Plant Genetic Engineering

This book on “Plant Genetic Engineering – Recent Methods and Protocols” has been written for the fresher's especially undergraduate, MSc and PhD students working in the field of Plant Molecular Biology and Biotechnology. The basic techniques used in Molecular Biology and Biotechnology has been presented in very simple and lucid manner along with flow-chart for better understanding of the methods. The protocols routinely used in our lab have been presented along with information on troubleshoot, so that the experiment can be performed by the students with repeatable results and high accuracy. A total of 19 experiments have been described in this book with introductory information, chemicals used, basic steps, calculation of results, troubleshoot and flow-chart. The book has been designed into five different parts - first part consist of detailed information about the evolution of Genetic Engineering and basic tools like electrophoretic techniques, isolation of plant genomic DNA and total RNA, cDNA synthesis, etc. Second part explains the techniques used for the cloning and expression analysis of genes like cloning, transformation, competent cell preparation, northern blotting and quantitative

Real-Time PCR expression analysis. Third part consists of most routinely used protein characterization techniques like SDS-PAGE and western blotting. Four parts has been dedicated to heterologous expression and purification of recombinant proteins, wherein all steps has been explained in simple and lucid manner. Last part consist of two chapters dedicated to genome editing – the advance tool of CRISPR/ Cas-9 followed by chapter on tissue culture. Lastly, a chapter has been included on biosafety and ethical issues in Genetic Engineering. A glossary has also been included in the last to comprehend the terms most commonly used in the area of Genetic Engineering.  All attempts have been made to make the book simple and user-friendly. This book will be a valuable piece of information for students, teachers, scientists, and researchers working in the area of Molecular Biology and Biotechnology.

Contents
About the Authors v
Preface ix
Chapter 1
An Introduction to Genetic Engineering 1-14
Chapter 2
Electrophoretic Techniques 15-50
Chapter 3
Plant Genomic DNA – Isolation and
Purity Analysis 51-60
Chapter 4
Southern Blotting 61-71
Chapter 5
Total RNA Isolation and its Quality
Analysis 72-81
Chapter 6
Northern Blotting 82-87
Chapter 7
Polymerase Chain Reaction 88-100
Chapter 8
Quantitative Real-Time PCR 101-114
Chapter 9
Cloning of DNA Insert 115-126
xii Contents
Chapter 10
Competent Cell Preparation 127-132
Chapter 11
Elution of DNA Fragments 133-136
Chapter 12
Isolation of Plasmid 137-140
Chapter 13
Heterologous Expression of Candidate Gene 141-150
Chapter 14
SDS – Polyacrylamide Gel Electrophoresis 151-156
Chapter 15
Western Blot Analysis 157-159
Chapter 16
Designing of Small Guide RNAs and Cloning
into Destination Vector 160-174
Chapter 17
Analysis of Genome Editing Efficiency and
Mutation Detection in Genome Edited Plants 175-181
Chapter 18
Tissue Culture: Plant Development and
Manipulation of Tissues 182-208
Appendix-I
Preparation of Buffered Phenol 209-210
Appendix-II
35S-labelled Nucleotides in Sequencing 211-213
Appendix-III
Common Conversion for Nucleic Acid 214-218
Glossary of Terms 219-270

Dr. Ranjeet Ranjan Kumar
Senior Scientist, Division of Biochemistry
Indian Agricultural Research Institute, New Delhi

Dr. Gyanendra K. Rai
Assistant Professor, School of Biotechnology
Sher-E-Kashmir University of Agricultural Sciences and Technology of Jammu
Chatha, Jammu

Dr. Vinutha T
Scientist, Division of Biochemistry
Indian Agricultural Research Institute, New Delhi

Dr. Suneha Goswami
Scientist, Division of Biochemistry
Indian Agricultural Research Institute, New Delhi

Dr. Shelly Praveen
Head and Principal Scientist, Division of Biochemistry
Indian Agricultural Research Institute, New Delhi