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Effect of 2, 4-Dichlorophenoxyacetic acid on the yield and metabolism of the bean plant Phaseolus vulgaris Chi, Chu-hsiang 1965

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EFFECT OF 2,4-DICHL0R0PHEN0XYACETIC ACID ON THE YIELD AND METABOLISM OF THE BEAN PLANT PHASEOLUS VULGARIS  by CHU-HSIANG CHI B. Sc., Tunghai University (Formosa), 1962  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n the Department of Biology and Botany  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1965  In the  requirements  British  mission  for  Columbia, I  available  for  for  p u r p o s e s may his  presenting an  extensive  representatives.  cation without  of my  this  written  and  by  It  thesis  that  for  the  the  of  Head  I  of  i s understood financial  Department Columbia.,  the  Library  this  permission*  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  in partial  degree at  study,  copying  granted  thesis  advanced  agree  reference  be  this  thesis my  make i t  agree for  that  or  c o p y i n g or  shall  not  of  of • freely per-  scholarly  Department  that  gain  University  shall  further  fulfilment  be  by publi-  allowed  ABSTRACT An i n v e s t i g a t i o n was designed to determine the e f f e c t of dusts and sprays o f 2,4-D on growth, y i e l d , enzyme a c t i v i t y , photosynthesis, r e s p i r a t i o n and photophosphorylation o f bush bean p l a n t s . Beans (Phaseolus v u l g a r i s , v a r . Top Crop) were grown under growth room c o n d i t i o n s i n f i v e - i n c h t i n cans f i l l e d w i t h s o i l , and i n s i x i n c h p l a s t i c pots f i l l e d w i t h v e r m i c u l i t e . Vermiculite-grown beans were i r r i g a t e d w i t h a modified Shive's three-salt nutrient solution.  Treatment u n i t o f 8 to 10 p l a n t s  was r e p l i c a t e d 4 to 6 times. Dusts c o n t a i n i n g 2,4-D, NCL, NH^.2,4-D and minerals a t a r a t e o f "8! pounds per acre were a p p l i e d to the f o l i a g e o f plants when 13 o r 14 days o l d .  Sprays c o n t a i n i n g NH^*2,4-D were  a p p l i e d to the p l a n t s u n t i l run o f f . The soil-grown beans t r e a t e d w i t h 2,4-D i n dust form were harvested when 8 weeks o l d . Growth (based on tops) and y i e l d (based on pods) were then determined, and enzyme a c t i v i t y was measured 2, 5, 7, i.?., and 14 days a f t e r treatment. miculite-grown beans were t r e a t e d w i t h both dusts 2,4-D.  The v e r -  and sprays o f  Growth and rates o f photosynthesis and r e s p i r a t i o n were  determined a t one-week i n t e r v a l s f o r s i x consecutive weeks a f t e r treatment.  Y i e l d was determined by c o l l e c t i n g pods a t one-week  i n t e r v a l s f o r three weeks s t a r t i n g a t the f o u r t h week a f t e r  ii treatment.  The gas exchange method was used f o r the deter-  mination o f photosynthesis and r e s p i r a t i o n .  E f f e c t s o f NCL i n  dust form and NH4/2,4-D i n spray form on c h l o r o p h y l l content and photophosphorylation  7 days a f t e r treatment were studied.  The r e s u l t s showed that dusts o f 2,4-D s i g n i f i c a n t l y increased the number o f pods, whereas sprays o f NH^.*2,4-D d i d not.  NCL 0.12 and NH^*2,4-D 1.0 ppm increased seed y i e l d by  18.52% and 12.667P over the c o n t r o l s .  With the exception o f NCL  0.12, a l l the 2,4-D treatments i n dust form f a i l e d to show a s i g n i f i c a n t e f f e c t on growth and y i e l d .  Sprays o f NH^*2,4-D a t  5 ppm s i g n i f i c a n t l y i n h i b i t e d growth and y i e l d .  A higher d r y  weight and percentage dry weight were obtained i n dust o f 2,4-D t r e a t e d vermiculite-grown beans.  Beans dusted w i t h NCL 0.12  showed a s i g n i f i c a n t i n c r e a s e i n f r e s h weight and dry weight o f tops, and f r e s h weight o f pods.  The increases were 41.73%,  48.84% and 28.30% over the c o n t r o l s . A p p l i c a t i o n o f NCL 0.12 and 2,4-D 0.12 r e s u l t e d i n a s i g n i f i c a n t s t i m u l a t i o n o f c a t a l a s e a c t i v i t y i n bean leaves but i n h i b i t e d c a t a l a s e a c t i v i t y i n bean stems i n 14 days.  I n the  case o f phosphatase, NCL 0.12 caused an i n h i b i t i o n i n the enzyme a c t i v i t y o f both leaves and stem i n 5 days and a s t i m u l a t i o n i n 14 days.  NCL 0.06 treated plants showed a lower c a t a l a s e  a c t i v i t y i n 2 days and higher a c t i v i t y i n 5, 7, and 14 days.  iii Phosphorylase a c t i v i t y was stimulated i n 2 days but was inhibited i n 5 days.  Minerals accelerated phosphorylase a c t i v i t y i n 2 and  5 days. NCL dusts and NH^'2,4-D sprays caused a stimulation i n the rate of photosynthesis one week a f t e r treatment.  This was  followed by an i n h i b i t i o n during the period from the second to s i x t h week a f t e r treatment.  A higher rate of r e s p i r a t i o n was  found i n NH^*2,4-D treated plants whereas i n the case of NCL treated plants a higher rate o f r e s p i r a t i o n at the f i r s t and s i x t h week and a lower rate at the second obtained.  to f i f t h , week were  Photophosphorylation i n plants treated with NCL dusts  and NH^*2,4-D sprays was s i g n i f i c a n t l y higher than that i n untreated plants except that NCL dust showed an uncoupling of phosphate uptake i n noncyclic photophosphorylation.  viti ACKNOWLEDGEMENT The writer wishes to extend her deepest gratitude to Dr. D. J . Wort, Professor, o f the Department o f Biology and Botany, f o r h i s s p e c i a l supervision and guidance. The writer also wishes to thank Dr. D. P. Ormrod, Assistant Professor of the D i v i s i o n of Plant Science i n the Faculty of Agriculture, f o r his suggestion i n experimental design and analysis o f data, Dr. J . R. H. Dempster, Chief Programmer o f the Computing Center at the University o f B r i t i s h Columbia f o r h i s help i n programming, and s t a f f members o f the electronic computing center f o r t h e i r guidance and correction. The writer g r a t e f u l l y acknowledges f i n a n c i a l assistance obtained  from the National Research Council of Canada and the  Department o f Biology and Botany.  iv TABLE OF CONTENTS PAGE Abstract . .  i  Table of Contents  iv  L i s t o f Tables L i s t of Figure  v i i . . . . . . . . . . . . . .  .viii  Acknowledgement.  ix  INTRODUCTION . . . .  1  LITERATURE REVIEW  3  A.  E f f e c t o f 2,4-D and 2,4-D-minerals on growth and y i e l d  B.  E f f e c t o f 2,4-D and 2,4-D minerals on photosynthesis  C.  E f f e c t o f 2,4-D on r e s p i r a t i o n . . . . .  D.  E f f e c t o f 2,4-D and 2,4-D minerals on enzyme a c t i v i t y  3 . .  EXPERIMENTAL METHODS  6 7 10  .  14  A.  Growth o f plants  15  B.  A p p l i c a t i o n o f formulations. . . . . . . . . . . .  16  1.  Dust method  16  2.  Spray method  17  C.  Determination o f growth and y i e l d . . . . . . . . .  17  D.  Determination o f enzyme a c t i v i t y  18  1.  P r e p a r a t i o n o f enzyme homogenate . . . . . . .  18  2.  Determination o f c a t a l a s e a c t i v i t y . . . . . .  18  3.  Determination o f phosphatase a c t i v i t y  19  4.  Determination o f phosphorylase a c t i v i t y . . . .  19  5.  Determination of r a t e o f photosynthesis and respiration Determination o f r a t e o f photosynthetic phosphorylation (photophosphorylation). . . .  6. 7.  S t a t i s t i c a l analysis . . . . . .  20 23 26  V PAGE EXPERIMENTS AND RESULTS  28  Experiment 1. E f f e c t o f NCL, 2,4-D and m i n e r a l s  on  growth, y i e l d and enzyme a c t i v i t y o f beans Experiment 2.  E f f e c t of NCL, 2,4-D and m i n e r a l s  28 on  growth, y i e l d and enzyme a c t i v i t y o f beans Experiment 3. E f f e c t o f NCL and stage o f of  37  application  treatment on growth and y i e l d o f beans  Experiment 4. 2,4-D,  42  E f f e c t o f NCL, 2 , 4 - D , m i n e r a l s , N H ^  NCL-NH^. 2,4-D on growth and y i e l d o f beans.  .  47  2,4-D and N C L - N H 4 » 2 , 4 - D on growth and y i e l d o f beans.  52  Experiment 5. E f f e c t o f NCL, 2,4-D m i n e r a l s , N H ^  Experiment 6. respiration,  E f f e c t o f NCL on p h o t o s y n t h e s i s , growth and y i e l d o f beans  62  Experiment 7. E f f e c t o f NH4*2,4-D on p h o t o s y n t h e s i s , respiration,  growth and y i e l d o f beans  74  Experiment 8. E f f e c t o f NCL and NH^*2,4-D on photophosphorylation  85  DISCUSSION  90  SUMMARY  96  REFERENCES  98  APPENDICES.  105  vi LIST OF TABLES TABLE  PAGE  1.  E f f e c t o f 2,4-D on enzymes o f beans . .  11  2.  E f f e c t o f NCL, 2,4-D and minerals on y i e l d and growth o f beans. Experiment 1  31  E f f e c t o f NCL, 2,4-D and minerals on enzyme a c t i v i t y o f beans. Experiment 1  32  E f f e c t o f NCL, 2,4-D, and minerals on y i e l d and growth o f beans. Experiment 2  39  5.  E f f e c t o f NCL, 2,4-D and minerals on enzyme a c t i v i t y . Experiment 2  40  6.  E f f e c t o f NCL and the stage o f a p p l i c a t i o n of treatment on growth o f beans. Experiment 3  45  7.  E f f e c t o f NCL and the stage o f a p p l i c a t i o n o f treatment on y i e l d o f beans. Experiment 3  46  8.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH4'2,4-D and NCL-NH4'2,4-D on growth o f beans. Experiment 4. . .  49  9.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH4»2,4-D, and NCL-NH4*2,4-D on y i e l d o f beans. Experiment 4 . . .  50  10.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH4.2,4-D, and NCL-NH4*2,4-D on growth o f beans. Experiment 5. . .  57  11.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH '2,4-D and NCL-NH '2,4-D « Experiment 5 . . .  3. 4.  4  o  n  v  i  e  l  d  o  f  b e a n s  4  12.  58  E f f e c t o f NCL on photosynthesis and r e s p i r a t i o n of beans. Experiment 6  65  13.  E f f e c t o f NCL on growth o f beans. Experiment 6. . .  67  14.  E f f e c t o f NCL on y i e l d o f beans. Experiment 6 . . .  69  15. 16.  E f f e c t o f NCL on seed y i e l d . Experiment 6 E f f e c t o f NH4»2,4-D on photosynthesis and r e s p i r a t i o n o f beans. Experiment 7  70  17.  E f f e c t o f NH «2,4-D on y i e l d o f beans  .  79  18.  E f f e c t o f NH4"2,4-D on seed y i e l d . Experiment 7 . .  82  19.  E f f e c t o f NCL and NH^*2,4-D on photophosphorylation of beans. Experiment 8 . . .  86  4  77  vii LIST OF FIGURES FIGURE  PAGE  1.  E f f e c t o f NCL 0.12, 2,4-D 0.12 and minerals on c a t a l a s e a c t i v i t y o f bean leaves. Experiment 1. . .  29  2.  E f f e c t o f NCL 0.12, 2,4-D 0.12 and minerals on c a t a l a s e a c t i v i t y of bean stems. Experiment 1 . . .  29  3.  E f f e c t o f NCL 0.12, 2,4-D 0.12 and minerals on phosphatase a c t i v i t y o f bean leaves. Experiment 1 .  30  4.  E f f e c t o f NCL 0.12, 2,4-D 0.12 and minerals on phosphatase a c t i v i t y o f bean stems. Experiment 2. .  30  5.  E f f e c t o f NCL 0.06, 2,4-D 0.06 and minerals on c a t a l a s e a c t i v i t y o f bean leaves. Experiment 2. . .  38  6.  E f f e c t o f NCL 0.06, 2,4-D 0.06 and minerals on phosphatase a c t i v i t y o f bean leaves. Experiment 2 .  38  7.  E f f e c t o f NCL and stage o f a p p l i c a t i o n of treatment on y i e l d and growth o f beans. Experiment 3  44  8.  E f f e c t o f NCL 2,4-D, m i n e r a l s , NH^2,4-D and NCL-NH4*2,4-D on growth o f beans. Experiment 4. . .  48  9.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH »2,4-D and NCL-NH^*2,4-D on y i e l d o f beans. Experiment 4 . . .  48  10.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH «2,4-D and NCL-NH4*2,4-D on growth o f beans. Experiment 5. . .  55  11.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH^.*2,4-D and NCL-NH »2,4-D on y i e l d o f beans. Experiment 5.. . .  56  13.  E f f e c t o f NCL on photosynthesis and r e s p i r a t i o n o f beans. Experiment 6 . . . . . . . . . E f f e c t of NCL 0.12 on growth o f beans  63 64  14.  E f f e c t o f NCL 0.12 on y i e l d o f beans. . . . . . . .  64  15.  E f f e c t o f NH^*2,4-D on photosynthesis and r e s p i r a t i o n o f beans. Experiment 7  75  16.  E f f e c t o f NH *2,4-D on y i e l d o f beans. Experiment 7  76  4  4  4  12.  4  INTRODUCTION During the past several years, a multitude o f problems associated with the e f f e c t s of 2,4-dichlorophenoxyacetic acid (2,4-D) on bean plants has have been published.  been investigated, and many a r t i c l e s  For many years, plant nutrients including  micro and macro elements have been applied to the leaves of plants as a dust or spray (71, 73, 75, 76, 77, 82).  I t was  known that f o l i a r application of 2,4-D with a complex o f microelements o r chelating agents, 2,4-D alone, and microelements alone at low levels as dusts or sprays increases the y i e l d (37, 38, 73, 77, 82) and r e s p i r a t i o n (26, 79) o f beans.  The mechanism by which  2,4-D stimulates vegetative and reproductive growth i s s t i l l unknown. Recently, a number of investigations haste' shown that meta l l i c ions play an important part i n the growth process (55). It was well known that growth stimulation of 2,4-D i s further increased by the presence o f metal ions.  The presence of i r o n ,  with the herbicide decreases the injurious e f f e c t of high levels of 2,4-D (37, 38, 73, 79). I t has been found that the 2,4-D-induced increase o f growth i s accompanied by an enhanced rate of r e s p i r a t i o n ( 5 , 72, 78, 79). French and Beevers (21) proposed that the respiratory increase by plant growth substances might be a consequence of growth promotion.  2. Measurement by gas exchange methods i n d i c a t e d that 2,4-D at;high c o n c e n t r a t i o n i n h i b i t s the r a t e o f photosynthesis (27, 34, 64).  Changes i n the photosynthetic r a t e and a v a i l a b l e  photosyn-  thate caused by the a p p l i c a t i o n o f 2,4-D may i n f l u e n c e growth r a t e , dry matter production, and y i e l d o f p l a n t s (37, 38, 65, 82). I t has been recognized that metabolic r e a c t i o n s w i t h i n a l i v i n g c e l l are c o n t r o l l e d by the enzyme system o f the c e l l . 2,4-D has been found to a f f e c t both biochemical and b i o p h y s i c a l r e l a t i o n s w i t h i n the c e l l .  Some enzymes are i n h i b i t e d and others  are stimulated (12, 74). The present study i s concerned  p r i m a r i l y w i t h the e f f e c t s  of f o l i a r a p p l i c a t i o n o f dusts o f 2,4-D i n combination w i t h microelements (NCL, see page 14), on growth, y i e l d , enzyme a c t i v i t i e s , photosynthesis, and r e s p i r a t i o n o f bean plant Phaseolus v u l g a r i s . The e f f e c t s o f sprays o f ammonium s a l t o f 2,4-D (NH -2,4-D) on 4  y i e l d , photosynthesis, and r e s p i r a t i o n , 2,4-D alone and minerals alone on growth, y i e l d , and enzyme a c t i v i t i e s are also i n v e s t i gated.  3. LITERATURE REVIEW The l i t e r a t u r e c i t e d i n t h i s review i s r e l a t e d to the e f f e c t o f 2,4-D and 2,4-D^minerals on growth, y i e l d , enzyme act i v i t y , photosynthesis, and r e s p i r a t i o n of bean p l a n t s . A number of books and reviews concerning the metabolic, compositional and chemical aspects o f s y n t h e t i c auxin a c t i o n have been published.  Synthetic auxin l i t e r a t u r e from 1900 to 1950 has been  reviewed by S c h e l l e t a l . (44).  Books concerned with the  chemistry and mode of auxin a c t i o n have been w r i t t e n by Leopold et a l . (32), Wain and Wightman (62), Skoog (46), and C r a f t (12). The metabolic and compositional e f f e c t s of 2,4-D are reviewed by Woodford (72), and by Wort (80, 81). The most recent reviews and books on the progress of auxin research are published by Audus ( 6 ) . A.  E f f e c t o f 2.4-D and 2.4-D-minerals on growth and y i e l d The growth of bean p l a n t i n n u t r i e n t s o l u t i o n s c o n t a i n i n g  s y n t h e t i c growth-regulating substances showed that low r a t e s o f 2,4-D i n n u t r i e n t s o l u t i o n increased the dry weight of soybean plants (53, 54). Swanson (51) reported that f o l i a r a p p l i c a t i o n o f 0.1 percent aqueous 2,4-D spray s i g n i f i c a n t l y increased the cambial a c t i v i t y i n young red kidney beans.  Weaver (63) s t a t e d  that the a p p l i c a t i o n o f aqueous s o l u t i o n s of 2,4-D a t r a t e s o f 0.001, 0.01, and 0.1 grams a c t u a l chemical per square yard, to the leaves o f young red kidney beans and soybeans r e s u l t e d i n  4.  reduced p l a n t weight and delayed pod appearance i n 31 days a f t e r treatment.  Wedding e t al.(65) reported that the a p p l i c a t i o n o f  10 ppm and 20 ppm o f 2,4-D e s t e r to lima beans two weeks a f t e r emergence r e s u l t e d i n a 35 percent increase i n the y i e l d o f s h e l l e d green beans.  The a p p l i c a t i o n o f 10 ppm 2,4-D e s t e r four  weeks a f t e r emergence s i g n i f i c a n t l y delayed maturation and the t o t a l y i e l d o f marketable s h e l l e d green beans was reduced to o n l y 267c o f that from untreated p l a n t s .  I n some years and i n some  l o c a t i o n s , the a p p l i c a t i o n o f 2,4-D sprays a t 5 ppm to 20 ppm produced a y i e l d increase as great as 70% i n the case o f green and dry lima beans and snap beans.  I n other experiments, however,  the same treatment r e s u l t e d i n a decrease o f as much as 757« o r , more f r e q u e n t l y , i n the lack o f any s i g n i f i c a n t  e f f e c t on y i e l d .  Leonard (31) reported that aqueous s o l u t i o n s o f the t r i e t h y l a m i n e s a l t o f 2,4-D (150 ppm) s l i g h t l y increased f r e s h weight o f bean plants 14 days a f t e r a p p l i c a t i o n to s e e d l i n g p l a n t s . Geranmayeh (23) reported that beans treated with 0.001% ammonium s a l t o f 2,4-D have l e s s d r y weight o f tops and s l i g h t l y l e s s t o t a l dry matter than the c o n t r o l s .  The a p p l i c a t i o n of 0.002% ammonium  s a l t o f 2,4-D showed a small*v decrease i n the dry matter o f the J  tops, and a smallKt increase i n t o t a l dry matter over that o f controls. A s e r i e s o f studies on the e f f e c t o f f o l i a r a p p l i c a t i o n of s u b l e t h a l concentrations o f 2,4-D and 2,4-D with n u t r i e n t dusts on the growth and y i e l d o f beans, has been c a r r i e d out and  5.  reviewed by Wort (75,  76,  77,  79,  82).  F o l i a r applied clay dusts  and sprays containing low concentration of 2,4-D elements, increased y i e l d and growth of beans. of dusts containing 0.17*  2,4-D  and microThe application  to Top Crop bean plants at the  rate of 12 pounds of dust per acre resulted i n an increase i n fresh weight of pods of as much as 55% while i n other experiments the same treatments did not show the effect on y i e l d and growth. Increases i n y i e l d of pods and threshed beans were also obtained by the application of NCL 0.1  to Masterpiece bush beans and  M i c h i l i t e navy beans. More frequently, the application of NCL 0.1  resulted i n an increase i n fresh weight of tops and of pods.  The increases were 13.97e and 23.1%  for tops and pods, respec-  The injurious effects of 2,4-D  tively.  sprays and dusts were  decreased or completely eliminated by previous, simultaneous or subsequent f o l i a r application of s a l t s or chelates of i r o n and copper, or chelating agents (27, M i l l e r et a l . (37, taining 0.5  or 1 ppm 2,4-D  38)  37,  73,  77,  79).  reported that f o l i a r sprays con-  stimulated the juvenile growth and Sprays which contained 5 ppm  seed production of f i e l d beans. above 2,4-D  38,  inhibited growth.  Incorporation of 2,4-D  with iron  as FeSO^ or i n the chelate form reduced the toxic effect of and ultimately increased growth.  F o l i a r treatments with 0.5  1 ppm, both with and without FESO4 and 100  ppm 2,4-D  with Fe  showed an increase i n dry matter production and i n seed y i e l d (37,  38).  and  2,4-D and  6.  B.  E f f e c t o f 2.4-D and 2,4-D-minerals on photosynthesis Freeland  (19) was among the f i r s t to study the e f f e c t o f  2,4-D on the photosynthetic  a c t i v i t y of plants.  By covering bean  plant tops with a b e l l j a r and analyzing the gas passing through an absorption tower every two hours, i t was shown that CO2 a s s i m i l a t i o n by bush beans was depressed to 70% of the o r i g i n a l rate for three days a f t e r the plants were sprayed with 0.01% 2,4-D. This r e s u l t has been confirmed by Aker and Fang (2).  Loustalot  et a l . (34) measured the rate of apparent photosynthesis by drawing the a i r through a posrometer cup attached  to the lower  surface o f a mature leaf and forcing this a i r through an absorption tower and a flow-meter for three hours.  The velvet bean  plants were treated with aqueous isopropyl ester of 2,4-D, ranging from 0.1%  to 0.0017c acid equivalent, u n t i l l i q u i d ran o f f .  Control plants were treated with d i s t i l l e d water.  The r e s u l t s  showed that 2,4-D 0.1% caused a sharp reduction of photosynthesis i n the leaves within f i v e hours a f t e r treatment.  The 0.057o 2,4-D  ester caused a decline within f i v e hours, then a further reduction within the next few days. plant died.  F i n a l l y CO2 a s s i m i l a t i o n ceased and the  The 2,4-D ester at 0.001% caused a s l i g h t reduction  a week or more a f t e r a p p l i c a t i o n and photosynthesis continued at a reduced rate for several weeks.  I n h i b i t i o n of  photosynthetic  rate was i n d i r e c t l y caused by destruction of mesophyll and o f stem phloem and impaired  translocation.  Loustalot etal.(34> found  7.  extensive p r o l i f e r a t i o n o f a l l the parenchyma c e l l s between the xylem and the i n n e r p o r t i o n o f the cortex formed a large sheath of abnormal t i s s u e , and sieve tubes and companion c e l l s were crushed and o b l i t e r a t e d .  This damage to the phloem may have been  a c o n t r i b u t i n g f a c t o r to the lower r a t e o f photosynthesis, because of accumulation of the photosynthate.  Huffaker e t a l . (26)  studied the e f f e c t o f 2,4-D, i r o n , and c h e l a t i n g agents on dark CO2 f i x a t i o n i n c e l l - f r e e homogenate o f beans.  They reported  that CC*2 f i x a t i o n was increased a t the l e v e l o f 2,4-D which stimulated p l a n t growth. t r a t i o n s o f 2,4-D increased.  treatment  S t i m u l a t i o n decreased as concenThey proposed that an increase i n  f i x a t i o n o f CO2 r e s u l t i n g from treatment w i t h 2,4-D could be important i n p r o v i d i n g both sugar and organic acids and could be a t t r i b u t e d to the increase i n phosphoenolpyruvate carboxylase, phosphoriboisomerase, phosphoribulokinase,  and ribulosediphosphate  carboxylase production, r e s u l t i n g i n increased metabolic  activity  and growth (26). This information points to a c o n c l u s i o n that 2,4-D a p p l i c a t i o n at high concentration i n h i b i t s the gas exchange o f photosynthesis. C.  E f f e c t o f 2.4-D on r e s p i r a t i o n Although numerous studies o f the e f f e c t o f 2,4-D on r e s -  p i r a t i o n have been made, few i n v e s t i g a t i o n s have i n v o l v e d 2,4-D  8. Brown (9) determined the  nutrient dusts and sprays on beans.  respiratory a c t i v i t y o f bean plants sprayed with 2,4-D by measuring CO2 output i n a respiratory chamber.  He concluded that  the rate of r e s p i r a t i o n of seedling bean plants sprayed with 1000 ppm 2,4-D was s i g n i f i c a n t l y greater than that o f the control plant during a period of 24 hours immediately a f t e r treatment. The production o f C0£ i n treated plants was approximately 19 to 20% greater than that i n untreated four days.  plants during a period o f  Smith (47) used the manometric method to determine  the aerobic r e s p i r a t i o n of bean stem t i s s u e .  Two drops of 0.1%  2,4-D were applied on the base o f each of the primary leaves and the f i r s t internodal tissue was cut into s l i c e s for manometric determination.  He reported that the rate of r e s p i r a t i o n i n  s l i c e s from 2,4-D-treated plants was s i g n i f i c a n t l y higher than i n those from untreated ones, and that the rate increased to a maximum by the seventh to ninth day.  Freeland  (19)  reported  that  the a p p l i c a t i o n of a spray of 0.0r% 2,4-D to bean leaves resulted i n a 70% reduction i n the respiratory rate on the f i r s t day and an increase to 1107e of the o r i g i n a l rate by the t h i r d day. Corns (11)  investigated the e f f e c t of 2,4-D and s o i l  moisture on the catalase a c t i v i t y , r e s p i r a t i o n and protein content o f bean plants.  He stated that stimulation o r reduction of  r e s p i r a t i o n of leaves depended upon concentration o f 2,4-D, s o i l moisture and time a f t e r treatment.  Avery (7) has shown that the  9. a p p l i c a t i o n o f 2,4-D to beans increased oxygen consumption and carbon d i o x i d e e v o l u t i o n .  He stated that high  concentrations  of 2,4-D may depress the r a t e of r e s p i r a t i o n . Linden (33) proposed that 2,4-D induced the increase i n the r a t e o f r e s p i r a t i o n by a f f e c t i n g phosphate metabolism and by combining w i t h products and enzymes. phosphorylation (ATP)  metabolic  This p r o p o s i t i o n , that 2,4-D i n f l u e n c e d  and the production o f adenosine  triphosphate  and thus subsequently enhanced the r a t e o f growth and r e s -  p i r a t i o n , supports the theory o f Bonner and Bandurski ( 8 ) , and i s confirmed by Switzer's i n v e s t i g a t i o n . Fang e t aj;. (15) reported that 2,4-D treatments g r e a t l y reduced the upward move32 ment o f P  to the leaves o f beans; the degree o f r e d u c t i o n  p r o p o r t i o n a l to the amount o f 2,4-D s u p p l i e d .  being  The i n c o r p o r a t i o n  o f p32 i n t o phosphate intermediate compounds i n the water f r a c t i o n , such as hexosediphosphate, was i n h i b i t e d by 2,4-D treatment. Switzer (52) reported t h a t mitochondria  i s o l a t e d from soy-  bean seedlings sprayed with 2,4-D had an increased o x i d a t i v e and phosphorylative mitochondria  a c t i v i t y , but 2,4-D added i n v i t r o to i s o l a t e d  i n h i b i t e d both o x i d a t i o n and phosphate uptake.  He  concluded that 2,4-D had e f f e c t s leading to the i s o l a t e d p a r t i c l e s r e t a i n i n g more o f t h e i r o r i g i n a l a c t i v i t y .  Fang et a l . (16) have  demonstrated that the bean stem t i s s u e pretreated with 2,4-D stimulated glucose uptake and r e s p i r a t o r y CCv* evolved from g l u cose.  They suggested that 2,4-D i n f l u e n c e s growth by a f f e c t i n g  10. glucose u t i l i z a t i o n to supply energy and substrates f o r synthesis of c e l l u l a r constituents.  Key (28) has i n v e s t i g a t e d the e l e c t r o n  micrographs o f mitochondria i s o l a t e d from soybean hypocotyl and i n d i c a t e d that a p p l i c a t i o n o f 2,4-D to soybean seedlings induced extensive growth and r e s p i r a t o r y and phosphorylatory o f the mitochondria.  During growth these mitochondria  activity increased  i n a c i d s o l u b l e n u c l e o t i d e s , phospholipid and n u c l e i c a c i d . concluded  He  that the normal growth o f soybean t i s s u e induced by  2,4-D i n v o l v e d an increase i n m i t o c h o n d r i a l a c t i v i t y and postul a t e d that t h i s growth i s regulated through m i t o c h o n d r i a l metabolism. D.  E f f e c t o f 2.4-D and 2.4-D-minerals on enzyme a c t i v i t y Much o f the work on the mechanism o f auxin-induced  growth  has i n v o l v e d attempts to study the connection between r e s p i r a t i o n and growth.  Smith (47) s t a t e d that the study o f the e f f e c t  of auxin on r e s p i r a t o r y enzyme a c t i v i t y might be a d i r e c t approach. The e f f e c t s o f auxins on enzyme a c t i v i t y have been reviewed by Freed (18), Bonner ( 8 ) , Woodford (72), and Wort (74, 81), among others.  Table I summarizes the information on the e f f e c t o f  2,4-D on various enzymes o f bean p l a n t s .  Wort (81) has s t a t e d  that the a d d i t i o n o f 2,4-D to c e l l - f r e e f i l t r a t e s o r to p u r i f i e d enzymes w i t h substrate r e s u l t s i n an i n h i b i t i o n o f enzyme a c t i v i t y while the a c t i v i t y o f c r y s t a l l i z e d enzymes has been both increased or decreased depending upon the concentration o f 2,4-D. The  11. Table 1.  E f f e c t o f 2,4-D on enzymes of beans  Enzyme  Plant Part  Method and Concentration  Amylase  stem s l i c e s  10 ppm  Amylase  S base o f blade  Effect  Ref. (22)  one drop 1000 ppm  6 day: stem -, leaf: 0  (39)  Ascorbic acid oxidase  leaf and root  0.005-0.0125M i n c e l l free extract  0.0125M: inhibit 5-30%  (60)  Catalase  castor bean  0  (24)  Catalase*  Top Crop bean leaves and stems  i n homo genate NCL 0*12,NCL 0.06,2,4-D 0.12, 2,4-D 0.06 f o l i a r dust  Glycolic acid oxidase  leaf and root  0.005-0.0125M in cell-free extrac t  decrease by 0.0/25M  5 day & 14 day: (24) NCL 0.12, NCL 0.06 = + 14 day: 2,4-D 0.12 = + Leaf 14 day: NCL 0.12, 2,4-D 0.12 = Stem (60)  Hydroxyacid castor bean oxidase  i n homogenate 0  (24)  IAA oxidase  2,4-D 5,10, 20 ppm  (72)  Invertase  Lipase  bean  kidney bean  Castor bean  prevent normal increase i n tissue  50 ug/plant (one drop 1000 ppm) 0.00303M 0.00303M i n substrate  (39)  0.000303M:-10% 0.00303M: -70%  (24)  12. Effect  Ref.  Enzyme  Plant Part  Method and Concentration  Pec t i n methyl esterase  bean  50 ug/plant  + (39) prevent normal increase i n (72) tissue  Peptidase  soybean  5 ppm i n nutrient solution f o r 25 hours  leaf: stem, root: +  Peroxidase  kidney bean  5.7 ue/plant 5xl0-5.M  increase leaf (17) protuberances prevent normal (72) increase i n tissue  Phosphatase* Top Crop bean NCL 0.12,NCL leaves and stems 0.06,2,4-D 0.12, 2,4-D 0.06 dusts on leaf  (20)  l e a f : NCL 0.12 stem: 2,4-D 0.12 = + i n 14 days, - i n 7 days  Phosphorylase  kidney bean  50 ug/plant (one drop 1000 ppm)  Po ly pheno 1 oxidase  castor bean  i n homogenate  Polypeptidase  soybean  5 & 500 ppm spray  0-8 day: + leaf: stem & root: +  (20)  Proteinase  soybean  leaf: stem & root: +  (20)  Proteolytic  kidney bean  5 ppm i n nutrient 50 ug/plant  stem: + root & l e a f : -  (42)  Note:  * results obtained from this + increase - decrease 0 no e f f e c t  (39)  0  investigation  (24)  13. a c t i v i t y i n m a t e r i a l from plants pretreated w i t h 2,4-D may be increased, decreased o r unaffected.  Freed e t a l . (18) have shown  that a marked s t i m u l a t i o n of the a c t i v i t y o f c r y s t a l l i n e  glycer-  aldehyde-3-phosphate dehydrogenase r e s u l t e d from using 180 ppm 2,4-D.  One thousand ppm caused a marked i n h i b i t i o n o f a c t i v i t y .  They suggested that 2,4-D i s adsorbed by p h y s i c a l forces on the surface o f enzyme p r o t e i n and thereby the s t r u c t u r e s o f the enzyme molecule change.  A low concentration o f 2,4-D might  modify the enzyme a c t i v i t y o f a p r o t e i n to make i t a more e f f i cient catalyst.  As the concentration o f 2,4-D increases, more  molecules o f 2,4-D are adsorbed to the p r o t e i n surface, the m o d i f i c a t i o n o f the s t r u c t u r e becomes severe, and a consequent l o s s of c a t a l y t i c property r e s u l t s .  Huffaker e t a l . (26) reported  that a p p l i c a t i o n o f 0.5 ppm and 1 ppm 2,4-D to bean plants i n creased the a c t i v i t y o f both phosphoenolpyruvate carboxylase and ribose-5-phosphate enzyme systems i n v i v o , but caused an i n h i bition i n vitro.  14. EXPERIMENTAL METHODS This i n v e s t i g a t i o n was i n i t i a t e d i n August 1963 to study growth, y i e l d , and various metabolic responses o f Top Crop green bush beans to f o l i a r a p p l i e d dusts and sprays c o n t a i n i n g 2,4-D and m i n e r a l s a l t s .  The f o l l o w i n g abbreviations are used to  i n d i c a t e the formulations. NCL 0.12. NCL 0.06. e t c . : NCL Leafeeder dusts c o n t a i n i n g elemental S and s u l f a t e s a l t s o f Fe, Cu, Zn, B, and Mn, to give 3.19% Fe, 2.75% Cu, 3.46% Zn, 1.10% B, 3.96% Mn, and 19.58% elemental S and 0.12%, 0.067. e t c . a c i d equivalent i s o p r o p y l e s t e r of 2,4-D, a l l i n attaclay. Minerals: As  NCL but no 2,4-D present.  2.4-D 0.12. 2.4-D 0.06. e t c . : 0.12%, 0.06% e t c . a c i d equivalent i s o p r o p y l e s t e r o f 2,4-D i n a t t a c l a y . NH&«2.4-D 0.12: Ammonium s a l t of 2,4-D i n a t t a c l a y to give 0.12% a c i d equivalent. NCL-NH »2,4-D 0.12: 4  Ammonium s a l t o f 2,4-D i n 'minerals' to g i v e 0.127. a c i d equivalent.  15. NH4'2,4-D 0.5, 1.0, and 5.0 ppm: Aqueous s o l u t i o n c o n t a i n i n g 0.5, 1.0 o r 5.0 ppm ammonium s a l t o f 2,4-D.  The dusts c o n t a i n i n g 2,4-D alone, minerals and  2,4-D w i t h minerals were supplied by N u t r i t i o n a l Consultants L t d . , (NCL) o f Winnipeg, Manitoba, Canada. A.  Growth o f plants F i v e to e i g h t bean seeds were grown i n each pot and were  thinned to two uniform plants when seven to nine days old.. Pots were placed i n a growth room w i t h the exception o f Experiment 1. I n t h i s case the pots were placed i n a greenhouse.  From E x p e r i -  ment 1 to Experiment 4, beans were grown i n sandy loam s o i l i n 5" t i n cans and i r r i g a t e d w i t h water u n t i l harvest.  I n Experi-  ments 5 to 8, beans were grown i n v e r m i c u l i t e - f i l l e d 6" p l a s t i c pots to e l i m i n a t e p o s s i b l e v a r i a t i o n s caused by l a c k o f uniform d i s t r i b u t i o n o f f e r t i l i z e r i n the s o i l .  Beans grown i n vermi-  c u l i t e were i r r i g a t e d w i t h a modified Shive's t h r e e - s a l t n u t r i e n t s o l u t i o n (36, 45) and flushed w i t h tap water once a week.  More  water as r e q u i r e d , was supplied during the l a t t e r part o f the growing period. In the green house the environmental f a c t o r s were somewhat v a r i a b l e and d i f f i c u l t to c o n t r o l . 16 hour photoperiod;  The growth room, provided a  62 - 74 F. i n the l i g h t period and 54 - 64 F.  i n the dark p e r i o d ; l i g h t i n t e n s i t y ranging from 800 to 1400 foot candles a t the tops o f the p l a n t s ; r e l a t i v e humidity 54% to 72% i n the l i g h t and 70% to 80% i n the dark.  The pot l o c a t i o n s were  16. changed a t appropriate i n t e r v a l s i n order to reduce l o c a l environmental e f f e c t s . B.  A p p l i c a t i o n o f formulations I t has been found that the most e f f e c t i v e time o f a p p l i -  c a t i o n o f 2,4-D dusts and sprays to bean plants was when the p l a n t s were 14 days o l d o r i n the t i g h t t r i f o l i a t e bud stage (65, 79). Since the advantage o f the dust form o f 2,4-D was the absence o f i n s o l u b l e p r e c i p i t a t e s , dusts o f 2,4-D were used i n Experiments 1 to 6. 1.  Dust method  NCL LeaFeeder dusts c o n t a i n i n g minerals w i t h 2,4-D, minerals alone, and 2,4-D alone were predried i n the oven a t 35 C f o r one hour.  The appropriate formulations were then prepared  from these dusts and kept i n a vacuum d e s i c c a t o r with Ca(OH) . 2  A plywood box (39.5" i n l e n g t h , 25" i n width and 24" i n height) w i t h two s m a l l holes d r i l l e d i n the upper part o f both ends was used f o r dusting.  Pots o f p l a n t s , a f t e r the s o i l o r  v e r m i c u l i t e surface was covered w i t h wet paper, were placed i n the box.  Using a g l a s s duster w i t h a flat-mouth g l a s s tube and long  rubber tube connected w i t h a bulb, the appropriate dust was blown through the holes to cover the l e a f surfaces with a t h i n l a y e r o f dust.  A weight o f 0.84 gm a p p l i e d to the box area o f 6.86 s q . f t .  was equivalent to 12 pounds per acre.  In this investigation,  0.56 gm o f dust was used and t h i s dosage was equivalent to 8  17. pounds per acre. 2.  Spray method  Aqueous s o l u t i o n s o f d i f f e r e n t concentrations o f NH4»2,4-D were a p p l i e d to the surface o f leaves by a sprayer u n t i l run o f f . Pots were covered w i t h paper before treatment. C.  Determination o f growth and y i e l d The measurement o f growth used i n t h i s i n v e s t i g a t i o n was  r e s t r i c t e d to f r e s h weight, dry weight, and percentage dry weight o f tops.  Y i e l d was based on the number, the f r e s h , dxyw^.r^^-rai.,'.  and percentage dry weight of pods and the seed production. Soil-grown bean p l a n t s were harvested on the s o i l l e v e l when they were 8 weeks o l d . Pod y i e l d and top growth were measured. In Experiment 5 and Experiment 7, y i e l d was determined by c o l l e c t i n g the green pods once a week f o r 3 consecutive weeks from the 4th week a f t e r treatment, and growth was determined when 8 weeks o l d . Seed y i e l d was determine when the p l a n t s were 12 weeks o l d . In Experiment 6, bean p l a n t s were harvested weekly f o r 6 consecutive weeks a f t e r treatment and the y i e l d and growth were measured.  For measuring seed production, seeds were gathered  from the 10th to 16th week a f t e r treatment.  18. A f t e r the measurement o f f r e s h weight, tops and pods were oven-dried a t 75 C f o r 24 hours.  Percentage dry weight was ob-  t a i n e d by c a l c u l a t i o n . D.  Determination o f enzyme a c t i v i t y (see Appendices I I and I I I ) The a c t i v i t y o f c a t a l a s e , phosphatase, and phosphorylase  was determined 2, 5, 7, 'U,, and 14 days a f t e r treatment. ;  I n the  f i r s t three t e s t s , primary leaves were used, and i n the l a s t test^  0  f i r s t t r i f o l i a t e leaves were used f o r enzyme determination. 1.  P r e p a r a t i o n o f enzyme homogenate  Twenty grams o f leaves o r stems was taken from ten p l a n t s and blended w i t h 100 ml precooled d i s t i l l e d water i n a Waring blendor. ring.  The homogenate was f i l t e r e d through the broadcloth on a  The f i l t r a t e was c o l l e c t e d i n a precooled f l a s k and kept  i n an i c e bath.  The f i l t r a t e was used f o r determining the  a c t i v i t y o f c a t a l a s e , phosphatase, and phosphorylase. 2.  Determination o f c a t a l a s e a c t i v i t y  Catalase a c t i v i t y was measured by the conventional manom e t r i c technique (56).  The r e a c t i o n mixtures i n a Warburg f l a s k ,  contained 3 ml 0.0001 M phosphate b u f f e r pH 6.8, 0.2 ml 0.2 M H 0 2  and 1.0 ml enzyme homogenate.  Reaction time was 3 minutes a t 25 C.  The u n i t o f c a t a l a s e a c t i v i t y was c a l c u l a t e d as u l 0 ml enzyme i n 3 minutes.  2  2  evolved per  19. 3.  Determination o f phosphatase a c t i v i t y  Phosphatase a c t i v i t y was measured by Sumner and Somer's (49) method which i s a m o d i f i c a t i o n o f the method used by Gottschalk.  I t c o n s i s t s o f i n c u b a t i n g an enzyme homogenate with  a phenyl phosphate substrate and estimating the amount o f l i b e r ated phenol.  The r e a c t i o n mixture c o n s i s t e d o f 1 ml enzyme  homogenate, 5 ml s u b s t r a t e , 2 ml F o l i n - C i o c a l t e i i reagent, 5 ml 10% Na2C03, and 100 ml water.  Reaction time was 1 hour a t 37 C.  A Klett-Summerson p h o t o e l e c t r i c c o l o r i m e t e r w i t h green f i l t e r was used i n the determination o f the amount o f phenol l i b e r a t e d . readings were r e f e r r e d to a phenol standard curve.  The  The u n i t o f  phosphatase a c t i v i t y was expressed as mg phenol released per 1 ml enzyme per hour. In p l o t t i n g the standard curve, 5 ml o f standard phenol s o l u t i o n and 1 ml o f d i s t i l l e d water were used i n s t e a d o f 5 ml s u b s t r a t e and 1 ml enzyme. 4.  Determination o f phosphorylase a c t i v i t y  Sumner's method (50), which i s a m o d i f i c a t i o n o f F i s k e and Subbrow's method, was used i n t h i s experiment to determine the amount o f inorganic phosphate l i b e r a t e d i n s o l u t i o n by the phosphorylase r e a c t i o n .  Reaction mixture contained 1 ml enzyme  homogenate, 5 ml s u b s t r a t e , 5 ml 6.66% ammonium molybdate, 5 ml 7.5 NH2SO4, 5 ml 4% F e S 0 and 10 ml water. 4  hour a t 30 C.  Reaction time was 1  The amount o f inorganic phosphate was measured by  20. a Klett-Summerson colorimeter with red f i l t e r .  The unit o f phos-  phorylase a c t i v i t y was expressed as mg phosphate released per 1 ml enzyme per hour. For standard curve preparation, 5 ml o f standard phosphate s o l u t i o n was used instead o f 5 ml of substrate. 5.  Determination o f rate of photosynthesis and r e s p i r a t i o n  Photosynthesis and r e s p i r a t i o n rates were determined by the manometric method (56).  The measurements o f photosynthetic  and respiratory rates were made at one week i n t e r v a l s f o r s i x weeks a f t e r treatment. of  The primary leaves were used f o r the test  f i r s t week, while the f i r s t and second t r i f o l i a t e leaves were  used f o r the 2nd to 4th week and 5th to 6th week respectively. Leaves taken from four bean plants were cut by a 1 cm cork borer to provide 100 discs avoiding the large veins.  The discs were  placed i n a P e t r i dish to maintain moisture and mixed thoroughly. Six  leaf d i s c s , which were picked up at random, were placed i n a  manometer f l a s k to provide a d e f i n i t e surface area.  Fresh weight  was used as a basis for the photosynthetic and respiratory rate measurement. A Warburg apparatus including water bath, manometric tube and one side-arm f l a s k was used i n measuring the rate of photosynthesis and r e s p i r a t i o n .  A 16" white 40-watt " C i r c l i n e "  fluorescent tube placed four inches above the f l a s k , and four  21. 150-watt white sprayed tungsten filament lamps spaced evenly eight inches above the f l a s k supplied a l i g h t i n t e n s i t y o f 560 foot-candles, within 5% at f l a s k l e v e l . constant at 25- 0.1 C.  The temperature was kept  This temperature was maintained by a  combination of a cooling tube through which cold water was flowing and a thermostatically controlled water bath. The determination of r e s p i r a t i o n by the manometric method i s based on the consumption of oxygen accompanied with the absorption o f carbon dioxide by potassium hydroxide as i t i s produced.  Warburg's i n d i r e c t method was used to check the e f f e c t of  C O 2 on the rate of r e s p i r a t i o n .  In this method, C 0 2 - b u f f e r was  placed i n the centre w e l l to maintain 1% C O 2 i n the f l a s k .  The  r e s u l t showed that this concentration of C O 2 did not cause a s i g n i f i c a n t e f f e c t on the rate of r e s p i r a t i o n .  This was on the  assumption that the C 0 2 - b u f f e r would have the same function as that o f KOH i n absorbing the CO2 liberated and would maintain CO2 i n the f l a s k at approximately 1%,  Since the presence or absence  of C O 2 d i d not a f f e c t the rate of r e s p i r a t i o n , Warburg's d i r e c t method f o r the determination of r e s p i r a t i o n was used i n this inves t i g a t i o n . A C 0 2 - b u f f e r was used to provide a constant percentage of carbon dioxide during measurement of photosynthesis.  The prepara-  t i o n of buffer and the a p p l i c a t i o n of manometric method i n the determination of photosynthesis were based on Pardee's and  22. Warburg's i n d i r e c t method.  The C0 -buffer was 2  t i o n of diethanoamine which binds constant  an aqueous solu-  r e v e r s i b l y and maintains a  pressure of CO2 i n the gas phase.  The e f f e c t of time  a f t e r preparation of CO2 buffer on i t s e f f i c i e n c y was measured. The r e s u l t s indicated that the use of the CC^-buffer two to 28 hours a f t e r i t s preparation had no s i g n i f i c a n t e f f e c t on the rate of photosynthesis.  The a c t i v i t y was  almost constant i n the i n -  t e r v a l 2 to 6 hours but decreased a f t e r 28 hours. was  The buffer  therefore f r e s h l y prepared f o r each run and was used 2 to 6  hours a f t e r preparation. For photosynthesis,  0.4 ml C02"buffer was  pipetted into "the  centre well and 0.3 ml into the side arm of each f l a s k , while for r e s p i r a t i o n , 0.2 ml of 207* KOH and 0.5 ml H 0 2  was  pipetted into the centre well  into the side arm of each f l a s t .  f l u t e d f i l t e r paper was  A 10 x 8 cm  inserted into the centre well to increase  the gas exchange surface.  Six leaf discs were taken and  trans-  ferred, bottom side uppermost, to a wet doughnut-shape f i l t e r paper around the centre well.  Respiratory flasks were covered  with aluminum f o i l to exclude a l l l i g h t .  The flasks were e q u i l i -  brated i n the water bath for 15 to 20 minutes with the l i g h t s on. The rate of photosynthesis  and r e s p i r a t i o n were measured at i n t e r -  vals of 20 minutes f o r 1 hour.  The f i r s t 20-minute reading  provided a check against leakage.  A thermobarometer, which  23. consists of a Warburg manometer with an attached f l a s k containing only water, was prepared to correct the change i n i n t e r n a l pressure caused by changed atmospheric pressure and  temperature  during the i n t e r v a l . The rate of photosynthesis was expressed i n terms of m i c r o l i t r e s of oxygen evolution by one gram fresh weight of tissue i n one hour.  The rate of r e s p i r a t i o n was expressed as  m i c r o l i t r e s of oxygen consumption by 1 gm fresh weight of tissue i n one hour.  The sum of the oxygen consumed i n r e s p i r a t i o n i n  the dark and the oxygen evolved i n photosynthesis i n the l i g h t gave an approximation of true photosynthesis. The measurement of photosynthesis and r e s p i r a t i o n was c a r r i e d out from 9:30  to 12:00  a.m.  and 1:00  r e p l i c a t e s were used i n each determination.  to 3:30  p.m.  Two  The rate of photo-  synthesis and r e s p i r a t i o n was measured at various harvest periods from 9:30  a.m.  to 5:30  p.m.  The results showed that the time of  harvest within this i n t e r v a l did not influence the photosynthetic and respiratory rate. 6.  Determination of rate of photosynthetic phosphorylation (photophosphorylation)  In order to study how 2,4-D  affects photosynthesis, i t was  decided that the evolution of oxygen, inorganic phosphate uptake, and chlorophyll content of the leaf tissue must be studied.  The  method of photophosphorylation determination used i n this experi-  24. ment was that of Whatley and Arnon (70) action was carried out under aerobic  except that the re-  conditions.  Chlorophyll content was measured by Arnon's (3) method, and was expressed as mg chlorophyll per ml of chloroplast suspension. Photophosphorylation can be represented by the following equations, and Arnon s proposed scheme (4^5). 1  NADP + NH3PO4  l i g h t , chloroplast -  . (1) v  > NATP  2NADP + 4H 0 + 2ADP + 2 H o P 0 - - ^ - - ^ - - - - - > .  2  4  L  PPNR*  2NADPH + O2 + 2ATP + 2H 0 2  (2)  2  phosphopyridine necleotide reductase. Reaction (1) was subsequently  c a l l e d c y c l i c pho to-  and reaction (2) was c a l l e d noncyclic photo-  phosphrylation, phosphorylation.  The terms " c y c l i c " and "noncyclic" r e f e r to the  electron-flow mechanisms proposed for these two reactions. Because no P-^2  w  a  s  used, the method applied i n this experiment  was Sumner's method i n measuring and the amount o f inorganic phosphate which was taken from the reaction mixtures.  The re-  action was carried out i n a Warburg f l a s k f o r 15 minutes.  The  temperature of water bath was maintained at 15- 0.1 C and l i g h t i n t e n s i t y was adjusted to 1400  foot-candles.  25 -(2)  U)  ass  i il "ki° m  a  n  •  Light  ADP - Adenosine diphosphate ATP - Adenosine triphosphate NADP - Nicotinamide'adenine jclinucleotide RDP - Ribulose-1, 5-diphosphate R-5-P  - Ribulose-5-phosphate  phosphate  •  26. The r a t e o f phosphate uptake was expressed as mg phosphate per ml i n 15 minutes.  Oxygen e v o l u t i o n was expressed as  m i c r o l i t r e s 0£ released by c h l o r o p l a s t which contained 2.0 mg c h l o r o p h y l l and 0.5 ml enzyme i n 15 minutes.  Phosphopyridine  n u c l e o t i d e reductase a c t i v i t y was based on oxygen e v o l u t i o n and phosphate uptake ( 4 3 ) . 7.  S t a t i s t i c a l analysis  A complete randomized block design w i t h and without subsamples was used i n t h i s i n v e s t i g a t i o n .  The bean plants were  separated i n t o groups according to t h e i r s i z e s to reduce the v a r i a t i o n among p l a n t s .  The number o f pots i n a group being  equal to the number o f treatments o r some m u l t i p l e o f i t . group i s c a l l e d a block.  A  The plants were r e - s e l e c t e d f o r u n i -  f o r m i t y , thus the observed d i f f e r e n c e s would be l a r g e l y due to treatment.  The treatments were a p p l i e d a t random.  Experimental  r e s u l t s were analyzed as f o l l o w s : (a)  The a n a l y s i s o f v a r i a n c e (48) was used to t e s t the  s i g n i f i c a n c e among treatments, groups, and p l a n t s .  The variance  r a t i o was compared w i t h the tabled F-value a t 1% and 5% s i g n i f i cant l e v e l s . (b)  Duncan's new m u l t i p l e range t e s t (14) was used to  compare the s i g n i f i c a n t d i f f e r e n c e between any two o f the t r e a t ments i f v a r i a n c e r a t i o showed a s i g n i f i c a n t d i f f e r e n c e among  27. treatments.  The d i f f e r e n c e s between two treatments means were  compared with the l e a s t s i g n i f i c a n t range a t 5% l e v e l . The s i g n i f i c a n c e  o f treatment was expressed i n the F-values  with a single asterisk(*) asterisks tUt:  as s i g n i f i c a n t a t 5% l e v e l and w i t h two  (**) as h i g h l y s i g n i f i c a n t a t 1% levels r  '  The use o f the term " s i g n i f i c a n t " i n the t e x t of  t h i s t h e s i s means s i g n i f i c a n t a t the 57o l e v e l .  Variance r a t i o  and Duncan's t e s t are shown a t the end o f Tables 2 to 19.  28. EXPERIMENTS AND RESULTS Top Crop bush beans were grown i n a l l experiments, with the exception of Experiment 2. Masterpiece was used.  In this case the v a r i e t y  Beans were grown i n growth room except  i n Experiment 1. Mean values of treated plants were calculated as T/C, and expressed as percent of control. Treatment u n i t and r e p l i cate s i z e are shown at the end of Tables 2 to 19. Experiment 1.  E f f e c t of NCL, 2,4-D, and minerals on  growth, y i e l d and enzyme a c t i v i t y of beans As has been stated by Wort (75, 76, 77, 82), NCL 0.1, 2,4-D 0.1, and minerals increased the y i e l d of Top Crop and Masterpiece bush beans. weight of pods.  The r e s u l t s were based on the fresh  Experiment 1 was undertaken to study the e f f e c t  of NCL 0.12, 2,4-D 0.12 and minerals on growth, y i e l d , and enzyme a c t i v i t y , and the relationships among them, when plants were grown under green house condition. Treatments:  control (untreated), NCL 0.12, 2,4-D 0.12, minerals.  Measurements:  growth and y i e l d - fresh weight of tops  and pods, and number of pods; enzyme a c t i v i t y - Catalase, phosphatase (both stems and leaves); 2nd, 5th, and 14th days a f t e r treatment.  Pig. 1. Effect of NCL 0.12, 2,4-D 0.12 and minerals on catalase a c t i v i t y of bean leaves. Experiment 1. T/C values varied with Time  -Xminerals .2,4-D 0.12 ___ NCL 0.12  130  T/C  110 100 90  70  2  5 7 14 Day after treatment'  Pig. 2. Effect of NCL 0.12, 2,4-D 0.12 and minerals on catalase a c t i v i t y of bean stems. Experiment 1. T/C values varied with Time 150 r  minerals . 2,4 D 0.12 NCL 0.12 r  125 T/C  (#) 100  2  5 7 Day after treatment  14  30. Pig. 3. E f f e c t of NCL O.12r'' 2';4-D'O^2;Haad^miaeEals.:i0a phosphatase activity- ~ of: >be'aa'i sterns^.. Experimeat ,  r  1.  T/C values varied with Time  70  1  0  2  |  |  5 7 Day after  ;  treatment  i  14  Pig. 4. E f f e c t of NCL 0.12, 2,4-D 0.12 and minerals on phosphatase a c t i v i t y of bean stems. Experiment 1. T/C values varied with Time  I  0  I  2  I  Day  5  i  7 1 after treatment t  31 Table 2.  E f f e c t o f NCL, 2,4-D and minerals on y i e l d and growth o f beans. Experiment 1, Aug. 9-0ct. 5, 1963. C o n t r o l M i n e r a l s 2,4-D 0.12  Tops F.Wt.  33.04  Pods F.Wt.  19.20  b  NCL T/C(%) 0.12 M i n e r a l s 2.4-D  NCL  a  28.41  29.78  31.63  85.99  90.13  95.73  17.21  16.43  17.25  89.64  85.57  89.84  (gm/plant)  (gm/plant)  No. o f pods  per p l a n t  Note:  1 0  .34  b  b 9  #  6  0  I2.50  a  12.07 92.89 120.95 116.72 a  a Treated p l a n t value expressed as percent o f the c o n t r o l p l a n t values b  Value i s the average f o r 30 p l a n t s . Three r e p l i c a t e s of 10 p l a n t s f o r each treatment. Variance r a t i o (F) o f Table 2 Tops F. Wt. Pods F. Wt. Pods No. 2.28 0.37  Treat Block  1.37 12.03**  7.54** 4.84*  Duncan's Test f o r Table 2 Pods No. Treat Eeaas  ' ' 0.12 _ 25.00 2  4  D  N  C  L  0.12 24.13  Control v20.67  Minerals 19.20  32. Table 3.  E f f e c t o f NCL, 2,4-D and minerals on enzyme a c t i v i t y of beans. Experiment 1, Aug. 9 - Oct. 5, 1963. C o n t r o l M i n e r a l s 2,4-D 0.12  NCL T/C (%) 0.12 Minerals 2 J h D  Catalase ( u l O2/3 min) Leaves 2 days 5 days 7 days 14 days  148.60* 153.47 136.83 121.72 138.27 111.85* 97.78 88.00  Stems 2 days 5 days 7 days 14 days  25.05 32.33 25.53 34.87  26.03 46.45** 24.70 27.35*  145.33 169.92 103.28 97.80 122.75 164.37** 88.95 89.71 127.52 117.55 80.89 92.23 117.90**113.12**111.12 133.98  114.35 120.12 85.02 128.54  24.60 103.93 86.69 35.87 143.66 76.64 29.97 96.74 102.15 29.92* 78.44 92.69  98.20 110.93 117.36 85.80  21.72 24.78** 26.08 32.32*  Phosphatase (ug phenol/hour) Leaves 2 days 5 days 7 days 14 days  227.33 320.33 247.00 223.00  241.50** 244.83** 252.67 229.17  250.17**223.00 106.23 110.04 265.33**260.33** 76.43 82.83 242.50 240.50 102.29 98.18 247.83**246.67**102.77 111.14  98.09 81.27 97.37 110.61  Stems 2 days 5 days 7 days 14 days  214.67 316.00 259.33 263.67  215.50 263.17** 232.00** 266.67  235.67**234.00**100.39 109.78 305.00 295.00 83.28 96.52 249.33**246.67** 89.46 96.14 265.67 291.19**101.11 100.76  109.01 81.96 95.12 110.43  Note:  a  average o f s i x t e s t s Variance r a t i o (F) o f Table 3.  Leaves (days a f t e r t r e a t ) 2 5 7 14  Catalase Treat 1.71 6.86** Block 3.65* 6.00** Phosphatase Treat 17.77**27.13** Block 3.05 14.36**  3.71* 9.64** 9.75** 0.40 1.51 0.79  Stems (days a f t e r t r e a t ) 2 5 7 14 3.22 2.56  19.45** 3.04 1.76 3.77  5.07* 2.92  78.97** 10.75**44.22**13.75**59.17** 1.14 1.78 0.37 1.09 0.88  Duncan's Test f o r Table 3. Catalase a c t i v i t y , leaves 5 days Treat  NCL 0.12  Means  164.37  Control  2,4-D 0.12  Minerals  122.75  121.72  - 136.83  Catalase a c t i v i t y , leaves 7 days Treat  Control  Means  138.27  2,4-D 0.12  NCL 0.12  127.52  117.55  Minerals 111.85  Catalase a c t i v i t y , leaves 14 days Treat Means  2,4-D 0.12  NCL 0.12  117.90  Minerals  113.12  99.78  Control 88.00  Catalase a c t i v i t y , stems 5 days Treat Block  Minerals 46.45  NCL 0.12 a  Control  35.87  32.33  2,4-D 0.12 24.78  Catalase a c t i v i t y , stems 14 days Treat  Control  Block  34.87  2,4-D 0.12  NCL 0.12  .32.32  Minerals  29.92  27.35  Control  NCL 0.12  Phosphatase a c t i v i t y , leaves 2 days Treat Block  2,4-D 0.12 250.17  Minerals 241.50  227.30  223.00  Phosphatase a c t i v i t y , leaves 5 days Treat  Control  NCL 0.12  Minerals  2,4-D 0.12  Block  320.30  265.30  260.30  244.80  34. Phosphatase a c t i v i t y , leaves 14 days Treat  2,4-0 0.12  NCL 0.12  Means  247.83  246.67  Minerals  Control  229.17  223.00  Minerals  Control  215.50  214.67  Phosphatase a c t i v i t y , stems 2 days Treat  2,4-D 0.12  NCL 0.12  Means  235.67  234.00  Phosphatase a c t i v i t y , stems 5 days Treat Means  Control 316.00  2,4-D 0.12  Minerals  305.00  263.17  NCL 0.12 259.00  Phosphatase a c t i v i t y , stems 7 days Treat  Control  Means  259.33  2,4-D 0.12  NCL 0.12  Minerals  264.67  232.00  249.33  Phosphatase a c t i v i t y , stems 14 days Treat  NCL 0.12  Minerals  2,4-D 0.12  Means  291.17  266.67  265.67  Control 263.67  35. Results A.  Growth and y i e l d The r e s u l t s shown i n Table 2 i n d i c a t e that NCL 0.12,  2,4-D 0.12, and minerals f a i l e d to a f f e c t growth and y i e l d o f beans s i g n i f i c a n t l y w i t h the exception o f the number o f pods. A p p l i c a t i o n o f NCL 0.12 and 2,4-D 0.12 h i g h l y s i g n i f i c a n t l y i n creased the t o t a l number o f pods 16.72% and 20.95% r e s p e c t i v e l y , but s l i g h t l y reduced the f r e s h weight o f tops and pods.  This  reduction i n f r e s h weight was higher i n 2,4-D 0.12 treated plants than that i n NCL 0.12 treated p l a n t s .  M i n e r a l treatment induced  a n o n - s i g n i f i c a n t decrease i n growth and y i e l d . B.  Enzyme a c t i v i t y Results o f enzyme a c t i v i t y , as shown i n Table 3 and Figures  1 to 4, are summarized as f o l l o w s . 1.  Catalase  activity  Leaves NCL 0.12 treatment increased the a c t i v i t y o f c a t a l a s e 2, 5, and 14 days, but decreased the a c t i v i t y 7 days a f t e r t r e a t ment compared w i t h that o f untreated p l a n t s .  A p p l i c a t i o n o f 2,4-D  0.12 r e s u l t e d i n an i n h i b i t i o n o f enzyme a c t i v i t y 2, 5, and 7 days, but an increase o f a c t i v i t y 14 days a f t e r treatment. M i n e r a l s induced a decrease i n a c t i v i t y 5, and 7 days a f t e r t r e a t ment and an increase i n a c t i v i t y 14 days a f t e r treatment.  Ob-  v i o u s l y , a l l treatments i n h i b i t e d c a t a l a s e a c t i v i t y i n primary  36.  leaves i n 7 days but stimulated the a c t i v i t y i n f i r s t leaves i n 14 days.  trifoliate  This phenomenon might be due to upward trans-  l o c a t i o n o f formulations i n the plant as stated by Huffaker (26). Stems Generally, 2,4-D  0.12 treatment caused an i n h i b i -  t i o n of catalase a c t i v i t y but o n l y 5 days a f t e r treatment was the i n h i b i t i o n s i g n i f i c a n t .  NCL 0.12 caused an increase i n - a c t i -  v i t y i n 5 and 7 days, and a decrease i n 14 days a f t e r treatment, but the changes lacked s i g n i f i c a n c e .  M i n e r a l s produced a s i g n i -  f i c a n t s t i m u l a t i o n i n 5 days and a s i g n i f i c a n t i n h i b i t i o n i n 14 days a f t e r treatment. Catalase a c t i v i t y i n stems was lower than i n leaves, and enzymatic response o f stems and leaves to a l l treatments was different. 2.  Phosphatase a c t i v i t y Phosphatase, both i n leaves and i n stems, showed a  s i m i l a r a c t i v i t y and response to the treatments. With the exc e p t i o n o f NCL 0.12-treated bean leaves i n 2 days, a l l the t r e a t ments produced an increase i n phosphatase a c t i v i t y i n 2 and 14 days, and decreased i n a c t i v i t y i n 5 and 7 days. NCL 0.12 s i g n i f i c a n t l y stimulated phosphatase a c t i v i t y i n leaves i n 14 days and i n h i b i t e d  a c t i v i t y i n 5 days.  I n stems,  NCL 0.12 treatment caused an increase i n phosphatase a c t i v i t y i n 2 and 14 days, and a decrease i n 5 and 7 days.  Untreated plants  37." showed the highest a c t i v i t y when 19 days o l d . I t appeared that a l l the treatments caused a smaller increase i n phosphatase a c t i v i t y when 19 days o l d than that o f the c o n t r o l s .  Experiment 2.  E f f e c t o f NCL, 2,4-D and minerals on  growth, y i e l d and enzyme a c t i v i t y o f beans Plants:  Masterpiece bush beans  Treatments: Measurements:  c o n t r o l , NCL 0.G6, 2,4-D 0.06 and m i n e r a l s . growth and y i e l d - f r e s h weight o f tops  and pods, and number o f pods; enzyme a c t i v i t y - c a t a l a s e , phosphatase, and phosphorylase o f leaves, a t 2nd, 5 t h , 7th, and 14th day a f t e r treatment. Results The r e s u l t s are tabulated i n Tables 4 and 5 and are a l s o shown i n Figures 5 and 6. A.  Growth and y i e l d A p p l i c a t i o n o f NCL 0.06, 2,4-D 0.06, and minerals increased  the number o f pods 24.09, 5.45, and 27.72% r e s p e c t i v e l y .  NCL 0.06  and 2,4-D 0.06 d i d not produce a s i g n i f i c a n t e f f e c t on f r e s h weight o f tops and pods.  The a p p l i c a t i o n o f minerals r e s u l t e d i n  a n o n s i g n i f i c a n t increase i n f r e s h weight o f tops, 5.217«, and i n f r e s h weight o f pods, 14.617o. I t can be seen from the r e s u l t s that minerals used i n both Experiments 1 and 2 caused various growth responses.  The v a r i a -  Pig. 5. E f f e c t of NCL 0.06, 2,4-»D 0.06 and minerals on catalase aactivity of bean leaves.  T/C (#)  150  r  130  -  110 100 90  * —  70  L  2  5 7 Day after  —  minerals 2,4-D 0.06 NCL 0.06  treatment  14  Pig. 6. Effect of NCL 0.06, 2,4-D 0.06 and minerals on phosphatase a c t i v i t y of bean leaves. minerals 2,4aD 0.06 NCL 0.06  110  100 T/C  90  80  0  2  j  L  5 7 11 Day after treatment  14  3&  Table 4. E f f e c t o f NCL, 2,4-D, and minerals on y i e l d and growth o f beans. Experiment 2, Oct. 10 - Dec. 5,1963. C o n t r o l Minerals 2,4-D 0.06 Tops F.Wt. (gm/plant) Pods F. Wt. (gm/plant)  16.30 15.47  Pods No. per p l a n t  Note:  a  6.88  NCL T/C (%) 0.06 Minerals 2.4-D NCL 0.06  17.75  16.12  16.01 105.21  17.73  15.62  15.75 114.61  100.97 101.81  7.25  8.53>127.72  105.45 124.09  8.78**  98.90  ** S i g n i f i c a n t l y d i f f e r e n t a t 1% l e v e l compared w i t h untreated p l a n t s . a  Value represents an average o f 32 p l a n t s . Four r e p l i c a t e s o f 8 plants f o r each treatment. -  Variance r a t i o (F) o f Table 4. Tops F. Wt.  Pods F.Wt.  Pods No.  Treat  0.74  2.51  5.25**  Block  0.13  0.39  0.56'  Duncan's t e s t o f Table 4. Pods No. Treat  Minerals  Means  8.78  NCL 0.06 8.53  2,4-D 0.06 7.25  Control 6.88  98.22  40. Table 5. E f f e c t o f NCL, 2,4-D and minerals on enzyme a c t i v i t y C o n t r o l M i n e r a l s 2,4-D 0.06 Catalase ( u l O2/3 min) ]Lcfl\^€ s  2 5 7 14  days days days days  153.15 142.43 130.45 89.25  a  NCL 0.06  T/C (%) M i n e r a l s 2.4-D  150.47*,. 152.4& 134.45* 98.25 99.53 113.22 183.11*178.38** 79.49 128.60 121.63 185.72 157.95** 93.24 142.37 100.37** 90.20 136.90**112.45 101.06  NCL 0.06 87.79 125.24 121.08 153.38  Phosphatase (ug phenol/hr) Leaves 2 5 7 14  days days days days  392.83 405.83 421.00 354.17  a  395.33 385.83 430.67 351.33  389.00 382.67 331.33*367.17 387.50 408.33 324.33 334.33  100.64 95.07 102.20 99.20  99.02 81.64 94.05 91.58  97.41 90.46 99.11 94.40  151.39 122.22 -  114.69 86.67 -  109.69 84.69  Phosphorvlase (ug P/ml/hr) Leaves 2 days 5 days 7 days 14 days Note:  a  36.33 67.50 Trace -  55.00** 41.67 82.50** 58.50 Trace Trace -  a  39.67 57.17 Trace -  Average o f s i x t e s t s , ten plants f o r each t e s t . Variance r a t i o (F) o f Table 5. Leaves (days a f t e r treatment)  Catalase Treat Block  2  4.06 * 1.32  5  7  17.72** 0.98  1 4  17.22 ** 42.68 ** 1.88 0.20  Phosphatase Treat  1.02  4.97*  2.07  0.89  Block  2.58  1.70  0.61  0.54  Phosphorvlase Treat Block  12.23** 1.86  19.61** 0.44  40a Duncan's t e s t f o r Table 5, Catalase a c t i v i t y , leaves 2 davs Treat  Control  2,4-D 0.06  Means  153.15  152.43  Mineral  NCL 0.06  150.47  134.45  Catalase a c t i v i t y , leaves 5 davs Treat  2,4-D 0.06  Means  183.17  NCL 0.06  Control  178.38  142.43  Minerals 113.22  Catalase a c t i v i t y , leaves 7 days Treat  2,4-D 0.06  Means  185.72  NCL 0.06  Control  157.95  130.45  Minerals 121.63  Catalase a c t i v i t y , leaves 14 davs Treat  NCL 0.06  Means  136.90  Minerals  2,4-D 0.06 C o n t r o l 90.20  100.37  89.25  Phosphatase a c t i v i t y , leaves 5 davs Treat M  e  a  n  s  Control 40.583  Minerals  NCL 0.06  2,4-D 0.06  367.17  331.33  385.83  Phosphorylase a c t i v i t y , leaves 2 days Treat  Minerals  Means  55.00  2,4-D 0.06  NCL 0.06  41.67  39.67  Control 36.33  Phosphorvlase a c t i v i t y , leaves 5 days Treat  Minerals  Means  82.50  Control * 67.50  2,4-D 0.06 58.50  NCL 0.06 57.17  41.  tion i n response might be v a r i e t a l or due to growth conditions. By comparing the control plants, the results indicate that beans grown i n the green house (Experiment 1) showed a higher rate of growth than those i n the growth room.  The s i g n i f i c a n t differences  among blocks obtained i n Experiment 1 but not i n Experiment 2 might be caused by growth conditions and sampling as well as randomization. B.  Enzyme a c t i v i t y 1.  Catalase a c t i v i t y The catalase a c t i v i t y showed a steady decline with  days i n untreated leaves, but not i n treated leaves.  NCL  0.06  caused a s i g n i f i c a n t i n h i b i t i o n i n catalase a c t i v i t y i n the seond day a f t e r treatment and a s i g n i f i c a n t stimulation i n a c t i v i t y on the f i f t h , seventh, and 14th day a f t e r treatment. 2,4-D  Application of  0.06 induced a s i g n i f i c a n t stimulation i n a c t i v i t y on the  5th and 7th day a f t e r treatment.  Minerals caused a s i g n i f i c a n t  reduction i n a c t i v i t y on the 5th day, and a s i g n i f i c a n t acceleration i n a c t i v i t y on the 14th day a f t e r treatment. 2.  Phosphatase a c t i v i t y A l l the treatments except 2,4-D  0.06  f a i l e d to  y i e l d a s i g n i f i c a n t e f f e c t on phosphatase a c t i v i t y i n leaves. 2,4-D  0.06 caused a s i g n i f i c a n t i n h i b i t i o n i n a c t i v i t y on the 5th  day after treatment.  In general, 2,4-D  0.06 and NCL 0.06  inhibited  42. the enzyme a c t i v i t y although there was no s i g n i f i c a n c e .  Minerals  produced a n o n s i g n i f i c a n t s t i m u l a t i o n o r i n h i b i t i o n o f a c t i v i t y at v a r i o u s times a f t e r treatment. 3.  Phosphorylase  activity  The presence o f a h i g h c o n c e n t r a t i o n of phosphate i n bean l e a f homogenate made determination of phosphorylase activity difficult.  I t caused a h i g h blank reading which  the accuracy of r e s u l t s .  reduced  The r e s u l t s , represented i n Table 5,  show that minerals s i g n i f i c a n t l y s t i m u l a t e d the a c t i v i t y on both the second and f i f t h day a f t e r treatment, whereas NCL 0.06, 2,4-D  and  0;06 n o n s i g n i f i c a n t l y increased the a c t i v i t y on the second  day, and decreased i t on the f i f t h day a f t e r treatment.  Experiment 3.  E f f e c t of NCL and stage of a p p l i c a t i o n o f  treatment on growth and y i e l d of beans I t was found by Wedding e t a l . (65) and Wort (79) that bean plants t r e a t e d w i t h 2,4-D  when they were 14 days o l d gave  b e t t e r growth and y i e l d response than those of untreated plants and plants t r e a t e d a t o t h e r stages.  Since Experiments 1 and 2  f a i l e d to show s i g n i f i c a n t growth and y i e l d e f f e c t s , i t was  de-  cided to study the e f f e c t of d i f f e r e n t concentration-age combinations.  A f a c t o r i a l design was used i n a n a l y z i n g the r e s u l t s .  Treatment:  c o n t r o l , NCL 0.12, NCL 0.06 and NCL  0.03  a p p l i e d to the p l a n t s when 14, 16, 23, and 30 days o l d .  43. Measurements:  growth and y i e l d - f r e s h weight o f tops  and pods, and number o f pods. Results The r e s u l t s as tabulated i n Tables 6 and 7, and Figure 7, are summarized as f o l l o w s . (1)  The s i g n i f i c a n c e o f i n t e r a c t i o n i n d i c a t e d that the  c o n c e n t r a t i o n o f NCL and the stage o f treatment a p p l i c a t i o n were not independent.  The e f f e c t o f NCL c o n c e n t r a t i o n a t the four  stages was s i g n i f i c a n t l y d i f f e r e n t , but the a p p l i c a t i o n o f three c o n c e n t r a t i o n l e v e l s o f NCL a t each stage f a i l e d to produce a significant effect. (2)  G e n e r a l l y , a p p l i c a t i o n o f NCL when p l a n t s were 14 o r  16 days o l d gave the best growth and y i e l d response i n comparison w i t h the stage means. (3)  NCL 0.03 treatment y i e l d e d a n o n s i g n i f i c a n t decrease  i n growth and y i e l d at a l l stages, w h i l e NCL 0.12 and NCL 0.06 increased the f r e s h weight o f tops and number of pods a t 14 and 16 days.  The e f f e c t s o f the v a r i o u s 2,4-D concentrations a t the  f o u r stages were not s i g n i f i c a n t . (4)  A l l the treatments caused a n o n s i g n i f i c a n t decrease  i n f r e s h weight o f pods.  NCL 0.06 was the o n l y formulation which  induced a n o n s i g n i f i c a n t increase i n f r e s h weight o f pods a t 14 days.  f  Pig. 7. Effect of NCL and the stage of application of treatment on y i e l d and growth of beans. Experiment 3. J 120 Fresh wt. 120 r I \ Fresh wt, of Pods / y of Tops  110  110 r  100  100 T/C  T/C  90  80  90  . 14 16  23  30  80  14 16  No. of Pods  NCL 0.03  r  NCL 0.06 NCL 0.12  T/C  14 16  30  Day after treatment  Day after "treatment 150  23  23  30  ;Day after it re at ment  45. Table 6. E f f e c t o f NCL and the stage o f a p p l i c a t i o n o f treatment on growth o f beans. Experiment 3, Feb. 11 - Apr. 17,1964 Tops Fresh Wt. T/C U) NCL 0.06  NCL 0.12  Stage Mean  NCL 0.03  NCL 0.06  NCL 0.12  20.6  24.6  23.8  22.6  97.17  116.04  112.26  Control NCL 0.03  Stage of Treat, (days) 14  21.2  16  21.2  21.1  22.9  26.0  22.8  99.53  108.02  122.69  23  21.2  19.2  18.4  20.6  19.9  90.56  86.79  97.17  30  21.2  19.9  21.4  22.4  21.2  93.87  100.94  105.66  21.2  20.2  21.8  23.2  a  NCL Mean • Note:  a  Average o f 48 p l a n t s . S i x r e p l i c a t e s o f 8 plants f o r each. Variance r a t i o s :  i ) Stage (4.54*), i i ) Cone. (1.73), i i i ) Stage X Cone. (9.97**).  46. Table 7. E f f e c t o f NCL and the stage o f a p p l i c a t i o n o f treatment on y i e l d o f beans. Experiment 3, Feb. 11 - Apr. 17, 1964  Stage of treat. (days)  Control NCL 0.03  NCL 0.06  Pods f r e s h wt. (gm/plant) T/C (%) NCL Stage NCL NCL 0.12 Mean 0.03 0.06  NCL 0.12  14  18.5  17.6  21.0  17.7  18.7  95.14  113.51  95.67  16  18.5  17.6  17.0  16.6  17.4  95.14  91.89  89.73  23  18.5  16.9  16.9  16.2  17.1  91.35  91.35  87.57  30  18.5  14.8  16.7  17.8  17.0  80.00  90.27  96.21  NCL Mean  18.5  16.7  17.9  17.1  No. Pods per plant Stage of treat. (days) 14  C o n t r o l NCL 0.03  NCL 0.06  NCL 0.12  Stage Mean  T/C (%) NCL NCL 0.06 0.03  NCL 0.12  6.7  7.0  8.3  8.0  7.5  105.09  123.50  120.21  16  6.7  5.9  8.1  9.6  7.6  87.87  121.56  143.71  23 30 NCL Mean  6.7 6.7  5.8 5.0  5.8 5.7  5.9  6.1  87.13  87.13  88.62  6.8  6.0  74.85  85.78  101.50  6.7  5.9  7.0  7.6  Note: Variance r a t i o s f o r Pods f r e s h wt.: i ) Stage (1.76), i i ) Cone. (1.89), i i i ) Stage X Cone. 2.01*). Variance r a t i o s for Pods No.: i ) Stage (3.87*), i i ) Cone. (2.51), i i i ) Stage X Cone. (3.96*). Value represents an average o f 48 p l a n t s . of p 8 plants f o r each treatment.  Six replicates  4.7. The r e s u l t s o f t h i s experiment provide a d d i t i o n a l evidence that s t i m u l a t o r y l e v e l s o f NCL a p p l i e d a t an e a r l y stage may i n f l u e n c e the growth o f bean p l a n t s .  I n a d d i t i o n , the most  e f f e c t i v e time o f treatment a p p l i c a t i o n was found to be, as r e ported by Wort (79), the t i g h t t r i f o l i a t e bud stage (about two weeks o l d ) .  Experiment 4.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH^*2,4-D,  NCL-NH^*2,4-D on growth and y i e l d o f beans I n a d d i t i o n to the formulations used i n Experiment 1, NH^*2,4-D 0.12 w i t h and without minerals was added i n t h i s experiment i n order to compare the e f f e c t o f the e s t e r and s a l t o f 2,4-D on growth and y i e l d .  Furthermore, i t was d e s i r e a b l e to  d i s c o v e r i f the preceding experimental r e s u l t s could be repeated. Measurement o f dry weight was added to t h i s experiment to determine the water content o r dry matter production. Treatment:  c o n t r o l , m i n e r a l s , 2,4-D 0.12, NCL 0.12,  2,4-D 0.12, NH '2,4-D 0.12, and NCL-NH «2,4©D 0.12. 4  Measurements:  4  growth - f r e s h , dry, and percentage dry  weight o f tops; y i e l d - f r e s h , dry, and percentage d r y weight o f pods, and number o f pods.  48. PigVig. Effect of NCL, 2,4-D, minerals, NIL,.2,4-D and NCL-NH4«2.4-D on growth of beans. Experiments,  | J |\\^  Presh weight of TopB Dry weight of TopB  110  /  100 T/C 90  80  7) / / / / /  7 ]  / / / / / / /  '/ A  Minerals  I  71 V  2  2 2 4 - D . i : ;oCNCL.'3 NH^'2,4-D 0.12 0.12 0.12 ?  r  'A y / / / / /  i  NCL-NH4.2,4-D  0.12  Pig. 9. E f f e c t of NCL, 2,4-D, i .minerals, NH4•2,4-D and . NCL-NH4•2.4-D on y i e l d of beans. Experiment 4, Fresh weight of Pods I W N  Dry weight of Pods I-  _  120  110-  100  / / / / / /  2  Minerals  / / / / / / / / 2,4-D  / /  v /  / / / / / /  NCL 0.12  V / / / /  z  / / / / / /  NH4*2,4-D NCL-NH4•2,4-D 0.12 0.12  49 Table 3.  E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH 2,4-D and NCL-NH4 2,4-D on growth o f beans. Experiment 4, May 25 J u l y 18, 1964. 4  C o n t r o l M i n e r a l s 2,4-D 0.12  NCL NH -2,4-D NCL-NH4-2,4-D 0.12 0.12 0.12 4  Tops F.Wt. (gm/plant) T/C (%)  12.91  12.96  13.15  12.73  14.06  99.92 102.87  98.61  108.91  2.23  2.04  2.12  98.13  97.41 102.20  93.82  97.41  16.67  16.56  17.16  16.22  15.21  98.92weight 98.29 101.96 Oven-dry  99.95  104.13  a  2.18  100.39  2.18  2.13  12.90  Tops D.Wt. (gm/plant) T/C (%)  2.12  Percentage D.Wt.  16.85  T/C (%) D.Wt. = Note:  *Va lue represents an average o f 32 p l a n t s . Four r e p l i c a t e s o f 8 plants f o r each treatment. Variance r a t i o (F) o f Table 8. F.Wt.  D. Wt.  Percentage D. Wt,  Treat  0.19  0.18  1.74  Block  0.48  2.05  1.56  T X B  4.58**  2.23*  0.99  50 Table 9. E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH 2,4-D, and NCL-NH4 2,4-D on y i e l d o f beans. Experiment 4. May 25 - J u l y 18, 1964. 4  C o n t r o l M i n e r a l s 2,4-D NCL 0.12 0.12 Pods F.Wt. (gm/plant)  20.53  T/C (%)  20.74 101.00  NH 2,4-D 0.12  21.22 220.93  4  NCL-NH 2,4-D 0.12 4  20.82  21.37  103.36 101.96 101.41  104.13  Pods D.Wt. (gm/plant)  4.02  T/C (%) D.Wt.  19.65  T/C (%)  4.67  4.49  4.46  4.44  4.72  116.17  111.74 110.89 110.42  117.42  22.52  21.49 21.60 21.33  22.16  114.63  109.36 109.89 108.55  112.77  Pods No. per plant  9.88  T/C (%) Note:  10.25  10.92 12.56 10.41  11.13  103.79  101.05 127.18 105.41  103.79  D. Wt. = Oven-dry weight.  Variance r a t i o (F) o f Table 9 F. Wt.  D. Wt.  Percentage D. Wt.  No.  Treat  0.13  0.47  0.33  1.69  Block  0.77  3.43*  1.35  1.05  T X B  3.05*  1.77*  3.00*  0.19  51. Results As shown i n Tables 8 and 9, and Figures 8 and 9, a l l treatments applied i n this experiment f a i l e d to produce a s i g n i f i c a n t e f f e c t on growth and y i e l d of beans.  Generally, a l l  treatments caused a s l i g h t decrease i n growth except that NCL 0.12 induced a s l i g h t increase i n top growth 2.3% (average o f 5 treatments means).  A l l treatments caused an increase i n fresh  weight, dry weight, percentage dry weight and number o f pods. This increase was approximately 23.377« i n fresh weight, 13.217o i n dry weight, 9.047. i n percentage dry weight, and 8.327. i n number of pods.  NCL 0.12 treatment increased the number of pods 27.18%  over the controls. According to the present r e s u l t s , the ester and s a l t o f 2,4-D produced a s i m i l a r e f f e c t on growth and y i e l d .  In the case  of the e f f e c t of treatments, a l l treatments f a i l e d to induce a s i g n i f i c a n t growth and y i e l d e f f e c t except the number o f pods. No s i g n i f i c a n t difference among the treatments might be explained as the portions o f v a r i a t i o n present i n the i n t e r a c t i o n of treatment and block.  In other words, a portion of the v a r i a -  t i o n was removed by the j o i n t e f f e c t s of treatments and blocks. The differences i n responses to the various treatments were not o f the same order o f magnitude from block to block.  52t.  In general, the natural v a r i a t i o n would be presented i n each plant.  This inherent v a r i a t i o n i s beyond a r t i f i c i a l control  even through the a p p l i c a t i o n of sampling methods. Comparing the untreated plants i n Experiments 1 to 4, d i f f e r e n t results for top growth and pod y i e l d were obtained i n each experiment.  These results showed an existence of natural  v a r i a t i o n among plants, and the d i f f e r e n t treatment effects were assumed to be from this v a r i a t i o n .  Various growth conditions  caused by the lack of uniformity i n the d i s t r i b u t i o n of f e r t i l i z e r i n s o i l might be another factor which affected treatment e f f e c t excluding inherent v a r i a t i o n .  Experiment 5.  E f f e c t o f NCL, 2,4-D, minerals, NH4*2,4-D and  NCL-NH^*2,4-D on growth and y i e l d of beans According to the results obtained from the preceding experiments, growth condition was considered to have affected the r e s u l t s and, therefore, made i t impossible to reproduce the experiment. The use o f only one harvest as a basis f o r measuring the growth and y i e l d assumes that the bean plants are uniform and would become mature at the same time, and therefore, the e f f e c t on growth and y i e l d i s due to treatment only.  53. Obviously, the influence of bean maturity i s one of the effects of 2,4-D on bean plants.  Wedding et a l . (65) reported  that higher concentration of 2,4-D tended to delay both i n i t i a t i o n and maturity of the f r u i t . ted  floral  M i l l e r e t a l . (38) repor-  that 0.5 ppm 2,4-D hastened harvest readiness 2 to 4 days  over the mean of the controls. s i g n i f i c a n t e f f e c t on maturity. delayed harvest readiness.  One ppm and 5 ppm 2,4-D had no Ten ppm and 100 ppm 2,4-D  Because pods attained marketable  size  at d i f f e r e n t stages, i t i s d i f f i c u l t to compare the differences i n t o t a l y i e l d e f f e c t by means o f a one harvest method. In the present experiment, Top Crop bush beans were grown i n v e r m i c u l i t e - f i l l e d p l a s t i c pots and i r r i g a t e d with a modified Shave's three-salt nutrient solution.  I t was assumed that growth  condition would be uniform i n each p l o t , and the difference i n e f f e c t would be caused by treatment. In addition to the dusts used i n Experiment 4, three levels of  2,4-D and NCL (0.015, 0.03, and 0.06%) were applied to the  plants.  Green pods were collected once a week as they reached  marketable  s i z e (about 4" i n length).  The t o t a l of three harvests  and grand t o t a l (G-total) including those of less than 4" were also calculated. Treatment:  control, minerals, NCL 0.015, NCL 0.03, NCL  0.06,  NCL 0.12, 2,4-D 0.15, 2,4-D 0.03, 2,4-D 0.06, 2,4-D  0.12,  NH *2,4-D 0.12, and NCL-NH4.2,4-D 0.12. 4  54. Measurements:  growth - f r e s h , d r y , and percentage dry  weight o f tops; y i e l d - f r e s h , d r y , percentage d r y weight and number o f pods i n each harvest (1-3 h a r v e s t ) , i n t o t a l o f three harvest (1-3 t o t a l ) , and i n grand total (G-total). Results (1)  With the exception o f NCL 0.06, NCL 0.12, and NCL-  NH *2,4-D 0.12, a l l the treatments caused a n o n s i g n i f i c a n t i n 4  crease i n f r e s h weight and dry weight a t the f i r s t harvest. Only the 2,4-D 0.0.5 y i e l d e d a s i g n i f i c a n t e f f e c t . (2)  A l l the treatments f a i l e d to y i e l d a s i g n i f i c a n t  e f f e c t on f r e s h weight and dry weight o f pods a t 2nd and 3rd harvest as w e l l as 1-3 T o t a l and G - t o t a l .  However, a l l the  treatments produced an increase i n dry weight o f pods a t 1-3 T o t a l and G - t o t a l .  P a r t i c u l a r l y , 2,4-D 0.12, 2,4-D 0.06, m i n e r a l s ,  and NCL 0.12 increased the 1-3 T o t a l dry weight o f pods by 53.77%, 38.51%, 37.90%, and 37.75%; and the G - t o t a l by 59.02%, 38.51%, 24.02%, and 64.22% o f the c o n t r o l , r e s p e c t i v e l y . (3)  I n the case o f percentage dry weight o f pods, the  a p p l i c a t i o n o f treatments r e s u l t e d i n a s t i m u l a t o r y e f f e c t which was not s i g n i f i c a n t . NCL 0.12, 2,4-D 0.06, and 2,4-D 0.12 i n creased the percentage dry weight o f pods f o r l - 3 T o t a l by 56.76%, 37.67%, and 42.46% over the c o n t r o l s ; and f o r G - t o t a l by 69.33%, 53.067<>, and 44.307o over the c o n t r o l s , r e s p e c t i v e l y .  3  h  F i g . 10. E f f e c t of NCL, 2,4-D, minerals, NH,*2,4-D, and NCL-NH^•2,4-D on growth of beans. Experiment 5,  NCL-NH.«2,4-D ///////// • 0.12/ Nh\.2,4-D 0.12  TV  /\  / / /  /I  1  4  NCL 0.12  /V  y / / / / / r r m  I i  NCL 0.06. NCL  0.03  v  /.////////y —'  ' ' ' ' of Tops 1  Fresh weight of Tops  / \ i  NCL 0.015  / / y / / /  2,4-D 0.12  V/.y  / /  {  /  /~r~r\  / T T T \ \  2,4-D 0.06  v //•///////  2,4-D 0.03  //.///\  2,4-D 0.015  //••////////]//////// /~7~7~h  Minerals  V / / /( / /\  y //  /m  J  r  r  I  80*  90  •I  100  110  T/c  m  120  130  S6*  F i g i 11. E f f e c t of NCL, 2,4-D, minerals, NH »2,4-D and NCL-NH^«2,4-D on y i e l d of beani. Experiment 5.  1 1  NCL-NHi• 2,4-D *0.12  /,/ n  ///y\ ~TTA  NH.'2,4-D 0.12 4  NCL  0,12  NCL  0.06  NCL  0.03  NCL 0.015  / / / /y\  weight of Pods (1-3 total)  U r v  j Fresh weight of Pods (1-3 total) / / / / / J  120 T/C  140 (*)  160  52. Table 10. E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH *2,4-D, and 4  NCL-NH *2,4-D on growth o f beans. Experiment 5. 4  C o n t r o l Minerals 2,4-D 0.015 Tops F.W. (gm/plant) T/C(%) Tops D.W. (gm/plant) T/C (%) % D.W. (T/C(%)  37.42  a  32.83 87.73  39.30 105.02  2,4-D 2,4-D 2,4-D 0.03 0.06 0.12 30.98 82.79  35.87 95.87  NCL NCL 0.015 0.03  33.15 31.47 31.09 88.58 84.1183.07  6.02  5.65 7.36 5.32 6.63 5.97 5.89 6.27 93.77 122.28 89.95 110.13 99.11 97.76104.07 16.05 17.13 18.67 * 17.57 18.29 * 17.97 18.81*19.97* 106.69 116.30 109.47 113.96 111.98 117.15124.39 NCL 0.06  NCL . 0.12  NH «2,4-D 0.12  Tops F.W. (gm/plant)  31.09  35.47  37.45  36.50  T/cm  94.09  94.78  100.08  97.53  Tops D.W. (gm/plant) T/C(%)  5.97 99.20  5.89 97.84  6.25 103.82  6.26 103.87  16.80 16.54 104.68 103.02  16.77 104.50  17.24 107.42  % D.W. (T/C(%) Note:  4  NCL-NH « 2.4-D 0.12 4  a - average o f s i x plants Variance r a t i o (F) o f Table 10 F. Wt.  D. Wt.  % D. Wt.  Treat  1.13  0.99  3.43*  Block  2.66*  4.96*  3.19*  Treat Means  Duncan's t e s t f o r Table 10 NCL 0.03 NCL 0.015 2,4-D 0.015 2,4-D 0.06 2,4-D 0.12 2,4-D 0.13 19.97 18.81 18.67 18.29 17.97 17.57  Treat Means  Minerals 17.13  NCL 0.06 NH »2,4-D 16.80 16.77 4  NCL 0.12 16.54  Control 16.05  Table 11. E f f e c t o f NCL, 2,4-D, m i n e r a l s , NH *2,4-D and 4  NCL-NH *2,4-D on y i e l d o f beans. 4  Experiment 5, Aug. 28-Qct.  22nd, 1964. Control  2,4-D .2,4-D 0.03 0.06  Minerals  2,4-D 0.015  15.98 14.64 13.03 43.65 115.09 46.82 103.21  26.25** 14.97 5.86 47.07 124.12 55.98 123.38  1.38 1.79 1.71 4.88 137.90 5.29 124.02  6.76 9.97 8.13 9.38 120.18 9.40  1.83 2.17 1.67 5.67  2,4-D 0.12  NCL 0.015  NCL 0.03  NCL 0.06  .NCL 0.12  NH ' 2,4-D 0.12 4  NCL-NH » 2.4-D 0.12 4  Pods F.W.^am/plant) 1 harvest 2 harvest 3 harvest 1-3 T o t a l T/C(%) G-To t a l T/C(%)  10.51 15.48 11.94 37.93 45.37  a  4.17 15.67 11.95 31.78 83.79 43.93 96.83  15.39 15.56 7.06 38.01 100.22 49.45 108.99  9.51 14.28 7.74 31.52 83.12 44.70 98.52  2.14* 1.39 1.69 1.10 0.94 1.19 1.73 1.82 0.70 1.92 1.48 2.53 3.78 4.50 4.90 5.44 106.69 127.03 138.51 153.77 4.77 5.30 5.91 6.79 111.72 124.10 138.59 159.02  1.71 1.46 0.83* 0.46 1.16 1.06 2.26 2.07 0.73 1.32 0.56 2.24 3.60 3.85 3.65 4.87 101.70 108.71 103.32 137.75 5.70 4.58 4.72 7.01 133.67 107.42 110.54 164.22  1.21 1.76 1.22 4.19 118.27 5.51 129.18  0.79 1.59 1.29 3.67 103.62 5.49 128.55  8.57 12.23 13.58 11.27 120.18 11.36 120.89  8.13 7.72 12.85 9.63 102.68 8.91 94.81  8.30 8.05 10.59 9.21 14.71*10.75 9.72-10.23 103.58 109.08 11.62 10.75 123.72 114.42  12.95 13.80 18.53 14.71 156.76 15.91 169.33  7.82 11.35 11.60 10.96 116.89 11.12 118.32  8.19 11.16 13.71 11.61 123.77 12.31 130.98  2.83 2.00 2.00 6.83 120.59 8.67 110.64  4.33* 2.67 4.00* 2.67 1.17 1.50 2.00 1.67 1.00 1.67 1.83 1.33 6.50 5.83 6.50 7.00 114.71 102.94 123.53 114.71 10.17 8.67 9.33 11.71 129.29 110.64 119.15 142.55  4.17* 3.50 2.00 * 1.33 1.50 1.50 3.00 2.33 1.50 1.50 0.50 1.67 6.50 6.50 5.50 5.33 114.71 97.06 94.12 94.12 11.17 10.50 8.33 9.17 134.08 106.38 117.02 121.28  2.33 2.00 1.00 5.33 94.12 9.50 114.89  1.83 2.17 1.17 5.17 91.18 9.00 114.89  16.31 20.43 13.63 10.22 13.99 13.86 7.85 13.95 10.62 37.15 42.27 41.44 97.25 111:46 109.27 43.62 46;02 50.14 96.14 101.43 110.52  20.55 10.40 5.33 36.27 95.64 49.30 108.66  18.11 9.41 10.02 37.27 98.28 42.16 92.93  10.38^ 21.19 4.47 36.04 95.02 44.62 98.36  Pods D.W.(em/plant) 1 harvest 2 harvest 3 harvest 1-3 T o t a l T/C(%) G- To t a l T/C(%)  0.86 1.52 1.16 3.54 4.27  Pods % D.W. 1 harvest 2 harvest 3 harvest 1-3 T o t a l T/C ( 7 o ) G-To t a l T/C ( 7 . ) Pods No. per p l a n t 1 harvest 2 harvest 3 harvest 1-3 T o t a l T/C(7o)  G-Total T/C ( 7 . )  7.83  7.09 8.23 8.02 11.67 12.54 13.16 14.34 18.77 18.64 12.27 12.91* 13.36 130.78 137.67 142.46 12.47 14.38 13.56 132.78 153.06 144.30  7.75 10.66 12.52 10.58 109.87 10.58 112.55  x  5ft. Variance Ratio (F) o f Table 11 Fresh Wt. 1st Wk. 3.51* 1.69  Treat Block  2nd Wk.  3rd Wk. 1-3 Wk.  1.22 1.29  Total  1.26 2.20  1.47 0.85  1.18 0.45  1.58 2.42  1.55 1.36  1.19 1.43  Dry Wt. 3.01* 1.66  Treat Block  1.55 0.45  Percentage Dry Wt. Treat Block  0.38 1.17  1.98* 1.79  1.29 1.39  2.33* 1.20  1.85 1.20  1.09 2.96  1.02 2.24  1.02 1.24  Pods No. 3.33* 1.74  Treat Block  1.96 0.36  Duncan's t e s t f o r Table 11 Fresh wt. o f pods. 1st week Treat Means Treat Means  2,4-D 0.015 NCL 0.015 2,4-D 0.06 NCL 0.03 2,4-D 0.03 M i n e r a l s 26.25  20.55  NH »2,4-D 2,4-D 4  15.39  20.43  Control  13.63  18.11  NCL 0.06  16.51  16.31  15.98  NCL-NH4.2,4-D NCL 0.12  10.38  9.51  4.17  Dry wt. o f pods. 1st week Treat  2,4-D 0.015  NCL 0.015  2,4-D 0.06  NCL 0.03  2,4-D 0.03  Means  2.14  1.71  1.69  1.46  1.39  Treat  2,4-D 0.12  Means  1.10  Control 0.86  NCL 0.06 0.83  M i n e r a l s NH «2,4-D 4  1.38  NCL-NH *2,4-D 4  0.79  1.21 NCL 0.12 0.46  60. Percentage dry wt. o f pods. 2 week Treat Means  NCL 0.12  2,4-D 0.12  2,4-D 2,4-D NH «2,4-D NCLNCL 0.06 M i n e r a l s 0.03 NH '2,4-D 0.06 4  4  13.80 13.16  12.54  Treat  NCL 0.06  Control  Means  10.59  9.97  12.23  11.64  NCL 0.03  11.35  11.16  10.66  2,4-D 0.015  9.21  7.72  Percentage dry wt. o f pods. 1 - 3 week Treat Means Treat Means  NCL 2,4-D 0.12 0.12  2,4-D 0.06  2,4-D NCL-NH * 0.03 2,4-D  14.71 13.36 12.91 12.27 NCL 0.015 9.72  Control  M i n e r a l s NH « NCL 2,4-D 0.06  4  11.61  2,4-D 0.015  9.63  4  11.27  10.96 10.58 10.23  Control  9.63  9.38  Pods No.. 1st week Treat  2,4-D 0.015  NCL 2,4-D 0.015 0.06  Means  4.33  4.17 4.00  Treat  NH « 2,4-d  Means  2.33  4  NCL 0.06 2.00  NCL 0.03 3.50  NCL-NH * 2,4-D 4  1.83  Minerals 2.83  Control 1.83  2,4-D 0.03  2,4-D 0.12  2.67  2.67  NCL 0.12 1.33  NCL 0.03  61. (4)  A t the f i r s t h a r v e s t , treatment by NCL 0.015 and  2,4-D 0.015 r e s u l t e d i n a s i g n i f i c a n t increase i n number o f pods, whereas the remaining treatments produced a n o n s i g n i f i c a n t increase. (5)  A p p l i c a t i o n o f 2,4-D 0.015 s i g n i f i c a n t l y increased the  f r e s h weight, d r y weight, and number o f pods a t the f i r s t harvest as w e l l as the percentage dry weight o f tops. A t the second harvest and 1-3 T o t a l , a n o n s i g n i f i c a n t decrease i n percentage dry  weight o f pods caused by 2,4-D 0.015 was found.  According to  the  r e s u l t s g i v e n i n Table 11, the s t i m u l a t o r y e f f e c t o f e s t e r  and a c i d o f 2,4-D on f r e s h weight, dry weight, and number o f pods was i n v e r s e l y p r o p o r t i o n a l to the concentration. (6)  Except percentage dry weight o f tops, i t was found  that a l l the treatments f a i l e d to produce a s i g n i f i c a n t e f f e c t on growth.  Treatments i n v o l v i n g 2,4-D 0.015, 2,4-D 0.06, NCL 0.015  and NCL 0.03 s i g n i f i c a n t l y increased the percentage dry weight of  tops by 16.30%, 13.96%, 17.15% and 24.39% o f the c o n t r o l s  respectively. (7)  I n comparison with Experiment 4, the beans planted  i n v e r m i c u l i t e had a higher r a t e o f growth than those planted i n soil.  The f r e s h weight o f pods o f vermiculite-grown beans was  h i g h e r than those o f soil-grown beans, but the t o t a l number o f pods o f vermiculite-grown beans was the same as that o f s o i l grown beans.  The f r e s h weight o f pods removed at the f i r s t har-  v e s t was approximately the same as that o f t o t a l f r e s h weight o f  62. pods i n Experiment 4.  The difference i n growth e f f e c t might  have come from various growth conditions and three-harvest method. I t i s known that water i s an important factor which governs the growth of plants, p a r t i c u l a r l y f o r f r u i t production. The use of vermiculite would tend to hold water more e f f e c t i v e l y i n the promotion of both top growth and pod y i e l d .  Experiment 6.  E f f e c t of NCL on photosynthesis, r e s p i r a t i o n  growth and y i e l d of beans The main purposes of this experiment were (1) to study the e f f e c t of NCL 0.12 on growth and y i e l d of Top Crop bush beans, and (2) to study the e f f e c t o f NCL 0.06 and NCL 0.12 on photosynthesis and r e s p i r a t i o n .  The growth and treatment procedures  are the same as those of Experiment 5.  I t was found from  Experiment 5 that NCL 0.12 stimulated the y i e l d of beans i n the case of dry weight, and percentage dry weight. The rate of photosynthesis and r e s p i r a t i o n , growth, and y i e l d were determined at weekly i n t e r v a l s f o r s i x consecutive weeks s t a r t i n g from the f i r s t week a f t e r treatment. For seed y i e l d determination, r i p e pods were harvested when the plants were 12 weeks o l d . Figures 12 and 13.  The results are given i n Tables 12 to 15 and  0* F i g . 12. E f f e c t of NCL on photosynthesis and respiration of beans. Experiment 6. Pho to synthe s i s 120  r-  NCL 0.12 NCL 0.06 100  W w 80  60  _L  2  6  3 4 5 Week after treatment Respiration  120 U  \ V100  V  w  \  / S  80  r  A  4 60  2  3 4 5 Week after treatment  6  6* P i g , 13. E f f e c t o f NCL 0.12 on growth o f beans.  }  Fresh weight o f Tops  140 Dry weight o f Tops  120 T/C  100  80 2  3  4  5  Week a f t e r treatment  130  F i g . 14. E f f e c t o f NCL 0.12 on y i e l d o f beans  r  F r e s h weight of Pods Dry weight of Pods  110 100 T/C 90  1  L  70  V  50  2  3 4 Week a f t e r treatment  5  6  65. Table 12. E f f e c t o f NCL on photosynthesis and r e s p i r a t i o n o f beans. Experiment 6.  Week a f t e r treatment 1 2 3 4 5 6  Photosynthesis ( u l 0 /gm F.W./hr) 2  •  Control 1207.38 1789.49 1722.58 1350.32 1569.51 1514.90  a  NCL 0.12  NCL 0.06  1338.04 1385.96* 1211.39* 1034.12* 1012.93** 1094.02**  1235.75 1342.02* 1501.92 1105.96* 1346.80** 1466.54  T/C (%) NCL 0.12 NCL 0.06 110.82 77.45 70.32 76.58 64.53 72.21  102.35 74.99 87.19 81.90 85.81 96.81  110.80 77.19 70.32 64.22 76.49 101.96  114.49 75.99 72.75 65.83 72.44 107.13  Respiration ( u l 02 /gm F.W./hr) 1 2 3 4 5 6 Note:  232.48 364.80 280.47 237.99 210.77 259.44  b  257.58* 281.58** 204.90* 152.85* 161.22** 264.53  266.17* 277.22** 204.03* 156.69* 152.70** 277.96  a ^ Average o f four t e s t s . k  e  Variance r a t i o of Table 12.  Treat Block  1  2  1.18 0.39  5.57* 6.48*  1 Treat Means  1.56 0.19  Photosynthesis (week a f t e r treatment) 3 4 5  2 14.17** 0.81  7.12* 1.00  5.76* 1.05  48.06** 8.20*  Respiration (week a f t e r treatment) 3 4 5 8.43* 0.92  4.38* 1.20  34.57** 31.55**  6 15.41** 0.67  6 0.15 9.76*  66. Duncan's t e s t f o r T a b l e 13 Photosynthesis. Treat  Control  Means  17 89.49  Photosynthesis, Treat  2 weeks a f t e r  Means  1722.58  Photosynthesis.  Control  Means  1350.32  NCL 0.06  Means  1569.51  1514.90  Respiration.  Control  Means  364.80  Respiration.  Control  Means  280.47  Respiration. Treat Means  NCL 0.06  NCL 0.12 1034.14 treatment  1012.93 treatment NCL 0.12  1466.54  1094.02  treatment  NCL 0.12  NCL 0.06  281.58  277.22  3 weeks a f t e r  Treat  treatment  846.80  2 weeks a f t e r  Treat  1211.39  NCL 0 . 1 2 .  6 weeks a f t e r  Means  NCL 0.12  1150.96  C o n t r o l NCL 0.06  Control  treatment  1501.92  5 weeks a f t e r  Treat  1342.02  NCL 0.06  Treat  Photosynthesis.  NCL 0.06  1385.96  4 weeks a f t e r  Treat  Photosynthesis.  NCL 0.12  3 weeks a f t e r  Control  treatment  treatment  NCL 0.12 204.90  NCL 0.06 204.03  4 weeks a f t e r treatment  C o n t r o l NCL 0.06 NCL 0.12 237.99 156.69 152.83  Respiration.  5 weeks  T r e a t C o n t r o l NCL 0.12 NCL0.06 Means 210.77 161.22 152.70  67. Table 13. E f f e c t o f NCL on growth o f beans. Experiment 6. Week a f t e r treatment F.W. (gm/plant)  C o n t r o l NCL 0.12 T/C(%)Wk.Control NCL 0.12 T/C(%) 31.32  35.32  112.76  97.50  4.29  4.25  99.02  10.12  100.40  13.48  11.98** 88.87  22.62  25.01  110.57  27.91  34.13  2.15  2.43  113.02  4.05  9.41  9.72  103.29  11.70  13.69  38.92  35.85  95.04  24.64  34.92* 141.73  88.62  3.87  5.76* 148.84  93.07  15.69  11.64  11.20  101.37  1.20  1.17  10.08  D.W. (gm/plant) %  D.W.  F.W. (gm/plant) D.W. (gm/plant) % D.W. F.W. (gm/plant) D.W. (gm/plant) % D.W. Note:  a  3.66**  4.13 10.96  10.20*  4  5  6  122.29  4.75* 117.28  16.66  117.00  106.18  a - average o f ten plants Variance r a t i o (F) o f Table 13 week a f t e r treatment  Fresh Wt. treat Block  1  0.09 1.76  0.93 0.23  27.50** 3.50*  0.01 0.04  0.40 0.10  6.44* 2.30  2.83 2.76  7.39* 0.84  11.67** 2.12  0.02 3.73*  6.10* 1.98  10.31** 1.98  Dry Wt. Treat Block  Percentage Dry Wt. Treat Block  0.03 2.78  3.59 1.61  8.97* 1.34  11.00** 1.14 1.11 0.70  0.71 0.56  68. Treatment: c o n t r o l , NCL 0.12, NCL 0.06 Measurements: growth and y i e l d - f r e s h weight, dry weight and percentage dry weight o f tops and pods a t one-week i n t e r v a l s from the 1st to the 6th week a f t e r treatment; seed y i e l d - weight and number o f seeds 12 weeks o l d (1 harvest o n l y ) ; photosynthesis and r e s p i r a t i o n - a t one-week i n t e r v a l s from the 1st to the 6th week a f t e r treatment. Results A.  Photosynthesis and r e s p i r a t i o n The beans treated w i t h NCL 0.12 and NCL 0.06 showed a  nonsignificant  increase i n the r a t e o f photosynthesis and a  s i g n i f i c a n t increase i n the r a t e o f r e s p i r a t i o n i n the f i r s t week a f t e r treatment.  The rates o f photosynthesis were found to be  s i g n i f i c a n t l y decreased from the second to s i x t h week a f t e r treatment.  An i n h i b i t i o n o f r e s p i r a t o r y r a t e i n NCL-treated  plants from second to f i f t h week a f t e r treatment was obtained. S i x weeks a f t e r treatment, the i n h i b i t i o n tended to disappear and the r e s p i r a t o r y r a t e rose above that o f the untreated p l a n t s .  Gen-  e r a l l y , NCL 0.12 showed a higher i n h i b i t i o n o f r e s p i r a t i o n than that o f NCL 0.06. B.  Growth The beans treated w i t h NCL 0.12 showed a s l i g h t increase  i n f r e s h weight, dry weight and percentage dry weight o f tops  69 Table 14.  E f f e c t o f NCL on y i e l d o f beans. Experiment 6. PODS  treatment  C o n t r o l NCL 0.12 0.34** 0.05* 13.78 6.8 3.9 4.7 15.4  53.13 62.50 102.61 74.73 111.14 121.88 97.47  26.41 2.32 18.73 10.00  21.40 1.47* 18.11* 12.50*  81.02 63.62 92.90 125.00  F.W.^gm/plant) D.W. (gm/plant) % D.W.(gm/plant) Pods No.(per p l a n t )  36.74 4.70 12.66 10.80  36.93 4.08 11.08* 11.70  100.51 86.81 87.52 108.33  F.W.(gm/plant) D.W.(gm/plant) % D.W. Pods No.(per p l a n t )  30.99 7.01 22.88 8.10  39.76* 6,28 15.50* 9.00  128.30 89.59 67.75 111.11  F.W, (gm/plant) D.W.(gm/plant) % D.W. Pods No. Flower No. Buds No. T o t a l No.  0.64 0.08 13.43 9.1 3.5 3.2 15.8  4  F.W.(gm/plant) D.W.(gm/plant) % D.W.(gm/plant) Pods No.(per p l a n t )  5  6  Note:  a  a - average o f ten plants Variance r a t i o (F) o f Table 14 week a f t e r treatment  Fresh Wt. Treat Block  3  4  37.50** 3.50*  5  6  4.42 2.30  0.001 1.46  7.79* 1.83  7.21* 2.04  0.82 1.15  1.09 1.57  Dry Wt. Treat Block  T/C (%)  9.86* 1.57  70 Table 15.  E f f e c t o f NCL on seed y i e l d *  Experiment 6.  Control  NCL 0.12  Pods No. per p l a n t  8.7  7.0  80.46  Seed No., per p l a n t  18.9  22.4  118.52  8.50  12.33  145.00  Weight o f seed (gm/1000 seeds)  452.70  55.9.29  123.55  Weight o f seed (mg/pod)  977.47  1761.43  180.20  2.17  3.20  147.46  Weight o f seed (gm/plant)  Seed No. per pod  a  T/C  (%)  Note: 1 - pods were harvested as they became r i p e when the plants were 12 weeks o l d . a - average o f ten plants Variance r a t i o (F) f o r Table 15 Pods No.  Seeds No.  Wt. o f Seed per p l a n t  Treat  2.76  0.67  2.37  Block  0.32  0.19  0.25  71. i n the second, f i f t h and s i x t h week a f t e r treatment.  A non-  s i g n i f i c a n t i n h i b i t i o n i n growth resulted at the t h i r d and fourth week a f t e r treatment.  The fresh weight o f tops was 12.76%  higher than the controls i n the fourth week a f t e r treatment. For the f i r s t week a f t e r treatment, there was a s l i g h t increase i n fresh weight tops which was not s i g n i f i c a n t .  The percentage dry  weight increased i n the f i f t h and s i x t h week a f t e r treatment, both f o r treated and untreated plants.  In c o n t r o l plants, the  t o t a l fresh weight i n the s i x t h week a f t e r treatment was less than that i n the f i f t h week, but more dry matter was produced i n the s i x t h than i n the f i f t h week a f t e r treatment.  This pheno-  menon indicated that evaporation of water reduced the t o t a l fresh weight, but the dry matter was added during growth process. In the case of NCL 0.12 treated plants, the s i g n i f i c a n t increase i n fresh and dry weight occurred i n the s i x t h week a f t e r treatment would be due to the increase i n water content and dry matter production. The results of this experiment revealed that NCL 0.12 induced a significant increase i n growth of the bean plants compared with c o n t r o l . dry  The fresh weight, dry weight and percentage  weight o f tops were increased by 22.29%, 17.28% and 17.00%  over the controls i n the s i x t h week a f t e r application. From Figure 13, the recovery l i n e showed the e f f e c t of NCL 0.12 on top growth against time a f t e r treatment.  A J lower  72. top growth i n treated plants than that i n untreated plants a t the t h i r d week a f t e r treatment was obtained.  This r e d u c t i o n i n top  growth i n the t h i r d week was recovered at the f o u r t h week and was followed by a s t i m u l a t i o n a t the f i f t h and s i x t h week.  The  percentage dry weight o f NCL 0.12 treated plants was s l i g h t l y lower than that o f c o n t r o l s a t the t h i r d and f o u r t h weeks. I t i s known that reproductive a c t i v i t y may c u r t a i l veget a t i v e growth, estimated as height, weight and number o f leaves. Some o f the food requirements o f the f r u i t may be s u p p l i e d from storage reserves, though most i s probably c u r r e n t l y synthesized by the leaves.  The r a t e of v e g e t a t i v e growth diminishes a t the  exact time and i n exact p r o p o r t i o n to the number o f flowers formed and the amount o f f r u i t s e t . of the flowers o r developing tion.  I t i s recognized  that removal  f r u i t permits f u r t h e r stem elonga-  I n t h i s experiment, however, decreased top growth and  decreased f r u i t production occurred simultaneously.  In this  experiment, however, decreased top growth and decreased f r u i t production were c o i n c i d e n t . C.  Yield According  to the r e s u l t s shown i n Table 14, the NCL 0.12  t r e a t e d plants had a lower dry weight and percentage dry weight than d i d the c o n t r o l s .  I t can be seen from Table 14 t h a t f r e s h  73. weight and dry weight of pods expressed as percentage o f controls showed a steady increase with time, while percentage dry weight showed a steady decline. The increase i n fresh weight tended to be faster than that of dry weight.  As a r e s u l t , the percentage dry weight  showed a decrease with time, while water content increased. Treatment with NCL 0.12 yielded an increase i n fresh weight of pods of 28.30% over the controls.  In the case of the number of  pods, NCL 0.12 stimulated the t o t a l number of pods (G-total) at the fourth week and the number of marketable-size at the s i x t h week by 257. and 11,11% respectively.  As shown i n Figure 14, the  i n h i b i t i o n of fresh weight of pods induced by NCL 0.12 was overcome at the f i f t h week. D.  Seed y i e l d The plants treated with NCL 0.12 produced nonsignificant  increase i n the number of seeds per plant (18.527o), weight of seeds per plant (45.00%), weight of 1000 seeds (23.55%), weight of seeds per pod (80.20%), and number of seeds per pod (47.46%). The lack of s i g n i f i c a n c e o f treatment e f f e c t might be due to the small number of plants involved o r perhaps to natural v a r i a t i o n among the plants.  74.  Experiment 7.  E f f e c t o f NH *2,4-D on photosynthesis, 4  r e s p i r a t i o n , growth and y i e l d o f beans  Up to Experiment 6, a l l the treatments were i n the form of dusts.  M i l l e r e t a l . (37,  38) reported that 1 ppm to 5 ppm  2,4-D f o l i a r sprays increased growth o f beans during the juveni l e stage and a l s o increased the y i e l d o f S u t t e r Pink beans. The present experiment was conducted to determine the e f f e c t of sprays c o n t a i n i n g NH «2,4-D a t 0.5 ppm, 1 ppm and 5 ppm on the 4  r a t e o f photosynthesis, r e s p i r a t i o n , growth and y i e l d o f Top Crop green bush beans.  Appropriate concentrations o f NH4«2,4-D  aqueous were sprayed on the leaves by an atomizer u n t i l run o f f . The growth c o n d i t i o n s i n t h i s experiment were the same as those i n Experiment 6.  Green pods were c o l l e c t e d a t one-week i n t e r -  v a l s as they a t t a i n e d marketable s i z e (4") f o r three consecutive weeks from the f o u r t h to s i x t h week a f t e r treatment.  For seed  y i e l d measurement, pods were harvested every two weeks from the 12th  to 16th week a f t e r treatment. Treatment:  c o n t r o l , NH4«2,4-D a t 0.5 ppm, 1.0 ppm and  5.0 ppm. Measurements:  y i e l d - f r e s h weight, d r y weight and  percent dry weight o f pods; seed y i e l d - weight and number o f seeds; photosynthesis and r e s p i r a t i o n - one week i n t e r v a l s from the 1st week to 5 t h week a f t e r treatment.  ?5* Pig. 15. Effect of NH »2,4-D on photosynthesis and respiration of beans. Experiment 4  120 -  Pho to synthesi s  * NH..2,4-D $ ppm  no  L  — NH -2,4-D /' 1 $pm / - NH -2,4-D/ 5 ppm / ^ 4  100  T/C 90  L  80  160  2 3 4 Week after treatment Respiration r  140  T/C  120  100  2  3  4  Week aftfrr treatment  76. Pig. 16. Effect of NH .2,4-D on y i e l d of beans Experiment . ' • •• 4  Fresh weight of 1-3, t o t a l 110  90 T/C (*) 70  50  | \ \ Nj  s  I  \ ppm  Dry weight of 1-3 t o t a l  I  I \  a. 5 ppm  1 ppm  \  77-. Table 16. E f f e c t o f NH »2,4-D on photosynthesis and 4  r e s p i r a t i o n o f beans. Experiment 7, Aug.28-Oct. 22, 1964. Photosynthesis ( l 0 /gm F.W./hr)  I/c Week a f t e r * treatment C o n t r o l 1/2 ppm 1 ppm 5 ppm 1/2 ppm 1 1032.50 1147.98 1245.06* 1154.32 111.18 2 1066.52 1228.62*1165.50 1088.55 115.20 3 1237.69 1265.58 1152.26 1003.22* 102.25 4 1466.12 1448.73 1416.65 1189.60** 98.81 5 1506.80 1577.37 1653.27**1245.27**104.68 U  2  a  (7.)  1 ppm 5 ppm 120.59 111.80 109.28 102.07 93.10 81.06 96.63 81.16 109.65 82.64  Respiration ( u l 0 /gm F.W./hr) 2  1 2 3 4 5 Note:  211.06 254.98 226.77 219.39 241.67  228.84 256.67* 380.68**270.31 311.79* 269.24* 248.47 299.56** 292.53**369.62**  253.82* 108.42 390.38**149.30 308.02* 137.49 282.70**113.38 311.63**121.04  a - average o f four t e s t s Variance r a t i o (F) o f Table 16  Treat Block  1 3.69* 1.61  Pho to syn t h e s i s (week a f t e r treatment) 2 3 4 5 3.12* 3.77* 14.57** 18.30** 1.81 2.16 0.78 2.16 Respiration  Treat Block  3.97* 1.54  18.68** 2.69  4.43* 0.71  5.88* 1.98  9.40* 0.42  121.56 106.01 118.72 136.54 152.94  120.26 153.10 135.83 128.86 128.95  78, Duncan's t e s t f o r Table 16 Photosynthesis. 3 weeks Treat  1/2 ppm  Control  1 ppm  5 ppm  Means  1265.58  1237.69  1152.26  1003.22  Photosynthesis. 4 weeks Treat  Control  1/2 ppm  1 ppm  5 ppm  Means  1466.12  1448.13  1416.75  1189.60  Photosynthesis. 5 weeks Treat  1 ppm  1/2 ppm  Contro1  5 ppm  Means  1653.27  1577.37  1506.80  1245.27  R e s p i r a t i o n . 2 weeks Treat  5 ppm  1/2 ppm  1 ppm  Control  Means  390.38  380.68  270.31  254.98  R e s p i r a t i o n . 4 weeks Treat  5 ppm  1/2 ppm  1 ppm  Control  Means  282.70  248.48  299.58  219.33  R e s p i r a t i o n . 5 weeks Treat  1 ppm  1/2 ppm  5 ppm  Control  Means  369.62  292.53  311.63  241.67  79. Table 17. E f f e c t o f NH^'2,4-0 on y i e l d o f beans  Pods 1st Harvest F.W. D.W. % D.W. Pods No. 2nd Harvest F.W. D.W. % D.W. Pods No.  C o n t r o l 1/2 ppm  3rd Harvest F.W. D.W. % D.W. Pods No. 1-3 T o t a l F.W. D.W. % D.W. Pods No. G-Total F.W. D.W. 7o D.W. Pods No.  1 ppm 5 ppm  T/C (7o) 1/2ppm 1 ppm  10.25 1.02 6.91 1.78  7.96 0.74 6.87 1.40  6.57 0.65 6.31 1.23  1.55** 77.61 0.14** 73.18 1.58** 99.41 0.28** 78.87  6.82 0.84 8.31 1.08  5.40 0.67 5.41* 0.85  9.59 1.21* 9.56 1.48  4.97 0.53 6.28* 0.88  79.22 79.52 65.09 79.07  6.97 0.98 12.39 1.38  7.57 1.12 11.05 1.40  4.38* 8.56 0.58* 0.97 7.72* 8.36* 0.78**1.60  108.66 114.10 89.13 101.82  24.03 2.84 12.17 4.24  20.92 2.53 11.97 3.65  20.53 15.08* 87.07 85.44 2.44 1.65* 89.22 86.13 12.56 9.54** 98.11 103.18 3.49 2.76** 86.39 82.25  28.21 3.44 12.47 6.43  25.15 3.05 12.23 6.03  24.86 21.68 3.00 2.44 12.48 11.72 6.13 5.60  64.10 15.09 63.64 14.01 91.31 32.86 69.01 15.49 140.60 72.92 144.78 63.67 115.00 76.75 137.21 81.40 62.86 122.93 59.32, 99.00 62.27 67.48. 56.36 116.36,  Pods No.  Treat Block T X B  7.08** 1.41 1.86*  (week a f t e r treatment) 2nd 3rd 1-3 3.10* 3.76* 1.02  4.80** 1.17 1.06  5.09** 0.38 1.28  62.75 58.10 78.41 65.09  89.14 88.12 76.87 88.69 87.24 79.91 98.08 100.03 93.96 93.77 95.33 87.16  Variance R a t i o n (F) f o r Table 17  1st  5 ppm  Total 0.45 0.69 1.79*  Pods f r e s h weight (week a f t e r treatment) 1st Treat Block T X B  6.67** 1.75 1.93*  2nd  3rd  3.97* 3.32 1.12  4.17* 1.43 1.20  1-3  Total  5.49* 0.20 1.60  1.35 0.28 1.89  Pods dry weight Treat Block T X B  4.38* 2.48 1.10  6.48** 2.21 2.06  4.01* 0.97 0.85  4.55*  0.08 1.81  2.06 0.19 2.16*  Pods percentage d r y weight Treat Block T X B  4.09* 3.78 1.43  13.70** 2.73 1.48  4.93* 1.53 0.75  4.98** 1.53 1.20  Duncan's t e s t f o r Table 17. Pods No.. 2nd week Treat  1 ppm  Means  1.48  Treat  5 ppm  1/2 ppm  Control  Means  1.60  1.40  1.38  Contro1  5 ppm  1/2 ppm  U08  0.88  0.85  1 ppm 0.78  Pods No.. 1-3 T o t a l treat  Control  1/2 ppm  1 ppm  5 ppm  Means  4.23  3.65  3.48  2.76  0.29 0.73 1.77*  81. Duncan's t e s t f o r Table 17 (cont'd) Pods f r e s h weight. 2nd week Treat  5 ppm  1/2 ppm  Control  1 ppm  Means  1.55  7.96  10.25  6.57  Pods f r e s h weight. 2nd week Treat  1 ppm  Control  1/2 ppm  5 ppm  Means  9.59  6.82  5.40  4.97  Pods f r e s h weight. 3rd week Treat  5 ppm  1/2 ppm  Control  1 ppm  Means  8.56  7.57  6.97  4.38  Pods f r e s h weight. 1-3 T o t a l Treat  Control  1/2 ppm  1 ppm  5 ppm  Means  24.03  20.93  20.53  15.08  Pods dry weight. 1st week Treat  Control  1/2 ppm  1 ppm  5 ppm  Means  I*£2.  0.74  0.65  0.14  Pods dry weight. 2nd week Treat  1 ppm  Control  1/2 ppm  5 ppm  Means  1.21  0.84  0.67  0.53  Pods dry weight. 3rd week Treat  1/2 ppm  Control  5 ppm  1 ppm  Means  1.12  0.98  0.97  0.58  Pods d r y weight. 1-3 T o t a l Treat  Control  1/2 ppm  1 ppm  5 ppm  Means  2.84  2.53  2.45  1.65  82'. Table 18. E f f e c t o f NH '2,4- D on seed y i e l d . Experiment 7, ,!  4  Seed No. /plant 1st harvest 2nd harvest 3rd harvest  C o n t r o l 1/2 ppm  1 ppm  5ppm  l/2ppm  1 ppm  5 ppm  15.20* 1.80 2.03  10.45 6.27** 2.82  10.00 5.20* 68.75 65.79 34.21 6.56** 5.70**348.33 364.44 316.67 4.89 7.60 138.92 240.89 374.38  T o t a l o f 1-2 harves ts  17.00  16.73  16.56  10.90  98.41  97.41  64.12  T o t a l o f 1-3 harvests  19.03  19.55  21.44  18.50 102.73  112.66  97.21  Wt. o f seed /plant(gm) 1st harvest 2nd harvest 3rd harvest  13.21 0.43 0.67  3.46 1.73** 0.65  4.29 1.91 26.19 32.48 14.46 1.70** 1.44**402.33 395.35 334.88 1.05 2.53** 97.01 156.72 376.12  1-2 harvests  13.64  5.18**  5.99** 3.36** 37.98  43.91  24.63  1-3 harvests  14.31  5.83**  7.04** 5.88** 40.74  49.20  41.09  Note:  a - average o f ten plants Variance r a t i o n (F) o f t a b l e 18  Treat Block  1st harvest 4.09* 2.14  2n  |eed No.  harvest 5.78** 2.60  1-2 harvest harvest 2.30 2.35 0.83 1.54 3 r d  1-3 harvest 0.30 5.06  Seed weight Treat Block  2.31 1.20  5.29** 0.81  3.92** 0.86  1.92 0.98  0.60 0.65  Seed No.. 1st harvest Treat  Control  1/2 ppm  1 ppm  5 ppm  Means  15.20  10.45  10.00  5.20  1/2 ppm  5 ppm  Control  5.70  1.80  1 ppm  5 ppm  Control  1.70  1.44  0.43  Control  1/2 ppm  0.67  0.65  Seed No.. 2nd harvest Treat  1 ppm  Means  6.56  6.27  Seed Weight. 2nd harvest Treat Means  1/2 ppm 1.73  Seed weight. 3rd harvest Treat  5 ppm  1 ppm  Means  2.52  1.05  .84. Results A.  Photosynthesis  and r e s p i r a t i o n  A p p l i c a t i o n o f sprays c o n t a i n i n g NH^*2,4-D a t 0.5 ppm, 1 ppm and 5 ppm induced a s i g n i f i c a n t increase on the r a t e o f r e s p i r a t i o n from the f i r s t to f i f t h week. plants had a higher photosynthetic week than that o f the c o n t r o l s .  The NH4»2,4-D treated  r a t e i n the f i r s t and second  The a p p l i c a t i o n o f 5 ppm showed  a s i g n i f i c a n t i n h i b i t i o n on the r a t e o f photosynthesis  i n the  t h i r d , f o u r t h and f i f t h weeks, while 1 ppm and 0.5 ppm induced a s t i m u l a t o r y e f f e c t i n the f i f t h week. 5.  Yield Generally, plants t r e a t e d w i t h NH »2,4-D had l e s s f r e s h 4  weight, d r y weight and percentage d r y weight o f pods than the controls. cant.  Only i n the case o f 5 ppm was the decrease s i g n i f i -  This decrease i n y i e l d might be the r e s u l t o f the stimu-  lation i n respiration. C.  Seed y i e l d A p p l i c a t i o n o f sprays c o n t a i n i n g NH4»2,4-D a t 1 ppm  induced an increase i n the number o f seeds by as much as 12.66%. A t 0.5 ppm, the number o f seeds y i e l d e d was s l i g h t l y increased and while a t 5 ppm, i t was decreased. plants contained  The NH4*2,4-D t r e a t e d  l e s s dry matter i n seed than d i d the c o n t r o l s .  On the whole, treatments c o n t a i n i n g NH4»2,4-D a t 0.5, 1 and 5 ppm delayed the maturity o f beans as f a r as top growth and pod y i e l d were concerned.  85. Experiment 8.  E f f e c t of NCL and NH 2,4-D on photo#  4  phosphorylation According  to the r e s u l t s obtained  i n the preceding  iments, the e f f e c t of 2,4-D on enzyme a c t i v i t y ,  exper-  photosynthetic  and respiratory rates o f beans revealed that the stimulatory e f f e c t of 2,4-D on metabolism can only be found the f i r s t week a f t e r treatment.  Aberg (1) reported that the plants are more  s e n s i t i v e to growth substances i n the early stages of development.  A p p l i c a t i o n of dusts containing NCL 0.12 and NCL 0.06, and  sprays containing NH4*2,4-D at 0.5, 1, and 5 ppm s l i g h t l y i n creased  the rate o f photosynthesis  i n the f i r s t week a f t e r treat-  ment . This experiment was designed to determine how NCL 0.12 and NCL 0.06, NH4»2,4-D at 0.5, 1, and 5 ppm would a f f e c t the rate o f photophosphorylation i n the f i r s t week a f t e r treatment. Chlorophyll content, phosphate uptake and oxygen evolution were measured. Treatment:  control, NCL 0.12, NCL 0.06, NH »2,4-D at 4  0.5 ppm, 1.0 ppm and 5.0 ppm. Measurements:  Chlorophyll content, phosphate uptake,  and oxygen evolution. Results (1)  Dusts containing NCL 0.06 and NCL 0.12 s i g n i f i c a n t l y  increased the chlorophyll content of treated plants by 45.87%  86. Table 19.  E f f e c t o f NCL and NH4*2,4-D on photophosphorylation  of beans. Experiment 8, Dec.19 - Jan. 2, 1965 Chlorophyll C o n t r o l 1/2 ppm mg/ml T/C (%)  57.32  a  59.35 103.54  1 ppm  content 5 ppm  NCL 0.12 NCL 0.06  70.76** 58.07 123.44 101.38  73.85** 83.62** 128.84 145.87  Noncvclic photophosphorylation Oxygen Evolved u l 02/15 min.191.03 206.10** T/C(%$ 107.89  223.47**222.20**266.85** 254.45** 116.98 116.31 139.69 133.20  Phosphate uptake ug P/ml/ 15 min T/C (%)  15.46  33.68** 217.82  27.84** 15.50 180.04 100.23  11.34** 73.34  9.62** 62.24  C y c l i c photophosphorylation Phosphate uptake ug P/ml/ 15 mm.  1 Q > 5 9  T/C(%)  3  3 < 7 8  **  172.47  32.65** 28.87** 31.27** 26.80** 166.67  147.37  159.66  Note: a - average o f s i x t e s t s Variance r a t i o (F) o f Table 19 Chlorophyll content  Oxygen evolved  Cyclic-P Uptake  Treat  621.77**  67.78**  8.31**  Block  1.96  16.32**  0.86  Noncyclic-P Uptake 36.06** 0.43  136.84  Duncan's t e s t f o r Table 19. C h l o r o p h y l l content Treat  NCL NCL 0.12 0.06  1 ppm  1/2 ppm  5 ppm  Means  836.17 738.53 707.60 593.53  Control  580.72  57^32  Oxygen evolved Treat  NCL 0.06  NCL 0.12  Means  266.85 254.45  1 ppm  5 ppm  1/2 ppm  Control  223.47  222.20  206.10  191.03  5 ppm  NCL 0.12  Cyclic-P-uptake Treat  1/2 ppm  1 ppm  Means  33.782  32.645  NCL 0.06  31.272 28.865 26.802  Control 19.587  Non c v c l i c - P - u p t a k e Treat  1/2 ppm  1 ppm  5 ppm  Means  33.678  27.837  15.497  C o n t r o l NCL 0.06 15.461  11.34  NCL 0.12 9.623  88. and 28.84% over the control plants.  Sprays containing  NH4*2,4-D at 1 ppm and 1/2 ppm induced s i g n i f i c a n t increases i n chlorophyll content (23.44% and 3.54%).  At 5 ppm,  the chloro-  p h y l l content was increased s l i g h t l y but not s i g n i f i c a n t l y . (2)  I t i s known that the electrons shot out of the  chlorophyll are captured by NADP which then becomes negatively charged.  This negatively charged NADP attracts a proton (H )  from water and then forms NADPH2.  A t the same time, oxygen i s  liberated by means of photolysis of water.  For noncyclic  photophosphorylation, a l l the treatments caused a s i g n i f i c a n t increase i n oxygen evolution. In other words, NADPH2 formation and photolysis of water might be stimulated by a l l treatments. Concentrations of 0.5 ppm and 1 ppm sprays on beans induced s i g n i f i c a n t increases i n phosphate uptake (117.827o and 80.04%). Dusts containing NCL 0.12 and NCL 0.06 had an i n h i b i t o r y  effect  upon phosphate uptake, but only NCL 0.12 yielded a s i g n i f i c a n t decrease e f f e c t . (3)  A l l the treatments caused a s i g n i f i c a n t stimulation  i n phosphate uptake i n c y c l i c photophosphorylation.  Dusts con-  taining NCL had a greater stimulatory e f f e c t than had sprays containing NH^*2,4-D. Since the chlorophyll content i n i s o l a t e d chloroplasts was adjusted to 2.0 mg/ml, the rate of phosphate uptake can be used as a measure of phosphopyridine nucleotide reductase (PPNR)  89.  activity.  The results of the e f f e c t of 2,4-D  the same as those of phosphate uptake.  on PPNR a c t i v i t y are  90 DISCUSSION A s e r i e s o f experiments was undertaken to study the e f f e c t s of dusts o f 2,4-D w i t h and without microelements, and sprays o f NH *2,4-D on growth, y i e l d , and metabolism o f Top Crop bush 4  beans.  A summary o f the e f f e c t s on growth and y i e l d i s shown  below. Treatments NCL 0.12 Exp.l Growth Yield  2,4-D 0.12  NCL 0.06  2,4-D M i n e r a l s NRy 0.06 2,4-D 0.12  F F"  F"  Ex l Growth Yield  NCLNH . 4  2,4-D  0.12  F F"  a  £ i  F Fo  Exp 3 Growth Yield  F F"  4 Growth Yield  F°D°d? F°D d  F ° D V  5 Growth Yield  F~D°d° F"D°d  F~D°d* F-D°d F D d F+D d  t  EXP.  +  F°D°d°  +  EXP.  Exp. 6 Growth Yeild Note:  +  0  +  F ° D V  *J>Z l I t t I I t* d  +  F°D"d° F°D d  F°D d°  +  +  +  F  D  +  +  d  F  D  F D d +  +  d  +  X !  +  d  F°D d +  +  F D°d +  F  D  +  d  F°D°d+ F"D°d +  _+*+*+  +* . -* F D d  F  D  d  F, D , d: f r e s h weight, dry weight, percentage dry weight of tops o r pods. +, -, o: i n c r e a s e , decrease, no e f f e c t . *:  s i g n i f i c a n t l y d i f f e r e n t a t 5% compared w i t h c o n t r o l s  a:  masterpiece bush beans were used.  91. Based upon the r e s u l t s o f a l l experiments, the o n l y c o n s i s t e n t e f f e c t of NCL treatment of the bean plants was an increase i n number o f pods ( s i g n i f i c a n t a t the 5% l e v e l ) .  In  only one case (Experiment 6) was the increase i n f r e s h and dry weight o f tops and the f r e s h weight of pods s i g n i f i c a n t a t the 5% l e v e l .  I t should be noted that o n l y i n Experiment 6 was  n u t r i e n t s o l u t i o n a p p l i e d i n s u f f i c i e n t volume that the vermicul i t e was saturated and excess s o l u t i o n r a n through the d r a i n holes o f the pots. As stated by Wort (81) and Aberg ( 1 ) , the e f f e c t s o f h e r b i c i d e s on the growth and metabolism o f a l i v i n g p l a n t are those associated w i t h the h e r b i c i d e s , the p l a n t i t s e l f , and i t s environment.  Wedding e^  (65) pointed out t h a t the v a r i a b i l i t y  encountered i n the case of p l a n t growth r e g u l a t o r s to increase y i e l d of beans can probably be a t t r i b u t e d to many environmental f a c t o r s , p a r t i c u l a r l y water and n u t r i e n t l e v e l .  In h i s experi-  ment, d e f i c i e n c y of water near the time when the f r u i t was maturing prevented pods from  reaching t h e i r maximum s i z e and  weight, thus r e s u l t i n g i n a no e f f e c t on y i e l d o r even decrease i n s p i t e o f the l a r g e r number o f pods produced.  As has already  been mentioned, i n h e r i t e d v a r i a t i o n among plants could be an important f a c t o r i n i n t r o d u c i n g u n c e r t a i n t y to treatment e f f e c t s . I t i s necessary to use an appropriate sample s i z e and r e p l i c a t e s i z e i n each experiment i n order to overcome the n a t u r a l v a r i a t i o n  92. and to o b t a i n the c o r r e c t r e s u l t s o f treatment e f f e c t s , i n the case o f growth and y i e l d .  I n a d d i t i o n , r o o t growth, root-shoot  and r o o t - f r u i t r e l a t i o n s h i p and i n t e r a c t i o n o f reproductive and v e g e t a t i v e growth have to considered p a r t i c u l a r l y i n determining the t o t a l growth and y i e l d . The r e s u l t s o f Experiment 6 i n d i c a t e d that dusts cont a i n i n g low l e v e l s o f 2,4-D w i t h minerals may be used to increase the f r e s h weight but not the dry weight y i e l d o f beans i f uniform bean plants with a s i m i l a r past h i s t o r y before a p p l i c a t i o n of treatment are used and s i m i l a r environmental c o n d i t i o n s e x i s t during growth. tial.  An adequate supply o f n u t r i e n t s o l u t i o n i s essen-  Pods should be harvested weekly during the f o u r t h , f i f t h  and s i x t h week a f t e r treatment. As shown i n Figure 14 a p p l i c a t i o n o f NCL 0.12 r e s u l t e d i n a decrease i n the average o f f r e s h weight o f pods harvested a t the t h i r d and f o u r t h week f o l l o w i n g treatment. diminished w i t h time a f t e r treatment.  The i n h i b i t i o n  A t the f i f t h week, the  recovery was completed and s t i m u l a t i o n o f the f r e s h weight of pods i n NCL-treated p l a n t s was obtained a t the s i x t h week a f t e r treatment. The s t i m u l a t i o n o r i n h i b i t i o n o f c a t a l a s e , phosphatase, and phosphorylase a c t i v i t y f o l l o w i n g 2,4-D treatment v a r i e d w i t h p l a n t organ and time a f t e r treatment.  A great many papers con-  cerning the i n f l u e n c e o f 2,4-D on enzyme system and p o s s i b l e  93. mechanism have been reported by Wort (74) and Freed(18).  2,4-D  might have increased the amount o f enzymes present and not the a c t i v i t y o f the enzymes.  Treatments o f NCL,  2,4-D  and  minerals  d i d not show an a d d i t i v e e f f e c t on growth, y i e l d and enzyme activity. I t i s w e l l known that a p p l i c a t i o n of 2,4-D an increase i n RNA  (28, 29).  RNA  i s a primary requirement f o r  growth, both i n p l a n t and i n animal, that the e x t r a growth induced by 2,4-D o f e x t r a RNA.  resulted i n  van Overbeek (59) stated i s caused by a  production  The abnormal q u a l i t y of t h i s e x t r a growth must be  a s c r i b e d to the hormonal imbalance r e s u l t i n g from the overpowering i n f l u e n c e of s a t u r a t i n g concentrations of 2,4-D.  I t was  recognized  t h a t p r o t e i n s occurred i n a large v a r i e t y of molecular species there were as many proteins i n a b i o l o g i c a l creature as were genes i n i t s chromosomes.  The f a c t t h a t 2,4-D  and  there  caused an  increase i n the number of pods by means of parthanogenesis, and abnormal p r o l i f e r a t i o n o f t i s s u e might be the r e s u l t of i n t e r a c t i o n between 2,4-D The e f f e c t of 2,4-D gene mutation.  and p r o t e i n , and 2,4-D  and n u c l e i c a c i d .  on chromosome behavior might have i n f l u e n c e d  As a r e s u l t , p r o t e i n and n u c l e i c a c i d synthesis  would be stimulated or i n h i b i t e d , thus s t i m u l a t i n g or i n h i b i t i n g enzyme a c t i v i t y . w e l l as 2,4-D  The i n t e r a c t i o n between 2,4-D  and p r o t e i n as  and n u c l e i c a c i d can be i n t e r p r e s t e d by Freed's  (18) theory of molecular l e v e l a c t i o n and Key's (29) nucleic acid action.  auxin-  94. As has already been s t a t e d , 2,4-D  treatment a t a high  c o n c e n t r a t i o n induced an almost e x c l u s i v e i n h i b i t o r y e f f e c t on the r a t e o f gas exchange i n photosynthesis.  This i n h i b i t i o n was  c o r r e l a t e d w i t h the d e s t r u c t i o n of mesophyll and stem phleom by 2,4-D, the reduced water supply to the leaves, and the concent r a t i o n o f u n d i s s o c i a t e d 2,4-D used f o r treatment (63). 2,4-D  a c i d molecules i n the s o l u t i o n  Morphological deformation i n leaves by  treatment appeared i n the f i r s t to f o u r t h t r i f o l i a t e  leaves.  The deformation was g r e a t e r at higher concentration. The r e s u l t s obtained i n t h i s i n v e s t i g a t i o n showed that  the r a t e of gas exhange i n photosynthesis was s t i m u l a t e d by treatments w i t h dusts and sprays of 2,4-D week.  at the end o f the f i r s t  Since morphological deformation was not evident i n the  primary leaves, t h i s increase i n gas exhcnage would be p o s s i b l e . A p p l i c a t i o n of 2,4-D respiration.  r e s u l t e d i n an i n c r e a s e i n the r a t e o f  The damage to l e a f t i s s u e , and unnatural condi-  t i o n s p r e v a i l i n g during the measurement of photosynthesis and r e s p i r a t i o n do not a l l o w d i r e c t comparison w i t h whole p l a n t under natural conditions. Since the number of l e a f d i s c s used i n these determinations was l i m i t e d by the n e c e s s i t y to keep the t o t a l oxygen evolved during photosynthesis to a value which could be e a s i l y determined i n the Warburg apparatus, and s i n c e the r e s p i r a t i o n values were approximately o n e - f i f t h o f those of photosynthesis, the s m a l l gas volumes are such t h a t i t i s d i f f i c u l t to draw v a l i d conclusions  95.  from s m a l l d i f f e r e n c e s which occurred during these  experiments.  As has been discussed by Jowett (27), a polarographic method might provide r e s u l t s o f greater accuracy than the Warburg apparatus. According to the r e s u l t s obtained i n t h i s i n v e s t i g a t i o n , a p p l i c a t i o n o f dusts o r sprays o f 2,4-D produced a s t i m u l a t i o n of photophosphorylation.  Sprays c o n t a i n i n g NH »2,4-D increased 4  the C<2 e v o l u t i o n and phosphate uptake by c h l o r o p l a s t s w h i l e dusts c o n t a i n i n g NCL increased C7 e v o l u t i o n but i n h i b i t e d phosphate uptake.  This r e s u l t might be explained on the b a s i s that  NCL i n t e r f e r r e d with the p a r t i c i p a t i o n o f water i n the n o n c y c l i c photosynthetic e l e c t r o n flow, but d i d not i n t e r f e r e w i t h the production o f NADP.  Since the present experiment was c a r r i e d  out under an aerobic c o n d i t i o n and production o f ATP and NADPH  2  was not measured, the r e s u l t s , while i n f o r m a t i v e , cannot be used to i n t e r p r e t the r a t e o f photosynthesis and subsequent phosphate metabolism.  96. SUMMARY In the past decade, most o f the i n v e s t i g a t i o n s concerned w i t h the metabolic response o f plants to 2,4-D i n v o l v e d aqueous sprays o f the compound.  This i n v e s t i g a t i o n was concerned w i t h  the e f f e c t o f 2,4-D i n both dust and spray form on growth and metabolism o f beans. Dusts c o n t a i n i n g i s o p r o p y l ester and ammonium s a l t o f 2,4-D w i t h o r without m i n e r a l s , and sprays c o n t a i n i n g ammonium s a l t o f 2,4-D were used to study t h e i r e f f e c t s on the growth p r o d u c t i v i t y and metabolism o f Top Crop bush beans.  Specifi-  c a l l y , e f f e c t s p e r t a i n i n g to y i e l d , growth, enzyme a c t i v i t y , photosynthesis, r e s p i r a t i o n and photophosphorylation were studied. The r e s u l t s o f 2,4-D treatment i n dust form showed that the dry weight o f pods and percentage dry weight o f tops and pods were increased w h i l e the f r e s h weight o f tops and pods were not.  Treatments such as NCL 0.12, 2,4-D 0.12, 2,4-D 0.06,  minerals and NCL 0.12 increased the y i e l d o f beans i n c e r t a i n instances.  Only NCL 0.12 produced a s i g n i f i c a n t e f f e c t on y i e l d  and growth.  I n most cases, a p p l i c a t i o n o f treatments f a i l e d to  produce a s i g n i f i c a n t e f f e c t on both growth and y i e l d . Beans treated w i t h NCL 0.12, NCL 0.06, 2,4-D 0.06 and minerals y i e l d e d e i t h e r an i n c r e a s e o r a decrease i n c a t a l a s e , phosphatase, and phosphorylase a c t i v i t y .  This s t i m u l a t i o n o f  97. i n h i b i t i o n was rather higher i n catalase a c t i v i t y . treatment appeared  NCL 0.12  to have the greatest stimulatory and i n h i b i -  tory effects on enzyme a c t i v i t y . The results showed that treatments with NCL i n dust form and NH «2,4-D i n spray form produced a s i g n i f i c a n t increase i n 4  the rate o f photosynthesis during the f i r s t week.  The rate of  photosynthesis, however, decreased s i g n i f i c a n t l y i n the t h i r d to s i x t h week.  In the case of r e s p i r a t i o n , treatment with  NH *2,4-D at 0.5 ppm to 5 ppm resulted i n a stimulatory e f f e c t , 4  while NCL 0.12 and NCL 0.06 showed an increase i n the f i r s t week followed by a decrease i n the second to f i f t h week. Rates of photophosphorylation were s i g n i f i c a n t l y increased by NH4"2,4-D treatment.  NCL 0.12 and NCL 0.06 resulted i n a  s i g n i f i c a n t stimulatory e f f e c t on phosphate uptake i n the c y c l i c photophosphorylation and oxygen evolution but an i n h i b i t i o n i n phosphate uptake i n noncyclic photophosphorylation.  98  REFERENCES 1.  Aberg, E. 1964. S u s c e p t i b i l i t y : Factors i n the p l a n t modifying the response o f a given species to treatment, i n The Physiology and Biochemistry o f h e r b i c i d e s . L. J . Audus Ed. pp. 401-422.  2.  Akers, T. J . and Fang, S.C. 1956. Studies i n metabolism. V I . E f f e c t o f 2,4-D on the metabolism o f a s p a r t i c a c i d and glutamic a c i d i n the bean p l a n t s . P l a n t P h y s i o l . 31:34-37.  • 3. 4. 5. 6.  Arnon, D. I . 1949. Copper enzymes i n i s o l a t e d c h l o r o p l a s t s . P l a n t P h y s i o l . 24:1-15. . 1960. The r o l e o f l i g h t i n photosynthesis. S c i . Amer. 203:104-118. . 1961. Changing concepts o f photosynthesis. B u l l . Torrey Bot. Club. 88:215-259. Audus, L. J . 1961. Metabolism and mode o f a c t i o n o f auxin, i n Handbuch der P f l a n z e n p h y s i o l o g i e . W. Ruhland, Ed. Springer-Verlag, B e r l i n . 14:1055-1083.  7.  Avery, G. S. 1951. S t i m u l a t i o n o f r e s p i r a t i o n i n r e l a t i o n to growth, i n P l a n t Growth Substances. F. Skoog, Ed. Univ. Wisconsin Press, Madison, pp. 105-109.  8.  Bonner, J . and Bandurski, R. S. 1952. Studies on the physiology and biochemistry o f the auxins. Ann. Rev. P l a n t P h y s i o l . 3:59-86.  9.  Brown, J . 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Studies i n p l a n t metabolism. IV. Comparative e f f e c t s o f 2,4Dichlorophenoxyacetic a c i d and other plant growth r e g u l a t o r s on phosphorus metabolism i n bean p l a n t s . P l a n t P h y s i o l . 29:365-368.  16.  Fang, S. C., Teeny, F., and B u t t s , J . S. 1960. Influence o f 2,4-D on pathways o f glucose u t i l i z a t i o n i n bean stem t i s s u e s . P l a n t P h y s i o l . 34:405-408.  17.  F e l b e r , I . M. 1948. The formation o f protuberances on bean leaves i n response to 2,4-D treatments. Amer. Journ. Bot. 35:555.  18.  Freed, V. H., R e i t h e l , F. L., and Remmert, L. F. 1961. Some physical-chemical aspects o f s y n t h e t i c auxins w i t h respect to t h e i r mode o f a c t i o n , i n P l a n t Growth Regulation. Iowa, p. 289-306.  19.  Freeland, R. 0. 1949. E f f e c t o f growth substances on photosynthesis. P l a n t P h y s i o l . 24:621-628.  20.  F r e i b e r g , S. R., and C l a r k , H. E. 1955. Changes i n n i t r o g e n f r a c t i o n s and p r o t e o l y t i c enzymes o f soybean plants treated w i t h 2,4-Dichlorophenoxyacetic a c i d . P l a n t P h y s i o l . 30:39-46.  21.  French, R. C., and Beevers, H., 1953. Respiratory and Growth responses induced by growth r e g u l a t o r s and a l l i e d compounds. Amer. Jour. Bot. 20:660-666.  22.  G a l l , H. J . F. 1949. Some e f f e c t s o f 2,4-D on s t a r c h d i g e s t i o n and reducing a c t i v i t y i n bean t i s s u e c u l t u r e . Bot. Gaz. 110:319-323.  100  23.  Geranmayeh, R. 1964. The e f f e c t o f 2,4-D on the growth formation o f root nodules and N-content o f V i c i a faba and Pisum sativum. P l a n t a . 62(1):66-87.  24.  Hagen, D. E., C l a g e t t , C. 0., and Helgeson, E. A. 1949. 2,4-Dichlorophenoxyacetic a c i d i n h i b i t i o n o f Castor bean l i p a s e . Science. 110:116.  25.  Housley, S. 1961. K i n e t i c s o f auxin-induced growth, i n Handbuch der P f l a n z e n p h y s i o l o g i e . W. Ruhland, Ed. Springer-Verlag, B e r l i n . 14:1007-1044.  26.  Huffaker, R. C., M i l l e r , M. D., and Mikkelsen, D. S. 1962. E f f e c t s o f 2,4-D, i r o n , and c h e l a t e supplements on dark C 0 f i x a t i o n i n c e l l f r e e homogenates o f f i e l d beans. Crop. S c i . 2:127-129. 2  27.  Jowett, P. A. 1962. A study o f the p o s s i b l e p r o t e c t i o n afforded by copper, f e r r o u s , and f e r r i c ions against the a c t i o n o f 2,4-D. M. Sc. D i s s e r t a t i o n . Univ. B r i t i s h Columbia.  28.  Key, L. J . , Hanson, J.B., and B i l s , R. F. 1960. E f f e c t of 2,4-D a p p l i c a t i o n on a c t i v i t y and composition o f mitochondria from soybeans. P l a n t P h y s i o l . 35:177-183.  29.  Key, J . L., and Wold, F. 1961. Some e f f e c t s o f 2,4Dichlorophynoxyacetic a c i d on the o x i d a t i o n - r e d u c t i o n s t a t e o f soybean s e e d l i n g . J . B i o l . Chem. 236: 549-553.  30.  Key, J . L. and Hanson, J . B. 1961. Some e f f e c t s o f 2,4-Dichlorophynoxyacetic a c i d on s o l u b l e nucleot i d e s and n u c l e i c a c i d on soybean s e e d l i n g s . P l a n t P h y s i o l . 36:145-152.  31.  Leonard, 0. A. 1958. Studies on the a b s o r p t i o n and t r a n s l o c a t i o n o f 2,4-D i n bean p l a n t s . H i l g a r d i a 28(5):115-160.  32.  Leopold, A. C. 1955. Auxins and p l a n t growth. Univ. C a l i f . Press. Berkeley and Los Angeles. 354 pp.  33.  Linden, G.  1954. Quoted i n Wort (1964).  101 34.  L o u s t a l o t , A. J . , and Muzik, T. J . 1954. E f f e c t o f 2,4-D on apparent photosynthesis and developmental morphology o f f i e l d beans. Bot. Gaz. 115j56-66.  35.  Meyer, B. S., and Anderson, D. B. 1955. P l a n t Physiology. D. van Nostrand Company, New York. 784 pp.  36.  M i l l e r , E. C. 1938. P l a n t Physiology. McGraw-Hill Book Company, New York. 1201 pp.. ,  37.  M i l l e r , M. D., M i k k e l s e n , D. S., and Huffaker, R. C. 1962. E f f e c t s of s t i m u l a t o r y and i n h i b i t o r y l e v e l s o f 2,4-D, i r o n , on growth and y i e l d o f f i e l d beans. Crop S c i . 2:114-116.  38.  M i l l e r , M. D., Mikkelsen, D. S., and Huffaker, R. C. 1962. E f f e c t s o f s t i m u l a t o r y and i n h i b i t o r y l e v e l s of 2,4-D, i r o n , and chelate supplements on j u v e n i l e growth o f f i e l d beans. Crop S c i . 2:111-113.  39.  Neely, W. B., B a l l , C. D., Hamner, C. L., and S e l l , A.M. 1950. E f f e c t o f 2,4-D on i n v e r t a s e , phosphorylase, and p e c t i n methixylase a c t i v i t y i n bean p l a n t s . P l a n t P h y s i o l . 25:525-528.  40.  Osborne, D. J . 1958.  41.  Pardee, A. B. 1949. Measurement o f oxygen uptake under c o n t r o l l e d pressures o f carbon d i o x i d e . Journ. B i o l . Chem. 179:1085-1091.  42.  Rebstock, T. L., Hamner, C. L., B a l l , C. D., and S e l l , H. M. 1952. E f f e c t o f 2,4-Dichlorophenoxyacetic a c i d on p r o t e o l y t i c a c t i v i t y o f red kidney bean p l a n t s . P l a n t P h y s i o l . 27:639-643.  43.  San P i e t r o , A. 1963. Photosynthetic p y r i d i n e n u c l e o t i d e reductase, i n Methods i n Enzymology. 6:439-445.  44.  S c h e l l , A. W., and Cartwright, D. R. 1955. B i b l i o g r a p h y o f plant growth substances 1900-1950. Quoted i n Jowett (1962).  45.  Shive, J . W. 1915. A t h r e e - s a l t n u t r i e n t s o l u t i o n f o r p l a n t s . Amer. Jour. Bot. 2:157-160.  46.  Skoog, F. 1951. P l a n t growth substances. Univ. Wise. Press, Madison.  Quoted i n Woodford e t a l . (1958).  47.  Smith, F. G. 1948. The e f f e c t o f 2,4-D on the r e s p i r a tory metabolism o f bean stem t i s s u e . Plant P h y s i o l . 23:70-83.  48.  S t e e l , R. G. D., and T o r r i e , J . H. 1960. P r i n c i p l e s and procedures o f s t a t i s t i c . McGraw-Hill Book Company, Inc., New York, 481 pp. Sumner, J . B., and Somers, G. F. 1953. Chemistry and methods o f enzymes. Academic Press I n c . , New York, 462 pp.  49.  50.  Sumner, J . B., Chou, T. C., and Bever, A. T. 1950. Phosphorylase o f the Jack bean: I t s p u r i f i c a t i o n , estimation, and p r o p e r t i e s . Arch* Biochem. 26:1-5.  51.  Swanson, C. A., and Whitney, L. B. 1953. Studies on the t r a n s l o c a t i o n o f f o l i a r a p p l i e d p32 d other r a d i o i s o t o p i c s i n bean p l a n t s . Amer. J . Bot. 40:816-823. a n  52.  Switzer, C. M. 1957. E f f e c t s o f h e r b i c i d e s and r e l a t e d chemicals on o x i d a t i o n and phosphorylation by i s o l a t e d soybean. P l a n t P h y s i o l . 32:42-44.  53.  Taylor, D. L. 1946. Observations on the growth o f c e r t a i n plants i n n u t r i e n t s o l u t i o n s containing s y n t h e t i c growth-regulating substances. I . Some e f f e c t s o f 2,4-D a c i d . Bot. Gaz. 107:597-611.  54.  . 1946. Observations on the growth o f c e r t a i n plants i n n u t r i e n t s o l u t i o n containing s y n t h e t i c growth-regulating substances. I I . the i n f l u e n c e o f presentation t i s s u e . Bot. Gaz. 107:611-619.  55.  Thimann, K. V., and Takahashi, N. 1961. I n t e r r e l a t i o n ships between m e t a l l i c ions and auxin a c t i o n , and the growth promoting a c t i o n o f c h e l a t i n g agents, i n P l a n t Growth Regulation, pp. 363-380.  56.  Umbreit, W. W., B u r r i s , R. H., and S t a n f f e r , J.F. 1957. Monometric methods. Quoted i n Burgess.  57.  van Overbeek, J . 1956. Quoted i n The chemistry and mode o f a c t i o n o f p l a n t growth substances. Wain, R. L., and Wightman, F. Ed., London, 205-219.  58.  . 1961. A p p l i c a t i o n o f auxins i n a g r i c u l t u r e and t h e i r p h y s i o l o g i c a l bases, i n Handbuch der Pflanzenphysiologie, W. Ruhland, Ed., 14:1137-1184.  1D.3  59.  van Overbeek, J . 1964. Survey o f mechanism o f h e r b i c i d e a c t i o n i n The physiology and biochemistry o f h e r b i c i d e s . Andus, L. J . , Ed., pp. 387-400.  60.  Wagenknecht, A. C , R i k e r , A. J . , A l l e n , T. C. and B u r r i s , R. H. 1951. P l a n t growth substances and the a c t i v i t y o f c e l l - f r e e r e s p i r a t o r y enzymes. Amer. Journ. Bot. 38:550-554.  61.  Wain, R. L., and Wightman, F. 1956. The chemistry and mode o f a c t i o n o f p l a n t growth substances. Butterworth's S c i e n t i f i c P u b l i c a t i o n s , London.  62.  Watson, D. P. 1948. An anatomical study o f the m o d i f i c a t i o n o f bean leaves as a r e s u l t o f treatment w i t h 2,4-D. Amer. Jour. Bot. 35:543-555.  63.  Weaver, R. J . 1956. E f f e c t o f spray a p p l i c a t i o n o f 2,4-D a c i d on subsequent growth o f various parts o f r e d kidney beans and soybean p l a n t s . Bot. Gaz. 107:532-539.  64.  Wedding, R. T., E r i c k s o n , L.C., and Brannaman, B.L. 1954. The e f f e c t o f 2,4-D on photosynthesis and r e s p i r a t i o n . P l a n t P h y s i o l . 29:64-69.  65.  Wedding, R. T., Kendrick, J . B., Stewart, W. S. and H a l l , B. J . 1956. Growth r e g u l a t o r s on beans. C a l i f o r n i a A g r i c u l t u r e . 10:4.  66.  Wedding, R. T. 1961. Uncoupling o f phosphorylation i n C h l o r e l l a by 2,4-D. P l a n t and S o i l . 14:242-248.  67.  Wedding, R. T. 1961. Respon§eriQfcdxidatibnrandteoupled phosphorylation i n p l a n t mitochondria to 2,4-D. P l a n t P h y s i o l . 37:364-370.  68.  Weintraub, R. L., Brown, J . W., F i e l d s , M.. and Rohan, J . 1952* Metabolism o f 2,-4-D I . C ^ production by bean plants t r e a t e d w i t h l a b e l l e d 2,4-D. P l a n t P h y s i o l . 27:293. 1  69.  Weintraub, R. L., Reinhart, J . H., S c h e r f f , R. A., and S c h i s l e r , L. C. 1954. Metabolism o f 2,4-D. I l l ; Metabolism and r e s p i r a t i o n i n dormant p l a n t t i s s u e . P l a n t P h y s i o l . 29:303-304.  104 70.  Whatley, F. R., and D a n i e l , I . A. 1963. Photosynthetic phosphorylation i n p l a n t s , i n Methods i n Enzymology. VI:308-313.  71.  Wittwer, S. H., 1964. F o l i a r absorption of p l a n t nut r i e n t s . Advancing F r o n t i e r s of P l a n t Sciences. Lokesh Chandra, Ed. 8:161-182.  72.  Woodford, E. K., H o l l y , K. and McCready, C C . 1958. Herbicides. Ann. Rev. P l a n t P h y s i o l . 9:311-358.  73.  Wort, D. J . i g a F o l i a r a p p l i c a t i o n o f sprays and dusts, c o n t a i n i n g major and minor elements w i t h and without 2,4-D. Proc. 7th West Can. Weed Cont. Conf. 7:93-101.  74. 75.  . 1954. Influence of 2,4-D Weeds. 3(2):131-135.  on enzyme systems.  'IbrSs Do J,,. 1956. The f o l i a r a p p l i c a t i o n o f minor elements w i t h 2,4-D. Pro. 9th Meeting West, S e c t i o n , N a t i o n a l Weed Committee, 1956. 22-28.  76.  . 1957. The f o l i a r a p p l i c a t i o n o f minor elements with 2,4-D. Proc. 10th Meeting West S e c t i o n , N a t i o n a l Weed Committee. 52-56.  77.  . 1959. The f o l i a r a p p l i c a t i o n o f 2,4-D and other growth r e g u l a t o r s w i t h and without added minor elements. Proc. IVth I n t . Cong, Crop P r o t , Hamburg. 1:497-502 (1957).  78.  1961, E f f e c t s on the composition and metabolism o f the e n t i r e p l a n t , i n Handbuch der P f l a n z e n p h y s i o l o g i e . W. Ruhland, Ed. 14:1110-1136.  79.  . 1962. The a p p l i c a t i o n o f s u b l e t h a l concent r a t i o n s o f 2,4-D and i n combination w i t h mineral n u t r i e n t s . World Review o f Pest C o n t r o l . 1(4):6-19.  80.  • 1964. Responses of plants to s u b l e t h a l concentrations o f 2,4-D, without and with added minerals, i n The Physiology and Biochemistry o f H e r b i c i d e s . Audus,L. J . , Ed., 335-342.  81.  . 1964. E f f e c t s o f h e r b i c i d e s on plant compos i t i o n and metabolism, i n The Physiology and Biochemistry o f Herbicides. Audus, L, J . , Ed. Academic Press, London and New York*  82.  Wort, D. J . , and LaBerge, D.E. E f f e c t s of 2,4-D n u t r i e n t dusts on the growth and y i e l d o f beans and sugar beets. Quoted i n Wort ( 1 9 6 4 ) .  A P P E N D I C E S  105. APPENDIX I A Modified Shive's Three-Salt  Nutrient  Solution (31, 44) A.  Stock solutions Macronutrients: Ca(N0 ) '4H 0 KH P04 MgS04.7H 0 FeEDTA 3  2  2  2  2  1M 1M 1M 5 mg  Fe/ml  Micronutrients: ( a l l i n 1 l i t e r ) H B0 3  2.86 gm  4  ZnS0 '7H 0 MnCl '4H 0 CuS0 »5H 0 Na Mo04»2H 0 4  2  2  2  4  2  2  B.  2  0.22 gm 1.81 gm 0.08 gm 0.025 gm  Nutrient solutions ( a l l i n 5 gallons) Ca(N0 ) «4H 0 3  KH P0 2  2  2  100 ml 50 ml  4  MgS0 »7H 0  50 ml  FeEDTA  30 ml  Micronutrients  30 ml  4  2  106 APPENDIX II Preparation of Chemicals A.  For determination of enzyme a c t i v i t y Phosphatase: Substrate - a phosphatase substrate i s prepared as follows: dissolve 1.09 gm phenol disodium phosphate i n 200 ml c i t r a t e buffer pH 6.0 and d i l u t e to one l i t e r with d i s t i l l e d water. F o l i n - C i o c a l t e r Reagent - A reagent used f o r phenol determination a f t e r phosphatase reaction. Standard 2N phenol reagent can be obtained from commercial, but, i n this experiment, i t was prepared as follows: (1) put 100 grams Sodium tungstate, 25 gm Sodium molybdate 700 ml H2O, 50 ml of 857o phosphoric acid, and 100 ml concentrated HC1 into a two-liter f l a s k . (2) r e f l u x f o r 10 hours. (3) Add 150 grams lithium s u l f a t e , 50 ml H2O and a few drops of bromine to the solutionaand b o i l f o r 15 minutes to remove excess Bu. (4) Cool and make to one l i t e r . (5) Dilute with water before use. Pho s pho ry las e: Substrate - (1) dissolve 1 gm glucose-l-P i n 50 ml d i s t i l l e d water. Shake with dry Ca(0H>2 to remove inorganic P. F i l t e r . Neutralize with drops of HC1 and measure the f i l t r a t e and add equal volume pH 6.0 c i t r a t e buffer. Keep i n d e f i n i t e l y i n r e f r i g e r a t o r . (2) Just before use, mix the buffered substrate with an equal volume of 1% starch s o l u t i o n and a c r y s t a l of thymol.  B.  For standard curves preparation Standard phosphate s o l u t i o n : Dissolve 1.0967 gm KH P04 i n d i s t i l l e d water and d i l u t e to 250 ml with d i s t i l l e d water. One ml of this s o l u t i o n equals 1 ml phosphorus. 2  10.7. Standard phenol solution: (1) dissolve 1 gm phenol i n one l i t e r 0.1 NHC1. (2) transfer 25 ml of this solution to a 250 ml f l a s k . (3) Add 50 ml 0.1 N NaOH and heat to 65 C. (4) Add 25 ml 0.1 N Standard iodine solution, stopper f l a s k , and stand f o r 30 minutes. (5) Add 5 ml concentrated HC1 and t i t r a t e excess iodine with 0.1 N Na S 04. (6) Each ml o f 0.1 N iodine solution used equals 1.507 ml of phenol. 2  C.  2  For photosynthetic and respiratory rate determination Carbon dioxide buffer: The buffer was made up as follows to supply 1% C0 :(4) 2  607o aqueous diethanoamine 6 N HCL H0  - 50 ml - 11 ml - 14 ml  KHCO3 powder  - 15 gm  2  D.  For photophosphorylation Magnesia mixture: Used f o r p r e c i p i t a t i o n of inorganic phosphate. I t was prepared by d i s s o l v i n g 5.5 gm MgCl and 10 gm NH4CI i n 50 ml H 0 and adding 10 ml 15 M ammonia. 2  2  108. APPENDIX I l l Procedures i n Determing Enzyme A c t i v i t y and Photophosphorylation (1)  .  Determination of catalase a c t i v i t y Catalase a c t i v i t y was measured by manometric technique  (56).  The procedure i s summarized as follows: a) place 3 ml 0.0001 M phosphate buffer pH 6.8 and 0.2 ml 0.2M #2^2 * * * - f l a s k and 1 ml enzyme homogenate i n the side arm of f l a s k . n  t  ie ma  n  b) Attach the f l a s k to the manometric tube and put into Warburg water bath with temperature maintained at 25± 0.1 C. c) A f t e r e q u i l i b r a t i n g i n water bath f o r 10 minutes,, the f l a s k i s closed and enzymes placed i n side arm is' tipped into the main f l a s k . d) A reading' i s then made at one, two and three minutes a f t e r adding catalase. e) The u n i t of catalase a c t i v i t y i s calculated as u l 0 evolved per ml enzyme i n 3 minutes. 2  (2)  Determination of phosphatase  activity  Phosphatase a c t i v i t y was measured by the Sumner and Somers' (49) method.  The procedure i s as follows:  A) Flasks are placed i n incubator f o r 30 minutes before s t a r t of measurement. b) Run 1 ml enzyme homogenate into each of two Erlenmeyer flasks by a volumetric pipette and then add 5 ml substrate into one f l a s k and mix, while the other f l a s k i s used as c o n t r o l .  109 c) The flasks are incubated at 37 C f o r one hour and 2 ml Folin-Ciocalten reagent i s added to each f l a s k . Add 5 ml substrate to the control blank. d) For color development 5 ml 107o Na2C03 i s added to each f l a s k , mix and allow to stand f o r 10 minutes and then d i l u t e by adding 100 ml d i s t i l l e d water. e) A reading i s made a t 10 to 20 minutes from d i l u t i o n , and i s referred to the phenol standard curve. f) The u n i t of phosphatase a c t i v i t y i s expressed as mg phenol released per 1 ml enzyme i n 1 hour. In p l o t t i n g a standard curve c a l i b r a t i o n of phenol concentration against colorimeter reading, the method used was the same as the above procedure except that 5 ml of standard phenol solution and 1 ml of d i s t i l l e d water were used instead of 5 ml substrate and 1 ml enzyme. (3)  Determination o f phosphorylase a c t i v i t y Sumner's (50) method, which i s used i n this experiment to  determine the amount of inorganic phosphate released i n the solut i o n a f t e r phosphorylase reaction (78).  The procedure i s l i s t e d  as follows: a) 5 ml substrate i s run into two test tubes and incubated i n water bath at 30 C f o r 15 minutes. b) Add one ml enzyme homogenate to the solution and incubate f o r 1 hour i n water bath at 30 C.  UG. c) Add 5 ml o f 6.66%ammonium molybdate, to stop enzyme reaction, 5 ml o f substrate i s then run to the blank tube, d) Add 5.ml o f 7.5N H S0 , and 4% percent FeS0 into each tube, and d i l u t e with 10 ml d i s t i l l e d water, 2  4  4  e) Stand f o r 10 minutes f o r color development, f) Reading i s made i n a Klett-Summerson colorimeter with red f i l t e r . For  standard phosphate curve preparation, the procedure  i s the same as that of Sumner's except that 5 ml of standard phosphate solution i s used, (4)  Determination o f rate of photophosphorylation Whatley and Arnon's (70) method i s summarized as follows: (1)  Chloroplast i s o l a t i o n :  a) Grind 25 gm bean leaves with 4 gm cold sand, 50 ml 0.35 M NaCl and 5 ml 0.2M T r i s - b u f f e r pH 8.0 f o r 2 minutes. F i l t e r . b) Centrifuge f o r 2 minutes at 200xg. c) Discard green supernatant and centrifuge a t lOOxg for 8 minutes. d) P r e c i p i t a t e i s suspended i n 10 ml 0.35M NaCl and used as chloroplast suspension. e) Chlorophyll content determined by Arnon's method(3) (2)  Phosphopyridine nucleotide reductase (PPNR) Preparation:  a) Add T r i s - b u f f e r to supernatant to give a f i n a l concentration of 0.05 M T r i s and add aceton to give a f i n a l concentration 35% acetone. Centrifuge at lOOOxg f o r 15 minutes. The c l e a r yellowgreen supernatant i s used f o r PPNR extract.  1<L1. (3)  Chlorophyll content  estimation(3):  a) Dilute 0.1 ml of chloroplast suspension to 20 ml with 807o acetone, and f i l t e r . b) Read the o p t i c a l density at 652 mu i n a Beckman model B spectrophotometer against 807o acetone. c) Multiply the o p t i c a l density by 5.8 to give mg chlorophyll per ml of o r i g i n a l suspension. (4)  C y c l i c photophosphorylation:  The reaction i s  carried out i n a Warburg f l a s k . a) Add to the main compartment of the f l a s k 0.5 ml each of the following solutions: Tris-HCl buffer pH 8.3 MgCl Sodium ascorbate ADP 100 Urn 0.5 ml FMN 0.3 uM 2  80 uM 10 uM 10 uM  b) Add 0.5 ml K H0P 250 uM to the side arm of f l a s k and c h i l l i n i c e . Add 0.1 ml chloroplast suspension which contains 2.0 mg chlorophyll by d i l u t i n g the s o l u t i o n from (3). 2  4  c) Attach f l a s k to Warburg and shake i n water bath at 15* 1 C with a l i g h t i n t e n s i t y of 1450 footcandles f o r 20 minutes. d) Close and t i p the phosphate from side arm.  Mix.  e) A f t e r 15 minutes, turn o f f the l i g h t s and add 0.3 ml 207o TCA. Centrifuge at lOOOxg for 10 minutes. f) Mix 1 ml f i l t r a t e with 1 ml Magnesia mixture and stand for 1 hour at room temperature. Centrifuge at 1300xg f o r 10 minutes. g) P r e c i p i t a t e i s used for inorganic phosphate determination. h) A control blank i s prepared by adding TCA reactions before pouring i n phosphate.  to stop  112 (5)  Noncyclic photophosphorylation  a) Add to the main compartment of the f l a s k 0.5 ml each o f the following solution: Tris-HCL-buffer ADP 100 uM MgCl 10 uM PPNR  pH 8.0, 80 uM  ?  and to the side arm 0.5 ml of K2HPO4, 250 uM' and TPN pH 8.4 uM. b) Pipette into the centre well 0.2 ml 20% K0H and i n s e r t a f i l t e r paper. c) C h i l l and then add 0.1 ml chloroplast suspension (containing 2.0 mg chlorophyll per ml) to the main f l a s k . d) Attach f l a s k to the manometric tube and place i n equilibrate i n 15* 0.1 C water bath with 1400 foot-candle l i g h t i n t e n s i t y for 20 minutes. e) Pour i n phosphate and TPN from side arm into the main f l a s k . Mix. f) After 15 minutes, measure 0 uptake (same as 4)).  2  evolved and phosphate  g) A control blank i s prepared as i n (4)(h). Oxygen evolution was expressed as m i c r o l i t e r O2 released by 2.0 milligram chlorophyll and 0.5 ml enzyme i n 15 minutes, while PPNR a c t i v i t y was based on O2 evolution and phosphate uptake. (6)  Determination of phosphate uptake  The amount o f inorganic phosphate uptake i n the s o l u t i o n a f t e r reaction i s measured by Sumner's method (50).  

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