CONTENTS

(note: page numbers correspond to original document)

 

CHAPTER

PAGE

DECLARATION

(i)

ACKNOWLEDGEMENTS

(ii)

LIST OF ABBREVIATIONS

(iv)

INTRODUCTION

1

1          REVIEW OF LITERATURE

4

     1.1  Introduction

4

     1.2  Radioimmunoassay

5

                 1.2.1  Terminology

5

                 1.2.2  Basic principle of RIA

6

                             1.2.2.1  Binder dose-response curves

8

                             1.2.2.2  Standard dose-response curves

9

                 1.2.3  The binder or antibody

11

                             1.2.3.1  Cellular events

11

                             1.2.3.2  Chemistry of antibodies

13

                             1.2.3.3  The immune response

15

                             1.2.3.4  Immunogens

16

                             1.2.3.5  Haptens

17

                             1.2.3.6  Adjuvants

19

                             1.2.3.7  Antibody production

20

                 1.2.4  The ligand

20

                             1.2.4.1  Purified ligand

21

                             1.2.4.2  Tracer ligand

21

                 1.2.5  Separation of bound and free ligand

23

                             1.2.5.1  The ideal separation technique

23

                                    1.2.5.2  Differential migration of bound and free fractions

24

                                                1.2.5.2.1  Paper chromato-electrophoresis

24

                                         1.2.5.2.2  Gel filtration

25

                                         1.2.5.2.3  Gel equilibrium

25

                             1.2.5.3  Adsorption methods

26

                                                1.2.5.3.1  Charcoal

26

                                         1.2.5.3.2  Silicates

27

                             1.2.5.4  Fractional precipitation

27

                             1.2.5.5  Double antibody method

28

                             1.2.5.6  Solid phase methods

29

                 1.2.6  Statistical analysis of RIA data

30

     1.3  Immunological Assays for PGSs

32

     1.4  Avocado Fruit Development and Senescence

34

                 1.4.1  PCS regulation of fruit growth

35

                        1.4.2  PCS regulation of senescence

38

2          RADIOIMMUNOASSAY FOR ISOPENTENYL-TYPE CYTOKININS

40

     2.1  Materials and Methods

40

                 2.1.1  Conjugation of IPA to BSA

40

                 2.1.2  Production of antisera

41

                             2.1.2.1  Immunization

41

                             2.1.2.2  Purification and storage

42

                             2.1.2.3  Titer determination

42

                 2.1.3  Radioimmunoassay

43

                             2.1.3.1  Optimization

43

                                                2.1.3.1.1  Effect of separation technique

43

                                         2.1.3.1.2  Effect of charcoal concentration

45

                                         2.1.3.1.3  Determination of incubation time

45

                                         2.1.3.1.4  Effect of pH

46

                                         2.1.3.1.5  Tracer preparation

46

                             2.1.3.2  Standard procedure

47

                             2.1.3.3  Statistical analysis and calculation of results

50

                 2.1.4  Validation

51

                             2.1.4.1  Extract dilution curves

51

                             2.1.4.2  Antibody selectivity (cross-reaction studies)

53

                 2.1.5  Biological sample preparation

53

                             2.1.5.1  Extraction

54

                             2.1.5.2  Purification

54

                             2.1.5.3  Isolation

55

     2.2  Results

56

                 2.2.1  IPA-BSA conjugate

56

                 2.2.2  Response to Immunization

58

                 2.2.3  Radioimmunoassay

61

                             2.2.3.1  Effect of separation technique

61

                             2.2.3.2  Effect of charcoal concentration

66

                             2.2.3.3  Effect of incubation time

66

                             2.2.3.4  Effect of pH

69

                             2.2.3.5  Tracer properties

69

                             2.2.3.6  Standard dose-response curve

69

                 2.2.4  Validation

73

                             2.2.4.1 Extract dilution, curves

73

                             2.2.4.2  Cross-reactivities to Anti-IPA

77

            2.3  Discussion

78

3          RADIOIMMUNOASSAY FOR ABSCISIC ACID

81

     3.1  Materials and Methods

81

                 3.1.1  Conjugation of (+)ABA to BSA

81

                 3.1.2  Production of antisera

82

                             3.1.2.1  Immunization

82

                             3.1.2.2  Purification and storage

82

                             3.1.2.3  Titer determination

83

                 3.1.3  Radioimmunoassay

83

                             3.1.3.1  Optimization

83

                                                3.1.3.1.1  Effect of separation technique

83

                                         3.1.3.1.2  Determination of incubation time

83

                                         3.1.3.1.3  Effect of charcoal concentration

84

                                         3.1.3.1.4  Effect of pH

84

                             3.1.3.2  Standard procedure

84

                 3.1.4  Validation

85

                             3.1.4.1  Extract dilution curves

85

                             3.1.4.2  Antibody selectivity (cross-reaction studies)

86

                 3.1.5  Biological sample preparation

86

                             3.1.5.1  Extraction

87

                             3.1.5.2  Purification

87

                             3.1.5.3  Isolation

87

     3.2  Results

88

                 3.2.1  ABA-BSA conjugate

88

                 3.2.2  Response to Immunization

90

                 3.2.3  Radioimmunoassay

90

                             3.2.3.1  Effect of separation technique

90

                             3.2.3.2  Effect of charcoal concentration

96

                             3.2.3.3  Effect of pH

96

                             3.2.3.4  Effect of incubation time

96

                             3.2.3.5  Standard dose-response curve

96

                 3.2.4  Validation

100

                             3.2.4.1 Extract dilution, curves

100

                             3.2.4.2  Cross-reactivities to Anti-ABA

100

            3.3  Discussion

100

4          RADIOIMMUNOASSAY FOR INDOLE-3-ACETIC ACID

106

     4.1  Materials and Methods

106

                 4.1.1  Conjugation of IAA to BSA

106

                 4.1.2  Production of antiserum

108

                             4.1.2.1  Immunization

108

                             4.1.2.2  Purification and storage

108

                             4.1.2.3  Titer determination

109

                 4.1.3  Radioimmunoassay

109

                             4.1.3.1  Optimization

109

                                                4.1.3.1.1  Effect of separation technique

109

                                         4.1.3.1.2  Effect of charcoal concentration

110

                                         4.1.3.1.3  Determination of incubation time

110

                                         4.1.3.1.4  Effect of pH

111

                                         4.1.3.1.5  Effect of methanol

111

                             4.1.3.2  Standard procedure

111

                 4.1.4  Validation

112

                             4.1.4.1  Extract dilution curves

112

                             4.1.4.2  Antibody selectivity

113

                 4.1.5  Biological sample preparation

114

                             4.1.5.1  Extraction

114

                             4.1.5.2  Purification

114

     4.2  Results

115

                 4.2.1  IAA-BSA conjugates

115

                 4.2.2  Response to Immunization

116

                 4.2.3  Radioimmunoassay

119

                             4.2.3.1  Effect of separation technique

119

                             4.2.3.2  Effect of charcoal concentration

119

                             4.2.3.3  Effect of pH

121

                             4.2.3.4  Effect of incubation

121

                             4.2.3.5  Effect of methanol

121

                             4.2.3.6  Standard dose-response curve

121

                 4.2.4  Validation

124

                             4.2.4.1 Extract dilution, curves

124

                             4.2.4.2  Cross-reactivities to Anti-IAA-75

124

            4.3  Discussion

126

5          PLANT GROWTH SUBSTANCE TRENDS IN AVOCADO FRUIT

129

     5.1  Fruit Growth and Development

129

                 5.1.1  Materials and methods

129

                             5.1.1.1  Fruit material

129

                             5.1.1.2  Preparation of material and RIA

130

                 5.1.2  Results

131

                             5.1.2.1  Fruit growth morphology

133

                             5.1.2.2  Auxin (Indole-3-acetic acid)

136

                             5.1.2.2  Isopentenyl adenine and isopentenyl adenosine

141

                             5.1.2.2  Free abscisic acid

141

                 5.1.3  Discussion

141

     5.2  Post-harvest PCS Trends in Avocado Fruit

148

                 5.2.1  Materials and methods

148

                             5.2.1.1  Avocado material

148

                             5.2.1.2  Preparation of material and RIA

148

                 5.2.2  Results

150

                             5.2.2.1  General

150

                             5.2.2.2  Abscisic acid

151

                             5.2.2.2  Isopentenyl-type cytokinins

154

                             5.2.2.2  Ethylene

157

                        5.2.3  Discussion

157

OVERALL DISCUSSION AND CONCLUSIONS

162

SUMMARY

169

LITERATURE CITED

172