u u 7‘“. ‘ 4 A, " r”: x. 7h}‘. v '. . ., uj" .‘..,,.’.. W.“ -...'.5¢, :- ‘J I . .t?’ ~ - 3 . r 45%.: ’ v .. m “M ' mifévsfg, £3935, ‘ PW?” ;*"=\Y 1...:- 19‘; -.- J”-'T" er‘VJ: a: v: u? 3 «.5333: . Q .3 . é“??? of rmjzva’; Vi’g‘: :V’KL'? i591 -. av “ 'G‘adyl‘r}; ‘3 y? “'5! A A ‘ . r , ‘1, 355.5 ’ 5.5? <53” .5 o 13:: . K 23; .3“: .W .5 r 595??“ ~34? “i .5175. JV”? {f5 T”? ‘5‘:- ‘2: ,‘ fi-n’ :4“ . l 'IH ”—Ei 7.9 If” {:{r‘ri' :jEA .5. . a fvwb 1‘ ¢~.:—.- up; ;":{L .37. 316:3: TE UNIVERSITY LIBRARIES Illllillllmnl mull ll it 3 1293 00902 4781 This is to certify that the dissertation entitled RELEASE OF DORMANCY IN APPLE (Malus domestica Borkh.) SEEDS AND EMBRYOS: RESPONSE TO TEMPERATURE, CYTOLOGICAL CHANGES, AND METABOLISM OF GIBBERELLIN A12 ALDEHYDE presented by ERIC H. C. CHILEMBWE has been accepted towards fulfillment of the requirements for Ph. D . degree in HORTI CULTURE @545 Major professor Date SEPTEMBER 26, 1991 MSU i: an Affirmative Action/Equal Opportunity Institution 0-12771 r \ LIBRARY Michigan State University K I 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 MSU Is An Affirmative Action/Equal Opportunity Institution cmmn.‘ RELEASE OF DORMANCY IN APPLE (nalgg ggmgsgiga Borkh.) SEEDS AND EMBRYOS: RESPONSES TO TEMPERATURE, CYTOLOGICAL CHANGES, AND METABOLISM OF GIBBERELLIN A12-ALDEHYDE. BY Eric Hetlason Chikafa Chilembwe A DISSERTATION Submitted to Michigan State University in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1991 ABSTRACT RELEASE OF DORMANCY IN APPLE (Mains domestic; Borkh.) SEEDS AND EMBRYOS: RESPONSES TO TEMPERATURE, CYTOLOGICAL CHANGES, AND METABOLISM OF GIBBERELLIN AlZ-ALDEHYDE. BY Eric Hetlason Chikafa Chilembwe The optimum temperature for breaking dormancy of ’Golden Delicious' and ’Paulared' apple embryos was 2.5 to 7C. Embryo germination was stimulated by alternating the stratification temperature between 5 and 10C (16/8 h) but temperatures higher than 100 either had no effect or were inhibitory. Chilling negation by high temperature was more dependent upon the temperature than upon cycle length. Seed germination was consistently inhibited by alternating between 5C and higher temperatures. Seeds chilled in fruit germinated poorly in comparison with those chilled in Petri dishes; this effect was much less pronounced following embryo excision. Holding seeds under water (anaerobiosis) failed to break embryo dormancy at 20C, but was effective at 2.5C. Gibberellin A4+7, benzyladenine (BA), Promalin (GA4+7 and BA) and cyanamide all significantly stimulated the germination of non-chilled embryos, but were ineffective on intact seeds. The effects of chilling vs. chemical treatments on cytological changes in the cells of the embryonic axes were compared. Dormancy release was always associated with degradation of protein, appearance of rough endoplasmic ii reticulum and Golgi apparati, and increases in nucleolar volume and numbers of mitochondria. l4C-GA12-aldehyde was incubated with apple embryos, cotyledons, or embryonic axes, and methanolic extracts were partially purified by high performance liquid chromatography (HPLC). Six l4C-labelled metabolites were recovered. One metabolite had the same retention time as GA12, but none was eluted at the retention time of GAl or 6A4. There were no qualitative differences in metabolite profiles between chilled vs. dormant embryos, cotyledons, or embryonic axes, but one metabolite was more abundant in dormant, and another in chilled embryos. The rate of metabolism was higher in chilled than in dormant tissues. iii DEDICATION This thesis is dedicated to my mother, ENEGESI, who passed away when I was very young, but is still with me in Spirit. iv ACKNOWLEDGMENTS I express my deep appreciation and indebtedness to my major professor, Dr. Frank G. Dennis Jr. for his encouragement, direction, dedication and sense of humor during my program of research and preparation of this thesis. I am also very grateful indeed to, in alphabetical order, Drs. D. Dickmann, J. A. Flore, K. Poff and K. Sink for their advice, encouragement and direction in the research, and critical review of the manuscript, as members of my guidance committee. My special gratitude goes to Drs. Karen L. Klomparens and J. Heckman for their guidance and assistance in Transmission Electron Microscopy and also Dr. J. Everard for his assistance in computer programs for data processing. My special thanks and appreciation go to the Malawi government and USAID for their financial and moral support which made this study possible. I would like to thank my wife, Catherine, and children Agnes, Olive, Limbani and Chifundo for their support and love during my graduate study. Finally, my thanks go to my father, grandmother, father-in-law and mother-in-law for their patience and understanding during my long study leave away from them. TABLE OF CONTENTS LIST OF TABLES O O O O O O O O O O O O ........ O O O O O OOOOOOOOOOOOOO LIST OF FIGURES. O O O O O O O O O O O O O O O O O ..... O O O O O O I O ....... INTRODUflION. O O O C O O O O O O O O O O O O O O O O O O O O O O O O O I O ......... LITERATURE REVIEW....... ..... ............ ............ Terminology..................................... Dormancy............................. ...... Stratification....................... ...... After-ripening............... .............. Seed vs Bud Dormancy............. ............... Induction of Dormancy.. ..... ...... .............. Temperature.......................... ...... Oxygen supply....................... ....... Growth inhibitors.......................... Low levels of growth promoters............. Cell membrane integrity........ ............ Dormancy Release................................ Chilling temperature.......... ..... ... ..... High temperature........................... Alternating temperatures................... Chemical treatments........................ Hormones................................... Gibberellins..................... ..... Cytokinins............................ Auxins and ethylene................... Enzymes.................................... Anaerobiosis............................... Ultrastructural changes....... ...... . ...... Summary ...................................... vi inbubcsp H Ul CHAPTER ONE, SECTION ONE THE EFFECTIVENESS OF CONSTANT VS. ALTERNATING TEMPERATURE IN BREAKING THE DORMANCY OF APPLE SEEDS AND EMBRYOS Abstract............................................. 23 Introduction......................................... 24 Materials and methods................................ 25 Results.............................................. 30 Discussion........................................... 33 Literature cited..................................... 53 CHAPTER ONE, SECTION TWO CHILLING NEGATION IN APPLE SEEDS AND EMBRYOS FOLLOWING STRATIFICATION OF THE SEEDS Abstract....................................... ...... 56 Introduction......................................... 57 Materials and Methods................................ 57 Results.............................................. 60 Discussion........................................... 62 Literature cited..................................... 73 CHAPTER TWO, SECTION ONE BREAKING OF APPLE EMBRYO DORMANCY WITH LOW TEMPERATURE, HORMONES, CHEMICALS, AND ANOXIA. I. EVALUATION OF METHODS Abstract............................................. 75 Introduction......................................... 76 Materials and Methods................................ 77 Results.............................................. 79 Discussion........................................... 82 Literature cited..................................... 92 CHAPTER TWO, SECTION TWO BREAKING OF APPLE EMBRYO DORMANCY WITH LOW TEMPERATURE, HORMONES, CHEMICALS, AND ANOXIA. II. CYTOLOGICAL CHANGES Abstract............................................. 95 Introduction......................................... 96 Materials and Methods................................ 98 Results.............................................. 100 Discussion........................................... 105 Literature cited..................................... 129 vii CHAPTER THREE METABOLISM OF 14C-GA12 ALDEHYDE BY APPLE EMBRYOS IN RELATION TO DORMANCY RELEASE Abstract..................................... ........ 134 Introduction.................... ..................... 135 Materials and Methods................... ............. 137 Results.............................................. 143 Discussion........................................... 146 Literature cited..................................... 180 SUMYANDCONCLUSIONOCOOO......OOOOOO OOOOOOOOOOOOOOO 183 BIBLImmHYOOCOOOO......OOOOOOOOO0....O 00000000000000 188 APPENDIXOOOOOOOOOOOOOO......OOOOOOOOOOOOOOOOOO ........ 204 viii 1.3.21: LIST OF TABLES CHAPTER ONE, SECTION ONE Effects of time of stratification of 'Golden Delicious’ and 'Paulared' seeds at SC on subsequent germination capacity (%) of the seeds and embryos and on germination rate (MDG) of Golden Delicious embryos................ 42 CHAPTER TWO, SECTION ONE The germination rate (mean days to germination) of ’Golden Delicious’ and 'Paulared' apple embryos excised from seeds stratified at 2.5C in petri dishes or within fruits................. 88 Effect of after-ripening 'Golden Delicious' apple seed at 2.5C under water with or without sparging with N2 on subsequent germination of the embryos at 20COOOOOOOOOCOCOO......OOOOOOOOOOO 89 CHAPTER TWO, SECTION TWO Summary of cytological changes induced by dormancy-breaking treatments..................... 128 CHAPTER THREE Retention times on HPLC of GA4, GAl, GA12 and 14C-GA12-aldehyde standards and of metabolites in apple embrYOSOOOOOOOOOOOOO0.00.00.00.000...... 149 ix LIST OF FIGURES Kim CHAPTER ONE, SECTION ONE Effects of stratifying seed at constant' temperatures on subsquent germination of the embryos at 20C.0.........OOOOOOOOOOOOOOOOO 0000000 Effects of stratifyng seeds at constant 5C vs. alternating temperatures in a diurnal cycle on subsequent germination of the embryos at 20C ..... Effects of stratifying seeds at constant 5G vs. alternating temperatures in a diurnal cycle on subsequent germination of the seeds at 20C....... Effects of stratifying seeds at constant 5C or alternating temperatures in a 24-hour or 3-week cycle on subsequent germination of the embryos at ZOCOOOOOOOOOOOOOO00............OOOCOOOOOOO.... Effects of stratifying seeds at constant 5C vs. alternating temperatures in a 6-wk cycle on subsequent germination of the embryos during 6 days at 20C...................................... Comparison of effects of constant vs alternating temepratures and of cycle length on the breaking of apple embryo dormancy......................... CHAPTER ONE, SECTION TWO Effects of method of stratification and length of exposure to 30C after 10 weeks at SC on the germination capacity of the embryos at 20C....... Effects of stratification period and length of exposure to 30C on the germination capacity of seeds at ZOCOOOOOO0............OOOOOOOOOOOOOOI... Effect of temperature of exposure after chilling on induction of secondary dormancy in apple embrYOSOOOOOOOOOOOOOOOO0.0.00.0...0.0.0.0....O..0 41 44 46 48 50 52 66 68 7O 4. E1913“: Effect of stratifying at 5C before or after a ' lo-day exposure to 30C on induction of secondary dormancy in apple seeds................... ....... 72 CHAPTER TWO, SECTION ONE Effect of stratifying 'Golden Delicious' and 'Paulared' apple seed at 2.5C within fruit vs. in petri dishes on germination of the seeds and embryos at 20C.............................. 87 Effect of hormone treatment of G. Delicious and Paulared apple embryos on subsequent germination at 20C.................... ........... 91 CHAPTER TWO, SECTION TWO Transmission electron micrographs of whole or portions of cells in the meristematic regions of dormant apple embryonic axes.. ..... ... ........ 113 Transmission electron micrographs, of whole or portions of cells in the meristematic regions of apple embryos chilled for 8 weeks in petri dishes at 2.5C................................... 115 Transmission electron micrographs of whole or portions of cells in the meristematic regions of apple embryos chilled within fruit for 8 weeks at 2.5C................................... 117 Transmission electron micrographs of whole or portions of cells in the meritematic regions of apple embryos chilled under water at 2.5C for 8 weeks...................................... 119 Transmission electron micrographs of whole or portions of cells in the meristematic regions of non-chilled apple embryos after treatment with gibberelin A4+7 at 40 mg/l for 24 hours..... 121 Transmission electron micrographs of whole or portions of cells in the meristematic regions of non-chilled apple embryos after treatment with benzyladenine at 20 mg/l for 24 hours....... 123 Transmission electron micrographs of whole or portions of cells in the meristematic regions of non-chilled apple embryos after treatment xi with Dormex (cyanamide) at 80 mg/l ............... Transmission electron micrographs illustrating organelles in selected cells in the meristematic regions of apple embryos chilled in petri dishes or under water at 2.5C.................... ....... CHAPTER THREE 21911:: High performance liquid chromatography (HPLC) of l4C-GA12 aldehyde with and without extract of killed embryos and of 14C-GA12, 3H-GA4 and 3H-GA1............OOOOOOOOO00.00.0000... ......... HPLC of 14-C-labelled metabolites in dormant apple embryos following 6 to 96 hours incubation with 14C-GA12-aldehyde at 20C......... HPLC of 14C-1abelled metabolites in chilled apple embryos following 6 to 96 hours incubation with 14C-GA12-aldehyde at 20C......... Relative quantities and profile of major 14C-labelled metabolites in eluates from HPLC column of extracts following 6 to 96 hours incubation of dormant and fully chilled apple embryos with 14C-GA12-aldehyde............ ....... Relative amounts of 14C-labelled metabolites in dormant apple cotyledons following 6 to 96 hour incubation with 14C-GA12-aldehyde at 20C......... Relative amounts of 14C-labelled metabolites in chilled apple cotyledons following 6 to 96 hour incubation with 14C-GA12-aldehyde at 2°C.... Relative amounts of 14C-labelled metabolites in dormant apple embryonic axes following 6 to 24 hours incubation with 14C-GA12-aldehyde at 20C... Relative amounts of 14C-labelled metabolites in chilled apple embryonic axes 6 to 24 hours incubation with 14C-GA12-aldehyde at 20C......... Relative quantities of 14C-GA12-aldehyde retained in eluates from HPLC column of extracts following 6 to 96 h incubation of embryos, cotyledons and embryonic axes excised from dormant and fully chilled seeds with l4C-GA12-aldehyde at 20C............. ............ xii 125 127 151 153 155 157 159 161 163 165 167 10. £191.11: 11. 12. 13. 14. 15. Relative quantities and profile of a 14C-labelled metabolite with retention time of 26 minutes in eluate from HPLC column of extracts following 6 to 96 h incubation with 14C-GA aldehyde of embryos, cotyledons and embryonic axes excised from seeds chilled for 0 and 10 weeks at 5C... ..... ........ ........ 169 Relative quantities of a 14C-labelled metabolite with retention time of 18 min. in eluates from HPLC column of extracts following 6 to 96 h incubation with 14C-GA12-aldehyde of embryos, cotyledons, and embryonic axes excised from seeds chilled for 0 or 8 weeks at 5C...................................... ...... 171 Relative quantities of a 14C-labelled metabolite with retention time of 12 minutes in eluates from HPLC column of extracts following 6 to 96 h incubation with 14C-GA12- aldehyde of embryos, cotyledons and embryonic axes axcised from seeds chilled for 0 or 8 weeks at 5C...................................... 173 Relative quantities of a 14C-labelled metabolite with retention time of,9 minutes in eluates from HPLC column of extracts following 6 to 96 hours incubation with 14C-GA12 aldehde of embryos, cotyledons and embryonic axes excised from seeds chilled for O to 8 weeks at 5C................... 175 Relative quantities of 14C-GA12-aldehyde ,remaining in eluate from HPLC column of extracts following 6 to 96 h incubation with 14C-GA12- aldehyde of dormant vs. chilled (4 or 8 wk) embryos, cotyledons and embryonic axes..... ...... 177 HPLC of 14C-labelled metabolites in apple embryos excised from seeds held moist at 20C for 8 wk following 6, 12, and 24 h incubation with 14C-GA12-aldehyde at 20C......................... 179 xiii APPENDIX Figure M 1. Relative quantities and profile of a 14C-labelled metabolite with retention time of 30 min. (GA12) in eluates from HPLC column following 6 to 9 h incubation with l4C-GA12-aldehyde of dormant vs. chilled embryos, cotyledon and embryonic axes.... 206 2. Relative quantities and retention times of 14-labelled metabolites and standards in eluates from HPLC column of embryos excised from seeds chilled for 8 wk at SC......... ....... 208 Table me 1. Distribution of radioactivity during extraction and purification of apple embryo tissues. Means forBreplicates.......OOOOOOOOOO... ........ 0.... 209 2. Retention times (minutes) of gibberellins in reverse phase High Performance Liquid Chromatagraphy using u-Bondapak C18 column.. ..... 210 xiv TO: The Guidance Committee, This thesis is organized in Journal-style in accordance with departmental and university requirements. The format is that of the Journal of the American Society for Horticultural Science. INTRODUCTION The need for commercial production of deciduous fruits in many tropical regions of the world (Australia, Asia, Southern Africa, South America) has recently been emphasised for both economic and nutritional reasons. Dormancy is the major limiting factor to the successful production of deciduous fruits under tropical conditions. Buds and seeds enter an annual dormancy period in order to survive low winter temperatures. Dormancy is naturally broken by 1,000 to 3,000 hours of chilling temperatures, between 0 and 7C in winter, and this leads to resumption of normal growth, development, flowering and fruiting. The process of breaking dormancy of seeds by cold temperature is called stratification or after-ripening (Bennett 1950; Powell 1986; Saure 1985; Westwood 1978). Under tropical conditions, buds and seeds do not receive sufficient chilling hours to break their dormancy fully; this results in delayed foliation or prolonged dormancy, erratic bud break and flowering. The trees may not come out of dormancy, hence they weaken and may eventually die. Alternative methods of breaking dormancy have been developed for commercial use under tropical 2 conditions; these include defoliation of trees soon after harvest, use of cultivars with low chilling requirements, use of dormancy-breaking chemical agents such as dinitro- ortho-cresol (DNOC), cyanamide, and plant growth regulators (Fuchigami and Nee 1987; Morimoto and Kumashiro 1978; Powell 1987). However, the application of these alternative methods for breaking dormancy has met with limited success. The apple embryo which is like a miniature plant, which exhibits dormancy and hence makes a good model system for studies on induction and release of dormancy under controlled conditions (Come and Thevennot 1982). Moderate temperatures of 10 to 15C can enhance the chilling effect of 5C , while high temperatures negate it (Erez and Lavee 1971; Erez et al. 1979a, 1979b ). Under sub- tropical conditions, the daily temperatures during winter fluctuate between 2 and 30C. The beneficial effects of moderate temperatures have only been demonstrated in buds of peach (Erez and Couvillon 1987) and the mechanism involved in not known. Little attention has been paid to the ultrastructural changes which take place during the breaking of dormancy by either chilling or by treatment with chemical agents. An intricate balance between growth- promoting and growth-inhibiting substances appears to regulate bud and seed dormancy (Wareing and Saunders 1971) and gibberellins (GAs) are believed to play a major role in this process. GAs undergo significant shifts in free and 3 bound forms during stratification. GA4 is believed to be involved in the dormancy release process, for it increases during chilling as a result of either release from its bound forms or g; 3929 synthesis (Sinska et al. 1973; Smolenska and Lewak 1975). The ability of the embryos to synthesize GA4 is dependent on the stage of the chilling period. (Sinska and Lewak 1977). Hence, dormancy may affect biosynthesis of GAs by influencing the biosynthetic pathway. The goals of this research were to: a) determine the effects of alternating temperatures during the after- ripening process on the breaking of dormancy in apple seeds and embryos; b) observe ultrastructural changes associated with dormancy release by both chilling and chemical agents; and c) compare the metabolism of GA12-aldehyde in chilled vs. non chilled embryos. LITERATURE REVIEW W DQINADEI- The temporary suspension or arrest of active growth in plant organs containing meristems under conditions suitable for growth is called dormancy (Lang 1987). Three types of dormancy are identified, namely a) groggrmgngy which is synonymous with quiescence or imposed dormancy and is regulated by environmental factors b) egrgggrmgngy which is synonymous with correlative inhibition or summer dormancy and is regulated by physiological factors outside the affected Structure, and lastly c) gnggggrmangy which is synonymous with rest, winter dormancy or deep dormancy and is regulated by physiological factors inside the affected structure. Endodormant seeds or buds require chilling for the resumption of growth. Primary ggrmgngy prevails during development and maturation of buds or seeds on the mother plant while ggggngary ggrmgngy is re-imposed in partially chilled seeds or buds under conditions unfavorable for germination or growth (Karssen 1980/81). Stratification The holding of seeds under moist conditions at any temperature is referred to as stratification (Pellet 1973), although some would restrict the term to any temperatures that break dormancy. Alien-ripening After-ripening refers to changes that occur within the seed during storage as a result of which germination can take place or is improved (Nikolaeva 1969). After-ripening may occur at room temperature (grains), or only at low temperature (apple), depending upon species. W The breaking of dormancy allows induction of active growth of an organ. Bud and seed dormancy have several common characteristics. The optimum temperature which breaks dormancy in buds and seeds is similar and so is the length of the chilling period required. Furthermore, secondary dormancy can be induced in both buds and seeds by high temperature during the early period of chilling. 111W Several external and internal factors are known to induce dormancy in fruit trees, including temperature, low oxygen levels, endogenous rhythms, growth inhibitors, and cell membrane integrity. IQEEQIRSEIE Little is known about how temperature induces dormancy. Various investigations have demonstrated that chilling may intensify dormancy in the fall although it breaks dormancy 6 later (Ben-Ismail 1989; Hatch and Walker, 1969; Lavarenne 1975; Walser et al 1981). Dormancy of peach and apricot buds increased as chill units accumulated in the autumn (Hatch and Walker 1969). Ben-Ismail (1989) reported that growth of vegetative buds of apple was inhibited by artificial chilling of cuttings sampled in early October. However, the response varied depending on time of collection of shoots and length of the period of exposure to low temperature. Some physiologists contend that summer temperatures induce dormancy while cold temperatures in fall intensify it (Samish, 1954). The knowledge that both cold and warm temperatures induce dormancy and the reported dual role of cold temperature i.e., its ability to both induce and break dormancy, complicate the interpretation of the model of action of temperature in the induction and release of dormancy. Low temperature may not be essential for the induction of dormancy, for deciduous fruit trees become dormant in the tropics in areas where chilling temperatures do not occur. W The seed coat presumably restricts diffusion of oxygen to the embryo, thereby limiting respiration and inducing embryo dormancy. Seeds displayed no dormancy if the seed coat was removed (Cracker 1916). Visser (1956b) observed that restriction of oxygen uptake by apple seed coats 7 increased with temperature. If intact, partially stratified seeds were germinated at 25C, about 60% of the seeds entered into secondary dormancy. However, if the seed coat was removed and the endosperm ruptured, high germination levels were obtained (Visser 1956a). Come, et al. (1972) suggested that phenolic compounds in the testa react with oxygen, thereby reducing oxygen supply to the embryo resulting in dormancy. However, Tissaoui and Come (1973) were able to break apple embryo dormancy in the absence of chilling by anaerobic conditions under pure nitrogen alone. If the testa induces anaerobiosis, one would expect that embryo dormancy could be broken by holding embryos at room temperature, but this is not the case. W Abscisic acid is an endogenous growth hormone presumably involved in the control of dormancy in seeds and buds. Attempts to correlate the level of ABA with the induction of dormancy have resulted in conflicting evidence. Seeds of species which exhibit dormancy, i.e, hazel nut (Qgrylng aggllana) and apple, contain relatively high levels of ABA (Williams et. a1. 1973, Balboa-Zavala and Dennis 1977; Dudarichi 1969). The ABA declines rapidly during chilling, but several studies have indicated that the decline occurs under both high and low temperature regimes, although only chilling alleviates dormancy (Borkowska and 8 Powell 1982-1983; Balboa-Zavala and Dennis 1977). Strong evidence for the involvement of ABA in inducing dormancy comes from studies with ABA-deficient mutants of Arabidopsis ragliana_L. Heynh. Mutant lines of Arabiggpsis characterized by high transpiration rates ( wilty mutant) and lack of seed dormancy contain low levels of ABA in leaves and mature seeds in comparison with the wild type (Karssen et al. 1983) which exhibits dormancy. Rudnicki (1969) found a good correlation between chilling and the disappearance of ABA in seeds during the first 3 weeks of stratification. Thus it appears that ABA can be relatively effective in inhibiting bud or seed growth where a) the potential for growth is low, thus allowing ABA to tip the balance towards a no-growth situation, and b) in situations where the initial events leading to growth are absent or have not progressed very far. Hence ABA may play a role in the early stages of dormancy but not in later stages; rather some kind of promoting force becomes dominant tending to override the possible effects of endogenous ABA. L9H_L2Ifilfl_QI_§IQ!§h_RIQEQ§§I§ Growth promoting hormones decrease to low levels in shoots late in the growing season and this alone could account for dormancy induction (Lewak 1985). This hypothesis is questionable because the application of growth-promoting substances might be expected to promote 9 growth in resting organs and this is not generally the case. Furthermore, rest gradually intensifies long after growth- promoting hormones have reached extremely low levels, suggesting the build-up of an inhibitory influence. This increasing intensity of dormancy could result from a gradual loss of an essential metabolic function rather than from the gradual increase of an inhibitory compound (Powell 1987). W The sites of metabolic activities in the living cell are separated from each other by membranes. The most important properties of the membranes are semipermeability, the activity of membrane-bound enzymes, and membrane potential (Bewley and Black 1982). Membranes undergo transitions in their physical state at specific temperatures, and the activity of enzymes associated with them also changes. Therefore, the factors that induce dormancy may do so through changes in membranes and membrane-bound enzymes (Bewley and Black 1982). Membrane repair mechanisms and membrane development are under hormonal control and there is evidence that GA is involved in changing the permeability and also in the synthesis of some essential membrane constituents (Wood and Paleg 1974 ; Ben-Tel and Varner 1979). Similarly, an increase in membrane permeability may be the cause of 10 dormancy release (Doorenbos 1953). 911W The optimum chilling temperature for breaking dormancy in buds and seeds varies with species. In most cases 5C is considered to be the optimum temperature (Erez and Lavee 1971; Perry 1971). Sarvas (1974) reported that 3.5C was the optimum for 25:91: nubesgens. In peach buds (Erez and Lavee 1971) and pear seeds (Westwood and Bjornstad 1968) the optimum was found to be 6C, and between 7 to 10C, depending upon species, respectively. The effect of temperatures below DC on overcoming rest is not clear. Generally temperatures just above freezing are more effective than lower temperatures. However, subfreezing temperatures are effective in overcoming rest in some species (Sparks 1976). Sarvas (1974) found that temperatures above 10C were not effective in satisfying the chilling requirement of certain forest trees. However, such temperatures are effective in peach (Erez and Lavee 1971). Kobayashi et al. (1987) reported that temperatures up to 20C were effective in meeting the chilling requirement of red-osier dogwood (Corns: mince)- 11W High temperatures can either prolong dormancy or induce 11 secondary dormancy in buds and seeds. Weinberger (1954) reported that longer chilling periods were required to overcome rest in peach during warm winters. In a few North American forest trees, a moderate temperature interruption can counteract all the previous chilling effects of low temperature (Nienstaedt 1966), but Erez and Lavee (1971) found that high temperature must occur within a few days after chilling in order to negate prior chilling; otherwise a fixation process will prevent reversal. Short periods of high temperature during a daily cycle can negate chilling. Erez et al. (1979b) reported that ’exposure of dormant peach to 6 hr at 21C to 24C negated 18 hours at 6C. Thus the length of the high temperature treatment following chilling is critical. In apple buds greater chilling negation was found if the high temperature occurred every 2 days rather than every 4 days (Thompson et a1. 1975). The effect of high temperature in negating chilling depends on both the stage of rest and the temperature. The temperature range for induction of secondary dormancy decreases progressively during the post-dormancy period (Vegis 1964). Alternatins_Tennsratnres Pluctuating temperatures are more effective in breaking dormancy of peach buds than are constant temperatures. In 12 peach, daily fluctuation between 6 and 15C was more effective in breaking rest than constant 6C (Samish 1954; Erez et al. 1979). Erez et al. (1979) demonstrated that constant 15C alone was ineffective in breaking dormancy of peach buds. However, alternating temperatures ( 15C for 8 hr, 6 for 16 hr) were more effective than constant 4C for the same number of chilling hours. Temperatures higher than 15C, i.e. 18C had no effect, while 21 and 24C were inhibitory. Erez and Couvillon (1987) also demonstrated that the most efficient moderate temperature for alternation was 13C; alternation was effective only during the latter part of the chilling period and could even be inhibitory during the initial third. Later, Erez et al.(1979a) showed that temperatures higher than 15C were capable of enhancing the effects of chilling temperatures on breaking rest provided exposure time was reduced to 4 hours or less during a 24-hour cycle. The promotive effects of alternating temperatures were confirmed in nectarine buds (Gilreath and Buchanan 1981) and peach seeds (Aduib and Seeley 1986; Mahhou 1991). However, in peach seeds, 10C was promotive while 15C was inhibitory. In contrast Sarvas (1974) found no beneficial effects of alternating temperatures in breaking rest of several forest species, and Del Real Laborde (1987) observed that alternating temperatures never released apple seed dormancy. 13 Erez et al. (1979, 1986) proposed a two-step model to explain temperature effects on rest completion in peach buds as follows: A ,_ s c where A a the resting state B a the product of low temperature exposure, which can revert to ”A” at high temperature; C = the product of B, which is fixed and thus irreversible; b = the chilling reaction (favored by low temperature); and a a the reverse reaction (favored by high temperature) c = the reaction converting B to C at moderate temperature, which fixes the chilling effect. Of the two steps, the first one is reversible while the second is irreversible. The reactions have different temperature response curves. The chilling reaction (b) occurs at temperatures between 0C and 13C. Chilling efficiency decreases as the temperature rises above 8C, reaching zero at 14C. Reaction 'a' occurs at temperatures above 16C, reaching high levels of activity at 24C. The influence of temperatures greater than 24C and less than 0C is unknown. Reaction 'c' occurs at a wide range of temperatures but is most rapid at moderate temperatures i.e., 13 to 15C, and thus overlaps reactions 'a' and 'b'. Reaction 'c' can occur at 0C since budbreak will occur on 14 plants chilled continuously at this temperature. Reactions 'a’ and 'c' compete for the same substrate '8’, but reaction 'c' has a much lower 010 than reaction ’a' which partly or fully masks it at a temperature of 13C. Reaction 'a’ seems to be affected by the level and duration of the high temperature. Short diurnal periods of exposure to 20C may divide the substrate 'B' between 'a' and 'c’ resulting in no negative effect. Erez and Couvillon (1986) demonstrated that the chilling efficiency of low temperature was indeed increased by cycling low temperature (5C) with moderate temperature (15C). They later suggested that the level of the day temperature in a diurnal cycle is of critical importance under marginal growing conditions with warm winters, i.e., the temperature can either negate or enhance budbreak depending on its level and duration. Fishman et al. (1987), developed a mathematical model to explain the mechanism of chilling promotion by moderate and high temperatures. Mahhou (1991), working with peach seeds, observed that cycling was unnecessary. Germination of seeds held at constant temperatures for three 3-week periods was promoted or inhibited by raising the temperature from SC to 10C or 15C, depending upon the time the higher temperature was applied. This casts doubt on the cyclical scheme proposed by Pishman et al. (1987) for peach buds. 15 W Several chemicals have been reported to break dormancy of fruit trees, including mineral oils and dinitro-ortho- cresol (DNOC) (Jeffrey 1951); nitrogen containing compounds like thiourea (Blommaert 1965); cyanamide (Morimoto and Kumashiro 1978; Shulman et al. 1983). How these Chemicals break rest in deciduous fruit plants is still unknown. It is highly unlikely that low temperature, heat, injury, oils, toxic substances and hormones all act at the same site in plants. One hypothesis is that agents which produce sublethal stress cause plants to produce 'necrohormones' (Doorenbos 1953; Erez and Lavee 1974). The ’necrohormone' may be ethylene (Fuchigami and Nee 1987). In crabapple and red-osier dogwood, a positive correlation between ethylene production and the breaking of rest has been established with several rest-breaking agents (Nee 1986). Therefore sublethal stresses may overcome rest by stimulating ethylene production and/or increasing membrane permeability. Increased production of ethylene due to sublethal stresses may be due to the release or activation of the ethylene forming enzyme (EFE) that is reported to be associated with membranes and is required for the conversion of ACC to ethylene (Mayak et al. 1981). 16 HQIEQDES gibberellins; Among the promoters of seed germination special emphasis has been given to gibberellin (GA) because of its pronounced germination-stimulatory effect and its common occurrence in seeds during dormancy removal. Endogenous gibberellins are presumed to play a role in the chill-related dormancy mechanism. In general, dormant seeds contain minute amounts of gibberellins and these increase during the chilling process. In contrast, Ross and Bradbeer (1971) reported that chilled hazel nut seeds had to be transferred to warm temperatures before substantial amounts of gibberellin were detectable, suggesting that chilling removes blocks to gibberellin biosynthesis. Several inhibitors of GA biosynthesis were ineffective during chilling but inhibited germination following chilling (Ross and Bradbeer 1971). The first evidence supporting the involvement of GA in dormancy release came from exogenous application of GA3, which breaks the dormancy of peach buds and hazel nuts (Walker and Donoho 1959, Jarvis et. al. 1968). In general, however, treatment with GAs cannot entirely replace the chilling requirement (Walker and Donoho 1959; Powell (1987). Werk with physiological dwarf plants has also implicated the involvement of gibberellins in chill-related dormancy. Seedlings developing from unchilled embryos are dwarfs, but normal growth can be stimulated by chilling the l7 seedlings or treating them with GA's (Barton 1956; Bloomaert and Hurter 1959). However, a single application of GA is not sufficient; a continuous supply is necessary for sustained growth, suggesting that the chilling process activates the biosynthesis of gibberellins (Powell 1987). Furthermore, the treated seedlings often exhibit abnormalities associated with insufficient chilling. Current evidence suggests that GA may may break dormancy through the stimulation of an enzyme, acid lipase, which breaks down reserve lipids. However, the gibberellin- mediated hydrolysis of reserve lipids cannot be the only mechanism involved in the removal of apple embryo dormancy (Zarska-Macikerska et al. 1980). The presence of acid lipase with an optimum at 5C was also observed in hazel nut (Ross 1983). ergkininfir Both bound and free cytokinin-like compounds occur in mature apple seeds and increase during stratification (Borkowska and Rudnicki 1974). Cytokinin- like activity reached a maximum in the 5th week, but the increased level of cytokinins was not directly correlated with the ability of the seeds to germinate (Borkowska and Rudnicki 1974; Kopecky et al. 1975). Among the 6- substituted purines tested, 6-benzylaminopurine (BA) was especially active in stimulating germination of seeds in a number of species (Van Overbeek 1966). Other investigators 18 have broken apple embryo dormancy with 10 to 25 mg/l BA (Badizadegan 1967; Zhang and Lespinasse 1991; Lewak and Bryzek 1974). Although application of cytokinins to dormant or partially chilled seeds has promoted growth in some cases, results have been inconsistent (Lewak and Bryzek 1974; Tzou et al. 1973). Auxin§_ang_grny1gngr There is no convincing evidence that these two hormones play an important role in regulating dormancy in buds or seeds in which chilling is required to break dormancy. In studies where a positive correlation between ethylene and dormancy release was obtained it was generally attributed to ethylene action on events after partial or full release from dormancy by chilling (Powell 1987; Ozga 1988). EBZIEEE Dormancy release has been closely associated with an increase in activity of enzymes. The activity of peroxidase, succinate dehydrogenase, lipases, and proteases in apple embryos increased seven-fold during chilling, and interruption of the cold stratification period caused a sharp decline in enzyme activity (Nikolaeva and Yankelevich 1974). The biosynthesis or release of hydrolytic enzymes is presumably under hormonal control (Burg and Burg 1962) and treatment of dormant seeds with GA4 or benzyladenine 19 increases the activity of lipase, phosphatase and peroxidases (Rychter and Lewak 1971; Rychter et al. 1971). Although a gradual rise in hydrolytic enzymes could be responsible for breaking of dormancy, such changes may be a result , rather than the cause of dormancy removal. Anaerobiosis Anaerobic conditions have been reported to break dormancy (Tissaoui and Come 1973). Apple embryo dormancy was eliminated at room temperature when imbibed embryos were held under nitrogen for 7 days (Barthe and Bulard 1983). More evidence was provided by Esashi et al. (1976) who broke the dormancy of cocklebur (XQBSBIBE) seed by anaerobic treatment. Anaerobiosis may induce changes in membrane permeability leading to the leaching of cell constituents including hormones implicated in the regulation of embryo dormancy. Hl§I§§§IB£§BI§l_£h§DQ§§E Dormant embryos have a minimal rate of metabolism and are highly resistant to adverse environmental conditions. Many reports are available concerning metabolic aspects of dormancy release but little is known about ultrastructural changes associated with them. Biosynthesis of and interconversions between the major reserves and the formation and proliferation of cellular organelles and 20 membranes that take place during stratification have been reported in many species (Villiers 1971; Lewak et al. 1975; Dawidowcez-Grzegorzewska and Maciejewska 1979; Bouvier- Durand et al. 1981; Kupila-Ahvenniemi et al. 1978). The following changes have been observed in the embryo meristem cells as a result of stratification: a) lipid and protein bodies decrease; b) development of organized endoplasmic reticulum lined with ribosomes develop d) Golgi bodies become numerous and active; e) plastids differentiate and starch grains appear and f) volume of the vacuole and nucleus increases. These changes take place during the breaking of dormancy by cold temperature, but little is known about the effects of other treatments which break dormancy, e.g., anaerobiosis or treatment with gibberellins, cytokinins, cyanamide or DNOC. Summary Mature apple seeds exhibit dormancy which may be released by either chilling at 5C for 2 to 3 months or treatment of the embryos with dormancy breaking chemicals. Many studies have emphasized seed dormancy and paid little attention to embryo dormancy which is the site of dormancy in seeds. Recent studies with peach have demonstrated that the chilling effects of low temperature (5C) can be enhanced by moderate temperatures (lo-15C) if alternated during 21 stratification. However the promotive effect of moderate temperatures has not been demonstrated in apple buds or embryos. High temperatures (20-30C) are known to negate the previous chilling effect, resulting in induction of secondary dormancy. Current evidence suggests that dormancy is regulated by a balance between growth inhibitors and promoters. Dormancy appears to be the result of low levels of gibberellins and cytokinins and high levels of ABA. Chilling apparently reduces the levels of ABA while promoting the synthesis of promoters and hydrolytic enzymes. GAs and cytokinins stimulate germination of non-chilled apple embryos and their effects are greater when the embryos are partially chilled. On the other hand ABA inhibits the germination of non- dormant embryos and counteracts the effects of promoters. Light and photoperiod appear to have little or no effect on dormancy release in apples. This thesis was designed to explore several aspects of dormancy in apple seeds, including response to alternating temperatures, comparison of the cytological effects of chilling with those of chemicals which break rest, and the effect of chilling on gibberellin metabolism. CHAPTER ONE, SECTION ONE THE EFFECTIVENESS OF CONSTANT VS ALTERNATING TEMPERATURES IN BREAKING THE DORMANCY OF APPLE SEEDS AND EMBRYOS THE EFFECTIVENESS OF CONSTANT VS. ALTERNATING TEMPERATURES IN BREAKING THE DORMANCY OF APPLE SEEDS AND EMBRYOS agrrrggr. The effects of constant vs. alternating temperatures in breaking dormancy of apple embryos and seeds were investigated. The germination response of the embryos at constant temperatures was bell-shaped, but skewed to the cold temperature side. Temperatures between 2.5 and 7C were the most effective in breaking dormancy, while -2.5, 0, and 15C had marginal effects. Temperatures higher than 15C either had no effect or were inhibitory. The chilling requirement of embryos was fulfilled earlier (6 weeks) than that of seeds (8 to 12 weeks). The rate of germination increased with increasing time of stratification. Mean time to germination was two days in fully chilled embryos and 7 days in non-chilled embryos. The chilling effect of 5C was enhanced by alternating with 10C on a daily cycle (16 h at 5C, 8 h at 10C), the effects of the two temperatures being additive. Higher temperatures either had no effect or were inhibitory. Parallel data were obtained with 3-week and 6- week cycles. The degree of negation by high temperature depended more on the temperature of alternation than on cycle length. Inhibition increased as temperature increased from 15 to 25C. Germination of seeds was nil following exposure to all alternating temperatures except 5/10C; in this case 10C negated the chilling effect of 5C. 23 24 Introduction Dormancy limits the production of deciduous fruits under tropical climates. The dormancy of both buds and seeds was broken by temperatures between 0 and 10C, with the optimum near 5C. (Abbott 1955; Gilreath and Buchanan 1981; Seeley and Damavandy 1985, Westwood and Bjornstad 1968; Erez et al. 1979a). Constant chilling temperatures broke dormancy while high temperatures interspersed with chilling temperatures negated the chilling effect (Couvillon and Erez 1985). However, moderate temperature (i.e, 15C), when alternated with a chilling temperature (SC), in a daily cycle, enhanced the chilling effect in peach buds. Constant 15C was ineffective while 21 or 24C was inhibitory. Moderate temperatures promoted rest completion only when applied at later stages of the chilling period. Temperatures of 21 and above negated the chilling effect if the exposure time was more than 4 hours per day, but became less effective as cycle time increased (Convillon and Erez 1985; Erez and Couvillon 1987; Erez et al. 1979a, 1979b). Aduib and Seeley (1985) and Mahhou (1991), using peach seeds, observed chilling enhancement by alternating so with 10C in a daily cycle, whereas chilling negation occurred when 5C was alternated with 15, 20 or 25C. Mahhou (1991) also observed that cycling was not necessary because a 3- week period at 10C given at the end of the stratification 25 period was as effective as the same total time of exposure to 10C on daily cycles. The effect of moderate temperatures on hastening dormancy release has been confirmed in nectarine buds (Ergnug pgrgiga negraring) (Gilreath and Buchanan 1981) and sour cherry (Ergngg geraggfi L.) (Felker and Robitaille 1985). The objectives of this study were : a) to determine the response of apple seeds and embryos to alternating temperatures in both daily and longer cycles; and b) attempt to explain the mechanisms involved during chilling enhancement or negation by alternating temperatures on the basis of the Erez et al. 1979, 1986 and Fishman et al. (1987a, 1987b) two-step model for induction and release of dormancy. Materials and Methods Elant_material Mature apple (Malgg ggmggriga, cv. Golden Delicious and Paulared) fruits were harvested from mature trees at the Horticultural Research center, Michigan State University, East Lansing, in 1989 and 1990. The seeds were extracted from the fruits, air-dried and stored at room temperature (21C). 26 E! !°El ll I . !° Seeds were soaked for 48 hours in distilled water, then placed in Petri dishes lined with Whatmann no.1 filter paper (Whatman No.1) wetted with 1% Captan [ N-(trichloromethyl thio)-4-cyclohexene-1,2-dicarboximide] solution. The Petri dishes were placed in growth chambers at various temperatures in the darkness. The germination capacity of the seeds or embryos was used as a measure of the degree of dormancy release. No germination occurred during stratification, regardless of treatment. At the end of each stratification period the seeds or excised embryos were germinated in Petri dishes lined with filter paper wetted with distilled water at 20C in darkness. Each Petri dish contained 20 seeds or embryos; 3 replicate dishes were used per treatment. The seeds or embryos were considered germinated when the radicle had grown at least 3 mm. Germination counts were made daily over a lO-day period and used to calculate germination percentages, and, in some cases, the rate of germination (mean days to germination or MDG) according to the formula of Tincker (1925) as follows:- 27 C Z" MDG 8 n 1 (X11) (n) X where Xn - number of seeds or embryos germinating on day n n a day on which germination occured in days c a duration of germination test in days X = total number of seeds or embryos which germinated over the germination period. m; If all seeds germinate on day 1, MDG = 1 x 20/20 = 1 day If all seeds germinate on day 10, MDG a 10 x 20/20 = 10 days 5 If 5, 10 and 5 seeds germinated on day 3, 4 and 5, respectively, MDG = 15/20 + 40/20 + 25/20 + 80/20 = 4 days EmrmentaLdeeisn Treatments were arranged factorially in a completely random design. The data were subjected to analysis of variance (ANOVA) and were analyzed both as percentages and as arcsin transformations. The data were also subjected to regression analysis, however no regression equation satisfactorily described the germination response curves. Consequently the data are presented as means of 3 replicates plus or minus standard error. 28 A", u‘! f - o. ‘ g I, '1- =--- 1 0 st. t :uv: -_ -. 6+ 01 1-..??‘9L‘! -:1I_1-_t-2 o 9- ‘1L3108 t 0C. ’Golden Delicious' and 'Paulared' apple seeds were stratified at -2.5, 0, 2.5, 5, 7, 10, 15 and 20C for 2, 4, 6, and 8 weeks. Embryos were excised from the seeds at the end of each stratification period and germinated at 20C for 10 days. A control treatment of non- stratified seeds was included. The germination percentage and rate of germination were calculated from the germination counts. A-‘ 'u-! - , .- ~ at- ',. ‘-ed 1 ’15 a t ‘,_.°=,°l_‘I ramp. 01! r wan-er 2”. :f--_‘ -_ 0 . 'Golden Delicious' and 'Paulared' seeds were stratified for 2, 4, 6, 8, 10, 12, 15 and 18 weeks at constant temperatures (5, 10, 15 and 200) and at alternating temperatures of 5/10, 5/15, 5/20 and 5/25 C in a diurnal cycle (16 hrs at SC and 8 hr at the higher temperature). An additional treatment of non-stratified embryos was included. Total stratification time for alternating temperatures was adjusted so that all seeds were exposed to SC for the same period of time. At the end of this time-the seeds were germinated at 200 over a 10-day period. 29 WW 0!; a! - 2 ’ z '1' 8u9‘ a -7‘: .1 1 4-90-_ 0 3- W at gm. Seeds of 'Golden Delicious' and 'Paulared' apple were held for 4 weeks at constant 5C, for 6 weeks at alternating temperatures in a 24-hour cycle (5/10) or for 6 weeks on a 3-week cycle with 5-5-10C or 10-5-5C for a total of 2 cycles. Total time at 5C was 4 weeks in all cases. At the end of this time, the embryos were excised from the seeds and germinated at 20C. W. ' n as ant ' ‘1 1 1'. !' '!'°5 . - ‘: .l . '-.“1 5, ‘ on ‘-!~‘°l_‘! .7191: “I “ l; "13": d. ° - °— ‘ -_ O 0 ’Golden Delicious' and 'Paulared' seeds were stratified for 4 weeks at constant 5C or for 6 weeks at alternating temperatures, to give an equivalent time of 4 weeks at 5C in a long cycle (six weeks total, two consecutive weeks at each temperature). The temperature was held constant at SC or alternated between SC and 10, 15, 20 or 25C, as follows : 5- 5-10, 5-10-5, 10-5-5; 5-5-15, 5-15-5, 15-5-5; 5-5-20, 5-20-5, 20-5-5; 5-5-25, 5-25-5, and 25-5-5. The embryos were excised at the end of the stratification period and germinated at 20c for 10 days. 30 Results m We 8 ti e onstant - -~ ., s-.-se-L -- .._ at'o; o rembgos t 20C. The germination capacity of non-chilled embryos of 'Golden Delicious' and 'Paulared' from 1990 harvest ranged from 28 to 38%. The germination response curves of the two cultivars at constant temperatures ranging from -2.5 to 20C were similar (Fig. 1). Temperatures most effective in releasing dormancy were between 0 and 10C with the optimum between 2.5 and 7C. Temperatures of -2.5C and 15C had a slight chilling effect while 20C was ineffective. The response of embryos chilled for 8 weeks was similar to that of embryos chilled for 6 weeks (data not shown) At the optimum temperature SC, germination capacity increased with increasing time of stratification, reaching 100% after 6 weeks. The germination rate increased significantly with increasing period of stratification (2 days for fully chilled embryos vs. 7 for non-chilled embryos). Non-chilled 'Golden Delicious’ embryos required seven days for germination while all embryos chilled for 6 or 8 weeks germinated within three days (Table l). Germination of 'Paulared' embryos after 2 weeks at 5b was 31 significantly enhanced when 5C was alternated with 10C, but the difference was non-significant in 'Golden Delicious’ (Fig. 2). The effect in 'Paulared' can be explained by the additive effects of exposure time at 10C. With more than 2 weeks at SC, 5/10C had no significant promotive effect. All other temperatures negated the chilling effect, and the degree of negation increased with increasing temperatures. In the case of seeds, the moderate to high temperatures either had no effect or negated the chilling effect of 5C during stratification in both 'Golden Delicious' and 'Paulared' (Fig, 3) No seeds germinated following exposure to 5/15, 5/20 or 5/25c. Ernsriment_3- Effest_2f_stratifxing_seegs_at_22n§tant - v. ‘1!— 10 ‘u9‘ -. _ ‘: '1: '0 ’."1 ‘ O! snbsssusnt_germinati2n.2f_shs_smbr22§_at_zgsi Germination of embryos was enhanced by alternating temperatures, regardless of cycle time, with one exception (10-5-5) for ’Paulared' (Fig. 4). However, there were no significant differences between 24-hr vs. 3-week cycles. germination response differed between cultivars (Fig. 5), therefore each will be discussed separately. No treatment 32 enhanced germination of 'Golden Delicious' embryos significantly; however, 20 or 25C in the last step of the cycle significantly inhibited germination. Exposure to 15C in the third step significanlty reduced germination in comparison with that of seeds exposed to 15C earlier in the cycle, but not in comparison with the control. Thus, a one week exposure to temperatures of 15 to 25C was inhibitory only when applied at the end of the 3-week cycle. Germination of 'Paulared’ embryos was promoted by only one treatment , 10C during the last week of the cycle and many other treatments significantly inhibited germination. Exposure to 25C inhibited germination regardless of timing, 20C was inhibitory only in the first or second weeks of the cycle, 15C inhibited only in the 2nd week, and 10C was not inhibitory at any time. .... -., . - - s .- .,~ -, -.[o; ;_ _ ;‘ ._,o_ e - “‘0‘, e! ,- 3 th,o 9 .39 emhrxe_dgrmansx goldgn_flgligigg§: Alternating temperatures of 5-5-10 in a 3- wk cycle significantly promoted germination (Fig 6). The effects of constant 5 and 10C were similar and the germination responses declined as temperatures increased from 10 to 25C. Daily alternating temperatures inhibited germination whenever the high temperature was 20 or 25C. The inhibitory effects of 15, 20 and 25C in the daily cycle 33 were similar to those in the 6-wk cycle. Exposure to 25C completely negated the effects of chilling regardless of cycle time. ngnlgrgdL: The data for' Paulared' generally paralleled those for 'Golden Delicious' (Fig. 6). However, differences between treatments were more pronounced on daily cycles and less pronounced on longer cycles. Alternating temperatures of 5-5-10 in a 3 or 6-wk cycle promoted germination significantly relative to the control. Discussion Seed dormancy is a complex phenomenon controlled by a large number of factors. Embryos were used in this study because they are more sensitive than seeds to factors that induce or release dormancy. About 35% of the embryos were not dormant at harvest time. This may be attributed to the effect of environmental factors such as temperature during seed development, or to genetic variability as a result of cross pollination, although both ’Golden Delicious' and 'Paulared' are self-fruitful. The chilling requirement of apple embryos was fulfilled earlier (6 to 8 weeks) than that of seeds (10 to 12 weeks). Thus, the seed coat has an inhibitory effect. A longer chilling period may be required for softening the seed coat, or for leaching of inhibitors from it, or may merely 34 increase the vigor of the radicle sufficiently to enable it to break through the seed coat. The effective constant chilling temperature range of 0- 10C was similar to that reported for apple seeds (Abbott 1955; Aduib and Seeley, 1985; Del Real Laborde 1987; Ozga 1989; Purwoko 1990; Seeley and Damavandy 1985). However, the optimum chilling temperature range (0-10C) for embryos was broader than that of seeds. It is known that the effectiveness of chilling temperatures is dependent on the chilling requirement of the cultivar (Gilreath and Buchanan 1981). The effective chilling temperature range is narrower for high than low chilling requirement cultivars. It is therefore suggested that low chilling requirement cultivars are adapted to mild climatic conditons due to the widening of the temperature range over which chilling is effective. It is also reported that species originating from warmer climates have a lower chilling requirement, broader temperature response curve and a higher optimum temperature range than those from colder climates (Gilreath and Buchanan 1981b; Seeley and Damavandy 1985; Westwood and Bjornstad 1968). These observations imply that the greater the intensity of dormancy the narrower the effective chilling temperature range. Embryo dormancy is less than that of whole seed dormancy which is attributed to both the embryo and seed coat. A temperature of 15C had a marginal chilling effect while 20C and 25C had none. The rate of germination 35 increased with increasing periods of stratification. Alternating 5C with 10C in a daily cycle promoted the germination of embryos but temperatures higher than 10C either had no effect or were inhibitory. Parallel data were obtained with 3- and 6- week cycles. The stimulatory effect of 5/10C was largely the result of additive effects of the two temperatures. The inhibition increased as temperatures increased from 15C to 25C, although 25C was often no more inhibitory than 20C. These data are in agreement with those obtained by Aduib and Seeley (1985) with apple seeds and Mahhou (1991) with peach seeds. Alternating 5 with 10C reduced the germination capacity of seeds whereas no seed germinated following exposure to 15, 20, 5/15, 5/20 and 5/25. These data corroborate those of Porwoko (1991) and Del Real Laborde (1987). Therefore the degree of negation by high temperatures depended more on the temperature of alternation than on cycle length, i.e., the higher the temperature the greater the chilling negation. Weinberger (1954) reported that the opening of peach leaf buds was reduced by 33% when the temperature was raised from 10C to 18C for 15 days while raising the temperature to 22.2C for the same period reduced the bud break by 80%. Furthermore chilling negation by high temperature is dependent on the chilling requirement of the cultivar. Chilling negation by high temperature is less pronounced in low than high chilling peach cultivars (Gilreath and Buchanan 1981a). As 36 a result, low chilling requirement species have a higher optimum chilling temperature than high chilling requirement species and such species could be more tolerant to high temperatures. Why embryo dormancy is broken by 5/10C while seed dormancy is not remains an enigma. Conceivably the seed coat could restrict respiration at the higher temperature, leading to the accumulation of toxic products. The intensity of whole seed dormancy is higher than that of embryo, therefore the latter has a broader effective chilling temperature range than the former. The beneficial effects of moderate temperatures during the last stages of chilling for both ’Golden Delicious' and 'Paulared' cultivars may be attributed to the promotion of germination processes, chilling effects due to a change in optimum temperature, and widening of the effective chilling temperature range as stratification proceeded. The inhibitory effect of moderate temperatures given in the middle of the chilling period is attributed to the sensitivity of partially chilled seeds or embryos to high temperatures. However, it is difficult to explain why the moderate temperatures would inhibit germination when applied at the beginning of the chilling period since such temperatures would be expected to have no effect on subsequent chilling. The following model was proposed by Erez and Couvillon (1986) to explain the promotive effect of moderate 37 temperatures on breaking dormancy of peach buds. a C A B C h. 45 ‘ b Chilling converts a precursor (A) to an intermediate (B) which , upon further chilling, is fixed by conversion to a dormancy breaking factor (C). High temperatures enhance dormancy release by promoting the conversion of B to C. Alternating temperatures enhance the effect of chilling in peach buds provided the higher temperature does not exceed 15C (Erez and Couvillon 1987). This scheme does not explain the inhibitory effect of moderate temperatures during the early stages of seed stratification as shown by these results. Similar effects were apparent but ignored in the studies with peach buds (Erez and Couvillon 1987). Fishman et al. (1987) provided a mathematical analysis which attempted to rationalize the effects of moderate temperatures during cycling. The scheme, as shown below, assumes the existence of a thermally unstable precursor (PDBF) formed from the dormant state. ko \‘ PDBF 4 DBF a/( Once PDBF reaches a critical level it undergoes an irreversible conversion to the dormancy breaking factor (DEF) by chilling temperatures. The basis for the promotive effects of alternating temperatures between 4 and 15C is 38 that the initial exposure to 15C causes PDBF to accumulate faster innitially. When the temperature is shifted to 4C, less time is needed to reach the critical level. Hence more DBF accumulates over a given time period. Cycle time is crucial in this scheme if dormancy is to be broken more . rapidly. The critical level of PDBP is reached faster when low temperature is alternated with moderate temperature than 5C, hence the chilling efficiency is enhanced. The total time required to accumulate the amount of chilling units is shorter at cycling temperatures than at constant low temperature. However, alternating temperatures do not enhance the effect of chilling in apple seeds and embryos supporting previous work with apple (Porwoko 1980; Del Real Laborde 1987; Aduib and Seeley 1985) and peach seeds (Mahhou 1991; Aduib and Seeley 1985). Therefore if these models are valid they must apply only to buds of peach. A somewhat different set of temperature conditions may be optimal, with seeds responding better to constant temperature. This hypothesis can only be tested by comparing seed and bud responses to alternating temperatures in the same experiment. These data show that cycling was either ineffective or inhibitory; the promotive effect of 10C on embryo germination was largely the result of additive effects of the two temperatures (5/10C). Saure (1985) suggested the existence of two distinct but overlapping temperature reactions; one producing a 39 dormancy breaking factor by chilling temperature while the other produces a factor which maintains and/or enforces dormancy by high temperature. The first reaction has a low temperature range at first, but as chilling accumulates the effective temperature range widens. The inhibitory potential is higher during the early stages of chilling hence could be negated by both moderate and high temperatures. As chilling proceeds the buds accumulate sufficient chilling units that the second reaction can proceed and the range of temperatures capable of negating subsequent chilling becomes narrower ( > 23C). These data partially support the hypothesis based on widening of the temperature range for the breaking of dormancy as the chilling process proceeds at low temperature. 40 Figure 1. Effect of stratifying seed at constant temperatures on subsquent germination of the embryos at 20C. (1990 seed source, Expt. 1) GERMINATION ($) 41 Figure 1. 1m .4 G. Delicious Chill M06 ("'0 '.' . ——o— o . ' —o— 2 I - -——