Auxin and haploid embryo induction – Molecular Biology

Plants are very special because they can continually differentiate new organs from the stem cell niche throughout their lifespan, but they are also capable of regenerating new cell types and organs after wounding or from explants in tissue culture.

Description

Microspore embryogenesis is a unique process that beautifully illustrates this developmental plasticity of plant cells. In this process, the immature male gametophyte (microspore) is induced by a simple and short heat stress treatment to form haploid embryos in culture. The haploid embryos that arise can be germinated and converted to homozygous doubled haploids (DHs). Microspore embryogenesis is therefore widely used as a means to generate homozygous plants in a single generation, allowing plant breeders to accelerate their breeding programs. However, despite the practical advances of this process, the (epi)genetic and molecular mechanisms underlying this process remain poorly understood.

The phytohormone auxin, which is essential for correct tissue patterning and organ formation, seems to play an early and important role in microspore embryogenesis: we observed the presence of an auxin response shortly after the heat stress treatment and this auxin response was specifically present in those cells that will eventually form haploid embryos in culture. Based on our current knowledge, we hypothesize that microspore embryogenesis is a two-step process comprising i) a stress‐related event needed for the switch from pollen development to embryo development, followed by ii) an endogenous auxin‐related event required for cell proliferation.

This project aims to examine the role of stress and early auxin signaling during microspore embryogenesis. To this end, we will use different fluorescent auxin reporters in combination with transcriptome analysis to identify the stress- and auxin-related events during microspore embryogenesis. Additionally, we use novel and existing auxin compounds to identify the specific role of this hormone in microspore embryo induction. The overall focus is to improve our understanding of the molecular basis of microspore embryogenesis

Used skills

  • Analysis of GFP reporter lines using Confocal Laser Scanning Microscopy
  • Fluorescent activated Cell sorting (FACS) of GFP-labelled cells
  • Gene expression analysis (RNA isolation, qRT-PCR)
  • Tissue culture (haploid embryo culture, brassica transformation)
  • Chemical screens (auxin-related assays)

Requirements

  • Good theoretical and practical basis in (plant) molecular biology