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I .filoyvflhr I IIIIIJQNIIIIUIIJQI I. .I v... ..I III. ‘IIH‘ND .III’\.IIIIJ I," Inc. I II.VIII«o’.llII'|IIIQ .1. I! I I I A . 1 IV I t a! I. I I. o c J THESIS "Am-«— —- I? 9:...“ n 73-» r .i-1 .~‘ dQ-(J’fi'. .31! IIIIIIIII III I III IIIIIIWII 74 6436 BS’JQl ' $.33; iiltate This is to certify that the dissertation entitled Hormonal Control of Fruit Development in Strawberry (Fragaria g ananassa Duch.) presented by Douglas D. Archbold has been accepted towards fulfillment of the requirements for Ph. D. Horticulture degree in Mfl/QW ”IL Major professor Date August 4, 1982 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 4 k —r—v———-‘-4 4‘ MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from .1; YOU!“ record. FINES Will be charged if book is J returned after the date 3 I 2. stamped below. HORMOHAL CONTROL OF FRUIT DEVELOPMENT IN STRAWBERRY (EBAGARIA X ANANASSA DUCH.) By Douglas 0. Archbold A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture l982 (ixlcw?<»o ABSTRACT HORMONAL CONTROL OF FRUIT DEVELOPMENT IN STRAWBERRY (FRAGARIA X ANANASSA DUCH.) by Douglas D. Archbold Indoleacetic acid (IAA) was identified by gas chromatography- mass spectrometry in extracts of achene and receptacle tissue of strawberry. Free, ester-conjugated and amide-conjugated IAA present in achene and receptacle tissue from anthesis to maturity were Quan- tified by a double-standard isotope dilution method using ‘4 and 14C-indolebutyric acid as internal standards. Whole fruit at C—IAA anthesis contained high concentrations of free IAA, small amounts of ester-conjugated IAA, and no amide-linked IAA. Maximum concentrations of free IAA in achenes were 4- (1980) and 1.2-fold (1981) as high as those in receptacle tissue; maxima occurred simultaneously in the two tissues 16 (1980) and 14 (1981) days after anthesis. The maximum concentration of ester-conjugated IAA in achene tissue was 3.4- (1980) and 17.5-fold (1981) as high as that in receptacle tissue with the maxima occurring at 6 (1980) and 8 (1981) days after anthesis with a second maximum at 22 days after anthesis in 1980. Amide-conjugated IAA was found in significant quantities in achene tissue at 11 days and again at maturity. Maximum concentrations in achenes were Douglas 0. Archbold 21- (1980) and 34-fold (1981) as great as those in receptacle tissue. Secondary fruit on the cyme contained higher concentrations of free IAA in the achenes, but not in the receptacle tissue, than did pri- mary fruit. Abscisic acid was detected in whole fruit at anthesis by electron capture gas chromatography. The concentration in the achenes declined until midway through development, then increased as fruit approached maturity. The total quantities in both achenes and receptacle increased as fruit ripened. The ratio of free IAA to free ABA changed during fruit development but was not well correlated with fruit growth rate. Treatment of emasculated flowers with aqueous solutions of naphthaleneacetic acid (NAA), gibberellins (6A3, GA4/7),NAA + 0A3, or NAA + GA4/7, each at 10'3 M in 2% dimethylsulfoxide (DMSO) and 0.1% Tween 80, induced parthenocarpy. A11 fruits stopped growing within 12 days of treatment, except those induced with NAA or NAA + 0A3. Re-treatment with the same or another growth regulator at 20 days after initial treatment stimulated continued growth as compared to no re-treatment. All induced fruits were considerably smaller than pollinated fruit; sizes ranged from 40 to 70% of pollinated fruit. Free IAA concentration in NAA—treated fruit was 5-fold as high as that in control flowers and 3-fold as great as that in GA4/7- treated fruit 6 days after treatment. By 14 days after treatment levels had declined in all treated fruit. These results are discussed in relation to the role of IAA in strawberry fruit development. Douglas 0. Archbold Complete achene removal 12 days after pollination stopped receptacle growth. Removal of achenes from half of the receptacle diminished growth as compared with that of intact fruit. Free IAA concentration in the receptacle tissue of intact fruit at 14 days after pollination was nearly equal to that in achenes. Removal of achenes significantly reduced the IAA content of receptacle tissue, complete removal being more effective than partial removal. The extent of achene removal and growth rates of receptacles were posi- tively correlated with free IAA content. Achenes were removed from some fruits 16 days after pollina- tion, and the receptacles were treated with aqueous solutions of 3 NAA, 6A3, or GA at 10' M in 2% DMSO and 0.1% Tween 80. None of 4/7 the growth regulators were as effective as were achenes in maintain- ing growth. NAA treatment of receptacles produced fruit 75% the size of controls, while fruit treated with 0A3 and GA4/7 did not grow. Removal of achenes 24 days after pollination had no effect on enlargement of receptacles. For Rhonda ii ACKNOWLEDGMENTS I would sincerely like to thank Dr. Frank G. Dennis, Jr., for six years of skillful guidance and support and for whom I have tremen- dous respect and admiration as a horticulturalist, scientist, and human being. Many thanks go to Drs. J. A. Flore and A. J. M. Smucker for their continuing encouragement and efforts to improve my scien- tific acumen during both my M.S. and Ph.D. programs. Also, I would like to thank Drs. M. J. Bukovac and C. J. Pollard for their helpful advice and critiques during the course of this project. This research would have been impossible without the willing cooperation of Dr. R. S. Bandurski for making his lab facilities available for the analyses. Thanks also go to Dr. J. D. Cohen for teaching me analytical methods used in this study, and to the other members of the lab, J. Chisnell, P. Hall, D. Reinecke, and Y. Momonoki, for their advice and assistance. I would like to recognize Don Gibbs of Gibbs Berryland, Onondaga, M1, for his friendly cooperation in the use of his field plantings of stawberries, and Ahrens Nursery, Huntingburg, IN, for their donation of strawberry plants used in this work. Last, but as important as all others, I thank my wife, Rhonda, and son, Shane, for their years of love and devotion without which I could not have maintained my pursuit of this degree. TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . V11 LIST OF FIGURES . . . . . . . . . . . . . . . viii LIST OF ABBREVIATIONS . . . . . . . . . . . . . X LITERATURE REVIEW 1 Role of Seeds in Fruit Development. . . 1 Association between seeds and fruit development . 1 Seeds as sources of hormones 2 Strawberry. A model system for studying the role of seeds in fruit development . 2 Strawberry Fruit Development and Morphology. 2 Role of Hormones in Fruit Development. 3 Exogenous control--in vivo studies 4 Exogenous control--1n v1tro studies . . 8 Endogenous control--significance of achenes and the. calyx . . . . . . . . . . . 9 Endogenous control-~hormones . . . . . . . . 11 Summary. . . . . . . . . . . . . . 15 Literature Cited. . . . . . . . . . . . . 18 SECTION I QUANTIFICATION OF FREE ABSCISIC ACID AND FREE AND CONJUGATED INDOLEACETIC ACID IN STRAWBERRY (FRAGARIA X ANANASSA DUCH.) ACHENE AND RECEPTACLE TISSUE DURING FRUIT DEVELOPMENT . . 24 Abstract . . . . . . . . . . . . . 25 Materials and Methods . . . . . . . . . . . 28 Plant material . . . . . . . . . . . . . 28 Tissue preparation . . . . . . . . . . . 29 IAA analysis . . . . . . . . . . . . . 29 Gas chromatography . . . . . 30 Calculation of endogenous IAA in sample . . . . 32 GC- MS identification . . . . . . . . . . . 32 ABA analysis . . . . . . . . . . . . . 33 iv Identification of IAA . . Effect of lyophilization on free IAA content Variability in IAA recovery among replicate samples . . . Levels of free and conjugated IAA during fruit development. . . . . . . . Free IAA in secondary fruit Free ABA in primary fruit Discussion . . . . Literature Cited SECTION II EFFECTS OF EXOGENOUS APPLICATION OF AUXIN AND/ORGIBBERELLIN ON STRAWBERRY FRUIT SET, GROWTH AND ENDOGENOUS INDOLEACETIC ACID CONTENT. . . . . Abstract . . Materials and Methods . Results . . . Effects of hormones on fruit development . Effects of hormones on free IAA content Discussion . . . . . . . . Literature Cited SECTION III EFFECTS OF STRAWBERRY ACHENE REMOVAL ON RECEPTACLE GROWTH AND FREE INDOLEACETIC ACID CONCENTRATION AND OF AUXINS AND GIBBERELLINS IN REPLACING ACHENES. . . Abstract . . Materials and Methods . Plant material . . Pollination and removal of achenes . Achene substitution . Results . Discussion . Literature Cited CONCLUSION Parallel variation Source excision . Substitution . Page 58 59 60 61 61 62 69 73 74 75 77 78 78 79 84 86 87 87 89 Isolation Generality Specificity . Literature Cited vi Table LIST OF TABLES SECTION I Rate of receptacle growth and quantities of free, ester- and amide-conjugated and total IAA in achene and receptacle tissue of primary 'Sparkle' strawberry (Fragaria x ananassa Duch.) fruits from anthesis to maturity, 1980 Rate of receptacle growth and quantities of free- ester- and amide-conjugated and total IAA in achene and receptacle tissue of primary 'Midway' straw- berry (Fragaria x ananassa Duch.) fruits from anthe- sis to maturity, 1981 . . . . . . Concentration of free IAA in achene and receptacle tissue of secondary strawberry (Fragaria x ananassa Duch.) fruits during their development Quantity of free ABA in achene and receptacle tissue of primary strawberry (Fragaria x ananassa Duch.) fruits from anthesis to maturity vii Page 38 39 48 49 LIST OF FIGURES Figure Page SECTION I 1. Flow diagram of procedure for purification and prepa- ration of strawberry achene and receptacle extracts for GLC analysis of free and conjugated IAA . . . . 3l 2. Mass spectra of putative Me-IAA in methylated extracts of achene (A) and receptacle (B) tissue from 'Midway' strawberry fruits harvested 14 days after anthesis, and of authentic Me-IAA (C) . . . . . . . . . 35 3. Receptacle and achene weights and concentrations of free, ester-conjugated and amide-conjugated IAA in primary 'Sparkle' strawberry fruit from anthesis to maturity, 1980 . . . . . . . . . . . . . . 4O 4. Receptacle and achene weights and concentrations of free, ester-conjugated and amide-conjugated IAA in primary 'Midway' strawberry fruit from anthesis to maturity, 1981 . . . . . . . . . . . . . . 42 5. Total free, ester- -conjugated and amide- conjugated IAA per primary strawberry fruit in achenes (A, C) and receptacle (B, D) . . . . . . . . . . . . . 44 6. Concentration (A,B) and total amount of ABA per pri- mary fruit (C,D) in achenes (A,C) or receptacle (B,D) of 'Sparkle' and 'Midway' strawberry from anthesis to maturity in 1980 and 1981 . . . . . . . . . . 50 SECTION II 1. Effect of application of aqueous solutions of NAA, , or GA3 at 10 3M in 2% DMSO and 0.1% Tween 80 to agglulated3 strawberry flowers on fruit growth from anthesis to maturity. . . . . . . . . . . . 63 2. Effect of re- -treatment of parthenocarpic strawberry fruit with NAA or GA4/7 at 20 days after initial treatment on subsequent growth . . . . . . 65 viii Figure Page 3. A. Effect of application of aqueous solutions of NAA or GA4{7 at 10' M in 2% DMSO and 0.1% Tween 80 to emascu ated strawberry flowers on fruit growth between 6 and 14 days after treatment . . . . . . . . 67 8. Free IAA concentration in induced fruits (recepta- cles plus achenes) 6 and 14 days after treatment . . 67 4. Log free IAA concentration vs growth rate of partheno- car ic strawberry fuit induced with NAA or GA4/ 7 at 10- M in 2% DMSO and 0.1% Tween 80 at 6 and 14/ days after treatment . . . 70 SECTION III 1. Effect of achene removal on growth of (A, C) and free IAA concentrations in (B, 0) 'Midway' (A, B) and 'Our Own' (C, D) strawberry receptacles . . . . . 80 2. Effect of removal of achenes 16 days after pollina- tion of 'Our Own' flowers and treatment of recepta- cles with aqueous solutions of growth regulators at 10'3M in 2% DMSO and 0.1% Tween 80 upon the ratio of diameter (Dx) at 18, 24, or 30 days after opollina- tion to diameter (016) at 16 days . . . . 82 ix ABA DMSO EC EtOH EtOAc FID GA IAA IBA MeOH NAA NAD,NAAm NOA NSD THF LIST OF ABBREVIATIONS abscisic acid dimethylsulfoxide electron capture ethyl alcohol ethyl acetate flame ionization detector denotes the series of gibberellins--use of a sub- script denotes a specific gibberellin, as GA3 indoleacetic acid indolebutyric acid methyl alcohol naphthaleneacetic acid naphthaleneacetamide naphthoxyacetic acid nitrogen sensitive detector tetrahydrofuran Guidance Committee: The journal-article format was adopted for this dissertation in accordance with departmental and university requirements. Three sections were prepared and styled for publication in the Journal of the American Society for Horticultural Science. xi LITERATURE REVIEW The tree fruit industry relies heavily on several plant growth regulators to control various aspects of fruit physiology (56), but with the exception of fruit thinning, only limited control of fruit set and early development has been realized (l4). Species and cultivar differences in response to applied compounds have contribu- ted to the problem; thus, the goal of regulated cropping has remained elusive since F. G. Gustafson demonstrated that plant hormones induce parthenocarpy (18). A better understanding of the role of endogenous plant hormones in fruit set and development could provide a. basis fOr developing effective means of controlling yield. This review will be concerned primarily with strawberry fruit set and development and the control of these phenomena by endogenous and exogenous growth regulators. Although many other species have been studied (9,10), strawberry represents a model system. Role of Seeds in Fruit Development Association between seeds and fruit development. Though not essential to fruit development, as evidenced by parthenocarpy, seeds play an important role in influencing the growth of surrounding fruit tissues. Seed distribution can affect both fruit shape and volume (32,37,54). Fruit weight is directly correlated with achene or seed number in strawberry and other multi-seeded fruit (1,37,38). Premature seed removal may result in cessation of both growth and nutrient accumulation in the surrounding tissues (28,38), or hasten ripening of fruit approaching maturity (38,53). Seeds as sources of hormones. Immature seeds are rich sources of hormones. Because these compounds affect fruit growth when exo- genously applied (26,38,58), they may be responsible for the effects of seeds. However, the presence of a multiplicity of hormones makes the determination of their precise roles difficult,for little is known about hormonal interactions. Strawberry: a model system for studying the role of seeds in fruit development. Strawberry, Fragaria spp., is not a true fruit. The fruit are the individual 'seeds' which lie on the surface of the fleshy receptacle; they are actually ovaries, termed achenes. Horti- culturally, the strawberry receptacle is a fruit; it accumulates sugars and ripens as do true fruits. Most fruits bear seeds within the fleshy tissue; seed removal incurs extensive damage to the flesh. Because the achenes are on the surface of the strawberry receptacle, they can be removed with minimal damage to the supporting tissue. Thus, a model system for studying the influence of seeds on fruit development exists. Further discussion will be confined to this system. Strawberry Fruit Development and Morphology The strawberry fruit is borne on an inflorescence with one primary, two secondary, four tertiary, and up to eight quaternary flowers (13). The flower has five sepals and petals, while stamens occur in multiples of five. The number of achenes on the surface of the receptacle ranges from 50 to 500. The petals and stamens senesce within 24 to 48 hrs after pollination and receptacle enlarge- ment begins within 48 hrs, most growth being due to cell enlargement, cell division occurring at a low rate (21). Receptacle enlargement continues for ca. 25 days and exhibits a sigmoidal pattern. Achene enlargement is almost linear until maturity (11). Change from the free nuclear to cellular endosperm occurs at 10 to 14 days after pollination (49). Fruit mature about 30 days after pollination but varietal differences and environmental factors can shorten or prolong this time. Unpollinated flowers remain attached to the plant; an abcission zone is absent from the pedicel. Primary fruit are the largest, followed by secondary, terti- ary, etc. Berry size is linearly proportional to achene number, and the larger the receptacle, the more achenes it bears (60). However, berry weight per achene is relatively constant across classes (34), and achene number and spacing can be used to calculate potential yields (2). Because primary flowers have the largest receptacles and quaternary flowers the smallest, both achene number and berry size decline with class (1?, 2°, etc.). Role of Hormones in Fruit Development Hormones may control two distinct yet related phenomena: fruit set and fruit development. Fruit set is considered an induce- tive phase beginning at pollination or chemical treatment. Growth of the ovary and/or associated structures begins within a short time, often prior to fertilization. Accompanying morphological changes include petal and stamen wilting and abscission (26). The transition from a flower into a young fruit is termed fruit set. Fruit develop- ment begins with the initial swelling of the fruit tissues and ends at maturity. It is characterized by increasing fresh and dry weights and changeS'h1sugar, starch, and organic acid content (8). Exogenous control--in vivo studies. Due to their external location on the receptacle, achenes could be considered as an exo- genous factor in controlling fruit development. However, their con- tinuity with the tissues of the receptacle preclude considering them as exogenous. This term will be reserved for influences arising out- side the plant. Lanolin paste was the carrier of choice in early experiments with applied growth regulators (30,31,36,37,48,50,51,52). Although lanolin provides a continuous supply of the hormone, it does not allow calculation of the precise dose. Aqueous applications have often been ineffective, possibly because of limited uptake by the treated tissues. Mudge et a1. (35) found that addition of DMSO doubled the response to auxin and resulted in 100% fruit set. Thus, penetration of the applied compound was rapid and effective, permitting the study of dosage effect without the uncertainty involved with the use of lanolin. Treatment of whole plants of pistillate varieties with aqueous or ethanolic solutions of growth regulators has had limited effect in inducing parthenocarpy. Gardner and Marth (16) reported the first success. Although high concentrations of IAA were effective when applied at full bloom, no more than one fruit per inflorescence was obtained. Achenes developed but were devoid of embryos. Vapors of methyl and ethyl esters of NAA (64) and single and repeated appli- cations of IBA, NOA, GA, or combinations thereof were relatively ineffective in inducing fruit set (51). Tafazoli and Vince-Prue (48) reported that GA3 treatment of perfect-flowered cultivars prior to anthesis inhibited fruit set by inducing sterility. Though NOA applied prior to anthesis can increase fruit set, it cannot overcome the inhibitory effect of GA3. Erratic or incomplete pollination, generally due to poor pollen production,although environmental factors cannot be excluded, results in low yields and small or misshapen fruit (5,19,25,29). Applications of aqueous sprays containing IAA, NOA, and NAA to Open- pollinated plants at bloom or thereafter have increased yields (6,45, 63,65); however, the investigators have seldom estimated the extent of pollination or distinguished between effects on fruit set and fruit development. Most reports of increased yields cite increased numbers of 'normal' fruit, although Swarbrick (45) states that increased fruit size rather than fruit number was responsible for the higher yield. Thus, spraying whole plants with auxin enhances fruit development, possibly substituting for those achenes not pollinated. Field applications of GA at or shortly after bloom to open-pollinated varieties may increase yields or fruit size (6,48), but reports are contradictory. The evidence suggests that low concentrations may be promotive while high concentrations have an inhibitory effect. Treatment of individual flowers has been more effective than applications to whole plants in most studies. Hunter (22) and Wong (62) reported that applications of NAA and IBA in 95% EtOH to blos- soms stimulated parthenocarpy, though percent set was not reported. Tukey (55) was unable to induce parthenocarpy in a single fruit in an extensive study utilizing 60 varieties of strawberry and several auxins. On the other hand, Lord and White (31) succeeded by using much higher concentrations of auxin in lanolin paste. IBA application resulted in 90% of the treated flowers setting fruit,NAD in 60%, and NOA, NAA, and IAA slightly less than 50%. Thompson (50,51,52) induced fruit set with GA3, GA4/7, and auxins, but subsequent development varied with the chemical treatment. Cytokinins alone are unable to induce fruit set and do not affect the response to auxins when applied simultane- ously with them (50). Parthenocarpic strawberry fruits induced by auxin applied in lanolin vary in size from 50 to 100% of that of pollinated fruit (51,52), the response depending on the specific auxins applied and their concentrations. Varieties and species differ with respect to response to specific auxins. Generally, IBA, NOA, NAA, and IAA all stimulate parthenocarpic development, but IBA produces small fruit, NOA large fruit, and NAA and IAA fruit of intermediate size. Parthenocarpic fruit are usually smaller than pollinated con- trols, although varieties differ in response. 'Freya' flowers treated with NOA produced fruit nearly equal in size to pollinated controls, while 'Tardive de Leopold' produced fruit only half the size of control fruit. Between 10 and 20 days after treatment, the growth rate of auxin-treated parthenocarpic fruit declines (51,52). This suggests that most of the initially-applied dose has been util- ized. Re-application of auxin or GA or both increased final fruit size, which is a function of both initial and final treatment. When flowers were treated with NOA, then retreated after 10 days with either NOA or 6A3, GA3 induced the greater response (52). Fruit induced with 6A3 responded equally well to retreatment with NOA or GA3. Mudge et al. (35) reported that parthenocarpic fruit ceased growth about 10 days after treatment with aqueous solutions of auxin containing DMSO and would not resume growth unless retreated. Retreatment was not necessary when lanolin paste was used. GA4/7 is more effective than GA3 at equivalent concentrations (51). At optimal concentrations, both yield fruit only slightly smaller than auxin-induced fruit. Synergism between auxins and GAs has been noted (51,52). NOA-induced fruit were half the size of pollinated controls in 'Tardive de Leopold'; adding GA3 increased fruit size to 75% of that of the controls. All results are complicated by the effect of the applied growth regulator on maturation. NAA- and GA-treated fruit mature early, IBA- and NOA-treated fruit mature late (50,51,52). Retreat- ment of auxin-induced fruit with GA shortens the time to maturity. The longer the developmental period, the larger the fruit. Thus, NOA produces large fruit but the growth rate is low. Removal of fertilized achenes before 8 days after anthesis prevents receptacle growth regardless of subsequent treatment (35,55), possibly because of wounding and desiccation. Nitsch (36,37) removed achenes 8 days after pollination and substituted lanolin paste con- taining IBA or NOA. He obtained fruit nearly as large as intact fruit. A more extensive study of 15 auxin analogs demonstrated that IBA and NAA were more effective than IAA and NOA (35). 6A3 is less effective than auxin (46). Exogenous control--in vitro studies. Culturing fruits in_ vitro has provided a useful means of examining more closely the effects of growth regulators on fruit set and development. Accord- ing to some investigators, inclusion of auxins such as NAA, NOA, and NAAm, and of 6A5 and kinetin in the supporting media stimulates receptacle growth; other workers have been unsuccessful in similar attempts (3,12,17,23). The reasons for these contradictions are not apparent unless varietal differences affected the results. Intact receptacles jg_ijg_respond to a variety of auxins as described earlier, thus the use of different auxins cannot be the explanation for these discrepancies. Other chemicals such as ABA and maleic hydrazide do not promote receptacle growth (3,17,23). There are no known reports of sub-optimal auxin concentrations promoting set when combined with another growth regulator, such as GA or cytokinin. Effects of growth regulators on parthenocarpic fruit growth, size, and length of development in_vitrg have also been noted. NAAm alone promotes receptacle enlargement and synergizes with GA3 (23). NAAm tends to delay ripening while 6A3 hastens it. Both the applica- tion of cytokinins and the presence of intact carpels delay both growth and ripening. Endogenous control--significance of achenes and the calyx. The discussion to this point has dealt with the application of growth regulators to intact flowers, i.e., achenes plus receptacles. The response differs following the removal of achenes, which appear to mediate fruit set and initiate fruit growth. Intact achenes are required for the receptacle to begin growing following fertilization or growth regulator application j_'vjvg, Achene removal during the first week after anthesis results in severe wounding of the receptacle and growth of the naked receptacle cannot be induced (35,56). This failure suggests that the achenes supply more than auxins or GAs to the receptacle. However, wounding may prevent response to applied compounds. Thompson (48) reported that unfertilized achenes inhibit growth of the adjacent receptacle tissue. Growth will not occur in this area even if all the surrounding achenes develop. One might assume that achene-derived hormones would diffuse in all directions to stimulate development, but the evidence indicates that there is a localization of the stimulus. Modification of the environment to stimulate production of receptacles with bracts in place of achenes, or treatment of inflorescences with maleic hydrazide to inhibit achene development, can result in naturally parthenocarpic fruit (48). Again, receptacle tissue adjacent to the few viable but unfertilized 10 achenes does not develOp. The mechanism responsible for this local~ ized effect of the achenes is not known. Receptacles cultured in vitro following achene removal at anthesis are capable of initiating growth when auxins or GA are supplied (12,17). Apparently, damage to the receptacle is slight, although this was not discussed. Intact achenes, whether viable or not, inhibit receptacle expansion significantly jn_vitrg (l7). Calyx removal accompanied flower excision in the cited studies. Although the calyx is not essential to fruit growth i_n _v_i_vg, i_n vitro work indicates that removal results in smaller fruit (3,57). The calyx may supply an unknown factor required for normal fruit growth which other plant parts supply in 1119. This factor cannot be replaced by auxin, GA, or cytokinin and thus would be absent in culture. Its absence may restrict embryo development, thereby reducing the pro- duction of growth promoter(s) and reducing fruit size. The inhibitor from achenes has been characterized to a limited extent. Crude aqueous extracts of achenes collected prior to bloom inhibit GA activity in the barley endosperm bioassay whereas extracts of fertilized achenes exhibit little or no inhibition (12). However, no attempt was made to separate promoters from inhibitors; therefore, an increase in GA content following fertilization could be responsible for the difference. Extracts of unfertilized and fertilized achenes are equally inhibitory to growth of receptacles jg_vjtrg (l7) and wheat coleoptile sections (46). Following normal fractionation procedures with ether, the inhibitor remains in the water fraction and is not adsorbed on cation exchange resin. 11 There is some evidence available that suggests that the fruit set stimulus may arise from locations other than the achene. Hunter (22) treated selected pistillate flowers on some inflorescences with IAA or NAA and noted that the receptacles of nontreated flowers on the same inflorescences grew. Accidental spraying was eliminated as a potential cause of the phenomenon. Thompson (48) observed a simi- lar effect. Following repeated application of 50 pg GA at weekly intervals to a mature leaf, swelling of the neck region of the pri- mary fruit occurred. Thus, the hormone must have moved from treated flowers or leaves to nontreated flowers. Endogenous control--hormones. Few studies have been made of growth hormones in strawberry fruit tissue. Nitsch (35,39) reported that auxin activity of achenes increased from 3 through 12 days after anthesis and then declined. Maximum levels detected by the .Aygga curvature bioassay were 0.3 to 0.5 ug IAA-equivalents per 100 mg dry weight. No activity was detected in receptacle tissue despite Nitsch's suggestion that auxin controlled receptacle growth and his demonstration that auxins could replace achenes in stimulating receptacle develOpment. Lis et a1. (29) reported that the peak in auxin activity in receptacle extracts coincided with that in achenes but that the maxi- mum concentration was lO-fold less per kg fresh weight. No activity was detected prior to 2 days after pollination. Although the devel- 0pmental period was longer in this study, the peak in auxin activity occurred at 7 to 8 days after pollination, earlier than that observed 12 by Nitsch. Activity in both tissues declined after 8 days but was detectable in achenes through maturation. The decline in auxin activity in the achenes occurs during berry swelling, and auxin production by the achenes correlates more closely with changes in the achene itself than with changes in recep- tacle growth. The peak in activity corresponds to the change from free to cellular endosperm about 10 days after pollination (49). The endosperm rather than the embryo may be responsible for auxin production. By periodic application of maleic hydrazide to fruits before and after pollination to inhibit achene development, Thompson (49) showed that a viable embryo was not required for receptacle enlargement to occur. He also noted that the greatest response to applied auxin occurred about 10 days after pollination. Thus, he suggested a 2-phase growth period. The first phase, pollination through 10 days, was characterized by low auxin sensitivity but the control mechanism was unknown. The second phase at 10 days and later was mediated by auxin. Nitsch (39) tentatively identified IAA as the major auxin present in the achenes, though several unknown compounds with auxin activity were present. He estimated the maximum concentration of IAA in the achenes at 1.5 to 2.0 pg per gram dry weight. This was less than half the total detectable activity. Thus, auxins other than IAA may be involved in controlling receptacle growth. The history, chemistry, and physiology of conjugated, or bound, auxins have been extensively reviewed (4,43). Though most 13 reviews deal with conjugates in relation to IAA metabolism and func- tion, a recent review presents evidence for other roles (7). IAA conjugates may participate in systems of (a) IAA transport, (b) stor- age and reuse of IAA, (c) protection of IAA from enzymatic degrada- tion, and (d) homeostatic control of the concentration of free IAA in the tissues. Because auxins, particularly IAA, are the most active growth regulators that promote fruit set and development in strawberry, IAA conjugates may play an essential role. Conjugated auxin extracted from maize kernels as well as the synthetic ethyl and methyl esters of IAA induce parthenocarpy in tomato (42,61). The ethyl ester of IAA has been tentatively identified in extracts of immature apple seeds (40,47), though it may be an artifact (33). Application of l4 14 14 C-IAA to strawberry fruit yields C-IAA aspartate jg_yitrg and C-IAA-glucose in vivg (27). The reason for this discrepancy is not apparent, but the mechanisms for conjugation are obviously pres- ent in strawberry fruit. GA-like activity has been quantified in extracts of straw- berry achenes and receptacles via the barley endosperm bioassay (29) and the lettuce hypocotyl bioassay (46). At peak levels the recepta- cle contained higher concentrations of such compounds than did the achenes. Activity in receptacles was highest at 5-6 days after pol- lination, then declined. Extracts of flowers and receptacles were active at 3 days after pollination. No pattern was apparent in achenes. Floral diffusates exhibited activity in the Rumex-leaf 14 senescence bioassay, but extracts of flowers showed little activity (15). Abscisic acid has been tentatively identified in extracts of both ripe and unripe fruit (41) and quantified by the wheat coleoptile bioassay (29). No inhibitory activity could be detected two days after pollination. In achenes, levels remained relatively constant until late stages of develOpment, then increased as fruit ripened. Concentration in achenes was 2- to lO-fold higher than in receptacle tissue until maturity, when levels in achenes rose lO-fold. The total quantity per fruit was approximately evenly divided between achenes and receptacles until maturity. Cytokinin-like activity, as measured by the Amaranthus (29) or soybean callus bioassay (24) was detected in flowers at anthesis and increased in achenes and receptacle through 7 or 15 days, depend- ing on the study, then declined. Activity in receptacle tissue paralleled that in achenes but was several-fold less per unit weight. Ethylene has been detected in the volatiles emanating from pollinated strawberry flowers (20). An increase in evolution was noted following pollination, perhaps associated with petal or stamen wilting or abscission. Strawberry fruit set may be governed by a balance of promoters and inhibitors (44). Evidence for the existence of an inhibitor in strawberry fruit has been presented. Auxin and cytokinin levels rise in whole fruit as soon as 2 days after pollination when a small growth increment is evident (29) and GA levels increase in receptacle tissue 15 within 3 days (46). Total GA activity is higher in the receptacle tissue than in the achenes (29). Levels prior to 2 days after pol- lination are unknown, though some activity is present at anthesis. The increase in promoter level(s) occurs when berry enlargement is first noted. Summary Seeds play a very important role in influencing growth of surrounding fruit tissues, an effect possibly mediated by seed- derived hormones. Strawberry, whose 'seeds' or achenes occur on the surface of the receptacle, provides an excellent model system with which to study the mechanisms of control. The strawberry fruit exhibits a sigmoidal growth pattern. Most receptacle growth is due to cell enlargement rather than cell division. Fruit mature about 30 days after pollination, though varieties differ. Primary fruit are the largest, and berry size is linearly pr0portional to achene number. Applied auxins and GAS elicit fruit set jg_vjvg, lanolin paste being more effective than aqueous solutions. Addition of DMSO to the aqueous solution nearly doubles the response as compared to aqueous solutions lacking DMSO. Work in vitro has not yielded clear- cut results; some investigators have reported positive effects of auxins, while others have reported no effects. Parthenocarpic fruit growth in strawberry is most effectively induced by applied auxins; GAs are capable of promoting limited development, while cytokinins have little effect. Synergism occurs 16 between auxins and 6A5. Compounds differ in their effects on the length of the developmental period. Auxins can replace achenes, sustaining growth nearly as well as achenes. Field applications of auxins reportedly increase yields, while GAs are ineffective. Auxins probably increase fruit size rather than fruit number. Unfertilized achenes inhibit growth of the receptacle in the region of attachment in vivo, whereas both fertilized and unfertilized achenes inhibit development in vitro. In vitro studies are con- founded by calyx removal, which reduces fruit growth. Cytokinins, auxins, or GAs cannot substitute for the calyx. Endogenous levels of auxin-like compounds parallel achene and receptacle development in early phases of growth, then decline. Maximum activity in achenes is lO-fold higher than in receptacle tissue, IAA having been tentatively identified as the major auxin present. GA-like activity is high soon after pollination in recep- tacles, while no pattern is evident in the achenes. ABA levels increase in both achene and receptacles as maturity approaches. Cytokinin-like activity increases through 1 to 2 weeks after pollina- tion and subsequently declines. Fruit set in strawberry may be governed by a balance of promoters and inhibitors. Rapid increases in cytokinins and auxins within13days and 6A5 within 5 days after pollination have been observed; these promoters may counteract the effect of an inhibitor present in achenes. Auxin conjugates may play a role in fruit development, for these compounds are capable of eliciting fruit set and growth and the 17 mechanisms for conjugation are present in strawberry fruit. These facts, together with the observation that free auxin levels are not well correlated with fruit set or development, warrant an investiga— tion of the levels of such conjugates in the strawberry during its development. 10. 11. Literature Cited Abbott, A.J., G.R. Best, and R.A. Webb. 1970. The relation of achene number to berry weight in strawberry fruit. J. Hort. Sci. 45:215-222. , and R.A. Webb. 1970. Achene spacing of strawberries as an aid in calculating potential yield. Nature 225:657- 664. Bajaj, Y.P.S. and W.B. Collins. 1968. Some factors affecting the jn_vitro develOpment of strawberry fruits. Proc. Amer. Soc. Hort. Sci. 93:325-333. Bandurski, R.S. 1979. Chemistry and physiology of conjugates of indole-3-acetic acid. pp. l-l7. 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Raussendorf-Barger, G. von. 1962. Indolderivate im apfel. Planta 58:471-482. Rudnicki, R., J. Pieniazek, and N. Pieniazek. 1968. Abscisin II in strawberry plants at two different stages of growth. Bull. Acad. Polon. Sci. 16:127-130. Sell, H.M., S.H. Wittwer, T.L. Rebstock, and C.T. Redemann. 1953. Comparative stimulation of parthenocarpy in the tomato by various indole compounds. Plant Physiol. 38: 481-487. Sembdner, G. 1974. Conjugates of plant hormones. pp. 283-302. In Biochemistry and chemistry of plant growth regulators. K. Schreiber, H.R. Schutte, and G. Sembdner, eds. Halle, GDR: Inst. of Plant Biochem. 432 pp. Sjut, V. and F. Bangerth. 1981. Effect of pollination or treatment with growth regulators on levels of extractable hormones in tomato ovaries and young fruits. Physiol. Plant. 53:76-78. Swarbrick, T. 1943. Progress report on the use of naphtho- xyacetic acid to increase the fruit set of the strawberry variety Tardive de Leopold. Annu. Rept. Long Ashton Hort. Res. Sta. 1943. pp. 31-32. Tafazoli, E. and D. Vince-Prue. 1979. Fruit set and growth in strawberry, Fragaria1 cmgz < ems: << Axmu\msv mwmmcucm Pouch muws< Lmumm mm»; Nmumm sazogo gmgwm mama _wm~ .xuwgapme op mwmmsucm soc» mpwzgm A.:u:o mmmmcmcm x mwgmmmuuv xggmazmgpm .Amzwwz. meEwLa we mammwg mpumuamumg ucm mcmzum cw <