SOME FACTORS AFFECTING, CONTROLLED ATMO$PHERE STORAGE DISORDERS OF JONATHAN APPLES Thesis for ”to Degree o§ pk. D. MICHIGAN STATE UN’EVERSITY William George Chace. Jr. 1959 n-[ESlS This is to certify that the thesis entitled Some Factors Affecting Controlled Atmosphere Storage Disorders of Jonathan Apples presented by WILLIAM GEORGE CHACE, JR. has been accepted towards fulfillment of the requirements for __PLP_°_degree in. Hort iCUlture Jams /% AW; Major professor Date October 11+, 1959 0.169 LIBRARY Michigan State University SOME FACTORS AFFECTING CONTROLLED ATMOSPHERE STORAGE DISORDERS OF JONATHAN APPLES By WILLIAM GEORGE CHACE, Jr. AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1959 pproved \ WILLIAM GEORGE CHACE, Jr. ABSTRACT The length of the storage period for the Jonathan variety of apple fruit usually is limited by the incidence of one or more storage disorders, such as soft scald, soggy breakdown, Jonathan spot and internal breakdowu. Controlled atmosphere (CA) storage reduces the incidence of these disorders, but some- times other injuries are encountered. Some of the factors influencing the devel- opment of CA disorders, such as voids, core browning and breakdown were in- vestigated. Prestorage treatments in 1957 included modification of the fruit/leaf ratio and enclosure of the fruit within a plastic film prior to harvest, maturity of the fruit at harvest, and holding the apples at 55° F for 10 days following harvest. Storage conditions employed were regular storage at 32° F and controlled atmos— pheres of 13 percent C02 - 3 percent 02 and 5 percent C02 — 3 percent 02 at 32 and 38°F for seven months. In 1958-59, the effects of maturity and fruit/leaf ratio of the fruit were studied in relation to regular and CA storage at 32°F. Core browning, although previously reported as a CA disorder, occurred in regular storage as well as in CA storage in one of the two seasons studied. This disorder was characterized by a slight to severe brown discoloration of the pith tissue near the cambial line. The cells of the affected tissues showed a vacuole-like sac containing many particles surrounded by a brown fluid. The highest incidences of this disorder were associated with advanced fruit maturity, and a high concentration of C02 (13 percent) and a low temperature (32°F) during WILLIAM GEORGE CHACE, Jr. ABSTRACT - 2 storage. Defoliation of the limbs bearing the test fruit two months prior to harvest in 1958 did not significantly affect the development of core browning in the fruit during storage. Core browning appeared after 156 and 76 days in storage during the first and second years, respectively. The development of voids or small spheroid air pockets in the pith and occasionally in the cortex of the fruit occurred only in controlled atmosphere storage. Factors associated with a high incidence of this disorder were large-sized fruits, the presence of water core in the apples at harvest, late picking, and storage in 13 percent carbon dioxide with 3 percent oxygen at 32° F. Defoliation of branches bearing the test fruit in 1958 significantly re- duced the incidence of this disorder in CA storage. Similar fruits from branches which were not defoliated were of higher soluble solids content and were affected with water core. Voids developed in susceptible fruit after 125 or 176 days of storage. Three types of fruit breakdown developed in these tests - internal break- down in regular and CA storage, soggy breakdown in regular storage, and brown heart in CA storage. A high concentration of carbon dioxide (13 percent) and low temperature (32° F) were most conducive to the development of this dis- order during storage. Over-maturity at harvest favored breakdown: whereas the other prestorage treatments had no consistent effect on its development. In 1957, fruit held in CA and regular storage at 32° F developed breakdown WILLIAM GEORGE CHACE, Jr. ABSTRACT - 3 after 176 days of storage and after 210 days when stored in controlled atmos- pheres at 38° F. Fruit held in CA and regular storage in 1958 developed break- down after 210 days of storage. Techniques were developed for the utilization of labeled carbon to possibly relate the distribution and movement of carbon dioxide in the fruit tissues to the development of CA disorders. These studies indicated that the properties of the fruit cells in respect to the accumulation and/or permeability to carbon diox— ide are altered by CA storage since CA apples, following exposure to C1402. evolved a greater total amount of C140 and at a higher rate than fruit from 2 regular storage. Fruit previously stored in 13 percent carbon dioxide also 140 2 than fruit stored in 5 percent carbon evolved a greater total amount of C dioxide. The greatest radioactivity accumulated in the seed cavities, cambial line, vascular bundles and pith tissues of the fruit. Also, the pith and cortex tissues, on a per gram basis, accumulated more C14 in large fruit than in small fruit. The recommended CA storage condition (5 percent C02 - 3 percent 02 at 32° F) for Jonathan apples proved sound, since it gave the best retention of eating quality with a minimum danger of core browning and void formation. These ex- periments demonstrated that fruit of advanced maturity or affected with water core at harvest time should be avoided when selecting Jonathan apples for CA storage. SOME FACTORS AFFECTING CONTROLLED ATMOSPHERE STORAGE DISORDERS OF JONATHAN APPLES By WILLIAM GEORGE CHACE, Jr. A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1959 ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. D. H. Dewey for his constant supervision, encouragement and guidance throughout this study. The writer is indebted to Doctors A. L. Kcnworthy, l. J. Pflug, L. W. Mericle and M. J. Bukovac, who reviewed the manuscript and J served as members of the committee. Grateful acknowledgement is accorded to Doctors F. G. Teubner and M. J. Bukovac for their interest and assistance in adapting Carbon techniques and applications; and to Michigan apple growers who provided the Jonathan apples for this study. TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW. . . . . . . . . . . . . . . . MATERIALS AND METHODS . RESULTS. Core Browning . . . . . . . . Voids . Brea Kdo‘k’n O O O O O O O O O O O O O O O 6 O O O O Other Symptoms of Controlled Atmosphere Injury. . Water Core. . . . . . . . . . . . . . Fruit Condition and Quality . . . . . . . . . . Labeled Carbon Dioxide Studies . . . . . . . . . . DISCUSSION.................... SUMMARY AND CONCLUSIONS . LITERATURE CITED . APPE NDIX TABLES . 12 37 37 48 61 7O 72 74 78 9O 99 103 109 Table 10 LIST OF TABLES Page The Effect of Storage Temperature on the Development of Core Browning in Controlled Atmospheres . . . 42 The Effect of Storage Atmospheres on the Development ofCoreBrowning................ 43 Mean Percentages of Jonathan Apples in Core Browning According to Time of Harvest and Subsequent Stor~ age for 210 Days in Controlled Atmosphere Regular Storage....................45 Percent Jonathan Apples with Core Browning. The Fruit Received Various Prestorage Treatments and were Subsequently Stored for 210 Days in Controlled Atmos- phere and Regular Storage - 1957-58 . . . . . . . 47 Periodic Observations on Percent Core Browning Devel- oped in Controlled Atmosphere and Regular Storage 49 The Effects of Storage Temperature on the Development of Voids in Fruit Stored in Controlled Atmosphere 53 The Effect of Storage Atmospheres on the Development ofVoids.................... 54 Mean Percentages of Jonathan Apples with Voids Accord- ing to Time of Harvest and Subsequent Storage for 210 Days in Controlled Atmosphere and Regular Stor— age 56 Percent of Jonathan Apples with Voids According to Vari- ous Prestorage Treatments and Subsequent Storage for 210 Days in Controlled Atmosphere and Regular Storage(l957-58). . . . . . . . . . . . . . . . 58 Periodic Observations on the Percent Voids Developed in Controlled Atmosphere and Regular Storage. . . . 59 Table 13 LIST OF TABLES CONT'D Page The Occurrence of Voids in Various Sized Fruits Stored in 13 Percent Carbon Dioxide with 3 Percent Oxygen at 32° F forSevenMonths.................. The Effect of Storage Temperature on the Development of Breakdown in Controlled Atmosphere Storage. . . The Effect of Storage Atmospheres on the Development of Breakdown.................... Mean Percentages of Jonathan Apples with Breakdown Accord- ing to Time of Harvest and Subsequent Storage for Seven Months in Controlled Atmosphere and Regular Storage . Percent Breakdown in Jonathan Apples from Various Prestor— age Treatments Subsequently Stored for Seven Months in Controlled Atmosphere and Regular Storage (1957-58) . Periodic Observations on the Percent Breakdown Developed in Controlled Atmosphere and Regular Storage . The Quantity of Cl4 Recovered in the Core, Flesh and Peel Tissue of CA Jonathan Apples Exposed for 14 Days to 50 Percent C02 Labeled with C1402, with 3 Percent 02 . . 60 63 65 66 68 69 79 Figure LIST OF FIGURES A schematic and flow diagram of the storage facilities used in 1957—58. The atmosphere of the master CA chambers A and B were circulated through the small (2-bushel capa- city) chambers in the refrigerated rooms C and D. . . . . A schematic and flow diagram of the storage arrangements for 1958—59. The atmosphere of the master chamber A was circulated through the small (two—bushel capacity) cham- bers in the refrigerated chamber B . . . . . . . . . . . A schematic diagram of the apparatus used for exposing apples to controlled atmospheres containingC 02 . . . . . Modification of an Orsat analyzer to enable collection of the C02 absorbing solution containingC ()2 . . . . . . . . . Reaction apparatus for measuring C14 content of blended radio— activetissues..................... Core browning of Jonathan apple fruit radiating from the pri- mary vascular bundles (see arrow.~.) into the pith tissue. . Severe core browning in which the affected pith tissues appear~ eddryandflakyintexture. . . . . . . . . . . . . . . The cross sectional drawings of fruit A through D, the shaded portions represent the areas developing core browning during controlled atmosphere storage. Drawing D also depicts a void or air pocket present in the browned tissue The shaded areas of the apple cross-sections E through H rep- resent the regions affected by core browning. Usually only the pith tissues were involved, but occasionally core brown- ing originated at the vascular bundles in the cortical tissue as in drawing F. O I O O O 0 O O O O O O O O O O 0 These drawings depict cells from the browned pith tissues of thefruit....................... Page 13 18 26 29 32 38 38 39 40 41 LIST OF FIGURES CONT'D Figure Page 11 Longitudinal view of 3 Jonathan apple having small voids in thepithtissue................... 50 12 A medial cross-section of a Jonathan apple from CA storage withseverevoids.................. 50 13 A medial cross—section of a jonathan apple showing a void in tissue which was also affected with brown heart . . . 50 14 Photomicrographs -(X 10) showing voids within the pith tissue of a jonathan apple. Note the smaller voids formed around the large void in the upper photograph. . . . . . 51 15 Photomicrograph (X 100) of the cells at one side of a void in the pith tissue of a jonathan apple. The walls of at least six cells close together at the left side of the photograph form- ed the side of the void . . . . . . . . . . . . . . . . 52 16 The darkened areas in the pith and cortical tissue of this jonathan apple are injured as a result of internal break- down. Note the voids in the pith tissue within the affected tissue 62 17 The shaded tissues within the pith and cortex of these fruits are affected with brown heart. The large air pockets or voids in the pith tis~..ue were not associated with brown heart 62 18 Epidermal injury of the jonathan apple stored in controlled at— mosphere conditions of 13 percent C02 - 3 percent 02 at 32°F 71 19 Injury of epidermal and subepidermal tissues of the jonathan apples stored in 5 percent carbon dioxide and 3 percent oxygen....................... 71 Figure 20 21 22 23 24 25 LIST OF FIGURES CONT'D Page The glassy, water-soaked areas of water core that persisted through seven months of CA storage are the darkest wedge- shaped discolorations around the vascular bundles. The larger and less darkened areas are affected with brown heart (which was not particularly associated with the pre- senceofwatercore)................. 73 The dark areas in these apples are brown and water-soaked pith tissue of apples exposed to 15 percent carbon dioxide with l. 3 percent oxygen for seven days at 32° F . . . . . 73 Cumulative evolution of carbon14 dioxide by storage apples following 17 days of exposure to 25 ercent carbon dioxide containing 433. 3 microcuries of C1 at 75°F . . . . . . 81 Cumulative evolution of C140 by Jonathan apples following ex- posure to 25 percent car on1 dioxide with 10 percent oxy- gen at 32° F and 75° F for seven days. l‘hese apples had been stored prior to C14 treatment in 13 percent carbon dioxide with 3 percent oxygen at 32° F for five months. . 83 Cumulative evolution of C140 from jonathan apples stored for four months in 13 percent carbon dioxide with 3 percent oxygen, 5 percent carbon dioxide with 3 percent oxygen and normal air at 32° F. This fruit was then exposed to 25 ercent carbon dioxide containing 457 microcuries of Cl , with 10 percent oxygen for seven days and the evolu- tion of CM’O2 measured when flushed with normal air. . 84 Diagram showing the distribution of C140 from the internal atmosphere of a 3/8 inch thick median transverse jona— than apple slice. The internal atmosphere was withdrawn from the apple slice onto a treated blotting paper. The paper was dried and cut into 1/4 inch squares for radio— active measurement . . . . . . . . . . . . . . . . 86 LIST OF FIGURES CONT'D Figure 26 A autoradiogram of a 3/8 inch thick jonathan apple slice taken from an intact apple that had been exposed to controlled ‘ . 14 . ‘ f . . atmosphere containing C 0 (llght areas radioactlve). The shaded area on the right of the photograph was due to fogging of the film during exposure to the X-ray film. 27 An autoradiogram showing the C14 distribution (light areas) from the internal atmosphere of a 3/8 inch thick median Jonathan apple slice. The internal atmosphere was with- drawn from the apple slice onto BaOH treated filter paper. The filter paper was exposed to X-ray film for 72 days. 28 Correlation of percent voids with soluble solids of fruit from non-defoliated branches after seven months storage in 13 percent carbon dioxide - 3 percent oxygen at 32° F and with percent water core at harvest — 1958-59. Page 88 88 94 APPENDIX TABLES Table ' Page I Experiment 1: The Effect of Time of Harvest on jonathan Apples During Storage in Controlled Atmospheres and Regular Air - 1957-58. . . . . . . . . . . . . . . 109 II Experiment 2: The Effect of Time of Harvest on Jonathan Apples in Controlled Atmosphere and Regular Storage - 1958-59...................... 110 III Experiments 3-8: The Effect of Prestorage Treatments on Jonathan Apples in Controlled Atmosphere and Regular Storage— 1957-58. . . . . . . . . . . . . . . . . 111 IV Percentage of Fruit with Core Browning from Defoliated and Non-defoliated Trees of Five Growers. Fruit Stored in Controlled Atmosphere and Regular Storage at 32° F for Seven Months (1958-59). . . . . . . . . . . . . . . 112 V Percentage of Fruit with Voids from Defoliated and Non—defol- iated Trees of Five Growers. Fruit Stored in Controlled Atmosphere and Regular Storage at 32° F for Seven Months(l958-59).................. 113 VI Percentage of Fruit with Breakdown from Defoliated and Non- defoliated Trees of Five Growers. Fruit Stored in Con- trolled Atmosphere and Regular Storage at 32°F (1958- 59). . . . . . . . . . . . . . . . . . . . . . . . 114 VII Percentage of jonathan Apples from Defoliated and Non—defol- iated Trees with Water Core at Harvest (1958-59) . . . 115 VIII Mean Flesh Firmness According to Pressure Tests of Jona- than Apples at Various Times of Harvest and Firmness Loss after 210 Days in Controlled Atmosphere and Regular Storage. . . . . . . . . . . . . . . . . . 116 Table IX XI XII XIII XIV XV XVI APPENDIX TABLES CONT' D Flesh Firmness According to Pressure Tests Given Vari- ous Prestorage Treatments at Harvest and After 210 Days in Controlled Atmosphere and Regular Storage (l957-58).................... Fruit Firmness (Pounds) According to Pressure Tests of Jonathan Apples at Harvest. Fruit Harvested from Defoliated and Non-defoliated Areas of Four Trees from Five Growers (1958) . . . . . . . . . . . Fruit Firmness (Pounds) According to Pressure Tests of jonathan Apples after 210 Days in Controlled Atmos— phere and Regular Storage. Fruit Harvested from Defoliated and Non-defoliated Areas of Four Trees from Five Orchards (1958-59) . Percentage Soluble Solids of juice from jonathan Apples at Various Times of Harvest and Percentage Loss After 210 Days of Controlled Atmosphere and Regular Stor— age (1958-59) . . . . . . . . Percentage Soluble Solids of juice from jonathan Apples from Various Prestorage Treatments at Harvest and Percentage Loss after 210 Days in Controlled Atmos- phere and Regular Storage (1957-58). . . . . . . . Percentage Soluble Solids of Juice from jonathan Apples at Harvest. Fruit Harvested from Defoliated and Non- defoliated Areas of Four Trees from Five Growers (l958-59)................... Percentage Soluble Solids of jonathan Apples after 210 Days in Controlled Atmosphere and Regular Storage. Fruit Harvested from Defoliated and Non-defoliated Areas of Four Trees from Five Growers (1958-59) . Total Carbon Dioxide (G rams) per Container at Standard Temperature and Pressure . . . . . . . . . . Page 117 118 119 120 121 122 123 124 INTRODUCTION The varied recommendations for extending the storage life of the jonathan variety of apple can be largely attributed to the numerous physiological dis- orders associated with the storage of this fruit. The high incidence and severity of one or more disorders, such as soft scald, soggy breakdown, jonathan spot and internal breakdown have limited the length of storage per— iod for this variety. The importance of the jonathan variety in Michigan, to- gether with the need for a longer storage life of the fruit, led to controlled atmosphere experimentation with this variety in 1956 in Michigan. Previously, it had been reported that certain storage disorders could be reduced by alter- ing the storage atmospheres, but also that other difficulties were then some- times encountered. Controlled atmospheres (CA) have been proven to be suitable means of storing jonathans in Michigan, and by 1959, the controlled atmosphere storage capacity was about 183, 000 bushels of jonathan apples, or 34. 4 percent of the total CA storage capacity in the State. The controlled atmosphere conditions utilized for commercial storage have not completely eliminated storage disorders. The amount and severity of CA injury has varied considerably from year to year, and from one lot of apples to another under the same storage conditions. Obviously, the inherent characteristics of the fruit are important; even though most of the difficulties in the past have been attributed to the storage conditions. In view of the increasing importance of the controlled atmosphere method for the storage of jonathans, this research was initiated to study the physiological behavior of the fruit to ascertain the possible factors influenc- ing the development of CA injury. This was approached by inducing CA dis- orders by several storage conditions, by ascertaining the effects of pre- storage treatments on the induction of these disorders, and by study of the affected fruit tissues in relation to accumulation or deficiency of carbon dioxide and oxygen within the fruit. The storage conditions employed in this study were normal air, the re- commended controlled atmosphere conditions for Michigan jonathans (5 per- cent carbon dioxide and 3 percent oxygen at 32° F), and controlled atmosphere conditions using high levels of carbon dioxide and higher temperatures which have tended to promote CA disorders. Since such preharvest treatments as defoliation, ringing, thinning and removing fruit from limbs bearing excess crops are known to alter the physiological behavior of the fruit, these treatments were included. Radioisotopic techniques were used to trace the distribution and pathways of carbon dioxide in the apples. For the latter, it was necessary to develop suitable techniques for the application and measurement of C14 in apple fruit tissues. LITERATURE REVIEW Functional or physiological disorders of jonathan apples during storage have been the subject of much research. For the most part, these disorders have been attributed to a complexity of factors with few single influences specifically identified. Some of the factors known to affect the development of functional disorders in storage will be discussed in this review. The effect of carbon dioxide in controlling the physiological disorders of jonathan apples was probably first observed by the Australians in connection with overseas shipments in 1928—29 (Carne and Martin, 1935). Since then studies in New Zealand by Mandeno and Padfield (1953), in Victoria by Trout _e_t§_l_. (1940), and in Tasmania by Carne and Martin (1935, 1935a, 1938) and Carne _e_t 531°. (1930, 1930a) have developed further advantages of storing jonathan apples in controlled atmospheres. Control of jonathan spot, reduced incidence of soft scald, preservation of eating quality, extended storage life and longer shelf life of the apples have been attributed in varying degrees to controlled atmosphere storage by the above authors as well as by Van Heile (1951) in the Netherlands, Rasmussen (1951) in Denmark, Phillips and Poapst (1952) in Canada, and by Dewey}: _a_l_. (1957) in this country. These workers have also reported varying degrees of susceptibility of jonathan apples to CA injuries. The disorders encountered in the storage of jonathan apples have included Jonathan spot, soft scald, soggy breakdown and various types of senescent breakdown (Daley, 1924; Kidd and West, 1925; Plagge, 1925; Brooksitaln 1935; and Heald, 1933). jonathan spot has been a factor in preventing the long term regular stor— age of jonathan apples. Plagge and Gerhardt (1930) found that 32° F was the best temperature for control of this disorder, but that low temperatures in- creased the incidence of soggy breakdown and soft scald. jonathan spot has been controlled in CA storage at temperatures above 32° F by Mandeno and Padfield (1953), Ballinger (1955), Dewey_et 31: (1957), Plagge (I942), Vickerygtal: (1951), Rasmussen (1951), Huelin and Tindale (1947), and Trout E31; (1947). The control of other disorders by CA storage has been less successful. Soggy breakdown has been reported by Plagge gal: (1935) and Plagge and Maney (1937) as an internal breakdown of the cortical tissue caused by low temperatures. The best control of this disorder was storage at 36° F rather than at the lower temperatures (Plagge, 1926: and Kidd and West, 1925). jonathan apples in 9 and 11 percent carbon dioxide and 11 and 9 percent oxygen at 32°F developed some soggy breakdown, however, concentrations of carbon dioxide below 9 percent reduced the incidence of this disorder (Plagge, 1942). Fruit had become predisposed by immediate storage at 32°F to the develop- ment of soggy breakdown, but was held practically free of this disorder as a result of prestorage exposures of carbon dioxide gas (Brooks and Harley, 1934). Carnegzil: (1930), Plagge and Maney (1928), and Phillips and Poapst (1952) consider "low temperature breakdown" (Kidd and West, 1925) identi- cal to soggy breakdown (Plagge and Maney, 1928; Plagge_e_t a_l_. , 1935). Carne and Martin (1935), Troutet 31: (1940) and Huelin and Tindale (1947) reported the appearance of low temperature breakdown in jonathan apples was not altered by the use of "gas storages". Low temperature breakdown increased in jonathan apples with the maturity at picking time, and the con- centration of carbon dioxide and the length of time in storage (Game and Martin, 1935). Huelin and Tindale (1947) found that when this disorder was prevalent in regular storage it was also a serious problem in gas storage. Soft scald appeared as a browning of the skin and flesh of the apple characterized by a sharp demarcation between the injured and healthy tissue (Plaggeet a1: , 1935; Plagge and Maney, 1937; and Brooks and Harley, 1934). Soft scald develops at low temperatures and was controlled to some degree by temperatures of 36° F (Plagge and Maney, 1937; Brooks and Harley, 1934). Brooks and Harley (1934) found that exposure of fruit to a concentration of carbon dioxide of 20 percent or more before storing at 32°F will sometimes reduce the incidence of soft scald. Trout gal. (1940) showed that jonathan apples stored in gas storage at 36 to 40°F controlled soft scald, as well as jonathan spot. Mandeno and Padfield (1953) reported that deep scald (soft . scald) occurred when the concentration of carbon dioxide reached 8 percent 6. or above at temperatures above 40°F. Soft scald did not appear when held in atmospheres of 5 percent carbon dioxide and 3 percent oxygen at 32°F and 38°F (Ballinger, 1955; and Dewey_e_t_a_l_. , 1957). The description of brown heart by Kidd and West (1925) is similar to that of Plagge and Maney (1928, 1937) for soggy breakdown. The latter authors (1928), however, found that brown heart developed in the cortex, pith or in both areas, whereas soggy breakdown arose only in the cortex. Soggy break- down tissue appeared also in more definite and regular patterns than brown heart. Brown heart may appear in storage at any time (Kidd and West, 1925), whereas soggy breakdown occurs only after a definite time interval in stor- age (Plagge and Maney, 1928). Brown heart has been associated with an ex- cessive accumulation of carbon dioxide, but soggy breakdown did not appear to be associated with this condition (Plagge and Maney, 1928). Kidd and West (1925) showed that jonathan apples and other English varieties were susceptible to brown heart at higher than normal levels of carbon dioxide. Carne and Martin (1935a) reported jonathan apples less suscepible to brown heart than Sturmer and French Crab. These authors also stated that increasing the carbon dioxide levels increased the possibility of brown heart. Huelin and Tindale (1947) and Trout gt a_l_. (1940) showed that the more mature jonathan apples were the more susceptible to brown heart. With concentrations higher than 7 percent carbon dioxide at 32° F brown heart occurred (Plagge, 1942). The appearance of internal breakdown in controlled atmosphere storage apples has been reported by several workers (Kidd and West, 1928 and Dewey it ill. , 1957). Kidd and West (1928) found that within the temperature range of susceptibility of Bramley's Seedling apples, high levels of carbon dioxide tended to increase the amount of internal breakdown to a greater extent than the decreased levels of oxygen. Dewey St 211; (1957) showed that storage of jonathan apples in various levels of carbon dioxide and oxygen reduced the appearance of internal breakdown over fruit stored in normal air. They also noted that the least internal breakdown was found in apples stored in 5 per— cent carbon dioxide with 3 percent oxygen at 32° F. Internal breakdown in jonathan apples stored at 2 1/2 percent carbon dioxide and 3 percent oxygen at 32°F was more prevalent than in regular storage at 35° F (Bunemann it 31:, 1959). Mandeno and Padfield (1953) reported that reduced oxygen and in- creased carbon dioxide increased the incidence of this disorder. Brooks and Fisher (1926), Harley (1938) and Smock and Neubert (1950) reported that watercore apples have a high degree of susceptibility to internal breakdown. Watercore had a non-parasitic disorder that developed in the fruit on the trees when exposed to strong sunlight or allowed to become over- mature (Brooks and Fisher, 1926). Fisher 339.1: (1930) found that watercore resulted in premature and non-uniform starch conversion within the fruit. In addition to the many disorders that appear in regular storage, several types of injury occurred as a result of storage under controlled atmosphere conditions. Smock (1949) reported that New York-grown jonathan apples stored in 5 percent carbon dioxide and 2 percent oxygen at 40°F were highly susceptible to internal carbon dioxide injury characterized by dry, brown, flaky areas or pockets in the flesh, particularly around the core. Ballinger (1955) found a similar type of injury as well as browning of the pith tissue and flesh brown— ing which started at the epidermis and extended to various depths within the flesh. He attributed these disorders to the above-normal levels of carbon dioxide. Smock (1946) suggested there were two separate types of internal brown- ing injuries on McIntosh apples. One was due strictly to low temperature and the other resembled brown core, but was definitely due to high carbon dioxide. Kidd and West (1928) employed 1°, 5° and 10°C with various concentrations of carbon dioxide for Bramley's Seedling apples and found that alternating the atmosphere within the temperature range of susceptibility increased internal browning. Atmosphere alterations, when utilized at temperatures above the critical temperature for browning, reduced the occurrence of this physiological disorder. Barker and Kidd (1935) believed that carbon dioxide toxicity gener- ally was greater at the lower temperatures than at higher temperatures. Many workers, such as Kidd and West (1934), Haller and Lutz (1937), Barker and Kidd (1935), Kidd and West (1926), have been unsuccessful in correlating respiration rate with the various storage disorders. Kidd and West (1934) reported that low temperature breakdown (soggy breakdown) occurred during storage in English apples only when they were placed in storage while at the respiration peak. They did not believe this to be serious since the beneficial effects of carbon dioxide were slight for fruit placed in storage at this stage of maturity. Haller and Lutz (1937) found no correlation between the respiration rate and the occurrence of soft scald in jonathan apples. Kidd and West (1926) found a positive correlation between the respiration rate of apples and softening and general storage life, but Ryall and Aldrich (1944) found that other factors may alter this relationship. Cox's Orange Pippin apples were highly susceptible to carbon dioxide in- jury during the period immediately after the climacteric rise (Kidd and West, 1939). Fruit harvested three weeks early and treated with 1/500 ppm ethylene for 24 hours at 12°C before exposure to atmospheres containing carbon dioxide showed marked carbon dioxide injury (brown heart). These authors concluded that even immature apples, if stimulated by ethylene in such a manner as to cause premature climacteric rise, will become susceptible to carbon dioxide injury. Many workers (Daley, 1924; Magness, 1929; Trout _e_t_ 31:, 1940; Smock and Neubert, 1950; Game and Martin, 1938) have reported a positive relation- 10. ship of size of fruit to the storage disorders of the fruit. Martin (1954) re- ported that light crop trees were more susceptible to breakdown in storage than the heavy crop trees, due to the larger average size of the cortical cells. Carne and Martin (1938) found a high positive correlation between the mean size of the fruit per tree and the incidence of disorders. Martin and Lewis (1952) indicated that in order to hold fruit in storage for longer periods, the size of the cell must be decreased and that the fruit size should be increased only by increasing the number of cells. Smith found that he late maturing varieties had a smaller number of cells per unit weight than earlier varieties. He associated the greater number of cells with lower respiration rate and good keeping qualities including fewer storage disorders. Studies of seasonal variations in relation to storage quality have been limited and have given conflicting results. Seasonal variations in storage quality have been attributed to mean fruit size. No relation of maturity and breakdown was dependent on the season, but modified by fertilizer treatments seemed to have some influence. Susceptibility to a given concentration of carbon dioxide may also depend upon the growing or climactic conditions in the orchard (Smock, 1944). Iowa- grown jonathan apples have been shown by Plagge (1942) to tolerate 5 percent carbon dioxide at 40° F, while Smock and Van Doren (1941) showed that New York-grown jonathan apples developed flesh browning when stored in the same conditions. 11. Histological and biochemical studies of the various types of carbon dioxide injuries have been limited. Although Bain (1956) found that browning in Granny Smith apples was associated with cells containing numerous chloroplasts, the browning did not appear to be caused by disorganization of the chloroplasts. The accumulation of succinic acid during controlled atmosphere storage was associated with carbon dioxide injury according to Hulme (1956). He believed that abnormal concentrations of this acid was toxic to the tissues. Some insight into the biochemistry of the CA effects is given by Allentoff gt a_l_. (1954, 1954a) who exposed mature McIntosh apples to C1402 for 18 hours in darkness. They found that the tissues incorporated C14 in the malic, aspartic and glutamic acids, and alanine and serine of the nitrogenous fraction. Low radioactivity was found in sucrose. They found a linear relationship between the rate of carbon dioxide fixation and the concentration of external carbon dioxide. The fixation rate at harvest was concurrent with the respiratory climacteric. Greater variations after storage through December appeared to be due to the higher incidence of breakdown. Rakitin_e_t_al. (1956) stored apples in 8 percent carbon dioxide labeled with radioactive C14 at 38° to 46° F. Autoradiograms of apple slices showed that stem, calyx, skin, seed embryo, vascular bundles. and cortical tissue contained the largest amount of radioactivity which apparently was due to inspired car- bon dioxide from the atmosphere surrounding the fruit. 12. MATERIALS AND METHODS StoragArrangement - 1957-58 Two gastight, insulated and refrigerated chambers, each containing 38 bushels of jonathan apples were used as master chambers to establish and maintain the desired atmospheres of 13 percent carbon dioxide - 3 percent oxygen and 5 percent carbon dioxide - 3 percent oxygen. The chamber with 13 percent carbon dioxide was held at 38° F, whereas the 5 percent carbon dioxide atmosphere was held at 32° F. Two refrigerated storage chambers were used to maintain the desired temperature of six two-bushel drums at 32° F, and six at 38°F. Each atmosphere from the master chamber was circulated through the six two-bushel drums (Figure 1) by a diaphram type pump at the rate of 1. 5 cubic feet per minute changing the atmospheres in the drums once every 27 minutes. The atmospheres in the main chambers were analyzed daily. The atmos- pheres in the two-bushel drums were analyzed weekly. The oxygen level in the master chambers was adjusted by adding measured amounts of oxygen from compressed gas cylinders. The excess carbon dioxide was removed with a column-type carbon dioxide absorber (Angelini, 1956) using 160 grams of caustic soda (NaOH) per gallon of water as the absorbing solution. Figure l A schematic and flow diagram of the storage facilities used in 1957—58. The atmosphere of the master CA chambers A and B were circulated through the small (2-bushel capacity) chambers in the refrigerated rooms C and D. - —-—-—-—q—.- -— 13% co2 - 3% 02 38°F 32°F 5% co2 - 39 02 32°F 38°F Pre-harvest Treatments - 1957-58 Forty-year-old jonathan trees at the Michigan State University Horticul- ture Farm were selected as the source of fruit for the 1957-58 studies. A NE, NW, SE or SW quadrant of each tree was selected for each treatment, with a different quadrant of each of the four trees used for a given treatment. 1. Defoliation. Branches bearing representative fruit crops with leaf/ fruit ratios of 30 to 50 leaves per fruit were completely defoliated back to i the main scaffold approximately two months before harvest. Leaves were removed from enough branches of each tree to provide approximately one bushel of fruit from the defoliated area at harvest. 2. Ringing. Approximately two months before harvest, enough two to three-inch diameter limbs to provide one bushel of fruit per tree were ringed by removing a 1/4 inch strip of bark approximately one foot from the scaf- f01d branches. The grooved area was scraped to insure that the cambium tiSSUe was completely destroyed. The leaf/fruit ratio of the ringed branches Was 40 to 50 leaves per fruit. 3. Thinning. Limbs bearing abnormally heavy crops (i. e., 3 to 5 apples per six inches of limb) were selected for the thinning treatments. Two months befOre harvest, fruits were removed so as to leave one apple for every six linear inches of the limb. The thinning increased the leaf/fruit ratio from 10 to 1 5 to approximately 40 to 60 leaves per fruit. One bushel from each of the f . Our trees was harvested from the thinned areas. 15. 4. Excess Crop. Limbs bearing a heavy crop in the same quadrants of the trees were used for the thinning treatments. The fruit was allowed to mature for harvest. These limbs bore three to five fruits for each six linear inches of the limb and the leaf/fruit ratio was 10 to 15 leaves per fruit. 5. Plastic Bag Fruit Covering. Approximately two weeks before harvest, five fruits from each tree were covered with white plastic film and five were covered with black plastic film. Thermocouples were inserted through the calyx end of the fruit into the seed cavity to measure the temperature. One copper-constantan thermocouple was placed in each of the two types of bags, one in a check fruit and one in air on each of the four trees. The tempera- tures were recorded at early morning and mid-day. 6. Checks. The fruit produced in the remaining quadrant of each tree served as control for all treatments. These fruits were stored in regular storage at 32 to 34° F in the Horticulture Building. Time of Harvest Studies - 1957-58 Different stages of maturity were obtained by harvesting fruit September 20 and 25, and October 1 and 5. The optimum date for commercial harvest for storage purposes was estimated to occur about October 1, according to past history of harvest dates in this orchard and the condition of the fruit. Two bushels were harvested from each of the four trees on each date from the check quadrant of the trees used for the preharvest studies. The fruit 16. of each harvest was composited and randomized into five one-bushel samples. One bushel was stored in 5 percent carbon dioxide and 3 percent oxygen at 32° F, 5 percent carbon dioxide and 3 percent oxygen at 38° F, 13 percent car- bon dioxide and 3 percent oxygen at 32° F, 13 percent carbon dioxide and 3 per- cent oxygen at 38° F, and regular storage at 32° F. The early harvested fruits were stored temporarily in sealed 55-gallon drums at the desired atmosphere and temperature until all harvests were completed. The atmospheres were artificially established and adjusted daily until the "master chambers" had established the desired atmospheres. When this atmosphere had been attained, the fruit was transferred to specially constructed two-bushel 15 x 17. 5 x 40 inch drums according to the arrangement shown in Figure 1. The temperature in each of the two master chambers was recorded daily from copper-constantan thermocouples located in front of the evaporator, one behind the evaporator and one in the fruit. The air temperature within the drums was checked at intervals of one to two weeks with a thermocouple inserted to the center of the drum. Comparable fruit samples were held in regular storage in small walk- in rooms regulated to a temperature of 32 to 34° F. Storage Arrangement - 1958-59 The 38-bushel CA chamber holding the sample fruits from the defoliated and non—defoliated areas at 13 percent carbon dioxide - 3 percent oxygen at 32°F was used as a master chamber to establish the atmosphere for the time of harvest studies. Six two-bushel drums (15 x 17. 5 x 40 inches) were maintained at 32°F in a small refrigerated storage. The atmosphere in the master chamber was circulated by a diaphragm type pump through plastic and rubber hose (1/4 inch inside diameter) to the six two-bushel drum, as shown in Figure 2. Each of the three diaphragm pumps supplied two drums at the rate of O. 5 cubic foot per minute so as to change the atmosphere of each drum once every 25 minutes. The atmosphere of the master chamber was analyzed daily and maintained as described for the 1957-58 storage arrangement. The atmospheres in the two-bushel drums were analyzed weekly. The temperatures of the master chamber and the six two-bushel drums were recorded daily with a recording potentiometer and copper—constantan thermocouples placed in the center of each drum, in front of the evaporator, behind the evaporator and in a fruit at the center of the stack of the master chamber. The sample lots stored in 5 percent carbon dioxide with 3 percent oxygen were placed near a removable window of the metal door of the tilt-up storage building (Pflug e_t 31; , 1957). The window was constructed to allow quick re- moval of samples through the storage season, without serious disruption of the atmosphere of the storage room. Figure 2 A schematic and flow diagram of the storage arrange- ments for 1958-59. The atmosphere of the master chamber A was circulated through the small (two-bushel capacity) chambers in the refrigerated chamber B. 1 -1 \ :~D\“D>- a. 2 5\ §\"\ \\ Ld\\\\x\‘ 3 :_¥ \ 6 \ A \\\\N 13% co2 - 3% 02 32°F 18. 19. The excess carbon dioxide was removed with a caustic soda absorber most of the season and by a water absorber during the final six weeks of storage. The oxygen level was adjusted by the addition of outside air from a pump and by metered amounts of nitrogen from a compressed gas cylinder. Temperatures in the tilt-up storage buildings were recorded daily from six copper-constantan thermocouples in the fruit and two in the air which were connected to a recording potentiometer. Defoliation Study — 1958—59 Defoliation studies in 1958 were made in commercial orchards located approximately five miles southwest of Sparta, Michigan. The orchards were selected for their similarity of climate and soil conditions and nutritional status so as to provide fruit of similar stages of maturity at a single harvest date. Four of the five trees used in previous studies (Bunemann, 1958) were selected for uniformity of appearance and size of crop. The age of the trees ranged from 14 to 22 years. The orchard soil types in the area were silt loam and silty loam soil. Two months before the estimated time of harvest, limbs in successive quadrants of each tree were completely defoliated. Three bushels of fruit from the non-defoliated and the defoliated areas of each tree were harvested October 5. The three bushels were composited and randomized into three 20. one-bushel samples; one was stored in 13 percent carbon dioxide and 3 per- cent oxygen, one in 5 percent carbon dioxide and 3 percent oxygen at 32° F, and one in regular storage at 32 to 33°F for approximately seven months. The fruit held in 5 percent carbon dioxide and 3 percent oxygen was stored in a 650-bushel capacity storage building (Pfluggt a_l. , 1957). The fruit stored in 13 percent carbon dioxide and 3 percent oxygen was held in a 38- bushel experimental controlled atmosphere storage in the Agriculture Engin- eering Building. The fruit held in regular storage was held in a walk-in storage in the Horticulture Building. Time of Harvest Studies - 1958-59 The time of harvest study conducted in 1957 was repeated in 1958 using fruit of the same four trees. Two bushels were harvested from different quadrants of each tree, on September 20, September 30 and October 14. Each tree's fruit was composited at random into three two-bushel samples for placement in 13 percent carbon dioxide and 3 percent oxygen, 5 percent carbon dioxide and 3 percent oxygen, and regular storage at 32°F. The early harvested fruit was handled similarly to that in 1957-58. The fruit stored in 13 percent carbon dioxide and 3 percent oxygen was placed in a storage according to the arrangement shown in Figure 2. The fruit stored in 5 percent carbon dioxide and 3 percent oxygen was placed in the tilt-up CA storage building. Sampling Size Thirty fruits were taken at random for evaluation of the effects of time of harvest, ringing, thinning, excess crop and defoliation treatments at harvest and after 33, 90, 125, 176 and 211 days of storage. Fruit covered with plastic bags were observed at harvest and after 210 days in regular and controlled atmosphere storage. In 1958—59, samples of each time of harvest were observed at harvest and from all storage treatments after 76, 120, 156, 176 and 210 days in storage. A 50-fruit sample was selected at harvest time from the defoliated and non-defoliated areas of each tree of the five locations for observations of fruit firmness, soluble solids, ground color and internal and external defects. Thirty-fruit samples were used for all observations after 210 days in regu- lar and CA storage treatments. Fruit Quality and Condition The quality and condition of the fruit were evaluated both years, as follows: Flesh firmness: Flesh firmness was determined with a Magness- Taylor pressure tester (Haller, 1941) equipped with a 7/16 plunger and applied at the pared surfaces of the blushed and non-blushed cheeks of each fruit. All readings were made by the author. Ground color: The ground color was rated numerically by com- 22. parison with the McIntosh Color Chart (Southwick and Hurd, 1948) which ranges from a yellow color, with a rating of l, to dark green color, having a rating of 5. Fruits with the entire surface red or striped red so as to mask the ground color were not measured. Soluble solids: The juice pressed from the fruit during pressure testing was tested for soluble solids with a Zeiss-Opton hand refractometer calibrated in percent sugars. Taste and texture: The fruit of all treatments were tasted and rated for flavor as highly acid, acid, sweet acid, sweet, lacking and alcoholic, and for texture as firm, crisp, melting and mealy. Fifteen fruits were tested to obtain an average judgment for each sample. Off-flavors were noted separ— ately. All samples were evaluated by the author. Exterior or skin blemishes: Disorders that had developed during the various storage treatments were observed and recorded from individual fruit inspections. Interior disorders: Each apple tested for flesh firmness was cut in half laterally at least twice and examined for internal disorders that had developed during storage. Statistical Methods The statistical methods employed to evaluate the results of the defolia- tion study of 1958, were in accordance with the methods prescribed by Cochran and Cox (1950) and Snedecor (1946) for the split plot technique and correlation coefficient. The results of evaluation of watercore, voids, core browning and break- down were calculated on the basis of the percent fruit free of the disorder and these values were converted to the arc sine for further statistical computa- tions. The actual values of percent soluble solids and pounds pressure of flesh firmness were used in all statistical comparisons. Due to the limitations of the storage facilities, it was impossible to replicate within storage treatments. Fruit from each treatment (defoliated and non-defoliated) of each tree from each grower were kept as separate sam- ples- They were placed in storage so that the fruit from the defoliated and non- defoliated limbs of each tree were in close proximity and therefore, used as replications within each storage treatment. Micro techniques Tissue from an apple of each tree of the five growers was placed into CR 1 / AF solution two days after harvest for comparison with the tissue of fruit stored in regular storage, 5 percent carbon dioxide and 3 percent oxygen and 13 perCent carbon dioxide and 3 percent oxygen at 32° F for seven months. _—~ \ 1/ SOluti 0n A - chromic acid 1 g Solution B - Formalin 30 cc glacial acetic acid 7 cc distilled water 70 CC distilled water 92 cc - ___ ——44—44 H ‘1 l l 24. A l x 1 cm sample of radial tissue was cut, impregnated in vacuo, and then fixed for 24 hours. The material was then transferred to 70 percent alco- h01 for holding until all materials were available for examination. The dehydration, infiltration and embedding methods of johansen (1940) were followed. The sections were cut with a Spencer Rotary Microtome at 12 to 15 microns thickness, then prepared for staining as set forth by johansen (1940). The Saf- ranin and Fast Green staining procedure of Cross (1937) was used. The slides were mounted in piccarite and allowed to dry thoroughly before examination. Samples of fruit from all storage conditions plus sections of tissues shoxving the various types of internal and external injury were prepared. The areas of tissues which showed typical types of CA injury were exam- ined and photographed. Both hand sections and sections made by using the freezing microtome technique were mounted (Sass, 1940; johansen, 1940) for further intracellular inspections. _ 14 Isotopic Procedures with C 02 The radioisotope of carbon (C14) was used as C1402 to determine the r . . . . . ates 0 f carbon diox1de evolution upon removal from storage and carbon diox- id ' . . . . . . . e dlstr ibutlon in apple tissue during controlled atmosphere condltlons. J()I‘Iathan apples harvested from the university orchard on October 1, 1957 and _ . September 30, 1958 stored in regular storage and controlled atmospheres . 25. of 13 percent carbon dioxide — 3 percent oxygen, 2 1/2 percent carbon dioxide - 3 percent oxygen, and 5 percent carbon dioxide - 3 percent oxygen at 32° F and which were free from blemishes, 2 1/2 to 3 inches in diameter and graded U. S. Fancy were utilized in these studies. Methods of Exposure to C140; - Procedure 1 A volume of fruit equal to approximately two liters (usually 12 fruits) was placed in a six-liter vacuum desiccator with a gas—tight seal. A weighed sample of BaC02 and BaC1402, precalculated to produce the desired carbon dioxide concentration (see Appendix Table XVI) in the volume of free atmos- Phere in the desiccator, was placed in a dry reaction flask and connected to the desiccator containing the fruit. The apparatus was prepared by opening all stopcoc ks with the exceptionof "A" (See Figure 3). The outlet from stopcock "D" was then connected to a water aspirator and the maximum vacuum obtained, then " D" was turned to the closed position. The system was charged by adding an excess amount of 10 percent H3P04 through "1 " which was allowed to react with the BaC03 and BaCMOZ in the reaction flask "2" until the reaction had gone to completion. Water was added through the graduated burette into the reaction flask to sweep the C02 and C1402 remaining in the reaction flask into the vacuum desiccator. A liquid was kept in the burette at all times to pre- Vent the Outside atmosphere from being swept into the system. A measured amount of oxygen was added to the desiccator through the aspirator tube (5) Figure 3 A schematic diagram of the apparatus used for exposing . . 1-1 apples to controlled atmospheres containing C 02, A graduated n burette " l was used to add acid to the reaction flash "2", and also . . .14 . . to add water to sweep the remaining L 02 into the vacuum desmcator "”.4 Tube "3" was used as a connection to a mercury manometer. Connection to the water aspirator for drawing a vacuum in the system was made at '5' 27. to attain the desired oxygen level. Nitrogen was added to equalize the pres- sure and dilute the atmosphere when necessary. The atmosphere was analyzed daily w ith an Orsat analyzer. The atmosphere was adjusted daily by adding a measured amount of water to the desiccator to displace a calculated amount of atmosphere. Meas- ured amounts of oxygen and nitrogen were added to replace the water and adjust the atmosphere in the desiccator. Additional C140 was supplied as 2 necessary according to Procedure 2, as described below. ' Although this method was satisfactory for exposure of fruit to radioactive carbon dioxide, the precalculated atmosphere was not always attained. Com- Plete evacuation of the system was impractical as there was a possibility of damage to the fruit tissues; therefore, it was difficult to precalculate the exact volume of normal air remaining in the system. mcedure 2 The same apparatus was used as in Procedure 1. All stopcocks were in the open position and a water source was connected to tube "5" (see Figure 3). The manometer was disconnected from tube ”".3 Water filled the vacuum desl'cca tor containing the fruit sample until it reached stopcock "C" and "B'; a\\ 3‘01) COC ks were then closed and the water source turned off. The mano- meter was reconnected and tubing was connected to point "5" to act as a Si ll '1 '1 '0 '1 " phon. StOpcocks D , C and B were opened and some water allowed to ¥ siphon Off to build up a slight vacuum. Stopcock "C" was opened to draw all water out of the tube "3". A precalculated volume of 10 percent H3P04 was added to an amount of BaC03 and BaCl403 to give the desired level of labeled carbon dioxide. The carbon dioxide remaining in the reaction flask was swept out by adding water. The vacuum in the desiccator was controlled by stopcoc k "D". By measuring the volume of water siphoned off and knowing the volume of the reaction flask, exact levels of carbon dioxide and oxygen could be obtained. An Orsat analyzer was modified, as shown in Figure 4, so that the amount of radioactivity of the atmosphere, as well as the percent carbon diOXide. could be determined. The carbon dioxide-containing atmosphere was forced from the burette through stopcock "2" into the capillary tube which Provided a means of bubbling the atmosphere into the C02-absorbing solution contained in bottle ""A. Bottle "A" was so constructed that as the atmosphere was bubbled in the solution could be forced out through the bottom into bottle "3'. By closing stopcock "2" and opening stopcock "1 and lowering of the leveling bottle, the atmosphere was drawn out and replaced by the absorbing SOlution. Once the analysis was complete, all stopcocks were opened and the SOIUCIQJD drained into bottle "B". Immediate analysis for the amount of radio- aCtiVitY Could then be made. Upon completion of the experiment, water was siphoned into the desic- cat . . . . . . or to S‘Neep the carbon diox1de atmosphere containing Cl4 into tWO Fisher- Figure 4 Modification of an Orsat analyzer to enable collection of . . , . 14 .. . . the C02 absorbing solution containing C 02. By lillmg the sampling line with water prior to connecting to desiccator, no dilution loss of sample was obtained. When flushing sample out of burette valve "2" is open, valve " 1 " closed. Upon returning the sample to the burette valve "2" is closed, valve "1 opened. Absorbing solution containing 14 C 02 is obtained by draining from container "A" into container "3'. 29. 3 -Woy Slop Coo? l 2 i Ox en "II 1 ' «Pipyegll’e Burette .- I ll \4 g A ll 1. Capillary 11 4* TUbe ‘”“wxll .. H 1’ Water Leveling . -'—|I" “i Bottle . ”4:“— + \ ' - ll '- ' gilt“: T. - _ _._. 14 ll 1:! id ii ii 8 ti Milligan gas washers in series attached to tube "".3 The gas washers con- tained 250 ml of 0. 1N NaOH. Platig and Measurement of Radioactivity Several procedures were used for plating and counting the radioactive samples utilizing the precipitated form of barium carbonate. Aronoff (1956) completely describes a technique using a glass filtering assembly which was found to be satisfactory, if comparatively large amounts of BaCO3 and BaC1403 precipitate were collected. When small amounts of BaC03 were plated, it was found that the BaC03 and BaCl403 precipitate would pass through Watman No. 42 filter paper. This procedure was used for all 1957 and 1958 determinations. In 1959, a slurry of BaCO3 and BaCH'O3 and 95 percent alcohol on a 2. 5 cm diameter by 0. 5 cm in depth stainless steel planchet was plated (Calvin eta—1. 1949) with this method repeatable results were easily obtained. The radioactive samples were counted by a Chicago Nuclear Model 47 gas flow counter— with 1 micro-mil window, and a Model 17 Scaling Unitse/ was used to record the num- ber of particles detected by the gas flow counter. 14 Distribution of C 02 in Peel, Core and Flesh k Eight jonathan apples were removed for 2 1/2 percent carbon dioxide - 3 percent oxygen after five months and exposed by Procedure 1 (above) to 50 1 r/Nuclear Instruments and Chemical Corporation, 223 West Avenue, Chicago 10, Illinois. 30. percent carbon dioxide labeled with (500 microcuries) of Cl4 and 10 percent oxygen for 14 days at 75° F. After removal of the labeled carbon14 dioxide normal air was passed through the desiccator at the rate of 210 cc per minute. The radioactive carbon dioxide given off by the fruit was collected in 0. 1N NaOH in 500 ml fritted gas washer. Periodic 10 ml samples were taken and measured for radioactivity as described. The remaining four fruits were peeled and cored mechanically. Peel, core and flesh were blended separately in Waring Blenders with 250 ml of 0. 1N NaOH for five minutes. These solutions were stored at 32°F for future pro- cessing. This experiment was repeated using four jonathan apples removed from 2 1/2 percent carbon dioxide - 3 percent oxygen after 5 1/2 months and exposed to 50 percent carbon dioxide containing 457 microcuries of C14, and 10 percent oxygen for 14 days at 75° F. The procedure for handling the fruit was the same as above after the mechanical peeler had been adjusted so less of the flesh was included with the peel. The blended solutions were reacted with 10 percent H3P04 solution to re- lease the carbon dioxide containing C14 in the blended solution in the apparatus shown in Figure 5. The released C1402 was collected in 50 mls of 0. 2N KOH. One milliliter samples were plated and counted, as previously described. 31. Figure 5 Reaction apparatus for measuring CH content of blended radioactive tissues. The blended solution was put into flask "2". 100 ml of 0. 2N KUH was put in flask "4" with magnetic stirring rod. Beaker "6" was filled with ice water and beaker "3" was heated slowly by a Bunsen burner. The graduate burette " l ", with stopcock "A" in the off position, was filled with 11 percent phosphoric acid. All joints were sealed with stopcock grease and vacuum taken at "B". When n: n blended solution in started to boil, "B" was turned off and the phos- phoric acid was added slowly. The magnetic stirrer "5" was started and the reaction allowed to continue for three hours. 32. .ll... '1 33. C02 Evolution Studies After approximately six months in storage, eight McIntosh apples from 5 percent carbon dioxide - 3 percent oxygen at 38° F and regular storage at 32°F, and eight jonathan apples from 2 1/2 percent carbon dioxide - 3 percent oxygen at 32°F and regular storage at 32° F were treated in a sealed 12-liter glass jar. The atmosphere was adjusted to 25 percent carbon dioxide and 10 percent oxygen using 10. 4 milligrams of BaCl403 to produce 433. 3 micro- 14 as described by Procedure 2 above. After 17 days of treatment curies of C the radioactive atmosphere was displaced with water and collected in fritted gas washers containing 500 m1 of O. 1N NaOH. The fruit samples were separated by variety and storage treatment and placed into separate 5-gallon jars and resealed. Air at the rate of 200 to 300 ml per minute was passed over the fruit and into a fritted gas washer containing 500 ml of 0. 2N NaOH. Ten ml aliquots were removed after 1/3, 1, 1. 5, 2. 5, 9. 25, 15, 24 and 39 hours. One milliliter samples were plated and dried for counting. Four jonathan apples removed from 2 1/2 percent carbon dioxide and 3 percent oxygen and regular storage at 32° F after six and one—half months stor- age and placed in 25 percent carbon dioxide containing 260 microcuries and 10 percent oxygen for 17 days, were also evaluated. In another test, eight jonathan apples stored four months in 13 percent 34. carbon dioxide - 3 percent oxygen at 32°F were placed in a two 4-liter con- tainer and sealed. The atmosphere in the container was adjusted to 25 percent carbon dioxide - 10 percent oxygen labeled with 166 microcuries of C14 02 (Procedure 2). After seven days at 75°C for one jar and 32°C for the other jar the radio- active atmospheres were removed and collected in a fritted gas washer contain- ing 0. 2N KOH. Two apples from each jar were left in the container at 75° F and the container resealed. An air flow of 200 to 300 cc of air per minute was passed over the fruit and washed with 500 ml of 0. 2N KOH. One ml samples were taken after 1, 3, 17, 23, 29 and 53 hours for radioactivity measurements. Four Jonathan apples, removed from 13 percent carbon dioxide with 3 per— cent oxygen, 5 percent carbon dioxide with 3 percent oxygen and regular stor- age at 32°F after five months, were exposed to 25 percent carbon dioxide con— taining 166 microcuries of C14, and 10 percent oxygen. After seven days of treatment the radioactive atmosphere was removed and the evolution of C1402 from the three storage treatments measured at I, 2, 6, 12, 20, 24 and 53 hours as previously described. C02 Distribution in Fruit - Blotting Paper Technique A 3/8 inch thick median cross sectional slice was removed from two fruits of each jar from the previous experiment. The slice was placed immediately between two pieces of blotting paper saturated with 0. IN BaOH and placed in a Buchner funnel. A slight suction was applied for five minutes. A stopper was placed over the seed cavity to prevent loss of suction. The blotting paper and fruit slices were separated and each placed between two five-pound metal plates. The plates containing the samples were dried in an oven at 150° F. The dried samples were removed and marked off into 1/4 inch square grids and each grid was counted directly for activity. C02 Distribution - Filter Paper Technique Sample fruits were removed from 13 percent carbon dioxide - 3 percent oxygen after five months. Three fruits were sealed into each of 14 one-gallon containers in which the atmosphere was adjusted to 20 percent carbon dioxide and 10 percent oxygen using 2. 9 milligrams of BaCMOz as the source to pro- vide 229, 5 microcuries of C14 labeled carbon. Single containers placed at 32°F and at 75°F for l, 4, 24, 168 and 384 hours. After the desired exposure period, the fruit was removed from storage and the radioactive atmosphere flushed and absorbed as before. Three cross sectional approximate median slices were taken from each fruit. One slice was immediately blended in a Waring Blender with 100 ml of 0. 2N KOH for three minutes. After each slice the cut surfaces were placed on a 0.1N barium hydroxide soaked No. 2 Whatman filter paper. The filter paper and fruits were held while the next slice was taken. The cut surfaces of the slice and apple were immediately placed on No. 2 filter paper saturated in 0. IN 36. BaOH. The fruit slice between the two pieces of filter paper was then put in a Buchner funnel and a slight suction applied for three to five minutes. The filter paper and fruit slices were separated, placed between two pieces of botanical blotting paper and dried at 147° C under sufficient weight to ensure filter paper and fruit would be flat when dry. The blended solution was placed in the reaction apparatus (Figure 5) and the evolved carbon dioxide collected in 100 ml of 0. 2N KOH. One set of dried filter paper and fruit slice samples were attached to botanLcal mounting boards (8 x 10 inches) and covered with Saran film. The mounted samples were placed in a light-tight box in contact with 8 x 10 inches Kodak Medical X-ray film, each sample being separated by 8 x 10 inches plastic board separators. Pressure was applied to the stac-. in the light-tight box to keep the film and mounted samples in close contact. After 73 days, the film was removed and developed with Kodak developer and fixer. The development time was varied to bring out the grain in the auto- graphs With a minimum of background fog. The developed film was washed for two hOUrs in tap water and then dried at room temperature. The second set of dried filter paper and fruit slices were treated with 10 per- cent phosphoric acid for two minutes and dried at 147°C. The samples were div- ided into 1 /4 inch square grids and counted for radioactivity. The filter paper method was considered a better technique than the blotting paper teQ hnique. Diffusion of the BaCl403 away from the point of absorption was reduce . . . d to a minimum by this method. RESULTS The data accumulated from the experiments for the two years in which these studies were conducted are tabulated in Appendix Tables I to XV. Por- tions of these data are presented in the text under the appropriate headings related to the storage disorders and their development. Core Browning Core browning was characterized by various degrees of brown discolor— ation in some or all cells in the pith and occasionally in the cortical tissue. The injured tissue varied from light to dark brown in color and from dry and flaky to moist in texture depending on the severity of the injury (Figures 6 and 7). Although core browning development varied from fruit to fruit, it generall y radiated from the vascular bundles and occurred around the cambial line in the pith tissue rather than in the tissue between the seed cavities (Figures 8 and 9). Microscopic observations showed the presence of particles in the vacuole- like sacs of the browned tissue (Figure 5, A and B). These sacs contained a brown fluid surrounding the particles which was responsible for the brown color of the tissue. Normal appearing cells without brown discoloration of the vacuole ~ 11 kc sacs were also present in the browned tissue (Figure 10, A). Data showing the effect of storage temperature upon the development of Figure 6 Core browning of Jonathan apple fruit radiating from the primary vascular bundles (see arrows) into the pith tissue. Figure 7 Severe core browning in which the affected pith tissues appeared dry and flaky in texture. Figure 8 The cross sectional drawings of fruit A through D, the shaded portions represent the areas developing core browning dur- ing controlled atmosphere storage. Drawing D also depicts a void or air pocket present in the browned tissue. Figure 9 The shaded areas of the apple cross-sections E through l-I represent the regions affected by core browning. Usually only the pith tissues were involved, but occasionally core browning origin- ated at the vascular bundles in the cortical tissue as in drawing F. Figure 10 These drawings depict cells from the browned pith tissues of the fruit. The shaded areas within the cells represent vacuole-like sacs containing particles. Drawing A (approximately 30X) shows that not all cells in the affected area have these sacs. Some of these sacs contained a brown fluid surrounding the particles which was re- sponsible for the brown color of the tissue. Drawing B (approximately 60X) is a single cell from the group of cells in A, showing the shape and position of the vacuole-like sacs within a cell. .. . :J , «i I, , unpla- Irflmfiflfin’ml; 9T] w . Fraimt it: lull. 42. core browning are summarized in Table 1. These data are the average per- centage of fruit with core browning upon removal from controlled atmosphere storage at 32 and 38° F. Some core browning appeared in fruit held at 38° F, although 32° F was considerably more favorable for its development in con- trolled atmospheres. Table l The Effect of Storage Temperature on the Development of Core Browning in Controlled Atmospheres Percent Fruit with Gore Browning 32° F 38° F Experiment 1, Appendix I 74. 3 32. 9 Experiment 3, Appendix III ’ 60.0 5. 0 Average 67. 2 19. O The effects of storage atmospheres upon core browning for the experi- ' ments from which comparisons are available are summarized in Table 2. The average values for storage at 32° F Show the atmosphere of 13 percent carbon dioxide and 3 percent oxygen (51. 8 percent) was more favorable for the development of this disorder than 5 percent carbon dioxide with 3 per- cent oxygen (49. 0 percent) and both atmospheres yielded more core browning than storage in normal air (24. 4 percent). The fruit in 1957 was considerably 43. m .3 m .fi - 823... o .2 o .o - $3 he 5 mam be 3% are S E m mm o .3. c. .E - $3 smote: we mac. H a w .3 o .3. a. as wage: a. .3 s .2 o .o 32 eecezoho m .2 m .8 o .o $2 smote: we meme 2 N o .5. o .8 m .3. mm $2 messes E a. o .8 o .2. e .0 32 he 5 "rem a. same are 2 E m m .Nw o .8 m .8 $2 sate: do 9:5 H a g N g o o «.8 see; moo can Ace as .02 058. _ 00 a2 o0 5m H2 ohdm Meow E9533. Epsom“? .oz woseammofiua. owengm -39th com EoECoaxm @555on 0.30 no Eefigflgoo on“ no moaocmmofiz owmwoum Ho Seam 23. N Sash. more susceptible to core browning than apples harvested in 1958. In the latter year, large amounts of core browning occurred in regular storage in two of these experiments, whereas none appeared in 1958. The occurrence of core browning in some of the individual experiments varies notably from the average for storage treatments. For instance, fruit from ringed limbs showed more core browning when stored at 5 percent car- bon dioxide and 3 percent oxygen (90 percent) than when stored in either air (41. 5 percent) or 13 percent carbon dioxide and 3 percent oxygen (47 percent). The controlled atmosphere with the highest carbon dioxide level (13 per- cent) produced more core browning than the atmosphere containing 5 percent carbon dioxide at a storage temperature of 38° F. The defoliation studies of 1958 (Appendix Table IV) enabled a statistical comparison of core browning in relation to storage atmospheres at 32° F. Significantly less core browning occurred in apples in regular storage (0. 0 percent) whereas those in 5 percent carbon dioxide and 3 percent oxygen had an average of 13. 2 percent and those stored in 13 percent carbon dioxide and 3 percent oxygen averaged 26. 1 percent. Highly significant interactions of atmospheres with growers and with defoliation were evident. Time of fruit harvest was of some influence on core browning. Accord- ing to the average values presented in Table 3, fruit harvested October 1, the date considered to be optimum for commercial harvest in 1957, developed the 44. 45. emote: EoHoEEoo .3..— oome EzECQo ceHoEmcoU I \« m .2 - - E t. o 22 .2 $880 2 and. x652... 8% w .2 - - S a 9 E22 .8 Heeeeaem N Eeaceexm m2 - - mm em 0 £2 .8. Heeeeseem 0.2 a. 2 as a... me $2 .m .88an m. .2. 2 8 me 5 S \Mem2 .2 $880 c and. 533% 89 0 .me S cm 2: ms ow $2 .3 seeeeaeem 2 Eeeaeexm :e S a. 3 ow 02 $2 .8 $8833 Ne em me can No .2 me was .2... .80 0st So can So .2: «8 .2 a... ”ram mam wficaoum oHoU 52> “Sum Eoohom Boa/.3: Ho 9:; owmuoom $33M osecemoEZ 8:93:00 5 gen— 03 new owwuoum “convomnzm was $95.5 mo eEC. 8 ”36.88.... mats/cum v.80 5:5 339.. cmnumcg Ho momwocoocom :32 m. oEmF least core browning in storage. This did not, however, hold true for all storage conditions. Namely, fruit picked on October 1 and stored in 13 per- cent carbon dioxide and 3 percent oxygen at 32° F developed 83 percent core browning, whereas fruit harvested on September 20 developed only 57 per- cent. Also, fruit harvested on October 5, stored in 5 percent carbon dioxide and 3 percent oxygen at 38° F yielded a lower percentage (16 percent) of core browning than fruit harvested on October 1 and stored under the same condi- tions (20 percent). The lowest average core browning in 1958-59 was for fruit picked at the earliest harvest date, but again there were exceptions according to the storage method and picking date. Fruit harvested on Sep- tember 30, for instance, showed a lesser amount (21 percent) of this dis- order than fruit harvested on September 20 (26 percent). None of the prestorage treatments (Table 4) were of consistent effect on core browning development in the first year. Holding the fruit for 10 days at 55° F after harvest prior to placement in storage, prevented this disorder from occurring in regular storage, but gave quantities in controlled atmos- pheres similar to that of untreated fruit. Defoliation and thinning, on the other hand, increased this disorder in regular storage, but decreased it in controlled atmosphere storage. Defoliation, when tested more thoroughly in the following year, did not affect core browning. There was no significant difference between fruits from branches with and without their leaves re- moved approximately 60 days before harvest (see Appendix Table IV). The 46. 47 o .2 o .2 o .2 o .3. o .S cameos: H 2 , o .o - - - o .0 was and E m o .N - - o .2. o 52 88 38$. 2 s o .e - - o .NN o .3 mafia? E e - - - o .N o .02 8:283 2 m. - o .o o .2. o .2 m 2. meme; 2 2. o .2 o .o o .2 o .2. o .o he E 2.2 a same was. 2 E m N No .2 N No 02 N No em N No es... a? o o o o 8 s2 oo 2 8 s2 oo 2 .oz Bees m owm m .Nm Em Samoa. ommsouwosm 598mg. 285 , wficBoum 0.80 :23 245m Eooaom mom 2.3de wméma I owwHSm Hefiswom cam muonmwofifi. @6225qu E 230 EN H8 noHSm Euuonwompsm A ml, i, a. 3an one? use munofiumohh «@396on 222.35 uofieoom “Sum 23. .muanwm 300 :23 mean? 285222. Econom thinning test was not repeated the following year. Periodic observations of the fruit condition were made during the stor- age season to determine the time of occurrence of core browning in storage (Table 5). The initial appearance of core browning in the 1957-58 storage season occurred after 90 days in fruit stored in 5 percent carbon dioxide and 3 percent oxygen at 32° F. Core browning first appeared in fruit stored at 32 and 38° F after 125 days. Generally core browning in all treatments for this year became more prevalent as the season progressed. In 1958—59, the fruit stored in controlled atmospheres showed core brown- ing after 76 days in storage. No core browning appeared in fruit held in regu- lar storage at 32° F. Voids Carbon dioxide injury often was evidenced by the formation of small spheroid air pockets or voids in the pith tissue and occasionally in the cortical tissue of Jonathan apples (Figure 11). These voids varied in size from barely visible (approximate size . 1 cm to 3 cm) to a complete hollowing of the pith tissue between the seed cavities (Figures 12 and 13). The tissues immediately surrounding the pockets were brown in color and spongy in texture and this condition sometimes extended into the surrounding areas, as shown in Figure 12. Compressed layers of cell walls from the collapsed cells, which previ- ously occupied the voided areas, formed around the voids (Figures 14 and 15). 49. o .o o .o o .o - o .o - o .o - HE Tmm mew. 0.x. - o .2. - s .mm - m 2 w .mm w .2 o .Nm - v .wm - H .3 - m m m omm $-me N .mm 0 .8 m .N - o - o m 2 m .3. o .2 - o N - o - o m m momm 0 .mn n .mm - H .2. - o - 0 ads N. .3 0 .0w - e .2. - o - o m 2 w .3 0 .mo - 2 .2. - m .2 i o m m m emm wméme @5555 9.80 :23 “Sum Eoouom SN o: 02 mg 2.4 oo on mm. No.35 moves oweuoum E 230 UMQHOHD .HNudwle 3:3. D...D:LOD:—an 3.111).}.1)’ :0 lihbri , - l .. a a Figure 11 Longitudinal view of a jonathan apple having small voids in the pith tissue. Figure 12 A medial cross-section of a jonathan apple from CA storage with severe voids. Figure 13 A medial cross-section of a jonathan apple showing a void in tissue which was also affected with brown heart. 50- 3 Figure 14 Photomicrographs (X 10) showing voids within the pith tissue of a jonathan apple. Note the smaller voids formed around the large void in the upper photograph. NM, 3. . . ; \, Figure 15 Photomicrograph (X 100) of the cells at one side of a void in the pith tissue of a jonathan apple. The walls of at least six cells close together at the left side of the photograph formed the side of the void. 53. 1p sed cells were free of contents other than an occasional sac Lg plastid-like particles. effects of storage temperature upon the development of voids in ed atmosphere storage (Table 6) are shown as the average percen- :curring in apples of comparable experiments when stored at 32 and The storage temperature of 32°F was considerably more favorable formation of voids than 38° F. Table 6 ffects of Storage Temperature on the Development of Voids in Fruit Stored in Controlled Atmospheres Percent of Fruit with Voids 32° F 38° F ’iment 1, Appendix I 43. 6 5. 6 :iment 3, Appendix III 60. 0 5. 0 Average 51. 8 5. 3‘ Data from comparable experiments are summarized in Table 7 .showing affects of storage atmospheres upon the development of voids. The .es, which are the average percentages of fruit with voids formed in :age temperatures of 32 and 38° F, consistently show that the storage lospheres of 13 percent carbon dioxide and 3 percent oxygen were more [111 ii i ,1» it ill} ., , .i . I: l 11111 ll 54. H .o m .2 ohsowaoQEoF mewm no“ owwso>< o .2 o .o - 2.. $2 he 5 a .m... a same was 2 E m N .w o .m - mm 53 emote: we oEC. H H 0 .mm 2. .2 o .0 332.3th m eNm .22 ommaoia. o .2. o .o o .0 NM mm: 20:23.60 N .m m .o o .o Nm wmofi emote: .6 6&5. E N e .5. o .o o .e Nm $2 erase E e o .2 o .2. e .0 NM $2 as. E firmm a same 3% 2 E m w .3 0 .ON 0 .o Nm $3 emote: do 2:5. H 2 Ace Age Age No as... No .2 a... a e . . N8 N2 N8 .2 . oz Bees. oz 0.3.2... .8on Eocbmourw fences: ucoE mmcmfimoEE mwmaozm -SQEmH 8m -Cmaxm L» l->.:L>..J»)\J D33 :3 nDHOCQWOEH< OMNHOHW HO uUOHum— DER. for the development of voids than 5 percent carbon dioxide with 3 xygen. Voids never develop in fruit stored -in normal air at 32° F. of the 1957 crop was more susceptible to the formation of voids es harvested in 1958. atistical comparison of void formation in relation to storage atmos- .t 32° F was made in 1958 for the defoliation experiment; the data of :e shown in Appendix Table V. Apples stored in 5 percent carbon with 3 percent oxygen and in normal air showed little or no void de- tnt. Voids developed in 3. 4 percent of the fruit stored in 13 percent dioxide with 3 percent oxygen, and this amount was significantly greater r the other storage atmospheres. me of harvest was of some influence on the susceptibility of fruit to the pment of voids in storage. According to the data in Table 8, fruit har- September 25 in the 1957 storage test developed the least amounts of in CA storage. The earliest harvested apples (September 20) in 1958 :he only fruits which remained free of voids in CA storage. Generally, uit became more susceptible to the development of voids as it became mature. Although this occurred for the average of each storage season, not hold true for all storage conditions. For example, fruit harvested :tober 1, 1957 showed more voids when held at 13 percent carbon dioxide °F (25 percent) than fruit harvested on October 5, 1957, and stored r the same conditions (8 percent). .r 1.1 .Illr . .UIWU... again: #2955200 so“ 88. :22an eoHoEmeoO I \m o .m - - 2 .2 w .o o 22 .2 $880 2 e38. insomnia. oomv ed - .. N .2 o 0 E22 .0... aeeeeaem N eeeaceexm ed - - o o o 2.2 .ON anagram msm m e 02 22 0 $2 .m 28880 m .Nm N e 02 em o \m$2 .2 Beebe a meme x6525. oomv ON 0 o 2 o 0 $2 .mN Seesaw 2 “56:0me NN o 0 mm m 0 $2 .ON seeeeaem 829:... No .2 No ,2 No 02 No .2 E. N802: Noe 0% Novena N8 ,2 m ewm mon Bot/Her we 95% mEo> :23 322m Eoouom mwmuoam Hflswom 28 8m: @8224. 8:93:00 5 230 SN H2 .- d, l lilbillnluo i) ))®3J4¢\I\I .10 d. dudDd>H 57. 'he prestorage treatments listed in Table 9 showed no conformable effects 1 development of voids. Holding apples for 10 days at 55°F prior to place- in storage prevented this disorder from occurring in 5 percent carbon ie at 38° F, but gave quantities about equal to the non-treated fruit when .n the other storage conditions. Thinning and excess crop were asso- d with few or no voids in 5 percent carbon dioxide with 3 percent oxygen ”‘F. Defoliation greatly increased the incidence of voids over that of on-treated fruit when stored in 5 percent carbon dioxide and 3 percent en at 32°F. When a more thorough defoliation study was conducted in , however, fruit from the defoliated braches responded in an opposite ner in respect to the development of voids when stored in CA conditions Appendix Table V). Fruit from non—defoliated branches had an average e of 2. 2 percent voids, which was significantly greater than the amount s than 0. 1 percent) of voided-fruits which had been picked from defoliated iches. Intermittent observations of void development during the storage season summarized in Table 10. Voids first appeared in the 1957—58 storage son at 125 days in fruit stored in 13 percent carbon dioxide with 3 percent 'gen at 32° F. The initial appearance of voids in fruit stored in 5 percent .‘bon dioxide and 3 percent oxygen at 32° F occurred at the inspection made 176 days. No voids were noted in fruit stored in CA conditions at 38° F F|L| ill 58. mm o 2: on o 858:5 H 2 mm - - - o 38 SEE E. w o - - o o no.8 wmooxm E N. N - - o o 2:539 E o - - - $ 0 83:88 E m - $ S o o mEmEm E w 2 o ow ea o 5 E ”7% a :28 3% 2 E m g as as g Q; No 0% mo 02 No 02 No 02. ..:< moo 052 moo “Rm moors? «8 0$ . .oz 038. .02 m .mm .9on EmEuonE ommuoumoum inseam? “:95. wow 359nm mEo> £23 tam Eoouom - l s Ir ironlt>a1 4.5430911 3413 94D=LODzduc JDHADHa—uaj :H Wk“: Du.“ oHOH QMNHOUW 59. o o o - o - o - Him N .m p .N w A .. o - o - m 2 N .o N .o o - o - o - m m momm @9me N. .w o - o - o - o m 2 m o . - o - o - o m m m owm o o - o - o - o .22 N .00 m be - N .om - . o - o m 2 o .8 a - o - o - o m m m on . wmémg mEo> 5T5 “Sum unmomom NOR NOUQB SN 0: cm: m3 HNH 00 cu mm mwwaoum E 225 @WGHOHD HQBMOVH 5:3 DHbu—IDDSSKN 3D4434433) 3... 3)L>4).)\J 13¢) . ill)!» I l--- l. 60. 11 210 days. Generally, voids became more prevalent as both seasons gressed. In the 1958-59 storage season, the fruit stored in 13 percent carbon tide with 3 percent oxygen showed voids after 156 days in storage. :13 first appeared in fruit stored in 5 percent carbon dioxide and 3 percent gen at the inspection made at 176 days of storage. Information on the occurrence of voids according to fruit size is avail- : from the defoliation study made in 1958 (Experiment 9). One hundred ts from each of four trees of the five growers were used to establish an 'age fruit size per grower. These average fruit sizes were found to fall four categories according to grower. Upon removal from seven months age in 13 percent carbon dioxide with 3 percent oxygen at 32° F, 30 fruits r the four trees of the five growers were inspected for the development lids. According to the data in Table 11, the larger sized fruits were a susceptible to the development of voids than the smaller fruits. This rvation was confirmed in fruit of the other experiments. Table 11 Occurrence of Voids in Various Sized Fruits Stored in 13 Percent Car- on Dioxide with 3 Percent Oxygen at 32°F for Seven Months I, |__—— meter of Fruits (Inches) 2-1/4 I 2-1/2 2-3/4 3 |——-—— :ntage fruits with voids 4 11. 5 9. 7 17. 0 H |.__—— 61. Breakdown tree types of breakdown developed in the Jonathan apples stored under rage conditions of this study. ternal breakdown was characterized by browning of the vascular bundles cortical tissue followed by a collapse and browning of the cortical tissue l the affected vascular bundles (Figure 16). In the later stages, the 2d tissues became soft, dry and crumbly, and light to dark brown in Internal breakdown in the advanced stages was evidenced externally ull waxy appearance of the epidermis over the affected areas. This {er developed in controlled atmosphere and in regular storage. )ggy breakdown developed in the inner part of the cortical tissue as , soft and spongy areas that were sharply delimited from the normal The affected and adjacent tissue often developed an alcoholic or fer- d flavor. Soggy breakdown was not evident externally until large areas tical tissue had become affected; then, the skin showed a brown dis- Ltion and the flesh seemed spongy when a slight pressure was applied. lisorder developed only in regular storage. rown heart, on the other hand, developed only in controlled atmosphere :ions. This disorder developed in the pith, or cortical tissue alone, or h tissues of the same fruit and often on numerous patches of brown y tissue that were sharply defined from the normal tissue (Figure 17). Figure 16 The darkened areas in the pith and cortical tissue of this Jonathan apple are injured as a result of internal breakdown. Note the voids in the pith tissue within the affected tissue. Figure 17 The shaded tissues within the pith and cortex of these fruits are affected with brown heart. The large air pockets or voids in the pith tissue were not associated with brown heart. 63. Llly a zone of the peripheral tissue remained sound; similar to that in ;y breakdown. Because of the difficulty of distinguishing either the very early stages or latter stages, which were often invaded by secondary pathogens; these sorders are reported as the combined percentages of breakdown present. The effects of storage temperature on the development of breakdown in )ntrolled atmosphere storage (Table 12) are shown as the average per- entages of fruit with breakdown upon removal from storage after seven months in CA storage at temperatures of 32 and 38° F. Both experiments included over-ripe fruit which were susceptible to breakdown in storage. Small amounts of breakdown appeared in fruit held at 38°F, whereas, large amounts occurred in storage at 32° F. Table 12 The Effect of Storage Temperature on the Development of Breakdown in Controlled Atmosphere Storage % Fruit with Breakdown 32° F 38° F Experiment 1, Appendix I 29. 0 2. 2 Experiment 3, Appendix III 58. O 1. 5 — Data showing the effect of storage atmospheres upon the development of breakdown for experiments from which comparisons are available, are summarized in Table 13. The average values for storage at 32°F show that the storage atmosphere of 13 percent carbon dioxide with 3 percent oxygen were more favorable for the developmait of breakdown than an atmosphere containing 5 percent carbon dioxide or normal air. Storage in normal air yielded slightly greater amounts of breakdown than fruit stored at 5 percent carbon dioxide at 32° F. The average percentage of breakdown at 38°F was relatively small within both CA atmospheres. Fruit in 1957 was considerably more susceptible to breakdown than apples harvested in 1958. Breakdown was affected by circumstances other than storage temperature and atmosphere. Fruit from the 1957 time of harvest studies and fruit de- layed 10 days at 55°F prior to storage, for example, showed more breakdown when stored at 5 percent carbon dioxide with 3 percent oxygen at 32°F than in normal air, whereas all other experiments stored in 32°F developed more breakdown when stored in normal air than in 5 percent carbon dioxide with 3 percent oxygen. Statistical comparison of breakdown in fruit of the defoliation studies in 1958 (Appendix Table VI) showed there was no significant effects of storage atmospheres, orchards, defoliation or their interactions. The effect of time of harvest on breakdown of fruit in storage is presented in Table 14. The greatest amount of breakdown developed in storage occurred in the more mature fruit (harvested on October 1 and 5 in 1957 and on October 64. 65. o .N 2 .2 - mmmue>< on... o .o - 2.. $2 28 222 ”2 .mm 22. 2are 92% 2 222 m N .N N .N - mm $2 23:2 20 9222s 2 2 m .2. 2 .m o .2. 2203.. m .2 2. .2 o .2. N... $2 252.2288 N .m o .o m .0. NM 22 2822.22 20 92229 22 N o .2 o .o o .2 Nm $2 92222222 222 2. o .02 o .2 o .2 N2... $2 222. 222 .mm 222 $22. 92% 2 222 m o .2 o .22 N .m Nm $2 2852.22 20 9222s 2 2 20% Age g No .222 No .2 22... 2.2; .02 2.22% .oz , Nov 0&2 Nov cam .2262. 28> 22525822. 3222282222... 225E W uohonawofiz omeBm mom -Emaxm :Booxwonm mo unofimo2o>oo 0:2 220 mesonamofifiN mwmwoum Ho uooflm 2E. m2 3an 66. smote: 220322222200 .28 32% 2222222222220 2520222200 I , \N 2. .w - - o .22 o 2.. .m2 $02 .22 28080 o - - o o 0 I302 .90. HonEmfiom 222 2an \m 2222225222222 89 o - - o o o 22 .ON 282223.22 N 2852692 22 .3 m. o 002 02 2 >32 6 2220.200 o .mm o m 002 02 o $2.362 .2 2022080 22 2an 2222252232 eemv o .o o . m o o 0 $02 .3 2222228228 2 2coE2Hoaxm o o o o o 0 $2 .ON 282228222 2.228222 No .2 No as... No .2 No .2 2222 N822 N8 0% N822 Noo em n2 .wm "2 2.6 28:32 20 02222.2. 2225232235 22223 2222.2.n2 2280.292 mwwHSm Hm222mom 22$ 302222882222 2522022250 222 22222022 22m>om 28 owe -HBm 226266326 225 232,222 20 0522.2. 8 9262802222 223025285 2.2223 mean/2. 22222222202. 20 mowficoouom 222222 2.2 0222.92. 14 in 1958). Breakdown development, when stored in normal air at 32°F appeared only in the fruit picked on October 5 in 1957, and October 14 in 1958. The percentages of breakdown occurring as a result of the various pre- storage treatments applied in 1957 appear in Table 15. No consistent effect of treatment occurred. Holding the fruit for 10 days at 55° F after harvest and prior to placement in storage, for example, increased the development of breakdown in regular storage, but gave quantities in CA storage similar to that of the non-treated fruit. Ringing, on the other hand, prevented this disorder from occurring in 5 percent carbon dioxide and 3 percent oxygen at 32° F, but yielded 30 percent breakdown when stored in the same atmos- phere at 38° F. Thinning and excess crop decreased the development of break- down in storage. The average value of breakdown from the plastic bag treat- ment markedly increased the development of breakdown in normal air at 32°F and 13 percent carbon dioxide and 3 percent oxygen at 38°F. Defoliation in— creased slightly the amount of breakdown when held in normal air and 5 per- cent carbon dioxide with 3 percent oxygen over that formed by non-treated fruit in comparable storage treatments. Fruit condition was inspected periodically during the storage season to determine the time of occurrence of breakdown in storage. According to the data in Table 16, breakdown appeared initially after 176 days in all storages 68. e m 222 2 22 28282225 2 2 m2 - - - mN .22 02232.2 222 22 m - - m m 20.8 mmooxm 222 2. 2. - - o m 22222222229 222 e r - - - ON 2 252222028 222 m r - on me o 2 22222222 222 2. m o 02 2 2 H2... 222 2.2.. a 222.2% 22.2. 2 222 m Age Age Age . 2&2 me No .2 No .2 No 0a... No.2 22... N8 .22 N8 .2 N8 .22 N8 .$ .oz 323 22.222 2.2 owm n2.on 2226 252322. 092.206on fincoWMM -Emaxm mc2o> 22223 22222.n2 2228.292 2 02222.2. AwmémoC owwhoum 222222322 222222 9292228224 25220222200 2.22 22.222022 2292mm .202 26.8% 32222225225 82222222822. «982023.222 $2.22; 22.20.22 8222?. 2222222222202 2 22263.85 2223.822 69. 2. .2. o o - o - - - 22... . m .2. o o - o - . - - m 2 o o o - o - - - m m m on omImm02 N .N o I O I o I o m m2 m .2 O I O I O I O m m "2 «mm N .m m .N , I o I o I o .2244. 0 .0m 0 .mm I O I O I o m. m2 0 .w N .2 I O I o I o m m m aNm wthmo2 :Bouxmohm 2223 23.2.12 22200292 No 05 Noooe O2N o: 02 m2 2N2 00 on mm owm202m :2 22mm mmfism 22.2232 2.2:. 8222288222 222222280 222 2.302969 2222282222822 22282022 222 28 22022222222220 2.252822 02 0222222. at 32°F during 1957. No breakdown occurred at 38° F in 1957, or at 32°F in 1958 until the inspection made at 210 days of storage. Other Symptoms of Controlled Atmosphere Injury Several types of injury were observed on fruit which has been stored in controlled atmospheres. One of these appeared as a yellowish—brown, de- pressed and wrinkled area of the epidermal layers quite similar in shape to the injury caused on fruit in regular storage by soft scald (Figure 18). Less than 0. 1 percent of the fruit stored in controlled atmospheres in either year of this study was affected. This disorder usually appeared on the most mature fruit, and was seen more often on fruit stored in 13 percent carbon dioxide than in the atmosphere containing 5 percent carbon dioxide. Another external injury was characterized by a blister-like disorder of the epidermal layers similar in size and shape to soft scald. Usually the blisters were slightly raised and sharply delimited from the normal tissue. The tissue below the epidermal layers were soft, brown and moist. Browned and blister-like indented areas that extended in irregular patterns over the fruit were more similar to soft scald than the injury des- cribed immediately above (Figure 19).. This disorder developed on fruit from one orchard employed for Experiment 14 in 1958 when stored in 5 per- cent carbon dioxide and 3 percent oxygen at 32° F. It also appeared occasionally on large fruit from trees bearing light crops when stored in controlled atmos- Figure 18 , Epidermal injury of the Jonathan apple stored in controlled atmosphere conditions of 13 percent C02 - 3 percent 02 at 32° F. Figure 19 Injury of epidermal and subepidermal tissues of the Jonathan apples stored in 5 percent carbon dioxide and 3 percent oxygen. 71. 72. pheres. Comparable fruit developed a high percentage of soft scald (37. 7 percent) when held in regular storage at 32° F. A dark brown, water-soaked area occurred in the pith tissue (Figure 21) of one lot of fruit. These apples had inadvertently been exposed to an at— mosphere of 15 percent carbon dioxide and 1. 3 percent oxygen for approxi- mately seven days. The normal and affected tissue had a musty and alcoholic flavor when this injury appeared. Water Core Water core had developed in some of the larger and more mature fruits at the time they were harvested for experimental storage. Since there were no external symptoms of the disorder it was detected only upon cutting the fruit. Internally it appeared usually around the cortical and primary vas- cular bundles of the fruit. This condition generally disappeared during con- trolled atmosphere and regular storage, but occasionally it was still present at the end of the storage season (Figure 20). In 1957, relatively large quantities (47 percent) of the fruit harvested from the defoliated branches had water core; whereas non-defoliated branches yielded fruit relatively free of water core. The fruit from the defoliated branches developed a high percentage (87 percent) of voids during seven months of storage in 5 percent carbon dioxide and 3 percent oxygen at 32° F. Further studies of the effect of defoliation on water core development Figure 20 The glassy, water—soaked areas of water core that persisted through seven months of CA storage are the darkest wedge-shaped dis- colorations around the vascular bundles. The larger and less darkened areas are affected with brown heart (which was not particularly associated with the presence of water core). Figure 21 The dark areas in these apples are brown and water~soaked pith tissue of apples exposed to 15 percent carbon dioxide with 1. 3 percent oxygen for seven days at 32° F. 74. the following year gave opposite results. Apples from the non-defoliated branches yielded 34. 9 percent water core, an amount significantly greater than the amount of water core in the fruit from the defoliated areas (4. 9 per- cent). These data, together with their statistical evaluations are given in Appendix Table VII. Fruit Condition and Quality The average flesh firmness of jonathan apples as affected by prestorage treatments, time of harvest, defoliation and storage atmospheres and tem— perature, is shown in Appendix Tables VIII through XI. The time of harvest somewhat influenced flesh firmness changes during storage. According to the average values presented in Appendix Table VIII, fruit harvest on October 1 and 5 in 1957 softened 2. 8 and 2. 7 pounds respec- tively, whereas earlier harvested fruit softened 2. 2 and 2. 4 pounds. Fruit harvested on October 14, the last harvest date in 1958, showed the greatest loss (5. 8 pounds), and the middle harvested fruit the least (3. 5 pounds). Flesh softening was more severe in 1958 than in 1957. Storage temperatures and atmospheres also affected the changes in flesh firmness. In 1957, fruit stored in 13 percent carbon dioxide with 3 percent oxygen at 38°F had the greatest average loss (3. 8 pounds), whereas in 1958 fruit stored in normal air at 32° F yielded the greatest change (5. 3) pounds. 75. Fruit stored in 5 percent carbon dioxide with 3 percent oxygen had the best average retention of flesh firmness in both years. The flesh firmness changes in fruit due to prestorage treatments are shown in Appendix Table IX. Small differences in flesh firmness occurred between the prestorage treatments and the non-treated fruit at harvest and upon removal from CA and regular storage. Fruit held for ten days in 55°F in air lost 2. 0 pounds prior to placement in CA and regular storage, but softened only a slightly more during the storage period than the non-treated fruit. A statistical comparison of flesh firmness in 1958 (Appendix Table X) showed there was no significant effect at harvest of orchards or defoliation treatment. When compared after storage at 32°F, it was found (Appendix Table XI) that apples stored in 13 percent carbon dioxide with 3 percent Oxygen were significantly firmer (14. 92 pounds) of flesh than fruit stored in 5 percent carbon dioxide with 3 percent oxygen (12. 90 pounds) or normal air (12. 3 pounds). The flesh firmness of fruit stored in 5 percent carbon dioxide with 3 percent oxygen and in normal air was similar. The soluble solid contents of fruit as affected by prestorage treatments, time of harvest, defoliation and storage atmospheres and temperatures are Presented in Appendix Tables‘XII through XV. , According to the time of harvest data for 1958 (Appendix Table XII), . .I .fl. :4 ..I4 ,. .— Uv , 2 76. soluble solids increased from 12 percent at the first harvest to 13. 5 percent at the second harvest and then leveled off. Small changes in soluble solids appeared within each harvest date as a result of storage atmospheres. Usually little change in soluble solids occurred during the storage con— ditions of this study due to the prestorage treatments (Appendix Table XIII). The soluble solids in jonathan apples from the non—defoliated branches had a significantly higher percentage soluble solids (13. 45 percent) than fruit from defoliated branches (12. 45 percent), see Appendix Table XIV. After seven months in CA and regular storage, fruit from the non-defoliated areas still had a significantly higher percentage soluble solids than fruit from the defoliated areas. Also fruit stored in 13% carbon dioxide with 3 percent oxygen had a significantly higher percent soluble solids than fruit stored in 5 percent carbon dioxide with 3 percent oxygen or from fruit in normal air. The number of fruits having complete red coloring, which masked the ground color, or striped red coloring, which partially masked the ground color, varied considerably from one treatment to another. Since this varia- tion made it impossible to accurately rate the ground color, these data were not presented. Fruit quality was judged by the author on the basis of the flavor and texture. The fruit harvested for the maturity studies had wide variation in fruit quality. The early harvested fruit was firm—crisp in texture and 77. acid in flavor at harvest; when removed from CA and regular storage, it was firm in texture, but lacking in flavor. Fruit picked on the optimum date for commercial harvest was generally firm and sweet-acid at harvest. After regular storage, it was slightly mealy and had very little flavor, -whereas that from controlled atmosphere storage at 32°F or 38°F maintained much of the quality. The most mature fruit, especially fruit harvested on} October 14, 1958, was melting and sweet and considered to be of high eating quality prior to storage. These apples after regular storage were melting with most of the large fruits mealy and sweet in flavor. Similar fruit stored in 5 percent carbon dioxide with 3 percent oxygen was firm in texture, but lacking in flavor. Fruit delayed for ten days at 55°F following harvest was of melting texture and sweet flavor prior to storage. All of these became mealy in regular storage, whereas in CA storage only the larger fruits became mealy. Fruit receiving the other prestorage treatments was generally of similar quality to the fruit picked on the optimum date for commercial harvest. The development of core browning and voids in storage did not affect the flavor and texture of the fruit. Apples with internal breakdown were generally mealy and dry with a musty flavor. Soggy breakdown, when severe, imparted an alcoholic or fermented taste to the fruit, whereas brown heart 1 gaVe a musty flavor to the fruit. 78. Labeled Carbon Dioxide Studies Much of the experimentation utilizing the radioisotope of carbon14 was devoted to developing techniques of application and measurement that would provide evidence for the distribution and movement of carbon dioxide in the fruit tissues. The uptake of labeled carbon dioxide by whole Jonathan apples which had been previously stored in controlled atmospheres was measured by C14 recovered in the recovery of C14 from the tissues. The quantities of peel, flesh and core of fruit exposed to an atmosphere of 50 percent carbon dioxide and 10 percent oxygen at 75° F labeled with C1402 (500 microcuries) for 14 days are given in Table 17. The fruit employed in the first trial (apples A through D) yielded great irregularities in the radioactivity per gram of peel, flesh and core. These variations were reduced in the second test by improving the cutting techniques to give more uniform thicknesses of peel and flesh tissues. In both experiments, the incorporation of C14 (counts/minute/gram of fresh weight) in the flesh and core of apples A through H was inversely related to the apple diameter. The average radioactivity per gram of flesh tissue was slightly greater than for the core, and considerably higher than for the peel. Controlled atmosphere and regular—stored McIntosh and Jonathan apples were exposed to 25 percent labeled carbon dioxide (433. 3 microcuries of C ) 79. N62 . m .222 .N $20 .N 2: - m: 82?... mos 0.0.0 .2 I Nos .N 000 .2.N , 2.2. .N 02. .22. 2.\2-N : 2222 .2 oom .N2 o22. .N ooo .22.N 222.”. .N oom .om N\2-N 0 moo oow .m2 N3 .N ooo .oom ooo .N oow .02. 2.\m-m , 22 N22 .2 oow .2 So .m oom .Nsm New .N omm .sm m m omm .2 oom .22 s: .N ooo .wom 09. .N oom .m2. 2.2-2.. D mos .2 ooo .om mom .2. oom .owm omm .m oom .oo N\2-N 0 owm .2. ooo .wm ooo .m ooo .202. m3 .N oom .om 2.\mIN m omm .2. ooo .3 So .m oom .2os 82 .0 ooo .Nm2 m < EmsMEhao Ego 2220.2. :2me\222220 c.2220 2020.2. 222me\an E220 2220.2. 2002202222 A Ii 230222225 oEEwm 2o®n2 22mm2n2 0200 229:2 22222.2 No 2228.202 m 22223 .No2.20 22223 00202222 N00 22285.2 on 02 230 2.2 .202 2230222222 002222.. 2222222202 <0 20 0252.2. 2002 new 228222 .0200 0222 2 22020220032 2.20 20 522220 02.2. 2.2 m222223.. and 10 percent oxygen at 75°F for 17 days. The subsequent cumulative evolution of carbon14 dioxide from the intact fruit is illustrated in Figure 22. In both varieties and from CA (150. 4 Cpm) and regular storage 43. 3 Cpm) C1402 was released rapidly for the first 2 1/2 hours following removal from the atmosphere containing C1402. Thereafter, the rates of evolution were approximately 0. 5 and l. 0 counts per minute for CA and regular stored fruit, respectively. After 24 hours the rates of evolution of CA and regular stored fruit were approximately the same (0. 5 Cpm). For both varieties the total 01402 evolved in 40 hours by CA fruit was approximately twice that evolved by fruit from regular storage. The experiment was repeated by exposure of controlled atmosphere and regular stored Jonathan apples to 25 percent carbon dioxide labeled with 260 microcuries of C14, and 10 percent oxygen at 75°F for 17 days. Measure- ment of C1402 evolved at 4, 24, and 56 hours following exposure gave cumu- lative totals for CA fruit of 12, 000, 15, 200 and 16, 400 cpm, respectively, and for fruit from regular storage of 6, 010, 9, 500 and 11, 000 cpm, respec— tively. The C1402 evolution pattern was similar to that of the previous ex- periment. 14 The cumulative release of C 02 by Jonathan apples which had been stored at 13 percent carbon dioxide and 3 percent oxygen for 5 months at 32° F and subsequently exposed to 25 percent carbon dioxide labeled atmosphere Figure 22 Cumulative evolution of carbonH dioxide by storage apples following 17 days of exposure to 25 percent carbon dioxide containing 433. 3 microcuries of C14 at 75° F. Chart A is for Jonathan apples previously stored in 2 l/2 percent carbon dioxide with 3 percent oxygen at 32° F and in regular storage at 32° F for four months. Chart B is for McIntosh apples previously stored in 5 percent carbon dioxide and 3 percent oxygen and in regular storage at 32 to 33°F for four months. Jonathan 20 - CA __________ ‘ ”'-.——-— *_ _______ #— 8 8 15 . f d l 3 1 g I, Air 5 g 8. 10 .; 3 l I § . O . l I 5 _ 0 10 20 30 40 Hours flushed with air after C14 treatment 13' McIntosh M CA 20 ‘ ”‘—--——-:-__--‘—-'-_--———— 8 o S’. .93 :1 .5 E u :5 A1r Q. U] E :3 o D lb 26 36 40 Hours flushed with air after C14 treatment -.u\_ 82. with 10 percent oxygen at 32 and 75°F for seven days is depicted in Figure 23. The C1402 evolved in the first ten hours after exposure to labeled carbon dioxide atmosphere was greatest from fruit which had been exposed at 32°F. By 24 hours, similar amounts had been evolved from . fruit at both temperatures, and the amounts remained similar (approxi- mately 0. 7 Cpm) throughout the test period. 14 dioxide from Jonathan apples stored The cumulative release of carbon for four months in normal air, 5 percent carbon dioxide and 3 percent oxy- gen and 13 percent carbon dioxide with 3 percent oxygen with subsequent exposure to 25 percent carbon dioxide (labeled with 457 microcuries of C14) with 10 percent oxygen is presented in Figure 24. The greatest amount of C1402 was released by fruit from all three storage treatments during the first ten hours. The fruit stored in 13 percent carbon dioxide, however, evolved a greater amount (60 cpm) during this time than fruit from 5 per- cent carbon dioxide and 3 percent oxygen (23. 3 Cpm) or normal air (16. 6 Cpm). Fruit previously stored in 5 percent carbon dioxide released a greater amount of C1402 than that stored in normal air. After ten hours, fruit from 13 percent carbon dioxide and 3 percent oxygen released C1402 at a steady rate until the experiment was terminated. Between 10 and 24 hours fruit from 5 percent carbon dioxide with 3 percent oxygen (4. 8 cpm) and 1 14 normal air (5. 9 Cpm), however, continued to evolve C 02 at a faster rate than fruit from 13 percent carbon dioxide and 3 percent oxygen (1. 2 Cpm) Figure 23 Cumulative evolution of C1402 by Jonathan apples following exposure to 25 percent carbon14 dioxide with 10 percent oxygen at 32°F and 75°F for seven days. These apples had been stored prior to Cl4 treatment in 13 percent carbon dioxide with 3 percent oxygen at 32° F for five months. am 8 Eocbmob 2‘20 Hoax 22m 523 conga $2022 on ow om om 2W2 . _ L0 (0001) GJHUIUI 19d snunog Figure 24 Cumulative evolution of C1402 from Jonathan apples stored for four months in 13 percent carbon dioxide with 3 percent oxygen. 5 percent carbon dioxide with 3 percent oxygen and normal air at 32'F. This fruit was then exposed to 25 percent carbon dioxide containing 14 457 microcuries of C , with 10 percent oxygen for seven days and 14 the evolution of C 02 measured when flushed with normal air. 84. 2:95on 220 .2325 be 52% 25233.2 $50212 om ow o.m ON 02. o ~ 3 O n m\ S \ \ d \ \ .2 m \ \\ w. x m ‘ \ ) II|\ \ X 0 \II I \ \ m . H222 \\ O \‘l‘ /\ II IIIIIIIIIIIIIIII 'IO“ N o I N o 0 an 0 am .8 No cam - Noe 0%: . 85. but at a slower rate than the first ten hours. After 24 hours, fruit from the three storage treatments evolved approximately the same amount of carbon14 dioxide until the experiment was terminated. The total C1402 evolved by the fruit stored in 13 percent carbon dioxide with 3 percent oxygen was 4000 and 6000 cpms greater than by fruit stored in 5 percent carbon dioxide with 3 percent oxygen and normal air, respectively; evolu- tion by the fruit in 5 percent carbon dioxide with 3 percent oxygen was 2000 cpms greater than from storage in normal air. The distribution of radioactivity in the internal atmosphere of a median Jonathan apple slice (3/8 inch thick) is diagrammed in Figure 25. The apples had been stored in 13 percent carbon dioxide with 3 percent oxygen at 32° F for four months prior to exposure to 25 percent labeled carbon dioxide (166 microcuries of C14), with 10 percent oxygen. The radioactivity in each 1/4 inch square of cross—section fruit surface was determined. The areas of activity greater than 90 cpm/l/4 inch square are outlined in diagram A, one occurred in the cortical tissue and the smaller area extended into the pith tissue. There was a wide variation in the radioactivity of the measured blocks; the greatest gradient between adjacent blocks was 89 cpm. ‘ As shown in diagram B of Figure 25 the greatest radioactivity concen— tration developed in the seed cavity, pith tissue and some of the cortical tissue surrounding the pith tissue. A sharper line of demarcation occurred between the areas of high and low activity. Figure 25 Diagram showing the distribution of C1402 from the. internal atmosphere of a 3/8 inch thick median transverse Jonathan apple slice. The internal atmosphere was withdrawn from the apple slice onto a treated blotting paper. The paper was dried and cut into 1/4 inch squares for radioactive measurement. 87. The distribution of radioactivity in an apple slice (3/8 inch thick) is portrayed in Figure 26. Although the resolution is poor, it may be seen that the greatest radioactivity occurred in the primary vascular bundles and the cambial line. The radioactivity pattern in the cortical tissues , 14 shows small pockets that contained no C . The distribution of the internal atmosphere on the treated filter paper (Figure 27) shows that the greatest radioactive concentration in the seed cavity, cambial line, with lesser amount in the pith and cortical tissue adjacent to the pith tissue. Some radioactivity outside the outline of the fruit indicated that a small amount of radioactive material diffused laterally in the filter paper (see upper left hand corner of Figure 27). By autoradiography, slight radioactivity was detected in the pith and cortical tissue when intact fruit had been exposed to labeled carbon dioxide for four hours. The greatest concentration occurred in the seed cavities with a lesser amount in the pith and cortical tissues. Fruit exposed to C1402 for one hour showed approximately equal radioactivity in the seed cavity, pith and cortical tissue (10-20 cpm). A second set of filter paper was treated with 10 percent phosphoric acid to release the C1402 to see if other materials containing C14 were withdrawn from the apple slice. Slight radioactivity (5-25 cpm) was found evenly dis- tributed on the filter paper. Figure 26 An autoradiogram of a 3/8 inch thick Jonathan apple slice taken from an intact apple that had been exposed to controlled atmos- phere containing C1402 (light areas radioactive). The shaded area on the right of the photograph was due to fogging of the film during expos- ure to the X-ray film. Figure 27 An autoradiogram showing the C14 distribution (light areas) from the internal atmosphere of a 3/8 inch thick median Jonathan apple slice. The internal atmosphere was withdrawn from the apple slice onto BaOH treated filter paper. The filter paper was exposed to X-ray film for 72 days. 88. 89. The total radioactivity of slices from which the atmosphere had not been withdrawn was determined by assay of the tissue. The total radio- activity in the complete slice for one hour of exposure was 3, 000 ppm, 10, 500 for four hours, 22, 000 for 24 hours, 30, 500 for 168 hours, and 880, 000 cpm for 384 hours. DISCUSSION The development of air pockets or voids in fruit tissues was the only important disorder noted in these experiments which could be attributed to controlled atmosphere storage. Other disorders, namely, core browning and breakdown, were not limited to fruit stored in controlled atmospheres, but were aggravated by the modified levels of carbon dioxide and oxygen em- ployed. Although surface injuries appeared only in controlled atmosphere fruit, they were of such'limited quantity that the causal factors could’not be ascertained. Quite often voids and breakdown were extensive so as to adver- sely affect the marketability of the fruit. Core browning, although frequently prevalent, was limited and was not considered to be detrimental to the fruit quality. A high level of carbon dioxide (13 percent) invariably increased the amounts of fruit with voids and core browning over the low levels (5 percent) as used in these tests. These concentrations of carbon dioxide were always employed in conjunction with 3 percent oxygen. Plagge (1942), Smock (1949) and Dewey e_t 31. (1957) found that the greatest amounts of voids developed in Jonathan apples when stored in relatively high levels of carbon dioxide. Dewey gt £11: (1957) also found dry pith areas in the flesh near the core, similar to the advanced stages of core browning, developed in the higher con- centrations of carbon dioxide. 91. Breakdown in controlled atmosphere storage has also been attributed to high carbon dioxide (Carne and Martin, 1935, 1938; Huelin and Tindale, 1947; Ballinger, 1955). This was found to be true for the 1957-58 tests, but in 1958- 59 no significant differences appeared in the amount of breakdown from the storage treatments. The quantities noted in fruit from regular storage was 4. 0 percent, in 13 percent carbon dioxide with 3 percent oxygen, 1. 2 per- cent, and in 5 percent carbon dioxide with 3 percent oxygen, 1. 4 percent. Low temperatures (32° F) in controlled atmosphere storage were more favorable for the development of voids, core browning and total breakdown than the higher controlled atmosphere temperature (38° F) in the one season in which temperatures were compared. This effect of low temperature on the formation of voids did not agree with the findings of Dewey e_t a}: (1957), but did agree with Trout e_t a_1_. (1940) and Huelin and Tindale (1940). Fruit of advanced maturity were considerably more susceptible to the development of voids and breakdown than fruit picked at earlier stages of maturation. Mandeno and Padfield (1953), Trout _e_t_ 31; (1940) and Huelin and Tindale (1947) also found that the more mature fruit was susceptible to CA disorders and breakdown in CA storage. However, the effects of fruit maturity on the development of core browning was not as clear cut. In 1957, factors other than maturity masked any distinct effect that maturity may have contributed. The most mature fruit of the 1958 study (harvested on 92. October 14) yielded the greatest amount of core browning in CA storage. The degree of susceptibility to core browning appeared to be due to a pre- disposition of the fruit tissue to core browning during the growing season. For example, some of the fruit stored in regular storage in 1957 developed core browning, whereas no core browning was found in fruit from regular storage of the 1958 storage season. Also, the fruit harvested for the 1957 storage tests was more susceptible to core browning than fruit picked for comparable storage tests in 1958. ) The effect, if any, of the various prestorage treatments on the develop- ment of disorders appeared to be concealed by some factor or factors other than the treatment itself. Where apparently opposite treatments had been applied, for example, thinning and excess crop, no differences in the occur- rence of voids, core browning and breakdown were observed between simi- lar storage conditions. The defoliation treatment when applied in 1957 greatly increased the incidence of voids in CA storage, but in 1958, when a more thorough experiment was conducted, defoliation had an opposite effect on the development of voids. Defoliation in 1957 increased the incidence of core browning in regular storage, but decreased it in CA storage; also, breakdown was increased slightly in regular and CA storage over the non- treated fruit held in comparable storage treatments. In 1958, defoliation had no significant effect on the development of core browning or breakdown. 93. In 1957, fruits having a high percentage of water core at harvest had a high percentage of voids upon removal from controlled atmosphere stor- age. jonathan apples from the non-defoliated branches in 1958 developed an amount of water core significantly greater than the amount in fruit from the defoliated limbs. After seven months of CA storage at 32° F, a significantly greater amount of voids developed in fruit from the non—defoliated limbs than from the defoliated limbs. From these data, a highly significant positive correlation occurred between the percent water core developed at harvest and the percent voids appearing in fruit held in 13 percent carbon dioxide with 3 percent oxygen at 32°F for seven months (Figure 28). This relationship did not hold true for the fruit held in 5 percen carbon dioxide with 3 percent oxygen since few voids were dormed in this atmosphere. Fruit at harvest from the non-defoliated limbs had a significantly higher soluble solids content that fruit from the defoliated limbs. A positive corre- lation was found between the percent soluble solids and the percent water core in the fruit from the non-defoliated limbs when measured at harvest (Figure 28). In turn, a significant positive correlation occurred between the percent solubls solids and the percent voids developed in the fruit stored in 13 percent carbon dioxide with 3 percent oxygen at 32° F for seven months (Figure 28). Although significantly correlated with the occurrence of water core, the Figure 28 Correlation of percent voids with soluble solids of fruit from non-defoliated branches after seven months storage in 13 percent carbon dioxide - 3 percent oxygen at 32° F and with percent water core at harvest - 1958-59. 94. 13 Soluble Solids 10 70. 60‘ 50- 40‘ ozoowom 20- 10" 25 20 15 Percent Voids 95. development of voids was not necessarily caused by water core. Voids developed in some fruits which did not show water core, and when both dis- orders appeared together in the same fruit, the same tissues were not always involved. The conditions of the cells responsible for susceptibility to water core may, however, cause them to be susceptible to controlled atmosphere injury. Other factors not determined here ultimately seemed to determine whether or not injury developed. The injury characterized by air pockets or voids occurred most often in the pith tissues. Microscopic examination of these tissues revealed that the tissues had collapsed to form the air pocket and the remaining affect ed cell walls had been ruptured or partially disintegrated. The autoradiograms of the C1402 activity of the internal atmospheres of the slice from intact fruits exposed to an atmosphere containing C1402, in most cases, showed the larg- er amounts of C1402 in the seed cavity, pith tissues, cambial line, vascular bundles and the cortical tissues immediately surrounding the pith tissues. With a carbon dioxide buildup in the pith tissues, it is possible that the meta— bolic processes of the tissue are disrupted so as to cause an accumulation of toxic materials in the cells. Hulme (1956) noted an accumulation of succinic acid in tissues which had been injured by higher than normal levels of carbon dioxide. Allentoff et a1. (1954) found that intact McIntosh apples exposed to an external source of C1402 incorporated C14 in the malic acid, aspartic 96. acid and glumatic acid. These authors also found that McIntosh apples in storage produced malic acid by fixation of carbon dioxide and the rate of fixation increased with a rising concentration of carbon dioxide in the ex- ternal atmosphere. It would, therefore, appear that there is a mechanism affected by a high external source of carbon dioxide which eventually could lead to the buildup of injurious amounts of a toxic material. The appearance of core browning in controlled atmosphere storage was not associated with the formation of voids or breakdown. Since it occurred in an area showing accumulation of C1402, and was more severe in CA than in regular storage, the accumulation of carbon dioxide may be the causal factor. As true for other disorders, the susceptibility of the fruit varied widely, being highly susceptible in 1957-58 so as to develop as a result of the normal accumulation of carbon dioxide even in regular storage. The three types of breakdown appeared to originate in the cortical tis- sues with internal breakdown and brown heart spreading to include the pith tissues. The points of origin of these disorders did not coincide with the areas found to have high C1402 activity. Over—maturity of the fruit upon placement in CA storage seemed to be the primary factor responsible for breakdown. This was found to be true also in the studies of Trout et 31: (1940) and Huelin and Tindale (1947). Several external injuries appeared as a result of storage of fruit in con- 97. trolled atmosphere conditions but none appeared in amounts greater than 0. 1 percent. Although different in appearance, these injuries were similar in size and shape to those on Jonathan apples in regular storage caused by soft scald. Trout _e_t a_l_. (1940) has reported soft scald on fruit stored in controlled atmospheres. The relatively small amounts of surface injuries that developed in the tests reported here did not permit direct correlations with soft scald developed in regular storage. Controlled atmospheres apparently affected the pith and cortical cells so as to change the diffusion rate of carbon dioxide. When controlled atmos- phere and regular stored Jonathan and McIntosh apples were exposed to an atmosphere containing C1402, the pattern of release was similar; however, fruit from controlled atmospheres evolved C140 at a much greater rate dur- 2 ing the first ten hours and released a greater total amount. Possibly the cell contents are changed under controlled atmosphere conditions so as to allow a greater adsorption of C1402; or possibly during controlled atmos- phere storage the permeability of the tissues to gases is altered. Either or both of these changes would account for the difference in the diffusion of carbon dioxide from these tissues. Evidence that these changes in cell properties are affected by carbon 14 dioxide content of the atmosphere was the differences in C 02 evolution from fruit previously stored in the different atmospheres (Figure 24). Apples pre- viously stored in 13 percent carbon dioxide with 3 percent oxygen released a greater total amount of C1402 and at a higher initial rate than fruit stored in 5 percent carbon dioxide with 3 percent oxygen and these in turn released a greater total amount of C1402 and at a greater initial rate than fruit which had been stored in normal air. The higher rate of evolution of C1402 from fruit released at 32°F than fruit treated at 75° F (Figure 23) can be attributed to the greater solubility of carbon dioxide in solutions at low temperature. For example, the solu- bility of carbon dioxide per 100 ml of water at 32°F is 0. 348 grams and 0. 145 grams at 77° F. SUMMARY AND CONCLUSIONS The purpose of these experiments was to investigate the effects of fruit maturity and various prestorage factors on the development of controlled atmosphere storage disorders in jonathan apples. Core browning, a form of brown discoloration in some or all of the pith tissues of the fruit, was aggravated by CA storage, but also appeared in fruit in regular storage during one of the two years in which these experi— ments were conducted. Cells injured by core browning showed a vacuole- like sac containing many particles surrounded by a brown fluid. High concentrations of carbon dioxide (13 percent) and low tempera- tures (32°F) increased the incidence of core browning in CA fruit over 5 per- cent carbon dioxide and a storage temperature of 38° F. Advanced fruit maturity, associated with delaying the time of harvest of fruit for storage, tended to increase the amount of fruit showing core browning in CA storage. Removal of the leaves from branches bearing the test fruit two months prior to fruit harvest did not have a significant effect on the development of core browning in storage. The time of appearance of core browning in controlled atmosphere and regular storage differed in the two years of this study. In 1957—58, core browning appeared after 156 days of storage, whereas, in 1958-59, it appeared after 76 days of storage. Air pockets or voids in the pith tissues and occasionally in the cortical 1 00. tissue of the fruit appeared only in fruit stored in controlled atmospheres. It was favored by high concentrations of carbon dioxide (13 percent) and low temperatures (32° F), and the larger and more mature fruits were more susceptible to this injury than small apples or apples harvested early in the picking season. Prestorage treatments of defoliation, delayed storage, thinning, ringing, excess crop, and the plastic bag fruit covering showed no consistent effect on the development of voids in controlled atmosphere stor- age. Voids first appeared between 125 days and 176 days after placement in CA storage. Three types of fruit breakdown developed in these tests. Internal break- down appeared in regular and controlled atmosphere storage, soggy break- down occurred only in regular storage, whereas brown heart developed only in controlled atmosphere conditions. Late-harvested fruit stored in 13 percent carbon dioxide with 3 percent oxygen at 32° F showed the greatest amount of breakdown. The early har- vested fruits were the least susceptible to breakdown in both controlled at— mosphere and regular storage. Prestorage treatments in 1957 had no con- sistent effects on the development of breakdown. Breakdown appeared initially after 175 days in controlled atmosphere and regular storage at 32°F; how- ever, fruit held at 38°F in CA storage and fruit stored in regular and con- trolled atmosphere storage in 1958 developed breakdown after 210 days. Several external injuries appeared on fruit held in CA storage, but none developed in amounts greater than 0. 1 percent. There was a positive linear correlation of water core in fruit at harvest, with the soluble solids contents of the fruit juice both before and after stor- age, and with the developm ent of voids in high concentrations of carbon diox— ide (13 percent) during storage. The best storage conditions for avoiding severe core browning, for min- imum development of voids and breakdown, and for retention of good eating quality was the controlled atmosphere containing 5 percent carbon dioxide and 3 percent oxygen at a temperature of 32°F. Fruit of advanced maturity or showing water core at harvest should be avoided when selecting Jonathan apples for controlled atmosphere storage. Techniques were developed for the utilization of labeled carbon to follow the movement and distribution of carbon dioxide in fruit tissues. More C14 accumulated in the core and flesh of the larger fruit, on a per gram basis, than in the smaller fruit. The flesh and the core contained a higher concen- tration of radioactivity than the peel of individual fruits. Controlled atmosphere Jonathan and McIntosh apples, following exposure to C1402, evolved a greater amount of C1402 and at a higher initial rate than fruit from regular storage. Fruit previously stored in 13 percent carbon di- oxide also evolved a greater total amount of C140.2 than fruit stored in 5 per- 101.. 102. cent carbon dioxide. Fruit stored in 5 percent carbon dioxide released a 14 . . greater total amount of C 02 than fru1t from normal air. The greatest radioactivity occurred in the seed cavities, cambial line, vascular bundles, pith tissues and the cortical tissues immediately surrounding the pith tissues of the fruit. These studies indicate that the properties of the fruit cells in respect to accumulation and/or permeability to carbon dioxide are altered as a result of storage in controlled atmospheres. Further study is needed to define these changes. 103. LITERATURE CITED Allentoff, N., w. R. Phillips, and F. B. Johnston. 1954. A 14C study of carbon dioxide fixation in the apple. 1. The distribution of incorpor- ated 14C in the detached McIntosh apple. Jour. Sci. Food Agric. 5: 231—234. . 1954a. A 14C study of carbon dioxide fixation in the apple. 11. Rates of carbon dioxide fixa- tion in the detached McIntosh apple. jour. Sci. Food Agric. 5: 234- 238. Angelini, P. 1956. A study of carbon dioxide absorption equipment for con- trolled atmosphere fruit storages. M. S. Thesis. Michigan State University. Aronoff, S. 1956. Techniques of Radiobiochemistry. Iowa State College Press, Ames, Iowa. Bain, J. M. 1956. A histological study of the development of superficial scald in Granny Smith apples. jour. Hort. Sci. 31: 234-238. Ballinger, W. E. 1955. Storage of jonathan apples in controlled atmos— pheres and film crate liners. M. S. Thesis, Michigan State Univer- sity 1-78. Barker, j. and F. Kidd. 1935. Injury to Australian apples due to carbon dioxide at low temperatures. (Gr. Br.) Dept. Sci. and Ind. Res. Food Inv. Bd., Rep. 1934: 109-110. Brooks, C. and D. F. Fisher. 1926. Water core of apples. Jour. Agr. Res. 32: 223-260. . , and C. P. Harley. 1934. Soft scald and soggy breakdown of apples. Jour. Agr. Res. 49: 55-69. , J. S. Cooley and D. F. Fisher. 1920. Diseases of apples in storage. U. S. Dept. Agr. Farmers Bul. 1160. Bunemann, G. 1958. The relation of the nutrient element content of the leaves and fruits to storage quality of jonathan apples in regular and controlled atmospheres. Ph. D. Thesis, Michigan State University. 1 04. Bunemann, G., D. H. Dewey and A. L. Kenworthy. 1959. The storage quality of Jonathan apples in relation to the nutrient levels of the leaves and fruit. Mich. Agr. Exp. Sta. Quart. Bul. 4;: 820-833. Calvin, M., C. Heidelberger, J. C. Reid, B. N. Tolbert and P. E. Yankwich. 1949. Isotopic Carbon, Techniques in its Measurements and Chemical Manipulations. John Wiley and Sons, Inc. Carne, W. M., and D. Martin. 1935. Breakdown in Tasmanian apples. Jour. Counc. Sci. Ind. Res. 5: 265-270. 1935a. Apple investigations in Tasmania: Miscellaneous notes. 7. The safe limit of carbon dioxide concentra- tion under ordinary cool storage conditions. Jour. Counc. Sci. Ind. Res. 5: 271-276. . 1938. The relation of crop size to the inci- dence of storage disorders in apples and to their chemical and physical characters. Jour. Counc. Sci. Ind. Res. 11: 83-86. . 1938a. Apple investigations in Tasmania: 8. 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Amer. Soc. Hort. Sci. 27: 276-280. Haller, M. H. 1941. Fruit pressure testers and their practical applications. U. S. Dept. Agr. 627. ' , and J. M. Lutz. 1937. Soft scald of Jonathan apples in rela- tion to respiration. Proc. Amer. Soc. Hort. Sci. 34: 173-176. Harley, C. P. 1938. Some associated factors in the development of water core. Proc. Amer. Soc. Hort. Sci. 36: 435-439. Heald, F. D. 1926. Manual of Plant Diseases. McGraw—Hill Book Co. , New York. Huelin, F. E. and G. B. Tindale. 1947. The gas storage of Victorian apples. Dept. Agr. Tech. Bul. No. 6. Hulme, A. C. 1956. Carbon dioxide injury and the presence of succinic acid in apples. Nature 178: 218—219. Johansen, D. A. 1940. Plant Microtechnique. McGraw-Hill Book Co., New York. Kidd, F. and C. West. 1923. Brown heart — A functional disease of apples and pears. (Gr. Br.) Dept. Sci.and Ind. Res. Food Inv. Rep. 12. 1925. Functional diseases of apples in cold storage. (Gr. Br.) Dept. Sci. and Ind. Res. Food Inv. Rep. 23. . 1926. A relation between the respiration activity and keeping quality of apples. (Gr. Br.) Dept. Sci. and Ind. Res. Food Inv. Ed. 1925: 37-41. . 1928. Atmospheric control in fruit storage. (Gr. Br.) Dept. Sci. and Ind. Res., Rep. Food Inv. Ed. 1927: 32-33. 1930. The gas storage of fruit. 11. Optimum tempera- tures and atmospheres. Jour. Hort. Sci. 8: 67—77. 1934. The cause of low temperature breakdown in apples. (Gr. Br.) Dept. Sci. and Ind. Res., Rep. Food Inv. Ed. 1933: 57-60. 106. Kidd, F. and C. West. 1939. Effect of oxygen and carbon dioxide on the chemical changes in stored apples. New Phytologist 38: 105-122. Magness, J. R. 1929. Relation of leaf area to size and quality in apples. Proc. Amer. Soc. Hort. Sci. 25: 285-288. ' Mandeno, J. L. and C. A. S. Padfield. 1953. Refrigerated gas storage of apples in New Zealand. New Zealand Jour. Sci. and Tech. , Sect. B. 34. Martin, D. 1954. Variation between apple fruits and its relation to keeping quality. 11. Between tree variations due to cropping factors. Austr. Jour. Agr. Res. 5: 9-30. . 1954a. Variations between apple fruits and its relation to keeping quality. 111. Between— season variations in relation to seasonal climate. Austr. Jour. Agr. Res. 5: 392-421. , and T. L. Lewis. 1952. The physiology of growth in apple fruits. III. Cell characteristics and respiratory activity of light and heavy crop fruits. Austr. Jour. Sci. Res. B5: 315—317. Overly, F. L. and E. L. Overholser. 1932. Some effects of fertilizer upon storage response of Jonathan apples. Proc. Amer. Soc. Hort. Sci. 28: 572—577. Pflug, I. J., M. W. Brandt and D. H. Dewey. 1957. An experimental tilt-up concrete building for the controlled atmosphere storage of apples. Mich. Agr. Exp. Sta. Quart. Bul. 39: 505-510. Phillips, W. R. and P. P. Poapst. 1952. Storage of apples. Canada Dep. Agr. Publ. 776. Plagge, H. H. 1925. Soft-scald and breakdown of apples as affected by stor- age temperature. Proc. Amer. Soc. Hort. Sci. 22: 58-66. . 1942. Controlled atmosphere storage for Jonathan apples ‘as affected by restricted ventilation. Refrig. Eng. 43: 215-220. , and F. Gerhardt. 1930. Acidity changes associated with the keeping quality of apples under various storage conditions. Iowa Agr. Exp. Sta. Res. Bul. 131. , and T. J. Maney. 1928. Soggy breakdown of apples and its control by storage temperature. Iowa Agr. Exp Sta. Res. Bul. 115. Plagge, H. H., and Maney. 1937. Factors influencing the development of soggy breakdown in apples. Jour. Agr. Res. 55: 739—763. , and B. S. Pickett. 1935. Functional diseases of apples in storage. Iowa Agr. Exp. Sta. Bul. 329. Rakitin, J. V., V. Krylov, and Kolenika. 1956. The gas regime of stored fruits and vegetables. Priroda 45 (10): 97—99. Rasmussen, P. M. 1951. Gas storage of apples in Denmark. Proc. Eighth Int. Cong. Refrig. 420—422. Ryall, A. L., and W. W. Aldrich. 1944. The effects of water deficits in the tree upon maturity, composition and storage of Bosc pears. Jour. Agr. Res. 68: 121-133. Sass, J. E. 1940. Elements of Botanical Microtechniques. McGraw-Hill Book Co. Inc., New York. Smith, W. H. 1940. The histological structure of the flesh of the apple in relation to growth and senescence. Jour. Pom. and Hort. Sci. 18: 249-260. Smock, R. M. 1944. The physiology of deciduous fruits in storage. Bot. Rev. 10: 560-598. 1946. Some factors affecting the brown core disease of McIntosh apples. Proc. Amer. Soc. Hort. Sci. 47: 67-74. . 1949. Controlled atmosphere storage in apples. Cornell Ext. Bul. 750: 1—36. , and A. M. Neubert. 1950. Apples and Apple Products. Inter- science Publishers, Inc. New York. , and A. Van Doren. 1941. Controlled atmosphere storage of apples. Cornell Agr. Exp. Sta. Bul. 762. Snedecor, G. W. 1946. Statistical Methods. Iowa State College Press, Ames, Iowa (Fourth Edition) pp. 485. Southwick, F. W., and M. Hurd. 1948. Harvesting, handling and packing apples. Cornell Ext. Bul. 750. Trout, S. A., G. B. Tindale, and F. E. Huelin. 1940. Investigations on the storage of Jonathan apples grown in Victoria. Coun. Sci. Ind. Res. (Aust.) Bul. 135: 1—96. 108. Van Doren, A. 1940. Physiological studies with McIntosh apples in modi- fied atmosphere cold storage. Proc. Amer. Soc. Hort. Sci. 37: 453—458. Van Hiele, T. 1951. Gas storage of fruits in the Netherlands. Proc. Eighth Int. Cong. Refrig. 416-420. , .I-r- . . Vickery, J. R., F. E. Huelin, E. F. Hall and G. B. Tindale. 1951. The gas storage of apples and pears in Australia. Proc. Eighth Int. Cong. Refrig. 416-420. APPENDIX TABLES umoafida #230588 H8 3% Esfifiumo cohogmnoo\w. naouxmoun awom can Emma 9595 .nBoEonn Essa: mo owmuofiqfim Emotes Ho oEU Hm omwhm><\m o .o; . o .2. o .2 o .Ntm 0-02 mm. HE . 7 o .3 o .3. o .m o .o o .0 mm m ma W . o .3 0 da o .03 o .o o .o 0 do an m. mg m H8800 W o .o o A: o .o o .o o .0 mm m m o .3. o .3. o .2 o .o o .0 mm m. m o .o o Km 0 .o o .o 0 .mo mm H5. 0 EN o .3 o .o o .o o .0 mm m. m; o .9: o .mw o .03 o .o o .o o .3» mm m. 2 \wA $2800 0 .o o .ow o .m o .o o .0 mm m. m o .om 0 Km. o A: o .o o .0 mm m m o .o o .3 o .o o .o o No mm H2 06 och 0.0 06 0.0 mm m 2 o A: o .03 o .o o .o o .o o .0 mm m m4 mm HonEoEom o .o o .om o .m o .o o .o mm m m o .o o .mw o .o o .o o 6 mm m m o .o o .02 o .o o .o o .3 mm H3. 0 .o o Hm o .o o .o o .0 mm m 2 o .mm 0 .sm 0 .o o .o o .o o .0 mm m 2 om HonEoEom o .o o .3. o .o o .o o .o mm m m o .m o .ow o .o o .o o .0 mm m m Ahmv ARV Gav 2% 8 g E .V N o m o mEo> wicaem \IEsou Emom Sam \mouoo 33m 0 5 o0 5 0.30 - «ohm flow :mfmcoH L335 35¢th mmhosaonjw “maxim: Ho oEE. $-me - b... “gamma use moaonmmoEz UoHHoficoO E ammuoum $3.30 $33. 533:2. no umotam Ho 05E. Ho “comm och. J Eofiioaxm H m4m

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N00 0000 N00 0% N00 0000 N00 0% 0820.00 0:0500000Lu .0 .00 “0 .N0 2 Awmémoc 0mm08m 003M0m E00 000000000500. 000000000000 0: 0000 SN 0000.00. 0000 0003000 00 0000000000000. 0mm0o0000n0 005000; 00030 00000. 0000000000 8 wc0u0ooo< 0000080000 0605 NH m4m<fi 502mm: .00000 0008 we 030> 0w000>0 000000 comm l \0 0N .o 00 30 0800 00 .o 00 0000000000000 30E 0 003000 .\ Nd 0 . 008000000003 05.585 2000 00 .0 N0 3 0800 mo .N 00 0003000 0 .o 0 05008000030 000000.005 50000.50 00 0000M0O 0o0=om 00000.0 002000.; ”00 0000000200 0 .00 o .00 0 .00 a .00 0 .00 00230 $380 0 .00 0 N0 0 .00 0 .00 o .00 0 .00 0080000000 0 N0 0 0.0 N .00 0 .00 o .00 \m0 .00 0020008002070 0000 0000 00% 00% 00% 00% .0030 0 0 m N 0 000050000 00 0002500 F E .m Ammo: 0003000 0>0m E000 0000B 005m 00 0000< 0000008000002 0000 0000:0030 5000 0000000003 002:0 000000001 00 00000.30 00050002. 00 00008 00000000m 8 mc€0ooo< A0959: 000000000000 005:0 N m4m0 000000 somm I \0 00.0 on A8 0000m 0.00 .0 w 0000000008000 0900000 00 0000050000 00000 00 0003000 0w .0 N 0000000000050 0w00000 00 0000050000 0000.00 iwm .N w 000000000000000 0900000 00 003000 0.0.3. .N0. 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E00m 0 0 0 N 0 0003000 $0-000: 00000900 2,000 880 880 006m .00 00050. 0000000000002 0000 00000000000 E000 0000050: 0000.5 0000000 003M050 00000 000000 -0082 00:000on 000 000D EN 00000 00000400 000000003 00 0000.0. 0000000000 00 wo000000< 300000006 00008.03 00000.00 HN mdmflb XHDZmEnz 120. 00000020 0000 0.000 00630 000>0000 00 0:000 000 0000 0 .o- N .o+ o .o m .o- 0 .00 00 000800 N .o- o .o m .o- m .o- m .M0 om. 0000800000m 0 .o+ m .0+ 0 .0+ 0 .0+ 0 .N0 0N 0000000000000 09000030. No 000.. No 00m .0000. N o N 0 CD 0000 00 00 0000/0000 0000/0000 00 0000.0. 0 .Nm 00 0w0000m M00005 0womao 0< AomémoS 0m00o0m 000sm0m 0000 000000000500 00000000000 00 00009 o0N 000000 00010 0900000000 0000 000:0: 00 00800. 00000005 00 0000000 0000000000 8000 000000 00 033m 000300 09300000000 00% M10950. 0000me00000 121. 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