ABSTRACT
Membranes ...............................................................................................633 36.2.3 Selection of Appropriate Vector, Promoter, Poly(A) Signal, Reporter Gene,
and Selectable Marker Gene ..................................................................................... 633 36.2.3.1 Reagents Needed ........................................................................................ 636
36.2.4 Preparation of Blunt-End DNA ................................................................................ 636 36.2.4.1 Reagents Needed ........................................................................................ 637
36.2.5 Preparation of Insert DNA Lacking the 5′ Phosphate Groups ................................. 637 36.2.5.1 Reagents Needed ........................................................................................ 637
36.2.6 Construction of Chimeric Gene Construct by Ligating Plasmid Vector and Insert DNA ......................................................................................................... 638 36.2.6.1 Reagents Needed ........................................................................................ 639
36.2.7 Transformation of an Appropriate Strain of Bacteria in Order to Amplify the Recombinant Plasmids........................................................................................ 639 36.2.7.1 Method 1: Introduction of Recombinant Plasmids into Agrobacterium
tumefaciens (Biotype 1: Ach5, A6, B6, C58, T37, Bo542, and 15955) by Transformation ...................................................................................... 639
36.2.7.2 Method 2: Introduction of Plasmids into Agrobacteria by Conjugation ....640 36.2.7.3 Method 3: Introduction of Plasmids into Agrobacteria
by Electroporation ...................................................................................640 36.2.7.4 Reagent Needed ......................................................................................... 641
36.2.8 Transformation of Plants with Recombinant Gene Constructs ................................ 641 36.2.8.1 Method 1: Gene Transfer by Agrobacterium tumefaciens ......................... 641 36.2.8.2 Method 2: Direct Gene Transfer to Protoplasts by Electroporation .......... 643 36.2.8.3 Method 3: Direct Gene Transfer by Microprojectile Bombardment .........646
36.2.9 Proof of Stable Transformation ................................................................................649 36.2.9.1 Evidence 1: Phenotypic and/or Functional Examination ...........................649 36.2.9.2 Evidence 2: Analysis of the Stable Integration of the Transgene .............. 650 36.2.9.3 Evidence 3: Analysis of the Expression of the Reporter Gene .................. 650
