WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 1 New Plant Breeding Techniques Group 3 Transgenic construct driven breeding Maria Lusser Joint Research Centre, European Commission Workshop Comparative situation of new plant breeding techniques 12-13 September 2011, Seville, Spain
Definition WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 2 Transgene encoding an RNAi construct or dominant-negative protein inserted in genome of inducer line Expression of transgene leads to the inhibition of gene expression or protein function Interference with plant biology induces formation of materials enhancing breeding (e.g. biodiversity, recombination, haploids) Inducer transgenes segregated out during further breeding
Techniques WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 3 RNA-dependent DNA methylation (RdDM) Reverse breeding Accelerated breeding following induction of early flowering Further techniques under development
WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 4 T-DNA Ti-Plasmid ds RNA Endonuclease Agrobacterium Plant cell Transformed plant cell Enzymes DNA Selection INTERMEDIATE PLANT Cultured plant cell SEGREGATION FINAL/COMMERCIALISED CROP e.g.: Matieu and Bender, J. Cell Science 117, 2004
Reverse breeding WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 5 x RNAi-mediated downregulation of meiotic recombination Achiasmatic gametes x Doubled haploid cells Reconstituted hybrid e.g. Dirks R., et al., Plant Biotechnology Journal (2009) 837-845
Accelerated breeding following induction of early flowering WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 6 Reduction of time of breeding cycle (e.g. fruit trees) through Reduction of juvenility/vegetative maintenance factors such as TFL1 by gene silencing Over-expression of BpMADS4, a flower initiation related transcription factor from silver birch Flachowsky H. et al., Acta Hort 763: 215-222 (2006) Flachowsky H. et al., Acta Hort 738: 307-312 (2007)
Commercial pipeline WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 7 RNA-dependent DNA methylation (RdDM) Maize - trait not disclosed (research phase) Oilseed rape - trait not disclosed (phase III) Reverse breeding Main crops and vegetable (research phase) PHASE I: Gene optimisation, crop transformation PHASE II: Trait development, pre-regulatory data, large-scale transformation PHASE III: Trait integration, field testing, regulatory data generation (if applicable) PHASE IV: Regulatory submission (if applicable), seed bulk-up, premarketing Source: Lusser M. et al., New Plant breeding Techniques: State-of-the art and prospects for commercial development, JRC Technical Report EUR 24760 EN (2011)
Changes in the genome WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 8 RNA-dependent DNA methylation (RdDM) Specific gene silencing through DNA methylation (promoter region) DNA methylation can also occur as natural process DNA sequence not modified Possible unintended effects: - Loss of silencing of the specific gene - Silencing of genes with homologous promoter sequence - Unintended manipulation of expression of other genes
Changes in the genome WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 9 Reverse breeding No changes are foreseen in the selected non-gmo offspring Possible unintended changes: - currently few data available - Silencing of genes with homologous promoter sequence (as for RdDM) - Unintended manipulation of expression of other genes (as for RdDM) - Incomplete suppression of meiosis Source: Glandorf B. et al., Evaluation of changes in the genome in plants through application of new plant breeding techniques in JRC Technical Report New plant Breeding Techniques, EUR 24760 EN
Detection/Identification WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 10 RNA-dependent DNA methylation (RdDM) Several methods are available for the analysis of DNAmethylation status such as - Methylation specific PCR-based techniques - Methylation-sensitive restriction enzymes - methylation-sensitive High-resolution Melting (MS- HRM) analysis However, differentiation of methylation through deliberate use of RdDM difficult to differentiate from naturally occurring methylation Therefore, identification of RdDM products is not possible
Detection/Identification WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 11 Reverse breeding End-products of reverse breeding are free of genetic modification-related DNA sequences Not possible to distinguish from products obtained through conventional breeding Therefore, identification of reverse breeding products is not possible Source: New plant breeding techniques: Challenges for detection and identification report of the New Techniques Task force (NTTF) in JRC Technical Report New plant Breeding Techniques, EUR 24760 EN (2011)
WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 12 Copyright: European Union, 2011