Ressources and skills in cell biology: Production of haploid and transgenic rapeseed lines

Production of haploid and doubled haploid rapeseed lines

The culturing of microspores has been implemented over the past twenty years in our lab for the production of haploid lines or doubled haploid (DH) lines (microspore culturing followed by chromosome doubling in the latter case), which are largely used in our research programs such as:

• genetic analyses of basic traits including homoeologous recombination (Cifuentes et al. 2010) or seed quality (seed composition in phenylpropanoids, fatty acids, …),
• gene cloning (cleistogamy, recombination), haplodiploidisation of plant material for different research programs on Brassica species.

Cifuentes M et al. Repeated polyploidy drove different levels of crossover suppression between homoeologous chromosomes in Brassica napus allohaploids. Plant Cell. 2010, 22(7):2265-76. DOI

Production of haploid rapeseed plants

A- Microspore culturing B- Microspore-derived embryos C- Regenerated plantlets

Functional validation of candidate genes by transgenesis

An efficient protocol for rapeseed transformation has been set up in our lab to generate stable rapeseed transgenic plants by agro-inoculation of cotyledons or hypocotyls. The plant selection and regeneration is fully optimized to recover F1 seeds over 6-9 months for spring or winter rapeseed types respectively. Examples for rapeseed transgenesis applications include the validation of the CLG gene (Lu et al. 2012), the identification of seed specific promoters in (Nesi et al. 2009, Auger et al. 2010).

We are now involved in the GENIUS project funded by the French Program “Investments for the Future” to investigate the potential of the nuclease-mediated gene targeting in rapeseed.

For more information, please contact Dr. Nathalie NESI.

Lu YH et al. A dominant point mutation in a RINGv E3 ubiquitin ligase homoeologous gene leads to cleistogamy in Brassica napus. Plant Cell. 2012, 24(12):4875-91. DOI
Auger B et al. Brassica orthologs from BANYULS belong to a small multigene family, which is involved in procyanidin accumulation in the seed. Planta. 2009, 230(6):1167-83. DOI
Nesi N et al. The promoter of the Arabidopsis thaliana BAN gene is active in proanthocyanidin-accumulating cells of the Brassica napus seed coat. Plant Cell Rep. 2009, 28(4):601-17. DOI

Production of rapeseed transgenic plants

A- Selection of agro-infiltrated cotyledons B- Rooted regenerated plantlets C- Activation pattern of a seed specific promoter in rapesed