Professional Documents
Culture Documents
Genetic Engineering in Plants
Genetic Engineering in Plants
Plants: Methodology
• Plant transformation with the Ti plasmid of
Agrobacterium tumefaciens
• Ti plasmid derived vector systems
• Physical methods of transferring genes to plants
(microprojectile bombardment)
• Chloroplast engineering
• Use of reporter genes in transformed plant cells
• Manipulation of gene expression in plants
• Production of marker-free transgenic plants
Why genetically engineer plants?
Figure 18.3
Ti plasmid structure
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.1
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Fig. 28-27
Crown Gall on
Tobacco
Figure 18.4
Conserved nucleotides at the right and left borders of the Ti plasmid are imperfect
direct repeats.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.6
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Fig. 18.7 The binary Ti plasmid system involves using a small
T-DNA plasmid (shown below) and a disarmed (i.e., no T-
DNA) Ti plasmid in A. tumefaciens
Clone YFG (your favorite gene) or
the target gene in the small T-DNA
plasmid in E. coli, isolate the plasmid
and use it to transform the disarmed
A. tumefaciens as shown.
(disarmed)
Disarmed
Ti plasmid
Table 18.1
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Table 18.2
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Fig. 18.10
Microprojectile
bombardment or
biolistic-mediated
DNA transfection
*
equipment
(a) lab version
(b) portable version
Figure 18.10
Microprojectile bombardment
(biolistics) apparatus
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.12
Figure 18.13
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Table 18.5
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Table 18.5 Some plant cell reporter and
selectable marker gene systems
Enzyme activity Selectable Reporter
marker gene
Neomycin phosphotransferase (kanr) Yes Yes
Hygromycin phosphotransferase (hygr) Yes Yes
Nopaline synthase No Yes
Octopine synthase No Yes
-glucuronidase (GUS) No Yes
Firefly luciferase No Yes
-galactosidase No Yes
Bromoxynil nitrilase Yes No
Green fluorescent protein (GFP) No Yes
Reporter Genes
• For how reporter genes work, see:
http://bcs.whfreeman.com/lodish7e/#800911__811966__
Figure 18.21
Rhizosecretion using a
plant promoter active in
roots and a signal peptide
sequence.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.26
Marker genes may be a safety issue, so it is best to remove them—here is one strategy.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Copyright © 2010 ASM Press
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904