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A study of the possible protection afforded by copper, ferrous and ferric ions against the actions of… Jowett, Philip Anthony 1962

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A STUDY OP THE POSSIBLE PROTECTION AFFORDED BY COPPER, FERROUS AND FERRIC IONS AGAINST THE ACTIONS OF 2,lj.-DICHL0R0PHEN0XY ACETIC ACID by t" PHILIP ANTHONY JOWETT B.Sc, University of Wales, I960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biology and Botany We accept t h i s theais as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1962 In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Biology and Botany The University of British Columbia, Vancouver 3, Canada. Date April 19, 1962 ABSTRACT The pos s i b i l i t y that iron and copper can ameliorate the effects of 2,lj-dichlorophenoxy acetic acid ^ i ^ D ) on the morphology and yield of plants has been investigated by Wort (61+). A study of the literature indicates that although there is considerable variation among plant species and also parts of the same plant, 2,lf-D concentrations below 50 p.p.m. promote or have no effect on respiration, while higher concentrations are generally inhibitory. Photosynthesis i s generally inhibited. An experiment was planned and executed to determine whether iron and copper protect against the effects of 2,l|.-D on photosynthesis and respiration, and on fresh, dry and ash weights. Each treatment was replicated six times. Potato (Solanum tuberosum L.) plants were grown to the age required for treatment (lj.-7 weeks) i n a hydroponic system. Treatment was applied either i n the form of 'Attaclay' dust including growth substance and/or metal, or as aqueous spray in which the metal was applied two hours before the growth substance. At given intervals after treatment, photosynthesis and respiration in the leaflets were determined by the diethanolamine buffer method (60) on the Warburg apparatus. After the leaflets had been removed for the final determination of photosynthesis i i and r e s p i r a t i o n , the plants were harvested and their fresh, dry and ash weights were determined, A comparison was made of photosynthetic values based on both area and f r e s h weight. The r e s u l t s were analysed f o r s t a t i s t i c a l s i g n i f i c a n c e by the analysis of variance and Duncan's new multiple range te s t . C o e f f i c i e n t s of v a r i a b i l i t y (C«V.$) were calculated for the various measurements. Xn most cases the c o e f f i c i e n t s were shown to decrease by h a l f during the course of the experiment. Results showed that 2,!^ -D was more i n h i b i t o r y to gas exchange than i t s copper s a l t and also caused greater deformation of the plants. Further r e s u l t s indicated that a p p l i c a t i o n of i r o n with 2tl\.-D i n h i b i t e d the effects measured for 2,1^-D. Gas exchange values for the 2,ij.-D treated plants were frequently s i g n i f i c a n t l y d i f f e r e n t from those of plants treated two hours e a r l i e r with an i r o n spray. The i r o n spray when followed by 2,l|.-D application caused a s i g n i f i c a n t height increase. This suggested that the e f f e c t i v e concentration of 2,ij.-D had been lowered by the i r o n . These observations are discussed i n the l i g h t of recent publications on the r o l e of metal chelation i n auxin action* i i i TABLE OP CONTENTS Page Introduction. Review of Respiratory E f f e c t s of and IAA. . . . 2 Review of Photosynthetic E f f e c t s of 2,lj.-D l\. Review of Other Physiological E f f e c t s of 2,1].-D. . . . 5 Experimental D e t a i l s . . . . . . . 7 (1 ) General Outline 7 (2) The Nutrient Solution 8 (3) Growth Procedure Followed i n t h i s Experiment. • . 11 Treatment Procedure . . . . . . . . . . 15 Sampling Procedure and Manometric Determination . . . . . 16 S t a t i s t i c a l Analysis 21 Results 2£> Outline of Treatments One to Five ..• 26 Tabulated Results of Treatments One to Four 28 Summary of Treatments One to Four . 36 Tabulated Results of Treatment Five . . . . . . . . . 37 Summary of Treatment Five . . . . . . . . . . . . . . 39 Outline of Treatments Six to Eight Ij.2 Tabulated Results of Treatments Six to Eight. . . . . Ijij. Summary of Treatments Six to Eight $1 Outline of Treatments Nine to Thirteen $k Tabulated Results of Treatments Nine to Thirteen. • • 56 Summary of Treatments Nine to Thirteen 69 Leaf Age Test 75 I n f i l t r a t i o n Tests 76 E f f e c t s of Ferrous and F e r r i c Ions Alone. • • • • • • 77 Discussion • • • • • • 79 Summary 8 3 Appendix. • • 85 S t a b i l i t y Constants 85 Table of Least S i g n i f i c a n t Ranges . 86 L i t e r a t u r e Cited 88 i v FIGURES Page 1 Treatment £ Graphs Showing Values Varying with Time w 1+0 2 Treatment 7 Graphs Showing Gas Exchanged Varying with Time if8 3 Treatment 12 Graphs Showing ± Values Varying with Time 5 63 Ij. Treatment 13 Graphs Showing 1 Values Varying with Time V 67 PLATES 1 The Growth and Treatment Tanks • 12 2 The Warburg Apparatus, 2,i|.-D Treated and Untreated Potato Lea f l e t s and the Photoelectric Planimeter. . . 19 3 The Plants from Treatment 5 • • 1+1 l± The Plants from Treatment 6 . . . 1+6 5 The Plants from Treatment 7 1+9 6 The Plants from Treatment 12 61{. 7 The Plants from Treatment 13 68 ACKNOWLEDGEMENTS I should l i k e to thank Dr. D.J, Wort for h i s help and encouragement throughout the course of t h i s work. I am indebted also to Dr. W.B. Schofield, Dr. D.P. Ormrod, Dr. J.R. Stein, Dr. B.R. James, Mr. R.C. Brooke, Mr. R.N. North and Mr. J.G. Thorpe f o r t h e i r help at different stages of the work. F i n a l l y T should l i k e to thank 1) Chemical Machines Ltd., Winnipeg, Manitoba. 2) General Foods Corporation, Caldwell, Tdaho. 3) J.i'R. Simplot Co., Caldwell, Tdaho. fo r t h e i r f i n a n c i a l help. v i 1 Introduction Considerable research has been devoted to 2,q.-D since the appearance of papers by Hitchcock and Zimmerman i n 19q2 (2l|., 2 5 , 26). Much of this research was applied to the more practical aspects of 2,i{.-D used as a herbicide, although a number of workers (for example Wedding (61) , Preeland (17) and S e l l et a l . (lj.7))have been concerned with i t s various effects on the physiology of the plant. In spite of the considerable work done, the fundamental effects of auxin, including 2,!+-D, remain an enigma ( 6 ) . The study of 2,it-D i s particularly d i f f i c u l t because i t appears to produce radically different effects under different conditions, i n different concentrations, on different parts of the plant and on different plant species. The whole subject of synthetic auxins has been reviewed recently i n Vol. XIV of Handbuch der Pflanzenphysiologie, and the metabolic effects of 2,t}.-D are summarized particularly i n the papers of Wort (63) and Cleland ( 11 ) . The literature from 1900 to 1950 on synthetic auxins has been reviewed and brought together by Scholl (i}.6). An account of progress i n auxin research was recently published i n the Proceedings of the Fourth  International Congress on Plant Growth Regulation ( 6 ) . The present study i s concerned with the effects of 2,ij.-D with metal ions, notably iron and copper. A study on 2,lj.-D injury and metal ion protection, based largely on morphological observations and yield, has been carried out and reviewed by Wort (61+), though l i t t l e has appeared in the literature on the physiological nature of such a protection. The fact that l i t t l e i s known of any fundamental 2,lf.-D action makes i t d i f f i c u l t to show protection by metal ions against such action, Xt was decided, therefore, to study the effects of 2,lj-D with and without metal ions on respi r a t o r y and photosynthetic rates, fresh, dry and ash weights. Percentages of the weight values were deter-mined and the weights themselves were compared with the o v e r a l l measured gas exchanges i n order to ascertain whether these gas exchanges corresponded with dry weight accumulation. The l i t e r a t u r e includes a large number of references to r e s p i r a t o r y e f f e c t s of indole acetic acid (XAA) and 2,lj-D but r e l a t i v e l y few to photosynthetic e f f e c t s : Respiration: The most convenient sources of information on re s p i r a t o r y e f f e c t s are two review a r t i c l e s by Avery and Smith i n Plant Growth Substances edited by Skoog (1+9 pp. 105-109) . The work has been done on both excised tissues and inta c t plants, Avena col e o p t i l e s being prominent i n much of the e a r l i e r work. Using a polarographic method, Du Buy and Olsen (l\+) f a i l e d to demonstrate that IAA produced any stimulation of r e s p i r a t i o n i n oat c o l e o p t i l e s . A low oxygen tension r e s u l t i n g from the i n f i l t r a t i o n method used has been suggested as the cause of thi s observation. For the same material XAA was most e f f e c t i v e i n stimulating r e s p i r a t i o n at 10 to 20 parts per m i l l i o n (p.p.m.) while 2,ij.-D (31) was most stimulatory at 100 p.p.m., increasing r e s p i r a t i o n by 2 5 $ . 2,i].-D was i n h i b i t o r y at 1000 p.p.m., decreasing oxygen uptake by 1+0%, The same workers 3 (31) showed pea stem tissue to Increase oxygen uptake by 1+0% with 0.1 to 10 p.p.m. 10$ with 100 p.p.m. 2,ij.-D and to decrease uptake by 1+0% with 1000 p.p.m. 2,1+-D. Work on in t a c t plants has produced more varied r e s u l t s : Pratt (1+1+) reported a 78$ stimulation of oxygen uptake by 50 p.p.m. IAA and 1+0% i n h i b i t i o n of oxygen uptake by 150 p.p.m. IAA i n barley seedlings. 2,1+-D at 1000 p.p.m. i n h i b i t e d oxygen uptake by 67-79$ i n the three days following treatment of barley seedlings, carbon dioxide evolution f a l l i n g from +36$ to -70$ (1+9), Oxygen uptake and carbon dioxide evolution are also reported as f a l l i n g 15 to 25$ with 2,1+-D at 5 p.p.m. applied to wheat and mustard seedlings (lj .9). M i t c h e l l et. a l . (39) using, M i n vitro", tissue s l i c e s of several species with 2,l+-D at 350 p.p.m. and IAA at 1+10 p.p.m., showed percentage i n h i b i t i o n to be greater i n roots than i n stems, except f o r IAA on tomato, and greater with 2,1+-D than IAA. The extent of i n h i b i t i o n was shown to be proportional to the oxygen tension, and to be reversed by washing. I n f i l t r a t i o n of c i t r u s l e a f discs with from 30-100 p.p.m. of 2,1+-D caused an i n i t i a l i n h i b i t i o n of oxygen uptake, followed by a stimulation a f t e r forty-eight hours ( 6 1 ) . French and Beevers (18) showed IAA and 2,lj.-D to stimulate r e s p i r a t i o n i n corn c o l e o p t i l e s when inducing growth i n length, i n d i c a t i n g the r e s p i r a t o r y e f f e c t to be a r e s u l t of growth rather than a di r e c t response to 2,ij.-D. Recently Oaborne and Hallaway (1+1) showed a respiratory increase i n Buonymus l e a f discs cut from areas treated with 2,q.~D butyl ester i n ethanol at 26 micrograms per m i c r o l i t r e twenty-four hours previously. The rate continued k to r i s e and the treated portion of the l e a f remained green while senescence set i n over the remainder of the l e a f . This stimulation mounted to 200-300$ of control and per s i s t e d f o r 13 days. The rate i n the h a l f of the same l e a f not treated d i r e c t l y increased to 170$, declining to 130$ of control a f t e r f i v e days. Thus there appears a general picture of re s p i r a t o r y increase at low concentrations and i n h i b i t i o n at high concentrations, though the evidence i s by no means consistent f o r t h i s generalization. Photosynthesis: photosynthesis appear to have been used as there are publications on auxin e f f e c t s on photosynthesis. Sugar translocation from the f i r s t t r i f o l i a t e leaves of soybean was found to Increase with a p p l i c a t i o n of 2£ micrograms of 2,lj-D to these leaves i n an i n translocation, i n d i c a t i n g a p a r a l l e l i s m with r e s p i r a t i o n (57). Loustalot and Muzik (3k-) drew a i r through leaves of velvet beans by means of a porometer cup and passed th i s a i r through absorption towers and a flowmeter for three hours. Duplicate treated and control leaves were used. 2,1|.-D at rates of 0.1$ - 0.05$ caused a rapid cessation of photosynthesis; 0.01$ caused a decline over f i v e hours, then a steady depressed l e v e l u n t i l death followed In a few days; 0.001$ caused a s l i g h t depression aft e r a week. I n h i b i t i o n was correlated with anatomical damage. Preeland (16) covered bean plant tops with a b e l l jar and analyzed the gas every two hours by passing i t through absorption towers. He showed that with 2,lj.-D a p p l i c a t i o n Almost as many techniques for the measurement of Higher dosages produced a decrease 5 there was a generally steady decline to 70$ of the o r i g i n a l rate of photosynthesis over three days; however, r e s p i r a t i o n showed a f a l l of 70$ of the o r i g i n a l rate i n one day, and a r i s e to 110$ reversing to 1 0 0 $ of the o r i g i n a l by the t h i r d day. Later, (17) using the Canadian pond-weed Elodea (Anacharls), he u t i l i z e d oxygen evolution measurements to demonstrate a decline to 60$ i n two days with 30 p.p.m. 2.,lj-D, and to 67$ i n one hour with 100 p.p.m. 2,i}.-D. Respiration declined, then rose to 130-114.0$ of the o r i g i n a l r a t e by the second day. Wedding ( 6 1 ) , using i n f i l t r a t e d c i t r u s l e a f discs, was able to show manometrically that the decline i n photosynthesis was a function of the degree of d i s s o c i a t i o n of the 2,q.-D i n the solutions used for i n f i l t r a t i o n . He buffered the solutions at di f f e r e n t pH values. Thus i n the case of photosynthesis there i s an almost exclusive i n h i b i t o r y e f f e c t as a resu l t of 2,[j.-D treatment at any concentration. The effects on production of fre3h and dry matter, and mineral content appear to depend very much on concentration, mode of a p p l i c a t i o n and plant material used ( 6 3 ) . Very recently Miller, Mikkelson and Huffaker ( i n press) have produced some i n t e r e s t i n g r e s u l t s showing that 0 . 5 and 1 p.p.m. of 2,i|.-D as a spray stimulated the growth of bean seedlings almost immediately, and that t h i s stimulation was increased by i r o n as sulphate or by ethylene diamine di-o-hydroxyphenyl acetate at IjpOO p.p.m. ferrous and f e r r i c ion r e s p e c t i v e l y . This proved to be the case also i n height increase and l e a f area; however, dry weight y i e l d was depressed by the i r o n supplements at low concentrations. 6 The i r o n supplements were able to overcome the i n h i b i t o r y e f fects of 2,li.-D at 5 and 10 p.p.m. I t would appear from other work (I4.7) that 2,lj.-D e f f e c t s on the composition of the entire plant are secondary to those of r e d i s t r i b u t i o n within the plant. The present experiment was designed to produce values for photosynthesis and r e s p i r a t i o n , fresh, dry and ash weights of a s e n s i t i v i t y s u f f i c i e n t to demonstrate any difference between the effects of IAA and 2,lj.-D on any of these by copper and i r o n . 7 Experimental Details 1. General Outline In order to obtain the greatest amount of information from the material available for study, a primary consideration i n the design of the experiment was to reduce v a r i a b i l i t y between plants to a low level, and then to allow for the inevitable residue of va r i a b i l i t y , arising both from the material and the experimental techniques, by replication. This permits an estimate of experimental error to be made and by the application of s t a t i s t i c a l methods, described later, allows one to determine both the significance of thae results and the sensitivity of the experiment as indicated by the coefficient of variation. The potato, Solanum tuberosum L., i n this case cv. •Idaho gem1, provides a useful starting point i n the reduction of va r i a b i l i t y ( 2 3 ) . Each tuber of reasonable size was found to provide up to twenty 'eyes' or 'seed pieces' from which the young clonal plants were grown. The apical dominance found i n whole potatoes was largely removed by cutting 'seed pieces' from the tuber. Though there was s t i l l considerable variation in the time of sprouting, i t was found possible, by planting three times as many pieces as were ultimately required, to obtain a population of plants essentially uniform i n size and habit. The potatoes were grown throughout the experiment i n a growth chamber provided with a 16 hour photoperiod, with temperature controlled at 72° F i n the light period and 62° F in the dark period. Relative humidity was i n the light and 8 6$% In the dark. Light i n t e n s i t y varied with the height of the plants at the three phases of growth, being 8 0 0 - 9 0 0 , 1200-lq.OO and 1 1 0 0 - 1 3 0 0 foot-candles. Light i n t e n s i t y was uniform over the growing area and was measured with a Weston Illumination Meter model 756. watering or l o c a l i z e d i o n i c concentrations i n s o i l , and to provide clean, whole plants f o r measuring fresh and dry weights, i t was considered desirable to grow the potato plants f o r as long as possible i n aerated nutrient solution. 2. The Nutrient Solution solutions are M i l l e r (37) and Hewitt ( 2 3 ) . The l a t t e r worker favours the four main nutrient s o l u t i o n of the same constituents with which calcium: potassium r a t i o s can be a l t e r e d . The formula used was that of Hoagland and Arnon (28) which was based o r i g i n a l l y on the r a t i o of the elements found on analysis of tomato plants. The d e t a i l s were obtained from Thomas ($L\). To eliminate the v a r i a b i l i t y a r i s i n g from uneven The two main sources of Information on nutrient as the ' c l a s s i c a l ' formula of Knop ( 3 7 ) , l a r g e l y for the ease The formula used was: A. Molar solutions of ma cr onu trIents M i s . / l i t r e of nutrient so l u t i o n K H 2 P 0 ^ KNO3 CafNO-^ 5 1 2 9 B. Solution of essential microelements 1 C. Iron as 0.7$ f e r r i c c i t r a t e 1.5 The s o l u t i o n of microelements, B, was of the following composition: Micronutrient Grams per l i t r e Experience gained during the use of the above solut i o n showed that demineralized water was more s a t i s f a c t o r y than the d i s t i l l e d water which was acid (pHif-5). Demineralized water wa3 used at Long Ashton (23) where several nutrient solutions of e s s e n t i a l l y the same composition as the above have been i n use. Another change was i n the form of i r o n supplied. I n i t i a l l y i r o n as 'versene' i r o n ethylene diamine tetra-acetate (PeEDTA) chelate was used at the rate of f i v e milligrams Pe/ml. of stock so l u t i o n and one ml. of stock sol u t i o n per l i t r e of nutrient. This proved s a t i s f a c t o r y u n t i l a fresh sample of PeEDTA with a lower proportion of i r o n was used, when considerable growth retardation at the early stages, followed by root discolouration and necrosis at the l e a f margins resulted. The i r o n c i t r a t e has been used subsequently, i n accordance with the general practice ( 2 3 ) , r e s u l t i n g i n a generally more s a t i s f a c t o r y growth. The observations of Heath and Clark ( 2 1 , 22) on the p a r a l l e l behaviour of EDTA and auxins may be further reason to avoid the 10 PeEDTA, though FeEDTA is said to have no effect on growth (53> 62). The pH problem proved d i f f i c u l t with the d i s t i l l e d water. The limitations of pH are set by root injury, i n a b i l i t y to take up calcium at pH values lower than four and by precipitation of calcium phosphate (19) at pH7 or above (3,1^,37). Calcium also becomes unavailable at pH values i n excess of 8 . 5 (3>K)» The PH d i f f i c u l t y was overcome when demineralized water was used. D i s t i l l e d water required pH correction with a l k a l i prior to preparing the nutrient; i f a l k a l i was added afterwards precipitation of calcium and/or magnesium resulted. Precipitation occurs commonly i n nutrient solutions and i s not generally considered deleterious. Tottingham (55) noticed that the precipitate formed i n Knop's solution was almost exclusively calcium sulphate. Knop's solution and other nitrate-based solutions tend to become more alkaline due to rapid uptake of NO3" relative to K*. Any residual K* neutralizes acids i n the medium. Attempts have been made to buffer nutrient solutions with HHj£:N03 ratios or HH^HgPO^ (2), though pH was found not to be c r i t i c a l between ij.,5 and 7 .5 by Arnon (2). Potato plants were shown to grow more satisfactorily i n Knop's solution than i n a number of other solutions tested (37)t and to present no special problems i n this regard. Houghland (29, 30) grew potatoes i n nutrient solution for studies of phosphorus levels using a nutrient with high N03~:NH^+ ratio and a solution pH of \+ to Continuous aeration was provided. 11 3« Growth Procedure Followed In this Experiment The f i r s t stage of growth, that of bud break and growth of the young plant to a state independent of the 'seed piece', was c a r r i e d out i n vermiculite watered with nutrient s o l u t i o n . A teaspoon was used to cut c i r c u l a r pieces of tuber from around the eye. The pieces were then placed on edge i n vermiculite contained i n enamel trays. F o r t y - f i v e pieces were sown i n each of two trays and covered with a t h i n layer of vermiculite. The time taken to reach a size suitable f o r transplanting to the second stage varied with the condition of the tubers but was generally 10-llj. days. I t was important that the vermiculite should not be over-watered at t h i s stage. When the plants reached the desired size they were removed from the vermiculite. The 'seed piece'.was c a r e f u l l y removed from the new plants and the roots were washed i n nutrient s o l u t i o n . The plants were then selected f o r uniformity and planted out between halves of lacquered corks i n the four covers of the main growth tank. The phase of growth intermediate between bud break and treatment was conducted i n a s p e c i a l l y designed growth tank (plate 1). I t was p a r t i c u l a r l y important that a l l plants should be i d e n t i c a l l y treated. For t h i s reason there was provided a large tank of 100 l i t r e s capacity with an aerating and s t i r r i n g mechanism i s o l a t e d from the plant growing part of the system. The tank was of wood (2' x 3' x 7") with a polythene sheet l i n i n g . A wooden cross-piece was provided above nutrient l e v e l to support four lacquered hardwood boards which were d r i l l e d to 12 PLATE 1 IIIIIIIIII ' Ni^iL\ JBP-ST I S S & S B I The Growth Tank D e t a i l of the F l o a t and M i c r o s w i t c h Assembly The Treatment Tanks hold s p l i t corks of 1 3/V di a» spaced s i x inches apart. Up-draught edge effects were eliminated by a nine inch flange on the tank. By spacing the r e l a t i v e l y small plants at such a distance the need f o r guard plants was eliminated, as each plant was ostensibly unaffected by the presence of i t s neighbour. The tank was placed on a cart, the lower shelf of which carried a f o i l - c o v e r e d carboy of $0 l i t r e s capacity. The carboy and tank were connected by a tube forming a continuous siphon. The rubber bung of the carboy was provided with three tubes, the siphon, a large diameter a i r l i n e connection with aerators running to the bottom of the carboy and a small diameter ttbleed" valve. The a i r l i n e was separated from the compressed a i r supply by a solenoid a i r valve. On the tank i t s e l f was mounted a microswitch, the 'on' pressure of which exceeded the ' o f f pressure. This was connected by copper wire to a glass b o t t l e , acting as a f l o a t and p a r t i a l l y submerged i n the nutrient, and so arranged as to turn the microswitch on and o f f as the l e v e l of nutrient reached a low and high l e v e l . With t h i s arrangement, the growth tank was charged with aerated nutrient every 20 minutes, the non-aerated nutrient siphoning back Into the carboy. This resulted i n a two inch f l u c t u a t i o n i n the l e v e l of nutrient i n the growth tank. The upper end of the siphon was c a r r i e d to the bottom of the tank and directed to s w i r l the nutrient gently as the tank was being charged. Aeration was thus provided by both p a r t i a l submergence and exposure of two inches of root, and the charging of the Ik nutrient s o l u t i o n i t s e l f with a i r while under pressure. As the nutrient passed from the lower to the upper l e v e l , a i r appeared from out of solu t i o n to form bubbles on the roots and walls of the tank. The excellent root growth achieved by the potatoes was s u f f i c i e n t i n d i c a t i o n that the aeration was adequate. The f o l i a r a p p l i c a t i o n of growth substances meant that control and treated plants had to be grown i n separate treatment tanks, since loss of the applied growth substances from the roots with consequent contamination of control plants would have been an ever present danger. For t h i s reason the f i n a l stage of growth, the treatment stage, was c a r r i e d on i n nearly rectangular polythene baths (21" x 1 3 " ) modified to take the covers from the main growth tank (Plate 1 ) . Holes were d r i l l e d Into the covers to allow d i r e c t aeration of each nutrient tank. Three tanks were supplied, each one sprayed with ' s i l v e r chrome' paint to keep i t l i g h t t i g h t . Each tank contained 10 l i t r e s of nutrient solution, and the covers were transferred from the main growth tank to the treatment tanks a f t e r a f i n a l s e l e c t i o n of 18 out of 2k morphologically uniform plants. The duration of growth i n the main tank depended on the age of potato plants required f o r treatment and t h e i r rate of growth i n the main tank. Xt was found that size was more useful than age as a c r i t e r i o n f o r time of treatment. The plants at t h i s stage were generally four to s i x inches high and three to s i x weeks o l d . The f i r s t treatments were applied to 2k day-old plants, l a t e r ones to plant about I4.O days old. 15 Treatment Procedure The growth substances and metal s a l t s were applied to the potato plants i n two forms at d i f f e r e n t times: 1. The quantities of growth substance and metal s a l t to be used were dissolved i n water and/or alcohol and taken up i n a weighed quantity of commercial 'attaclay' dust to give the required percentages. When dry the dusts were ground to th e i r o r i g i n a l f i n e consistency. A polythene cover, with 3ix small holes d r i l l e d i n the upper edges, was placed over the plot of plants, and O.lj. gras. of the appropriate dust was blown through the holes, to cover the whole area under the cover (2 sq. f t . ) i n a t h i n layer of dust. This dosage was equivalent to 20 l b s . per acre. 2. Aqueous solutions of growth substance, and/or s a l t , were made up and a h o r t i c u l t u r a l sprayer was used f o r t h e i r a p p l i c a t i o n . The plants were sprayed to dr i p . Three p l o t s , each of s i x similar-plants, were treated. As a rule the sequence growth substance, growth substance and metal s a l t , and control was followed for the ap p l i c a t i o n of each treatment. 16 Sampling Procedure and Manometric Determination At suitable time .intervals a f t e r the ap p l i c a t i o n of treatment to the three plots of plants, four l e a f l e t s , as f a r as possible of the same size and p h y s i o l o g i c a l age, were taken from each plant. These were used to determine the rates of photo-synthesis and r e s p i r a t i o n . The determination of r e s p i r a t i o n by manometric methods i s a simple procedure requiring only the absorption of carbon dioxide as i t i s produced (56). However, i f photosynthesis Is to be determined at the same time, a more elaborate method must be used to provide an atmosphere with a constant percentage of carbon dioxide. One of the most widely used methods i s based on Pardee's (1+2) work. This method has been used by Wedding (60,61), and checked by Krebs ( 3 3 ) $ who observed that no solution appreciably more e f f e c t i v e than Pardee's had yet been found f o r carbon dioxide retention. Warburg et a l . (8) appraised the method, observing: "This type of reagent has a special advantage In photosynthetic studies i n that i t provides a v i r t u a l l y Inexhaustible supply of carbon dioxide so that experiments at high l i g h t i n t e n s i t i e s can be performed for days i n a given manometer vessel without danger of C02 depletion...." They also compared r e s p i r a t o r y rates indicated by the diethanolamine and sodium hydroxide. They observed: n•.• the exact agreement of Xog = -80 Is happenstance... Good r e s u l t s were obtained aft e r a duration of a week or more. 17 The following buffer was made up (see 1+2, 56, 60) t 60$ aqueous dlethanolamine 50 mis. 6N HCl 15 mis. Water 10 mis. Powdered KHCO3 15 gms. The bicarbonate dissolves overnight and the buffer keeps i n d e f i n i t e l y under r e f r i g e r a t i o n (Ij2). Krebs (32) found that autooxidation of the dlethanolamine occurred at high oxygen tensions and temperatures and recommended the addition of 0.1$ thiourea to i n h i b i t the oxygen uptake. In the present experiment the thermobarometer correction was found adequate, though addition of thiourea may be desirable as a matter of course. This buffer gives a constant carbon dioxide concen-t r a t i o n of approximately 1$ and the observed gas exchange r e s u l t s from the u t i l i z a t i o n or production of oxygen by the l e a f l e t s . Portions ( 0 . 5 ml.) of the buffer were pipetted into the centre well of each Warburg f l a s k . The l e a f l e t s were then harvested from the experimental plants by pinching them o f f at the rachis, and weighed on a to r s i o n balance. I n l a t e r runs the area of the l e a f l e t s was determined i n terms of the l i g h t intercepted by thera i n an improvised photoelectric planimeter. The l e a f l e t s were then spaced evenly, without overlap and with a drop of water, on the bottom of the Warburg f l a s k . A f i l t e r paper accordion was Inserted into the centre well to Increase the exchange surface of the dlethanolamine buffer. To f a c i l i t a t e gas exchange the l e a f l e t s were set i n the flask with the abaxial surface uppermost. When attached to the manometers the flasks were spaced around the Warburg bath with the 18 treatments alternating, and two thermobarometers set up with buffer and no l e a f l e t s on opposite sides of the bath. The flasks were equ i l i b r a t e d f o r 15-20 minutes with the l i g h t s on, and the p o s i t i o n of the manometers was changed at in t e r v a l s to ensure uniform i l l u m i n a t i o n . Taps were closed i n s t r i c t r o t a t i o n at 10 second i n t e r v a l s and readings taken at 20 minute i n t e r v a l s i n the same sequence. The 20 minute reading provided a check against leakages, the I4.O minute reading being noted and corrected f o r thermobarometer changes. A f t e r 1+0 minutes with the l i g h t s on, a 15-20 minute e q u i l i b r a t i o n period and a f o r t y minute dark run followed, the readings again being corrected for thermobarometer changes. Corrections for tissue weight, area of l i g h t interception and f l a s k constants were made. The l i g h t and dark manometer deflections were summed to give a true photosynthesis value, and respiratory and photo-synthetic gas exchanges entered on the analysis of variance sheets as m i c r o l i t r e s of oxygen exchanged by 100 milligrams of tis s u e i n 1+0 mins. Lighting on the Warburg was provided by a 16" white c i r c l i n e fluorescent tube of 1+0 watts placed four inches from the f l a s k s , and four 150 watt white sprayed tungsten filament lamps spaced evenly eight inches above the f l a s k s . This combined l i g h t i n g gave 1+00 foot-candles, within ten percent, at f l a s k l e v e l . Temperature wa3 maintained at 30°C both thermostatically and by a fan blowing a i r across the l i g h t s and water surface (Plate 2). 19 PLATE 2 A Potato Leaflet Treated An Untreated Potato Leaflet with 25% 2,4-D i n Attaclay 216 Hours Treatment 5 216 Hours Treatment 5 * 1. Light tube, s l i d e and photoreceptor 3. Light meter scale 2. Rheostat 4. Torsion balance 20 The use of f r e s h weight as a basis for the expression of photosynthetic r a t e can be c r i t i c i z e d i n that i t presupposes the l e a f material to be of uniform thickness and hence the leaves of an area to be proportional to the fresh weight. This d i f f i c u l t y i s l e s s apparent i n l e a f material amenable to the cutting of l e a f d i s c s , for by t h i s method a d e f i n i t e surface area i s included i n the f l a s k . However, the compound leaves of Solanum tuberosum provide too few l e a f l e t s of the same physio-l o g i c a l age to cater for the l e a f disc method, thus entire l e a f l e t s were used, which in e v i t a b l y varied i n surface area. This surface area discrepancy was p a r t i c u l a r l y pronounced afte r a p p l i c a t i o n of the growth substance:. (Plate 2 ) , as d i s t o r t i o n of l e a f growth i s one of the most obvious effects of these substances on the whole plant and has even been suggested as a sensitive assay ( 9 , 10, 27 , 3^) • With these considerations i n mind, a simple form of photoelectric planlmeter was devised (Plate 2), This was not as elaborate as those of Maggs and Milthorpe (36, 3 8 ) , but i t d i d give an estimate of the l i g h t intercepted by the l e a f l e t s as It passed down from v i r t u a l l y a point-source through a matt black cylinder to the photoreceptor of the Weston model 7^6 i l l u m i n a t i o n meter. The l i g h t strength was adjusted by a rheostat to an 80 foot-candle d e f l e c t i o n and the s p e c i a l l y designed s l i d e , with the l e a f l e t s spaced evenly on the glass, Inserted. The d e f l e c t i o n from 80 foot-candles was recorded and that due to the glass was subtracted. Tests with pieces of graph paper and aluminium f o i l showed that within l i m i t s of experimental error there was a d i r e c t p r o p o r t i o n a l i t y 21 between area of material exposed to l i g h t and def l e c t i o n , and that a d e f l e c t i o n of eight foot-candles was equivalent to a surface area of one cm. Thus the photosynthesis values from l a t e r r e s u l t s are expressed as m i c r o l i t r e s of oxygen produced per unit d e f l e c t i o n i n lj.0 rain,, f o r comparison with the weight-based values, to determine how fa r the observed photosynthetic values were a function of l e a f d i s t o r t i o n . The potato plants were noted to grow at a rate s u f f i c i e n t to supply uniform l e a f l e t s about every t h i r d day. As fa r as was possible estimations of photosynthesis and r e s p i r a t i o n were made on days one, four and seven. Afte r the f i n a l estimation of gas exchange, the entire plants were harvested, the roots blotted, and the plants weighed i n d i v i d u a l l y . Height measurements and morphological observations were made, and the plants were oven-dried at 90°C and reweighed to give dry weight and, by c a l c u l a t i o n , percentage dry weight values. Xn some cases the plants, or parts thereof, were ashed and ash weights determined. A l l weighings, percentages and height measurements were entered on the analysis of variance sheets, as were the photo-synthesis and r e s p i r a t i o n values. Analysis of variance was ca r r i e d out as follows: S t a t i s t i c a l analysis Xt has been observed, i n the discussion of growth procedure and experimental evaluation of treatment e f f e c t s , that though considerable trouble has been taken to reduce the 22 v a r i a b i l i t y of the experiment at each step, a recognized residue of v a r i a b i l i t y remains which i s not e a s i l y eradicated by Improvement i n experimental technique. Such sources of v a r i a b i l i t y may be summarized as follows: 1* Differences between the several potato tubers used at each planting. 2. Differences i n the time of bud break. 3. Possible undetected l o c a l environment e f f e c t s i n the growth room. Ij.. Possible l o c a l treatment differences within p l o t s . Possible nutrient differences between p l o t s . 6. Differences i n s i z e , shape, weight and physio-l o g i c a l age of l e a f l e t s sampled for manometric determinations• 7. Local environmental differences i n the Warburg apparatus. (These would be p a r t i c u l a r l y evident i n the l i g h t phase). 8. The generally recognized (£1+) error l e v e l of the Warburg apparatus at 9. Inevitable differences i n stature and morphology of the plants. 10. Local temperature differences i n oven and muffle furnace. Observed differences may be a t t r i b u t e d to treatment effects i f the plants are reselected for uniformity and a l l experimental and treatment steps are properly randomized. Only numbers three and f i v e remain as v a l i d c r i t i c i s m s i f these precautions are followed and the c r i t e r i o n of s t a t i s t i c a l s i g n i f i c a n c e i s used. No evidence has been obtained to indicate l o c a l environment and nutrient differences as causes f o r any of the r e s u l t s . 2 3 Six r e p l i c a t i o n s were used i n every run, and the r e s u l t s of each run were analyzed by two s t a t i s t i c a l t e s t s . 1 . The analysis of variance, culminating i n the variance r a t i o , or 'F' test, for the significance of the whole run (5>1 para 7 . 3 ) . 2 . Duncan's new multiple range t e s t , to decide exactly the si g n i f i c a n c e between the three means ($1 para 7 « 5 , and appendix). This test was used i n preference to the l e a s t s i g n i f i c a n t difference as use was made of non-independent comparisons (£l para 7»i+)« The calculations Involved i n these analyses are i l l u s t r a t e d i n the specimen c a l c u l a t i o n enclosed: 2k March 2nd 1962  Analysis of Variance Treatment 13 Ferrou3-24-D  21+ hrs. Reap. Control 24-D Fe++2,I}.-D Total 1 2 I 6 5.21 1 5.12 5.99 1+.73 5.08 5 4 6 5.77 6 4 9 5.kli 5.57 6 4 5 7.05 5.76 6.05 5.98 6.19 Total 31.59 32.63 3748 101.71 Sura of square3 (Total) 2/R 167.23 166.32 180.16 177.56 235.16 231+.13 582.55 578.01 Error term 0.91 2.60 I..03 k*5k Mean 5.27 s.kk 6.25 5.65 1 Microlitres of oxygen used by 100 milligrams of tissue in forty minutes. f r p o t a l 1 2 r 1 0 1 7 l l 2 Correction term LTotajj = q u i . (U = 5 7 ^ . 7 1 Total sum of squares - 582.55 - 571+.71 = 7.81+ Treatment sura of squares = 578.01 - 5 4.71 = 3.3 Source of variation O.F. Sura of sqs. Mean Var. aq. ratio Among plots (treat- 2 3.30 1 .65 5.1+5* ment) k-$k Within plots (error) 15 0.303 Total 17 7.81+ F levels for 2/l5 D.F. 0.05 = 3 .68 0.01 = 6.36 Duncan1a Test Sx = /error mean square/R = ^/°*|°3 = 0.225 S.S.R1 (from table) Limit Number of Means L.S.R' Ranked Means Mean Diff. 0.05 0.01 2 3 2 3 3.06 3.21 k.26 1+.1+8 0.690 a 5.27 0.716 b 5.1+q- h-a 0.17 0.952 c 6.25 c-a 0.98* 1.000 c-b 0.81* 1 = Significant studentized ranges. 2. Least significant ranges, Means connected by a vertical line at the side of the rank are not significantly different. / _ Coefficient of v a r i a b i l i t y (CV$) = ^ t o t a l ^ e l u i " yOOlxlOO _ n € 57bT" " 9 , 7 5 & Results The r e s u l t s of each treatment and analysis are presented i n the following pages. The in d i v i d u a l treatments are described together with the reasons for them. Details are given f o r any departure from the procedure outlined i n the preceding pages. Morphological observations are made where appropriate, and each treatment and r e s u l t i s evaluated. Mean values for photosynthesis and r e s p i r a t i o n are given, as they were determined and analyzed, as m i c r o l i t r e s of oxygen exchanged per 100 mg. (per unit of photometer deflection) per 1+0 minutes. Also tabulated are error mean square values (E.M.S.) from the analysis of variance tables and corresponding l e a s t s i g n i f i c a n t ranges at 5$ f o r three means (L.S.R. 5% 3 mns.). Some other pertinent l e a s t s i g n i f i c a n t ranges are given i n the appendix and a l l other mean values are given as inches, grams, percentages or Individual counts. The means are ranked i n columns and significance i s shown, as described on the previous page, both as the variance r a t i o and as indicated by Duncan's t e s t . The variance r a t i o s exceeding 3»68 and $.36 are s i g n i f i c a n t at the $% (.05) and 1% (.01) l e v e l s r e s p e c t i v e l y . These are designated 3»68"^  and 6.36**. Overall mean gas exchange values are given, and for every error mean square term the corresponding c o e f f i c i e n t of v a r i a b i l i t y (C.V.$) i s tabulated. 26 Treatments One to Five The f i r s t two runs were made using indole - 3-acetic a c i d (IAA) and copper sulphate, i n attaclay dust. Indications were that t h i s combination was the most l i k e l y one to involve chelation, and this was considered a l i k e l y explanation of a protective action ( 1 , 1 2 , 2 1 , 2 2 , 3 5 ,lj . 3 ) . Treatment 1 . 1) 50 p.p.m. IAA and 50 p.p.m. copper (as sulphate) - i n attaclay dust, 2) 50 p.p.m. IAA i n attaclay dust. 3) Control (untreated). Dust applied at 5 gm. to each plot of 2 square feet (250 l b s / a c r e ) . Treatment 2 1) 1000 p.p.m. XAA and 2000 p.p.m. copper (as _ sulphate) i n attaclay dust. 2) 1000 p.p.m. XAA i n attaclay dust. 3) Control (treated with pure attaclay dust). Dust applied at O.if. gms. to each plot (20 l b s / a c r e ) . A l l remaining dust applications other than i n treatment 7 were made at O.ij. gms. per p l o t . A l l control plots i n the remaining dust applications were treated with pure attaclay dust. In preparation, a l l of the dusts were treated i n the same way to eliminate any ef f e c t s from changes i n the consistency of the powder. The following three runs were made using 2,1^ -D and a copper s a l t of 2,l|-D from Pittsburg A g r i c u l t u r a l Chemicals Corporation. Portions (O.ij. gms.) of dust of the following compositions were applied to each p l o t : 27 Treatment 3 1) 0 . 2 9 $ cupric 2,1^-D. 2) 0 . 2 5 $ ' 2 ,IL-D . 3) Control, Treatment l\. 1) 2 . 9 $ cupric 2,4 . - D . 2) 2 . 5 $ 2 , 4 - D . 3) Control. Treatment 5 1) 29$ cupric 2,1{.4D. 2 ) 2 5 $ 2 , I | . - D . 3) Control. Only i n the l a s t treatment was the dosage of 2 , l j .-D • s u f f i c i e n t to cause any noticeable morphological effects within nine days. o 2k Day Old Plants Treatment 1 June 7th-June llith 1961 Results Photosynthesis 2k hrs. 1+8 hrs. 96 hrs. ±kk nr3. 192 hrs. Mean Means and 11.10 C u ^ L Treatments 13 .70 IAAj~{ Ranked 16.00 Con(3) 12 .80 Con 13.1+0 Cu 1ZJ..00 IAA 12.20 IAA 12.1+0 Cu 12.90 Con 9 . 8 8 IAA 10.1+0 Cu 11 .50 Con 9 .1 )5 Con 9 . 9 5 IAA 10.20 Cu 11 .50 Cu 11.91+ IAA 12 .53 Con Variance Ratio 0.62 E.M.S. 6 5 . 3 5 Total Mean 13 .51 L.S.R. 5% 3 mns. 10 .51 C.V.g 5 9 . 8 0 .27 9 . 1 4 13.1A 3 . 9 3 2 . 7 o.ok 22.2 12.1+8 5 . 8 37.8 0 .31 13.2 10 .59 5.1 31+.3 0.11+ 6 . 0 3 9.86 2^9 Respiration Meahs and 3 . 1 1 Cu Treatments 3.1+3 IAA Ranked 3 . 6 5 Con 5 . 0 3 Con 5 . 8 3 Cu 5 . 9 7 IAA 3.81+ Con 3 . 9 Cu 3 . 9 8 IAA 3.62 3 . 9 3 I+.33 IAA Cu Con 1+.08 XAA J+.21 Cu 1+.1+0 Con 1+.20 Cu 1+.22 IAA 1+.25 Con Variance Ratio 0 . 0 7 E.M.S. "" 1+.55 Total Mean 3*1+0 L.S.R. 5 $ 3 mns. . 2 . 7 C.V.g 62.7 1+.50" 1 . 0 5 . 6 1 1 . 3 17.8 0 . 0 1 1 . 3 3 3 . 9 1 1.1+ 2 9 . 5 0 . 6 6 1 .16 3 . 9 6 1.1+ 2 7 . 2 0 . 7 3 0 . 2 2 1+.23 0 . 5 7 1 1 . 1 Results (Continued) Other Measurements Means and Treatments . Ranked E.M.3. Total Mean L.S.R. % 3 mns. O.V.fo Fresh Wt. Dry Wt. % Dry Wt. % Ash 16.59 Cu 1 6 . 6 9 Con 1 7 . 7 7 IAA 1.17 Cu 1 . 2 k IAA 1 . 2 5 Con 6 . 8 9 IAA I 6 . 9 8 Cu || 7 . 4 I Con 1 1 9 . 1 5 Con 2 0 . 0 7 IAA 2 1 . 6 6 Cu > 0.35 55.34-1 7 . 0 2 0 . 0 0 3 0 . 3 5 1 . 2 2 3 . 5 9 0 . 1 3 7 . 1 0 8 . 3 7 * * 1 .17 2 0 . 2 9 9.5 4.3.7 0 . 7 7 1+8.5 0 . k 6 5 .1 1 .37 5.3 Footnote: (1) IAA + CuSO^ p l o t . (2) IAA p l o t . (3) Control p l o t . 2ll Day Old Plants Treatment 2 June 20th-June 2 6 t h 1961 Results Photosynthesis 2l+ hrs. 1+8 hrs. 96 hrs. XI4J4. hrs. 192 h r a Mean Means and 1 0 . 7 8 Cu^p. Treatments 1 0 . 9 5 IAA;~i Ranked 1 1 . 9 5 Con(3) 9 . 1 7 IAA 9 . 7 3 Cu 1 0 . 2 9 Con 6.31* IAA 7 . 3 0 Con 8.8I4. Cu 8 . 5 8 IAA 8 . 6 1 Con 8 . 7 2 Cu 9 . 3 0 IAA 9.82 Con 1 0 . 1 5 Cu 8 . 8 7 IAA 9 . 5 9 Con 9.61+ Cu Variance Ratio 0 . 8 l E.M.S* 2 . 7 0 T o t a l Mean 11.28 L.S.R. 5 $ 3 mns. 2 . 1 0 c.v.# i i+.6 1 .11 1 .67 9 . 7 3 1 . 6 5 1 3 . 3 1+.59* 2 . 0 6 7.1+9 1 . 8 2 19.2 0 . 0 3 3 . 2 0 8 . 6 3 2 . 2 8 2 0 . 7 0 . 2 1 2 . 9 0 9 . 7 7 2 . 1 6 17.1+ Respiration Means and Treatments Ranked 5 . 3 8 Cu 5 . 8 6 Con 6.1+2 IAA Variance Ratio 1 . 5 0 E.M.S. Tot a l Mean L.S.R. 5 $ 3 mns. C.V.$ 1 . 1 3 5 . 8 9 1 .37 1 8 . 0 5 . 7 9 Cu 5 . 9 9 Con 6.1+1 IAA 1 .07 0 .61 6 .09 1 .03 12.9 1+.23 IAA 1+.68 Cu 1+.68 Con 0 . 7 8 0 . 5 1 1+.53 0 . 9 1 1 5 . 8 1+.20 Cu 1+.32 Con 1+.38 IAA 0.011+ 1 .11 1+.30 1 . 3 1 21+.5 5 . 1 3 Con 5 . 3 8 Cu 5 . 5 0 XAA 0 . 1 7 1.1+9 5 . 3 3 1 . 5 6 2 2 . 9 5 . 1 2 Cu 5 . 2 0 Con 5 . 3 9 XAA Results (Continued) Other Measurements Preah Wt. Dry Wt. Means and Treatments Ranked 18.68 Cu 19.95 Con 21.63 IAA Variance Ratio 0 . 2 5 E.M.S. 5 3 . 3 Total Mean 2 0 . 0 9 L.S.R. $% . 3 mns. 9 . 3 0 C.V.fo 3 6 . 3 1.35 Con l . k 7 Cu 1.61 IAA 0.31 0.3k 1.1+8 0.73 39.1+ % Dry Wt. % Ash 6 . 8 3 Con 7.1+5 IAA 7.67 Cu 16.73 Cu 18.5k IAA 18.85 Con 1+.35* 0 . 2 5 7.32 6.8** 1 .07 18.01+ 0.66 6 . 8 3 1.33 5.7 Footnote: (1) IAA + CuSO^ p l o t . (2) IAA plot . (3) Control p l o t . 2k Day Old Plants Results Photosynthesis Treatment 3 July 3rd-llth 1961 21L hrs. Means and Treatments Ranked -D1} 2) 13.68 ConO) 10.27 2,k A 10.k6 Cu(?l.6i Variance Ratio 3 . i f 1 E.M.S. Total Mean L.S.R. 5% _ 3 mns. C.V.J* n . k7 3 . 3 22.1 1+8 hrs. 12.62 24-D i l l .58 Cu 26 Con 3.0 13.20 1 3 4 8 1+.7 27.0 96 hrs. 12.81+ Con 13.55 Cu 11+.36 2,1+-D ff. 3 21+.9 ll+q. hrs. 10.72 Con 11.91 Cu 12.8 2,IL-D 2.19 2.97 11.81 2.2 11+.6 192 hrs. 10.61 Con 10.67 Cu 12.72 2,q.-D; 1 .19 7.1 11 .33 2 3 . 5 Mean 12.0 Cu 12.k Con 12.6 2,l+-D Respiration Means and Treatments Ranked Variance Ratio E.M.S. Total Mean L.S.R. 5$ . 3 mns. • C.V.$ 2.7 Cu 3.12 2,1+-D 3.9 Con 1.57 1.39 3 . 2 i 1.50 3 6 4 1+.68 Cu k .77 24-D 6 .5 Con 0.31 2.11 5 .32 1.86 2 7 . 3 5 4 6 24-D 6.72 Cu 7.52 Con 1.02 6 . k l 6.56 3-21 38.6 5 . 6 Cu 5 . 6 3 Con 6.12 2 4-D 0.17 3.00 5 .78 2 .23 32.1 l+.9lj: cu li.99 Cor 6.28 24-D .93 Cu .15 24-D 5.71 Con 0 . 5 6 3.03 5 4 0 2.30 32.2 Results (Continued) Other Measurements Fresh Wt. Dry Wt. % Dry Wt. Ash Means and Treatments Ranked 5-91*. 2,1|.-D 7 . 2 0 Cu 7 . 3 7 Con Variance Ratio 0.28 E.M.S. Total Mean L.S.R. 5 $ 3 mns. C.V.$ 1 2 . 8 1 6.8I4. 1+.6 5 2 . 3 0 . 5 1 2,1|.-D 0 . 6 0 Cu 0 . 6 7 Con 0 . 3 9 0 . 1 0 0 . 5 9 O . l p . 51+.1 9 . 0 2,14,-D 9 . 5 Con 1 1 . 1 Cu 2 . 5 3 2 . 9 2 9 . 8 7 2 . 2 0 1 7 . 3 3 5 . 3 Con 1 5 . 6 2,1{.-D 1 5 . 8 Cu 0 . 1 7 2 . 5 0 1 5 . 5 6 1 . 3 2 1 0 . 2 Footnote: (1) Control p l o t . (2) 2,1+-D p l o t . (3) Cupric 2,i+-D p l o t . 2k Day Old Plants  Result s Photosynthesis Treatment k J u l y 17th-25th 1961 2i+ hrs. 72 hrs. 120 hrs. 168 hrs. 216 hrs. Mean Means and Treatments Ranked 12.97 ConfJ) 11+.26 2,k-f-15.26 Cu(3J Variance Ratio 0.50 E.M.S. 15.5 Total Mean 11+.16 L.S.R. 5$ 3 mns. 1+.99 c.v.g 27 .8 11+.56 Con Ik.56 2,1+-D 16.55 Cu 1.05 7.91+ 15.15 k .15 18 .6 7.70 2,1+-D 9.58 Con . 9.99 Cu 2.36 3.79 8 .93 2 .5 21.8 10.31+ 2,1+-D 10.80 Con . 12.97 Cu 1.38 8.55 11.37 3.73 25 .6 3.75 Con 3.85 2,k-D 1+.35 Cu 0.1+2 1.22 3.98 1.1+4 27.7 10.11+ 2,1+-D 10.33 Con 11.82 Cu Respiration Means and Treatments Ranked I+.76 Con 5 .50 2,1+-D 5.88 Cu Variance E.M.S. Total Mean L.S.R. 5$ 3 mns. Ratio 0 .52 23 38 k .  5*. 2.57 38.23 6.9I+ Con 8.02 2,1+-D 8.22 Cu 0.91+ 3.71 7.11+ 2.1+7 27.21 ' 3.1+1 Con 3.60 2,i+-D 3.83 Cu 0 .55 0.k7 3.61 0.88 19.00 1 Cu 5 Con .20 2,1+-D 1.09 1.1 1+.72 1 .36 22.2 5 . 6 6 Cu 5 . 6 8 Con 6.]6 2,1+-D 0.8k 0.51+ 5 . 8 3 0.91+ 12.6 5.07 Con 5.60 Cu 5.70 2 J+-D Other Measurements Fresh Wt. Dry Wt, % Dry Wt. Ash Wt Ash Wt. Means and Treatments Ranked . 3 Cu . 1 6 Con 5 .92 2,4-D Variance Ratio 2 . 5 E.M.S. ii . 2 9 Total Mean 4.1+6 L.S.R. S% 3 mns. 2 . 6 7 C.V.$ 1+6.1+ O.kl Cu 0.$1 Con 0 . 6 3 2,1+-D 2 . 3 0 . 0 3 0 . 5 2 0.21+ 35.1+ 11.3 2,i+-D 12.3 Con . 13.8 Cu 0.71+ 0.06 1 . 2 5 0 .31 19.1 0 . 0 5 Cu 0 . 0 8 Con I 0 . 1 0 2,1+-D| 2 . 7 8 0 . 0 0 1 3 0 . 0 8 0.01+7 1+6.9 1 5 . 6 Cu 1 5 . 9 Con 1 6 . 1 2,1+-D 0 . 2 2 . 0 1 5 . 8 8 1 . 8 2 8 . 9 Footnote: ( 1 ) Control plot. (2) 2,1+-D plot. (3) Cupric 2,1+-D plot. 36 Summary of Treatments One to Pour The f i r s t two treatments were made with no knowledge of what constituted an e f f e c t i v e concentration of growth substance or, i n the f i r s t case, a desirable rate of dust a p p l i c a t i o n . The actual amounts of XAA applied were 2j?0 micrograms i n treatment one and i+000 micrograms i n treatment two. The plants were quite small, therefore only about 5$ of t h i s amount can have landed on each plant. This low dosage f a i l e d to produce any e f f e c t . In only one case was a s i g n i f i c a n t difference observed i n the gas exchange means, and t h i s was probably the one chance i n twenty of a 'Type I e r r o r 1 (5l). The percentage dry and ash weight values i n treatment two show the reverse order of those i n treatment one, and were possibly due to poor randomization of samples i n the oven and furnace. Treatments three and four also f a i l e d to produce many s i g n i f i c a n t differences between means. Only photosynthesis at 21+ hours i n treatment three and the ash weights of treatment four were s i g n i f i c a n t . The actual amounts of 2,Ij-D applied were 1000 micrograms for treatment three and 10,000 micrograms f o r treatment four. Again only a small percentage of t h i s substance could have reached the plants. The high c o e f f i c i e n t s of v a r i a b i l i t y f o r gas exchange were caused by the low tissue weights used, which gave only s l i g h t manometer deflections. 26 Day Old Plants Results Treatment 5 Aug. lst-12th 1961 Photosynthesis Means and Treatments Ranked Variance Ratio E.M.S. Total Mean L.S »R• . 3 mns. C.V.$ -2l+ hrs. . 3 9 (2,1+-D) .26 (Con) . 5.7*4, (Cu). 1.09 2 .58 5.13 2.13 31.3 21+ hrs. 72 hrs. 120 hrs. 168 hrs 216 hrs. 2.91+ Con 3 . 0 1 Cu 3 . 7 2 2,i+-D 3 . 2 3 2,1+-D 3 . 3 9 Cu 3.1+1 Con 2 . 8 3 Con 2 . 8 6 2,1+-D 2 . 9 2 Cu 2 . 2 7 2,i+-D 2 . 8 0 Con 1 3 . 2 5 Cu 1 0 . 6 2>1+-D 2 . 1 2 Con 2.21+ Cu o . k 5 1 .62 3 .19 .011+ O.36 3 . 3 5 . 0 2 5 1 . 3 9 2 . 8 7 3 . 0 8 0.1+7 2 . 7 7 1 0 . 2 2 * * 0.1+1 1 . 62 1 .62 0 . 7 6 1 . 5 0 0 . 8 7 0 . 8 2 3 9 . 9 1 8 . 0 1 3 . 0 3 9 . 5 Respiration Means and Treatments Ranked Variance Ratio E.M.S. Total Mean L.S.R* 3 mns, C.V.fo 1+.19 (2,1+-D) 1+.1+2 (Con) . 1+.1+7 (Cu) . 0 . 0 5 3 . 2 5 1+.36 2 . 3 3 1+1.1+ 5 . 8 9 Con 6 . 8 3 2,1+-D 7.07 Cu 1 . 6 6 i.y+ 6 . 5 9 1 . 5 3 1 8 . 2 5.1+9 Con 5 . 5 8 2,1+-D 6.20 Cu 0.1+5 5 . 7 6 0.85; 1 1 . 6 1+.11+ Con 1+.77 Cu 1+.81+ 2,1+"D 0 . 9 1 . 0 1+.58 1 . 2 7 2 1 . 8 3 . 9 8 2,1+-D I 2.61+ 2,1+-D 1+.21 Con 11 3 .57 Con ' 5 .06 Cu I 3 . 8 2 Cu 3 . 0 0 . 6 5 1+.1+2 1 . 0 5 1 8 . 2 11. 0.21 3.31+ 0 .58 13.7 -0 Results (Continued) Other Measurements P.Syn.Mean Resp n Mean Means and Treatments Ranked 2.85 2 4-D 3.23 Con 3 4 3 Cu Variance Ratio E.M.S. Total Mean L.S.R. 5$ 3 mns. C.V.$ 1+. 62 Con " .68 2 4-D . 2 3 Cu Presh Wt. Dry Wt. % Dry Wt. % Ash Wt. 28.6 Cu 33.5 Con 3k'2. 24-D 2 .18 Cu 2.L.0 24-D 2.62 Con 7.3 24-D 8.2 Con 8 . 3 Cu 17.01 Cu 17.60 2,1+-D 17.96 Con 0.12 i+lO 32.2 0.17 1 .81 2.1+1 1.31 1.53 7.9k 1.91+ 0.72 17.5 26.0 63.O 1.71 55.8 1.58 15.6 0.98 1+.8 Curl 168<b> Curl 216 Means and Treatments Ranked 11+.0 C o n ^ l 20 .5 cuC 2);U 28.0 2 4 - D U ; Variance Ratio 8.18"* 36.0 20.9 7.61+ 28.7 E.M.S. Tot a l Mean L.S.R. %% 3 mns. C.V.$ Footnote: 2 0 . 5 Con I 20.5 Cu I 36.0 2 4-D 1 3 . 8 * * 37.0 25.5 7.71+ 23.8 Height Height * Presh Wt/ 6.87 Con 2 8 . 5 Con 6.87 Cu 30.2 24-D 8 .1 24-D 3k*2 Cu k . 0 7 * 0.31 0.92 319.8 7.28 31.1 1.23 22.7 23.8 5 7 . 5 Curl HexgnT 286 Cu 296 Con 1+1+8 2 4-D 1 0 . 3 * * 1+581. 3k3 8 6 . 1 19.7 Ash Wt. 0.38 It 0.16 0.06 0.1+3 0 . 3 5 6 . 9 Cu 2 4 - D Con a) (1) Control p l o t . (2) Cupric 2 4-D p l o t . (3) 2 4-D p l o t . b) Curl 168 and Curl 216 .are mean counts of curled l e a f l e t s at 168 hours and . 216 hours. 39 Summary The dosage applied i n this case was 25$ 2,lj.-D and 29$ cupric 2,4,-D i n O.I4. gms. attaclay, equivalent to 0 .1 gms. 2,q.-D per pl o t (or f i v e pounds of 2,I|.-D per acre). This high dosage was the f i r s t to produce morphological e f f e c t s . The gas exchange rates are graphed as g o n ^ r o ^ r a t i o s on the following page. Photographs of the plants are given i n Plate ( 3 ) . A determination of gas exchange was ca r r i e d out 2i+ hours before the treatment to indicate changes r e s u l t i n g d i r e c t l y from the treatment. The photographs and tabulated l e a f c u r l i n g and height values c l e a r l y show the copper s a l t of 2,lj.-D to be less e f f e c t i v e than the acid; i n these cases the copper s a l t means cl o s e l y correspond to the control means. After 168 hours the gas exchange expressed on a fre s h weight basis i s s i g n i f i c a n t l y reduced i n the 2,i+-D treated p l o t . 1+0 T R E A T M E N T 5 FIG. I | 4 F f i r o p h j s h o w i n g — v c l u c s v a r y i n g wi th t i m e . P h o t o s y n t h e s i s . 24 O 2 4 72 120 168 t ime a f t e r t r e a t m e n t (hours). 1-4 r re sP i r a t i o n . 72 1 2 0 168 t ime a f t e r t r e a t m e n t (hours). legend. 2 5 / 2 , 4 - D 2 9 / C u 2 / - D • • PLATE 3 Control and Cu2,4-D Plots Control and 2,4-D Plots The Plots from Treatment £ 216 Hours a f t e r Treatment 1+2 Treatments Six to Eight In the previous treatment the high dosage of 2,1+-D needed to produce morphological e f f e c t s l e d to a t r i a l of three methods of app l i c a t i o n : as a dust, as an aqueous spray and i n the nutrient solution. I t was known that a high percentage of the dust applied to the tops of the plants f e l l on the hardboard cover, and only a small percentage reached each plant. For t h i s reason more 2,I+-D was applied to the tops of the plants i n the aqueous spray and dust than was applied to the roots. Treatment 6 1) 0 . 1 gms. 2,1+-D i n 0.J+ gms. dust. 2) 0 . 1 gms. 2,1+-D i n water. 3) 0 . 0 1 gms. 2,1+-D i n the nutrient solution. Plots, 1 and 2 were both treated at a rate of 5 l b s . 2,1+-D per acre. Treatment seven was designed to compare ir o n as sulphate and ethylene diamine tetra-acetate-in a dust with 2,1+-D with the 25$ 2,1+-D dust used i n treatments f i v e and s i x . The same amount of 2,]+-D was applied to each p l o t , though i n order to provide a suitable molecular r a t i o of i r o n to 2,I+-D, the t o t a l weight of powder applied i n the iro n + 2,1+-D treated plots was greater than i n the plot treated with 2,1+-D alone: Treatment 1 1) 0 . 1 gms. 2,l+-D i n 0.1+ gms. dust. 2) 0 . 1 gms. 2,1+-D + 0 . 5 gms. FeS0v7H 2 0 i n 0 .8 gms. - dust. 3) 0 . 1 gms. 2,1+-D + 1 .25 gms. FeEDTA i n 1 . 6 5 S"13* . dust. Considerable reduction of l e a f area occurred one week af t e r the ap p l i c a t i o n of 25$ 2,1+-D. This, together with the epinasty produced i n the f i r s t few hours, suggested the use of the lower concentration of a 5 $ 2,1+-D dust for treatment eight. To discover whether the ferrous and f e r r i c forms of i r o n d i f f e r e d i n t h e i r protective capacity, s a l t s were chosen which had a sim i l a r s t a b i l i t y constant (j? and appendix) and a common anion. Ferrous and f e r r i c chlorides were used for treatment eight. Treatment 8 1) 0.02 gms. 2,1+-D in'0.1+ gms dust. 2) 0.02 gms. 2,1+-D + O.O967 gms. ferrous chloride FeCl3.6H20 i n 0.1+ gms. dust. 3) 0.02 gms. 2,1+-D + 0.0712 gms. f e r r i c chloride FeClg.l+R^O i n 0.1+ gms. dust. 47 Day Old Plants Results Treatment 6 Sept, 12th-20th 1961 Photosynthesis 2ij. hrs. 72 hrs. 120 hrs. 168 hra 216 hrs. Mean Mean and 1 2 . 2 7 D\\\ Treatments 1 2 . 3 2 SJ 2) Ranked 1 5 . 9 7 R<3) 8 . 0 7 S 9 . 1 0 R 9 . 7 1 D 5.11+ R 5 . 5 0 S 6 . 6 0 D 3 . 4 9 R 3 . 5 9 D 4 . 0 7 S 4 . 3 0 D 1+.72 R 7 . 0 0 S 7 . 2 5 D 7 . 3 9 S 7 . 6 8 R Variance Ratio O .83 E.M.S. 3 2 . 9 Total Mean 1 3 . 5 L.S.R. 5 $ 3 mns. 7 . 3 c . v . $ 1+2.5 0 . 5 1 3 8 . 9 6 8 . 9 9 3 . 8 3 3 . 3 1.08 2 . 0 9 5 . 6 8 1 . 8 6 2 5 . 5 0 . 2 2 2 . 7 1 3 . 7 1 2 . 1 1 4 . 4 5 * 2 . 9 0 5.31+ 2 . 1 9 3 1 . 9 Respiration Means and Treatments Ranked 4.28 D 5 . 0 7 S 6 . 9 0 R Variance Ratio 2.1+1 E.M.S.. 4 » 4 8 Total Mean 5.1+2 L.S.R. 5 $ 3 mns. 2 . 7 C.V.$ 39.1 5.70 s 6.60 R 7.10 D 1+.07 D 1+.19 S 5.38 R 1 . 8 7 SI 2 . 9 4 D 1 5.37 R • 3.65 D 3.70 S 5.0 R 4 . 1 1 S 2.01+ 2.60 6.65 2.68 1 .25 1+.53 6 . 9 * * 2.58 3 . 4 0 3.61 1.29 4.01 2.1 21+.2 2.09 4 7 . 2 1 . 4 9 28.3 Results (Continued) Other Measurements Fresh Wt. % Dry Wt. Height Dry Wt. Root Length Height Dry*Wt. Means and k.0$ S 7.70 D 5.90 S 0.3i|- S 7.16 R 71 . if S Treatments 6.52 R 7.78 R 7.08 R 0.50 D 10.92 S 91.6 R| Ranked 6.83 D 8.20 S 7.67 D o .5o R 12.91 D 100 DI Variance Ratio 2 .6 0.83 5 . 3 * 1.92 25.9** 1+.09* E.M.S. 5 . 2 5 0 . 3 0.92 0.03 1.97 313 Total Mean 5 . 8 0 7.91 6.88 0.1+5 10.33 87 .6 L.S.R. % 1.80 3 mns. 2.92 0.22 1.23 0.07 22.7 C.V.$ 39.5 6.93 13.95 37.2 13.5 2 0 . 1 Footnote: (1) Dust application. (2) Spray application. (3) Root application. PLATE 4 0.1 gms. 2,4-D A p p l i e d as a 2% Aqueous S o l u t i o n 0.1 5ms. 2,4-D A p p l i e d i n 0.4 gms A t t a c l a y Dust Plants kk Days Old  Results Photosynthesis Treatment 7 Sept. 2 3 r d-0ct l 3 t 1961 Respiration 18 hrs. 120 hrs. 216 hrs. 18 hrs. 120 hrs. Means and Treatments Ranked Variance Ratio E.M.S. 0.314. T o t a l Mean 5 . 2 1 L.S.R.5$ 3 mns. 0 . 7 2 C.V.$ 1 1 . 2 4 . 6 5 2 , 4 - D J 1 ^ 3 . 0 2 FeE I 5,1+2 PeE(2J j' 3 A 6 FeS I 5 . 5 6 Pes (3) I 5.C33 2 , 4-D 11.-5** O.I4.6 3 .77 O.87 1 8 . 0 2 . 6 5 PeE 2 . 6 8 PeS 5 . 3 0 2,4-D 2 7 . 0 * * 0 . 5 1 3 . 5 4 0 . 9 1 2 0 . 2 . 8 7 2,q.-D| • 64 PeS || 5 . 3 5 PeE I 6.69** 0 . k 9 4-62 0 . 9 0 15.2 i 3 . 7 9 2,4-D . 9 5 PeS . 3 1 PeE 0 . 0 9 4.02 0 . 3 8 7.4 216 hrs. I 1 I 2.94 PeE I . 0 0 PeS I . 0 2 2,4-D 7 . 5 3 * * 0 . 2 9 3 . 3 2 0 . 6 9 1 6 . 2 Other Measurements P.Syn.Mean Means and Treatments Ranked 3 . 6 9 PeE 3 . 9 0 PeS 4.93 2,4-D Variance Ratio E.M.S. Tota l Mean L.S.R.5$3 mns. C.V.$ Reap.Mean Fresh Wt. Dry Wt. $ Dry Wt. Height 3 . 8 6 FeS . 8 9 2,4-D .17 PeE . i 1 7 . 9 2 2,4-D 2 6 . 6 8 PeS 3 2 . 7 6 FeE 7 . 5 6 * * 44.24 25.8 25 8 : i r 1 . 3 8 2,4-D 2 . 1 6 PeS 2 . 8 0 PeE 1 2 . 2 5 * * 0 . 2 5 2 . 1 1 0 . 6 4 2 3 . 6 7.67 2,4-D 9 . 5 3 FeE I 8 . 2 2 PeS I 1 0 . 6 PeS | 8 . 5 8 PeE I 1 2 . 1 2,4-D 6 . 2 8 * 0 . 1 5 8 . 1 6 0 . 5 4 . 7 1 4 . 7 3 * 1 . 6 1 0 . 7 3 1 . 6 2 1 1 . 8 Dry l e a f material was ashed giving no s i g n i f i c a n t differences between treatments: 1 6 . 2 3 (FeE) 16.42 (2,4-D) 1 6 . 7 8 (PeS) ~ I J . B . K . . (.0i?) 3 means = 1.0 CV = l+,9% Footnote: The means are d i f f e r e n t i a t e d by the following symbols 1. Plot treated 2,k-D (25$) i n attaclay "" = 2,4"D. 2. Plot treated 25% 2,4-D + FeEDTA. i n attaclay = PeE. 3 . Plot treated 25$ 2,4"D + FeSOij. i n attaclay = PeS. TREATMENT 7 F I G. 2 graphs showing gas exchanged v a r y i n g wi th t i m e . P h o t o s y n t h e s i s . 250/o 2,4-D.x-l e g e n d , F e S 0 4 -f- 2,4-D. • • F e EDTA + 24-D. ° o -x 18 48 96 144 192 t ime a f t e r t reatment (.hours). 240 — r e s p i r a t i o n . \ o s \ \ \ \ \ V \ \ \ \ \ \ \ \ \ \ \ \ -\ — o 18 48 96 144 192 240 time a f t e r t r e a m e n t Chours) . 4-8 c o «A T R E A T M E N T 7 F I G . 2 graphs showhg gas exchanged v a r y i n g with t i m e . 6 0 5 0 E V a> c o c o X V c *> CP >» X o o M 4 0 3 0 6-Q-i 5-Oh u 6 4 0 3 0 . 2 0 p h o t o s y n t h e s i s . l e g e n d , 2 5 % 24-D. x x F e S 0 4 -f- 2,4-D. • • F e E D T A + 24-D. o o 18 4 8 96 144 192 t ime a f t e r t reatment (.hours). r e s p i r a t i o n . 2 4 0 •o 18 4 8 96 144 192 2 4 0 time a f t e r t r e a m e n t Chours) . k9 PLATE 5 2,4-D + FeEDTA 2,4-D Treated Plant Treated Plant 2,4-D + FeSC-4 Treated Plant A l l Nine Days After Treatment Plants 1+1+ Days Old  Results Photosynthesis Treatment 8 Oct. l 8 t h - 2 6 t h 1961 Respiration Means and Treatments Ranked Variance Ratio E.M.S. Total Mean L.S.R. 5 $ . 3 mns. C.V.$ 1+8 hrs. 120 hrs. 192 hrs . 1+8 hrs. 120 hrs. 5 . 1 5 5 . 3 0 F© ++< 2>-5 . 6 8 2,i+-D(3) 1.29 0.31+ 5.37 0.73 10.9 2.88 F e + + + 3.1+7 Fe*1"-3 . 6 0 2,1+-D 2 . 8 2 0 . 3 1 3 . 3 2 0 . 7 2 1 6 . 8 2,67 F e " 1 ^ 2.78 Fa*1"* 3.16 2,1+-D 1 . 13 0 . 3 5 2 . 8 6 0.71+ 2 0 . 6 5.1+5 F e ^ 5 . 6 9 Fa*** 6 . 0 7 2,1+-D 1 . 4 7 0.1+0 5 . 7 3 0 . 7 8 1 1 . 0 +++ 1+.51 Fe' 1+.57 2,|.-D 1+.61 Fe++ 0 . 2 0 . 7 6 1+.56 1 .7 19.1 192 hrs. 2 . 9 2 Fe 3.12 Fe + ++ 3.1+2 2,4-D 1 . 2 1 0 . 3 1 3 . 1 5 0.72 17.7 Other Measurements Means and Treatments Ranked P.Syn.Mean +++ 3.57 Fe 3.85 Fet+-1+.11+ 2,lj.-D Variance Ratio E.M.S. Tota l Mean L.S.R.5$3 mns, C.V.$ Resp. Mean Fresh Wt. Dry Wt, % Dry Wt. Height 1+.1+1+ Fe? 1+.69 2,i+-D +++ +++ 1+3.0 F e+ + 4 9 . 5 F e + ^ + 5 6 . 8 2,1+-D 1 . 6 3 172 1+9.8 16 2 6 . 3 3.i+8 F e + + 3 . 8 3 Fe 1+.1+8 2,1+-D 1 . 2 7 1 . 2 3 3.92 1.1+0 28.2 :+++ 7 . 7 3 Fe, 7 . 9 0 2,1+-D 8.01+ Fe 3 . 1 7 0 . 1 7 7 . 8 9 0 . 5 3 5 . 2 1 2 . 6 2,1+-D 1 3 * 5 Fe+++ 1 5 . 2 Fe^"+ 0 . 8 1+.31 1 3 . 7 8 2.71 15.1 Footnote: (1) F e r r i c chloride + 2,1+-D p l o t . (2) Ferrous chloride + 2,1+-D p l o t . (3) 2,1+-D p l o t . . . . o 51 Summary of Treatments Six to Eight The a p p l i c a t i o n t r i a l c l e a r l y showed the aqueous form of 2,lj . -D to be the most e f f e c t i v e i n terms of morphological e f f e c t s , height and weight (Plate Jj.). Height was s i g n i f i c a n t l y reduced by the aqueous application, and fresh and dry weights were reduced by about y~>%, though, since there was high error term, this was not s i g n i f i c a n t . Root growth was i n h i b i t e d by the 2,1J.-D. The gas exchange r e s u l t s showed a highly s i g n i f i c a n t depression i n res p i r a t o r y rate i n both the sprayed plo t and the dusted p l o t at 168 hours and a general pattern of depression at the other times. Photosynthesis was s i g n i f i c a n t l y higher i n the sprayed p l o t at 216 hours, though i t i s probable that a sampling error was induced by the stunted growth i n t h i s p l o t , there being f a r fewer l e a f l e t s produced than i n the other p l o t s . This l a s t d i f f i c u l t y and the high c o e f f i c i e n t of v a r i a b i l i t y for both forms of gas exchange l e d to the use of a t o t a l t i s s u e weight of 150-200 mg. i n subsequent runs. This increase replaced the e a r l i e r use of 50 mg. of t i s s u e i n each f l a s k . Four l e a f l e t s were sampled i n subsequent treatments instead of two i n t h i s and e a r l i e r treatments. In order to provide t h i s quantity of material, i t was necessary to use older plants and i t proved possible to sample f o r photosynthesis and r e s p i r a t i o n on three occasions only. In treatment seven both the i l l u s t r a t i o n s and a l l other values measured strongly suggest that the i r o n as both ethylene diamine tetra-acetate and sulphate ameliorated the 5 2 e f f e c t s of 2,lj.-D. The 2,4-D treated p l o t , with one exception, was always s i g n i f i c a n t l y d i f f e r e n t from the two i r o n + 2,4-D treated p l o t s . In only one case was there a s i g n i f i c a n t difference between the two i r o n + 2,4-D treated p l o t s . In the 2,4-D treated p l o t s fresh and dry weights were lower than i n the Iron + 2,4-D treated plots by over 1+0%, while height was 20$ greater and percentage dry matter 10$ lower than i n the i r o n + 2,4-D treated p l o t s . The gas exchange values are represented graphically (Figure 2). Although no control plot was available there was a strong i n d i c a t i o n that both photosynthetic and r e s p i r a t o r y Inhibitions were more rapid i n the 2,4-D treated p l o t than i n the two i r o n + 2,4-D treated p l o t s . This could suggest a delayed entry of 2,4-D, possibly r e s u l t i n g from i r o n s a l t formation i n the preparation of the powders. Again, the apparent r i s e i n photosynthetic rate i n the 2,4~D treated plot a f t e r 18 hours may be a sampling error r e s u l t i n g from the reduced l e a f s i z e , the mean sample weight In the 2,4~D treated plot being only one t h i r d of that i n the other plots at 216 hours, and two thirds at 120 hours. The e f f e c t of increasing the sample size at the expense of lowering the frequency of sampling was most s t r i k i n g . The c o e f f i c i e n t s of v a r i a b i l i t y ranged from 7$ to 20$ i n this run, compared to 2\+%A+1% i n the previous run. The low v a r i a b i l i t i e s were produced by the larger manometric deflections i n the Warburg apparatus. When the actual manometer deflections were lowest (120 hrs.) the CV$ was highest. 5 3 . Treatment with 5 $ 2,lj.-D i n number eight f a i l e d to produce any s i g n i f i c a n t differences i n spite of the lowered CV$« The consistently higher rate of carbon dioxide a s s i m i l a t i o n by the 2,Ij.-D treated p l o t was r e f l e c t e d i n both the gas exchange and fresh and dry weight values. The height values indicated that plants In the ferrous + 2,i+-D p l o t were the t a l l e s t and those i n the 2,1+-D treated p l o t shortest. 54 Treatments Nine to Thirteen I t was evident from treatment s i x that an aqueous spraj was the most e f f e c t i v e form of 2,4-D a p p l i c a t i o n . The d i f f i c u l t y of making up suitable dusts and the use of a p r o h i b i t i v e l y high concentration of 2,4~D together with the d i f f i c u l t y of evenly dusting the larger plants, l e d to the use of aqueous sprays for the remaining treatments. This change caused some trouble because i t was necessary to ascertain the soluti o n concentrations required f o r the l e a s t morphological e f f e c t . Further trouble was encountered i n the a c i d i t y of the i r o n solutions used, p a r t i c u l a r l y f e r r i c , and i n the necrosis and black f l e c k i n g produced by these solutions at concentrations of over £00 p.p.m. E a r l i e r workers (for example Wort (6ij.) and M i l l e r et a l . " ( i n press)) indicated that a concentration of 1$00 p.p.m. of i r o n as the sulphate was a reasonable concentration f o r i r o n protection. However, i n the present experiments i t was found that these concentrations produced necrosis and other undesirable side e f f e c t s , r t was found ultimately that a l l sprays had to be corrected with the parent a c i d to pH 1.5 to 2 , which was the pH of the f e r r i c solutions at 300 p.p.m. Xn one case over-correction of the control sprays caused considerable a c i d necrosis. I t was only i n treatments twelve and thi r t e e n that a suitable rate of 2,4-D a p p l i c a t i o n and protective i r o n concen-t r a t i o n was used without other side e f f e c t s . # M i l l e r , M .D., Mikkelson, D . S . , and Huffaker, R.C. 55 Treatment 9 1) 500 p.p.m. 2,4-D. 2) 500 p.p.m. 2,4-D + 1500 p.p.m. ferrous i r o n . applied as the chloride. 3) 500 p.p.m. 2,4-D + 1500 p.p.m. f e r r i c i r o n . applied as the chloride. Iron treatment preceded 2,4-D treatment by 24 hours. Treatment 10 1) Control plot sprayed with water. 2) 1500 p.p.m. ferrous i r o n applied as the sulphate . + 200 p.p.m. 2,4-D. 3) 200 p.p.m. 2,4-D. These.were based on the optimal figures from (64). Treatment 11 1) Control p l o t sprayed with water a c i d i f i e d with . HCl. 2) 150 p.p.m. f e r r i c i r o n applied as the chloride + 100 p.p.m. 2,4-D. 3) Treated as p l o t (1) then sprayed with 100 p.p.m. ., 2,4-D. . . Treatment 12 1) Control p l o t sprayed with water at pH 1 .5 . 2) 150 p.p.m. f e r r i c i r o n applied as the sulphate . + 100 p.p.m. 2,4-D. 3) Treated as p l o t (1) then sprayed with 100 p.p.m. , 2,4-D. Treatment 13 1) Control p l o t sprayed with water at pH 1*5. 2) 150 p.p.m. ferrous i r o n applied as sulphate + . 100 p.p.m. 2,4-D. 3) Treated as pl o t (1) then sprayed with 100 p.p.m. - 2,4-D. A l l plants were sprayed to d r i p . In treatments ten to thir t e e n i r o n treatment preceded 2,4-D treatment by two hours. hh- Pay Old Plants  Results  Photosynthesis Treatment 9 Respiration Nov. 7th-17th 1961 2k hra. Means and Treatments Ranked (Control Rate)£.17( J Variance Ratio3 .27 E.M.S. 3 . 3 Total Mean 1+.72 L.S.R.5$3 mns. 2 .38 c.v.$ 38.1 120 hrs. 2lj.0 hrs. 1 3.05 2,4-D 3.76 P e + + 1 3.33 Fe+++ 5.01 F e * + + 3.47 Fe++ 5.90 2,k-B 3.95 6.02 0.08 7 . 0 3 * * 2.8 1.0 3.28 4.91 2.23 1,31 51 .0 2 0 . 3 2k hrs. 120 hrs. 2l+0 hrs. 7.00 P e + + 8.25 F e * + + 8.55 2,4-D 7.25 2.80 1 4 7.91+ 1.55 4.9 4.76 k . 8 3 24-D 5.71 Pe+++ 1+.92 1 .59 2 . 3 5 . 0 8 2 .03 3 0 . 0 3.51 24-D 3.82 Pe++ k .36 Feft+ 5 . 7 0 . 78 1. k 1.55 3 o 4 Other Measurements P.Syn.Mean Resp. Mean P.Syn.A.120 P.Syn.A.2l+0 Dry Wt. Means and Treatments Ranked 3.94 F6+++ 547 Pe++-448 24-D Variance Ratio E.M.S. Total Mean L.S.R.5$ 3 mns C.V.? % Dry Wt. 5.19 Fe++ 5 . 6 3 24-D 6.11 Pe+++ 16.8 F e + + + 18.6 24-D 21.9 Pe++ 0 .L9 " 79.8 19.1 11.8 1+6.5 19.2 2 4-D 21 .1 Pe+++ 2 3 . 0 Fe++ 0.61 36.2 2 0 . 1 8 .07 28.1+ 1.03 2 4-D 1.71 Fe+++ 1.98 Fett, 0.72 1.99 1.57 1.85 8 9 . 8 9.15 Fe++ 9.38 Fe+++ 9 4 5 2l+,-D 0 . 2 3 2 . 3 9 .33 1.98 16 .3 Footnotes: (1) Ferric chloride and 24-D sprayed plot. (2) 24-D sprayed plot. (3) Ferrous chloride and 24"D sprayed plot. (1+) The control rates were determined at a different time from the others and are therefore of limited value. 5 5 Day Old Plants  Results Photosynthesis (Wt.) Treatment 10 Photosynthesis (Area) Dec. l8-Dec. 2 7 t h 1961 27 hrs. 120 hrs. 216 hrs. Means and Treatments Ranked .(1) I*.*27 ^ fo\ 5.16 Fe++ 5.29 2 , 4-D^ ; 5 . 1 4 Con. 6 .58 Con(3) - 5 . 4 7 2,4-D Variance Ratio 2 5 - 7 * * E.M.S. 0 . 3 1 Total Mean 5 . 3 8 L.S.R.5$ 3mns. 0 . 6 8 1 0 . 3 0.18 1 .15 5 . 3 6 1.26 20.0 . 2 2 Con . 9 9 P e + + 6.I4.6 2,4--D 8.06** 1 . 0 k 5 . 5 6 1 . 2 0 1 8 . 3 27 hrs. 1 9 . 7 Fe +* 2 2 . 6 Con I 2l+.7 2,k-D| i . l 2 2 . 3 3 . 7 1 2 . 8 120 hrs. 1 8 . 8 P e + + 2 0 , 3 2,4-D 2 1 . 9 Con 3 . 9 * 3 . 8 5 2 0 . 3 2 . 5 9 . 7 216 hrs. 19.2 Pe"^ 20.2 Con 20.9 2,4-D 1.18 2.83 20.1 2.2 8 .4 Respiration Means and 5 . 2 8 Con k.92 2,Ji-D 3 . 5 5 P e + + Treatments 6 . 3 0 2,4-D 4*92 Fe++ I 3 . 6 5 2,4-D Ranked 8 . 8 2 Pe++ 6 . 1 3 Con 5 . 2 5 Con Variance Ratio 2 9 . 3 * * 2 9 . 2 * * 41.3** E.M.S. 0 . 6 8 0 . 1 0 . 1 3 Total Mean 6 . 8 0 5 . 3 2 if. 15 L.S.R.5$ 3 m n s l . 0 0 0 . 3 ? 0 . k 6 C.V.$ 1 2 . 1 5 . 9 4 8 . 6 8 -0 Reaulta (Continued) Other Measurementa P.Syn.Mean P.Syn.A.Mean Reap.Mean Height Dry ¥t. % Dry Wt. Meaha and' Treatment3 Ranked 5 . l 4 Pe + + Con 5 . 7 4 2,4-D Variance Ratio E.M.S. Total Mean' L.S.R. % 3 mns. C.V.$ 19.2 P e + + 21.6 COn 22.0 2,4-D 4 . 9 6 2,4-D £ . 5 5 " 5 . 7 6 Con 1 0 . 8 3 Con 1 0 . 9 1 2,4-D 1 1 . 8 Pe++ 0 . 6 8 2 . 2 1 1 : 2 1 . 8 7 1 3 . 2 5 1 . 8 3 Pe++ 2 . 0 5 2,4-D 2 . 7 8 Con 2 . 8 0 . 5 2 2 . 2 2 0 . 9 4 3 2 . 5 8 . 2 5 P e + + 8.92 2,4-D 9 . 0 4 Con 2, 0, 8, 0 . 8 1 7 . 3 2 14 Footnotes: (1) Ferrous sulphate and 2,4-D sprayed plot. (2) 2,4-D sprayed plot. (3) Control. 1+2 Day Old Plants  Results Photosynthesis (Wt.) Treatment 11 Jan. 2nd-Jan 12th 1962 Photosynthesis (Area) Means and Treatments Ranked Variance Ratio E.M.S* Total Mean L.S.R.% 3 mns. C.V.$ 20 hrs. 1^2 hrs. 2l+0 hrs. 20 hrs. 152 hrs. k.1+7 2 ,4-Dj^ 5.00 Con(2) 5 . 6 5 Fe+++(3) 2.15 1.0 5.01+ 1.28 19.8 k.85 2,1+-D 6.26 Con 6.57 Fe+++ 5.81+* 0.67 5 .90 i .o5 13.9 ,68 Fe Con |.5k , 6.if 2,1+-D 8.2** 0.1+ 5.1+6 0.81 11.6 35.2 2,1+-D 15.9 Con 19.5 Fe+++ 1+.03* 7.8 16.87 3.61 16.6 19.1 Con 20 .5 Fe+++ 20.9 2,1+-D 1 .93 2.9 20.16 2.22 8.1+5 21+0 hrs. 19 .05 2,1+-D 20.1 Con I 2 2 . 3 Fe+++ l+.k7* 3.8 20.5 2.59 9 . 5 Respiration Means and Treatments Ranked Variance Ratio E.M.S. Total' Mean L.S.R. $% 3 mns. C.V.$ 1+.95 Fe+++ 5.77 2,i+-D 5.8 Con 6 . 0 1 * 0.25 5.51 0 . 6 3 9.07 3.6 2,1+-D 1+.16 Fe+++ 1+.73 Con 12.9** 0.11+ 1+.17 0.1+7 8.97 3; 56 Fe+++ . 7 2,1+-D .67 Con 5 . 3 * 0.1+ 3.98 0.81 15.9 Results (Continued) Other Measurements P.Syn.Mean P.Syn.A.Mean Resp.Mean Height Dry Wt. % Dry Wt. Means and Treatments Ranked 5 . 1 6 2,ij,-D 5 . 6 0 Con 5 . 6 3 Fe+++ 18.q. 2,Ii-D 18.a. Con 2 0 . 8 Pe+++ i}..22 P6+++ iL . 3 6 2,1L-D 5 . 0 7 Con 6 . 7 5 2,ij.-D| " 7 . 4 I Con 1 1 . 7 Fe+++ 0 . 7 7 2,1L-D I 1 . 18 Con 1 1 .87 Pe+++ 1 7 . 7 6 P e + + + 1 8 . 7 3 Con | 9 . 5 3 2 ,1L-D| Variance Ratio E.M.-S. Tota l Mean L.S.R. 5 $ 3 mns. C.V.$ 6 . 5 * * 0 . 6 7 8 . 6 1 1 . 0 9 . 5 1 6 . 6 * * 0 . 3 , 1 . 2 8 0 : 6 8 1L2.8 5 . 4 * O .87 8 . 6 7 1 . 19 1 0 . 8 Footnotes: (1) 2, i i-D sprayed p l o t . (2) Control p l o t . (3) Ferrous sulphate and 2 ,IL-D sprayed p l o t . 30 Day Old Plants  Results Photosynthesis (Wt.) Treatment 12 Jan. 23rd-31st 1962 Photosynthesis (Area) 2!L hrs. 118 hrs. Means and Treatments Ranked Variance Ratio E.M.S. Total Mean L.S.R.~ $% 3 mns. C.V.$ 190 hrs. 2li hrs. 118 hrs. 7-45 2,1L-D ( 1 ) 7.54 Con(2r 7.55 Pe+++(3) 0.06 O.kO 7.52 0.81 8.4 6.31 2,4-D 6.69 Con 7.30 re*** 2.03 0.73 6.77 1.1 12.6 5.40 6.05 Con I 6.97 2,4-D I 4 .98* 0.74 6.l4 "1.1 14.0 23.5 Con 23.8 2,k-D 24.0 Pe+++ 0.16 2.5 23.78 2.05 6.6 24.1 P e + + + 2k.5 Con 2?.4 2,4-D 0.43 6.9 24.7 Y.kl ).6 "3.  10, 190 hrs. 2 2 . 3 2,4-D 2 3 . 0 Con 26.4 Pe+++i 3.33 7.8 23.9 3.62 11.7 Respiration Means and Treatments Ranked Variance Ratio E.M.S. Total Mean L.S.R." 5$ 3 mns. C.V.fo 1+.68 F e + + + 5.40 2,li-D 5.49 Con 3.69* 0.28 5.19 0.68 10.2 4.68 PeH . 8 2 2,4-D .89 Con 6.02 0.1+2 5.13 0.82 12.6 4.81 P e + + + 5.35 2,4-D 5.69 Con 2.24 o.5 5.28 0.93 13.4 Results (Continued)  Other Measurements Height Dry Wt. Dry Wt. P.Syn.Mean P.Syn.A.Mean Resp.Mean Means and' Treatments Ranked 6.1+6 2,1+-D 6.75 Con 9.08 Pe+++ 0.92 2/1+-D 1.22 Fe+++ 1.70 Con 6;1+1+ 2;!+-D 6.75 Con 7.05 Fe+++ 6.75 6.76 Con 6.91 2,1+-D 2 3 . 7 Con 2 3 . 8 2,1+-D 21L.8 Pe+++ 1+.72 Fe+++ 5.19 2,1+-D 5 . 6 9 Con Variance Ratio E.M.S. Tota l Mean L.S.R. 3 mns. G.Y.% U+.5** 0.86 7 4 3 1.18 12 .5 9;69** 0.10 1 .28 0 . 3 9 21+.3 1 .61 0.35 6.7I+ 0.71+ 8 .8 Footnotes: (1) 2 4-D sprayed p l o t . (2) Control p l o t . (3) F e r r i c sulphate and 2,!+-D sprayed p l o t . 63 T C T R E A T M E N T 12. F I G . 3 grophsshowinf j values varying with t ime. K> 7=- O 8 • r e s p i r a t i o n . 06 48 96 144 192 hours. photosynthes is (weighty. 0-81 1 i i l 4 8 9 6 1 4 4 1 9 2 h o u r s . legend. 24-D. x x F e * ^ 4 - D . • _ - • PLATE 6 - P l a n t s from Treatment 12 ILO Day Old Plants  Results Photosynthesis (Wt.) Treatment 13 Feb. 23rd-Mar. 2nd 1962 Photosynthesis (Area) 2IL hrs. 96 hrs. 160 hrs. 2ii hrs. 96 hrs. 165 hrs. Means and" Treatments Ranked Variance Ratio E.M.S. Total Mean L.S.R. 5$ 3 mns. Q.V.% 6.39 2,4,-D^ 1^ 6.89 Fe++(2) 7.4.9 Gon(3) 2.18 0.8LL 6.91 1.18 13.3 4-53 Fe++ I4..88 Con 0.85 0.k8 4.59 0.91 15.1 5.17 Con 5 4 8 Fe++ 6 . 2 0 2 4 - D 2.58 0.68 5.61 "1.08 4.7 23.8 Fe ++ 24.. 3 Con 2 5 . 0 2 4 - D 0.58 2.71 8.5 184 Con 19.9 Fe++ 20.3 24-D 1 . 0 5 5 4 19.5 3 . 0 2 11.9 18.6 Con 23.2 Fe++ 2IL.9 24-D 7.98** 7.7 22.2 3.61 12.5 Respiration Means and Treatments Ranked Variance Ratio E.M.S. Total Mean L.S.R. 5$ 3 mns. C.V.$ 5.27 Con 5.4I}. 24-D 6.27 Fe++ 5 4 5 * 0.3 5.65 0.7 9.7 k.35 24-D 4.4 1 4.. 77 Con 1 . 5 4 0.2 4 . 5 1 0.56 9.9 .7$ Fe +* .11 24-Dj 5.35 Con 4-59* 0.12 5.07 0.4.6 6.8 Results (Continued)  Other Measurements P.Syn.Wt.Mn. P.Syn.A.Mn. Resp.Mn. Height Dry Wt, % Dry Wt. Means and Treatments Ranked 5 . 6 3 P e + + 5 . 6 5 2,4-D 5 . 8 5 Con Variance Ratio E.M.S. Total Mean L.S.R. 5$ 3 mns, C.V.$ 20.4 Con 22.3 Fe ++ 23.4 2,4-D 4.97 24,-D 5.13 Con 5.14 Fa** 6.63 Con I 8.04 2,4-D| 1 0 . 0 F e + + 45.1** 2 . 3 7.59 I . 8 9 19.9 1.59 Pe++ 1.65 2,4-D 1.94 Con 0.45 0 . 5 1.73 0.93 4 0 . 9 7.55 Pe++ 7.72 Con 7.92 2,4-D 2.13 0.1 7.73 0.38 4 . 1 Footnotes: (1) 2;4~D sprayed p l o t . (2) Ferrous sulphate and 2,4-D sprayed plot, (3) Control p l o t . 67 T R E A T M E N T 13. F I G . 4 1-2 r r 1 ° 08 12 X C l O g r a p h s showing T v a l u e s v a r y i n g w i th t i m e r e s p i r a t i o n . 0-8 1-3 L I-I c 0-9 4 8 48 96 144 192 h o u r s p h o t o s y n t hes is (weight). 7 96 144 photo s y n t h e s i s careg). 192 h o u r s , 1-46 96 144 l e g e n d . 192 h o u r s . 2 4 - D . x Fe**24-D. . 69 Summary of Treatments Nine to Thirteen As has already been stated, the solutions of f e r r i c s a l t s were acid and at 1500 p.p.m. markedly damaged the plants when sprayed to d r i p . This i s evident i n treatment nine, where photosynthesis i s depressed i n the f e r r i c - t r e a t e d p l o t at two hours, the difference between the ferrous and f e r r i c - p r e t r e a t e d p l o t s being s i g n i f i c a n t . Control values were obtained for t h i s run though at d i f f e r e n t times from the treatment values because treatment samples completely f i l l e d the Warburg at one run. The high c o e f f i c i e n t s of v a r i a b i l i t y for photosynthesis are caused by necrotic patches on the leaves sampled f o r determination. Comparison of area and weight-based photosynthetic values i n treatment nine indicates the r e s u l t s of the acid necrosis. At 120 hours the necrotic spots contributed l i t t l e to the fresh weight but continued to contribute to the area term, r e s u l t i n g i n a lower area-based photosynthesis value for the f e r r i c -treated p l o t . Dry weight y i e l d was lower i n the 2 ,1L-D-treated than iron-pretreated p l o t s , though the high c o e f f i c i e n t of v a r i a b i l i t y prevented t h i s difference from being s i g n i f i c a n t . In treatment ten the ferrous sulphate a p p l i c a t i o n caused black f l e c k s to appear on the leaves. This produced lowered photosynthesis values. The 2,ii-D p l o t had a s i g n i f i c a n t l y lower photosynthetic rate than the control when the rate was based on weight. The lower photosynthetic rate was due to l e a f curling, as indicated by the lack of significance i n the corresponding area-based values. At 216 hours, the rates i n the 2 ,ii-D-treated 70 plots were s i g n i f i c a n t l y higher, r e f l e c t i n g the d i f f e r e n t sizes of samples yielded at this time. Respiration values indicated an i n i t i a l stimulation, p a r t i c u l a r l y i n the iron-pretreated p l o t , but such values were lower i n both of the 2,1}.-D treated p l o t s than i n the control on days f i v e and nine. A l l of these differences were s i g n i f i c a n t . Height and weight values were not s i g n i f i c a n t l y d i f f e r e n t but the weight y i e l d bears out the picture given f o r carbon dioxide as s i m i l a t i o n . The lowered photosynthetic rates at 20 hours i n treatment eleven are a r e s u l t of the acid necrosis caused by the over-correction of the spray water f o r pH. The high photo-synthetic rate i n the 2,i+-D p l o t was again caused by the low y i e l d of l e a f tissue f o r the determination. This i s borne out by the complete reversal of the sequence of means when area i s used as a basis i n place of weight. The respiratory rate i s again s i g n i f i c a n t l y depressed i n the two plots treated with 2,I}-D. The dry weight y i e l d supports the sequence given f o r carbon dioxide a s s i m i l a t i o n . Height was s i g n i f i c a n t l y greater i n the p l o t treated with Iron and 2,lj.-D, t h i s e f f e c t i s also found i n treatments twelve and th i r t e e n . The only photosynthetic s i g n i f i c a n t differences i n treatment twelve show the i r o n + 2,1+-D-treated plot to be s i g n i f i c a n t l y d i f f e r e n t from the p l o t treated with 2,lt-D alone, and that the cause i s probably to be found i n the sampling difference noted In the previous experiments, since the weight-and area-based values show the opposite sequence. Respiration i s s i g n i f i c a n t l y depressed i n the f e r r i c + 2,1+-D-treated plot at 71 a l l times and i n the 2,li - D treated pl o t at 118 hours. The reason f o r t h i s apparent synergism i s not cle a r . Dry weight was s i g n i f i c a n t l y lowered i n both of the 2,U . - D -treated plots and, as i n treatments eleven and th i r t e e n , height i s s i g n i f i c a n t l y greater i n the i r o n + 2, 1L-D-treated p l o t . Treatment t h i r t e e n showed an i n i t i a l depression of photosynthesis, when based on weight, with an apparent stimulation at 168 hours. None of these differences were s i g n i f i c a n t , though a s i g n i f i c a n t stimulation of photosynthesis based on area i n both of the 2,Ij.-D treated plots was shown at 168 hours. The ferrous-2, l i - D treated p l o t declined from a s i g n i f i c a n t l y higher rate of r e s p i r a t i o n to one s i g n i f i c a n t l y lower than control during the experiment. The control and 2,IL-D values were never s i g n i f i c a n t l y d i f f e r e n t . Again plant height i n the 2,1L-D + i r o n treated plot i s s i g n i f i c a n t l y greater by over twenty percent; there was no concomitant dry weight increase. The dry weight values correspond to the o v e r a l l values for photosynthesis and p a r t i c u l a r l y to the net ass i m i l a t i o n when the corresponding respiratory rates are considered. The most consistent feature of these treatments has been the s i g n i f i c a n t increase i n height produced by i r o n + 2,IL-D treatment. This corresponds to the increased height when 2,li - D was applied alone as a dust, and suggests the int e r p r e t a t i o n that the i r o n was lowering the e f f e c t i v e concentration of 2 ,1L-D. The advantage of carrying out an experiment planned on s t a t i s t i c a l l i n e s i s to be seen not only i n the a t t r i b u t i o n of 72 quite d e f i n i t e values to the significance of the r e s u l t s , but also i n the Indication of experimental accuracy at each stage of the experiment afforded by the values of c o e f f i c i e n t of v a r i a b i l i t y . Major changes i n method were avoided as f a r as possible as they would render comparisons between d i f f e r e n t treatments i n v a l i d . The manometric c o e f f i c i e n t s of v a r i a b i l i t y showed the most s t r i k i n g improvement. In order to obtain reasonably uniform r e s u l t s f o r photosynthesis i n the f i r s t runs, i t was necessary continuously to move the manometer. As a r e s u l t , the c o e f f i c i e n t s of v a r i a b i l i t y were lowered from about 35$ to 20-25$. Prom treatment f i v e onward, the l i g h t i n g was altered from the three reflector-neck bulbs, used i n i t i a l l y , to a C i r c l i n e fluorescent tube and four white-sprayed tungsten filament lamps. The improvement was not immediately apparent, since the l i g h t i n t e n s i t y was increased only i n stages and runs were made i n i t i a l l y without turning the manometer (Tr. 5* -21+ hrs. and 2l+ h r s . ) . But when the manometers were moved, two or three places each minute, a lowering of v a r i a b i l i t y was achieved (Tr. 5 , 72 to 168 h r s . ) . However, I t was also found that the y i e l d of l e a f material sampled on alternate days was very low. This resulted i n small manometer deflections and increased error (Tr. 6 ) . Frequency of sampling was reduced and the number of l e a f l e t s In each f l a s k was increased from two to four. This gave a t o t a l tissue weight of approximately 200 mg. per f l a s k , replacing the e a r l i e r use of 50 to 100 mg. Con-sequently, a greater d e f l e c t i o n on the manometers resulted In a 73 lowering of the c o e f f i c i e n t s of v a r i a b i l i t y f o r both photo-synthesis and r e s p i r a t i o n from about 35$ i n treatment 6 , to below ll+$ i n the remaining treatments. Occasionally high c o e f f i c i e n t s of v a r i a b i l i t y for manometric determinations between treatments seven and thi r t e e n are caused by p a r t i c u l a r treatment effects (Tr. 9 ) . Coefficients of v a r i a b i l i t y for the weight deter-minations showed some Improvement because more uniform plants were used i n l a t e r runs. However, the c o e f f i c i e n t s were r a r e l y lower than 3 0 $ , hence few of these values were s i g n i f i c a n t . Ash weights r a r e l y had c o e f f i c i e n t s of v a r i a b i l i t y lower than 5 0 $ because of the small quantities involved; as a r e s u l t exper-imental error was added to the i n i t i a l non-uniformity. The expression of plant weights as percentages necessarily reduces the c o e f f i c i e n t of v a r i a b i l i t y , frequently to l e s s than 1 0 $ . However, i t was noticed that plants of d i f f e r e n t size d i f f e r e d considerably i n percentage dry matter, and furthermore the temperatures of the oven and furnace used for determining dry and ash weights were f a r from uniform. It appears that i f the error terra i s a r t i f i c i a l l y reduced when percentage values are used, these other causes of non-uniformity can be exaggerated so that they appear s i g n i f i c a n t . Thus In treatments one and two, where treatment dosages were too low to produce any apparent e f f e c t s , the percentage weight values were s i g n i f i c a n t , but i n p r e c i s e l y the opposite sense i n the two runs. Clearly these could not be treatment e f f e c t s . For these reasons the percentage values should not be considered s i g n i f i c a n t 71+ unless the fresh and dry weight c o e f f i c i e n t s of v a r i a b i l i t y are very low. I t can be seen that the absolute values for gas exchanges vary considerably between consecutive treatments. This e f f e c t was observed by Wedding (60,61) and appears to be a property of l i v i n g material, which cannot e a s i l y be avoided. I t i s possible that changes i n l i n e voltage or background l i g h t i n t e n s i t y could contribute to these v a r i a b i l i t i e s i n the case of photosynthesis, and any differences i n time between harvesting and determination of gas exchange could add to the v a r i a t i o n i n both photosynthesis and r e s p i r a t i o n . Hoi-fever, t h i s should not detract from the significance of the r e s u l t s i f comparisons are made within each time of harvesting and comparisons between di f f e r e n t times are treated as r a t i o s rather than absolute values. In general i t has been assumed that a l l plants were drawn from a single population, and that the control plot most cl o s e l y r e f l e c t s the population mean. 75 Tn addition to the main protection experiments ca r r i e d out, a number of small tests were run. Three of these tests are described below. 1 . Leaf age test 4 number of untreated plants were sampled f o r l e a f l e t s of d i f f e r e n t physiological age (judged s o l e l y by appearance and p o s i t i o n on the p l a n t ) . Three manometers were set up f o r each age and the remaining manometers were used as thermobarometers. There was l i t t l e scatter about the mean values given below, though once again the two runs (three days apart) showed a d i f f e r e n t t o t a l gas exchange. The second run had mean values which were only 7 0 $ of those obtained i n the f i r s t run. Results Nov. 7 t h Leaves: Old Mature Just Expanded Young Photosynthesis (wt.j 8 . 0 5 . 8 5 . 2 3 . 8 Respiration 3«q. 5 . 7 7 . 3 7 . 9 Nov. 1 0 t h Photosynthesis (wt.) 5 . 1 5 . 2 i+.O 2 . 7 Photosynthesis(area) 1 .7 2 . 2 2 . 3 2 . 1 Respiration . 1 .9 3 . 9 1+.9 5 . 8 expressed as m i c r o l i t r e s 02 exchanged/lOO rag. CCm2} /1L0 min. The discrepancy between area- and weight-based photosynthetic values was again apparent and made more clear by expression as r a t i o s of the mature rate: Nov. 7 t h Photosynthesis (wt.) 1 . 3 9 1 0 . 9 0 0 . 6 5 Nov. 1 0 t h Photosynthesis (wt.) 0 . 9 7 1 0 . 7 5 0 . 5 1 Nov. 1 0 t h Photosynthesis (area) O .76 1 1 . 0 5 O .96 The apparent increase i n photosynthesis, from 5 0 - 6 0 $ to over 1 0 0 $ , on a weight basis and the corresponding decline i n r e s p i r a t i o n (from 11+6$ to 1+7$) may indicate that the eff e c t of phy s i o l o g i c a l age was the greatest single source of v a r i a b i l i t y i n t h i s method. A subconcious yet consistent sampling of older or younger leaves could e a s i l y y i e l d a r e s u l t , highly s i g n i f i c a n t for t h i s cause alone• One can note from the above table that expression of res u l t s f o r photosynthesis on an area basis indicated sub-s t a n t i a l l y the same photosynthetic rate i n a l l leaves up to f u l l y mature ones. In contrast, the weight-based expression att r i b u t e d a f a r higher photosynthetic rate to the mature leaves than to the young leaves. 2. I n f i l t r a t i o n tests In an e f f o r t to overcome the c r i t i c i s m of morphological changes with treatment and to observe short term e f f e c t s , a method more l i k e that of Wedding (60) was employed. Several experiments were run, one of which i s described below. A large number of l e a f l e t s of approximately the same size and physiological age were taken and sorted, on the basis of s i m i l a r s i z e , into three groups. These three groups were divided so that one l e a f l e t from each was a l l o t t e d to a single treatment and manometer. The l e a f l e t s were vacuum i n f i l t r a t e d twice with pH-corrected water, with or without ferrous or f e r r i c sulphate at 300 p.p.m. Af t e r an i n t e r v a l of two hours i n which the l e a f l e t s were drained but kept i n a moist environment, pH-corrected water with or without 2,1+-D at 100 p.p.m. was i n f i l t r a t e d twice. Afte r the second i n f i l t r a t i o n , the selected l e a f l e t s were placed i n the Warburg f l a s k s and photosynthesis 77 r e s p i r a t i o n rates determined simply as the mean of three manometer def l e c t i o n s . The r e s u l t s are expressed as liO minute reading, a f t e r f i v e hours, as percentage of i n i t i a l reading, i n d i c a t i n g whether there was a stimulatory or i n h i b i t o r y e f f e c t and whether ir o n i n any way protected against the e f f e c t of 2 , 1L-D . Results Treatment Photosynthesis % f a l l Respiration % f a l l water only 6 8 . 0 2 9 . 7 water + 2 , 1L-D 7 2 . 2 2 8 . 8 ferrous + water 6 8 . 0 3 0 . 9 ferrous + 2 , 1L-D lk.7 3 2 . 0 f e r r i c + water 5 5 . 0 3I4..7 f e r r i c + 2 , ! L -D 8 0 . q. 31.O It appears that 2 , l i - D had a s l i g h t i n h i b i t o r y e f f e c t on photo-synthesis and l i t t l e e f f e c t on r e s p i r a t i o n . Ferrous i r o n had no e f f e c t on photosynthesis, but with 2 ,1L-D, i t i n h i b i t e d more strongly than d i d 2 , I L - D alone. Respiration was s l i g h t l y i n h i b i t e d by the ferrous i r o n and more strongly by ferrous i r o n plus 2,q . - D . These effects are perhaps att r i b u t a b l e to osmotic causes, as only pH was controll e d . However, the effects of f e r r i c i r o n seem s i g n i f i c a n t , i n greatly stimulating photosynthesis and i n h i b i t i n g r e s p i r a t i o n when alone, yet greatly i n h i b i t i n g photosynthesis i n company with 2 , 1L-D , r e s p i r a t i o n s being l e s s i n h i b i t e d . This at l e a s t provides reason for further i n v e s t i g a t i o n of the ferrous and f e r r i c effects when alone. 3 . E f f e c t s of ferrous and f e r r i c ions alone Ap p l i c a t i o n of 15>0 p.p.m. of ferrous and f e r r i c sulphates (both at pH 1 . 5 ) > to plots i n the same manner as f o r 78 treatments twelve and thirteen, failed to produce any significant differences between these plots and one treated with water at pH 1 . 5 . Results Photosynthesis , 1 Respiration 21+ hrs. 9b hrs. ll+8 hrs. 2l+ hrs. 96 hrs. ll+8 hrs. lj..69 Con if . 8 9 Con 5.73 Fe+++ Ji .89 Pe++ 5 . 1 6 Fe++ 5 . 2 5 Pe+++ 3 . 8 3 Con 6 . 3 3 Con 6 . 2 3 P e + + + 6.71 Pe+++ 6.kp Fe++ 6 . 9 9 P e + + 6.81+ Con 5.1+5 Con 5 . 5 5 Fe+++ 5 . 5 9 Pe++ Fresh ¥t. Height ll+8 hrs. ll+8 hrs. 59.3 Con 12.1+ Con 60.0 P e + + + 12.5 P e + + + 60.2 F e + + 12.6 P e + + 1 Gas exchanges are as microlitres O2 exchanged/100 milligrams F.W./1+0 minutes. 79 Discussion A series of experiments such as the one described here w i l l i n e v i t a b l y leave i n i t s wake a number of unanswered questions, a number of pointers to new and possibly f r u i t f u l l i n e s of research. I t i s not possible i n the few experiments described to clear up a l l the points which arose but a few questions remain which may bear closer examination. F i r s t there i s the question of the whole experimental method used: was the manometric approach most suitable? How f a r was this responsible f o r the r e l a t i v e l y few s i g n i f i c a n t differences observed? What was the cause of the v a r i a t i o n i n gas exchange i n consecutive determinations observed both here and by Wedding (60,61)? How f a r can storaatal control be disregarded i n experiments of t h i s kind ( 2 0 ) ? Was there any discrete or synergistic action by the i r o n i n the l a t e r exper-iments? I f i n t e r a c t i o n between the metal ions and 2,q.-D i n separate applications i s the case, then what could be i t s nature? I t has already been observed that there are many ways of measuring photosynthesis and r e s p i r a t i o n . The polarographic method i s one which could e a s i l y provide r e s u l t s of greater accuracy than can the Warburg apparatus (lii> 5 2 ) , though the r e l a t i v e l y complex equipment required would make r e p l i c a t i o n a more complex matter than with the Warburg apparatus. An advantage of the polarographic method i s that i t w i l l record almost continuously. Another method fo r the detection of carbon dioxide concentration changes i s described 80 by Decker (13)• He used a Beckman LB lj? i n f r a - r e d gas analyzer and recording potentiometer and was able to show that r e s p i r a t i o n during the l i g h t period, while photosynthesis was continuing, was three times that i n the dark. This may mean that the so-called 'actual' photosynthetic rate, derived from addition of alternate l i g h t and dark Warburg readings, i s too low by a factor of three. This method also could be made continuously-recording. Thus, there are two methods which could detect carbon dioxide and oxygen concentrations i n an a i r stream continuously and with automatic recording i f needed. Decker expressed his r e s u l t s i n terms of l e a f area, using the f o i l -weight method, A continuous-flow method, perhaps an elaboration of that used by Preeland (16) with more sophisticated analysis methods, would provide an i n t e r e s t i n g comparison with the Warburg methods. One might expect greater accuracy purely because a far l arger proportion of the t o t a l gas exchange of the plant would be available f o r analysis. The Warburg apparatus i t s e l f has been considerably modified for photosynthesis determin-ations, with new departures In f l a s k design and l i g h t source (1+0), The Barcroft manometer would be a considerable improve-ment i n eliminating changes i n atmospheric pressure, which the present worker found troublesome i n changeable weather. Methods are r e a d i l y available f o r i t s use for photosynthesis deter-mination ( 5 6 ) . The whole question of the r o l e of metal chelation i n the effects of plant growth substances i s of considerable i n t e r e s t at the present time, and i n the l i g h t of the preceding 81 r e s u l t s j u s t i f i e s some discussion. The work has centred on two main questions: f i r s t , whether plant growth substances are able to chelate metal ions, and second, whether chelating agents w i l l p a r a l l e l the behaviour of plant growth substances. The f i r s t question seems to be one of degree. That the growth substances derived from acetic acid can form s a l t s with i r o n and copper i s not i n dispute; the question at issue i s whether the chelating action i s any stronger than that of acetic a c i d . Fawcett (l£) and P e r r i n (qj) r e p l i e d negatively to this question, Cohen et a l . (12), Recaldin and Heath (1+5) and Armarego et a l . i n the aff i r m a t i v e , Armarego et a l . (1) claimed to have formed copper complexes of 2,1L-D and i t s methyl substituted a l l i e s , and of naphthaleneacetic acid, quite d i f f e r e n t from anything derived from acetic acid. However, they found no c o r r e l a t i o n between the pK values of t h e i r chelates and auxin a c t i v i t y , and doubted that chelation i s s i g n i f i c a n t i n explaining the effects of 2,1+-D. Cohen (12) claimed complexing between IAA, WAA and metals, and that the metals were sequestered by the aromatic r i n g rather than as a bidentate complex. They showed Cu**, P e + + + and C o + + to complex, i n decreasing order of strength, and found no complexing with Oa"1"1" or Mg*4". Thus i f there i s a growth e f f e c t by chelation, i t i s with the t r a n s i t i o n metals. P e r r i n (ij.3)> with more re f i n e d techniques, claimed that the substituted acetic acid growth substances show no more tendency to chelate than does the parent acid. His explanation of the consequent improbability of the chelation theory i s l e s s convincing. 82 The claims that ethylene diamine te t r a - a c e t i c acid and 8-hydroxy quinoline have growth effects are strong. Heath and Clark ( 2 1 , 2 2 ) demonstrated growth e f f e c t s . Weinstein et a l . (62) demonstrated the i r o n EDTA to be inactive and even growth-i n h i b i t o r y , where uncomplexed EDTA was active i n Lupinus hypocotyl elongation. Iron EDTA stimulated r e s p i r a t i o n but to a much les s marked degree than EDTA alone. The observation by Recaldin and Heath ( l ^ ) that f e r r i c i r o n and IAA complexed, but that the IAA carboxyl was destroyed, i n a reaction thought by Thimann (52) to be akin to the Salkowski reaction; and that of Shibaoka (1+8), that ferrous suphate has a discrete growth e f f e c t ; i n company with the chelation controversy, strongly suggest that the question of i r o n protection against the effects of 2,1+-D i s probably not a simple one. 83 Summary Certain effects of XAA with and without cupric ions, of 2 , J + - D and i t s cupric s a l t and of 2 , 1L-D with and without various forms of i r o n have been studied. The effects i n question have been those on photosynthesis and r e s p i r a t i o n , fresh, dry and ash weights, and morphology of potato plants. The applications of L M i n dust form were i n s u f f i c i e n t to produce any departures from the control, but these treatments provided useful practice i n lowering the c o e f f i c i e n t of v a r i a b i l i t y of the experiment. The copper s a l t of 2 , 1L-D was shown to have l e s s e f f e c t on these measurements than the acid alone, when they were applied i n dust form at a rate of f i v e pounds per acre. Gas exchange was s i g n i f i c a n t l y lowered by the a c i d at 168 and 216 hours aft e r treatment. Leaf cu r l i n g and height were s i g n i f i c a n t l y increased by the aci d . The effects of 2 , ! L - D applied at a rate of f i v e pounds per acre were i n every case s i g n i f i c a n t l y d i f f e r e n t from the effects of the same rate of 2 , 1L-D when ferrous sulphate or f e r r i c EDTA was added to the dust. Frequent s i g n i f i c a n t differences i n gas exchange were obtained between plots sprayed with various aqueous concen-trations of 2 , I L - D and plots sprayed with the same concentrations a f t e r being sprayed with i r o n solutions. Dry weights i n the plots treated with l£0 p.p.m. 2,q.-D with or without pretreatment with i r o n were lower than the control, though not s i g n i f i c a n t l y so. Height was s i g n i f i c a n t l y increased i n the iron-pretreated p l o t s , and there was no s i g n i f i c a n t difference between 2,1|-D-treated and control p l o t s . This l a s t observation i s taken as an i n d i c a t i o n that the e f f e c t i v e concentration of ,2,1+-D was lowered by i r o n pretreatment. 8 5 APPENDIX A Comparison of Stability Constants of Iron Compounds  after Bjerrum, Schwarzenbach et a l . (5) The choice of salts for experimental treatments 8-13 was governed by the desirability of a common anion and st a b i l i t y constant. This ideal is approached by the sulphate and chloride salts of iron. The citrate has a sta b i l i t y constant of a quite different order. 1 ~ CMTITJ K 2 [MLg] £ML]£L] (as logarithms) eg. [Fe+ ++Cl) £Pe)£C13 1.0 0.76 pK- PK2 Ferrous citrate Ferric citrate (MH 2L) 2.12 (MH 2L) 6.3 (MHL)3.08 (MHL)11.85 Ferrous chloride Ferric chloride 0.36 0.76 O.Olj. 0.30 0.06 Ferrous sulphate Ferric sulphate 3.02) O.0I4,) ) under different conditions. Appendix (Continued) Table of Least Signif i c a n t Ranges. Calculated from Tables (50 pp kk2-kk3) Error Mean Sq. Mean Square Level: 5$ 1$ Root Means involved: 2(3.06) . 3(3-21) 2(l+.26) 3(4»48) 0.01 0.02 0 . 0 3 O.OIL 0.05 0.06 0.07 0.08 0.09 0 . 1 . 0 . 2 0 . 3 O.k 0.5 0.6 0.7 0.8 0 .9 1.0 1.2 li 1.8 8 10 0.0016 0.0033 0.0050 0.0066 0.0083 0.0100 0.0116 0.0133 0.015 0.0166 0.033 0.050 0.066 0.083 0.100 0.116 0.133 0.150 0.166 0.200 0 .233 0.266 0.300 0.333 0.666 1.00 1.33 1.66 O.OljX) 0.057 0.0707 0.080 0.091 0.100 0.108 0.1153 0.1225 0.129 0.182 O.22J1 0.258 0.288 0.316 0 . 3 k l 0.365 0.387 O.I4.07 0.1+47 0.1+53 0.5165 0.51+8 0.577 0.8165 1.000 l.ll+O 1.290 0.122 0.176 0.216 0.21+5 0.278 0.306 0.330 0.353 0.375 0.395 0.556 0.685 0.789 0.881 0.967 1.01+3 1.117 1.18k 1.21+5 1.368 I . 4 7 8 1.580 1.677 1.766 2.498 3.060 3 « 4 8 8 3.947 0.128 0.184 0.227 0.257 0.292 0.321 0.347 0.370 0.393 O.I+14 0.581+ 0.719 0.828 0.924 1 . 0 l 4 1.095 1.172 1.21+2 1.306 I . 4 3 5 1.550 1.658 1.759 1.852 2.621 3.210 .659 .ll+l i 0.170 0.245 0.301 0.341 0.388 0.1+26 0.460 O.49I 0.522 0.549 0.775 0.954 1.099 1.227 1.555 1.649 1.734 1.90k 2.058 2.200 i : » 3.478 4.260 4.856 5.495 0.179 0.257 0.317 0.358 0 .408 0.1+48 0 .484 0.517 0 .549 0.578 0.815 1.004 1.15& 1.290 1.1+16 1.528 1 .635 1.734 1.823 2.003 2 .164 VM 2 .585 3.658 4.4^0 5.107 5.779 Appendix (Continued) Error Mean Square Level: 5$ 1% Mean Sq. 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