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Light quality effects on in vitro shoot proliferation of Spiraea nipponica Herrington, Edward John 1990

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LIGHT QUALITY EFFECTS ON IN VITRO SHOOT PROLIFERATION OF SPIRAEA NIPPONICA B . S c . ( B o t a n y ) , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1990 (c) Edward John H e r r i n g t o n , 1990 By Edward John H e r r i n g t o n MASTER OF SCIENCE i n PLANT SCIENCE In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract i i The work on Spiraea i n v i t r o shoot c u l t u r e s was done t o determine the f e a s i b i l i t y of u s i n g l i g h t q u a l i t y t o modify endogenous phytohormone balances t o decrease a p i c a l dominance. Such an e f f e c t would enable a r e d u c t i o n i n the high l e v e l s of exogenous c y t o k i n i n benzyladenine (BA) a p p l i e d i n c u l t u r e and thus reduce p o t e n t i a l s i d e - e f f e c t s . The Spiraea i n v i t r o l i g h t q u a l i t y response was c h a r a c t e r i z e d by examining the e f f e c t s of d i f f e r e n t l i g h t wavelengths on growth. A mixture of red/FR induced r a t e s of shoot p r o l i f e r a t i o n w i t h 0.25 mg/1 BA t h a t were as h i g h as r a t e s obtained under white l i g h t w i t h 0.5 mg/1 BA. Shoot q u a l i t y , as determined by the p r o p o r t i o n of shoots 1 cm or longer ( u s e f u l s h o o t s ) , was h i g h e s t under red/FR l i g h t . The lowest shoot p r o l i f e r a t i o n r a t e was observed under blue l i g h t . When l i g h t wavelengths intermediate between blue and red l i g h t (green, y e l l o w , and orange) were a p p l i e d t o explants only minor growth m o d i f i c a t i o n s occurred. Green l i g h t d i d not i n h i b i t shoot i n i t i a t i o n but i n h i b i t e d shoot e l o n g a t i o n at the 0.5 mg/1 BA l e v e l . The e f f i c a c y of the l i g h t s o u r c e - f i l t e r combinations i n the f i r s t experiment was s t u d i e d i n two f u r t h e r experiments. With the t h r e e l i g h t sources (tungsten f i l a m e n t , f l u o r e s c e n t , and metal h a l i d e ) together w i t h a blue f i l t e r , r e s u l t s supported the p u t a t i v e blue l i g h t i n h i b i t o r y e f f e c t suggested i n the f i r s t l i g h t q u a l i t y experiment. Under the red f i l t e r , the tungsten f i l a m e n t source induced the highest shoot number means at both BA l e v e l s used (0.25 and 0.5 mg/1). Two f a c t o r s may have c o n t r i b u t e d t o the red/FR e f f e c t observed i n the f i r s t experiment; the time under an i n c u b a t i o n l i g h t regime before t r a n s f e r t o the treatment regime, and the photon f l u e n c e r a t e of each regime. In the subsequent study t o examine these f a c t o r s , shoot i n i t i a t i o n was optimized at the lower BA l e v e l s of 0.25 and 0.4 mg/1 when c u l t u r e s under low f l u e n c e red/FR were t r a n s f e r r e d a f t e r f o u r weeks t o white l i g h t of a h i g h e r f l u e n c e f o r one more week. Glyphosate, a known promoter of IAA o x i d a t i o n , was used t o i n v e s t i g a t e the presumed e f f e c t of lowered I A A - c y t o k i n i n i n t e r a c t i o n s . Two types of responses t o glyphosate occurred, each one dependent on the glyphosate c o n c e n t r a t i o n . At the lower glyphosate l e v e l (0.087 mg/1), c u l t u r e s under both l i g h t regimes w i t h 0.25 mg/1 of BA, showed a s t r o n g i n h i b i t i o n of shoot i n i t i a t i o n . This i n h i b i t o r y e f f e c t was overcome i n c u l t u r e s w i t h 0.5 mg/1 of BA and an o v e r a l l s t i m u l a t o r y response occurred as shoot i n i t i a t i o n r a t e s were as much as f o u r - f o l d higher than i n the previous experiments. For both BA l e v e l s , changes i n shoot number were g r e a t e r under white l i g h t than under red/FR. At the h i g h e r glyphosate l e v e l (0.2 67 mg/1), the shoot i n i t i a t i o n r a t e s were g r e a t e r than g l y p h o s a t e - f r e e c o n t r o l s f o r both BA l e v e l s under white l i g h t although under red/FR the r a t e s were v i r t u a l l y unchanged from c o n t r o l s . The glyphosate e f f e c t i n v e s t i g a t e d f o r S p i r a e a c u l t u r e s appears t o be i n f l u e n c e d by the l e v e l s of the c y t o k i n i n BA r e s u l t i n g i n p l e i o t r o p i c e f f e c t s which depend on the s p e c i f i c c o n c e n t r a t i o n s of each component. Table of Contents Abstract i i L i s t of Tables v i i L i s t of Figures x i Acknowledgement x i i 1 Introduction 1 2 L i t e r a t u r e Review 9 2.1 The c o n t r o l o f i n v i t r o p l a n t growth by t h e e n v i r o n m e n t a l f a c t o r o f l i g h t 9 2.2 Phytohormones as used f o r i n v i t r o p l a n t c u l t u r e 15 2.2.1 A u x i n s 15' 2.2.2 C y t o k i n i n s 17 2.2.3 C y t o k i n i n s i d e - e f f e c t s 18 2.3 G l y p h o s a t e as a growth r e g u l a t o r f o r i n v i t r o p l a n t c u l t u r e 19 3 Materials and Methods 22 3.1 C u l t u r e medium and i t s p r e p a r a t i o n 22 3.2 L i g h t S o u r c e s 24 3.3 Photon f l u e n c e r a t e s 2 6 3.4 F i l t e r s 28 3.5 E x p l a n t p r e p a r a t i o n and i n c u b a t i o n c o n d i t i o n s 28 3.6 H a r v e s t i n g p r o c e d u r e 34 3.7 E x p e r i m e n t a l d e s i g n f o r each experiment 34 3.8 Growth v a r i a t e s measured and s t a t i s t i c a l a n a l y s e s ... 37 4 Results and Discussion 38 4.1 C h a r a c t e r i z a t i o n o f t h e S p i r a e a n i p p o n i c a i n v i t r o l i g h t q u a l i t y r e s p o n s e 38 v i 4.1.1 E x p e r i m e n t I - D e t e r m i n i n g t h e e f f e c t s o f b l u e , red/FR, and w h i t e l i g h t on i n v i t r o shoot p r o l i f e r a t i o n ... 38 4.1.2 E x p e r i m e n t I I - D e t e r m i n i n g t h e e f f e c t s on i n v i t r o s h o ot p r o l i f e r a t i o n o f t h r e e i n t e r m e d i a t e w a v e l e n g t h ranges between b l u e and r e d w a v e l e n g t h s 51 4.1.3 E x p e r i m e n t I I I - D e t e r m i n i n g t h e e f f e c t s on i n v i t r o shoot p r o l i f e r a t i o n o f t h r e e l i g h t s o u r c e - b l u e f i l t e r c o m b i n a t i o n s 59 4.1.4 E x p e r i m e n t IV - D e t e r m i n i n g t h e e f f e c t s on i n v i t r o s h o o t p r o l i f e r a t i o n o f t h r e e l i g h t s o u r c e - r e d f i l t e r c o m b i n a t i o n s 68 4.1.5 E x p e r i m e n t V D e t e r m i n i n g t h e e f f e c t o f a s h o r t - t e r m f a r - r e d l i g h t e x p o s ure a p p l i e d a t t h e end o f a w h i t e l i g h t p h o t o p e r i o d 78 4.2 F a c t o r s c o n t r i b u t i n g t o t h e red/FR l i g h t growth r e s p o n s e , 87 4.2.1 E x p e r i m e n t VI -The e f f e c t s o f l e n g t h o f exposure t o a low photon f l u e n c e r a t e , and t r a n s f e r t i m e t o a h i g h e r f l u e n c e r a t e , on t h e red/FR l i g h t growth response 87 4.3 D e t e r m i n i n g i f g l y p h o s a t e and l i g h t q u a l i t y i n t e r a c t t o m o d i f y i n v i t r o growth 98 4.3.1 E x p e r i m e n t V I I - G l y p h o s a t e - l i g h t q u a l i t y e x p e r i m e n t 98 5 General D i s c u s s i o n and Conclusions 114 5.1 C h a r a c t e r i z a t i o n o f t h e S p i r a e a n i p p o n i c a i n v i t r o l i g h t q u a l i t y r e s p o n s e 114 5.2 F a c t o r s c o n t r i b u t i n g t o t h e red/FR l i g h t growth r e s p o n s e 124 5.3 D e t e r m i n i n g i f g l y p h o s a t e and l i g h t q u a l i t y i n t e r a c t t o m o d i f y i n v i t r o growth 127 Summary 131 B i b l i o g r a p h y 133 V I I L i s t o f T a b l e s 3.1 S p e c t r a l d i s t r i b u t i o n of the energy i n the v i s i b l e r e g i o n of the r a d i a t i o n of tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t l i g h t sources 25 3.2 Photon f l u e n c e r a t e s used i n the experiments 27 3.3 I n c u b a t i o n c o n d i t i o n s f o r s t o c k c u l t u r e s 32 4.1 Red l i g h t : f a r - r e d l i g h t and blue l i g h t : red l i g h t r a t i o s f o r u n f i l t e r e d tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t l i g h t sources 40 4.2 Shoot and node number means under blue, red/FR, and white l i g h t , w i t h and without BA 42 4.3 Summarized an a l y s e s of v a r i a n c e . E f f e c t s of blue, red/FR, and white l i g h t , and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry weights 4 4 4.4 Shoot l e n g t h s as a f f e c t e d by BA and t h r e e l i g h t q u a l i t i e s : b lue, red/FR, and white 47 4.5 F r e s h and dry weight means under blue, red/FR, and white l i g h t , w i t h and without BA 49 4.6 Shoot and node number means under t h r e e i n t e r m e d i a t e wavelengths (green, yellow, and orange) between blu e and r e d l i g h t . 52 4.7 Summarized an a l y s e s of v a r i a n c e . E f f e c t s of t h r e e l i g h t q u a l i t i e s between b l u e and red l i g h t (green, yellow, and orange) and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry weights 54 « • • V 1 n 4-8 Shoot l e n g t h s as a f f e c t e d by BA and t h r e e l i g h t q u a l i t i e s between blue and red: green, yellow, and orange 56 4.9 F r e s h and dry weight means under t h r e e i n t e r m e d i a t e wavelengths (green, yellow, and orange) between blue and red l i g h t 57 4.10 Shoot and node number means under t h r e e l i g h t sources (tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t ) combined wi t h a blue a c e t a t e f i l t e r 60 4.11 Summarized analyses of v a r i a n c e . E f f e c t s of l i g h t s o u r c e - b l u e f i l t e r combination and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry weight 62 4.12 Shoot l e n g t h s as a f f e c t e d by t h r e e l i g h t s ource-blue f i l t e r combinations 65 4.13 F r e s h and dry weight means under t h r e e l i g h t sources (tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t ) combined with a blue a c e t a t e f i l t e r 66 4.14 Shoot and node number means under t h r e e l i g h t sources (tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t combined wi t h a red a c e t a t e f i l t e r 69 4.15 Summarized analyses of v a r i a n c e . E f f e c t s of l i g h t s o u r c e - r e d f i l t e r combination and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry weights 71 4.16 Shoot l e n g t h s as a f f e c t e d by three l i g h t s ource-red f i l t e r combinations 74. 4.17 F r e s h and dry weight means under t h r e e l i g h t sources (tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t ) combined with a red a c e t a t e f i l t e r 77 4.18 Shoot and node number means as a f f e c t e d by a short - t e r m f a r - r e d l i g h t exposure a p p l i e d at the end of a white l i g h t p h o t o p e r i o d 81 4.19 Summarized a n a l y s e s of v a r i a n c e . E f f e c t s of a short - t e r m f a r - r e d l i g h t exposure a p p l i e d a t the end of a white l i g h t p h o t o p e r i o d and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry weights 8 3 4.20 Shoot l e n g t h s as a f f e c t e d by a sh o r t - t e r m f a r - r e d l i g h t exposure a p p l i e d a t the end of a white l i g h t photoperiod.85 4.21 Fre s h and dry weight means as a f f e c t e d by a sh o r t - t e r m f a r - r e d l i g h t exposure a p p l i e d a t the end of a white l i g h t p h o t o p e r i o d 86 4.22 Shoot and node number means as a f f e c t e d by exposure t o a low photon f l u e n c e r a t e and the time of t r a n s f e r t o a h i g h e r f l u e n c e r a t e 89 4.23 Summarized a n a l y s e s of v a r i a n c e . E f f e c t s of exposure to a low photon f l u e n c e r a t e , t r a n s f e r time t o a h i g h e r f l u e n c e , and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry weights 92 4.24 Shoots l e n g t h s as a f f e c t e d by a low photon f l u e n c e r a t e and the t r a n s f e r t o a h i g h e r f l u e n c e r a t e 94 4.25 F r e s h and dry weight means as a f f e c t e d by exposure t o a low photon f l u e n c e r a t e and the time of t r a n s f e r t o a h i g h e r f l u e n c e r a t e 96 4.2 6 P r o p o r t i o n of each treatment's f o u r samples showing g l y p h o s a t e e f f e c t s 102 X 4.27 Shoot and node number means as a f f e c t e d by gly p h o s a t e and l i g h t q u a l i t y 104 4.27a Node number/shoot means 107 4.28 An a l y s e s of v a r i a n c e summary. E f f e c t s o f l i g h t q u a l i t y , g l y p h o s a t e , and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry weights 108 4.29 Shoot l e n g t h s as a f f e c t e d by BA, glyphosate, and l i g h t q u a l i t y I l l 4.30 F r e s h and dry weight means as a f f e c t e d by gl y p h o s a t e and l i g h t q u a l i t y 112 L i s t of Figures 3.1 T r a n s m i s s i o n c h a r a c t e r i s t i c s o f t h e a c e t a t e f i l t e r s u s e d i n E x p e r i m e n t I 29 3.2 T r a n s m i s s i o n c h a r a c t e r i s t i c s o f t h e a c e t a t e f i l t e r s u s e d i n E x p e r i m e n t I I 30 3.3 S p e c t r a l energy d i s t r i b u t i o n o f t u n g s t e n f i l a m e n t l i g h t w i t h and w i t h o u t a Lee medium r e d a c e t a t e f i l t e r 32 4.1 The e f f e c t o f 3.56 mg/1 o f g l y p h o s a t e on S p i r a e a shoot p r o l i f e r a t i o n 100 \ x i i Acknowledgement I would l i k e t o thank Dr. C o l i n N o r t o n f o r p l a n t i n g t h e seed o f t h i s t h e s i s . A l s o , Dr. George Eaton's d e f t hand a t s t a t i s t i c s was g r e a t l y a p p r e c i a t e d . A s p e c i a l thank you goes t o Dr. J o a n McPherson. Her knack f o r p e r c e i v i n g p a t t e r n s i n my d a t a , t y i n g l o o s e t h r e a d s , and r a i s i n g h y p o t h e t i c a l q u e s t i o n s , made me r e a l i z e how s c i e n c e works. Committee members, Dr. J o l l i f f e , Dr. V i d a v e r , Dr. E l l i s , and Dr. I . E . P . T a y l o r a r e g r a t e f u l l y acknowledged f o r t h e i r i n p u t . I g i v e warm kudos t o my f e l l o w g r a d u a t e s t u d e n t s . Our c a m a r a d e r i e was a g r a d s c h o o l b e n e f i t I had n o t a n t i c i p a t e d . F i n a l l y , I am i n d e b t e d t o my f a m i l y f o r t h e i r unwavering s u p p o r t . W i t h o u t them t h e f r u i t s o f my l a b o u r would l o n g ago have w i t h e r e d on t h e v i n e . 1 Chapter 1 Introduction T r a d i t i o n a l v e g e t a t i v e propagation techniques i n v o l v e u s i n g such m a t e r i a l as stem and l e a f c u t t i n g s . These can be viewed as 'macropropagation 1 techniques (George and Sherrington, 1984). In v i t r o or t i s s u e c u l t u r e propagation techniques i n v o l v e r e l a t i v e l y s m a l l p l a n t pieces termed explants. Such explants can be used t o i s o l a t e p r o t o p l a s t s , induced t o form c a l l u s , or used as a d i r e c t source f o r shoot p r o l i f e r a t i o n . This l a s t o p t i o n , where s m a l l shoots are made t o a r i s e from " p r e - e x i s t i n g v e g e t a t i v e meristems" (Hutchinson and Zimmerman, 1987), i s the most common meaning f o r the term 'micropropagation 1 and i s o f t e n used t o d i s t i n g u i s h t h i s method from other t i s s u e c u l t u r e methods such as somatic embryogenesis. In v i t r o c u l t u r e methods o f f e r s e v e r a l advantages over t r a d i t i o n a l propagation methods. These i n c l u d e a means t o e l i m i n a t e d isease-causing v i r u s e s , b a c t e r i a and fu n g i v i a the a s e p t i c techniques used, the p o t e n t i a l t o r a p i d l y s c a l e up new v a r i e t i e s and c u l t i v a r s f o r commercial s a l e s , and a way t o propagate p l a n t s r e c a l c i t r a n t t o other methods of propagation (George and Sher r i n g t o n , 19 84). Furthermore, true-to-form clones 2 can be maintained ( e s p e c i a l l y v i a the a x i l l a r y - d e r i v e d shoots of micropropagation) (George and Sherrington, 1984). The commercial p o t e n t i a l of propagating p l a n t s by i n v i t r o methods i s good. Although labour c o s t s are hig h , the small space requirements needed f o r m a i n t a i n i n g l a r g e p l a n t i n v e n t o r i e s and the year-round production c a p a b i l i t y can o f f s e t h i g h i n i t i a l c a p i t a l expenditures (George and S h e r r i n g t o n ) . Production f i g u r e s f o r the United States i n 1985 i n d i c a t e d a wholesale market v a l u e of 22.51 t o 24.35 m i l l i o n d o l l a r s f o r 60.7 t o 65.7 m i l l i o n i n v i t r o d e r i v e d p l a n t s (Jones, 1986). Of t h i s 1985 U.S. pr o d u c t i o n t o t a l , 20.5 t o 25.5 m i l l i o n p l a n t s were c a t e g o r i z e d as woody ornamentals and f r u i t crops (Jones, 1986). A Dutch study ( P i e r i k , 1985) concluded t h a t roses and landscape ornamentals would be the p l a n t types showing the g r e a t e s t f u t u r e market share. This i s p a r t i a l l y due to an i n c r e a s i n g c a p a b i l i t y t o grow woody ornamentals i n v i t r o , p l a n t s p r e v i o u s l y o f t e n d i f f i c u l t t o propagate and having slow growth r a t e s . With the good prospects f o r woody ornamental micropropagation, developing methods t o improve the e f f i c i e n c y of woody p l a n t micropropagation may enable production c o s t s t o be lowered. The p l a n t chosen f o r the present study i s the woody ornamental Spiraea n i p p o n i c a . S p i r a e a nipponica i s a member of a genus whose species are wi d e l y p r a i s e d f o r t h e i r f lowers and f i n e shrub form (Hudak, 1984) . The genus belongs t o the f a m i l y Rosaceae which i n c l u d e s many genera of ornamental and f r u i t - y i e l d i n g p l a n t s c u r r e n t l y grown i n v i t r o such as the rose, apple, plum, and strawberry (George and Sherrington, 1984). I f s u c c e s s f u l techniques t o improve Spiraea i n v i t r o c u l t u r e are developed, they may be a p p l i c a b l e t o the other more economically important rosaceous p l a n t s . In v i t r o p l a n t growth can be r e g u l a t e d by a r t i f i c i a l l y imposed c o n d i t i o n s . Those f a c t o r s which c o n t r o l growth such as environment and c u l t u r e medium c o n s t i t u e n t s are e a s i l y modified. Environmental f a c t o r s i n c l u d e temperature, l i g h t , and atmosphere. C u l t u r e medium c o n s t i t u e n t s i n c l u d e i n o r g a n i c and organic components. The i n o r g a n i c components are the m i c r o n u t r i e n t s and the macronutrients. The organic components i n c l u d e v i t a m i n s , carbohydrates and phytohormones. The purpose of the c u r r e n t t h e s i s i s t o improve micropropagation shoot c u l t u r e techniques by modifying phytohormone metabolism i n the shoot explant. Auxins and c y t o k i n i n s are the two types of phytohormones most commonly used t o i n f l u e n c e i n v i t r o p l a n t growth. E a r l y work on tobacco c a l l u s (Skoog and M i l l e r , 1957) i n d i c a t e d t h a t v a r y i n g the r a t i o of c y t o k i n i n t o auxin concentrations i n the growth medium could determine the course of organogenesis. Taking the tobacco work i n t o account, the o p t i m i z a t i o n of micropropagation production has depended on determining a p p r o p r i a t e c y t o k i n i n t o auxin r a t i o s . T y p i c a l l y , a range of auxins and c y t o k i n i n s and v a r i o u s c o n c e n t r a t i o n s of a u x i n - c y t o k i n i n p a i r s are t e s t e d , such as the p i o n e e r i n g work on the i n v i t r o shoot c u l t u r e of Rhododendron 4 where two a u x i n s and t h r e e c y t o k i n i n s were s c r e e n e d (Anderson, 1975). A r a t i o where c y t o k i n i n p r e d ominates u s u a l l y r e s u l t s i n o p t i m a l s h o o t p r o d u c t i o n . F o r example, work on lowbush b l u e b e r r y i n d i c a t e d t h a t as t h e c y t o k i n i n c o n c e n t r a t i o n i n c r e a s e d t h e s h o o t number i n c r e a s e d , w h i l e i n c r e a s e d a u x i n c o n c e n t r a t i o n s d e c r e a s e d t h e s h o o t number ( F r e t t and Smagula, 1983). As a r e s u l t o f t h e s e and o t h e r f i n d i n g s i t has been g e n e r a l l y assumed i n s t u d i e s on t h e hormonal c o n t r o l o f p l a n t development t h a t a " c o r r e l a t i o n e x i s t s between hormone l e v e l s and q u a n t i t a t i v e a s p e c t s o f development" (Barendse e t a l . , 1987). The mechanism o f a p i c a l dominance i s g e n e r a l l y assumed t o i n v o l v e t h e t r a n s p o r t o f a u x i n from a s h o o t ' s g r o w i n g t i p down t h e s h o o t , t h e a u x i n e i t h e r d i r e c t l y o r i n d i r e c t l y i n h i b i t i n g a x i l l a r y bud growth ( H i l l m a n , 1984). W i t h a r e d u c t i o n i n a u x i n t r a n s p o r t , a x i l l a r y buds can grow, as was d e m o n s t r a t e d by an e x p e r i m e n t i n v o l v i n g t r i i o d o b e n z o i c a c i d ( T I B A ) , an a u x i n t r a n s p o r t i n h i b i t o r ( T u c k e r , 1978). One e f f e c t o f a u x i n - i n d u c e d growth i n h i b i t i o n may be t o i n h i b i t c y t o k i n i n s y n t h e s i s w i t h i n t h e s u p p r e s s e d bud (Lee e t a l . , 1974) . I n t h e case o f s h o o t c u l t u r e s i n a m i c r o p r o p a g a t i o n system t h e t y p i c a l a pproach has been t o supplement t h e growth medium w i t h h i g h l e v e l s o f c y t o k i n i n t o overcome t h e growth s u p p r e s s i o n (George and S h e r r i n g t o n , 1984). U n t i l r e c e n t l y , a t t e m p t s t o m o d i f y t h e endogenous a u x i n c o n c e n t r a t i o n i n t h e s h o o t e x p l a n t . have been few. Thus i n t h e c u r r e n t work, f a c t o r s w h i c h may s p e c i f i c a l l y a f f e c t a s h o o t 5 explant's endogenous auxin metabolism have been studied. The underlying purpose i n a l t e r i n g endogenous auxin metabolism i s to provide a way of reducing a p i c a l dominance without the use of high exogenous l e v e l s of cytokinins. I f the amount of exogenous cytokinin needed to e l i c i t l a t e r a l bud release can be decreased, i t i s hoped that known deleterious side e f f e c t s of high cytokinin l e v e l s such as v i t r i f i c a t i o n (Leshem et a l . , 1988), and r e s i d u a l bushiness (Pierik, 1982) may be decreased. Also, reduction i n the l e v e l of cytokinin may help elucidate the putative r o l e of cytokinins i n contributing to i n v i t r o plants' often open stomata (cytokinins are known to promote stomatal opening (Jewer and I n c o l l , 1980) ). The approach taken to modify endogenous auxin metabolism i n t h i s study has been to manipulate an environmental factor, l i g h t ; s p e c i f i c a l l y , l i g h t q u a l i t y . The use of l i g h t q u a l i t y to produce s p e c i f i c desired e f f e c t s without having to increase the exogenous cytokinin l e v e l s has not previously been applied to i n v i t r o plant culture. Spiraea was chosen as a t e s t plant because of i t s growth, i n nature, i n exposed sunny locations. Such plants are l i k e l y to possess a shade-avoidance response which involves phytochrome e q u i l i b r i a (Smith, 1982). These plants when even s l i g h t l y shaded, detect the change i n l i g h t conditions v i a a phytochrome sensor and respond by increasing the rate of stem extension growth to regain a p o s i t i o n under a more favourable l i g h t regime. The possession of a developed phytochrome system for i n i n v i t r o plant may allow f o r s i g n i f i c a n t red and far-red 6 l i g h t m o d i f i c a t i o n o f growth. W h i l e e x t e n s i o n growth, e s p e c i a l l y o f newly i n i t i a t e d s h o o t s , i s d e s i r a b l e , i t a l s o seemed p o s s i b l e t h a t known r e d and f a r - r e d l i g h t e f f e c t s on a u x i n l e v e l s would be b e t t e r e x p r e s s e d u s i n g S p i r a e a . Red and f a r - r e d l i g h t has been shown t o reduce a u x i n l e v e l s i n e x t e n d i n g s h o o t s ( l i n o , 1982); i n some c a s e s r e d l i g h t e x p o s u r e s have l e d t o a l e s s e n i n g o f a p i c a l dominance w i t h subsequent s h o o t growth from l a t e r a l buds ( K a s p e r b a u e r , 1971; H e a l y and W i l k i n s , 1979) . A l s o , S p i r a e a , s i m i l a r t o many o t h e r woody o r n a m e n t a l s c u l t u r e d i n v i t r o (Lane, 1978), does n o t r e q u i r e exogenous a u x i n ; t h u s , e f f e c t s on endogenous a u x i n l e v e l s would n o t be compounded w i t h any exogenous a u x i n i n t e r a c t i o n s f o r t h e S p i r a e a system. The S p i r a e a s t u d y i s d i v i d e d i n t o t h r e e s e c t i o n s . The f i r s t s e c t i o n i s a c h a r a c t e r i z a t i o n o f t h e S p i r a e a i n v i t r o l i g h t q u a l i t y r e s p o n s e . Red l i g h t and f a r - r e d l i g h t a r e t h e p r i m a r y m o d i f i e r s o f phytochrome, w h i l e b l u e l i g h t , v i a b l u e - u l t r a -v i o l e t (U.V.) l i g h t a b s o r b i n g p i g m e n t s , can a l s o a f f e c t i n v i t r o g r o w t h (George and S h e r r i n g t o n , 1984). The f i r s t e x p e r i m e n t i n d i c a t e d w h i c h o f r e d and f a r - r e d l i g h t and b l u e l i g h t was more e f f e c t i v e i n m o d i f y i n g S p i r a e a i n v i t r o growth. Then t h r e e l i g h t q u a l i t i e s i n t e r m e d i a t e between b l u e and r e d l i g h t were t e s t e d t o d e t e r m i n e i f m i n o r changes i n phytochrome e q u i l i b r i a c o u l d m o d i f y growth. L i g h t s o u r c e c o m b i n a t i o n s w i t h b l u e and r e d f i l t e r s were used t o v e r i f y t h a t l i g h t s o u r c e - f i l t e r c o m b i n a t i o n s used i n t h e f i r s t e x p e r i m e n t were o p t i m a l and a l s o t o s t u d y t h e e f f e c t s on growth, o f v a r y i n g t h e p r o p o r t i o n s o f r e d 7 and blue l i g h t . F i n a l l y , the leng t h of exposure needed f o r l i g h t of each q u a l i t y t o cause a growth response was examined. The e f f e c t on growth of a sh o r t end-of-day l i g h t q u a l i t y exposure was compared t o the e f f e c t produced by a f u l l photoperiod of l i g h t q u a l i t y exposure. The second s e c t i o n i s concerned w i t h the e f f e c t on growth of the sequence of exposure t o l i g h t q u a l i t y . The f i r s t l i g h t q u a l i t y experiment i n v o l v e d the t r a n s f e r of explants from a two week white l i g h t regime t o the s p e c i f i c l i g h t q u a l i t y regimes of a much lower photon fl u e n c e r a t e f o r another three weeks. This i d e n t i f i e d two problems. F i r s t , there was a need to r e s o l v e whether or not any e f f e c t was caused by d i f f e r e n t fluence r a t e s ; thus, the e f f e c t on growth of an i n i t i a l low photon fluence r a t e was examined. The other problem concerned the time at which c u l t u r e s were t r a n s f e r r e d t o d i f f e r e n t l i g h t regimes. A study on a d v e n t i t i o u s bud formation from Douglas f i r c a l l u s i n d i c a t e d t h a t c u l t u r e s were most s e n s i t i v e t o red l i g h t during the t h i r d , f o u r t h , and f i f t h week of growth (Kadkade and Jopson, 1978) . This suggested the p o s s i b i l i t y t h a t the age of Spiraea c u l t u r e s when t r a n s f e r r e d t o d i f f e r e n t l i g h t q u a l i t y regimes might be important i n o p t i m i z i n g any l i g h t q u a l i t y enhancement of growth. T r a n s f e r of the explants from an i n i t i a l low photon fluence r a t e to a h i g h e r r a t e , and t r a n s f e r at s p e c i f i c i n t e r v a l s / was thus s t u d i e d i n an attempt t o understand the extent t o which these two f a c t o r s a f f e c t e d the observed l i g h t q u a l i t y response i n the f i r s t l i g h t q u a l i t y experiment. 8 The t h i r d s e c t i o n deals w i t h a study done t o determine i f the a c t i o n of a known m o d i f i e r of auxin metabolism c o u l d be optimized by i n t e r a c t i n g w i t h the l i g h t q u a l i t y found most e f f e c t i v e i n the f i r s t s e c t i o n . The compound, glyphosate, can decrease auxin l e v e l s (Lee, 1982). The p r e c i s e mode of gl y p h o s a t e 1 s a c t i o n i s not c l e a r ; one idea i s t h a t by d i s r u p t i n g phenol b i o s y n t h e s i s t h e r e i s a decrease i n s p e c i f i c p h e n o l i c compounds which i n h i b i t an auxin oxidase and thus a g r e a t e r auxin o x i d a t i o n can occur (Lee, 1982), another e f f e c t may be t o i n h i b i t a u xin b i o s y n t h e s i s (Kudoyarova et a l . , 1988). Glyphosate may a l s o provide an i n d i r e c t means of co n f i r m i n g t h a t l i g h t q u a l i t y i s modifying auxin metabolism; f o r example, i f red and f a r - r e d l i g h t do decrease auxin l e v e l s a glyphosate enhancement of the e f f e c t may y i e l d a shoot p r o l i f e r a t i o n p a t t e r n t y p i c a l of a hi g h e r c y t o k i n i n t o auxin r a t i o . Chapter 2 9 Literature Review 2.1 The control of i n v i t r o plant growth by the environmental f a c t o r of l i g h t The d i v e r s e e f f e c t s of l i g h t on p l a n t growth a r i s e from three l i g h t c h a r a c t e r i s t i c s : wavelength ( l i g h t q u a l i t y ) , f l u x d e n s i t y ( i r r a d i a n c e or i n t e n s i t y ) , and d u r a t i o n of l i g h t exposure (photoperiod) (George and Sherrington, 1984). The e f f e c t s of l i g h t on i n v i t r o p l a n t growth and development are i n f l u e n c e d by components of the growth medium. For example, photosynthesis i s only a minor determinant of i n v i t r o growth because of i t s i n h i b i t i o n by the exogenously s u p p l i e d carbohydrate source ( u s u a l l y sucrose) i n the growth medium. The sucrose supplement r e s u l t s i n the r e d u c t i o n of both c h l o r o p h y l l s y n t h e s i s and p h o t o s y n t h e t i c carbon f i x a t i o n ; the photosynthesis t h a t occurs i s minimal (George and S h e r r i n g t o n , 1984). The s m a l l c o n t r i b u t i o n to growth from photosynthesis a l l o w s i n v i t r o p l a n t s t o be grown under low l i g h t i n t e n s i t i e s . Under low i n t e n s i t i e s the r o l e of l i g h t q u a l i t y i n determining p l a n t 10 growth and development i s gr e a t e r than at higher l i g h t i n t e n s i t i e s (Grimstad, 1987)- Thus i n v i t r o p l a n t c u l t u r e s o f f e r the p o t e n t i a l t o observe s p e c i f i c photomorphogenic e f f e c t s which are dependent on l i g h t q u a l i t y . These e f f e c t s can be c l a s s i f i e d i n t o two groups: blue-U.V. absorbing pigment-mediated e f f e c t s and phytochrome-mediated e f f e c t s (George and Sherrington, 1984). Se v e r a l s t u d i e s have shown t h a t blue-U.V. e f f e c t s are most evident on c a l l u s growth and on morphogenesis. In one study ( S e i b e r t e t a l . , 1975), tobacco c a l l u s was exposed t o e i t h e r near-U.V. l i g h t (371 nm) or blue l i g h t (420 and 467 nm) . Near U.V. l i g h t s t i m u l a t e d maximum growth and shoot formation at the low i r r a d i a n c e of 24 uW/cm*- w h i l e f o r blue l i g h t the much higher i r r a d i a n c e of 300 uW/cm at 420 nm and 600 uW/cm at 467 nm was needed. The blue-U.V. s t i m u l a t i o n , however, was of t e n l o s t when higher i r r a d i a n c e s of blue or near U.V. were used. For example, when Haplopappus g r a c i l i s c a l l u s was exposed t o e i t h e r white, blue, red or f a r - r e d l i g h t , each at about 1000 uW/cm , blue had an i n h i b i t o r y e f f e c t on growth ( S t i c k l a n d and Sunderland, 1972). Reports of blue l i g h t e f f e c t s on shoot c u l t u r e s i n d i c a t e a d i v e r s i t y of responses. P a r t of the d i v e r s i t y can be a t t r i b u t e d t o d i f f e r e n t p l a n t species and d i f f e r e n t explant t i s s u e responses, but d i f f e r e n t medium formulations and l i g h t i n c u b a t i o n c o n d i t i o n s may a l s o confound any s p e c i f i c blue l i g h t e f f e c t . The f o l l o w i n g survey i s intended t o i l l u s t r a t e the range of responses noted. Juneberry shoot t i p c u l t u r e s s u p p l i e d w i t h an exogenous auxin and c y t o k i n i n and exposed t o white, blue , red, and green l i g h t , produced the g r e a t e s t number of shoots under blue and white l i g h t (Behrouz and Lineberger, 1981). When no exogenous hormones were a p p l i e d none of the l i g h t q u a l i t i e s improved growth. With a grape shoot c u l t u r e only an exogenous c y t o k i n i n was a p p l i e d ; the i n t e r a c t i o n of l i g h t q u a l i t y w i t h two growth medium m i c r o n u t r i e n t s , MnSOij. and KI, was s t u d i e d (Chee and Po o l , 1985). Both m i c r o n u t r i e n t s are m o d i f i e r s of auxin metabolism; when the MnSOi^. l e v e l was decreased and KI excluded, blue l i g h t caused a g r e a t e r shoot p r o l i f e r a t i o n than red l i g h t . I t was proposed t h a t the blue l i g h t - M n S O i j - i n t e r a c t i o n caused a r e d u c t i o n of a p i c a l dominance. Studies on a w i l l o w shoot c u l t u r e system showed t h a t when blue l i g h t was a p p l i e d t o shoots which had t h e i r apex removed, a x i l l a r y bud growth was suppressed (Letouze, 1974). This growth suppression was overcome when a c y t o k i n i n was a p p l i e d t o the uppermost bud. I n t e r p r e t i n g the blue l i g h t e f f e c t s on the Juneberry, grape, and w i l l o w c u l u r e s i s complicated by the f a c t t h a t d i f f e r e n t photoperiods were used f o r each c u l t u r e . Explant t i s s u e as a p o s s i b l e f a c t o r determining the degree of a l i g h t q u a l i t y response i s shown by work on A f r i c a n V i o l e t l e a f e x p lants ( L e r c a r i et a l . , 1986). On a medium without exogenous phytohormones, blue l i g h t induced a higher bud r e g e n e r a t i o n frequency than e i t h e r red, f a r - r e d , or U.V. l i g h t . However, the frequency under darkness was s i m i l a r t o t h a t under white l i g h t which suggests t h a t bud i n d u c t i o n may have been 12 determined before the explant was c u l t u r e d . Thus the extent of l i g h t q u a l i t y e f f e c t s , i n t h i s l e a f explant system, may have depended on f a c t o r s i n f l u e n c i n g the stock p l a n t . Red and f a r - r e d l i g h t e f f e c t s on p l a n t growth are mediated v i a the photochromic pigment phytochrome (George and Sh e r r i n g t o n , 1984). U n l i k e the s t i l l nebulous nature of the blue-U.V. pigment, phytochrome has been c h a r a c t e r i z e d i n great d e t a i l . However, the s i g n a l t r a n s d u c t i o n chain l e a d i n g from the l i g h t - i n d u c e d conformational changes i n the phytochrome molecule t o the observed p h y s i o l o g i c a l response remains t o be r e s o l v e d . B a s i c a l l y , phytochrome e q u i l i b r a t e s between two forms; one form has i t s a b s o r p t i o n maximum i n the red region of the spectrum (about 660 nm) and the other i n the f a r - r e d r e g i o n (about 73 0 nm). A s h i f t t o the f a r - r e d form can a l s o occur under c o n d i t i o n s of prolonged darkness and hence t h i s form i s i m p l i c a t e d i n p h o t o p e r i o d i c p l a n t responses. C l a s s i c r ed and f a r - r e d l i g h t - i n d u c e d p h y s i o l o g i c a l responses r e q u i r e b r i e f low energy exposures of l i g h t . Red l i g h t leads t o the p h y s i o l o g i c a l l y a c t i v e phytochrome form; when there i s a subsequent exposure t o f a r - r e d l i g h t the response i s negated. However, there i s a c l a s s of responses which depends on a s o - c a l l e d "high i r r a d i a n c e " of red and f a r - r e d l i g h t . These responses are .induced by long term exposure t o r e d / f a r - r e d l i g h t and are o f t e n , but not always, f l u e n c e - r a t e dependent (Kronenberg and Kendrick, 1986). The use of red and f a r - r e d l i g h t t o modify i n v i t r o growth 13 has concentrated on the low energy phytochrome system. Lettuce c a l l u s growth and shoot formation was improved by d a i l y f i v e minute exposures t o red l i g h t (Kadkade and S e i b e r t , 1977). When f a r - r e d l i g h t was a p p l i e d , the p o s i t i v e e f f e c t was l o s t . Pine embryo c a l l u s had a higher r a t e of shoot bud formation under red l i g h t (Kadkade et a l . , 1978). Root c u l t u r e s seem p a r t i c u l a r l y r e c e p t i v e t o the low energy-induced responses. Convolvulus  a r v e n s i s r o o t c u l t u r e s e x h i b i t e d enhanced shoot bud development w i t h an exposure of only ten seconds of red l i g h t (655 nm) per day; a response again negated by f a r - r e d l i g h t (Bonnett, 1972). Root formation on c u l t u r e d r o o t s of sunflower was enhanced by red l i g h t (Letouze and Beauchesne, 1969). Red l i g h t e f f e c t s have been d e t a i l e d e x t e n s i v e l y ; an i n t e r e s t i n g o b s e r vation t h a t has a r i s e n i s t h a t i n c u l t u r e s where red l i g h t has a p o s i t i v e i n f l u e n c e , the presence of a c y t o k i n i n i n the medium o f t e n i n t e n s i f i e s the e f f e c t . For example, duckweed grown on a c y t o k i n i n - f r e e medium e x h i b i t e d no growth i n the dark, but when a c y t o k i n i n was added or p e r i o d i c exposures of red l i g h t were given, some growth was induced ( C l e l a n d , 1976). When both a c y t o k i n i n was present and the red l i g h t exposures were given, the gr e a t e s t growth response occurred. Red l i g h t or a c y t o k i n i n was shown t o reduce a p i c a l dominance i n w i l l o w shoot c u l t u r e s (Letouze, 1974). In a study on somatic embryogenesis i n v o l v i n g c a r r o t suspension c u l t u r e s , i t was shown t h a t supplying a source of a d d i t i o n a l c y t o k i n i n t o the medium could, a t l e a s t over the short term, maintain the 14 c u l t u r e s ' s embryogenic p o t e n t i a l (Fujimura and Komamine, 1975). A study of l i g h t q u a l i t y e f f e c t s on c a r r o t somatic embryogenesis showed t h a t red l i g h t c o u l d a l s o a i d i n ma i n t a i n i n g a high embryogenic c a p a c i t y ( M i c h l e r and Lineberger, 1987). As w i l l be mentioned i n the s e c t i o n on auxins, red l i g h t can decrease auxin l e v e l s ( l i n o , 1982). Thus, any apparent red l i g h t - c y t o k i n i n synergy may be simply due t o red l i g h t causing a h i g h e r c y t o k i n i n - a u x i n r a t i o which i s f u r t h e r enhanced by an exogenous c y t o k i n i n . I t has a l s o been conjectured t h a t red l i g h t may i n f l u e n c e endogenous c y t o k i n i n l e v e l s (Read, 1987) . The e f f e c t s noted f o r red l i g h t and c y t o k i n i n s provide a promising means of modifying i n v i t r o growth. No work has been done t o e x p l o i t the i n t e r a c t i o n of red l i g h t and c y t o k i n i n s i n order t o e s t a b l i s h s u i t a b l e l e v e l s of exogenous c y t o k i n i n f o r cytokinin-dependent c u l t u r e s . The micropropagation of many woody p l a n t s o f t e n i n v o l v e s o n l y an exogenously s u p p l i e d c y t o k i n i n i n the growth medium r a t h e r than both an auxin and a c y t o k i n i n . Thus, i f red l i g h t e f f e c t s can be induced i n such a system the b e n e f i t s of n a t u r a l l y induced c y t o k i n i n s and/or reduced auxin l e v e l s c o u l d r e p l a c e the high l e v e l s of exogenous c y t o k i n i n s used, which, as w i l l be o u t l i n e d i n a l a t e r s e c t i o n , have p o t e n t i a l l y d e l e t e r i o u s e f f e c t s . The p o s s i b i l i t y of usi n g prolonged r e d / f a r - r e d l i g h t exposures t o e l i c i t s i m i l a r responses t o those noted f o r s h o r t e r red l i g h t exposures ( i n combination w i t h a c y t o k i n i n ) a l s o m e r i t s a t t e n t i o n . This i s f o r two reasons: f i r s t , s i n c e i t i s easy t o o b t a i n such l i g h t from 15 commercially a v a i l a b l e tungsten f i l a m e n t l i g h t sources and secondly, f o r any f u r t h e r i n f o r m a t i o n t h a t may be gained to help e l u c i d a t e the poo r l y understood mechanism of the "high i r r a d i a n c e " response. 2.2 Phytohormones used f o r i n v i t r o plant culture Auxins and c y t o k i n i n s are the phytohormones predominantly used f o r modifying i n v i t r o p l a n t growth and morphogenesis (George and Sherrington, 1984) . These two phytohormones i n t e r a c t i n a very complex way; exogenously a p p l i e d n a t u r a l or s y n t h e t i c forms of the two i n t e r a c t w i t h the endogenous phytohormone counterparts of the explant i t s e l f . Each one, though, does have c h a r a c t e r i s t i c p h y s i o l o g i c a l a c t i v i t i e s . 2.2.1 Auxins The auxins are d e f i n e d by t h e i r a b i l i t y to conr o l c e l l growth and c e l l e l o n g a t i o n (George and Sherrington, 1984). The most common n a t u r a l auxin i s 3 - i n d o l e - a c e t i c a c i d (IAA). Often f o r maximal i n v i t r o growth responses, chemical analogues of auxin such as 2,4-dichlorophenoxyacetic a c i d (2,4-D) and 1-naphthyTacetic a c i d (NAA) are used (George and Sherrington, 1984). One of the main uses of auxin i n t i s s u e c u l t u r e i s f o r c a l l u s i n d u c t i o n ; f o r d i c o t s a high l e v e l of auxin i s used w i t h a much lower l e v e l of a c y t o k i n i n , w h i l e f o r monocots f r e q u e n t l y only a high l e v e l of auxin i s used (George and Sherrington, 1984) . Embryogenesis i s another process c o n t r o l l e d by added auxins; i t i s i n i t i a t e d by high l e v e l s of auxin but subsequent embryo development r e q u i r e s a lowering of the a p p l i e d auxin l e v e l (George and She r r i n g t o n , 1984). The most common treatment i s t o vary the l e v e l s of a p p l i e d auxins w i t h respect to the l e v e l s of a p p l i e d c y t o k i n i n s i n order t o c o n t r o l the d i r e c t i o n of morphogenesis. The general r u l e i s t h a t w i t h a high r a t i o of c y t o k i n i n t o auxin, shoot pr o d u c t i o n occurs, w h i l e a high r a t i o of auxin t o c y t o k i n i n induces r o o t formation. The u l t i m a t e e f f e c t t h a t an a p p l i e d auxin has on an explant depends on how i t i n t e r a c t s w i t h the e x p l a n t 1 s endogenous auxin. Such an i n t e r a c t i o n depends on many f a c t o r s ; f o r i n s t a n c e , the r a t e of auxin uptake by the explant and the p a t t e r n of i t s subsequent metabolism. Furthermore, the i n t e r a c t i o n of the a p p l i e d auxin w i t h any a p p l i e d c y t o k i n i n and any endogenous c y t o k i n i n must be considered. One n a t u r a l m o d i f i e r of IAA l e v e l s i s l i g h t q u a l i t y . I t has been shown t h a t both red and f a r - r e d l i g h t can decrease the a v a i l a b i l i t y of p h y s i o l o g i c a l l y a c t i v e endogenous IAA (free IAA) i n c o l e o p t i l e s ( l i n o , 1982). Under r e d / f a r - r e d l i g h t i t may be p o s s i b l e t o reduce f r e e IAA l e v e l s and thus a f f e c t the r a t i o of 17 c y t o k i n i n t o auxin. For woody p l a n t s , o f t e n only an exogenous c y t o k i n i n i s necessary t o cause shoot p r o l i f e r a t i o n , s i n c e as f o r apple, "the shoots probably produce s u f f i c i e n t endogenous auxin f o r growth" (Lane, 1978). Thus, any lowering of endogenous auxin l e v e l s may permit a lowering of exogenous c y t o k i n i n l e v e l s s i n c e any c o m p l i c a t i n g i n t e r a c t i o n s w i t h exogenous auxins can be minimized. 2.2.2 Cytokinins C y t o k i n i n s are c h i e f l y c h a r a c t e r i z e d by t h e i r a b i l i t y t o s t i m u l a t e c e l l d i v i s i o n (Skoog e t a l . , 1965). Two n a t u r a l l y o c c u r r i n g c y t o k i n i n s used i n t i s s u e c u l t u r e are z e a t i n and N-(2-isopentyl) adenine ( 2 - i p ) . Two s y n t h e t i c c y t o k i n i n analogues commonly used are k i n e t i n and 6-benzyladenine (BA) . The s y n t h e t i c c y t o k i n i n s have a gr e a t e r a c t i v i t y than the n a t u r a l ones (George and Sh e r r i n g t o n , 1984) . The use of s y n t h e t i c c y t o k i n i n s made p o s s i b l e the development of e f f i c i e n t micropropagation systems. E x p l o i t i n g the observations made concerning the a b i l i t y of c y t o k i n i n s t o r e l e a s e l a t e r a l buds from a p i c a l dominance (Sachs and Thimann, 1964), workers used c y t o k i n i n s i n shoot t i p c u l t u r e s t o p r o l i f e r a t e m u l t i p l e shoots. BA was f i r s t observed t o s t i m u l a t e shoot e l o n g a t i o n of apple shoots i n v i t r o (Jones, 1967), before 18 i t s use t o enhance shoot p r o l i f e r a t i o n of apple shoot t i p s ( P i e n i a z e k , 1968). C u r r e n t l y BA i s the most common c y t o k i n i n used f o r the micropropagation of temperate f r u i t and nut t r e e s p e c i e s (Hutchinson and Zimmerman, 1987), as w e l l as f o r many woody ornamental shrubs and t r e e s . 2.2.3 C y t o k i n i n S i d e - e f f e c t s As b e n e f i c i a l as BA and other exogenous c y t o k i n i n s have been f o r o p t i m i z i n g shoot p r o l i f e r a t i o n , t h e i r use can have p o t e n t i a l d e l e t e r i o u s e f f e c t s . Recent work has i m p l i c a t e d BA as a cause of v i t r i f i c a t i o n (Leshem et a l . , 1988). V i t r i f i c a t i o n i s most commonly d e f i n e d as "an abnormal development of c u l t u r e d shoot t i p s i n t o t r a n s l u c e n t stunted shoots and thickened, t u r g i d and b r i t t l e leaves w i t h a g l a s s y appearance" (Leshem e t a l . , 1988). The c o n d i t i o n has been noted i n both herbaceous and woody p l a n t c u l t u r e s ; i t i s thought to be a response t o an excess of c y t o k i n i n (Leshem et a l . , 1988) . The t y p i c a l l y d i f f i c u l t c u l t u r e of many c e r e a l p l a n t s i s compounded by t h e i r s e n s i t i v i t y t o exogenously a p p l i e d c y t o k i n i n s (Wernicke and M i l k o v i t s , 1984). A c c l i m a t i z a t i o n , the process of t r a n s f e r r i n g i n v i t r o p l a n t s t o a p o t t i n g medium and preparing them f o r f r e e l i v i n g c o n d i t i o n s (Hutchinson and Zimmerman, 1987), a l s o may depend on exogenously s u p p l i e d c y t o k i n i n s . The main problem w i t h a c c l i m a t i z a t i o n i s 19 the poor water' r e g u l a t i v e a b i l i t i e s of ex v i t r o p l a n t s due t o d y s f u n c t i o n a l stomata (Hutchinson and Zimmerman, 1987). The ma l f u n c t i o n may be due t o carry-o v e r e f f e c t s of c y t o k i n i n s , s i n c e c y t o k i n i n s are known t o promote stomatal opening (Jewer and I n c o l l , 1980). Another general phytohormone carry- o v e r e f f e c t i s the m o d i f i c a t i o n of the growth h a b i t of p l a n t s f o r months a f t e r they have been removed from c u l t u r e ( P i e r i k et a l . , 1982). C y t o k i n i n s i n p a r t i c u l a r , by promoting l a t e r a l bud break, may cause unwanted branching ( P i e r i k et a l . , 1982). F i n a l l y , s p e c i f i c t o woody ornamental shrubs and t r e e s , i s the tendency f o r some shoot c u l t u r e s t o produce heavy c a l l u s on the shoot bases due t o high c y t o k i n i n l e v e l s (McCown, 1986). The r e s u l t i s a slowing of growth, s i n c e the shoot i s no longer i n d i r e c t c ontact w i t h the medium. 2.3 Glyphosate as a growth r e g u l a t o r f o r i n v i t r o p l a n t c u l t u r e Glyphosate i s the a c t i v e compound of the commercial h e r b i c i d e 'Roundup'. Work on tobacco c a l l u s suggests t h a t one f a c t o r c o n t r i b u t i n g t o glyphosate-mediated growth i n h i b i t i o n i s the glyphosate e f f e c t of i n c r e a s i n g IAA o x i d a t i o n (Lee, 1982). The mechanism of t h i s increased IAA o x i d a t i o n i s unclear. One proposal i s t h a t glyphosate, by i n h i b i t i n g aromatic amino a c i d b i o s y n t h e s i s ( v i a the s h i k i m i c a c i d pathway), causes a decrease 20 i n the l e v e l of secondary aromatic m e t a b o l i t e s , s p e c i f i c a l l y c e r t a i n p h e n o l i c compounds (Lee, 1982). These s p e c i f i c p h e n o l i c compounds can i n h i b i t IAA oxidase, and thus a decrease i n such p h e n o l i c compounds' l e v e l s may r e s u l t i n a higher IAA o x i d a t i o n . An a l t e r n a t i v e to increased IAA o x i d a t i o n , or perhaps a process o c c u r r i n g i n concert w i t h the o x i d a t i o n , i s the p o s s i b i l i t y t h a t IAA b i o s y n t h e s i s i s i n h i b i t e d (Kuoyarova et a l . , 1988). A study on soybean and pea s e e d l i n g s i n d i c a t e d t h a t glyphosate was e f f e c t i v e i n r e l e a s i n g l a t e r a l buds from a p i c a l dominance (Lee, 1984). Stem s e c t i o n s of the seedlings were fed w i t h r a d i o l a b e l l e d IAA. The glyphosate e f f e c t was d i f f e r e n t f o r each p l a n t species. Treated pea p l a n t s metabolized 61% more of the r a d i o l a b e l l e d IAA over a 4 hour i n c u b a t i o n p e r i o d than the c o n t r o l p l a n t s . For soybeans the i n c r e a s e i n IAA metabolism was only 4.4% f o r t r e a t e d p l a n t s . .This work suggested t h a t glyphosate might have the p o t e n t i a l t o i n c r e a s e shoot production i n micropropagation systems. As a growth r e g u l a t o r f o r i n v i t r o c u l t u r e , glyphosate has been t e s t e d at m i l l i m o l a r c o n c e n t r a t i o n s . A study on a x i l l a r y shoot p r o l i f e r a t i o n of i n v i t r o cranberry node explants i n d i c a t e d glyphosate's a b i l i t y t o i n c r e a s e shoot number, but there were unwanted s i d e - e f f e c t s , such as b a s a l s w e l l i n g of shoots and deformed leaves (Scorza et a l . , 1984). Another study i n v o l v e d b l u e b e r r y p l a n t s where the source p l a n t f o r shoot t i p explants was sprayed w i t h glyphosate ( F r e t t and Smagula, 1981). The shoot t i p explants when put i n t o c u l t u r e produced more a x i l l a r y - d e r i v e d shoots than explants taken from c o n t r o l p l a n t s . Perhaps glyphosate may be more e f f i c i e n t l y used i n d i r e c t l y on source p l a n t s than d i r e c t l y on i n v i t r o p l a n t l e t s ; i t s p o t e n t i a l as a growth r e g u l a t o r f o r i n v i t r o p l a n t c u l t u r e needs t o be c l a r i f i e d by t e s t i n g a wider range of micropropagated s p e c i e s . 22 Chapter 3 Materials and Methods 3.1 Culture Medium and i t s Preparation The medium used was developed by Linsmaier and Skoog (Linsmaier and Skoog, 1965) ; i t i s a m o d i f i c a t i o n of the Murashige and Skoog medium (Murashige and Skoog, 19 62). In t h i s m o d i f i c a t i o n o n l y one vi t a m i n i s used, thiamine at 0.4 m i l l i g r a m s per l i t r e (mg/1); a l l other medium c o n s t i t u e n t s are the same as i n the Murashige and Skoog medium. Stock s o l u t i o n s were made f o r the i n o r g a n i c s a l t s ; one f o r the m i c r o n u t r i e n t s , one f o r the major macronutrients, and one f o r the i r o n and EDTA macronutrient components ( t h i s s o l u t i o n was kept i n a c o n t a i n e r wrapped i n aluminum f o i l ) . The m i c r o n u t r i e n t stock s o l u t i o n was prepared at a 2 00X co n c e n t r a t i o n ; the c o n s t i t u e n t s , i n mg/1 of medium, were: H 3 B O 3 6.2, MnS0^-4H^0 22.3, ZnSCy 7H^0 8.6, KI 0.83, Na^MoCf2HjjD 0.25, CuSO^SHjjD 0.025, CoClgceH^O 0.025. The major macronutrient stock s o l u t i o n was prepared at a 10X concentration;- the c o n s t i t u e n t s , i n mg/1 of medium, were: NH^N03 1650, KN0 3 1900, CaCl-^H^O 440, MgSOq. 370, KH^POq. 170. The i r o n and EDTA stock s o l u t i o n was prepared at a 100X c o n c e n t r a t i o n ; these two components, i n mg/1 of medium, were: 23 FeS04.-7HJL0 27.8, and Na^ EDTA 37.3. For organic c o n s t i t u e n t s thiamine was kept as a stock s o l u t i o n at a 50X c o n c e n t r a t i o n ; 0.4 mg/1 was used i n the medium. The s y n t h e t i c c y t o k i n i n BA was prepared at a stock c o n c e n t r a t i o n of 100X, kept i n a c o n t a i n e r wrapped i n aluminum f o i l , and prepared f r e s h monthly. The BA l e v e l s used f o r each experiment were as s p e c i f i e d i n s e c t i o n 3.7 of t h i s M a t e r i a l s and Methods chapter. A l l stock s o l u t i o n s were kept as l i q u i d s and r e f r i g e r a t e d i n a standard r e f r i g e r a t o r . M y o - I n o s i t o l (100 mg/1 of medium) and sucrose (30 g/1 of medium) were added f r e s h f o r each medium p r e p a r a t i o n . A f t e r each batch of medium (1 l i t r e ) had been prepared the pH was ad j u s t e d t o 5.8 and 7 g/1 of D i f c o Bacto agar was added. The medium was dispensed i n 15 ml p o r t i o n s t o 25 x 150 mm g l a s s t e s t tubes which were stoppered w i t h cotton batten and p l a s t i c Kaput caps. One l i t r e batches were autoclaved f o r 2 0 minutes at o 121 C. A f t e r a u t o clavmg, t e s t tubes were h e l d a t a s l a n t of about 3 0°so t h a t when explants were c u l t u r e d any excess moisture c o u l d be dr a i n e d , and a l s o t o provide a gr e a t e r s u r f a c e area f o r shoot p r o l i f e r a t i o n . For the glyphosate study, the a d d i t i o n of glyphosate t o the medium d i f f e r e d from the procedure used f o r BA. U n l i k e BA, which was added p r i o r to a u t o c l a v i n g , glyphosate was added t o t h e medium a f t e r a u t o c l a v i n g . The glyphosate was f i l t e r s t e r i l i z e d and added when the medium had cooled t o 3 5 - 39 C. 24 3.2 L i g h t Sources Three l i g h t sources were used. Table 3.1 i n d i c a t e s the s p e c t r a l energy d i s t r i b u t i o n of each l i g h t source. H a l f the number of 4 0 watt Cool White f l u o r e s c e n t tubes i n a Conviron E7 growth chamber were used t o provide white l i g h t i n f o u r experiments. For the short term f a r - r e d and the i n i t i a l low photon f l u e n c e r a t e experiments, metal h a l i d e l i g h t was used. Cool White f l u o r e s c e n t tubes are t y p i c a l l y used as the s o l e l i g h t source i n both research and commercial micropropagation l a b o r a t o r i e s . S p e c t r a l output from such a white l i g h t source has proven t o be adequate i n promoting the growth of many micropropagated p l a n t s . Fluorescent l i g h t has a r e l a t i v e l y wide s p e c t r a l d i s t r i b u t i o n , w i t h almost equal energy outputs of blue and red wavelengths. In the experiment where intermediate wavelengths between blue and red were s t u d i e d , a Johns S c i e n t i f i c growth chamber w i t h two 2 0 watt Cool White f l u o r e s c e n t tubes per s h e l f (with t h r e e shelves) was used i n c o n j u n c t i o n w i t h the r e s p e c t i v e f i l t e r s t o provide green, yellow, and orange l i g h t . Two tungsten f i l a m e n t lamps (500 watts each) were used t o p r o v i d e red/FR l i g h t . Such lamps have a poor s p e c t r a l d i s t r i b u t i o n of blue wavelengths; they are c h a r a c t e r i z e d by h i g h p r o p o r t i o n s of red wavelengths and, although not shown i n Table 3.1, f a r - r e d wavelengths. The h i g h i n t e n s i t y lamps were used i n order t o minimize the lowering of photon f l u e n c e r a t e s t y p i c a l 25 Table 3.1 S p e c t r a l d i s t r i b u t i o n of the energy i n the v i s i b l e r e g i o n of the r a d i a t i o n of tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t l i g h t sources' S p e c t r a l L i g h t A c t u a l % of t« r e g i o n source wavelength r e g i o n range (nm) BI.- V i o . •3k Tun. 405-495 11.5 M.H. 400-510 28 . 4 F l . * 400-500 22.9 Ye.- Gr. Tun. 505-595 29.1 M.H. 510-610 43.0 F l . 500-600 49.3 Red-Or. Tun. 605-705 59.4 Red-Or. M.H. 610-700 • 18.8 F.R. M.H. 700-750 9.8 Red-Or. F l . 600-700 26.6 F.R. F l . 700-750 1.2 Legend: 1 - Except i n c l u d i n g p a r t s of the f a r - r e d r e g i o n f o r the metal h a l i d e and f l u o r e s c e n t l i g h t sources 2 - From (Moshkov, 19 69) 3 - From (Grimstad, 1987) 4 - From (Chee and P o o l , 1989) BI.- V i o . = b l u e - v i o l e t Ye.- Gr. = yellow-green Red- Or. = red-orange F.R. = f a r - r e d Tun. = tungsten f i l a m e n t M.H. = metal h a l i d e F l . = f l u o r e s c e n t w i t h f i l t e r use. The lamps were k e p t on a s h e l f o u t s i d e t h e g rowth chamber; l i g h t was t r a n s m i t t e d i n s i d e t h e g r o w t h chamber by t h e use o f l i g h t p i p e s . L i g h t p i p e s e l i m i n a t e t h e p r oblems o f h e a t b u i l d - u p t h a t would o c c u r i n s i d e t h e growth chamber i f t h e h i g h i n t e n s i t y lamps had been e n c l o s e d . Two m e t a l h a l i d e lamps (400 w a t t s each) were u s e d t o p r o v i d e b l u e l i g h t i n t h e f i r s t l i g h t q u a l i t y e x p e r i m e n t and w h i t e l i g h t f o r t h e two e x p e r i m e n t s as n o t e d i n t h e s e c t i o n on f l u o r e s c e n t l i g h t . Such lamps e m i t a h i g h p r o p o r t i o n o f b l u e w a v e l e n g t h s as w e l l as r e l a t i v e l y h i g h o u t p u t s o f o t h e r w a v e l e n g t h s . M e t a l h a l i d e l i g h t was chosen f o r t h e b l u e l i g h t s o u r c e because o f t h e p o o r t r a n s m i s s i o n o f t h e b l u e f i l t e r ; once l i g h t p a s s e d t h r o u g h i t t h e photon f l u e n c e r a t e was g r e a t l y r e d u c e d . Thus, because m e t a l h a l i d e was r i c h i n b l u e w a velengths and d i s c h a r g e d a h i g h p h o t o n f l u e n c e r a t e , i t was t h o u g h t t h a t t h e b l u e l i g h t o b t a i n e d would be a t a photon f l u e n c e r a t e c a p a b l e o f s u s t a i n i n g growth. As w i t h t h e t u n g s t e n f i l a m e n t lamps, m e t a l h a l i d e lamps were used i n c o n j u n c t i o n w i t h l i g h t p i p e s . 3.3 Photon Fluence Rates The photon f l u e n c e r a t e s used i n t h e e x p e r i m e n t s a r e g i v e n i n T a b l e 3.2. To r e d u c e t h e photon f l u e n c e r a t e o f f l u o r e s c e n t w h i t e l i g h t , n y l o n s c r e e n i n g w i t h 42 p e r f o r a t i o n s p e r cm was u sed. Depending on t h e f l u e n c e r e q u i r e d , two t o t h r e e s h e e t s were used. T h i s f l u e n c e r e d u c t i o n was i n a d d i t i o n t o t h a t Table 3.2 Photon fluence r a t e s used i n the experiments Experiment L i g h t q u a l i t y * Photon f l u e n c e r a t e (uM rn^s'1 ) I- blue, red/FR and white l i g h t e f f e c t s blue red/FR white 0.8 7 . 0 12 . 0 1.8 17 . 0 17 . 0 I I - e f f e c t of 3 intermediate l i g h t q u a l i t i e s between blue and red green yellow orange 6.0 32 . 0 21.0 13 .2 55.0 46.0 I I I - blue f i l t e r -l i g h t source e f f e c t s metal halide tungsten filament fluorescent 4.1 2.1 4.4 2 . 3 2.3 - 2.5 IV- red f i l t e r -l i g h t source e f f e c t s V- e f f e c t of short-term far-red l i g h t applied at the end of a white l i g h t photoperiod VI- e f f e c t s on the red/FR response caused by a low photon fluence and the time of tra n s f e r to a higher fluence metal halide tungsten filament fluorescent white l i g h t control white l i g h t + far-red l i g h t - before far-red extension - during far-red extension red/FR vS low l e v e l white" xN incubation level'' white 6.6 3.2 3.8 35.0 14.0 8.7 15.0 47 . 0 8.8 4.7 4 . 6 66.0 21.0 - 75.0 40.0 15. 9 23.0 62 .0 VII- glyphosate-l i g h t q u a l i t y i n t e r a c t i o n red/FR white 10.2 - 18.6 10.8 - 13.8 Legend: * - except for experiments III and IV where the l i g h t source i s s p e c i f i e d 28 p r o v i d e d by u t i l i z i n g o n l y h a l f o f t h e f l u o r e s c e n t t u b e s i n t h e growth chamber. The n y l o n s c r e e n i n g was a l s o used t o r e d u c e p h o t o n f l u e n c e r a t e s f o r t h e s i x t h e x p e r i m e n t where m e t a l h a l i d e l i g h t a c t e d as t h e low f l u e n c e w h i t e l i g h t s o u r c e . 3.4 F i l t e r s A l l f i l t e r s used were a c e t a t e f i l t e r s p r o d u c e d by Lee F i l t e r s . The t r a n s m i s s i o n c h a r a c t e r i s t i c s o f t h e f i l t e r s u s ed i n E x p e r i m e n t I a r e g i v e n i n F i g u r e 3.1 and t h o s e f o r E x p e r i m e n t I I a r e g i v e n i n F i g u r e 3.2. I n t h e i n i t i a l l i g h t q u a l i t y e x p e r i m e n t b l u e w a v e l e n g t h s were o p t i m i z e d w i t h t h e b l u e f i l t e r , Congo B l u e . I n e v e r y e x p e r i m e n t i n v o l v i n g red/FR l i g h t , t h e Medium Red f i l t e r was used. The t h r e e f i l t e r s i n F i g u r e 3.2 were used i n E x p e r i m e n t I I t o o p t i m i z e g r e e n , y e l l o w , and orange w a v e l e n g t h s . S i n c e f o r S p i r a e a , red/FR l i g h t was t h e l i g h t q u a l i t y w h i c h promoted i n v i t r o growth, a f u r t h e r a n a l y s i s o f t h e s p e c i f i c red/FR l i g h t q u a l i t y o b t a i n e d was done. To g a i n a more p r e c i s e c h a r a c t e r i z a t i o n , a s p e c t r o r a d i o m e t e r was used; t h e gr a p h o f t h e s p e c t r a l energy d i s t r i b u t i o n i s g i v e n i n F i g u r e 3.3. 3.5 Explant P r e p a r a t i o n and Incubation C o n d i t i o n s Shoot e x p l a n t s used f o r each e x p e r i m e n t were o b t a i n e d from 8 t o 10 w e e k - o l d s t o c k c u l t u r e s . The e x p l a n t s were t a k e n from t h e most a c t i v e l y g r o w i n g s e c t i o n s o f t h e s t o c k c u l t u r e s . 300 400 500 • 600 700 WAVELENGTH-NANOMETRES 800 3O0 400 &O0 6O0 700 800 WAVELENGTH-NANOMETRES Ltztz Filters HT027 MEDIUM RED (Y = 3.64%) 181 CONGO BLUE (Y = 0.78%) F i g u r e 3.1 T r a n s m i s s i o n c h a r a c t e r i s t i c s o f t h e a c e t a t e f i l t e r s u s e d i n E x p e r i m e n t I 30 300 4O0 SO0 600 700 800 WAVELENGTH-NANOMETRES 124 DARK G R E E N (Y = 29.71%) 100 300 400 500 600 700 800 WAVE LENGTH-NANOMETRES 105 ORANGE (Y = 41.28%) I I i L 80 | eo a 40 £ 2 20 < 300 4O0 500 6O0 700 800 WAVE LENGTH-NANOMETRES 101 YELLOW (Y = 80.00%) Figure 3.2 Transmission c h a r a c t e r i s t i c s of the acetate f i l t e r s used i n Experiment I I -Terminal segments, approximately 1 cm long i n c l u d i n g the apex, w i t h 3 t o 5 l e a f nodes, were ex c i s e d w i t h a s c a l p e l and t r a n s f e r r e d i n t o 25 x 150 mm c u l t u r e tubes c o n t a i n i n g 15 ml of n u t r i e n t medium, 1 explant per tube. The explants were placed u p r i g h t on the medium w i t h the cut end j u s t below the s u r f a c e of the medium. A l l experiments, except the short term f a r - r e d experiment, had l i g h t q u a l i t y regimes of 16 hours d u r a t i o n w i t h an 8 hour dark p e r i o d . The short term f a r - r e d experiment had a 6 hour dark p e r i o d ; the c o n t r o l white l i g h t regime had an 18 hour photoperiod w h i l e the treatment regime had a 16 hour photoperiod of white l i g h t f o l l o w e d by 2 hours of tungsten f i l a m e n t l i g h t . The experiments were c a r r i e d out i n growth chambers where the temperature was maintained between 21 and 2 3 C. For the f i r s t s et of experiments done t o c h a r a c t e r i z e the Spiraea l i g h t q u a l i t y response, explants were grown f o r two weeks under the c o n d i t i o n s used t o maintain stock c u l t u r e s . This two week growth p e r i o d was used t o o b t a i n p l a n t s of uniform s i z e and v i t a l i t y f o r the experimental treatments. Two i n c u b a t i o n l o c a t i o n s were used; one from September to May and the other from June t o August, due t o u n c o n t r o l l a b l e summer he a t i n g a t the September to May l o c a t i o n . The temperatures and photon fluence- r a t e s f o r both l o c a t i o n s are shown i n Table 3.3. In the experiment done to determine how a low photon fl u e n c e r a t e and t r a n s f e r time t o a higher fluence r a t e a f f e c t e d the l i g h t q u a l i t y response, there was no two week pre-experimental 32 T a b l e 3 . 3 I n c u b a t i o n c o n d i t i o n s f o r s t o c k c u l t u r e s L o c a t i o n T e m p e r a t u r e ( ° C ) P h o t o n f l u e n c e r a t e ( u M m s " 1 ) - S e p t . - M a y 3 - 3 2 ° d u r i n g p h o t o p e r i o d 5 2 - 7 4 J u n e - A u g u s t 2 0 - 2 2 d u r i n g d a r k p e r i o d 2 6 ° - 2 8 ° d u r i n g p h o t o p e r i o d 4 0 - 6 2 2 0 - 2 2 d u r i n g d a r k p e r i o d growth p e r i o d ; explants were immediately t r a n s f e r r e d t o the growth chambers used f o r the experiments. Here, s i n c e e x p l a n t s c o u l d not be i n i t i a l l y screened f o r uniform s i z e and v i t a l i t y , s e l e c t i o n of the most vigourous explants was done at the end of the experiment. For the glyphosate concentration t r i a l s , e xplants were grown f o r f i v e weeks under the stock c u l t u r e i n c u b a t i o n c o n d i t i o n s . In the g l y p h o s a t e - l i g h t q u a l i t y experiment, explants f o r the two-week pre-experimental growth period were grown on media w i t h the s p e c i f i c benzyladenine (BA) concentrations used i n the experiment but not c o n t a i n i n g any glyphosate. This was done because i t was thought t h a t the i n i t i a l explant, w h i l e a b l e t o grow a t the glyphosate l e v e l s used, might e x h i b i t a heightened s e n s i t i v i t y t o glyphosate a f t e r the s t r e s s of e x c i s i o n and i n i t i a l s u b c u l t u r e . Thus, i t was only a f t e r two weeks t h a t e x p l a n t s were t r a n s f e r r e d t o tubes w i t h f r e s h media c o n t a i n i n g glyphosate and BA (and glyphosate-free c o n t r o l c u l t u r e s were t r a n s f e r r e d t o tubes w i t h f r e s h media c o n t a i n i n g the r e s p e c t i v e BA c o n c e n t r a t i o n s used). These c u l t u r e s were l e f t f o r one f u r t h e r week under the stock c u l t u r e i n c u b a t i o n c o n d i t i o n s , s i n c e the o c c a s i o n a l contaminated tube due t o the t r a n s f e r needed t o be discarded. For the f i n a l three weeks, c u l t u r e s were t r a n s f e r r e d t o the growth chambers used f o r the a c t u a l l i g h t q u a l i t y experiment. Thus while a l l other experiments i n v o l v e d the h a r v e s t of five-week o l d c u l t u r e s , the c u l t u r e s from the g l y p h o s a t e - l i g h t q u a l i t y experiment were s i x weeks o l d a t 34 harvest. 3.6 H a r v e s t i n g P r o c e d u r e At the end of each experiment, cultures were kept i n the dark i n a growth chamber at room temperature u n t i l observations could be made. Growth variates for a l l the samples i n an experiment were measured within one week of an experiment's conclusion (due to the large number of samples). The order of treatments analyzed was randomized to reduce e f f e c t s due to any minimal growth during the one week harvest period. 3.7 E x p e r i m e n t a l D e s i g n f o r e a c h E x p e r i m e n t D e t e r m i n i n g t h e e f f e c t o f b l u e , r e d / F R , a n d w h i t e l i g h t o n i n  v i t r o g r o w t h A s p l i t p l o t experiment with three completely randomized blocks was used. Each block consisted of f i v e sub-samples for each of the two concentrations of BA (0.25 mg/1 and 0.5 mg/1) and the BA-free treatment, under each of the three l i g h t regimes (red/FR, blue and white). Each l i g h t regime, represented by a growth chamber, was a main plot, with the BA treatments and the BA-free treatment acting as sub-plots. The blocks represented experimental runs and thus were blocks over time. Experiment on three intermediate l i g h t wavelengths between blue and red A two-way f a c t o r i a l experiment was designed w i t h two completely randomized blocks over time. There were four sub-samples f o r each of the two BA concentrations (0.25 mg/1 and 0.5 mg/1) under each of the three l i g h t regimes (green, y e l l o w and orange). Light source experiments - blue f i l t e r and red f i l t e r For both the blue f i l t e r and red f i l t e r experiments, s p l i t p l o t s w i t h two completely randomized b l o c k s were used. Each block c o n s i s t e d of e i g h t sub-samples f o r each of the two c o n c e n t r a t i o n s of BA used (0.25 mg/1 and 0.5 mg/1) under each of the t h r e e l i g h t sources (tungsten f i l a m e n t , metal h a l i d e and f l u o r e s c e n t ) . Each l i g h t source was a main p l o t and the BA c o n c e n t r a t i o n s were sub-plots. The blocks represented experimental runs and thus were blocks over time. Determining the e f f e c t of a short-term far-red l i g h t exposure A s p l i t p l o t experiment w i t h f o u r completely randomized b l o c k s was used. Each block c o n s i s t e d of three sub-samples f o r each of the four concentrations of BA used (0.1 mg/1, 0.25 mg/1, 0.4 mg/1 and 0.5 mg/1) under each of the two l i g h t regimes (metal h a l i d e , and metal h a l i d e with end-of-day tungsten f i l a m e n t ) . Each l i g h t regime was a main p l o t and the BA c o n c e n t r a t i o n s were s u b - p l o t s . The n e c e s s i t y of u s i n g a d i f f e r e n t growth chamber f o r each l i g h t regime meant t h a t each bl o c k was s p l i t between the chambers. Factors contributing to the red/FR l i g h t growth response: low photon fluence rate and transfer time to higher fluence rate A s p l i t - s p l i t - p l o t experiment w i t h two completely randomized b l o c k s was used. Transfers of p l a n t s were made from two chambers w i t h low photon fl u e n c e s (one f o r tungsten f i l a m e n t l i g h t and one f o r metal h a l i d e l i g h t ) t o a f l u o r e s c e n t l i g h t chamber w i t h the higher photon f l u e n c e r a t e s used f o r m a i n t a i n i n g stock c u l t u r e s . Each block c o n s i s t e d of f i v e sub-samples f o r each treatment; of these f i v e the f o u r most healthy (as determined by the h i g h e s t shoot number) were used i n the ANOVA. The main p l o t s were the two l i g h t q u a l i t i e s of low photon flu e n c e r a t e . The sub - p l o t s were the three p e r i o d s under the low photon f l u e n c e regimes (a t r a n s f e r done a f t e r two weeks, one a f t e r f o u r weeks, and a s e t of p l a n t s remaining the whole f i v e weeks i n each chamber). Nested w i t h i n the sub-plots were the three BA c o n c e n t r a t i o n s used, 0.25 mg/1, 0.4 mg/1 and 0.5 mg/1. 37 Glyphosate-light q u a l i t y experiment A three-way f a c t o r i a l experiment was designed w i t h f o u r completely randomized b l o c k s . The n e c e s s i t y of u s i n g one growth chamber f o r red/FR l i g h t and another chamber f o r white l i g h t meant t h a t each block was s p l i t between the chambers. There were two c o n c e n t r a t i o n s of BA, 0.25 mg/1 and 0.5 mg/1, two c o n c e n t r a t i o n s of glyphosate, 0.089 mg/1 and 0.267 mg/1, and a g l y p h o s a t e - f r e e treatment counted as one of the glyphosate l e v e l s used. No sub-samples were used f o r the glyphosate treatments. The glyphosate-free (or c o n t r o l BA) treatments had two sub-samples per block. Each s e t of two sub-samples was averaged and the mean used f o r the ANOVA. 3 . 8 Growth Variates measured and S t a t i s t i c a l Analyses At the end of the c u l t u r e p e r i o d f o r each experiment, f i v e growth v a r i a t e s were recorded. These were shoot number/explant, node number/block of sub-samples, f r e s h weight/explant, dry weight/explant, and shoot length. To t e s t f o r the s t a t i s t i c a l s i g n i f i c a n c e of the f i r s t four v a r i a t e s , analyses of v a r i a n c e (ANOVA) were done. The f i f t h v a r i a t e , shoot l e n g t h , was analyzed by comparing the p r o p o r t i o n of shoots ranked as u s e f u l (1 cm or longer) w i t h the p r o p o r t i o n ranked not u s e f u l (below 1 cm). To t e s t f o r any s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s the C h i -square t e s t was used. 38 Chapter 4 Results and Discussion 4.1 Characterization of the Spiraea nipponica i n v i t r o l i g h t q u a l i t y response 4.1.1 Experiment I: Determining the e f f e c t s of blue, red/FR, and white l i g h t on i n v i t r o shoot p r o l i f e r a t i o n The f i r s t l i g h t q u a l i t y experiment was done to examine the response of an i n v i t r o shoot c u l t u r e t o the three l i g h t q u a l i t i e s , b l u e , red, and f a r - r e d , t h a t have been shown t o s i g n i f i c a n t l y modify the growth p a t t e r n s of both i n v i t r o and n a t u r a l l y grown p l a n t s . The experiment was done i n growth chambers. Blue l i g h t (400 nm - 500 nm) was obtained by u s i n g a metal h a l i d e l i g h t source and a blue acetate f i l t e r . Although i n most experiments red l i g h t (600 nm - 700 nm) and f a r - r e d l i g h t (700 nm - 750 ..nm) are s t u d i e d s e p a r a t e l y , the ease of o b t a i n i n g a high i n t e n s i t y mixture of both from a tungsten f i l a m e n t l i g h t source provided an o p p o r t u n i t y t o determine i f growth promoting e f f e c t s a t t r i b u t e d t o each s e p a r a t e l y could s t i l l occur when both l i g h t q u a l i t i e s were a p p l i e d together. A red acetate f i l t e r was used t o decrease the output of b l u e - v i o l e t and yellow-green wavelengths emitted by a tungsten f i l a m e n t l i g h t source. White l i g h t was obtained from Cool White f l u o r e s c e n t tubes, which emit l i g h t of a wide s p e c t r a l composition w i t h a r a t i o of blue t o red l i g h t of 0.9 (Table 4.1). C o n f l i c t i n g r e p o r t s i n the l i t e r a t u r e on the e f f e c t s of blue and red/FR l i g h t on i n v i t r o p l a n t growth made t h i s f i r s t experiment necessary t o determine which of the l i g h t q u a l i t i e s was optimal f o r the Spiraea i n v i t r o system. Once r e s o l v e d , the l i g h t q u a l i t y chosen could be more e x t e n s i v e l y s t u d i e d and p o s s i b l y have i t s growth e f f e c t s optimized, i n subsequent experiments. The main goal i n u s i n g l i g h t q u a l i t y t o enhance growth was t o lower the amount of exogenous c y t o k i n i n needed i n the medium. Thus, two BA l e v e l s were used, the normal BA l e v e l (0.5 mg/1) as a c o n t r o l , and h a l f t h i s l e v e l (0.25 mg/1). In a d d i t i o n c u l t u r e s were grown on BA-free media t o a s c e r t a i n whether or not BA was necessary f o r l i g h t q u a l i t y growth enhancement t o be expressed. Shoot numbers were determined and g i v e n a q u a l i t y r a t i n g mainly by determining the p r o p o r t i o n of u s e f u l shoots (defined as shoots 1 cm or l o n g e r ) . D e f i n i n g such shoots as u s e f u l i s meant as a measure of the shoots' probable s u r v i v e a b i l i t y f o r e i t h e r f u r t h e r s u b - c u l t u r e or f o r the subsequent phase of root i n d u c t i o n . The t o t a l node number f o r a l l the shoots from each b l o c k of f i v e i n i t i a l e xplants was taken as another q u a l i t y r a t i n g ; each node represents p o t e n t i a l 40 TABLE 4 . 1 Red l i g h t : far-red l i g h t and blue l i g h t : red l i g h t r a t i o s f o r u n f i l t e r e d tungsten filament, metal halide, and fluorescent l i g h t sources Licrht Source R:FR* B: R: Tungsten f i l a m e n t 0.7* 0. 2 Metal h a l i d e 2.7 1. 5 Flu o r e s c e n t 13 .1 0. 9 Legend: 1 - R:FR = 2 - B:R = Red l i g h t t o f a r - r e d l i g h t r a t i o Blue l i g h t t o red l i g h t r a t i o * - R:FR s p e c t r a l l y d e f i n e d and l i g h t source r a t i o s taken from (Hart, 1988). 41 f o r f u r t h e r a x i l l a r y shoot p r o l i f e r a t i o n . General Appearance P l a n t s grown under red/FR l i g h t g e n e r a l l y had l a r g e r leaves than p l a n t s grown under white l i g h t or blue l i g h t . A l s o red/FR l i g h t p l a n t s had v i s i b l y longer shoots than p l a n t s grown under white or blue l i g h t . Blue l i g h t p l a n t s g e n e r a l l y had a more compact growth p a t t e r n than p l a n t s from the other l i g h t regimes. Growth V a r i a t e s Shoot number/explant For a l l t h r e e l i g h t regimes the shoot number means f o r the BA-free c u l t u r e s were very low compared to the shoot number means at the 0.25 mg/1 BA l e v e l and the 0.5 mg/1 BA l e v e l (Table 4.2). These r e s u l t s i n d i c a t e the n e c e s s i t y of supplying BA i n the c u l t u r e medium t o induce s u b s t a n t i a l growth. A l s o , the s i m i l a r means recorded under each l i g h t regime suggests t h a t l i g h t q u a l i t y alone cannot s i g n i f i c a n t l y modify Spiraea i n v i t r o growth; l i g h t q u a l i t y e f f e c t s on i n v i t r o growth can only be f u l l y expressed i n concert w i t h the exogenously a p p l i e d c y t o k i n i n BA. The g r e a t e s t shoot p r o d u c t i o n , 21.6 shoots/explant, occurred under white l i g h t a t the 0.5 mg/1 BA l e v e l . A s i m i l a r l e v e l of Table 4 .2 42 Shoot, and node number means under blue, red/FR, and white l i g h t , w i th and without BA L i g h t BA 1 Shoot 2 Node 3 q u a l i t y l e v e l number number Blue 0 2.87 ± 0.25 58 ± 7 0.25 11.73 ± 0.93 255 ± 49 0.5 14.80 ± 1.66 310 ± 56 Red/FR 0 3.27 ± 0.36 79 ± 11 0.25 20.87 ± 1.84 518 ± 71 0.5 16.87 ± 1.89 413 ± 4 White 0 2.80 ± 0.44 75 ± 18 0.25 16.93 ± 1.63 419 ± 72 0.5 21.60 ± 2.18 485 ± 55 Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean + standard er ror 3 - node number/block of 5 sub-samples (mean of three blocks ± standard e r r o r ) 20.87 shoots/explant was obtained f o r red/FR c u l t u r e s a t the reduced c y t o k i n i n c o n c e n t r a t i o n of 0.25 mg/1. Red/FR c u l t u r e s had a mean of 16.87 at the 0.5 mg/1 BA l e v e l . Thus under red/FR l i g h t the maximal mean was found a t the 0.25 mg/1 BA l e v e l ; the decrease at the higher BA l e v e l suggests t h a t red/FR l i g h t e s t a b l i s h e s a c y t o k i n i n t o auxin r a t i o more favourable f o r shoot i n i t i a t i o n when c u l t u r e s are grown on medium c o n t a i n i n g 0.25 mg/1 of BA than when on medium w i t h the 0.5 mg/1 BA l e v e l . Blue l i g h t c u l t u r e s showed the lowest shoot number means at both BA l e v e l s . A n a l y s i s of Variance (ANOVA) showed th a t the only e f f e c t of l i g h t q u a l i t y was the lowering of mean shoot and node numbers under the blue l i g h t compared t o those under both red/FR and white l i g h t regimes (b/rw c o n t r a s t under l i g h t q u a l i t y i n Table 4.3). The BA c o n c e n t r a t i o n had the g r e a t e s t i n f l u e n c e on shoot number. There was a s t r o n g l i n e a r r e l a t i o n s h i p between shoot number and BA c o n c e n t r a t i o n i n both white and blue l i g h t c u l t u r e s . The h i g h e s t shoot number mean at the 0.25 mg/1 BA i n the red/FR l i g h t c u l t u r e s f o l l o w e d by a drop i n the mean at the 0.5 mg/1 BA l e v e l , may be i n t e r p r e t e d i n the ANOVA by the strong l a c k of f i t r e l a t i o n s h i p ( t h a t i s , a more complex mathematical model than a simple l i n e a r one i s needed to e x p l a i n the t r e n d ) . T h i s r e s u l t emphasizes the degree of d i f f e r e n c e between the red/FR l i g h t c u l t u r e s and both the white and blue l i g h t c u l t u r e s . The higher shoot number mean at the 0.25 mg/1 BA l e v e l under 44 Table 4.3 Summarized analyses of v a r i a n c e . E f f e c t s of blue, red/FR, and white l i g h t , and BA conc e n t r a t i o n on shoot and node numbers, and f r e s h and dry weights • Shoot 1 Node 2 F r e s h 3 Dry 4 Source of v a r i a t i o n df number number weight weight Light q u a l i t y ( 2 ) 5 * * * * * * * * b/rw 5 1 * * * * * * * ***** r/bw 1 NS NS * * * * * * r/w 1 NS NS NS NS BA concentration (2) ***** ***** ***** ***** l i n e a r ' 1 ***** ***** ***** ***** lack of f i t 1 ***** ***** ***** ***** Light q u a l i t y and BA i n t e r a c t i o n (4) * * * * * * * b/rw X li n e a r . 1 NS NS ** * * * b/rw X lack of f i t 1 * * * ** * r/bw X l i n e a r 1 - NS NS NS NS r/bw X lack of f i t 1 *** * * * * * * * r/w X l i n e a r 1 NS NS NS NS . r/w X lack of f i t 1 * * * NS Legend: 1 - shoot number/explant 2 - node number/block of 5 sub-samples ( t o t a l nodes of shoots of 5 explants) 3 - fresh weight/explant 4 - dry weight/explant 5 - e x t r a c o n t r a s t e x p l a i n s why c o n t r a s t df add to 3 instead of 2 6 - contrasts are: b/rw = blue versus red/FR and white l i g h t c u l t u r e s , r/bw = red/FR versus blue and white l i g h t c u l t u r e s , r/w = red/FR v e r s u s white l i g h t cultures 7 -- p o l y n o m i a l r e s p o n s e models: l i n e a r = amount of v a r i a t i o n explained by a s t r a i g h t l i n e r e l a t i o n s h i p , l a c k of f i t = amount of v a r i a t i o n e x p l a i n e d by a higher order-termed r e l a t i o n s h i p df = degree of freedom * = s i g n i f i c a n t at p < 0.1, ** = at p < 0.05, *** = at p < 0.025, **** = at p < 0.01, ***** = at p < 0.005, NS = non- s i g n i f i c a n t red/FR l i g h t i s s i g n i f i c a n t l y d i f f e r e n t than t h a t f o r white l i g h t ; t h i s r e f l e c t s the s i g n i f i c a n t i n t e r a c t i o n between l i g h t q u a l i t y and BA co n c e n t r a t i o n ( s p e c i f i c a l l y the r/w c o n t r a s t , Table 4.3). Although the s t a t i s t i c a l s i g n i f i c a n c e of t h i s i n t e r a c t i o n i s low (p < 0.1), the extent of the d i f f e r e n c e i s shown by the 23% gr e a t e r t o t a l shoot p r o d u c t i o n of red/FR l i g h t e x p l a n t s a t the 0.25 mg/1 BA l e v e l as compared t o the white l i g h t e x p l a n t s at the same l e v e l . A l s o i n c l u d e d i n t h i s i n t e r a c t i o n i s the s i g n i f i c a n t d i f f e r e n c e between the lower mean under red/FR l i g h t at the 0.5 mg/1 BA l e v e l and the maximal mean under white l i g h t at the 0.5 mg/1 BA l e v e l . One p o s s i b l e i n t e r p r e t a t i o n of these r e s u l t s i s t h a t red/FR l i g h t may be modifying c y t o k i n i n t o auxin r a t i o s i n the explant when the s y n t h e t i c c y t o k i n i n BA i s s u p p l i e d i n the growth medium. Node number/block of f i v e sub-samples Mean node numbers (Table 4.2) g e n e r a l l y f o l l o w e d the trends of the mean shoot numbers. The h i g h e s t value obtained was under red/FR l i g h t a t the 0.25 mg/1 BA c o n c e n t r a t i o n and t h i s s l i g h t l y exceeded t h a t f o r the higher BA l e v e l (0.5 mg/1) under white l i g h t . The mean shoot numbers were s i m i l a r l y high f o r these c o n d i t i o n s demonstrating t h a t p r o l i f e r a t i v e growth at the 0.25 mg/1 BA l e v e l under red/FR was comparable t o t h a t f o r white at the h i g h e r BA l e v e l . The ANOVA r e s u l t s a l s o g e n e r a l l y i n d i c a t e d the trends 46 observed f o r shoot number means (Table 4.3). Shoot l e n g t h For the BA-free c u l t u r e s and the c u l t u r e s t r e a t e d w i t h e i t h e r of the two BA l e v e l s , the range of shoots 1 cm or longer (considered " u s e f u l " shoots) was wide; f o r each BA l e v e l where the c u l t u r e s under a l l three l i g h t regimes were compared, blue l i g h t c u l t u r e s had the lowest p r o p o r t i o n of shoots 1 cm or longer, white l i g h t c u l t u r e s the median p r o p o r t i o n , and red/FR l i g h t c u l t u r e s the h i g h e s t u s e f u l shoot p r o p o r t i o n (Table 4.4). For t h i s experiment, as noted i n other s t u d i e s (Stimart and Harbage, 1989), i n c r e a s i n g the BA l e v e l i n h i b i t e d shoot e l o n g a t i o n ; f o r a l l three l i g h t regimes the highest p r o p o r t i o n s of u s e f u l shoots were at the lower BA l e v e l . The red/FR l i g h t regime caused the g r e a t e s t shoot e l o n g a t i o n f o r both the BA-free c u l t u r e s and the c u l t u r e s where the two l e v e l s of BA were used. When p a i r - w i s e comparisons of u s e f u l shoot p r o p o r t i o n s were done between white and red/FR l i g h t c u l t u r e s , with and without BA, o n l y BA-free c u l t u r e s showed no s i g n i f i c a n t d i f f e r e n c e . At the 0.25 mg/1 BA l e v e l the d i f f e r e n c e between red/FR and white l i g h t c u l t u r e s i s shown by the number of u s e f u l shoots produced under each l i g h t regime; f o r red/FR l i g h t c u l t u r e s t h i s amounted to 13 6 shoots w h i l e f o r white l i g h t c u l t u r e s u s e f u l shoots numbered 79. Even at the 0.5 mg/1 BA l e v e l , where red/FR c u l t u r e s had a lower shoot number t o t a l than white l i g h t 47 Table 4.4 Shoot l e n g t h s as a f f e c t e d by BA and three l i g h t q u a l i t i e s : b l u e , r e d / F R , a n d w h i t e t r e a t s .' B A 1 S a m p l e Lf U s e f u l 5 ^ A c t u a l u s e f u l X t e s t e d c o n . s i z e s h o o t V s h o o t number B l u e 0 43 9 4 ***** W h i t e 42 21 9 Red/FR 49 35 17 B l u e 0.25 176 23 40 ***** W h i t e 254 31 79 Red/FR 313 43 136 • B l u e 0 . 5 221 15 33 ***** W h i t e 324 23 75 Red/FR 262 40 105 B l u e 0 43 9 4 NS W h i t e 42 21 9 W h i t e 0 42 21 9 NS Red/FR 49 35 17 B l u e 0.25 176 23 40 * W h i t e 254 31 79 W h i t e 0.25 254 31 79 * * * * * Red/FR 313 43 136 B l u e 0.5 221 15 33 *** W h i t e 324 23 75 W h i t e 0 . 5 324 23 75 ***** Red/FR 262 40 105 W h i t e 0 . 5 324 23 75 * * * * * Red/FR 0.25 313 43 136 W h i t e 0 . 25 254 31 79 ** Red/FR 0 . 5 262 40- 105 Red/FR 0 . 25 313 43 136 NS Red/FR 0 . 5 262 40 105 W h i t e 0.25 254 31 79 ' * * " W h i t e 0.5. 324 23 75 L e g e n d : 1 - e a c h p a i r r e p r e s e n t s t h e s e t o f s h o o t l e n g t h , p r o p o r t i o n s a n a l y z e d xn a C h i - s q u a r e t e s t 2 - BA l e v e l i n mg/1 3 - t o t a l number o f s h o o t s p r o d u c e d i n a l l e x p e r i m e n t a l b l o c k s 4 - a u s e f u l s h o o t i s d e f i n e d as a s h o o t 1 cm o r l o n g e r i n l e n g t h ; t h u s t h e u s e f u l s h o o t % i s t h e H o f t h e s a m p l e s i z e r a n k e d u s e f u l 5 - t h e c o n v e r s i o n o f t h e u s e f u l s h o o t % i n t o number o f s h o o t s 6 - X i s t h e s y m b o l f o r t h e C h i - s q u a r e t e s t ; t h e a s t e r i s k s d e f i n e t h e d e g r e e o f s i g n i f i c a n c e : *** = s i g n i f i c a n t a t p<0.025, * * * * = a t p<0.01, * * * * * = a t p<0.oos, NS = n o n - s i g n i f i c a n t 7 - l i g h t s o u r c e s ; Tun.= t u n g s t e n f i l a m e n t , Me.H.= m e t a l h a l i d e , F l . = f l u o r e s c e n t 48 c u l t u r e s , the higher p r o p o r t i o n of longer shoots under red/FR l i g h t meant t h a t red/FR l i g h t c u l t u r e s had 105 u s e f u l shoots w h i l e white l i g h t c u l t u r e s had 75 u s e f u l shoots. Although the u s e f u l shoot p r o p o r t i o n s under red/FR l i g h t at both BA l e v e l s f o l l o w e d the tre n d of the other two l i g h t regimes, the d e c l i n e a t the higher BA l e v e l was the l e a s t of the three l i g h t regimes. The minor change i s confirmed by the C h i -square t e s t where the d i f f e r e n c e was found t o be not s i g n i f i c a n t (Table 4.4). Thus, red/FR l i g h t seems capable of reducing the i n h i b i t o r y e f f e c t on shoot e l o n g a t i o n of the higher BA l e v e l . Fresh weight/explant and Dry weight/explant The f r e s h weight means g e n e r a l l y f o l l o w e d the same trends f o r each l i g h t regime as f o r the shoot number means (Table 4.5) . However, the o v e r a l l f r e s h weight means under red/FR were g r e a t e r than under e i t h e r blue or white. Thus, w i t h a greater f r e s h weight per shoot i t i s p o s s i b l e t h a t shoot v i g o u r was a l s o i n c r e a s e d under red/FR l i g h t . When an ANOVA was done (Table 4.3), the f r e s h weight means g e n e r a l l y showed s i m i l a r r e s u l t s t o the shoot and node number means. Thus, d i f f e r e n c e s between f r e s h weight means occurred f o r each l i g h t regime according t o d i f f e r e n t BA concentrations. Although the degree of d i f f e r e n c e due t o l i g h t q u a l i t y was gr e a t e r f o r s e v e r a l comparisons (b/rw and r/bw c o n t r a s t s i n Table 4.3) than for-shoot and node number means, the d i f f e r e n c e Table 4.5 Fresh and dry weight means under blue, red/FR, and white l i g h t , w i t h and without BA Li g h t BA1 Fresh 2 Dry 3 q u a l i t y l e v e l weight weight Blue 0 17 + 1 3 0.25 50 + 4 8 + 1 0.5 68 ± 9 7 ± 1 Red/FR 0 26 + 2 6 0.25 146 + 14 19 + 1 0.5 127 + 19 17 + 2 White 0 22 ± 2 5 0.25 98 ± 9 15 + 1 0.5 119 ± 11 16 + 1 Legend: 1 - BA l e v e l i n mg/1 2 - fr e s h weight/explant mean ( i n mg) + standard e r r o r 3 - dry weight/explant mean ( i n mg) + standard e r r o r between red/FR and white l i g h t c u l t u r e s was s t i l l not s i g n i f i c a n t . Again i t was w i t h the l i g h t q u a l i t y - B A c o n c e n t r a t i o n i n t e r a c t i o n t h a t the red/FR l i g h t e f f e c t was apparent (r/w X l a c k of f i t i n Table 4.3). The dry weight means r e f l e c t the trends shown by f r e s h weight means (Table 4.5). However, the narrow range of dry weight means between the 0.25 mg/1 BA l e v e l and the 0.5 mg/1 BA l e v e l f o r both the red/FR and white l i g h t regimes d i d r e s u l t i n a l o s s of a s i g n i f i c a n t d i f f e r e n c e between red/FR and white l i g h t c u l t u r e s when the l i g h t q u a l i t y - B A c o n c e n t r a t i o n i n t e r a c t i o n was t e s t e d (r/w X l a c k of f i t i n Table 4.3). This r e s u l t c o n t r a s t s w i t h the f r e s h weight mean a n a l y s i s and i n d i c a t e s t h a t dry matter accumulation was only s l i g h t l y higher under red/FR l i g h t than under white l i g h t . 51 4.1.2 Experiment I I : Determining the e f f e c t s on i n v i t r o shoot p r o l i f e r a t i o n of three intermediate wavelength ranges between blue and red wavelengths The wavelengths most capable of modifying phytochrome are the b l u e , red, and f a r - r e d wavelengths. Minor s h i f t s i n phytochrome p h o t o e q u i l i b r i a can a l s o be caused by other wavelengths. I f such s h i f t s can induce endogenous phytohormone balances favourable f o r shoot p r o l i f e r a t i o n , then the requirement f o r exogenous c y t o k i n i n could be reduced. Thus, f o r t h i s experiment three wavelength ranges between the blue and red wavelengths were t e s t e d t o determine t h e i r e f f e c t on shoot p r o l i f e r a t i o n . The three wavelength ranges were green, y e l l o w , and orange. A f l u o r e s c e n t l i g h t source was combined w i t h green, y e l l o w , and orange acetate f i l t e r s . The f l u o r e s c e n t l i g h t source was chosen because of i t s h i gher energy output of green, y e l l o w , and orange wavelengths compared t o the metal h a l i d e source. Growth Variates Shoot number/explant At the 0.25 mg/1 BA l e v e l the shoot number means are q u i t e s i m i l a r (Table 4.6). At the 0.5 mg/1 BA l e v e l t h e r e i s a t r e n d of i n c r e a s e d shoot number means as the wavelengths approach the i I 52 Table 4.6 Shoot and node number means under three intermediate wavelengths (g'reen, yellow, and orange) between blue and red l i g h t . Light BA 1 Shoot 2 Node 3 Quality Level Number Number Green 0 25 8 63 + 2 12 152 + 1 0 5 11 38 ± 1 86 204 + 34 Yellow 0 25 7 13 + 1 10 153 + 28 0 5 . 15 75 + 2 01 299 + 57 Orange 0 25 8 38 + 2 27 180 + 2 0 5 18 38 + 6 13 303 + 50 Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean ± standard error 3 - node number/block (mean of two b l o c k s ± standard error) red wavelength range. However, the ANOVA (Table 4.7) i n d i c a t e s t h a t the only f a c t o r t h a t caused s i g n i f i c a n t changes i n shoot number was BA c o n c e n t r a t i o n . There was no e f f e c t due t o e i t h e r l i g h t q u a l i t y alone or t o a l i g h t q u a l i t y - B A c o n c e n t r a t i o n i n t e r a c t i o n . Thus, the u n d e r l y i n g goal of reducing exogenous c y t o k i n i n l e v e l s cannot be achieved by usi n g e i t h e r green, y e l l o w , or orange l i g h t , as i s i n d i c a t e d by the s i m i l a r shoot number means at the 0.25 mg/1 BA l e v e l . The ANOVA a l s o i n d i c a t e s t h a t w h i l e growth promotion at the higher BA l e v e l d i d occur f o r a l l three l i g h t regimes, to the g r e a t e s t extent under y e l l o w and orange l i g h t , h i g h v a r i a b i l i t y among i n d i v i d u a l c u l t u r e s under orange l i g h t meant t h a t any d e f i n i t e t r e n d could not be a f f i r m e d . The f a c t t h a t green l i g h t c u l t u r e s grew at s i m i l a r r a t e s t o c u l t u r e s under the other two l i g h t regimes provides evidence f o r the s t r o n g promotive e f f e c t of BA on shoot i n i t i a t i o n i n the Spiraea system; i n a study on another i n v i t r o p l a n t , Juneberry, green l i g h t g r e a t l y reduced shoot i n i t i a t i o n even when BA was used (Behrouz and Lineberger, 1981). Node number/block of 4 sub-samples Node number means f o l l o w e d the p a t t e r n of shoot number means (Table 4.6). However, at the 0.5 mg/1 BA l e v e l the node number means f o r ye l l o w and orange l i g h t were much more s i m i l a r than the corresponding shoot number means. The ANOVA (Table 4.7) 54 Table 4.7 Summarized analyses of variance. E f f e c t s of three l i g h t q u a l i t i e s between blue and red l i g h t (green, yellow, and orange) and BA concentration on shoot and node numbers, and fresh and dry weights. Shoot 1 Node 2 F r e s h 3 Dry 4 Source of v a r i a t i o n df. number number weight weight Light Quality (2) NS NS NS NS O/GY5 1 NS NS NS NS 0/Y 1 NS NS NS NS BA concentration 1 * * * * * NS Light q u a l i t y and BA i n t e r a c t i o n (2) NS NS NS NS O/GY X BA 1 NS NS NS NS 0/Y X BA 1 NS NS NS NS Legend: 1 2 3 4 5 df * shoot number/explant node number/block of 4 sub-samples ( t o t a l nodes of shoots of 4 explants) fresh weight/explant dry weight/explant contrasts are: O/GY = orange versus green and yellow l i g h t c u l t u r e s , O/Y = orange v e r s u s y e l l o w l i g h t cultures degree of freedom s i g n i f i c a n t at p < 0.01, ** = at p < 0.05, NS = non-s i g n i f i c a n t 55 i n d i c a t e s t h a t , as f o r shoot number means, node number means only d i f f e r e d due t o BA con c e n t r a t i o n . Shoot l e n g t h At both BA l e v e l s the p r o p o r t i o n of u s e f u l shoots i s lowest under green l i g h t (Table 4.8). While there i s no s t a t i s t i c a l d i f f e r e n c e between any u s e f u l shoot p r o p o r t i o n at the 0.25 mg/1 BA l e v e l , t h e r e i s a d i f f e r e n c e at the 0.5 mg/1 BA l e v e l . The comparison between y e l l o w and green l i g h t a t the 0.5 mg/1 BA l e v e l i n d i c a t e s t h a t i t i s the p r o p o r t i o n s of u s e f u l t o non-u s e f u l shoots under these two l i g h t q u a l i t i e s t h a t d i f f e r . Thus, i n the Spiraea system green l i g h t seems capable of e x e r t i n g an i n h i b i t o r y e f f e c t on shoot e l o n g a t i o n . Fresh weight/explant and Dry weight/explant The.increase i n the f r e s h weight mean from the lower t o the high e r BA l e v e l i s s i m i l a r f o r both y e l l o w and orange l i g h t c u l t u r e s (Table 4.9). The mean under green l i g h t i s s l i g h t l y lower at the lower BA l e v e l but i s much lower than the other two l i g h t q u a l i t y means at the higher BA l e v e l . The low mean a t the hi g h BA l e v e l i n d i c a t e s a lower f r e s h weight per shoot under green l i g h t than f o r shoots under the other two l i g h t q u a l i t i e s ; t h i s suggests a lower v i g o u r f o r the green l i g h t shoots. The s t r e n g t h of the f r e s h weight mean inc r e a s e from the Table 4.8 Shoot l e n g t h s as a f f e c t e d by BA and three ! q u a l i t i e s between blue and r e d : green, yellow, orange treats'. 3 Sample if U s e f u l ^ A c t u a l 5 a& u s e f u l X* t e s t e d con. s i z e shoot % shoot number Green 0.25 69 33 23 NS Yellow 57 39 22 Orange 67 37 25 Green 0.5 91 31 28 * Yellow 126 43 54 Orange 137 38 52 Yellow 0.25 57 39 22 NS Orange 67 37 25 Yellow 0.25 57 39 22 NS Green 69 33 23 Orange 0.25 67 37 25 NS Green 69 33 23 Yellow 0.5 126 _ 43 54 ^ NS Orange 137 38 52 Yellow 0.5 126 43 54 * Green 91 31 28 Orange 0.5 137 38 52 NS Green 91 31 28 Orange 0.25 67 37 25 NS Green 0 . 5 91 31 28 Yellow 0.25 57 39 22 NS Green 0.5 91 31 28 Yellow 0.25 57 39 22 NS Orange 0.5 137 38 52 Orange 0.5 137 38 52 NS Green 0.25 69 33 23 Yellow 0.5 126 43 54 NS Green 0.25 69 33 23 Yellow 0.5 126 43 54 NS Orange 0.25 67 37 25 Legend: 1 - each p a i r r e p r e s e n t s the s e t of shoot l e n g t h p r o p o r t i o n s analyzed i n a Chi-square t e s t 2 - BA l e v e l i n mg/1 3 - t o t a l number of shoots produced i n a l l experimental b l o c k s 4 - a u s e f u l shoot i s d e f i n e d as a shoot 1 cm or lo n g e r i n l e n g t h ; thus the u s e f u l shoot % i s the % of the sample s i z e ranked u s e f u l 5 - the c o n v e r s i o n of the u s e f u l shoot H i n t o number of shoots 6 - X i s the symbol f o r the Chi-square t e s t ; the a s t e r i s k s d e f i n e the degree of s i g n i f i c a n c e : *** = s i g n i f i c a n t at p<0.025, **** = at p<0.01, ***** = at p<0.005, NS = n o n - s i g n i f i c a n t Table 4.9 Fresh and dry weight means under three intermediate wavelengths (green, yellow, and orange) between blue and red l i g h t . Light BA 1 F r e s h 2 Dry 3 Quality Level Weight Weight Green 0 .25 51 9 0 . 5 59 + 4 9 Yellow 0.25 67 + 16 15 + 3 0 . 5 143 + 31 23 + 3 Orange 0.25 61 + 1 14 0 . 5 128 + 17 21 + 2 Legend: 1 2 3 BA l e v e l i n mg/1 fresh weight/explant mean ( i n mg) ± standard error dry weight/explant mean ( i n mg) + standard error 58 lower t o the higher BA l e v e l under y e l l o w and orange l i g h t was enough t o o v e r r i d e the low green l i g h t means; thus, as f o r shoot and node number means, BA c o n c e n t r a t i o n caused the l a r g e s t d i f f e r e n c e (ANOVA, Table 4 . 7 ) . Dry weight means fo l l o w e d the p a t t e r n of f r e s h weight means (Table 4 . 9 ) . However, i n the dry weight ANOVA (Table 4.7) the low green l i g h t means seem t o have negated the s i g n i f i c a n c e of any BA c o n c e n t r a t i o n e f f e c t i n causing dry weight mean d i f f e r e n c e s s i n c e there was no s i g n i f i c a n t d i f f e r e n c e due t o any f a c t o r . Thus, the evidence provided by dry weight means suggests t h a t green l i g h t a l s o e x e r t s an i n h i b i t o r y e f f e c t on growth by reducing dry matter accumulation. 59 4.1.3 Experiment I I I : Determining the e f f e c t s on i n v i t r o shoot p r o l i f e r a t i o n of three l i g h t source-blue f i l t e r combinations In the f i r s t l i g h t q u a l i t y experiment, metal h a l i d e l i g h t was chosen as the blue l i g h t source ( i n combination w i t h a blue a c e t a t e f i l t e r ) because of i t s high p r o p o r t i o n of blue wavelengths and a l s o because of i t s small f a r - r e d l i g h t component. Fluo r e s c e n t l i g h t a l s o has a h i g h p r o p o r t i o n of blue wavelengths; thus, t o t e s t t h a t a maximal blue l i g h t e f f e c t was achieved u s i n g metal h a l i d e , f o r t h i s experiment the f l u o r e s c e n t l i g h t source was a l s o combined w i t h the blue f i l t e r . Combining the blue f i l t e r w i t h the tungsten f i l a m e n t l i g h t source, as w e l l as w i t h the other l i g h t sources, provided an o p p o r t u n i t y t o study the e f f e c t on i n v i t r o growth of v a r y i n g the r a t i o s of b l u e , red, and f a r - r e d wavelengths (which d i f f e r f o r each l i g h t source). The r a t i o s t y p i c a l f o r each l i g h t source are shown i n Table 4.1. Using a blue f i l t e r w i t h the three l i g h t sources would decrease the red l i g h t t o f a r - r e d l i g h t r a t i o and i n c r e a s e the blue l i g h t t o red l i g h t r a t i o . Growth Variates Shoot number/explant As i s shown i n Table 4.10, at the lower BA l e v e l the metal 60 Table 4.10 Shpot and node number means under three l i g h t sources (tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t ) combined with a blue acetate f i l t e r . L ight . BA 1 Shoot 2 Node 3 Source Level Number Number Tungsten 0.25 9.19 ± 0.84 254 ± 17 filament 0.5 9.50 ± 0.84 241 + 17 Metal 0.25 7.63 ± 0.86. 230 h a l i d e 0.5 11.06 ± 1.69 306 ± 8 Fluorescent 0.25 7.75 ± 0.84 218 ± 4 0.5 10.81 ± 1.71 270 ± 11 Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean ± standard error 3 - node number/block (mean of two b l o c k s ± standard error) h a l i d e - b l u e f i l t e r treatment caused a s l i g h t l y lower shoot number mean than the f l u o r e s c e n t - b l u e f i l t e r treatment. The tungsten f i l a m e n t - b l u e f i l t e r treatment had the h i g h e s t mean at the lower BA l e v e l but the d i f f e r e n c e was s m a l l . At the higher BA l e v e l the metal h a l i d e mean was s l i g h t l y g r e a t e r than the f l u o r e s c e n t mean. Tungsten f i l a m e n t a t the higher BA l e v e l caused a s i m i l a r mean as the tungsten f i l a m e n t treatment at the lower BA l e v e l . As the ANOVA i n Table 4.11 i n d i c a t e s , the o v e r a l l d i f f e r e n c e s i n shoot number means due t o the l i g h t sources were not s i g n i f i c a n t . S e v e r a l r e s u l t s of t h i s experiment m i r r o r those seen i n the f i r s t experiment. F i r s t there are the s i g n i f i c a n t i n c r e a s e s i n shoot number means under metal h a l i d e and f l u o r e s c e n t l i g h t due to BA c o n c e n t r a t i o n . Then t h e r e i s the hi g h e s t shoot number mean at the lower BA l e v e l produced under tungsten f i l a m e n t l i g h t . In the f i r s t experiment the blue l i g h t source showed the g r e a t e s t r e d u c t i o n of growth. In t h i s experiment the metal h a l i d e - b l u e f i l t e r regime induced v i r t u a l l y the same range of shoot number means as the f l u o r e s c e n t - b l u e f i l t e r regime which suggests t h a t both caused an e q u i v a l e n t blue l i g h t e f f e c t on growth. A d i f f e r e n c e between the r e s u l t s of t h i s experiment and those of Experiment I was the g e n e r a l l y lower shoot number means, suggestive of a lower growth r a t e , a t both BA l e v e l s f o r a l l three l i g h t sources. This may have occurred i f the source c u l t u r e s used had a lower morphogenetic p o t e n t i a l than i n the f i r s t experiment, a f a c t o r which may have modified the e x p l a n t s ' Table 4.11 62 Summarized analyses of variance. E f f e c t s of l i g h t source-blue f i l t e r combination and BA concentration on shoot and node numbers, and fr e s h and dry weights Source of V a r i a t i o n df Shoot 1 number Node 2 number Fresh 3 wt. Dry 4 wt. L i g h t source T / M e F 5 T / F (2) 1 1 NS NS NS NS NS NS NS NS NS NS BA c o n c e n t r a t i o n * * * * * NS L i g h t source and BA i n t e r a c t i o n (2) NS T/MeF X BA 1 NS T / F X BA 1 NS NS * * NS NS NS NS NS NS Legend: 1 2 3 4 5 df shoot number/explant node number/block of 8 sub-samples ( t o t a l nodes o f shoots of 8 exp lant s ) f r e s h w e i g h t / e x p l a n t dry w e i g h t / e x p l a n t c o n t r a s t s a r e : T/MeF = tungsten f i l a m e n t v e r s u s meta l h a l i d e and f l u o r e s c e n t l i g h t c u l t u r e s , T / F = tungs ten f i l ament versus f l u o r e s c e n t l i g h t c u l t u r e s degree of freedom s i g n i f i c a n t at p < 0.1 , ** = at p < 0.05 , * * * = a t p < 0.025 , NS = n o n - s i g n i f i c a n t response t o l i g h t q u a l i t y . For example, another d i f f e r e n c e i n t h i s experiment, the s i m i l a r shoot number means at both BA l e v e l s under tungsten f i l a m e n t l i g h t , may be p a r t i a l l y due t o a lower growth r a t e . The main f a c t o r r e s p o n s i b l e f o r the r e d u c t i o n of growth under the tungsten f i l a m e n t - b l u e f i l t e r regime i s probably the poor energy output from the r e s t of the spectrum once the m a j o r i t y of red l i g h t i s f i l t e r e d out by the blue f i l t e r . Another reason f o r the g e n e r a l l y lower shoot numbers under each l i g h t source may be the changes i n the blue t o red r a t i o . This e f f e c t may be e s p e c i a l l y apparent f o r the f l u o r e s c e n t and metal h a l i d e blue f i l t e r regimes, w i t h much higher u n f i l t e r e d l e v e l s of b lue wavelengths than u n f i l t e r e d tungsten f i l a m e n t l i g h t . For the metal h a l i d e and f l u o r e s c e n t sources the use of a blue f i l t e r would cause an i n c r e a s e i n the B:R r a t i o i n a d d i t i o n t o a l a r g e r e d u c t i o n i n both the q u a n t i t y of red l i g h t and the R:FR r a t i o . E i t h e r of these m o d i f i e d l i g h t r a t i o s may play a r o l e i n the i n h i b i t i o n of shoot i n i t i a t i o n . Node number/block of eight sub-samples The p a t t e r n of node number means (Table 4.10) was s i m i l a r t o the shoot number means. The ANOVA (Table 4.11) i n d i c a t e s t h a t , as f o r shoots, node number means under the f l u o r e s c e n t and metal h a l i d e blue f i l t e r regimes d i f f e r e d most due t o BA co n c e n t r a t i o n , w i t h fewer nodes at 0.25 mg/1 BA than at 0.5 mg/1 64 BA (as f o r shoot number means, the mean node numbers under the tungsten f i l a m e n t - b l u e f i l t e r was v i r t u a l l y the same at both BA l e v e l s except t h a t the s l i g h t decrease noted at the higher BA l e v e l was not s i g n i f i c a n t ) . The degree of the increase i n node number from the lower t o the higher BA l e v e l f o r the f l u o r e s c e n t - b l u e f i l t e r regime i n p a r t i c u l a r , i s made apparent by the i n t e r a c t i o n between l i g h t source and BA c o n c e n t r a t i o n i n the ANOVA t a b l e (T/F c o n t r a s t i n Table 4.11), where tungsten f i l a m e n t - b l u e f i l t e r node number means s i g n i f i c a n t l y d i f f e r from f l u o r e s c e n t - b l u e f i l t e r means. Shoot l e n g t h None of the p a i r comparisons showed s i g n i f i c a n t d i f f e r e n c e s (Table 4.12). The hi g h e s t p r o p o r t i o n of u s e f u l shoots, 30%, occurred under the metal h a l i d e - b l u e f i l t e r w i t h 0.25 mg/1 BA; however, the range of u s e f u l shoots f o r a l l treatments was narrow, w i t h 20% f o r 0.5 mg/1 BA under tungsten f i l a m e n t l i g h t being the lowest. F r e s h w e i g h t / e x p l a n t and Dry w e i g h t / e x p l a n t The range of f r e s h weight/explant means i s q u i t e narrow f o r each BA l e v e l under each l i g h t source (Table 4.13). As f o r shoot and node number means, t h e r e was a change i n f r e s h weight/explant means due t o BA co n c e n t r a t i o n under f l u o r e s c e n t Table 4.12 Shoot lengths as affected by three light source-blue ^filter combinations. P a i r s 1 tested BA2 con. Sample3 s ize Useful 4 Actual shoot % shoot 6 use fu l 5 X 2 number T u n . 7 0.25 147 23 • 34 NS Me.H. 122 30 37 Tun. 0.25 147 23 34 NS F I . 124 22 27 F I . 0.25 124 22 27 NS Me.H. 122 30 37 Tun. 0.5 152 20 31 NS F I . 173 22 38 Tun. 0.5 152 20 31 NS Me . H . 177 23 40 F I . 0.5 173 22 38 NS Me.H 177 23 40 Tun. 0.25 147 23 34 NS Tun. 0.5 152 20 31 Tun. 0.25 147 23 34 NS Me.H. 0.5 177 23 40 Tun. 0.25 147 23 34 NS F I . 0.5 173 22 38 Legend: 1 - < each pair represents the set of shoot length proportions analyzed in a Chi-square test 2 - BA l e v e l in mg/1 3 - t o t a l number of shoots produced in a l l experimental blocks 4 - a useful shoot i s defined as a shoot 1 cm or longer in length; thus the useful shoot % is the % of the sample s ize ranked useful 5 - the conversion of the useful shoot % into number of shoots 6 - X i s the symbol for the Chi-square test; NS = non-s ignif icant 7 - l i g h t sources; Tun.= tungsten f i lament, Me.H.= metal ha l ide , F l . = fluorescent 66 Table 4.13 Fresh and dry weight means under three l i g h t sources (tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t ) combined with a blue acetate f i l t e r . Light BA 1 F r e s h 2 Dry 3 Source Level Weight Weight Tungsten 0 .25 45 + 3 6 + 1 filament 0 . 5 45 + 3 7 Metal 0.25 43 + 4 6 + 1 halide 0.5 50 ± 6 7 + 1 Fluorescent 0.25 37 + 3 6 0.5 46 + 6 6 + 1 Legend: 1 2 3 BA l e v e l i n mg/1 fresh weight/explant mean (in mg) ± standard error dry weight/explant mean (in mg) ± standard error 67 and metal h a l i d e l i g h t . Fresh weight/explant means d i f f e r from shoot and node number means by having a l i g h t source e f f e c t . I n t e r e s t i n g l y , although under metal h a l i d e l i g h t the shoot number means were lower than i n the f i r s t experiment, the f r e s h weight/explant means were s i m i l a r (43 mg and 50 mg f o r the 0.25 mg/1 and 0.5 mg/1 BA l e v e l s i n t h i s experiment compared t o 50 mg and 68 mg i n the f i r s t experiment). The f r e s h weight/explant means under the other two l i g h t sources were much lower than i n the f i r s t experiment. Thus, s i n c e the photon fluence r a t e f o r the metal h a l i d e - b l u e f i l t e r combination was s i m i l a r i n both experiments, but much lower f o r the tungsten f i l a m e n t and f l u o r e s c e n t l i g h t combinations (see Table 3.2), there i s the need t o take photon fluence r a t e e f f e c t s i n t o account as w e l l as the lower morphogenetic p o t e n t i a l of source c u l t u r e s t o e x p l a i n the lower growth r a t e under these two l i g h t sources. The range of dry weight/explant means was very narrow; between 6 and 7 mg over a l l the treatments (Table 4.13). The ANOVA (Table 4.11) confirms the n e g l i g i b l e d i f f e r e n c e s ; none were s t a t i s t i c a l l y s i g n i f i c a n t . These low dry weight/explant means emphasize the g e n e r a l l y low p r o l i f e r a t i o n r a t e c h a r a c t e r i s t i c of t h i s experiment. A l s o , as f o r f r e s h weight/explant means, dry weight/explant means under metal h a l i d e l i g h t were s i m i l a r t o the f i r s t experiment, w h i l e under tungsten f i l a m e n t and f l u o r e s c e n t l i g h t there was a re d u c t i o n , suggesting t h a t the photon f l u e n c e r a t e may be a f a c t o r reducing growth under these two l i g h t sources. 68 4.1.4 Experiment IV: Determining the e f f e c t s on i n v i t r o shoot p r o l i f e r a t i o n of three l i g h t source-red f i l t e r combinations Tungsten f i l a m e n t l i g h t was used i n t h e f i r s t e x p e r i m e n t as a s o u r c e o f r e d and f a r - r e d l i g h t because i t e m i t s a h i g h energy o u t p u t o f t h e s e two l i g h t q u a l i t y r a n g e s . I n t h i s l i g h t s o u r c e -r e d f i l t e r e x p e r i m e n t , t h e main p u r p o s e was t o d e t e r m i n e i f t h e r e d f i l t e r c o u l d o p t i m i z e t h e s m a l l e r p r o p o r t i o n o f r e d l i g h t under m e t a l h a l i d e and f l u o r e s c e n t l i g h t . The i m p o r t a n c e o f t h e r e d l i g h t t o f a r - r e d l i g h t r a t i o (R:FR) i n d e t e r m i n i n g t h e S p i r a e a i n v i t r o growth r e s p o n s e c o u l d be t e s t e d s i n c e t h e r a t i o v a r i e s w i d e l y between m e t a l h a l i d e and f l u o r e s c e n t l i g h t ( T a b l e 4.1). The r e d f i l t e r , by f i l t e r i n g o u t most o f t h e b l u e - v i o l e t and y e l l o w - g r e e n w a v e l e n g t h s , s h o u l d h e i g h t e n any R: FR r a t i o e f f e c t on g r o w t h . F o r m e t a l h a l i d e l i g h t t h e r e d f i l t e r w i l l r e d u ce t h e R:FR r a t i o . F o r f l u o r e s c e n t l i g h t , w i t h t h e h i g h e s t R:FR r a t i o ( T a b l e 4.1), t h e m o d i f i e d R:FR r a t i o w i l l s t i l l r e main much h i g h e r t h a n t h a t o f f i l t e r e d m e t a l h a l i d e l i g h t . Growth Variates Shoot number/explant As i s shown i n T a b l e 4.14, a t t h e 0.25 mg/1 BA l e v e l , t h e 69 Table 4.14 Shpot and node number means under three l i g h t sources (tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t ) combined with a red acetate f i l t e r . Light BA Shoot^ Node J Source Level Number Number Tungsten 0 25 9 50 + 1 16 322 + 5 filament 0 5 16 06 + 2 24 473 + 25 Metal 0 25 6 81 + 1 14 252 + 20 halide 0 5 11 31 + 1 43 349 + 45 Fluorescent 0 25 7 25 + 0 81 216 + 11 0 5 13 81 + 0 96 352 + 18 Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean ± standard error 3 - node number/block (mean of two blocks) ± standard error 70 tungsten f i l a m e n t - r e d f i l t e r combination had the highest shoot number mean, f o l l o w e d by f l u o r e s c e n t l i g h t , w i t h the metal h a l i d e l i g h t treatment being the lowest. At the 0.5 mg/1 BA l e v e l the same p a t t e r n was e x h i b i t e d . The in c r e a s e s i n shoot number means under metal h a l i d e and f l u o r e s c e n t l i g h t due t o BA c o n c e n t r a t i o n (Table 4.15) r e f l e c t the t r e n d observed f o r these l i g h t sources i n the f i r s t l i g h t q u a l i t y experiment. But f o r both l i g h t sources the d i f f e r e n t i a l between the lower and higher BA l e v e l s i s much higher i n t h i s experiment than i n the f i r s t experiment ( i n t h i s experiment an incr e a s e of 66.1% under metal h a l i d e and 90.5% under f l u o r e s c e n t l i g h t compared t o the f i r s t experiment i n c r e a s e s of 26.2% under metal h a l i d e and 27.6% under f l u o r e s c e n t l i g h t ) . Nevertheless, the o v e r a l l shoot number l e v e l s f o r metal h a l i d e and f l u o r e s c e n t l i g h t w i t h the red f i l t e r were q u i t e s i m i l a r i n t h i s experiment and much c l o s e r than the l e v e l s observed i n Experiment I . Since the R:FR r a t i o s of the two l i g h t sources remained q u i t e d i f f e r e n t u s i n g the red f i l t e r , i t seems u n l i k e l y that these r a t i o s caused the s i m i l a r p r o l i f e r a t i o n r a t e . Another p o s s i b l e f a c t o r i s the blue t o red r a t i o (B:R) . The B:R r a t i o of metal h a l i d e w i t h the red f i l t e r w i l l be lower than i n Experiment I and c l o s e r t o the B:R r a t i o under f l u o r e s c e n t l i g h t with the red f i l t e r . Perhaps then the B:R r a t i o i s an important f a c t o r causing the s i m i l a r i t i e s between the metal h a l i d e and f l u o r e s c e n t shoot number means. F i n a l l y , the q u a n t i t y of red l i g h t may a l s o be i m p l i c a t e d as Table 4.15 71 Summarized a n a l y s e s o f v a r i a n c e . E f f e c t s o f l i g h t s o u r c e - r e d f i l t e r c o m b i n a t i o n and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and d r y weigh t s Source o f V a r i a t i o n df Shoot 1 number Node 2 number F r e s h 3 wt. D r y 4 wt. L i g h t s o u r c e (2.) * * * * ** T/MeF5 1 * * * * * * * * * * T/F 1 * * * * * * * * * BA c o n c e n t r a t i o n 1 ***** ***** * * * * L i g h t s o u r c e and BA i n t e r a c t i o n 2 NS NS NS NS Legend: 1 - shoot number/explant 2 - node number/block of 8 sub-samples ( t o t a l nodes o f shoots of 8 e x p l a n t s ) 3 - f r e s h w e i g h t / e x p l a n t 4 - dry w e i g h t / e x p l a n t 5 - c o n t r a s t s a r e : T/MeF = t u n g s t e n f i l a m e n t v e r s u s m e t a l h a l i d e and f l u o r e s c e n t l i g h t c u l t u r e s , T/F = tu n g s t e n f i l a m e n t v e r s u s f l u o r e s c e n t l i g h t c u l t u r e s df = degree o f freedom * = s i g n i f i c a n t a t p < 0.1, ** = a t p < 0.05, *** a t p <. 0.025, **** = a t p < 0.01, ***** = a t p < 0.005, NS = n o n - s i g n i f i c a n t an important shoot i n i t i a t i o n f a c t o r . As shown i n Table 3.1, f l u o r e s c e n t l i g h t emits 26.6% of i t s energy output as red wavelengths, metal h a l i d e 18.8% and tungsten f i l a m e n t 59.4%. Thus, the hi g h e s t shoot number means which were caused by tungsten f i l a m e n t l i g h t may suggest t h a t t h i s l i g h t source's high red wavelength output promoted shoot i n i t i a t i o n . The s l i g h t l y h i g h e r means under the f l u o r e s c e n t - r e d f i l t e r regime compared t o the metal h a l i d e - r e d f i l t e r regime could then be a t t r i b u t e d t o f l u o r e s c e n t l i g h t ' s higher red wavelength output. Node n u m b e r / b l o c k o f e i g h t s u b - s a m p l e s The node number means d i f f e r i n two ways from the shoot number means (Table 4.14). F i r s t , a t the 0.25 mg/1 BA l e v e l , metal h a l i d e l i g h t has a s l i g h t l y higher node number mean than f l u o r e s c e n t l i g h t . Secondly, at the 0.5 mg/1 BA l e v e l , both metal h a l i d e and f l u o r e s c e n t l i g h t means are v i r t u a l l y the same. These r e s u l t s suggest t h a t the modified wavelength r a t i o s produce a g r e a t e r e f f e c t on shoot i n i t i a t i o n than on nodal d i f f e r e n t i a t i o n . The h i g h e s t node number mean at both BA l e v e l s , i s , as f o r shoot number means, under tungsten f i l a m e n t l i g h t . Again, as f o r shoots, the degree of d i f f e r e n c e between the tungsten f i l a m e n t node number means and those of the other two l i g h t sources i s i n d i c a t e d by the ANOVA where the l i g h t source f a c t o r caused s i g n i f i c a n t d i f f e r e n c e s ( c o n t r a s t s of T/MeF and T/F i n 73 Table 4.15) . The l a r g e s t d i f f e r e n c e i n node number means was due t o BA c o n c e n t r a t i o n (Table 4.15), w i t h fewer nodes a t the 0.25 mg/1 BA l e v e l than a t the 0.5 mg/1 BA l e v e l . Shoot length The shoot l e n g t h comparisons at both BA l e v e l s of tungsten f i l a m e n t l i g h t w i t h f l u o r e s c e n t l i g h t , and of metal h a l i d e l i g h t w i t h f l u o r e s c e n t l i g h t , are a l l s t a t i s t i c a l l y s i g n i f i c a n t (Table 4.16). However, when tungsten f i l a m e n t c u l t u r e s are compared to metal h a l i d e c u l t u r e s a t both BA l e v e l s , there are no s i g n i f i c a n t d i f f e r e n c e s . This i n d i c a t e s t h a t the u s e f u l shoot p r o p o r t i o n s f o r both tungsten f i l a m e n t l i g h t and metal h a l i d e l i g h t are s i m i l a r (at the 0.25 mg/1 BA l e v e l 45% f o r tungsten f i l a m e n t l i g h t and 4 9% f o r metal h a l i d e l i g h t , and at the 0.5 mg/1 BA l e v e l 37% f o r tungsten f i l a m e n t l i g h t and 41% f o r metal h a l i d e l i g h t ) . Thus, under f l u o r e s c e n t l i g h t the u s e f u l shoot percentages are much lower than the percentages under tungsten f i l a m e n t and metal h a l i d e l i g h t ( f o r f l u o r e s c e n t l i g h t at the 0.25 mg/1 BA l e v e l i t i s 28% and at the 0.5 mg/1 BA l e v e l i t i s 26%) . The high u s e f u l shoot p r o p o r t i o n s under metal h a l i d e and tungsten f i l a m e n t l i g h t , e s p e c i a l l y under metal h a l i d e l i g h t , emphasize the importance of f a r - r e d l i g h t i n promoting shoot e l o n g a t i o n . Although the red f i l t e r reduces the small amount of T a b l e 4.16 Shoot l e n g t h s as a f f e c t e d by t h r e e l i g h t s o u r c e -r e d f i l t e r c o m b i n a t i o n s . P a i r s 1 BA 2 Sample 3 U s e f u l 4 A c t u a l u s e f u l 5 6 X 2 t e s t e d con. s i z e shoot % shoot number Tun. 7 0.25 152 45 68 NS Me.H. 109 49 53 Me.H. 0. 25 109 49 53 ***** F l . 116 28 28 Tun 0.25 152 45 68 * * * 116 28 33 Tun. 0.5 257 37 94 * * * F l . 221 26 57 Tun. 0.5 257 37 94 NS Me.H. 181 41 74 Me.H. 0.5 181 41 74 ***** F l . 221 26 57 Tun. 0.25 152 45 68 NS Tun. 0 . 5 257 37 94 Tun. 0.25 152 45 68 NS Me.H. 0.5 181 41 74 > Tun. 0.25 152 45 68 ***** F l . 0.5 221 26 57 Legend: 1 - each p a i r r e p r e s e n t s the s e t of shoot l e n g t h p r o p o r t i o n s a n a l y z e d i n a C h i - s q u a r e t e s t 2 - BA l e v e l i n mg/1 3 - t o t a l number of shoots produced i n a l l e x p e r i m e n t a l b l o c k s 4 - a u s e f u l shoot i s d e f i n e d as a shoot 1 cm or l o n g e r i n l e n g t h ; thus the u s e f u l shoot % i s the % of the sample s i z e ranked u s e f u l 5 - the c o n v e r s i o n of the u s e f u l shoot % i n t o number of shoots 6 - X i s the symbol f o r the C h i - s q u a r e t e s t ; the a s t e r i s k s d e f i n e the degree of s i g n i f i c a n c e : *** = s i g n i f i c a n t a t p<0.025, **** = a t p<0.01, ***** = a t p<0.005, NS = n o n - s i g n i f i c a n t 7 - l i g h t s o u r c e s ; Tun.= tun g s t e n f i l a m e n t , Me.H.= m e t a l h a l i d e , F l .= f l u o r e s c e n t f a r - r e d l i g h t under metal h a l i d e l i g h t , enough was s t i l l t r a n s m i t t e d t o promote shoot e l o n g a t i o n . Metal h a l i d e l i g h t , u n f i l t e r e d , emits 9.8% of i t s energy output from the v i s i b l e spectrum and f a r - r e d r e g i o n as f a r - r e d wavelengths. In comparison u n f i l t e r e d f l u o r e s c e n t l i g h t emits 1.2% as f a r - r e d which would be reduced u s i n g the red f i l t e r and thus t h i s may account f o r the lower u s e f u l shoot percentages under t h i s regime. In economic terms, although under metal h a l i d e l i g h t the p r o p o r t i o n s of u s e f u l shoots were s l i g h t l y higher than the p r o p o r t i o n s under tungsten f i l a m e n t l i g h t f o r both c o n c e n t r a t i o n s of BA, the g r e a t e r shoot p r o d u c t i o n under tungsten f i l a m e n t l i g h t compensated f o r the lower p r o p o r t i o n s . Thus, w h i l e a t the 0.2 5 mg/1 BA l e v e l 53 u s e f u l shoots were produced under metal h a l i d e l i g h t , tungsten f i l a m e n t l i g h t produced 68 shoots. At the 0.5 mg/1 BA l e v e l , under metal h a l i d e l i g h t , 74 shoots were produced, w h i l e tungsten f i l a m e n t l i g h t produced 94. An i n t e r e s t i n g o bservation i s t h a t f o r a l l t h r e e l i g h t sources higher u s e f u l shoot percentages were found at the lower BA c o n c e n t r a t i o n ; t h i s supports previous work where decreasing the c y t o k i n i n l e v e l was found t o enhance shoot e l o n g a t i o n i n Pyrus (Stimart and Harbage, 1989). Fresh weight/explant and Dry weight/explant The f r e s h weight means (Table 4.17) r e f l e c t the node number t r e n d s . But u n l i k e the shoot and node number means, where BA c o n c e n t r a t i o n caused the g r e a t e s t e f f e c t , f o r f r e s h weight i t was the l i g h t source f a c t o r (ANOVA c o n t r a s t s of T/MeF and T/F i n Table 4.15). The range of dry weight means was narrow (Table 4.17); d e s p i t e t h i s , i t was g r e a t e r than the range observed i n the l i g h t source-blue f i l t e r experiment. This d i f f e r e n c e i s i n d i c a t e d by the ANOVA (Table 4.15), where the d i f f e r e n c e s due to l i g h t source were as s i g n i f i c a n t as f o r the f r e s h weight means. 77 Table 4.17 Fresh and dry weight means under three l i g h t sources (tungsten f i l a m e n t , metal h a l i d e , and f l u o r e s c e n t ) combined with a red acetate f i l t e r . Light BA 1 F r e s h 2 Dry 3 Source Level Weight Weight Tungsten 0 .25 56 ± 7 9 ± 1 filament 0. 5 89 ± 11 11 ± 1 Metal 0 . 25 43 ± 6 7 + 1 hali d e 0.5 58 ± 6 8 ± 1 Fluorescent 0.25 34 ± 3 5 0.5 57 + 4 7 ± 1 Legend: 1 2 3 - BA l e v e l i n mg/1 - fresh weight/explant mean ( i n mg) ± standard error - dry weight/explant mean ( i n mg) + standard error 78 4.1.5 E x p e r i m e n t V: D e t e r m i n i n g t h e e f f e c t o f a s h o r t - t e r m f a r - r e d l i g h t e x p o s ure a p p l i e d a t t h e end o f a w h i t e l i g h t p h o t o p e r i o d In the f i r s t l i g h t q u a l i t y experiment a red/far-red l i g h t mixture was shown to a f f e c t an explant's u t i l i z a t i o n of the applied cytokinin BA. The red/far-red l i g h t - c y t o k i n i n i n t e r a c t i o n was such that at the BA l e v e l of 0.25 mg/1 (one-half the BA l e v e l previously established as optimal for maintaining cultures) red/far-red l i g h t stimulated s i g n i f i c a n t l y more shoot production than i n white control or blue l i g h t treatments. The photoperiod used was 16 hours. One possible explanation i s that the photoperiod of continuous red/far-red l i g h t reduced endogenous free IAA l e v e l s and thus modified shoot cytokinin to auxin r a t i o s . The present end-of-day experiment was done to determine the importance of the red and far-red l i g h t photoperiod i n the f i r s t experiment. A t y p i c a l end-of-day treatment involves exposing plants to a far-red l i g h t i n t e r v a l (far-red extension) before the beginning of the dark period to cause shoot elongation (Downs et a l . , 1957). For t h i s experiment the end-of-day growth chamber had a metal halide l i g h t on for 16 hours followed by 2 hours of l i g h t supplied by a tungsten filament source. The control chamber had a metal halide l i g h t on for an 18 hour photoperiod. For the end-of-day exposure u n f i l t e r e d tungsten filament l i g h t was used. This choice was based on work where end-of-day stem e x t e n s i o n of s o i l grown p l a n t s was st u d i e d under a mixture of r ed and f a r - r e d l i g h t (Buck and Vince-Prue, 1985). The mixture of red and f a r - r e d l i g h t was shown to be about h a l f as e f f e c t i v e as f a r - r e d l i g h t alone i n causing increases i n the i n i t i a l e l o n g a t i o n of an expanding internode. However, as su c c e s s i v e internodes developed, the response t o f a r - r e d decreased. With l a t e r expanding internodes tungsten f i l a m e n t end-of-day exposures increased extension compared t o f a r - r e d ; the response appeared t o i n c r e a s e as d u r a t i o n of tungsten f i l a m e n t exposure increased. Since there was a gr e a t e r f a r - r e d l i g h t p r o p o r t i o n under the u n f i l t e r e d tungsten f i l a m e n t l i g h t , a shoot e l o n g a t i o n response would perhaps predominate over any shoot i n i t i a t i o n response f o r t h i s Spiraea i n v i t r o experiment. A l a c k of e i t h e r of these two responses, or a l a c k of both, would lend credence t o the proposal t h a t a photoperiod of continuous red and f a r - r e d l i g h t i s r e q u i r e d t o modify jln v i t r o growth. 80 Growth Variates Shoot number/explant For t h i s experiment a g r e a t e r range of BA concentrations was used t o determine i f there were any d e v i a t i o n s from the BA concentration-shoot number trends observed i n the previous experiments. As can be seen i n Table 4.18, the shoot number means under metal h a l i d e l i g h t w i t h a f a r - r e d extension, e x h i b i t e d a l i n e a r r e l a t i o n w i t h BA c o n c e n t r a t i o n ; as the BA c o n c e n t r a t i o n increased, the shoot number increased. This general t r e n d was observed i n the previous experiments f o r a l l l i g h t q u a l i t y regimes except f o r red/FR l i g h t . The absence of a maximal shoot number mean a t the 0.25 mg/1 BA l e v e l , as noted under red/FR l i g h t i n the f i r s t experiment, suggests t h a t prolonged exposures of red/FR l i g h t are r e q u i r e d t o enhance shoot i n i t i a t i o n a t lower c y t o k i n i n c o n c e n t r a t i o n s . Under metal h a l i d e without a f a r - r e d extension, the l i n e a r t r end i s d i s r u p t e d ; the maximal mean was at the 0.4 mg/i BA l e v e l i n s t e a d of at the 0.5 mg/1 BA l e v e l . This r e s u l t suggests t h a t under metal h a l i d e l i g h t a maximal B A - l i g h t i n t e r a c t i o n occurs a t the 0.4 mg/1 BA l e v e l . Such a peak would not have been observed i n the f i r s t experiment due t o t h a t experiment's l a r g e r incremental change i n BA c o n c e n t r a t i o n , i . e . , from 0.25 mg/1 t o 0.5 mg/1. The l a c k of the 0.4 mg/1 peak, when a f a r - r e d extension was 8 1 Table 4.18 Shoot ,.and node number means as af f e c t e d by a short-term f a r - r e d l i g h t exposure applied at the end of a white l i g h t photoperiod. Light BA 1 Shoot 2 Node 3 Quality Level Number Number Metal halide 0. 1 6 . 58 + 1. 28 87 + 14 + Red/FR 0. 25 10 .75 + 2. 08 139 + 24 0. 4 16 . 25 + 2. 01 205 + 31 0. 5 19 .50 + 2 . 94 247 + 32 Metal halide 0 . 1 7 . 08 + 0. 85 89 + 11 0. 25 11 . 58 + 1. 65 154 + 14 0. 4 20 .33 + 3. 27 251 + 35 0 . 5 17 . 33 + 3. 11 199 + 22 Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean ± standard error 3 - node number/block (mean of four blocks) ± standard error 82 a p p l i e d , provides evidence t h a t a weak red/FR e f f e c t occurred. The two hour exposure t o f a r - r e d l i g h t may have s h i f t e d the phytochrome e q u i l i b r i a induced by metal h a l i d e l i g h t t o a s t a t e l e s s conducive t o a B A - l i g h t q u a l i t y i n t e r a c t i o n at the 0.4 mg/1 BA l e v e l . T h i s weaker i n t e r a c t i o n may thus have l e d t o a l o s s of the 0.4 mg/1 BA peak. As i n d i c a t e d by the ANOVA (Table 4.19) a h i g h l y s i g n i f i c a n t l i n e a r r e l a t i o n was e x h i b i t e d between BA c o n c e n t r a t i o n and shoot number f o r metal h a l i d e l i g h t w i t h a f a r - r e d extension. The ANOVA a l s o i n d i c a t e s t h a t there are no shoot number mean d i f f e r e n c e s due t o e i t h e r a l i g h t q u a l i t y e f f e c t or a B A - l i g h t q u a l i t y i n t e r a c t i o n ; t h i s provides f u r t h e r evidence t h a t only prolonged red/FR l i g h t exposures are capable of promoting shoot i n i t i a t i o n a t lower BA c o n c e n t r a t i o n s . N o d e n u m b e r / b l o c k o f t h r e e s u b - s a m p l e s The same p a t t e r n f o l l o w e d by the shoot number means was e x h i b i t e d by the node number means (Table 4.18). Again, the ANOVA emphasizes a strong l i n e a r r e l a t i o n ; t h i s time between the BA co n c e n t r a t i o n s and the node number means (Table 4.19). As f o r shoot number means there i s no other f a c t o r a f f e c t i n g shoot i n i t i a t i o n (no l i g h t q u a l i t y e f f e c t s or B A - l i g h t q u a l i t y i n t e r a c t i o n s ) . Table 4.19 Summarized analyses of variance. E f f e c t s of a short-term far-red light-i exposure applied at the end of a white l i g h t photoperiod and BA concentration on shoot and node numbers, and fresh and dry weights I 3v 3 if Shoot Node Fresh Dry Source of v a r i a t i o n df number number wt. wt. L i g h t q u a l i t y 1 NS NS •NS NS BA c o n c e n t r a t i o n (3) ***** ii "k it it k ie it it it it ***** l i n e a r 5 1 ***** •k -k ie ~k ic it it it -k it * * * * * q u a d r a t i c 1 NS NS NS NS c u b i c . 1 NS NS NS ' NS BA and l i g h t q u a l i t y i n t e r a c t i o n 3 NS NS NS NS Legend: 1 - s h o o t number/explant 2 - node number/block, o f 3 sub-samples ( t o t a l nodes o f s h o o t s o f 3 e x p l a n t s ) 3 - f r e s h w e i g h t / e x p l a n t 4 - d r y w e i g h t / e x p l a n t 5 - p o l y n o m i a l r e s p o n s e models: l i n e a r = amount o f v a r i a t i o n e x p l a i n e d by a s t r a i g h t l i n e r e l a t i o n s h i p , q u a d r a t i c = amount o f v a r i a t i o n e x p l a i n e d by a q u a d r a t i c - t e r m e d r e l a t i o n s h i p , c u b i c = amount o f v a r i a t i o n e x p l a i n e d by a c u b i c - t e r m e d r e l a t i o n s h i p d f = de g r e e o f freedom * = s i g n i f i c a n t a t p < 0.1, ** = a t p < 0.05, *** = a t p < 0.025, **** = a t p < O.'OI, ***** = a t p < 0.005, NS = n o n - s i g n i f i c a n t 84 Shoot l e n g t h U s e f u l shoot p r o p o r t i o n s r e f l e c t e d the p a t t e r n of shoot and node number means w i t h s i m i l a r l i n e a r trends (Table 4.20). Again the metal h a l i d e l i g h t without a f a r - r e d extension e x h i b i t e d the same peak at the 0.4 mg/1 BA l e v e l . Although the comparisons of the four BA l e v e l s under metal h a l i d e l i g h t w i t h a f a r - r e d extension d i d show a s i g n i f i c a n t d i f f e r e n c e , i t was due t o the p r o p o r t i o n s a t the 0.1 mg/1 BA l e v e l and the 0.5 mg/1 BA l e v e l (49% and 59% r e s p e c t i v e l y ) . None of the p a i r comparisons of metal h a l i d e p l u s a f a r - r e d extension w i t h the metal h a l i d e c o n t r o l showed any d i f f e r e n c e . These r e s u l t s suggest t h a t the t y p i c a l end-of-day f a r - r e d response of incr e a s e d shoot l e n g t h does not occur i n the Spiraea i n v i t r o system. Thus, i t appears t h a t only prolonged red/FR l i g h t exposures are capable of enhancing growth v a r i a t e s , i . e . , shoot e l o n g a t i o n i n a d d i t i o n t o shoot i n i t i a t i o n , a t lower BA l e v e l s . Fresh weight/explant and Dry weight/explant Both the f r e s h and dry weight means fol l o w e d the same p a t t e r n as shoot and node number means (Table 4.21). A l s o , a s t r o n g l i n e a r r e l a t i o n between BA c o n c e n t r a t i o n and both f r e s h and dry weights was shown (Table 4.19). Table 4.20 Shoot lengths as affected by a short-term f a r - r e d l i g h t exposure appl ied at the end of a white l i g h t photoperiod P a i r s 1 BA2 Sample3 Useful 4 Actual use fu l 5 6 X 2 tested con. s ize shoot % shoot number MH+FR7 0.1 79 49 39 * MH+FR 0.25 129 50 64 MH+FR 0.4 0195 56 109 MH+FR 0.5 234 59 137 MH 0.1 85 49 42 NS MH 0 . 25 139 53 73 MH 0.4 244 57 140 MH 0.5 208 53 110 MH+FR 0.1 79 49 39 NS MH 85 49 42 MH+FR 0.25 129 50 64 NS MH 139 53 73 MH+FR 0.4 195 56 109 NS MH 244 57 140 MH+FR 0.5 234 59 137 NS MH 208 53 110 MH+FR 0.1 79 49 39 NS MH 0 . 5 208 53 110 MH+FR 0 .25 129 50 64 NS MH 0 . 5 208 53 110 MH+FR 0.4 . 195 56 109 NS MH 0.5 208 53 110 Legend: 1 - each pair represents the set of shoot length proportions analyzed in a Chi-square test 2 - BA leve l in mg/1 3 - to ta l number of shoots produced in a l l experimental blocks 4 - a useful shoot is defined as a shoot 1 cm or longer in length; thus the useful shoot % is the % of the sample s ize ranked useful 5 - the conversion of the useful shoot % into number of shoots 6 - X i s the symbol for the Chi-square test; the aster isks define the degree of s ign i f i cance: * = s ign i f i cant at p<0.1, NS = non-s ignif icant 7 - l i g h t treatments: MH = metal ha l ide , MH+FR = metal halide + f a r - r e d l i g h t exposure Table 4.21 Fresh ,and dry weight means as a f f e c t e d by a short-term f a r - r e d l i g h t exposure applied at the end of a white l i g h t photoperiod. Light BA Fresh^ Dry 3 Quality Level Weight Weight Metal h a l i d e 0.1 '+ Red/FR 0.25 0.4 0.5 Metal h a l i d e 0.1 0.25 0.4 0.5 71 + 12 14 ± 2 138 + 26 23 + 3 213 ± 29 32 + 4 268 + 38 36 + 4 86 + 10 17 + 2 155 + 20 25 + 2 285 + 35 41 + 4 241 + 40 32 + 4 Legend: 1 2 3 BA l e v e l i n mg/1 fresh weight/explant mean (in mg) + standard' error dry weight/explant mean (in mg) ± standard error 8 7 4.2 Factors contributing to the red/FR l i g h t growth response 4.2.1 Experiment VI: The e f f e c t s of length of exposure to a low photon fluence rate and tr a n s f e r time to a higher photon fluence rate In the f i r s t set of experiments where the l i g h t q u a l i t y response of Spiraea explants was characterized, a two week incubation period was used so that i n v i t r o p l a n t l e t s of s i m i l a r s i z e and vigour could be selected for the further three week l i g h t q u a l i t y treatments. This s e l e c t i o n procedure was an attempt to minimize v a r i a b i l i t y due to explants 1 d i f f e r i n g growth rates and vigour, so that any subsequent differences under the l i g h t q u a l i t y treatments could be assumed to be l i g h t q u a l i t y e f f e c t s . The incubation period was under white l i g h t of a higher photon fluence rate than that of the experimental l i g h t q u a l i t y regimes. Thus, i n t h i s second section of the the s i s two factors i n f l u e n c i n g shoot i n i t i a t i o n were examined; these included an i n i t i a l incubation period under red/FR or white l i g h t of a low photon fluence rate and v a r i a t i o n of the time of tr a n s f e r to a higher fluence rate. The experiment to examine the two factors, u t i l i z e d three d i f f e r e n t growth chamber conditions. Two chambers were used for an i n i t i a l low photon fluence rate, one u t i l i z i n g white l i g h t and the other red/FR l i g h t . The t h i r d chamber, a white l i g h t 88 chamber, had a higher photon fluence r a t e s i m i l a r t o t h a t used f o r m a i n t a i n i n g stock c u l t u r e s and f o r the i n i t i a l i n c u b a t i o n p e r i o d of the f i r s t s e t of experiments. To determine the e f f e c t t h a t t r a n s f e r time and d i f f e r e n t l i g h t treatments had, c u l t u r e s were t r a n s f e r r e d a f t e r two and four weeks from the low photon f l u e n c e r a t e chambers to the t h i r d chamber. One s e t of c u l t u r e s was kept under each low fl u e n c e regime f o r the e n t i r e f i v e week experiment t o determine i f the t r a n s f e r s t o a higher f l u e n c e r a t e were necessary t o optimize growth. Growth V a r i a t e s Shoot number/explant Each treatment u t i l i z i n g white l i g h t only, r e s u l t e d i n lower shoot numbers than treatments i n which red/FR l i g h t was used (Table 4.22). C u l t u r e s under white l i g h t , where a t r a n s f e r t o a higher photon fl u e n c e r a t e was used, produced shoot numbers which incr e a s e d w i t h the l e v e l of BA. The f i v e week low f l u e n c e white l i g h t gave r e l a t i v e l y low shoot i n i t i a t i o n at each BA l e v e l ; the l e v e l s were s i m i l a r , i n d i c a t i n g t h a t i n c r e a s i n g BA co u l d not i n c r e a s e p r o l i f e r a t i o n under these l i g h t c o n d i t i o n s . The g r e a t e s t d i f f e r e n c e s i n shoot p r o l i f e r a t i o n seen between d i f f e r e n t l i g h t treatments were those i n c l u d i n g 0.25 and 0.4 mg/1 BA. At these lower BA l e v e l s the highest shoot numbers were obtained a f t e r four weeks of low fluence red/FR f o l l o w e d by one Table 4.22 89 Shoot and node number means as affected by exposure to a low photon fluence rate and the time of transfer to a higher fluence rate L i g h t BA2 Shoot 3 Node 4 Q u a l i t y Wks.1 Level Number Number White 2 0.25 16 ± 1.48 256 ± 42 0.4 19 ± 3.24 297 ± 92 0.5 21 ± 2.63 300 ± 18 Red/FR 2 0.25 22 ± 2.13 309 ± 32 0.4 20 ± 3.07 303 + 55 0.5 32 ± 4.53 464 + 103 White 4 0.25 15 ± 2.87 178 ± 60 0.4 16 + 2.24 203 ± 37 0.5 19 + 3.20 248 + 33 Red/FR 4 0.25 26 + 3.91 357 ± 83 0.4 33 ± 3.88 395 ± 58 0.5 33 ± 3.87 419 ± 86 White 5 0.25 16 ± 1.76 198 ± 42 0.4 15 ± 3.04 197 ± 61 0.5 15 ± 1.64 174 ± 39 Red/FR 5 0.25 21 ± 2.13 265 + 29 0.4 19 + 2.67 234 + 75 0.5 30 ± 4.24 388 + 119 Legend: 1 - Weeks of exposure to low photon fluence r a t e . 2 - BA l e v e l i n mg/1. 3 - Shoot number mean ± standard e r r o r . 4 - Node number/block (mean of two blocks ± standard e r r o r ) . week of higher f l u e n c e white l i g h t . The lowest l e v e l s of shoot i n d u c t i o n under red/FR occurred at the 0.25 and 0.4 BA l e v e l s f o r the f i v e week low f l u e n c e or f o r the two week low red/FR p r o t o c o l . Red/FR c u l t u r e s w i t h the highest BA l e v e l of 0.5 mg/1 produced a s i m i l a r high l e v e l of shoots whether or not a t r a n s f e r t o a higher f l u e n c e white l i g h t was used. This suggests t h a t f o r red/FR l i g h t both the length of exposure t o the s p e c i f i c l i g h t q u a l i t y and the t r a n s f e r t o a higher photon f l u e n c e r a t e are important. S p e c i f i c a l l y , the data suggest t h a t a c r i t i c a l l e n g t h of exposure t o red/FR l i g h t of between two and f o u r weeks i s r e q u i r e d t o maximize shoot i n i t i a t i o n . The d e c l i n e i n shoot i n i t i a t i o n under a f i v e week p e r i o d of red/FR a t the lower BA l e v e l s i n d i c a t e s t h a t t r a n s f e r t o the higher f l u e n c e r a t e i s needed t o optimize growth f o l l o w i n g the e f f e c t of red/FR. The 0.5 mg/1 BA gave the highest l e v e l s of growth f o r a l l the l i g h t treatments t h a t i n c l u d e d red/FR i n c u b a t i o n . These l e v e l s were s i m i l a r t o the optimal l e v e l obtained w i t h 0.4 mg/1 BA under the f o u r week red/FR i n i t i a l treatment. This i n d i c a t e s t h a t t h i s c o n c e n t r a t i o n of BA (0.5 mg/1) can achieve the r e q u i r e d t h r e s h o l d f o r optimal growth even i n the absence of a h i g h f l u e n c e l i g h t exposure. Thus, the higher BA l e v e l , by o p t i m i z i n g the i n t e r a c t i o n or synergy w i t h red/FR, may be able t o overcome any modulating i n f l u e n c e r e s u l t i n g from the low f l u e n c e red/FR exposure time. Lower BA l e v e l s produced o p t i m a l growth w i t h one s p e c i f i c l i g h t regimen, i . e . four weeks of red/FR f o l l o w e d by one week of higher fl u e n c e white. The r e s u l t s emphasize an i n t e r a c t i o n or synergy of BA and red/FR l i g h t which i s dependent on the exposure time of c u l t u r e s t o s p e c i f i c l i g h t q u a l i t y . At lower BA l e v e l s , perhaps the two weeks of low fl u e n c e red/FR was an i n s u f f i c e n t s p e c i f i c wavelength s t i m u l u s whereas the f i v e week red/FR lacked a subsequent p e r i o d of higher photon f l u e n c e . The ANOVA (Table 4.23) i n d i c a t e s a s i g n i f i c a n t i n t e r a c t i o n between l i g h t q u a l i t y and weeks of exposure ( t r a n s f e r time) t o low photon f l u e n c e l i g h t . This i n t e r a c t i o n , and the s i g n i f i c a n c e of weeks of exposure ( t r a n s f e r time) as an i n d i v i d u a l f a c t o r , emphasizes the importance of t r a n s f e r time t o high photon f l u e n c e l i g h t . The e f f e c t on shoot number due t o BA co n c e n t r a t i o n can be mainly a t t r i b u t e d t o the u s u a l wide d i f f e r e n c e i n shoot number means between the 0.25 and 0.5 mg/1 BA l e v e l s . The BA e f f e c t was modulated by i n t e r a c t i n g w i t h l i g h t q u a l i t y and weeks of exposure t o the s p e c i f i c wavelength and fl u e n c e r a t e . Node number/block of 4 sub-samples The trends noted f o r shoot number means were more s t r o n g l y expressed w i t h the node number means (Table 4.22). The importance of the t r a n s f e r from the lower t o higher photon f l u e n c e r a t e f o r the white treatments was emphasized. There were white l i g h t node number decreases at a l l BA l e v e l s from the two 9 2 ; Table 4.23 Summarized analyses of v a r i a n c e . E f f e c t s of exposure to a low photon f l u e n c e r a t e , t r a n s f e r time t o a higher f l u e n c e , and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry weights Shoot 1 Node 3 Fresh Dry Source of V a r i a t i o n df number number wt. wt. L i g h t q u a l i t y 1 NS NS NS NS T r a n s f e r time (2) * ***** ***** ***** l i n e a r $ 1 NS ***** ***** ***** l a c k of f i t 1 ** **** * ***** I n t e r a c t i o n between l i g h t q u a l i t y and t r a n s f e r time (2) * **** *** ***** l i n e a r 1 NS ** ** * * * l a c k of f i t 1 * ***** *** ***** BA c o n c e n t r a t i o n (2) **** ** ***** ***** l i n e a r 1 **** *** ***** ***** l a c k of f i t 1 NS NS * ** I n t e r a c t i o n between l i g h t q u a l i t y and BA c o n c e n t r a t i o n (2) NS NS * * l i n e a r 1 NS NS * NS l a c k of f i t 1 NS NS NS * * I n t e r a c t i o n between l i g h t q u a l i t y , t r a n s f e r time, and BA c o n c e n t r a t i o n 1 * NS NS * shoot number/explant, 2 - node number/block, 3 -f r e s h weight/explant, 4 - dry weight/explant, 5 -polynomial response models: l i n e a r = amount of v a r i a t i o n e x p l a i n e d by a s t r a i g h t l i n e r e l a t i o n s h i p , l a c k of f i t = amount of v a r i a t i o n explained by a higher order-termed r e l a t i o n s h i p degree of freedom t e s t of s i g n i f i c a n c e s i g n i f i c a n t at p < 0.1 s i g n i f i c a n t a t p < 0.05 s i g n i f i c a n t at p < 0.025 s i g n i f i c a n t at p < 0.01 s i g n i f i c a n t at p < 0.005 n o n - s i g n i f i c a n t Legend: 1 df F * ** *** **** ***** NS week . t r a n s f e r regime t o the four week t r a n s f e r regime, and a f u r t h e r decrease f o r two BA l e v e l s from the four t o f i v e week regime. For red/FR c u l t u r e s at the 0.25 and 0.4 mg/1 BA l e v e l s the o p t i m a l means were again reached w i t h the four week exposure. The ANOVA (Table 4.23) confirms the stronger t r e n d s . The l i n e a r node number decreases f o r white l i g h t c u l t u r e s as len g t h of exposure t o the lower photon flu e n c e r a t e i n c r e a s e d , may-e x p l a i n the h i g h l y s i g n i f i c a n t l i n e a r t r e n d f o r weeks of exposure ( t r a n s f e r t i m e ) . The much more s i g n i f i c a n t l i g h t quality-weeks of exposure ( t r a n s f e r time) i n t e r a c t i o n f o r node number means as compared t o shoot number means, i s f u r t h e r evidence f o r the greater e f f e c t of weeks of exposure ( t r a n s f e r time) on node numbers. This g r e a t e r e f f e c t due t o weeks of exposure may e x p l a i n the l e s s s i g n i f i c a n t e f f e c t of BA c o n c e n t r a t i o n on node numbers than t h a t observed f o r shoot numbers. Shoot l e n g t h A comparison of the nine treatments under white l i g h t w i t h those under red/FR l i g h t , shows t h a t f o u r treatments under white l i g h t r e s u l t e d i n u s e f u l shoot p r o p o r t i o n s 40% or over, w h i l e of the red/FR l i g h t treatments, e i g h t had u s e f u l shoot p r o p o r t i o n s over 40% (Table 4.24). For treatments where the t r a n s f e r was done a t f o u r weeks, and where p l a n t s were kept under a low T a b l e 4.24 S h o o t l e n g t h s a s a f f e c t e d b y a l o w p h o t o n f l u e n c e r a t e a n d t h e t r a n s f e r t o a h i g h e r f l u e n c a r a t e T r e a t s .' BA l e v e l ^  o , 3 Sample U s e f u l " * A c t u a l ^ X t e s t e d (mg/1) s i z e s h o o t % u s e f u l s h o o t number W h i t e 2 wks. 0.25 58 40 23 * 0 . 4 89 39 35 0.5 75 28 21 Red/FR 2 wks. 0 . 25 76 28 21 * * * * * 0 . 4 74 47 35 0.5 128 46 59 W h i t e 4 wks. 0.25 71 37 2 6 NS 0.4 57 47 27 0.5 78 47 37 R e d / F R 4 wks. 0.25 115 44 51 NS 0.4 132 45 59 0.5 131 50 65 W h i t e 5 wks. 0 .25 62 34 21 * 0.4 75 48 36 0 . 5 58 38 22 Red/FR 5 wks. 0 .25 81 41 33 * 0 . 4 82 45 37 o.s 129 53 69 W h i t e 2 wks. 0.5 75 28 21 NS Red/FR 2 wks. 0.25 76 28 21 W h i t e 2 wks. O.S 75 28 21 * * * Red/FR 2 wks. 0.4 74 47 35 W h i t e 4 wks. 0.5 78 47 37 NS Red/FR 4 wks. 0.25 115 44 51 W h i t e 4 wks. 0.5 78 47 37 NS R e d / F R 4 wks. 0 . 4 132 45 59 W h i t e 5 wks. 0 . 5 58 38 22 . NS Red/FR 5 wks. 0 .25 81 41 33 W h i t e 5 wks. 0 . 5 58 38 . 22 NS Red/FR 5 wks. 0 . 4. 82 4 5 '; 37 L e g e n d : 1 - e a c h p a i r r e p r e s e n t s t h e s e t o f s h o o t l e n g t h p r o p o r t i o n s a n a l y z e d i n a C h i - s q u a r e t e s t 2 - BA l e v e l i n mg/1 3 - t o t a l number o f s h o o t s p r o d u c e d i n a l l ' e x p e r i m e n t a l b l o c k s i 4 - a u s e f u l s h o o t i s d e f i n e d a s a s h o o t 1 | cm o r l o n g e r i n l e n g t h ; t h u s t h e u s e f u l ' s h o o t V i s t h e % o f t h e s a m p l e s i z e - r a n k e d u s e f u l 5 - t h e c o n v e r s i o n o f t h e u s e f u l s h o o t % i n t o number o f s h o o t s 5 - X i s t h e s y m b o l f o r t h e C h i - s q u a r e t e s t ; t h e a s t e r i s k s d e f i n e t h e d e g r e e o f s i g n i f i c a n c e : *** = s i g n i f i c a n t a t p<0.025, **** = a t p<0.01, * * * * * = a t p<0.005, NS = n o n - s i g n i f i c a n t f l u e n c e r a t e f o r f i v e weeks, the red/FR c u l t u r e s w i t h 0.25 mg/1 BA had a s i m i l a r or greater p r o p o r t i o n of longer shoots than the corresponding white l i g h t c u l t u r e s w i t h 0.5 mg/1 BA. When the u s e f u l shoot percentages are compared f o r the 0.25 mg/1 BA red/FR and the 0.5 mg/1 BA white l i g h t c u l t u r e s at the fo u r week exposure, there i s l i t t l e d i f f e r e n c e (47% f o r white and 44% f o r red/FR) . Thus i t appears t h a t the e f f e c t of t h i s f o u r week red/FR exposure was more c r i t i c a l i n determining shoot i n i t i a t i o n than i n determining shoot e l o n g a t i o n . However, because of the optimal shoot i n i t i a t i o n response at the four week exposure f o r red/FR c u l t u r e s the a c t u a l number of longer shoots i s g r e a t e r (51 f o r red/FR and 37 f o r w h i t e ) . Fresh weight/explant and Dry weight/explant The p a t t e r n of f r e s h weight means (Table 4.25) g e n e r a l l y f o l l o w e d t h a t of the node number means. The s i g n i f i c a n c e of the e f f e c t s caused by the f a c t o r s of weeks of exposure ( t r a n s f e r time) and BA c o n c e n t r a t i o n , and the l i g h t quality-weeks of exposure ( t r a n s f e r time) i n t e r a c t i o n were g r e a t e r f o r f r e s h weight means (Table 4.2 3)- than f o r shoot number means. Another d i f f e r e n c e was the s i g n i f i c a n t l i g h t q u a l i t y - B A i n t e r a c t i o n f o r f r e s h weight means, an i n t e r a c t i o n not seen w i t h shoot number means. The amount of change explained by the l i g h t quality-weeks of exposure ( t r a n s f e r time) i n t e r a c t i o n and the a d d i t i o n a l l i g h t q u a l i t y - B A i n t e r a c t i o n , i n d i c a t e s the importance of l i g h t Table 4.25 96 Fresh and dry weight means as a f f e c t e d by exposure to a low photon fluence rate and the time of t r a n s f e r to a higher fluence rate L i g h t BA2 Fresh 3 Dry 4 Q u a l i t y Wks.1 Level Weight Weight White 2 0.25 0.4 0.5 Red/FR 2 0.25 0.4 0.5 White 4 0.25 0.4 0.5 Red/FR 4 0.25 0.4 0.5 White 5 0.25 0.4 0.5 Red/FR 5- 0.25 0.4 0.5 179 ± 20 29 ± 2 219 ± 30 34 ± 3 267 + 38 36 ± 4 216 + 25 35 ± 3 218 + 37 31 ± 5 392 + 53 49 ± 5 104 + 21 16 ± 2 131 ± 18 18 ± 2 166 + 23 24 ± 3 240 ± 48 33 ± 6 297 ± 45 36 ± 4 317 + 50 39 ± 4 105 + 13 14 ± 1 118 + 23 16 ± 2 115 + 17 14 ± 1 163 + 20 20 ± 2 176 ± 35 19 ± 3 284 ± 56 30 ± 5 Legend: 1 - Weeks of exposure to low photon fluence rate 2 - BA l e v e l i n mg/1 3 - Fresh weight mean ± standard error (per explant) i n mg 4 - Dry weight mean ± standard error (per explant) i n mg q u a l i t y as a c o - f a c t o r i n modifying an exp l a n t ' s f r e s h weight. The dry weight means fol l o w e d the p a t t e r n of f r e s h weight means (Table 4.25). The degrees of s i g n i f i c a n c e were, however, g r e a t e r f o r the two l i g h t q u a l i t y i n t e r a c t i o n s (Table 4.23). The s t r e n g t h of trends f o r white l i g h t c u l t u r e s probably account f o r much of t h i s d i f f e r e n c e . This i s because the red/FR means underwent l i t t l e change from the two t o four week exposure, e s p e c i a l l y at the 0.25 and 0.4 mg/1 BA l e v e l s . The percentage i n c r e a s e i n f r e s h weight under red/FR from the two t o four week regime a t these BA l e v e l s was much g r e a t e r than f o r the corresponding treatments' dry weights. 98 4.3 Determining i f glyphosate and l i g h t q u a l i t y i n t e r a c t to modify i n v i t r o growth 4.3.1 Experiment VII: Glyphosate-light q u a l i t y experiment The red/FR l i g h t e f f e c t of enhancing Spiraea shoot i n i t i a t i o n may i n v o l v e a combination of f a c t o r s . Two mechanisms i n v o l v i n g hormone metabolism may be i n o p e r a t i o n . F i r s t , red l i g h t may i n t e r a c t w i t h an exogenously a p p l i e d c y t o k i n i n t o i n c r e a s e the c y t o k i n i n ' s a b i l i t y t o promote growth as suggested by C l e l a n d ( C l e l a n d , 1976). Secondly, red/FR l i g h t may decrease l e v e l s of endogenous IAA ( l i n o , 1982). One p o s s i b l e method to examine the extent of a m o d i f i c a t i o n of auxin metabolism would be to use a s p e c i f i c compound known t o d i s r u p t such metabolism. Glyphosate, the a c t i v e i n g r e d i e n t of the h e r b i c i d e 'Roundup' i s j u s t such a compound. At s u b l e t h a l m i l l i m o l a r c o n c e n t r a t i o n s , glyphosate has been shown t o i n c r e a s e the r a t e of IAA o x i d a t i o n i n soybean and pea s e e d l i n g s l e a d i n g t o a r e l e a s e of l a t e r a l buds from a p i c a l dominance (Lee, 1984). The i n c r e a s e i n IAA o x i d a t i o n i s proposed t o occur due t o a glyphosate-induced d i s r u p t i o n of the s y n t h e s i s of c e r t a i n p h e n o l i c compounds. Such phe n o l i c s i n h i b i t IAA oxidase and thus w i t h reduced l e v e l s of these p h e n o l i c s a g r e a t e r IAA o x i d a t i o n occurs. For the c u r r e n t study, the e f f e c t of glyphosate on c u l t u r e s i n a d d i t i o n t o the red/FR e f f e c t may be p r e d i c t e d t o g i v e f u r t h e r i n c r e a s e s i n c y t o k i n i n t o auxin r a t i o s which may a f f e c t p r o l i f e r a t i o n . In t h i s experiment the p o t e n t i a l of glyphosate t o act as an i n v i t r o growth r e g u l a t o r f o r Spiraea, e i t h e r alone or i n combination w i t h a red/FR e f f e c t , was determined. Two p r e l i m i n a r y c o n c e n t r a t i o n t r i a l s were done t o determine glyphosate l e v e l s t h a t , w h i l e modifying Spiraea growth, would not cause gross a b n o r m a l i t i e s . In the f i r s t t r i a l the two l e v e l s chosen were 3.56 and 17.80 mg/1. At the higher l e v e l t h e r e was p l a n t m o r t a l i t y ; the lower l e v e l was c h a r a c t e r i z e d by r o s e t t e masses of unextended shoots ( F i g . 4.1). In the second t r i a l , 0.356 and 0.712 mg/1 were t e s t e d ; here a more c o n t r o l l e d r e l e a s e of a p i c a l dominance occurred but masses of unextended shoots co u l d s t i l l a r i s e . Two f i n a l l e v e l s were chosen f o r the l i g h t q u a l i t y experiment, 0.087 and 0.267 mg/1. These two l e v e l s appeared t o cause only o c c a s i o n a l s i d e e f f e c t s . + g l y + gly + g l y + gly - g l y Figure 4.1 The e f f e c t of 3-5° mg/l of glyphosate on Spiraea shoot p r o l i f e r a t i o n ( - g l y = no glyphosate, + g l y = glyphosate) General appearance G e n e r a l l y , glyphosate-treated p l a n t s under both r ed and white l i g h t had normal leaves and shoots. However, the co n c e n t r a t i o n s of glyphosate used i n t h i s experiment s t i l l o c c a s i o n a l l y caused growth m o d i f i c a t i o n s . The most common fe a t u r e was s m a l l e r , narrower leaves than those from untreated p l a n t s . Sometimes s m a l l f a s c i a t e d shoots ( l e s s than 1 cm.) were a l s o present. There was some v a r i a t i o n i n the c h a r a c t e r i s t i c s expressed f o r s p e c i f i c treatments under the two l i g h t regimes (see Table 4.26). Red l i g h t c u l t u r e s w i t h the 0.5 mg/1 BA + 0.087 mg/1 glyphosate treatment produced three of the f o u r samples w i t h the l e a f e f f e c t ; the same treatment f o r white l i g h t c u l t u r e s caused a l l f o u r samples t o show the l e a f e f f e c t and i n a d d i t i o n two of the four showed s m a l l f a s c i a t e d shoots. The treatment of 0.5 mg/1 BA + 0.267 mg/1 glyphosate under red l i g h t was s i m i l a r but a l l f o u r samples showed the l e a f e f f e c t as occurred w i t h white l i g h t which a l s o produced two c u l t u r e s w i t h shoot e f f e c t s . Table 4.26 Proport ion of each treatment's four samples showing glyphosate effects BA-GLY 1 Combination Red/FR l i g h t White l i g h t 0.25 BA 0.087 GLY 1/4 LVS.+ S H . 2 1/4 LVS. 0.25 BA 0.267 GLY 4/4 L V S . , 3 2/4 LVS.+ SH. 3/3 L V S . , 4 2/3 LVS.+ SH. 0.5 BA 0.087 GLY 3/4 LVS. 4/4 L V S . , 2/4 LVS.+ SH. 0.5 BA 0.267 GLY 4/4 LVS. 4/4 L V S . , 2/4 LVS.+ SH. Legend: BA - benzyladenine, GLY - glyphosate 1 - BA-GLY combinations measured in mg/1 2 - proportion of samples showing glyphosate effect on both leaves (LVS.) and shoots (SH.) 3 - proportion of samples showing glyphosate effect on leaves 4 - only 3 samples due to one discarded contaminated sample 103 Growth v a r i a t e s Shoot number/explant For both the glyphosate-free and 0.267 mg/1 glyphosate treatments, the 0.5 mg/1 BA c u l t u r e s had s l i g h t l y h i gher shoot number means than the 0.25 mg/1 BA c u l t u r e s (Table 4.27). However, a t the 0.087 mg/1 glyphosate l e v e l t h e r e was a much g r e a t e r i n c r e a s e i n shoot number means at the higher BA l e v e l . The 0.087 mg/1 glyphosate r e s u l t s were extreme a t both BA l e v e l s , w i t h the lowest shoot number means of a l l the treatments produced at the lower BA l e v e l and the highest means produced a t the higher BA l e v e l . To i n t e r p r e t any g l y p h o s a t e - l i g h t q u a l i t y e f f e c t s , the glyphos a t e - f ree c u l t u r e s were used as markers of the l i g h t q u a l i t y response i n t h i s experiment. The experimental design d i f f e r e d s l i g h t l y from the previous ones as t h i s one l a s t e d s i x weeks i n s t e a d of f i v e and a l s o there was a t r a n s f e r of p l a n t l e t s t o f r e s h medium a f t e r two weeks. Cul t u r e s under red/FR l i g h t had hig h e r shoot number means than white l i g h t c u l t u r e s . The mean shoot numbers under both red/FR and white l i g h t a t the 0.5 mg/1 BA l e v e l were g r e a t e r than the means at the 0.2 5 mg/1 BA l e v e l . In the presence of 0.087 mg/1 of glyphosate w i t h 0.25 mg/1 of BA, p r o l i f e r a t i o n was s t r o n g l y i n h i b i t e d compared t o the c o n t r o l f o r both white and red/FR (the d i f f e r e n c e was g r e a t e r f o r red/FR). Very l a r g e increases i n shoot number (up t o f o u r -f o l d ) were observed i f the c u l t u r e s under the same glyphosate c o n c e n t r a t i o n (0.087 mg/1) were grown w i t h higher exogenous Table 4.27 Shoot and node number means as a f f e c t e d by glyphosate and l i g h t q u a l i t y Light, BA1 GLY2 Shoot 3 Node4 q u a l i t y l e v e l l e v e l number number Red/FR 0 . 25 0.087 13.75 ± 3.86 59 ± 19 0.267 31.00 ± 3.14 104 ± 15 0 30.00 ± 2.84 128 ± 11 0.5 0.087 47.75 ± 10.92 168 + 38 0.267 33.75 ± 6.17 109 + 22 0 34.00 ± 3.90 123 ± 16 White 0.25 0.087 12.50 ± 1.03 49 + 8 0.267 40.67 + 7 . 51 128 + 15 0 23.50 ± 2.33 96 + 11 0.5 0.087 50 . 25 ± 12.83 151 ± 38 0.267 43.75 + 7.65 130 ± 22 0 29.75 + 4.79 107 + 18 Legend: 1 - BA l e v e l i n mg/1 2 - GLY= glyphosate, GLY l e v e l i n mg/1 3 - shoot number/explant mean ± standard error 4 - node number/block (mean of four b l o c k s , except mean of four blocks with two sub-samples for the "0" GLY treatment) ± standard error 105 l e v e l s of BA ( i . e . 0.5 mg/1). The increases occurred f o r both red/FR and white and reached s i m i l a r high l e v e l s . They represented much higher i n c r e a s e s than had been observed i n any pre v i o u s t h e s i s experiments w i t h m o d i f i c a t i o n of l i g h t q u a l i t y and c y t o k i n i n l e v e l s . The o v e r a l l l e v e l s of shoot p r o l i f e r a t i o n reached were a l s o higher than observed with 0.5 mg/1 of BA alone, e i t h e r i n t h i s experiment or i n those r e p o r t e d p r e v i o u s l y . Thus an i n h i b i t o r y e f f e c t of glyphosate on p r o l i f e r a t i o n can be overcome by the a d d i t i o n of exogenous BA t o 0.5 mg/1. A c r i t i c a l l e v e l between 0.25 and 0.5 mg/1 i s r e q u i r e d , presumably t o overcome the i n h i b i t o r y t h r e s h o l d e f f e c t e xerted by 0.087 mg/1 of glyphosate i n the medium. The f i n d i n g t h a t red/FR l i g h t i n the presence of glyphosate, w i t h 0.25 mg/1 of BA, d i d not increase the growth over white l i g h t , suggests t h a t glyphosate i s i n t e r f e r i n g w i t h the p u t a t i v e BA-red/FR l i g h t i n t e r a c t i o n or synergy; only the a d d i t i o n of the e f f e c t i v e l e v e l (0.5 mg/1) of the c y t o k i n i n BA can overcome the i n t e r f e r e n c e . The l a r g e increases i n p r o l i f e r a t i o n over t h a t seen i n the presence of 0.5 mg/1 of BA alone, r e f l e c t a s t i m u l a t o r y e f f e c t of glyphosate on growth which augments t h a t seen w i t h h i g h BA alone. I t may be p r e d i c t e d t h a t another e f f e c t which occurs due t o 0.087 mg/1 of glyphosate i n v o l v e s the r e d u c t i o n of a p i c a l dominance thus r e s u l t i n g i n an increase of shoot numbers. The s t i m u l a t o r y e f f e c t on growth at the 0.087 mg/1 glyphosate c o n c e n t r a t i o n may depend on the i n t e r a c t i o n w i t h 0.5 mg/1 of BA. 106 A mechanism of glyphosate a c t i o n , i . e . an i n c r e a s e i n IAA metabolism p u t a t i v e l y mediated by d i s r u p t e d p h e n o l i c compound s y n t h e s i s , has been demonstrated (Lee 1982; Lee 1984). At h i g h e r l e v e l s of glyphosate (0.2 67 mg/1) growth r a t e s under red/FR were s i m i l a r t o the c o n t r o l s w i t h BA alone whereas s t i m u l a t i o n occurred under white. I f the glyphosate e f f e c t a t t h i s h i g h e r c o n c e n t r a t i o n i s predominantly causing lowering of auxin then reduced a p i c a l dominance could r e s u l t i n i n c r e a s e d p r o l i f e r a t i o n . The lower values under red/FR than white l i g h t may be due t o the i n t e r f e r e n c e of intermediates of a c y t o k i n i n m e t abolic pathway which accumulate as an i n d i r e c t r e s u l t of glyphosate's block of c e r t a i n p h e n o l i c compounds' s y n t h e s i s (see r e s u l t s a t the lower glyphosate (0.087mg/l) and BA (0.25 mg/1) l e v e l s ) . However, the o v e r a l l growth i n h i b i t i o n seen a t lower glyphosate c o n c e n t r a t i o n s was not manifested because the predominant lowering of auxin would r e s u l t i n a s i g n i f i c a n t change i n the c y t o k i n i n / a u x i n r a t i o at higher glyphosate l e v e l s . The ANOVA (Table 4.28) i n d i c a t e s t h a t the f a c t o r s causing d i f f e r e n c e s i n shoot number were BA c o n c e n t r a t i o n and an i n t e r a c t i o n of BA and glyphosate c o n c e n t r a t i o n s . The changes i n shoot number observed i n the presence of glyphosate were manifested by a BA-glyphosate i n t e r a c t i o n . This r e s u l t supports data from a p r e l i m i n a r y experiment where glyphosate alone was added t o BA-free medium; here glyphosate was unable t o modify growth. Thus as a growth r e g u l a t o r i n the Spiraea i n v i t r o system the e f f e c t i v e n e s s of glyphosate a c t i o n depends on an Table 4.27a. Node number/shoot means 107 L i g h t q u a l i t y BA1 l e v e l GLY2 l e v e l Node number3 per shoot Red/FR 0.25 0.087 0. 267 0 4.3 3.4 4.3 0 . 5 0.087 0. 267 0 3.5 3.2 3.6 White 0.25 0.087 0.267 0 3.9 3.1 4.1 0.5 0.087 0. 267 0 3.0 3.0 3.6 Legend: 1 - BA l e v e l i n mg/1 2 - GLY= g l y p h o s a t e , GLY i n mg/1 3 - node number/shoot means c a l c u l a t e d by d i v i d i n g shoot and node number means i n T a b l e 4.27 T a b l e 4 . 28 A n a l y s e s o f v a r i a n c e summary. E f f e c t s o f l i g h t q u a l i t y , g l y p h o s a t e , and BA c o n c e n t r a t i o n on shoot and node numbers, and f r e s h and dry we ights . Source o f i df Shoot 1 Node 2 F r e s h 3 D r y 4 v a r i a t i o n number number weight weight G l y p h o s a t e l e v e l 2 NS NS NS NS BA l e v e l 1 ***** * * * **** * * L i g h t q u a l i t y 1 NS NS * * G l y p h o s a t e and l i g h t q u a l i t y i n t e r a c t i o n 2 NS NS NS NS G l y p h o s a t e , BA and l i g h t q u a l i t y i n t e r a c t i o n 2 NS NS NS NS G l y p h o s a t e and BA i n t e r a c t i o n (2) *** *** * ** G l y p h o s a t e l i n e a r 5 X BA 1 ** *** * ** G l y p h o s a t e l a c k o f f i t X BA 1 * * * * * * * BA and l i g h t q u a l i t y i n t e r a c t i o n 1 NS NS NS NS Legend: df * shoot number/explant node number/block f r e s h w e i g h t / e x p l a n t dry w e i g h t / e x p l a n t p o l y n o m i a l response models: l i n e a r = amount of v a r i a t i o n e x p l a i n e d by a s t r a i g h t l i n e r e l a t i o n s h i p , l a c k of f i t = amount of v a r i a t i o n e x p l a i n e d by a h igher o r d e r - t e r m e d r e l a t i o n s h i p degree of freedom s i g n i f i c a n t at p < 0 .1 , ** = at p < 0 .05 , * * * = at p < 0 .025, * * * * = at p < 0 .01 , * * * * * = at p < 0 .005, NS = n o n - s i g n i f i c a n t 109 i n t e r a c t i o n w i t h BA. Node number/block Node number means d i d not r e f l e c t as great a red/FR modulation of glyphosate's e f f e c t as d i d shoot number means. One d i f f e r e n c e was the d e c l i n e of the node number means under red/FR l i g h t at both BA l e v e l s i n combination w i t h the 0.267 mg/1 glyphosate l e v e l (Table 4.27). Since shoot numbers under red/FR remained almost the same at t h i s glyphosate l e v e l , the r e d u c t i o n i n node number i n d i c a t e s fewer nodes per shoot. However, t h i s decrease a l s o occurred f o r the white l i g h t shoots (Table 4.27a.). The l a r g e increase i n shoot numbers accompanying the i n c r e a s e i n node numbers t r a n s l a t e s i n t o fewer nodes per shoot. These r e s u l t s could suggest glyphosate-induced i n h i b i t i o n of shoot e l o n g a t i o n ; as discussed i n the "shoot l e n g t h " s e c t i o n t h i s i n h i b i t i o n d i d i n f a c t occur. The ANOVA supports the s m a l l e r d i f f e r e n c e s observed f o r node numbers w i t h a l e s s s i g n i f i c a n t e f f e c t a t t r i b u t e d t o BA c o n c e n t r a t i o n (Table 4.28). Despite the weaker BA e f f e c t , the s t r e n g t h of the glyphosate BA i n t e r a c t i o n remained the same or b e t t e r . 110 Shoot length Shoot e l o n g a t i o n was i n h i b i t e d by glyphosate. The 0.087 mg/1 glyphosate l e v e l e f f e c t on shoot e l o n g a t i o n was much l e s s extreme than i t s shoot i n i t i a t i o n e f f e c t . I t was the 0.2 67 mg/1 glyphosate l e v e l t h a t caused the g r e a t e s t r e d u c t i o n i n shoot lengths f o r both BA l e v e l combinations. The g r e a t e s t d i f f e r e n c e s between treatment comparisons i n Table 4.29 were thus between treatments where the 0.267 mg/1 glyphosate l e v e l was in v o l v e d . The p r o p o r t i o n of u s e f u l shoots under red/FR l i g h t remained higher than under white f o r every BA-glyphosate combination except one. This general r e t e n t i o n of increased shoot e l o n g a t i o n under red/FR l i g h t suggests t h a t any red/FR-glyphosate i n t e r a c t i o n has a gre a t e r e f f e c t on shoot i n i t i a t i o n than on shoot e l o n g a t i o n . When both shoot i n i t i a t i o n and shoot e l o n g a t i o n are considered, i t i s apparent t h a t the glyph o s a t e - f r e e c u l t u r e s w i t h 0.2 5 mg/1 BA under red/FR had higher numbers of u s e f u l shoots than any 0.2 5 mg/1 BA-glyphosate combination. Fresh weight/explant and Dry weight/explant The f r e s h and dry weight means were more g r e a t l y a f f e c t e d by l i g h t q u a l i t y than was shoot i n i t i a t i o n . This can be i l l u s t r a t e d when mean t o t a l s of the three treatments at each BA l e v e l are compared (from Table 4.30) f o r each l i g h t regime. The red/FR Table 4 . 2 9 Shoot l e n g t h s as a f f e c t e d by BA, glyphosate , and : l i g h t q u a l i t y P a i r s * BA 3- Gly3 f Sample Us e f u l ^ A c t u a l t e s t e d con. con. s i z e shoot % number Red/FR 0 .25 0 119 52 62 NS White 89 47 42 Red/FR 0.5 0 135 52 70 ** White 118 38 45 Red/FR 0.25 0 119 52 62 ** White 0.5 • 0 118 38 45 Red/FR 0 . 25 0. 087 55 49 . 27 NS White 50 34 17 Red/FR 0.25 0.267 124 27 34 NS White 120 32 38 Red/FR 0.5 0. 087 191 39 75 NS White 201 34 69 Red/FR 0.5 0.267 135 36 48 *** White 175 23 40 Red/FR 0 . 25 0. 087 55 49 27 NS White 0.5 0 118 38 45 Red/FR 0.25 0.087 55 49 27 ***** White 0.5 0.267 175 23 40 Red/FR 0.25 0.087 55 49 27 ** White 0.5 0.087 201 34 69 Red/FR 0.25 0.267 124 27 34 * White 0.5 0 118 38 45 Red/FR 0.25 0. 267 124 27 34 ***** White 0. 25 0 89 47 42 Legend: 1 - each p a i r represents the s e t of shoot l e n g t h p r o p o r t i o n s analyzed i n a Chi-square t e s t 2 - BA l e v e l i n mg/1 3 - g l y = glyphosate, glyphosate l e v e l i n mg/1 4 - t o t a l number of shoots produced i n a l l experimental blocks 5 - a u s e f u l shoot i s de f i n e d as a shoot 1 cm or longer i n length; thus the u s e f u l shoot % i s the % of the sample s i z e ranked u s e f u l 6 - the co n v e r s i o n of the u s e f u l shoot % i n t o number of shoots 7 - X i s the symbol f o r the Chi-square t e s t ; the a s t e r i s k s d e f i n e the degree of s i g n i f i c a n c e : . * = s i g n i f i c a n t a t p<0.1, ** = at p<0.05, *** = a t p<0.025, ****' = a t p<0.01, ***** = a t p<0.005, NS = n o n - s i g n i f i c a n t Table 4.30 Fresh and dry weight means as a f f e c t e d by glyphosate and l i g h t q u a l i t y Light^ BA1 GLY2 Fresh 3 Dry 4 q u a l i t y l e v e l l e v e l weight weight Red/FR 0.25 0.087 0.267 0 0.5 0.087 0.267 0 White 0.25 0.087 0.267 0 0.5 0.087 0.267 0 162 ± 60 26 ± 6 282 ± 45 42 ± 4 398 ± 39 53 ± 4 541 ± 128 62 + 13 320 ± 90 39 ± 9 433 + 59 51 + 6 124 ± 12 22 + 2 306 ± 60 40 + 7 199 ± 19 30 + 2 406 + 89 47 + 8 333 + 63 38 + 6 270 ± 39 35 + 4 Legend: 1 - BA l e v e l i n mg/1 2 - GLY= g l y p h o s a t e , GLY l e v e l i n mg/1 3 - f r e s h w e i g h t / e x p l a n t mean ± s tandard e r r o r 4 - dry w e i g h t / e x p l a n t mean ± s tandard e r r o r 113 c u l t u r e s had a f r e s h weight t o t a l over 25% g r e a t e r a t both BA l e v e l s than the white l i g h t c u l t u r e s . Dry weight t o t a l s were over 33% hi g h e r under red/FR at the lower BA l e v e l and were 20% g r e a t e r a t the higher BA l e v e l . These l i g h t q u a l i t y dependent s t a t i s t i c a l d i f f e r e n c e s (ANOVA, Table 4.28), are g r e a t e r than those observed f o r shoot numbers. The glyphosate-BA i n t e r a c t i o n i n the ANOVA was l e s s f o r the f r e s h weights than f o r the dry weights. Both were l e s s than the same i n t e r a c t i o n f o r shoot number means. This r e s u l t suggests t h a t w h i l e glyphosate can i n t e r a c t w i t h BA t o cause l a r g e d i f f e r e n c e s i n shoot numbers, i t s e f f e c t on dry matter accumulation i s l e s s important; here the f a c t o r of l i g h t q u a l i t y p l a y s a g r e a t e r r o l e . 114 Chapter 5 General D i s c u s s i o n and Conclusions 5.1 C h a r a c t e r i z a t i o n of the Spiraea nipponica i n v i t r o l i g h t q u a l i t y reponse E s t a b l i s h i n g a system f o r the micropropagation of a s p e c i f i c p l a n t g e n e r a l l y i n v o l v e s e m p i r i c a l t r i a l s i n which c u l t u r e medium c o n s t i t u e n t s and environmental f a c t o r s are v a r i e d . Once a b a s i c system has been designed, p h y s i o l o g i c a l concepts gleaned from the study of n a t u r a l l y grown p l a n t s can be a p p l i e d t o optimize the e f f i c i e n c y of i n v i t r o growth. In these s t u d i e s , e f f i c i e n c y has been de f i n e d as o b t a i n i n g maximal shoot p r o l i f e r a t i o n w h i l e m i n i m i z i n g exogenous c y t o k i n i n l e v e l s . The approach taken to optimize e f f i c i e n c y i n v o l v e d using l i g h t q u a l i t y as a p o s s i b l e means of modifying growth hormone metabolism i n the explant i t s e l f . Experiments were done to c h a r a c t e r i z e the optimal l i g h t q u a l i t y f o r propagation i n the Spiraea system. The i n i t i a l experiment i n v e s t i g a t e d which l i g h t q u a l i t y was most e f f i c i e n t i n promoting i n v i t r o growth. The r e s u l t s i n d i c a t e d t h a t at a BA l e v e l of 0.25 mg/1, which i s h a l f t h a t normally used f o r sub-c u l t u r i n g stock c u l t u r e s , tungsten f i l a m e n t l i g h t with a high p r o p o r t i o n of red/FR l i g h t produced the most e f f i c i e n t growth. The h i g h shoot production was accompanied by high mean node 115 numbers per shoot and g r e a t e r shoot lengths than at other wavelengths. Both these c r i t e r i a are important f o r e i t h e r s u c c e s s f u l s u b - c u l t u r i n g f o r shoot p r o l i f e r a t i o n or the next step of r o o t i n g shoots. The f a c t t h a t on BA-free medium none of the l i g h t q u a l i t i e s were capable of i n c r e a s i n g a low growth r a t e i n d i c a t e s t h a t any i n v i t r o l i g h t q u a l i t y e f f e c t (at l e a s t f o r Spiraea) can only be expressed i n concert w i t h an exogenous hormone, i n t h i s case the c y t o k i n i n BA. M o d i f i c a t i o n of the endogenous c y t o k i n i n / a u x i n r a t i o t o a s t a t e conducive t o i n t e r a c t i o n w i t h lower exogenous BA l e v e l s c l e a r l y depended on the s p e c i f i c l i g h t q u a l i t y exposure. The maximal growth e f f e c t under white l i g h t had p r e v i o u s l y been shown to occur at a standard BA s u b - c u l t u r e l e v e l of 0.5 mg/1 (Norton and Norton, 1986), and above t h i s l e v e l shoot production decreased. Using a high p r o p o r t i o n of red/FR l i g h t optimal growth was achieved at a lower BA l e v e l of 0.25 mg/1. Such f i n d i n g s suggest t h a t under red/FR l i g h t the endogenous c y t o k i n i n / a u x i n r a t i o was very favourable f o r the i n d u c t i o n of both shoot p r o l i f e r a t i o n and shoot e l o n g a t i o n . Blue l i g h t induced the lowest shoot production at BA l e v e l s of 0.25 mg/1 and 0.5 mg/1, suggesting t h a t i f increases i n shoot p r o d u c t i o n could occur under blue l i g h t they would r e q u i r e h i g h e r BA l e v e l s (which might be accompanied by unwanted s i d e -e f f e c t s ) . The lower shoot production under blue l i g h t may be a s c r i b e d to s e v e r a l f a c t o r s . One f a c t o r may be blue l i g h t ' s a b i l i t y t o maintain a p i c a l dominance. This was noted i n an e a r l i e r study-where shoot t i p s of w i l l o w explants were removed; a subsequent blue l i g h t exposure i n h i b i t e d l a t e r a l bud growth (Letouze, 1974). Another p o s s i b i l i t y i s t h a t there was a photon fluence r a t e e f f e c t . Plum shoot e x p l a n t s , when exposed t o red and blue l i g h t at 37 u mol photons m^s' d i s p l a y e d s i m i l a r growth r a t e s ( B a r a l d i et a_l. , 1988) . When the photon fluence r a t e was reduced to 9 u mol photons m'^ s,1 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 the p r o l i f e r a t i o n r a t e f o r red l i g h t w h i l e the r a t e under blue l i g h t s i g n i f i c a n t l y d e c l i n e d . In our Spiraea study the photon - l h i g h e s t fluence r a t e of red/FR was 17 u mol photons m s while f o r blue i t was 1.8 u mol photons m'^ s.' Thus i f i t had been p o s s i b l e t o ob t a i n higher f l u e n c e r a t e s of f i l t e r e d blue i n our system, the blue l i g h t i n h i b i t i o n might have been l o s t . Under red/FR l i g h t c o n d i t i o n s , i n c r e a s i n g the BA l e v e l reduced shoot p r o l i f e r a t i o n , suggesting t h a t the r e s u l t i n g endogenous c y t o k i n i n / a u x i n r a t i o i n h i b i t e d shoot i n i t i a t i o n . In a study on plum ex p l a n t s , a pl a t e a u of maximum shoot i n i t i a t i o n occurred at one BA l e v e l f o r red, f a r - r e d , and blue l i g h t c u l t u r e s and then showed a d e c l i n e at a higher BA l e v e l ( B a r a l d i et al. , 1988). Thus f o r Spiraea. a more pronounced e f f e c t of s p e c i f i c l i g h t q u a l i t y i n t e r a c t i o n w i t h BA seems t o be i n f l u e n c i n g growth c h a r a c t e r i s t i c s . Although blue and red/FR wavelengths were assumed to be most capable of modifying phytochrome e q u i l i b r i a , perhaps i n a high i r r a d i a n c e response (HIR), i t was p o s s i b l e t h a t any minor changes caused by intermediate wavelengths (between blue and red l i g h t ) might a l s o promote e f f i c i e n t _in v i t r o growth. However, when green, y e l l o w , and orange wavelengths were used f o r the second experiment only minor m o d i f i c a t i o n s of growth occurred. An i n v e s t i g a t i o n i n v o l v i n g Juneberry explants suggested t h a t green l i g h t suppresses shoot i n i t i a t i o n (Behrouz and Lineberger, 1981) . Our r e s u l t s i n d i c a t e t h a t f o r the i n v i t r o Spiraea system, under the s p e c i f i c exogenous BA c o n d i t i o n s , green l i g h t d i d not i n h i b i t shoot i n i t i a t i o n but caused lower shoot lengths than at other wavelengths at the higher BA l e v e l . Thus, under y e l l o w l i g h t 43% of the shoots were 1 cm or longer, orange l i g h t had 38% whi l e green had 31%. These comparative r e s u l t s under green l i g h t i n d i c a t e p o s s i b l e d i f f e r e n c e s i n growth response between d i f f e r e n t p l a n t s pecies c u l t u r e d i n v i t r o . The yellow l i g h t shoot lengths were the h i g h e s t of a l l the l i g h t q u a l i t i e s at both BA l e v e l s , a r e s u l t which agrees w i t h a study on a Prunus s e r o t i n a i n v i t r o system where y e l l o w l i g h t enhanced shoot lengths (Fuernkranz et a l . , 1988). The two experiments i n v o l v i n g l i g h t s o u r c e - f i l t e r combinations (using blue and red f i l t e r s ) were done t o v e r i f y t h a t the l i g h t sources chosen t o provide s p e c i f i c l i g h t q u a l i t y ranges i n the f i r s t experiment were the most e f f e c t i v e w i t h the a v a i l a b l e f i l t e r s . They a l s o provided an opportunity t o study the e f f e c t on i n v i t r o growth of v a r y i n g the r a t i o s of blue to red/FR l i g h t . The shoot means f o r these f i l t e r experiments were lower than 118 i n the f i r s t l i g h t q u a l i t y experiment. Thus, an a d d i t i o n a l f a c t o r c o u l d be s t u d i e d , the degree of response t o l i g h t q u a l i t y determined by p r o l i f e r a t i o n r a t e ; a r a t e f o r these experiments much lower than i n the f i r s t experiment. The most probable e x p l a n a t i o n f o r the lower growth r a t e i s t h a t the p a r t i c u l a r source c u l t u r e s chosen f o r explants had a lower morphogenic p o t e n t i a l than other a v a i l a b l e source c u l t u r e s . Wide v a r i a t i o n s i n p r o l i f e r a t i o n r a t e s between sub-cultures have been reported f o r other i n v i t r o grown species (Druart, 1987). A f a c t o r a f f e c t i n g morphogenetic p o t e n t i a l i s the endogenous hormone balance w i t h i n the p a r t i c u l a r explants used. For example, i f the source c u l t u r e s used had grown at a f a s t e r r a t e than other c u l t u r e s , a d e p l e t i o n of exogenous BA might have r e s u l t e d i n a lower c y t o k i n i n / a u x i n r a t i o i n the mother p l a n t s ; e x plants from these would be l e s s responsive t o the p r e v i o u s l y used l e v e l s of exogenous BA and would undergo l e s s shoot i n i t i a t i o n . The p u t a t i v e i n h i b i t o r y e f f e c t of blue l i g h t on shoot i n i t i a t i o n noted i n the f i r s t l i g h t q u a l i t y experiment was again apparent i n the experiment where the blue f i l t e r was combined w i t h each l i g h t source. In t h i s experiment the blue f i l t e r combined w i t h the tungsten f i l a m e n t l i g h t source reduced t h i s l i g h t source's a b i l i t y to enhance growth at the lower BA l e v e l . Although shoot i n i t i a t i o n was s t i l l h i ghest under tungsten f i l a m e n t l i g h t at the lower BA l e v e l , the shoot mean was not s t a t i s t i c a l l y d i f f e r e n t from those under the other l i g h t regimes. The decrease i n the q u a n t i t y of red/FR l i g h t reaching the c u l t u r e s under the tungsten f i l a m e n t - b l u e f i l t e r regime w i t h an acdompanying r e d u c t i o n i n shoot i n i t i a t i o n suggests that the amount of red/FR l i g h t i s an important f a c t o r c o n t r i b u t i n g to the p r e v i o u s l y observed red/FR promotion of growth i n Experiment I. Any o p t i m i z a t i o n of the small blue wavelength component of tungsten f i l a m e n t l i g h t by the blue f i l t e r c ould a l s o reduce growth; t h i s p o s s i b i l i t y then supports the idea of blue l i g h t being an i n h i b i t o r y f a c t o r . D i f f e r e n c e s f o r shoot p r o l i f e r a t i o n were observed at the d i f f e r e n t BA l e v e l s w i t h the exception of tungsten filament l i g h t which gave s i m i l a r growth p a t t e r n s at both BA l e v e l s . The lower amounts of red/FR passing through the blue f i l t e r under tungsten f i l a m e n t l i g h t may represent a t h r e s h o l d l e v e l f o r growth under these c o n d i t i o n s . One of the reasons f o r using the metal h a l i d e source f o r the blue l i g h t regime of the i n i t i a l experiment was to. compensate f o r the poor t r a n s m i s s i o n of the blue f i l t e r . Thus, because metal h a l i d e was r i c h i n blue wavelengths and discharged a high photon f l u e n c e r a t e , i t was thought t h a t the blue l i g h t obtained would be at a photon fluence r a t e capable of s u s t a i n i n g growth. There i s a g r e a t e r p r o p o r t i o n of blue wavelengths under the metal h a l i d e l i g h t source than under the f l u o r e s c e n t l i g h t source. However, the shoot means at both BA l e v e l s were v i r t u a l l y the same f o r both l i g h t sources. This suggests t h a t the e f f e c t of blue l i g h t i s achieved at a s p e c i f i c l e v e l of blue wavelength output above which d i f f e r e n c e s are not observed. Other wavelength outputs, such as a higher p r o p o r t i o n of red under 'fluorescent l i g h t i n g do not appear t o be modulating the blue e f f e c t at t h i s r e l a t i v e blue l e v e l . The shoot lengths under each l i g h t source were s i m i l a r . This may i n d i c a t e t h a t shoot e l o n g a t i o n occurs at a s i m i l a r r a t e above a low fluence r a t e t h r e s h o l d of red/FR l i g h t . F i n a l l y , the lower p r o l i f e r a t i o n r a t e i n the blue f i l t e r experiment may have a f f e c t e d the response of the c u l t u r e t o l i g h t q u a l i t y . I t may be t h a t the proposed i n h i b i t o r y e f f e c t of blue l i g h t i n the f i r s t experiment was p a r t l y due to the higher growth r a t e i n t h a t experiment. A higher growth r a t e could imply a higher metabolism of BA and perhaps a s t a t e more conducive t o o p t i m i z i n g any i n t e r a c t i o n or synergy between BA and r e d / f a r - r e d l i g h t . Slower growing c u l t u r e s which may have a lower BA metabolism could have a reduced r e d / f a r - r e d l i g h t - B A i n t e r a c t i o n or synergy and a lower s e n s i t i v i t y t o blue l i g h t . This would then e x p l a i n s i m i l a r l e v e l s of growth under the metal h a l i d e and f l u o r e s c e n t - b l u e f i l t e r regimes. The p r o l i f e r a t i o n r a t e s of plum c u l t u r e s ( B a r a l d i et aJL. , 1988) were lower than f o r S p i r a e a ; t h i s lower growth r a t e may have i n f l u e n c e d t h e i r response to s p e c i f i c l i g h t q u a l i t i e s . In the plum system, blue and red l i g h t caused s i m i l a r growth r a t e s which were higher than under white l i g h t . Thus although a red l i g h t - B A i n t e r a c t i o n or synergy may have been suppressed by a lower growth r a t e i t could s t i l l promote growth. S i m i l a r l y , the shoot number means f o r Spiraea under tungsten f i l a m e n t l i g h t may be p a r t l y due to a reduced BA 1 2 1 i n t e r a c t i o n w i t h the minimal amount of red/FR l i g h t passing through the blue f i l t e r . In the red f i l t e r experiment the highest p r o l i f e r a t i o n r a t e was a t the higher BA l e v e l (0.5 mg/1) under tungsten f i l a m e n t l i g h t . This c o n t r a s t w i t h the drop under the higher BA l e v e l i n Experiment I suggests t h a t the low o v e r a l l p r o l i f e r a t i o n r a t e i n the f i l t e r experiments a f f e c t e d the i n t e n s i t y of the red/FR l i g h t - B A i n t e r a c t i o n or synergy. Perhaps the lower photon f l u e n c e r a t e i n t h i s experiment was not enough to enhance growth at the lower BA l e v e l as had occurred p r e v i o u s l y i n Experiment I w i t h an increased photon fluence r a t e under tungsten f i l a m e n t l i g h t . The tungsten f i l a m e n t l i g h t had high p r o p o r t i o n s of both red and f a r - r e d wavelengths optimized under the red f i l t e r and thus induced the highest shoot number means of a l l l i g h t regimes at both BA l e v e l s . Fluorescent and metal h a l i d e red f i l t e r regimes induced s i m i l a r l e v e l s of shoot i n i t i a t i o n at both BA l e v e l s . These r e s u l t s suggest t h a t any p o s s i b l e growth e f f e c t s due to d i f f e r e n c e s between f l u o r e s c e n t and metal h a l i d e p r o p o r t i o n s of red and f a r - r e d wavelengths were negated by these l i g h t sources' r e l a t i v e l y lower output of such wavelengths compared to tungsten f i l a m e n t l i g h t . The red f i l t e r r e d u c t i o n of blue wavelengths under f l u o r e s c e n t and metal h a l i d e l i g h t d i d not seem e f f e c t i v e i n enhancing shoot i n i t i a t i o n ; perhaps enough blue passed through the f i l t e r t o maintain shoot numbers below those obtained under tungsten f i l a m e n t l i g h t . 122 An e f f e c t which may be p a r t i a l l y due t o the low p r o l i f e r a t i o n r a t e i s the lower shoot l e n g t h p r o p o r t i o n under the tungsten f i l a m e n t - r e d f i l t e r regime compared t o the metal h a l i d e - r e d f i l t e r regime. The proposed decrease i n the red/FR l i g h t - B A i n t e r a c t i o n or synergy, due t o the lower growth r a t e , may have reduced any red/FR l i g h t enhancement of shoot e l o n g a t i o n . In the f i r s t q u a l i t y experiment red/FR l i g h t was a p p l i e d over a 16 hour photoperiod. I f f a r - r e d enriched l i g h t promotes the growth of i n v i t r o Spiraea shoots v i a the same mechanism as i t s growth promotion of n a t u r a l l y grown p l a n t s , then a short f a r - r e d enriched l i g h t exposure given at the end of a white l i g h t photoperiod should e l i c i t a s i m i l a r i n v i t r o growth response as the 16 hour red/FR photoperiod. Since the tungsten f i l a m e n t l i g h t source has a higher p r o p o r t i o n of f a r - r e d l i g h t than red l i g h t , the red f i l t e r was omitted f o r t h i s s h ort term f a r - r e d enriched l i g h t experiment. A t y p i c a l end-of-day f a r - r e d l i g h t response of n a t u r a l l y grown p l a n t s i s an .increase i n shoot l e n g t h ; thus, i t was assumed t h a t the growth e f f e c t most l i k e l y t o be observed w i t h the i n v i t r o c u l t u r e s was an i n c r e a s e i n shoot l e n g t h . Comparisons between t r e a t e d and untreated explants i n d i c a t e d only one d i f f e r e n c e . Under the white l i g h t source (metal h a l i d e ) , c u l t u r e s showed a s l i g h t l y higher percentage of longer shoots a t the 0.4 mg/1 BA l e v e l than at the 0.5 BA l e v e l . C u l t u r e s under metal h a l i d e w i t h a f a r - r e d a d d i t i o n d i d not 123 e x h i b i t such a peak at the 0.4 mg/1 BA l e v e l . The same p a t t e r n was present under each l i g h t regime f o r shoot i n i t i a t i o n . The probable e x p l a n a t i o n i s t h a t f o r metal h a l i d e l i g h t a maximal l i g h t - B A i n t e r a c t i o n occurs at the 0.4 mg/1 BA l e v e l . Such a peak would not have been observed i n the f i r s t experiment due t o t h a t experiment's l a r g e r incremental change i n BA c o n c e n t r a t i o n , i . e . , from 0.25 mg/1 t o 0.5 mg/1. The l a c k of the 0.4 mg/1 BA peak under metal h a l i d e w i t h the f a r - r e d a d d i t i o n suggests t h a t the minimal f a r - r e d exposure was s t i l l capable of e x e r t i n g a minor e f f e c t on growth. S p e c i f i c a l l y the f a r - r e d l i g h t may have s h i f t e d the phytochrome e q u i l i b r i a induced by metal h a l i d e l i g h t t o a s t a t e l e s s conducive t o a l i g h t - B A i n t e r a c t i o n or synergy at the 0.4 mg/1 BA l e v e l , thus e l i m i n a t i n g the peak. The r e s u l t s i n d i c a t e t h a t d e s p i t e a p o s s i b l e minor f a r - r e d e f f e c t a t the 0.4 mg/1 BA l e v e l , the p r e v i o u s l y s u b s t a n t i a l red/FR e f f e c t s on growth were absent. Thus, i t appears t h a t f o r the S p iraea i n v i t r o system prolonged exposures of f a r - r e d enriched l i g h t and/or the red/FR l i g h t p r e v i o u s l y used, are r e q u i r e d t o enhance shoot i n i t i a t i o n and shoot e l o n g a t i o n . At l e a s t one other study supports our Spiraea shoot e l o n g a t i o n r e s u l t . In a c o n i f e r study (Zel e t a l . , 1988), a 16 hour photoperiod of f a r - r e d l i g h t given over e i g h t days followed by ten days w i t h a 16 hour white l i g h t photoperiod was r e q u i r e d t o produce g r e a t e r shoot e l o n g a t i o n than observed under white l i g h t c o n t r o l s . 124 5.2 Factors contributing to the red/FR l i g h t growth response y The experimental design of the f i r s t l i g h t quality-experiment i n v o l v e d growing explants f o r the f i r s t two weeks under a higher photon f l u e n c e r a t e than the treatment l i g h t regimes. Thus, two stage l i g h t treatments of s p e c i f i c wavelength and f o l l o w e d by higher f l u e n c e were examined i n a subsequent experiment. The time r e q u i r e d under a s p e c i f i c regime and the combination of s p e c i f i c wavelengths and photon fluence r a t e s were examined. There appears to be a c r i t i c a l i n t e r a c t i o n between the amount of exposure to red/FR and the t r a n s f e r t o a higher photon f l u e n c e r a t e . T h i s experiment suggests t h a t a t r a n s f e r t o a h i g h e r f l u e n c e r a t e i s necessary t o optimize the p u t a t i v e red/FR-BA i n t e r a c t i o n or synergy at 0.25 and 0.4 mg/1 of BA. Higher l e v e l s of BA (0.5 mg/1) can overcome the need f o r the h i g h e r photon fluence step. The expression of such an i n t e r a c t i o n or synergy was dependent on the time of t r a n s f e r a t the lower BA l e v e l s of 0.25 mg/1 and 0.4 mg/1, whereas the h i g h e r BA l e v e l of 0.5 mg/1 c o u l d overcome the high fluence requirement. This suggests t h a t the e x t r a BA, by o p t i m i z i n g the i n t e r a c t i o n or synergy, was able t o overcome any modulating i n f l u e n c e of e i t h e r or both, the red/FR exposure time and the t r a n s f e r t o a higher fluence r a t e . The shoot number means at the 0.5 mg/1 BA l e v e l f o r each t r a n s f e r regime under red/FR were c o n s i s t e n t l y high and exceded those under white a l s o i n d i c a t i n g 12 5 the e f f e c t of a red/FR-BA i n t e r a c t i o n or synergy f o r p r o l i f e r a t i o n of Spiraea. The f a c t t h a t there was a g r e a t e r shoot p r o l i f e r a t i o n at the f o u r week red/FR exposure compared t o the two week exposure suggests t h a t the age of the c u l t u r e may determine explant r e c e p t i v i t y t o red/FR l i g h t . Such a c u l t u r e age e f f e c t has been observed i n at l e a s t two other i n v i t r o systems. One i n v o l v e d l e t t u c e cotyledons where a d v e n t i t i o u s shoot formation was optimal when d a i l y pulses of red l i g h t were given dur i n g e i t h e r the second week of a f i v e week c u l t u r e p e r i o d or durin g the whole c u l t u r e p e r i o d (Kadkade and S e i b e r t , 1976). The other study i n v o l v e d a d v e n t i t i o u s bud formation from embryo-derived c a l l u s of Douglas f i r (Kadkade and Jopson, 1978). Here a s i x t e e n hour photoperiod of red l i g h t , when given dur i n g the t h i r d , f o u r t h , and f i f t h week of growth of an e i g h t week c u l t u r e p e r i o d , promoted optimal growth. Although c u l t u r e age may p l a y a r o l e i n determining the red/FR l i g h t e f f e c t , i t i s apparent t h a t even w i t h a two week red/FR exposure c u l t u r e s e x h i b i t e d h i gher shoot number means at a l l BA l e v e l s than d i d white l i g h t c u l t u r e s . T h i s suggests a general promotive red/FR l i g h t e f f e c t . In a d d i t i o n , such a f i n d i n g p rovides f u r t h e r s u p p o r t i v e data f o r the idea of a red/FR l i g h t - c y t o k i n i n i n t e r a c t i o n or synergy. In terms of the t h e s i s o b j e c t i v e of f a c i l i t a t i n g the use of lower exogenous c y t o k i n i n c o n c e n t r a t i o n s , i t was found t h a t shoot i n i t i a t i o n was optimized a t the lower BA l e v e l s of 0.25 12 6 and 0.4 mg/1 when four weeks of low fluence r a t e red/FR was f o l l o w e d by a t r a n s f e r t o white l i g h t of a higher fluence f o r one week. The i n i t i a t i o n r a t e was c o n s i s t e n t l y g r e a t e r than t h a t observed under the analogous white l i g h t regimes. Even wi t h the h i g h e r l e v e l of 0.5 mg/1 BA, the optimal i n i t i a t i o n r a t e was not achieved w i t h white l i g h t , but was c o n s i s t e n t l y observed under each regime which had i n v o l v e d red/FR. This f i n d i n g i n d i c a t e s the s t r o n g s i g n i f i c a n c e of the red/FR-BA i n t e r a c t i o n or synergy f o r o p t i m i z i n g growth. 127 5.3 Determining i f glyphosate and l i g h t q u a l i t y i n t e r a c t to modify i n v i t r o growth Responses t o glyphosate have been r e l a t e d t o each p l a n t s p e c i e s ' r a t e of IAA metabolism (Lee, 1984). Thus, although an e f f e c t on IAA metabolism f o r the Spiraea explants could be expected, the extent of the e f f e c t could not be p r e d i c t e d . The t r i a l s t o determine s u i t a b l e glyphosate concentrations i n d i c a t e d the severe growth m o d i f i c a t i o n s t h a t glyphosate can cause. Shoots were almost e l i m i n a t e d ; what remained were masses of compact r o s e t t e s of reduced le a v e s . The c o n c e n t r a t i o n s f o r these t r i a l s ranged from 17.80 mg/1 t o 0.356 mg/1. The glyphosate c o n c e n t r a t i o n s used f o r the l i g h t q u a l i t y experiment (0.087 mg/1 and 0.2 67 mg/1) d i d not cause any explant m o r t a l i t y . T h i s l a c k of m o r t a l i t y i s noteworthy s i n c e i n a study w i t h cranberry node explants (Scorza et a l . , 1984) s i m i l a r glyphosate l e v e l s caused m o r t a l i t y . Furthermore, w h i l e p e r s i s t e n t a b n o r m a l i t i e s were observed f o r shoots and leaves from the cranberry e x p l a n t s , Spiraea explants only o c c a s i o n a l l y e x h i b i t e d growth m o d i f i c a t i o n s at the s e l e c t e d glyphosate c o n c e n t r a t i o n s . Perhaps the r a p i d growth of Spiraea promotes a h i g h e r metabolism of glyphosate, thus l e s s e n i n g i t s t o x i c i t y . There appear to be two types of i n t e r a c t i o n w i t h glyphosate, each dependent on the glyphosate c o n c e n t r a t i o n . At the lower l e v e l of glyphosate, 0.087 mg/1, w i t h 0.25 mg/1 of BA, shoot p r o l i f e r a t i o n was s t r o n g l y i n h i b i t e d under both white and red/FR 128 l i g h t . A g r e a t e r d e c l i n e under red/FR may suggest t h a t a p u t a t i v e red/FR r e l a t e d decrease i n IAA l e v e l s exacerbated a glyphosate-induced IAA decrease. The s t i m u l a t i o n of shoot p r o l i f e r a t i o n under both l i g h t regimes when 0.5 mg/1 of BA was used i n d i c a t e s the complexity of glyphosate's a c t i o n . With t h i s glyphosate-BA treatment shoot p r o l i f e r a t i o n r a t e s were much hi g h e r than i n previous t h e s i s experiments. I t appears t h a t w i t h the 0.087 mg/1 glyphosate l e v e l , two d i s t i n c t e f f e c t s are expressed. The i n h i b i t i o n e f f e c t noted on growth at 0.2 5 mg/1 of BA i s overcome at higher BA l e v e l s (0.5 mg/1) and under these c o n d i t i o n s , another e f f e c t of glyphosate g i v e s augmentation of growth. The r e s u l t s obtained w i t h the hi g h e r glyphosate l e v e l (0.2 67 mg/1) suggest t h a t a presumed g r e a t e r auxin o x i d a t i o n than w i t h the lower glyphosate l e v e l i s determining the growth p a t t e r n . T h i s i s e s p e c i a l l y apparent under white l i g h t , a t both BA l e v e l s , where shoot p r o l i f e r a t i o n was much higher than c o n t r o l s . Shoot numbers under red/FR, at both BA l e v e l s , were lower than under white and v i r t u a l l y unchanged from c o n t r o l s . Here, an e f f e c t of lowered auxin l e v e l s may have been t o i n t e r f e r e w i t h the p u t a t i v e r e d / F R - c y t o k i n i n i n t e r a c t i o n or synergy by causing i n t e r m e d i a t e s of a c y t o k i n i n metabolic pathway t o accumulate because of a p o t e n t i a l b l o c k i n d i r e c t l y induced by glyphosate's d i s r u p t i o n of c e r t a i n p h e n o l i c compounds' s y n t h e s i s . A l t e r n a t i v e l y , red/FR l i g h t may be exacerbating a glyphosate d i s r u p t i o n of an auxin r e g u l a t i o n of c y t o k i n i n metabolism. Auxin 129 r e g u l a t i o n of c y t o k i n i n metabolism has p r e v i o u s l y been demonstrated f o r a tobacco stem p i t h - e x p l a n t system ( P a l n i e t a l . , 1988). Glyphosate i n h i b i t e d shoot e l o n g a t i o n ; however, the extreme e f f e c t of the 0.25 mg/1 BA-0.087 mg/1 glyphosate treatment seen on shoot i n i t i a t i o n was l o s t . For shoot l e n g t h s , the 0.267 mg/1 glyphosate treatment caused the g r e a t e s t d i f f e r e n c e s . This r e d u c t i o n i n shoot l e n g t h c o r r e l a t e d w i t h the fewer number of nodes observed per shoot under both red/FR and white l i g h t . Although under both l i g h t regimes shoot lengths were reduced, the red/FR c u l t u r e s s t i l l had g e n e r a l l y g r e a t e r shoot l e n g t h s . This r e t e n t i o n of g r e a t e r shoot lengths under red/FR supports the f a c t t h a t other f a c t o r s besides auxin c o n t r o l e l o n g a t i o n , f o r example, the g i b b e r e l l i n s (George and S h e r r i n g t o n , 1984). F i n a l l y , i t has p r e v i o u s l y been shown t h a t at the h e r b i c i d a l c o n c e n t r a t i o n of glyphosate (0.8 l b s . / a c r e ) the enzyme EPSP synthase (which i s c e n t r a l i n the s h i k i m i c a c i d pathway) i s i n h i b i t e d . Such i n h i b i t i o n may have many r a m i f i c a t i o n s f o r s e v e r a l metabolic pathways. At lower glyphosate l e v e l s , some such e f f e c t s may be o c c u r r i n g and c o u l d e x p l a i n the p l e i o t r o p i c e f f e c t s observed i n t h i s study. In c o n c l u s i o n , the use of glyphosate i n t h i s study suggested t h a t the red/FR e f f e c t may i n v o l v e a m o d i f i c a t i o n of auxin metabolism. A l s o i t i s apparent t h a t glyphosate, at the lower l e v e l , c a n b e u s e d t o a u g m e n t s h o o t p r o l i f e r a t i o n i f s u f f i c i e n t 130 BA i s present (even under white l i g h t ) . Although o c c a s i o n a l abnormal shoots appeared and shoot lengths were sub-optimal, f u r t h e r m anipulation of glyphosate l e v e l s may optimize shoot p r o l i f e r a t i o n and e l i m i n a t e abnormal shoot morphology. Summary The r e s u l t s of t h i s study i n d i c a t e t h a t f o r Spiraea  ni p p o n i c a l i g h t q u a l i t y can be used t o lower exogenous c y t o k i n i n requirements. A mixture of red and f a r - r e d (red/FR) l i g h t emitted from a tungsten f i l a m e n t l i g h t source (combined w i t h a red a c e tate f i l t e r ) promoted shoot p r o l i f e r a t i o n r a t e s at 0.25 mg/1 BA s i m i l a r t o those a t 0.5 mg/1 BA under white l i g h t p r ovided by f l u o r e s c e n t l i g h t . The q u a l i t y of shoots under red/FR l i g h t , as measured by shoot l e n g t h , was higher than under white l i g h t . Blue l i g h t , from a f i l t e r e d metal h a l i d e l i g h t source, produced fewer shoots than under white l i g h t . Intermediate wavelengths (green, y e l l o w , and orange) between blue and red wavelengths produced s i m i l a r p r o l i f e r a t i o n r a t e s which were not g r e a t e r than those produced under white l i g h t . For o p t i m i z a t i o n of the red/FR l i g h t e f f e c t at 0.25 and 0.4 mg/1 BA, a p r o t o c o l i n v o l v i n g 4 weeks of low photon f l u e n c e red/FR (8.7 - 15.9 u mol photons m s ) and t r a n s f e r t o a h i g h e r photon f l u e n c e of white l i g h t (47.0 - 62.0 u mol photons m^s*) f o r 1 f u r t h e r week i s r e q u i r e d . For Spiraea the e f f e c t of the i n i t i a l 4 week, low photon f l u e n c e exposure t o red/FR l i g h t was more c r i t i c a l on shoot p r o l i f e r a t i o n than on shoot e l o n g a t i o n . A l s o , i t i s important t o note t h a t the red/FR l i g h t was a p p l i e d c o n t i n u o u s l y over a 16 hour photoperiod. Two hours of red/FR a p p l i e d at the end of a 16 hour white photoperiod was i n s u f f i c e n t t o cause any s i g n i f i c a n t change from white l i g h t . 132 The use of glyphosate, which has been shown t o promote IAA o x i d a t i o n l e a d i n g t o a r e d u c t i o n i n a p i c a l dominance (Lee, 1984) , has the p o t e n t i a l f o r o p t i m i z i n g shoot p r o l i f e r a t i o n . The e f f e c t of glyphosate on the red/FR l i g h t response was a s i g n i f i c a n t i n c r e a s e i n shoot p r o l i f e r a t i o n w i t h a 0.087 mg/1 glyphosate-0.5 mg/1 BA treatment. The white l i g h t , 0.087 mg/1 glyphosate-0.5 mg/1 BA treatment a l s o produced an increase i n shoot p r o l i f e r a t i o n . In a d d i t i o n , the glyphosate treatments w i t h white l i g h t i n c r e a s e d shoot p r o l i f e r a t i o n at a higher glyphosate l e v e l of 0.267 mg/1 at both the 0.25 and 0.5 mg/1 BA c o n c e n t r a t i o n s , w h i l e under red/FR t h i s h i g h e r glyphosate l e v e l produced l i t t l e or no change. With j u d i c i o u s manipulation of glyphosate and BA l e v e l s , any p o s s i b l e e f f e c t s on shoot morphology should be minimized and should a l l o w glyphosate t o be used as a growth r e g u l a t o r i n i n v i t r o c u l t u r e . 133 B i b l i o g r a p h y 1. Anderson, W.C. 1975. Propagation of rhododendrons by t i s s u e c u l t u r e : P a r t 1. Development of a c u l t u r e medium f o r m u l t i p l i c a t i o n of shoots. The I n t e r n a t i o n a l Plant Propagators' S o c i e t y . Combined Proceedings. 25:129-135. 2. B a r a l d i , R., F. R o s s i , and B. L e r c a r i . 1988. 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