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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

2020 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.

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
Progress was made on Objective 1 to generate new molecular tools and new genetic strategies for effectively introducing and pyramiding multiple disease defense 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 of an array of other desirable priority traits like improved stress tolerance and/or improved end-use quality. Previously, multiple transformation vectors containing different candidate defense genes were assembled, validated, and transformed into potato and citrus plants. Approximately 100 transgenic citrus plants from 11 different defense gene combinations were shipped to the U.C. Davis containment greenhouse facility for HLB challenge. Tissue is being collected on a monthly basis and will be processed for the HLB titer once the ‘shelter in place’ order is lifted. Approximately 60 transgenic potato plants were produced from six different defense gene combinations and were shipped to Wapato, Washington, for zebra chip inoculation. To date, none of the defense gene combinations has shown improved tolerance to disease. Previously, six novel candidate rice promoters with callus and/or embryo-specific expression were selected and cloned in support of Objective 2. These isolated promoter control elements were then fused to a reporter gene and used for transformation into rice and the model grass species Brachypodium distachyon. Experiments qualitatively evaluating and quantifying the performance of the selected promoters in transgenic plants has been initiated and preliminary results suggest that several of the promoters control strong expression in the expected plant tissues. Further characterization will be conducted when sufficient transgenic plant material becomes available and lab work is allowed to resume. Progress was also made on Objective 3. The overall goal of this objective is to develop new methods that enable biotechnology-based germplasm improvement in prioritized target crops and varieties. For Sub-objective 3A, 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 new technology provides an efficient means of assembling large multigene constructs and transforming them into 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 (BAC) vectors that carry between ~10 kilobase pairs (kb) to more than 70 kb pairs of plant genomic DNA sequence, were inserted into a modified Donor vector and assembled into GAANTRY plant transformation constructs. The transformation of several of these large constructs into plants was initiated and transgenic Arabidopsis plants were recovered. The molecular and phenotypic characterization of these transgenic plants has been initiated. Progress was also made on Sub-objective 3B to design and deploy a site-specific recombinase system that enables targeted transgene integration and marker removal in crop plants. Transformation of potato and citrus lines was performed with a “Founder Line” construct. The resulting transgenic citrus and potato lines were screened and plants with low or single-copy transgene insertion events were identified. These lines have since been cultivated for gene targeting assays and were re-transformed and examined for the introduction of a new plant antibiotic resistance trait and the expected loss of reporter gene expression (which indicates the desired targeted gene integration and marker removal occurred). Multiple events were identified as having achieved a complete gene targeting event. Currently, the potato events with the expected traits are being grown in culture, awaiting molecular analysis to confirm genome targeting. The citrus experiments had initially generated encouraging results, but the effort is currently halted due to the ‘shelter in place’ order. These experiments will be reinitiated when active lab research is allowed to resume.


Review Publications
Hotton, S., Kammerzell, M., Chan, R., Hernandez, B.T., Young, H.A., Tobias, C.M., Mckeon, T.A., Brichta, J.L., Thomson, N.J., Thomson, J.G. 2020. Phenotypic examination of camelina sativa (L.) crantz accessions from the USDA – ARS National Genetics Resource Program. Plants. 9(5).
Dasgupta, K., Hotton, S., Belknap, W., Syed, Y., Dardick, C.D., Thilmony, R.L., Thomson, J.G. 2020. Isolation of novel citrus and plum fruit promoters and their functional characterization for fruit biotechnology. BioMed Central (BMC)Biotechnology. 20.