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Assessment of Genetic Variability and Development of Hybrids in Rosa Species

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Assessment of Genetic Variability and Development of Hybrids in Rosa Species ASSESSMENT OF GENETIC VARIABILITY AND DEVELOPMENT OF HYBRIDS IN ROSA SPECIES MUHAMMAD FAISAL KHAN 14-arid-980 DEPARTMENT OF HORTICULTURE FACULTY OF CROP & FOOD SCIENCES PIR MEHR ALI SHAH ARID AGRICULTURE UNIVERSITY RAWALPINDI PAKISTAN 2020 ASSESSMENT OF GENETIC VARIABILITY AND DEVELOPMENT OF HYBRIDS IN ROSA SPECIES by MUHAMMAD FAISAL KHAN (14-arid-980) A thesis submitted in partial fulfillment of the requirement for degree of Doctor of Philosophy in Horticulture DEPARTMENT OF HORTICULTURE FACULTY OF CROP & FOOD SCIENCES PIR MEHR ALI SHAH ARID AGRICULTURE UNIVERSITY RAWALPINDI PAKISTAN 2020 i ii iii iv v DEDICATION Oh, Allah Almighty open our eyes, To see what is beautiful, Our minds to know what is true, Our heart to love what is Allah Blessing of Almighty ALLAH Always help me to complete task positively with truth and Prayers of family Whose encouragement, spiritual inspiration, well wishes, sincere prayers and an atmosphere that initiate me to achieve high academic goals iii CONTENTS List of Tables X List of Figures Xi Acknowledgements Xiv Chapter 1 INTRODUCTION 1 Chapter 2 REVIEW OF LITERATURE 9 2.1 ROSA HYBRIDA CLASSIFICATION AND HISTORIC 9 BACKGROUND 2.1.1 Breeding History and Classification 10 2.1.2 Reproductive Physiology and Factors Effecting Breeding 11 Success 2.1.3 Fertility Status and Associated Factors 12 2.1.4 Rose Seeds Collection 16 2.1.5 Seed Germination Problem 17 2.1.6 Genetic Diversity Evaluation 19 2.1.7 Growth Performance 23 2.1.8 Physiological Response 24 2.1.9 Water Requirement and Transpiration 26 2.1.10 Stomatal Conductance 26 Chapter 3 MATERIALS AND METHODS 28 3.1 PLANTING MATERIAL 28 3.1.1 Field Preparation 31 3.2 DATA COLLECTION FOR CHARCTERIZATION OF ROSA 31 HYBRIDA VARIETIES 3.2.1 Morphological Characters 31 3.2.1.1 Plant height (cm) 31 3.2.1.2 Primary branches per plant 31 3.2.1.3 Number of prickles 32 3.2.1.4 Number of flowers per plant 32 3.2.1.5 Flower diameter per flower (cm) 32 3.2.1.6 Number of petals per flower 32 3.2.1.7 Flower persistency life 32 3.2.2 Physiological Response of Rosa Hybrida Varieties 32 3.2.2.1 Leaf area (cm2) 33 3.2.2.2 Photosynthesis rate (Pn) 33 3.2.2.3 Transpiration rate (E) 33 3.2.2.4 Stomatal conductance (gs) 33 3.2.3 Experimental Design for Statistical Analysis 33 3.3 NUMBER OF ANTHERS, INVITRO POLLEN VIABILITY 34 AND POLLEN GERMINATION STUDY OF ROSA HYBRIDA VARIETIES 3.3.1 Preparation of Material for Pollen Testing 34 3.3.2 Pollen Viability Test 35 3.3.3 Pollen Diameter (µm) 35 3.3.4 Germination Capacity of Pollens 35 iv 3.3.5 Experimental Design for Statistical Analysis 36 3.4 NEW CROSS COMBINATIONS FOR CONVENTIONAL 36 BREEDING 3.4.1 Pollination Process 36 3.4.2 Data Collection for Successful Cross Combinations 37 3.4.2.1 Hip set percentage (%) 37 3.4.2.2 Days taken to maturity of hips (days) 37 3.4.2.3 Hip fresh weight (g) 38 3.4.2.4 Number of seeds per hip 38 3.4.2.5 Seed dry weight (g) 38 3.4.2.6 Seeds length and width (mm) 38 3.4.3 Experimental Design for Statistical Analysis 38 3.5 MITIGATION OF SEED DORMANCY IN NEW 38 COMBINATIONS AMONG ROSA SPECIES 3.5.1 Seed Germination Treatments 38 3.5.2 Data Collection After Seed Germination 39 3.5.2.1 Germination percentage 39 3.5.2.2 Germination days 39 3.5.2.3 Growth performance of seedlings at the time of transplantation 40 3.5.3 Experimental Design for Statistical Analysis 40 3.6 MORPHOLOGICAL DATA OF PROGENIES IN FIELD 40 3.6.1 Number of Petals per Flower 41 3.6.2 Flower Diameter (cm) 41 3.6.3 Height of Progeny Plant (cm) 41 3.6.4 Number of Prickles 41 3.6.5 Flower Persistency Life (days) 41 3.6.6 Experimental Design for Statistical Analysis 41 3.7 DETERMINATION OF GENETIC VARIABILITY AMONG 42 DEVELOPED HYBRIDS AND THEIR PARENTS 3.7.1 Collection of Leaf Samples 42 3.7.2 DNA Extraction Protocol 42 3.7.3 DNA Quantification 43 3.7.4 Microsatellite Molecular Markers for Genetic Diversity Analysis 43 3.7.5 Electrophoresis and Analysis of Amplified Samples 45 3.7.6 Experimental Design for Data Analysis 45 Chapter 4 RESULTS AND DISCUSSION 46 4.1 MORPHOLOGICAL EVALUATION OF ROSA HYBRIDA 46 VARIETIES 4.1.1 Plant Height (cm) 46 4.1.2 Number of Primary Branches per Plant 48 4.1.3 Number of Flowers per Plant 49 4.1.4 Flower Diameter (cm) 50 4.1.5 Number of Petals per Flower 51 4.1.6 Petal Length of Flowers 54 4.1.7 Number of Prickles on Stem 55 v 4.1.8 Flower Persistence Life 55 4.1.9 Correlation Among Morphological Character 56 4.1.10 Discussion on Morphological Attributes 57 4.2 ESTIMATION OF LEAF GAS EXCHANGE DATA OF ROSA 68 HYBRIDA VARIETIES 4.2.1 Leaf Area (cm2) of Rosa Hybrida Varieties 68 4.2.2 Photosynthesis Rate of Rosa Hybrida Varieties (Pn = μmol CO2 69 m-2s-1) -2 - 4.2.3 Transpiration Rate of Rosa Hybrida Varieties (E= mol H2O m s 70 1) 4.2.4 Stomatal Conductance of Rosa Hybrida Varieties (gs= mol m-2s- 71 1) 4.2.5 Discussion on Physiological Attributes 74 4.3 NUMBER OF ANTHERS, INVITRO POLLEN VIABILITY 75 AND POLLEN GERMINATION STUDY 4.3.1 Number of Anthers per Flower 75 4.3.2 Pollen Viability Percentage (%) 76 4.3.3 Pollen Invitro Germination Percentage (%) 77 4.3.4 Pollen Diameter (µm) 79 4.3.5 Discussion on Pollen Behavior for Success of Breeding 80 4.4 SEED SETTING SUCCESS IN NEW CROSS 82 COMBINATIONS 4.4.1 Hip Set Percentage (%) 83 4.4.2 Hip Fresh Weight (g) 83 4.4.3 Hip Size (cm) 84 4.4.4 Days to Maturity of Hips (days) 84 4.4.5 Number of Seeds per Hip 85 4.4.6 Dry Weight of Seeds (mg) 85 4.4.7 Length and Width of Seeds (mm) 85 4.4.8 Discussion on Seed Setting Among Successful Cross 86 Combinations 4.5 MITIGATION OF SEED DORMANCY IN DEVELOPED 93 NEW COMBINATIONS AMONG ROSA SPECIES 4.5.1 Control T0: Effect of Seed Storage at Room Temperature 93 4.5.2 Effect of Warm and Cold Temperature on Seed Germination 93 4.5.3 T2: Effect of Cold Stratification Combined with GA3 94 4.5.4 T3: Effect of Warm and Cold Temperature in Combination with 94 GA3 4.5.5 Seedling Germination Days 94 4.5.5.1 Seedling leaf at the time of transplantion 98 4.5.5.2 Seedling length at the time of transplantion 99 4.5.5.3 Seedling root length at the time of transplantion 99 4.5.5.4 Seedling shoot at the time of transplantion 100 4.5.5.5 Discussion on germination of rose seed 101 4.5.6 Nursery Seedling Growth Performance 103 4.5.6.1 Results of hybrid seedlings growth 104 vi 4.5.6.2 Discussion of seedling plants 105 4.6 MORPHOLOGICAL DATA OF HYBRID PROGENY 106 POPULATION 4.6.1 Parent Varieties and Developed Progenies Description 117 4.7 EVALUATION OF GENETIC DIFFERENCES IN 118 DEVELOPED PROGNIES WITH THEIR PARENTS 4.7.1 Genetic Diversity Analysis 118 4.7.2 Genetic Diversity Analysis by Using Microsatellite SSR Markers 119 4.7.3 Discussion on Genetic Diversity Among Parents and Developed 120 Hybrid Population SUMMARY 124 LITERATURE CITED 128 vii Abbreviations R Rosa DNA Deoxyribonucleic acid RAPD Random amplified polymorphic AFLP Amplified fragment length polymorphism UAE United Arab Emirates PHDEB Pakistan Horticulture Development and Export Board ° C Celsius ROS Reactive oxygen species RNS Reactive nitrogen species %age Percentage Μm Micrometer ABA Abscisic acid GA3 Gibberellic acid SSR Simple sequence repeats RAPD Random Amplified Polymorphic DNA SCARs Sequence characterized amplified regions ISSR Inter simple sequence repeat SCoT Start codon targeted PCA Principal component analysis HCA High-content screening Umol m- Micromole: per second and square meter 2 -1 s CO2 Carbon dioxide RCBD Randomized Complete Block Design Cm Centimeters ANOVA Analysis of variance IRGA InfraRed Gas Analyzer mm Millimeter PAR Photosynthetically active radiation Pn Photosynthesis LSD Least Significant Difference URF Universty Research Farm PMAS Pir Mehr Ali Shah AAUR Arid Agriculture University Rawalpindi CTAB Cetyltrimethylammonium bromide RNAase Ribonuclease Rpm Revolutions per minute UV Ultraviolet PCR Polymerase chain reaction ddH2O Double Distilled Water µl Microliter viii S Seconds Min Minutes Bp Base pair UPGMA Unweighted pair group method with arithmetic mean Cm Centimeters ix List of Tables Table No. Page 3.1 List of Rosa hybrida varieties with their parentage and characters 29 grown in Pothwar climate of Pakistan 3.2 Varieties with their cross combinations used in hybridization study 37 3.3 List of microsatellite markers (SSR) used in genetic diversity 44 among Rosa hybrida population and parents 3.4 Thirty-four hybrid population and ten parent plant DNA extraction 45 4.1 Morphological foliage characters of varieties grown in Pothwar 59 climate 4.2 Growth parameters of Rosa hybrida varieties during the years 60 2016-2017 in Pothwar climate 4.3 Correlation study among examined morphological traits by 62 principal component analysis 4.4 Correlation among examined morphological characters 62 4.5 Leaf area, photosynthesis rate, transpiration rate and stomatal 73 conductance of Rosa hybrida varieties for years 2016-2017 4.6 Rosa hybrida varieties for parentage characters 81 4.7 Detail description of collected data from successful hip set up to 87 extraction of seed of successful crosses 4.8 Seed germination percentage resulted from different treatments 95 4.9 Successful seedlings after transplantation screened for 96 morphological traits 4.10 Examined morphological characters of hybrid progenies of Rosa 107 hybrida varieties 4.11 Morphological characters of the Rosa hybrida parents used in rose 115 breeding programme in Pothwar climate 4.12 Morphological characters of the hybrid seedlings successfully 115 shifted in field x List of Figures Figure No.
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