Progress 07/15/07 to 07/14/10
Outputs
OUTPUTS: The goal of this project is to develop and test Direct Repeat Induced Gene Silencing (DRIGS) as a tool for functional genomics approaches in tomato. Experiments were designed to devise strategies for practical application of DRIGS in tomato. We have generated a large number of Micro-Tom transformants using a DRIGS based universal vector for random gene knock down / gene inactivation. These transformants were visually screened for mutant phenotypes. Many putative mutants ranging from very early flowering and fruit formation to abnormal morphology were identified. We have also tested DRIGS for targeted silencing of known genes. During the course of this project we have identified a total of 121 phenotypic tomato mutants, 56 of which appear to be silencing induced. Obtained flanking DNA sequences of T-DNA insertion sites from 25 mutants. We have also established the utility of DRIGS SIL vectors for targeted silencing: Targeted silencing of Phytoene desaturase gene (PDS) produced all white photosynthesis deficient T1 progeny, while silencing of lycopene produced smaller yellow-green tomato fruits. However, silencing of 7 EST sequences did not generate obvious visual phenotype. PARTICIPANTS: Project Personnel: Amber Davis: Lab technician, worked on tomato tissue culture. Heather Janzen: Lab technician, worked on plant cell culture. Leny Galvez: Graduate student, worked on Tomato transformation. Trung Nguyen: Undergraduate student, worked on media preparation and Green House plant care. Collaborations: I have established new collaborations with labs at USDA, Beltsville, and Colorado State University, Fort Collins, to test and use our DRIGS based silencing system. TARGET AUDIENCES: Target audiences for this project were research scientists and plant breeders. Data from the project were used in various seminars, class room teaching and training of graduate and under graduate students. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Mutant phenotype-driven approach using DRIGS represent a potentially efficient route for generation of large number of transformants that can be screened for novel phenotypes. With the help of our DRIGS vectors silencing can be used to associate gene sequences with function. Utilization of a unique and efficient DRIGS based system is invaluable for functional genomic analysis in tomato. Successful completion of this proposal will allow quick analysis of gene function and may help generate functional maps of tomato genome.
Publications
- Mitra, A., Ma, C., Han, Jigang, H., and Galvez, L (2010) Direct Repeat Induced Gene Silencing: A Versatile Functional Genomics Tool, Journal of Agricultural Biochemistry (in press).
- Mitra, A, Han, J., Zhang, J.J., Mitra, A (2009) The intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes functions as a bidirectional promoter. Planta 229:1015-1022.
- R. S. Ranu and Mitra, A (2009): Gene Expression by Translational-Coupling in Plant and Animal Viruses. Indian Phytopathology S:11
- Mitra, A and Galvez, L (2009) Transgenic Plants for Disease Resistance: A Review, In M. K. DasGupta (Ed) Principles of Plant Pathology, Allied Publishers.
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Progress 07/15/08 to 07/14/09
Outputs
OUTPUTS: The goal of this project is to develop and test Direct Repeat Induced Gene Silencing (DRIGS) as a tool for functional genomics approaches in tomato. Experiments were designed to devise strategies for practical application of DRIGS in tomato. We have generated a large number of Micro-Tom transformants using a DRIGS based universal vector for random gene knock down / gene inactivation. These transformants were visually screened for mutant phenotypes. Many putative mutants ranging from very early flowering and fruit formation to abnormal morphology were identified. To determine if the mutations were silencing induced, the selected mutants were crossed with two tomato lines containing two different silencing suppressors. Seeds from these crosses were used to obtain plants that were screened for the suppression of the mutant phenotype observed before. If the mutations are silencing-linked, the genes involved can be identified by sequencing the chromosomal DNA flanking the T-DNA insertion sites and BLASTing against plant genome databases. We have obtained over 600 transgenic tomato lines using the DRIGS vector that generates random silencing based mutants. We have identified 78 putative mutants from these 600 transgenic lines. The mutants have a wide array of phenotypes affecting growth, flowering and fruit formation. All of these 78 mutants were crossed with silencing suppressor tomato lines expressing two different viral silencing suppressors. Forty nine mutants showed either partial or full reversion in the suppressor background. The flanking sequences at the point of insertion of these mutants are being determined by TAIL and Inverse PCR. The deduced sequences will be used to search databases for match with known genes. We have made the silencing vectors freely available to the research community. We will prepare a poster for the 2010 PAG that will have a description of all the putative mutants. We also have a limited supply of seeds of these mutants. PARTICIPANTS: The project was used to train a graduate student and a undergraduate student. In addition, two part-time technicians were also supported. Following is a list of project participants: Amber Davis: Lab technician, worked on tomato tissue culture. Heather Janzen: Lab technician, worked on plant cell culture. Leny Galvez: Graduate student, worked on Tomato transformation. Trung Nguyen: Undergraduate student, worked on media preparation and Green House plant care. TARGET AUDIENCES: Target audiences for this project are scientists and breeders that are working on functional genomics and tomato breeding. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Plant mutagenesis is a key element of gene function studies. We have established that a phenotype-driven approach using DRIGS represents a potentially efficient route for generating a large number of transformants that can be screened for novel phenotypes. With the help of our DRIGS vectors silencing can be used to associate gene sequences with function. Hence, DRIGS can be used for functional genomic analysis in tomato. On going analyses of the mutants will also identify genes associated with the observed phenotypes. These genes might be useful for modulating agronomic traits of tomato.
Publications
- Mitra, A., Han, J., Z, Zhang and A. Mitra (2009). The intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes functions as a bidirectional promoter. Planta 229: 509-516
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Progress 07/15/07 to 07/14/08
Outputs
OUTPUTS: The goal of this project is to develop and test DRIGS as a tool for functional genomics approaches in tomato. Experiments were designed to devise strategies for practical application of DRIGS in tomato. We have generated a large number of Micro-Tom transformants using a DRIGS based universal vector for random gene knock down / gene inactivation. These transformants were visually screened for mutant phenotypes. Many putative mutants ranging from very early flowering and fruit formation to abnormal morphology were identified. Twenty mutants were selected from this pool for further study. To determine if the mutations were silencing induced, the selected mutants were crossed with two tomato lines containing two different silencing suppressors. Seeds from these crosses were used to obtain plants that will be screened for the suppression of the mutant phenotype observed before. If the mutations are silencing-linked, efforts will be made to identify genes from the phenotypic mutants. A poster is being prepared to describe this technique and outputs for an upcoming international scientific meeting. PARTICIPANTS: This project was used to train a graduate student Leny Galvez, and an undergraduate student Trung Nguyen. TARGET AUDIENCES: Target audiences for this project are scientists and breeders that are working on functional genomics and tomato breeding. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We obtained seeds from forty crosses with two suppressor lines. Seeds from all these crosses uniformly produced very big tomato plants. This is surprising, as wild type Micro-Tom plants as well as primary Micro-Tom transformants only grow to a height of about six inches. Although we purchased Micro-Tom seeds from a reputable and well known commercial outlet, it seems that the Micro-Tom lines were unstable and reverted to a tall phenotype. We have obtained seeds of a stable Micro-Tom line from a colleague and are repeating all the transformation experiments. We might, however, be able to use some of the existing crosses to determine the role of silencing in some mutants such as early flowering and fruit-set. Preliminary observations suggest that very early flowering and fruit formation in mutant Micro-Tom are induced by DRIGS. Plant mutagenesis is a key element of gene function studies. Phenotype-driven approaches using DRIGS represent a potentially efficient route for generation of large number of transformants that can be screened for novel phenotypes. With the help of our DRIGS vectors silencing can be used to associate gene sequences with function. Accordingly, utilization of a unique and efficient DRIGS based system is invaluable for functional genomic analysis in tomato.
Publications
- No publications reported this period
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