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Capturing the results of gene editing in plants: regeneration from protoplasts (in collaboration with Dr. Martijn Fiers)
CRISPR/Cas action on a plant genome can have a wide variety of outcomes, but only a fraction of these make it into a mutant plant if transformation by Agrobacterium tumefaciens and regeneration from explant-derived callus is used. Regeneration from individual protoplasts would allow to pick and select interesting low-frequency events in an early stage. This allows the screening of many more events in a high-throughput fashion and reducing the amount of labour and resources needed for producing gene-edited plants.
In tomato, we have multiple times successfully produced mutations in genes using CRISPR/Cas, which knock out their function and allows the study of their function. However, the route to these plants, using stable transformation by A. tumefaciens and regeneration is long and laborious, which with limited personnel input can only produce a relatively small number of mutants. While this works fine for knockout mutations in a single or a few genes, less frequent events (~1-2% of plants) such as gene-editing by homology-directed repair or by prime editing requires a lot of effort and resources. We know that we can achieve and detect all kinds of events in protoplasts (cell wall-less plant cells), even if their frequency is low. So wouldn’t it be great if we had a reliable regeneration procedure for protoplasts and only continue to regenerate and grow the plants with the desired edits?
In this project, we aim at developing and optimizing regeneration protocols for tomato protoplasts by testing several peptides that are involved in cell division and cell differentiation in order to induce cell division of the protoplasts into small clumps of genetically uniform cells (microcalli ). These will have to be subcultured to create macroscopic calli, which can then be induced to differentiate in shoots and roots, and finally viable plants.
All these steps have been achieved in other species, but are not routine in tomato. The emphasis will be on optimizing tissue culture conditions to achieve cell division in first untransfected and later the transfected individual tomato protoplasts, leading to undifferentiated callus and eventually new plantlets.
All work is performed in tomato, which makes the acquired data directly applicable to one of the world’s most important crops. Would you like to work on one of these projects with me? Don’t hesitate to send me or firstname.lastname@example.org an e-mail!
- Willingness to work precisely and using sterile conditions
- Protoplast isolation and transfection with DNA constructs or Ribonucleoprotein particles
- Plant tissue culture
- Basic molecular biology techniques such as DNA extraction, cloning, PCR, restriction enzyme assays, etcetera
Molecular regulation of vegetative branching in tomato (Gül Hatinoglu)
Plants continuously grow from the top of their main shoot, known as the apical meristem. Certain signals during this growth will lead to the budding of additional lateral shoots from leaf axils. In tomato, these so-called axillary shoots compete for energy with fruit production, and they need to be manually removed. Therefore, this feature is undesired.
In my project, I aim to uncouple molecular regulation of vegetative branching. I am studying both upstream and downstream transcription factors involved in this pathway and using CRISPR/Cas9-mutagenesis to generate different mutants of branching-regulating genes. In addition, my work uses high throughput techniques such as Y1H-seq and RNA-seq. Altogether, we aim to contribute to the knowledge of increased apical dominance in tomato reducing manual labour and make tomato cultivation more sustainable in the future.
- Basic bioinformatics skills
- Stable tomato transformation
- CRISPR/Cas9 mediated mutagenesis
- Plant tissue culture
- Molecular biology techniques such as DNA extraction, cloning, PCR, restriction enzyme assays, RNA extraction, gene expression analysis and possibly others!
Are you interested in my topic and would like to learn more? Please contact me via e-mail!
Tomato development and ripening: Regulatory network of MADS-box transcription factors (Xiaowei Wang)
MADS-domain proteins are important transcription factors involved in many biological processes of plants, but an in-depth characterization of their unique and redundant functions is lacking for tomato fruit. By applying CRISPR/Cas and RNAi technologies, we can generate single knockout mutants and combinations thereof to unveil the molecular regulatory network of MADS-domain proteins with other TFs in fruit development and ripening.
In tomato (Solanum lycopersicum), several MIKC-type MADS-domain proteins, such as FUL1, FUL2, MADS-RIN, TAGL1, and MADS1, playing a role in fruit development and ripening have been identified via CRISPR/Cas or RNAi. We aim to elucidate further the tomato fruit development and ripening regulation exerted by MADS-box TFs and their interactions among each other and with other related genes. Detailed phenotyping for macroscopic, microscopic, and molecular fruit aspects of different MADS-box mutants is needed to test their redundancy in the regulation of fruit development, and we also will analyze the temporal and spatial expression of candidates to identify specific targets and co-factors. In addition, the effects of different interaction complexes on target promoter activity will be tested in various combinations, and four natural variations in FUL1 and different splicing variants in MADS1, and RIN are of interest to us. Altogether, we aim to develop a refined regulatory model for tomato fruit.
All work is performed on tomato, which is a lovely, fresh and important model crop! So if you’d like to work on one of these projects with me, please do not hesitate to send me an e-mail!
Basic molecular biology techniques (PCR, cloning, restriction enzyme etc.)
Plant transformation and tissue culture
Mutant analysis & plant phenotyping (macroscopic and microscopic)