Optimization of acclimatization and storage conditions to improve graft take ratio and seedling quality in tomato plug production syste

The high relative humidity 90% for first 2 or 3 days and afterwards reduced low relative humidity 70% at 23oC condition during healing and acclimatization promoted the graft-take and qua

Dissertation for the Degree

of Doctor

Optimization of Acclimatization and Storage Conditions to

Improve Graft-take Ratio and Seedling Quality

in Tomato Plug Production System

by

Vu Ngoc Thang

Department of Horticulture

Graduate School Kangwon National University Republic of Korea

August, 2015

Supervised by

Professor Kim Il Seop

Optimization of Acclimatization and Storage Conditions to

Improve Graft-take Ratio and Seedling Quality

in Tomato Plug Production System

Approved by Committee of the Graduate School of Kangwon National University in Partial Fulfillment of the

Requirement for Degree of Doctor of Agriculture

i

Optimization of Acclimatization and Storage Conditions to

Improve Graft-take Ratio and Seedling Quality

in Tomato Plug Production System

Vu Ngoc Thang

Department of Horticulture Graduate School, Kangwon National University

Abstract

Although many grafting methods and techniques for environmental control during grafting process are widely recognized, many other factors must be carefully considered to ensure successful grafting process with this technology In order to enhance graft-take ratio and quality of tomato seedlings, control environmental conditions were studied in second chapter In this chapter, control temperature, humidity was examined in first study The higher graft-take ratios (84.0-87.4%) were showed at 23oC compared to 20oC and 26oC in all rootstock cultivars Graft-take ratios decreased and percentage of infected plants increased at high temperature The graft-take ratios increased with increasing relative humidity in all temperature levels However, increasing relative humidity significantly increased percentage of infected plants The graft-take ratio increased with increasing period

of 90% relative humidity, but it decreased with continued increasing 90% relative humidity for 10 days The high relative humidity (90%) for first 2 or 3 days and afterwards reduced low relative humidity (70%) at 23oC condition during healing and acclimatization promoted the graft-take and quality of grafted tomato seedlings From above optimal temperature and humidity, effect of water content in substrate,

ii

grafting position and different cultivars on the graft-take ratio and quality of tomato seedlings were investigated The graft-take ratio and seedling quality were improved by control of water content in the substrate The maximum graft-take ratio (100%) with the highest compactness value was observed in seedlings which were grafted by scion mid water content and rootstock high water content in the substrate There was no significant difference in graft-take ratio between two grafting positions but grafting position effected on growth characteristics of grafted tomato seedlings There was significant different among scion cultivars but not significant different among rootstock cultivars with graft-take ratio In addition, influence of short-term irradiation by light quality on the graft-take ratio and quality of tomato seedlings was also investigated When short-term irradiation was applied before grafting, the graft-take ratios (27.8-66.7%) were considerably low in all light treatments as compared with natural light (96.7%) The graft-take ratio of red LED was not statically different with WFL treatment, but higher than far-red and blue LED treatments The lowest graft-take ratio (27.8%) was observed in darkness treatment Changing light intensity before grafting was the cause of reduced graft-take ratios in this study There was no significant difference among natural light, WFL, and red LED treatments in growth parameters, except for leaf chlorophyll level, leaf width, and fresh weight of root, but decreased in seedlings treated with far-red LED, blue LED, and darkness Graft-take ratios (68.5-100.0%) were enhanced when short-term irradiation was applied after grafting The maximum (100%) graft-take ratio was recorded in red LED treatment, but was not statistically different with the WFL treatment The lowest graft-take ratio was also observed in the darkness treatment Plant growth responses to red LED were also similar with those to WFL after grafting However, when short-term irradiation was applied after grafting, the lowest values of plant growth were observed in far-red LED treatment The plant growth parameters were similar in seedlings treated with darkness and blue LED, but lower than red LED and WFL treatments The root

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morphology was improved in seedlings treated with red LED after grafting by increasing total root surface, total root length, and number of root tips Seedling quality increased at 35 days after transplanting in the red LED treatment by increased plant growth parameters, especially compactness and root morphology,

as compared with other treatments In order to confirm effect of red LED on take ratio and seedling quality, red LED and no light (darkness) were examined on three rootstock cultivars The result showed that graft-take ratios in red LED were higher than those in no light treatment Significant variation on graft-take ratio of rootstock cultivars was observed in no light treatment but there was not significant variation in red LED treatment Red LED treatment also reduced the percentage of infected plants Seedling quality in red LED was better than that in no light treatment by improving growth parameters

graft-Tomato seedling quality is highly valued in Korea vegetable production Ideally, tomato seedlings are transplanted when they reach the correct size, but seedlings are often ready before grower can transplant them Therefore, growth must be slowed or delayed Low temperature storage can be used to stop or suppress the growth and development of tomato seedlings while preserving its quality and not adversely affecting future growth Therefore, effect of low storage temperature on growth and quality of tomato seedlings was investigated Inhibition

of seedling elongation was observed in all low storage temperature levels However,

4oC temperature was not suitable for long-term storage of tomato seedlings Plant height and number of leaves decreased with decreasing storage temperature Leaf chlorophyll values decreased with increasing storage duration However, above 10 days storage duration leaf chlorophyll value of seedlings at nature condition was lower than that at 13oC temperature level, but it was higher than that at 10oC and

7oC temperature level Chilling injury and curly leaf were observed in seedlings at

10, 7oC temperature after 10 or 20 days storage, but they were not observed in seedlings at 13oC temperature The time of expanding leaf and chilling injury index

iv

of seedlings at 7 and 10oC temperatures increased with increasing storage duration and decreasing storage temperature The delay flowering was observed in all low temperature treatments Delay flowering time increased with increasing storage duration and decreasing storage temperature After 10 or 20 days storage, growth parameters of tomato seedlings decreased significantly with decreasing storage temperature, except leaf chlorophyll value, specific leaf area and average root diameter However, there were not statistically different in seedlings at 10 and 7oC with growth parameter except plant height, leaf area and specific leaf area Although growth parameters decreased significantly with decreasing storage temperature, but after 10 days storage the compactness of seedlings at 13, 10 and

7oC were similar to that at nature condition However, after 20 days storage the compactness of seedlings decreased significantly in low storage temperature

In order to improve seedling quality of tomato in long-term storage duration

at low temperature, effect of silicon and ABA on growth and abiotic stresses and examination of silicon and ABA’s ability to maintain the seedling quality of tomato

at low storage temperature were investigated in the fourth chapter Although silicon (Si) has not been listed among the essential elements for plant growth, however, the beneficial role of silicon in stimulating the growth and development of many plant species has been generally recognized Silicon is also known to effectively mitigate various abiotic stresses Based on this study, silicon stimulated the growth and development of tomato seedlings by increasing growth parameters, root morphology Transpiration rate decreased and stomatal diffusive resistance increased with increasing silicon concentrations to 32 mM Silicon could migrate to reduce chilling injury index during low temperature by reducing damage of leaf area The plant hormone abscisic acid (ABA) is an important regulator in many aspects of plant growth and development, as well as stress resistance Therefore, in the other studies, foliar application of ABA affected in reducing growth characteristics of tomato seedlings However, ABA enhanced cold tolerance in

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tomato seedlings by reducing relative ion leakage and chilling injury index in low temperature In addition, ABA also enhanced drought tolerance in tomato seedlings

by decreasing transpiration rate in the leaf therefore ABA delaying the starting time

of wilting point in drought condition Although lower transpiration rate and higher stomatal diffusive resistance in 50 mg•L-1 treatment compared with control but transpiration rate and stomatal diffusive resistance in 50 mg•L-1 of ABA treatment were much higher and lower than those of 100 mg•L-1 of ABA treatment Therefore

in the control treatment the start time of wilting point was observed on the third day, but in 50 and 100 mg•L-1 of ABA treatments the start time of wilting point were observed on the fifth and seventh day after without irrigation, respectively Based

on above results, 16 mM of silicon and 100 mg•L-1 of ABA were applied for tomato seedlings before storage The result shown that during storage duration plant height

of tomato seedlings increased in silicon but decreased in ABA treatment Number

of leaf was similar between non-treatment and silicon treatment in the same storage duration, but they decreased significantly in ABA treatment The leaf chlorophyll content of seedlings at 7oC was lower than those at 10oC in all treatments during storage duration, but leaf chlorophyll content of seedlings in ABA treatment was higher than that in silicon and non-treatment Silicon and ABA improved seedlings quality in low temperature by reducing damage of leaf area However, chilling injury index of seedlings in ABA treatment was lower than that in silicon treatment Silicon and ABA improved number of flowers at second, third and fourth cluster and also improve number of fruits at second and third cluster in all two levels of temperature However, number flowers and fruits of plants treated with ABA were greater than those in silicon treatment

Keywords: Environment, grafting, seedling quality, storage condition, tomato

to him for the guidance and financial support throughout the period of my study

I would like to express my sincere appreciation to my thesis committee Prof Kim Jong Hwa, Prof Kang Ho Min, Prof Kang Won Hee of Kangwon National University, and Prof Lee Yong Beom of University of Seoul for their valuable suggestions and helpful comments They encouraged and discussed many problems with me My sincere thank to Prof Jeong Cheon Soon, and Prof Park Sung Min of Kangwon National University for offering their kind and valuable advice

I would like to thank to all leaders of Vietnam National University of Agriculture for providing me study leave

The past three years working in Kangwon National University, I always obtained comments and helps from a lot of Professors, Doctors, Researchers, and other lab members in there Although I can’t list all of their names but I would like

to thank for their advices in my PhD study

Finally, I thank my wife and daughter who have given me generous support and understanding in every part of my life And I would also like to express the deepest gratitude to my father, mother and sister for all of the visible and invisible supports they have given to me in long-time

Author

Vu Ngoc Thang

vii

Content

Abstract ……… iv

Acknowledgments ……… ix

Content ……… x

List of Tables ……… xiv

List of Figures ……… xviii

List of Abbreviations ……… xxi

Chapter I General Introduction ……… 1

Chapter II Improving the Graft-take Ratio and Quality of Grafted Tomato Seedlings by Controlling Environmental Condition 5 2.1 Abstract ……… …… 5

2.2 Introduction ……… 9

2.3 Materials and methods ……… … 12

2.3.1 Plant material and growing scions and rootstocks…… 12

2.3.2 Grafting method, healing and acclimatization process … 12 2.3.3 Temperature and humidity treatments ……… 13

2.3.4 Water content in substrate, grafting position and different cultivars ……….……… ……… 14

2.3.5 Light quality treatment……… 15

2.3.6 Data collection and analysis ……… ………… 16

2.4 Results ……… ………… 18

2.4.1 Enhanced graft-take ratio and quality of grafted tomato seedlings by controlling temperature and humidity conditions 18

2.4.1.1 Effect of temperature on graft-take ratio and infected plants in four rootstock cultivars……… … 18

2.4.1.2 Effect of humidity on graft-take ratio and infected

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plants in different temperature levels ……… 20

2.4.1.3 Effect of humidity period on graft-take ratio and

2.4.2 Effect of water content in substrate, grafting position and different cultivars on the graft-take ratio and quality of grafted

2.4.2.1 Effect of water content in substrate during pre- and post-grafting on the graft-take ratio and quality of grafted

2.4.2.2 Effect of grafting position on the graft-take ratio and

2.4.2.2 Effect of different cultivars on the graft-take ratio and

2.4.3 Improvement of graft-take ratio and quality of grafted tomato seedlings by light quality application……….… 37

2.4.3.1 Effect of short-term irradiation before grafting on the graft-take ratio and quality of grafted tomato seedlings… 37

2.4.3.2 Effect of short-term irradiation after grafting (during the healing and acclimatization period) on the graft-take ratio and quality of grafted tomato seedlings… 41

2.4.3.3 Effect of red LED during the healing and acclimatization period on the graft-take ratio and quality of grafted tomato seedlings… 48

Chapter III Effect of Low Temperature on Growth and Quality of

Stored Tomato Seedlings ……… 63

ix

3.2 Introduction ……… 65

3.3 Materials and methods ……… 66

3.3.1 Plant material and growing conditions ……… 66

3.3.2 Temperature treatments ……… …… 66

3.3.3 Data collection and analysis ……… … 67

3.4 Results ……… 68

3.4.1 Effect of low temperatures on growth characteristics and cold stress of tomato seedlings during storage duration ……… 68

3.4.2 Effect of low temperatures on growth characteristics and quality of tomato seedlings after 10 days in storage….….…… 71

3.4.3 Effect of low temperatures on growth characteristics and quality of tomato seedlings after 20 days in storage 74

3.5 Discussion ……… ……… … 78

3.6 Conclusion ……… ……… … 79

Chapter IV Improvement of Seedling Quality in Stored Tomato by Application of Silicon and ABA……… 80

4.1 Abstract ……… 80

4.2 Introduction ……… … 83

4.3 Materials and methods ……….… 85

4.3.1 Plant material and growing conditions …… 85

4.3.2 Silicon application and chilling stress treatments ……… 86

4.3.3 ABA application and abiotic stresses treatments……… 86

4.3.4 Silicon and ABA application in stored tomato seedlings 88

4.3.5 Data collection and analysis ……… 88

4.4 Results ……… 90

4.4.1 Effect of silicon on growth, physiology, and chilling stress of tomato seedlings……… ………… 90

4.4.1.1 Effect of silicon concentrations on growth and

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physiology of tomato seedlings ……….…… … 90

4.4.1.2 Effect of number of silicon treatments on growth and chilling injury of tomato seedlings……… … 95

4.4.2 Effect of abscisic acid (ABA) on growth, physiology, and abiotic stress tolerance of tomato seedlings………… … 99

4.4.2.1 Effect of different ABA concentrations on growth and abiotic stress tolerance (cold and drought) of tomato seedlings 99 4.4.2.2 Effect of ABA application number on growth and abiotic stress tolerance (cold and drought) of tomato seedlings 105 4.4.2.3 Effect of ABA on physiology and drought stress of tomato seedlings ……… 110

4.4.3 Effect of silicon and ABA on quality of tomato seedlings in low storage temperature……… ………… ………… 116

4.4.3.1 Effect of silicon and ABA on growth of tomato seedlings in different temperature ……….……… 116

4.4.3.2 Effect of silicon and ABA on chilling injury of stored tomato seedlings ……… 119

4.4.3.3 Effect of silicon and ABA on flowers and fruits of stored tomato seedlings after transplanting ……… 120

4.5 Discussion ……… ……….… 124

4.6 Conclusion ……… ……….… 129

Chapter V General Discussion ……… 130

Literature Cited ……… 135

Publications in this thesis……… ……… 150

Abstract in Korea……… 152

xi

List of Tables

Table 1 Effect of temperature on graft-take ratio and infected plants of

four rootstock cultivars……… 19 Table 2 Effect of humidity on graft-take ratio and infected plants in

different temperature levels……… 21 Table 3 Effect of humidity periods on the graft-take ratio and growth

of grafted tomato seedlings……… 22 Table 4 Effect of humidity periods on fresh and dry weight of shoot

and root, T/R ratio, and compactness of grafted tomato

Table 5 Effect of water content in the substrate during pre-and

post-grafting on graft-take ratio of grafted tomato seedlings……… 27 Table 6 Effect of water content in the substrate during pre-and post-

grafting on growth characteristics of grafted tomato seedlings 28 Table 7 Effect of water content in the substrate during pre-and post-

grafting on leaf area, dry weight of shoot and root, T/R ratio, and compactness of grafted tomato seedlings……… 29 Table 8 Effect of grafting position on the graft-take ratio and growth

characteristics of grafted tomato seedlings……… 32 Table 9 Effect of grafting position on fresh and dry weight of shoot

and root, T/R ratio and compactness of grafted tomato

Table 10 Effect of different scion cultivars on the graft-take ratio of

grafted tomato seedlings……… 35 Table 11 Effect of different rootstock cultivars on the graft-take ratio of

grafted tomato seedlings……… 36

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Table 12 Effect of short-term irradiation before grafting on the

graft-take ratio and growth characteristics of grafted tomato seedlings at 15 days after grafting……… 37 Table 13 Effect of short-term irradiation before grafting on fresh and

dry weight of shoot and root, T/R ratio and compactness of grafted tomato seedlings at 15 days after grafting……….…… 38 Table 14 Effect of short-term irradiation after grafting on the graft-take

ratio and growth characteristics of grafted tomato seedlings at

15 days after grafting……… …… 42 Table 15 Effect of short-term irradiation after grafting on fresh and dry

weight of shoot and root, T/R ratio and compactness of grafted tomato seedlings at 15 days after grafting……….…… 43 Table 16 Effect of short-term irradiation after grafting on growth

characteristics of grafted tomato seedlings at 35 days after

Table 17 Effect of short-term irradiation after grafting on fresh and dry

weight of shoot and root, SLA, T/R ratio, and compactness of grafted tomato seedlings at 35 days after transplanting…….… 46 Table 18 Effect of red LED during healing and acclimatization period

on the graft-take ratio and infected plant of grafted tomato

Table 19 Effect of red LED during healing and acclimatization period

on growth characteristics of grafted tomato seedlings (measured on 15 days after grafting)……… 50 Table 20 Effect of red LED during healing and acclimatization period

on fresh weight, dry weight, T/R ratio and compactness of grafted tomato seedlings (measured on 15 days after grafting) 51 Table 21 Effect of low temperatures on survival rate, chilling injury

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index, time of expanding leaf, and day of first flowering of

Table 22 Effect of low temperatures on growth characteristics of tomato

seedlings after 10 days in storage ……….… 72 Table 23 Effect of low temperatures on fresh and dry weight of shoot

and root, T/R, and compactness of tomato seedlings after 10

Table 24 Effect of low temperatures on growth characteristics of tomato

seedlings after 20 days in storage ……… ………… 75 Table 25 Effect of low temperatures on fresh and dry weight of shoot

and root, T/R, and compactness of tomato seedlings after 20

Table 26 Effect of silicon concentrations on growth characteristics of

Table 27 Effect of silicon concentrations on fresh and dry weight of

shoot and root, T/R, and compactness of tomato seedlings… 92 Table 28 Effect of number of silicon treatments on growth of tomato

Table 29 Effect of number of silicon treatments on fresh and dry weight

of shoot and root, T/R and compactness of tomato seedlings… 96 Table 30 Effect of silicon on the growth and chilling injury of tomato

seedling at 7 and 10oC……… 98 Table 31 Effect of ABA concentrations on growth characteristics of

Table 32 Effect of ABA concentrations on fresh and dry weight of shoot

and root, T/R ratio and compactness of tomato seedlings…… 101 Table 33 Effect of ABA concentrations on percentage of wilted plant of

tomato seedling after without irrigation at 25oC……… 103

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Table 34 Effect of ABA concentrations on relative ion leakage and

chilling injury index of tomato seedling in low temperature… 104 Table 35 Effect of ABA application number on growth of tomato

Table 36 Effect of ABA application number on fresh and dry weight of

shoot and root, T/R, and compactness of tomato seedlings…… 106 Table 37 Effect of ABA application number on percentage of wilted

plant of tomato seedlings after without irrigation at 25oC…… 108 Table 38 Effect of ABA application number on relative ion leakage and

chilling injury index of tomato seedlings in low temperature… 109 Table 39 Effect of ABA concentrations on growth characteristics of

Table 40 Effect of ABA concentrations on fresh and dry weight of shoot

and root, T/R and compactness of tomato seedlings………… 111 Table 41 Effect of ABA concentrations on plant wilting in tomato

seedling after without irrigation at 25oC……… 115 Table 42 Effect of silicon and ABA on chilling injury of stored tomato

Table 43 Effect of silicon and ABA on number of flowers of stored

tomato after transplanting ……… 122 Table 44 Effect of silicon and ABA on number of marketable fruits of

stored tomato after transplanting……… 123

xv

List of Figures

Figure 1 Grafting position above the rootstock cotyledons……… 15 Figure 2 Grafting position below the rootstock cotyledons……… 15 Figure 3 Effect of humidity periods on root morphology of grafted

Figure 4 Effect of water content in the substrate during pre- and

post-grafting on root morphology of grafted tomato seedlings…… 30 Figure 5 Grafted tomato seedlings were treated with different water

content in the substrate during pre- and post-grafting………… 31 Figure 6 Effect of grafting position on root morphology of grafted

Figure 7 Grafted seedlings were treated with light quality before

Figure 8 Effect of short-term irradiation before grafting on root

morphology of tomato seedlings at 15 days after grafting…… 40 Figure 9 Grafted seedlings were treated with light quality after grafting 43 Figure 10 Effect of short-term irradiation after grafting on root

morphology of tomato seedlings at 15 days after grafting…… 44 Figure 11 Effect of short-term irradiation during the healing and

acclimatization period on root morphology of grafted tomato seedlings at 35 days after transplanting……… ……… 47 Figure 12 Grafted tomato seedlings after 10 days in red LED and no

Figure 13 Effect of red LED during healing and acclimatization period on

root morphology of tomato seedlings……… 52 Figure 14 Effect of low temperatures on plant height of tomato seedlings

during storage duration……… 69 Figure 15 Effect of low temperatures on number of leaves of tomato

xvi

seedlings during storage duration……… 69 Figure 16 Effect of low temperatures on leaf chlorophyll content of

tomato seedlings during storage duration……… 70

Figure 17 Seedlings after 10 days in storage……… 73 Figure 18 Effect of low temperatures on root morphology of tomato

seedlings (10 days after storage)……… 74 Figure 19 Effect of low temperatures on internode length of tomato

Figure 20 Seedlings after 20 days in storage……….…… 76 Figure 21 Effect of low temperature on root morphology of tomato

seedlings (20 days after treating)……… 77

Figure 22 Scheme of time treatments of ABA……… 87 Figure 23 Effect of silicon concentrations on plant height of tomato

Figure 24 Effect of silicon concentrations on root morphology of tomato

Figure 25 Effect of silicon concentrations on transpiration rate, stomatal

diffusive resistance, and leaf temperature of tomato seedlings 95 Figure 26 Effect of number of silicon treatments on root morphology of

Figure 27 Tomato seedlings and root after 10 days treatments………… 100 Figure 28 Effect of ABA concentrations on root morphology of tomato

Figure 29 Seedlings on third day after without irrigation……… 103

Figure 30 Seedlings on three day after treating at low temperature… … 104 Figure 31 Effect of ABA application number on root morphology of

Figure 32 Tomato seedlings and root morphology after 10 days treating

xvii

different ABA application number……… 108 Figure 33 Seedlings on three day after treating at low temperature…… 109 Figure 34 Effect of ABA concentrations on root morphology of tomato

Figure 35 Effect of ABA on transpiration rate, stomatal diffusive

resistance, and leaf temperature of tomato seedlings …….… 113 Figure 36 Effect of ABA concentrations on relative water content of

Figure 37 Effect of ABA concentrations amount transpiration of tomato

Figure 38 The pots of seedlings were covered with vinyl film to prevent

water loss from the substrate surface……….……… 115 Figure 39 Tomato seedlings after 10 days treatment (leaf) Tomato

seedlings at fourth day after without irrigation (right)……… 115 Figure 40 Effect of silicon and ABA on plant height of tomato seedlings

stored at 10oC and 7oC……… 116 Figure 41 Effect of silicon and ABA on number of leaves of tomato

seedlings stored at 10oC and 7oC……… 117 Figure 42 Effect of silicon and ABA on leaf chlorophyll value of tomato

seedlings stored at 10oC……….……… 117 Figure 43 Effect of silicon and ABA on leaf chlorophyll value of tomato

seedlings stored at 7oC……… 118 Figure 44 Stored seedlings after 3 weeks at 10oC……… 118 Figure 45 Stored seedlings after 3 weeks at 7oC……… 118

Figure 46 Stored tomato plants after transplanting in plastic house…… 121

Figure 47 Strategy for grafting process of tomato seedlings……… 132

mg.cm-1 Milligram per centimeter

µg.cm-2.s-1 Microgram per square centimeter per second

s.cm-1 Second per centimeter T/R Shoot dry weight/root dry weight

SLA Specific leaf area

SD Standard deviation

SPAD Leaf chlorophyll value

1

Chapter I General Introduction

Tomato (Lycopersicon esculentum Mill) is one of the most important

vegetable crops grown throughout the world under field and greenhouse conditions (Kaloo, 1986) In the terms of human health, tomato is major component in the daily diet in many countries, and constitutes and important source of minerals, vitamins, and antioxidants (Grierson and Kader, 1986)

Plug seedlings production technology was introduced to Korea in the early 1990s and has been widely used in recent years especially for vegetable crops because it save labor for raising seedlings, facilitates mass production of uniform seedlings, and allows division of production labor (Jeong, 1998, 2000) Although vegetable growers previously have grown their own seedlings, commercial plug seedling production is becoming more prevalent The quality of both grafted and non-grafted tomato plug seedlings is crucial in vegetable production in Korea because it supports the success of final crop production both open fields and greenhouses However, there are problems in supplying for growers with high-quality seedlings in commercial tomato plug seedling production Many environmental factors affect on graft-take ratio, growth and quality of tomato seedlings in during healing process and nursery stages Tomato seedlings are able

to modify their growth, development and physiology according to their environment Therefore, control of environmental conditions could enhance graft-take ratio and improve seedling quality (Oda, 1999) Moreover, proper management of environmental conditions could minimize cost for seeds, improve seedling quality, and reduce labor cost for sowing and raising of seedlings (Jeong, 1998)

Since grafting is considered an important technique for the sustainable production of fruit vegetables in Korea, Japan and several European countries (Lee,

2

1994; Oda, 1995), the use of grafted tomato for commercial production has increased in order to improve resistance to biotic and abiotic stresses (Lee and Oda, 2003; Rivero et al., 2003) The successful production of grafted seedlings requires grafting methods, grafting skills, and environmental control pre-and post-grafting, especially during healing and acclimatization period (Lee, 1994) Although many grafting methods and techniques for environmental control during grafting process are widely recognized, many other factors must be carefully considered to ensure successful grafting process with this technology such as temperature, humidity during healing process, or irradiation and water stress before and after grafting

Tomato seedling quality is highly valued in Korea vegetable production (Lee and Oda, 2003) Ideally, tomato seedlings are transplanted when they reach the correct size, but seedlings are often ready before grower can transplant them Therefore, growth must be slowed or delayed Traditional methods of slowing seedlings growth include using water and nutrient stress and chemical growth regulators (Cutler and Schneider 1990) All of these methods can stress plants and potentially delay growth after transplanting However, seedling storage with low temperature is a useful technique to solve this problem Low temperature storage can be used to stop or suppress the growth and development of seedlings while preserving its quality and not adversely affecting future growth However, long-term stored duration in low temperature affected seedling morphology and yield after transplanting (Leskovar and Cantliffe, 1991)

Although silicon (Si) is the second most abundant element both on the surface of the Earth’s crust and in soils, it has not yet been listed among the essential elements for higher plants However, the beneficial role of Si in stimulating the growth and development of many plant species has been generally recognized The results of initial Si experiments indicate that silicon affects plant growth and crop quality, stimulates photosynthesis, reduces transpiration rate, and

3

enhances plant resistance to a series of both abiotic and biotic stresses such as manganese, aluminum and heavy metal toxicities, and salinity, drought, chilling and freezing stresses (Ma and Takahashi, 1990; Cherif et al., 1992; McAvoy and Bible, 1996; Agrie et al., 1998; Liang et al., 2001; Lu and Cao, 2001; Seebold et al., 2001; Hodson and Sangster, 2002; Savvas et al., 2002; Zhou et al., 2002)

Abscisic acid (ABA) is very important agent in the mechanisms of resistance and adaptation in plants against various abiotic stress conditions (Li et al., 2010; Bakhsh et al., 2011).It plays an essential role in many physiological processes, including seed development, dormancy, germination and reproduction (Finkelstein et al., 2002) In addition, it also plays a pivotal role in abiotic stress tolerance (Leung and Giraudat, 1998) ABA mediates responses to environmental stresses such as heat, cold, salt, drought and high irradiance (Taylor et al., 2000; Larkindale and Knight, 2002; Cousson, 2009; Pospisilova et al., 2009)

Based with important aforementioned background, the following specific objectives are set for the thesis research

1 Improving the graft-take ratio and quality of grafted tomato seedlings by controlling environmental condition

To determine the effects of temperature and humidity on graft-take ratio and quality of grafted tomato seedlings

To investigate the effects of water content in substrate, grafting position and different cultivars on the graft-take ratio and quality of grafted tomato seedlings

To investigate the effect of light quality on the graft-take ratio and quality of grafted tomato seedlings

5

Chapter II Improving the Graft-take Ratio and Quality of Grafted Tomato

Seedlings by Controlling Environmental Condition

2.1 Abstract

Three temperature levels (20, 23, and 26oC) were carried out to determine optimal temperature on four rootstocks In addition, twelve combinations of three relative humidity levels (70, 80, and 90%) and four temperature levels (17, 20, 23, and 26oC) were set up to evaluate the effect of relative humidity and temperature

on the graft-take ratio of grafted seedlings In the other hand, five relative humidity periods (H0, H1, H2, H3, and H4: 90% relative humidity for first 0, 1, 2, 3 and 10 days and afterwards relative humidity was reduced to 70%, respectively) were examined effect of relative humidity periods on the graft-take and quality of grafted seedlings The higher graft-take ratios (84.0-87.4%) were showed at 23oC compared to 20 and 26oC in all rootstocks Graft-take ratios decreased and number

of diseased plants increased at high temperature The graft-take ratios increased with increasing relative humidity in all temperature levels on the 3rd and 7th day after grafting However, increasing relative humidity significantly increased percent of diseased plants The graft-take ratio reduced at (26oC) and (17oC) temperature under all relative humidity conditions The graft-take ratio increased with increasing period of 90% relative humidity Maximum graft-take ratios were observed in H2 and H3 treatments Graft-take ratio decreased with increasing 90% relative humidity for 10 days (H4) Infected plants had not been found in H0, H1, H2, and H3 treatments Seedling quality was improved through increasing fresh and dry weight of root, compactness, and root morphology of tomato seedlings in H2 and H3 treatments Therefore, high relative humidity (90%) for first 2 or 3 days and afterwards reduced low relative humidity (70%) at 23oC condition during

in substrate of rootstock The maximum graft-take ratios (100%) were observed in treatment with high water in substrate of rootstock Graft-take ratios decreased with decreasing water content in substrate of rootstock There are not statically different among water content in substrate of scion with growth characteristics but statically different among water content in substrate of rootstock Growth characteristics decreased with decreasing water content in substrate of rootstock The maximum graft-take ratio (100%) with the highest compactness value was observed in seedlings which were grafted by scion mid and rootstock high water content in the substrate There was no significant difference in graft-take ratio between two grafting positions but grafting position effected on growth characteristics of grafted tomato seedlings The higher values of growth characteristics were observed in above the rootstock cotyledons treatment There was no significant different among scion cultivars with graft-take ratio on the 3rd day after grafting but significant different among scion cultivars with graft-take ratio on the 7th day The higher infected plants were observed in cherry tomato group compared other groups There was significant different among rootstock cultivars with graft-take ratio on the 3rd day after grafting but not significant different among rootstock cultivars on the 7th day and final graft-take ratio

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On the other hand, influence of short-term irradiation during pre- and grafting period on the graft-take ratio and quality of tomato seedlings were carried out to determine suitable light for grafting process Irradiation by six light qualities, darkness, white fluorescent lamps (WFL), red LED, far-red LED, blue LED, and natural light, were used to treat seedlings for 10 days before grafting And irradiation by five light qualities, darkness, WFL, red LED, far-red LED, and blue LED, were used to treat seedlings for 10 days after grafting, during healing and acclimatization periods When short-term irradiation was applied before grafting, the graft-take ratios (27.8 - 66.7%) were considerably low in all light treatments as compared with natural light (96.7%) The graft-take ratio of red LED was not statically different with WFL treatment, but higher than far-red and blue LED treatments The lowest graft-take ratio (27.8%) was observed in darkness treatment Changing light intensity before grafting was the cause of reduced graft-take ratios

post-in this study There was no significant difference among natural light, WFL, and red LED treatments in growth parameters, except for leaf chlorophyll level, leaf width, and fresh weight of root, but decreased in seedlings treated with far-red LED, blue LED, and darkness Graft-take ratios (68.5 - 100.0%) were enhanced when short-term irradiation was applied after grafting The maximum (100%) graft-take ratio was recorded in red LED treatment, but was not statistically different with the WFL treatment The lowest graft-take ratio was also observed in the darkness treatment Plant growth responses to red LED were also similar with those to WFL after grafting However, when short-term irradiation was applied after grafting, the lowest values of plant growth were observed in far-red LED treatment The plant growth parameters were similar in seedlings treated with darkness and blue LED, but lower than red LED and WFL treatments The root morphology was improved

in seedlings treated with red LED after grafting by increasing total root surface, total root length, and number of root tips Seedling quality increased at 35 days

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2.2 Introduction

Since grafting is considered an important technique for the sustainable production of fruit bearing vegetables in Korea, Japan and several European countries (Lee, 1994; Oda, 1995), the use of grafted tomato for commercial production has increased in the world in order to improve resistance to biotic and abiotic stresses (Lee and Oda, 2003; Rivero et al., 2003) The successful production of grafted transplants requires highly technical grafting skill and environmental control pre- and post-grafting, especially during healing and acclimatization period Proper acclimatization is critical for grafted plants to survive (Lee et al., 2010) After grafting, it is important to control the environmental conditions to facilitate the union of the rootstock and scion In conventional cultures, healing and acclimatization of the grafted plants were done

in tunnels that were made by plastic-film and shaded by cloth to avoid heat buildup and to maintain high relative humidity and low light intensity until the union is formed (Oda, 1999; Davis et al., 2008) It is quite difficult to control optimal environment for healing and acclimation of grafted seedlings under the natural condition However, recently several types of acclimatization chambers have been developed and widely used by commercial plug seedling growers in Korea and Japan In healing chambers under highly controlled healing conditions, higher survival ratios, faster growth, and higher quality of grafted plants were reported in some papers (Kim et al., 2001; Nobuoka et al., 2005; Vu et al., 2013)

Regarding temperature and relative humidity conditions during healing and acclimatization for grafted plants, Liang (1990) suggested that the suitable temperature for grafted watermelon post-grafting curing environment was 25-30oC Mii et al (1994) pointed out the appropriate curing environment for grafted tomato seedlings was 20-30oC for temperature, more than 80% for relative humidity, and

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50-70% shading of the daylight Nishiura (1999) also proposed the air temperature, relative humidity, and light and dark interval period as 25-28oC, 90%, 12 hrs respectively for the healing and acclimatization of grafted tomato seedlings Chang

et al (2003) suggested that the survival rate and quality of grafted seedlings were promoted if the relative humidity in the acclimatization chamber was maintained at 80-90% In contrast, Jang et al (2009) reported that the growth of grafted peppers was greater under low temperature and low relative humidity conditions The low relative humidity (65%) and light condition at 25oC during healing and acclimatization promoted the survival of grafted seedlings Although there is general consensus that controlling of temperature and humidity is required for successful grafting of herbaceous plants However, there is not so much information on control of relative humidity period for grafted tomato seedlings Oda (1999) just suggested that keeping the grafted plants at about 30oC temperature and with more than 95% relative humidity for three days of healing promotes the survival ratio and then the relative humidity was lowered and the light intensity increased

Beside temperature and humidity, moisture in the substrate is also important factor that affect on survival rate of grafted seedlings Oda, (1999) suggested that in order to improve survival rate of grafted plants, the seedlings should be exposed to sunshine and withhold water from plant to avoid spindly growth before grafting Furthermore Lee et al (2010) suggested that maintenance of proper moisture content before and after grafting is critical for the production of uniform grafted seedlings However, information on effect of moisture content in the substrate on survival rate and quality of grafted seedlings is limited

Various methods for grafting vegetable crops have been developed such as cleft grafting (also known as apical grafting or wedge grafting), side grafting (also known as tongue approach grafting or side-by-side grafting) (Lee, 1994), and splice

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grafting (also known as top grafting, tube grafting, or slant-cut grafting) (Lee and Oda, 2003) Nowadays, splice grafting is the most commonly method for tomato seedlings because this method is relatively simple (Oda, 1995), high survival rates and also saves labor (Kubota et al., 2008) Furthermore, this method can be performed by hand, semi-automatic, and with automatic robots (Kurata, 1994; Lee and Oda, 2003) Using splice grafting method, tomato seedlings could be grafted above or below the rootstock cotyledons Although the exact role of the cotyledons

in the formation of the graft union is not fully understood, it is possible that the cotyledon produces compounds required for the formation of the graft union (Asahina et al., 2002; Asahina et al., 2007) However, there is not much research activities focused on grafting position on survival rate and quality of grafted tomato seedlings

Plant development and physiology are strongly influenced by the light spectrum (McNellis and Deng, 1995) Light-emitting diodes (LED) are the new, fourth-generation light source with good spectral characteristics and spectral width (wavelength) of emission peak of ± 15nm, and can be assembled to shed the light quality which plants need (Goins et al., 1997) The LED has been proposed as a photosynthetic radiation source for space flight growing systems and as more efficient sources for terrestrial controlled-environment agriculture facilities (Bula et al., 1991) Moreover, LED has the advantages such as low energy consumption, small size, durability, long lifetime, cool emitting temperature, and option to select specific wavelengths for target Therefore, LED is expected to be widely used as a new type of good light source effective for the propagation and growth of plants Many studies have shown such effects of LED on growth and development of plants as elongation, axillary shoot formation, leaf anatomy, and morphogenesis (Tennessen et al., 1994; Kim et al., 2004; Miyamoto et al., 2006) However, most studies have focused on growth, photosynthesis, metabolism, and gene expression

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(Neff et al., 2000; Yu and Ong, 2003; Wang et al., 2009), while less is known about how irradiation of LED affects the graft-take ratio and seedling quality If LED is used as a lighting source for graft-taking of grafted seedlings, the light intensity and photoperiod can be easily controlled Especially, the space efficiency of the healing chambers will be increased considerably by using vertical surface areas

Therefore, the objective of this study was conducted in order to enhance graft-take ratio and quality of grafted tomato seedlings through controlling temperature and humidity during healing and acclimatization period In addition, when optimal condition in healing and acclimatization period was selected, effects

of water content in the substrate, grafting position, and different cultivars were investigated On the other hand, the effect of light quality during pre- and post-grafting period on the graft-take ratio and quality of grafted tomato seedlings was also investigated

2.3 Materials and methods

2.3.1 Plant material and growing scions and rootstocks

Both scion and rootstock of tomato seeds were sown in the 128-cell plug trays (Bumnong Co., Ltd., Jeongeup, Korea) that was filled with commercial growing substrate (BM2, Berger Group Ltd., Quebec, Canada) During the plastic house, seedlings were watered daily or as required One week after sowing, seedlings started to receive a fertilization based on Wonder Grow fertilizers (Wonder Grow Fertilizers, Chobi Co., Ltd., Seoul, Korea) twice a week through an overhead irrigation Twenty-two days after sowing, seedlings were used for grafting

2.3.2 Grafting method, healing and acclimatization process

Grafting was done by splice grafting method Plants were grafted by first making approximately 45o cuts on both the rootstock and scion seedlings using a

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sharp razor blade After placing the scion on the rootstock, ordinary grafting clips were used to fix the grafted position tightly together (Lee and Oda, 2003) Rootstock and scion plants with similar stem diameters (2.3∼2.4mm) were chosen

to increase the grafting success rate After grafting, grafted plants were placed in healing rooms for 10 days After 10 days in healing room, grafted seedlings were transferred to plastic house in natural condition for 5 days The grafting clips were removed 2 days after plants were out of the healing room Healing rooms were also equipped with an auto-control air conditioning system for healing the grafting Light, relative humidity and temperature were set up with different values for the individual purpose of each experiment

2.3.3 Temperature and humidity treatments

Experiment 1 The effect of temperature and rootstock on the graft-take ratio was examined by treating grafted tomato seedlings with three temperature levels (20, 23, and 26oC) on four rootstock cultivars (‘Kanbarune’, ‘B-Blocking’,

‘Magnet’, and ‘Solution’) In this experiment, ‘Super-Top’ cultivar was used as scion and relative humidity was maintained at 85-90% Light intensity was approximately 30µmol.m-2.s-1 provided by fluorescent lamps

Experiment 2 The effect of relative humidity and temperature on the take ratio was examined by treating grafted tomato seedlings with three relative humidity levels (70, 80, and 90%) on four temperature conditions (17, 20, 23, and

graft-26oC) Light intensity was approximately 30µmol.m-2.s-1 provided by fluorescent lamps ‘Myrock’ cultivar was used as scion while ‘Support’ cultivar was used as rootstock for this experiment

Experiment 3 The effect of humidity period on graft-take ratio and quality of grafted tomato seedlings was studied by treating grafted tomato seedlings with five treatments of humidity period H0 (70% relative humidity for 10 days), H1 (90%

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relative humidity for first one day and then relative humidity was reduced to 70% for next 9 days), H2 (90% relative humidity for first two days and then relative humidity was reduced to 70% for next 8 days), H3 (90% relative humidity for first

3 days and then relative humidity was reduced to 70% for next 7 days), H4 (90% relative humidity for 10 days) Light intensity was approximately 30µmol.m-2.s-1provided by fluorescent lamps ‘Unicorn’ cultivar was used as scion and rootstock

‘Self-grafted’ and temperature was maintained at 23oC for this experiment

2.3.4 Water content in substrate, grafting position and different cultivars

From optimal temperature and humidity above studies, three experiments were performed

Experiment 4 The effect of water content in the substrate during pre- and post-grafting was studied by treating tomato seedlings by three levels (High, Mid, and Low) of water content in the substrate in both scion and rootstock for 6 days (3 days before and 3 days after grafting) Three levels of water content (high, medium, and low) were applied overhead irrigation 3 times, twice, and once a day about 30,

15, 10 ml of water/cell/day, respectively Treatments with medium water were applied twice a day with sufficient water (similar irrigation schedule of Seedling Company) ‘Unicorn’ cultivar was used as scion and rootstock ‘Self-grafted’ for this experiment

Experiment 5 The effect of grafting positions on graft-take ratio and quality

of tomato plug seedlings was studied by using splice grafting method for two grafting positions such as above (Fig 1) and below (Fig 2) the rootstock cotyledons ‘Unicorn’ cultivar was used as scion and rootstock “Self-grafted” for this experiment

Experiment 6 Effect of different cultivars on the graft-take ratio was studied Eleven cultivars included three groups such as group from Japan, Europe, and

Fig 1 Grafting position above the rootstock cotyledons

Fig 2 Grafting position below the rootstock cotyledons

2.3.5 Light quality treatments

Experiment 7 The effect of short-term irradiation before grafting was examined by treating tomato seedlings for 10 days before grafting in a growth chamber (Hanbaek Co., Ltd., Bucheon, Korea) with either darkness, WFL (Orex Co., Ltd., Goyang, Korea), blue LED with peak wavelength 450 nm (Led Plant Radiation System, Joeun LED Co., Ltd., Seongnam, Korea), red LED with peak wavelength 660 nm (Led Plant Radiation System, Joeun LED Co., Ltd., Seongnam,

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Korea), or far-red LED with peak wavelength 730 nm (Led Plant Radiation System, Joeun LED Co., Ltd., Seongnam, Korea), or natural light in a greenhouse The light intensity in the growth chamber was approximately 15 µmol.m-2.s-1 PPFD for light treatments Ten days after treating, seedlings were used directly for grafting, and grafted plants were placed in healing room for 10 days And then grafted seedlings were transferred to a plastic house in a natural condition and grown for five days

‘Lapito’ cultivar was used as scion and rootstock “Self-grafted” for this experiment Experiment 8 The effect of short-term irradiation after grafting was studied

by treating grafted tomato seedlings in a healing room with either darkness, WFL, blue LED, red LED, or far-red LED for 10 days during the healing and acclimatization period The light intensity in the growth chamber was approximately 15 µmol.m-2.s-1 PPFD for light treatments After 10 days in the healing room, grafted seedlings were transferred to a plastic house in a natural condition and grown for five days And then grafted seedlings were transplanted to 32-cell plug trays ‘Lapito’ cultivar was used as scion and rootstock “Self-grafted” for this experiment

Experiment 9 Red LED and no light (darkness) were used for treating three rootstock cultivars ‘B-Blocking’, ‘Kanbarune’ and ‘High-Power’ while ‘Choice’ cultivar was used as scion in healing room for 10 days The healing room was equipped with an automatically controlled air conditioning system Relative humidity was maintained 85-90%, temperature set up point was 23oC, and light intensity of red LED was approximately 15 µmol.m-2.s-1 PPFD After 10 days in the healing room, grafted seedlings were transferred to plastic house in natural condition for 5 days

2.3.6 Data collection and analysis

All grafted seedlings were evaluated for signs of graft failure on the 3rd and

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7th day or 10th day after grafting Infected plants were evaluated on the 10th after grafting Grafted seedlings on the 15th day after grafting and on the 35th day after transplanting were harvested for analysis of growth characteristics Seedling height (cm) and number of leaves of grafted seedlings were measured Leaf area (cm2) was measured by leaf area meter (Delta-T Device Ltd., Burwell, Cambridge, UK) Leaf chlorophyll value was measured by using a chlorophyll meter (SPAD-502 Plus, Konica Minolta Sensing Inc., Osaka, Japan) Fresh and dry weights of shoot and root were measured The fresh shoot and root were dried in an oven (MOV-212F, Sanyo Electric Co., Ltd., Osaka, Japan) at 80oC for 72 hrs before measuring the dry mater T/R ratio (shoot dry weight/root dry weight ratio) and compactness (shoot dry weight/plant height) were calculated according to Kim et al (2008) For the root system morphology, the WinRHIZO Pro 2009c (Regent Instruments, Inc, Quebec, Canada) images analysis system was used, coupled with professional scanner Epson 10000XL (Seiko Epson Corporation, Nagano, Japan) according to Arsenault et al (1995) The roots were detached from their shoots and then placed

in a tray (15 x 30 x 2 cm) with water and placed on the scanner Scanned images were analyzed by the WinRHIZO program for total root surface area, total root length, average root diameter, and number of root tips

The experimental design was a split-plot for first, second, fourth and ninth experiment Temperature was the main plot and rootstock variety was the sub plot

in first experiment Humidity was the main plot and temperature was the sub plot in second experiment Water contents in the substrate of rootstock were the main plot and water contents in the substrate of scion were the sub plot in fourth experiment Light was the main plot and rootstock cultivars were the sub plot in ninth experiment Third, fifth, sixth, seventh and eighth experiments were arranged in completely randomized design In each replication, one 128-cell plug tray with 64 plants was measured For the statistical analysis of growth parameters, ten

On the 3rd day after grafting, the graft-take ratios were decreased at 20oC and

26oC compared with 23oC in all rootstock cultivars except ‘B-Blocking’ But the graft-take ratios were not statistically different among temperature levels and cultivars However, 7 days after grafting, graft-take ratios significantly decreased at

20oC and 26oC temperatures in all rootstock cultivars The higher graft-take ratios (84.0-90.0%) were observed at 23oC temperature compared with 20oC and 26oC temperatures (64.6-83.4% and 64.6-78.0%, respectively) In each temperature levels, there was no significant difference in graft-take ratio among different rootstock cultivars From the 3rd to 7th day after grafting, high percentage of graft failure was observed in ‘B-Blocking’ (28.2%) and ‘Kanbarune’ (16.6%) at low temperature (20oC), whereas high percentage of graft failure of ‘Magnet’ (20.0%) and ‘Solution’ (16.6%) was observed at high temperature (26oC) (Table 1)

Percentage of infected plants increased significantly with increasing temperature to 26oC during the healing and acclimatization processes in all rootstock cultivars High percentage of infected plants of the all rootstock cultivars was found at (26oC) Therefore, final graft-take ratios at 26oC were affected by percentage of infected plants High final graft-take ratios (84.0-87.4%) were

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showed at (23oC) temperature in all rootstock cultivars The final graft-take ratios were not statistically different among rootstock cultivars at (23oC) temperature, whereas there were statistically different on graft-take ratio among different rootstock cultivars at 20oC and 26oC (Table 1)

Table 1 Effect of temperature on graft-take ratio and infected plants of four rootstock cultivars

plants (%)

Final take ratio (%)

graft-3 days 7 days

20oC

Kanbarune 91.4 bcdz 75.4 d 0.0 f 75.3 c B-Blocking 92.8 abc 64.6 e 5.3 c 59.3 f Magnet 89.4 b-e 83.4 c 2.0 ef 81.3 b Solution 78.0 gh 74.0 d 3.4 c-e 70.6 d

23oC

Kanbarune 94.0 ab 87.0 abc 0.0 f 87.0 a B-Blocking 90.0 b-e 90.0 a 2.6 de 87.4 a Magnet 98.6 a 88.6 ab 3.0 cde 85.3 ab Solution 87.4 c-f 84.0 bc 0.0 f 84.0 ab

26oC

Kanbarune 75.0 h 68.6 e 4.6 cd 64.0 e B-Blocking 82.6 fg 78.0 d 8.6 b 69.4 d Magnet 84.6 ef 64.6 e 12.0 a 52.6 g Solution 86.0 def 69.4 e 10.6 ab 58.7 f Significance

Temperature (T) NSy

zMean separation within columns by Duncan’s multiple range test at P = 0.05

yNS, *, ** indicates Non-significant; significant at P≤ 0.05 and P≤ 0.01, respectively