Plant Biotechnology and Molecular Biolog : A Laboratory Manual
Contents: Preface. 1. Organisation of Plant Tissue Culture Laboratory. 2. Media Preparation, Sterilization and Storage. 3. Aseptic Seedlings Production and Sterilization of In vivo Grown Plant Material and Excised Explants. 4. Micropropagation. 5. Initiation and Maintenance of Callus and Suspension Cultures. 6. Ovule Culture in Wheat. 7. Anther Culture for Haploid and Dihaploids (DH) Production in Sunflower (Helianthus annuus L) 30. 8. Embryo Culture for Crop Improvement. 9. Somatic Embryogenesis in Alfalfa (Madicago sativa). 10. Production of Synthetic Seeds. 11. In Vitro Selection for Salt Tolerance. 12. In Vitro Selection for Disease/Toxin (Verticillium Wilt) Resistance Cell Lines in Alfalfa. 13. Cryopreservation for Germplasm Conservation. 14. Determination of Endogenous Content of Cytokinins in Plant Material. 15. Identification of Viruses and/or Nucleocapsid Protein by ELISA. 16. Isolation and Culture of Plant Protoplasts. 17. Production of Somatic Hybrids Through Protoplast Fusion. 18. Electroporation of Protoplasts. 19. Genetic Transformation of Plants by Using Agrobacterium tumefaciens Vector System. 20. Agrobacterium tumefaciens Mediated Transformation. 21. Assaying Marker Genes in Plants-GUS (β-Glucuronidase) Gene. 22. Detection of Neomycin Phosphotransferase II (NPT II) Activity in Transformed Plant Tissues. 23. Genetic Transformation of Plants Using Agrobacterium rhizogenes Vector System. 24. Tobacco Chloroplast Transformation using Micro Projectile Bombardments (Biolistic Gun). 25. Micro Projectile Mediated Transformation of Rice Using Biolistic Gun. 26. Inoculation and Storage of Escherichia coli from Overnight Culture. 27. Construction of Genomic DNA Library. 28. Isolation of Bacteriophage Lambda DNA. 29. Screening of Genomic and cDNA Libraries. 30. Isolation of Plasmid DNA (Alkaline Lysis Method, Miniprep). 31. Preparation of Probe. 32. Isolation of High Molecular Weight Plant Genomic DNA and Mitochondrial (mt.) DNA. 33. Restriction Fragment Length Polymorphism (RFLP). 34. Isolation of Molecular Markers Using Random Amplified Polymorphic DNA (RAPD). 35. SCARS and CAPS, PCR-Based Markers for Genetic Analysis. 36. Site Directed Mutagenesis. 37. Isolation of RNA and Northern Blotting. 38. Isolation of Chloroplast DNA.39. DNA Sequencing. 40. Identification of Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis (PAGE) - Based analysis and Western Blotting. Appendices. The book, “A Laboratory Manual of Plant Biotechnology and Molecular Biology” comprises of workable laboratory protocols for a large number of techniques related to plant biotechnology, genetic engineering and molecular biology. This includes plant cell and tissue culture, callus and suspension culture, anther culture, ovule culture, embryo culture, Cryopreservation, Isolation of Plant protoplasts, Protoplast culture and regeneration, production of somatic hybrids through protoplast fusion, gene transformation using Agrobacterium as vector, direct gene transfer using biolistic gun, Isolation of plant and organells DNA, construction and screening of genomic DNA libraries, Molecular markers like RFLP, RAPD, SCARS and CAPS, DNA sequencing, RNA isolation and northern blotting, Isolation of proteins and western blotting etc. The manual is prepared with the objective to cater the needs of post- graduate students as well as for scientists working in the disciplines of Plant Breeding, Genetics, Botany, Plant physiology, Biochemistry, Plant Biotechnology, Molecular Biology etc. It gives an update on some well established methods and presents reliable protocols.