The Effect of Nutrient Ratios on Plant Height

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1 The Effect of Nutrient Ratios on Plant Height by Laura Anne Wiser A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Plant Agriculture Guelph, Ontario, Canada Laura Wiser, January,

2 ABSTRACT THE EFFECT OF NUTRIENT RATIOS ON PLANT HEIGHT Laura Anne Wiser University of Guelph, Advisors: Professor T.J. Blom Professor B.J. Micallef Environmental concerns surround the use of plant growth retardants (PGRs) for plant height control. Studies suggest that plant height control may be achievable through modifications in plant nutrition. The primary objective of this study was to evaluate an alternative means of plant height control through adjustments the ratios of the three macroelements: N, P and K. Ratios of NO /NH +, NO /H PO, NO /K +, K + /H PO, NH + /H PO and NH + /K + were tested at different levels, and approximately 5 hydroponic nutrient solutions were compared to quantify their effects on plant height of species, namely marigolds, sunflowers and tomatoes. Solutions were tested at an electrical conductivity (EC) of cm in the first objective, and retested at four ECs (.6,.,. cm ) in a second objective. Marigold height decreased while tomato height increased with increasing EC. Responses to ratio modifications were largely season and species dependent, suggesting that modifications in plant nutrition may not effectively control plant height in certain hydroponic systems. ii

3 ACKNOWLEDGEMENTS I would like to express my sincerest gratitude to my supervisor, Dr. Theo Blom, for his guidance, encouragement and support in carrying out this project. This project would not have been possible without his extensive knowledge of the greenhouse industry, and his profound understanding of the problems facing it. I would also like to offer special thanks to my committee members, Dr. Barry Micallef and Dr.Youbin Zheng, for their guidance and advice. They provided valuable feedback and suggestions, and asked questions that challenged me to achieve a better understanding of certain aspects of this project. The assistance and guidance provided by David Kerec was also greatly appreciated. I would like to thank him for the countless hours he spent assisting with, amongst other things, treatment creation and application, greenhouse setup and data collection. He was also instrumental in helping me become familiar with the various methodologies and greenhouse tasks needed to complete this project. I would also like to acknowledge and offer my appreciation for the generous financial support provided by the Canadian Ornamental Horticulture Alliance (COHA) and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA). iii

4 Table of Contents ABSTRACT... ii ACKNOWLEDGEMENTS... iii List of Tables... vii List of Figures... xii CHAPTER LITERATURE REVIEW.... Introduction to the Canadian greenhouse industry.... Importance of plant height control.... Plant growth retardants for plant height control..... Human health and the environment Crop production PGR persistence in the environment Nonchemical means of plant height reduction Temperature management as an environmental cue Light management as an environmental cue..... Water and humidity stress..... Mechanical stress....5 The role of GAs in nonchemical methods....6 Problems with nonchemical methods of height control....7 Introduction to plant nutrition Hydroponics Nutrient solutions Nutrient solution ph Nutrient solution EC.... The effect of EC on plant height.... Phosphorous nutrition and height control.... Nitrogen nutrition and height control Ammonium versus nitrate in plant nutrition and height management Potassium nutrition and height control Conclusion, hypothesis and objectives....materials AND METHODS.... Introduction to treatment composition..... Treatment composition for objective #... iv

5 .. Treatment composition for objective # Hydroponic system and experimental setup Plant species used in this study Seeding Greenhouse setup Treatment creation and application Crop Evaluation Statistical analysis... CHAPTER FIGURES... CHAPTER TABLES... 6 CHAPTER RESULTS Modifications in NO /NH +, NO / H PO and NO / K + ratios at an EC cm (objective #, experiment #) NO /NH + results NO / H PO results NO / K + results Modifications in K + /H PO +, NH + /H PO and K + / NH + ratios at an EC cm (objective #, experiment #) Modifications in NO /NH + ratios at four EC levels (objective #, experiment #) Modifications in NO / H PO ratios at four ECs (objective #, experiment #) Modifications in NO /K + ratios at four ECs (objective #, experiment #) Modifications in K + /H PO ratios at four ECs (objective #, experiment #) Modifications in NH + /H PO ratios at four ECs (objective #, experiment #5) Modifications in K + /NH + ratios at four ECs (objective #, experiment #6) CHAPTER TABLES Results: NO /K + ratio modifications repeated at four... 8 CHAPTER FIGURES... CHAPTER DISCUSSION AND CONCLUSIONS General overview Effect of NO / H PO ratios on plant height Effect of NO / K +, K + /H PO, NH + /H PO and K + / NH + ratios on plant height Effect of EC on height Changes in EC and ph over time....7 Conclusions and recommendations for future studies... v

6 References... 5 APPENDIX I Tables corresponding to the analysis of experiment #, objective # data.... APPENDIX II Tables corresponding to the analysis of experiment #, objective # data.... APPENDIX III Experiment #, Objective # Results: NO /NH + ratio modifications repeated at four ECs APPENDIX IV Experiment #, Objective # Results: NO /H PO ratio modifications repeated at four ECs APPENDIX V Experiment #, Objective # Results: NO /K + ratio modifications repeated at four ECs APPENDIX VI Experiment #, Objective # Results: K +/ H PO ratio modifications repeated at four ECs APPENDIX VII Experiment #5, Objective # Results: NH + /H PO ratio modifications repeated at four ECs APPENDIX VIII Experiment #6, Objective # Results: K + /NH + ratio modifications repeated at four ECs APPENDIX IX Initial ph of solutions APPENDIX X Examples of weekly EC and ph changes in experiment #, objective # solutions APPENDIX XI ly EC and ph changes in experiment #, objective # solutions APPENDIX XII ly EC and ph changes in experiment #, objective # solutions APPENDIX XIII ly EC and ph changes in experiment #, objective # solutions.... APPENDIX XIV ly EC and ph changes in experiment #5, objective # solutions APPENDIX XV ly EC and ph changes in experiment #6, objective # solutions vi

7 List of Tables CHAPTER TABLES Table. + Ionic composition of NO /NH ratios (objective, experiment )... 6 Table. Ionic composition of NO /H PO ratios (objective, experiment ). 7 Table. Ionic composition of NO /K + ratios (objective, experiment )... 8 Table. Ionic composition of K + /H PO ratios (objective, experiment ) 9 Table.5 Ionic composition of NH + /H PO ratios (objective, experiment ). 5 Table.6 Ionic composition of K + / NH + ratios (objective, experiment )... 5 Table.7 Table.8 Ionic concentrations of N, P, K, Ca, Mg and S for different NO /NH + ratios at four ECs... 5 Ionic concentrations of N, P, K, Ca, Mg and S for different NO /H PO ratios at four different ECs... 5 Table.9 Ionic concentrations of N, P, K, Ca, Mg and S for different NO /K + ratios at four different ECs... 5 Table. Ionic concentrations of N, P, K, Ca, Mg and S for different K + /H PO ratios at four different ECs Table. Ionic concentrations of N, P, K, Ca, Mg and S for different NH + /H PO ratios at four different ECs Table. Ionic concentrations of N, P, K, Ca, Mg and S for different K + /NH + at four different ECs CHAPTER TABLES. Tables corresponding to the analysis of objective #, experiment #, data 68 Table.. The effect of nutrient solution NO /NH + ratio modification at an EC of cm on marigold height and dry weight, as part of objective #, experiment #, replication # vii

8 Table.. Table.. Table.. Table..5 Table..6 Table..7 Table..8 The effect of nutrient solution NO /NH + ratio modification at an EC of cm on tomato plant dry weight, as part of objective #, experiment #, replication # The effect of nutrient solution NO / H PO ratio modification at an EC of cm on marigold height, number of buds and dry weight, as part of objective #, experiment #, replication #... 7 The effect of nutrient solution NO / H PO ratio modification at an EC of cm on tomato dry weight, as part of objective #, experiment #, replication #... 7 The effect of nutrient solution NO / H PO ratio modification at an EC of cm on tomato dry weight, as part of objective #, experiment #, replication #... 7 The effect of nutrient solution NO / K + ratio modification at an EC of cm on marigold dry weight, as part of objective #, experiment #, replication #... 7 The effect of nutrient solution NO /K + ratio modification at an EC of cm on tomato dry weight, as part of objective #, experiment #, replication #... 7 The effect of nutrient solution NO /K + ratio modification at an EC of cm on tomato dry weight, as part of objective #, experiment #, replication # Results: NO /NH + ratio modifications repeated at four ECs (objective #, experiment #). 76 Table.. Table.. Overall effect of nutrient solution NO /NH + ratio modifications repeated at four ECs of ~.6,.,. cm on marigold, sunflower and tomato height and dry weight as part of objective #, experiment # Height, in marigolds supplied with modified NO /NH + ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment # Results: NO /H PO ratio modifications repeated at four ECs (experiment #, objective #). 78 Table.. Overall effect of nutrient solution NO /H PO ratio modifications repeated at four ECs of ~.6,.,. cm on marigold, viii

9 sunflower and tomato height and dry weight as part of objective #, experiment # Table.. Height and dry weights in marigolds supplied with modified NO /H PO ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment # Table.. Table.. Height and dry shoot weight in tomatoes supplied with modified NO /H PO ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment #... 8 Height and dry shoot weight in sunflowers supplied with modified NO /H PO ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment #... 8 Table..5 Dry root weights from sunflowers supplied with modified NO /H PO ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment # Results: NO/K+ ratio modifications repeated at four ECs (objective #, experiment #) 8 Table.. Overall effect of nutrient solution NO /K + ratio modifications repeated at four ECs of ~.6,.,. cm on marigold, sunflower and tomato height, stem diameter and dry weight as part of objective #, experiment #... 8 Table.. Height and dry weights in marigolds supplied with modified NO /K + ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment #... 8 Table.. Dry weights in tomatoes supplied with modified NO /K + ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment #, replication # Table.. Dry weights in tomatoes supplied with modified NO /K + ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment #, replication #, January to May Results: K +/ H PO ratio modifications repeated at four ECs (objective #, experiment #). 87 Table.5. Overall effect of nutrient solution K + /H PO ratio modifications repeated at four ECs of ~.6,.,. cm on marigold, sunflower and tomato height and dry weight as part of objective #, experiment #, January to May ix

10 Table.5. Table.5. Height and dry weights in marigolds supplied with modified K + /H PO ratios repeated at four ECs of ~.6,.,. cm, as part of objective #, experiment #, January to May Dry weights in tomatoes supplied with modified K + / H PO ratios repeated at four ECs of ~.6,.,. cm, as part of objective #, experiment #, January to April Table.5. Dry root weights in sunflowers supplied with modified K + /H PO ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment #, January to March Results: NH + /H PO ratio modifications repeated at four ECs (objective #, experiment #5). 9 Table.6. Overall effect of nutrient solution NH + /H PO ratio modifications repeated at four ECs of ~.6,.,. cm on marigold, sunflower and tomato height, number of buds, stem diameter and dry weight, as part of objective #, experiment # Table.6. Height and dry weights in marigolds supplied with modified NH + /H PO ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment # Table.6. Stem diameter and dry weights in tomatoes supplied with modified NH + /H PO ratios repeated at four ECs of ~.6,.,. cm, as part of objective #, experiment # Table.6. Stem diameter and dry weights in sunflowers supplied with modified NH + /H PO ratios repeated at four ECs of ~.6,.,. cm, as part of objective #, experiment # Table.6.5 Effect of NH + /H PO ratios on sunflower height, as part of objective #, experiment # Results: K + /NH + ratio modifications repeated at four ECs (objective #, experiment #6). 96 Table.7. Overall effect of nutrient solution K + /NH + ratio modifications repeated at four ECs of ~.6,.,. cm on marigold, sunflower and tomato height, number of buds, stem diameter and dry weight as part of objective # Table.7. Height, number of buds and dry weights in marigolds supplied with modified K + /NH + ratios repeated at four ECs of ~.6,.,. x

11 cm as part of objective #, experiment #6, replication # Table.7. Height, number of buds, and dry shoot weights in marigolds supplied with modified K + /NH + ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment #6, replication # Table.7. Stem diameter and dry weights in tomatoes supplied with modified K + /NH + ratios repeated at four ECs of ~.6,.,. cm as part of objective #, experiment # Table.7.5 K + /NH + ratio modifications on sunflower stem diameter and dry root weight as part of objective #, experiment #6... xi

12 List of Figures CHAPTER FIGURES Figure. One of the three benches used in this study... Figure. Components involved in the hydroponic setup for each plant... Figure. Hydroponic components in place... 5 CHAPTER FIGURES Figure. Linear relationship between marigold height and NO /H PO ratios at an EC of.6ms cm, as part of objective #, experiment #, replication #... Figure. Figure. Bar graph showing mean ±se marigold height at four ECs as part of objective #, experiment #... Bar graph showing mean ±se marigold height at four ECs as part of objective #, experiment #... Figure. Bar graphs showing mean ±se sunflower height at NO /H PO ratios of (a), (b) 5, (c), (d) and (e) 6 at four ECs as part of objective #, experiment #... Figure.5 Figure.6 Bar graph showing mean ±se tomato height at four ECs as part of objective #, experiment #... 5 Relationship between sunflower height and NO /H PO ratios at an EC of.6ms cm, as part of objective #, experiment #... 6 Figure.7 Relationship between marigold shoot dry weight and NO /H PO ratios at an EC of.6ms cm, as part of objective #, experiment #... 7 Figure.8 Relationship between tomato shoot dry weight and NO /H PO ratios at an EC of.6ms cm, as part of objective #, experiment #... 8 Figure.9 Relationship between sunflower shoot dry weight and NO /H PO ratios at ECs of. (a) and.6ms cm (b), as part of objective #, experiment #... 9 xii

13 Figure. The mean height of marigold (±se) at four ECs as part of objective #, experiment #... Figure. Figure. Height of marigold (±se) marigold at four ECs as part of objective #, experiment #... Mean ±se of marigold shoot weight (a) root weight (b) and shoot to root ratios (c) at four ECs as part of objective #, experiment #... Figure. Mean ±se of marigold height at ratios of. (a),.5 (b),. (c),. (d),. (/) (e), and.(/.5) (f), at four ECs as part of objective #, experiment #5... Figure. Figure.5 Mean ±se of sunflower height at six nutrient ratios as part of objective #, experiment #5... Mean ±se of marigold (a) shoot weight (b) root weight and (c) shoot to root ratios at four ECs as part of objective #, experiment # xiii

14 List of Abbreviations DIF EC GAs PGR S/R Temperature differential between daytime and nightime Electrical Conductivity Gibberellins Plant Growth Retardant Shoot/Root Ratio xiv

15 CHAPTER LITERATURE REVIEW. Introduction to the Canadian greenhouse industry As a valuable part of the Canadian economy, the Canadian greenhouse industry generated approximately $.5 billion in sales in (Statistics Canada, ). Of this, roughly $6 million was attributed to the sale of ornamental and vegetable bedding plants. To generate a profit from bedding plant production, the greenhouse industry must consistently adapt to changes in governmental regulations and consumer expectations. One such change is that consumers and government organizations are becoming increasingly focused on environmentally friendly products while greenhouse operational and labor costs are simultaneously continuing to climb. The greenhouse industry must therefore adapt by implementing new environmentally friendly technologies and strategies without increasing cost.. Importance of plant height control A considerable amount of research has been devoted to exploring new methods of reducing environmental impacts while concurrently reducing associated costs in the greenhouse. For instance, environmentally friendly alternatives to rockwool substrates (Shinohara et al, 997), improvements in water use efficiency (Pardossi and Marzialetti, 8) and nutrient use efficiency (Grewal et al., ) have been evaluated in order to lessen associated environmental impacts and expenditures. Bedding plant height is an additional aspect of plant quality that must be controlled in a costeffective and environmentallyfriendly manner. Plant height is often artificially controlled in commercial greenhouses, as shorter plants are considered

16 favorable over their taller counterparts by both producers and consumers. Consumers tend to find shorter bedding plants more aesthetically appealing, and the relative ease at which they can be packaged and shipped adds to their profitability. Shorter plants are also easier to maintain in both greenhouse and outdoor environments. For instance, reduced transplant height is advantageous in that it assures ease of acclimatization to harsher garden and field conditions (Liptay and Sikkema, ). Additionally, many greenhouse setups require closely spaced plants, which often promote stem elongation. This observation motivated researchers to study height restriction in Nicotiana tabacum L. (tobacco) seedlings (Rideout and Gooden, 998) and Lycopersicon esculentum Mill. (tomato; Rideout and Overstreet, ) seedlings. Plant height control is of great value to both consumers and producers for various reasons. Because of this, researchers have conducted numerous studies in an attempt to develop new methods of artificially controlling plant height in various plant species. New methods of plant height control should be commercially viable; that is, methods should produce favorable results in an energy efficient way, with little need for additional equipment or extensive labor. Methods should be environmentally friendly; their use should not result in an accumulation of toxic substances within the environment, impact nontarget organisms or be hazardous to human or animal health. Plant height control should be achieved with little adverse effects to target species, such as changes in flower morphology, delays in flowering or significant decreases in yield or keeping quality. Ideally, plant height control should also be achieved in a consistent and dependable manner; that is, one should expect similar results within different seasons and in a range

17 of plant species. Several methods of plant height control, including examples of their respective benefits and drawbacks, will be detailed in the following sections.. Plant growth retardants for plant height control Growers customarily turn to plant growth retardant (PGR) application as a preferred means of height control. PGRs function by altering the biosynthesis of certain phytohormones that are essential in mediating stem elongation. At low concentrations, these compounds regulate aspects of plant growth and development through complex signaling mechanisms (Arteca, 996; Taiz and Zeiger, 6b). They can do so locally, or in tissues further away from their site of synthesis. The use of phytohormones to alter plant growth and development is not new; their use by agriculturists and horticulturists dates back to the 9s (Vivanco and Flores, ). Most PGRs function by altering gibberellin (GA) biosynthesis to control plant height. Other heightreducing compounds exist, however these are less prevalent and less well understood compared to GA biosynthesis inhibitors (Arteca, 996). At a molecular level, GAs are a group of structurally similar terpenoid phytohormones (Taiz and Zeiger, 6b). Each is composed of a tetracyclic skeleton containing 9 or carbon atoms, referred to as norentgibberellane in the case of 9C gibberellins and entgibberellane in the case of C gibberellins. Bioactive GAs regulate plant physiological processes, such as stem elongation, bolting, seed germination and flower induction. Regulated largely by environmental cues and genetics, GA biosynthesis occurs in three stages via the mevalonic acid pathway (Taiz and Zeiger, 6b). The first stage of GA biosynthesis occurs in the plastids and is initiated when geranylgeranyl diphosphate

18 (GGPP), a precursor for both carotenoids and diterpenes, forms the tetracyclic skeleton of gibberellins, entkaurene, via terpene cyclases. In the second stage, occurring in the endoplasmic reticulum, entkaurene is oxidized to the first GA in the pathway, GA, via Cyt P5 monooxygenases. GA biosynthesis is identical in all plants up until this point (Vivanco and Flores, ; Yamaguchi and Kamiya, ). The third stage, which ultimately forms different GAs depending on plant species, occurs in the cytosol, where GA is converted into bioactive GAs via oxoglutaratedependent dioxygenases. PGRs generally function by targeting enzymes involved in the pathways listed above (Luster and Miller 99; Fletcher et al., ). For instance, triazole growth retardants such as paclobutrazol (Bonzi) inhibit reactions mediated by the cytochrome (Cyt) P5 oxygenase enzyme. This reduces the conversion of entkaurene, formed in the initial stage, to entkaurenoic acid, the first intermediate of the second stage in biosynthesis. Although PGRs, which are classified as pesticides in Ontario, are effective in controlling plant height, they are increasingly becoming restricted. In April, 9, The Ontario Ministry of the Environment imposed a ban on the use of pesticides, including PGRs, for noncommercial cosmetic purposes (Pesticides Act: Ontario Regulation 6/9). Pesticides have been overwhelmingly associated with environmental damage and human health concerns, making environmentally friendly and pesticide free products more desirable. As the general public becomes more opposed to pesticide usage and chemical companies become less interested in selling PGRs, PGR availability will likely decline.

19 .. Human health and the environment Only recently has research started to document the environmental and human health effects associated with PGRs. To help protect against potential health effects, the Ontario Ministry of the Environment () has incorporated PGRs into the Ontario pesticide classification system, ranking PGRs according to their associated risks. For instance, Bonzi (paclobutrazol), Sumagic (uniconazole) and BNine (daminozide), belong to class pesticides, indicating that they are considered moderately hazardous to human health. The nature of the human health hazards related to PGR exposure is not entirely clear. Studies have suggested that PGRs may have damaging reproductive effects in animals. For instance, researchers found that ng/l of paclobutrazol inhibited spermatogenesis in Sebastiscus marmoratus, a fish species (Li et al., ), a finding which further highlighted the risks associated with triazole accumulation in external water supplies. PGR exposure may also result in delitirious effects in human reproductive systems. One study found that pregnant women who were occupationally exposed to pesticides, including the PGRs daminozide, paclobutrazol, chlormequat chloride and ethephon, had sons with decreased penile length, smaller testicles, and lower concentrations of certain male sex hormones compared to sons of nonexposed mothers (Andersen et al., 8). Although it should be noted that these mothers were also exposed to additional nonpgr pesticides, these results nonetheless incite further research into the potential human health hazards associated with PGRs. 5

20 .. Crop production Not only do PGRs have associated environmental and health concerns, they can also deleteriously affect plant growth and development, especially when applied incorrectly. Misapplication of PGRs can result in extensive crop damage. The potential for application related errors is exaggerated by the fact that the method and rate of application depend on multiple factors, including type of growth retardant, plant species, plant cultivar, greenhouse temperature and relative humidity (Gent and McAvoy, ). Phytotoxicity is one possible result of PGR overapplication, where the degree of phytotoxic response depends largely on species and cultivar. The PGR concentration needed to control height may also cause phytotoxicity in certain cases. For instance, concentrations of Bas 6 and Sd89 that significantly reduced height of Dianthus L. cultivars Snowfire, Persian Carpet, and Indian Carpet, also resulted in foliar damage (Messinger and Holcomb, 986). Researchers produced a similar outcome when chlormequat chloride and ancymidol were applied to Persian Carpet, and Indian Carpet. However, no damage was observed on the third cultivar, Snowfire. These findings emphasize the importance of not assuming that PGRs will produce similar effects throughout closelyrelated species or cultivars. A potential for negative effects on nontarget plant species also exists (Million et al., 999). Leachate from pots and spray drift can contaminate recirculating subirrigation systems, which can ultimately lead to PGR effects on nontarget organisms. Ancymidol and paclobutrazol, which are both xylem systemic, are especially problematic as low concentrations can have considerable effect on plant growth and development. 6

21 Studies have also shown that PGRs can reduce, delay or stop floral development (Gent and McAvoy, ). For instance, applications of the PGR S7 to Zinnia elegans L. delayed flowering by as much as six days (Kim et al., 989). S7 also affected flower type, as S7 treatments produced single or halfdouble flowers with many ray florets, or female flowers. S7 application resulted in the conversion of ray florets into tubular florets, or hermaphrodite flowers. Delays or changes in flowering have been documented in several species. Flowering in Hibiscus rosasinesis L. was delayed by roughly seven days due to Bonzi applications (Gogoláková and Štrba, 9). In another example, applications of ethephon on tomato plants grown in a float system delayed maturity to a point where only 8% of tomato fruit were harvested in the first three harvests, compared to 69% for those plants not treated with the PGR (Rideout and Overstreet, )... PGR persistence in the environment Problems associated with PGRs, especially triazoles, are of greater concern when one considers their persistence within plants, soils, and greenhouse equipment. PGR application can have long term effects on plant growth, especially in woody plants (Gent and McAvoy, ). For instance, at an application rate of g per cm of trunk diameter, paclobutrazol was shown to affect Acer saccharinum and A. rubrum biomass ten years after application. Effects have not been found to be as persistent in herbaceous plants, but should nonetheless be taken into consideration. For instance, paclobutrazol, at spray concentrations greater than 6ppm, inhibited plant growth for twelve weeks after application in Pelargonium Ringo White and Ringo Rose. The persistence of triazoles within plant tissues was found to be largely determined by application rate, whereas, the halflives of triazoles within soils were found to be additionally dependent on substrate 7

22 composition and triazole type (Andriansen and Odgaard, 997). PGRs can also accumulate in nonorganic substrates. For instance, when overdosing plants with uniconazole ( mg l ), uniconazole residues were absorbed into greenhouse ebb/flood tanks on concrete floors, even after equipment had been flushed with acetic acid and water. Triazole accumulation within the greenhouse and surrounding environment has been shown to have long lasting effects on plant growth. However, the potential longterm effects of PGR accumulation on animal and human health have not been investigated.. Nonchemical means of plant height reduction To efficiently apply PGRs, one must have a detailed understanding of the various PGR types as well as the responses of different species and cultivars to these PGRs, all while taking into account environmental conditions and stage of plant development. One must also acknowledge the fact that PGR application may produce effects that are not necessarily short lived. Finding substitutes to plant growth retardants that pose less danger to the environment and human health without additional logistical challenges is therefore imperative. Rather than relying solely on PGRs, growers are increasingly using environmental cues and stressors to trigger hormonal and physiological changes within plants. These cues and stressors are vital components of plant height management, as researchers and growers can obtain desired physiological responses, such as height control, via their manipulation. When exposed to stress, plants modify their growth and developmental habits to increase their chance of survival (Skirycz and Inze, ). One mechanism that increases the likelihood of survival under certain environmental conditions is growth 8

23 retardation. As cell division and expansion slow in response to stress, energy can be stored or reallocated to areas in the plant where it is more urgently needed. However, even in controlled environments, stressors can damage plants, resulting in lower yields, less commercially desirable plants, and lower profits. One must therefore mimic stress in a way that controls plant height without inducing the associated negative effects of plant stress. There are many nonchemical methods of plant height control that use modified environmental signals to induce abiotic stress and produce shorter plants... Temperature management as an environmental cue The effect of modifying the temperature differential between daytime and nighttime (DIF) is commonly used to control height of potted plants. Erwin et al. (989) found that Easter lily (Lilium longiflorum Thunb.) height was affected by the difference between day and nighttime temperature, more so than by day or night temperature alone, or by different constant temperature regimes. Shortest plants were produced when daytime temperature was lower than nighttime temperature (DIF). DIF responses have been studied most extensively in ornamental bedding plants, including campanulas (Campanula isophylla; Moe, 99) and pot chrysanthemums (Chrysanthemum morifolium; Sach, 995). This technique has also been documented in numerous other species, such as poinsettias (Euphorbia pulcherrima; Moe et al., 99) peppers (Capsicum annuum; Si and Heins, 996), and cucumber (Cucumis sativus; Grimstad and Frimanslund 99). Height responses to temperature depend on timing, duration and magnitude of temperature changes. DIF treatments also present problems in that they require extensive greenhouse cooling during the day and heating during the night, and therefore 9

24 could result in expending a great deal of energy. To help mitigate this problem, many growers open energy curtains to cool the greenhouse during the day while closing the curtains at night. An additional technique, termed morning drop, similarly employs temperature management as a means of plant height control, however, uses less energy than DIF treatments (Grimstad, 99). Grimstad found that a temperature reduction of ºC during the first two hours of the day resulted in significantly shortened cucumber and tomato plants, and, in doing so, provided a means of minimizing greenhouse cooling. Although temperature management is typically associated with modifications in air temperature, temperature stress can also be induced using cold water, termed cold shock. Blom et al. () found that overhead irrigation with cold (ºC and 5ºC) water resulted in plants that were as much as 6% shorter than controls, thereby providing more height control than the temperature management practices mentioned above. Timing of overhead cold water irrigation was not important; plants were shortest when cold water was applied during the morning or afternoon, as long as the water was applied to the apical meristem... Light management as an environmental cue Plants undergo various morphological and physiological modifications in response to changes in light intensity and quality. Because one such response is stem elongation, growers can manipulate various features of the light perceived by plants in order to produce the desired height limiting effects. For instance, higher light intensities have been shown to reduce stem elongation (Kefeli, 978). When pea plants (Pisum sativum L.) were grown under light intensities of 5 W m for days, both stem length and leaf area decreased at the higher light intensities.

25 Plant height is also dependent on the wavelenghts of light perceived by phytochromes. Plants perceive light with low red (R) to farred (FR) ratios as shade, likely from competing vegetation, which triggers a series of shade avoidance responses, such as stem elongation (Pierik et al., ). Plants grown under incandescent lamps that emmitted light with low R:FR ratios (.7) were therefore taller than those grown with fluorescent lamps with higher R:FR ratios (; Moe et al, 99). Light during endoftheday (EOD) twilight period also has low R:FR ratios (.7). Plant height can therefore be controlled to some extent by using a blackout cloth prior to the EOD twilight period in order to limit a plant s exposure to light with low R:FR ratios (Blom et al., 995)... Water and humidity stress Just as plants respond to changes in light quality and intensity in order to withstand low light conditions, they must also adapt to changes in water availability in order to survive drought conditions. One adaptation to drought is inhibition of stem elongation. Baher et al. () found that Satureja hortensis L. plants that had undergone severe water stress were % shorter than their control counterparts. Control plants were watered to full field capacity (FC) where, on average, water potential within leaves was.5 MPa. Moderate water stress was induced by reducing irrigation to 66% FC, and severe water stress was induced by reducing irrigation to % FC. Water potential had decreased to an average of.6 MPa in plants that had undergone the severe water stress treatments. Those plants from the severe water stress treatments were, on average, % shorter than controls. Humidity stress has also been shown to induce inhibition of stem elongation. To study the effects of humidity on plant growth, Gislerod and Mortensen (99) grew

26 begonia (Beginia hiemalis Fotsch) plants for ten weeks at either 6±5% or 9±5% relative humidity (RH) and found that plants grown at a higher RH (9±5%) were significantly taller than those grown at a lower RH (6±5%). Similar results were obtained in chrysanthemum, poinsettia and kalanchoe (Mortensen, ). Plants were grown at three vapour pressure deficits (VPDs) of 66, and 55 Pa (equivalent to 7, 8 and 9% RH respectively). Just as in the previously mentioned experiment, results showed that plants were taller with increasing RH... Mechanical stress Studies have also indicated that mechanical stress can be useful in plant height control. For instance, brushing or clipping plants can reduce plant height (Rideout, ). Clipping is often accomplished by moving a suspended lawn mower over the plant canopy, whereas brushing involves physically bending stems. Furthermore, subjecting plants to high air movement via a fan can produce plants with shorter stems (Rideout and Overstreet, )..5 The role of GAs in nonchemical methods Just as PGRs disrupt GA biosynthesis to control plant height, evidence suggests that modifications in environmental stimuli and/or the induction of stress influence GA biosynthesis, however, without the use of synthetic chemicals. To provide evidence for this, many studies have correlated the use of nonchemical height promoting practices with an increased production of bioactive GAs. Alternately, nonchemical heightlimiting practices have often been associated with a decrease in bioactive GA production. For instance, Jensen et al. (996) found that stem elongation in +DIF treated Campanula isophylla correlated positively with increases in endogenous concentrations of GA, a

27 bioactive GA commonly associated with stem elongation. The ratio of GA 9 :GA was lower in +DIF plants, suggesting that the rate of conversion of GA 9 to GA was higher in +DIF compared to DIF plants. An interaction between GA biosynthesis and light quality may also exist. Beall et al. (996) found that Phaseolus vulgaris L. plants with FRrelated stem elongation also had augmented bioactive GA activity, suggesting that FR promotes GA biosynthesis. Compared to the mode of action of PGRs, little is known about the precise biochemical pathways involved in plant height reduction by nonchemical means. These pathways are likely quite complex, and additional studies have suggested that other phytohormones, such as ethylene, interact with GAs to reduce plant height (Pierik et al., )..6 Problems with nonchemical methods of height control Nonchemical methods of plant height reduction can significantly decrease plant height. However, these are not without their shortcomings in commercial practices. Nonchemical methods tend to involve added labor, equipment and cost. As previously mentioned, DIF requires greenhouse cooling during the day and, to maintain the same overall hour temperature (T ), extra heating during the night, which would require extra energy costs when not using an energy curtain (Erwin et al., 989). Low temperature pulses have proven useful in lessening the costs associated with this technique, however, this technique is less effective, and certain species require daily temperature reductions by as much as ºC (Grimstad, 99). Too great of a temperature decrease may cause deleterious effects on plant growth and development.

28 Light quality management and cold shock techniques also often involve added cost. Light quality management may require modifying previously established greenhouse lighting systems, and implementing lights with higher R:FR ratios, such as high pressure sodium or metal halide lamps, and/or installing a blackout curtain (Blom et al., 995; Moe et al., 99). On the other hand, overhead irrigation with cold water would require maintaining a substantial body of cold water (Blom et al., ). This practice would also result in overlymoist leaves that would be prone to fungal infection. Mechanical stress can be labor intensive, damaging to plants and may promote the spread of pathogens (Rideout, ). For instance, the high air movement technique described by Rideout and Overstreet () could result in fungal spores and small insects blowing from plant to plant, making disease localization and containment difficult. In addition to this, to produce significant effects using this technique, Rideout and Overstreet placed plants a minimum distance of m from fans, which is not a practical distance in most greenhouses. In this experiment, researchers also observed salt injury on those plants that had been significantly shortened. Rideout and Overstreet hypothesized that augmented capillary motion of the nutrient solution within the media, resulting from increased evaporation of the solution from the media s surface, led to these injurious effects. Although these nonchemical methods influence plant height, they present numerous challenges, as described above. One potential alternative to these methods as well as PGR application is plant nutrient management. Nutrient modification, by either supplying a plant with an excessive or inadequate amount of nutrients, may be a way to induce growth reduction as a stress response. If modifications in plant nutrition were

29 effective in reducing plant height, growers could implement modified nutrient solutions with little added labor or cost, without needing to replace preestablished greenhouse systems..7 Introduction to plant nutrition Plant nutrient management is a littleevaluated means of height control. If one was able to achieve plant height control by modifying previouslyexisting nutrient solutions, one could control plant height with little added cost, labor or impact to the environment. Few studies have concretely quantified the relationship between plant height and plant nutrition. Historically, researchers believed that plants arose solely from water, a theory suggested by a Belgian physician by the name of J.B. van Helmont, who lived in the 7 th century (Epstein and Bloom, 5). Our understanding of plant nutrition has undoubtedly expanded in the last years. Presently, plant nutrition is primarily associated with a plant s ability to absorb inorganic elements, referred to as nutrients, and to structurally integrate these nutrients into cells or chemically integrate them into energyproducing reactions (Mengel and Kirkby, ). Essential nutrients, such as phosphorous (P), nitrogen (N) and potassium (K), are vital constituents of compounds necessary for plant survival (Epstein and Bloom, 5). For instance, N is a critical component of all amino acids, whereas P is a critical component of adenosine triphosphate (ATP). If not a part of a vital compound, a nutrient is nevertheless considered essential if deficiency of that element causes severe irregularities in a plant s life cycle. Nutrients that are required in greater quantities are referred to as macronutrients. These are nitrogen (N), phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg) 5

30 and sulfur (S). There are several nutrients that are needed in comparatively lower quantities, which are referred to as micronutrients. These are iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), molybdenum (Mo), boron (B), chloride (Cl) and nickel (Ni). Both macronutrients and micronutrients are extracted from the environment and ultimately integrated into the plant s cellular structure and/or metabolic activity..8 Hydroponics In studying the effects of plant nutrient levels on plant growth and development, having control over factors that could potentially interact with nutrients is crucial. For this reason, studies evaluating the effect of these nutrients on particular plant parameters are normally conducted in hydroponic systems. Hydroponics, a word coined by Dr. W.F. Gericke in 96, describes a soilless system that supports plant growth and development. Rather than deriving nutrients from soil, roots are immersed within and obtain nutrients from a solution containing dissolved nutrients and water (Roberto, ). Plant growth within a soilless system is not a recent development. Interest in hydroponics was sparked in the mid9s, largely because growers, due to the gradual nutritional depletion of their soils, had to routinely replace or add large quantities of fertilizers to greenhouse soils. Historically, hydroponics benefitted growers as a time saving and cost cutting measure. Today, the primary benefit of hydroponics is that it provides added control over the root environment (Jones, 5). In a hydroponic system, one can minimize unknown interactions between inorganic nutrients, organisms and organic matter within the solution and rhizophere. This added control means that one can better restrict the outflow of pollutants from the greenhouse. For this reason, hydroponics is considered a more 6

31 environmentally friendly growing system compared to conventional agriculture (Jensen 997). Because hydroponic systems provide added control over the growing environment, hydroponics is also often considered a valuable research tool. In addition to minimizing unknown interactions between inorganic and organic matter, water culture also allows researchers to exclude effects associated with soil matrixes (Tavakkoli et al., ). Nutrient modification will produce different responses in plants grown in soil versus those grown in aerated nutrient solutions. Water uptake in soil solutions is largely dependent on both matrix and osmotic potential, whereas uptake in hydroponic solutions is dependent on osmotic potential alone. Compared to aerated nutrient solutions, the ionic composition of soil solutions is heterogeneous. This heterogeneity is due, in part, to the adsorption and exchange that occurs between positive ions and predominantly negative soil colloids, where, depending on concentration and valency, cations form a diffuse double layer (Evangelou and Mcdonald, 999). Soil colloids provide a buffer for the various cations. There are various types of hydroponic systems. In most systems roots are anchored to media, such as sand, rockwool, peatmoss or vermiculite (Jensen, 997). Adequate oxygen supply to roots is also crucial (Epstein and Bloom, 5). Some methods involve nutrient solution oxygenation via a tube connected to an air pump. Other examples use passive movement of the nutrient solution across the roots via capillary action (Roberto, ). Researchers use various different hydroponic systems in plant nutritional experiments. Rideout () used a float system to evaluate effects of reduced 7

32 phosphorous supply, mechanical stress and PGR application on tomato transplant height. In this system, nutrient solutions were contained within polyethylenelined wooden frames. Seedlings were sown in flats containing media made up of peat and vermiculite, which floated on the surface of the nutrient solution. The solution moved passively via capillary action up into the media. The float system is efficient in terms of water and nutrient use, and limits leaching of nutrients into groundwater. However, because this system involves plants being tightly packed together, excessive stem elongation is common. The float system was chosen by Rideout () primarily for this reason; it allowed researchers to evaluate height limiting practices in a system known to produce elongated transplants. Although the float system is useful in certain contexts, it would not be useful in quantifying the direct effect of nutrients on plant height, as results may reflect interactions between components of the system and plant nutrient levels, rather than nutrient levels alone. The goal of Rideout s () study was therefore not necessarily to determine exactly how different mechanisms control plant height, but how these mechanisms can mediate certain problems associated with an otherwise efficient system. A second problem with this system is that solid media was used, which, as noted above, can provide a buffering system. Other studies have used simpler hydroponic setups. Unlike Rideout (), the goal of a study conducted by Kavanova et al (6) was to determine the direct effect of P limitation on cell elongation in L. perenne L. leaves. Seeds were sown in pots filled with a medium composed of quartz sand. Seedlings were irrigated with a modified onehalfstrength Hoagland solution (which will be discussed in later sections). Although P 8

33 could have been distributed unevenly throughout the solid media, researchers determined the concentration of P within the tissues of representative plants per treatment to show that exogenous P applications accurately reflected P deficiency within plant tissues. Findings were therefore less likely the result of uneven nutrient accumulation. One system that promotes nutrient uniformity is the use of standing aerated nutrient solutions (Jones, 5). Unlike previously mentioned methods, roots in this method are not anchored to solid media. Rather, roots from each plant are immersed directly into a container filled with nutrient solution, and an air pump bubbles oxygen directly into the solution through a tube. Although this system can promote solution homogeneity, it is disadvantageous in a commercial setting as it requires manually dumping and refilling solutions every 5 to days, as changes in electrical conductivity (EC), ph and nutrient composition change over time. Containers must also be frequently topped up to compensate for solution uptake by plants. Lastly, the researcher must devise a technique to support and stabilize plants, as media is not used for support..9 Nutrient solutions Solutions of ionic essential nutrients dissolved in water, commonly referred to as nutrient solutions, are often considered the foundation of modern hydroponic culture (Taiz and Zeiger, 6a). A nutrient solution must contain all essential elements, however, nutrients can be present in various combinations. One common nutrient formulation is the Hoagland solution, named after Dennis R. Hoagland (8899), a prominent figure in the development of modern mineral nutrition research (Epstein and Bloom, 5). Nutrient solutions designed by Hoagland and Arnon (95) are wellknown worldwide. The first solution, deemed Solution #, provided nitrate (NO ), an 9

34 anionic form of N, as the only source of N (Epstein and Bloom, 5). A second solution, deemed Solution # was later developed. Solution # included ammonium (NH + ), a cationic form of nitrogen, in addition to NO as a N source. Supplying N in both cationic and anionic forms stabilizes rapid fluctuations in solution ph that can occur when only NO N is used. As NO ions are absorbed into the root system, roots release OH ions into the nutrient solution to balance the negative charge, thus increasing the ph of the surrounding solution. When adding NH +, roots will release H + into the surrounding solution, potentially neutralizing the effect of the OH ions, or by oxidizing NH + to NO and H +. When creating a nutrient solution formulation, many considerations must be kept in mind. Anions and cations must be balanced in the formula on an equivalent basis (Epstein and Bloom, 5). K, Ca and Mg are macronutrient cations, whereas P and S are macronutrient anions. N, as previously mentioned, can be supplied as either an anion or cation. There are different guidelines and recommendations for the appropriate concentrations of each nutrient. Each essential ion is provided from a source reagent (Jones, 5). These salts dissociate into at least two ions. Because of this, one cannot simply source each nutrient from a single reagent, as one or more additional ions must always be added in conjunction with this nutrient. The appropriate reagents are dissolved into distilled water, which is later applied to roots (Hershey and Sand, 99). At present, researchers commonly use Hoagland s Solution # in hydroponic systems. This solution provides a total of 5mM N. Of this, mm is from NH +, whereas mm is from NO (Hoagland and Arnon, 95). Hoagland and Arnon used ammonium

35 dihydrogen phosphate (NH H PO ) as a source of NH +, and potassium nitrate (KNO ) and calcium nitrate [Ca(NO ) H O] as sources of nitrate. Other common sources of N are ammonium nitrate (NH NO ) and nitric acid (HNO ) (Jones, 5). It is recommended that P concentration be at concentrations from. to.5mm P, but can be maintained at lower concentrations of. to.6mm without significantly affecting plant growth (Jones, 5). P is supplied as either monohydrogen phosphate (HPO ) or dihydrogen phosphate (H PO ).The form of P depends on the ph of the solution. At a neutral ph, HPO is the more prevalent form, whereas at a lower ph H PO is more common. In Hoagland s Solution #, the concentration of P is mm (Hoagland and Arnon, 95). In terms of S concentration, Hoagland s Solution # calls for mm of SO (6ppm S). Hoagland used magnesium sulphate (MgSO 7H O) as a source of both Mg + and SO. Most formulations contain approximately 5mM of K + and Ca +, whereas Hoagland solution # provides 6mM K + and mm Ca + (Hoagland and Arnon, 95; Jones, 5). The source of K + in Hoagland s Solution # is KNO, however one can also use potassium sulphate (K SO ). The source of Ca + in Hoagland s Solution # is calcium nitrate Ca(NO ) H O. Hoagland s Solution # provides approximately mm Mg Nutrient solution ph Differential absorption of cations versus anions by plant roots causes continual changes in the nutrient solution. Solutions must therefore be frequently adjusted or replaced to prevent significant changes in nutrient concentrations, ph and electrical conductivity (EC) (Taiz and Zeiger, 6a).

36 Optimal growth in most plant species occurs between the ph of 5 and 7, however, ph optima often vary from species to species (Epstein and Bloom, 5; Jones, 5). ph influences many factors, such as the ionic form, solubility and oxidationreduction equilibria of nutrients. When initially created, a nutrient solution should have a ph between 5 and 6. However, the ph of a solution will change as roots absorb and exude substances that alter the initial cation to anion ratio of the solution (Savvas et al., ). Roots release H +, HCO and OH as well as organic anions into the nutrient solution (Hershey, 99). If roots absorb more cations than anions, roots will excrete H +, lowering ph until equilibrium is restored. If the opposite occurs, where more anions are absorbed than cations, the roots will excrete anions, HCO and OH, to restore electroneutrality. Because ph has an effect on nutrient uptake, monitoring and regulating ph is, to a certain degree, important. Some control over nutrient solution ph is required, however, fluctuations in ph are inevitable, especially in nonbuffering hydroponic solutions (Jones, 5). Extensive ph monitoring and control can be time consuming and expensive, and may not effect overall plant growth..9. Nutrient solution EC EC is a measure of a solution s electric conductivity, and is somewhat proportional to the overall ion concentration of a solution (Hershey, 99). It is therefore used to estimate ionic concentration, using two platinum electrodes which are cm in size each and cm apart. If water evaporates or is absorbed at a faster rate than nutrients in a solution, the ionic concentration and EC of the solution will increase (Roberto, ). Likewise, if nutrients are absorbed at a faster rate than water in a solution, the

37 ionic concentration and EC will decrease. The units used in measuring EC values are normally ds m or ms cm (Hershey, 99).. The effect of EC on plant height Studies have suggested that excess ions in nutrient solutions, measured using an EC meter, reduces plant growth (Taiz and Zeiger, 6a). Height reduction may occur when ion concentrations increase to a point where osmotic stress is induced and water availability becomes limited. To evaluate the role of medium EC on growth of Impatiens hybrids or I. platypetala Lindl. plants of Selenia, Judd and Cox (99) treated plants with fertilizer solutions at different EC levels. Plants were potted in plastic pots with Pro Mix BX (Premier Brands, New Rochelle, N.Y.) and were irrigated with fertilizer treatments of N.P6.6K at.5,.,.5, or. g litre with initial EC readings of.6,.8,.77, and. ms cm, respectively. The two highest fertilizer levels, namely at.5 and. g liter, suppressed plant growth, where plant growth was defined as the sum of height plus the average width of two plants. This finding did provide some evidence that higher EC levels reduce plant height. However, this experiment investigated overall plant growth rather than height alone. Tavakkoli et al. () noted that responses to osmotic stress are generally assumed to be identical in both hydroponics and soil. To investigate this assumption, Tavakkoli et al. grew two varieties of barley (Hordeum vulgare L.), namely Clipper, which can exclude Na + and Sahara which cannot, in both soil (sandy loam), and hydroponics (standing aerated nutrient solution). Different levels of osmotic stress were induced by adding NaCl or CaCl + KCl. In hydroponics, the greatest reduction in growth occurred at 5mS cm. Osmotic stress was determined to be the principle cause of growth

38 reduction, with some added ionspecific effects. The two varieties did not differ in shoot dry matter when grown in nonsaline solution, nor were there differences in how varieties responded to EC. Results were different when plants were grown in soil. For instance, one difference was that responses differed depending on basil variety.. Phosphorous nutrition and height control In addition to overall ionic concentration, studies suggest that concentrations or ratios of individual plant nutrients affect plant height. Of all the research associated with the effect of macronutrient levels on plant height, the majority has specifically focused on P. Phosphorous, which comprises roughly.% of a plant s dry weight, is a component of many compounds that are fundamental to plant metabolism, such as nucleic acids, ATP, phospholipids and phosphoproteins (Epstein and Bloom, 5; Jain et al., 7). P is absorbed mainly as HPO in alkaline soils or as H PO in acidic soils. The Ontario Ministry of Agriculture and Food (Publication 7: Guide to Greenhouse Floriculture Production, ) suggested that growers limit P supply to reduce plant height. This strategy may be effective as studies show that plant height reduction may be an adaptive response to P starvation. P is, after N, often considered the most limiting plant nutrient in nature due to its general immobility in the soil and its unavailability when in its organic form (Schachtman et al., 998). Unfertilized soils also seldom release P at a fast enough rate to sustain rapid plant growth. To adapt to such stress, plants undergo a distinct series of phosphate starvation responses. One of these responses is height reduction, but other examples include anthocyanin accumulation and changes in root morphology (Devaiah et al., 9).

39 Many studies have evaluated the molecular mechanisms associated with P starvationinduced morphological changes. For instance, Devaiah et al. (9) showed that transcripts of the MYB6 gene in Arabidopsis thaliana L., were induced only in response to P starvation. To produce deficiency symptoms, plants were given treatments without P and treatments with.5mm P, both at the 5 to 7 leaf stage of development. They found that increased MYB6 expression suppressed GA biosynthesis. This suggests that one influencing factor in P starvationrelated height reduction is overexpression of MYB6, which results in repression of GA biosynthesis genes and ultimately morphological changes. Although the molecular mechanisms behind Prelated height reduction are likely more complex than suppression of one hormone via the induction of one gene, this study at least suggests that the molecular mechanisms behind height reduction via P starvation have similarities to the molecular mechanisms behind height reduction via PGR application. Rideout and Gooden (998) achieved significant height reduction in pretransplant tobacco (Nicotiana tabacum L.) seedlings by limiting the mass of P to / the mass of N. They found that this practice had no effect on field performance. In, Rideout and Overstreet continued from the previous tobacco trial, however this time using tomatoes seedlings. 5N.P.5K was used as a high P treatment, where P was /7 the mass of nitrogen. This can be compared to plant tissues, where the mass of N is ten times greater than that of P. 5N.87P6.6K was used as a low P treatment where P was /7 the mass of nitrogen. The low P treatment caused height limitation, however, these treatments also had elevated K compared to the high P treatment. It is therefore 5

40 impossible to determine whether height reduction was a result of P deficiency alone, and/or due to the higher K rate. Rideout () grew tomato transplants in a float system, which, as previously mentioned, often induces stem elongation. Stem elongation is especially detrimental once transplants are planted in a field for many reasons. For instance, workers often complain that taller plants are more difficult to maintain as they break more easily. Rideout () therefore decided to evaluate certain methods of alleviating this problem, either via pretransplant reduced phosphorous supply, mechanical stress, PGR application, or delayed fertilization. After growing seedlings for three weeks in the float system, plants were transplanted into the field to determine how they would grow. Plants were treated with high (.6mg L ) and low (.5mg L ) P fertilizers to determine the effect on shoot height after plants were transplanted into a field. P concentrations of.5mg L produced plants that were only slightly shorter, and not significantly so, compared to those at.6mg L. For instance, 8 days after planting in the field, high P plants were, on average 5.5cm, whereas the low P plants were 5.8cm. When repeated one year later, high P plants were, on average, the same height as low P. This could indicate that height reduction may not correlate directly with reduced P, or that P from previous years may have accumulated within the substrate. Results did show, however, that P level did not affect overall fruit yield. Liptay and Sikkema () conducted a similar experiment using pretransplant tomatoes. Pretransplant tomato seedlings were irrigated with solutions containing P concentrations of,,,, 5 and mg L. They found that P treatments produced the shortest seedlings (cm), and, at mg L height had doubled, indicating that small 6

41 differences in P concentration can generate large variations in shoot height. Seedlings grown with low P treatments had fewer nodes, smaller leaves and less massive shoots compared to high P treatments. However, after being transplanted into a complete nutrient solution, plants that had been subjected to P regimens developed slower than those that had been supplied with higher P concentrations. Nine days after transplanting, leaf growth from P treatments had only increased by 5%, compared to those from mg L treatments, which had increased by %. The only exception was dry root weight, which was not affected by pretransplant P regimen. Morphological changes in response to Pstarvation have rarely been studied at a cellular level. To provide some insight into these changes, Kavanova et al. (6) studied the effect of P deficiency on cell production and division in the meristematic zone of the grass Lolium KH PO was used to induce P deficiency, compared to a control of mm KH PO. Kavanova et al. determined that the overall decrease in leaf elongation (9%) could be attributed to both a decrease in cell division rate and a decrease in final length by 9 and % respectively. Pdeficiency, however, did not affect the total number of cell divisions. Ideally, P concentration should be reduced to a point where adequate height reduction is achieved, but where no detrimental deficiency symptoms occur. For instance, according to previously mentioned research by Liptay and Sikkema (), Prestriction in the seedling stage can lead to a longterm slowed developmental rate. Rideout and Gooden, in their 998 research on tobacco plants, found that other plant parameters were not adversely affected until P rate was / the rate of N. 7

42 . Nitrogen nutrition and height control Due primarily to its assimilation into amino acids, the role of nitrogen in plant growth and development is paramount (Epstein and Bloom, 5). Nitrogen is therefore required in higher quantities than any other nutrient, and, like P, is a limiting factor in most environments. However, unlike P, little research has been done to quantify the relationship between plant height and nitrogen concentration. In one study, to determine the effect of N levels on height and phytohormone levels, Rajagopal and Roa (97) used Hoagland and Arnon Solution no. # as their control, and used this formulation with /8N and no N as Ndeficient treatments. They found that control plants were taller than /8N and N plants. Control plants also had significantly more IAA and GA activity compared to Nstarved plants, suggesting that growth promoting phytohormones are linked to N concentration... Ammonium versus nitrate in plant nutrition and height management Plants use either NO or NH + as a nitrogen source (GonzálezGarcía et al, 9; Taiz and Zeiger, 6a). NO can be stored or translocated throughout the plant without + injurious effects. Alternatively, NH is toxic and is quickly assimilated into amino acids. To prevent further toxic effects, excess amounts are stored in plant vacuoles. Growers therefore typically use NO as their predominant nitrogen source (Epstein and Bloom, 5). There is little research documenting the effects of NO versus NH + on plant height, however, a common belief exists amongst growers that NO to NH + ratios can affect height. For instance, according to the Ontario Ministry of Agriculture and Food (Publication 7: Guide to Greenhouse Floriculture Production, ) one should use 8

43 NO as a principal N source, as NH + promotes stem elongation. However, González García et al (9), found that Allium schoenoprasum L. (chives) were generally taller when grown with a solution containing significantly more NO N than NH + N, which contradicts suggestions provided by OMAF. Rather, height, leaf area and biomass generally decreased as NH + + N was added. However, augmented concentrations of NH may not be a viable means of height control in this example as this concentration also increased instances of leaf chlorosis. This experiment was also repeated in basil (Ocimum basilicum L.) but no significant effects on plant height were recorded, suggesting that the effect of NO N to NH + N ratios may be speciesdependent.. Potassium nutrition and height control Potassium makes up 6% of a plant s dry weight and is the most abundant cation found in plants (Epstein and Bloom, 5). Unlike N and P, K is not a vital component of any metabolicallycrucial plant compound, such as ATP or amino acids. Rather, it is present as an ion in vacuoles and in the cytosol to maintain osmotic potential and turgor. Very few studies have evaluated the effects of K deficiency on plant height (Peuke et al., ; Guruprasad and Guruprasad, 988). In one of the few existing studies, Purves (966) studied the effect of various monovalent and divalent cation salts on Cucumis sativus L. hypocotyl segments. Five different externally applied K + salts, KNO, KI, KBr, K SO, and K HPO, promoted hypocotyl elongation up until a maximal promotion at.m of K. In a second study, Peuke et al () supplied Ricinus communis plants with a control solution K and a K deficient solution containing K. The K deficient solution resulted in an increased root to shoot ratio (+7% compared 9

44 to control) but with no reduction in overall biomass. The increased ratio was due to both an increase in root growth (%) and decrease in shoot growth (75%). K may also interact with gibberellins to alter plant growth. Cycocel, or ( chloroethyl)trimethylammonium chloride (CCC) is an inhibitor of gibberellin biosynthesis which inhibited chlorophyll synthesis in Lactuca sativa (Knypl and Chylinska, 97; Guruprasad and Guruprasad, 988; Fletcher et al., ). The K salts, KCl and KNO reversed this effect, suggesting that K may interact with GAs (Knypl and Chylinska, 97). Guruprasad and Guruprasad (988) applied M K and GA to elongated etiolated, Amaranthus caudatus L. seedlings and found that GA and K synergistically promoted hypocotyl length by more than % compared to distilled water. GA alone only promoted elongation by 5%, and KCl alone only promoted elongation by %. When CCC was applied to both dark and light grown seedlings, it produced 7% inhibition of hypocotyl length. Both KCl and GA treatments were able to reverse this effect. Research into the molecular and physiological changes that occur in response to modifications in plant nutrition is limited. Studies suggest that modifications in P, K and N nutrition bring about changes in height. Just as PGRs and certain nonchemical methods of height control reduce plant height through disruptions in GA biosynthesis, research suggests that nutrition modifications may do the same. However, before delving further into molecular research, it is important to concretely quantify the effects of nutrient modification on plant height control.

45 . Conclusion, hypothesis and objectives Operational costs in the commercial greenhouse industry are continually increasing along with expectations for environmentally friendly products. Many aspects of plant growth and development, such as stem elongation, must be controlled in costeffective and environmentallyfriendly manners. PGRs, which inhibit GA biosynthesis, have traditionally been used to control plant height. However, their availability is diminishing because of associated environmental and human health concerns; it would therefore be best to develop an alternative means of controlling plant height. Nonchemical methods of plant height control manipulate environmental cues and/or induce stress to manage plant height. Although these methods can produce significant reductions in plant height, they also have associated disadvantages. For instance, most of these techniques require added labor and cost. Plant nutrient management is one potential alternative to nonchemical methods and to PGR application. If plant nutrition could be managed to effectively control plant height, it could be an ideal alternative method of plant height control. Implementing this practice would require little in terms of added equipment, labor or cost. This method would also likely not cause any environmental damage beyond what is already associated with greenhouse fertilization programs. Although plant nutrient management could be an ideal option for plant height control, few studies have evaluated this alternative. Evidence does suggest that increases in overall ionic concentration, or EC, may reduce plant height. Several studies have also looked into the effects of P limitation on plant height. However, these studies have had

46 mixed results. Very few studies have evaluated the effects of N and K ratios or concentration on plant height. Because so few studies have evaluated plant nutrient management as a means of plant height control, the general objective of this study is to further explore this option. The hypothesis of this study is that modified macronutrient (N, P and K) ratios in hydroponic nutrient solutions can be used as a viable means of plant height control. his study will evaluate all possible combinations of N,P,K (NO /NH +, NO /H PO, NO /K +, K + /H PO, NH + /H PO, and K + / NH + ), at several ratios per combination, to establish any potential effects on plant height. The first specific objective of this study is to quantify the effects of P, K, NH + and NO ratios on plant height using the same total ionic strengths in solution (EC). In a second objective, the effects of these same ratios will be evaluated at four ECs to help establish any potential links between EC, nutrient ratio and plant height. In order to best quantify the direct effects of these ratios on plant height, all experiments will be performed in a hydroponic aerated nutrient solution setup. To both limit interactions between nutrients and substrates and to promote solution homogeneity, this setup will not include a solid substrate. As mentioned in section., plant height control should be achieved in a consistent and dependable manner, meaning that an ideal method of plant height control should produce similar results independent of season or plant species. To determine whether plant nutrient management agrees with this principle, experiments will be replicated over time, using three different species....

47 .MATERIALS AND METHODS. Introduction to treatment composition The focus of this study was to quantify the effects of four components on plant height: N (two forms: NO + and NH ), P (H PO ) and K. To evaluate all possible combinations of these four components, six sets of nutrient ratios were required. Treatments were therefore created by varying NO /NH +, NO /H PO, NO /K +, K + /H PO, NH + /H PO, and K + / NH + ratios. Control treatments were comprised of a modified Hoagland s solution # with an EC of cm. This formulation provided meq L anions and meq L cations. The control solution contained 5mM NO and mm NH +, mm H PO, mm SO, 6mM K +, mm Ca + and mm Mg +. All solutions were provided with the same amount of micronutrients at g/l (Plant Products Ltd. Chelated Micronutrient Mix, Bramalea, Ontario). Treatment formulations were created by adjusting ion ratios (mm/mm). Six sets of treatments were created, where the ratio of two specific ions was adjusted in each set. For instance, NO + /NH were modified in the first set of ratios. This involved adjusting the control NO + /NH ratio of 5: by lowering the concentration of NO by mm and + increasing the concentration of NH by mm, resulting in a / ratio, while maintaining the same amount of N. For many ratios, either the nominator or denominator was kept constant. The modification of these nutrients inevitably resulted in an alteration in the anion/cation balance and EC. To compensate for losing one anion (NO ) and gaining one cation (NH + ), slight modifications were made to the concentrations of other ions.

48 For instance, to compensate for the loss of one anion,.5mm SO was added to the formulation. To compensate for the extra cation,.5mm K + and.5mm Ca + was subtracted from the formulation. Modifications in ions were also done so that Ca/ Mg/ K ratios remained as constant as possible. All formulations, despite differing nutrient ratios, were modified so that they provided meq L anions and meq L cations at a similar EC... Treatment composition for objective # In the first objective of this study, the Hoagland s solution listed above was used at onehalf strength, resulting in the control solution having an EC of approximately. ms cm and containing 7.5mM NO and mm NH + as sources of N,.5mM H PO as a source of P, mm SO as a source of S, as well as mm K +, mm Ca + and mm Mg +. The ultimate goal of the first objective was to compare all treatment combinations. However, limited bench and greenhouse size restricted the total number of treatments to 6 at any given time. For this reason, the first objective was divided into two separate experiments (experiment #, experiment #). Twentysix treatments with varying NO /NH +, NO /H PO and NO /K + ratios were initially tested in experiment #, followed by 6 treatments with varying K + /H PO, NH + /H PO, and K + + / NH ratios tested in experiment # of objective #. NO /NH +, NO /H PO, NO /K +, K + /H PO, NH + /H PO, and K + + / NH ratios were modified from the ½ Hoagland solution control. NO + /NH was modified in a set of 7 different ratios (table.), NO /H PO was modified in a set of 8 ratios (table.), NO /K + was modified in a set of ratios (table.), K + /H PO was modified in a set of ratios (table.), NH + /H PO was modified in a set of different ratios (table

49 .5) and K + /NH + was modified in five ratios (table.6). At least one control was included in each set of ratios... Treatment composition for objective # Treatment formulations used in objective # were repeated in the second objective by using ½,,, or times the concentrations of macronutrients resulting in ECs of.6,.,. and. ms cm. This objective was divided into six different experiments (experiments # to #6), where one set of ratios was tested in a given experiment. The first experiment tested five variations of NO + /NH ratios at the four ECs listed above (table.7), the second experiment tested five NO /H PO ratios (table.8), the third experiment tested 5 NO /K + ratios (table.9), the fourth experiment tested five K + /H PO ratios (table.), the fifth experiment tested six NH + /H PO (table.) and the sixth experiment tested six NH + /K + ratios at the four ECs (table.).. Hydroponic system and experimental setup Experiments were conducted using three benches, where each crop was allocated to one bench. Crops used in this study were tomatoes (Lycopersicon esculentum Mill., cv. Roma), marigolds (Tagetes erecta L., cv. Vanilla) and sunflowers (Helianthus annuus L., cv. Sunrich Orange). Onelitre round ceramic pots, cm in diameter and cm high, were spaced roughly cm apart center to center and were arranged in 6 rows down the length of each bench with six plants per row, resulting in a total of 56 pots per bench (figure.). This study was conducted as a randomized complete block design with two blocks that were replicated once over time (resulting in two replications per experiment). As such, benches were divided into two equal halves of 6 rows with three pots per row, where each half was considered one block and each row of three plants was allocated to 5

50 one treatment. Each block was comprised of a complete set of treatments, which were randomized between blocks and replications. Rows were tagged with numbers indicating the appropriate treatment. The same treatment was manually applied to all three pots within a given row, however, the outermost plant in each row was considered a guard plant and was therefore not included in statistical analysis. After rows were marked with their corresponding treatment number, pots were manually filled with the appropriate nutrient solution. Nutrient solutions were the only source of fertilizer and water supplied to plants. To aerate the solutions, compressed air was provided from a central utility facility at the University of Guelph. Compressed air ran from this facility through an air line to the research greenhouse, where it was attached to a regulator. The air pressure was rated at 75 to 5psi. Air was distributed to each pot through additional networks of air lines, three of which ran down the center of each bench as well as between the two outer rows. One smaller spaghetti air line, which was connected to the larger lines via emitters, was placed into each pot approximately days after transplanting to aerate solutions (figure.). Air lines were fed through a short section of plastic tubing that had previously been wedged between each pot and disk (figures. and.). These short sections of plastic tubing also kept disks in place. Healthy seedlings of similar height and overall appearance were transplanted after seedling roots had grown through drainage holes at the bottom of plug trays and after the appearance of first true leaves. This was typically days after initial seeding for sunflowers and days after seeding for both marigolds and tomatoes. Transplanting 6

51 involved inserting roots, still attached to either rockwool or growing mix, into a tapered hole of roughly.5cm in diameter at the center of a styrofoam disk. Disks were approximately.5cm in diameter and.5cm thick. Disks were then floated on top of the nutrient solution contained within each pot and were stabilized with a piece of tubing.. Plant species used in this study Plant species with short generation times, long central shoots and little branching were considered for this study. The hydroponic setup used in this study greatly restricted our choice of plant species as few species grew adequately in trial runs of this system. Based on these criteria we chose to use tomatoes and marigolds for the first objective of this study and tomatoes, marigolds and sunflowers for the second objective.. Seeding Tomato and marigold seeds were sown in rockwool cell plug trays that had been soaked in deionized (DI) water. After sowing, rockwool plugs were covered in a thin layer of finegrained vermiculite. Sunflower seeds germinated inadequately in rockwool plugs, and were therefore sown in standard plastic cell plug trays containing Sunshine Professional Growing Mix LP5 (Sun Gro Horticulture Canada Ltd., Vancouver, British Columbia), covered with finegrained vermiculite. Seeds were germinated in plastic misting chambers inside a glass greenhouse compartment. After germination, the seedlings were hardened off gradually and transplanted..5 Greenhouse setup All experiments were conducted in a m glass greenhouse compartment with top ventilation and water chilling during summer months at the University of Guelph, 7

52 Guelph Ontario, from April to November. The greenhouse was ventilated and cooled with water chillers when temperatures reached ºC and heated when temperatures declined to ºC. To provide some control of light intensity, a 5% shade curtain was set to open entirely when outside light levels were less than 5W m, to close 8% when light levels were 6 W m and to close 9% when light levels reached or surpassed 7 W m. No artificial sources of light or CO were used..6 Treatment creation and application Treatments listed in tables. to. were comprised of a combination of seven greenhousegrade salts. Four different sources of NO were used: potassium nitrate (KNO ), calcium nitrate [5Ca(NO ) NH NO 8H O], ammonium nitrate (NH NO ) and magnesium nitrate [Mg(NO ) 6H O]. Ca(NO ) was also the only source of Ca +, and NH NO was the principal source of NH +. KNO was also one source of K +, in addition to monopotassium phosphate (KH PO ) and potassium sulphate (K SO ). Sources of SO were magnesium sulphate [MgSO 7H O] and [Mg(NO ) 6H O]. After the salt concentrations for each treatment were calculated and weighed out, one gram of micronutrient (Plant Prod, Bramalea, ON) mix/l was added to each solution. For organizational purposes, all solutes were placed in plastic bags marked with their appropriate treatment numbers. The contents of each bag were dissolved into L DI H O in L sealed plastic bins labeled with the corresponding treatment number. When solutes had completely dissolved, samples were obtained from each solution and sent to AgriFood Laboratories (Guelph, Ontario) to provide a report detailing the ph, EC and concentrations of each ion. This helped assure that treatments had been properly 8

53 prepared, and provided initial EC (EC i ) and ph (ph i ) readings in order to better track changes in EC and ph. Treatment application involved manually filling pots of a given treatment with nutrient solution from the marked L bins. Pots were manually refilled or topped up as needed with the appropriate nutrient solution. Pots were manually flushed out and refilled once every week. Treatment samples were taken prior to the weekly flushes and EC and ph measurements were obtained using a Hanna Instruments HI 87 conductivity meter for EC readings and a ph8 Aqualytic meter for ph readings. The endofweek EC and ph readings (EC f and ph f ) were taken so that increases or decreases from EC i and ph i values could be determined for each week of growth (= EC and ph). After the L of solution stored in the bins had been depleted, a new set of solutions was created using the same method as explained above. New batches of solution were created once every four to six weeks, depending on season and stage of plant growth. EC and ph measurements were recorded from newlymade solutions and were compared to initial measurements obtained from AgriFood Laboratories. These new measurements then replaced previous EC i and ph i values in any calculations used to deternine the EC and ph for the weeks of growth after the creation of the new batch..7 Crop Evaluation Data were collected when plants were in flower (approximately four to six weeks after transplanting into the ceramic pots). Each species was evaluated separately. Prior to data collection, root size and colour was noted, and photographs of roots were taken. Although plant height was the primary parameter of interest in this study, plant root and shoot dry weight, number of buds and stem diameter were also recorded and included in 9

54 analysis. In this study, shoot dry weight refers to the dry weight of all plant parts above the styrofoam plate, including the weight of the stem, leaves, petioles and flowers. Root dry weight refers to the dry weight of all roots below the styrofoam plate. Length and dry weights of all nonguard plants were measured. To obtain dry weights, roots from the two plants making up one experimental unit (two plants/ treatment /block) were cut from the plants and placed in a paper bag. Shoots from one experimental unit were also cut and added to a separate paper bag. Each bag was labelled with the appropriate block, replication and treatment numbers. Plants were then dried for weeks in a drying room, at which point these were removed, allowed to cool and weighed. Paper bag weight was obtained prior to data collection and was subtracted from weight readings to obtain net plant dry weight prior to statistical analysis..8 Statistical analysis A Type I error rate of α=.5 was used in all statistical analyses. Analyses were performed using SAS version 9. (SAS Institute Inc., Cary, N.C.). The sampling plan used in this study was a randomized complete block design. As mentioned above, one experimental unit consisted of two plants per block that were allocated to the same treatment. Both plants were always located in the same row. Each plant of a given experimental unit was considered a subsample of that experimental unit. To account for background variables, two blocks were set up and experiments were repeated once over time (each experiment was therefore made up of two replications). Blocks were composed of to 6 rows, depending on experiment, where one treatment was applied to each row. Each row had plants, where the outermost plant was considered a guard plant. Treatment order was randomized between blocks. The

55 factor of interest in the first objective was nutrient ratio and factors of interest in the second objective were nutrient ratio and EC. The response variables of interest were plant height, number of buds, stem diameter and dry shoot and root weights. Initial assumptions were that errors were normally distributed, independent, homogeneous and summed to zero. PROC UNIVARIATE was used to generate means of residuals and a Shapiro Wilk s test was used to determine if errors were normally distributed. Scatterplots of residuals over predicted values, blocks, ratios, replications, and ECs were also generated and analyzed to determine if errors were homogeneous and independent. If assumptions were not met, data were logtransformed, however, untransformed means were reported in tables. Lund s tests (Lund, 975) of studentized residuals were conducted to detect the presence of outliers. When critical values were larger than those supplied by Lund, values computed by Rotondi and Koval (9) were used. Outliers were removed when a recording error had been made, or when plant disease had been noted while recording data. PROC MIXED was used to perform variance analyses and the Tukey s method for multiple comparisons was applied to make multiple comparisons. Estimate statements and best linear unbiased estimators were used to estimate the difference between treatments of interest. The sources of variance accounted for in the first objective were nutrient ratios, blocks, nutrient ratios blocks and blocks nested within replications. Nutrient ratios were the only fixed effect, whereas blocks and replications were random effects. A similar model was applied in the second objective, however rather than analyzing the effect of only one factor, this objective was analyzed as a factorial with EC as an additional

56 source of variation. Nutrient ratio, EC and nutrient ratio EC were fixed effects, whereas replication, block nested within replication, ratio replication, EC replication, ratio block and EC block were random effects. A mixed model variance analysis combined across replications was performed using PROC MIXED to determine whether significant variation could be attributed to nutrient ratio, EC, or an interaction between both. Regression analysis was also performed in both objectives to determine whether there were linear or quadratic relationships between nutrient ratios and dependent variables. Because treatments were unevenly spaced, orthogonal coefficients were produced using PROC IML. When a significant linear or quadratic relationship was found, PROC REG was used to generate an equation of the trend. Regression analysis was performed on each set of ratios in the first objective. In the second objective, regression analysis was performed at each EC. Regression analysis was not performed on K + + /NH experiments as this set of ratios was tested at only four different levels (,, and 6).

57 CHAPTER FIGURES Figure.. One of the three benches used in this study. Three larger air lines ran down the length of the bench, of which only the central line is visible.

58 Figure.. Components involved in the hydroponic setup for each plant.

59 Figure.. Hydroponic components in place. A section of tube (not visible) was wedged between each pot and disk to keep components in place. 5