Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): Objective 1. Characterize, evaluate, breed, select, and release improved woody landscape plant germplasm such as ornamental cherry, hemlock, catalpa, elm, boxwood, crapemyrtle, and redbud with superior disease and pest resistance, non-invasiveness, climate resilience, and improved ornamental traits. Sub-objective 1a: Develop and evaluate intra- and interspecific hybrids and breeding populations to use as genetic mapping families and for use in cultivar development, with a focus on Prunus (flowering cherry), Ulmus (elm), Buxus (boxwood), Lagerstroemia (crapemyrtle), and Cercis (redbud). Sub-objective 1b: Propagate and evaluate advanced selections of Prunus, Tsuga, Ulmus, Buxus, Lagerstroemia, and Cercis developed in previous breeding cycles through multi-location, replicated field trials. Sub-objective 1c: Name, release, distribute, and promote new cultivars. Objective 2. Develop genomic resources for genetic mapping of key traits and phenotypic methods for trait characterization and quantification to accelerate breeding and selection in Buxus, Cercis, Prunus, and Lagerstroemia. Sub-objective 2a: Develop and utilize quantitative high-throughput phenotyping methods for ornamental traits and evaluate disease resistance of boxwood hybrids and germplasm. Sub-objective 2b: Create reference genomes by sequencing and assembling genomes of accessions, cultivars, and parental lines of Buxus. Sub-objective 2c: Establish a foundation for genetic mapping, gene identification, and marker-trait analysis by genotyping germplasm and hybrid progeny in Buxus, Cercis, Prunus, and Lagerstroemia. Objective 3. Develop and apply biotechnology to accelerate characterization, identification, selection, or breeding of priority plant materials for key traits, with a focus on disease and pest resistance in Buxus and plant architecture and flowering traits in Prunus. Sub-objective 3a: Establish efficient micropropagation, regeneration, and transformation systems for selected flowering cherry (Prunus) and boxwood (Buxus) genotypes. Sub-objective 3b: Identify genes or genetic elements in flowering cherry and boxwood for targeted traits and use transgenic and gene editing approaches to modify gene expression. Sub-objective 3c: Characterize and evaluate selected transgenic or gene- edited plants. Approach (from AD-416): The approach for Objective 1 uses traditional methods of plant breeding, selection, and evaluation. We will focus on Prunus, Ulmus, Buxus, Lagerstroemia, and Cercis to develop plants that are disease resistant, have superior ornamental or growth characteristics, and are tolerant of abiotic stresses. Controlled intra- and interspecific hybridizations will be carried out by hand or by insects to produce hybrid progeny, to determine compatibility among parents, and to study breeding systems and inheritance of traits of interest. Resultant progeny will be evaluated, and selections will be evaluated in replicated field trials. We will introduce new plants in consultation with ARS�s Office of Technology Transfer (OTT) and stakeholders following standard ARS administrative approval procedures. Promotional materials (fact sheets, press kits for garden writers, information on the USNA website) will be prepared and distributed. Propagation material in the form of rooted cuttings (preferably) or cuttings will be sent to nurseries upon request until the cultivar is routinely available in the trade. The approach for Objective 2 will be based on molecular breeding, genetics, and machine learning methodologies to develop mapping tools and to facilitate trait discovery. We will develop a large image data set of boxwood blight-infected plants to develop a convolutional neural network (CNN) to assist in detecting and monitoring infected boxwood plants. We will also develop a 3D imaging pipeline for measuring plant architecture traits to develop marker-trait associations for breeding and selection. We will use next-generation sequencing technologies to create reference genomes of two boxwood genotypes (B. harlandii 60705 and B. microphylla �Little Missy�). We will map QTLs, establish multi-environmental genome- wide association (GWA) mapping trials of B. sempervirens, and develop SNP markers based on genotyping-by-sequencing (GBS) methodologies. In addition to Buxus, we will use GBS or de-novo sequencing and assembly for novel trait discovery in Cercis, Prunus and Lagerstroemia. These traits include growth habit, leaf and flower color, and reblooming traits that can be used for gene-editing technologies in Objective 3. The approach for Objective 3 focuses on developing and utilizing new breeding tools and technologies to characterize, select, and develop superior cultivars of ornamental plants, particularly establishing and optimizing transgenic and gene-editing methods in Prunus and Buxus. We will first establish an efficient in vitro axillary proliferation and regeneration system, including testing selectable markers. We will establish a protoplast isolation and culture protocol and test various developmental regulator genes and growth-regulating factors for their effects on promoting regeneration. Finally, we will develop a genetic transformation system in both genera, focusing on Agrobacterium-mediated transformation and biolistic particle bombardment methods. We will characterize these new transformed and gene-edited plants by PCR and sequencing, propagation to ensure stability, and in growth chambers or greenhouses to assess phenotype. Under Objective 1, interspecific crosses in boxwood that were previously developed are being propagated for replicated evaluation for desirable horticultural traits and boxwood blight resistance. Fifteen promising genotypes have been sent to an industry cooperator under MTRA agreement #68731 and have completed year 1 of evaluations. New accessions and hybrids have been evaluated for boxwood blight using our established detached leaf assays. Advanced selections of Tsuga and Prunus were propagated for further evaluation, and ~1,000 rooted liners of Tsuga �Crossroad� and �Traveler� were sent to nursery cooperators. A new xChitalpa hybrid (�Strawberry Moon�) was released to the trade. Under Objective 2, we developed a quantitative imaging pipeline to detect boxwood blight by generating images in-house and from crowd sourcing images from collaborators and online image databases. A custom Convolutional Neural Network (CNN) was developed and tested against an industry standard deep learning model. A manuscript has been prepared and will be submitted for publication for FY24. Under Objective 3, we established two more flowering cherry genotypes in tissue culture in addition to the previously established three genotypes. An organogenesis-based regeneration system has been established for two of these genotypes, and somatic embryogenesis-based regeneration has been tested in two genotypes. We established a protoplast isolation method for flowering cherry and are applying it to our gene editing pipeline for optimizing editing components. Also in flowering cherry, genes related to flower morphology, flowering time, flower color, plant height and plant architecture were identified using a homology-based approach that will be used as targets for genetic improvement. Collaborating with Texas A&M University, we are developing an Agrobacterium-mediated transformation method for Lagerstroemia speciosa and other crapemyrtle genotypes in our breeding program. We have established in vitro cultures for two genotypes of boxwood, as well as a protoplast isolation protocol, and are optimizing the medium for micropropagation of boxwood. We identified essential genes in boxwood blight for developing blight resistance in boxwood using host-induced gene silencing strategy. We have established a floral tissue-based transient expression system using petunia as a model system that can be used for testing gene function and gene editing efficiency across many ornamental species. One of the Lead scientists on this Project took a position with another ARS location in January 2024, which had an impact on progress, especially in Objectives 1 and 2. Artificial Intelligence (AI)/Machine Learning (ML) Artificial intelligence (AI) methods were used for this project during FY2024 for computer vision detection of disease presence. We trained a Convolutional Neural Network (CNN) model to detect boxwood blight on stem and leaf images from artificial inoculations in controlled laboratory settings and from naturally infected plants in the nursery, field, and garden landscapes and achieved 97% accuracy on a separate set of test images. The analyses were implemented on SCINet�s HPC clusters Ceres using the machine learning node and was done in collaboration with Mississippi State University. All image data and code has been backed up on JUNO and data has been archived on AgData Commons with a permanent DOI. The quality of this computer vision data enables better precision and discovery of resistance gene loci that might not be detectable using manual visual detection and disease ratings. ACCOMPLISHMENTS 01 Introduction of a new ornamental shade tree �Strawberry Moon�. Growers, landscapers, and city planners are always looking for trees that offer shade, have long-lasting ornamental appeal, and can withstand the unique stresses of the urban environment. ARS scientists in Washington, D.C., released a new hybrid shade tree that has all these traits. �Strawberry Moon� xChitalpa is a hybrid involving species of Chilopsis (desert willow) and Catalpa (cigar tree), and is characterized by long- lasting showy pink blooms, resistance to powdery mildew, heat and drought tolerance, large leaves for increased shade, and superior street tree structure over the parental species. It is also seed- sterile, which means it has reduced litter, an important quality in public green spaces. �Strawberry Moon� is currently in the hands of commercial propagators with limited wholesale availability. 02 Development of a floral organ-based transient gene expression toolbox. Biotechnology, including transgenic technologies and gene-editing, is a promising tool that can complement traditional breeding in ornamental crops. However, the application of biotechnology requires knowledge of the function of the target genes which is unknown for many ornamental species. In addition, for many traits, the effects of gene manipulation can only be observed after the lengthy and labor-intensive process of generating stable engineered plants. ARS scientists in Beltsville, Maryland, developed a fast, effective transient assay to narrow down the target genes and gene combinations to optimize bioengineering strategies. Using petunia flower petals as a model, they devised a system that can be applied broadly across other ornamental species to test gene function, metabolic engineering strategies, and to optimize gene editing components.
Impacts
(N/A)
Publications
- Tang, D., Sun, H., Kalluri, A., Ding, J., Zhai, L., Gu, X., Li, Y., Yer, H. , Yang, X., Tuskan, G., Deng, Z., Duan, H., Gmitter, F., Kumar, C., Li, Y. 2024. Engineered DsRNA-protein nanoparticles for effective systemic gene silencing in plants. Horticulture Research. https://doi.org/10.1093/hr/ uhae045.
- Wu, B., Zhang, N., Dixon, B., Sierra, I., Kan, S., Layton, A., Gu, M., Pooler, M.R., Duan, H., Qin, H. 2024. Reliable callus-induced plantlet regeneration from leaf explants of Lagerstroemia speciosa and genetic fidelity assessment through ISSR markers. Plant Cell Tissue and Organ Culture. https://doi.org/10.1007/s11240-024-02801-w.
- Duan, H. 2023. Transient gene expression in Petunia flower petals as a toolbox. Journal of Horticultural Science and Biotechnology. https://doi. org/10.1080/14620316.2023.2248127.
- Tang, D., Li, Y., Zhai, L., Wang, X., Li, W., Kumar, R., Yer, H., Duan, H., Cheng, B., Deng, Z., Li, Y. 2023. Root predominant overexpression of iaaM and CKX genes promotes root initiation and biomass production and initiation in citrus. Plant Cell Tissue and Organ Culture. https://doi.org/ 10.1007/s11240-023-02557-9.
- Guo, Y.H., Zhou, B., Pooler, M.R. 2024. Evaluation of a novel fungicidal extract from blue spruce, Picea pungens. Plant Health Progress. https:// doi.org/10.1094/PHP-10-23-0089-RS.
- Li, X., Weiland, J.E., Ohkura, M., Luster, D.G., Daughtrey, M.L., Gouker, F.E., Chen, G., Kong, P., Hong, C. 2024. Cultivars and production environments shape shoot endophyte profiles of boxwood with different blight resistance. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-03-24- 0023-R.
- Alexander, L.W., Wu, X., Gouker, F.E. 2024. Production and verification of novel Osmanthus hybrids. Frontiers in Horticulture. https://doi.org/10. 3389/fhort.2024.1382450.
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