Progress 04/01/24 to 03/31/25

Outputs
Target Audience:The target audience for the project included undergraduate and graduate students, attendees at national conferences including the 1890 Association of Research Directors (ARD) conference, the Society for In Vitro Biology (SIVB) annual conferences, the American Society of Plant Biologist (ASPB) conferences and international grape conferences. Additional target audiences included, grape growers, companies involved in grape breeding and new variety development, and the table and wine grape production industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PIworked with undergraduate students to provide hands on trainingin areas of plant cell culture, gene editing, genomic DNA and total RNA isolation. How have the results been disseminated to communities of interest?The UMES Ag Showcase event was held on 7th August 2024. Interested stakeholders including farmers, backyard and home grape growers and students were provided information on the various table and wine grapes varieties suitable for cultivation in the Mid-Atlantic region, the different trellis systems used for training and pruning grapevine varieties and management strategies for minimizing sunburn damage in grapevines. What do you plan to do during the next reporting period to accomplish the goals?Independent lines are currently being propagated to produce replicate clones and will be screened in a greenhouse for studying fruit production and quality traits. Once fruit is produced from independent lines, quality and post-harvest shelf life will be analyzed using HPLC and GC-MS techniques.

Impacts
What was accomplished under these goals? 1) identification and isolation of Vitis-derived Myb transcription factors, polyphenol oxidase genes and their related genetic elements for the development of precision bred and editing constructs, 2) establishment of embryogenic cultures and transformation for the development of precision bred (PB) and edited plant lines. The Pinot Noir genome from the NCBI database was analyzed to obtain the Vitis PPO gene sequences. Guide RNA (gRNA) sequences were designed that could simultaneously target the large regions of the PPO genes. Following off target analyses to prevent unintended editing, the dual gRNA sequences were inserted into a cloning vector under the control of the Arabidopsis thaliana U-26 and U-29 promoters. The CRISPR construct was then inserted into a binary vector that also contained the hygromycin gene under the control of a CaMV35S promoter for constitutive and the Cas9 gene under the control of an Arabidopsis egg cell promoter for Cas9 expression in cell cultures. Vector sequence confirmation was obtained by DNA sequencing and then the binary vector was transformed into Agrobacterium strain 'EHA 105' by the freeze-thaw method. Similarly, the VvMybA genes in the grapevine genome were analyzed to develop constructs for the constitutive expression of these genes. Embryogenic cultures were obtained from grapevine varieties 'Thompson Seedless', Seyval Blanc' and 'Bronx Seedless'. Cultures at the mid-cotyledonary stage of development were transformed with Agrobacterium harboring the CRISPR/Cas9 and constitutive expression constructs. Independent plant lines expressing the MybA1 genes and lines edited for the PPO genes were generated. Genomic DNA was isolated from independent plant lines and PCR analysis was conducted to determine transgene presence and expression. Amplified products were cloned into a cloning vector and sent for sequencing. PPO edited lines were confirmed by DNA sequencing while MybA1 lines were confirmed by PCR analysis to detect the presence of the MybA1 and the NPT II genes.

Publications