Search staff/organisations dr. RA de Maagd
Namedr. RA de Maagd

Job details
Descriptionsenior researcher
OrganizationWageningen Plant Research
Organization UnitBioscience
Phone+31 6 44058173
Secretarial phone+31 317 480 964
Phone 2+31 317 480 548
Fax+31 317 418 094
Note for telephonist
Note by telephonist
Visiting addressDroevendaalsesteeg 1
Postal addressPostbus 16
Regular availability
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Interested in doing a BSc or MSc thesis, or doing an internship with us? This is possible through the chair group Molecular Biology (Prof. G.C. Angenent). Contact me or check out the link to "Projects" (to the right of this text) for subjects and for the work of our PhD students Gül Hatinoglu, Victor Aprilyanto, Xiaowei Wang, Ellen Slaman, and postdoc Xinping Yang

I also collaborate with colleagues from Bioscience (a.o. Prof. Richard Immink) in a project on the regulation of shoot branching in tomato plants, with Dr. Martijn Fiers on the regeneration of tomato from protoplasts, and with others in a part of the EU project "CHIC", where we use CRISPR-mutagenesis of genes encoding fructan-degrading enzymes to increase the inulin content of Chicory. Students interested in an internship in the latter project may contact  Ingrid van der Meer.





Fruits and their products are an indispensable part of the human diet. They provide us with calories and many essential nutrients, vitamins and other health-sustaining compounds and, importantly, with many pleasant taste sensations. Although not consistently recognized by the casual observer, all flowering plants produce fruits in some form or another. However, it is mainly the juicy, fleshy fruits that are cultivated for human consumption.

Since the beginning of agriculture some 10,000 years ago, farmers have domesticated, selected, and improved fruit-producing plant species for higher production and better taste. In the past century, this task has been gradually taken over by professional breeders. For wild plants, the fruits are the organs that protect the developing seeds and, when mature, promote the dispersal of mature seeds. Fleshy fruits often enhance seed dispersal through their attractiveness to the animals that eat them. During their development, a fleshy fruit grows, through cell division and cell expansion, often to a final size many times that of the ovary that gave rise to it.

Additionally, many fleshy fruits undergo ripening at the end of their development, when seeds are mature and ready for dispersal. Ripening comprises many visible and not-so visible changes, such as changes in colour, taste and firmness. In our group, we are studying the regulation of fruit growth and ripening at the molecular level. We focus particularly on transcription factors, which are proteins that regulate the activity of genes through the activation or inhibition of their expression. Many of these transcription factors work in concert or have opposing activities and often regulate each other’s activities positively or negatively. This concerted action forms a regulatory network of considerable complexity. Studying how this network functions and its variations might contribute to obtaining higher yields or better quality of cultivated fruits. We use tomato as the primary model in most of our studies but are also interested in studying other fruits.

Transcription factors

Tomato is a model plant for research in fleshy fruit development and ripening. Ripening in the cultivated tomato comprises a series of biochemical and physiological events, including softening, pigment change, development of flavour components. Driven by autocatalytic ethylene production and climacteric respiratory behaviour, these result in ripe fruits. Several naturally occurring ripening mutants have been characterized. These include, for example, Colorless non-ripening (Cnr), ripening-inhibitor (rin), and non-ripening (nor), all of which have been identified by positional cloning or by genetic mapping. All three loci encode or are near genes for transcription factors, providing the first insights into the fruit-specific transcriptional control of ripening (Wang, R. et al., 2020, Plant Sci. 294: 110436). Another transcription factor that has been shown to be involved in tomato fruit ripening is SlAP2a, which was characterized in our group (Karlova, R. ... de Maagd, R.A. (2011), Plant Cell 23: 923-941). SlAP2a was shown to regulate different aspects of ripening in seemingly opposing ways, being required for both the proper progression of ripening and simultaneous inhibition of ethylene production.

Our group showed two other transcription factors, SlFUL1 and SlFUL2, to play more subtle and redundant roles in tomato fruit ripening (Bemer, M. ... de Maagd, R.A. (2012), Plant Cell 24: 4437-4451). Curiously all these transcription factor genes are orthologs (derived from a common ancestral gene) of genes that were shown to be involved in flowering and floral architecture in the model plant Arabidopsis thaliana. However, arabidopsis lacks fleshy fruits and lacks most developmental changes during tomato ripening, indicating that these similar genes were recruited for different purposes depending on their evolutionary path. The relative position of these proteins in the fruit ripening regulatory network and how their functions interact remains largely unknown and is the subject of our research.

CRISPR/Cas-mutagenesis and gene editing

Site-specific nucleases such as the highly versatile CRISPR/Cas9 nucleases offer many possibilities for precise genome modification. This may happen either by introducing mutations due to erroneous repair of double-stranded DNA breaks (non-homologous end joining, NHEJ) or by genome editing through homology-directed repair (HDR). In their most elegant form, targeted mutations or deletions introduced by these nucleases are indistinguishable from variations found in nature or introduced after well-established chemical or radiation-induced mutagenesis. This observation fuels the public debate whether organisms with precise mutations made via the novel nucleases would need to be considered and regulated as genetically modified organisms (GMOs), while those obtained by crossings or after treatment with mutagens are not. While this discussion is ongoing, novel methods using nucleases with properties that are complementary to Cas9 (such as Cas12a), improving Cas9, or using the targeting capacity of Cas9 for additional modifications are being studied. These include regulating gene expression by epigenetic modifications, base-editing, and prime editing and have been developed in animal systems. These techniques are currently finding their way to crop plants. Gene editing, gene replacement, or knock-in using homology-driven recombination around a nuclease-induced DSB is possible in plants but much less effective than in animal cells or yeast, and therefore not yet much used. However, the rewards in terms of  fundamental insights and applied plant research rewards make them likely to be further developed as practical tools.

Already our use of CRISPR/Cas-mutagenesis for gene function knock-out, as well as the work of others, has wholly overturned our understanding of the function of the transcription factors MADS-RIN, NAC-NOR, and SPL-CNR in tomato ripening regulation (Wang, R., .. de Maagd, R.A. (2019). Sci. Rep. 9: 1696). The natural mutations (rin, nor, Cnr) in these genes were revealed all be dominant-negative or gain-of-function mutations. True knock-out mutations depict a completely different picture of their function (Wang, R., Maagd, R.A. (2020). Trends Plant Sci. 25: 291–301).

We are developing applications of Cas9 and Cas12a to answer fundamental questions in our research on the regulation of fruit development and ripening and for practical applications in improved fruit quality, extended shelf life or yield. We have been successfully using Cas9 for targeted knock-out of regulatory genes and for the creation of promoter deletion mutants in tomatoes for over six years and are currently applying Cas12a for similar applications. Additionally, we have a project addressing questions of efficacy and specificity of different CRISPR technologies in tomato in order to, among others, be better able to predict the occurrence of and mitigate unwanted effects of these technologies (if any). This project involves testing and optimizing Cas9 and Cas12a activity and different delivery methods in plants, and single protoplasts for short term, multiplexed, and high throughput mutagenesis assays. Furthermore, we aim to harnass homology-directed repair and other allele replacement methods for the improvement of tomatoes.


In plants, expression levels of many transcription factors are also regulated at the post-transcriptional level by microRNAs. microRNAs negatively regulate protein levels by either slicing the messenger RNAs that they bind to or blocking their translation into a protein. In tomatoes, SPL-CNR and AP2a are members of two different, conserved miRNA-targeted gene families, and miRNA activity on their mRNAs has been demonstrated. Many other tomato targets of miRNAs are transcription factors, and a yet unknown number might be involved in fruit development or ripening (Karlova, R. Maagd, R.A.. (2013), J. Exp. Bot. 64: 1863-1878). miRNA action allows for a new layer of regulatory interactions d(uring fruit ripening, including regulating miRNA expression itself. We have identified the active miRNA genes during ripening and are modifying their expression to see what role they play.

Natural variation

Tomato and its wild relatives, which are crossable, comprise a stunning variety of fruit colours, sizes and shapes, tastes, shelf life, metabolite profiles, disease resistance and other traits. These are the starting material for further study on the improvement of tomato varieties by breeding. The availability of many sequenced genomes and advances in genome sequencing technology and throughput allows us to study in detail the molecular mechanisms underlying the variety of tomato fruit traits. We are studying a collection of cultivated tomatoes and wild relatives (Aflitos et al. 2014, Plant J. 80:136-148) by phenotyping them for yield and quality traits and correlating them to DNA sequence variation in genes known to be involved in these traits (Roohanitaziani, R., de Maagd, R.A., et al. (2020). Genes (Basel). 11: 1–22).


Expert Profile

Key publications
Publication lists


CRISPR: opportunities and safety aspects (Ellen Slaman)


The CRISPR/Cas9 system has made precise genome editing much more easily attainable. It is now possible to induce targeted insertions or deletions at virtually every position in the genome. This innovation has already been shown to be able to significantly speed up plant breeding, which may help us feed a growing population in a changing world.

Recently, besides Cas9, the nuclease enzyme Cas12a (also known as Cpf1) was hown to be able to induce mutations at specific positions in the plant genome. However, for both Cas9 and Cpf1 no large screens have yet been performed in plants regarding both the mutagenic spectrum and off-target effects of these two nucleases. In my research, I aim to compare the mutational spectra and off-target effects of Cas9 and Cas12a in a high-throughput fashion At the same time, while also comparing different delivery methods for these nucleases, such as stable transformation, protoplast transfection with DNA constructs, and protoplast transfection with ribonucleoproteins: the Cas9 enzyme pre-loaded with a gRNA. Additionally, a non-biased detection system for off-targets will be developed and applied to know precisely how on-target Cas9 and Cas12a make their breaks in the DNA.

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? Do not hesitate to send me an e-mail!

Used skills:

  • CRISPR/Cas9 and CRISPR/Cas12a mediated mutagenesis through:
    • Stable tomato transformation
    • Protoplast isolation and transfection
  • Plant tissue culture
  • High throughput sequencing and analysis
  • Basic molecular biology techniques such as DNA extraction, cloning, PCR, restriction enzyme assays, etcetera


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 an e-mail!

Used skills:

  • 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

Investigating the molecular regulation of fruit ripening and shelf life (Victor Aprilyanto)

see the project on Victor's page

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.

Used skills

  • 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! 

Used skills 

  • Basic molecular biology techniques (PCR, cloning, restriction enzyme etc.)  

  • Plant transformation and tissue culture 

  • Mutant analysis & plant phenotyping (macroscopic and microscopic) 

  • Interaction analysis (Promoter-reporter assay, Y3H, etc.) 

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