:wmmmmmmmrma meals 310713 0647 LIBRARY Michigan Sn. UM ' This is to certify that the thesis entitled CHILLING SENSITIVITY 0F PREGERMINATED PEPPER SEED presented by CHRISTINE CAYER IRWIN has been accepted towards fulfillment of the requirements for MASTER'S , HORTICULTURE ._____degree in Mdor professor Date 11/20/79 0-7839 ovmnm: FINES ARE 25¢ PER DAY . PER ITEM Return to book drop to remove this checkout from your record. CHIILING SENSITIVTI‘Y OF PREGWA’IED PEPPER SEED By Christine Cayer Irwin A THESIS Submitted to Michigan State University in partial fUlfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1979 ABSTRACT CHILLING SENSITIVITY OF PREGERMINATED PEPPER SEED I. Effect of Low Temperature on Emergence II. Effect of Low Temperature, Potassium, Calcium and EDTA on Solute Leakage By Christine Cayer Irwin Section I The effect of storage of pregerminated pepper seeds (Capsicum EEEEEE;L- cv. Hungarian wax) at 0°C and 5°C for 2 to 21 days on total percent emergence, time to 50% emergence (T50), and viability was examined. An emergence assay at 25°/20°C showed that only dry seed and seed held at 0°C fer 4 days were slower in emerging than fresh pregerminated seed. Assays at 15°/10°C showed that all treatments but 50C for H days resulted in slower seedling emergence. Percent emergence data and tetrazolium.chloride assay results supported the T50 data. Section II Storage of pregermdnated pepper seed (Capsicum annuum L. cv. Calwonder) at 5°C fer 4 days did not result in increased solute leakage. Exposure to 0°C fer the same period, however, resulted in rapid leakage of solutes into distilled water. Calcium.chloride in the incubation solution reduced the level of leakage to that of fresh, unchilled seed. Potassium chloride did not reduce leakage, while EDTA increased leakage. to my mother and father for their support and encouragement ACMOWMEN’I‘S I would like to acknowledge the assistance and direction of Drs. Robert Herner, Stanley Ries and Lawrence Copeland, and to express my appreciation to them for serving as committee members. Special acknowledgment goes to Dr. Hugh C. Price, who, in his role as major professor, was patient, always good-natured (even at 6 arm. on the way to Sodus), and full of suggestions, yet very open to mine. iii TABLE OF CONTENTS LISTOFTABLES LISTOFFIGURES SECTIONI EFFECT OF LOW TEMPERATURE 0N EIVIERGENCE Introduction...................... MaterialsandMethods Results........................ Discussion....................... Literature Cited . . . . . . . . . . . . . . . . . . . . SECTIONII m0? OF LOW TEMPERATURE, POTASSIUM, CALCIUM AND EDI'A ON SOLUTE LEAKAGE Introduction...................... MaterialsandMethods Results........................ Discussion....................... Literature Cited . . . . . . . . . . . . . . . . . . . . APPENDICES Appendix Al - Chilling Sensitivity of Various Radicle Lengths of Pregerminated Pepper Seeds . . Appendix A2 — The Emergence of Chilled and Unchilled Pregenninated Seed and Dry Pepper Seed in Field Soil and Greenhouse Soil Mix. . . . iv Page vii Ill 20 21 22 29 33 3’4 35 36 39 LII ‘43 51 Page Appendix A3 -- Determination of the Optimum Tetrazolium Chloride ('I'I‘C) Concentration and Duration of the TTC Soak for Tetrazolium Testing of Pregerminated Pepper Seed . . . . . . . . . 57 Appendix All - The Effect of 'Priming' with Polyethylene Glycol on Germination of Pepper Seeds . . . 62 Table Al—l. A2-l. A3—l. Ali-1 . LIST OF TABLES SECTION II Absorbance of incubating solutions after treatment of chilled pregenminated pepper seed with water, CaClg, KCl and EDTA. Fresh pregerminated seed are included as a control. Delta absorbance is a comparison of all treatments to the chilled seeds incubated in water. Absorbance values at 3 and 7.5 hours were analyzed fer statistical significance. . . . . . . . . . . . . . . . APPENDIX Percent emergence of Staddon's Select pepper seeds germinated for A to 7 days, exposed to 0°C for 2 days, then grown at 25°/20°C or 15°/10°C. . . . . . . . . . . The effect of chilling and soil type on the time to 50% emergence (T50) of pepper seedlings . . . . . . . . The effect of'ITC concentration and number of hours seeds were soaked in the ITC solution on absorbance (1480 nm) of formazan in 2-methoxyethanol extracts . . . Effect of number of days in a PEG 6000 solution with an osmotic potential of -ll.5 bars on percent emergence of A cultivars of pepper seeds . . . . . . . . . . . . Page 36 A14 55 58 63 Figure Al—l. Al.2 o A2-l. LIST OF FIGURES SECTION I Absorbance (A80 nm) of’2-methoxyethanol extracts from.tetrazolium1chloride treated fresh pregerminated pepper seeds (unstored) and seeds held at 0°C and 5°C fer 2 to 21 days. . . . . . . . . . . . . . . . . . . The relationship between days at 5°C and absorbance (A80 nm) of 2amethoxyethanol extracts from tetrazolium chloride treated pregerminated pepper seeds 0 O O I O O O O O C C O C C O O O O O O O O 0 Effect of days at 0°C (A) and 5°C (B) on time to 50% emergence (T50). Emergence assays were conducted at 25°/20°C and ISO/10°C. Dry seed was significantly slower in emerging than fresh (unstored) pregerminated seed under both assay temperatures . . . . . . . . . Effect of days at 0°C and 5°C on percent emergence. Emergence assays were conducted at 25°/20°C and lSO/lOOC O O O C O O C O O C C O O O C O O O C O O 0 SECTION II Solute leakage, measured as absorbance at 262 nm, from pregerminated pepper seeds stored at 0°C or 5°C for A days, or not stored. Measurements were taken over a 9.5 hour period. The fresh (unstored) and 5°C treatments are not significantly different . . . APPENDICES Effect of exposing pepper seeds germinated fpr A to 7 days to 0°C for 2 days (A) and A days (B) on time to 50% emergence. (A) represents the average T50 values from.the 250/200C and 15°/lO°C growth chambers. (B) represents T50 data from the 25°/20°C chamber only. Effect of exposing pepper seeds germinated for A to 7 days to 0°C fer A days on percent emergence. values represented are the average of values from the 250/2000 growth chamber and the ISO/10°C growth chamber . . . The effect of chilling and soil type on the percent emergence of pepper seedlings . . . . . . . . . . . . vii Page 2A 26 28 31 38 A6 A8 5A Figure Page AA-l. Effect of number of days in a PEG 6000 solution of -ll. 5 bars on the time to 50% emergence (T50) of A cultivars of pepper seeds. . . . . . . . . . . . 65 viii Readers: The paper format which was utilized in this thesis neets the requirements stipulated by the Horticulture Department and the University. The thesis body was separated into two sections. Both Section I and Section II were prepared for the Journal of the American Society for Horticultural Science, and follow the manuscript style of that Journal. LITERATURE REVIEW LITERATURE REVIEW Seed germination is often a slow and sporadic process, resulting in non-uniform crop stands. Much effort has been directed towards improving the speed and uniformity of germination with the use of various seed pretreatments. "Hardening" involves cycles of wetting and drying the seed prior to planting. Kanchan and Jayachandra (197A) found that 3 cycles of hardening by soaking in ascorbic acid for 3 hours and drying for 21 hours resulted in quicker germination of pepper seeds. A—As-Saqui and Corleto (1978) improved both the percentage of emergence and the speed of emergence of A forage species by soaking the seed for 2A hours in water, then drying at room temperature. Damage to the seed results, however, if it is soaked for too long a time prior to drying. Heydecker and Coolbear (1977) reported no damage to wheat embryos if dried back after 2A hours of inhibition, but drying after 72 hours of inhibition damaged newly synthesized RNA and no recovery occurred. Polyethylene glycol (PEG) has been used as an osmotic agent, both for simulating water stress and as a 'priming' agent for seeds. Heydecker, Higgins and Gulliver (197A) reported quicker and more uniform seedling emergence when seeds were pretreated, or 'primed' , with a PEG solution. Heydecker and Hendy (1975) reported no effect on percent germination (in general), but a large increase in speed of germination with PEG 'priming'. Salter and Derby (1976) found celery seed also benefitted from 'priming'. The concentration of the PEG solution must be high enough to prevent complete germination, but allow sufficient water absorption for initiation of germination processes. 1 2 PEG is an inert, large molecule, which does not react with plant material to any appreciable degree, as other osmotic agents such as mannitol. The germination-prompting properties of potassium nitrate (KNO3) have resulted in its use in routine germination testing of many creps (COpeIand, 1976). Ells (1963) found that treating tomato seed with KNO3 in combination with K3POL¢ stimulated germination when seeds were exposed to a 10°C night temperature. More recently, work by Rumpel and Szudyga (1978) has shown a reduced time to germination at 8, 12 and 15°C when tomato seed was presoaked in a PEG solution or a solution of KNO3 + K3P0u. Larger increases in speed and uniformity of emergence than those obtained with seed pretreatments have been seen with the use of pregerminated seed. Much work in England by Gray (197A), Currah, Gray and Thomas (197A) and Bussell and Gray (1976) has shown that sowing pregerminated seed in a fluid gel can increase the speed of emergence and reduce the spread of emergence of many crops. In the U.S. , Taylor (1977) and others have reported similar benefits of pregermination. The advantages of using pregerminated seeds are particularly evident when soil temperatures are suboptimal (in the case of early direct- seeding of warm season vegetables) or supraoptimal (eg. celery transplant production in Florida) for germination of dry seed. Research into fluid-drilling raised the question of how to handle the pregerminated seed in the event of delayed planting, and how to slow its growth until planting could take place. Low temperature storage has long been used for dry seed, nursery root stock and some fresh produce, since it effectively slows metabolic processes. The applicability and limits of low temperature storage for use with 3 pregerminated pepper seeds was therefore examined in this research. Overexposure of plant material of tropical or subtropical origin to low temperature can result in chilling injury. This differs from freezing injury, and occurs in the range of 0 to 15°C. Symptoms of chilling injury manifest themselves in many ways and all plant parts are susceptible. Inhibitional chilling injury can affect not only seed germination, but subsequent growth as well, and can occur very quickly. Pollock and Toole (1966) found that lima beans were most severely injured when chilling occurred during the first 10 minutes of inhibition. Chilling reduced seedling survival and seedling size. Harrington and Kihara (1960) showed with pepper and musknelon seeds that exposure to 0°C and 5°C during inhibition resulted in collapse and necrosis of tissue behind the radicle. Occurrence of a stelar lesion during inhibitional chilling of a corn strain has been reported by Cohn and Obendorf (1978). Soybeans are also very sensitive to low temperature inhibition. Germination and elongation of embryonic axes were reduced when soybean seeds were imbibed at 5°C (Bramlage, Leopold and Parrish, 1978). Extensive work by Christiansen (1963) with cotton showed that exposure to 5°C resulted in aborted primary roots in 60% of the seedlings examined, with subsequent development of lateral roots. The abnormal root tip failed to stain with tetrazolium chloride. Later studies (196A) showed that early chilling results in a developmental lag, though after some time, development may assume a normal rate. Christiansen also found (1967) there to be two periods of chilling sensitivity in cotton seed - the first during the initial few hours of inhibition, and the second after 18 to 30 hours of inhibition. Along with Thomas, Christiansen (1969) reinforced some L} earlier findings and showed that chilling cotton seed during germination reduced plant height, delayed fruiting and reduced fiber quality in a direct relation to the length of exposure to cold. ‘Wiles and Downs (1977) used the term.'nub root' to describe the short, blunt root tip which results from.chilling cotton seed, a condition earlier Observed by Christiansen (1963). Chilling can have many complex effects on the metabolism of chilling-sensitive tissue. Lewis (1956) fpund that protoplasmic streaming ceased after short exposure to about 11°C in Cucurbita pepo and Lycopersicon esculentum.(chillingrsensitive), whereas streaming proceeded in chilling-resistant radish (Raphanus sativus) and carrot (Daucus carota) at temperatures as low as 0°C. Chilling-sensitive plants also differ fromichilling—resistant plants in their respiratory response to chilling. Lyons and Raison (1970) reported that the Arrhenius plots of the respiration rate of mitochondria isolated from Chillingeresistant tissue (cauliflower buds, potato tubers and beet roots) showed a linear decrease over a temperature range of 25°C to l.5°C. The respiration rates of chillingysensitive tissues (tomato and cucumber fruit, and sweet potato roots), on the other hand, showed a linear decrease from 25°C to about 9 to 12°C, at which point there was a sharp increase in the slope of the line as temperatures were drOpped to l.5°C. This 'break' in the line of the Arrhenius plot is believed to represent a phase change from fluid to gel in the membrane phospholipids (Kumamoto, Raison and Lyons, 1971). Simon (197A) in a review of the subject states that at the temperature at which the break occurs, the phospholipids can exist in either of two alternative states, whereas at the other temperatures, they can only exist in one state. 5 The explanation for the differing respiration responses of chilling— sensitive and chillingeresistant plants lies in the degree of unsaturation of the membrane fatty acids. -Lyons, Wheaten and Pratt (196A) reported that chillingeresistant plants have more unsaturated fats in the mitochondrial membranes than do chillingesensitive plants. Therefore, the mitochondrial.membranes of chillingeresistant plants have the capacity to swell at low temperatures, whereas those of chillingesensitive plants do not have such a capacity. Dogras, Dilley and Herner (1977) found that peas and broad beans (both chillinge resistant) imbibed at 10°C incorporated a larger percentage of labelled glycerol into phosphatidylcholine, which is highly unsaturated, than did lima beans (chillingesensitive). Lima beans incorporated.more glycerol into less unsaturated phosphatidylethanolamine and phosphatidylglycerol. The amounts of total phospholipids and the degree of unsaturation has been found to change in some plants in relation to temperature. Gerloff, Richardson and Stahmann (1966) found an increase in both total fatty acids and the degree of unsaturation in alfalfa roots during cold hardening. Grenier and Willemot (197A) reported similar results with alfalfa roots, and noted that coldphardy cultivars showed.greater increases than did less cold-hardy cultivars. DeLaRoche, Andrews and Pomeroy (1972) reported similar increases in the degree of unsaturation in winter wheat seedlings grown at 2°C, but did not find sudh increases in seedlings grown at 2A°C. These changes, however, are gradual, and for the most part limited to tissues which become cold-hardened in preparation for overwintering. The inability of membranes of chillingrsensitive plants to withstand low temperatures results in many metabolic disturbances. Processes catalyzed by 6 membraneebound enzymes show an increase in activation energy. Raison, Lyons and Thomson (1971) observed this increased activation energy in the mitochondrial succinate oxidase system.of chilling-sensitive plants when exposed to temperatures below 9°C. Raison (1973) later showed this to hold true fpr other enzyme systems. Shneyour, Raison and Smillie (1973) reported a sharp increase in the activation energy needed for photoreduction of NADP+ when tomato plants were exposed to temperatures below 11°C. In lettuce, a chillingeresistant plant, the activation energy remained constant under the same temperatures. Later work by Raison and Chapman (1976) witijEE a radiata substantiated earlier work with other chillingesensitive plants. Towers, et. al. (1973) showed that'membraneebound ribosomes are also adversely affected by low temperature, resulting in decreased protein synthesis. Arrhenius plots of the protein.synthetic activity of membraneebound ribosomes displayed characteristic 'breaks', whereas Arrhenius plots for protein synthesis by free ribosomes were linear. As a result of the increased activation energy of many enzymatic reactions, some substances may accumulate within the tissue. Murata (1969), in chilling studies with green bananas, found that the acetaldehyde , (X —keto acid and ethanol contents of both peels and fruits increased with the onset of chilling injury. Polyphenols can also accumulate, as shown by Liebermann et. al. (1958). The accumulation of polyphenols is believed to be due, in part, to the destruction of ascorbic acid (ASA) at low temperatures. Miller and Heilman.(l952) showed that chilling reduces the ASA content of pineapple fruits. Storage of pepper fruits at 1°C caused a large decrease in the ASA content of the pepper seeds, as Observed.by Yamauchi, Inaba and Ogata (1978). Simon (197A) indicates that such 7 'imbalances' of metabolismimay be responsible for the appearance of some chilling injury symptoms, such as the appearance of browning with phenol accumulation. In addition, as a result of the disrupted respiratory pathway, large decreases in the ATP content of the tissue may ensue, as seen by Stewart and Guinn (1969) and Wilson (1978). Another serious consequence of the phospholipid phase change incurred at chilling temperatures is increased membrane permeability. This can be determined by measuring leaked solutes, either spectrophotometrically or with the use of a conductivity bridge. Increased solute leakage has been shown to be a.symptom of chilling injury in seeds, leaf and fruit tissue. Christiansen, Carns and Slyter (1970) reported.large steady increases in solute leakage from radicles of cotton seeds exposed to 5°C. Guinn (1971) fpund cotton cotyledons to also suffer increased solute leakage when exposed to 5°C for 3 hours of'more. Chilling greatly increased the amounts of reducing sugars, ninhydrin—positive material, and ionic material which subsequently leaked fromlthe cotyledons. Powell (1969) measured the resistance of the solution surrounding chilled and unchilled cotton leaf discs, and found increased conductivity after chilling. Bramlage, Leopold and Parrish (1978) found that dry soybeans, placed in water, leak solutes profusely at first, but leakage quickly subsides to a much slower rate as the membranes reorganize. In chilled cotyledons, however, the decline in leakage takes considerably longer to occur. Nflnchin and Simon (1973) found increased leakage of water and electrolytes from cucumber leaf discs, resulting in wilting. Some later work by Tanczos (1977) with cucumber leaf discs showed.no increase in conductivity with 3 days or less at 2°C, but longer exposure 8 resulted in increased conductivity. Phaseolus vulfls leaves also suffer increased solute leakage with chilling, as observed by Wright (197A). Lewis and Workman, in some early work (196A) showed that exposure of mature green tomato fruits to 0°C for A weeks caused a 3-fold increase in cell membrane permeability, but had no effect on the permeability of cabbage leaves. Recently, Tatsumi and Murata (1978) found that Arrhenius plots of ion leakage from cucumber and pepper fruits showed a dramatic increase in leakage around 7 to 10°C, indicated by a change in s10pe, but a straight line response from potato tubers was observed. Some other recent work (1978) by Fukushima and Yamazaki revealed that phospholipid phase changes are not the only factors involved in increased membrane permeability with chilling. They found in cucumbers, bananas and sweet potato roots that chilling results in an increase in hot water insoluble pectin and a decrease in hot water soluble pectin, contributing to increased cell wall rigidity. The significance of solute leakage was recogiized as early as 1928, when Hibbardand Miller reported that leakage and percent seed germination showed a negative correlation. Matthews and Bradnock (1967 , 1968) showed a sigiificant negative correlation between solute leakage and field emergence of peas and French beans, and believe that a measurement of seed exudation is a better indicator of seed vigor than standard laboratory germination tests. If chilling injury has not been too severe, it can be reversed by subsequent exposure to warmer temperatures. Work by Brand, Kirchanski and Ramirez-Mitchell (1979) has recently shown with a blue-green alga, Anacystis nidulans, that cells grown at 25°C, then exposed to 0°C for 30 minutes underwent a phase separation which was totally reversed by 9 re-exposing the cells to 25°C. However, growth at 39°C prior to exposure to 0°C resulted in considerably more morphological alterations than.the 25°C growth temperature, and subsequent exposure to 39°C resulted in only partial reversal of the phase separation. Ibanez (1963), working with cacao seed, showed that if seeds were exposed to A°C for 10 minutes or less, cold inhibition of germination could be reversed by exposure to 37°C, but longer exposure to A°C resulted in irreversible injury. Creencia and Bramlage (1971) observed warm temperature reversal of chilling injury incurred during short term exposure to O.3°C in corn seedlings. A decrease in solute leakage from cucumber leaf discs exposed to 2°C for A days or less was noted by Tanczos (1977) when the discs were transferred to 25°C. Five days or more, however, did not allow for recovery. Cold hardening refers to the gradual process which plants undergo in preparation for winter. Wheaton and Morris (1967) succeeded in applying the same principles to tomato plants and reduced their chilling sensitivity by exposing the plants to l2.5°C, slightly above the chilling range for tomatoes, for A8 hours. Higher temperatures provided less protection than did l2.5°C. various treatments have been used, both to prevent chilling injury and to reduce the symptoms, with varying degrees of success. Pollock and Toole (1966) found they could reduce imbibitional chilling injury in lima beans by imbibing them for a brief period at 2500, prior to continued imbibition at 5°C. Miller and Corns (1957) reported increased low temperature resistance in sugar beet seedlings treated with Dalapon or trichloroacetic acid. Ilker, et. al. (1976) found ethanolamine lended some increased low temperature resistance to tomato seedlings, 10 perhaps by lowering the temperature of the phospholipid phase transition. Bartowski., Katterman and Buxton (1978) were able to increase the germination of some cotton cultivars at lA°C by applying exogenous fatty acids, but failed to increase the germination of all cultivars tested. Another approach has been taken by Christiansen and Ashworth (1978), who observed a reduction in chilling injury of cotton seedlings by placing plants in plastic bags to elevate humidity or by the use of antitranspirants. Since water uptake by roots of cotton and other tropical species is considerably restricted at temperatures below 15°C, reducing transpiration reduces foliar necrosis and other injury symptoms associated with water loss. Spraying cucumber seedlings with abscisic acid (ABA) was shown by Rikin and Richmond (1976) to reduce chilling injury. They found in other work (1976) that subjecting the seedlings to water stress, which resulted in an elevated ABA content, had the same effect as an ABA spray application. The role of ABA in reducing water loss was suggested as the mechanism of ABA—induced chilling resistance. Calcium has long been lmown to be an integral part of plant cell walls, and Marinos (1962) showed in some submicroscopic studies of calcium deficient barley, that lack of calcium results in membrane disorganization. With this information in mind, several researchers have utilized Ca to reduce or prevent leakage resulting from membrane damage. Van Steveninck (1965) found he could reverse EDI‘A-induced leakage from beet root tissue with equivalent amounts of CaClz. He observed reduced leakage to some degree with several other divalent cations, but Ca was most effective. Sucrose leakage from corn scutellum slices was reduced with as low as .OOJM Ca by Garrard and Humphreys 11 (1967). Using artificial membranes, Gary-Bobo (1970) showed that CaCl2 decreases pore size, fUrther evidence of calcium's role in controlling membrane permeability. Christiansen, Cams and Slyter (1970) found that Ca was effective in reducing chill-induced membrane leakage from cotton radicles. They suggested that part of calciumfis function in membrane stabilization may be in influencing cell surface charge. Poovaiah and Leopold (1976) reduced leakage fromibeet root tissue and Rumex Obtusifolius leaf discs with CaC12 solutions. Most recently, Poovaiah (1979) has been successful in preventing ethephon—induced membrane»leakage fromibeet root tissue with CaClZ. Other divalent cations were somewhat less effective, and monovalent cations were not effective. Chilling injury is a time-temperature response, ie. the lower the temperature or the longer the period of exposure, the greater the injury. Christiansen (1968) points this out in some of'his work with imbibing cotton seed. Chilling incurred in the field may be cumulative with chilling incurred in storage, as may occur with produce going into post-harvest storage. The reverse situation could be the case as well, with transplants stored prior to field planting. ‘With pregerminated seed, this is also a consideration if it is to be stored prior to planting. Other stresses may accentuate injury caused by low temperature exposure. Soil compaction has been shown to adversely affect emergence and growth of many crops, including calabrese (Hegarty and Royle, 1976), barley (Wilson.and Rebardo, 1977) and pepper (Fawusi, 1978), and combined with chilling, could produce more injury than either stress alone. The need for accurate assessment of the viability and vigor of 12 seeds has resulted in the development of several types of tests. The biochemical test most widely used today, according to Copeland (1976) is the tetrazoliumrchloride (TTC) test for seed vigor and viability. In contact with actively respiring tissue, the tetrazolium salt forms a red, water insoluble substance, formazan. Nonviable tissue does not stain. Kittock and Law (1968) found with wheat seeds that vigor, TTC reduction and rate of respiration all were positively correlated. However, Lobanov (1967) warns that chemical seed viability determinations of spring crops are only approximations and cannot be used for determining sowing rates. Heydecker (1973) contends this may be because such tests as the TTC test reflect only a measurement of seed condition and.not the interaction between seed and environment that occurs in the field. Germination tests are also routinely used for assessing seed viability. Standard germination tests are carried out under Optimpmlconditions, and results are often not as closely correlated with field performance as are results of the cold test (Johnson and wax, 1978), or other stress-imposing tests. The cold test involves germinating the seeds in cool, moist soil, then moving them to warmer conditions. This test more accurately reflects the seed/ environment interaction which Heydecker (1973) discusses than either the standard germination test or the TTC test. In light of all this, the research for this thesis was undertaken with the following Objectives: 1. assessment of the performance of pregerminated pepper seed vs. dry seed under various conditions. 2. determination of the chilling sensitivity of pregerminated pepper seed, and the limits of cold tolerance. 13 application of such knowledge to development of a cold storage technique for pregerminated pepper seed. application of such knowledge to a better understanding of what happens with early sowings of pregerminated pepper seed. definition of some of the symptoms of chilling injury in pregerminated pepper seed. examination of the effectiveness of some preventative and remedial measures for reducing chilling injury. assessment of the value of the'TTC test and modifications of other vigor tests for use with pregerminated pepper seed. BIBLIOGRAPHY BIBLIOGRAPHY A-As-Saqui, M. and A. Corleto. 1978. Effect of seed presowing hardening on seedling emergence of four forage species. Seed Sci. and Tech. 6:701—709. Bartowski, E.J., F. Katterman and D.R. Buxton. 1978. Influence of exogenous fatty acids on cotton seed germination. Physiol. Plant. Bramlage, W.J., A.C. Le0pold, and O.J. Parrish. 1978. Chilling stress to soybeans during imbibition. Plant Physiol. 61:525-529. Brand, J .J ., S.J. Kirchanski and R. Ramirez-Mitchell. 1979. Chill- induced morphological alterations in Anac stis nidulans as a function of growth temperature. Planta 1A5:63—68. Bussell, W.T. and D. Gray. 1976. Effects of pre-sowing seed treatments and temperatures on tomato seed germination and seedling emergence. Scientia Hort . 5 : 101-109 . Christiansen, M.N. 1963. Influence of chilling upon seedling development of cotton. Plant Physiol. 38:520-522. Christiansen, M.N. 196A. Influence of chilling upon subsequent growth and morphology of cotton seedlings. Crop Sci. A:58A-586. Christiansen, M.N. 1967. Periods of sensitivity to chilling in germinating cotton. Plant Physiol. A2:A31-A33. Christiansen, M.N. 1968. Induction and prevention of chilling injury to radicle tips of imbibing cotton seed. Plant Physiol. A3:7A3—7A6. Christiansen, M.N. and E.N. Ashworth. 1978. Prevention of chilling injury to seedling cotton with anti-transpirants. Crop Sci. 18: 907-908. Christiansen, M.N., H.R. Cams and D.L. Slyter. 1970. Stimulation of solute loss from radicles of Gossypium hirsutum L. by chilling, anaerobiosis, and low pH. Plant Physiol. A6:53-56. Christiansen, M.N. and R.O. Thomas. 1969. Season-long effects of chilling treatments applied to germinating cotton seed. Crop Sci. 9:672-673. - Cohn, M.A. and R.L. Obendorf. 1978. Occurrence of a stelar lesion during imbibitional chilling of _Z_ea_1_ gays L. Amer. J. Bot. 65:50-56. lA 15 Copeland, L.O. 1976. Principles of seed science and technology. Burgess Publ. 00., Minneapolis, Minn. Creencia, R.P. and W.J. Bramlage. 1971. Reversibility of chilling injury to corn seedlings. Plant Physiol. A7:389-392. Currah, I., D. Gray and T.H. Thomas. 197A. The sowing of germinating vegetable seeds using a fluid drill. Ann. Appl. Biol. 76:311-318. DeLaRoche, I. , O.J. Andrews and M.K. Pomeroy. 1972. Lipid changes in winter wheat seedlings at temperatures inducing cold hardiness. Can. J. Bot. 50:2A01-2AO9. Dogras, C.C., D.R. Dilley and R.C. Herner. 1977. Phospholipid biosynthesis and fatty acid content in relation to chilling injury during germination of seeds. Plant Physiol. 60:897-902. Ells, J. 1963. The influence of treating tomato seed with nutrient solutions on emergence rate and seedling growth. Proc. Amer. Soc. Hort. Sci. 83:68A-687. Fawusi, M.O. 1978. Emergence and seedling growth of pepper as influenced by soil compaction, nutrient status and moisture regime. Scientia Hort . 9: 329-335 . Fukushima, T. and M. Yamazaki. 1978. Chilling-injury in cucumbers. V. Polysaccharide changes in cell walls. Scientia Hort. 8:219-227. Garrard, L.A. and T.E. Humphreys. 1967. The effect of divalent cations on the leakage of sucrose from corn scutellum slices. Phytochem. 6: 1085-1095 . Gary-Bobo, C.M. 1970. Effect of Ca on the water and non-electrolyte permeability of phospholipid membranes. Nature 228:1101-1102. Gerloff, E.D., T. Richardson and M.A. Stahmann. 1966. Changes in fatty acids of alfalfa roots during cold hardening. Plant Physiol. A1: Gray, D. 197A. Some developments in the establishment of drilled vegetable crops. )CLXth Int'l. Hort. Congress. Grenier, G. and C. Willemot. 197A. Lipid changes in roots of frost hardy and less hardy alfalfa varieties under hardening conditions. Cryobiol. 11:32A-33l. Guinn, G. 1971. Chilling injury in cotton seedlings: changes in permeability of cotyledons. Crop Sci. 11:101-102. Harrington, J.F. and G. M. Kihara. 1960. Chilling injury of germinating muskmelon and pepper seed. Proc. Amer. Soc. Hort. Sci. 75:A85-A89. Hegarty, T.W. and S.M. Royle. 1976. Impedance of calabrese seedling emergence from ligmt soils after rainfall. Hort. Res. 16:107-11A. l6 Heydecker, W. (ed). 1973. Seed ecology - proceedings of the 19th Easter School in Agricultural Science, Univ. of Nottingham, 1972. The Pennsylvania State Univ. Press, Univ. Park, Penn. Heydecker, W. and P. Coolbear. 1977. Seed treatments for improved performance - survey and attempted progiosis. Seed Sci. and Tech. 5:353—A25. Heydecker, W. and A. Hendy. 1975. Pre-treating bedding plant seed for 'instant' germination. Comm. Grower Oct. 1975. Heydecker, W. , J. Higgins and R. Gulliver. 197A. Instant germination - a method of brinkmanship. Comm. Grower Jan. 197A. Hibbard, R.P. and E.V. Miller. 1928. Biochemical studies on seed ' viability. I. Measurements of conductance and reduction. Plant Physiol . 3: 335-352 . Ibanez, M.L. 1963. A reversal of cacao seed sensitivity to cold. Turrialba 13:31-32. Ilker, R., A.J. Waring, J.M. Lyons and R.W. Breidenbach. 1976. The cytological responses of tomato-seedling cotyledons to chilling and the influence of membrane modifications upon these responses. Protoplasma 99:229-252. Johnson, R.R. and L.M. Wax. 1978. Relationship of soybean germination and vigor tests to field performance. Agron. J. 70:273-278. Kanchan, S.D. and Jayachandra. 197A. Influence of light and temperature on seed hardening in sweet pepper. Curr. Sci. A3:520-521. Kittock, D.L. and A.C. Law. 1968. Relationship of seedling vigor to respiration and tetrazolium chloride reduction by germinating wheat . Agron. J. 60:286-288. Kunamoto, J., J.K. Raison and J.M. Lyons. 1971. Temperature 'breaks' in Arrhenius plots: a thermodynamic consequence of a phase change. J. Theor. Biol. 31:A7-51. Lewis, D.A. 1956. Protoplasmic streaming in plants sensitive and insensitive to chilling temperatures. Science l2A:75-76. Iewis, T.L. and M. Workman. 196A. The effect of low temperature on phosphate esterification and cell membrane permeability in tomato fruit and cabbage leaf tissue. Aust. J. Biol. Sci. 17:1A7-152. Liebermann, M., C.C. Craft, W.V. Audia and M.S. Wilcox. 1958. Biochemical studies of chilling injury in sweet potatoes. Plant Physiol. 33: 307-311. Lobanov, V.Y. 1967. Quality determination of seeds. Israel Program for Scientific Translations. l7 Lyons, J.M. and J.K. Raison. 1970. Oxidative activity of mitochondria isolated from plant tissues sensitive and resistant to chilling injury. Plant Physiol. A5:386-389. Lyons, J.M., T.A. Wheaton and H.K. Pratt. 196A. Relationship between the physical nature of mitochondrial membranes and chilling sensitivity in plants. Plant Physiol. 39:262-268. Marinos, N.G. 1962. Studies on submicroscopic aspects of mineral deficiencies. I. Calcium deficiency in the shoot apex of barley. Amer. J. Bot. A9:83A—8Al. Matthews, 8. and W. Bradnock. 1967. The detection of seed.samples of wrinkle-seeded peas (Pisum sativum L.) of potentially low planting value. Proc. Intl. Seed Test. Assoc. 32:553-563. Matthews, S. and W. Bradnock. 1968. Relationship between seed exudation and field emergence in peas and French beans. Hort. Res. 8:89-93. Miller, E.Vk and A.S. Heilman. 1952. Ascorbic acid and physiological breakdown in the fruits of the pineapple (Ananas comosus L. Merr.). Science 116:505-506. Miller, S.R. and W.G. Corns. 1957. The constitution of sugar beet seedlings associated with chemically induced improvement in their low temperature resistance. Can. J. Bot. 35:5-8. Minchin, A. and E.W} Simon. 1973. Chilling injury in cucumber leaves in relation to temperature. J. Exp. Bot. 2A:123l-1235. Murata, T. 1969. Physiological and biochemical studies of chilling injury in bananas. Physiol. Plant. 22:A01-All. Pollock, B.M. and'V.K. Toole. 1966. Imbibition period as the critical temperature sensitive stage in germination of lima bean seed. Plant Physiol. Al:221-229. Poovaiah, B.W. 1979. Effects of inorganic cations on ethephon- induced increases in membrane permeability. J. Amer. Soc. Hort. Sci. lOA:l6A-l66. Poovaiah, B.W. and A.C. Leopold. 1976. Effects of inorganic salts on tissue permeability. Plant Physiol. 58:182—185. Powell, R.D. 1969. Permeability changes in leaf discs as affected by low temperatures. Plant Physiol. Suppl. AA:16. Raison, J.K. 1973. The influence of temperature-induced phase changes on the kinetics of respiratory and other membrane-associated enzyme systems. Bioenergetics A:285-309. Raison, J.K. and E.A. Chapman. 1976. Membrane phase changes in chillingesensitive'yigg§_radiata and their significance to growth. 18 Raison, J.K., J.M. Lyons and W.W. Thomson. 1971. The influence of membranes on the temperature-induced changes in the kinetics of some respiratory enzymes of’mitochondria. Arch. Biochem. Biophys. lA2:83-90. Rikin, A., A. Blunenfeld and A.E. Richmond. 1976. Chilling resistance as affected by stressing environments and abscisic acid. Bot. Gaz. 137:307-312. Rikin, A. and A.E. Richmond. 1976. Amelioration of chilling injuries in cucumber seedlings by abscisic acid. Physiol. Plant. 38:95-97. Rumpel, J. and I. Szudyga. 1978. The influence of pre—sowing seed treatments on germination and emergence of tomato 'New Yorker' at low temperatures. Scientia Hort. 9:119—125. Salter, P.J. and R.J. Darby. 1976. Synchronization of germination of celery seeds. Ann. Appl. Biol. 8A:A15-A2A. Shneyour, A., J.K. Raison and REM. Smillie. 1973. The effect of temperature on the rate of photosynthetic electron transfer in chloroplasts of chilling-sensitive and chilling-resistant plants. Biochim. Biophys. Acta 292:152-161. Simon, E.W. 197A. Phospholipids and plant membrane permeability. New Phytol. 73:377-A20. Stewart, J.M. and G. Guinn. 1969. Chilling injury and changes in adenosine triphosphate of cotton seedlings. Plant Physiol. AA:605- 608. Tanczos, O.G. 1977. Influence of chilling on electrolyte permeability, oxygen uptake and 2,A-dinitrophenol stimulated oxygen uptake in leaf discs of the thermophilic Cucumis sativus. Physiol. Plant. Al:289-292. Tatsumi, Y. and T. Murata. 1978. Studies on chilling injury of fruits and vegetables. Part 1. Chilling injury of cucumber fruits with special reference to permeability of'membrane of tissues. J. Jap. Soc. Hort. Sci. A7:105-110. Taylor, A.G. 1977. Comparative performance of pregerminated, high moisture content and dry vegetable seed in greenhouse and field studies. J. Seed Tech. 2. Towers, N.R., G.M. Kellerman, J.K. Raison and A.W. Linnane. 1973. Effects of temperature-induced phase changes in membranes on protein synthesis by mitochondria. Biochim. BiOphys. Acta 299:153-161. 'Van Steveninck, R.F. 1965. The significance of calcium on the apparent permeability of cell membranes and effects of substitution with other divalent cations. Physiol. Plant. 18:5A-69. l9 Wheaton, T.A. and L.L. Morris. 1967. Medification of chilling sensitivity by temperature conditioning. Proc. Amer. Soc. Hort. Sci. 91:529-533. ‘Wiles, E.L. and R.J. Downs. 1977. Determination of chilling sensitive pegiods during the germination of cotton seed. Seed Sci. and Tech. 'Wilson, A.J. and A.W. Robardo. 1977. Effects of mechanical impedance on root growth in barley, Hordeum vulgare L. II. Effects on cell development in seminal roots. J. Exp. Bot. 28:1226-1227. Wilson, J.M. 1978. Leaf respiration.and ATP levels at chilling temperatures. New Phytol. 80:325-33A. wright, M. 197A. The effect of chilling on ethylene production, membrane permeability and water loss of leaves of Phaseolus vulgari . Planta 120:63-69. Yamauchi, N., M. Inaba and K. Ogata. 1978. Physiological and chemical studies on ascorbic acid of fruits and vegetables. IV. Effect of ascorbic acid on the metabolism of phenolic compound in the seed of sweet pepper fruit associated with the incidence of chilling injury. (Part 1). J. Japan. Soc. Hort. Sci. A7:273-281. SECTION I EFFECT OF LOW TEMPERATURE ON EMERGENCE Chilling Sensitivity of Pregerminated Pepper Seed I. Effect of Low Temperature on Emergence. Christine Cayer Irwin and Hugh C. Price1 Department of Horticulture Michigan State University East lensing, MI A882A Additional index words: Capsicum.annuum, tetrazolium chloride assay, cold storage Abstract. The effect of storage of pregerminated pepper seeds (Capsicum annuum L. cv. Hungarian wax) at 0°C and 5°C for 2 to 21 days on total percent emergence, time to 50% emergence (T50), and viability was examined. An emergence assay at 250/200C showed that only dry seed and seed held at 0°C for A days were slower in emerging than fresh pregerminated seed. Assays at 150/1000 showed that all treatments but 5°C for A days resulted in slower seedling emergence. Percent emergence data and tetrazolium chloride assay results supported the T50 data. Fluid-drilling is a direct-seeding technique utilizing seed pregerminated under controlled conditions. The seed is dispersed in a fluid gel just prior to planting. The gel acts as a carrier for the seed and minimizes the amount of mechanical damage during the planting process. The advantages of this technique over conventional direct- seeding techniques include quicker and.more uniform seedling emergence (A,ll), especially when temperatures are suboptimal for germination of dry seed. To develop a means of holding pregerminated seed in the event of delayed planting, low temperature storage was examined. For successful storage of pregerminated seed, the temperature must be low enough to temporarily halt radicle growth, but not so low as to cause chilling 1Graduate research assistant and ggofessor, respectively 21 injury. Elongated radicles are more subject to damage during planting. A further objective was to study the effect of low soil temperature on stored and unstored pregerminated pepper seed. Materials and Methods Pepper seeds, Capsicum m L. cv. Hungarian Wax, were germinated for 5 days in aerated glass water columns at room temperature. Radicles after 5 days were 2 to A m in length. Germinated seeds were removed from the columns and placed in cold storage wrapped in 2 thicknesses of moist cheesecloth 15 x 15 cm and loosely wrapped in polyethylene bags. Storage temperatures of 0°C and 5°C were used since preliminary experiments had shown that 10°C did not stop radicle elongation. Based on preliminary experimentation, seeds were stored at 0°C for 2 and A days, and 5°C for A, 8, l2 and 21 days. Dry seed and fresh pregerminated seed (unstored) were used as controls. Four replicates of 10 seeds/treatment were used for the tetrazolium chloride (TI‘C) assay for seed viability. Replicates were placed in glass test tubes, and covered with A ml of a 0.5% 2,3 ,5-triphenyl tetrazolium chloride solution. The tetrazolium salt had been dissolved in a .01 M phosphate buffer solution of pH 7.0. Reduction of the tetrazolium salt by actively respiring tissue results in the formation of water-insoluble formazan, which is red. Test tubes were kept at room temperature in darkness for 2A hours. The TC solution was decanted, the seeds rinsed with distilled water, and each replicate was then covered with A ml of 2-methoxyethanol for extraction of the formazan. After A hours, each sample extract was measured for absorbance at A80 nm in a Bausch & Iomb Spectronic 20. Tetrazolium chloride reduction has been shown to be 22 positively correlated with vigor (9). For the emergence assay, 8 replicates of 25 seeds/treatment were planted at a 1.5 cm depth in flats containing moistened vermiculite. Treatments were arranged in a completely randomized design. Four replicates were placed in a Percival environmental growth chamber with a 2500 1A hour day and 20°C 10 hour night, and the other A in an identical growth chamber with a 1500 1!: hour day and 10°C 10 hour night. Flats were subirrigated as needed, and daily emergence counts were taken. The experiment was terminated Al days after planting, and time to 50% emergence, an index of seedling vigor (10), and percent emergence were calculated for each treatment. Data were analyzed using analysis of variance and.LSD was used for mean separation. Results The tetrazolium.assay showed a significant reduction in formazan absorbance compared with fresh pregerminated seed after 2 days storage at 0°C, and an even greater reduction with A days at 0°C (Fig. 1). Examination of the seeds prior to color extraction showed that the tips of the radicles had been.injured by the 0°C treatments, indicated by the lack of formazan in the tips. Though the assay showed no significant reduction in viability with storage at 5°C, formazan absorbance and days at 5°C showed a significant negative correlation (Fig. 2). Emergence assay results obtained in the 25°/20°C growth chamber indicated that only dry seed, and pregerminated seed stored at 0°C for A days is slower in emergence than fresh seed, as seen by the higher T50 values (Fig. 3). Storage of seeds for 21 days at 5°C did not increase the T50 emergence. In the 15°/10°C assay, however, all Fig. l. 23 Absorbance (A80 nm) of 2-methoxyethanol extracts from tetrazolium.chloride treated fresh pregerminated pepper seeds (unstored) and seeds held at 0°C and 5°C for 2 to 21 days. (LSD.05 = 0.25) 2A H .wE uG<¢Ohm Z. m> cam-I u .— 00; (“"1087) I DNVEUOSGV Fig. 3. 27 Effect of days at 0°C (A) and 500 (B) on time to 50% emergence (T O). Emergence assays were conducted at 250/2000 and 150/1000. Dry seed was significantly slower in emerging than fresh (unstored) pregerminated seed under both assay temperatures. (LSD.05 = 1.89) 28 Pa booze. uon ._.< m> $00.".- h (SAVCI)°91 Q (SAVINGSL xmmélvoduua a .mmf ..:. O P < or A7 Figure 2. Effect of exposing pepper seeds germinated for A to 7 days to 00C for A days on percent emergence. values represented are the average of values from the 250/200C growth chamber and the lSO/lOOC growth chamber. PERCENT EMERGENCE A8 100 80 O 60 . . = - 94* 4o ' - y: 123.6 -10.05x 20 o \_L_l___|___l 4 5 6 7 NO. OF DAYS GERMINATED Figure 2 A9 considerably less than the percentage of any other treatment. An additional 2 days at 00C, fer a total of A days, however, resulted in number of days of germination and percent emergence having a high negative correlation (Figure 2) in both growth chambers. Discussion Early exposure to low temperature can adversely affect subsequent performance of pregerminated pepper seeds. Similar effects have been seen by Pollack and.Tbole (1966) with lima'beans and with cotton seed by Christiansen (196A), and Christiansen and Thomas (1969) after exposure to low temperature during imbibition. Storing pregerminated pepper seeds for A days at 00C resulted in slower emergence and a reduced stand. Exposure to 000 for 2 days did not appear to cause injury. It has been noted in other work by this researcher that severe chilling compounded with low growing temperatures can have such an adverse effect on the emergence of pepper seedlings that the effect of other variables is masked. This might explain the poor correlation of T50 values for seeds held at 0°C fer A days with radicle length at 150/1000. The greater the number of days pepper seeds have been germinated, the longer the radicle. When uninjured, the more advanced seeds with the longer radicles are the first ones up. The longer the radicle, however, the greater the amount of tissue susceptible to chilling injury, thus the negative correlation seen between radicle length.and speed and percent of emergence after A days at 00C. In view of this data, it appears that a short radicle is desirable, not only to minimize damage during planting, but to minimize the adverse effects of chilling during storage or in the field. BIBLIOGRAPHY BIBLIOGRAPHY Christiansen, M.N. 1963. Influence of chilling upon seedling development of cotton. Plant Physiol. 38:520—522. Christiansen, M.N. 196A. Influence of chilling upon subsequent growth and morphology of cotton seedlings. CrOp Sci. A:58A—586. Christiansen, M.N. and R.O. Thomas. 1969. Season-long effects of chilling treatments applied to germinating cotton seed. Crop Sci. 9 : 672-673. Harrington, J.F. and G.M. Kihara. 1960. Chilling injury of germinating muskmelon and pepper seed. Proc. Amer. Soc. Hort. Sci. 75:A85—A89. Orchard, T.J. 1977. Estimating the parameters of plant seedling emergence. Seed Sci. and Tech. 5:61-69. Pollock, B.M. and V.K. Toole. 1966. Imbibition period as the critical temperature sensitive stage in germination of lime bean seed. Plant Physiol. Al:221—229. Wiles, E.L. and R.J. Downs. 1977. Determination of chilling sensitive periods during the germination of cotton seed. Seed Sci. and Tech. 5:6A9-657. 50 APPENDIX A2 THE EMERGEN CE OF CHILLED AND UNCHILLED PREGEH‘HNATED SEED AND DRY PEPPER SEED IN FIELD SOIL AND GREENHOUSE SOILMIX The Emergence of Chilled and Unchilled Pregerminated Seed and Dry Pepper Seed in Field Soil and Greenhouse Soil Mix Poor seedling emergence can be a problem in field planting situations, and is often a result of soil compaction, crusting, chilling injury or various combinations of these. The objectives of this experiment were to assess the effects of soil type and chilling and the interaction of the two on the speed of emergence and percent emergence of pregerminated and dry pepper seed. Materials and Methods Capsicum annuum L. cv. Hungarian Wax pepper seeds were germinated for 5 days in aerated water columns at room temperature. Pregerminated seeds were removed from the columns and stored at 00C for 2 days, wrapped in moist cheesecloth. Fresh (unstored) pregerminated seed and dry seed were used for controls. Three replicates of 25 seeds per treatment were planted at a 1.5 cm depth in a standard greenhouse potting mix of peat and sand, and 3 replicates were planted at the same depth in Miami silt loam from the Horticultural Research Center, East Lansing. Flats were placed in a Scherer environmental growth chamber with a 1A hour 15°C day and 10 hour 120C night, and watered as necessary. Daily emergence counts were taken, and at the termination of the experiment, time to 50% emergence, an indicator of seedling vigor (Orchard, 1977) and percent emergence were calculated for each treatment. 51 52 Data were analyzed using standard analysis of variance techniques and LSD was used for mean separation. Results The time to 50% emergence (T50) of fresh pregerminated seed was lower than that of chilled or dry seed from both field and greenhouse soil (Table 1). With all treatments, field soil reduced the speed of Table l. The effect of chilling and soil type on the time to 50% emergence (T50) of pepper seedlings. (LSD.O5 = 1.78) T50 (mean of 3 reps) Treatment Greenhouse mix Field soil Fresh 10.75 days 17.7A days 00C for 2 days 17.38 19.67 Dry 16.65 21.AA emergence when compared with emergence from.the greenhouse soil. The field soil had become compacted soon after initiation of the experiment, while the greenhouse mix remained friable. Field soil increased the T50 by as much as 7 days. The effect of soil type on percent emergence is quite clear. Although there was no significant difference in percent emergence of chilled and unchilled pregerminated seed and dry seed when grown in greenhouse soil, large differences can be seen with field soil (Figure 1). Fresh pregerminated seed suffered the least reduction in percent emergence. Field soil reduced the dry seed stand from.8A to 37%, and the chilled pregerminated seed stand from 67 to A%. 53 Figure l. The effect of chilling and soil type on the percent emergence of pepper seedlings. (LSD 05 = 2A.7l) 5A new MIX 100 IFIELD sou. //////////7777777L , . ,V . , . ///////,/// o o o 8 6 4 m 0 uUZuGflwEm hZuuzun 0°C 2 DAYS FRESH DRY TREATMENTS Figure l 55 Discussion Based on the results of this experiment, it appears that pregerminated seed is better able to emerge through field soil than dry seed, provided it has not been chilled. Storage of pregerminated seed at 000 for 2 days, in combination with planting in field soil, resulted in a larger reduction in percent emergence than.any other combination of conditions. The adverse effect of 2 days storage at 00C on emergence has been seen in other work with pregerminated pepper seed, especially when raised at the suboptima1.temperatures of 150/1200 (Irwin and Price, 1979). Imposing an additional stress in the ferm.of heavy field soil accentuates the weakened condition of the chilled seed, resulting in the poor stand. Pepper seedling emergence has been observed to be adversely affected by soil compaction (Fawusi, 1978). Fritz (1965) recognized the correlation between vigor and the ability of seedlings to emerge through a layer of paper and sand, and standardized such a test for use with small grains. The emergence ferce exerted by seedlings of small-seeded legumes has been shown by Williams (1956) to be a direct function of seedling vigor. Therefbre, the reduction in vigor resulting from storage at 00C was reflected in the reduced ability of the pregerminated pepper seed to penetrate the compacted field soil. When unchilled, however, pregerminated pepper seed emerges considerably faster and produces a better stand in field soil than does dry seed. BIBLIOGRAPHY BIBLIOGRAPHY Fawusi,.M.O. 1978. Emergence and seedling growth of pepper as influenced by soil compaction, nutrient status and moisture regime. Scientia Hort. 9:329—335. Fritz, T. 1965. Germination and vigor tests of cereal seed. Proc. Intl. Seed Test. Assoc. 30:923-927. Irwin, C. and H.C. Price. 1979. Unpublished results. Orchard, ThJ. 1977. Estimating the parameters of plant seedling emergence. Seed Sci. and Tech. 5:61-69. Williams, W.A. 1956. Evaluation of the emergence ferce exerted by seedlings of small-seeded legumes using probit analysis. Agron. J. A8:273—27A. 56 APPENDIX A3 DETERMINATION OF THE OPTIMUM TETRAZOLIUM CHLORIDE (TTC) CONCENTRATION AND DURATION OF THE TTC SOAK FOR TETRAZOLIUM TESTING OF PREGERMINATED PEPPER SEED Determination of the Optimum Tetrazolium Chloride (TTC) Concentration and Duration of TI‘C Soak for Tetrazolium Testing of Pregerminated Pepper Seed Tetrazolium testing has been used in the U.S. since shortly after WWII, and is now a routine test for determining seed viability of many crops (Grabe, 1970). The test is a non-specific indicator of dehydrogenase activity. In contact with actively respiring tissue, the water soluble tetrazolium salt is reduced to a water insoluble red substance, formazan. Non-respiring tissue, indicative of non-viability, does not stain. Tetrazolium testing normally is performed on non- germinated seed, which is then examined individually, and termed germinable or non-germinable, depending on the location and extent of unstained tissue. For pregerminated seed, however, a modification of the technique used by Kittock and Law (1968) with germinating wheat was made. The objective of this experiment was to determine the optimum concentration of the tetrazolium chloride solution, and the shortest length of time the pregerminated seeds could be soaked in the solution to get good staining results. Materials and Methods Capsicum annuum L. cv. Hungarian Wax seeds were germinated for 5 days in aerated glass water columns at room temperature. Pregerminated seeds were removed from the columns and placed in .l%, .5% or 1% solutions of 2,3 ,S-triphenyl tetrazolium chloride in .01 M phosphate buffer of pH 7.0. Intact seeds were soaked in the TTC solutions for 57 58 6, 12, 18 or 2A hours. Four replicates of 10 seeds per treatment were placed in test tubes, covered with A ml of solution, and.maintained in darkness in a randomized block design. At the end of the soaking period, the TTC solution was decanted and discarded, seeds were rinsed with distilled water, and A ml of 2+methoxyethanol were added to each tube for extraction of the fermazan. After A hours, the 2-methoxyethanol was decanted, and each sample measured for absorbance at A80 nm in a Bausch and Lomb Spectronic 20. Data were analyzed using standard analysis of variance techniques and LSD was used for mean separation. Results Use of a .l% tetrazolium chloride solution did not result in sufficient coloration for routine use with pregerminated pepper seed (Table 1). Although a 2A hour soak in a .1% solution did produce an acceptable absorbance of 0.70, this concentration would not be high enough were chilled seeds soaked in it, since chilling reduces the amount of stainable tissue. A .5% solution, however, produced Table 1. The effect of TTC concentration and number of hours seeds were soaked in the'TTC solution on absorbance (A80 nm) of formazan.in 2—methoxyethanol extracts. (LSD.01 = 0.27). TTC concentration (%) Hours in solution .1% .5% 1% 6 0.00 0.26 D.AA 12 0.16 0.78 1.08 18 0.30 0.93 1.20 2A 0.70 1.28 1.38 satisfactory staining of the pregerminated seed. Results of the .5% concentration and 1% concentration were not significantly different at 59 the 1% level except when the seeds were soaked fer 12 hours. Differences between absorbance values obtained with .1% solution and .5% solution were, in general, considerably larger than between the .5% and the 1% solutions. Soaking seeds for 12 hours or more in TI‘C produced acceptable results. Six hours in TI‘C were not long enough for detectable coloration to occur at all concentrations. The interaction between percent concentration and length of soaking period was sigiificant . Discussion The results indicate that the reduction of TTC is a time/ concentration response. The longer the seeds are soaked, or the higher the concentration of the TTC solution, the greater the formazan development. Although the recommended time in solution (Grabe, 1970) for any crop on which TTC testing is routinely used does not exceed 6 to 8 hours (some of the grasses), it is obvious that this is not a sufficient length of time for use with pregerminated seeds. Recommended preparation for T'I‘C testing usually involves dissecting, piercing or removing the seed coat to facilitate quicker staining. None of these were done with the pregerminated seed, however, and the presence of the seed coat may slow the staining reaction. Sato (1962) working with embryos of Phaseolus vulgaris found that the rate of TTC reduction and the number of embryos in a tube of TIC solution were directly proportional when up to 12 embryos per tube were used. Above 12 embryos, the linear relationship no longer existed. It may be that routine testing involves larger numbers of seeds than what were used in this experiment (10 seeds/tube), therefore a shorter soaking period 60 is necessary. Other factors enter into the rate of TI‘C reduction - eg. temperature (Grabe, 1970) and pH (Sato, 1962), however, recommendations regarding these two were followed in this work. Based on the obtained results, use of a .5% solution or greater for at least 12 hours is necessary for detectable and measurable levels of formazan production. BIBLIOGRAPHY BIBLIOGRAPHY Grabe, D.F. (ed). 1970. Tetrazolium testing handbook. Assoc. Off. Seed Anal. PUbl. Kittock, D.L. and A.G. Law. 1968. Relationship of seedling vigor to respiration and tetrazolium.chloride reduction of germinating wheat. Agron. J. 60:286-288. Sato, S. 1962. Studies on the reduction of tetrazolium salt by plant tissues. 11. Effect of plasmolysis on the reduction of TTC in plant cells. Cytologia 27:158-171. 61 APPENDIX AA THE EFFECT OF 'PRIMING"WITH POLYETHYLENE GLYCOL ON GERMINATION OF PEPPER SEEDS The Effect of 'Priming' with Polyethylene Glycol on Germination of Pepper Seeds Soaking seeds in a concentrated solution of polyethylene glycol (PEG), termed 'prirning' , prior to planting has been shown by several researchers to be beneficial to germination. Heydecker, Higgins and Gulliver (197A) reported quicker and more uniform seedling emergence when seeds were 'primed' with a PEG solution. Heydecker and Hendy (1975) found that percent germination was usually unaffected by PEG pretreatment. Celery is a crop characterized by non-uniform germination, but Salter and Darby (1976) were able to greatly improve the uniformity by 'priming' the seed. The PEG solution allows enough imbibition to initiate germination processes, but not enough for radicle emergence to occur. The objective of this experiment was to determine the effect of PEG 'priming' on the germination and emergence of A cultivars of pepper seeds. Materials and Methods Four replicates of 50 seeds of Capsicum w L. cvs. Atlas, Sonnette, Shepard and Yolo Wonder L were placed in 9.0 cm Petri dishes containing 2 pieces of Whatman #2 filter paper. The filter paper was moistened with 6 ml of a solution of Carbowaxl (polyethylene glycol) 6000 with an osmotic potential of -1l.5 bars. Seeds were 'primed' for 1Union Carbide Corporation 62 63 A, 8, or 12 days, and dry seed was used as a control. During the 'priming', the seeds were maintained at room.temperature. At the end of the 'priming' period, 25 seeds from each replicate were planted in a completely randomized design at a 1.5 cm depth in horticultural grade vermiculite. Flats were placed under Gro-Lux lights and watered as necessary. Daily emergence counts were taken until the number of emerged seedlings had.not changed fer 3 days. Percent emergence and time to 50% emergence, an indicator of seedling vigor (Orchard, 1977), were calculated for each treatment within each cultivar. Data were analyzed using standard analysis of variance techniques and LSD was used for mean separation. Results PEG pretreatment reduced the T50 emergence for all A pepper cultivars (Figure 1). The decrease was linear until 8 days of soaking, with no additional decrease at 12 days. Percent emergence exhibited very little change with any of the 'priming' treatments (Table 1). There was no significant interaction between cultivars and the PEG treatments. Table 1. Effect of number of days in a PEG 6000 solution with an osmotic potential of -11.5 bars on percent emergence of A cultivars of pepper seeds. (LSD.01 = 12.12) Number of days in PEG solution Cultivar 0 (dry) A 8 12 Atlas 77 7A 76 70 Sonnette 91 95 96 97 Shepard 8A 89 89 91 Yolo wonder L 86 78 85 83 Figure 1. 6A Effect of'number of days in a PEG 6000 solution of -11.5 bars on the time to 50% (T50) emergence of A cultivars of pepper seeds. (LSD.01 = 1.09) 150mm!» 15 N . \ 1o * . 5 o 65 ATLAS '— SONNETTE '- SHEPARD ‘- YOLO WONDER L H \‘2... . .\ \‘\“‘ z m. 0 s“““““‘ . \ o — — — . 4 8 12 DAYS IN PEG Figural 66 Discussion The results of this experiment substantiate the benefit of PEG 'priming' proclaimed by Heydecker and coworkers (197A,l975) and Salter and Darby (1976). In all A cultivars tested, the T50 emergence was reduced by nearly 50% with an 8 day soak in a PEG 6000 solution. There appears to be no added advantage to an additional A days of 'priming', however, as T50 values differed very little fromlthose obtained with an 8 day soak. Salter and Darby (1976), working with celery seed, found germination to be very rapid after a 1A day treatment with a PEG solution of -12.5 bars. The results of shorter treatment periods were not discussed. Heydecker, Higgins and Gulliver (197A) also recommend 'priming' periods of 2 to 3 weeks for onion, carrot, beet and celery seed. Though the osmotic potential of the PEG solutions used by these researchers was in the range of -11.5 bars (-10 to -l2.5), the temperature used fer 'priming' by Salter and Darby and Heydecker et. al. was different. Both groups worked with 1590, while the 'priming' of pepper seeds was carried out at roomltemperature (20-2590). The higher temperature could be responsible for those processes initiated within the seed proceeding at a faster rate than at 150C. This could result in the maximmm1benefit to be obtained from 'priming' showing up in 8 days at 2590 versus 1A or 21 days at 1500. Another factor could be simply the difference between pepper seeds and seeds of onion, beet, carrot and celery. 'Priming"with a PEG solution of -11.5 bars is beneficial to germination of pepper seeds. An 8 day soak was most successful in reducing the number of days to 50% emergence in all A cultivars tested, but 'priming' had no effect on percent emergence of any cultivar. BIBLIOGRAPHY BIBLIOGRAPHY Heydecker, W., J. Higgins and R. Gulliver. 197A. Instant germination - a method of brinkmanship. Comm. Grower Jan. 197A. Heydecker, W. and A. Hendy. 1975. Pre-treating bedding plant seed for 'instant' germination. Comm. Grower Oct. 1975. Orchard, T.J. 1977. Estimating the parameters of plant seedling emergence. Seed Sci. and Tech. 5:61-69. Salter, P.J. and R.J. Darby. 1976. Synchronization of germination of celery seeds. Ann. Appl. Biol. 8A:A15-A2A. 67 HICHIan STATE UNIV. LIBRARIES 111111111111111111“11111111111111111111 31293107130647