Location: Crop Improvement and Genetics Research
Project Number: 2030-21220-002-00-D
Project Type: In-House Appropriated
Start Date: Mar 26, 2018
End Date: Mar 25, 2023
The long-term goal of this project is to develop useful biotechnology tools that enable the effective and precise genetic improvement of crop plants. Specifically, during the next five years we will focus on the three following objectives: Objective 1: Generate new molecular tools and new genetic strategies for effectively introducing and pyramiding multiple disease defense genes into citrus and potato to combat Huánglóngbìng and Zebra Chip diseases along with other priority traits. Objective 2: Identify and characterize new transcriptional control sequences (promoters and terminators) chosen for the precise control of gene transcription (tissue and/or developmental/environmental specificity) in crop plants containing single or multiple transgenes. Objective 3: Develop new methods that permit ARS biotechnology tools to be used for germplasm improvement in prioritized target crops and varieties. Subobjective 3A: Examine the capacity of the GAANTRY gene stacking system to enable the assembly and transfer of large arrays of sequences into transgenic plants. Subobjective 3B: Design and deploy a site-specific recombinase system that enables targeted transgene integration and marker removal in crop plants.
Candidate plant defense response genes will be introduced into potato and citrus plants using established methods for Agrobacterium–mediated transformation. The defense genes will either be constitutively expressed throughout the plant or expressed specifically in the phloem, the site of infection. Ten or more independent events for each candidate defense gene will have their susceptibility to zebra chip (in potato) or Huánglóngbìng (in citrus) evaluated. Candidate promoters with useful cell-type/organ expression specificities will be identified from crop plants. The candidate promoters will be fused to a reporter gene and transformed into rice, using Agrobacterium and/or other established transformation methods. Novel transcription terminator sequences will also be isolated from crop plants and fused to a reporter gene. The functionality of these promoter and terminator testing constructs will be examined in transient expression assays and stably transformed transgenic plants. Reporter gene expression levels will be quantitatively measured in major organs and compared to identify the sequences that provide the highest levels of transgene products while preserving promoter expression specificity. Plant molecular biological techniques will be used to further develop sophisticated biotechnology tools and methods for the improvement of crops. Transformation constructs of various large sizes (greater than 20 kilo base pairs) will be assembled using the site-specific recombinase-based GAANTRY gene stacking system. These constructs will be evaluated for their stability in bacteria and used to generate transgenic plants. The resulting genetically engineered plants will be molecular characterized to determine the effective capacity of the gene stacking technology. In parallel, technology enabling targeted integration and precise marker removal in transgenic plants will be developed and evaluated. “Exchange” T-DNA vectors will be constructed and transformed into “target” transgenic plants. Selection and molecular screening will be used to identify plants in which the incoming DNA has replaced the original transgenic locus (Recombinase-Mediated Cassette Exchange or RMCE). The efficiencies of different combinations of the unidirectional recombinases in performing RMCE will be compared.