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Polyploidy and its application in forestry and a preliminary study of aberrant Douglas-fir seedlings Bolotin, Moshe 1958

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POLYPLOIDY AND ITS APPLICATION IN FORESTRY AND A PRELIMINARY STUDY OF ABERRANT DOUGLAS-FIR SEEDLINGS by MOSHE BOLOTIN B.S.F., U n i v e r s i t y o f Washington, 1951 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Ma s t e r o f F o r e s t r y i n t h e Department o f F o r e s t r y We a c c e p t t h i s t h e s i s as co n f o r m i n g to the r e q u i r e d s t a n d a r d . THE UNIVERSITY OF BRITISH COLUMBIA F e b r u a r y , 1958 I ABSTRACT The paper reviews the l i t e r a t u r e on polyploidy In respect to i t s possible application i n forest tree breeding. The occurrence of spontaneous polyploidy i n various species i s enumerated and i t s q u a l i t i e s outlined. An account i s presented of the success and f a i l u r e thus far attained i n the search for improved v a r i e t i e s of forest tree species through polyploidy. F i n a l l y , the p o t e n t i a l i t i e s of polyploidy i n forestry are summarized and some recommendations concerning future l i n e s of research are outlined. The experimental work i n connection with t h i s thesis was the preliminary inve s t i g a t i o n of Douglas f i r (Pseudotsuga menziesii (Mirob) Franco) aberrants found each year among the seedlings at the B.C. Forest Service nursery i n Duncan. These abberrants have been thought to be spontaneous autopolyploids because they resemble such polyploids found among nursery stock of other . coniferous species elsewhere. This study, however, indicated beyond any doubt that these abberrants are not polyploids. Other p o s s i b i l i t i e s which might have caused the aberrant form are discussed, . I n 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 Columbia, I agree 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 t 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 t h e 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 . 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 o r e s t r y  The U n i v e r s i t y o f B r i t i s h C olumbia, Vancouver 8, Canada. Date F e b r u a r y , 1958  ACKNOWLEDGEMENTS The writer i s indebted to Dr. P. G. Haddock and Dean G. S. A l l e n for advice and c r i t i c i s m of the present t h e s i s . To Dr. A. H. Hutchinson for advice and help i n taking most of the microphotographs. To Mr. H. E. Sweet for advice on microtechnique work. To Mr. W. V. Hancock for advice and help i n taking of microphotographs, to Mr. K. A. Helium for taking the photographs of seedlings, and to Miss L. Bowers for typing t h e s i s . I I TABLE OF CONTENTS Abstract I L i s t of i l l u s t r a t i o n s I l l Introduction 1 PART A:, REVIEW OF LITERATURE Meaning of polyploidy 3 Causes of polyploidy . . . . . . . . . . . . . . . . . 5 Changes caused by polyploidy 8 Limitations of polyploidy . 12 V a r i a b i l i t y of polyploids . 13 Polyploids and environment 1*+ T r i p l o i d y i n woody plants ,\ 16 Allopolyploidy i n woody plants . 19 Polyploids among the conifers 20 Spontaneous coniferous polyploids i n nursery stock • 23 P o t e n t i a l i t i e s of polyploidy i n fore s t r y . 25 PART B: PRELIMINARY STUDY OF ABERRANTS IN DOUGLAS FIR Introduction 27 Materials and methods 29 Results • 33 Discussion 35 I l l u s t r a t i o n s 37 Bibliography *+8 Acknowledgements 56 I l l LIST OF ILLUSTRATIONS F i g . 1. Normal and aberrant seedlings one-year o l d . . 37 F i g . 2. Normal and aberrant seedlings two-year o l d . . 37 F i g . 3« Stem d i f f e r e n c e s in*normal and aberrant seed l i n g s 38 F i g . k. An aberrant s e e d l i n g w i t h a normal branch. . 38 F i g . 5» Pro-metaphase i n an aberrant s e e d l i n g . . . . 39 F i g . 6. Pro-metaphase i n a normal seedling 39 F i g . 7« Metaphase i n an aberrant s e e d l i n g *f0 F i g . 8. Metaphase i n a normal seedling ho F i g . 9, E a r l y anaphase i n an aberrant s e e d l i n g . . . >+l F i g . 10. E a r l y anaphase i n a normal seedling ' f l Fig.11. Pro-metaphase i n a normal twig on ah aberrant,h2 F i g . 12. Metaphase i n a normal W i g on ah aberrant. . .if2 Fig.13. Anaphase i n an aberrant seedling ^3 F i g . l * f . Anaphase i n a normal s e e d l i n g . . . h'Q F i g . 15. Anaphase i n an aberrant seedling Mf F i g . 16. Pro-metaphase i n a normal se e d l i n g hh Fig.17. Telophase i n an aberrant s e e d l i n g . . . . . . '.^ 5 F i g . 18. Telophase i n a normal seedling ^5 Fig.19. Telophase i n an aberrant seedling.' *+6 Fig.20. Telophase i n a normal seedling ^6 Fig.21. Telophase i n an aberrant s e e d l i n g . . . . . . . A 7 Fig.22. Telophase i n a normal seedling k-J PART A REVIEW OF LITERATURE ON POLYPLOIDY I N FOREST TREES 1 INTRODUCTION During the past few decades considerable work has been carried out i n the i n v e s t i g a t i o n of polyploidy i n plants. Many superior polyploid strains have been developed either by selecting them from natural populations of d i p l o i d s or by a r t i f i c i a l induction. With the discovery of the e f f e c t of colchicine i n the induction of polyploidy i n plants, a new t o o l has been added by which many more polyploids have been produced. These polyploids have replaced the former d i p l o i d s i n cases where their superiority or usefulness has been demonstrated. In forestry the i n v e s t i g a t i o n of polyploidy lags considerably behind other f i e l d s which deal with plant breeding. This lag i s understandable because there i s much les s opportunity for introducing new strains of plants i n forestry than i n a g r i c u l t u r e . Forestry i s a less intensive form of plant, culture than h o r t i c u l t u r e or the r a i s i n g of f i e l d crops; the l i f e span of the ..' i n d i v i d u a l plant before harvest, i s much, longer,,.and the practice of a r t i f i c i a l methods of regeneration not as. common. Another reason for the lack,of intensive re- . search on polyploidy i n f o r e s t trees may be the f a c t that only a l i m i t e d amount of success has been attained i n finding superior polyploids. The purpose of the present study i s to survey the f i e l d of polyploidy research i n plants i n general and i t s 2 a p p l i c a t i o n i n f o r e s t r y , with p a r t i c u l a r emphasis on work carried out on coniferous trees. The p r a c t i c a l work consisted of the study of abnormal Douglas-fir (Pseudotsuga  menziesii (Mirob.) Franco) seedlings vhich were suspected to be autopolyploids. During the course of the study a schedule f o r squash technique i n vegetative buds of Douglas-fir was developed and a solution to the problem of breaking dormancy i n that species was undertaken. 3 MEANING OF POLYPLOIDY Most animals and plants are d i p l o i d s . A d i p l o i d organism car r i e s two homologous sets of chromosomes i n each c e l l nucleus, one set having come from each parent. Reproductive c e l l s (gametes) are haploid i . e . , they carry only one set of chromosomes. In rare instances the somatic nuclei of plants may contain only one set of chromosomes. Such monoploid plants are usually s t e r i l e and do not become established i n nature. Living organisms with n u c l e i containing three or more sets of chromosomes are known as polyploids. A polyploid with three sets of chromosomes i n the somatic nucleus i s c a l l e d a t r i p l o i d , with four sets a t e t r a p l o i d , and so on. An autopolyploid i s a polyploid which originated from one parental species. An a l l o p o l y p l o i d on the other hand, has i t s o r i g i n i n the hybridization of two or more species. A euploid plant has one or more complete sets of chromosomes. An aneuploid plant has a normal complement (genome) plus some extra chromosomes or with some chromosomes missing. Polyploids may be s t a b i l i z e d and capable of s e l f - perpetuation, or unstabilized, i n which cases vegetative reproduction i s the only possible means of propagation. P a r t i c u l a r l y among unstabilized and a r t i f i c i a l l y induced polyploids, several forms of retrogression to diploidy may occur. In mixoploids, d i p l o i d c e l l s are encountered alongside polyploid c e l l s . Chimeras are plants composed of tissues of d i f f e r e n t chromosomal count. In p e r i c l i n a l h chimeras several layers of tissue are polyploid whereas adjacent layers are d i p l o i d . In s e c t o r i a l chimeras c e r t a i n sectors of the plant are polyploid and other sectors are d i p l o i d . The wide d i s t r i b u t i o n of mixoploids, chimeras, and aneuploids, p a r t i c u l a r l y among a r t i f i c i a l l y induced polyploids indicates that c y t o l o g i c a l examination of the various tissues of plants i s necessary before these plants can be i d e n t i f i e d as true polyploids. 5 CAUSES OF POLYPLOIDY The occurrence of spontaneous polyploidy has been attributed to extreme temperature changes and other sudden and severe changes i n the environment. Experim ental evidence indicates that polyploidy may be induced by rather moderate temperature changes i n some genera, but that sudden changes are more e f f e c t i v e . Extreme temperature changes encountered by d i p l o i d races that extend into unfavourable t e r r i t o r y , or a change i n the l o c a l climate, might cause polyploidy. A survey of the natural polyploids indicates that they have a more northerly d i s t r i b u t i o n or a more alpine habitat than t h e i r d i p l o i d ancestors. In such habitats plants are subjected to greater and sharper temperature variat i o n s which might be the cause of polyploidy (Sax,1936). Love and Love (1957) believe that the higher frequency of polyploids i n a r c t i c regions i s an i n d i c a  t i o n of the a b i l i t y of polyploids to survive under adverse conditions. This theory, however, does not support the findings of Bowden (19^0) who investigated the hardiness of polyploids as compared to that of di p l o i d s and did not f i n d any greater hardiness i n the polyploids. Several abnormalities i n meiosis, such as asyndesis (Andersson, 19^-7) have been observed to favour the formation of:.polyploidy i n p l a n t s B Abnormalities i n 6 the reproductive organs of plants might also favour the production of unreduced gametes and thus cause polyploidy. This suggestion finds support i n a study made by Seitz (195*+) on a hermaphrodite clone of grey poplar (Populus  canescens Smith) with abnormalities i n the development of the flowering parts. One per cent of the progeny produced by s e l f p o l l i n a t i o n i n such plants were t r i p l o i d . Upon c y t o l o g i c a l examination of the anthers i t was found that some of the pollen mother c e l l s remained i n the d i p l o i d stage due to f a i l u r e i n the reduction d i v i s i o n , and i n t h i s way d i p l o i d pollen grains are believed to a r i s e and produce polyploid progeny. It i s possible also that s e l f i n g i n plants increases the chances of the occurrence of polyploids since there i s more chance for the accumulation of chromosome abnormalities due to a lower v a r i a b i l i t y i n the genetic make-up i n such a method of f e r t i l i z a t i o n . The fact that s e l f i n g i s rare among the conifers (Allen,19^2) might explain i n part the r a r i t y of polyploidy i n those species. Andersson (19^7) considered polyploidy to a r i s e i n nature through the formation of unreduced gametes due to f a i l u r e i n the reduction d i v i s i o n . Kiellander (1950), on the other hand, believed that, at l e a s t i n the conifers, doubling of the chromosome number i s more l i k e l y to take place a f t e r f e r t i l i z a t i o n than before i t . This was found i n Datura stramonium by Blakeslee (Vide Muntzing,1936), and might also be true i n the gymnosperms. 7 Several workers (Namikawa,193^) (Johnsson,19^6) have reported a higher percentage of polyploids among twin seedlings of several genera than i n the corresponding normal seedling populations. It i s also conceivable that, i n nature, polyploids may arise through the spontaneous development of chimeras and the subsequent development of inflorescences on the polyploid sectors (Andersson,19!+7) • 8 CHANGES CAUSED BY POLYPLOIDY Polyploids may d i f f e r from d i p l o i d s i n the following ways: a) vigor of vegetative growth (may be greater or lower), b) size of c e l l s and c e l l n u c l e i (may be l a r g e r ) , c) f r u i t size and shape (may be shorter and stouter), d) chemical and physical properties (may be quite d i f f e r e n t ) , e) size and number of stomata (may be larger and fewer), f) f e r t i l i t y of pollen grain and seed (may be lower i n autopolyploids and higher i n a l l o p o l y p l o i d s than i n the corresponding parent species). g) colour and shape of leaves (may be darker and th i c k e r ) . These changes are extremely variable however, and, when polyploidy i s to be induced a r t i f i c i a l l y , i t s effects cannot be predicted. In forestry applications desirable changes would include greater rate of growth and an improvement i n the chemical and physical properties of the woody parts. An increased rate of growth has been reported i n such Angiosperm-plants as poplar (Johnsson,19^2,1953? and others), and b i r c h (Johnsson,19^6,1956, and others). Among the conifers, good re s u l t s have been obtained with a l l o t r i p l o i d l a r c h which was obtained as the only progeny 9 from the cross of two species (Larsen and Westergaard, 1 9 3 8 ) , and a giant t e t r a p l o i d fprm has been reported i n seedlings of Cryptomeria .iaponica (Chiba , 1952 ). A l l other instances of polyploidy reported i n forest tree species resulted i n retarded growth rates compared with those of the corresponding d i p l o i d s . As f a r as s t r u c t u r a l changes i n the wood are concerned, Kanezawa (1951) reported an increase i n f i b e r length of 2,0% and i n f i b e r thickness of h0% i n induced polyploids of Japanese cypress (Chamaecyparis obtusa Endl). Several reports have been made of an increased water content i n autopolyploids as compared with d i p l o i d s . Johnsson (1950) made a study of t h i s problem i n Alnus glutinosa and found that dry matter content of the wood i s lower i n the t r i p - l o i d than i n the control and the s p e c i f i c weight of 'the wood i n the dry state i s lower. No difference i n the shrinkage properties of the wood was observed between the two s t r a i n s . On the basis of the measured data the pore volume was determined and found to be 7 0 . 1 3 $ of the t o t a l volume i n the t r i p l o i d s as compared with 68.1*+$ i n the d i p l o i d s . This difference i n pore volume i s explained by Johnsson as res u l t i n g from the larger elements i n the wood of the t r i p l o i d s . Several external features have been suggested by various workers as aids i n i d e n t i f y i n g polyploids and 10 distinguishing them from d i p l o i d s of the same species. None of these has proved to be so r e l i a b l e as a d i r e c t chromosomal count. Sax (1938) studied the number of stomata per square millimeter of l e a f surface i n herbarium material of a number of plant genera i n the d i p l o i d and polyploid condition. He found, with some exceptions, a co r r e l a t i o n between chromosomal counts and the size of stomata. In his opinion,.:stomatal size cannot be used as an absolute index of polyploidy, but i n many cases i t may be of use i n preliminary surveys. Johnsson (19^0) did not f i n d a di r e c t c o r r e l a t i o n between stomatal length and chromosome number i n the genus Populus but, on the average, aneuploids seemed to have larger stomata than d i p l o i d s , and t e t r a - ploids larger stomata than aneuploids and d i p l o i d s . The study of polyploidy i n forest trees up to now dealt only with the Co (Co i s the generation treated with colchicine) and Cl (Cl i s the f i r s t progeny generation of plants treated with colchicine) generations and conse quently l i t t l e i s known at present about the properties of subsequent generations i n forest trees. The findings of Wettstein (1937) i n polyploid races of mosses might throw l i g h t on the subject. Following the development of these mosses for several generations, he found that there was a decrease i n size of c e l l s and of stomata with the passage 11 of generations so that, a f t e r 11 years, the polyploids had the same c e l l and stomata sizes as the d i p l o i d s . F e r t i l i t y was also gradually restored yet the chromosome number rem ained the same and the polyploid races did not cross with the o r i g i n a l races. Kanezawa (19*+8) considered that a similar development i s l i k e l y to take place i n forest trees and that with the passage of generations, as the polyploids become adapted to the environment, differences i n c e l l and stomatal sizes w i l l tend to be eliminated. The size and f e r t i l i t y of the pollen grains have been suggested by some as aids i n distinguishing between poly ploidy and diploidy. The pollen grains usually tend to be considerably larger and to have a much lower f e r t i l i t y i n polyploid races than i n the corresponding d i p l o i d s . Peto (1938) i n his researches on spontaneous t r i p l o i d poplars arrived at the conclusion that no correlation existed between chromosome number, the size of the pollen grain, and the quality of the pollen. Furthermore, i n one of the polyploids (Populus alba var. auerointertexta) he found the f e r t i l i t y of the pollen to be as high as 9*+ per cent. 12 LIMITATIONS OF POLYPLOIDY The findings of various workers i n the f i e l d of polyploidy are summarized below as' to i t s e f f e c t on the vigour of plants. Plants show a varying degree of s e n s i t i v i t y to polyploidy. For every species there i s an optimal number of chromosomes with which i t exhibits i t s highest vigor and rate of growth. When th i s optimal number i s exceeded, the growth rate and v i t a l i t y decrease. Darlington (1937) considered that the optimal number i s conditioned by the size of the metaphase plate and the a v a i l a b i l i t y of extra space to permit an increase i n the number of chromosomes. According to him, plants with a r e l a t i v e l y small number of chromosomes and with small chromosomes are, generally speaking., more suitable to polyploid induction than- plants with a large basic chromosome number i n their genome or with large chromosomes. For t h i s reason he considered the angiosperms more l i k e l y to react favourably to i n - duction than the other classes of plants. Pauley (19*+9) stated that t r i p l o i d y seems to be the natural l i m i t i n the genus Populus and, therefore, the optimal number. Liellander (1950) believed that the optimal number of chromosomes i n Norway spruce (Picea  Abies (L) Karst) i s 2n = 2h and any increase of the number of chromosomes above th i s number would r e s u l t i n poor growth and lowering of v i t a l i t y . 13 VARIABILITY OF POLYPLOIDS Polyploidy, -which i s e s s e n t i a l l y the quantitative increase of the -chromosome complement of plants, produces various r e s u l t s i n d i f f e r e n t i n d i v i d u a l s of the same species. This i s p a r t i c u l a r l y true i n c r o s s - f e r t i l i z e d plants be cause i n such cases there tends to be an e f f e c t i v e re combination of the genetic material. One source of the increased v a r i a t i o n i n autopolyploids can be sought i n the disturbances during meiosis which may lead to numerical aberrants and aneuploids. There i s , however, the possib i l i t y that the e f f e c t s of such disturbances may be eliminated to a great degree at the gametic stage. Another source of increased v a r i a t i o n i n polyploids may be the increased number of l o c i and consequently the greater p o s s i b i l i t y of recombination afforded. I f the character i s t i c investigated i s determined by several genes simult aneously with extreme f a c t o r i a l combinations, an increased v a r i a t i o n i s doubtless also to be expected i n the poly ploids (Johnsson,1950). That the v a r i a b i l i t y of polyploids i s greater than that of the corresponding d i p l o i d s i n various types of plants i s a f a c t stated by many workers. Genetic d i f f  erences of d i p l o i d plants are accentuated i n the polyploids. a In order to produce/superior s t r a i n of polyploids, s e l e c t i o n i s necessary p r i o r to induction as well as a f t e r i t . Ih POLYPLOIDS AND ENVIRONMENT It i s widely accepted at present that the process of chromosome doubling i n all o p o l y p l o i d s has played a great r o l e i n the evolution of plant species. There i s however, s t i l l a great d i v e r s i t y of opinion among the various workers with regard to the evolutionary s i g n i f i c  ance of chromosome doubling i n autopolyploids. Muntzing (1933) considered autopolyploidy to be of importance i n plant evolution because i t ; a) a f f e c t s both the morphology and the physiology of plants. b) causes sexual i s o l a t i o n of polyploids. c) may give r i s e to polyploid races and species i n nature. Stebbins (19^7), on the other hand, considered that the evolutionary processes i n nature are l i k e l y to l i m i t the importance of autopolyploidy because of the reduced f e r t i l i t y and the i n a b i l i t y to give r i s e to anything new among the derivatives of autopolyploids. According to him, the importance of chromosome duplication i n the evolution of plants i s , i n the main, i n f i x i n g and spreading hybrid combinations, either i n t e r v a r i a t e or i n t e r s p e c i f i c . The fate of a spontaneous autopolyploid within a population of a d i p l o i d prototype from which i t arose has not been investigated for forest trees but two studies conducted on barley species throw l i g h t on the problem. Sakai and Suzuki (1955a,b) have found that, autotetraploid 15 strains of barley were almost always poor competitors against the d i p l o i d s . On the other hand, a l l o p o l y p l o i d s were found to be superior to both parental s t r a i n s . These findings might explain the su r v i v a l of Sequoia sempervirens, an autoallopolyploid with 2n = M+ (Stebbins ,19 i +8), and Aesculus carnea, an a l l o p o l y p l o i d with 2n = 80 (Upcott, 1 9 3 6 ) . I f the above findings are univ e r s a l l y true for plants, i t i s apparent that there are good prospects for future a l l o p o l y p l o i d s which could be developed a r t i f i c i a l l y or n a t u r a l l y . It i s also l i k e l y that autopolyploids would have to be propagated vegetatively for each generation i n cases where they do not produce f e r t i l e seeds. 16 TRIPLOIDY IN WOODY PLANTS Tr i p l o i d s of several woody plants exhibit an increased growth rate i n comparison with d i p l o i d s of the same species. *n the genus Populus both spontaneous and induced t r i p l o i d s are much faster growing and bigger than the corresponding diploids (Muntzing, 1936 , and others). T r i p l o i d y also pro duced good r e s u l t s i n A l n u s glutinosa (L) G aertn; young seed l i n g s of t h i s species were studied by Johnsson (1950) and found to be 50% t a l l e r on the average than the d i p l o i d s . The r e a l difference i n height attributable to t r i p l o i d y was pro bably higher since the t r i p l o i d seedlings included many aneu ploids with retarded growth, i n Betula verucosa t r i p l o i d y produced robust seedlings with a larger average diameter than that of the dip l o i d s "but with a somewhat reduced height (Johnsson, 1 9 5 6 ) . In the conifers, examples of spontaneous au t o t r i p l o i d y are unknown. In these the p o t e n t i a l i t i e s of induced autotrip loidy' are at present unknown. In a private communication to the author, Kiellander (1957) expressed the opinion that: " a r t i f i c i a l t r i p l o i d s of such hardwoods as Betula, Populus, Alnus, and Ulmus possess good growing capacities. Autotripl - r oids among the conifers have not, however, been produced as yet; consequently we know nothing about a u t o t r i p l o i d conifers". A l l o t r i p l o i d l a r c h (Larix decidua/Larix pccidentalis) was obtained by Larsen and Westergaard ( 1 9 3 8 ) . This i s 17 the only mention found i n t h i s l i t e r a t u r e survey of t r i p l o i d y of any kind i n the conifers. This a l l o t r i p l o i d was the only progeny obtained from a cross between the two species of l a r c h . Soegard (1957) reported on the development of the above a l l o t r i p l o i d : " i t has an ex c e l l e n t rate of growth and i n January, 1957 was l*+.5 metres t a l l which i s almost the height of a hybrid l a r c h Larix decidua/Larix l e p t o l e p i s of the same age." The main d i f f i c u l t y i n the production of t r i p l o i d s of both hardwoods and conifers i s that they cannot be induced d i r e c t l y from d i p l o i d s . The case of the a l l o  t r i p l o i d l a r c h c i t e d above i s uncommon and probably took place because the two parent species were mutually i n  compatible and could not produce a d i p l o i d progeny of the cross. Normally a t r i p l o i d has to be produced through a cross between a t e t r a p l o i d and a d i p l o i d . The fact that tetraploids bear f r u i t at an older age than d i p l o i d s makes the problem more serious i n woody perennials. One way of overcoming this d i f f i c u l t y i s to graft tetraploids on d i p l o i d stock. Larson, (1956) and others, with the development of new techniques i n connection with seed orchard work, found i n recent years that grafting i s con ducive to f r u i t bearing at a younger age. T r i p l o i d s , owing to their degree of s t e r i l i t y , present another d i f f i c u l t y i n the f a c t that they are incapable of bearing a progeny true to type. At l e a s t two types of gametes, univalents and bivalent, and i n many cases unreduced t r i v a l e n t gametes, have been reported i n t r i p -18 l o i d s . The high frequency of occurrence of aneuploids, i n addition to the variety of euploids with d i f f e r e n t chromo some count, which have been reported as the progeny of t r i p l o i d s (Johnsson,19^2) makes i t clear that vegetative propagation would be the only p r a c t i c a l means of repro ducing t r i p l o i d s . The extra cost involved i n such propa gation would be a l i m i t i n g factor i n undertaking c u l t i v a t i o n of t r i p l o i d s . In the case of t r i p l o i d clones of poplar such a means of propagation was found worthwhile and has been employed i n Sweden for some time. Whether such a method also would be worthwhile i n other species w i l l have to be decided i n d i v i d u a l l y for each species a f t e r the added value of the t r i p l o i d crop has been compared with the extra cost involved i n vegetative propagation. 19 ALLOPOLYPLOIDY IN WOODY PLANTS Allopolyploidy i s d i r e c t l y e f f e c t i v e i n producing new species since such polyploids r e s u l t from species hybridization followed by a chromosomal increase. The duplication of each of the parental genomes restores f e r t i l i t y and the hybrid breeds true to type. Thus the al l o p o l y p l o i d i s a constant species hybrid which has ch a r a c t e r i s t i c s of a true species, and i n the case of generic crosses may merit a generic rank (Sax,1936) . A l l o p o l y p l o i d s are known to occur i n nature and under c u l t i v a t i o n , and have been produced experimentally i n several d i f f e r e n t f a m i l i e s . Examples of natural a l l o  polyploid woody perennials are Sequoia sempervirens (2n = k-k) among the conifers (Stebbins ,19 l +8) and Aesculus  carnea (2n = 80) among the hardwoods (TJpcott ,1936) . A l l o p o l y p l o i d s with superior growth q u a l i t i e s have been produced a r t i f i c i a l l y and i n numerous cases rendered i n f e r t i l e crosses f e r t i l e . I t i s s i g n i f i c a n t that many of the most important c u l t i v a t e d plants today are of known or supposed a l l o p o l y p l o i d o r i g i n . Very l i t t l e use has been made i n forestry thus f a r of allopolyploidy i n the propaga t i o n of hybrid v a r i e t i e s of trees. 20 POLYPLOIDY AMONG THE CONIFERS Changes i n the chromosome number have been of l i t t l e importance in the differentiation between families and genera of most coniferous trees. Sax and Sax (1933) believe that the stability of the conifers with respect to polyploidy indicates that evolution in this group has passed i t s climax and that the existing forms are survivors of long natural selection. In another study, Sax (1932) stated that the comparatively high number of chiasmata with the prevalence of i n t e r s t i t i a l chiasmata account for the great uniformity of chromosome number and the general sta b i l i t y of that group. He attributes the rarity of polyploids to the large number of chiasmata i n the diploid stage. There is an average of 2 A i n t e r s t i t i a l chiasmata per bivalent and this number seems to be remarkably constant i n a l l the conifers covered in his study. Any autopolyploids produced would be lik e l y to form closely paired tetravalents and the segregation of homologous chromosomes would be too irregular to produce a high degree of f e r t i l i t y . Muntzing (1933) explained the rarity of polyploids among the conifers by the absence of double f e r t i l i z a t i o n i n this group and the fact that diploid and polyploid races can cross readily and, consequently, the polyploid forms are not isolated and developed independently. This hypothesis might also explain the differentiation of relatively few species and genera 21 among t h e c o n i f e r s . The mechanism of e v o l u t i o n i n the c o n i f e r s has i n v o l v e d a g a i n o r l o s s o f one chromosome, s t r u c t u r a l r e a r r a n g e m e n t , and gene m u t a t i o n s , but p o l y  p l o i d y has p l a y e d a s m a l l r o l e . The l o s s o f a chromosome has been r e s p o n s i b l e f o r the e v o l u t i o n o f T a x o d i a c e a e and and Cupressaceae. T h i s i n v o l v e d a l o s s o f a centromere w h i c h f o l l o w s t r a n s l o c a t i o n s o f a l l e s s e n t i a l genes t o t h e r e s t o f t h e c e n t r o m e r e s . I n s t a n c e s o f a g a i n o f a chromo some a r e few and oc c u r o n l y i n the genus Pseudotsuga and i n t h e f a m i l y A r a u c a r i a c e a e . T h i s always i n v o l v e s dup- , l i c a t i o n o f a centromere and c o u l d be a c h i e v e d by a system o f t r a n s l o c a t i o n s as proposed by D a r l i n g t o n (1937)• The m a j o r i t y of t h e c o n i f e r s a r e thus found t o have a h a p l o i d chromosome number o f 12 (n = 12). The o n l y known genera w i t h d e v i a t i n g chromosome numbers a r e Taxodium, T h u j a , and J u n i p e r u s , each o f w h i c h has. a h a p l o i d comp lement o f 11 (n = 11) and Pseudotsuga and A r a u c a r i a w i t h a h a p l o i d number o f 13 (n = 13). Spontaneous s t a b i l i z e d p o l y p l o i d s among the c o n i f e r s a r e v e r y r a r e . The o n l y cases known a r e t h a t o f J u n i p e r u s  c h i n e s i s v a r . P f i t z e i a n i a , an a u t o t e t r a p l o i d w i t h a 2n = hh number o f chromosomes, and Sequo i a s e m p e r v i r e n s , an a u t o - a l l o p o l y p l o i d a l s o w i t h a chromosome complement o f 2n = M+. O c c a s i o n a l o c c u r r e n c e o f spontaneous p o l y p l o i d s among the c o n i f e r s i s r e p o r t e d f r o m time t o t i m e but none o f t h e s e has proved t o be s t a b i l i z e d . The o l d e s t r e p o r t e d such p o l y p l o i d i s t h a t found by C h r i s t i a n s e n (1952) i n Denmark. 22 This a u t o t e t r a p l o i d , L a r i x decidua M i l l e r , which was "between 56 and 58 years o l d at the time of d i s c o v e r y , was 15.2 metres t a l l and 97.5 cms. at breast h e i g h t . The t e t r a v a l e n t chromosome complement was found to act very i r r e g u l a r l y during meiosis and most of the seeds produced were found to be hollow. 23 SPONTANEOUS CONIFEROUS POLYPLOIDS IN NURSERY STOCK The occurrence of spontaneous p o l y p l o i d s among coniferous nursery stock has been reported f o r s e v e r a l species. K i e l l a n d e r (1950) reported such an occurrence i n two-year-old Picea Abies. The seed m a t e r i a l f o r these seedlings was gathered from a l a r g e number of t r e e s and i t was found t h a t the p o l y p l o i d aberrants were a r a r e occurrence among the progeny of many d i f f e r e n t t r e e s . The c a l c u l a t e d frequency of occurrence f o r P i c e a Abies was 8 per 100 ,000 s e e d l i n g s . The p o l y p l o i d aberrants resembled the p o l y p l o i d s of the same species which were produced by means of c o l c h i c i n e treatment. They e x h i b i t e d the same slowness of growth and t h i c k needles. The r a t i o of the t o t a l height of these two-year-old seedlings to the t o t a l height of normal seedlings of the same age was found to be 0 . 3 . Z i n n a i (1955) made a study of spontaneous p o l y p l o i d y i n nursery stock of Cryptomeria .iaponica and found, an occurrence of 5 . 1 5 per 10 ,000 i n unthinned seedbeds and only 1.6*+ i n thinned seedbeds. Z i n n a i d i d not give any f i g u r e s on leng t h r a t i o of p o l y p l o i d and normal s e e d l i n g s , but s t a t e d that among seedlings w i t h heights from 6 . 5 to 7 . 5 cm. there were 2 . 2 3 p o l y p l o i d s compared w i t h 1 .65 per 1 0 , 0 0 0 seedlings with a height of 7 . 5 to 9 . 0 cm, and only 0 ,56 per 10 ,000 i n the 9 . 0 to 1 5 . 0 cm. height group. He a l s o found that there was a high occurrence of p o l y p l o i d s 2h among the seedlings removed by t h i n n i n g . This agrees w i t h K i e l l a n d e r ' s e x p l a n a t i o n f o r the r a r i t y of p o l y p l o i d spruce and the reason that p o l y p l o i d s of t h i s and other c o n i f e r s have h i t h e r t o escaped observation. Apparently a u t o t e t r a p l o i d s i n the c o n i f e r s are poor competitors of the d i p l o i d s and i n a d d i t i o n , they do not reproduce true to type. Chiba and Watanabe (1952) have i n v e s t i g a t e d the occurrence of a u t o t e t r a p l o i d s i n nursery stock of L a r i x  kaempferi and found that they lacked the gian t form of the a u t o t e t r a p l o i d Cryptomeria .iaponica s e e d l i n g s , reported e a r l i e r by Chiba (1950). No account has been found i n the l i t e r a t u r e of the occurrence of spontaneous p o l y p l o i d y i n Douglas f i r . Only one in s t a n c e , (Meyer,195D i s known of i n d u c t i o n of p o l y p l o i d y i n that s p e c i e s . Meyer immersed seeds f o r four days i n a 0.2% s o l u t i o n of c o l c h i c i n e i n water and obtained seedlings which proved to be t e t r a p l o i d s . Dean George; S. A l l e n , F a c u l t y of F o r e s t r y , U n i v e r s i t y of B r i t i s h Columbia, and Mr. R. R. S i l e n e , U. S. Forest S e r v i c e , P a c i f i c N. ¥. For e s t Experiment S t a t i o n , C o r v a l l i s , Oregon, t r i e d to induce p o l y p l o i d y i n Douglas f i r by t r e a t i n g seed w i t h c o l c h i c i n e and have obtained abnormal seedlings s i m i l a r to those described f o r other coniferous s p e c i e s . 25 P O T E N T I A L I T I E S OF P O L Y P L O I D Y I N FORESTRY I n c o n c l u d i n g t h i s r e v i e w o f l i t e r a t u r e , i t c a n h e s a i d t h a t , d e s p i t e t h e many f u t i l e a t t e m p t s t o f i n d o r i n d u c e s u p e r i o r v a r i e t i e s o f p o l y p l o i d s i n f o r e s t t r e e s , a l l t h e p o s s i b i l i t i e s h a v e b y no means b e e n e x h a u s t e d . I t i s b e l i e v e d t h a t p o l y p l o i d y c a n p r o v i d e a g o o d means b y w h i c h e x i s t i n g v a r i e t i e s c a n b e i m p r o v e d . A l l o p o l y p l o i d y , i n p a r t i c u l a r , c a n be u s e f u l i n d e v e l o p i n g f e r t i l e h y b r i d s . The c h e m i c a l a n d p h y s i c a l c h a n g e s t h a t t a k e p l a c e i n p o l y  p l o i d s o f v a r i o u s s p e c i e s s h o u l d b e i n v e s t i g a t e d i n m o r e d e t a i l . I t i s p o s s i b l e t h a t t h e s e m o d i f i e d c h a r a c t e r i s t i c s m i g h t be m o r e d e s i r a b l e t h a n t h o s e o f t h e c o r r e s p o n d i n g d i p l o i d v a r i e t i e s . On t h e w h o l e , much m o r e i n v e s t i g a t i o n o f p o l y p l o i d y i s n e c e s s a r y b e f o r e t h i s f i e l d i s a b a n d o n e d . I m p r o v e d m e t h o d s o f i n d u c t i o n w o u l d h a v e t o b e f o u n d i n o r d e r t o p r o d u c e t r u e p o l y p l o i d s i n w h i c h t h e i n c r e a s e d n u m b e r o f c h r o m o s o m e s t e n d s t o p r o d u c e b e t t e r v a r i e t i e s . The o p t i m a l n u m b e r o f c h r o m o s o m e s w o u l d h a v e t o b e d e t e r m i n e d f o r e a c h s p e c i e s . I n a n y e v e n t , s e l e c t i o n i s a s e s s e n t i a l i n a p o l y p l o i d p o p u l a t i o n a s i n t h e v a r i a n t s o f d i p l o i d f o r m s . The a d d e d , v a l u e r e s u l t i n g f r o m t h e p r o d u c t i o n o f s u p e r i o r p o l y p l o i d v a r i e t i e s w o u l d h a v e t o be d e t e r m i n e d a n d w e i g h e d a g a i n s t t h e e x t r a c o s t i n v o l v e d i n t h e i r p r o d u c t i o n a n d c u l t i v a t i o n . I m p r o v e d m e t h o d s o f i n d u c i n g p o l y p l o i d y i h a l l t i s s u e s o f t h e p l a n t w o u l d e n t a i l d i f f e r e n t a p p l i c a t i o n s o f c o l c h i c i n e o r a c o m b i n a t i o n o f t h i s a n d o t h e r c h e m i c a l s t o 26 ensure p e n e t r a t i o n . I n t h e p a s t , c o l c h i c i n e t r e a t m e n t was c o n f i n e d t o g e r m i n a t i n g seeds and s h o o t s . One attempt t o i n d u c e p o l y p l o i d y i n e x c i s e d embryos o f p i n e was c a r r i e d out w i t h o u t s u c c e s s , by Hyun (195^). I n e x c i s e d embryos the m e r i s t e m a t i c t i s s u e i s more exposed t h a n i n seeds and t r e a t m e n t o f e x c i s e d emryos i n c u l t u r e s h o u l d g i v e b e t t e r r e s u l t s . Haddock (195*+) d e s c r i b e d a method o f grow i n g e x c i s e d embryos o f sugar p i n e i n c u l t u r e , and such a method, i f i t proves to be s u c c e s s f u l i n o t h e r c o n i f e r s , c o u l d be used i n p o l y p l o i d a l i n d u c t i o n work. The f a c t t h a t t h e r e i s no d o u b l e f e r t i l i z a t i o n i n th e c o n i f e r s c o u l d be used i n a t t e m p t s t o i n d u c e d o u b l i n g of t h e chromosomes i n the gametic s t a g e and might prove t o be u s e f u l i n the p r o d u c t i o n o f c o n i f e r o u s a u t o t r i p l o i d s . I n t h e angiosperms, on t h e o t h e r hand, d o u b l i n g o f the chromosomes i n t h e gametic s t a g e i s l e s s l i k e l y t o succeed s i n c e d o u b l e f e r t i l i z a t i o n and t h e d i f f e r e n t chromosomal count i n t h e embryo and the endosperm t i s s u e s might cause d i f f i c u l t i e s i n the development o f the seed. PART B A PRELIMINARY STUDY OF ABERRANT SEEDLINGS IN DOUGLAS FIR 27 INTRODUCTION The occurrence of aberrants i n nursery stock at the B.C. Forest Service nursery at Duncan has been noticed for several years. Mr. Jack Long, superintendent of the nursery, has separated some of these aberrants and planted them i n two rows on the nursery grounds. The oldest of these aberrants i s now ten years old, but none has pro duced any cones thus f a r . The aberrants are characterized by thicker, darker needles than those i n normal seedlings and by a retarded growth. The buds of the aberrants are more round and smaller than the normal buds (Figs. 1,2,3>^)» The frequency of occurrence of the aberrants has been estimated by Mr. Long as from 6 to 10 per m i l l i o n seed l i n g s . I t i s possible that some were unnoticed and the frequency of occurrence may actually be higher. The di f f e r e n t phenotypes that are found among the aberrants and t h e i r presence each year suggest that they are of rare occurrence among the progeny of various trees. The external features of these aberrants and t h e i r spontaneous and rare occurrence every year strongly suggest auto polyploidy. They resemble autopolyploids which have been described for other coniferous species (Kiellander, 1950; Chiba, 1952; and others). The presence of normal twigs on some of the aberrants (Fig. h) resemble s e c t o r i a l chimeras i n autopolyploids. The in v e s t i g a t i o n to determine whether these were 28 r e a l l y polyploids was undertaken i n the summer 1957> following a suggestion by Dr. P. G. Haddock and Dr. A. Orr-E\ri.ng. The study consisted of: a) The measurement of the length of aberrant seed l i n g s and comparing them to normal seedlings. b) The embedding of cross-sections"of aberrant needles and detecting any abnormalities i n them. c) The comparison of- stomata and guard c e l l lengths i n the aberrants and i n normal seedlings. d) The breaking of dormancy i n aberrant and normal seedlings to f a c i l i t a t e a c y t o l o g i c a l study. e) The finding of a suitable squash technique for examining vegetative buds of Douglas f i r . f) The preparation of s l i d e s of chromosomes i n the dividing c e l l s of buds i n aberrant and normal Douglas f i r seedlings. 29 MATERIALS AND METHODS The study was c o n f i n e d m o s t l y t o 22 a b e r r a n t s e e d l i n g s (5 O n e - y e a r - o l d and 17 t w o - y e a r - o l d ) and an e q u a l number o f normal s e e d l i n g s o f the same age groups w h i c h were shippe d t o Vancouver, thanks t o t h e c o u r t e s y o f Mr. J . £ong. The normal s e e d l i n g s were p i c k e d a t random from among n u r s e r y s t o c k . A measurement o f the l e n g t h o f t h e a b e r r a n t s and normal s e e d l i n g s was c a r r i e d out p r i o r t o p l a n t i n g them i n p o t s i n the U n i v e r s i t y o f B r i t i s h Columbia green-house on November 25, 1957* Needles o f a b e r r a n t and normal s e e d l i n g s were embed ded i n p a r a f f i n , c u t i n t o c r o s s - s e c t i o n s , mounted, and s t a i n e d w i t h s a f r a n i n and f a s t g r e e n . f o u n t s o f stomata from a b e r r a n t and normal n e e d l e s were p r e p a r e d by p e e l i n g o f f the l o w e r e p i d e r m i s o f n e e d l e s a f t e r b o i l i n g i n water f o r a few m i n u t e s , t h e n i n s e r t i n g i n c o l d w ater (Johanssen, 19^0). T h i s method of p e e l i n g o f f t h e e p i d e r m i s was found t o be more s a t i s  f a c t o r y t h a n t h e method employing c o l l o d i o n f i l m s (Long . and e l e m e n t s , 193*+) • The stomata were s t a i n e d w i t h a c e t o - a carmine. The l e n g t h o f the g u a r d - c e l l s i n the a b e r r a n t and i n the normal s e e d l i n g s was measured and compared. I n o r d e r t o break t h e dormancy o f the s e e d l i n g s t he f o l l o w i n g t r e a t m e n t s were t r i e d : a) C h i l l i n g t h e s e e d l i n g s f o r 2h hours p r i o r t o p l a n t i n g i n the green-house by p l a c i n g the seed-30 l i n g s i n a r e f r i g e r a t o r with th e i r roots covered with wet s o i l contained i n a p l a s t i c bag. (6 aberrants and 6 normal seedlings). b) Coating some of the buds of seedlings grown i n the green house with g i b e r e l l i c acid i n l a n o l i n paste (Marth et a l , 1956). 0+ aberrants and k normal seedlings). c) Increasing the photoperiod i n the green house by 2 hours d a i l y . (5 aberrants and 7 normal seedlings). d) Increasing the photoperiod i n the green house by h hours d a i l y . (5 aberrants and 7 normal seedlings). e) Treating plants grown indoors, i n a heated labora tory, with a r t i f i c i a l l i g h t from ordinary table lamps for alternate periods of 2>+ hours, i n addition to f e r t i l i z i n g with small amounts of ammon ium s u l f a t e . 0+ aberrant seedlings). During the course of the study i t was found that there was no completely s a t i s f a c t o r y squash technique for Douglas f i r . Neither the conservative method with aceto-carmine (Johanssen,191+0) nor the method suggested by Mergen and Novotny (1956), using c r y s t a l v i o l e t s t a i n , gave good r e s u l t s . By a process of t r i a l and error, various other schedules were used and i t was found that an adaptation of the aceto- lacmoid squash method suggested by Mikaelson (1952) f o r staining spruce and bi r c h was the most suitable for Douglas f i r . The f i x a t i v e used by him, however, was found to r e s u l t sometimes i n plasmolysis of Douglas f i r c e l l s and did not give as good r e s u l t s as acetic alcohol. Only temporary s l i d e preparations, sealed with n a i l p o l i s h , were used. Mikaelson suggested that these s l i d e s could be made permanent 31 by removing the seal a f t e r four days and mounting i n well aged (acid) euparal. The following schedule has been t r i e d i n chromosome staining of vegetative buds of Douglas f i r and found to be most suitable: Step 1, A c t i v e l y dividing buds are cut o f f the seedling or tree and dissected l o n g i t u d i n a l l y . Step 2. (This step i s carried out only when an i n h i b i t i o n of the spindle formation and shortening of the chromosomes are desired; otherwise i t may be omitted). Dissected buds are treated as follows: a) Placed i n a solution of 1%> colchicine i n water for one hour under a r t i f i c i a l l i g h t from an ordinary table lamp. (Mergen and Novotny, 1957) b) Placed i n a 0.002M solution of 8 hydro-oxyquinoline i n water for 2k hours. ( l i l i e s , 1952) c) Placed i n a saturated solution of paradichloro- benzene i n water for 2k hours. (Hyun, 195*0 • Step 3 . Dissected buds are k i l l e d and fix e d i n acetic alcohol 3*1 ( G l a c i a l acetic acid to 96% ethanol i n the r a t i o of 3:1) (Darlington,1950). Step k. Single needles are excised and hydrolized i n a mixture of 96% ethanol and concentrated hydrochloric acid i n the r a t i o of 1:1 for 5 to 15 minutes, depending on hardness of the material. (Mikaelson,195*0 • Step 5. Hydrolizing f l u i d i s washed of f with water. (Mikaelson, 195*0. Step 6. Single needles are placed on a s l i d e and stained 32 with standard acetic lacmoid stai n (1% r e s o r c i n blue i n \ % acetic a c i d ) . (Mikaelson, 195*+)• Step 7. Needles are squashed with a cover glass. The s l i d e i s placed i n a bibulous book and pressure i s applied to i t . Tapping of the s l i d e may help to obtain polar views and scatter the chromosomes. (Darlington and La Cour, 1950). Step 8. S l i d e i s examined under a microscope and, i f under- stained more st a i n i s applied, (Darlington and La Cour,1950). 33 RESULTS The measurements of average length of aberrants and normal seedlings are presented i n Table 1. TABLE 1. Comparison of stem and root lengths of one-and two-year-old aberrants with those of an equal number of normal seedlings picked at random. No.of Type Length, inches Range of Ratio of seed- Stem Root Total T o t a l f Length l i n g s Length^ Aberrant/ inches' normal One-year-old 5 Normal h»9 *+.7 9.6 8.0-10.1 5 Aberrant 2.9 3.0 5*9 ^.5-6.0 0.6 Two-year-old I B N o r m a l 12.5 9.0 21.5 13.5-25.*+ 17 Aberrant 6.6 6.0 12.6 8.5-21,5 0.6 From the table i t can be seen that the r a t i o of the t o t a l length of aberrants to that of normal seedlings re mained constant on the average, f o r the one-and the two- year-old seedlings. The ranges of the one-year-old seedlings did not overlap and, i n the two-year-old seedlings, only three aberrants overlapped the range of the normal seedlings. A " t " test conducted on the two-year-old seedlings indicated that the normal and aberrant seedlings were from d i f f e r e n t populations with a confidence l e v e l of much less than 1 per cent. 3^ No i r r e g u l a r i t i e s i n the epidermal layer and the size of the mesophyll c e l l s were noted i n the cross-sections of the aberrant needles. Such i r r e g u l a r i t i e s were des cribed i n autopolyploids of pine (Hyun, 195k)• This was the f i r s t i n d i c a t i o n that the aberrants might not be polyploids. A subsequent study of the size of stomata and guard c e l l s f a i l e d to reveal any s i g n i f i c a n t difference between the aberrants and the normal seedlings. The length of the average guard c e l l i n both the aberrants and the normal seedling was around 57 microns. Of the f i v e various treatments to break the dormancy of the seedlings a l l the four aberrant seedlings grown indoors and f e r t i l i z e d with ammonium sulfate (treatment e) resumed t h e i r growth ten days a f t e r the st a r t of the treatment. Of the 5 aberrants and 7 normal seedlings grown under increased photoperiod of k hours (treatment d) only two aberrants and one normal seedling resumed their growth three weeks afte r the s t a r t of the treatment. The other three treatments f a i l e d to break dormancy i n any of the seedlings treated. Cytological study of six aberrants and four normal seedlings f a i l e d to show any increase above the normal complement of 26 chromosomes (Zenke, 1953 and others). This finding (Figs. 5,7,9,11,13,15,17,19,21) was consistent for a l l plants studied and i n various tissues of the bud. 35 DISCUSSION In spite of the f a c t that the external features of the aberrant seedlings strongly suggest polyploidy i t i s obvious that the cause for the aberrant shape must be other than polyploidy. l i l i e s (1952) described a s i m i l a r pheno menon i n dwarf spruce with a l l the external features of a polyploid but with a regular d i p l o i d number of chromosomes i n the somatic t i s s u e . The cause of the aberrant form i n such cases must be due to some sort of a mutation and t h i s would require a further study. The f a c t that some of the aberrants have normal branches below the aberrant ones suggests that at l e a s t i n some of the aberrants the appearance of the mutation took place i n the somatic ti s s u e s . Some of the older aberrants i n the Duncan nursery have normal branch whorls above aberrant whorls which suggests that the mutation can be normalized as well as formed i n the somatic tissues of the plant. It i s of i n t e r e s t to note that the aberrants studied strongly resemble the f o l i a g e of trees expressing the phenomenon of brooming which was described i n older Douglas f i r trees (Buckland and K u i j t , 1957)* This broom ing appears at a l a t e stage i n the development of the tree. The p o s s i b i l i t y that the same causes underlie the formation of brooming as well as the aberrant form under.study cannot be excluded. During the course of the foregoing study i t has been 36 noted that at anaphase only the haploid number of chromo somes i s di s c e r n i b l e i n a l l Douglas f i r bud c e l l s studied and t h i s p e c u l i a r i t y would require further study. 37 ' F i g . 1. Normal and aberrant one-year-old s e e d l i n g s : a, b, aberrant s e e d l i n g s ; c, d, normal seedlings F i g . 2. Normal and aberrant two-years-old seed l i n g s : a, d, normal seedl i n g s ; b, c, aberrants 38 F i g . 3 otera d i f f e r e n c e s i n aberrant and normal seedlings a, normal s e e d l i n g ; b, aberrant s e e d l i n g . (Note d i f f e r e n c e i n green hue of the needles and i n the shape of buds) F i g . A Aberrant se e d l i n g w i t h a normal branch: a, normal branch; b, c, aberrant branches F i g . 6. Prometaphase i n a normal s e e d l i n g . (X23C0) F i g . 7. Metaphase i n an a b e r r a n t s e e d l i n g . (X3000) F i g . 8. Metaphase (polar view) i n a normal s e e d l i n g . (X2500) hi * i g . 10. ^ a r l y anaphase i n a normal s e e d l i n g . (X2900) P i g . 12. Metaphase i n a normal tv.dg on an aberrant seed l i n g . (X1350) F i g . 16. fro-metaphase i n a normal seedling. (X2900) F i g . 17. Telophase ( t i l t e d p o l a r view) i n an aberrant s e e d l i n g . ( X 2 0 2 0 ) F i g . 18. Telophase ( t i l t e d p o l a r view-' i n a normal seed l i n g . (X1950) 2 0 . Telophase i n a normal s e e d l i n g . (X2730) F i g . 22. Telophase i n a normal s e e d l i n g . (X1900) »+8 BIBLIOGRAPHY A l l e n , G. S., 19*+2. Parthenocarpy, parthenogenesis, and s e l f - s t e r i l i t y of Douglas f i r . Jour. Forestry A l l e n , G. S., 19*+6. Emhryogeny and development of the api c a l meristem of Pseudotsuga. Amer. Jour. Botany 3 3 : 6 6 6 - 6 7 6 Andersson, E., 19*+7» A case of asyndesis i n Picea Abies Hereditas 33:301-3 1 +7 Bergstrom, I., 19*+0. On the progeny of d i p l o i d and t r i  p l o i d Populus tremula with sp e c i a l reference to the occurrence of tetraploidy. Hereditas 2 6 : 1 9 1 - 2 0 1 Buckland, D. C. and J . K u i j t , 1957 . Unexplained brooming of Douglas f i r and other conifers i n B r i t i s h Columbia and Alberta. Forest Science 3:236-2,1+3 Bowden, W. M., 19*+0. Diploidy, polyploidy, and winter hardiness i n flowering plants. Amer. Jour. Botany 2 7 : 3 5 7 - 3 7 1 Chiba, S., 1950 . T r i p l o i d s and tetraploids of Sugi (Crypt- omeria .japonica D. Don) coll e c t e d i n the forest nursery. B u l l . Govt. For. Expt. Sta., Meguro, Japan Chiba, S. and M. Watanabe, 1952 . Tetraploids of Larix kaempferi i n the nurseries. B u l l . Govt. For. Expt. Sta., Meguro, Japan Chiba, S. and M. Watanabe, 1952b . T\etraploids of Larix l e p t o l e p i s that have appeared i n the nurseries. Jour. Jap. For. Soc. 3>+: 276-278 Christiansen, H. A., 1952 Tetraploid Larix decidua M i l l e r Danske Vidensk Selsk B i o l . Medd. 18 Dallimore, W. and A. R. Jackson, 19*+8. A handbook of coniferae. London, Edward Arnold & Co. Darlington, C. D., 1929* Meiosis i n polyploids. Jour. Genetics 2 1 : 1 7 - 5 6 Darlington, C. D., 195 l« Recent advances i n cytology. P. Balkinson & Co., Philadelphia. Darlington, C. D., 1956 . Chromosome botany. A l l e n & Unwin, London Darlington, C. D. ;and L. F. La Cour. (2nd e d i t i o n ) , 1950 Handling of chromosomes. London, A l l e n & Unwin Dark, S. 0 . S., 1938 . Chromosomes of Taxus, Sequoia and Cryptomeria. Ann. of Bot. ^ 6 : 9 6 5 - 9 7 7 D u f f i e l d , J . W., 1 9 ^ 2 . The c y t o l o g i c a l basis of f o r e s t tree improvement. Jour. Forestry I f 0 : 859 -86 lf D u f f i e l d , J . W., 1 9 5 ^ . The importance of species hybrid i z a t i o n and polyploidy i n f o r e s t tree improvement. Jour. Forestry 52:6*+5-6*1-6 E i g e s t i , 0 . J . 19*+6. Literature on c o l c h i c i n e . Evanston, I l l i n o i s Faberge, A. C , 1936 . The physiological sequence of polyploidy. Jour. Genetics 3 3 * 3 8 3 - ^ 0 0 Flory, W. S., 1936 . Chromosome number and physiology i n the gymnosperms. Jour. Arn. Arb. 1 7 : 8 3 - 8 9 Goodspeed, T. H. and M. P. Carney, 1920 . Chromosome number i n the Sequoias. Bot. Gazette 69:3 l f8-3 1 +9 Gyorffy, B., l^kO. Die colchicinemethode zur erzeugung polyploider pflanzen. Der Zuchter 1 2 : 1 3 0 - 1 ^ 9 5Q Haddock, P. G., 1951+. Sapling sugar pines grown from excised embryos. Jour. Forestry 52:LK3 l+-1+37 Hunter, A. ¥. S., 19^-0. Tetraploidy i n vegetative shoots of apple. Jour. Heredity 1+5:130-l 1+9 Hutchinson, A. H., 1915 . F e r t i l i z a t i o n i n Abies balsamea Bot. Gaz. 60:>+57-1+72 Hyun, K. S., 195*+. Induction of polyploidy i n pines by means of col c h i c i n e . Z. Forstgenetik 3 * 2 5 - 3 3 l i l i e s , Z. M., 1952 . Colchicineversuche an Larix decidua M i l l e r und Picea abies (L) Karst. Z. Forstgenetik 1:58 Isaac,Leo A., 19*+9. Better Douglas f i r forests from better seed. University of Washington Press, Seattle Johansen, D. A., 19*+0. Plant microtechnique. McGraw H i l l , London Johnsson, H., 19*+0. Cytological studies on d i p l o i d and t r i p l o i d Populus tremula and crosses between them. Hereditas 2 6 : 3 2 1 - 3 5 2 Johnsson, H., 19*+2. Cytological studies of t r i p l o i d progenies of Populus tremula. Hereditas 2 8 : 3 0 6 - 3 1 2 Johnsson, H., 19*+6. Progeny of t r i p l o i d Betula verucosa Botaniska Notiser 2 : 2 8 5 - 2 9 0 Johnsson, H., 1950 . On the Co and CI generation i n Alnus glutinosa. Hereditas 3 6 : 2 0 5 - 2 1 9 Johnsson, H., 1 9 5 3 . Development of Populus tremula during the juvenile period. Z. Forstgenetik 2 : 7 3 - 7 7 Johnsson, H., 1956 . Auto and a l l o t r i p l o i d Betula families derived from colchicine treatment. Z. Forstgenetik 5 : 6 5 - 7 0 51 Johnsson, H. and C. Eklundh, 1 9 ^ 0 . Colchicinbehandling som metod v i d vaxtforandling som metod vid vaxt- forandling av lovtrad (Summary i n English). Svensk Papperstinding Medd.fr. Foren. Skogstrad Johnson, L.B. and H.W. Holtz, 191+6. Colchicine treatment for sprouted seeds and seedlings. Can. Jour. Res. Coun. 2 ^ : 3 0 3 - 3 0 ^ Kalmus, H., 195*+. Genetics. Pelican Books, London Kanezawa, R., 1 9 ^ 9 . A l i s t of chromosome numbers i n woody plants. La Kromosomo 5:2^+9-260 Kanezawa, R., 1951* Induced tetraploidy i n Japanese cypress (Chamaecyparis obtusa Endl.). B u l l . Tokyo Univ. For. 3 9 : 2 1 - 3 0 Kanezawa, R.,and M. Go, 1 9 ^ 8 . On the morphological changes i n some polyploid forest trees which were induced by colchicine treatments. Jap. Jour. Genet. 2 3 : 7 7 - 8 1 Khan, M.I., 1955 . Forest tree breeding. Z. Forstgenetik ^f: 2 1 - 2 5 Kiellander, C.I., 19^9» Demonstrations of the conifer department. Proc. 8 t h Int. Cong. Genet. Hereditas Spp. Vol. I9I+9 Kiellander, C.I., 1 9 5 0 . Polyploidy i n Picea abies Hereditas 3 6 : 5 1 3 - 5 1 6 Kiellander, C.I., 1957'• Private communication dated March 3 , 1957 Larsen, Syrach C., .1956. Genetics i n s i l v i c u l t u r e . Oliver and Boyd Larsen, Syrach C. and M. Westergaard, 1938 . Contributions to the cytology of forest trees. I. T r i p l o i d hybrid between Larix decidua M i l l e r & L. occidentalis Nutt. Jour. Genet. 3 6 : 5 2 3 - 5 3 0 Long, F.A. and F.E.. Clements, 193*+. The method of collodion films for stomata. Amer. Jour. Bot. 2 1 : 7 - 1 7 Love, A. and D. Love, 1957 . A r c t i c polyploidy. Proc. Genetic Soc. Can. 2 : 2 3 - 2 7 Marth, P.C. and W.V. Audia, 1 9 5 6 . G i b e r e l l i c Acid-a plant regulator'.' Hort. Cr.^Hes. Br. #6 U.S. Dept. Agr. B e l t s v i l l e , Maryland Mehra, P.N.. and T.N. Khoosho, 1 9 5 5 . Cytology of conifers Jour.. Genet. 5 L M l 6 5 - l 8 5 Mirov, N.T. and. P. Stockwell, 1 9 3 9 . Colchicine t r e a t  ment of pine seeds. Jour. Hered. 3 0 : 3 8 9 - 3 9 0 s Mergen, F. and H.M. Novotny, 1957* Squah technique for .chromosome studies i n pine needles and root t i p s of slash pine. Forest Science 3556-60 Meyer, H., 1 9 5 1 . Aufgeben und wege der Douglasien zuchtung. Aig, F o r s t z e i t . 6 : 2 8 1 - 2 8 3 Mikelson, K., 1952 . 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Cytology of poplar species and natural hybrids. Can. Jour. Res. Council l6: Lf L f5- 1+55 Sato, K., 19*1-9. Tree breeding. Tokyo. (vide Khan i n Z, Forstgenetik k:21-25 Sax, H. J . , 1932 . Chromosome pairing i n Larix species. Jour. Arn. Arb. 1 3 : 3 6 8 - 3 7 3 Sax, H. J . , 1938 . The r e l a t i o n between stomata counts and chromosome number. Jour. Arn. Arb. 19:*+37- l f^l Sax, K., 1936 . The experimental production of polyploidy. Jour. Arn. Arb. 1 7 : 1 5 3 - 1 5 8 Sax, H. J. and K. Sax, 1933* Chromosome number and morphology i n the conifers. Jour. Arn. Arb. l*f:356-375 Sax, H. J . and K. Sax, 1937 . Stomata size and d i s t r i b u t i o n i n d i p l o i d and polyploid plants. Jour. Arn. Arb. I8 : l 6*f - 173 Sakai, K. I. and Y. Suzuki, 1955a . Competition between d i p l o i d and autotetraploid plants of barley. Jour. Genetics 5 3 : 1 1 - 2 0 Sakai, K. I. and Y. S u z u k i , 1955b . Competition between all o p o l y p l o i d s and t h e i r d i p l o i d parents. Jour. Genetics 5 3 : 5 8 5 - 5 9 0 S e i t z , F. ¥., 195k. Uber das autrefen von t r i p l o i d e n nach der selbstung anomaler zwitterbluten einer grupappel- form. Z. Forstgenetik 3 : 1 - 6 S e i t z , F. W. , 1952 . Chromosomevenzahlerhaltjnisse bei holz pflanzen. Z. Forstgenetik 1 : 22 -32 Simamura, T., 1938 . C y t o l o g i c a l studies of polyploidy induced by c o l c h i c i n e . Cytologia 9:1+86-1+91+ Soegard, B., 1957 . Hersholm Arboretum, Denmark. Private communication dated Jan. 2 3 , 1957 Stebbins,G. L., 19*+7. Types of polyploid: t h e i r c l a s s  i f i c a t i o n and s i g n i f i c a n c e . Adv. Genetics l : 1+03- l+29 Stebbins,G. L., 191+8. The chromosomes and rel a t i o n s h i p of Meta-sequoia and Sequoia. Science 1 0 8 : 9 5 - 9 8 Swaminathan, M. S., 1 9 5 2 . Polyploidy and plant breeding. Hew Siology 13:31-*+8 Upcott, M., 1936 . The parents and progeny of Aesculus carnea. Jour. Genetics 33:135-l 1 +9 Walker, D. R. and C. W. Donoho, 1957 . E f f e c t of G i b e r e l l - i c acid on breaking of rest period i n Elberta peaches. Science 126 :1178 -1179 55 Wetts-tein, F., 1927. Die erscheinung der heteroploidie, besonders i n pflanzenreich. Ergebniss der Biologie Bd. 2:311-356 Zinnai, I., 1953* The morphological character and the f e r t  i l i t y of the pollen of Japanese red pine induced by the colchicine method. Jour. Jap. For. Soc. 35:2H-5r-21+8 Zinnai, I., 1957. Colchicine induced polyploidy innsome coniferous trees. B u l l . Tokyo Univ. Forests 35:232-233 Zinnai, I., 1955. Frequency of occurrence of polyploid Cryptomeria i n forest nursery. Jour. Jap. For. Soc. 37:513-51^ Zenke, U., 1953* Untersuchen uber den ablauf der Meiosis bei Pseudotsuga t a x i f o l i a B r i t t o n . Z. Forstgenetik 2:96-106 

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