Module 1
- Microspore isolation and culture – Mária Braun Šimášková;
- • Flower bud selection
- • Microspore isolation
- • Cell Counting and concentration determination
- • Microspore stress treatment
- • Cell stage assessment (confocal microscopy)
- • Cell viability assessment (Amphasis)
- Microspore embryo analysis and conversion – Robin Lardon
- Microspore embryo imaging and automated quantification
- Microspore cell sorting and enrichment for embryogenic populations
- Detecting Polyploidy with Flow Cytometry
Module 2
- Isolation and culturing of protoplasts – Tiago Monteiro Rodrigues
- Enzymes and incubation conditions
- Gradient centrifugation
- Embedding protoplasts in alginate
- Protoplast fusion – Hoang Khai Trinh
- Protoplast quality and density assessment
- Preparing asymmetric fusion: UV and iodoacetate treatment
- PEG-based and electrofusion
The two modules are supplemented with 2 lectures:
Lecture 1: Optimization of media and incubation conditions – Steffen Vanneste, Tiago Monteiro Rodrigues.
Lecture 2: Protoplast and microspore cell sorting – Andre Skirtach, Robin Lardon, Mária Braun Šimášková.
There will be a timeslot dedicated for discussing the protocol and experimental results.