A LABORATORY GUIDE FOR GENERAL BOTANY
Chapter 1 Organisation of Plant Tissue Culture Laboratory 1 2 Media Preparation, Sterilization and Storage 5 3 Aseptic Seedlings Production and Sterilization of In vivo Grown Plant Material and Excised Explants 9 4 Micropropagation 12 5 Initiation and Maintenance of Callus and Suspension Cultures 15 6 Ovule Culture in Wheat 28 7 Anther Culture for Haploid and Dihaploids (DH) Production in Sunflower (Helianthus annuus L) 30 8 Embryo Culture for Crop Improvement 32 9 Somatic Embryogenesis in Alfalfa (Madicago sativa) 35 10 Production of Synthetic Seeds 39 11 In Vitro Selection for Salt Tolerance 44 12 In Vitro Selection for Disease/Toxin (Verticillium Wilt) Resistance Cell Lines in Alfalfa 46 13 Cryopreservation for Germplasm Conservation 55 14 Determination of Endogenous Content of Cytokinins in Plant Material 68 15 Identification of Viruses and/or Nucleocapsid Protein by ELISA 73 16 Isolation and Culture of Plant Protoplasts 77 17 Production of Somatic Hybrids Through Protoplast Fusion 94 18 Electroporation of Protoplasts 101 19 Genetic Transformation of Plants by Using Agrobacterium tumefaciens Vector System 103 20 Agrobacterium tumefaciens Mediated Transformation 111 21 Assaying Marker Genes in Plants-GUS (?-Glucuronidase) Gene 115 22 Detection of Neomycin Phosphotransferase II (NPT II) Activity in Transformed Plant Tissues 119 23 Genetic Transformation of Plants Using Agrobacterium rhizogenes Vector System 122 24 Tobacco Chloroplast Transformation using Micro Projectile Bombardments (Biolistic Gun) 128 25 Micro Projectile Mediated Transformation of Rice Using Biolistic Gun 133 26 Inoculation and Storage of Escherichia coli from Overnight Culture 136 27 Construction of Genomic DNA Library 139 28 Isolation of Bacteriophage Lambda DNA 151 29 Screening of Genomic and cDNA Libraries 156 30 Isolation of Plasmid DNA (Alkaline Lysis Method, Miniprep) 164 31 Preparation of Probe 170 32 Isolation of High Molecular Weight Plant Genomic DNA and Mitochondrial (mt) DNA 176 33 Restriction Fragment Length Polymorphism (RFLP) 179 34 Isolation of Molecular Markers Using Random Amplified Polymorphic DNA (RAPD) 192 35 SCARS and CAPS, PCR-Based Markers for Genetic Analysis 196 36 Site Directed Mutagenesis 200 37 Isolation of RNA and Northern Blotting 207 38 Isolation of Chloroplast DNA 216 39 DNA Sequencing 228 40 Identification of Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis (PAGE) - Based analysis and Western Blotting 235