Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1990_A6_7 H47.pdf [ 7.34MB ]
Metadata
JSON: 831-1.0302364.json
JSON-LD: 831-1.0302364-ld.json
RDF/XML (Pretty): 831-1.0302364-rdf.xml
RDF/JSON: 831-1.0302364-rdf.json
Turtle: 831-1.0302364-turtle.txt
N-Triples: 831-1.0302364-rdf-ntriples.txt
Original Record: 831-1.0302364-source.json
Full Text
831-1.0302364-fulltext.txt
Citation
831-1.0302364.ris

Full Text

LIGHT QUALITY EFFECTS ON IN VITRO SHOOT PROLIFERATION OF SPIRAEA NIPPONICA By Edward J o h n B.Sc.  (Botany),  University  Herrington of  B r i t i s h Columbia,  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES PLANT SCIENCE We a c c e p t t h i s to  the  t h e s i s as  required  conforming  standard  THE UNIVERSITY OF BRITISH COLUMBIA September (c)  1990  Edward J o h n H e r r i n g t o n , 1990  1983  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)  ii  Abstract  The  work on  determine  the  Spiraea i n v i t r o  feasibility  of  s h o o t c u l t u r e s was  using l i g h t  quality  done t o  to  modify  endogenous phytohormone b a l a n c e s t o d e c r e a s e a p i c a l dominance. Such an e f f e c t would e n a b l e a r e d u c t i o n i n t h e h i g h l e v e l s o f exogenous c y t o k i n i n b e n z y l a d e n i n e (BA) a p p l i e d thus reduce p o t e n t i a l The  Spiraea  characterized wavelengths  by  on  i n culture  and  response  was  side-effects.  in  vitro  examining  growth.  light the  quality  effects  of  A m i x t u r e o f red/FR  s h o o t p r o l i f e r a t i o n w i t h 0.25 mg/1  different  light  induced rates  of  BA t h a t were as h i g h as r a t e s  o b t a i n e d under w h i t e l i g h t w i t h 0.5 mg/1  BA. Shoot q u a l i t y ,  as  d e t e r m i n e d by t h e p r o p o r t i o n o f s h o o t s 1 cm o r l o n g e r ( u s e f u l shoots),  was  h i g h e s t under  proliferation wavelengths  r a t e was  red/FR  light.  o b s e r v e d under  The  lowest  blue l i g h t .  i n t e r m e d i a t e between b l u e and  red  shoot  When  light  (green,  y e l l o w , and orange) were a p p l i e d t o e x p l a n t s o n l y minor modifications initiation  occurred.  but  inhibited  Green  light  did  not  growth  inhibit  s h o o t e l o n g a t i o n a t t h e 0.5  light  shoot  mg/1  BA  level. The e f f i c a c y o f t h e 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 i n t h e first the  e x p e r i m e n t was  three l i g h t  s t u d i e d i n two  sources  f u r t h e r experiments. With  (tungsten filament,  fluorescent,  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 putative blue l i g h t light  quality  inhibitory  effect  suggested  e x p e r i m e n t . Under t h e r e d f i l t e r ,  i n the  first  the tungsten  f i l a m e n t s o u r c e i n d u c e d t h e h i g h e s t s h o o t number means a t b o t h BA l e v e l s used Two  (0.25 and 0.5  factors  may  have  mg/1). contributed  to  the  red/FR  effect  o b s e r v e d i n t h e f i r s t e x p e r i m e n t ; t h e t i m e under an i n c u b a t i o n l i g h t regime b e f o r e t r a n s f e r t o t h e t r e a t m e n t regime, and photon f l u e n c e r a t e o f each regime. I n t h e subsequent examine t h e s e f a c t o r s , l o w e r BA  l e v e l s o f 0.25  shoot and  initiation 0.4  mg/1  was  the  study t o  optimized at the  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 w h i t e l i g h t o f a h i g h e r f l u e n c e f o r one more week. G l y p h o s a t e , a known promoter o f IAA o x i d a t i o n , was used t o investigate  the  interactions.  presumed  Two  effect  of  types of responses  lowered  IAA-cytokinin  to glyphosate occurred,  each one dependent on t h e g l y p h o s a t e c o n c e n t r a t i o n . A t t h e l o w e r g l y p h o s a t e l e v e l (0.087 mg/1), c u l t u r e s under b o t h l i g h t regimes with  0.25  mg/1  of  BA,  showed  a  strong i n h i b i t i o n  of  shoot  i n i t i a t i o n . T h i s 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  o f BA and an o v e r a l l s t i m u l a t o r y r e s p o n s e o c c u r r e d as  s h o o t 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 h i g h e r t h a n i n t h e p r e v i o u s e x p e r i m e n t s . F o r b o t h BA l e v e l s , changes i n shoot number were g r e a t e r under w h i t e l i g h t t h a n under red/FR. A t t h e h i g h e r g l y p h o s a t e l e v e l (0.2 67 mg/1), t h e shoot i n i t i a t i o n r a t e s were g r e a t e r t h a n 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 b o t h BA  levels  under w h i t e l i g h t a l t h o u g h under red/FR t h e 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 g l y p h o s a t e 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 t h e l e v e l s o f t h e 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 w h i c h depend  on  t h e s p e c i f i c c o n c e n t r a t i o n s o f each component.  Table of Contents Abstract  i i  L i s t of Tables  v i i  L i s t of Figures  xi  Acknowledgement  xii  1 Introduction  1  2 L i t e r a t u r e Review 2.1 The c o n t r o l o f i n v i t r o factor of light  9 p l a n t growth by t h e e n v i r o n m e n t a l 9  2.2 P h y t o h o r m o n e s a s u s e d f o r i n v i t r o p l a n t c u l t u r e  2.3  2.2.1  Auxins  15'  2.2.2  Cytokinins  17  2.2.3  Cytokinin  side-effects  Glyphosate as a growth r e g u l a t o r plant  18 f o ri n v i t r o  culture  19  3 M a t e r i a l s and Methods 3.1  15  Culture  22  medium a n d 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 3.3 P h o t o n f l u e n c e  24 rates  26  3.4 F i l t e r s 3.5 E x p l a n t  28 preparation  3.6 H a r v e s t i n g  and i n c u b a t i o n c o n d i t i o n s  procedure  3.7 E x p e r i m e n t a l d e s i g n  28 34  f o r each experiment  3.8 G r o w t h v a r i a t e s m e a s u r e d a n d s t a t i s t i c a l 4 R e s u l t s and D i s c u s s i o n 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 i n v i t r o l i g h t q u a l i t y response  34 analyses  ... 37 38  nipponica 38  vi  4.1.1  Experiment  I - Determining the e f f e c t s of blue, r e d / F R , 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  Experiment  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 of t h r e e i n t e r m e d i a t e wavelength r a n g e s b e t w e e n b l u e and r e d wavelengths 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 of 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 combinations  4.1.4  4.1.5  4.2  Experiment  Experiment  I V - 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 of t h r e e l i g h t source-red f i l t e r combinations V  Factors contributing response 4.2.1  59  68  Determining the e f f e c t of a short-term f a r - r e d l i g h t exposure 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 photoperiod 78 t o the red/FR l i g h t  growth  ,  87  Experiment VI The e f f e c t s o f l e n g t h o f e x p o s u r e t o a l o w p h o t o n 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 r e d / F R l i g h t g r o w t h r e s p o n s e 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 t o modify i n v i t r o growth 4.3.1  Experiment VII - G l y p h o s a t e - l i g h t experiment  interact  98  quality 98  114  5 G e n e r a l D i s c u s s i o n and C o n c l u s i o n s 5.1  C h a r a c t e r i z a t i o n of the S p i r a e a n i p p o n i c a i n v i t r o q u a l i t y response  5.2  Factors contributing response  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 modify i n v i t r o growth  t o the red/FR l i g h t  light 114  growth  quality  interact  124 to 127  Summary  131  Bibliography  133  V I I  List  3.1  Spectral distribution of  the  radiation  of  Tables  of the  energy  of tungsten  i n the  visible  f i l a m e n t , metal  region  halide,  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  Incubation conditions for stock cultures  32  4.1  Red  light:  ratios  far-red  light  for unfiltered  and  blue  tungsten  light:  red  light  f i l a m e n t , metal  halide,  and f l u o r e s c e n t l i g h t s o u r c e s 4.2  Shoot  and  40  node number means u n d e r b l u e ,  red/FR,  and  white  l i g h t , w i t h and w i t h o u t BA 4.3  Summarized red/FR,  analyses  and  white  of variance.  light,  and node numbers, and 4.4  Shoot  lengths  as  42  and  BA  Effects  of  blue,  c o n c e n t r a t i o n on  shoot  f r e s h and d r y w e i g h t s  affected  by  BA  and  three  44 light  q u a l i t i e s : b l u e , r e d / F R , and w h i t e 4.5  Fresh  and  47  d r y w e i g h t means u n d e r b l u e ,  red/FR,  and  white  l i g h t , w i t h and w i t h o u t BA 4.6  Shoot  and  49  node number means u n d e r t h r e e  wavelengths  (green,  yellow,  and  intermediate  orange) between b l u e  and  red l i g h t 4.7  . 52  Summarized qualities orange) and  analyses  of variance.  between b l u e  and  BA  and  red  c o n c e n t r a t i o n on  f r e s h and d r y w e i g h t s  Effects  light shoot  of three  (green, and  light  yellow,  node  and  numbers, 54  « • •  V  4-8  Shoot  lengths  as a f f e c t e d  by BA and t h r e e  b e t w e e n b l u e and r e d : g r e e n , 4.9 F r e s h  red  (green,  yellow,  56  intermediate  and o r a n g e ) b e t w e e n b l u e and  light  4.10 S h o o t  57  and node number means u n d e r t h r e e  (tungsten  f i l a m e n t , metal  halide,  light  sources  and f l u o r e s c e n t )  combined w i t h a b l u e a c e t a t e f i l t e r 4.11 Summarized  analyses  source-blue shoot 4.12 S h o o t  filter  of variance. combination  60 Effects  of l i g h t  and BA c o n c e n t r a t i o n on  and node numbers, and f r e s h and d r y w e i g h t lengths  as a f f e c t e d  by t h r e e  light  62  source-blue  f i l t e r combinations 4.13 F r e s h  65  and d r y w e i g h t means u n d e r t h r e e  (tungsten  f i l a m e n t , metal  halide,  light  sources  and f l u o r e s c e n t )  combined w i t h a b l u e a c e t a t e f i l t e r 4.14 S h o o t  66  and node number means u n d e r t h r e e  (tungsten  f i l a m e n t , metal  halide,  light  sources  and f l u o r e s c e n t  combined w i t h a r e d a c e t a t e f i l t e r 4.15 Summarized source-red shoot 4.16 S h o o t  analyses filter  of variance.  combination  69 Effects  of light  and BA c o n c e n t r a t i o n on  and node numbers, and f r e s h and d r y w e i g h t s lengths  as a f f e c t e d  by t h r e e  light  74.  and d r y w e i g h t means u n d e r t h r e e  (tungsten  f i l a m e n t , metal  71  source-red  f i l t e r combinations 4.17 F r e s h  n  qualities  y e l l o w , and o r a n g e  and d r y w e i g h t means u n d e r t h r e e  wavelengths  light  1  halide,  combined w i t h a r e d a c e t a t e f i l t e r  light  sources  and f l u o r e s c e n t ) 77  4.18 S h o o t  a n d node number means a s a f f e c t e d  far-red  light  exposure a p p l i e d  by a  short-term  a t t h e end o f a w h i t e  l i g h t photoperiod 4.19 Summarized far-red light  analyses  light  81 of variance.  exposure a p p l i e d  Effects  of a  short-term  a t t h e end o f a w h i t e  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 s h o o t and node  numbers, a n d f r e s h and d r y w e i g h t s 4.20 S h o o t  lengths  as a f f e c t e d  83  by a s h o r t - t e r m  far-red  light  e x p o s u r e 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 . 8 5 4.21 F r e s h  a n d d r y w e i g h t means a s a f f e c t e d  far-red  light  exposure a p p l i e d  by a s h o r t - t e r m  a t t h e end o f a w h i t e  l i g h t photoperiod 4.22 S h o o t  86  a n d node number means a s a f f e c t e d  low p h o t o n  fluence  rate  by e x p o s u r e t o a  and t h e t i m e o f t r a n s f e r  to a  higher fluence rate 4.23  Summarized a  analyses  low photon  fluence,  89 of variance.  fluence rate,  Effects  transfer  a n d BA c o n c e n t r a t i o n  o f exposure t o  time t o a higher  on s h o o t a n d node  numbers,  and f r e s h and d r y w e i g h t s 4.24 S h o o t s  lengths  92  as a f f e c t e d  by a l o w p h o t o n  fluence  rate  and 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 e r a t e 4.25 F r e s h  a n d d r y w e i g h t means a s a f f e c t e d  low p h o t o n  fluence  rate  94  by e x p o s u r e t o a  and t h e t i m e o f t r a n s f e r  to a  higher fluence rate 4.2 6 P r o p o r t i o n  o f each t r e a t m e n t ' s f o u r  glyphosate effects  96 samples  showing 102  X  4.27  Shoot  and node number means as a f f e c t e d  by g l y p h o s a t e and  light quality  104  4.27a Node n u m b e r / s h o o t means 4.28 A n a l y s e s  o f v a r i a n c e summary.  glyphosate,  Effects  l e n g t h s as a f f e c t e d  and node 108  by BA, g l y p h o s a t e ,  a n d d r y w e i g h t means a s a f f e c t e d  light quality  quality,  and d r y w e i g h t s  quality 4.30 F r e s h  of light  and BA c o n c e n t r a t i o n on s h o o t  numbers, and f r e s h 4.29 S h o o t  107  and  light I l l  b y g l y p h o s a t e and 112  L i s t of F i g u r e s 3.1 3.2  Transmission characteristics used i n Experiment I  of the acetate  Transmission characteristics used i n Experiment I I  of the acetate  filters 29 filters 30  3.3  S p e c t r a l energy d i s t r i b u t i o n of tungsten 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  4.1  The e f f e c t o f 3.56 proliferation  \  mg/1  of glyphosate  on S p i r a e a  32  shoot 100  xii Acknowledgement  I would l i k e t o thank Dr. C o l i n Norton f o r p l a n t i n g t h e s e e d o f t h i s t h e s i s . A l s o , Dr. George E a t o n ' 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 t h a n k y o u g o e s t o Dr.  J o a n M c P h e r s o n . H e r k n a c k f o r p e r c e i v i n g p a t t e r n s i n my  data,  tying  loose threads,  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 w o r k s . C o m m i t t e e members, D r . Jolliffe,  Dr. V i d a v e r ,  Dr. E l l i s ,  g r a t e f u l l y acknowledged f o r t h e i r I  and Dr. I . E . P . T a y l o r a r e input.  g i v e warm k u d o s 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 . O u r  c a m a r a d e r i e was a g r a d  school b e n e f i t I had n o t a n t i c i p a t e d .  Finally,  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  support.  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 w o u l d l o n g ago  have w i t h e r e d  on t h e v i n e .  unwavering  1  Chapter 1  Introduction  T r a d i t i o n a l vegetative propagation techniques involve using such m a t e r i a l a s stem and l e a f c u t t i n g s . These c a n be viewed as 'macropropagation vitro  or  1  t e c h n i q u e s (George and S h e r r i n g t o n , 1984). I n  tissue  culture  propagation  techniques  involve  r e l a t i v e l y s m a l l p l a n t p i e c e s termed e x p l a n t s . Such e x p l a n t s c a n be used t o i s o l a t e p r o t o p l a s t s , i n d u c e d t o form c a l l u s , o r used as a d i r e c t s o u r c e f o r shoot p r o l i f e r a t i o n . T h i s l a s t where  small  shoots  v e g e t a t i v e meristems"  a r e made  to arise  from  option,  "pre-existing  ( H u t c h i n s o n and Zimmerman, 1987), i s t h e  most common meaning f o r t h e term ' m i c r o p r o p a g a t i o n and i s o f t e n 1  used  to distinguish  this  method  from  other t i s s u e  culture  methods such as s o m a t i c embryogenesis. In  vitro  traditional  culture  methods  offer  p r o p a g a t i o n methods.  several  These  advantages  include  over  a means t o  e l i m i n a t e d i s e a s e - c a u s i n g v i r u s e s , b a c t e r i a and f u n g i v i a t h e a s e p t i c t e c h n i q u e s used, t h e 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 varieties  and c u l t i v a r s  f o r commercial  sales,  and a way t o  p r o p a g a t e p l a n t s r e c a l c i t r a n t t o o t h e r methods o f p r o p a g a t i o n (George and S h e r r i n g t o n , 19 84). F u r t h e r m o r e , t r u e - t o - f o r m c l o n e s  2 can be m a i n t a i n e d  ( e s p e c i a l l y v i a t h e a x i l l a r y - d e r i v e d shoots o f  micropropagation)  (George and S h e r r i n g t o n , 1984).  The c o m m e r c i a l p o t e n t i a l o f p r o p a g a t i n g p l a n t s by i n v i t r o methods i s good. A l t h o u g h l a b o u r c o s t s a r e h i g h , t h e s m a l l space requirements the  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  year-round  capital figures  production  expenditures  capability  (George  f o r the United  States  and  can o f f s e t h i g h Sherrington).  i n 1985 i n d i c a t e d  initial  Production a  wholesale  market v a l u e o f 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. production t o t a l , as  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  woody o r n a m e n t a l s and f r u i t  study  (Pierik,  1985)  crops  concluded  (Jones,  that  roses  1986). A and  Dutch  landscape  o r n a m e n t a l s would be t h e p l a n t t y p e s showing t h e g r e a t e s t f u t u r e market s h a r e . T h i s i s p a r t i a l l y due t o an i n c r e a s i n g c a p a b i l i t y to  grow woody o r n a m e n t a l s  difficult  in vitro,  t o p r o p a g a t e and h a v i n g  plants previously often  slow growth r a t e s . W i t h t h e  good p r o s p e c t s f o r woody ornamental m i c r o p r o p a g a t i o n , methods  to  improve  micropropagation plant  chosen  the  efficiency  of  developing  woody  plant  may enable p r o d u c t i o n c o s t s t o be lowered.  f o r the present  study  i s t h e woody  The  ornamental  Spiraea nipponica. S p i r a e a n i p p o n i c a i s a member o f a genus whose s p e c i e s a r e widely praised f o r t h e i r  f l o w e r s and f i n e shrub form  (Hudak,  1984) . The genus b e l o n g s t o t h e f a m i l y Rosaceae w h i c h i n c l u d e s many g e n e r a o f o r n a m e n t a l and f r u i t - y i e l d i n g p l a n t s  currently  grown i n v i t r o (George  and  such as  the  Sherrington,  improve S p i r a e a  1984).  in vitro  a p p l i c a b l e t o the  other  rose,  apple, If  plum, and  successful  c u l t u r e are  techniques  developed,  more e c o n o m i c a l l y  strawberry  t h e y may  important  to be  rosaceous  plants. In  vitro  plant  growth  can  be  regulated  by  artificially  imposed c o n d i t i o n s . Those f a c t o r s w h i c h c o n t r o l growth such as e n v i r o n m e n t and c u l t u r e medium c o n s t i t u e n t s a r e e a s i l y Environmental  factors  atmosphere. C u l t u r e organic  and  temperature,  medium c o n s t i t u e n t s  components.  micronutrients  include  The  inorganic  purpose  of  micropropagation phytohormone  the  shoot  metabolism  in  the  and  inorganic  and  The  are  organic  the  components  phytohormones.  current culture  light,  components  the macronutrients.  i n c l u d e v i t a m i n s , c a r b o h y d r a t e s and The  include  modified.  thesis  is  techniques shoot  to  improve  by  explant.  modifying Auxins  and  c y t o k i n i n s a r e t h e two t y p e s 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 t o b a c c o (Skoog and  Miller,  cytokinin to  auxin  1957)  indicated that varying  concentrations  i n the  the  callus  ratio  growth medium  of  could  d e t e r m i n e t h e c o u r s e o f o r g a n o g e n e s i s . T a k i n g t h e t o b a c c o work i n t o account, the has  depended  on  o p t i m i z a t i o n of m i c r o p r o p a g a t i o n determining  appropriate  cytokinin  production to  r a t i o s . T y p i c a l l y , a range of a u x i n s and c y t o k i n i n s and concentrations pioneering  auxin various  of a u x i n - c y t o k i n i n p a i r s a r e t e s t e d , such as  work on t h e  the  i n v i t r o shoot c u l t u r e o f 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 w e r e 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 o m i n a t e s u s u a l l y  results in  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 e x a m p l e , w o r k on l o w b u s h indicated shoot  that  number  decreased  the  as  the  cytokinin  increased, shoot  while  number  concentration  increased  (Frett  and  blueberry  increased  auxin  the  concentrations  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 h o r m o n a l "correlation  exists  between  a s p e c t s o f development" The  c o n t r o l of p l a n t development hormone  levels  (Barendse e t a l . ,  mechanism o f a p i c a l  and  that  a  quantitative  1987).  dominance i s g e n e r a l l y  assumed  to  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 f r o m a s h o o t ' s g r o w i n g t i p down the  shoot, the auxin e i t h e r  axillary  bud growth  transport,  axillary  experiment  growth the  buds  grow, a s  can  triiodobenzoic  was  demonstrated  acid  ( T u c k e r , 1 9 7 8 ) . One  (TIBA),  et  a l . , 1974) . I n t h e  case  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  been  supplement  cytokinin  to  Sherrington, Until  current  work,  the  overcome  growth the  medium  growth  an  auxin  with  of  shoot  approach  high  suppression  within  levels  (George  has of and  1984).  recently,  concentration  an  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  (Lee  by  e f f e c t of auxin-induced  cultures to  inhibiting  1984). W i t h a r e d u c t i o n i n a u x i n  i n h i b i t i o n may  s u p p r e s s e d bud  or i n d i r e c t l y  (Hillman,  involving  transport inhibitor  directly  attempts  to  modify  the  endogenous  i n t h e s h o o t e x p l a n t . h a v e b e e n few. factors  which  may  specifically  auxin  Thus i n t h e  affect  a  shoot  5  explant's  endogenous a u x i n metabolism  underlying  purpose i n a l t e r i n g endogenous a u x i n metabolism i s t o  p r o v i d e a way o f r e d u c i n g high  have been  studied.  The  a p i c a l dominance without t h e use o f  exogenous l e v e l s o f c y t o k i n i n s . I f t h e amount o f exogenous  c y t o k i n i n needed t o e l i c i t l a t e r a l bud r e l e a s e can be decreased, it  i s hoped  cytokinin and  that  known  l e v e l s such as v i t r i f i c a t i o n  r e s i d u a l bushiness  reduction putative often  effects  of  of cytokinin  stomata  (cytokinins  (Jewer and I n c o l l ,  high  (Leshem e t a l . , 1988),  may  help  Also,  elucidate the  r o l e of cytokinins i n contributing to i n v i t r o  opening  this  side  ( P i e r i k , 1982) may be decreased.  i n the l e v e l  open  The  deleterious  a r e known t o promote  plants' stomatal  1980) ) .  approach taken t o modify endogenous a u x i n metabolism i n  study  has been  light; specifically,  t o manipulate  an environmental  factor,  l i g h t q u a l i t y . The use o f l i g h t q u a l i t y t o  produce s p e c i f i c d e s i r e d e f f e c t s without h a v i n g t o i n c r e a s e t h e exogenous c y t o k i n i n l e v e l s has not p r e v i o u s l y been a p p l i e d t o i n v i t r o p l a n t c u l t u r e . S p i r a e a was chosen as a t e s t p l a n t because of  i t s growth,  plants  i n nature,  are l i k e l y  i n exposed  sunny  locations.  Such  t o possess a shade-avoidance response which  i n v o l v e s phytochrome e q u i l i b r i a (Smith, 1982). These p l a n t s when even s l i g h t l y shaded, d e t e c t t h e change i n l i g h t c o n d i t i o n s v i a a phytochrome sensor and respond by i n c r e a s i n g t h e r a t e o f stem extension light  growth t o r e g a i n  regime. The p o s s e s s i o n  a p o s i t i o n under a more  favourable  o f a developed phytochrome system  f o r i n i n v i t r o p l a n t may allow  f o r s i g n i f i c a n t r e d and f a r - r e d  6 light  modification  especially seemed auxin  of  of  newly  initiated  possible that levels  growth.  known  w o u l d be  While  shoots,  red  and  extension  growth,  i s desirable, i t also  far-red light  b e t t e r expressed  using  effects  Spiraea.  Red  on and  f a r - r e d l i g h t h a s b e e n shown t o r e d u c e a u x i n l e v e l s i n e x t e n d i n g shoots to  ( l i n o , 1 9 8 2 ) ; i n some c a s e s  r e d l i g h t exposures have l e d  a l e s s e n i n g o f a p i c a l dominance w i t h subsequent shoot growth  from l a t e r a l buds (Kasperbauer,  1971;  Healy  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 in  vitro  (Lane,  1978), does not r e q u i r e exogenous a u x i n ;  thus,  e f f e c t s on e n d o g e n o u s a u x i n l e v e l s w o u l d 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 the S p i r a e a The  Spiraea study  section  is a  quality  response.  modifiers violet growth  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  c h a r a c t e r i z a t i o n of  of  system.  Red  light  phytochrome,  and while  the  Spiraea  in vitro  far-red light blue  first  are  light,  the  via  light primary  blue-ultra-  (U.V.) l i g h t a b s o r b i n g p i g m e n t s , c a n a l s o a f f e c t i n v i t r o (George  and  Sherrington,  i n d i c a t e d w h i c h o f r e d and  1984).  The  first  experiment  f a r - r e d l i g h t and b l u e l i g h t was  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  more light  q u a l i t i e s i n t e r m e d i a t e b e t w e e n b l u e and r e d l i g h t w e r e t e s t e d t o determine modify filters  i f minor  growth. were  combinations  changes  Light  source  used  to  in  combinations  verify  used i n the f i r s t  phytochrome  that  equilibria  with light  blue  and  could red  source-filter  e x p e r i m e n t w e r e o p t i m a l and  also  t o s t u d y t h e e f f e c t s on g r o w t h , 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 b l u e l i g h t . F i n a l l y , t h e l e n g t h o f exposure needed f o r l i g h t of  each q u a l i t y t o cause a growth r e s p o n s e was examined.  e f f e c t on growth o f a s h o r t end-of-day  light quality  The  exposure  was compared t o t h e e f f e c t produced by a f u l l p h o t o p e r i o d o f 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 t h e e f f e c t on growth o f the  sequence  o f exposure  to light  quality.  The f i r s t  light  q u a l i t y e x p e r i m e n t i n v o l v e d t h e t r a n s f e r o f e x p l a n t s from a two week w h i t e l i g h t regime t o t h e s p e c i f i c l i g h t q u a l i t y regimes o f a much l o w e r photon f l u e n c e r a t e f o r a n o t h e r t h r e e weeks. T h i s identified  two p r o b l e m s .  First,  t h e r e was a need t o r e s o l v e  whether o r n o t any e f f e c t was caused by d i f f e r e n t f l u e n c e r a t e s ; t h u s , t h e e f f e c t on growth o f an i n i t i a l low photon f l u e n c e r a t e was examined.  The o t h e r problem  concerned  t h e time a t w h i c h  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 s t u d y on a d v e n t i t i o u s bud f o r m a t i o n from Douglas  f i r callus  indicated  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 r e d l i g h t d u r i n g t h e t h i r d , fourth,  and f i f t h  week o f growth  (Kadkade  and Jopson,  1978) .  T h i s s u g g e s t e d t h e p o s s i b i l i t y t h a t t h e age o f S p i r a e a c u l t u r e s when t r a n s f e r r e d  to different  light  quality  regimes might  be  i m p o r t a n t 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 o f growth. T r a n s f e r o f t h e e x p l a n t s from an i n i t i a l low photon f l u e n c e r a t e t o a h i g h e r r a t e , and t r a n s f e r a t s p e c i f i c i n t e r v a l s / was t h u s s t u d i e d i n an a t t e m p t t o u n d e r s t a n d t h e e x t e n t t o which t h e s e two f a c t o r s a f f e c t e d t h e o b s e r v e d l i g h t q u a l i t y response i n t h e 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 the  s e c t i o n d e a l s w i t h a s t u d y done t o d e t e r m i n e i f  a c t i o n o f a known m o d i f i e r o f a u x i n m e t a b o l i s m c o u l d  optimized  by  interacting  e f f e c t i v e i n the f i r s t decrease  auxin  with  the l i g h t  quality  found  most  s e c t i o n . The compound, g l y p h o s a t e ,  levels  (Lee,  1982).  The  precise  be  can  mode  of  g l y p h o s a t e s a c t i o n i s n o t c l e a r ; one i d e a i s t h a t by d i s r u p t i n g 1  phenol b i o s y n t h e s i s there compounds  which  inhibit  a u x i n o x i d a t i o n can occur  i s a decrease i n s p e c i f i c  an a u x i n  oxidase  and t h u s  also provide  a  greater  (Lee, 1982), a n o t h e r e f f e c t may be t o  i n h i b i t a u x i n b i o s y n t h e s i s (Kudoyarova e t a l . , 1988). may  phenolic  Glyphosate  an i n d i r e c t means o f c o n f i r m i n g t h a t  light  q u a l i t y i s m o d i f y i n g a u x i n m e t a b o l i s m ; f o r example, i f r e d and f a r - r e d l i g h t do d e c r e a s e a u x i n l e v e l s a g l y p h o s a t e enhancement o f t h e e f f e c t may y i e l d a s h o o t 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 o f a higher c y t o k i n i n t o auxin  ratio.  9 Chapter 2  L i t e r a t u r e Review  2.1  The  c o n t r o l of i n v i t r o p l a n t  f a c t o r of  The  density  effects  of l i g h t  characteristics:  (irradiance  exposure  the  environmental  light  diverse  three l i g h t  growth by  or  (photoperiod)  on p l a n t  wavelength  intensity),  and  from  quality),  flux  duration  Sherrington,  of  light The  e f f e c t s o f l i g h t on i n v i t r o p l a n t growth and development  are  by  and  (light  arise  1984).  influenced  (George  growth  components o f t h e growth  medium. F o r  example,  p h o t o s y n t h e s i s i s o n l y a minor d e t e r m i n a n t o f i n v i t r o because  of  its  inhibition  by  the  exogenously  growth  supplied  c a r b o h y d r a t e s o u r c e ( u s u a l l y s u c r o s e ) i n t h e growth medium.  The  s u c r o s e supplement r e s u l t s i n t h e r e d u c t i o n o f b o t h 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 c a r b o n f i x a t i o n ; t h e p h o t o s y n t h e s i s t h a t o c c u r s i s m i n i m a l (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 t o growth from p h o t o s y n t h e s i s 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 . low i n t e n s i t i e s  Under  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 greater  than  a t higher  light  i n t e n s i t i e s ( G r i m s t a d , 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 t h e p o t e n t i a l t o o b s e r v e s p e c i f i c photomorphogenic e f f e c t s which a r e dependent on l i g h t q u a l i t y . i n t o two g r o u p s :  These e f f e c t s can be c l a s s i f i e d  blue-U.V. a b s o r b i n g pigment-mediated  effects  and phytochrome-mediated e f f e c t s (George and S h e r r i n g t o n , 1984). S e 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 a r e most evident  on c a l l u s  growth  and on morphogenesis.  I n one s t u d y  ( S e i b e r t e t a l . , 1975), t o b a c c o c a l l u s was exposed t o e i t h e r near-U.V. l i g h t U.V.  (371 nm) o r b l u e l i g h t  (420 and 467 nm) . Near  l i g h t s t i m u l a t e d maximum growth and shoot f o r m a t i o n a t t h e  low i r r a d i a n c e o f 24 uW/cm* w h i l e f o r b l u e l i g h t t h e much h i g h e r -  i r r a d i a n c e o f 300 uW/cm  a t 420 nm and 600 uW/cm  a t 467 nm was  needed. The blue-U.V. s t i m u l a t i o n , however, was o f t e n l o s t when h i g h e r i r r a d i a n c e s o f b l u e o r near U.V. were used. F o r 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 w h i t e ,  b l u e , r e d o r f a r - r e d l i g h t , each a t about 1000 uW/cm  , b l u e 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 S u n d e r l a n d , 1972). R e p o r t s o f b l u e 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 o f r e s p o n s e s . P a r t o f t h e d i v e r s i t y can be a t t r i b u t e d to  different  responses,  plant  species  but d i f f e r e n t  and d i f f e r e n t medium  explant  formulations  and  tissue light  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 b l u e l i g h t e f f e c t . The f o l l o w i n g s u r v e y i s i n t e n d e d t o i l l u s t r a t e t h e range of responses noted. Juneberry  shoot t i p c u l t u r e s  supplied  with  an  exogenous  a u x i n and c y t o k i n i n and exposed t o w h i t e , b l u e , r e d , and green light,  produced  white l i g h t  t h e g r e a t e s t number o f s h o o t s under b l u e and  (Behrouz and L i n e b e r g e r , 1981). When no exogenous  hormones were  applied  none  of  the  light  qualities  improved  growth. W i t h a grape shoot c u l t u r e o n l y an exogenous c y t o k i n i n was  a p p l i e d ; t h e i n t e r a c t i o n o f l i g h t q u a l i t y w i t h two  medium m i c r o n u t r i e n t s ,  MnSOij.  growth  and K I , was s t u d i e d (Chee and P o o l ,  1985). B o t h m i c r o n u t r i e n t s a r e m o d i f i e r s o f a u x i n m e t a b o l i s m ; when t h e caused  l e v e l was d e c r e a s e d and KI e x c l u d e d , b l u e l i g h t  MnSOi^.  a greater  proposed  that  shoot p r o l i f e r a t i o n  the  blue  than red l i g h t . interaction  light-MnSOij-  r e d u c t i o n o f a p i c a l dominance.  It  caused  was a  S t u d i e s on a w i l l o w shoot c u l t u r e  system showed t h a t when b l u e l i g h t was a p p l i e d t o s h o o t s which had  their  (Letouze,  apex  removed,  axillary  1974). T h i s growth  c y t o k i n i n was  bud  growth  was  s u p p r e s s i o n was  a p p l i e d t o t h e uppermost  suppressed  overcome when a  bud.  I n t e r p r e t i n g the  b l u e l i g h t e f f e c t s on t h e J u n e b e r r y , grape, and w i l l o w c u l u r e s i s c o m p l i c a t e d by t h e f a c t t h a t d i f f e r e n t p h o t o p e r i o d s were used f o r each c u l t u r e . E x p l a n t t i s s u e as a p o s s i b l e f a c t o r d e t e r m i n i n g t h e degree of a l i g h t q u a l i t y r e s p o n s e i s shown by work on A f r i c a n V i o l e t leaf  explants  exogenous  (Lercari  phytohormones,  et  a l . , 1986). blue  light  On  a  induced  medium w i t h o u t a  higher  r e g e n e r a t i o n f r e q u e n c y t h a n e i t h e r r e d , f a r - r e d , o r U.V.  bud  light.  However, t h e f r e q u e n c y under d a r k n e s s was s i m i l a r t o t h a t under white  light  which  s u g g e s t s t h a t bud  i n d u c t i o n may  have been  12 d e t e r m i n e d b e f o r e t h e e x p l a n t was c u l t u r e d . light  quality effects,  i n this  Thus t h e e x t e n t o f  l e a f e x p l a n t system, may have  depended on f a c t o r s i n f l u e n c i n g t h e s t o c k 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 a r e m e d i a t e d via  the  photochromic  Sherrington, blue-U.V.  1984).  pigment  Unlike  phytochrome  the s t i l l  pigment, phytochrome  (George  nebulous  nature of the  has been c h a r a c t e r i z e d  d e t a i l . However, t h e s i g n a l t r a n s d u c t i o n  and  i n great  c h a i n l e a d i n g from t h e  l i g h t - i n d u c e d c o n f o r m a t i o n a l changes i n t h e phytochrome m o l e c u l e t o t h e o b s e r v e d p h y s i o l o g i c a l r e s p o n s e remains t o be B a s i c a l l y , phytochrome has  resolved.  e q u i l i b r a t e s between two forms; one form  i t s a b s o r p t i o n maximum i n t h e r e d r e g i o n  o f t h e spectrum  (about 660 nm) and t h e o t h e r i n t h e f a r - r e d r e g i o n  (about 73 0  nm). A s h i f t t o t h e f a r - r e d form c a n a l s o o c c u r under  conditions  of  prolonged darkness  and hence t h i s  form  i s implicated  in  photoperiodic plant responses. Classic  red  and  far-red  light-induced  physiological  r e s p o n s e s r e q u i r e b r i e f l o w energy exposures o f l i g h t . Red l i g h t l e a d s t o t h e p h y s i o l o g i c a l l y a c t i v e phytochrome is  a  subsequent  exposure  to far-red  light  form; when t h e r e t h e response i s  n e g a t e d . However, t h e r e i s a c l a s s o f responses w h i c h depends on a so-called  o f r e d and f a r - r e d l i g h t .  These  r e s p o n s e s a r e .induced by l o n g t e r m exposure t o r e d / f a r - r e d  light  and  are  "high i r r a d i a n c e "  often,  but  not  always,  fluence-rate  dependent  (Kronenberg and K e n d r i c k , 1986). The u s e o f r e d 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 c o n c e n t r a t e d on t h e low energy phytochrome c a l l u s growth  system.  Lettuce  and shoot f o r m a t i o n was improved by d a i l y  minute e x p o s u r e s t o r e d l i g h t  five  (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 , t h e 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 h i g h e r r a t e o f shoot bud f o r m a t i o n under r e d light  (Kadkade  receptive  e t 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  t o t h e 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 o f o n l y t e n seconds o f r e d l i g h t day; a r e s p o n s e a g a i n negated by f a r - r e d l i g h t Root red  (655 nm) p e r  ( B o n n e t t , 1972).  f o r m a t i o n on c u l t u r e d r o o t s o f s u n f l o w e r was enhanced by light Red  ( L e t o u z e and Beauchesne, light  interesting  have  observation that  where r e d l i g h t cytokinin  effects  been  detailed  has a r i s e n  has a p o s i t i v e  i n t h e medium  1969).  often  extensively;  i s that  influence, intensifies  an  i n cultures  t h e presence o f a the effect. 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 t h e d a r k , b u t when a c y t o k i n i n was added o r p e r i o d i c exposures  of red light  were g i v e n ,  some growth  was i n d u c e d  ( C l e l a n d , 1976). When b o t h a c y t o k i n i n was p r e s e n t and t h e r e d light  exposures  were  given,  the greatest  growth  response  o c c u r r e d . Red l i g h t o r a c y t o k i n i n was shown t o reduce dominance i n w i l l o w shoot c u l t u r e s on s o m a t i c embryogenesis  apical  ( L e t o u z e , 1974). I n a s t u d y  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 s u p p l y i n g a s o u r c e o f a d d i t i o n a l c y t o k i n i n t o the  medium c o u l d , a t l e a s t o v e r t h e s h o r t term, m a i n t a i n t h e  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 s t u d y o f 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 s o m a t i c embryogenesis showed t h a t  red light  embryogenic c a p a c i t y  could  also  a i d i n maintaining  ( M i c h l e r and L i n e b e r g e r ,  a  high  1987).  As w i l l be mentioned i n t h e s e c t i o n on a u x i n s , r e d l i g h t can decrease  auxin  levels  (lino,  1982).  l i g h t - c y t o k i n i n s y n e r g y may be s i m p l y a higher  cytokinin-auxin  Thus,  any a p p a r e n t r e d  due t o r e d l i g h t  causing  r a t i o w h i c h 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 c o n j e c t u r e d  that 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 r e d l i g h t  and c y t o k i n i n s p r o v i d e  a  p r o m i s i n g means o f m o d i f y i n g i n v i t r o growth. No work has been done t o e x p l o i t t h e i n t e r a c t i o n o f r e d l i g h t and c y t o k i n i n s i n o r d e r 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 o f exogenous c y t o k i n i n f o r c y t o k i n i n - d e p e n d e n t c u l t u r e s . The m i c r o p r o p a g a t i o n o f 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 e x o g e n o u s l y 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  t h a n b o t h an a u x i n  and a c y t o k i n i n .  Thus, i f r e d l i g h t e f f e c t s c a n be i n d u c e d i n such a system t h e benefits levels used,  o f n a t u r a l l y i n d u c e d c y t o k i n i n s and/or reduced  could which,  potentially prolonged  replace as w i l l  the high  be o u t l i n e d  deleterious red/far-red  l e v e l s o f exogenous  effects. light  i n a later The  exposures  responses t o those noted f o r s h o r t e r  cytokinins  section,  possibility to  elicit  red l i g h t  auxin  of  have using  similar  exposures ( i n  combination with a cytokinin) a l s o merits a t t e n t i o n . This i s f o r two  r e a s o n s : 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  filament  light  sources  and  s e c o n d l y , 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 g a i n e d t o h e l p elucidate  the poorly  irradiance"  understood  mechanism  used  the  "high  response.  2.2 Phytohormones used f o r i n v i t r o p l a n t  Auxins  of  culture  and c y t o k i n i n s a r e t h e phytohormones  f o r modifying  i n vitro  plant  growth  predominantly  and morphogenesis  (George and S h e r r i n g t o n , 1984) . These two phytohormones i n t e r a c t i n a v e r y complex way; e x o g e n o u s l y a p p l i e d n a t u r a l o r s y n t h e t i c forms  o f t h e two i n t e r a c t  with  t h e endogenous  phytohormone  c o u n t e r p a r t s o f t h e e x p l a n t i t s e l f . Each one, though, does have characteristic physiological activities.  2.2.1 Auxins  The a u x i n s  are defined  by t h e i r  ability  to conrol  g r o w t h and c e l l e l o n g a t i o n (George and S h e r r i n g t o n , 1984). most common n a t u r a l a u x i n i s 3 - i n d o l e - a c e t i c a c i d (IAA). for  maximal i n v i t r o  growth r e s p o n s e s ,  a u x i n such as 2 , 4 - d i c h l o r o p h e n o x y a c e t i c  chemical acid  cell The Often  analogues o f  (2,4-D) and  1-naphthyTacetic  acid  (NAA) a r e used  (George and S h e r r i n g t o n ,  1984). One o f t h e main u s e s o f a u x i n 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 h i g h l e v e l o f a u x i n i s used w i t h a much l o w e r l e v e l o f 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  level  of auxin  1984) . Embryogenesis  i s used  i s another  (George and S h e r r i n g t o n ,  process  controlled  by added  a u x i n s ; i t i s i n i t i a t e d by h i g h l e v e l s o f a u x i n b u t subsequent embryo development r e q u i r e s a l o w e r i n g level is  of the applied  (George and S h e r r i n g t o n , 1984). The most common  t o vary  the levels  o f a p p l i e d auxins  with  auxin  treatment  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 o f morphogenesis. The g e n e r a l r u l e i s t h a t w i t h a h i g h r a t i o o f c y t o k i n i n t o auxin, shoot p r 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 root 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 a u x i n has on an e x p l a n t depends on how i t i n t e r a c t s w i t h t h e e x p l a n t s endogenous a u x i n . 1  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 , t h e rate  of auxin  subsequent applied  uptake by t h e e x p l a n t  metabolism.  auxin  with  Furthermore,  and t h e p a t t e r n o f i t s the interaction  of the  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 c o n s i d e r e d . One n a t u r a l m o d i f i e r o f 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 b o t h  r e d and f a r - r e d l i g h t  can d e c r e a s e t h e  a v a i l a b i l i t y o f p h y s i o l o g i c a l l y a c t i v e endogenous IAA ( f r e e IAA) in coleoptiles  ( 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 r e d u c e f r e e IAA l e v e l s and t h u s a f f e c t t h e r a t i o o f  17 c y t o k i n i n t o a u x i n . F o r woody p l a n t s , o f t e n o n l y an exogenous c y t o k i n i n i s n e c e s s a r y t o cause s h o o t p r o l i f e r a t i o n , f o r a p p l e , " t h e s h o o t s p r o b a b l y produce s u f f i c i e n t auxin  f o r growth"  endogenous cytokinin  (Lane,  auxin l e v e l s levels  since  1978).  Thus,  may p e r m i t any  any  a lowering  complicating  s i n c e as endogenous  lowering  of  o f exogenous  interactions  with  exogenous a u x i n s c a n be m i n i m i z e d .  2.2.2  Cytokinins  Cytokinins are c h i e f l y stimulate  cell  division  characterized  (Skoog  et al.,  by t h e i r 1965).  Two  ability to naturally  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 a r e z e a t i n and N-(2-isopentyl)  adenine  (2-ip).  Two  synthetic  cytokinin  a n a l o g u e s commonly used a r e k i n e t i n and 6 - b e n z y l a d e n i n e The  synthetic  cytokinins  have  a  greater  activity  (BA) .  than t h e  n a t u r a l ones (George and S h e r r i n g t o n , 1984) . The  use  of  synthetic  cytokinins  made  possible  development o f e f f i c i e n t m i c r o p r o p a g a t i o n systems. t h e o b s e r v a t i o n s made c o n c e r n i n g t h e a b i l i t y  the  Exploiting  of cytokinins 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  cytokinins  i n shoot  t i p cultures  to  p r o l i f e r a t e m u l t i p l e s h o o t s . BA was f i r s t o b s e r v e d 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), b e f o r e  18 its  u s e t o enhance  (Pieniazek,  1968).  shoot  p r o l i f e r a t i o n of apple  Currently  BA i s t h e most common  used f o r t h e m i c r o p r o p a g a t i o n o f temperate species  shoot  tips  cytokinin  f r u i t and n u t t r e e  ( H u t c h i n s o n and Zimmerman, 1987), as w e l l as f o r many  woody o r n a m e n t a l shrubs and t r e e s .  2.2.3  Cytokinin  Side-effects  As b e n e f i c i a l as BA and o t h e r 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 c a n 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 o f vitrification  (Leshem  commonly d e f i n e d  e t a l . , 1988).  as "an abnormal  t i p s into translucent  Vitrification  development  of cultured  s t u n t e d s h o o t s and t h i c k e n e d ,  b r i t t l e l e a v e s w i t h a g l a s s y appearance"  i s most shoot  t u r g i d and  (Leshem e t a l . ,  1988).  The c o n d i t i o n has been noted i n b o t h herbaceous and woody p l a n t cultures;  i t i s thought  t o be a r e s p o n s e  t o an excess o f  c y t o k i n i n (Leshem e t 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  plants  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 plants t o a p o t t i n g medium and p r e p a r i n g  them f o r f r e e l i v i n g  conditions  ( H u t c h i n s o n and Zimmerman, 1987), a l s o may depend on exogenously supplied  c y t o k i n i n s . The main problem w i t h  acclimatization i s  19 t h e poor water' r e g u l a t i v e a b i l i t i e s o f ex v i t r o p l a n t s due t o dysfunctional malfunction  stomata  ( H u t c h i n s o n and Zimmerman,  may be due t o c a r r y - o v e r  effects  1987).  of cytokinins,  s i n c e c y t o k i n i n s a r e known t o promote s t o m a t a l o p e n i n g and  Incoll,  effect  1980).  Another  The  g e n e r a l phytohormone  (Jewer  carry-over  i s t h e m o d i f i c a t i o n o f t h e growth h a b i t o f p l a n t s f o r  months a f t e r t h e y have been removed from c u l t u r e ( P i e r i k e t 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 p r o m o t i n g l a t e r a l bud break, may cause unwanted b r a n c h i n g ( P i e r i k  e t a l . , 1982).  Finally,  s p e c i f i c t o woody o r n a m e n t a l shrubs and t r e e s , i s t h e tendency f o r some s h o o t c u l t u r e s t o produce heavy c a l l u s  on t h e shoot  bases due t o h i g h 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 s l o w i n g o f growth,  s i n c e t h e shoot i s no l o n g e r i n d i r e c t  c o n t a c t w i t h t h e medium.  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  Glyphosate herbicide  i s the  active  compound  of  the  commercial  'Roundup'. Work on t o b a c c o c a l l u s s u g g e s t s 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 g l y p h o s a t e - m e d i a t e d growth i n h i b i t i o n i s t h e g l y p h o s a t e e f f e c t o f i n c r e a s i n g IAA o x i d a t i o n The mechanism o f t h i s  (Lee, 1982).  i n c r e a s e d IAA o x i d a t i o n i s u n c l e a r . One  p r o p o s a l i s t h a t g l y p h o s a t e , by i n h i b i t i n g a r o m a t i c amino a c i d b i o s y n t h e s i s ( v i a t h e s h i k i m i c a c i d pathway), causes a d e c r e a s e  20 in  the  level  of  secondary a r o m a t i c  metabolites,  specifically  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 o x i d a s e , and t h u s a d e c r e a s e 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 h i g h e r IAA o x i d a t i o n .  An a l t e r n a t i v e t o i n c r e a s e d IAA o x i d a t i o n , o r 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 A  study  glyphosate  on  was  soybean  and  (Kuoyarova e t a l . , pea  seedlings  1988). indicated  that  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 o f t h e s e e d l i n g s were f e d w i t h r a d i o l a b e l l e d IAA. The g l y p h o s a t e  e f f e c t was  different for  each p l a n t s p e c i e s . T r e a t e d pea p l a n t s m e t a b o l i z e d  61% more of  t h e r a d i o l a b e l l e d IAA o v e r a 4 hour i n c u b a t i o n p e r i o d t h a n t h e c o n t r o l p l a n t s . For soybeans t h e i n c r e a s e i n IAA m e t a b o l i s m only  4.4%  for  treated  plants. .This  work  suggested  was that  g l y p h o s a t e might have t h e p o t e n t i a l t o i n c r e a s e shoot p r o d u c t i o n i n micropropagation culture,  systems. As a growth r e g u l a t o r f o r i n v i t r o  glyphosate  c o n c e n t r a t i o n s . A study  has  been  tested  on a x i l l a r y  at  s h o o t p r o l i f e r a t i o n of i n  v i t r o c r a n b e r r y node e x p l a n t s i n d i c a t e d g l y p h o s a t e ' s increase  s h o o t number, but  there  millimolar  a b i l i t y to  were unwanted s i d e - e f f e c t s ,  s u c h as b a s a l s w e l l i n g o f s h o o t s and deformed l e a v e s a l . , 1984). A n o t h e r s t u d y  i n v o l v e d b l u e b e r r y p l a n t s where t h e  s o u r c e p l a n t f o r shoot t i p e x p l a n t s was ( F r e t t and Smagula, 1981). The culture  produced  (Scorza e t  sprayed w i t h  glyphosate  s h o o t t i p e x p l a n t s when put  more a x i l l a r y - d e r i v e d  shoots  than  into  explants  taken  from  control  plants.  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 s o u r c e p l a n t s t h a n d i r e c t l y on i n vitro  p l a n t l e t s ; i t s p o t e n t i a l as  vitro  plant  c u l t u r e needs t o be  range o f m i c r o p r o p a g a t e d  species.  a growth  clarified  regulator  for in  by t e s t i n g a w i d e r  22 Chapter 3  M a t e r i a l s and  3.1  C u l t u r e Medium and  The  medium  (Linsmaier  used  and  i t s Preparation  was  Skoog,  Methods  developed  1965) ;  by  Linsmaier  i t is a  and  modification  Skoog of  the  M u r a s h i g e and Skoog medium (Murashige and Skoog, 19 62). I n t h i s modification  only  one  milligrams per l i t r e  vitamin  is  used,  thiamine  at  0.4  (mg/1); a l l o t h e r medium c o n s t i t u e n t s a r e  t h e same as i n t h e Murashige and Skoog medium. S t o c k s o l u t i o n s were made f o r t h e i n o r g a n i c s a l t s ; one f o r t h e m i c r o n u t r i e n t s , one f o r t h e major m a c r o n u t r i e n t s , macronutrient  and one f o r t h e i r o n and EDTA  components ( t h i s s o l u t i o n was k e p t 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 s t o c k s o l u t i o n was p r e p a r e d a t a 2 00X c o n c e n t r a t i o n ; t h e c o n s t i t u e n t s , i n mg/1  of  medium, were:  KI  H3BO3  0.83, Na^MoCf2HjjD major  6.2, MnS0^-4H^0  0.25, CuSO^SHjjD  macronutrient  stock  22.3, ZnSCy 7 H ^ 0  0.025, CoClgceH^O  solution  was  c o n c e n t r a t i o n ; - t h e c o n s t i t u e n t s , i n mg/1 1650, K N 0 iron  and  3  1900, CaCl-^H^O EDTA  concentration;  stock  440, MgSOq.  solution  was  prepared  8.6, 0.025.  The  at  10X  a  o f medium, were: NH^N0 370, KH^POq. 170. The prepared  t h e s e two components, i n mg/1  at  a  100X  o f medium, were:  3  23 FeS0 .-7H 0 4  JL  thiamine  27.8, and Na^ EDTA  37.3. F o r o r g a n i c  constituents  was k e p t as a s t o c k s o l u t i o n a t a 50X c o n c e n t r a t i o n ;  0.4 mg/1 was used i n t h e medium. The s y n t h e t i c c y t o k i n i n BA was p r e p a r e d a t a s t o c k c o n c e n t r a t i o n o f 100X, k e p t i n a c o n t a i n e r wrapped i n aluminum f o i l ,  and p r e p a r e d f r e s h monthly. The BA  l e v e l s u s e d 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 o f t h i s M a t e r i a l s and Methods c h a p t e r . A l l s t o c k s o l u t i o n s were k e p t as l i q u i d s Myo-Inositol  and r e f r i g e r a t e d i n a s t a n d a r d  (100 mg/1 o f medium) and s u c r o s e  refrigerator.  (30 g/1 o f 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 b a t c h o f medium (1 l i t r e ) had been p r e p a r e d t h e pH was a d j u s t e d t o 5.8 and 7 g/1 o f D i f c o B a c t o agar was added. The medium was d i s p e n s e d  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 t u b e s w h i c h were s t o p p e r e d  w i t h c o t t o n b a t t e n and p l a s t i c  Kaput c a p s . One l i t r e b a t c h e s were a u t o c l a v e d  f o r 2 0 minutes a t  o  121  C.  A f t e r a u t o c l a v m g , t e s t t u b e s were h e l d a t a s l a n t o f  about 3 0°so t h a t when e x p l a n t s were c u l t u r e d any e x c e s s m o i s t u r e c o u l d be d r a i n e d , and a l s o t o p r o v i d e a g r e a t e r s u r f a c e a r e a f o r shoot p r o l i f e r a t i o n . For t h e g l y p h o s a t e  study, the a d d i t i o n of glyphosate  to the  medium d i f f e r e d from t h e p r o c e d u r e used f o r BA. U n l i k e BA, w h i c h was added p r i o r  to autoclaving,  glyphosate  medium a f t e r a u t o c l a v i n g . The g l y p h o s a t e and  was added t o t h e  was f i l t e r  added when t h e medium had c o o l e d t o 3 5  - 39  C.  sterilized  24 3.2  L i g h t Sources  Three spectral  light energy  s o u r c e s were distribution  used.  Table  o f each  3.1  light  indicates  the  source. Half  the  number o f 4 0 w a t t C o o l White f l u o r e s c e n t t u b e s i n a C o n v i r o n E7 growth  chamber  experiments. photon  used  to  provide white  F o r t h e s h o r t term  far-red  light  and  the  in  initial  f l u e n c e r a t e e x p e r i m e n t s , m e t a l h a l i d e l i g h t was  C o o l White light  were  fluorescent  tubes a r e t y p i c a l l y  used  as  four low used.  the  sole  s o u r c e i n b o t h r e s e a r c h and commercial m i c r o p r o p a g a t i o n  l a b o r a t o r i e s . S p e c t r a l output from such a w h i t e l i g h t s o u r c e has proven  to  be  adequate  in  promoting  the  growth  of  m i c r o p r o p a g a t e d p l a n t s . F l u o r e s c e n t l i g h t has a r e l a t i v e l y spectral distribution,  many wide  w i t h a l m o s t e q u a l energy o u t p u t s o f b l u e  and r e d w a v e l e n g t h s . In  t h e e x p e r i m e n t where i n t e r m e d i a t e w a v e l e n g t h s  b l u e and  r e d were s t u d i e d , a Johns  Scientific  growth  between chamber  w i t h two 2 0 w a t t C o o l White f l u o r e s c e n t t u b e s p e r s h e l f three  s h e l v e s ) was  used  i n conjunction  with  f i l t e r s t o p r o v i d e g r e e n , y e l l o w , and orange Two provide  the  (with  respective  light.  t u n g s t e n f i l a m e n t lamps (500 w a t t s each) were used t o red/FR  distribution  light.  Such  lamps  have  a  poor  spectral  o f b l u e w a v e l e n g t h s ; t h e y a r e 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 o f r e d wavelengths and, a l t h o u g h n o t shown i n T a b l e 3.1, f a r - r e d w a v e l e n g t h s . The h i g h i n t e n s i t y lamps were used i n o r d e r t o m i n i m i z e t h e l o w e r i n g o f photon f l u e n c e r a t e s  typical  25  T a b l e 3.1 S p e c t r a l d i s t r i b u t i o n o f t h e energy i n t h e 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 s o u r c e s ' Spectral region  Light source •3k  Actual wavelength range (nm)  % o f t« region  Tun. M.H. Fl. *  405-495 400-510 400-500  11.5 28 . 4 22.9  Ye.- Gr.  Tun. M.H. Fl.  505-595 510-610 500-600  29.1 43.0 49.3  Red-Or. Red-Or. F.R. Red-Or. F.R.  Tun. M.H. M.H. Fl. Fl.  605-705 610-700 700-750 600-700 700-750  59.4 • 18.8 9.8 26.6 1.2  BI.- V i o .  Legend: 1 - E x c e p t i n c l u d i n g p a r t s o f t h e f a r - r e d r e g i o n f o r t h e 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 s o u r c e s 2 - From (Moshkov, 19 69) 3 - From ( G r i m s t a d , 1987) 4 - From (Chee and P o o l , 1989) BI.- Vio. = b l u e - v i o l e t Ye.- Gr. = y e l l o w - g r e e n Red- Or. = red-orange F.R. = f a r - r e d Tun. = t u n g s t e n f i l a m e n t M.H. = m e t a l h a l i d e F l . = fluorescent  with  filter  use.  The  lamps were k e p t  g r o w t h c h a m b e r ; l i g h t was  on  a  shelf  outside  the  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 u s e 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 o b l e m s o f heat build-up t h a t would occur  i n s i d e t h e g r o w t h chamber i f t h e  high  enclosed.  intensity Two  lamps had  been  m e t a l h a l i d e l a m p s (400 w a t t s e a c h ) w e r e u s e d t o  provide  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 for  t h e two  light. well  e x p e r i m e n t s a s n o t e d i n t h e s e c t i o n on  light  fluorescent  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  relatively  h a l i d e l i g h t was  high  outputs  of  other  wavelengths.  chosen f o r the blue l i g h t source  as  Metal  because of  the  poor t r a n s m i s s i o n of the b l u e f i l t e r ; once l i g h t p a s s e d  through  it  because  the  photon f l u e n c e  m e t a l h a l i d e was  rich  r a t e was  g r e a t l y reduced.  i n b l u e w a v e l e n g t h s and  p h o t o n f l u e n c e r a t e , i t was  at a photon fluence r a t e capable  As  the  tungsten  filament  Photon F l u e n c e  The in  To  white  light,  halide  lamps  were  pipes.  Rates  used.  Depending used.  reduce the photon f l u e n c e r a t e of  nylon  This  on  high  obtained  photon f l u e n c e r a t e s used i n the experiments are  T a b l e 3.2.  were  a  of s u s t a i n i n g growth.  lamps, m e t a l  used i n conjunction w i t h l i g h t  3.3  discharged  thought t h a t the blue l i g h t  w o u l d be with  Thus,  screening the  with  fluence  fluence  42  reduction  fluorescent  p e r f o r a t i o n s per  required, was  two in  to  given  cm  three  addition  was  sheets to  that  T a b l e 3.2 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 Experiment  Light quality*  Photon f l u e n c e r a t e (uM rn^s' ) 1  I - b l u e , red/FR and white l i g h t effects  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 light qualities between b l u e and r e d  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 effects  metal h a l i d e tungsten filament fluorescent  4.1 2.1  4.4 2.3  IV- r e d f i l t e r l i g h t source effects  metal h a l i d e tungsten filament fluorescent  V- e f f e c t o f short-term far-red light a p p l i e d a t the end o f a white l i g h t photoperiod  white l i g h t control  V I - e f f e c t s on the red/FR response caused by a low photon f l u e n c e and the time o f t r a n s f e r to a higher fluence VII- glyphosatelight quality interaction  white l i g h t + far-red light - before far-red extension - during far-red extension vS  xN  2.3  -  2.5  6.6 3.2  8.8 4.7  3.8  4.6  35.0  66.0  21.0 14.0  -  75.0 40.0  red/FR low l e v e l white" incubation level'' white  15.0  23.0  47 . 0  62 .0  red/FR white  10.2 - 18.6 10.8 - 13.8  8.7  15. 9  Legend: * - except f o r experiments I I I and IV where the source i s s p e c i f i e d  light  28 p r o v i d e d by u t i l i z i n g growth  chamber.  The  only h a l f of the f l u o r e s c e n t tubes i n the nylon  screening  was  also  used  to  reduce  photon f l u e n c e r a t e s f o r t h e s i x t h experiment 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  3.4  source.  Filters  All  filters  used  were  acetate  filters  produced  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 e d 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 a n d 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  light quality  blue  the blue  wavelengths  were  optimized  with  experiment  filter,  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 r e d / F R l i g h t , t h e Medium R e d filter  was u s e d .  The t h r e e  filters  i n Figure  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 , r e d / F R l i g h t was t h e l i g h t q u a l i t y promoted  in vitro  growth,  red/FR l i g h t q u a l i t y  a further  analysis  of the  which  specific  o b t a i n e d was d o n e . 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 u s e d ; t h e g r 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 given i n Figure  3.5  3.3.  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  Shoot e x p l a n t s used f o r each experiment were o b t a i n e d 8 t o 10 w e e k - o l d the  most  stock  actively  cultures.  growing  The e x p l a n t s w e r e t a k e n  sections  of  the  stock  from from  cultures.  Ltztz Filters HT027 MEDIUM RED  300  400  500 • 600  700  800  (Y = 3.64%)  WAVELENGTH-NANOMETRES  181  CONGO BLUE 3O0  400  &O0  6O0  700  800  (Y = 0.78%)  WAVELENGTH-NANOMETRES  Figure  3.1  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  30  124 DARK G R E E N  300  4O0  SO0  600  700  (Y = 29.71%)  800  WAVELENGTH-NANOMETRES  100  105 ORANGE  300  400  500  600  700  (Y = 41.28%)  800  WAVE LENGTH-NANOMETRES  I  I i L  80  |  eo  a  40  101 YELLOW  £ 2  20 < 300  4O0  500  6O0  700  800  (Y = 80.00%)  WAVE LENGTH-NANOMETRES  F i g u r e 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 used i n Experiment I I -  T e r m i n a l segments, a p p r o x i m a t e l y 1 cm l o n g i n c l u d i n g t h e with  3  to  5  leaf  nodes,  were  t r a n s f e r r e d i n t o 25 x 150 mm  excised  with  a  apex,  scalpel  and  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 e x p l a n t per tube. The e x p l a n t s were p l a c e d u p r i g h t on t h e medium w i t h t h e c u t end j u s t below t h e s u r f a c e o f the  medium. All  e x p e r i m e n t s , e x c e p t the s h o r t 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 d a r k p e r i o d . The s h o r t term f a r - r e d experiment had a 6 hour d a r k period;  the  control  white  light  regime  had  an  18  hour  p h o t o p e r i o d w h i l e t h e t r e a t m e n t regime had a 16 hour p h o t o p e r i o d of w h i t e l i g h t f o l l o w e d by 2 hours of t u n g s t e n f i l a m e n t The  light.  e x p e r i m e n t s were c a r r i e d out i n growth chambers where t h e  t e m p e r a t u r e was m a i n t a i n e d between 21 and 2 3 C. For  the f i r s t  Spiraea  light  weeks under  s e t o f experiments done t o c h a r a c t e r i z e t h e  quality  response,  the conditions  e x p l a n t s were grown f o r  used  to maintain stock  two  cultures.  T h i s 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 o f u n i f o r m size  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 t o May  and  the  and  vitality  for  the  o t h e r from June t o August,  h e a t i n g a t t h e September t o May  experimental  due  to uncontrollable  summer  l o c a t i o n . The t e m p e r a t u r e s  photon fluence- r a t e s f o r both l o c a t i o n s a r e shown i n T a b l e  and 3.3.  I n t h e e x p e r i m e n t done t o determine how a low photon f l 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 r e s p o n s e , t h e r e was no two week p r e - e x p e r i m e n t a l  32  T a b l e  3 . 3  I n c u b a t i o n  L o c a t i o n  - S e p t . -  M a y  c o n d i t i o n s  f o r  T e m p e r a t u r e  3  -  3 2 °  s t o c k  (°C)  d u r i n g  c u l t u r e s  P h o t o n  f l u e n c e  ( u M  m  s"  5 2  -  7 4  4 0  -  6 2  1  p h o t o p e r i o d  2 0  -  d a r k J u n e  -  A u g u s t  2 6 ° -  2 2  d u r i n g  p e r i o d  2 8 ° d u r i n g  p h o t o p e r i o d 2 0  -  d a r k  2 2  d u r i n g  p e r i o d  )  r a t e  growth  period;  explants  were  immediately  transferred to the  growth chambers used f o r t h e e x p e r i m e n t s . Here, s i n c e c o u l d n o t be i n i t i a l l y screened f o r u n i f o r m  s i z e and  explants vitality,  s e l e c t i o n o f t h e most v i g o u r o u s e x p l a n t s was done a t t h e end o f the experiment. For t h e g l y p h o s a t e c o n c e n t r a t i o n t r i a l s , e x p l a n t s were grown for  f i v e weeks under t h e s t o c k 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 . I n  t h e g l y p h o s a t e - l i g h t q u a l i t y experiment, e x p l a n t s week p r e - e x p e r i m e n t a l specific  growth p e r i o d were grown on media w i t h t h e  benzyladenine  experiment  f o r t h e two-  (BA)  but not containing  concentrations  used  any g l y p h o s a t e .  This  in was  the done  because i t was t h o u g h t t h a t t h e i n i t i a l e x p l a n t , w h i l e a b l e t o grow a t t h e g l y p h o s a t e sensitivity initial  l e v e l s used, might e x h i b i t a h e i g h t e n e d  t o glyphosate  subculture.  a f t e r the s t r e s s  Thus, i t was  only  o f e x c i s i o n and  a f t e r two weeks  that  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 g l y p h o s a t e - f r e e  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 t u b e s w i t h f r e s h media c o n t a i n i n g t h e r e s p e c t i v e BA  concentrations  further since  used).  These  week under t h e s t o c k  the occasional  cultures  culture  contaminated  were  left  incubation  tube  due  f o r one  conditions,  t o the t r a n s f e r  needed t o be d i s c a r d e d . F o r t h e f i n a l t h r e e weeks, c u l t u r e s were t r a n s f e r r e d t o t h e growth chambers used f o r t h e a c t u a l quality the  e x p e r i m e n t . Thus w h i l e  harvest  a l l other  experiments  light  involved  o f f i v e - w e e k o l d c u l t u r e s , t h e c u l t u r e s from t h e  glyphosate-light  quality  experiment  were  s i x weeks  old at  34 harvest.  3.6  Harvesting  At  Procedure  t h e end of each experiment,  c u l t u r e s were kept  i n the  dark i n a growth chamber a t room temperature u n t i l o b s e r v a t i o n s could  be made.  Growth  variates within  f o r a l l t h e samples one week o f an  i n an  experiment  were measured  experiment's  conclusion  (due t o t h e l a r g e number o f samples). The o r d e r of  t r e a t m e n t s a n a l y z e d was randomized t o reduce e f f e c t s due t o any minimal growth d u r i n g t h e one week h a r v e s t p e r i o d .  3.7  Experimental  Determining vitro  the  Design  effect  for  of  each  blue,  Experiment  red/FR,  and  white  light  on  in  growth  A s p l i t p l o t experiment w i t h t h r e e c o m p l e t e l y randomized b l o c k s was used. Each b l o c k c o n s i s t e d of f i v e  sub-samples f o r  each o f t h e two c o n c e n t r a t i o n s o f BA (0.25 mg/1 and 0.5 mg/1) and t h e BA-free treatment, under each o f t h e t h r e e l i g h t (red/FR, b l u e and w h i t e ) . Each l i g h t  regime,  regimes  r e p r e s e n t e d by a  growth chamber, was a main p l o t , w i t h t h e BA treatments and t h e BA-free treatment a c t i n g as s u b - p l o t s . The b l o c k s r e p r e s e n t e d e x p e r i m e n t a l runs and thus were b l o c k s over time.  Experiment on t h r e e i n t e r m e d i a t e l i g h t wavelengths between b l u e and  red  A  two-way  factorial  c o m p l e t e l y randomized  experiment  was  designed  with  two  b l o c k s o v e r t i m e . There were f o u r  sub-  samples f o r each o f t h e two BA c o n c e n t r a t i o n s (0.25 mg/1 mg/1)  and  0.5  under each of t h e t h r e e l i g h t regimes ( g r e e n , y e l l o w and  orange).  L i g h t s o u r c e experiments - b l u e f i l t e r  For  red  filter  b o t h t h e b l u e f i l t e r and r e d f i l t e r e x p e r i m e n t s , s p l i t  plots  with  block  consisted  two  c o m p l e t e l y randomized of  eight  three  light  b l o c k s were used.  sub-samples  c o n c e n t r a t i o n s o f BA used (0.25 mg/1 the  and  f o r each  and 0.5 mg/1)  of  Each  the  two  under each o f  sources (tungsten f i l a m e n t , metal h a l i d e  f l u o r e s c e n t ) . Each concentrations  light  were  s o u r c e was  sub-plots.  a main The  plot  blocks  and  and  the  BA  represented  e x p e r i m e n t a l r u n s and t h u s were b l o c k s o v e r t i m e .  Determining the  A  split  e f f e c t of a s h o r t - t e r m f a r - r e d l i g h t  plot  experiment w i t h  four  exposure  c o m p l e t e l y randomized  b l o c k s was used. Each b l o c k c o n s i s t e d o f t h r e e sub-samples f o r each o f t h e f o u r c o n c e n t r a t i o n s o f BA used (0.1 mg/1, 0.4  mg/1  and  0.5  mg/1)  under  each o f t h e two  0.25  light  mg/1,  regimes  (metal  halide,  filament) .  and  Each  concentrations  metal  light  were  halide  regime  with  was  sub-plots.  end-of-day  a main  The  plot  necessity  tungsten  and of  the  BA  using  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 b l o c k was s p l i t between t h e chambers.  Factors  c o n t r i b u t i n g to  photon f l u e n c e r a t e and  the  red/FR l i g h t  growth response:  t r a n s f e r time to h i g h e r f l u e n c e  A s p l i t - s p l i t - p l o t e x p e r i m e n t w i t h two c o m p l e t e l y  low photon f l u e n c e s  (one f o r t u n g s t e n  rate  randomized  b l o c k s was used. T r a n s f e r s o f p l a n t s were made from two with  low  chambers  filament l i g h t  and  one f o r m e t a l 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  fluence  block  rates  consisted  used  f o r maintaining  cultures.  Each  of f i v e  sub-samples  treatment;  o f t h e s e f i v e t h e f o u r most h e a l t h y  stock  f o r each  (as d e t e r m i n e d by  t h e h i g h e s t s h o o t number) were used i n t h e ANOVA. The main p l o t s were t h e two sub-plots  light qualities  o f low photon f l u e n c e  rate.  were t h e t h r e e p e r i o d s under t h e low photon  The  fluence  r e g i m e s (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  chamber).  Nested  remaining  within  the  c o n c e n t r a t i o n s used, 0.25 mg/1,  t h e whole  five  sub-plots  were  0.4 mg/1  and 0.5  weeks i n each the  three  mg/1.  BA  37 Glyphosate-light  q u a l i t y experiment  A three-way f a c t o r i a l e x p e r i m e n t was  designed with  four  c o m p l e t e l y randomized b l o c k s . The n e c e s s i t y o f u s i n g one growth chamber f o r red/FR  light  and a n o t h e r chamber f o r w h i t e  light  meant t h a t each b l o c k was s p l i t between t h e chambers. There were two  concentrations  of  BA,  0.25  mg/1  and  c o n c e n t r a t i o n s of g l y p h o s a t e , 0.089 mg/1 glyphosate-free levels  used.  t r e a t m e n t counted  No  sub-samples  as  were  0.5  mg/1,  and 0.267 mg/1, one  used  of  two and a  the glyphosate  f o r the glyphosate  t r e a t m e n t s . The g l y p h o s a t e - f r e e (or c o n t r o l BA) t r e a t m e n t s had two  sub-samples  per  block.  Each  s e t o f two  sub-samples  was  a v e r a g e d and t h e mean used f o r t h e ANOVA.  3.8  Growth V a r i a t e s measured and  At  S t a t i s t i c a l Analyses  t h e end of t h e c u l t u r e p e r i o d f o r each e x p e r i m e n t , f i v e  growth v a r i a t e s were r e c o r d e d . These were s h o o t number/explant, node  number/block  of  sub-samples,  fresh  weight/explant, dry  w e i g h t / e x p l a n t , and s h o o t l e n g t h . To t e s t f o r t h e s i g n i f i c a n c e of the f i r s t  statistical  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 a n a l y z e d by comparing t h e p r o p o r t i o n o f s h o o t s ranked as u s e f u l (1 cm o r l o n g e r ) w i t h t h e p r o p o r t i o n ranked n o t u s e f u l test  f o r any  statistically  s q u a r e t e s t was used.  significant  (below 1 cm).  differences  To  the C h i -  38  Chapter 4  R e s u l t s and  4.1  Characterization quality  4.1.1  of the  Discussion  Spiraea nipponica i n v i t r o  light  response  Experiment I : Determining the and  e f f e c t s of b l u e , red/FR,  white l i g h t on  i n v i t r o shoot  proliferation  The  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 was done t o examine t h e  response  of  qualities,  an  shoot  b l u e , r e d , and  significantly naturally  in vitro  culture  far-red,  that  to  the  three  have been  light  shown t o  m o d i f y t h e growth p a t t e r n s o f b o t h i n v i t r o  grown  plants.  chambers. B l u e l i g h t  The  experiment  (400 nm - 500 nm)  was  done  in  and  growth  was o b t a i n e d by u s i n g a  m e t a l h a l i d e l i g h t s o u r c e and a b l u e a c e t a t e f i l t e r . A l t h o u g h i n most e x p e r i m e n t s r e d l i g h t  (600 nm - 700 nm)  and f a r - r e d  light  (700 nm - 750 ..nm) a r e s t u d i e d s e p a r a t e l y , t h e ease o f o b t a i n i n g a h i g h i n t e n s i t y m i x t u r e o f b o t h from a t u n g s t e n f i l a m e n t l i g h t s o u r c e p r o v i d e d an o p p o r t u n i t y t o d e t e r m i n e i f growth p r o m o t i n g effects  attributed  t o each  separately  could  still  o c c u r when  b o t h l i g h t q u a l i t i e s were a p p l i e d t o g e t h e r . A r e d a c e t a t e f i l t e r was used t o d e c r e a s e t h e o u t p u t o f b l u e - v i o l e t and wavelengths  yellow-green  e m i t t e d by a t u n g s t e n f i l a m e n t l i g h t s o u r c e . White  l i g h t was o b t a i n e d from C o o l White f l u o r e s c e n t t u b e s , which  emit  l i g h t o f a wide s p e c t r a l c o m p o s i t i o n w i t h a r a t i o o f b l u e t o r e d l i g h t o f 0.9  ( T a b l e 4.1).  C o n f l i c t i n g r e p o r t s i n t h e l i t e r a t u r e on t h e e f f e c t s o f b l u e and  red/FR  experiment was  light  on  in vitro  p l a n t growth  made t h i s  n e c e s s a r y t o d e t e r m i n e which o f t h e l i g h t  first  qualities  o p t i m a l f o r t h e S p i r a e a i n v i t r o system. Once r e s o l v e d , t h e  light  quality  possibly  chosen  have  experiments.  could  i t s growth  The  be  more e x t e n s i v e l y s t u d i e d  effects  optimized,  main g o a l i n u s i n g l i g h t  and  in  subsequent  quality  t o enhance  growth was t o l o w e r t h e amount o f exogenous c y t o k i n i n needed i n t h e medium. Thus, two BA l e v e l s were used, t h e normal BA (0.5 mg/1) addition whether  as a c o n t r o l , cultures  or  not  were  BA  was  and  half this  grown  on  level  BA-free  necessary  for  (0.25  media  light  to  level  mg/1). I n ascertain  quality  growth  enhancement t o be e x p r e s s e d . Shoot numbers were d e t e r m i n e d  and  g i v e n a q u a l i t y r a t i n g m a i n l y by d e t e r m i n i n g t h e p r o p o r t i o n o f u s e f u l s h o o t s ( d e f i n e d as s h o o t s 1 cm o r l o n g e r ) . D e f i n i n g such s h o o t s as u s e f u l i s meant as a measure of t h e s h o o t s '  probable  surviveability  for  for  either  further  sub-culture  subsequent phase o f r o o t i n d u c t i o n . The all  the  shoots  from  each b l o c k o f f i v e  or  the  t o t a l node number f o r initial  explants  was  t a k e n as a n o t h e r q u a l i t y r a t i n g ; each node r e p r e s e n t s p o t e n t i a l  40  TABLE 4 . 1 Red  light:  ratios  far-red light  for unfiltered  light  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  Licrht S o u r c e  and b l u e l i g h t : red  sources  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  13 .1  0. 9  Fluorescent Legend: 1 - R:FR 2 - B:R * - R:FR taken  = 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 r e d l i g h t r a t i o s p e c t r a l l y d e f i n e d and l i g h t s o u r c e from ( H a r t , 1988).  :  ratios  41 for  f u r t h e r a x i l l a r y shoot  proliferation.  G e n e r a l 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 l e a v e s t h a n p l a n t s grown under w h i t e l i g h t o r b l u e 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 l o n g e r s h o o t s than p l a n t s grown under white  or blue  light.  Blue  light  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 t h a n p l a n t s from t h e o t h e r l i g h t  regimes.  Growth V a r i a t e s  Shoot number/explant  F o r a l l t h r e e l i g h t regimes t h e shoot number means f o r t h e BA-free  c u l t u r e s were v e r y  low compared t o t h e shoot  means a t t h e 0.25 mg/1 BA l e v e l  and t h e 0.5 mg/1 BA  number  level  ( T a b l e 4.2). These r e s u l t s i n d i c a t e t h e n e c e s s i t y o f s u p p l y i n g BA i n t h e c u l t u r e medium t o i n d u c e s u b s t a n t i a l growth. A l s o , t h e s i m i l a r means r e c o r d e d  under each l i g h t  regime s u g g e s t s  that  l i g h t q u a l i t y a l o n e cannot s i g n i f i c a n t l y modify S p i r a e a i n v i t r o growth; l i g h t fully  quality  expressed  in  effects concert  on i n v i t r o with  the  growth can o n l y be exogenously  applied  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 s h o o t s / e x p l a n t , o c c u r r e d under w h i t e l i g h t a t t h e 0.5 mg/1 BA l e v e l . A s i m i l a r l e v e l o f  42  Table 4.2 Shoot, and node number means under b l u e , red/FR, and w h i t e w i t h and w i t h o u t BA Light quality  BA level  Shoot number  Blue  0 0.25 0.5  2.87 ± 0.25 11.73 ± 0.93 14.80 ± 1.66  58 ± 7 255 ± 49 310 ± 56  Red/FR  0 0.25 0.5  3.27 ± 0.36 20.87 ± 1.84 16.87 ± 1.89  79 ± 11 518 ± 71 413 ± 4  2.80 ± 0.44 16.93 ± 1.63 21.60 ± 2.18  75 ± 18 419 ± 72 485 ± 55  White  Legend:  1  0 0.25 0.5  2  Node number 3  1 - BA l e v e l i n mg/1 2 - s h o o t number/explant mean + s t a n d a r d er r o r 3 - node number/block o f 5 s u b - s a m p l e s (mean of three b l o c k s ± standard e r r o r )  light,  20.87 s h o o t s / e x p l a n t was  o b t a i n e d f o r red/FR  r e d u c e d c y t o k i n i n c o n c e n t r a t i o n o f 0.25 had a mean o f 16.87  a t t h e 0.5 mg/1  c u l t u r e s at the  mg/1.  Red/FR c u l t u r e s  BA l e v e l . Thus under  red/FR  l i g h t t h e maximal mean was found a t t h e 0.25 mg/1  BA l e v e l ; t h e  decrease  red/FR  at the  h i g h e r BA  level  suggests  that  light  e s t a b l i s h e s a c y t o k i n i n t o a u x i n r a t i o more f a v o u r a b l e f o r shoot initiation mg/1  when c u l t u r e s  a r e grown on medium c o n t a i n i n g  o f BA t h a n when on medium w i t h t h e 0.5 mg/1  0.25  BA l e v e l .  Blue  l i g h t c u l t u r e s showed t h e l o w e s t s h o o t number means a t b o t h BA levels. A n a l y s i s o f V a r i a n c e (ANOVA) showed t h a t t h e o n l y e f f e c t o f light  q u a l i t y was  t h e l o w e r i n g o f mean shoot and node numbers  under  the blue l i g h t  compared t o t h o s e under  b o t h red/FR  and  w h i t e 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 T a b l e 4.3). The BA c o n c e n t r a t i o n had t h e g r e a t e s t i n f l u e n c e on shoot number. There number  and  was  BA  a strong linear  r e l a t i o n s h i p between  concentration i n both  white  and  c u l t u r e s . The h i g h e s t shoot number mean a t t h e 0.25 the  shoot  blue  light  mg/1  BA i n  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 t h e mean a t t h e  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 t h e ANOVA by t h e s t r o n g  lack 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 m a t h e m a t i c a l  model t h a n a s i m p l e l i n e a r one i s needed t o e x p l a i n t h e t r e n d ) . This  result  red/FR  light  emphasizes cultures  t h e degree and  both  of difference the  white  and  between  the  blue  light  BA l e v e l  under  cultures. The h i g h e r shoot number mean a t t h e 0.25 mg/1  44 Table  4.3  Summarized a n a l y s e s o f v a r i a n c e . w h i t e l i g h t , and BA c o n c e n t r a t i o n f r e s h and dry weights •  E f f e c t s o f b l u e , red/FR, and on shoot and node numbers, and  Shoot number  Node number  Fresh weight  Dry weight  * **  * **  NS NS  NS NS  *** *** ***  ** * ***** ***  NS  NS  ***** ***** *****  ***** ***** *****  ***** ***** *****  1  2  3  4  Source o f v a r i a t i o n  df  Light quality b/rw r/bw r/w  (2) 1 1 1  BA c o n c e n t r a t i o n linear' l a c k of f i t  (2) 1 1  ***** ***** *****  L i g h t q u a l i t y and BA interaction b/rw X l i n e a r . b/rw X l a c k of f i t  (4) 1 1  **  *  NS  NS  **  ** ** **  ** *** *  5  5  *  r/bw X l i n e a r r/bw X l a c k of f i t  1 1  NS  NS  NS  NS  r/w X l i n e a r r/w X l a c k o f f i t  1 1  NS  NS  NS  NS . NS  Legend: 1 2 -  ***  *  ***  *  ***  *  *  shoot number/explant node n u m b e r / b l o c k o f 5 s u b - s a m p l e s ( t o t a l nodes o f shoots o f 5 e x p l a n t s ) 3 - fresh weight/explant 4 - dry w e i g h t / e x p l a n t 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 d f add t o 3 instead of 2 6 - c o n t r a s t s a r e : b/rw = b l u e v e r s u s red/FR and white l i g h t c u l t u r e s , r/bw = red/FR v e r s u s b l u e and w h i t e l i g h t c u l t u r e s , r/w = r e d / F R v e r s u s w h i t e 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 m o d e l s : 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 , l a c k o f f i t = amount o f 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 o f freedom * = s i g n i f i c a n t a t p < 0.1, ** = a t p < 0.05, *** = at p < 0.025, **** = a t p < 0.01, ***** = a t p < 0.005, NS = non-significant  red/FR  light  i s significantly  light; this reflects quality  the significant  and BA c o n c e n t r a t i o n  Table  4.3). Although  interaction  different  than  that  interaction  (specifically  the s t a t i s t i c a l  f o r white  between l i g h t  t h e r/w c o n t r a s t ,  significance  of  this  i s low (p < 0.1), t h e e x t e n t o f t h e d i f f e r e n c e i s  shown by t h e 23% g r e a t e r t o t a l shoot p r o d u c t i o n o f red/FR l i g h t e x p l a n t s a t t h e 0.25 mg/1 BA l e v e l light  explants  interaction  a t t h e same  as compared t o t h e w h i t e  level.  Also  included  in  this  i s t h e s i g n i f i c a n t d i f f e r e n c e between t h e l o w e r mean  under red/FR l i g h t a t t h e 0.5 mg/1 BA l e v e l and t h e maximal mean under  white  light  interpretation modifying  a t t h e 0.5 mg/1  o f these  cytokinin  results  BA  level.  One p o s s i b l e  i s t h a t red/FR  t o auxin r a t i o s  light  may be  i n t h e e x p l a n t when t h e  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 t h e growth medium.  Node number/block o f 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 t h e t r e n d s o f t h e mean s h o o t numbers. The h i g h e s t v a l u e o b t a i n e d was under red/FR l i g h t a t t h e 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 t h e h i g h e r BA l e v e l light.  The mean shoot  c o n d i t i o n s demonstrating  (0.5 mg/1) under  numbers were s i m i l a r l y that proliferative  high  white  f o r these  growth a t t h e 0.25  mg/1 BA l e v e l under red/FR was comparable t o t h a t f o r w h i t e a t t h e h i g h e r BA l e v e l . The  ANOVA  results  also  generally  indicated  the trends  46 o b s e r v e d f o r s h o o t number means ( T a b l e 4.3).  Shoot l e n g t h  For  the  BA-free  cultures  e i t h e r o f t h e two BA l e v e l s ,  and  the  cultures  treated  with  t h e range o f s h o o t s 1 cm o r l o n g e r  ( c o n s i d e r e d " u s e f u l " s h o o t s ) was w i d e ; f o r each BA l e v e l  where  t h e c u l t u r e s under a l l t h r e e l i g h t regimes were compared, b l u e light  cultures  had  the  lowest p r o p o r t i o n of  shoots  1 cm  or  l o n g e r , w h i t e l i g h t c u l t u r e s t h e 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 t h e h i g h e s t u s e f u l s h o o t p r o p o r t i o n ( T a b l e 4.4). For t h i s  experiment,  Harbage,  1989),  as n o t e d  increasing  i n other studies  the  BA  level  (Stimart  inhibited  and  shoot  e l o n g a t i o n ; f o r a l l t h r e e l i g h t regimes t h e h i g h e s t p r o p o r t i o n s of  u s e f u l s h o o t s were a t t h e l o w e r BA l e v e l .  The red/FR  light  regime caused t h e g r e a t e s t shoot e l o n g a t i o n f o r both t h e B A - f r e e c u l t u r e s and t h e c u l t u r e s where t h e two l e v e l s o f BA were used. When p a i r - w i s e comparisons o f u s e f u l s h o o t p r o p o r t i o n s were done between w h i t e and red/FR l i g h t c u l t u r e s , w i t h and w i t h o u t BA,  o n l y B A - f r e e c u l t u r e s showed no s i g n i f i c a n t  t h e 0.25  mg/1  difference.  At  BA l e v e l t h e d i f f e r e n c e between red/FR and w h i t e  l i g h t c u l t u r e s i s shown by t h e number o f u s e f u l s h o o t s 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  while f o r white  numbered  79.  Even  cultures  had  a  at  lower  the  0.5  shoot  light mg/1  number  cultures BA  level,  total  than  useful  shoots  where  red/FR  white  light  47 T a b l e 4.4 S h o o t 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 : red/FR,  and  t r e a t s .'  BA con.  tested  white  1  Sample size  Lf Useful shoot V  Actual useful shoot number  5  X  *****  Blue White Red/FR  0  Blue White Red/FR  0.25  176 254 313  23 31 43  40 79 136 •  *****  Blue White Red/FR  0 . 5  221 324 262  15 23 40  33 75 105  *****  43 42 49  9 21 35  4 9 17  Blue White  0  43 42  9 21  4 9  NS  White Red/FR  0  42 49  21 35  9 17  NS  *  Blue White  0.25  176 254  23 31  40 79  White Red/FR  0.25  254 313  31 43  79 136  Blue White  0.5  221 324  15 23  33 75  White Red/FR  0 . 5  324 262  23 40  75 105  White Red/FR  0 . 5 0.25  324 313  23 43  75 136  White Red/FR  0 . 25 0. 5  254 262  31 40-  79 105  Red/FR Red/FR  0 . 25 0. 5  313 262  43 40  136 105  White White  0.25 0.5.  254 324  31 23  79 75  Legend:  1  2 3 4  5 6  7  ^  ***** *** *****  ***** **  NS  '  ** "  - each p a i r represents the s e t o f shoot length, p r o p o r t i o n s a n a l y z e d xn a Chi-square test - BA l e v e l i n mg/1 - 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 experimental blocks - 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 shoot % i s t h e H of the sample size ranked useful - the c o n v e r s i o n o f the useful shoot % i n t o number o f s h o o t s - 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 define the degree 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 . o o s , NS = n o n - s i g n i f i c a n t - l i g h t s o u r c e s ; Tun.= t u n g s t e n filament, 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  blue,  48 c u l t u r e s , t h e h i g h e r p r o p o r t i o n o f longer shoots light  meant t h a t red/FR l i g h t  under red/FR  c u l t u r e s had 105 u s e f u l  shoots  w h i l e w h i t e l i g h t c u l t u r e s had 75 u s e f u l s h o o t s . Although both  BA  t h e u s e f u l s h o o t p r o p o r t i o n s under red/FR l i g h t a t  levels  followed  the trend  of the other  two  light  r e g i m e s , t h e d e c l i n e a t t h e h i g h e r BA l e v e l was t h e l e a s t o f t h e t h r e e l i g h t r e g i m e s . The m i n o r change i s c o n f i r m e d  by t h e C h i -  square t e s t where t h e d i f f e r e n c e was found t o be n o t s i g n i f i c a n t (Table 4.4).  Thus, red/FR l i g h t  seems c a p a b l e  of reducing the  i n h i b i t o r y e f f e c t on s h o o t e l o n g a t i o n o f t h e h i g h e r BA l e v e l .  Fresh weight/explant  The  and D r y w e i g h t / e x p l a n t  f r e s h w e i g h t means g e n e r a l l y f o l l o w e d t h e same t r e n d s  f o r each l i g h t regime as f o r t h e s h o o t number means (Table 4.5) . However,  the overall  fresh  weight  means  g r e a t e r t h a n under e i t h e r b l u e o r w h i t e .  under  red/FR  were  Thus, w i t h a g r e a t e r  f r e s h w e i g h t p e r s h o o t 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),  t h e f r e s h w e i g h t 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 t h e s h o o t and node number means. Thus, d i f f e r e n c e s between f r e s h w e i g h t means o c c u r r e d f o r each  light  Although greater  regime  according  t h e degree  to different  BA  concentrations.  o f d i f f e r e n c e due t o l i g h t  f o r s e v e r a l comparisons  quality  was  (b/rw and r/bw c o n t r a s t s i n  T a b l e 4.3) t h a n f o r - s h o o t and node number means, t h e d i f f e r e n c e  T a b l e 4.5 F r e s h and d r y weight means under b l u e , red/FR, and w h i t e w i t h and w i t h o u t BA Light quality  BA level 1  Fresh weight 2  Dry weight 3  Blue  0 0.25 0.5  17 + 1 50 + 4 68 ± 9  Red/FR  0 0.25 0.5  26 + 2 146 + 14 127 + 19  6 19 + 1 17 + 2  White  0 0.25 0.5  22 ± 2 98 ± 9 119 ± 11  5 15 + 1 16 + 1  Legend:  light,  3 8 + 1 7 ± 1  1 - BA l e v e l i n mg/1 2 - f r e s h w e i g h t / e x p l a n t mean ( i n mg) + s t a n d a r d e r r o r 3 - d r y w e i g h t / e x p l a n t mean ( i n mg) + s t a n d a r d e r r o r  between  red/FR  and  white  significant.  Again  concentration  interaction  apparent The weight  i t  light  cultures  was  with  the  that  t h e red/FR  was  still  not  light  quality-BA  light  effect  was  (r/w X l a c k o f f i t i n T a b l e 4.3). d r y weight  means  means r e f l e c t  t h e t r e n d s shown by  ( T a b l e 4.5). However, t h e narrow  range  fresh  of dry  w e i g h t means between t h e 0.25 mg/1 BA l e v e l and t h e 0.5 mg/1 BA level a  f o r b o t h t h e red/FR and w h i t e l i g h t regimes d i d r e s u l t i n  loss  light  of a s i g n i f i c a n t cultures  when  i n t e r a c t i o n was t e s t e d result  contrasts  with  d i f f e r e n c e between red/FR and w h i t e  the  light  quality-BA  concentration  (r/w X l a c k o f f i t i n T a b l e 4.3). T h i s the fresh  weight  mean  analysis  and  i n d i c a t e s t h a t d r y m a t t e r a c c u m u l a t i o n was o n l y s l i g h t l y h i g h e r under red/FR l i g h t t h a n under w h i t e l i g h t .  51 4.1.2  Experiment I I : Determining the  e f f e c t s on  shoot p r o l i f e r a t i o n of  in vitro  three  i n t e r m e d i a t e wavelength ranges between b l u e and  red wavelengths  The w a v e l e n g t h s most c a p a b l e o f m o d i f y i n g phytochrome the  blue,  phytochrome  red,  and  far-red  photoequilibria  wavelengths. can  also  be  Minor caused  are  shifts by  in  other  w a v e l e n g t h s . I f such s h i f t s can i n d u c e endogenous phytohormone balances  favourable  for  shoot  proliferation,  then  the  r e q u i r e m e n t f o r exogenous c y t o k i n i n c o u l d be r e d u c e d . Thus, f o r t h i s e x p e r i m e n t t h r e e w a v e l e n g t h ranges between t h e b l u e and r e d wavelengths  were  proliferation.  tested  to  determine  their  effect  on  shoot  The t h r e e w a v e l e n g t h ranges were g r e e n , 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 s o u r c e was combined w i t h g r e e n , y e l l o w , and orange a c e t a t e f i l t e r s . The f l u o r e s c e n t l i g h t s o u r c e was chosen because of i t s h i g h e r energy o u t p u t o f g r e e n , y e l l o w , and orange wavelengths compared t o t h e m e t a l h a l i d e s o u r c e .  Growth  Variates  Shoot number/explant  A t t h e 0.25 similar  mg/1  BA l e v e l t h e shoot number means a r e q u i t e  ( T a b l e 4.6). A t t h e 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 s h o o t number means as t h e w a v e l e n g t h s approach t h e  i 52  Table 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 (g'reen, y e l l o w , and orange) between blue and r e d l i g h t . Light Quality  BA Level  Shoot Number  Node Number  Green  0 25 0 5  8 63 + 2 12 11 38 ± 1 86  152 + 1 204 + 34  Yellow  0 25 0 5  7 13 + 1 10 . 15 75 + 2 01  153 + 28 299 + 57  Orange  0 25 0 5  8 38 + 2 27 18 38 + 6 13  180 + 2 303 + 50  1  2  3  Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean ± s t a n d a r d e r r o r 3 - node n u m b e r / b l o c k (mean o f two b l o c k s ± error)  standard  I  r e d w a v e l e n g t h range. However, t h e ANOVA (Table 4.7) i n d i c a t e s t h a t t h e o n l y 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 s h o o t 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 light  quality  interaction. cytokinin  alone  or to a light  Thus, t h e u n d e r l y i n g  levels  cannot  quality-BA  goal  be a c h i e v e d  concentration  of reducing  by u s i n g  exogenous  either  green,  y e l l o w , o r orange l i g h t , a s i s i n d i c a t e d by t h e s i m i l a r number  means  a t t h e 0.25 mg/1  BA l e v e l .  The ANOVA  shoot also  i n d i c a t e s t h a t w h i l e growth p r o m o t i o n a t t h e h i g h e r BA l e v e l d i d o c c u r f o r a l l t h r e e l i g h t r e g i m e s , t o t h e g r e a t e s t e x t e n t under yellow  and orange  light,  high  variability  among  individual  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 c o u l d not be a f f i r m e d . The  f a c t t h a t g r e e n l i g h t c u l t u r e s grew a t s i m i l a r r a t e s t o  c u l t u r e s under t h e o t h e r two l i g h t regimes p r o v i d e s e v i d e n c e f o r the  s t r o n g p r o m o t i v e e f f e c t o f BA on shoot i n i t i a t i o n i n t h e  S p i r a e a system; i n a s t u d y on a n o t h e r i n v i t r o p l a n t ,  Juneberry,  g r e e n l i g h t g r e a t l y r e d u c e d s h o o t i n i t i a t i o n even when BA was used (Behrouz and L i n e b e r g e r ,  1981).  Node number/block o f 4 sub-samples  Node number means f o l l o w e d t h e p a t t e r n o f shoot number means ( T a b l e 4.6). However, a t t h e 0.5 mg/1 BA l e v e l t h e node number means f o r y e l l o w and orange l i g h t were much more s i m i l a r  than  the  4.7)  corresponding  shoot number means. The ANOVA  (Table  54  Table  4.7  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 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 r e d l i g h t ( g r e e n , y e l l o w , and o r a n g 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 w e i g h t s .  df.  Shoot number  Node number  Fresh weight  Dry weight  (2) 1 1  NS NS NS  NS NS NS  NS NS NS  NS NS NS  1  **  *  **  NS  (2) 1 1  NS NS NS  NS NS NS  NS NS NS  NS NS NS  1  Source  of  variation  Light Quality O/GY 0/Y 5  BA c o n c e n t r a t i o n L i g h t q u a l i t y and interaction O/GY X BA 0/Y X BA Legend: 1 2 3 4 5 df *  2  3  4  BA  shoot number/explant node n u m b e r / b l o c k o f 4 s u b - s a m p l e s ( t o t a l nodes o f shoots of 4 e x p l a n t s ) f r e s h weight/explant dry w e i g h t / e x p l a n t contrasts are: O/GY = orange v e r s u s green and y e l l o w 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 light cultures degree of freedom s i g n i f i c a n t a t p < 0.01, ** = at p < 0.05, NS = nonsignificant  55 indicates that,  as f o r shoot number means, node number means  o n l y d i f f e r e d due t o BA c o n c e n t r a t i o n .  Shoot l e n g t h  A t b o t h BA l e v e l s t h e p r o p o r t i o n o f u s e f u l s h o o t s i s l o w e s t under g r e e n l i g h t  ( T a b l e 4.8). W h i l e t h e r e 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 a t t h e 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 a t t h e 0.5 mg/1 BA l e v e l . The  c o m p a r i s o n between y e l l o w and g r e e n l i g h t level  a t t h e 0.5 mg/1 BA  i n d i c a t e s t h a t i t i s t h e p r o p o r t i o n s o f u s e f u l t o non-  u s e f u l s h o o t s under t h e s e 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 t h e S p i r a e a system g r e e n l i g h t seems c a p a b l e o f 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 s h o o t e l o n g a t i o n .  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  T h e . i n c r e a s e i n t h e f r e s h w e i g h t mean from t h e l o w e r t o t h e higher  BA l e v e l  cultures  i s similar  f o r both y e l l o w  and orange  ( T a b l e 4 . 9 ) . The mean under g r e e n l i g h t  light  i s slightly  l o w e r a t t h e l o w e r BA l e v e l b u t i s much l o w e r t h a n t h e o t h e r two l i g h t q u a l i t y means a t t h e h i g h e r BA l e v e l . The low mean a t t h e h i g h BA l e v e l  i n d i c a t e s a l o w e r f r e s h w e i g h t p e r shoot under  g r e e n l i g h t t h a n f o r s h o o t s under t h e o t h e r two l i g h t  qualities;  t h i s suggests a lower v i g o u r f o r t h e green l i g h t shoots. The  strength  o f t h e f r e s h w e i g h t mean i n c r e a s e  from t h e  treats'. tested  Table 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 ! q u a l i t i e s between b l u e and r e d : g r e e n , y e l l o w , orange if 3 5 a& Actual Useful^ Sample useful X* con. size shoot % s h o o t number  Green Yellow Orange  0.25  Green Yellow Orange  0.5  Yellow Orange  69 57 67  33 39 37  23 22 25  91 126 137  31 43 38  28 54 52  0.25  57 67  39 37  22 25  Yellow Green  0.25  57 69  39 33  22 23  NS  Orange Green  0.25  67 69  37 33  25 23  NS  Yellow Orange  0.5  126 _ 137  43 38  54 52  Yellow Green  0.5  126 91  43 31  54 28  Orange Green  0.5  137 91  38 31  52 28  NS  Orange Green  0.25 0.5  67 91  37 31  25 28  NS  Yellow Green  0.25 0.5  57 91  39 31  22 28  NS  Yellow Orange  0.25 0.5  57 137  39 38  22 52  NS  Orange Green  0.5 0.25  137 69  38 33  52 23  NS  Yellow Green  0.5 0.25  126 69  43 33  54 23  NS  Yellow Orange  0.5 0.25  126 67  43 37  54 25  NS  NS  *  NS  ^  NS  *  Legend: 1 - each p a i r r e p r e s e n t s the s e t o f shoot length proportions 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 o f s h o o t s p r o d u c e d i n a l l experimental blocks 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 % o f t h e sample s i z e ranked 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 shoot H i n t o number o f s h o o t s 6 - X i s t h e symbol f o r t h e 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 t h e degree 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  T a b l e 4.9 F r e s h and d r y weight means under t h r e e i n t e r m e d i a t e wavelengths (green, y e l l o w , and orange) between b l u e and r e d l i g h t . Light Quality  BA Level  Green  0 .25 0. 5  51 59 + 4  Yellow  0.25 0. 5  67 + 16 143 + 31  15 + 3 23 + 3  Orange  0.25 0. 5  61 + 1 128 + 17  14 21 + 2  Legend: 1 2 3  1  Fresh Weight  2  Dry Weight 3  9 9  BA l e v e l i n mg/1 f r e s h w e i g h t / e x p l a n t mean ( i n mg) ± s t a n d a r d e r r o r dry w e i g h t / e x p l a n t mean ( i n mg) + s t a n d a r d e r r o r  58  l o w e r t o t h e h i g h e r 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 t h e low g r e e n l i g h t means; t h u s , as f o r shoot and  node number means, BA  d i f f e r e n c e (ANOVA, T a b l e  concentration  caused  the largest  4.7).  Dry w e i g h t means f o l l o w e d t h e p a t t e r n o f f r e s h w e i g h t means (Table 4 . 9 ) .  However, i n t h e d r y w e i g h t ANOVA ( T a b l e 4.7)  the  low g r e e n l i g h t means seem t o have negated t h e s i g n i f i c a n c e o f any  BA  concentration  effect  i n causing  dry weight  mean  d i f f e r e n c e s s i n c e t h e r e 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, t h e e v i d e n c e p r o v i d e d by d r y w e i g h t means s u g g e s t s t h a t g r e e n 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  effects  shoot p r o l i f e r a t i o n  of  source-blue f i l t e r  In was  the f i r s t  acetate  filter)  wavelengths  and  because also  of  its  because  of  in  three  vitro light  combinations  l i g h t q u a l i t y experiment,  chosen as t h e b l u e l i g h t s o u r c e  on  metal h a l i d e l i g h t  ( i n combination w i t h a blue high  proportion  i t s small  of  far-red  blue light  component. F l u o 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 o f b l u e wavelengths;  t h u s , t o t e s t t h a t a maximal b l u e l i g h t e f f e c t  was  a c h i e v e d u s i n g m e t a l h a l i d e , f o r t h i s experiment t h e f l u o r e s c e n t l i g h t s o u r c e was  a l s o combined w i t h t h e b l u e  filter.  Combining t h e b l u e f i l t e r w i t h t h e t u n g s t e n f i l a m e n t l i g h t source,  as w e l l as w i t h t h e o t h e r l i g h t  sources, provided  an  o p p o r t u n i t y t o s t u d y t h e e f f e c t on i n v i t r o growth o f v a r y i n g the r a t i o s  o f b l u e , r e d , and  f a r - r e d w a v e l e n g t h s (which  f o r each l i g h t s o u r c e ) . The r a t i o s t y p i c a l  f o r each l i g h t  differ source  a r e shown i n T a b l e 4.1. U s i n g a b l u e f i l t e r w i t h t h e t h r e e 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 increase the blue l i g h t to red l i g h t  and  ratio.  Growth V a r i a t e s  Shoot number/explant  As i s shown i n T a b l e 4.10,  a t t h e l o w e r BA l e v e l t h e  metal  60  T a b l e 4.10 Shpot and node number means under t h r e e l i g h t s o u r c e s ( t u n g s t e n f i l a m e n t , m e t a l h a l i d e , and f l u o r e s c e n t ) combined w i t h a b l u e acetate f i l t e r . Light Source  . BA Level  Shoot Number  Tungsten filament  0.25 0.5  9.19 9.50  Metal halide  0.25 0.5  Fluorescent  0.25 0.5  1  Node Number  2  3  ± 0.84 ± 0.84  254 241  7.63 11.06  ± 0.86. ± 1.69  230 306 ± 8  7.75 10.81  ± 0.84 ± 1.71  218 270  Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean ± s t a n d a r d e r r o r 3 - node n u m b e r / b l o c k (mean o f two b l o c k s ± error)  ± 17 + 17  ± 4 ± 11  standard  halide-blue  filter  number mean t h a n  treatment  caused  a  the fluorescent-blue  slightly filter  lower  shoot  treatment.  The  t u n g s t e n f i l a m e n t - b l u e f i l t e r t r e a t m e n t had t h e h i g h e s t mean a t t h e l o w e r BA l e v e l b u t t h e d i f f e r e n c e was s m a l l . A t t h e h i g h e r BA l e v e l  t h e m e t a l h a l i d e mean was s l i g h t l y  fluorescent  mean.  Tungsten  filament  greater than the  at the higher  BA  level  caused a s i m i l a r mean as t h e t u n g s t e n f i l a m e n t t r e a t m e n t a t t h e lower  BA  overall  level.  As t h e ANOVA  differences  i n shoot  i n Table  4.11  number means  indicates,  the  due t o t h e l i g h t  s o u r c e s were n o t 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 o f t h i s e x p e r i m e n t m i r r o r t h o s e seen i n t h e f i r s t experiment. F i r s t t h e r e are the s i g n i f i c a n t i n c r e a s e s i n s h o o t number means 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 due t o BA c o n c e n t r a t i o n . Then t h e r e i s t h e h i g h e s t s h o o t number mean a t t h e l o w e r BA l e v e l produced under t u n g s t e n f i l a m e n t l i g h t . I n t h e f i r s t e x p e r i m e n t t h e b l u e l i g h t s o u r c e showed t h e g r e a t e s t r e d u c t i o n o f growth.  I n t h i s experiment t h e m e t a l  halide-blue  f i l t e r regime i n d u c e d v i r t u a l l y t h e same range o f s h o o t number means as t h e f l u o r e s c e n t - b l u e f i l t e r regime which s u g g e s t s t h a t both  caused  an  equivalent blue  light  effect  on  growth.  A  d i f f e r e n c e between t h e r e s u l t s o f t h i s e x p e r i m e n t and t h o s e o f Experiment  I  was  the generally  s u g g e s t i v e o f a l o w e r growth three  light  sources.  lower  rate,  T h i s may  shoot  number  a t b o t h BA l e v e l s  have  occurred  c u l t u r e s used had a l o w e r morphogenetic  means, for a l l  i f the source  p o t e n t i a l than i n the  f i r s t e x p e r i m e n t , a f a c t o r which may have m o d i f i e d t h e e x p l a n t s '  62  T a b l e 4.11 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 - b l u e 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 weights Shoot number 1  Source o f V a r i a t i o n  df  Light  (2) 1 1  source T/MeF T/F 5  BA  concentration  L i g h t s o u r c e and BA i n t e r a c t i o n T / M e F X BA T / F X BA Legend:  1 2 3 4 5 df  (2) 1 1  Node number 2  NS NS NS  NS NS NS  **  ***  NS NS NS  NS **  Fresh wt.  NS  3  Dry wt. 4  NS NS NS NS  NS NS NS  NS NS NS  shoot number/explant node n u m b e r / b l o c k o f 8 s u b - s a m p l e s ( t o t a l nodes o f shoots of 8 explants ) fresh weight/explant dry weight/explant 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 = t u n g s t e n filament 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 d e 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 , NS = n o n - s i g n i f i c a n t  response t o l i g h t this  experiment,  quality.  F o r example, a n o t h e r d i f f e r e n c e i n  the similar  shoot  number means a t b o t h BA  l e v e l s under t u n g s t e n 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 l o w e r growth The  rate.  main  factor  r e s p o n s i b l e f o r t h e r e d u c t i o n o f growth  under t h e t u n g s t e n f i l a m e n t - b l u e f i l t e r regime i s p r o b a b l y t h e poor  energy  majority  output  from  of red l i g h t  the rest  o f t h e spectrum  i s filtered  once t h e  o u t by t h e b l u e  filter.  A n o t h e r r e a s o n f o r t h e g e n e r a l l y l o w e r shoot numbers under each l i g h t s o u r c e may be t h e changes i n t h e b l u e t o r e d r a t i o .  This  e f f e c t may be e s p e c i a l l y a p p a r e n t f o r t h e f l u o r e s c e n t and m e t a l h a l i d e b l u e f i l t e r r e g i m e s , w i t h much h i g h e r u n f i l t e r e d  levels  of b l u e w a v e l e n g t h s t h a n u n f i l t e r e d t u n g s t e n f i l a m e n t l i g h t . F o r the metal  halide  and f l u o r e s c e n t  sources  t h e use o f a blue  f i l t e r would cause an i n c r e a s e i n t h e B:R r a t i o  i naddition t o  a l a r g e r e d u c t i o n i n b o t h t h e q u a n t i t y o f r e d l i g h t and t h e R:FR r a t i o . E i t h e r o f t h e s e m o d i f i e d l i g h t r a t i o s may p l a y a r o l e i n the i n h i b i t i o n  o f shoot i n i t i a t i o n .  Node number/block o f e i g h t  sub-samples  The p a t t e r n o f node number means ( T a b l e 4.10) was s i m i l a r t o t h e s h o o t number means. The ANOVA (Table 4.11) i n d i c a t e s t h a t , as f o r s h o o t s , node number means under t h e f l u o r e s c e n t and m e t a l halide  blue  filter  regimes  differed  most  due  to  BA  c o n c e n t r a t i o n , w i t h fewer nodes a t 0.25 mg/1 BA t h a n a t 0.5 mg/1  64 BA (as f o r s h o o t number means, t h e mean node numbers under t h e t u n g s t e n 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 t h e same a t b o t h BA levels  except t h a t t h e s l i g h t decrease  noted a t t h e h i g h e r BA  l e v e l was n o t s i g n i f i c a n t ) . The degree o f t h e i n c r e a s e i n node number  from  the  lower  to  the  higher  BA  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 , by t h e i n t e r a c t i o n t h e ANOVA t a b l e  level  f o r the  i s made apparent  between l i g h t source and BA c o n c e n t r a t i o n i n (T/F c o n t r a s t i n T a b l e  4.11), where t u n g s t e n  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 o f t h e p a i r comparisons (Table  4.12).  showed s i g n i f i c a n t d i f f e r e n c e s  The h i g h e s t p r o p o r t i o n o f u s e f u l  s h o o t s , 30%,  o c c u r r e d under t h e m e t a l 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,  was  t h e range  of useful  shoots  f o r a l l treatments  narrow, w i t h 20% f o r 0.5 mg/1 BA under t u n g s t e n f i l a m e n t l i g h t being the lowest.  Fresh weight/explant  and Dry w e i g h t / e x p l a n t  The range o f f r e s h w e i g h t / e x p l a n t 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 s o u r c e (Table 4.13). As f o r shoot and  node  number  means,  there  was  a  change  in  fresh  w e i g h t / e x p l a n t means due t o BA c o 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 l i g h t sourceblue ^filter combinations. 6  Pairs tested  BA con.  Sample size  Tun. Me.H.  0.25  147 122  23 30  • 34 37  NS  Tun. FI.  0.25  147 124  23 22  34 27  NS  FI. Me.H.  0.25  124 122  22 30  27 37  NS  Tun. FI.  0.5  152 173  20 22  31 38  NS  Tun. Me . H .  0.5  152 177  20 23  31 40  NS  FI. Me.H  0.5  173 177  22 23  38 40  NS  Tun. Tun.  0.25 0.5  147 152  23 20  34 31  NS  Tun. Me.H.  0.25 0.5  147 177  23 23  34 40  NS  Tun. FI.  0.25 0.5  147 173  23 22  34 38  NS  1  7  2  3  Useful Actual useful shoot % shoot number 4  5  X  2  Legend: 1 - <each p a i r represents the set of shoot length 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 blocks 4 - a u s e f u l shoot i s defined as a shoot 1 cm or longer 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 conversion of the u s e f u l shoot % i n t o number of shoots 6 - X i s the symbol for the Chi-square t e s t ; NS = n o n - s i g n i f i c a n t 7 - l i g h t sources; Tun.= tungsten f i l a m e n t , Me.H.= metal h a l i d e , F l . = f l u o r e s c e n t  66  T a b l e 4.13 F r e s h and d r y weight means under t h r e e l i g h t s o u r c e s ( t u n g s t e n f i l a m e n t , m e t a l h a l i d e , and f l u o r e s c e n t ) combined w i t h a b l u e acetate f i l t e r . Light Source  BA Level  Fresh Weight  Dry Weight  Tungsten filament  0 .25 0. 5  45 + 3 45 + 3  6 + 1 7  Metal halide  0.25 0.5  43 50  ± 6  4  6 + 1 7 + 1  Fluorescent  0.25 0.5  37 + 3 46 + 6  6 6 + 1  Legend: 1 2 3  1  2  +  3  BA l e v e l i n mg/1 f r e s h w e i g h t / e x p l a n t mean ( i n mg) ± s t a n d a r d e r r o r dry w e i g h t / e x p l a n t mean ( i n mg) ± s t a n d a r d e r r o r  67 and m e t a l h a l i d e l i g h t . F r e s h w e i g h t / e x p l a n t means d i f f e r shoot and node number means by h a v i n g a l i g h t s o u r c e  from  effect.  I n t e r e s t i n g l y , a l t h o u g h under m e t a l h a l i d e l i g h t t h e shoot number means were l o w e r t h a n i n t h e f i r s t e x p e r i m e n t , w e i g h t / e x p l a n t means were s i m i l a r  the fresh  (43 mg and 50 mg f o r t h e 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 t h e f i r s t  experiment).  The f r e s h  weight/explant  means under t h e o t h e r two l i g h t s o u r c e s were much l o w e r t h a n i n the f i r s t  experiment.  the metal  halide-blue f i l t e r  experiments, fluorescent  Thus, s i n c e t h e photon f l u e n c e r a t e f o r  b u t much light  lower  combination  was s i m i l a r  f o r the tungsten  combinations  (see T a b l e  i n both  f i l a m e n t and  3.2), t h e r e i s the  need t o t a k e photon f l u e n c e 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 o f source c u l t u r e s t o e x p l a i n t h e l o w e r growth r a t e under t h e s e two l i g h t The  range  of dry weight/explant  sources.  means was v e r y  narrow;  between 6 and 7 mg o v e r a l l t h e t r e a t m e n t s  (Table 4.13). The  ANOVA  differences;  ( T a b l e 4.11) c o n f i r m s t h e n e g l i g i b l e  were s t a t i s t i c a l l y  significant.  means  the  emphasize  characteristic weight/explant  of  means,  These low d r y w e i g h t / e x p l a n t  generally  this  none  low  experiment.  proliferation Also,  dry weight/explant  as  means  h a l i d e l i g h t were s i m i l a r t o t h e f i r s t e x p e r i m e n t ,  rate  for  fresh  under  metal  w h i l e under  t u n g s t e n 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 t h e r e was a r e d u c t i o n , s u g g e s t i n g t h a t t h e photon f l u e n c e r a t e may be a f a c t o r r e d u c i n g growth under t h e s e two l i g h t s o u r c e s .  68  4.1.4  Experiment IV:  Determining the  e f f e c t s on  in vitro  shoot p r o l i f e r a t i o n of t h r e e l i g h t source-red f i l t e r  Tungsten  f i l a m e n t l i g h t was  used  combinations  i n the f i r s t  experiment  a s o u r c e o f r e d and f a r - r e d l i g h t b e c a u s e i t e m i t s a h i g h 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 ranges. In t h i s  red  f i l t e r e x p e r i m e n t , t h e m a i n p u r p o s e was  red  filter  could optimize the  red  light  far-red  sourcei f the  smaller proportion of red  f l u o r e s c e n t l i g h t . The  to  energy  t o determine  u n d e r m e t a l h a l i d e and light  light  ratio  (R:FR)  light  importance of the  i n determining  S p i r a e a i n v i t r o g r o w t h 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 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 (Table  fluorescent  the ratio  light  4.1).  The r e d f i l t e r , b y f i l t e r i n g o u t m o s t o f t h e b l u e - v i o l e t yellow-green wavelengths,  s h o u l d h e i g h t e n any R: FR r a t i o  ratio.  (Table  4.1),  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 the  m o d i f i e d R:FR  ratio  will  higher than that of f i l t e r e d metal halide  still  and  effect  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 c e R:FR  as  remain  the  ratio much  light.  Growth V a r i a t e s  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  level,  the  69  Table 4.14 Shpot and node number means under t h r e e l i g h t s o u r c e s ( t u n g s t e n f i l a m e n t , m e t a l h a l i d e , and f l u o r e s c e n t ) combined w i t h a r e d acetate f i l t e r . Light Source  BA Level  Shoot^ Number  Node Number  Tungsten filament  0 25 0 5  9 50 + 1 16 16 06 + 2 24  322 473  + +  5 25  Metal halide  0 25 0 5  6 81 + 1 14 11 31 + 1 43  252 349  + +  20 45  Fluorescent  0 25 0 5  7 25 + 0 81 13 81 + 0 96  216 352  + +  11 18  J  Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean ± s t a n d a r d e r r o r 3 - node n u m b e r / b l o c k (mean o f two b l o c k s ) ± error  standard  70 tungsten filament-red number mean, halide  light  followed  f i l t e r c o m b i n a t i o n had t h e h i g h e s t shoot by  fluorescent  light,  with  the metal  t r e a t m e n t b e i n g t h e l o w e s t . A t t h e 0.5 mg/1  BA  l e v e l t h e same p a t t e r n was e x h i b i t e d . The i n c r e a s e s  i n shoot number means 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 due t o BA c o n c e n t r a t i o n the t r e n d quality  observed  ( T a b l e 4.15)  reflect  f o r these l i g h t sources i n the f i r s t  light  e x p e r i m e n t . But f o r b o t h l i g h t s o u r c e s t h e d i f f e r e n t i a l  between t h e l o w e r and h i g h e r BA l e v e l s experiment than i n t h e f i r s t  i s much h i g h e r i n t h i s  experiment  ( i n t h i s experiment an  i n c r e a s e o f 66.1% under m e t a l 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 t h e f i r s t e x p e r i m e n t i n c r e a s e s o f 26.2% under m e t a l 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 ) . N e v e r t h e l e s s , the o v e r a l l  s h o o t number l e v e l s  f o r 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 w i t h t h e r e d f i l t e r were q u i t e s i m i l a r  i n this  experiment  and much c l o s e r t h a n t h e l e v e l s  o b s e r v e d i n Experiment I . S i n c e  the  light  R:FR  different ratios  ratios using  of  t h e two  the red f i l t e r ,  caused t h e s i m i l a r  sources  remained  i t seems u n l i k e l y  proliferation  quite  that  r a t e . Another  these  possible  factor i s the blue t o red r a t i o  (B:R) . The B:R r a t i o o f m e t a l  halide with the red f i l t e r w i l l  be l o w e r t h a n i n Experiment  I  and c l o s e r t o t h e B:R r a t i o under f l u o r e s c e n t l i g h t w i t h t h e r e d filter. causing  Perhaps the  then  t h e B:R  similarities  ratio  between  i s an the  important  metal  factor  halide  and  f l u o r e s c e n t s h o o t number means. F i n a l l y , t h e q u a n t i t y o f r e d l i g h t may a l s o be i m p l i c a t e d as  71  T a b l e 4.15  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 o n s h o o t a n d node n u m b e r s , a n d f r e s h a n d d r y w e i g h t s  Light  of Variation source T/MeF T/F 5  Dry wt.  df  Node number  (2.) 1 1  * ** *  * ** **  ** ** * ***  ** *** ***  1  Source  Fresh wt.  Shoot number  2  3  4  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 n u m b e r / b l o c k o f 8 s u b - s a m p l e s ( t o t a l n o d e s o f shoots of 8 explants ) 3 - fresh weight/explant 4 - dry weight/explant 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 a n d 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 = tungsten f i l a m e n t versus fluorescent 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 = non-significant  an  i m p o r t a n t s h o o t i n i t i a t i o n f a c t o r . As shown i n T a b l e 3.1,  fluorescent wavelengths, Thus,  emits  metal  26.6% o f i t s energy  halide  t h e h i g h e s t shoot  tungsten high  light  filament  light  r e d wavelength  output  as  red  18.8% and t u n g s t e n f i l a m e n t 59.4%. number  means  may s u g g e s t  output  which  were caused by  that this  promoted  shoot  light  source's  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 t h e f l u o r e s c e n t - r e d  filter  compared t o t h e m e t a l h a l i d e - r e d  c o u l d t h e n be  filter  regime  regime  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 h i g h e r r e d wavelength output.  Node  number/block of  eight  sub-samples  The node number means d i f f e r number means  i n two ways from t h e s h o o t  ( T a b l e 4.14). F i r s t ,  a t t h e 0.25 mg/1 BA l e v e l ,  m e t a l h a l i d e l i g h t has a s l i g h t l y h i g h e r node number mean t h a n fluorescent  light.  Secondly,  a t t h e 0.5 mg/1 BA l e v e l ,  both  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 means a r e v i r t u a l l y t h e same. These  results  produce  suggest  that  a greater effect  the modified  on s h o o t  wavelength  initiation  than  ratios  on n o d a l  differentiation. The h i g h e s t node number mean a t b o t h BA l e v e l s , i s , a s f o r shoot number means, under t u n g s t e n f i l a m e n t l i g h t . A g a i n , as f o r s h o o t s , t h e degree o f d i f f e r e n c e between t h e t u n g s t e n f i l a m e n t node number means and t h o s e o f t h e o t h e r two l i g h t s o u r c e s i s indicated  b y t h e ANOVA where t h e l i g h t  source  factor  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 o f T/MeF and T/F i n  caused  73 T a b l e 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  concentration  due t o BA  ( T a b l e 4.15), w i t h fewer nodes a t t h e 0.25 mg/1  l e v e l t h a n a t t h e 0.5 mg/1  BA  BA  level.  Shoot l e n g t h  The s h o o t l e n g t h comparisons  a t b o t h BA l e v e l s 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 f l u o r e s c e n t l i g h t , and o f m e t a l h a l i d e l i g h t with fluorescent 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 t u n g s t e n f i l a m e n t c u l t u r e s a r e compared t o metal  halide  cultures  at  both  BA  levels,  there  are  significant  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  proportions  f o r both tungsten filament  l i g h t are s i m i l a r filament mg/1  ( a t t h e 0.25  l i g h t and 4 9%  BA l e v e l  mg/1  l i g h t and m e t a l  BA l e v e l  no  shoot halide  45% f o r t u n g s t e n  f o r m e t a l h a l i d e l i g h t , and a t t h e  0.5  37% f o r t u n g s t e n f i l a m e n t l i g h t and 41% f o r m e t a l  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  p e r c e n t a g e s a r e much l o w e r t h a n t h e p e r c e n t a g e s under t u n g s t e n filament 0.25  mg/1  and m e t a l h a l i d e l i g h t BA l e v e l  (for fluorescent  i t i s 28% and a t t h e 0.5 mg/1  l i g h t a t the BA l e v e l i t i s  26%) . The  high useful  tungsten filament  shoot p r o p o r t i o n s  light,  emphasize t h e i m p o r t a n c e  under m e t a l h a l i d e  e s p e c i a l l y under m e t a l h a l i d e of f a r - r e d l i g h t  i n promoting  and  light, shoot  e l o n g a t i o n . A l t h o u g h t h e r e d f i l t e r reduces t h e s m a l l amount of  T a b l e 4.16 S h o o t l e n g t h s a s a f f e c t e d by t h r e e red f i l t e r combinations. Pairs tested  BA con.  Sample size  Tun. Me.H.  0.25  152 109  45 49  1  7  2  3  Useful shoot % 4  light  source-  Actual useful s h o o t number  5  68 53  X  6 2  NS *****  Me.H. Fl.  0. 25  109 116  49 28  28  Tun  0.25  152 116  45 28  68 33  Tun. Fl.  0.5  257 221  37 26  94 57  ***  Tun. Me.H.  0.5  257 181  37 41  94 74  NS  Me.H. Fl.  0.5  181 221  41 26  74 57  *****  Tun. Tun.  0.25 0.5  152 257  45 37  68 94  Tun. Me.H.  0.25 0.5  152 181  45 41  68 74  Tun. Fl.  0.25 0.5  152 221  45 26  57  53  ***  68  NS NS >  *****  Legend: 1 - each p a i r represents t h e s e t o f shoot length proportions analyzed i n a Chi-square test 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 experimental blocks 4 - a u s e f u l shoot i s d e f i n e d as a shoot 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 % o f t h e sample s i z e ranked u s e f u l 5 - the conversion o f the u s e f u l shoot % i n t o number o f s h o o t s 6 - X i s t h e symbol f o r t h e 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 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 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  far-red  light  under  metal  halide  light,  transmitted  t o promote shoot e l o n g a t i o n .  unfiltered,  e m i t s 9.8%  spectrum  and  of i t s energy  far-red  region  comparison u n f i l t e r e d  as  enough  was  still  Metal halide  light,  o u t p u t from t h e far-red  fluorescent light  visible  wavelengths.  e m i t s 1.2%  as  far-red  w h i c h would be r e d u c e d u s i n g t h e r e d f i l t e r and t h u s t h i s account  f o r the  lower  useful  shoot  percentages  In  under  may this  regime. In  economic t e r m s ,  proportions  of  proportions  useful  under  concentrations  a l t h o u g h under m e t a l h a l i d e  of  shoots  were  tungsten BA,  the  slightly  filament  greater  light  the  higher than  the  light  shoot  for  both  production  under  t u n g s t e n f i l a m e n t l i g h t compensated f o r t h e l o w e r p r o p o r t i o n s . Thus, w h i l e a t t h e 0.2 5 mg/1  BA  produced  light,  under  metal  halide  produced 68 s h o o t s . A t t h e 0.5 mg/1 light,  53  useful  tungsten  s h o o t s were  filament  light  BA l e v e l , under m e t a l h a l i d e  74 s h o o t s were produced, w h i l e t u n g s t e n f i l a m e n t  produced An  level  light  94. interesting  observation i s that  for a l l three  light  s o u r c e s h i g h e r u s e f u l shoot p e r c e n t a g e s were found a t t h e l o w e r BA c o n c e n t r a t i o n ; t h i s the  cytokinin  level  was  s u p p o r t s p r e v i o u s work where d e c r e a s i n g found t o enhance s h o o t e l o n g a t i o n i n  P y r u s ( S t i m a r t and Harbage, 1989).  F r e s h w e i g h t / e x p l a n t and Dry weight/explant  The f r e s h w e i g h t means ( T a b l e 4.17) r e f l e c t t h e node number trends.  B u t u n l i k e t h e s h o o t and node number means, where BA  c o n c e n t r a t i o n caused t h e 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 t h e l i g h t s o u r c e f a c t o r (ANOVA c o n t r a s t s o f T/MeF and T/F i n T a b l e 4.15). The  range  despite t h i s , light  o f d r y weight  means was narrow  i t was g r e a t e r t h a n t h e range  source-blue  filter  experiment.  This  (Table  4.17);  observed i n t h e difference  is  i n d i c a t e d by t h e ANOVA ( T a b l e 4.15), where t h e d i f f e r e n c e s due to  light  means.  s o u r c e were as s i g n i f i c a n t  as f o r t h e f r e s h  weight  77 T a b l e 4.17 F r e s h and dry weight means under t h r e e l i g h t s o u r c e s ( t u n g s t e n f i l a m e n t , m e t a l h a l i d e , and f l u o r e s c e n t ) combined w i t h a r e d acetate f i l t e r . Light Source  BA Level  Fresh Weight  Dry Weight  Tungsten filament  0 .25 0. 5  56 ± 7 89 ± 11  9 ± 1 11 ± 1  Metal halide  0 . 25 0.5  43 ± 6 58 ± 6  7 + 1 8 ± 1  Fluorescent  0.25 0.5  34 ± 3 57 + 4  5 7 ± 1  1  2  3  Legend: 1 - BA l e v e l i n mg/1 2 - f r e s h w e i g h t / e x p l a n t mean ( i n mg) ± s t a n d a r d e r r o r 3 - d r y w e i g h t / e x p l a n t mean ( i n mg) + s t a n d a r d e r r o r  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 far-red  light  exposure a p p l i e d  end o f a w h i t e l i g h t  In t h e f i r s t mixture  was  applied  light  shown  a tthe  photoperiod  q u a l i t y experiment a r e d / f a r - r e d  t o a f f e c t an e x p l a n t ' s  cytokinin  of a short-term  BA.  The  utilization  red/far-red  BA l e v e l p r e v i o u s l y e s t a b l i s h e d as optimal  cultures) red/far-red l i g h t stimulated production  of the  light-cytokinin  i n t e r a c t i o n was such t h a t a t t h e BA l e v e l of 0.25 mg/1 the  light  (one-half  f o r maintaining  s i g n i f i c a n t l y more shoot  than i n white c o n t r o l or b l u e  l i g h t treatments. The  p h o t o p e r i o d used was 16 hours. One p o s s i b l e e x p l a n a t i o n  i s that  the  reduced  photoperiod  of  continuous  red/far-red  endogenous f r e e IAA l e v e l s and thus m o d i f i e d auxin  light  shoot c y t o k i n i n t o  ratios.  The p r e s e n t  end-of-day experiment was done t o determine t h e  importance o f t h e r e d and f a r - r e d l i g h t p h o t o p e r i o d i n t h e f i r s t experiment. plants the  A  typical  end-of-day  to a far-red l i g h t  beginning  treatment  interval  o f t h e dark  period  involves  exposing  ( f a r - r e d extension) t o cause  shoot  before  elongation  (Downs e t a l . , 1957). F o r t h i s experiment t h e end-of-day growth chamber had a metal h a l i d e l i g h t on f o r 16 hours f o l l o w e d hours  of  control  light  chamber  supplied had  by  a metal  a  tungsten  halide  filament  light  on  source.  f o r an  18  by 2 The hour  photoperiod. For  t h e end-of-day  exposure  u n f i l t e r e d tungsten  filament  l i g h t was used. T h i s c h o i c e was based on work where  end-of-day  stem e x t e n s i o n o f s o i l grown p l a n t s was s t u d i e d under a m i x t u r e of  r e d and  far-red  light  (Buck  and V i n c e - P r u e , 1985).  The  m i x t u r e o f r e d and f a r - r e d l i g h t was shown t o be about h a l f as e f f e c t i v e as f a r - r e d initial  elongation  successive  the  of  internodes  decreased. With end-of-day  light  later  an  alone i n causing increases i n the expanding  developed, expanding  internode.  the  response  However,  as  to  far-red  internodes tungsten  filament  e x p o s u r e s i n c r e a s e d e x t e n s i o n compared t o f a r - r e d ;  response  appeared  to  increase  as  duration  of tungsten  f i l a m e n t e x p o s u r e i n c r e a s e d . S i n c e t h e r e was a g r e a t e r  far-red  l i g h t p r o p o r t i o n under t h e u n f i l t e r e d t u n g s t e n f i l a m e n t  light,  a s h o o t e l o n g a t i o n r e s p o n s e would perhaps predominate o v e r any shoot i n i t i a t i o n response f o r t h i s S p i r a e a i n v i t r o experiment. A l a c k o f e i t h e r o f t h e s e two r e s p o n s e s , o r a l a c k o f b o t h , would  lend  credence  to the proposal that  a  photoperiod of  c o n t i n u o u s r e d 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  Shoot  Variates  number/explant  F o r t h i s e x p e r i m e n t a g r e a t e r range o f BA c o n c e n t r a t i o n s was used  t o determine  concentration-shoot  i f there number  were any d e v i a t i o n s trends  e x p e r i m e n t s . As can be seen means  under  exhibited  metal  a linear  concentration  halide  observed  from  i n the  previous  i n T a b l e 4.18, t h e shoot number light  with  a  far-red  extension,  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 ;  increased,  t h e BA  the  shoot  number  as t h e BA  increased.  This  g e n e r a l t r e n d was o b s e r v e d i n t h e p r e v i o u s e x p e r i m e n t s f o r a l l l i g h t q u a l i t y regimes e x c e p t f o r red/FR l i g h t . The absence o f a maximal s h o o t number mean a t t h e 0.25 mg/1 under  red/FR  prolonged  light  exposures  i n the f i r s t o f red/FR  BA l e v e l ,  experiment,  light  as noted  suggests  are required  that  t o enhance  shoot i n i t i a t i o n a t l o w e r c y t o k i n i n c o n c e n t r a t i o n s . Under m e t a l h a l i d e w i t h o u t a f a r - r e d e x t e n s i o n , t h e l i n e a r trend  i s d i s r u p t e d ; t h e maximal mean was a t t h e 0.4 mg/i BA  level  i n s t e a d o f a t t h e 0.5 mg/1 BA l e v e l . T h i s r e s u l t s u g g e s t s  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  interaction  o c c u r s a t t h e 0.4 mg/1 BA l e v e l . Such a peak would n o t have been o b s e r v e d i n t h e f i r s t e x p e r i m e n t due t o t h a t e x p e r i m e n t ' s l a r g e r i n c r e m e n t a l 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 o f t h e 0.4 mg/1 peak, when a f a r - r e d e x t e n s i o n was  8 1  T a b l e 4.18 Shoot ,.and node number means as a f f e c t e d by a s h 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 o f a white l i g h t p h o t o p e r i o d . Light Quality  BA Level  Shoot Number  Metal h a l i d e + Red/FR  0. 1 0. 25 0. 4 0. 5  6 . 58 10 .75 16 . 25 19 .50  + + + +  1. 28 2. 08 2. 01 2 .94  87 139 205 247  + + + +  14 24 31 32  Metal  0 .1 0. 25 0. 4 0 .5  7 . 08 11 . 58 20 .33 17 . 33  + + + +  0. 85 1. 65 3. 27 3. 11  89 154 251 199  + + + +  11 14 35 22  halide  1  Node Number  2  3  Legend: 1 - BA l e v e l i n mg/1 2 - shoot number/explant mean ± s t a n d a r d e r r o r 3 - node number/block (mean o f f o u r b l o c k s ) ± s t a n d a r d error  82 applied,  p r o v i d e s e v i d e n c e t h a t a weak red/FR e f f e c t  The two h o u r exposure t o f a r - r e d  l i g h t may have s h i f t e d t h e  phytochrome e q u i l i b r i a i n d u c e d by m e t a l h a l i d e l e s s conducive t o a BA-light  occurred.  l i g h t t o a state  q u a l i t y i n t e r a c t i o n a t t h e 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 t h u s have l e d t o a l o s s o f the  0.4 mg/1 BA peak. As  i n d i c a t e d by t h e ANOVA ( T a b l e 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 m e t a l h a l i d e ANOVA  also  differences quality  indicates  l i g h t with  that  there  a far-red  a r e no  e x t e n s i o n . The  shoot  number mean  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 o r a  i n t e r a c t i o n ; t h i s provides further  BA-light  evidence that  only  p r o l o n g e d red/FR l i g h t e x p o s u r e s a r e c a p a b l e o f p r o m o t i n g shoot i n i t i a t i o n a t l o w e r BA c o n c e n t r a t i o n s .  Node  number/block  of  three  The same p a t t e r n exhibited  sub-samples  followed  by t h e s h o o t number means was  by t h e node number means  (Table  4.18). A g a i n , t h e  ANOVA emphasizes a s t r o n g l i n e a r r e l a t i o n ; t h i s t i m e between t h e BA c o n c e n t r a t i o n s and t h e node number means  (Table 4.19).  As  f o r s h o o t number means t h e r e i s no o t h e r f a c t o r a f f e c t i n g shoot initiation  (no l i g h t  interactions).  quality  effects  or BA-light  quality  Table  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 o f a s h o r t - t e r m f a r - r e d l i g h t - i exposure a p p l i e d a t the end o f 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 Source o f v a r i a t i o n  df  Light  1  BA  quality  concentration linear quadratic cubic 5  BA a n d l i g h t interaction  (3) 1 1 . 1  I Shoot number NS ***** ***** NS NS  3v  Node number  Fresh wt.  NS  •NS  ii "k it it k  ie it it it it  •k -k ie ~k ic  it it it -k it  NS NS  NS NS '  NS  NS  3  Dry wt.  if  NS ***** **** * NS NS  quality 3  NS  NS  Legend: 1 - shoot number/explant 2 - node number/block, o f 3 s u b - s a m p l e s ( t o t a l nodes o f shoots of 3 explants ) 3 - fresh weight/explant 4 - dry weight/explant 5 - p o l y n o m i a l r e s p o n s e m o d e l s : 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 b y 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 = 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 < O.'OI, ***** = a t p < 0.005, NS = n o n - s i g n i f i c a n t  84 Shoot  length  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 t h e p a t t e r n o f shoot and node  number means w i t h  Again  the  metal  similar  linear  light  without  halide  e x h i b i t e d t h e same peak a t t h e 0.4 mg/1  trends a  (Table  far-red  4.20).  extension  BA l e v e l . A l t h o u g h t h e  comparisons of t h e f o u r BA l e v e l s under m e t a l h a l i d e l i g h t w i t h a f a r - r e d e x t e n s i o n 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 , due t o t h e p r o p o r t i o n s a t t h e 0.1 mg/1 BA  level  (49%  comparisons metal  59%  BA l e v e l and t h e 0.5  respectively).  None  of  mg/1  the  pair  of m e t a l h a l i d e p l u s a f a r - r e d e x t e n s i o n w i t h t h e  halide  suggest  and  i t was  that  control the  showed  typical  any  difference.  end-of-day  far-red  These  results  response  of  i n c r e a s e d shoot l e n g t h does n o t o c c u r i n t h e S p i r a e a i n v i t r o system.  Thus,  i t appears  that  only prolonged  red/FR  light  e x p o s u r e s a r e c a p a b l e o f e n h a n c i n g 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 s h o o t i n i t i a t i o n , a t l o w e r BA  levels.  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  Both  the  fresh  and  dry  weight  means  followed  the  same  p a t t e r n as shoot and node number means ( T a b l e 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 b o t h f r e s h and d r y w e i g h t s was shown ( T a b l e 4.19).  T a b l e 4.20 Shoot lengths as a f f e c t e d by a s h 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 at the end of a white l i g h t photoperiod 6  Pairs tested  BA con.  Sample size  MH+FR MH+FR MH+FR MH+FR  0.1 0.25 0.4 0.5  79 129 0195 234  49 50 56 59  39 64 109 137  *  MH MH MH MH  0.1 0 . 25 0.4 0.5  85 139 244 208  49 53 57 53  42 73 140 110  NS  MH+FR MH  0.1  79 85  49 49  39 42  NS  MH+FR MH  0.25  129 139  50 53  64 73  NS  MH+FR MH  0.4  195 244  56 57  109 140  NS  MH+FR MH  0.5  234 208  59 53  137 110  NS  MH+FR MH  0.1 0. 5  79 208  49 53  39 110  NS  MH+FR MH  0 .25 0. 5  129 208  50 53  64 110  NS  MH+FR MH  0.4 0.5  . 195 208  56 53  109 110  NS  1  7  2  3  Useful shoot % 4  Actual useful shoot number  5  X  2  Legend: 1 - each p a i r represents the set of shoot length 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 blocks 4 - a u s e f u l shoot i s defined as a shoot 1 cm or longer 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 conversion of the u s e f u l shoot % i n t o number of shoots 6 - X i s the symbol for the Chi-square t e s t ; the a s t e r i s k s define 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<0.1, NS = n o n - s i g n i f i c a n t 7 - l i g h t treatments: MH = metal h a l i d e , MH+FR = metal h a l i d e + f a r - r e d l i g h t exposure  T a b l e 4.21 F r e s h ,and d r y weight means as a f f e c t e d by a s h 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 o f a white l i g h t p h o t o p e r i o d . Light Quality  BA Level  Fresh^ Weight  Metal h a l i d e '+ Red/FR  0.1 0.25 0.4 0.5  71 138 213 268  12 26 29 38  14 23 32 36  ± + + +  2 3 4 4  Metal h a l i d e  0.1 0.25 0.4 0.5  86 + 10 155 + 20 285 + 35 241 + 40  17 25 41 32  + + + +  2 2 4 4  Legend: 1 2 3  + + ± +  Dry Weight 3  BA l e v e l i n mg/1 f r e s h w e i g h t / e x p l a n t mean ( i n mg) + standard' e r r o r dry w e i g h t / e x p l a n t mean ( i n mg) ± standard e r r o r  87  4.2  F a c t o r s c o n t r i b u t i n g t o the red/FR  4.2.1  l i g h t growth  response  Experiment V I : 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 time t o a higher photon f l u e n c e  In  the f i r s t  response  s e t of experiments  of Spiraea  explants  was  rate  where t h e l i g h t  characterized,  a  quality two week  i n c u b a t i o n p e r i o d was used so t h a t i n v i t r o p l a n t l e t s o f s i m i l a r size  and v i g o u r  light  quality  attempt growth  could  treatments.  t o minimize rates  be s e l e c t e d f o r t h e f u r t h e r t h r e e This  variability  and v i g o u r ,  so t h a t  selection  procedure  due t o e x p l a n t s any subsequent  1  week  was  an  differing differences  under t h e l i g h t q u a l i t y treatments c o u l d be assumed t o be l i g h t quality The  effects. incubation  period  was under  white  light  photon f l u e n c e r a t e than t h a t o f the experimental regimes. Thus, influencing initial  of a  higher  light quality  i n t h i s second s e c t i o n o f t h e t h e s i s two f a c t o r s  shoot i n i t i a t i o n were examined; t h e s e i n c l u d e d an  i n c u b a t i o n p e r i o d under red/FR o r white l i g h t o f a low  photon f l u e n c e r a t e and v a r i a t i o n of t h e time o f t r a n s f e r t o a higher  fluence rate.  The experiment t o examine t h e two f a c t o r s , u t i l i z e d  three  d i f f e r e n t growth chamber c o n d i t i o n s . Two chambers were used f o r an i n i t i a l  low photon  and t h e o t h e r  red/FR  f l u e n c e r a t e , one u t i l i z i n g white light.  The t h i r d  chamber, a white  light light  88  chamber, had a h i g h e r photon f l u e n c e r a t e s i m i l a r t o t h a t for maintaining  stock  c u l t u r e s and f o r t h e i n i t i a l  p e r i o d of the f i r s t s e t of experiments.  used  incubation  To d e t e r m i n e t h e e f f e c t  t h a t t r a n s f e r t i m e and d i f f e r e n t l i g h t t r e a t m e n t s had,  cultures  were t r a n s f e r r e d a f t e r two and f o u r weeks from t h e low f l u e n c e r a t e chambers t o t h e t h i r d chamber.  photon  One s e t o f c u l t u r e s  was k e p t under each low f l u e n c e regime f o r t h e 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 to a higher  r a t e were n e c e s s a r y t o o p t i m i z e  fluence  growth.  Growth V a r i a t e s  Shoot  number/explant  Each t r e a t m e n t u t i l i z i n g w h i t e l i g h t o n l y , r e s u l t e d i n l o w e r s h o o t numbers t h a n t r e a t m e n t s i n which  red/FR l i g h t was  used  (Table 4.22). C u l t u r e s under w h i t e l i g h t , where a t r a n s f e r t o a higher  photon  fluence  rate  was  used,  produced  shoot  w h i c h i n c r e a s e d w i t h t h e l e v e l o f BA. The f i v e week low white  light  level;  gave r e l a t i v e l y  low  t h e l e v e l s were s i m i l a r ,  shoot  initiation  greatest  different mg/1  differences  light  fluence  a t each  BA  i n d i c a t i n g that increasing  BA  c o 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 t h e s e l i g h t The  numbers  i n shoot p r o l i f e r a t i o n  conditions. seen  t r e a t m e n t s were t h o s e i n c l u d i n g 0.25  between and  0.4  BA. A t t h e s e lower BA l e v e l s t h e h i g h e s t shoot numbers were  obtained  a f t e r f o u r weeks of low f l u e n c e red/FR f o l l o w e d by  one  89  Table 4.22 Shoot and node number 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  BA Level  Shoot 3 Number  2  0.25 0.4 0.5  16 19 21  ± ± ±  1.48 3.24 2.63  256 297 300  ± ± ±  42 92 18  Red/FR  2  0.25 0.4 0.5  22 20 32  ± ± ±  2.13 3.07 4.53  309 303 464  ± + +  32 55 103  White  4  0.25 0.4 0.5  15 16 19  ± + +  2.87 2.24 3.20  178 203 248  ± ± +  60 37 33  Red/FR  4  0.25 0.4 0.5  26 33 33  + ± ±  3.91 3.88 3.87  357 395 419  ± ± ±  83 58 86  White  5  0.25 0.4 0.5  16 15 15  ± ± ±  1.76 3.04 1.64  198 197 174  ± ± ±  42 61 39  Red/FR  5  0.25 0.4 0.5  21 19 30  ± + ±  2.13 2.67 4.24  265 234 388  + + +  29 75 119  Light Quality  Wks.  White  Legend:  2  1 2 3 4  -  1  Node 4 Number  Weeks o f exposure t o low photon f l u e n c e r a t e . BA l e v e l i n mg/1. Shoot number mean ± standard e r r o r . Node number/block (mean o f two b l o c k s ± s t a n d a r d e r r o r ) .  week of h i g h e r f l u e n c e w h i t e  l i g h t . The  i n d u c t i o n under red/FR o c c u r r e d f o r the  f i v e week low  lowest l e v e l s of shoot  a t t h e 0.25  and  f l u e n c e or f o r t h e two  0.4  BA  levels  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 t h e h i g h e s t BA l e v e l of 0.5 produced  a  similar  high  level  of  shoots  whether  or  mg/1 not  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 w h i t e l i g h t was used. T h i s s u g g e s t s that  for  red/FR  specific  light  light  quality  both and  f l u e n c e r a t e are important.  the the  length  of  transfer to  exposure a  to  higher  the  photon  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 s h o o t 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 lower  BA  rate  is  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 h i g h e r needed  to  optimize  growth  f o l l o w i n g the  the  fluence  effect  of  red/FR. The the  0.5  light  mg/1  BA  treatments  gave t h e h i g h e s t l e v e l s of growth f o r a l l that  included  red/FR  incubation.  These  l e v e l s were s i m i l a r t o t h e o p t i m a l l e v e l o b t a i n e d w i t h 0.4 BA under t h e f o u r week red/FR i n i t i a l t r e a t m e n t . that  this  concentration  of  BA  (0.5  mg/1)  mg/1  This i n d i c a t e s  can  achieve  the  r e q u i r e d t h r e s h o l d f o r o p t i m a l growth even i n t h e absence of a high  fluence  light  exposure.  Thus,  the  higher  BA  level,  o p t i m i z i n g t h e i n t e r a c t i o n o r synergy w i t h red/FR, may to  overcome any  modulating  influence resulting  f l u e n c e red/FR exposure t i m e . Lower BA growth  with  one  specific  light  be  able  from t h e  l e v e l s produced  regimen,  by  i . e . four  low  optimal  weeks  of  red/FR f o l l o w e d by one week o f h i g h e r f l u e n c e w h i t e . The r e s u l t s emphasize an i n t e r a c t i o n o r synergy o f BA and red/FR l i g h t w h i c h i s dependent quality.  on t h e exposure t i m e o f c u l t u r e s t o s p e c i f i c l i g h t  A t lower  BA l e v e l s ,  perhaps  t h e two weeks o f low  f l 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 w a v e l e n g t h whereas t h e f i v e h i g h e r photon  week red/FR  lacked  a subsequent  stimulus period of  fluence.  The ANOVA (Table 4.23) i n d i c a t e s  a significant interaction  between l i g h t q u a l i t y and weeks o f 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 . T h i s i n t e r a c t i o n , and t h e s i g n i f i c a n c e o f weeks o f exposure emphasizes fluence  t h e importance  light.  concentration difference  ( t r a n s f e r time) as an i n d i v i d u a l  The  of transfer  effect  on  c a n be m a i n l y  time  shoot  attributed  factor,  t o high  number  due  photon t o BA  t o the usual  wide  i n shoot number means between t h e 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 quality  and weeks o f exposure t o t h e s p e c i f i c w a v e l e n g t h and  fluence rate.  Node number/block  o f 4 sub-samples  The t r e n d s n o t e d f o r shoot number means were more s t r o n g l y expressed importance  with  t h e node  of the transfer  number from  means  (Table  4.22).  t h e lower t o h i g h e r  The  photon  f l u e n c e r a t e f o r t h e w h i t e t r e a t m e n t s was emphasized. There were w h i t e l i g h t node number d e c r e a s e s a t a l l BA l e v e l s from t h e two  92 ;  T a b l e 4.23 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 exposure t o a low p h o t o n f l u e n c e r a t e , 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 , 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 w e i g h t s Shoot number 1  Source o f V a r i a t i o n Light quality  df 1  NS  Transfer time linear $ lack of f i t  (2) 1 1  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 t r a n s f e r time linear lack of f i t  (2) 1 1  Node number NS  NS  NS  ***** ***** *****  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 and BA c o n c e n t r a t i o n linear lack of f i t  (2) 1 1  NS NS NS  NS NS NS  I n t e r a c t i o n between light quality, t r a n s f e r t i m e , and BA c o n c e n t r a t i o n  1  *  df F * ** *** **** ***** NS  Dry wt.  ***** ***** *  (2) 1 1  Legend: 1  F rwt. esh  ***** ***** ****  *  concentration linear lack of f i t  BA  3  NS  NS  NS  NS  **  *  s h o o t number/explant, 2 - node number/block, 3 fresh weight/explant, 4 - dry weight/explant, 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 , l a c k o f f i t = amount o f 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 d e g r e e o f freedom t e s t of significance s i g n i f i c a n t a t 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 a t p < 0.025 s i g n i f i c a n t a t p < 0.01 s i g n i f i c a n t a t p < 0.005 non-significant  week . t r a n s f e r regime t o t h e f o u r week t r a n s f e r regime, and a f u r t h e r d e c r e a s e f o r two BA l e v e l s from t h e f o u r t o f i v e regime. F o r red/FR c u l t u r e s a t t h e 0.25 and 0.4 mg/1 BA the  optimal  means  were  again  reached  with  the  week  levels  four  week  trends.  The  exposure. The ANOVA  (Table  4.23) c o n f i r m s  the stronger  l i n e a r node number d e c r e a s e s f o r w h i t e l i g h t c u l t u r e s as l e n g t h of  exposure t o t h e lower  explain  the highly  exposure  fluence  significant  (transfer time).  quality-weeks number  photon  The  linear much  rate trend  more  increased,  may-  f o r weeks  significant  of  light  o f 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  means as compared t o s h o o t  number  means,  i s further  e v i d e n c e f o r t h e g r e a t e r e f f e c t o f weeks o f exposure ( t r a n s f e r time) on node numbers. exposure  may  concentration  explain  This the  greater less  on node numbers  e f f e c t due t o weeks o f  significant  than  that  effect  of  BA  observed  f o r shoot  under w h i t e  l i g h t with  numbers.  Shoot l e n g t h  A comparison of the nine treatments  t h o s e under red/FR l i g h t , shows t h a t f o u r t r e a t m e n t s under w h i t e l i g h t r e s u l t e d i n u s e f u l s h o o t p r o p o r t i o n s 40% o r o v e r , w h i l e o f t h e red/FR l i g h t t r e a t m e n t s , o v e r 40% (Table  e i g h t had u s e f u l shoot p r o p o r t i o n s  4.24). F o r t r e a t m e n t s  where t h e 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 k e p t under a low  Table  4.24  Shoot lengths r a t e and t h e BA  T r e a t s .' tested  White  2  Red/FR  White  4  Red/FR  White  wks.  wks.  4  5  Red/FR  0.25 0. 4 0.5  wks.  2  wks.  wks.  5  l e v e l^ (mg/1)  wks.  as a f f e c t e d t r a n s f e r to Soa m p l,e size  3  by a low photon a higher fluenca  Useful"* shoot %  fluence rate  Actual^ useful shoot number  X  *  58 89 75  40 39 28  23 35 21  76 74 128  28 47 46  21 35 59  0.25 0.4 0.5  71 57 78  37 47 47  26 27 37  NS  0.25 0.4 0.5  115 132 131  44 45 50  51 59 65  NS  0 .25 0.4 0.5  62 75 58  34 48 38  21 36 22  0 .25 0. 4  81 82 129  41 45 53  33 37 69  0 . 25 0 . 4 0.5  o.s  *****  *  *  W h i t e 2 wks. Red/FR 2 wks.  0.5 0.25  75 76  28 28  21 21  W h i t e 2 wks. Red/FR 2 wks.  O.S 0.4  75 74  28 47  21 35  W h i t e 4 wks. R e d / F R 4 wks.  0.5 0.25  78 115  47 44  37 51  NS  W h i t e 4 wks. R e d / F R 4 wks.  0.5 0 . 4  78 132  47 45  37 59  NS  W h i t e 5 wks. R e d / F R 5 wks.  0. 5 0 .25  58 81  38 41  22 33  . NS  W h i t e 5 wks. R e d / F R 5 wks.  0 . 5 0 . 4.  58 82  38 . 45 '  22 37  NS  Legend:  1 -  2 3  -  4  -  5  -  5  -  '  ;  each p a i r represents the s e t of shoot length proportions analyzed i n a Chi-square test BA l e v e l i n mg/1 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 experimental blocks i a u s e f u l shoot i s defined as a s h o o t 1 | cm o r l o n g e r i n length; thus the useful' shoot V i s the % o f t h e sample s i z e ranked useful the conversion of the useful shoot % i n t o number o f s h o o t s X i s t h e symbol f o r the C h i - s q u a r e test; the asterisks define the degree 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 . 0 0 5 , NS = non-significant  NS  ***  f l u e n c e r a t e f o r f i v e weeks, t h e 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 o r g r e a t e r p r o p o r t i o n o f l o n g e r s h o o t s t h a n t h e c o r r e s p o n d i n g w h i t e l i g h t c u l t u r e s w i t h 0.5 mg/1 BA. When t h e u s e f u l shoot p e r c e n t a g e s a r e compared f o r t h e 0.25 mg/1 BA red/FR and t h e 0.5 mg/1 BA w h i t e l i g h t c u l t u r e s a t t h e f o u r week exposure, t h e r e i s l i t t l e and  difference  (47% f o r w h i t e  44% f o r red/FR) . Thus i t appears t h a t t h e e f f e c t o f t h i s  f o u r week red/FR exposure was more c r i t i c a l i n d e t e r m i n i n g shoot initiation because  than  i n determining  shoot  o f t h e o p t i m a l shoot i n i t i a t i o n  elongation.  However,  response a t t h e four  week e x p o s u r e f o r red/FR c u l t u r e s t h e a c t u a l number o f l o n g e r s h o o t s 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 ) .  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 p a t t e r n  o f f r e s h w e i g h t means ( T a b l e 4.25)  generally  f o l l o w e d t h a t o f t h e node number means. The s i g n i f i c a n c e o f t h e effects time)  caused by t h e f a c t o r s o f weeks o f exposure  and BA  exposure  concentration,  (transfer  time)  and t h e l i g h t  interaction  (transfer  q u a l i t y - w e e k s of  were g r e a t e r  f o r fresh  w e i g h t means ( T a b l e 4.2 3)- t h a n f o r shoot number means.  Another  d i f f e r e n c e was t h e 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 w e i g h t means, an i n t e r a c t i o n n o t seen w i t h s h o o t number means. The amount o f change e x p l a i n e d by t h e l i g h t q u a l i t y - w e e k s of e x p o s u r e ( t r a n s f e r time) i n t e r a c t i o n and t h e a d d i t i o n a l l i g h t quality-BA  interaction,  indicates  the  importance  of  light  96  T a b l e 4.25 F r e s h and d r y 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 o f t r a n s f e r t o a higher fluence rate Light Quality  BA Level  Fresh Weight  Wks.  White  2  0.25 0.4 0.5  179 219 267  ± ± +  20 30 38  29 34 36  ± ± ±  2  Red/FR  2  0.25 0.4 0.5  216 218 392  + + +  25 37 53  35 31 49  ± ± ±  3 5 5  White  4  0.25 0.4 0.5  104 131 166  + ± +  21 18 23  16 18 24  ± ± ±  2 2 3  Red/FR  4  0.25 0.4 0.5  240 297 317  ± ± +  48 45 50  33 36 39  ± ± ±  6 4 4  White  5  0.25 0.4 0.5  105 118 115  + + +  13 23 17  14 16 14  ± ± ±  1 2 1  Red/FR  5-  0.25 0.4 0.5  163 176 284  + ± ±  20 35 56  20 19 30  ± ± ±  2 3 5  Legend: 1 2 3 4  2  -  1  Dry Weight  3  4  3 4  Weeks o f exposure t o low photon f l u e n c e r a t e BA l e v e l i n mg/1 F r e s h weight mean ± s t a n d a r d e r r o r (per e x p l a n t ) i n mg Dry weight mean ± s t a n d a r d e r r o r (per e x p l a n t ) i n mg  q u a l i t y as a c o - f a c t o r i n m o d i f y i n g an e x p l a n t ' s f r e s h The  weight.  d r y weight means f o l l o w e d t h e p a t t e r n o f f r e s h  weight  means ( T a b l e 4.25). The degrees o f s i g n i f i c a n c e were, however, greater  f o r t h e two l i g h t  quality  interactions  (Table  The s t r e n g t h o f t r e n d s f o r w h i t e l i g h t c u l t u r e s p r o b a b l y  4.23). account  f o r much o f t h i s d i f f e r e n c e . T h i s i s because t h e red/FR means underwent l i t t l e  change from  t h e two t o f o u r week  e s p e c i a l l y a t t h e 0.25 and 0.4 mg/1 BA l e v e l s . The  exposure, percentage  i n c r e a s e i n f r e s h w e i g h t under red/FR from t h e two t o f o u r week regime  a t these  BA  levels  was  much  corresponding treatments' dry weights.  greater  than  f o r the  98 4.3  D e t e r m i n i n g i f glyphosate and t o modify i n v i t r o  4.3.1  Experiment V I I :  The  red/FR  i n i t i a t i o n may  may  quality  Glyphosate-light q u a l i t y  light  effect  of  experiment  enhancing  Spiraea  i n v o l v e a c o m b i n a t i o n of f a c t o r s .  i n t e r a c t with  interact  growth  i n v o l v i n g hormone metabolism may light  light  an  be  Two  mechanisms  i n operation.  exogenously  applied  shoot  First,  red  cytokinin  to  i n c r e a s e t h e c y t o k i n i n ' s a b i l i t y t o promote growth as s u g g e s t e d by C l e l a n d  ( C l e l a n d , 1976). S e c o n d l y , red/FR l i g h t may  l e v e l s o f endogenous IAA One  (lino,  decrease  1982).  p o s s i b l e method t o examine t h e e x t e n t of a m o d i f i c a t i o n  of a u x i n m e t a b o l i s m would be t o 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 the h e r b i c i d e  active ingredient  'Roundup' i s j u s t such a compound. A t  sublethal  m i l l i m o l a r c o n c e n t r a t i o n s , g l y p h o s a t e has been shown t o t h e r a t e o f IAA  o x i d a t i o n i n soybean and pea  increase  seedlings  leading  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, The  increase  i n IAA  glyphosate-induced  oxidation  i s proposed t o  disruption  of  the  1984).  o c c u r due  synthesis  of  occurs.  For  the  current  study,  the  a g r e a t e r IAA e f f e c t of  c u l t u r e s i n a d d i t i o n t o t h e red/FR e f f e c t may give  further increases  i n c y t o k i n i n to auxin  to  a  certain  p h e n o l i c compounds. Such p h e n o l i c s i n h i b i t IAA o x i d a s e and w i t h r e d u c e d l e v e l s of t h e s e p h e n o l i c s  of  thus  oxidation  glyphosate  on  be p r e d i c t e d  to  r a t i o s which  may  affect  proliferation.  In t h i s  experiment  the p o t e n t i a l of  g l y p h o s a t e t o a c t as an i n v i t r o growth r e g u l a t o r f o r S p i r a e a , either  alone  or i n combination  with  a  red/FR  effect,  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 d e t e r m i n e glyphosate l e v e l s that, while  modifying  Spiraea  g r o w t h , would  not cause g r o s s a b n o r m a l i t i e s . I n t h e f i r s t t r i a l t h e two l e v e l s chosen were 3.56 and 17.80 mg/1. A t t h e h i g h e r  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 ; t h e 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 o f unextended s h o o t s  ( F i g . 4.1). I n t h e second  trial,  0.356 and 0.712 mg/1 were t e s t e d ; h e r e 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 o c c u r r e d  could  still  quality  a r i s e . Two f i n a l  experiment,  0.087  b u t masses o f unextended  shoots  l e v e l s were chosen f o r t h e l i g h t  and 0.267 mg/1.  These two l e v e l s  appeared t o cause o n l y o c c a s i o n a l s i d e e f f e c t s .  + gly F i g u r e 4.1  + gly  + gly  + gly  The e f f e c t of 3-5° mg/l of glyphosate on S p i r a e a shoot p r o l i f e r a t i o n ( - g l y = no glyphosate, + g l y = glyphosate)  - gly  G e n e r a l appearance  Generally, white  light  glyphosate-treated plants had  concentrations occasionally  normal  leaves  of glyphosate caused  growth  and  used  under  shoots.  i n this  modifications.  both  r e d and  However,  experiment The  most  the still  common  f e a t u r e was s m a l l e r , narrower l e a v e s t h a n t h o s e from u n t r e a t e d p l a n t s . Sometimes s m a l l f a s c i a t e d s h o o t s ( l e s s t h a n 1 cm.) were a l s o p r e s e n t . There was some v a r i a t i o n i n t h e c h a r a c t e r i s t i c s e x p r e s s e d f o r s p e c i f i c t r e a t m e n t s under t h e two l i g h t (see T a b l e 4.26). 0.087 mg/1  Red l i g h t  glyphosate  regimes  c u l t u r e s w i t h t h e 0.5 mg/1  treatment  produced  three  BA +  of the four  samples w i t h t h e l e a f e f f e c t ; t h e same t r e a t m e n t f o r w h i t e 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 t h e l e a f e f f e c t and i n addition  two o f t h e f o u r showed s m a l l f a s c i a t e d  s h o o t s . The  t r e a t m e n t o f 0.5 mg/1 BA + 0.267 mg/1 g l y p h o s a t e under r e d l i g h t was s i m i l a r  b u t a l l f o u r samples showed t h e l e a f  effect  as  o c c u r r e d w i t h w h i t e 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 P r o p o r t i o n of each t r e a t m e n t ' s showing glyphosate e f f e c t s BA-GLY Combination 1  Red/FR  light  LVS.+ S H .  four  samples White  light  0.25 BA 0.087 GLY  1/4  0.25 BA 0.267 GLY  4/4 L V S . , 2/4 LVS.+ SH.  3/3 L V S . , 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.  3  2  1/4 LVS. 4  Legend: BA - benzyladenine, GLY - glyphosate 1 - BA-GLY combinations measured i n mg/1 2 - p r o p o r t i o n of samples showing glyphosate e f f e c t on both leaves (LVS.) and shoots (SH.) 3 - p r o p o r t i o n of samples showing glyphosate e f f e c t on leaves 4 - only 3 samples due to one d i s c a r d e d contaminated sample  103  Growth v a r i a t e s Shoot number/explant For  both  t h e g l y p h o s a t e - f r e e and 0.267  mg/1  glyphosate  t r e a t m e n t s , t h e 0.5 mg/1 BA c u l t u r e s had s l i g h t l y h i g h e r shoot number  means  than  t h e 0.25 mg/1  BA  cultures  However, a t t h e 0.087 mg/1 g l y p h o s a t e l e v e l  (Table  4.27).  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 a t t h e h i g h e r BA l e v e l . The  0.087  mg/1  glyphosate  results  were  extreme  a t both  BA  l e v e l s , w i t h t h e l o w e s t shoot number means o f a l l t h e t r e a t m e n t s produced a t t h e lower BA l e v e l and t h e h i g h e s t means produced a t t h e h i g h e r BA l e v e l . To  interpret  glyphosate-f ree quality  any g l y p h o s a t e - l i g h t q u a l i t y cultures  response  i n this  were used  as markers  experiment.  effects,  the  of the l i g h t  The e x p e r i m e n t a l  design  d i f f e r e d s l i g h t l y from t h e p r e v i o u s ones as t h i s one l a s t e d s i x weeks i n s t e a d o f f i v e and a l s o t h e r e was a t r a n s f e r o f p l a n t l e t s t o f r e s h medium a f t e r two weeks. C u l t u r e s under red/FR l i g h t had h i g h e r s h o o t number means t h a n w h i t e l i g h t c u l t u r e s . The mean s h o o t numbers under b o t h red/FR and w h i t e l i g h t a t t h e 0.5 mg/1 BA l e v e l were g r e a t e r t h a n t h e means a t t h e 0.2 5 mg/1 BA l e v e l . I n t h e p r e s e n c e o f 0.087 mg/1 o f g l y p h o s a t e 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  c o n t r o l f o r b o t h w h i t e and red/FR  inhibited  compared t o t h e  ( t h e d i f f e r e n c e was g r e a t e r  f o r r e d / F R ) . V e r y l a r g e i n c r e a s e s i n shoot number (up t o f o u r f o l d ) were o b s e r v e d concentration  i f t h e c u l t u r e s under t h e same g l y p h o s a t e  (0.087 mg/1)  were grown w i t h h i g h e r  exogenous  T a b l e 4.27 Shoot and node number means as a f f e c t e d by g l y p h o s a t e and l i g h t q u a l i t y Node number  Light, quality  BA level  GLY level  Red/FR  0 . 25  0.087 0.267 0  13.75 31.00 30.00  ± ± ±  3.86 3.14 2.84  59 104 128  ± ± ±  19 15 11  0.5  0.087 0.267 0  47.75 33.75 34.00  ± ± ±  10.92 6.17 3.90  168 109 123  + + ±  38 22 16  0.25  0.087 0.267 0  12.50 40.67 23.50  ± + ±  1.03 7 . 51 2.33  49 128 96  + + +  8 15 11  0.5  0.087 0.267 0  50 . 25 ± 12.83 43.75 + 7.65 29.75 + 4.79  151 130 107  ± ± +  38 22 18  White  1  2  Shoot number  4  3  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 - shoot number/explant mean ± s t a n d a r d error 4 - node number/block (mean o f f o u r b l o c k s , except mean o f f o u r b l o c k s w i t h two sub-samples f o r the "0" GLY treatment) ± standard error  105  l e v e l s o f BA ( i . e . 0.5 mg/1). The i n c r e a s e s o c c u r r e d red/FR  and  white  and  reached  similar  high  f o r both  levels.  They  r e p r e s e n t e d much h i g h e r i n c r e a s e s t h a n had been o b s e r v e d i n any p r e 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 o f l i g h t  quality  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 o f shoot p r o l i f e r a t i o n reached  were a l s o h i g h e r  than  alone,  either  experiment  previously.  in  Thus  this an  observed  inhibitory  with  or  0.5 mg/1  i n those  effect  of  o f BA  reported  glyphosate  on  p r o l i f e r a t i o n can be overcome by t h e a d d i t i o n o f exogenous BA t o 0.5  mg/1.  A  critical  level  between  0.25  and  0.5  mg/1 i s  r e q u i r e d , presumably t o overcome t h e 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 x e r t e d by 0.087 mg/1 o f g l y p h o s a t e  i n t h e medium.  The f i n d i n g t h a t red/FR l i g h t i n t h e presence o f g l y p h o s a t e , with  0.25 mg/1 o f BA, d i d n o t i n c r e a s e t h e growth o v e r  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 t h e 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 o r s y n e r g y ; o n l y t h e a d d i t i o n o f t h e e f f e c t i v e l e v e l (0.5 mg/1) o f t h e c y t o k i n i n BA c a n overcome t h e interference. The the  l a r g e i n c r e a s e s i n p r o l i f e r a t i o n o v e r t h a t seen i n  p r e s e n c e o f 0.5 mg/1  e f f e c t of glyphosate high  BA a l o n e .  o f BA a l o n e ,  reflect  a stimulatory  on growth which augments t h a t seen  I t may be p r e d i c t e d t h a t another e f f e c t  with which  o c c u r s due t o 0.087 mg/1 o f g l y p h o s a t e i n v o l v e s t h e r e d u c t i o n o f a p i c a l dominance t h u s r e s u l t i n g i n an i n c r e a s e o f shoot numbers. The  s t i m u l a t o r y e f f e c t on growth a t t h e 0.087 mg/1  glyphosate  c o n c e n t r a t i o n may depend on t h e i n t e r a c t i o n w i t h 0.5 mg/1 o f BA.  106 A  mechanism  metabolism  of  glyphosate  action,  p u t a t i v e l y mediated  s y n t h e s i s , has been demonstrated At higher  levels  i . e . an  increase  in  IAA  by d i s r u p t e d p h e n o l i c compound (Lee 1982;  of glyphosate  Lee  1984).  (0.2 67 mg/1)  growth r a t e s  under red/FR were s i m i l a r t o t h e c o n t r o l s w i t h BA a l o n e whereas s t i m u l a t i o n o c c u r r e d under w h i t e . I f t h e g l y p h o s a t e e f f e c t t h i s higher c o n c e n t r a t i o n i s predominantly  at  causing l o w e r i n g of  a u x i n t h e n reduced  a p i c a l dominance c o u l d r e s u l t i n i n c r e a s e d  proliferation.  lower v a l u e s under red/FR t h a n w h i t e  may  The  light  be due t o t h e i n t e r f e r e n c e of i n t e r m e d i a t e s o f a c y t o k i n i n  metabolic  pathway which  accumulate  as  an  indirect  result  of  g l y p h o s a t e ' s b l o c k 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 t h e lower g l y p h o s a t e levels).  (0.25  mg/1)  However, t h e o v e r a l l growth i n h i b i t i o n seen a t  lower  glyphosate  concentrations  predominant  l o w e r i n g of  was  (0.087mg/l) and BA  not  manifested  a u x i n would r e s u l t  because  in a  the  significant  change i n t h e c y t o k i n i n / a u x i n r a t i o a t h i g h e r g l y p h o s a t e l e v e l s . The  ANOVA ( T a b l e 4.28)  differences  in  shoot  i n d i c a t e s that the f a c t o r s  number  were  BA  causing  concentration  and  an  i n t e r a c t i o n o f BA and g l y p h o s a t e c o n c e n t r a t i o n s . The changes i n shoot  number  observed  in  m a n i f e s t e d by a B A - g l y p h o s a t e  the  presence  of  glyphosate  interaction. This r e s u l t  d a t a from a p r e l i m i n a r y experiment  were  supports  where g l y p h o s a t e a l o n e  was  added t o B A - f r e e medium; here g l y p h o s a t e was  unable t o  growth.  Spiraea i n v i t r o  system  Thus as the  a growth r e g u l a t o r i n t h e  e f f e c t i v e n e s s of  glyphosate  action  modify  depends on  an  107  T a b l e 4.27a. Node number/shoot means Light quality  BA level  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  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  White  Legend:  1  GLY level 2  Node number per shoot  3  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 n u m b e r / s h o o t means c a l c u l a t e d by d i v i d i n g s h o o t and node number means i n T a b l e 4.27  Table 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 s h o o t and node n u m b e r s , and f r e s h and d r y w e i g h t s . Source of variation  idf  Shoot number 1  Node number  Fresh weight  Dry weight  2  3  4  Glyphosate level  2  NS  NS  NS  NS  BA  1  *****  ***  ****  **  Light quality  1  NS  NS  *  *  Glyphosate and light quality interaction  2  NS  NS  NS  NS  Glyphosate, BA and l i g h t quality interaction 2  NS  NS  NS  NS  ***  ***  *  **  **  ***  *  **  level  Glyphosate and BA i n t e r a c t i o n (2) Glyphosate linear X BA 1 Glyphosate lack of fit X BA 1  **  **  BA and l i g h t quality interaction 1  NS  NS  5  Legend:  df *  *  NS  **  NS  shoot number/explant node n u m b e r / b l o c k fresh weight/explant dry weight/explant p o l y n o m i a l response 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 , lack of f i t = amount o f 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 d e g r e e o f 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 . 0 1 , * * * * * = a t p < 0 . 0 0 5 , 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 g r e a t a red/FR modulation One  of glyphosate's e f f e c t  d i f f e r e n c e was  glyphosate l e v e l  red/FR remained reduction  number means.  t h e d e c l i n e o f t h e node number means under  red/FR l i g h t a t b o t h BA mg/1  as d i d shoot  l e v e l s i n c o m b i n a t i o n w i t h t h e 0.267  (Table 4.27). S i n c e shoot numbers under  almost t h e same a t t h i s g l y p h o s a t e l e v e l ,  i n node  number  indicates  fewer  nodes  per  However, t h i s d e c r e a s e a l s o o c c u r r e d f o r t h e w h i t e l i g h t  the  shoot. shoots  ( T a b l e 4.27a.). The l a r g e i n c r e a s e i n shoot numbers accompanying t h e i n c r e a s e i n node numbers t r a n s l a t e s  into  fewer  nodes p e r  s h o o t . These r e s u l t s c o u l d suggest g l y p h o s a t e - i n d u c e d i n h i b i t i o n o f s h o o t e l o n g a t i o n ; as d i s c u s s e d i n t h e "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 s u p p o r t s t h e s m a l l e r d i f f e r e n c e s o b s e r v e d f o r node numbers  with  concentration  a  less  significant  effect  attributed  (Table 4.28). D e s p i t e t h e weaker BA e f f e c t ,  s t r e n g t h o f t h e g l y p h o s a t e BA i n t e r a c t i o n remained better.  to  BA the  t h e same o 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 g l y p h o s a t e . The 0.087 mg/1 glyphosate  level  effect  on shoot  e l o n g a t i o n was much  less  extreme t h a n i t s shoot i n i t i a t i o n e f f e c t . I t was t h e 0.2 67 mg/1 glyphosate  level  t h a t caused  t h e g r e a t e s t r e d u c t i o n i n shoot  l e n g t h s f o r b o t h BA l e v e l c o m b i n a 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 between t r e a t m e n t comparisons  i n T a b l e 4.29 were t h u s between  t r e a t m e n t s where t h e 0.267 mg/1 g l y p h o s a t e l e v e l was i n v o l v e d . The p r o p o r t i o n o f u s e f u l s h o o t s under red/FR l i g h t higher  than  under w h i t e  f o r every  BA-glyphosate  remained  combination  e x c e p t one. T h i s g e n e r a l r e t e n t i o n o f i n c r e a s e d s h o o t e l o n g a t i o n under  red/FR  light  suggests  that  any  i n t e r a c t i o n h a s a g r e a t e r e f f e c t on shoot  red/FR-glyphosate i n i t i a t i o n t h a n on  shoot e l o n g a t i o n . When  both  considered, with  shoot  initiation  i t i s apparent  and shoot  elongation are  that the glyphosate-free cultures  0.2 5 mg/1 BA under red/FR had h i g h e r numbers o f u s e f u l  s h o o t s t h a n any 0.2 5 mg/1 B A - g l y p h o s a t e  combination.  Fresh weight/explant and Dry weight/explant  The f r e s h and d r y w e i g h t 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 t h a n was shoot i n i t i a t i o n . T h i s can be i l l u s t r a t e d when mean t o t a l s o f t h e t h r e e t r e a t m e n t s a t each BA l e v e l a r e compared  (from T a b l e 4.30) f o r each  l i g h t regime.  The red/FR  Table 4 . 2 9 S h o o t l e n g t h s as a f f e c t e d by BA, g l y p h o s a t e , and :l i g h t quality Pairs * tested  BA con.  Gly3  Red/FR White  0 .25  Red/FR White  f  Actual number  Sample size  Useful ^ shoot %  0  119 89  52 47  62 42  0.5  0  135 118  52 38  70 45  Red/FR White  0.25 0.5 •  0 0  119 118  52 38  62 45  Red/FR White  0 . 25  0. 087  55 50  49 . 34  27 17  NS  Red/FR White  0.25  0.267  124 120  27 32  34 38  NS  Red/FR White  0.5  0. 087  191 201  39 34  75 69  NS  Red/FR White  0.5  0.267  135 175  36 23  48 40  Red/FR White  0 . 25 0.5  0. 087 0  55 118  49 38  27 45  Red/FR White  0.25 0.5  0.087 0.267  55 175  49 23  27 40  Red/FR White  0.25 0.5  0.087 0.087  55 201  49 34  27 69  Red/FR White  0.25 0.5  0.267 0  124 118  27 38  34 45  Red/FR White  0.25 0. 25  0. 267 0  124 89  27 47  34 42  3  con.  NS  ** **  *** NS  ***** ** * *****  Legend: 1 - each p a i r r e p r e s e n t s the s e t o f shoot length proportions 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 = g l y p h o s a t e , g l y p h o s a t e l e v e l i n mg/1 4 - 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 experimental blocks 5 - a u s e f u l shoot i s d e f i n e d as a shoot 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 % o f t h e sample s i z e ranked u s e f u l 6 - the conversion of the u s e f u l shoot % i n t o number o f s h o o t s 7 - X i s t h e symbol f o r t h e C h i - s q u a r e t e s t ; the a s t e r i s k s define 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, ** = a t p<0.05, *** = a t p<0.025, ****' = t p<0.01, ***** = a t p<0.005, NS = n o n - s i g n i f i c a n t a  T a b l e 4.30 F r e s h and d r y weight means as a f f e c t e d by g l y p h o s a t e and l i g h t q u a l i t y Light^ quality  BA level  Red/FR  0.25  0.087 0.267 0  162 ± 60 282 ± 45 398 ± 39  0.5  0.087 0.267 0  541 ± 128 320 ± 90 433 + 59  62 + 13 39 ± 9 51 + 6  0.25  0.087 0.267 0  124 ± 12 306 ± 60 199 ± 19  22 + 2 40 + 7 30 + 2  0.5  0.087 0.267 0  406 + 89 333 + 63 270 ± 39  47 + 8 38 + 6 35 + 4  White  Legend:  1  GLY level 2  Fresh weight 3  Dry weight 4  26 ± 6 42 ± 4 53 ± 4  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 t a n d a r d error 4 - d r y w e i g h t / e x p l a n t mean ± s t a n d a r d error  113  cultures  had a f r e s h w e i g h t t o t a l over 25% g r e a t e r a t b o t h BA  l e v e l s than the white l i g h t  cultures.  Dry w e i g h t  t o t a l s were  o v e r 33% h i g h e r under red/FR a t t h e lower BA l e v e l and were 20% g r e a t e r a t t h e h i g h e r BA l e v e l . These l i g h t q u a l i t y statistical  differences  dependent  (ANOVA, T a b l e 4.28), a r e g r e a t e r t h a n  t h o s e o b s e r v e d f o r s h o o t numbers. The g l y p h o s a t e - B A i n t e r a c t i o n i n t h e ANOVA was l e s s f o r t h e f r e s h w e i g h t s t h a n f o r t h e d r y w e i g h t s . B o t h were l e s s t h a n t h e same i n t e r a c t i o n f o r shoot number means. T h i s r e s u l t that  while  differences  glyphosate in  shoot  can i n t e r a c t numbers,  with  BA  i t s effect  suggests  t o cause on  dry  large matter  accumulation i s l e s s important; here t h e f a c t o r o f l i g h t q u a l i t y plays a greater role.  114 Chapter 5  G e n e r a l D i s c u s s i o n and  5.1  C h a r a c t e r i z a t i o n o f the S p i r a e a  Conclusions  nipponica  in vitro  light  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 t h e m i c r o p r o p a g a t i o n of a s p e c i f i c plant  generally  involves  medium c o n s t i t u e n t s and  empirical  trials  in  which  culture  e n v i r o n m e n t a l f a c t o r s a r e v a r i e d . Once  a b a s i c system has been d e s i g n e d , p h y s i o l o g i c a l c o n c e p t s g l e a n e d from  the  optimize  study the  efficiency  of  n a t u r a l l y grown p l a n t s  quality  has  taken as  been  defined  a  to  optimize  possible  metabolism i n the explant Experiments quality  for  were  obtaining  proportion The  high  mg/1,  stock  maximal  e f f i c i e n c y involved  means  of  to  shoot  modifying  using  growth  The  light hormone  itself. characterize  i n the  i n p r o m o t i n g i n v i t r o growth. The  culturing  applied  exogenous c y t o k i n i n l e v e l s .  done t o  propagation  as  Spiraea  the  optimal  system.  e x p e r i m e n t 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  l e v e l o f 0.25  be  e f f i c i e n c y of i n v i t r o growth. I n t h e s e s t u d i e s ,  p r o l i f e r a t i o n while minimizing approach  can  The  light initial  most e f f i c i e n t  r e s u l t s i n d i c a t e d t h a t a t a BA  which i s h a l f t h a t normally  c u l t u r e s , tungsten filament  used f o r  sub-  l i g h t with a  high  o f red/FR l i g h t produced t h e most e f f i c i e n t growth. shoot production  was  accompanied by  high  mean node  115 numbers  per  shoot  wavelengths.  Both  successful  and  g r e a t e r shoot  these  sub-culturing  criteria  lengths than  are  important  f o r shoot p r o l i f e r a t i o n  at  for  other either  or the  next  step of r o o t i n g shoots. The f a c t t h a t on B A - f r e e medium none o f t h e l i g h t  qualities  were c a p a b l e 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 ( a t l e a s t f o r S p i r a e a ) can o n l y be e x p r e s s e d i n c o n c e r t w i t h an exogenous hormone, i n t h i s case t h e cytokinin  BA.  Modification  of the  endogenous  cytokinin/auxin  r a t i o t o a s t a t e c o n d u c i v e t o i n t e r a c t i o n w i t h l o w e r exogenous BA  levels  exposure. previously  clearly The  depended  maximal  on  growth  the  effect  been shown t o o c c u r mg/1  specific  (Norton and  under  light  white  quality  light  had  a t a s t a n d a r d BA s u b - c u l t u r e  level  o f 0.5  Norton,  level  shoot p r o d u c t i o n decreased.  1986),  and  above  this  Using a high p r o p o r t i o n of  red/FR l i g h t o p t i m a l growth was a c h i e v e d a t a l o w e r BA l e v e l o f 0.25  mg/1.  Such f i n d i n g s  suggest  endogenous c y t o k i n i n / a u x i n  t h a t under red/FR l i g h t  ratio  was  very  the  favourable f o r the  i n d u c t i o n o f b o t h 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 . B l u e l i g h t i n d u c e d t h e l o w e s t shoot p r o d u c t i o n a t BA l e v e l s o f mg/1  and  production  0.5  mg/1,  suggesting  could occur  h i g h e r BA l e v e l s  under  that  blue  i f increases i n  light  they  would  0.25 shoot  require  (which might be accompanied by unwanted s i d e -  effects) . The l o w e r shoot p r o d u c t i o n under b l u e l i g h t may be a s c r i b e d t o s e v e r a l f a c t o r s . One  f a c t o r may  be b l u e l i g h t ' s a b i l i t y  to  maintain apical  dominance. T h i s was noted  i n an e a r l i e r  study-  where shoot t i p s o f w i l l o w e x p l a n t s were removed; a subsequent blue  light  exposure  inhibited  lateral  bud growth  (Letouze,  1974). A n o t h e r p o s s i b i l i t y i s t h a t t h e r e was a photon f l u e n c e rate effect. light  Plum shoot e x p l a n t s , when exposed t o r e d and b l u e  a t 37 u mol photons m^s' d i s p l a y e d s i m i l a r  (Baraldi  e t a_l. , 1988) . When t h e photon f l u e n c e r a t e was reduced  t o 9 u mol photons m'^s, t h e r e was no s i g n i f i c a n t 1  the p r o l i f e r a t i o n light  growth r a t e s  difference i n  r a t e f o r r e d l i g h t w h i l e t h e r a t e under b l u e  significantly  d e c l i n e d . I n o u r S p i r a e a study t h e photon -l  h i g h e s t f l u e n c e r a t e o f red/FR was 17 u mol photons m s w h i l e for  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 o b t a i n h i g h e r f l u e n c e r a t e s o f f i l t e r e d blue i n our system, t h e b l u e l i g h t i n h i b i t i o n might have been l o s t . Under reduced  red/FR  shoot  light  c o n d i t i o n s , i n c r e a s i n g t h e BA  proliferation,  suggesting  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  that  the  a t one BA l e v e l  f o r red,  resulting  shoot i n i t i a t i o n . I n  a s t u d y on plum e x p l a n t s , a p l a t e a u o f maximum shoot occurred  far-red,  initiation  and b l u e  c u l t u r e s and then showed a d e c l i n e a t a h i g h e r BA l e v e l et  level  light  (Baraldi  al. , 1988). Thus f o r S p i r a e a . a more pronounced e f f e c t o f  specific  light  quality  i n f l u e n c i n g growth  interaction  with  BA  seems  t o be  characteristics.  A l t h o u g h b l u e and red/FR w a v e l e n g t h s were assumed t o be most c a p a b l e o f m o d i f y i n g phytochrome e q u i l i b r i a , perhaps i n a h i g h irradiance  response  (HIR),  i t was p o s s i b l e t h a t  any minor  changes caused by i n t e r m e d i a t e w a v e l e n g t h s (between b l u e and r e d light)  might a l s o promote e f f i c i e n t _in v i t r o  when g r e e n , y e l l o w ,  growth.  and orange w a v e l e n g t h s were used  However, f o r the  second e x p e r i m e n t o n l y minor m o d i f i c a t i o n s o f growth o c c u r r e d . An  investigation  involving  Juneberry e x p l a n t s suggested  that  g r e e n l i g h t s u p p r e s s e s shoot i n i t i a t i o n (Behrouz and L i n e b e r g e r , 1981) .  Our r e s u l t s  indicate  that  f o r the i n v i t r o  Spiraea  system, under t h e s p e c i f i c exogenous BA c o n d i t i o n s , g r e e n l i g h t d i d n o t i n h i b i t shoot i n i t i a t i o n b u t caused l o w e r s h o o t l e n g t h s t h a n a t o t h e r w a v e l e n g t h s a t t h e h i g h e r BA l e v e l . Thus, under y e l l o w l i g h t 43% o f t h e s h o o t s were 1 cm o r l o n g e r , orange l i g h t had  38% w h i l e green had 31%.  green  light  indicate  These c o m p a r a t i v e r e s u l t s under  possible  differences  between d i f f e r e n t p l a n t s p e c i e s c u l t u r e d  i n growth  i n vitro.  The y e l l o w  l i g h t shoot l e n g t h s were t h e h i g h e s t o f a l l t h e l i g h t at  b o t h BA l e v e l s ,  Prunus  serotina  a result  in vitro  which  system  agrees w i t h  where y e l l o w  response  qualities  a s t u d y on a light  enhanced  s h o o t l e n g t h s ( F u e r n k r a n z e t a l . , 1988). The  two  combinations  experiments  involving  ( u s i n g b l u e and r e d f i l t e r s )  light  source-filter  were done t o v e r i f y  t h a t t h e l i g h t s o u r c e s chosen t o p r o v i d e s p e c i f i c l i g h t q u a l i t y r a n g e s i n t h e f i r s t e x p e r i m e n t were t h e most e f f e c t i v e w i t h t h e available the  filters.  They a l s o p r o v i d e d an o p p o r t u n i t y t o s t u d y  e f f e c t on i n v i t r o growth o f v a r y i n g t h e r a t i o s o f b l u e t o  red/FR l i g h t . The shoot means f o r t h e s e f i l t e r e x p e r i m e n t s were l o w e r t h a n  118 in  the f i r s t  light  quality  experiment.  Thus,  an  additional  f a c t o r c o u l d be s t u d i e d , t h e degree o f response t o l i g h t q u a l i t y d e t e r m i n e d 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 t h e s e much  lower  than  explanation source  i n the f i r s t  experiment.  f o r t h e l o w e r growth  cultures  chosen  experiments  The most  probable  rate i s that the p a r t i c u l a r  f o r e x p l a n t s had a l o w e r  morphogenic  p o t e n t i a l t h a n o t h e r a v a i l a b l e s o u r c e 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 s u b - c u l t u r e s have been r e p o r t e d for  other  affecting  i n vitro  grown  morphogenetic  species  potential  ( D r u a r t , 1987).  A  i s t h e endogenous  factor hormone  b a l a n c e w i t h i n t h e p a r t i c u l a r e x p l a n t s used. F o r example, i f t h e source  cultures  used  had grown a t a f a s t e r  rate  than  other  c u l t u r e s , a d e p l e t i o n o f exogenous BA might have r e s u l t e d i n a l o w e r c y t o k i n i n / a u x i n r a t i o i n t h e mother p l a n t s ; e x p l a n t s from t h e s e would be l e s s r e s p o n s i v e t o t h e p r e v i o u s l y used l e v e l s o f exogenous BA and would undergo l e s s shoot The  putative  inhibitory  effect  initiation.  of blue  light  on  shoot  i n i t i a t i o n n o t e d i n t h e f i r s t l i g h t q u a l i t y experiment was a g a i n apparent with  each  i n t h e e x p e r i m e n t where t h e b l u e f i l t e r was combined light  source.  In t h i s  experiment  the blue  combined w i t h t h e t u n g s t e n f i l a m e n t l i g h t s o u r c e reduced  filter this  l i g h t s o u r c e ' s a b i l i t y t o enhance growth a t t h e l o w e r BA l e v e l . Although  shoot  filament l i g h t statistically  initiation  was  still  a t t h e l o w e r BA l e v e l , different  from  those  h i g h e s t under  tungsten  t h e shoot mean was n o t under  the other  light  r e g i m e s . The d e c r e a s e i n t h e q u a n t i t y o f red/FR l i g h t r e a c h i n g  t h e c u l t u r e s under t h e t u n g s t e n 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 s u g g e s t s t h a t t h e amount o f red/FR l i g h t i s an i m p o r t a n t f a c t o r c o n t r i b u t i n g t o t h e p r e v i o u s l y o b s e r v e d red/FR p r o m o t i o n o f growth i n Experiment I . Any o p t i m i z a t i o n o f t h e s m a l l b l u e w a v e l e n g t h tungsten growth;  filament this  light  could also  reduce  p o s s i b i l i t y then supports the idea of blue  b e i n g an i n h i b i t o r y Differences different  by t h e b l u e f i l t e r  component o f  light  factor.  f o r shoot p r o l i f e r a t i o n  BA l e v e l s  were o b s e r v e d  with the exception of tungsten  at the filament  l i g h t w h i c h gave s i m i l a r growth p a t t e r n s a t b o t h BA l e v e l s . The l o w e r amounts o f red/FR p a s s i n g t h r o u g h t h e b l u e f i l t e r tungsten  filament  light  may  represent a threshold  under  level for  growth under t h e s e c o n d i t i o n s . One o f t h e r e a s o n s f o r u s i n g t h e m e t a l h a l i d e s o u r c e f o r t h e b l u e l i g h t regime for  t h e poor  o f t h e i n i t i a l e x p e r i m e n t was t o . compensate  transmission of the blue  filter.  Thus,  because  m e t a l h a l i d e was r i c h i n b l u e wavelengths and d i s c h a r g e d a h i g h photon 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. There  i s a g r e a t e r p r o p o r t i o n o f b l u e wavelengths  metal  halide  source.  light  However,  virtually  source  than  t h e shoot  under  means  under t h e  the fluorescent  a t both  BA  levels  t h e same f o r b o t h l i g h t s o u r c e s . T h i s s u g g e s t s  light were that  t h e e f f e c t o f b l u e l i g h t i s a c h i e v e d a t a s p e c i f i c l e v e l of b l u e wavelength  output  above which  differences  a r e n o t observed.  Other wavelength  o u t p u t s , such as a h i g h e r p r o p o r t i o n  of red  under ' f l u o r e s c e n t l i g h t i n g do not appear t o be m o d u l a t i n g t h e 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 T h i s may  shoot  l e n g t h s under  each  light  s o u r c e were  similar.  i n d i c a t e t h a t shoot e l o n g a t i o n o c c u r s a t a s i m i l a r r a t e  above a low f l u e n c e r a t e t h r e s h o l d o f red/FR Finally,  the lower p r o l i f e r a t i o n  e x p e r i m e n t may  have a f f e c t e d  rate  light. i n the blue  the response  filter  of the c u l t u r e  to  l i g h t q u a l i t y . I t may be t h a t t h e p r o p o s e d i n h i b i t o r y e f f e c t o f b l u e l i g h t i n t h e f i r s t experiment was p a r t l y due t o t h e h i g h e r growth r a t e i n t h a t e x p e r i m e n t . A h i g h e r growth r a t e c o u l d i m p l y a h i g h e r m e t a b o l i s m o f BA and perhaps a s t a t e more c o n d u c i v e t o o p t i m i z i n g any i n t e r a c t i o n o r s y n e r g y between BA and r e d / f a r - r e d light.  Slower  growing  cultures  which  may  have  a  lower  BA  m e t a b o l i s m c o u l d 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  s y n e r g y and a l o w e r s e n s i t i v i t y t o b l u e l i g h t .  T h i s would  t h e n e x p l a i n s i m i l a r l e v e l s o f growth under t h e m e t a l 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 r e g i m e s . The p r o l i f e r a t i o n r a t e s o f plum cultures this  ( B a r a l d i e t aJL. , 1988)  l o w e r growth r a t e may  were l o w e r t h a n f o r S p i r a e a ;  have i n f l u e n c e d  their  response t o  s p e c i f i c l i g h t q u a l i t i e s . I n t h e plum system, b l u e and r e d l i g h t caused s i m i l a r growth r a t e s w h i c h were h i g h e r t h a n under w h i t e l i g h t . Thus a l t h o u g h a r e d l i g h t - B A i n t e r a c t i o n o r synergy have been  s u p p r e s s e d by  a  promote growth. S i m i l a r l y ,  l o w e r growth  rate  i t could  may  still  t h e shoot number means f o r S p i r a e a  under t u n g s t e n f i l a m e n t l i g h t may be p a r t l y due t o a reduced BA  121  interaction  with  the minimal  amount o f red/FR  l i g h t passing  through the blue f i l t e r . In was  the red f i l t e r experiment the h i g h e s t p r o l i f e r a t i o n r a t e  a t t h e h i g h e r BA l e v e l  (0.5 mg/1)  under t u n g s t e n f i l a m e n t  l i g h t . T h i s c o n t r a s t w i t h t h e drop under t h e h i g h e r BA l e v e l i n E x p e r i m e n t I s u g g e s t s t h a t t h e 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  filter  light-BA  experiments  interaction  affected  or  the  synergy.  intensity  Perhaps  o f the  red/FR  lower  photon  the  f l u e n c e r a t e i n t h i s e x p e r i m e n t was n o t enough t o enhance growth at  t h e l o w e r BA l e v e l as had o c c u r r e d p r e v i o u s l y i n Experiment  I w i t h an i n c r e a s e d photon f l u e n c e r a t e under t u n g s t e n f i l a m e n t light. The t u n g s t e n f i l a m e n t l i g h t had h i g h p r o p o r t i o n s of b o t h r e d and f a r - r e d w a v e l e n g t h s o p t i m i z e d under t h e r e d f i l t e r and t h u s i n d u c e d t h e h i g h e s t shoot number means o f a l l l i g h t regimes a t b o t h BA l e v e l s . F l u o r e s c e n t and m e t a l h a l i d e r e d f i l t e r  regimes  i n d u c e d s i m i l a r l e v e l s o f shoot i n i t i a t i o n a t b o t h BA  levels.  These r e s u l t s s u g g e s t 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 m e t a l h a l i d e p r o p o r t i o n s o f red  and f a r - r e d w a v e l e n g t h s were negated by t h e s e l i g h t s o u r c e s '  r e l a t i v e l y l o w e r o u t p u t o f such w a v e l e n g t h s compared t o t u n g s t e n filament  light.  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 m e t a l h a l i d e l i g h t d i d n o t seem e f f e c t i v e in  enhancing  through  the  shoot filter  initiation; to  perhaps  maintain  o b t a i n e d under t u n g s t e n f i l a m e n t  shoot  light.  enough numbers  blue  passed  below  those  122 An  effect  proliferation  which  may  be  due  to  the  low  r a t e i s t h e l o w e r s h o o t l e n g t h p r o p o r t i o n under  the tungsten filament-red f i l t e r halide-red  partially  filter  regime. The  regime  proposed  compared t o t h e m e t a l d e c r e a s e i n t h e red/FR  l i g h t - B A i n t e r a c t i o n o r s y n e r g y , due t o t h e l o w e r growth  rate,  may  shoot  have  reduced  any  red/FR  light  enhancement  of  elongation. In  the f i r s t  quality  experiment  red/FR l i g h t  was  applied  o v e r a 16 hour p h o t o p e r i o d . I f f a r - r e d e n r i c h e d l i g h t promotes t h e growth o f i n v i t r o S p i r a e a s h o o t s v i a t h e same mechanism as its  growth  far-red light  promotion  o f n a t u r a l l y grown p l a n t s ,  enriched l i g h t photoperiod  exposure  should  then a short  g i v e n a t t h e end  elicit  a  similar  of a  white  in vitro  growth  r e s p o n s e as t h e 16 hour red/FR p h o t o p e r i o d . S i n c e t h e t u n g s t e n f i l a m e n t l i g h t s o u r c e has a h i g h e r p r o p o r t i o n o f f a r - r e d t h a n r e d l i g h t , t h e r e d f i l t e r was o m i t t e d f o r t h i s far-red enriched l i g h t A typical grown p l a n t s  light  s h o r t term  experiment.  end-of-day  far-red  light  i s an . i n c r e a s e i n s h o o t  response length;  of  naturally  thus,  i t  was  assumed t h a t t h e growth e f f e c t most l i k e l y t o be o b s e r v e d w i t h the  in  vitro  cultures  was  Comparisons between t r e a t e d only  one  difference.  Under  an and the  increase  in  shoot  untreated explants white  light  length. indicated  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 h i g h e r p e r c e n t a g e o f l o n g e r shoots  at the  Cultures  0.4  mg/1  under m e t a l  BA  halide  level  than  with a  at the  far-red  0.5  BA  addition  level. d i d not  123 e x h i b i t such a peak a t t h e 0.4 was  p r e s e n t under each l i g h t  mg/1  BA l e v e l . The same p a t t e r n  regime  f o r shoot i n i t i a t i o n .  The  p r o b a b l e e x p l a n a t i o n i s t h a t f o r m e t a l h a l i d e l i g h t a maximal light-BA  interaction  o c c u r s a t t h e 0.4  mg/1  BA  level.  Such a  peak would n o t have been o b s e r v e d i n t h e f i r s t experiment due t o t h a t e x p e r i m e n t ' s l a r g e r i n c r e m e n t a l 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  The l a c k o f t h e 0.4 mg/1  mg/1. BA peak under m e t a l h a l i d e w i t h t h e  f a r - r e d a d d i t i o n s u g g e s t s t h a t t h e m i n i m a l f a r - r e d exposure s t i l l c a p a b l e o f e x e r t i n g a minor e f f e c t on growth. the  f a r - r e d l i g h t may  have s h i f t e d t h e phytochrome  i n d u c e d by m e t a l h a l i d e l i g h t  was  Specifically equilibria  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 o r s y n e r g y a t t h e 0.4 mg/1  BA l e v e l ,  thus  e l i m i n a t i n g t h e 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 effect  at the  0.4  mg/1  BA  level,  the p r e v i o u s l y  far-red  substantial  red/FR e f f e c t s on growth were a b s e n t . Thus, i t appears t h a t f o r the  Spiraea i n v i t r o  enriched  light  and/or  system the  prolonged  red/FR  light  exposures previously  of  far-red  used,  are  r e q u i r e d t o enhance s h o o t i n i t i a t i o n  and shoot e l o n g a t i o n .  least  S p i r a e a shoot  one  result.  In  o t h e r s t u d y s u p p o r t s our a  conifer  study  (Zel et  al. ,  1988),  At  elongation a  16  hour  p h o t o p e r i o d o f f a r - r e d l i g h t g i v e n o v e r e i g h t days f o l l o w e d by ten  days w i t h a 16 hour w h i t e l i g h t p h o t o p e r i o d 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 t h a n o b s e r v e d under w h i t e l i g h t controls.  124 5.2  Factors contributing  to the  red/FR l i g h t growth response  y  The  experimental  experiment  design  i n v o l v e d growing  under a h i g h e r photon  of  the  first  light  explants f o r the f i r s t  fluence rate  qualitytwo  weeks  than the treatment  light  r e g i m e s . Thus, two s t a g e l i g h t t r e a t m e n t s o f s p e c i f i c  wavelength  and  subsequent  f o l l o w e d by h i g h e r f l u e n c e were examined i n a  experiment.  The  t i m e r e q u i r e d under a s p e c i f i c regime  combination  of s p e c i f i c  wavelengths  and  photon  and  the  fluence rates  were examined. There  appears  t o be  a critical  interaction  between  the  amount o f exposure t o red/FR and t h e t r a n s f e r t o a h i g h e r photon fluence higher  rate.  T h i s experiment  fluence rate  suggests  i s necessary  red/FR-BA i n t e r a c t i o n o r s y n e r g y H i g h e r l e v e l s o f BA higher  photon  (0.5 mg/1)  fluence  to  that  a transfer  optimize  a t 0.25  and  the  0.4  to  a  putative  mg/1  of  BA.  can overcome t h e need f o r t h e  step.  The  expression  of  such  an  i n t e r a c t i o n o r synergy was dependent on t h e t i m e o f t r a n s f e r a t the  lower  higher  BA  BA  levels  level  of  o f 0.5  0.25  mg/1  and  0.4  mg/1,  whereas  the  mg/1  c o u l d overcome t h e h i g h f l u e n c e  r e q u i r e m e n t . T h i s s u g g e s t s t h a t t h e e x t r a BA, by o p t i m i z i n g t h e interaction  or  synergy,  was  a b l e t o overcome any  i n f l u e n c e o f e i t h e r o r b o t h , t h e red/FR exposure  modulating  t i m 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 . The shoot number means a t t h e 0.5  mg/1  BA  level  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 h i g h and exceded t h o s e under w h i t e a l s o  indicating  12 5  the  effect  of  a  red/FR-BA  interaction  or  synergy  for  p r o l i f e r a t i o n of Spiraea. The f a c t t h a t t h e r e was a g r e a t e r s h o o t p r o l i f e r a t i o n a t t h e f o u r week red/FR suggests  that  exposure  compared t o t h e two week  t h e age o f t h e c u l t u r e  may  exposure  determine e x p l a n t  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 o b s e r v e d i n a t l e a s t two o t h e r i n v i t r o systems. One i n v o l v e d lettuce  cotyledons  where  adventitious  shoot  formation  was  o p t i m a l when d a i l y p u l s e s o f r e d l i g h t were g i v e n d u r i n g e i t h e r t h e second week o f a f i v e week c u l t u r e whole  culture  period  (Kadkade  period  and S e i b e r t ,  1976).  s t u d y i n v o l v e d a d v e n t i t i o u s bud f o r m a t i o n from c a l l u s o f Douglas f i r hour  and f i f t h  The o t h e r  embryo-derived  (Kadkade and J o p s o n , 1978). Here a s i x t e e n  photoperiod of red l i g h t ,  fourth,  or during the  when g i v e n d u r i n g  week o f growth  o f an e i g h t  the t h i r d ,  week  culture  p e r i o d , promoted o p t i m a l growth. Although red/FR  culture  light effect,  age may p l a y  a role  i n determining the  i t i s a p p a r e n t t h a t even w i t h a two week  red/FR e x p o s u r e c u l t u r e s e x h i b i t e d h i g h e r shoot number means a t all  BA l e v e l s t h a n d i d w h i t e l i g h t  general  promotive  red/FR  finding  provides further  light  cultures.  effect.  supportive  T h i s suggests a  In addition,  data  such  f o r the idea  a  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 o r s y n e r g y . I n terms o f t h e t h e s i s o b j e c t i v e o f f a c i l i t a t i n g t h e u s e o f lower  exogenous  cytokinin  concentrations,  i t was  found  that  s h o o t i n i t i a t i o n was o p t i m i z e d a t t h e l o w e r BA l e v e l s o f 0.25  12 6  and  0.4  mg/1  f o l l o w e d by one week. The  when f o u r weeks o f  low  a t r a n s f e r to white l i g h t i n i t i a t i o n r a t e was  fluence  rate  of a higher  red/FR  was  fluence  for  c o n s i s t e n t l y greater than t h a t  o b s e r v e d under t h e analogous w h i t e l i g h t regimes. Even w i t h  the  h i g h e r l e v e l o f 0.5 mg/1  not  BA, t h e o p t i m a l i n i t i a t i o n r a t e was  a c h i e v e d w i t h w h i t e l i g h t , but was each regime w h i c h had  c o n s i s t e n t l y o b s e r v e d under  i n v o l v e d red/FR. T h i s  finding indicates  t h e s t r o n g s i g n i f i c a n c e o f t h e red/FR-BA i n t e r a c t i o n o r s y n e r g y f o r o p t i m i z i n g growth.  127 5.3  Determining i f g l y p h o s a t e and interact  to modify i n v i t r o  Responses t o  glyphosate  light  quality  growth  have been r e l a t e d  to  each  plant  s p e c i e s ' r a t e o f IAA m e t a b o l i s m (Lee, 1984). Thus, a l t h o u g h  an  effect  could  be  t h e e x t e n t o f t h e e f f e c t c o u l d not be p r e d i c t e d .  The  on  expected,  IAA  metabolism  f o r the  Spiraea  explants  t r i a l s to determine s u i t a b l e glyphosate concentrations i n d i c a t e d the  severe  growth  modifications that  S h o o t s were a l m o s t  glyphosate  can  cause.  e l i m i n a t e d ; what remained were masses  of  compact r o s e t t e s o f r e d u c e d l e a v e s . The c o n c e n t r a t i o n s f o r t h e s e t r i a l s ranged from 17.80 The  glyphosate  mg/1  t o 0.356  concentrations  e x p e r i m e n t (0.087 mg/1  mg/1.  used  and 0.2 67 mg/1)  f o r the  light  quality  d i d not cause any  explant  m o r t a l i t y . T h i s l a c k o f m o r t a l i t y i s noteworthy s i n c e i n a s t u d y with  cranberry  glyphosate persistent  node  levels  explants caused  (Scorza  et  a l . , 1984)  mortality.  a b n o r m a l i t i e s were observed  similar  Furthermore, f o r shoots  and  while leaves  from t h e c r a n b e r r y e x p l a n t s , S p i r a e a e x p l a n t s o n l y o c c a s i o n a l l y exhibited  growth  modifications  at  the  selected  glyphosate  c o n c e n t r a t i o n s . Perhaps t h e r a p i d growth o f S p i r a e a 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 t o be two t y p e s o f i n t e r a c t i o n w i t h g l y p h o s a t e , each dependent on level  the  of glyphosate,  glyphosate 0.087 mg/1,  c o n c e n t r a t i o n . At the  lower  w i t h 0.25  shoot  mg/1  o f 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 under b o t h w h i t e and red/FR  128  light.  A  putative  greater decline red/FR r e l a t e d  glyphosate-induced  IAA  under  decrease  red/FR  may  suggest  i n IAA l e v e l s  decrease.  The  that  a  exacerbated a  stimulation  of  shoot  p r o l i f e r a t i o n under b o t h l i g h t regimes when 0.5 mg/1 o f BA was used i n d i c a t e s t h e c o m p l e x i t y o f g l y p h o s a t e ' s a c t i o n . W i t h t h i s glyphosate-BA  treatment  shoot  proliferation  rates  were  much  h i g h e r t h a n i n p r e v i o u s t h e s i s e x p e r i m e n t s . I t appears t h a t w i t h the  0.087  mg/1  glyphosate  level,  two d i s t i n c t  effects are  e x p r e s s e d . The i n h i b i t i o n e f f e c t n o t e d on growth a t 0.2 5 mg/1 o f BA i s overcome a t h i g h e r BA l e v e l s  (0.5 mg/1) and under t h e s e  c o n d i t i o n s , a n o t h e r e f f e c t o f g l y p h o s a t e g i v e s augmentation o f growth. The r e s u l t s o b t a i n e d w i t h t h e h i g h e r g l y p h o s a t e l e v e l (0.2 67 mg/1) s u g g e s t t h a t a presumed g r e a t e r a u x i n o x i d a t i o n t h a n w i t h the lower glyphosate l e v e l This  i s especially  i s d e t e r m i n i n g t h e growth  apparent  under  white  light,  pattern.  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 h i g h e r t h a n c o n t r o l s . Shoot numbers under red/FR, a t b o t h BA l e v e l s , were l o w e r t h a n under w h i t e  and v i r t u a l l y  unchanged  from  controls.  Here,  an  e f f e c t o f l o w e r e d a u x i n l e v e l s may have been t o i n t e r f e r e w i t h t h e 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 o r synergy by c a u s i n g intermediates  o f a c y t o k i n i n m e t a b o l i c pathway t o  accumulate  because o f 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 i n d u c e d by g l y p h o s a t e ' s disruption  of  Alternatively,  certain  phenolic  compounds'  synthesis.  red/FR l i g h t may be e x a c e r b a t i n g a g l y p h o s a t e  d i s r u p t i o n o f an a u x i n r e g u l a t i o n o f c y t o k i n i n metabolism. A u x i n  129 regulation  of  cytokinin  metabolism  has  previously  been  d e m o n s t r a t e d f o r a t o b a c c o stem p i t h - e x p l a n t system ( P a l n i e t a l . , 1988). G l y p h o s a t e i n h i b i t e d shoot e l o n g a t i o n ; however, t h e extreme e f f e c t o f t h e 0.25  mg/1  BA-0.087 mg/1  on s h o o t i n i t i a t i o n was glyphosate  treatment  reduction  glyphosate  t r e a t m e n t seen  l o s t . For s h o o t l e n g t h s , t h e 0.267 caused  the  greatest  differences.  i n shoot length c o r r e l a t e d w i t h the  under  both  light  regimes  r e d u c e d , t h e red/FR c u l t u r e s s t i l l lengths.  This r e t e n t i o n of greater  supports  the  fact  that  elongation,  for  Sherrington,  1984).  other  example,  shoot  light.  lengths  were  had g e n e r a l l y g r e a t e r  shoot  shoot lengths  f a c t o r s besides  the  This  fewer number o f  nodes o b s e r v e d p e r shoot under b o t h red/FR and w h i t e Although  mg/1  under red/FR auxin  gibberellins  control  (George  and  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 a t t h e h e r b i c i d a l concentration synthase  (which  inhibited. several  of  glyphosate  lbs./acre)  i s c e n t r a l i n the  Such  inhibition  metabolic  such e f f e c t s may  (0.8  may  pathways. At  red/FR  metabolism. A l s o l e v e l ,  can  be  used  have  many  ramifications  lower glyphosate  levels,  for some  pleiotropic  study.  e f f e c t may  involve  a  i n t h i s study  suggested  modification  of  auxin  at the  lower  i t i s apparent t h a t glyphosate, to  EPSP  s h i k i m i c a c i d pathway) i s  I n c o n c l u s i o n , t h e use o f g l y p h o s a t e the  enzyme  be o c c u r r i n g and c o u l d e x p l a i n t h e  e f f e c t s observed i n t h i s  that  the  augment  shoot  p r o l i f e r a t i o n  if  s u f f i c i e n t  130  BA  i s present  abnormal  shoots  (even  under w h i t e  appeared  and  light).  shoot  f u r t h e r m a n i p u l a t i o n of g l y p h o s a t e  Although o c c a s i o n a l  l e n g t h s were levels  may  sub-optimal,  optimize  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.  shoot  Summary  The  results  of  this  study  indicate  that  f o r Spiraea  n i p p o n i c a l i g h t q u a l i t y can be used t o l o w e r exogenous c y t o k i n i n requirements.  A  mixture  of  red  and  far-red  (red/FR)  light  e m i t t e d from a t u n g s t e n f i l a m e n t l i g h t s o u r c e (combined w i t h a red  acetate f i l t e r )  mg/1  BA  similar  provided  by  promoted  t o those  fluorescent  shoot p r o l i f e r a t i o n r a t e s a t  at  0.5  light.  mg/1  The  BA  under  quality  of  0.25  white  light  shoots  under  red/FR l i g h t , as measured by s h o o t l e n g t h , was h i g h e r t h a n under white  light.  source,  Blue l i g h t ,  produced  fewer  Intermediate wavelengths  from a shoots  filtered than  metal h a l i d e  under  (green, y e l l o w ,  and  light  white  light.  orange)  between  b l u e and r e d w a v e l e n g t h s produced s i m i l a r p r o l i f e r a t i o n  rates  w h i c h were n o t g r e a t e r t h a n t h o s e produced under w h i t e l i g h t . For mg/1  o p t i m i z a t i o n o f t h e red/FR l i g h t e f f e c t a t 0.25  BA,  red/FR  a protocol  involving  0.4  fluence  (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 o f w h i t e l i g h t for  4 weeks o f low photon  and  (47.0 - 62.0 u mol photons  m^s*)  1 f u r t h e r week i s r e q u i r e d . F o r S p i r a e a t h e e f f e c t o f t h e  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 more c r i t i c a l  on s h o o t p r o l i f e r a t i o n t h a n on shoot e l o n g a t i o n .  A l s o , i t i s i m p o r t a n t t o n o t e t h a t t h e red/FR l i g h t was  applied  c o n t i n u o u s l y o v e r a 16 hour p h o t o p e r i o d . Two  hours o f  applied  photoperiod  at  was  the  end  of  a  16  hour  white  red/FR 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 w h i t e l i g h t .  132  The u s e o f g l y p h o s a t e , w h i c h has been shown t o promote IAA oxidation  leading to a reduction i n apical  dominance (Lee,  1984) , has t h e 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 effect  of  significant  glyphosate  on  t h e red/FR  light  response  was  a  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 t r e a t m e n t . The w h i t e l i g h t ,  glyphosate-0.5  mg/1 BA t r e a t m e n t a l s o produced  0.087 mg/1  an i n c r e a s e i n  shoot p r o l i f e r a t i o n . I n a d d i t i o n , t h e g l y p h o s a t e t r e a t m e n t s w i t h w h i t e 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 a t a h i g h e r g l y p h o s a t e level  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 g l y p h o s a t e l e v e l produced  little  glyphosate  and  o r no change. W i t h BA  levels,  any  judicious manipulation of  possible  effects  on  shoot  morphology s h o u l d be m i n i m i z e d and s h o u l d a l l o w g l y p h o s a t e 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  culture.  133 Bibliography  1. Anderson, W.C. 1975. P r o p a g a t i o n of rhododendrons by t i s s u e c u l t u r e : P a r t 1. Development o f 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 o f s h o o t s . The I n t e r n a t i o n a l P l a n t P r o p a g a t o r s ' S o c i e t y . Combined P r o c e e d i n g s . 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. In v i t r o s h o o t development o f Prunus GF 655-2: I n t e r a c t i o n between l i g h t and b e n z y l a d e n i n e . P h y s i o l . P l a n t . 74:440-443. 3. Barendse, G.W.M., A.F. C r o e s , M. B o s v e l d , W.M. van der K r i e k e n , and G.J. Wullems. 1987. Uptake and metabolism o f NAA and BAP i n e x p l a n t s o f t o b a c c o i n r e l a t i o n t o i n v i t r o f l o w e r bud f o r m a t i o n . J . P l a n t Growth R e g u l . 6:193-200. 4. Behrouz, M. and R.D. L i n e b e r g e r . 1981. I n f l u e n c e of l i g h t q u a l i t y on i n v i t r o s h o o t m u l t i p l i c a t i o n of A m e l a n c h i e r l a e v i s . H o r t S c i e n c e 16(3):406. Abs. 52. 5. B o n n e t t , H.T. 1972. Phytochrome r e g u l a t i o n o f endogenous bud development i n r o o t c u l t u r e s o f C o n v o l v u l u s a r v e n s i s . P l a n t a 106:325-330. 6. Buck, M.F. and D. V i n c e - P r u e . 1985. P h o t o m o d u l a t i o n of stem e x t e n s i o n i n l i g h t - g r o w n p l a n t s : E v i d e n c e f o r two r e a c t i o n s . P l a n t C e l l P h y s i o l . 26(7):1251-1261. 7. Chee, R. and R.M. P o o l . 1986. I n v i t r o c u l t u r e o f V i t i s : The e f f e c t s o f l i g h t spectrum, manganese s u l f a t e , and p o t a s s i u m i o d i d e on morphogenesis. P l a n t C e l l , T i s s u e and Organ C u l t . 7:121-134. 8. Chee, R. and R.M. P o o l . 1989. Morphogenic responses t o p r o p a g u l e t r i m m i n g , s p e c t r a l i r r a d i a n c e , and photoperiod of g r a p e v i n e s h o o t s r e c u l t u r e d i n v i t r o . J . Amer. Soc. H o r t . S c i . 114 (2) :350-354. 9. C l e l a n d , C F . 1976. S y n e r g i s t i c i n t e r a c t i o n of r e d l i g h t and c y t o k i n i n i n p r o m o t i o n o f growth i n Lerana q i b b a G3. P l a n t P h y s i o l . 57 (Suppl.) 75 (Abst. 390). 10.  Downs, R.J., S.B. H e n d r i c k s , and H.A. B o r t h w i c k . 1957. P h o t o r e v e r s i b l e c o n t r o l of e l o n g a t i o n o f p i n t o beans and o t h e r p l a n t s under normal c o n d i t i o n s of growth. Bot. Gz. 118:199-208.  134 11. D r u a r t , P. 1987 * R e g u l a t i o n o f a x i l l a r y b r a n c h i n g i n m i c r o p r o p a g a t i o n o f woody f r u i t s p e c i e s . I n : L o r e t i , F. ( e d . ) . I n t e r n a t i o n a l Symposium on V e g e t a t i v e P r o p a g a t i o n o f Woody S p e c i e s , pp. 369-380. A c t a H o r t . 227. D r u k k e r i j J a n s e n B.V., Leiden. 12. F r e t t , J . J . , and J.M. Smagula. 1981. E f f e c t of s t o c k p l a n t p r e t r e a t m e n t on lowbush b l u e b e r r y e x p l a n t shoot m u l t i p l i c a t i o n . H o r t S c i e n c e 16:418 (Abst. 138). 13. F r e t t , J . J . , and J.M. Smagula. 1983. I n v i t r o shoot p r o d u c t i o n o f lowbush b l u e b e r r y . Can. J . P l a n t S c i . 63:467-472. 14. F u e r n k r a n z , H., J . M i l l e r , and C. Maynard. 1988. L i g h t e f f e c t s on a d v e n t i t i o u s r o o t f o r m a t i o n i n a x i l l a r y shoots o f mature Prunus s e r o t i n a . I n V i t r o 24(3) ( P t . I I ) 51A (Abst. 160). 15. F u j i m u r a , T., and A. Komamine. 1975. E f f e c t s o f v a r i o u s growth r e g u l a t o r s on t h e embrogenesis i n a c a r r o t c e l l s u s p e n s i o n c u l t u r e . P l a n t S c i . L e t t . 5:359-3 64. 16. George, E.F. and P.D. S h e r r i n g t o n . 19 84. P l a n t P r o p a g a t i o n by T i s s u e C u l t u r e . E x e g e t i c s L t d . , E v e r s l e y , Hants. 17. G r i m s t a d , S.O. 1987. The e f f e c t o f s u p p l e m e n t a l i r r a d i a t i o n w i t h d i f f e r e n t l i g h t s o u r c e s on growth f l o w e r i n g o f G l o x i n i a . S c i e n t i a H o r t . 32:297-305. 18. H a r t , J.W. 1988. Hyman, London.  and  L i g h t and P l a n t Growth, p. 41. Unwin  19. H e a l y , H.W. and H.F. W i l k i n s . 1979. The e f f e c t o f l i g h t q u a l i t y on r o s e s h o o t p r o d u c t i o n . P l a n t P h y s i o l . (Supp.) 63:126 ( A b s t . 6 9 2 ) . 20. H i l l m a n , J.R. 1984. A p i c a l dominance. I n : W i l k i n s , M.B. ( e d . ) . Advanced P l a n t P h y s i o l o g y , pp. 127-148. P i t m a n , London. 21. Hudak, J . 1984. H i l l , I n c . , New  Shrubs i n t h e Landscape, p. 241. McGrawYork.  22. H u t c h i n s o n , J.F. and R.H. Zimmerman. 1987. T i s s u e c u l t u r e o f f r u i t and nut t r e e s . I n : J a n i c k , J . ( e d . ) . H o r t i c u l t u r a l Reviews, V o l . 9., pp. 273-327. Van N o s t r a n d R e i n h o l d Company I n c . , New York.  135 23. l i n o , M. 1982. A c t i o n o f r e d l i g h t on i n d o l e - 3 - a c e t i c - a c i d s t a t u s and growth i n c o l e o p t i l e s o f e t i o l a t e d maize s e e d l i n g s . P l a n t a 156:21-32. 24. Jewer, P.C. and L.D. I n c u l l . 1980. Promotion o f s t o m a t a l o p e n i n g i n t h e g r a s s Anthephora pubescens by a range o f n a t u r a l and s y n t h e t i c c y t o k i n i n s . P l a n t a 150:218-221. 25. J o n e s , J.B. 1986. D e t e r m i n i n g m a r k e t s and market p o t e n t i a l of h o r t i c u l t u r a l c r o p s . I n : Zimmerman, R.H., R.J. G r i e s b a c h , B.A. Hammerschlag, and R.H. Lawson. ( e d s . ) . T i s s u e C u l t u r e as a P l a n t P r o d u c t i o n System f o r H o r t i c u l t u r a l Crops, pp. 175-182. M a r t i n u s N i j h o f f P u b l i s h e r s , Dordrecht. 26. J o n e s , O.P. 1967. E f f e c t o f b e n z y l a d e n i n e on i s o l a t e d a p p l e s h o o t s . Nature 215:1514-1516. 27. Kadkade, P.G. and M. S e i b e r t . 1976. u n p u b l i s h e d d a t a , c i t e d i n : S t a b a , E . J . (ed.) P l a n t T i s s u e C u l t u r e as a Source o f B i o c h e m i c a l s , pp. 123-141. CRC P r e s s I n c . , Boca Raton, F l o r i d a . (1980). 28. Kadkade, P.G. and M. S e i b e r t . 1977. Phytochrome-regulated o r g a n o g e n e s i s i n l e t t u c e t i s s u e c u l t u r e . N a t u r e 270:49-50. 29. Kadkade, P.G. and H. Jopson. 1978. I n f l u e n c e o f l i g h t q u a l i t y on o r g a n o g e n e s i s from t h e embryo-derived c a l l u s o f Douglas f i r (Pseudotsuga m e n z i e s i i ) . P l a n t S c i . L e t t . 13:67-73. 30. Kadkade, P.G., P. Wetherbee, H. J o p s o n , and C. B o t t i c e l l i . 1978. P h o t o r e g u l a t i o n o f Organogenesis i n P i n e Embryo and S e e d l i n g T i s s u e C u l t u r e s . Abs. 4 t h I n t . Congr. P l a n t T i s s . C u l t . Calgary.. 108, 29. 31. K a s p e r b a u e r , M.J. 1971. S p e c t r a l d i s t r i b u t i o n o f l i g h t i n a t o b a c c o canopy and e f f e c t s o f end-of-day l i g h t q u a l i t y on growth and development. P l a n t P h y s i o l . 47:775-778. 32. Kronenberg, G.H.M. and R.E. K e n d r i c k . 1986. P a r t 2. Phytochrome. The p h y s i o l o g y o f a c t i o n . I n : K e n d r i c k , R.E. and G.H.M. Kronenberg ( e d s . ) . Photomorphogenesis i n P l a n t s , p. 101. M a r t i n u s N i j h o f f / D r . W. Junk P u b l i s h e r s , Dordrecht. 33. Kudoyarova, G.R., E.P. Cheredova, V.Z. G y u l i - Z a d e , A.R. M u s t a f i n a , and V.M. Krut'Kov. 1988. E f f e c t o f g l y p h o s a t e on c o n t e n t o f f r e e and bound a u x i n s i n c o r n s e e d l i n g s . F i z i o l o g i y a R a s t e n i i 35(5):888-892.  136 34. Lane, W.D. 1978. R e g e n e r a t i o n o f a p p l e p l a n t s from m e r i s t e m - t i p s . P l a n t S c i . L e t t . 13:281-285.  shoot  35. Lee, P.K.-W., W.B. K e s s l e r , and K.V. Thimann. 1974. The e f f e c t o f h a d a c i d i n on bud development and i t s i m p l i c a t i o n s f o r a p i c a l dominance. P h y s i o l . P l a n t . 31:1114 . 36. Lee, T.T. 1982. P r o m o t i o n o f i n d o l e - 3 - a c e t i c a c i d o x i d a t i o n by g l y p h o s a t e i n t o b a c c o c a l l u s t i s s u e . J . P l a n t Growth Reg. 1:37-48. 37. Lee, T.T. 1984. R e l e a s e o f l a t e r a l buds from a p i c a l dominance by g l y p h o s a t e i n soybean and pea s e e d l i n g s . J . P l a n t Growth Reg. 3:227-235. 38. L e r c a r i , B., F. Tognoni, G. Anselmo, and D. C h a p e l . 1986. P h o t o c o n t r o l o f i n v i t r o bud d i f f e r e n t i a t i o n o f Saintpaulia i o n a n t h a l e a v e s and L v c o p e r s i c o n exculentum c o t y l e d o n s . P h y s i o l . P l a n t . 67:340-344. 39. Leshem, B., D.P. S h a l e y , and S. I z h a r . 1988. C y t o k i n i n as an i n d u c e r o f v i t r i f i c a t i o n i n melon. Ann. B o t . 61:255260. 40. L e t o u z e , R., and G. Beauchesne. 1969. A c t i o n d ' e c l a i r e m e n t s monochromatiques s u r l a r h i z o g e n e s e de topinambour. Comp. Rend.- Aca. S c i . P a r i s 2 69: pp. 152.81531. 41. L e t o u z e , R. 1974. E f f e c t s o f monochromatic l i g h t on t h e development o f w i l l o w c u t t i n g s i n v i t r o . I n : Chouard, P., and N. de B i l d e r l i n g ( e d s . ) . P h y t o t r o n i c s i n A g r i c u l t u r a l and H o r t i c u l t u r a l R e s e a r c h , pp.4 6-58. Bordas, P a r i s . 42. L i n s m a i e r , E.M. and F. Skoog. 1965. O r g a n i c growth f a c t o r .requirements of tobacco t i s s u e c u l t u r e s . P h y s i o l . P l a n t . 18:100-127. 43. McCown, B.H. 1986. Woody o r n a m e n t a l s , shade t r e e s , and c o n i f e r s . I n : T i s s u e C u l t u r e as a P l a n t P r o d u c t o r y System f o r H o r t i c u l t u r a l Crops. R.H. Zimmerman, R.J. G r i e s b a c h , F.A. Hammerschlag and R. H. Lawson (eds.) pp. 175-182. Martinus N i j h o f f P u b l i s h e r s , Dordrecht. 44. M i c h l e r , C H . and R.D. L i n e b e r g e r . 1987. E f f e c t s o f l i g h t on s o m a t i c embryo development and a b s c i s i c a c i d l e v e l s i n c a r r o t s u s p e n s i o n c u l t u r e s . P l a n t C e l l , T i s s u e and Organ C u l t . 11:189-207.  137 45. Moshkov, B.S. 1969. Growing P l a n t s i n A r t i f i c i a l L i g h t , pp. 69-7 0. N a t i o n a l L e n d i n g L i b r a r y f o r S c i e n c e and Technology, Y o r k s h i r e , England. 46. M u r a s h i g e , T and F. Skoog. 1962. A r e v i s e d medium f o r r a p i d growth and b i o a s s a y s w i t h t o b a c c o t i s s u e c u l t u r e s . P h y s i o l . P l a n t . 15:473-497. 47. N o r t o n , M.E. and C.R. N o r t o n . 1986. E x p l a n t o r i g i n as a d e t e r m i n a n t o f i n v i t r o shoot p r o l i f e r a t i o n i n Prunus and Spiraea. Journal of H o r t i c u l t u r a l Science. 61(l):43-48. 48. P a l n i , L.M.S., L. Burch, and R. Horgan. 1988. The e f f e c t o f a u x i n c o n c e n t r a t i o n on c y t o k i n i n s t a b i l i t y and m e t a b o l i s m . P l a n t a 174:231-234. 49. P i e n i a z e k , J . 1968. Growth i n v i t r o o f i s o l a t e d a p p l e s h o o t t i p s from young s e e d l i n g s on media c o n t a i n i n g growth r e g u l a t o r s . B u l l . Acad. P o l . S c i . , S e r . S c i . B i o l . 16:179183. 50. P i e r i k , R.L.M., H.H.M. Steegmans, J.A.M. Verhaegh, and A.N. Wouters. 1982. E f f e c t o f c y t o k i n i n and c u l t i v a r on shoot f o r m a t i o n of Gerbera i a m e s o n i i i n v i t r o . Netherland J o u r n a l o f A g r i c u l t u r a l S c i e n c e . 30:341-346. 51. P i e r i k , R.L.M. 1985. V e r m e e r d e r i n g i n kweekbuizen s t i j g t t o t 30 m i l j o e n . V a k b l a d Voor De B l o e m i s t e r i j 26:44-45. 52. Read, P.E. 1987. S t o c k p l a n t s i n f l u e n c e m i c r o p r o p a g a t i o n s u c c e s s . I n : von H e n t i g , W.-U. and G. Gruber ( e d s . ) . V o l . 1 I n t e r n a t i o n a l Symposium on P r o p a g a t i o n o f Ornamental P l a n t s . A c t a H o r t i c u l t u r a e 226:41-52. 53. Sachs, T., and K.V. Thimann. 1964. R e l e a s e o f l a t e r a l buds from a p i c a l dominance. N a t u r e 201:939-940. 54. S c o r z a , R., W.V. Welker, and L . J . Dunn. 1984. The e f f e c t s o f g l y p h o s a t e , a u x i n , and c y t o k i n i n c o m b i n a t i o n s on i n v i t r o development o f c r a n b e r r y node e x p l a n t s . H o r t S c i e n c e 19(1):66-68. 55. S e i b e r t , M., P . J . Wetherbee, and D.D. J o b . 1975. The e f f e c t s o f l i g h t i n t e n s i t y and l i g h t q u a l i t y on growth and s h o o t i n i t i a t i o n i n t o b a c c o c a l l u s . P l a n t P h y s i o l . 56:130139. 56. Skoog, F. and C O . M i l l e r . 1957. Chemical r e g u l a t i o n o f growth and organ f o r m a t i o n i n p l a n t t i s s u e s c u l t i v a t e d i n v i t r o . I n : B i o l o g i c a l A c t i o n o f Growth S u b s t a n c e s . 11th Symp. Soc. Exp. B i o l . 11:118-131.  138 57. Skoog, F., F.M. S t r o n g , and C O . S c i e n c e 148:532-533.  Miller.  1965. C y t o k i n i n s .  58. S m i t h , H. 1982. L i g h t q u a l i t y , p h o t o p e r c e p t i o n and p l a n t s t r a t e g y . Ann. Rev. P l a n t P h y s i o l . 33:481-518. 59. S t i c k l a n d , R.G. and N. S u n d e r l a n d . 1972. P h o t o c o n t r o l o f growth, and o f a n t h o c y a n i n and c h l o r o g e n i c a c i d p r o d u c t i o n i n c u l t u r e d c a l l u s t i s s u e s o f Haplopappus g r a c i l i s . Ann. Bot. 36:671-685. 60. S t i m a r t , D.P. and J . F . Harbage. 1989. I n v i t r o shoot p r o l i f e r a t i o n o f P y r u s c a l l e r y a n a from v e g e t a t i v e buds. H o r t S c i e n c e 24 (2):298-299. 61. T u c k e r , D.J. 1978. A p i c a l dominance i n t h e tomato: t h e p o s s i b l e r o l e s o f a u x i n and a b s c i s i c a c i d . P l a n t S c i . L e t t . 12:273-278. 62. W e r n i c k e , W. and L. M i l k o v i t s . 1984. Developmental g r a d i e n t s i n wheat l e a v e s - r e s p o n s e o f l e a f segments i n d i f f e r e n t genotypes c u l t u r e d i n v i t r o . J . P l a n t P h y s i o l . 115:49-58. 63. Z e l , J . , N. Gogala, and M. Camloh. 1988. M i c r o p r o p a g a t i o n o f P i n u s s y l v e s t r i s . P l a n t C e l l , T i s s u e and Organ C u l t . 14:169-175.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0302364/manifest

Comment

Related Items