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Storage quality of lettuce leaves as affected by kinetin and abscisic acid Hemapat, Thosporn 1973

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C- I  STORAGE QUALITY OF LETTUCE LEAVES AS AFFECTED BY KINETIN AND ABSCISIC ACID  by Thosporn Hemapat B.Sc, Kasetsart University, Thailand, 1966  A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in the Department of Plant Science  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1973  ii  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes  may be granted by the Head of my Department or  by his representatives.  It is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of  PLANT SCIENCE  The University of British Columbia Vancouver 8, Canada  Date  October 3, $973.  in Abstract  Some effects of post-harvest treatments of abscisic acid  (ABA)  and kinetin on the maintenance of quality and consumer appeal were studied on young lettuce plants.  The treatments employed two concentrations of  abscisic acid (1 and 5 ppm), one concentration of kinetin (20 ppm) and a combination of 5 ppm abscisic acid and 20 ppm kinetin.  The plants were  sprayed to the run-off point and placed in a storage chamber at 3±1°C with relative humidity close to 100%.  After 6 weeks of storage a l l lettuce  including untreated controls were in good condition.  The chemical  treatments did not have any distinct effect on the quality of lettuce as evaluated by a panel of observers for visual quality rating.  The 20 ppm  kinetin retarded chlorophyll degradation when compared to the control or the ABA-only treatments.  Considering chlorophylls A and B separately, the  kinetin-treated plants showed a significantly higher chlorophyll A content than other treatments, including the control.  The differences in chlorophyll  B content followed the same trend but only approached the 5% level of significance.  ABA in the 5 ppm + 20 ppm kinetin treatment had a mild antagonistic  activity to kinetin, and hence reduced the effect of kinetin on both chlorophyll Aiand B.  Measurement of chlorophyll contents and adjustment  to the original fresh weight before the samples were put in storage, provided a common basis to make comparisons for the study of chlorophyll degradation as functions of storage time and chemical treatment.  Means of chlorophyll  contents reported on this basis showed a trend of degradation from the 5th week to the 7th week. Temperature at 3tl°C and high relative humidity in the storage appear to be favourable for keeping lettuce.  Hygenic pre-  paration of the storage chamber also resulted in disease-free product even at the end of 7 weeks in storage.  iv Acknowledgements  This study was made possible by grants from the Canadian International Development Agency, and the author is deeply grateful to the Agency for the opportunity to study at the University of British Columbia. Special gratitude is due to Dr. C.A. Hornby, Associate Professor, Department of Plant Science, University of British Columbia, for supervision, helpful guidance and encouragement. Grateful acknowledgement is also extended to Dr. V.C. Runeckles, chairman of the committee, Dr. G.W.  Eaton,  Dr. P.A. Jolliffee and Dr. R.L. Taylor, members of the committee - whose criticism and suggestions were most valuable for the accomplishment of this study. Sincere thanks is offered to Mr. F.L. Billings, Hoffman-LaRoche Ltd., 1956 Bourdon Street, St. Laurent, Montreal 378, P.Q. for the supply of abscisic acid; Mr. A. Ploadpliew, Mr. Tom L i , Mr. T. Greenberg, Mr. L. Spraul, Ms. C. McConnell and Ms. C. Wisdom for invaluable help during study and thesis preparation; and also to the staff of the Department of Plant Science, particularly Ms. D. Green for all f a c i l i t i e s and kind co-operation.  V  CONTENTS Page INTRODUCTION  1  LITERATURE REVIEW  3  A. Abscisic Acid 1. Effects on abscission and senescence  4  2. Effects on growth, dormancy, and seed germination  5  3. Effects on RNA, DNA, enzyme and protein  6  synthesis  4. Effects on transpiration and stomatal activity  7  5. Effects on other physiological behavior  9  B. Kinetin 1. Effects on senescence  11  2. Effects on RNA, DNA, enzyme and protein synthesis  13  3. Effects on respiration and stomatal activity  14  4. Effects on transpiration  14  5. Effects on other physiological behavior  15  C. Inteaction of abscisic acid with kinetin, and with other hormones MATERIALS AND METHODS  15 20  A. Materials 1. Test plants 2. ' Chemical treatments 3. Cold storage f a c i l i t i e s  21 22 23  B. Methods 1. Experimental design  24  2. Treatment procedure  24  3. Visual quality rating  25  4. Total weight loss measurement  26  vi  Page 5. Moisture content measurement  26  6. Chlorophyll content measurement  27  RESULTS 1. Visual quality rating  30  2. Total weight loss  32  3. Moisture content  33  4. Chlorophyll content  35  5. Correlation and simple linear regression (1) Chlorophyll A and chlorophyll B content  46  (2) Percent weight loss and storage time  47  (3) Percent moisture content and storage time  48  DISClUSS.-IONW AND CONCLUSION  50  LITERATURE CITED  55  APPENDICES  61  LIST OF TABLES  Table 1A Means of quality ratings by a panel of observers, of lettuce subjected to 5 treatments, after 2,3, 4,5,6, and 7 weeks in storage Table IB Analysis of variance of numerical quality ratings of lettuce under 5 treatments after 5,6, and 7 weeks in storage Table 2A Percentages of total weight loss (means of 8 observations in each.i experimental lot) of lettuce under 5 treatments after 5,6 and 7 weeks in storage Table 2B Analysis variance of percent total weight loss of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 3A Percentages of moisture content (means of 8 observations in each experimental lot) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 3B Analysis of variance of percent moisture content of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 4A Chlorophyll A contents (means of 8 observations in ea.ch experimental lot) in mg/gm FW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 4B Analysis of variance of chlorophyll A content (mg/gm FW) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 5A Chlorophyll  A contents (means of 8 observations  in each experimental lot) in mg/gm OFW of lettuce  under 5 treatments after 5, 6 and 7 weeks in storage Table 5B Analysis of variance of chlorophyll A content (mg/gm OFW) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 6A Chlorophyll B contents (means of 8 observations in each experimental lot) in mg/gm FW of lettuce under 5 treatments after 5, 6 and 7 weeks in >  storage  Table 6B Analysis of variance of chlorophyll B content (mg/gm FW) of lettuce under 5 treatments after 5,6 and 7 weeks in storage Table 7A Chlorophyll B contents (means of 8 observations in each experimental lot) in mg/gm OFW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 7B Analysis of variance of chlorophyll B content (mg/gm OFW) of lettuce under 5 treatments after 5,6 and 7 weeks in storage Table 8A Chlorophyll (A+B) contents (means of 8 observations in each experimental lot) in mg/gm FW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 8B Analysis of variance of chlorophyll (A+B) content (mg/gm FW) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 9A Chlorophyll (A+B) contents (means of 8 observations in each experimental lot) in mg/gm OFW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Table 9B Analysis'of variance of chlorophyll (A+B) content  ix  Page (mg/gm OFW) of lettuce under. 5 treatments after 5, 6 and 7 weeks in storage  41  Table 10 Tabulation of the means (mg) of chlorophyll A, B and (A+B) contents, based on 1 gm fresh weight (gm FW) and 1 gm original fresh weight (gm OFW), for 2 replications, under 5 treatments and iafter 5, 6 and 7 weeks in storage Table 11  42  Linear regression equations of chlorophyll B content (Y) on chlorophyll A content (X)  47  Table 12 Linear regression equations of percent weight loss (Y) on storage time in weeks (X)  48  Table 13 Linear regression equations of percent moisture content (Y) on storage time in weeks  49  X  LIST OF FIGURES Figure  Page  1. Structure of (S)- abscisic acid 2. Structure of kinetin 3. Modified vacuum f i l t r a t i o n apparatus  3 11 28  4. Chlorophyll A and B in mg/gm OFW as affected by treatment and storage time 5. Chlorophyll  45  (A+B) in mg/gm FW and mg/gm OFW of lettuce  leaves under control, 20 ppm kinetin and 5 ppm ABA+ 20 ppm kinetin treatments at the end of 5, 6 and 7 weeks in storage  46  1  INTRODUCTION  Minimizing losses and conserving quality of vegetable crops in the post-harvest period is a challenge for growers, shippers and merchants who wish to get good quality produce to the consumer. Consequently, improved methods are constantly being sought for retarding the rates of transpiration, respiration, and chlorophyll degradation, thus lessening wilting and senescence, and extending the post-harvest salabi1ity of vegetable crops. Present methods of fresh vegetable preservation include precooling, cold storage and special processing such as waxing and prepackaging; however, recent reports on the use of kinetin or abscisic acid suggested that these chemicals along with conventional cooling methods might be valuable to extend the post-harvest l i f e of those vegetable crops even further. Abscisic acid is known to induce stomatal closure and inhibit transpiration in some plants at the normal room temperature range (Little and Eidt, 1968; Mittelheuser and Van Steveninck, 1969; Horton, 1971; Cummin et a l , 1971), and might be expected to inhibit transpiration in fresh vegetable crops and so extend their post-harvest l i f e .  If used in conjunction with  conventional cold storage, the quality l i f e of produce might then be s i g n i f i cantly lengthened. Kinetin was demonstrated by El-Mansy e_ al_. (1967) to be an effective senescence-retardation agent under cold storage conditions, therefore, this chemical was also used in the present study.  Furthermore, abscisic acid and  kinetin have been known to interact in many physiological systems (Addicott and Lyon, 1969), therefore the effect of these two chemicals together on post-harvest quality was included.  2  In the present study, lettuce (Lactuca sativa L. var. capitata L.) was selected as the test vegetable. The rapid development and perishability of lettuce make i t a convenient test plant for this type of research. Additionally, El-Mansy e_t al_. (1967) used lettuce in his experiments with kinetin and these studies provide a valuable background for reference and comparison for the present study. An experiment was planned to observe some effects of abscisic acid and kinetin, both separately and in combination, on the post-harvest quality of young lettuce plants.  3  LITERATURE REVIEW  A. Abscisic Acid  Figure 1. Structure of (S)-abscisic acid  The structural formula shown above is for a 3-methyl-5(l-hydroxy-4-  i t/  f  '  oxo-2-6-6-trimethyl-2-cyclohexen-l-yl)-cis, trans-2,4-pentadienoic hormone now known under the name  acid - a  "abscisic acid" and usually designated as  ABA. This hormone is a relatively recent discovery; nevertheless, its physiological importance may rank with auxins, gibberellins or cytokinins (Addicott and Lyon, 1969). The substance was f i r s t isolated by Ohkuma et aJL (1963) from young cotton fruit (Gossypium hirsutum L.). II" because i t promoted abscission activity.  It was then named "abscisin  Almost at the same time, a group  led by Wareing and Cornforth, being interested in dormancy-inducing substances, isolated an active substance from sycamore leaves (Acer pseudoplatanus). This substance was named "dormin" and later i t was found to be the same substance as abscisin II (Cornforth et_ al_. 1965a; Robinson and Wareing, 1964). This chemical was f i r s t synthesized by Cornforth et al_. (1965b) and more contributions were added on its physical and biological activities (Cornforth et a l . 1966). After the Sixth International Conference on Plant Growth Substances  4  in 1967, the present term "abscisic acid", by mutual consent, is being used in place of the names "dormin" and "abscisin II" (Addicott et a l . 1968).  Figure 1 was derived from the latest revision on this chemical by  Ryback (1972). ABA is widely distributed in plants ( i f not ubiquitous), and mostly found in very low concentrations, such as 40 yg/kg dry weight from Gossypium fruits (Ohkuma et al_. 1963), and 9 ug/kg dry weight from Acer leaves (Cornforth e_t al_. 1965a). The natural enantiomer of ABA has been found to be (S)-(+)-abscisic acid (Cornforth e_ al_. 1966). The synthetic racemis substance is (RS)-(t)-abscisic acid, and this compound, on bioassay, showed approximately one-half the inhibitory activity of the natural hormone (Cornforth e_t al_. 1965b). ABA, like a l l other hormones, induces a wide spectrum of plant responses.  Besides its activities in abscission and senescence, i t is well  recognized in various other significant phenomena including germination, dormancy, enzyme a c t i v i t i e s , and flowering.  The general physiology of ABA,  as well as its chemistry, historical discovery and development, is well reviewed by Addicott and Lyon (1969).  Lately, i t has been found that ABA  affected stomatal diffusion resistance and transpiration (Little and Eidt, 1968; Mittelheuser and Van Steiveninck, 1969; Mizrahi e_ al_. 1970; Horton, 1971; Jones and Mansfield, 1970). This particular effect has given rise to the idea of using ABA as an antitranspirant which may be useful in prolonging post-harvest quality of some horticultural crops.  A.I.  Effect of ABA on abscission and senescence Leaf or fruit abscission is a common response to ABA treatment.  This  response is accepted as a part of the bioassay technique for ABA (Addicott  5  and Lyon, 1969).  Bornman e_t al_. (1967) studied the nature of ABA-induced  abscission in 14-day-old cotton explants.  Comparisons were made among the  1  effects caused by ABA, an abscission accelerant GA^, and an abscission 2  retardant IAA.  ABA was found to cause a breakdown of cells in a weakly  defined separation layer and the separation could be commenced either ador abaxially, but i t occurred abaxially in the control and in IAA-treated plants.  The breakdown in a well defined separation layer of three or more  rows of cells in width was observed in GA^-treated plants.  Cracker and  Abeles (1969), working with explants of cotton and bean, suggested that the effect of ABA on abscission was two-fold. ABA appeared to cause an increase of ethylene production from explants which was found to account, at least in part, for the ability to accelerate abscission.  There was also an  increase in cellulase activity simultaneously, leading to an acceleration of abscission.  Galston and Davies (1970, p.167) do not attribute the whole  process of abscission ,to ABA only, but rather to the more complex system involving other hormones such as auxin and ethylene.  Much evidence of  hormone balance in connection with abscission has been reported (Salisbury and Ross, 1969, p.652). Acceleration of senescence is another effect of ABA.  Sankhla and  Sankhla (1968a) demonstrated that ABA treatment proved a potent accelrator of senescence of Arabidopsjs leaf disks.  Within 24 hours, leaf disks floated  on 5 ppm ABA lost 3 times more chlorophyll than the control.  The mechanism  whereby ABA promotes senescence is not yet clearly explained.  A.2.  Effects of ABA on growth, dormancy and seed germination Growth inhibition is the basis of several bioassays for ABA content.  Such assays include growth inhibition of coleoptiles (Robinson and Wareing, 1964), hypocotyls (Aspinall et a]_. 1967; Eagles and. Wareing, 1964), 1  GAo = Gibberellic acid;  2  IAA = Indole acetic acid.  6 radicles (Aspinall ejt al_. 1967), and leaf sections (Eagles and Wareing, 1964).  ABA-induced dormancy in deciduous trees was reported by Eagles and  Wareing (1964). ABA treatments by means of dipping, soil drench or spraying showed the same response by prolonging the bud break in several coniferous trees (Little and Eidt, 1968).  Buds on potato tubers could be  induced to go into their rest period by applying ABA (Shih and Rappaport, 1971). Seed dormancy in many plants has been found to be associated with ABA.  Aspinall et al_. (1967) showed the inhibitory effect of ABA on the ger-  mination of lettuce seed.  Germination of Xanthiurn seed was inhibited by the  same chemical (Khan, 1967a).  It is of interest that this effect on seed  germination is relatively transient; that i s , germination can be promptly resumed after washing away the inhibitor (Sumner and Lyon, 1967, as cited by Addicott and Lyon, 1969).  A.3.  Effects of ABA on RNA, DNA, enzyme and protein synthesis ABA has been found to influence some of the fundamental biochemical  mechanisms in plants.  Crispeels and Varner (1967), working on isolated  aleurone layer, found that the GA-promoted synthesis of the hydrolytic enzymes  a-amylase and ribonuclease were inhibited by ABA within 2 to 3  hours after treatment.  It was suggested that ABA might act by inhibiting  the synthesis of enzyme-specific RNA molecules, or by preventing the incorporation of RNA into an active enzyme-synthesising unit.  Working on intact  barley seed,. Khan and Downing (1968) reported inhibitions of growth response and a-amylase synthesis in treated seed. Van Overbeek e_t al_. (1967) reported a blocking effect on specific DNA synthesis caused by ABA; this effect, as observed, seemed to precede the inhibition effect on RNA. Khan and Heit (1969) demonstrated that ABA inhibited the labelling of  32  P  7  into soluble RNA, DNA-RNA hybrid and light-nibosomal RNA fractions of germinating pear embryos. Khan and Anojulu (1970) found a greatly altered nucleotide composition in the rapidly labelled RNA species after ABA treatment in pear embryos. Khan e_t al_. (1970) found the same response in the composition of rapidly labelled RNA species of excised lentil root. Also, Pi let (1970) showed that ABA caused a strong inhibition of total accumulation and accelerated ribonuclear activity.  RNA  ABA (10" M) was found  to inhibit an increase of a- and B-amylase in excised bean cotyledons without 14 affecting the  C-leucine incorporation activity or rate of respiration of  cotyledons, and no inhibition occurred i f the cotyledons were excised 3 days after germination (Yomo, 1971). Besides those inhibitors observed, promotions of some activities were reported, e.g. the increased development of invertase in slices of sugar beet, an increase of a-amylase activity (but not 3-amylase) in a commercial enzyme preparation (Addicott and Lyon, 1969) and phenylalanine ammonia lyase in Phaseolus (Walton and Sondheimer, 1968). De Leo and Sacher (1970) reported that ABA accelerated the increase in activity of acid phosphate resulting in increase in free space of Rhoeo leaf sections. Srivastava (1968) also found the accelerated increase in chromatin-associated nuclease in senescing f i r s t leaves from 7-day-old barley seedlings which were floated on 10 ppm ABA in the dark. A.4.  Effects of ABA on transpiration and stomatal activity ABA induced bud dormancy and simultaneously inhibited transpiration  in red maple, white ash, balsam f i r , and white spruce (Little and Eidt, 1968). Mittelheuser and Van Steveninck (1969) found the same inhibitory effect of ABA on transpiration in excised leaves of wheat, barley, oats and Nasturtium; and their studies of stomatal imprints from wheat and  8  barley showed that ABA treatment induced stomatal closure.  Jones and  Mansfield (1970) demonstrated the same effect in Xanthium and tobacco leaves and found that the effect could not be reversed by flushing the leaves with COg-free a i r .  They suggested that the effect was not due  simply to an increase in the intercellular C0^ concentration but a more direct effect on the stomatal apparatus i t s e l f .  Horton (1971) sought to  determine whether ABA changes stomatal aperture indirectly by altering water relations throughout the leaf or by acting directly on the mechanism of stomatal movement. He showed that ABA can inhibit stomatal opening in isolated epidermal strips of Vicia faba; thus, i t was likely that ABA acted directly on the guard c e l l s . Activities of endogenous ABA have also been investigated.  Wright and  Hiron (1969) found that wilting induced a higher level of ABA in detached leaves of wheat, cotton, pea and dwarf bean; thus ABA may be acting as a part of a protective mechanism against drought.  Mizrahi e_t al_. (1970)  found an increase of inhibitors (with similar chromatographic properties to ABA) while transpiration was inhibited through an osmotic stress applied to the roots.  A wilty mutant of tomato "flacca" which tends to lack an ability  to close i t s stomata was found to contain a much lower amount of the substance. Loveys and Kriedemann (1971) found that stomatal closure due to water stress was accompanied by an increased level of ABA. Closure caused by exogenous ABA was found to be initiated within minutes after treatment and completed within half an hour.  This response appeared to be specific for ABA. They  also found that exogenous applications of ABA caused stomatal closure in both attached and detached leaves, and the amount needed to trigger the response was dependent on species and was in the same order as the endogenous levels of those plants.  Cummins et a l . (1971) found that foliar application  of ABA initiated stomatal closure within 5 minutes, and withdrawal of the  9  hormone reversed the effect within 5 minutes, suggesting a rapid metabolism of ABA..  They also suggested that ABA affected the stomatal apparatus  directly.  A.5.  Effects of ABA on other physiological behavior ABA was found to inhibit flowering in long-day plants (Evans, 1966).  Heide (1968) found that ABA stimulated-the formation of adventitious buds in begonia leaves but reduced the number of roots,«J(the inhibitory effect on root formation occurred at high concentration only); root length was not significantly affected, but lamina expansion and petiole extension were reduced with increasing concentration of ABA.  Sloger and Caldwell (1970)  found that different cultivars of soybean had a different physiological response to applied ABA, and there was evidence that responsiveness genetically controlled.  was  Lichtenthaler and Becker (1970) found that ABA  inhibited the synthesis of vitamin KJ., chlorophyll, and carotenoids in etiolated barley seedlings under illumination.  They suggested that ABA  interfered with thykaloid formation which then resulted in a reduced isoprenoid synthesis. Glinka (1971) found that ABA markedly raised the permeability to water of xylem disks from root of Daueus and stem tissue of Pelargonium. Gamborg and LaRue (1971) found that the ethylene production which actually occurred in rose and Ruta cell cultures was inhibited in the presence of ABA.  Zeevaart (1971) found that when long-day spinaches were  transferred from short-day to long-day condition, ABA content of the spinaches increased up to threefold during the f i r s t long day.  It was found that ABA  content was higher at the end of 8 hours high intensity light period than at the beginning in. both short- and long-day conditions. Lieberman and Kunishi (1971) found that ABA, like ethylene, inhibited growth of isolated pea seedlings, but did not promote the "triple response" characteristic of  10  ethylene.  Application of both ABA and ethylene resulted in an increased  inhibition of epicotyl growth. The results suggested that the inhibitory action of ABA and ethylene on growth of etiolated pea seedling was to different mechanisms.  due  11  B. Kinetin HC  HN  CH 2  CH  C  CH  H Figure 2. Structure of Kinetin  Miller found this chemical in 1954 and named i t "kinetin" after its first-observed activity in association with cytokinesis. was  The substance  identified in 1955 as 6-furfurylaminopurine (Salisbury and Ross, 1969,  p.461), the structure of which is shown in Figure 2.  Kinetin itself has  never been found in plants, although many other related purine derivatives do exist.  Kinetin promotes cell division, and this activity in certain  plants has been used in bioassay procedures. There is a large body of literature on physiological aspects of kinetin, and only selected works are reviewed here.  B.I.  Effects of kinetin on senescence The treatment of 20 ppm  kinetin as a pre-harvest  spray or ppst-harvest  dip on mature head lettuce was shown to prolong the fresh appearance of lettuce heads under storage conditions of 40°F and 85% R.H. and extend the shelf-life period (El Mansy et al_. 1967). Better  chlorophyll retention  and higher moisture content were also observed. Abdel-Kader et a\. (1966)  12  demonstrated a similar effect of kinetin in tomato f r u i t , where ripening of mature green fruits was delayed by 5 and 7 days with treatments of 10 ppm and 100 ppm kinetin respectively.  Treatments on mature green  tomatoes were more effective than on pink-ripe with both concentrations, and the higher concentration was more effective than the lower one.  However,  once the fully ripened stage was reached, those tomatoes with prior exposure to higher concentration deteriorated more rapidly than those with lower concentration.  Von Abram and Pratt (1966) found that senescence was  strongly retarded by kinetin and slightly influenced by NAA in broccoli leaves; the effect was markedly reduced by NAA when both kinetin and NAA were applied simultaneously.  Boasson (1967) found chloroplast maturation  in tobacco tissue culture to depend, in part, on kinetin activity. Kinetin was essential to, but kinetin alone would not support, chlorophyll synthesis unless sucrose was present, suggesting sucrose as a source of energy for the process. Shibaoka and Thimann (1970) experimented the mode of action of cytokinins and found evidence that the primary action of kinetin is to inhibit proteolysis rather than to promote protein synthesis. A correlation between senescence-postponing capability and the endogenous cytokinin was found in rose petals by Mayak and Halevy (1970). The endogenous cytokinin concentration in petals of a long-lived rose variety was higher than in a short-lived variety, and higher in young petals than in the g old ones of the same variety.  Application of N -benzyladenine lengthened  the vase-life of a short-lived variety.  This chemical had been tried and  proved to yield similar effects to kinetin on post-harvest handling of many crops such as prolonging fresh appearance, reduced transpiration rate and weight loss in celery stalks (Zink, 1961; Wittwefr e_t al_. 1962), lettuce (Bessey, 1960; Zink, 1961; Lipton and Ceponis, 1962), cauliflower (Kaufman and Ringel, 1961), endive escarole, Brussels sprouts, sprouting broccoli,  13  mustard greens, radish tops, parsley, green onions, and asparagus (Zink, 1961).  Senescence was delayed and display-life of many cut flowers was  prolonged by N -benzyladenine, e.g. carnations (MacLean and Dedolph, 1962; Waters, 1964; Heide and 0ydvin, 1969), chrysanthemums (MacLean and Dedolph, 1962; Waters, 1964), asters and gerberas (Waters, 1964).  Although the  effectiveness of N^-benzyladenine was widely demonstrated in the previous works, the work by El-Mansy e_ al_. (1967) showed that kinetin was more effective than N -benzyladenine in prolonging storage l i f e and subsequent shelf l i f e of lettuce.  B.2.  Effects of kinetin on RNA, DNA, enzyme and protein synthesis Osborne (1962), working with detached Xanthium leaves and excised  leaf disks, reported a kinetin-induced increase of 14 ation into protein and of  C-orotic acid into RNA.  stimulate both RNA and protein synthesis.  14  C-leucine incorporThus, kinetin can  Osborne suggested that the  retardation of senescence by kinetin is mediated through i t s action in sustaining nucleic and protein synthesis.  Kuraishi (1968) obtained a  similar effect of kinetin on Brassica rapa. He floated leaf disks on a 14 medium containing kinetin.  C-L-leucine in both the presence and absence of  The increase in  radioactivity in the protein fraction of treated  disks was almost linear with time, whereas the control, lacking kinetin, started to slow down. With leaf disks f i r s t incubated on ^C-L-leucine then transferred to either solution or water, the radioactivity of treated disks decreased slower than in the case of the control.  The slower decrease  in radioactivity caused by kinetin was not due to an increased turnover rate, since the same phenomena were observed in the presence of cold leucine or casein hydrolysate solution.  These results suggest that kinetin  retards the decomposition rather than stimulates the synthesis of protein.  14  B.3.  Effects of kinetin on respiration and stomatal activity After kinetin treatments, a slight reduction in respiratory evolution  of carbondioxide was observed by Dedolph et al_. (1962); Katsumi (1963) as cited by Meidner (1967); and El-Mansy e_t al_. (1967).  Livne and Vaadia  (1965) treated the excised, mature primary leaves of barley with kinetin -fi (3 x 10  M) and observed an increased opening of stomatal apertures (the  response in young leaves was not as noticeable as in mature leaves). They suggested that the subsequent increase in opening of stomatal aperture might be due to a lower carbondioxide concentration in the leaves. Meidner (1967) treated mature primary leaves of barley with kinetin and observed the increased rates of assimilation of carbondioxide. He suggested that the resulting reduction in the concentration of carbondioxide inside the leaves be considered as one factor causing the observed decrease in stomatal resistance, but, in addition, kinetin appeared to affect the stomatal mechanism directly.  Tal et aj_. (1970) studied a kinetin-like activity in  a wilty mutant of tomato using labelled leucine and a soybean callus bioassay. This specific mutant "flacca" wilts easily because its stomata resist closure.  They found that kinetin-like activity in both leaf and  root exudate was higher in the mutant than in the normal variety.  It was  also found that the actual decreased resistance to closure with age of this plant coincided with the decrease of kinetin-like activity in the leaf and root exudate at the time.  Ben-Zioni et al_. (1967) found evidence suggesting  a lower level of endogenous cytokinin in osmotic stressed tobacco leaf disks. B.4.  Effects of kinetin on transpiration An increase in stomatal aperture accompanied by a higher transpiration  rate are reported by Livene'and Vaadia (1965) in excised mature barley leaves treated with 10"^M  and 10~^M  kinetin.  Luke and Freeman (1968), using  15  cytokinins (including kinetin), observed the same phenomena in many gramineous, but not in dicotyledonous species. They also suggested that the increased transpiration of species of Gramineae should be considered as one of the biological activities specific to cytokinin.  Besides, i t was  noticeable that the kinetin effect on stomata was relatively quick in comparison with the other known effects caused by kinetin (which always showed a time lag in the order of several hours). This increase in opening of the stomatal aperture discussed above (Section B.3.) might be one explanation for a higher rate of transpiration caused by kinetin and other cytokinins.  B. 5. Effects of kinetin on other physiological behavior Kinetin possessed the capability of retarding leaf abscission in Phaseolus (Chatterjee and Leopold, 1964). Wade and Brady (1971) found that, in transverse slices of green banana, kinetin hastened the peak of ethylene evolution and maximum rates were also 30% higher than the control.  The  respiration rate of kinetin-treated slices was found to exceed that of the control throughout the 48 hour period after slicing; peel degreening was also retarded. Street et al_. (1967) found that the growth response of cultured sycamore cell suspensions to added kinetin depended on adequate carbohydrate (glucose) as the source of carbon energy.  C.  Interaction of abscisic acid with kinetin, and with other hormones Aspinall et al_. (1967) exposed lettuce seed to far-red light in the  presence of ABA and GA^, or kinetin, and found that the effect of low concentration of ABA in suppressing GA^-promoted germination was completely overcome by a high concentration of GA~ and, in the case of kinetin, ABA  16  was inhibitory only in the presence of a high concentration of this promoter.  Khan (1967a) found that kinetin reversed the action of ABA  inhibition of germination in lettuce and nondormant seed of Xanthium; also dormancy breaking action of kinetin on dormant seed of Xanthiurn was found to be affected by ABA.  Khan (1968) found that an inhibitory effect of ABA on  dark germination of Grand Rapids lettuce seed was reversed by kinetin but not by excess GA3#  Sankhla and Sankhla (1968b) showed that inhibition of  seed germination caused by ABA was completely overcome by kinetin in both dark and light, whereas gibberellie acid and IAA showed no interaction with ABA. Auxin-mediated growth of Avena coleoptile was found to be inhibited by ABA (Addicott  1964, cited by Aspinall ___!_•  Thomas e_t al_.  (1965) demonstrated that such an inhibition could be overcome by GAg but not by auxin, although the coleoptiles were responsive to auxin in the presence of ABA.  They also found that ABA reduced the elongation of tall  (but not dwarf) maize leaf sections, and GA^ could overcome this effect. Aspinall e_t al_. (1967) found that elongation of cucumber radicle, on the other hand, was promoted by ABA in the presence of a mixture of GA^ and GA^. Khan and Downing (1968) reported an inhibitory effect of ABA on the growth of barley coleoptile and the effect was reversed by kinetin.  On the  contrary, a synergistic inhibition of root growth was observed as affected by the combinations of kinetin and ABA.  Khan (1969) also demonstrated that  ABA inhibited coleoptile growth to a greater extent than the root growth, and although the increase in coleoptile growth by gibberel1 in plus ABA over ABA alone was observed, he did not think there was an interaction effect.  Blumenfeld and Gazit (1970) reported that, in soybean callus  culture, ABA (10 mg/1) acted as inhibitor when the kinetin level was low, but this inhibition was cancelled and changed to synergism when the kinetin  17  level in the medium was raised.  They stated that both the absolute  quantities and ABA-kinetin ratio were important in the transition from inhibition to synergism. Pi let (1970) found that ABA inhibited the growth of lentil roots, but the effect was less noticeable than for IAA, and when both chemicals were applied simultaneously, ABA acted as a growth antagonist to IAA.  IAA was found to have a synergistic effect on ABA-induced callus  formation in the culture of citrus explants while ABA was much less effective.  IAA and ABA alone or in combinations induced no callus formation  in the absence of ABA (Altman and Goren, 1971).  Blaydes found that ABA  inhibited elongation of Avena coleoptile and the inhibition was lessened by kinetin. Chrispeel and Varner (1967) found that GA enhanced the synthesis of a-amylase and ribonuclease in isolated aleurone layers of barley, and this process was inhibited by ABA.  They suggested that ABA might exert its  action by inhibiting the synthesis of a-amylase-specific RNA molecules or by preventing their incorporation into an active enzyme-synthesizing unit. Khan and Downing (1968) found that GA was far less effective than kinetin in reversing ABA inhibition of  a-amylase synthesis in intact seed of barley,  and a combination of GA and kinetin caused nearly complete reversal of ABA inhibition of a-amylase synthesis.  They suggested that kinetin might act  by removing the ABA inhibition of enzyme specific sites thereby allowing GA to function on a-amylase synthesis.  Khan (1969), working on both intact  and embryoless seeds, found that kinetin effectively reversed inhibition of a-amylase by ABA, but there was no reversal effect caused by excess GA or kinetin in the embryoless endosperm, thus cytokinin reversal of inhibition of enzyme synthesis probably depended on some factor(s) in the embryo. Srivastava (1968) found that ABA increased the chromatin-associated nucleases in excised barley leaves, and kinetin completely reversed the ABA  18  effect with results comparable to the activity of these enzymes of barley leaves floated on solutions of kinetin alone.  Pi 1et (1970) found  that the IAA-induced RNA accumulation and inhibition of ribonuclease activities were reversed by ABA.  Khan e_t __]_. (1970) showed that ABA  induced  changes in the nucleotide composition of rapidly labelled RNA species in excised lentil roots, and that the effect was reversed by kinetin.  De Leo  and Sacher (1970) found that ABA increased ribonuclease activity and inhibited the incorporation of uridine and leucine in leaf sections removed from plants grown under stress, and these effects were suppressed by  NAA.  A study of RNA synthesis in the Avena coleoptile by Blaydes (1971) showed that ABA decreased RNA synthesis (as measured by the incorporation of radioactive uracil and adding kinetin lessened the inhibition). found that ABA inhibited the increase of  Yomo (1971)  a-amylase and 3-amylase a c t i v i t i e s ,  but not of ^C-leucine incorporation or the respiration of excised bean and pea cotyledons during incubation. The inhibition was not reversed by kinetin, GA, or IAA. Aspinall e_ al_. (1967) found that high concentrations of kinetin overcame the capability of ABA to hasten senescence in radish leaf disks. Bhardwaj (1967) found the acceleration of abscission by ABA to be counteracted almost completely by IAA and to a lesser extent by GA3.  Sankhla and  Sankhla (1968a) also demonstrated that kinetin reversed the senescence accelerating effect of ABA on both leaf disks and whole leaves of Arabidopsis. Srivastava (1968) found the same kind of interaction between kinetin and ABA in excised barley leaves.  Gamborg and La Rue (1971) found, in cell  culture of rose and Ruta, that ABA inhibited growth and ethylene production in rose cells but only ethylene production in Ruta cells; and the addition of kinetin reversed the ABA inhibitory effect in rose cells but not in Ruta  19  cells.  Glinka (1971) found that ABA raised the permeability of tissue  while kinetin decreased i t and the effect of ABA dominated the system when both chemicals were applied simultaneously.  Concerning ethylene production  in plants, Lieberman and Kunishi (1971) found that ABA suppressed the IAAand kinetin-induced stimulation of ethylene evolution in etiolated pea seedlings.  20  MATERIALS'AND METHODS A. Materials Preceeding the present study, a small preliminary test had been carried out to investigate the effects of kinetin and ABA on the postharvest quality of 5-week-old "Great Lakes" lettuce rosettes.  The plants  were cut, trimmed, and treated with 3 separate-,solutions; distilled water, 20 ppm kinetin, and 5 ppm ABA.  The lettuce rosettes were dipped in the  specified solution for one minute then allowed to drain and kept in a cold storage chamber with the temperature setting at 3+1°C (no supplementary humidification). no replication.  Twelve plants were used for each treatment and there was Post-harvest quality was observed once a week up to 5 weeks  in storage, and the results revealed that the 5 ppm ABA-treated plants remained fresher and greener in comparison with the control and 20 ppm kinetin-treated plants.  The results seemed encouraging for a further  study of the potential use of these chemicals for retention of the fresh appearance of lettuce. The interest of the present study was directed toward the uses of ABA (and/or kinetin) in preserving the post-harvest quality of lettuce in actual practice. .".Nevertheless, the present study could not be carried out to the fullest extent for 2 reasons.  F i r s t l y , the head lettuce industry uses the  Great Lakes variety which in the Lower Mainland of British Columbia requires about 10 weeks from seeding to edible maturity in the growing season.  That  time period increases when lettuce is grown in the off-season in greenhouses. For example, even with supplementary lighting, i t takes 7 weeks to reach the 10-leaf rosette stage, and the plants are s t i l l weeks away from head formation. Thus, i t was expedient to use young plants in order to conserve time.  Secondly, only a limited amount (25mg) of ABA was available at the  21  time and this was not enough to establish an experiment using fully mature lettuce heads.  Also, the chemical is very expensive.  The antitranspirational activity of this chemical has not been confirmed from any vegetable crops, thus, with the need to economize on time and quantity of ABA, i t was logical at this i n i t i a l stage to use a small laboratory model to gain more evidence before considering experiments in field production.  A.l.  Test plants The experiment was undertaken at The University of British Columbia  from October 26, 1972 to February 3, 1973 at which time the field growing of lettuce was not feasible. conditions.  The plants were instead grown under greenhouse  "Great Lakes" head lettuce (Lactuca sativa L. var. capitata, L.)  seeds were sown in steam-sterilized soil in 3" x 12" x 18" wooden flats on two different days ( October 26 and 28, 1972) to provide 2 sets of seedlings for two replications.  The plants were grown under a temperature setting of  20°C by day and 18°C by night and approximately 800 lux of supplementary light from fluorescent light banks 16 hours a day.  Three weeks after  sowing, seedlings were transplanted into standard flats using 2V1 x 2%" spacing.  Watering was done once a day and no f e r t i l i z e r , pesticide or  herbicide was used throughout the growing period.  Because of the limiting  factors of time and supply of chemicals as previously described, the plants were harvested when 7 weeks old, at which time they had already formed about 10 true leaves and weighed an average of 2.94 gm.  No watering was done on  the day of harvest to avoid possible inaccuracy in weight due to the extra water that might adhere to the leaf surface of the plants;  Plants were  harvested in the evening by cutting at root level just below the soil surface.  Cotyledons, the f i r s t and the second outer leaves, and undesirable  22  root portions were trimmed away. Foreign matter and soil were eliminated by means of a soft brush.  A.2. Chemical treatments Concentrations of 1 ppm and 5 ppm ABA were chosen since they were within a range comparable to that used by previous researchers studying the effects of ABA on plant transpiration and stomatal activities. The selection of 20 ppm kinetin concentration parallels the study by El-Mansy (1967) on lettuce post-harvest quality.  Finally, to study possible inter-  action of the two chemicals, a combination of 20 ppm kinetin plus 5 ppm ABA was used. Aqueous solutions of ABA, kinetin or a combination of the two were made from the anhydrous forms of ABA and kinetin (bought from Sigma Chemical Company, St. Louis, Mo., U.S.A.). The chemicals were f i r s t made into stock solutions of 10 ppm ABA and 40 ppm kinetin, and then diluted to 500 ml each of the following with the corresponding designated abbreviations: Treatment  Abbreviation  1. Control (distilled water)  0  2. 1 ppm ABA  Al  3. 5 ppm ABA  A2  4. 20 ppm kinetin  K  5. 5 ppm ABA + 20 ppm kinetin  A2K  The diluted solutions were prepared in the morning and kept in 500 ml flasks wrapped with aluminum foil and stored in a refrigerator (approximately 4°C) until the time of application in the evening of the same day. The solutions were sprayed on the plants which were spread on plastic sheets. The plants were sprayed thoroughly to the run-off point and let drain before they were shifted into cold storage. The spray application was  23  chosen in preference to the dipping procedure used in the preliminary experiments, to conserve the chemical solutions, and thus permitting larger numbers of plants within treatments.  A. 3.  Cold storage f a c i l i t y and instruments A Bell-Craft walk-in growth chamber was used as a cold storage  facility.  This chamber could be manipulated for conditions ranging from  -20°C to 50°C approximately. Temperature for the experiment was maintained between 3° and 5°C. Humidity was kept as high as possible up to the saturation point using continuous humidification; a certain amount of water was fed into the humidifier via the pre-adjusted regulator valve.  A thermo-  hygrograph was placed in the chamber for continuous recording of temperature and humidity during the experiment. meters were used as a further check.  Ordinary dry-bulb and wet-bulb thermoThe cold storage was tested and  adjusted to meet the required conditions until there were 3 days of steady and reliable performance.  B. Methods In addition to the quality observed by a panel, the study sought to investigate any correlation that might exist between this quality rating and other measurable phenomena which occurred during the post-harvest period, e.g. percent weight loss, percent moisture content, and chlorophyll content, since these might relate to the wilting and yellowing of the stored lettuce. follow.  Details of the experimental procedures and measurements  24  B.I.  Experimental design A s p l i t plot design was employed. The experiment was composed of  3 main plots (40 plants each) which were randomly designated to be kept for 3 different lengths of time (5, 6 and 7 weeks) under storage.  Each main  plot was then subdivided into 5 groups of eight plants each and these groups were randomly assigned to the 5 different treatments. The experiment was replicated twice and each used plants from only one of the two  B.2.  seedings.  Treatment procedure Randomization was exercised throughout the experiment wherever  applicable.  One of four flats of one seeding was randomly selected and  set aside, and the entire population of 120 plants in the remaining 3 flats was used as one replication.  In order to minimize physiological differences  between plants within a treatment, plants were harvested in lots of 40. Each plant within the lot was weighed rapidly before being labelled and treated.  Thus, time lapses between i n i t i a l harvest and final weighing were  reduced by using the lot of forty plants rather than harvesting the entire replication at one time. The longest lapse of time occurred in the weighing of individual plants. A pre-arranged randomization scheme was used to get plants distributed within a replication considering length of storage time, chemical treatment and plant number, and thence to determine where each plant was placed in the storage chamber. Plants were spread on 5 separate plastic sheets according to the groups to which the plants were assigned.  Each group was then sprayed with the treatment solution  (distilled water in the case of the control) up to the run-off point, then allowed to drain before being placed in the cold storage.  The same  procedure was repeated for the second and third sets (40 plants each), which represented the second and third main plots respectively.  25  The second replication was handled in the same manner using the succeeding sowing.  B.3. Visual quality rating Rating of plants was done by a panel of 3 observers.  The plants  examined at the end of weeks 2, 3 and 4 in storage were used again at the end of 5, 6, and 7 weeks in storage in the following manner. Five groups of eight pi ants(under 5 different treatments) with the same length of time in storage were inspected at a time.  Numerical values  were given to those groups for a pooled or group quality manifestation according to the following scheme. Numerical Rating  Quality Description  9  excellent: field fresh, bright green appearance, free from a l l defects.  7  good:  green colour slightly decreased, s t i l l good retail sale appeal.  5  fair:  slightly wilted, some minor defects.  3  poor:  severely wilted, unsaleable.  1  very poor: some decay, yellowing, would not be eaten.  In addition to the numerical record of quality, a representative plant from each treatment was photographed using a 35 mm single lens reflex camera. All settings (exposure time, aperture, distance) were fixed and all pictures were taken on the same roll of colour film.  Plants subjected  to photography were s t i l l kept continuously under the cold and humid experimental conditions and were disturbedtci only by a slight touch during arrangement, since the photography was done in the same cold storage chamber. The chamber also served as a light-seal studio and helped eliminate a l l sources of light except the electronic flash equipment on the camera.  26  Domestic 110 volts A.C. electricity supplied power to the flash unit.  Thus,  i t was assumed that there was a fairly constant illumination so that the photograph can be used as a valid record of visual comparison.  B.4.  Measurement of weight loss Each plant was weighed, as described already, at harvest. Then fresh  weights were obtained after the storage periods. A modified styrofoam case was used to provide a low temperature and humid condition for the plant sample during transfer from the cold chamber to laboratory. A twolayer screen box was put in the middle of the styrofoam container and surrounded with at least 1%" layer of crushed ice in the bottom and a l l side-walls. The two-layer screen box provided good circulation of cold air and separated the samples from the melting ice.  Plants were taken  individually from the case and were rapidly weighed. Fresh weight after storage and original fresh weight were subsequently used to calculate the percent weight loss for each plant.  B.5.  Moisture content measurement Following the recording of fresh weight of a plant after storage, the  fourth leaf (counting in spiral order from the outside in) of that plant was detached at a petiole base and kept in a plastic weighing dish with its identification tag attached. extraction.  This leaf was set aside for chlorophyll  The remaining portion of the plant was then weighed again for  a fresh weight before drying. This portion was placed in a pre-labelled position in an aluminum foil tray. to be dried simultaneously. 15 hours at 70°C.  Five trays were used for all 40 plants  Samples were placed in a vacuum dryer for  27  Samples were removed from the dryer one tray at a time, and each dried plant was weighed as quickly as possible (the remaining samples were s t i l l in the dryer with the heater on but no more vacuum). Fresh weight before drying and the dry weight were used to calculate the percent moisture content.  B.6.  Chlorophyll content measurement The leaf samples from individual plants were used to measure the  chlorophyll content in the following manner. A half gram sample  was cut  from the mid section of each leaf (eliminating leaf tip and base). Each sampleswas placed in an osterizer for Ih minutes with approximately 30 ml of refrigerated-cold 80% acetone to yield a crude extract which was then filtered through 2 layers of Whatman No. 1 f i l t e r paper in a modified suction filtration apparatus as shown in Figure 3. Additional acetone was used to wash down the chlorophyll left on the f i l t e r papers and funnel to make up a final volume of 50 ml f i l t r a t e .  The apparatus allowed the  f i l t r a t e to flow directly into the 50 ml volumetric flask thus bypassing a few steps of the conventional method (that is no removal of stopper and funnel from the suction flask, no transferring and using acetone to wash down the f i l t r a t e from the suction flask into a volumetric flask and replacement of equipment to handle the next sample). This procedure made more efficient use of the acetone in that a larger volume was available for extraction and efficient washing of extract into the collective volumetric flask.  The modified procedure allowed a large reduction in  surface area of f i l t r a t e when the volumetric flask was used and thus lessened evaporation of the highly volatile acetone due to exposure to low pressures during f i l t r a t i o n .  The volumetric flask containing  28  Figure 3 Modified vacuum filtration apparatus  chlorophyll extract was then plunged in crushed ice until the absorbancy measurement was made. Liquefaction and filtration were done in groups of 5 samples and each group was handled as quickly as possible. A Perkin-Elmer double bean spectrophotometer (Model 124) was used to obtain the absorbancy measurements. Samples were read in a s i l i c a cell at 647, 664 and 700 my wavelengths with s l i t size of 0.5 my.  Chlorophyll  contents were calculated using the equations given by Ziegler and Egle (1965) as cited by Sestak (1971) in the following:  29  Chlorophyll A  =  (11.78 A g64 - 2.29 A g 4 ? ) mg/1  Chlorophyll B  = (20.05 A g 4 7 - 4.77 A g 6 4 ) mg/1  Chlorophyll A+B = (7.01 A g 6 4 + 17.76 A g 4 7 ) mg/1 The results were subsequently calculated and are reported as mg of chlorophyll per gm fresh weight of leaf at the time of chlorophyll observation (designated as mg/gm FW), and also are reported as a mg of chlorophyll in mg per gm original fresh weight (designated as mg/gm OFW) the fresh weight immediately before the same leaf sample had been treated and put in storage. The content based on gm FW is more or less parallel to the greenness of the leaf tissue, while the other based on gm OFW, is for the purpose of following the chlorophyll degradation with time.  Using the  OFW basis, the effect of weight loss is used in calculating chlorophyll contents, and these derived values make a common basis between original and subsequent determinations in spite of tissue shrinkage during the experiment. Hence the chlorophyll values on OFW basis measured at different periods of time, reveal the real picture of the possible degradation of chlorophyll from the original 1 gm samples regardless of the shrinkage due to weight loss.  30 RESULTS  1. Visual quality rating The numerical values for lettuce quality were derived from the visual ratings made by the panel of observers, and only the values obtained after 5, 6 and 7 weeks in storage are presented.  The panel made quality  ratings at the end of 2, 3 and 4 weeks in storage, and the same plants were utilized in the same sequence for the 5, 6 and 7 week storage periods.  The f i r s t cycle of observations for weeks 2, 3 and 4 showed no  significant differences for treatments, and the inclusion of such data with the second cycle of observations presented a problem in statistical methods which would not permit sensible comparisons: therefore, data for weeks 2, 3 and 4 were omitted, and the values for weeks 5, 6 and 7 only were used to demonstrate the differences observed by the panel. It is obvious in Table 1A that the quality of lettuce was decreasing as the storage period continued from 5 to 7 weeks. At the end of 7 weeks, all lettuce had reached an unsaleable condition with obvious yellowing of leaf tissue and severe wilting.  The Duncan's multiple range test  at the 5% level (Table IB) shows no significant difference in quality between two replications, but time in storage and chemical treatment did have some effect on quality.  After 5 weeks in storage, quality was  significantly higher than that for 7 weeks but not for the 6 weeks of storage.  The control and the Ippm ABA-treated lettuce had a significantly  higher quality than the 20 ppm kinetin and 5 ppm ABA + 20 ppm kinetin treatments.  The 5 ppm ABA treatment did not differ significantly from any  of the other treatments.  31 Table 1A Means of quality ratings''".by a panel of 3 observers, of lettuce subjected to 5 treatment^, after 5, 6 and 7 weeks in storage Week Replication  Treatment . . . . . .  r l c d il  5  1 2  8.0 7.8  Al 7.8 8.7  6  1 2  6.7 6.2  6.0 6.2  4.3 5.7  3.8 4.0  4.2  7  1 2  3.3 3.6  3.8  3.7  3.8  3.3  4.0  3.0  5.0 •  4.0.  5.9  6.1  5.5  5.0  5.1  Mean  A2 8.5 7.7  K 7.3 6.2  A2K 7.7 6.2  Mean of replication 1 =5.4889  7.6 5.3  5.3  '5.5 5.5  Mean of replication 2 = 5.5667  Numerical ratings: 1 = very poor, 3 = poor, 5 = f a i r , 7 = good, " _ . ; 9 = excellent Treatments: 0 = control, Al = 1 ppm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin, A2K = 5 ppm ABA + 20 ppm kinetin Table IB Analysis of variance of numerical quality ratings of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F.  Replication Week (A) Error a Treatment (B) Week x Treatment (AB) Error b Error TotaldS  1 2 2 4 8 12 60 89  Duncan's test Replication 1 Mean 5.48 a * Time under cr O storage (weeks) Mean 7.6 b Treatment Mean  0 5.9 b  S.S.  . M.S.  0 .136 0.136 231 .810 115.900 5 .739 2.869 16 .361 4.090 15 .056 1.882 10 .417 0.868 51 .667 0.861 331 .180  F 0.16 40.39 3.33 4.71 2.17 1.01  Prob. 0.6936 0.0348 0.0414 0.0163 0.1095 0.4533  2 5.6 a c O  "7  1  o5.3 b . Al 6.1 b  3.7 a \~>  A2 5.5 ab  K ....5.0.a.  A2K 5.1 a  * Mean separation in row by Duncan's multiple range t e s t , 5% level  32  2. Total weight loss Changes in weight of lettuce after 5, 6 and 7 weeks in storage were measured and calculated as percentage of total weight loss (% TWL). The record of these 240 observations is shown in Appendix 2. The data shown in Table 2A areaave.rag.es of 8 Observations in the experimental unit. The comparisons among., the 5 treatment means show only small and nonsignificant differences.  The more obvious ones are % TWL of lettuce in the f i f t h week  in comparison with the sixth or the seventh week of time under storage. The lettuce had a relatively lower percent weight loss in the f i f t h week than in the sixth or the seventh week. There was a very small difference between the latter two weeks. The analysis of variance shown in Table 2B reveals no significant differences in the % TWLiat the 5% level between the replications, or among the treatments and different periods of time under storage Table 2A Percentages,: of total weight loss (means of 8 observations in each experimental lot) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage^" ~a.: J' •"z r..:i jndc • 3 •"• .o-'z. "it-.r- 5 G i >J • •' . B  storag 2 Week "Replication  Treatment 0  Al  A2  M r**n n  K  A2K  1  -0.809  5.660  -5.716  -1.030  1.789  2  7.844  4.061  8.407  '9.540  11.106  1  14.146  17.968  20.731:  20.707  12.164  2  15.690  15.121  15.800  il.625  14.738  1  21.005  22.724  16.384  16.075  14.101  2  17.183  12.694  10.522  14.506  18.704  12.510  13.038  11.021  11.904  12.101  rlccul  5  4.085  6  15.869  7 Mean  16.390  Mean of.replication 1 =11.727  12.115  Mean of replication 2 = 12.503  Treatments: 0 = control, Al = 1 ppm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin, A2K = 5 ppm ABA + 20 ppm kinetin  33  Table 2B Analysis of variance of total weight loss of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F.  Replication 1 Week (A) 2 Error a 2 Treatment (B) 4 Week x Treatment (AB) 8 Error b 12 Error 210 Total 239  S.S.  M.S.  36.14 36.14 7747.60 3873.80 1665.30 832.66 107.94 29.98 627.52 78.44 1388.70 115.73 4569.40 21.76 16143.00  F  Prob.  1.66 4.65 388277 0.23 0.68 5.32  0.1958 0.1795 0.0000 0.9132 0.7043 0.0000  3. Moisture content Moisture content was fairly uniform for all the lettuce plants regardless of replications, treatments or different lengths of time under storage.  The data for these observations are in appendix 2, and the means  for eight-plant experimental units are in Table 3A. Uniformity can be observed throughout for every treatment and every week. The overall mean of 240 observations is 94.46% with a standard deviation of 0.6630%. The analysis of variance (Table 3B) showed no significant differences at the 5% level for replications, treatments, or times under cold storage.  34  Table 3A Percentages of moisture content (means of 8 observations in each experimental lot) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Week  5  6  Replication  Treatment 0  r i c a 11  A2K  K  1  95.33  Al 94.76  A2 95.44  95.36  94.99  2  94.66  94.97  94.65  94.62  94.61  1  94.35  93.88  93.22  93.78  94.50  2  94.21  94.32  94.41  94.44  94.53  1  94.02  93.72  94.26  94.56  94.48  :.94.02  94.53  94.27  94.42  94.35  94.43  . 94.37  94.38  94.53  94.58  94.94  94.17  7 Mean  94.26 94.46  Mean of replication 2 =94.47  Mean of replication 1 =94.44  pm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin Treatments: 0 = control, Al = 1 p| A2K = 5 ppm ABA + 20 ppm kinetin  Table 3B Analysis of variance of percent moisture content of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F.  Replication 1 Week (A) 2 ErroY a 2 Treatment (B) 4 Week x Treatment (AB) 8 12 Error b Error 210 Total 239  S.S.  M.S.  F  0.035 28.265 8.483 1.688 5.655 9.695 51.253 105.070  0.035 14.132 4.242 0.422 0.707 0.808 0.244  0.15 3.33 17.38 0.52 0.87 3.31  Prob. 0.7020 0.2320 0.0000 0.7232 0.5629 0.0002  35  4. Chlorophyll content Measurements and calculations of the contents of chlorophyll A and B and the total were based on two different fresh weights: 1 . one gm fresh weight of the sample leaf at the time of chlorophyll extraction after chemical and storage treatments. 2. one gm of the original fresh weight (before that same sample was treated and put into the storage). The above two fresh weights are differentiated hereafter as gm FW and gm OFW A+B  respectively.  Analyses of variance for chlorophyll A, B and  (appendices 4-9) were done on each of those two bases, and the  results are summarized in Table 4B to 9B inclusive. (Tables 4A to 9A are means from 8 plant experimental l o t s , shown in appendices  4-9).  36  Table 4A Chlorophyll A contents (means of 8 observations in each experimental lot) in mg/gm FW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Week ^ e P^ l c a " t l 0 n  0  Al  Treatment A2  mean K  A2K  1  0.3409  0.5012  0.4229  0.4385  0.4734  2  0.5356  0.4730  0.4822  0.5769  0.5960  1  0.4421  0.5229  0.5576  0.5661  0.5333  2  0.5328  0.4858  0.4836  0.5785  0.5435  1  0.4729  0.4055  0.4131  0. 481:6  0.4676  2  0.4799  0.4103  0.4798  0.5710  0.5197  0.4673  0.4664  0.4732 . 0.5354  0.5223  5  0.4840  6  0.5246  7 Mean  0.4702  Mean of replication 1 =014693  0.4929  Mean of replication 2 = 0.5166  Treatments: 0 = control, Al = 1 ppm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin, A2K = 5 ppm ABA + 20 ppm kinetin  Table 4B Analysis of variance of chlorophyll A content (mg/gm FW) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F.  Replication 1 Week (A) 2 Error a 2 Treatment (B) 4 Week x Treatment (AB) 8 Error b 12 Error 210 Total 2393?  S.S.  M.S.  F  Prob.  0.1341 0.1281 0.0943 0.2118 0.0870 0.1996 1.8367 2.6915  0.1341 0.6403 0.0472 0.0530 0.0109 0.0166 0.0087  15.33 1.36 5.39 3.18 0.65 1.90  0.0002 0.4239 0.0054 0.0531 0.7219 0.0356  37  Table 5A Chlorophyll A contents (means of 8 observations in each experimental lot) in mg/gm OFW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Week  Replica^, tion  5  Treatment K  r i c a ii  1  0 0.3442  Al 0.4690  A2 0.4498  0.4417  A2K 0.4663  2  0.4957  0.4542  0.4423  0.5235  0.5307  1  0.3823  0.4297  0.4440  0.4493  0.4671  2  0.4417  0.4122  0.4093  0.5124  0.4628  1  0.3740  0.3160  0.3475  0.4030  0.4036  2  0.3984  0.3599  0.4281  0.4884  0.4225  0.4060  . 0.4068  0.4202  0.4697  0.4588  0.4617  6  7 Mean  0.4411  0.3941  Mean of replication 1 =0.4125  0.4323  Mean of replication 2 = 0.4521  Treatments: 0 = control, Al = 1 ppm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin, A2K = 5 ppm ABA + 20 ppm kinetin  Table 5B Analysis of variance of chlorophyll A content (hig/gm OFW) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F.  Replication 1 Week (A) 2 Error a 2 Treatment (B) 4 Week x Treatment (AB) 8 Error b 12 Error 210 Total 239  S.S.  M.S.  CT  Prob  0.0942 0.1919 0.0211 0.1724 0.0464 0.1237 1.6207 2.2704  0.0942 0.0960 0.0106 0.0431 0.0058 0.0103 0.0077  12.21 9.08 1.37 4.18 0.56 1.34  0.0007 0.1053 0.2553 0.0240 0.7897 0.1998  38  Table 6A Chlorophyll B contents (means of 8 observations in each experimental lot) in mg/gm FW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Week  Replication  5  Treatment II c a 11  0  Al  A2  1  0.1735  0.2613  0.2110  0.2188  0.2405  2  0.2713  0.2550  0.2369  0.2963  0.2915  1  0.2615  0.3170  0.3049  0.3234  0.2973  2  0.2884  0.2649  0.2540  0.3012  0.2853  1  0.2641  0.2183  0.2133  0.2577  0.2450  0.2759  0.3118  0.2843  0.2493  0.2849  0.2740  K  A2K 0.2456  0.2898  6  0.2551  7  .1 ...0.2534 . 0.2275 Mean  0.2520  0.2573  Mean of replication 1 = 0.2538  0.2635  Mean of replication.2.= 0.2732  Treatments: 0 = control, Al = 1 ppm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin, A2K = 5 ppm ABA + 20 ppm kinetin .  Table 6,B Analysis of variance of chlorophyll Bcc6ntenfo(mg/;gm^F/W:). of-lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F.  Replication 1 Week (A) 2 Error a 2 Treatment (B) 4 Week x Treatment (AB) 8 Error b 12 Error 210 Total 239  S.S.  M.S.  F  0.0224 0.0865 0.0549 0.0450 0.0301 0.0594 0.8173 1.1156  0.0224 0.0432 0.0275 0.0112 0.0038 0.0050 0.0039  5.77 1.57 7.05 2.27 0.76 1.27  Prob. 0.0164 0.3882 0.0012 0.1213 0.6436 0.2364  39  Table 7A Chlorophyll B contents (means of 8 observations in each experimental lot) in mg/gm OFW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Week  Replication  Treatment 0  Al  A2  1  0.1750  0.2436  0.2249  0.2187  0.2366  2  0.2515  0.2450  0.2166  0.2687  0.2594  1  0.2255  0.2595  0.2432  0.2566  0.2600  2  0.2386  0.2248  0.2150  0.2665  0.2428  1  0.2090  0.1700  0.1795  0.2156  0.2112  2  0.2103  0.1994  0.2462  0.2668  0.2319  0.2183  0.2237  0.2209  0.2488  0.2403  K  A2K  5  0.2340  0.2432  6  0.2140  7 Mean  Mm n ii ecu i  Mean of replication 1 .=0.2219  0.2304  Mean of replication 2 = 0.2389  Treatments: 0 = control, Al = 1 ppm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin, A2K = 5 ppm ABA + 20 ppm kinetin  Table 7.B Analysis of variance of chlorophyll B content (mg/gm OFW) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F.  Replication 1 Week (A) 2 Error a 2 Treatment (B) 4 Week x Treatment (AB) 8 Error b 12 Error 210 Total 239  S.S.  M.S.  F  Prob.  0.0172 0.0358 0.0245 0.0345 0.0200 0.0377 0.6856 0.8553  0.0172 0.0179 0.0122 0.0086 0.0025 0.0031 0.0033  5.28 1.46 3.75 2.74 0.80 0.96  0.0214 0.4060 0.0246 0.0782 0.6180 0.4861  40  Table 8A Chlorophyll (A+B) contents (means of 8 observations in each experimental lot) in mg/gm FW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Week  Replication  Treatment 0  Al  A2  1  0.5143  0.7624  0.6339  0.6573  0.7138  2  0.8068  0.7280  0.7191  0.8733  0.8875  1  0.7036  0.8399  0.8625  0.8895  0.8306  2  0.8211  0.7504  0.7376  0.8796  0.8288  1  0.7370  0.6238  0.6264  0.7393  0.7126  0.7333  0.6379  0.7557  0.8829  0.8041  0.7194  0.7237  0.7225  0.8203  0.7962  K  A2K  0.7296  .5  0.8144  6  0.7253  7 2 Mean  llcO-M  Mean of replication 1 = 0.7231  0.7564  Mean of replication 2 = 0.7897  Treatments: 0 = control, Al = 1 ppm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin, A2K = 5 ppm ABA + 20 ppm kinetin  Table 8B Analysis of variance of chlorophyll (A+B) content (mg/gm FW) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F.  Replication 1 Week (A) 2 2 Error a Treatment (B) 4 Week x Treatment (AB) 8 12 Error b Error 210 239 Total  S.S.  M.S.  F  Prob.  0.2663 0.4035 0.2853 0.4444 0.2155 0.4655 4.7147 6.7951  0.2663 0.2018 0.1426 0.1111 0.0269 0.0388 0.0225  11.86 1.41 6.35 2.86 0.69 1.73  0.0008 0.4140 0.0023 0.0702 0.6918 0.0625  41  Table 9A Chlorophyll (A+B) contents (means of 8 observations in each experimental lot) in mg/gm OFW of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Week ReplIcation tion  Treatment K  A2K  1  0.5192  Al 0.7126  2  0.7471  0.6992  0.6590  0.7922  0.7900  1  0.6077  0.6891  0.6872  0.7060  0.7271  20.6803  0.6369  0.6243  0.7789  0.7056  1  0.5831  0.4861  0.5270  0.6181  0.6148  2  0.6087  0.5593  0.6743  0.7552  0.6543  0.6244  0.6305  0.6411  0.7185  0.6991  o  A2 0.6747  0.6604  0.7029  5  0.6957  6  0.6843  7  0.6081  Mean  Meani7o.f repl ication 1 = 0.6344 Treatments:  0.6627  Mean of.repl ication 2 = 0.6910  0 = control, Al = 1 ppm ABA, A2 = 5 ppm ABA, K = 20 ppm kinetin, A2K = 5 ppm ABA + 20 ppm kinetin Table 9B  Analysis of variance of chlorophyll (A+B) content (mg/gm OFW) of lettuce under 5 treatments after 5, 6 and 7 weeks in storage Source  D.F..  Replication 1 2 Week (A) 2 Error a 4 Treatment !(iB) 8 Week x Treatment (AB) 12 Error b 210 Error 239 Total  S.S.  M.S.  0.1920 0.3629 0.0901 0.3561 0.1251 0.2898 4.0785 5.4945  0.1920 0.1814 0.0451 0.0890 0.0156 0.0241 0.0194  F 9.89 4.03 2.32 3.69 0.65 1.24 .  Prob. 0.0021 0.2008 0.0986 0.0351 0.7270 0.2547  42 Table 10 Tabulation of the means (mg) of chlorophyll A,.:B and A+B contents, based on 1 gm fresh weight (gm FW) and 1 gm original fresh weight (gm OFW), for 2 replications, under 5 treatments, and after 5, 6 and 7 weeks in storage Basis of measurement  OFW  FW  Chlorophyll 1  G„ ,oA3i\  A+B  [1 0.4693A1 Repl icatiom 12 0.5166B  0.2538a  0.7231A  0.4125A  0.2219a  0.6344A  0.2732b  0.7897B  0.4521B  0.2389b  0.6910B  5  0.4840ad  0.2456a  0.7296a  0.4617a  0.2340a  0.6957a  6  0.5246a  0.2898a  0.8144a  0.4411a  0.2432a  0.6843a  17 0.4702a  0.2551a  0.7253a  0.3941a  0.2140a  0.6081a 0.6244a  Time in storage (weeks)  B  A+B  0Z5B3e.  0  0.4673!3  0.2520a  0.7194!,  0.4060a  0.2183!  Al  0.4664.§.  0.2573a  0.72371  0.4068a  0.2237!. 0.6505ab  0.47321  0.2493a  0.7225^  0.4202ab  0.2209§;  0.6411ab  K 0.5354&  0.2849a  0.8203S  0.4697c  0.2488!  0.7185c  0.5223t\ji 0.2740a  0.7962S  0.4588bc  0.24031  0.6991bc  Treatment • A2  A2K  Meanr-separation in column by Duncan's multiple range test: 1 separation by upper case letter- significant at 1% level 2  separation by lower case letter- significant at 5% level 3 separation by Gr§;e< symbol- approaching the b% level of significance Treatments: 0 = control Al = 1 ppm ABA A2 = 5 ppm ABA K = 20 ppm kinetin A2K = 5 ppm ABA + 20 ppm kinetin  43 Chlorophyll A, B and A + B contents of replication 2 were significantly higher than that of replication 1 in a l l respects regardless of pigment or basis of measurement, but the differences in chlorophyll A and total chlorophyll (A + B) contents were highly significant at the 1% level, whereas differences in chlorophyll B contents were significant at the 5% level. There were small differences among the contents of chlorophylls in lettuce kept under storage for 5, 6 and 7 weeks in both gm FW and gm OFW bases, but the differences were not significant.. Nevertheless, the two bases showed different characteristics as illustrated in Figure 5 where the content based on gm OFW showed a gradual decrease of contents from week 5 to week 7, whereas the other base (gm FW) had high contents at week 6 and lower contents at weeks 5 and 7. Treatment effects were revealed only in cases of chlorophyll A and A + B contents as measured on gm OFW basis, and some of the differences were significant at the 5% level.  In the case of chlorophyll B, some of  the differences approached the 5% level of significance. Considering chlorophyll A content per gm OFW, there was a significantly higher content in the 20 ppm kinetin treatment than in the lppm ABA, 5 ppm ABA, and the control, but not significantly higher than the 5ppm ABA + 20 ppm kinetin treatment.  The 5 ppm ABA + 20 ppm kinetin treatment had  a higher chlorophyll A content than 1 ppm ABA and the control, but the difference between the 5ppm ABA + 20 ppm kinetin and 5 ppm ABA alone was not significant. The total chlorophyll (A + B) contents per gm OFW showed the same response to the treatments as did chlorophyll A alone with the exception that the combination treatment of 5 ppm ABA + 20 ppm kinetin was not significantly different from either of the ABA treatments.  44  Chlorophyll A contents were roughly twice those of chlorophyll B. Figure 4 shows the relative comparison and also shows the trend of chlorophyll contents under the 5 different treatments which varied from 5 to 7 weeks in storage.  45 Figure 4 Chlorophyll A and B in mg/gm OFW as affected by treatment and storage time  Chlorophyll A  -• iX  X-  • • . ,.#  control 1 ppm ABA 5 ppm ABA 5 ppm ABA + 20 ppm kinetin • 20 ppm-;kinetin  Chlorophyll B  5  6  TIME UNDER STORAGE (WEEKS)  7  46 Figure 5 Chlorophyll (A+B) in mg/gm FW and mg/gm OFW of lettuce under control, 20 ppm kinetin and 5ppm ABA + 20 ppm kinetin treatments at the end of 5, 6 and 7 weeks in storage 0.9  0.8  S 0.7 >—  a. o c_ O _i  c_> 0.64 _x  content based on gm FW  _  content based on gm OFW  0.5  TIME UNDER STORAGE (WEEKS)  5. Correlation and simple linear regression The data were further studied using correlation and linear regressions. Only results which were deemed useful are presented. are three correlation and regression values.  In each case, there  One for the total experiment  using 240 pairs of observations and the other two are for the individual replications each employing 120 paired observations.  5.1. Chlorophyll A and chlorophyll B contents Correlations of chlorophyll A with chlorophyll B within the same leaf sample showed very high correlation coefficients, as can be seen in  47  the r  (coefficient of determination) values in Table 11. Arbitrarily  designating chlorophyll A as the dependent variable X, and chlorophyll B as the independent Y, the simple linear regression equations were obtained as shown in Table 11.  Table 11 Linear regression equations for chlorophyll B content* (Y) on chlorophyll A content* (X)  F-Prob (b)  Standard error (b) (Y)  (a)  p r  Source  Regression equation  N  2 Reps.  Y = 0.5551X - 0.01014  0.0  0.0107  0.0211  0.0347  0.7435 240  Rep. 1  Y = 0.6480X - 0.05026  0.0  0.0152  0.031)5  0.0356  0.7820 120  Rep. 2  Y = 0.4702X - 0.03028  0.0  0.0143 0.0272  0.0301  0.7167 120  * content in mg/gm FW The regression equations show a very high degree of association between these 2 chlorophylls within the same sample. Nevertheless, the three linear regression equations are not identical.  This means that in spite  of the strong correlation, a different quantity of chlorophyll B in association with a changed quantity of chlorophyll A is different when the affecting conditions are different, as in this,ease of the two replications producing different effects.  5.2. Percent weight loss andystorage time The simple regression equations (Table 12) show a high coefficient of regression (F probability = 0) which means that such a linear relation  48  existed between percent weight loss and the storage time in weeks. The coefficient of determination was not very high in these cases but a l l the equations imply that the percent weight loss of a plant tended to increase with time in storage. Table 12 Linear regression equations of percent weight loss (Y) on storage time in weeks (X)  Source  FrProb. RegressiohQequat,iono;,(b)  standard error (a) (b) (Y)  _ r  N  2 Reps.  Y = 6.152X - 24.80  0.0  3.116  0.5147  6.510  0.3752  240  Rep. 1  Y = 9.040X - 42.51  0.0  4.804  0.7933  7.096  0.5239  120  Rep. 2  Y = 3.265X - 7.09  0.0  3.283  0.5422  4.850  0.2351  120  5.3. Percent moisture content and storage time The equations in Table 13 imply a progressive loss in moisture content of lettuce during storage, but provide no information on possible differences which might exist among the treatments.  49  Table 13  Linear regression equations of percent moisture content (Y) on storage time in weeks (X)  Source  Regression equation  F-Prob. (b)  2 Reps.  Y = 96.48 - 0.3372X  0.000  0.2892  0.0477  0.6042  0.1732 240  Rep. 1  Y = 97.34 - 0.4825X  0.000  0.4556  0.0752  0.6729  0.2584 120  Rep. 2  Y = 95.62 - 0.1920X  0.001  0.3416  0.0564  0.5045  0.0894 120  (a)  standard error (b) (Y)  2  r  N  50  DISCUSSION AND CONCLUSION  Lettuce held at 3_1°C and relative humidity close to 100% in the experiment maintained the marketing quality of lettuce satisfactorily up to 5 or 6 weeks in the storage, regardless of the chemical treatment used. At the end of 6 weeks in storage, the numerical quality rating of a l l lettuce in the experiment averaged 5.3, and in the scale employed, this valued indicated "fair condition".  A severe wilting and yellowing occurred  only in the seventh week. No disease was observed on any lettuce plant throughout the seven week period of storage.  This freedom from disease  might be due to the growing conditions in the greenhouse, the hygenic handling of the specimens and clean cold storage f a c i l i t i e s .  The above  mentioned conditions which are generally recommended for storage of mature lettuce appeared to be favourable for the juvenile, 7-week-old lettuce used in this experiment. Kinetin, which has been shown effective in prolonging the storage and shelf l i f e of various vegetables (as previously described in the literature review) did not result in any significant improvement in lettuce quality as observed by the rating panel.  The other  treatments, 1 ppm ABA, 5 ppm ABA and 5ppm ABA + 20 ppm kinetin showed no effects which the panel could observe. The lettuce lost weight with time, but the percentage of total weight loss varied greatly from plant to plant within the same treatment. All lettuce under 5 different treatments lost an average of 16.4% of its original fresh weight at the end of week 7 in storage.  In contrast with  the great variability in percentage of total weight loss, a l l plants tended to have a percent moisture content around 94.45% (standard deviation of 240 observations = 0.66%) regardless of storage times (5, 6 and 7 weeks)  51  treatments, or the subsequent variability in terms of percent total weight loss.  These particular results, i f not just a coincidence, imply  that a certain relationship and some harmony between the transpiration and other biological processes, particularly respiration, existed so that the plant could maintain i t s level of percent moisture content at about 94.5% throughout the period of 5, 6 and 7 weeks in storage. Apparently, the expected antitranspirant characteristic of ABA showed no beneficial effect under the conditions of this experiment. Other experiments using the same chemical treatments at room temperatures or the conditions normally existing on the shelf of a retail store may be useful because the value of ABA as an antitranspirant was observed by Mittelheuser and Van Steveninck (1969), Jones and Mansfield (1970) in experiments carried out under normal room temperatures. Hofstra and Hesketh (1969) found that the change of air temperature affected stomatal opening and transpiration in various  pjlrahtispecies:'  Stomata closed  and transpiration was reduced at a low temperature (the experiment was carried out in the 15° to 36°Or.ange). Under the conditions of the present experiment, i t was likely that the low temperature of the cold storage affected stomatal activity to favour moisture conservation. This low temperature plus the high relative humidity in the cold storage provided such good storage conditions for the lettuce that the applications of ABA were ineffective and unnecessary. Furthermore, ABA has been reported as highly subject to rapid biological breakdown - an inactivation process (Walton and Sondheimer, 1971; Milborrow, 1970).  Also, most of the previous work on the antitranspirant  effect of ABA was studied under short periods of time such as a few days up to one week, thus i t was possible that the ABA effect did not last as long as 6 or 7 weeks. No attempt was made to investigate the breakdown of  52  ABA in the present study.  The only conclusion is that ABA at the  concentrations used in this experiment was ineffective as a qualitypreservation agent. It is possible that additional ABA was not needed to conserve quality in the present experiment. Wright and Hiron (1969) reported an increase in ABA content in detached wheat leaves induced by wilting; (they also found similar increases in excised leaves of cotton, pea and dwarf bean).  This  phenomenon may reduce the severity of wilting in nature, and similarly ABAtreated lettuce may thus appear to be l i t t l e different from the untreated. High humidity is definitely recommended for storage of young lettuce. In this experiment, extra moisture which sometimes condensed on lettuce leaves did no harm. However, this condition might be questionable i f the subsequent shelf l i f e quality was studied. Keeping relative humdity within a range of 93-95% with no fluctuation to the saturation point, could eliminate excess moisture within a few days.  Certainly the storage of  lettuce is dependent largely on the time lapse between cutting at harvest and being put in a cold storage, and obviously the shortest time lapse is best.  The lack of large differences between treatments and storage time  was undoubtedly due to the rapid placement of freshly harvested lettuce in high humidity storage. The chlorophyll analyses showed roughly av 2.ctdl!ratio of chlorophyll A to chlorophyll B.  Regression equations of chlorophyll B on A (Table 11)  show a high association between these two substances.  Nevertheless, the  relationship was subject to alteration to some degree by' exogenous factors and surroundings. The chemical treatment, particularly kinetin, retarded the degradation of chlorophyll A and possibly chlorophyll B.  The treatment effect on  chlorophyll A content was apparent at the 5% level but, for chlorophyll B,  53  the difference was just approaching the 5% level of significance. ABA in the 5 ppm ABA + 20 ppm kinetin treatment appeared to have mild antagonistic activity against kinetin so that the chlorophyll content in the combination treatment was lower than that of the kinetin only treatment, but the difference was not significant.  The differences of  chlorophyll contents between the two replications were also less obvious in the case of chlorophyll B than A. Comparisons of chlorophyll contents (A, B or A + B) of lettuce with 5, 6 and 7 weeks under storage showed no significant differences among the three different periods of storage, regardless of the basis (mg/gm FW or mg/gm OFW)  used. The mg/gm OFW basis was more useful in following the  degradation trend of chlorophyll content with storage time. The quantitative measurements of chlorophyll were more objective than the subjective visual ratings of green colour as a quality component. Nevertheless, the small differences in the chlorophyll measurements could not be detected visually; therefore such differences cannot be of any importance to influence consumer acceptance. Slight differences in green colour do exist in lettuce on the market, but of greater concern is the freshness of appearance and crispness of the commodity on sale. The present investigation indicates that the use of abscisic acid and kinetin were of l i t t l e practical value to maintain quality in leaf lettuce beyonq what is commonly achieved in conventional cold storage, and that good storage conditions including good hygiene would prolong the post harvest l i f e of lettuce for periods up to 6 weeks. It is also significant that the present experiment is far from simulating the actual lettuce production conditions which involve different environmental conditions and cultivation practices, (e.g. f e r t i l i z a t i o n , herbicide and pesticide applications) - factors that might complicate  54  the effect of the intended post-harvest quality prolonging agent. Up to this stage, ABA is unlikely to work as an antitranspirant under the low temperature and high humidity of cold storage but i t is s t i l l possible that i t might be beneficial in retarding transpiration rate and help prolong the quality of the commodity under  normal room temperature  ranges in places and under certain situations where cold storage f a c i l i t i e s are not available. Further studies should be considered and carried out before concluding that ABA, as well as kinetin, has any value in the post-harvest handling of lettuce.  The response of the chemicals may be affected by (1) age and  maturity of plant tissue, (2) concentrations of chemicals, (3) mode of application (spraying, dipping, single- or multi-application), (4) temperature, and (5) relative humidity.  All these factors should be studied, and  particularly in the variable environments encountered in the handling, storing and retailing of lettuce.  55 LITERATURE! CITED Abdel-Kader, A.S., L.L. 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Influence of chemical p r e s e r v a t i v e s on keeping q u a l i t y of a s t e r s , c a r n a t i o n s , chrysanthemums, and gerbera d a i s i e s . Proc. F l a . St. h o r t . Soc. 77: 466-470. Wittwer, S.H., R.R. Dedolph, V. T u l i and D. G i l b a r t . 1962. R e s p i r a t i o n and s t o r a g e . d e t e r i o r a t i o n i n celegy (Apium graveolens L.) as a f f e c t e d by post-harvest treatments w i t h N -benzylaminopurine. Proc. Amer. Soc. Hort. S c i . 80: 408-416. Wright, S.T.C. and R.W.P. Hiron. 1969. ( i ) - a b s c i s i c a c i d , the growth i n h i b i t o r induced t o detached wheat leaves by a period of w i l t i n g . Nature 224: 719-720. Ycmo, H. 1971. I n h i b i t i o n of amylase formation by a b s c i s i c a c i d i n ^ excised pea and bean cotyledons. P l a n t P h y s i o l . 47: supplement NOD.. 1IS> Zeevaart, J.A.D. 1971. (+)-Abscisic a c i d content of spinach i n r e l a t i o n • t o photoperiod and water s t r e s s . P l a n t P h y s i o l . 48: 86-90. 6  Zink, F.W. 1961. N -benzyladenine, a senescence i n h i b i t o r f o r green vegetables. J . Agr. Food Chem. 9: 304-307.  61 APPENDIX 1 Visual rating of lettuce quality under 5 different treatments after 2,3,4,5,6, and 7 weeks in storage. Week  Treatment  Don  r\cL».  0  Al  A2  K  A2K  1  9.0 9.0 9.0  8.0 8.5 8.0  9.0 9i0 9.0  9.0 9.0 9.0  9.0 8.0 8.0  2  8.0 8.0 8.0  9.0 9.0 9.0  9.0 9.0 9.0  9.0 9.0 9.0  8.0 8.5 9.0  7,0 7.0 7.0  7.0 7.0 7.0  9.0 8.0 8.0  8.0 8.0 8.0  9.0 8.0 8.0  2  6.0 6.0 6.0  7.0 7.0 7.0  7.0 7.0 7.0  5.0 6.0 6.0  5.0 6.0 6.0  1  6.5 4.5 5.0  7.0 7.0 7.0  5.5 4.0 5.0  5.5 4.0 5.0  6.0 5.0 5.0  2  7.0 6.0 6.0  5.0 7.5 7.0  5.5 4.0 5.0  5.5 6.0 5.0  7.0 5.0 5.0  1  9.0 7.0 8.0  8.0 7.5 8.0  9.0 7.5 ,9.0  4.5 .3.0 4.0  7.0 8.0 8.0  2  8.5 7.0 8.0  9.0 8.0 9.0  9.0 7.0 7.0  7.0 4.0 4.0  7.5 5.0 6.0  1  7.0 7.0 6.0  7.0 6.0 5.0  5.0 3.0 5.0  5.0 3.5 3.0  4.5 4.0 4.0  2  7.5 6.0 5.0  7.5 6.0 5.0  7.0 5.0 5.0  4.0 4.0 4.0  6.5 4v5 5.0  1  3.0 4.0 3.0  4.5 3.0 4.0  4.0 3.0 4.0  5.0 3.5 3.0  4.0 3.0 3.0  2  4.0 4.0 3.0  5.0 4.0 3.0  3.0 3.0 3.0  4.0 4.0 4.0  5.0 4.0 3.0  ?  1 o  A  R •J  u  7  .  62  APPENDIX 2 Percent total weight loss of lettuce under 5 d i f f e r e n t treatments after 5,6, and 7 weeks i n storage . 0 0.255 9.459 -1.027 1.916 -5.740 -1.190 -9.914 -0.235 3.182 8.571 880095 11.480 7.692 7.179 12.605 3.947 15.288 17.355 22.187 20.231 12.315 6.590 8.140 11.060  Al 027 2.239 -3.341 1.881 15.790 8.585 9.366 4.235 77237 2.198 £677115 0.000 2.491 5.351 3.965 4.530 12.245 16.432 15.471 30.153 12.800 16.320 19.333 20.988  A2 -0.784 -9.145 -7.527 -6.000 -4.947 -16.466 -1.435 0.580  13.514 15.849 15.522 15.938 9.259 11.507 36.905 26.525 21.622 17.608 18.400 23.724 19.802 20.120 20.238 3.774 13.873 21.073 21.000 20.648 19.167 14.545 23.383  12.462 10.638 15.816 18.142 15.970 16.964 13.693 2T720T 25.347 18.779 27.386 23.922 21.429 16.794 23.936 25.630 13.566 8.929 13.014 10.791 12.602 12.459 4.563  14.222 15.790 13.208 11.917 21.519 16.337 20.000 19.198 10.749 17.266 21.401 18.919 20.488 6.941 16.110 9.091 12.158 13.636 10.163 10.853 7.738 10.109 10.425  777055  T772S4"  7.347 8.537 10.860 10.432 9.091 5.098 6.539 24.561 18.692 24.242 24.092 11.314 18.944 17.472 26.531  T374"0l>  K BT433 12.987 -13.123 0.000 -2.222 -4.018 0.443 -7.772 11.470 11.245 10.622 9.705 7.960 6.731 7.023 11 .562 26.720 24.101 18.214 24.939 19.098 18.983 19.626 13.975  A2K 7.837 4.051 4.955 -0.357 -1.522 0.431 5.856 -6.936 14.220 10.933 11 .524 14.057 9.662 12.500 10.476 5.479 6.468 17.624 7.018 9.554 13.008 7.750 19.355 16.538  ToTSTS  14.783  12.544 11.312 10.945 9.730 9.524 16.235 11.832 151686 15.517 14.563 12.625 12.523 14.173 22.247 21.265 17.365 12.202 9.247 13.043 15.041 17.699 16.908 14.545  13.726 15.306 14.286 10.891 12.081 19.324 17.508 11 .027 10.928 21.116 14.727 14.011 17.003 11.350 12.648 15.723 25.714 20.588 27.132 14.395 15.152 12.217 18.750  63  APPENDIX 3 Percent'moisture contents of lettuce under 5 d i f f e r e n t treatments after 5,6, and 7 weeks i n storage.  0 95.635 94.628 95.227 95.655 95.825 94.996 95.618 95.053 94.040 95.267 94.831 94.776 94.352 94.837 94.596 94.582 94.251 94.412 93^911 94.016 94.632 94.355 94.988 94.260 94.676 94.415 94.219 94.368 94.075 93.964 94.530 93.464 93.781 93.596 94.168 94.190 94.509 94.000 93.886 94.057  "9X264 93.879 94.179 94.258 93.711 93.921 94.469 94.455  i  Al 94.793 94.898 95.233 95.454 94.231. 94.326 94.274 94.894 95.891 93.345 94.647 95.832 95.189 94.966 94.712 95.185 94.243 94.092 93.884 93.016 94.436 94.107 93.835 93.439. 94.744 94.856 93.916 93.980 94.318 94.315 94.246 94.215 93.388 93.721 93.770 93.870 93.935 93.972 93.583 93.537 947^09" 94.902 94.678 94.250 94.108 94.993 94.377 94.511.  A2 95.128 95.332 95.914 95.423 95.642 95.650 95.338 95.053  9B7T2c1  95.782 95.474 95.007 94.503 92.225 93.995 95.095 93.125 93.718 93.815 . 92.467 93.869 92.973 93.007 92.813 94.177 94.115 94.471 94.472 94.340 94.785 94.160 94.783 93.940 '93.966 94.134 94.074 94.155 93.756 95.553 94.512 •  K 95.181 94.779 95.897 95.061 95.556 95.517 95.055 95.810  A2k 94.945 94.947 94.985 95.274 94.814 94.947 94.806 95.146  95.079 95.469 94.557 94.242 94.182 94.071 94.600 93.303 94.494 94.057 93.127 93.751 94.122 93.235 94.150 9l~9Tl 93.960 94.488 94.317 94.701 94.294 94.445 94.443 94.240 94.618 94.254 96.851 94.251 94.381 94.103 .193.809  94.826 95.119 95.089 93.841 94.861 94.969 93.314 95.016 94.532 95.226 95.242 93.585 94.473 93.821 94.110 94.484 95.238 94.638 94.404 94.789 94.391 94.604 93.683 94.570 94.772 94.020 94.923 94.547 93.852 94.107 95.020  9TT7515  W^U  9476T8"  9T3T8  94T22F  93.985 94.079 94.472 94.065 94.951 94.268 93.711 1  94.913 94.318 93.952 94.019 94.184 94.737 94.690  94.287 93.911 94.379 94.57.0 94.478 94.398 94.560  64 APPENDIX 4 Chlorophyll A contents (mg/gmFW) of lettuce under 5 d i f f e r e n t treatments after 5,6, and 7 weeks i n storage. 0 0.3512 0.3269 0.2944 0.3704 0.3315 0.4664 0.3764 0.2096 0.7138 0.4220 0.5320 0.4175 0.7149 0.4974 0.4585 0.5284 0.3995 0.3519 0.3836 0.4726 0.3621 0.5969 0.5083 0.4622 0.4859 0.5420 0.4883 0.5342 0.4226 0.5295 0.4747 0.7848 0.3697 0.6393 0.4960 0.3563 0.4420 0.5179 0.6501 0.3118 0.5091 0.5266 0.5120 0.4857 0.5731 0.4424 0.4298 0.3602  Al A2 K A2K 0509 0.3055 0412 0.2859 0.4589 0.4647 0.4858 0.6374 0.4383 0.4841 0.3974 0.4111 0.4067 0.4192 0.4083 0.5671 0.6125 0.3653 0.4127 0.4060 0.5067 0.5330 0.4807 0.4383 0.7032 0.3745 0.5913 0.5165 0.4240 0.43720.3854 0.5245 0.3799 0.4787 0.5130 0.6145 0.8271 0.4518 0.4348 0.4379 0.4526 0.3426 0.5315 0.4628 0.3598 0.4496 0.5637 0.5926 0.3689 0.4763 0.6710 0.8713 0.5203 0.5236 0.6215 0.5979 0.4379 0.6008 0.7625 0.5317 0.4372 0.5340 0.5174 0.6593 0.6007 0.4713 0.5206 0.5450 0.4345 0.5570 0.5355 0.5142 0.4642 0.6301 0.6502 0.4046 0.4660 0.6025 0.6207 0.4730 0.5436 0.6946 0.5253 0.6407 0.5050 0.4797 0.5502-2 0.5481 0.5351 0.5591 0.5418 0.5487 0.6339 0.4662 0.5845 0.5921 0.4718 0.4462 0.5249 0.4681 0.4264 0.6000 0.6789 0.5336 0.5304 0.4831 0.6060 0.5978 0.4532 0.5899 0.6561 0.5304 0.5672 0.5417 0.5884 0.5007 0.4742 0.3370 0.6418 0.5312 0.4756 0.4463 0.3999 0.5211 0.4872 0.4245 0.5319 0.6651 0.4970 0.4008 0.4601 0.7249 0.2647 0.3722 0.4363 0.3745 0.5587 0.3723 0.5566 0.3196 0.2548 0.4575 0.4048 0.3182 0.4698 0.2616 0.4666 0.5094 0.4555C 38310.;3834 0.4559 0.5514 0.4751 0.5706 0.5713 0.5709 0.2686 0.48650.50130.3722 0.3468 0.3380 0.4775 0.4383 0.4751 0.5385 0.4539 0.4328 0.4400 0.5901 0.6588 0.5106 0.3600 0.4835 0.5202 0.5964 0.4453 0.5077 0.6436 0.5103 0.4574 0.3580 0.5851 0.5603 0.3016 0.4615 0.6480 0.5371 0.4566 0.5614 0.5815 0.5720  65  APPENDIX 5 Chlorophyll A contents (mg/gmOFW) of lettuce under 5 d i f f e r e n t treatments after 5,6, and 7 weeks in storage.  0 0.3503 0.2960 0.2974 0.3633 0.3505 0.4719 0.4137 0.2101 0^9TTj 0.3858 0.4889 0.3696 0.6599 0.4617 0.4007 0.5076 0.3385 0.2909 0.2985 0.3770 0.3175 0.5576 0.4669 0.411V: 0.4518 0.4687 0.4109 0.4513 0.3552 0.4804 0.4201 0.4952 TJ727T7 0.5011 0.4087 0.2907 0.3371 0.4154 0.5193 0.2487 0.4899 0.4536 0.4041 0.3837 0.4548 0.3576 0.3673 0.2760  Al 0.4290 0.4486 0.4529 0.3991 0.5158 0.4632 0.6373 0.4060 073B74" 0.8089 0.4222 0.3598 0.3597 0.4924 0.4205 0.4174 075771 0.3631 0.3924 0.3255 0.4740 0.4226 0.4317 0.5009 0.3903 0.3732 0.4740 0.3815 0.4643 0.3985 0.3949 0.4205 073757 0.1976 0.4538 0.1850 0.3574 0.3579 0.3953 0.2043 0.2579 0.4107 0.4007 0.3131 0.3973 0.3998 0.2640 0.4358  A2 0.3079 0.5072 0.5205 0.4443 0.3833 0.6208 0.3799 0.4346 074135 0.4186 0.3134 0.4008 0.4266 0.4760 0.5702 0.4991 0.3556 0.4529 0.4773 0.4574 0.6160 0.3888 0.4614 0.3425 0.3864 0.5147 0.4068 0.5120 0.477T 0.2645 0.3734 ' 0.3396  OS39  0.3322 0.3080 0.3596 0.2121 0.3049 0.5310 0.4081 0.3073 0.4730 0.5096 0.4344 0.4526 0.3303 0.4149 0.5028  K 0.3273 0.4227 0.4495 0.4083 0.4219 0.5000 0.5887 0.4153  0.4542  0.3859 0.4751 0.5090 0.6176 0.5796 0.7090 0.4576 0.3815 0.4064 0.5318 0.4659 0.4250 0.4458 0.4354 0.5028 0.4678 0.5937 0.5374 0.5843 0.5311 0.5807 0.3350 0.4689 073879 0.3686 0.4756 0.3537 0.4082 0.3913 0.4442 0.3947 0.3946 0.3985 0.5979 0.4523 0.5468 0.4816 0.5384 0.4969  A2K 0.2635 0.6116 0.3907 0.5691 0.4121 0.4364 0.4863 0.5609 07S27T 0.3900 0.4095 0.5093 0.7872 0.5232 0.4760 0.6232 0.5097 0.4236 0.3762 0.4278 0.5573 0.5056 0.4425 0.4942 0.3989 0.4604 0.5064 0.4546 0.4462 0.4671 0.4204 0.5486 075430" 0.3336 0.2521 0.2713 0.4380 0.4577 0.5061 0.3251 0.3694 0.3215 0.4055 0.4346 0.4370 0.4754 0.4715 0.4648  66  APPENDIX 6 Chlorophyll B contents (mg/gmFW) of lettuce under 5 d i f f e r e n t treatments after 5,6, and 7 weeks in storage  0 0.1714 0.1181 0.1275 , 0.2000 0.1373 0.2548 0.1574 0.2211 0.3612 0.2498 0.2607 p.1713 ? 0.3607 0.2193 0 ??5 0.2255 0.3215 0.2398 0.1845 0.2711 , 0.2886 0.1938 0.3511 0.2914 0.072718 0.2507 0.3504 0.1905 0.3000 0.2152 0.2828 0.2706 0.4471 1  v o  c  r  1  r  ?  c  o^rrsi  7  i 1  0  | '  ? c  0.3339 0.3002 0.2057 0.2322 0.2856 0.073'774 0.1625 0.2547 0.2878 0.2689 0.2623 0.2842 0.2091 0.2600 0.2002  Al 0.2310 0.2119 0.1859 0.2751 0.3620 0.1773 0.4438 0.2031 0.2152 0.4510 0.2297 0.1947 0.2331 0.2846 0.2090 0.2245 0.3477 0.2486 0.2538 0.2875 0.2771 0.3004 0.3513 0.4700 0.2564 0.2213 0.2748 0.2028 0.2675 0.2746 0.2625 0.3573  A2 0.1287 0.2497 0.1884 0.2548 0.2154 0.2814 0.1639 0.2054 OBT1 0.2741 0.1481 0.2539 0.2565 0.2853 0.1698 0.2427 0.2604 0.3367 0.3759 0.2628 0.4110 0.2074 0.3186 0.2664 0.2247 0.3537 0.2557 0.2851 0.2740 0.1771 0.2438 0.2182  K 0.1658 0.2718 0.1603 0.1636 0.2230 0.1687 0.4299 0.1671 0.3086 0.2064 0.2648 0.2881 0.3421 0.2838 0.4064 0.2705 0.2520 0.3186 0.4060 0.3988 0.3189 0.2878 0.2931 0.3121 0.3229 0.3715 0.3015 0.3348 0.2589 0.3023 0.2167 0.3009  A2K 0.1328 0.4494 0.1586 0.2484 0.1836 0.1859 0.2880 0.2772 0.2560 0.1994 0.2562 0.3299 0.4274 0.2933 0.2627 0.3067 0.2650 0.2985 0.1957 0.2655 0.3716 0.3460 0.3114 0.3244 0.2694 0.2658 0.2742 0.2748 0.2983 0.2477 0.3168 0.3350  our?  orrwi  ^mr-  OTW  0.1223 0.2902 0.1397 0.2247 0.2516 0.2570 0.1571 0.2000 0.2665 0.2789C 0.1832 0.2423 0.2546 0.1628 0.2320  0.1839 0.1935 0.2240 0.1197 0.2138 0.3063 0.2658 0.1882 0.3021 0.3424 0.2919 0.2763 0.2203 0.2874 0.2986  0.2325 0.2909 0.2032 0.2528 0.2380 0.3194 0.2541  0.1734 0.1823 0.1752 0.2718 0.2873 0.3138 0.1839 0.2642 0.2435 0.2809 0.2767 0.2733 0.3181 0.3141 0.3038  OTBT  0.2560 0.3796 0.2655 0.3456 0.3463 0.3075 0.3191  67  APPENDIX 7 Chlorophyll B contents (mg/gmOFW) of lettuce under 5 d i f f e r e n t treatments after 5,6, and 7 weeks in storage  0 0.1710 0.1069 0.1288 0.1961 0.1452 0.2578 0.1730 0.2216 0.3497 0.2284 0.2396 0.1517 0.3329 0.2036 0.1971 0.3088  Al  " 0.'24'5l  0.2072 0.1921 0.2699 0.3048 0.1621 0.4023 0.1945 0.1997 0.4411 0.2126 0.1947 0.2273 0.2694 0.2007 0.2144 0.3051 0.2077 0.2145 0.2oo8 0.2416 0.2513 0.2834 0.3713 5T2T21 0.1937 0.2455 0.1707 0.2190 0.2307 0.2180 0.3083 0.2287 0.0913 0.2371 0.1014 0.1710 0.1976 0.2138 0.1195  •'• b'1487  A2 0.1297 0.2726 0.2025 O.270JI 0.2261 0.3277 0.1662 0.2042 0.2403 0.2539 0.1355 0.2264 0.2298 0.2593 0.1611 0.2268 0.1965 0.2737 0.2848 0.1995 0.3645 0.1681 0.2629 0.1957 0.1946 0.3034 0.2153 0.2475 0.2414 0.1390 0.2040 0.1746 0.1609 0.1642 0.1642 0.1760 0.0971 0.1700 0.2850 0.2230  K A2K 0'.1567 0'.1224" 0.2365 0.4312 0.1814 0.1508 0.1636 0.2493 0.2279 0.1864 0.1755 0.1851 0.4280 0.2712 0.1801 0.2964 0.2732 0.2196 0.1832 0.1776 0.2366 0.2367 0.2601 0.2835 0.3149 0.3861 0.2647 0.2567 0.3779 0.2352 0.2392 0.2899 0.1846 0.2479 0.2418 0.2459 0.3321 0.1819 0.2994 0.2401 0.2580 0.3233 0.2331 0.3192 0.2356 0.2511 0.2685 0.2707 0.2878 0.2296 0.3249 0.2293 0.2674 0.2323 0.2981 0.2355 0.2338 0.2658 0.2735 0.2177 0.1815 0.2556 0.2653 0.2763 0.2283 . 0.3309 0.1965 0.1545 0.1965 0.1545 0.1776 0.1494 0.2211 0.2337 0.2043 0.2384 0.2483 0.2782 0.2000 0.1607  0.2478 0.2122 0.2072 0.2256 0.1690 0.2222 0.1534  0.2304 0.2540 0.1593 0.2161 0.2225 0.1425 0.2214  0.2654 0.2957 0.2622 0.2463 0.2033 0.2583 0.2674  0.2248 0.3445 0.2309 0.2936 0.2850 0.2555 0.2727  072750"  0.1525 0.2110 0.2302 0.1699 0.3280 0.2677 0.2417 0.2331 0.3031 0.1603 0.2534 0.1809 0.2566 0.2394 0.2821 0.1584 u0i2617 0.2473 0.1678 0.1771 0.2290 0.3014 0.1296  *  "  l,  oTTTTi  572774"  O22T  0.1809 0.2231 0.2016 0.2341 0.2699 0.2757 0.2469  68 APPENDIX 8 Chlorophyll (A+B) contents (mg/gmFW) of lettuce under 5 different treatments after 5,6, and 7 weeks in storage Week  T r e a t m e n t  Rep. 0  ~™~  S3226"  Al  09T5U  ' A2 .  0332  1  0.4451 0.4219 0.5704 0.4688 0.7212 0.5338 0.4307  0.6c708 0.6241 0.6819 0.9745 0.6840 1.1470 0.6271  0.7144 0.6724 0.6740 0.5807 0.8144 0.5384 0.6426  TT0719  075951  0.7438  £  0.6718 0.7927 0.5888 1.0756 0.7167 0.6840 0.8499 0.6393 0.5364 0.6547 0.7612 0.5559 0.9480 0.7996 0.7340  1 1  0  9 L  , 1  o  0 > 7 3 6 6  9 c  , 1  2 '  9 L  0.8924 0.6788 0.8342 0.6377 0.8123 0.7452 1.2319 0.5853 0.9733 0.7962 0.5619 0.6742 0.8035 1.0275 0.4743 0.7638 0.8144 017808 0.7480 0.8574 0.6514 0.6899 0.5605  1.2781 0.7259 0.6805 0.4907 0.5545 0.7036 0.6020 0.7328 0.8049 0.8088 0.6469 0.7706 0.6618 0.7767 0.9484 0.7318 0.6831 0.8937 0.7180 1.0060 0.7535 0.8653 0.8207 1.1056 0.8053 . 0.6870 0.8864 0.8777 1.1039 0.7325 0.7282 0.6709 0.6477 0.9537 0.8052 0.7388 0.6560 0.8750 0.8347 0.8157 0.7488 0.5142 0.7381 0.6901 0.8445 0.6427 0.7987 0.5999 0.3870 0.5561 0.8507 0.5657 0.3945 0.6815 016945 0.3813 0.7071 0.5972 0.7321 0.8769 0.4252 0.7523 0.5468 0.5262 0.7417 0.8406 0.7189 0.9325 0.5431 0.7753 0.6876 0.7804' 0.7120 0.5783 0.4644 0.7489 0.68860.8599  K  0~oT5o"  0.7576 0.5577 0.5719 0.6357 0.6494 1.0212 0.5525  A2K  O W  1.0868 0.5697 0.8155 0.5896 0.6241 0.8045. 0.8017  0.8216  0.8705  0.6412 0.7963 0.8518 1.0131 0.9053 1.1689 0.7879 0.7726 0.8541 1.0562 l.ol95 0.8442 0.8380 0.8349 0.8967 0.8478 1.0503 0.9075 0.9909 0.8473 0.9441 0.6166 0.8328 0.7308 0.6689 0.8475 0.6081 0.7194 0.6939 0.8907 0.7553 0.7527 0.7099 1.0384 0.7857 0.9892 0.9314 0.9555 0.9066  0.6373 0.7191 0.9225 1.2987 0.8912 0.7945 0.9660 0.8100 0.8127 0.6003 0.7385 1.0123 0.8941 0.8600 0.9165 0.7375 0.7995 0.8721 0.8052 0.7990 0.7789 0.8379' 1.0000 1.0968 0.5480 0.5019 0.4934 0.7812 0.8387 0.9947 0.5561 0.7026 0.6764 0.7915 0.8732 0.7837 0.8784 0.8512 0.8758  69 APPENDIX 9 Chlorophyll (A+B) contents (mg/gmOFW) of lettuce under 5 different treatments after 5,6, and 7 weeks in storage Week  Treatment  Rep. 0  ?  1  ?  r  9  Al_  A2  0.5213 0.4030 0.4262 0.5594 0.4957 0.7289 0.5867 0.4317 1.0407 0.6142 0.7286 0.5212 0.9929 0.6652 0.5978 0.8163 0.5416 0.4433 0.5095 0.6072 0.4874 0.8855 0.7346 0.6528 0.6849 0.7718 0.5712 0.7047 0.5361 0.7370 0.6595 0.7773  0.6450 0.6558 0.6450 0.6690 0.8207 0.6253 1.0396 0.6005 075572 1.2500 0.6348 0.5545 0.5870 0.7618 0.6212 0.6318 0.8322 0.5708 0.6069 0.5263 0.7156 0.6739 0.7150 0.8722 0.6024 0.5670 0.7195 0.5522 0.6833 0.6292 0.6129 0.7298  0.4367 0.7798 0.7230 0.7144 0.6094 0.9485 0.5461 0.6389 0737T3" 0.6726 0.4488 0.6271 0.6564 0.7352 0.7313 0.7259 0.5520 0.7266 0.7621 0.6568 0.9805 0.5569 0.7243 0.5382 0.5810 0.8180 0.6221 0.7594 0.7185 0.4035 0.5774 0.5142  0.4840 0.3859 0.6592 1.0428 0.6309 0.5415 0.5719 0.8184 0.6498 0.5986 0.6755 0.6215 1.0166 0.7574 0.5954 0.8573 077274" 0.7467 0.5691 0.5676 0.7117 0.6362 0.7691 0.7928 0.9325 1.1732 0.8443 0.7798 1.0868 0.7113 0.6968 . 0.9131 0.5662 0.7576 0.6482 0.6695 0.8638 0.5581 0.7653 0.6680 0.6830 0.8806 0.6789 0.8248 0.6710 0.6936 0.7714 0.7649 0.7556 0.6285 0.9186 0.6897 0.8048 0.7386 0.8824 0.6901 0.7649 0.7120 0.8542 0.6848 0.5165 0.6706 0.7343 0.8249  OT61  079759  0.7628 0.6560 0.4585 0.5142 0.6444 0.8207 0.3783 0.7350 0.7014 0.6163 0.5909 0.6803 0.5266 0.5895 0.4294  0.2289 0.6910 0.2865 0.5284 0.5555 0.6092 0.3237 0.4066 0.6411 0.6547 0.4724 0.6134 0.6223 0.4065 0.6572  0.4964 0.4681 0.5356 0.3092 0.4749 0.8160 0.6311 0.4784 0.7384 0.8053 0.6965 0.6989 0.5335 0.6732 0.7703  0.5651 0.7241 0.5313 0.6293 0.5956 0.6926 0.5947 0.6220 0.6233 0.9424 0.6832 0.8404 0.7666 0.7939 0.7696  0.4881 0.3959 0.4207 0.6717 0.6961 0.7843 0.4858 0.5921 0.5024 0.6285 0.6363 0.6711 0.7453 0.7472 0.7116  0~4T0D  7 /  9 c  075051  OM§  K  A2K  

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