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II IIIIHII .I IIUHH I .I h [I I IIIII I Iu._I II b I. III I II I I III III I I. I IIIIIWIIII I I! l . I III I II n. Ith4.IIII““III I II pl I Iquf 11-“:ng 1““"“‘ f. ."" ”‘ .'-" L--..- , {Mich £3 ' ‘ L'-‘.I -: :11; This is to certify that the thesis entitled Environmental and Physiological Factors Influencing the Response of Chrysanthemum morifolium cvs. "Bri ht Golden Anne" and fiCircus" To Daminozide presented by John Enos Erwin has been accepted towards fulfillment of the requirements for Master' 8 degree in Horticulture K , c/ao/ylééw Date @M/// W; 6 0-7639 MS U i: an Waive Action/Equal Opportunity Institution llll\\\lllll\lllllllll§ll1llll L 3 1293 00995 MSU LIBRARIES ”- RETURNING MATERIALS: ace n 00 rop to remove this checkout from your record. FINES will be charged if 500? is returned after the date stamped below. h— m h.‘ VOQQ 3133 3“: $3? lnvironnental And Physiological tactora Influencing the Beaponae 0f Chrzaagthegun aorifoliun cva. 'Bright Golden Anne’ and 'Circna’ To Daninozide 3! John Bnoa Irvin rheaia Submitted To Michigan State University In Partial Fulfillment 0f the Require-onto For The Degree 0! MASTER OF SCIIICI Department Of Horticulture 1986 Abstract Environmental and Physiological Factors Influencing the Response of Chrysanthemum morifolium Ramat. cvs. 'Bright Golden Anne’ and 'Circus’ to Daminozide Applications of daminozide to Chrysanthemum morifolium shoots early in development (less than 7 days after an in- ternode reached 0.5 cm in length) stimulated internode cell division by as much as 120 X. However, cell elongation was retarded to such a degree that final internode length was reduced 30 to 40 8. Proximal internodes responded more strongly to daminozide than more distal internodes. Retardation of final shoot length by a 5000 mg 1‘1 a.i. daminozide application to 'Bright Golden Anne’ and 'Circus’ shoots decreased 76 and 83 x, respectively, as day tempera- ture increased from 10°C to 26°C. Increasing day tempera- ture from 26°C to 30°C reversed this trend and increased daminozide retardation of shoot length. Photosynthetic photon flux did not influence plant response to daminozide. Three methods were evaluated for modeling the effect of day temperature and daminozide concentration on shoot elongation. Method II predicted parameter estimates for the gompertz function through multilinear regression. When the parameter estimates were evaluated within the gompertz func- tion shoot elongation over time was predicted. hethod II showed the greatest potential for modeling stem elongation as influenced by day temperature and daminozide. ii Table Of Contents List Of Tables . . . . . . . . . . . . . . . . . . . . v List Of Figures . . . . . . . . . . . . . . . . . . . vii Dedication . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . xi Literature Review I. Cell Mall Structure and Expansion . . . . . Plant Cell Wall Composition . . . . . . . Cell Hall Expansion . . . . . . . . . Dynamics of Cell Wall Extension . . . . . . . Viscoelastic Extension . . . . . . . . . . . Biochemical Modifications . . . . . . . . . . Turgor Pressure . . . . . . . . . . . . . . II. Roromonal Responses . . . . . . . . . . . . l Auxin . . . . . . . . . . . . . . . l Gibberellins . . . . . . . . . . . . . . . . . . l Ethylene . . . . . . . . . . . . . . . . . 1 III. Plant Cell Differentiation and Development . . . 1 Apical Meristem . . . . . . . . . . . . . . 1 Cell Development . . . . . . . . . 1 IV. The Influence of Light 0n Stem Elongation . l The Effect of Light Intensity on Stem Elongation l The Effect of Light Quality on Stem Elongation . 2 Stimulation of Elongation by Par-red Light . . . 26 Roromonal Basis for the Effects of Light on Stem Elongation . . . . . . . 2 The Effects of Light on Growth Inhibitors . . . 2 V. The Effects of Temperature on Stem Elongation . . 3 VI. The Influence of Mater on Stem Elongation . . . . 3 VII. Retardation of Stem Elongation with Daminozide . . 35 VIII. The Dynamics and Analysis of Plant Stem Elongation 3 Linear Regression . . . . . . . . . . . . . . . 40 Nonlinear Regression . . . . . . . . . . . . . . 40 Monomolecular . . . . . . . . . . . . . . . . 41 Logistic . . . . . . . . . . . . . . . . . . . 42 Gomperts . . . . . . . . . . . . . . . . . . . 43 Richard . . . . . . . . . . . . . . . . . . . 44 Meibull . . . . . . . . . . . . . . 45 Stochastic Distributions and Time Series Analysis . . . . . . . . . . . . . . . . . . . 46 IX. Bibliography . . . . . . . . . . . . . . . . . . . 49 iii I. II. III. IV. V. VI. VII. Section I Factors Influencing the Response of Chrysanthemum morifolium Ramat. cvs. ’Bright Golden Anne’ and ’Circus’ to Daminozide. Abstract . . . . . . . . . . . . . . . . . . . . . 60 Introduction . . . . . . . . . . . . . . . . . 61 Materials and Methods . . . . . . . . . . . . . . 62 General Procedures . . . . . . . . . . . . . . . 62 Individual Experiment Procedures . . . 63 Application to a Whole Plant During Development . . . . . . . . . . . . 63 Application to a Single Internode During Development . . . . . . 64 Additive Responses of Multiple Applications 65 Results . . . . . . . . . . . . . 65 Application to a Whole Plant During Development. 65 Application to a Single Internode During Development . . . . . . . . . 66 Additive Responses to Multiple Applications . . 67 Discussion . . . . . . . . . . . . . . . . . . . . 68 Conclusions . . . . . . . . . . . . . . . . . . . 71 Bibliography . . . . . . 73 Section II The Influence of Day Temperature and Photosynthetic Photon Flux on the Response of Chrysanthemum morifolium I. II. III. IV. V. VI. Ramat. ’Bright Golden Anne’ To Daminozide cvs. and ’Circus’ Abstract . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . General Procedures . . . . . . . . . . . . . . Individual Experimental Procedures . . . . . . . Day Temperature Experiment . . . . . . . . Photosynthetic Photon Flux Experiment . . . Data Analysis of Elongation . . . . . . . . . . Technique I . . . . . . . . . . . . Technique II . . . . . . . . . . . . Technique III . . . . . . . Results and Discussion . . . . . . . . . Shoot Length versus Time . . . . . . . . . . . Rate of Elongation . . . . . . . . . . . . . . Day Temperature Experiment Photosynthetic Photon Flux Model Evaluation . . . . . . Conclusions . . . . . . . . . Bibliography . . . . . . . . . Table Table List Of Tables Page Section I Treatment combinations designed to study the effect of daminozide applications to internodes at different positions on the plant and responses to multiple applications. . . . . . . . . . . . . . . 75 The effect of a single daminozide application at different stages in the development of Chrysanthe- mum morifolium 'Bright Golden Anne’ and ’Circus’ on final length of the second lateral shoot. Damino- zide was applied as a foliar application to the whole plant till foliage was wet. . . . . . . . . 76 The effect of daminozide application time on cell size and number in Chrysanthemum morifolium. Two hundred ninety two ug SADR was applied as five 25ul droplets of a 2333 mg 1‘1 solution to the leaf at the base of the second stem segment. Tissue samples were taken from the treated internodes at the cessation of elongation . . . .77 Percent retardation of Chrysanthemum morifolium cv. 'Bright Golden Anne’ stem segments resulting from single and multiple applications of 292 ug of daminozide delivered as three 75u1 droplets applied to the leaf immediately below the stem segment when it was 0.5 cm in length. . . . . . 78 Percent retardation of Chrysanthemum morifolium cv. ’Circus’ stem segments resulting from single and multiple applications of 292 ug of daminozide delivered as three 75 ul droplets applied to the leaf immediately below the stem segment when it was 0.5 cm in length. . . . . . . . . . . . . . 79 Page Section II Effect of day temperature (DT) and daminoside concentration on maximum likelihood parameter estimates of final shoot length (a), initial length determinant (b), and mean relative stem extension rate (c), of gompertz functions representing Chrysanthemug morifolium cv. ’Bright Golden Anne’ lateral shoot elongation over time. The influence of DT and SADR on the V mean absolute stem extension rate (MAER) and inflection point (IP) are also presented. . . .115 2. Effect of day temperature (DT) and daminozide concentration on maximum likelihood parameter estimates of final shoot length (a), initial length determinant (b), mean relative stem extension rate (c), of gompertz functions representing Chrysanthemum morifolium cv. ’Circus’ lateral shoot elongation over time. The influence of DT and daminozide on the mean absolute stem extension rate (MAER) and inflect- ion point (IP) are also presented. . . . . . . 116 3. Regression coefficients predicting the multiplication factor as influenced by day temp- erature (DT), time, and daminoside conc- entration (CN) . . . . . . . . . . . . . . . .117 4. Multilinear regression coefficients predict ing gompertz function parameters as influenced by day temperature (DT), daminoside concentration, and the ’a’ parameter . . . . .118 5. Multilinear regression function parameters which describe the influence of day temperature and time on the rate of growth of Chrysanthemum morifolium cvs. ’Bright Golden Anne’ and 'Circus’ . . . . . . . . . . . . . . . . . . .119 6. Multilinear regression coefficients which predict g(x), or the inhibition function, as influenced by DT, daminozide concentration, and time. . . . . . . . . . . . . . . . . . .120 vi Figure Figure 1. List Of Figures Page Section I Diagram identifying stem segment partitioning of the second lateral shoot. The first and sixth stem segments are single internodes. The remaining segments are composed of two adjacent internodes. Experimental plants consisted of three shoots. One daughter shoot is shown. . . . . . . . . . . . . . 81 Effect of the time of a daminozide application on final second lateral shoot length of BGA (a) and Circus (b). Plants were sprayed with a 2500 mg 1'1 solution of SADR till foliage was wet . . . 83 Effect of the time of a daminoside application on the final stem segment length of BGA (a) and Circus (b). The leaf at the base of the second stem segment received an application of 292 ug of daminoside delivered as five 25u1 droplets of a 2333 mg 1'1 solution . . . . . . . . . . . . . . . 85 Effect of stem segment position of BGA (a) and Circus (b) on the response of that segment to a 292 ug daminozide application delivered as three 25 ul droplets of a 7500 mg 1" solution . . 87 Page Section II Schematic diagram representing the procedures followed for prediction of daminoside retardation of shoot elongation ofC Chryganthemug gorifolig; for MethOd I O O O O O O I O I O 121 Influence of day temperature on second lateral shoot elongation of Chrysanthemum morifolig; Ramat. 'Bright Golden Anne’(a) and ’Circus’(b). Lines represent gompertz function estimates (Table l and 2) fit to each data set. 123 Schematic diagram representing the procedures followed for prediction of daminozide retardation of shoot elongation of Chrysanthemgg morifolium for Method II . . . . . . . . . . . . . . . . . 125 vii 10. 11. Schematic diagram representing the procedures followed for prediction of daminozide retardation of shoot elongation of Chrysanthemum morifolium for "ethOd III. 0 O O O O O O 0 O O O O 0 0 a O 127 Response surface calculated from method II indicating the absolute shoot length of Chrysanthegum morifolium ‘Bright Golden Anne’ as influenced by time and day temperature. . . . . 129 The influence of a daminoside application on day 15 after the start of short days on second lateral shoot elongation over time on Chrysanthemum morifolium cv. 'Bright Gloden Anne. 0 O O O O O O O O O O 0 O I O O O O O 0 O 131 Response surfaces calculated from method II describing Chrysanthemum morifolium cvs. ‘Bright Golden Anne’(a) and ’Circus’ (b) final shoot length as influenced by daminozide conc- entration, and day temperature . . . . . . . . 133 Response surface calculated from method II describing the influence of daminozide conc- entration and day temperature on percent retardation of shoot length resulting from a daminozide application 15 days after the start.of short days on Chrysanthemum morifolium cv. ’Bright Golden Anne’. . . . . . . . . . . . . 135 The influence of photosynthetic photon flux on shoot elongation over time of Chrysanthemum morifolium cvs. ’Bright Golden Anne’ (a) and 'Circus’ (b). Lines represent gompertz function estimates (Tables 1 and 2) fit to each data set . . . . . . . . . . . . . . . . . . . . 137 The influence of day temperature on the rate of shoot elongation of Chrysanthegum morifoliu; cvs. 'Bright Golden Anne’ (a) and 'Circus’ (b). Lines represent first derivatives of gompertz functions fit to shoot elongation data over time at each day temperature treatment . . . . . . . 139 Diagram identifying the three phases of daminozide influenced elongation following an application of daminozide 15 days after the start of short days to Chrysanthemum morifoliug cv. ’Bright Golden Anne’ grown with day and night temperatures of 30°C and 16°C, respectively. The solid line represents the first derivative of a gompertz function (Table 1) fit to shoot elongation over time. The segmented line rep- resents the first derivative of retarded shoot viii 12. 13. 14. 15. elongation calculated at four day intervals . . 141 Response surface calculated from method I identifying the influence of time and daminozide concentration on the percent retardation of shoot elongation resulting from an application of 2500 mg 1‘1 daminoside 15 days after the start of short days to Chrysanthemum morifolium cv. ’Bright Golden Anne’. . . . . . . . . . . . 143 The influence of day temperature and time on the retardation remaining from a given application following an application of 2500 mg l"1 15 days after the start of short days to Chrysanthemum morifolium cv. ’Bright Golden Anne’ as calculated from method II. . . . . . .145 The influence of photosynthetic photon flux and daminozide concentration as calculated from method I on the percent retardation resulting from an application of 2500 mg 1"1 daminozide 15 days after the start of short days to Chrysanthemum morifolium cvs. ’Bright Golden Anne’ (a) and ’Circus’ (b) . . . . . . . . . . .147 Predicted versus actual response of Chrysanthemum morifolium cv. ’Bright Golden Anne’ grown with 18°C day temperatures and an application of 5000 mg 1'1 daminoside 15 days after the start of short days using Method I (a), Method II (b), and Method III (c) . . . . .149 ix Dedication This thesis is dedicated to my grandparents without whos guidance and encouragement the completion of this work would not have been possible. It is only my hope that I may be as good a grandparent as my grandparents have _been for me. I love them all very much. Edward Enos Erwin Sophie Blendina Erwin Janis Albert Gedrovics Zinaida Gedrovics Acknowledgments As with everything I have undertaken, my family has supported and encouraged me throughout this project. I am deeply grateful to all of them. I have been very fortunate throughout my life to have good friends which have always been there to help me when I need encouragement. I would especially like to thank Candice Shoemaker, Rob Berghage, Mike McCaffery, and Jim Eppink. In addition, I would also like to express my gratitude to Royt Emmons, whose enthusiasm and encouragement have always, and will always, be an inspiration to me. I have also been fortunate to work with a wonderful major professor and friend, Royal Reins. Royal’s patience and willingness to give support have been tremendous. Lastly, I would like to thank the rest of my graduate committee, John Bukovac, William Carlson, and C. Alan Rots, for their efforts and encouragement. xi Literature Review Cell Mall Structure and Expansion Plant Cell Compggitiog The primary plant cell wall of higher plants is composed of cellulose, a matrix of various compounds, and water (82,125). The cellulose molecule is composed of long chains of B- 1,4 linked glucose units (82,125). These chains of glucose are organized into highly ordered crystalline lattices which are held together by hydrogen bonds (82,125). When approximately one hundred cellulose molecules are organized into a lattice, they are collectively called a 'micelle’. Twenty or more micelles interwoven into a helical structure are called a microfibril. These microfibrils are imbedded in a matrix composed of pectins, protopectins (with a high hydroxyproline content), lignins, and hemicelluloses con- sisting of pentoses, arabinose, xylose, hexose, glucose, .galactose and mannose (82,125). The hemicellulose sugars are organised into chains which form a highly hydrated hydrophilic gel. The degree of hydration of the gel is variable. Cell Hall Expansion: For growth of a cell to occur the wall or microfibril network must yield to an applied stress (19.41.72.88,125). The degree of stress a microfibril net- work can tolerate before it yields is determined by' the 1 coherence between, and the alignment of, the the microfibrils (19,70,88) . Coherence is dependent on the number of crosslinkages between the microfibrils. (19,70). Crosslinkages consist of both covalent and hydrogen bonds which contain sufficient potential energy to retain the in- tegrity of the system. As the number of crosslinkages increases, cell wall rigidity increases and resistance to an applied force increases. Alignment of the microfibrils also influences resis- tance to an applied force. Resistance to elongation is greatest on a plane parallel to the microfibril alignment (96) and least in a plane perpendicular to microfibril alignment. Extension of a cell wall would therefore be ,easiest in a plane with a low microfibril density, and per- pendicular to the alignment of the microfibrils. Since cell wall elongation is influenced by microfibril orientation in the cell wall, cell shape is determined to a great extent by microfibril orientation (96). Early in differentiation, all plant cells have a multinet, i.e. random, micrifibril orientation. When the microfibrils are deposited in a multinet manner isodiameteric growth results (19,96). However, as maturation progresses, differential placement of microfibrils may occur resulting in non- isodiameteric growth. (19,37,96). Cells with a transverse microfibril distribution elongate more longitudinally than laterally and appear as a cylinder (37,96). Cells with an oblique orientation of microfibrils expand on that side of the cell with the least stress tolerance and elongate as a helix or spiral (37.88.96). Examples of spiraling growth are seen in ghygglygg; sporangiophore cells and Trandscatig staminal hairs (37). The shape of a cell may also be influenced by the stress from adjacent cells. The growth of a cell influenced by the stress from surrounding cells is said to undergo 'passive growth’ (Fig. 1 (96)) (19). For instance, if an epidermal cell is isolated and allowed to expand in a suspension, it would develop a ’U’ shape due to an oblique distribution of the microfibrils. Axial stress placed on the cell by adjacent cells in the whole plant results in a cylinderically shaped cell. '. res-elm, 0'" l i l Cm FIG. 20. Diagram of examples of passive growth. Figure 1 D n Ce 1 Mel o The cell wall with its microfibril network has a structure similar to a polymer (19,70). Therefore, a polymer system may be used to help understand the mechanical properties of the plant cell wall. When a force is applied to a polymer, it undergoes a deformation (102,110,125). Upon release of the force the polymer may be permanently deformed or it may return to its original shape (19,70,102). The degree and mode of deforma- tion is dependent on the extent of polymerization and crosslinkage (110). Linear polymers (i.e. vegetable oil) have few, if any, crosslinkages and the deformation is a linear function of time and is proportional to the mag- nitude of the stress (70). lhen the deformation of a material is both irreversible and a linear function of time (i.e. linear polymers), it is said to undergo viscous flow (19,70,110). In contrast to linear polymers, deformation of rubbers which have a high degree of crosslinking is not a linear function of time and is often temporary, or elastic (19,70,102). Elastic extension involves the reorientation of configurations from a minimum potential energy to a con- figuration which has a greater potential energy (70). The energy used in changing a configuration is regained when the stress is removed. (19,110). Long polymers which are crosslinked such as cellulose microfibrils often undergo an intermediate type of deformation called ’viscoelastic extension’. Viscoelastic extension contains both a viscous flow and an elastic component (19,70,102,125). Viscoelgstic Extension Viscoelastic substances, when placed under stress, undergo an initial phase of rapid elongation which is elastic (19,70). This elastic exten- sion phase persists until a 'yield point’ is reached (19,70). After the ’yield point’ is reached the material will undergo a phase of extension called ’viscoelastic creep’ (19,20,110). This phase is characterized by both a viscous flow component and an elastic component. The rate of elongation during this phase is constantly diminishing which ultimately approaches a horizontal asymptote (Fig. 2 (19)) (19,70). / C /' z .........eleeeeeeee B 9 ...e' / g ."L. / A 3 / "J : / E / E / iMeAppled TIME memu.Thydubuflphmnuunumbnudmhmkwhuummmms duneu@mn¢5).visoodudceatsnsion(3).andvisoousflow(¢). Figure 2 6 Extension beyond the yield point often results in scis- sion of bonds (19,70,102). Scission occurs as a result of a bond configurations inability to retain the potential energy which it is receiving (70). Excessive scission of crosslinkages ultimately results in a tearing of the material (19,70). The stress required to tear a material is called the 'ultimate yield stress’. If the force is removed prior to tearing the material, the material un- dergoes an ’instantaneous elastic deformation’ (19,70). The material will not, however, return to its original length if scission of bonds has occurred (70). The difference between the original length, and the length after the force has been applied is referred to as the ‘plastic component’ (19,70,125). ' Mhether a viscoelastic extension is reversible depends upon the history of the material (19,20,70). If a material has been extended previously, subsequent extension will be entirely elastic if the extension does not exceed the .1ength of the previous Extension (19,20,70). If an Exten- sion is beyond the length of the previous Extension both an elastic and a plastic component will occur (19,20,70). The ability of a polymer system to change from irreversible to reversible Extension is known as ’mechanical conditioning’ (19). The physical concepts of viscoelastic Extension studied in rheology have been found to apply to the behavior of physical cell wall section extension. An intact plant cell can maintain a constant rate of Extension for up to twenty four hours (19). Viscoelastic Extension alone results in a continuously diminishing rate of elongation (19,70). Therefore, constant plant growth rate is composed of a series of small viscoelastic extensions with each sub- sequent Extension being greater than the previous one (is). Bioc c l Modif ca ion Cell elongation is composed of not only a viscoelastic Extension but also biochemical modification (19.46.90). This is best demonstrated by the sensitivity of elongation to excessively high temperatures, and metabolic inhibitors (20,46). Ray determined that the .rate of elongation of Avena coleoptile segments increased 3.5 times in response to a temperature increase from 2 to 23 degrees centigrade. Metabolic inhibitors such as cyanide inhibit the increase in rate of elongation due to tempera- ture (20.90.91). Rager determined that the elongation response seen when Reliagthgs gnnnns hypocotyl segments were placed in a buffer at pH 4 was completely inhibited by addition of 5 mM Cuu ions. Re concluded that elongation must be a result of enzyme catalysed reactions with a p! optima of approximately 4 (46). In contrast. Cleland has found that neither temperature or metabolic inhibitors had any significant effect on physical cell wall Extension of Avegg (19). Turgor Pressugg The force which drives cell elongation is the internal hydrostatic pressure of the cell, or tugor pressure (19.70.80). This force is variable and dependent on the size, shape, and water potential of the cell (82). The larger the size or diameter of a cell the greater the total stress which is placed on the cell wall (19,82). This relationship is given by the equation: r P ----- = Cell Wall Stress 245? Where r = radius of the cell P = internal pressure of the cell (bars) Ar = wall thickness The relationship is demonstrated by comparing a cell with a four centimeter diameter (Valonia) and one with a 4 micrometer diameter (Chlorella) (82). The total pressure exerted on the Valonia cell wall is one thousand times greater than the pressure exerted on the Chlorella cell wall (82). The shape of the cell determines if the hydrostatic pressure will be distributed equally or preferentially to any side of the cell (70.82.96). For instance. the stress on the side walls and end walls of a cylinder shaped cell (Nitellg) may be separated into a transverse and a lon- gitudinal stress: Transverse r P Longitudinal r P Cell Wall = ----- Cell Wall = ------ Stress 2Ar Stress Ar The force exerted on the side walls is twice that which is exerted on the end walls (82). The water potential of a cell is inversely proportional to the force which is exerted on the cell wall. Therefore, those cells with a lower osmotic potential (more negative) will have a greater tendency to elongate than cells with higher osmotic potentials due the greater turgor pressure and the resulting increase in stress on the cell wall (79). Cell elongation will only occur if the turgor pressure inside the cell is greater than some critical value (19.41.42). In an expanding call this critical value is variable and dependent on the turgor pressure, i.e. the critical value changes as the turgor pressure changes (19.41.42). Typically the difference between the critical value and the turgor pressure is approximately .2 bars. If the turgor pressure in the cell is dropped via an external osmoticum, growth of a cell will initially cease (19.41.42). After a period of time, which is proportional to the drop in turgor pressure, the critical value drops and growth resumes at a rate similar to its previous rate (19.41.42). If’ the turgor pressure is increased to its original value, ,growth acceleration will occur for a short time (41.42). 'The additional length 'gained’ during this burst of growth 10 will often be equal to that which was initially lost with the drop in turgor. The dependence of cell elongation on a critical turgor suggests that wall loosening and/or wall Extension may be dependent on a critical turgor pressure (19). The absence of a critical turgor pressure for physical cell wall extension of Aygg; coleoptile sections suggests that it is not the wall extension process which is the limiting factor (19). Auxin induced wall loosening has, however, been found to have a critical turgor suggesting that the biochemical modification of the cell wall may be dependent on a critical turgor (19). The dependence of cell elongation on a critical turgor pressure has led to the hypothesis that cell elongation in- volves a reversible crosslinkage cleavage which requires a minimum pressure for scission to occur and subsequent slid- ing of the microfibrils along each other and reformation of bonds (19). If that pressure is not present. the bonds will reform in their original positions. Similar behavior is seen in rubbers where some catalysts are only effective if the material is in an extended state (110). Cogclusiog The plant cell wall has physical properties which make it structurally similar to polymers. Basic con- cepts of rheology may be used to describe the behavior of a cell wall sections when placed under stress. The plant cell elongates through. a series of small viscoelastic 11 extensions. If a stress is greater than the yield stress the cell may undergo plastic deformation followed by a biochemical modification resulting in a permanent increase in size. For cell elongation to occur the turgor pressure of the cell must be greater than a critical value needed for elongation. Roromonal Responses 435;; Auxin increases cellular elongation by increasing the plasticity of the cell wall (9.34.79.86.107). The mechanism involved in induction of accelerated elongation by auxin is still controversial. The theory, which has gained widest acceptance is the ’acid-growth hypothesis’(91). A number of recent studies have supported that both the low p8 induced growth observed by Bonner (9.32.90), and auxin induced growth as having the same mechanism for induc- -ing elongation, i.e. acidification of the cell wall and/or cytoplasm (33,46,53,90.92). This acidification may stimu- late the activity of acid labile enzymes with low pH optima to cleave bonds between microfibrils (46). This reduction in coherence facilitates cell enlargement by allowing the microfibrils to slide along each other. The theory is sup- ported by the following: 12 l. the discovery that auxin causes a drop in the pH of both the cytoplasm and the cell wall (21,32.34.53,9l,lll). 2. the pH drop occurs immediately before auxin stimulated elongation (20,45,84,89,109). 3. Specific wall bound enzymes, glycosidases, have been found to have low pH optima (45,77,108). 4. Metabolic inhibitors and reduced temperatures inhibit both auxin induced cell elongation and low pH induced elongation (19,21,32,46,90). A secondary effect of auxin induced wall loosening is _to decrease the cell wall pressure (34.62.79). This results in an increase in the turgor pressure which allows for continued Extension of the cell. This increase in tur- gor pressure occurs only when the turgor pressure is above the critical threshold value to enable bond cleavage to occur (18). The acid growth theory is not supported by Pope who has observed that the auxin induced elongation and the acid growth stimulation of elongation have different pH optima which suggests that they may act through two separated mechanisms (86). In addition, Barkley and Leopold have shown that green pea stems respond strongly to an auxin treatment but not to an acidic buffer treatment (33). l3 Gibberellins Gibberellic acid has been shown to increase cell wall extensibility (55) in Avena internode (1,55), lettuce hypocotyl (55,106). and etiolated pea epicotyl seg- ments (55). In addition, gibberellins may in some cases cause an increase in synthesis of IAA or decrease in destruction of IAA oxidase. Gibberellins promote cell wall synthesis. The promotion of cell wall synthesis has been found to occur simultaneously with the initiation of gib- berellin induced elongation (55). 0f the cell wall constituents, polysaccahides are preferentially synthesized in response to gibberellins. The mechanism by which gib- berellins increase Extension has not been found to involve acidification as is seen with auxin (55). Instead gib- berellins have been found to increase the concentration of calcium ions in the protoplast (55). Since the presence of calcium ions in the cell wall is believed to decrease the extensibility of the well, an influx of such ions into the protoplast from the cell wall is thought to increase the extensibility of the cell wall (55). There is also evidence that GA may reduce the osmotic potential of the cell cytoplasm . and as a result, increase the turgor pres- sure and as a result, the stress applied to the cell wall (55). 14 Ethylene Ethylene affects cell division (67). cell expansion. auxin transport, auxin synthesis (27), GA response to tissue (27). and crosslinking of microfibrils (27) in plant cells (82). Cell division and D.R.A. syn- thesis (27) are inhibited in the apical hook of etiolated pea seedlings after an application of 50 mg 1'1 ethylene (67) while in some aquatic plants ethylene stimulated cell division (82). In terrestrial plants, ethylene has been shown to in- hibit longitudinal expansion of cells, while promoting isodiameteric or radial expansion (4.27.61.67.82). The decrease in longitudinal expansion results from reorienta- tion of microtubules from radial to longitudinal (27.67.82). Therefore. radial expansion was favored to 1on- gitudinal (67). Ethylene also retarded both lateral auxin transport (67) and polar auxin transport (4,27,66,67) and enhanced indoleacetic acid oxidase activity (67). Ethylene also reduced both the amount of auxin present within the plant tissue and the mobility of that auxin. This was ac- complished through ethylene enhanced IAA oxidase activity and retardation of polar auxin transport (4,67). 15 Plant Cell Differentiation and Development The Apical Meristem The apical meristem is the progenitor of all aerial plant parts (97,98). It is lo— cated at the distal tip of a stem and is composed of a group of meristematic cells organized into a hemisphereical or conical dome from a few microns to several millimeters in size. Two distinct layers are present in the meristem (97,98): the tunic. and the corpus (Fig. 3 (98)). Outer tunica layer Antlcllnal dmsian Inner tunica layer _ Corpus ‘00 \ @9399 o harassingo °§osboseéeaeo 8n, 2n, 2n , Periclinal division In corpus Figure 7-2. Diagrams of longitudinal sections of the promeristem region of shoot spices of before. A: Normal. untreated plant with all cells normal diploid. I—D: Different types of periclinal chimeras in plants after recovery from treatment with colchicine. Polyplaidy occurs in the outer tunica layer only (I), in the inner tunica layer only (C), or in the corpus (0). Note orientation of anticlinol cell division in A and periclinal division in C. [Adapted from S. Satina, A. F. Blakeslee, and A. G. Avery, in American Journal of Botany, 27.- 895, 1940.] Figure 3 16 The tunic is composed of the outermost one to four cell layers of the meristem. Cell division within the tunic is through aniticlinal division. i.e. perpendicular to the nearest outer wall, and results in an increase in the sur- face area of the meristem (98). Periclinal division. parallel to the nearest outer wall. only occurs when leaves are being initiated. Differentiation within this tissue ul- timately leads to the development of the epidermis. The corpus is located below the tunic and is composed of randomly organized cells which undergo both anticlinal and periclinal division (82). Division in the corpus leads to an increase in volume of the meristem. As differentia- tion occurs in the corpus, the cells are organized into longitudinal files. It 1. this tissue which leads .to the formation of the internal tissues of the plant. Cell Development The growth of any multicellular or- ganism is the result of both cell division. and cell elon- gation (29). These two processes are separate and distinct as demonstrated by the ability of specific factors, i.e. low fluence rates of light, to inhibit one, cellular division, but not the other (97). Typically both processes occur simultaneously within growing systems although Exten- sion of the atom in the early phases of internode develop- ment is predominantly a result of cell division (97). The later phases of extension are primarily a result of col- 17 lular elongation. The duration of both cell division and cell elongation is determined by genetic predetermination, and external factors which influence these processes through internal growth regulators (63.97). Two external environ- mental factors of particular importance are light and temperature. The Influence Of Light On Stem Elongation The Effect Of Light Iptepsity 0n Step Elopgptiop Plant shoot length is reduced by exposure to white light (64.79.109.116.118). Went determined that an exposure for _100 minutes to orange light ( >750 nm) with a fluence of 3 ergs cm"1 sec‘2 retarded elongation of the first and second internodes of etiolated Avena seedlings while stimulating elongation of the third and fourth internodes. Overall elongation was retarded as the final total stem length was 468 less than that of plants grown in the dark. The stimulation of the more distal internodes was attributed to a ’compensatory mechanism’ in the plant which responded to a retardation of the more proximal internodes (116). Kinetic growth studies by Thompson determined the response of an internode was not dependent on its position on the plant as Ment had suggested. but rather, on the internode’s developmental stage when it was exposed to 18 light (40.111). Exposure of an actively dividing cells to white light reduced the duration of cell division in the internode, but increased the rate at which division occurred. Exposure of older elongating internodes to light where elongation was primarily occurring through cell elon- gation was found to reduce the duration of the cell elonga- tion phase, and as a result, the length of the individual cells (39,109). Therefore, the overall reduction of elonga- tion observed by Rent may have been a result of. an ac- celeration in maturity of the individual cells (39,109), which resulted in fewer (Fig. 4 (11)) and shorter (Fig. 5 (11)) cells within an internode. 5W- ‘ F—q \ “x ’o” .. ‘c I’ m; .‘L’ ‘ _ ' ‘ \ ‘m l... § h \ .. g " \ 5 a 3’ \ 2 3 3 zoo - 1' t, 3 int 2 i “ 3 2 2 — g l a S 3 i 200 - g ' int. i 2 5 I - Int.) Int. I int! '00 " .1- . . . .M l l l I L]. L_ 1 1 l J 0 or i It too Loon c or i to I00 loco Light irxensaty (Lc) Light intensity (i.e.) Fig. 5. Slice: of light intensity on mean internode cell number. I‘m. 6. l‘lllcct «of light intensity on mean internals cell length. Vertical bun indicate the liducial limits at the P - o'os level. Figure 4 Figure 5 19 An actively growing plant is composed of several internodes, each of which may be in a different developmen- tal stage at any given time (29,64). Therefore. exposure to light may influence both cell elongation and cell division in different degrees, with the overall response depending on the developmental stage of the individual internodes (Table l (109)) (109). 'l' ..\m r: .Ilimcuxmux mul immbcrs of phloem cells (sieve elements). Values are means for 8—10 cells in each of 5 plants. Miller «In. ijimdiom ux fur ”labia 3 am 9-... . 1o.— .. --——-.¢_a--—”- .. a .- :—_.-—— - - -— . -..--..e.e.- --._-- -m .9 . .-—. u. o. . . -‘A- - .—~ . _.—.—._- ..-. - . ..-_— -- —~_“-‘-¢. -.--__ 31:11 ilimr: minus (.1) Int. l . Int. 2 Int. 3 Int. -1 Int. 5 Int. 6 “Into 82.0 X 11 J 91.1 X 10 I 87. 0 X 10.3 (34.3 X ll. 13 4-1.0 X 7.‘.l 35.7 X 6.1 1ch 130.8 X 11 .1 125.7 X 10 0 84. 4 X 9.1 67.8 X 8. (i 49.9 X 7.3 37.0 X 6.5 Dark 119.4 X 12.8 120.4 X 10 0 I49. 7 X 10.2 63.3 X 8. 3 41.0 X 7.4 29.2 X 6.8 Calculated number 01’ cells per internmlc White 207.7 169.9 208.0 59.1 10.5 9.0 Red 243.1 288.0 631.3 126.8 26.1 7.2 Dark (31:1 8 745.8 605.2 62. I 18.8 5.3 Table 1 The irradiance which the plant perceives throughout the day plays a major role in stem elongation .(Fig. 6 (117)). Seventy to ninety percent of tomato stem elongation occurred during the night (117,118). The slowest rate of 20 Extension occurred at noon (117), and the maximum occurred just prior to dawn (117). The maximum rate of elongation that occurs in the dark is dependent on the light fluence prior to the dark period (65). An abrupt drop in the rate of elongation occurs as soon as the plant is exposed to daylight, which is consistent with the theories of blue and red light retardation (117,118). In contrast to stem elongation, Van Volkenburgh deter- mined that white light increases cell wall extensibility and rapid cell elongation in Phaseolus vulgaris leaves. Re sug— gested that light stimulated Rt excretion from leaf cells in the presence of light and elongation via the acid growth hypothesis (112.113). i r ' ' ' l ' r ' 9 10 II I? l3 DEC. Fig. 1. Growth rate of tomato stains (ordinate in nun. per hour. nwnn of two plants), kept. continuously at 2635'“, either in natural daylight or in darkness. Periods of darkness indicated by heavy line along top of graph. Dates of _ consecutive days marked at noon on abscissa. Smooth llnc gives the mean of all lllt'milll’t'llli’lltl from December 5—8 and l)cc\-Inbcr 121-14; Iinc conunocting the circles given the actual growth rnli‘. Figure 6 21 The Effects Of Light Quality on Stem Elongation Stem elongation can be profoundly influenced by both light fluence (38.108) and light quality (28.38). Until recently. most plant photomorphogenic responses were believed to be influenced only by phytochrome. It has become increasingly apparent that there are two photoreceptor pigments: a photoreceptor in the blue region and phytochrome. Retardation of stem elongation can be achieved by ex- posure of plant tissues to blue light (400-500nm)(Fig. 7 ' (28)) (15.28.38.94.108). nor 7. E '3' 6-. WOMC’IOB at U [74. g "'7' test ninth I: a. :17.» o—0 inhibitm of ' stern growth mi (photon; coal for 5 a F -L06 (premier?) tor 201. mmamom “L06 5 le_i ._'— m‘ L J A 440 :60 450 500 WAVELENGTH (nml ...o » l I . - \/ \ Figure 7 22 Retardation of cucumber hypocotyls began five minutes after an exposure to white light and persisted for thirty minutes (38). Growth resumed at a rate similar to that previous to the exposure (38) . The extent of the retardation may range from 50- 95s with a fluence of 11 umol s‘1 m" blue light and is species dependent. Retardation increased with increasing fluence rate. The relationship between fluence rate and retardation is log linear in cucumber and lettuce (39,64,108) (Fig. 8). 1001 Tomato 0 50‘ go o 3’." /. C 3. 100i Cucumber '9 é /;8 5 50‘ p.43/ 5 0/ o G . 3’ 1001 Lettuce . /° 50‘ . 9/ _/ 0- - - - 0%. (16 (18 ii) Blue fluence rote ' (Log pmol photons m‘2 s‘ l . Fig. 2. Flucnce-rcspoasc curves for blue light inhibition of hy- pocotyl extension in dc-cliolalcd cucumber. lettuce and tomato Ill the presence of 80X at ZIOpmol photons m' ’s" 0—0 or ISOpmol photons m" s" e—e. Results were calculated as Figure 8 23 Exposure of plant tissues to red light (600-710nm) also leads to retardation of elongation (28,39)(Pig. 9 (28)). In most cases, the degree of retardation is equivalent to that seen with blue light (lb,25,28,38,39,108). The period be- tween termination of an exposure and initiation of retarda- tion is, however, considerably longer than that seen with blue light (39). Exposure of Cucugis sativug seedlings to 8 I .-2 red light for 1 hour did not produce any sig- nificant response until 5-6 hours after the exposure (38). The degree of retardation increased from 5 to 10 hours after an exposure, there after the plant returned to a growth rate equal to that prior to a red light exposure. ".5 l' ITO 0-0 PROMOTION OF LEAF GROWTH I0.0 r- I7.CI' H INHIBITION OF STEM GROWTH .§ 3 g seqL figure» 8 2 a 5 8 fi 0 tea l- o I0.0 > - N E E “5’ no.2 , ‘3 no.2 - 1% 3 3 o g ‘ 3 39.5 b 3 IOAV _ £9 a \ ‘..\ 3 3 \_ l o . 3‘ 20.0I’ IBJ -. ‘ .. f \~.——\~ .“_‘ l "it? 660 030 030 " 330 no INELENOTH Inn) Figure 9 24 lxposure of seedlings to both blue and red light, i.e. white light.- for 1 hour resulted in a dual kinetic response (Fig. 10). The seedlings underwent an immediate retardation of elongation which persisted for 20 minutes (64). After which growth returned to the rate previous to the exposure (39). Approximately two hours after the exposure a second period of retardation of elongation occurred (39). s 8 2 .. - . , —I— 3 -U— -'| s b e O 2 s 's s 5 ”000%. * -.- 8 l2 ws*fir‘1i Time lb) "3.1 maswmumimmmum ammoniumNflwauvamusamnusnuumflhus llCmmssmsrkthsmesngrowthmesesleulstedevsrydli .muymmmwioruamym ' Mbynler(u-l2).g\sinflglbthmindicstethst influuunbnuwuuuggzzhnuummwunoamfiufl Figure 10 The location of the blue and red photoreceptors are believed to be in two separate areas, the hypocotyl and the cotyledons, (39). The blue light retardation response acts directly on the hypocotyl (38.39.64). Exposure of the 25 cotyledons to blue light does not enhance or prolong the response (38). In contrast, the phytochrome, or red light retardation response is reduced in magnitude but not dura- tion if tissue other than the cotyledons are exposed to white light (38). Conclusion There appears to be two photoreceptor pig- ments involved in retardation of stem elongation by light, the blue photoreceptor and phytochrome (ll,38,39,64,108). This conclusion is supported by the following observations: 1. Retardation of stem elongation by both blue light (400-500nm) and red light (600-710nm) (39). 2. Response kinetics differ between red and blue light (39). 3. Differential sensitivity of specific tissues (cotyledon, hypocotyl) to either blue or red light (39,108). This suggests different locations of the photoreceptors. 4. The species specific sensitivity seen in some cases (lettuce, mustard) to only blue light or red light (39,81). The fluence dependence of the blue light retardation and the lack of a fluence dependence of the retardation response from phytochrome suggests that the purpose of a blue photoreceptor may be to detect changes in fluence rate (94). The photomorphogenic responses seen with small shifts in the Pfr/Pr ratio of phytochrome suggests that the primary role of phytochrome may be in perceiving changes in light quality (80). 26 Stimulation of Elongation by Far Red Light Far red light (710-800nm) stimulates stem elongation in a number of plant species (l5,26,39,83)(!ig. ll (25)). A five minute exposure to far red light after an eight hour exposure to white light increased internode elongation 4008 in Phaseolus. ‘5‘ a“... .smd'alsd Samotunumaudsu ’4‘ 29¢ 3.01. 5 3w 3‘“’ \ ‘ fi \ h a \ \ . \ \ " ‘ I \ Eel liliiilzpp go I: :Ii ~ 3 ~ a a t: a a a m soon To..." emu. can ”we man. an m Ses' w-m mm" 3.22.. «no, we use) . 3 Second Internode - Been Venous: was“ “2:3“, Dee-mt Fig.5 O40 - Fig 7 '2a .FUMLOI' DOW". E BMW. 3'20 .FOI H0. IOQ b '5- 00 2 “d u ‘0 g .3 u“; ‘344 3 5 3‘0 ‘ §1° g t 32° 0 9 9 7 a e. s o m. m. I. m'_ m. c i o 9 1 '-' 1 Y ' ::::::: £233.32 :33: NW 0! Treatment: with Tmereturs l'CI Wflsdmfisd s s I I M has. 5-7.—Mesn In a oi Indicated parts. Pk. 5. Pinto indicated “radiations at beginning of IO-hoer pariah. lms plum treated with 53'»... numlnenot alternating S-mia- lug. 7. hate hean phat: grown with diluent caribou:- el twink“ lar-ralssdmlndiant energies; trestmeeugiven day and night tesnuetatsres and quoted to S tau-ute- tar stegina'stgeldsrkperiedwithlunds'miaeschinstasa. ndortoSmnstesetredradistiueatbqhsmgoilbhsur htlasasnusflewmsndming-ghry plants trestcdwith dark pariah. Figure 1 1 27 Elongation increased as a log linear function of a decrease in the photoequilibria to a Pfr/Pr ratio of 0.3 (39). The magnitude of the elongation response was reduced as the time between the termination of the white light ex- posure and initiation of the far red exposure prior to a far-red exposure increased. The elongation response was also reduced as the length of the white light exposure prior the a far-red exposure increased (115), and the alon- gation response was enhanced as the white light fluence rate increased. The stimulation ofelongation was reversible if the 5 minute exposure to far-red light was followed by a five minute exposure to red light (600-710nm) (25). Mor- gan has determined that the importance of the end of day ex- posure increases as the photoperiod decreases (80). The rapidity of this induction and its reversibility strongly suggests the involvement of phytochrome as a photoreceptor (25). Far-red stimulation of elongation is not restricted to an end of day exposure. Comparisons between plants grown under white light supplemented with far red light and plants treated with an end of day exposure showed that 803 of the resulting elongation response was a result of the far-red to red ratio during the day (80). The magnitude of the elongation response to the red far red ratio is species specific (81). Species native to open grasslands such as Veronica persica and Senecio vul- 28 gggig where phytochrome photoequilibria tend to be higher below the canopy tend to respond more strongly to a reduc- tion in photoequilibria than species native to lower canopies of woodlands such as Oxalis gcetosella and Geug ur- bamgg where phytochrome photoequilibria tend to be lower (81). Rormonal Easis For The Effects of Light on Stem Elongation Application of red light to Avena coleoptile segments retarded elongation after 120 minutes, and reduced cell wall plasticity after 180 minutes (96). Exposure of 512g; coleoptile cell walls has shown that a 4-hour exposure to red light reduced auxin induced proton excretion (96). Galston determined that Avena coleoptile segments contain less extractible auxins after an exposure to red light than before the exposure (40). Lockhart suggested that light reduces the a decrease in the level of endogenous gibberellins within Phaseolus vul- gggig cv. Mores’s Progress #9 (69). Bands and Lang compared the gibberellin content of dwarf (only expressed in the presence of light) and normal Bhgzbjtjg (60) and concluded that light influenced the response of plant tissues to gib- berellins and not the total gibberellin content of the plant (55,60). 29 T e t 0f Li 0 Growt Inhibitors Until recently, it was believed that responses to environmental stimuli were primarily dependent on the concentration of growth 'promoters’ present within a system at any par- ticular time i.e. auxins and gibberellins. It is now un- derstood that there are substances within a plant which in- hibit ‘promoter’ response (102.64.59,57,58,48). Therefore, plant growth results from a coordinated interaction between promoter and inhibitor concentrations (102.64.57.58,48). The process of cell elongation promotes synthesis of peroxidase by the cell (58). Peroxidase has at least two functions, deactivation of indolacetic acid and the par- ticipation in polymerization of phenols into lignins (58). Lignins are the primary component of a cell wall which make its size permanent. Secondaryeffects of peroxidase promo- tion include reduction of internal auxin levels, reduced influence of intracellular auxin upon cellular elongation and stimulation of biosynthesis of phenolic compounds. The discovery of natural phenolic activity within the cell promoted research into the differences in the con- centrations of phenols that may exist in either genetically or environmentally dwarfed plants. In studies on g;;;; sgtivum, the concentration of quercetin-3-glycosyl-p- coumarate (000), a phenolic precursor, was found to in- crease significantly under environments of high light in- tensity (57,40). Dramatic increases in phenolic precursors 30 were also noted in dark versus light grown plants (ll). Phenols can be divided into two groups, the polyphenols such as chlorogenic, caffeic, ferulic, and protocatechuic acids which suppress IAA oxidase acivity, and monophenols such as paracoumaric, parahydroxybensoic, vanillic, stringic, phloretic, and guercietic acids which stimulate I.A.A. oxidase synthesis (78). When stimulatory and inhibitory phenols are present together in the plant the inhibitory function is found to predominate (78). Light stimulates cell elongation in younger tissues but inhibits elongation in older tissues. Polyphenols are produced initially in the cell (57). However, as the cell ages, monophenols are synthesized (57). This would result ,in stimulation of I.A.A. oxidase synthesis and a resultant cessation of growth (40,57). The rapidity of monophenol synthesis is dependent upon genetic predetermination and/or environmental factors. Environmental factors which have been found to induce phenol synthesis are nitrogen deficiency, and cool temperatures, and light (103). In each of these situations, monophenols are synthesised (57). Phenols may also reduce the ability of a plant to translocate gibberellins and/or cytokinins and reduce the ability of the plant tissue to respond to gibberellins (57,58); 31 The Effects Of Temperature 0n Stem Elongation The optimal temperature for development of different parts of a plant or the whole plant at any given time in its development varies (22,114). Cline observed that the shoot- root ratio declined as both day and night temperature decreased with Scrophularia (22). In ghgzgggthggg; a decline in the shoot-root ratio was found to be primarily dependent on a decrease in night temperature and was en- hanced by decreasing day temperature (56). Want determined that chopersicum esculentum plants had a temperature optima for stem elongation of 26 C day and night temperature early in their development ((30 cm). As the plants matured (>30 cm), the optimal night temperature for stem elongation decreased to 18° C (117). A similar decline in optimal night temperature with maturity was observed with Capsicum, Phaseolus, Antirrhinug, and Chrysanthemum (117). This indi- cated that the processes which may be limiting for elonga- tion late in plant development that occur during the day had a higher temperature optima than those processes which occur during the night (Fig. 12 (117)). Although the night tem- perature is more critical with stem elongation in Lycopegsicum, day temperature has been found to be more critical than night temperature with stem elongation of Phaseolgg, Chrysanthemum| agd Lilia; (31,56,117). ' When a cool day temperature (15° C) is given in com- 32 bination with a warm night temperature (24° C) stes elonga- tion has been found to be severely reduced (93). In addition, a chlorosis of the leaves may occur. Warm climate plants such as corn, sorghum, and hemp exhibited a more severe leaf chlorosis response than cool climate plants such as rye, nasturtium, and bluegrass (95). MWog deg tempe ratun 165' / night temperature as indicated on abscissa 2N— t’ernperehre employ day and nighl’ 1°1— .fi*. 1 . 7L 1 L 25 20' 15' f? goc Figure 12 Miller proposed that the change in temperature optima for stem elongation as a plant matures was related to the change in the available photosynthates within the plant. As plants grow the proportion of plant tissues respiring to plant tissues photosynthesizing increased (116). As a result, respiration may become a more limiting factor in an older plant growth than on younger plants (116). Since the 33 One of respiration ranges from 2 to 30 a small decline in temperature would result in a significant reduction in respiration (116). Rent and Eonner observed that chopersicum grown in daylight and placed in the dark elongated for approximately 16 to 24 hours after which the growth rate declined sharply. This decline in elongation was reversible by submerging in- dividual leaves in a 103 sucrose solution (116,118). The resultant growth rate was proportional to the number of leaves submerged. An exposure to daylight was also found to stimulate elongation after an extended period of darkness (116). They concluded that the carbohydate content and/or translocation may be the limiting processes for stem elon- gation during the night on Lycopersicg; greater than 30 cm tall (116). The Influence 0f later On Stem Elongation later stressing a plant to such a point that cell tur- gor pressure decreases below some critical turgor pressure necessary for cell expansion reduces shoot length (2,8,104). The reduction in shoot length associated with water stress occurs primarily through a decrease in cell length and not call number within a shoot (8,104). Reduc- tion in cell length by.a short period of water stress was 34 reversed within seconds after rewatering Maize plants (2). The speed of recovery following rewatering indicated that no metabolic processes were involved and that, initially, the retardation response was physical and not biochemical (2). Prolonged water stress resulted in long term retarda- tion of elongation (2,121,124). On a horomonal basis, water stress stimulated both ethylene and abscisic acid (ARA) biosynthesis (121,124). Ethylene reduces cell elongation through reorientation of microfibrils which results in isodiametric cell expansion (68). ABA induces stomatal closure which ultimately results in a reduction in available assimilates necessary for elongation (104). In addition to a reduction in assimilates, the transport of these assimi- lates was also restricted (104). The opposite of water stress, or waterlogging, also in- hibits shoot elongation (122). In contrast to water stress, waterlogging produces anaerobiosis in the roots which results in the transport of an ethylene precursor (ACC) from .the roots to the shoots where ethylene is formed. As with water stress, isodiametric cell expansion is induced which ultimately results in a reduction in elongation (122). 35 Retardation 0f Stem Elongation lith Daminozide The compound butanedioic acid mono (2,2-dimethyl hydraside) (daminoaide) is used as a commercial growth retardant on a number of ornanental crops (5,9,10,14,16.24.73.74,93,101,119,123). The plant response is typically characterised by a reduction in the length and increase in breath of the internodes and darker greener foliage (10). Daminozide is typically delivered as a foliar spray. loliar absorption of daminoside is greatest on the newly expanding leaves (23). Studies by Dicks with C r sa t em mo 1 o i showed that leaves on the lateral shoots absorbed 393 more daminoside than the older leaves .on the mother shoot (23). Transport of daminozide from the leaves on the mother shoot was predoninantly to those areas of the plant with the greatest mitotic activity, i.e. lateral shoots and roots (23,24). The movement of daminozide to the regions of more active growth suggests that daminozide is transported in the phloem (23). However, studies by Rothenberger showed that when daminozide was ap- plied to the roots, it was transported to the apical meris- tem within one hour. This suggests that both phloem and xylem transport may occur (23). Undurraga observed 1°C labeled daminozide noved readily from the phloem to the xylem. The leakage appeared to initiate with an increase in cytoplasmic permeability (76). The daminozide induced 36 leakage may explain, in part, the ability of daminozide to depress utilization of respiratory energy necessary for the retention of solutes in the cell vacuole (76). Retardation of stem elongation by daninozide in apple occurs primarily through a reduction of cell division (119). Wilde and Edgerton found a 662 reduction in mitotic activity 3 hours after an application of daminozide to the lateral shoots of apple (119). Subapical meristem activity was reduced 148 and after 24 hours, the mitotic. activity in the subapical neristem was reduced an additional 542. After 14 days, the number of cells in the apical meristem was 692 less than those plants which did not receive daminoside. Cell number in the subapical meristem was 283 of that of plants which did not receive daminozide. Therefore, daminozide is initially transported to the api- cal seristem after which it accumulates in the subapical meristems where it has been found to substantially reduce cell division (119). In contrast, an application of daminozide to an apple leaf increases the length of the palisade cells within the leaf. The exact mode of action of daminozide is still under question (24,76). Dennis et al found that 100 ug/ml daminozide resulted in a 40% inhibition of the synthesis of kaurene from navelonic acid (24). Ryugo and Sachs have determined that kaurene-l9-ol synthesis from melavonic acid in peach ovules is also inhibited by daminozide (24). In 37 addition, an application of 10 mM daminozide to Pisum epicotyl tips has been found to block the conversion of trans- geranylgeranyl pyrophosphate to kauren-lS-al. Ross and Brand determined that the total accumulation of gib- berellins in vernalised hasel seeds was reduced by daminoside. The conclusion of the previous studies was that daninozide may reduce the total gibberellin content of the tissue through interfering with the biosynthetic path- way (24). Menhennett has alternatively suggested that daminozide may modify the biological activity of the gibberellins in the plant (76,77). This theory is based on the ability of daminoside to reduce the activity and/or block the response of GA. (76). It was hypothesized that daninoside prevents the hydroxylation of CA9 to CAao (76), a more biologically active form. Daminozide is not readily degraded in the plant (23,24). Dicks has determined that the quantity of daminoside in the plant remained constant for a minimum of 5 weeks in Chrysanthemum morifolium (24). Despite the rela- tive stability of this compound in plant tissue, its dura- tion of effectiveness is often limited (16.23.24.123). Zeevaart treated arbitis 9;; cotyledons with 5000 ng l‘1 daminoside as a foliar application and following a period of retardation observed a resumption of growth rate similar to that previous to the application after approximately 25 38 days of retarded elongation (23.123). Only a small portion of the daminozide present in the plant was present in the upper shoot portion of the plant after 25 days (23.123). Zeevaart concluded that cell division in the upper shoot region diluted the concentration of the daminozide below a critical threshold concentration needed for retardation of elongation (123). Dicks called this loss of response to daminozide 'growing out’ and quantified this response in Chrysanthemum with the equation: 1 (dmI°/dt) = ------------------- (dm/dt) 1 + E( I - It) Where: (dmI‘/dt) = Retarded growth rate I = Concentration of inhibitor 1' = Threshold level of inhibitor E = Retardation constant (dm/dt) = Growth rate of controls This equation was initially used for describing retardation of exponential growth in bacteria cultures (24). The studies with Chrysagthemum were not conducted under constant environmental conditions. Other factors which have been shown to influence the response of a plant are physiological age (47), temperature and light levels (12.73.100.101). Schonherr and Duckovac determined that daminozide absorption increased as light in- tensity increased to 1.82 I .2 and temperature increased to 35°C with Phaseolus vulgaris (100). 39 The Dynamics and Analysis of Plant Stem Elongation Growth curves can describe the behavior of growth processes as they vary with time (30). The underlying form of a growth process over an extended period of tine is nor- mally curvilinear. and increasing with time (the first derivative varies and is positive) (17). The rate of in- crease in size for all growth processes ultimately ap- proaches a horizontal asymptote as sone factor necessary for growth becomes lisiting or genetic predetermination causes a cessation of growth. Typically the objective of fitting a growth curve to data is to obtain information from the growth curve parameters which may give insight into the growth process itself or to simply predict sole factor as it increases with time (51). Insight into the true underlying growth process is often of primary isportance to the biologist. Conversely. a statistician is concerned with the ability of -an equation to describe a data set (51). A modeler of a living organism’s growth must incorporate both views and Judge a function’s fulfillment of the ’biological expectations’ of the modeler and the ’statistical exactitude' of a model in describing a data set. The degree to which either of these qualifiers is emphasized depends on the data set itself. 40 Linear Regression Linear regression is not based on un- derlying biological assumptions, i.e. proportionality be- tween growth that has occurred and that which has yet to oc- cur (26,30.36.89). Polynomials are also unable to predict an independent variable as it approaches an asymptotic value with an increase in the dependent variable to infinity (36.50.51). As a result, linear regression has tradi- tionally been used to describe an independent variable within a limited range for the dependent variable. Nonlinear Regression The difficulties associated with linear regression have led to an increase in the popularity of a group of functions which are based on biological as- _sumptions of growth (26.30.89). In addition they are non- linear in form and display asymptotic behavior (105). Some nonlinear models which have been found to be useful in describing stem elongation are: General form: Nhere: Characterized by: Previously used in describing 41 Monomglecular (26.35.50.89) -cT a ( l - be ) asymptotic value at(l-b) at (t=0) = initial size C rate constant No point of inflection Curve is convex to the dependent variable axis Relative growth rate declines linearly with time Describes irreversible growth Rate of growth is proportional to the growth yet to occur The relationship between leaf area and temperature (43) First order chemical reactions (35) Cell expansion (35) Regrowth studies on Pestuca arundinacea (52) General form: Where: Characterized by: Previously used to describe: 2. 3. 2. 3. 4. 42 Logigtic (26.89.17.36) -cT a / ( l + be ) asymptote at t=0 is a(l-b) = initial size . Hhen t rate constant Symmetric curve Inflection at a / 2 Relative growth rate declines with tile Growth is proportional to the product n o. s: u a/(1+b) t Single leaf growth data on Cucumis sativus (44) Animal population studies(52) Disease progression (71) Flower number analysis (87) Seedling germination and growth (52) General form: "here: Characterized by: Previously used to describe: C 43 Gompertz (17.26.35.85,89) -cT asymptote -b at t=0 is initial size rate constant Nonsymneteric sigmoid function Inflection at a / a Relative growth rate is a declining linear function of the logarithm of the indep- endent variable I Faster earlier growth with a slower approach to an asymptote Pelargonium leaf expansion(3) Animal population studies(7l) Cotton hypocotyl elongation (71) Germination studies (50) 44 Richard’s (17.26.35.89.92) (b-cT) -l/d General form: H = a ( l + be ) Ihere: a = asynptote b = initial size c = rate constant d inflection point determinant Characterized by: l. signoid curve with variable inflection 2. Inflection point moves up the curve as the value of d increases from -1 d = 0 monomolecular - d = l gompertz d = 2 logistic 3. Does not fit data with insufficient curvature approaching the upper asymptote Previously used to describe: 1. Individual leaf growth (35.92) General form: Where: Characterized by: Previously used to describe: 1. 45 Weibull’s(12,71.72.84.89) -(T/b) c a ( l - e ) independent variable intercept scale parameter inversely related to the rate of increase shape parameter Sigmoid curve with a variable point of inflection Shape parameter is independent of a and b parameters When c< 3.6 curve skewed right 3.6 Symmetric 0 II inflection c> 3.6 curve skewed left The curve may pass through the origin Disease progression in Phaseglus (13) 'Time-to-failure’ quality control investigations (70) Cotton hypocotyl rot (84) Differential pathogenicity of vectors (72) 46 The computational difficulties associated with the iterative techniques necessary for estimation of nonlinear parameters have limited their use. Recent advances in com- puter technology have elisinated many of these limitations. While the use of nonlinear functions has become more widespread. appropriate techniques for their evaluation has fallen behind the use of the functions themselves (52). When evaluating a nonlinear function on its ability to describe a data set. .the common assumptions employed with linear regression, i.e. norsality and unbiased. indepen- dently distributed parameters. are not valid unless the sample population is very large (26,89). As a result, regression statistics based on t and F distributions may not be valid for small sample sizes typically seen in experi- ments (26,89). Dates and Watts and the Box methods are two sethods which have been developed to evaluate nonlinear functions (89). From the statistics generated by these pro- cedures the validity of conclusions based on t and P statis- tics can be determined (89). In addition. the confidence which may be placed in the parameter estimates can be calculated. Stochastic distributions and Time Series Analysis Growth models for stem elongation typically make predictions for a specific quantity at some point in time, i.e. length. dry 47 weight (99). The validity of this quantity is, however, un- der question unless an entire population is sampled. Therefore, when developing models based on population samples. it is often of benefit to introduce a probability distribution to help account for variability (99). These probability distributions, often referred to as stochastic distributions. predict an exact value and an associated variance (99). The importance of such models increases as the variability of the data increases. Another method of analysis which may be used to sum- marize a growth process with longitudinal studies, or studies in which a series of measurements are taken on the same subjects. is discrete time series analysis (49,52). Time series analysis. is of particular benefit in determin- ing if there are any subtle relationships in the kinetics of growth of an individual plant, i.e. cyclic events, cor- relations between points, random components (49,52). Often these relationships may go unnoticed. when data from in- dividual plants is combined for standard regression analysis (49). A model resulting from a time series analysis may com- bine a number of other sodels. e.g. Gompertz, logistic etc.. The paraneters in these equations are not fixed as in standard regression analysis (49). Instead, they very slowly and at random, and may be based on a previous pre- dictions value (48). 48 Conclusion In conclusion. nonlinear regression functions are useful when prediction of sone dependent variable. when growth is asymptotic within the time range studied. 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The authors ap- preciate the technical assistance of James Eppink and Cathy Fredenburg during this project. Plant material was donated by Yoder Brothers of Barberton. Ohio. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations. this paper must be hereby marked 'advertisement’ solely to indicate this fact. 1Research Assistant. 2Associate Professor 59 60 Abstract Factors Influencing The Response Of Chrysanthemum morifolium Ramat. To Daminozide Applications of daminozide to Chrysanthemum morifolium shoots early in internode developement stimulated 'internode cell division by as much as 120 percent however cell elonga- tion was inhibited to such a degree that final internode length was reduced 30 to 40 percent. Response of an internode to daminozide depended on the internode location on the shoot. The second stem segment from the shoot base was the most responsive segment to an daminozide application. daminozide effectiveness decreased as much as 70 percent as stem segments became more distal. Multiple applications of daminozide to the same shoot were not additive in their retardation response. In general, inhibiition of internode elongation by daminozide was increased if the shoot had received a previous applica- tion of daminozide. 61 Introduction The chemical daminozide reduces stem elongation in a number. of plants (1) through retardation of cell division and elongation (8.11). The degree of _reduction in final shoot length is dependent in part on the physiological age of the plant at the time of treatment (1.4.9). The maximum response of chrysanthemum to an daminozide application is reported to occur when an application is made 14 days after the initiation of short days (9). The relationship between the physiological age of a shoot and response to an daminozide has not been quantified in chrysanthemum under controlled environmental conditions. In addition, the response of an internode to multiple daminozide applications and the influence of internode position on the response to daminozide are not known. This paper quantifies the relationships between plant age. and the position of ac- tively elongating internodes on a lateral shoot to the num- ber and timing of daminozide applications in chrysanthemum. 62 Materials and Methods General Progedures: Rooted cuttings of Chrysanthemum morifolium ’Dright Golden Anne’ and ’Circus’ were planted in a commercial potting mix (sphagnum peat, perlite. vermiculite) in 10.2 cm plastic pots. The cultivars ’Rright Golden Anne’ and ’Circus’ were chosen because of their com- mercial significance and to compare responses to daminozide of cultivars with different flower forms: decorative and daisy. All plants were placed in controlled environment chambers and grown with a photosynthetic photon flux (PPF) of 325 umol s"1m'2 supplied from cool white fluorescent lamps for 16 hr d'l. Day and night temperatures were main- tained at 20°C and 16°C. respectively. Plants were fertil- ized at each irrigation with 200 mg 1"1 of N and E. After 7 days, the apical growing point was removed from each plant to promote branching and the photoperiod was reduced to 8 hr (1'1 to induce flowering. Twelve days after the initiation of short days, lateral shoots other than the apical 3 of each plant were removed. In experiments where daminozide was applied to a single leaf, the daminozide solution was applied as five 25 ul droplets to the leaf immediately below the stem segment: which was being studied (Figure 1). A stem segment was defined as two adjacent internodes. Two internodes were 63 chosen as an experimental unit to reduce variability. Variability in length among internodes was great. especially in the more proximal internodes of the lateral shoot. By ad- ding the lengths of two adjacent internodes. some of this variability was removed while still maintaining a system which would display more sensitivity than an entire shoot system (6). The second lateral shoot_ was used 'for all single leaf experiments. The droplets were applied using a digital Finnipippette (Cole-Palmer). In whole plant experiments. the entire plant was sprayed to wet the foliage. Data were collected on total shoot length every five days with a ’MaxCal’ Max-15 digital micrometer. . In all experiments treatments were applied and data were collected on both ’Hright Golden Anne’ (BGA), and 'Circus’ to study cultivar specificity. .Individua Ex er ent Procedures: Experiment 1: Application To A Whole Plgpt During Development: Plants were treated with a foliar application of a 2500 mg 1’1 a.i. daminozide solution 10. 20. 30, 40, or 50 days after the initiation of short days. The average dosage for each treatment date was 7.25, 16.25, 25.13, 27.30, or 27.30 mg plant'l. respectively. The total length 64 of the second lateral shoot was measured every five days un- til elongation ceased. Cessation of elongation was cultivar and treatment dependent and occurred between 48 and 62 days after the initiation of short days. The plants were randomized within the chambers at in- itiation of short days and every measurement day thereafter. The data were analyzed as a completely randomized design with six treatments and five plants per treatment. Expgrigentyyg: Application To g Single Internode During ve e : The leaf below the second stem segment from the stem base was treated with 125 ul of a 2500 mg 1'1 ,daminozide solution on 16, 19, 22, 25. 28, 31, 34. or 37 days from the initiation of short days. Data were collected on the length of the second stem segment every 3 days from day 15 to day 40 after the initiation of short days. At flower. the treated stem segments were removed and fixed in a 708 FAA solution. Cell length and number determinations were made on the xylem cells. Plants were randomized within the chambers at the in- itiation of short days and every measurement day thereafter. The data were analyzed in a completely randomized design with nine treatments and five plants per treatment. 65 Experiment 3: Additive Responses 0f Multiple Applications: One or two stem segments on a shoot were treated with 75 ul of a 3890 mg 1'1 solution of daminozide in the combinations shown in Table l. The leaf at the base of a stem segment was treated with the daminozide solution when the segment reached 0.5 cm in length. Data were collected on all stem segments every 4 days for 60 days from, the initiation of short days. The plants were randomized at the initiation of short days and every measurement day thereafter. Data were analyzed as a completely randomized design with 16 treat- ments and five plants per treatment. Results Experiment 1: Application To A Whole Plant During Development: Retardation of lateral shoot elongation decreased as the time of daminozide application occurred later in plant development (Table 2). With ’Circus’. greatest total shoot retardation (43 8) occurred with an ap- plication on day 10 after which retardation associated with a daminozide application decreased rapidly until day 50 (Fig. 2). With EGA retardation from applications made on days 10. 20 and 30 were similar. Retardation of elongation 66 decreased rapidly fros day 30 to day 60. Retardation per microgram daminozide decreased faster than retardation of total elongation. The lack of response to an daminozide application after day 50 with Circus and day 60 with BGA was expected as elongation had ceased on plants which had received no daminozide application after these days. In contrast to total retardation, post application elongation retardation. defined as the percent retardation of elongation that occurred following an daminozide application, increased for both Circus and BGA as the time of application occurred later in plant development (Table 2). Experiment 2: Application To A Single Internode During Development: Retardation of elongation decreased as the time of daminozide application occurred later in the development of the second stem segment (Table 3). The retar- dation response was asymptotic for daminozide applications after day 13 with no difference in the final lengths of the second stem segments compared with plants which received no daminozide (Fig. 3). Although both cultivars responded to daminozide in a similar manner (Fig. 3), elongation of DGA stem segments was inhibited approximately 12-138 more than stem segments of Circus. 67 Final cell length decreased as applications of daminozide occurred earlier in development (Table 3). The retardation of cell elongation was accompanied by an in- crease in cell number. No stimulation of total stem length was observed with an application of daminozide to BGA on day 19, whereas. stem extension was stimulated 188 with Circus. The cell number and length data indicate that the stimulation result- ing from a late application of daminozide resulted from an increase in cell elongation. not number. The magnitude of cell elongation retardation or cell number promotion in response to daminozide was at least 208 greater with BGA than Circus. Experiment 3: Additive Responses To Multiple Applications: Response of a stem segment to daminozide applied to the leaf immediately below that segment decreased as a stem segment became more distal, then increased with the fifth stem seg- ment (Table 4 and Fig. 4). The stem segments most sensitive to daminozide were the second and fifth. Applications of daminozide made to the second stem segment were three times more effective in inhibiting elongation than applications to the fourth stem segment (Table 4 and 5). The retardation resulting from multiple applications was not additive. Eighty percent of the plants from treat- 68 ments receiving multiple treatments had greater overall retardation than the sum of the retardation resulting from applications made to separate stem segments on separate plants. Discussion Retardation of shoot elongation by daminozide varied with the physiological age of the shoot’s component internodes. the position of the internodes on the shoot, and if previous applications of daminozide had been made to the shoot. Applications of daminozide early in internode development stimulated cell division, but inhibited cell elongation to such a degree that the ultimate length of the internodes was reduced. Applications late in development did not increase cell number but stimulated cell elongation in Circus but not HGA. To achieve any retardation of inter- inode expansion these results indicate that it was necessary to apply daminozide no later than 10 days after an internode elongated to 0.5 cm in length. Distal internodes displayed a smaller response to daminozide than more proximal internodes with the exception of stem segment 5 (Fig. 4). Some factor either enhanced the response of more proximal internodes to daminozide or .some factors reduced the effectiveness of applications to distal 69 internodes. Hanks and Rees (3) reported the removal of a tulip flower early in development reduced total stem length by 59 percent. Retardation of elongation resulted from pedicel and more distal internode retardation (3). Applica- tion of indoleacetic acid (1AA) to the cut surface reversed the retardation while application of gibberellins did not (7). Studies by Op den Helder showed that removal of the gynoecium alone could produce the same retardation of stem elongation associated with flower removal (7). Stem elongation of tulip was also found to be inhibited by removal of leaves prior to anthesis (2). Retardation of stem elongation due to leaf removal resulted from retarda- .tion of the more proximal internodes. Application of ancymidol, a known gibberellin biosynthesis retardant (10), retarded internode expansion in the first internode but not the fourth (10). Based on these results Hanks suggested that two mechanisms control stem extension in tulip: 1) an auxin mediated system which is dependent on gynoecium auxin production and 2) a gibberellin mediated system dependent on stem and leaf gibberellin production (3). Jeffcoat showed chrysanthemum flowers produce GA: and 0A9 and an auxin similar to IAA (5). Gibberellins similar to those produced .by the flower were found in the leaves. while a different gibberellin complex (GAe. GAs, GAe, or GA?) was found in the stem. The activity of the latter gib- 70 berellins decreased with shoot age. These findings suggest the presence of a differentially distributed growth promoter. i.e. auxin and/or gibberellins. present in higher concentrations in the more distal inter- nodes of chrysanthemum. The increase in the response to daminozide of the fifth stem segment indicates that the con- centration of the elongation promoter may decline late in development of the shoot. Jeffcoat observed that the growth promotive activity of the flower decreased significantly from stage 3 of flower development to stage 5 (from the time when the flower bud is spherical with 12 bracts around the rim to when the bud is flattened with 2 or 3 rows of floret primordia)(5). This period corresponds to the time period just prior to day 60 with BGA and 50 with Circus when the fifth stem segment is developing. These studies plus the data presented in this paper suggests that the flower, young leaves and the stem of the chrysanthemum produce growth promotive substances which are present in higher concentra- tions in the distal internodes. These substances reduce daminozide induced retardation. As the shoot develops and as the flower approaches stage 5, growth promoter production of the flower probably declines. and as no new leaves are produced. gibberellin activity probably declines. This decease in gibberellin activity may be responsible for the increase in retardation response observed in the fifth stem 71 segment as compared to the fourth stem segment. The results of experiment 3 indicate that multiple ap- plications of daminozide are not additive in their retarda- tion of internode extension. The sum of the retardation of plants which received single applications to a stem segment was less than the total retardation resulting from plants which received multiple applications to the same segments. Conclusions 1. Applications of daminozide to internodes in excess of 10 days old did not inhibit internode elongation and in some cases stimulated elongation primarily through stimulation of cell elongation. 2. Daminozide stimulated cell division but inhibited cell elongation to such a degree that the final length of an internode was less than if it had received no daminozide application. 3. Applications of daminozide to more distal internodes resulted in less retardation of elongation than that seen with a similar application to more proximal internodes. It was hypothesized that differential levels of a growth promoter present in higher concentrations in more distal internodes interfered with daminozide retardation of cell 72 elongation. Multiple applications of daminozide were not additive in their response. 73 Bibliography 1. Cathey. ELM. 1975. Comparative plant growth retarding activities of Ancymidol with ACPC, Phosphon. Chlormequat, and daminozide on ornamental plant species. Hortscience 10:204-216. 2. De MMnk, WkJ. 1979. Influence of plant regulators on the development of bulbous plants with special reference to organ relationships. Acta Hort. 91:207-213. 3. Hanks, G.R. and A.R. Rees. 1977. Stem elongation in tulip and narcissus; .the influence of floral organs and growth regulators. New Phytol. 78:579-591. 4. Rentig,‘W.U.v. 1979. Early treatment of ornamental plants with retarding substances. Acta Horticulturae 91: 353-364. 5. Jeffcoat. B. and R.E. Cockshull. 1972. Changes in the levels of endogenous growth regulators during develop- ment of the flowers of cpgyganthemum.morifolium. J. of Exp. Bot. 23(76): 722-732. 6. 74 Eobayashi. S. 1975. Growth analysis of plant as an assedflage of internodal segments - a case of sunflower plants in pure stands. Japanese Journal of Ecolm 25(2) : 61-70. 7. Op den Eelder. P. and M. Denschop, and A.A. DeHertough. 10. 11. 1971. Factors affecting floral stalk elongation of flowering tulips. J. Amer. Hort. Soc. &(5):603-605. Riddell. J.A.. H.A.Hageman, C.M.J’Anthony, and 11.1.. Hubbard. Retardation of plant growth by a new group of ch-icals. Science 136:391. Seeley.J. Interpetation of growth regulator research with floriculture crops. Acta Hort. 91:83- 92. Shoub, J. and A.A. DeHertough. 1974. Effects of An- ' cymidol and gibberellins Aa and AM? on Tulim ges- neriana L. cv. 'Paul Richter’ during development in the greenhouse. Scientcia Hort. 2:55-67. Wilde, M.H.. and L.J.Edgerton. 1969. Some effects of a growth retardant on shoot meristems in apple. J. “Amer. Soc. Hort. Sci. 94:118-122. 75 Table 1. Treatment combinations designed to study the effect of daminozide applications to internodes at different positions on the plant and responses to multiple applications. Segment ' Stem Segment Number First Second 0 l 2 3 4 5 Application Application None None - - - - - - 1 None - + - - - - l 2 - + + - - - l 3 - + - + - — l 4 - + — - + - 1 5 - + - - - + 2 None - - + - - - 2 3 - - + + - - 2 4 - - + - + - 2 5 - - + - - + 3 None - - - + - - 3 4 - - - + + - 3 5 - - - + - + 4 None - - - - + - 4 5 - - - - + + 5 None - - - - - + ' Applications were made when stem segments were 0.5 cm in length. 76 Table 2. The effect of a single daminozide application at different stages in the development of Chgypanthemum morifolium “Bright Golden Anne’ and ’Circus’ on final length of the second lateral shoot. Duinozide was applied as a foliar application to the whole plant until runoff. Retardation(mm0 Retardation of Day Of Total Per Microgram Total Post Application Application 3 Daminozide applied Retardation(8)y Elongation ‘Bright Golden Anne’ 10 10.0 1.37 25 28 20 11.0 0.67 28 34 30 11.4 0.45 29 44 40 8.8 0.32 22 53 50 4.3 0.16 11 66 so x - - _ _ Linear ' at: set 888 sex Quadratic as: are xxx xxx Cubic N.S. N.S. N.s. N.S. 'Circus’ 10 7.8 1.08 43 51 20 4.5 0.28 25 42 30 4.4 0.18 24 52 40 3.1 0.11 17 62 50 0.7 0.03 4 42 60 x _ - _. - Linear 888 888 888 888 Quadratic N.s. N.s. N.s. W.S. Cubic 88 88 88 88 ' Days after pinch and start of short days. V Based on total second lateral shoot length. 5 No application was given to these plants. ' Significant at P=.05(#). P=.01(8t). P=.001(¥88), not significant(N.S.) 77 Table 3. The effect of daminozide application time on cell size and number in Chpyganthemum morifolium. Two hundred ninety two ug daminozide was applied as five 25ul droplets of a 2333 mg/l solution to the leaf at the base of the second stem segment. Tissue samples were taken from the treated internodes at the cessation of elongation. Percent Cell Length Cell Number Internode'l Day Of Retardation of Application Final Length Finalx 8 Change' Final" 8 Change Y 'Bright Golden Anne’ -5 41 164 - 65 246 + 84 1 24 179 - 62 285 + 113 4 18 189 - 60 293 + 119 7 13 198 - 58 293 + 119 10 10 234 - 50 252 + 88 13 -2 262 - 44 255 + 90 16 -6 309 - 34 211 + 57 19 9 328 - 30 184 + 37 Control - 469 - 134 - Linear ' 888 ' 888 888 Quadratic 888 888 888 Cubic 888 888 888 'Circus’ -5 30 200 - 9 150 + 78 l 12 159 - 27 101 + 20 4 3 180 - 18 99 + 18 7 8 210 - 4 79 - 6 10 4 229 + 5 77 - 8 13 -2 216 - l 87 + 4 16 -6 235 + 7 83 + l 19 -18 197 - 10 100 + 19 22 - 219 - 84 - Linear 8 888 888 88 Ouadrat lo 888 888 888 Cubic 888 888 888 3 Significant at P=.05(8). P=.01(88), P=.001(888),not significant(N.S.) ’ Percent change relative to untreated control. " Mean cell length (microns) " Cellw m is calculated frm the mean internode length divided by the mean cell length 78 Table 4. Percent retardation of C anthemnm morifolium cv. ’Bright Golden Anne’ stem segments resulting from single and multiple applications of 292 ug of daminozide delivered as three 75u1 droplets applied to the leaf immediately below the stem segment when it was 0.5 cm in length. Percent Retardation Segment ' Stem Segment Number First Second 1 2 3 4 5 Application Application 1 None 2 l4 3 6 l l 2 - 26 7 l5 _ 18 l 3 2 l9 15 23 18 l 4 6 26 16 18 14 l 5 11 16 10 ll 4 2 None 2 27 14 17 2 2 3 2 31 24 22 12 2 4 14 23 5 21 l 2 5 5 25 6 7 -4 3 None 0 20 15 17 7 3 4 10 28 26 15 12 3 5 -8 18 20 15 2 4 None ll 14 0 8 6 4 5 -7 25 l4 l5 7 5 None 0 12 l4 13 11 Position versus Percent Retardation Linear V 8 Quadratic 8 Cubic 8 ' Applications were made when the stem segments were 0.5 cm in length. V Significance at P = .05 (8). P = .01 (88). P = .001 (888), not significant (W.S.) . 79 Table 5. Percent retardation of C anthemum morifolium cv. ‘Circus’ shes segments resulting from single and multiple applications of 292 ug of daminozide delivered as three 75ul droplets applied to the leaf’immediately below the stem segment when it was 0.5 cm in length. Percent Retardation Segment ' Stem Segment Number First Second 1 2 3 4 5 Application Application ‘Circus’ 1 None -7 15 6 -2 5 l 2 -3 25 6 3 ll 1 3 6 25 19 10 6 l 4 -9 5 l 6 2 1 5 -6 26 15 5 7 2 None 6 28 7 2 21 2 3 -12 16 10 14 12 2 4 5 24 13 16 18 2 5 -3 25 25 15 23 3 None 4 15 11 -l 2 3 4 3 3 l7 12 16 3 5 -14 0 8 5 18 4 0 -10 11 7 9 23 4 5 -6 6 3 7 23 5 None 0 12 17 20 37 Position versus Percent Retardation Linear V 888 Quadratic 888 Cubic 888 ' Applications were made when the stem segments were 0.5 cm in ‘ length. V Significance at P = .05 (8), P = .01 (88). P = .001 (888). not significant (N.S.) Figure 1. 80 Diagram identifying stem segment partitioning of the second lateral shoot. The first and sixth stem segments are single internodes. The remaining segments are composed of two adjacent internodes. Experimental plants consisted of three shoots. One daughter shoot is shown. €51.55” Segment 6 f“ Segment 5 are 4"» Segment 4 / l". giék Segment 3 . I: an“ ) E 46*: g: ' Segment 2 82 Figure 2. Effect of the time of a daminozide application on final second lateral shoot length of BGA (a) and Circus (b). Plants were sprayed with a 2500 mg 1‘1 solution of daminozide till ’runoff’. Shoot Length (cm) Shoot Length (cm) 83 'Bright Golden Anne' 0 42-)! - 31/(1+Exp(32+a3-x+34:x:x+a4oa4.x«3/(3-33))) D B R'- .332 . I, 35... 34- 30- 26- 22 'Circus' b 20- . . m- .349 z 3 3 134 a 4 D 13~ J . 149 2 Circus . 3(1) 39.1354 39.0901 ,2- 3(2) -.46517 1.3365 0 . 3 3(3) -.73433-1 4.03544 ‘ I . 3 - e . . - f , r 0 1'0 2'0 3'0 4'0 5'0 3'0 7'0 3'0 Time Of Application (days) 84 Figure 3. Effect of the time of a daminozide application on the final stem segment length of BGA (a) and Circus (b). The leaf at the base of the second stem segment recieved an application of 292 ug of daminozide delivered as five 25 ul droplets of a 2333 mg 1'1 solution. Length (cm) Length (cm) 85 'Bright Golden Anne' 5' YHAT - 3(1) . EXP(B(2) . x) + 3(3) 0 d a 7- o g a n o t-%--&--a D a ‘9’" 5-l ’,s’ n a O,o’ I D g 1 ’a D . 5-l a ,4, n 9 4 ° ‘1 I, a 8 3' a — Control 2 "' 'i' SAD" 'Circus' 2.4- ‘ I U 2.0d u ”may“- ' 5' 1:3d’--. n .~.: 0 'v" a 1e6‘ ’o’”lo ' ’4’ 0 :.2~ ° ’x’ . a" BGA Circus 0,3J 0 8(1) 4.95425 1.41757 4 8(2) .I 709390 .0801 E-I . R3 .54 .77 0.0 -‘I0 -'3 0 73 1'0— 1'5 2'0 2': Day Of Application 86 Figure 4. Effect of stem segment position of BGA (a) and Circus (b) on the response of that segment to a 292 ug dainozide application delivered as three 25 ul droplets of a 7500 mg 1'1 solution. Inhibition Of Elongation (2) Inhibition Of Elongation (7;) 87 'Bright Golden Anne' 40-1 Y-8(0)+B(t)4X+8(2)eXes2+B(3)eX«3 R'- .736 ° 30.. 20- .l 104‘ cl 0 'Circus' so. 4 R‘- e874 0 40-1 9 . J , 30d 20- 10- 8 i D D 0- ° ° . BGA. ° Circus --104 3(0) -59.1431 -123.922 . 8 3(1) 102.3733 133.7293 '2°‘ 3(2) -37.3237 -57.3200. -30 ‘ 3(3) 3.0759 . 6.2276 0 i 2 3 i i ' 3 Stem Segment Number Section II the Influence of Day Tenperetnre end Photosynthetic Photon flux on the Reopen-e of Chrzeenthennn norifolig; Benet. cvs. 'Brixht Golden Anne’ and 'Circnn’ to Deninozide The influence of dey tenpereture end photosynthetic photon flux on the response of Chgzsenthgggl gogifoliun Benet. cvs. Bright Golden Anne’ end Circus’ to deninozide. John B. Irwin1 end Royel D. Reins2 Depertnent of Horticulture Michigen Stete University lest Lensing. MI 48824-1112 Additionel Index words: deninozide, Aler, gonpertz function, sten elongetion, dey tenpereture, photosynthetic photon flux. fleceived for publicetion . Mich. Agr. Expt. Ste. J. Article No. . The euthors eppreciete the technicel essistence of Cethey lredenburg. Jenes Eppink, end Sheron Strned during this project. Plent neteriel wes doneted by Yoder Brothers of Berberton, Ohio. The cost of publishing this peper wes defreyed in pert by the peynent of page cherges. Under postel reguletions, this peper lust be hereby nerked edvertisenent’ solely to indi- cete this fect. 1Reseerch Assistent. 3Associete Professor 88 89 Abstract The Influence of Day Tenpereture end Photosynthetic Photon Flux on the Response of Chrysanthenu; gozjtgliun Benet. cvs. ‘Bright Golden Anne’end ‘Circus’ to deninozide Retardation of final shoot length by e 5000 ng l"1 deninoeide epplicetion to 'Bright Golden Anne’ end 'Circus’ decreased 76 end 83 percent as day tenperature (DT) in- creased fron 10 to 26°C. Increesing BT fron 26 to 30°C reversed this trend end increesed deninozide retardation of final shoot length. Photosynthetic photon flux (PPF) did not influence plent response to deninozide at low reterdent concentretions. At high deninozide concentrations (>2500 ng ‘1) reterdetion of elongetion increased 10 to 17 percent ae PPP increased from 200 unol s“ n" to 600 unol s"l n'z. Three nethode were evelueted for quantifying the response of shoot elongetion over tine to 0? end deninozide concentretion. Method I end III were of the fore f(x) 3 g(x) = retarded elongetion: where g(x) = en in— hibitor function. and f(x) defined the rete of shoot elonge- 90 tion (Method I) or absolute shoot length over tine (Method III). Method II predicted gonpertz function peraneter es- tinetes through nultilineer regression; when evaluated within a gonpertz function absolute shoot elongation over tine was predicted. Method II had the greatest potentiel for nodeling sten elongation as influenced by daninozide Introduction The chenicel butanedioic acid nono (2,2-dinethyl hydrazide) (daninozide) has been found to reduce internode elongation in a nunber of plant species (1.3.4.14,16,17). Once applied to a sensitive plant species. sten elongation is depressed for sone period of tine after which elongation nay resune at a rate sinilar to control plants (6,19). Studies of daninozide persistence within the plant have in- dicated that breakdown is very slow and is negligible during the life of nost herbaceous plants (5,19). Zeeveert at- tributed the loss in response to daninozide over tine to be caused by deninozide dilution within the plant tissue during growth to sone concentration below which no retardation oc- curred (19). The length of tine fron a growth retardant application to the resunption of unretarded elongation is influenced by the environnent (3.6.13.17). Reductions in effectiveness 91 associated with variations in the environnent have been noted with the response of Chrzgggthengg to daninozide (9). The extent to which either tenperature or PPP deternine the response of a plant to an daninozide application has not been quantified. The results presented in this paper quantify the in- fluence of both PPP and day tenperature (DT) on the duration of daninozide induced retardation of sten elongation in c r nt e u norifoliu . Research by Karlson and Reins (11) indicated that there was no significant interaction be- tween PPP and daninozide effectiveness, therefore, this fac- tor was not studied. The ultinate goal of this research is to .predict deninozide effectiveness within Chrzggntheggn norifgliu; over tine under a variety of environnentel conditions. The ability to predict daninozide effectiveness would enable daninozide reapplication prior to the loss of effectiveness .of a previous application if additional height control is necessary e Materials and Methods flaggggl [ggcedgreg: Rooted cuttings of Chgzgggthenug norifoliu; 'Bright Golden Anne’ (BGA) and 'Circus’ were planted in a connercial potting nix (sphagnun peat, verniculite, perlite) in 10.2 on plastic pets. The cul- 92 tivers 'Bright Golden Anne’ and 'Circus’ were were chosen because of their connercial significance and to conpare responses to daninozide of cultivars with different flower ferns: decorative and daisy. All plants were placed in controlled environnent chenbers under a photosynthetic photon flux (PP!) of 326 unol s'1 n'3 delivered with cool white fluorescent lanps for 16 hour d'l. Day and night ten- peratures were nainteined at 20°C and 16°C. respectively. Plants were fertilized at each irrigation with 200 ng l"1 of N and B. After 7 days, the apical neristen of the plants were renoved and the photoperiod was reduced to 8 hours d“! to induce flowering. Twelve days after the initiation of .short days, lateral breaks other than the apical 3 of each plant were renoved. daninoside was applied as a foliar ap- plication to the whole plant 16 days after the start of short days. All treetnents were applied to both 'Bright Gloden Anne’(BGA) and 'Circus' and data were collected on total shoot length every 4 days with a Mexcal 'Max-l5’ digital caliper. a x e ta ed Wiesel; Twenty plants of each cultivar were placed in 5 controlled environnent chenbers. At the initiation of short days the day tenperature (DT) in each of 93 the chenbers was changed to either 10°, 18°, 22°, 26°, or 30°C. The plants within each DT treetnent were separated into four groups of five plants each. Each group received a foliar application of deninozide with a concentration of either 0, 1250, 2500, or 5000 ng 1'1. The daninozide was delivered as a foliar application until 'runoff’. This resulted in a dose applied to the plants of 0.0, 5.63, 11.25, or 22.50 ng, respectively. Final shoot length data were analyzed at the cessation of elongation of the second lateral shoot as a split-plot statistical design with DT as the nain plot and daninozide concentrations as the subplots. The experinent consisted of twenty treetnents with 5 replicates per treetnent. Photogznthetic Photon Flux Bxgerinegt: Twenty plants of each cultivar were placed in 4 controlled environnent chenbers. At the initiation of short days (8 hours) the PP! within each environnentel chanber was changed to either 50, 200, 400, or 600 unol s'1 n'z. Plants within each PPF treetnent were separated into 4 groups of 5 plants each. Bach group received an application of daninoside with a con- centration of either 0, 1250, 2500, or 5000 ng 1". The average dose applied with each application was the sane as in the DT experinent. Final shoot length data were analyzed as e split-plot 94 statistical design with PPF as the nain plot and daninozide concentrations as the subplots. Twenty treetnent conbina- tions were used with 5 replicates per treetnent. D t A o t on Tech 1 ue Pi re 1 : A nonlinear regression function was selected as a neens of quantifying shoot elongation over tine for three reasons: 1) nonlinear regression functions are based on the prenise that growth which has yet to occur is in sone way related to the growth which has previously occurred (this would be expected with nost eucaryotic or- ganisns where growth rate is often influenced by an organisn’s size) 2) nonlinear function paraneter estinetes can have real biological neaning, i.e. when significant they nay represent the rate of growth, initial length, or the asynptote of a fit date set 3) nonlinear functions are capable of approaching an asynptote. Based on the charac- teristics of the sten elongetion over tine rete , i.e. non- syneteric point of inflection and a slow approach in final shoot length (PSI), the gonpertz function (10) was fit to the shoot length data (Figure 2). The gonpertz function takes the forn of: -CT -be Shun nummh==ae Where a = esynptote (FSL) 95 ae'b = initial at- length c==reuscmmnant T==t1ne (1n b)/c = inflection point (IF) a/(ln IF) = new absolute extension rate (MAER) Paraneter estinetes were calculated fron the data for the gonpertz function for elongation of unretarded shoots as DT or PP! varied using the software progran ’Plotit’. First derivatives for each function were calculated at 5 day in- tervals and were subnitted to nultilinear regression analysis as the dependent variable with tine and DT or tine and PP! level as the independent variables. The ‘All pos- sible subsets’ subroutine of the BMDP statistical software package (7) was used to deternine the best possible predict- ion function based on r3. Maxinun likelihood paraneter es- tinetes were calculated fron the data for the gonpertz func- tion using ’Plotit’ for daninozide retarded elongation after day fifteen. First derivatives for each function were cal- culated and a nultiplication factor, i.e. a nunber which when nultiplied by the rate of growth of untreated plants yielded the rate of retarded elongation, was calculated at five day intervals. The predicted nultiplication factors were subnitted to nultilinear regression analysis with tine, daninozide concentration, and day tenperature or PPF as the independent variables. The ’All possible subsets’ sub- routine of the BMBP statistical software package (7) was used to deternine the best possible prediction function 96 based on r3. h i I 3 : The gonpertz function was fit to shoot elongation data over tine of both untreated and daninozide treated plants fron day 0 to 60, and day of ap- plication .15. to day 80, respectively (figure 3). All paraneter estinetes were tested for significance using the Student’s t value. If the paraneters within a nodal were not significant, the FBI (or asynptote) was weighted and the data were resubnitted to nonlinear regression analysis. The naxinun nunber of tines that this procedure was enployed was twice. The ’a’ paraneter estinate was then subnitted to nultilinear regression analysis as the dependent variable with RT and daninozide concentration as‘ the independent variables. The ’All possible subsets’ subroutine of the BMDP statistical software package was used to deternine the best possible prediction function based on r“. The ’b’ and ’c’ paraneters were predicted in a sinilar .fashion to the ’a’ paraneter with the exception that the ’a’ paraneter was used, in addition to BT and daninozide concentration. as an independent variable. Prediction of daninozide retarded elongation on an ab- solute length basis was acconplished by using the predicted absolute length of untreated plants prior to the day of an daninozide epplicetion (day 15) and the predicted retarded absolute shoot length after day 15. 97 Technigge III (Figure 4): The gonpertz function was fit to shoot elongation data over tine for both untreated plants and daninozide treated plants as described for Method II. Bowever, rather than predicting daninozide retarded growth through prediction of paraneter estinetes, absolute shoot length was predicted through nultiplication by an inhibitor function (figure 4). The inhibitor function was derived by dividing the predicted retarded absolute shoot length by the 3 untreated shoot length and subnitting these data to the ‘All possible subsets’ subroutine of the BMBP statistical software package as the dependent variable and DT, daninozide concentration, and tine as the independent ,variebles. Results and Discussion Shoot Length Versus Ting: Final lateral shoot length (FSL) increased 1358 for both BGA and Circus as DT increased fron 10 to 26°C, then decreased as DT increased fron 26 to 30°C (Table l and 2, figure 2). Of the change in BGA shoot length, 87 percent of the increase in shoot length was as- sociated with an increase in OT fron 10 to 18°C. Mith Cir- cus only 422 of the increase in shoot length occurred as DT increased fron 10 to 18°C; 93 percent of the increase in shoot length occurred with an increase in DT fron 18 to 98 26°C. As DT was further increased fron 26 to 30C shoot length decreased 22* for Circus, but renained unchanged for BGA. As DT increased fron 10 to 26°C the nean absolute sten extension rate (MABR) increased and the inflection point of the fit gonpertz function occured earlier; this indicated that the tine at which shoots reached their final length oc- curred earlier, and the rate at which they approached their final length increased (Table l and 2). As DT further in- creased fron 26 to 30°C, the tine at which shoots attained their final length was delayed and the MABR decreased. These results suggest the optinal RT for sten elonga- tion in Ch s the u nor 0 iun cvs. BGA and Circus occurs between 26 and 30°C when the night tenperature is 16°C (Figure 5). ’Circus’ responded to an increase or decrease in OT about the optinal BT nore strongly than BGA. BGA ap- peared to have a slightly higher optinal DT (28-29)°C than Circus (26-27)°C. The existence of an optinal tenperature is expected since an increase in OT nust eventually result in denaturation of proteins and a subsequent decrease in final shoot length and increase the rate of respiration to a level which nay linit elongation through a reduction in the total carbohydrates available for elongation (18). Want reported optinal day tenperatures for sten elongation for a nunber of plant species including Pisgg. Achilles, 99 Cypridedigg.agd ngbidiun (18). larlsson and Reins (12) suggested that shoot length in BGA increased linearly as DT increased fron 10 to 30°C. Since no experinental treetnents above 26°C DT had night tenperatures below 20°C in their work, the lack of an op- tinal or naxinun sten length as BT increased appeared to be a result of a delay in flower initiation and an increase in node nunber. Internode length did not decrease in their work at tenperatures above 26°C. Daninozide decreased the ’a’ paraneter of the gonpertz function and the absolute sten extension rate in all DT treetnents, i.e. FSL decreased and the tine when the PSI was reached was delayed as evidenced by the delay in the day the inflection point occurred (Fig. 6, Tables 1 and 2). Overall daninozide reduction of FSL increased under conditions which were not optinal for elongetion, i.e. RT of 10 and 30°C (Figure 7). At optinal DT, i.e. 26 to 29°C the response to an daninozide application was linited indicating that the daninozide nay be diluted nore rapidly at these tenperatures due to increased sten growth and loose effectiveness nore rapidly than at less than optinal tenperatures (Fig. 7). In general, BGA was nore responsive to daninozide applica- tions than Circus with tenperatures below 26°C. Circus was nore responsive to daninozide applications with day tenpera- tures above 26°C (18) (Table l and 2) 100 As DT increased, daninozide response decreased. A 50 percent reduction in daninozide effectiveness was seen as DT was increased fron 10 to 30°C with a 5000 ng 1‘1 application (Figure 8). Schonherr and Bukovec deternined that an in- crease in tenperature fron 20-25°C to 25-30°C increased daninozide penetration into Phaseolus vulgaris leaves by 482 (15). This increase in perneability was believed to occur through an increase in flexibility of the cutin natrix through dissociation of weak bonds within the nenbrane. Results presented in our research suggest that sone internal factor is influencing plant response to daninozide near a day tenperature of 26°C which resulted in a overall reduc- tion in response despite of the presuned high absorbence of daninozide through the leaf cuticle due to the relatively high day tenperature. Applications of daninozide increased the length of tine which a shoot elongated as deternined fron the delay in the point of inflection (Tables 1 and 2) of the fitted growth functions. Previous studies on the influence of daninozide on cell elongation and division in Chrysanthegun indicated that daninozide increased cell nunber within an internode, but decreased cell elongation to such an extent that overall internode length was reduced (8). Mornal shoot developnent through both cell division and elongation is hypothesized to result in a dilution of daninozide below sone critical con- 101 centration needed for retardation. It is suggested that those cells which had forned as a result of daninozide stinulated division which had not yet expended prior to a loss of responsiveness, develop nornally after daninozide is no longer effective. This cell elongation results in an extended period of elongation late in developnent. The nag‘ nitude and duration of the extended period was enhanced with nonoptinal day tenperatures. otos nthetic Photo F : Shoot length was not influenced by an increase in PPF (Fig. 9). The reduction in shoot length associated with an daninozide application was PPF dependent. daninozide effectiveness decreased as PPF in- creased fron 50 to 400 unol s"1 n‘z, then increased as PPF increased fron 400 to 600 unol s" n". II Rate Of Bloggation Shoots of BGA plants had a higher rate of elongation than shoots of Circus for all 0T treetnents except at 26°C where the naxinun rate of elongation for Circus was sinilar to that of BGA ( 1.01 on day'1 vs. 1.08 on day‘l) (Figure 10). Mean sten elongation rate increased as DT increased fron 10 to 26°C. Circus exhibited a greater change in the rate of elongation than BGA as DT increased above '22°C 102 (Table l and 2). An increase in the DT fron 22 to 26°C resulted in a 45 percent increase in the naxinun rate of elongation for Circus as conpared to only a 20 percent in- crease with BGA. Further increasing 0T fron 26 to 30°C decreased the naxinun rate of elongation by 33 and 3 percent for Circus and BGA. respectively. . The day when the naxinun rate of elongation occurred (inflection point) was cultivar and DT dependent (Tables 1 and 2). With a 26°C DT, the naxinun rate of elongation oc- curred on day 15 and 20 for Circus and BGA, respectively. As the DT either increased or decreased fron 26°C, the day when the naxinun rate of elongation occurred was delayed. The _naxinun delay under these experinental conditions was greater with BGA (14 days at 10°C) than Circus (8 days at 10°C). The length of tine a shoot elongated was shortest with a 0T of 26°C (Fig. 10). As tenperatures deviated fron 26°C the length of tine which a shoot elongated increased. Shoots fron BGA plants elongated 6 to 21 days longer than shoots of Circus plants for all BT treetnents. Deninoz de In d Retardat on: Application of daninozide resulted in a discontinuity in the rate of shoot elongation at the tine of an application (Fig. 11). Follow- ing an daninozide application, three distinct post applica- tion phases of elongation were evident: 1) elongation fron 103 the tine of an application to the start of a brief burst in elongation (Phase I), 2) the brief burst in elongation (Phase II), 3) and elongation following Phase II (Phase III) (Fig. 11). The nature of Phase II nede the developnent of a single function to describe postapplication elongation difficult. Since the overall influence of Phase II on the absolute length was not great, it was not included in the nodal. Mhen Phase II was not included it was possible to develop a single function which described the percent retardation of both Phase I and III as they were influenced by BT over tine. Day Tenperature Bxperinent The relationship between tine and percent retardation due to an daninozide application was quadratic irrespective of DT (Figure 12). The percent retar- dation initially increased at high daninozide concentrations for about ten days after an application (fron day 15 to 25) then decreased to day 55. The nagnitude of the quadratic response increased as daninozide concentration increased and was greater with BGA than Circus. Percent retardation decreased as BT increased fron 10 to 26°C irrespective of tine then increased as 0T increased fron 26 to 30°C at high daninozide concentrations (Figure 8). The response to daninozide was linear and increasing as daninozide concentration increased irrespective of DT. 104 An increase in DT fron 10 to 26°C decreased the tine that an daninozide application was useful in retarding elongetion. Increesing DT fron 26 to 30°C increased the tine which an application was effective (Figure 13). Photo t c Ph ton F1 Bx erin A change in PPF did not influence percent retardation of elongation at low concentrations of daninozide. As the concentration of daninozide increased percent retardation of elongation was greater at 100 unol s'1 n'3 than at 500 unol s"1 n‘3 (Figure 14). With BGA, no difference in the percent retardation was seen between different PPF levels until concentrations of daninozide were above 2500 ng 1‘1. odel Bva atio : Retardation of sten elongation by daninozide is an episodic event, i.e. sten elongation is in- fluenced by sone stinulus which varies in nagnitude with tine. One forn a nodel describing an episodic event nay take is: Shoot length = f(x)*¢(8) where: f(x)= unretarded elongation g(x)= sane nultiplication factor which when nultiplied by f(x) yields the retarded absolute shoot length, or shoot elongation nun. Method I and II (Figures 2 and 3) are based on this forn where g(x)= function defining the retardation of the 105 rate of elongation by daninozide (Table 3) and retardation of absolute shoot length over tine (Table 4), respectively. The use of an inhibitor function is attractive in that, the influence of a stinulus such as DT, daninozide concentration, or tine on the response of a plant to a growth inhibitor can be studied independently. Models which are conposed of a single function quantifying absolute shoot elongation over tine as influenced by one or nore stinuli nay be nore difficult to interpet, since the kinetics of the elongation response, as one or nore stinuli change, is in- herent within the function. Model I (Figure 2 and 15) is based on the prediction of growth rate over tine (Table 5). The benefit of this type of systen is the increased sensitivity to snall changes in elongation thereby gaining a better insight into the kinetics of the loss of daninozide response. The liniting factor with this type of nodal is the flexibility required by the predicted function which is not necessarily possible when nultilinear regression techniques are used. This problen is conpounded by two successive nultilinear regres- sions on a given function, and nultiplication of two functions, thus conpounding any previous errors in prediction. As a result this nethod was not found to be ac- ceptable as a neens for predicting daninozide retarded elon- gation over tine. 106 Method I was found to yield acceptable results when a variation of this nethod was used to quantify the influence of daninozide on Chgzsanthenun norifgliun shoot nass over tine; where g(x) was derived fron a function previously used to quantify retardation of an exponentially growing bacteria culture by tetracycline (6). Leith and Reynolds also used a variation of Method I to quantify the influence of a short tern ozone exposure on plant dry weight (11). Method II (Figure 3 and 15) uses nultilinear regression of paraneter estinetes resulting fron gonpertz functions fit to elongation data at different DT and daninozide concentra- tions to predict gonpertz paraneter estinetes under a variety of OT and daninozide concentrations (Table 6). The resulting paraneter estinetes are then used to predict elon- gation over tine through a second gonpertz function. The gonpertz function was chosen for two reasons: 1) the func- tion fit the data well 2) there were indications that non- -1inearity of the data/nodal relationship was low, i.e. low iteration count to convergence and significant paraneter estinetes. This suggested that the paraneter estinetes were not variable and that interpetations nay be nade fron the paraneter estinetes with confidence . The benefits of Method II are twofold: 1) only one step involving nultilinear regresssion is used which does not involve division of the dependent variable prior to nul- 107 tilinear regression analysis 2) the resulting nodel nay be interpeted through paraneter estinetion evaluation. The success of Method II depends on the validity of the initial paraneter estinetes calculated fron the raw data. Snell errors in the initial paraneter estinetes nay result in poor prediction of absolute shoot length, especially if they occur in the ’b’ and ’c’ paraneters, or exponentials. Despite the potential for error, nethod II predicted final shoot length within 5x of the raw data values. Method III (Figure 4 and 15) is sinilar to Method I with the exception, that absolute lengths are used instead of elongation rates. Since Method III regressed absolute _shoot lengths and not elongation rate it was not as sensi- tive to snall changes as Method I, however, it still con- tains an retardation nultiplier (Table 4) which aids in evaluation of daninozide effectiveness kinetics as in- fluenced by tine, daninozide, and DT. Disadvantages of this nethod are sinilar to those of Method I. Bach nethod nay lend itself to nultiple application studies. Method I and III would be the easiest to adapt where g(x) nay be nultiplied by a second g(x) function for each subsequent application. Each of these functions would dinish in nagnitude since daninozide has not been found to be additive in its reduction of elongation with nultiple ap- plications (8). Method II could be adapted by developing 108 sets of gonpertz functions as tine of application, DT, and daninozide concentration vary. A nultilinear function could then be used to predict the final paraneter estinetes. The three nodeling nethods has specific benefits which nake each one nore appropriate for specific types of episodic studies than the others: Method I) Studies where all factors are held constant and the stinulus is varied, i.e. application of a retardant, change in tenperature, water stress. This is particularly useful when recovery kinetics are of interest. Method II) Methods where changes in plant response to a single stinulus at different physiological stages in plant developenent are of interest. Method III) Studies where the influence of nultiple stinuli on a plant syten at different tines in plant developnent are of interest. l) 2) 3) 4) 109 Conclusions An optinal DT ranging fron 26 to 30°C existed for sten elongetion on Chrzganthenun norifoliun with a night tenperature of 16°C. As DT deviated fron this optina, final shoot length was reduced, and the tine at which shoots reached their final length occurred later. An application of daninozide reduced final shoot length and delayed the tine when a shoot reached its final length. Plant response to an daninozide application was quadratic at high daninozide concentrations. As 0T increased fron 10 to 30°C. The nininun retardation of sten elongation associated with a 5000 ng 1“ application of daninozide occurred at 26°C. At low daninozide concentrations, the percent retardation of sten elongation was not influenced by DT. Retardation of sten elongation increased linearly as daninozide concentration increased fron 0 to 5000 ng '1. 5) 6) 110 PPF had little effect on plant response to a daninozide application except at high PPF levels (500 unol s“ n“), where PPF reduced daninozide response slightly. Three nethods for nodeling the influence of tine, DT, and daninozide concentration are presented for, both BGA and Circus. Method II showed the greatest potential for quantifying episodic processes involving a growth retardant application. 111 Biblical-cellar Bukovac, M.J., R.P. Larsen, and M.R. Robb. 134. Effect of R.M- Dinethyl-inosuccinuic acid on shoot elongation and nutrient comosition of Vitis labrusca 1.. Cv. Concord. Quart. Bull. Mich. Agr. Bxpt. Sta. 46(4):488—494. Cathay, R.M., and M.W. Stuart. 1%1. Generative plant growth- retarding activity of uni-1618, phosfon, and COO. Bot.lGaz. 123:51-57. Cathay, R.M. 1%4. Physiology of growth rear-ding chaicals. Ann. Cathay, R.M. 1975. Generative plant growth-retarding activities of midol with ACPC, phosfon, chlolnaquat, and dainozide on ornuental species. Bortscienoe 10(3) : 204-216. Dicks, J .W. 1972. Uptake and distribution of the growth reterd- ent, uinozide, in relation to control of latera shoot elong- ation in Chrysanthatn norifoliu. Ann. Appl. Biol. 72:313- 326. 10. ll. 12. 112 Dicks, J.R. and D.A. Charles-BM. 1973. A Quantitative description of inhibition of st- growth in vegetative lateral shoots of W by M-Dinethyl-inosuccinuic Acid (D-inozide), Planta (Berl.)112:7l-82. Dixon, M.J. 183. MP statistical software. Univ. of Calif. Press, Berkley, Calif. pp. 264-278. Irwin, J.R. 1933. Section I, Master’s Thesis. Glockner, F.C., 1933. Chrysanthem- nanual. Runt, R.H. 1&2. Plant growth curves. University Park Press. Belt. me we 1‘1“. Leith, J.R. and J.F. Reynolds. 1986. Plant growth analysis of discontinuous growth data: a nodified Richard’s function. unpiblished. Karlsson, M.G. and R.D. Reins. 1&6.Response Surface Analysis of flowering in Chrysanthu- ’Bright Golden Anne’. J. Aner. Soc. Bart. Sci. 111(2)::anc-sarx. l3. l4. 15. 16. 17. 18. 19. 113 Ouisfling, R.C., and R.E. Mylund. Influence of shading, tine of application and tanerature on the response of pea (Pie:- satin- I... Cv. Green Giant No. 447) to 3—993 sprays. Phillip. Agr. Riddell. J.A., R.A. Begaan, C.M. J’Anthony, and M.L. Hubbard. 1962. Retardation of plant growth by a new group of chenicals. Science 136:391. Schhonherr, J. and M.J. Buckovac. 1978. Foliar penetration of Succinic acid-2,2-dinethylhydrazide: nechanisn and rate liniting step. lesiol. Plant. 42:243-251. Seeley, J .G.. 1979. Interpetation of growth regulator research with floriculture crops. Acta Bort. 91:83-92. Shem, M. and LG. Rillyer. 1976. D-inozide reduces eta elongation of ’Grand hpids Forcing’ leaf lettuce M at high greenhouse tqeratures. Rortscience ll(6):607-608. lent, F.W., 1&7. The experinental control of plant growth. Chrontica Botsnica, Malthu, Mass. 'Zeevaart, J.A.D. 1&6. Inhibition of eta growth and flower fern- 114 ation in Pharbitis nil with R.M-Dinethylaninosuccinuic acid (ls—993). Planta(Berl.) 71:68-80. 115 Table 1. Effect of day tenperature (DT) and daninozide concentration on naxin- likelihood parueter estinetes of final shoot length (a), initial length deterninent (b), and.nean relative sten extension rate (c), of gonpertz functions representing Chgygnathggg! norifoliun cv. ’Bright Golden Anne’ lateral shoot elongation over tine. The influence of UT and d-inozide on the nean absolute eta extension rate (MAER) and inflection point (IP) are also presented. Final Paranater Day daninozide Shoot Tenperature' conc.’ Length! a b c MARE! IF" 10 0000 11.68 - - 1250 9.41 - - 2500 8.26 — - 5000 6.16 - - 18 0000 21.66 7.05 .404 1250 19.67 6.07 .332 2500 18.52 5.32 .332 5000 14.79 4.20 .272 22 0000 24.12 26.81 7.30 .085 .425 23 1250 22.51 25.76 5.08 .071 .408 23 2500 20.15 23.57 4.27 .065 .393 22 5000 17.05 21.64 3.44 .051 .332 24 26 0000 ‘31.10 32.22 7.97 .106 .596 20 1250 29.61 31.03 6.50 .095 .575 20 2500 28.55 30.39 5.83 087 .563 20 5000 27.43 29.72 5.77 088 .550 20 30 0000 30.87 31.17 4.18 .059 .479 24 1250 27.22 28.97 3.80 .053 .426 25 2500 27.77 29.07 3.80 .053 .427 25 5000 24.07 27.98 3.52 044 .354 29 ' day tenperature (°C) 3' dainozide concentration is in ng a.i. dainozide l"1 ' 60 days divided by total increase in length (a)(cn dey'l),IF#e ' calculated es la (b)/ (c) in days. “ second lateral shoot length on day 60 (on) 116 Table 2. Effect of day tenperature (UT) and daninozide concentration on naxinun likelihood paraneter estinetes of final shoot length (a), initial length deterninant (b), nean relative sten extension rate (c), of gonpertz functions representing C anthenun norifoli cv. ’Circus’ lateral shoot elongation over tine. The influence of UT and dainozide on the neen absolute sten extension rate (MAER) and inflection point (IP) are also presented. ---------------------------------------—*------------------—------‘—- ------“_-------------------------C---------------------------------- Final Paraneter Day Dandnozide Shoot Tenp°' Conc.’ Lengthfi a b c MABRF IF" 10 0000 7.24 9.59 4.36 .063 .152 23 1250 6.29 7.86 4.50 .061 .116 25 2500 6.33 10.03 3.37 .040 .122 30 5000 5.75 8.26 2.92 .039 .113 27 18 0000 ‘ 10.27 11.00 - 5.91 .087 .203 20 1250 9.27 10.92 3.95 .059 .173 23 2500 8.78 10.28 3.62 .061 .180 21 5000 8.01 9.50 3.01 .056 .176 20 22 0000 11.38 13.84 4.99 .077 .242 21 1250 9.41 9.95 6.63 .095 .191 19 2500 8.93 9.76 4.47 .084 .199 18 5000 8.21 9.92 3.49 .063 .129 20 26 0000 16.98 20.00 7.72 .138 .487 15 1250 16.02 16.62 9.72 .155 .405 15 2500 16.21 16.81 9.02 .150 .410 15 5000 16.43 16.99 7.44 .136 .414 15 30 0000 13.15 13.32 6.80 .098 .247 20 1250 11.92 13.50 5.06 .080 .250 20 2500 10.99 11.59 4.55 .083 .236 18 5000 9.64 10.29 4.88 .082 .197 19 3 day tenperature (°C) V daninozide concentration is in ng a.i. daninozide 1"1 F 60 days divided by total increase in length (a) (on day'l),,IFte ' calculated es la (b)/ (c) in days “ second lateral shoot length on day 60 (on) 117 ram 3. Regression coefficients predicting the nultiplication factor as influenced by day tqerature (DT), tine, and duinozide concentration (CM). Regression Coefficients Tern BGA Circus Tine - . 041602100 - Tinea . 000884756 - Tina“ - . 14168733-08 - CM - -. 0004559180 CNtTine - . 00000897330 ' . 0000224419 ClitTinez . 160$528-06 -. 20310008-06 CflatTine - -. 36616703-12 CIF’ItTine3 - . 38123518-16 CMIIDT - -. 0000170769 ClitD‘l'° - . 32453OI-09 0M3 tDT - . 60644398—08 Cl? Ikll'l'a - - . 35310143-11 DT° tTine . 28026788-06 - DT°tTine3 -. 88824878—10 - Intercept l . 34911000 1 . 0004200 Re . 82 . 54 118 Table 4. m1tilinear regression coefficients predicting gonpertz function paraneters as influenced by day tqerature (DT), d-inozide concentration, and the ’a’ per-star. Regression Coefficients Tern a b c ’Bright Golden Anne’ a - .5218310000 .00769129000 DT 2.562130 - - DT° -.00102908 -.0002768460 -.0000033l345 CM -.00088206 .0020788200 .00006669700 DT'CM - - -.00000509957 DT'ICN - -.0000114014 .97661948-07 DT°tCM’ - .30984573-06 - Intercept -17.35120000 -4.3764200000 -.08681720000 R? .87 .92 .95 'Circus’ a - .534140 .0100378 DT -9.08978000 - - DTa .53045800 - - DT° -.00913851 - - DTICM -.00001915 - - Intercept 55.67280000 -1.11064 -.0345133 Tdale 5. 119 Mltilinear regression function paraneters which describe the influence of day tqerature and tine on the rate of growth of Chmantha- norifoli:- cvs. ’Bright Golden Anne’ and ’Circus’. Tern Regression Coefficient BGA Circus DT - -. 0438070 01" - . 13480278-07 DT” - - . 42055463-09 Tine - . 0381609 - Tine2 . 000898661 - Tine? - . 9608732008-13 - DTtTine . 005842440 . 00274723 DTtTine3 -. 000129120 -. 0000694193 DTtTine' - . 1 1900323-09 DTtTinefi . 84% 1663-12 - 0T? tTine“ . 17558903-09 - D‘l'° *Tine" - . 32578413-18 - 111's #Tine -.27l580.-08 - DT‘tTinea .4fi6577B-10 - Intercept - . OIWS . 4320320 R3 . 91 . 58 120 Table 6. Multilinear regression coefficients which predict g(x), or the inhibition function, as influenced by DT d-inozide concentration, and tine. Regression Coefficients Tern BGA Circus DT - . 78582200 - D‘P . 00177616 - Tine - . 01597210 - DTtTine . 00123335 . 000510675 DT° *Tine - . 79408743-06 - CNtTine - . 00000428564 - . 000001314$ Clllerine2 . 49149543-07 . 16571858-07 CNtDT . 82765708-06 - . 00000123030 CPIDT . 16623373-13 - Intercept l . 77103 . $673 lla . 92 . 78 121 Figure 1. Schenatic diagran representing the proce- dures followed for prediction of daninozide retardation of shoot elongation of Chrysanthenun norifoliun for Method I. 122 CONTROL SADR DATA INRIBITED DATA FIT TO FIT T0 3mm FUNCTION WERTZ FUNCTION FIRST MULTIPLICATION FIRST DERIVATIVE f ‘ FACTOR DERIVATIVE MULTILINEAR i MULTILINEAR REGRESS ION REGRESS ION INRID ITED am RATE ‘ INHIBITOR FUNCTION (g(x)) CONTROL FUNCTION (f(x)) INTEGRATION 123 Figure 2. Influence of day tenperature on second lateral shoot elongation of Chrysantheng; norifoliun Ranat. 'Bright Golden Anne’ (a), and 'Circus’ (b). Lines represent gonpertz function estinetes (Tables 1 and 2) fit to each data set. 124 A58 596.. Hoozm .233 0 a 6 I. . I . [r a. a — l .a . w z A” . ’ I K .. I II I m .1 e. . / ., s” a 1” o I,” .- l l 1. 1. ll / 0 (II VI. r2 I [I]. 7 3.3. J” -m mmum < u — . _m._ m . m . . . 3 2 m 0 Time (days) 24 1 _ . _ . . u. _ n .. . a _a . — 0' o .. J A.» a V) A; .M II ( e l I m A n I I I moment». mum» . - . __.. I o- ! 6W 1 q I 2 1. 2 8 4. 0 AEan £98.. woocm .933 125 Figure 3. Schenatic diagran representing the proce- dures followed for prediction of daninozide retardation of shoot elongation of Chrysanthenun norifoliun for Method II. 126 CONTROL DATA FIT T0 ur in mm'z mum _ mm nmcuou MULTILINEAR REGRESSION PARAMETER ESTIMATES ESTIMATED GOMPERTZ FUNCTION 127 Figure 4. Schenatic diagran representing the proce- dures followed for prediction of daninozide retardation of shoot elongation of Chrysanthegun norifoliu; for Method III. 128 CONTROL DATA FIT TO GOMPERTZ FUNCTION. MULTILINEAR REGRESSION CONTROL FUNCTION (f(x)) m'npucurou ' moron MULTILINEAR REGRESSION INHIBITOR FUNCTION (g(x)) SADH INHIBITED DATA FIT TO GOMPERTZ FUNCTION INHIBITED SHOOT LENGTH 129 Figure 5. Response surface calculated fron nethod 11 indicating the absolute shoot length of Chrysantheng; norifoliun ’Bright Golden Anne’ as influenced by tine and day tenperature. 131 Figure 6. The influence of a daninozide application on day 15 after the start of short days on second lateral shoot elongation over tine on Chrysanthenun norifoliun cv. “Bright Golden Anne’. 132 cm on A983 95% 0* P l- on r ON 0P H .. \ cozoo=oo< :93 7. us 88 To 19m 7. 9: 8mm in :3 1. as one. one .9550 013 Imp TNN ImN (we) 14151191 1,0qu [0.19101 133 Figure 7. Response surfaces calculayted fron nethod II describing Chrysanthenum norifoliun cvs. ‘Bright Golden Anne’ (a) and ‘Circus’ (b) final shoot length as influenced by daninozide concentration, and day tenperature. 134 3: ‘W :S \ \ é,” 2° ‘ “ j 16 - \‘\ \ § :0 . \\\\\\\\\\\\\\\k w .1 \\\\\\\\\\\\\\\ o .N \\\\\\\\\\\\\\\\\k 00:: 22 \\ e m K 4000 Oral, 14 \ 1000 2000 . m: n (mg r.) Cont:6mm ‘0 OI. GI Sh°°t Length (cm) 3 3 °o1 \\\ \ \\\\\ ' W \\ .. ”~K\ W‘fi 135 Figure 8. Response surface calculated fron nethod II describing the influence of daninozide concentration and day tenperature on percent inhibition of shoot length resulting fron a daninozide application 15 days after the start of short days to Chrysanthenun norifoliun cv. ‘Bright Golden Anne’. .1. .U m. .I M» d d 1 t o 0 an. “I r G h In C a uogiopJoiaa waxed 137 Figure 9. The influence of photosynthetic photon flux on shoot elongation over tine of Chrzgggthengg norifoliu; cvs. ‘Bright Golden Anne’(a) and ‘Circus’(b). Lines repre- sent gonpertz function estinetes (Tables 1 and 2) fit to ,each data set. 138 70 Time (days) AEoV £98.. Hoocm .233 0 7 lo 6 -m . 1.» I“ W . w 0 -m m 4... «I 4mmm .2... m .m mmm m mg _m. q q q 1 u q d a - 4 We. mm a 5 4. 2 o AEov 59.6.. 3oocm .833 139 Figure 10. The influence of day tenperature on the rate of shoot elongation of Chrysanthenun norifoliu; cvs. ‘Bright Golden Anne’(a) and ‘Circus’(b). Lines represent first derivatives of gonpertz functions fit to shoot elongation data over tine at each day tenperature. 140 — 10 'C ... 18 'C "' 25 'C q .... Alan 1 d J 4 d 1 1 H 1 d u o o .3 3 .A a. o 1 0 o o o O .u Ea. cozamcofi co 3am Time (days) .0 meme man» I: 9: y a .Iax e .m. .I -2 1 ounce Eav cozomcom mo 33. 141 Figure 11. Diagran identifying the three phases of daninozide influenced elongation following an application of daninozide 15 days after the start of short days to Chrzgan- thenun norifoliun cv. ‘Bright Golden Anne’ grown with day and night tenperatures of 30°C and 16°C, respectively. The solid line represents the first derivative of a gonpertz function (Table 1) fit to shoot elongation over tine. The segnented line represents the first derivative of retarded shoot elongation calculated at four day intervals. 142 A983 oEP cm on 9. on cm 3 o — p p b b b _ — p 0.0 1N6 136 cowomcom 3353 .. .. / -md — — . s .. .. 558.3% . . ~ . __.. -3 = . .9550 ll . d/ . 7. up. coon .i cozomcom. 5 «93m grist (,-Kop we) 11011050013 )0 9103 143 Figure 12. Response surface calculated fron nethod I identifying the influence of tine and daninozide concentra- tion on the percent inhibition of shoot elongation resulting fron an application of 2500 ng 1‘1 daninozide 15 days after the start of short days to Chrysanthenun norifoliun cv. ‘Bright Golden Anne’. Percent Retardation O) . O .p. 01 30 15 144 0 145 Figure 13. The influence of day tenperature and tine on the retardation of shoot elongation renaining following an application of 2500 ng 1'1 daninozide 15 days after the start of short days to Chrysanthenun norifoliu; cv. ‘Bright Golden Anne’as calculated fron nethod II. 146 me 8.33 5.60.33. oo.~oc.Eoo .32. 35... m 0... mm 0.. e O— _ (Z) 5“!“10‘1198 UORDpJoiaa 147 Figure 14. The influence of photosynthetic photon flux and SADR concentration as calculated fron nethod I on the percent inhibition resulting fron and application of 2500 ng 1"1 daninozide 15 days after the start of short days to .Chrysanthenun norifoliu; cvs. ‘Bright Golden Anne’(e) and ‘Circus’ (b). Inhibition of Rate (2) Inhibition (Z) -..d—- f r r7 r §§ at? \ \ r—fi Ifr r r r 1000 2000 3000 4000 5000 6000 Concentration SADH (mg l") Day 15 l—tOOumol --5000mol ---300umol H160 F26orabof4bo'soro'600 Concentration SADH (mg l") 149 Figure 15. Predicted versus actual response of C r sa thenun nor oliun cv. ‘Bright Golden Anne’ grown with 18°C day tenperatures and an application of 5000 ng l"1 daninozide 15 days after the start of short days using Method I (a), Method II (b), and Method III (e). b o w a, 5 a .1 I.O\ ld\ . e m m Tl. _H H. w W. _. m m. m w m e. co m w w m w ... mo .53 59.3 .85.. .223 A58 59:... «Sen .233 —Contrel -- saoomsl" . m u q q u 2 w m 5 AEov 50:3. «025 .233 Time (days) ‘71111111111111.1111“