Groups of new plant breeding techniques

WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN Groups of new plant breeding techniques Maria Lusser Joint Research Centre, European Commission Workshop Comparative situation of new plant breeding techniques 12-13 September 2011, Seville, Spain 1

Techniques (JRC NPBT report) WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 2 1. ZINC FINGER NUCLEASE TECHNOLOGY (ZFN-1, ZFN-2 and ZFN-3) 2. OLIGONUCLEOTIDE DIRECTED MUTAGENESIS (ODM) 3. CISGENESIS/INTRAGENESIS 4. RNA-DEPENDENT DNA METHYLATION (RdDM) 5. GRAFTING ON GM ROOTSTOCK 6. AGRO-INFILTRATION (Agro-infiltration sensu-strictu, Agro-infection, Floral dip) 7. REVERSE BREEDING

RdDM WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 3 1. TISSUES/CELLS MANIPULATED T-DNA Agrobacterium Ti-Plasmid Integrated gene ds RNA Endonuclease Plant cell 2. DELIVERY OF TECHNOLOGY Transformed plant cell Enzymes DNA 3. INITIAL PROCESS AT MOLECULAR LEVEL Selection INTERMEDIATE PLANT 4. SEGREGATION Cultured plant cell 5. FINAL/COMMERCIALISED CROP e.g.: Matieu and Bender, J. Cell Science 117, 2004

1. Tissues/cells manipulated WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 4 Delivery of technology to explant - tissue cuttings - cultured cells -embryos - protoplasts, etc. Local delivery to intact plant - infiltration of non germline tissue (e.g. leaves) - delivery to germline tissue (e.g. immersion of flowers)

2. Method of delivery of technique into cell WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 5 Delivered as DNA vector construct (e.g. Plasmid, viral vector) - Agrobacterium tumefacience, biolistic, etc. Delivered as DNA, RNA, spec. Oligonucleotides - mrna encoding ZFN - linear DNA as repair template (ZFN-2) - chimeric oligonucleotides (DNA + RNA), singlestranded DNA (ODM) Delivered as protein - research in possibility to deliver ZFNs directly as proteins

ZFN-2 WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 6 Delivery of repair template - as DNA Gene insertion in the case of delivery as DNA vector construct encoding ZFN? DSB Delivery of ZFN - as DNA vector construct encoding ZFN - as RNA encoding ZFN - as protein Repair by HR Transgenic intermediate plant Regeneration/ Selection for trait Segregation Regeneration/ Selection for trait Final crop

3. Initial process at molecular level WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 7 Stable insertion of gene Transient gene expression - (gene construct eventually degrades) Other - Mutation - DNA methylation

4. Intermediate crop/segregation WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 8 Inserted gene construct - Segregated out Not applicable - In the case of transient gene expression, the gene construct eventually degrades (absence has to be proved)

5. Commercialized/final product WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 9 No intended change in genome Stably inserted gene Other effects - Mutagenesis - Gene silencing through methylation of promoter region

Possible criteria for clustering WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 10 1. Tissues/cells manipulated 2. Method of delivery of technique into cell 3. Process in genome at molecular level 4. Intermediate crop/segregation 5. Commercialized/final product

Groups of NPBTs WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 11 ZFN ODM Meganuclease TALE (Transcription activator like effector) proteins GROUP 1: Targeted Mutagenesis Cisgenesis/Intragenesis GROUP 2: Cisgenesis/ Intragenesis

Groups of NPBTs WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 12 Reverse Breeding RdDM Accelerated breeding following induction of early flowering GROUP 3: Transgenic construct driven breeding Grafting on GM rootstock Agro-infiltration GROUP 4: Others Agro-infection

WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES 12-13 SEPTEMBER 2011 SEVILLE, SPAIN 13 Copyright: European Union, 2011