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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Research Project #434430

Research Project: Molecular Genetic Tools Advancing the Application of Biotechnology for Crop Improvement

Location: Crop Improvement and Genetics Research

2022 Annual Report

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. Objective 4: Proposed research will create a new Objective 4: Understand the biochemical processes involved in smoke taint and apply plant biotechnology and genome editing to reduce smoke taint in commercial wine grape varietals. (NP301, C3, PS3A) Objective 4 will utilize existing resources for fast and efficient strategies to engineer wine grapes with reduced smoke-derived phenolic compounds and will coordinate findings with a growing network of smoke taint researchers in the U.S. Anticipated products include new transcriptomics and metabolomics studies as well as biotechnology-based approaches to genetically modify pathways involved.

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.

Progress Report
Substantial progress was made on Objective 1. New molecular tools and new genetic strategies were generated to effectively introduce and stack multiple disease resistance genes into citrus and potato. This technology will be potentially useful in combatting Huanglongbing (HLB; citrus) and zebra chip (potato) diseases as well as the engineering an array of other desirable traits like improved stress tolerance and/or improved end-use quality. A series of five unique multi-gene transgenic stacks have been designed and assembled to increase pathogen detection and transformed into potato and/or citrus. The gene stacks are designed to increase the sensing of pathogen-derived molecules and trigger defense responses. The engineered plants have been validated and sent to collaborators for further evaluation. Limited progress was made on Objective 2. Research focused on the characterization of candidate transcriptional terminator sequences has been pursued with several novel regulatory sequences. Selected terminators were isolated and cloned into a test construct and initially tested in a transient expression assay in tobacco leaves, but the results from both control constructs and the test sequences displayed little variation in expression in the transient expression assay. Efforts were made to generate alternative test constructs that would allow the functional characterization of the terminator sequences within stably genetically engineered plants, but efforts pursuing that goal were delayed due to the pandemic. This research effort will be reinitiated in the future. For Objective 3 progress was made in developing new methods that enable biotechnology-based crop improvement. For Sub-objective 3A, previously a strategy was designed and successfully implemented to insert large cargo sequences into the GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) gene stacking system. This technology provides an efficient means of assembling large multigene constructs and transforming them into crop plants. The transformation of several large assemblies into plants and the molecular and phenotypic characterization of the resulting transgenic plants is complete demonstrating that the GAANTRY system can produce transgenic plants with large constructs greater than 50 kilobases in size. Further technology development is needed to make the generation of biotech crops with constructs larger than 80 kilobases a more efficient process. Sub-objective 3B deploys the use of recombinase-based technology to precisely integrate sequences into the crop genome. The technology provides an efficient means of inserting genes of interest into a target location. This is advantageous because the use of a predetermined location removes the genomic ‘position effect’ and provides stable, predictable expression of the introduced genes, simplifying the testing and utilization of the genetically engineered plants. For this purpose, ‘Founder lines’ in potato and citrus were previously created and molecularly verified. More than 10 founder lines have been examined for their capacity for receiving a targeted integration construct. The characterization of resulting potato plants is currently underway.


Review Publications
Mehlferber, E., McCue, K.F., Ferrel, J.E., Koskella, B., Khanna, R. 2022. Temporally selective modification of the tomato rhizosphere and root microbiome by volcanic ash fertilizer containing micronutrients. Applied and Environmental Microbiology. 88(7). Article e00049-22.
Huo, N., Gu, Y.Q., McCue, K.F., Alabed, D., Thomson, J.G. 2021. Draft genome sequence of Agrobacterium fabrum strain 1D1104. Microbiology Resource Announcements. 10(48). Article e00996-21.