dosage to the pollen up to 600 and 800 r. Also the seed germination was lower at 400 r and higher at 600rrr and again lower at 800 r when compared to the control. Stimul-atory effects of low doses of irradiation on seed v i a b i l i t y in Xg were reported again by Rudolph in 1967. Our germination experi-ment was based on unequal number of replications since the amount of f i l l e d seeds was variable from one pollen treatment to another. However, the test revealed interesting results. There was no significant difference among the treatments. This may be due to 2 1 0 . the fact that only the f i l l e d seeds were used. It also indicates that while doses up to 5 ,000 R seemed to increase the number of f i l l e d seeds/cone they had no effect on seed germination or at least did not reduce i t . Another interesting point is that the germination percentage of seeds from a r t i f i c i a l pollination i (Tree E x Tree 11) was considerably higher than that of open pollinated seeds. This agrees with the results of Sziklai (1964). Pollen germinability in vitro differed significantly depending upon irradiation treatment. 500 R reduced the germination but not significantly more than the control. Pollen irradiated with 2 ,000 R had significantly higher v i a b i l i t y than any other treatment and 1 0 , 0 0 0 R reduced the germination below a l l other exposures. A reduction of germination of irradiated pollen in vitro was found by Mergen and Johansen, (1963) for Pinus riglda. Brewbaker and Emery (1962) reported the inhibition of pollen germination by ionizing radiation required high dose levels ranging up to 800 kR for 95$ inhibition in 19 species of angiosperms. The relatively high radiation resistance of pollen is usually understood by supposing that pollen germination and pollen tube elongation are possibly attributed to a simple physiological mechanism, and that these phenomena are not accompanied by successive nuclear divisions or c e l l divisions. Another possibility is the lower nuclear volume and haploid chromosome number of the gametes compared to the somatic tissues. However, variation in radiosensitivity among 18 pollen types was not correlated with gametic chromosome number, pollen size or nuclear volumes, (Brewbaker et a l . 1 9 6 5 ) ' There appears to be a close resemblance between the results of pollen germination in vitro and the number of f i l l e d seeds 2 1 1 . resulted from a r t i f i c i a l pollination with pollen given the same dosages. The lack of s t a t i s t i c a l l y significant difference between the control and 1 0 , 0 0 0 R treatments in their effects on the number of f i l l e d seeds/cone indicates that such similarity could be coincidental. A comparison between the data of the two experiments may shed some light on the mechanisms by which ionizing radiation at certain dosages may increase or decrease pollen v i a b i l i t y . The increase in number of f i l l e d seeds/cone found here could be explained, at least partially, in terms of increased pollen germ-inability at these particular exposures. Similarly the reduction in the number of f i l l e d seeds at higher dosages could be discussed on the same basis. But how ionizing radiation could stimulate pollen germination in vitro and in vivo is not completely under-stood. A subject of interest to radiobiologists which provides conflicting results is the stimulation of pollen tube growth by irradiation. Nikitin (1934) reported the most extensive X-ray data interpreted to indicate radiostimulation of pollen germin-ation and tube growth. Similar results were reported by some others using different species, Breslavetz (1946), and Swaminathan and Murty ( 1959 ) - Brewbaker and Emery (1962) considered that the evidence for stimulation of germination and growth by irradiation is inclusive. Comparable studies on pollen radiobiology in gymnosperms are very few, Fujimata et a l . ( 1 9 6 4 ) , Mergen and Johansen ( 1 9 6 3 ) « Much of the work was conducted on Angiosperms and number of studies, Armstrong (1959), Uematsu and Nishiyama (1967) and several others, 2 1 2 . have shown tha t seed p roduc t ion and format ion are a f f ec t ed by-p o l l e n i r r a d i a t i o n . In g e n e r a l , p o l l e n i r r a d i a t i o n reduces seed se ts and a f f e c t s seed v i a b i l i t y by damage to embryo and endosperm. The evidence i n d i c a t e d tha t seed set f o l l o w i n g p o l l e n i r r a d i a t i o n d i f f e r s from one spec ies to another and decreases over a wide range of i r r a d i a t i o n exposure . Furthermore, the exposure which leads to 50$ r e d u c t i o n i n seed v i a b i l i t y ranges from 2 ,000 to 1 0 , 0 0 0 R, (Shap i ro , 1 9 6 6 ) . In a study of embryogenesis f o l l o w i n g X - i r r a d i a t i o n of tomato p o l l e n , Nishiyama and Uematsu (1967) repor ted tha t p o l l e n tube from i r r a d i a t e d p o l l e n grew and penetra ted i n t o i t h e embryo sacs n e a r l y as w e l l as those of the c o n t r o l , excep t ing those t r ea t ed w i t h 100 kR. Delay of embryo and endosperm growth was u s u a l l y observed i n i r r a d i a t e d samples. They a l s o noted tha t most of the normal ly deve lop ing ovules might cease t h e i r growth l a t e r and subsequently d i s i n t e g r a t e . At 50 and 100 kR zygote format ion and growth of embryo and endosperm were almost complete ly i n h i b i t e d . C y t o l o g i c a l observa t ions were made by Gave and Brown (195^) on the f a i l u r e of seed set i n L i l i u m formosum a f t e r p o l l i n a t i o n w i t h 4 kR X - i r r a d i a t e d p o l l e n . Major c y t o l o g i c a l a b n o r m a l i t i e s e x h i b i t e d i r r e g u l a r l y - l o b e d g i an t n u c l e i , m i c r o - n u c l e i from fragmented chromosomes, and chromosome b r i d g e s . They s t a t ed f u r t h e r tha t these i r r e g u l a r i t i e s mechan ica l ly p r o h i b i t e d the success ive m i t o s i s i n the endosperm and embryo, which l a t e r became n e c r o t i c and degenerated. U s u a l l y no seed was formed. Th i s was a t t r i b u t e d to the f a i l u r e of f u s i o n of the female and male n u c l e i or to the degenera t ion of young embryos and endosperm f o l l o w i n g m i t o s i s i n t h e i r development. 213. Based on the evidence presented above and the experiments by Brewbaker and Emery ( 1 9 6 2 ) , McQuade (1952) ?and several others, one can conclude that pollen irradiation leads to the impairment of viable seed production primarily by i t s effects on (l) pollen germination, (2) sperm formation and tube penetration to ovules, (3) zygote formation and embryo growth, (4) endosperm development, (5) v i a b i l i t y of mature seed, and (6) delayed death in seedlings. The least sensitive of these systems is that of pollen germination. Brewbaker and Emery ( 1 9 6 2 ) , concluded that pollen irradiation at high doses produces impairment of seed and seedling development in a diverse assortment of dominant lethal effects in both embryo and endosperm. The v i a b i l i t y of f i l l e d seeds perhaps provides a more precise c r i t e r i a of dominant lethality and drops exponent-i a l l y with dose. Although they were taken at the age of three months, the results of survival and growth of the seedlings in height from controlled pollination indicated that pollen irradiation with 500 R has some stimulatory effects. There was not much difference between the control, 2 ,000 and 5 .000 R irradiation treatments, while 1 0 , 0 0 0 R dosage reduced the survival and height growth markedly. Such stimulatory response at the lower dosages of irradiation are in harmony with the results of Vidakovic ( 1 9 6 7 ) , in Pinus sylvestris. This also favours the hypothesis of Osborne (195? a fe) that the use of irradiated pollen causes an increase in the number of heterozygotic plants and that the induction of heterosis is possible. The average height of a r t i f i c i a l l y pollinated seedlings is greater than that for open pollinated seedlings, and although 2]>. this is beyond the objectives of this investigation, i t is an apparent extension of the trend found in the germination data. This also confirms the similar findings of Sziklai ( 1 9 6 4 ) , in which he concluded that tree " 1 1 " has a high general specific combining a b i l i t y . 2 1 5 . 6 SUMMARY AND CONCLUSIONS A study of some e f f e c t s of i o n i z i n g r a d i a t i o n on three Western con i fe rous spec ies was c a r r i e d out . The spec ies weres 1. Pseudotsuga m e n z i e s i i ( M i r b . ) Franco , D o u g l a s - f i r , (from two d i f f e r e n t provenances r ep re sen t i ng the C o a s t a l and I n t e r i o r forms of the s p e c i e s ) , 2 . P i c e a s i t c h e n s i s (Bong.) C a r r . , S i t k a spruce , and 3 . Tsuga h e t e r o p h y l l a (Ra f . ) S a r g . , Western hemlock The work was d i v i d e d i n t o three main phases: A . E f f e c t s of Gamma-Irradia t ion on Seed Germinat ion and S e e d l i n g  Growth and S u r v i v a l : The f i l l e d seeds were separated from the empty ones u s i n g X - r a y f l u r o s c o p y . Only the f i l l e d seeds were g iven the f o l l o w i n g i r r a d i a t i o n dosages: 0 ( c o n t r o l ) , 500 , 2 ,000, 5,000 and 10,000 R of gamma r a d i a t i o n a t 12:3 R/sec from a 6200 c u r i e C o b a l t - 6 0 - s o u r c e , (220 gamma c e l l manufactured by Atomic Energy of Canada L t d . ) . A f t e r i r r a d i a t i o n the seed l o t s were d i v i d e d i n t o two groups: the f i r s t was sown immediately and the second was s t r a t i f i e d before sowing. F i v e r e p l i c a t i o n s were ass igned f o r each subtreatment . Seeds were germinated on two Jacobsen germinators and germina t ion counts were made a t two-day i n t e r v a l s . Germinants were t r a n s -p lan ted i n t o J i f f y pots i n sandy-loam s o i l , kept i n a greenhouse and t h e i r s u r v i v a l was fo l l owed and r eco rded . S i x seed l ings r e p r e s e n t i n g each r e p l i c a t i o n were t r a n s f e r r e d i n t o two growth chambers under constant environmental c o n d i t i o n s . The he igh t measurements of these seed l ings were taken every l 4 days f o r 182 days . Morpho log i ca l express ions of r a d i a t i o n e f f e c t s were recorded 216. throughout the study as well as visible mutations. B. Gytogenetical and Biochemical Investigations: An attempt was made to correlate the differential radio-sensitivity of the species with some nuclear characteristics viz. nuclear volume (N.V.) interphase chromosome volume, (I.C.V.), DNA content/cell, and DNA content/chromosome. The nuclear volumes were measured in embryos stained with leucobasic fuchsin. The I.C.V.'s were estimated by dividing N.V.'s by somatic chromosome number. DNA was extracted from the embryos using hot Na CI solution and i t s content was determined by the diphenylamine reaction. Embryos were disintegrated in chromic acid and a haemocytometer was used to estimate the number of cells/embryo. The amount of DNA/embryo was divided by the number of cells/embryo in order to get the DNA content/cell. When the last value was divided by the somatic chromosome number, the DNA content/chromosome was obtained. Root tips from germinated irradiated and non-irradiated seeds were excised, pretreated with colchicine and processed through Feulgen's technique. Chromosomes in different phases of c e l l division were examined and aberrations were recorded. C, Intraspecific Hybridization with Douglas-fir Irradiated Pollen: Two trees, (designated "E" and " 1 1 " ) , located on the campus of the University of British Columbia were selected because a good deal of information on their phenology and progenies were available. Tree "E" was used as the mother tree. Pollen grains from tree " 1 1 " were given 5 different dosages: 0 , 500 , 2 , 0 0 0 , 5 ,000 and 1 0 , 0 0 0 R of gamma radiation using the. same Co^° source mentioned before. When the female conelets were ready a r t i f i c i a l 217. pollination was carried out. Mature cones were collected and seeds were extracted by hand. The number of f i l l e d seeds/cone was counted for each pollen irradiation treatment. Seeds from a r t i f i c i a l pollination were germinated in Jacobsen germinators and the seedlings were raised in the green-house. Seedling growth and survival were recorded for three months. The effect of pollen irradiation on germination in vitro was tested using a nutritive medium. The following points could be drawn from the results: 1. Gamma-irradiation affected seed germination and seedling growth and survival in different ways, depending upon the species. Generally, increasing the dosage decreased the germination percentage, increased the germination time and decreased survival and height growth of the seedlings. 2 . Lower dosages of irradiation, (500 and 2 , 000 R) appeared to stimulate germination and survival in Interior Douglas-fir only, and in Coastal Douglas-fir height growth was stimulated by similar exposures, 3 . The differences among the species were highly significant, Interior Douglas-fir proved to be the most radioresistant of a l l , followed by Coastal Douglas-fir, Western hemlock and Sitka spruce. The LD^Q (germination) for the same species were 7500 , 5595 , 2525 and 1915 R, and the LD^Q for the survival of 182-days-old seedlings were 5950 , 2500 , 1290 and lk00 R respectively. The slight discrepancy between seed germin-ation and seedling survival data in Western hemlock and Sitka spruce indicated that the former was more susceptible to environmental stresses than the latter. 2 1 8 . 4 . Post-Irradiation stratification of seed reduced the germin-ation percentage, survival and height growth significantly-compared with immediate sowing. The differences were more pronounced in the irradiated seeds than the controls. 3 5 . Sitka spruce had the largest nuclear volume of 8 5 0 . 0 9 1 p^ , followed by Western hemlock 5 3 2 . 6 8 7 , Coastal Douglas-fir 5 2 1 . 3 3 1 . and fina l l y Interior Douglas-fir 4 5 0 . 9 9 8 y?. The interphase chromosome volumes were 3 5 . 4 2 1 , 2 2 . 1 9 5 , 2 0 . 0 5 1 •a and 1 7 . 3 4 6 p in the same order. Only Sitka spruce was significantly different from the other species. 6 . The correlation coefficient for the relationship between N.V. and LD^Q (germination) was - 0 . 7 4 9 4 and for I.C.V. and LD^0 (germination), - 0 . 7 9 4 6 which were not significant. 7 . The amounts of DNA per c e l l and per chromosome differed significantly among the species. Sitka spruce had the highest DNA content/cell of 1 1 1 . 3 3 g x 1 0 " 1 2 , followed by Western hemlock 9 7 * 9 5 . Coastal Douglas-fir 6 9 - 3 3 and Interior 1 2 Douglas-fir 4 2 . 2 8 g x 1 0 . The DNA content/chromosome for - 1 2 the same species were 4 . 6 3 9 , 4 . 0 8 1 , 2 . 6 6 7 and 1 . 6 2 6 g x 1 0 8 . Highly significant inverse correlations were found between the amount of DNA per c e l l and per chromosome and L D ^ Q (germination). The correlation coefficients were - 0 . 9 9 4 8 and - 0 . 9 9 7 3 respectively. 9 . No correlation was found between N.V. and DNA content/cell on one hand, nor between I.C.V. and DNA content/chromosome on the other. 1 0 . Chromosome breaks, micro-nuclei and chromosome erosion were detected in root tips from Douglas-fir irradiated seeds, but 219. ' no such a b e r r a t i o n s cou ld be seen i n S i t k a spruce nor Western hemlock. 1 1 . P o l l e n i r r a d i a t i o n up to 5,000 R seemed to increase the number of f i l l e d seeds/cone, but 10,000 R reduced i t d r a s t i c a l l y . 12. S e e d l i n g he igh t growth and s u r v i v a l a f t e r 3 months were not s i g n i f i c a n t l y d i f f e r e n t from one p o l l e n i r r a d i a t i o n t r e a t -ment to another however, 500 R showed some s t i m u l a t i v e e f f e c t s . 1 3 . AjLremarkable resemblance was found between the data of p o l l e n ge rmina t ion i n v i t r o and the r e s u l t i n g f i l l e d seeds i n each t rea tment . I t cou ld be concluded that f o r future mutat ion breeding work the f o l l o w i n g dosages cou ld be a p p l i e d : f o r I n t e r i o r D o u g l a s - f i r 500 to 6,000 R, Coas t a l D o u g l a s - f i r : 500 to 3,000 R and f o r S i t k a spruce and Western hemlock: 'Up to 2,000 R. The v i s i b l e mutat ions were very ra re and to inc rease t h e i r r a t e perhaps a combinat ion of i o n i z i n g i r r a d i a t i o n and chemical mutagenesis may be t r i e d . S t i m u l a t i o n responses of he igh t growth i n Coas t a l Douglas-f i r seen under c o n t r o l l e d c o n d i t i o n s , (growth chambers), are encouraging . The next s tep would be to observe the performance of such seed l ings i n the f i e l d . The d i f f e r e n c e s i n r a d i o s e n s i t -i v i t y between the c o a s t a l and i n t e r i o r forms of D o u g l a s - f i r , and the v a r i a t i o n i n t h e i r nuc l ea r volumes and DNA conten t s , are p a r t i c u l a r l y i n t e r e s t i n g and subs t an t i a t e the p r e v i o u s l y repor ted m o r p h o l o g i c a l , ana tomica l and p h y s i o l o g i c a l d i f f e r e n c e s . 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Biochem. B i o p h y s . , 76 : 1 9 6 - 2 0 3 . Z i r k l e , R . E . 1 9 3 2 . Some e f f e c t s of a lpha r a d i a t i o n upon p l a n t c e l l s . J. C e l l u l a r comp. P h y s i o l , 2 : 2 5 1 - 2 7 4 . 249 APPENDIX 1 List of Some Common Names Used in the Text Hinokl white cedar Cryptomerla Maidenhair tree European larch Japanese larch Siberian larch Norway spruce White spruce Black spruce Red spruce Sitka spruce Jack pine Japanese red pine Shortleaf pine Slash pine Alepo pine Singleleaf pine Western white pine Mountain pine Austrian pine Longleaf pine Stone pine Ponderosa pine Red pine Pitch pine Chir pine Eastern white pine Scotch pine Loblolly pine Japanese black pine Virginia pine Giant sequoia Northern white cedar Gymnosperms > Chamaecyparls obtusa (S. and Z.), Endl. Cryptomerla japonlca (Llndl.), Decne. Ginkgo blloba L. Larlx decldua M i l l . Larlx leptolepls Sieb. and Zucc. Larlx s l b l r l c a Ledeb. Plcea ables L. Plcea glauca (Moench.), Voss. Plcea marlana (Mill.) BSP. Plcea rubrens Sarg. Plcea sitchensis (Bong.), Carr. Plnus banksiana Lamb. Plnus denslflora Sieb. and Zucc. Plnus echlnata M i l l . Plnus e l l l o t t l l Englem. Plnus halepensls M i l l . Plnus monophylla Torr. and Frem. Plnus montlcola Dougl. Plnus mugo Turra. Plnus nigra Arnold Plnus palustrls M i l l . Plnus plnea L. Plnus ponderosa Laws. Plnus reslnosa A i t . Plnus rlgida M i l l . Plnus roxburghll Sarg. Plnus strobus L. Plnus sylvestris L. Plnus taeda L. Plnus thunbergll Pari. Plnus vlrglnlana M i l l . Sequoia glgantla (Lindl.), Decne. Thuja occldentalls L. European alder White birch White ash European ash Black woolnut Sweet gum Yellow poplar Mock orange American sycamore Eastern cottonwood Angiosperms Alnus glutlnosa (L.) Gaertu Betula pubescens Ehrn. Fraxlnus amerlcana L. Fraxlnus excelsior L. Juglans nigra L. Llquldamber styraclllua L. Lirlodenderon tuliplfera L. Philadelphia caucaslca Koehne Platanus occldentalls L. Populus X deltoldes Marsh. 250 White oak Red oak Scarlet oak Bear oak Black oak Black locust American elder Honduran mahogany Silver linden American elm Quercus alba L. Quereus borealis Michx. Quercus cocclnea Muenchh. Quercus i l l c l f o l i a Wangh. Quercus velutlna Lamb. Roblnla pseudoacacla L. Sambucus canadensis L. Swletenla macrocarpa King. T l l l a tomentosa Moench. Ulmus amerlcana L. 

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