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Agrobacterium Mediated Gene Transfer

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Puranjaya Pancholi

Agrobacterium-mediated gene transfer predominantly results in the integration of foreign genes at a single locus in the host plant, without associated vector backbone, and is also known to produce marker-free plants, which are the prerequisites for the commercialization of transgenic crops.

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Agrobacterium Mediated T-DNA Transfer 1
HISTORY 1907: Smith and Townsend demonstrated that Agrobacterium tumefaciens causes crown gall tumors. Crown gall tumor on an oak tree 1958: Arnim Braun (Rockfeller Univ) showed that tumors could be excised and propagated on in vitro culture media without addition of plant hormones. It was also demonstrated that tumor could propagate even when the bacteria was removed from the tumor. He proposed that the bacteria is able to transfer “Tumor Inducing Principle” (TIP) into plant cells 2
1968: Georges Morel (France) found that the tumors released compounds (opines) that Agrobacteria use as nutrients. Therefore, he proposed that: (1) Bacteria transfer opine synthesis genes into plant cell, and (2) The synthesized opine is transported back into the bacterial cell. Transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge- like connection between two cells 1971: Hamilton and Fall (University of Pennsylvania) reported that a virulent strain of Agrobacterium, when grown at 37oC lost virulence irreversibly. Allen Kerr (Adelaide, Australia) co-inoculated “avirulent” and “virulent” strains on sunflower, and re-isolated the “avirulent” strain. He found that the “avirulent” strain had become virulent. These results indicate that the TIP in Agrobactrium probably resides on a plasmid, which can be transferred between bacterial strains by conjugation. 1974: Ivo Zaenan isolated the megaplasmids of Agrobacterium. He called them Ti plasmids, which was later proven to be the TIP. 3
Features of Ti Plasmid • About 200 kb. • Opine catabolism region. • T-DNA segment (transferred into plant cell). • T-DNA carries gene for phytohormones (Cytokine and Auxin) and opines that are expressed in plant cell. • Vir region for T-DNA transfer (virA, virB, virC, virD, virE, virF, virH, and virG). • virD have five genes (virD1-virD2) and virB have eleven genes (virB1-virB11). 4
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Recognition and attachment Vir Genes Expression by host signals T-strand produce T-complex export into host Transport through cytoplasm and Nuclear T-DNA uncoating and integration 7
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9 Reference • Păcurar DI, Thordal-Christensen H, Păcurar ML, Pamfil D, Botez C, Bellini C. Agrobacterium tumefaciens: From crown gall tumors to genetic transformation. Physiological and Molecular Plant Pathology. 2011 Aug 1;76(2):76-81. • Tzfira T, Citovsky V. Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Current opinion in biotechnology. 2006 Apr 1;17(2):147-54. • Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J. Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic acids research. 1985 Jul 11;13(13):4777-88. • Zambryski P. Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annual review of genetics. 1988 Dec;22(1):1-30. • Wu H, Sparks C, Amoah B, Jones HD. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant cell reports. 2003 Mar;21(7):659-68.

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Agrobacterium Mediated Gene Transfer

  • 2. HISTORY 1907: Smith and Townsend demonstrated that Agrobacterium tumefaciens causes crown gall tumors. Crown gall tumor on an oak tree 1958: Arnim Braun (Rockfeller Univ) showed that tumors could be excised and propagated on in vitro culture media without addition of plant hormones. It was also demonstrated that tumor could propagate even when the bacteria was removed from the tumor. He proposed that the bacteria is able to transfer “Tumor Inducing Principle” (TIP) into plant cells 2
  • 3. 1968: Georges Morel (France) found that the tumors released compounds (opines) that Agrobacteria use as nutrients. Therefore, he proposed that: (1) Bacteria transfer opine synthesis genes into plant cell, and (2) The synthesized opine is transported back into the bacterial cell. Transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge- like connection between two cells 1971: Hamilton and Fall (University of Pennsylvania) reported that a virulent strain of Agrobacterium, when grown at 37oC lost virulence irreversibly. Allen Kerr (Adelaide, Australia) co-inoculated “avirulent” and “virulent” strains on sunflower, and re-isolated the “avirulent” strain. He found that the “avirulent” strain had become virulent. These results indicate that the TIP in Agrobactrium probably resides on a plasmid, which can be transferred between bacterial strains by conjugation. 1974: Ivo Zaenan isolated the megaplasmids of Agrobacterium. He called them Ti plasmids, which was later proven to be the TIP. 3
  • 4. Features of Ti Plasmid • About 200 kb. • Opine catabolism region. • T-DNA segment (transferred into plant cell). • T-DNA carries gene for phytohormones (Cytokine and Auxin) and opines that are expressed in plant cell. • Vir region for T-DNA transfer (virA, virB, virC, virD, virE, virF, virH, and virG). • virD have five genes (virD1-virD2) and virB have eleven genes (virB1-virB11). 4
  • 5. 5
  • 6. 6
  • 7. Recognition and attachment Vir Genes Expression by host signals T-strand produce T-complex export into host Transport through cytoplasm and Nuclear T-DNA uncoating and integration 7
  • 8. 8
  • 9. 9 Reference • Păcurar DI, Thordal-Christensen H, Păcurar ML, Pamfil D, Botez C, Bellini C. Agrobacterium tumefaciens: From crown gall tumors to genetic transformation. Physiological and Molecular Plant Pathology. 2011 Aug 1;76(2):76-81. • Tzfira T, Citovsky V. Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Current opinion in biotechnology. 2006 Apr 1;17(2):147-54. • Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J. Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic acids research. 1985 Jul 11;13(13):4777-88. • Zambryski P. Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annual review of genetics. 1988 Dec;22(1):1-30. • Wu H, Sparks C, Amoah B, Jones HD. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant cell reports. 2003 Mar;21(7):659-68.