Principles and Methods of Plant Molecular Biology Biochemistry and Genetics

Contents: Chapter: 1A. General Laboratory Principles i. Laboratory records ii. Laboratory requirements and safety iii. Laboratory techniques 1. Preparation of solutions 2. Preparation of buffers iv. Use of equipment 1. Use and calibration of micropipettes 2. Safe handling of microorganisms 3. Storage of cultures 4. Storage of DNA and handling of enzymes 5. Use and care of the pH meter 6. Centrifuge and centrifugation v. Methods in molecular biology 1. Spectrophotometry a. Beer's Law b. ambert's Law 2. Chromatography vi. Paper chromatography vii. Thin-layer chromatography (TLC) viii. Gas liquid chromatography (GLC) ix. Column Chromatography x. Columns used in HPLC and LPLC xi. Electrophoresis xii. Microscopy xiii. Micrometry xiv. Radioactivity and autoradiography xv. Isolation of nucleic acids xvi. Electrophoresis to separate DNA molecules xvii. Southern and Northern hybridization xviii. DNA sequencing xix. Cloning and genetic engineering xx. Polymerase chain reaction xxi. References 2. Chapter: 2 A. Plant Biochemistry And Physiology i. Sample preparation ii. Qualitative tests 1. Qualitative tests for carbohydrates a. General tests for presence of carbohydrates i. Molisch's test ii. Anthrone's reaction b. Reactions for presence of reducing sugars i. Fehling's test ii. Benedict's test iii. Barfoed's test iv. Bial'stest v. Seliwanoff s test c. Test for sucrose: d. Test for starch i. Iodine test iii. Qualitative tests for amino acids 1. General tests a. Ninhydrin reaction 2. Reactions for sulphur amino acids a. Lead sulphide test b. Nitro prusside test i. Test for cysteine ii. Test for cystine c. Reactions of aromatic amino acids i. Xantho proteic reaction ii. Millon's reaction iii. Glyoxylic test iv. Pauli's test v. Ehrlich's test vi. Sakaguchi test d. Test for proline and hydroxy proline iv. Quantitative estimations 1. Estimation of carbohydrates by anthrone method 2. Estimation of reducing sugars (glucose) by 3,5-dinitro salicylate method 3. Estimation of fructose by Roes resorcinol method 4. Estimation of reducing sugars in fruits by Benedict's quantitative method 5. Estimation of starch by modified method of Mc Cready et al 6. Estimation of soluble amino acids by ninhydrin method (phenyl alanine) 7. Estimation of proline in leaf material 8. Separation of amino acids and identification by paper chromatography 9. Separation of amino acids by Thin Layer Chromatography 10. Estimation of total nitrogen (and proteins) by Micro-Kjeldahl's method 11. Estimation of proteins by Biuret method 12. Estimation of protein content by Lowry's method 13. Estimation of proteins by Bradford's method 14. Determination of iso electric point of proteins (IP) 15. Electrophoresis of proteins: SDS - PAGE of proteins 16. Dtermination of iodine number of oils 17. Determination of saponification number of oil 18. Estimation of phosphate 19. Estimation of RNA 20. Estimation of DNA by dipheny1 amine method 21. Estimation of ascorbic acid in fruit 22. Determination of total and titrable acidity 23. Estimation of total chlorophyll content and ratio of chlorophyll a and b 24. Estimation of carotenoids 25. Chemical separation of chloroplast pigments 26. Column chromatography of photosynthetic pigments 27. Determination of water potential of tissue by falling drop method 28. Estimation of indoleacetic acid (IAA) 29. Determination of IAA oxidase activity 30. Determination of amylase activity 31. Determination of catalase activity (Permanganate titration method) 32. Determination of polyphenol oxidase activity 33. etermination of peroxidase activity v. References 3. Chapter: 3 A. Cytology i. Introduction ii. Cytological procedures 1. Mitosis a. Prophase b. Metaphase c. Anaphase d. Telophase 2. Study of mitosis in onion root tips 3. Study of C - mitosis (colchicine treated root tips) 4. Study of karyotype of Allium cepa 5. Effects of chemical mutagens on somatic chromosomes 6. Localization of DNA by Feulgen method 7. Orcein-banding of somatic chromosomes 8. Meiosis a. Study of meiosis in maize sporocytes b. Study of translocation heterozygosity at meiosis in plants c. Cytology of cell suspension culture 9. Preparation of fixatives 10. Preparation of stains a. Preparation of Feulgen reagent: b. Preparation of temporary seal 11. References 4. Chapter: 4 A. Plant Genetics i. Principle of segregation ii. Principle of independent assortment iii. Terminology in Genetics 1. Phenotype 2. Genotype 3. Dominant and recessive alleles 4. Co-dominant alleles 5. Multiple alleles 6. Incomplete dominance 7. Lethal alleles iv. Single gene inheritance 1. Fl and F2 generations 2. Test cross 3. Back Cross 4. Inheritance of two or more genes 5. Dihybrid F2 ratio v. Predicting out come of crosses and probability formulations vi. Chi - Square Test vii. Exercises and Problems 1. Exercises: Study of monohybrid cross by using maize cobs 2. Problems viii. Genetic interaction 1. Dominant epistasis: (Ratio 12:3:1) 2. Recessive epistasis: (Ratio 9:3:4) 3. Duplicate genes with cumulative effect: (Ratio 9:6: 1) 4. Duplicate dominant genes: (Ratio 15 : 1) 5. Duplicate recessive genes: (Ratio 9:7) 6. Dominant and recessive interaction: (Ratio 13 : 3) 7. Problems ix. Polygenic inheritance x. Sex related inheritance 1. Sex chromosome mechanisms a. Heterogametic males b. Heterogametic females c. Genie balance d. Haplodiploidy e. Single gene effects 2. Inheritance related to sex a. Sex linked inheritance b. Sex-influenced traits c. Sex-limited traits d. Sexuality in plants xi. Chromosome mapping a. Procedure of finding the gene order and mapping b. Interference and coincidence 1. Problems xii. Mutation breeding 1. Methodology of mutation breeding xiii. Statistical analysis 1. The binomial expansion 2. Problems on Binomial distribution 3. The Poisson distribution a. Problems on Poisson distribution xiv. The Normal distribution 1. Problems on Normal distribution xv. Comparison of means of two samples xvi. The t - test 1. Problems on 't' test xvii. X2 (Chi - square) test 1. Problems on Chi-square test xviii. Correlation xix. Problems on correlation xx. Population genetics (Hardy-Weinberg's equilibrium) 1. Gene and genotype frequencies a. Calculation of gene frequencies 2. Problems in population genetics xxi. References 5. Chapter: 5 A. Plant Molecular Genetics i. An overview of the methods of molecular genetics ii. Isolation of DNA 1. Isolation of plant genomic DNA 2. Isolation and purification of plasmid DNA iii. Isolation of RNA from plant tissue iv. Quantification of DNA and RNA v. Restriction analysis of DNA 1. Digestion of DNA 2. Preparation of agarose gel for electrophoresis 3. Electrophoresis of the digested DNA 4. Viewing the gel and photographing it 5. Analysis of the gel 6. Purification of DNA fragments from agarose gels a. Elution of DNA fragments by gel slicing and melting b. Elution of DNA fragment from agarose gels (crush method) c. Recovery of DNA from agarose gel by electroelution vi. Southern blotting vii. DNA hybridization with radioactive probes 1. Isolation of the DNA sequence to be used as the probe 2. DNA labeling: (Preparation of radioactive probes) 3. DNA hybridization 4. Autoradiography and X - ray film developing a. Xray Film development viii. DNA hybridization with non - radioactive probes ix. Northern blotting 1. Electrophoresis of RNA and Northern transfer 2. RNA hybridization x. Dot and Slot blottig 1. Dot blotting of RNA xi. Western blotting xii. PCR amplification of DNA xiii. cDNA cloning by RT - PCR xiv. RAPD analysis xv. Gene cloning and recombinant DNA techniques 1. Isolation of plasmid DNA 2. Digestion of plasmid DNA and insert DNA with the restriction 3. endonuclease Eco Rl 4. Ligation of plasmid and insert DNA to produce a recombinant 5. plasmid 6. Dephosphorylation of the digested vector (plasmid) DNA 7. Ligation of plasmid and insert DNA 8. Preparation of competent cells of the E. coli host 9. Transformation of E. coli cells 10. Selection of transformants 11. Analysis and confirmation of the recombinant DNA xvi. References 6. Chapter: 6 A. Plant Genetic Engineering i. Plant cell and tissue culture 1. Tissue culture laboratory 2. Tissue culture media 3. Preparation of culture medium a. MS basal medium 4. Sterilization and inoculation of plant material 5. Incubation and maintenance of the cultures 6. In vitro culture of plant tissues 7. Protoplast isolation and culture 8. Protoplast fusion (somatic cell hybridization) 9. Preparation of synthetic seeds ii. Gene transfer and expression in plants 1. Genetic transformation of plants using Agrobacterium system a. Induction of crown gall or hairy roots using wild strains of Agrobacterium b. Leaf disc transformation t echnique using Agrobacterium constructs 2. Genetic transformation of plants using direct methods 3. Gene transfer through electroporatioli 4. Gene transfer through micro-projectile bombardment iii. Confirmation of the transformants 1. GUS assay :Method for transient (3 - glucuronidase (GUS) gene expression by enzyme assays a. Histochemical assay b. Fluorometric assay (semi-quantitative assay) iv. Selection of stably transformed plants v. References 7. Appendixes 8. Appendix - 1: Guidelines for the preparation of a scientific research article 9. Appendix - 2: Electronically accessible data for Biotechnologists and Molecular biologists (Sequence Database Searching and Some Interesting World Wide Web Sites) 10. Appendix - 3: Cell and tissue culture needs 11. Appendix - 4: Preparation of Phenol 12. Saturation of phenol: 13. Water-saturation of phenol 14. Buffer-saturation of phenol 15. Appendix - 5: Suggested reading

This book was written to help researchers, teachers, and students to properly understand and use the major techniques and methods employed in cellular and molecular biology.

Each chapter covers an outline of theoretical concepts and principles of various protocols followed by a detailed practical methodology. Aspects on General Laboratory Principles together with a brief description of various instruments and methods are dealt with in the first chapter followed by the second chapter on qualitative and quantitative estimations in Plant Biochemistry and Physiology. The chapter on Cytology concerns crisp methods for cytological study, followed by the chapter on Genetics, which includes theory and problems besides statistical analysis. The chapter concerning Plant Molecular Genetics includes a sizeable collection of protocols dealing with various aspects of recombinant DNA technology and gene cloning. The sixth chapter deals with Plant Genetic Engineering, which includes tissue culture methods followed by protocols of gene transfer and molecular analysis of the putative transformants.