Chapter 18 Genetic Engineering of Plants Methodology Plant

Why genetically engineer plants? • To improve the agricultural or horticultural value of plants

Plant transformation with the Ti plasmid of Agrobacterium tumefaciens • A. tumefaciens is a

The Ti plasmid of Agrobacterium tumafaciens and the transfer of its T-DNA to the

Fig. 18. 3 The Ti plasmid of Agrobacterium tumafaciens and its T-DNA region containing

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 3 Ti plasmid structure Molecular

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 1 Infection of a plant

Fig. 28 -27 Crown Gall on Tobacco Fig. 18. 1 Infection of a plant

Figure 18. 2 and 18. 3 Ti plasmid structure and function. Note the woundinduced

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 4 Conserved nucleotides at the

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 6 Chemical structures of three

Fig. 18. 7 The binary Ti plasmid system involves using a small T-DNA plasmid

Clone YFG (your favorite gene) or the target gene in the small T-DNA plasmid

Chapter 18 Genetic Engineering of Plants: Methodology Table 18. 1 Molecular Biotechnology: Principles and

Chapter 18 Genetic Engineering of Plants: Methodology Table 18. 2 Molecular Biotechnology: Principles and

Fig. 18. 10 Microprojectile bombardment or biolistic-mediated DNA transfection equipment (a) lab version (b)

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 10 Microprojectile bombardment (biolistics) apparatus

Chapter 18 Genetic Engineering of Plants: Methodology Chloroplasts can be genetically engineered using microparticle

Chapter 18 Genetic Engineering of Plants: Methodology Table 18. 5 Molecular Biotechnology: Principles and

Table 18. 5 Some plant cell reporter and selectable marker gene systems Enzyme activity

Reporter Genes • For how reporter genes work, see: http: //bcs. whfreeman. com/lodish 7

Manipulation of gene expression in plants • Strong, constitutive promoters (35 S Cauliflower mosaic

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 21 Rhizosecretion using a plant

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 26 Marker genes may be

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Chapter 18 -Genetic Engineering of 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? • To improve the agricultural or horticultural value of plants • To serve as living bioreactors for the production of economically important proteins or metabolites • To provide a renewable source of energy (biofuels) • To provide a powerful means for studying the biological action of genes and gene products

Plant transformation with the Ti plasmid of Agrobacterium tumefaciens • A. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in crown gall (cancer) tumors • Tumor formation is the result of the transfer, integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the TDNA (transferred DNA) • The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found in A. tumefaciens

The Ti plasmid of Agrobacterium tumafaciens and the transfer of its T-DNA to the plant nuclear genome

Fig. 18. 3 The Ti plasmid of Agrobacterium tumafaciens and its T-DNA region containing eukaryotic genes for auxin, cytokinin, and opine production.

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 3 Ti plasmid structure Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press American Society for Microbiology 1752 N St. NW, Washington, DC 20036 -2904

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 1 Infection of a plant with A. tumefaciens and formation of a crown gall tumor. Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press American Society for Microbiology 1752 N St. NW, Washington, DC 20036 -2904

Fig. 28 -27 Crown Gall on Tobacco Fig. 18. 1 Infection of a plant with A. tumefaciens and formation of crown galls

Figure 18. 2 and 18. 3 Ti plasmid structure and function. Note the woundinduced plant phenolics induce the vir genes on the Ti plasmid. The infection process: 1. Wounded plant cell releases phenolics and nutrients. 2. Phenolics and nutrients cause chemotaxic response of A. tumefaciens 3. Attachment of the bacteria to the plant cell. 4. Certain phenolics (e. g. , acetosyringone, hydroxyacetosyringone) induce vir gene transcription and allow for T-DNA transfer and integration into plant chromosomal DNA. 5. Transcription and translation of the T-DNA in the plant cell to produce opines (food) and tumors (housing) for the bacteria. 6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use opines as a C, H, O, and N source.

Chapter 18 Genetic Engineering of Plants: Methodology 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 Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press American Society for Microbiology 1752 N St. NW, Washington, DC 20036 -2904

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 6 Chemical structures of three opines produced by plants. Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press 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 TDNA) 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 Plant genetic engineering with the binary Ti plasmid system Transgenic plant

Chapter 18 Genetic Engineering of Plants: Methodology Table 18. 1 Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press 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 Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press 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 When the helium pressure builds to a certain point, the plastic rupture disk bursts, and the released gas accelerates the flying disk* with the DNAcoated gold particles on its lower side. The gold particles pass the stopping screen, which holds back the flying disk, and penetrate the cells of the plant. *

Chapter 18 Genetic Engineering of Plants: Methodology Figure 18. 10 Microprojectile bombardment (biolistics) apparatus Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press American Society for Microbiology 1752 N St. NW, Washington, DC 20036 -2904

Chapter 18 Genetic Engineering of Plants: Methodology Chloroplasts can be genetically engineered using microparticle bombardment. Figure 18. 12 Figure 18. 13 Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press 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 Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press 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 marker Reporter gene Neomycin phosphotransferase (kanr) Yes Hygromycin phosphotransferase (hygr) Yes Nopaline synthase No Yes Octopine synthase No Yes b-glucuronidase (GUS) No Yes Firefly luciferase No Yes b-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/lodish 7 e/#800911__811966__ • GFP Researchers Win Nobel Prize (October 8, 2008) Osamu Shimomura, Martin Chalfie, and Roger Tsien won the Nobel Prize in chemistry for their work on green flourescent protein, a tool that has become ubiquitous in modern biology as a tag and molecular highlighter, vastly improving our ability to understand what goes on inside cells. • Perhaps you may even want to see a 10 minute You. Tube video on GFP; if so please see http: //www. youtube. com/watch? v=Sl 2 PRHGp. Yu. U

Manipulation of gene expression in plants • Strong, constitutive promoters (35 S Cauliflower mosaic virus promoter or 35 S Ca. MV or 35 S) • Organ and tissue specific promoter (e. g. , the leaf-specific promoter for the small subunit of the photosynthetic enzyme ribulosebisphosphate carboxylase or rbc) • Promoterless reporter gene constructs to find new organ- and tissue-specific promoter (see Fig. 18. 15) • Inducible promoters • Secretion of transgene products by inclusion of a signal peptide sequence between a root promoter and YFG and growing the transgenic plant hydroponically (YFG product will be secreted)

Chapter 18 Genetic Engineering of Plants: Methodology 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 Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press 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 Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Copyright © 2010 ASM Press American Society for Microbiology 1752 N St. NW, Washington, DC 20036 -2904