State-of-the-art of new plant breeding techniques Maria Lusser European Commission - Joint Research Centre Workshop on NBT for Regulatory Considerations Tokyo, 15 October 2013 Contact: maria.lusser@ec.europa.eu www.jrc.ec.europa.eu Serving society Stimulating innovation Supporting legislation
JOINT RESEARC CENTRE (JRC) IE Petten, The Netherlands Institute for Energy IRMM Geel, Belgium Institute for Reference Materials and Measurements ITU Karlsruhe, Germany Institute for Transuranium Elements www.jrc.ec.europa.eu IES/ IHCP/ IPSC Ispra, Italy Institute for Environment and Sustainability Institute for Health and Consumer Protection Institute for the Protection and Security of the Citizen IPTS Sevilla, Spain Institute for Prospective Technological Studies Serving society Stimulating innovation Supporting legislation
NPBT - DEFINITION NEW PLANT BREEDING TECHNIQUES (NPBTs) Breeding techniques which deploy biotechnology Fall product derived through these techniques under the GMO legislation? Developed during the last 10 (20) years 10 October 2013 3
ACTIVITIES OF THE EUROPEAN COMMISSION ON NEW PLANT BREEDING TECHNIQUES 10 October 2013 4
EC ACTIVITIES ON NPBTs (1) WORKING GROUP ON NEW TECHNIQUES (NTWG) - experts from Member States managed by the European Commission (DG SANCO) Evaluated whether NPBTs constitute techniques of genetic modification and Whether the resulting organisms falls under EU GMO legislation Evaluation started in 2008 Report was finalised in January 2012 (not public) 10 October 2013 5
REGULATORY FRAMEWORK EUROPEAN UNION Regulatory system for GM crops since 1990 Amended in 2001 Expanded to food and feed in 2003 GMO definition: Directive 2001/18/EC, Article 2 (2) Genetically modified organism (GMO) means an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination
DIRECTIVE 2001/18/EC ANNEX 1 A, PART 1 Techniques which are considered to result in genetic modification such as recombinant nucleic acid techniques, micro- and macroinjection and cell fusion ANNEX 1 A, PART 2 Techniques which are NOT considered to result in genetic modification such as in vitro fertilization, natural processes such as: conjugation, transduction, transformation and polyploidy induction ANNEX 1 B Techniques of genetic modification yielding organisms to be excluded from the Directive such as mutagenesis and cell fusion TRANSGENESIS MUTAGENESIS RISK ASSESSMENT
EC ACTIVITIES ON NPBTs (2) EFSA Panel on Genetically Modified Organisms (Managed the European Food Safety Authority) Mandate of DG SANCO in 2011 to address the safety aspects of NPBTs Scientific opinion addressing the safety assessment of plants developed through cisgenesis and intragenesis (2012) Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function (2012) 10 October 2013 8
EC ACTIVITIES ON NPBTs (3) European Commission Joint Research Centre (JRC) Study on New plant breeding techniques: state-of-theart and prospects for commercial development (2010 published 2011) Study on Comparative regulatory approaches for new plant breeding techniques (2011 published 2012) 10 October 2013 9
WHAT IS THE EC DOING? JRC Project team Maria Lusser Claudia Parisi Damien Plan Emilio Rodríguez Cerezo JRC-IPTS JRC-IPTS JRC-IHCP (now: JRC-headquarters) JRC-IPTS
GROUPS OF NEW PLANT BREEDING TECHNIQUES 10 October 2013 11
GROUPS OF NPBTs GROUP 1: SITE DIRECTED MUTAGENESIS OLIGONUCLEOTIDE DIRECTED MUTAGENESIS (ODM) ZINC FINGER NUCLEASE (ZFN) TECHNOLOGY MEGANUCLEASE TECHNOLOGY TAL-EFFECTOR NUCLEASE (TALEN) TECHNOLOGY GROUP 2: CISGENESIS & INTRAGENESIS CISGENESIS/INTRAGENESIS GROUP 3: BREEDING WITH TRANSGENIC INDUCER LINE RNA-DEPENDENT DNA METHYLATION (RdDM) REVERSE BREEDING ACCELERATED BREEDING FOLLOWING EARLY FLOWERING 10 October 2013 12
GROUPS OF NPBTs GROUP 4: GRAFTING TECHNIQUES GRAFTING ON GM ROOTSTOCK GROUP 5: AGRO-INFILTRATION TECHNIQUES AGRO-INFILTRATION "SENSU-STRICTO" AGRO-INFECION FLORAL DIP 10 October 2013 13
GROUPS OF NPBTs GROUP 1: SITE DIRECTED MUTAGENESIS SITE DIRECTED NUCLEASE TECHNIQUES OLIGONUCLEOTIDE DIRECTED MUTAGENESIS ZINC FINGER NUCLEASE TECHNIQUE MEGANUCLEASE TECHNIQUE TALEN TECHNIQUE
GROUP 1 ODM Uses Oligonucleotides share homology with target sequence with the exception of the nucleotides to be modified Oligonucleotides target the homologous sequence in the genome Create one or more mismatch base pairs corresponding to the non-complementary nucleotides
GROUP 1 ZFN TECHNIQUES Zinc finger nucleases are chimeric proteins consisting of Zinc finger domain (recognising specific DNA sequence) and nuclease cutting double-stranded DNA ZFN-1 technology Leads to mutation in one or a few bp, short deletions or insertions (site-specific but non-specific change) ZFN-2 technology Leads to mutation in one or a few bp, short deletions or insertions (mutations of the repair template are introduced site-specific in site-specific way ZFN-3 technology Leads to site-specific insertion of the target gene
GROUP 1 ZFN-1 TECHNIQUE ZFN - DNA cutting domain ZFN- DNA binding domain Double strand break (DSB) Repair by NHEJ (error prone repair) Source: e.g.: http://www.sigmaaldrich.com Point mutation
GROUP 1 ZFN-2 TECHNIQUE Zinc Finger Nuclease (ZFN) Double strand break (DSB) Repair template with one bp mismatch Repair by homologous recombination (HR) Source: e.g.: http://www.sigmaaldrich.com Point mutation
GROUP 1 MEGANUCLEASE TECHNIQUES Meganucleases are natural proteins acting as "DNA-scissors" recognise specific DNA sequences (12-30 pb) cause site-specific DNA breaks possible applications as for ZFN technology http://www.cellectis.com/science
GROUP 1 TALEN TECHNIQUES Transcription activator like effector (TALE) proteins are natural proteins that bind to DNA in a sequencespecific way are converted into DNA-scissors by binding to a nuclease (catalytic domain of FokI) can be modified for targeting a given sequence possible applications as for ZFN technology http://www.cellectis.com/science Miller, J C, Nature Biotechnology 29,143-148 (2011)
GROUPS OF NPBTs GROUP 2: CISGENESIS & INTRAGENESIS CISGENESIS INTRAGENESIS OWN PROMOTER GENE (INCL. INTRONS) OWN TERMINATOR PROMOTER GENE (SENSE OR ANTISENSE) TERMINATOR
GROUP 2 CISGENESIS INTRAGENESIS Cisgenesis/Intragenesis deploy the same technology as transgenesis. DNA fragment from the species itself or from crosscompatible species inserted in the plant genome. Cisgenesis: Inserted gene is unchanged with its own introns and regulatory sequences in the normal sense orientation Intragenesis: Inserted DNA can be a new combination of DNA fragments from the species itself or from a crosscompatible species in sense or antisense orientation.
GROUPS OF NPBTs GROUP 3: BREEDING WITH TRANSGENIC INDUCER LINE REVERSE BREEDING RNA DIRECTED DNA METHYLATION ACCELERATED BREEDING FOLLOWING EARLY FLOWERING FURTHER TECHNIQUES UNDER DEVELOPMENT
GROUP 3 BREEDING WITH TRANS- GENIC INDUCER LINE Transgene encoding an RNAi construct or dominantnegative protein inserted in genome of inducer line Expression of transgene leads to the inhibition of gene expression or protein function Effect of transgene is used during several breeding cycles Inducer transgenes finally segregated out (negative segregant)
GROUP 3 RdDM T-DNA Ti-Plasmid ds RNA Agrobacterium Endonuclease 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
GROUP 3 REVERSE BREEDING 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
GROUP 3 EARLY FLOWERING In initial step transgenic plants produced using gene silencing for reduction of juvenility/vegetative maintenance factors Or over-expression constructs to reach over-expression of a flower initiation related transcription factor Leads to reduction of time required for breeding cycle After several breeding cycles (when breeding target is reached) transgene will be crossed out Flachowsky H. et al., Acta Hort 763: 215-222 (2006) Flachowsky H. et al., Acta Hort 738: 307-312 (2007)
GROUPS OF NPBTs GROUP 4: GRAFTING TECHNIQUES GRAFTING ON GM ROOTSTOCK
GROUP 3 GRAFTING ON GM ROOTSTOCK Nontransgenic scion Transgenic rootstock Classification of fruits?
GROUPS OF NPBTs GROUP 5: AGRO-INFILTRATION TECHNIQUES AGROINFILTRATION 'sensu-stricto' FLORAL DIP AGROINFECTION
GROUP 5 AGROINFILTRATION "SENSU STRICTO Infiltration with suspension of Agrobacterium sp. containing the gene of interest Local, transient gene expression at high levels AGRO-INFECTION Infiltration with suspension of Agrobacterium sp. containing the gene of interest inserted into a full-length virus vector Facilitates the spreading and expression of the target gene in the plant FLORAL DIP Germline tussue (typically flowers) is immersed into a suspension of Agrobacterium sp. containing a T-DNA construct to transform female gametocyte GM seeds are obtained
GROUP 5 AGROINFILTRATION 'SENSU SRTICTO' Classification of progeny? Detached plant part infiltrated Intact plant locally infiltrated
STATE-OF-THE ART OF NEW PLANT BREEDING TECHNIQUES 10 October 2013 33
N. scientific publications LITERATURE SEARCH Development over time of scientific publications on NPBTs 30 25 20 15 10 5 0 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 ODM ZFN MEGA TALEN CISG-INTRA RdDM REV BREED GRAFT
LITERATURE SEARCH Country of origin of institutions authoring scientific publications on NPBTs Sector of institutions authoring scientific publications on NPBTs Other countries 12.1% EU-27 42.6% Asia 12.6% Sector N. publications % Academia 143 77% Industry 24 13% Joint 19 10% Total 186 North America 32.7%
LITERATURE SEARCH ODM ZFN MGN TALEN RdDM REVERSE BREEDING CISGENESIS- INTRAGENESIS GRAFTING ON GM ROOTSTOCK Commercial development: oilseed rape (HT + other traits), maize (HT), flax, potato, tomato, tobacco, Petunia Additional info from literature: HT rice Commercial dev.: maize (HT), oilseed rape, tomato, tobacco, sugar beet, potato (starch quality), trees (lunber/paper), lettuce, Petunia, Agyranthemum, poplar Commercial dev.: maize (HT) and tobacco Literature: HT tobacco Commercial dev.: maize, oilseed rape Literature: maize (male sterility), potato (starch content), tomato (no ripening), Petunia (reduce pigmentation) Commercial dev.: tomato (taste), broccoli Literature: oilseed rape Commercial dev.: potato (fung. res. and starch content), maize, oilseed rape, barley (less phytate) Literature: potato (sev. traits), apple (fung. res and red flesh), grapevine and melon (fung. res.), alfalfa (reduced lignine), poplar (wood properties) Commercial dev.: grapevine (virus res.), apple and pear (root. ab.), citrus (dwarfing and fung. res.) Literature: apple, rose and walnut (root. ab.), watermelon, pea, potato and cucumber (virus res.)
LITERATURE SEARCH R&D on NPBT is active since 2000, with the exception of grafting on GM rootstock EU public institutions have largest share of publications (esp. for cisgenesis/intragenesis, reverse breeding, RdDM and grafting on GM rootstock), USA plays the second most important role NPBT are mainly applied for pest resistance and herbicide tolerance. Some techniques (like cisgenesis) have been already tested in many crop plants, while others (like ZFN) have been tested mainly in model plants
N. Patents PATENT SEARCH Development over time of patents on NPBTs 25 20 15 10 5 0 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 ODM ZFN MEGA TALEN CISG-INTRA RdDM REV BREED GRAFT
PATENT SEARCH Country of origin of patent assignees on NPBTs Sector of institutions of patent assignees on NPBTs Other countries Asia 5.1% 7.0% EU-27 37.6% Sector N. patents % Academia 27 20% Industry 104 76% Joint 6 4% Total 137 North America 50.3%
PATENT SEARCH Most active institutions in NPBTs in plants, based on number of patents produced and number of techniques claimed. O:ODM, Z:ZFN, M:MGN, T:TALEN, C: Cisgenesis/Intragenesis, R:RdDM, B:Reverse Breeding, G:Grafting. Institution Sector Country N. patents Techniques Cellectis Industry FR 17 M Sangamo Biosciences Industry US 13 Z Dow Agrosciences Industry US 11 Z Keygene Industry NL 8 O,Z J.R. Simplot Industry US 7 C University of Delaware Academia US 7 O BASF Industry DE, NL, CN 6 O,Z,M Bayer Industry BE, FR 6 Z,M Cornell Research Foundation Industry US 5 G Pioneer Hi Bred Industry US 4 O,M
PATENT SEARCH Patents in NPBT mostly filed since 2000 USA private companies have largest share of patents, followed by EU-based applicants USA particularly dominant for grafting, ODM and ZFN 50 companies are active in patenting on NPBT (mostly active only in one to two of the techniques) Crops/traits identified for NPBT in line with findings of literature search
COMMERCIAL DEVELOPMENT NPBT Patent owner Product name Licensee Plants ODM ZFN Cibus Company (US) RTDS TM rape (BrettYoung), Flax (Flax Council of HT Oilseed rape (BASF), oil quality oilseed Canada), potato (NEU seed) Keygene (NL) KeyBase TM HT tobacco, tomato and Petunia Sangamo (US) EXZACT TM Dow (exclusive) HT maize, sugar beet (KWS Company), quality potato (Wageningen U.), lumber/paper prod. Trees (Oregon State U.) Danziger Innovations (IL) MemoGene TM tobacco, tomato, Petunia, Arabidopsis, lettuce, Agyranthemum and Populus tremula Toolgen (KR) GeneGripR Arabidopsis MGN Cellectis Plant Sciences Monsanto n.d. (FR) (non-excl.) n.d. Precision Dupont, BASF DNE BioSciences (US) (non excl.) Arabidopsis, tobacco, maize and more Cellectis Plant Sciences TALEN (FR) n.d. n.d. Two Blades Foundation n.d. n.d. CISGENESIS Wageningen University potatoes with late bligh res., apples with n.d. (NL) apple scab res. and red flesh INTRAGENESIS Company Simplot (US) Innate TM potatoes with reduced black spot bruise and lower acrylamide
COMMERCIAL DEVELOPMENT Drivers for commercial development of crops via NPBT Technical potential of NPBT Targeted mutagenesis (ZFN, ODM) Site specific insertion (ZFN 3) Gene silencing (RdDM) No transgene integration Segregating out of transgene Method of selection Constraints Technical constraints of NPBT Efficiency issues Regeneration of plants from cuttings, protoplasts, etc. Method for selection of successfully modified plants Economic advantage: Faster breeding process Regulatory costs Uncertainty of regulatory status High costs if regulated
THANK YOU FOR THE ATTENTION 10 October 2013 44
REFERENCES 10 October 2013 45
JRC Scientific and Technical Reports Lusser, M., Parisi, C., Plan, D. and Rodriguez-Cerezo, E. (2011) New plant breeding techniques: State-of-the-art and prospects for commercial development. JRC Technical Report EUR 24760 http://ipts.jrc.ec.europa.eu/publications/pub.cfm?id=4100 Lusser, M. and Rodríguez-Cerezo, E., (2012). Comparative regulatory approaches for new plant breeding techniques. Workshop proceedings. European Commission. JRC Technical Report EUR 25237 EN. http://ipts.jrc.ec.europa.eu/publications/pub.cfm?id=4959 10 October 2013 46
Scientific papers Lusser, M., Parisi, C., Plan, D. and Rodriguez-Cerezo, E. (2012) Deployment of new biotechnologies in plant breeding. Nat. Biotechnol., 30, 231-239. Lusser, M., Davies, H.V. (2013) Comparative regulatory approaches for groups of new plant breeding techniques. New Biotechnol., 30, 437-446. Parisi, C., Rodríguez-Cerezo, E., Thangaraj, H., 2013. Analysing patent landscapes in plant biotechnology and new plant breeding techniques. Transgenic Res. 22, 15-29. 10 October 2013 47
EFSA Opinions Scientific opinion addressing the safety assessment of plants developed through cisgenesis and intragenesis. EFSA Journal 2012: 10(2):2561. http://www.efsa.europa.eu/en/efsajournal/doc/2561.pdf Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function. EFSA Journal 2012: 10(10):2943. http://www.efsa.europa.eu/en/efsajournal/doc/2943.pdf 10 October 2013 48
Thesis Parisi, C. (2013) New plant breeding techniques: state of the art, potential and challenges http://helvia.uco.es/xmlui/handle/10396/9492 EFSA Opinions Scientific opinion addressing the safety assessment of plants developed through cisgenesis and intragenesis. EFSA Journal 2012: 10(2):2561. http://www.efsa.europa.eu/en/efsajournal/doc/2561.pdf Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function. EFSA Journal 2012: 10(10):2943. http://www.efsa.europa.eu/en/efsajournal/doc/2943.pdf 10 October 2013 49