Grant Information

IMPROVED METHODS OF CITRUS GENETIC TRANSFORMATION

Sponsoring Institution Agricultural Research Service/USDA
Status ACTIVE
Funding Source USDA COOPERATIVE AGREEMENT
Reporting Frequency Annual
Project Director BLECHL A E
Accession Number 432083
Project Number 2030-21000-020-23S
Dates 2016-12-01 - 2017-09-30
Recipient Organization UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Keywords agrobacterium-mediated
budwood
citrus
recombination
site-specific
transformation
Research Effort Applied (0%)
Basic (100%)
Developmental (0%)
Classification Parameters
Knowledge AreaSubject of InvestigationField of SciencePercent
201 - Plant Genome, Genetics, and Genetic Mechanisms 1510 - Corn (for sweetcorn use 1480) 1040 - Molecular biology 30%
201 - Plant Genome, Genetics, and Genetic Mechanisms 1530 - Rice 1040 - Molecular biology 20%
201 - Plant Genome, Genetics, and Genetic Mechanisms 2410 - Cross-commodity research--multiple crops 1040 - Molecular biology 20%
201 - Plant Genome, Genetics, and Genetic Mechanisms 1540 - Hard red winter wheat 1040 - Molecular biology 10%
201 - Plant Genome, Genetics, and Genetic Mechanisms 1541 - Hard red spring wheat 1040 - Molecular biology 10%
201 - Plant Genome, Genetics, and Genetic Mechanisms 1542 - Soft red wheat 1040 - Molecular biology 10%
Goals / Objectives
Improve transformation methods for citrus. In particular, decrease the time it takes to obtain flowering transformed citrus plants and broaden the range of varieties that can be transformed.
Methods (unparsed)

Standard methods of Agrobacteria transformation will be applied to mature meristematic tissues such as budwood. This tissue type tends to flower/fruit more rapidly, allows a monoembryonic citrus cultivar to be transformed, and provides an avenue for Recombinase-Mediated Cassette Exchange (RMCE) to stack genes in genomes of citrus plants. Research will consist of overexpressing novel and proven embryo induction genes during transformation to stimulate regeneration and looking for increased rates of callus formation and/or shoot regeneration. In addition, novel and proven anti-apoptotic genes will be co-transformed to repress the programed cell death response to Agrobacterium infection. During the assay development process, potential Huanglongbing (HLB) disease resistance genes will also integrated into the genome. The mature tissue explant will be trimmed down to expose the pluripotent cells and incubated with Agrobacteria under conditions that facilitate transfer of the T-DNA to plant cells. Initial studies will focus on four cultivars: ¿Carrizo¿, cocktail grapefruit, Mexican lime, and ¿W. Murcott¿. After transformation, various parameters will be evaluated during regeneration of plantlets from the cells. These will include comparisons among types and concentrations of plant hormones. Transformants that are recovered will contain DNA sequences that will facilitate future research for targeted integration of new DNA into the same genome locations. Those that contain potential HLB disease resistance genes will be tested by a collaborator at the University of California, Davis for disease resistance in a containment greenhouse.

Methods
Standard methods of Agrobacteria transformation will be applied to mature meristematic tissues such as budwood. This tissue type tends to flower/fruit more rapidly, allows a monoembryonic citrus cultivar to be transformed, and provides an avenue for Recombinase-Mediated Cassette Exchange (RMCE) to stack genes in genomes of citrus plants. Research will consist of overexpressing novel and proven embryo induction genes during transformation to stimulate regeneration and looking for increased rates of callus formation and/or shoot regeneration. In addition, novel and proven anti-apoptotic genes will be co-transformed to repress the programed cell death response to Agrobacterium infection. During the assay development process, potential Huanglongbing (HLB) disease resistance genes will also integrated into the genome. The mature tissue explant will be trimmed down to expose the pluripotent cells and incubated with Agrobacteria under conditions that facilitate transfer of the T-DNA to plant cells. Initial studies will focus on four cultivars: 'Carrizo', cocktail grapefruit, Mexican lime, and 'W. Murcott'. After transformation, various parameters will be evaluated during regeneration of plantlets from the cells. These will include comparisons among types and concentrations of plant hormones. Transformants that are recovered will contain DNA sequences that will facilitate future research for targeted integration of new DNA into the same genome locations. Those that contain potential HLB disease resistance genes will be tested by a collaborator at the University of California, Davis for disease resistance in a containment greenhouse.