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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 i i 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. i n 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 all 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 signif-icance. 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. Acknowledgements iv This study was made possible by grants from the Canadian Inter-national 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 facilities 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 synthesis 6 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 15 MATERIALS AND METHODS 20 A. Materials 1. Test plants 21 2. ' Chemical treatments 22 3. Cold storage facilities 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 42 Table 11 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 3 2. Structure of kinetin 11 3. Modified vacuum filtration apparatus 28 4. Chlorophyll A and B in mg/gm OFW as affected by treatment and storage time 45 5. Chlorophyll (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 li f e of those vegetable crops even further. Abscisic acid is known to induce stomatal closure and inhibit trans-piration 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 life of produce might then be signifi-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 acid - a hormone now known under the name "abscisic acid" and usually designated as ABA. This hormone is a relatively recent discovery; nevertheless, its physiol-ogical importance may rank with auxins, gibberellins or cytokinins (Addicott and Lyon, 1969). The substance was first isolated by Ohkuma et aJL (1963) from young cotton fruit (Gossypium hirsutum L.). It was then named "abscisin II" because it promoted abscission activity. 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 it was found to be the same substance as abscisin II (Cornforth et_ al_. 1965a; Robinson and Wareing, 1964). This chemical was first synthesized by Cornforth et al_. (1965b) and more contrib-utions were added on its physical and biological activities (Cornforth et al. 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 (if 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 all other hormones, induces a wide spectrum of plant responses. Besides its activities in abscission and senescence, it is well recognized in various other significant phenomena including germination, dormancy, enzyme activities, 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 ad-or 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 2 GAo = Gibberellic acid; 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 incor-poration 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 32P 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 RNA accumulation and accelerated ribonuclear activity. 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 first 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 air. 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 itself. 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 cells. 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 its 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 al. (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 was genetically controlled. 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 iso-prenoid synthesis. Glinka (1971) found that ABA markedly raised the per-meability 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 first 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 due to different mechanisms. 11 B. Kinetin HC CH HN CH2 C CH H Figure 2. Structure of Kinetin Miller found this chemical in 1954 and named it "kinetin" after its first-observed activity in association with cytokinesis. The substance was 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 fruit, 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 endog-enous 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 life and subsequent shelf li 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 14 leaf disks, reported a kinetin-induced increase of C-leucine incorpor-14 ation into protein and of C-orotic acid into RNA. Thus, kinetin can stimulate both RNA and protein synthesis. Osborne suggested that the retardation of senescence by kinetin is mediated through its 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 C-L-leucine in both the presence and absence of kinetin. 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 first 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 con-centration 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 radio-active uracil and adding kinetin lessened the inhibition). Yomo (1971) found that ABA inhibited the increase of a-amylase and 3-amylase activities, 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 counter-acted 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 it 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 IAA-and 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 post-harvest 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). Twelve plants were used for each treatment and there was no replication. 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. Firstly, 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 initial 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. The plants were instead grown under greenhouse conditions. "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 fertilizer, 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 first 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 first 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 facility 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 satur-ation 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. Ordinary dry-bulb and wet-bulb thermo-meters were used as a further check. The 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. Details of the experimental procedures and measurements follow. 24 B.I. Experimental design A split 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 seedings. B.2. 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 initial 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 all 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 all 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, it 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 two-layer 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 all 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. This leaf was set aside for chlorophyll extraction. 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. Five trays were used for all 40 plants to be dried simultaneously. Samples were placed in a vacuum dryer for 15 hours at 70°C. 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 fi 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 filtrate. The apparatus allowed the filtrate 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 filtrate 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 filtrate when the volumetric flask was used and thus lessened evaporation of the highly volatile acetone due to exposure to low pressures during filtration. 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 silica 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 Ag64- 2.29 Ag4?) mg/1 Chlorophyll B = (20.05 Ag47 - 4.77 Ag64) mg/1 Chlorophyll A+B = (7.01 Ag64 + 17.76 Ag47) 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. RESULTS 30 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 first 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 Replica- Treatment tion . . . . . . r l c d il Al A2 K A2K 5 1 2 8.0 7.8 7.8 8.7 8.5 7.7 7.3 6.2 7.7 6.2 7.6 6 1 2 6.7 6.2 6.0 6.2 4.3 5.7 3.8 4.0 4.2 5.3 5.3 7 1 2 3.3 3.6 3.8 4.0 3.7 3.0 3.8 5.0 • 3.3 4.0. ' 5 . 5 Mean 5.9 6.1 5.5 5.0 5.1 5.5 Mean of replication 1 =5.4889 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. S.S. . M.S. F Prob. Replication 1 0 .136 0.136 0.16 0.6936 Week (A) 2 231 .810 115.900 40.39 0.0348 Error a 2 5 .739 2.869 3.33 0.0414 Treatment (B) 4 16 .361 4.090 4.71 0.0163 Week x Treatment (AB) 8 15 .056 1.882 2.17 0.1095 Error b 12 10 .417 0.868 1.01 0.4533 Error 60 51 .667 0.861 TotaldS 89 331 .180 Duncan's test Replication 1 2 Mean 5.48 a * 5.6 a Time under cr c "7 storage (weeks) O O 1 Mean 7.6 b o5.3 b . 3.7 a Treatment 0 Al \~> A2 K A2K Mean 5.9 b 6.1 b 5.5 ab . . . . 5 . 0 . a . 5.1 a * Mean separation in row by Duncan's multiple range test , 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 fifth 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 fifth 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 B G i >J • •' . storag Week 2 "Replica- Treatment M r**n n tion 0 Al A2 K A2K rlccul 5 1 2 -0.809 7.844 5.660 4.061 -5.716 8.407 -1.030 '9.540 1.789 11.106 4.085 6 1 2 14.146 15.690 17.968 15.121 20.731: 15.800 20.707 il.625 12.164 14.738 15.869 7 1 2 21.005 17.183 22.724 12.694 16.384 10.522 16.075 14.506 14.101 18.704 16.390 Mean 12.510 13.038 11.021 11.904 12.101 12.115 Mean of.replication 1 = 11.727 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. S.S. M.S. F Prob. Replication 1 36.14 36.14 1.66 0.1958 Week (A) 2 7747.60 3873.80 4.65 0.1795 Error a 2 1665.30 832.66 388277 0.0000 Treatment (B) 4 107.94 29.98 0.23 0.9132 Week x Treatment (AB) 8 627.52 78.44 0.68 0.7043 Error b 12 1388.70 115.73 5.32 0.0000 Error 210 4569.40 21.76 Total 239 16143.00 3. Moisture content Moisture content was fairly uniform for all the lettuce plants regard-less 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 Replica- Treatment tion K A2K r i c a 11 0 Al A2 5 1 2 95.33 94.66 94.76 94.97 95.44 94.65 95.36 94.62 94.99 94.61 94.94 6 1 2 94.35 94.21 93.88 94.32 93.22 94.41 93.78 94.44 94.50 94.53 94.17 7 1 94.02 :.94.02 93.72 94.53 94.26 94.27 94.56 94.42 94.48 94.35 94.26 Mean 94.43 . 94.37 94.38 94.53 94.58 94.46 Mean of replication 1 = 94.44 Mean of replication 2 =94.47 Treatments: 0 = A2K control, = 5 ppm Al = 1 | ABA + 20 ppm ABA, A2 = ppm kinetin 5 ppm ABA, K = 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. S.S. M.S. F Prob. Replication 1 0.035 0.035 0.15 0.7020 Week (A) 2 28.265 14.132 3.33 0.2320 ErroY a 2 8.483 4.242 17.38 0.0000 Treatment (B) 4 1.688 0.422 0.52 0.7232 Week x Treatment (AB) 8 5.655 0.707 0.87 0.5629 Error b 12 9.695 0.808 3.31 0.0002 Error 210 51.253 0.244 Total 239 105.070 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 respectively. Analyses of variance for chlorophyll A, B and A+B (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 lots, 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 ^eP^lca" Treatment mean t l 0 n 0 Al A2 K A2K  1 0.3409 0.5012 0.4229 0.4385 0.4734 5 0.4840 2 0.5356 0.4730 0.4822 0.5769 0.5960 1 0.4421 0.5229 0.5576 0.5661 0.5333 6 0.5246 2 0.5328 0.4858 0.4836 0.5785 0.5435 1 0.4729 0.4055 0.4131 0. 481:6 0.4676 7 0.4702 2 0.4799 0.4103 0.4798 0.5710 0.5197 Mean 0.4673 0.4664 0.4732 . 0.5354 0.5223 0.4929 Mean of replication 1 =014693 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. S.S. M.S. F Prob. Replication 1 0.1341 0.1341 15.33 0.0002 Week (A) 2 0.1281 0.6403 1.36 0.4239 Error a 2 0.0943 0.0472 5.39 0.0054 Treatment (B) 4 0.2118 0.0530 3.18 0.0531 Week x Treatment (AB) 8 0.0870 0.0109 0.65 0.7219 Error b 12 0.1996 0.0166 1.90 0.0356 Error 210 1.8367 0.0087 Total 2393? 2.6915 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^, Treatment tion K r i c a i i 0 Al A2 A2K 5 1 2 0.3442 0.4957 0.4690 0.4542 0.4498 0.4423 0.4417 0.5235 0.4663 0.5307 0.4617 6 1 2 0.3823 0.4417 0.4297 0.4122 0.4440 0.4093 0.4493 0.5124 0.4671 0.4628 0.4411 7 1 2 0.3740 0.3984 0.3160 0.3599 0.3475 0.4281 0.4030 0.4884 0.4036 0.4225 0.3941 Mean 0.4060 . 0.4068 0.4202 0.4697 0.4588 0.4323 Mean of replication 1 =0.4125 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. S.S. M.S. CT Prob Replication 1 0.0942 0.0942 12.21 0.0007 Week (A) 2 0.1919 0.0960 9.08 0.1053 Error a 2 0.0211 0.0106 1.37 0.2553 Treatment (B) 4 0.1724 0.0431 4.18 0.0240 Week x Treatment (AB) 8 0.0464 0.0058 0.56 0.7897 Error b 12 0.1237 0.0103 1.34 0.1998 Error 210 1.6207 0.0077 Total 239 2.2704 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 Replica- Treatment tion 0 Al A2 K A2K II c a 11 5 1 2 0.1735 0.2713 0.2613 0.2550 0.2110 0.2369 0.2188 0.2963 0.2405 0.2915 0.2456 6 1 2 0.2615 0.2884 0.3170 0.2649 0.3049 0.2540 0.3234 0.3012 0.2973 0.2853 0.2898 7 1 .1 ... 0.2641 0.2534 . 0.2183 0.2275 0.2133 0.2759 0.2577 0.3118 0.2450 0.2843 0.2551 Mean 0.2520 0.2573 0.2493 0.2849 0.2740 0.2635 Mean of replication 1 = 0.2538 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. S.S. M.S. F Prob. Replication 1 0.0224 0.0224 5.77 0.0164 Week (A) 2 0.0865 0.0432 1.57 0.3882 Error a 2 0.0549 0.0275 7.05 0.0012 Treatment (B) 4 0.0450 0.0112 2.27 0.1213 Week x Treatment (AB) 8 0.0301 0.0038 0.76 0.6436 Error b 12 0.0594 0.0050 1.27 0.2364 Error 210 0.8173 0.0039 Total 239 1.1156 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 Replica- Treatment M m n tion 0 Al A2 K A2K ii ecu i 5 1 2 0.1750 0.2515 0.2436 0.2450 0.2249 0.2166 0.2187 0.2687 0.2366 0.2594 0.2340 6 1 2 0.2255 0.2386 0.2595 0.2248 0.2432 0.2150 0.2566 0.2665 0.2600 0.2428 0.2432 7 1 2 0.2090 0.2103 0.1700 0.1994 0.1795 0.2462 0.2156 0.2668 0.2112 0.2319 0.2140 Mean 0.2183 0.2237 0.2209 0.2488 0.2403 0.2304 Mean of replication 1 .= 0.2219 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. S.S. M.S. F Prob. Replication 1 0.0172 0.0172 5.28 0.0214 Week (A) 2 0.0358 0.0179 1.46 0.4060 Error a 2 0.0245 0.0122 3.75 0.0246 Treatment (B) 4 0.0345 0.0086 2.74 0.0782 Week x Treatment (AB) 8 0.0200 0.0025 0.80 0.6180 Error b 12 0.0377 0.0031 0.96 0.4861 Error 210 0.6856 0.0033 Total 239 0.8553 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 Replica- Treatment tion 0 Al A2 K A2K l l c O - M .5 1 2 0.5143 0.8068 0.7624 0.7280 0.6339 0.7191 0.6573 0.8733 0.7138 0.8875 0.7296 6 1 2 0.7036 0.8211 0.8399 0.7504 0.8625 0.7376 0.8895 0.8796 0.8306 0.8288 0.8144 7 1 2 0.7370 0.7333 0.6238 0.6379 0.6264 0.7557 0.7393 0.8829 0.7126 0.8041 0.7253 Mean 0.7194 0.7237 0.7225 0.8203 0.7962 0.7564 Mean of replication 1 = 0.7231 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. S.S. M.S. F Prob. Replication 1 0.2663 0.2663 11.86 0.0008 Week (A) 2 0.4035 0.2018 1.41 0.4140 Error a 2 0.2853 0.1426 6.35 0.0023 Treatment (B) 4 0.4444 0.1111 2.86 0.0702 Week x Treatment (AB) 8 0.2155 0.0269 0.69 0.6918 Error b 12 0.4655 0.0388 1.73 0.0625 Error 210 4.7147 0.0225 Total 239 6.7951 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 Treatment tion K A2K o Al A2 5 1 0.5192 2 0.7471 0.7126 0.6992 0.6747 0.6590 0.6604 0.7922 0.7029 0.7900 0.6957 6 1 0.6077 2 0 . 6 8 0 3 0.6891 0.6369 0.6872 0.6243 0.7060 0.7789 0.7271 0.7056 0.6843 7 1 0.5831 2 0.6087 0.4861 0.5593 0.5270 0.6743 0.6181 0.7552 0.6148 0.6543 0.6081 Mean 0.6244 0.6305 0.6411 0.7185 0.6991 0.6627 Meani7o.f repl ication 1 = 0.6344 Mean of.repl ication 2 = 0.6910 Treatments: 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.. S.S. M.S. F Prob. Replication 1 0.1920 0.1920 9.89 0.0021 Week (A) 2 0.3629 0.1814 4.03 0.2008 Error a 2 0.0901 0.0451 2.32 0.0986 Treatment !(iB) 4 0.3561 0.0890 3.69 0.0351 Week x Treatment (AB) 8 0.1251 0.0156 0.65 0.7270 Error b 12 0.2898 0.0241 1.24 . 0.2547 Error 210 4.0785 0.0194 Total 239 5.4945 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 FW OFW Chlorophyll1 G„ ,oA3i\ 0Z5B3e. A+B B [1 0.4693A1 0.2538a 12 0.5166B 0.2732b A+B Repl icatiom 0.7231A 0.4125A 0.2219a 0.6344A 0.7897B 0.4521B 0.2389b 0.6910B Time in storage (weeks) 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 Treatment 0 0.4673!3 0.2520a 0.7194!, 0.4060a 0.2183! 0.6244a Al 0.4664.§. 0.2573a 0.72371 0.4068a 0.2237!. 0.6505ab • A2 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 A2K 0.5223t\ji 0.2740a 0.7962S 0.4588bc 0.24031 0.6991bc 1 Meanr-separation in column by Duncan's multiple range test: 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 signif-icantly higher than that of replication 1 in all 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 0.5 _x content based on gm FW _ content based on gm OFW 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. In each case, there are three correlation and regression values. 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 Standard error p Source Regression equation (b) (a) (b) (Y) r 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 replic-ations 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 all 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) FrProb. standard error _ Source RegressiohQequat,iono;,(b) (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) F-Prob. standard error 2 Source Regression equation (b) (a) (b) (Y) r N 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 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 all 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 facilities. 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 prolon-ging the storage and shelf li 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, all 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 its 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 tem-perature 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 bio-logical 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 quality-preservation 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 ABA-treated 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 li 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 life 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. fertilization, 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 it 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 facilities 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) temper-ature, 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. Morris, and E.C. 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Week D o n Treatment 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 1 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 o 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 A 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 R 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 •J 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 u 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 7 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 62 APPENDIX 2 Percent total weight loss of lettuce under 5 different treatments after 5,6, and 7 weeks in storage . 0 Al A2 K A2K 0.255 0 2 7 -0.784 BT433 7.837 9.459 2.239 -9.145 12.987 4.051 -1.027 -3.341 -7.527 -13.123 4.955 1.916 1.881 -6.000 0.000 -0.357 -5.740 15.790 -4.947 -2.222 -1.522 -1.190 8.585 -16.466 -4.018 0.431 -9.914 9.366 -1.435 0.443 5.856 -0.235 4.235 0.580 -7.772 -6.936 3.182 77237 11.470 14.220 8.571 2.198 7.347 11.245 10.933 880095 £677115 8.537 10.622 11 .524 11.480 0.000 10.860 9.705 14.057 7.692 2.491 10.432 7.960 9.662 7.179 5.351 9.091 6.731 12.500 12.605 3.965 5.098 7.023 10.476 3.947 4.530 6.539 11 .562 5.479 15.288 12.245 24.561 26.720 6.468 17.355 16.432 18.692 24.101 17.624 22.187 15.471 24.242 18.214 7.018 20.231 30.153 24.092 24.939 9.554 12.315 12.800 11.314 19.098 13.008 6.590 16.320 18.944 18.983 7.750 8.140 19.333 17.472 19.626 19.355 11.060 20.988 26.531 13.975 16.538 777055 T772S4" T374"0l> ToTSTS 14.783 13.514 12.462 14.222 12.544 13.726 15.849 10.638 15.790 11.312 15.306 15.522 15.816 13.208 10.945 14.286 15.938 18.142 11.917 9.730 10.891 9.259 15.970 21.519 9.524 12.081 11.507 16.964 16.337 16.235 19.324 36.905 13.693 20.000 11.832 17.508 26.525 2T720T 19.198 151686 11 .027 21.622 25.347 10.749 15.517 10.928 17.608 18.779 17.266 14.563 21.116 18.400 27.386 21.401 12.625 14.727 23.724 23.922 18.919 12.523 14.011 19.802 21.429 20.488 14.173 17.003 20.120 16.794 6.941 22.247 11.350 20.238 23.936 16.110 21.265 12.648 3.774 25.630 9.091 17.365 15.723 13.873 13.566 12.158 12.202 25.714 21.073 8.929 13.636 9.247 20.588 21.000 13.014 10.163 13.043 27.132 20.648 10.791 10.853 15.041 14.395 19.167 12.602 7.738 17.699 15.152 14.545 12.459 10.109 16.908 12.217 23.383 4.563 10.425 14.545 18.750 63 APPENDIX 3 Percent'moisture contents of lettuce under 5 different treatments after 5,6, and 7 weeks in storage.  0 Al A2 K A2k 95.635 94.793 95.128 95.181 94.945 94.628 94.898 95.332 94.779 94.947 95.227 95.233 95.914 95.897 94.985 95.655 95.454 95.423 95.061 95.274 95.825 94.231. 95.642 95.556 94.814 94.996 94.326 95.650 95.517 94.947 95.618 94.274 95.338 95.055 94.806 95.053 94.894 95.053 95.810 95.146 94.040 95.891 9B7T2c1 9TT7515 W^U 95.267 93.345 95.782 95.079 94.826 94.831 94.647 95.474 95.469 95.119 94.776 95.832 95.007 94.557 95.089 94.352 95.189 94.503 94.242 93.841 94.837 94.966 92.225 94.182 94.861 94.596 94.712 93.995 94.071 94.969 94.582 95.185 95.095 94.600 93.314 94.251 94.243 93.125 93.303 95.016 94.412 94.092 93.718 94.494 94.532 93^911 93.884 93.815 94.057 95.226 94.016 93.016 . 92.467 93.127 95.242 94.632 94.436 93.869 93.751 93.585 94.355 94.107 92.973 94.122 94.473 94.988 93.835 93.007 93.235 93.821 94.260 93.439. 92.813 94.150 94.110 94.676 94.744 94.177 9l~9Tl 94.484 94.415 94.856 94.115 93.960 95.238 94.219 93.916 94.471 94.488 94.638 94.368 93.980 94.472 94.317 94.404 94.075 94.318 94.340 94.701 94.789 93.964 94.315 94.785 94.294 94.391 94.530 94.246 94.160 94.445 94.604 93.464 94.215 94.783 94.443 93.683 93.781 93.388 93.940 94.240 94.570 93.596 93.721 '93.966 94.618 94.772 94.168 93.770 94.134 94.254 94.020 94.190 93.870 94.074 96.851 94.923 94.509 93.935 94.155 94.251 94.547 94.000 93.972 93.756 94.381 93.852 93.886 93.583 95.553 94.103 94.107 94.057 93.537 94.512 • .193.809 95.020 "9X264 i 947^ 09" 9476T8" 9 T 3 T 8 9 4 T 2 2 F 93.879 94.902 93.985 94.913 94.287 94.179 94.678 94.079 94.318 93.911 94.258 94.250 94.472 93.952 94.379 93.711 94.108 94.065 94.019 94.57.0 93.921 94.993 94.951 94.184 94.478 94.469 94.377 94.268 94.737 94.398 94.455 94.511. 93.711 1 94.690 94.560 64 APPENDIX 4 Chlorophyll A contents (mg/gmFW) of lettuce under 5 different treatments after 5,6, and 7 weeks in storage. 0 Al A2 K A2K 0.3512 0 5 0 9 0.3055 0 4 1 2 0.2859 0.3269 0.4589 0.4647 0.4858 0.6374 0.2944 0.4383 0.4841 0.3974 0.4111 0.3704 0.4067 0.4192 0.4083 0.5671 0.3315 0.6125 0.3653 0.4127 0.4060 0.4664 0.5067 0.5330 0.4807 0.4383 0.3764 0.7032 0.3745 0.5913 0.5165 0.2096 0.4240 0 . 4 3 7 2 0 . 3 8 5 4 0.5245 0.7138 0.3799 0.4787 0.5130 0.6145 0.4220 0.8271 0.4518 0.4348 0.4379 0.5320 0.4526 0.3426 0.5315 0.4628 0.4175 0.3598 0.4496 0.5637 0.5926 0.7149 0.3689 0.4763 0.6710 0.8713 0.4974 0.5203 0.5236 0.6215 0.5979 0.4585 0.4379 0.6008 0.7625 0.5317 0.5284 0.4372 0.5340 0.5174 0.6593 0.3995 0.6007 0.4713 0.5206 0.5450 0.3519 0.4345 0.5570 0.5355 0.5142 0.3836 0.4642 0.6301 0.6502 0.4046 0.4726 0.4660 0.6025 0.6207 0.4730 0.3621 0.5436 0.6946 0.5253 0.6407 0.5969 0.5050 0.4797 0.5502-2 0.5481 0.5083 0.5351 0.5591 0.5418 0.5487 0.4622 0.6339 0.4662 0.5845 0.5921 0.4859 0.4718 0.4462 0.5249 0.4681 0.5420 0.4264 0.6000 0.6789 0.5336 0.4883 0.5304 0.4831 0.6060 0.5978 0.5342 0.4532 0.5899 0.6561 0.5304 0.4226 0.5672 0.5417 0.5884 - 0.5007 0.5295 0.4742 0.3370 0.6418 0.5312 0.4747 0.4756 0.4463 0.3999 0.5211 0.7848 0.4872 0.4245 0.5319 0.6651 0.3697 0.4970 0.4008 0.4601 0.7249 0.6393 0.2647 0.3722 0.4363 0.3745 0.4960 0.5587 0.3723 0.5566 0.3196 0.3563 0.2548 0.4575 0.4048 0.3182 0.4420 0.4698 0.2616 0.4666 0.5094 0.5179 0.4555C 38310.;3834 0.4559 0.5514 0.6501 0.4751 0.5706 0.5713 0.5709 0.3118 0.2686 0 . 4 8 6 5 0 . 5 0 1 3 0 . 3 7 2 2 0.5091 0.3468 0.3380 0.4775 0.4383 0.5266 0.4751 0.5385 0.4539 0.4328 0.5120 0.4400 0.5901 0.6588 0.5106 0.4857 0.3600 0.4835 0.5202 0.5964 0.5731 0.4453 0.5077 0.6436 0.5103 0.4424 0.4574 0.3580 0.5851 0.5603 0.4298 0.3016 0.4615 0.6480 0.5371 0.3602 0.4566 0.5614 0.5815 0.5720 65 APPENDIX 5 Chlorophyll A contents (mg/gmOFW) of lettuce under 5 different treatments after 5,6, and 7 weeks in storage.  0 Al A2 K A2K 0.3503 0.4290 0.3079 0.3273 0.2635 0.2960 0.4486 0.5072 0.4227 0.6116 0.2974 0.4529 0.5205 0.4495 0.3907 0.3633 0.3991 0.4443 0.4083 0.5691 0.3505 0.5158 0.3833 0.4219 0.4121 0.4719 0.4632 0.6208 0.5000 0.4364 0.4137 0.6373 0.3799 0.5887 0.4863 0.2101 0.4060 0.4346 0.4153 0.5609 0^9TTj 073B74" 074135 0.4542 07S27T 0.3858 0.8089 0.4186 0.3859 0.3900 0.4889 0.4222 0.3134 0.4751 0.4095 0.3696 0.3598 0.4008 0.5090 0.5093 0.6599 0.3597 0.4266 0.6176 0.7872 0.4617 0.4924 0.4760 0.5796 0.5232 0.4007 0.4205 0.5702 0.7090 0.4760 0.5076 0.4174 0.4991 0.4576 0.6232 0.3385 075771 0.3556 0.3815 0.5097 0.2909 0.3631 0.4529 0.4064 0.4236 0.2985 0.3924 0.4773 0.5318 0.3762 0.3770 0.3255 0.4574 0.4659 0.4278 0.3175 0.4740 0.6160 0.4250 0.5573 0.5576 0.4226 0.3888 0.4458 0.5056 0.4669 0.4317 0.4614 0.4354 0.4425 0.411V: 0.5009 0.3425 0.5028 0.4942 0.4518 0.3903 0.3864 0.4678 0.3989 0.4687 0.3732 0.5147 0.5937 0.4604 0.4109 0.4740 0.4068 0.5374 0.5064 0.4513 0.3815 0.5120 0.5843 0.4546 0.3552 0.4643 0.477T 0.5311 0.4462 0.4804 0.3985 0.2645 0.5807 0.4671 0.4201 0.3949 0.3734 ' 0.3350 0.4204 0.4952 0.4205 0.3396 0.4689 0.5486 TJ727T7 073757 OS39 073879 075430" 0.5011 0.1976 0.3322 0.3686 0.3336 0.4087 0.4538 0.3080 0.4756 0.2521 0.2907 0.1850 0.3596 0.3537 0.2713 0.3371 0.3574 0.2121 0.4082 0.4380 0.4154 0.3579 0.3049 0.3913 0.4577 0.5193 0.3953 0.5310 0.4442 0.5061 0.2487 0.2043 0.4081 0.3947 0.3251 0.4899 0.2579 0.3073 0.3946 0.3694 0.4536 0.4107 0.4730 0.3985 0.3215 0.4041 0.4007 0.5096 0.5979 0.4055 0.3837 0.3131 0.4344 0.4523 0.4346 0.4548 0.3973 0.4526 0.5468 0.4370 0.3576 0.3998 0.3303 0.4816 0.4754 0.3673 0.2640 0.4149 0.5384 0.4715 0.2760 0.4358 0.5028 0.4969 0.4648 66 APPENDIX 6 Chlorophyll B contents (mg/gmFW) of lettuce under 5 different treatments after 5,6, and 7 weeks in storage  0 Al A2 K A2K 0.1714 0.2310 0.1287 0.1658 0.1328 0.1181 0.2119 0.2497 0.2718 0.4494 0.1275 0.1859 0.1884 0.1603 0.1586 , 0.2000 0.2751 0.2548 0.1636 0.2484 1 0.1373 0.3620 0.2154 0.2230 0.1836 0.2548 0.1773 0.2814 0.1687 0.1859 0.1574 0.4438 0.1639 0.4299 0.2880 v 0.2211 0.2031 0.2054 0.1671 0.2772 o 0.3612 0.2152 OBT1 0.3086 0.2560 0.2498 0.4510 0.2741 0.2064 0.1994 0.2607 0.2297 0.1481 0.2648 0.2562 ? p.1713 0.1947 0.2539 0.2881 0.3299 c 0.3607 0.2331 0.2565 0.3421 0.4274 0.2193 0.2846 0.2853 0.2838 0.2933 0 ??5 r 0.2255 0.2090 0.1698 0.4064 0.2627 0.3215 0.2245 0.2427 0.2705 0.3067 0.2398 0.3477 0.2604 0.2520 0.2650 0.1845 0.2486 0.3367 0.3186 0.2985 0.2711 0.2538 0.3759 0.4060 0.1957 , 0.2886 0.2875 0.2628 0.3988 0.2655 1 0.1938 0.2771 0.4110 0.3189 0.3716 0.3511 0.3004 0.2074 0.2878 0.3460 0.2914 0.3513 0.3186 0.2931 0.3114 r 0.072718 0.4700 0.2664 0.3121 0.3244 0.2507 0.2564 0.2247 0.3229 0.2694 0.3504 0.2213 0.3537 0.3715 0.2658 0.1905 0.2748 0.2557 0.3015 0.2742 ? 0.3000 0.2028 0.2851 0.3348 0.2748 c 0.2152 0.2675 0.2740 0.2589 0.2983 0.2828 0.2746 0.1771 0.3023 0.2477 0.2706 0.2625 0.2438 0.2167 0.3168 0.4471 0.3573 0.2182 0.3009 0.3350 o^rrsi our? orrwi ^mr- OTW 0.3339 0.1223 0.1839 0.2325 0.1734 0.3002 0.2902 0.1935 0.2909 0.1823 7 i 0.2057 0.1397 0.2240 0.2032 0.1752 1 0.2322 0.2247 0.1197 0.2528 0.2718 0.2856 0.2516 0.2138 0.2380 0.2873 0.073'774 0.2570 0.3063 0.3194 0.3138 0 | 0.1625 0.1571 0.2658 0.2541 0.1839 ' 0.2547 0.2000 0.1882 OTBT 0.2642 0.2878 0.2665 0.3021 0.2560 0.2435 0.2689 0.2789C 0.3424 0.3796 0.2809 ? 0.2623 0.1832 0.2919 0.2655 0.2767 c 0.2842 0.2423 0.2763 0.3456 0.2733 0.2091 0.2546 0.2203 0.3463 0.3181 0.2600 0.1628 0.2874 0.3075 0.3141 0.2002 0.2320 0.2986 0.3191 0.3038 67 APPENDIX 7 Chlorophyll B contents (mg/gmOFW) of lettuce under 5 different treatments after 5,6, and 7 weeks in storage  0 Al A2 K A2K  0.1710 072750" 0.1297 0'.1567 0'.1224" 0.1069 0.2072 0.2726 0.2365 0.4312 0.1288 0.1921 0.2025 0.1814 0.1508 0.1961 0.2699 O.270JI 0.1636 0.2493 0.1452 0.3048 0.2261 0.2279 0.1864 0.2578 0.1621 0.3277 0.1755 0.1851 0.1730 0.4023 0.1662 0.4280 0.2712 0.2216 0.1945 0.2042 0.1801 0.2964 0.3497 0.1997 0.2403 0.2732 0.2196 0.2284 0.4411 0.2539 0.1832 0.1776 0.2396 0.2126 0.1355 0.2366 0.2367 0.1517 0.1947 0.2264 0.2601 0.2835 0.3329 0.2273 0.2298 0.3149 0.3861 0.2036 0.2694 0.2593 0.2647 0.2567 0.1971 0.2007 0.1611 0.3779 0.2352 0.3088 0.2144 0.2268 0.2392 0.2899 0.3051 0.1965 0.1846 0.2479 0.1525 0.2077 0.2737 0.2418 0.2459 0.2110 0.2145 0.2848 0.3321 0.1819 0.2302 0.2oo8 0.1995 0.2994 0.2401 0.1699 0.2416 0.3645 0.2580 0.3233 0.3280 0.2513 0.1681 0.2331 0.3192 0.2677 0.2834 0.2629 0.2356 0.2511 0.2417 0.3713 0.1957 0.2685 0.2707 0.2331 * 5T2T21 0.1946 0.2878 0.2296 0.3031 0.1937 0.3034 0.3249 0.2293 0.1603 0.2455 0.2153 0.2674 0.2323 0.2534 0.1707 0.2475 0.2981 0.2355 0.1809 0.2190 0.2414 0.2338 0.2658 0.2566 0.2307 0.1390 0.2735 0.2177 0.2394 0.2180 0.2040 0.1815 0.2556 0.2821 0.3083 0.1746 0.2653 0.2763 0.1584 " 0.2287 0.1609 0.2283 . 0.3309 u0i2617 0.0913 0.1642 0.1965 0.1545 0.2473 0.2371 0.1642 0.1965 0.1545 0.1678 0.1014 0.1760 0.1776 0.1494 0.1771 0.1710 0.0971 0.2211 0.2337 0.2290 0.1976 0.1700 0.2043 0.2384 0.3014 0.2138 0.2850 0.2483 0.2782 0.1296 0.1195 0.2230 0.2000 0.1607 " 0.'24'5,ll •'• b'1487 oTTTTi 572774" O22T 0.2478 0.2304 0.2654 0.2248 0.1809 0.2122 0.2540 0.2957 0.3445 0.2231 0.2072 0.1593 0.2622 0.2309 0.2016 0.2256 0.2161 0.2463 0.2936 0.2341 0.1690 0.2225 0.2033 0.2850 0.2699 0.2222 0.1425 0.2583 0.2555 0.2757 0.1534 0.2214 0.2674 0.2727 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 Rep. T r e a t m e n t  0 Al ' A2 . K A2K ~ ™ ~ S3226" 09T5U 0332 0~oT5o" O W 0.4451 0.6c708 0.7144 0.7576 1.0868 0.4219 0.6241 0.6724 0.5577 0.5697 1 0.5704 0.6819 0.6740 0.5719 0.8155 1 0.4688 0.9745 0.5807 0.6357 0.5896 0.7212 0.6840 0.8144 0.6494 0.6241 0.5338 1.1470 0.5384 1.0212 0.8045. 1 0.4307 0.6271 0.6426 0.5525 0.8017 0 TT0719 075951 0.7438 0.8216 0.8705 0.6718 1.2781 0.7259 0.6412 0.6373 0.7927 0.6805 0.4907 0.7963 0.7191 9 0.5888 0.5545 0.7036 0.8518 0.9225 L 1.0756 0.6020 0.7328 1.0131 1.2987 0.7167 0.8049 0.8088 0.9053 0.8912 0.6840 0.6469 0.7706 1.1689 0.7945 0.8499 0.6618 0.7767 0.7879 0.9660 0.6393 0.9484 0.7318 0.7726 0.8100 0.5364 0.6831 0.8937 0.8541 0.8127 0.6547 0.7180 1.0060 1.0562 0.6003 , 0.7612 0.7535 0.8653 l.ol95 0.7385 1 0.5559 0.8207 1.1056 0.8442 1.0123 0.9480 0.8053 . 0.6870 0.8380 0.8941 0.7996 0.8864 0.8777 0.8349 0.8600 £ 0.7340 1.1039 0.7325 0.8967 0.9165 o 0 > 7 3 6 6 0.7282 0.6709 0.8478 0.7375 0.8924 0.6477 0.9537 1.0503 0.7995 0.6788 0.8052 0.7388 0.9075 0.8721 9 0.8342 0.6560 0.8750 0.9909 0.8052 c 0.6377 0.8347 0.8157 0.8473 0.7990 0.8123 0.7488 0.5142 0.9441 0.7789 0.7452 0.7381 0.6901 0.6166 0.8379' 1.2319 0.8445 0.6427 0.8328 1.0000 0.5853 0.7987 0.5999 0.7308 1.0968 0.9733 0.3870 0.5561 0.6689 0.5480 0.7962 0.8507 0.5657 0.8475 0.5019 , 0.5619 0.3945 0.6815 0.6081 0.4934 1 0.6742 016945 0.3813 0.7194 0.7812 0.8035 0.7071 0.5972 0.6939 0.8387 1.0275 0.7321 0.8769 0.8907 0.9947 2 0.4743 0.4252 0.7523 0.7553 0.5561 ' 0.7638 0.5468 0.5262 0.7527 0.7026 0.8144 0.7417 0.8406 0.7099 0.6764 017808 0.7189 0.9325 1.0384 0.7915 9 0.7480 0.5431 0.7753 0.7857 0.8732 L 0.8574 0.6876 0.7804' 0.9892 0.7837 0.6514 0.7120 0.5783 0.9314 0.8784 0.6899 0.4644 0.7489 0.9555 0.8512 0.5605 0.68860.8599 0.9066 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 Rep. Treatment 0 Al_ A2 K A2K 0.5213 0.6450 0.4367 0.4840 0.3859 0.4030 0.6558 0.7798 0.6592 1.0428 0.4262 0.6450 0.7230 0.6309 0.5415 0.5594 0.6690 0.7144 0.5719 0.8184 0.4957 0.8207 0.6094 0.6498 0.5986 0.7289 0.6253 0.9485 0.6755 0.6215 0.5867 1.0396 0.5461 1.0166 0.7574 0.4317 0.6005 0.6389 0.5954 0.8573 1.0407 075572 0737T3" 077274" 0.7467 0.6142 1.2500 0.6726 0.5691 0.5676 0.7286 0.6348 0.4488 0.7117 0.6362 ? 0.5212 0.5545 0.6271 0.7691 0.7928 0.9929 0.5870 0.6564 0.9325 1.1732 0.6652 0.7618 0.7352 0.8443 0.7798 0.5978 0.6212 0.7313 1.0868 0.7113 0.8163 0.6318 0.7259 0.6968 . 0.9131 0.5416 0.8322 0.5520 0.5662 0.7576 0.4433 0.5708 0.7266 0.6482 0.6695 0.5095 0.6069 0.7621 0.8638 0.5581 0.6072 0.5263 0.6568 0.7653 0.6680 1 0.4874 0.7156 0.9805 0.6830 0.8806 0.8855 0.6739 0.5569 0.6789 0.8248 0.7346 0.7150 0.7243 0.6710 0.6936 r? 0.6528 0.8722 0.5382 0.7714 0.7649 0.6849 0.6024 0.5810 0.7556 0.6285 0.7718 0.5670 0.8180 0.9186 0.6897 0.5712 0.7195 0.6221 0.8048 0.7386 9 0.7047 0.5522 0.7594 0.8824 0.6901 0.5361 0.6833 0.7185 0.7649 0.7120 0.7370 0.6292 0.4035 0.8542 0.6848 0.6595 0.6129 0.5774 0.5165 0.6706 0.7773 0.7298 0.5142 0.7343 0.8249 0~4T0D 075051 OM§ OT61 079759 0.7628 0.2289 0.4964 0.5651 0.4881 0.6560 0.6910 0.4681 0.7241 0.3959 0.4585 0.2865 0.5356 0.5313 0.4207 0.5142 0.5284 0.3092 0.6293 0.6717 0.6444 0.5555 0.4749 0.5956 0.6961 0.8207 0.6092 0.8160 0.6926 0.7843 7 0.3783 0.3237 0.6311 0.5947 0.4858 / 0.7350 0.4066 0.4784 0.6220 0.5921 0.7014 0.6411 0.7384 0.6233 0.5024 0.6163 0.6547 0.8053 0.9424 0.6285 9 0.5909 0.4724 0.6965 0.6832 0.6363 c 0.6803 0.6134 0.6989 0.8404 0.6711 0.5266 0.6223 0.5335 0.7666 0.7453 0.5895 0.4065 0.6732 0.7939 0.7472 0.4294 0.6572 0.7703 0.7696 0.7116 

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