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Growth and biochemical responses of the tomato (Lycopersicum esculentum var. Bonny Best) to K naphthenates Chu, Soong-ming 1969

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GROWTH AND BIOCHEMICAL RESPONSES OF THE TOMATO (Lycopersicum  esculentum var. Bonny Best) to K NAPH 3-HENATES by SOONG-MING CHU B . S c , New As ia Col lege , Chinese U n i v e r s i t y of Hong Kong, Hong Kong, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Botany We accept th i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t of Botany The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date A p r i l 18, . 1969 ABSTRACT Recent reports, e s p e c i a l l y those of Russian s c i e n t i s t s , have emphasized that application of stimulatory concentrations of naphthenates (Naps) induced greater and better growth and productivity of a number of species of plants. This stimulatory action of Naps has been found to r e s u l t from seed soaking and spraying seeds or developing plants once or repeatedly. However, no systematic attempt has so far been made to investigate physio-l o g i c a l and biochemical changes induced i n a r e l a t i v e short period following immediately these treatments. A c o r r e l a t i o n of such changes with f i n a l improvements i n growth and y i e l d may provide a better understanding of the mechanism of action of Naps It was therefore ess e n t i a l and s i g n i f i c a n t to investigate these aspects. Seeds of tomato (Lycopersicum esculentum var. Bonny Best) were germinated i n wooden f l a t s containing s t e r i l i z e d s o i l and were transplanted when 10 days old to p l a s t i c pots of 6 inche diameter containing s t e r i l i z e d s o i l . The plants were grown i n a growth room. In separate experiments, potassium naphthenate (KNap) aqueous solutions, 2,500 ppm and 5,000 ppm, were sprayed onto tomato leaves when plants were 2, 3, and 4 weeks old. Measurements of vegetative growth, based on fresh and dry weights of plant tops, indicated that maximum stimulation was induced by the 5,000 ppm KNap solution applied to plants when 3 weeks o l d . I t was then decided to invest igate the biochemical and p h y s i o l o g i c a l responses of the tomato plants to 5,000 ppm KNap when treated at the age of 3 weeks. Determinations of pigment content, i n t e n s i t i e s of photo-synthesis and r e s p i r a t i o n , a c t i v i t y of enzymes involved i n nitrogen metabolism, such as n i t r a t e reductase (NRase) and glutamic-pyruvic transaminase (transaminase), and of enzymes involved i n carbo-hydrate metabolism, such as succ in ic dehydrogenase, phosphorylase, and phosphoglyceryl kinase were made three times at 2-week i n t e r -v a l s , beginning 2 weeks af ter the spraying. Number and fresh weight of tomato f r u i t s , q u a l i t y of tomato f r u i t s i n terms of sugars, t i t r a t a b l e a c i d i t y and ascorbic a c i d were also invest igated at scheduled.v.'-. i n t e r v a l s . Results ind icated the fo l lowing: (1) In the treated p lants , the content of the pigments c h l o r o p h y l l a and b, and e s p e c i a l l y carotenoid, i n the l ea f blades was higher than i n c o n t r o l p lants , (2) Measurements made with i n t a c t plants using an i n f r a r e d CC>2 analyzer revealed increases i n i n t e n s i t i e s of photo-synthesis and r e s p i r a t i o n of the a e r i a l port ions 4 weeks af ter treatment but the opposite was true 2 weeks af ter treatment, (3) Under the inf luence of KNap, of the 5 enzymes examined only phos-phorylase a c t i v i t y was found to be st imulated at a l l three observation times. Transaminase a c t i v i t y was greater 6 weeks af ter treatment. A c t i v i t i e s of succ in ic dehydrogenase, NRase, and phosphoglyceryl kinase were a l l reduced by treatments, (4) In a subsequent experiment, leaf blades of plants treated when 2 weeks old were analyzed for succinic dehydrogenase a c t i v i t y 4, 8, 12, 16, 20, and 24 days afte r spraying. The e f f e c t on succinic dehydrogenase a c t i v i t y fluctuated with the age of the plant. P a r a l l e l changes i n the protein content of the enzyme extract could not be detected, (5) Tomato f r u i t y i e l d , based on number and fresh weight, was decreased by 2,500 ppm KNap treat-ment but increased by 5,000 ppm KNap. In addition, 5,000 ppm KNap-treated plants were more re s i s t a n t to blossom-end rot and showed better and quicker recovery when the deficiency disease was treated with CaC^. E a r l i e r maturity was found i n 5,000 ppm KNap-treated plants, (6) The mature tomato f r u i t s from 5,000 ppm KNap-treated plants contained larger amounts of sugars (re-ducing sugar and sucrose) than the controls, and the sugars i n mature jjomato f r u i t s were l o s t at a lower rate during the storage period. The treatment resulted i n decreased t i t r a t a b l e acid and ascorbic acid content. I t afforded no protection against loss of t i t r a t a b l e acid and ascorbic acid during storage. i v ACKNOWLEDGEMENT The author wishes to take th i s opportunity to extend her deepest thanks and apprec iat ion to Dr. D . J . Wort who not only suggested the o r i g i n a l idea for th i s study, but who has given h is guidance, advice, encouragement, and patience to the author throughout the progress of th i s i n v e s t i g a t i o n . Further-more, the author wishes to thank him for h i s construct ive r e -view of the ent i re manuscript. Sincere grat i tude i s a lso due to Dr. G.H.N. Towers, Dr. D.P. Ormrod, and Dr. B .A. Bohm for the i r moral support and advice through th i s study. P a r t i c u l a r apprec iat ion i s owed to Dr. G.W. Eaton for h i s generous help i n s t a t i s t i c a l analys i s for th i s thes i s . C o r d i a l thanks go to Mr. K. Pate l who has helped the author i n many ways. F i n a l l y , acknowledgement i s expressed to the Department of Botany for the use of a l l ava i lab le f a c i l i t i e s and equipment to make the experiments poss ib le . V TABLE OF CONTENTS PAGE Abstract i Acknowledgment i v Table of Contents v L i s t of Figures •••• v i i i E i s t of Tables x i CHAPTER I. NAPHTHENIC ACIDS 1 A. Introduction 1 B. Composition 1 C. Properties 1 D. Uses .... 1 II. EFFECTS OF NAPHTHENATES ON LIVING ORGANISMS.. 3 A. Introduction 3 B. Effects on animals 3 C. Eff e c t s on plants 4 I I I . MATERIALS AND METHODS 7 A. Plant material 7 B. Preparation of the potassium naphthenate aqueous solution from naphthenic acids (HNap) 8 C. Spray treatments 8 D. Experimental design 8 E. Measurement time 9 v i TABLE OF CONTENTS (cont'd) Page F. Vegetative growth measurement 9 G. Determination of pigments 10 H. Determination of photosynthesis and r e s p i r a t i o n 11 I. Determination of enzyme a c t i v i t i e s 12 1. Preparation of Tris-HCl buffer solution 13 2. Preparation of the crude extract 13 3. Nitrate Eeductase (NRase) 14 4. Glutamic-pyruvic transaminase (transaminase) 14 5. Phosphoglyceryl kinase.. 15 6. Phosphorylase .. 17 7. Succinic dehydrogenase 18 J. Determination of protein 19 K. P o l l i n a t i o n 20 L. Blossom-end rot 20 M. Y i e l d measurement 20 N. Quality of tomato f r u i t s 20 1. Determination of reducing sugars and sucrose .. 21 2. T i t r a t a b l e acid 22 3. Ascorbic acid 23 IV. RESULTS 24 A. Vegetative growth 24 1. Experiment 1 25 v i i TABLE OF CONTENTS (cont'd) Page 2. Experiment II 29 3. Experiment III 32 4. Experiment IV 34 B. Chlorophy l l and carotenoid c o n t e n t . . . . 41 C. Photosynthesis and r e s p i r a t i o n 41 D. Enzyme a c t i v i t i e s 46 1. N i t r a t e reductase 47 2. Glutamic-pyruvic transaminase 47 3. Phosphoglyceryl kinase 54 4. Phosphorylase 54 5. . Succinic dehydrogenase 54 E . Prote in content 55 F . Tomato y i e l d 55 G. Qual i ty of tomato f r u i t 63 1. Sugars 63 2. T i t r a t a b l e a c i d 63 3. Ascorbic a c i d . . . 63 V. DISCUSSION 69 VI . CONCLUSIONS 78 VII . REFERENCES 80 VIII . APPENDICES 86 v i i i LIST OF FIGURES FIGURE PAGE 1 E f f e c t of 2,500 ppm and 5,000 ppm KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment I) 28 2 E f f e c t of 2,500 ppm and 5,000 ppm KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment II) 31 3 E f f e c t of 2,500 ppm and 5,000 ppm KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment III, f i r s t harvest) 36 4 E f f e c t of 2,500 ppm and 5,000 ppm KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment III , second harvest) 38 5 E f f e c t of 5,000 ppm KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment IV).... .-. 4 0 6 E f f e c t of 5,000 ppm KNap on the contents of chlorophyll a and b and carotenoid of tomato leaf, blades 43 7 E f f e c t of 5,000 ppm KNap on the rates of photosynthesis and r e s p i r a t i o n of tomato plants, and on the contents of pigments of tomato le a f blades 44 8 E f f e c t of 5,000 ppm KNap on the rates of apparent photosynthesis, r e s p i r a t i o n and true photo-synthesis of tomato plants 45 9 E f f e c t of 5,000 ppm KNap on the a c t i v i t y of n i t r a t e reductase i n tomato leaf blades 49 10 E f f e c t of 5,000 ppm KNap on the a c t i v i t y of phos-phorylase i n tomato leaf blades 49 11 E f f e c t of 5,000 ppm KNap on the a c t i v i t y of phos-phoglyceryl kinase i n tomato leaf blades 50 ix LIST OF FIGURES (cont'd) FIGURE PAGE 12 E f f e c t of 5,000 ppm KNap on the a c t i v i t y of glutamic-pyruvic transaminase in tomato lea f blades 51 13 E f f e c t of 5,000 ppm KNap on the a c t i v i t y of succinic dehydrogenase i n tomato leaf blades (Experiment IV) 52 14 E f f e c t of 5,000 ppm KNap on the enzyme a c t i v i t y of tomato leaf blades 53 15 E f f e c t of 5,000 ppm KNap on the a c t i v i t y of succinic dehydrogenase i n tomato leaf blades (Experiment V) 57 16 E f f e c t of 5,000 ppm KNap on the a c t i v i t y of succinic dehydrogenase i n tomato leaf blades (Experiment V) 58 17 Protein content of the enzyme extracts of the lea f blades of KNap-treated and control plants.. 59 18 E f f e c t of 5,000 ppm KNap on protein content of enzyme extracts of leaf blades of tomato plants 59 19 E f f e c t of KNap on number and fresh weight of tomato f r u i t s (Experiment II and IV) 62 2 0 E f f e c t of 5,000 ppm KNap on content of sucrose, reducing sugar, and t o t a l sugars i n mature tomato f r u i t s 65 21 E f f e c t of 5,000 ppm KNap on the content of t i t r a t a b l e acid i n mature tomato f r u i t s 66 22 E f f e c t of 5,000 ppm KNap on content of ascorbic ac i d i n mature tomato f r u i t s 67 23 E f f e c t of 5,000 ppm KNap on the contents of sugars, ascorbic acid and t i t r a t a b l e acid i n mature tomato f r u i t s . . . 68 X LIST OF FIGURES (cont'd) FIGURE PAGE 24 Standard chart for n i t r a t e reductase 87 25 Standard chart for phosphorylase 88 26 Standard chart for succ in ic dehydrogenase 89 27 Standard chart for ascorbic ac id 90 x i LIST OF TABLES TABLE PAGE Ia-e The e f f e c t of KNap on fresh and dry weights of leaves and stems of tomato plants. a Experiment I 2 7 b Experiment II 30 c Experiment I I I , f i r s t harvest 35 d Experiment I I I , second harvest 3 7 e Experiment IV 39 II The e f f e c t of 5,000 ppm KNap on the rates of • photosynthesis and re s p i r a t i o n of tomato plants and on the pigment contents of tomato le a f blades . . .42 III The e f f e c t of 5,000 ppm KNap on the a c t i v i t i e s of succinic dehydrogenase, phos-phoglyceryl kinase, glutamic-pyruvic trans-aminase, n i t r a t e reductase, and phosphory-lase of tomato l e a f blades 48 IV The ef f e c t of 5,000 ppm KNap on the a c t i v i t i e s of succinic dehydrogenase and the protein content of the enzyme extract of the leaf blades of tomato plants over a period of 2 0 days be-ginning when 18 days old. 56 V The e f f e c t of KNap on numbers and fresh weights of ripe and rotten tomato f r u i t s (Experiment II) 61 VI The e f f e c t of KNap on numbers and fresh weights of ripe and green tomato f r u i t s (Experiment IV) 61 VII The e f f e c t of 5,000 ppm KNap on the content of sugars, ascorbic acid, and t i t r a t a b l e acid i n mature tomato f r u i t s 64 I . NAPHTHENIC ACIDS A. Introduct ion The name, naphthenic acids , was suggested i n 1883 by Markovnikoff and Oglobl in for the C -QH . 2 0 ^ 2 a c i d s ° f unknown structure which H e l l and Medinger had discovered from Rumanian o i l . Current ly , the term i s used to denote the carboxy l i c acids occurr ing i n and recovered from petroleum. They are frequently termed "petroleum acids", because subsequent work has shown that phenols and a l i p h a t i c acids are also present i n some crudes. Commercial naphthenic a c i d (HNap) i s a product which contains a l l the a c i d i c components of the crude, and varying amounts, usua l ly less than 10%, of " o i l " , that i s to say nonacidic compounds mostly hydrocarbons. B. Composition Chemical ly , HNaps are monocarboxylic acids of naphthene ( a l i c y c l i c ) ser ies of hydrocarbons. Their general formula may be wri t ten .R(CH 2 ) nC00H, where R i s a c y c l i c nucleus composed of one or more r i n g s . These r ings are usua l ly 5-membered (cyclopentane) and may be a l k y l a t e d . The simplest a c i d conforming to th i s d e f i n i t i o n when n= 1 i s cyclopentane-acet ic a c i d , C H 0 — C H CHoCOOH The l i t e r a t u r e on the composition of the acids occurring in petroleum leads to the following generalizations: 1. They are natural components of the crude and not formed during r e f i n i n g . 2. They are predominantly monocarboxylic acids. 3. Generally, the carboxyl group i s not d i r e c t l y attached to the r i n g but through a methylene group or a chain containing up to 5 or more methylene groups. 4. Cyclopentane rings predominate but cyclohexane rings are present i n some cases. Aromatic rings or fused aromatic-naphthene rings may be expected i n the high molecular weight acid 5. The types of acids i n petroleum and the approximate carbon range i n which they have been found i s as follows: acids n a l i p h a t i c , C nH2 n0 2 < 7 monocyclic, C nH2 n-202 7-12 b i c y c l i c , C nH2 n- 4 02 12-20 t r i c y c l i c , C nH2 n-6°2 ^ 2 0 p o l y c y c l i c , C nH2 n-802 to C nH 2 n-140 2 > 20 C. Properties Naphthenic acids are o i l y l i q u i d s of a c h a r a c t e r i s t i c odor which varies with the acid source and degree of refinement. Phenols and sulfur compounds are responsible for most of the odor D. Uses Naphthenic acid can be used i n lubricants, driers and 3 catalysts, perservatives, emulsifiers, corrosion, i n h i b i t o r s , and fungicides. Besides, HNaps and their s a l t s of sodium, potassium, n i c k e l , and copper have been found to act as growth stimulators. More detailed information about the HNaps was given by J o l l y (1967) i n Encyclopedia of Chemical Technology Vol 13. II. EFFECTS OF NAPHTHENATES ON  LIVING ORGANISMS A. Introduction I t has been reported by a number of workers, most of which are Russians, that HNaps or their s a l t s (Naps) can act as growth stimulators. Nap has been found to stimulate or promote growth, y i e l d , and biochemical and physiological a c t i v i t i e s of a number of species of animals and plants. B. Effects on animals Glushkov and Yakovlev (1963) reported that the bee group which received the NaNap fed i n a dose of 2 00 g/day/family for 3 weeks showed 18.30% more growth than controls. Probosces were 1.97% longer than those of the controls. There are several additional reports which bear out the stimulative e f f e c t on growth of animals. A l i e v (1963) reported that NaNap fed to swine, c a t t l e , and calves at the rate of 2 to 20 mg/kg of feed resulted i n an increase i n body weight over that of control animals. Starkova et a l . (1963) reported that 3 mg NaNap/kg of feed raised the weight increments of pigs by 10 to 17%, 4 and that of poultry by 15%. Dimitrov and Popoff (1966) reported that Naps fed to hens showed encouraging r e s u l t s i n increased egg-laying. C. E f f e c t on plants Some of the l i t e r a t u r e concerning the stimulatory e f f e c t of Nap on d i f f e r e n t species of plants i s summarized as follows. I t has been shown by Burachevskii (1965) that the application of NaNap to cultures of Aspergillus usamii could promote both growth and amylase formation by the mold. Cotton plants treated with NaNap have shown to be increased i n the rate of photosynthesis, content of chlorophyll and ascorbic a c i d of the leaves. It has also been demonstrated that the effects of NaNap on cotton plants appeared to in t e r a c t with the amount of applied f e r t i l i z e r s (Agakishier and Bazanova, 1965; Bazanova and Akapova, 1966; and Naghibin, 1966). Under the influence of NaNap, corn has been observed to be stimulated i n the development of vegetative and generative organs>- and increased i n y i e l d . (Starkova and Sevast'yanova, 1963; Popoff et a l . , 1966; and Eyubov and Issaeva, 1966). The e f f e c t of Nap on potatoes has been examined by d i f f e r e n t workers including Starkova et a l . (1963), Abolina and Ataullaev (1966), and Ladygina (1965). The l a t t e r investigator demonstrated that higher.chlorophyll content i n leaves stimulated photosynthetic and respiratory i n t e n s i t i e s , more protein and starch content i n 5 the tuber, and increased y i e l d were found i n potatoes soaked i n 0.0005% Nap for 1 hour before planting. Promotive effects of Nap on the growth and development of tobacco plants were noticed by Zamanov (1966) and Popoff et a l . (1966). However, no clear e f f e c t was established with respect to the q u a l i t y of the tobacco leaves. Different investigators postulated that Nap has a favorable influence on both the q u a l i t y and quantity of a number of d i f f e r e n t f r u i t s . For instance, Kulieva (1964) showed that when o l i v e trees were sprayed with a 0.01% solution of the petroleum growth-promoter NaNap during f r u i t formation, f r u i t y i e l d was increased approximately 70%. The average number of f r u i t s which matured increased 1.50 to 2.60%. Work of Abolina and Ataullaev (1966) indicated that Nap spraying gave a considerable y i e l d increase of d i f f e r e n t sorts of melons up to 10 to 15 tons per ha. The sugar content of the f r u i t was also increased by the treat-ment. The application of petroleum growth-promoting substance to the grape plant led to increased photosynthesis, increased catalase a c t i v i t y , and increased sugar content i n grapes (Kolesnik, 1965). Extensive studies on the e f f e c t of Nap on tomato plants have been made by several investigators. Szekely (1966) demon-strated that the crop y i e l d of tomato was increased s i g n i f i c a n t l y by seed treatment with Nap and Nap + trace elements. 6 Pakhomova (1965) showed that spraying tomato plants with 0.005% aqueous petroleum growth-promoting stubstance caused changes i n proteins and nucleic acids i n leaves. Besides, an increase of 18.80% i n tomato y i e l d was found. A l i e v (1965) accelerated the development of tomato plants by the application of NaNap i n combination with mineral f e r t i l i z e r s . He suggested that the most e f f e c t i v e ways of application of NaNap were into the s o i l and as a spray. An increase of 3 0 to 3 7% i n y i e l d was found. In addition, the composition of the tomato f r u i t changed sharply, increased amounts of sugars, dry substances and ascorbic acid were observed. Popoff (1966) obtained early ripeness by spraying tomato plants with Nap. Other investigators at the University of B r i t i s h Columbia also got enhanced growth i n Nap-treated plants. For example, maize, sugar beet, sunflower, radish, spinach, tomato and tobacco (Wort,, unpublished), and bush bean (Wort, Patel and Fattah, unpublished) were found to be stimulated by Nap treatment. Patel and Fattah (unpublished) observed higher pigment contents, greater photosynthesis and r e s p i r a t i o n i n t e n s i t i e s , and increased a c t i v i t i e s of transaminase, phosphoglyceryl kinase, and phos-phorylase i n l e a f blades of KNap-treated bush bean plants. 7 III. MATERIALS AND METHODS A. Plant material A single variety of tomato (Lycopersicum esculentum var. Bonny Best) was used i n a l l the experiments. Seeds were obtained from Buckerfield 1s Limited, New Westminster, B r i t i s h Columbia. The seeds were placed,equally spaced,in the wooden f l a t s contain-ing s t e r i l i z e d garden s o i l to germinate. The seeds, and later the plants, were watered d a i l y with tap water. When the seedlings were about two weeks old, one or two uniform seedlings were trans-planted into each of the p l a s t i c pots, of 6 inches diameter, containing s t e r i l i z e d garden s o i l . The transplantation was conducted very c a r e f u l l y to minimize damage to roots of the seedlings. The pots were put into a growth room wherein the photoperiod was 16 hr, the l i g h t i n t e n s i t y was 1,400 foot candles at the tops of the plants. The temperature was 20 to 2 5°C, and the r e l a t i v e humidity was 55 to 72% during l i g h t period. In the dark period, the temperature was maintained at 18 to 22°C, and the r e l a t i v e humidity 70 to 85%. The pot locations were changed at inte r v a l s i n order to average l o c a l environmental v a r i a b i l i t y . Plants which were raised for mature growth were transplanted into a bench of s o i l i n a green house. The seedlings were spaced 16 inches apart. In the green house the environmental factors were somewhat variable and d i f f i c u l t to control. Soluble f e r t i l i z e r , N:P:K,20:20:20, at the concentration 8 of 0.5 g/1 was applied weekly to the s o i l at the rate of 50 ml/ plant beginning when plants were 3 weeks old. B. Preparation of the potassium naphthenate aqueous solution  from naphthenic acid (HNap) KOH solution (2.1 g KOH dissolved i n 17 ml d i s t i l l e d water) was added to a fl a s k containing 5 g HNap and shaken vigorously for 15 minutes. The solution was allowed to stand and then the upper portion was made to a volume of 24 ml with d i s t i l l e d water. The solution obtained was the stock solution which contained 2 5o mg of KNap/ml. By d i l u t i n g 1 ml of the stock solution to 100 and 50 ml with d i s t i l l e d water containing the wetting agent Tween 20, 0.3%, 2,500 ppm and 5,000 ppm KNap solutions were obtained. The pH value of the dil u t e d solution was adjusted to about 10 with d i l u t e HCl. C. Spray treatments The 2,500 ppm and 5,000 ppm KNap solutions at the rate of 2 ml/plant were sprayed onto the tomato leaves when the plants were 2, 3, and 4 weeks old, i n d i f f e r e n t experiments. The solutions were allowed to dry on the surface of the leaves and thereafter the pots were transferred to the growth room again. D. Experimental design Five experiments were c a r r i e d out. For the f i r s t two experiments, the treated and controlled plants were randomized i n each of three blocks. In the l a s t three experiments, the 9 layout was a complete randomized design. E. Measurement time For the f i r s t experiment, harvests were made 2 and 5 weeks aft e r treatment. There were three harvests made on the 14th, 28th and 90th days after spraying i n the second experiment. Plants were harvested when 33 and 47 days o l d i n the t h i r d experiment. A l l these experiments were concerned primarily with the fresh and dry weights of leaves and stems of the plants. In the fourth experiment, a l l measurements of juvenile growth, chemical composition, photosynthesis, r e s p i r a t i o n , and e n z y m e a c t i v i t i e s were made 2, 4, and 6 weeks after treatment. The fourth harvest, for mature tomato analysis, was made when the plants were 100 to 110 days old. In the l a s t experiment, determinations of succinic dehydrogenase a c t i v i t y were made 4, 8, 12, 16, 20, and 24 days after the application of KNap to 2-week-61d plants. F. Vegetative growth measurement The stems of each plant were cut just above the s o i l sur-face. The leaf blades from each plant were grouped together to get the fresh weight of leaves. Stem and leaf petioles were weighed together to give the fresh stem weight. After fresh weights were recorded, the plant materials were dried at 75°C for 24 hours. 10 G. Determination of pigments Chlorophyll a and b, and carotenoid contents of le a f blades were determined spectrophotometrically (Beckman Model B). The pigments were extracted according to the procedure suggested by Frank and Kenny (1954). Leaf materials were cut into small pieces and mixed thoroughly. One-gram aliquots were blended i n 80 ml of 85% acetone i n a Waring f l a s k for 2 minutes. The homogenate was f i l t e r e d through Whatman No. 1 f i l t e r paper using a Buckner funnel, and made to 100 ml volume with 85% acetone. The o p t i c a l density (O.D.) of each pigment solution was read at 663 mu, 645 mu, and 440.5 mu against 85% acetone as blank i n a 1-cm c e l l . The concentrations of chlorophyll a and b were calculated following formulas of McKinney (1940) and that of carotenoid using the equation of von Wettstein (1957). The resul t s were, expressed as mg of pigment/g fresh leaf blade. The formulae used were as follows: c"= ( 1 2 - 3 °663 - ° ' 8 6 D645> V  a d x 1000 x w C = ( 1 9 - 3 D645 - 3 ' 6 D663J v  b d x 1000 x w where c — concentration i n mg/g fresh weight a = chlorophyll a b = chlorophyll b 11 D = o p t i c a l denisty at wavelength indicated d = length of l i g h t path i n cm V = f i n a l volume of extract W = fresh weight of leaf material used i n extraction C c = 4.695 D 4 4 Q > 5 x V _ - 0.268 C 1000W where C c = concentration of carotenoid i n mg/g fresh weight H. Determination of photosynthesis and re s p i r a t i o n The same two potted control and the same two treated plants were used for photosynthesis and re s p i r a t i o n measurements 2 and 4 weeks afte r treatment. The rate of C0 2 exchange was measured i n the growth room using an open system with a Beckman inf r a r e d analyzer IR215, and a Heath B u i l t Servo-Recorder, Model EUW-20A. The pot of the plant was enclosed i n a 2-mil polythene bag sealed around the plant stem to prevent C0 2 escape from the s o i l . The potted plant was set c a r e f u l l y into the chamber, which consisted of a 20-lb capacity polythene bag of 3-mil thick ness and the opening of the chamber was sealed t i g h t l y around a three-holed rubber stopper which provided i n l e t and ex i t for the gas and the thermistor probe.- This chamber was connected to the analyzer by tygon tubing. The a i r i n a tank containing about 300 ppm of CC>2 was passed into the chamber through tygon tubing a constant rate of 2,000 ml/min. 12 For photosynthesis measurement the chamber was exposed to l i g h t of 1,400 f . c . For determination of the rate of r e s p i r a t i o n the chamber was covered with three layers of b lack c l o t h to ex-clude l i g h t . In the i l luminated system, a drop i n the CC>2 concentration compared with that i n the tank was considered to be due to the CO2 f i x a t i o n i n apparent photosynthesis. In the dark system the increase i n CO2 concentration was considered as due to the CO2 l i b e r a t i o n by the p lant i n dark r e s p i r a t i o n . The S inn of the CC^ consumed i n apparent photosynthesis and the CO evolved during.dark r e s p i r a t i o n gave an approximation of true photosynthesis . The product of the flow rate by the di f ference i n the CO2 concentration of a i r before and af ter passing through the chamber gave the rate of CO2 exchange. The resu l t s were expressed as 2 m i c r o l i t e r s CX^/hour/dm of l ea f blade area. Sens i t i zed paper was used to determine the l ea f area. Since the shapes of tomato leaves were very i r r e g u l a r , use of the r e l a t i o n between weight and area of the s ens i t i z ed paper, gave the approximate l ea f area of tomato l ea f . I . Determination of enzyme a c t i v i t i e s The enzymes which were invest igated were n i t r a t e reductase (NRase), g lutamic-pyruvic transaminase (transaminase), phospho-g l y c e r y l kinase, succ in ic dehydrogenase, and phosphorylase. The f i r s t two enzymes are involved i n N metabolism while the l a s t 13 three r e l a t e to carbohydrate metabolism. The above enzyme a c t i v i t i e s were assayed 2, 4, and 6 weeks after spraying. Succinic dehydrogenase a c t i v i t y was determined 4, 8, 12, 16, 20, and 24 days after treatment i n Experiment V. Leaf blades were detached from petioles and washed with cold o d i s t i l l e d water. The enzyme a c t i v i t i e s were determined i n homogenates prepared from freshly harvested tissue. 1. Preparation of Tris-HCl buffer solution D i s t i l l e d water, 0.2 M trihydroxmethylamine methane ( T r i s ) , and 0.1 N HCl were mixed i n the r a t i o 7:8:5 by volume. The pH value of th i s solution, 0.05 M, was adjusted to 7.4 to 7.5. 2. Preparation of the crude extract The crude extract was prepared by grinding one weight of f i n e l y chopped leaf blades with 4 weights of cold 0.05 M Tris-HCl buffer of pH 7.4 to 7.5 i n a Waring blendor at f u l l speed for 2 minutes i n a cold room at 0 to 4°C. The homo-genate was decanted through four layers of cheesecloth and then centrifuged i n a Servall centrifuge at 2,000 x g for 20 minutes at 0 to 4°C. The r e s u l t i n g c e l l - f r e e supernatant solution was used for the assay of the enzyme a c t i v i t i e s . The protein content i n the enzyme preparation was determined by the method of Lowry et a l . (1951). 14 3. Nitrate reductase (NRase) A modification of the procedure of Evans and Nason (1953) was followed to determine the NRase a c t i v i t y . At zero time, 0.4 ml of the enzyme preparation was added to a reaction -5 mixture containing 0.1 ml of 0.1 M KN03, 0.05 ml of 2 x 10 M -3 FAD, 0.05 ml of 2 x 10 M DPNH, and 0.1 M phosphate buffer, pH 7.0, to give a t o t a l volume of 0.5 ml. Incubation was continued for 30 minutes i n a constant temperature water bath at 30°C, after which 1 ml of ^ 0 , 1 ml of 1% (W/V) sulfanilamide, and 1 ml of 0.22% (W/V) N-(1-naphthyl)-ethylene diamine hydrochloride reagent were added and the contents mixed by inverting the tube. Fifteen minutes were allowed for the development of the pinkish color. F i n a l l y , the o p t i c a l density of each solution and i t s blank (complete except for DPNH) was measured at 540 mu by the Beckman Model B spectrophotometer. The actual amount of the n i t r i t e f o r -med was determined from a standard curve (see Appendix Fig. 24) prepared i n advance with known quantities of n i t r i t e . The s p e c i f i c a c t i v i t y was defined as ug of n i t r i t e formed/mg protein/hour. 4. Glutamic-pyruvic, transaminase (transaminase) Transaminase a c t i v i t y was measured by following the method of Reitman and Frankel (1957). One ml of -keto-glutarate-alanine substrate was pipetted into a dry, clean test tube and t h i s was placed i n a water bath at 3 7°C for 10 minutes. 15 Upon the addition of 0.2 ml of the crude extract, the contents were mixed and incubated for exactly 30 minutes i n the water bath. One ml of 2,4-dinitrophenylhydrazine reagent (prepared by d i s s o l v i n g 19.8 mg of 2,4-dinitrophenylhydrazine i n 100 ml of 1 N HCl) was added to the tubes immediately aft e r being removed from the water bath. This reagent stopped fur-ther transaminase a c t i v i t y . After the tubes were allowed to stand at room temperature for 20 minutes, 10 ml of 0.4 N NaOH was added. A clean rubber stopper was inserted i n each tube and the contents were mixed by inversion. At the end of exactly 30 minutes, the color i n t e n s i t y of the solution was measured by a Klett-Summerson colorimeter equipped with a green f i l t e r . While the samples were incubating, a control for each homogenate was prepared. The only difference i n control tubes was that the 2,4-dinitrophenylhydrazine reagent was added to the reaction mixture before incubation. The s p e c i f i c a c t i v i t y was defined as an increase of 0.05 i n o p t i c a l density/mg protein/hour. 5. Phosphoglyceryl kinase The method described by Axelrod and Bandurski (1953) was employed. 3-Phosphoglyceric acid i n the presence of ATP, was used as the substrate. The diphosphoglycerate was trapped with hydroxylamine, and the anhydride thus formed measured 16 c o l o r i m e t r i c a l l y by the hydroxamic test of Lippman and Tuttle (1945). Reactions were run i n 10 ml test tubes. To each tube were added 1 ml of 0.1 M succinate buffer (succinic acid, 4.724 g, was dissolved i n d i s t i l l e d water and made to a volume of 100 ml.. Twenty-five ml of the succinate and 46 ml of 0.4 M NaOH were mixed and adjusted to pH 6.2 with NaOH solution), 1 ml of 2 M hydroxylamine hydrochloride (equal volumes of 4% hydroxylamine-HCl and 3.5% NaOH were mixed and adjusted to pH 6.2), 0.5 ml of 0.01 M ATP, 0.25 ml of the crude enzyme preparation, 1 drop of NaF and 1 drop of 0.01 M MgCl 2, i n the above order. The tube was shaken after the addition of each reagent. The control tube at this time received 2 ml of FeCl2-TCA-HCl reagent to stop the enzyme a c t i v i t y . (The FeCl^-TCA-HCl reagent was prepared by d i s -solving 9.3 g FeCl3.6H2 0 i n 42 ml cone. HCl and then 20 g TCA was added. The mixture was made to a volume of 500 ml with d i s t i l l e d water.) The reaction i n the experimental tube was then i n i t i a t e d by the addition of 1 ml of 0.01 M 3-phosphoglyceric acid (barium s a l t ) . The reaction was allowed to continue for 1 hour at 30°C i n a constant temperature water bath and was terminated by the addition of 2 ml of FeC^-TCA-HCl reagent to the experimental tubes. Thirty minutes was allowed for color development. The op t i c a l density of the solution from each experimental tube, and i t s blank, was determined by a Beckman Model B spectrophotometer at 430 raji. Spec i f i c a c t i v i t y was. defined as the' increase of 0.05 i n o p t i c a l density/mg prote in /hour . 6. Phosphorylase Sumner's (1950) method, which i s a modif icat ion of Fiske and Subbarow's method (1925), was employed to assay the enzyme phosphorylase by running the reac t ion i n the d i r e c t i o n of s tarch synthesis and measuring the amount of inorganic phos-phate l i b e r a t e d . The reac t ion mixture consisted of 1 ml of enzyme preparat ion of 2 ml buffered substrate (the buffered sub-s t ra te was prepared by d i s s o l v i n g 1 g G - l - P i n 50 ml d i s t i l l e d water. The so lu t ion was shaken with dry Ca(0H)2 or CaO to remove inorganic phosphate and then was f i l t e r e d . The f i l t r a t e was n e u t r a l i z e d with drops of H C l . Equal volumes of f i l t r a t e , and c i t r a t e buf fer , pH 6.0, were mixed. This buffered substrate was kept i n a r e f r i g e r a t o r . Before use, the buffered substrate was mixed with an equal volume of 1% potato s tarch s o l u t i o n , and a c r y s t a l of thymol i s added). The tes t tubes were kept i n a 30°C water bath for one hour. At the end of the incubation per iod , the reac t ion was terminated with 5 ml of 6.66% ammonium molybdate. Addi t ion of 5 ml of 7.5 N H 2 S 0 4 followed by 5 ml of 4% a c i d i c FeS0 4 developed a deep blue c o l o r . The so lu t ion was d i l u t e d with 10 ml of d i s t i l l e d water and the o p t i c a l density of each so lu t ion and i t s corresponding blank (ammonium molybdate was added before the addi t ion of substrate) was read i n a K l e t t -18 Summerson colorimeter equipped with a red f i l t e r . The actual amount of phosphate formed was read from a standard curve (see appendix Fig. 25) prepared with known amounts of phosphate using the same reagents as above. Spec i f i c a c t i v i t y was defined as ug of inorganic phosphate liberated/mg protein/hour. 7. Succinic dehydrogenase The procedure of Kun and Abood (1949) was followed e s s e n t i a l l y , with the modification that the incubation period was prolonged to 20 hours, as suggested by Isenberg et a l . (1951). Into a 15 ml test tube were pipetted 0.5 ml 0.1 M phosphate buffer of pH 7.4 (prepared by mixing 81 ml 0.1 M Na2HP04 and 19 ml of 0.1 M NaH 2P0 4 and adjusting the pH to 7.4), 0.5 ml 0.2M sodium succinate, 2 ml of homogenate and f i n a l l y 1 ml of fre s h l y prepared 0.1% triphenyltetra-zolium chloride solution. After shaking, the tubes were stoppered and were kept i n a constant temperature water bath at 38°C. The incubation was allowed to continue for 20 hours, at the end of which 7 ml of pure acetone was added to each tube. The contents were shaken vigorously to l e t the red pr e c i p i t a t e dissolve i n the acetone. The solutions were then centrifuged for 3 minutes at 2000 x g and the clear red solution was decanted o f f . The o p t i c a l density of each test solution, and i t s blank (with homogenate heated at 80°C for 5 minutes before assaying) was determined using a Klett-Slimmer son colorimeter 19 equipped with a blue f i l t e r . A standard curve was prepared i n advance by using known amounts of formazan disso lved i n 10 ml of acetone. The amount of formazan formed i n each experimental tube was read from the standard curve (see appendix F i g . 26) . Spec i f i c a c t i v i t y was defined as the ug of formazan formed/mg prote in /hour . In the f i f t h experiment, the a c t i v i t y of succ in ic dehydrogenase was assayed by using the method described by Zieg ler • i and Rieskie (1967). However, the s p e c i f i c a c t i v i t y was modified and expressed as 0.05 o p t i c a l density increased/mg prote in /hour . J . . Determination of pro te in The method of Lowry et a l . (1951) was employed to determine the prote in content of a l l the enzyme preparations mentioned above. To 0.4 ml of the enzyme was added 2 ml of a l k a l i n e copper so lu t ion (prepared by mixing 50 ml 2% N a 2 C 0 3 i n 0.1 N NaOH and 1 ml 0.5% CuSO 4 .5H 2 0 i n 1% sodium potassium t a r t a r a t e ) . . The contents were mixed. After 10 minutes 0.2 ml of 1 N F o l i n -Cioca l teau phenol reagent (Fisher S c i e n t i f i c C o . , Fair lawn, N . J . ) was added and the contents were mixed and allowed to stand for 30 minutes for co lor development. The o p t i c a l density of each experimental so lut ion and i t s blank (phenol reagent was replaced by d i s t i l l e d water) was read at 500 mp with a Beckman Model B spectrophotometer. Prote in content as mg/ml was obtained by comparison with a standard curve which was prepared with known 20 amounts of Bovine albumin. The values expressed as mg/ml were then converted to mg protein/g fresh leaf blade. K. P o l l i n a t i o n As the plants were grown i n either growth room or green house, a r t i f i c i a l p o l l i n a t i o n was achieved by f l i p p i n g the plant body l i g h t l y . L. Blossom-end rot Some of the tomato f r u i t s exhibited blossom-end rot. This rot i s caused by undesirable environmental conditions, such as a sudden shortage of water and flu c t u a t i n g humidity, or by calcium deficiency. The leaves, flowers, and developing f r u i t s of the plants were sprayed with CaCl2 aqueous solution at a rate of 0.5 g CaCl 2/plant. M. Y i e l d measurement For the t h i r d harvest i n Experiment I I , and the fourth harvest i n Experiment IV, number and weight of both green and mature tomato f r u i t s were recorded. For chemical composition determination at the fourth harvest of Experiment IV, tomato f r u i t s were picked as soon as they were ripe, that i s , r e d and firm. After being weighed they were stored i n a r e f r i g e r a t o r at 5°C. N. Quality of tomato f r u i t s The measurements of sugars, t i t r a t a b l e acid, and ascorbic acid of the ripe tomato f r u i t s were made. The tomato f r u i t s were stored at 5°C for 10, 4, and 1 day before the actual assays were car r i e d out i n order to check the e f f e c t of KNap on the loss of these compounds during storage. 1. Determination of reducing sugars and sucrose Soxhlet extraction i n ethanol, c l e a r i n g and deleading of extracts were done according to the method of Loomis and Shull (1947). Nitrophenol (DNP) reagent was prepared by di s s o l v i n g 7.145 g sodium 2,4-dinitrophenol i n 23 0 ml 5% NaOH. The mixture was heated on a water bath u n t i l the DNP dissolved completely. Then 2.5 g phenol was added. If the solution did not remain clear, i t was heated further. One hundred g sodium potassium tartarate was dissolved i n ca 500 ml d i s t i l l e d water. The two above-mentioned solutions were transferred to a 1 - l i t e r volumetric f l a s k and made to a volume of 1 - l i t e r . For sucrose determination, 25 ml of the cleared and deleaded extract was pipetted into a 400 ml beaker. Two drops methyl red, 5 drops of 10% acetic acid and 4 drops of invertase solution were added to each beaker. The mixture was rotated and was allowed to stand overnight at room temperature. The reducing sugars of both hydrolyzed and unhydrolyzed solutions were measured c o l o r i m e t r i c a l l y using the dinitrophenol reagent method. A standard curve was prepared with graded solutions of glucose. The difference between hydrolyzed and unhydrolyzed extract, a f t e r 22 m u l t i p l i c a t i o n with the correction factor 0.95 gave the amount of the sucrose. The DNP reagent method for the measurement of reducing sugar was as follows: six ml DNP reagent was pipetted into 3 large test tubes. Two ml d i s t i l l e d water was pipetted i n tube 1, and 2 ml of clear deleaded extract was pipetted into tubes 2 and 3. The tubes were kept i n cold, running water u n t i l a l l samples were ready. A l l tubes were stoppered loosely with glass stoppers and were placed i n b o i l i n g water for exactly 6 minutes. The tubes were then transferred to cold,running water again. The absorbance of each solution was read i n a K l e t t -Summerson colorimeter with a red f i l t e r , 3 to 20 minutes l a t e r . Sugar contents were expressed as percentage of dry weight of tomato f r u i t . 2. T i t r a t a b l e acid The methods described by Lepper et a l . (1945) for preparation of the extract and analysis of t i t r a t a b l e acid were followed. The f r u i t s were cleaned, dried, chopped, and quickly blended i n a Waring blendor. One hundred and f i f t y g of the sample and 400 ml of d i s t i l l e d water were put into a 2 - l i t e r beaker. The mixture i n the beaker was b o i l e d for one hour and water l o s t by evaporation was replaced at i n t e r v a l s . The solution was made to a volume of one l i t e r and f i l t e r e d . Twenty-five ml of the above-mentioned solution was dil u t e d to 250 ml with recently b o i l e d water. The dil u t e d solution was t i t r a t e d with 0.1 N KOH using 0.3 ml of 1% phenolphthalein solution (prepared by dissolving 1 g phenolphthalein i n 50 ml of 95% ethanol and then 50 ml water were added) for each 100 ml of solution being t i t r a t e d . The re s u l t s were reported as ml of 0.1 N KOH/100 g of fresh tomato f r u i t . 3. Ascorbic acid The extraction procedure used was e s s e n t i a l l y the same as described by Lo e f f l e r and Ponting (1942) except that 0.5% oxalic acid was used rather than metaphosphoric acid. Twenty g of tomato i n 150 ml 0.5% oxalic acid was blended i n a Waring blendor. The extract was decanted through two layers of cheesecloth. The method for determination of ascorbic acid i n the extract followed the indophenol reduction technique as modified by Schuster (1952) for use with a Klett-Summerson color-imeter. One ml of tomato extract was pipetted into each of the two colorimeter tubes. Nine ml of dye solution was pipetted into a colorimeter tube. (The dye solution was prepared by dissolving 14 mg Na-2, 6-dichlorophenol indophenol i n warm d i s t i l l e d water. The solution was f i l t e r e d and made to a volume of one l i t e r with cold d i s t i l l e d water.) The solution was mixed by inversion and read within 20 seconds with a Klett-Summerson colorimeter v equipped with a green f i l t e r . The blank was prepared by mixing 1 ml 0.5% oxa l i c acid, 9 ml dye solution and a tiny 24 c r y s t a l of pure ascorbic acid (Nutritional Biochemical Corporation, Cleveland). A standard regression l i n e (see appendix Fig. 27) was drawn from readings obtained with graded solutions of pure ascorbic acid. Results were expressed as mg of ascorbic acid/100 g of fresh tomato f r u i t . IV. RESULTS e In the text, reference to an increase or decrease i s to be taken to mean an increase or decrease compared with the appropriate value found i n untreated (or control) plants. Results, unless s p e c i f i c a l l y stated otherwise, were subjected to s t a t i s t i c a l analysis. In most instances, no s t a t i s t i c a l l y s i g n i f i c a n t difference was found. The re s u l t s did show that . there was a posi t i v e tendency of KNap to stimulate growth, y i e l d , and some of the biochemical and physiological aspects observed i n tomato plants, i f the general concept that 10% changes was regarded as- of some significance was considered. A. Vegetative growth Data on fresh and dry weights of leaves, and petioles and stem are given i n Tables l a to Ie, and also i l l u s t r a t e d i n Figures 1 to 5. In the text, stem includes the peti o l e s , while "leaves" means leaf blades. 25 1. Experiment I Results are given i n Table l a and Fig. 1. No s t a t i s t i c a l l y s i g n i f i c a n t difference was found i n this exper-iment. For the f i r s t harvest, plants were harvested 2 weeks after treatment. Based on dry weight magnitude, plants sprayed with 2,500 ppm KNap showed a s l i g h t increase, 0.91%, i n stem weight, but decreases of 4.68% and 1.30% i n le a f and t o t a l weights. In the 5,000 ppm KNap-treated plants, both the leaves and stem and therefore the t o t a l were increased by 3.28%, 10.77%, and 7.80% respectively. When dry weight was under consideration, 2,500 ppm KNap caused an increase of 2.31% i n stem but i n h i b i t e d growth of leaves 4.11%, consequently the t o t a l weight was reduced 1.3 0%. In the case of 5,000 ppm KNap-sprayed plants, increases of 5.70%, 13.21% and 8.30% were observed i n leaves, stem and t o t a l respectively. The second harvest was done 5 weeks after treatment. Fresh weight results revealed decreases of 0.21%, 3.62%, and 3.50% i n leaves, stem and t o t a l i n the 2,500 ppm KNap-treated plants. In 5,000 ppm KNap-treated plants, only a s l i g h t drop of 0.61% was observed i n stem. The leaves and t o t a l showed increases of 4.84% and 1.10% respectively. E f f e c t of the treatment on dry weight followed c l o s e l y the pattern of the fresh weight changes. Leaves, stem, and also the t o t a l weight were a l l decreased by the 26 2,500 ppm KNap to the extent of 5.07%, 9.14%, and 5.70% respec t ive ly . Increases of 6.15% and 1.10%, and a decrease of 1.57% were found i n leaves, t o t a l and stem of 5,000 ppm KNap-treated p lants . TABLE l a The e f f e c t of KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment I ) . Treatment Weight (g) T/C%  (ppm) Leaves Stems Total Leaves Stems Total C 317. 04* 476. 12 793.16 Fresh 2,500 302. 19 480. 43 783. 6.2 95.32 100. 91 98. 70 F i r s t Weight 5,000 327. 45 527. 40 854.88 103.28 100. 77 107. 80 Harvest* C 47. 91 36. 80 84. 71 Dry 2, 500 45. 94 . 37. 65 .83.59 . . 9.5. 89 .102. 31 98. 70 Weight 5,000 50. 68 41. 66 92.34 105.78 113. 21 108. 30 c 466. 70 1001. 10 1476.80 Fresh 2,500 451. 70 .964. .90 1416.60 99. 79 96. .3.8 96. 50 Second Weight 5, 000 489. 30 995. 00 1484.30 104.84 99. 39 101. 10 Harvest C 87. 75 128. 60 216.35 Dry 2, 500 83. 3 0 120. 7 0 2 04.00 94. 93 ...90. 86 94. 30 Weight 5, 000 93. 15 125. 60 218.80 106.15 98. 43 101. 10 x : In the f i r s t harvest plants were harvested 2 weeks after treatment or at the age of 6 weeks. Each reading i s the pooled value of 24 plants/treatment. In the second harvest, plants were harvested when 9 weeks old or 5 weeks after treatment. Each value i s the pooled value of 15 plants/treatment. No s t a t i s t i c a l l y s i g n i f i c a n t difference was found i n this experiment. 100 80 % of 60_J Control Value 4Q_ 20_ 0 Treatment(ppm) Weight Weeks afte r Treatment Fig. 1 Fresh [ I 5, 000 Fresh V / / / / / / K 2, 500 Dry / / / / / / / / / / ! 5 , 0 0 0 Dry Total / / / / / / / / / / / / 2, 500 Fresh /] / / / / / / / / / / / / / / / / / / / OTJ R / / / / / / / / / / / / / / / / / / / Fresh T 7 5 U T 7 Dry 71 / / / / / / / / / / / / / / / / / / / / / / b, UuU Dry 2 . 2 2 2 5 5 5 5 Effect of 2 , 5 0 0 ppm and 5 , 0 0 0 ppm KNap on fresh and dry weights of leaves, and stems of tomato plants (Experiment I ) . 29 2. Experiment II The re s u l t s are given i n Table lb and i l l u s t r a t e d i n Fig. 2. S t a t i s t i c a l l y s i g n i f i c a n t differences were not found in Experiment II. On the f i r s t harvest, both 5,000 ppm and 2,500 ppm KNap treatments enhanced growth of plants 2 weeks afte r spraying, that i s when 6 weeks old. In the sequence of leaves, stems, and t o t a l , increases of fresh weight of 8.32%, 4.34%, and 6.00% were observed i n 2,500 ppm KNap-treated plants, whereas 5.02%, 0.22%, and 2.00% increases resulted from the application of 5,000 ppm KNap. Increases of 7.16%, 10.05%, and 8.30% were observed i n the dry weights of plants which had been sprayed with 2,500 ppm KNap. Less pronounced increases of 1.31%, 2.85% and 1.90% were observed i n the 5,000 ppm KNap-treated plants. The second harvest was c a r r i e d out 2 weeks after the f i r s t . The 2,500 ppm KNap induced augmentation i n fresh weights of leaves, stem, and t o t a l by 4.40%, 2.52%, and 3.20% respectively. The 5,000 ppm KNap stimulated growth of stems only, 2.48% i n fresh weight, but decreased those of leaves and t o t a l by 5.62% and 0.80%. The e f f e c t of 2,500 ppm on leaf dry weight production was found to be i n h i b i t o r y , though not s i g n i f i c a n t l y so, by showing a s l i g h t decrease of 0.42%, while the weights of the stem and t o t a l were increased by 1.50% and 0.50% respectively. Under the influence of 5,000 ppm KNap treatment, leaves and t o t a l dry weights were decreased by 4.85% and 1.60% respectively. The stem TABLE lb The e f f e c t of KNap on fresh and dry weights of leaves - and stems of tomato plants (Experiment ID-Treatment Weight (g) T/C % (ppm) Leaves Stems Total Leaves Stems Total F i r s t x Fresh Weight C 2,500 5,000 3 03.47±A 328.72 B 318.70 AB 472.40 492.90 473.49 775.87 821.62 792.15 108.32 105.02 104.34 100.22 106.00 102.00 Harvest Dry Weight C 2,500 5, 000 37.41 40. 09 37.90 27.12 30.12 28.15 64.21 70. 20 66. 05 107.16 101.31 110.05 102.85 108.30 101.90 Second Fresh Weight C 2, 500 5,000 475.25 496.15 448.55 688.25 705.60 705.30 1163.50 1201.75 1153.80 104.40 94.38 102.52 102.48 103.20 99.20 Harvest Dry Weight C 2,500 5, 000 82. 50 82.15 78. 50 78. 85 77. 00 77.20 158.35 159.15 155.70 99. 58 95.15 101.52 101.78 100.50 98.40 x : In th i s experiment, plants were sprayed at the age of 4 weeks. The f i r s t harvest was done 2 weeks after treatment. The second harvest was done 4 weeks after treatment. Each value i n t h i s experiment i s the pooled value of 9 plants/treatment. ± : In Tables Ia-e to III, and VII, means d i f f e r i n g s i g n i f i c a n t l y from the respective control mean at the 0.01% l e v e l are indicated by **, and sample means sharing the same l e t t e r do not d i f f e r s i g n i f i c a n t l y according to Duncan's New Multiple Range Test at the 5% protection l e v e l . T Value in treated plants ± = — - ; -— x 100 Q Value in control plants c o o \/////\ Leaves 100 80 _ % of Control Value 60 40 20 0 Treatment(ppm) Weight Weeks after treatment Fig. 2 / / Stems l l l l l l l l Total 2, 500 Fresh / 1 I / / i 5, 000 Fresh 2, 500 Dry / i 5, 000 Dry 7 / / / 7 2, 500 Fresh 5, 000 Fresh 2, 500 Dry 5, 000 Dry 2 2 2 Eff e c t of 2,500 ppm and 5,000 stems of tomato plants (Exper ppm KNap on iment I I ) . 4 4 4 4 fresh and dry weights of leaves and 32 dry weight was increased by 1.78%. 3. Experiment III In this experiment, the plants were divided into 5 groups. One of these groups was untreated; the second group received 2 , 5 0 0 ppm KNap spray at the age of 12 days; and the t h i r d group received 5,000 ppm KNap spray at the same age of 12 days. The other two groups were treated 7 days l a t e r , that i s when 19 days old, with 2,500 ppm and 5,000 ppm KNap respectively. Half of each group was harvested when plants were 33 days old, and the other h a l f two weeks l a t e r at which time the plants were 4 7 days old. The re s u l t s of the f i r s t harvest are tabulated i n Table Ic and represented graphically i n Fi g . 3. The res u l t s indicated that treatment fresh weight means were s i g n i f i c a n t l y d i f f e r e n t from the respective control means at 5% or 1 % l e v e l , but no s t a t i s t i c a l l y s i g n i f i c a n t difference among treatment means was found according to Duncan's New Multiple Range Test. Fresh weights greater by 3 . 8 5 % i n leaves, 7 . 6 4 % i n stems, and 6 . 1 1 % i n t o t a l were induced by 5,000 ppm KNap applied to plants when 1 9 days old. The growth of the other three groups was in h i b i t e d by the KNap. Differences i n percentage are given i n the order of leaves, stems, and t o t a l i n the following statements. The fresh weights of plants treated at the age of 12 days by 2,500 ppm KNap were decreased by 7.02%, 1 0 . 6 3 % , and 9.24%, while 33 the fresh weights of plants which received 5,000 ppm KNap at the same stage, were less by 3.33%, 5.99%, and 4.91%. The plants which were sprayed with 2,500 ppm KNap when 19 days o ld had weight values which were lowered by 3.78%, 7.20%, and 5.82%. Results based on dry weight showed that both the 2,500 ppm and 5,000 ppm KNap, sprayed either when plants were 12 or 19 days old, increased the dry weights of leaves, stems and the t o t a l top weights. The increases range from 0.83% to 9.92% but no s t a t i s t i c a l l y s i g n i f i c a n t difference was observed. Data on the second harvest are included i n Table Id and Fig. 4. The ef f e c t on fresh weights of 2,500 ppm KNap applied to 12-day old plants showed 9.94% decrease i n leaves, 2.56% increase i n stem and 4.40% decrease i n t o t a l weight, while that of 5,000 ppm showed a decrease of 7.37% i n leaves, but 9.18% and 1.70% increases i n stem and t o t a l weight respectively. The plants which received 2,500 ppm KNap at 19 days of age showed 1.11% de-crease, 12.29%, and 6.20% increases i n leaves, stems and t o t a l weight, while those under the- influence of 5,000 ppm KNap at the same age showed increases of 8.66%, 24.03%, and 17.70% i n leaves, stems, and t o t a l weight respectively. The differences i n dry weights of leaves and stems are s t a t i s t i c a l l y s i g n i f i c a n t at 5% l e v e l . Increases of 20.13%, 8.66%, and 14.95% i n leaves, stems, and t o t a l dry weight were observed i n plants treated with 2,500 ppm when 12 days old. Increases of 26.79%, 18.18%, and 5.20% i n 34 leaves, stems and t o t a l dry weights were observed i n plants sprayed with 5,000 ppm when 12 days old. Higher dry weights of 36.11%, 18.83%, and 9.90% were found i n leaves, stems, and t o t a l tops i n plants sprayed with 2,500 ppm KNap when 19 days old. More s t r i k i n g increases of 43.63%, 32.47%, and 18.70% i n leaves, stems, and t o t a l dry weights were found i n plants sprayed with 5,000 ppm KNap when 19 days old. 4. Experiment IV In this experiment 5,000 KNap was applied to 3-week-old piiants. The res u l t s obtained from the f i r s t three harvests made 2, 4, and 6 weeks after treatment are summarized i n Table Ie and shown i n Fig. 5. No s t a t i s t i c a l l y s i g n i f i c a n t difference was found i n this experiment. For the f i r s t harvest, decreases i n fresh weight of 4.52%, 4.93%, and 4.70% i n leaves, stems, and t o t a l were found. Increases of 0.48%, and 2.55% and 0.64% of decreases of dry weight were found i n leaves, stems and t o t a l top respectively. The 5,000 ppm KNap resulted i n an i n h i b i t o r y e f f e c t on both fresh and dry t o t a l top weights when plants were harvested 4 weeks afte r spraying. The decreases of 6.82%, 6.45%, and 6.60% on fresh weight, and 7.34%, 3.28%, and 5.50% on dry weight were observed i n leaves, stems, and t o t a l respectively. In the t h i r d harvest, a lessening of 4.07% i n leaf fresh weight, and increases of 2.74% and 0.25% i n stem and t o t a l TABLE IC The e f f e c t of KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment I I I , f i r s t harvest) x Treatment Weight (g) T/C% (ppm) Leaves Stems Total Leaves Stems Total C 144.20 A* 214.55 B 358.00 C 2,500-1 133.85 A 191.75-- •B 325.60 C 92.98 89.3 7 90. 76 Fresh 5,000-1 139.40 A 201. 70/ :B 341.10 C - 96.67 94. 01 95. 09 Weight 2,500-2 138. 75 A 199.10 >; B 337. 85 C 96.22 92. 80 94.18 5,000-2 149.75 230.95' 380.70 103.85 107.64 106.11 C 19.40 12.10 31. 50 Dry 2,500-1 20. 60 12.20 32. 80 106.19 100.83 104.12 Weight 5,000-1 21.00 12. 50 33. 50 108.25 103.31 106.34 2,500-2 20. 70 12.65 33.35 106.70 104.55 105.85 5,000-2 21.20 13.30 34. 50 109.28 109.92 109.52 x : Each value i s the pooled value of 12 plants/treatment and plants were harvested when 33 days old. In Tables l c and Id, 2,500-1 = 2,500 ppm KNap applied when plants were 12 days old; 5,000-1= 5,000 ppm KNap applied when plants were 12 days old; 2,500-2= 2,500 ppm KNap applied when plants were 19 days old; and 5,000-2= 5,000 ppm KNap applied when plants were 19 days old. *: In a column, values not followed by a common letter are significantly different at the 0.05 level. c o c n 120 100 % of Control Value 80 _ 60 40 20 0 Treatment (ppnrf: Weeks a f t e r treatment': Fig . 3 x. Leaves I \ Stems Total Fresh Weight-9 / / \ / / / / I 2 / / / / / / / / / / / / / i / Dry Weight-/ I fl A / / i 2,500-1 5,000-1 2,500-2 5,000-2 2,500-1 5,000-1 2,500-1 5,000-2 2 3 . 3 2 2 5,000 ppm KNap on fresh and dry weights tomato plants (Experiment III, f i r s t 3 3 2 E f f e c t of 2,500 ppm and of leaves, and stems of harvest). -1= sprayed on 12 days o l d tomato plants; and -2= sprayed on 19 days ol d tomato plants. U) TABLE Id The ef f e c t of KNap on fresh weights and dry weights of leaves, peti o l e s , and stems of tomato plants (Experiment I I I , second harvest). Treatment Weight (g) T/C% (ppm) Leaves Stems Total Leaves Stems Total C 194.10 234.25 428.35 2,500-1 174.80 240.25 415.05 90. 06 102.56 95. 60 Fresh 5,000-1 179.80 255.75 435.55 92.63 109.18 101.70 Weight 2,500-2 191.95 263.03 454.90 98. 89 112.29 106.20 5,000-2 210.90 290.55 501.45 108.66 124.03 117.70 C 30. 05 A 23.10 C 53.15 2,500-1 36.10 A 25.10 C 61. 20 120.13 108.66 114.95 Dry 5,000-1 38.10 AB 27.30 C 65.40 126.79 118.18 105.20 Weight 2,500-2 40.90 AB 27.45 CD 68.35 136.11 11.8.83 . 109.90 5,000-2 43.16 B 30. 66 D 73. 76 143.63 132.47 118.70 x : Each value i s the pooled value of 6 plants/treatment. Plants were harvested when 47 days old. LO 140 % of Control Value 120 100 _ 80 60 _ 40 20 I" 1111111 T O T A L  f | Stems I W A V M Leaves Treatment(ppm) x: Weeks af t e r treatment : Fig. 4 : I I / A k - / / / / A n / / / ./ / / / / / / / / / / / PI / A 2,500-1 5,000-1 2,500-2 5,000-2 5 5 4 4 2,500-1 5,000-1 2,500-2 5,000-2 5 5 4 4 L O 00 Fresh Weight Dry Weight E f f e c t of 2,500 ppm and 5,000 ppm KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment III, second harvest) TABLE Ie The ef f e c t of KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment IV). Treatment Weight (q) T/C% (ppm) Leaves Stems Total Leaves Stems Total F i r s t Fresh Weight C 5, 000 105.00 100.25 130.90 124.45 230.90 . 224.70 95.48 95.07 95. 30 Harvest Dry Weight C 5,000 2.0.80 20.90 11. 75 11.45 32. 55 32.35 100.48 97.45 99.36 Second Fresh Weight C 5 , 000 146.70 136.90 235.50 220.30 382.20 357.00 93.18 93. 55 93.40 Harvest Dry Weight C 5, 000 36. 80 34.10 30. 50 29. 50 67.30 63. 60 92.66 96. 72 94. 50 Third Fresh Weiqht C 5,000 3240.50 3108.50 5650.00 5805.00 8890.50 8913.50 95.93 102.74 100.25 Harvest Dry Weight C 5,000 78.90 73. 50 91.00 94.35 169.90 167.85 93.16 103.68 98. 80 x : Values t h i r d i n f i r s t harvest 10 and second harvests are pooled values of plants are pooled i n each value. 6 plants/treatment. and i n the ± : Plants were 5, 7 , and 9 weeks old (or 2, 4, and 6 weeks after treatment) i n f i r s t , second, and t h i r d harvest respectively. 0) > o n JJ c o u o 5s-104 102 100 98 96 94 92 fO^XNT Leaves ] ] Stems fl Ml 11 in Total 6. fo / / / / 0 2 4 6 2 4 6 Weeks after treatment Fresh Weight Dry Weight Fig. 5: Eff e c t of 5,000 ppm KNap on fresh and dry weights of leaves and stems of tomato plants (Experiment IV). 4^  O 41 fresh weights were caused by the 5,000 ppm KNap treatment. On a dry weight basis, 6.84% and 1.20% decreases, and a 3.68% increase were found i n leaves, t o t a l and stems respectively. B. Chlorophyll and carotenoid content Plants were sprayed with 5,000 ppm KNap when 3 weeks old. Three measurements of chlorophyll a and.b, and carotenoid content of leaf blades were conducted 2, 4, and 6 weeks after treatment. Results are shown i n Table II and Fig. 6 and 7. No s t a t i s t i c a l l y s i g n i f i c a n t difference was found. In a l l the 3 observations, both chlorophyll a and b contents were increased rather i n e f f e c t i v e l y i n the leaf blades by the KNap spray. Chlorophyll a content was found to show small increases of 2.70% and 1.00% i n the treated plants i n the f i r s t and t h i r d measurements. Chlorophyll b content was found to be increased by the 5,000 ppm KNap to 2.10% and 1.80% i n the f i r s t and second observations, but a decline of 1.50% was noticed i n the t h i r d measurement. The stimulatory e f f e c t of KNap on carotenoid content was more pronounced. A l l observations showed increases i n carotenoid content i n treated plants, 6.50%, 13.80% and 1.40% increases were detected for the three measurements. C. Photosynthesis and r e s p i r a t i o n The rates of photosynthesis and r e s p i r a t i o n are presented i n Table II and also represented graphically i n Fig. 7 and 8. Five thousand ppm KNap applied to 3-week-old plants resulted i n a , TABLE II The e f f e c t of 5,000 ppm KNap on the rates of photosynthesis and r e s p i r a t i o n of tomato plants, and on the pigment contents of tomato leaf blades. Harvest time Rate or content (weeks after 5,000 T % Unit treatment) C ppm C Apparent 2 1740.00 x 1500. 00 86. 20 u l c o 2 photosynthesis 4 945.00 985. 00 104. 20 exchanged/hr/ Respiration 2 1640.00 1500. 00 91. 50 dm^  of leaf 4 1345.00 1475. 00 109. 70 blade area True 2 3380.00 3000. 00 88. 50 photosynthesis 4 2290.00 2460. 00 107. 40 2 0. 75 0. 77 102. 70 Chlorophyll a 4 0. 84 0. 84 100. 00 mg/g of 6 0.96 0. 97 101. 00 fresh 2 0.47 0. 48 102. 10 leaf Chlorophyll b 4 0. 55 0. 56 101. 80 blade 6 0.66 0. 65 98. 50 2 0.61 • o . 65 106. 50 Carotenoid 4 0.65 0. 74 113. 80 6 0. 73 0. 74 101. 40 : Rates of photosynthesis and re s p i r a t i o n are the average of two measurements. Each value of pigment content i s the average value of 6 samples. 4^  to 1.0 Chlorophy l l a 0.8 rcS C H (1) cn cu u C H C H O CT> \ e 0. 6 0.4 0.2 _ F i g . 6: I NVVAM Control ] 5,000 ppm Chlorophyl l b I 4 2 4 6 Weeks af ter treatment Carotenoid / / / / / Effec t of 5,000 ppm KNap on the contents of ch lorophyl l s a and b, and carotenoid of tomato l ea f blades . 4i> (JO 2 weeks afte r treatment (D rd > o u +J c o u o 120 100 80 60 40 _ 20 I W 4 weeks a f t e r treatment I ~~l 6 weeks af t e r treatment 0 I ', I I \ I I i / / / / / i ', i i i I / / / / / / / / / / I / / I / / / L "PI" / / / / / / / / / / / / / ; / i ± / / / t / / / / / / / / / Apparent Respiration True Chlorophyll a Chlorophyll b Photosynthesis Photosynthesis Carotenoid Fig. 7: E f f e c t of 5,000 ppm KNap on the rates of photosynthesis and r e s p i r a t i o n of tomato plants, and on the contents of pigments of tomato leaf blades. 45 ro cu u rd o C N M O TJ CD cn C rd u X cu C N O u 3,500 True Photosynthesis 3,000 _ Control I I 5,000 ppm 2, 500 2,000 _. Apparent Photosynthesis 1,500 _. 1,000 _ 500 Respiration / -2 4 2 4 2 4 Weeks after treatment Fig. 8: E f f e c t of 5,000 ppm KNap on the rates of apparent photosynthesis, r e s p i r a t i o n and true photosynthesis of tomato plants. 4:6 slowing down of both apparent photosynthesis and r e s p i r a t i o n at the early stage, that i s 2 weeks after treatment, and the values were 13.80% and 8.50% below those of the untreated plants. However, the treatment resulted i n higher rates i n both apparent photosynthesis and r e s p i r a t i o n 4 weeks afte r treatment by showing 4.20% and 9.70% increases respectively. In treated plants, the rate of true photosynthesis, equal to the sum of net CC>2 f i x a t i o n and C0 2 l i b e r a t i o n i n the dark, showed an increase of 7.40% 4 weeks after treatment, whereas a decrease of 11.40% was detected 2 weeks after spraying. However, no difference i n photosynthesis, or i n respiratory rates, was found to be s i g n i f i c a n t s t a t i s t i c a l l y . D. Enzyme a c t i v i t i e s The f i v e enzymes under study were NRase, transaminase, phosphoglyceryl kinase, phosphorylase and succinic dehydrogenase. The a c t i v i t i e s of these f i v e enzymes were determined i n c e l l - f r e e homogenates of freshly harvested l e a f blades 2, 4, and 6 weeks after spraying with 5,000 ppm KNap, applied when plants were 3 weeks old. Additional measurements of the succinic dehydrogenase were also made 4, 8, 12, 16, 20 and 24 days after the treatment which was applied to plants 14 days old i n another experiment. The s p e c i f i c a c t i v i t i e s of the enzymes are presented i n Tables III and IV and also by Fig. 9 to 16. A l l data, excluding those for succinic dehydrogenase a c t i v i t i e s examined i n Experiment V, were 47 subjected to s t a t i s t i c a l analysis and no difference was found to be s i g n i f i c a n t s t a t i s t i c a l l y . The 5,000 ppm KNap treatment had di f f e r e n t e f fects on the a c t i v i t i e s of d i f f e r e n t enzymes and also had d i f f e r e n t e f fects on the a c t i v i t y of an enzyme i n plants at d i f f e r e n t ages. A stimulation was found i n the case of transaminase 6 weeks after treatment, and phosphorylase at a l l three times of obser-vation. Inhibitory effects on enzyme a c t i v i t i e s of other enzymes studied were recorded. 1. Nitrate reductase (NRase) Results are shown i n Table III and Fig. 9 to 14. NRase a c t i v i t i e s i n both control and treated plants measured 4 weeks after treatment were very similar, but a decrease i n a c t i v i t y i n the leaf blades of treated plants of 24.80% was ob-served 6 weeks after treatment. Incorrect a n a l y t i c a l procedures were used i n the assay c a r r i e d out 2 weeks after treatment; hence the results have not been tabulated. 2. Glutamic-pyruvic transaminase (transaminase) Results included i n Table III and Fig. 12 and 14 show that the transaminase a c t i v i t y was retarded most e f f e c t i v e l y i n the leaves harvested 2 weeks after spraying and a decrease of 54.80% occurred. A decrease of 7.70% was observed 4 weeks after treatment. On the other hand, 6 weeks after application, an appreciable increase, 20.40%, resulted from treatment. Both the TABLE III The e f f e c t of 5,000 ppm KNap on the a c t i v i t i e s of succinic dehydrogenase, phosphoglyceryl kinase, glutamic-pyruvic transaminase, n i t r a t e reductase, and phosphorylase of tomato leaf blades. Weeks after Specific a c t i v i t y x T % Enzyme Unit treatment C 5,000 ppm C pg formozan 2 5.820 0.900 18. 00 Succinic formed/ mg protein/ 4 1.640 1.480 90. 25 dehydrogenase hour 6 1. 740 1.150 66. 10 An increase of 0.05 2 0. 024 0.018 75. 00 Phosphoglyceryl o p t i c a l density/ 4 0. 039 0. 036 92. 30 kinase mq protein/hour 6 0. 067 0.066 98. 50 An increase of 0.05 2 0.376 0. 843 45. 20 (Glutamic-pyruvic o p t i c a l density/ 4 3.900 3. 600 92. 30 transaminase mq protein/hour 6 4. 760 5.730 120. 40 Nitrate p.g of n i t r i t e 4 0. 556 0.554 99. 70 reductase formed/ mg protein/hour 6 1. 010 0. 760 75. 20 pg of inorganic 2 33.920 41.400 122. 05 Phosphorylase phosphate formed/ 4 5. 040 5. 670 114. 00 -A,., mg protein/hour 6 10.660 19.070 178. 89 : Each value of s p e c i f i c a c t i v i t y i s the average value of 6 samples. 4 6 Weeks a f ter treatment F i g . 9: E f f e c t of 5,000 ppm KNap on the a c t i v i t y of n i t r a t e reductase i n tomato l e a f blades . 50 I V C o n t r o l i i5 ,000 ppm 45 _ 10 F i g . 2 4 6 Weeks af ter treatment 10: Ef fec t of 5,000 ppm KNap on the a c t i v i t y of phosphorylase i n tomato l e a f blades . 42> 50 0. 07 _ 4 J •H > -H -U U rd u -H CH •H u <u CO M Control I I 5,000 ppm 0.06 0. 05_ 0. 04. 0. 03 _ 0. 02 ._ 0. 01 / / / z: / / / / / / / / / / / / / / / / / / / / / y Weeks after treatment Fig. 11: Effect of 5,000 ppm KNap on the activity of phosphoglyceryl kinase in tomato leaf blades. 51 F i g . 12: Ef fec t of 5,000 ppm KNap on the a c t i v i t y of g lutamic-pyruvic transaminase i n tomato l ea f blades. 52 G 3 X 3 c o n t r o l ' i^nnn p p m 2 4 6 Weeks af ter treatment F i g . 13: Ef fec t of 5/000 ppm KNap on the a c t i v i t y of succ in ic dehydrogenase i n tomato l ea f blades (Experiment IV) . 200 180 160 _ <D 140 _ 3 120 _ 100 80 60 40 20 2 weeks af ter treatment s^o-^v^q 4 weeks af ter treatment i ~) 6 weeks af ter treatment 1 / / / / / / / / / / / / 7 / / / / / / / / / / / L PI / / / / / / / / Succinic dehydrogenase Phosphoglyceryl kinase Glutamic-pyruvic Transaminase / / / / / / / / / / / / / _ _ / . / / / / / / / / / / / / / Nitra te Reductase Phosphory as F i g . 14: E f f e c t of 5,000 ppm KNap on the enzyme a c t i v i t y of tomato l ea f b lades . 54 treated and control plants exhibited a steady increase i n transaminase a c t i v i t y over a period of 4 weeks beginning when plants were 3 weeks old. 3. Phosphoglyceryl kinase Results are l i s t e d i n Table III and Fig. 11 and 14. The phosphoglyceryl kinase a c t i v i t y i n leaf blades of treated plants was decreased by 25.00%, 7.70% and 1.50% 2, 4, and 6 weeks after the treatment. 4. Phosphorylase Data are given i n Table III and Fig. 10 and 14. This was the only enzyme found to be stimulated i n a c t i v i t y by the 5,000 ppm KNap spraying i n the three observations made, although the extents of stimulation were d i f f e r e n t . Increases of 22.05% and 14.00% were found i n the treated plants i n the f i r s t and second observations. A sharp increase of 78.89% was detected i n the l a s t assay made 6 weeks after the treatment. 5. Succinic dehydrogenase. Results given i n Table III and Fig. 13 and 14 show that the a c t i v i t y of succinic dehydrogenase was i n h i b i t e d markedly by the 5,000 ppm KNap treatment. The maximum reduction, 72.00%, was found 2 weeks afte r treatment. As the plants became older, the i n h i b i t i o n was less, being 9.35% and 33.90% at 4 and 6 weeks after the application of KNap. In the subsequent experiment (Experiment V), a di f f e r e n t technique was employed to determine the a c t i v i t y of succinic dehydrogenase i n order to shorten the 20-hour incubation period. Plants sprayed when 2 weeks old were analyzed for succinic dehydrogenase a c t i v i t y 4, 8, 12, 16, 20, and 24 days after treatment. The res u l t s showed that succinic dehydrogenase a c t i v i t y i n treated plants was lower than i n the controls i n the f i r s t 4 measurements. The reductions measured at these times were 35.11%, 36.32%, 41.53%, and 17.14%. However, in the l a s t two determinations, enhanced succinic dehydrogenase a c t i v i t y of 6.90% and 14.23% were obtained. E. Protein content Protein content of each enzyme extract was determined. Only those of Experiment V are tabulated i n Table IV and pre-sented graphically i n Fig. 17 and 18. Increases due to treatment, 48.48%, 12.72%, and 29.79%, were measured 4, 12, and 20 days a f t e r application of KNap. However, treatment resulted i n drops of 14.82%, 4.20%, and 23.41% as determined 8, 16, and 24 days after treatment. These quantitative changes i n enzyme protein suggest that the growth stimulation may a f f e c t the synthesis of the enzyme at the ribosomal l e v e l . F. Tomato Y i e l d Data of Experiment I I , i n which the plants were sprayed when 4 weeks old, are recorded i n Table V and i l l u s t r a t e d i n Fig. 19. In this experiment, malformation and/or darkened d i s t a l end of the tomato f r u i t were observed. Up to the age of 111 days, res u l t s based on fresh weight of f r u i t showed that the treated TABLE IV The e f f e c t of 5,000 ppm KNap on the s p e c i f i c a c t i v i t y of succinic dehydrogenase and the protein content of the enzyme extract of the leaf blades of tomato plants over a period of 20 days beginning when 18 days old. Succinic : dehydrogenase Age of s p e c i f i c activity- 1- Protein content plants 1 % (day) C 5,000 ppm C C 5,000 ppm C 18 8.38 ' 5.44 64.89 4.95 7. 35 148.48 22 4. 68 2.98 63.68 4.86 4.14 85.18 26 1.43 0. 83 58.47 5.11 5. 76 112.72 30 2.94 2.44 82,86 7.62 7.30 95. 80 34 5.47 5. 85 106.90 8.46 10.88 129.79 38 1.82 2. 07 114.23 7.05 5.40 76. 59 x : Protein content: expressed as mg protein/100 mg of fresh l e a f blades. *: Sp e c i f i c a c t i v i t y : a 0.05 o p t i c a l density increase/mg protein/hour. cn 57 7 / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / K \ \ \ t Contro l J 5,000 ppm 7 / / / / / / / / / / / / / / 7 / / / / / / / / / / / / / / / / / 8 24 12 16 20 Days af ter treatment Ef fec t of 5,000 ppm KNap on the a c t i v i t y of succ in ic dehydrogenase i n tomato l ea f blades (Experiment V ) . 120 100 80 60 40 20 0 |„ I I I I I I I I 1 1 I 4 8. 12 16. 20 24 Days af ter treatment F i g . 16: Ef fec t of 5,000 ppm KNap on the a c t i v i t y of succ in ic dehydrogenase i n tomato l ea f blades (Experiment V ) . 59 4-1 O tn Q) rd O H O -H 0) O 5-1 rd H 42 CO . S-l tn i n 12 _ 10 8 5,000 ppm KNap Contro l Ti "Ti 20" Days af ter treatment F i g . 17: Prote in content of the enzyme extracts of the l ea f blades of KNap-treated and contro l tomato p lants . 140 _ 120 100 80 60 JL 4 8 12 16 20 24 Days af ter treatment F i g . 18: Ef fec t of 5,000 ppm KNap on prote in content of enzyme extracts of l ea f blades of tomato plants . 60 plants were less suscept ible to th i s disease. On 2,500 ppm-and 5,000 ppm-treated p lants , the r i p e tomatoes free of blossom-end rot were 7.70% and 62.50% more than those of the contro l plants by weight. Results based on f r u i t number showed that good tomato f r u i t s from 2,500 ppm KNap-treated plants were 16.00% fewer but were 40% more i n the case of 5,000 ppm KNap-treated p lants . In the case of the diseased tomato f r u i t s , an increase of 16.13% and a decrease of 35.48% i n number were induced by the 2,500 ppm and 5,000 ppm KNap treatments. For Experiment IV, the t o t a l number and fresh weight of both green and r i p e tomato f r u i t s are l i s t e d i n Table VI and i l l u s t r a t e d i n F i g . 19. There were 67 r i p e tomato f r u i t s i n both contro l and treated p lants , although the fresh weights of r ipe tomato f r u i t s from 5,000 ppm KNap-treated plants were 24.60% more than that of the contro l s . The fresh weight of green tomato f r u i t s from the 5,000 ppm KNap-treated plants was almost i d e n t i c a l with that of the contro l s . The number of green tomato f r u i t s from the treated plants was decreased by 38.00%. If a l l green and r ipe tomatoes were considered together, the treatment appeared to r e s u l t i n a favorable e f fec t on both number and fresh weight of tomatoes, by showing 16.92% and 14.71% promotions respec t ive ly . Each of the values here represented the pooled values of 10 plants / treatment . S t a t i s t i c a l analys is for f r u i t y i e l d has not been done due 61 TABLE V The e f fect of KNap on number and fresh weight of r ipe and rot ten tomato f r u i t s (Experiment I I ) . Fresh T/C% Tomato Treatment Weight Fresh F r u i t ppm Number (g) Number 'Wei'giht C 50 847.20 Ripe 2,500 42 712.50 84. 00 107.70 5,000 70 1376.90 140.00 162.50 C 31 346.50 Rotten 2,500 36 752.80 116.13 188.30 5,000 20 173.50 64. 52 50.40 C 81 1193.70 Ripe + 2,500 78 1465.30 96.29 122.74 Rotten 5,000 90 1550.40 111.11 121.48 x : A l l tomato f r u i t s were harvested at the same time when plants were 111 days o l d . Each value i s the pooled value of ' 9 plants per treatment. TABLE VI The e f fec t of KNap on number and fresh weights of r ipe and green tomato f r u i t s (Experiment IV) . T/C % Fresh Tomato Treatment Weight Fresh'. F r u i t (ppm) Number (g) Number Weight C 67 5635.65 Ripe 5,000 67 7022.28 100.00 124.60 C 63 3882.64 Green 5,000 87 3908.17 138. 00 100.66 Ripe + C 130 9518.29 Green 5,000 154 10930.45 116.92 114.71 : Ripe tomato f r u i t s were picked as soon as they were f irm and red. Green tomato f r u i t s were picked when plants were 110 days o l d . Each value represents the pooled value of 10 p lants . 62 Ripe tomato f r u i t s Rotten tomato f r u i t s Green tomato f r u i t s Ripe + rotten tomato f r u i t s (Experiment II) or Ripe + green tomato f r u i t s (Experiment IV) 200 180 160 140 120 100 80 60 40 20 . 0 Experiment II-A F i 2,500 5,000 2,500 5,000 ppm KNap Number Number Weight Weight of tomato f r u i t s g. 19: Ef f e c t of KNap on number and fresh f r u i t s (Experiments II & IV). Experiment IV 0 5, 000 5,000 Number Weight weight of tomato 63 to the fact that there were not enough rep l i c a t e s i n the experiments. However, the r e s u l t s showed that there was an evident promotive e f f e c t of KNap on f r u i t y i e l d . G. Quality of tomato f r u i t s Reducing sugar, sucrose, t o t a l sugars, t i t r a t a b l e acid, and ascorbic acid (vitamin C) of the mature tomato f r u i t s were determined to see i f the KNap treatment affected the chemical composition of tomato f r u i t s . 1. Sugars Sugar content data are summarized i n Table VII and i l l u s t r a t e d graphically i n Fig. 20 and 23. The mature tomato f r u i t s from the treated plants had higher sucrose content, being 7.91%, 20.75%, and 5.48% i n f r u i t s stored for 10, 4, and 1 day. Of course, t o t a l sugars behaved i n a similar pattern and were increased by 11.08%, 23.64% and 4.33% i n tomato f r u i t s stored for 10, 4, and 1 day respectively. 2. T i t r a t a b l e acid At the end of 10 days of storage, the t i t r a t a b l e acid content of f r u i t s of treated plants was lower than that of the a controls by 4.30%, but an increase of 5.60% i n t i t r a t a b l e acid was found when the storage period was shortened to 4 days. These results can be seen i n Table VII and Fig. 21 and 23. 3. Ascorbic acid The results tabulated i n Table VII and i l l u s t r a t e d i n 64 TABLE VII The e f fect of 5,000 ppm KNap on the content of sugars, ascorbic a c i d , and t i t r a t a b l e a c i d i n mature tomato f r u i t s . Days of storage af ter Content T '% harvest C 5,000 ppm C. 10 1 0 . 0 7 x x 12.25 121. 60 Sucrose . 4 10.17 13. 58 125. 40 1 11. 87 11.97 100. 85 10 35.40 38.20 107. 90 Reducing 4 37.10 44. 80 120. 70 sugar 1 38. 30 40.40 105. 50 10 46. 00 51.10 111. 08 Tota l 4 47. 80 59.10 123. 60 sugars 1 50. 80 53. 00 104. 30 10 34.40 25.30 73. 60 Ascorbic 4 32.60 29. 60 90. 80 ac id 1 33. 50 28.90 86. 30 T i t r a t a b l e 10 115.00 110.00 95. 70 ac id 4 118.00 125.00 105. 60 X; Content of sugars: Percentage of dry tomato f r u i t weight. Ascorbic ac id content: mg/100 g of fresh tomato f r u i t T i t r a t a b l e ac id content: ml of 0.1.N KOH/100 g of fresh tomato f r u i t x x ; Each value i s the average value of 6 samples. Total sugars o 4-1 m B o -U d) H -U rd e 4-) O 60 50 40 4-1 Xi cn •H — (U CQ >,3 30 u u Tj CH CH O b 20 c 4-1 c. O u u rd CP co 10 0 Pig. IS \ \ SI Control l I 5,000 ppm Sucrose / / / / 110 4 Reducing sugar / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / A . 1 10 4 1 10 4 Days of Storage 20: E f f e c t of 5,000 ppm KNap on content of sucrose,reducing sugar, and t o t a l sugars i n mature tomato f r u i t s . cn 66 140 -P • H U 4- 1 O -P rd e o 4J CQ CD 5- l m CM O in o o rH \ X o CM o >1 +J • H T3 • H U fd <u rH r Q rd J J rd M 4J • H 120 100 . _ 80 60 40 20 I W \ M Control l l5,000 ppm 10 4" Days of storage F i g . 21: Ef fec t of 5,000 ppm KNap on the content of t i t r a t a b l e ac id i n mature tomato f r u i t s . 67 Fig. 22: Effe c t of 5,000 ppm KNap on content of ascorbic acid i n mature tomato f r u i t s . 10 days o f s t o r a g e 120 _ 100 _ . 80 60 _ 40 20 FT / / / / / / / / / / / / / / / / / / / i T I I --/-I I I I I I I I / / / / I / l / / 7' / / / / / / / / / / / / / / / / / / / / 4 days o f s t o r a g e I 3 1 day o f s t o r a g e / / / / / / / / / / / / / / / / / / A. I • / / / / / / / / / / / / / / / / / / / / / / / / F i g . 23 Sucrose Reducing sugar T o t a l sugar A s c o r b i c a c i d T i t r a t a b l e a c i d i t y E f f e c t o f 5,000 ppm KNap on the contents o f sugars, a s c o r b i c a c i d and t i t r a t a b l e a c i d i n mature tomato f r u i t s . 69 F i g . 22 and 23, ind icated that the ascorbic a c i d content was decreased by the treatment. Decreases of 26.40%, 9.20% and 13.73% were detected i n tomato f r u i t s stored for 10, 4, and 1 day respec t ive ly . Although there was no s t a t i s t i c a l l y s i g n i f i c a n t dif ference found i n sugars, t i t r a b l e a c i d i t y , and ascorbic ac id content, a d i s t i n c t tendency toward change caused by KNap treatment i s evident. V. DISCUSSION A. Vegetative growth It has been shown by Wort (unpublished) that the f o l i a r app l i ca t ion of aqueous solut ions of KNap and NaNap, 2,500 and 5,000 ppm, to 2-week-01d bush bean plants (Phaseolus vulgaris var. Top Crop) resu l ted i n a 20% increase i n juveni le growth and i n y i e l d of green pods and r i p e seeds. In the experiments described i n th is thesis KNap so lu t ions , 2,500 and 5,000 ppm, were appl ied to tomato (Lycopersicum esculentum var. Bonny Best) p lants . In the f i r s t two experiments tomato plants were treated at the age of 4 weeks and no d e f i n i t e and consistent growth responses were obtained. I t was thought that the age of the plant at the time of treatment, 4 weeks o l d , might have been respons ib l for the inconsis tent responses. The t h i r d experiment was designed to examine the 70 in terac t ion between concentration of KNap and the treatment time and the tomato plant growth. The re su l t s suggested that the 5,000 ppm KNap appl ied to tomato plants when 19 days o l d gave encouraging resu l t s i n terms of fresh and dry weights of plant tops. The app l i ca t ion of 5,000 ppm KNap to 12-day-old plants and of 2,500 ppm KNap to plants when 12 and 19 days o ld gave i n -consistent r e s u l t s . Data of the fourth experiment i n which plants were sprayed when 3 weeks o l d with 5,000 ppm KNap ind icated that the ef fects of treatment again on fresh and dry weights measured 2, 4, and 6 weeks after treatment had no s t a t i s t i c a l s i g n i f i c a n c e . Based on the resu l t s of the four experiments, i t appears that both the 2,500 ppm and the 5,000 ppm KNap treatment resu l ted i n inconsis tent ef fects on growth of tomato p lants . Szekely (1966) reported that HNap could increase growth of tomato p lants . Yureva (1965) proved that Nap favorably af fected the growth of corn plants when appl ied at strength of 0.005%. More concentrated solut ions were i n h i b i t o r y . Members i n the team working with KNap at the Univers i ty of B r i t i s h Columbia observed considerable s t imulat ion of growth of a number of species of plants such as maize, sugar beet, sunflower, bush bean, r a d i s h , spinach, tobacco, and tomato (Wort, unpublished). The growth of tomato plants was found to be increased by 12.50% by 2,500 ppm KNap sprays, i n terms of fresh and dry weights of p lant tops. Fattah and Patel (unpublished) got enhanced growth and y i e l d of bush bean plants by the application of KNap. Bazanova and Akopova (1966) reported that effects of HNap interac t with l e v e l of N-P f e r t i l i z e r s applied at the same time, therefore the growth appeared to depend on the amount of applied f e r t i l i z e r . Popoff, Dimitrov, and Stefanova stated that Naps markedly stimulated the growth of wheat and corn. However, most of the above-named investigators studied the e f f e c t of Nap on species of plants other than tomato and for t h i s reason a d i r e c t comparison of the present data with above-mentioned reports would not seem very v a l i d . B. Chlorophyll and carotenoid content It was noticed that the contents of chlorophyll a and b, and carotenoid, increased as both the cmtrol and treated plants became older. However, when the pigment content i n leaf blades of treated and control plants of the same age were compared, the chlorophyll content (both a and b) was increased to a small extent but the carotenoid content was increased more. This might be the reason for the increased photosynthetic rate observed simultaneously. Bazanova et a l . (1966) reported that Nap could either increase or decrease the chlorophyll content of the cotton plant depending on the l e v e l of applied N-P f e r t i l i z e r . Higher chlorophyll content was found i n the Nap-treated plants when the usual amount of N-P was applied. Under the influence of Nap, increases i n chlorophyll and/or carotenoid content have been 72 found i n other species of plants, for example, potato (Lydygina, 1965, Abolina and Ataullaev, 1966), corn, sugar beet (Yureva, 1965), and bush bean (Fattah, and Patel, unpublished). In the experiments, soluble N:P:K, 20:20:20, f e r t i l i z e r was applied weekly to the s o i l during the growth period of the tomato plants. The s l i g h t increases i n chlorophyll content observed i n this experiment might have resulted from the i n t e r -action of the f e r t i l i z e r applied and the e f f e c t of KNap. C. Photosynthesis and r e s p i r a t i o n The data suggest that the 5,000 ppm KNap-treated plants proceded more energetically i n both photosynthesis and res-p i r a t i o n 4 weeks after treatment, but the opposite was observed 2 weeks after treatment. This suggests that a rather long time (for example, 4 weeks i n thi s experiment) i s needed for KNap to exhibit it's stimulatory e f f e c t on photosynthesis and r e s p i r a t i o n of tomato plants. The higher photosynthetic rate was found to be accompanied by increases i n pigment content and both fresh and dry weights of plant tops. The stimulatory e f f e c t of Nap on the i n t e n s i t i e s of photo-synthesis and r e s p i r a t i o n has been reported by several investigators i n d i f f e r e n t species of plants, for example, potatoes (Ladygina, 1965; Abolina and Ataullaev, 1966); grape plants (Kolesnik, 1965); melons and other vegetables (Abolina and Attaullaev, 1966). Besides, Fattah (unpublished) observed 73 similar e ffects i n bush bean plants under d i f f e r e n t l i g h t i n t e n s i t y regimes. D. Enzyme a c t i v i t i e s The effects of Nap on a c t i v i t i e s of several enzymes have been reported by some s c i e n t i s t s . Nap was found to promote both a c t i v i t y and formation of amylase i n the mold A s p e r i g i l l u s  usamii (Burachevskii, 1965). A c t i v i t i e s of ascorbic acid oxidase and peroxidase i n leaves of Nap-treated cotton plants were found to be higher than the controls (Agakishier and Bazanova, 1965). Effects of Nap on catalase a c t i v i t y of cotton (Akopova & Bazanova, 1966) and grape plant (Kolesnik, 1965) were found to be stimulative. Fattah (unpublished) showed that a c t i v i t i e s of transaminase, phosphoglyceryl kinase, and phosphorylase were higher i n the leaf blades of KNap-treated bush bean plants. The results of the present experiment indicated that the e f f e c t of KNap on enzyme a c t i v i t i e s depended on the s p e c i f i c enzyme and the age of plants. Among the f i v e investigated enzymes, only phosphorylase i n a l l three observations, and transaminase i n the l a s t observation made 6 weeks afte r treat-ment, were increased i n a c t i v i t y by the treatment. However, NRase, succinic dehydrogenase,and phosphogfryceryl kinase were decreased i n a c t i v i t i e s by .the KNap application. Experiment V was done to follow more c l o s e l y the immediate e f f e c t of KNap on succinic dehydrogenase a c t i v i t y and the protein 74 content of the corresponding enzyme extract. The results indicated that there was no p a r a l l e l i s m between the changes of protein content and s p e c i f i c a c t i v i t y of succinic dehydrogenase. The a c t i v i t i e s of phosphoglyceryl kinase, succinic dehydro-genase and NRase were lessened by the KNap treatment. This might be because the KNap solution used was too concentrated. Indeed, catalase a c t i v i t y was found to be decreased by high concentrations of Nap or double spraying with a lower concen-t r a t i o n by Kolesnik (1965). E. Protein content The e f f e c t of KNap on protein content i n leaf blades of tomato plants was found to be quite inconsistent. Maximum amount of protein was measured 4 days after spraying by showing 50.00% increase. Higher protein content i n corn and sugar beets treated by Naps was reported by Yureva (1965), and similar r e s u l t s were obtained by Abolina and Ataullaev (1966) on d i f f e r e n t sorts of potatoes. Subbotina (1965) found that 0.1% Naps increased protein content i n apple tree leaves 1 month after treatment, but 1.5 months after the application the concentration of N compound decreased. Furthermore, Pakhomova (1965) reported that the protein and nucleic acids contents i n Nap-treated tomato plants were d i f f e r e n t from the controls. 75 F. F r u i t Y i e l d Several investigators i n the past have reported the e f f e c t of Naps on f r u i t quantity and q u a l i t y of d i f f e r e n t species of plants. Kulieva (1964) reported that o l i v e f r u i t y i e l d was en-hanced by approximately 70.00% under the influence of NaNap. Marshaniya, Sharashenidze, and Dumbadze (1965) found that 0.05% Nap treatment increased the tangerine f r u i t weight. The fact that Naps accelerated the ripening of the cotton plant and opening of b;olls was confirmed by Naghibin (1965) . Working on the same plant, Bazanova and Akopova (1966) discovered that the maturation and crop y i e l d of cotton depended on the amount of applied f e r t i l i z e r . More valuable information stating that an increase of 6 to 13% i n t o t a l y i e l d of Nap-sprayed tomato plants was contributed by Popoff and Boikov (1962). Increased y i e l d up to 18.80% i n Nap-treated tomato plants was reported by Polikarpova (1965). Results of Experiments II and IV indicated that the KNap at the concentrations of 2,500 ppm or 5,000 ppm could favor the tomato f r u i t number and fresh weight and also e a r l i e r maturity. In Experiments II and i v , tomato f r u i t s were found to be rotten at the d i s t a l end. This blossom-end rot may be due to the poor v e n t i l a t i o n system of both the growth room and green house. Besides, the plants were placed quite close together due to the l i m i t a t i o n of space. It was noticed that the 2,500 ppm 76 KNap treatment induced a greater incidence of the disease, but the 5,000 ppm treatment seemed to induce resistance to the rot, and also the plants reacted more favorably to the CaCl2 spray. .It was suggested that Ca deficiency, rather than water shortage was the primary cause of the disorder. In view of t h i s , and the experimental r e s u l t s , KNap at the strength of 5,000 ppm might influence the calcium uptake from the s o i l s and/or f a c i l i t a t e the translocation of the rather immobile Ca to the leaves and consequently decrease the blossom-end rot to a certain extent. G. Quality of tomato f r u i t s Different.workers have claimed that Nap treatment could a f f e c t the chemical composition such as sugar, ascorbic acid and t i t r a t a b l e acid of d i f f e r e n t plants and plant organs. Agakishier and Bazanova (1965) reported that KNap heightened the ascorbic acid content of cotton plant leaves. Bazanova and Akopova (1966) observed that Nap increased the ascorbic acid and free carbohydrate i n leaves of cotton plants only i f low con-centration or no f e r t i l i z e r was applied. Sugars and vitamin C content were raised i n cabbage plants by Nap i n the experiment, done by Asadov (1965). In tangerine f r u i t s of Nap-treated plants as stated by Marshaniya and Sharashenidze (1965), increasing sugar content accompanied by decreasing acid content were observed. Higher sugar content i n grapes (Kolesnik, 1965), higher sugar content i n melon f r u i t s , and higher vitamin C i n cabbage, carrots, 77 and onions (Abolina and Ataullaev, 1966) were observed under the influences of Naps. Aliev (1965) reported that with the introd-uction of NaNap, the composition of the tomato f r u i t s changed sharply; increased amount of sugar and ascorbic acid. In the present experiment, 5,000 ppm KNap did exert a p o s i t i v e e f f e c t on reducing sugar, sucrose, and t o t a l sugars i n the mature tomato f r u i t s and also the loss of sugar content during the course of storage was less. T i t r a t a b l e acid of the mature tomato f r u i t under the influence of KNap was higher at the end of 4 days storage but lower as the duration.of storage was longer. Apparently, 5,000 ppm KNap increased t i t r a t a b l e acid in tomato f r u i t s , but during storage the acid content disappeared . quicker and more. Ascorbic acid content was lessened by the treatment which also did not exhibit favorable e f f e c t on the protection against ascorbic acid loss during storage. H. Overall statement In view of the above-mentioned.experimental r e s u l t s , i t seems too early to propose any mechanism for the action of KNap on plants. The mechanism concerning how the KNap treatment af f e c t s d i f f e r e n t biochemical and physiological aspects of the tomato plants i s s t i l l up i n the a i r and therefore furthe r studies are essential to gain more knowledge about t h i s . In fact, radioactive KNap l a b e l l e d at the carboxylic C was proved to form conjugates with glucose and aspartic acid (Severson, unpublished). 78 However, i t can be pointed out that since the a c t i v i t i e s of NRase and transaminase were suppressed the quantity of NH-^ , and synthesis of glutamic acid, and i n turn other amino acids synthesized by transamination would be less. Acids involved i n the Kreb Cycle then would not be drawn away for amino acid and protein synthesis. In addition, succinic dehydrogenase a c t i v i t y declined. Under these situations, sugar content would be accumulated more and perhaps th i s was the reason why higher sugar content i n mature tomato f r u i t s was detected under the influence of 5,000 ppm KNap. Higher pigment content was found to associate with the increased photosynthetic rate i n KNap-treated plants. The KNap treatment might increase the absorption and/or translocation of Ca and/or water and consequently protected tomato plants from blossom-end rot which was supposed to r e s u l t from Ca deficiency and sudden water shortage. How the KNap affected the enzyme a c t i v i t i e s could not be explained by the resu l t s obtained. VI. CONCLUSIONS KNap aqueous solution of the concentrations 2,500 ppm and 5,000 ppm were sprayed on tomato leaves to study the effects on the growth, productivity, and metabolism of tomato (Lycopersicum esculentum var. Bonny Best) plants. S p e c i f i c a l l y , effects pertaining to growth, y i e l d , enzyme a c t i v i t y , pigment content, photosynthesis, r e s p i r a t i o n and qual i t y of tomato f r u i t s 79 i n terms of sugar, t i t r a t a b l e acid and ascorbic acid were studied. Based on the re s u l t s obtained, i t i s possible to draw the following conclusions: (1) Contents of pigments including chlorophyll a and b, and carotenoid were increased i n the leaf blades of the 5,000 ppm KNap-treated plants. (2) Higher photosynthesis and r e s p i r a t i o n rates were found under the influence of KNap. (3) Increase i n photosynthetic rate might be the cause of promoted growth and higher y i e l d of tomato f r u i t s . Besides, the 5,000 ppm KNap treatment also hastened the maturity of tomato f r u i t s , protected them against blossom-end rot to a certain extent and also increased the recovery frequency after receiving CaCl 2 spray. •(4) The treatment induced considerable a l t e r a t i o n s i n chemical composition of tomato f r u i t s . Reducing sugar, sucrose, and t o t a l sugars were more i n mature tomato f r u i t s of the treated plants. T i t r a t a b l e acid and ascorbic acid, however, were lessened under the influence of the KNap. (5) Only phosphorylase up to 6 weeks after treatment and NRase at 6 weeks after treatment were found to be increased i n a c t i v i t y . Other enzymes, succinic dehydrogenase, phosphoglyceryl kinase and transaminase were found to have lower a c t i v i t i e s caused by the treatment. 80 VII. REFERENCES Abolina, G., and N. Ataullaev.. 1966. Influence of naphthenic growth substance (NGS) upon the physiological and biochemical progresses and the productivity of potatoes, melons and vegetables i n the conditions of Uzbekistan. Symposium on Plant Stimulation. Sofia, Bulgaria. Oct. 25-30. Abstracts. Aliev , A.A.. 1963. Petroleum nutrients, Quoted from Chem. Ab. 64:10158a. Ali e v , A.Y.. 1965. Ef f e c t of NRV (sodium naphthenate) on growth, development, and composition of tomatoes. Quoted from Chem. Ab. 66:45849n. Ali-Zade, M.A., and Z.B. Guseinov. 1965. Ef f e c t of petroleum growth-promoting substances, gibber-e l l i n , and heteroauxin on the growth and development of egg plants. 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Activation of the synthesis of amylase by Aspergillus  usamii. Quoted from Chem. Ab. 62:16920h. Chi, CH. . 1964. Ef f e c t of 2,4-dichlorophenoxyacetic acid on the y i e l d and metabolism of the bean plant. M.Sc. Thesis, Univ. of B r i t i s h Columbia. Dimitrov, D.I., M. Popoff, and A.D. Stefanova. 1966... Testing the physiological a c t i v i t y of synthetic acids obtained from naphtha and other surface-active substances. Symposium on Plant Stimulation. Sofia, Bulgaria. Oct. 25-30 Abstracts. Dixon, M., and E.C. Webb. 1964. Enzymes. Acad. Press Inc., N.Y. Eul'fugarly, D.I., D.M. Guseinov, and A.M. Ataullaev.„ 1967. B i o l o g i c a l action of Ni, Mn, Cu naphthenate. Quoted from Chem. Ab. 68:94771f. Evans, H.J., and A. Nason. 1953. Pyridine nucleotide-nitrate reductase from extracts of higher plants. Plant Physiol. 28:233-254. Eyubov, R., and F. Issaeva. 1966. Influence of naphthenic growth substances upon the growth, development and y i e l d of maize and lucerne. Symposium on Plant Stimulation. Sofia, Bulgaria. Oct. 25-30. Abstracts. Fiske, C.H., and Y. Subbarow.192 5. The colorimetric determination of phosphorus. Jour. B i o l . Chem. 66:375-400. Frank, S., and A.L. Kenney. 1955. Chlorophyll and carotenoid destruction i n the absence of l i g h t i n seedlings of Zea mays L.. Plant Physiol. 30:413-41 Glushkov, N.M., A.S. Yakovlev. 1963. Bee feeding with growth substances. Quoted from Chem. Ab. 63:60806 Guseinoiv, D.M., and F.G. Isaeva. 1965. Eff e c t of combined delivery of petroleum growth-promoting substances and radioactive phosphorus-on the a l f a l f a crop. Quoted from Chem. Ab. 67:20836p. 82 Hubert, G.. 1966. Treatment of naphthenic o i l to sugar cane to increase sugar content. Quoted from Chem. Ab. 65:17655a. Isenberg, F.M.R., M.L. Odland, H.W. Popp, and CO. Jensen. 1951. The e f f e c t of maleic hydrazide on certa i n dehydrogenases"in tissue of onion plants. Science. 113:58-60. J o l l y , S. E.. 1967. "Naphtenic acids", i n Enclyclopedia of Chemical Technology 13 edited by Mark, H. et a l . John Wiley and Sons, Inc. pp 727-734. Kosobokov, V. I.. 1965. Ef f e c t of petroleum growth-promoting substance on growth and development of feed cultures. Quoted from Chem. Ab. 67:20839s. Kazakova, I . I . . 1965. E f f e c t of growth stimulants on the germination and growth of weed seeds. Quoted from Chem. Ab. 2083 7q. Kolesnik, Z.V.. 1965. Ef f e c t of petroleum growth-promoting substance on the biochemistry of the grape, plant. Quoted from Chem. Ab. 62:20838v. , Krasnova,.E.M., V.I. Kotyashkina, and G.S. Ilichev. 1965. B i o l o g i c a l c h a r a c t e r i s t i c s of petroleum growth-promoting substances as plant activators. Quoted from Chem. Ab. 67:20840K. Kulieva, N.A.I 1964. Ef f e c t of petroleum growth promoters on the o l i v e f r u i t drop. Quoted from Chem. Ab. 66:27894h. Kun, E., and L.G. Aboo.d. 1949. Colorimetric estimation of succinic dehydrogenase by triphenyltetrazolium chloride. Science. 109:144-146. Ladygina, E.A.. 1965. Ef f e c t of petroleum growth-promoting substance on growth, development, and y i e l d of potatoes. Quoted from Chem. Ab. 67-20833K. 83 Lepper, H.A.. 1945. O f f i c i a l and Tentative Methods of Analysis of the Association of O f f i c i a l A g r i c u l t u r a l Chemists. Published by The Association of O f f i c i a l A g r i c u l t u r a l Chemists. 6th Edition. Lipmann, I., and L.C. Tuttle. 1945. A s p e c i f i c micromethod for determination of acyl phosphates. Jour. B i o l . Chem. 159:21-28. Lo e f f l e r , H.J., and J.D. Ponting. 1942. Ascorbic acid - rapid determination i n fresh frozen, or dehydrated f r u i t s and vegetables. Ind. Eng. Chem. Anal. 14:846-849. Loomis, W. E. i and CA. Shull. 1937. Methods i n Plant Physiology. McGraw H i l l , Inc. N.Y. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with F o l i n phenal reagent. Jour. B i o l . Chem. 193:265-275. Mans, R.J.. 1967. Protein synthesis i n higher plants. Ann. Rev. Plant Physiol. 18:127-146. Markovnikoff, W., and W. Ogloblin. 1883. Naphthenic acids. J. Russ. Phys. Chem. Soc. 13:34. Marshaniya, I.I., M.G. Sharashenidze, and A.I. Dumbadze. 1965. Ef f e c t of petroleum gcowth-promoting substances on the tangerine crop and qual i t y . Quoted from Chem. Ab. 67:20834m. McKinney, G.. 1940. C r i t e r i a for purity of chlorophyll preparations. Jour. B i o l . Chem. 132:91-107. Naghibin, I a . D . . 1966. Influence of naphthenic growth substances (NGS) upon the development and p r o d u c t i v i t y of the cotton-plan t i n the condit ions of T a d j i k i s t a n . Symposium on Plant St imulat ion. Sof ia , Bu lgar ia . Oct. 25-30. Abstracts . Pakhomova, G . I . . 1965. Ef f ec t of treatment with petroleum growth-promoting sub-stance on biochemical processes i n tomato leaves. Quoted from Chem. 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Hort. Sci. 47:285-293. Wittwer, S.H 1964. F o l i a r absorption of plant nutrients. Adv. Frontiers Plant Sci. 8:161-182. Yureva, K.V.. 1965. Results of using petroleum growth-promoting substances i n plant growing. Quoted from Chem. Ab. 67:20846S. Zamanov, P.. 1966. Influence of naphthenic growth substances upon the growth and productivity development of tobacco plant. Symposium on Plant Stimulation. Sofia, Bulgaria. Oct. 25-30. Abstracts. Ziegler, D., and J.S. Rieske. 1967. Preparation and Properties of Succinate Dehydrogenase-coenzyme Q Reductase (Complex II) i n Methods i n Enzymology X. edited by Estabrook and Pullman, Academic Press, New York and London, pp 231-235. 86 VIII. APPENDICES 0.25 0. 50 0. 75 1.00 Microgram n i t r i t e / m l F i g . 24: Standard chart for n i t r a t e reductase. 88 F i g . 25: Standard chart for phosphorylase. 09 F i g . 26: Standard chart for succ in ic dehydrogenase. 90 120 10 20 30 40 50 mg ascorbic acid/1 Fig. 27: Standard chart for ascorbic acid. 

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