Thesis subjects Haploid embryo culture: a model for understanding plant cell puripotency
Microspore-derived embryo (MDE) development is a unique process in plants where the immature male gametophyte (microspore) is induced by a simple and short heat stress treatment to form a haploid embryo in culture. The haploid embryos that arise can be germinated and converted to homozygous doubled haploids (DHs) by colchicine treatment. DHs are widely used in crop improvement as parents for F1 hybrid seed production, to facilitate backcross conversion and as segregating populations in molecular mapping studies. Historically more emphasis has been placed on producing DHs for breeding purposes than on understanding the mechanism underlying haploid embryo induction. Limited studies on MDE induction in model systems such as Brassica napus, tobacco and barley have provided insight into the cellular and molecular changes that are associated with this process. However these studies are largely plagued by the use of fixed material for cell biology studies and cultures that contain a mix of responsive and non-responsive cells for molecular and biochemical studies. The link between the observed changes and DH induction remains correlative at best.
In this project fluorescent protein reporter lines will be used in combination with microarray-based transcript profiling to define the cellular and molecular events that occur in individual cells in real time as Brassica napus microspores are induced to become haploid embryos. The project will contribute to our general understanding of plant pluripotency, specifically how differentiated plant cells acquire the competence to regenerate.
- Analysis of GFP reporter lines using Confocal Laser Scanning Micrsoscopy
- Fluorescent activated Cell sorting (FACS) of GFP-labelled cells
- Gene expression analysis (RNA isolation, microarrays, qRT-PCR)
- GFP reporter- and functional analysis constructs (PCR, Gateway Cloning, arabidopsis transformation)
- Tissue culture (haploid embryo culture, brassica transformation)
- Theoretical and practical basis in (plant) molecular biology
- Affinity for (plant) tissue culture