Alternate Plant Transformation Strategies Using Chemical-Inducible Promoters
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• Agbiotech 99 Speakers ❖ 27 Speakers Prof. Nam-Hai Chua, Ph.D. Andrew W. Mellon Professor and Head, Laboratory of Plant Molecular Biology The Rockefeller University 1230 York Avenue New York, NY 10021-6399 United States 1962-1965 University of Singapore, B.Sc. in Botany and Biochemistry 1967 A.M. in Biology, Harvard University 1969 Ph.D. in Biology, Harvard University 1969-1971 Lecturer, Biochemistry Department, University of Singapore Medical School 1971-1973 Research Associate, Cell Biology Department, The Rockefeller University 1973-1977 Assistant Professor, Cell Biology Department, The Rockefeller University 1977-1981 Associate Professor, Cell Biology Department, The Rockefeller University 1981-1988 Professor and Head, Laboratory of Plant Molecular Biology, The Rockefeller University 1988-present Andrew W. Mellon Professor and Head, Laboratory of Plant Molecular Biology, The Rockefeller University Honors 1965-1967 Fulbright Fellow, U.S. Government Exchange Program 1966-1967 Harvard Arts and Sciences Fellowship, Harvard University 1967-1969 Maria Moors Cabot Foundation Fellowship, Harvard University 1967-1971 National Institutes of Health Research Career Development Award 1988 Appointed Andrew W. Mellon Professor, The Rockefeller University 1988 Elected Fellow of The Royal Society, UK 1992 Gadsby Flying Fellow, The Sainsbury Laboratory, John Innes Institute, UK 1998 Elected Fellow, Singapore National Academy of Science, Singapore Alternate Plant Transformation Strategies Using Chemical-Inducible Promoters Present methods for Agrobacterium-mediated plant transforma- tion generally employ an antibiotic selectable marker to distin- guish transgenic from non-transgenic plant regenerants. Because of concerns over the use of antibiotic resistance genes in the field, we sought to develop alternate plant transformation strategies that would obviate the use of such genes. We have previously constructed a chemical-inducible expression system (GVG) that is responsive to dexamethasone (DEX) 1 . This GVG system consists of a hybrid transcription factor that, after acti- vation by DEX, induces transcription of target genes placed downstream of cis-acting DNA elements. We have used this sys- tem to conditionally express the isopentenyl-transferase gene (ipt) from the Ti-plasmid of Agrobacteirum tumefaciens. In the presence of DEX, expression of ipt promoted shoot regeneration from tobacco and lettuce leaf discs on hormone-free media 2 . Over 40% of the transgenic tobacco shoots developed a strong root system within 20 days of transfer to root-inducing media without DEX. With very few exceptions (< 2%), the morpholo- gy of the transgenic tobacco plants appeared normal. The plants could be transferred easily to soil and they developed normal leaves, flowers, and seeds. We have used this combined system to transfer multiple transgenes (35S-luc and NOShpt) into tobacco, and segregation analysis demonstrated that the trans- genes were inherited along with GVG-ipt in a 3-1 manner. Moreover, ipt expression can be reversibly induced by DEX in the F1 progeny. Our results demonstrate the feasibility of using a chemical-inducible ipt to promote transgenic regenerants and recover transgenic plants without the use of antibiotic resistance markers. Extending this method further should allow selection of transformants using conditioned expression of plant genes to promote development of shoots, roots, embryos, or other plant organs. 1. Aoyama, T, et al. The Plant J. 11: 605-612 (1997). 2. Kunkel, T., et al. Nature Biotechology 17: 916-919 (1999). © 1999 Nature America Inc. • http://biotech.nature.com © 1999 Nature America Inc. • http://biotech.nature.com
Transcript of Alternate Plant Transformation Strategies Using Chemical-Inducible Promoters
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Agbiotech 99 Speakers ❖ 27
Speakers
Prof. Nam-Hai Chua, Ph.D.
Andrew W. Mellon Professor and Head,Laboratory of Plant Molecular BiologyThe Rockefeller University1230 York AvenueNew York, NY 10021-6399United States
1962-1965 University of Singapore, B.Sc. in Botany and Biochemistry1967 A.M. in Biology, Harvard University1969 Ph.D. in Biology, Harvard University1969-1971 Lecturer, Biochemistry Department, University of
Singapore Medical School1971-1973 Research Associate, Cell Biology Department,
The Rockefeller University1973-1977 Assistant Professor, Cell Biology Department,
The Rockefeller University 1977-1981 Associate Professor, Cell Biology Department,
The Rockefeller University 1981-1988 Professor and Head, Laboratory of Plant Molecular
Biology, The Rockefeller University 1988-present Andrew W. Mellon Professor and Head, Laboratory of
Plant Molecular Biology, The Rockefeller University
Honors1965-1967 Fulbright Fellow, U.S. Government Exchange Program1966-1967 Harvard Arts and Sciences Fellowship, Harvard University1967-1969 Maria Moors Cabot Foundation Fellowship,
Harvard University 1967-1971 National Institutes of Health Research Career
Development Award1988 Appointed Andrew W. Mellon Professor,
The Rockefeller University1988 Elected Fellow of The Royal Society, UK1992 Gadsby Flying Fellow, The Sainsbury Laboratory,
John Innes Institute, UK1998 Elected Fellow, Singapore National Academy of Science,
Singapore
Alternate Plant Transformation StrategiesUsing Chemical-Inducible Promoters
Present methods for Agrobacterium-mediated plant transforma-tion generally employ an antibiotic selectable marker to distin-guish transgenic from non-transgenic plant regenerants.Because of concerns over the use of antibiotic resistance genesin the field, we sought to develop alternate plant transformationstrategies that would obviate the use of such genes. We havepreviously constructed a chemical-inducible expression system(GVG) that is responsive to dexamethasone (DEX)1. This GVGsystem consists of a hybrid transcription factor that, after acti-vation by DEX, induces transcription of target genes placeddownstream of cis-acting DNA elements. We have used this sys-tem to conditionally express the isopentenyl-transferase gene(ipt) from the Ti-plasmid of Agrobacteirum tumefaciens. In thepresence of DEX, expression of ipt promoted shoot regenerationfrom tobacco and lettuce leaf discs on hormone-free media2.Over 40% of the transgenic tobacco shoots developed a strongroot system within 20 days of transfer to root-inducing mediawithout DEX. With very few exceptions (< 2%), the morpholo-gy of the transgenic tobacco plants appeared normal. The plantscould be transferred easily to soil and they developed normalleaves, flowers, and seeds. We have used this combined systemto transfer multiple transgenes (35S-luc and NOShpt) intotobacco, and segregation analysis demonstrated that the trans-genes were inherited along with GVG-ipt in a 3-1 manner.Moreover, ipt expression can be reversibly induced by DEX inthe F1 progeny. Our results demonstrate the feasibility of usinga chemical-inducible ipt to promote transgenic regenerants andrecover transgenic plants without the use of antibiotic resistancemarkers. Extending this method further should allow selectionof transformants using conditioned expression of plant genes topromote development of shoots, roots, embryos, or other plantorgans.
1. Aoyama, T, et al. The Plant J. 11: 605-612 (1997).2. Kunkel, T., et al. Nature Biotechology 17: 916-919 (1999).
© 1999 Nature America Inc. • http://biotech.nature.com©
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