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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

2023 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
This is the final report for project 2030-21220-002-000D, Molecular Genetic Tools Advancing the Application of Biotechnology for Crop Improvement, which has been replaced by new project 2030-21220-003-000D, Molecular Resources for Enhanced Crop Biotechnology. For additional information, see the new project report. Objective 1 involved the generation of new molecular tools and new genetic strategies for effectively pyramiding multiple disease defense genes in citrus and potato to combat Huanglongbing and Zebra chip diseases. Fifteen plant disease defense-related genes have been inserted into various constructs in multiple configurations. These constructs were introduced into citrus and potato plants via genetic engineering and molecularly characterized. Four sets of ten independent transgenic verified potato events were generated, propagated and subsequently challenged with Zebra Chip disease. The tested plants were susceptible to the disease challenge. An additional four more sets of transgenic potato events carrying different defense gene constructs were generated, molecularly characterized and are undergoing disease susceptibility testing. Five citrus transgenic events were generated for four unique constructs carrying multiple defense genes. The resulting trees were verified to carry the introduced genes and were challenged with Huanglongbing. Although the tested citrus trees allowed pathogen growth and were susceptible to the disease, the trees carrying a construct with the hrf1 gene survived months longer than the control trees suggesting a modest level of disease tolerance. ARS researchers in Albany, California, made progress on Objective 2 through the identification and characterization of novel crop promoters that control gene expression in plants. Six rice promoters with callus and/or embryo-specific expression were identified. These promoters were introduced into rice and Brachypodium distachyon plants and shown to confer reporter gene expression specifically within callus or embryo tissues of multiple independent transgenic events. Also, three candidate promoters with floral-specific expression were also identified and two of these promoters exhibited the expected reproductive tissue-specific expression in multiple transgenic Brachypodium plants. In addition, twenty candidate transcriptional terminators sequences from various plant species were isolated and demonstrated to confer expression when combined with a functional promoter and gene of interest in a rapid testing assay using infiltrated plant leaves. These promoter and terminator sequences will be useful components for controlling the expression of desirable traits in genetically engineered crops. Progress was also made on Objective 3. The overall goal is to develop new methods that enable biotechnology-based crop improvement. For Sub-objective 3A, ARS researchers designed a strategy to insert very large cargo sequences into the Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY (GAANTRY) gene stacking system. This novel technology provides an efficient means of assembling large multigene constructs and genetically engineering crop plants. A modified GAANTRY “Donor” plasmid vector was designed and constructed to successfully receive and stably maintain large fragments of DNA. Selected Bacterial Artificial Chromosome vectors that carry between 16 kilobase pairs (kb) to more than 110 kb of cargo DNA sequence were successfully inserted into a stable GAANTRY construct. These large assemblies were used to generate transgenic plants and their molecular and phenotypic characterization demonstrated that the system was able to deliver complete constructs up to 77 kb in size. Although even larger constructs can be successfully assembled using the GAANTRY system, further technology development is needed to make the generation of biotech crops with constructs larger than 80 kb a feasible and efficient process. The GAANTRY gene stacking system was also shown to be useful for the genetic improvement of the important crops of potato and rice. Transgenic potato events carrying a 28kb construct were generated and characterized and transgenic rice plants were created using a novel 37 kb construct. The system was shown to efficiently generate high-quality transgenic rice plants expressing all the introduced traits. In addition, two novel strains, JGT105 (a derivative of Agrobacterium strain EHA105) and 1416G were developed and shown to enable GAANTRY gene assembly. These new strains will diversify the capability of the GAANTRY gene stacking system. ARS researchers in Albany, California, also made progress on Sub-objective 3B by designing and deploying a site-specific recombinase system that enables targeted transgene integration and marker removal in crop plants. This technology will allow gene insertion within a predetermined genomic location and remove variability due to genomic position effects. The technology will help provide stable, predictable expression of introduced traits, simplifying the testing and development of future genetically engineered crops. Transformation of potato and citrus lines was performed with a “founder line” construct and the resulting plants were screened to identify high quality transgenic events. The vector constructs for targeted integration were designed, assembled, and molecularly validated. More than 10 founder lines were transformed and examined for their capacity to receive a targeted integration construct. Candidate events were identified as having potentially achieved targeted integration and their molecular characterization is underway. To improve the efficiency of the process, a new strategy utilizing a novel selection scheme for targeted integration was designed and the related constructs assembled. In addition, improved expression systems for delivery of the recombinase enzymes that perform the desired integration and excision have also been developed.


Review Publications
Thilmony, R.L., Dasgupta, K., Shao, M., Harris, D., Hartman, J., Harden, L.A., Chan, R., Thomson, J.G. 2022. Tissue-specific expression of Ruby in Mexican lime (C. aurantifolia) confers anthocyanin accumulation in fruit. Frontiers in Plant Science. 13. Article 945738.
Huynh, J.N., Hotton, S.K., Chan, R., Syed, Y., Thomson, J.G. 2022. Evaluation of novel surfactants for plant transformation. BMC Research Notes. 15. Article 360.
Alabed, D., Huo, N., Gu, Y.Q., Thomson, J.G. 2023. Complete genome of Agrobacterium fabrum strain 1D1416. Microbiology Resource Announcements. 12(7).