KEEPING QUALITY AND ABSCISSION OF ILEX VERTICILLATA (L.) GRAY (AQUIFOLIACEAE) FRUIT Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY HUGH CHRISTGPHER BOYLAN I 1975 LIBRAR 1' ‘3"? Michigan State University This is to certify that the thesis entitled Keeping quality and abecieeion of flex verticillata (L.) Gray (equifoliaceae) fruit presented by Hugh Christopher Boylen has been accepted towards fulfillment of the requirements for _. _ Eh. .n._degree in We me: at: Major professor Date Ma 12 1 ‘ I 0-7639 K III . I; ‘ LIBRARY muons . t: M 33min!!- , A '7 I) ABS'IRAOT KEEPING QUALITY AND ABSGISSION 0F ILEX VERTIOILLATA (L.) GRAY (AQUIFOLIACEAE FRUIT 3! Hugh ChristOpher Bovlsn SECTION I. KEEPING QUALITY. ABSCISSION AND LEVELS OF 02. 002 AND ITHYLENE DURING STORAGE OF FRUITING BRANCHES 0F ILBX mrrcuu'm (L.) can "'— Erniting hrenches of Ilex verticillsts (L.) Grey were stored st 9 environnsnt conbinstions of low. internediste end high relstiwe hnnidities (RI-l) in 0°; 8.8° or 10° end 20°C chsnbers for 0. period of 8 weeks in 1973 end repented in 197k. In 1973. wster loss es indexed by weight loss of fruit. twigs and lesves wss nininised with mic/991 RH. Leswes present on these twigs st 20°C end high hunidities turned brown end were shed within 3 weeks. Fruit stored in high.RH st 00 end 8.8°c reneined in good condition for the entire eXperinent. The noisture content of fruit. in both 1973 end 197“. wss only ssrginslly reduced when stored in high RH st 0°C wheress fruit quslity deterior- sted before week 2 st s11 other conbinstions of tenpersture end low or internediste an. By week 5 fruit stored st 20°0/9ux an m shrivelled. It wee observed thst red fruit when dried. in en oven for noisture detersinstions. fsiled to turn brown if the soisture was 203 or less. Abscission. indexed by reduction in trait rencvel force (FRI). wes correlsted with vepor pressure deficits (VPD) during stcrsge. Hugh Christopher Boylan Usually the fruit separated froa the pedicel (distal type) with 60% frequency. pedicel separation fro: the star (proxinal type) was 30% and breakage at any point along the pedicel (internedial type) was 10%. Frequencies of the distal type were least and that of the proxinal type were largest at 20°C and high hulidities. Ethylene concentration (due to fungal contuinstion) in chaabers at high hunidity and 20°C reached 315 ppb at the end of week it. Reduced values of m were recorded for fruit in these chanbers. Carbon dioxide concentrations renained below 0.5% in all chaabers and the ninilun levels of oxygen were 20%. A study on ethylene production showed that green. red-green and red fruit produced peaks of 5.8. 16.6 and 1.1 pl/kg/hr at 54}. 6'} and 9} days after harvest respect- ively. MION II. THE mwr OF ANTIMSPMTS 0N KEEPING QUALITY 01' FRUITING BRANCHES O? 1181 VEHCILLATA (L.) W! Studies on the effect of wax and plastic antitranspirant naterials at concentrations of 2.5 and 5% were conducted on fruiting branches of W (L.) Gray. Vapor Gard treataents reduced loss in fresh we ight. water uptake and transpiration. Hater uptake for 5% Vapor Gard treataents was less than those at 2. 55. Twigs treated with Fri-afresh 23 at both concentrations. showed an increase in water uptake. Fruit treated with Vapor Gard renained in excellent condition until day 23 when shrivelling occurred. .11 other treateents shrivelled by day 17. Respiration rates of fruit treated with Vapor Card and Concord Alcoat 88-12 did not differ fron that of the control. Hugh Christcpher Doylan Cross sectional views of the epicarp (exocarp) under plane polar- ised light identified the cuticle as being isotrOpic. Scanning electron nicrcgraphs of Vapor Gard treatuents confirled its presence on the surface of I. verticillata fruit. Surfaces of young I. cpgg; fruit were heavily coated with a snooth wax which was covered with another war in the fore of spheres. These spheres were absent free old fruit of I. 0332; and the continuity of the underlying surface was was broken. SICTION III. PHYSIOLOGY or man nscissron IN Irrx mmmxn (L.) GRAY Iield.and laboratory experinents were conducted to deter-ins the fruit abecission process of Ilex verticillata (L.) Gray. Eruit renoval force (In!) of fruit free branches treated with naphthaleneacetic acid (NAA). cyclchexiaide (CH). gibberellic acid A3 (GA) . sodius aside and (z-chloroethyl) phosphonic acid (ethephon) and non treated branches were sinilar. Fruit and leaf abscission occurred on hranches treated with ethephon and CH. Histological and histochenical studies revealed the presence of sclereids along the entire length of the pedicel with no structural or staining differences indicative of abscission layer fornation. FRF'increased fros July to Septesber. then decreased to July values. Approrinately 50% of fruit with pedicels attached abscised Just before leaf fall. Proxinal applications of CH. NAA. sodius aside. calciua chloride. sucrose. 8 hydreryquincline sulfate (8 308) and alars and distal appli- ‘ cations of CH. NAA. sodius aside. calciun chloride. kinetins and bensyladenine caused loss in fresh weight, for detached fruiting branches. Hugh Christopher Doylan which was sieilar to or greater than the control. Loss in fresh weight was increased when polyvinylpyrrolidone ( a phenol inhibitor) was included with proximal treat-cuts. And antitranspirant applications reduced loss in fresh weight. MICK IV. THE m 0" mm. RELATIVE mm AND "IND ON ”011' “301881011 0" 1m MCIIMTA (In) (RAY Killing tenperatures for fruit and pedicels of Ilex verticillata (1...) Gray were between 40°C and ~11°C and -12°C and -13°C respectively. Fruit reacval force (an) of fruiting branches exposed to -18°C were less than those exposed to -9°C. m for branches subsequently stored at 20°C was lower than 0°C. F8! was significant for the inter- action between storege teaperature and high and low relative huaidities (RI-I) after 16 days. Fruiting branches when subjected to a nexisue wind fame of 20.“ leters per second retained 1005 of their fruit. It was considered that Ilex verticillata fruit and pedicels are killed by low teaperatures abscise when tenperatures fluctuate above 0°C and heavy winter stores occur. KEEPING QUALITY AND ABSCISSION OF ILEX VERTICILLATA (L.) (BAY (AQUIFOLIACEAE) FRUIT By Hugh ChristOpher Boylan A DISSETATION Submitted to Michigan State University in partial fulfillmnt of the require-ants for the degree of DOOM OF PHILOSOPHY Departaent of Horticulture i975 Dedicated to my daughter Hichelle ii ACKNOWLEDGMENTS The author expresses his sincere appreciation to Dr. Harold Davidson for valuable suggestions and direction during the course of uy graduate progras. Gratitude is extended to Drs. H.J. Bukovac, R. Herner. I.H. Knobloch, R.A. Necklenburg and H.P. Raseussen for counsel and assistance on my guidance cceeittee. For suggestions. help and use of laboratory facilities I an indebted to Drs. A. De Hertogh. D.H. Dewey. DJl. Dilley. JJ'. Foss. 0.1!. Hocper, H.P. Raseuseen and Hr. Philip Nott. Appreciation is also extended to Hidden Lake Gardens Trust Fund for financial aid. without which this study would not have been possible. I A special thanks is extended to ny wife Hairead for her patience. encouragesent and assistance during this prograa of study. 111 TABLE OF CONTENTS Page LIST OF TABLES e e e e e e e e e e e e e e e '1 LIST OF FIGURES e e e e e e e e e e e e e e e V11 INTRODUCTION e e e e e e e e e e e e e e e 1 menm 1mm . . . . . . . . A. . . . . . 3 SECTION I KEEPING QUALITY ' ABSCISSION AND LEVELS OF 02, 002 AND ETI‘IYLENE DURING STORAGE OF FRUITING BRANCHES OF ILEX VETICILLATA (L.) CRAY e e e 5 MATERIALS AND METHODS e e e e e e e e e e e e 8 RESULTS e e e e e e e e e e e e e e e e e 11 DISCUSSION e e e e e e e e e e e e e e e 29 SECTION II THE EFFECTS OF ANTI'IBANSPIRANTS ON KEEPING QUALITY OF FRUITING BRANCHES OF IIEX VERTICILLATA (L.) GRAY . . . . . . . . 36 MATERIALS AND MODS e e e e e e e e e e e e 39 REULTS e e e e e e e e e e e e e e e "'1 DISCUSSION e e e e e e e e e e e e e e e 61 SECTION III PHYSIOImY 0!“ FRUIT ABSCISSION IN ILEX VHH'ICILLATA (L.) GIAI e e e e e e e e 63 MATERIALS AND METHODS e e e e e e e e e e e e 66 RESULTS e e e ' e e e e e e e e e e e e 70 DISCUSSION e e e e e e e e e e e e e e e 88 SECTION IV THE EFFECT OF TEWATURE. RELATIVE HUMIDITY AND "IND ON FRUIT ABSCISSION OI" ILEX VERT- ICILLATA(L.)GRAY e e e e e e e e . 91 iv HATFRIALS AND METHODS . RESULTS e DISCUSSION . SUMMARY AND CONCLUSIONS LIST 0]" REFERENCES Page 95 103 105 108 Thble 1. 2. 3. u. 1. 2. 3. u. 5. 1. 2. LIST OF TABLES SECTION I Loss in fresh weight 0%) of fruiting branches at weeks 3. h} and 6 as affected by tenperature. relative husidity and vapor pressure deficit . . . . . . . . . . . Noisture content 0%) of fruit at weeks 1. 2 and 8 of storage as affected by teaperature. relative husidity and vapor pressure deficit . . . . . . . . . . . Fruit reeoval force as affected by vapor pressure deficit, temperature and relative humidity . . . . . . moisture content Ci) of fruit as affected by vapor pressure deficit, temperature and relative humidity . . . SECTION III Fruit removal force as affected by growth regulators in a field eXperiuent e e e e e e e e e e e Loss in fresh weight (i) of fruiting branches as ‘ffOCLOd by various chemical media e e e e e e e e Loss in fresh weight CI) of fruiting branches as affected by addition of polyvinylpyrrolidone to the media . Loss in fresh weight 0%) of fruiting branches as affected by Vapor Gard. Coon Latex and control . . . . Loss in fresh weight (%) of fruiting branches for the interaction between polyvinylpyrrolidone and various media. SECTION IV Freezing and killing temperatures °C of fruit when super COOIOd St 10°C/hour e e e e e e e e e e e Number of pedicels alive after subfreezing teeperature treataents e e e e e e e e e e e e _e e e e vi 12 12 16 23 71 80 81 81 83 LIST OF FIGURES Figure 1. 2. 3. h. 5. 6. 7. 8. 1. 2. SECTION I lose in fresh weight (5) of fruiting branches and aoisture content (%) of fruit in 1973 . . . . . . . Loss in fresh weight (5) of fruiting branches after 1+} weeks and soisture content (9‘) of fruit at weeks 2 and 8 as affected by temperature and relative hunidity . Fruit renoval force (g) and frequency (5%) of distal separation as affected by temperature and relative hunidity e e e e e e e e e e e e e e e e Moisture content (S) of fruit and twigs in 1971; as affected by temperature and relative hunidity . . . . Fruit removal force (g) at weeks 1 and 6 and soisture content (x) of fruit at weeks 2 and 5 as affected by teeperature and relative humidity . . . . . . . . Ethylene concentrations (ppb) at 20°C: fruit reeoval force (g) and C02 (95) concentrations at high relative hueidity as affected by teaperature . . . . . . . Ethylene production rate of fruit . . . . . . . The pedicel viewed under the dissecting eicroscOpe . . SECTION II Loss in fresh weight (g) and water uptake {-1) of fruiting branches as affected by Vapor Gard. Concord Alcoat and COfltIOl e e e e e e e e e e e e Loss in fresh weight (g). water uptake (el) and trans- piration (ml) of fruiting branches as affected by Vapor Gard, Concord Alcoat. other treatments and control. vii 13 19 21 2a 26 33 “3 “5 Figure Puss 3. Respiration rate of fruit as affected by Vapor Card. Concord Alcoat and control . . . . . . . . 47 a. Cross sectional views of fruit epicarp . . . . . . 50 5. Asphondylia illicifolia Foote . . . . . . 52 6. Morphology of fruit surface (SEM) . . . . . . 53 7. Morphology of fruit surface (SEM) . . . . . . 55 8. Morphology of fruit surface (SEM) . . . . . . 57 9. Morphology of fruit surface (SEM) . . . . . . 59 SECTION III 1. Longitudinal sections of the pedicel . . . . . . 73 2. Longitudinal sections of the pedicel . . . . . . 75 3. Frequency 0%) of abscission types and fruit resoval force (a) fro. July 8 through October 13. 1970 . . . 77 h. Loss in fresh weight 0%) of fruiting branches as affected by the interaction between polyvinylpyrr- olidone and chemical eedia; Vapor Card and control . . 80 5. Loss in fresh weight (%) of fruiting branches for the interaction between Vapor Card and cheuical nedia OhleIOlflddlYIJeeeeeeeeeeeeeSé SECTION IV 1. Fruit reeoval force as affected by subfreesing and storagetenperatures............97 2. Eruit renoval force for the interaction between storage tesperature and relative huuidity on day 16 . . 99 3. Effect of subfreezing temperatures on pedicel and fruit. 101 viii Guidance Coaaitteei The Paper-Format for this dissertation was adopted.in accordance with departeental and university regulations. The dissertation body was separated into 0 sections. Each section was styled for public- ation in the Journal of the Aaerican Society for Horticultural Science. INTRODUCTION Ilex. coaaonly called holly, is a genus within the plant faaily Aquifoliaceae. Soae evergreen species (Ilex agnifoliua L. English holly and Ilex opgca Ait. Aaerican holly) are coasercially produced for the use of out branches in Christ-as decorations. This utilis- ation of holly is not restricted to evergreen types. aany deciduous species can be used for floral arrangesents. One such species- Ilg§_ verticillata (L.) Gray (co-non winterberry) is coaaon in low lying areas throughout the state of Michigan.where it fruits abundantly. In the past. fruiting branches were obtained by destructive cutting of the plants in their natural habitat. Land owners then prohibited trespassers and the popularity of this shrub declined. Sole people (along then Mr. Harry A. Fee, for-er owner of Hidden Lake Gardens in the Irish Hills near Tipton. Michigan) transplanted several plants into landscapes and others atteapted to cultivate speciaens as a row crop. The nest recent local entrepreneur in this area is Mr. Philip Mott of Kala-asoo, a veteran of the old holly harvesting systee. He has successfully propagated over 25,000 plants and has plans for further expansion. Very little scientific inforaation on the prop- agation of this species and post harvest physiology of the fruit was available. The need for research in this area was sensed by Dr. Harold Davidson who had been interested in Michigan holly for acne years when he becaae faeiliar with Mr. Mott's efforts. An M.S. prograa by the present author was devoted to propa- gation aethods and keeping quality. The present Ph. D. dissertation 1 ',,.- 2 deals with keeping quality and fruit abscission of detached branches. When harvested and placed in floral arrange-ants. the fruit on cut branches dried and shrivelled within 7 to 10 days. Moreover. the fruits abscised easily during handling. The following experieents were designed to detersine the optieua storage environsent and to increase the period of keeping quality at rooa conditions. hper- iaents were also conducted to discover the physiology of fruit abscission in the hope that abscission could be delayed by use of growth regulators. In the wild. the aajority of I. verticillata plants reaain heavily fruited until aid winter. Experisents were conducted to evaluate the influence of environsental factors on absc iesion in aid-winter. LITERATURE REVIEW Harvesting of fruiting branches of Ilex verticillgtg'(L.) Gray (con-on winterberry) in the wild was coaaon practice in the early part of this century. Although Ricker in 1920 (86) urged the culti- vation of this species as a row crop. it was not until 19h8 that attespte were aade by Fee (3“). Haablin (an). Neal (72) and.Reinseith (85) to sake this a business siailar to any other fruit crap. Research on post harvest utilisation of fruiting branches of cos-on winterberry was never conducted because their use in Christ-as decorations declined due to unavailability of plant aaterial. In recent years attention has again been directed toward cultivation of cos-on winterberry. Thus infornetion froa research on keeping quality and abscission of fruit is necessary for the grower. florist and consuaer. Production of Ilex gguifoliua L. (English holly) on the northwest coast of the U.S.A. and Ilex opgg; Ait. (Aaerican holly) on the eastern.coast is a coasercial venture for use of holly branches in Christ-es floral arrange-cute. These hollies are popular aainly because of their glossy green evergreen leaves. Discoloration of the leaves followed by abscission occurred during storage at high teap- eratures and high relative huaidities (RH) (87. iiu). This was overcoee by partial drying before packing or by cold storage and application of «(naphthaleneacetic acid at 37 to #0 ppa (27. 35. 68, 69. 87). Storage reconsendations for fruiting branches were 0°c and 85 to 90% an 3 (12. 82. no). The above studies dealt with saintaining keeping quality of ever- green leaves but no infer-ation was available on quality and abscission of fruit. Since I. verticillata is a deciduous holly, the fruit is of aejor iepertance. The objective of the following experisents was to deteraine storage requireaents of fruiting branches of coaeon winter- berry; evaluate the influence of antitranspirants on aaintenance of keeping quality of the fruit and detersine the process of fruit abscission by use of growth regulators and environsental stresses. SECTION I KEEPING QUALITY. ABSCISSION AND ISVELS 01" 02, C02 AND mm HIRING STCBAGI 01" FRUITINC BRANCHES OF IIIX VETICILLATA (L.) GRAY Most horticultural products are stored at low temperature and high relative humidity (RH) whereby moisture loss is reduced to a minimum because the vapor pressure deficit (VPD) is small. In addition, plant material stored at low temperature has a low rate of respiration: aging and ripening processes are slowed down (58). Addit- ionally, spoilage and changes in texture and color are minimised. At a constant temperature. evaporation of water from plants increases with decreasing RH. resulting in wilting or shrivelling of the product (108). To prevent this. high RH is desirable but control of pathogenic organisms becomes necessary (58). As RH decreased from 100 to 65%, water loss fron apples increased and then reached a plateau (97). In grapes, this plateau occurred at uOSZRH. The drying of the outer fruit layers was considered as being the explanation of this phenomenon (9). Most fruits and vegetables contain 80 to 95% water by weight (2“, 58). During storage. loss in fresh weight results principally from transpiration (109). Host fruits shrivel when they lose 5 to 2% of their weight (22). In addition to factors already discussed. the extent of this loss is influenced by a number of factors including product type. sise. cosposition and structure. Hhen environmental conditions exist which cause plants to lose moisture. the initial loss is rapid but eventually stabiliees (97). At constant RH (excluding low values) within a temperature range of 3 to 1590. water loss froa plants is directly related to VPD (24. 97). Recommendations for storage of ornamental plant saterial are contained in handbooks prepared by the American Association of Nursery-en (60) and the U. S. Department of Agriculture (58). Rooted > 7 cuttings and seedlings of marry plant species can be stored at tenperatures slightly above 0°C for h to 6 months (58). To prevent injury to florist greens. bulbous plants and some nursery stock they should not be stored with ripening fruit or other sources of ethylene (58). Since evergreens are in full leaf. particular care is essential to prevent dessication (60). Cut branches of Ilex aquifolium L. (anglish holly) and Ilex 925. Ait. (American holly) can be stored up to '6 weeks at 0°C and 85 to 90% RH (12. 82. 114). Discoloration of the leaves. followed by abscission. was found to occur at high temper- atures and high hunidities (87. 111+). This was overcome by partial drying before packing or by cold storage and application of or. naphthal- eneacetic acid (27. 35. 68. 69. 87). Treated leaves should not be packed with untreated holly to avoid the adverse effect on the untreated leaves of increased ethylene production resulting from auxin treatments (27)- Holh fruit were reported to produce very small amounts of etivlene but the quantity was not specified (68). Defoliation can occur in 3 to it days in response to ethylene levels as low as 5 ppm (68. 69. 82. 87). Despite the presence of ethylene provided by ripening apples. defoliation was prevented for 30 days at 0°c and high RH storage but similar storage at 20% resulted in 255 leaf drop. The objective of this study was to determine how much ethylene is produced by the fruit and to evaluate the effects of temperature. RH and VPD during storage on levels of 02. C02. ethylene and keeping quality of fruiting branches of Ilex verticillata (L.) Gray (com-on winterberry). MATERIAIS AND METHODS Moisture 103M. Fruiting branches with leaves were stored in 9 environnent combinations. 3 temperatureea(O°C. 10°C and 20°C) and within each temperature 3 an (low. intermediate and high) which varied with temperature (Table 1). Low RH (20 to 35%) were achieved utilizing air previously passed through a calcium chloride filter. Intermediate RH (30 to 45%) were obtained using air and high RH (89 to 98%) by first passing air through water. Three replications of each RH were obtained by using 7.5 liter plastic containers within each temperature chamber. The lid was equipped with 2 air-line connectors for an inlet and an outlet. Calculations of RH were obtained with a potentiometer equipped with wet and dry thermocouples. On September 27 branches of Ilex verticillata were harvested from a single plant. In each container. 3 uniformly fruited twigs. approxima- tely 15 cm in length. were inserted in 3 bottles (2.3 cm diameter) containing approximately 75 m1 of water. Each twig was weighed initially and again at 3. W} and 6 weeks to determine lose in fresh weight. The condition of the leaves and fruit were rated each tine. Moisture content of the fruit 1 . Experimental conditions were identical as previously reported. Defoliated plant material was collected on November 23. Five twigs. supported by a wire mesh franc. were placed in each dry container. Data consisted of fresh and. dry weight measurements for which moisture content of fruit in S was calculated at time zero and at 1. 2. 1+. 6 and 8 weeks of storage. For dry weight determinations. fruit were placed in an oven at 65°C for 2 days. 9 Fruit removal force (FRF), moisture content of fruit and twigs and gag analysis 122“. Treatment combinations and replications were similar to the 1973 experiments. Environmental replications were enclosed within cardboard boxes. approximately 0.0“ m3 in volume. encased in 10 um opaque polyethylene bags. These were fitted with a serum cap for withdrawl of gas samples. Low RH within each teap- erature were obtained by placement of dry calcium chloride inside the boxes. Saturated salt solutions of this material at 0° and 8.8°C and lithiua chloride at 20°C were used for establishsent of inter- mediate RH. High humidities occurred in boxes containing distilled water. RH and temperature parameters recorded with thermohydrographs are shown in Table 3. Sasples of air were extracted weekly from each container and ethylene concentrations determined by injection of 1 cc into a Varian deregraph 1700 gas chromatograph. The stainless steel column (0.32 X #6 cm) contained activated alumina at 50°C. The carrier gas was nitrogen and detection was by flame ionisation. Oxygen and 002 levels were determined by similar methods onha vapor fractometer using a parallel column system: a molecular sieve for oxygen and silica gel for 002 with He as the carrier gas. Observation of the separation point and ER! were measured for 20 fruit on a single twig from each replication with a Hunter Mechanical Force Gauge (Hunter Springs. Hatfield. Pa.). Abscission at the point of contact between the fruit and the pedicel was termed distal: between the pedicel and the twig proximal and between any parts of the pedicel intermedial. Moisture content of fruit and twigs was deterained as previously stated as was a subjective rating of fruit quality. At the 10 end of week 8. December 7. the experisent was terminated. VPD calculated for treatment combinations were correlated with data on ERR. Statistical analysis employed was a split plot design with temperature being the main split and 3 replications of RH. Significant differences of means were determined by Tukey's HSD test. (98). Determination of ethylene production rate. On September 22 samples of 2 to a green. red-green and red fruit were placed in 10 m1 syringes. at room temperature. Treatments. including controls. were replicated 3 times. Filter paper soaked in 10% NaOH (for absorption of C02) was attached to the plunger with a pin. An oxygen supply (drawn in by negative partial pressure) was provided from a reservoir and passed through asscnium sulfate. Samples of gas (1 cc) were withdrawn at intervals of 3. 6 or 12 hours and analysed by gas chromatography as described above. The system was flushed with ethylene free air for 15 minutes after each sample was taken and the data plotted on semi- logarithmic paper in pl/kg/hr over a time period from Septeaber 23 to October 13. RESULTS Moisture loss 1222. Eruiting branches stored at 0°C/9811RH showed an increase in fresh weight (by an average of b.9fl) at the end of week 3. all other treatment combinations lost weight (Table 1). Treatments at high tesperature or low RH resulted in greater weight losses than those at low or intermediate temperatures and intermediate or high RH (Figs. 1A and 13). The effect of’RH at the 3 tesperatures for week h} is graphically presented in Fig. 2A. Leaves were blackened and loosely attached in high humidity chasbers at 10° and 20°C on week 3. In 11 contrast. sisilar symptoms were not apparent under 0°C and high husidity until iv} weeks. At 0° and 10°C and high an, fruit remained in excell- ent condition throughout the experiment. The 20°C/89% an treatments develOped fungal growth and were discontinued after 4} weeks. In intermediate and low humidity chambers at 10° and 20°C the fruits were shrivelled and dehydrated and the twigs had lost their leaves by week 3. At 0°C some leaves turned brown but the majority remained green and held well. Fruit quality deteriorated by week 6 in intermediate and low RH at all temperatures. Hoisture content of the fruit 1221. Fruit at 0°c retained more moisture then those at 10° or 20% (rig. in. Table 2). Dehydration at 20°C was rapid and the fruit were brittle with the result that routine handling of the containers on week 2 caused fruit abscission. Low and intermediate humidity effects were sisilar but differed from those of high husidity throughout the experiment (Fig. 1D). The combination of 0°C/98% an consistently maintained more moisture than other combinations such as 35.6% on week 8. The effect of RH at the 3 temperatures for weeks 2 and 8 is graphically presented in Figs. 23 and 20. During oven drying. fruit with a moisture content of 20% or less remained red. while fruit with higher moisture contents turned brown. FRI. moisture content of the fruit and twigs. and gas augeis 1222. Values for proximal. distal and internedial separation were similar. hence overall values were used in computations. Throughout the experimental period. FRF was inversely related to temperature: higher values were consistently recorded for the lowest temperature of 0°c (Fig. 3A). 12 Table 1. Loss in fresh weight (5) of fruiting branches of Ilex vert- icillata (1..) Gray at weeks 3. ‘3' and 6 as affected by temperature ,. (temp). relative humidity (RH) and vapor pressure deficit (VPD). Loss in fresh weight of fruiting branches {5) Teap °C fill-I VPD mm Hg wk.3 whit} wk.6 . _, 7 O 98 0e01 sh.9 9e1 9e2 10 97 0e28 13e4 31e0 37.2 20 89 2.07 31.6 56.6 --- 0 “5 2e52 7e2 10e1 12e2 0 35 2e98 7e3 22e5 2#.l+ 10 “O 5e52 31e5 “2e2 5h.6 10 30 6am 37e2 “e8 56s” 20 30 13e1h 50e“ 63.u -- 20 20 15e01 52e1 72e2 , --- s- a positive value weight increase due to water uptake -- - missing data Table 2. Hoisture content (S) of fruit of Ilex verticillata (L.) Gray at weeks 1. 2 and 8 of storage as affected by temperature (temp). relative humidity (RH) and vapor pressure deficit (VPD). Moisture content of the fruit (2) Tempe °C m VPD mm Hg wkei wk.2 wk.8 0 98 0e01 61e9 61e3 35e6 10 97 0e23 60e2 58e5 25e1 20 89 2e07 31e9 11e2 -- 0 “5 2052 59-7 53.7 19.1 0 35 2e98 60e3 58e2 20e8 10 “0 5e52 52e6 39e5 1e5 10 30 e 52e3 38.h 1e? 20 30 13A.“ 22e8 3e8 --- 20 20 15e01 25e8 a “.8 -- -- - missing data 13 Figure 1 A-D Loss in fresh weight (x): moisture content (I) of fruiting branches of Ilex verticillata (L.) Gray in 1973. as affected by temperature (temp) and relative humidity (RH). A-- loss in fresh weight as affected by temperature sB-- loss in fresh weight as affected by RH C- moisture content of fruit as affected by temperature sD- moisture content of fruit as affected by RH s - means of low and high RH were significantly different at the 0.01 level MOIST. CONTENT OF FRUIT (°o) LOSS IN FRESH WEIGHT (°/o) 80 60 4O 20 1h TEMP RH IA IB - n ,e - .” z A - g, A/ - / .. /A I l I 1 1 l I 1 '0 20°C —u—— ID Low —0— I0°c------ INTERM ------0- 0°C —A—- HIGH --A-- " *' RH -FA‘A A .. \ . ' t \\ "ks - \\. \ - \\A\A \\ A\ \ x \\ - in \ A \8 A\A - .\\ - Q n z\\. - KB 119111111 llanllllfiLj O|2345678 O|2345678 WEEKS 15 At the end of weeks 1 and 2 (Fig. 3B) FRF at high RH were greater than those at low RH. For the following 3 weeks FRF for intermediate RH treatments decreased to levels similar to those of low RH (150 g) and the RF for high RH remained above 200 g. The interaction of humidity and temperature at weeks 1. 5 and 6 was significant (Table 3). At 0°C/ 98% RH (Fig. 63) there was no reduction in my and while at 20°C/91a an there was an initial increase. it later was followed by a rapid decrease. RH effects at the 3 temperatures for weeks i and 6 are shown in Figs. 5A and SB. Estimated VPD correlated with the mean of m are shown in Table 3. In general over 60% of the fruits separated from the pedicel (distal). 30% were reaoved with the pedicel attached (proximal) and the remaining 10% were separated by a beak in the pedicel (inter- medial). Frequencies of the interledisl type did. not fluctuate as much as those of the distal and proximal. Minimum distal frequencies at 0°. 8.8° and 20°C were 69.2. 61.1 and “9.8% respectively (Fig. 30). Similar frequencies for low. intermediate and high humidities were 62.5. 68.3 and 53.9% respectively (Fig. 3D). hoistur. content of the fruit was highest for 0°c treatments (Fig. 1%. Table 15). The levels at week 7 fell to 35.0. 26.0 and 21.95 for 0°. 8.8° and 20°C respectively. Hinisum values for high. intermediate and low humidities were 53.8. 16.0 and 9.“ respectively (Fig. 1&3) and. ssch level from week 2 onward was highly significant. Interactions occurred on weeks 2. 3. lb and 5. while intermediate values of VDD did not demonstrate a consistent trend the combinations of 00/98% an contained 61.9 and 58. six moisture and 20°C/25% an treat- ments retained only 13.2 and 15.6% moisture after 2 and 5 weeks respec- tively (Figs. SC and 5D. Table ‘5). 16 an; up you essences.- eeseuuee I «a soaveseuuoo uses: no useaeameoo I u even goods I 1.... «mo-G 5.2— 639335». I w nwu I can H.333 ensure." «0.0 2.3. as archeHMd—e 3.51330 PM. seabed anachmdu s 3 consume.“ gaoo e 558: case: no.0 «s.e «n.o en.o «n.o ne.o «a as.o eo.o em.e mm.e em.o an.e u -u. one" ens" «on. «me «on some mm e.o~ ma.ma one some ammo men an” emu enemmm ms o.o~ ee.m we" seen eras can man are scenes on m.» mo.m and some emu“ and «us new carom om o.m a~.e «ea ens” echo and so" emu seesaw mm o.e mo.~ was era“ some new «on new cones mm e.e eo.~ omen semen A needs new «mm ens earn no o.o~ no.“ «on eons smnu «mm mmw new oeeoem em n.m en.o ohm nosu seem «am new «em serum no e.o oo.o a.ee e.ae n.as e.xe n.ar ~.xe a.er new no area en en one Auwuummmmummnmuumummmmm .35 guinea—n Redefine“ one Ame—3v changes... saggy finance eflfieean some» 3 uevoemme no .38.. a «o venues e use e83. 33d sens 7.5 3323?... xefi son .88 Hero...» 3.5 .n edfiea 17 Figure 2 A-C A“ Loss in fresh weight (%) of fruiting banshee after 1+4} weeks; Bo- moisture content (%) of fruit at week 2: C- and week 8 of Ilex vert- icillata (L.) Gray as affected by temperature (temp) and relative humidity (RH). Vertical lines indicate standard deviations above the 18 00000 864.2 node #1653 .mm 2. wwo... E H R D E M. R E T W O O O O O O 6 4 2 4 2 noxov .EDmC do PZMFZOQ mm:...w.02 20°C I0°C 19 Figure 3 A-D Fruit removal force (F‘RF) (g) and frequency (%) of distal separation of Ilex verticillata (L.) Cray as affected by temperature (temp) and relative humidity (RH). A— FRF as affected by temp sB- FRI as affected by RH C-D— frequency as affected by temp and RH respectively a - means of low and. high HR were significantly different at the 0.01 level F RR?) FREQUENCY 96 300 250 200 ISO l00 50 IOO 80 60 40 20 20 TEMP RH 3A 38 Cl -§9\u -N \\ \\\ O D n bk Vi“ \. n— \ \\“9\Ka .. V:\\\ U \9\~~2 \§\ _ av? _ EV? .. 0-0°c—n— _. HIGH—D- e-e°c----- INTERMED ----- 20~0°c--v—— LOW--V-- lllLlllll llllllljl 30 30 .. n u T. ~.. KEM‘jfi‘: B‘B fibrin. ' r V\Vr;_ ‘ ‘33 " gay/:25 I— v ' — [Illlllll . Illllllll 0l2345678 O|2345678 WEEKS 21 Figure it A-D Hoisture content (%) of fruit and twigs of Ilex vert- icillata (L.) Cray as affected by temperature (temp) and relative humidity (RH) in 19715 experiment. A--- moisture content of fruit as affected by tesp sR- moisture content of fruit as affected by RH C-- moisture content of twig as affected by temp yD- moisture content of twig as affected by RH s - means of low and high RH were significantly differ- ent at the 0.01 level y - means of all RH were significantly different at the 0.01 level MOISTURE CONTENT OF FRUIT (C70) TEMP RH 4A 0-0°c + 43 HIGH -0- ' 8'8°C"'" " INTERM "'" 20'O°C-V- W- - \ - \p e\ n - - &\\\ D \"~ ' " 3\ ‘h ' §V ‘Q " \e \‘\. \\ V t IIIIIIII IIIIIIII 4C 4D - “ ‘\ _ \ ‘:~ \\ ' W43. W l I I I I I 2 3 4 5 6 7 533553.06 cargo: 8m .6..ch none 35 .«o Hoaoo do.” no agendas veflm I h 3.5 usages mo confluence the .. x Ram I need essence finance .33ch doses 36 .5 e. Housman Regency. one use: oregano e P eeaofioo 5.38 e 552. uses: 23 as Rm 8.: . ené on; 8.9» V 2.8x mm o.o~ 3.9 7.2 Qua 93.3 £53» 8.8 oases 5.8x we o.o~ 8d a; 84. no.3 2.2» no.8 no.8 98 x on on we.“ 03» Tea 8.x onén some: 808.. 8.8 on m6 8.: he“ may.» 25.8. "than onto: 3.8x 98 mm e5 8d Tau Tau 8.8 8.? e213 . 8.8x can 8 o.o 8.~ See «.8 8.me 8.8 8:8 3.8 ~.8 so o6... no." TN... TR 8.8 3.8 8.8 8.8 3.8 8 . n.» :00 m8 ”.8 8.8 8.8 8.8 no.8 «.8 8 ed 8.0 as? we... no? sin man. «a... 3:. RE no area or as an; Asqerfie no «scenes ebenwo: .35 5.5355 gape—Hen one 3.3.5 993898.... Aggy «done... c.5393 some» he. veneer? no .372. b no weaken e non dense ecu?“ b9.5 A25 denudaoaah: wean no any «sauces 0930: .4 capes 214 Figure 5 A-D A- halt reml farce (m) (5) et weeke 13 Ba- week 6; c— lot-tare content (I) of fruit et week 23 Do- veek 5 of Ilex muggy; (L.) Grey ea effected by temperature (temp) end relat- ive huntdity (RH). Verticel linee indicete etend- erd devietione above the new. 25 OOON U rm :2: comm 000 E Im ou:mm...2_ ..M 1m 30.. UOON Comm 000 W o m S 1. ON q. 0 co m. 0 om 4. J H m 0 I.— am on. w OONJJ Cour/w 00m 26 Figure 6 A4: A-- Concentrations (ppb) of ethylene during etcrege of Ilex verticillete (L.) Grey fruiting trenches et 20% u effected by reletive hunidity (RH) B- trait renovel force (5) (FRF) of Ilex verticillete (L.) Gray et high reletive hunidity (RH) ee effected by storage tenpereture. c-- Concentration (X) of carbon dioxide (002) during etcrege 0f Ilex verticilletn (Le) Grey fruiting hrenchee et high an et 8.8°c end 20°C. 6“ HIGH + D ( INTERM ----... D Low--v-- 20°C 0 RH D ./ \., I C O 2 (9’0) 0 N v---v-"° Wino I I I l I I I I I GB - V . '3 l3"Fm-"1:10 0°C _ VII—r 0 9\V\".‘------___.___8. o _ \e\\ O 8 C V \V F- HIGH RH ZO'OOC I I I I I I I I I - 6C _ HIGH RH 20. 000 L- 8/5 , Jig/”88°C L. 9---v--- I I I I I I 0 I 2 S 7 8 28 Red fruit with 20% or less noisture did not turn brown during oven drying (‘l‘sble u). Shrivelled dehydrsted fruit had It noisture content of 52.81 or less. The lowest moisture content (with the exception of 20°C/9M RH in week 2) recorded for good quslity fruit wss 53.%. Thus the threshold vslue for good quslity fruit lies between these points. With the exception of nsterisl stored st 20°C/9M an. which boos-e conteninsted with fungi in week 3. s11 fruit stored st high RH rennined in excellent condition throughout the entire experinent. Shrinlling of knit wss observed following 2 weeks of low or inter- nedinte RH storage in 0.11 3 tenperstures. Tenpereture differences for twig noisture were null (Fig. lMl). By week 6, s seen between 8 snd 16% soisture wss recorded for sll tenperstures. Twigs stored ct high RH hed s nesn wslue of 30.2% soisture st the end of the experinent (Fig. an). hoisture content of twigs st low snd internediste RH dropped to 2.1 snd 1+.6fl respect- ively by week 6 when the lsst dsts was recorded. Mini-u- lewels of oxygen in s11 chesbers were 20%. Ethylene concentrstions in the chsnbers st 0° snd 8.8% fluctusted between 50 snd 100 ppb. Sinilnr vslues were recorded for 30 and 50% RH at 20°C. but fungsl contuimticn in the 910% RH chsnber wss sssused to be responsible for neon levels of 317 ppb st week h (Fig. 6A). Conc- entretions of 602 were generslly in the region of 0.23. Vslues incressed to 0.x; st week it in cosbinstions of 20°C/9M RH snd et week 7 in ccnbinstions of 8.8°c/96% RH (Fig. 6c). Detersinstion of etfllene Egguct ion rste . The red fruit were larger then the red-green or green fruit. Consequently occssionsl injury occurred from contact with the slimline Nam. Such injury 29 was none cannon with it fruit than with 2 fruit per syringe. Data fron danaged fruit was excluded. Peaks of ethylene production were highest and occurred earlier for green fruit (5.8 pl/kg/hr at 5} days after harvest)(Fig. 7). Red-green fruit olinaxed with u.6 pl/kg/hr at 6} days fron harvest. Red fruit began to increase ethylene production at the sane tine as green and red-green. The peak (1.1 pl/kg/hr) was much snaller and delayed until 9} days fron harvest. DISCUSSION In the 1973 experinents, RH control and neasurenent proved quite difficult to obtain. w.t bulb depressions at 0°c were very .ggn conp- ared with those at 20°C for equivalent RH. Additional technological variations spurred the deve lop-ent of an alternative systen in 1971}. Containers sufficiently large to contain thernohydrographs were found sore satisfactory. Abscission in this experinent. as neasured by a reduction in m, was shown to be related to VPD of the environnent (Table 3). Fruit stored in low and internediate RH showed greater reduction in FRI" than those in high RH (Fig. 3B). This difference could have been such greater if it were not for fungal contanination gt 20°C/9m an result- ing in ethylene production values greater than 300 PPb (Fig. 6A). The consequences can be seen in Fig. 68. where m with this oonbination declined fron 251 g in week 3 to 180 g at the end of week 6. This occurrence was responsible for the low coefficient of linear correlation (0.1.1) on week 6. on. best treatnent conbination was 0°c/985 RH where M did not significantly change throughout the experinent (Figs. 5A. SB and 63, Table 3) and noisture loss fron fruit was least (Figs. 50 30 Figure 7. Ethylene production rate in pl/lrg/hr for Ilex verticillata (L.) Gray fruit. 31 7.59%: 20.5803 M5425 r ._ V a e u _ . I ''''' U T e\e\e vvu ecleleuelelelenbno V m Vobbb.‘ D\ R // I a U N o , chm afifl9kPL F Wu; R W55- 0“”. E — ’ V’s. deli. .l.‘||D|I-|III|I .‘I’.l.l.l VII. \Illn‘UI‘l W): Role V euOIe QIOleIOIOIO 0“)! || Ielele neIeOIe 0| Ill..- IIO‘P' / v....H-N.-. . I allll....n... a lelel el en eleleaerIelOlele e O I/ 7 F_ _ _ _ _ _ _ _ _ _ _ _ . _ _ _ _ 0 00 O O O O O 0 O O O O 8 6 5 4 000000 00.06542 .noooo 8 65 4 3 2 I O O O O O O O O O 0 l3 OCTOBER l3579 SEPT 23 25 27 29 32 and 59. Table u). Moisture loss fron fruit in the 1973 experinent was also nininised with the 0°c/96% 1m conbination (Figs. 23 and 2c). Differences due to tenperature were such greater in 1973 (Fig. 10) than in 197“ (Fig. an). Because of the previously nentioned variat- ions in 1973 and the greater precision of variable control in 197k this difference was assuned due to orperinental error. This sane error was present for the 1973 experinent on loss in fresh weight of fruiting branches with attached leaves. Abscissicn at the point of contact between the fruit and the pedicel (distal type) occurred.605 of the tine. Separation of the pedicel fron the twig (prorinal) occurred with 30% frequency and the internedial type nade up the renaining 10%. Internedial fruit absc- ission was characterised by separation of the central xylen core fron the cortex tissue. Breakage occured at an point along the pedicel with the fruit retaining the xylen core and leaving the tubular cortex on the twig (Figs. 8A-0). High tenperatures and high hunidities produced lowest frequencies of distal separation (Figs. 30 and 3D). Interpretation of data corresponds with those who postulated that a lack of neisture stress results in stronger cell walls which act to delay abscission and loss of noisture causes shrinkage which aids separation (8). This shrinkage of fruit and branches was evident in low and internediate RH treatnents no later than week 2 (Table 3). Iron this period on. routine handling of such naterial resulted in fruit abscission. Good quality fruit had 53.“ noisture and greater, whereas shrunken fruit were recorded as having a naxinun of 52.81 noisture. Assuning a threshold value of 531 in fruit containing 63% initial noisture the calculated point of shrinkage was 8% loss in fresh weight. sinilar to values for other fruits (9. 22). The 33 Figure 8 A4: Ilex verticillata (L.) Gray pedicels A and Du Internedial abscission; fracture of cortex c— shrivelled cortex exposing xylen core 35 occurrence of brown leaves which abscised at high tenperatures and high RH agrees with infornation available on evergreen holly (68. 69, 11h). Eruit of Ilex verticillathwere reported to contain 3-glucoside and 3-xylosylglucoside pelargonidin types of anthocyanin pignents (90). Stability of this red color is decreased at high tenperatures where browning occurs by ensynatic or non enzynatic (Maillard) reactions (53. 96). This reaction was prevented when the noisture content was decreased to approxinately 20% where the water soluble anthocyanins at this level were not free for hydrolysis. Fruit of this species (at roon temperature) produced a definite peak of ethylene production (Figure 7). The highest peak (5.8 pl/kg/ hr) occurred with green fruit after 5% days. Red-green fruit reached u.6 pl/kg/hr one day later. The peak for red fruit was approxinately 20% the height of the previous peaks (1.1 pl/kg/hr) and was delayed until 99 days free harvest. The cranberry Vacciniun nacrocarpon Ait. has been reported to produce increased anounts of ethylene as the fruit natured (36). Respiration neasured by 002 production did not show an increase. yet these fruit were classified as being clinacteric. In absence of data on respiration and because a clinacteric fruit is defined by an increase in this process the author prefers not to place the fruit of connon winterberry in any category until nore data is accunulated. SECTION II THE EFFECT OF ANTITRANSPIRANTS 0N KEEPING QUALITY OF FRUITING BRANCHES OF ILBX MTICILLATA (L.) (BAY 36 37 If water loss fron plants (transpiration) is considerably reduced. sons researchers suggest that cooling and nutrient uptake in plants is not significantly altered (10. 38, 80). Diffusion of water vapor occurs nainly through stonata and lenticels. Guticular water loss becones inportant when the stonata are closed (38). Therefore a critical area of water loss fron plants is the leaf-air interface. and retardation proxinal to this point will cause the develOpnent of noisture stress in the distal region (38). The prevention of this occurrence is possible by use of antitranspirants. There are at least 4 approaches to artificial reduction of trans- piration. These conprise A) use of naterials which increase reflectance (prinarily for leaves) (63. 80), B) windbreaks. C) enclosures surrounding the plant and D) by increasing resistance to vapor diffusion (80). This last nethod involves the use of filn forning naterials for example silicone, or compounds which cause stonatal closure such as abscisic acid (38. 63, 80, 105). The list of filn forming naterials includes wax, latex. various plastics. silicones, polyterpenes and higher alcohols (77). Earlier work dealt with nultinolecular layers of wax (23. #3, 50. 63, 79, 95, 101). which were difficult to use because they required heating before application and were inclined to chip when hard (79). Hedern antitranspirants fora nononolecular filns on plant surfaces by nolecule coalescence after solvent evaporation (63). Perneation of liquids and gases through these polyners occurs by a process called activated diffusion (56). The 3 steps involved are sorption by the polyner and diffusion through it. followed by desorption on its other 38 side. An ideal filn forming antitranspirant. not yet develOped. would have convenient and inexpensive application. greater perneability to gases than to water. and conplete coverage which should be effective for long periods of tine (38. 63. 80). The selective perneability is sonetines said to be an attribute of polyethylene and some other polyners (38. 77). Evidence presented.by'lebovits 1966 (56) and Hooley 1967 (113) has proven otherwise and such references are only relevant to other polyners.. In all polyners studied. perneability to water was greater than to 002. Reductions of 20 to 50% transpiration were obtained with antitran- spirant applications during the growing season of vegetable crops and trees and prior to transplanting (29. 30. #1. 61. 62. 78). but because of inpeded gas exchange counteractiye effects of photosynthesis inhibition have also been reported (28. 31. 37. #9. 73. 105). Phytotoxicity was a result of inconplete cover and toxin accunulation. In post harvest utilisation of plant naterial. photosynthesis is rarely a concern. Thus antitranspirant applications have naintained keeping quality of leaves and fruit by decreasing noisture loss (30. #2. 67. 93. 9n. 101). Reduction of water loss was greatest for fruit varieties such as Golden Delicious apples and.Eur0pean forcing cucunbers which have little natural wax and shrivel during long tern storage. Effects on some other varieties have been mainly to inpart a lustre and increase consumer acceptance. The study reported here was designed to deternine the effectiveness of various antitranspirants on the keeping quality of fruiting branches of Ilex verticillata (L.) Gray. MATERIALS AND METHODS Eggs in fresh weight. water uptake. transpiration and respiration. Fruiting branches of Ilex verticillata were harvested on October 17. 197a fron which unifornly fruiting twigs. approxinately 15 cn in length were selected. Ten twigs were randonly selected for each of 9 treatnents. These conprised 2 plastic fornulations of antitranspirants: Vapor Gard (Miller Ghenical and Fertiliser Corp.. Hanover. Pa.) (polyterpene) and Polyethylene Emulsion (American Machinery Corp.. Orlando. Florida). and 2 wax naterials: Concord Alcoat 33—12 (Concord Chenical Gonpany. Ganden. New Jersey) (anionic wax emulsion) and Prinafresh 23 (Johnson flax.ZRacine. Vise.) (a shellac base). Fruiting twigs were nonentarily dipped in O. 2.5 or 5% aqueous solutions of these naterials. when the fruit surfaces appeared dry the sten bases were out under water and transferred to test tubes which contained distilled water. Treatnents were assigned to a conpletely randomized design under conditions of 22°C and “0% relative hunidity to sinulate roon conditions. The test tubes with and without the fruiting twigs were weighed at intervals of 3 to a days. fron tine zero to day 26. The loss in fresh weight of twigs was transforned to a per cent of the original weight and water uptake fron the test tubes was deternined. The latter was adjusted for evaporation by deduction of water loss fron test tubes not containing twigs. The contribution of respiration was considered negligible and therefore water loss was assumed due to loss in fresh weight of twigs and fruit. Thus transpiration estimates were achieved by the sun of water uptake and loss in fresh weight of fruit and twigs 39 #0 and both were standardised for a 10 g fruiting twig. In addition. ratings of fruit quality were nade at each recording date. Extra sanples of control. Vapor Gard and Concord Alcoat 83-12 concentrations were used for respiration rate deterninations. At intervals of 3 to b days sanples of fruit were renoved and placed in a flnrburg Constant Volune Respironeter (Gibson Medical Electronics). Carbon dioxide evolved was absorbed with 10% XOR and oxygen uptake in pl/g/hr was deternined at 25°C (103). At connencenent of the experi- nent. paraneters of noisture content in fruit expressed in per cent and fruit renoval force were obtained as an indication of naterial quality. Vhen treatnents were significant the following orthogonal contrasts were conducted: control vs treatnents; Vapor Gard vs other treatnents: Concord Alcoat 83-12 vs Prinafresh 23 and Polyethylene Rnulsion; Prinafresh 23 vs Polyethylene Enulsion; and 2.5% vs 5% concentrations of each naterial. Fruit surface norpholggz. Epicarp sanples of 5 untreated fruit of Ilex verticillata and Ilex opgga Ait. were prepared for observation with an Advanced Metals Research Model 900 scanning electron nicroscope (83R). Sanples were placed in liquid nitrogen and innediately transferred to a Virtis (nodel 10-010) autonatic freeze-dryer where water was renoved by sublination. Five other sanples were critical point dried using a graded series of ethanol. anyl acetate and liquid 002. The CO; was released as a gas in a Denton (DCP-i) critical point apparatus. In 197”. observations on the SIR were repeated for I;35_ verticillata and 5 specinens fron fruit treated with 5% Vapor Gard were inc IMCd e Lu Fruit surface in cross section. Fruit epicarp specinens were enbedded in Anss our conpound at -2o°c and sectioned at 2 pn on an International Equipnent nodel CTD cryostat. Vhen placed on slides these sections were stained with Sudan IV and IKI/xzson for lipids and cellulose (5“). A test for birefringence by polarised light was also carried out. RESUDTS Holly fruit treated with Vapor Card 5% and 2.5% was naintained in an excellent condition for 23 to 26 days. Vhereas in all other treatnents. including control. the quality deteriorated by day 17. The fruit and twigs contained 60 and my noisture respectively at connencenent of the experinent. Fruit renoval force was 2&0 g with 70% separation between the fruit and the pedicel. 20% between the pedicel and twig and the renaining 10% were breaks through the pedicel. Sons fruit in a uniforn distribution throughout the experinent were contalinated by a nidge. These fruit turned black and a larvae energed (Figure 5). Taxononic research. by Hr. John Newnan of the Entonology Departnent. Michigan State University. identified this insect as being Alggoggllia illicioOIa Foot. (the Holly Ridge). After day 7 treatnent differences for loss in fresh weight were highly significant and orthogonal contrasts showed that fruiting twigs dipped in Vapor Gard were superior to all other treatnents (Figures 1A and 20). Effects of Prinafresh 23 and Poltethylene Rnulsion were sinilar to Concord Alcoat SI-12 (Figure 13). The effects of Vapor Gard #2 reduced loss in fresh weight for example a scan of 3.7 g compared with 5.“ g for the control on day 23 (Figure 1A). Effects fron 5% and 2.5% ' concentrations were sinilar. Hater uptake for all treatnents was greater during the first 3 days than at any period later on but treatnent differences were highly signi- ficant throughout the experiment. Uptake for twigs treated with Primafresh 23 was greater than control. but uptake for twigs of other treatments were less than or sinilar to the control. The least anount of water uptake was 6.“ nl for twigs treated with 5% Vapor Card by day 20 (Figure 10). The effects of Primafresh 23 and Polyethylene Enulsion were similar to Concord Alcoat 88-12 (Figure 1D). While the effects of Vapor Card at 2.5%Iwere similar to those of the control (Figure 10). the combined nean of Vapor Gard treatnents was signific- antly different from the mean of other antitranspirants (Figure 2D). Transpiration differences were highly significant during the experinent. The effects of Prinafresh 23 and.Polyethylene Enulsion were sinilar to Concord Alcoat SE-12 (Figure 23). The lowest cunul- ative values: 13.2 and 10.1 nl. were recorded for twigs treated with Vapor Gard 2.5 and 5% on day 20 (Figure 2A). These conbined treat- nents were superior to all other antitranspirants in reducing transp- iration. The largest amount of cumulative transpiration on day 20 was 15.h nl recorded for 5% Prinafresh 23 and the control was 1h.7 nl. In general an equal decline in the respiration rate was observed for the control and Vapor Gard treatnents (Figures 3A and 33). Fruit treated with Concord Alcoat SE-iz reached lower values earlier. but experienced an increase fron day 17 to day 19 and then declined (Flam BC)- “3 Figure 1 A-D A-B loss in fresh weight (g) C-D water uptake (ll) of fruiting branches of Ilex verticillata (L.) Gray as affected by Vapor Gard. Concord Alcoat and control. m>\>\D\ .a .. . m M com ._.<004< omoozoo n .. ohm om<0 mom<> a mm acne 58.2 omoozoo . some 92o moses . J m m. .6528 D . <_ nomazoo pg 9% “5 Figure 2 A-D Loss in fresh weight (g). water uptake (n1) and transpiration (n1) of fruiting branches of Ilex verticillata (L.) Gray. A--transpiration as affected by control. Vapor Card 5% and 2.5% B-transpiration as affected by control. Concord Alcoat 5% and 2.5% C-loss in fresh weight as affected by control. Vapor Gard. other treatnents D-uwater uptake as affected by control. Vapor Gard. other treatnents d) V 0 WATER UPTAKE (ML)- N w>< o mNONt 90.5 no MN 0N N. n. 0. N n nlIuI... -- - _.- u- D. .D- -D..\.D\ ON 024.0 mom<> n whzmsikmm... muff—.0 e O ... ..J .1 \c\ \\m.. \ \\ D D\ Q' Dm<0 m0m<> 0 ON mhzmibdimmk mMIkO e - - o u u d d oemN ._.<004< .0200 a 060.0 ._.<00.._< .0200 e \m \ \ a \ D \ \D mtxmx mN 4010.200 D d u u q u - - ormN om<0 m0m<> e \qumwm ‘0‘ J \D‘ \ D .t\\ \CMWX e\D\\D I D.\D\D oemomé m0d<> e (N Jomhzoob .. 0 as N q Q) N O (1W)NO|.LV8|dSNVH.L (BllHDIBM H8383 NI SSO‘I £2 “7 Figure 3 A-C Respiration rate in p1 Oz/g/hr uptake of Ilex verticillata (L.) Cray fruit as affected by control. Vapor Card and Concord Alcoat. CONTROL - p _ VAPOR CARD SC CONCORD ALCOAT O O 0 0 R043 mqus zo_._.— (a) 60 - .\. :3 4° ' 1:: XXX}; 0 \\ ”’ \ ’ Lu 20 - /‘~\ ,,,, ’\ a v/ 0” LL 0 l l I I m 8 23 3| I4 30 I3 JULY AUG SEPT OCT 300 r PROXIMAL-e— /u‘n INTERMED ---u--- V I 33 ._ __ I DIS TAL -v /va\_§q/ ’, \\ /// v \DI \b 0:. I Ilaljll 823 3|l430l3 JULY AUG SEPT OCT FRUIT REMOVAL FORCE (9) 1oz; ./ / ./, 79 “0% frequency from July 23 to September 8. Proximal abscission (between twig and pedicel) declined from.60% frequency in July to 28% in late August. By September 14 frequency was again 60% and then declined to 12% on October 13. FRF for intermedial and distal abscission increased from a mean value of 235 g in July to 295 8 on September 22 and then decreased to 218 g on October 13 (Figure 3B). FRF‘for proximal abscission. which were in the region of 20 g less than intermedial or distal. followed a similar pattern except on September 1“ when they declined to 210 g expanding this difference to 65 g. In anatomical studies of the pedicel in longitudinal section. structural or staining differ- ences were not detected. Laboratggz studies. Hater loss indexed by % loss in fresh weight of fruiting branches as affected by 10‘3. 10'“ and 10'5M concentrat- ions of alar and all antitranspirant treatments were similar to the control. Keeping quality of the fruit was maintained for approximately 1 week with controls; 9 to 10 days with alar and 14 to 21 days with antitranspirants. Loss in fresh weight with NaN3 10'4flitreatments was 18.b% on day 8 and insignificant from the control (2h.6%). On day 8. means of CH 10-311 (32. 9%) and NaN3 io-3w (29.2%) solutions were larger than and significantly different from the control. and on day 20 the same was true for an 10.311 (111.25%) and 8 HQS (116.5%) solutions. All other treatments were similar to the control on day 20 (Table 2). By day 8 the addition of PVP caused greater loss of weight compared with a control but by day 20 values for both treatments reached similar levels (Table 3). The interaction between media treat- ment and PVP was significant on day 8 but not on day 20. The control 80 Table 2. Loss in fresh weight (f) of Ilex verticillata (Le; Gray fruiting branches when proximal ends-weren'minm—sertedm— ' "in" 10- w and 10414 concentrations of acnaphthaleneacetic acid (NAA). cycloheximide (OH) and sodium aside (H.113): calcium chloride 4%: sucrose 2% and 8 hydro- xyquinoline sulfate (8 HQS) 200 ppm and water (control) at days 8 and20. Loss in fresh weight (x) Treatment Day 8 Day 20 mu 10'311 23.0. 36.1. 1m 10% 26.11. 39.6. on 10-3w 32. 5b ui.2b on 104111 27.2. 38.1. NaN3 10-3w 29.211 37.». NaN3 104m 18.l1a 37.0. Calcium chloride be: 23.11. 30.2. Sucrose a 28s“ We“ 8 HQB 200 PP‘ 2708.: “ssh Control 24.6a 37s9‘ Keane within a column followed by a different letter are significantly different at the 0.05 level with Tukey's HSD test (98). 81 Table 3. Loss in fresh weight 0%) of Ilex verticillata (L.) Gray fruiting branchss when the proximal ends were inserted in various media containing polyvinylpyrrolidone (PVP) 1% and control on days deZOe W Loss in fresh weight C5) Treatment Day 8 Day 20 P” 1% 30e7l. 39s 1‘ Control 21.5b 37.9a means within a column followed by a different letter are significantly different at the 0.05 level with Tukey's HSD test (98). Table 0. Loss in fresh weight 0%) of Ilex verticillata (L.) Gray fruiting branches when the proximal ends were inserted in various media and the distal ends dipped in water (control) and 5% concen- trations of Vapor Gard and Geon Latex. Loss in fresh “4513 15L Treatment 1).: 8 Day 20 Vapor Gard 5% 20e8b 33e3¢ Geon Latex 5% 27.3a 39.5b Control 30.3a 02.6a leans within a column followed by a different letter are signifisantly different at the 0.05 level with Tukey's use test (98). 82 plus PVP combination on day 8 lost 12.2% of its weight whereas without PVP. weight loss was 38.8% (Table 5). Some treatments showed the oppo- site effect for example GK 10'311 and NaN3 10'“)! (Figure 15A). Antitranspirants significantly reduced weight loss. Vapor Gard 5% treatments resulted in 20.8% weight loss on day 8 compared with 30.3% for the control and 33.3% versus h2.6% on day 20. Geon latex . treatments were similar to the control on day 8 but were statistically separated from it on m 20 (Table 0). Fruit in H.213 104m and a HQS treatments were in good condition for 12 days: all other treatments for 6 to 9 days. Treatment combinations. with and without PVP maintained keeping quality for 7 and 12 days respectively. Antitranspirant treatments prevented loss of fruit turgidity for 1b to 21 days and the control treatments shrivelled in 7 days. In the experiment on top dipping of fruiting twigs. the effects of various chemicals were similar to the control. On days 10. 17 and 22 significant differences were recorded for antitranspirant treat- ments and the interaction between chemical dips and the antitrans- pirants. Hater loss indexed by loss in fresh weight of fruiting branches was reduced by application of Vapor Gard (Figure 11B). Loss in fresh weight of fruiting twigs when dipped in water or most chemicals and later dipped in Vapor Gard was usually less than those not treated with Vapor Gard. Nalg 100ppn and the control in Figures 5A and 53 are an example. 011 45 ppm and BA 10"“)! treatments on days 10 and 17 respectively lost more weight when treated with Vapor Gard than when untreated (Figures 5A and SB). Keeping quality. evidenced by fruit condition. lasted approximately 10 days for controls and 23 83 Table 5. Loss in fresh weight (%) of Ilex verticillata (L.) Gray fruiting branches for the interaction between proximal insertion in 10’3l and 10'“)! concentrations of ocnaphthaleneacetic acid (NAA). cycloheximide (an) and sodium aside (m3): calcium Chloride as. sucrose 2% and 8 hydroxyquinoline sulfate (8 HQS) 200 ppm and water (control) with and without the addition of polyvinylpyrrolidone (NP) 1% on day 8. Loss in fresh weight (%) ncdi. without m with we 1m 10-311 19.7abcd 26.1abcde NAA 10% 16.0.1: 39.11. on 10%: 29.0mm 39.2. ca 10401 22.7abcde 33.6de 11.213 10-311 31.1cd. 36.3“ NaN3 10411 12.0. 32.7cd. Calcium chloride 34% 20.3abcd 26.9abcde 811320.. a 22e8‘b0d0 33s”. 8 HQS 200 pp! 12e3‘b “6e” Control Sass. 12e2‘b Means within a column followed by a different letter are significantly different at the 0.05 level with Tukey's HSD test (98). Figure l} A-B Loss in fresh weight (%) of Ilex verticillata (L.) Gray fruiting branches. A-8 days after proximal ends were inserted in cyclo- heximide (cu) 10-314. sodium amide (nun) 10-311 and control with and without the addition of polyviwl- pyrrolidone (PVP). Vertical lines indicate standard deviations above the mean. B--when the teps were treated with Vapor Gard 2. 5% and compared with a control dipped in water. Vertical lines indicate standard deviations. 35 4A E] MINUS PVP 40 .. 771, PLUS PVP 30 - : __'s:e FE, ,8 20 ,- : ::.0 :e. O~ — :... 0's V '0 - _ _.'. .e .— ..._ :... e.e I O CONTROL 1 NEW 16314 S2 CH 10% 3 Lu 3 70 . T 48 2 I v ‘60 / 1.. v i; E 50 CONTROL Jr i : O .7: 4o— e / 1 (D l (0 30 /’? J. O T,” J. -J 20 ; VAPOR GARD 25% I I0 E O ‘ - l l 86 Figure 5 A-B Loss in fresh weight (5) of Ilex verticillata (L.) Gray fruiting branches A- 10 days and B- 17 days after proximal ends were inserted in water (control). sodium aside (NaN3) 100 ppm. 8 hydrory- quinoline sulfate (8 HQS) 300 ppm. cycloheximids (CH) 1.5 pp- and bsnsyladenine (3.) 10411 and the tops dipped in water and Vapor Gard 2.5%. Vertical lines indicate standard deviations above the mean. 87 PLUS VAPOR GARD 5A MINUS VAPOR GARD E N! O DAY I? (9b) 04 b 01 a) O O O O N O o m 0 O s O O O m 0 O s O s s e m 0 O m s s o m 0 o 5 L088 IN FRESH WEIGHT '2; ‘6‘ 8 8 o 5 O CONTROL 1 SHQS-BOO 1‘ BA 104M 88 days for treatments receiving Vapor Gard. DISCUSSION Evergreen holly. when harvested for Christmas displays. is immersed in a bath of #0 ppm NAA (68) to prevent leaf fall. when fruiting branches of Ilex verticillata are harvested for the same purpose some loss of fruit occurs during the handling process. A preharvesting treatment which would reduce this loss was envisioned. But studies , in the field show that FRF could not be increased by application of GA. CH. NAA or NaN3 (Table 1). FRF for untreated branches increased slightly over time. Ethephon increased fruit fall but did not change FRF when recorded one week later (Table 1). A response from ethephon occurred within 1 week thus FR! measurements after 1 week were obtained from the remaining unresponsive fruit on these branches. CH also caused fruit abscission. probably by increasing ethylene production (1. 26). Some plants of Ilex verticillata are noted to lose their fruit Just before leaf fall. For cropping purposes one would not propagate such a plant. Instead. selection of plants. which retain their fruit until December or January. was desirable. Such were the type of plants selected.for this study. It is understandable therefore that staining or structural differences indicative of abscission layer for-ation were not detected.in histological and histochemical studies. The fruit was firmly held with no tendency towards reduction in.FRF3 as winter approached. The ability of the fruit to remain attached to the plant must relate to a discontinuous ring of sclereid tissue in the cortex 89 outside the vascular tissue. In apple (65) and cherry (112) there was a reduction in sclerenchyma in the abscission zone. Surrounding the layer of lignified cells the cortex loosened and cell separation was visible along the entire length of the pedicel. Vith the paraffin technique this was thought to be part of the torn section artifact. but glycol methacrylate resin (which was also stained) proved otherwise (Figure 23). ' ‘ The following year FRF'data (for a different plant) was measured over time beginning approximately 2 weeks after fruit set. FRF increased from July to September but decreased in October (Figure 3A). On Septe- mber 14. a large quantity of proximal type abscission occurred naturally and when abscised.samples were studies only cell loosening (previously described) was observed. All histological and histochemical studies recorded similar findings to the previous experiment. There are 2 possibilities for the reduced FRF for proximal abscission on September 14 (Figure 3A). The first is low temperature effect. On September 4. 2°C was recorded in the Lansing area. The temperature could have been much less in the low area where the plant was growing. The other possibility is a natural abscission process. If this is true. then abscission occurred with little change in pedicel structure . Former abscission studies on cherry. cotton. bean etc. were conducted with varieties of known performance (13. 83. 112). Elg§_ verticillata plants are very variable in fruit sise. color and pedicel length etc. from one habitat to another. This diversification limits conclusions of the fruit abscission process. Further studies should be conducted with individual plants all propagated from 1 selected specimen. In spite of the present limitations. a generalisation can 90 be formulated from the present study. There are at least 2 classes of fruit abscission within the native I. verticillata plants. Some lose most of their fruit before leaf fall and the process may be similar to that of other fruits for exasple sweet cherry (111). Other plants retain their fruit until December or January; no abscission occurs and eventually fruit fall is a result of the fruit and pedicel being killed by sub sero temperatures (17). Proximal application (insertion of stem ends) of NAA. CH. alar or new; did not alter weight loss of fruiting branches. The ease results occurred with sucrose (an energy source); 8 HQS (a fungicide) and calcium chloride (for sell firansss). Distal applications of similar compounds and cytokinins had no effect on fruit fall. Use of inhibitors such as laN3 were expected to cause browning of the fruit and a phenol inhibitor (PVP) was included to prevent this occurrence. Fruit color was not affected but PVP increased weight loss and negatively affected all treatments (Thble 3). Fruit abscission and weight loss were reduced when moisture loss was impeded with antitranspirants and Vapor Gard at 5% and 2.5%iwas better than Geon Latex (Table 4). SECTION IV THE EFFECT OF TEMPERATURE. RELATIVE HUMIDITY AND WIND ON FRUIT ABSCISSION OF ILEX VETICILLATA (L.) CIMY 91 92 The effects of ecological factors on abscission of plant parts were reviewed by Addicott in 1968 (5) and by Addicott and Lyon in 1973 (8). Changes in temperature affected abscission. Abscission was increased in sour cherry fruit when the temperature was increased from 15 to 35°C (112) and in olive fruit when the temperature was increased from 0.5 to 29. 5°C (46). In g_i_tr__u_s_,. subfreesing temperatures of -5°c increased abscission (115). Petioles exposed to low temperatures become brittle and break easily in strong winds (52). Abscission of buds. flowers. fruit or leaves (8) can be initiated by moisture stress. And the process is accelerated during warm weather (7). Abscission of cotton leaves induced by ethylene was enhanced (55) and abscission of olive fruit induced by cycloheximide was inhibited by water stress (46). noisture stress can also result from drying winds (8): and strong winds remove leaves and branches from plants. Shaking of 10 day old Cucurbita mslgpopo plants inhibited the growth of stems and petioles (102) and seedlings of Robinia pseggr oacacia were smaller. had less dry weight and fewer leaves after being subjected to . wind velocity of 3.7 m/sec for 8 weeks (92). The following experiments were designed to test the effects of temperature and relative humidity (RH) on fruit abscission of Il2§_ verticillata (L.) Gray (common winterberry). Fruit fall during the winter months was hypothesised to be due to a combination of low temperature killing of the pedicel and fruit tissue followed by periods of high temperature and low RH which cause dessication and the shruhken browned fruit and pedicels fall during heavy windstorms. MATERIALS AND METHODS Tempgrature and relative himidity. Two experiments were conducted to determine the effect of temperature and RH on abscission. Plant material for both experiments was harvested on September 18. 1974 and stored in sealed 10 pm opaque polyethylene bags. On December 15. fruiting twigs approximately 10 cm in length. were selected and the number of fruit on each twig recorded for the first experiment. The twigs were exposed to -9°C and -18°C temperatures. followed by storage at 0°C or 20°C at low or high RH. Four twigs packed in aluminum foil. were inserted in a vacuum cylinder flask fitted with thermocouple wire and placed in a.Revco freeser. The temperature was monitored with a potentiometer; the sensor was a thermocouple inserted into a separate fruit and enclosed with the twigs. This comprised 1 of 2 replications for low temperature treatments. The temperature was decreased .t 10°C/hour until the desired level of -9°C or -18°C was reached and immediately the fruiting twigs were removed from the flasks and transferred to the storage environ- ment chambers. These were cardboard boxes approximately 0.04 m3. encased in 10 um opaque polyethylene bags. Low RH at 0°C (35%) and at 20°C (25%) were obtained by placement of dry calcium chloride within the chamber. High an at 00c (98%) and at 20°C (94%) were obta- ined by placement of distilled water in the chambers. Fruit removal force (FRF) (g) was determined for 4 fruit on each twig with a Hunter Mechanical Force Gauge (Hunter Springs. Hatfield. Pa.) before. at time sore and at 2. 4. 6 and 16 days after low temperature treatment. Abscission between the pedicel and the fruit 93 9a was termed distal: between the pedicel and the stem proximal: breakage through the pedicel intermedial. Analyses of variance for a split plot design were conducted with data on FRI. Data on fruit fall in a wind tunnel and FRF were correlated. The proximal ends of randomly selected fruiting twigs were placed through holes (0.5 cm in diaseter) in a plexiglass sheet and inverted in the ceiling of a wind tunnel (0.75 m in cross section). Quantitative measurements of fruit fall from each twig were made at wind velocities (supplied by a turbine engine) of 6.1. 12.2. 16.3 and 20.1. m/sec .nd qualitative observations of the condition of the fruit were recorded. The second experiment was begun on January 2 and designed to confirm the findings of the foraer experiment. The fruiting twigs were gradually exposed to -9°c or -18°c for 24 hours within sealed flasks which were later removed and the foil covered twigs left in the freezer for an additional 5 hours. Storage temperatures were 0°C and 20°C with high RH combinations as in the first experiment. Data on FRF was determined for 10 fruit on each twig before. at time sero and at 2. 4 and 8 days after treatment. Determination of killigg pgin . A thersocouple was inserted in a fruit; wrapped in aluminum foil and enclosed in a vacuum cylinder flask. The flask was placed in a freezer and the temperature decrease (10°C/hour) monitored with a potentiometer. The first and third exotheras (66. 107) were determined for 5 fruit. Ten excised pedicels were attached to masking tape. A micro- thermocouple (76 um) was inserted into an eleventh pedicel. The tape. covered with foil. was inserted in a flask and placed in a freezer. Temperature decrease (10°C/hour) was monitored with a potentiometer. lil.’ Illil! L\| t u is, ' I 95 Two flasks were used for each treatment of -9°C. -12°C. —15°C. -18°C. Hhen the desired temperature was reached and the foil removed. the pedicels and masking tape were placed in high humidity chaabers at 22°C. Four days later sectioned pedicels were examined for browning under the dissecting microscope. This process was repeated with temperatures of -11.1°C. -12.2°C. -13.3°C. -14.4°C and -155C. RESUDTS FRF’for -18°C temperature treatments were lower than those recorded for -9°C treatments (Figures 1A and 1B). Treated branches stored at 20% had lower in? than those stored at 0°c (Figures 10 and 1D). Both low temperature and storage temperature treatment differences were significant at 2. 4 and 6 days in the first experiment. The interaction between storage tesperature and.RH was highly significant on day 16 (Figure 2). Differences for’RH and all other interactions were insignificant. In both experiments. abscission at the distal end fluctuated between 50 to 80%: at the proximal end 15 to 45% and the intermedial type was always less than 10%. Fruit fall for all treatments at wind speed velocities of 6.1. 12.2. 16.3 and 24.4 m/sec was zero. host fruit exposed to -18°C and some exposed to -9°C turned brown. Browning had occurred by day 2 and reached a maximum by day 6 or 8 (Figure 30). Determination of killing points. The fruit when super-cooled. produced 2 significant exotherms (Table 1). The mean freezing point . was -3.7°C and the fruit were killed at a mean of -10.8°C. Samples of pedicel tissue were alive at -9°C and -12°C (Figure 3B) but were 96 Table 1. Freezing and killing temperatures °C (exotherms) of Ilex verticillata (L.) Gray fruit when super-cooled at 10°C/hour. # Freezing temperature Killing temperature 1 '3e3 ‘10e8 2 -3.5 -10.6 3 -4.2 -10e7 u .3e9 .11e0 5 -3.6 -10.8 "OCR '3e7 .IOeE Table 2. Number of Ilex verticillata (L.) Gray pedicels alive after subfreesing temperature treatments of ~11.1“C. -12.2°C. -13.3°C. -14.4°C and ‘15e506e Tempgrature °C # .11e1 '12e2 ‘13e3 ~14.4 ’15e5 ..... Figure 1 A-D Fruit removal force (g) of flex verticillat_a_ (L.) Gray A- before. at time zero. 2. 4. 6. and 16 days B-— before. at time zero. 2. 4 and 8 days after exposure of fruiting branches to subfreesing temperatures of -9°C and -20°C. C- before. at time sero. 2. 4. 6 and 16 days D-- before. at time mere. 2. 4 and 8 days of storage at 0°C and 20°C after exposure of fruit- ing branches to subfreesing temperatures. Subfree- sing and storage temperature differences were significantly different at days 2. 4 and 6. w>