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Varietal evaluation and quality enhancement of cut tulips by exogenous application of growth promoting substances
By
Mohsin Bashir M.Sc. (Hons.) Horticulture
A thesis submitted in the partial fulfillment of the requirement for the degree of
Doctor of Philosophy In Horticulture Institute of Horticultural Sciences, Faculty of Agriculture, University of Agriculture, Faisalabad, Pakistan 2017
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To
The Controller of Examinations, University of Agriculture, Faisalabad.
We the supervisory committee, certify that after necessary revision in accordance to the suggestions made by external reviewers, the contents and forms of the thesis of Mr. Mohsin Bashir (Regd. No. 2004-ag-1447) have been found satisfactory. The final thesis is therefore may kindly be submitted for the notification of the PhD degree.
SUPERVISORY COMMITTEE
1. Supervisor ______(Prof. Dr. Muhammad Aslam Khan)
2. Member ______(Prof. Dr. Muhammad Qasim)
3. Member ______(Prof. Dr. Shahzad M. A. Basra)
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DECLARATION
“I hereby declare that contents of thesis, entitled “Varietal evaluation and quality enhancement of cut tulips by exogenous application of growth promoting substances” are the product of my own research and no part has been copied from any published source
(except the references, standard mathematical or genetic models/equation/formulae etc.). I further declare that this work has not been submitted for award of any other diploma/degree.
The university may take action if the information provided is found inaccurate at any stage
(In case of any default the scholar will be proceeded against as per HEC plagiarism policy).”
.
Mohsin Bashir
Reg. No. 2004-ag-1447
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WHO ALWAYS RAISED THEIR HANDS FOR MY SUCCESS
AND HAPPINESS, SO MUCH OF WHAT I AM TODAY IS BECAUSE OF THEM AND I WANT TO TELL THEM THAT I THANK THEM, APPRECIATE THEM AND LOVE THEM FROM THE CORE OF MY HEART, EVER AND FOREVER
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ACKNOWLEDGMENTS
All praises and thanks are for Almighty Allah, the Merciful, the only creator of the universe and source of all knowledge and wisdom, who blessed me with health, thoughts, talented teachers, helping friends and opportunity to complete this study. I am honor to express my deepest sense of gratitude and indebtedness to my honorable supervisor Dr. Muhammad Aslam Khan, Professor Institute of Horticultural Sciences, U.A.F, for his supervision and help in writing of this manuscript together with thankful to the members of my supervisory committee, Dr. Muhammad Qasim , Professor, Inst. of Horticultural Sciences, U.A.F., and Dr. Shahzad M.A. Basra, Professor, Department of Agronomy for their kind help and constructive criticism during the course of study for the accomplishment of the work presented in this thesis. I take this opportunity to record my deep sense of gratefulness and appreciation to my honorable teacher Prof. Dr. Asif Ali, Vice Chancellor, Muhammad Nawaz Sharif University of Agriculture, Multan for his constant encouragement, inspiring guidance, constructive criticism and keen interest throughout these investigations. I deem it my utmost pleasure in expressing my cardise gratitude with the profound benedictions to my post graduate supervisor Dr. Iftikhar Ahmad, Asstt. Prof. Inst. of Horticultural Sciences, U.A.F. for his deep concern and guidance in my research and carrier. Dr. C.M. Ayyub, Associate. Prof. Inst. of Horticultural Sciences, U.A.F. for his kind cooperation and moral support in providing chemical materials and instruments used in the execution of this research and data handling. Dr. Khurram Ziaf, Asstt. Prof. Inst. of Horticultural Sciences, U.A.F. for his guidance in drafting and interpretation of the results. Dr. Muhammad Shahid, Associate. Prof. Department of Biochemistry, U.A.F. for his support in conducting biochemical analysis and their interpretation in data analysis. Efforts of the above mentioned scientists have always inculcated the spirit of hard work and maintenance of professional integrity besides other valuable suggestions will always serve as a beacon of light through the course of my life. No acknowledgement could ever adequately express my obligations to my affectionate and adoring parents whose hands always rose in prayers for me and without whose moral and financial support; the present distinction would have merely been a dream.
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They always acted as a light house for me in the dark oceans of life path. No words can really express for the feelings that I have for my beloved parents. The name of my parents will always be in front of my eyes, as I will look on the cover of my thesis, even though my name may be printed on it. I pay my thanks for my family who helped me in all the process of completing this manuscript for keeping my esteem rocket high, especially my Father and mother who through their motivational statements helped me whenever I felt low, without their help, support and prayers this thesis would have never been completed. Special love and thanks to my loving friends for their kind sincerity, supportive and joyful company Raheel Sultan Khan, Sheikh Asim Shahzad, Sheikh Ahsan Jamal, Sheri Bhai, Bobby Bhai, Qb, Tada, Hanu and Mian Rehan Khalid. Likewise, I am thankful to my friends of all the time Hafiz Nazar Faried, Asstt. Prof. (M.N.S.U.A.) and M. Rizwan Asghar for their motivational support. They always stood by me mentally and spiritually, their encouraging love boosted my moral and pursues higher ideas in my life. Moreover, I would like to extend my gratitude to my postgraduate students particularly Abbad Shaukat, Usman Nadeem and Faheem Anjum for their cooperation during this study period. Finally, I would like to convey my admiration to highly caring and helping Mrs. Maryam Ali. Conclusively, without their constant encouragement and moral support, this work would have not been possible.
(Mohsin Bashir)
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TABLE OF CONTENTS Chapter CONTENTS Page No. No. Acknowledgements v Table of contents vi List of tables xv List of figures xvi List of abbreviations xxiv Abstract xxv 1 Introduction 1 2 Review of Literature 2 2.1 Agriculture and Climate Change 5 2.2 Plant growth in unfavorable environment 6 2.3 Effect of plant growth modulating substances in floriculture 7 2.4 Source of chitosan 7 2.5 General features of chitosan 8 2.6 Biochemical features of chitosan 8 2.7 Physiological effects of chitosan in plants 9 2.8 Chitosan and plant defense mechanism 11 2.9 Use of chitosan in floriculture 13 2.10 Use of chitosan in some horticultural crops 16 2.11 Plants and malevolent environment 20 2.12 Tailoring a response to specific stress condition 20 2.13 Glycine betaine and acclimation of plants 22 2.14 Biosynthetic pathway of glycine betaine 23 2.15 Foliar application of glycine betaine and its physiological effects 25 2.16 Polyamines 30 2.17 Polyamines and their role in abiotic stress 31 2.18 Foliar application of PAs and abiotic stress 32 2.19 ROS and polyamine catabolism 33 2.20 Stress retention and polyamines function 34 2.21 Putrescine and induction of stress tolerance 35 2.22 Spermidine, an essential polyamine for plant growth 37 2.23 Spermine and plant stress response 38 2.24 Thermospermine induces stem elongation 40 2.25 Polyamines their oxidation and defence mechanism 41 2.26 Prospects of polyamines as exogenous application 41 3 Materials and Methods 43 3.1 Experiment # 1: Screening of different tulip cultivars under 43 Faisalabad conditions based upon their growth response 3.1.1 Materials and methods 43 3.1.2 Treatments 43 3.1.3 Plant material and growth conditions 44 3.1.4 Data collection 46
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3.1.5 Calculation of time to reach 50% sprouting (E50) 46 3.1.6 Calculation of mean emergence time (MET) 46 3.1.7 Calculation of final emergence percentage (FEP) 46 3.1.8 Calculation of emergence index (EI) 46 3.1.9 Measurement of plant height (cm) 47 3.1.10 Measurement of plant fresh mass (g) 47 3.1.11 Measurement of plant dry mass (g) 47 3.1.12 Measurement of leaf area (cm2) 47 3.1.13 Measurement of leaf chlorophyll contents (SPAD Value) 47 3.1.14 Measurement of days to flower bud emergence (days) 47 3.1.15 Measurement of tepal diameter (mm) 47 3.1.16 Measurement of flower diameter (mm) 47 3.1.17 Measurement of length of petals (cm) 47 3.1.18 Measurement of width of petals (cm) 48 3.1.19 Measurement of scape diameter (mm) 48 3.1.20 Measurement of flower quality 48 3.1.21 Calculation of vase life (days) 48 3.1.22 Calculation of days to start petal senescence (days) 48 3.1.23 Calculation of tepal abscission (days) 48 3.1.24 Calculation of mass of bulbils per clump (g) 48 3.1.25 Calculation of diameter of bulbils per clump (mm) 48 3.1.26 Calculation of number of bulbils per clump 48 3.1.27 Experimental Design and Statistical Analysis 48 3.2 Experiment # 2: Exogenous application of chitosan for the 49 assessment of growth and quality of different tulip cultivars 3.2.1 Materials and methods 49 3.2.2 Treatments 49 3.2.3 Plant material and growth conditions 50 3.2.4 Exogenous application of chitosan 50 3.2.5 Data Collection and sampling 50 3.2.6 Parameters studied 3.2.6.1 Morphological attributes 51 3.2.6.1.1 Measurement of plant height (cm) 51 3.2.6.1.2 Measurement of plant fresh mass (g) 51 3.2.6.1.3 Measurement of plant dry mass (g) 51 3.2.6.1.3 Measurement of leaf area (cm2) 51 3.2.6.2 Physiological attributes 51 3.2.6.2 Photosynthesis rate (A), transpiration rate (E), stomatal 51 conductance (gs) and sub-stomatal CO2 (Ci) 3.2.6.2.1 Instantaneous water use efficiency (WUE instantaneous) 52 3.2.6.2.2 Intrinsic water use efficiency (WUE intrinsic) 52 –2 –1 3.2.6.2.3 Mesophyll conductance (gm) to CO2 assimilation(mol CO2 m s ) 52 3.2.6.2.4 Leaf’s total chlorophyll contents (SPAD value) 52 3.2.6.3 Biochemical attributes 52 3.2.6.3.1 Malondialdehyde (MDA) contents 53
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3.2.6.3.2 Total phenolic contents (mg GAE/100g fresh mass) 53 3.2.6.3.3 Hydrogen peroxide (H2O2) Scavenging Capacity (%) 53 3.2.6.3.4 DPPH (1, 1-Diphenyl-2-picrlhydrazyl) radical scavenging activity 54 (% inhibition) 3.2.6.4 Measurement of antioxidative enzymes 54 3.2.6.4.1 Superoxide dismutase activity (Units/mg Protein) 54 3.2.6.4.2 Catalase activity (Units/mg Protein) 54 3.2.6.4.3 Peroxidase activity (Units/mg Protein) 55 3.2.6.5 Measurement of flower attributes 55 3.2.6.5.1 Calculation of days to flower bud emergence (days) 55 3.2.6.5.2 Measurement of tepal diameter (mm) 55 3.2.6.5.3 Measurement of flower diameter (mm) 55 3.2.6.5.4 Measurement of length of petals (cm) 55 3.2.6.5.5 Measurement of width of petals (cm) 55 3.2.6.5.6 Measurement of scape diameter (mm) 56 3.2.6.5.7 Measurement of flower quality (Copper and Spokas, 1991) 56 3.2.6.6 Measurement of shelf-life attributes 56 3.2.6.6.1 Calculation of vase life (days) 56 3.2.6.6.2 Calculation of days to start senescence (days) 56 3.2.6.6.3 Calculation of tepal abscission (days) 57 3.2.6.7 Measurement of bulb attributes 57 3.2.6.7.1 Calculation of mass of bulbils per clump (g) 57 3.2.6.7.2 Calculation of diameter of bulbils per clump (mm) 57 3.2.6.7.3 Calculation of number of bulbils per clump 57 3.2.7 Experimental Design and Statistical Analysis 57 3.3 Experiment # 3: Exogenous application of glycine betaine for 58 the assessment of growth and quality of different tulip cultivars 3.3.1 Materials and methods 58 3.3.2 Treatments 58 3.3.3 Plant material and growth conditions 58 3.3.4 Exogenous application of glycine betaine 58 3.3.5 Data Collection and sampling 59 3.3.6 Parameters studied 59 3.3.7 Experimental Design and Statistical Analysis 59 3.4 Experiment # 4: Comparative effect of exogenous application 59 of different levels of polyamines (putrescine, spermine and spermidine) on two tulip cultivars to assess cut flower quality 3.4.1 Materials and methods 59 3.4.2 Treatments 60 3.4.3 Plant material and growth conditions 60 3.4.4 Exogenous application of glycine betaine 61 3.4.5 Data Collection and sampling 61 3.4.6 Parameters studied 61
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3.4.7 Experimental Design and Statistical Analysis 61 4 Results 62 4.1 Experiment # 1 Screening of different tulip cultivars under 62 Faisalabad climatic conditions based upon their growth response 4.1.1 Time to start sprouting 63 4.1.2 Time to reach 50% sprouting (E50) 63 4.1.3 Mean Emergence Time (MET) 65 4.1.4 Final Emergence Percentage (FEP) 66 4.1.5 Emergence Index (EI) 67 4.1.6 Plant height (cm) 68 4.1.7 Plant fresh mass (g) 69 4.1.8 Plant dry mass (g) 70 4.1.9 Leaf area (cm2) 71 4.1.10 Chlorophyll contents (SPAD Value) 72 4.1.11 Days to flower bud emergence (d) 73 4.1.12 Tepal diameter (mm) 74 4.1.13 Flower diameter (mm) 75 4.1.14 Length of petals (cm) 76 4.1.15 Width of petals (cm) 77 4.1.16 Scape diameter (mm) 78 4.1.17 Flower quality (Copper and Spokas, 1991) 79 4.1.18 Vase life (d) 80 4.1.19 Days to start petal senescence (d) 81 4.1.20 Tepal abscission (d) 82 4.1.21 Mass of bulbils per clump (g) 83 4.1.22 Number of bulbils 84 4.1.23 Diameter of bulbils (mm) 85 4.1.24 Images showing growth response of different cultivars of tulip 87 grown under Faisalabad conditions 4.2 Experiment # 2 Exogenous application of chitosan for growth 89 and quality assessment of different tulip cultivars 4.2.1 Plant height (cm) 89 4.2.1.1 Images showing response of various concentrations of chitosan on 90 plant height of five tulip cultivars 4.2.2 Plant fresh mass (g) 92 4.2.3 Plant dry mass (g) 93 4.2.4 Leaf area (cm2) 94 4.2.5 Photosynthetic rate (A) (µmol m-2s-1) 95 4.2.6 Transpiration rate (E) (mmol m-2s-1) 96 4.2.7 Instantaneous Water Use Efficiency (A/E) 97 4.2.8 Intrinsic Water Use Efficiency (A/gs) 98 -1 4.2.9 Sub-stomatal CO2 (Ci) (µmol mol ) 99 4.2.10 Stomatal Conductance (gs) (mmol m-2s-1) 100 4.2.11 Chlorophyll contents (SPAD Value) 101
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4.2.12 Mesophyll Conductance (A/Ci) 102 4.2.13 Superoxide dismutase (SOD) activity (Units/mg Protein) 103 4.2.14 Catalase (CAT) activity (Units/mg Protein) 104 4.2.15 DPPH Scavenging Activity (% inhibition) 105 4.2.16 Peroxidase (POD) activity (Units/mg Protein) 106 4.2.17 Malondialdehyde (MDA) contents (µmol g-1 fresh weight) 107 4.2.18 Total Phenolic Compounds (mg GAE/100g FW) 108 4.2.19 H2O2 Scavenging Activity (%) 109 4.2.20 Days to flower bud emergence (d) 110 4.2.21 Tepal diameter (mm) 111 4.2.22 Flower diameter (mm) 112 4.2.23 Length of petals (cm) 113 4.2.24 Width of petals (cm) 114 4.2.25 Scape diameter (mm) 115 4.2.26 Flower quality (Copper and Spokas, 1991) 116 4.2.27 Vase life (d) 117 4.2.28 Images depicting the response of various concentrations of 118 chitosan on vase display of five tulip cultivars 4.2.29 Days to start petal senescence (d) 121 4.2.30 Tepal abscission (d) 122 4.2.31 Diameter of bulbils (mm) 123 4.2.32 Mass of bulbils per clump (g) 124 4.2.33 Number of bulbils 125 4.3 Experiment # 3 Exogenous application of glycine betaine for 128 growth and quality assessment of different tulip cultivars 4.3.1 Plant height (cm) 128 4.3.1.1 Images showing the response of various concentrations of glycine 129 betaine on plant height of five tulip cultivars 4.3.2 Plant fresh mass (g) 131 4.3.3 Plant dry mass (g) 132 4.3.4 Leaf area (cm2) 133 4.3.5 Photosynthetic rate (A) (µmol m-2s-1) 134 4.3.6 Transpiration rate (E) (mmol m-2s-1) 135 4.3.7 Instantaneous Water Use Efficiency (A/E) 136 4.3.8 Intrinsic Water Use Efficiency (A/gs) 137 -1 4.3.9 Sub-stomatal CO2 (Ci) (µmol mol ) 138 4.3.10 Stomatal Conductance (gs) (mmol m-2s-1) 139 4.3.11 Chlorophyll contents (SPAD Value) 140 4.3.12 Mesophyll Conductance (A/Ci) 141 4.3.13 Superoxide dismutase (SOD) activity (Units/mg Protein) 142 4.3.14 Catalase (CAT) activity (Units/mg Protein) 143 4.3.15 DPPH Scavenging Activity (% inhibition) 144 4.3.16 Peroxidase (POD) activity (Units/mg Protein) 145 4.3.17 Malondialdehyde (MDA) contents (µmol g-1 fresh weight) 146 4.3.18 Total Phenolic Compounds (mg GAE/100g FW) 147
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4.3.19 H2O2 Scavenging Activity (%) 148 4.3.20 Days to flower bud emergence (d) 149 4.3.21 Tepal diameter (mm) 150 4.3.22 Flower diameter (mm) 151 4.3.23 Length of petals (cm) 152 4.3.24 Width of petals (cm) 153 4.3.25 Scape diameter (mm) 154 4.3.26 Flower quality (Copper and Spokas, 1991) 155 4.3.27 Vase life (d) 156 4.3.28 Images showing the response of various concentrations of glycine 157 betaine on vase display of five tulip cultivars 4.3.29 Days to start petal senescence (d) 160 4.3.30 Tepal abscission (d) 161 4.3.31 Diameter of bulbils (mm) 162 4.3.32 Mass of bulbils per clump (g) 163 4.3.33 Number of bulbils 164 4.4 Experiment # 4 Comparative effect of exogenous application of 167 different levels of polyamines (Putrescine, spermine and spermidine) on two tulip cultivars to assess the cut flower quality 4.4.1 Plant height (cm) 167 4.4.2 Plant fresh mass (g) 168 4.4.3 Plant dry mass (g) 169 4.4.4 Leaf area (cm2) 170 4.4.5 Photosynthetic rate (A) (µmol m-2s-1) 171 4.4.6 Transpiration rate (E) (mmol m-2s-1) 172 4.4.7 Instantaneous Water Use Efficiency (A/E) 173 4.4.8 Intrinsic Water Use Efficiency (A/gs) 174 -1 4.4.9 Sub-stomatal CO2 (Ci) (µmol mol ) 175 4.4.10 Stomatal Conductance (gs) (mmol m-2s-1) 176 4.4.11 Chlorophyll contents (SPAD Value) 177 4.4.12 Mesophyll Conductance (A/Ci) 178 4.4.13 Superoxide dismutase (SOD) activity (Units/mg Protein) 179 4.4.14 Catalase (CAT) activity (Units/mg Protein) 180 4.4.15 DPPH Scavenging Activity (% inhibition) 181 4.4.16 Peroxidase (POD) activity (Units/mg Protein) 182 4.4.17 Malondialdehyde (MDA) contents (µmol g-1 fresh weight) 183 4.4.18 Total Phenolic Compounds (mg GAE/100g FW) 184 4.4.19 H2O2 Scavenging Activity (%) 185 4.4.20 Days to flower bud emergence (d) 186 4.4.21 Tepal diameter (mm) 187 4.4.22 Flower diameter (mm) 188 4.4.23 Length of petals (cm) 189 4.4.24 Width of petals (cm) 190 4.4.25 Scape diameter (mm) 191
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4.4.26 Flower quality (Copper and Spokas, 1991) 192 4.4.27 Vase life (d) 193 4.4.28 Images showing the response of various concentrations of 194 polyamines on vase life display of two tulip cultivars 4.4.29 Days to start petal senescence (d) 195 4.4.30 Tepal abscission (d) 196 4.4.31 Diameter of bulbils (mm) 197 4.4.32 Mass of bulbils per clump (g) 198 4.4.33 Number of bulbils 199 5 Discussion 202 5.1 Conclusion 221 6 Summary 223 6.1 Disclaimer 226 6.2 Recommendations 226 6.3 Future prospects 226 Literature cited 227 Appendices 265
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LIST OF TABLES
Table # Title Page # 2.1 Response of foliar application of glycine betaine on various plants 29 3.1 Physical and chemical properties of soil 44 3.2 Macronutritional status of soil 44 3.3 Various tulip cultivars tested for their adaptability 45 4.1 Pearson’s correlation among various attributes of tulip cultivars 86
4.2 (a) Pearson’s correlation matrix among various morpho-physiological 126 attributes of tulip cultivars in response to chitosan application
4.2 (b) Pearson’s correlation matrix among various antioxidant enzymes 127 activity and biochemical changes induced on floral attributes of tulip cultivars in response to chitosan application 4.3 (a) Pearson’s correlation matrix among various morpho-physiological 165 attributes of tulip cultivars in response to glycine betaine application 4.3 (b) Pearson’s correlation matrix among various antioxidant enzymes 166 activity and biochemical changes induced on floral attributes of tulip cultivars in response to glycine betaine application 4.4 (a) Pearson’s correlation matrix among various morpho-physiological 200 attributes of two tulip cultivars in response to Polyamines application 4.4 (b) Pearson’s correlation matrix among various antioxidant enzymes 201 activity and biochemical changes induced on floral attributes of two tulip cultivars in response to Polyamines application
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LIST OF FIGURES
Figure # Title Page # 2.1 Different pathways explaining glycine betaine biosynthesis 24 2.2 Biosynthetic pathway of polyamines 36 4.1.1 Time to start sprouting of different tulip cultivars 64 4.1.2 Time to reach 50% sprouting of different tulip cultivars 64 4.1.3 Mean Emergence Time (MET) of different tulip cultivars 65 4.1.4 Final Emergence Percentage (FEP) of different tulip cultivars 66
4.1.5 Emergence Index (EI) of different tulip cultivars 67 4.1.6 Plant height of different tulip cultivars 68
4.1.7 Plant fresh mass of different tulip cultivars 69 4.1.8 Plant dry mass of different tulip cultivars 70 4.1.9 Leaf area of different tulip cultivars 71 4.1.10 Chlorophyll contents (SPAD Value) of different tulip cultivars 72 4.1.11 Days to flower bud emergence of different tulip cultivars 73 4.1.12 Tepal diameter of different tulip cultivars 74 4.1.13 Flower diameter of different tulip cultivars 75 4.1.14 Length of petals of different tulip cultivars 76 4.1.15 Width of petals of different tulip cultivars 77 4.16 Scape diameter of different tulip cultivars 78 4.1.17 Flower quality of different tulip cultivars 79 4.1.18 Vase life of different tulip cultivars 80 4.1.19 Days to start petal senescence of different tulip cultivars 81 4.1.20 Tepal abscission of different tulip cultivars 82
4.1.21 Mass of bulbils per clump of different tulip cultivars 83 4.1.22 Number of bulbils of different tulip cultivars 84
4.1.23 Bulbil diameter of different tulip cultivars 85 4.1.24.1 Apeldorn 87 4.1.24.2 Barcelona 87 4.1.24.3 Clear water 87
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Figure # Title Page # 4.1.24.4 Golden Apeldorn 87 4.1.24.5 Ile de France 87 4.1.24.6 Leen Vander Mark 87 4.1.24.7 Parade 88 4.1.24.8 Purple Flag 88 4.1.24.9 Queen of night 88 4.1.24.10 Queen of night at harvesting stage 88 4.1.24.11 Spring green 88 4.1.24.12 Spring green on bloom 88 4.2.1 Response of various concentrations of chitosan on plant height of 89 five tulip cultivars
4.2.1.1 Plant heights of Apeldorn cultivar in response to various levels of 90 chitosan 4.2.1.2 Plant heights of Barcelona cultivar in response to various levels of 90 chitosan 4.2.1.3 Plant heights of Ile de France cultivar in response to various levels 90 of chitosan 4.2.1.4 Plant height of Leen Vander Mark cultivar in response to various 91 levels of chitosan 4.2.1.5 Plant height of Parade cultivar in response to various levels of 91 chitosan 4.2.2 Response of various concentrations of chitosan on plant fresh 92 mass of five tulip cultivars
4.2.3 Response of various concentrations of chitosan on plant dry mass 93 of five tulip cultivars 4.2.4 Response of various concentrations of chitosan on leaf area of five 94 tulip cultivars 4.2.5 Response of various concentrations of chitosan on photosynthetic 95 rate of five tulip cultivars
4.2.6 Response of various concentrations of chitosan on transpiration 96 rate of five tulip cultivars 4.2.7 Response of various concentrations of chitosan on Instantaneous 97 water use efficiency (A/E) of five tulip cultivars
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Figure # Title Page # 4.2.8 Response of various concentrations of chitosan on Intrinsic water 98 use efficiency (A/gs) of five tulip cultivars 4.2.9 Response of various concentrations of chitosan on Sub-stomatal 99 CO2 (Ci) of five tulip cultivars 4.2.10 Response of various concentrations of chitosan on Stomatal 100 conductance (gs) of five tulip cultivars 4.2.11 Response of various concentrations of chitosan on chlorophyll 101 contents of five tulip cultivars 4.2.12 Response of various concentrations of chitosan on Mesophyll 102 conductance (A/Ci) of five tulip cultivars 4.2.13 Response of various concentrations of chitosan on Superoxide 103 dismutase activity of five tulip cultivars 4.2.14 Response of various concentrations of chitosan on Catalase 104 activity of five tulip cultivars 4.2.15 Response of various concentrations of chitosan on DPPH 105 Scavenging Activity of five tulip cultivars 4.2.16 Response of various concentrations of chitosan on Peroxidase 106 activity of five tulip cultivars 4.2.17 Response of various concentrations of chitosan on 107 Malondialdehyde contents of five tulip cultivars 4.2.18 Response of various concentrations of chitosan on Total Phenolic 108 compounds of five tulip cultivars
4.2.19 Response of various concentrations of chitosan on H2O2 109 Scavenging Activity of five tulip cultivars 4.2.20 Response of various concentrations of chitosan on days to flower 110 bud emergence of five tulip cultivars
4.2.21 Response of various concentrations of chitosan on tepal diameter 111 of five tulip cultivars 4.2.22 Response of various concentrations of chitosan on flower diameter 112 of five tulip cultivars 4.2.23 Response of various concentrations of chitosan on length of petals 113 of five tulip cultivars 4.2.24 Response of various concentrations of chitosan on width of petals 114 of five tulip cultivars 4.2.25 Response of various concentrations of chitosan on scape diameter 115 of five tulip cultivars
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Figure # Title Page # 4.2.26 Response of various concentrations of chitosan on flower quality 116 of five tulip cultivars 4.2.27 Response of various concentrations of chitosan on vase life of five 117 tulip cultivars 4.2.28.1 Vase display of Apeldorn cultivar on 13th day after harvest 118 4.2.28.2 Vase display of Barcelona cultivar on 7th day after harvest 118 4.2.28.3 Vase display of Ile de France cultivar on 12th day after harvest 119 4.2.28.4 Vase display of Leen Vander Mark cultivar on 14th day after 119 harvest th 4.2.28.5 Vase display of Leen Vander Mark cultivar on 11 day after 120 harvest
4.2.29 Response of various concentrations of chitosan on days to start 121 petal senescence of five tulip cultivars 4.2.30 Response of various concentrations of chitosan on tepal abscission 122 of five tulip cultivars
4.2.31 Response of various concentrations of chitosan on diameter of 123 bulbils of five tulip cultivars 4.2.32 Response of various concentrations of chitosan on mass of bulbils 124 per clump of five tulip cultivars 4.2.33 Response of various concentrations of chitosan on number of 125 bulbils of five tulip cultivars
4.3.1 Response of various concentrations of glycine betaine on plant 128 height of five tulip cultivars
4.3.1.1 Plant heights of ‘Apeldorn’ in response to various levels of glycine 129 betaine
4.3.1.2 Plant heights of Barcelona in response to various levels of glycine 129 betaine
4.3.1.3 Plant heights of Leen Vander Mark in response to various levels of 129 glycine betaine
4.3.1.4 Plant heights of Ile de France in response to various levels of 130 glycine betaine
4.3.1.5 Plant heights of Parade in response to various levels of glycine 130 betaine
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Figure # Title Page # 4.3.2 Response of various concentrations of glycine betaine on plant 131 fresh mass of five tulip cultivars 4.3.3 Response of various concentrations of glycine betaine on plant dry 132 mass of five tulip cultivars 4.3.4 Response of various concentrations of glycine betaine on leaf area 133 of five tulip cultivars
4.3.5 Response of various concentrations of glycine betaine on 134 photosynthetic rate (A) of five tulip cultivars 4.3.6 Response of various concentrations of glycine betaine on 135 transpiration rate (E) of five tulip cultivars 4.3.7 Response of various concentrations of glycine betaine on 136 Instantaneous water use efficiency (A/E) of five tulip cultivars
4.3.8 Response of various concentrations of glycine betaine on Intrinsic 137 water use efficiency (A/gs) of five tulip cultivars 4.3.9 Response of various concentrations of glycine betaine on Sub- 138 stomatal CO2 (Ci) of five tulip cultivars 4.3.10 Response of various concentrations of glycine betaine on Stomatal 139 conductance (gs) of five tulip cultivars 4.3.11 Response of various concentrations of glycine betaine on 140 chlorophyll contents of five tulip cultivars
4.3.12 Response of various concentrations of glycine betaine on 141 Mesophyll conductance (A/Ci) of five tulip cultivars 4.3.13 Response of various concentrations of glycine betaine on 142 Superoxide dismutase activity of five tulip cultivars
4.3.14 Response of various concentrations of glycine betaine on Catalase 143 activity of five tulip cultivars
4.3.15 Response of various concentrations of glycine betaine on DPPH 144 Scavenging Activity of five tulip cultivars 4.3.16 Response of various concentrations of glycine betaine on 145 Peroxidase activity of five tulip cultivars 4.3.17 Response of various concentrations of glycine betaine on 146 Malondialdehyde contents of five tulip cultivars 4.3.18 Response of various concentrations of glycine betaine on Total 147 Phenolic compounds of five tulip cultivars
4.3.19 Response of various concentrations of glycine betaine on H2O2 148
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Figure # Title Page # Scavenging Activity of five tulip cultivars 4.3.20 Response of various concentrations of glycine betaine on days to 149 flower bud emergence of five tulip cultivars 4.3.21 Response of various concentrations of glycine betaine on tepal 150 diameter of five tulip cultivars
4.3.22 Response of various concentrations of glycine betaine on flower 151 diameter of five tulip cultivars 4.3.23 Response of various concentrations of glycine betaine on length of 152 petals of five tulip cultivars
4.3.24 Response of various concentrations of glycine betaine on width of 153 petals of five tulip cultivars 4.3.25 Response of various concentrations of glycine betaine on scape 154 diameter of five tulip cultivars 4.3.26 Response of various concentrations of glycine betaine on flower 155 quality of five tulip cultivars 4.3.27 Response of various concentrations of glycine betaine on vase life 156 of five tulip cultivars
th 4.3.28.1 Vase display of Apeldorn cultivar on 12 day after harvest 157
4.3.28.2 Vase display of Barcelona cultivar on 6th day after harvest 157 4.3.28.3 Vase display of Ile de France cultivar on 10th day after harvest 158 4.3.28.4 Vase display of Ile de France cultivar on 12th day after harvest 158 4.3.28.5 Vase display of Parade cultivar on 11th day after harvest 159 4.3.29 Response of various concentrations of glycine betaine on days to 160 start petal senescence of five tulip cultivars
4.3.30 Response of various concentrations of glycine betaine on tepal 161 abscission of five tulip cultivars 4.3.31 Response of various concentrations of glycine betaine on diameter 162 of bulbils of five tulip cultivars 4.3.32 Response of various concentrations of glycine betaine on mass of 163 bulbils per clump of five tulip cultivars 4.3.33 Response of various concentrations of glycine betaine on number 164 of bulbils of five tulip cultivars 4.4.1 Response of various concentrations of polyamines on Plant height 167 of two tulip cultivars
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Figure # Title Page # 4.4.2 Response of various concentrations of polyamines on Plant fresh 168 mass of two tulip cultivars
4.4.3 Response of various concentrations of polyamines on Plant dry 169 mass of two tulip cultivars
4.4.4 Response of various concentrations of polyamines on Leaf area of 170 two tulip cultivars
4.4.5 Response of various concentrations of polyamines on 171 Photosynthetic rate of two tulip cultivar 4.4.6 Response of various concentrations of polyamines on 172 Transpiration rate of two tulip cultivars
4.4.7 Response of various concentrations of polyamines on 173 Instantaneous water use efficiency of two tulip cultivars 4.4.8 Response of various concentrations of polyamines on Intrinsic 174 water use efficiency of two tulip cultivars 4.4.9 Response of various concentrations of polyamines on Sub- 175 stomatal CO2 of two tulip cultivars 4.4.10 Response of various concentrations of polyamines on Stomatal 176 conductance of two tulip cultivars 4.4.11 Response of various concentrations of polyamines on Mesophyll 177 conductance of two tulip cultivars 4.4.12 Response of various concentrations of polyamines on SPAD value 178 of two tulip cultivars
4.4.13 Response of various concentrations of polyamines on Superoxide 179 dismutase activity of two tulip cultivars 4.4.14 Response of various concentrations of polyamines on Peroxidase 180 activity of two tulip cultivars 4.4.15 Response of various concentrations of polyamines on Catalase 181 activity of two tulip cultivars
4.4.16 Response of various concentrations of polyamines on DPPH 182 Scavenging activity of two tulip cultivars 4.4.17 Response of various concentrations of polyamines on 183 Malondialdehyde contents of two tulip cultivars
4.4.18 Response of various concentrations of polyamines on Total 184 Phenolic compounds of two tulip cultivars
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Figure # Title Page #
4.4.19 Response of various concentrations of polyamines on H2O2 185 Scavenging activity of two tulip cultivars 4.4.20 Response of various concentrations of polyamines on days to 186 flower bud emergence of two tulip cultivars 4.4.21 Response of various concentrations of polyamines on plant fresh 187 mass of two tulip cultivars
4.4.22 Response of various concentrations of polyamines on plant fresh 188 mass of two tulip cultivars
4.4.23 Response of various concentrations of polyamines on Length of 189 petals of two tulip cultivars
4.4.24 Response of various concentrations of polyamines on width of 190 petals of two tulip cultivars
4.4.25 Response of various concentrations of polyamines on Scape 191 diameter of two tulip cultivars
4.4.26 Response of various concentrations of polyamines on Flower 192 quality of two tulip cultivars
4.4.27 Response of various concentrations of polyamines on vase life of 193 two tulip cultivars
4.4.28.1 Vase display of Apeldorn cultivar at 8th day (a-d), (e-g) at 13th day 194 and (h-j) at 10th day after harvesting 4.4.28.2 Vase display of Clear water cultivar at 8th day (a-d), (e-g) at 13th 194 day and (h-j) at 11th day after harvesting 4.4.29 Response of various concentrations of polyamines on days to start 195 petal senescence of two tulip cultivars
4.4.30 Response of various concentrations of polyamines on tepal 196 abscission of two tulip cultivars
4.4.31 Response of various concentrations of polyamines on Mass of 197 bulbils per clump of two tulip cultivars
4.4.32 Response of various concentrations of polyamines on Diameter of 198 bulbils per clump of two tulip cultivars 4.4.33 Response of various concentrations of polyamines on Number of 199 bulbils per clump of two tulip cultivars
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LIST OF ABBREVIATIONS
Abbreviation Description °C Degree celsius A Photosynthetic rate A/E Instantaneous Water Use Efficiency A/gs Intrinsic Water Use Efficiency CAT Catalase Ci Sub-stomatal CO2 cm Centimeter DD Degree of deacetylation DW Distilled Water E Transpiration rate E50 Time to reach 50% emergence EI Emergence index FEP Final emergence percentage Fig. Figure g Gram GABA Gamma-Aminobutyric acid GB Glycine betaine gm Mesophyll conductance gs Stomatal conductance kDa Kilodalton LSD Least significant difference LVM Leen Vander Mark MDA Malondialdehyde MET Mean emergence time mgL-1 Miligram per litre mM Milimolar mm Milimeter PEG Polyethylene glycol PLBs Protocorm like bodies POD Peroxidase Put Putrescine S.E Standard Error SOD Superoxide dismutase Spd Spermidine Spm Spermine TPC Total Phenolic contents TSP Time to start sprouting
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ABSTRACT
Tulips are one of the most popular springs of all the time and possess eminent status among various cut flowers cultivated world-wide next to rose and chrysanthemum in the global floriculture trade. However, they are not as productive in subtropical areas as they are in temperate zones. This is attributed by short span of winter season and other abiotic stress factors that are aggravated by climate change. All these factors impede tulip production in subtropical regions. Therefore, a study, comprised of four experiments was conducted to investigate the varietal response of different cultivars of cut tulips under Faisalabad conditions and role of various bioregulators on morpho-physiological, biochemical, enzymatic and ionic attributes that can be used for characterization of inducing tolerance and adaptability in tulip cultivars. Bulbs were planted in open field having sandy loam soil provided with supplemental doze of macronutrients. In first experiment, screening of 10 different tulip cultivars for their growth response and adaptability was carried out on the basis of their sprouting, morphological, floral and bulb attributes. Cultivars were categorized into best performing and least performing on the basis of their performance in 1st experiment. Five better adapted cultivars namely Apeldorn, Barcelona, Ile de France, Leen Vander Mark and Parade were selected for further study. In 2nd and 3rd experiment exogenous application of chitosan and glycine betaine, respectively were carried out to assess the performance of various attributes for quality enhancement. Moreover, in 3rd experiment, response of different polyamines (putrescine, spermine and spermidine) was studied in two cultivars Apeldorn and Clear water by their exogenous application at different concentrations. Results of 1st experiment depicted highly significant difference among all the tested cultivars that all varieties behaved differently and some of them showed better adaptability that have potential for production on commercial basis in the country. Furthermore, optimized levels of chitosan and glycine betaine were also identified that enhanced the growth response of different tulip cultivars. Inferences were developed on the basis of standards needed for any cut flower crop like increased quality parameters flower size, stem length, freshness and postharvest attributes. Results depicted exogenous application of chitosan and glycine betaine improved plant fresh mass, height, leaf area, chlorophyll contents, photosynthetic rate (A), transpiration rate (E), water use efficiency (WUE), stomatal conductance (gs), sub-stomatal CO2 (Ci), antioxidant enzymes and all other attributes associated to flower morphology. Furthermore,
xxv these compounds also improved the postharvest longevity by causing significant decrease in MDA contents. Though the chemicals used did not improve the mass, diameter and number of perrenating organs, thus they imparted negative effects on bulb attributes necessary for the growers to use them in the preceding year. In 4th experiment, 0.03 mM spermine (Spm) concentration proved to be the best for enhancing flower quality attributes in Apeldorn cultivar while low level of spermine (Spm) 0.01 mM proved to be best amongst all other tested dozes of polyamines in Clear water cultivar. Overall best performing traits were found in the plants that were exogenously applied by spermine followed by spermidine while putrescine improve petal lengths and width while other attributes remained less affected. Furthermore, findings of 4th experiment clearly indicated that spermine significantly enhanced postharvest potential of both the tulip cultivars by sustaining vase life and improving physiological, biochemical and enzymatic attributes. Overall, it can be extracted that suboptimal environmental conditions were found to be injurious for tulip growth and productivity and exogenous application of chitosan and glycine betaine specific concentration for particular cultivars were found effective tool for enhancing growth potential. However, effect of polyamines was more pronounced in both the cultivars reflecting that it may induce similar effects on other tulip cultivars as in majority of the parameters varietal effect was found to be non-significant. Besides, one can establish that A, E, WUE, gs, gm and protein might directly be linked with growth and flower quality attributes as they are severely affected by malicious environmental conditions. Thus it can be concluded from the current findings that by planting better performing cultivars, identified in the research, the growth potential of tulip can be expanded. The application of optimized levels of chitosan and glycine betaine particularly at lower levels proved effective in extending display life of tulips. Both these strategies when applied together could lead to a breakthrough in tulip cut flower production by improving the flower quality achieved through minimizing the life cycle in short duration of winters in sub-tropical terrains.
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Chapter # 1 Introduction
Floriculture industry is a dynamic enterprize in its product varieties, trade volumes and global factors with an annual increasing trend of 6 to 9%. It has demonstrated tremendous increase during last two decades in its geographical sprawl. There are about 120 countries actively involved as global manufactures in this business with the Netherlands as an epicenter of production and distribution (Martsynovska, 2011). Cut flower production in Pakistan is estimated about ten to twelve thousand tons per annum and floriculture is fast emerging sector as a profitable venture for the farmers of small land holdings. Yet it is still in state of inception which is likely to become a booming enterprize in the near future (Manzoor et al., 2001). Tulip, a perennial geophyte from Liliaceae family belongs to genus Tulipa having 150- 160 species. In 16th century, 1st cultivation of tulip is reported as a garden flower in the Netherlands (Singh, 2006). It is one of the most popular springs of all the times and occupies 4th position amongst all cut flowers in the international flower trade (Jhon and Neelofar, 2006). The largest production area of tulips is in the Netherlands that accounts 87% of the area globally. Tulip bulbs are also produced in 14 other countries, headed by Japan, France and Poland (Buschman, 2005). Flowering bulbs show immense range in their morphology, development, developmental biology and responses of physiology due to environmental factors. They add a lot in ornamental business worldwide and are used as industrial bulbs, fresh cut flower and pot plants (Lawson, 1996). The bulbs of this crop require twelve to sixteen weeks of cold temperature exposure to increase the length of flowering stalk. Temperature fluctuations especially at elevated levels, impart negative impacts on growth and development (Kamerbeek et al., 1972). Cold temperature is essential for the adequate growth and successive development of tulip flower stems. Short duration of winter and abrupt weather changes at the onset of spring leads to the production of inferior quality stems with shorter vase life. In winter season, parts of flower develop slowly and quick elongation of stalk in spring season takes place (de Hertogh and le Nard, 1993). Short phase of bloom and production of valuable bulbs for the preceding year are major constraints in the commercial cultivation of tulips. Similarly, significant physiological alterations like cell division, expansion, stimulation and encouragement of flowering are established by the use of growth regulators (Vieira et al., 2010).
1
The structure and function of multicellular organism is organized for effective signaling among various parts like cells, tissues, and organs. In all higher plants, control and coordination of metabolic processes, growth, and organ development depends upon chemical signals among different organs of plants. Plant growth regulators play an important role in response to various environmental stimuli which influence flowering, production behavior and plant development when applied exogenously (Ashraf and Foolad, 2007). Developmental procedures like flower size, number, synchronization of early and regular flowering and stem elongation are directly influenced by primary growth regulators (Al-Khassawneh et al., 2006). In the bulbs of tulip, stem expansion is controlled by flower buds which are modified internodes, reconciled by auxins and gibberellins at the lowest ones (Rees, 1992). The standard and quality of cut flowers is evaluated by its overall appearance and longevity. Disease spots, blemishes, scratches and weak growth is undesirable in this regard. For commercial production of tulips in the country, protocols are needed to be streamlined for their commercial production, capable of presenting fresh produce right from grower to end consumer. For this purpose, plant growth modulating substances are keenly focused by the scientists and they are in the center of their interest because they are concerned in regulating various growth and developmental processes by demonstrating their biological activity even at low concentrations ranging from 10-9- 10-6 M. All biological systems are considered open systems driven by multifaceted energy dynamism intricately regulated either by genetic or physiological mechanisms must adapt to certain degree of control to homeostatic, stationary and high level of functioning even under inimical environmental conditions. Biopolymers like chitosan are useful compounds to synchronize the plant life cycle and may achieve a quality produce. They are advantageous as they promote plant growth by acting as a source of carbon and growth modulator, they also impart resistance against malicious environmental conditions and different pathogens in many crop plants (Fry et al., 1993; Lee et al., 1999). Moreover, it is effective in regulating rate of transpiration and conserves water in plants which could be useful for cut tulips (Bittelli et al., 2001). Oligomeric chitosans exhibit a better antifungal activity which is more effective in many prokaryotes other than bacteria (Savard et al., 2002). Stimulating features of this substance leads to variety of defense mechanism in many plants in response to microbial activity, including the deposition of phytoalexins, pathogenic proteins, inhibitors, lignin biosynthesis, and callose production
2
(Hadrami et al., 2010). In low and non- accumulating plants, chitosan may prove to be useful to combat damaging properties caused by environmental factors (Agboma et al., 1997a,b,c; Makela et al., 1998; Yang and Lu, 2005). Plant life is continuously exposed to environmental vagaries like heat shock, cold damage, light intensity, drought stress, acidity, alkalinity, oxidative and metal injury, which influences growth, development and yield (Ahmad et al., 2008; Ahmad et al., 2010 a and b). Plant growth modulating substances may play cardinal roles in the potential of plants to adapt in varying environments, by facilitating growth, development, nutrient acquisition, and source-sink relationships (Peleg and Blumwald, 2011). However, they are independent and interrelated in their synergistic and antagonistic effect by modulating each other’s biosynthesis and responses. Glycine betaine (GB), an osmolyte, is produced by plants against these malevolent environmental factors (Hanson et al., 1994). Exogenous application of this compound is reported to enhance the resistance ability of plants by behaving as an osmoprotectant and preventing cell components by functioning as a molecular chaperone during unfavorable conditions (Mahouachi et al., 2012). It works by preserving normal cellular turgor tht play role in respiration and photosynthesis and its exogenous application enhances the endurance of wide variety of plant life under various unfavorable conditions (Hoque et al., 2007; Park et al., 2006; Chen and Murata, 2008). By considering the potential of tulip as an innovative cut flower in the local market being an unaccustomed crop by the growers in the country, this study will provide an insight for exploring the varietal response for commercial production in Faisalabad region, Pakistan. Apart from this, exogenous application of various growth promoting substances will be useful tool for the production of high quality stems with enhanced postharvest quality to hit the market window. As most of the abiotic environment related studies are performed under controlled conditions which do not portray the actual field conditions. A noticeable gap may exist between the knowledge rummaged by the findings and the required knowledge to unveil the growth performance in the field conditions. A focus on morpho-physiological, biochemical and metabolic aspects of tulips and their acclimation is needed, to establish protocol for its production and bridge the gap to facilitate the cultivation of this new crop in the region with enhanced tolerance in field conditions.
3
Foliar application of growth promotors could be advantageous to mitigate various unexpected weather changes in the environment. Abiotic stresses like temperature fluctuations, frost, drought and other biotic factors like disease incidence and disorders will be addressed for establishing production protocols that demand sophistication and accuracy hence difficult to cultivate by the growers. Furthermore, no recommended varieties are evaluated for cut flower production on scientific basis. Currently, no appreciable work has been done in Pakistan on tulips especially screening of different varieties in our climate and use of growth promoting substances to achieve quality produce. So, this research would help scientists and growers to get acquaintance with this crop in the climatic conditions of Faisalabad, Pakistan. Objectives:
Varietal evaluation of different tulip cultivars for cut flower production in climatic conditions of Faisalabad, Pakistan.
Induction of tolerance against environmental vagaries by exogenous application of different bio-regulators.
Quality enhancement and doze optimization of bio-regulators for pre and postharvest attributes.
Investigation of different physiological and biochemical attributes influencing the growth and postharvest longevity
4
Chapter # 2 Review of literature
Tulips are unsurpassed in the bulb world as a source of subdued and brilliant colors and regarded as one of the charming cut flower. However, in sub-tropical climatic conditions, it is relentlessly affected by its uneven sprouting of bulbs, time of flowering and shorter stem length accompanied with diminished vase life (Ramzan et al., 2014). For prolific flowering, most of the geophytes require particular period of exposure to low temperature which is usually delivered by unadorned winter season as seen in temperate zones across the globe. Among various geophytes, same is the case with tulip bulbs that require prolonged phase of low temperature condition to induce quality flowering having marketable stem length (de Hertogh and le Nard, 1993). By considering the rapid increase in demand of flowers, tulip has been seen as emerging and a promising cut flower in commercial trade in the field of agriculture and graded as lucrative and profitable enterprise that may provide trade and currency especially in developing countries (Rani and Singh, 2014). 2.1. Agriculture and Climate Change Agriculture is an utmost sector that is exposed to impulses of climate change. Rapid industrialization and urbanization are principle cause of unusual climatic variations leading to threatened agriculture land and desertification (Malik et al., 2012). Unfavorable abiotic environmental factors afflict negative patterns on plant growth and development that leads to profit reduction due to low yield with inferior quality. These factors drastically alter morpho- physiological, anatomical and biochemical changes especially in horticultural crops (Diaz et al., 2010) impeding in expressing overall genetic potential (Vahdati and Lotfi, 2013). Climate change accompanied with saline water and improper irrigation management result in deposition of higher soluble salts that leaches ions ultimately degenerating soil quality, crop growth, productivity and yield (Mahajan and Tuteja, 2005). These environmental stresses are becoming more prevalent due to extreme and instant variations particularly in arid and semiarid zones across the globe by increasing temperature, soil erosion, flooding and decreased precipitation rates (Olusegun and Samuel, 2014; Jbir-Koubaa et al., 2014). Weather extremes with scarce water resources in arable lands are needed to be addressed particularly in the developing countries of Asia and Africa (Abdallah et al., 2014). Changing patterns in seasons, like late onset or late ending of winters, instant temperature rise
5 and fall are affecting growth phases and development of winter crops. On the other hand, intensity and duration of summer season has been increasing to 3°C rise in temperature with the probability of 10-30% reduction in the availability of freshwater by 2040 in most of the dry areas. Consequently, higher evapo-transpiration, loss of soil moisture and accumulation of salts in rhizosphere are observed (Waqas et al., 2015). 2.2. Plant growth in unfavorable environment Each plant has its peculiar set of environmental requirements needed for adequate growth and development. These set of conditions may be favorable for one plant or could be traumatic for another. Many plants, particularly those, which are native to cooler habitats, exhibit symptoms of damage when exposed to higher temperature conditions (Lynch, 1990). Plant growth and efficiency is undesirably affected by nature’s rage in the form of various environmental vagaries. They are often exposed to superfluity of unfavorable environments such as heat, salt, flooding, flooding, drought and heavy metal toxicity. These abiotic stresses in fact are the major cause of global crop botch, plunging regular yields to more than 50% for most of the crops (Bray et al., 2000). Crop plants when raised in saline patches have to endure this environmental stress that limits plant growth and yield (Allakhverdiev et al., 2000) triggering vacuolation and partial swelling of endoplasmic reticulum, deteriorating mitochondrial cristase, mitochondrial swelling, vesiculation and fragmentation of tonoplast, and cytoplasmic degradation by the amalgamation of their matrices in the leaves (Mitsuya et al., 2000). In fact these stresses are threatening to the sustainability of agriculture industry. Hence, unfavorable environmental conditions are the major reason of field crop damage in global scenerio (Saini and Westgate, 1999; Xiong et al., 2002). Amongst various environmental stresses, plants when exposed to drought, show drastic change in plant growth pattern that ultimately decrease productivity (Campos et al., 2004; Zhang et al., 2008). In nutshell, nature’s wrath shows a decisive influence in diminishing growth and productivity of plants in countering various biotic and abiotic harmful factors that show pronounced symptoms when exposed to plethora of unfavorable conditions. The reason for all these factors could be different anthropogenic activities that have been accentuated by malicious conditions. Plant growth modulating substances could be an effective tool to combat abiotic stresses that may influence signaling with respect to increase in productivity. Therefore, these substances need understanding of their response in inducing adaptability and compatibility in
6 various crop plants to rule out different stresses. As plants are non-motile organisms it is intractable to tolerate adverse environmental conditions as motile organisms can. In order to withstand in such conditions they show certain alterations in their metabolic processes. One such alteration is the deposition of certain organic metabolites and growth regulators in plant tissues in unfavorable environment (Rhodes and Hanson, 1993). Plant treatment with various physiologically active compounds, as a chemical method for regulating productivity and resistance is successfully applied for long time. Exogenous application of these substances in crop plants induce tolerance against various environmental stresses that has been epicenter of many researchers from past few years (Iqbal and Ashraf, 2007; Iqbal et al., 2005) 2.3. Effect of plant growth modulating substances in floriculture The term ‘Plant growth regulators’ was suggested by Tukey et al. (1954) which include natural and synthetic compounds. They are actually the organic compounds apart from nutrients, which are effective in very little quantities that may promote, impede or otherwise transform any physiological process occurring in plants. This term is not only confined to synthetic substancess but also comprises naturally occurring hormones as well (Nickell, 1982). Basic aim of these compounds in floriculture is to regulate plant height, enhance branching, propagation, flowering, enhancing stress tolerance or increasing post-harvest longevity during marketing channel (Janowska et al., 2009). In most cases, reduced plant height is desirable as in bedding, potted and woody ornamentals and in some cases height improvement is needed as in case of cut flowers (Das et al., 1999; Al-Khassawneh et al., 2006; Mackay and Sankhla, 2006). Polysaccharides are reported to be an effective tool to improve the plants ability against different stresses (Ludlow and Muchow, 1999; Bautista-Banos et al., 2006). 2.4. Source of chitosan Chitosan is widely applied in agriculture commodities because of its pragmatic effects on plant growth and developmental patterns. It is structurally and chemically similar to cellulose and chitin (Hadwiger and McBride 2006; Freepons 1991) and hold strong positive charge which attract negative ions. Chitosan is a biopolymer derivative of chitin, a polysaccharide, sourced from exoskeleton of inexhaustible marine organisms from the phylum arthropoda class crustaceans (Palpandi, et al., 2009). It is usually extracted from the shells of prawns and crabs making it easily available as an organic waste from seafood industry on sustained basis. It is also present in cuticles of insects and also in the cell walls of some algae and fungi (Sandford 2002;
7
Sandford and Hutchings 1987; EPA 1995). In recent years, it has extensively been researched and applied as efficient green pesticide and plant growth modulator in agricultural fields for its non-toxic, non- pollution and biodegradation abilities (Ma, 2001; Ma et al., 2010). It is extracted by deacetylation of chitin with diversified uses in crop plants (Sandford, 2002; Larez, 2008). Its solutions are readily soluble in weak acids and water that impart constructive effects on plant tissues (Raygosa, 2001) in fruits (Salvador et al., 1999; Hernandez et al., 2001) and in soil (Ohta et al., 1999). 2.5. General features of chitosan Chitosan possesses antiviral, antibacterial, and antifungal properties which has potential to be used for crop production (Felix et al., 1993). They are utilized to prevent disease incidence and works by chelating nutrient and minerals. Moreover, it enhances plant innate defense mechanism by inhibiting pathogenic access particularly in monocots and dicots (Felix et al., 1998). These responses include lignification (Barber et al., 1989), ionic instability, cytoplasmic acidification, protein phosphorylation, membrane depolarization, (Kuchitsu et al., 1997; Kikuyama et al., 1997), glucanase and chitinase activation (Kaku et al., 1997; Roby et al., 1987), phytoalexin biosynthesis (Yamada et al., 1993; Ren et al., 1992), synthesis of reactive oxygen species (ROS) (Kuchitsu et al., 1995), jasmonic acid biosynthesis (Nojiri et al., 1996), and manifests early responsive and defense associated inheritable factors (Takai et al., 2001; Minami et al., 1996; Nishizawa et al., 1999). Chitosan when used as seed coating improved the germination percentage as well as the seed vigor in wheat (Bhaskara Reddy et al., 1999) while vegetative growth in pearl millet (Sharathchandra et al., 2004). However, it is important to search replacements to increase the efficacy of chitosan in plants and flower crops in particular. These crops are highly sensitive and are more prevalent to global warming which differs regionally especially in developing countries that are vulnerable to greater extent (Rosenzweig and Parry, 1994). Currently, there are limited economic and technological means to ameliorate abiotic stress conditions. Biodegradable features of natural substances obtained from animal and plant sources have fascinated many researchers to focus upon and chitosan being nontoxic bioactive agent has gained much popularity because of its efficient defense mechanism in plants (Terry and Joyce, 2004; Wilson et al., 1994). 2.6. Biochemical features of chitosan
8
Phytoalexins are accumulated by chitosan application that results in antifungal activity and safeguards against contagious toxicities (Vasyukova et al., 2001; Hadwiger 2013). It further increases the phenolic substances in plant tissues to induce defense mechanism (Bautista-Banos 2006). Foliarly applied chitosan and oligomers of chitin in soybean leaves and in sweet basil (Kim et al., 2005) enhanced the enzymatic activities of phenylpropanoid pathway particularly phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) (Khan 2003). Moreover, it also improved polyphenol oxidase (PPO) activity and induced resistance against certain diseases in millets (Raj et al., 2006; Thipyapong et al., 2004). Thus, it plays key role in signaling pathway for the biosynthesis of phenolics. Similarly, pathogenesis related enzymes like chitinase and chitosanase are also synthesized by chitosan (Collinge et al., 1993; van Loon et al., 1994). Systemic acquired resistance (SAR) is further aggravated by chitosan application which is long lasting and confers broad-based resistance against various pathogens to improve plant immune system. SAR usually develops in non-infected organs of plants; thus the whole plant acquires resistance against secondary infections. β-1, 3-glucanases, PR-1 and PR-5 are amongst the most effective proteins synthesized as a result of SAR, possessing antifungal chitinases (Sathiyaba and Balasubramanian 1998). Many studies have demonstrated that exogenously applied chitosan triggers the expression of various genes that play role in plant resistance responses like gene encoding protease inhibitors and PAL (Notsu et al., 1994; Vander et al., 1998; Doares et al., 1995). 2.7. Physiological effects of chitosan in plants Rate of transpiration in pepper plants is found to be reduced at adequate levels causing 26-43% decrease in water by chitosan application though it sustained the biomass and yield. There results clearly depicted the efficacy of chitosan as a useful anti-transpirant that could be helpful in unavailability of water to the plants (Bittelli et al., 2001). Transpiration rate is further reduced when ther is elevated level of abscissic acid (ABA) resulting in stomatal closure accompanied with lessened transpiraration by regulating water use (Willmer and Pricker 1996). Sembdner and Parthier (1993) revealed ABA presented some features similar to jasmonic acid. Thus, closure of stomata impacts various pathways relating to abscisic acid and jasmonic acid.
9
In crops like tomatoes, pears and tangerines chitosan reduced inter cellular CO2 in mesophyll cells produced as a result of respiration when stored at ambient temperature at 13°C than at 20°C (El Ghaouth et al., 1992e; Du et al., 1997; Salvador et al., 2003). Similarly, in cucumbers and bell peppers electron microscopy and histochemical analysis revealed efficient water use efficiency and stomatal conductance, either by partial or complete stomatatal closure with improved biomass to water ratio in controlled and in field conditions where plants are more prone to environmental maladies (El Ghaouth et al., 1991a). Furthermore, ethylene production was inhibited in peaches because of low respiration induced by chitosan coatings (Li and Yu, 2000). As, opened stomata facilitate prompt access to leaf anatomy to many plant pathogens. Therefore, narrow stomatal aperture is useful in plant defense. This aspect is observed in tomato and commelina as chitosan covered the layer of epidermal cells (Lee et al., 1999). Chitosan along with jasmonic acid induced transcriptional activation, of genes that encode PAL and protease inhibitors influencing jasmonic acid pathways (Walker-Simmons et al., 1983; Farmer and Ryan, 1990; Doares et al., 1995). Jasmonates possess some attributes same like to abscisic that matters water use regulation in many plants (Sembdner and Parthier, 1993). As, higher endogenous level of abscisic acid results in stomatal closure with reduced transpiration (Willmer and Fricker, 1996; Leung and Giraudat, 1998), it manipulated signaling pathway of ABA that checks plant water relations (Grill and Ziegler, 1998). It is recently reported that addition of 1-carbon group of farnesyl to protein caused stomatal closure in Arabidopsis thalliana (Pei et al., 1998). Moreover, light-induced opening of stomatal aperture in tomato and commelina is inhbited by chitosan by causing H2O2 synthesis in guard cells. These features make chitosan a valuable anti-transpirant in scarcity of water. The water conservation attribute in pepper plants was considerable, and similar trend is seen in tomato, wheat and barley when treated with anaogs of ABA (Rademacher et al., 1989). Evapotranspiration estimation is one of the reliable parameter that reveals plant water use, it is calculated by Penman- Monteith calculation that shows an ardent relationship between actual and calculated rate of evapotranspiration for chitosan-treated and control plants. Chitosan treated plants revealed threefold increase in stomatal resistance in comparison to non-treated ones. Stomatal resistance at 210 sm−1 caused by evapotranspiration resulted in partial closure of stomata while at 840 sm−1 stomata got completely closed (Allen et al., 1998). So, this physiological feature of chitosan grades it as an effective anti-transpirant for crops making it
10 economically viable (Muzzarelli, 1989). Thus, in limited resources of water, it has potential to reduce transpiration to an optimized level, especially in irrigated agriculture zones. 2.8. Chitosan and plant defense mechanism Chitosan exhibits the potential to induce plant defence mechanism by expressing various genes involved in synthesis of secondary metabolites in ample quantity in plants (Walker- Simmons et al., 1983; Loschke et al., 1983). This activated many genes that played role in defense mechanism in the octadecanoid pathway (Doares et al., 1995) as seen in tomatoes where wound responsive proteins increase their synthesis (Stratmann and Ryan, 1997), same is the case reported in slash pine (Mason and Davis, 1997) and in oats (Tada et al., 2001). This induction of defense gene activity that induces disease resistance is highly specific in context to plant cultivar and pathogen attack (Eikemo et al., 2003) resulting in antifungal activity. It is reported that 50 mgL-1 chitosan almost completely inhibited Botrytis cinerea conidia germination in in vitro, and also controlled gray mold that is caused by B. cinerea, particularly in cucumbers (Ben-Shalom et al., 2003). The unique features of chitosan, like bioactivity and bio-compatibility (Dias et al., 2013) depicts that, when used in plants, it has potential to improve yield (Mondal et al., 2012), reduced transpiration (Dzung et al., 2011) and induction of various metabolic changes making plants more resistant to viral, bacterial and fungal infections (Al-Hetar et al., 2011). Moreover, plants treated with chitosan may be less prone to stresses, evoked by stressful conditions, like drought, salt stress or temperature abberations (Lizarraga-Pauli et al., 2011, Jabeen and Ahmad 2013, Pongprayoon et al., 2013). The stimulatory effect of chitosan is worth considering in vital processes of plants on every level of biological organization, from single cells and tissues, in physiological and biochemical reactions, till the changes at molecular level (Limpanavech et al., 2008; Hadwiger, 2013; Nguyen Van et al., 2013). Chitosan activates de-novo production of phenolics as a first defensive line to impede fungal development by synthesising of β-1, 3-glucans acting as a secondary mechanical obstruction preventing invasion of fungal cells by shielding plant tissues from phytotoxicity (Lafontaine and Benhamou, 1996; Benhamou et al., 1994). They further reported that to initiate plant signaling against defense system, pathogenic contact is necessary and plants treated with chitosan were more expressive to defense responses at higher pace and extent rather than pathogens only after septicity. However, in cucumber plants chitosan alone didn’t induce defense
11 response against fungal activity to reduce gray mold (Ben- Shalom et al., 2003). Although in hydroponic system, antifungal hydrolases are found in roots and aerial plant parts of cucumber treated with chitosan when inoculated with Pythium aphanidermatum artificially (El Ghaouth et al., 1994a). Exogenous application of chitosan against F. oxysporum when applied as root and seed dressing, responded by restricting pathogen sprawl in the external root tissues by provoking various defense responses, together with structural obstructions (Benhamou et al., 1998). This effect might have been occurred due to accumulation of fungitoxic compounds at an adequate level at the sites of pathogen penetration. Filmogenic property of chitosan is another feature that acts as an obstruction to outward fluctuation of nutrients that may impede the access to nutrients needed beyond the threshold level of pathogenic growth to a sustained level. This can be proved as fungal cells commonly display the symptoms of nutritional deficiency when exposed to chitosan (Ait Barka et al., 2004). In chitosan treated peanuts, resistance response is reported, where pronounced increase in endogenous SA, glucanase activities and intercellular chitinase were existed (Sathiyabama and Balasubramanian, 1998). Moreover, chitosan treated wheat plants when inoculated with B. cinerea showed moderate cell wall lignification after the period of 48 and 72 hours (Pearce and Ride, 1982). Observations made from electron microscopy evidenced the formation of typical structures and some new materials in the roots and leaves of tomatoes that were infected by F. oxysporum and treated with chitosan. They brought about series of changes in the anatomical pattern as constriction of xylem tissues by an impervious fibro-granular structures forming a bubble like shape, coated with secondary swellings and pitted membrane, formation of papillae (wall appositions) in cortical cells and endodermal layer of cells (Benhamou and Theriault, 1992; Lafontaine and Benhamou, 1996). Moreover, twisted epidermal cells with contortions were also seen in host cells on chitosan-treated roots in tomato (Benhamou et al., 1994). Structural transformation was observed in bell pepper fruit anatomy due to defense response that showed thickening of host cells only in the exterior most tissue layer below the ruptured cells, by forming curved and globular protuberances all around the cell walls, and constriction of intercellular spaces and fibrous structures (El Ghaouth et al., 1997). Many other studies were manifested to compare the efficacy of chitosan application to treat fusarium wilt in tomato; one by foliarly applied chitosan and another by biological method with Bacillus pumilus,
12 revealed the increased defense reaction in tomato roots by chitosan (Benhamou et al., 1998). Plants of cucumber when raised in nutrient solutions containing chitosan, inoculated by P. aphanidermatum, same reactions of hosts were found as that in chitosan treated roots in tomato like constriction of intercellular spaces with impervious fibrous material and formation of papillae all around the cell walls of host cells (El Ghaouth et al., 1994b). In another study, it was found that alginate chitosan reduced the disease incidence caused by mycorrhizal fungi in encapsulated seeds of Spathoglottis plicata (Tan et al., 2004). Chitosan and oligomers of chitin are reported to kindle in inducing resistance, like the enzymatic activity conferred by ammonia lyase, lipoxygenase, phenylalanine or lignin formation as seen in the leaves of wheat crop (Vander et al., 1998; Bohland et al., 1997). Pathogen invasion due to fungal penetration results in the formation of structural barriers along with suberization and lignification of cells provoked during infection process in various plant parts. Coatings with chitosan are also illustrious in its capacity to enhance postharvest longevity of many fruits and vegetable crops. It develops partially permeable layer that controls gaseous exchange attributes by lowering transpiration accompanied with reduced rate of respiration during fruit ripening. The same fact is reported in numerous horticultural crops like strawberries, longan, tomatoes, apples, bell peppers, mangoes, bananas, etc. (El Ghaouth et al., 1992e; Du et al., 1997, 1998; Jiang and Li, 2001; Kittur et al., 2001). 2.9. Use of chitosan in floriculture The utility of chitosan in flower crops may play a constructive role regarding different morphological, physiological and yield attributes. In cut orchids, particularly in dendrobium, chitosan having 45 kDa mol. mass with more than 90% DD when foliarly applied at 1-100 mgL-1 conc., enhanced quantitative and qualitative traits especially in inflorescence yield (Limpanavech et al., 2008). Nge et al. (2006) reported the same fact for its role in increased number of plantlets in dendrobium and Phalaenopsis orchids that were reinforced from protocorm like body (PLB). Spraying with chitosan, not only inhibited disease incidence of leaf-spot disease but also supported plant development in other plants from orchidaceae family, by improving the florets vigor and inflorescence size (Win et al., 2005). Moreover, foliar application of chitosan at 600 mgL-1 increased the petals width at harvest in them (Uthairatanakij et al., 2007). Limpanavech et al. (2003) elucidated the chitosan effects at various conc. DD, and polymerisation at deflasking, on morphological aspects of dendrobium orchid. He reported that
13 chitosan at 10 mgL-1 improved the growth and number of PLBs of D. formosum and P. sanderianum. The initiation of root and leaf growth in D. formosum completely depended upon chitosan composition in culture medium. The forms of chitosan either oligomeric or polymeric with 70, 80, and 90% (DD) with conc. of 1, 10, 50 and 100 mgL-1 had non-significant effect on vegetative attributes, but resulted in early flowering. In an in vitro experiment, 6 forms of chitosan were compared; polymer (P) and oligomer (O) with DD of 70%, 80% and 90%, and mol. mass (MW) of P=400,000-500,000 and O=30,000-100,000 at 6 various conc., 0, 10, 20, 40, 80, and 160 mgL-1. The results reported that PLBs were completely faded and destroyed at 160 mgL-1 irrespective of form, while conc. at 80 mgL-1 withdrawn the growth of PLBs. Although, all molecular forms of chitosan prominently increased the growth of PLBs at 10 and 20 mgL-1 (Pornpeanpakdee et al., 2006). Moreover, chitosan sprays at 10 mgL-1 increased growth of young orchid plantlets while its sprays at 2.5-40 mgL-1 increased the length of leaves in P. sanderianum orchid (Chandrkrachang 2002). Chitosan application when mixed with soil medium remarkably influenced the time of sowing and enhanced the plant development patterns particularly flowering time was hastened 15 days earlier as compared to controls. It further improved the mass of flowers as well as promoted seedling growth in Torenia fournieri, Begonia hiemali, Sinningia speciosa, Lobelia erinus, Mimulus hybridus, Calceolaria herbeohybrida and Campanula fragilis. The first flowering date in chitosan treated T. fournieri was expressively prior than the other treatments and not in C. herbeohybrida and C. fragilis (Ohta et al., 2004). Hasegawa et al. (2005) reported that corms of Arisaema ternatipartitum with an increased diameter and height as a result of cultivation in a substrate with an addition of chitosan. Pichyangkura (2002) reported that chitosan efficacy depends upon molecular mass, sugar carbons : glucosamine and level of N-acetyl-glucosamine, concentration and frequency of application. Chitosan-treated orchids especially (Dendrobium cv. Sensational Purple) developed more shoots and inflorescences as compared to non-treated ones. Chandrkrachang et al. (2005) observed premature inflorescences in orchids as a result of over dosing of chitosan, when sprayed 6 times; at 0 (water), 200, 400, or 600 mgL-1 with weekly intervals, produced higher fresh mass of florets (29 g) as compared to non-treated ones (26.8 g). Chitosan also influenced silica uptake or silica metabolism in orchids reported by Hodson and Sangster, (2002) who found accumulation of silica bodies play effective role in inducing thermotolerance, wind and salinity.
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Freesia, an imperative geophyte, of substantial economic importance is cultivated as cut flower and potted plant. In 2012, about 300 million cut freesia flowers worth €50 million were traded and touched the sale growth of 6.5% in comparison to 2011 in Dutch market (FloraHolland 2012). This increased growth lead to the assortment of freesia cultivars extended to include new genetically low heighted cultivars that became more demanding as pot plants (Ehrich et al., 2009). In its production chain improvement of cultivation methods, corm production and health of plants is desired for pot production that remained unheaded (Vaira et al., 2009; Ruamrungsri et al., 2011). In an experiment chitosan was made into use as biostimulator for potted freesia production. Regardless of its molecular weight, chitosan treated plants developed more leaves and shoots, exhibited earlier flowering and yielded more flowers and corms. Moreover, high-molecular-weight chitosan resulted in increased plant height with relatively higher SPAD values for chlorophyll contents (Salachna and Zawadzinska, 2014). Commercial grade chitosan increased the flower number and prolonged the post-harvest display in cut lizianthus (Eustoma glandiflorum). Moreover, vegetative growth with some improved processes in developing and induction of flower buds with chitosan application is also reported in them (Ohta et al., 1999; Uddin et al., 2004). Ohta et al. (2001) also reported pronounced increase in the flower count with better quality and associated this fact to high chitinase enzyme activity, when seeds of this crop were soaked in commercial grade chitosan at 1% for the duration of 1 hour and raised in a gowing mixture containing chitosan. In further investigations, chitosan at 10 mgL-1 with nutrients (NPK) in soil mixture prolonged the display life of curcuma flowers cv. ‘Laddawas’ demonstrated by Tamala et al. (2007), flowers grown in chitosan treated media lasted 3 days more as compared to the media containing fertilizer only. Other evidences as reported by Kim et al. (2005a), mentioned the increased display life of 3-4 days more in lilies either dipped or sprayed with chitosan solution containing colloidal particles of silver. Similar trend of its application is reported in cut gerbera where it significantly improved growth attributes like flower stalk length, leaves number, leaf dimensions as well as flower number borne per cluster of plants (Wanichpongpan et al., 2000). In the studies regarding vase solution recipe Azian et al. (2004) found that vase solutions comprising of chitosan with 25 and 50 mgL-1 improved display life of chrysanthemum cut flowers to 13 and 15 d, respectively, compared to non-treated ones that sustained their freshness for 6.8 days. On the other hand, in
15 the findings by Luan et al. (2005), the stimulating effect of low-molecular weight chitosan (16 kDa) was shown when it was added in vitro to the medium culture, where it increased the height of shoots and fresh biomass of Chrysanthemum morifolium, Limonium latifolium, Eustoma grandiflorum and Fragaria ananasa. Gladiolus is one of the most popular and highly demanded cut flower worldwide and possesses a pivotal status in the cut flower industry of Pakistan (Bashir et al., 2015). In an investigation by Margarita et al. (2009) gladiolus corms of cultivar ‘Blanca Borrego’ were dipped in chitosan Biorend® a reagent and in warm water at varying temperatures with treatment combinations with Biorend® at 1.5% and warm water. Results revealed corm sprouting, flower count per spike, cormlets count and display life as the most influenced variables. This compound exacerbated corm emergence to 4 days earlier, increased flower number by 2-7 and display life was increased up to 3 days beyond normal. Corms dipped in Biorend® at varying intervals were not significantly pretentious except for the sprouting and cormlets count. However, significant increase in flower and leaf count was observed in interaction between chitosan and dipping time. The mixing of the two treatments the Biorend® compound and warm water proved to be a useful choice for boosting the spike quality, vase life and overall plant vigor. 2.10. Use of chitosan in some horticultural crops There is vast repertoire of literature that advocated the use of chitosan as an alternative to fungicide in agriculture because of its elicitation in boosting defense mechanism in plant tissues particularly in many fruits, vegetables and various seeds as well (Zhang and Quantick 1998; Jiang and Li 2001; Lee et al., 2005; Photchanachai et al., 2006). It’s use as coating material in bell pepper and strawberry enhanced postharvest longevity by limiting fungal decay (Terry and Joyce 2004). Moreover, it altered growth performance in root and shoots development in radish (Raphanus sativus L.) in an effective way (Tsugita et al., 1993) and boosted defense mechanism in tomato (Benhamou et al., 1994). In tomatoes, synthesis of phenolics and phytoalexins enhanced the activity of hydrolytic enzymes to defend against F. oxysporum (Benhamou and Theriault, 1992). It also impeded the virulence factor of fungi by synthesizing cell wall digesting enzymes like polygalacturonase and pectate lyase, various organic acids like fumaric and oxalic acid and host specific toxic compounds like alternariol monomethyl ether and prompted the rishitin production (Bhaskara Reddy et al., 1998). Studies on peanut seeds also revealed the
16 synthesis of free and bound phenolics in seed tissues that improved their viability by the use of chitosan (Fajardo et al., 1995). In table grapes, synthesis of enzyme phenylalanine ammonialyase improved postharvest longevity by inhibiting decay when chitosan dissolved in glacial acetic acid. When applied on berries individually and stored at 15 °C and on bunches stored at 0°C for the duration of 60 days (Romanazzi et al., 2009). Enzymatic activity of POD and PPO were elicited in the roots of date palm when injected with chitosan solution (El Hassni et al., 2004). This mode of chitosan application resulted in the production of caffeoylshimick acids like sinapic, p-coumaric and feluric derivatives in the roots as a major phenolic compound to induce anti-fungal role and lignin precursors. Synthesis of several defense associated enzymes by chitosan use has been reported by Bohland et al. (1997) and Vander et al. (1998) that play role in immediate and rapid defense mechanisms to avert pathogenic infections. In perishable fruit crops like passion fruit (Passiflora edulis Sims) Utsunomiya and Kinai (1994) reported early flowering with increased flower number when soil application of chitosan was carried out. Similar response is seen in some cole crops like cabbage (Brassica oleracea L.) as reported by Hirano (1988), soybean sprouts (Lee et al., 2005) and sweet basil as reported by Kim et al., (2005b) in which it boosted the growth attributes. A similar effect on potato yield of mini tubers was observed by Asghari-Zakaria et al. (2009) in in vitro cultures. Plants grown on a medium with the addition of 500 mgdm-1 chitosan produced 3.33 mini tubers on average, while non treated plants yielded 2.44 mini tubers. Studies on response of varying conc. and application frequency of chitosan are also steered in crops like chillies, celery, bitter gourd, Chinese cabbage and agronomic crop like rice (Chandrkrachang et al., 2003; Boonlertnirun et al., 2005). Findings revealed pronounced increase in growth pattern of chili and yield of Chinese cabbage at various concentration and frequency. Boonlertnirun et al. (2005) envisaged that 4 exogenous sprays of chitosan at 20 mgL-1 resulted in slight increase in rice yield. Sauerwein et al. (1991) reported that adding of chitosan to culture medium improved the shoot growth of Lippia dulcis. However, the imprecise mode of action of this compound is needed to be explored. Dzung et al. (2011) reported that as a result of spraying with chitosan on coffee seedlings with the solutions of molecular weight of 2 kDa, the total chlorophyll contents and carotenoids in the leaves increased up to 15.36% in plants grown in the field and by 46.38–73.5% in plants grown in the greenhouse in contrast to control. The increased
17 level of chlorophyll contents might because of enhanced uptake of nutrients that are reported in the findings of Nguyen Van et al. (2013) on coffee seedlings. Chitogel, a chitosan derivative, is reported to improve vegetative growth attributes in grapevine plantlets, when the culture medium was complemented with chitosan solution 1.75% (v/v) that boosted the biomass of root and shoot, photosynthetic rate and other variables in in vitro culturing (Barka et al., 2004). Moreover, the average O2 production by plantlets was amplified 2-folds, while CO2 fixation improved 1.5 folds only, depicting the efficacy of chitogel on grapevine physiology. Chitosan coatings on freshly harvested strawberries and raspberries showed a pronounced increase in the activities of chitinase and β-1, 3-glucanase as compared to uncoated ones (Zhang and Quantick, 1998). Morover, it partially introverted the excessive POD activity, a leading factor to cause browning of tissues (Zhang and Quantick, 1997). Several other examples of chitosan treated fruits like tomatoes, peaches and papayas maintained their firmness while softening during their storage was delayed. Similar findings are also reported by El Ghaouth et al. (1991b), (1992b), Li and Yu (2000), Bautista-Banos et al. (2003) that describes sustained firmness of various fruits even at the end of storage. Pre harvest application of chitosan sprays on strawberry fruit, along with ambient storage temperature and adequate harvest stage, resulted in persistent firmness (Bhaskara Reddy et al., 2000). Hence, organoleptic properties like fruit color, firmness and overall appearance of various fruits and vegetable crops were generally improved by chitosan coatings. However, in case of Japanese pear and papaya there was a minor off color expression, though deep green fruit color is reported in cucumber and bell peppers (El Ghaouth et al 1991a, 1992e; Woods et al., 1996; Du et al., 1997; Jiang and Li., 2001; Luna et al., 2001). Similarly, chitosan when applied in combination with ammonium carbonate offered a commercially and economically viable approach that was found to be a useful alternative for preventing stored papaya from anthracnose. Dipping papaya fruits in the solution of chitosan mixed with ammonium carbonate, delayed color development of skin and mesocaarp ultimately enhanced the postharvest longevity because of maintained firmness and fruit mass (Sivakumar et al., 2005). Generally, anthocyanin degeneration in chitosan treated fruits was arrested that has been seen in litchi, raspberries and strawberries (Li and Chung, 1986; Zhang and Quantick, 1997, 1998). However, some findings mention a contradictory effect in which anthocyanin production continued even after its application like in strawberries (El Ghaouth et al., 1991b). It could be
18 due to cultivar specificity or chitosan source. Titrable acidity is reported to increase in tomatoes, peaches and strawberries generally at the end of storage period in chitosan treated fruits, while it reduced slowly in mangoes and longan resulting in the loss of eating quality (El Ghaouth et al., 1992e; Li and Yu, 2000; Jiang and Li, 2001; Srinivasa et al., 2002). Total soluble solid (TSS) of chitosan treated fruits, even after storage, differed significantly depending upon the fruit type. Low level of TSS was reported in non-treated mangoes and bananas coated with chitosan while higher levels in treated peaches. However, chitosan dipped papayas and zucchinis have no effect on their TSS as compared to non treated ones (Du et al., 1997; Kittur et al., 2001; Constantino et al., 2001; Srinivasa et al., 2002; Bautista-Banos et al., 2003). Chitosan coatings also affected the reducing sugar contens in some fruit crops. Low levels of reducing sugars are reported in chitosan coated fruits than the untreated ones, particularly in bananas at the end of storage (Kittur et al., 2001). However, paradoxical reports regarding the levels of reducing sugars of chitosan-treated mangoes are there, might be due to mode of its application. In a study, when mango fruits were packed in a carton and shielded with chitosan coating exhibited higher levels of reducing sugars than the control treatment while, in another study, when mango fruits dipped in chitosan solution, possessed lower levels of reducing sugars than the control ones (Kittur et al., 2001; Srinivasa et al., 2002), depicting reduced metabolic rate in the dipped than in non-dipped fruits. Similarly, ascorbic acid contents were also evaluated by Li and Yu, (2000) and Srinivasa et al. (2002) in chitosan treated mangoes and peaches. They found out, the level of vitamin-C in the treated mangoes slowly declined during storage and were lower than in untreated fruits. However, in peaches, higher contents of ascorbic acid are reported in chitosan-treated fruits even after 12 days of storage as compared to untreated fruits of peaches. Although few studies clearly express the effect of chitosan on sensory attributes, commonly, the parameters like flavor, taste and aroma remain unchanged. In this regard, mango and strawberry when treated with chitosan, acquired the significant superiority as compared to untreated ones even after the storage duration of 21 and 15 days, correspondingly (Li and Chung, 1986; Kittur et al., 2001). Moreover, chitosan along with some Ca++ salts showed the best sensory attributes for strawberry even after 21 days of storage (Li and Chung, 1986; Kittur et al., 2001). In another study chitosan coated strawberries when packed for 7days developed an unpleasant even at 0 day (Devlieghere et al., 2004) while pawpaws when treated with chitosan
19 developed less off flavor that might be due to delayed ripening (Luna et al., 2001). These reports concomitantly indicate that pre-harvest chitosan application affected fruit quality and yield at harvest. For example, there exists a positive and significant correlation with increase in tomato yield at harvest with adequate conc. of chitosan when applied to soil and inoculated with F. oxysporum before the transplanting of seedlings (Lafontaine and Benhamou, 1996). However, when fungal inoculation was not done, fruit yield from chitosan treated soil and untreated was found to be the same, depicting the disease reduction by chitosan highly correlated with improved fruit yield. 2.11. Plants and malevolent environment Plants are sessile organisms that typically need to regulate their growth and development in response to various external stimuli and continually changing environment (Wolters and Jurgens 2009). Abiotic stresses impart massive losses in agricultural yields globally (Bray et al., 2000) particularly to field crops and other plants in general that are imperiled to the effect of these stresses (Moffat, 2002). Most of the plant species are unable to produce naturally or accumulate anti stress compounds like glycine betaine and others in response to unfavorable conditions. Varieties of compatible organic solutes are also produced by various plants in relation to environmental maladies (Serraj and Sinclair, 2002). Trehalose, polyols, proline, sucrose and quaternary ammonium compounds (QACs) such as glycine betaine, choline O- sulfate, proline, alanine betaine, pipecolate betaine & hydroxyproline betaine are amongst them (Rhodes and Hanson, 1993). These solutes possess low molecular mass, high solubility and non- toxicity even at higher cellular concentrations. They boost plant defense mechanism in different ways including cellular osmotic regulation, ROS detoxification, stabilizing membrane stability and by maintaining globular structure of enzymes (Yancey et al., 1982; Bohnert and Jensen, 1996). It is reported that even genetically engineered plants having transgene of GB production show limitation to synthesize appropriate amounts of them to cope with unfavorable effects of abiotic factors (Ashraf and Foolad, 2007). 2.12. Tailoring a response to specific stress condition Specific responses are tailored to typical environmental conditions that the plants usually encounter to acclimate in particular abiotic stress. These responses include molecular, biochemical and physiological mechanisms that come into action caused by stresses which may vary from those, activated by somewhat different configuration of environmental attributes.
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Studies related to transcriptome profiling illustrate this view point subjected to various abiotic unfavorable conditions. Various stress conditions prompt somewhat distinctive responses and minute overlapping in transcript expression prevails among different plants to malicious environmental conditions like temperature extremes, drought, cold, salinity, intense light or mechanical aberrations (Rizhsky et al., 2004; Kreps et al., 2002; Cheong et al., 2002). Although, reactive oxygen species are closely linked to variety of biotic and abiotic stresses, gene network of ROS in Arabidopsis was observed to affect differently in various stress conditions (Mittler et al., 2004). As every abiotic stress needs a peculiar reaction for acclimation, tailored to particular requirement of plant, combined effect of two or more stresses may show a specific effect. Moreover, the basic difference between the response of acclimation acquired by the plants to various unfavorable conditions (Fowler et al., 2002; Rossel et al., 2002) is coined with different hazardous elements that may need some conflicting or antagonistic effects. Plants facing high temperature stress keep their stomata open to lower their temperature by increasing the rate of transpiration. However, when heat stress accompanied with water scarcity, plants fail to open their stomatal pores, hence increase their leaf temperature (Rizhsky et al., 2002). Stress caused by salinity or heavy metals may bring in its wake similar maladies in plants along with heat stress because exacerbated transpiration enhances uptake of salts and heavy metals. Similarly, chilling stress or water scarcity, in the presence of intense light hastens ROS production by photosynthetic apparatus because availability of CO2 for dark reaction is hindered, evolving O2 as one of the decisive reduced product of photosynthesis reaction (Mittler 2002). A typical example of antagonistic effect of various environmental stresses is seen in water deficiency and heavy metals that boosts the consequence of each other (Barcelo and Poschenrieder 1990). Plant acclimation needs energy and resources for the synthesis of HSPs and LEA proteins to combat malicious conditions and in this situation nutrient deficiency may pose a lethal threat to endure. Likewise, insufficient availability of elements like Fe, Cu, Zn or Mn that are co- enzymes involved in the activity of various defence enzymes, like superoxide dismutase (SOD) or ascorbate peroxidase (APX), may increase the oxidative stress subjected to various environmental stresses (Ranieri et al., 2001). Acclimation combined with different abiotic stress conditions would, therefore, need an efficient feedback customized to distinct stress condition tailored to adjust for some antagonistic aspect of stress combination.
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2.13. Glycine betaine and acclimation of plants Glycine betaine is an exceptionally efficient compatible solute that scavenges oxidative stress causing molecules and safeguards the photosynthetic system in plants (Cheng et al., 2013). Different stresses caused by soil salinity and drought negatively affect various aspects of plant physiology, causing significant losses in crop productivity and biodiversity (Joseph et al., 2013). Acclimation of various plants to incessantly varying environment encompasses the buildup of various compounds, collectively termed as compatible solutes, in the cytoplasmic fluid of the cells. This is evident from different findings of plant physiology, biophysics and biochemistry that adamantly suggest (GB) as an amphoteric quaternary compound, to play pivotal role as compatible solute in plant physiology exposed to various hazardous environmental conditions like salinity and varying temperature regimes. Plant species have specific potential in their ability to make GB while some plants enmasse its relatively higher levels in their chloroplasts and others, like tobacco and Arabidopsis are unable to synthesize it (Sakamoto and Murata, 2002). Many plants accumulate compounds exhibiting cryoprotectant activity when exposed to low temperature regimes (Guy, 1990; Rajashekar, 2000). GB is amongst such organic solutes (Stushnoff et al., 1997) that possess osmoprotective function, and guard proteins, membrane stability and enzymatic activities during water deficiency (Rhodes and Hanson, 1993). Accumulation of this compound in low temperatures is reported in wheat, rye, barley and strawberry (Allard et al., 1998; Koster and Lynch, 1992; Kishitani et al., 1994; Rajashekar et al., 1999). Endogenous accumulation of GB induces freezing tolerance (Kishitani et al., 1994). Moreover, after its exogenous application in many plants, substantial increase in freezing tolerance is reported (Allard et al., 1998; Rajashekar et al., 1999). These findings suggest accumulation of GB play role in inducing freezing tolerance during cold acclimation. Water stress has long been known to induce freezing tolerance in variety of plant species including Arabidopsis (Guy et al., 1992; Mantyla et al., 1995). In many of these studies, this stress proved effective in cold acclimation treatment in developing freezing tolerance (Cloutier and Siminovitch, 1982; Mantyla et al., 1995) and imparted same effects in plants as in low temperature conditions (Shinozaki and Yamaguchi-Shinozaki, 1996; Guy et al., 1992). At genetic level, various genes have also been reported to be activated in response to environmental
22 adaptability particularly in cold acclimation and water deficit conditions (Curry et al., 1991; Holappa and Walker Simmons, 1995). Plants grown in arid to semiarid zones of the world have become more prevalent to elevated levels of salinity, which is amongst most important constraint in crop production. The more intensive cultivation is practiced, the lesser agricultural lands will be there for crop production due to salinity. For sustainable agricultural practice, this can be mitigated by genetic modification of plants capable to tolerate the environment with minimized inputs rather than manipulating the environment according to plant requirement (Duncan, 1994). Many studies have proven the expression of particular genes (John and Bohnert, 2000) and metabolic changes (Hasegawa et al., 2000) that are induced by salinity though inadequate success has been accomplished in enlightening the mechanism of salinity tolerance. Hence, to improve the tolerance of plants to elevated levels of salts either by irrigational water or soil salinity, physiological approaches are needed to be focused that enlighten the functions of intricate systems involved in defense. The cryoprotective features of GB are exhibited because of its functions association with macromolecular structure. It strengthens the protein tertiary structure by safeguarding their disruption caused by non-compatible solutes (Bateman et al., 1992). In spinach plants it protected thylakoids against frost stress, due to its fragile association between positively charged ammonium cations and anionic carboxyl groups of conjugated structures of proteins in cell membranes (Coughlan and Heber, 1982). In addition, ABA is found to amalgamate in relation to low temperature conditions and water scarcity (Chen et al., 1983; Cornish and Zeevaart, 1984) where it induces chilling tolerance in variety of plants, when exogenous application of GB is done (Chen and Gusta, 1983; Lang et al., 1989). Various genes are also induced by ABA in response to cold and drought that suggests its pathway in tempting chilling tolerance that may overlap in response to low temperature condition. 2.14. Biosynthetic pathway of glycine betaine Among many QACs present in plants, GB occurs most abundantly in response to water deficiency (Venkatesan and Chellappan, 1998; Mohanty et al., 2002;) in chloroplast where it plays key function in thylakoid membrane stability and adjustment, thereby supporting photosynthetic apparatus (Robinson and Jones, 1986; Genard et al., 1991). In most of the higher plants, GB is synthesized in chloroplast from serine via ethanolamine, choline, and betaine
23 aldehyde (Hanson and Scott, 1980; Rhodes and Hanson, 1993). Choline is converted into betaine aldehyde, by an enzyme choline monooxygenase (CMO), which then forms GB by an enzyme namely betaine aldehyde dehydrogenase (BADH) as shown in (Fig. 2.1) below.
Fig. 2.1 Different pathways explaining glycine betaine biosynthesis COD, choline oxidase; GSMT, glycine sarcosine methyltransferase; SDMT, sarcosine dimethyltransferase Fd(red) and Fd(ox), ferredoxin in reduced and oxidized forms, respectively; SAH, S adenosylhomocysteine; SAM, S- adenosylmethionine. Although many other pathways like N-methylation of glycine are also reported, in GB accumulating plant species, though the pathway from choline to GB has been identified
24
(Weretilnyk et al., 1989). It is reported to accumulate in response to various environmental stresses in different plants, like sugar beet, spinach, barley, wheat, and sorghum (Weimberg et al., 1981; Fallon and Phillips, 1989; Mc Cue and Hanson, 1990; Rhodes and Hanson, 1993; Yang et al., 2003). These plant species usually accumulate more quantity of GB than sensitive ones in abiotic stressful conditions. This condition is however, not customary as no substantial relationship is observed between accumulation of GB and salt tolerance in plants from Triticum, Agropyron, and Elymus genus (Wyn Jones et al., 1984), though elevated levels of choline and betaine are found in salt sensitive than in salt tolerant lines of Egyptian clover when exposed to saline conditions (Varshney et al., 1988). Generally, GB is present at the conc. of 4-40 μmol g-1 fresh weight in naturally accumulating plants e-g in spinach and sugar beet where it serves as osmoconformer in abiotic stressful conditions (Chen and Murata, 2011). However, it is anticipated that the relation between GB accumulation and stress tolerance is highly plant specific. Some plant species like rice, mustard, Arabidopsis and tobacco do not naturally synthesize GB under stress or non-stress situations (Rhodes and Hanson, 1993). Transgenic plants of these exhibit ability to produce GB in ample quantity and possess greater tolerance level to salinity, freezing, drought or extreme temperatures (Rhodes and Hanson, 1993). The stored levels of GB in these species, however, were much lesser than in naturally accumulating plant species in unfavorable abiotic factors (Rhodes and Hanson, 1993). It might be due to metabolic flux or less available substrate i-e choline or diminished translocation of choline in chloroplasts of the cells (Nuccio et al., 1998; Huang et al., 2000; McNeil et al., 2000). An alternative, rather more effective approach to increasing plant adaptability to ameliorate environmental stress tolerance could be through exogenous application of such compounds. 2.15. Foliar application of glycine betaine and its physiological effects Some plants synthesize GB in relatively lesser quantities than the adequate levels needed to mitigate the harmful effects of dryness generated by different abiotic stress factors (Wyn Jones and Storey, 1981; Yancey, 1994; Subbarao et al., 2001). Foliar application of GB in less or non- accumulating plants may prove to be useful to combat the malicious effects of certain environmental hazards (Agboma et al., 1997a,b,c; Makela et al., 1998a; Yang and Lu, 2005). Foliar application of GB seeps through the leaves actively and translocated to various organs, where it contributes to perform its role in inducing tolerance factor (Makela et al., 1998a). The efficacy of absorption is further hastened by mixing it with surfactants like kinetic, lus-50 or
25 sito+ (Subbarao et al., 2001). As naturally synthesized GB do not readily break down in plant cells (Bray et al., 2000), and can easily be used as an inexpensive alternative. This may make a cautious and feasible approach to respond environmental vagaries in crop production Many proteins are denatured in excessive heat and saline conditions thereby, disrupting actual structure and their mode of action. Chaperone proteins are involved to play role in protein aggregation and help in refolding of proteins under unfavorable conditions. These osmolyte chemical chaperons possess low molecular weight hence revamp and stabilize the native structure of proteins (Diamant et al., 2001). GB activates ClpB, a fragment of chaperone network that increases the efficacy in disaggregation of chaperone-mediated proteins under inadequate growth conditions in E. coli (Diamant et al., 2003). During various abiotic stresses, protein synthesis involved in the repair of PSII is negatively affected that cause photoinhibition (Takahashi and Murata 2008; Allakhverdiev and Murata 2008). Here GB provokes biosynthesis of reticent proteins and thus boosts the plant ability to combat stress. Reactive oxygen species production recurrently takes place as a derivative of metabolism in chloroplast and mitochondria. However, they are produced abundantly under abiotic stressful conditions leading to photoinhibition of PSII. GB protects photosynthetic apparatus by maintaining the repair of proteins (Murata et al., 2007). Therefore, GB detoxifies ROS production which was found at negligible levels in transgenic plants under scarcity of water (Kathuria et al., 2009). Endogenous genes present in transgenic plants contribute to combat abiotic stresses. Thus, the mechanisms that confer GB mediated stress tolerance exhibit stabilization of protein structure, enzymatic activity, osmoregulation, integrity of membranous structures, and optimum rate of photosynthesis and detoxification of ROS. GB has another feature, the destabilization of double-helical structure of DNA by lowering its melting temperature in vivo. This makes GB to regulate the genes being expressed under various abiotic stresses by activating the process of replication and transcription
(Rajendrakumar et al., 1997). Other than GB, H2O2 is also evolved as a byproduct of codA gene expression in transgenic plants. Accumulation of H2O2 expressing codA gene was more pronounced in Arabidopsis and tomato plants than untransformed ones (Park et al., 2004; Alia et al., 1999). H2O2 is an illustrious regulator of gene expression during stress signaling (Mittler
2002). In transgenic rice, fungal glucose oxidase gene produces high levels of H2O2 and shows tolerance to many biotic stresses (Kachroo et al., 2003). Although the H2O2 evolved in transgenic
26 tomatoes was just 17-21 percent more than wild-type tomato plants under optimal conditions (Park et al., 2007). GB treated plants remarkably mollified the deleterious environmental factors and induced tolerance, though a better acquaintance of its mechanism, mode of action and extent of its impact in different plant species is needed. In bean plants, under water deficit conditions when exogenously applied, it improved CO2 immersion and ability of chlorophyll to harvest light while it had negligible impact on increase in biomass and yield of legumes. Similarly, in tomato plants GB mitigated the adverse effects of temperature extremes and salinity by increasing the fruit yield up to 40% as compared to non-treated ones (Makela et al., 1998a and b). A varying trend of exogenously applied GB was seen in rice seedling while studying ultrastructural damages where it improved the shoot growth while root growth remained unaffected in salt induced regimes (Rahman et al., 2002). Moreover, plants when exposed to salt stress and were treated with GB had lower Na+ conc. and greater K+ conc. in aerial parts. At sub cellular level, saline conditions cause structural damage to the leaf anatomy, by swelling thylakoid membrane; disruption of grana stacks, inter granal lamellac and plasmolysis of mitochondria. All these damages were prevented by GB application by producing additional vacuoles in root cells. This factor impedes the transportation of accumulated Na+ ions in the roots, to shoot. In the roots a change in structure due to salinity stress was found with an increased number and clumping of mitochondria in cytosol of root tip and cap. In another study, foliar application in rice plants grown in saline condition, resulted in abated Na+ deposition and retained normal conc. of K+ ions in the plant shoots (Lutts et al., 1999). In maize crop, GB application sustained the growth, water relations, and photosynthetic rate in plants grown in salt affected soils (Yang and Lu, 2005). Though, it did not affect the efficacy of PSII. The improved and stable redox reaction of photosynthesis even in salt affected maize plants treated with GB is coined with stabilized stomatal conductance and substantial efficiency of PSII. Similarly, foliar application of GB on Arabidopsis lessened the chilling temperature limiting from -3.1 to -4.5 °C (WeiBing and Rajashekar, 2001). Moreover, low temperature endurance was also observed by foliar application of GB in some cultivars of potato (Somersalo et al., 1996). In strawberry plants it induced cold tolerance in unhardened and cold- hardened seedlings as well (Rajashekar et al., 1999). 2 mM of GB application to unhardened strawberry plants supported chilling tolerance by almost twice within 72 hours of application,
27 depicting its practical use for open conditions for cultivating strawberries (Rajashekar et al., 1999). Besides the direct protective features of GB, either by supporting enzymatic activities, membrane stability or as an osmoprotectant, it also safeguards the plant cells from various environmental stresses directly or indirectly in signal transduction. It may have influence in maintaining Na+, K+ balance, which substantially contribute to combat salt tolerance by signal transduction and ionic homeostasis (John, 2002; Yilmaz, 2004). Different membrane transport systems govern ionic homeostasis in plants: recently, cation carriers that keep up ion homeostasis during salinity are characterized. Amongst them salt overly sensitive (SOS) is an avant-grade signal pathway (Chinnusamy et al., 2005) regulated by MAP kinases and its expression is highly influenced by GB. In turfgrass and Arabidopsis, GB up-regulated the expression of more than 360 genes amongst them about 6% play role in signal transduction (John, 2002) with the typical examples of lipoxygenase, osmotin, monodehydro ascorbate reductase, protein kinase, putative receptor kinase, calmodulin and receptor protein kinase. Exogenous application of glycine betaine alleviates lethal effects of salts present in the rhizosphere. Rice plants grown in saline environment was studied by Demiral and Turkan (2004) who reported increased levels of catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), and glutathione reductase (GR) in view of relative water content and melondialdehyde in leaves of Pokkali, a cultivar of rice. In another cultivar of rice, IR-28, the activities of POX, APX and SOD were increased, whereas GR and CAT were reduced upon exposure to different salt levels. Moreover, many evidences suggest that abiotic environmental maladies, particularly salt and drought, intensified the activated oxygen radicals (AOR) 1 concentrations like O2 (singlet oxygen), O2 (superoxide), H2O2 (hydrogen peroxide), and ·OH (hydroxide ions), by increasing the electron leakage in electron transport chain (Hernandez et al., 2001). The response of GB application on plants is highly specific in its quality inducing parameters. For instance, in turnip, rapeseed or spring cereals no improvement was found in growth under drought stress and GB remained unaffected (Makela et al., 1996). Similarly, it had no effect on yield, physiological parameters, or its endogenous level in cotton crop when exposed to drought conditions (Meek et al., 2003). These contrary findings however, could have been due to varying experimental differences among genotypes. Moreover, currently, the
28 mechanism by which exogenous application of GB exerts its effect at molecular level is still unclear. In a recent study of two crucifer species, B. napus (canola) and Arabidopsis, its destabilizing effects on photorespiration process at the mitochondrial step of the glycolate pathway are reported (Sulpice et al., 2002). There are many reports depicting its efficacy on plant morphology and yield obtained by its foliar application in various crops as shown in table 2.1. Table 2.1. Response of foliar application of glycine betaine on various plants Plant Species Stress Role of GB Reference Nicotiana tobacum Water deficiency Improved growth and Agboma et al. (1997b) yield Phaseolus vulgaris Water deficiency Reduction in leaf water WeiBing and potential at slow pace Rajashekar (1999) Glycine max Water deficiency Improved growth Agboma et al. (1997c)
Triticum aestivum Water deficiency (i) Maintained growth Borojevic et al (1980) (ii)No effect on growth Agboma et al. (1997a) Brassica napus Water deficiency Normal growth Makela et al. (1996)
Zea mays Water deficiency Improved growth of Agboma et al. (1997a) plants under stress Lycopersicon Salnity and high Improved growth of Makela et al. (1998a,b) esculentum temperature stressed plants Oryza sativa Salinity (i) Improved plant Harinasut et al. (1996) growth in salinity and Lutts (2000) (ii) Hastened shoot Lutts (2000) and growth but not roots Rahman et al. (2002) (iii) Lower levels of Na+ Lutts (2000) and and higher levels of K+ Rahman et al. (2002) in shoots in saline soils
Arabidopsis Chilling Improved freezing WeiBing and Rajashkar thaliana temperature tolerance from -3.1 to - (2001) 4.5o C Solanum tuberosum Low temperature Enhanced growth of Somersalo et al. (1996) stressed plants Gossypium Water deficiency Improved growth and Naidu et al. (1998) and hirsutum yield under stressful Gorham et al. (2000) condition Meek et al. (2003) No effect on growth and yield
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Moreover, the growth of F. graminearum, a pathogenic fungus of wheat and some cereal crops, was observed to be aggravated due to choline and GB application (Nkongolo et al., 1993; Engle et al., 2004). Similarly, aphids attack was found to be higher because of more GB accumulation under stressful conditions in barley and other cereals (Araya et al., 1991). Therefore, it is sagacious, before making any recommendation to its commercial use, the extent of possible collateral damages caused by biotic factors must be examined in different plant species to improve plant stress tolerance and ultimate adaptability. 2.16. Polyamines Polyamines, a group of plant growth modulating substance appears to be involved in variety of processes in plants like organogenesis, leaf senescence and other environmental challenges (Malmberg et al., 1998). They include putrescine, spermine, spermidine and cadavarine which possess low molecular weight and are organic polycations displaying high biological activity (Kaur-Sawhney et al., 2003). They modify roles of RNA, DNA, proteins and nucleotide triphosphates by shielding macromolecules under unfavorable conditions by modulating stress regulated genes and exhibiting antioxidant properties (Kuznetsov and Shevyakova, 2007). Polyamines and ethephon compounds are modulators involved in plant growth and development causing varying responses (Wang et al., 2002). Ethylene production radically increases in plants under, abnormal temperature, drought, wounding and ultra violet irradiation (Rakitina, 1994). However, roles of polyamines and ethylene on plants caused by abiotic stresses are still needed to be focussed (Kuznetsov et al., 2007). Similarly, it is reported that main naturally occurring polyamines like putrescine, spermidine and spermine act together with ethylene in monitoring functions (Galston et al., 1997; Kaur-Sawhney et al., 2003). These organic molecules are ubiquitous in majority of living organisms. Their multitude of biological functions is based upon simplicity in structure, universal distribution in cell organelles presumed to be involved in physiological roles by considering structural stability of macromolecules and cell membranes. They accumulate in ample quantity in cell and sequest extra nitrogen by reducing the levels of NH3 toxicity. This maintains balance in overall nitrogen distribution in various pathways. It’s unsurprising that fluctuations in their levels in cell organelles are related to differential effects in plants in various stresses influencing different stages in plant life. They are deemed important in inducing stress tolerance in plants by directly ameliorating the stress causing factors. Similarly, catabolic products synthesized by them may
30 play role in causing stress impairment. Several features that exist between polyamines and abiotic stresses seem to play contradictory roles in their mode of action (Galston and Sawhney, 1990; Alcazar et al., 2006a, 2010, 2011a; Kusano et al., 2007; Liu et al., 2007; Bachrach, 2010; Alet et al., 2011; Hussain et al., 2011; Shi and Chan, 2014). 2.17. Polyamines and their role in abiotic stress The ubiquitous nature, presence in ample quantity (millimolars) in plants and absolute roles in cell survival issues related to their functions during unfavorable environment make them focussed and difficult to study. The most confounding problem in their role is the dearth of understanding their mechanism principle. Hussain et al. (2011) graded them as “mysterious modulator in response to stresses,” possibly because of their diversified activities at cellular level and poorly understood mode of action. Several findings (Alcazar et al., 2006a; Takahashi and Kakehi, 2010; Alet et al., 2011; Hussain et al., 2011; Gupta et al., 2013; Shi and Chan, 2014) in a classy manner have demonstrated their various roles in inducing tolerance. These features cover: (i) a compatible solute with Proline, GB and GABA; (ii) interaction with conjugated molecules like DNA or RNA or translational complexes, cellular and organelle membrane stability; (iii) - role in direct rummaging of O2 and OH radicals that promote the synthesis of antioxidant enzymes and secondary metabolites; (iv) serving as a signaling molecule in abscisic acid regulated pathway by producing H2O2; (v) regulator of various ionic channels; and, ultimately (vi) role in programmed cell death (PCD).
Moreover, they are known to serve function in controlling ammonia (NH3) toxicity, nitric oxide (NO) evolvement, and harmonizing organic nitrogen metabolism at cellular level (Nihlgard, 1985; Moschou et al., 2012; Guo et al., 2014). Biosynthesis of polyamines require glutamine, a vital amino acid needed in large quantities for nitrogen assimilation as starting material cause leading homeostatic shifts in cell metabolism. Thus, under stress conditions they perform these functions in an efficient way when changes in metabolic reactions are inevitable and exist in narrower limits. In this way, they combat catastrophic perturbations that takeplace during homeostasis of carbon and nitrogen (Minocha et al., 2000; Bhatnagar et al., 2001; Bauer et al., 2004; Majumdar et al., 2013). Higher plants when face continuous ecological stresses like air pollutants results in changing soil chemical characteristics while altered metabolic homeostasis stabilizes the elevated levels of PAs and classify them as biochemical indicators of stressful conditions (Minocha et al., 2000, 2010). They become counteractive in minimizing
31 stress injury rather inducing temporary remedy. The four basic aspects that envisage the importance of PAs in developing the response against stress are as follows (Galston and Sawhney, 1990; Alcázar et al., 2006a, 2010; Kusano et al., 2007; Liu et al., 2007; Bachrach, 2010; Alet et al., 2011; Hussain et al., 2011; Shi and Chan, 2014). (i) Upregulation of polyamine; (ii) increased accumulation of PAs accompanied by the activity of biosynthetic enzymes and gene expression; (iii) mutation in genes for synthesis of PAs that are mildly tolerant against abiotic stress; (iv) while their exogenous application induce tolerance to malicious environment, inhibited biosynthesis of PAs make the plants more prone to stress calamities. 2.18. Foliar application of PAs and abiotic stress Exogenous or foliar application of polyamines exhibit positive results against increased stress tolerance, besides its transgenic upregulation, their chemical inhibition in biosynthesis exacerbates the damages caused by various stresses. Exogenous application presumably develops direct interaction with membrane bounded structures of the cells, causing reduced oxidant activity by serving as attuned osmolite and favoring ionic features (Hu et al., 2005; Ndayiragije and Lutts, 2006; Wang et al., 2007; Afzal et al., 2009). In accordance with their use as foliar application, many studies have revealed them as a useful tool in in vitro studies, especially in improved callus formation from leaf explants that involved in short term stress mitigation. Following are some of the findings of exogenously applied polyamines on inducing stress tolerance in plants: Exogenous application of putrescine at 0.1 mM at the time of anthesis in wheat (Triticum aestivum L.) expressively improved the parameters associated to photosynthesis by increasing the contents of proline, amino acids, soluble sugars, water related attributes and reduced membrane damage, these features ultimately increased the total grain yield (Gupta et al., 2012). In in vitro, explants of tobacco, pre-treatment with 1mM putrescine 1hour prior to PEG (25%) Kotakis et al. (2014) found pronounced prevention in osmotic stress, by reducing water loss and sustaining the maximum efficiency of photosystem II. Exogenous application of putrescine or arginine at 2.5 mM increased the tolerance against high temperature ranging from 35±2°C for the duration of 4-8 hours in 30-35 days old wheat plants. These plants synthesized higher levels of endogenous putrescine, spermidine, after five days of application (Hassanein et al., 2013).
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Foliarly applied spermidine for 7days at initial stage in rice, prior to inducing salinity stress prominently enhanced grain yield, calcium contents in grains, and improved K+/Na+ ratio (Saleethong et al., 2013). Foliarly applied spermidine while inducing salinity hastened the endogenous spermidine, spermine in the seedlings of Panax ginseng by activating the defense mechanism in the
presence of antioxidants, by reducing the levels of H2O2 and superoxides as well (Parvin et al., 2014). Exogenous application of polyamines in mitigating salinity stress induced by NaCl in 5 months old sour orange was studied by Tanou et al. (2014). They concluded, genes producing antioxidant enzymes became more expressive by revamping the oxidation status of cells. Similarly, proteomic studies revealed lesser protein carbonylation with improved protein S-nitrosylation. In Bermuda grass, Shi et al. (2013) reported significant increase in antioxidant enzymes by foliar application of polyamines against drought and salinity. Similar findings are expressed by Zhao et al. (2010) who also found that water deficiency affected the proteins involved in photosynthesis and defense associated pathways mediating antioxidant system. Thus, putrescine positively affects the photosynthetic machinery as explained by Sobieszczuk-Nowicka and Legocka (2014). 2.19. ROS and polyamine catabolism There exists the most intrinsic and multidimensional interaction between polyamines, antioxidants and ROS. This association is amongst one of the most complex physicochemical relationship in plant kingdom. The relationship between ROS and abiotic stresses has been known since age’s right from their discovery. Although, ROS is illustrious for its widespread damage in various cellular metabolites (Pottosin et al., 2014), but some responses particularly to water scarcity, salinity and other environmental threats presumably need to get rid of it and other free radicals having oxygen. This is achieved by maintaining water potential of the cells and amplified synthesis of antioxidants and different associated enzymes. Various studies revealed the complexity of this relationship, especially under unfavorable conditions in plants (Bhattacharjee, 2005; Gill and Tuteja, 2010; Velarde-Buendia et al., 2012; Pottosin et al., 2014).
At higher endogenous levels of polyamines, their catabolism, the level of H2O2, various ROS and antioxidant system and metabolites, increases. Hence, they possess the preventing roles
33 from stress (Bouchereau et al., 1999; Groppa et al., 2007; Thangavel et al., 2007; Mapelli et al., 2008; Rakitin et al., 2009; Jantaro et al., 2011; Pothipongsa et al., 2012; Chmielowska-Bak et al., 2013; Mandal et al., 2013; Scoccianti et al., 2013). These diversified roles of polyamines elucidate the fact that increased inter cellular levels contribute to the both, ROS and antioxidant under damaging conditions. This is so because, ROS contribute in biotic and abiotic stress as a part of signal transduction pathway to persuade protective responses (Moschou et al., 2012), while on the other hand they cause prompt membrane destruction, degradation of chlorophyll and oxidation of various metabolites present in cells. Polyamines have also been involved in variety of protective responses in cells (Bouchereau et al., 1999; Zepeda-Jazo et al., 2011; Pothipongsa et al., 2012; Tanou et al., 2012, 2014). This includes membrane and macromolecules protection that are ultimate target of damage caused by ROS. Moreover, the catabolism of polyamines directly contributes towards cell damage, most prominently by the synthesis of H2O2 and acrolein found cells of tobacco plants (Kakehi et al., 2008; Mano, 2012; Takano et al., 2012). Here, H2O2 evolved as a result of polyamine catabolism is importantly needed for lignin synthesis in the plant cells (Moschou et al., 2008b). As the polyamines possess strong +ve charge, they efficiently block the cation channels embedded in the cell surface membrane (Dobrovinskaya et al., 1999a, b; Zepeda-Jazo et al., 2008; Bose et al., 2011; Zepeda-Jazo et al., 2011). The specificity in action for partial blocking of sodium channels from outside versus potassium channels in tonoplast of vacuoles apparently assists vacuoles to amalgamate sodium in it, thus altering the actual K+/Na+ ratio in cytosol of the cells under unfavorable conditions (Pottosin et al., 2012, 2014). 2.20. Stress retention and polyamines function Plants have ability to respond stresses in both aspects i-e short term and long term. These aspects are governed by the combination of their inherent ability that could either be genetically controlled or of evolutionary nature. It is further envisaged by the fact that the plants might have been exposed to stresses in very initial stages of life or during early developing stages. This idea was termed as “priming” or “stress retention” (Bruce et al., 2007). Although from evolution point of view, plants are distributed in various micro and macro ecosystems based upon their adaptation to multifaceted biotic and abiotic stress responses. There exists vast repertoire of examples depicting physiological effects in plants induced by repeated exposure against variety
34 of stresses (Jisha et al., 2013). These abiotic factors include the adaptive features of halophytes, xerophytes, hydrophytes and the plants that have ability to face the temperature of both the extremes, and ozone. Priming is a useful approach practiced prior to seed germination or sprouting of geophytes that may have long term effects when grown under unpredictable or abrupt environmental changes inducing stress e-g the presence of heavy metals or accumulation of salts in the soil. Overall growth rate might be stunted or at slower pace but it still sustains by priming. Adaptation of various ecotypes to diversified environments presents typically the collective role of priming and genetic selection in the continuum to different agro-climatic conditions. Moreover, the priming because of early exposure to various abiotic stresses may involve epigenetic alterations that strengthen with the passage of time. These epigenetic effects in altering environment, changes the gene expression best suited to that particular environment; however, the mode of action or mechanism is still obscure. Though, epigenetic alterations usually occur at chromatin level with specific sequence of DNA methylation, acetylation of histone and other modifications. Most of these alterations provide stability to organisms; others may have changeable effect because of exposure to growth modulating substances that appeared to be diffused in next generations induced by sexual duplication (Sano, 2010; Shao et al., 2014; Sharma, 2014). There are various external physical and chemical factors that are responsible to control epigenetic changes that manipulate the fate of cells. These factors include hormonal control of totipotency in cells and tissue culture. So, it can be envisioned that polyamines play critical roles that influence epigenetic changes in relation to priming for unfavorable environment. This response associated to short-term effect to epigenetic changes is coined with alterations of DNA molecules and histone proteins and chromatin interaction (Pasini et al., 2014). The promising aspects of this premise includes: (i) the basic role of polyamines in these changes normally occurring in metabolic activities by particular interaction with DNA preceding or through methylation process (Krichevsky et al., 2007; Sharma et al., 2012), (ii) the improvement in specific alterations that occur in circumstances of priming specifically for chilling or salt exposure of seeds to induce tolerance against seed desiccation or dormancy of buds. 2.21 Putrescine and induction of stress tolerance
35
Endogenous synthesis of putrescine is mediated by different enzymes though ADC enzyme is of foremost importance (Fig. 2.2). The genome of Arabidopsis thaliana has two genes (ADC1 and ADC2) that encode ADC. ADC1 play pivotal role in developing structural features and express in all cells and tissues while ADC2 responds to unfavorable environmental conditions like wounding, mechanical damage or osmotic stress (Soyka and Heyer, 1999; Perez- Amador et al., 2002). The mutant ADC2 gene is unsuccessful to combat the osmotic stress in the wild-type plants of Arabidopsis and had no pronounced phenotype in ordinary growth conditions although it had higher levels of endogenous putrescine (Soyka and Heyer, 1999). It was later found that mutant allele of ADC2 gene was proved sensitive to saline conditions as compared to wild Arabidopsis (Urano et al., 2004). This mutant type had low endogenous putrescine and was restored by foliar application of putrescine to ameliorate various stresses, though it contained adequate levels of endogenous spermidine and spermine.
Fig. 2.2 Biosynthetic pathway of polyamines. Abbreviations: ACL5, ACAULIS5; ADC, arginine decarboxylase; AIH, agmatine iminohydrolase; CPA, N- carbamoyl putrescine amidohydrolase; ODC, ornithine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase; SPDS, spermidine synthase; SPMS, spermine synthase; TSPMS, thermospermine synthase.
36
These findings clearly depict protective roles of putrescine against abiotic stress tolerance, as well as premise it as precursor of biosynthesis of other PAs. A threshold model in transgenic plants explained transformed levels of putrescine (Capell et al., 2004), that it must surpass to a certain level to exacerbate the production of spermidine and spermine needed by the plants under stress to combat with. There is evidence that mutants of ADC1 and ADC2 gene, fails to synthesize polyamines as they die earlier at embryo development phase (Urano et al., 2005). Moreover, putrescine is also a precursor of biosynthesis for various alkaloids like pyridine and tropane as nicotine contained in tobacco and hyoscyamine in D. stramonium. 2.22. Spermidine, an essential polyamine for plant growth In E. coli, the mutants’ deficit in spermidine synthesis undergoes normal growth and development. Contrary to that, there is no spermidine synthase activity in SPE3 mutant of yeast and needs exogenous spermidine to carry out its normal growth pattern (Tabor and Tabor, 1999). In Arabidopsis genome, 2 genes encode spermidine synthase enzyme i-e SPDS1 and SPDS2. Both of these genes and each of their single mutants exhibited normal growth, while, the embryo development of double mutant was observed to be impeded at heart stage, indicating the spermidine a necessary polyamine in the process of embryogenesis (Imai et al., 2004a). Conversely, spermine proved to be non-functional for viability in Arabidopsis plant (Imai et al., 2004b). It is worth considering, whether spermine is functionally the alternate for spermidine or not, as the later may specifically be needed and may play role in development. As spermidine is substrate for the synthesis of an amino acid, deoxyhypusine. This particular amino acid is synthesized by an enzyme deoxyhypusine synthase (DHS) that shifts half of the butylamine of spermidine to 1-amino group of lysine-50 of dormant eukaryotic translation instigation factor 5A precursor. Moreover, this deoxyhypusine-eIF5A is transformed into active hypusine-eIF5A, that play role in the transport of mRNA from nucleus to cytoplasm. This proactive eIF5A is important for mitosis and proliferation in eukaryotes (Park, 2006) and consistently require spermidine for the development of plant embryo. Any sort of mutation in DHS gene may result in arrested development of embryo sac as reported in Arabidopsis plant (Pagnussat et al., 2005) as deoxyhypusine-eIF5A cannot be shared by maternal tissues. Plant PAs exist as free particles as well as conjugated to cinnamic acids like caffeic, p- coumaric and ferulic acids (Bagni and Tassoni, 2001). These conjugated molecules are termed as hydroxylcinnamic acid amides. Currently, 2 encoding genes that intercede the syntheses of
37 spermidine conjugate are recognized in Arabidopsis namely spermidine disinapoyltransferase (SDT) and spermidine dicoumaroyltransferase (SCT) (Luo et al., 2009). Both of these genes are prominently expressive in emerging embryos (SDT) and in root tips (SCT). Moreover, another tapetum-specific gene was cloned from modal plant that encoded spermidine hydroxycinnamoyltransferase (Grienenberger et al., 2009). Conjugates of spermidine synthesized are concerned in inducing various protective features like pathogenic defense, detoxification of phenolic compounds or rationing as a backup of polyamines, made available to actively burgeoning tissues. In most of the eukaryotes, polyamines also covalently bond to the residues of glutamine of specific proteins by the activity of transglutaminase (TGase) enzyme. The pervasive existence of TGase activity in the tissues of all plants envisage the connotation of inter and intra particular cross-link creation of different proteins by polyamines activity (Serafini-Fracassini and Del Duca, 2008). Many species from Asteraceae, Boraginaceae and Orchidaceae family, synthesize an unusual polyamine namely homospermidine. This polyamine is a prime intermediate of biosynthesis of pyrrolizidine alkaloids which work as a shielding compound in the plants of aforementioned families. Homospermidine is formed by the transference of aminobutyl group of spermidine - putrescine by accomplishment of an enzyme homospermidine synthase (HSS), that catalyses its synthesis. This HSS gene was first cloned from Senecio vernalis and was found resultant from DHS gene (Ober and Hartmann, 1999). 2.23. Spermine and plant stress response The genome of E. coli does not contain gene for spermine synthesis, though, yeast SPE4 mutant that dearths spermine synthase activity has shown usual growth even in non-existence of spermine (Tabor and Tabor, 1999). Many findings depict the interaction of spermine with functionally dissimilar receptors and ion channels (Williams, 1997). Generally their efficiency in blocking or modulating these kinds of proteins falls in order of spermine ˃ spermidine ˃ putrescine. Under optimal growth conditions most of the plants don’t require spermine, this concept has been proved as a loss in the function of SPMS mutant in Arabidopsis (Imai et al., 2004b) though the reality of temporary structural-functional complex formed between sequential enzyme in the metabolic pathway (metabolon) of polyamine contains spermidine and spermine synthase in Arabidopsis plant, responsible for proficient synthesis of spermine in the plant cells
38
(Panicot et al., 2002). SPMS mutants are relatively sensitive to drought and salinity stress than wild-types (Yamaguchi et al., 2007). This phenotypic expression may be due to inward flow of potassium ions across the guard-cells that get jammed by the intracellular polyamines (Liu et al. 2000). This condition of obstruction or blocking of ionic channels is reported in barley and red beet for vacuolar cationic channels (Dobrovinskaya et al., 1999b) and in the mesophyll cells of the pea plants (Shabala et al., 2007). Many evidences support the modulating role of spermine in regulating receptor activities and ion channels; furthermore it also protects the DNA molecules in the nucleus from succeeding mutation and free attack radical even at low concentrations of millimolar (Ha et al., 1998). Similarly, it plays a typical function as a mediator in defence signalling pathways against various plant pathogens (Yamakawa et al., 1998; Takahashi et al., 2003). The most pronounced effect of spermine is hypersensitive response, which is brought about by its accumulation in apoplast during ‘spermine signalling pathway’ and involves the activity of genes related to defence and those encoding for pathogenesis associated proteins, MAP kinases, and ultimately PCD.
This hypersensitive response is elicited by H2O2 derived by spermidine, generated by the activity of an enzyme contained in the apoplast polyamine oxidase (PAO) (Cona et al., 2006; Kusano et al., 2008; Moschou et al., 2008b). In nut shell, the two roles of spermine that are of foremost importace in cell survival are: scavenger of free radicals and the source of these radicals in apoplast, though there exists the chance of negative interaction of spermine with some molecules causing cell death cannot be ruled out. The ironic features of polyamines discussed earlier are indicative to those found in mammals where disproportionate accumulation of polyamines instigated apoptosis in various types of cells, either by direct activity or by the synthesis of H2O2 by intracellular spermine/spermidine acetyltransferase or PAO pathway (Seiler and Raul, 2005). It is still sbscure that how plant pathogens accumulate spermine in apoplast. 2.24. Thermospermine induces stem elongation Thermospermine, an essential isomer of spermine was first revealed in thermophilic bacteria (Oshima, 2007) and evolved from spermidine in the same process as of spermine synthesis (Fig. 2.2). The ACL5 gene encodes for thermospermine synthase and was identified as a mutant spermine synthase from acaulis5 (ACL5) that exhibited dwarf phenotype with excessive proliferation of vascular bundles (xylem tissues) in Arabidopsis (Hanzawa et al.,
39
2000). The orthologue of ACL5 was cloned from a diatom Thalassiosira pseudonana that revealed thermospermine synthase as an encoding enzyme (Knott et al., 2007). Studies on the evolutionary genetic pathways reported that genes involved in polyamine biosynthesis acquire the capacity to produce thermospermine at initial stages of evolution by transferring the gene from prokaryotes (Minguet et al., 2008). Foliar spray of thermospermine to ACL5 mutant increased plant height to some extent though it reduced the level of ACL5 transcript level and was found at elevated levels in its mutants (Kakehi et al., 2008). Decrease in the expression of ACL5 gene by thermospermine application is observed in wild-type Arabidopsis where it indicated negative feedback mechanism of thermospermine synthesis. ACL5 shows better countenance in emerging xylem vessels that particularly depicts its role in inhibiting premature cell expiry seen in xylem cells during differentiation (Clay and Nelson, 2005; Muniz et al., 2008). In some experiments designed to envisage the thermospermine activity causing stem elongation, it was found that suppressed mutants of ACLl5 gene, namely SAC showed repossession from dwarf phenotype in ACL5 that were isolated. SAC51 plays key role in stem elongation as it possesses a transcription factor that encrypts basic-helix-loop-helix (bHLH). SAC51-d allele shows point mutation at 4th upstream open reading frame (uORF) amongst five uORFs that exist in 5 leader sequence (Imai et al., 2006). This allele has dominant nature in ACL5 that attributes high translational efficiency seen in SAC51 as the 4th uORF shows inhibitory effect during translation process of main ORF. Similarly, another gene SAC52-d for a mutant, encrypts ribosomal protein namely, L10 (RPL10A) that also enhances translational efficacy of SAC51 core ORF (Imai et al., 2008). Moreover, a recently found mutant, SAC56-d, showed defect in the gene encrypting RPL4 and depicts synonymous effect to that of SAC52-d on SAC51 during translation. These findings clearly reveal that SAC51 is an eminent transcription factor involved in controlling the stem elongation and the pivotal role of thermospermine in translational regulation of its uORF cannot be ruled out. 2.25. Polyamines their oxidation and defence mechanism Catabolism of spermine, spermidne is carried out by PAOs, same is the case with putrescine in which catabolism is carried out by Cu containing diamine oxidase to 4- aminobutanal with the release of ammonia (NH3) and hydrogen peroxide (H2O2) (Bagni and
40
Tassoni, 2001). The classification of PAOs is done on the basis of their degree of deamination. Spermidine is deaminated to 4-aminobutanal while spermine to N-(3-aminopropyl), together with 1,3-diaminopropane and hydrogen peroxide, resulting in converting back spermine→spermidine→ putrescine with simultaneous release of 3-aminopropanal and hydrogrn peroxide (Moschou et al., 2008b).
The site of occurrence of these deamination reactions is apoplast and resulting H2O2 induces a complex array of defence against microbes or other pathogenic attack. Moreover, H2O2 derived by polyamines causes cell-wall maturation, strengthening and lignification in different phases of development particularly in wound-healing by reinforcing the cell walls during pathogenic invasion (Cona et al., 2006). Plants secrete spermidine in apoplast and oxizes to H2O2 when they come across to unfavorable environmental conditions (Moschou et al., 2008a). The physiological and biochemical roles of PAOs are still obscure in plants and their converting back activity is seen to be sorted into peroxisomes in Arabidopsis (Moschou et al., 2008b). 2.26. Prospects of polyamines as exogenous application Polyamines possess miraculous features regarding overall development of plants when exposed to suboptimal environmental conditions. Their physiological roles are also elucidated especially at molecular level. Exogenously applied polyamines serve as a source of hydrogen peroxide most prominently of spermine that causes stem elongation seems to be matchless regarding cut flower production, in which longer stem length is always demanded and appreciated. Moreover, the catabolism of polyamines includes degradation and conjugation that may prove to be a useful tool in unveiling the homeostatic function of polyamines in plants at cellular level. Although the mode of absorption of exogenosly applied polyamines and translocation of them in plants is needed to be explored. As seen in Arabidopsis, the synthesis of thermospermine is regulated by feedback mechanism of ACL5 gene, the mode of action and detailed mechanis of which remains unclear. Characterization of SAC mutants and associated genes that aid in cracking the defined mode of of spermine action particularly thermospermine causing stem elongation is needed to be explored in detail. Further exploration of responsive genes may also be helpful to apprehend the efficacy of each polyamine. The mode of action and molecular mechanism of foliarly applied polyamines is obviously poles apart from that of endogenous plant hormones, where the pathway of ubiquitin
41 proteasome is involved (Santner and Estelle, 2009), and certainly will provide a novel understanding in modulating plant growth and various stress responses.
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Chapter # 3 Materials and Methods
Plant growth promoting substances are designed to affect plant growth and development. They are applied for particular purposes to elicit typical phases in plant growth. Although there is plenty of scientific information on their use in greenhouses, however there use in open fields cannot be ruled out. Achieving the optimum results with them is the combination of art and science with a lot of trial and error and good understanding in their response befitting to plant type. They are considered as integrated part of plant production cycle and graded as best production tool like water and fertilizers. On the other hand they should not be mediated as crutches for weak management or neglect in cultural practices. However, they must be used in conjunction with number of nonchemical control options to manipulate well- proportioned plant growth. Therefore, a project was planned with the title of ‘Varietal evaluation and quality enhancement of cut tulips by exogenous application of growth promoting substances” using following materials and methods for morpho-physiological and, biochemical analysis. The studies were carried out at Institute of Horticultural Sciences (IHS), University of Agriculture, Faisalabad, Pakistan comprising of following experiments. 3.1. Experiment # 1
Screening of different tulip cultivars under Faisalabad conditions based upon their growth response.
3.1.1. Materials and Methods
Location: Shade house (IHS), latitude=31°-26´ N, longitude=73°-06´ E, altitude=184.4m
No. of cultivars: 10
Replications: 03
Growth medium: Sweet sand in open field
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3.1.2. Treatments: 10 cultivars
T1 = Apeldorn
T2 = Barcelona
T3 = Clear water
T4 = Golden Apeldorn
T5 = Ile de France
T6 = Leen Van der Mark
T7 = Parade
T8 = Purple flag
T9 = Queen of night
T10 = Spring green 3.1.3. Plant material and growth conditions The experiments were executed in open field conditions and were repeated twice. Forced bulbs of 5-6 cm in circumference of ten promising tulip cultivars regarded as best for cut flower production were purchased from Greenworks Pvt. Ltd. a stoop flower bulb company of Holland in Pakistan. All field practices were very well maintained to support optimal growth conditions favorable for plant growth and development. Bulbs were planted on raised beds in sweet sand as a soil medium having properties mentioned in Table 3.1 and 3.2. The cultivars considered for research along with their unique flower color are mentioned in Table 3.3. Table 3.1 Physical and chemical properties of soil SOIL ANALYSIS Soil characteristics Soil depth 0-6 (inch) 6-12 (inch) Texture Loamy Loamy pH 8.30 8.10 EC(dS m-1) 0.42 0.37 Exchangeable Sodium (mmol/100g) 0.90 0.70 Organic matter (%) 0.51 0.54
Table 3.2 Macronutritional status of soil
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Macronutrient Status
Nitrogen (mgL-1) 0.049 Weak
Phosphorus (mgL-1) 4.3 Too weak
Exchangeable potassium (mgL-1) 105 Medium
Table 3.3 Various tulip cultivars tested for their adaptability
Serial # Cutivar Name Flower color 1 Apeldorn Yellow 2 Barcelona Pink 3 Ile de France Maroon 4 Clear water White 5 Golden apeldorn Mustard 6 Leen Vander Mark Red with yellow edge 7 Parade Orange 8 Purple flag Purplish black 9 Queen of night Deep maroon 10 Spring Green White with light green edge
Each cultivar was considered as treatment to assess their adaptability and growth response in climatic conditions of Faisalabad (Pakistan). Bulbs were planted at raised beds at a distance of fifteen centimeters bulb to bulb and at depth of twice the size of bulbs with three replicates. Fifty same sized bulbs of each cultivar per replication were planted in the month of November. The experiment was repeated twice i-e in the year 2013 and 2014 to increase the precision and accuracy of the results. Prior to planting in the soil, bulbs were treated with fungicide Topsin-M 70WP at 1mgL-1 of water. Bulbs were kept dipped in the solution for five minutes for adherence of fungicide on bulbs surface. Cultivars were evaluated on the basis of their morphological, floral and postharvest attributes. Basal dose of NPK (17:17:17) 100 gram per bed was applied prior to planting because of inadequate nutritional status of the soil as revealed in the above mentioned tables. Meteorological data during the course of experiment for both the years was monitored and recorded to assess different growth stages as influenced by the
45 environmental factors. First irrigation with canal water was applied after planting and further irrigations were maintained according to requirement. In total three irrigations were applied each year. The field was kept free from weeds, bird attack and other unfavorable biotic factors. Data regarding following parameters was recorded at relevant growth stages during the life cycle of crop. 3.1.4. Data Collection
Data regarding time to start sprouting, time to reach 50% sprouting and sprouting percentage was assessed by visiting the field on daily basis early in the morning and counting the number of sprouted bulbs each day. This practice was continued for thirty days after planting of the bulbs.
3.1.5. Calculation of time to reach 50% sprouting (E50)
Time taken to reach 50% sprouting (E50) of bulbs was calculated by the following formula of Coolbear et al. (1984) modified by Farooq et al. (2005):
푁 −푛푖 2 E50 = 푡푖 + (tj-ti) 푛푗−푛푖
Where N is the final number of sprouted bulbs, and ni, nj are the cumulative number of bulbs sprouted by adjacent bulb counts at times ti and tj respectively, when ni < N/2 < nj.
3.1.6. Calculation of mean emergence time (MET)
Mean emergence time (MET) was calculated by the following equation of Ellis and Roberts (1981) as under:
∑ 퐷푛 Mean emergence time (MET) = ∑ 푛
Where n is the number of bulbs, which were emerged on day D, and D is the number of days counted from the beginning of emergence.
3.1.7. Calculation of final emergence percentage (FEP)
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Data were recorded daily for sprouting count by following seedling evaluation protocol given in the handbook of the Association of Official Seed Analysis (AOSA, 1990). Final germination percentage (FEP) was calculated at the end of experiment by the following equation.
Total numbers of bulbs sprouted Final emergence percentage (FEP) (%) = × 100 Total numbers of bulbs planted
3.1.8. Calculation of emergence index (EI)
Data regarding emergence index (EI) was calculated using the formula of Scott et al., 1984:
푇푖푁푖 Emergence index (EI) = 푆
Here Ti is the number of days after sowing, Ni is the number of bulbs sprouted on day i, and S is the total number of bulbs planted. Final emergence was calculated as a percentage of the number of bulbs planted.
3.1.9. Measurement of plant height (cm)
Plant height was measured as described in 3.2.6.1.1.
3.1.10. Measurement of plant fresh mass (g)
Plant fresh mass was measured as described in 3.2.6.1.2.
3.1.11. Measurement of plant dry mass (g)
Plant dry mass was measured as described in 3.2.6.1.3.
3.1.12. Measurement of leaf area (cm²)
Leaf area was measured as described in 3.2.6.1.4.
3.1.13. Measurement of leaf chlorophyll contents (SPAD Value)
Leaf chlorophyll contents were recorded as described in 3.2.6.2.4.
3.1.14. Calculation of days to floral bud emergence (days)
Days to floral bud emergence were recorded as described in 3.2.6.5.1.
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3.1.15. Measurement of tepal diameter (mm)
Tepal diameter was measured as described in 3.2.6.5.2.
3.1.16. Measurement of flower diameter (days)
Flower diameter was measured as described in 3.2.6.5.3.
3.1.17. Measurement of length of petals (cm)
Length of petals was measured as described in 3.2.6.5.4.
3.1.18. Measurement of width of petals (cm)
Width of petals was measured as described in 3.2.6.5.5.
3.1.19. Measurement of scape diameter (mm)
Scape diameter was measured as described in 3.2.6.5.6.
3.1.20. Measurement of flower quality
Flower quality was measured as described in 3.2.6.5.7.
3.1.21. Calculation of vase life (days)
Vase life was calculated as described in 3.2.6.6.1.
3.1.22. Calculation of days to start senescence (days)
Days to start senescence were recorded as described in 3.2.6.6.2.
3.1.23. Calculation of tepal abscission (days)
Tepal abscission was recorded as described in 3.2.6.6.3.
3.1.24. Calculation of mass of bulbils per clump (g)
Mass of bulb clump was calculated as described in 3.2.6.7.1.
3.1.25. Calculation of diameter of bulbils (mm)
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Diameter of bulbs was recorded as described in 3.2.6.7.2.
3.1.26. Calculation of number of bulbils
Number of bulbils produced per bulb was counted as described in 3.2.6.7.3.
3.1.27. Experimental Design and Statistical Analysis Experimental unit having three replications and ten treatments was designed in a simple Randomized Complete Block Design (RCBD). The obtained featured values, as the average, were statistically verified by means of variance analysis method (ANOVA). The difference between the means was assessed by Tukey test at the significance level of α = 0.05 were computed using Statistix 8.1 computer package. Differences among treatments were considered significant only when p ≤ 0.05 after statistical analysis.
3.2. Experiment # 2
Exogenous application of chitosan for the assessment of growth and quality of different tulip cultivars 3.2.1. Materials and Methods
Location: Shade house (IHS), latitude = 31°-26´ N, longitude = 73°-06´ E, altitude = 184.4m
No. of cultivars: 05 (Best performing cultivars screened from experiment # 1)
Cultivars: Apeldorn, Barcelona, Ile de France, Leen Vander Mark and Parade
Replications: 03
3.2.2. Treatments: 06
T0 = Control (Distilled water) -1 T1 = 30 mgL (Chitosan) -1 T2 = 60 mgL (Chitosan) -1 T3 = 90 mgL (Chitosan) -1 T4 = 120 mgL (Chitosan) -1 T5 = 150 mgL (Chitosan) 3.2.3. Plant material and growth conditions
49
The experiment was executed in open field conditions and repeated twice. Fifteen same sized bulbs (5-6cm in circumference) were planted against every treatment in each three replications. Forced bulbs of five screened cultivars were purchased from the same firm as mentioned earlier. Bulbs were planted at raised beds at a distance of fifteen centimeters bulb to bulb and at depth of twice of their size. In total 90 similar sized bulbs of each cultivar per replication were planted in the month of November 2014 and November 2015. Prior to planting in the soil, bulbs were treated with fungicide Topsin-M WP 70 at 1mgL-1 of water. Bulbs were kept dipped in the solution for five minutes for adherence of fungicide on bulbs surface. Basal dose of NPK (17:17:17) 100 gram per bed was applied prior to planting to support the macro-nutritional requirements of the crop. Meteorological data during the course of experiment for both the years was monitored and recorded to assess different growth stages as influenced by the environmental factors. First irrigation with canal water was applied after planting and further irrigations were maintained according to field requirement. In total three irrigations were applied each year. The field was kept free from weeds, bird attack and other unfavorable biotic factors. 3.2.4. Exogenous application of chitosan The growth promoting substance biopolymer chitosan of low molecular weight (2 kDa) was purchased from Musaji Adam & Sons, an authorized dealer of products for healthcare, industry and research labs in Lahore, Pakistan. Chitosan was delivereded by the Yuhuan Ocean Biochemical Co. Ltd. (China) with the deacetylation degree (DD) of 85% defined by UV method. This chitosan was water soluble, off white and flaky. Ultra-pure water (Maxima Ultra- Pure Water, Elga-Prima Corp, UK) with a resistivity greater than 18 MΩcm-1 was used to prepare chitosan concentrations. Chitosan was used without further purification and freshly prepared solutions were used when foliar application was to be conducted. Exogenous foliar application of chitosan solutions was carried out twice in the plant life cycle, once it was applied at two leaf stage and the next was applied right after flower bud emergence. A day before its foliar application, plants were showered with mist of water in the evening to move away any dust or dirt particle on foliage of plants. The very next day early in the morning, plants were sprayed with chitosan concentrations, Half hour prior to its foliar application plants were sprayed with surfactant, Tween-20 with the strength of 0.1% (v/v) for maximum retention and absorption of chitosan by the epidermal cells of the leaf tissues. The
50 convergent type nozzle was used for carrying out foliar spray and was adjusted in full cone position at an angle of 110° fitted at the hand held container of 2L capacity. 3.2.5. Data Collection and sampling
Data was collected from fifteen randomly selected plants (observational units) from every treatment in each replication. Data regarding various morphological, physiological and biochemical attributes of cultivars screened from experiment # 1 against various concentrations of chitosan was recorded. 3.2.6. Parameters studied Parameters regarding plant morphology, physiology, biochemical analysis, enzymatic antioxidants, flower attributes, shelf-life attributes and bulb indices were recorded as under. 3.2.6.1. Morphological attributes 3.2.6.1.1. Measurement of plant height (cm)
Plant height was measured with the help of measuring rod after harvesting the stems. Fifteen plants from each replication of every treatment were randomly selected and their heights were recorded. Plant height was measured from the base of stem till the tip of flower head.
3.2.6.1.2. Measurement of plant fresh mass (g)
After measuring the plant height, same twenty plants from each replication were selected and their fresh mass was measured on digital balance and their average was calculated.
3.2.6.1.3. Measurement of plant dry mass (g)
After measuring the fresh mass, flowers stem were chopped into pieces and were packed in paper bags. They were then placed in drying oven (Memmert-110, Schawabach) at the temperature of 72°C for 72 hours. After this they were taken out and then weighed again on an electric balance to record their dry mass. In this way, dry mass of flowers was recorded in grams.
3.2.6.1.4. Measurement of leaf area (cm²)
Two well developed leaves preferably from the base of the plant were selected. After finding their length with the help of measuring tape and maximum width with the help of digital calipers, leaf area was worked out by the formula devised by Kemp (1960) for monocots:
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Leaf area = Maximum length × maximum width×0.68
3.2.6.2. Physiological attributes
3.2.6.2.1. Photosynthesis rate (A), transpiration rate (E), stomatal conductance (gs) and sub-stomatal CO2 (Ci) Data regarding physiological attributes such as photosynthesis rate (A), transpiration rate
(E), stomatal conductance (gs) and sub-stomatal CO2 (Ci) were assessed from fully mature leaf of intact tulip plant with an open system LCi Portable Photosynthetic System (InfraRed Gas Analyzer (IRGA) (ADC Bio-Scientific Ltd. Hoddesdon, Herts, EN11, England) from 10:30 am to 12.30 pm. IRGA was operated at a light intensity (P.A.R.) of 797.45 µmol m-2 s-1, Leaf 2 -1 surface area 6.25 cm , ambient CO2 concentration (Cref) 380-450 µmol , temperatures of leaf chamber (20.6-31.4 ᵒC) and surface (20.63 ᵒC), flow rate of air per unit leaf area (U) 200.78 µmol -1 s , ambient atmospheric pressure (P) 995 mBar, H2O partial pressure 14.6 mBar and boundary 2 -1 -1 resistance to H2O at full flow (rb) was 0.17 m s mol . These gaseous exchange attributes were taken four times every year. After foliar application, two times before and after the emergence of flower bud.
3.2.6.2.1. Instantaneous water use efficiency (WUE instantaneous)
Instantaneous WUE is the ratio between photosynthetic rate (An) and the amount of water transpired (E) by plant leaf. In this study instantaneous WUE was measured as described by Polley (2002) by using the following equation: A WUE instantaneous = 퐸
3.2.6.2.2. Intrinsic water use efficiency (WUE intrinsic)
Intrinsic WUE is the ratio between photosynthetic rate (An) and the stomatal conductance
(gs) by plant leaf. In this study instantaneous WUE as described by (Polley) 2002 was measured by using the following equation: A WUE intrinsic = gs –2 –1 3.2.6.2.3. Mesophyll conductance (gm) to CO2 assimilation (mol CO2 m s ) Mesophyll conductance, the diffusion component of photosynthetic pathway was estimated by the equation of Pons et al. (2009). A Mesophyll conductance (gm) to CO2 assimilation = Ci
Where A is Photosynthetic rate and Ci is Sub-stomatal CO2.
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3.2.6.2.4. Leaf’s total chlorophyll contents (SPAD value)
Portable chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc., Japan) was used to measure leaf greenness in tulip plant. SPAD-502 chlorophyll meter can estimate total chlorophyll amounts in the leaves of a variety of species with a high degree of accuracy, which is a nondestructive method (Neufeld et al., 2006). Measurements were taken from the abaxial surface of fully expanded second to third leaf from base of the plant (Khan et al., 2003).
3.2.6.3. Biochemical attributes 3.2.6.3.1. Malondialdehyde (MDA) contents To estimate Lipid peroxidation, Malondialdehyde (MDA) contents was determined by following the method described by Li (2000). A leaf sample (1g) of each observational unit from every treatment in three replicates was homogenized in 5mL of 10% (w/v) trichloro acetic acid (TCA). The homogenate was centrifuged at 10,000g for 20 minutes and then 2mL supernatant of sample was reacted with 2mL of 0.6% (w/v) 2-thiobarbituric acid (TBA). This mixture of leaf extract, TBA and TCA was kept at water bath at 100 ᵒC for 15 minutes and then rapidly cooled in a cooling bath filled with ice. Then mixture was centrifuged at rpm 3000g for 10 minutes. After centrifugation supernatant was separated and absorbance was noted. The absorbance was monitored at 600 nm, 532 nm and 450 nm. Calculation of MDA was based on the following equation: -1 Malondialdehyde (MDA) nM L = 6.45(A532- A600) - 0.56A450 3.2.6.3.2. Total phenolic contents (mg GAE/100g fresh mass) Total phenolic contents were determined by using protocol of Julkunen and Tiitto (1985). 50 mg fresh leaf sample was homogenized and centrifuged with 80% acetone at 10,000g for ten minutes. After centrifugation, the 100 μL supernatant was mixed with 2.0 ml of water along with
1 ml of Folin–Ciocalteau’s phenol reagent and then 5.0 ml of 20% Na2CO3 solution was added in this mixture. Finally, volume was elevated to 10 ml by the addition of distilled water. Final mixture was shaken scrupulously and absorbance was taken at 750 nm by using spectrophotometer (Hitachi-120, Japan).
3.2.6.3.3. Hydrogen peroxide (H2O2) Scavenging Capacity (%) Hydrogen Peroxide Scavenging Capacity of the different tulip cultivars when applied by various growth promoting substances was determined according to the method of Ruch et al.
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(1989). A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4). Extracts (100µg/mL) in distilled water were added to a hydrogen peroxide solution (0.6 mL, 40mM). Absorbance of hydrogen peroxide at 230 nm was determined 10 minutes later against a blank solution containing the phosphate buffer without hydrogen peroxide. The percentage of hydrogen peroxide scavenging of both tulip extracts and standard compounds were calculated by the following equation:
(퐴c – 퐴s) Percentage scavenging of H2O2 = × 100 (퐴c)
Where AC is the absorbance of control and AS is the absorbance in the presence of extract sample. 3.2.6.3.4. DPPH (1, 1-Diphenyl-2-picrlhydrazyl) radical scavenging activity (% inhibition) The ability of the extracts to annihilate DPPH radical (1, 1-diphenl-2 picrylhydrazyl) was investigated by the method described by (Blois, 1958). Stock solution of the leaf extracts was prepared to the concentration of 1 mg/mL. 100 µg of each extract was added, at an equal volume, to methanolic solution of DPPH (0.1 mM). The reaction mixture was incubated for 30 minutes at room temperature. The absorbance was recorded at 517 nm. The annihilation activity of free radicals was calculated in % inhibition according to the following formula:
(퐴c – 퐴s) Percentage inhibition = × 100 (퐴c)
Where AC is the absorbance of control and AS is the absorbance in the presence of extract sample. 3.2.6.4. Measurement of antioxidative enzymes For the estimation of antioxidative enzymes, fresh leaves (0.5 g) randomly selected were grounded in an ice-cooled tissue grinder in 5 ml of 50 mM cooled phosphate buffer (pH 7.8). The homogeneous mixture was centrifuged at 15000g for 20 min at 4 °C. The supernatant was used for determining activities of the following enzymes. 3.2.6.4.1. Superoxide dismutase activity (SOD) (Units mg-1 protein) The activity of SOD was analyzed according to the protocol of Giannopolitis and Ries (1977) by calculating its potential to hinder the photo reduction of nitroblue tetrazolium (NBT). The reaction solution (3 mL) contained 50 mM NBT, 1.3 mM riboflavin, 13 mM methionine, 75 mM EDTA, 50 mM phosphate buffer (pH 7.8) and 20–50 ml of enzyme extract. The test tubes having the reaction solution were irradiated under light (15 fluorescent lamps) at 78 mmol m-2 s-1 for 15 min. Absorbance of irradiated solution was noted at 560 nm by using a spectrophotometer
54
(Hitachi-650, Japan). One unit of SOD activity was explained as the amount of enzyme that restrained 50% of NBT photo decline. 3.2.6.4.2. Catalase activity (CAT) (Units mg-1 protein) Catalase (CAT) and peroxidase (POD) activities were measured by the procedure of Chance and Maehly (1955) with some alteration. The CAT reaction mixture (2 mL) was comprised of 500µL phosphate buffer (pH 5.0), 100µL 5.9 mM H2O2 and 100µL of enzyme extract. Changes in absorbance of reaction solution were recorded after every 20s at 240 nm. One unit CAT activity was specified as an absorbance change of 0.01 units per min.
3.2.6.4.3. Peroxidase activity (POD) (Units mg-1 protein) Peroxidase (POD) reaction mixture was comprised of 800µl phosphate buffer (pH 5.0),
100 µL 20 mM guaiacol, 100µL 40 mM H2O2 and 100 µL of enzyme extract. Variations in absorbance of the reaction solution at 470nm were calculated after every 30 seconds. One unit POD activity was assigned as an absorbance change of 0.01 units per min. Activity of each enzyme was expressed on the basis of protein content. 3.2.6.5. Measurement of flower attributes 3.2.6.5.1. Calculation of days to flower bud emergence (days)
Days to floral bud emergence were recorded by calculating the days from the time of planting till the emergence of flower bud on the plant by visiting the field on daily basis and individually observing the observational units.
3.2.6.5.2. Measurement of tepal diameter (mm)
Tepal diameter was measured in millimeters with the help of digital calipers from the middle of the flower bud when it completely showed its color and was in tight bud stage.
3.2.6.5.3. Measurement of flower diameter (mm)
Flower diameter was measured in millimeters with the help of digital calipers from the middle of the flower when it was completely opened and flower heads had attained their maximum bloom best in the visually acceptable condition.
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3.2.6.5.4. Measurement of length of petals (cm)
Length of petals was measured in centimeters with the help of measuring tape. Petals from fully developed flowers were randomly plucked and were measured from the point of attachment at floral axis till the tip of petals.
3.2.6.5.5. Measurement of width of petals (cm)
Width of petals was measured in centimeters with the help of measuring tape. Petals from fully developed flowers were randomly plucked and were measured across where maximum width was observed.
3.2.6.5.6. Measurement of scape diameter (mm)
Diameter of stem was measured in millimeters with the help of digital caliper from three different positions (top, middle and base) of tulip cut flower stem after harvesting from fifteen randomly selected samples from each replication of every treatment and their average was worked out.
3.2.6.5.7. Measurement of flower quality (Copper and Spokas, 1991)
Flower quality was rated by different judges (postgraduate floriculture students and researcher himself) adapting the method described by Copper and Spokas (1991) and average was worked out. Flower quality was rated in numbers using scale ranging from 1 to 9.
1= poor quality, 2= medium quality and 3= good quality
3.2.6.6. Measurement of shelf-life attributes In this study shelf life attributes consisted of three parameters describing the post-harvest longevity of tulip cut flowers. 3.2.6.6.1. Calculation of vase life (days)
The flowers were harvested early in the morning at the stage when color was completely attained by the perianth. They were plucked at tight bud stage, placed in buckets containing distilled water and were shifted to Postharvest and Floriculture laboratory. The lower most leaf and base of the stem was trimmed to avoid air embolism and placed individually in the glass vases containing 200 ml of distilled water. After every two days, vase water was replaced with
56 fresh distilled water and lower half inch of them was given a slanting cut with sharp secateurs to avoid any bacterial clogging. Twenty flowers per treatment were used for vase life evaluation. Lab. temperature 20° ± 2°C, 80 ± 20% relative humidity, with a 0.2 – 0.4 m s–1 air speed, under a photosynthetic photon flux density (PPFD) of 8–12 μmol m–2 s–1 provided at stem level from white florescent tubes set to a daily 12 h photoperiod was maintained in the laboratory. Average vase life of the stems was estimated by the method of Gul and Tahir (2013) counted from the day of transfer of cut stems to the holding solution and terminated when 50% flowers had senesced. 3.2.6.6.2. Calculation of days to start senescence (days)
Days to start flower senescence were calculated by the method of Desai et al. (2012). Cut flower stems while in glass vases containing distilled water were visually observed daily. Days to start senescence were calculated when flower showed symptoms of color change, desiccation of tepal, blemishes or transparent area of tepal after harvesting.
3.2.6.6.3. Calculation of tepal abscission (days)
Tepal abscission was recorded by the method of Van doorn and Stead (1997) when first petal got abscised or shed naturally while assessing the shelf life display.
3.2.6.7. Measurement of bulb attributes 3.2.6.7.1. Calculation of mass of bulbils per clump (g)
After harvesting of flower stems, bulbs were left in the soil for their conditioning. They remained in the soil for about a month and then uprooted washed and dried in lab. Mass of bulbils produced per plant was weighed on an electric balance and the average of observational units was worked out.
3.2.6.7.2. Calculation of diameter of bulbils per clump (mm)
Diameter of bulbils was recorded from the center of bulbs with the help of digital calipers and their average was worked out.
3.2.6.7.3. Calculation of number of bulbils per clump
Number of bulbils produced per bulb was counted manually right after harvesting from each treatment. Twenty bulbs from each replication of every treatment were randomly selected
57 and bulbils attached at the base of mother bulb were manually counted prior to washing and their average was worked out.
3.2.7. Experimental Design and Statistical Analysis Experimental unit having three replications and six treatments was designed in a two factor factorial experiment under Randomized Complete Block Design (RCBD). The obtained featured values, as the average, were statistically verified by means of variance analysis method (ANOVA). The difference between the means was assessed by Tukey test at the significance level of α = 0.05 computed by using Statistix 8.1 computer package. Differences among treatments were considered significant only when p ≤ 0.05 after statistical analysis.
3.3. Experiment # 3 Exogenous application of glycine betaine for the assessment of growth and quality of different tulip cultivars 3.3.1. Materials and Methods Location: Shade house (IHS), latitude = 31°-26´ N, longitude = 73°-06´ E, altitude = 184.4m
No. of cultivars: 05 (Best performing cultivars screened from experiment # 1)
Cultivars: Apeldorn, Barcelona, Ile de France, Leen Vander Mark and Parade
Replications: 03
3.3.2. Treatments: 06
T0 = Control (Distilled water)
T1 = 30 mM (Glycine betaine)
T2 = 60 mM (Glycine betaine)
T3 = 90 mM (Glycine betaine)
T4 = 120 mM (Glycine betaine)
T5 = 150 mM (Glycine betaine) 3.3.3. Plant material and growth conditions
Plant growth conditions for the experiment were same as mentioned in 3.2.3. 3.3.4. Exogenous application of glycine betaine
58
The amphoteric quaternary amine, glycine betaine (N,N,N-trimethyl glycine) + - (CH3)3N CH2COOH·Cl , molecular weight (117.1), melting point 241-242 °C with an assay level of 99% of Sigma- Aldrich, Japan was purchased from Musaji Adam & Sons, an authorized dealer of products for healthcare, industry and research labs in Lahore, Pakistan. It was highly water soluble and made clear, transparent solution when dissolved in surfactant Tween-20 0.01% (v/v) to make concentrations. For making 1 mM solution of Glycine betaine, 0.117g of GB was dissolved in 1L (1000 ml) of solvent, treatment concentrations were made by calibrating accordingly. Freshly prepared solutions were used when foliar application was to be conducted. Exogenous foliar application of glycine betaine was carried out twice in the plant life cycle, once it was applied at two leaf stage and the next application was done right after flower bud emergence. A day before its foliar application, plants were showered with mist of distilled water in the evening to move away any dust or dirt particle on foliage of plants. The very next day early in the morning, plants were sprayed with glycine betaine concentrations. The convergent type nozzle was used for carrying out foliar spray and was adjusted in full cone position at an angle of 110° fitted at the orifice of hand held container of 2L capacity. 3.3.5. Data Collection /Sampling
Data was collected from fifteen randomly selected plants (observational units) from every treatment in each replication. Data regarding various morphological, physiological and biochemical attributes of cultivars screened from experiment # 1 against various concentrations of glycine betaine was recorded. 3.3.6. Parameters studied Parameters regarding plant morphology, physiology, biochemical analysis, enzymatic antioxidants, flower attributes, shelf-life attributes and bulb indices were studied in complete detail as explained in 3.2.6. 3.3.7. Experimental Design and Statistical Analysis Experimental unit having three replications and six treatments was designed in a two factor factorial experiment under Randomized Complete Block Design (RCBD). The obtained featured values, as the average, were statistically verified by means of variance analysis method (ANOVA). The difference between the means was assessed by Tukey test at the significance level of α = 0.05 computed by using Statistix 8.1 computer package. Differences among treatments were considered significant only when p ≤ 0.05 after statistical analysis.
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3.4. Experiment # 4 Comparative effect of exogenous application of different levels of polyamines (putrescine, spermine and spermidine) on two tulip cultivars to assess cut flower quality 3.4.1. Materials and methods Location: Shade house (IHS), latitude = 31°-26´ N, longitude = 73°-06´ E, altitude = 184.4m
No. of cultivars: 02 (One well adapted and other weakly adapted cultivar)
Cultivars: Apeldorn and Clear water
Replications: 03
3.4.2. Treatments: 10
T0 = Distilled Water (Control)
T1 = 1 mM (Putrescine)
T2 = 2 mM (Putrescine)
T3 = 3 mM (Putrescine)
T4 = 0.01 mM (Spermine)
T5 = 0.02 mM (Spermine)
T6 = 0.03 mM (Spermine)
T7 = 0.5 mM (Spermidine)
T8 = 1.5 mM (Spermidine)
T9 = 2.5 mM (Spermidine) 3.4.3. Plant material and growth conditions The experiment was executed in open field conditions and repeated twice i-e in the year 2014 and 2015. Fifteen same sized bulbs (5-6cm in circumference) were planted against every treatment in each three replications. Forced bulbs of two tulip cultivars namely, Apeldorn and Clear water were purchased from the same firm as mentioned earlier. Bulbs were planted at raised beds at a distance of fifteen centimeters bulb to bulb and at depth of twice of their size. In total 150 similar sized bulbs of each cultivar per replication were planted in the month of November 2014 and November 2015. Prior to planting in the soil, bulbs were treated with fungicide Topsin-M WP 70 at 1mgL-1 of water. Bulbs were kept dipped in the solution for five minutes for adherence of fungicide on bulbs surface. Basal dose of NPK (17:17:17) 100 gram per bed was applied prior to planting to support the macro-nutritional requirements of the crop.
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Meteorological data during the course of experiment for both the years was monitored and recorded to assess different growth stages as influenced by the environmental factors and treatment effect. First irrigation with canal water was applied after planting and further irrigations were maintained according to field requirement. In total three irrigations were applied each year. The field was kept free from weeds, bird attack and other unfavorable biotic factors.
3.4.4. Exogenous application of polyamines Putrescine (1,4-Butanediamine dihydrochloride: C H N .2HCl, molecular mass 161.07 4 12 2 gmol-1, Wettable powder with ≥ 98% activity with solubility level of 100 mg/ml), Spermine
( N,N'-Bis(3-aminopropyl)-1,4-butanediamine tetrahydrochloride: C H N .4HCl, molecular 10 26 4 mass 348.18 g mol-1, colorless, liquid crystals with ≥ 97% activity and solubility level of ≥ 100 mg/ml), Spermidine (1,8-Diamino-4-azaoctane: C H N molecular mass 145.25 g mol-1 , 7 19 3, transparent amorphous semi solid crystals with ≥ 99% activity with solubility level of 1.45g/10ml) were obtained from Sigma- Aldrich Chemical Company. All these three polyamines were water soluble and treatment combinations (mM) were made for 1 litre of solvent below 20°C. Fresh solutions were made twice; first spray was done at two leaf stage while second spray was done right after flower bud emergence.
A day before its foliar application, plants were showered with mist of distilled water in the evening to remove any dust or dirt particle on foliage of plants. The very next day early in the morning, plants were sprayed with various polyamine concentrations, Half hour prior to its foliar application plants were sprayed with surfactant, Tween-20 with the strength of 0.01% (v/v) for maximum retention and absorption of solutions by the epidermal cells of the leaf tissues. The convergent type nozzle was used for carrying out foliar spray and was adjusted in full cone position at an angle of 110° fitted at the orifice of hand held container of 2L capacity. 3.4.5. Data Collection /Sampling
Data was collected from ten randomly selected plants (observational units) from every treatment in each replication. Data regarding various morphological, physiological, enzymatic antioxidants and biochemical attributes of two cultivars (apeldorn and clear water) against different levels of various polyamines was recorded. 3.4.6. Parameters studied
61
Parameters regarding plant morphology, physiology, biochemical analysis, enzymatic antioxidants, flower attributes, shelf-life attributes and bulb indices were studied in complete detail as explained in 3.2.6. 3.4.7. Experimental Design and Statistical Analysis Experimental unit having three replications and 10 treatments was designed in a two factor factorial experiment under Randomized Complete Block Design (RCBD). The obtained featured values, as the average, were statistically verified by means of variance analysis method (ANOVA). The difference between the means was assessed by Tukey test at the significance level of α = 0.05 computed by using Statistix 8.1 computer package. Differences among treatments were considered significant only when P ≤ 0.05 after statistical analysis.
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Chapter # 4 Results
During the course of investigation, an attempt was made to assess the response of different cultivars of tulip for cut flower production under agro-climatic conditions of Faisalabad (Pakistan). They were further evaluated by exogenous application of different bio-regulators to induce tolerance against sub-optimal environmental conditions to enhance quality. Moreover, various physiological and biochemical attributes affecting this innovative crop were the focus of study. Data obtained for various indices of each experiment were statistically analyzed and results obtained from this study are presented and interpreted in this chapter. 4.1. Experiment # 1 Screening of different tulip cultivars under Faisalabad climatic conditions based upon their growth response 4.1.1. Time to start sprouting Results regarding time to start sprouting were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.1). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent. The earliest sprouting (5 days) was found in Apeldorn cultivar, followed by Golden apeldorn (6 days), Ile de France (9 days), Clear water (10.66 days) and Leen Vander Mark (12 days). Similarly, the cultivar Purple flag took the maximum time (17.33 days) to initiate sprouting followed by Queen of night (14.66 days), Barcelona (13.66 days), Spring green (13 days) and cultivar Parade (12.66 days) that were statistically at par with each other.
4.1.2. Time to reach 50% sprouting (E50) Results regarding time to reach 50% sprouting were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.2). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent. The minimum time to reach 50% sprouting (7.76 days) was found in Apeldorn cultivar, followed by Golden
63 apeldorn (7.84 days), Ile de France (13.67 days), Clear water (14.01 days) and Parade (16.16 days). Similarly, the cultivar Barcelona took the maximum time (28.20 days) to ascertain 50% sprouting followed by Purple flag (25.66 days), Queen of night (21.33 days), Spring green (18.93 days) and cultivar Leen Vander Mark (17.37 days) that were statistically at par with each other.
21
17.5
14
10.5
7
3.5
Time to start sprouting (days) to sprouting start Time 0
Cultivars Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.1. Time to start sprouting of different tulip cultivars
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) ) 35 50 30 25
20
15 (days) 10 5
0 Time to reach 50% sprouting (E sprouting 50% to reach Time
Cultivars Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.2. Time to reach 50% sprouting of different tulip cultivars 4.1.3. Mean Emergence Time (MET) Mean emergence time is the measure of rate and time spread of sprouting. It is not the mean time to sprout but just an index of sprouting speed and does not depict the time to specific sprouting percentage. Results regarding mean emergence time were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.3). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to mean emergence time. The minimum MET (5 days) was found in Apeldorn cultivar, followed by Golden apeldorn (16 days), Parade (24 days), Clear water (26.00 days) and Leen Vander Mark (27 days). Similarly, the cultivar Ile de France took the maximum MET (28 days) followed by Purple flag (32 days), Spring green (32 days), Queen of night (34 days) and cultivar Barcelona (35 days) that were statistically at par with each other.
65
40
35 30 25 20 15 10 5
0 Mean emergence (MET) (days) emergence time Mean
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.3. Mean Emergence Time (MET) of different tulip cultivars
4.1.4. Final Emergence Percentage (FEP)
Final emergence percentage is the concrete indicator of vigor and quality of any seed lot. It is pre-requisite to forecast the potential of the seed and crop stand. Higher the FEP more the acclimation response or adaptability can be inferred. Results regarding final emergence percentage (FEP) were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.4). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to FEP. The maximum emergence percentage (98.66%) was found in Apeldorn cultivar, followed by Clear water (94%), Leen Vander Mark (92%), and Queen of night (90.66%). Similarly, the cultivar Purple flag exhibited minimum percentage (11.33%) followed by Ile de France (72.66 %), Parade (75.33 %) while the cultivar, Golden apeldorn and Spring green ascertained same emergence percentage (77.33%) that were statistically at par with each other.
66
120
100
80
60
40
20
0 Final Emergence Percentage (FEP) Percentage Emergence Final
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.4. Final Emergence Percentage (FEP) of different tulip cultivars
4.1.5. Emergence Index (EI) The estimation of vigor tests develop a good judgement for predicting the planting value of propagating material and provide results for ranking based upon their performance. This is usually a helpful tool for indicating the planting value of seed lots as compared to ordinary germination tests. Results regarding emergence index were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.5). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to EI. Among ten cultivars maximum value of emergence index was obtained by the cultivar clear water (44.29) followed by Golden apeldorn (42.41), Leen Vander Mark (36.73), Spring green (34.74), Ile de France (34.45) while minimum emergence index was observed in Purple flag (2.83) followed by Barcelona (16.42), Parade (22.58), Apeldorn (31.27) and Queen of night (34.41) that were statistically at par with each other.
67
54
45
36
27
18
Emergence Index Emergence 9
0
Variety (Treatments) Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.5. Emergence Index (EI) of different tulip cultivars
4.1.6. Plant height (cm)
Plant height is an important attribute and the indicator of quality in cut flower crops. Thus, plant height is one of the imperative criteria indicating the growth standard. Different cultivars of tulips differ in plant height that varies when grown in different climatic zones. Results regarding plant height were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.6). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to plant height. The maximum plant height (42.06 cm) was found in Apeldorn cultivar, followed by Parade (39.93 cm), Ile de France (38.53 cm), Clear water (33.26 cm) and Leen Vander Mark (33.20 cm). Similarly, the cultivar Purple flag exhibited the least plant height (22.13 cm) followed by Spring green (24.33 cm), Queen of night (26 cm) while the cultivar, Golden apeldorn and Apeldorn ascertained same plant height (28.40cm) that were statistically at par with each other.
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45
40
35 30 25 20
Plant height (cm) heightPlant 15 10 5 0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.6. Plant height of different tulip cultivars 4.1.7. Plant fresh mass (g)
Plant fresh mass depicts about the quantity of living matter i-e protoplasm along with adsorbed water contents that are physically bound with biomass. It has significant importance to assess the quality in cut tulips and an indicator of ultimate value in flower crops. Results regarding plant fresh mass was statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.7). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to plant height. The maximum plant fresh mass (36.22 g) was found in Parade cultivar, followed by Leen Vander Mark (33.54 g), Barcelona (32.91 g), Clear water (29.32 g) and Ile de France (26.22 g). Similarly, the cultivar Queen of night exhibited the least plant fresh mass (20.36 g) followed by Golden apeldorn (20.81 g), Spring green (23.58 g) while the cultivar, Purple flag and Apeldorn ascertained the plant fresh mass 23.60 g and 24.48 g respectively, that were statistically at par with each other.
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40 35
30 25 20 15
10 Plant fresh mass (g) mass fresh Plant 5 0
Cultivars Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.7. Plant fresh mass of different tulip cultivars
4.1.8. Plant dry mass (g)
Dry mass is the actual bio mass of any living organism. Generally, different classes of tulips differ in plant dry mass when grown in different environmental conditions. Actual biomass (dry mass) of a living organism depicts the presence of constitutive organic molecules like proteins, fats and carbohydrates. Results regarding plant dry mass were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.8). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to plant dry mass. The maximum plant dry mass (3.28 g) was found in Parade cultivar, followed by Leen Vander Mark (3.24 g), Barcelona (2.99 g), Clear water (2.89 g) and Ile de France (2.59 g). Similarly, the cultivar Golden apeldorn exhibited the least plant dry mass (2.02 g) followed by Queen of night (2.12 g), Purple flag (2.23 g) while the cultivar, Spring green and
70
Apeldorn ascertained the plant dry mass 2.37 g and 2.41g respectively, that were statistically at par with each other. 4
3.5
3
2.5
2
1.5
Plant dry mass (g) mass dry Plant 1
0.5
0
Cultivars Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.8. Plant dry mass of different tulip cultivars 4.1.9. Leaf area (cm2)
Leaves are the main source for light interception, absorption and harvesting needed for photosynthesis. Plants solely depend on leaves for metabolism, larger the leaf area more will be the exposure to sunlight and ultimately the pronounced growth. Results regarding leaf area were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.9). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to leaf area. The maximum leaf area (71.15 cm2) was found in Leen Vander Mark cultivar, followed by Parade (69.19 cm2), Ile de France (67.33cm2), Golden apeldorn (56.88 cm2) and Apeldorn (56.66 cm2). Similarly, the cultivar Purple flag exhibited the minimum leaf area (32.80 cm2) followed by Queen of night (36.10 cm2), Spring green (40.93
71 cm2) while the cultivar, Clear water and Barcelona ascertained the leaf area 48.41 cm2 and 54.25 cm2 respectively, that were statistically at par with each other.
80
70
) ² 60 50 40
30 Leaf area (cm area Leaf 20 10 0
Cultivars Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.9. Leaf area of different tulip cultivars
4.1.10. Chlorophyll contents (SPAD Value)
Chlorophyll is a vital biomolecule, needed for photosynthesis, which allows plant to absorb energy from light. Mesophyll cells have numerous chloroplasts that contain specialized light absorbing pigments, the chlorophyll. The complex series of reactions that culminate in the reduction of CO2 include the thylakoid reactions and the carbon fixation reactions. Results regarding chlorophyll contents were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.10). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to chlorophyll contents. The maximum SPAD value (63.20) was found in Leen Vander Mark cultivar, followed by Barcelona (59.85), Ile de France (54.47), Parade (45.45) and Golden apeldorn (36.34). Similarly, the
72 cultivar Queen of night exhibited the minimum SPAD value (11.36) followed by Spring green (15.12), Purple flag (21.37) while the cultivar, Clear water and Apeldorn ascertained the SPAD value 33.36 and 35.06 respectively, that were statistically at par with each other.
70 60 50 40 30 20 10
0 Chlorophyll Contents (SPAD Value) (SPAD Contents Chlorophyll
Cultivars Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.10. Chlorophyll contents (SPAD Value) of different tulip cultivars 4.1.11. Days to flower bud emergence (d)
Flower bud emergence takes place at shoot apical meristem and is a multifaceted morphological event in the plant life cycle. Plants differ considerably in the age at which they become sensitive to reach flowering stage. Consequently, they may alter the time to complete both, the vegetative and reproductive phases. Results regarding days to flower bud emergence were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.11). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to flower bud emergence. The minimum number of days to emerge flower bud emergence (34.53 d) was found in cultivar Parade, followed by Ile de France (41.26 d), Barcelona (44.26 d), Leen Vander Mark (45.00 d) and Golden apeldorn (50.93
73 d). Similarly, the cultivar Spring Green exhibited maximum number of days to flower bud emergence (68.40 d) followed by Purple flag (67.66 d), Queen of night (63.66 d) while the cultivar, Clear water and apeldorn ascertained the 55.86 d and 54.53 d respectively, that were statistically at par with each other.
80 70 60 50 40 30 20 10
0 Days to flower bud emergence (days) emergence bud flower to Days
Cultivars Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.11. Days to flower bud emergence of different tulip cultivars 4.1.12. Tepal diameter (mm)
Tepal diameter is an imperative standard of quality in cut tulips and the indicator of ultimate value particularly in bulbous flower crops. Results regarding tepal diameter were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.12). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to tepal diameter. The maximum tepal diameter (28.39 mm) was found in Parade cultivar, followed by Leen Vander Mark (25.50 mm), Ile de France (21.60 mm), Barcelona (17.85 mm) and Clear water (16.56 mm). Similarly, the cultivar Purple flag exhibited the smallest of all tepal diameter (12.53 mm) followed Apeldorn (13.65 mm), Queen of night
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(13.79 mm) while the cultivar, Spring green and Golden apeldorn ascertained the tepal diameter 14.71 mm and 15.44 mm respectively, that were statistically at par with each other.
35
30
25
20
15
10 Tepal diamter (mm) diamter Tepal 5
0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.12. Tepal diameter of different tulip cultivars
4.1.13. Flower diameter (mm)
Flower size is a pivotal factor that decides the value of cut flowers in both the stages i-e pre and postharvest longevity. Optimized and pronounced flower vigor of cut flower reflects the response of plants to the environment in which they are grown, particularly the abiotic factors. Results regarding Flower diameter were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.13). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to flower diameter. The maximum flower diameter (74.29 mm) was found in Parade cultivar, followed by Leen Vander Mark (64.35 mm), Ile de France (57.25 mm), Barcelona (57.12 mm) and Clear water
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(51.17 mm). Similarly, the cultivar Apeldorn exhibited the smallest of all flower diameter (40.87 mm) followed by Purple flag (48.52 mm), Golden apeldorn (48.78 mm) while the cultivar, Queen of night and Spring green ascertained the flower diameter 49.04 mm and 50.09 mm respectively, that were statistically at par with each other. 90
80
70 60 50 40 30
Flower diameter (mm) diameter Flower 20 10 0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.13. Flower diameter of different tulip cultivars 4.1.14. Length of petals (cm)
Petals are modified leaves that surround the reproductive parts of flowers. They are often brightly colored or unusually shaped to attract viewer’s choice. Length of petals is an essential standard of quality in cut tulips. Longer the length of petals more their appreciations will be. Results regarding petal length were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.14). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to petal length. The maximum petal length (8.64 cm) was found in Parade cultivar, followed by Leen Vander Mark (8.09 cm), Barcelona (6.23 cm), Clear water (6.15 cm) and Ile de France (6.07 cm). Similarly,
76 the cultivar Apeldorn exhibited the smallest of all petal length (4.82 cm) followed by Queen of night (4.95 cm), Purple flag (5.00 cm) while the cultivar, Spring green and Golden apeldorn ascertained the petal length 5.02 cm and 5.38 cm respectively, that were statistically at par with each other. 10 9
8 7 6 5 4 3
Length of petals (cm) petals Lengthof 2 1 0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.14. Length of petals of different tulip cultivars 4.1.15. Width of petals (cm)
The promising function of flower petals is to attract pollinators. They typically have bright and vivid colors that assist in pollination by drawing the attention of insects. Some petals have markings, such as spots and stripes, to help pollinators and provide enchanting attraction to the viewer. Results regarding petal width were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.15). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to petal width. The maximum width of petals (4.74 cm) was found in Parade cultivar, followed by Leen Vander Mark (4.62 cm), Ile de France (3.73 cm), Barcelona (3.15 cm) and Clear water (2.84 cm).
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Similarly, the cultivar Purple flag and Queen of night exhibited the smallest and same width of petals amongst the all cultivars (2.72 cm) followed by Apeldorn (2.58 cm), Spring green (2.76 cm) and Golden apeldorn (2.78 cm) that were statistically at par with each other.
6
5
4
3
2 Width of petals (cm) petals of Width 1
0
Cultivars Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.15. Width of petals of different tulip cultivars
4.1.16. Scape diameter (mm)
Girth of flower stem provides strength to support the flower head as well as mechanical strength by the deposition of vascular bundles. Results regarding tepal diameter were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.16). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to scape diameter. The maximum scape diameter (6.64 mm) was found in Barcelona cultivar, followed by Parade (6.14 mm), Leen Vander Mark (5.80 mm), Ile de France (5.31 mm) and Clear water (5.17 mm). Similarly, the cultivar Spring green exhibited the smallest
78 of all scape diameter (4.71 mm) followed by Golden apeldorn (4.57 mm), Queen of night (4.64 mm) while the cultivar, Purple flag and Apeldorn ascertained the scape diameter 4.81 mm and 4.88 mm respectively, that were statistically at par with each other.
8
7 6 5 4 3
Scape diameter (mm) diameter Scape 2 1 0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.16. Scape diameter of different tulip cultivars
4.1.17. Flower quality (Copper and Spokas, 1991)
Flower quality is most promising parameter that is directly related to longevity and crop status in cut flower crops. Results regarding flower quality were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.17). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to flower quality. Maximum score (8.20) was achieved by Parade cultivar, followed by Leen Vander Mark (7.93), Ile de France (7.40), Barcelona (6.80) and Clear water (5.80). Similarly, the cultivar Purple flag acquired the minimum score (3.40) for flower quality
79 parameter followed by Apeldorn (3.66), Queen of night (4.20) while the cultivar, Golden apeldorn and Spring green ascertained the score for quality standard 4.73 and 5.50 respectively, that were statistically at par with each other.
10 9 8 7 6 5 4
Flower Quality Flower 3 2 1 0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.17. Flower quality of different tulip cultivars
4.1.18. Vase life (d)
Vase life is the period during which a cut flower retains its usable and appreciable appearance in a vase. The vase life of flowers is generally determined by the time to abscission of petals that are still turgid, or by the time to petal wilting or withering. In many cultivars, high rates of stem elongation result in stem bending during vase life. Results regarding vase life were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.18). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found
80 to be inconsistent in context to their display life. The longest display life (9.4 d) was shown by the cultivar Leen Vander Mark, followed by Ile de France (8.4 d), Parade (7.6 d), Barcelona (5.7 d) and Queen of night (5.6 d). Similarly, the cultivar Purple flag exhibited the shortest display life (4.7 d) followed by Clear water (5.1 d), apeldorn (5.2 d) while the cultivar, Golden apeldorn and Spring green ascertained the same display life 5.3 d that were statistically at par with each other.
12
10
8
6
4 Vase life (days) Vase
2
0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.18. Vase life of different tulip cultivars 4.1.19. Days to start petal senescence (d)
Day to start petal senescence is an essential indicator of postharvest life of cut tulips and its importance cannot be denied from consumer point of view. Results regarding days to start petal senescence were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.19). The response of all the tested cultivars grown under agro-climatic conditions of Faisalabad in open field was found to be inconsistent in context to the initiation of petal senescence. The maximum number of days to start senescence (10.33 d) was found in cultivar
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Leen Vander Mark, followed by Ile de France (9.46 d), Parade (8.60 d) and Queen of night (6.60 d) while the cultivar Apeldorn and Barcelona showed the same number of days (6.53 d) to start petal senescence. Similarly, the cultivar Purple flag exhibited the earliest (5.73 d) senescence followed by Clear water (6.10 d), Spring green (6.33 d) and Golden apeldorn (6.46 d), that were statistically at par with each other.
12
10
8
6
4
2
0 Days to start petal senescence senescence petal start (days) to Days
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.19. Days to start petal senescence of different tulip cultivars 4.1.20. Tepal abscission (d)
Abscission is a well-characterized developmental process, occurring in leaves, fruit, and floral organs. In cut flowers, it plays decisive role in assessing their postharvest quality. Petal wilting or abscission terminates the life of the flower. However, the way in which wilting and abscission are coordinated, is not fully understood. There is wide variation in the extent to which petals wilt before abscission, even between cultivars of the same specie. Results regarding tepal abscission were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig.
82
4.1.20). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to abscission. The maximum number of days to abscission (12.26 d) was found in cultivar Leen Vander Mark, followed by Ile de France (11.13 d), Parade (10.60 d) and Queen of night (7.60 d) and Barcelona (7.53 d) while the cultivar Apeldorn and Golden apeldorn showed the same number of days (7.46 d) to start tepal abscission. Similarly, the cultivar Purple flag exhibited the earliest (6.73 d) abscission followed by Clear water (7.20 d) and Spring green (7.33 d) that were statistically at par with each other.
14
12
10
8
6
4 Tepal abscision (days) abscision Tepal 2
0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.20. Tepal abscission of different tulip cultivars 4.1.21. Mass of bulbils per clump (g)
Storage organs usually grow underground, where they are better protected. In geophytic and other vegetatively propagated plants, growth and development is influenced by the amount of stored reserves present in the tuber, corm, rhizome, or bulb. Results regarding mass of bulbs per clump were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.21). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to bulb mass per clump. The maximum mass
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(25.20 g) was found in Clear water cultivar, followed by Barcelona (21.44 g), Golden apeldorn (21.47 g), Ile de France (19.76 g) and Apeldorn (16.91 g). Similarly, the cultivar Queen of night exhibited the minimum bulb mass (7.56 g) followed by Purple flag (8.70 g), Spring green (10.13 g) while the cultivar, Leen Vander Mark and Parade ascertained the bulb mass 15.68 g and 16.50 g respectively, that were statistically at par with each other.
30
25
20
15
10
5 Mass of bulbils per clump (g) clump bulbilsof per Mass 0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.21 Mass of bulbils per clump of different tulip cultivars 4.1.22. Number of bulbils
Number of bulbils is an important attribute for perrenating organs and the indicator of ultimate value in geophytes. Results regarding number of bulbils were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.22). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to number of bulbils per clump to some extent. The maximum number of bulbils (2.8) was found in Leen Vander Mark cultivar, followed by (2.3) in Ile de France and Clear water,
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(2.0) in Parade and Apeldorn, (1.4) in Barcelona, Golden apeldorn and Queen of night. Similarly, the cultivar Purple flag exhibited the least number (1.2) of bulbils followed by cultivar Spring green (1.3), that were statistically at par with each other.
3.5
3
2.5
2
1.5
1
0.5 Number of bulbils per clump per bulbilsof Number
0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.22. Number of bulbils per clump of different tulip cultivars
4.1.23. Diameter of bulbils (mm)
Diameter of bulbils is an essential attribute of bulb quality in tulips like other geophytes that possesses ultimate value for using in the next growing season. More the diameter of bulbs more the reserves of food they will have. Results regarding diameter of bulbils were statistically significant (P ≤ 0.05) for all Tulipa gesneriana cultivars (Fig. 4.1.23). The response of all the tested cultivars grown under climatic conditions of Faisalabad in open field was found to be inconsistent in context to bulbil diameter. The maximum diameter (24.51 mm) was found in
85
Apeldorn cultivar, followed by Ile de France (22.28 mm), Leen Vander Mark (21.32 mm), Clear water (19.64 mm) and Barcelona (19.25 g). Similarly, the cultivar Purple flag exhibited the minimum bulbil diameter (9.79 mm) followed by Queen of night (10.40 g), Spring green (13.53 mm) while the cultivar, Parade and Golden apeldorn ascertained the bulbil diameter 14.31 mm and 14.60 mm respectively, that were statistically at par with each other.
30
25
20
15
10 Diameter of bulbils (mm) ofbulbils Diameter 5
0
Cultivars
Cultivar (**); Each value is the mean of three replicates ± S.E. Significant (*) = P ≤ 0.05; Highly Significant (**) = P ≤ 0.01
Fig. 4.1.23. Bulbils diameter of different tulip cultivars
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Table 4.1. Pearson’s correlation matrix among various attributes of tulip cultivars
PFM PDM PH LA SV TD DFBE TD FD LP WP SD FQ VL DSPS TA MBC
PDM 0.915** 1
PH 0.617** 0.534** 1
LA 0.522** 0.468** 0.592** 1
SV 0.630** 0.550** 0.713** 0.735** 1
BD 0.153 0.172* 0.278** 0.400** 0.401** 1
DFBE -0.59** -0.47** -0.72** -0.75** -0.79** -0.31** 1
TD 0.640** 0.621** 0.538** 0.648** 0.603** 0.146 -0.697** 1
FD 0.629** 0.530** 0.561** 0.476** 0.479** 0.004 -0.616** 0.723** 1
LP 0.723** 0.647** 0.544** 0.625** 0.608** 0.146 -0.694** 0.775** 0.789** 1 WP 0.636** 0.608** 0.507** 0.610** 0.589** 0.168* -0.665** 0.794** 0.780** 0.831** 1
SD 0.579** 0.494** 0.615** 0.415** 0.618** 0.169* -0.596** 0.509** 0.543** 0.607** 0.500** 1
FQ 0.394** 0.345** 0.388** 0.450** 0.448** 0.208* -0.475** 0.469** 0.464** 0.482** 0.431** 0.405** 1
VL 0.485** 0.486** 0.466** 0.632** 0.612** 0.229** -0.554** 0.675** 0.570** 0.649** 0.729** 0.349** 0.401** 1
DSPS 0.467** 0.473** 0.449** 0.643** 0.588** 0.233** -0.545** 0.666** 0.541** 0.617** 0.712** 0.307** 0.377** 0.979** 1
TA 0.503** 0.498** 0.461** 0.674** 0.601** 0.232** -0.592** 0.723** 0.604** 0.682** 0.761** 0.349** 0.412** 0.966** 0.981** 1
MBC 0.308** 0.295** 0.490** 0.424** 0.509** 0.340** -0.496** 0.203* 0.096 0.237** 0.100 0.285** 0.213** 0.127 0.118 0.114 1
NOB 0.307** 0.322** 0.228** 0.420** 0.371** 0.742** -0.330** 0.303** 0.241** 0.397** 0.377** 0.198* 0.282** 0.441** 0.450** 0.471** 0.244**
Values indicate Pearson’s correlation coefficient; *significant (P ≤ 0.05); **highly significant (P ≤ 0.01);
PFM, Plant fresh mass; PDM, Plant dry mass; PH, Plant height; LA, Leaf area; SV, SPAD Value; DFBE, Days to flower bud emergence; TD, Tepal diameter; FD, Flower diameter; LP, Length of petals; WP, Width of petals; SD, Stem diameter; FQ, Flower quality; VL,Vase life; DSPS, Days to start petal senescence; TA, Tepal abscission; MBC, Mass of bulb clump; NOB, Number of bulbs; BD, Bulb diameter
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4.1.24. Images showing growth response of different cultivars of tulip grown under Faisalabad conditions
Fig. 4.1.24.1. Apeldorn Fig.4.1.24.2. Barcelona
Fig. 4.1.24.3. Clear water Fig. 4.1.24.4. Golden apeldorn
Fig. 4.1.24.5. Ile de France Fig. 4.1.24.6. Leen Vander Mark
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Fig. 4.1.24.7. Parade Fig. 4.1.24.8. Purple flag
Fig. 4.1.24.9. Queen of night Fig. 4.1.24.10. Queen of night at harvesting stage
Fig. 4.1.24.11. Spring green Fig. 4.1.24.12. Spring green on bloom
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4.2. Experiment # 2 Exogenous application of chitosan for growth and quality assessment of different tulip cultivars
Objective of this experiment was to evaluate and identify the optimized doze for improving morphological, physiological, biochemical and enzymatic changes in five tulip cultivars (Apeldorn, Barcelona, Ile de France, Leen Vander Mark and Parade) by exogenous application of various levels of chitosan. 4.2.1. Plant height (cm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.1 that tulip variety ‘Parade’ superseded all other varieties by producing tallest plants, while reciprocal trend was observed in variety ‘Leen Vander Mark’. Results envisaged that various levels of chitosan induced different plant heights while untreated plants were shortest among all treatments. It is evident from results that tallest plants were noticed in variety ‘Parade’ where chitosan was applied at 150 mgL-1, exhibiting 76.04% increases over the respective control. Moreover, Apeldorn (45.68 cm), Barcelona (50.30 cm), Ile de France (47.47 cm) and Leen Vander Mark (41.84 cm) showed maximum plant heights in response to 90, 60, 120 and 60 mgL-1 chitosan applications, which correspond to 55.76, 101.60, 79.50 and 42.75% increase, respectively.
60 Apeldorn Barcelona Ile de France LVM Parade
50
40
30
20 Plant height (cm) heightPlant 10
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05. Fig. 4.2.1. Response of various concentrations of chitosan on plant height of five tulip cultivars
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4.2.1.1. Images showing response of various concentrations of chitosan on plant height of five tulip cultivars
Fig. 4.2.1.1. Plant heights of Apeldorn cultivar in response to various levels of chitosan
Fig. 4.2.1.2. Plant heights of Barcelona cultivar in response to various levels of chitosan
Fig. 4.2.1.3. Plant heights of Ile de France cultivar in response to various levels of chitosan
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Fig. 4.2.1.4. Plant height of Leen Vander Mark cultivar in response to various levels of chitosan
Fig. 4.2.1.5. Plant height of Parade cultivar in response to various levels of chitosan
(a) = T0 = Control (Distilled water)
-1 (b) = T1 = 30 mgL (Chitosan)
-1 (c) = T2 = 60 mgL (Chitosan)
-1 (d) = T3 = 90 mgL (Chitosan)
-1 (e) = T4 = 120 mgL (Chitosan)
-1 (f) = T5 = 150 mgL (Chitosan)
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4.2.2. Plant fresh mass (g)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.2 that tulip variety ‘Apeldorn’ superseded all other varieties by producing maximum fresh mass, while plants with the minimum amongst best were noticed in variety ‘Leen Vander Mark’. Results envisaged that various levels of chitosan induced varying values for plant fresh mass, while untreated plants yielded the least fresh mass among all treatments. It is evident from results that heaviest plant (45.33 g) was noticed in variety ‘Apeldorn’ when chitosan was applied at 90 mgL-1, exhibiting 76.89% increases over the respective control. Moreover, Parade (40.19 g), Barcelona (38.72 g), Ile de France (37.54 g) and Leen Vander Mark (36.54 g) exhibited maximum plant fresh mass in response to 150, 60, 120 and 60 mgL-1 chitosan application, which correspond to 45.87, 41.41, 59.83 and 49.50% increase, respectively.
50 Apeldorn Barcelona Ile de France LVM Parade 45
40
35 30 25 20
15 Plant fresh mass (g) mass fresh Plant 10 5 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.2. Response of various concentrations of chitosan on plant fresh mass of five tulip cultivars
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4.2.3. Plant dry mass (g)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.3 that tulip variety ‘Apeldorn’ superseded all other varieties by producing maximum dry mass, while the least amongst best was noticed in variety ‘Parade’. Results envisaged that various levels of chitosan induced varying values for plant dry mass, while untreated plants yielded the least dry mass among all treatments. It is evident from results that highest value (4.05 g) for dry mass was noticed in variety ‘Apeldorn’ when chitosan was applied at 90 mgL-1, exhibiting 76.74% increase over the respective control. Moreover, Parade (3.68 g), Barcelona (3.40 g), Ile de France (3.38 g) and Leen Vander Mark (3.34 g) exhibited maximum plant dry mass in response to 150, 60, 120 and 60 mgL-1 chitosan application, which correspond to 52.06, 44.35, 71.28 and 57.05% increase, respectively.
4.5 Apeldorn Barcelona Ile de France LVM Parade 4
3.5
3
2.5
2
1.5 Plant dry mass (g) mass dry Plant 1
0.5
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.3. Response of various concentrations of chitosan on plant dry mass of five tulip cultivars
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4.2.4. Leaf area (cm2)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.4 that tulip variety ‘Parade’ superseded all other varieties by producing maximum leaf area, while the least amongst best was noticed in variety ‘Ile de France’. Results envisaged that various levels of chitosan induced different values for leaf area while untreated plants were shortest among all treatments. It is evident from results that maximum leaf area (94.64 cm2) was noticed in variety ‘Parade’ when chitosan was applied at 150 mgL-1, exhibiting 74.37% increase over the respective control. Moreover, Apeldorn (84.46 cm2), Leen Vander Mark (81.86 cm2), Barcelona (66.18 cm2) and Ile de France (59.08 cm2) showed maximum leaf area in response to 90, 60, 60 and 120 mgL-1 chitosan application, which correspond to 178.18, 48.86, 47.52 and 78.25% increase, respectively.
120 Apeldorn Barcelona Ile de France LVM Parade
100
)
2 80
60
Leaf area (cm area Leaf 40
20
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.4. Response of various concentrations of chitosan on leaf area of five tulip cultivars
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4.2.5. Photosynthetic rate (A) (µmol m-2s-1)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.5 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by achieving highest photosynthetic rate, while the minimum rate was noticed in variety ‘Apeldorn’. Results envisaged that various levels of chitosan induced different rate of photosynthesis while untreated plants possessed the least values among all treatments. It is evident from results that maximum rate (18.10) of photosynthesis was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 60 mgL-1, exhibiting 113.38% increase over the respective control. Moreover, Barcelona (18.05), Parade (16.25), Ile de France (15.15) and Apeldorn (13.42) exhibited maximum photosynthetic rate in response to 60, 150, 120 and 90 mgL-1 chitosan application, which correspond to 72.53, 468.19, 254.87 and 247.28% increase, respectively.
25
Apeldorn Barcelona Ile de France LVM Parade
)
1
-
s
2 -
20
mol m mol µ 15
10
5 Photosynthetic rate (A) ( rate Photosynthetic 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.5. Response of various concentrations of chitosan on photosynthetic rate (A) of five tulip cultivars
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4.2.6. Transpiration rate (E) (mmol m-2s-1)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.6 that tulip variety ‘Apeldorn’ superseded all other varieties by possessing highest transpiration rate, while the lowest rate was noticed in variety ‘Ile de France’. Results envisaged that various levels of chitosan induced different rates of transpiration while untreated plants had the minimum values among all treatments. It is evident from results that highest rate (7.95) of transpiration was noticed in variety ‘Apeldorn’ when chitosan was applied at 90 mgL-1, exhibiting 52.28% increase over the respective control. Moreover, Barcelona (6.95), Leen Vander Mark (6.08), Parade (4.83) and Ile de France (4.51) showed maximum rates of transpiration in response to 90, 60, 150 and 90 mgL-1 chitosan application, which correspond to 35.81, 25.32, 60.34 and 35.78% increase, respectively.
9 Apeldorn Barcelona Ile de France LVM Parade
) 1
- 8
s
2 - 7
6 mmol mmol m
(E) ( (E) 5
4
3
2
Transpiration rate rate Transpiration 1
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.6. Response of various concentrations of chitosan on transpiration rate (E) of five tulip cultivars
97
4.2.7. Instantaneous Water Use Efficiency (A/E)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.7 that tulip variety ‘Ile de France’ superseded all other varieties by showing maximum efficiency of instantaneous water use, while minimum efficiency was noticed in variety ‘Parade’. Results envisaged that various levels of chitosan induced different efficiency level of instantaneous water use while untreated plants had the least efficiency among all treatments. It is evident from results that highest ratio (3.49) of instantaneous water use was noticed in variety ‘Ile de France’ when chitosan was applied at 120 mgL-1, exhibiting 152.12% increase over the respective control. Moreover, Parade (3.39), Leen Vander Mark (3.00), Barcelona (2.91) and Apeldorn (1.71) showed maximum ratio of instantaneous water use efficiency in response to 150, 60, 60 and 90 mgL-1 chitosan application, which correspond to 263.82, 64.18, 41.05 and 128.05% increase, respectively.
4 Apeldorn Barcelona Ile de France LVM Parade 3.5
3
2.5
2
1.5
1
0.5
Instantaneous Water (A/E) Use Efficiency Water Instantaneous 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.7. Response of various concentrations of chitosan on Instantaneous water use efficiency (A/E) of five tulip cultivars
98
4.2.8. Intrinsic Water Use Efficiency (A/gs)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.8 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by producing maximum ratio of intrinsic water use efficiency, while minimum efficiency was noticed in variety ‘Parade’. Results envisaged that various levels of chitosan induced different ratios for intrinsic water use efficiency while untreated plants had least levels among all treatments. It is evident from results that maximum ratio of intrinsic water use efficiency (26.68) was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 60 mgL-1, exhibiting 73.58% increase over the respective control. Moreover, Barcelona (26.49), Apeldorn (20.33), Ile de France (18.88) and Parade (18.02) showed maximum ratio of intrinsic water use efficiency in response to 60, 90, 120 and 150 mgL-1 chitosan application, which correspond to 45.53, 212.80, 144.82 and 253.85% increase, respectively.
35
Apeldorn Barcelona Ile de France LVM Parade 30
25
20
15
10
5 Intrinsic Water (A/gs) Efficiency Use Water Intrinsic 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.8. Response of various concentrations of chitosan on Intrinsic water use efficiency (A/gs) of five tulip cultivars
99
-1 4.2.9. Sub-stomatal CO2 (Ci) (µmol mol )
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.9 that tulip variety ‘Parade’ superseded all other varieties by possessing maximum sub stomatal CO2, while minimum values for sub stomatal
CO2 was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different values for sub stomatal CO2 while untreated plants had the least values among all treatments. It is evident from results that maximum (437.4) sub stomatal CO2 were noticed in variety ‘Parade’ when chitosan was applied at 150 mgL-1, exhibiting 23.95% increase over the respective control. Moreover, Leen Vander Mark (416.26), Ile de France (408.2), Apeldorn
(389.26) and Barcelona (334.6) showed highest values for sub stomatal CO2 in response to 60, 120, 90 and 30 mgL-1 chitosan application, which correspond to 17.50, 15.81, 13.60 and 18.40% increase, respectively.
500
Apeldorn Barcelona Ile de France LVM Parade
) 1
- 450 400 350
300
(Ci) (µmol (Ci) mol (µmol
2 250 CO 200 150
stomatal stomatal 100 -
Sub 50 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.9. Response of various concentrations of chitosan on Sub-stomatal CO2 (Ci) of five tulip cultivars
100
4.2.10. Stomatal Conductance (gs) (mmol m-2s-1)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.10 that tulip variety ‘Parade’ superseded all other varieties by exhibiting maximum stomatal conductance, while the least value for stomatal conductance in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different values for stomatal conductance while untreated plants had the minimum values among all treatments. It is evident from results that maximum (0.90) stomatal conductance was noticed in variety ‘Parade’ when chitosan was applied at 150 mgL-1, exhibiting 49.97% increase over the respective control. Moreover, Ile de France (0.80), Apeldorn (0.76), Leen Vander Mark (0.72) and Barcelona (0.71) showed maximum stomatal conductance in response to 120, 90, 60 and 60 mgL-1 chitosan application, which correspond to 45.22, 24.80, 25.38 and 17.24% increase, respectively.
1
) ) Apeldorn Barcelona Ile de France LVM Parade
1 -
s 0.9
2 - 0.8
mol mol m 0.7 0.6 0.5 0.4 0.3 0.2 0.1
Stomatal Conductance (gs) (m (gs) Conductance Stomatal 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.10. Response of various concentrations of chitosan on Stomatal conductance (gs) of five tulip cultivars
101
4.2.11. Chlorophyll contents (SPAD Value)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.11 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by possessing maximum SPAD value, while minimum amongst best was noticed in variety ‘Parade’. Results envisaged that various levels of chitosan induced different SPAD values while untreated plants had minimum among all treatments. It is evident from results that maximum (61.37) SPAD value was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 60 mgL-1, exhibiting 48.02% increase over the respective control. Moreover, Barcelona (53.62), Apeldorn (52.72), Ile de France (52.28) and Parade (51.81) showed maximum SPAD values in response to 60, 90, 120 and 150 mgL-1 chitosan application, which correspond to 42.14, 49.81, 29.56 and 44.47% increase, respectively.
70
Apeldorn Barcelona Ile de France LVM Parade
60
50
40
30
20
10 Chlorophyll contents (SPAD Value) contents (SPAD Chlorophyll
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.11. Response of various concentrations of chitosan on chlorophyll contents of five tulip cultivars
102
4.2.12. Mesophyll Conductance (A/Ci)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.12 that tulip variety ‘Barcelona’ superseded all other varieties by exhibiting maximum mesophyll conductance, while minimum was noticed in variety ‘Apeldorn’. Results envisaged that various levels of chitosan induced different mesophyll conductance while untreated plants had minimum values among all treatments. It is evident from results that maximum (0.0540) mesophyll conductance was noticed in variety ‘Barcelona’ when chitosan was applied at 60 mgL-1, exhibiting 45.55% increase over the respective control. Moreover, Leen Vander Mark (0.0434), Ile de France (0.0372), Parade (0.0371) and Apeldorn (0.0352) showed maximum mesophyll conductance in response to 60, 120, 150 and 90 mgL-1 chitosan application, which correspond to 80.48, 206.37, 359.14 and 204.66% increase, respectively.
0.07 Apeldorn Barcelona Ile de France LVM Parade
0.06
0.05
0.04
0.03
0.02
Mesophyll Conductance Conductance (A/Ci) Mesophyll 0.01
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.12. Response of various concentrations of chitosan on Mesophyll conductance (A/Ci) of five tulip cultivars
103
4.2.13. Superoxide dismutase (SOD) activity (Units/mg Protein)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.13 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by possessing maximum SOD activity, while minimum activity was noticed in variety ‘Parade’. Results envisaged that various levels of chitosan induced different levels of SOD activity while untreated plants had minimum among all treatments. It is evident from results that maximum (1454.42) SOD activity was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 60 mgL-1, exhibiting 90.05% increase over the respective control. Moreover, Apeldorn (1429.20), Ile de France (1344.61), Barcelona (1327.20) and Parade (1238.15) showed maximum SOD activity in response to 90, 120, 60 and 150 mgL-1 chitosan application, which correspond to 83.67, 80.78, 120.03 and 64.45% increase, respectively.
1800 Apeldorn Barcelona Ile de France LVM Parade 1600
Units/mg Units/mg 1400
1200
1000
Protein) 800 600
400
200 Superoxide dismutase activity activity ( dismutase Superoxide 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.13. Response of various concentrations of chitosan on Superoxide dismutase activity of five tulip cultivars
104
4.2.14. Catalase (CAT) activity (Units/mg Protein)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.14 that tulip variety ‘Ile de France’ superseded all other varieties by possessing maximum CAT activity, while minimum was recorded in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different levels of CAT activity while untreated plants had the least among all treatments. It is evident from results that maximum (1021.29) CAT activity was noticed in variety ‘Ile de France’ when chitosan was applied at 90 mgL-1, exhibiting 58.30% increase over the respective control. Moreover, Leen Vander Mark (994.72), Apeldorn (977.45), Parade (926.83) and Barcelona (861.30) showed maximum CAT activity in response to 90, 90, 150 and 60 mgL-1 chitosan application, which correspond to 50.10, 70.58, 39.24 and 45.55% increase, respectively.
1200
Apeldorn Barcelona Ile de France LVM Parade
1000
800
(Units/mg Protein) (Units/mg 600
400
200 Catalase Catalase activity 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.14. Response of various concentrations of chitosan on Catalase activity of five tulip cultivars
105
4.2.15. DPPH Scavenging Activity (% inhibition)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.15 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by exhibiting maximum DPPH scavenging activity, while minimum was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different levels of percentage inhibition of DPPH ions while untreated had the least percentage inhibition among all treatments except Ile de France. It is evident from results that maximum (82.95%) DPPH ion scavenging activity was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 60 mgL-1, exhibiting 19.40% increase over the respective control. Moreover, Ile de France (82.44%), Parade (80.34%), Apeldorn (78.95%) and Barcelona (62.40%) showed maximum percentage inhibition of DPPH ion scavenging activity in response to 120, 120, 30 and 60 mgL-1 chitosan application, which correspond to 2.82, 18.19, 17.63 and 44.87% increase, respectively.
90 Apeldorn Barcelona Ile de France LVM Parade 80 70
60
50
40 inhibition) 30 20
DPPH Scavenging Activity (% DPPH Scavenging 10 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.15. Response of various concentrations of chitosan on DPPH Scavenging Activity of five tulip cultivars
106
4.2.16. Peroxidase (POD) activity (Units/mg Protein)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.16 that tulip variety ‘Parade’ superseded all other varieties by exhibiting maximum POD activity, minimum activity was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different levels of POD activity while untreated plants had least among all treatments. It is evident from results that maximum (4379.40) POD activity was noticed in variety ‘Parade’ when chitosan was applied at 150 mgL-1, exhibiting 140.01% increase over the respective control. Moreover, Leen Vander Mark (3688.91), Ile de France (3129.15), Apeldorn (2621.32) and Barcelona (2265.96) showed maximum POD activity in response to 90, 90, 60 and 60 mgL-1 chitosan application, which correspond to 83.93, 103.79, 91.56 and 135.69% increase, respectively.
5000
Apeldorn Barcelona Ile de France LVM Parade 4500 4000 3500 3000 2500 2000 1500 1000
500 Peroxidase activity (Units/mg Protein) (Units/mg activity Peroxidase 0 Control 30 60 90 120 150 Chitosan concentrations (mg L-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.16. Response of various concentrations of chitosan on Peroxidase activity of five tulip cultivars
107
4.2.17. Malondialdehyde (MDA) contents (µmol g-1 fresh weight)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.17 that tulip variety ‘Barcelona’ superseded all other varieties by exhibiting minimum levels of MDA, while maximum was noticed in variety ‘Apeldorn’. Results envisaged that various levels of chitosan induced different levels of MDA contents while untreated plants had highest levels among all treatments. It is evident from results that minimum (1.00) MDA contents were noticed in the variety ‘Barcelona’ when chitosan was applied at 60 mgL-1, exhibiting 64.82% decrease over the respective control. Moreover, Leen Vander Mark (0.862), Parade (0.879), Ile de France (0.886) and Apeldorn (1.00) showed minimum MDA contents in response to 90, 150, 120 and 90 mgL-1 chitosan applications, which correspond to 41.33, 43.41, 47.71 and 33.46% decrease, respectively.
2
Apeldorn Barcelona Ile de France LVM Parade
1.8
1.6
1.4
fresh weight) fresh
1 - 1.2
1
µmol µmol g ( 0.8
0.6
0.4
Malondialdehyde 0.2
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.17. Response of various concentrations of chitosan on Malondialdehyde contents of five tulip cultivars
108
4.2.18. Total Phenolic Compounds (mg GAE/100g FW)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.18 that tulip variety ‘Apeldorn’ superseded all other varieties by exhibiting maximum total phenolic compounds, while minimum were noticed in variety ‘Parade’. Results envisaged that various levels of chitosan induced different levels of total phenolics while untreated plants had least of them among all treatments. It is evident from results that maximum (258.04) level of phenolics was noticed in variety ‘Apeldorn’ when chitosan was applied at 60 mgL-1, exhibiting 83.03% increase over the respective control. Moreover, Ile de France (252.56), Leen Vander Mark (238.22), Barcelona (236.62) and Parade (213.98) showed maximum level of phenolic contents in response to 120, 90, 60 and 150 mgL-1 chitosan application, which correspond to 92.24, 10.11, 44.76 and 74.86% increase, respectively.
300 Apeldorn Barcelona Ile de France LVM Parade
250
GAE/100g GAE/100g 200
150
FW) 100
50
0 Total Phenolic Compounds (mg (mg Compounds Phenolic Total Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.18. Response of various concentrations of chitosan on Total Phenolic compounds of five tulip cultivars
109
4.2.19. H2O2 Scavenging Activity (%)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.19 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by maximum scavenging percentage of H2O2, while less was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different levels of percentage scavenging of H2O2 while untreated plants possessed the least among all treatments. It is evident from results that maximum (43.02%) H2O2 scavenging activity was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 90 mgL-1, exhibiting 154.03% increase over the respective control. Moreover, Parade (41.09%), Apeldorn (40.99%),
Ile de France (39.53%) and Barcelona (32.71%) showed maximum percentage for H2O2 scavenging in response to 150, 90, 120 and 60 mgL-1 chitosan application, which correspond to 65.25, 125.60, 160.90 and 200.19% increase, respectively.
50 Apeldorn Barcelona Ile de France LVM Parade
45
40 35 30 25 20
Scavenging Activity Activity (%) Scavenging 15
2 O
2 10 H 5 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.19. Response of various concentrations of chitosan on H2O2 Scavenging Activity of five tulip cultivars
110
4.2.20. Days to flower bud emergence (d)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.20 that tulip variety ‘Ile de France’ superseded all other varieties by earliest emergence of flower buds, while late emergence was noticed in variety ‘Leen Vander Mark’. Results envisaged that various levels of chitosan induced different days to flower bud emergence while untreated plants had extremely late emergence among all treatments. It is evident from results that earliest (29.53 d) emergence of flower buds was noticed in variety ‘Ile de France’ when chitosan was applied at 120 mgL-1, exhibiting 19.16% earlier over the respective control. Moreover, Parade (31.2 d), Apeldorn (36.8 d), Barcelona (37.8 d) and Leen Vander Mark (40.53 d) showed earliest emergence in response to 60, 90, 30 and 60 mgL-1 chitosan application, which correspond to 9.47, 14.15, 15.75 and 10.98% respectively, earliness in flower bud emergence.
50 Apeldorn Barcelona Ile de France LVM Parade
45 40 35 30 25 20 15 10
Days to flower bud emergence (d) emergence bud flower to Days 5 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.20. Response of various concentrations of chitosan on days to flower bud emergence of five tulip cultivars
111
4.2.21. Tepal diameter (mm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.21 that tulip variety ‘Ile de France’ superseded all other varieties by exhibiting maximum tepal diameter, while minimum was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different dimensions in tepal diameter while untreated plants had the least values for tepal diameter among all treatments. It is evident from results that maximum diameter (36.92 mm) was noticed in variety ‘Ile de France’ when chitosan was applied at 120 mgL-1, exhibiting 30.94% increase over the respective control. Moreover, Parade (36.27 mm), Apeldorn (32.03 mm), Leen Vander Mark (28.58 mm) and Barcelona (24.17 mm) showed maximum tepal dimeter in response to 150, 90, 60 and 60 mgL-1 chitosan application, which correspond to 16.38, 32.64, 20.42 and 51.25% increase, respectively.
45 Apeldorn Barcelona Ile de France LVM Parade 40
35
30
25
20
15 Tepal diameter (mm) diameter Tepal 10
5
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.21. Response of various concentrations of chitosan on tepal diameter of five tulip cultivars
112
4.2.22. Flower diameter (mm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.22 that tulip variety ‘Parade’ superseded all other varieties by producing largest diameter of flower, while shortest was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different flower diameters while untreated plants had the least among all treatments. It is evident from results that largest diameter (63.76 mm) of flowers was noticed in variety ‘Parade’ when chitosan was applied at 150 mgL-1, exhibiting 17.11% increase over the respective control. Moreover, Leen Vander Mark (62.79 mm), Ile de France (56.75 mm), Apeldorn (53.40 mm) and Barcelona (46.46 mm) showed maximum values for flower diameter in response to 60, 120, 90 and 60 mgL-1 chitosan application, which correspond to 19.71, 41.17, 35.47 and 38.62% increase, respectively.
70 Apeldorn Barcelona Ile de France LVM Parade
60
50
40
30
Flower diameter (mm) diameter Flower 20
10
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.22. Response of various concentrations of chitosan on flower diameter of five tulip cultivars
113
4.2.23. Length of petals (cm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.23 that tulip variety ‘Apeldorn’ superseded all other varieties by producing largest petals, while shortest were noticed in variety ‘Ile de France’. Results envisaged that various levels of chitosan induced different petal lengths while untreated plants had shortest petals among all treatments. It is evident from results that largest petals (10.8 cm) were noticed in variety ‘Apeldorn’ when chitosan was applied at 90 mgL-1, exhibiting 63.96% increase over the respective control. Moreover, Parade (8.57 cm), Leen Vander Mark (8.48 cm), Barcelona (6.90 cm) and Ile de France (6.76 cm) showed maximum petal lengths in response to 150, 60, 60 and 120 mgL-1 chitosan applications, which correspond to 30.82, 15.74, 13.22 and 11.67% increase, respectively.
12 Apeldorn Barcelona Ile de France LVM Parade
10
8
6
4 Length of petals (cm) petals Lengthof
2
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.23. Response of various concentrations of chitosan on length of petals of five tulip cultivars
114
4.2.24. Width of petals (cm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.24 that tulip variety ‘Apeldorn’ superseded all other varieties by producing largest petal width, while shortest were noticed in variety ‘Ile de France’. Results envisaged that various levels of chitosan induced different petal widths while untreated plants were shortest among all treatments. It is evident from results that maximum (5.88 cm) width of petals was noticed in variety ‘Apeldorn’ when chitosan was applied at 150 mgL-1, exhibiting 22.63% increase over the respective control. Moreover, Leen Vander Mark (5.49 cm), Parade (4.77cm), Barcelona (4.71 cm) and Ile de France (4.56 cm) showed maximum width of petals in response to 90, 120, 30 and 90 mgL-1 chitosan application, which correspond to 23.53, 11.52, 27.61 and 34.31% increase, respectively.
7 Apeldorn Barcelona Ile de France LVM Parade 6
5
4
3
Width of Petals (cm) Petals of Width 2
1
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.24. Response of various concentrations of chitosan on width of petals of five tulip cultivars
115
4.2.25. Scape diameter (mm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.25 that tulip variety ‘Apeldorn’ superseded all other varieties by producing maximum scape diameter, while the least was noticed in variety ‘Parade’. Results envisaged that various levels of chitosan induced different scape diameters while untreated plants were shortest among all treatments. It is evident from results that maximum (6.65 mm) scape diameter was noticed in variety ‘Apeldorn’ when chitosan was applied at 120 mgL-1, exhibiting 36.37% increase over the respective control. Moreover, Barcelona (6.5 mm), Ile de France (6.24 mm), Leen Vander Mark (5.83 mm) and Parade (5.45 mm) showed maximum scape diameters in response to 60, 90, 90 and 120 mgL-1 chitosan application, which correspond to 10.50, 19.00, 6.10 and 10.46% increase, respectively.
8 Apeldorn Barcelona Ile de France LVM Parade 7
6
5
4
3
Scape diameter (mm) diameter Scape 2
1
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.25. Response of various concentrations of chitosan on scape diameter of five tulip cultivars
116
4.2.26. Flower quality (Copper and Spokas, 1991)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.26 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by ascertaining maximum score regarding visual appearance, while minimum score was attained in variety ‘Apeldorn’. Results envisaged that various levels of chitosan influenced different score points while untreated plants had minimum among all treatments. It is evident from results explained in ranked order of varieties Leen Vander Mark, Parade, Ile de France, Barcelona and Apeldorn. Similarly, ‘Leen Vander Mark’ scored maximum (8.2) when chitosan was applied at 60 mgL-1, exhibiting 41.37% increase over the respective control. Moreover, Parade (7.4), Ile de France (7.4), Apeldorn (6.6) and Barcelona (6.0) showed maximum scores in response to 150, 120, 90 and 60 mgL-1 chitosan application, which correspond to 40.50, 48.00, 130.23 and 28.16% increase, respectively.
10 Apeldorn Barcelona Ile de France LVM Parade 9 8
7 6 5
4 Flower quality Flower 3 2 1 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.26. Response of various concentrations of chitosan on flower quality of five tulip cultivars
117
4.2.27. Vase life (d)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.27 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by exhibiting maximum display life, while minimum was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan influenced vase life with improved post-harvest longevity while untreated plants displayed minimal shelf life display among all treatments. It is evident from results that longest display life (14 d) was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 60 mgL-1, exhibiting 43.83% increase over the respective control. Moreover, Apeldorn (12.7 d), Ile de France (11.8 d), Parade (11.2 d) and Barcelona (7.0 d) showed longest vase life display in response to 90, 120, 150 and 60 mgL-1 chitosan application, which correspond to 72.07, 36.15, 47.36 and 40.0% increase, respectively.
16 Apeldorn Barcelona Ile de France LVM Parade 14
12
10
8
6 Vase life (days) Vase 4
2
0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.27. Response of various concentrations of chitosan on vase life of five tulip cultivars
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4.2.28. Images depicting the response of various concentrations of chitosan on vase display of five tulip cultivars
Fig. 4.2.28.1. Vase display of Apeldorn cultivar on 13th day after harvest
Fig. 4.2.28.2. Vase display of Barcelona cultivar on 7th day after harvest
119
Fig. 4.2.28.3. Vase display of Ile de France cultivar on 12th day after harvest
Fig. 4.2.28.4. Vase display of Leen Vander Mark cultivar on 14th day after harvest
120
Fig. 4.2.28.5. Vase display of Leen Vander Mark cultivar on 11th day after harvest
(a) = T0 = Control (Distilled water)
(b) = T1 = 30 mgL-1 (Chitosan)
(c) = T2 = 60 mgL-1 (Chitosan)
(d) = T3 = 90 mgL-1 (Chitosan)
(e) = T4 = 120 mgL-1 (Chitosan)
(f) = T5 = 150 mgL-1 (Chitosan)
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4.2.29. Days to start petal senescence (d)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.29 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by ascertaining largest number of days to start petal senescence, while minimum number of days was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan remarkably influenced the number of days to initiate petal senescence while petals of untreated plants showed earliest senescence among all treatments. It is evident from results that prolonged freshness (17 d) was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 60 mgL-1, exhibiting 33.50% increase over the respective control. Moreover, Apeldorn (14.73 d), Ile de France (13.8 d), Parade (12.2 d) and Barcelona (8.0 d) showed maximum days to start petal senescence in response to 90, 120, 150 and 60 mgL-1 chitosan application, which correspond to 75.39, 29.37, 41.86 and 33.33% increase, respectively.
20
Apeldorn Barcelona Ile de France LVM Parade
18 16 14 12 10 8 6 4
Days to start petal senescence senescence petal start (days) to Days 2 0 Control 30 60 90 120 150 Chitosan concentrations (mg L-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.29. Response of various concentrations of chitosan on days to start petal senescence of five tulip cultivars
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4.2.30. Tepal abscission (d)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.30 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by exhibiting late abscission of tepals, while earliest abscission was noticed in variety ‘Barcelona’. Results envisaged that various levels of chitosan influenced different days to tepal abscission while untreated plants had rapid abscission among all treatments. It is evident from results that largest number of days (18.0 d) to abscission was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 60 mgL-1, exhibiting 31.06% increase over the respective control. Moreover, Apeldorn (16.73 d), Ile de France (14.8 d), Parade (14.2 d) and Barcelona (9.0 d) showed late abscission in response to 90, 120, 150 and 60 mgL-1 chitosan application, which correspond to 46.78, 26.85, 33.96 and 28.57% increase, respectively.
20 Apeldorn Barcelona Ile de France LVM Parade 18
16
14 12 10 8
6 Tepal abscission (days) abscission Tepal 4 2 0 Control 30 60 90 120 150 Chitosan concentrations (mg L-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.30. Response of various concentrations of chitosan on tepal abscission of five tulip cultivars
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4.2.31. Diameter of bulbils (mm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.31 that tulip variety ‘Ile de France’ superseded all other varieties in developing bulbils with largest diameter, while smallest bulbils were formed in variety ‘Barcelona’. Results envisaged that various levels of chitosan induced different sizes of bulbils in context to their diameter and surprisingly untreated plants produced relatively lager bulbils among all tested treatments. It is evident from results that largest bulbils (42.37 mm) were developed in variety ‘Ile de France’ when chitosan was applied at 120 mgL-1, exhibiting 44.90% increase over its respective control. Moreover, Parade (41.21 mm), Apeldorn (38.48 mm) and Leen Vander Mark (35.50 mm) developed largest diameter of bulbils in untreated plants except Barcelona (33.59 mm) in which response of very low level of chitosan at 30 mgL-1 application corresponded to 9.54% increase in diameter as compared to control.
50 Apeldorn Barcelona Ile de France LVM Parade 45
40 35 30 25 20 15
Diameter (mm) bulbilsof Diameter 10 5 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.31. Response of various concentrations of chitosan on diameter of bulbils of five tulip cultivars
124
4.2.32. Mass of bulbils per clump (g)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.2.32 that tulip variety ‘Ile de France’ superseded all other varieties by ascertaining maximum mass of bulbils per clump, while minimum mass was noticed in variety ‘Leen Vander Mark’. Results envisaged that various levels of chitosan induced different values for the mass of bulbils per clump while untreated plants had less mass in both of these cultivars. It is evident from results that highest mass (25.56 g) was noticed in variety ‘Ile de France’ followed by Leen Vander mark (15.48 g) when chitosan was applied at 30 mgL-1, exhibiting 85.54 and 11.29% increase respectively over its corresponding control. Moreover, Barcelona (25.48 g), Apeldorn (16.12) and Parade (15.62 g) showed maximum values for mass of bulbils per clump in untreated plants while the levels of chitosan application revealed decreasing trend regarding mass of bulbils per clump.
30 Apeldorn Barcelona Ile de France LVM Parade
25
20
15
10
Mass of bulbils per clump (g) clump bulbilsof per Mass 5
0 Control 30 60 90 120 150 Chitosan concentrations (mg L-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.32. Response of various concentrations of chitosan on mass of bulbils per clump of five tulip cultivars
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4.2.33. Number of bulbils
Results depict significant (P ≤ 0.05) differences for various treatments and varieties while their interaction was non-significant. It is obvious from results in Fig. 4.2.33 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by producing maximum number of bulbils, while minimum number of bulbils was noticed in variety ‘Parade’. Results envisaged that various levels of chitosan induced different number of bulbils while untreated plants had minimum number. It is evident from results that maximum number (3.7) was noticed in variety ‘Leen Vander Mark’ when chitosan was applied at 120 mgL-1, exhibiting 47.36% increase over the respective control. Moreover, Apeldorn (3.6), Ile de France (3.2), Barcelona (2.8) and Parade (2.7) showed maximum number of bulbils in response to 120, 90, 90 and 90 mgL-1 chitosan application, which correspond to 92.85, 50, 95.45 and 70.83% increase, respectively.
5 Apeldorn Barcelona Ile de France LVM Parade 4.5 4
3.5
3 2.5 2
1.5 Number bulbilsof Number 1 0.5 0 Control 30 60 90 120 150 Chitosan concentrations (mgL-1)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.2.33. Response of various concentrations of chitosan on number of bulbils of five tulip cultivars
126
Table 4.2 (a) Pearson’s correlation matrix among various morpho-physiological attributes of tulip cultivars in response to chitosan application
PH PFM PDM LA TD DFBE BD MBC NOB SV A E InsWUE IntWUEInt Ci gs
PFM 0.686** 1
PDM 0.683** 0.973** 1
LA 0.638** 0.567** 0.572** 1
TD 0.421** 0.303** 0.311** 0.378** 1
DFBE -0.43** -0.23** -0.23** -0.23** -0.589 1
BD -0.12** -0.096* -0.077 -0.20** 0.109 -0.12** 1
MBC -0.17** -0.091 -0.118* -0.36** -0.175 -0.042 0.23** 1
NOB 0.172** 0.153** 0.144** 0.162** 0.177 -0.040 0.047 -0.139** 1
SV 0.368** 0.322** 0.327** 0.337** 0.094 -0.023 -0.14** -0.141** 0.085 1
A 0.543** 0.364** 0.334** 0.395** 0.012 -0.072 -0.17** 0.054 0.096* 0.349** 1
E 0.215** 0.403** 0.400** 0.197** -0.296 0.379** -0.23** -0.086* 0.130** 0.208** 0.217** 1
InsWUE 0.430** 0.154** 0.133** 0.268** 0.242 -0.34** -0.034 0.104* 0.058 0.190** 0.786** -0.36** 1
IntWUE 0.295** 0.184** 0.151** 0.215** -0.139 0.059 -0.099* 0.083 0.039 0.239** 0.769** 0.190** 0.576** 1
Ci 0.522** 0.316** 0.346** 0.571** 0.742 -0.52** 0.010 -0.307** 0.191** 0.252** 0.173** -0.24** 0.354** -0.022 1
gs 0.381** 0.260** 0.269** 0.319** 0.213 -0.21** -0.115* -0.074 0.073 0.198** 0.302** 0.076 0.265** -0.29** 0.284** 1 gm 0.386** 0.263** 0.223** 0.216** -0.216 0.075 -0.18** 0.151* 0.030 0.259** 0.948** 0.277** 0.686** 0.783** -0.14** 0.210**
Values indicate Pearson’s correlation coefficient; *significant (P ≤ 0.05); **highly significant (P ≤ 0.01);
PFM, Plant fresh mass; PDM, Plant dry mass; PH, Plant height; LA, Leaf area; SV, SPAD Value; DFBE, Days to flower bud emergence; TD, Tepal diameter; MBC, Mass of bulb clump; NOB, Number of bulbs; BD, Bulb diameter; A, Photosynthetic rate; E, Transpiration rate; InsWUE,
Instantaneous water use efficiency; IntWUE, Intrinsic water use efficiency; Ci, Sub-stomatal CO2; gs, Stomatal conductance; gm, Mesophyll conductance.
127
Table 4.2 (b) Pearson’s correlation matrix among various antioxidant enzymes activity and biochemical changes induced on floral attributes of tulip cultivars in response to chitosan application
FD VL LP WP FQ SD DSPS TA SOD CAT DSA POD MDA TPC VL 0.620** 1 LP 0.408** 0.470** 1 WP 0.272** 0.376** 0.556** 1 FQ 0.286** 0.264** 0.141** 0.059 1 SD -0.105* 0.094* 0.122** 0.098* 0.050 1 DSPS 0.612** 0.979** 0.434** 0.353** 0.279** 0.097* 1 TA 0.610** 0.984** 0.516** 0.442** 0.241** 0.065 0.975** 1 SOD 0.328** 0.408** 0.335** 0.228** 0.236** 0.231** 0.383** 0.357** 1 CAT 0.372** 0.485** 0.305** 0.271** 0.216** 0.157** 0.467** 0.455** 0.631** 1 DSA 0.562** 0.592** 0.311** 0.305** 0.176** -0.098* 0.591** 0.626** 0.221** 0.375** 1 POD 0.562** 0.495** 0.311** 0.224** 0.256** -0.005 0.462** 0.460** 0.392** 0.456** 0.412** 1 MDA -0.22** -0.33** -0.326** -0.331** -0.17** -0.15** -0.31** -0.33** -0.39** -0.37** -0.21** -0.34** 1 TPC 0.083 0.271** 0.239** 0.257** 0.110* 0.312** 0.236** 0.244** 0.444** 0.366** 0.031 0.315** -0.36** 1 HSA 0.520** 0.497** 0.447** 0.300** 0.250** 0.103* 0.437** 0.468** 0.496** 0.537** 0.353** 0.534** -0.42** 0.445*
Values indicate Pearson’s correlation coefficient; *significant (P ≤ 0.05); **highly significant (P ≤ 0.01);
FD, Flower diameter; VL, Vase life; LP, Length of petals; WP, Width of petals; FQ, Flower quality; SD Stem diameter; DSPS, Days to start petal senescence; TA, Tepal abscission; SOD, Superoxide dismutase activity; CAT, Catalase activity; DSA, DPPH ion scavenging activity; POD,
Peroxidase activity; MDA, Malondialdehyde contents; TPC, Total Phenolic Contents; HSA, H2O2 Scavenging Activity
128
4.3. Experiment # 3 Exogenous application of glycine betaine for growth and quality assessment of different tulip cultivars
Objective of this experiment was to evaluate and identify the optimized doze for improving morphological, physiological, biochemical and enzymatic changes in five tulip cultivars (Apeldorn, Barcelona, Ile de France, Leen Vander Mark and Parade) by exogenous application of various levels of glycine betaine. 4.3.1. Plant height (cm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.1 that tulip variety ‘Parade’ superseded all other varieties by producing tallest plants, while reciprocal trend was observed in variety ‘Leen Vander Mark’. Results envisaged that various levels of glycine betaine induced different plant heights while untreated plants were shortest among all treatments. It is evident from results that tallest plants were noticed in variety ‘Parade’ where glycine betaine was applied at 90 mM, exhibiting 52.00% increase over the respective control. Moreover, Barcelona (45.60 cm), Ile de France (45.37 cm), Apeldorn (43.28 cm) and Leen Vander Mark (39.64 cm) showed maximum plant heights in response to 30, 90, 60 and 120 mM glycine betaine application, which correspond to 70.81, 86.36, 60.73 and 46.22% increase, respectively.
60 Apeldorn Barcelona Ile de France LVM Parade
50
40
30
20
Plant height (cm) heightPlant 10
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM) Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05. Fig. 4.3.1. Response of various concentrations of glycine betaine on plant height of five tulip cultivars
129
4.3.1.1. Images showing the response of various concentrations of glycine betaine on plant height of five tulip cultivars
Fig. 4.3.1.1. Plant heights of ‘Apeldorn’ in response to various levels of glycine betaine
Fig. 4.3.1.2. Plant heights of Barcelona in response to various levels of glycine betaine
Fig. 4.3.1.3. Plant heights of Leen Vander Mark in response to various levels of glycine betaine
130
Fig. 4.3.1.4. Plant heights of Ile de France in response to various levels of glycine betaine
Fig. 4.3.1.5. Plant heights of Parade in response to various levels of glycine betaine
(a) = T0 = Control (Distilled water)
(b) = T1 = 30 mM (Glycine betaine)
(c) = T2 = 60 mM (Glycine betaine)
(d) = T3 = 90 mM (Glycine betaine)
(e) = T4 = 120 mM (Glycine betaine)
(f) = T5 = 150 mM (Glycine betaine)
131
4.3.2. Plant fresh mass (g)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.2 that tulip variety ‘Apeldorn’ superseded all other varieties by producing maximum fresh mass, while plants with the minimum amongst best were noticed in variety ‘Leen Vander Mark’. Results envisaged that various levels of glycine betaine induced varying values for plant fresh mass, while untreated plants yielded the least fresh mass among all treatments. It is evident from results that heaviest plant (43.03 g) was noticed in variety ‘Apeldorn’ when glycine betaine was applied at 60 mM, exhibiting 84.48% increase over the respective control. Moreover, Parade (37.59 g), Barcelona (35.62 g), Ile de France (35.44 g) and Leen Vander Mark (35.04 g) exhibited maximum plant fresh mass in response to 60, 30, 30 and 120 mM glycine betaine application, which correspond to 50.65, 21.12, 65.71 and 52.74% increase, respectively.
50 Apeldorn Barcelona Ile de France LVM Parade 45
40
35 30 25 20
15 Plant fresh mass (g) mass fresh Plant 10 5 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.2. Response of various concentrations of glycine betaine on plant fresh mass of five tulip cultivars
132
4.3.3. Plant dry mass (g)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.3 that tulip variety ‘Apeldorn’ superseded all other varieties by producing maximum dry mass, while the least amongst best was noticed in variety ‘Parade’. Results envisaged that various levels of glycine betaine induced varying values for plant dry mass, while untreated plants yielded the least dry mass among all treatments. It is evident from results that highest value (4.12 g) for dry mass was noticed in variety ‘Apeldorn’ when glycine betaine was applied at 90 mM, exhibiting 91.54% increase over the respective control. Moreover, Parade (3.66 g), Barcelona (3.16 g), Ile de France (3.37 g) and Leen Vander Mark (3.35 g) exhibited maximum plant dry mass in response to 90, 30, 90 and 120 mM glycine betaine application, which correspond to 52.54, 14.95, 71.38 and 56.43% increase, respectively.
4.5 Apeldorn Barcelona Ile de France LVM Parade 4
3.5
3
2.5
2
1.5 Plant dry mass (g) mass dry Plant 1
0.5
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.3. Response of various concentrations of glycine betaine on plant dry mass of five tulip cultivars
133
4.3.4. Leaf area (cm2)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.4 that tulip variety ‘Parade’ superseded all other varieties by producing maximum leaf area, while the least amongst best was noticed in variety ‘Ile de France’. Results envisaged that various levels of glycine betaine induced different values for leaf area while untreated plants were shortest among all treatments. It is evident from results that maximum leaf area (89.37 cm2) was noticed in variety ‘Parade’ when glycine betaine was applied at 90 mM, exhibiting 48.12% increase over the respective control. Moreover, Leen Vander Mark (78.71 cm2), Apeldorn (65.67 cm2), Barcelona (62.01 cm2) and Ile de France (55.87 cm2) showed maximum leaf area in response to 120, 90, 30 and 90 mM glycine betaine application, which correspond to 46.19, 150.22, 25.55 and 86.64% increase, respectively.
100 Apeldorn Barcelona Ile de France LVM Parade 90 80
70
) 2 60 50 40
Leaf area (cm area Leaf 30 20 10 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.4. Response of various concentrations of glycine betaine on leaf area of five tulip cultivars
134
4.3.5. Photosynthetic rate (A) (µmol m-2s-1)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.5 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by achieving highest photosynthetic rate, while the minimum rate was noticed in variety ‘Apeldorn’. Results envisaged that various levels of glycine betaine induced different rate of photosynthesis while untreated plants possessed the least values among all treatments. It is evident from results that maximum rate (19.24) of photosynthesis was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 120 mM, exhibiting 123.13% increase over the respective control. Moreover, Parade (17.39), Ile de France (16.29), Barcelona (15.91) and Apeldorn (12.28) showed maximum photosynthetic rate in response to 90, 30, 60 and 60 mM glycine betaine application, which correspond to 334.80, 201.15, 35.27 and 350.69% increase, respectively.
25
Apeldorn Barcelona Ile de France LVM Parade
)
1
-
s
2 -
20
mol m mol µ 15
10
5 Photosynthetic rate (A) ( rate Photosynthetic 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.5. Response of various concentrations of glycine betaine on photosynthetic rate (A) of five tulip cultivars
135
4.3.6. Transpiration rate (E) (mmol m-2s-1)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.6 that tulip variety ‘Barcelona’ superseded all other varieties by possessing highest transpiration rate, while the lowest rate was noticed in variety ‘Ile de France’. Results envisaged that various levels of glycine betaine induced different rates of transpiration while untreated plants had the minimum values among all treatments. It is evident from results that highest rate (7.33) of transpiration was noticed in variety ‘Barcelona’ when glycine betaine was applied at 60 mM, exhibiting 19.78% increase over the respective control. Moreover, Leen Vander Mark (6.48), Apeldorn (6.45), Parade (5.43) and Ile de France (4.80) showed maximum rates of transpiration in response to 120, 60, 90 and 90 mM glycine betaine application, which correspond to 26.44, 24.27, 31.00 and 29.11% increase, respectively.
8
Apeldorn Barcelona Ile de France LVM Parade
)
1 -
s 7
2 - 6
mmol mmol m 5
(E) ( (E) 4
3
2
1 Transpiration rate rate Transpiration 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.6. Response of various concentrations of glycine betaine on transpiration rate (E) of five tulip cultivars
136
4.3.7. Instantaneous Water Use Efficiency (A/E)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.7 that tulip variety ‘Parade’ superseded all other varieties by showing maximum efficiency of instantaneous water use, while minimum efficiency was noticed in variety ‘Apeldorn’. Results envisaged that various levels of glycine betaine induced different efficiency level of instantaneous water use while untreated plants had the least efficiency among all treatments. It is evident from results that highest ratio (3.21) of instantaneous water use was noticed in variety ‘Parade’ when glycine betaine was applied at 90 mM, exhibiting 234.35% increase over the respective control. Moreover, Ile de France (3.10), Leen Vander Mark (2.99), Barcelona (2.17) and Apeldorn (1.94) showed maximum ratio of instantaneous water use efficiency in response to 30, 120, 60 and 60 mM glycine betaine application, which correspond to 112.22, 76.87, 12.05 and 268.21% increase, respectively.
4 Apeldorn Barcelona Ile de France LVM Parade 3.5
3
2.5
2
1.5
1
0.5
Instantaneous Water (A/E) Use Efficiency Water Instantaneous 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.7. Response of various concentrations of glycine betaine on Instantaneous water use efficiency (A/E) of five tulip cultivars
137
4.3.8. Intrinsic Water Use Efficiency (A/gs)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.8 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by producing maximum ratio of intrinsic water use efficiency, while minimum efficiency was noticed in variety ‘Apeldorn’. Results envisaged that various levels of glycine betaine induced different ratios for intrinsic water use efficiency while untreated plants had least levels among all treatments. It is evident from results that maximum ratio of intrinsic water use efficiency (23.57) was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 120 mM, exhibiting 55.17% increase over the respective control. Moreover, Ile de France (21.91), Barcelona (19.10), Parade (18.90) and Apeldorn (11.85) showed maximum ratio of intrinsic water use efficiency in response to 30, 60, 90 and 60 mM glycine betaine application, which correspond to 114.95, 23.72, 147.21 and 2.48% increase, respectively.
30 Apeldorn Barcelona Ile de France LVM Parade
25
20
15
10
5
Instrinsic Water (A/gs) Efficiency Use Water Instrinsic 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.8. Response of various concentrations of glycine betaine on Intrinsic water use efficiency (A/gs) of five tulip cultivars
138
-1 4.3.9. Sub-stomatal CO2 (Ci) (µmol mol )
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.9 that tulip variety ‘Parade’ superseded all other varieties by possessing maximum sub-stomatal CO2, while minimum values for sub-stomatal
CO2 was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different values for sub stomatal CO2 while untreated plants had the least values among all treatments. It is evident from results that maximum (417.4) sub-stomatal CO2 were noticed in variety ‘Parade’ when glycine betaine was applied at 60 mM, exhibiting 25.39% increase over the respective control. Moreover, Leen Vander Mark (416.26), Ile de France (398.2), Apeldorn
(380.26) and Barcelona (314.6) showed highest values for sub-stomatal CO2 in response to 120, 90, 60 and 30 mM glycine betaine application, which correspond to 12.72, 16.27, 7.50 and 4.65% increase, respectively.
450
Apeldorn Barcelona Ile de France LVM Parade
) 1 - 400
350
300
(Ci) (µmol (Ci) mol (µmol 250
2
200
CO
150
100
stomatal stomatal -
50 Sub 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.9. Response of various concentrations of glycine betaine on Sub-stomatal CO2 (Ci) of five tulip cultivars
139
4.3.10. Stomatal Conductance (gs) (mmol m-2s-1)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.10 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by exhibiting maximum stomatal conductance, while the least value for stomatal conductance in variety ‘Ile de France’. Results envisaged that various levels of glycine betaine induced different values for stomatal conductance while untreated plants had the minimum values among all treatments. It is evident from results that maximum (1.14) stomatal conductance was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 120 mM, exhibiting 92.80% increase over the respective control. Moreover, Apeldorn (1.12), Parade (0.92), Barcelona (0.84) and Ile de France (0.74) showed maximum stomatal conductance in response to 60, 90, 60 and 30 mM glycine betaine application, which correspond to 118.06, 66.10, 9.23 and 40.65% increase, respectively.
1.4
) ) Apeldorn Barcelona Ile de France LVM Parade
1
-
s 2 - 1.2
mol mol m 1
0.8
0.6
0.4
0.2
Stomatal Conductance (gs) (m (gs) Conductance Stomatal 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.10. Response of various concentrations of glycine betaine on Stomatal conductance (gs) of five tulip cultivars
140
4.3.11. Chlorophyll contents (SPAD Value)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.11 that tulip variety ‘Ile de France’ superseded all other varieties by possessing maximum SPAD value, while minimum amongst best was the variety ‘Parade’. Results envisaged that various levels of glycine betaine induced different SPAD values while untreated plants had minimum among all treatments. It is evident from results that maximum (63.28) SPAD value was noticed in variety ‘Ile de France’ when glycine betaine was applied at 90 mM, exhibiting 39.25% increase over the respective control. Moreover, Barcelona (55.38), Leen Vander Mark (52.42), Apeldorn (52.24) and Parade (52.16) showed maximum SPAD values in response to 30, 90, 90 and 60 mM glycine betaine application, which correspond to 16.78, 39.04, 35.98 and 34.24% increase, respectively.
80
Apeldorn Barcelona Ile de France LVM Parade
70
60
50
40
30
20
10 Chlorophyll contents (SPAD Value) contents (SPAD Chlorophyll
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.11. Response of various concentrations of glycine betaine on chlorophyll contents of five tulip cultivars
141
4.3.12. Mesophyll Conductance (A/Ci)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.12 that tulip variety ‘Barcelona’ superseded all other varieties by exhibiting maximum mesophyll conductance, while minimum was noticed in variety ‘Apeldorn’. Results envisaged that various levels of glycine betaine induced different mesophyll conductance while untreated plants had minimum values among all treatments. It is evident from results that maximum (0.0524) mesophyll conductance was noticed in variety ‘Barcelona’ when glycine betaine was applied at 60 mM, exhibiting 33.87% increase over the respective control. Moreover, Leen Vander Mark (0.0461), Parade (0.0433), Ile de France (0.0424) and Apeldorn (0.0322) showed maximum mesophyll conductance in response to 120, 90, 30 and 60 mM glycine betaine application, which correspond to 96.81, 260.95, 167.91 and 317.53% increase, respectively.
0.07 Apeldorn Barcelona Ile de France LVM Parade
0.06
0.05
0.04
0.03
0.02
Mesophyll Conductance Conductance (A/Ci) Mesophyll 0.01
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.12. Response of various concentrations of glycine betaine on Mesophyll conductance (A/Ci) of five tulip cultivars
142
4.3.13. Superoxide dismutase (SOD) activity (Units/mg Protein)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.13 that tulip variety ‘Ile de France’ superseded all other varieties by possessing maximum SOD activity, while minimum activity was noticed in variety ‘Parade’. Results envisaged that various levels of glycine betaine induced different levels of SOD activity while untreated plants had minimum among all treatments. It is evident from results that maximum (2664.061) SOD activity was noticed in variety ‘Ile de France’ when glycine betaine was applied at 90 mM, exhibiting 248.68% increase over the respective control. Moreover, Leen Vander Mark (2494.07), Barcelona (1881.84), Apeldorn (1419.59) and Parade (1346.95) showed maximum SOD activity in response to 120, 30, 90 and 90 mM glycine betaine application, which correspond to 196.17, 95.81, 64.93 and 35.71% increase, respectively.
3500 Apeldorn Barcelona Ile de France LVM Parade 3000
Units/mg Units/mg 2500
2000
1500 Protein) 1000
500
Superoxide dismutase activity activity ( dismutase Superoxide 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.13. Response of various concentrations of glycine betaine on Superoxide dismutase activity of five tulip cultivars
143
4.3.14. Catalase (CAT) activity (Units/mg Protein)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.14 that tulip variety ‘Apeldorn’ superseded all other varieties by possessing maximum CAT activity, while minimum was recorded in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different levels of CAT activity while untreated plants had the least among all treatments. It is evident from results that maximum (951.62) CAT activity was noticed in variety ‘Apeldorn’ when glycine betaine was applied at 120 mM, exhibiting 73.91% increase over the respective control. Moreover, Leen Vander Mark (943.89), Ile de France (914.38), Parade (876.00) and Barcelona (762.67) showed maximum CAT activity in response to 90, 90, 120 and 30 mM glycine betaine application, which correspond to 54.26, 28.04, 42.48 and 5.86% increase, respectively.
1200
Apeldorn Barcelona Ile de France LVM Parade
1000
800
(Units/mg Protein) (Units/mg 600
400
200 Catalase Catalase activity 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.14. Response of various concentrations of glycine betaine on Catalase activity of five tulip cultivars
144
4.3.15. DPPH Scavenging Activity (% inhibition)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.15 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by exhibiting maximum DPPH scavenging activity, while minimum was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different levels of percentage inhibition of DPPH ions while untreated had the least percentage inhibition among all treatments. It is evident from results that maximum (79.45%) DPPH ion scavenging activity was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 30 mM, exhibiting 50.72% increase over the respective control. Moreover, Ile de France (79.32%), Apeldorn (74.77%), Parade (72.95%) and Barcelona (58.77%) showed maximum percentage inhibition of DPPH ion scavenging activity in response to 90, 90, 60 and 30 mM glycine betaine application, which correspond to 21.99, 18.80, 28.58 and 23.61% increase, respectively.
90 Apeldorn Barcelona Ile de France LVM Parade 80 70
60
50
40 inhibition) 30 20
DPPH Scavenging Activity Activity (% Scavenging DPPH 10 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.15. Response of various concentrations of glycine betaine on DPPH Scavenging Activity of five tulip cultivars
145
4.3.16. Peroxidase (POD) activity (Units/mg Protein)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.16 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by exhibiting maximum POD activity while minimum activity was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different levels of POD activity while untreated plants had least among all treatments. It is evident from results that maximum (3389.04) POD activity was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 60 mM, exhibiting 123.13% increase over the respective control. Moreover, Parade (3178.95), Apeldorn (2972.19), Ile de France (2910.32) and Barcelona (2066.10) showed maximum POD activity in response to 90, 120, 60 and 60 mM glycine betaine application, which correspond to 104.36, 72.88, 43.80 and 109.85% increase, respectively.
4000
Apeldorn Barcelona Ile de France LVM Parade 3500
3000
2500
2000
1500
1000
500 Peroxidase activity (Units/mg (Units/mg Protein) activity Peroxidase 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.16 Response of various concentrations of glycine betaine on Peroxidase activity of five tulip cultivars
146
4.3.17. Malondialdehyde (MDA) contents (µmol g-1 fresh weight)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.17 that tulip variety ‘Barcelona’ superseded all other varieties by exhibiting minimum levels of MDA contents, while maximum was noticed in variety ‘Apeldorn’. Results envisaged that various levels of glycine betaine induced different levels of MDA contents while untreated plants had highest quantity among all treatments. It is evident from results that minimum (0.64) MDA contents were noticed in variety ‘Barcelona’ when glycine betaine was applied at 30 mM, exhibiting 34.93% decrease over the respective control. Moreover, Ile de France (0.95), Leen Vander Mark (0.96), Parade (1.07) and Apeldorn (1.13) showed minimum MDA contents in response to 90, 90, 90 and 120 mM glycine betaine application, which correspond to 45.14, 42.67, 26.17 and 30.91% decrease, respectively.
2 Apeldorn Barcelona Ile de France LVM Parade 1.8 1.6
fresh weight) fresh 1.4
1 - 1.2
1
µmol µmol g ( 0.8 0.6 0.4 0.2
Malondialdehyde 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.17. Response of various concentrations of glycine betaine on Malondialdehyde contents of five tulip cultivars
147
4.3.18. Total Phenolic Compounds (mg GAE/100g FW)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.18 that tulip variety ‘Apeldorn’ superseded all other varieties by exhibiting maximum total phenolic compounds, while minimum were noticed in variety ‘Parade’. Results envisaged that various levels of glycine betaine induced different levels of total phenolics while untreated plants had least of them among all treatments. It is evident from results that maximum (284.13) level of phenolics was noticed in variety ‘Apeldorn’ when glycine betaine was applied at 90 mM, exhibiting 70.18% increase over the respective control. Moreover, Leen Vander Mark (264.05), Ile de France (260.72), Barcelona (221.27) and Parade (219.17) showed maximum level of phenolic contents in response to 60, 90, 30 and 90 mM glycine betaine application, which correspond to 35.06, 29.70, 22.49 and 12.95% increase, respectively.
350 Apeldorn Barcelona Ile de France LVM Parade 300
250
200
150 GAE/100g FW) GAE/100g 100
Total Phenolic Compounds (mg (mg Compounds Phenolic Total 50
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.18. Response of various concentrations of glycine betaine on Total Phenolic compounds of five tulip cultivars
148
4.3.19. H2O2 Scavenging Activity (%)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.19 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by maximum scavenging percentage of H2O2, while less was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different levels of percentage scavenging of H2O2 while untreated plants possessed the least among all treatments. It is evident from results that maximum (48.49%) H2O2 scavenging activity was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 90 mM, exhibiting 119.72% increase over the respective control. Moreover, Ile de France (46.04%), Parade (45.40%), Apeldorn (35.86%) and Barcelona (30.40%) showed maximum percentage for
H2O2 scavenging in response to 90, 120, 120 and 30 mM glycine betaine application, which correspond to 79.99, 55.60, 179.32 and 43.26% increase, respectively.
60 Apeldorn Barcelona Ile de France LVM Parade
50
40
30
20
Scavenging Activity Activity (%) Scavenging
2
O 2
H 10
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.19. Response of various concentrations of glycine betaine on H2O2 Scavenging Activity of five tulip cultivars
149
4.3.20. Days to flower bud emergence (d)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.20 that tulip variety ‘Ile de France’ superseded all other varieties by earliest emergence of flower buds, while late emergence was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different days to flower bud emergence while untreated plants had extremely late emergence among all treatments. It is evident from results that earliest (33.53 d) emergence of flower buds was noticed in variety ‘Ile de France’ when glycine betaine was applied at 90 mM, exhibiting 17.26% earliness over the respective control. Moreover, Parade (34.2 d), Leen Vander Mark (37.53 d), Apeldorn (40.8 d) and Barcelona (47.8 d) showed earliest emergence in response to 90, 120, 60 and 30 mM glycine betaine application, which correspond to 8.71, 11.75, 10.39 and 3.23% respectively, earliness in flower bud emergence.
60
Apeldorn Barcelona Ile de France LVM Parade
50
40
30
20
10 Days to flower bud emergence (d) emergence bud flower to Days
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.20. Response of various concentrations of glycine betaine on days to flower bud emergence of five tulip cultivars
150
4.3.21. Tepal diameter (mm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.21 that tulip variety ‘Ile de France’ superseded all other varieties by exhibiting maximum tepal diameter, while minimum was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different dimensions in tepal diameter while untreated plants had the least values for tepal diameter among all treatments. It is evident from results that maximum diameter (34.77 mm) was noticed in variety ‘Ile de France’ when glycine betaine was applied at 90 mM, exhibiting 19.91% increase over the respective control. Moreover, Parade (33.10 mm), Apeldorn (28.78 mm), Leen Vander Mark (26.83 mm) and Barcelona (22.92 mm) showed maximum tepal dimeter in response to 90, 90, 60 and 30 mM glycine betaine application, which correspond to 11.39, 37.72, 19.89 and 47.35% increase, respectively.
40 Apeldorn Barcelona Ile de France LVM Parade 35
30
25
20
15
Tepal diameter (mm) diameter Tepal 10
5
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.21. Response of various concentrations of glycine betaine on tepal diameter of five tulip cultivars
151
4.3.22. Flower diameter (mm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.22 that tulip variety ‘Parade’ superseded all other varieties by producing largest diameter of flower, while shortest was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different flower diameters while untreated plants had the least among all treatments. It is evident from results that largest diameter (66.97 mm) of flowers was noticed in variety ‘Parade’ when glycine betaine was applied at 90 mM, exhibiting 19.87% increase over the respective control. Moreover, Leen Vander Mark (57.62 mm), Ile de France (54.49 mm), Apeldorn (50.93 mm) and Barcelona (41.14 mm) showed maximum values for flower diameter in response to 60, 90, 90 and 30 mM glycine betaine application, which correspond to 29.40, 27.47, 46.15 and 40.38% increase, respectively.
80 Apeldorn Barcelona Ile de France LVM Parade 70
60
50
40
30
Flower diameter (mm) diameter Flower 20
10
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.22. Response of various concentrations of glycine betaine on flower diameter of five tulip cultivars
152
4.3.23. Length of petals (cm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.23 that tulip variety ‘Apeldorn’ superseded all other varieties by producing largest petals, while shortest were noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different petal lengths while untreated plants had shortest petals among all treatments. It is evident from results that largest petals (9.4 cm) were noticed in variety ‘Apeldorn’ when glycine betaine was applied at 60 mM, exhibiting 81.23% increase over the respective control. Moreover, Parade (7.7 cm), Leen Vander Mark (6.9 cm), Ile de France (6.5 cm) and Barcelona (6.2 cm) showed maximum petal lengths in response to 90, 60, 90 and 30 mM glycine betaine application, which correspond to 26.46, 13.68, 27.47 and 6.88% increase, respectively.
12 Apeldorn Barcelona Ile de France LVM Parade
10
8
6
4 Length of petals (cm) petals Lengthof
2
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.23. Response of various concentrations of glycine betaine on length of petals of five tulip cultivars
153
4.3.24. Width of petals (cm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.24 that tulip variety ‘Apeldorn’ superseded all other varieties by producing largest petal width, while shortest were noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine induced different petal widths while untreated plants were shortest among all treatments. It is evident from results that maximum (5.5 cm) width of petals was noticed in variety ‘Apeldorn’ when glycine betaine was applied at 90 mM, exhibiting 24.14% increase over the respective control. Moreover, Leen Vander Mark (5.09 cm), Parade (4.47 cm), Ile de France (4.46 cm) and Barcelona (4.31 cm) showed maximum width of petals in response to 60, 60, 30 and 0 mM glycine betaine application, which correspond to 12.39, 7.90 and 39.37 % increase, respectively, while reduction in petal width was observed in Barcelona variety on either of the glycine betaine application.
6 Apeldorn Barcelona Ile de France LVM Parade
5
4
3
2 Width of Petals (cm) Petals of Width
1
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.24. Response of various concentrations of glycine betaine on width of petals of five tulip cultivars
154
4.3.25. Scape diameter (mm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.25 that tulip variety ‘Apeldorn’ superseded all other varieties by producing maximum scape diameter, while the least was noticed in variety ‘Parade’. Results envisaged that various levels of glycine betaine induced different scape diameters while untreated plants were shortest among all treatments. It is evident from results that maximum (6.35 mm) scape diameter was noticed in variety ‘Apeldorn’ when glycine betaine was applied at 90 mM, exhibiting 38.75% increase over the respective control. Moreover, Barcelona (6.25 mm), Ile de France (5.39 mm), Leen Vander Mark (5.33 mm) and Parade (5.27 mm) showed maximum scape diameters in response to 30, 90, 60 and 90 mM glycine betaine application, which correspond to 6.04, 22.67, 4.52 and 6.20% increase, respectively.
8 Apeldorn Barcelona Ile de France LVM Parade 7
6
5
4
3
Scape diameter (mm) diameter Scape 2
1
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.25. Response of various concentrations of glycine betaine on scape diameter of five tulip cultivars
155
4.3.26. Flower quality (Copper and Spokas, 1991)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.26 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by ascertaining maximum score regarding visual appearance, while minimum score was attained in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine influenced different score points while untreated plants had minimum among all treatments. It is evident from results explained in ranked order of varieties Leen Vander Mark (8.2), Parade (7.4), Apeldorn (6.6), Ile de France (6.3) and Barcelona (6.0). The best levels of glycine betaine application and percentage increase as compared to their respective controls are as follows; ‘Leen Vander Mark’ at 120 mM with 41.37%, Parade at 90 mM with 33.73%, Apeldorn at 60 mM with 130.23%, Ile de France at 30 mM with 26.66% and Barcelona at 30 mM with 22.97% increase was obtained.
10 Apeldorn Barcelona Ile de France LVM Parade 9 8
7 6 5
4 Flower quality Flower 3 2 1 0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.26. Response of various concentrations of glycine betaine on flower quality of five tulip cultivars
156
4.3.27. Vase life (d)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.27 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by exhibiting maximum display life, while minimum was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine influenced vase life with improved post-harvest longevity while untreated plants displayed minimal shelf life display among all treatments. It is evident from results that longest display life (12 d) was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 60 mM, exhibiting 35.33% increase over the respective control. Moreover, Apeldorn (11.7 d), Parade (10.2 d), Ile de France (8.9 d) and Barcelona (6.0 d) showed longest vase life display in response to 60, 90, 60 and 30 mM glycine betaine application, which correspond to 83.33, 34.21, 33.00 and 42.85% increase, respectively.
14 Apeldorn Barcelona Ile de France LVM Parade 12
10
8
6 Vase life (d) Vase 4
2
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.27. Response of various concentrations of glycine betaine on vase life of five tulip cultivars
157
4.3.28. Images showing the response of various concentrations of glycine betaine on vase display of five tulip cultivars
Fig. 4.3.28.1. Vase display of Apeldorn cultivar on 12th day after harvest
Fig. 4.3.28.2. Vase display of Barcelona cultivar on 6th day after harvest
158
Fig. 4.3.28.3. Vase display of Ile de France cultivar on 10th day after harvest
Fig. 4.3.28.4. Vase display of Ile de France cultivar on 12th day after harvest
159
Fig. 4.3.28.5. Vase display of Parade cultivar on 11th day after harvest
(a) = T0 = Control (Distilled water)
(b) = T1 = 30 mM (Glycine betaine)
(c) = T2 = 60 mM (Glycine betaine)
(d) = T3 = 90 mM (Glycine betaine)
(e) = T4 = 120 mM (Glycine betaine)
(f) = T5 = 150 mM (Glycine betaine)
160
4.3.29. Days to start petal senescence (d)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.29 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by ascertaining maximum number of days to start petal senescence, while minimum number of days was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine remarkably influenced the number of days to initiate petal senescence while petals of untreated plants showed earliest senescence among all treatments. It is evident from results that prolonged freshness (14 d) was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 60 mM, exhibiting 28.83% increase over the respective control. Moreover, Apeldorn (12.73 d), Parade (11.20 d), Ile de France (9.80 d) and Barcelona (6.0 d) showed maximum days to start petal senescence in response to 60, 90, 60 and 60 mM glycine betaine application, which correspond to 72.07, 30.23, 28.69 and 15.83% increase, respectively.
16
Apeldorn Barcelona Ile de France LVM Parade
14
12
10
8
6
4
Days to start petal senescence senescence petal start (d) to Days 2
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.29. Response of various concentrations of glycine betaine on days to start petal senescence of five tulip cultivars
161
4.3.30. Tepal abscission (d)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.30 that tulip variety ‘Leen Vander Mark’ superseded all other varieties by exhibiting late abscission of tepals, while earliest abscission was noticed in variety ‘Barcelona’. Results envisaged that various levels of glycine betaine influenced different days to tepal abscission while untreated plants had rapid abscission among all treatments. It is evident from results that largest number of days (15 d) to abscission was noticed in variety ‘Leen Vander Mark’ when glycine betaine was applied at 60 mM, exhibiting 26.40% increase over the respective control. Moreover, Apeldorn (13.73 d), Parade (12.22 d), Ile de France (10.86 d) and Barcelona (8.00 d) showed late abscission in response to 60, 90, 60 and 30 mM glycine betaine application, which correspond to 63.49, 27.08, 25.38 and 29.03% increase, respectively.
16 Apeldorn Barcelona Ile de France LVM Parade 14
12
10
8
6
Tepal abscission (d) abscission Tepal 4
2
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.30. Response of various concentrations of glycine betaine on tepal abscission of five tulip cultivars
162
4.3.31. Diameter of bulbils (mm)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.31 that tulip variety ‘Ile de France’ superseded all other varieties in developing bulbils with largest diameter, while smallest bulbils were formed in variety ‘Leen Vander Mark’. Results envisaged that various levels of glycine betaine induced different sizes of bulbils in context to their diameter. It is evident from results that largest bulbils (38.09 mm) were developed in variety ‘Ile de France’ when glycine betaine was applied at 150 mM, exhibiting 5.54% increase over its respective control. Moreover, Parade (36.84 mm), Apeldorn (33.27 mm), Barcelona (31.22 mm) and Leen Vander Mark (29.37 mm) developed largest diameter of bulbils in response to 0, 120, 120 and 150 mM glycine betaine application, which correspond to 0, 52.98, 12.89 and 29.37% increase, respectively.
45 Apeldorn Barcelona Ile de France LVM Parade 40
35
30
25
20 15
Diameter (mm) bulbilsof Diameter 10
5
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.31. Response of various concentrations of glycine betaine on diameter of bulbils of five tulip cultivars
163
4.3.32. Mass of bulbils per clump (g)
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.32 that tulip variety ‘Ile de France’ superseded all other varieties by ascertaining maximum mass of bulbils per clump, while minimum mass was noticed in variety ‘Parade’. Results envisaged that various levels of glycine betaine induced different values for the mass of bulbils per clump while untreated plants had maximum mass in all of the cultivars except Barcelona. It is evident from results that highest mass (23.30 g) was noticed in variety ‘Ile de France’ followed by Barcelona (18.61 g) when glycine betaine was applied at 120 mM, exhibiting 35.27% increase over its corresponding control. Moreover, Leen Vander Mark (14.18 g), Apeldorn (12.93) and Parade (12.66 g) showed maximum values for mass of bulbils per clump in untreated plants while different concentrations of glycine betaine application revealed decreasing trend regarding mass of bulbils per clump.
30 Apeldorn Barcelona Ile de France LVM Parade
25
20
15
10
Mass of bulbils per clump (g) clump bulbilsof per Mass 5
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.32. Response of various concentrations of glycine betaine on mass of bulbils per clump of five tulip cultivars
164
4.3.33. Number of bulbils
Results depict significant (P ≤ 0.05) differences for various treatments, varieties and their interaction. It is obvious from results in Fig. 4.3.33 that tulip variety ‘Barcelona’ superseded all other varieties by producing maximum number of bulbils, while minimum number of bulbils was noticed in variety ‘Parade’. Results envisaged that various levels of glycine betaine induced different number of bulbils while maximum number (3.8) of bulbils was found in Barcelona variety when glycine betaine was applied at 60 mM, exhibiting 41.46% increase over its respective control. Moreover, Leen Vander Mark (2.73), Apeldorn (2.66), Ile de France (1.86) and Parade (1.73) showed maximum number of bulbils in response to 90, 90, 90 and 60 mM glycine betaine application, which correspond to 156.25, 207.69, 12.00 and 73.33% increase, respectively.
4.5 Apeldorn Barcelona Ile de France LVM Parade 4
3.5
3
2.5
2 1.5
Number bulbilsof Number 1
0.5
0 Control 30 60 90 120 150 Glycine betaine concentrations (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.3.33. Response of various concentrations of glycine betaine on number of bulbils of five tulip cultivars
165
Table 4.3 (a) Pearson’s correlation matrix among various morpho-physiological attributes of tulip cultivars in response to glycine betaine application
PH PFM PDM LA DFBE TD SV BD MBC NBC A E InsWUE IntWUE Ci gs
PFM 0.645** 1
PDM 0.672** 0.961** 1
LA 0.647** 0.530** 0.576** 1
DFBE -0.365** -0.141** -0.203** -0.368** 1
TD 0.428** 0.243** 0.295** 0.310** -0.699 1
SV 0.280** 0.231** 0.216** 0.072 -0.099* 0.201** 1
BD -0.210** -0.158** -0.126** -0.216** 0.013 0.004 -0.043 1
MBC -0.359** -0.317** -0.347** -0.446** 0.226** -0.141** -0.063 0.274** 1
NOB 0.176** 0.184** 0.154** 0.082 0.309** -0.209** 0.170** -0.077 0.088 1
A 0.513** 0.329** 0.325** 0.454** -0.212** 0.189** 0.272** -0.197** -0.127** 0.226** 1
E 0.251** 0.322** 0.287** 0.260** 0.442** -0.393** 0.063 -0.275** -0.205** 0.374** 0.282** 1
InsWUE 0.438** 0.219** 0.228** 0.348** -0.415** 0.374** 0.275** -0.100* -0.050 0.074 0.909** -0.106* 1
IntWUE 0.265** 0.089** 0.086 0.213** -0.212** 0.162** 0.222** -0.103* -0.010 0.089 0.762** 0.088 0.744** 1
Ci 0.453** 0.314** 0.355** 0.515** -0.706** 0.578** 0.099* -0.188** -0.323** -0.243** 0.277** -0.156** 0.361** 0.238** 1
gs 0.322** 0.378** 0.377** 0.388** -0.033** 0.046 0.105* -0.162** -0.233** 0.162** 0.393** 0.322** 0.269** -0.056 0.152** 1 gm 0.408** 0.258** 0.240** 0.314** -0.007 0.030 0.264** -0.148** -0.039 0.315** 0.955** 0.350** 0.831** 0.723** -0.004 0.357**
Values indicate Pearson’s correlation coefficient; *significant (P ≤ 0.05); **highly significant (P ≤ 0.01);
PFM, Plant fresh mass; PDM, Plant dry mass; PH, Plant height; LA, Leaf area; SV, SPAD Value; DFBE, Days to flower bud emergence; TD, Tepal diameter; MBC, Mass of bulb clump; NOB, Number of bulbs; BD, Bulb diameter; A, Photosynthetic rate; E, Transpiration rate; InsWUE,
Instantaneous water use efficiency; IntWUE, Intrinsic water use efficiency; Ci, Sub-stomatal CO2; gs, Stomatal conductance; gm, Mesophyll conductance
166
Table 4.3 (b) Pearson’s correlation matrix among various antioxidant enzymes activity and biochemical changes induced on floral attributes of tulip cultivars in response to glycine betaine application
FD LP WP SD FQ DSPS TA VL SOD CAT POD DSA MDA TPC LP 0.427** 1 WP 0.260** 0.465** 1 SD -0.083 0.266** 0.146** 1 FQ 0.260** 0.187** 0.035 -0.021 1 DSPS 0.622** 0.510** 0.474** 0.026 0.252** 1 TA 0.622** 0.510** 0.474** 0.026 0.252** 1.000** 1 VL 0.638** 0.533** 0.475** 0.041 0.239** 0.992** 0.992** 1 SOD 0.285** 0.273** 0.220** 0.191** 0.298** 0.420** 0.420** 0.410** 1 CAT 0.293** 0.258** 0.218** 0.088 0.204** 0.327** 0.327** 0.339** 0.450** 1 POD 0.494** 0.368** 0.369** 0.022 0.131** 0.503** 0.503** 0.531** 0.330** 0.397** 1 DSA 0.594** 0.332** 0.380** 0.011 0.116* 0.518** 0.518** 0.562** 0.306** 0.405** 0.613** 1 MDA 0.027 -0.130** -0.039 -0.318** -0.110* 0.026 0.026 0.029 -0.379** -0.137** -0.039 0.077 1 TPC 0.305** 0.327** 0.361** 0.215** 0.084 0.414** 0.414** 0.437** 0.364** 0.310** 0.525** 0.457** -0.072 1 HSA 0.703** 0.312** 0.209** 0.001 0.227** 0.494** 0.494** 0.498** 0.416** 0.441** 0.399** 0.463** -0.086 0.276**
Values indicate Pearson’s correlation coefficient; *significant (P ≤ 0.05); **highly significant (P ≤ 0.01);
FD, Flower diameter; VL, Vase life; LP, Length of petals; WP, Width of petals; FQ, Flower quality; SD Stem diameter; DSPS, Days to start petal senescence; TA, Tepal abscission; SOD, Superoxide dismutase activity; CAT, Catalase activity; DSA, DPPH ion scavenging activity; POD,
Peroxidase activity; MDA, Malondialdehyde contents; TPC, Total Phenolic Contents; HSA, H2O2 Scavenging Activity
167
4.4. Experiment # 4 Comparative effect of exogenous application of different levels of polyamines (Putrescine, spermine and spermidine) on two tulip cultivars to assess the cut flower quality
Objective of this experiment was to evaluate and identify the optimized doze that improve morpho-physiological and biochemical attributes with adequate level of changes in antioxidant enzymes in two tulip cultivars (Apeldorn and Clear water) by exogenous application of various levels polyamines namely putrescine (Put), spermidine (Spm) and spermine (Spd). 4.4.1. Plant height (cm) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.1 that tulip variety ‘Clear water’ performed comparatively better than ‘Apeldorn’ by producing tallest plants. Results envisaged that various levels of polyamines induced different plant heights while untreated plants were found to be shortest among all treatments. It is evident from results that tallest plants were noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 83.83% increase as compared to control while Spd at 1.5 mM and Put at 2 mM resulted in 36.69 and 47.96 cm which correspond to 65.64 and 26.73% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum plant height was observed in plants in which Spm at 0.03 mM (48.38 cm), Spd at 2.5 mM (44.04 cm) and Put at 2 mM (37.85 cm) was applied that corresponded to 51.06, 37.53 and 18.19% increase, respectively.
60 Apeldorn Clear Water 50 40 30 20
Plant height (cm) heightPlant 10 0
Polyamines levels (mM) Fig. 4.4.1. Response of various concentrations of polyamines on Plant height of two tulip cultivars
168
4.4.2. Plant fresh mass (g) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction while varieties revealed non-significant difference. It is obvious from results in Fig. 4.4.2 that various levels of polyamines induced highly significant difference in plant fresh mass while untreated plants were found to be lightest among all treatments irrespective of varieties. It is evident from results that heaviest plants were noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 73.35% increase as compared to control while Spd at 2.5 mM and Put at 2 mM resulted in 42.24 and 38.97 g which correspond to 42.67 and 31.10% increase respectively. Similarly, in ‘Clear water’ cultivar maximum fresh mass of plant was observed in plants in which Spm at 0.01 mM (49.39 g), Spd at 0.5 mM (42.36 g) and Put at 3 mM (37.75 g) was applied that corresponded to 50.80, 29.33 and 15.26% increase, respectively.
60 Apeldorn Clear Water
50
40
30
20 Plant fresh mass Plant mass fresh (g)
10
0
Polyamines levels (mM) Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means.
Fig. 4.4.2. Response of various concentrations of polyamines on Plant fresh mass of two tulip cultivars
169
4.4.3. Plant dry mass (g) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.3 that tulip variety ‘Apeldorn’ performed comparatively better than ‘Clear water’ by possessing maximum dry mass. Results envisaged that various levels of polyamines induced significant difference in plant dry mass while untreated plants were found to be least among all treatments. It is evident from results that highest dry mass (5.58) were noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 35.86% increase as compared to control while Spd at 2.5 mM and Put at 2 mM resulted in 5.15 and 4.6 g which correspond to 35.86 and 21.26% increase respectively. Similarly, in ‘Clear water’ cultivar maximum dry matter contents were observed in plants in which Spm at 0.01 mM (4.88 g), Spd at 1.5 mM (4.32 g) and Put at 3 mM (3.88 g) was applied that corresponded to 47.64, 30.69 and 17.38% increase, respectively.
8 Apeldorn Clear Water
7
6
5
4
3
Plant dry mass (g) mass dry Plant 2
1
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.3. Response of various concentrations of polyamines on Plant dry mass of two tulip cultivars
170
4.4.4. Leaf area (cm2) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.4 that tulip variety ‘Clear water’ performed comparatively better than ‘Apeldorn’ by exhibiting maximum leaf area. Results envisaged that various levels of polyamines induced different values for leaf area while untreated plants were found to exhibit the least among all treatments. It is evident from results that highest value (90.83 cm2) for area were noticed in variety ‘Clear water’ where Spm was applied at 0.02 mM, exhibiting 100.97% increase as compared to control while Spd at 0.5 mM and Put at 2 mM resulted in 75.57 and 63.05 cm2 which correspond to 67.21 and 39.51% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum leaf area was observed in plants in which Spm at 0.03 mM (87.63cm2), Spd at 2.5 mM (68.84 cm2) and Put at 2 mM (59.60 cm2) was applied that corresponded to 134.73, 84.40 and 59.01% increase, respectively.
Apeldorn Clear Water 100 90
80
) 70 2
cm 60 50 40
Leaf area ( area Leaf 30 20 10 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.4. Response of various concentrations of polyamines on Leaf area of two tulip cultivars
171
4.4.5. Photosynthetic rate (A) (µmol m-2s-1) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction while non- significant for varieties. It is obvious from results in Fig. 4.4.5 that various levels of polyamines induced different values for photosynthetic rate while untreated plants were found to exhibit least among all treatments. It is evident from results that maximum rate (18.63) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 255.59% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 14.05 and 10.11 which correspond to 168.21 and 93.04% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum photosynthetic rate was observed in plants in which Spm at 0.03 mM (18.38), Spd at 2.5 mM (13.22) and Put at 2 mM (9.39) was applied that corresponded to 261.43, 160.04 and 84.80% increase, respectively.
24 Apeldorn Clear Water
)
1
- s
2 21 -
18
mol mol m µ 15
12
9
6
3 Photosynthetic rate (A) ( 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.5. Response of various concentrations of polyamines on Photosynthetic rate of two tulip cultivar
172
4.4.6. Transpiration Rate (E) (mmol m-2s-1) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.6 that tulip variety ‘Apeldorn’ had relatively higher transpiration rate as compared to ‘Clear water’. Results envisaged that various levels of polyamines induced transpiration at different rates while untreated plants had the least among all treatments. It is evident from results that maximum rate (8.07) of transpiration was noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 91.20% increase as compared to control while Spd at 2.5 mM and Put at 2 mM resulted in 7.19 and 6.36 which correspond to 70.32 and 50.71% increase respectively. Similarly, in ‘Clear water’ cultivar highest rate of transpiration was observed in plants in which Spm at 0.01 mM (6.43), Spd at 1.5 mM (5.55) and Put at 3 mM (4.51) was applied that corresponded to 83.06, 58.17 and 28.39% increase, respectively.
10 Apeldorn Clear Water
)
1 -
s 9
2 - 8
7 mmol mmol m 6 5 4 3 2
1 Transpiration Rate ( (E) Rate Transpiration 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.6. Response of various concentrations of polyamines on Transpiration rate of two tulip cultivars
173
4.4.7. Instantaneous Water Use Efficiency (A/E) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.7 that tulip variety ‘Clear water’ was found to be more efficient regarding instantaneous water use. Results envisaged that various levels of polyamines induced highly significant differences in A/E values compared to untreated plants. It is evident from the results that highest instantaneous A/E ratio (2.91) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 96.20% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 2.55 and 2.27 which correspond to 71.97 and 52.60% increase respectively. Similarly, in ‘Apeldorn’ cultivar highest A/E ratio was observed in plants in which Spm at 0.03 mM (2.29), Spd at 2.5 mM (1.85) and Put at 3 mM (1.53) was applied that corresponded to 84.24, 48.67 and 23.16% increase, respectively.
3.5 Apeldorn Clear Water
3
2.5
2 (A/E) 1.5
1
0.5 Instantaneous Water Use Efficiency Water Instantaneous 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.7. Response of various concentrations of polyamines on Instantaneous water use efficiency of two tulip cultivars
174
4.4.8. Intrinsic Water Use Efficiency (A/gs) Results depict significant (P ≤ 0.05) differences for various treatments in both varieties and their interaction while non-significant difference for varieties. It is obvious from results in Fig. 4.4.8 that various levels of polyamines induced different values for A/gs ratio among all treatments as compared to untreated plants. It is evident from results that highest A/gs ratio (20.78) was noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 151.05% increase as compared to control while Spd at 2.5 mM and Put at 2 mM resulted in 18.19 and 14.39 which correspond to 119.77 and 73.89% increase respectively. Similarly, in ‘Clear water’ cultivar highest A/gs ratio was observed in plants in which Spm at 0.01 mM (20.46), Spd at 1.5 mM (17.21) and Put at 3 mM (15.79) was applied that corresponded to 108.16, 75.04 and 60.58% increase, respectively.
30
Apeldorn Clear Water
25
20
15 (A/gs)
10 Intrinsic Water Efficiency Use Water Intrinsic 5
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.8. Response of various concentrations of polyamines on Intrinsic water use efficiency of two tulip cultivars
175
-1 4.4.9. Sub-stomatal CO2 (Ci) (µmol mol ) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction while non-significant for varieties. It is obvious from results in Fig. 4.4.9 that various levels of polyamines induced significant difference in the values of Ci while untreated plants exhibited the least among all treatments. It is evident from results that highest Ci value (412.4) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 31.81% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 385.4 and 365.4 which correspond to 23.18 and 16.79% increase respectively. Similarly, in ‘Apeldorn’ cultivar highest level of Ci was observed in plants in which Spm at 0.03 mM (407.93), Spd at 2.5 mM (379.93) and Put at 2 mM (356.06) was applied that corresponded to 29.99, 21.07 and 13.46% increase, respectively.
525 Apeldorn Clear Water
)
1 -
450
375
(Ci) (µmol (Ci) mol (µmol
2 CO
300 stomatal stomatal
- 225 Sub
150
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.9. Response of various concentrations of polyamines on Sub-stomatal CO2 of two tulip cultivars
176
4.4.10. Stomatal Conductance (gs) (mmol m-2s-1) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties while the varieties and their interaction was found to be non-significant. It is obvious from results in Fig. 4.4.10 that tulip variety ‘Clear water’ when provided with various levels of polyamines induced different values for stomatal conductance as compared to untreated plants. It is evident from results that highest gs values (0.918) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 54.19% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 0.823 and 0.705 which correspond to 38.36 and 18.47% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum gs was observed in plants in which Spm at 0.03 mM (0.894), Spd at 2.5 mM (0.747) and Put at 3 mM (0.691) was applied that
corresponded to 41.79, 18.44 and 9.51% increase, respectively.
) Apeldorn Clear Water 1
- 1.2
s
2 -
1
mol mol m m 0.8
0.6
0.4
0.2 Stomatal ( conductance (gs) Stomatal 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.10. Response of various concentrations of polyamines on Stomatal conductance of two tulip cultivars
177
4.4.11. Mesophyll conductance (gm) (A/Ci) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction while varietal effect was found to be non-significant. It is obvious from results in Fig. 4.4.11 that various levels of polyamines induced different values for gm as compared to untreated plants among all treatments. It is evident from results that highest value for gm (0.045187) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 169.94% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 0.036 and 0.027 which correspond to 117.80 and 65.25% increase respectively. Similarly, in ‘Apeldorn’ cultivar highest value for gm was observed in plants in which Spm at 0.03 mM (0.045104), Spd at 2.5 mM (0.0348) and Put at 3 mM (0.0264) was applied that corresponded to 176.76, 113.60 and 62.41% increase, respectively.
0.05 Apeldorn Clear Water
0.04
0.03
0.02
0.01 Mesophyll conductance (gm) conductance (A/Ci) (gm) Mesophyll
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.11. Response of various concentrations of polyamines on Mesophyll conductance of two tulip cultivars
178
4.4.12. Chlorophyll contents (SPAD Value) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.12 that tulip variety ‘Apeldorn’ performed comparatively better than ‘Clear water’ by exhibiting highest SPAD value. Results envisaged that various levels of polyamines induced different SPAD values while untreated plant acquinted minimum among all the tested treatments. It is evident from results that highest SPAD value (79.17) was noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 73.76% increase as compared to control while Spd at 2.5 mM and Put at 2 mM resulted in 65.59 and 56.75 which correspond to 43.96 and 24.55% increase respectively. Similarly, in ‘Clear water’ cultivar maximum SPAD value was observed in plants in which Spm at 0.02 mM (72.91), Spd at 0.5 mM (57.71) and Put at 2 mM (47.34) was applied that corresponded to 88.18, 48.89 and 22.13% increase, respectively.
90 Apeldorn Clear Water
80 70 60 50 40 30 20
10 Chlorophyll contents (SPAD Value) contents (SPAD Chlorophyll 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.12. Response of various concentrations of polyamines on SPAD value of two tulip cultivars
179
4.4.13. Superoxide dismutase (SOD) activity (Units/mg Protein) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.13 that tulip variety ‘Apeldorn’ performed comparatively better than ‘Clear water’ by exhibiting highest SOD activity. Results envisaged that various levels of polyamines induced different levels of SOD activity while untreated plants were found to be at least level among all treatments. It is evident from results that highest rate of SOD activity (1998.2) was noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 156.82% increase as compared to control while Spd at 1.5 mM and Put at 2 mM resulted in 1501.76 and 1220.64 which correspond to 93.01 and 56.88% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum SOD activity was observed in plants in which Spm at 0.01 mM (1474.33), Spd at 1.5 mM (1261.48) and Put at 3 mM (1083.71) was applied that corresponded to 101.88, 72.74 and 48.39% increase, respectively.
2500 Apeldorn Clear Water
Units/mg Units/mg 2000
) 1500
Protein 1000
500
Superoxide dismutase activity activity ( dismutase Superoxide 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.13. Response of various concentrations of polyamines on Superoxide dismutase activity of two tulip cultivars
180
4.4.14. Peroxidase (POD) activity (Units/mg Protein) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction while varietal effect was found to be non-significant. It is obvious from results in Fig. 4.4.14 that various levels of polyamines induced different levels of POD activity while untreated plants were found to exhibit least among all tested treatments. It is evident from results that highest (4179.02) POD activity was noticed in variety ‘Clear water’ where Spm was applied at 0.02 mM, exhibiting 253.14% increase as compared to control while Spd at 1.5 mM and Put at 2 mM resulted in 2978.57 and 1992.49 which correspond to 151.70 and 68.37% increase respectively. Similarly, in ‘Apeldorn’ cultivar highest POD activity was observed in plants in which Spm at 0.02 mM (2822.19), Spd at 2.5 mM (2430.03) and Put at 3 mM (2006.81) was applied that corresponded to 68.62, 45.19 and 19.90% increase, respectively.
5000 Apeldorn Clear Water 4500 4000 3500 3000 2500 2000 1500 1000
Peroxidase activity (Units/mg (Units/mg Protein) activity Peroxidase 500 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.14. Response of various concentrations of polyamines on Peroxidase activity of two tulip cultivars
181
4.4.15. Catalase (CAT) activity (Units/mg Protein) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction while varietal effect was found to be non-significant. It is obvious from results in Fig. 4.4.15 that various levels of polyamines induced different levels of CAT activity while untreated plants were found to exhibit least among all tested treatments. It is evident from results that highest (1062.83) CAT activity was noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 61.43% increase as compared to control while Spd at 2.5 mM and Put at 2 mM resulted in 956.06 and 871.00 which correspond to 45.21 and 32.29% increase respectively. Similarly, in ‘Clear water’ cultivar highest CAT activity was observed in plants in which Spm at 0.01 mM (976.38), Spd at 1.5 mM (876.45) and Put at 3 mM (756.59) was applied that corresponded to 58.70, 42.45 and 22.97% increase, respectively.
1400 Apeldorn Clear Water
1200
1000
800
600
400
200 Catalase activity (Units/mg Protein) (Units/mg Catalase activity 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.15. Response of various concentrations of polyamines on Catalase activity of two tulip cultivars
182
4.4.16. DPPH Scavenging Activity (% inhibition) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.16 that tulip variety ‘Clear water’ performed comparatively better than ‘Apeldorn’ regarding DPPH ion scavenging activity. Results envisaged that various levels of polyamines induced different level of percentage inhibition while untreated plants were found to be least among all treatments. It is evident from results that highest percentage inhibition (84.09%) was noticed in variety ‘Clear water’ where Spm was applied at 0.02 mM, exhibiting 54.03% increase as compared to control while Spd at 0.5 mM and Put at 2 mM resulted in 76.89 and 69.20% which correspond to 40.84 and 26.75% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum scavenging activity was observed in plants in which Spm at 0.03 mM (82.08%), Spd at 2.5 mM (73.03%) and Put at 2 mM (58.24%) was applied that corresponded to 84.55, 64.20 and 30.95% increase, respectively.
100 Apeldorn Clear Water 90 80 70 60 50 40 30 20 10
DPPH Scavenging Activity inhibition)Activity (% Scavenging DPPH 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.16. Response of various concentrations of polyamines on DPPH Scavenging activity of two tulip cultivars
183
4.4.17. Malondialdehyde (MDA) contents (µmol g-1 fresh weight) Results depict significant (P ≤ 0.05) differences in treatment effect while varietal effect and their interaction were found to be non-significant. It is obvious from results in Fig. 4.4.17 that various levels of polyamines induced different levels of MDA contents while untreated plants exhibited highest levels of MDA in comparison to all tested treatments. It is evident from results that minimum quantity (0.93) of MDA contents were noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 46.86% decrease as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 1.08 and 1.34 which correspond to 38.35 and 23.45% decrease respectively. Similarly, in ‘Apeldorn’ cultivar minimum quantity of MDA contents was observed in plants in which Spm at 0.03 mM (1.00), Spd at 1.5 mM (1.19) and Put at 2 mM (1.39) was applied that corresponded to 36.14, 24.20 and 11.66% decrease, respectively.
Apeldorn Clear Water
2
fresh weight) fresh
1 -
1
µmol µmol g (
Malondialdehyde 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.17. Response of various concentrations of polyamines on Malondialdehyde contents of two tulip cultivars
184
4.4.18. Total Phenolic Compounds (TPC) (mg GAE/100g fresh weight) Results depict significant (P ≤ 0.05) differences for various treatments and varieties while their interaction was found to be non-significant. It is obvious from results in Fig. 4.4.18 that tulip variety ‘Apeldorn’ performed comparatively better than ‘Clear water’ by exhibiting more total phenolic compounds. Results envisaged that various levels of polyamines induced different levels of TPC while untreated plants were found to exhibit minimum of them among all treatments. It is evident from results that highest TPC (304.66) were noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 88.46% increase as compared to control while Spd at 1.5 mM and Put at 2 mM resulted in 264.56 and 238.73 which correspond to 63.66 and 47.68% increase respectively. Similarly, in ‘Clear water’ cultivar maximum TPC was observed in plants in which Spm at 0.01 mM (80.44), Spd at 0.5 mM (50.68) and Put at 3 mM (39.10) was applied that corresponded to 80.44, 50.68 and 39.10% increase, respectively.
350 Apeldorn Clear Water
300
250
mg mg GAE/100g
200
150 fresh weight) fresh 100
50
Total Phenolic Compounds ( Compounds Phenolic Total 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.18. Response of various concentrations of polyamines on Total Phenolic compounds of two tulip cultivars
185
4.4.19. H2O2 Scavenging Activity (%) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.19 that tulip variety ‘Clear water’ performed comparatively better than ‘Apeldorn’ by exhibiting maximum percentage of H2O2 scavenging activity. Results envisaged that various levels of polyamines induced different percentage scavenging while untreated plants were found to exhibit least among all treatments. It is evident from results that maximum scavenging (61.68%) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 104.60% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 51.97 and 41.33% which correspond to 72.39 and
37.09% increase respectively. Similarly, in ‘Apeldorn’ cultivar highest H2O2 scavenging was observed in plants in which Spm at 0.03 mM (51.37%), Spd at 2.5 mM (41.24%) and Put at 2 mM (32.58%) was applied that corresponded to 128.81, 83.68 and 45.12% increase, respectively.
70 Apeldorn Clear Water
60
50
40
30
Scavenging Activity (%) Scavenging 20
2 2
O 2
H 10
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.19. Response of various concentrations of polyamines on H2O2 Scavenging activity of two tulip cultivars
186
4.4.20. Days to flower bud emergence (d) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.20 that tulip variety ‘Clear water’ performed better than ‘Apeldorn’ in earliest emergence of flower buds. Results envisaged that various levels of polyamines induced different days to emerge flower bud while untreated plants exhibited late emergence among all treatments. It is evident from results that earliest emergence (34.46 d) was noticed in variety ‘Clear water’ where Spm was applied at 0.03 mM, exhibiting 2.45% decrease as compared to control while Spd at 2.5 mM and Put at 1 mM resulted in 42.8 and 51.2 d which correspond to 21.13 and 44.90% increase respectively. Similarly, in ‘Apeldorn’ cultivar earliest emergence was observed in plants in which Spm at 0.01 mM (36.8 d), Spd at 0.5 mM (44.86 d) and Put at 1 mM (51.8 d) was applied that corresponded to 5.15% decrease, 15.63 and 33.50% increase, respectively.
Apeldorn Clear Water
60
50
40
30
20
10
Days to flower bud emergence (days) emergence bud flower to Days 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.20. Response of various concentrations of polyamines on days to flower bud emergence of two tulip cultivars
187
4.4.21. Tepal diameter (mm) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.21 that tulip variety ‘Apeldorn’ performed better than ‘Clear water’ by producing larger tepal diameter. Results envisaged that various levels of polyamines induced different tepal diameters while untreated plants were found to exhibit shortest among all treatments. It is evident from results that largest tepal diameter (32.03 mm) was noticed in variety ‘Apeldorn’ where Spm was applied at 0.03 mM, exhibiting 54.56% increase as compared to control while Spd at 2.5 mM and Put at 2 mM resulted in 27.63 and 25.12 mm which correspond to 33.33 and 21.22% increase respectively. Similarly, in ‘Clear water’ cultivar maximum tepal diameter was observed in plants in which Spm at 0.01 mM (33.02), Spd at 2.5 mM (28.93 mm) and Put at 3 mM (22.12 mm) was applied that corresponded to 76.88, 54.97 and 18.48% increase, respectively.
40 Apeldorn Clear Water
35
30
25
20
15
Tepal diameter diameter (mm) Tepal 10
5
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.21. Response of various concentrations of polyamines on plant fresh mass of two tulip cultivars
188
4.4.22. Flower diameter (mm) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.22 that tulip variety ‘Clear water’ performed better than ‘Apeldorn’ by producing larger diameter of flowers. Results envisaged that various levels of polyamines induced different flower diameters while untreated plants were found to exhibit shortest among all treatments. It is evident from results that largest flower diameter (66.97 mm) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 49.93% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 59.99 and 52.12 mm which correspond to 34.31 and 16.68% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum diameter of flowers was observed in plants in which Spm at 0.03 mM (55.79), Spd at 2.5 mM (48.32 mm) and Put at 2 mM (44.21 mm) was applied that corresponded to 33.44, 15.57 and 6.22% increase, respectively.
Apeldorn Clear Water 80
70
60
50
40
30
Flower diameter (mm) diameter Flower 20
10
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.22. Response of various concentrations of polyamines on plant fresh mass of two tulip cultivars
189
4.4.23. Length of Petals (cm) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.23 that tulip variety ‘Apeldorn’ performed better than ‘Clear water’ by producing longest petals. Results envisaged that various levels of polyamines induced different petal lengths while untreated plants were found to exhibit shortest among all treatments. It is evident from results that longest petal length (10.90 cm) was noticed in variety ‘Apeldorn’ where Put was applied at 2 mM, exhibiting 68.03% increase as compared to control while Spm at 0.03 mM and Spd at 2.5 mM resulted in 9.24 and 7.64 cm which correspond to 42.54 and 17.88% increase respectively. Similarly, in ‘Clear water’ cultivar maximum petal length was observed in plants in which Spm at 0.01 mM (8.27 cm), Spd at 1.5 mM (7.44 cm) and Put at 3 mM (6.64 cm) was applied that corresponded to 51.71, 36.43 and 21.88% increase, respectively.
12 Apeldorn Clear Water
10
8
6
4 Length of Petals (cm) Petals Lengthof 2
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.23. Response of various concentrations of polyamines on Length of petals of two tulip cultivars
190
4.4.24. Width of Petals (cm) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.24 that tulip variety ‘Apeldorn’ performed better than ‘Clear water’ by exhibiting higher petal widths. Results envisaged that various levels of polyamines induced different widths while untreated plants were found to have least among all treatments. It is evident from results that largest width of petals (5.68 cm) was noticed in variety ‘Apeldorn’ where Put was applied at 2 mM, exhibiting 18.47% increase as compared to control while Spm at 0.03 mM and Spd at 2.5 mM resulted in 5.44 and 5.20 cm which correspond to 13.47 and 8.33% increase respectively. Similarly, in ‘Clear water’ cultivar maximum width was observed in plants in which Spm at 0.01 mM (5.41 cm), Spd at 1.5 mM (4.75 cm) and Put at 3 mM (3.99 cm) was applied that corresponded to 46.83, 28.87 and 8.31% increase, respectively.
8 Apeldorn Clear Water
6
4
Width of Petals (cm) Petals of Width 2
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.24. Response of various concentrations of polyamines on width of petals of two tulip cultivars
191
4.4.25. Scape diameter (mm) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.25 that tulip variety ‘Apeldorn’ performed better than ‘Clear water’ by exhibiting maximum scape diameter. Results envisaged that various levels of polyamines induced difference in scape diameters while untreated plants were found to possess the least among all treatments. It is evident from results that maximum (6.61 mm) scape diameter was noticed in variety ‘Apeldorn’ where Put was applied at 2 mM, exhibiting 30.15% increase as compared to control while Spm at 0.03 mM and Spd at 2.5 mM resulted in 5.71 and 6.09 mm which correspond to 12.45 and 19.88% increase respectively. Similarly, in ‘ Clear water’ cultivar maximum scape diameter was observed in plants in which Put at 3 mM (5.95 mm), Spm at 0.01 mM (5.54 mm) and Spd at 2.5 mM (5.14 mm) was applied that corresponded to 25.15, 16.56 and 8.17% increase, respectively.
Apeldorn Clear Water 7.5
6
4.5
3
Scape diameter (mm) diameter Scape 1.5
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.25. Response of various concentrations of polyamines on Scape diameter of two tulip cultivars
192
4.4.26. Flower quality (Copper and Spokas, 1991) Results depict significant (P ≤ 0.05) differences for various treatments and both the varieties while their interaction was found to be non-significant. It is obvious from results in Fig. 4.4.26 that tulip variety ‘Clear water’ performed better in rating than ‘Apeldorn’. Results envisaged that various levels of polyamines acquired different scores while untreated plants scored the least among all treatments. It is evident from results that maximum score (8.2) was ascertained by the variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 55.69% increase as compared to control while Put at 2 & 3 mM scored the same (5.8) and Spd at 1.5 mM scored 6.8 which correspond to 10.12 and 30.37% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum score was ascertained in plants in which Spm at 0.03 mM (7.4), Spd at 2.5 mM (6.0) was applied that corresponded to 117.64 and 78.43% increase respectively. Put at 2 & 3 mM got the same score (5) with 47.05% increase as compared to untreated plants.
10 Apeldorn Clear Water 9 8 7 6 5
Flower QualityFlower 4 3 2 1
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.26. Response of various concentrations of polyamines on Flower quality of two tulip cultivars
193
4.4.27. Vase life (d) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.27 that tulip variety ‘Clear water’ performed better than ‘Apeldorn’ by exhibiting maximum days for shelf life display. Results envisaged that various levels of polyamines induced increased vase life while untreated plants were found to possess least among all treatments. It is evident from results that prolonged vase life (13.2 d) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 100% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 11.6 and 9.8 d which correspond to 76.76 and 49.49% increase respectively. Similarly, in ‘Apeldorn’ cultivar maximum vase life was observed in plants in which Spm at 0.03 mM (12.73 d), Spd at 2.5 mM (10.33 d) and Put at 2 mM (45.83 d) was applied that corresponded to 98.95, 61.45 and 45.83% increase, respectively.
16 Apeldorn Clear Water 14
12
10 8
6 Vase life (days) Vase 4 2 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.27. Response of various concentrations of polyamines on vase life of two tulip cultivars
194
4.4.28. Images showing the response of various concentrations of polyamines on vase life display of two tulip cultivars
Fig. 4.4.28.1. Vase display of Apeldorn cultivar at 8th day (a-d), (e-g) at 13th day and (h-j) at 10th day after harvesting
Fig. 4.4.28.2. Vase display of Clear water cultivar at 8th day (a-d), (e-g) at 13th day and (h-j) at 11th day after harvesting
To: Control (Distilled water) (a), T1: Put 1mM (b), T2: Put 2 mM (c), T3: Put 3 mM (d), T4: Spm 0.01mM (e), T5: Spm 0.02 mM (f), T6: Spm 0.03 mM (g), T7: Spd 0.5 mM (h), T8: Spd 1.5 mM (i), T9: Spd 2.5 mM (j)
195
4.4.29. Days to start petal senescence (d) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction. It is obvious from results in Fig. 4.4.29 that tulip variety ‘Clear water’ performed better than ‘Apeldorn’ for retaining prolonged freshness ultimately late senescence. Results envisaged that various levels of polyamines induced different days to initiate senescence while untreated plants exhibited early senescence among all treatments. It is evident from results that delayed senescence (14.66 d) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 92.98% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 12.86 and 10.53 d which correspond to 69.29 and 38.59% increase respectively. Similarly, in ‘Apeldorn’ cultivar delayed senescence was observed in plants in which Spm at 0.03 mM (13.73 d), Spd at 2.5 mM (11.33 d) and Put at 2 mM (10.33 d) was applied that corresponded to 85.58, 53.15 and 39.63% increase, respectively.
20 Apeldorn Clear Water
16
12
8
4
Days to start petal senescence senescence petal start (days) to Days 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.29. Response of various concentrations of polyamines on days to start petal senescence of two tulip cultivars
196
4.4.30. Tepal abscission (d) Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties and their interaction while non-significant difference in varieties. It is obvious from results in Fig. 4.4.30 that various levels of polyamines induced different days to tepal abscission while untreated plants exhibited early abscission among all treatments. It is evident from results that delayed tepal abscission (16.66 d) was noticed in variety ‘Clear water’ where Spm was applied at 0.01 mM, exhibiting 93.79% increase as compared to control while Spd at 1.5 mM and Put at 3 mM resulted in 13.86 and 12.53 d which correspond to 61.24 and 45.73% increase respectively. Similarly, in ‘Apeldorn’ cultivar delayed abscission was noticed in plants in which Spm at 0.03 mM (15.73 d), Spd at 2.5 mM (13.33 d) and Put at 2 mM (12.33 d) was applied that corresponded to 51.28, 28.20 and 18.58% increase, respectively.
20 Apeldorn Clear Water
16
12
8
Tepal abscission (days) abscission Tepal 4
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.30. Response of various concentrations of polyamines on tepal abscission of two tulip cultivars
197
4.4.31. Mass of bulbils per clump (g) Results depict significant (P ≤ 0.05) differences for various treatments and both the varieties while their interaction was found to be non-significant. It is obvious from results in Fig. 4.4.31 that tulip variety ‘Clear water’ performed better than ‘Apeldorn’ by producing higher values for mass of bulbils per clump. Results envisaged that various levels of polyamines induced decreased values for bulbil mass while untreated plants were found to possess maximum among all treatments. Although maximum mass (19.16 g) in untreated plants next was noticed in variety ‘Clear water’ followed by (20.42 g) where Put was applied at 1 mM, exhibiting 6.17% decrease as compared to untreated plants while Spd at 2.5 mM and Spm at 0.03 mM resulted in 16.80 and 15.20 g which correspond to 17.69 and 25.53% decrease respectively. Similarly, in ‘Apeldorn’ cultivar untreated plants exhibited maximum mass (15.92 g) of bulbils per clump followed by Put at 1 mM (14.78 g), Spd at 0.5 mM (13.23 g) and Spm at 0.01 mM (8.61 g) was applied that corresponded to 7.16, 16.89 and 45.91% decrease, respectively.
30 Apeldorn Clear Water 25
20
15
10
5 Mass of bulbils per clump (g) clump bulbilsof per Mass
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.31. Response of various concentrations of polyamines on Mass of bulbils per clump of two tulip cultivars
198
4.4.32. Diameter of bulbils per clump (mm) Results depict significant (P ≤ 0.05) differences for various treatments while varietal effect and interaction was found to be non-significant. It is obvious from results in Fig. 4.4.32 that tulip variety ‘Clear water’ developed relatively larger diameter than in ‘Apeldorn’. Results envisaged that various levels of polyamines induced decreased values for bulbil diameter while untreated plants were found to possess maximum among all treatments. Although maximum diameter (38.40 mm) in untreated plants in tulip variety ‘Clear water’ was noticed followed by (35.33 mm) where Put was applied at 1 mM, exhibiting 8% decrease as compared to untreated plants while Spd at 2.5 mM and Spm at 0.03 mM resulted in 27.50 mm and 24.81 mm which correspond to 28.39 and 35.40% decrease respectively. Similarly, in ‘Apeldorn’ cultivar untreated plants exhibited maximum diameter (36.11 mm) of bulbils per clump followed by Put at 1 mM (33.75), Spd at 0.5 mM (28.05 mm) and Spm at 0.01 mM (25.04 mm) was applied that corresponded to 6.54, 22.32 and 30.64% decrease, respectively.
Apeldorn Clear Water 50
40
30
20
10
Diameter of bulbils per clump (mm) clump per bulbilsof Diameter 0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.32. Response of various concentrations of polyamines on Diameter of bulbils per clump of two tulip cultivars
199
4.4.33. Number of bulbils per clump Results depict significant (P ≤ 0.05) differences for various treatments in both the varieties while their interaction was found to be non-significant. It is obvious from results in Fig. 4.4.33 that tulip variety ‘Apeldorn’ performed better than ‘Clear water’ by producing more number of bulbils per clump. Results envisaged that various levels of polyamines induced decreased values for number of bulbils while untreated plants were found to possess maximum among all treatments. Although maximum number (4.6) next to control (4.4) was noticed in variety ‘Apeldorn’ where Put was applied at 1 mM, exhibiting 2.8% decrease as compared to control while Spd at 0.5 mM and Spm at 0.01 mM resulted in 3.2 and 2.4 which correspond to 28.98 and 46.37% decrease respectively. Similarly, in ‘Clear water’ cultivar untreated plants exhibited maximum number (3.73) of bulbils per clump followed by Put at 1 mM (3.6), Spd at 2.5 mM (3) and Spm at 0.03 mM (1.6) was applied that corresponded to 1.7, 19.64 and 55.35% decrease, respectively.
6 Apeldorn Clear Water
5
4
3
2
1 Number of bulbils per clump per bulbilsof Number
0
Polyamines levels (mM)
Every value in above figures is the mean of 3 replicates and vertical bars give standard error (SE) of means. HSD (Tukey Test) for cultivar and treatments were significant at P ≤ 0.05.
Fig. 4.4.33. Response of various concentrations of polyamines on Number of bulbils per clump of two tulip cultivars
200
Table 4.4 (a) Pearson’s correlation matrix among various morpho-physiological attributes of two tulip cultivars in response to Polyamines application
PH PFM PDM LA DFBE TD MBC BD NOB SV A E Ins WUE Int WUE Ci gs PFM 0.681** 1 PDM 0.515** 0.786** 1 LA 0.742** 0.703** 0.478** 1 DFBE -0.489** -0.432** -0.261** -0.460** 1 TD 0.672** 0.612** 0.548** 0.683** -0.509** 1 MBC -0.223** -0.325** -0.483** -0.284** 0.167** -0.362** 1 BD -0.512** -0.580** -0.443** -0.531** 0.328** -0.454** 0.227** 1 NOB -0.547** -0.502** -0.294** -0.564** 0.468** -0.513** 0.219** 0.378** 1 SV 0.565** 0.665** 0.679** 0.519** -0.363** 0.574** -0.398** -0.420** -0.397** 1 A 0.805** 0.770** 0.633** 0.793** -0.509** 0.703** -0.323** -0.573** -0.511** 0.625** 1 E 0.490** 0.626** 0.816** 0.487** -0.278** 0.579** -0.498** -0.437** -0.290** 0.621** 0.592** 1 Ins 0.618** 0.451** 0.138* 0.606** -0.346** 0.417** -0.006 -0.376** -0.388** 0.257** 0.762** -0.040 1 WUE Int 0.525** 0.506** 0.456** 0.500** -0.230** 0.409** -0.228** -0.415** -0.304** 0.363** 0.715** 0.397** 0.572** 1 WUE Ci 0.797** 0.726** 0.608** 0.768** -0.517** 0.703** -0.324** -0.547** -0.558** 0.577** 0.836** 0.614** 0.561** 0.571** 1 gs 0.419** 0.389** 0.279** 0.448** -0.335** 0.431** -0.190** -0.219** -0.334** 0.409** 0.435** 0.285** 0.313** -0.265** 0.417** 1 gm 0.772** 0.742** 0.614** 0.762** -0.447** 0.670** -0.313** -0.556** -0.469** 0.605** 0.988** 0.565** 0.777** 0.720** 0.756** 0.419**
Values indicate Pearson’s correlation coefficient; *significant (P ≤ 0.05); **highly significant (P ≤ 0.01);
PFM, Plant fresh mass; PDM, Plant dry mass; PH, Plant height; LA, Leaf area; SV, SPAD Value; DFBE, Days to flower bud emergence; TD, Tepal diameter; MBC, Mass of bulb clump; NOB, Number of bulbs; BD, Bulb diameter; A, Photosynthetic rate; E, Transpiration rate; Ins WUE,
Instantaneous water use efficiency; Int WUE, Intrinsic water use efficiency; Ci, Sub-stomatal CO2; gs, Stomatal conductance; gm, Mesophyll conductance.
201
Table 4.4 (b) Pearson’s correlation matrix among various antioxidant enzymes activity and biochemical changes induced on floral attributes of two tulip cultivars in response to Polyamines application
FD LP WP SD FQ VL DSPS TA SOD POD CAT DSA MDA TPC LP 0.009 1 WP 0.043 0.540** 1 SD -0.228** 0.407** 0.201** 1 FQ 0.351** 0.050 0.054 -0.083 1 VL 0.601** 0.168** 0.209** -0.129* 0.327** 1 DSPS 0.607** 0.227** 0.266** -0.059 0.291** 0.902** 1 TA 0.548** 0.244** 0.301** -0.006 0.302** 0.916** 0.925** 1 SOD 0.326** 0.266** 0.314** 0.039 0.269** 0.535** 0.515** 0.561** 1 POD 0.485** 0.207** 0.181** -0.124* 0.310** 0.508** 0.514** 0.494** 0.351** 1 CAT 0.285** 0.285** 0.376** 0.032 0.182** 0.478** 0.473** 0.491** 0.674** 0.350** 1 DSA 0.681** 0.088 0.167** -0.134* 0.383** 0.706** 0.669** 0.643** 0.614** 0.499** 0.555** 1 MDA -0.470** -0.158** -0.248** 0.046 -0.237** -0.519** -0.495** -0.500** -0.423** -0.361** -0.351** -0.539** 1 TPC 0.103 0.388** 0.443** 0.234** 0.169** 0.420** 0.431** 0.490** 0.550** 0.285** 0.373** 0.389** -0.320** 1 HSA 0.742** 0.013 0.104 -0.167** 0.344** 0.624** 0.617** 0.574** 0.397** 0.466** 0.354** 0.705** -0.468** 0.226**
Values indicate Pearson’s correlation coefficient; *significant (P ≤ 0.05); **highly significant (P ≤ 0.01);
FD, Flower diameter; VL, Vase life; LP, Length of petals; WP, Width of petals; FQ, Flower quality; SD Stem diameter; DSPS, Days to start petal senescence; TA, Tepal abscission; SOD, Superoxide dismutase activity; CAT, Catalase activity; DSA, DPPH ion scavenging activity; POD, Peroxidase activity; MDA, Malondialdehyde contents; TPC, Total Phenolic Contents; HSA, H2O2 Scavenging Activity
202
Chapter # 5 Discussion
Tulips possess powerful verdancy echoing with rich colors are produced commercially for cut flower markets across the globe but its productivity is jeopardized with abrupt environmental changes coined with malicious abiotic factors. Unfavorable environment is a serious threat for any crop that impedes its growth and productivity. Therefore studies are needed to be carried out regarding the response of exotic and new plant species in diversified climatic zones and induce adaptability to support in expressing their full genetic potential. A comprehensive research was planned for tulips that demonstrated its response in terms of various aspects of growth, physiology and enzymatic activity when grown in open field under Faisalabad, Pakistan conditions. The outcome of these findings will play a pivotal role to overcome the harmful effects caused by abiotic environmental factors on flower quality of cut tulips, because the cultivars screened in these studies will be recommended to the grower community that can successfully be cultivated in subtropical zones of Pakistan. Moreover, the application of chitosan, glycine betaine and different polyamines proved to be an effective tool in inducing adaptability and combat harmful effects of environment. The findings of present study are discussed here in the light of evidences in relation to other plants.
Experiment # 1
The grower community, have little knowledge concerning unaccustomed, non-native and new crops for cultivation because of their unacquaintance with their adaptability response. Each potential cultivar needs evaluation in the light of target market, consumer demand, and sale potential. Convenience in production, harvesting, and handling methods are of deep concern in this regard. Moreover, the crop’s adaptability to local growing conditions, disease resistance, vase-life and flowering patterns are needed to be addressed in precise paraphernalia according to prevailing conditions. Developing new introductions, as well as old favorites, increases the market appeal but it is hard to alter parochial approach of growers towards new crops and to provide a due status of them until complete production potential is accepted by the grower community. Crop’s production expenditure, especially seed cost and comparison of estimates with their market value and expected revenue are of foremost consideration from grower point of view. Cut stems of bulbous crops particularly tulips have usually shorter vase life and long
203 distance transportation further reduces it. Therefore, it is easier to break this high value crop into local markets, particularly wholesale florists, where there exists a niche for rare cut flowers in the market at competitive price.
In this experiment, growth potential of 10 cultivars against their adaptability response was studied. The parameters like time to start sprouting, time to reach 50% sprouting, mean emergence time, sprouting percentage, emergence index and other morphological parameters like plant height, fresh mass, leaf area, chlorophyll contents days to flower bud emergence, tepal diameter, stem diameter and vase life were focused to estimate their growth potential and adaptability. It was found that all cultivars responded differently under Faisalabad conditions which explained the fact that cultivars are environment specific in their growth performance and potential under different environmental conditions, the results of this finding second to that of Ahmad et al. (2013) who reported the trend of adaptability of various gladiolus cultivars. It was further observed that earlier sprouting of bulbs was recorded as indicated by lower values of E50 and MET in the bulbs of Apeldorn, Golden Apeldorn and Ile de France cultivars (Fig. 4.1.2 and 4.1.3). However, the higher EI and FEP indicated the sprouting spread and final emergence percentage over time (Fig. 4.1.5 and 4.1.4). This study revealed that some cultivars responded in earlier and more uniform sprouting and emergence as indicated by lower MET, E50, higher EI and higher FEP that aggravated the shorter life cycle that was more conducive in lower temperature duration in the compendious winter season that prevails in experimental site.
The results of this study indicated highly significant differences among varietal response towards other growth characteristics in contrast to the sprouting pattern of tulip bulbs. The longest plant height was exhibited by Barcelona followed by Parade and Ile de France while the shortest plant height was ascertained by Purple flag followed by Queen of night, Spring green, and Golden apeldorn. Varieties with more growth rate than others depict those longer plant heights could be due to the low temperature exposure that was sufficient enough for their growth and development. On the other hand, the varieties that exhibited shorter heights may depend upon further lower temperature needed to cater their optimized growth and development process. Similar findings are reported by Shafique et al. (2011) when they tested different cultivars of Snapdragon for cut flower production. Some cultivars superseded for vegetative growth and developed quality flower spikes and proved their superiority over other cultivars. This explains
204 that the varieties with more growth have ability to acclimate in the temperature regimes of Faisalabad conditions. There exists strong positive correlation of plant height with leaf area and SPAD value while negative correlation of plant height was found in days to flower bud emergence although highly significant and positive correlation existed between flower diameter and scape diameter (Table 4.1). Similarly, positive but weak and highly significant correlation was found between flower quality and days to initiate petal senescence that depict growth and quality indices are interlinked traits and cultivars that are capable of maintaining net carbon assimilation under particular environmental conditions could have potential to envisage better adaptability. Plant fresh mass is an ultimate indicator of growth and quality indices based on biomass production, influenced by favorable environment conditions best suited for particular plant species. Higher fresh mass were seen in cultivar Parade followed by Leen Vander Mark, Barcelona and Clear water, while the least values for plant fresh mass was found in Queen of night followed by Golden apeldorn, Purple flag and Spring green. Plants when grown in adapted and suitable environment undergo more mitotic division and other metabolic processes at exacerbated rates giving them vigorous growth and homeostatic conditions for development. Higher fresh mass is an index that depicts growth vigor and overall quality status for a particular crop plants especially for cut flower crops. Similarly, the plants that exhibited higher fresh mass had the higher dry matter contents which clearly reveals that they have developed more structural entities providing with adequate spatial structures and conjugated molecules like proteins, fats and other assimilates that induced quality parameters associated to acclimation process.
This trend is further advocated by the Pearson’s correlation matrix among various morphological attributes. There existed strong, positive and highly significant correlation between plant fresh mass and dry mass, while other morphological traits had also shown the similar trend i-e highly significant positive correlation (Table 4.1) except for days to flower bud emergence and plant fresh mass where negative but moderate and highly significant correlation existed between them. These findings indicate that more the plant fresh mass earlier will be the flower bud emergence and crop will complete its life cycle in lesser time ultimately the harvest will be achieved in minimum time. As in the said location, winters are short duration so shorter the life cycle; the better quality produce will be achieved. On the other hand tulip cultivars that
205 had lesser fresh mass may develop into weaker stems that may not be able to fetch the due status in the market, as the florists as well as the consumer may reject them for being unappealing.
Leaves are the principal or presumably the largest factories on the earth with maximum outcome of its produce synthesized by the process of photosynthesis in the form of food and its translocation in respective organs. In cut flower crops photosynthates induce strong source-sink relationship that is particularly seen by the overall structural appearance of flower heads. So, leaf area is of pivotal importance that is desirous for optimum photosynthetic rate. Cultivars like Parade, Leen Vander Mark, Ile de France and Barcelona had maximum dimensions for leaf area and highest SPAD values. This further resulted in increased dimensions for flower, scape, length and width of petals and ultimately the prolonged display life. There existed positive and highly significant correlation between leaf area and other floral attributes with improved features. On the other hand, the cultivars like Purple flag, Queen of night and Spring green exhibited smaller leaf area and had negatively influenced the floral traits. In this context, chlorophyll might have played an important role in harvesting light for the process of photosynthesis. So their SPAD values must be at optimized levels that are best suited for adequate metabolic rate. Highest SPAD value was noticed in Leen Vander Mark cultivar followed by Barcelona, Ile de France and Parade, that had positive correlation with other morphological and floral attributes.
Days to flower bud emergence decides the harvesting time for tulip cut flower that ranged more or less between 5 to 10 days after its emergence. Earliest the flower bud emergence early will be the harvesting so, the tulip crop shall have completed and ascertained the harvesting stage in exposure to low temperature conditions. These findings are in concomitant with those of Noy- Porat et al. (2009) who reported that flower initiation and development require a particular duration of low temperature exposure. Although some geophytes develop flowers within the underground storage organs particularly in bulbs during summer quiescence period hence they don’t need low temperature for florogenesis. This low temperature is highly favorable to achieve the quality produce meeting the market standards when grown in subtropical regions where winters are short duration. In this regard cultivar Parade exhibited earliest flower bud emergence followed by Ile de France, Leen Vander Mark and Barcelona. Contrary to this trend, cultivars like Purple flag, Queen of night, Spring green and Golden apeldorn responded in another way where late emergence of flower bud was observed. Optimal temperature for induction of flower
206 meristem at for floral organogenesis at initial phases vary between 9-25 °C, in the autumn, by a week period of lower temperature (4-9 °C) needed for stem elongation (Khodorova and Boitel- Conti, 2013). The absence of low temperature at optimized levels might have led to slow shoot growth in spring and severe flowering disorders So, the cultivars in which flower bud emergence was delayed had to mature in relatively higher temperature regimes that hinder optimum growth and development. This finding is further supported by moderate, negative but highly significant correlation that existed between the days to flower bud emergence and other attributes like scape length, leaf area and SPAD value and bulbils diameter. Moreover, late flower bud emergence resulted in diminished tepal and flower diameter, length and width of petals that negatively influenced the flower quality rating. Consequently, these attributes shortens the display life by causing early senescence and abscission of tepals. This trend is unacceptable from growers, florists and consumer’s points of view who are interested in early maturation, prolonged storage life and sustained freshness accompanied with longest display life. These findings are supported by correlation matrix as explained in (Table 4.1).
In their native regions tulips perform a highly influential pattern regarding production potential of perrenating organs in a more pronounced and normal way. As the bulbs are curious plants, while other plants go dormant during winters, bulbs are actually growing underground. Winter is when they send their roots deep into the soil and begin to sprout. Then, in earliest spring, while all the other plants are just beginning to stir, bulbs burst into bloom. Unfortunately, shorter winter duration and higher temperature regimes in very early spring in subtropical terrain aggravates the agony of weak development of parrenating structures. In most of the geophytes when summer arrives, other plants come into their prime in the abundant sunlight, but the bulbs lose their leaves and go fully dormant. Tulip bulbs proved to be highly sensitive in this regard when grown in subtropical region. They failed to develop adequate mass, diameter and number of bulbils in this geographic proximity. It was further noticed that the cultivars which performed extravagantly regarding their aerial structures responded in an inverse trend for bulbils attributes.
Growers are keenly interested in the quality of crop as well as in seed quality that can be used for the next year to save the seed cost. Tulips when grown in subtropical climate were found to be successful for cut flower production for some better adapted varieties but none of them was successful enough to develop useable storage organs. They were either inferior in
207 quality or remained under developed. Though, Cultivars like Leen Vander Mark, Ile de France, Clear water and Parade developed relatively more number of bulbils yet they were premature enough to be used in the next season to achieve a commercial crop while conditioning lead to rotting because of higher temperature. Singh and Dohare (1994) reported the similar response in gladiolus crop in which low rate of multiplication of propagules and higher corm spoilage resulted in insufficient availability of planting material. Similarly, results regarding bulbils diameter revealed that the cultivar Apledorm developed the maximum diameter of bulbils followed by Ile de France, Leen Vander Mark and Clear water, yet they were not large enough to be used in the next season to achieve the commercial crop. Similarly highest bulb mass was observed in Clear water followed by Golden apeldorm, Barcelona, Apeldorm, Leen Vander Mark and Parade. It was found that they had insufficient energy reserves to develop a uniform and healthier crop stand in the succeeding year. These results are in line with the findings of Memon et al. (2009) who reported that cormel mass varies from variety to variety in gladiolus crop. Current findings are further supported by correlation matrix (Table 4.1) among various attributes that depicted weak positive correlation between plant fresh mass, leaf area, SPAD value against bulbils attributes while negative and highly significant relationship was found between days to flower bud emergence and mass of bulbils. Similar trend is observed in number of bulbils and diameter of bulbils as well that accounts for lower quality of bulbils when grown in subtropical climate. Conclusively, all phases of growth and development process in tulips are highly regulated by seasonal thermoperiodicity. These findings clearly depict that variations in cultivars response are temperature dependent that regulated the growth cycle. We can certainly develop further inferences by considering the growth response of other geophytes that show temperature dependent growth patterns. However, more keen research on other tulip cultivars is needed to address complete understanding in the correlation between seasonal thermoperiodicity and growth cycle.
Experiment # 2
In this experiment, five cultivars of tulip namely, Apeldorn, Barcelona, Ile de France, Leen Vander Mark and Parade were tested against various concentrations of exogenous application of chitosan (30,60,90,120 and 150 mgL-1) along with control in which distilled water
208 was foliarly applied in open field conditions. The objective of this experiment was to test the response of these cultivars against various chitosan levels to achieve the optimized dose best suited for particular cultivar that have enhanced the growth and quality attributes of tulips for cut flower production. The results revealed significant differences among the tested cultivars and levels of chitosan. The parameters like plant height, plant fresh mass, dry mass, leaf area and other physiological, biochemical and antioxidant enzyme activity was significantly increased differently in different cultivars.
The study indicated that for the cultivar Apledorn optimized level for the above mentioned parameters was found to be 90 mgL-1 of chitosan that improved all morphological, gaseous exchange attributes and other biochemical features. Beyond and below this level a declining trend in these traits were found. Similarly in the cultivar Barcelona relatively low level of chitosan at 30 mgL-1 was found to be optimum. At higher levels this cultivar exhibited negative response in achieving the quality produce. In the cultivar, Ile de France it was found to be compatible even at higher doses of chitosan and at 120 mgL-1, it exhibited the optimized performance that ended in a decline beyond that level, while a consecutive increasing trend was found to attain the optimum level for various attributes studied. For the cultivar Leen Vander Mark, the optimized level was found to be 60 mgL-1 of chitosan. Beyond this level attributes started to depict the declining trend. The cultivar Parade has depicted entirely the different response against various levels of chitosan and optimized level was found to be at 150 mgL-1. This cultivar was found to be highly compatible even at higher levels of chitosan it may start to show declining trend beyond this level and recommended to be tested against them. A steep inclining trend was observed till the optimized level had been achieved.
The above mentioned findings of all the morphological attributes are coined with other physiological attributes like photosynthetic rate, SPAD value, transpiration rate, instantaneous WUE, intrinsic WUE, Ci, gs and gm. There existed a weak, positive and highly significant correlation amongst these physiological attributes with that of plant height, fresh and dry mass and leaf area (Table 4.2 a). Some evidences revealed that photosynthetic rate and stomatal conductance of leaves are correlated across diversified environmental conditions. The correlation between A and gs has led to postulate them as a "messenger" from mesophyll that controls the stomatal behavior. This is so because (A) is a function of sub-stomatal CO2 (Ci) concentration
209 which is in turn a response of gs, such a correlation may be partially mediated by Ci if (gs) is to some extent an independent variable (Radin et al., 1988). In cultivar Apeldorn, the optimized levels of chitosan at 90 mgL-1 also improved biochemical attributes like reduced levels of MDA contents while higher levels of the antioxidants like SOD and CAT while POD and TPC at 60 mgL-1 were also exhibited their maximum activity at this level. Moreover, in this cultivar maximum percentage inhibition of DPPH ions scavenging was found at relatively lower level of chitosan i.e at 30 mgL-1. Although -1 the highest scavenging of H2O2 inhibition percentage was also found at 90 mg L , depicting that SOD and CAT activity was proved to be more efficient in inducing higher quality parameters. The levels of antioxidant vary during development, senescence and in many biochemical systems as well as they bring about variations in molecular mechanisms that activates to counteract the elevated level of reactive oxygen species and free radicals and there exists a peculiar correlation between these antioxidants and senescence process (Cavaiuolo et al., 2013). In cultivar Barcelona, relatively lower level of chitosan at 60 mgL-1 showed maximum activity of SOD, POD, TPC and DPPH, while maximum activity of CAT and H2O2 inhibition percentage was also observed at this level that decreased further beyond this level. This trend shows the relationship that higher activity of CAT increased the H2O2 scavenging that was involved to inducing better quality in this cultivar. This could be due to the fact that when the experiences stressful conditions, activities of antioxidant enzyme increases that results in improved cellular protection (Kaur et al., 2009). In cultivar Ile de France, chitosan at 120 mgL-1 was proved to be maximum in TPC, SOD and CAT activity while POD at 90 mgL-1 had its maximum activity followed by the optimized level. Percentage scavenging of DPPH ion was proved to be in consistent in this regard where it had more or less same percentage inhibition -1 found in at 60 mgL and untreated plants. The scavenging of H2O2 was found to be maximum at optimized level that is 120 mgL-1 that makes this concentration the most influencing and adequate level for this cultivar.
Similarly in Leen Vander Mark the optimized level of chitosan was found to be at 90 mgL-1 at which the maximum enzyme activity of CAT and POD was seen accompanied with TPC and maximum H2O2 scavenging at extremely lowered contents of MDA were observed at this level, while SOD activity and percentage inhibition of DPPH ions scavenging was found at 60 mgL-1. In this cultivar, the activity of all the enzymatic antioxidants like SOD, POD and
210
CAT were found to be maximum at 120 mgL-1 while non enzymatic antioxidants like TPC also performed at peak. While the maximum percentage inhibition of DPPH ions scavenging was at its highest level at relatively lowered level of chitosan i.e 120 mgL-1 followed by the optimized level that is 150 mgL-1 of chitosan application in which the maximum scavenging activity of H2O2 and the minimum occurrence of MDA contents was also found supported by the correlation matrix (Table 4.2 b). This could be due to the fact that free radicals are generated when cells consume oxygen to generate energy. These by-products are usually ROS such as super oxide anion, hydroxyl radical and hydrogen peroxide that result from the cellular redox process. At low or moderate concentrations, ROS exert beneficial effects on cellular responses and immune function but at high levels, free radicals and oxidants generate oxidative stress, a deleterious process that can damage cell structures, including lipids, proteins, and DNA (Pham- Huy et al., 2008).
In the above mentioned discussion the optimized levels also possessed the higher quality parameters regarding flower morphology that was found to be showing their desired features for appreciable crop response from grower, florist and consumer point of view who are interested in the prolonged postharvest survival of cut tulips. The correlation matrix (Table 4.2 a) among different morphological and physiological attributes further affirms that tepal diameter is positively correlated with SPAD value, photosynthetic rate, instantaneous WUE, stomatal conductance while tepal diameter was found to be negatively correlated to E, gm and intrinsic WUE. Similarly, days to flower bud emergence was found to be positively correlated with transpiration rate intrinsic WUE and gm while negatively correlated with A and Ci. Moreover, the SPAD value was found to be positively correlated with gm and A. Furthermore, E had negative correlation with Ci and instantaneous WUE possessed by this cultivar (Table 4.2 a).
Floral attributes are of greatest concern in any cut flower crop with improved and extra ordinary traits regarding their quality. In this study it was moreover found out that antioxidants either enzymatic or non-enzymatic and biochemical attributes had the influence on all morphological attributes relative to flower response in its postharvest life span. It was found that there existed positive correlation between flower diameters, vase life, petal length, petal width with associated flower quality parameter while it was observed that flower diameter proved to be negatively correlated with scape diameter. There existed strongly positive and highly significant
211 effect of SOD, CAT and POD activity with all attributes related to flower morphology and postharvest longevity. On the other hand negative correlation of scape diameter with DPPH and POD activity was found. A very interacting finding was achieved in this study was negative correlation of MDA contents with floral morphology and its postharvest longevity attributes.
Furthermore negative correlation of MDA contents with TPC and H2O2 scavenging activity was also found in this experiment where chitosan was foliarly applied on different cultivars of tulip (Table 4.2 b).
In this experiment bulbils proved to be unresponsive of chitosan application and no precise or reliable results were obtained when grown in this region. They failed to develop adequate mass, diameter and number of bulbils in this geographic proximity this might be due to inadequate duration of cold temperature needed for conditioning process. Prolonged low temperature exposure of tulip bulbs simultaneously induces the stimulation of shoot growth and bulbing in axillary buds. Moreover, faster elongation of stem is also associated with more rapid bulbing of axillary buds (Le Nard and De Hertogh, 1993). Thus, extended duration of cold results in rapid expression of bulbing (Aoba, 1976; Le Nard and De Hertogh, 1993). Bulbs were lifted up two months later after the harvesting of crop. In Apeldorn cultivar low level of chitosan at 30 mgL-1 exhibited maximum diameter while mass of bulbils was found to be at highest in untreated plants. Moreover, the number of bulbils was found maximum at higher levels of chitosan at 120 mgL-1. Similar trend was observed in Barcelona cultivar in which the bulbils of untreated plants possessed maximum diameter while the mass and number of bulbils was highest 30 and 90 mgL-1 respectively. In Ile de France cultivar highest diameter and mass of bulbils was found in much higher doses of chitosan at 120 mgL-1 and number of bulbils was found to be highest in 90 mgL-1. Flavonoids, mainly quercetin and kaempferol, as endogenous auxin transport inhibitors (Murphy et al., 2000), inhibitors of lipid peroxidation (Alcaraz et al., 1986; Torel et al., 1986), strong antioxidants (Pietta, 2000), and substrates for peroxidase (Takahama and Oniki, 2000) plays an important role in many physiological processes, including dormancy release and daughter-bulb enlargement in mother tulip bulbs as a result of low temperature treatment.
In Leen Vander Mark cultivar diameter and mass was at its maximized level in untreated plants and at lower level of chitosan application at 30 mgL-1 respectively. In cultivar Parade
212 maximum diameter and mass of bulbils was ascertained by untreated plants while number of bulbils was found to be highest in relatively higher levels of chitosan at 90 mg L-1. Limited information is available regarding hormonal control of daughter bulb development in field- grown tulips. Aung and Rees (1974) determined the changes in the endogenous gibberellins content of the developing bulblets derived from field-grown bulbs cv. Apeldoorn. They showed that the gibberellin activity of bulblets increased dramatically in November-March and declined sharply in April-July. The increase in gibberellin-like substances was considered to be derived primarly by synthesis within the bulblets with possible contributions via translocation from the mother bulb scales, roots, and shoots.
Experiment # 3
In this experiment, five cultivars of tulip namely, Apeldorn, Barcelona, Ile de France, Leen Vander Mark and Parade were tested against various concentrations of exogenous application of glycine betaine (30, 60, 90, 120 and 150 mM) along with control in which distilled water was foliarly applied in open field conditions. The objective of this experiment was to test the response of these cultivars against various levels of glycine betaine to achieve the optimized dose best suited for particular cultivar that had potential to enhance the growth and quality attributes of tulips for cut flower production under sub-optimal environmental conditions to support adaptability response.
The results revealed significant differences among the tested cultivars and levels of glycine betaine application. The parameters like plant height, plant fresh mass, dry mass, leaf area and other physiological, biochemical and antioxidant enzyme activity was significantly increased differently in different cultivars when studied in detail. The longest plant height was observed in the cultivar Parade at 90 mM when glycine betaine was exogenously applied. Similarly this level also exhibited the maximum leaf area and dry matter contents, contrary to dry mass, the highest quantity of plant fresh mass was found in relatively lower level at 60 mM of GB. In this cultivar highest photosynthetic rate, transpiration rate, instantaneous and intrinsic WUE and mesophyll conductance were at their peak when GB was applied at 90 mM except for sub-stomatal CO2 and stomatal conductance that had their maximum values at relatively lower levels of GB application 60 mM GB . This depicted that stomatal conductance decreased with
213 high sub-stomatal CO2 and photosynthetic rate. This trend is in line with Fick’s first law of diffusion in which net photosynthetic flux at steady rate (A) was maximum and expressed as:
A = gs (Ca-Ci) = gm (Ci-Cc)
Where: Ca is atmospheric CO2; Cc is chloroplast stroma CO2 and Ci is sub-stomatal CO2.
Similarly, GB at 60 mM exhibited maximum tepal diameter and width of petals. Although, other floral attributes (flower diameter, length of petals, scape diameter, flower quality, vase life, days to start petal senescence and tepal abscission) performed best at 90 mM of GB. Moreover, enzymatic antioxidants like SOD and POD had their highest activity at this level of GB while
CAT exhibited its maximum activity at 120 mM of GB likewise highest percentage of H2O2 scavenging with respect to CAT. Hence, it was also found that minimum quantity of MDA contents was also found 90 Mm GB and highest percentage inhibition of DPPH ion scavenging was maximum at 60 mM GB. In the view of above evidences and discussion, 90 mM GB can be presumed as the optimized dose of GB for this cultivar that performed best to make it better adapted in the environmental conditions of Faisalabad.
In Apeldorn cultivar 60 mM GB application exhibited maximum plant height while 90 mM GB of this substance gave the maximum dry and fresh mass. Similarly, all the gaseous exchange attributes (Photosynthetic rate, transpiration rate, instantaneous WUE, intrinsic WUE, sub stomatal CO2 and SPAD value) were at their highest level when GB was applied as 60 mM. This trend depicts that at this particular level, attributes of gaseous exchange and morphological traits responded at an adequate level. Although fresh and dry matter contents at relatively higher level (90 mM GB) improved them. Moreover, the activity of enzymatic antioxidant (SOD) was also at its peak at 60 mM GB while CAT and POD activity was maximum at 120 mM of GB. On the other hand, non-enzymatic antioxidant like TPC and DPPH ion scavenging activity had their maximum percentage inhibition at 90 mM of GB. Although H2O2 scavenging activity had its maximum impact at 120 mM GB corresponded to CAT activity. This shows that, the activity of
CAT in context to H2O2 scavenging was at par.
The floral attributes like (tepal diameter, flower diameter, stem diameter) were highest at 90 mM while, the parameters like length of petal, flower quality, vase life, days to start petal senescence and tepal abscission had their adequate performance at 60 mM GB. So, this can be
214 inferred that 60 mM GB is found to be an optimized dose for this cultivar. Similarly, days to flower bud emergence were also earliest at this level. This was further depicted in negative correlation with LA, DM, FM and PH (Table 4.3 a). Moreover, negative correlation of days to flower bud emergence is also seen with tepal diameter and SPAD value as well. An entirely inconsistent trend of GB application was found in bulbous attributes, where it performed inadequate performance regarding mass of bulbils that were highest in untreated plants and in 120 mM GB. Likewise, number and diameter of bulbils was at their maximal potential for 90 and 120 mM GB respectively.
In Barcelona cultivar, 30 mM GB application exhibited maximal plant height, dry mass and fresh mass. Similarly, all the gaseous exchange attributes (Photosynthetic rate, transpiration rate, stomatal conductance, sub stomatal CO2, mesophyll conductance and SPAD value) were at their highest level when 30 mM GB was applied as foliar spray except for instantaneous WUE, instantaneous WUE, intrinsic WUE and transpiration rate had their optimal response at relatively higher level (60 mM) of GB. This trend depicts that at this specific levels of gaseous exchange attributes plants morphological traits were responded at an adequate level. Moreover, enzymatic antioxidants (CAT and POD) were also at their peak at 60 mM GB while SOD activity was maximum at 30 mM. On the other hand, non-enzymatic antioxidant like TPC and DPPH ion scavenging activity had their maximum percentage inhibition at 30 mM of GB. Although, H2O2 scavenging was found to be maximum at 30 mM GB, while the minimum MDA contents were also found at this concentration. This trend is further affirmed by negative correlation of MDA with TPC and H2O2 scavenging activity.
The floral attributes like (tepal diameter, flower diameter, stem diameter, length of petal, flower quality, vase life, days to start petal senescence and tepal abscission) were highest at 30 mM GB. So, this can be inferred that 30 mM GB is found to be an optimized dose for this cultivar. Similarly, days to flower bud emergence were also earliest at this level of GB application. This is depicted in negative correlation of this parameter with all gaseous exchange attributes except for E in which positive correlation was occurred (Table 4.3a). Moreover, negative correlation of days to flower bud emergence is also seen with tepal diameter and SPAD value as well. An entirely inconsistent and non-precise trend of GB application was found in bulbous attributes, where it performed inadequate performance regarding mass of bulbils that
215 were highest in untreated plants and in 150 mM GB. Likewise, number and diameter of bulbils was at their maximal potential for 60 and 120 mM GB respectively.
In Ile de France cultivar 90 mM GB application exhibited maximal plant height, dry mass and leaf area while fresh mass was at its peak at relatively lower level (30 mM) of GB. Similarly all the gaseous exchange attributes (Photosynthetic rate, transpiration rate, instantaneous WUE, stomatal conductance and mesophyll conductance) were at their highest level when 30 mM GB was applied as foliar spray except for intrinsic WUE, sub-stomatal CO2 and SPAD value had their optimal response at 90 mM GB. This trend depicts that at this specific levels of gaseous exchange attributes plants morphological traits were responded at an adequate level. Moreover, the activity of enzymatic antioxidants (CAT and SOD) was also at their peak at 90 mM GB while POD activity was maximum at 60 mM. On the other hand, non-enzymatic antioxidant like
TPC, DPPH and H2O2 ion scavenging activity had their maximum percentage inhibition at 90 mM of GB. Same response in acquisition of MDA contents at minimal level was also exhibited at this concentration of GB. Likewise this trend is further affirmed by negative correlation of MDA with petal length and width, scape diameter and flower quality and positive correlation with days to senescence, vase life and tepal abscission. It is also observed that enzymatic antioxidants had negative but highly significant correlation with MDA (Table 4.3 b).
The floral attributes like (tepal diameter, flower diameter, stem diameter, length of petal, flower quality, vase life, days to start petal senescence and tepal abscission) were highest at 90 mM GB while, the width of petal had adequate performance at 30 mM. So, this can be inferred that 90 mM GB is found to be an optimized dose for this cultivar. Similarly, days to flower bud emergence were also earliest at this level. This is depicted in negative correlation with all gaseous exchange attributes except for E in which positive correlation was found (Table 4.3 a). Moreover, negative correlation of days to flower bud emergence also existed with tepal diameter and SPAD value. An entirely inconsistent trend of GB application was found in bulbils attributes, where it performed inadequate performance regarding mass and diameter that were found to be highest in untreated plants. Although, number of bulbils exhibited their maximal potential for 60 mM of GB application. This response is further affirmed by negative correlation of LA, DM, FM and PH with diameter of bulbils. Similarly, all the morphological attributes had the negative correlation with mass of bulbils as well.
216
In Leen Vander Mark cultivar, 120 mM GB application exhibited maximal plant height, dry mass, leaf area and fresh mass. Similarly, all the gaseous exchange attributes (Photosynthetic rate, transpiration rate, instantaneous WUE, mesophyll conductance, intrinsic WUE, sub stomatal CO2 and stomatal conductance) were at their highest level when 120 mM GB was applied as foliar spray except for SPAD value that had optimal response at 90 mM GB.
Moreover, enzymatic antioxidants SOD, CAT and POD were at their maximal activity at 120, 90 and 60 mM GB. On the other hand, non-enzymatic antioxidant like TPC and DPPH showed their maximum activity at 30 and 60 mM GB respectively. Similarly, H2O2 scavenging activity and percentage inhibition of DPPH ion scavenging had their maximum values at 90 mM of GB application along with lowest levels of MDA contents at this concentration. This trend is further affirmed by negative correlation of MDA with petal length and width, scape diameter and flower quality, while, positive correlation with days to senescence, vase life, tepal abscission and flower diameter was also noticed at this concentration. It is also observed that TPC and H2O2 scavenging activity had highly significant but negative correlation with MDA.
The floral attributes like (tepal diameter, flower diameter, width of petal, stem diameter, length of petal, vase life, days to start petal senescence and tepal abscission) were highest at 60 mM GB, while, flower quality acquired highest ranking when 120 mM GB was applied. So, this can be inferred that 120 mM GB is found to be an optimized dose for this cultivar. Similarly, days to flower bud emergence were also earliest at this level. This depicted negative correlation with all gaseous exchange attributes except for E in which positive correlation was exhibited (Table 4.3 a). Moreover, negative correlation of days to flower bud emergence was also occurred with tepal diameter and SPAD value. An entirely inconsistent trend of GB application was found in bulbous attributes, where it performed inadequate performance regarding mass and diameter of bulbils that were highest in untreated plants and at 120 mM GB. Although number of bulbils show their maximal potential at 90 mM GB. This is further affirmed by negative correlation of LA, DM, FM and PH with diameter of bulbils. Similarly, all morphological attributes had the negative correlation with mass of bulbils as well.
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Experiment # 4
In this experiment, two cultivars of tulip namely, Apeldorn, and clear water were tested against different polyamines (Putrescine, spermine and spermidine) at various levels of their exogenous application. The objective of this experiment was to test the response of polyamines for the quality enhancement in cut tulips under sub-optimal environmental conditions to support adaptability response. Results revealed significant effect of all the tested treatments of various polyamines in the tulip cultivars. More or less the overall effect of polyamines was found to be similar irrespective of the variety. The morphological attributes like plant height, plant fresh mass, plant dry mass and leaf area were found to be at their maximum level when spermine was applied at 0.03 mM in Apeldorn cultivar while in clear water spermine at 0.01 mM produced maximum plant height and dry matter contents but spermine at 0.02 mM exhibited the maximum fresh mass and leaf area. So this can be inferred that spermine at relatively at higher levels is more responsive in enhancing morphological traits in Apeldorn cultivar while its lower levels induced morphological quality in Clear water. Likewise, Spd at 2.5 mM improved morphological traits in Apledorn cultivar next to Spm. Similarly, Spd at relatively lower levels like 1.5 mM improved the height and dry matter contents in Clear water cultivar. Spd at 0.6 mM gave the maximum fresh mass and leaf area in the same cultivar. When comparing the response of Put it revealed the least performance compared to Spm and Spd. For cultivar Apeldorn Put was found to be better performing at 2 mM. Although, the tested doses of this polyamine resulted in the least standards affecting the quality. In Clear water cultivar Put at 3 mM improved plant height and dry matter contents while at 2 mM it had its maximum potential for fresh mass and leaf area of the cultivars.
In cultivar Apeldorn Spm at 0.03 mM exhibited its maximum response in various attributes (Photosynthetic rate, transpiration rate, instantaneous WUE, intrinsic WUE, sub- stomatal CO2, SPAD value and mesophyll conductance) while stomatal conductance has the same effect when Spm was applied at 0.03 and 0.02 mM. Likewise Spd at 2.5 mM exhibited the maximum levels of above mentioned gaseous exchange attributes. Moreover Put at 2 mM had improved these attributes, but they were at the least standard in comparison to Spd and Spm except for instantaneous WUE that was found to be maximum in this polyamine at 3 mM. This trend is afformed by the positive correlation of morphological traits with all the gaseous
218 exchange attributes (Table 4.4 a). Exogenous application of polyamines gave non-significant findings with respect to the varietal effect for various gaseous exchanges attributes (A, intrinsic WUE, Ci, gs and gm. This shows that gaseous exchange attributes perform in a similar manner irrespective of the cultivar in tulips.
In Clear water cultivar Spm was found to be the best performing polyamine in improving gaseous exchange attributes that were found to be at their peak when exogenously applied at relatively lower level at 1.5 mM. Except for instantaneous WUE that were highest in this polyamine with the similar effect at 0.5 and 1.5 mM while SPAD value exhibited its maximum potential at 2.5 mM of Spd. Put at 3 mM resulted in the maximal performance of gaseous exchange attributes for this cultivar except for SPAD value that was maximum when this polyamine was applied at 2 mM. So, the attributes of gaseous exchange can be used as a module in crop simulation model (Kim and Lieth, 2003) that can assist tulip growers to predict useful conditions needed for tulip crop.
The enzymatic antioxidants like SOD and CAT had their maximum activity amongst all the polyamines in Spm at 0.03 mM in the cultivar Apeldorn. The highest activity of these antioxidants resulted in the least amalgamation of MDA contents at the same level except for POD activity that was found to be highest in Spm at 0.02 mM. The non-enzymatic antioxidants like TPC and percentage inhibition of DPPH ions scavenging activity was highest when Spm at 0.03 and 0.02 mM was exogenously applied respectively. Moreover the maximum percentage scavenging of H2O2 was also observed at Spm 0.03 mM with respect to CAT activity that was also at its peak at this level. Similarly Spd at 2.5 mM had the maximum activity of CAT, POD and percentage inhibition of DPPH ion scavenging activity, while Spd at 1.5 mM had the maximum activity of SOD and the least MDA contents at 1.5 mM. Moreover TPC were also highest when Spd was applied at 1.5 mM. The H2O2 scavenging was also found to be at its highest level when Spd was applied at 2.5 mM in relation to highest CAT activity. By considering above discussed polyamines (Spd and Spm) putrescine was least effective as compared to them. It indicated its maximum potential in all the enzymatic attributes at 2 mM except for POD activity which was highest when applied at 3mM. Hence the best performing dose is of Spm at 0.03 mM that can be regarded as the optimized dose amongst the all the
219 polyamines tested for tulip cut flowers. This is because spermidine and putrescine improved the quality by depicting weak responses as compared to Spm.
In cultivar Clear water all the antioxidants and their counter parts exhibited their maximum potential when Spm was applied at 0.01 mM except for DPPH and POD that was best in their response at relatively higher dose (Spm at 0.01 mM). All these attributes when tested for spermidine, it exhibited its peak potential when applied at 1.5 mM except for Spd that performed best at 0.5mM. Putrescine at 3 mM depicted highest responses for the antioxidants except for percentage inhibition of DPPH ion and POD activity that was highest in relatively lowered level when applied at 2 mM. The nonsignificant effect of POD activity, CAT activity and MDA contents revealed that the tested doses were irrespective of different cultivars of tulips and will exhibit the same response for other varieties. So, the level of these antioxidants is important for flower quality parameters as senescence is considered as developmental change that decides the life of plant tissue. This is generally believed for being genetically programmed. Petals, that primarily determine the commercial longevity of flowers and as a consequence, it is essential to study physiological and biochemical processes that take place during flower senescence. Similar trend for these antioxidants activity is reported by Chakrabarty et al. (2009) in hemerocalis flowers. All the floral attributes exhibited their best performance in the treatment where spermine was applied, the earliest emergence of floral bud was seen when Spm at 0.03 mM was applied in Apeldorn cultivar while Spm at 0.01 mM in Clear water hastened the flower bud emergence. Delayed flower bud emergence was seen in spermidine and putrescine treatments. The floral attributes (Tepal diameter, flower diameter, length of petals, stem diameter, flower quality, vase life, days to start petal senescence and tepal abscission) were at their best level in Spm at 0.03 mM in Apeldorn cultivar. Similarly Spd at 2.5 mM improved all the floral attributes except for width of petals and days to start petals senescence. Similarly Put at 2 mM had the synonymous effect for floral traits. Width of petal amongst all other floral attributes was found to be differently influenced in response to the tested polyamines. Maximum width of petal was observed in putrescine treated flowers that gave the maximum width when applied at 2 mM in Apeldorn cultivar and at 2 mM followed by Spd at 2.5 mM. This was somewhat undesirable respond that led to expansion of flower in more horizontal axis disturbing the length to width ratio the petals and ultimately misshaping flower heads. But the normal shape of flower heads
220 was retained in Clear water cultivar with the application of either if the polyamine. Similarly the flower attributes (DFBE, TD, FD, LP, WP, SD, FQ, VL, DSPS and TA) were at their highest levels when Spm at 0.01 mM was applied in Clear water cultivar. Moreover, the attributes (FQ, VL, DSPS and TA) at this level were highest in comparison to Apeldorn cultivar. Similarly Spd at 1.5 mM and Put at 3 mM had their best performance among the tested treatments hence it can be inferred that Spm at 0.01 mM was the most useful dose for Clear water cultivar in enhancing the quality for cut tulips and recommend has an optimized dose.
The above discussed attributes are further affirmed by the correlation pattern existed between different floral attributes like, petal length and width was highly significant and positively correlated with each other, similar trend was seen for vase life as well. Moreover, scape diameter was negatively correlated to flower quality and vase life while it was also negatively correlated to days to start senescence and tepal abscission. Length and width of petals was positively correlated to senescence and abscission. Furthermore, vase life and flower quality was highly significant and positively correlated to SOD activity while POD activity was found to be negatively correlated with scape diameter. On the other hand scape diameter was also negatively correlated to DPPH and H2O2 scavenging. It was also found that the antioxidants had significant but negative correlation with all the floral attributes except for scape diameter.
Likewise CAT activity had highly significant positive correlation with H2O2 scavenging activity (Table 4.4 b).
Morphological attributes like plant height and negative correlation with days to flower bud emergence while positive with photosynthetic rate. Leaf area had negative correlation with days to flower bud emergence highly significant with SPAD value. Days to flower bud emergence had positive correlation with all the gaseous exchange attributes while tepal diameter had positive correlation with them. SPAD value and transpiration rate had positive correlation with all the gaseous exchange attributes, except for transpiration rate with instantaneous WUE (Table 4.4 a).
Bulbils attributes like diameter, mass and number were not affected by either of the polyamine treatment and gave the best results in untreated plants followed by the treatments of putrescine and spermidine. Spermine gave unbeatable combination with its treatments in both the tested cultivars regarding cut flower quality though it delivered in negative for bulbils attributes.
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Hence it can be conceded as the most decisive and quality inducing polyamine for tulip cut flowers. This is further confirmed by the negative correlation that existed between the morphological attributes and the bulbil attributes (Table 4.4 a).
5.1 Conclusion
From this study, it can be concluded that
1. Time to start sprouting, time to reach 50% sprouting, mean emergence time and emergence index are the efficient indices for assessing adaptability at preliminary level for different cultivars of tulip (Tulipa gesneriana L.). 2. Growth promoting substances like chitosan, glycine betaine and polyamines are effective tools that had significant effect on various attributes that depicted improved morphological and floral traits. 3. Enzymatic activity of various antioxidants (SOD, CAT and POD) was found to be more pronounced at optimized doses of growth promoting substances while MDA contents were at their minimal level. 4. Comparison among various cultivars regarding their quality standards for cut flower production was directly linked with concentration and compatibility of tested cultivars with growth promoting substances. 5. Apeldorn, Barcelona, Clear water, Ile de France, Leen Vander Mark and Parade are amongst elite cultivars that strongly combat against environmental vagaries. 6. Overall, it can be concluded that intensity of gaseous exchange attributes, biochemical analysis and antioxidant enzyme activities are the true indicators of assessing the growth potential. 7. Uncertain abiotic factors of environment explicit toxic effects that can significantly be alleviated by exogenous application of low concentration of glycine betaine while chitosan at relatively higher levels responded well. Although their optimized level differed significantly among various cultivars. 8. However, glycine betaine at higher concentration aggravated the negative response regarding flower quality with damaging effects on plant parts that might be due to ROS production, hampered Rubisco synthesis, reduced chlorophyll contents but enhanced chloroplast volume, coagulation of stroma along-with swelling of thylakoid membranes.
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9. Polyamines also significantly improved growth of tulips particularly spermine had the most profound response with improved postharvest longevity might associated with pre- harvest treatment. 10. Polyamines mitigated harmful abiotic effects by improving water relations by enhancing the osmotic adjustment potential of both the tested tulip cultivars. 11. Since, all the tested substances significantly accelerated enzymatic activities and organic solute’s accumulation. Therefore, it can be concluded that efficient performance of above mentioned cultivars was due to enhanced antioxidant enzymatic (SOD, CAT, POD) activities and increased deposition of osmolytes like, total phenolic contents. 12. Non-significant effect of all the tested substances was found in all tulip cultivars regarding bulbils production due to inadequate source sink relationship. 13. Finally, from the results of this study it is extracted that exogenous application of growth promoting substances play decisive role and develop unbeatable combination for producing cut tulips of extra ordinary standard.
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Chapter # 6 Summary
The research work reported in this manuscript was conducted in open field conditions at two different locations, Rose project and floriculture area of Institute of Horticultural sciences, University of Agriculture Faisalabad. The chemical analysis was performed in floriculture lab and chemistry lab, Department of chemistry, University of Agriculture Faisalabad, Pakistan. The objectives were to identify the growth response of different cultivars of tulip for cut flower production and to investigate the biochemical and physiological characters associated with exogenous application of different growth promoting substances to enhance the pre-harvest and post-harvest longevity. The execution of experiments in open field conditions were laid out in randomized complete block design each with three replications. The obtained results are briefly summarized below; Study-1: Screening of different cultivars of tulip to test their growth and adaptability response. Screening was done on the basis of emergence attributes of bulbs, morphological traits and floral parameters. It was found that all cultivars exhibited different responses regarding the studied parameters. The cultivars that exhibited earliest emergence and maximum response for various morphological and floral attributes were conceded better adapted. Moreover, the life cycle of the plants that exhibited minimal duration were keenly focused as shortest duration makes the plant complete their life cycle in short duration winter season that prevails in subtropical region of the country. Likewise the cultivars that had highest plant heights, more vigorous growth, prolonged post-harvest display life and strengthened plant parts were also graded better adapted. Growers have great interest in the underground storage organs for utilizing them in the next season was also considered the integral part of the study. It was found that the cultivars that superseded in high quality morphological, and floral attributes behave negatively regarding bulbils production. Cultivars like Apeldorn, Barcelona, Ile de France, Leen Vander Mark and Parade were found to possess extraordinary features and are highly adapted in Faisalabad environmental conditions. Study-2: Exogenous application of chitosan for growth and quality assessment of different cultivars of tulip.
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Chitosan application improved the quality parameters in all tested cultivars. Improved plant height, leaf area, earlier flower bud emergence, flower diameter, length and width of petals, stem diameter, flower quality, prolonged shelf display and late senescence and abscission was noticed in chitosan treated plants. However number of leaves remained unaffected by its application likewise it also reduced the transpiration rate beyond the optimized level. There existed positive correlation of morphological traits with the floral attributes. Exogenous application of chitosan was found to be promising approach to achieve high quality cut flowers of tulip, however traditional sowing of bulbs resulted in inferior performance than that of chitosan treated bulbs. Exogenous application of chitosan improved the physiological and antioxidant enzyme activity as well that vary differently in different tulip cultivars. Moreover chitosan application remarkably reduced the disease incidence and infestation of any fungal growth on bulbs or foliage hence boosted the ability to combat diseases. The optimized dose of chitosan for Apeldorn cultivar was found to be at 90 mg L -1 while this substance at relatively lower level performed best in Barcelona that had optimal growth response at 60 mg L-1. Similarly, Ile de France cultivar responded best at 120 mg L -1 while Leen Vander Mark had its optimized level at 60 mg L -1 while the cultivar Parade showed its compatibility at highest level of chitosan application that was at 120 mg L -1. Study-3: Exogenous application of glycine betaine for growth and quality assessment of different cultivars of tulip. Glycine betaine behaved like an effective osmo-protectant and growth promoting substance that ameliorated the malicious environmental effects induced by abiotic environmental factors. Exogenous application of GB improved the quality parameters in all tested cultivars. Improved plant height, leaf area, earlier flower bud emergence, flower diameter, length and width of petals, stem diameter, flower quality, prolonged shelf display and late senescence and abscission was noticed in GB treated plants. It showed somewhat in compatible behavior in some tulip cultivars at higher levels that resulted in damaged and blemished quality features on foliage and stunted floral growth. However number of leaves remained unaffected by its application likewise it also reduced the transpiration rate beyond the optimized level. There existed positive correlation of morphological traits with the floral attributes. Exogenous application of GB was
225 found to be promising approach to achieve high quality cut flowers of tulips, however, traditional sowing of bulbs resulted in inferior performance than that of GB treated bulbs. Exogenous application of GB improved the physiological and antioxidant enzyme activity particularly higher activity of SOD, CAT, POD, DPPH ions scavenging, higher TPC and higher percentage inhibition of H2O2 in different tulip cultivars. Moreover GB application remarkably improved the physiological attributes like SPAD value, photosynthetic rate, transpiration rate, WUE, stomatal conductance, mesophyll conductance and the levels of substomatal conduction of CO2. The optimized dose of GB for Apeldorn cultivar was found to be 60 mM while GB at very low concentration i.e 30 mM resulted in achieving highest quality in Barcelona cultivar. Similarly, Ile de France cultivar responded best when GB was exogenously applied at 90 mM while Leen Vander Mark had its optimized level for its improved morphological and physiological traits at 120 mM while the antioxidant activities and all the post-harvest attributes performed best at relatively lowered dose that was 60 mM while the cultivar Parade showed its compatibility at highest level of GB application that was at 90 mM. Study-4: Exogenous application of different levels of polyamines (Putrecine, spermine and spermidine) to assess pre and postharvest attributes in two cultivars of tulips. Polyamines were found to improve various growth phases in plants like organogenesis, senescence and combating hazardous environmental factors because of their extremely high biological activity. This trend was clearly evident in the current study in which polyamines dramatically improved the morphological, physiological, biochemical and floral attributes. Exogenous application of polyamines exhibited positive results in inducing increased tolerance in unfavorable environmental conditions. It was found that polyamines had similar effect on both the cultivars so they impart no significant difference in different varieties. Though they are very expensive compounds beyond the reach of conventional grower yet their remarkable performance cannot be ignored in delivering remarkable finesse in growth attributes of plants. The present study concludes that spermine amongst other tested polyamines improved the pre and postharvest attributes particularly enhanced performance in floral traits in both the cultivars. It was observed that for most of the physiological and morphological parameters, the response of polyamines was same irrespective of the variety. Although, best performance was
226 exhibited by spermine, when exogenously applied at 0.03 mM in Apeldorn cultivar and Spm at 0.01 mM exhibited unbeatable combination with Clear water cultivar. 6.1 Disclaimer These recommendations are purely based on the field experiments conducted under controlled conditions. The discrepancies may arise in the outcomes under commercial situations. The project investigator, Mohsin Bashir and University of Agriculture, Faisalabad, accept no liability whatsoever reason of negligence or otherwise arising from the reliance or use of these recommendations. 6.2 Recommendations
Exogenous application of chitosan, glycine betaine and spermine at optimized doses should be employed to achieve high quality cut tulips. Cultivars shall be selected that have shortest life cycle and best suited to acquire a commercial crop accompanied with uniform crop stand. Early sowing in mid winters performs excellent regarding cut flower production in open field conditions. Handling of bulbs prior to sowing must be done in adequate temperatures by storing them at 2°C.
6.3 Future prospects
Growers should be provided adequate training courses regarding modern techniques of handling sensitive geophytes and their management. More cultivars based on their color preferences should be tested for catering the florist’s and consumer demand. Studies on other growth modulating substances and planting geometry for the sowing should be conducted. Weed management strategies and other cultural practices that may enhance the plant response should be studied. Response of different micronutrients like zinc, boron, and iron should be tested as foliar sprays along with enhanced macro nutritional status. Further studies on postharvest behavior of Tulipa gesneriana should be continued to hit the market window with prolonged display life.
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