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Physiology of the terminal bud of Vaccinium macrocarpon Ait. cultivar McFarlin in relation to winter… Eady, Francis Charles 1971

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PHYSIOLOGY OF THE TERMINAL BUD OF VACGINIUM MAGROGARPON AIT. CULTIVAR McFARLIN IN RELATION TO WINTER DORMANCY by FRANCIS CHARLES EADY B.S.A., U n i v e r s i t y of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of P l a n t Science We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1971 In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ivers i t y of B r i t i s h Columbia, I agree that the L ib ra ry sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th i s thes is fo r s cho la r l y purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l i ca t i on o f th i s thes is fo r f i nanc i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department of P l a n t Science The Univers i ty o f B r i t i s h Columbia Vancouver 8, Canada Date A p r i l 23, 1971 i i ABSTRACT A study of the r e l a t i o n s h i p between the wi n t e r dormant p e r i o d and sub-sequent growth and development i n Vaccinium macrocarpon A i t . c u l t i v a r Mc-F a r l i n was c a r r i e d out. The main o b j e c t i v e s of the study were t o ob t a i n data on t h i s r e l a t i o n s h i p , t o i n v e s t i g a t e the p o s s i b l e r o l e of g i b b e r e l l i n s i n the i n d u c t i o n of f l o w e r i n g , t o c o r r e l a t e the data w i t h development of the t e r m i n a l bud under f i e l d c o n d i t i o n s , and t o provide b a s i c i n f o r m a t i o n r e l a t i n g t o the problem of f r o s t i n j u r y . C o n t r o l l e d environment f a c i l i t i e s were used t o i n v e s t i g a t e the e f f e c t of c h i l l i n g on the subsequent growth and development of the t e r m i n a l bud. Exposure t o an accumulation of c h i l l i n g temperatures below 7°C was r e q u i r e d t o break dormancy of the t e r m i n a l bud. Longer p e r i o d s of c h i l l i n g were needed t o induce flower development. The f a s t e r r a t e of v e g e t a t i v e response was apparently due t o development of the p l a n t d u r i n g the warmer p o r t i o n of the d a i l y temperature c y c l e . The best responses were obtained when the c h i l l i n g c o n d i t i o n s i n c l u d e d a p e r i o d of approximately 10 eC during the day. The f l o r a l p r i m o r d i a of t e r m i n a l buds i n the f i e l d recommenced d e v e l -opment i n l a t e February or e a r l y March, and were w e l l d i f f e r e n t i a t e d by the end of A p r i l under B r i t i s h Columbia c o n d i t i o n s . A p p l i c a t i o n of g i b b e r e l l i c a c i d t o dormant, u n c h i l l e d t e r m i n a l buds s t i m u l a t e d only v e g e t a t i v e growth. G i b b e r e l l i n i s probably not i n v o l v e d i n the i n d u c t i o n of f l o r a l development. Cold methanol e x t r a c t i o n of g i b b e r e l l i n - l i k e substances and subsequent p u r i f i c a t i o n was c a r r i e d out on the leaves and t e r m i n a l buds of V. macro- carpon grown i n the f i e l d . F r a c t i o n s of 0.1 were scraped from t h i n -l a y e r chromatography sheets and bioassayed f o r g i b b e r e l l i n - l i k e a c t i v i t y u s i n g a l e t t u c e hypocotyl bioassay. An increase i n such a c t i v i t y on March 9, 1970 was observed f o r both buds and lea v e s . On A p r i l 6, the a c t i v i t y i i i i n the l e a v e s was c o n s i d e r a b l y reduced but the bud l e v e l was i n c r e a s e d , thus suggesting a t r a n s l o c a t i o n of g i b b e r e l l i n - l i k e substances from the leaves t o the t e r m i n a l buds. Some p o s s i b l e i m p l i c a t i o n s of the study i n r e l a t i o n t o f r o s t i n j u r y were dis c u s s e d . The c h i l l i n g requirement may be a f a c t o r i n determining both the southern and northern l i m i t s of the geographic range of the s p e c i e s . i v TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 5 P l a n t M a t e r i a l . ••• •••• 5 Anatomy of the Terminal Bud. . 5 F i e l d Temperature 6 C o n t r o l l e d Environment S t u d i e s . . . . . 6 1968- 1969 Experiment 6 1969 (Summer) Experiment 7 1969- 1970 Experiment 9 G i b b e r e l l i c A c i d Experiment 13 G i b b e r e l l i n - l i k e A c t i v i t y 13 E x t r a c t i o n , P u r i f i c a t i o n and Separation of G i b b e r e l l i n - l i k e Compounds. • 13 Bioassay. .x.... • • 16 G i b b e r e l l i n Recovery. * • • 16 RESULTS 17 Anatomy of the Terminal Bud, 17 F i e l d Temperature. 17 C o n t r o l l e d Environment S t u d i e s . 20 1968- 1969 Experiment 20 1969 (Summer) Experiment • 20 1969- 1970 Experiment 23 G i b b e r e l l i c A c i d Experiment 30 G i b b e r e l l i n - l i k e A c t i v i t y 30 DISCUSSION.... 36 SUMMARY 5^ LITERATURE CITED ^8 APPENDIX 1. L i g h t Sources of C o n t r o l l e d Environment F a c i l i t i e s 52 APPENDIX 2. A n a l y s i s of Variance 5^ APPENDIX 3. Dry Weights and Minimum Detectable L e v e l s of G i b b e r e l l i n -l i k e A c t i v i t y i n Terminal Bud and Leaf Samples...... 62 v i LIST OF TABLES Table Page 1 Treatment Durations of C o n t r o l l e d Environment Experiments....... 8 2 F i e l d Temperatures f o r 1969-1970 21 3 Mean Number of Days t o Bud Break, Shoot Length, and Flowers per P l a n t i n V, macrocarpon, 1968-1969 C h i l l i n g Treatments 22 k Number of P l a n t s I n i t i a t i n g New Terminal Growth F o l l o w i n g Completion of C h i l l i n g Treatments, 1969 (Summer) Experiment 23 5 Average Percent Bud Break of V. macrocarpon, 1969-1970 C h i l l i n g Treatments Zk 6 Growth Rates (cm/day) f o r the L i n e a r P o r t i o n of the Growth Curve of V . macrocarpon, 1969-1970 C h i l l i n g Treatments 29 7 Mean Shoot Length (cm) of V, macrocarpon 100 Days a f t e r Removal from 1969-1970 C h i l l i n g Treatments 31 8 Mean Number of Flowers per 20 P l a n t s , 1969-1970 C h i l l i n g Treatments 31 v i i LIST OF FIGURES Figure Page 1 D a i l y Temperature Curves f o r C h i l l i n g Regimes, 1969-1970 C o n t r o l l e d Environment Experiment 11 2 Flow Diagram f o r E x t r a c t i o n , P u r i f i c a t i o n and Separation of G i b b e r e l l i n - l i k e Compounds from P l a n t T i s s u e . . . . . . . . . . . . . . . . . . . 15 3 Vaccinium macrocarpon Flower Bud ( l . s . ) t o show Winter Dormant Stages 18 4 Vaccinium macrocarpon Flower Bud ( l . s . ) t o show Stages of S p r i n g Development ................................... • 19 5 Number of Days t o Bud Break F o l l o w i n g Termination of C h i l l i n g Treatments, Regimes 1969-1970 C o n t r o l l e d Environment Experiment 26 6 Shoot Growth F o l l o w i n g Termination of C h i l l i n g Treatments, 1969-1970 C o n t r o l l e d Environment Experiment 27 7 G i b b e r e l l i n - l i k e A c t i v i t y of Substances E x t r a c t e d from Leaves and Buds of V. macrocarpon. 33 8 Histograms of G i b b e r e l l i n - l i k e A c t i v i t y of Substances E x t r a c t e d from Leaves and Buds of V, macrocarpon 35 ACKNOWLEDGEMENTS I wish t o thank my research s u p e r v i s o r , Dr, G.W, Eaton, f o r h i s support of t h i s work and h i s encouragement of independent re s e a r c h , I am a l s o g r a t e f u l f o r the help provided by the f o l l o w i n g members of my t h e s i s committee: Dr. V.C. B r i n k , Dr, A. Kozak, Dr. J.R. Maze, Dr. V.C. Runeckles, Dr. J.G. W o r r a l l . The i n t e r e s t and a s s i s t a n c e of Dr. R.P. P h a r i s and h i s a s s o c i a t e s a t the U n i v e r s i t y of Calgary w i t h r e g a r d t o the a n a l y s i s of p l a n t m a t e r i a l f o r endogenous g i b b e r e l l i n - l i k e a c t i v i t y i s g r a t e f u l l y acknowledged. I wish t o thank Mr. J . Thomas and Mr. F. Shaw of B i g Red Cranberry Co., Richmond, B.C. f o r s u p p l y i n g the cranberry p l a n t s used i n these experiments. The t e c h n i c a l a s s i s t a n c e of Mrs. A. Gammel and Miss C. Meehan i s g r e a t -l y a p p r e c i a t e d . I a l s o wish t o acknowledge the support, encouragement, t e c h n i c a l h e l p , and the t y p i n g of t h i s t h e s i s by my w i f e Karen. The f i n a n c i a l support f o r t h i s p r o j e c t was provided by a N a t i o n a l Research C o u n c i l of Canada operating grant A-2023 made t o Dr. G.W. Eaton, a N a t i o n a l Research C o u n c i l of Canada Bursary, and teac h i n g a s s i s t a n t s h i p s from the Department of P l a n t Science. 1 INTRODUCTION The commercial cranberry, Vacciniuro macrocarpon A i t . , i s a low growing p e r e n n i a l woody v i n e w i t h p e r s i s t e n t l e a v e s . The stems, or runners, range from 0.3 t o gr e a t e r than 6 m i n l e n g t h . Short v e r t i c a l branches, commonly c a l l e d u p r i g h t s , are produced from the runners. Most of the f r u i t i s borne on the u p r i g h t s . A mixed bud forms a t the apex of the upri g h t during the summer. These t e r m i n a l buds undergo some d i f f e r e n t i a t i o n before the onset of w i n t e r dormancy. Under normal c o n d i t i o n s , they complete t h e i r develop-ment the f o l l o w i n g s p r i n g , producing new stems, leaves and f l o w e r s . The anatomy of the t e r m i n a l bud of V. macrocarpon has been s t u d i e d by s e v e r a l authors. Flower bud pri m o r d i a have been d i s t i n g u i s h e d as e a r l y as J u l y 29 under f i e l d c o n d i t i o n s i n Wisconsin (Roberts and Struckmeyer, 19^8). Development of these p r i m o r d i a continued u n t i l the onset of win t e r dormancy. Dormant cranberry f l o w e r buds c o l l e c t e d from Nova S c o t i a ( B e l l and B u r c h i l l , 1955). Massachusetts ( L a c r o i x , 1926), and Wisconsin (Goff, 1901) were r e -p o r t e d as having d i s c e r n i b l e c a l y x and p e t a l s w i t h r e c o g n i z a b l e , but r u d i -mentary, stamen and c a r p e l p r i m o r d i a . I n most of the major cranberry grow-i n g areas, i t i s apparent t h a t the f l o w e r buds overwinter i n a somewhat u n d i f f e r e n t i a t e d stage. A general review of experimental s t u d i e s of the shoot apex and shoot morphogenesis, i n c l u d i n g the e f f e c t of g i b b e r e l l i n s , has been done by Cut t e r (1965). Growth and development of meristems i n woody p l a n t s have been reviewed by Romberger (1963). The f l o r a l morphology of V. macrocarpon i n r e l a t i o n t o other members of the E r i c a l e s has been discussed by P a l s e r ( l 9 6 l ) . Flower numbers and f r u i t set of M c F a r l i n c r a n b e r r i e s have been recorded f o r B r i t i s h Columbia ( G a r l i c k , 1966), Massachusetts (Bergman, 1950), 2 New Jersey (Bergman, 195*0, and Wisconsin ( B a i n , 19^). There has been considerable i n t e r e s t i n the r e l a t i o n s h i p between w i n t e r c o n d i t i o n s and f r u i t p r o duction and q u a l i t y . Bergman (19^7, 19^9, 1950) s t u d i e d the problem of w i n t e r i n j u r y and concluded t h a t much of the i n j u r y was a r e s u l t of oxygen d e f i c i e n c y caused by the c u l t u r a l p r a c t i c e of f l o o d -i n g the bogs. Gross (1969) r e p o r t e d t h a t the t e r m i n a l buds had t h e i r best growth d u r i n g the months of September and October, but d i d not d i s c u s s bud anatomy. He a l s o r e p o r t e d a strong c o r r e l a t i o n between January sunshine and berry s i z e , which he suggested i s r e l a t e d t o the oxygen d e f i c i e n c y prob-lem. Doughty* approached the problem of winter i n j u r y on a degreerhour b a s i s . He r e p o r t e d t h a t new growth may be i n i t i a t e d a f t e r kOO hr below ^.5°C under Washington St a t e c o n d i t i o n s . Growth and development of V. macrocarpon i n d i r e c t r e l a t i o n t o the wi n t e r r e s t requirement were i n v e s t i g a t e d by Chandler and Demoranville (196*0. They r e p o r t e d t h a t below 7°C 2500 hr of c h i l l i n g were necessary t o f u l f i l the r e s t p e r i o d requirement of M c F a r l i n cranberry p l a n t s , w h i l e l e s s c h i l l i n g (1500 hr) r e s u l t e d i n abnormal v e g e t a t i v e growth of the t e r m i n a l shoot. The present author^ found t h a t a t 5-6°C under nine hour photoperiods, 650 hr were s u f f i c i e n t f o r the i n d u c t i o n of normal growth of the t e r m i n a l shoot. Long photoperiods have been found t o cause i n c r e a s e d v e g e t a t i v e growth of u n c h i l l e d cranberry p l a n t s , but f l o w e r bud d i f f e r e n t i a t i o n was re p o r t e d as r a r e (Eaton and Ormrod, 1968), The l i t e r a t u r e r e l a t i n g t o the dormancy of woody p l a n t s has r e c e n t l y 1 C.C, Doughty, p e r s o n a l communication, 1971. 2 The work p u b l i s h e d by Eady and Eaton (1969) forms a p a r t of t h i s t h e s i s . 3 been reviewed ( P e r r y , 197l). Other reviewers have d i s c u s s e d the subject of dormancy and v e r n a l i z a t i o n of p l a n t s i n d e t a i l ( H i l l m a n , 1969; Wareing, 1969; Lang, 1965; Vegis, 1964; Ghouard, I960; Samish, 195^5 Doorenbos, 1953). The morphogenetic e f f e c t s of the g i b b e r e l l i n s have been di s c u s s e d by B r i a n ( l959)» who noted t h a t " g i b b e r e l l i n w i l l u s u a l l y r e p l a c e v e r n a l i z a t i o n and a l l o w development t o proceed normally i n p l a n t s kept i n long day photo-p e r i o d s . " G i b b e r e l l i c a c i d has been found t o overcome the dormancy of buds i n many t r e e species (Eagles and Wareing, 1963). Smith and K e f f o r d (1964) have suggested t h a t completion of c h i l l i n g f o l l o w e d by long days s t i m u l a t e the production of g i b b e r e l l i n s which are i n v o l v e d i n r e l e a s e from dormancy. "The p o s s i b l e r o l e of n a t i v e g i b b e r e l l i n s i n dormancy must be i n v e s t i g a t e d by determining the g i b b e r e l l i n content of dormant and non-dormant t i s s u e " (Wareing, 196l). G i b b e r e l l i c a c i d a p p l i e d t o V. macrocarpon during and a f t e r bloom caused a s i g n i f i c a n t i n c r e a s e i n f r u i t set but a l s o i n t e r f e r e d w i t h t e r m i n a l bud development ( D e v l i n and Demoranville, 1967). However, the l a t t e r pheno-menon was r e p o r t e d t o have r e s u l t e d i n a marked r e d u c t i o n i n f l o w e r i n g i n the year a f t e r a p p l i c a t i o n (Mainland and Eck, 1968), The importance of the wi n t e r dormant p e r i o d t o the o v e r a l l growth and development of V, macrocarpon i s g e n e r a l l y recognized, but the a c t u a l e f f e c t of the dormant p e r i o d has been l i t t l e s t u d i e d . The o b j e c t i v e s of the pres-ent study are: l ) t o i n v e s t i g a t e the e f f e c t of winter c o n d i t i o n s on the growth and subsequent development of the t e r m i n a l bud through the use. of c o n t r o l l e d environment f a c i l i t i e s , 2) t o i n v e s t i g a t e the p o s s i b l e r o l e of g i b b e r e l l i n i n the i n d u c t i o n of f l o w e r i n g , 3) t o c o r r e l a t e the above data w i t h t e r m i n a l bud development under f i e l d c o n d i t i o n s , and 4) t o provide some b a s i c i n f o r m a t i o n r e l a t i n g t o the problem of f r o s t i n j u r y . In order t o make maximum use of the a v a i l a b l e f a c i l i t i e s , the present study was co n f i n e d t o one c u l t i v a r of V. macrocarpon. The use of a d d i t i o n a l c u l t i v a r s would have r e s u l t e d i n a r e d u c t i o n of e i t h e r treatments or r e p l i c -a t i o n s . The c u l t i v a r M c F a r l i n was chosen because i t comprises 65 percent of the B r i t i s h Columbia p l a n t i n g s (Eaton, 1970) and t h e r e f o r e i s both commer c i a l l y important and r e a d i l y a v a i l a b l e . Because of v a r y i n g usage, i t i s necessary t o d e f i n e some of the terms as used i n t h i s - study. Dormancy i s a "general term f o r a l l i n s t ances i n which a t i s s u e predisposed t o elongate (or grow i n some other manner) does not do so" (Romberger, 1963). C h i l l i n g requirement i s used t o r e f e r t o a need f o r the p l a n t or organ t o be exposed t o a p e r i o d of low temperature before the c o n t i n u a t i o n of normal development f o l l o w i n g the onset of dor-mancy. There has been considerable v a r i a t i o n i n the use of the term v e r n a l i z a t i o n . Many workers favour l i m i t i n g the term t o apply only t o low temperature promotion of f l o w e r i n g ( S a l i s b u r y , 1963); t h i s i s the usage adopted i n the present study. 5 MATERIALS AND METHODS P l a n t M a t e r i a l Vaccinium macrocarpon A i t . c u l t i v a r M c F a r l i n was used i n a l l of the f o l l o w i n g experiments. The p l a n t s were obtained from a commercial p l a n t i n g ^ i n Richmond, B r i t i s h Columbia. T h i s p l a n t i n g was e s t a b l i s h e d i n 1955 u s i n g c u t t i n g s of M c F a r l i n p l a n t m a t e r i a l obtained from the Greyland area of Washington S t a t e , and has been maintained i n commercial p r o d u c t i o n s i n c e t h a t time. M c F a r l i n cranberry v i n e s were o r i g i n a l l y a s e l e c t i o n from the w i l d made by Thomas H. M c F a r l i n i n 1874 a t a n a t u r a l bog near South Carver, Mass-ach u s e t t s . I n 1885, Charles Dexter M c F a r l i n , h i s b r o t h e r , e s t a b l i s h e d the f i r s t commercial cranberry p l a n t i n g near Coos Bay, Oregon u s i n g mainly Mc-F a r l i n v i n e s (Chandler and Demoranville, 1958). The commercial p r a c t i c e i s t o propagate cranberry v i n e s by c u t t i n g s , which r o o t e a s i l y ; thus i n the absence of d e t a i l e d taxonomic or gen e t i c s t u d i e s , i t i s assumed t h a t the present p l a n t m a t e r i a l i s a reasonably d i r -e c t descendent of the o r i g i n a l s e l e c t i o n , although some s e e d l i n g mixtures are p o s s i b l e . Anatomy of the Terminal Bud One hundred s i x t y - t w o buds, c o l l e c t e d between January and June 1969, and 53 buds c o l l e c t e d between October 1969 and June 1970 were f i x e d , s e c t -ioned, s t a i n e d and examined. The f i r s t s e t of buds were f i x e d i n a f o r m a l i n , a c e t i c a c i d , a l c o h o l s o l u t i o n (FAA); those of the second set were f r e e z e - d r i e d . Dehydration and 1 B i g Red Cranberry Co., Richmond, B r i t i s h Columbia. 6 p a x a f f i n i n f i l t r a t i o n were done according t o Jensen (1962). The m a t e r i a l was s e c t i o n e d a t a t h i c k n e s s of 10 microns. The s t a i n i n g procedure f o r a l l s e c t -ions was t h a t d e s c r i b e d by .Sharman (1943), using t a n n i c a c i d and i r o n alum w i t h s a f r a n i n and orange G. A f t e r s t a i n i n g , the s e c t i o n s were examined and compared on the b a s i s of gross anatomical d i f f e r e n c e s . F i e l d Temperature Temperatures i n the f i e l d from which the p l a n t s were c o l l e c t e d were recorded u s i n g a Short and Reed thermograph of the revolving-drum type. The instrument was p l a c e d i n a Stevenson screen l o c a t e d d i r e c t l y on the bog s u r f a c e ; the sensor was thus l o c a t e d approximately at the l e v e l of the t e r m i n a l buds. C o n t r o l l e d Environment St u d i e s Three set s of experiments were done u s i n g a v a r i e t y of c o n t r o l l e d environment f a c i l i t i e s . The d e t a i l s f o r the l i g h t i n g systems of these f a c i l i t i e s are d e s c r i b e d i n Appendix 1. 1968-1969 Experiment Budded u p r i g h t s were c o l l e c t e d on October 4, 1968. Each u p r i g h t was trimmed 10 cm below the bud. The lower 4 cm of leaves were removed and the p l a n t s set i n commercial peat moss. The c o l l e c t i o n was maintained i n the greenhouse under a m i s t i n g system u n t i l November 20, 1968 when the p l a n t s were t r a n s p l a n t e d t o 15 cm pots, one p l a n t per pot. These remained on a greenhouse bench u n t i l i n i t i a t i o n of treatments on December 18, 1968. A s e l e c t i o n of uniform p l a n t s were then assigned treatment numbers and 7 a l l o c a t e d random p o s i t i o n s i n the growth chambers. Treatments were a p p l i e d i n two Sherer Model CEL-266-6 growth chambers us i n g one-half l i g h t i n g f o r a nine hour photoperiod. S e l e c t i o n of the c h i l l -i n g temperature was based on the observation of Chandler and Demoranville (1964) t h a t V. macrocarpon responded t o temperatures below 7°C. A l i m i t a t -i o n on the d u r a t i o n of the treatment temperature was imposed by the f a c t t h a t the chambers were not designed f o r continuous low temperature o p e r a t i o n . Treatment temperature was e s t a b l i s h e d as 20 hr a t 5-6°C w i t h a 4 hr midday p e r i o d of 13°C t o a l l o w the machinery time t o d e f r o s t . Treatment d u r a t i o n s are l i s t e d i n Table 1 . F o l l o w i n g the treatment p e r i o d s , the p l a n t s were t r a n s f e r r e d t o another Sherer growth chamber w i t h 15 hr of f u l l l i g h t per day. The temperature was maintained a t 19°C day and 13°C n i g h t . Frequent i n s p e c t i o n s were made t o r e c o r d the i n i t i a t i o n of new shoot growth, f o l l o w i n g which shoot l e n g t h was recorded. Numbers of f l o w e r s were a l s o recorded. T h i s experiment was set up as a randomized complete b l o c k design w i t h each of treatments 2-6 r e p l i c a t e d t h ree times i n each chamber. The s i x untreated p l a n t s c o u l d not be i n c l u d e d i n t h i s design as they had not been exposed t o e i t h e r of the chambers ( b l o c k s ) . When the data were analysed u s i n g the completely random design w i t h s i x treatments and s i x r e p l i c a t e s f o r a t o t a l of 36 p l a n t s , the un t r e a t e d set c o u l d be i n c l u d e d . In a l l the experiments, Duncan's new m u l t i p l e range t e s t was used t o compare mean values when the F value i n the a n a l y s i s of v a r i a n c e was s i g n i -f i c a n t . 1969 (Summer) Experiment The p l a n t s used i n these experiments were from the same c o l l e c t i o n as f o r the previous experiment. They had been maintained i n the greenhouse a t 8 Table 1 . Treatment Durations of Controlled Environment Experiments Experiment 1968-1969 1969 (Sumner) Treatment No, 1 (cont ro l ) 2 3 4 5 6 1 (contro l ) 2 3 4 5 6 7 Days Treated 0 33 65 65 105 125 0 15 33 65 85 105 125 Aecunulateij hr' belo i 7 C 0 650 1300 1700 2100 2500 0 300 650 1300 1700 2100 2500 1969-1970 Regius 1 Regime 2 Regius 3 Regius 4 RegUs S 1 (cont ro l ) 2 3 4 5 6 7 1 (cont ro l ) 2 3 4 5 6 7 1 (cont ro l ) 2 3 4 5 6 1 (contro l ) 2 3 4 5 6 1 (cont ro l ) 2 3 4 5 6 0 25 50 75 100 125 150 0 25 50 75 100 125 150 0 25 50 75 100 125 0 25 50 75 100 125 0 25 50 75 100 125 0 175 350 525 700 875 1050 0 194 387 581 775 969 1162 0 262 525 787 1050 1312 0 600 1200 1803 2400 3000 0 0 0 0 0 0 1 Conditions and treatments lers the sane for the l i g h t and dark experiments. 9 temperatures above 18°G from November 20, 1968 u n t i l June 20, 1969 when they were p l a c e d i n t o the treatment chambers. Immediately p r i o r t o being p l a c e d i n the chambers, a l l l a t e r a l growth was removed from the p l a n t s . Treatment c o n d i t i o n s were e x a c t l y the same as i n the previous experiment except t h a t one of the chambers was operated without l i g h t s , thus p r o v i d i n g a dark treatment. Treatment d u r a t i o n s are l i s t e d i n Table 1 , The u s e f u l c a p a c i t y of the treatment chambers was in c r e a s e d by t r a n s -f e r r i n g the p l a n t s t o a set of photoperiod c a b i n e t s i n the greenhouse f o l l -owing treatment. From O83O hr t o I63O hr d a i l y , the p l a n t s r e c e i v e d n a t u r a l d a y l i g h t , the remainder of a 15 hr photoperiod being s u p p l i e d by the photo-.period c a b i n e t s . Observations were recorded i n the same manner as i n the previous experiment. The l i g h t c h i l l e d p l a n t s and the dark c h i l l e d p l a n t s were considered as two separate experiments. The completely randomized design w i t h seven treatments and f i v e r e p l i c a t i o n s was used w i t h a t o t a l of 35 p l a n t s i n each experiment. 1969-1970 Experiment Budded u p r i g h t s were c o l l e c t e d on October 17, 1969. P l a n t s were prep-a r e d as i n 1968 and were set 30 per box i n 30 cm x 20 cm x 7 cm cedar f l a t s . These were allowed t o r o o t i n the greenhouse u n t i l i n i t i a t i o n of treatments on October 31 , 1969. Two growth chambers and two mod i f i e d c o l d rooms were used t o provide f o u r d i f f e r e n t c h i l l i n g regimes. A warm temperature regime was provided i n the photoperiod c a b i n e t s . A nine hour photoperiod was used f o r a l l t r e a t -ment regimes. Temperature Regimes 1 and 2 were s u p p l i e d i n P e r c i v a l Model PGG-78 growth chambers equipped w i t h Partlow Model RG-15 thermal c o n t r o l l e r s . 10 Both regimes had maxima of 10°C and minima of 2°C, Regime 1 reached the minimum once i n 24 hr and Regime 2 reached i t three times ( F i g . l ) ^ Temperature Regimes 3 and 4 were a p p l i e d i n the two c o l d rooms. I n each case, a corner area approximately 1.0 m x 1.6 m was p a r t i t i o n e d from the r e s t of the room by a b l a c k p l a s t i c c u r t a i n suspended from the c e i l i n g , thus e n c l o s i n g the bench on which the p l a n t s were l o c a t e d . Temperatures f o r Regime 3 were 13°C day, 4.5°C n i g h t ; f o r Regime 4, 3°C day and 0°C n i g h t ( F i g . 1). Regime 5 was a p p l i e d i n the photoperiod c a b i n e t s . The ambient temp-era t u r e was maintained above 18°G f o r the d u r a t i o n of the experiment. P l a n t s r e c e i v e d n a t u r a l d a y l i g h t from 0830 hr t o I63O hr d a i l y , w i t h an a d d i t i o n a l hour of low i n t e n s i t y i l l u m i n a t i o n given as one h a l f hour before and a f t e r the n a t u r a l d a y l i g h t . F o l l o w i n g the treatments, the p l a n t s were t r a n s f e r r e d t o a greenhouse bench. To ensure a minimum 15 hr daylength, supplemental l i g h t i n g was prov-i d e d from O63O hr t o 2130 h r . Observations were recorded as f o r the 1968-1969 experiment. I n a d d i t -i o n , the mean shoot growth of the p l a n t s w i t h i n each box was c a l c u l a t e d and p l o t t e d a g a i n s t time. The data f o r the l i n e a r p o r t i o n of the r e s u l t i n g growth curves were used t o c a l c u l a t e the simple r e g r e s s i o n c o e f f i c i e n t , b, from the formula y = a + bx, where y <= shoot l e n g t h and x = time (days) f o l l o w i n g t e r m i n a t i o n of c h i l l i n g treatments. These b values were then used i n an a n a l y s i s of variance t o compare d i f f e r e n c e s In growth r a t e s . Two boxes of p l a n t s were used f o r each regime-treatment combination. Twenty p l a n t s from each box were measured; thus a t o t a l of 1080 p l a n t s were i n v o l v e d i n t h i s experiment. With the omission of the 150 day t r e a t s ments i n Regimes 1 and 2 and the u n c h i l l e d p l a n t s (which d i d not show a 1 Regime 2 was used t o i n v e s t i g a t e the e f f e c t of a f l u c t u a t i n g minimum. 11 IO 5^ 1200 REGIME I 1800 2 4 0 0 0 6 0 0 1200 10 5 -\ REGIME 2 1200 1800 2 4 0 0 Time (hr) 0 6 0 0 1200 Fig. 1. Daily Temperature Curves for Chilling Regimes, 1969-1970 Controlled Environment Experi merit. 12 Fig. 1. (Continued) 54 OH REGIME 3 I200 I800 2 4 0 0 0600 I200 51 OA REGIME 4 1200 1200 1800 2400 Time (hr) 0 6 0 0 13 response), the data were analysed u s i n g a two-way a n a l y s i s of v a r i a n c e . Be-cause of the s i g n i f i c a n t i n t e r a c t i o n "between regime and treatment, a one-way a n a l y s i s of varia n c e w i t h i n each treatment was used t o compare the e f f e c t of the d i f f e r e n t regimes and v i c e versa t o compare the e f f e c t of the d i f f e r -ent treatments. G i b b e r e l l i c A c i d Experiment Three s e t s of f i v e p l a n t s , each c o l l e c t e d October 4, 1968 and handled as p r e v i o u s l y d e s c r i b e d , were used f o r t h i s experiment. Treatments were i n i t i a t e d i n the greenhouse on May 15, 1969 as f o l l o w s : Set 1 g i b b e r e l l i c a c i d aqueous spray 100 ppm Set 2 " " " 1000 ppm Set 3 water c o n t r o l (0 g i b b e r e l l i c a c i d ) D a i l y a p p l i c a t i o n from May 15 t o May 23, 1969 f a i l e d t o have any v i s i b l e e f f e c t on the p l a n t s . On June 8, 1969, a sm a l l i n c i s i o n was made 1 cm below the bud of each p l a n t . One hundred m i c r o l i t r e s of the app r o p r i a t e treatment s o l u t i o n f o r the set was a p p l i e d t o each p l a n t by means of repeated i n j e c t -i o n s w i t h a 10 m i c r o l i t r e s y r i n g e . The response of each p l a n t was recorded. G i b b e r e l l i n - l i k e A c t i v i t y A n a l y s i s of the q u a n t i t y of compounds e x h i b i t i n g g i b b e r e l l i n - l i k e a c t i v i t y i n V . macrocarpon was done on fie l d - g r o w n m a t e r i a l f o r the 1969-1970 winter season. E x t r a c t i o n , P u r i f i c a t i o n and Separation of G i b b e r e l l i n - l i k e Compounds Budded u p r i g h t s were c o l l e c t e d a t va r i o u s dates between October 1969 and June 1970. The m a t e r i a l was c o l l e c t e d d i r e c t l y onto dry i c e and was s t o r e d a t -20°C both before and a f t e r f r e e z e - d r y i n g . The quantity of terminal buds used for each extraction depended on the number present i n the sample. Extraction of leaf tissue was made from 15-gram samples. A total of 11 bud samples and 12 leaf samples were done. The extraction and purification of gibberellin-like compounds from the plant tissue i s outlined i n a flow diagram (Fig. 2) . The f i r s t part i s as adapted by Crozier et a l . (1969) from Hayashi and Rappaport (1962). A l l solvents with the exception of methanol were r e d i s t i l l e d prior to use as a precaution against impurities which could cause gibberellin-like growth promoting activity (Hartley et a l . , 1969). Concentration to dryness was done in vacuo with a water bath temperature of 32°C. The polyvinylpyrrolidone (PVP) column was adapted from that of Pharis*. A slurry of PVP was prepared in 0.1 M, pH 8,0 phosphate buffer, and a 19 mm glass column f i l l e d to a height of 20 cm. A layer of small glass beads was sprinkled on top of the PVP. The crude acidic fraction was taken up in phosphate buffer and transferred to the column. Three hundred ml of buffer were used to elute the column. The charcoal-celite column* was prepared by mixing 10 g of Celite 5^ 5 (Fisher) with 5 g of Darco G-60 activated charcoal (Matheson). This was slurried with 80% acetone and placed in a 19 mm glass column. Glass beads were again used to receive the sample. The column was eluted with 250 ml of 80% acetone. To remove the water, i t was necessary to add methanol when concentrating the sample. Thin layer chromatography was done according to Reid et a l . (1969)» using Eastman-Kodak Si l i c a - g e l thin layer chromatography sheets. Aliquots of the acetone residue were taken up in methanol and streaked onto the sheets. The chromatogram was developed with ethyl acetate»chloroforms formic acid (50.:50:1).. The sheets were air-dried and cut into ten equal strips 1 R.P. Pharis, personal communication, 19?0. FREEZE—DRI ED PLANT TISSUE Homogenized with 200 ml 80? aqueous methanol below 0° C. Fi l ter . Stir residue with 801 aqueous methanol for 24 hr at 4 C. Fi l ter combine methanolic f i l t ra tes . I 1 Methanol extract Discard residue. Evaporate to aqueous phase, add equal vol. 0.5 M phosphate buffer, pH 8.0, Adjust extract to pH 9.0 with K0H. Partition 3 X at pH 9.0 with 1/3 vol. diethyl ether. Discard ether phase Aqueous phase Adjust to pH 3.0 with HCl. Partition 5 X with 2/5 vol. ethyl acetate. Ethyl acetate phase Discard aqueous phase, (crude acidic fraction) Concentrate to dryness at 32° C. Chromatograph on PVP column with 0.1 M phosphate buffer, pH 8.0. Adjust eluate to pH 3.0 with HCl and partition 5 X with 2/5 vol. ethyl acetate. Discard aqueous phase Ethyl acetate phase Concentrate to dryness at 32° C. Chromatograph on charcoal-cel i te column with 80$ acetone. Concentrate eluate to dryness. Residue Chromatograph aliquots of residue on thin-layer sheets. Scrape residue from thin-layer sheets in bands at Rf 0.1. BSoassay all fractions Fig. 2. Flow Diagram for Extraction, Purification and Separation of Gibberellin-like Compounds from Plant Tissue. . between the l i n e of a p p l i c a t i o n and the solvent f r o n t ; thus each s t r i p r epresented = 0.1. The s i l i c a - g e l was scraped from the s t r i p s ( i n c l u d i n g the s t a r t i n g l i n e as a separate s t r i p , R^ = 0) and used i n the bioassay. Bioassay The l e t t u c e hypocotyl bioassay (Frankland and Wareing, I960) was used t o estimate the amount of g i b b e r e l l i n - l i k e a c t i v i t y i n each f r a c t i o n . L e t t -uce seeds c u l t i v a r A r c t i c were used. The s i l i c a - g e l from the chromatograms was p l a c e d i n 6.0 cm x 1.5 cm p e t r i d ishes l i n e d w i t h f i l t e r paper. These were moistened w i t h 3*0 ml of a one-quarter s t r e n g t h n u t r i e n t s o l u t i o n ( M a c h l i s and Torrey, 1956). A s e r i e s of g i b b e r e l l i c a c i d (GA-j) standards was made up u s i n g the same n u t r i e n t s o l u t i o n and were a l s o added t o p e t r i d i shes i n 3.0 ml q u a n t i t i e s . The l e t t u c e seeds were germinated i n the dark f o r two days. Ten of these s e e d l i n g s were added t o each p e t r i d i s h . Both the unknowns and the , standards were kept under continuous l i g h t f o r t h r e e days, a f t e r which the hyp o c o t y l l e n g t h s were measured. G i b b e r e l l i n Recovery The recovery of known amounts of g i b b e r e l l i c a c i d (GA-j) was checked u s i n g the above methods. The r a t e of recovery u s i n g g i b b e r e l l i c a c i d alone or mixed i n w i t h p l a n t m a t e r i a l was 100%. 17 RESULTS Anatomy o f t h e T e r m i n a l B u d E x a m i n a t i o n o f t h e t e r m i n a l b u d s e c t i o n s f o r t h e two w i n t e r p e r i o d s r e v e a l e d t h a t t h e f l o w e r b u d s h a d o v e r w i n t e r e d a t d i f f e r e n t s t a g e s o f d e v e l -opment . T h e u p p e r p o r t i o n o f F i g . 3 shows a l o n g i t u d i n a l s e c t i o n o f a f l o w e r b u d c o l l e c t e d J a n u a r y 28 , 1969. T h e l o w e r p o r t i o n o f t h e same f i g u r e shows a f l o w e r b u d c o l l e c t e d O c t o b e r 17, 1969. On O c t o b e r 17 , 1969, t h e s tamen a n d c a r p e l p r i m o r d i a were more d e v e l o p e d t h a n t h o s e o f t h e p r e v i o u s w i n t e r , e v e n t h o u g h t h e p r e v i o u s w i n t e r ' s f l o w e r b u d s were c o l l e c t e d a t a l a t e r t i m e . I n e a c h o f t h e two w i n t e r s f o r w h i c h t e r m i n a l buds were e x a m i n e d , t h e f l o w e r b u d s a p p e a r e d t o be dormant u n t i l e a r l y M a r c h . F i g , 4 shows t h e s t a g e s o f d e v e l o p m e n t o b s e r v e d f o r b u d s c o l l e c t e d on M a r c h 9, May 4 a n d May 19, 1970. I n c o m p a r i n g t h e March 9, 1970 s e c t i o n t o t h a t o f O c t o b e r 17, 1969, i t i s r e a d i l y s e e n t h a t t h e g r o u n d m e r i s t e m below t h e f l o w e r i n F i g . kA h a s more c e l l e l o n g a t i o n . C e l l e l o n g a t i o n i s most l i k e l y a phenomenon w h i c h marks t h e o n s e t o f f l o w e r d e v e l o p m e n t . H e n c e , i t w o u l d a p p e a r t h a t f l o w e r b u d d e v e l o p m e n t b e g i n s i n l a t e F e b r u a r y o r e a r l y M a r c h . By t h e e n d o f A p r i l i n e a c h y e a r , t h e f l o w e r b u d s h a d d i f f e r e n t i a t e d t o a b o u t t h e same e x t e n t . T h e May 19, 1970 s e c t i o n shows t h e s t a g e o f d e v e l o p m e n t r e a c h e d j u s t p r i o r t o t h e e l o n g a t i o n o f t h e t e r m i n a l b u d s i n t h e f i e l d . F i e l d T e m p e r a t u r e F i e l d t e m p e r a t u r e d a t a were r e c o r d e d f r o m O c t o b e r 20, 1969 t o May 4, 1970. O n l y t h e i n f o r m a t i o n r e l a t i n g t o t h e p l a n t m a t e r i a l c o l l e c t e d f r o m t h e f i e l d i s p r e s e n t e d h e r e . A c c u m u l a t e d h o u r s below 7°C f r o m O c t o b e r 20, 1969 ( r e a d f r o m t h e r m o g r a p h c h a r t s ) , a l o n g w i t h t h e maximum a n d minimum 18 F i g . 3« Vacclnium macrocarpon flower bud ( l . s . ) t o show w i n t e r dormant stages. A. C o l l e c t e d from f i e l d January 2 8 , 1969; B. C o l l e c t e d October 17, 1 9 6 9 . A b b r e v i a t i o n s : c = c a r p e l , s = stamen F i g . 4, Vaccinium macrocarpon flower bud ( l . s . ) t o show stages of s p r i n g development. A. C o l l e c t e d from f i e l d March 9, 1970 B. C o l l e c t e d May 4, 1970; C. C o l l e c t e d May 19, 1970. A b b r e v i a t i o n s ; c = c a r p e l , s = stamen temperatures f o r the week preceding each p l a n t c o l l e c t i o n date, are presented i n Table 2. Records are missing from March 24 t o 26, 1970 and were not a v a i l a b l e f o r the p e r i o d immediately f o l l o w i n g May 4, 1970. C o n t r o l l e d Environment S t u d i e s  1968-1969 Experiment The p o r t i o n of the data d e a l i n g w i t h the mean number of days t o bud break has been p u b l i s h e d (Eady and Eaton, 1969), along w i t h some observat-i o n s on the growth of the t r e a t e d p l a n t s . The data from t h i s experiment are summarized i n Table 3« No macroscopic evidence of t e r m i n a l bud a c t i v i t y was observed i n the u n c h i l l e d p l a n t s . The mean number of days t o bud break was decreased from 44 days a f t e r 65O h r c h i l l i n g t o 9 days a f t e r 2500 hr c h i l l i n g ( s t a t i s t i c -a l l y s i g n i f i c a n t , P = 0.05)' 1 -. Mean shoot l e n g t h a t the completion of the observation p e r i o d (100 days a f t e r t e r m i n a t i o n of c h i l l i n g ) and number of f l o w e r s per p l a n t were not s i g n i f i c a n t l y d i f f e r e n t , w i t h the exception of the u n c h i l l e d treatment. The number of p l a n t s f l o w e r i n g under these c o n d i t -i o n s reached 100% a f t e r 1300 hr of c h i l l i n g . There was no s i g n i f i c a n t d i f f e r e n c e i n any of the p l a n t responses between the two treatment chambers. 1969 (Summer) Experiment P l a n t response t o both the l i g h t and the dark c h i l l i n g treatments was poor. I n i t i a t i o n of new shoot growth f o l l o w i n g treatments occurred only i n a few in s t a n c e s (Table 4), Buds h e l d f o r months without c h i l l i n g had thus 1 The 5 percent l e v e l of s i g n i f i c a n c e was used throughout t h i s s e c t i o n . The term " s i g n i f i c a n t " t h e r e f o r e i n d i c a t e s P = 0.05. The a n a l y s i s of variance t a b l e s are presented i n Appendix 2. T a b l e 2 . 1 F i e l d Temperatures f o r 1969-1970 Accumulated h r below 7 C Maximum Temp. Minimum Temp. P l a n t C o l l e c t i o n Date ( f r o m O c t . 20/69) ( ° C ) ( ° C ) . O c t . 17/69 not a v a i l a b l e Nov. 18/69 194- 1 0 . 0 0 . 0 Dec. 18/69 680 9 . 0 0 . 0 F e b . 3/70 1564 8 . 5 - 5 . 0 F e b . 23/70 1934 1 2 . 0 - 5 . 0 M a r . 9/70 2177 1 2 . 0 - 7 . 5 A p r i l 6/70 2 4 3 8 2 1 4 . 0 - 3 . 5 A p r i l 27/70 2688 1 1 . 0 0 . 5 May 4/70 2777 1 8 . 0 1.0 May 19/70 not a v a i l a b l e June 22/70 not a v a i l a b l e 1 Maximum and minimum t e m p e r a t u r e s are f o r a one week p e r i o d p r e c e d i n g each p l a n t c o l l e c t i o n d a t e . 2 Records were not a v a i l a b l e f o r March 2 4 , 25 and 2 6 , 1970. T a b l e 3 . Mean Number of Days t o Bud B r e a k , Shoot L e n g t h , and F l o w e r s per P l a n t i n V. m a c r o c a r p o n , 1968-1969 C h i l l i n g Treatments C h i l l i n q Days 2 Shoot Lenqth F l o w e r s / P l a n t P l a n t s F l o w e r i n g P l a n t s T r e a t e d ( h r ) (mean n o . ) (cm) (mean n o . ) (%) ( n o . ) 0 no bud break 0 0 0 6 650 44 d 7 . 9 1.1 50 6 1300 27 c 8 . 4 1 . 7 100 6 1700 23 c 7 . 8 1 . 3 100 6 2100 14 b 9.1 1 . 3 100 6 2500 9 a 11*8 1 . 3 100 6 1 A f t e r Eady and Eaton ( 1 9 6 9 ) . 2 Means s h a r i n g the same l e t t e r d i d not d i f f e r s i g n i f i c a n t l y (P = 0 . 0 5 ) . 23 apparently l o s t the a b i l i t y t o respond t o the c h i l l i n g treatments provided. Table 4. Number of P l a n t s I n i t i a t i n g New Terminal Growth F o l l o w i n g Completion of C h i l l i n g Treatments, 1969 (Summer) Experiment No. of P l a n t s I n i t i a t i n g Experiment C h i l l i n g New Growth (out of 5) (hr) l i g h t 1700 1 2100 1 dark 650 2 1300 2 1700 1 No macroscopic evidence of f l o w e r bud development was found on any of the p l a n t s which had broken dormancy, t h e i r growth being e n t i r e l y vegetat-i v e . The l i g h t c h i l l e d p l a n t s r e t a i n e d t h e i r leaves both d u r i n g and a f t e r c h i l l i n g . Most of the dark c h i l l e d p l a n t s l o s t t h e i r leaves e i t h e r d u r i ng or s h o r t l y a f t e r the completion of the c h i l l i n g treatments. 1969-1970 Experiment I n a l l of the c h i l l i n g treatment regimes, there was an increase i n the number of t e r m i n a l buds breaking dormancy i n response t o longer c h i l l -i n g times (Table 5)# The a n a l y s i s of variance i n d i c a t e d a s t a t i s t i c a l l y s i g n i f i c a n t i n t e r a c t i o n between the c h i l l i n g regime and the le n g t h of time c h i l l e d . T h i s i n d i c a t e s t h a t the p l a n t s d i d not respond a t the same r a t e f o r each regime. When the data were reanalyzed, the only s i g n i f i c a n t d i f f -erence between regimes occurred f o r the 75 day treatment, where Regimes 1 24 Table 5. 1 Average Percent Bud Break of V. macrocarpon, 1969-1970 Ch i l l i ng Treatments Standard Error Regime Total Number of Days Chi l led of the Mean 25 50 75 100 125 150 1 32,5 52.5 77.5 85.0 100 92.0 8.00 a ab be c c c 2 10.0 52.5 52.5 90.0 100 98.0 4.86 a b b c c c 3 0 15.0 37.5 82.5 87.5 _ 4.33 a a b c c 4 20.0 62.5 85.0 82.5 90.0 9.33 a b '•b b b 5 5.0 7.5 5.0 10.0 5.0 1 Means within rows sharing the same l e t t e r or le t ters did not d i f f e r s ign i f i can t l y (P = 0.05). Data from Regime 5 were not included in the s t a t i s t i c a l analysis . 25 and k were s i g n i f i c a n t l y d i f f e r e n t from Regimes 2 and 3. Some of the t e r m i n a l buds i n Regime 5 i n i t i a t e d new growth (Table 5 ) • I n a l l of these p l a n t s , the r e s u l t i n g growth was v e g e t a t i v e only and d i s -p l a y e d the c h a r a c t e r i s t i c s of runner growth r a t h e r than t h a t of u p r i g h t s . T h i s would i n d i c a t e t h a t from 5-10% of the t e r m i n a l buds were v e g e t a t i v e r a t h e r than mixed buds. The data f o r Regime 5 were not i n c l u d e d i n any of the s t a t i s t i c a l analyses. The mean number of days from t e r m i n a t i o n of the c h i l l i n g treatment u n t i l the f i r s t 25 percent of the t e r m i n a l buds had broken dormancy ( a l l of the t e r m i n a l buds where the percent bud break was l e s s than 25) was p l o t t e d a g a i n s t length of time i n the c h i l l i n g treatments f o r each regime ( F i g . 5)» Again there was a s t a t i s t i c a l l y s i g n i f i c a n t i n t e r a c t i o n . The mean values f o r each of the f o u r regimes changed i n t h e i r r e l a t i v e order depending on the l e n g t h of c h i l l i n g . The means t h a t d i d not d i f f e r s i g n i f i c a n t l y a f t e r the same len g t h of c h i l l i n g are i n d i c a t e d i n F i g , 5« A s i g n i f i c a n t r e d u c t -i o n i n the number of days t o bud break occurred w i t h each increased c h i l l -i n g p e r i o d f o r Regimes 1 and 2 , In Regime 3t added c h i l l i n g up t o 100 days caused a s i g n i f i c a n t decrease. I n Regime k, the treatment d i f f e r e n c e s were not as pronounced; the 25 day treatment was s i g n i f i c a n t l y d i f f e r e n t from a l l the others, 50 days was not from 75, 75 days was not from 100, and 100 days was not from 125. In Regimes 1 and 2 , a few p l a n t s i n i t i a t e d new t e r m i n a l growth w h i l e s t i l l i n the growth chambers. Growth of the p l a n t s f o l l o w i n g removal from c h i l l i n g was l i n e a r w i t h time during the f i r s t phase ( F i g , 6). The growth r a t e s , which were c a l c u l -a t e d as the l i n e a r r e g r e s s i o n c o e f f i c i e n t f o r t h i s p e r i o d , are l i s t e d i n Table 6, There was not a s i g n i f i c a n t i n t e r a c t i o n between regimes and c h i l l i n g times, and the d i f f e r e n c e between regimes was not s i g n i f i c a n t . 26 2 Time in chil l ing treatment (days) Fig. 5. Number of Days to Bud Break Following Termination of Chilling Treatments, Regimes 1-4, 1969-1970 Controlled Environment Experiment. Means of symbols encircled by the same dotted line did not di f fer significantly (P * 0.05). 20-i 2 0 -.10 0 REGIME 2 i 100 0 25 Time from termination of chil l ing (days) Fig. 6. Shoot Growth Following Termination of Chilling Treatments, 1969-1970 Controlled Environment Experiment. Legend; 1=25 days chi l l ing, 2 = 50 days, 3 = 75 days, h = 100 days, 5 = 125 days, 6 = 150 days. i 5 0 —I— 7 5 I00 - N 3 00 29 Table 6. Growth Rates (cm/day) for the Linear Portion of the Growth Curve of j / . macrocarpon, 1969-1970 Ch i l l i ng Treatments1 Standard Error Regime Total Number of Days Chi l led of the Mean 25 50 75 100. 125 150 1 .151 .164 .271 .279 .241 .407 a ab be c abc d 2 .113 .171 .228 .263 .364 .344 a ab ab be c c •3 ' - .241 .190 .249 .291 _ a a a a 4 .062 .180 .212 .214 .290 a b b b b Average .082 ..189 .225 .251 .296 — a b be cd d 1 Means within rows sharing the same l e t t e r or l e t te rs did not d i f f e r s ign i f i can t l y (P = 0.05). 30 There was a ge n e r a l i n c r e a s e i n growth r a t e as the treatment time was i n c -reased. There was no bud break a f t e r only 25 days i n Regime 3. The remain-i n g treatment times i n t h i s regime 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 each other. I n Regime 4, the only d i f f e r e n c e was t h a t the 25 day treatment r e s u l t e d i n slower growth r a t e s than any of the other treatments. The mean l e n g t h of the new shoot growth a t the end of the experiment (100 days a f t e r removal from the c h i l l i n g treatment) g e n e r a l l y increased w i t h longer c h i l l i n g p e r i o d s (Table ? ) . There was a s i g n i f i c a n t i n t e r a c t i o n between regimes and the c h i l l i n g times. The only s i g n i f i c a n t d i f f e r e n c e i n response between regimes f o r the same treatment times occurred f o r the 75 day treatment where the p l a n t s in,Regime 1 had a mean len g t h s i g n i f i c a n t l y g r e a t e r than those of the other regimes. Flowering d i d not occur a f t e r any of the 25 or 50 day treatment times and only occurred i n the c o l d e s t regime (4) a f t e r 75 clays (Table 8). The number of p l a n t s f l o w e r i n g was s i g n i f i c a n t l y i n c r e a s e d w i t h time c h i l l e d f o r Regimes 1, 2 and 3. The f l o w e r i n g response i n Regime 4 was not in c r e a s e d s i g n i f i c a n t l y w i t h the a d d i t i o n a l c h i l l i n g . G i b b e r e l l i c A c i d Experiment There was no response t o the aqueous f o l i a r spray of g i b b e r e l l i c a c i d . When the same m a t e r i a l was i n j e c t e d i n t o i n c i s i o n s i n the stems, the te r m i n -a l buds of the g i b b e r e l l i c a c i d t r e a t e d p l a n t s responded w i t h i n 10 days. The t e r m i n a l buds of the responding p l a n t s elongated and leaves developed, but there was no v i s i b l e f l o w e r bud development. G i b b e r e l l i n - l i k e A c t i v i t y The recovery of known amounts of g i b b e r e l l i c a c i d by the methods used Table 7. Mean Shoot Length (cm) of V. macrocarpon 100 Days a f t e r Removal from 1969-1970 C h i l l i n g Treatments 1 Standard E r r o r of the Mean 1.83 0.83 1.42 1.44 Reqime Tota l Number of Days C h i l l e d 25 50 75 100 125 150 ,1 7.9 6.8 12.3 14.5 12.3 16.1 a a a a a a • 2 5.1 7.9 9.3 15 .0 19.2 15.5 a ab b c d c 3 - 5.7 7.6 14.9 15.2 a a b b 4 3.1 6.7 9.3 13.2 12.2 _ a ab be c c 1 Means w i t h i n rows sha r ing the same l e t t e r or l e t t e r s d i d not d i f f e r s i g n i f i c a n t l y (P = 0 . 0 5 ) . : . . Table J _ • . ._. . • • 1 • I Mean Number of F lowers per 20 P l a n t s , 1969-1970 C h i l l i n g Treatments- •'• { ~~ ~ TbTa"Oum5eT~of~Davs ChTTIecT ' ^ " " S t a n d l r d E r r o r Reqime 25 50 75 100 125 150 of the Mean 1 0 0 0 10.5 12.5 29 .0 2.48 a a b 2 0 0 0 2.5 15 .0 39 .0 6.07 a ab b 3 0 0 0 6.0 14 .0 1.00 a b 4 0 0 4 .0 2.0 14 .0 - 6.07 a a a 1 Means w i t h i n rows sha r ing the same l e t t e r or l e t t e r s d id not d i f f e r s i g n i f i c a n t l y (P = 0 . 0 5 ) . 32 was 100 %, The minimum q u a n t i t i e s of g i b b e r e l l i n - l i k e a c t i v i t y detected w i t h the l e t t u c e hypocotyl bioassay were eq u i v a l e n t t o 5 x 10 ^ micrograms of g i b b e r e l l i c a c i d (GA^) a c t i v i t y . The standard e r r o r of the estimate was _2 approximately 2 x 10 micrograms of g i b b e r e l l i n - l i k e a c t i v i t y . I n most of the samples, a c t i v i t y was e s t a b l i s h e d u s i n g l / l O t h and l/20th a l i q u o t s . The use of l/k a l i q u o t s gave u n s a t i s f a c t o r y r e s u l t s probably be-cause of the presence of i n h i b i t o r s which were d i l u t e d out at the lower l e v e l s . The minimum d e t e c t a b l e a c t i v i t y of any one sample was based on a l / l O t h a l i q u o t , and was d i r e c t l y r e l a t e d t o the o r i g i n a l dry weight of the sample. Sample dry weights and the c a l c u l a t e d minimum de t e c t a b l e l e v e l s are presented i n Appendix 3. A c o n s i d e r a b l e r e d u c t i o n i n the dry weight of the sample a f t e r methan-o l e x t r a c t i o n was obtained through the use of PVP and c h a r c o a l - c e l i t e chrom-atography columns (see Appendix 3) . F i g , 7 shows the g i b b e r e l l i n - l i k e a c t i v i t y of e x t r a c t s from both term-i n a l buds and l e a v e s . Where the a c t i v i t y was too low t o be detected, the c a l c u l a t e d value f o r the minimum de t e c t a b l e q u a n t i t y was s u b s t i t u t e d t o i n d i c a t e t h a t the t r u e value was below t h a t f i g u r e . The a c t i v i t i e s f o l l o w -ed the same gene r a l p a t t e r n , w i t h the notable exception of the March 9 and A p r i l 6 samples where a r e d u c t i o n i n the l e a f l e v e l was accompanied by an increase i n the bud l e v e l . The r e d u c t i o n i n the l e v e l s on February 23 c o i n c i d e d w i t h a p e r i o d of low n i g h t and f a i r l y high day temperatures f o r the previous week, but there i s no evidence f o r a d i r e c t c a u s a l r e l a t i o n -s h i p . The g i b b e r e l l i n - l i k e a c t i v i t y d i d not always occur at the same R f values ( t h i n - l a y e r chromatography) f o r each sample. Much of the a c t i v i t y d i d occur between R f 0.2 and 0.6, but a c t i v i t y i n other f r a c t i o n s was not 34 uncommon. Known samples of g i b b e r e l l i c a c i d (GA^) showed a c t i v i t y between 0 . 2 and 0 . 4 w i t h the s o l v e n t system used. G A r , showed a c t i v i t y between R^ 0 . 3 and 0 . 5 . Histograms of a c t i v i t y from the t e r m i n a l bud and l e a f e x t r a c t s f o r March 9 and A p r i l 6 are shown i n F i g . 8 . The l e a f sample f o r March 9 showed h i g h a c t i v i t y a t R^ 0 . 2 t o 0 . 4 , which decreased considerably by A p r i l 6 w i t h a corresponding r i s e i n the same r e g i o n f o r the bud samples. I t i s p o s s i b l e t h a t a t r a n s f e r of these substances from the leaves t o the t e r m i n a l bud had taken p l a c e . TERMINAL BUDS March 9/70 0.5 R, 10 10" 0 1.0 A p r i l 6/70 •~oV _ ^ . 0 LEAVES March 9/70 10" 10 A p r i l 6/70 0.5 R, 1.0 Histograms of G i b b e r e l l i n - l i k e A c t i v i t y of Substances Ex t rac ted f rom Leaves and Buds of V. macrocarpon. Samples i l l u s t r a t e d were 1/10 a l i q u o t s , dry weights of t i s s u e were as f o l l o w s : Buds; March 9/70 = 0.3429 g , A p r i l 6/70 = 0.7051 g . Leaves; March 9/70 = 15.00 g , A p r i l 6/70 - 15.00 g . 36 DISCUSSION The post-dormant response of the t e r m i n a l bud of V, macrocarpon t o c h i l l i n g temperatures during the dormant p e r i o d i s q u i t e e v i d e n t . The o b l i g a t o r y nature of the c h i l l i n g requirement was demonstrated by the f a i l -ure of the mixed t e r m i n a l buds t o break dormancy when maintained under non-c h i l l i n g c o n d i t i o n s d u r ing the w i n t e r p e r i o d . The f a i l u r e of the t e r m i n a l bud t o respond t o v a r y i n g photoperiods under n o n - c h i l l i n g c o n d i t i o n s was r e p o r t e d by Eaton and Ormrod ( 1 9 6 8 ) , who i n v e s t i g a t e d the e f f e c t of photo-p e r i o d on the non-terminal v e g e t a t i v e growth. Of the v e g e t a t i v e responses measured, the most evident e f f e c t of c h i l l -i n g was a decrease i n the number of days t o bud break. T h i s response i s the f i r s t v i s i b l e r e s u l t of the c h i l l i n g treatment and i s t h e r e f o r e probably the one which has the l e a s t number of i n t e r v e n i n g f a c t o r s a f f e c t i n g i t . The g r e a t e s t responses were obtained i n those c h i l l i n g treatments which had a s u i t a b l e balance between i n d u c t i v e c h i l l i n g temperatures and a warm p a r t of the c y c l e , which would a l l o w a c e r t a i n amount of a c t i v i t y t o occur w i t h -i n the p l a n t . T h i s a c t i v i t y was evident i n those p l a n t s which broke dormancy before completion of the 150 day c h i l l i n g treatments. There i s an apparent l e v e l l i n g o f f i n the number of days t o bud break w i t h longer c h i l l i n g times i n Regime 4 . One p o s s i b l e e x p l a n a t i o n i s t h a t i n the absence of longer per-i o d s of higher temperatures, very l i t t l e a c t i v i t y can occur d u r i n g the c h i l l -i n g treatment p e r i o d ; t h e r e f o r e the time l a g between t e r m i n a t i o n of the c h i l l i n g treatment and bud break i s not reduced as r a p i d l y as i n the other regimes. The f a c t t h a t some of the p l a n t s i n i t i a t e d new shoot growth be-f o r e the t e r m i n a t i o n of the 150 day c h i l l i n g treatments i n the 1969-1970 experiment i s i n i t s e l f evidence t h a t some development took p l a c e under these p a r t i c u l a r c h i l l i n g c o n d i t i o n s . I t i s p o s s i b l e t h a t as f a r as the 37 v e g e t a t i v e development of the p l a n t i s concerned, the c h i l l i n g requirement i s f u l f i l l e d q u i t e e a r l y and t h a t the a d d i t i o n a l response t o f u r t h e r time i n the c h i l l i n g treatment i s due t o e x t r a growth and development t h a t took place d u r i n g t h a t time and not as a d i r e c t r e s u l t of the a d d i t i o n a l c h i l l i n g i t -s e l f . The r a t e of growth and the shoot l e n g t h a t t a i n e d were l e s s dependent on the c h i l l i n g treatment r e c e i v e d than was the r e d u c t i o n i n the number of days t o bud break. The more r a p i d r a t e of growth a f t e r some of the longer c h i l l i n g p eriods i s again more l i k e l y a r e s u l t of the a c t i v i t y of the p l a n t s d u r i n g warm pe r i o d s i n the c h i l l i n g c y c l e r a t h e r than a d i r e c t e f f e c t of c h i l l i n g i t s e l f . Both the r a t e of growth and shoot l e n g t h a t t a i n e d would be extremely dependent on the post-dormant c o n d i t i o n s , which were maintained reasonably uniform f o r these experiments. The f l o r a l p r i mordia responded somewhat d i f f e r e n t l y t o the c h i l l i n g treatments than d i d the v e g e t a t i v e p a r t s of the t e r m i n a l bud. I n a l l of the c h i l l i n g treatments given i n 1969-1970, the s h o r t e r treatment times r e s u l t e d only i n v e g e t a t i v e growth of the t e r m i n a l bud, A longer p e r i o d was necess-ary t o induce f u r t h e r development of the f l o r a l p r i m o r d i a . T h i s d i f f e r e n c e was not as evident i n the 1968-1969 treatments. There are at l e a s t two p o s s i b l e reasons f o r t h i s . F i r s t , there may have been an e f f e c t of spacing on the response of the p l a n t s and, secondly, the warm par t of the c h i l l i n g c y c l e was q u i t e short i n the 1968-1969 treatments, thus a l l o w i n g f o r more a c t u a l hours of c h i l l i n g i n a day while s t i l l p r o v i d i n g a p e r i o d of reason-a b l y h i g h temperature. I t i s p o s s i b l e t h a t the f a i r l y long exposure of the p l a n t s t o higher temperatures during the c h i l l i n g c y c l e i n Regime 3 was r e s u l t i n g i n a p a r t i a l d e v e r n a l i z a t i o n . D e v e r n a l i z a t i o n has been r e p o r t e d i n other p l a n t 38 species as a r e s u l t of exposure t o high temperatures immediately a f t e r vern-a l i z a t i o n . S a l i s b u r y (1963) noted t h a t temperatures above 3 5 ° G are most e f f e c t i v e f o r d e v e r n a l i z a t i o n , but t h a t i n rye temperatures above 15°C be-g i n t o have a d e v e r n a l i z i n g e f f e c t . The exposure of the p l a n t s t o 13°C during the l i g h t p e r i o d could be a major f a c t o r i n e x p l a i n i n g the marginal response patterns f o r Regime 3« Flowering occurred i n V. macrocarpon f o l l o w i n g as few as 65O hr below 7°C during the 1968-1969 experiment. The longest a c t u a l exposure t o temp-eratur e s below 7°C before i n d u c t i o n of flo w e r development occurred i n Reg-ime 4, where 1800 hr of exposure was r e q u i r e d . The a d d i t i o n a l c h i l l i n g requirement f o r f l o w e r formation suggests t h a t the v e g e t a t i v e and f l o r a l p o r t i o n s of the t e r m i n a l bud may not have c l o s e l y i n t e g r a t e d c h i l l i n g requirements. There i s a l s o the p o s s i b i l i t y of a consecutive r e l a t i o n s h i p whereby pa r t or a l l of the v e g e t a t i v e requirement must be f u l f i l l e d before the f l o r a l p o r t i o n of the bud becomes r e c e p t i v e t o the c h i l l i n g s t i m u l u s . Examination of the development of t e r m i n a l buds from the f i e l d r e v e a l -ed t h a t a considerable amount of d i f f e r e n t i a t i o n of the f l o r a l p r i m o r d i a occurred p r i o r t o bud break during periods when the temperatures i n the f i e l d were q u i t e low. I t has been suggested t h a t v e r n a l i z a t i o n takes p l a c e only i n c e l l s which are undergoing m i t o s i s (Wellensiek, 1962, 1964). I t i s p o s s i b l e t h a t the i n i t i a l development of the f l o w e r bud pr i m o r d i a of V, macrocarpon must take place when the p l a n t i s s t i l l being exposed t o c h i l l i n g temperatures. I f t h i s i s t r u e , i t would e x p l a i n the l a c k of f l o w e r i n g i n p l a n t s t h a t were removed from the c h i l l i n g treatments prematurely. The f a c t t h a t a longer p e r i o d of exposure t o a c t u a l temperatures below 7°G was r e q u i r e d f o r the p l a n t s c h i l l e d i n Regime 4 t o flower could be r e l a t e d t o the r e t a r d i n g 39 e f f e c t of the o v e r a l l temperatures of t h a t regime. The maximum temperature of 7°C would not a l l o w r a p i d development t o take place w i t h i n the p l a n t . For both v e g e t a t i v e and f l o r a l c h i l l i n g requirements t o be e f f i c i e n t l y f u l f i l l e d , i t would appear t h a t not only i s the amount of low temperature exposure of importance but a l s o the f l u c t u a t i n g nature of the temperatures d u r i n g the c h i l l i n g p e r i o d . I n g e n e r a l , i f the o v e r a l l temperatures are too low, the c h i l l i n g reauirement appears t o become saturated, and a d d i t i o n -a l c h i l l i n g w i l l not r e s u l t i n as great an increase i n the response t h a t would occur under warmer c o n d i t i o n s . I f o v e r a l l temperatures are too h i g h , even though a good p o r t i o n of the d a i l y c y c l e i s low enough t o s a t i s f y c h i l l i n g requirements from a thermal standpoint, t h ere may be an e f f e c t o f d e v e r n a l i z a t i o n , A balance between exposure t o low temperatures and a warm p a r t of the c y c l e t o a l l o w some a c t i v i t y i n the p l a n t appears t o be best s u i t e d t o f i l l i n g the dormancy requirement. Because of the v a r y i n g e f f e c t of the c h i l l i n g temperature c y c l e s , i t i s not p o s s i b l e t o say t h a t e i t h e r the v e g e t a t i v e or f l o r a l c h i l l i n g requirement appears t o be f u l l y met by a c e r t a i n time p e r i o d under so many degrees G, unless the a c t u a l treatment c o n d i t i o n s are s p e c i f i e d . For t h i s reason, i t i s extremely d i f f -i c u l t t o p r e d i c t when these requirements would be f u l f i l l e d under f i e l d c o n d i t i o n s . That the f l o r a l and v e g e t a t i v e c h i l l i n g requirements may be separate mechanisms i n V, macrocarpon i s supported by the r e s u l t s of the experiment i n which u n c h i l l e d p l a n t s were induced t o i n i t i a t e t e r m i n a l growth i n r e s -ponse t o a p p l i c a t i o n of g i b b e r e l l i c a c i d . I f the two c h i l l i n g requirements were one and the same, i t would be expected t h a t f l o w e r i n g would a l s o occur i n response t o the g i b b e r e l l i c a c i d . The f a c t t h a t the induced growth was only v e g e t a t i v e a l s o suggests t h a t g i b b e r e l l i c a c i d i s r e l e a s i n g a mechanism 40 t h a t i s separate from t h a t i n v o l v e d i n f l o w e r i n d u c t i o n . The r o l e of an a p p l i e d p l a n t growth substance i n breaking dormancy may or may not be r e l a t e d t o the n a t u r a l mechanism. There i s a p o s s i b i l i t y t h a t the a c t i o n of a p p l i e d g i b b e r e l l i c a c i d was a s u b s t i t u t i o n e f f e c t . In order t o determine the p o s s i b l e r o l e of g i b b e r e l l i n s i n breaking dormancy, i t was necessary t o have some i n f o r m a t i o n on the amounts of endogenous g i b b e r e l l i n - l i k e a c t i v i t y i n V . macrocarpon f o r the dormant and immediate post-dormant p e r i o d . There i s a general i n c r e a s e i n the l e v e l s of a c t i v i t y i n both buds and leaves apparent i n e a r l y March. The continued i n c r e a s e of t h i s a c t i v i t y i n the buds and the decrease i n a c t i v i t y i n the leaves demon-s t r a t e d f o r the A p r i l 6, 1970 sample suggest a t r a n s l o c a t i o n of the gibb-e r e l l i n - l i k e substances. F u r t h e r support f o r t h i s hypothesis i s obtained by examining the histograms of a c t i v i t y i n leaves and buds f o r the March 9, 1970 and A p r i l 6, 1970 sample dates. The increased a c t i v i t y of the buds on A p r i l 6 was i n the same as was the h i g h a c t i v i t y f o r the leaves on March 9. Because t h i s apparent t r a n s l o c a t i o n occurs a f t e r f l o w e r develop-ment has begun, i t seems u n l i k e l y t h a t g i b b e r e l l i n s are i n v o l v e d i n flower production f o l l o w i n g dormancy. T h i s i s f u r t h e r s u b s t a n t i a t e d by the f a i l u r e of u n c h i l l e d V , macrocarpon p l a n t s t o f l o w e r i n response t o g i b b e r e l l i c a c i d , Fontes et a l . (1970) a l s o have r e p o r t e d t h a t g i b b e r e l l i n s seem not t o be i n v o l v e d i n f l o w e r i n d u c t i o n i n b r o c c o l i . Halaban et a l . (1965) con-cluded t h a t the response of Qrnithogalum a p i c e s t o g i b b e r e l l i n i s c o n d i t i o n -ed by the thermal regime. Jones and Stoddart (1970) concluded t h a t the primary a c t i o n of g i b b e r e l l i c a c i d i n the shoot apex of r e d c l o v e r was not f l o w e r i n d u c t i o n but the i n d u c t i o n of s y n t h e s i s of s p e c i f i c p r o t e i n s . The delay observed between a p p l i c a t i o n of g i b b e r e l l i c a c i d t o dormant u n c h i l l e d V , macrocarpon p l a n t s and the v i s i b l e growth response c o u l d 41 occur i f the immediate a c t i o n of a p p l i e d g i b b e r e l l i c a c i d was t o induce p r o t e i n s y n t h e s i s i n the shoot apex. I t I s d i f f i c u l t t o p r e d i c t how long a delay between the occurrence of in c r e a s e d amounts of endogenous g i b b e r e l l -i n - l i k e substances i n the t e r m i n a l bud and the i n i t i a t i o n of v i s i b l e a c t i v -i t y would t a k e , but some delay should be expected. Jones and Stoddart (1970) r e p o r t e d a delay of 4 days between a p p l i c a t i o n of g i b b e r e l l i c a c i d and p r o t e i n s y n t h e s i s . The d i f f e r e n t stages of development reached by the flo w e r bud primor-d i a p r i o r t o the onset of dormancy i n 1968 and 1969 suggest t h a t t h e i r c e s s a t i o n of development i s not under i n t e r n a l c o n t r o l , and t h a t the i n i t -i a t i o n of dormancy i n the t e r m i n a l bud r e l i e s on the response of the veget-a t i v e p o r t i o n t o an e x t e r n a l s t i m u l u s . Once t h i s stimulus has caused the bud t o enter the dormant s t a t e , the c o n t r o l of dormancy i s i n t e r n a l u n t i l the c h i l l i n g requirement has been s a t i s f i e d . Vegetative growth i s i n i t i a t e d f o l l o w i n g s h o r t e r c h i l l i n g periods than are r e q u i r e d f o r the i n i t i a t i o n of f l o r a l growth under a r t i f i c i a l c o n d i t i o n s . I n the f i e l d , however, v e g e t a t i v e growth i s not i n i t i a t e d u n t i l a f t e r the fl o w e r bud pr i m o r d i a have begum t h e i r development. T h i s f l e x i b i l i t y i n the order of ve g e t a t i v e and f l o r a l responses t o c h i l l i n g suggests t h a t t h e i r r e s p e c t i v e p r i m o r d i a are under independent c o n t r o l mechanisms. The apparent t r a n s l o c a t i o n of g i b b e r e l l i n - l i k e sub-stances t o the t e r m i n a l bud p r i o r t o el o n g a t i o n f u r t h e r suggests t h a t w h i l e the c o n t r o l of dormancy may remain w i t h i n the bud i t s e l f , there i s under n a t u r a l c o n d i t i o n s a r e l i a n c e on the leaves t o supply t h e necessary s t i m -u l u s t o i n i t i a t e v e g e t a t i v e growth, Vegis (1964) suggested t h a t " f o r b e t t e r understanding of the o r i g i n of the dormant c o n d i t i o n i n p l a n t organs one should always take i n t o c o n s i d e r a t i o n t h a t dormancy i s the r e s u l t of a h i g h l y u s e f u l adaptation t o the environmental c o n d i t i o n s which p r e v a i l where the species or v a r i e t y o r i g i n a t e s . " V. macrocarpon i s indigenous t o Eastern North America ( P o r s i l d , 1938); thus i t i s not s u r p r i s i n g t h a t the p l a n t has a c h i l l i n g requirement which ensures t h a t dormancy i s maintained throughout the w i n t e r . The e f f e c t of the c h i l l i n g requirement on the range of t h i s species has been t w o f o l d . The obvious e f f e c t i s t h a t c h i l l i n g has been a f a c t o r i n l i m i t i n g the southern extent of the n a t u r a l range of V. macrocarpon, which occurs o c c a s i o n a l l y as f a r south as Arkansas ( F e r n a l d , 195°)• The c h i l l i n g requirement may a l s o p l a y a r o l e i n l i m i t i n g the northern range of the s p e c i e s . N e i t h e r of these range l i m i t a t i o n s , as such, has been of concern i n the cranberry production i n d u s t r y , and t h e r e f o r e they have hot been exten-s i v e l y s t u d i e d . The problem of southern range l i m i t a t i o n s due t o l a c k of c h i l l i n g has been s t u d i e d i n a number of other h o r t i c u l t u r a l crops such as b l u e b e r r i e s (Darrow, 19^2), grapes (Magoon and D i x , 19^3), and peaches (Weinberger, 1950a, 1950b), The general c o n c l u s i o n f o r a l l of these crops was t h a t l a c k of c h i l l i n g was the major f a c t o r i n l i m i t i n g the southern range f o r commercial f r u i t p r oduction. T h i s i s a l s o t r u e f o r V. macrocarpon. The r o l e of the c h i l l i n g requirement as a f a c t o r i n l i m i t i n g the northern range of the species i s somewhat more complex. I n the more northern l a t i t u d e s , the absolute minimum temperatures would most l i k e l y be r e s p o n s i b l e f o r the p l a n t s not s u r v i v i n g , but t h i s may not be the case near the border of the northern l i m i t f o r the s p e c i e s . Doughty* observed 1 C.C. Doughty, personal communication, 1971 t h a t a f t e r 557 hr below 4,5°C i n the f i e l d , V, macrocarpon could withstand temperatures as low as -20.5°C before more than 25 percent of the buds were i n j u r e d . T h i s occurred on January 30, 1966 under Washington S t a t e c o n d i t i o n s . By February 25, the temperature which would cause 25 percent i n j u r y was -12.5°C, and by March 17 was -4.0°C. The s u s c e p t i b l e p e r i o d t o f r o s t i n -j u r y apparently occurs a f t e r the flower bud primordia have recommenced t h e i r development i n the s p r i n g . T h i s development c o u l d be induced by r e l a t i v e l y warm day temperatures a f t e r the minimum c h i l l i n g requirement has been met. I t i s q u i t e p o s s i b l e t h a t the northern range of the p l a n t could be l i m i t e d not by the absolute minimum temperatures encountered d u r i n g the wint e r months but by the occurrence of low s p r i n g temperatures which would cause i n j u r y a f t e r e a r l y f u l f i l m e n t of the c h i l l i n g requirements. What may then happen i s t h a t the s u s c e p t i b i l i t y of the pl a n t t o f r o s t i n j u r y may i n -crease a t a f a s t e r r a t e than the r i s e i n minimum temperatures f o r the r e g i o n . Considerable damage t o the buds could r e s u l t i f the s u s c e p t i b l e temperature was higher than the r e g u l a r minimum temperatures. In B r i t i s h Columbia, development of the f l o r a l p r i m ordia i s w e l l under way i n March and by the end of A p r i l considerable d i f f e r e n t i a t i o n has taken pl a c e ; thus the crop i s q u i t e s u s c e p t i b l e t o f r o s t damage at a time of year when minimum temperatures s u f f i c i e n t l y low t o cause damage are not uncommon. The c u r r e n t l y recommended p r a c t i c e i n Canada i s t o i r r i g a t e u n t i l the danger of f r o s t damage i s past ( H a l l , 1969). Because of the complex nature of the f a c t o r s i n v o l v e d , determination of the s u s c e p t i b i l i t y of the crop t o f r o s t i n j u r y i s d i f f i c u l t , and f o r t h i s reason there are no r e a l g u i d e l i n e s as t o when f r o s t p r o t e c t i o n should be i n i t i a t e d . T h i s i s of concern t o the comm-e r c i a l grower i n t h a t the operation of the i r r i g a t i o n equipment w h i l e the p l a n t s are s t i l l hardy u n n e c e s s a r i l y removes from h i s p r o f i t s . 44 There axe two p o s s i b l e approaches t h a t might be used i n decreasing the p e r i o d of i r r i g a t i o n t o correspond more c l o s e l y t o the a c t u a l minimum r e q u i r e -ments c o n s i s t e n t w i t h s u f f i c i e n t crop p r o t e c t i o n . The f i r s t approach i n v o l v e s studying the response of the p l a n t t o the complexity of environmental f a c t -ors and t r y i n g t o p r e d i c t the behaviour of t e r m i n a l bud a c t i v i t y i n the f i e l d . The second approach i s t o examine the p l a n t i t s e l f f o r sig n s of a c t i v i t y and determine the minimum temperatures which w i l l cause damage once t h i s a c t i v i t y has been i n i t i a t e d . More i n v e s t i g a t i o n i s needed t o define a c c u r a t e l y the f r o s t p r o t e c t i o n requirements f o r commercial cranberry bogs. I t may be p o s s i b l e t o devise a simple yet r e l i a b l e method f o r measuring enlargement of the t e r m i n a l buds a c c u r a t e l y i n the f i e l d . I f bud enlargement co u l d be shown t o i n t e g r a t e both c h i l l i n g and d e v e r n a l i z a t i o n e f f e c t s , i t c o u l d be used i n conjunction w i t h environmental records t o p r e d i c t when f r o s t p r o t e c t i o n becomes necess-ar y . 4 5 SUMMARY The purposes of t h i s research were t o ob t a i n data on the e f f e c t of wint e r c o n d i t i o n s on the subsequent growth and development of V. macrocarpon through the use of c o n t r o l l e d environment f a c i l i t i e s , t o i n v e s t i g a t e the p o s s i b l e r o l e of g i b b e r e l l i n s i n the i n d u c t i o n of f l o w e r i n g , t o c o r r e l a t e the data obtained w i t h development of the t e r m i n a l bud under f i e l d c o n d i t -i o n s , and t o provide some b a s i c i n f o r m a t i o n r e l a t i n g t o the problem of f r o s t i n j u r y . The main r e s u l t s are summarized belows 1. The overwintering stage of the f l o r a l p r i m o r d i a i n the t e r m i n a l bud of V, macrocarpon was more advanced i n 1969-1970 than i n 1968-1969. 2. Development of the f l o r a l p r i m o r d i a recommenced i n l a t e February or e a r l y March under B r i t i s h Columbia f i e l d c o n d i t i o n s . These s t r u c t u r e s were w e l l d i f f e r e n t i a t e d by the end of A p r i l i n each of the two years s t u d i e d . 3. Dormant mixed t e r m i n a l buds t h a t were not exposed t o c h i l l i n g tempera-t u r e s d i d not i n i t i a t e new growth even under long photoperiods. 4. The number of days t o bud break f o l l o w i n g t e r m i n a t i o n of the c o n t r o l l e d environment c h i l l i n g treatments was reduced as the le n g t h of the c h i l l -i n g treatments was incre a s e d . Rate of growth and f i n a l shoot l e n g t h were a l s o a f f e c t e d , but were l e s s dependent on the le n g t h of c h i l l i n g . The i n c r e a s e d responses a f t e r longer c h i l l i n g were p o s s i b l y the r e s u l t of other p l a n t a c t i v i t i e s i n a d d i t i o n t o the f u l f i l m e n t of the c h i l l i n g requirement. 5. Longer c h i l l i n g p e r i o d s were r e q u i r e d f o r the i n d u c t i o n of f l o r a l d e v e l -opment; sho r t e r c h i l l i n g p e r i o d s r e s u l t e d i n v e g e t a t i v e growth only. I t i s suggested t h a t the f l o r a l p r i mordia may need t o undergo a c e r t a i n amount of development while c y c l i c a l l y exposed t o r e l a t i v e l y low temperatures. 46 6. I t was not p o s s i b l e t o define e i t h e r v e g e t a t i v e or f l o r a l c h i l l i n g requirements i n the form of a c e r t a i n number of hours below a c e r t a i n given temperature r e g a r d l e s s of other f a c t o r s . There was an apparent a c t i o n of the warmer pa r t of the c h i l l i n g regimes t h a t caused an a c c e l -e r a t i o n of the p l a n t responses q u i t e a s i d e from the f u l f i l m e n t of the c h i l l i n g requirements. 7. A p p l i c a t i o n of g i b b e r e l l i c a c i d t o u n c h i l l e d p l a n t s r e s u l t e d only i n veg e t a t i v e growth of the t r e a t e d p l a n t s . 8. The a c t i v i t y of endogenous g i b b e r e l l i n - l i k e substances i n both the leaves and buds of V. macrocarpon was found t o have i n c r e a s e d by March 9 of the sample year (1970). The continued increase of a c t i v i t y i n the A p r i l 6 bud sample and the r e d u c t i o n of a c t i v i t y i n the correspond-i n g l e a f sample suggested t h a t a t r a n s l o c a t i o n of the g i b b e r e l l i n - l i k e substances had occurred. The t i m i n g of t h i s t r a n s l o c a t i o n was such t h a t these substances are probably more important f o r v e g e t a t i v e than f l o r a l development. 9. The combination of r e s u l t s r e g a r d i n g the i n d u c t i o n of v e g e t a t i v e and f l o r a l development i n the t e r m i n a l bud suggested t h a t these two pro-cesses are under independent c o n t r o l . S horter c h i l l i n g p eriods r e s u l t e d i n only v e g e t a t i v e growth of the t e r m i n a l bud, but under f i e l d c o n d i t -i ons development of the f l o r a l p rimordia takes place p r i o r t o the i n i t -i a t i o n of v e g e t a t i v e growth. I t i s probable t h a t once the v e g e t a t i v e c h i l l i n g requirement has been s a t i s f i e d , a stimulus from another p l a n t organ, such as g i b b e r e l l i n - l i k e compounds from the l e a v e s , i s needed t o i n i t i a t e e l o n g a t i o n of the t e r m i n a l bud, 10. Once the b a s i c c h i l l i n g requirement has been met, the f l o r a l p r i m o r d i a w i l l , under warm daytime c o n d i t i o n s , begin t o develop. As they develop, they may become s u s c e p t i b l e t o f r o s t i n j u r y a t a r a t e f a s t e r than the 47 progressive r i s e of minimum temperatures. The r e s u l t i n g i n j u r y c o u l d be one of the prime f a c t o r s i n l i m i t i n g the northern range of V. macrocarpon. 1 1 . Further study of the morphological and p h y s i o l o g i c a l development of V, macrocarpon c u l t i v a r s under f i e l d c o n d i t i o n s i s needed t o develop ade-quate g u i d e l i n e s f o r commercial f r o s t - p r o t e c t i o n programs. 48 LITERATURE CITED Bain, H.F. 1946, Blooming and f r u i t i n g h a b i t s of the cranberry i n Wiscon-s i n , Cranberries 10(9): 11, 14. B e l l , H.P. and Jane B u r c h i l l , 1955* Winter r e s t i n g stages of c e r t a i n E r i c -aceae. Can. J . Bot. 33: 547-561. Bergman, H.F. 1947. Bud, f l o w e r , and f r u i t production by cranberry v i n e s i n r e l a t i o n t o depth of wi n t e r f l o o d i n g . Cranberries 12(3): 9-10. Bergman, H.F. 1949. Winter c o n d i t i o n s i n cranberry bogs i n r e l a t i o n t o fl o w e r and f r u i t production. Rev. Can. B i o . 7: 629-641. Bergman, H.F, 1950. Cranberry f l o w e r and f r u i t p roduction i n Massachusetts. Cranberries 15(4); 6-10, Bergman, H.F. 195^. Flowering and f r u i t i n g c h a r a c t e r i s t i c s of the cran-berry i n New Je r s e y , Proc. Amer. Cranberry Growers' Ass. P.17-27. B r i a n , P.W. 1959. Morphogenetic e f f e c t s of the g i b b e r e l l i n s . J . Linnean Soc. London Bot. 56: 237-248. Chandler, F.B. and I . Demoranville. 1958* Cranberry v a r i e t i e s of North America. Mass. Agr. Exp. S t a . B u l l . 513• Chandler, F.B. and I.E. Demoranville. 1964. Rest p e r i o d f o r c r a n b e r r i e s . Proc. Amer. Soc. Hort. S c i . 85: 307-311. Chouard, P, I960. V e r n a l i z a t i o n and i t s r e l a t i o n s t o dormancy. Annu. Rev. Pl a n t P h y s i o l . 11: 191-238. Cross, C.E. 1969. R e l a t i o n of weather c o n d i t i o n s t o production and q u a l i t y . I n Modern c u l t u r a l p r a c t i c e i n cranberry growing. Mass. Univ. Coop. Ext. Serv. P u b l . 39. P.38-40. C r o z i e r , A., H. A o k i and R.P. P h a r i s . 1969. E f f i c i e n c y of countercurrent d i s t r i b u t i o n , sephadex G-10, and s i l i c i c a c i d p a r t i t i o n chromatography i n the p u r i f i c a t i o n and se p a r a t i o n of g i b b e r e l l i n - l i k e substances from p l a n t t i s s u e . J . Exp. Bot. 20: 786-795. C u t t e r , E l i z a b e t h G. 1965. Recent experimental s t u d i e s of the shoot apex and shoot morphogenesis. Bot. Rev, 31: 7-113. Darrow, G.M, 1942. Rest p e r i o d requirement f o r b l u e b e r r i e s . Proc. Amer. Soc. Hort. S c i . 4 l : 181-194. D e v l i n , R.M, and I.E. Demoranville. 1967. Influence of g i b b e r e l l i c a c i d and G i b r e l on f r u i t set and y i e l d i n Vaccinium macrocarpon cv. E a r l y Black. P h y s i o l . P l a n t . 20: 587-592. Doorenbos, J . 1953. Review of the l i t e r a t u r e on dormancy i n buds of woody p l a n t s . Meded. Landbouwhogesch. Wageningen 51: 1-24. 49 Eady, F. and G.W. Eaton. 1969. Reduced c h i l l i n g requirement of M c F a r l i n cranberry buds. Can. J . P l a n t S c i . 49: 637-638. Eagles, C.F. and P.F. Wareing. 1963. Dormancy r e g u l a t o r s i n woody p l a n t s . Nature 199: 874-875. Eaton, G.W. 1970. Cranberry production i n B r i t i s h Columbia. C r a n b e r r i e s 35(2): 13-14. Eaton, G.W. and D.P. Ormrod, 1968. Photoperiod e f f e c t on p l a n t growth i n cranberry. Can. J . P l a n t S c i . 48: 447-450. F e r n a l d , M.L. 1950. Gray's Manual of Botany. 8th E d i t . American Book Co. New York, Fontes, M.R., J.L, Ozbun and L.E. P o w e l l , 1970. Are endogenous g i b b e r e l l i n -l i k e substances i n v o l v e d i n f l o r a l i n d u c t i o n . Nature 228: 82-83. Frankland, B, and P.F, Wareing, I960. E f f e c t of g i b b e r e l l i c a c i d on hypo-c o t y l growth of l e t t u c e s e e d l i n g s . Nature I85: 255-256. G a r l i c k , D.H. 1966. The growth, f l o w e r i n g and f r u i t i n g c h a r a c t e r i s t i c s of the cranberry. Unpublished B.S.A. T h e s i s , Univ. of B.C. Goff, E.S, 1901. I n v e s t i g a t i o n of flower-buds. Ann, Rept. Wis, Agr, Exp. S t a . 18: 304-319. Halaban, Ruth, E. Galun and A.H. Halevy. 1965. Experimental morphogenesis of stem t i p s of Ornithogalum arabicum L. c u l t u r e d i n v i t r o . Phytomorphology 15: 379-387. H a l l , I.V. 1969. Growing c r a n b e r r i e s . Can. Dep. Agr. P u b l . 1282. H a r t l e y , R.D., T.A. H i l l , G.F, Pegg and G.G. Thomas. 1969. Solvent and chemical i m p u r i t i e s as sources of g i b b e r e l l i n - l i k e growth promoting a c t i v i t y . J . Exp. Bot. 20: 276-287. Hayashi, F, and L, Rappaport, 1962. G i b b e r e l l i n - l i k e a c t i v i t y of n e u t r a l and a c i d i c substances i n the potato t u b e r . Nature 195* 617-618. H i l l m a n , W.S. 1969. Photoperiodism and v e r n a l i z a t i o n . In M.B. W i l k i n s (Ed.), Physiology of p l a n t growth and development. McGraw-Hill, London. P.560-601. Jensen, W.A. ' 1962. B o t a n i c a l h i s t o c h e m i s t r y . W.H, Freeman and Co., San F r a n c i s c o , Jones, T.W.A. and J.L, Stoddart. 1970. G i b b e r e l l i n - i n d u c e d changes i n p r o t e i n s y n t h e s i s and enzyme a c t i v i t y i n shoot a p i c e s of T r i f o l i u m  pratense. J . Exp. Bot. 21s 452-461. L a c r o i x , D.S, 1926, Cranberry flower-bud i n v e s t i g a t i o n s . J . A g r i c . Research 33: 355-363. 50 Lang, A. 1965. Physiology of flower i n i t i a t i o n . I n Encyclopedia of Pl a n t Physiology. S p r i n g e r - V e r l a g , B e r l i n . X V / l : 138O-1536. M a c h l i s , L. and J.G, Torrey. 1956. P l a n t s i n a c t i o n . W.H. Freeman and Co., San F r a n c i s c o . Magoon, C.A. and I.W. Di x , 1943. Observations on the response of grape vi n e t o winter temperatures as r e l a t e d t o t h e i r dormancy requirements. Proc. Amer. Soc. Hort. S c i . 42: 407-412. Mainland, CM. and P. Eck. 1968. Cranberry f r u i t s e t , growth, and y i e l d as i n f l u e n c e d by g i b b e r e l l i c a c i d alone and i n combination w i t h A l a r . Proc. Amer. Soc. Hort. S c i . 92: 296-300. P a l s e r , Barbara F, 1961. S t u d i e s of f l o r a l morphology i n the E r i c a l e s . V, Organography and v a s c u l a r anatomy i n s e v e r a l United States s p e c i e s of the Vacciniaceae. Bot. Gaz, 123: 79-111. P e r r y , T.O. 1971. Dormancy of t r e e s i n w i n t e r . Science 171: 29-36. P o r s i l d , A.E. 1938. The cranberry i n Canada. Can. F i e l d Natur. 51s 116-117. Reid, D.M., A. C r o z i e r and Barbara M.R. Harvey. 1969. The e f f e c t s of f l o o d i n g and the export of g i b b e r e l l i n s from the r o o t t o the shoot. P l a n t a 89: 376-379. Roberts, R.H. and B, Esther Struckmeyer. 1948. Blossom i n d u c t i o n of the cranberry. P l a n t P h y s i o l . 18: 534-536. Romberger, J.A, 1963. Meristems, growth and development i n woody p l a n t s . U.S. Dep. Agr. Tech. B u l l . 1293. S a l i s b u r y , F.B, 1963. The f l o w e r i n g process. Pergamon Press, New York, Samish, R.M. 195^. Dormancy i n woody p l a n t s . Annu. Rev. P l a n t P h y s i o l , 5: 183-204. Sharman, B.C. 1943. Tannic a c i d and i r o n alum w i t h s a f r a n i n and orange G i n s t u d i e s of the shoot apex. S t a i n Technol. 18: 105-111. Smith, H. and N.P. K e f f o r d . 1964. The chemical r e g u l a t i o n of the dormancy phases of bud development. Amer. J . Bot, 51: 1002-1012. Vegis, A. 1964. Dormancy i n higher p l a n t s . Annu. Rev. P l a n t P h y s i o l . 15: 185-224. Wareing, P.F. 1961, Dormancy of woody p l a n t s . In Recent advances i n botany. Ninth I n t e r n a t i o n a l B o t a n i c a l Congress, Montreal. Univ. of Toronto Press. P.1212-1219. Wareing, P.F. 1969. Germination and dormancy. In M.B. W i l k i n s (Ed.). Physiology of p l a n t growth and development. McGraw-Hill, London, P.605-644. 51 Weinberger, J.H, 1950a. Chilling requirements of peach varieties. Proc. Amer. Soc. Hort. S c i . 56: 122-128. Weinberger, J.H. 1950b, Prolonged dormancy of peaches. Proc. Amer. Soc. Hort. Sci. 56: 129-133. Wellensiek, S.J, 1962. Dividing c e l l s as the locus for vernalization. Nature 195: 307-308. Wellensiek, S.J, 1964. Dividing cells as the prerequisite for vernalizat-ion. Plant Physiol. 39: 832-835. APPENDIX 1. L i g h t Sources of C o n t r o l l e d Environment F a c i l i t i e s Facility Number of Lamps  Tungsten filament Fluorescent Intensity (klux) at plant level  Sherer Model CEL-266-6 Full l ight One-half l ight Percival Model PGC-78 Cold Room Photoperiod Cabinets 4 • 8 7.1 4 4 3.6 10 16 7.9 3 4 2.1 1 2 1.1 1 All lamps listed were 40 watt. APPENDIX 2. A n a l y s i s of Variance 1968-1969 Experiment Days to Bud Break Source Chilling time Chamber Residual Total Shoot Length Source Chilling time Chamber Residual Total Floviersrper iPlaht; . Source Chilling time Chamber Residual Total 1969-1970 Experiment Percent Bud Break - Factorial Source Chilling time (CT) Regime Regime x CT Residual Total Percent Bud Break - Regime 1 Source Chilling time Residual Total Percent Bud Break - Regime 2 Source Chilling time Residual Total d.f. SS f_. 4 4381.53 119.6* 1 0.30 0.03 24 228.87 29 4610.70 d.f. SS_ £ 4 63.139 2.23 1 6.912 0.98 24 170.05 29 240.10 d.f. SS. F_ 4 3.199 0.26 1 3.332 1.07 24 74.658 29 81.190 d.f. SS. I 4 31888.0 76.99* 3 3927.5 12.64* 12 3104.7 2.50* 20 2071.0 39 40991.0 d.f. SS F_ 5 6628.8 10.34* 6 769.5 11 7398.2 d.f. SS F_ 5 12497.0 52.99* 6 283.0 11 12780.0 * Statistically significant P = 0.05 56 Percent Bud Break - Regime 3 Source d.f. SS Chilling time 4 12367.0 82.45* Residual 5 187.5 Total 9 12555.0 Percent Bud Break - Regime 4 Source d.f. SS _F_ Chilling time 4 6625.4 9.51* Residual 5 871.0 Total 9 7496.4 Percent Bud Break - 25 Days Chilling Source d.f. SS J_ Regime 3 1154.0 1.61 Residual 4 958.5 Total 7 2112.5 Percent Bud Break - 50 Days Chilling Source d.f. SS F_ Regime 3 2634.4 4.46 Residual 4 787.5 Total 7 3421.9 Percent Bud Break - 75 Days Chilling Source d.f. SS F_ Regime 3 2909.4 16.33* Residual 4 237.5 Total 7 3146.9 Percent Bud Break - 100 Days Chilling Source d.f. SS F_ Regime 3 75.0 4.00 Residual 4 25.0 Total 7 100.0 Percent Bud Break - 125 Days Chilling Source d.f. SS J_ Regime 3 259.4 5.53 Residual 4 62.5 Total 7 321.9 Percent Bud Break - 150 Days Chilling Source d.f. SS £_ Regime 1 36.0 1.80 Residual 2 40.0 Total 3 76.0 * Statistically significant P = 0.05 Days to Bud Break - Factorial Source Chilling time (CT) Regime Regime x CT Residual Total d.f. 4 3 12 156 175 SS 456500.0 177590.0 2239800.0 1126.9 2875000.0 15798.5* 8194.5* 25838.7* Days to Bud Break - Regime 1 Source Chilling time Residual Total Days to Bud Break - Regime 2 Source Chilling time Residual Total Days to Bud Break - Regime 3 Source Chilling time Residual Total Days to Bud Break - Regime 4 Source Chilling time Residual Total Days to Bud Break Source Regi me Residual Total Days to Bud Break Source Regime Residual Total d.f. 5 52 . 57 d.f. 5 48 53 d.f. 4 37 41 d.f. 4 40 44 25 Days Chilling d.f. 2 14 16 50 Days Chilling d.f. 3 32 35 SS_ 3109.1 324.6 3433.7 SS_ 5082.3 156.5 5238.8 SS_ 3514.6 395.9 3910.5 SS. 481.5 283.7 765.2 511.7 359.8 871.5 ss 1018.5 544.5 1563.0 F_ 311.7* F_ 82.f F_ 16.9* F_ 9.9* 19.9* Statistically significant P = 0.05, Days to Bud Break - 75 Days Chilling 58 Source d.f. _SS_ £ Regime 3 193.5 40.38* Residual 36 57.5 Total 39 251.0 Days to Bud Break - 100 Days Chilling Source d.f. SS_ F_ Regime 3 22.1 3.02* Residual 36 87.8 Total 39 109.9 Days to Bud Break - 125 Days Chilling Source d.f. SS F_ Regime 3 45.7 7.09* Residual 36 77.3 Total 39 123.0 Days to Bud Break - 150 Days Chilling Source d.f. SS _F_ Regime 1 1.8; 1.23 Residual 18 26.4 Total 19 28.2 Growth Rates - Factorial Source d.f. SS _F_ Chilling time (CT) 4 0.21072 20.61* Regime 3 0.010176 1.33 Regime x CT 12 0.04946 1.61 Residual 20 0.051109 Total 39 0.32147 Growth Rates - Regime 1 Source d.f. SS J_ Chilling time 5 0.08647 9.33* Residual 6 0.01112 Total 11 0.09759 Growth Rates - Regime 2 Source d.f. SS F_ Chilling time 5 0.09505 9.12* Residual 6 0.01251 Total 11 0.10756 * Statistically significant P = 0.05 Growth Rates - Regime 3 59 Source Chilling time Residual Total Growth Rates - Regime 4 Source Chilling time Residual Total Mean Shoot Length - Factorial Source Chilling time (CT) Regime Regime x CT Residual Total Mean Shoot Length - Regime 1 Source Chilling time Residual Total Mean Shoot Length - Regime 2 Source Chilling time Residual Total Mean Shoot Length - Regime 3 Source Chilling time Residual Total Mean Shoot Length - Regime 4 Source Chilling time Residual Total d.f. SS. £ 4 0.10400 4.37 5 0.02975 9 0.13375 d.f. SS_ £ 4 0.05504 7.03* 5 0.00978 9 0.06482 d.f. SS. £ 3 703.59 49.07* 4 50.98 4.74* 12 110.92 2.58* 20 71.69 39 937.18 d.f. SS £ 5 131.83 3.94 6 40.15 11 171.98 d.f. . SS. £ 5 289.72 42.12* 6 8.25 11 297.97 d.f. SS £ 4 332.08 20.65* 5 20.11 9 352.19 d.f. SS_ £ 4 137.50 8.31* 5 20.68 9 158.19 * Statistically significant P = 0.05 Mean Shoot Length - 25 Days Chilling Source d.f. SS Regime 3 66.91 Residual 4 29.02 Total 7 95.93 Mean Shoot Length - 50 Days Chilling Source d.f. SS Regime 3 4.96 Residual 4 9.97 Total 7 14.93 Mean Shoot Length - 75 Days Chilling • Source d.f. SS Regime 3 21.33 Residual 4 2.41 Total 7 23.74 Mean Shoot Length - 100 Days Chilling Source d.f. SS Regime 3 3.75 Residual 4 12.95 Total 7 16.70 Mean Shoot Length - 125 Days Chilling Source d.f. SS Regime 3 64.94 Residual 4 17.34 Total 7 82.28 Mean Shoot Length - 150 Days Chilling Source d.f. SS Regime 1 0.35 Residual 2 17.50 Total 3 17.85 Mean Number of Flowers per 20 Plants - Regime 1 Source d.f. SS Chilling time 2 -412.33 Residual 3 37.00 Total 5 449.33 * Statistically significant P = 0.05 Mean Number of Flowers per 20 Plants - Regime 2 Source d.f. SS Chilling time 2 1417.0 Residual 3 221.0 Total 5 1638.0 Mean Number of Flowers per 20 Plants - Regime 3 Source d.f. SS Chilling time 1 64.00 Residual 2 4.00 Total 3 68.00 Mean Number of Flowers per 20 Plants - Regime 4 Source d.f. SS Chilling time 2 180.28 Residual 3 36.42 Total 5 216.70 * Statistically significant P = 0.05 APPENDIX 3. Dry Weights and Minimum Detectable L e v e l s of G i b b e r e l l i n - l i k e A c t i v i t y i n Terminal Bud and Leaf Samples Sample C o l l e c t i o n Date I ni t i al A f t e r MeOH E x t r a c t i on Oc t . 17/69 0.1873 0.1359 Nov. 18/69 0.4990 0.0610 Dec. 18/69 0.4860 0.0323 Feb. 3/70 0.3864 0.1982 . Feb. 23/70 0.4930 0.0745 Mar. 9/70 •0,3429 . 0.0632 Apr . 6/70 0.7051 0.1146 Apr . 27/70 0.5335 0.1350 May 4/70 0.5025 0.1216 May 19/70 0.5460 0.1461 June 22/70 1.6596 0.1656 Bud Samples Dry Weight (g ) A f t e r PVP Col umn 0.0177 0.0273 0.0110 0.0220 0.0073 0.0117 0.0072 0.0224 0.0427 0.0244 0.0160 A f t e r Charcoal - C e l i t e column 0.0102 0.0110 0.0093 0.0138 0.0052 0.0097 0.0057 0.0109 0.0163 0.0114 0.0010 Minimum De tec tab le Level of GA—1 i ke A c t i v i t y t " q GA/q t i s s u e )  2 .6 X 10 1.0 X 1 0 " 1.0 X 1 0 " 1.3 X 1 0 " 1.0 X 1 0 " 1.5 X 1 0 " 7.1 X 1 0 " 9.4 X 1 0 " 9 .9 X 1 0 " 9.2 X 1 0 " 3 .0 X 1 0 " ON Sample Colliection \ Date I ni t i al After MeOH Extraction Nov. 18/69 15.00 1.4705 Dec. 18/69 15.00 1.3340 Feb. 3/70 15.00 1.4647 Feb. 23/70 15.00 1.2520 Mar. 9/70 15.00 1.5090 Apr. 6/70 15.00 1.6434 Apr. 27/70 15.00 1.4729 . May 19/70 15.00 1.3771 Leaf Samples Dry Weight (g) After PVP Col umn 0.1219 0.1190 0.0906 0.0794 0.0982 0.1216 0.1157 0.0586 After Charcoal -Celite column 0.0504 0.0563 0.0520 0.0377 0.0641 0.0879 0.0866 0.0443 Minimum detectable Level of GA-like activity (*«g GA/g tissue)  3.3 x 10"3 3.3 x 10"3 3.3 x 10"3 3.3 x 10"3 3.3 x 10"3 3.3 x 10"3 3.3 x 10"3 3.3 x 10' 3 ON 

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