THE EXPERIMENTAL USE OF AGRO-BASED PLASMIDS FOR TRANSFER OF NOVEL GENES INTO PLANTS

THE EXPERIMENTAL USE OF AGRO-BASED PLASMIDS FOR TRANSFER OF NOVEL GENES INTO PLANTS

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LEARNING MODULE 2: THE EXPERIMENTAL USE OF AGRO-BASED PLASMIDS FOR TRANSFER OF NOVEL GENES INTO PLANTS OUTLINE Modifications Made to the Agro Ti Plasmid to Make it A Useful Plant Transformation Vector Steps Required for Preparing Agro For Plant Transformation Modifications to the Plant Host to Broaden the Host Range of Agro LEARNING OBJECTIVES Understand what key changes had to be made to the Agro Ti plasmid for the experimental transfer of novel genes into plants Understand (in basic terms) the binary plasmid transformation system Understand some mechanisms of modifying plant hosts so that they become susceptible to Agro-mediated gene transfer: increasing the host range of Agro to agronomically useful plant spp. New research (early stages) 1 BISC 457LM2
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Features of Ti plasmid: Amenable to Exploitation in the Construction of Plant Transformation Vectors The onc genes are not required for transfer of T-DNA to plant cell nor for T-DNA integration within the nuclear genome The vir region of the Ti plasmid functions in trans (can be present on a separate plasmid) T-DNA vir RB LB The DNA inserted between the 24 bp border repeats of the T-DNA (whether the borders are natural or synthetic) is transferred to the plant cell The foreign DNA integrated into the genome can be stably inherited in a Mendelian manner 2 BISC 457LM2
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Major Modifications to Ti Plasmid to Make it A Useful Plant Transformation Vector 1. REMOVAL OF ONC GENES FROM T-DNA (KEEPING BORDER REPEAT SEQUENCES INTACT) WHY? Cannot regenerate a whole plant from tumor cells Tumor = undifferentiated plant cells continously making auxin/cytokinin Excised all these genes LB RB opine synthase onc genes 3 BISC 457LM2
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2. REPLACEMENT OF ONC GENES WITH PLANT SELECTABLE MARKER GENES Kanamycin resistance Insertion of neomycin phosphotransferase II gene (bacterial antibiotic resistant gene) flanked by 5’ 3’ NPT II gene LB RB WHY? Mechanism to differentiate between transformed plant cells and untransformed plant cells Direct selection is possible by placing selectable marker agent (antibiotic) in culture medium used for plant regeneration 4 BISC 457LM2
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Basis of Kanamycin Resistant Phenotype Plant cells – normally sensitive to Kan (the antibiotic inhibits protein synthesis and protein translocation across membranes) Successfully transformed plant cells will have integrated the NPTII gene Æ synthesis of NPT II enzyme NPT II enzyme phosphorylates Kan thereby inactivating it Æ no longer able to inhibit plant growth
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This note was uploaded on 08/01/2009 for the course HORT hor-11-12 taught by Professor Park during the Spring '09 term at A.T. Still University.

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THE EXPERIMENTAL USE OF AGRO-BASED PLASMIDS FOR TRANSFER OF NOVEL GENES INTO PLANTS

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