Progress 06/15/21 to 06/14/24
Outputs
Target Audience:The target audience for the project included undergraduate and graduate students involved in genome editing research, public institution researchers involved in grapevine genetic improvement efforts, and industry personnel working on the development of new table grape cultivars. Research presentations were made by students from the Louis Stokes Alliance for Minority Participation (LSAMP) chapter of UMES on the utilization of the RUBY gene for transient gene expression in tobacco and grapevine. Research presentations were made on gene editing for grapevine genetic improvement at the 1890 Association of Research Directors (ARD) conferences in 2022 and 2024. The PI made a research presentation at the European Horticultural Congress while a graduate student presented her work at the 2024 American Society of Plant Biologists conference. Changes/Problems:The research programs for both PI's were severely affected by the COVID-19 pandemic. The Zhao lab was shut down during 2021. The entire 2021, we mostly worked remotely. In January 2022, two students who work in the laboratory tested positive for Covid, thereby impacting the entire laboratory personnel. Physical presence in the labs was resumed by April 2022 but there were interruptions by infections to other lab members. The Dhekney lab was less impacted by COVID but a majority of the undergraduate and graduate students started routine in person lab work starting in Spring 2022. This significantly delayed our experiments and ability to complete the work in time. We continue to test the NIET constructs for optimizing their efficiency and use in transient gene expression for genome editing. What opportunities for training and professional development has the project provided?Undergraduate and graduate students made research presentations and the UMES research symposium and the American Society for Plant Biologists conference. Students were trained in the lab in cell culture, genetic transformation and genome editing techniques. How have the results been disseminated to communities of interest?Research presentations were made at local, national and international conferences. Results from the research project were also presented to small and minority farmers at the UMES small farm conference and the Agshow Case events in 2023 and 2024. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Establish protocols for efficient Agrobacterium-mediated transient gene expression Embryogenic cultures for Vitis vinifera cultivars 'Thompson Seedless' and 'Bruce Sport' and Vitis interspecific hybrids were initiated from leaves of in vitro micropropagation cultures. Leaves were placed in the dark for 10 weeks followed by tranfer to light for 6 weeks. Resulting cultures were transferred to embryo development medium for the production of somatic embryos. To test,Agrobacterium-mediated transient editing, the VVGAI gene was selected as the target sequence. Successful editing of this candidate gene would result in a dwarf phenotype. We adopted a dual guide RNA strategy where two guide RNAs encompassing a 988 bp region of the GAI gene were designed and were inserted into a binary vector downstream of an Arabidopsis U26 and U29 promoter. The binary vector also contained the Cas9 gene under the control of an embryo specific promoter. Somatic embryos at the mid-cotyledonary stage of development were co-cultivated with Agrobacterium GV 3101 containing the binary vector. Cultures were then transferred to callus production medium for 30 days followed by transfer to embryo development medium for the development of edited embryo lines. Embryo lines were germinated to obtain plants. Genomic DNA from embryo lines was isolated and used in PCR and sequencing reactions to determine the presence of the hygromycin selectable marker, the Cas9 sequence and whether editing occurred at the target locus. PCR amplification of 1026 bp and 4.1 kb fragments corresponding to the Hyg and Cas9 gene were observed in all ten transgenic lines and the positive plasmid control. In contrast, no amplification was observed from the non-transformed plant line. Sequencing results indicated insertions and deletions at both guide RNA sites, however there was no significant phenotypic differences between the edited lines and non-transformed control plants. We are currently conducting whole genome sequencing to further characterize the mutations and analyze our results. 2) Utilize a newly developed Nonsexual and Induced Elimination of Transgenes (NIET) CRISPR/Cas-9 mediated gene editing technology to generate transgene free edited plants. We have improved the NIET Technology (Non-sexual and Induced Elimination of Transgenes for CRISPR/Cas9mediated gene editing) in several aspects. NIET will be very useful for generating transgene free plants that are propagated asexually. The basic design is to use two temporally separated guide RNA production units to edit the target gene and to eliminate any transgenes including gene editing components such as the nuclease gene Cas9. The gRNA for target gene is produced from a U6 promoter, an RNA Polymerase III promoter. The guide RNA for eliminating T-DNA insertion is produced by our ribozyme-based strategy, which allows to usage of RNA Polymerase II promoter. We optimized the promoter for producing the gRNA for eliminating the T-DNAs by using a light-inducible promoter in conjugation with our ribozyme based technology. We added an exonuclease to the vector, which can improve the efficiency of eliminating the transgenes. The modified vectors are currently being tested to determine their efficiency for the NIET technology. 3) Utilizing the RUBY gene to monitor the development of edited cells and plant lines. We used our newly developed visible marker to monitor the presence of transgenes. Previously, we relied on antibiotic resistance gene and fluorescence markers to monitor the transgenes, a process that is very labor-intensive. RUBY does not need special equipment and chemical treatments. RUBY uses tyrosine as a substrate to produce betalians, which have a vivid and bright color. The RUBY gene was tested for its efficiency as a reporter gene in grapevine transformation. In the first construct, the RUBY gene was placed under the control of a CaMV 35S promoter (RUBY-35S) while in the other construct, it was placed under the control of anArabidopsisubiquitin promoter (RUBY-Ubq). Both constructs also contained the hygromycin selectable marker for identification of transformed cells and plants. A high level of RUBY expression was observed from both constructs in grapevine embryogenic cultures and plant lines. RUBY-35S expression was observed uniformly in all plant tissues and abnormal plant development was observed. In contrast, RUBY-Ubq expression was only observed in the roots of transgenic plant lines and normal growth and development of regenerated plant lines occurred. Thus, the use of the RUBY gene would have greater efficiency as a reporter gene when placed under the control of the ubiquitin promoter.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Sardaru, P., Jackson, C.L., Wood, C., Dai, X., Zhao, Y., and S.A. Dhekney. 2024. Precision breeding and genome editing of wine and table grape cultivars for improving disease resistance. Acta Horticulturae, In Press
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Applications of precision breeding and genome editing for Vitis trait improvement. Association of Research Directors (ARD) symposium, Nashville, TN, April 6-10.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Jackson, C.L., Sardaru, P., and S.A. Dhekney. 2024. Optimizing in vitro regeneration protocols for Vitis precision breeding and genome editing. Annual conference of the American Society of Plant Biologists, Honolulu, HI June 22-27.
|
Progress 06/15/22 to 06/14/23
Outputs
Target Audience:The target audience for the project included undergraduate and graduate students involved in genome editing research, public institution researchers involved in grapevine genetic improvement efforts, and industry personnel working on the development of new table grape cultivars. Research presentations were made by students from the Louis Stokes Alliance for Minority Participation (LSAMP) chapter of UMES on the utilization of the RUBY gene for transient gene expression in tobacco and grapevine. Changes/Problems:The research programs for both PI's were severely affected by the COVID-19 pandemic. This has put us behind schedule for achieving some of the objectives for the project. What opportunities for training and professional development has the project provided?Undergraduate and graduate students were trained in plant tissue culture and genome editing techniques. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Genetic constructs will be developed the express the RUBY gene under the control the CAMV35S and the ubiquitinconstitutive promoters. The constructs will be tested in grape to determine their efficiency and suitability for recovery of edited cells following transient expression.We optimized the promoter for producing the gRNA for eliminating the T-DNAs by using a light-inducible promoter in conjugation with our ribozyme based technology. We added an exonuclease to the vector, which can improve the efficiency of eliminating the transgenes. The modified vectors will be tested to determine their efficiency for the NIET technology.
Impacts
What was accomplished under these goals?
To test, Agrobacterium-mediated transient editing, the VVGAI gene was selected as the target sequence. We adopted a dual guide RNA strategy where two guide RNAs encompassing a 988 bp region of the GAI gene were designed and were inserted into a binary vector downstream of an Arabidopsis U26 and U29 promoter.The binary vector also contained the Cas9 gene under the control of an embryo specific promoter. Somatic embryos at the mid-cotyledonary stage of development were cocultivated with Agrobacterium GV 3101 containing the binary vector. Transgenic emrbyo and plant lines are currently being recovered. The use of the RUBY reporter gene was tested for transient and stable gene expression in tobacco cultures. Transgenic plant lines expressing the RUBY gene were recovered.
Publications
|
Progress 06/15/21 to 06/14/22
Outputs
Target Audience:The target audience included grape growers involved in the production of wine, table and juice grapes, private table grape companies that are involved in the development of improved, disease resistant cultivars, undergraduate and graduate students involved in the research activities. Changes/Problems:Although we made decent progress, our research was significantly delayed by the Covid pandemic. The Zhao lab at UC San Diego was adversely affected and through most of 2021, the work was carried out remotely. In January 2022, two students who work in the lab tested positive for Covid, thereby impactingthe entire laboratory personnel. The research activities have returned to normalcy since April, 2022. Due to this loss of time, we anticipate some delay in the transformation work with the NIET constructs. What opportunities for training and professional development has the project provided?A postdoc was hired to work on the project and is being trained in grapevine molecular biology and biotechnology. An undergraduate student received training on in vitro culture and plant regeneration. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will estimate the efficiency of transient edicint based on dwarf phenotypes that are observed after plant regeneration. The NIET constructs which are currently being constructed will be used to study editing efficiency following transformation.
Impacts
What was accomplished under these goals?
Objective 1. Establish protocols for efficientAgrobacterium-mediated transient gene expression Embryogenic cultures forVitis viniferacultivars 'Thompson Seedless' and 'Bruce Sport' andVitisinterspecific hybrids were initiated from leaves of in vitro micropropagation cultures. Leaves were placed in the dark for 10 weeks followed by tranfer to light for 6 weeks. Resulting cultures were transferred to embryo development medium for the production of somatic embryos. To test,Agrobacterium-mediated transient editing, the VVGAI gene was selected as the target sequence. We adopted a dual guide RNA strategy where two guide RNAs encompassing a 988 bp region of the GAI gene were designed and were inserted into a binary vector downstream of an Arabidopsis U26 and U29 promoter. The binary vector also contained the Cas9 gene under the control of an embryo specific promoter. Somatic embryos at the mid-cotyledonary stage of development were co-cultivated withAgrobacteriumGV 3101 containing the binary vector. Cultures were then transferred to callusing medium without any selection antibioticsand are currently being monitored for embryo development. Once plant regeneration occurs, we should be able to identify edited lines based on a dwarf phenotype. 2)Utilize a newly developed Nonsexual and Induced Elimination of Transgenes (NIET) CRISPR/Cas-9 mediated gene editing technology to generate transgene free edited plants. We have been improving the NIET Technology (Non-sexual and Induced Elimination of Transgenes for CRISPR/Cas9-mediated gene editing) in several aspects. NIET will be very useful for generating transgene free plants that are propagated asexually. The basic design is to use two temporally separated guide RNA production units to edit the target gene and to eliminate any transgenes including gene editing components such as the nuclease gene Cas9. The gRNA for target gene is produced from a U6 promoter, an RNA Polymerase III promoter. The guide RNA for eliminating T-DNA insertion is produced by our ribozyme-based strategy, which allows to usage of RNA Polymerase II promoter. We conducted the following experiments to improve the technology a. We used our newly developed visible marker to monitor the presence of transgenes. Previously, we relied on antibiotic-resistance gene and fluorescence markers to monitor the transgenes, a process that is very labor-intensive. RUBY does not need special equipment and chemical treatments. RUBY uses tyrosine as a substrate to produce betalians, which have a vivid and bright color. b. We have optimized the promoter for producing the gRNA for eliminating the T-DNAs. We now use a light-inducible promoter in conjugation with our ribozyme based technology. c. We added an exonuclease to the vector, which can improve the efficiency of eliminating the transgenes.
Publications
|
|