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Influence of Plant Growth Regulator Application on Grafted Tomato Transplant Production

Permanent Link: http://ufdc.ufl.edu/UFE0045477/00001

Material Information

Title: Influence of Plant Growth Regulator Application on Grafted Tomato Transplant Production
Physical Description: 1 online resource (89 p.)
Language: english
Creator: Teo, Shuan Hao
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: aba -- contego -- solanaceae -- sumagic -- uniconazole
Horticultural Sciences -- Dissertations, Academic -- UF
Genre: Horticultural Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The objective of this project was to determine if plant growth regulator (PGR) application can enhance efficiency of grafted tomato seedling production. Abscisic acid (ABA) causes stomatal closure, and can prevent excessive scion water loss when applied prior to grafting. Uniconazole competitively inhibits ABA 8’-hydroxylase, which regulates ABA catabolism. Hence, uniconazole application can increase plant endogenous ABA content, and result in stomatal closure. PGR solutions at different concentrations were applied to ‘Florida 47’ tomato scions prior to grafting onto ‘Maxifort’ or ‘RST-04-106-T’ tomato rootstock, and newly grafted plants were healed inside or outside of the healing chamber. ABA spray application was more effective compared to root application. ABA application delayed wilting as a result of the reduction in stomatal conductance, and aided faster recovery from wilting in grafted plants kept outside the healing chamber. However, the ultimate graft survival was not improved by ABA application. In plants kept outside of the chamber, ABA spray application at 2000 mg/L reduced plant chlorophyll content compared to those with water application. Scion length reduction was also observed in plants healed outside when 1200 mg/L ABA was sprayed compared to those kept inside and sprayed with water. Uniconazole application delayed initial wilting of grafted seedlings kept outside, but did not improve subsequent recovery. Graft survival was not enhanced with uniconazole application, while plant height reduction was observed at 2.1 mg/L. ABA appeared to be a better PGR for use in vegetable grafting in contrast to uniconazole, although future studies involving combinations of ABA and uniconazole applications may yield synergistic effects. PGR use in grafting of other vegetable species also warrants further research.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Shuan Hao Teo.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Zhao, Xin.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045477:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045477/00001

Material Information

Title: Influence of Plant Growth Regulator Application on Grafted Tomato Transplant Production
Physical Description: 1 online resource (89 p.)
Language: english
Creator: Teo, Shuan Hao
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: aba -- contego -- solanaceae -- sumagic -- uniconazole
Horticultural Sciences -- Dissertations, Academic -- UF
Genre: Horticultural Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The objective of this project was to determine if plant growth regulator (PGR) application can enhance efficiency of grafted tomato seedling production. Abscisic acid (ABA) causes stomatal closure, and can prevent excessive scion water loss when applied prior to grafting. Uniconazole competitively inhibits ABA 8’-hydroxylase, which regulates ABA catabolism. Hence, uniconazole application can increase plant endogenous ABA content, and result in stomatal closure. PGR solutions at different concentrations were applied to ‘Florida 47’ tomato scions prior to grafting onto ‘Maxifort’ or ‘RST-04-106-T’ tomato rootstock, and newly grafted plants were healed inside or outside of the healing chamber. ABA spray application was more effective compared to root application. ABA application delayed wilting as a result of the reduction in stomatal conductance, and aided faster recovery from wilting in grafted plants kept outside the healing chamber. However, the ultimate graft survival was not improved by ABA application. In plants kept outside of the chamber, ABA spray application at 2000 mg/L reduced plant chlorophyll content compared to those with water application. Scion length reduction was also observed in plants healed outside when 1200 mg/L ABA was sprayed compared to those kept inside and sprayed with water. Uniconazole application delayed initial wilting of grafted seedlings kept outside, but did not improve subsequent recovery. Graft survival was not enhanced with uniconazole application, while plant height reduction was observed at 2.1 mg/L. ABA appeared to be a better PGR for use in vegetable grafting in contrast to uniconazole, although future studies involving combinations of ABA and uniconazole applications may yield synergistic effects. PGR use in grafting of other vegetable species also warrants further research.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Shuan Hao Teo.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Zhao, Xin.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045477:00001


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1 INFLUENCE OF PLANT GROWTH REGULATOR APPLICATION ON GRAFTED TOMATO TRANSPLANT PRODUCTION By SHUAN HAO TEO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR T H E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Shuan Hao Teo

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3 ACKNOWLEDGMENTS I would like to thank members of my graduate committee: Dr. Xin Zhao, Dr. Stephen Olson, and Mr. Craig Campbell for their advice and guidance; Jason Neumann and members from the same laboratory for their invaluable assistance s

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 3 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 Overview, History and Current Status ................................ ................................ ..... 12 Grafting Method ................................ ................................ ................................ ...... 14 Graft Healing Process and Survival ................................ ................................ ........ 14 Structures and Conditions for Graft Healing ................................ ........................... 15 Plant Growth Regulators (PGRs) ................................ ................................ ............ 18 Overview of PGRs Use and Effects ................................ ................................ .. 18 Gibberellin (GA) ................................ ................................ ................................ 19 Uniconazole ................................ ................................ ................................ ...... 20 Abscisic Acid (ABA) ................................ ................................ .......................... 21 PGR Application Methods ................................ ................................ ....................... 26 Water Application Method during Graft Healing ................................ ...................... 26 Monitoring of Graft Survival ................................ ................................ .................... 28 Stomatal Conductance Measurements ................................ ................................ ... 28 Chlorophyll Fluorescence ................................ ................................ ....................... 29 Objectives and Hypotheses ................................ ................................ .................... 30 2 EFFECTS OF EXOGENOUS ABSCISIC ACID APPLICATION ON RECOVERY, SURVIVAL, AND QUALITY OF GRAFTED TOMATO TRANSPLANTS ................. 31 Background ................................ ................................ ................................ ............. 31 Materials and Methods ................................ ................................ ............................ 34 Location ................................ ................................ ................................ ............ 34 Grafting Experiments ................................ ................................ ........................ 34 Plant Materials ................................ ................................ ................................ .. 34 Tomato Seedling Production ................................ ................................ ............ 35 Growth Regulator Applications ................................ ................................ ......... 36 Grafting Method ................................ ................................ ................................ 36 Graft Healing ................................ ................................ ................................ .... 37 Acclimation ................................ ................................ ................................ ....... 38 Transplanting ................................ ................................ ................................ .... 38 Measurements ................................ ................................ ................................ .. 39 Experimental Design and Statistical Analyses ................................ .................. 40 Results and Discussion ................................ ................................ ........................... 40 Stomatal Conductance after ABA Application ................................ .................. 40 Wilting and R ecovery from Wilting ................................ ................................ .... 40

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5 Graft Survival ................................ ................................ ................................ .... 42 Quality Parameters at 10 or 11 Days after Grafting ................................ .......... 43 Chlorophyll content ................................ ................................ .................... 43 Stomatal conductance ................................ ................................ ............... 46 Scion length ................................ ................................ ............................... 47 Leaf area ................................ ................................ ................................ .... 48 Scion diameter ................................ ................................ ........................... 49 Scion fresh weight ................................ ................................ ...................... 49 Scion dry weight ................................ ................................ ......................... 50 Chlorophyll fluorescence ................................ ................................ ............ 51 Quality Parameters at 3 or 4 Weeks after Transplanting ................................ .. 51 Leaf area ................................ ................................ ................................ .... 51 Scion length ................................ ................................ ............................... 52 Scion fresh weight ................................ ................................ ...................... 52 Scion dry weight ................................ ................................ ......................... 52 Other measurements ................................ ................................ ................. 53 Summary ................................ ................................ ................................ ................ 53 3 EFFECTS OF EXOGENOUS UNICONAZOLE APPLICATION ON RECOVERY, SURVIVAL, AND QUALITY OF GRAFTED TOMATO TRANSPLANTS ................. 63 Background ................................ ................................ ................................ ............. 63 Materials and Methods ................................ ................................ ............................ 65 Results and Discussions ................................ ................................ ......................... 66 Stomatal Conductance after Uniconazol e Application ................................ ...... 66 Wilting and Recovery from Wilting ................................ ................................ .... 67 Graft Survival ................................ ................................ ................................ .... 68 Quality of Grafted Tomato Seedlings at 11 Days after Grafting ........................ 69 Chlorophyll content ................................ ................................ .................... 69 Leaf number ................................ ................................ ............................... 70 Leaf area ................................ ................................ ................................ .... 70 Scion length ................................ ................................ ............................... 71 Scion fresh weight ................................ ................................ ...................... 72 Scion dry weight ................................ ................................ ......................... 72 Visual rating ................................ ................................ ............................... 72 Chlorophyll fluorescence ................................ ................................ ............ 73 Other plant quality parameters ................................ ................................ ... 73 Grafted Tomato Plant Quality at 3 Weeks after Transplanting ......................... 74 Leaf number ................................ ................................ ............................... 74 Leaf area ................................ ................................ ................................ .... 74 Other plant quality parameters ................................ ................................ ... 75 Summary ................................ ................................ ................................ ................ 75 4 CONCLUSIONS ................................ ................................ ................................ ..... 83 LIST OF REFERENCES ................................ ................................ ............................... 85

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6 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 89

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7 LIST OF TABLES Table page 2 1 Analysis of variance on the effects of ABA concentration, heali ng location, rootstock type, and ABA application method on grafted transplant quality at 11 days after grafting in Spring 1. ................................ ................................ ....... 55 2 2 Analysis of variance on the effects of ABA concentration, he aling location, rootstock type, and ABA application method on grafted transplant quality at 10 days after grafting in Spring 2. ................................ ................................ ....... 55 2 3 Analysis of variance on the effects of ABA concentration, healing location, and water application method on grafted transplant quality at 11 days after grafting in Fall 1. ................................ ................................ ................................ 56 2 4 Analysis of variance on the effects of ABA concentration, healing loca tion, and water application method on grafted transplant quality at 11 days after grafting in Fall 2. ................................ ................................ ................................ 56 2 5 ABA folia r and root applications in Spring 2. ................................ ...................... 57 2 6 Stomatal conductance (mmol/m 2 12 h after ABA spray applications in Fall 1 and Fall 2. ................................ ....... 57 2 7 Effects of ABA concentration, application method and location of healing on proportion of non wilted grafted tomato plants on the 4 th day after grafting in Spring 1 and Spring 2. ................................ ................................ ........................ 57 2 8 Effects of ABA concentration and location of healing on proportion of non wilted grafted tomato plants on the 1 st and 4 th day after grafting in Fall 1. .......... 58 2 9 Effects of ABA concentration, application method, location of healing, and rootstock type on chlorophyll content (chlorophyll concentration index) at 11 and 10 days after grafting in Spring 1 and Spring 2, respectively. ...................... 58 2 10 Effects of ABA concentration and location of healing on chlorophyll content (chlorophyll concentration index) at 11 days after grafting in Fall 1 and Fall 2. .. 59 2 11 Effects of ABA concentration, application method and location of healing on stomatal conductance, dry weight and scion diameter at 10 days after grafting in Spring 2. ................................ ................................ ............................ 59 2 12 Effects of ABA concentration, application method and location of healing on leaf area at 11 days after grafting in Spring 1. ................................ .................... 60

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8 2 13 Effects of ABA concentration and location of healing on stomatal conductance, dry weight, fresh weight and leaf area at 11 days after grafting in Fall 1 and Fall 2. ................................ ................................ ............................. 60 2 14 Effects of ABA concentration and location of healing on scion length (m m) at 11 days after grafting in Fall 1 and Fall 2. ................................ ........................... 61 2 15 Average air temperature, relative humidity and light intensity inside and outside of the chamber during graft healing (day 1 to 4) in the spring studies. ... 61 2 16 Average air temperature, relative humidity and light intensity inside and outside of the chamber during graft healing (day 1 to 4) in the fall studies. ........ 61 2 17 Average maximum and minimum air temperature, relative humidity and light intensity inside and outside of the chamber during graft healing (day 1 to 4) in the spring studies. ................................ ................................ .............................. 62 2 18 Average maximum and minimum air temperature, relative humidity and light intensity inside and outside of the chamber during graft healing (day 1 to 4) in the fall studies. ................................ ................................ ................................ .... 62 3 1 Analysis of variance on the effects of uniconazole (UN) concentration, healing location, and water application method during healing on grafted tomato transplant quality at 11 days after grafting in the Fall 1 study. ................ 77 3 2 Analysis of variance on the effects of uniconazole (UN) concentration, healing location, and water application method during healing on grafted tomato transplant quality at 11 days after grafting in the Fall 2 study. ................ 77 3 3 Analysis of variance on the effects of uniconazole (UN) concentration, healing location, and water application method during healing on grafted tomato tra nsplant quality at 3 weeks after transplanting. ................................ .... 78 3 4 Effects of uniconazole concentration on stomatal conductance (mmol/m 2 s) of ole spray application in both fall studies. ................................ ................................ ............ 78 3 5 Effects of uniconazole concentration and location of healing on proportion of non wilted tomato plants after grafting in both fall studies. ................................ 7 9 3 6 Effects of uniconazole concentration and healing location on quality parameters of grafted tomato seedlings at 11 days after grafting in both fall studies. ................................ ................................ ................................ ............... 80 3 7 Effects of uniconazole concentration, healing location, and water application method during healing on scion length (mm) of grafted tomato seedlings at 11 days after grafting in the Fall 2 study. ................................ ............................ 81

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9 3 8 Effects of uniconazole concentration and healing location on visual ratings z of grafted tomato seedlings at 11 days after grafting in both fall studies. ............... 81 3 9 Effects of uniconazole concentration, location of healing, and water application method during healing on leaf number and leaf area of grafted tomato plants at 3 weeks after transplanting. ................................ ..................... 82

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science INFLUENCE OF PLANT GROWTH REGULATOR APPLICATION ON GRAFTED TOM ATO TRANSPLANT PRODUCTION By Shuan Hao Teo May 2013 Chair: Xin Zhao Major: Horticultural Sciences The objective of this project was to determine if plant growth regulator (PGR) application can enhance efficiency of grafted tomato seedling production. Abscisic acid (ABA) causes stomatal closure, and can prevent excessive scion water loss when hydroxylase, which regulates ABA catabolism. Hence uniconazole application can increase plant endogenous ABA content, and result in stomatal closure. PGR solutions at different RST 04 106 T or outside of the healing chamber. ABA spray application was more effective compared to root application. ABA application delayed wilting as a result of the reduction in stomatal conductance, and aided faster recovery from wilting in grafted plants kept ou tside the healing chamber. However the ultimate graft survival was not improved by ABA application. In plants kept outside of the chamber, ABA spray application at 2000 mg/L reduced plant chlorophyll content compared to those with water application. Scion length reduction was also observed in plants healed outside when 1200 mg/L ABA was sprayed compared to those kept inside and sprayed with water. Uniconazole application

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11 delayed initial wilting of grafted seedlings kept outside, but did not improve subsequ ent recovery. Graft survival was not enhanced with uniconazole application, while plant height reduction was observed at 2.1 mg/L. ABA appeared to be a better PGR for use in vegetable grafting in contrast to uniconazole, although future studies involving c ombinations of ABA and uniconazole applications may yield synergistic effects. PGR use in grafting of other vegetable species also warrants further research.

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12 CHAPTER 1 INTRODUCTION Overview, History and Current Status Grafting is the co mbination of a shoot system, known as the scion, with a root system, known as the rootstock. In this way, desirable characteristics of both the scion and rootstock can be combined in a single composite plant, thereby complementing breeding and artificial s election programs. It is commonly used in fruit and nut tree production beginning from 1560 B.C. (Oda, 2002) as a form of clonal propagation, while vegetable grafting did not begin until the late 1920s in Japan and Korea when watermelon was grafted onto bo ttle gourd (Lee, 1994 ; Lee et al., 2010 ; Oda, 2002 ) to combat problems of soilborne diseases (Oda, 2002). Some of these soilborne diseases include Fusarium Verticillium Pseudomonas and nematodes; while vegetable grafting also improves stress tolerance, increases plant growth, yield and fruit quality (Lee, 1994; Oda, 2002). By the early 1990s, grafted vegetables accounted for about 50% of total field production acreage, and approximately 70% of greenhouse production acreage in Japan (Lee, 1994). Grafting was more widely adopted in Korea, accounting for approximately 80% of both field and greenhouse production acreage (Lee, 1994). In 1995, it was reported that grafted plants were used for more than 50% of the total acreage for production of major fruiting v egetables like watermelon, cucumber, eggplant, and tomato (Oda, 1995). This widespread adoption of vegetable grafting was due to problems associated with intensive cultivation because of limited arable lands (Kubota et al., 2008). Vegetable grafting began to attract attention in Europe from the early 1990s as a form of sustainable agriculture (Lee et al., 2010 ; Oda, 2002 ), with aims of conferring resistance/tolerance to soilborne diseases and nematodes, improving fruit

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13 yield and quality, and coping with adv erse environmental stresses (Kubota et al., 2008). It became especially popular in the Mediterranean areas of Europe, as seen by the enhanced adoption of vegetable grafting in Italy with an increase of 10 million grafted seedlings used in production betwe en 1997 and 2000 (Leonardi and Romano, 2004). This technique was also widely used in Spain (Leonardi and Romano, 2004), and has been disseminated to countries in Northern Africa, Middle East, Central America and other parts of Asia (Kubota et al., 2008). Despite its benefits and widespread adoption in Asia and Europe, vegetable grafting is still a novel technique in North America, especially in open field production systems. However, research in this area is increasing as vegetable grafting may provide a partial solution for the phasing out of methyl bromide as a soil fumigant (Kubota et al., 2008). Currently, most of the grafted seedlings are utilized in hydroponic tomato production under protected structures in North America (Kubota et al., 2008; Lee et al., 2010). The main barriers to wider adoptions of vegetable grafting include: intensive labor requirement, management of graft healing, high costs of grafted transplant production, limited availability of rootstock information, and graft incompatibilitie s ( Aloni et al., 2010 ; Kubota et al., 2008; Lee, 1994 ). According to Kubota et al. (2008), the majority of grafted tomato transplants used by growers in the United States and northern Mexico are produced by Canadian propagators in Ontario and British Colu mbia. Growers who wish to use grafted transplants in their production systems will need to bear the risks of transporting the transplants over long distances. On the other hand, some producers are interested in grafting their own plants to meet specific ne eds. Developing methods and systems

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14 suited for grafted transplant production in local conditions would be beneficial to growers looking for reduced costs and improved quality in grafted transplants. Grafting Method The tube grafting method is commonly us ed in North America for grafted tomato transplant production (Johnson and Miles, 2011 ; Kubota et al., 2008 ). Using this met hod, rootstock and scion plants with similar stem diameters are cut at approximately plants can be easily grafted 3 times faster than conventional methods (Oda, 1995). It helps reduce the space and time required to grow the seedlings to a minimum size before they can be grafted. Graft survival rate was also reported to be high (Oda, 1995). However, because smaller plants are used, precise control of the conditions insid e the healing chamber is essential for optimum survival (Oda, 1995). Graft Healing Process and Survival According to Hartmann et al. (2002), stages of graft union formation in general can be described as follows: Cambial regions of both rootstock and sci on must be in contact with one another before proliferation of parenchyma cells from both rootstock and scion tissues can occur, resulting in the formation of a callus bridge that connects both rootstock and scion. Differentiation of vascular tissue from c allus tissue follows the following order: firstly, wound repair xylems; secondly, wound repair phloems; and finally, new cambial cells. This new cambial layer then assumes the typical role of differentiating into more xylems and phloems, thereby establishi ng further vascular connections between the scion and rootstock. In the morphological and developmental study conducted on self grafted tomato plants by Jeffree and Yeoman (1983), initial cohesion of piths of the rootstock and scion

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15 by pectic secretions into the graft union was determined to be the first step in the formation of a graft union. Cells in the vascular cambium, endodermis, phloem parenchyma, xylem parenchyma, and the outer layers of the cortex then began to divide and filled in the space betw een the rootstock and scion, thereby forming the callus bridge. As the callus cells of the rootstock and scion approached each other, pectinaceous beads were produced on their surfaces, which helped to cement rootstock and scion cells together, thereby fun ctioning like middle lamellae. This secretion and accumulation of pectic substances by cemented cells in the graft union was also reported by Asahina et al. (2002). Localized thinning of sections of the cemented cell walls then occurred, followed by plasmo desmata formation in the thinned cell wall regions. The formation of plasmodesmata between the rootstock and scion cells enabled the exchange of molecules for cell recognition, which would in turn determine compatibility between the scion and rootstock. Di fferentiation of the callus tissue into wound vessels began approximately on the fourth day after grafting in tomato. A grafted tomato study by Fernndez Garca et al. (2004) indicated that formation of new vascular strands in the callus bridge and their subsequent connections with the vascular tissues of the scion and rootstock began between 4 to 8 days after grafting, with complete development 15 days after grafting. Results from root hydraulic conductance studies also showed that graft unions were func tional by the eighth day. Structures and Conditions for Graft Healing After grafting, vegetable transplants must be kept in an environment with high relative humidity and appropriate temperature for approximately 1 week, so as to reduce scion transpira tion rate until the graft union has successfully formed, and water

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16 transport from the roots to the scion is resumed (Johnson and Miles, 2011). Failure to maintain such conditions may result in low graft survival. According to Leonardi and Romano (2004), n ewly grafted plants can be kept on covered benches inside greenhouses for graft union formation, whereby temperatures radiation should be reduced to approximately 60 to 70% thr ough shading to prevent high temperatures inside the covered benches. Excessive shading is discouraged, as that will interfere with photosynthesis, resulting in weak seedlings. Kubota et al. (2008) reported that conditions most favorable for healing graft ed tomato seedlings are: 1) high relative humidity of more than 95%, 2) temperatures of 27 2 s 1 photosynthetic photon flux. In the United States, a relatively simple chamber consisting of an enclosed structure located wi thin a shaded greenhouse is frequently used. Such chambers are preferred as they provide for cheaper alternatives to maintain the necessary environmental conditions for graft healing (Johnson and Miles, 2011). According to Johnson and Miles (2011), the 3 types of commonly used healing chambers frequently constructed in North America include: 1) research design, 2) industry design, and 3) small farm design. In a typical research design, the chamber consists of double layered high density woven polypropyle ne shade cloths, a 6 mil polythene plastic cover, and a humidifier that turns on for 20 seconds every 5 minutes. Only the shade cloths and plastic are used in the typical industry design, with hand misting of the plastic inner surfaces. In a typical small farm design, only the shade cloths are utilized, with hand misting of the plants. They reported diurnal fluctuations in

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17 temperature in different systems, with opposite diurnal fluctuations in humidity. Mean relative humidity was highest with the industry d esign, followed by the research design, with the small farm design having the lowest mean relative humidity of 52.6%, which is much lower than that recommended for grafting healing by other authors. Johnson and Miles (2011) also suggested that the use of a humidifier in the healing chamber may not be necessary, although its use may help to lower temperatures inside the healing chamber. According to Kubota et al. (2008), healing of grafted tomato plant is relatively easy compared to other crops. This was su pported by the study conducted by Johnson and Miles (2011), whereby a 96% survival rate in self relative humidity of 53% and temperatures bet out that this study was conducted in western Washington from January to February, 29 to 68%, with an average solar radiation of 92.7 W/ m 2 Hence, such results may not be directly applicable to places in the southeastern U.S., such as Florida, where high temperature and light intensity inside greenhouses often occur. The use of commercial rootstocks instead of self grafting might also affe ct survival rates under such conditions. Moreover, in their study, grafted plants kept under the small farm design healing chamber were misted twice daily. It is not clear whether similar survival rates can be achieved without misting, or with lower mistin g frequencies (Johnson and Miles, 2011).

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18 Plant Growth Regulators (PGRs) Overview of PGRs Use and Effects Use of PGRs in practical vegetable grafting has been limited (Aloni et al., 2010), although there have been numerous studies on how different PGRs can affect graft union formation and differentiation of vascular tissues. It was reported that auxins and cytokinins promoted differentiation of vascular tissues, thereby enhancing graft healing (Aloni et al., 2010). For instance, it was observed that low ind ole 3 acetic acid (IAA) levels led to differentiation of sieve elements, whereas differentiation of phloem and xylem were promoted at high IAA levels in calli of Syringa Daucus and Glycine in vitro (Aloni, 1980). Dayan et al. (2012) demonstrated that in t obacco plants, undifferentiated cells in the cambial region can be induced to differentiate into xylem vessels by the application of auxins. Differentiation of vascular strands was reported to occur where there were polar movements of auxins from developin g leaves to roots via the procambium, cambium or parenchyma tissues (Aloni, 1987). Xylem regeneration was also found to be correlated to auxins produced from leaves and buds above wounded internodes in Coleus blumei (Jacobs, 1952). According to an in vitro grafting study conducted by Parkinson and Yeoman (1982), apical application of IAA was found to be essential for the differentiation of vascular systems at the graft union in tomato. Callus formation was also reported to be induced by IAA in Pinus contort a (Savidge, 1983). Parkinson and Yeoman (1982) also reported that kinetin resulted in an increased production of vascular connections in the graft union only in the presence of exogenous IAA in tomato. This was supported by Aloni (1993), whereby in general cytokinins were found to promote early phases of vascular differentiation when exogenous auxins were present.

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19 Gibberellin (GA) Application of gibberellin to undifferentiated tobacco cambial cells resulted in the differentiation of xylem fibers (Dayan et al., 2012). Lack of gibberellin was also reported to inhibit cell division in the cortex during graft union formation in a GA deficient mutant reported to inhibit vascular c onnection and callus formation in tomato (Parkinson and Yeoman, 1982). Similar effects on inhibition of tracheid formation were reported by Savidge (1983) in Pinus contorta cuttings when gibberellin was applied to the basal sections. In the study on Pinus pinea by Kalev and Aloni (1998), gibberellin application did not result in tracheid differentiation, but promoted tracheid elongation when applied together with an auxin. These tracheids were differentiated from parenchyma cells induced by auxin applicati on. Effects of gibberellin application observed in gymnosperms, however, may differ from angiosperms. In Arabidopsis thaliana xylogenesis was found to be promoted with the application of gibberellin independent of auxin, while fiber differentiation was th ought to be a secondary effect (Ragni et al., 2011). Synergistic effects of GA were reported in lettuce pith cultures whereby GA 1 in combination with IAA and kinetin resulted in enhanced xylogenesis (Pearce et al., 1987). These studies indicated that the e ffects of gibberellin on vascular differentiation are complex and dependent on the plant species, presence of other hormones, and the conditions of the study. Little applied research has been conducted to study the effects of gibberellin, or the lack of it in vegetable grafting. Gibberellin was also reported to have an antagonistic effect with abscisic acid (ABA) with respect to regulation of stomatal opening. Aharoni et al. (1977) reported that in desiccating lettuce leaves with increasing ABA contents, exogenous applications of

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20 GA slowed down the rates of stomatal closure. Moreover, application of 9 M GA 3 to broad bean leaves increased stomatal conductance (Yuan and Xu, 2001). Since it is imperative to reduce transpiration and prevent excessive water l oss from the scion during graft healing, application of a GA biosynthesis inhibitor may help to reduce stomatal conductance and improve graft survival. Uniconazole Uniconazole inhibits the oxidation of ent kaurene to ent kaurenoic acid in the gibberellin biosynthesis pathway (Pressman and Shaked, 1991), thereby preventing GA biosynthesis. In a study on tomato seed germination and subsequent seedling height and hypocotyl length by Shin et al. (2009), soaking seeds in 100 mg/L uniconazole for a day was found to suppress seedling height and hypocotyl length. Seed emergence percentage was also reduced. These observed effects were probably related directly to the lack of endogenous GA, as GA is widely known to promote stem elongation and enhance germination. Acc ording to Shin et al. (2009), soaking tomato seeds in 100 and 200 mg/L uniconazole resulted in lower fresh and dry shoot weights. Uniconazole treatments also produced seedlings with smaller leaf area, lower leaf number, and reduced chlorophyll content. Thi s is contrary to that reported by Thetford et al. (1995), which may be attributed to different endogenous levels of cytokinins and accumulations of uniconazole in plant tissues in different studies (Shin et al., 2009). In the study conducted by Thetford et al. (1995) on Forsythia x intermedia cuttings, application of uniconazole was found to increase leaf chlorophyll content and decrease the chlorophyll a to chlorophyll b ratio, which are similar to typical characteristics of shade grown leaves. They also r eported higher stomata density, stomatal water vapor conductance, and net photosynthesis in cuttings treated with

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21 uniconazole. Uniconazole application inhibited initiation of phloem cells but not xylem cells. However, both phloem and xylem cell expansions were reduced by uniconazole applications. According to Saito et al. (2006), application of uniconazole inhibited the activity of hydroxylase in Arabidopsis thaliana by acting as a competitive inhibitor to ABA. hydroxylase is a key enzy me in regulating the catabolism of ABA to phaseic acid, inhibition of the enzyme by uniconazole resulted in enhanced accumulation of endogenous ABA within the plant, thereby leading to reduced transpiration, and improved drought tolerance. Application of G A 4 together with uniconazole yielded the same result, indicating that the observed increase in endogenous ABA level was not affected by the lack of endogenous gibberellin. Inhibition of phaseic acid production, and hence ABA catabolism, was reported to occ ur 1.5 h after treatment with 25 M uniconazole. However, it is not known if the same effects could be observed in tomato plants. Based on the results from these previous studies, it is likely that uniconazole application may be utilized in vegetable graf ting to reduce transpiration rates of newly grafted tomato plants, thereby improving survival rate and possibly quality. Abscisic Acid (ABA) Abscisic acid (ABA) is widely known to play a vital role in regulating stomatal opening, and hence, transpiration in plants, although hydraulic signals like alterations in xylem pressure potential and water content in guard cells and other epidermal cells can also regulate stomatal conductance (Pospisilova, 2003b). We hypothesized that exogenous application of ABA can help lower transpiration in grafted tomato seedlings during the healing process, thereby improving survival and quality of grafted transplants.

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22 However, applied research has been rather limited regarding the role of ABA in graft healing and its long term impacts on growth of grafted transplants. At present, much of the ABA application studies are focused on plant responses to salinity and drought stresses. In a study conducted by Wilkinson et al. (1998) on tomato plants, ABA was found to be essential for s tomatal closure. It was also reported by the authors that at very low concentrations of ABA (up to 0.03 mmol/m 3 ), an increase in xylem pH from 6.0 to 7.75 resulted in stomatal closure, indicating that at certain pH conditions very small amount of the hormo ne is required to be present in plant tissues to induce stomatal closure. Moreover, their study led to the conclusion that presence of ABA in small amounts is necessary for proper stomatal functioning, as lack of, or extremely low concentrations of ABA com bined with high xylem pH resulted in widening of stomata in flacca tomato (a mutant that biosynthesizes less ABA than a normal wild type tomato) leaves. Interestingly, in the study conducted by Pospisilova and Batkova (2004), French bean, sugar beet and ma ize plants pre treated with ABA exhibited higher stomatal conductance, net photosynthesis and transpiration rates compared to plants pre treated with water during drought stress. It must be noted that sensitivity of stomata to xylem ABA may be related to m any other factors such as water potential of the leaf, temperature, carbon dioxide concentration, leaf age and genotype (Dodd et al., 1996; Pospisilova, 2003a). On a physiological basis, ABA induces stomatal closure and/or prevents stomatal opening by inh ibiting K + channels that will otherwise allow for inward movement of K + ions into the guard cells, and by activating channels that facilitate the outward

PAGE 23

23 movement of K + and other anions from the guard cells (Pospisilova, 2003a ; Pospisilova, 2003b ). These w ill lower water potential inside the guard cells, resulting in stomatal closure. ABA can also affect expression of genes coding for proteins responsible for carbon metabolism, and for ion and water transport in guard cells (Pospisilova, 2003a; Pospisilova, 2003b; Webb et al., 2001 ). Lastly, ABA can also affect organization of cell cytoskeleton in guard cells (Pospisilova, 2003a; Pospisilova, 2003b ). It had been demonstrated by Bradford et al. (1983) that applications of up to 30M ABA resulted in effects of stomatal closure that lasted for about 4 days on both flacca flacca was derived. In the study by Goreta et al. (2007), application of 2 g/L ABA led to approximately 50% decrease in stomatal co nductance 1 day after treatment, and such an effect lasted for up to 8 days after treatment in pepper seedlings. Pospisilova and Batkova (2004) also demonstrated that net photosynthetic rate, transpiration rate and stomatal conductance decreased 1 h after application of 100 M ABA in maize, French bean and sugar beet plants. In another study conducted by Pospisilova (2003b), similar effects on stomatal conductance, transpiration and net photosynthesis were observed in French bean leaves with spray applicati on of 100 M ABA after 1 h; however, the ABA effects did not last for more than 24 h after application. In the same study, when plants pretreated with 100 M ABA were subjected to drought stress by withholding water, ABA application was reported to reduce gas exchange parameters in the plants for the initial stage of the experiment, and delayed the onset of water stress symptoms in the plants during the later stage of the experiment.

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24 Decreased stomatal conductance results in reduced carbon dioxide concentr ation and diffusion within the leaf, which ultimately lead to a decreased carbon dioxide concentration in chloroplasts and hence, reduce carbon dioxide fixation rate (Haisel et al., 2006 ; Pospisilova and Batkova, 2004 ). This might adversely affect plant gr owth in terms of dry matter accumulation. Also, lowered carbon dioxide levels within chloroplasts coupled with high irradiance can cause photoinhibition (Pospisilova and Batkova, 2004). Height of pepper seedlings was reduced with 2 g/L ABA application com pared to those sprayed with tap water in a study conducted by Goreta et al. (2007). In their study, seedlings treated with ABA also had higher relative water content and leaf water potential, with lesser electrolyte leakage and leaf abscission. However, lo wer net carbon dioxide assimilation was also observed in plants treated with ABA for up to 5 days after treatment, after which it slowly increased, and was similar to the control 8 days after treatment. Total relative growth rate was also reduced with ABA application. Results from this study indicate that although ABA application resulted in improved water status and enhanced drought tolerance in pepper seedlings, certain growth parameters might be negatively affected. It would be interesting to assess the influence of ABA application on grafted tomato seedlings. In the study conducted by Haisal et al. (2006), application of ABA before imposing a water stress period was found to prevent degradation or stimulate production of chlorophyll and carotenoid in b ean, sugar beet, maize and tobacco plants. Levels of zeaxanthin, antheraxanthin and violaxanthin were also increased in plants treated with ABA, which may serve to protect leaves from photoinhibition under drought

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25 stress conditions coupled with high light levels by dissipating extra heat energy as part of the xanthophyll pigment cycle, thereby reducing damage to photosystem II. In another study, maize, sugar beet and French bean plants pre treated with 100 M ABA and exposed to drought stress showed enhance d recovery in transpiration rate, net photosynthetic rate and stomatal conductance after rehydration compared to plants pre treated with water (Pospisilova and Batkova, 2004). Given that ABA application helped to alleviate some negative impacts caused by d rought stress, use of ABA may be advantageous in grafted tomato transplant production by improving graft recovery, especially when the ideal healing condition of high humidity is not conveniently available. Exogenous ABA application in Arabidopsis thalia na resulted in upregulation of hydroxylase, which converts ABA to phaseic acid thereby increasing ABA catabolism (Saito et al., 2004; Saito et al., 2006). In other words, an increase in the level of ABA within Arabidopsis thaliana also activates a feedback reaction for ABA catabolism, thereby regulating the level of ABA within the plant. Hence, physiological changes brought about by ABA application may not be a long lasting effect. Moreover, it is unclea r as to how long the influence of ABA will last in grafted tomato seedlings, which we will seek to elucidate from our research. Meanwhile, it is noteworthy that application of uniconazole for enhanced ABA accumulation, as mentioned above, might be a better choice if longer lasting effects are desired. In an in vitro study on Pinus contorta 10 ppm ABA was reported to prevent callus formation in explant chips (Savidge, 1983). When ABA was applied to the bottom

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26 sections of Pinus contorta cuttings, formation of tracheids was affected (Savidge, 1983). However, since these studies were conducted on a gymnosperm, it is uncertain that ABA will have the same effects on tomato, which is an angiosperm. PGR Application Methods Two methods of ABA application, namely f oliar spray or soil application, can be used to alter stomatal behavior in plants, which will ultimately depend on uptake, transport and breakdown of the hormone (Dodd, 2003). The rate of ABA catabolism is in turn affected by the environment and plant wate r content (Dodd et al., 1996). Pospisilova (2003b) reported that in French bean plants, spray application of 100 M ABA resulted in decreased gas exchange parameters for up to 24 h, whereas dipping of French bean roots in ABA solution of the same concentra tion resulted in stomatal closure that lasted for more than 24 h. In other words, soil application of ABA was found to be more effective compared to spray application in terms of prolonging the duration of lowered gas exchange parameters in French bean pla nts. That was attributed to low cuticle permeability in French bean plants. However, cuticle permeability may differ in tomato plants. Hence, in our study, we seek to compare the effectiveness of the 2 different application methods in terms of survival an d quality of grafted tomato transplants. Water Application Method during Graft Healing southeastern United States, whereby transplants are grown in soilless me dia in a expanded poly styrene foam tray floated on a layer of nutrient solution (Frantz and Welbaum, 1998 ; Frantz et al., 1998 ). In this system, the float bed usually consisted of water or nutrient solution on a plastic sheet lined inside of a rectangular wooden frame

PAGE 27

27 (Frantz a nd Welbaum, 1998). It had since been researched on, and adapted for transplant production of many vegetable crops. In the study on cabbage transplants by Frantz et al. (1998), the flotation system was found to result in faster growth and greater fresh weig ht accumulation in seedlings compared to those that were overhead irrigated. system produced plants that had higher water content. After transplanting into the field, seedlings from the floatation system, when sufficiently hardened, also showed enhanced growth during the first 3 weeks. Nevertheless, oxygen content of the water used for floatation was found to decrease from 70 to 10 mg/L between the second and fourth we ek. However, it must be noted that the rate of oxygen depletion is also influenced by other factors such as the depth of the water, and the shape of the bed (Frantz and Welbaum, 1998). The floatation system is advantageous in that water is supplied direc tly to the roots, and foliage of transplants will not get wet, thereby reducing the problem of fungal and bacterial diseases (Frantz et al., 1998). Also, labor cost associated with hand watering of the plants can be reduced (Frantz et al., 1998). Despite t hese advantages, using the floatation system may result in plants that are too succulent and therefore, are more difficult to establish after transplanting (Frantz et al., 1998). Moreover, the layer of water might facilitate the movement of diseases (Frant z et al., 1998). High survival rates of greater than 90% had been achieved in previous experiments when grafted tomato transplants were floated on a shallow layer of water in the healing chamber. However, in addition to the disadvantages discussed above, floating the grafted transplants on a layer of water may leach nutrients away from the

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28 substrate, resulting in nutrient deficiencies and a subsequent reduction in quality of the transplants. Also, it is uncertain if floatation is necessary to achieve high survival rates, as in some small scale operations and commercial settings in North America, grafted tomato plants are simply misted during the healing period (Johnson and Miles, 2011). Monitoring of Graft Survival It was reported by Johnson and Miles (2 011) that newly grafted plants may appear completely wilted right after grafting, but may recover and produce new growth some time later. Therefore, they concluded that graft survival will vary over time, and that complete wilting of the grafted plants was the most convenient and logical method to determine graft survival on a daily basis. Hence, in our study, we chose to record the number of non wilted plants over a 10 day period, as studies by by Fernndez Garca et al. (2004) showed that completely funct ional graft unions were formed 8 days after grafting in tomato plants. Stomatal Conductance Measurements Stomatal conductance (g s ) is a measurement of the rate at which carbon dioxide or water vapor moves through the stomata in leaves, and is influenced by the number of stomata in a given unit area, dimension of stomata, as well as the degree of stomatal opening as regulated by guard cells (Bradford et al., 1983; Decagon Devices, 2012). In our study, stomatal conductance will be monitored using the SC 1 l eaf porometer manufactured by Decagon Devices. Using a sensor head that can be clipped onto a leaf surface, temperature and relative humidity at 2 points along the diffusion path are measured for 30 s, after which a steady state g s will be extrapolated fro m those variables (Decagon Devices, 2012).

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29 Chlorophyll Fluorescence Light absorbed by chlorophyll molecules can follow one of three pathways: 1) be used for photosynthesis, 2) be dissipated as heat, and 3) be re emitted by chlorophyll molecules at longer wavelengths, a phenomenon known as chlorophyll fluorescence (Maxwell and Johnson, 2000). These 3 pathways are antagonistic to one another, and therefore, measurements of chlorophyll fluorescence can help provide insights into the efficacies of photosynthe sis (Maxwell and Johnson, 2000). In our study, dark adapted F 0 (chlorophyll fluorescence without actinic light) and F m (maximum chlorophyll fluorescence) measurements were taken using the OS1p modulated chlorophyll fluorometer (Opti Sciences) after which F v /F m (maximum quantum yield of photosystem II) can be calculated using the formula below to provide us with the maximum efficiency of photosystem II (Maxwell and Johnson, 2000). F v /F m = (F m F 0 )/F m Tomato leaves were dark adapted for different durations in various studies conducted by different authors. For instance, a period of 5 min was used in the study by Jung and Steffen (1997), whereas in the study by Al aghabary et al. (2004), tomato leaves were exposed to darkness for 15 min. In our study, a 15 m in dark period was chosen for dark adaptation of tomato leaves. F v /F m values will enable us to determine the extent of damage to photosystem II and the effects on photosynthesis efficiency caused by different PGRs applications, healing of grafted plants o utside the healing chamber, and/or water application methods during healing. According to Haisel et al. (2006), bean plants pre treated with ABA did not exhibit decreases in F v /F m after a drought stress treatment compared to those

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30 treated with water. In ou r study, we seek to determine if ABA applications will have similar effects on grafted tomato transplants. Objectives and Hypotheses The first objective of our study was to investigate the influence of ABA and uniconazole applications on the recovery fro m wilting, graft survival, and quality of grafted tomato seedlings, without facilitation of a healing chamber. Since exogenous ABA and uniconazole applications had been reported to reduce plant stomatal conductance, which can prevent excessive scion water loss during the graft healing period, we hypothesized that ABA and uniconazole applications will aid production of grafted tomato seedlings in the absence of a healing chamber. Secondly, we would like to determine if applications of uniconazole or ABA wi ll affect post transplant growth of grafted tomato seedlings, and hypothesized that growth of grafted tomato seedlings following transplanting will not be negatively impacted by PGR applications. Our third objective was to compare the efficiency of the 2 ABA application methods, i.e., foliar spray vs. root application. Root application was expected to result in greater reduction in plant stomatal conductance, and/or lengthen the period of stomatal closure compared to spray application, as previous studies had suggested. These will in turn improve graft recovery, survival and quality of grafted tomato transplants. Lastly, the effectiveness of different water application methods during graft healing will be studied. Floatation was hypothesized to be the pr eferable water application method compared to daily misting during the graft healing period for improved recovery, survival and quality of grafted plants.

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31 CHAPTER 2 EFFECTS OF EXOGENOUS ABSCISIC ACID APPLICATION ON RECOVERY, SURVIVAL, AND QUALITY OF GRAF TED TOMATO TRANSPLANTS Background Vegetable grafting is widely practiced in East Asia and in European countries in the Mediterranean region to combat problems commonly associated with intensive cultivation (Kubota et al., 2008; Leonardi and Romano, 2004) Some of these problems include soilborne diseases caused by Fusarium Verticillium Pseudomonas and nematodes (Lee, 1994; Oda, 2002). Despite the advantages of vegetable grafting, it began to gain attention in North America only recently. High cost of pr oduction has often been cited as one of the major reasons that hinders wider adoption of grafting in vegetable production systems (Aloni et al., 2010; Kubota et al., 2008; Lee, 1994). Part of the high production cost stems from the fact that newly grafte d plants need to be kept in a controlled environment with appropriate temperature and relative humidity to prevent excessive loss of water via transpiration from the scion until the graft has healed, and water can be transported from the roots to the scion (Johnson and Miles, 2011). Fernndez Garca et al. (2004) reported that a functional graft union required at least 8 days to form in tomato. To maintain optimum conditions during this period, healing chambers of different designs are used in North America (Johnson and Miles, 2011). However, construction and maintenance of such chambers will incur extra cost to growers, in addition to labor cost of grafting, and cost of rootstock seeds and plant care, etc. Johnson and Miles (2011) reported that a high survi val percentage of 96% was achieved in self a healing chamber. According to them, the tomato plants were healed under 2 layers of shade cloth in the greenhouse with a temperature range of 23 to 2

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32 relative humidity of 53%. Kubota et al. (2008) also reported that grafted tomato plants were relatively easy to heal in comparison with other crops. Hence, in our study, we seek to determine if a healing chamber is necessary for healing of grafted tomato seedlings under typical greenhouse conditions in north Florida, and also evaluate the quality of the resultant grafted transplants. Although there have been numerous studies on how plant growth regulators (PGRs) can affect graft healing, p ractical use and research involving PGRs in vegetable grafting is rather limited (Aloni et al., 2010). Abscisic acid (ABA) at 2g/L resulted in about 50% decrease in stomatal conductance in pepper seedlings a day after treatment, with effects that lasted fo r up to 8 days (Goreta et al., 2007). Similar effects of ABA on lowering stomatal conductance were also reported in tomato and other crop species, with effects that lasted for varying periods of time depending on the concentrations applied (Bradford et al. 1983; Pospisilova, 2003b; Pospisilova and Batkova, 2004). Reduction in stomatal conductance, and hence transpiration, can prevent excessive loss of water from the scion during graft healing when kept outside of the healing chamber. On the other hand, low ered stomatal conductance can decrease rate of carbon dioxide diffusion into leaves, thereby reducing carbon dioxide fixation rate, and eventually affecting net photosynthesis (Haisel et al., 2006; Pospisilova and Batkova, 2004). Decreased carbon dioxide l evels in leaves might also result in photoinhibition if leaves are exposed to high light levels (Pospisilova and Batkova, 2004), although Haisal et al. (2006) observed increased levels of zeaxanthin, antheraxanthin and violaxanthin in bean, sugar beet, mai ze and tobacco plants following ABA applications, which can help protect leaves from photoinhibtion. ABA might also adversely affect plant height

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33 and total relative growth rate as indicated by Goreta et al. (2007) in pepper seedlings. It would be of intere st to evaluate the use of ABA in healing grafted tomato seedlings, especially without facilitation of a healing chamber. According to Dodd (2003), ABA can be applied through foliar sprays or provided directly to the soil for absorption by roots. The exte nt of the effects of ABA application will be determined upon the uptake, transport and breakdown of the hormone (Dodd, 2003). Environmental conditions and plant water content also affect ABA catabolism (Dodd et al., 1996). Pospisilova (2003b) illustrated t hat soil application of ABA was more effective in reducing stomatal conductance in French bean plants compared to foliar application. It is uncertain how tomato plants will respond to different methods of ABA application. many systems utilized to irrigate vegetable transplants, although it was originally developed for tobacco production in the southeastern United States (Frantz and Welbaum, 1998 ; Frantz et al., 1998 ). Cabbage transplants produced using this method exhibite d faster growth and greater fresh weight compared to those that were watered overhead (Frantz et al., 1998). The floatation system is also advantageous in that labor cost of hand watering the plants can be eliminated, and chances of transplants getting inf ected by fungi and bacteria will be lower as their foliage will hardly get wet (Frantz et al., 1998). Despite these advantages, seedlings produced via the floatation system might be too succulent, thereby reducing their survival after being transplanted in to the field (Frantz et al., 1998). The maintenance of a layer of water in the system might also hasten the spread of diseases (Frantz et al., 1998).

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34 According to Johnson and Miles (2011), grafted vegetables are often misted during the healing period in some small scale operations in North America. However, tomato seedlings since expanded polystyrene foam flats are commonly utilized in tomato transplant production by man y growers in Florida. The objectives of this study are to: 1) determine the effects of ABA application on recovery, survival, and quality of grafted tomato seedlings when healed outside of the healing chamber, 2) assess the long term effect of ABA applicat ion on tomato plant growth, 3) evaluate the different ABA application methods, and 4) compare water application methods during the healing period with respect to their impacts on recovery, survival, and subsequent quality in grafted tomato transplants. Ma terials and Methods Location Our studies were conducted in the research greenhouse at the University of Florida, Gainesville campus. Grafting Experiments Two grafting experiments were conducted in both the spring 2012 and fall 2012 seasons. They were re ferred to as Spring 1, Spring 2, Fall 1 and Fall 2 for the first spring trial, second spring trial, first fall trial, and second fall trial, respectively. Plant Materials De Ruiter Seeds, Bergschenhoek, The Netherlands 04 106 Vegetable Seeds, Inc., Oxnard, CA) was used as the scion in the spring studies. In the

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35 These rootstocks were chosen as they are commonly used in grafted tomato production systems in the United States. The tomato scion is a widely grown field cultivar in Florida. Tomato Seedling Production In the first spring grafting trial, seeds were sown in 12 8 cell expanded polystyrene foam flats (Speedling, Inc., Sun City, FL) containing M etro M ix 200 (Sun Gro Horticulture, Bellevue, WA). Heating mats were used to maintain soil temperatures When the first true leaf appeared, tomato seedlings were fertilized with 20.0N 8.7P 16.6K fertilizer (Scotts Sierra Horticultural Products Company, Marysville, OH) at 53 mg/L N. Plants were watered daily or as required. 04 106 and fertilization regimes were similar to Spring 1. Plants were sprayed on 6 Apr il 2012 and 13 April 2012 with O rganocide (Organic Laboratories, Inc., Stuart, FL) for thrips control. In the first fall study, rootstock seeds were planted on 4 October 2012, while the scion seeds were planted a day later. In Fall 2, rootstock and scion seeds were planted on 31 October 2012 and 1 Novem ber 2012, respectively. Management of plan ts was similar to Spring 1. BT Worm & Caterpillar K iller (Schultz Company, Bridgeton, MO) was sprayed as needed to control caterpillars.

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36 Growth Regulator Applications gs were sprayed with 0, 1000, or 2000 mg/L ABA solution; or watered with 0, 118, or 236 mg/L ABA solution through root application, at least 12 hours before grafting. ABA solut ions were prepared by diluting ConT ego (Valent BioSciences Corporation, Liberty ville, IL ), which consisted of 10% S ABA, with deionized water. All spray solutions contained 0.05% Latron B 1956 ( Dow AgroSciences LLC Indianapolis, IN ) as an adjuvant. For the spray treatments, each plant received approximately 363 L of solution, acco rding to the recommended spray volume of 2 quarts per 100 ft 2 (204 mL/m 2 ). For the root application treatments, each plant received 3 mL of solution so that the amounts of active ingredient were equivalent between the 1000 mg/L ABA foliar and 118 mg/L root application treatments; and between the 2000 mg/L ABA spray and 236 mg/L ABA root application treatments. ABA solution, but spray volume per plant was doubled to 720 L for better coverage. For the root application treatments, scion seedlings were individually watered with 3 mL of 0, 236 or 473 mg/L ABA solutions to match the higher amounts of active ingredient in the spray treatments. In both fall studies, only the spray a pplication method was used. Scions were sprayed with 0, 400, 800, or 1200 mg/L ABA at the recommended rate of 2 quarts per 100 ft 2 (204 mL/m 2 ) at least 12 hours before grafting. Grafting Method Tomato seedlings were grafted in the morning in the greenhou se using the tube rootstock below the cotyledons using a double edged razor blade. A similar angled cut

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37 was then made on the epicotyl of the scion above the cotyledons. The rootstock and scion were then held together in place using a silicone grafting clip (Hydro Gardens, Colorado Springs, CO). Grafting was conducted on 16 March 2012 and 17 April 2012 for the Spring 1 and Spring 2 studies, respectively. In the fall season seedlings were grafted on 5 November 2012 and 3 December 2012. For the fall studies, both rootstock and scion were cut at the hypocotyls below the cotyledons. Graft Healing The healing chamber consisted of PVC pipes as its frame, set on a greenhouse b ench. The frame was covered with a layer of clear plastic, followed by a layer of black plastic, and finally by a layer of shade cloth for insulation. For the spring studies, non treated plants (i.e. the control treatment) were floated on water inside the healing chamber equipped with an air conditioning system and a humidifier. Grafted seedlings sprayed or watered with different ABA concentrations were also floated on water outside of the healing chamber inside the shaded greenhouse. Temperature, light in tensity and relative humidity (RH) were recorded using HOBO dataloggers (Onset Computer Corporation, Inc., Pocasset, MA). Average temperature, RH and light intensity inside and outside of the chamber during graft healing (day 1 to 4) were presented in tab le 2 15. In the fall studies, the healing chamber was partitioned into two, whereby half of it accommodated plants that were floated, while the other half held plants that were hand misted daily. A humidifier was used in each side of the partitioned chamb er. Only plants treated with water were kept inside the chamber. Grafted plants sprayed with water and various ABA concentrations were kept in the greenhouse outside of the chamber, and were either floated or hand misted daily. Table 2 16 showed the averag e temperature,

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38 RH and light intensity at different healing locations from day 1 to 4 after grafting. The greenhouse was shaded in Fall 1 only. Acclimation For the Spring 1 study, the healing chamber was opened approximately way on both sides on the 5 th day after grafting. On the 6 th day, the openings were enlarged to about halfway on both sides. The air conditioning system and humidifier were also turned off. On the 7 th day, the plants were brought out from the chamber and floated on water in the greenh ouse. Plants were also sprayed with O rganocide in the evening, as thrips damages were evident. In the second spring trial, acclimation procedures were similar to Spring 1 for the 5 th and 6 th day after grafting. However, on the 7 th day, the sides were lo wered, leaving openings that were of the chamber height. On the 8 th day, the openings were widened to about halfway on both sides; and on the 9 th day, the plants were removed from the chamber. These differences in acclimation treatment were due to differ ences in ambient air temperatures and light levels in the greenhouse between the 2 trials, for plants in the second spring trial started to show moderate wilting on the 7 th day. For Fall 1 and Fall 2, both sides of the healing chamber were opened way o n the 5 th day, and enlarged to halfway on the 6 th day. On the 7 th day, the chamber was opened to of its height on both sides, and the air conditioning system and humidifiers were switched off. The plants were removed from the chamber on the 8 th day. T ransplanting To mimic field production, grafted seedlings were transplanted into 0.4 L s quare plastic pots filled with Metro M ix 200 10 days after grafting for the Spring 1 study. Plants were grown for another 4 weeks in the greenho use and fertilized wit h Peters

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3 9 P rofessional 20.0N 8.7P 16.6K fertilizer at 264 to 317 mg/L N twice weekly. Grafting clips were removed 7 days after transplanting. Plants were transplanted 10 days after grafting in the first fall study. Grafting clips were removed 1 week afte r transplanting. However, plants were only grown for 3 weeks. Measurements In Spring 1, number of turgid plants was counted daily until the 10 th day after grafting. On the 11 th day, quality of the grafted transplants was determined by taking measurements of chlorophyll content (using the CCM 200 chlorophyll content meter by Opti Sciences, Hudson, NH), fresh weight, scion stem diameter, scion length, and leaf area (using the LI 3100 area meter by LI COR Environmental, Lincoln, NE). Four weeks after transpl anting, quality of the grafted plants was assessed again by taking measurements of stomatal conductance ( using the SC 1 leaf porometer by Decagon Devices, Pullman, WA), chlorophyll content, fresh weight, scion stem diameter, leaf area, scion length and dr y weight. Similar data were taken for Spring 2. In addition, stomatal conductance of non applications. Stomatal conductance and dry weight measurements were also taken on top of similar parameters recorded in Spring 1 to determine grafted transplant quality at 10 days after grafting. For the fall studies, measurements similar to the spring studies were recorded. However, final measurements of transplant quality were tak en at 3 weeks after transplanting. Dark adapted maximum and minimum chlorophyll fluorescence readings

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40 were measured in the Fall 2 study using an OS1p modulated chlorophyll fluorometer (Opti Sciences, Hudson, NH) to calculate the F v /F m ratio. Experimental Design and Statistical Analyses Randomized complete block designs were used for all the experiments. Eight plants per treatment per replicate were grafted, while 3 plants per treatment per replicate were transplanted. There were 4 replicates in each study Grafted transplant wilted plants per day was analyzed using logistic regression. All statistical analyses were conducted using the SAS statistical software package for Windows (version 9.3, Cary, NC). Results and Discussion Stomatal Conductance after ABA Application Spring 2. Plants sprayed with either 1000 or 2000 mg/L ABA exhibited significantly lower stomatal conductance compar ed to those that were sprayed with 0 mg/L ABA, at least 12 h after ABA application (Table 2 5). The same trend was also observed for plants that received root applications. Fall. For both fall studies, 400, 800 and 1200 mg/L ABA spray applications resulte d in lower stomatal conductance in plants compared to those treated with 0 mg/L ABA (Table 2 6). These results concurred with those reported by Goreta et al. (2007), Pospisilova (2003b), and Pospisilova and Batkova (2004). Wilting and Recovery from Wiltin g Spring 1. On the fourth day after grafting, among plants that were kept outside of the chamber, only those that received 1000 mg/L ABA spray application had a proportion of non wilted plants that was similar to those kept inside the chamber (Table

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41 2 7). For the foliar treatments, higher proportions of non wilted plants were observed for those treated with either 1000 or 2000 mg/L ABA compared to those that were treated with 0 mg/L ABA (Table 2 7). On the other hand, root applications of 118 or 236 mg/L AB A resulted in proportions of non wilted plants that were comparable to those that received 0 mg/L root application (Table 2 7). These results suggested that ABA foliar application was more effective than root application, and helped with faster plant recov ery from wilting when grafted seedlings were healed outside of the healing 04 106 (data not shown) Spring 2. At 4 days after grafting, the highe st proportions of non wilted plants were recorded in plants that received ABA foliar applications at 1000 or 2000 mg/L ABA, and those that were kept inside the chamber without any ABA application (Table 2 7). Among the foliar and root applications, proport ions of recovered plants followed the same trend as in Spring 1, which further indicated that grafted tomato plants sprayed with ABA had faster recovery from wilting when kept outside of the healing chamber in contrast to those with ABA root application. Fall 1. On the first day after grafting, plants that received 1200 mg/L ABA had similar proportion of turgid plants as those that were kept inside the chamber (Table 2 8). When kept outside of the chamber, proportion of non wilted plants decreased signific antly as ABA spray concentration decreased (Table 2 8). At day 4, plants kept inside the chamber had significantly higher recovery compared to those that were kept outside (Table 2 8). Among plants that were kept outside, those that were sprayed with 400 o r 800 mg/L ABA had similar recovery as those that received 0 mg/L ABA (Table 2

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42 8). When 1200 mg/L ABA was sprayed, plants showed greater recovery as compared to when 0 mg/L ABA was applied (Table 2 8). These results showed that ABA application, particularl y at 1200 mg/L, helped to delay the onset of wilting symptoms in the newly grafted plants that were kept outside of the chamber, and also resulted in better recovery from wilting later on. These were in accordance to that reported by Pospisilova (2003b) an d Pospisilova and Batkova (2004). Graft Survival High survival rates of more than 90% were observed for almost all treatments regardless of ABA applications and where the plants were kept during the healing process. For instance, in Fall 2, plants kept o utside the chamber with 0 mg/L ABA application had an average of 95% survival, while those with 1200 mg/L ABA application had about 98% survival. These results were comparable to when plants were healed inside the chamber, which had approximately 95% survi val. Therefore, ABA applicat ions did not improve survival. Moreover our study indicated that grafted tomato seedlings can be healed without facilitation of a healing chamber with economically viable survival rates of more than 90%, as suggested by Johnson and Miles (2011). This was surprising as grafted plants kept outside of the chamber were subjected to average maximum air temperature s that w ere much higher than the recommended range as suggested by Kubota et al. (2008) (Table 2 17 and table 2 18) Mean minimum humidity outside the chamber during the healing period was also a lot lower than the optimum as recommended by Kubota et al. (2008) (Table 2 17 and table 2 18) However, lower temperature and higher humidity during the night might have aided in gra ft survival. In general, grafted tomato seedlings appeared to be tolerant of sub optimal healing conditions.

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43 Quality Parameters at 10 or 11 Days after Grafting Chlorophyll content Spring 1. Interactions between the rootstock type, location of healing, me thod of ABA application and ABA concentration were present in the Spring 1 study (Table 2 1). Overall, the effects of healing location and exogenous ABA application on chlorophyll the rootstock, spray applications of 1000 and 2000 mg/L ABA resulted in reduced chlorophyll content compared to when 0 mg/L was used (Table 2 9), indicating signs of phytotoxicity as a result of high ABA levels. With respect to the root application of ABA, content (Table 2 9). Plants kept inside the healing chamber had chlorophyll content that was similar to when plants were kept outside and sprayed or watered with 0 mg /L ABA (Table 2 9), which suggested that healing grafts without a healing chamber will not 04 106 comparable to those that were placed outside with 118 or 236 mg/L ABA root applications, or 1000 mg/L ABA foliar application (Table 2 9). In contrast, plants kept outside the healing chamber and watered with 0 mg/L ABA, or sprayed with 0 or 2000 mg/L ABA exhibited lower chlorophyll content as compared to plants kept inside the chamber (Table 2 9). These results suggested that absorption of ABA by the scion was probably better via spray application compared to root application, and therefore, phytotoxicity was more likely to occur in scions that received the spray applications. Moreover, root application of ABA at 236 mg/L resulted in a similar level of chlorophyll content as the treatment inside the chamber. This was in accordance with the report by

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44 Haisal et al. (2006) that ABA application prior to drought stress helped to prevent degradation of chlorophyll in bean, maize, sugar beet and tobacco plants. Spring 2. Interactions between the application method, location of healing and ABA concentration were detected (Table 2 2). Plants that were healed in the chamber had the highest level of chlorophyll content, which did not differ significantly from that of plants kept outside with 473 mg/L ABA root application and 0 mg/L ABA foliar application (Table 2 9) Chlorophyll content in plants was similar between different concentrations of the root treatment (Table 2 9). For plants healed outside with foliar treatments, application of 2000 mg/L ABA resulted in a lower chlorophyll level compared to the 0 mg/L ABA application, whereas applications of 1000 and 0 mg/L ABA resulted in similar plant chlorophyll content (Table 2 9). In this study, root application of 473 mg/L ABA appeared to be useful in maintaining chlorophyll content of grafted plants left outside of t he healing chamber. Tomato plants seemed to absorb greater amounts of ABA via foliar sprays compared to root applications, and the ABA spray application rate at 2000 mg/L may cause phytotoxicity. This was especially so in the second spring study when the a mount of solution received by each plant was doubled. Given the toxicity symptoms observed in the spring studies, concentrations of ABA application were reduced for the fall studies. Sp r ay application was also chosen as it was more effective compared to ro ot application, and therefore, lower amounts can be applied for similar effects, which will translate to cost savings in terms of lesser chemical usage for growers. In addition, spraying seems to be a more practical ABA application method compared to root application.

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45 Fall 1. There were significant interactions between the water application method during healing, healing location and ABA concentration in the first fall study (Table 2 3). Floatation or misting did not have any impact on chlorophyll content at different ABA concentrations except when 400 mg/L ABA was applied, whereby floated plants had significantly higher chlorophyll content than that of misted plants (Table 2 10). When floated, tomato plants kept inside of the healing chamber with 0 mg/L AB A application, and those that were kept outside with 400 mg/L ABA application had similar chlorophyll levels, which were higher than other treatments (Table 2 10). When misted, plants kept inside of the chamber exhibited significantly higher chlorophyll co ntent than all other treatments (Table 2 10). Fall 2. Interactions similar to F all 1 were observed in the second fall study (Table 2 4). Water application method during healing did not affect chlorophyll content except when 1200 mg/L ABA was applied, in w hich misting resulted in plants with higher chlorophyll content (Table 2 10). Among plants that were floated, those that were kept outside of the chamber with 800 or 1200 mg/L ABA applications exhibited significantly lower chlorophyll content compared to t he other treatments (Table 2 10), which were similar to results obtained in Fall 1, indicating that these concentrations of applied ABA led to reductions in plant chlorophyll content possibly due to phytotoxicity. For the mist treatments, plants that were kept inside the chamber had similar chlorophyll content as those that were healed outside with 0, 400 or 1200 mg/L ABA applications (Table 2 10). The differences in results between the mist treatments in the 2 fall studies could be due to higher average RH and reduced mean light intensity outside the chamber (Table 2 16) in the second fall study, which resulted in less environmental stresses imposed on

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46 the plants. Overall, results from the 2 fall studies suggested that 400 mg/L ABA spray application in comb ination with floatation could be used for healing grafted tomato plants outside of the healing chamber with no adverse effect on chlorophyll content. Stomatal conductance Spring 2. In the Spring 2 study, plants that were sprayed with 1000 or 2000 mg/L ABA exhibited lower stomatal conductance compared to the other treatments (Table 2 11), indicating that the ABA effect of lowering stomatal conductance lasted for at least 10 days after foliar application, which was contrary to that reported by Bradford et al. (1983) and Pospisilova (2003b). However, it should be noted that in our study, relatively high concentrations of ABA were used, which could account for the longer lasting effects on lowering stomatal conductance. Similar results were reported by Goreta et al. (2007) in pepper seedlings with reduced stomatal conductance that lasted for at least 8 days when plants were sprayed with 2000 mg/L ABA. Fall 1. Plants sprayed with 0 mg/L ABA and kept either inside or outside of the healing chamber had similar stom atal conductance measurements, which were also not significantly different from when 400 mg/L ABA was applied (Table 2 13). Application of 800 or 1200 mg/L ABA resulted in reduced stomatal conductance in grafted plants compared to when 0 mg/L ABA was appli ed at 11 days after grafting (Table 2 13). However, plants sprayed with 1200 mg/L ABA had stomatal conductance that was similar to plants that received 400 mg/L ABA applications; while plants that received 800 mg/L ABA had significantly lower stomatal cond uctance compared to those that received 400 mg/L ABA (Table 2 13). Response of stomata to ABA application is dependent on a variety of other factors such as leaf age and leaf water potential other than the ABA concentrations that were applied (Dodd et al., 1996; Pospisilova, 2003a),

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47 which might account for the observed responses in this study. It might also seem plausible that ABA application concentrations beyond 800 mg/L could not increase stomatal closure further. However, it is certain that effect of AB A on stomatal closure lasted for at least 11 days after spray application at 800 and 1200 mg/L. Fall 2. No significant treatment differences were observed in terms of stomatal conductance in the s econd fall study (Table 2 4 ). This might be due to greater rates of ABA catabolism triggered by increases in endogenous ABA levels as seen in Arabidopsis thaliana (Saito et al., 2004; Saito et al., 2006). Different environmental conditions between the studies i.e., higher mean maximum temperature outside the cham ber in Fall 2 (Table 2 18), might also affect ABA catabolism rate, as suggested by Dodd et al. (1996) Scion length Spring. 04 106 (d ata not shown) Plants kept inside the chamber also had greater scion length as compared to those kept outside of the chamber, regardless of ABA concentrations and application methods (data not shown) However, in the second spring study, treatments did no t result in any significant difference in scion length (Table2 2) Fall 1. In the first fall study, plants kept inside the chamber exhibited similar scion length as those kept outside, except for the 1200 mg/L ABA spray treatment which resulted in signifi cantly shorter scion length (Table 2 14). This indicated that when grafted seedlings were kept outside of the chamber, exogenous application of 1200 mg/L ABA may result in reduced scion length. Plant height reduction following ABA spray application was als o reported by Goreta et al. (2007) in pepper seedlings.

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48 Fall 2. Grafted tomato seedlings that were kept inside the healing chamber had significantly longer scion length compared to those kept outside (Table 2 14). This may be due to etiolation as a result of the lack of light inside the chamber, ABA application, or a combination of both factors. It might be advantageous to have transplants with shorter heights, as they will less likely be damaged by wind in the field. Leaf area Spring. In the S pring 1 st udy, highest leaf area was observed in plants kept inside the chamber, which was similar to those kept outside and received 118 mg/L ABA root application (Table 2 12). For the foliar applications, plants did not exhibit differences in leaf area when differ ent concentrations of ABA were used (Table 2 12). The same trend was observed in the root applications (Table 2 12). Leaf area was greater in plants that were watered with 118 mg/L ABA compared to those that were sprayed with 1000 mg/L ABA (Table 2 12). Ou r results suggested that keeping plants outside of the healing chamber may slow down the rate of leaf expansion. In the second spring study, only the method of application showed a significant effect on leaf area, with a larger leaf area observed for plant s that received the spray treatment (data not shown). Fall. No significant difference in leaf area was observed in the Fall 1 study (Table 2 3) In the second fall study, leaf area of plants kept inside the chamber was similar to that of all treatments ke pt outside, except when 1200 mg/L ABA was applied, which resulted in a significant reduction in plant leaf area (Table 2 13). When kept outside of the chamber, application of 1200 mg/L ABA reduced leaf area compared to the 800 mg/L ABA treatment (Table 2 1 3), indicating that 1200 mg/L ABA application may reduce rate of leaf expansion.

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49 Scion diameter Spring. Plants that were kept outside of the healing chamber had similar scion diameter in S pring 2 (Table 2 11). Plants that were healed inside the chamber h ad the smallest scion diameter, which did not differ significantly from that of plants kept outside with 2000 mg/L ABA foliar application (Table 2 11). These results indicated that healing of grafted tomato plants without utilizing a healing chamber may re sult in scions with thicker stems that are sturdier and more resistant to wind damage. This will facilitate transplanting into the field without the need for extra hardening as compared to plants healed inside the chamber. Two thousand mg/L ABA, when spray ed, can negatively impact scion diameter. In the Spring 1 study, no significant differences were detected (Table 2 1) Fall. Treatments resulted in no significant effects on scion diameter in both fall studies (Table 2 3 and table 2 4) Scion fresh weight Spring. In the first spring study, fresh weight of plants kept inside the healing chamber was only significantly higher than those that were kept outside of the chamber with 0 mg/L ABA application (data not shown). This may imply that application of ABA improved fresh weight of grafted transplants when healed without a healing chamber. As reported in pepper seedlings by Goreta et al. (2007), ABA application could result in higher relative water content and leaf water potential, thereby increasing plant fr esh weight. However, no significant differences were found in the second spring study (Table 2 2) Fall. Fresh weight was not affected by the treatments in Fall 1 (Table 2 3) However, in Fall 2, plants kept inside the chamber had the greatest fresh weigh t

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50 compared to those kept outside, except for the 800 mg/L ABA treatment, which resulted in comparable fresh weights (Table 2 13). These inconsistent results in fresh weight between the different studies may be related to different greenhouse conditions tha t the plants were exposed t o during healing (Table 2 15, table 2 16 table 2 17, and table 2 18 ). Scion dry weight Spring 2. Scion dry weight was significantly lower in plants that were placed inside the chamber compared to plants kept outside, except fo r those that received 2000 mg/L ABA foliar application (Table 2 11). No significant differences were observed among plants receiving root application with different ABA concentrations (Table 2 11). However, for the foliar ABA application treatments, plants treated with 1000 or 2000 mg/L had significantly lower dry weights compared to the 0 mg/L ABA treatment (Table 2 11). The low dry weight observed for plants healed inside the chamber could be attributed to the fact that plants were kept under low light co nditions for 5 days during the healing process, resulting in negligible photosynthesis and net carbon accumulation. Also, foliar ABA applications of 1000 or 2000 mg/L may negatively impact dry matter accumulation by causing prolonged stomatal closure, resu lting in reduced net photosynthesis (Haisel et al., 2006; Pospisilova and Batkova, 2004). Fall 1. Plants kept outside of the chamber with 800 mg/L ABA application had the highest scion dry weight, which was comparable to when plants were kept outside with 0 or 400 mg/L ABA applications (Table 2 13). Compared to plants kept inside the chamber and those kept outside with 1200 mg/L ABA application, plants kept outside with 800 mg/L ABA application exhibited significantly higher dry weight (Table 2 13). These r esults concurred with those obtained in Spring 2, indicating that high ABA

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51 concentration inhibited dry matter accumulation. Also, lack of light inside the chamber during healing (Table 2 16) might adversely affect dry weight of grafted seedlings. Fall 2. No significant differences were observed in the second fall study in terms of scion dry weight (Table 2 4) Chlorophyll fluorescence Fall 2. No significant differences were detected among treatments with regards to Fv/Fm ratio ( Table 2 4) indicating that healing of grafted tomato transplants outside the healing chamber and ABA application did not affect maximum efficiency of photosystem II. On the other hand, it should be stressed that average air temperature and light intensity were lower in the greenhou se during the second fall study compared to the spring studies (Table 2 15 and table 2 16), which reduced the amount of environmental stresses imposed on the newly grafted transplants while healing outside of the chamber. Different results may be observed when newly grafted plants are exposed to higher temperatures and light levels, as reported in the drought stress study on bean plants by Haisel et al. (2006) that plants treated with ABA did not exhibit a significant decrease in Fv/Fm ratio compared to tho se treated with water. Quality Parameters at 3 or 4 Weeks after Transplanting Leaf area Spring. 04 106 pla nt vigor between the 2 rootstocks, resulting in different rates of water and nutrient uptake. Fall. have any impact on leaf area at 3 weeks after transplanting.

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52 Scion length Sprin g. Plants that received either 1000 mg/L ABA spray application or 118 mg/L ABA root application had longer scion length compared to those that received 2000 mg/L ABA spray application or 236 mg/L ABA root application (data not shown) indicating that highe r concentration of ABA application may have long lasting effect of plant height suppression. Fall. No significant differences were observed in terms of scion length. This was expected as lower ABA concentrations were used in the fall study. Scion fresh w eight Spring. There was no significant difference in terms of scion fresh weight. Fall. At 3 weeks after transplanting, plants that were misted exhibited greater fresh weight compared to those that were floated during the healing period (data not shown) This was surprising as the water application method during healing did not affect scion fresh weight at 11 days after grafting. It might be due to excessive leaching of nutrients from the media of floated plants compared to those of misted plants during healing, which resulted in slower growth rate of the floated plants later on. Scion dry weight Spring. Plants that received root application of ABA solution had higher dry weight compared to those that were treated with foliar ABA application (data not sh own) This might indicate that tomato plants absorbed ABA better via foliar application, which resulted in longer lasting effect of lowered stomatal conductance, and ultimately reduced dry weight accumulation. This was in contrast to that reported by Pospi silova (2003b) in French bean, which might be explained by higher leaf cuticle permeability in tomato compared to French bean.

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53 Fall. Treatments did not result in any significant difference in dry weight at 3 weeks after transplanting. Other measurements There were no significant differences between treatments in terms of chlorophyll content, stomatal conductance and scion diameter for both seasons. Summary Due to the differences in environmental conditions, particularly temperature, relative humidity, a nd light intensity between the 4 studies, some variations and differences between results were expected. However, for newly grafted tomato plants kept outside of the chamber, it was clear that ABA application prior to grafting helped to delay the onset of wilting, and hastened their recovery from wilting during graft healing. Faster recovery from wilting may mean less time required for grafted transplant production, which can ultimately lower production costs for growers. Although our results suggested tha t grafted tomato seedlings can be healed successfully without facilitation of a healing chamber, and without ABA application in some instances, low concentration (i.e. 400 mg/L) of exogenous ABA application is recommended under conditions of high temperatu res and intense radiation, which are typical greenhouse conditions in Florida, especially during late spring and early fall seasons. High ABA concentration (i.e. 1200 mg/L) should be avoided as it may negatively affect chlorophyll content. Scion length may be reduced by ABA application, which will be advantageous for transplanting into the field. Spray application was also found to be more efficient compared to root application, although appropriate ABA concentration needs to be used to prevent phytotoxicit y. ABA application did not appear

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54 to have significant negative impacts on post transplant growth of grafted tomato seedlings when used at low concentration.

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55 Table 2 1. Analysis of variance on the effects of ABA concentration, healing location, rootstock type, and ABA application method on grafted transplant quality at 11 days after grafting in Spring 1. Effects Chlorophyll content Scion diameter Fresh weight Scion length Leaf area Rootstock NS NS NS ABA concentration x ABA application method x healing location NS NS NS Rootstock x ABA concentration x ABA application method x healing location NS NS NS NS and NS indicate significance and non Table 2 2. Analys is of variance on the effects of ABA concentration, healing location, rootstock type, and ABA application method on grafted transplant quality at 10 days after grafting in Spring 2. Effects Chlorophyll content Scion diameter Fresh weight Dry weight Scion l ength Leaf area Stomatal conductance Rootstock NS NS NS NS NS NS NS ABA concentration x ABA application method x healing location NS NS NS Rootstock x ABA concentration x ABA application method x healing location NS NS NS NS NS NS NS and NS indicate significance and non

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56 Table 2 3. Analysis of variance on the effects of ABA concentration, healing location, and water application method on grafted transplant quality at 11 da ys after grafting in Fall 1. Effects Chlorophyll content Scion diameter Fresh weight Dry weight Scion length Leaf area Stomatal conductance ABA concentration x healing location NS NS NS Water application method NS NS NS NS NS N S ABA concentration x healing location x water application method NS NS NS NS NS NS and NS indicate significance and non significance, respectively, Table 2 4. Analysis of variance on the effects of ABA concentration, healing location, and water application method on grafted transplant quality at 11 days after grafting in Fall 2. Effects Chlorophyll content Scion diameter Fresh weigh t Dry weight Scion length Leaf area Stomatal conductance Chlorophyll fluorescence ABA concentration x healing location NS NS NS NS Water application method NS NS NS NS NS NS NS NS ABA concentration x healing location x water ap plication method NS NS NS NS NS NS NS and NS indicate significance and non significance, respectively,

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57 Table 2 ABA foliar and root applications in Spring 2. ABA concentration (mg/L) Application method Stomatal conductance (mmol/m 2 s) 0 Foliar 79.3 a 1000 Foliar 34.2 b 2000 Foliar 29.7 b 0 Root 78.2 a 236 Root 42.2 b 473 Root 23.7 b LSD test. Table 2 6. Stomatal conductance (mmol/m 2 at least 12 h after ABA spray applications in Fall 1 and Fall 2. ABA concentration (mg/L) Fall 1 Fall 2 0 379.1 a 102.5 a 400 53.6 b 23.5 b 800 47.2 b 19.5 b 1200 40.6 b 18.1 b Different letters within a column indicate significant differences at P LSD test. Table 2 7. Effects of ABA concentration, application method and location of healing on proportion of non wilted grafted tomato plants on the 4 th day after grafting in Spring 1 and Spring 2. ABA concentration (mg/L) Applicat ion method Location z Spring 1 Spring 2 0 Foliar Outside 0.60 c w 0.70 b 1000 Foliar Outside 0.86 ab 0.92 a 2000 Foliar Outside 0.84 b 0.89 a 0 Root Outside 0.70 bc 0.66 b 118/236 y Root Outside 0.62 c 0.65 b 236/473 x Root Outside 0.70 bc 0.58 b Non treated Non treated Inside 0.97 a 0.95 a z Grafted plants were kept inside or outside of the healing chamber during the healing period. y 118 mg/L ABA was applied in Spring 1, while 236 mg/L ABA was applied in Spring 2. x 236 mg/L ABA was applied in Spr ing 1, while 473 mg/L ABA was applied in Spring 2. w 60% of grafted plants was not wilted for this treatment. LSD test.

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58 Table 2 8. Effects of ABA concentration a nd location of healing on proportion of non wilted grafted tomato plants on the 1 st and 4 th day after grafting in Fall 1. ABA concentration (mg/L) Location z Day 1 Day 4 0 Inside 0.96 a y 0.88 a 0 Outside 0.03 d 0.09 c 400 Outside 0.35 c 0.11 c 800 Outsi de 0.57 b 0.21 bc 1200 Outside 0.86 a 0.27 b z Grafted plants were kept inside or outside of the healing chamber during the healing period. y 96% of grafted plants was not wilted for this treatment. Different letters within a column indicate significant LSD test. Table 2 9. Effects of ABA concentration, application method, location of healing, and rootstock type on chlorophyll content (chlorophyll concentration index) at 11 and 10 days after grafting in Spring 1 and Spring 2, respectively. ABA concentration (mg/L) Application method Location z Spring 1 Spring 2 Maxifort RST 04 106 T 0 Foliar Outside 8.3 a 4.8 e 6.6 ab 1000 Foliar Outside 5.0 e 5.7 cde 5.5 bc 2000 Foliar Outside 5.6 cde 5.1 de 4.6 c 0 Root Outside 6.2 bcde 4.7 e 6.2 bc 118/236 y Root Outside 5.6 cde 5.8 cde 6.0 bc 236/473 x Root Outside 6.6 bcd 6.7 abc 7.4 ab Non treated Non treated Inside 7.8 ab 6.8 abc 8.3 a z Grafted plants were kept inside or outside of the healing chamber durin g the healing period. y 118 mg/L ABA was applied in Spring 1, while 236 mg/L ABA was applied in Spring 2. x 236 mg/L ABA was applied in Spring 1, while 473 mg/L ABA was applied in Spring 2. Different letters within and between columns in Spring 1, and withi n a column in Spring 2 indicate significant differences at P

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59 Table 2 10. Effects of ABA concentration and location of healing on chlorophyll content (chlorophyll concentration index) at 11 days after grafting in Fall 1 and Fall 2. ABA concentration (mg/L) Location z Fall 1 Fa ll 2 Floatation Misting Floatation Misting 0 Inside 9.6 a 9.4 a 6.5 a 6.1 a 0 Outside 6.6 b 6.3 b 6.4 a 5.7 ab 400 Outside 9.7 a 5.4 b 6.0 a 5.6 ab 800 Outside 6.3 b 5.2 b 4.0 c 4.7 bc 1200 Outside 6.4 b 6.7 b 4.1 c 5.6 ab z Grafted plant s were kept inside or outside of the healing chamber during the healing period. Different letters within and between columns in Fall 1 and Fall 2 indicate significant differences at P Table 2 11. Effects of ABA concentration, application method and location of healing on stomatal conductance, dry weight and scion diameter at 10 days after grafting in Spring 2. ABA concentration (mg/L) Application metho d Location z Stomatal conductance (mmol/m 2 s) Dry weight (g) Scion diameter (mm) 0 Foliar Outside 118.2 a 0.26 a 2.8 a 1000 Foliar Outside 54.4 b 0.21 bc 2.7 a 2000 Foliar Outside 55.9 b 0.19 cd 2.5 ab 0 Root Outside 109.8 a 0.23 abc 2.6 a 236 Root O utside 101.4 a 0.24 ab 2.7 a 473 Root Outside 106.5 a 0.23 abc 2.6 a Non treated Non treated Inside 116.1 a 0.17 d 2.2 b z Grafted plants were kept inside or outside of the healing chamber during the healing period. Different letters within a column in LSD test.

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60 Table 2 12. Effects of ABA concentration, application method and location of healing on leaf area at 11 days after grafting in Spring 1 ABA concentration (mg/L) Application method Location z Leaf area (cm 2 ) 0 Foliar Outside 32.09 c 1000 Foliar Outside 32.64 c 2000 Foliar Outside 33.83 bc 0 Root Outside 33.41 bc 118 Root Outside 37.38 ab 236 Root Outside 34.28 bc Non treated Non treated Inside 41.86 a z Grafted plants wer e kept inside or outside of the healing chamber during the healing period. Different letters within a column indicate significant differences at P LSD test. Table 2 13. Effects of ABA concentration and location of healing on stomatal conductance, dry weight, fresh weight and leaf area at 11 days after grafting in Fall 1 and Fall 2. ABA concentration (mg/L) Location z Fall 1 Fa ll 2 Stomatal conductance (mmol/m 2 s) Dry weight (g) Fresh weight (g) Leaf area (cm 2 ) 0 Inside 86.4 a 0.10 b 1.04 a 20.95 ab 0 Outside 90.6 a 0.13 ab 0.77 bc 15.91 bc 400 Outside 76.0 ab 0.12 ab 0.76 bc 17.94 abc 800 Outside 50.1 c 0.15 a 0.90 ab 22.02 a 1200 Outside 52.9 bc 0.10 b 0.60 c 14.29 c z Grafted plants were kept inside or outside of the healing chamber during the healing period. Different letters within a column indicate significant differences at P LSD test.

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61 Table 2 14. Effects of ABA concentration and location of healing on scion length (mm) at 11 days after grafting in Fall 1 and Fall 2. ABA concentration (mg/L) Location z Fall 1 Fall 2 0 Inside 52 a 60 a 0 Outsid e 46 ab 47 bc 400 Outside 46 ab 46 c 800 Outside 52 a 52 b 1200 Outside 41 b 45 c z Grafted plants were kept inside or outside of the healing chamber during the healing period. Different letters within a column indicate significant differences at P LSD test. Table 2 15. Average air temperature, relative humidity and light intensity inside and outside of the chamber during graft healing (day 1 to 4) in the spring studies. Location z Spring 1 Spring 2 Temperature Relative humidity (%) Light intensity (lum/ft 2 ) Temperature Relative humidity (%) Light intensity (lum/ft 2 ) Inside 21.3 89.9 0 20.8 91.0 Outside 24.7 69.8 1106 26.5 78.9 1194 z Grafted plants were kept inside or outside of the healing chamber during th e healing period. Table 2 16. Average air temperature, relative humidity and light intensity inside and outside of the chamber during graft healing (day 1 to 4) in the fall studies. Location z Fall 1 Fall 2 Temperature Relative humidity (%) Lig ht intensity (lum/ft 2 ) Temperature Relative humidity (%) Light intensity (lum/ft 2 ) Inside, Misting 18.8 68.8 0 19.1 88.6 0 Inside, Floatation 18.6 75.3 0 18.7 89.7 0 Outside 20.3 61.0 137 9 21.5 71.8 81 2 z Grafted plants were kep t inside or outside of the healing chamber with different type s of water application method provided during the healing period.

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62 Table 2 17 Average maximum and minimum air temperature, relative humidity and light intensity inside and outside of the cham ber during graft healing (day 1 to 4) in the spring studies. Location z Spring 1 Spring 2 Tempera ture Relative humidity (%) Light intensity (lum/ft 2 ) Temperature Relative humidity (%) Light intensity (lum/ft 2 ) Max Min Max Min Max Min Max Min Max Min Max Min Inside 25.9 17.9 100.0 63.8 8 0 23.6 19.0 99.7 67.1 0 0 Outside 38.4 17.1 86. 5 50.5 4928 0 39.7 19.3 91.4 57.2 5888 0 z Grafted plants were kept inside or outside of the healing chamber during the healing period. Table 2 18 Average maximum and minimum air temperature, relative humidity and light intensity inside and outside of the chamber during graft he aling (day 1 to 4) in the fall studies. Location z Fall 1 Fall 2 Tempera ture Relative humidity (%) Light intensity (lum/ft 2 ) Temperature Relative humidity (%) Light intensity (lum/ft 2 ) Max Min Max Min Max Min Max Min Max Min Max Min Inside Misting 24.5 15.6 86.1 37.1 4 0 25.1 16.2 99.3 59.1 0 0 Inside, Floa tation 23.4 16.0 90.2 49.6 4 0 23.0 16.4 98.0 68.5 0 0 Outside 34.1 14.9 78.4 31.3 9728 0 40.6 15.4 92.6 26.3 4864 0 z Grafted plants were kept inside or outside of the healing chamber with different type s of water application method provided during th e healing period.

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63 CHAPTER 3 EFFECTS OF EXOGENOUS UNICONAZOLE APPLICATION ON RECOVERY, SURVIVAL, AND QUALITY OF GRAFTED TOMATO TRANSPLANTS Background Although much research has been conducted on how plant growth regulators (PGR) can affect healin g and physiological status of graft union in several plant species, limited information is available with respect to the practical application of PGRs in production of grafted vegetable seedlings (Aloni et al., 2010). In a study by Parkinson and Yeoman (19 82), gibberellin (GA) was found to inhibit callus and vascular formation in tomato. Moreover, GA was reported to be antagonistic to the effect of stomatal closure induced by abscisic acid (ABA). In the study by Aharoni et al. (1977), exogenous application of GA was reported to slow down the rate of stomatal closure in desiccating lettuce leaves with increasing ABA contents. In broad bean leaves, GA application also increased stomatal conductance (Yuan and Xu, 2001). Hence, application of a GA biosynthesis i nhibitor may help to lower stomatal conductance in newly grafted tomato plants, so as to prevent excessive water loss from the scions via transpiration during the healing process. This is especially useful when grafted seedlings are healed without a healin g chamber, or under less desirable environmental conditions. Uniconazole is a GA biosynthesis inhibitor that prevents the oxidation of ent kaurene to ent kaurenoic acid in the GA biosynthesis pathway (Pressman and Shaked, 1991). Saito et al. (2006) repor hydroxylase in Arabidopsis thaliana was competitively inhibited by uniconazole, which resulted in increased accumulation of ABA hydroxylase is a key

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64 enzyme that controls the ca tabolism of ABA to phaseic acid. This increase in endogenous ABA content can reduce stomatal conductance, which could be beneficial during the healing period of grafted tomato transplant production. However, very few researches have examined such an effect in tomato plants. Uniconazole spray applications at 90, 120, 170, and 210 mg/L resulted in higher leaf chlorophyll content in Forsythia x intermedia (Thetford et al., 1995), whereas Shin et al. (2009) reported reduced chlorophyll content following unico nazole application in tomato seedlings. According to Shin et al. (2009), uniconazole application also reduced seedling height, hypocotyl length, fresh weight, dry weight, leaf area, and leaf number. However, in their study, uniconazole was applied by soaki ng tomato seeds, and similar effects may not be present if uniconazole solution is sprayed onto tomato seedlings. Also, relatively high concentrations of 100 and 200 mg/L uniconazole were utilized in their study for seed treatment. The study on Forsythia x intermedia (Thetford et al., 1995) showed that uniconazole application inhibited initiation of xylem and expansion of both phloem and xylem cells. If this is the case in tomato plants, it may affect graft healing and survival in grafted tomato seedlings. This study was aimed to determine the effects of uniconazole application on recovery, survival and quality of grafted tomato seedlings when healed without the use of a healing chamber. Effects of water application method on graft recovery during healing, as mentioned in Chapter 2, will also be evaluated in combination with uniconazole application. In addition, long term effects of uniconazole application on grafted transplant quality will be examined.

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65 Materials and Methods the first and second fall studies, respectively. Scion seeds were sown a day after the rootstock seeds for both studies. Afte r approximately 4 weeks, tomato scions were sprayed with uniconazole solutions at least 24 hours before grafting at a rate of 2 quarts per 100 ft 2 Uniconazole solutions at 0, 0.5, 1.0 and 1.5 mg/L were applied in the Fall 1 study; while concentrations of 0, 0.7, 1.4 and 2.1 mg/L were used in the Fall 2 experiment. Uniconazole solut ions were prepared by diluting S umagic ( Valent U.S.A. Corporation Walnut Creek, CA) with deionized water. All spray solutions contained 0.05% Latron B 1956 as adjuvant. Differ ent uniconazole concentrations were utilized in the second fall trial to determine if further improvements in quality can be achieved at higher uniconazole concentrations, as suggested by results in Fall 1. Tomato plants were splice grafted and placed eit her inside or outside of the healing chamber in the research greenhouse located in Gainesville, FL. Plants kept inside the healing chamber were used as the control, and consisted of scions that received the water application. Scions of grafted plants kept outside received 0, 0.5, 1.0, and 1.5 mg/L uniconazole in Fall 1; and 0, 0.7, 1.4, and 2.1 mg/L uniconazole in Fall 2. Half of those kept inside the chamber was floated, while the other half was misted daily. Plants kept outside were treated similarly. Acc limation of plants healed inside the chamber began on day 5. The number of turgid plants was counted daily for the first 10 days after grafting, while plant quality parameters were measured on day 11. Plant quality parameters included: visual rating (0 Completely wilted, 1 more than 75% of leaf area showing

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66 yellowing and/or severe necrosis, 2 Between 50 and 75% of leaf area showing yellowing and/or moderate necrosis, 3 Between 25 to 50% of leaf area showing yellowing and/or slight necrosis, 4 Les s than 25% of leaf area showing yellowing and/or slight necrosis, 5 No yellow and necrosis), stomatal conductance (using the SC 1 leaf porometer by Decagon Devices, Pullman, WA), chlorophyll content (using the CCM 200 chlorophyll content meter by Opti Sc iences, Hudson, NH), fresh weight, scion stem diameter, leaf area (using the LI 3100 area meter by LI COR Environmental, Lincoln, NE), scion length, dry weight, rootstock stem diameter and leaf number. In Fall 2, dark adapted maximum and minimum chlorophyl l fluorescence readings were determined using an OS1p modulated chlorophyll fluorometer (Opti Sciences, Hudson, NH). In the Fall 1 study, successfully healed seedlings were transplanted into 0.4 L plastic pots and grown on for another 3 weeks in the greenh ouse, after which plant quality parameters mentioned above were recorded again. Eight plants per treatment per replication were grafted for both studies, while 3 plants per treatment per replication were transplanted in Fall 1. There were a total of 4 rep lications. Recovery data were analyzed via logistic regression, while quality data via ANOVA followed by Fisher SAS statistical software package for Windows (version 9.3, Cary, NC). Results and Discussions Stomatal Conductance after Uniconazole Application Fall 1. Plants sprayed with uniconazole solutions at 0.5 and 1.0 mg/L showed significantly lower stomatal conductance at least 24 h after uniconazole application compared to the water control (Table 3 4). This may be due to the reduction of

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67 antagonistic effect on ABA caused by GA (Aharoni et al., 1977) as unico nazole inhibited hydroxylase in the catabolism pathway of ABA (Saito et al.,2006). However, plants that received 1.5 mg/L uniconazole had comparable stomatal conductance as those that received 0 mg/L unic onazole (Table 3 4). This could be a result of the variable nature of stomatal conductance measurement, for it can be affected by other factors like density and size of stomata, in addition to the extent of stomatal opening as determined by uniconazole app lication (Bradford et al., 1983; Decagon Devices; 2012). Fall 2. Similar to the Fall 1 study, application of 0.7, 1.4 and 2.1 mg/L uniconazole solutions in the Fall 2 study also resulted in a significant reduction in stomatal conductance in tomato scion seedlings in contrast to the water control (Table 3 4). Wilting and Recovery from Wilting Fall 1. On the first day after grafting, the greatest proportion of non wilted tomato plants was observed for plants kept inside the chamber compared to all the oth er treatments that were kept outside (Table 3 5). Among plants kept outside, the water spray control treatment had the lowest proportion of turgid plants (Table 3 5), indicating that uniconazole application delayed the onset of drought stress symptoms when grafted tomato seedlings were healed without a healing chamber, which was in line with the lowered stomatal conductance observed in the tomato scion plants after treatment with uniconazole prior to grafting. However, high uniconazole concentration should be avoided as plants that received 1.5 mg/L uniconazole application exhibited significantly lower proportion of non wilted plants compared to the 1.0 mg/L uniconazole treatment (Table 3 5). At day 5, all plants kept outside of the chamber had similar propo rtions of

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68 non wilted plants, which were significantly lower than those of plants kept inside the chamber (Table 3 5). Floated plants also showed better recovery from wilting compared to misted plants (data not shown) At day 6, the recovery of grafts was s imilar between plants kept inside the chamber and those outside the chamber with uniconazole application at 0, 0.5, and 1.0 mg/L. Grafted plants outside the chamber did not differ significantly in recovery, whereas the 1.5 mg/L uniconazole treatment result ed in a significant reduction in recovery compared with plants inside the chamber and plants outside the chamber receiving 1.0 mg/L uniconazole application (Table 3 5). These results implied that although uniconazole application postponed plant wilting ini tially after grafting, it did not show a significant advantage towards the end of graft healing. Nevertheless, the 1.0 mg/L uniconazole treatment appeared to achieve a more comparable result to the control inside the chamber. Moreover, 1.5 mg/L uniconazole application may interfere with recovery. Similar to day 5, floated plants exhibited better recovery compared to misted plants at day 6 (data not shown) Fall 2. Similar results were observed on the day of grafting (day 0) in the Fall 2 study as on the f irst day after grafting in Fall 1. Plants kept inside the chamber had significantly higher proportion of turgid plants compared to those kept outside (Table 3 5). In addition, plants kept outside with 0.7, 1.4 and 2.1 mg/L uniconazole treatments exhibited higher proportions of non wilted plants in comparison with the water control treatment kept outside the chamber (Table 3 5), indicating that uniconazole application delayed wilting. Graft Survival Fall 1. High graft survival rates of more than 90% were o bserved in the Fall 1 study, regardless of healing location and uniconazole application. For instance, about

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69 92% survival was obtained for plants kept inside the chamber, while those kept outside with 0 mg/L uniconazole application had a survival rate of a pproximately 97%. Plants kept outside with 1.0 mg/L uniconazole application achieved about 95% survival. Fall 2. High survival of grafted tomato plants (more than 90%) was also observed in the Fall 2 study. Plants kept inside the chamber and those kept o utside with 0 mg/L uniconazole application had around 95% survival, while plants kept outside with 1.4 mg/L uniconazole application achieved approximately 98% survival. Hence, our results indicated that grafted tomato seedlings can be healed without facili tation of a healing chamber with economically viable survival rates of at least 90%. According to a previous greenhouse study by Johnson and Miles (2011) in Mount Vernon, western Washington, self grafted tomato plants showed a high level of survival regard less of the healing condition differences between shaded open structured chamber and well maintained closed chamber. Also, uniconazole application did not appear to interfere with vascular tissue fo r mation as opposed to that suggested by Thetford et al. (1 995). Quality of Grafted Tomato Seedlings at 11 Days after Grafting Chlorophyll content Fall 1. In the first fall study, plants kept inside the healing chamber had the highest level of chlorophyll content (Table 3 6). Among plants that were kept outside, application of 1.5 mg/L uniconazole resulted in chlorophyll content that was similar to the water spray control, while the 0.5 mg/L uniconazole treatment significantly reduced chlorophyll content as compared to the water control and the 1.5 mg/L uniconazo le treatment (Table 3 6). Moreover, chlorophyll contents of plants sprayed with 0.5 and 1.0 mg/L uniconazole were lower than that of plants which received 1.5 mg/L uniconazole. Results from this study indicated that chlorophyll content appeared to increase with

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70 increasing uniconazole concentration, although it is unclear if concentrations above 1.5 mg/L can result in plant chlorophyll levels that are comparable to that of plants kept inside the chamber. In order to explore the higher concentration effect of uniconazole application, the concentrations of uniconazole were increased in the second fall study. Fall 2. In contrast to the Fall 1 study, no significant treatment differences were detected in chlorophyll content among different treatments in the Fall 2 study. This discrepancy may not be surprising given that the average and mean maximum light intensity was lower while the average and mean maximum relative humidity was higher in Fall 2 than in Fall 1 (Table 2 16 and table 2 18 ). According to Shin et al. (2009), the effect of uniconazole application may also vary depending upon the endogenous concentrations of cytokinins and disparate accumulation of uniconazole in leaf tissues. Leaf number Fall 1. Leaf number was not affected by the treatments in the fi rst fall study (Table 3 1). Fall 2. Leaf number was similar among plants kept inside the chamber, and those kept outside with 0 and 1.4 mg/L uniconazole applications (Table 3 6). Plants kept outside exhibited similar leaf numbers regardless of uniconazole application (Table 3 6), indicating that uniconazole application did not negatively impact leaf number. Leaf area Fall 1. Treatments did not affect leaf area in the first fall study (Table 3 1). Fall 2. Plants kept inside the chamber and those kept out side with 1.4 mg/L uniconazole application had comparable leaf areas that were larger than that of plants kept outside with 0.7 and 2.1 mg/L uniconazole applications (Table 3 6). High concentration of uniconazole application (i.e. 2.1 mg/L) might reduce le af area in grafted

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71 tomato seedlings. This was similar to that reported by Shin et al. (2009) and Wang and Gregg (1990) in non grafted tomato. Scion length Fall 1. In the first fall study, plants that were kept outside and sprayed with either water or 0.5 mg/L uniconazole had similar scion length compared with those that were kept inside the chamber (Table 3 6). Scion lengths of plants kept outside of the chamber and sprayed with either 1.0 or 1.5 mg/L uniconazole were significantly lower than that of plan ts kept inside (Table 3 6). This effect of plant height suppression after uniconazole application was also reported by Shin et al. (2009) and Wang and Gregg (1990) in tomato seedlings, and by Wang and Gregg (1991) in hibiscus. Fall 2. Interactions betwee n uniconazole concentration, healing location and water application method during healing were observed in the Fall 2 study (Table 3 2). For plants kept inside the chamber, those that were floated had significantly greater scion length compared to those th at were misted (Table 3 7). Among plants that were floated, those kept inside the chamber had significantly longer scion length compared to those kept outside (Table 3 7). For plants that were kept outside and floated, those that received 0, 0.7 and 1.4 mg /L uniconazole did not differ significantly in scion length, which were longer than when 2.1 mg/L uniconazole was applied (Table 3 7). Among the misting treatments, plants that were kept outside had similar scion lengths, which were lower than that of plan ts kept inside the chamber (Table 3 7). These results indicated that uniconazole application at 2.1 mg/L in combination with floatation during healing inhibited scion stem elongation. This is not surprising as uniconazole is a GA biosynthesis inhibitor (Pr essman and Shaked, 1991).

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72 Scion fresh weight Fall 1. Scion fresh weight did not differ significantly among different treatments (Table 3 1). Fall 2. In the second fall study, fresh weight of scions kept inside the chamber was the highest among all the tr eatments, while plants kept outside with 1.4 mg/L uniconazole application showed a similar level of fresh weight as plants kept inside the chamber (Table 3 6). Plants kept outside of the chamber with 2.1, 0.7 or 0 mg/L uniconazole applications exhibited si gnificantly lower scion fresh weights compared to those kept inside (Table 3 6). Spray application of high uniconazole concentration (e.g. 2.1 mg/L) may reduce scion fresh weight of grafted tomato seedlings. Wang and Gregg (1990) reported that fresh weight of tomato seedlings was reduced 2 weeks after soil uniconazole applications at 12.5, 25, 50, 100, 200 and 400 g per plant compared to when 0 g per plant was applied. Scion dry weight Fall 1. No significant treatment differences were observed in terms of scion dry weight in the first fall study (Table 3 1). Fall 2. Plants that received 1.4 mg/L uniconazole application and kept outside exhibited significantly higher scion dry weight compared to all other treatments (Table 3 6). The increase in plant dr y weight as a result of uniconazole application observed in this study was contrary to the dry weight reductions reported by Shin et al. (2009) and Wang and Gregg (1990) in tomato seedlings following uniconazole applications. Visual rating Fall 1. In the Fall 1 study, grafted plants that were kept inside the healing chamber had the highest visual rating compared to the other treatments (Table 3 8).

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73 When plants were placed outside, those that received 0 and 1.5 mg/L uniconazole had similar ratings that wer e significantly higher than the 0.5 and 1.0 mg/L uniconazole treatments (Table 3 8). The visual rating results were similar to that of chlorophyll measurements. Floated plants also had higher ratings compared to plants that were misted (data not shown) F all 2. Similar to the first fall study, plants kept inside the chamber had the highest visual rating compared to those kept outside in the second fall study (Table 3 8). Among plants that were kept outside, those treated with 0.7, 1.4 and 2.1 mg/L uniconaz ole exhibited similar ratings (Table 3 8). Without facilitation of a healing chamber, uniconazole application at 0.7 mg/L resulted in a significant improvement in visual quality of grafted plants as compared to the water control. Plants that were kept outs ide with 0 mg/L uniconazole application had comparable visual rating to those kept outside with 1.4 or 2.1 mg/L uniconazole applications (Table 3 8). Chlorophyll fluorescence Fall 2. Chlorophyll fluorescence was only measured in the Fall 2 study. The F v / F m ratio did not differ significantly among different treatments (Table 3 2) indicating that healing grafted plants outside the chamber and uniconazole application did not affect the maximum efficiency of photosystem II. However, it must be stressed that the light intensity in the greenhouse during this study was relatively low, and different results may be observed under higher levels of solar radiation during the spring season. Other plant quality parameters In both fall studies, the uniconazole treatm ents did not show any significant impact on scion diameter, rootstock diameter, and stomatal conductance of grafted tomato plants at 11 days after grafting (Table 3 1 and table 3 2)

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74 Grafted Tomato Plant Quality at 3 Weeks after Transplanting Leaf number The leaf number of grafted tomato plants at 3 weeks after transplanting was affected by significant interactions between uniconazole concentration, healing location and water application method during healing (Table 3 3). Plants kept outside and sprayed wi th 0.5, 1.0 or 1.5 mg/L uniconazole had similar leaf number, regardless of water application method (Table 3 9). When compared to floated plants kept inside the chamber, plants that were floated and kept outside with 1.0 mg/L uniconazole application had si gnificantly higher leaf number (Table 3 9). However, when plants were misted, all the treatments kept outside the chamber resulted in similar plant leaf numbers as those kept inside the chamber (Table 3 9). Leaf area Interactions between uniconazole con centration, healing location and water application method during healing were observed with respect to the leaf area of grafted tomato plants at 3 weeks after transplanting (Table 3 3). Plants kept inside the chamber had similar leaf area as those kept out side with 0.5, 1.0 or 1.5 mg/L uniconazole applications, regardless of water application method (Table 3 9). This suggested that uniconazole applications at 0.5, 1.0 and 1.5 mg/L did not affect leaf area of plants healed outside the chamber at 3 weeks post transplanting. When floated, plants kept inside the chamber with 0 mg/L uniconazole application exhibited significantly higher leaf area compared to the water control treatment kept outside the chamber (Table 3 9). Conversely, when misted, plants kept out side the chamber with 0 mg/L uniconazole application had significantly higher leaf area compared to plants that were kept inside (Table 3 6). Interestingly, among plants that were kept outside the chamber with 0 mg/L

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75 uniconazole application, those that rec eived the mist treatment had significantly higher leaf area compared to those that were floated during healing (Table 3 9). Water application method during healing may have long term impacts on plant leaf area in the absence of growth regulator application when grafted tomato plants are healed without facilitation of a healing chamber. Other plant quality parameters Uniconazole application, healing location and water application method during healing did not show any significant impacts on chlorophyll conte nt, scion length, scion diameter, rootstock diameter, fresh weight, dry weight, and stomatal conductance of grafted tomato plants at 3 weeks after transplanting (Table 3 3) Summary Uniconazole application reduced stomatal conductance in tomato scions pri or to grafting, which helped delay wilting of grafted seedlings, thereby reducing the period of drought stress during the graft healing process. However, uniconazole application did not aid in subsequent recovery from wilting in the transplants, nor did it improve graft survival rate. Although plants healed outside the chamber with 1.4 or 1.5 mg/L uniconazole applications achieved comparable results to those kept inside the chamber for a few quality parameters 11 days after grafting, improvements in plant q uality, as a whole, were not as apparent as when ABA was used. One of the benefits of uniconazole application is the reduction of scion length, which may enable grafted seedlings to better withstand wind damage after being transplanted into the field. Unic onazole application prior to grafting had negligible negative effects on post transplant growth of grafted tomato seedlings. The synergistic effects of combined ABA

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76 and uniconazole applications on graft survival and quality of grafted tomato transplants ma y be explored in future studies.

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77 Table 3 1. Analysis of variance on the effects of uniconazole (UN) concentration, healing location, and water application method during healing on grafted tomato transplant quality at 11 days after grafti ng in the Fall 1 study. Effects Chlorophyll c ontent Scion d iameter Rootstock d iameter Leaf n umber Fresh w eight Dry w eight Scion l ength Leaf a rea Stomatal c onductance Visual r ating UN concentration x healing location NS NS NS NS NS NS NS Water application method NS NS NS NS NS NS NS NS NS UN concentration x healing location x water application method NS NS NS NS NS NS NS NS NS NS and NS indicate significance and non significance, respectively, at P Table 3 2. Analysis of variance on the effects of uniconazole (UN) concentration, healing location, and water application method during healing on grafted tomato transplant quality at 11 days after grafting in the Fall 2 study. Effects Chlorophy ll c ontent Scion d iameter Rootstock d iameter Leaf n umber Fresh w eight Dry w eight Scion l ength Leaf a rea Stomatal c onductance Visual r ating Chlorophyll f luorescence UN concentration x healing location NS NS NS * NS NS Water application method NS NS NS NS NS NS NS NS NS NS NS UN concentration x healing location x water application method NS NS NS NS NS NS NS NS NS NS and NS indicate significance and non significance, respectively, at P

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78 Table 3 3. Analysis of variance on the effects of uniconazole (UN) concentration, healing location, and water application method during healing on grafted tomato transplant quality at 3 weeks after transplanting. Effects Chloroph yll c ontent Scion d iameter Rootstock d iameter Leaf n umber Fresh w eight Dry w eight Scion l ength Leaf a rea Stomatal c onductance UN concentration x healing location NS NS NS NS NS NS NS NS NS Water application method NS NS NS NS NS NS NS NS UN concentration x healing location x water application method NS NS NS NS NS NS NS and NS indicate significance and non significance, respectively, at P Table 3 4. Effects of uniconazole concentration on stomatal conductance (mmol/m 2 s) of application in both fall studies. Uniconazole concentration (mg/L) Fall 1 Fall 2 0 3 79.1 a 102.5 a 0.5/0.7 z 255.9 b 53.7 b 1.0/1.4 y 272.5 b 66.7 b 1.5/2.1 x 296.6 ab 64.9 b z 0.5 mg/L uniconazole was applied in Fall 1, while 0.7 mg/L uniconazole was applied in Fall 2. y 1.0 mg/L uniconazole was applied in Fall 1, while 1.4 mg/L uniconaz ole was applied in Fall 2. x 1.5 mg/L uniconazole was applied in Fall 1, while 2.1 mg/L uniconazole was applied in Fall 2. Different letters within a column indicate significant differences at P LSD test.

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79 Table 3 5. Effects of uniconazole concentration and location of healing on proportion of non wilted tomato plants after grafting in both fall studies. Uniconazole concentration (mg/L) Location z Fall 1 Fall 2 Day 1 Day 5 Day 6 Day 0 0 Inside 0.96 a v 0.81 a 0.84 a 0.94 a 0 Outside 0.03 d 0.28 b 0.69 ab 0.14 d 0.5/0.7 y Outside 0.26 bc 0.23 b 0.67 ab 0.42 c 1.0/1.4 x Outside 0.42 b 0.34 b 0.82 a 0.59 bc 1.5/2.1 w Outside 0.18 c 0.30 b 0.62 b 0.64 b z Grafted tomato plants were kept inside or outside of the healing chamber during the healing period. y 0.5 mg/L uniconazole was applied in fall 1, while 0.7 mg/L uniconazole was applied in Fall 2. x 1.0 mg/L uniconazole was applied in fall 1, while 1.4 mg/L uniconaz ole was applied in Fall 2. w 1.5 mg/L uniconazole was applied in fall 1, while 2.1 mg/L uniconazole was applied in Fall 2. v 96% of grafted plants was not wilted for this treatment. Different letters within a column indicate significant differences at P LSD test.

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80 Table 3 6. Effects of uniconazole concentration and healing location on quality parameters of grafted tomato seedlings at 11 days after grafting in both fall studies. Uniconazole concentration (mg/L) Lo cation z Fall 1 Fall 2 Chlorophyll content (CCI y ) Scion length (mm) Leaf number Fresh weight (g) Leaf area (cm 2 ) Dry weight (g) 0 Inside 9.5 a 52 a 3.7 a 1.04 a 20.95 a 0.12 b 0 Outside 6.5 bc 46 ab 3.3 ab 0.77 bc 15.91 bc 0.11 b 0.5/0.7 x Outside 4.4 d 46 ab 3.1 b 0.73 bc 15.11 c 0.11 b 1.0/1.4 w Outside 5.2 cd 42 b 3.4 ab 0.90 ab 20.25 ab 0.15 a 1.5/2.1 v Outside 6.9 b 42 b 3.1 b 0.65 c 13.65 c 0.10 b z Grafted plants were kept inside or outside of the healing chamber during the healing period. y Chlorophyll concentration index x 0.5 mg/L uniconazole was applied in Fall 1, while 0.7 mg/L uniconazole was applied in Fall 2. w 1.0 mg/L uniconazole was applied in Fall 1, while 1.4 mg/L uniconazole was applied in Fall 2. v 1.5 mg/L uniconazole was appl ied in Fall 1, while 2.1 mg/L uniconazole was applied in Fall 2. LSD test.

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81 Table 3 7. Effects of uniconazole concentration, healing location, and water application method during healing on scion length (mm) of graf ted tomato seedlings at 11 days after grafting in the Fall 2 study. Uniconazole concentration (mg/L) Location z Floatation Misting 0 Inside 63 a 57 b 0 Outside 48 c 45 cd 0.7 Outside 45 cd 4 0 de 1.4 Outside 43 cd 42 de 2.1 Outside 36 e 46 cd z Grafted plants were kept inside or outside of the healing chamber during the healing period. Different letters within and between columns indicate significant differences at P Table 3 8. Effects of uniconazole concentration and healing location on visual ratings z of grafted tomato seedlings at 11 days after grafting in both fall studies. Uniconazole concentration (mg/L) Location y Fall 1 Fall 2 0 Inside 4.0 a 4.0 a 0 Outside 3.2 b 2.9 c 0.5/0.7 x Outside 2.6 c 3.4 b 1.0/1.4 w Outside 2.7 c 3.3 bc 1.5/2.1 v Outside 3.3 b 3.0 bc z Plants were rated on a scale from 0 to 5 with 5 being assigned to plants with the best visual quality y Grafted plant s were kept inside or outside of the healing chamber during the healing period. x 0.5 mg/L uniconazole was applied in Fall 1, while 0.7 mg/L uniconazole was applied in Fall 2. w 1.0 mg/L uniconazole was applied in Fall 1, while 1.4 mg/L uniconazole was appl ied in Fall 2. v 1.5 mg/L uniconazole was applied in Fall 1, while 2.1 mg/L uniconazole was applied in Fall 2. LSD test.

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82 Table 3 9. Effects of uniconazole concentration, location of healing, and water application method during healing on leaf number and leaf area of grafted tomato plants at 3 weeks after transplanting. Uniconazole concentration (mg/L) Location z Leaf number Leaf area (cm 2 ) Floatation Mis ting Floatation Misting 0 Inside 10.8 cd 11.5 abc 346.06 ab 321.45 bc 0 Outside 10.5 d 12.3 a 296.43 c 376.17 a 0.5 Outside 11.5 abc 11.3 bcd 315.68 bc 320.34 bc 1.0 Outside 11.9 ab 12.0 ab 340.70 abc 336.61 abc 1.5 Outside 11.4 abcd 11.1 bcd 31 9.31 bc 324.26 bc z Grafted plants were kept inside or outside of the healing chamber during the healing period. Different letters within and between columns under leaf number and leaf area indicate

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83 CHAPTER 4 CONCLUSIONS Tomato seedlings can be easily grafted, as demonstrated by high survival rates of more than 90% when newly grafted seedlings were placed outside of the healing chamber in the greenhouse without any growth regulator application. This suggested that grafted tomato tra nsplants can be healed successfully under sub optimal healing conditions although provision of shade is advisable to prevent excessive wilting and water loss following grafting. Based on our studies, tom ato plants appeared to absorb ABA better via their leaves compared to from their roots. Moreover, ABA ap plied to the roots can be broken down by microbes in the media, resulting in lesser amount of ABA available for uptake by the scions Hence, foliar appl ication of ABA is recommended, for lesser quantity of ABA will be required to bring about the desired effects, which will translate to greater cost savings for growers. Foliar application may also be more convenient to administer. Although ABA application to the tomato scion prior to grafting did not enhance the final survival rate of grafted tomato transplants, it resulted in delayed wilting due to the decrease in stomatal conductance, and subsequent faster recovery from wilting in grafted seedlings kept o utside of the healing chamber. This will reduce the period of drought stress experienced by the scions, and may also reduce the time needed for complete healing. It would be interesting to monitor the effect of ABA application in future studies with respec t to its role in formation of vascular connection between scion and rootstock. Results from this project also suggested that ABA spray concentrations below 800 mg/L should be used to avoid negative impacts on tomato transplant quality

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84 due to phytotoxicity. ABA application can result in reduced scion length, which may help grafted tomato seedlings better withstand wind damage following field transplant. Uniconazole application to the tomato scion prior to grafting did not improve recovery, but delayed the i nitial wilting of newly grafted transplants. No significant improvements in transplant quality were observed following uniconazole application. Treating grafted tomato seedlings with uniconazole at 2.1 mg/L may shorten scion length due to the inhibition of GA biosynthesis. Growth regulator application did not impact grafted seedling growth after transplanting. Field studies may be conducted in the future to assess the effect of growth regulation application on yield potential of grafted plants. Combination of both ABA and uniconazole applications may yield interesting results in terms of recovery, survival and quality of grafted tomato transplants. In addition, since grafted tomato seedlings are relatively easy to heal, improvements in grafted transplant re covery, survival and quality following growth regulator application may be more apparent in vegetable species that are considered to be more difficult to graft, such as watermelon, melon, and cucumber in the Cucurbitaceae family.

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85 LIST OF REFERENCES Aha roni, N., A. Blumenfeld, and A. E. Richmond. 1977. Hormonal activity in detached lettuce leaves as affected by leaf water content. Plant Physiol. 59: 1169 1173. Al aghabary, K., Z. Zhu, and Q. Shi. 2007. Influence of silicon supply on chlorophyll con tent, chlorophyll fluorescence, and antioxidative enzyme activities in tomato plants under salt stress. Journal of Plant Nutrition 27:2101 2115. Aloni, R. 1980. Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tis sue cultures. Planta 150:255 263. Aloni, R. 1987. Differentiation of vascular tissues. Am. Rev. Plant Physiol. 38:179 204. Aloni, R. 1993. The role of cytokinin in organiz ed differentiation of vascular tissues. Aust. J. Plant Physiol. 20:601 608. Alo ni, B., R. Cohen, L. Karni, H. Aktas, and M. Edelstein. 2010. Hormonal signaling in rootstock scion interactions. Scientia Horticulturae 127:119 126. Asahina, M., H. Iwai, A. Kikuchi, S. Yamaguchi, Y. Kamiya, H. Kamada, and S. Satoh. 2002. Gibberellin pr oduced in the cotyledon is required for cell division during tissue reunion in the cortex of cut cucumber and tomato hypocotyls. Plant Physiology 129:201 210. Bradford, K.J., T.D. Sharkey, and G.D. Farquhar. 1983. Gas exchange, stomatal 13 C values of the flacca tomato mutant in relation to abscisic acid. Plant Physiol. 72:245 250. Dayan, J., N. Voronin, F. Gong, T. Sun, P. Hedden, H. Fromm, and R. Aloni. 2012. Leaf induced gibberellin signaling is essential for internode elongation, cambi al activity, and fiber differentiation in tobacco stems. Plant Cell 24:66 79. Decagon Devices. 2012. Leaf porometer user manual. Decagon Devices, Inc., Pullman, WA Dodd, I.C. 2003. Hormonal interactions and stomatal responses. J. Plant Growth Regul. 22 :32 46. Dodd, I.C., R. Stikic, and W.J. Davies. 1996. Chemical regulation of gas exchange and growth of plants in drying soil in the field. Journal of Experimental Botany 47:1475 1490. Fernandez Garcia, N., M. Carvajal, and E. Olmos. 2004. Graft union formation in tomato plants: peroxidase and catalase involvement. Annals of Botany 93:53 60.

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86 Frantz, J.M. and G.E. Welbaum. 1998. Producing horticultural crops using hydroponic tobacco transplant systems. HortTechnology 8:392 395. Frantz, J. M., G.E. We lbaum, Z. Shen, and R. Morse. 1998. Comparison of cabbage seedling growth in four transplant production systems. HortScience 33:976 979. Goreta, S., D.I. Leskovar, and J.L. Jifon. 2007. Gas exchange, water status, and growth of pepper seedlings exposed t o transient water deficit stress are differentially altered by antitranspirants. J. Amer. Soc. Hort. Sci. 132:603 610. Haisel, D., J. Pospisilova, H. Synkova, R. Schnablova, and P. Batkova. 2006. Effects of abscisic acid or benzyladenine on pigment conte nts, chlorophyll fluorescence, and chloroplast ultrastructure during water stress and after rehydration. Photosynthetica 44:606 614. Hartmann, H.T., D.E. Kester, F.T. Davies, and R.L. Geneve. 2002. Plant propagation principles and practices. 7 th ed. Pren tice Hall, Upper Saddle River, NJ. Jacobs, W .P. 1952. The role of auxin in differentiation of xylem around a wound. American Journal of Botany 39:301 309. Jeffree, C.E. and M.M. Yeoman. 1983. Development of intercellular connections between opposing ce lls in a graft union. New Phytol. 93:491 509. Johnson, S.J. and C.A. Miles. 2011. Effect of healing chamber design on the survival of grafted eggplant, tomato, and watermelon. HortTechnology 21:752 758. Jung, S. and K.L. Steffen. 1997. Influence of pho tosynthetic photon flux densities before and during long term chilling on xanthophyll cycle and chlorophyll fluorescence quenching in leaves of tomato ( Lycopersicon hirsutum ). Physiologia Plantarum 100:958 966. Kalev, N. and R. Aloni. 1998. Role of auxin and gibberellin in regenerative differentiation of tracheids in Pinus pinea seedlings. New Phytologist 138:461 468. Kubota, C., M.A. McClure, N. Kokalis Burelle, M.G. Bausher, and E.N. Rosskopf. 2008. Vegetable grafting: history, use, and current techno logy status in North America. HortScience 43:1664 1669. Lee, J. 1994. Cultivation of grafted vegetables I. Current status, grafting methods, and benefits. HortScience 29:235 239. Lee, J., C. Kubota, S.J. Tsao, Z. Bie, P. Hoyos Echevarria, L. Morra and M. Oda. 2010. Current status of vegetable grafting: diffusion, grafting techniques, automation. Scientia Horticulturae 127:93 105.

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87 Leonardi, C. and D. Romano. 2004. Recent issues on vegetable grafting. Acta Hort. 631:163 174. Maxwell, K. and G. N. John son. 2000. Chlorophyll fluorescence a practical guide. Journal of Experimental Botany 51:659 668. Oda, M. 1995. New grafting methods for fruit bearing vegetables in Japan. JARQ 29:187 194. Oda, M. 2002. Grafting of vegetable crops. Sci. Rep. Agric. & Biol. Sci., Osaka Pref. Univ. 54:49 72. Parkinson, M. and M.M. Yeoman. 1982. Graft formation in cultured, explanted internodes. New Phytologist 91:711 719. Pearce, D., A.R. Miller, L.W. Robert, and R.P. Pharis. 1987. Gibberellin mediated synergism of xylogenesis in lettuce pith cultures. Plant Physiol. 84:1121 1125. Pospisilova, J. 2003a. Participation of phytohormones in the stomatal regulation of gas exchange during water stress. Biologia Plantarum 46:491 506. Pospisilova, J. 2003b. Interaction o f cytokinins and abscisic acid during regulation of stomatal opening in bean leaves. Photosynthetica 41:49 56. Pospisilova, J. and P. Batkova. 2004. Effects of pre treatments with abscisic acid and/or benzyladenine on gas exchange of French bean, sugar beet, and maize leaves during water stress and after rehydration. Biologia Plantarum 48:395 399. Pressman, E. and R. Shaked. 1991. Interactive effects of GAs, CKs and growth retardants on the germination of celery seeds. Plant Growth Regulation 10:65 72. Ragni, L., K. Nieminen, D. Pacheco Villalobos, R. Sibout, C. Schwechheimer, and C.S. Hardtke. 2011. Mobile gibberellin directly stimulates Arabidopsis hypocotyl xylem expansion. Plant Cell 23:1322 1336. Saito, S., N. Hirai, C. Matsumoto, H. Ohigashi, D Ohta, K. Sakata, and M. Mizutani. 2004. Arabidopsis CYP707As encode (+) hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid. Plant Physiol. 134:1439 1449. Saito, S., M. Okamoto, S. Shinoda, T. Kushiro, T. Koshiba, Y. Kamiya, N. Hirai, Y. Todoroki, K. Sakata, E. Nambara, and M. Mizutani. 2006. A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis Biosci. Biotechnol. Biochem. 70: 1731 1739.

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88 Savidge, R.A. 1983. The role of plant hormones in higher plant cellular differentiation. II. Experiments with the vascular cambium, and sclereid and tracheid differentiation in the pine, Pinus contorta Histochemical Journal 15:447 466. Shin, W.G., S.J. Hwang, I. Sivanesan, and B.R. Jeong. 2 009. Height suppression of tomato plug seedlings by an environment friendly seed treatment of plant growth retardants. African Journal of Biotechnology 8:4100 4107. Thetford, M., S.L. Warren, F.A. Blazich, and J.F. Thomas. 1995. Response of Forsythia in termedia and photosynthesis. J. Amer. Soc. Hort. Sci. 120:983 988. Wang, Y. and L.L. Gregg. 1990. Uniconazole controls growth and yield of greenhouse tomato. Scientia Horticulturae 43: 55 62. Wang, Y. and L.L. Gregg. 1991. Modification of hibiscus growth by treating unrooted cuttings and potted plants with uniconazole or paclobutrazol. J. Plant Growth Regul. 10:47 51. Webb, A.A.R., M.G. Larman, L.T. Montgomery, J.E. Taylor, and A.M. Hetherington. 2001. The role of calcium in ABA induced gene expression and stomatal movements. The Plant Journal 26:351 362. Wilkinson, S., J.E. Corlett, L. Oger, and W.J. Davies. 1998. Effects of xylem pH on transpiration from wild type and flacca tomato leaves. Plant Physiol. 117:703 709. Yuan, L. and D. Xu. 2001. Stimulation effect of gibberellic acid short term treatment on leaf photosynthesis related to the increase in Rubisco content in broad bean and soybean. Photosynthesis Research 68:39 47.

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89 BIOGRAPHICAL SKETCH He began his undergraduate study at the University of Florida in 2009, and wa s awarded a Bachelor of Science (Summa Cum Laude) in Horticultural Sciences in 2011. His undergraduate thesis research was on how different grafting positi ons can affect graft survival and production of rootstock suckers in grafted tomato seedlings.