Preparation and fusion of protoplasts from the phytopathogenic fungus Sclerotium rolfsii (Sacc.)

Abstract

1.11. Epidemiology 1.12. Control of Disease 1.13. Aims of Thesis 2. Materials and Methods 2.1. Organisms and Growth conditions 2.2. Preparation of semi-permeable cellophane membrane 2.3. Pretreatment of mycelia 2.4. Digestion mixtures 2.4.1. Osmotic stabilizers 2.4.2. Lytic enzymes 2.5. Protoplast formation 2.6. Determination of protoplast number 2.7. Purification of protoplast 2.8. Determination of protoplast size 2.9. Regeneration of protoplasts 2.9.1. Regeneration on solid medium 2.9.2. Regeneration in liquid medium 2.10. Fusion of protoplast 2.11. Study of stability of fusants 2.12. Growth rate of fusants 2.13. Pathogenic properties of fusants in green house pot experiment 2.13.1. Inoculum preparation 2.13.2. Soil preparation 2.13.3. Selecetion of host crops 66 2.13.4. Seed soaking and planting 66 2.13.5. Study of growth parameters of tomato, carrot and beans 67 2.13.5.1 Growth parameters studied 67 2.13.6. Disease incidence and mortality 68 2.13.7. Chlorophyll fluorescence 68 2.13.8. Estimation of chlorophyll a, b and carotenoids 69 2.14. Statistical analysis 70 3. Results 71 3.1.Strains A and R of Sclerotium rolfsii 71 3.2. Optimization of protoplast yields from Sclerotium rolfsii 71 3.2.1. Effect of pretreatment of mycelia 71 3.2.2. Effect of the osmotic stabilizers on protoplast yields and morphology74 3.2.2.1. Effect of different molarities of MgSO 4 on protoplast yields 76 3.2.3. Effect of different lytic enzymes on protoplast yields 76 3.3. Measurement of size of protoplast 3.4. Mechanism of protoplast release from the hyphae of Sclerotium rolfsii 3.5. Regeneration of protoplast into hyphae 3.6. Fusion of protoplasts of strains A and R 3.6.1. Recovery of the fusants 88 3.6.2. Morphological characters of fusants 3.6.3. Stability of the fusants 92 3.6.4. Growth rate of fusants 92 3.7. Assessment of pathogenicity of fusants and parent strains A and R of Sclerotium roflii 94 3.8. Physiological parameters 3.8.1.Chlorophyll fluorescence 3.8.2. Estimation of chlorophyll a, b and carotenoids 4. Discussion 4.1. Investigation into optimization for protoplast yields 4.3. Protoplast release mechanism 4.4. Protoplast regeneration 4.5. Protoplast fusion 4.6. Properties of fusants 4.7. Pathogenicity of fusants 5. References