r'i'fi 11:11.17 "L... -Lu." '..;..l I 0 Michigan 53:23 3‘33””. 7‘34 L I..."t'1;¥i';-;7 This is to certify that the thesis entitled CORRELATION 0F SUBJECTIVE AND QUANTIATIVE TECHNIQUES T0 MEASURE CHILLING INJURY. OF SELECTED LYCOPERSICON SPECIES AND SOLANUM LYCOPERSICOIDES presented by Terry L. Kamps has been accepted towards fulfillment of the requirements for M.S. Horticulture degree in (’1 Major professor Date Feb. 28, 1986 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES gag-.5. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. _..____ _._ ,, mrmwmmmmmmoms mmmmwsflmLmromspmns mmwroom. By Terry L. Kanps A‘IHESIS Submitted to Michigan State University in partial fulfilment of the requirenents for the degree of mwscm Department of Horticulture 1986 ABSTRACI‘ comrw or 5mm AND QUANTITATIVE 'IEXIHNIQUES 10 reasons Gamma INJURY a? ammo Lmaasrcm SPECIES AND 501mm WICOIIFS. By Terry L. Kanps Intact plants of Solanum lmrsicoides and Mrsicon species ecotypes between the four and eleven leaf developmental stage were subjected to 20°C or a chilling stress of 2.5°C for 72 hours. Chilling injury was assayed sequentially on each plant by visually rating damage of specified leaflets (VRL). chlorophyll fluorescence (CF), electrolyte leakage (EL), and visually rating entire plants (VRP). Correlation estimates of genotypic effects were significant between CF and VRL, CF and VRP, VRL and VRP, and VRP and EL. Correlation estimates of the interaction of temperature by genotypic effects were highly significant between CF and VRL, CF and VRP, and VRL and VRP. A relationship between chilling tolerance and collection altitude of wild species ecotypes was apparent. The chilling resistant intergeneric hybrid of Mrsicon esculentum Mill. cv. Sub-Arctic Maxi x Solanum 1mg rsicoides suggested dominant nuclear gene control. 'Ilemperature by genotype effects corresponded with the terminal or either near proximal leaflet position. ACKNJEEDGNQHS I would like to extend my appreciation to my major professor Dr. K. C. Sink for his guidance and support throughout this research program. Appreciation also goes to the members of my guidance committee: Drs. T. G. Isleib, R. C. Herner and G. S. Howell for their direction and assistance. A special thank you to Mrs. L. Kent for her assistance in the typographical completion of this thesis. I also wish to extend my sincere appreciation to my family, Stephen, and Cas for their love and encouragement throughout this educational program. Financial support for this study was provided in part by a grant fran the H. J. Ikinz Co. Glidance Calmittee: The paper format was adopted for this thesis is in accordance with departmental and university regulations. The paper is to be submitted to the Journal 9f the American Society _f_gr_ Horticultural Science. 'EBIEG'CINI'ENI'S 1‘19er 0 O O O O O O O O O O O O O 0 LIST m Elm O O O C O O O O C O O O O . LI'ERATURE REVIEW Introduction . . . . . . Seed germination and emergence Seedlings and tranmlants . Flower and fruit produticn . Pollen and fruit set . . . ksponses of the nabranes . Responses of the chloroplast Selection criteria for resistance to chilling injury LiteratureCited................ WIWWWMWWIMIVEMNIQES ‘IOMEASURECHIILMDUURYCFSELECIEDLWIGNSPEIES ANDSCIAMMIWICIDIIIS Abstract . . . . Introduction . . . Materials and Methods Plant material . Grilling tenperature treatment Evaluation procedures . . . Visual rating of leaflets Chlorophyll fluorescence Electrolyte leakage . . Plant visual rating (VRP) Statistical methods . . . . msults . . Linear correlation of chilling injury assays - genotypic .....§...... 0 O C O O V. O O 0 0 0 effects . . . . . . . . . . . . . Linear correlation of chilling injury assays - taperature x genotype effects . . . . . Genotypic responses - chlorophyll fluorescence assay . . . Effect of leaflet position . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . Literature Cited . . . . . . . . . . . . . . . . iv GE'S’BEmNH N N Table l. IJSPCI‘MES Page Collection sites and sources for selected Solanaceousspecies............. 34 Estimated correlation coefficients (r) of genotypic effects between assays evaluating chilling injury: CF - chlorophyll fluorescence; VRL - visual rating of leaflets; VRP - visual rating of plants; EL - electrolyte leakage................. 42 Estimated correlation coefficients (r) of the taperamre by genotype treatment effects between assays evaluating chilling injury: CF - chlorophyll fluorescence; VRL - visual rating of leaflets; V1!J - visual rating of plants ................. 44 Effects of tenperature and genotype on chlorophyll f1uorescence...............45 Effects of tenperature, genotype. and leaflet position '(see materials and methods) on variable fluorescence . . 46 LISTCFFIGJRFS Figure Page - 11W mvmw 1. Schematic pathway of the events leading to injury in sensitive plant tissues. Lyons, J. M. (1973) . . . . . l7 PUBLICATIG‘I MIG] 1. Representative sanpling patterns of leaflets of selected Solanaceous species. Sanpling pattern of Mrsicon pinpinellifolimn was the sane as h emlentm O O O O O O O .0 O O O O O O O 37 2. Genotypic response to two taperatures as measures by eachof the four assays;VRL, CF, EL, andVRP . . . . 40 3. Effect of tenperature, genotype, and leaflet position on variablefluorescenoe ............ 48 vi LI'IERATUIEREVIEW LI'ERATUREREVIEW Introduction Temperature is a major environmental stress factor limiting crop distribution and production (Sutcliffe, 1977; Thompson, 1970). Genetic manipulation and cultural technology have permitted expanded production of many crops to more diverse environments. However, marginally adapted crops remain subject to reductions in vigor and yield as a result of environmental conditions experienced during the growing season. The length of a growing season may be defined by the temperature requirements of the crop. For example, low temperatures in late spring and early fall shorten the growing season of crops which may be injured if exposed to these tenperatures. The deleterious effects of low, nonfreezing temperatures on several plant species were reported by Bierkander in 1778 (Levitt, 1980). Researchers have continued to investigate plant responses to nonfreezing temperatures, but it was not until 1897 that Molisch (Lyons, 1973) suggested that the injury caused by low temperatures (above 0°C) be referred to as ”chilling injury“ (Erk'altung); thereby, differentiating it from freezing (below 0°C) injury (Erfrieren). Currently, chilling injury is considered a physiological dysfunction resulting fran a low taperature exposure (Lyons, 1973) . (milling sensitive species are categorized as those which sustain physiological injury when exposed to temperatures within the o - 12°C range (Levitt, 1980; Lyons, 1973). Sensitivity and symptom expression vary with a number of factors including 1) center of origin of the species, 2) genetic differences, 3) physiological age, 4) tissue or cell type, 5) environmental conditions prior to and during exposure to chilling temperatures, and 6) the interaction of time and temperature (Lyons, 1973). Direct injury may result from an irreversible, qualitative physical change; whereas, indirect injury may be due to slower quantitative changes in metabolism. Chilling sensitive crops, including tomato, primarily originated from trcpical and sub—tropical lowlands. The cultivated tomato (Lyccpegicon esculentum Mill.) and related species are native to the western slopes of the Andes, a region extending from northern Chile through western Bolivia and Peru to Ecuador (Luckwill, 1943). The tomato has become widely accepted as an edible “vegetable” and has gained worldwide importance as a horticultural crop (Herner and Ramps, 1983). The popularity of the tomato can be attributed to the fruit's attractive color, flavor, and versatility (Rick, 1978). Tomatoes are grown as a fresh market commodity and for many processed products. In the United States both categories rank among the top five vegetable crops grown based on acreage planted, harvested, and economic value (U.S.D.A. Crop mpcrting Board, 1984). Sensitivity to chilling injury resulting in shorter growing seasons limits tomato production in the more northern, temperate climates. All stages of develqament, from seed to harvested fruit, are susceptible to injury induced by low temperatures (Kemp 1968) . Seed Germination a_ng Emergence The use of transplants in tomato production is a common cultural technique employed to avoid the erratic and reduced germination and emergence of tomato seeds associated with cold soil temperatures below 8 - 10°C. Direct seeding is an economical alternative to the use of transplants for establishment of tomato fields (Oyer and Koehler, 1966; Sullivan and Wilcox, 1971). Sullivan and Wilcox (1971) reported that in addition to reduced costs at planting, direct seeding offered a number of other advantages. Among these are: 1) increased probability of more vigorous and disease-free seedlings, 2) elimination of scheduling problems during critical planting seasons, 3) greater production flexibility, 4) earlier completion of planting operations, 5) increased yield potentials, and 6) the ability to establish high density populations necessary for an economical mechanical harvest. This technology has been widely used in California since 1966 (DeVos et 31., 1981). Nearly 100% of the California acreage planted in processing tomatoes is direct seeded (El Sayed and John, 1973; Smith and Millett, 1964; Sullivan and Wilcox, 1971). In contrast, the short growing season and cold, wet soil conditions in early spring limit the use of direct tomato seeding in the midwestern and eastern United States. Unlike California, direct seeding has not become a standard practice in these areas (DeVos et a1., 1981; Sullivan and Wilcox, 1971). The short growing season in these northern areas necessitates early spring seeding to allow sufficient time for the crop to mature (Herner and Ramps, 1983). However, soil temperatures below 10 - 15°C are considered sub-(ptimal for tomato seed germination and emergence which either fails to take place or becomes increasingly erratic (Bussell and Gray, 1976; DeVos et a1., 1981; El Sayed and John, 1973; Jaworski and Valli, 1964; Kctowski, 1926b; Lorenz and Maynard, 1980; N9 and Tigchelaar, 1973; Smith and Millett, 1964; Thompson, 1974). Soil temperature and water are major factors influencing germination of viable seed (Dubetz et a1., 1962); however, these may be compounded by light. Mancinelli et a1. (1966;1967), demonstrated phytochrome control of tomato seed germination in the dark. Phytochrome efficacy varied in response to temperature, and at low temperatures (17 - 20°C) sensitivity to PFR influence increased. The probability of seed mortality due to soil saprophytes and pathogens occurs during prolonged periods in cool soils (Harper et a1., 1955; Harrington and Kihara, 1960; Pinthus and Ibsenblum, 1961; Tlocle et a1., 1951), sometimes resulting in serious reductions in plant populations. These problems arising from direct seeding in cool soils are most important when the field is to be mechanically harvested. Dense populations necessary to obtain a high marketable yield (Bussell and Gray, 1976; DeVos et a1., 1981; Wer and Kcehler, 1966), are reduced by the failure of some seeds to germinate and by seedling mortality. Also, cold temperatures delay emergence; thereby, expanding the germination period over several weeks (Cannon et a1., 1973; Dchs et a1., 1981; N9 and Tigchelaar, 1973). This results in a less uniform stand which matures irregularly. Stand uniformity is necessary to obtain a high concentration of mature fruit for a once-over harvest. Plants developing at variable physiological ages confound the determination of a harvest date when yields will be maximized. Increased probabilities of weed establishment, insect injury, and soil crusting are additional complications associated with a prolonged emergence period (DeVos et a1., 1981; Sullivan and Wilcox, 1971). Cultural techniques and plant breeding provide means of increasing the rate and uniformity of seed germination and seedling emergence in cold soils. Cultural techniques (Bussell and Gray, 1976) have consisted of seed hardening (Christiansen, 1968; Hegarty, 1970), seed priming (Heydecker et a1. 1973; Kotcwski, 1926a; Qer and Koehler, 1966), and pregerminated seed (Sachs, 1977). Variable success has been attained. Genetic studies have shown sufficient variability in seed germination under cold conditions within L_. esculentum and wild Lyccpersicon species that may be utilized in breeding programs (Cannon et a1. 1973; DeVos et a1., 1981; 31 Sayed and John, 1973; ng and Tigchelaar, 1973; Patterson and Payne, 1983; Webb, 1973). The number of genes controlling resistance to chilling injury of seeds, or the ability to germinate at sub-(ptimal temperatures, is not clear. Work by Cannon et a1. (1973), implied that low temperature germinatim of tomato seed was controlled by a recessive gene. Their conclusion conflicts with reports by other researchers who claimed a minimum of 3 - 24 gene pairs determined this characteristic (DeVos, et a1., 1981; E1 Sayed and John, 1973; N9 and Tigchelaar, 1973). According to DeVos et a1. (1981) the study conducted by Cannon et a1. (1973) was not designed to detect continuous polygenic variation. Furthermore, DeVos et a1. (1981) and Mg and Tigchelaar (1973) detected a significant maternal inheritance effect. Abdul-Saki and Stoner (1978) found physiological evidence of a maternal ccntr ibuticn after experimenting with the leachates of seeds that had varying abilities to germinate under cold conditions. Genotypes that performed poorly in the cold contained a germination inhibitor; whereas, those more cold tolerant contained a promoter. Estimates of nuclear heritability of low temperature seed germination in tomato indicate significant additive gene effects (DeVos et a1., 1981; El Sayed and John, 1973; N9 and Tigchelaar, 1973) Also, dominance and partial dominance were reported by Ng and Tigchelaar, (1973) and DeVos et a1., (1981) respectively, to contribute to a proportion of the total nuclear genetic variance. While seed germination and emergence may be genetically improved for tolerance to low temperature conditions, such tolerance is not necessarily correlated to the plant response to chilling temperatures at other developmental stages (Herner and Ramps, 1983; Kemp, 1968; Patterson and Payne, 1983). Seed11_r_ig' s _ag Trmlants The widespread use of transplants in the midwest and eastern United States shifts the focus of the effects of chilling temperatures in the spring from the seed to the developing plant. Tomato seedlings may be transplanted to the field well before the danger of frost has passed. Placing caps or row covers over the plants protects them from frost and chilling injury. When future frost probabilities are sufficiently low the protective covers are removed. However, the threat of chilling night temperatures often persists until mid-June. Within this period, tomatoes can shift from the vegetative growth phase to the reproductive stage. Therefore, the effects of chilling temperatures early in the growing season may significantly affect seedling survival, growth, flowering, fruit set, and early fruit develcpment. Seedlings often respond rapidly to low temperatures (King et a1., 1982) and thus provide an in v__iy2 system for ascertaining the temperature sensitivity of some physiological processes. The benefit of response research. A variety of qualitative and quantitative techniques have been used in attempts to assess chilling injury in seedlings. Techniques range from simple, though sometimes subjective, visual evaluations to quantitative measurements indicative of physiological changes. “Chilling injury evaluated by qualitative visible symptoms is a function of both physiological injury & s_e_, and rate of symptom development in the particular tissue“ (Lyons, 1973). A change of physical structures within the cell may cause the disruption of normal metabolic processes, resulting in physiological injury. Visible changes in ultrastructmre of chilled cells of sensitive species have been microscopically detected (Ilker et a1., 1979; Moline, 1976; Patterson and Graham, 1979). The more apparent macroscopic symptoms of general concern are loss of turgor, tissue. necrosis, external discoloration, reductions in growth and vigor, flowering, and fruit set, abnormal fruit development, surface pitting, (Lyons, 1973) and increased susceptibility to decay organisms (McColloch and Worthington, 1952). Loss of turgor, an irreversible symptom of chilling injury is caused by water loss and entry of air into the cells (Wright and Simon, 1973). At chilling temperatures turgor loss may be difficult to identify, but becomes more evident once tissue is warmed to non- chilling temperatures (Patterson et a1., 1978). Furthermore, subjecting tissue to warmer temperatures following a chilling exposure usually results in rapid development of additional symptoms (Lyons, 1973 ; Patterson et a1., 1978). Chilling injury of seedlings is determined by low temperature and interactions with other parameters. The role of water in the develcpment of chilling injury has been examined from several aspects. Studies of the effects of relative humidity at chilling temperatures, and abscisic acid levels, have demonstrated the relationship between water and the expression of chilling injury (Herner and Ramps, 1983; King et a1., 1982; Rikin and Richmond, 1976; Rikin et a1.,1976; Rikin et a1., 1979; Rikin and Richmond, 1979; Rikin et a1., 1981; Sasson and Bramlage, 1981; Wright and Simon, 1973). High (100%) humidities or increases in ABA levels inhibit or delay dehydration of cells during a chilling stress. Cell water loss is generally a predecessor to other symptoms of injury such as tissue necrosis. Diurnal responses of tomato seedlings (Homer and Ramps, 1983; King et aL, 1982; Patterson et a1., 1979) to chilling temperatures illustrates the significance of light as another interacting component of the develcpment of chilling injury. Subjective evaluation of tissue necrosis is a simple and common method to test interacting environmental and genetic parameters of chilling stress. Genetic applications have been demonstrated by Herner and Ramps (1983) and Patterson et a1. (1978) in studies of natural genetic adaptation of selected species in the Solanaceae family. Such a visual test may have limited value, however, due to low sensitivity (Patterson and Payne, 1983), and inherent subjectivity. Nondestructive and destructive measurements of growth and development are quantitative and perhaps more sensitive assays of the temperature response of plants. Nondestructive measurements, for example, permit repeated observations on the same specimen over time. Collection of fresh and/or dry weight values of specified plant parts Collection of fresh and/or dry weight values of specified plant parts during the coirse of an experiment or at its completion are examples of widely used but destructive techniques. With regard to growth and developmental research the aforementioned technologies, evaluation of tissue damage and the collection of fresh and/or dry weights are inherently limited, even when used in conjunction. Plants of the same chronological age often are not the same physiological age, resulting in a large source of variability that can obscure results (Erickson and Michelini, 1957). Utilization of Erickson's and Michelini's (1957) non-destructive plastochron index (PI), a numerical index of the develqamental age of plants, can often minimize this type of variability. In 1880 Askensay proposed the term plastcchron to designate the time interval between formation of two successive internode cells, or more broadly defined as the interval between corresponding stages of development of successive leaves (Erickson and Michelini, 1957). The index indirectly relates observations of each experimental unit to time. Therefore, appearance of leaves at successive intervals is one criterion that must be met for the PI to be reliable (Coleman and Greyson, 1976; Lamoreaux et a1., 1978). In tomato, flower bud production has been shown to change this time interval, thus limiting application in tomato research to approximately the eleventh leaf stage (Coleman and Greyson, 1976; Stevens et a1., 1984; Vallejos et a1., 1983). Growth rate changes can be measured by prcper implementation of the aforementioned methods (Coleman and Greyson, 1976; Jaworski and Valli, 1964; Kemp, 1968; Learner and Wittwer, 1953; Martin and Wilcox, 1963; Patterson and Payne, 1983; Rikon and Richmond, 1976; Stevens et 10 a1., 1984; Vallejos et a1., 1983; Went, 1944). Detection of these changes or differences can be used to estimate temperature minima, optima, and maxima for growth. The optimum is the temperature range most conducive to rapid growth; an increase or decrease of temperature outside this range reduces the growth rate. In general, the optimal temperature range for tomato is 65 - 75°F (Lorenz and Maynard, 1980). Differences have been reported between tomato varieties and other Mgsicon species (Kemp, 1968; Learner and Wittwer, 1953; Patterson and Payne, 1983; Vallejos et a1., 1383). Species of Mrsiccn which grow naturally at high altitudes are exposed to lower mean temperatures, therefore they would be expected to have evolved a lower temperature optimum. Varieties which undergo a slower rate of change of growth at lower temperatures or have lower temperature optimum should be more tolerant of chilling temperatures than those with a higher optima (Learner and Wittwer, 1953; Patterson et a1., 1978; Sutcliffe, 1977). For the vegetative developmental stage several different azproaches may be used to detect tolerance to chilling temperatures. Utilizing PI, Vallejos et a1. (1983) demonstrated differential reductions in growth rate, at low temperatures, of selected tomato species. Kemp (1968) detected differences between varieties grown at a sub-optimal temperature (10°C) for two weeks. Went (1944) reported cessation of differentiation of the growing point and stem elongation in a canning variety of tomato subjected to a constant 5°C. Patterson and Payne's (1983) screening technique identifies genotypes which can grow during a light period at 20°C following the 16 hour dark period at 0°C. Temperature optima for growth has been correlated with 11 Plants more tolerant of low temperatures usually have lower temperature cptima (Learner and Wittwer, 1953; Patterson et a1., 1978; Sutcliffe, 1977). Went (1957) proposed that the optimal temperatures for stem elongation equaled the optimal temperature for fruit production in tomato, contradicting an earlier report by Learner and Wittwer (1953) which noted varietal interaction. Flower and Fruit Production Fruit production is a function of flower formation, successful pollination (fruit set), and fruit development. The effects of low temperatures on these characters has been studied to improve early market tomato production. Went (1957) observed that the tomato inflorescence size and the number of nodes between each inflorescence were related to the night temperature. Warmer night temperatures reduced the size of, and increased the number of leaves between, inflorescences. Experiments initiated after the expansion of the cotyledons of tomato were conducted by Calvert (1957), Lewis (1953) , Phatak et a1. (1966), and Wittwer and Teubner (1956;1957) to ascertain the temperature sensitive period in flower initiation and development. These researchers obtained results similar to Went (1957). Lewis (1953) suggested three main factors affecting the size of the inflorescence in tomatoes, one of which is environment Exposure to what is considered a low (14°C) but non-chilling temperature after the expansion of the cotyledons increases the production of flowers in an inflorescence as compared to plants raised at higher temperatures (25 - 30 0C). A decrease in node number to first inflorescence resulting from a low temperature treatment was also confirmed. Whether this is a result of slower growth is unclear. Clarification is necessary since the number of nodes below the first inflorescence is considered an index for earliness of flowering in tomato (Phatak, 1964; Phatak and Wittwer, 1965). The temperature sensitive period for changes in node number precedes slightly the temperature sensitive period for changes in floral number. Maintenance of cool temperatures can extend the increased flower effect through the fifth inflorescence in some varieties. Studies by Phatak et a1. (1966) and Wittwer and Teubner (1956;1957) included temperatures in the upper range (100C) of chilling. No mention was made of chilling injury pe_r _se_ except in fruit development (Wittwer and Teubner, 1956). Went (1944) observed that apparently normal flowers were produced at 5°C, but fruit set failed to occur. Pollen and Fruit get Successful pollination is required for non-parthenocarpic fruit set. Poor fruit set on early flowers of tomato can be ascribed to low temperatures adversely affecting the pollination process. Inhibition of pollination by temperature can be attributed to the development of inviable pollen under low temperature conditions, germination failure, or the inability of the pollen tube to successfully grow through stylar tissue to the ovary (Kemp, 1965a). Went (1957) reported that night temperatures below 12.8°C resulted in formation of abnormal and empty pollen grains. Charles and Harris (1972) attributed poor fruit set at low temperatures (10 and 12.80C) primarily to poor pollen viability and germination. Pollen produced at 10°C was inviable, failing to produce pollen tubes in germination tests. Normal pollen development increased with increasing temperatures. Reductions in 13 development increased with increasing temperatures. Reductions in percent germination of pollen and rate of pollen tube growth were observed by Smith and Cochran (1935). _I_n .Vi_tl'_.'2 germination at 5°C inhibited pollen germination of tomato (Zamir et a1., 1981). Some growth regulating substances have been effective in overcoming low temperature restrictions (Mann and Minges, 1949), but plant injury reduces the appeal of such application of these substances to improve early fruit set in field tomatoes. The possibility of genetic improvement woild be a preferable alternative. Differential responses to chilling temperatures of pollen and fruit set of chcpersiccn moies and varieties have been observed by Daubeny (1961), Huner and VanHuystee (1982), Kemp (1965a; 1965b), Maisonneuve (1983), Ward (1956), and Zamir et a1. (1981; 1982). Maisonneuve (1983) examined pollen developed at 7°C and noted better quality of pollen from accessions of high altitude l_:._ hirsutum. Zamir et a1. (1981; 1982) reported a selective advantage of high altitude h hirsutum pollen for fertilization at low temperatures. Their research concluded that the genes expressed by the haploid pollen grains are responsible for differential fertilization at low temperatures and selection of these gametes could have a corresponding effect on the spcrophyte. Studies by Huner and VanHuystee (1982) disagreed, noting there was no vegetative difference in repcnse to chilling temperatures of the cultivars tested but was observed for the ability to set fruit. Some varieties of L; esculentum have the ability to set fruit at low night temperatures (Kemp,l965a; 1965b). Kemp (1965b) identified a recessive gene in the variety Earlinorth which permitted fruit set to occur at night temperatures of 40°F. Inheritance was prqaosed to be 14 simple with corrplete dominance. Pollination at low temperatures may result in a reduction in numbers of fertilized seeds within the fruit thereby producing abnormal, 'catfaced" fruit (Ward, 1956). same 2: me. _._Membranes The physiological responses discussed above are generally considered secondary, or indirect, events of chilling temperatures (Lyons et a1., 1979). Comparative physiological studies of chilling- sensitive and -resistant species have provided evidence to suggest cell membranes are the primary temperature sensor (Lyons, 1972; 1973; Lyons and Raison, 1976; Lyons et a1. 1979; Raison, 1974). At tenperatures critical for chilling injury of intact plants (9-12°C in tomato) “breaks”, deviations from expected linearity, occured in Arrhenius plots measuring respiration activity of mitochondria from chilling sensitive tissue (Lyons and Raison, 1976). The "breaks" are a relatively consistant phenomenon of chilling sensitive species and represent an increase in activation energy (Ea) of the membrane bound enzymes (Graham and Patterson, 1982). Corresponding results of electron spin resonance (esr) have been observed; thus, indicating a membrane phase change from a liquid-crystalline form to a less flexible solid gel (Lyons, 1972; Lyons et a1., 1979). Chilling resistant species generally maintain a linear tenperature dependency to near or below 0°C. The phase change experienced by chilling sensitive species is viewed as the primary mechanism of tenperature response followed by physiological dysfunction. Prolonged periods of dysfunction lead to the develcpment of permanent injuries (Lyons et a1., 1979). The various membrane systems within the cell may exhibit differential sensitivity to chilling (Ilker et a1., 1979; Thompson, Jr., 1979). Ilker et a1. (1979) studied the sequence of ultrastructural changes in tomato cotyledons during a chilling stress. They concluded that “the ultrastructural chilling synptoms of tomato seedling cotyledons (held at 5°C for 2 to 24 hours) manifested themselves primarily as a progression of membrane deter iorations.” Damage sustained by the different organelles was dependent upon the period of chilling exposure. A tenperature-induced phase transition from a liquid-crystalline to a coagel form requires a greater order of the lipid molecules composing the membrane (Levitt, 1980). Therefore, a change in the semi-permeable properties characteristic of the membranes could be expected. Levitt (1980) proposed that increased permeability of the plasmalemma measured by solute leakage or ion accumulation in the cell wall and intercellular spaces may result from mechanical and/or metabolic stresses. Solidification of the membranes is often accompanied by contraction and loss of flexibility (Levitt, 1980; Lyons, 1972; 1973). Nam-uniform contraction caused by sudden chilling or chilling combined with dehydration subjects the membranes to mechanical stresses likely to produce fractures causing the membrane to become leaky (Ievitt, 1980). The enhancement of cellular dehydration on solute leakage and chilling injury was demonstrated by Wright and Simon (1973). Simon (1974) aggested that dehydration of water from the cells is necessary to create a mass flow of electrolytes to the apoplast. The rate of electrolyte leakage from the cell has been shown to increase with increased periods of chilling stress. This may be attributed to the ultimate degeneration (Ilker et a1., 1979; Moline, 1976) of the membranes as phosphorylative activity and the energy to 16 (1973) presented a schematic summarization of the events leading to chilling injury in sensitive plants (Figure 1). Since the amount of electrolyte leakage is dependent on the duration of the chilling stress (Wright and Simon, 1973) it would be expected that measurement of this parameter world be indicative of the injury incurred. Electrolytes in the cell wall and intercellular spaces will leak from injured tissue submersed in an appropriate liquid medium, commonly water. Subsequent measurement of conductivity of the water prov ides evidence of the leakiness of the membranes. Increased amounts of leakage have been reported in chilling sensitive tissues as conpared to chilling resistant tissues (Nobel, 1974). Differences such as these suggest the possibility of detecting genotypic variation to chilling within species generally considered susceptable (Paull et a1. 1979). Van De Dijk et aL (1985) reported differences with respect to electrolyte leakage among 10 genotypes of tomato. Conversly, reports by Miltau et a1. (1984) and Stevens et a1. (1984) concluded that measurement of electrolyte leakage was not sufficiently reliable as a selection criteria. Coiflicting results may be due to differences in treatment methodologies. Consequently, the interaction of treatment parameters and electrolyte leakage should be carefully scrutinized prior to its employment as a selection criteria. Egsppn—seg _o_f_ go; Q’nlorglast Further evidence of conformational changes in membranes may be acquired by examining charges in the physiological processes directly associated with specific membrane systems. A variety of assays have been utilized to investigate temperature effects on the thylakoid l7 .Amnm—V .2 moans .mcoxh .moamm_u acopa o>_uwm:om op shown? op mcpooop muco>o oz» to museum; o_uoeozom ._ ot=m_m mmammmh oz< mohmu in no 2hm=wzH l ot:mooxo oomco—ota .8o.~955o mocopoo :ow omuaom_u .mo>;oepouooo .m.o use omoxoop waspcm mmavponoume upxop we :o_uo_:s:oo< \ Em__ooou9= :F oooN on cannot use otamooxo oo_tm . x—onam oucoponeo op< twosomm Emwpoooume /(’/ _ sEo: m=_sowtum on eczema owamopoonoco Co moea~ew ocsoo :o_pommou -mcotoan to >oamzm zo~h<>~pu< ommomtoco auw Q_aom mz_44P== oueum eeapeoez mp>eo .opctoew_ou to xummto>pea toucou gooum mopuocow oueeop ecooeou Need: .w .: ecoaeou Need: .w .: xeoosou Nepm: .5 .: mp>eo .o_:toew_ou co eupmtm>pcz toucmu sooum moppocmw cause» mp>oo .chtoempmu co xuwmtm>Pca toucmu gooum mowumcmo cameo» me>oo .opctoewpou co eupmto>wea toucou xuoum moeuoeou oueeoe moe< ..>p:= oueum azom .eowueum .oeueo «cope _ocomaom potucmu eutoz area .ocome .oopad .memmmm _ao some .emou=< .eNopoutou eo_< .ntoooe comm some .emeu:< .osocmd some Ex .5 .osmmu ow: .mtouoe oomfinooofi toooaou .woocoz .ooonpowa .mtouos oofiv seed Aommp <4v moopoo_mtmooue_ Encepom x Aeop he smzv exoz owuto .ppwz saucepaomo cooemtwoowem loos. <4. mwowouvmtooooxp seem—om loo. on smzc lxez ovot<-a=m >o ._p_z Sauce—sumo coopmtonooxa ANNA =1 ~e_o:~ .ppwz Esacopsomo coupmtmooueq AmmoN 1e neeoxy .prz Eaucopaomm.mmontm oo 4 Anon. «Lo .pocom a .oe:: sausage; eooemeooooxJ lemeel eee .pocom a .ae:: Sausage; coowmtmaooaa Memmp <4~ .pocom a .ossz antenna. .e Eauzmtwe coo_mtoaouxh lemeo~_ .l.ec .__.zn»m_maev sap—oepppm=_nsnm coopmtuoooaa motsom ooen eolooo__oo moxuocmu .mo_omom maoooecepom oouoopom toe mootaom oco moupm copped—poo .p o—ace 35 plant. Asexual propagation was accomplished by terminal shoot cuttings, insuring genetic uniformity within each species ecotype. Cuttings were rooted in perlite under an intermittent mist system and after three weeks rooted cuttings were transplanted to 10.2 cm clay pots as described above. Potted plants were grown in a glasshouse maintained at 17 i 2°C minimum night temperature, fluctuating day temperatures and natural photoper iodic conditions present at East Lansing, Michigan from January through March 1985. Standard insect control practices were practiced and the plants were fertilized daily with a solution of 110.9, 92.1, and 61.5 mg/l of N, P, and K respectively through a drip-tube irrigation system. Six plants of each genotype were selected for controlled chilling temperature treatments based hon similarity in developmental age. The multiple meristematic growth habit of P. I. 126430 and LA 1363 produced plants with an average of 9-11 expanded leaves compared to 4-8 leaves typical of the other genctypes. Senescencing leaves and visible flower clusters were removed within 15 hours of the controlled temperature experiment. The youngest 70% (approximately) expanded leaf to be evaluated from each plant was identified and tagged prior to the chilling treatment. Chilll_rg' Mature Treatment At the end of a daily natural dark period, the test plants were watered to saturation and subsequently transferred from the greenhonse to two Percival controlled environment chambers (CEC). Each one was set to maintain a constant temperature of 20 i 2°C (control) or 2.5 i 1.0% (chilling), providing 10 )1E m-ZS-l light intensity (Westinghouse Econ-O-Watt F40cw/rs/ewII) on a 10 hour photoperiod and relative 36 humidities of 68 - 83% (2.5°C) and 80 - 100% ( 20°C). Three plants of each genotype were held in each chamber for a 72 hour period and subsequently returned to the greenhouse. Both temperature treatnments were replicated folr times. Evaluation Procedures Five leaflets of the selected youngest leaf were sampled in a terminal to proximal pattern (Figure 1), therefore, leaflets reflected a within leaf position effect. Subsequently, each leaflet was subjected to one qualitative and two quantitative assays to evaluate chilling injury. 1. Visual lhtirg 0_f Leaflets (VRL) Immediately following transfer of the plants from the GEES to the greenhonse the leaflets were visually rated on a 1 - 9 scale: 1 - no injury 3 - slight injury, some wilting and dehydration of the leaflet margins - 30% of the leaf affected 5 - moderate injury, 50% of the leaflet wilted and dehydrated 7 - severe injury, 70% of the leaflet wilted and dehydrated \D I entire leaflet affected, probable death 2. Chlorflyll Fluorescence _(g_r)_ Visually rated leaflets were detached from the leaf petiole, briefly washed oce in deionized distilled water and placed on mcist filter paper in covered plastic petri dishes. The petri dishes were then placed in the dark for a minimum of 30 minutes at room temperature. Fluorescence was measured using the Branker model SF-lO 3'7 ’6 ‘4; .3, 154:, M.,... ,3! W "i: L esculentum x s. Iyeoponlcoidu Figure l. Reprensentative sampling patterns of leaflets of selected Solanaceous species. Sampling pattern of P. I. 126430 was the same as I: esculentum. 38 plant prodnctivity fluorometer described by Ahrens et a1. (1). A 1 mm hole was cut in black cardboard fitted to the sensing probe partially occluding the opening to accommodate narrow and deeply dissected leaves. Acclimated leaflets were surface-dried with Rimwipes and placed with their abax ial sides up on a black piece of cardboard. The above described restriction form was placed on top of the leaflet, taking care to avoid the midrib. Fluorescence signals were displayed on a Nicollet Explorer III oscillisccpe and recorded on magnetic disks (Verbatim MD 525-01-18158). Variable fluorescence was calculated by the formula: mere : fo = the initial level 0 (origin) in the fluorescence transient of Chl a yield fnm = the high peak in the fluorescence transient of Chl a fluorescence yield 3. Electrolyte Ieakgge _(E_:I.-)_ Following the fluorescence measurement, leaflets were prepared to measure electrolyte leakage. Leaflets were immersed in 10 ml deionized distilled water in 18 x 150 mm glass test-mbes which were stcppered with foam plugs. Electrolytes were allowed to leak for 24 hours at room temperature. Samples were placed on a Vortex Geni-mixer for a few secods and subsequently conductivity was measured at room temperature with the YSI (Yellow Springs Instrument Co., Inc.) Model 32 Coductance Meter. Samples were subsequently autoclaved for 20 minnutes, placed 39 on gyratory shakers and allowed to leak for another 24 hours to obtain total electrolytes leaked. Coductivity is expressed as a percent of the total. 4. Plant Visual Rating (VRP) Four days after the plants were returned to the greenhouse, they were given a visual rating using the scale described by Herner and Ramps (5). Statistical Methods An analysis of variance was calculated for each of the assays. Correlations between the assays were estimated for the treatment effects of l) genotype, and 2) the temperature by genotype interaction. Coefficients of variation were calculated for each assay to compare precision. msults Ling; correlation o_f chillgg' i_njp_n_:y m - genot_:ypic effects Plants exposed to 2.5°C compared to those exposed to 20°C resulted in increased values for visual ratings and electrolyte leakage and reduced values of variable chlorophyll fluorescence (Figure 2). Estimated correlations of genotypic effects (Table 2) were significant between the pairs of assays of: l) chlorophyll fluorescence (CF) and visual rating of leaflets (VRL), 2) CF and visual rating of entire plants (VRP), 3) VRL and VRP, and 4) VRP and electrolyte leakage (EL). EL did nnot significantly correlate with either CF or VRL. 40 Figure 2. Gennotypic response to two temperatures as measured by each of four assays; VRL, CF, EL, and VRP. 41 VlSUAL EVALUATION OF PLANTS m 20.0‘0 1150 IL 104 pH 722 {H 2653 P1 126430 LA 1624 GENOTYPE LA 1775 LA1363 1:50 In. 104 an LA 1990 LA 1990 f v w 9.00 8,001 m 2.5‘C v ééé Ti? 4.00- (N099) ONLLVH VISUAL EVALUATION OF LEAFLETS . “20.01: 1150 IL 104 {H 722 'H 2115:: PI. 120430 LA 1824 GENOTYPE M1775 M1363 USU IL104 In LA 1990 [A1990 5,00. m are v ‘v é (WWI) ONLLVH 0.00-1 i ELECTROLYTE LEAKAGE m 20.0‘0 i m 2.512 .................. ......... .......... oooooooooooooooooo .................. cccccccccccccccccccccccccc .......................... ooooooooooooooooooooooo oooooooooooooooooooo ....................... cccccccccccccccccccccccccccccccc oooooooooooooooooooooooo ................................ ........ ...... oooooooooooooooooooooooooooooo oooooooooooooooooooooooooooooooo ........................................ oooooooooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooooooo oooooooooooooo ............................. ...................... ............................. \\\\\\\\\\\\\\\\\\\ 1 0000| 1 T T' T ' T ' asséé so 8 8 9 (nuamd) All/\LLODONOO CHLOROPHYLL FLUORESCENCE I... o as. . uuuuuuuuuuuuu oooooooooooo ooooooooooooo ooooo ..... oooooooooooooooooooooo oooooooooooooooooooooo ccccccccccccccccccccccccc I O D C oooooooooooooooo .00....AIODIOnlbhoo x\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\V ..... u. cccccccccccccccccccccccccc ooooooooooooooooooooooooooooooo ooooooooooooooooooooooooooo oooooooooooooooooooooooooooooooooo ccccccccccccccccccccccccccccccccccccccc 1.00- sue/11mm EONBOS 8001;! WGVIMVA 0.50-l MSU 'L 104 'H 722 [H 2653 PI. 126430 LA 1624 LA 1775 LA 1363 1130 IL 104 in LA 1990 LA 1990 1150 IL 104 {H 722 [H 2653 PJ. 126430 LA 1024 LA 1775 [A1363 MSU IL 104 an LA 1990 M1990 GENOTYPE GENOTYPE 42 Table 2. Estimated correlation coefficients (r) of genotype effects between assays evaluating chilling injury: CF - chlorophyll fluorescence; VRL - visual rating of leaflets; VRP - visual rating of plants; EL - electrolyte leakage. CF VRL VRP EL CF - -0.926** -l.000** NS VRL - 0.989** NS VRP - -0.7l0* EL - * Significant at the 5% level. ** — Significant at the I% level. NS - Nonsignificant. 43 _L_inear corregtion of chillirg injury assays - temrature 3g genotym effects Estimates of correlations of the interaction of temperature by genotypic effects were highly significant between 1) CF and VRL, 2) CF and VRP, and 3) VRL and VRP (Table 3). Temperature and genotype interaction effects were not discerned with the EL assay as indicated by a nonsignificant F-test. Genotypic gm - chlorqghyll fluorescence flay Plants held at the non-chilling temperature of 20 C showed no difference between genotypes with regard to CF activity. Tolerance/sensitivity to the chilling temperature was identified by variable fluorescence reductions following exposure to 2.5°C. Genotypes with detectable differences between the 20 and 2.5°C temperature treatments are chilling-sensitive (Table 4). Significant differences between LA 1990 and 1480 L 104 are indicative of chill- resistant and -sensitive genotypes, respectively (Table 4). The response of the intergeneric hybrid MSU L 104 x LA 1.990 was resistant, significantly different from MSU L 104, the female parent, but not from LA 1990, the pollen parent. Egg; gr; leaflet gition Within each leaflet position, genotypic differences were detected between plants treated at 2.5°C, but not at 20°C. Differences associated with the terminal or either of the near proximal positions (Table 5) closely compared with the interaction of temperature by genotype effects at 2.5°C (Table 4). Sensitive genotypes (Table 4) showed significant reductions in CF between corresponding non-stressed 44 Table 3. Estimated correlation coefficients (r) of the temperature by genotype treatment effects between assays evaluation chilling injury; CF - chlorophyll fluorescence; VRL - visual rating of leaflets; VRP - visual rating of plants. CF VRL VRP CF - -O.885 ** -I.059 ** VRL - 1.012 ** VRP ** Significant at the l% level. 45 Table 4. Effects of temperature and genotype on chlorophyll fluorescence. Treatment Variable No. of CEC Temperature Genotype meansy fluorescence observations (0C) (millivolts) 20° (1]) LA 1990 1.30 60 MSU #L 104 x LA 1990 1.22 60 LA 1363 1.36 60 LA 1775 1.29 60 LA 1624 1.23 60 P.I. 126430 1.25 60 Heinz #H 2653 1.17 60 Heinz #H 722 1.20 60 MSU #L 104 1.20 60 2.5° (12) LA 1990 1.26 a 60 MSU #L 104 x LA 1990 1.19 ab 60 LA 1363 1.12 abc 60 LA 1775 1.07 abc 60 LA 1624 1.01 bed 60 P.I. 126430 0.89 cde 55 Heinz #H 2653 0.80 def 60 Heinz #H 722 0.74 ef 60 MSU #L 104 0.61 f 60 Standard deviation of the means (sd) = 0.12 T1 ' T2 LA 1990 MSU #L 104 x LA 1990 LA 1363 LA 1775 LA 1624 P.I. 126430 Heinz #H 2653 Heinz #H 722 MSU #L 104 Standard deviation of the means (sd) = 0.14 yMean separation by LSD at the 5% level NS - Nonsignificant by LSD at the 5% level * Significant by LSD at the 5% level ** Significant by LSD at the 1% level 46 ..o>o— am 0:“ as and a: messpoo =.;u_3 eeALegcaom coma» «_.o u Away menu: «so so eopuep>mo orcveaum m no.9 u c~.o w om.e o mm.c o _m.o cap 4. am: moo ~m.o u oa.c we 59.: o mo.o o .m.c NNN 2‘ ~:.m: on oa.c on mm.o we e~.c we -.c we ~o.c «mom z. Nepmz can sm.o on mm.o so mm.= moo ~m.o to _w.o oneo~_ ._.a tone mc._ one cc.— 03 _c._ eon sm.c on am.c «No. <4 cone me.— one w... one cc.p one ~c.. one mo.p m-p ueosuemgh .ouewommgo:_$ w—aepem> :o eopupmoa uupumo— wee .mnxuoemu .mgzaogmasma co mucouum .m open» 47 and stressed leaflets (Figure 3). Discussion Intact tomato and §_._ ficopersicoides plants at the vegetative growth stage subjected to a 2.5°C temperature stress for 72 hours developed visible symptoms typical of chilling injury. These symptoms included discoloration of plant tissue, loss of turgor, and necrosis, hereafter referred to as chilling symptoms. Results of the VRL and VRP assays showed that the average amount of chilling symptoms ranged from very slight (<15%) to moderate (55%), depending upon genotype. Since all senescencing and necrotic leaf tissues were removed prior to the chilling exposure, it is reasonable to assume that the injury which occurred afterward reflected actual chilling injury. Nonsignificant r values estimated between the EL assay and either VRL or CF assays for genotype effects indicated that EL was not an accurate method to quantify chilling injury. mrthermore, the nonsignificant F-test for the temperature by genotype interaction suggested that all genotypes responded similarly to the chilling temperature. Ion leakage has been proposed as a screening criteria for chilling injury, (8, 16, 31, 34) but the studies to date are conflicting on the reliability of this method. Ion leakage has been used to detect the increases in membrane permeability associated with chilling injury and can be measured either as an increase in rate or relative percent of leakage. Patterson et a1. (16) measured rates of EL over a 15 day chilling period and reported a relationship between the sensitivity/resistance to chilling temperatures and the natural environments of Passiflora species. Stevens et a1. (31) used 48 Figure 3. Effect of temperature, genotype, and leaflet position on variable fluorescence. VARIABLE FLUORESCENCE (millivolts) VARIABLE FLUORESCENCE (millivolm VARIABLE FLUORESCENCE (millivolts) 1.4< 1.3-1 1.2-1 1.1-4 1.0- 0.9‘ 0.8- 0.7-1 0.6- 0.5-1 A H H H H H E- POSITION 1 POSITION 2 POSITION 3 POSITlON 4 POSITION 5 uV 20.0'C 2.5'C LA 1990 1.4-1 1.3“ 1.2-1 1.0« 0.9-1 0.89 0.7+ 0.51 0.5 A .‘7 200°C ' 2.S'C LA 1775 1.4-1 1.31 1.2-1 1.0-1 0.9-1 0.84 0.74 0.6-1 0.5-4 A -" 200°C - 2.S'c Heinz {H 2653 VARIABLE FLUORESCENCE (miliivolts) VARIABLE FLUORESCENCE (millivolts) VARIABLE FLUORESCENCE (millivolts) 49 1.41 1.3« 1.2~ 1.1-I 1.o« o.9~ o.8~ 0.7-1 O.6~ 0.54 A .W 20.0'C 2.S‘C MSU #L104 x LA 1990 1.0-1 0.9-4 0.8- 0.7-4 0.6 0.1 0.4 200°C - 2.5-c LA 1624 200°C 2.5‘C Heinz {H 722 VARIABLE FLUORESCENCE (miHivoHs) VARIABLE FLUORESCENCE (millivolts) VARIABLE FLUORESCENCE (millivolts) 205°C Y 2.5°C LA 1363 1.4. 1.3-4 1.2-1 1.1-J 1.01 0.94 0.84 0.7-4 0.6-1 0.5-4 A .1 ‘K 200°C 2.5°C P.I. 126430 Ill 1_4.. 1.3-4 1.2-4 1-1-1 1.0-1 0.9-1 0.8-1 0.7-1 0.6-4 0.54 A .V 200°C - 2.S‘C MSU {L 104 50 techniques similar to those described by Patterson et a1. (16) to measure chilling resistance of Solanaceous species, but concluded the assay was unreliable for identifying chilling tolerance. In the present study EL was represented as a percent of the total electrolytes after leaf tissue was allowed to leak for 24 hours. A comparison between the EL of chilled and non-chilled leaf tissue indicated that this leakage period was too long to determine actual injury. Failure to detect temperature response differences after a 24 hour leakage period and reports by other researchers of significant response differences of plant tissue to chilling temperatures using leakage periods of only 1-6 hours (8, 9, 23, 26, 32, 34, 36) would also imply that the 24 hour period was too long. Visual evaluations of chilling symptoms and CF accurately reflected the response of tomato leaf tissue to the chilling exposure as indicated by significant correlation estimates between the three assays. Although rating visible damage is a simple procedure, subjectiveness in evaluating late manifestations of cellular damage, and low precision may considered inherent limitations. Reliability and accuracy of subjective assays, as opposed to objective assays, largely depends upon the experience and the ability of the researcher to make unbiased observations. Comparisons of coefficients of variation indicated that the subjective assays were the least precise (sensitive) relative to the quantitative assays. CF was the most precise assay in this study and readily detected significant differences between the temperature effects; whereas, this was not possible with either the VRL, VRP or EL assays. Reductions in variable chlorophyll fluorescence in response to temperature stresses have been shown to be indicative of cell damage 51 (6, 20, 29). The rates of reduction during the course of a chilling exposure have been related to the degree of chilling sensitivity/tolerance, such that, rapid reduction rates were indicative of chilling sensitive plants. In this study the measurement of chlorophyll fluorescence immediately after completion of the chilling exposure also detected genotypic sensitivities to chilling temperatures. As noted above, the CF assay was significantly correlated with VRL and VRP for genotype effects and effects due to the interaction of temperature by genotype. This indicated that CF is an accurate and reliable means to quantitate the responses of intact plants to a chilling exposure. Results from the CF assay were used to evaluate genotype response to temperature. Plants subjected to the 20°C temperature exhibited no genetic differences with regard to fluorescence activity. These results suggest that chilling-sensitivity and -resistance can be detected by evaluating the responses of plants to low temperature treatments alone. This has apparently been an assumption in earlier studies since non-chilling treatments were not included (15, 20, 31). Results of the genotypic response to chilling stress showed a clear distinction between the extremes of resistant LA 1990 and sensitive MSU L 104. The intergeneric hybrid produced from the cross MSU L 104 (female parent) x LA 1990 was determined to be chilling- resistant. This suggests that chilling tolerance is primarily controlled by dominant nuclear gene(s), which is in agreement with an earlier report by Robinson and Phillis (25). Differences between the other genotypes were less obvious, but the apparent trend suggested a correlation between the altitude at which the wild genotypes were 52 collected and the relative ranking of genotype means of the chill- stressed plants. k hirsutum is naturally distributed over a range of altitudinal elevations from coastal lowlands in Equador to 3,300 m in Peru (33). L_.; pimpinellifolium is found in coastal Peru (22). Throughout this range the mean temperature decreases approximately 4°C for each increase in elevation of 1000 m (28). Genetic adaptation to this natural thermal gradient would be expected, producing ecotypes with increasing tolerance to low temperatures as altitude increased. This study in conjunction with others which correlated temperature responses of such parameters as growth responses (33), pollen germination and fertilization (38), protcplasmic streaming (1?), seed germination and chlorophyll development (18), seedling growth and survival (19), electrolyte leakage (16), and photoreductive activity (30) to altitudinal origin, are supportive of this expectation. Genotypic responses to chilling temperatures were determined by assaying five leaflets, which represented five positions of a leaf, for each plant (see materials and methods). The question arises, does the genotypic response of a particular leaflet to the chilling stress correspond to the low temperature genotypic effects? Our results showed a significant interaction between leaflet position and genotype of plants subjected to the 2.5 but not to the 20°C treatment. The differential response of the leaflets of a genotype to the chilling stress were proportional, such that, the overall effect was a change in the scale of measurement, not a change in rank. Genetic differences associated with each leaflet position corresponded with the terminal or either of the near proximal leaflets, which would permit a reduction in the number of leaflets per plant needed to make an accurate 53 assessment of chilling tolerance. The results of this study show that a single CF measurement per sample after the completion of a chilling stress is an accurate and sensitive assay to quantify chilling injury. Furthermore, the number of leaflets per plant required to assess the genetic response to chilling temperatures may be reduced from five to one, but sampling position of all plants should be consistent. LI'IERA'IURECI'IED 4. 10. 54 Literature Cited Ahrens, W. E., C. J. Arntzen and E. W. Stoller. 1981. Chlorophyll fluorescence assay for the determination of triazine resistance. Weed Sci. 103:684—686. Charles, W. B. and R. E. Harris. 1972. Tanato fruit-set at high and low temperatures. Can. J. Plant Sci. 52:497-506. DeVos, D. A., R. R. Hill, Jr., R. W. Hepler and D. L. Garwood. 1981. Inheritance of low tenperature sprouting ability in F1 bauato crosses. J. Amer. Soc. Hort. Sci. 106:352-355. Graham, D. and B. D. Patterson. 1982. ksponses of plants to low, non-freezing tenperatures: proteins, netabolisn, and acclimation. Ann. Rev. Plant Physiol. 33:347-372. Herner, R. C. and T. L. Kanps. 1983. Chilling injury tolerance of wild bamto species. Proc. 4th 'Ibmato Quality Workshop. Larch '7-10. Veg. Crops res. RPT VEC-83-l. Hetherington, S. E., R. M. Smillie, P. Malaganba and Z. Huanan. 1983. Heat tolerance and cold tolerance of cultivated potatoes measured by the chlorcphyll fluorescence method. Planta. 159:119-124. Kanuiga, A., F. Sochanowicz, J. Zabek and K. Krzystyniak. 1978. Photosynthetic apparatus in chilling-sensitive plants. I. Reactivation of Hill reaction activity inhibited on the cold :23 631k 1ggorage of detached leaves and intact plants. Planta. King, A. I. and P. M. Ludford. 1983. (milling injury and electrolyte leakage in fruit of different tanato wltivars. Jo MEI. ”0 bit. kio 108374-77. King, A. 1., M. 8. kid and B. D. Patterson. 1982. Diurnal changes in the chilling sensitivity of seedlings. Plant Physiol. Levitt, J. 1980. Responses of plants to envirormental stresses 2nd Ed. Academic Press. ll. 14. 16. 17. 18. 20. 21. 22. 23. 55 Lewis, T. L. and M. Workman. 1964. The effect of low tenperature on phosphate esterification and cell membrane permeability in tanabo fruit and cabbage leaf tissue. Aust. J. Biol. 17:147-152. Lyons, J. M. 1973. Chilling injury in plants. Ann. Rev. Plant , J. K. Raison and P. L. Stepcnkus. 1979. The plant menbrane in response to low tenperature: an overview. p. In: Lyons, J. M., D. Graham and J. K. Raison (eds. 1-24. ) . low temperature stress in crop plants: The role of the neubrane. Academic Press. New York. Melcarek, P. K. and G. N. Brown. 1977. The effects of chilling stress on the chlorophyll fluorescence of leaves. Plant and Cell Miltau, 0., D. Zamir and J. Rudich. 1984. p. 45-50. Breeding for chilling tolerance in tomato: an examination of selection criteria. Eucarpia; Synopsis IXth Meeting. Wageningen, The Netherlands. Patterson, B. D., T. wrata and D. Graham. 1976. Electrolyte leakage induced by chilling in Passiflora species tolerant to different climates. Aust. J. Plant Physiol. 3:435-442. and D. Graham. 1977. Effect of chilling teuperatures on the protoplasmic streaming of plants fran different climates. J. M. BOt. 28:736-743. , R. Paull and R. M. Smillie. 1978. Chilling resistance in Lycogrsiccn hirsutum limp. & Bonpl., a wild tomato with a wide altitudinal distribution. Aust. J. Plant Physiol. and L. A. Payne. 1983. Screening for chilling resistance in tanato seedlings. IbrtSci. 18:340-341. 5 : 609-617 . Potvin, C. 1985. Effect of leaf detachment on chlorophyll fluorescence during chilling experiments. Plant Physiol. 78:883-886. Thison, J. K. 1974. A biological explanation of low temperature stress in trcpical and sub-trqaical plants. p. 487-497. In: Bieleski, R. L., A. R. Ferguson and M. M. Cresswell (eds. ). Mechanisms of regulation of plant growth, Bull. 12. The lbyal Soc. of New Zeeland, Wallington. Rick, C. M. 1976. Natural variability in wild species of rsicon and its bearing on tomato breeding. Genet. Agr. 30 : 249-259 . Rikon, A. and A. Richmond. 1976. Amelioration of chilling injuries in cucunber seedlings by abscisic acid. 38 :95-97. Physiol. Plant. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 56 and . 1979. Factors affecting leakage from cucunber cotyledons during chilling stress. Plant Science Letters. 14: 263-268. lbbinson, R. W. and B. R. Phillis. 1977. Solgnmn lmersicoides: A source of tolerance to low tenperature. Tau. Genet. Coop. Rep. No. 27. Sasson, N. and W. J. Bramlage. 1981. Effects of chemical protectants against chilling injury of young cucumber seedlings. J. 3119!. me Ibrto k1. 106:282-2840 Schreider, V. and J. A. Berry. 1.977. Beat induced changes of chlorophyll fluorescence in intact leaves correlated with damage of the photosynthetic apparatus. Planta. 136:233-238. Schwerdtfeger, W. 1976. World survey of climatology. Vol. 12. Elsevier, New York. Smillie, R. M. 1979. The useful chloroplast: A new approach for investigating chilling stress in plants. p. 187-202. In: Lyons, J. M., D. Graham and J. K. Raison (eds.). Low tenperamre stress in crop plants: The role of the neubrane. Academic Press, New York. and R. Nott. 1979. Assay of chilling injury in wild and domestic tomatoes based on photosystem activity of chilled leaves. Plant Physiol. 68:796-801. Stevens, M. A., S. Wolf and D. Yakor. 1984. p. 51-56. Introgression of cold tolerance fran high altitude wild talato qaecies into processing cultivars. In: Eucarpia; Synopses IXth Meeting. Wageningen, The Netherlands. Tanczos, 0. G. 1977. Influence of chilling on electrolyte permeability, oxygen uptake and 2,4-dinitrophenol stimulated oxygen uptake in leaf discs of the thermophillic Cucumis sativus. Physiol. Plant. 41:289-292. Vallejos, C. E., J. M. Lyons, R. W. Briedenbach and M. Miller. 1983. Guaracterization of a differential low tenperature growth response in two species of Mrsicon: the plastochron as a tool. Planta. 159 :487-496. Van De Dijk, S. J., J. A. Maris and P. R. VanHasselt. 1985. Genotypic variation in chilling-induced leakage of electrolytes fran leaf tissue of talato (Lyccpersicon esculentum Mill.) in relation to growth under low energy conditions. J. Plt. Physiol. 120 :39-46. Ward, K. M. 1956. Tenperature and fruit set in the tanato. Queensland Agr. J. 82:641-644. 36. 37. 38. 57 Wright, M. and E. W. Simon. 1973. Chilling injury in cucunber leaves. J. Exp. Bot. 24:400-411. Yakir, D., J. Rudich and Ben-Ami Bravdo. 1985. Photoacoustic and fluorescence neasurenents of the chilling response and their relationship to carbon dioxide uptake in tomato plants. Planta. 164 : 345-353 . Zamir, D., S. D. Tanksley and R. A. Jones. 1981. Low tenperature effect on selective fertilization by pollen mixtures of wild and cultivated tanato species. Theor. Appl. Genet. 59:235-238.