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The auxin-like properties of potassium naphthenates and their effect on indole-3-acetic acid biosynthesis… Loh, John Wai-Choong 1972

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THE AUXIN-LIKE PROPERTIES OF POTASSIUM NAPHTHENATES AND THEIR EFFECT ON INDOLE-3-ACETIC ACID BIOSYNTHESIS AND DEGRADATION.  by  John Wai-Choong Loh Dip. A g r i c , B.Agr.Sc. (Hons),  (Malaysia), (Malaysia),  A t h e s i s submitted i n p a r t i a l  1965. 1970.  fulfillment  o f t h e requirements f o r t h e degree o f MASTER OF SCIENCE i n the Department o f BOTANY  We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA, VANCOUVER, BRITISH COLUMBIA, CANADA.  June  1972  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 by his representatives.  be granted by the Head of my Department or It is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of  Botany  The University of British Columbia Vancouver 8, Canada  Date  22nd August,  1972  i  ABSTRACT  The  auxin-Tike  (KNap), and acid  (IAA)  p r o p e r t i e s o f potassium naphthenates  the e f f e c t o f these compounds on i n d o l e a c e t i c biosynthesis  Chapter I.  The  and  d e g r a d a t i o n were examined.  a u x i n - l i k e p r o p e r t i e s o f KNap.  Cucumber seeds were t r e a t e d with. 1000 significant  (at the 0.05  ppm  e f f e c t s o f KNap and  i n i t i a t i o n o f r o o t s by were examined.  The  obtained.  i n d o l e b u t y r i c a c i d on  treatments w i t h 10 and root  100  ppm  The was  100,  elongation  s t i m u l a t e d by  and  1000  ppm  ppm  pea  KNap at 100  and  ppm  1000  stem segments  KNap (279% over the c o n t r o l ) .  S u r p r i s i n g l y , t h i s s t i m u l a t i o n d i d not d i f f e r from t h a t caused by 0.1  IAA  ppm,  significantly  (339% over the c o n t r o l ) . a p p l i e d to the d i s t a l  o f debladed p e t i o l e s , d i d not a f f e c t a b s c i s s i o n . required 1000  ppm  f o r 50%  significantly  KNap.  o f dark-grown A l a s k a  1.0  o f both  initiation.  Root i n i t i a t i o n of a z a l e a stem c u t t i n g s was 10,  the  stem c u t t i n g s o f Phaseolus v u l g a r i s L.  compounds s i g n i f i c a n t l y s t i m u l a t e d  augmented by  a  l e v e l ) i n h i b i t i o n o f r o o t growth  (91%) , compared w i t h c o n t r o l s e e d l i n g s was The  KNap and  The  end times  a b s c i s s i o n of p e t i o l e s t r e a t e d w i t h 10  cyclohexanecarboxylic  a c i d were s i g n i f i c a n t l y  and greater  than t h a t f o r a b s c i s s i o n o f c o n t r o l p e t i o l e s , b u t n o t from t h a t r e q u i r e d by p e t i o l e s t r e a t e d w i t h 100 ppm naphthaleneacetic  acid.  Chapter I I .  The e f f e c t o f KNap on IAA b i o s y n t h e s i s and  degradation. When t h e seeds' o f Phaseolus v u l g a r i s L. were soaked f o r 12 hours i n a s o l u t i o n o f 100 ppm KNap immediately p r i o r t o sowing,  t h e r e was a s i g n i f i c a n t i n c r e a s e (140%  over c o n t r o l p l a n t s )  i n the c o n t e n t o f IAA i n the a p i c a l  5-8 cm o f the stems o f 14-day-old The immersion grown bean p l a n t s  plants.  o f the r o o t systems o f 13-day-old darki n a s o l u t i o n o f 100 ppm KNap f o r 24 hours  resulted i n a s i g n i f i c a n t stimulation o f the a c t i v i t y o f the IAA oxidase  (4% over the c o n t r o l )  system.  The evidence p r e s e n t e d i s i n t e r p r e t e d as s u p p o r t i n g the view t h a t KNap has some a u x i n - l i k e p r o p e r t i e s . of t h i s i n t e r p r e t a t i o n i s discussed.  The v a l i d i t y  iii  TABLE OF CONTENTS  page Abstract  i  Table o f contents  i i i  Abbreviations  vi  L i s t of tables  v i i  List of figures L i s t of plates  viii .  ix  Dedication Acknowledgements Introduction  x .  xi .  xii  Naphthenic a c i d s  1  CHAPTER I. INVESTIGATION OF THE AUXIN-LIKE PROPERTIES POTASSIUM NAPHTHENATES.  OF  8  a) Growth o f i n t a c t r o o t s o f cucumber s e e d l i n g s . LITERATURE REVIEW  9  MATERIALS AND METHODS  13  RESULTS  15  DISCUSSION  18  iv  page b)  i. ii.  Adventitious root i n i t i a t i o n i n succulent and woody stem c u t t i n g s . LITERATURE REVIEW  20  MATERIALS AND METHODS  23  Succulent  23  stem c u t t i n g s u s i n g bean p l a n t s .  Woody stem c u t t i n g s u s i n g  azalea plants.  RESULTS i. ii.  c)  d)  26 28  Rooting o f s u c c u l e n t  stem c u t t i n g s o f bean. 28  Rooting o f woody stem c u t t i n g s o f a z a l e a .  33  DISCUSSION  37  Elongation  o f pea stem  segments.  LITERATURE REVIEW  39  MATERIALS AND METHODS  42  RESULTS  44  DISCUSSION  47  A b s c i s s i o n o f debladed p e t i o l e s o f bean plants. LITERATURE REVIEW  49  MATERIALS AND METHODS  58  RESULTS  61  DISCUSSION  63  V  page CHAPTER I I . THE EFFECT OF POTASSIUM NAPHTHENATES ON IAA BIOSYNTHESIS AND DEGRADATION. a)  b)  65  The e f f e c t o f KNap on IAA b i o s y n t h e s i s . LITERATURE REVIEW  66  MATERIALS AND METHODS  70  RESULTS  75  DISCUSSION  77  The e f f e c t o f KNap on IAA d e g r a d a t i o n . LITERATURE REVIEW  79  MATERIALS AND METHODS  84  RESULTS  88  DISCUSSION  90  Conclusions  92  Bibliography  93  vi'.  ABBREVIATIONS  HNap  Naphthenic  KNap  Potassium  CHCA  Cyclohexanecarboxylic  Sh-8  acids naphthenates acid  ' A compound o f t h e c y c l o h e x y l b u t a n o l c l a s s  NAA  Naphthaleneacetic  acid  IAA  Indoleacetic acid  IBA  Indolebutyric acid  2,4-D  2,4-dichlorophenoxyacetic  2,4,5-T  2,4,5-trichlorophenoxyacetic acid  acid  vii  LIST OF TABLES  Table I II  Page Response o f i n t a c t r o o t s o f cucumber t o KNap.  16  Rooting response o f bean stem c u t t i n g s t o KNap and IBA treatments.  30  III  Response o f stem c u t t i n g s o f a z a l e a t o KNap. 34  IV  Response o f dark-grown A l a s k a pea stem segments t o exogenously a p p l i e d IAA, CHCA, and KNap.  46  A b s c i s s i o n o f debladed p e t i o l e s o f bean (Phaseolus v u l g a r i s L.) i n response t o KNap, CHCA, and NAA treatments.  62  IAA c o n t e n t o f t h e 5-8 cm t i p s o f e p i c o t y l s o f 14-day-old dark-grown Phaseolus v u l g a r i s L. s e e d l i n g s f o l l o w i n g treatment w i t h 100 ppm KNap.  76  S p e c i f i c a c t i v i t y o f the enzymes i n the c o n v e r s i o n o f tryptophan t o IAA i n the e p i c o t y l s o f dark-grown bean p l a n t s .  76  V  VI  VII  V I I I The a c t i v i t y o f the IAA oxidase system i n the 14-day-old dark-grown bean e p i c o t y l s f o l l o w i n g treatment w i t h 100 ppm KNap.  89  LIST OF FIGURES  Figure  Page  1  Growth response o f i n t a c t r o o t s o f cucumber.  2  S t i m u l a t i o n o f r o o t i n i t i a t i o n o f bean stem c u t t i n g s by KNap and IBA treatments. 31  3  Stimulation of root i n i t i a t i o n i n azalea stem c u t t i n g s by KNap treatments.  17  35  LIST OF PLATES Plate  Page  1  Rooting response o f bean stem c u t t i n g s t o KNap and IBA treatments. 32  2  Rooting response o f a z a l e a stem c u t t i n g s t o KNap treatments.  36  3  Determination o f a b s c i s s i o n o f a p e t i o l e b y t h e use o f a f i v e gramme pressure a p p l i c a t o r .  60  X  DEDICATION  This t h e s i s i s dedicated devotion,  t o my w i f e , Trudy, f o r h e r  a s s i s t a n c e , and p a t i e n c e  t h a t have made my  study p o s s i b l e .  xi  ACKNOWLEDGEMENTS  G r a t e f u l thanks a r e extended D.J. W o r t , f o r c o u n s e l , the W.B.  t o my a d v i s o r ,  guidance, and c r i t i c a l  e n t i r e m a n u s c r i p t ; t o D r s . R.F. S c a g e l , S c h o f i e l d , a n d R. J . B a n d o n i ,  advice;  Professor  review o f  I.E.P.  Taylor,  f o r a s s i s t a n c e and  t o M r . S.M. S m i t h o f t h e F a c u l t y o f F o r e s t r y ,  for  advice  i nstatistical  for  technical assistance  a n a l y s i s ; a n d t o M r . D.R. i n making p h o t o g r a p h i c  Peirson,  prints.  INTRODUCTION  The und  p u b l i c a t i o n o f Went's c l a s s i c a l p a p e r  W a c h s t u m " i n 1928  i n w h i c h he  "Wuchsstoff  described a quantitative  method o f u s i n g Avena s e e d l i n g s f o r a s s a y i n g p l a n t growth substances, 1933  o p e n e d a new  era of research.  K o g l e t a_l i n  i s o l a t e d a c r y s t a l l i n e growth-inducing  human u r i n e .  They a l s o d e t e r m i n e d t h e c h e m i c a l  the growth substance,  and,  together with the  Went, c o i n e d t h e t e r m " a u x i n " The results  substance  f r e q u e n t use i n the  to confusion.  (from Greek  nature  late  to  to a s i n g l e substance, to the confusion,  IAA  the term "auxin" to  indole-3-acetic acid i s the  substance  grow).  researchers  l a c k o f u n i f o r m i t y i n n o m e n c l a t u r e , and Many w o r k e r s u s e  of  F.A.F.C.  "auxein",  of the term "auxin" by  from  (IAA).  referred to  leads  refer To  add  as  " h e t e r o a u x i n " b y many i n v e s t i g a t o r s i n R u s s i a a n d B u l g a r i a . Tukey e t a l  (1954)recommended t h e t e r m " a u x i n " be  used  as  a g e n e r i c t e r m o r g r o u p - n a m e f o r "compounds c h a r a c t e r i z e d by The  t h e i r c a p a c i t y to induce  elongation i n shoot  name " a u x i n " u s e d h e r e i s t a k e n  cells".  i n the generic  sense.  xiii  The d e f i n i t i o n o f auxin by Tukey e t a l (1954) r e q u i r e s a r e v i s i o n i n view o f s i m i l a r c h a r a c t e r i s t i c s o t h e r compounds such as g i b b e r e l l i c : . a c i d s .  exhibited  by  A good d e f i n i t i o n  f o r auxin i s l a c k i n g . The g e n e r a l b e l i e f t h a t Went's Avena c o l e o p t i l e c curvature t e s t  (Went, 1928) i s a c o n c l u s i v e b i o a s s a y f o r  auxin i s q u e s t i o n e d by the f i n d i n g o f Huseinov (1960) "hybberellic  acid"  (probably g i b b e r e l l i c a c i d ) caused bending  o f the Avena c o l e o p t i l e , g i b b e r e l l i c acid.  that  s u g g e s t i n g p o l a r movement o f  Huseinov's f i n d i n g has been supported  by Jacobs and Kaldeway  (1970) who demonstrated p o l a r move-  ment o f g i b b e r e l l i c a c i d  (GA^) i n young p e t i o l e s  of Coleus.  The l a c k o f p o l a r movement o f IAA i n some p l a n t s has been shown by E s c h r i c h Vicia  (1968) u s i n g r a d i o a c t i v e  IAA on  faba. The b i o a s s a y s commonly used t o demonstrate auxin a c t i v i t y  are: 1.  Wents Avena c o l e o p t i l e c u r v a t u r e t e s t  2.  S p l i t pea stem c u r v a t u r e t e s t  3.  Root growth t e s t  (Went, 1928).  (Went, 1934).  (Moewus, 1948; Audus, 1949,  Ready e t a l , 1947; Aberg, 1957).  1951;  xiv  4.  S t r a i g h t growth t e s t , u s i n g Avena c o l e o p t i l e segments  (Bonner, 1933, Hand, 1949; and 5.  1949); u s i n g pea  Galston  stem segments  and Baker, 1951,  1953;  (Galston  and  Christiansen  Thimann, 1950). P e t i o l e a b s c i s s i o n t e s t ( L u c k w i l l , 1956;  Mitchell et a l ,  1968). 6. A d v e n t i t i o u s  root i n i t i a t i o n t e s t (Libbert,  1956;  M i t c h e l l e t a l , 1968). The  d e t e c t i o n o f IAA  (Klambt, 1961; Good, 1955;  complexes such as  Zenk, 1964), and  Zenk, 1964)  IAA-aspartate  complexes may  (Andreae  and  i s an important c o n t r i b u t i o n towards  the understanding o f the mechanism o f IAA IAA  IAA-glucose  action i n plants.  escape d e t e c t i o n because they do not  give  t y p i c a l i n d o l e c o l o u r r e a c t i o n s w i t h the Salkowski and E h r l i c h reagents which are commonly used by research. f r e e IAA  auxin  While workers are becoming i n c r e a s i n g l y aware t h a t accounts f o r o n l y a p o r t i o n o f the auxin  i n p l a n t s , and the time has  t h a t bound IAA  should be geared to i s o l a t i o n ,  q u a n t i t a t i v e determination  activity  i s of " p h y s i o l o g i c a l s i g n i f i c a n c e " ,  come f o r a t u r n i n g p o i n t i n auxin  Future research and  investigators in  o f IAA  research. identification,  complexes.  The  f a c t t h a t a p p l i c a t i o n o f naphthenic a c i d s . (HNap) t o p l a n t s  r e s u l t s i n i n c r e a s e d growth, and t h e r e p o r t t h a t petroleum growth substances caused bending o f t h e Avena c o l e o p t i l e •,2>y Huseinov joi  (1960), r a i s e s t h e q u e s t i o n o f t h e q u a l i f i c a t i o n  -HNap and t h e i r s a l t s as a u x i n .  In t h i s i n v e s t i g a t i o n ,  some o f the a u x i n - l i k e p r o p e r t i e s o f naphthenates were d e t e r mined, as were t h e i r e f f e c t s on the b i o s y n t h e s i s and degradation !Phe the  o f IAA.  f i r s t phase o f t h e p r e s e n t  i n v e s t i g a t i o n deals  e v a l u a t i o n o f t h e a u x i n - l i k e p r o p e r t i e s o f potassium  naphthenates  (KNap), u t i l i s i n g t h e f o l l o w i n g  1. Growth o f i n t a c t r o o t s o f cucumber 2.  with  Adventitious  bioassays:  (Cucumis s a t i v a L)  root i n i t i a t i o n i n succulent  and woody  stem c u t t i n g s . 3. E l o n g a t i o n o f pea stem segments. 4. A b s c i s s i o n o f p e t i o l e s o f bean The  (Phaseolus v u l g a r i s ) p l a n t s .  second phase o f t h e p r e s e n t  investigation i s  concerned w i t h t h e e f f e c t o f KNap on IAA b i o s y n t h e s i s and degradation. In the pages t h a t f o l l o w , t h e term " s i g n i f i c a n t " s t a t i s t i c a l s i g n i f i c a n t d i f f e r e n c e a t t h e 0.05 l e v e l , otherwise stated.  implies unless  1  NAPHTHENIC ACIDS  The and  name, n a p h t h e n i c a c i d , was  Oglobin  The  p l a n t growth  h a v e b e e n named a n d  substances  naphthenates growth  structure  s t i m u l a t o r s o b t a i n e d from  petroleum  substances  (P.G.S.),  (N.G.S.),  petroleum  n a p h t h e n i c a c i d s (HNap),  o i l hormone s u b s t a n c e s  potassium  (O.H.S.),  naphtha  matter, o i l growth matter, petroleum n u t r i e n t ,  (an o x i d i s e d p e t r o l e u m p r o d u c t ) , Sh-8  H.T.I.  The  l a t t e r two  In the Russian language,  mean " g r o w t h - h e l p i n g Naphthenic  and  (P.R.V. o r N . R . V . ) , are used by  t h e a b b r e v i a t i o n s R.V.  from petroleum by  from d i e s e l o i l by  a complex mixture o f a l k y l a t e d  monocarboxylic  some o f w h i c h  cyclopentane, cyclohexane,  and  acid-  adsorption.  a c i d s a r e known t o b e acids,  investigators  substance".  acids are i s o l a t e d  base e x t r a c t i o n ,  sintovit  (a c y c l o b u t a n o l ) ,  petroleum or growth-helping substances  in Bulgaria.  crude  a b b r e v i a t e d d i f f e r e n t l y by v a r i o u s r e s e a r c h e r s  (KNap),  H.P.B., K h . T . I . ,  Markovnikoff  (1874) r e c o v e r e d f r o m R u m a n i a n  namely: naphthenic growth growth  suggested by  (1883) f o r t h e C,,H 0 a c i d s o f unknown 11 20 2  w h i c h H e l l and M e d i n g e r oil.  first  are d e r i v a t i v e s  cycloheptane.  Jolly  noted t h a t c y c l o p e n t y l d e r i v a t i v e s predominate a c i d m i x t u r e , f o l l o w e d b y c y c l o h e x y l compounds.  Naphthenic alicyclic of (1967)  i n the  naphthenic  Recently,  Kazanis  (1971) by mass s p e c t r o m e t r i e s n u c l e a r magnetic  and i n f r a r e d a n a l y s e s , d e t e c t e d methyl  resonance  2-DL-4D-dimethyl  heptanoate, methyl o c t a n o a t e , methyl nonanoate,  and c i s  1 , 3 - d i m e t h y l c y c l o h e x y c a r b o x y l a t e i n the n a p h t h e n i c a c i d m i x t u r e i s o l a t e d from crude petroleum. The m o l e c u l a r weights and b o i l i n g p o i n t s o f HNap v a r y w i t h t h e source o f the a c i d s . weights o f 214 (Tanchuk,  HNap w i t h an average m o l e c u l a r  (Cason and Khodair, 1966); 211,  1971); 216  (Nametkin, 1971) have been  A b o i l i n g p o i n t range from 30° t o 150° C was Kazanis  239, and  303  reported.  r e p o r t e d by  (1971), and 45° t o 170° by Artaraonov (1971).  The  c o m m e r c i a l l y a v a i l a b l e naphthenic a c i d s , used i n the p r e s e n t i n v e s t i g a t i o n s , have an average m o l e c u l a r weight o f 230. The c a r b o x y l group o f most i n d i v i d u a l members o f the n a p h t h e n i c a c i d m i x t u r e i s not a t t a c h e d d i r e c t l y t o the alicyclic  r i n g , b u t i s s e p a r a t e d from the r i n g by an  s i d e c h a i n c o n t a i n i n g one t o f i v e o r more methylene According to J o l l y  aliphatic groups.  (1967), a g e n e r a l formula may be w r i t t e n  as R t C ^ ^ C O O H , where R i s an a l i c y c l i c n u c l e u s composed o f one o r more r i n g s .  An e x c e p t i o n t o t h i s g e n e r a l  rule  i s c y c l o h e x a n e c a r b o x y l i c a c i d and i t s K s a l t which have been  shown t o e x h i b i t b i o l o g i c a l a c t i v i t y  (Wort and P a t e l , 1 9 7 0 ;  Severson, 1 9 7 1 ; Padmanabhan, 1 9 7 2 ; P e i r s o n , 1 9 7 2 ) and a l s o i n the present investigations. The n a p h t h e n i c a c i d s , o b t a i n e d from Baku have an a c i d number o f  mg KOH/g (Nametkin,  259  1971).  Naphthenic a c i d s have a c h a r a c t e r i s t i c rubbery odour which v a r i e s w i t h t h e a c i d source, degree o f r e f i n e m e n t , and c o n t e n t o f p h e n o l i c and suphur compounds.  The a c i d s  are r e a d i l y s o l u b l e i n n o n - p o l a r s o l v e n t s , and t h e lower m o l e c u l a r weight members, such as c y c l o h e x a n e c a r b o x y l i c and c y c l o p e n t a n e c a r b o x y l i c a c i d  acid  are s p a r i n g l y s o l u b l e i n  water. In low c o n c e n t r a t i o n s  (5000  ppm o r l e s s ) ,  naphthenates  have been found t o promote v e g e t a t i v e and r e p r o d u c t i v e (e.g. Severson,  1971).  growth  There a r e a l s o r e p o r t s t h a t when  a p p l i e d a t h i g h c o n c e n t r a t i o n s naphthenates a c t as h e r b i c i d e s (Mailov,  1968;  Zhukova,  1965).  In t h i s r e s p e c t , t h e b e h a v i o u r  o f naphthenates i s s i m i l a r t o t h a t o f '2,4-D. The mode o f a c t i o n by which a p p l i e d naphthenates p l a n t growth  and metabolism i s o b s c u r e .  Wort  (1969)  influenc suggested  4  the anion,  naphthenate was  of vegetative e s t e r and than the  and  reproductive  f o r the  stimulation  growth i n beans.  The  a s p a r t i c a c i d amide o f naphthenic a c i d s f r e e a c i d were r e s p o n s i b l e  g l u c o s e uptake and t o Severson designate  responsible  (1972).  one  glucose  rather  f o r the s t i m u l a t i o n  i t s metabolism i n bean r o o t  tips  according  A t t h i s time, i t i s not p o s s i b l e  to  or more naphthenic a c i d s as . . s p e c i f i c a l l y  f o r s t i m u l a t i o n o f p l a n t growth and  development b u t  of  responsible  some  s t r u c t u r a l c h a r a c t e r i s t i c s o f e f f e c t i v e a c i d s have been suggested (Wort and  P a t e l , 1970).  Following  t h e i r a p p l i c a t i o n to  bean p l a n t s , naphthenates s t i m u l a t e many p h y s i o l o g i c a l and biochemical The  s t i m u l a t i o n of photosynthesis  (Fattah, 1972;  processes.  1969;  dark r e s p i r a t i o n  Wort e t a l , 1970), p r o t e i n s y n t h e s i s  Wort e t aJL, 1971), and  the  specific activities  numerous enzymes i n crude e x t r a c t s Fattah  and  and Wort, 1970;  (Chu,  Wort e t al_, 1971)  1969;  (Severson, of  Fattah,  1969;  suggest t h a t naphthenate  s t i m u l a t i o n o f p l a n t growth i s the r e s u l t o f the a c t i o n o f the c h e m i c a l s or t h e i r m e t a b o l i t e s levels  a t the g e n e t i c  and  (Wort e t a l , 1971).  A method f o r q u a n t i t a t i v e d e t e r m i n a t i o n naphthenates i n p l a n t s was  g i v e n by Guseinov  o f sodium (1970).  metabolic  5  Responses  t o naphthenate t r e a t m e n t s , i n c l u d i n g  greater  v e g e t a t i v e and r e p r o d u c t i v e growth have been r e p o r t e d f o r maize, tomato, potato, w i n t e r c e r e a l s g r a p e s , and o t h e r c r o p t  plants  (Popoff and Boikov, 1966).  The e f f e c t s o f naphthenates  on p l a n t growth, y i e l d and c o m p o s i t i o n were summarised by Severson  (1971).  Petroleum growth substances a r e v e r s a t i l e i n c a u s i n g s t i m u l a t i o n o f growth and metabolism.  The s t i m u l a t o r y  effects  o f P.G.S.. a r e n o t o n l y observed i n p l a n t s b u t a l s o i n a n i m a l s . P.G.S. when a d m i n i s t e r e d o r a l l y a t 1.0 mg/kg body weight o f c a s t r a t e d rams, i n c r e a s e d the serum p r o t e i n l e v e l by 3.647.2% w i t h a maximum a f t e r 40 days, and i n c r e a s e d serum l e v e l by 12.92% (Mekhtiev, 1970).  Gorshkova  albumin^  (1970) r e p o r t e d  p r o l i f e r a t i o n o f b o t h i n t e r and i n t r a l o b u l a r t i s s u e s i n the l i v e r o f r a b b i t s 10 days a f t e r a d m i n i s t r a t i o n o f 20 mg P.G.S. p e r kg body weight.  T h i r t y - d a y doses o f 5 mg/kg  i n c r e a s e d r a b b i t body weight by 18-20%, w h i l e 20 mg/kg d e c r e a s e d t h e body weight by 20-22% (Loshmanova,  1970).  The n o n - b i o l o g i c a l uses o f naphthenic a c i d s and t h e i r s a l t s are varied.  They a r e used as l u b r i c a n t s i n r e f r i g e r a t i o n  and  a i r - c o n d i t i o n i n g , d r i e r s i n the p a i n t i n d u s t r y , c a t a l y s t s  preservatives,  emulsifiers  (Jolly,  1967), and  as  lead  naphthenate, a c o n s t i t u e n t i n a mixture t h a t i s r e s i s t a n t t o s a l t water c o r r o s i o n .  They are a l s o used as copper  naphthenates i n f u n g i c i d a l p r e p a r a t i o n s . The  i n f o r m a t i o n -accumulated thus f a r i s s u f f i c i e n t  t o j u s t i f y the  i n c l u s i o n o f naphthenic a c i d s and  their  as one  o f the p l a n t growth substances along w i t h the  called  " e s t a b l i s h e d " p l a n t growth r e g u l a t o r s  such  salts  so-  as:  i n d o l e - 3 - a c e t i c a c i d , g i b b e r e l l i c a c i d s , c y t o k i n i n s , 2,4-D, 2,4,5-T, n a p h t h a l e n e a c e t i c maleic and  hydrazide,  and  c y c o c e l , and  t h e i r s a l t s may  r e g u l a t o r s due animals.  a c i d , 2,3,6  be  tri-iodobenzoic acid,  phosfon.  s u p e r i o r to other  Naphthenic  acids  p l a n t growth  to t h e i r d u a l s t i m u l a t o r y e f f e c t s on  plants  7  NAPHTHENIC ACIDS  CO OH  COOH  cyclopentanecarboxylic  :  C  6 10°2 H  -  acid  cyclohexanecarboxylic C H 0 7  , m o l e c u l a r weight 114  1 2  2  m o l e c u l a r weight 128  COOH  3-methyl-cyclopentyl-n-propionic 9 16 2 m o l e c u l a r weight 156  acid  acid  CHAPTER I  INVESTIGATION OF THE AUXIN-LIKE PROPERTIES OF POTASSIUM NAPHTHENATES.  9  i a ) Growth o f i n t a c t r o o t s o f cucumber  seedlings.  LITERATURE REVIEW  A s u r v e y o f t h e l i t e r a t u r e on t h e i n f l u e n c e o f naphthenates on r o o t -growth i n d i c a t e s t h e s c a n t y n a t u r e o f r e s e a r c h i n this  area. The  auxin  prevalent  response o f i n t a c t r o o t s t o exogenous  i s retardation of elongation.  t o respond t o low c o n c e n t r a t i o n s s l i g h t i n c r e a s e i n growth r a t e 1961).  Roots have been  of indoleacetic acid  reported (IAA) by  (e.g. Burstrom, 1951; Larpen,  The use o f r o o t measurements o f r e d c l o v e r t o determine  the presence o f 2,4-D i n s o i l was d e s c r i b e d Nutman e t a l (1945).  i n a paper by  They found t h a t 2,4-D a t c o n c e n t r a t i o n s  o f 1.0, 10, and 100 ppm were i n h i b i t o r y t o r o o t growth. I n v e s t i g a t i o n s on t h e e f f e c t o f auxins on r o o t growth have shown t h a t , i n g e n e r a l , i n t h e Avena c o l e o p t i l e , (e.g. B e n t l e y  substances which cause growth promotion cause i n h i b i t i o n o f r o o t growth  and B i c k l e , 1952).  Moewus i n 1949 who d e v i s e d  t h e c r e s s r o o t t e s t as a  q u a n t i t a t i v e method o f e s t i m a t i n g growth substances,  found  10  t h a t 2.4 x 10 r o o t growth.  M IAA was n e c e s s a r y f o r 50% i n h i b i t i o n o f Audus  (1949) o b t a i n e d 50% i n h i b i t i o n o f r o o t  -7 growth an c r e s s , r a d i s h , garden pea, and maize i n 6.8 x 10 M 2,4-D  i n s o l u t i o n c u l t u r e a t pH 6.8.  on r o o t growth u s i n g Moewus  On t h e e f f e c t o f IAA  (1949) c r e s s t e s t , B e n t l e y (1951)  showed t h a t IAA i n h i b i t e d r o o t growth i n Lepidium sativum o v e r a range o f c o n c e n t r a t i o n s from 1-0 t o 10  mg/1.  A  s l i g h t growth promotion a t lower c o n c e n t r a t i o n s , t o a maximum -7 "8 , o f c a 30% a t 10 t o 10 mg/1 was a l s o found. Grant  Ready and  (1947) found s e e d l i n g s o f Cucumis s a t i v u s  t o 2,4-D.  t o be s e n s i t i v e  The growth o f p r i m a r y r o o t s was i n h i b i t e d t o 50%  -7 by 2.1 x 10 M s o l u t i o n o f 2,4-D.  Using s e e d l i n g s  of'Avena  s a t i v a grown on f i l t e r paper moistened w i t h s o l u t i o n s o f growth substances, Lane  —6 (1936) found t h a t 1.7 x 10 M IAA  was n e c e s s a r y f o r 50% i n h i b i t i o n o f r o o t growth,  w h i l e Bonner  -7 and K o e p f l i  (1939) r e p o r t e d 3 x 10  -6 M IAA and 7 x 10  M  NAA were r e q u i r e d t o b r i n g about t h e same i n h i b i t i o n . A r e d u c t i o n o f c a 10% i n growth o f t h e r o o t  system  f o l l o w i n g treatment w i t h sprays o f 0.005% P.G.S. on.eggplants was demonstrated by A l i - Z a d e and Guseinov  (1965).  11  Bentley  (1958) f o u n d an  a v e r a g e maximum s t i m u l a t i o n o f  20%  -3 with  IAA  1948,  a t 5.7  x 10  M u s i n g Moewus r o o t t e s t  (Moewus,  1949). The  e f f e c t o f t e m p e r a t u r e on  i n a paper by  P i l e t and  Went  r o o t g r o w t h was  (1956).  described  They o b s e r v e d  that  -4 IAA  a t 10  M r e t a r d e d growth o f o l d r o o t s o f Lens  and  the degree o f r e t a r d a t i o n i n c r e a s e d markedly w i t h  w h e r e a s t h e g r o w t h o f y o u n g r o o t s was a t low  t e m p e r a t u r e s and  accentuated  inhibited at high  culinaris, temperature,  slightly  temperatures.  -8 At  1.0  x 10  M IAA,  g r o w t h o f b o t h o l d and  stimulated at a l l temperatures Huseinov  (1960), u t i l i s i n g  naphthenic growth substances ( 0 . 1 , 0.01,  0 . 0 0 1 , and  young r o o t s  (5-27° C) .  .-  similar concentrations  (N.G.S.) a n d  0.0001%) o b t a i n e d  IAA  ( 0 . 1 % ) and  s i m i l a r trends  s t i m u l a t o r y a t weak  ( 0 . 0 0 0 1 % N.G.S., 0 . 0 0 1 % I A A ) . periods and  0.05%  o f seed soak N.G.S., a n d  (6, 12,  He and  of  (heteroauxin)  response o f r o o t growth o f wheat i . e . i n h i b i t o r y a t concentration  was  high  concentrations  a l s o employed  different  24 h o u r s ) i n 0.005,  0.01,  found t h a t r o o t lengths of w i n t e r  were g r e a t e r than t h a t o f the c o n t r o l s .  in  Seed w e t t i n g  wheat  with  12  weak s o l u t i o n s o f N.G.S. (0.004 and 0.0004%) p r i o r t o sowing, enhanced t h e r o o t l e n g t h s o f c o t t o n , onion, w i n t e r wheat. b y soaking 1966).  cucumber, and  The growth o f c o t t o n r o o t s was  stimulated  t h e seeds i n s o l u t i o n s o f 10 mg/1 HNap  (Babaev,  13  MATERIALS AND METHODS  Seeds o f cucumber (Cucumis s a t i v u s L.) were washed w i t h tap water and f i f t e e n uniform p e t r i dish  seeds were p l a c e d i n each  (diameter 9 cm) c o n t a i n i n g t h r e e p i e c e s o f Whatman  No. 1 f i l t e r paper. test  size  E i g h t ml volumes o f each o f the f o l l o w i n g  s o l u t i o n s were s e p a r a t e l y t o each d i s h :  KNap 1000 ppm KNap 100 ppm KNap 10 ppm KNap 1.0 ppm D i s t i l l e d water served The  as c o n t r o l .  p e t r i d i s h e s were covered and p l a c e d  at a c o n s t a n t  i n an i n c u b a t o r  temperature o f 20° C f o r 72 hours.  A t the  end o f t h e i n c u b a t i o n p e r i o d , t h e lengths o f the primary r o o t s were determined.  The average l e n g t h o f the r o o t s was used  t o compute the p e r c e n t  i n h i b i t i o n o f root  Average c o n t r o l r o o t l e n g t h minus average t r e a t e d r o o t l e n g t h Average c o n t r o l r o o t l e n g t h  elongation:  14  Each treatment was experiment was design.  l a i d down i n a randomised complete b l o c k  The r e s u l t s o f t h e experiment were s u b j e c t e d t o  statistical (Duncan,  r e p e a t e d f o u r times, and the whole  a n a l y s i s and Duncan's New  M u l t i p l e Range T e s t  1955).  P r e p a r a t i o n o f potassium naphthenates . (KNap) aqueous s o l u t i o n from naphthenic a c i d s Seventeen ml o f a 12.3% in  (w/v)  17 ml d i s t i l l e d water) was  KOH  (HNap) solution  (2.1 g  added t o an Erlenmeyer  KOH  flask  c o n t a i n i n g 5 g HNap ( P r a c t i c a l grade, average m o l e c u l a r weight 230> Eastman Organic Chemicals, Rochester, New Y o r k ) . The f l a s k was  shaken f o r 10-15  minutes and t h e s o l u t i o n  made t o a volume o f 25 ml w i t h d i s t i l l e d water. thus p r e p a r e d was KNap per ml.  The  By d i l u t i n g 1.0 ml o f the s t o c k s o l u t i o n w i t h the f i n a l  c o n c e n t r a t i o n o f KNap was  -2 M  s o l u t i o n was  solution  the s t o c k s o l u t i o n c o n t a i n i n g 250 mg o f  49 ml o f d i s t i l l e d *  2 x 10  was  (5000 ppm  o r 0.5%).  The pH o f the d i l u t e d  a d j u s t e d t o c a 10 w i t h 1.0  N  HC1.  RESULTS  Low KNap c o n c e n t r a t i o n s o f 1.0 and 10 ppm treatment o f cucumber seeds s t i m u l a t e d r o o t growth respectively),.  However, t h i s -stimulatory e f f e c t o f KNap  was n o t s i g n i f i c a n t . 1000  High KNap c o n c e n t r a t i o n s  (100 and  ppm) had an i n h i b i t o r y e f f e c t on r o o t growth  and 91% r e s p e c t i v e l y ) . 1000  (ca 3 and 4%  Only the i n h i b i t o r y e f f e c t o f  ppm was s i g n i f i c a n t  presented g r a p h i c a l l y  (ca 21  (Table I ) .  i n figure  1.  The r e s u l t s a r e  16  T a b l e I.  Response o f i n t a c t  Concentration (ppm)  0  (control)  r o o t s o f cucumber t o KNap.  Average l e n g t h o f r o o t s from 60 d e t e r m i n a t i o n s (mm)  93.18  a  1.0  96.32 a  10  97.37 a  100  73.30 a  1000  8.37 **  Values f o l l o w e d by the same l e t t e r do not d i f f e r s i g n i f i c a n t l y from one another, a c c o r d i n g t o Duncan's New M u l t i p l e Range T e s t (Duncan, 1955). ** Value d i f f e r s s i g n i f i c a n t l y from the c o n t r o l v a l u e a t the 0.01 l e v e l .  17  18  DISCUSSION  The t r e n d i n response o f cucumber r o o t s t o KNap i s t y p i c a l o f auxin e f f e c t ,  i . e . prevalent retardation of  e l o n g a t i o n i n h i g h c o n c e n t r a t i o n s and s l i g h t promotion i n low c o n c e n t r a t i o n s  (e.g. Burstrom, 1951;" Larsen,  1961).  The i n h i b i t i o n o f cucumber r o o t growth by 1000 ppm KNap i s s i m i l a r t o t h a t r e p o r t e d by Huseinov N.G.S. i n wheat.  (1960) u s i n g  In t h e p r e s e n t experiment, t h e t r e a t m e n t s  w i t h low c o n c e n t r a t i o n s o f KNap  (1.0 and 10 ppm) on  cucumber r e s u l t e d i n a s l i g h t promotion o f r o o t growth. S i m i l a r i n c r e a s e s have been r e p o r t e d by Huseinov utilising  (1960)  5, 10, and 50 ppm N.G.S. seed soak o f wheat.  The f a i l u r e t o o b t a i n s i g n i f i c a n t s t i m u l a t i o n o f r o o t growth i n 1.0 and 10 ppm KNap c o n c e n t r a t i o n s suggests t h a t the e f f e c t i v e p r o p e r t y o f KNap i s p r e d o m i n a n t l y t h a t o f i n h i b i t i o n o f r o o t growth which i s c h a r a c t e r i s t i c o f a u x i n (e.g. Burstrom, 1951; Larsen,  1961).  The r e t a r d a t i o n o f r o o t growth caused by IAA i s e x h i b i t e d i n much lower c o n c e n t r a t i o n s than KNap, f o r i n s t a n c e , a t c a 0.3 ppm on Avena s a t i v a  (Lane, 1936);  a t a range from 1.0 t o 10  ppm on Lepidium sativum  -7  (Bentley,  1951); a t 10  M on wheat r o o t  (Burstrbm,  1942). The f a c t t h a t i t r e q u i r e s a r e l a t i v e l y concentration  high  o f KNap (1000 ppm) t o b r i n g about a  s i g n i f i c a n t r e t a r d a t i o n o f r o o t .growth as compared to IAA (e.g.,  1.0 t o 10 ^ ppm, B e n t l e y , 1951) suggests  t h a t the degree o f e f f e c t i v e n e s s o f KNap i s l e s s than t h a t o f IAA.  20  ib)  Adventitious root i n i t i a t i o n  i n s u c c u l e n t and woody  stem c u t t i n g s .  LITERATURE REVIEW  The  auxin-induced  f o r m a t i o n o f r o o t s on stem and  leaf  c u t t i n g s , and on i n t a c t p l a n t s p r o v i d e s a good b i o a s s a y for  auxin a c t i v i t y .  That the f o r m a t i o n o f a d v e n t i t i o u s  r o o t s on a c u t t i n g always o c c u r s a t the p h y s i o l o g i c a l base, was  first  shown i n w i l l o w by H. V o c h t i n g  e x t e n s i v e c r i t i c a l review  i n 1878.  An  o f the r o l e o f hormones i n the  f o r m a t i o n o f a d v e n t i t i o u s r o o t s was  p u b l i s h e d by L i b b e r t  (1957a). The  o r i g i n a l q u a n t i t a t i v e method was  developed  by Went  (1934b), u t i l i s i n g e t i o l a t e d Pisum stem c u t t i n g s . L a t e r , Went's method was  m o d i f i e d by o t h e r i n v e s t i g a t o r s f o r the  use w i t h d i f f e r e n t s p e c i e s o f p l a n t s Luckwill, The was  (e.g., L i b b e r t , 1957b;  1956). p a r t i c i p a t i o n o f auxin i n c o n t r o l l i n g r o o t  amply demonstrated by Thimann  branching  (1936) i n experiments  21  t r e a t i n g t h e r o o t s o f Avena and Pisum w i t h  IAA. The  a p p l i c a t i o n o f indolebutyric acid i n concentrations o f 12.5  and 25 mg/1 t o t h e base o f r o o t c u t t i n g s o f Taraxacum  and C i c h o r i u m  s t i m u l a t e d abundant r o o t p r o d u c t i o n  (Warmke  and Warmke, 1950). Polikarpova root formation  (1963) r e p o r t e d t h e use o f P.G.S. d u r i n g  i n green c u t t i n g s o f c h e r r i e s , g o o s e b e r r i e s ,  and b l a c k c u r r a n t s , f a v o u r a b l y a f f e c t e d t h e formation o f r o o t s and subsequent development o f the c u t t i n g s .  The b e s t  c o n c e n t r a t i o n was found t o be 0.01% (100 ppm) P.G.S. The p h y s i o l o g i c a l a c t i v i t i e s o f sodium s a l t s o f low and h i g h m o l e c u l a r weight naphthenic  acids obtained  from  d i f f e r e n t sources were i n v e s t i g a t e d w i t h r e s p e c t t o r o o t formation  ( P o r u t s k i i e t aJL, 1963) .  f o r m a t i o n decreased HNap o b t a i n e d  They found  that root  w i t h i n c r e a s i n g m o l e c u l a r weights o f  from d i f f e r e n t sources  as i n d i c a t e d i n t h i s  order: d i e s e l f u e l < transformer  o i l < l u b r i c a t i o n o i l < motor o i l .  Root f o r m a t i o n was s t i m u l a t e d by P.G.S. i n c u t t i n g s o f c r a n b e r r y , phlox,  vine, p r i v e t ,  and t h e l e a v e s o f  p e r i l l a , b u t no s t i m u l a t i o n i n c u t t i n g s o f s p i n d l e - t r e e and p o p l a r was observed  (Yusufov,  1963).  The p h y s i o l o g y o f r o o t p r i m o r d i a i n i t i a t i o n has been e x t e n s i v e l y d i s c u s s e d f o l l o w i n g t h e i n t r o d u c t i o n o f the r h i z o c a l i n e h y p o t h e s i s by B o u i l l e n n e and Went i n 1933.  22  The  name, r h i z o c a l i n e , was f i r s t suggested by  B o u i l l e n n e and Went i n 1933 f o r the r o o t i n g  substance  produced by the l e a v e s i n the presence o f l i g h t . substance i s s t o r e d  This  i n the c o t y l e d o n s and buds, and  transported b a s i p e t a l l y . A c c o r d i n g t o the r h i z o c a l i n e h y p o t h e s i s Went, 1938), the b a s i p e t a l  transport  o f auxin causes i t  t o accumulate a t t h e base o f a c u t t i n g , auxin g r a d i e n t  and t h e r e s u l t i n g  causes a downward movement o f r h i z o c a l i n e  t o t h e base o f the c u t t i n g .  Auxin then " a c t i v a t e s " o r  "reacts" with r h i z o c a l i n e to bring The  (Cooper, 1936;  about r o o t  formation.  e x i s t e n c e o f r h i z o c a l i n e o r r h i z o c a l i n e - l i k e substances  was amply demonstrated  (Bouillenne  G a l s t o n , 1948; Hess, 1962).  and B o u i l l e n n e ,  1952;  The l a t e s t m o d i f i c a t i o n  the r h i z o c a l i n e h y p o t h e s i s by B o u i l l e n n e  of  (1964) d e s c r i b e s  r h i z o c a l i n e as a complex o f t h r e e f a c t o r s , namely 1) A highly  s p e c i f i c , mobile f a c t o r w i t h o r t h o d i p h e n o l i c  which i s s y n t h e s i s e d i n the l e a v e s . r e q u i r i n g enzyme l o c a t e d Hess  The c o f a c t o r s  (1964) added the concept o f  were c o n s i d e r e d t o be endogenous  substances capable o f a c t i n g rooting  of cuttings  Haissig  2) Auxin. 3) An oxygen-  i n s p e c i f i c c e l l s and t i s s u e s .  (1957) and Kawase  cofactors.  groups  s y n e r g i s t i c a l l y w i t h IAA i n the  o f mung bean and b r i t t l e  willow.  (1971) suggested t h a t RNA i s n e c e s s a r y f o r the  i n i t i a t i o n o f root  primordia.  MATERIALS AND METHODS  i ) S u c c u l e n t stem c u t t i n g s u s i n g bean  plants.  Uniform s i z e seeds o f bush bean p l a n t , Phaseolus •vulgaris L. c u l t i v a r Top Crop New  Westminster, B.C.)  composted  were sown on  s o i l i n s i x wooden f l a t s  U n i f o r m i t y o f p l a n t s was of  runts.  (Buckerfield's Ltd., steam-sterilised (30 x 47 x 7 cm).  a c h i e v e d by p e r i o d i c  culling  The p l a n t s were grown i n a greenhouse w i t h  a 14-hour p h o t o p e r i o d under S y l v a n i a Gro-Lux  phototube  which gave 10,000 l u x l i g h t i n t e n s i t y a t the top o f the  p l a n t s a t 25° + 1° C. The stems o f 14-day-old s e e d l i n g s were c u t a t ca  cm above s o i l  l e v e l and washed w i t h running t a p water.  One-half o f each o f the two primary l e a v e s was to the  reduce t r a n s p i r a t i o n a l l o s s o f water.  removed  The bases o f  stem c u t t i n g s thus prepared were soaked f o r s i x  hours t o a depth o f f o u r cm i n one o f the f o l l o w i n g solutions:  1) D i s t i l l e d water served  as c o n t r o l .  2) KNap 10 ppm 3) KNap 100 ppm 4) I n d o l e b u t y r i c a c i d 10 ppm 5) I n d o l e b u t y r i c a c i d 100 ppm A c i r c u l a r Pyrex g l a s s c o n t a i n e r  (diameter 27 cm)  s u f f i c i e n t t o h o l d one l i t r e o f s o l u t i o n , was p l a c e d i n a wooden f l a t .  Care was e x e r c i s e d n o t t o a l l o w the  l e a v e s t o come i n c o n t a c t w i t h the s o l u t i o n . At the c o n c l u s i o n o f the soak p e r i o d , the c u t t i n g s were removed from the s o l u t i o n , and the b a s a l  portions  o f t h e i r stems i n s e r t e d t o a depth o f f o u r cm i n evenly spaced rows i n the r o o t i n g medium k e p t i n the propagation chamber.  The r o o t i n g medium c o n s i s t e d o f 1:1 (v/v) o f  f i n e sand and peat.  The sand was s i e v e d s u c c e s s i v e l y  through % - i n c h mesh and 1/8-inch mesh s i e v e s , and then washed  thoroughly.  25  The p e a t was wetted w i t h t a p water and s c r e e n e d % - i n c h mesh s i e v e .  through  The r o o g i n g medium was s t e r i l i s e d a t  248° C f o r 15 minutes a t a p r e s s u r e o f 15 l b p e r square  inch.  The p r o p a g a t i o n chamber was covered w i t h a p o l y t h e n e l i d t o a l l o w a d a i l y 14-hour l i g h t o f i n t e n s i t y 5200 l u x t o r e a c h the c u t t i n g s .  The chamber had a i r temperatures  of  20.5 + 1° C, and a r e l a t i v e h u m i d i t y o f 90 ± 5%. I n d o l e - 3 - b u t y r i c a c i d , a r o o t i n g hormone, was i n c l u d e d i n t h i s experiment  t o serve as a s t a n d a r d f o r comparison  ( M i t c h e l l e t a l , 1968). The  stem c u t t i n g s were allowed t o r o o t f o r f i v e  days,  then t h e r o o t systems were t h o r o u g h l y washed i n r u n n i n g water. The  The number and l e n g t h o f r o o t s were experiment  was conducted  block design with three b l o c k s .  determined.  i n a randomised complete Each treatment c o n s i s t e d  o f 24 c u t t i n g s , w i t h e i g h t c u t t i n g s randomly p l a c e d i n each b l o c k .  The r e s u l t s  were s u b j e c t e d t o a n a l y s i s o f  v a r i a n c e and Duncan's M u l t i p l e Range T e s t  (Duncan, 1955).  26  i i ) Woody stem c u t t i n g s u s i n g  azalea  Stem c u t t i n g s o f "Chichibu", a z a l e a o f the Wadai group  plants.  an evergreen Japanese  (Rhododendron  eriocarpum x k a e m p f e r i , a c c o r d i n g  indicum v a r .  t o Lee e t al_, 1952),  were c o l l e c t e d on 20th August, 1971 from s e v e r a l bushes i n t h e Nursery on campus.  Uniform stem c u t t i n g s were  taken from the a p i c a l r e g i o n s  (12-15 cm) o f the p l a n t s .  The stem c u t t i n g s c o l l e c t e d from t h e f i e l d ,  were washed  and were c u t under water below the second node. t o f o u r leaves were l e f t on each c u t t i n g .  Three  The p r e p a r e d  stem c u t t i n g s were s u r f a c e s s t e r i l i s e d by immersing i n 1.0% Captan f u n g i c i d e  f o r 15 minutes.  Thereafter, the  c u t t i n g s were soaked f o r s i x hours i n one o f the f o l l o w i n g s o l u t i o n s i n the greenhouse a t a l i g h t i n t e n s i t y o f 6000 l u x , and a i r temperatures o f 25° + 1° C: 1) KNap 1000 ppm 2) KNap .100 ppm 3) KNap 10 ppm 4) D i s t i l l e d  water served as c o n t r o l .  27  F o l l o w i n g the s i x - h o u r soak p e r i o d , t h e b a s a l ends o f the c u t t i n g s were i n s e r t e d i n t o s t e r i l i s e d medium,consisted  o f 1:1  as d e s c r i b e d i n ( i ) ,  rooting  (v/v) f i n e sand and peat  prepared  c o n t a i n e d i n wooden f l a t s .  were a l l o w e d t o r o o t i n a greenhouse. a 16-hour p h o t o p e r i o d , a i r temperatures a l i g h t i n t e n s i t y o f 4,400 l u x . o f the e x p e r i m e n t a l a r e a was  The  cuttings  The greenhouse p r o v i d e d o f 22 + 1° C,  and  T h e . r e l a t i v e humidity  m a i n t a i n e d a t ca 100%  means o f an overhead water s p r a y o p e r a t e d by an  by  automatic  humidity sensing device. The experiment randomised  complete  was  s e t up i n accordance  t o the  bloc3c d e s i g n c o m p r i s i n g f o u r treatments  arranged i n f o u r b l o c k s w i t h t e n c u t t i n g s o f each  treatment  per b l o c k . The  stem c u t t i n g s remained  a t o t a l o f 35 d a y s  4  The  i n the r o o t i n g medium f o r  r o o t e d c u t t i n g s were then  carefully  removed, washed, and the number and. l e n g t h o f the r o o t s determined.  An a n a l y s i s o f v a r i a n c e and Duncan'-s  M u l t i p l e Range T e s t r e s u l t s obtained.  (Duncan, 1955)  were performed  New on t h e :  RESULTS  i ) Rooting o f s u c c u l e n t  stem c u t t i n g s o f bean.  The average number o f r o o t s on a stem c u t t i n g i s used t o r e p r e s e n t  the degree o f r o o t i n i t i a t i o n .  This  i s based on the assumption t h a t the number o f r o o t s i s analogous t o the number o f r o o t  primordia.  In a p r e l i m i n a r y experiment, l o g a r i t h m i c o f KNap c o n c e n t r a t i o n s  dilutions  o f a range from 1000 t o 0.0001  ppm were used t o determine t h e e f f e c t o f d i f f e r e n t concentrations  o f KNap on r o o t i n i t i a t i o n  and t o a c e r t a i n the o p t i m a l investigations.  concentrations  and growth, f o r subsequent  I t was found t h a t 1000 ppm KNap was  i n j u r i o u s t o stem c u t t i n g s , and caused a profound i n h i b i t i o n of root i n i t i a t i o n .  The p o r t i o n o f the stem  c u t t i n g s immersed i n 1000 ppm KNap gave a "water appearance.  soaked"  The g r e a t e s t s t i m u l a t o r y e f f e c t was observed  w i t h 10 and 100 ppm.  29  The growth acid  induction  (IBA) w e r e s i g n i f i c a n t l y  100  ppm  a t t h e 0.01  10 a n d  100 ppm  KNap a n d  level.  increases  (152.94%) b y  from the s t i m u l a t i o n  100 ppm  The  The  cuttings  o f 164.54 a n d  297.41%  stimulation of  KNap d i d n o t d i f f e r  (164.54%) b y  10 ppm  IBA  significantly  stem  c u t t i n g s o v e r c o n t r o l c u t t i n g s w e r e o b s e r v e d , b u t no d i f f e r e n c e s b e t w e e n K N a p - t r e a t e d . and The  stimulation of root  detect-  IBA-treated  cuttings.  i n i t i a t i o n as p e r c e n t a g e  control,  The  e f f e c t s o f t h e t r e a t m e n t s on t h e s t e m c u t t i n g s c a n i n Plate  1.  represented schematically  in figure  of  the  seen  is  root  (Table I I ) .  Greater s w e l l i n g at the bases o f KNap-treated  able  10  i n i t i a t i o n o f 46.35  over c o n t r o l plants.  IBA h a d  of  treatments with  i n root  r e s p e c t i v e l y over c o n t r o l plants. induction  The  root  indolebutyric  d i f f e r e n t from t h a t  KNap g a v e i n c r e a s e s  and 152.94% r e s p e c t i v e l y •in  and t h e p r o m o t i o n o f  following treatments with  control plants and  of roots,  2. be  30  Table I I .  Rooting response  o f bean stem c u t t i n g s t o  KNap and IBA t r e a t m e n t s . Treatment  +Average number o f .roots ; p e r cutting 1  Distilled H 0 (control)  28.20  KNap 10 ppm  41.27  KNap 100 ppm IBA 10 ppm  Increase over control (%)  „  +Average t o t a l length of roots per c u t t i n g  Increase over control (%)  231.33  2  IBA 100 ppm  46.35  400.00  72.91  71.33 a  152.94  507.40  119.34  74.60 a  164.54  722.67  212.40  297.41  1257.33  443.52  112.06  +Value from 15 stem c u t t i n g s . V a l u e s f o l l o w e d by t h e same l e t t e r do n o t d i f f e r s i g n i f i c a n t l y from each o t h e r , a c c o r d i n g Duncan's New M u l t i p l e Range T e s t (Duncan, 1955).  31  40d n  300 •  Average number of " roots per c u t t i n g (% c o n t r o l ) 2  0  0  100 •  control  KNap  10 ppm F i g u r e 2.  KNap'  100 ppm  IBA  IBA  10 ppm  100 ppm  S t i m u l a t i o n o f r o o t i n i t i a t i o n o f bean c u t t i n g s by KNap and IBA t r e a t m e n t s .  stem  P l a t e 1.  Rooting response o f bean stem c u t t i n g s KNap and IBA treatments.  Legend: 1  C u t t i n g i n d i s t i l l e d water.  2  C u t t i n g i n 10 ppm KNap.  3  C u t t i n g i n 100 ppm KNap.  4  C u t t i n g i n 10 ppm IBA.  5  C u t t i n g i n 100 ppm IBA.  33  ii)  Rooting o f woody stem c u t t i n g s o f a z a l e a . The  (10,  treatments o f a z a l e a stem c u t t i n g s w i t h KNap  100, and 1000 ppm) s t i m u l a t e d b o t h i n d u c t i o n and  growth o f r o o t s .  High c o n c e n t r a t i o n s  o f KNap (100 and 1000  ppm) r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e  i n number o f r o o t s  (ca 50 and c a 134% r e s p e c t i v e l y ) over t h e c o n t r o l c u t t i n g s a t t h e 0.01 l e v e l .  However, t h e low c o n c e n t r a t i o n  o f KNap had n o . s i g n i f i c a n t s t i m u l a t i o n o f r o o t over c o n t r o l v a l u e s .  A l l concentrations  (10 ppm)  initiation  o f KNap  and  1000 ppm) s i g n i f i c a n t l y augmented r o o t growth  66,  and 220% r e s p e c t i v e l y ) a t t h e 0.01 l e v e l .  (10, 100, (ca 36,  The treatments  w i t h 10 and 100 ppm KNap. d i d n o t d i f f e r s i g n i f i c a n t l y each o t h e r .  A slight  "scorching"  from  o f t h e p o r t i o n o f t h e stems  immersed i n 1000 ppm KNap was observed. The and  r e s u l t s o f t h i s i n v e s t i g a t i o n are given  the stimulatory  i n P l a t e 2.  i n Table i n  e f f e c t o f t h e t r e a t m e n t s can be seen  Root i n i t i a t i o n c a l c u l a t e d as percentage o f  c o n t r o l values  are represented  i n a h i s t o g r a m i n - f i g u r e 3.  34  Table I I I .  Response o f stem c u t t i n g s o f a z a l e a t o KNap.  Concentration o f KNap (ppm)  0  (control)  Average number of roots per cutting +  29.62 a  Increase over control (%)  —  Average t o t a l length of roots per cutting + 279.75  Increase over control (%)  —  33.37 a  12.66  382.17 b  36.61  100  44.46  50.10  464.12 b  65.90  1000  69.29  133.93  10  894.58  219.77  + V a l u e from 24 stem c u t t i n g s . V a l u e s f o l l o w e d by t h e same l e t t e r do n o t d i f f e r s i g n i f i c a n t l y from each o t h e r , a c c o r d i n g t o Duncan's New M u l t i p l e Range T e s t (Duncan, 1955).  250 i  200  150 Average number of roots per c u t t i n g {% c o n t r o l ) 100  50  control  F i g u r e 3.  10 ppm  100  ppm  1000 ppm  Stimulation of root i n i t i a t i o n i n azalea c u t t i n g s by KNap treatments.  stem  P l a t e 2.  Rooting response ,of a z a l e a  stem c u t t i n g s  KNap treatments.  The c o n c e n t r a t i o n s  o f KNap employed as i n d i c a t e d .  37  DISCUSSION  The  L  r o o t i n g o f stem c u t t i n g s i n d i s t i l l e d water i s  e v i d e n c e o f t h e presence o f endogenous a u x i n , predominance o f i t s b a s i p e t a l t r a n s p o r t .  and t h e  The average number  o f r o o t s formed p e r t r e a t e d stem c u t t i n g i s taken as a measure o f r o o t - f o r m i n g The  marked i n c r e a s e s  a c t i v i t y o f KNap and IBA t r e a t m e n t s .  i n t h e average number o f r o o t s p e r c u t t i n g  i n KNap- and I B A - t r e a t e d possess high  root-forming  c u t t i n g s suggest t h a t these compounds activity.  The g r e a t e r number o f  r o o t s i n KNap t r e a t e d c u t t i n g s may be t h e r e s u l t o f a c c e l e r a t e d m i t o s i s which l e a d s i n i t i a t i o n o f r o o t meristems. i n i t i a t i o n and t h e i n c r e a s e d  t o an i n c r e a s e d  rate of  The s t i m u l a t i o n o f r o o t s w e l l i n g s a t t h e extreme t i p s  o f t h e bases o f KNap-treated stem c u t t i n g s over t h a t o f the c o n t r o l s may be accounted f o r on t h r e e  bases:  1) The p o s s i b l e augmentation o f endogenous auxin by KNap. 2) The r e l a t i v e l y low m o b i l i t y , and g r e a t e r c h e m i c a l  stability  o f KNap f o l l o w i n g a p p l i c a t i o n t o stem c u t t i n g s which r e s u l t e d i n t h e accumulation and r e t e n t i o n o f KNap near t h e s i t e o f application. 3) A m o b i l i z a t i o n o f r o o t - f o r m i n g  substances o r c o f a c t o r s  38  f o r example r h i z o c a l i n e , t o the s i t e o f KNap a p p l i c a t i o n . Chemical s t a b i l i t y and low m o b i l i t y a r e two important properties o f root-forming The  substances  (e.g., Audus, 1959).  stimulation o f rooting o f azalea cuttings i s i n  agreement w i t h the o b s e r v a t i o n s  o f Polikarpova  100  ppm P.G.S. on woody c u t t i n g s o f c h e r r i e s ,  and  blackcurrants. In v e r y h i g h KNap c o n c e n t r a t i o n  (1963) u s i n g gooseberries,  (1000 ppm), a t o x i c  e f f e c t on bean c u t t i n g s s e t i n and o f f s e t s t i m u l a t i o n r e s u l t i n g i n a "water soaked" appearance i n the t r e a t e d stem p o r t i o n s .  The optimum r o o t - f o r m i n g  was found t o be i n the c o n c e n t r a t i o n s  property  o f 100 ppm i n s u c c u l e n t  bean c u t t i n g s , and 1000 ppm i n woody a z a l e a The  increases  are i n t e r p r e t e d as  o f KNap  cuttings.  i n r o o t growth o f t r e a t e d stem c u t t i n g s m a i n l y due t o a r e l a t i v e l y e a r l y r o o t  i n i t i a t i o n compared w i t h the c o n t r o l c u t t i n g s . The  l a c k o f s i g n i f i c a n t d i f f e r e n c e between the e f f e c t s  o f 100 pprn KNap and 10 ppm IBA treatments on bean c u t t i n g s i n d i c a t e s t h a t the r o o t - i n i t i a t i o n e f f e c t o f IBA i s s i m i l a r t o t h a t o f KNap. and  IBA i s an e s t a b l i s h e d r o o t i n g hormone,  proved t o be s u p e r i o r t o IAA (e.g., Audus, 1959). As  i s t y p i c a l o f the e f f e c t o f auxin,  of adventitious  r o o t i n i t i a t i o n by KNap.  there  i s a stimulation  39  i c ) E l o n g a t i o n o f pea stem  segments.  LITERATURE REVIEW  The b e h a v i o u r o f stem segments o f Pisum under t h e i n f l u e n c e o f s y n t h e t i c auxin i n v a r i o u s e x p e r i m e n t a l c o n d i t i o n s has been s t u d i e d by G a l s t o n and Hand Nitsch  (1956\ and o t h e r s .  (1949); N i t s c h and  The response o f such segments  has been" employed t o d e t e c t and c h a r a c t e r i s e o c c u r r i n g auxin incubated  (Larsen, 1961).  naturally-  G a l s t o n and Hand  8-day-old dark-grown A l a s k a pea stem  (1949) who segments  i n v a r i o u s c o n c e n t r a t i o n s o f IAA a t 25° C f o r 24 hours, found t h a t h a l f maximal growth was g e n e r a l l y produced by 0.01 mg/1, and maximal growth by 0.1 and 1.0 mg/1. segments immersed appearance.  The  i n 10 mg/1 IAA had a "water soaked"  Maximum response i n t h e s t r a i g h t growth  was o b t a i n e d by t h e use o f 0.1 mg/1 IAA, pH 6.1  test  (K^PO^ -  Na^HPO^) i n 2% sucrose, and a t 30° C i n darkness (Galston and Hand, 1949).  40  White l i g h t r e t a r d s the e l o n g a t i o n of e t i o l a t e d stem segments i n v a r y i n g c o n c e n t r a t i o n s  of  IAA,  and  l i g h t - i n d u c e d i n h i b i t i o n o f growth c o u l d n o t be i n t e r m s o f p h o t o i n a c t i v a t i o n o f IAA. IAA  levels,  there  u t i l i s a t i o n of  IAA b y  the  segments.  e f f e c t o f s u g a r on  debatable.  C h r i s t i a n s e n and  sucrose  no  has  whereas G a l s t o n  e f f e c t on and  The  are-taken.  ( G a l s t o n and  Thimann  Hand,  (1950) r e p o r t e d  (1950) r e p o r t e d a n  the p o r t i o n of the  t o e x o g e n o u s IAA.  Hillman  than  stem  that  segments,  increase  in  otherwise. depends  stem from w h i c h the  segments  (1958) s u g g e s t e d t h a t  apex o f the  C h r i s t i a n s e n and  o f segments t a k e n  1949).  However, more i n v e s t i g a t o r s  in their trials  P u r v e s and  the  This e f f i c i e n c y i s  e l o n g a t i o n o f pea  Hand  f u r t h e s t from the  t h e use  exogenous  r e s p o n s e o f segments t o t e s t m a t e r i a l s  g r e a t l y on  taken  sucrose  explanied  e l o n g a t i o n o f stem segments i s  e l o n g a t i o n v / i t h 2% s u c r o s e . i n c l u d e 2%  low  this  i s relatively greater efficiency i n  g r e a t e r i n the dark than i n l i g h t The  At  pea  from t h i r d  internodes Thimann  segments  respond w e l l  (1950)  internodes.  suggested  Nitsch first  and  Nitsch  (1956) u t i l i s e d  segments t a k e n  from  the  internodes. -9 The  Bentley, To  lower  l i m i t o f t h i s t e s t i s 10  mg/1  IAA  (e.g.,  1958). date,  no  literature i s available  HNap o n t h e g r o w t h o f s t e m s e g m e n t s .  on  the e f f e c t  of  42  MATERIALS AND METHODS  The procedure adopted i n t h i s i n v e s t i g a t i o n was m o d i f i e d from C h r i s t i a n s e n and Thimann (1968). of  Pisum  Uniform seeds  sativum L c u l t i v a r A l a s k a were soaked f o r s i x hours  i n d i s t i l l e d water and sown i n v e r m i c u l i t e i n wooden  flats.  The p l a n t s were grown i n darkness a t a i r temperatures o f 23° + 0.5° C, and a r e l a t i v e h u m i d i t y o f 95% + 2%. seven days, u n i f o r m p l a n t s were s e l e c t e d and 19.5  After mm  segments were c u t from t h e s u b a p i c a l p o r t i o n o f the t h i r d i n t e r n o d e s w i t h r a z o r b l a d e s mounted 19.5 mm p l a s t i c block. to  dry  T h i s r e g i o n has been shown t o respond w e l l  a u x i n (Purves and H i l l m a n , 1958).  excision  a p a r t on a  Immediately  following  the segments were washed i n d i s t i l l e d water,  blotted  w i t h paper towels, and d i v i d e d i n t o groups o f f i v e .  Each group was weighed one o f t h e f o l l o w i n g  and p l a c e d i n p e t r i d i s h e s c o n t a i n i n g  solutions:  1) IAA (K s a l t ) 0.1  ppm  2) IAA (K s a l t ) 1.0  ppm  3) CHCA (K s a l t ) 0.1  ppm  43  4) CHCA  (K s a l t )  1.0 ppm  5)  CHCA  (K s a l t )  10.0 ppm  6)  KNap  0.1 ppm  7)  KNap  1.0 ppm  8)  KNap  10.0  ppm  9) D i s t i l l e d w a t e r s e r v e d With the exception o f the control,  as c o n t r o l . t h e pl-l o f t h e  a b o v e s o l u t i o n s was a d j u s t e d t o 10 w i t h I N KOH. The  stem segments were i n c u b a t e d  f o r 24 h o u r s i n  d a r k n e s s a t a n a i r t e m p e r a t u r e o f 29° C. The  e x p e r i m e n t was c o n d u c t e d i n a r a n d o m i s e d  bio k design,  arranged i n three r e p l i c a t i o n s , with  segments i n each  complete five  replication.  At the completion  of the specified  incubation period,  the  l e n g t h s o f t h e segments were measured w i t h a m i c r o m e t e r .  All  observations  two 4 0 - w a t t The  and m a n i p u l a t i o n s  red Sylvania  were performed  under  lamps.  r e s u l t s were s u b j e c t e d t o a n a l y s i s o f v a r i a n c e and  D u n c a n ' s New M u l t i p l e Range T e s t c o m p a r i s o n o f t r e a t m e n t means.  (Duncan,  1955) f o r m u l t i p l e  44  RESULTS  The  e f f e c t o f treatment on e l o n g a t i o n o f stem segments  was a s s e s s e d  using t h i s  formula:  Increase o f t r e a t e d over c o n t r o l — Increase i n C o n t r o l The  x 100  s t i m u l a t o r y e f f e c t o f t h e t r e a t m e n t s on segment  elongation,  and on i n c r e a s e s  i n weight o f segments was  s i g n i f i c a n t a t t h e 0.01 l e v e l . A r i s e i n concentrations  o f t h e IAA, CHCA, and KNap .  t r e a t m e n t s r e s u l t e d i n an i n c r e a s e i n e l o n g a t i o n o f t h e segments, and a c o r r e s p o n d i n g segments The  i n c r e a s e i n weight  o f the  (Table i v ) . e l o n g a t i o n o f segments i n 0.1 ppm IAA (ca 339%  i n c r e a s e over c o n t r o l segments) d i d n o t d i f f e r  significantly  from t h a t o f 1.0 ppm KNap ( c a 279% over c o n t r o l segments), and  a l s o from t h e induced  e l o n g a t i o n by 1.0 and 10 ppm CHCA  (ca 327 and 360% i n c r e a s e over c o n t r o l segments).  This  suggests t h a t t h e s t i m u l a t o r y e f f e c t s o f CHCA and KNap are s i m i l a r t o t h a t o f IAA. In low KNap c o n c e n t r a t i o n  (0.1 ppm), 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 o f induced c o n t r o l segments.  e l o n g a t i o n over t h e  45  KNap a t 10 ppm had g r e a t e r s t i m u l a t o r y e f f e c t on elongation IAA  (ca 473% over c o n t r o l segments) than t h a t o f  (ca 339% over c o n t r o l  segments).  The i n c r e a s e s i n weights o f segments r e c e i v i n g 0.1 ppm IAA  (ca 70%), 10 ppm KNap (ca 67%), and 1.0 ppm KNap d i d  not d i f f e r s i g n i f i c a n t l y  from one another, a c c o r d i n g t o  Duncan's New M u l t i p l e Range T e s t  (Duncan,  1955).  46  T a b l e IV. Response o f dark-grown A l a s k a pea stem segments t o exogenously a p p l i e d IAA, CHCA, and KNap.  Treatment (ppm)  Average Average length increase o f 15 in segments l e n g t h (mm) (mm)  Increase in length over control  Average increase in total weight o f 5 segments + (mg)  (°/o)  Increase in weight over control <<&)  1 Control  19.83*  0.33  2 IAA 0.1  20.95  1.45  339.39  35.67  69.85  3 IAA 1.0  22.10  2.60  687.87  74.67  255.57  4 CHCA 0.1  20.24  0.74  124.24  21.67  3.19  5 CHCA 1.0  20.91  1.41  327.27  27.00  28.57  6 CHCA 10  21.02  1.52  360.60  29.67  41.28  7 KNap 0.1  20.16  0.66  100.00  25.33  20.62  8 KNap 1.0  20.75  1.25  278.78  31.00  47.61  9 KNap 10  21.39  1.89  472.72  35.00  66.66  21.00  Segment A T r e a t m e n t number:  3  9  6  2  Increase A T r e a t m e n t number:  3  2  9  8  elongation 5  8  4  7  1  i n weight 6  5  7  4  1  A Corresponds t o the treatment i n Table V. Treatments u n d e r s c o r e d by t h e same l i n e do not d i f f e r s i g n i f i c a n t l y , a c c o r d i n g t o Duncan's New M u l t i p l e Range T e s t . + Value from t h r e e r e p l i c a t i o n s . * I n i t i a l l e n g t h was 19.5 mm f o r a l l segments.  DISCUSSION  The w i d e l y h e l d concept o f IAA-induced c e l l  elongation  i s t h a t the primary e f f e c t o f IAA i s on the c e l l w a l l , and t h a t IAA r e g u l a t e s c e l l expansion by l o o s e n i n g walls  the c e l l  (Heyn, 1931, 1940; C l e l a n d and Bonner, 1956;  Burstrom, 1961; Preston  and Hepton, 1960).  Burstrom  (1942)  suggested, i n the case o f wheat r o o t s , the i n f l u e n c e o f IAA  on r o o t growth was mainly due t o i t s e f f e c t on c e l l  elongation  r a t h e r than c e l l m u l t i p l i c a t i o n .  concept may apply  t o the e f f e c t o f o t h e r  The same  auxin-like  substances on stem segments. The  l a c k o f s i g n i f i c a n t d i f f e r e n c e i n the e f f e c t s  o f 0.1 ppm IAA, 1.0,and 10 ppm CHCA, and 1.0 ppm KNap on augmentation o f e l o n g a t i o n  o f pea stem segments  provides  evidence t h a t the b e h a v i o u r o f CHCA and KNap a t the s a i d concentrations  was analogous t o t h a t o f IAA.  The  consequence o f t h i s analogy leads t o the suggestion i n t h e promotion o f c e l l e l o n g a t i o n ,  that  CHCA and KNap f i r s t  r e l a x the c e l l w a l l s , and, as a r e s u l t o f t h i s r e l a x a t i o n , water i s taken i n o s m o t i c a l l y . p r o c e s s i n v/ater uptake d u r i n g has been amply demonstrated  The absence o f an a c t i v e IAA-induced c e l l  expansion  ( C l e l a n d and Bonner, 1956;  48  Ordin cell is  e t a l , 1956;  Levitt,  1953;  Thimann, 1954).  That  e n l a r g e m e n t i s l a r g e l y due t o i n c r e a s e d w a t e r  uptake  s u b s t a n t i a t e d i n t h i s e x p e r i m e n t by marked i n c r e a s e s i n  w e i g h t i n KNap- a n d C H C A - t r e a t e d  segments.  F r o m t h e r e p o r t s o f i n c r e a s e s i n de n o v o  synthesis  a n d / o r a c t i v i t y o f c e r t a i n enzymes i n b u s h b e a n s f r o m KNap a n d CHCA t r e a t m e n t s ( F a t t a h and W o r t ,  resulting 1970;  S e v e r s o n , 1 9 7 2 ) , a n d t h a t KNap a c t s a t t h e g e n e t i c (Wort e t a l , 1 9 7 1 ) , i t i s t e m p t i n g t o s p e c u l a t e KNap- and CHCA-augmented i n c r e a s e s i n c e l l  level  that the  elongation  may  be m e d i a t e d t h r o u g h t h e s t i m u l a t i o n o f t h e s y n t h e s i s o f h y d r o x y p r o l i n e - r i c h p r o t e i n , and t h e s u b s e q u e n t i n c o r p o r a t i o n of t h i s p r o t e i n i n t o the c e l l w a l l i n order elongation to occur. the  suggestion  This explanation  by C l e l a n d  e l o n g a t i o n o f Avena  for cell  i s i n agreement  with  (1967) w i t h r e s p e c t t o I A A - i n d u c e d  coleoptiles.  49  id) Abscission  o f debladed p e t i o l e s o f bean  LITERATURE  There i s s t i l l u n c e r t a i n t y time w i t h r e s p e c t 1933,  plants.  REVIEW  p r e v a i l i n g a t the p r e s e n t  t o the p h y s i o l o g y o f a b s c i s s i o n .  Since  v/hen L a i b a c h found t h a t the a b s c i s s i o n o f debladed  p e t i o l e s was d e l a y e d by the a p p l i c a t i o n o f a u x i n - r i c h o r c h i d p o l l i n i a t o the c u t s u r f a c e s it  i s recognised that auxin plays  abscission.  o f t h e debladed p e t i o l e s ,  a regulatory  role i n  I n h i b i t i o n o f a b s c i s s i o n b y the a p p l i c a t i o n  o f 5 mg/1  IAA t o debladed p e t i o l e s o f Coleus b l u m e i Benth.  by La Rue  (1936) c o n f i r m e d the c l a s s i c a l work o f L a i b a c h .  Early investigations age  showed t h a t l e a f a u x i n decreased w i t h  (Avery, 1935; Goodwin,  Working w i t h Coleus, Myers between age o f l e a v e s , times r e q u i r e d  1937; Went and Thimann, 1937). (1940) found p o s i t i v e c o r r e l a t i o n s  amount o f d i f f u s i b l e auxin, and  f o r a b s c i s s i o n o f i n t a c t o r debladed  S h o j i e t a l (1951) r e p o r t e d h e a l t h y l e a f l e t s o f bean  that  leaves.  f r e e a u x i n i n the mature  (Phaseolus v u l g a r i s L ) , i s about  50  t h r e e times t h a t i n the l e a f s t a l k .  Immediately b e f o r e  a b s c i s s i o n , l e a f l e t auxin f e l l t o a l e v e l  approximately  equal t o t h a t o f the l e a f s t a l k , w h i l e s t a l k auxin remained unchanged. cotton  S i m i l a r changes were observed  (Jacobs, 1955;  Carns,  1957,  i n Coleus  and  respectively).  The process o f a b s c i s s i o n i s a f f e c t e d by a m u l t i t u d e o f compounds and environmental  factors:  1) Auxin Auxin r e t a r d s o r promotes a b s c i s s i o n depending on the s i t e o f a p p l i c a t i o n w i t h r e s p e c t t o the a b s c i s s i o n zone (e.g., A d d i c o t t e t a l , 1955). The  regulatory e f f e c t  o f auxin on a b s c i s s i o n i s governed by the time o f i t s a p p l i c a t i o n t o p e t i o l e s a f t e r the removal o f the  leaf  blades  by  ( R u b i n s t e i n and Leopold,  .the c o n c e n t r a t i o n o f auxin  1962,  1963), and  (e.g., Gaur and Leopold,  1955).  The r o l e o f auxin i n the r e g u l a t i o n o f a b s c i s s i o n i s discussed. 2) A b s c i s i c a c i d  (Abscisin  Ohkuma e t a l , 1963,  1965;  Eagles and Wareing, 1963;  I I : A d d i c o t t e t al_,  1964;  Dormin: C o r n f o r t h e t a l , 1965; Wareing e t a l , 1964).  51  A b s c i s i c a c i d was thought t o be an a b s c i s s i o n - r e g u l a t i n g hormone based on the f a c t t h a t the amounts o f t h i s hormone i n c r e a s e d u r i n g p e r i o d s o f ageing, c o r r e l a t e d with  abscission..  a c i d to explants 3) G i b b e r e l l i c  and t h a t ageing i s  The a p p l i c a t i o n o f a b s c i s i c  accelerates abscission.  acids  G i b b e r e l l i c a c i d s promote a b s c i s s i o n C a m s e t a t , 1951; C h a t t e r j e e  (Bornman,  and Leopold, 1964;  1965;  Greenblatt,  1965). 4)  Ethylene Ethylene  i s a potent  Zimmerman e t a l , 1931) .  accelerant of abscission  I t s e f f e c t on t h e a b s c i s s i o n  has been s t u d i e d by Gawadi and Avery Ethylene  (e.g.,  (1950); H a l l  process  (1952).  a c c e l e r a t e s a b s c i s s i o n through i t s i n h i b i t o r y  i n f l u e n c e on the b a s i p e t a l t r a n s p o r t o f a u x i n i n i t i a t i o n o f s p e c i f i c RNA, and Morgan,  and  on t h e  and p r o t e i n s y n t h e s i s  (Beyer  1971) .  5) Amino a c i d s and c a r b o h y d r a t e s Rubinstein and  glutamic  and Leopold  (1962) r e p o r t e d t h a t  alanine  a c i d promoted a b s c i s s i o n o f bean p e t i o l e s .  52  Yager and Muir promoter  (1958) found t h a t methionine was a p o t e n t  o f abscission.  . Carbohydrates have been shown t o r e t a r d o r promote a b s c i s s i o n under a wide range o f e x p e r i m e n t a l c o n d i t i o n s (Biggs and Leopold, 1957; Brown and A d d i c o t t , 6)  Environmental  1950).  factors  An e x t e n s i v e review o f t h e i n f l u e n c e o f e n v i r o n m e n t a l f a c t o r s on a b s c i s s i o n i s d e s c r i b e d i n a paper by A d d i c o t t  * (1968).With bean l e a f l e t e x p l a n t s , t h e r a t e o f a b s c i s s i o n i s v e r y low below 5° C, r i s e s w i t h temperature t o a maximum o  o  between 25  and 30  C, and f a l l s  a t h i g h e r temperatures  (Yamaguchi,  1954).  T h i s phenomenon may be a s s o c i a t e d w i t h  enzyme a c t i v i t y as c e l l u l a s e h a s been found t o break down c e l l walls  (e.g., Abeles and Leather, 1971).  r e l a t i v e l y extreme temperatures  Exposure t o  i s o f t e n f o l l o w e d by  i n c r e a s e d a b s c i s s i o n o f l e a v e s , f l o w e r s , and f r u i t s (Chandler, 1925).  The autumn l e a f a b s c i s s i o n o f deciduous  t r e e s i s c o r r e l a t e d w i t h s h o r t e n i n g o f p h o t o p e r i o d , e.g., sugar maple  (Acer saccharum)  (Wiesner, 1904).  Under reduced  oxygen t e n s i o n s , a b s c i s s i o n i s r e t a r d e d (Sampson, * An e x p l a n t i s a s m a l l segment o f a l e a f an a b s c i s s i o n zone.  1918).  containing  53  Oxygen a c c e l e r a t i o n o f a b s c i s s i o n may i n a c t i v a t i o n o f auxin by and H a l l , The  be  the r e s u l t o f  IAA. o x i d a s e system  (e.g.,  Morgan  1963). auxin a c c e l e r a t i o n o f a b s c i s s i o n was  f o l l o w i n g a p p l i c a t i o n s o f 10,  105,  and  525  first  mg/1  observed  IAA  to  the p r o x i m a l s i d e o f the a b s c i s s i o n zones o f e x c i s e d o f bean  ( A d d i c o t t and  o f a range o f IAA  Lynch, 1951).  concentrations  However, a p p l i c a t i o n s  10-1000 mg/1  the a b s c i s s i o n zones were shown to r e t a r d They o b t a i n e d s i m i l a r r e s u l t s w i t h 2,4-D Retardation  o f a b s c i s s i o n was  p o r t i o n to the c o n c e n t r a t i o n The and  work o f . A d d i c o t t  extended by  and  Lynch  s e v e r a l workers.  distal  to  abscission. and  found t o be o f IAA  leaflets  2,4,5-T.  i n d i r e c t pro-  applied. (1951) has been confirmed  Jacobs  (1955) working  w i t h Coleus, i n d i c a t e d t h a t auxin moving from the a p i c a l bud  and  from i n t a c t leaves  accelerated  the  lower debladed p e t i o l e s .  accelerated leaves.  , and  abscission  With f i e l d c o t t o n ,  (1960) found t h a t removal o f the abscission  the  a p i c a l bud  a p p l i c a t i o n o f IAA  of  Louie  retarded  t o the stem stump  a b s c i s s i o n o f debladed p e t i o l e s o f the  upper  The  employment o f p e t i o l e a b s c i s s i o n s e r v e s a s u i t a b l  bioassay t o evaluate the a u x i n - l i k e properties o f t e s t compounds. No l e s s t h a n s i x t h e o r i e s have been p r o p o s e d i n an attempt t o e l u c i d a t e the physiology o f a b s c i s s i o n . The more r e c e n t t h e o r i e s a r e : 1) A u x i n g r a d i e n t  ( A d d i c o t t , Lynch, and C a m s , 1955)  T h i s t h e o r y i s f o r m u l a t e d on t h e f o l l o w i n g p r i n c i p l e s a) A g r a d i e n t o f a u x i n e x i s t s a c r o s s t h e a b s c i s s i o n zone. b) T y p i c a l l y , t h e g r a d i e n t appears t o be from high auxin d i s t a l l y , t o r e l a t i v e l y  relatively  low a u x i n p r o x i m a l l y .  c) A l o w e r i n g o f t h e g r a d i e n t i n i t i a t e s a b s c i s s i o n . d) The degree o f l o w e r i n g o f t h e a u x i n g r a d i e n t d e t e r m i n e s the r a t e o f a b s c i s s i o n . T h i s t h e o r y i s s u p p o r t e d b y T e r p s t r a (1956) and L o u i e (1960). 2) Two-stage t h e o r y  ( R u b i n s t e i n and L e o p o l d ,  1962, 1963)  T h i s t h e o r y a p p a r e n t l y supersedes t h e a u x i n g r a d i e n t t h e o r y o f A d d i c o t t e t a l , (1955).  -4 U t i l i s i n g 5 x 10 M  55  r a d i o a c t i v e NAA Leopold  on bean p e t i o l e e x p l a n t s , R u b i n s t e i n  and  (1963) were a b l e t o demonstrate the l a c k o f an  a u x i n g r a d i e n t a c r o s s the a b s c i s s i o n zone.  Stage I i s  the i n d u c t i o n stage o f about s i x h o u r s o r more, and i s i n h i b i t e d by a u x i n .  Stage I I i s a c c e l e r a t e d by a u x i n o f  similar concentration.  Gaur and  t h a t low c o n c e n t r a t i o n s o f NAA and h i g h c o n c e n t r a t i o n s whether the. auxin was  (1-10  ppm)  (100-1000 ppm)  (1955) r e p o r t e d promoted a b s c i s s i o n  retarded abscission  a p p l i e d d i s t a l l y or proximally with  r e s p e c t t o the a b s c i s s i o n zone. t o NAA  Leopold  The  "two-stage"  i s i n t e r p r e t e d by R u b i n s t e i n and Leopold  a t t r i b u t a b l e t o the "two-stage" e f f e c t . t h e promotion o f a b s c i s s i o n by d i s t a l  response (1962) as  They proposed t h a t  a p p l i c a t i o n s of  low  a u x i n c o n c e n t r a t i o n s i s "a consequence o f an amount o f a u x i n j u s t low enough t o a l l o w the i n d u c t i o n stage t o proceed completion  y e t h i g h enough t o s t i m u l a t e the second  to  stage..."  T h i s "two-stage" e f f e c t o f a u x i n on a b s c i s s i o n i s i n c o r r o b o r a t i o n w i t h the two-phase scheme o f a u x i n a c t i o n on growth as proposed by Thimann  (1951), whereby low  auxin  c o n c e n t r a t i o n promotes growth, w h i l e h i g h a u x i n c o n c e n t r a t i o n is  inhibitory.  56  3) A q e i n q - e t h y l e n e  theory  (Abeles,  1968)  A b e l e s (1968) n o t e d t h a t e t h y l e n e i n i t i a t e s RNA  and p r o t e i n s y n t h e s e s n e c e s s a r y  for c e l l  specific  separation.  As t h e t i s s u e ages, i t becomes i n c r e a s i n g l y s e n s i t i v e t o ethylene.  Ethylene production by p l a n t t i s s u e s i n response  t o wounding was  r e p o r t e d by B u r g  (1962), and  suggested  t h a t e t h y l e n e produced by f r e s h l y - c u t bean e x p l a n t s  (ca 3  m i c r o l i t r e s p e r g p e r hour) i s s u f f i c i e n t t o s t i m u l a t e abscission  (Burg, 1968).  B e y e r and Morgan (1971) i n d i c a t e d  t h a t endogenously p r o d u c e d e t h y l e n e may to  function i n part  regulate l e a f a b s c i s s i o n through i t s i n h i b i t o r y  effect  on a u x i n t r a n s p o r t and t h e i n d u c t i o n o f t h e s y n t h e s i s o f the c e l l wall-degrading  enzyme c e l l u l a s e .  (•1967) r e p o r t e d t h a t c e l l u l a s e was l a y e r , and t h a t e t h y l e n e  H o r t o n and  l o c a l i s e d i n the  Osborne  separation  increased c e l l u l a s e a c t i v i t y ,  while  2,4,5-T d e c r e a s e d i t . The  l a t t e r two t h e o r i e s have s u b s t a n t i a l b a s e s ,  and  o f f e r s a t i s f a c t o r y e x p l a n t i o n s of the process  of abscission.  The  d i s c r e p a n c i e s i n the f i n d i n g s o f v a r i o u s  researchers  may  be due  t o t h e use o f d i f f e r e n t s p e c i e s o f p l a n t s .  57  P e t i b l a r explant in this field.  technique i s popular with i n v e s t i g a t o r s  S i n c e t h i s t e c h n i q u e does not account f o r  the c o r r e l a t i v e i n f l u e n c e o f o t h e r p l a n t p a r t s on  the  a b s c i s s i o n zone i n q u e s t i o n ,  from  such s t u d i e s may  the r e s u l t s o b t a i n e d  not r e f l e c t the t r u e o r  situation in intact plants.  close-to-true  In t h i s r e s p e c t ,  the  o f i n t a c t p l a n t s t o t e s t the a c t i v i t y o f v a r i o u s  employment compounds  i n an e f f o r t t o g a i n i n s i g h t i n t o the p r o c e s s o f a b s c i s s i o n may  prove t o be No  superior.  information  with regard  i n the  l i t e r a t u r e has been encountered  t o the i n f l u e n c e o f HNap on  abscission.  58  MATERIALS AND METHODS  Four seeds o f bush bean  (Phaseolus v u l g a r i s L. c u l t i v a r  Top Crop), were sown i n composted s o i l i n 15-cm p l a s t i c pots.  U n i f o r m i t y o f p l a n t s was o b t a i n e d by r e d u c i n g t o  two s e e d l i n g s i n each p o t seven days a f t e r sowing, and f i n a l l y t o one plant,two days b e f o r e treatment.  The p l a n t s  were arranged i n r o w s o f four, and grown i n a greenhouse under n a t u r a l l i g h t c o n d i t i o n s .  The number o f sunshine hours  f o r the months o f September and October, 1971 were: September: t o t a l 162.8 hours, average 5.43 hours,  range  0.00-11.7 hours. October: t o t a l 121.2 hours, average 3.90 hours, 0.00-9.7 hours.  range  The l i g h t i n t e n s i t y v a r i e d from 12,000  to 20,000 l u x . The procedure used i n t h i s i n v e s t i g a t i o n was m o d i f i e d from M i t c h e l l e t a l , (1968). sowing,  Ten days  from  the b l a d e o f a primary l e a f o f each p l a n t was  severed j u s t below the p u l v i n u s .  The debladed p e t i o l e s  were t r e a t e d by a p p l y i n g , w i t h a wooden t o o t h p i c k , l a n o l i n Tween 20 c o n t a i n i n g one o f the t e s t compounds namely: KNap, CHCA (K s a l t ) ,  NAA,  and lanolin-Tween 20.  The con-  c e n t r a t i o n s employed i n each case, except the lanoline-Tween 20, were 1.0, 10, 100, and 1000 ppm.  59  The treatments were a p p l i e d around the debladed p e t i o l e as a band o f about f i v e mm wide, and a t a d i s t a n c a o f about f i v e mm  from the stem.  The d e t e r m i n a t i o n o f a constant  o f a b s c i s s i o n was made by a p p l i c a t i o n  f i v e g pressure  once each day i n a downward  d i r e c t i o n a g a i n s t the upper s u r f a c e o f each debladed p e t i o l e , a t a d i s t a n c e o f 8-10  mm  from the stem.  by p r e s s i n g the metal r o d o f a p r e s s u r e  T h i s was  accomplished  applicator against  the p e t i o l e as i n d i c a t e d i n P l a t e 3. The number o f p e t i o l e s a b s c i s e d each day upon a p p l i c a t i o n o f the f i v e g p r e s s u r e was  recorded.  The number o f  days r e q u i r e d f o r 50% o f the p e t i o l e s i n each treatment t o a b s c i s e was  taken f o r comparison o f the t r e a t e d and c o n t r o l  plants. T h i s i n v e s t i g a t i o n was b l o c k design  s e t up i n a randomised  i n v o l v i n g three blocks.  Four p l a n t s o f each  treatment were arranged i n a row i n each b l o c k . of variance 1955)  and Duncan's New  complete  M u l t i p l e Range T e s t  Analysis (Duncan,  f o r m u l t i p l e treatment comparisons were performed  on the r e s u l t s o b t a i n e d .  P l a t e 3.  D e t e r m i n a t i o n o f a b s c i s s i o n o f a p e t i o l e by t h e use o f a f i v e gramme p r e s s u r e a p p l i c a t o r .  RESULTS  The a p p l i c a t i o n o f l a n o l i n c o n t a i n i n g 1.0 ppm KNap t o t h e d i s t a l end o f d e b l a d e d p e t i o l e s o f t e n - d a y - o l d bean p l a n t s r e s u l t e d i n a s i g n i f i c a n t a c c e l e r a t i o n o f abscission plants  (9 days) compared w i t h about 11 days i n c o n t r o l  (Table V ).  The t i m e s r e q u i r e d f o r 5 0 % a b s c i s s i o n i n p l a n t s w h i c h r e c e i v e d h i g h KNap c o n c e n t r a t i o n s  (10, 100, and 1000 ppm)  were n o t s t a t i s t i c a l l y d i f f e r e n t from t h a t o f t h e c o n t r o l p l a n t s , o r from each o t h e r .  The t i m e s r e q u i r e d f o r 5 0 %  a b s c i s s i o n i n p l a n t s t r e a t e d w i t h 10 and 1000 ppm CHCA (13 and 12.67 days, r e s p e c t i v e l y ) were s i g n i f i c a n t l y d i f f e r e n t from t h a t o f c o n t r o l p l a n t s , b u t n o t from p l a n t s t r e a t e d w i t h 100 ppm NAA. In p l a n t s t r e a t e d w i t h 1000 ppm NAA, t h e p e r i o d r e q u i r e d f o r 5 0 % a b s c i s s i o n was 25 days w h i c h was s i g n i f i c a n t l y d i f f e r e n t from t h a t o f c o n t r o l p l a n t s a t t h e 0.01 l e v e l .  62  Table V .  Abscission  o f d e b l a d e d p e t i o l e s o f bean  (Phaseolus  v u l g a r i s L) i n r e s p o n s e t o KNap, CHCA, and NAA treatments. Treatment  +Average t i m e t o 50% a b s c i s s i o n (days)  (ppm) Lanolin alone (control)  10.67  2  KNap 1.0  3  KNap 10  12.00  4  KNap 100  11.67  5  KNap 1000  12.00  6  CHCA (K s a l t ) 1.0  12.00  7  CHCA (K s a l t ) 10  13.00 *  8  CHCA (K s a l t ) 100  12.33  9  CHCA (K s a l t ) 1000  12.67 *  10  NAA .1.0  12.00  11  NAA ;10  12.33  12  NAA ;100  13.00 *  13  NAA :1000  25.00 **  A Treatment  number:  9.00  13  12  7  9  8  11  3  5  6  10  4  + V a l u e from 12 p l a n t s . A T r e a t m e n t number c o r r e s p o n d s t o t r e a t m e n t i n T a b l e V I . The t r e a t m e n t s u n d e r s c o r e d b y t h e same l i n e do n o t d i f f e r s i g n i f i c a n t l y from one a n o t h e r . * V a l u e s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l v a l u e . ** V a l u e d i f f e r s from t h e c o n t r o l v a l u e a t t h e 0.01 l e v e l .  1  2  DISCUSSION  I t has been r e c o g n i s e d f o r a l o n g time t h a t a u x i n regulates abscission.  The two-phase t h e o r y o f a b s c i s s i o n  proposed by R u b i n s t e i n and Leopold  (1962) g i v e s a s a t i s f a c -  t o r y e l u c i d a t i o n o f the p r o c e s s o f a b s c i s s i o n .  According  t o these two workers, phase I o f the a b s c i s s i o n p r o c e s s i s i n h i b i t e d by auxin,and  phase I I i s a c c e l e r a t e d by a u x i n o f  similar concentration. In r e c e n t y e a r s , the involvement a b s c i s s i o n process has researchers.  o f e t h y l e n e i n the  received considerable interest  by  That e t h y l e n e i s a p o t e n t a c c e l e r a n t o f  a b s c i s s i o n has been suggested by, P r a t t and Goeschl  (1969).  The  promote a b s c i s s i o n i s i n d i r e c t .  f o r example, Burg  (1962);  action of ethylene to I t may  a c t through i t s  e f f e c t s on auxin l e v e l .  E t h y l e n e has been shown t o  p o l a r transport of auxin  (Beyer and Morgan, 1971), t o  i n c r e a s e a u x i n d e s t r u c t i o n ( H a l l and Morgan, 1964), t o i n i t i a t e the s y n t h e s i s o f s p e c i f i c RNA c e l l s e p a r a t i o n (Abeles, 1968).  inhibit  and  and p r o t e i n f o r  64  The  r e t a r d a t i o n e f f e c t o f 10 a n d 1000 ppm CHCA o n  a b s c i s s i o n may b e a t t r i b u t e d t o t h e e n h a n c e m e n t o f a u x i n a c t i o n and/or IAA b i o s y n t h e s i s .  I t was d e m o n s t r a t e d i n  the  100 ppm KNap  present investigations, that  IAA b i o s y n t h e s i s difference and  i n bean e p i c o t y l s .  i n delaying  stimulated  The l a c k o f s i g n i f i c a n t  a b s c i s s i o n b y 10 a n d 1000 ppm CHCA,  100 ppm NAA s u g g e s t s t h a t t h e r e t a r d a t i o n e f f e c t o f  CHCA i s s i m i l a r t o t h a t o f a u x i n . CHCA a c t e d  that  i n t h e f i r s t phase o f t h e two-phase t h e o r y o f  abscission of Rubinstein possibility  I t i spossible  and Leopold  (1962).  Another  i s t h a t CHCA i n h i b i t e d e t h y l e n e s y n t h e s i s  or  action. The (1.0  a c c e l e r a t i o n o f a b s c i s s i o n b y t h e l o w KNap  concentration  ppm) may b e i n t e r p r e t e d a s t h e e n h a n c e m e n t e f f e c t o f  KNap o n t h e b i o s y n t h e s i s  and/or a c t i o n o f  ethylene.  CHAPTER I I  EFFECT OF POTASSIUM NAPHTHENATES ON IAA BIOSYNTHESIS AND  DEGRADATION.  66  iia)  E f f e c t o f KNap on  IAA  biosynthesis.  LITERATURE REVIEW  IAA  i s w e l l e s t a b l i s h e d as t h e p r i n c i p a l hormone  higher  plants.  Yet  higher  plants are  the pathways o f  o b s c u r e , and  much i n v e s t i g a t i o n and pathways o f species.  are  IAA b i o s y n t h e s i s still  controversy.  IAA b i o s y n t h e s i s  differ  the  p l a n t s as w e l l as  (Moore and  S h a n e r , 1967,  S h e r w i n and  P u r v e s , 1969;  Sequira,  I t appears t h a t  the  i n different plant  1967;  1968;  Zenk a n d  M i u r a and  Gordon  preparations,  The  1971;  IAA  D - t r y p t o p h a n was  1963; Phelps  conversion  L-enantiomer  demonstrated by  (1961) n o t e d t h a t w i t h  t h a n L - t r y p t o p h a n as  Scherf,  Mills,  Wightman, 1963).  c u l t u r e s o f t o b a c c o was  of  i n numerous m i c r o o r g a n i s m s  the D-enantiomer o f t r y p t o p h a n t o the  (1971).  of  There i s ample e v i d e n c e t o e s t a b l i s h t h a t  i n higher  cell  in  subject  tryptophan i s the predominant n a t u r a l precursor  and  of  of in  M i u r a and  several  Mills  plant  e q u a l t o o r more e f f e c t i v e  a precursor  of  IAA.  Similarly,  Kim  "67.  and Rohringer  14  (1969) r e p o r t e d t h a t D-tryptophan-methylene-C  14 was  i n c o r p o r a t e d more e f f i c i e n t l y i n t o IAA-C than 14  methylene-C by e x c i s e d wheat l e a v e s .  Several  L-tryptophan-  researchers  q u e s t i o n the v a l i d i t y o f the c l a i m t h a t t r y p t o p h a n n a t u r a l p r e c u r s o r o f IAA  ( L i b b e r t e t a l , 1966;  Thimann and Grochowska, 1968;  Winter,  B l a c k and Hamilton,  The c o n v e r s i o n o f t r y p t o p h a n  t o IAA  p l a n t s p e c i e s has been worked out, and, workers i n t h i s area, t h i s c o n v e r s i o n  i s the 1966;  1971).  i n various according  f o l l o w s two  to major  proposed pathways: i n d o l e p y r u v i c a c i d and  indoleacetaldehyde,  and  (Larsen, 1951;  indoleacetaldehyde  Fawcett, 1961; Kim  Wightman, 1962,  and Rohringer,  1969;  1968;  or  tryptamine  Gordon, 1956, Moore and  S r i v a s t a v a and  1961;  Shaner,  Shaw, 1962;  1968;  Erdmann  and Schiewer, 1971). The  f o l l o w i n g pathway o f IAA b i o s y n t h e s i s has been  demonstrated i n the t i p s o f green pea (Moore and  Shaner, 1968;  i n oat c o l e o p t i l e s  Erdmann and  (Erdmann and  seedlings  (Pisum sativum)  Schiewer, 1971);  Schiewer, 1971);  68  i n r u s t fungus, Melampsora l i n i  ( P e r s . ) Lev. ( S r i v a s t a v a  and Shaw, 1962); and i n mung bean (Wightraan,  (Phaseolus  aureus)  1968).:  t r y p t o p h a n —*»indolepyruvic a c i d  indoleacetaldehyde — I A A  A n o t h e r pathway: t r y p t o p h a n - * t r y p t a m i n e - • i n d o l e a c e t a l d e h y d e - * I A A was found t o o c c u r i n tomato p l a n t s ( L y c o p e r s i c u m esculentum) (Wightman,  1963).  The e x i s t e n c e o f y e t a n o t h e r pathway: t r y p t o p h a n - ^ i n d o l e a c e t o n i t r i l e - * - i n d o l e a c e t a l d e h y d e -••IAA was d e t e c t e d i n cabbage b y Wightman  (1962).  A n o t h e r h y p o t h e t i c a l r o u t e f o r IAA b i o s y n t h e s i s i n Avena c o l e o p t i l e s i s g i v e n by Winter  (1966):  a n t h r a n i l o n i t r i l e - * a n t h r a n i l i c acid-*» i n d o l e - * t r y p t a m i n e - * IAA Tryptamine  r a t h e r than tryptophan i s the p r e c u r s o r  o f IAA i n Avena c o l e o p t i l e s was t h e c o n c l u s i o n r e a c h e d b y Thimann and Grochowska  (1968).  These two w o r k e r s  emphasised t h a t t h e c o n v e r s i o n o f t r y p t o p h a n t o t r y p t a m i n e d i d n o t o c c u r , and s u g g e s t e d t h a t t h e c o n v e r s i o n o f t r y p t o phan t o IAA was due t o b a c t e r i a l c o n t a m i n a t i o n . substantiated by Winter  T h i s was  (1966) and B l a c k and H a m i l t o n  (1971).  69  The  f a i l u r e o f exogenously  s u p p l i e d t r y p t o p h a n t o promote  e l o n g a t i o n o f Avena c o l e o p t i l e s was t h e r e s u l t o f i t s i n c o r p o r a t i o n i n t o p r o t e i n and consequent  unavailablity  f o r c o n v e r s i o n t o IAA under s t e r i l e c o n d i t i o n s , a c c o r d i n g t o B l a c k and Hamilton t o IAA i n bean  (1971).  The c o n v e r s i o n o f t r y p t o p h a n  (Phaseolus v u l g a r i s L) shoot experiments  with  14  tryptophan-C was r e p o r t e d b y B l a c k and Hamilton The  (1971).  f i n d i n g s o f P e r l e y and Stowe (1966) w i t h b a c t e r i a l  c u l t u r e s o f B a c i l l u s cereus s t r a i n KVT, showed t h a t  direct  decarboxylation o f tryptophan t o tryptamine i s a very r a r e reaction. To t h e b e s t o f my knowledge, t h e r e i s o n l y one p u b l i c a t i o n on t h e e f f e c t o f HNap on t h e c o n t e n t o f a u x i n in plants.  Bazanova  (1970) found t h a t Sh-8 (or HNap) a t  0.005% caused r e d i s t r i b u t i o n o f endogenous a u x i n and i n h i b i t o r s i n v a r i o u s organs  of f i n e - f i b r e d cotton, increased  the t r a n s l o c a t i o n o f g r o w t h - r e g u l a t i n g substances v e g e t a t i v e t o r e p r o d u c t i v e organs,  from  and enhanced t h e a c t i v i t y  o f n a t u r a l g r o w t h - r e g u l a t i n g substances and i n h i b i t o r s i n r e p r o d u c t i v e organs.  70  MATERIALS AND METHODS  Bush bean seeds ( P h a s e o l u s v u l g a r i s L, c u l t i v a r Top Crop) were s u r f a c e s t e r i l i s e d b y w a s h i n g w i t h 95% e t h a n o l f o r ten  minutes, then w i t h s t e r i l e water.  f o r 12 h o u r s i n 100 ppm  The seeds were soaked  (0.01%) s t e r i l i s e d KNap (Wort and  P a t e l , ' 1 9 7 0 ; N a g h i b i n , 1966; E j u b o v , 1966).  The seeds used  as c o n t r o l were soaked i n s t e r i l e w a t e r f o r t h e same l e n g t h of time. culite  The seeds were sown i n rows i n s t e r i l i s e d v e r m i -  ( " T e r r a - l i t e " , Grace C o n s t r u c t i o n M a t e r i a l s L t d . ,  V a n c o u v e r ) , s a t u r a t e d w i t h 1.0% Captan f u n g i c i d e .  Steriliza-  o t x o n was c a r r i e d o u t a t 248 of for  15 l b p e r s q u a r e i n c h .  C f o r 15 m i n u t e s a t a p r e s s u r e The p l a n t s were a l l o w e d t o grow  14 days i n t h e d a r k , a t a i r t e m p e r a t u r e s o f 22.5 + 1° C,  and 95 + 2% r e l a t i v e h u m i d i t y . A p i c a l regions  (5-8 cm) i n c l u d i n g l e a v e s were e x c i s e d  and washed v/ith c o l d d i s t i l l e d w a t e r .  Immediately a f t e r  e x c i s i o n , 20 g f r e s h w e i g h t o f p l a n t t i s s u e s was  homogenised  i n a W a r i n g b l e n d o r a t f u l l speed f o r t e n m i n u t e s i n 40 ml o o f KH P0 -Na_HP0 b u f f e r i n a c o l d room a t 4 C (Moore and 9  A  A  71  Shaner, 1968). a t pH 7.4  The c o n c e n t r a t i o n o f the phosphate b u f f e r was  (Moore and Shaner, 1968)  c o n t a i n i n g 0.1 M s u c r o s e  (Wightman, 1968), t e n u n i t s per ml p e n i c i l l i n G, and u n i t s per ml s t r e p t o m y c i n s u l p h a t e  100  (Valdovinos and P e r l e y ,  1966).  The crude homogenates were f i l t e r e d through f o u r l a y e r s o f c h e e s e c l o t h and k e p t i n an i c e b a t h throughout the p r e p a r a t i v e procedures.  The f i l t r a t e thus o b t a i n e d , was  fuged a t 10,000 g_ f o r 20 minutes a t 4 ° C 1968)  and the r e s u l t i n g s u p e r n a t a n t was  extract.  centri-  (Moore and Shaner, used as t h e enzyme  Enzyme e x t r a c t s were d i a l y s e d a g a i n s t phosphate o  b u f f e r f o r 24 hours a t 4  C w i t h c o n s t a n t a g i t a t i o n and  one change o f e x t e r n a l b u f f e r . R e a c t i o n m i x t u r e s , m o d i f i e d from Moore and Shaner c o n t a i n e d t h r e e ml enzyme e x t r a c t and t h r e e ml o f 0.1 KH P0 -Na HP0 2  4  2  4  b u f f e r , pH 7.4,  30 micromoles  containing:  alpha-ketoglutaric  acid  0.6 micromoles  pyridoxal  0.6 micromoles  t h i a m i n e pyrophosphate  0.6 micromoles  n i c o t i n a m i d e adenine d i n u c l e o t i d e  0.03 M 0.0005 M  0.1  phosphate  D-tryptophan diethyldithiocarbamic  acid  (1968), M  72  The above c h e m i c a l s were purchased from Sigma Chemical Company, S t . L o u i s , M i s s o u r i . The D-enantiomer p r e c u r s o r o f IAA  o f t r y p t o p h a n was  found t o be the b e s t  (Kim and Rohringer, 1969).  t i o n o f D-tryptophan  (0.03 M) was  The c o n c e n t r a -  adopted from Wightman and  Cohen (1968), w h i l e the i n c l u s i o n o f 0.0005 M d i e t h y l d i t h i o carbamic a c i d which i s a p o t e n t i n h i b i t o r o f IAA oxidase, was  adopted from Wagenknecht and B u r r i s  (1950).  The r e a c t i o n mixtures, h e l d i n t e s t tubes, were i n c u b a t e d i n darkness f o r s i x hours a t 35° C  (Wightman and Cohen,  i n a water b a t h w i t h t h e r m o s t a t i c c o n t r o l s Company, P o r t l a n d , Oregon).  1968)  (National Appliance  Enzymatic r e a c t i o n s were stopped  by l o w e r i n g the pH t o 3.0 w i t h two drops o f 85% orthophosphoric acid. • The e s t i m a t i o n s o f p r o t e i n i n enzyme e x t r a c t s were made w i t h a view o f a s s e s s i n g the s p e c i f i c a c t i v i t y o f the enzymes i n v o l v e d i n IAA b i o s y n t h e s i s .  The method o f Lowry e_t a l (1951)  as m o d i f i e d by E g g s t e i n and Kreutz (1955) was ml o f enzyme e x t r a c t was solution  added  (50 ml o f 2% N a C 0 2  0.5% CuSO^.SB^O i n 1.0%  3  used.  To  one  f i v e ml o f a l k a l i n e copper  i n 0.1  N NaOH p l u s 1.0 ml o f  sodium c i t r a t e ) .  The mixture  was  t h o r o u g h l y mixed  and allowed t o stand f o r t e n minutes a t  room temperature  (23-24° C ) .  T h e r e a f t e r , 0.5 ml o f 1.0  N  F o l i n - C i o c a l t e a u reagent (commercial reagent 2 N d i l u t e d w i t h  73  water t o g i v e a s o l u t i o n o f 1.0 N i n a c i d ) was p i p e t t e d r a p i d l y i n t o t h e mixture w i t h thorough mixing.  The o p t i c a l  d e n s i t y was measured a t 650 nm a f t e r 30 minutes.  The amount  o f p r o t e i n i n t h e enzyme e x t r a c t s was c a l i b r a t e d by r e f e r e n c e t o a standard curve developed u s i n g c r y s t a l l i n e b o v i n e  albumin.  E x t r a c t i o n and q u a n t i t a t i v e d e t e r m i n a t i o n o f IAA. A l l m a n i p u l a t i o n s were performed temperature  (23-24° C ) .  i n red light,  a t room  P r i o r t o e x t r a c t i o n , t e n microgrammes  o f a u t h e n t i c IAA were added t o each r e a c t i o n mixture, and each r e a c t i o n mixture was e x t r a c t e d t w i c e w i t h s i x ml o f methylene c h l o r i d e b y t h e method o f Moore and Shaner  (1968).  The combined e x t r a c t s o f each r e a c t i o n mixture were evaporated to dryness i n a r o t a r y - f i l m vacuum e v a p o r a t o r a t 30° C.  The  •residue was d i s s o l v e d i n two ml o f 95% e t h a n o l and a 0.5 ml a l i q u o t o f each e x t r a c t was s p o t t e d on Whatman No. 1 chromatographic  paper  (26 x 36 cm) and  w i t h t e n microgrammes o f a u t h e n t i c IAA. were developed i n 10:1:1 h y d r o x i d e : d i s t i l l e d water and Chkanikov,  co-chromatographed The chromatograms  (v/v) 95% i s o p r o p a n o l : 2 8 % ammonium ( K u r a i s h i and Muira, 1963; Artemenko  1970) i n an ascending manner  f o r 16 hours, a t 4 ° C i n t h e dark.  (Wightman, 1963)  The spots developed on  the chromatograms were l o c a t e d under u l t r a v i o l e t l i g h t , and immediately sprayed w i t h E h r l i c h ' s reagent.  E h r l i c h ' s reagent  was prepared by mixing 2% p-dimethylaminobenzaldehyde  i n 10 N  74  HC1 w i t h acetone  i n t h e p r o p o r t i o n o f 1:1  b e f o r e use (Wightman, 1963).  (v/v) immediately  Q u a n t i t a t i v e determinations o f  IAA were made 15 minutes f o l l o w i n g treatment w i t h E h r l i c h ' s reagent.  The o p t i c a l d e n s i t y o f t h e spots was  by means o f a densitometer  determined  (Photovolt Densitometer,  C o r p o r a t i o n , New York C i t y , Model 501 A ) .  Photovolt  Maximum t r a n s m i s s i o n  v a l u e s o f t h e sopts o b t a i n e d w i t h the densitometer were used to compute the amount o f IAA p r e s e n t i n p l a n t t i s s u e s by c a l i b r a t i o n w i t h v a l u e s o f a standard c u r v e .  Transmission  v a l u e s were employed c h i e f l y because o f t h e i r l i n e a r  relation-  s h i p t o t h e l o g a r i t h m o f IAA c o n c e n t r a t i o n ( V l i t o s and Meudt, 1953). A standard curve was developed by d e l i v e r i n g 1.0, 2, 5, and 20 microgrammes o f a u t h e n t i c IAA t o a chromatogram w i t h a m i c r o l i t r e syringe.  The chromatogram was developed and  t r e a t e d i n the same manner as d e s c r i b e d i n the p r e c e d i n g paragraph. Meudt  The c o n c e n t r a t i o n s were adopted  from V l i t o s and  (1953). A l l s o l v e n t s used i n chromatography were  redistilled.  A randomised complete b l o c k d e s i g n was used i n t h i s i n v e s t i g a t i o n i n v o l v i n g t h r e e b l o c k s w i t h two d e t e r m i n a t i o n s per treatment  i n each b l o c k .  The r e s u l t s o b t a i n e d were  subjected to analysis of variance.  75  RESULTS  S o a k i n g the bean seeds i n a 100  ppm  s o l u t i o n o f KNap  f o r 24 h o u r s p r i o r t o sowing r e s u l t e d i n a 140.5% i n t h e c o n t e n t o f IAA  i n t h e e p i c o t y l s o f dark-grown bean  p l a n t s , d e t e r m i n e d 14 days a f t e r t r e a t m e n t . was  s i g n i f i c a n t a t t h e 0.01 The  amount o f p r o t e i n  The  (1.26  than t h a t i n the c o n t r o l p l a n t s  mg  per g f r e s h w e i g h t )  c o n v e r s i o n o f t r y p t o p h a n t o IAA,  was  (1.96  mg  1.34  plants  synthesised  ).  for  the  e x p r e s s e d i n terms o f per mg  i n the c o n t r o l p l a n t s and (Table V I I  less  per g f r e s h weight)..  s p e c i f i c a c t i v i t y o f the enzymes r e s p o n s i b l e  microgrammes o f IAA  increase  l e v e l (Table VI..:).  i n t h e e p i c o t y l s o f K N a p - t r e a t e d bean p l a n t s was  The  increase  5.03  p r o t e i n per i n the  hour,  KNap-treated  76  T a b l e VI-'.  I A A c o n t e n t o f t h e 5-8 cm t i p s  of epicotyls  of 1 4 - d a y - o l d dark-grown P h a s e o l u s v u l g a r i s s e e d l i n g s f o l l o w i n g t r e a t m e n t w i t h 100 ppm  Treatment  +Microgrammes o f IAA per g fresh weight  Distilled H 0 (control)  KNap.  Increase over c o n t r o l (%)  5.28  2  KNap 100 ppm + **  L  12.70**  140.5  Mean o f s i x d e t e r m i n a t i o n s . Value d i f f e r s s i g n i f i c a n t l y from t h e c o n t r o l v a l u e a t t h e 0.01 l e v e l .  Table V I I .  S p e c i f i c a c t i v i t y o f t h e enzymes i n t h e c o n v e r s i o n o f t r y p t o p h a n t o IAA i n t h e e p i c o t y l s o f dark-grown bean  Treatment  IAA c o n t e n t + P r o t e i n c o n t e n t microgrammes microgrammes per g fresh per g fresh weight weight  plants.  Specific activity o f enzymes microgrammes IAA p e r mg p r o t e i n per hour  Distilled H 0 2  (control)  5.28  KNap 100 ppm 12.70 + Mean o f s i x d e t e r m i n a t i o n s .  1.96  1.34  1.26  5.03  DISCUSSION  In view o f the absence o f r e p o r t e d pathways on the b i o s y n t h e s i s o f IAA from t r y p t o p h a n i n Phaseolus v u l g a r i s L p l a n t s , i t i s tempting t o s p e c u l a t e t h a t the pathway o p e r a t i n g i n P. v u l g a r i s i s s i m i l a r t o t h a t o f Phaseolus aureus L (mung bean).  I t was r e p o r t e d t h a t IAA i s synthe-  s i s e d i n e x c i s e d E\_ v u l g a r i s shoots v i a t r y p t o p h a n (Black and Hamilton, 1971).  The pathway o f . e n z y m a t i c c o n v e r s i o n  o f t r y p t o p h a n t o IAA i n P. aureus has been worked out by Wightman and Cohen  (1968), and, a c c o r d i n g t o them, the  pathway i s i n the f o l l o w i n g Tryptophan  sequence:  Indolepyruvic acid I  I n d o l e a c e t a l d e h y d e —> IAA II III  The enzymes r e s p o n s i b l e f o r the c o n v e r s i o n o f t r y p t o p h a n t o IAA have been i d e n t i f i e d ,  and the p r o p e r t i e s o f the  i n d i v i d u a l enzymes i n v o l v e d i n each s t e p o f t h i s pathway were determined by Wightman  and Cohen  biosyntheti  (1968).  A c c o r d i n g t o these two i n v e s t i g a t o r s , t h e enzyme c a t a l y s i n g the p a r t i c u l a r s t e p i n the pathway i s i n d i c a t e d as f o l l o w s :  78  Tryptophan transaminase i n s t e p I; I n d o l e p y r u v i c  acid  d e c a r b o x y l a s e i n s t e p I I ; and Aldehyde dehydrogenase i n step I I I . The plants  i n c r e a s e i n the content  (140.5%) compared w i t h  o f IAA i n the KNap-treated  control plants  suggests  KNap s t i m u l a t i o n o f IAA b i o s y n t h e s i s i n v i t r o . stimulated  T h i s KNap-  IAA b i o s y n t h e s i s i n P. v u l g a r i s e p i c o t y l s may  be mediated by e i t h e r t h e i n d u c t i o n o f de_ novo o f t h e enzymes i n v o l v e d i n t h e c o n v e r s i o n IAA  (mentioned i n t h e p r e c e d i n g  synthesis  o f tryptophan t o  paragraph), o r b y t h e  s t i m u l a t i o n o f the a c t i v i t y o f these enzymes, o r i n combination. This suggestion  i s made i n t h e l i g h t o f r e p o r t s  on bush  bean p l a n t s f o l l o w i n g KNap treatments, on the s t i m u l a t i o n o f p r o t e i n s y n t h e s i s b y Severson on t h e augmentation o f s p e c i f i c enzymes i n crude e x t r a c t s  (1972) and Wort e t a l (1971); a c t i v i t i e s o f numerous  (Chu, 1969; F a t t a h ,  1969; F a t t a h  and Wort, 1970); and on i n c r e a s e s i n RNA and DNA  content  (Wort e t a l , 1971). The  i n c r e a s e s i n p l a n t growth and c r o p y i e l d  a p p l i c a t i o n s o f KNap r e p o r t e d  i n the l i t e r a t u r e  following  can be  i n t e r p r e t e d as due t o KNap-induced IAA b i o s y n t h e s i s i n these p l a n t s .  79  i i b ) E f f e c t o f KNap on  IAA  degradation.  LITERATURE REVIEW  The metabolism o f IAA  i n p l a n t s has been i n t e n s i v e l y -  d i s c u s s e d but not w e l l understood, even t i l l  today.  The  s t a r t i n g p o i n t o f d i s c u s s i o n on t h i s s u b j e c t dates back t o 1934 faba and  when Thimann found t h a t l e a f e x t r a c t s o f Helianthus  c o u l d i n a c t i v a t e IAA.  But,  Vicia he,  d i d not demonstrate t h a t the d i s a p p e a r a n c e o f IAA enzymatic.  Later,  i n 1936,  i n a c t i v a t i o n o f IAA was The  Larsen discovered  c a t a l y s e d by  oxidase, i n e t i o l a t e d pea by  the  Bonner i n  1947  IAA-degrading enzyme,  e p i c o t y l s was  quoted  IAA  repeatedly  i n v e s t i g a t o r s which l e d r e a d e r s t o b e l i e v e t h a t IAA  i s the o n l y enzyme r e s p o n s i b l e The  was  an o x i d i s i n g enzyme.  c l a s s i c a l p u b l i c a t i o n o f Tang and  on some c h a r a c t e r i s t i c s o f the  that  however,  enzyme i s d e s i g n a t e d  i n a c t i v a t i o n o f IAA oxygen, and  IAA  IAA.  o x i d a s e c h i e f l y because  t a k e s p l a c e o n l y i n the presence o f  about one  molecule o f IAA  as  f o r the d e s t r u c t i o n o f  oxidase  molecule o f oxygen i s consumed  i n a c t i v a t e d , and  one  per  m o l e c u l e o f carbon  80  dioxide i s liberated  (Tang a n d B o n n e r , 1 9 4 7 ) .  Evidence  s u p p o r t i n g t h e c l a i m t h a t IAA o x i d a s e i s n o t a s i n g l e enzymed b u t a m i x t u r e o f enzymes i s f o u n d i n t h e work o f S e q u i r a and Mineo  (1966); Lee (1971); Meudt  (1960); Hinman a n d Lang  (1965).  (1967); Ray  T h a t I A A o x i d a s e may  c o n s i s t o f a m i x t u r e o f p l a n t p e r o x i d a s e s , and t h a t IAA o x i d a s e t o g e t h e r w i t h p e r o x i d a s e s may e x i s t a s was s u g g e s t e d b y S e q u i r a a n d M i n e o  (1966).  aggregates  Ray  (1960)  c o n s i d e r e d b o t h p e r o x i d a s e a n d IAA o x i d a s e a c t i v i t i e s due  t o o n e enzyme.  IAA o x i d a s e h a s b e e n i d e n t i f i e d  heme p r o t e i n p e r o x i d a s e b y G o l d a c r e inferred  (1951).  were  as a  I t may b e  from t h e s e f i n d i n g s t h a t IAA o x i d a s e i s an isoenzyme  of peroxidase.  The a b i l i t y o f p e r o x i d a s e s t o o x i d i s e I A A  has been r e p o r t e d b y Meudt  (1967), u s i n g c r y s t a l l i n e  r a d i s h p e r o x i d a s e ; Hinman a n d Lang  horse-  (1965); Ray (1960),  p e r o x i d a s e from t h e fungus Omphalia f l a v i d a .  using  Seven p e r o x i d a s e s  i s o e n z y m e s were d e t e c t e d i n h o r s e r a d i s h r o o t s , w i t h no i n t e r c o n v e r s i o n s among t h e i s o e n z y m e s J e r m y a n d Thomas  (Shannon e t aJL, 1966) .  (1954) r e p o r t e d t h a t t h e i s o e n z y m e s o f  h o r s e r a d i s h p e r o x i d a s e have s u b s t r a t e s p e c i f i c i t i e s .  Enzymes o f the p o l y p h e n o l o x i d a s e systems a r e a l s o known t o o x i d i s e IAA  (Leopold and Plummer, 1961;  That t h e IAA o x i d a s e system p o s s e s s e d a c o n s i d e r a b l e degree was  Skoog,  i n e t i o l a t e d pea  1944)  epicotyls  of substrate s p e c i f i c i t y ,  one o f the c o n c l u s i o n s reached by Tang and Bonner  They r e p o r t e d t h a t the IAA o x i d a s e system indoleacetamide, i n d o l e b u t y r i c acid,  (1947).  d i d not a t t a c k  indolepyruvic acid,  i n d o l e c a r b o x y l i c a c i d or t r y p t o p h a n . The p r o d u c t s o f IAA o x i d a t i o n have been s t u d i e d , b u t no c l e a r - c u t c o n c l u s i o n s a r e a v a i l a b l e . (1965) suggested  Hinman and  3-methylene o x i n d o l e , and a n e u t r a l  Lang indole,  and emphasised t h a t the p r o d u c t c o m p o s i t i o n i s h i g h l y depende n t on  IAA c o n c e n t r a t i o n s .  aidehyde,  That the end p r o d u c t i s i h d o l e -  and the i n t e r m e d i a t e s , i n d o l e - 3 - g l y c o l l i c  and  i n d o l e - 3 - g l y o x y l i c a c i d s were suggested by G o l d a c r e  (1951).  A g e n e r a l agreement among i n v e s t i g a t o r s i s t h a t the  indole  r i n g remains 1965;  intact  Meudt, 1967).  (Tang and Bonner, 1947;  Hinman and  The o x i d a t i o n p r o d u c t s are  Lang,  inactive  i n the Avena t e s t o r i n c h e m i c a l methods o f IAA d e t e r m i n a t i o n  82  Meudt  (1967) i n d i c a t e d t h a t the o x i d a t i v e t r a n s f o r m -  a t i o n o f IAA  l e a d s t o the f o r m a t i o n o f b i o l o g i c a l l y  active  p r o d u c t s , p r o v i d e d p r e v a i l i n g c o n d i t i o n s a r e such t o p r e v e n t the f o r m a t i o n o f secondary o x i d a t i o n p r o d u c t s which i n a c t i v a t e IAA. Meudt and G a l s t o n  (1962) suggested a mechanism by which  IAA a t t a i n s b i o l o g i c a l a c t i v i t y i n p l a n t s . p r o d u c t o f IAA o x i d a t i o n i s o x i n d o l e  The  primary  (Hinman and Lang, 1965),  and i s spared from d e s t r u c t i o n by b i n d i n g t o RNA.  The-  oxindole-RNA complexes then s t i m u l a t e p l a n t growth. complexes gave p o s i t i v e r e a c t i o n s i n Salkowski and reagents  (Meudt and G a l s t o n , 1962).  h y p o t h e s i s comes from £he  These Ehrlich  A support f o r t h i s  s t u d y o f the d i s t r i b u t i o n p a t t e r n  o f peroxidase a c t i v i t y i n p l a n t s .  Meudt  (1967) found  the d i s t r i b u t i o n p a t t e r n o f p e r o x i d a s e a c t i v i t y w i t h the growth c e n t r e s o f the p l a n t .  that  correlates  Peroxidase  activity  i s h i g h e r i n a c t i v e l y - g r o w i n g t i s s u e s than i n mature and dormant t i s s u e s . In t h e l i g h t o f evidence p r e s e n t e d ,  IAA o x i d a s e s h o u l d  n o t be thought o f as a s i n g l e component, b u t r a t h e r as a  m i x t u r e o f p e r o x i d a s e isoenzymes.  I t i s appropriate to  c o n s i d e r IAA o x i d a s e as a system o f enzymes, hence t h e t e r m "IAA o x i d a s e system"  s h o u l d be used.  has been used b y i n v e s t i g a t o r s f o r example, Goren, and M o n s e l i s e  The l a t t e r  term  Goldschmidt,  (1967); Lee (1971).  Babaev (1966) r e p o r t e d t h a t s o a k i n g c o t t o n seeds i n s o l u t i o n s o f HNap (10 mg/1), and t h e a p p l i c a t i o n o f t h e acid t o the s o i l  (20 mg/kg o f d r y s o i l ) a c t i v a t e d p e r o x i d a s e  a c t i v i t y i n the roots.  84  MATERIALS AND  METHODS  Uniform seeds o f Phaseolus v u l g a r i s c u l t i v a r Top Crop were s u r f a c e minutes.  s t e r i l i s e d by washing  i n 95% e t h a n o l  The seeds were sown i n s t e r i l i s e d v e r m i c u l i t e ,  s a t u r a t e d v/ith 1.0% Captan f u n g i c i d e , c o n t a i n e d flats,  i n wooden  and a l l o w e d t o grow i n darkness a t an a i r temperature  o f 22.5 + 0.5° C and a r e l a t i v e h u m i d i t y o f 95 + Thirteen-day-old  dark-grown  Treatment was  2%.  s e e d l i n g s were c a r e f u l l y  removed from t h e v e r m i c u l i t e and washed v/ith t a p  100 ppm  f o r ten  water  e f f e c t e d by immersing t h e r o o t systems i n  (0.01%) KNap s o l u t i o n f o r 24 hours i n darkness a t o  room temperature  (23-24  C).  The r o o t systems o f c o n t r o l  p l a n t s were soaked i n d i s t i l l e d water under s i m i l a r c o n d i t i o n s . Subsequent h a n d l i n g  of plant materials  i n r e d l i g h t a t room temperature. l e a v e s , were h a r v e s t e d ,  was  Epicotyls  washed v/ith i c e c o l d  performed  (5-8 cm) distilled  water, and immediately f r o z e n w i t h l i q u i d n i t r o g e n . f o l l o w i n g procedure was m o d i f i e d (1966).  from S e q u e i r a  including  The  and Mineo  The f r o z e n p l a n t t i s s u e s were ground t o a f i n e  85  powder i n a motar w i t h p e s t l e .  The powder was  suspended  i n c o l d 0.02 M K^PO^-Na^PC^ b u f f e r , pH 6.1, i n t h e proportion o f ten g o f frozen stirred  t i s s u e t o 50 ml b u f f e r , and  f o r 1.5 hours i n t h e c o l d room a t 4 ° C.  was c e n t r i f u g e d  a t 18,000 £ f o r t e n minutes  The m i x t u r e  a t 4° C.  s u p e r n a t a n t was decanted i n t o an 150-ml Erlenmeyer and s o l i d  flask  (NH.).SO, was added g e n t l y t o 35% s a t u r a t i o n 4 2 4  (Green e t a l , 1955). 4° C i n  The  The p r e c i p i t a t e which was formed a t  24 hours, was removed by c e n t r i f u g a t i o n a t 18,000 £  f o r t e n minutes.  The s u p e r n a t a n t was then brought t o 70%  saturation with s o l i d  (NH^^SO^, and t h e p r e c i p i t a t e which  was formed a t 4° C i n . 24 hours, was r e c o v e r e d by c e n t r i f u g a t i o n a t 18,000 2. f ° buffer. room  r t  e  n  minutes,  and resuspended  The s o l u t i o n was d i a l y s e d  i n 12.5 ml  f o r 24 hours i n t h e c o l d  (4° C) a g a i n s t 3000 ml b u f f e r v/ith one change o f  external buffer.  T h i s d i a l y s e d s o l u t i o n , which had b o t h  IAA o x i d a s e and p e r o x i d a s e a c t i v i t i e s , was r e f e r r e d t o as crude enzyme.  No attempts were made t o s e p a r a t e t h e s e two  enzymes as b o t h were found t o degrade S e q u e i r a and Mineo, 1966; Meudt,  IAA (Ray, I960;  1970).  86  Assay f o r enzyme a c t i v i t y  The a c t i v i t y o f the I A A - d e s t r o y i n g enzymes was determined by t h e Salkowski r e a c t i o n  (Tang and Bonner, 1947;  Gordon and Weber, 1951; Gordon and P a l e g , 1957). r e a c t i o n mixture  The  ( f i v e ml) was p r e p a r e d by t h e method o f  S e q u e i r a and Mineo  (1966), and c o n s i s t e d o f the f o l l o w i n g :  0.5 ml enzyme e x t r a c t 0.25 ml 1.0 mM Na s a l t o f 2 , 4 - d i c h l o r o p h e n o l 1.00 ml 1.0 mM  IAA i n 0.5 mM M n C l  3.25 0.02 M KH P0 -Na HP0 , 2  4  2  4  The r e a c t i o n mixture was shaken  2  (1:1, v/v)  pH 6.1  i n a water b a t h  (Dubnoff  M e t a b o l i c Shaking Incubator, P r e c i s i o n S c i e n t i f i c ,  Chicago)  f o r two hours a t 30° C i n darkness, and then 1.0 ml o f Salkowski reagent was added.  The m i x t u r e was shaken f o r  an a d d i t i o n a l t h r e e hours and absorbance determined w i t h a Bausch  a t 525 nm was  and Lomb S p e c t r o n i c 20 c o l o r i m e t e r  u s i n g a b l u e - s e n s i n g phototube  , type CEA-59RX.  The  Salkowski reagent was p r e p a r e d a c c o r d i n g t o Gordon and Weber (1951), b y mixing 1.0 ml o f 0.5 M F e C l HC10.. 4  3  t o 50 ml o f 35%  87  The  r e s i d u a l IAA  i n the r e a c t i o n m i x t u r e s was  by r e f e r e n c e t o a s t a n d a r d c u r v e .  The  determined  s t a n d a r d curve  was  developed by mixing a s e r i e s o f two ml known q u a n t i t i e s o f a u t h e n t i c IAA t o f o u r ml Salkowski minutes. 20  The absorbance was  reagent f o r 35  r e a d a t 525  withaSpectronic  spectrophotometer. The e x p e r i m e n t a l setup used i n t h i s  was  nm  investigation  t h e randomised complete b l o c k d e s i g n , c o m p r i s i n g  two  r e p l i c a t i o n s w i t h f i v e d e t e r m i n a t i o n s per r e p l i c a t i o n i n each treatment. variance.  The  r e s u l t s were s u b j e c t e d t o a n a l y s i s o f  RESULTS  When the r o o t systems o f 13-day-old dark-grown bean p l a n t s were immersed i n a 100 ppm s o l u t i o n o f KNap f o r 24 hours, t h e r e was a 4% i n c r e a s e i n the a c t i v i t y o f the IAA oxidase  system compared w i t h t h a t o f c o n t r o l p l a n t s .  T h i s s t i m u l a t o r y e f f e c t o f KNap was s i g n i f i c a n t a t the 0.01 level  (Table V I I I ) .  The a c t i v i t y o f the IAA oxidase system  was measured i n terms o f microgrammes g f r e s h weight o f e p i c o t y l t i s s u e .  o f IAA destroyed p e r  89  T a b l e v i l l . T h e a c t i v i t y o f t h e IAA o x i d a s e system i n t h e 1 4 - d a y - o l d d a r k - g r o w n bean e p i c o t y l s  following  t r e a t m e n t w i t h 100 ppm KNap.  Treatment  I n i t i a l IAA concentration in reaction mixture (ugm/ml)  +Residual IAA Amount o f Amount o f concentration IAA IAA i n r e a c t i o n destroyed destroye'd mixture (% i n c r e a s e (ugm/ml) (ugm/g f r wt) over c o n t r o l )  Control  175  26.27  371.82  KNap 100 ppm  175  20.20  386.00**  + Mean o f 15 d e t e r m i n a t i o n s . ** V a l u e d i f f e r s s i g n i f i c a n t l y t h e 0.01 l e v e l .  from t h e c o n t r o l v a l u e a t  3.81  90  DISCUSSION  I t i s r e c o g n i s e d t h a t t h e IAA o x i d a s e system i s i n v o l v e d i n t h e d e g r a d a t i o n o f IAA. The i n c r e a s e (ca 4% over c o n t r o l p l a n t s ) i n the a c t i v i t y o f t h e IAA o x i d a s e system  i n bean p l a n t s f o l l o w i n g  treatment  w i t h 100 ppm KNap s u p p o r t s t h e r e p o r t o f i n c r e a s e s i n peroxidase a c t i v i t y i n the roots o f cotton p l a n t s r e s u l t e d from seed soak w i t h 10 ppm HNap b y Babaev  (1966).  That  p e r o x i d a s e i s a component o f t h e IAA o x i d a s e system was suggested  by, f o r example, S e q u e i r a and Mineo  (1966).  The s t i m u l a t i o n o f IAA b i o s y n t h e s i s i n bean e p i c o t y l s by 100 ppm KNap r e p o r t e d b y t h e author i n t h e p r e s e n t i n v e s t i g a t i o n , and t h e a c t i v a t i o n o f t h e IAA o x i d a s e  system  by s i m i l a r KNap c o n c e n t r a t i o n , suggests t h a t the a c t i v i t y o f t h e IAA o x i d a s e system  i s induced b y t h e s u b s t r a t e , IAA.  I t i s d i f f i c u l t t o r e s o l v e the p.aradox t h a t IAA o x i d a s e system  i s capable o f i n a c t i v a t i n g  o f t h i s system  IAA; and t h a t t h e a c t i v i t y  i s induced b y the s u b s t r a t e , IAA, and  c o r r e l a t e s v/ith the a c t i v e growth c e n t r e s o f p l a n t s i . e .  91  g r e a t e r a c t i v i t y i n a c t i v e l y grov/ing t i s s u e s than i n mature and  dormant t i s s u e s  q u e s t i o n : how  can  (Meudt, 1967).  t h e r e be  c o n t i n u a l l y destroyed ?  a c t i v e growth i f IAA  It i s possible  a c t i v e l y growing p l a n t t i s s u e s , to s u s t a i n d e s t r u c t i o n , a c t i v e growth. further  T h i s poses a fundamental  and,  IAA  a t the  that i n  is the  i s i n excess and  able  same time, m a i n t a i n s  A s o l u t i o n t o t h i s q u e s t i o n must await  research.  92  CONCLUSIONS  1) Potassium naphthenates auxin-like a)  (KNap) e x h i b i t e d the f o l l o w i n g  properties:  I n h i b i t i o n o f r o o t growth KNap a t 1000 ppm i n h i b i t e d t h e growth o f i n t a c t r o o t s  o f cucumber. b)  Stimulation The  100  o f root  initiation  treatment o f bean stem c u t t i n g s w i t h 10 and  ppm KNap r e s u l t e d i n t h e s t i m u l a t i o n o f r o o t  compared w i t h c o n t r o l c u t t i n g s .  initiation  The treatment o f a z a l e a  stem c u t t i n g s w i t h 100 and 1000 ppm KNap s i g n i f i c a n t l y increased  r o o t i n i t i a t i o n compared w i t h c o n t r o l c u t t i n g s .  c) S t i m u l a t i o n  of elongation  o f pea stem segments  KNap a t 10 ppm had a g r e a t e r elongation  e f f e c t on  o f pea stem segments than 0.1 ppm IAA.  d) R e t a r d a t i o n The  stimulatory  of petiole abscission  a p p l i c a t i o n s o f 10 and 1000 ppm  a c i d t o the d i s t a l  cyclohexanecarboxylic  end o f debladed p e t i o l e s o f bean, r e s u l t e d  in retardation of abscission. 2) When a p p l i e d t o t h e seeds f o r 12 hours p r i o r t o sowing, 100  ppm KNap augmented the b i o s y n t h e s i s  o f IAA from t r y p t o p h a n  i n t h e 5-8 cm t i p s o f dark-grown e p i c o t y l s o f bean. 3) When a p p l i e d t o t h e r o o t s o f 13-day-old dark-grown bean plants  f o r 24 hours, 100 ppm KNap s t i m u l a t e d  i n t h e 5-8 cm t i p s o f e p i c o t y l s .  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