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Univemsty [2; This is to certify that the thesis entitled Alleviation of imbibitional chilling injuny in snap bean (Phaseolus vulgaris) seeds presented by Rufaro Magnus Makoni has been accepted towards fulfillment of the requirements for Wdegree in HortiCU1tUY‘e Major professor iDaugDecember 22, 1988 0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE Ava—U85, ' MSU is An Affirmative ActiorVEqual Opportunity Institution MLE'M'HJfl fr Hunting“ “gum m 53 it? PM 'Pbum'a www- ‘1 m ‘ 1 I i I l xv . ‘21 ‘ i v. A rim. ‘ .\ 1 butc‘. that u. - egg-$13 11. Meaty" 39w: extra-run: 1 .1 1 ,v' in pct-tin! fulfiiimnt at the ft¢.nMQ — 3:; x, . ‘ - ‘ for the we of . ,. 1 mm or mm _ ‘ Department of Horticulture ms ALLEVIATION 0F INBIBITIONAL CHILLING INJURY IN SNAP BEAN (Enasgglus xulganig) SEEDS By RUFARO M. HAKONI A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1988 5973643 ABSTRACT ALLEVIATION 0F INBIBITIONAL CHILLING INJURY IN SNAP BEAN (Phaseolus vulgaris) SEEDS By Rufaro M. Makoni Coating snap bean seeds with antitranspirants, Vapor Gard, Hiltpurf, and a postharvest fruit wax, Pacrite 383, reduced imbibition at 5, 10, and 20C. These waxes also reduced damage to cotyledons and axes and improved germination of imbibitionally chilled snap bean seeds in pots. Raising the initial moisture content to 15-20% and 25-30% from 6-9% reduced damage to axes and cotyledons of snap beans chilled during imbibition. Germination of snap beans chilled 24 hours during imbibition was improved by initial moisture contents greater than 15%. Polyethylene glycol solutions of -5 and -15 bars reduced water uptake in the first four hours of imbibition at 5, 10 and 20C. Priming solutions of -5 and -15 bars also reduced damage to cotyledons and axes and improved germination of seeds chilled during imbibition. Assessment of damage by both staining with tetrazolium chloride and growth assays of the axis and cotyledon proved to be reliable methods. To my husband, Casper, whose love and encouragement facilitated this accomplishment and to my daughter, Rutendo, for being 56 patient. ACKNOWLEDGEMENTS I am grateful to Dr. Robert C. Herner, my advisor and committee chairman, for his guidance and support in everything I did, especially this thesis. I would also like to express my appreciation to Drs. Hugh Price, Irvin Hidders, and Lawrence Copeland for their assistance in both the research and the writing of this thesis. I would also like to thank the graduate students in the postharvest laboratory, especially Dr. Bill Wolk, for their assistance throughout this project. My parents, Maynard and Faith Makoni, deserve a special thank you for being staunch supporters of my educational goals and for patiently looking after our daughter, Rutendo, making it possible for me to finish this research. I am also indebted to my husband Casper and to Melba Lacey for their efforts and patience in typing this thesis. I would like to thank the Faculty of Agriculture, University of Zimbabwe, and Michigan State University exchange programm and U.S. Agency for International Development for providing the scholarship that made it possible for this research and my graduate career at Michigan State University. iii Finally, I would like to thank the faculty and graduate students in the Department of Horticulture for creating an environment suitable for learning and for their assistance in my graduate studies. TABLE OF CONTENTS List of Tables List of Figures Chapter I. Literature Review A. Introduction 8. Causes of Imbibitional Chilling C. Explanations (Theories) for Imbibitional Chilling Injury D. Alleviation of Imbibitional Chilling Injury Chapter II. Materials and Methods Seedcoating with Haxes and Oils 8. Moisture Content Experiments C. Priming Experiments Chapter III. Results A Effect of Seedcoating with Waxes B. Effect of Initial Seed Moisture Content C. Effect of Osmotic Potential Solutions Chapter IV. Discussion Seedcoating with Haxes 8. Raising Initial Seed Moisture Content C. Effect of Osmotic Potential Solutions D. Conclusions References Appendices vi vii wt—Iu—n LIST OF FIGURES Figure Page I The effect of coating snap bean seeds with waxes on water uptake in the first few hours of imbibition. 37 2 The effect of temperature and time on the rate of water uptake in wax-coated snap bean seeds 39 3 The effect of wax coating and temperature on the number of normal embryos (measured as the sum of seeds with uniform TTC stain, normal axes and cotyledons after 24 hours of imbibition) in snap bean. Small letters on bar graphs indicate levels of significance at 5%, comparing wax treatments at each temperature. 42 4 Effect of wax treatments and temperature on . germination of snap bean seeds after 24 hours of imbibition at the indicated temperatures. Small letters on bar graphs indicate levels of significance at 5%, comparing wax treatments at each temperature. 46 5 Effect of varying initial seed moisture content at different temperatures on the number of normal embryos in snap bean (as shown by number of seeds with uniform TTC stain, normal axes and cotyledons) after 24 hours of imbibition. Small letters on bar graph indicate the 5% level of significance comparing moisture content at the same temperature. 48 6 Effect of varying initial seed moisture content and temperature on the number of damaged cotyledons in snap beans after 24 hours of imbibition. Small letters on bar graph indicate the 5% level of significance comparing moisture content at the same temperature. 50 7 Effect of osmotic potentials and temperature on water uptake in the first four hours of imbibition in snap beans 55 vi Effect of osmotic potential and temperature on the number of normal embryos in snap beans after 24 hours of imbibition. Small letters on bar graph indicate 5% level of significance comparing osmotic solutions at each temperature. 57 Effect of osmotic potential and temperature on the number of damaged cotyledons and axes in snap beans after 24 hours of imbibition. Small letters on bar graph indicate 5% level of significance comparing osmotic solutions at each temperature. 59 LIST OF TABLES IéDlQ Page 1 Seed priming conditions for crops (modified from Bradford, 1986) 17 2 The amount of water evaporated from different waxes of different formulations 25 3 The effect of seedcoating on damaged axes, axes and cotyledon weight increases and total cotyledon chlorophyll content in snap been 43 4 The effect of wax coating and temperature on axes germination and radicle length 43 5 Effect of initial seed moisture content on axes germination, radicle and plumule length, axes and . cotyledon weight increase and total cotyledon chlorophyll content in snap bean 51 6 Effect of initial seed moisture content on germination of snap bean seeds in pots 52 7 Effect of osmotic solutions on axes germination, and axes and cotyledon weight increase 53 8 Effect of osmotic solutions and temperature on total chlorophyll content in snap beans 60 9 Effect of osmotic solutions on germination of snap beans in pots 60 viii 1. LITERATURE REVIEW mm Chilling injury is a physiological disorder occurring in some crops at temperatures between 0 and 10-15C. Chilling sensitive crops originate from tropical and subtropical regions and chilling injury affects all stages of growth and development in plants, from germination to senes- cence. Chilling injury during germination results in inhibition of germination or abnormal seedling development (Christiansen, 1967; Pollock and Toole, 1966; Holk and Herner, 1982). Two types of chilling injury prevent seeds of chilling sensitive crops from germinating (Herner, 1986). The first occurs when the seed has already germinated and affects the radicle of the plants. In this type of chilling injury, the seed does not germinate at the low temperatures but only germinates when the tempera— tures rise. Exposure of the germinating seeds to chilling temperatures causes necrosis of the tissue just behind the radicle tip and injures the root cortex. The second type is where the chilling injury occurs in the initial stages of germination when the seed is taking in water. This is called imbibitional chilling injury. Legume seed, especially snap beans (Phaseolus vulgaris L.) and lima beans (Phaseolus lunatus L.), are susceptible to imbibitional chilling injury. 2 This greatly reduces germination of these two legume crops if they are planted in cool spring soils. Pollock and Toole (1966) reported that lima bean seeds and excised embryonic axes can be injured during imbibition at temperatures below 2°C. Christiansen (1968) and Bramlage gt a1. (1978) reported the same thing for cotton (Gossypium hjrsgtum) and soybean (Glycine max), respectively. If seeds of cotton or lima bean are first imbibed at 31C and then transferred to SC, they are less severely damaged than if they had started imbibition at 5C. Four hours at 5C is enough to cause imbibitional chilling injury in cotton (Christiansen, 1968). The early imbibition stage is the critical stage in imbibitional chilling injury (Pollock and Toole, 1966). The symptoms of imbibitional chilling injury in snap beans and lima beans are transverse cotyledon cracking, abnormal germination and failure to germinate due to increased decay. Poor and slow germination result if these crops are planted in cold soils (Kooistra, 1971). Snap beans and lima beans originate from tropical and subtropical regions High temperatures are required both for germination and growth of these crops. Many factors affect imbibitional chilling injury including temperature, relation between timing of temperature exposure and stage of germination, initial seed moisture content, speed of imbibition, seed coat characteris- tics and integrity, seed vigor and cultivar or species. The lower the temperature the greater the imbibitional chilling injury. If exposure to low temperature occurs after the seed has already taken in some water, then the seed will not be affected. Seed with a high initial moisture content (12-20%) is less susceptible to imbibitional chilling injury than 3 that with a low initial moisture content (6-7%) (Herner, 1986). A rapid imbibitional rate enhances imbibitional chilling injury. Firmly attached and undamaged seed coats reduce imbibitional chilling injury. The causes, theories and methods of alleviating imbibitional chilling injury are hereby reviewed. B. CAUSES OF M IBITIONAL CH IMG Rapid rate of water uptake, solute leakage and seed coat characteristics may cause imbibitional chilling injury. 1. Rapid rate of water uptake Dickson (1971) and Powell and Matthews (1978) showed that the rapid rate of cold water uptake during the early stages of imbibition results in imbibitional chilling injury. Tully, g; a1. (1981) also reported that the sensitivity to imbibitional chilling injury is a consequence of the imbibition rate of pea (Eisum satiyum) (chilling resistant crop) and soybean (a chilling sensitive crop). They reported that the imbibition of pea seeds proceeds slowly in cold water, whereas soybean seeds imbibe cold water rapidly and suffer significant vigor loss. Hhen the imbibition rate of peas was increased by nicking the seed coats, the peas became susceptible to imbibitional chilling injury. This was probably due to the fast rate of imbibition in the nicked seeds. When the rate of soybean imbibition was slowed with a solution of polyethylene glycol, its susceptibility to imbibitional chilling was lessened. Cold imbibition of intact soybeans reduced vigor by about 83%. In nicked peas, the vigor as 4 a result of cold imbibition was also reduced by about 80% (Tully gt al, 1981). As a result of this observation, Tully and coworkers attributed the resistance of chilling injury in peas to the rate of imbibition. Duke gt g1. (1986) also reported that the rapid imbibition of soybean seeds greatly increases the leakage of intracellular substances and decreases seedling survival. They also reported that the testa epidermis of the soybean seed decreases the leakage of intracellular substances both during rapid and slow imbibition and increases survival. They found testa tissues other than the epidermis to have little effect on the intracel- lular components during slow imbibition but these tissues greatly decreased leakage of seeds during rapid imbibition and increased subsequent seedling survival. Holk (1988) studied effects of imbibition rates on germination of low moisture (7.5-9%) 2. vulgaris seeds (cv. "Tendercrop" and "Kinghorn Wax") using four different ways to control water uptake rate. These were positioning the seed relative to the hilum, incremental addition of water, varying number of layers of filter paper on which seeds were imbibed and applying wax to the seed’s hilum region. He found that each imbibition method affected the rate of water entry into the seeds; those methods that reduced imbibition rate were significantly correlated with increased germination. He then proposed that a hydrophobic compound, which when applied to a seed retards imbibition rates, would be beneficial and might be important on a commercial scale. Walk (1988) also reported that cotyledon tissue is more readily injured by rapid imbibition than the 5 axes. Injury to the cotyledon is deleterious to axes germination. He also suggested that an uninjured, fully imbibed axes might confer a degree of protection to the cotyledon and that the sequence in which embryo tissues imbibe might be a factor in the injury process. Powell and Matthews (1978) did not observe any staining with tetrazolium chloride on the outer surfaces of the cotyledons of imbibed pea seeds. These researchers concluded that the unstained cells were killed possibly by the rush of water into the seed. However, later work showed that the unstained cells are not necessarily killed because they stain if imbibed in succinate solution or if imbibed in water and then treated with succinate and tetrazolium salt (Powell and Matthews, 1981). Simon and Mills (1983) suggested that the incomplete staining of imbibed embryos indicate that succinate and other dehydrogenase substrates are lost from the cells. This was supported by work by Duke gt g1. (1983) which showed that imbibing soybean seeds lose dehydrogenase enzymes. The tetrazolium test distinguishes between viable and dead or injured tissues of the embryo on the basis of their relative respiration rates in the hydrated state. The test utilizes the activity of dehydrogenase enzymes as an index to the respiration rate and seed viability (Copeland, 1976). The dehydrogenase enzymes reduce tetrazolium chloride to formazan, a red pigment which causes the red stain on live tissue. This further supports the fact that the unstained outer pea cells in Powell and Matthews’ (1978) work was possibly due to loss of dehydrogenase enzymes and not necessarily due to a breakdown of cell organization. 6 The absolute rate of imbibition is not the only factor correlated with chilling injury. Hhen soybean seeds of different initial moisture content are imbibed, those with 44% initial moisture content imbibe more rapidly than those with 6% initial moisture content (Bramlage gt gl., 1978). Pollock (1969) showed the same response in lima beans. Even though seeds with high initial water content imbibe moisture more rapidly, these seeds are protected from chilling injury. Also, the rate of imbibition of seeds at low temperature is slower than at high temperature yet damage to the seeds is greater when seeds are imbibed at low temperature (Leopold, I980). The most critical stage of imbibition is the first few minutes or hours (Herner, 1986). 2. §glute leakage Solute leakage is also one of the possible causes of imbibitional chilling injury. It is also possible that solute leakage is just a symptom of chilling injury. The legume seed testa protects the embryo from massive cellular rupture and leakage of intracellular substances during imbibition (Duke and Kakefuda, 1981; Duke gt gl., 1983). Various intracellular substances leak from imbibing legume seeds which is negatively associated with seed vigor (Larson, 1968; Matthews and Bradnock, 1968; Perry and Harrison, 1970; Bramlage gt gl., 1978; Yaklich gt _l., 1979). Leachates may reflect a general deterioration of seed tissues which results in loss of seed vigor (Parrish and Leopold, 1978). Seed leachates may also serve as substrates for pathogen growth (Simon, 1974). Cells ruptured by imbibition may serve 7 as infection sites for seed pathogens (Duke gt gl., 1983). Substances and ions leaking out of seeds during imbibition include amino acids, sugars, organic acids, gibberellic acids, phenolics, phosphates (Simon, 1974), succinate (Powell and Matthews, 1981) and enzymes (Duke gt gl., 1983). Duke gt g1. (1983) reported that embryos with intact testa from soybean, navy bean, pea and peanut leak detectable activities of either intracellular enzymes of the cytosol (glucose-6—phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate, trans- aminase, and NADP-isocitrate dehydrogenase) after six hours imbibition at 2.5C. They unequivocally stated that the testa of legume seeds inhibit the leakage of high molecular weight intracellular substances. Leakage of intracellular substances from legume seeds during imbibition can involve two processes: (i) passive diffusion of low molecular weight substances through cell membranes during a period of membrane reorganization (Chabot and Leopold, 1982; Duke gt gl., 1983; Simon, 1978) and (ii) release of the entire cellular contents of ruptured testa and/or embryo cells (Duke and Kakefuda, 1981; Duke gt g1., 1983). Testa integrity has a large effect on both phenomena in legume seeds (Larson, 1968; Simon, 1974; Powell and Matthews, 1978; Duke and Kakefunda, 1981; Tully gt gl., 1981; Duke gt gl., 1983). Other factors which affect the leakage of intracellular substances from legume seeds during imbibi- 8 tion include seed moisture content (Hobbs and Obendorf, 1972; Parrish and Leopold, 1977; Simon and Hiebe, 1975), temperature (Pollock gt gl., 1969; Bramlage gt gl., 1978; Leopold, 1980; Duke gt 31., 1983; Marbach and Meyer, 1985), water potential (Knypl gt gl., 1980; Woodstock and Taylor- son, 1981a; Duke gt gl., 1983) and seed aging (Parrish and Leopold, 1978). Duke gt g1. (1986) found that anoxia has little or no effect on soybean seed leakage during imbibition and on subsequent seedling survival. Duke and Kakefuda (1981) suggested that leakage of electrolytes and ultra violet (u.v.) absorbing compounds, the two most commonly used methods of measuring leakage, do not accurately reflect the state of membrane integrity because many smaller molecules diffuse freely through the membrane. Instead they assert that determination of enzymatic activity in seed leachate should be used as an assay for membrane damage. 3. §eed goat chgracteristics Seed coat characteristics are not really causes of imbibitional chilling but compound both the rate of solute leakage out of the seed and the rate of water entrance into the seed. Imbibition may also be rapid, resulting in damage and poor seed germination if the seed coat is damaged during seed development or during harvesting and handling. The peanut (Atgghig nyngggg) seed coat is very thin and its presence or absence has little effect on embryo imbibition. Leakage from the embryo increases greatly when the seed coat is removed, decreasing the vitality and vigor of the embryo (Abdel Samad and Pearce, 1978). Powell and Matthews (1978) showed that if pea seeds are nicked with a razor blade water uptake is more rapid 9 and germination at 2C is much poorer than in controls imbibed at 25C. They concluded that the seed coat protects pea embryos from rapid imbibition, prevents extensive leakage and stops chilling damage. Tully gt g1. (1981) reported that cold tolerance during imbibition relates to the seed coat condition or to its pigmentation, both of which affect the rate of imbibition. Powell gt 31. (1986) tested differences in the field emergence of 30 commercial seed lots of dwarf French bean associated with the color of the testa. They reported that eleven lots with a white testa had a lower mean field emergence of 67% compared with 91% for lots with black or brown testa. The white seeded lots also had higher leakage conductivities and imbibed more rapidly than black or brown seed lots. Dickson (1971) also showed that cultivars with white seeds produce weaker and less vigorous seedlings than those with colored seeds, especially under unfavorable field conditions. This susceptibility of white seeded cultivars to imbibitional chilling injury has been associated with the degree of seed coat adherence to the cotyledons (Powell and Matthews, 1981). However, Holk’s (1988) work with "Kinghorn Wax" (with white seed coat) and "Tendercrop" (with pigmented seed coat) shows that the opposite can be true as far as pigmentation is concerned. "Kinghorn iriax'I had more resistance to imbibitional chilling injury than "Tendercrop." The relationship to injury and adherence of the seed coat to the cotyledons, however, still holds. "Kinghorn Wax" has a semi-hard seed compared with variety I'Tendercrop" which has a loosely bound and pigmented seed coat. 10 Taylor and Dickson (1987) determined that the semi-hard seed coat characteristic in snap beans delayed the onset of imbibition at low initial moisture levels and reduced imbibitional chilling injury. Water entry into the semi-hard seeds takes place primarily through the chalaza and raphe rather than the hilum and micropyle resulting in a slower rate of water uptake (Holubowicz gt gl., 1988). C. EXELANATIONS (THEORIES) FOR lMBIBITIONAL CfllLLING INQUR! Many theories have been proposed to explain imbibitional chilling injury. These ideas include membrane compositional differences, seed coat characteristics, membrane disruption, membrane reorganization and the water replacement hypothesis. 1. Nembrgne cgmgositional differences Chilling resistance is correlated with a greater degree of unsaturation in the lipid components of membranes. Lyons gt g1. (1964) showed that membrane lipids of chilling sensitive plants contain less unsaturated fatty acids than those of chilling resistant plants. Dogras gt 31. (1977) reported differences in percent"C glycerol incorporated into phosphotidyl choline (PC), phosphotidyl ethanolamine (PE) and phosphitidyl glycerol (PG) in imbibing seeds between chilling resistant species (broad beans and peas) and the chilling sensitive species (lima beans). The type of phos- pholipid synthesized may be related to sensitivity to chilling injury at 10C in the imbibition stage. This could be taken as evidence for the role of membrane compositional differences in affecting chilling injury. 11 In order to eliminate the species differences Holk (1980) studied the fatty acid composition of two cultivars of E. vulgarig which differ significantly in their resistance/sensitivity to imbibitional chilling injury. The differences in membrane fatty acid saturation between the two cultivars were very slight and did not support the membrane phase transition hypothesis as the mechanism of low temperature injury in imbibing seeds. Priestley and Leopold (1980) also reported the same observation and suggested that the difference in chilling sensitivity between pea and soybean is not related to compositional differences in the major lipid components of the seed membranes. Stewart and Bewley (1981) also did not find any correlation between membrane compositional differences and imbibitional chilling injury. These analyses were all performed on bulk membrane lipids extracted from seed and may not reflect the true membrane lipid composition of individual membranes such as the plasma membrane. In addition, the synthesis of new membrane lipids may be quite different between chilling sensitive and resistant species or cultivars. The differences in membrane composition (if there are any) may result in a change in the physical state of the chilling sensitive membranes at low temperatures which adversely affects energy supply and metabolism and results in the build-up of such toxins as acetaldehyde and ethanol. Mem- branes also increase in leakiness as a result of this phase change. 2. Mgmbrane gigruptjgn Larson (1968) proposed that cell membranes are ruptured by the rapid in- 12 rush of water as a seed imbibes, increasing leakage from the tissue. Powell and Matthews (1978) showed damage to a layer of cells on the abaxial surface through failure to stain with triphenyl tetrazolium chloride (TTC). Seeds with high rates of water uptake had cracks in the seed coats. A high proportion of seeds with cells not stained by TTC were low in vigor, had high amounts of solute leakage and exhibited poor field emergence (Powell and Matthews, 1979). Powell gt g1. (1986) noted that the pattern of leakage over time is the same for both living and dead seeds and concluded that it was a purely physical phenomenon and reflected disruption of cell membranes caused by imbibition. 3. Mgmttane rgozganization The theory of membrane reorganization is supported by the time course of initial water entry into dry cotyledons. It shows a period of rapid non- linear entry lasting 30 minutes or more (Leopold, 1980). The initial time course of solute leakage from the imbibing cotyledon also shows a period of rapid non-linear leakage followed by a linear phase beginning at the same time that water entry becomes linear. Leopold (1980) suggested that the initial rapid phases of these two events (imbibition rate and solute leakage) represent a period during which the membranes were relatively disorganized. The linear phases represented steady state processes through the reorganized membranes. Simon and Mills (1983) also proposed that membranes are disorganized in dry seeds no longer forming an intact barrier around the cytoplasm of each cell but regain their normal semi- permeable condition during imbibition. In a short period at the start of imbibition the membrane constituents in each cell may be going through a 13 phase of reorganization, and solutes could leak out. During imbibition, the water front penetrates slowly into the body of a seed or embryo, wetting each layer of cells, in turn, and allowing leakage from all the wet cells. Simon and Hiebe (1974) have suggested that the membrane of developing seed corresponds to the bilayer structure seen in bulk phospholipid/water mixtures. As the seed matures, the plasma membrane dries out. The molecular architecture of the membrane constituents is thought to change when its water content falls below 20% (Simon and Hiebe, 1974). The membrane forms a hexagonal phase, holding the remaining water more tightly and at the same time leaving gaps or leaks in the membrane. Solutes leak from these gaps. Recently several reports have been published refuting the theory that the membrane changes structure in dry seeds (McKersie and Stinson, 1980; Seewaldt gt g1., 1981). Investigations of membrane structure in dry seeds ~ by X-Ray diffraction of both extracted (McKersie and Stinson, 1980) and igggitg (Seewaldt gt gl., 1981) seed membrane lipids refuted the hexagonal array hypothesis. 31P-NMR studies of membrane structure in dehydrated pollen grains also indicated that the bilayer configuration is maintained even under conditions of extreme desiccation. With the doubts discussed above of the events taking place during imbibitional chilling a new hypothesis has been suggested to explain the phenomenon. 4. Ngtgr peplgcgmgnt hypothesis Vertucci and Leopold (1983) studied the wetting reaction of soybean embryo tissue. They found that PEG acts not only as an osmotic agent retarding imbibition rate, but also as a surfactant and, in dilute solutions (<2%), actually increased the rate of imbibition in embryos with initial moisture contents less than 24%. They suggested that the wetting reaction, a purely physical process, was the critical step in the injury process. This led them into the area of anhydrobiosis. These studies are focussed on the maintenance of cell structure during dehydration. The central hypothesis is the "water replacement hypothesis" as reviewed by Clegg (1986), one of its principal proponents. The water replacement hypothesis states that certain polyhydroxyl compounds such as the sugar trehalose are structured in a way that allows their hydroxyl groups to form hydrogen bonds to macromolecular sites in dehydrating tissues that would normally be occupied by water. In the presence of trehalose the membrane bilayer structure is maintained at low water activities, the fusion of vesicles is prevented and the activity of membrane bound enzyme systems is retained upon rehydration (Rudolp and Crowe, 1985; Crowe, Crowe and Jackson, 1983). Seeds do not contain trehalose (Kandler and Hope, 1966), but Caffrey gt _1. (1988) have recently demonstrated in-vitro the ability of raffinose/sucrose mixtures, sugars common in seeds, to preserve the bilayer configuration of phospholipid mixtures at low water activities. Holk (1988) indicated that the injury incurred by low moisture seeds during imbibition is the same at non-chilling temperatures. He also 15 reported that imbibition rate maxima of embryo tissue reflect transitions in the states of seed water. The experiments he did with NMR T1 support the application of the water replacement hypothesis to seeds. 0. ALLEIIATION 0E IMBlBITIONAL CHILLING INJURY The causes of imbibitional chilling injury and the theories explaining it are not fully understood. This makes the alleviation of imbibitional chilling injury difficult. Genetic improvements, including good pod and plant types that can germinate at 8 to 10C (Dickson, 1971) and seed coat characteristics allowing slow imbibition (Taylor and Dickson, 1987), are viable methods of preventing imbibitional chilling injury in seeds. The other methods tried include seed priming, raising initial moisture content and coating of seeds. l-M Osmotic conditioning or priming is a technique based upon controlled hydration of seeds to a level that permits pre-germinative metabolic activity to proceed, but prevents actual emergence of the radicle (Bradford, 1986). Early attempts to achieve this relied on alternately imbibing the seeds then redrying prior to the completion of germination (May gt gl., 1962; Henckel, 1964). This process was termed hardening. A consistent result of such treatments was more rapid germination (Heydecker and Coolbear, 1977). Henckel (1964) proposed that it was the dehydration that was responsible for the hardening effect but Hanson (1973) showed that the effective invigoration of seed occurs in the 16 imbibition period and is subsequently "fixed" by drying. Heydecker gt 31. (1973, 1975) achieved the same end by using an osmotic solution to inhibit radicle emergence, but allowing the maintenance of sufficient hydration for metabolism to proceed. Salt solutions had previously been used for this purpose (Ells, 1963; Koehler, 1967) but Heydecker gt g1. (1973) used PEG of high molecular weight (average 6000) as an inert osmoticum. The basic process, which Heydecker _t _l. (1975) called priming, consisted of imbibing seeds in an aerated PEG 6000 solution of sufficient osmotic strength to prevent visible germination. The seeds were held under these conditions for periods of up to several weeks, then rinsed and redried to the original water content. When planted, such seeds often germinated much more rapidly and uniformly, particularly under adverse temperature or moisture conditions. A considerable amount of success has been achieved by the use of PEG to improve germination of several crop species under adverse conditions. Table I shows some of the crop species in which PEG has had an effect on germination, the water potentials and the sources of information. l7 IA§L£_1: Seed priming conditions for crops (modified from Bradford, 1986) TEMP. DURATION DAYS CROPS SOLUTION C RESULTS SOURCE Barley PEG 10 1-8 accelerated germin— Bodsworth (ngdgpm (-0.5 to ation and improved and vplgarg) -1.5 MPa) uniformity Bewley(1981) Corn PEG 10 1-8 accelerated germina- Bodsworth (Zgg (-0.5 to tion and improved and mgys) -l.5 MPa) uniformity Bewley(1981) PEG(ZSOg/kg 15 8 improved germination; Khan gt g1. +0.2% thiram) increased seedling (1978) growth rate Pea PEG(250-5009 15 4;8 accelerated germina- Khan gt gt. tfiigpm per kg) tion;increased root) (1978) ' ) and shoot growth Hheat PEG(-0.5 10 1-8 improved germination Bodsworth to -1.5MPa) and aegtjvgm) Bewley(1981) Soybean PEG(-0.5 10 1—8 accelerated germina- Bodsworth Gl i e to -1.5MPa) tion;improved and mtg) uniformity Bewley(1981) PEG(250-3509 15 4-10 accelerated emergence; Khan gt g1. per kg+0.2% increased seedling (1978) thiram growth rate PEG(ZSOg/kg) 15 ll accelerated emergence Khan gt g1. (1980-81) PEG(ZSOg/kg 10 4 accelerated Knypl gt g1. + 0.2% thiram germination (1980) + 1200 U penicillin 18 The major obstacle to commercial application of seed priming is the variability of results among species, varieties, and even seed lots (Heydecker, 1977). The specific treatment conditions must be optimized essentially by trial and error for each species, cultivar or seed lot. Priming results in increasing seed initial moisture content before planting and thus has a beneficial effect. Drying the seed reduces seed initial moisture content and therefore loss of the beneficial effect of priming (Heydecker and Hainwright, 1976). Primed seeds can be stored at low temperatures to maintain the priming effect (Irwin and Price, 1981). Storing the primed seed at low temperatures and then planting it at subtropical temperatures has no benefit. Heydecker (1977) reported one major disadvantage of PEG 6000. PEG 6000 reduces oxygen availablity within the solution compared with that in water which in some cases may already be limiting. In PEG 6000, oxygen solubility is 50% that of water and oxygen mobility is only 10%, depressing relative oxygen availability to the order of 5% (Mexal gt gl., 1975). Despite the success of seed priming only a few studies have been carried out on the biochemistry and physiology of seed priming. Osmoconditioning of lettuce seeds reduces the time of imbibition required for the onset of ribonucleic acid (RNA) and protein synthesis, polyribosome formation and increases the total amount of RNA and protein synthesized (Khan gt 31., 1978; Khan gt gl., 1980-81). They also reported increased activity of enzymes like acid phosphatase and esterase following osmoconditioning. Osmoconditioning also caused qualitative changes in soluble proteins, acid phosphatases, esterases and 3-phoshoglyceraldehyde dehydrogenses as 19 indicated by electrophoresis on polycrylamide gels. Complete disap- pearance of abscisic acid (ABA) was also reported after osmoconditioning (Khan gt g1. (1978). From their results Khan gt g1. (1978; 1980-81) suggested that the increased synthesis of RNA, protein and enzymes in treated seeds may be due either to the removal of certain inhibiting factors such as ABA or to the production of promotive factors. They also suggested that mobilization of storage materials such as sugars, fats and proteins by activation or gggpgyg synthesis of key enzymes may underlie the mechanism of osmoconditioning. Hegarty (1978) concluded that while some work has been done on the physiology and biochemistry of seed priming "the physiology of incomplete- ly hydrated seeds is a field in which very little information is available." The information that is available does not provide easily measurable parameters that can be correlated with successful priming treatments. Hhile seed priming depends on the control and manipulation of seed hydration, little attention has been paid to the water relationships of the seed during and after treatment. Bradford (1986) put together some basic information on water relations of seed priming. In seeds the water content equilibrium attained at any given water potential (v0 depends upon solute potential (up) and pressure potential (4%) of the embryo cells. Solutes present in the cells lower‘+; and provide the driving force for water uptake in a relatively high \V range. A 20 Resistance of the cell walls to expansion as water is taken up results in turgor pressure, which increases theHVof the cell and reduces the driving force for water uptake. At the water content plateau, there is no net movement of water and the external water potential (Hg) is equal to the water potential of the cell (Hue) which is the sum of 9; and H; . In seed priming Hg is either set sufficiently low that radicle expansion cannot occur, or the duration of priming at higher‘i’o is shortened to be within the plateau period. Damage often results from dehydration as radicle growth has begun (that is, water content has begun to increase from the plateau level). Effective priming treatments are concerned with achieving and maintaining a near equilibrium between‘rg and‘fg. 2. ai i i tur c te t Experiments with seeds at different water contents have shown that there is no injury in cotton and soybean seeds at moisture contents above 13% (Christiansen, 1968; Hobbs and Obendorf, 1972). The corresponding figure for lima bean is 20% (Pollock, 1969; Cal and Obendorf, 1972). Simon and Raja Harun (1972) reported that pea embryos that have first been allowed to imbibe some water through a small part of their surface leak relatively slowly when subsequently immersed in water. The greater the initial imbibition the slower the subsequent leakage. Embryos taken from peas that were harvested when succulent and tender only showed slow leakage. Simon and Niebe (1975) showed that leakage from embryos with an initial water content of 17% is reduced and there is little sign of rapid leakage from embryos already containing 48% water. The rate declines steadily as 21 water content rises from air-dry value to levels of between 17-25% and then declines further as the embryos become more hydrated. At water contents above 30-35% leakage is reduced to a relatively low rate. Cohn and Obendorf (1976) measured enzyme metabolism in corn with 5% and 13% initial moisture content measured at 0, 12, 24 and 48 hours at 25C subsequent to imbibition at 5C. The interaction of low kernel moisture with imbibition at 5C resulted in reduced radicle growth in seedlings. Oxygen uptake of whole kernels after imbibitional chilling was independent of initial kernel moisture. Differences in initial moisture did not alter ATP levels of embryos and embryonic axes after chilling. Mitochondria isolated from embryos of low moisture kernels exhibited slightly higher respiratory rates 24 hours after cold imbibition but not at other sampling times. This showed that a disruption of energy metabolism was not the primary cause of kernel moisture mediated imbibitional chilling injury. Respiration after imbibitional chilling was the same for both low and high initial moisture kernels. 3. Haxing o: cogtipg of geeds Imbibitional chilling of seeds is generally associated with a rapid entry of water at low temperatures. A way of alleviating chilling injury would be to retard the entry of cold water into the seed. Attempts to slow the rate of imbibition of soybean, corn and cotton seeds through the application of a thin coat of lanolin (20-30g per kg seed) provided alleviation of chilling injury in the susceptible soybean and cotton (Priestly and Leopold, I986). Lanolin coated soybean seeds imbibed water 22 at a greatly reduced rate compared to untreated seeds. Hhen subjected to an imbibitional chilling stress (18 hours at 20) coated seeds had higher percentage emergence and individual weight of the emerged seedlings than the controls. Very little work has been done elsewhere on the use of waxes to control imbibitional chilling injury. The hypothesis in this study was that imbibitional chilling injury is caused by a rapid rate of cold water uptake. The objectives of this study were: 1. To try to alleviate imbibitional chilling injury in snap beans and lima beans by: (a) seed coating with waxes to reduce rate of celd water uptake by the seeds (b) allowing slow imbibition in aqueous solutions of PEG of different osmotic potential (c) gradually increasing the initial seed moisture content at room temperature before imbibition. 2. To determine the effectiveness of the above methods by assessing (a) damage to axes and cotyledons, and (b) germination of treated seed in pots II. MATERIALS AND METHODS Three methods to control imbibitional chilling injury in snap bean (Ehgggglpg vulgaris L.) and lima bean (Phageolus lppgtpg L.) were carried out. These were coating seeds with hydrophobic waxes to reduce rate of water uptake by the seed during imbibition, raising initial seed moisture content before imbibition and finally, imbibing the seed at different temperatures in polyethylene glycol 6000 to reduce the rate of water uptake by the seed. Seeds of snap bean, cv. Tendercrop, and lima bean cv. Fordhook 242, were obtained from Harris Moran Seed Company, New York. The stated seed quality characteristics were: germination percentage 84, purity 99% and 1328 seeds per pound for Tendercrop (lot number 83-3745); 86% germination and 388 seeds per pound for Fordhook 242 (lot number 26-3622). Both seed lots were prepared for commercial use and therefore pretreated with the fungicide Captan. In the laboratory the seed was stored in paper bags at 5C and 35% relative humidity until needed. Seeds in all experiments were presorted by hand and excessively small, large and/or damaged seeds were discarded. The three methods used in this study are described separately below. 23 24 A. 5 0mm mu AX 5 AND ons l. §elggtion apd prepgtation of waxes Twenty different waxes and oils were screened for their ability to effectively coat snap and lima bean seeds, and to reduce the rate of water uptake by the seeds. These waxes and oils were also selected for their ability to dry on the seed without leaving a sticky residue that impedes easy handling. The waxes and oils were applied 24 hours before the imbibition. Twenty different seed lots (corresponding to the twenty waxes/oils), each of about 30 seeds, were hand sorted for both lima and snap bean. During application the wax/oil was added to a seed lot in a Petri dish and gently stirred to allow complete coverage of the seed by the wax/oil. The seeds were then spread out on paper towels to dry. Two experimental units of ten seeds each were counted from each waxed/oiled seed lot. Each unit was weighed to the nearest hundredth of a gram on a Mettler PJ4000 balance. For imbibition treatments, each experimental unit (10 seeds per replica- tion) was immersed in 20ml distilled water in a 57mm aluminum foil dish at 2°C. The amount of water uptake was determined every 20 minutes for the first 2 hours of imbibition. At the end of each 20 minute period the seeds were removed from the water, thoroughly blotted on paper towels and weighed on the Mettler PJ4000 balance. The seeds were then reimmersed in water for the next 20 minute period. Amount of water uptake was based on the seed fresh weight increments. 25 The following four waxes were chosen for further study: (a) Vapor Gard from Miller Chemical and Fertiliser Corporation, Hanover, Pennsylvania, (b) Hiltpruf from Hiltpruf Products Inc., Greenwich, Connecticut, (c) Sta-fresh 320 manufactured by FMC corporation Florida and California, and (d) Pacrite 383 manufactured by American Machinery Corporation, Orlando, Florida. Preliminary work indicated that most wax formulations contained enough water that allowed some imbibition of water to occur during wax applica- tion. To prevent this from occurring all wax formulations were placed in a rotovap to remove water for l-2.5 hours at 45-50C. SinCe the four waxes selected were of different formulations and therefore different water contents, the amount of excess water removed (percentage of original wax volume placed in the rotovap) varied as shown in Table 2. The waxes/oils were applied by mixing them with the seed 24 hours before the imbibition studies. After thoroughly mixing the seed and wax the seeds were dried at air temperature. A completely randomized design was used to analyze the differences in treatments. TABLE 2: The amount of water evaporated from different waxes of different formulations Hax Hater Removed (% priginal wax vglume) Hiltpruf 80 Sta-fresh 320 84 Pacrite 383 60 Vapor Gard 60 26 2. Amgppt gf water uptgke Amounts of water uptake (water imbibition) were determined at three different temperatures, 5, 10 and 20C, for the first 4 hours of imbibition for both snap and lima beans. The determinations were done 15, 30, 60, 120 and 240 minutes (4 hours) after the beginning of imbibition. Ten seeds were used as one experimental unit and each unit was weighed before the start of imbibition. There were three replications for each treatment. For imbibition the seed was placed between two wet Hhatman No.1 filter papers in 15 x 100ml plastic Petri dishes. The filter papers were wetted by water equilibrated to the respective temperature regime. Hater was added to wet the filter papers as needed. At the end of each observational period (15, 30, 60, 120 and 240 minuteS) the seed was removed from the filter papers, dried on paper towels and weighed. 3. T ti f a e with 5- ri hen l- H-te re 0 I o 1 11m Maxed seeds imbibed at 5, 10 and 20C were assessed for damage 24 hours after the beginning of imbibition using triphenyl tetrazolium chloride (Aldrich Chemical Company, Milwaukee, Wisconsin). Ten seeds were placed in 20ml of 2% TTC solution for four hours followed by a thorough rinse in distilled water. The seed was then checked for uniform stain, cotyledon cracks, and damage to axes under a lighted magnifying glass (x3 magnifi- cation). Seed with greater than three cotyledon cracks and large trans- verse cotyledon cracks on both cotyledons, damaged axes and seed that did not take up any stain was considered nonviable and unable to germinate. 27 4. Growth assgy of axes and cotyledon tissue and chlorophyll determinations in cotyledons Haxed seed (and non-waxed control seed) imbibed at 5, 10 and 20C for 24 hours was assayed for growth. The assay used required growth of separate axes and cotyledons at 20C for ten days. After imbibition for 24 hours the axes and cotyledons were separated using a scalpel blade. The seed coat was also removed from the cotyledons. In snap bean five axes were used as one experimental unit in the axes growth assay while twenty cotyledons comprised one experimental unit in the cotyledon growth assay. The corresponding figures in lima beans were five axes and ten cotyledons. The large size of the lima bean cotyledons restricted the number of cotyledons per experimental unit to ten. Each experimental unit was weighed before the beginning of the assay. Three replications were used for each treatment combination in both the axes and cotyledon growth assays. The axes were placed on one Hhatman No. 1 filter paper in 15 x 100ml plastic Petri plates and the cotyledons were carefully placed flat side down between two filter papers also in 15 x 100ml Petri plates. Two percent sucrose solution was added to wet the filter papers in both the Petri plates with the axes and cotyledons. These were then kept in a growth chamber under continuous flourescent light at 20C for ten days. The nutrient solution (2% sucrose solution) was replenished on a daily basis by keeping the filter papers wet. After ten days the axes were checked for growth using fresh weight increases and length of the radicle and plumule. The cotyledons were also checked for growth using fresh 28 weight increases. Damage to the cotyledons was determined by measuring chlorophyll content in the cotyledons. This was based on the presumption that only living cotyledon tissue produces chlorophyll and that the amount of chlorophyll extracted from each cotyledon experimental unit was related to the extent of damage inflicted by the respective temperature regime. The amount of chlorophyll in the cotyledons was extracted using N,N- dimethyl formamide (DMF) as described by Moran and Porath (1980). For both snap and lima bean the cotyledons were cut into small pieces and weighed before being placed in medium sized test-tubes containing 20ml of DMF. The test-tubes were then wrapped in aluminum foil and placed at SC in the dark for 48 hours. The aluminum foil was to ensure darkness .at all times. After 48 hours, absorbance was read for the extracted chlorophyll solutions at 664, 647 and 625nm on a Beckman recording quartz spectrophotometer. A blank of 98% DMF was used in all cases and before each absorbance reading. Chlorophyll "a", "b" and "p' chlorophyll determinations were computed in ug/ml/g of cotyledon material according to the following equations by Moran (1982): Chlorophyll "a" - 12.65A6“ - 2.99Aw - 0.04A625 Chlorophyll "b" - -5.48A6“ + 23.44AM7 - 0.97A625 "p" Chlorophyll - '3°49Aw. - 5.25AM7 + 28.3A625 where A6“, A“7 and Afi25 are absorbance readings at 664, 647 and 625nm, respectively. 29 5. fiermipatjgn of treated seeds in pots Treated snap and lima bean seed was planted in 15cm diameter plastic pots with Bacto (from Michigan Peat Company, Sandusky, Michigan) as the planting mix. The pots were prepared, watered and transferred to the respective constant temperature rooms (5, 10 and 20C) 24 hours before planting to equilibrate the soil with the required temperature. Five replications for each treatment combination were completed. Five seeds were planted per pot. Immediately after planting, the pots were watered with water equilibrated to the required temperature and left in the constant temperature rooms for 24 hours after which they were transferred to the greenhouse. In the greenhouse the pots were watered every day. Germination percentage and heights of the germinated plants were recorded two weeks after planting for snap beans and three weeks after planting for lima beans. Germination was considered as emergence from the soil. 8. TUR 0 XP R MENT Three different seed moisture contents were used for both snap and lima beans. The moisture contents used were 6-9%, 15-20% and 25-30%. For all experiments moisture content was determined by drying seeds for 48 hours at 95C in a forced draft drying oven. All moisture contents are expressed on a fresh weight basis. 1. Mgistupe gdjpstmgnts Seed moisture contents were adjusted to the desired levels in humidity chambers made by placing some distilled water in air tight glass desiccators. The seeds were placed on netted wires above the distilled 30 water. Six to nine percent was the original moisture content of the seed from storage. For snap bean 300 seeds were kept in the humidity chamber for 48 hours to raise the moisture content to 15-20% and for five days to obtain 25-30% moisture content. For lima beans 300 seeds were kept in the humidity chamber for three and a half days to obtain 15-20% moisture content and for eight days to obtain 25-30% moisture content. 2. T st for dama e with 3 5- ri hen l-2H-tetrazo i 1 Seeds of three different moisture contents (6-9%, 15-20% and 25-30%) were imbibed for 24 hours at 5, 10 and 20C between two wet Hhatman No.1 filter papers in 15 x 100ml plastic Petri plates. Ten seeds per Petri plate were used as one experimental unit. Five replications were completed for each treatment combination. The low moisture and 20C combination was the control treatment. After 24 hours of imbibition the seed was then assessed for damage using 20ml of 2% TTC solution as described in section A(3). 3. Growth agsays and chlorophyll determination in cotyledons Assays for the amount of damage to the axes and cotyledons after imbibition at the three temperatures were also carried out for the three moisture content levels. For both snap bean and lima bean five axes made up one experimental unit in the axes growth assay while ten cotyledons were one experimental unit in the cotyledon growth assay. Five replica- tions were completed for each treatment combination. The method described in section A(4) was used in growing the axes and 31 cotyledons. Fresh weight increases and the length of radicle and plumule were used to assess growth. Chlorophyll content per gram of cotyledon was used to reflect imbibitional chilling damage of the cotyledon tissue. 4. rmi o f seeds of different moisture content 0 Both snap bean and lima bean seeds at three different moisture contents were planted in pots at 5, 10 and 20C. The pots were prepared, watered and equilibrated to the respective temperature as outlined in section A(5). Five seeds were planted per pot and there were five replications (with one pot as the experimental unit) for all treatment combinations. The pots were left in the constant temperature rooms for 24 hours after planting followed by transfer to the greenhouse. In the greenhouse the pots were watered every day. Germination percent and heights were measured two weeks after planting for snap beans and three weeks after planting for lima beans. C. ERIMING EXPERIMENTS Polyethylene glycol (PEG) 6000 obtained from Sigma Chemical Company (St. Louis, Missouri) was used for all the priming experiments. PEG at two osmotic potentials, -5 MPa and -15 MPa, was used to control rate of water uptake in the seeds. Distilled water at 0 MPa was used as the control. The osmotic potentials of PEG were calculated according to the two equations below adopted from Michel (1983); (a) Relationship between PEG 6000 osmotic potential and concentration within a temperature range of 5-40C is expressed as follows: 32 - 1.29 [PEGJZT - 140 [PEG]2 - 4.0 [PEG] (b) The quadratic solution for PEG concentration of equation (a) is as follows: [PEG] - r4 - (5.16 T - §_o +16)°-5i 2.58T - 280 where T is the temperature, is the osmotic potential desired in bars and, [PEG] is the concentration of polyethylene glycol. 1. Hater uptake Amounts of water uptake were determined at 5, 10 and 20C for the first four hours of imbibition using the three solutions of different osmotic potential (-1.5, -0.5 and 0 MPa). The experimental unit was ten seeds and each unit was weighed before the beginning of imbibition. Amount of water uptake was determined 15, 30, 60, 120 and 240 minutes after the start of imbibition. There were three replications in the experiment. For imbibition, seed from each experimental unit was placed between two wet filter papers in plastic Petri plates. The filter papers were then wetted with the respective solutions. At the end of each observation period (15, 30, 60, 120 and 240 minutes) the seed was removed from the Petri dish, quickly dried on paper towels, then weighed. 2. Testing for damage with TTC Snap bean and lime bean seeds imbibed with solutions of different osmotic potential at 5, 10 and 20C were assessed for damage with TTC as described in sections A(3) and 8(2). Ten seeds were used as one experimental unit 33 and three replications were made for all treatment combinations. After 24 hours of imbibition all the seed were thoroughly rinsed with distilled water before being placed in the TTC solution. 3. a o d i t o 1 co Seed imbibed in the three different solutions (of different osmotic potential) and at 5, 10 and 20C was also assayed for growth. The axes and cotyledons were separated as outlined in section A(4). In both snap bean and lima bean, five axes and ten cotyledons were used as experimental units in the axes and cotyledon growth assay, respectively. The assay was replicated three times for all treatment combinations. Fresh weight increases and radicle and plumule length were used in the assessment of growth. In the cotyledon assay chlorophyll content showed the extent of imbibitional chilling injury. L w wmh old G‘l'v'ntr%PGSMU° o‘s =4 WM Snap bean and lima bean seeds imbibed in the three osmotic potential solutions at 5, 10 and 20C in Petri dishes for 24 hours were also planted in pots with Bacto planting mix at the three temperatures (5, 10 and 20C). The pots were prepared as in sections A(5) and 8(4). Five seeds were planted in each pot and three replications were used for each treatment combination. The pots were kept in the constant temperature rooms for 24 hours and then transferred to the greenhouse where they were watered everyday. Germination percent and heights were taken two weeks after planting for snap beans and three weeks after planting for lima beans. 34 A completely randomized design was used to analyze the results. Except for the water uptake experiments, in which the treatments were arranged in a three factor factorial, all the experiments were arranged in a two factor factorial. Analysis of variance was carried out and 5% level of significance was considered as significant in all cases. All mean separations were carried out by least significance differences. III. RESULTS The lima bean seed used in these experiments showed a lot of inconsistency and poor germination even under ideal conditions. The results reported in this study pertain mostly to snap bean. Summaries of the lime bean results are included in the appendix. A. A G N N 1- W Max—coated snap bean seeds imbibed water at reduced rates compared to untreated seeds. In snap bean Vapor Gard and Pacrite 383 reduced water uptake by as much as 45% and were better (p>0.01) than Hiltpruf and Sta- fresh 320 whose corresponding figure was 35%. Figure 1 shows the effect of different waxes in reducing water uptake with time. There was clear separation between the waxes and control as well as among the waxes themselves as imbibitional time increased, giving rise to wax x time interaction (p>0.001). For all the treatments, water uptake increased with temperature. The highly significant (p>0.001) temperature x time interaction is represented in Figure 2. The high water uptake at 20C compared to the chilling injury inducing temperatures (5 and 10C) suggests that the rate of water uptake pg; gg might not be the only cause of imbibitional chilling injury. 35 36 ML}. The effect of coating snap bean seeds with waxes on water uptake in the first few hours of imbibition. 37 $8358 .5 m2: zoEmas: OWN OPN OW— omp OWP Om _ p — X03 O: u\\\\ 8n 8:03 can 53.:le 3.9:; Pam Loao> Hill roe on 393 1ou161Jo )o z n J91DM to iunowv 92M 38 FIGURE 2. The effect of temperature and time on the rate of water uptake in wax-coated snap bean seeds. 39 \\ H 1 240 210 r 180 IMBIBITION TIME (in minutes) O V 393 lougbuo 10 % snip), n J91DM 1o iunouuv \ 00 000 Ins-N l l l l 1.0 O in O I’D N 40 2.5.5.5355me In a preliminary study there was a high positive correlation (r-0.87) between germination percent of bean seeds imbibed at 5 and 1°C with the sum of seeds with uniform TTC stain, normal axes and cotyledons with at most three small cracks. This indicated that these three measurements were reliable estimates of normal (viable) embryos after imbibition at low temperatures. At 5 and 10C all waxes increased the number of normal embryos compared to nonwaxed controls (Figure 3). Niltpruf, Vapor Gard, Pacrite 383, and the nonwaxed controls were similar at 20C while Sta-fresh 320 reduced the number of normal embryos. There was no effect of temperature and waxes on the number of damaged cotyledons. Niltpruf, Pacrite 383 and Vapor Gard reduced the number of damaged axes while Sta-fresh 320 had no effect (Table 3). More axes were damaged at 5 and 10C than at 20C. This is expected since there is im- bibitional chilling injury at these temperatures. Assessment of seed damage by uniform TTC stain, normal axes and cotyledon cracks is very subjective and can be inaccurate. Growth perfomances of axes and cotyledons were also used to assess seed damage after imbibition at low temperatures. Cotyledon and axes weight increases were improved by all waxes (Table 3). Only Vapor Gard, Hiltpruf and Pacrite 383 increased total chlorophyll content. The temperature effect was only significant for axes germination (p>0.01) and radicle length (p>0.001). The wax temperature interaction for these two parameters was also signif- icant (Table 4). Niltpruf and Pacrite 383 increased axes germination FIQU E 3. 41 The effect of wax coating and temperature on the number of normal embryos (measured as the sum of seeds with uniform TTC stain, normal axes and cotyledons after 24 hours of imbibi- tion) in snap bean. Small letters on bar graphs indicate levels of significance at 5%, comparing wax treatments at each temperature. 42 @ PACRITE 383 Z STA—FRESH 320 m N0 WAX X WlLTPRUF Q VAPOR CARD .o £03333;910x(do:61914101010314:61010141910:(0191410191410)2 UNENUUHNNODRMNUDHMMUDRMNOURMNUUHMNU NANNDNNRNARNANNARNDNNRRNRND VUUUUUUUUUUUUUUUWVUU"UVVUUUUI AAAAAAAAAAAAAAAAAAAA.A.AAAAAAAA ahX\\\\\\\\\\\\\\\V33\\\\\\\Y\VK\\\\ ~ {OI01020102920201919101919310:10192 .. L91910191010333;lit1tillililililtllllllltltltI(91929191920263):1 .. 1\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\V VOWHVOtflflflflviflf'OWHVOBHVOI' DIIAAAAAIAIAIIIAAAIAI ..€§§§fififia MHEMUUURMMUUHNNUUNMNN AINURNNDNNNDNNNRNND .0 °IWNVIWHVOIHVOWHVOWNVOIHVO'lVONHVI IAAAAAAAAAAAAAAAAAAA.AAAAA :i\\\\\\\\\\\\\\\\X\\\\\\ 100 I l I O O O O O O O O) ID V' I0 (%)so/0.01) effect on all the growth assay measurements. Initial seed moisture content above 15%.showed a consistent increase in axes germination, radicle length, axes and cotyledon weight increases. Plumule length and total cotyledon 45 Effect of wax treatments and temperature on germination of snap bean seeds after 24 hours of imbibition at the indicated temperatures. Small letters on bar graph indicate significance at the 5% level comparing wax treatments at each temperature. 46 [XI WILTPRUF a VAPOR CARD E PACRITE 383 @ STA—FRESH 320 22 NO WAX a {o1919101919{9:9{919193191910331919;010:9101?ozoxqfimxd Itlililtliltltliltltlilt2929191919lilillllilltliltltlililtIiItItIiItIiltIllll .0 i .0 0101010101010IIIIIIIIIIIIII101.102.101.201“ e m\\\\\\\\\\\\\\\\\\\\\I\\\\ ~— 14191919191010);It){titltllltltltllltltlt1610191920ItIii .\\\\\\\\\\\\\\\\\\\‘ .D m "' nIX\\\\\\\\\\I\\\\\\\\\\\ o 91919:.19191019101933)!“OI .0 ‘3 01020101020101.1020:IIOIII0101.10.01.10!!! -° BX\\\\\\\\\\L\\\\\Y\\\\\ \\\\\ 100 904 I I O 1 O O I’D N '- 404 T T l O O O i\ LO L0 80—1 amour/mums TEMPERATURE °c EIQURE 5. 47 Effect of varying initial seed moisure content at different temperatures on the number of normal embryos in snap been (as shown by number of seeds with uniform TTC stain, normal axes and cotyledons) after 24 hours of imbibition. Small letters on bar graph indicate the 5% level of significance comparing moisture content at the same temperature. 48 m 25-30% MC 8 15—20% MC 52 6-9% MC ‘° N\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\' O lIIIIIIIIIIIII.IIIIIIIIIIIIZIIIIIIIIIIIXIII o L\\\\\\\\\\\\\\\\\\\\\ n .\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\V ° lIIIIIIIZI101.201.201.101.II ° “1 0 m\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\‘ PW, US$531 100 I I O O 0 V' F) N 50-1 1 0'0 0 o (O (z) soxaawa vaaon 20 O TEMPERATURE ‘0 P L0 FIGURE §. 49 Effect of varying initial seed moisture content and temperature on the number of damaged cotyledons in snap beans after 24 hours of imbibition. Small letters on bar graph indicate the 5% level of significance comparing moisture content at the same temperature. SO 0 -° x x x ' D \\\\\\\ . cum . DW‘ ° \\\\\\\X\\\\\“\\\\\\\\\\\ as ° 0 .01 ass NNro 0 mil ' limo (Dr-N V WI T l T T T O O O O O O O to L0 sl- n N .— (%)suope|/0.001) osmotic potential x tempera- ture x time interaction suggests that the greatest reductions in water uptake are achieved by low osmotic potential at low temperatures after long periods of imbibition. 2. Assessins_seed_daaase Both PEG priming solutions and temperature significantly (p>0.001) affected the number of normal embryos. The number of normal embryos was increased by osmotic solutions of -O.5 and -1.5 MPa and were reduced at 53 5 and 10C (Figure 8). More axes and cotyledons were damaged at low temperatures (5 and 10C) and priming solutions of -0.5 and -1.5 MPa significantly alleviated this damage (Figure 9). Osmotic solutions of -0.5 and -1.5 MPa increased axes germination, axes weight increase, and cotyledon weight increase in snap beans (Table 7). IABL£_1; Effect of osmotic solutions on axes germination and axes and cotyledon weight increase Osmotic Axes Axes wt. Cotyledon wt. Egtgntjg] (MPa) gem % 111ch 0 65.7“ 0.05°“ 0.084“ -o.5 93.3b 0.066b 0.158b -1.5 88.9” 0.068b 0.197c Axes germination increased with increases in temperature. Low tempera- tures reduced cotyledon weight increase and total cotyledon chlorophyll content. There was no osmotic solution by temperature interaction for axes germination, axes weight increase, and cotyledon weight increase. For all the priming solutions, total chlorophyll content increased with increases in temperature (Table 8). At 5C, -O.5 MPa osmotic solution reduced chlorophyll content but for 10 and 20C, chlorophyll content increased with increase in osmotic potential of the priming solution. 54 E1§UB£_Z. Effect of osmotic potentials and temperatures on water uptake in the first four hours of imbibition in snap beans. 1 1 cPtoke (Z of original see wt.) Amount of water u 1 1 uptake (7. of original see wt.) Amount of water 1 1 crtoke (Z of original see wt.) Amount of water u 55 30 g TH OMPo 5C 1 H -O.SMP0 1 25-. H -1.5MPo ‘ i 20- I I 151 I lO-i 54 I 17" I' If 0 30 60 90 120 150 180 210 240 IMBIBITION llME (in minutes) o—e OMPo , 10C 35.1 H .°.3“P° H -1.5MPo .304 T o 30— ab 90 1120 150 18? 2io' 240 IMBIBITION TIME (in minutes) 60 o—e OMPo 20C H -O.5MPo SO-J H -l.5MPo 40d // 1/ / -J m-w--»m.~——- 20-1 J .‘L 10-1 0 3'0 50 9'0 150 130 183 2io 24o IMBIBITION TIME (in minutes) 56 Effect of osmotic potential and temperature on the number of normal embryos in snap beans after 24 hours of imbibition. Small letters on bar graph indicate 5% level of significance comparing osmotic solutions at each temperature. 57 [X -O.5MPo 2 0MPo a -1.5MPo ° .\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\V ° l\\\I\\\\\\\\L\\X\\\\\\\X\\\\\X\“\\ .\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\‘ ‘° 'IIIIIIZIIIIIIIIIIIIIIIIIIOIIIIIIZIIIII IO TEMPERATURE °C '° .\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\‘ 100 I 1 I I I I I O O O O O O O O 05 no I\ (D ID '3‘ V) N (34) 801(1qu 1ouuou 10 0N 58 FIGURE 9. Effect of osmotic potential and temperature on the number of damaged cotyledons and axes in snap beans after 24 hours of imbibition. Small letters on bar graph indicate 5% level of significance comparing osmotic solutions at each temperature. 59 20 TEMPERATURE °c m. A. m. L oww a mmw W 4. ... o N W ._1 ... a? . 25. ac E a 26% o m g a 555.. m ELSE b Biggie/€25 m” gag m. .. Egg/g .. Egg . ggfixgxgg s b922/2229;5265 gamma w. m... m... w... m o m m ANvmconotfioo oomoEoo .6 .oz .. ANVmoxo noooEov Lo .02 60 IABLE_§: Effect of osmotic solutions and temperature on total chlorophyll content in snap beans Osmotic Temperature (°C) solutientnflal 5, 10 29 0 0.186 0.408 0.535 -0.5 0.123 0.490 0.786 4.1 0.339 04614 0.978 3. Sera1nat19n_9£_nrlmed_seeds_in_nets In snap beans, both the priming solutions and temperature affected seed germination but not plant height. Priming solutions of -0.5 and -l.5 MPa increased germination percent compared to the untreated controls (Table 9). There was a general increase in germination with increase in tempera- ture. There was no PEG priming solution by temperature interaction on germination of snap beans in pots. IABL£_2: Effect of osmotic solutions on germination of snap bean in pots PEG priming Germination % 591911901023) in pots 0 42.2' -0.5 56.7b -1.5 71.1b CHAPTER IV. DISCUSSION A. H AXES The results obtained indicate that imbibitional chilling injury in snap beans can be alleviated by using hydrophobic materials (waxes) that reduce the rate of water uptake during the first few hours of imbibition. The waxes act as physical barriers to water uptake. The reduced water uptake in seeds coated with Vapor Gard, Hiltpruf and Pacrite 383 consistently increased the number of normal embryos and germination in pots. Priestley and Leopold (1986) reported similar results with theuse of lanolin coating in soybean; however, lanolin is very difficult to handle because it is thick and sticky. It, therefore, had to be melted and diluted with acetone for ease of application to the seed. waxes used in this experiment were all thin, easy to handle and apply, and all dried on the seed without stickiness; properties that encourage their widespread use. Although Sta-Fresh 320 reduced water uptake by the seed, it decreased germination in pots and the number of normal embryos especially at 20C. It is quite possible that this wax had some toxic effects. No further comment can be given because the wax compositions were not known. The beneficial effects of these waxes were shown at 5 and 1°C, the chilling injury inducing temperatures. Apart from Sta-Fresh 320, these 61 62 waxes showed no deleterious effects at 20C which is an advantage since they can still be applied when one is not sure imbibitional chilling inducing temperatures will prevail. The relationship between the wax mediated reduction in‘water uptake and the reduced number of'damaged axes, increased cotyledon and axes weight and increased total cotyledon chlorophyll content suggested that both axes and cotyledons are injured during imbibition at low temperatures. The reduction in water uptake rate by waxed seeds may result in less injury because there is less free water causing damage between cotyledons and between the testa and cotyledons (Hulk, 1988). Several authors have tried to explain the mechanism of imbibitional chilling injury (Dickson, 1971; Powell and Mathews, 111978 and 1981; Leopold, 1980; Tully gt 31., 1981; and Holk, 1988). The apparent consensus is that the rate of cold water uptake is a major cause of imbibitional chilling injury. The current study supports this general understanding. However it remains unresolved as to whether imbibitional chilling injury is the physical damage from water entrance or is a result of some biological phenomenon triggered by cold water uptake. 8. N D MD S C0 Raising the initial seed moisture content before imbibition at low temperatures has the desired effects of reducing seed damage and increasing germination. The absence of any statistical differences between 15-20%.and 25-30%.initial moisture content treatments supports the existence of a critical moisture content above which seed is protected 63 from imbibitional chilling injury. Thirteen percent initial seed moisture has been suggested as the critical value for snap bean (Holk, 1988) and soybean (Christiansen, 1968; Hobbs and Obendorf, 1972). A higher figure of 20% has been suggested for lima bean (Pollock, 1969; Cal and Obendorf, 1972). How the raising of initial seed moisture content alleviates imbibitional chilling injury is not clear. It cannot be explained by the reduction of rate of cold water uptake because seeds of high initial moisture content imbibe water more rapidly than those of low initial moisture contents (Bramlage gt 11., 1978). C. F 0L N Osmotic solutions reduced seed water uptake. The consistent relationship between the reduction in water uptake and the reduced number of damaged seeds together with enhanced germination indicates that osmotic solutions alleviate imbibitional chilling injury by reducing cold water uptake. The use of priming solutions reduced the number of damaged axes and cotyle- dons, increased axes and cotyledon weights as well as cotyledon chloro- phyll content. This supports an earlier inference that both axes and cotyledons are damaged when seeds are imbibed at low temperatures. The reduced rate of' cold water uptake explains the alleviation of imbibitional chilling injury by osmotic solutions. PEG solutions reduced water uptake because they are of higher osmotic potential (hold water'more tenaciously) and therefore make less water available for uptake by the 64 seed. The fact that osmotic priming with PEG increases germination under cold temperatures has already received attention from other researchers (Khan gt g1.,1978; 1980-81; Bodsworth and Bewley, 1981). In a series of experiments involving priming seeds for several days these researchers concluded that the tolerance to imbibitional chilling injury imparted through PEG priming is a result of seed physiological and biochemical changes. Although physiological and biochemical changes were not monitored in this study, it is quite reasonable to accept that these changes contributed to the alleviation of imbibitional chilling injury. The data from this study suggests that there are several causes of imbibitional chilling injury. Results from the use of waxes and PEG solutions support the idea that rapid cold water uptake is one of the major causes. The fact that high initial moisture content in the seed imparts protection against imbibitional chilling injury suggests phenomena other than just rapid rate of cold water uptake. Vertucci and Leopold (1983) suggested that the wetting reaction was the critical step in the injury process, an observation supported by Christiansen (1968) who elucidated that the effects of imbibitional chilling injury are set within the first few minutes of imbibition. The water replacement hypothesis propounded by Clegg (1986) and supported by Holk (1988) can explain how high initial seed moisture contents impart tolerance to imbibitional chilling injury in seeds. Using NMR techniques Holk (1988) showed that imbibition rate~maxima of embryo tissue reflects transitions in the states of seed water. It is probable that the state of seed water in high initial seed moisture contents imparts resistance to imbibitional chilling 65 injury. The high leakage of solutes at low temperatures (Bramlage gt 31., 1978; Duke gt 31., I983; Marbach and Meyer, 1985) and initial seed moisture content (Hobbs and Obendorf, 1972; Simon and Niebe, 1975; Parrish and Leopold, 1977) is a phenomenon which can also be used in explaining imbibitional chilling injury. The three methods used to control imbibitional chilling injury in snap bean in this study were all successful. The use of wax coating of seeds seems to be the most practical and easiest method to use. The successful application of the waxes on Captan treated seeds suggests that some pesticides could be incorporated into the waxes before application to the seed. The success of this method, however, depends on the waxes chosen. Although raising the initial moisture content reduced imbibitional chilling injury in both snap and lima beans, there are several problems with this method. Raising initial moisture content of the seed enhances faster deterioration in seed quality and therefore high moisture seeds cannot be stored for long periods. Seeds of high initial moisture content also lose moisture if stored under dry conditions and therefore lose the ability to protect seeds against imbibitional chilling injury. Raising the initial moisture content and coating seeds with waxes could be combined to reduce the moisture loss problem and enhance seed germination. Priming with PEG was also effective in reducing imbibitional chilling injury in snap beans, but this method has several disadvantages. The first is that after priming, the seed has to be dried for easy handling. This, however, has shown to result in some retardation of germination or 66 loss of some of the beneficial effects of priming (Heydecker and Rain- wright, 1976). Storage of primed seed also results in loss of the beneficial effects of priming (Irwin and Price, 1981). For large seeded crops like soybean there is a limit to the number of days the seed can be primed, since there is disintegration of the testa and cotyledon separation with handling (Bodsworth and Bewley, 1981). W 1) Three of the waxes used (Hiltpruf, Vapor Gard and Pacrite 383) reduced the rate of water uptake and damage to cotyledons and axes and improved germination of imbibitionally chilled snap beans. Sta- fresh 320 also reduces rate of water uptake, but it was phytotoxic to the seed. 2) Raising the initial seed moisture content reduced damage to cotyledons and axes and improved germination of imbibitionally chilled snap bean seeds. 3) Polyethylene glycol of -5 and -15 bars reduced water uptake and damage to cotyledons and axes and also improved germination of imbibitionally chilled snap beans. 4) Assessment of damage with TTC and growth assay of separate axes and cotyledons showed good consistency with germination of imbibitional- ly chilled snap beans and can therefore be used to assess damage in seed. REFERENCES Abdel Samad, I. M. and R. S. Pearce (1978). Leaching of ions, organic molecules, and enzymes from seeds of peanut (Argghjg hypgggg L.) imbibing without testa or with intact testas. Jt_£xptt_figt. 29:1471- 1478 Bodsworth, S and J. D. Bewley (1981). 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The effect of temperature change on leakage from pea seeds. Qt_£yptt_figt. 36:353-358 Matthews, S. and H.T. Bradnock (1968). Relationship between seed exudation and field emergence in peas and French beans. Hort. Bg§. 8:89-93 May L.H.; E.J. Milthorpe and F.L. Milthorpe (1962). Pre-sowing hardening of plants to drought. An appraisal of the contributions by P.A. Genckel. E1g13_§rgp_Apgtp. 15(2):93-98 McKersie, 8.0. and R. H. Stinson (1980). Effect of dehydration on leakage and membrane structure in Lgtgg,gg_gig313tgg L. seeds. £13nt_Ehygig1. 66: 316- 320 Mexal, J.; J.T. Fisher; J. Osteryoung and C.P. Patrick-Reid (1975). Oxygen availability in polyethylene glycol solutions and its implications in plant water relations. £13pt_£hy31g1. 55:20-24 Michel, B. E. (1983). Evaluations of the water potentials of solutions of polyethylene glycol 8000 both in the absence and presence of other solutes. £133t_£hygigl. 72:66-70 Moran, R. (1982). 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Bpt. 30:193-197 Powell, A.A. and S. Matthews (1980). The significance of damage during imbibition to the field emergence of pea (£13m gatjygm L.) seeds. J_,_ Agrigt_§§1. 95:35-38 Powell, A.A. and S. Matthews (1981). A physical explanation for solute leakage from dry pea embryos during imbibition. J,_£zpt_,_B_ot. 32:1045- 1050 Priestley, D.A. and A.C. Leopold (1986). Alleviationof imbibitional chilling injury by use of lanolin. grgp_§g1gngg 26:1252-1256 Rudolp, A.S. and J.H. Crowe (1985). Membrane stabilisation during freezing: the role of two natural cryoprotectectants, trehalose and proline. Ctygptgl. 22:367-377 Seewaldt, V.; D.A. Priestley; A.C. Leopold; G.H. Feigenson, and F. Good- said-Zalduondo (1981). Membrane organisation in soybean seeds during hydration. £13pt3 152:19-23 Simon, E.H. (1974). Phospholipids and plant membrane permeability. Ngy Ehytgl. 73:377-420 Simon, E.H. (1978). Membranes in dry and imbibing seeds. In: Qty . J.H. Crowe and J.S. Clegg (Eds.). pp.205-244. Academic Press. NY. Simon, E.H. and R.M. Raja Harun (1972). Leakage during seed imbibition. J1_£3ptt_flgt. 23:1076-1085 Simon, E.H. and L.K. Mills (1983). Imbibition, leakage and membrane. WW. 17:9-27 Simon, E.H. and H.H. Hiebe (1975). Leakage during imbibition, resistance to damage at low temperature and the water content of peas. Ngw Ehytgl. 74:407-411 72 Stewart, R.R.C. and J.D. Bewley (1981). Protein synthesis and phospholipids in soybean axes in response to imbibitional chilling. £13pt_£pygtgl. 68:516-518 Taylor, A.G. and M.N. Dickson (I987). Seed coat permeability in semi-hard snap bean seeds: Its influence on imbibitional chilling injury. J1 ngtg_§§1. 62:183-189 Tully, R.E.; M.E. Musgrave and A.C. Leopold (I981). The seed coat as a control of imbibitional chilling injury. Qrgp §gj. 21:312-317 Vertucci, C.H. and A.C. Leopold (1983). Dynamics of soybean imbibition. £13nt_Ehy§ig1. 17:190-193 Holk, H.D. (1980). Chilling injury and membrane fatty acid saturation in imbibing and germinating seeds of 21133951111; yu1ggr_i_s. MS Thesis. Michigan State University, East Lansing, MI. Holk, H.D. (1988). Imbibitional chilling injury in Engggglpgflyglggrig, PhD Dissertation. Michigan State University, East Lansing, MI. Holk, H. D. and R. C. Herner (1982). Chilling injury of germinating seeds and seedlings. flgpt§g1. 17: 169-173 Hoodstock, L.H. and R.B. Taylorson (I981). Soaking injury and its reversal with polyethylene glycol in relation to respiratory metabolism in high and low vigor soybean seeds. Ehygjgl, Elgpt. 53:263-268 Yaklich, R.H.; M.N. Kulik and J.D. Anderson (1979). Evaluation of vigor tests in soybean seeds; relationship of ATP, conductivity and radioactive tracer multiple criteria tests to field performance. tpgp figl. 19:806-810 APPENDIX A Screening_9£_uaxes Results IABL£_1: Effect of waxes on reduction of water uptake. Mean water uptake ng/oil C5 of origjn3! gegg wgight Sunflower oil 39.44b Vegetable oil 37.05b Corn oil 39.77“ Peanut oil 38.53“ Safflower oil 39.37“ Vapor Gard 29.17' Hiltpruf 29.67“ Apple wax 35.94b Olive oil 44.82° Sta-fresh 560 33.71' Sta-fresh 350 37.72“ Sta-fresh 320 27.528 Pacrite Sunshine wax 38.65b Lustre Dry 38.74b Prime Shine 43.91c Sealbrite 74 52.82d Citrashine 801 36.14“ Pacrite 383 25.728 Sealbrite 65 39.14“ Control (no wax) 44.79° Vapor Gard, Hiltpruf, Sta-fresh 320, Pacrite 383 and Sta-fresh 560 reduced the rate of water uptake by the seed in the first two hours of imbibition. Sta-fresh 560 was not chosen because it does not dry on the seed. 73 APPENDIX B ea GU T' A. ngect of waxy seedcoating on imbibitional chilling injury in lima ans IABLLZ; Effect of waxes on water uptake, number of damaged cotyledons, number of normal embryos, and germination in pots Hater Number of Damaged Uptake cotyledons Number of normal Germination H. - . . . o -f I e .r 0 t 1 .- Vapor Gard 5.53' 1.22' 61.1c 13.33 Hiltpruf 6.27‘ 1.33' 60.0c 5.33 Sta-fresh 320 5.72“ 1.79“ 45.6“ 8.00 Pacrite 383 5.05' 1.67' 57.8c 14.67 No wax 7.87“ 2.44“ 32.2“ 18.67 All the waxes reduced water uptake and number of damaged cotyledons to the same extent and increased the number of normal embryos. The waxes had no effect on germination of lima beans in pots. 74 75 mm: Effect of temperature on water uptake, number of damaged cotyledons, number of normal embryos, and germination in pots Hater Number of Damaged Temperature Uptake cotyledons Germination ° ' f 10) in pgt; (t) 5 5.53“ 2.53“ 6.67“ 10 5.75“ 1.80“ 8.55“ 20 ' 6.87“ 0.73“ 28.76“ 5 and 10C reduced water uptake, increased the number of damaged cotyle- dons, and reduced germination percent in pots, but generally there was very poor germination of lima beans. The waxes had no effect on axes germination, axes weight increase, cotyledon weight increase, total chlorophyll content and plumule and radicle length. 76 B. ngect of moisture contents on imbitional chilling injury in lime ans IABLEJ: Effect of initial seed moisture content on the number of damaged cotyledons, normal embryos, and germination of lime beans in pots Moisture Number of Damaged Normal Germination Content Cotyledons Embryos in Pots 12) (9113.9.me 4%) (7.4.; 6-9 40.67“ 7.61' 15-20 43.33“ 64.69“ 25-30 59.33“ 71.23“ Initial moisture content of greater than 15%.increased germination percent in pots but only 25-30%1initial moisture content increased normal embryos. Raising the initial moisture content had no effect on the number of damaged axes and cotyledons. 77 IABL£_§. Effect of initial seed moisture content on axes germination, radicle and plumule length, axes and cotyledon weight increase and total cotyledon chlorophyll content in lime bean Moisture Axes Radicle Plumule Axes Total Cotyledon content germ. length length weight chlorophyll 6-9 47.0“ 2.16“ 0.47“ 0.072“ 0.105“ 15-20 72.8“ 2.99“ 0.56“ 0.105“ 0.179“ 25-30 62.2“ 3.24“ 0.55“ 0.098“ 0.194“ Initial moisture content greater than 15% consistently increased axes germination, radicle length, plumule length, axes weight increase and total chlorophyll content. Raising the initial moisture content had no effect on cotyledon weight increase. In all the moisture content experiments, 5 and 10C decreased germination percent and the number of normal embryos and all the growth assay measurements made. 78 C. Effect of osmotic solutions on imbibitional chilling injury in lima beans IABLLQ. Effect of osmotic solutions on water uptake, damages axes and cotyledons, number of normal embryos and germination of lime beans in pots Osmotic Hater Uptake Normal Germination solutions (% of original embryos in pots (M23) sggg ygjght) 1%1. 1%) o 6.11“ 34.4“ 4.4“ -0.5 2.80“ 46.7“ 20.0“ -1.5 2.00“ 47.8“ 17.8“ PEG osmotic solutions of -O.5 and -1.5 MPa reduced water uptake and increased both the number of normal embryos and germination percent in pots. Generally germination percent of the lima beans in pots was very poor. The primary solutions did not, however, have any effect on the number of damaged axes and cotyledons. 79 IABLLZ. Effect of temperature on water uptake, the number of damaged axes and cotyledons, number of normal embryos and germination of lima beans in pots Hater uptake Number of Number of Normal Germination Temperature (% original damaged damaged embryos in pots 5 3.51“ 3.22“ 3.0“ 34.4“ 0.00“ 10 3.01“ 3.22“ 2.0“ 25.6“ 0.00“ 20 4.40“ 1.22“ 1.1“ 68.9“ 42.20“ 5 and 10C reduced water uptake, the number of normal embryos and resulted in no germination in pots; these temperatures also increased the number of damaged cotyledons and axes. Priming solutions of -0.5 and -1.5 MPa only increased axes germination and cotyledon weight increase in the growth assay. 1115421551911 The poor and erratic germination obtained from work with lima beans probably indicates that the batch of seeds used in this experiment was very poor quality and, therefore, these results cannot be interpreted properly. GRN STATE UNIV. LIBRARIE mIlIIIII1I 2I||I1111II11II|II||1I|II7|lI|IIII|I1III|