One of the major bottlenecks in plant breeding programs is the recalcitrance for in vitro embryogenesis in some species or genotype of common crops, which limits the use of modern biotechnology tools.
Progress in tissue culture procedures by using growth regulators and culture media combinations have been time consuming and inefficient for a large number of crops, which is why there is an urgent need to develop novel, generic tools to improve plant regeneration processes in a germplasm-independent manner. Embryo-expressed transcription factor like the AP2 domain protein BABY BOOM and CAAT-box binding factor LEAFY COTYLEDON1 have been used to enhance plant regeneration in a range of crops when expressed from a constitutive promoter, resulting in transgenic lines. This project aims to examine the extent to which BBM and LEC1 can be used to transiently promote in vitro regeneration without genomic integration of nucleic acids and without genomic DNA mutation. Different approaches will be used to transiently induce BBM/LEC1 protein in plant cells. One approach is to use cell-penetrating peptides to introduce those proteins into the plant. Additionally, we aim to activate endogenous BBM/LEC1 gene expression by using CRISPR-dCas9 technology and small chemical compounds. The overall focus lies on improving in vitro regeneration in haploid embryo induction for doubled-haploid production as well as somatic embryogenesis for clonal propagation.
- CRISPR-Cas9 mutagenesis (in silico sgRNA design, plasmid construction)
- Protein-DNA interactions (yeast 1 hybrid analysis)
- Genetic analysis of arabidopsis mutants
- Gene expression analysis (RNA isolation, qRT-PCR)
- GFP reporter- and functional analysis consructs (PCR, Gateway cloning, Golden Gate cloning, arabidopsis transformation)
- Analysis of GFP reporter lines using confocal laser scanning microscopy
- Transient protoplast transfections (firefly luminescence measurements)
- Somatic embryo culture
- Good theoretical and practical basis in (plant) molecular biology
- Please contact me if you are interested in working on CRISPR-Cas9 mutagenesis on promoters, transient protoplast transfections or chemical screens to study gene regulation of BABYBOOM or LEAFY COTYLEDON 1