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Safety assessment and regulation of new plant breeding
technologies
Joachim Schiemann & Frank Hartung
Institute for Biosafety in Plant Biotechnology
Plant Biotech Denmark (PBD)
Annual Meeting
Copenhagen, January 31 – February 1, 2013
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EU Legislation on GMOs
“ ... now in 2013 with more research into GM technology than almost any other area of food research, there is no evidence to suggest that the GM technology per se poses any unique risk compared to any other plant breeding technology.”
“To be provocative - can we meet the demand for food to feed 9 billion citizens by 2050 without using every tool in the tool box? Is it ethical to reject technology without evidence but on the grounds of preference when 1 billion global citizens every single day are starving?”
“Our obligations as citizens is to look at the evidence presented and have the courage to reposition our views as that evidence accumulates.”
Editorial EPSO NL29
Anne Glover,
Chief Scientific Adviser to the President
of the European Commission
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EU Legislation on GMOs
For clarification:
GM technology is a necessary and indispensible part of new breeding technologies.
In contrast to the other technologies to be described later, GM technology is regulated worldwide.
A regulation based on the technology and not on the trait/product is scientifically not justified, but reality.
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Experiences from experimental field
releases and placing on the market
Commercial cultivation of GM plants:
2011: world wide on 160 million ha
by 16.9 million farmers*
in 29 countries (import in 60 countries)
1996-2011: world wide on 1258 million ha
(Source: ISAAA Briefs No. 43-2011)
Experimental field releases with GM plants:
more then 200 species
several ten thousands since 1987
* >90% small farmers in developing and newly industrialyzing countries
Negative (GMP-specific) ecological or health effects have not
been observed.
But: Case-by-case assessment (trait-oriented) according to the
state of the art !
Comparison with base line !
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Biosafety research - EU
Funding by EU Commission (DG Research):
130 research projects in 25 years, EU funding 300 million €,
500 independent research groups
Commission conclusions:
GM technology – no higher risk than other breeding methods
Commissioner for research:
GM plants can provide solutions – improving agricultural production,
global food security
A decade of EU-funded GMO research [ 2001 - 2010 ]
Chapter 1. Environmental Impacts of GMO
Chapter 2. GMO and Food Safety
Chapter 3. GMOs for biomaterials and biofuels –
Emerging technologies
Chapter 4. Risk assessment and management –
Policy support and communication
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EU Legislation on GMOs
• GMO defined as “an organism… in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination”
• Annex IA Part 1 of Directive 2001/18/EC: Non- exhaustive list of techniques that lead to genetic modification
• Techniques not considered to result in genetic modification or excluded from the scope of the GMO legislation also listed
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New techniques & GMO Legislation
Working group:
Established at request of 2001/18/EC CAs in October 2007
Two experts per Member State (nominated by CAs)
Objective: examine new techniques in the context of the GMO legislation
Final report provided in February 2012 (grey literature)
Safety aspects - EFSA mandate: Is current guidance appropriate?
Risks related to the use of new techniques
- Compared to conventional breeding
- Compared to current GMOs
Cisgenesis/intragenesis (EFSA Journal 2012;10(2):2561)
Zinc Finger Nucleases (ZFN-3) (EFSA Journal 2012;10(10):2943)
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New plant breeding techniques
Zinc Finger Nuclease (ZFN) Technology (restriction and manipulation of DNA, new: TALENs)
Oligonucleotide Directed Mutagenesis (precisely directed point mutations, ODM)
Cisgenesis/Intragenesis (genes/cDNAs of sexual compatible species)
RNA-dependent DNA methylation (miRNA, RNAi and hairpin RNA for DNA methylation)
Grafting (grafting on a GMO rootstock)
Reverse Breeding (isolate parental genotypes by suppression of meiosis)
Agro-Infiltration (transient expression by Agrobacterium transformation)
Synthetic Genomics (Biology) (transplantation of synthesized genes and genomes)
Evaluation of new technologies, defined by the EU-Comission in 2007
* Lusser et al., 2011. JRC Scientific and technical report.
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ODM
Oligo directed mutagenesis
An oligonucleotide modified for stable binding induces a point mutation
Works like a natural point mutation
stabilised mismatch, repair via MMR leads to point mutation
Creation of herbicide resistance and disruption of catalytic centers
Only small alterations are possible
Oligonucleotide Original gene
modified gene
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Cisgenesis
Only genes which are present in close relatives are transformed
Utilisation of the natural gene pool (e.g. genes from wild type species)
So far the transformation procedure is a GMO technique
Instead of a transgenic plant this is called a cisgenic plant
Intragenesis = Cloning of regulatory areas or the cDNA of a cisgene
The plants used for cisgenesis must be sexually compatible in nature
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Grafting on GM rootstock
Like a conventional grafting, but a transgenic plant is used as rootstock
Is there any stable integration of the effector molecule into the genome of the scion?
New side effects are not to expect
GM rootstock
non-GM scion
Offspring and fruits are non-GMOs
e.g. effector-RNA
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Reverse Breeding
A superior heterozygous hybrid plant is used to reproduce the parental lines
• meiotic recombination in the heterozygous hybrid is suppressed
• chromosomes segregate without new recombination but randomly
• some of the haploid microspores possess the exact genotype of the parents
• diploidization of the haploid cells is leading to homozygous parental genomes
Meiotic suppression is achieved by RNAi or deletion of SPO11
+ No side effects to expect, there is no change in the genomic sequence
GMO
- Only possible for plants with 10 or less chromosomes in the haploid set
+ No back-crossing and selection necessary
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Agro-Infiltration
UV
UV
Magnifection: Infiltration of Leaves or Whole Plants
Source: NOMAD
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Agro-Infiltration
‘Spray-N-Trait’: the Future Process Source: NOMAD
Growing
bacteria
Field agroinfiltration
Biomass processing
Limitations today:
technical
regulatory
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Synthetic Genomics
whole new genomes synthesized in vitro & transplanted into an empty host cell performed by Craig Venters group with a Mycoplasma mycoides genome (Gibson et al., 2008, 2010, Science)
One or more genes syntesized & transformed into a living cell for production
Ideas
• synthesizing of enzymatic pathways for a specific chemical • transplantation of these into plant cells • e.g. as chromosomal integration or extrachromosomal (mini chromosome)
• Developing of a new chloroplast genome as a factory for new products -first tests with Synechococcus elongatus injected into animals and cells (Agapakis et al., 2011. PLoS One 6(4) e18877)
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RNA dependent DNA-Methylation
By expression or cell to cell transport of double stranded RNA, a specific sequence (e.g. promotor) in the target cell is silenced These dsRNAs in plants are usually hairpin RNAs, but also other RNA forms can be used (siRNA, asRNA, miRNA)
Source: www.scbio.de/gene_silencers.html
RdDM
Histone modification
DNA methylation
Stable inheritable silencing
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Nuclease technology
Nuclease technology includes meganucleases, Zinc Finger Nucleases (ZFN) and Transcription Activator-Like Effector Nucleases (TALEN)
The principle for all three kinds of nucleases are similar:
• a DNA binding domain is either encoded or fused to a restriction domain
• the nuclease is stably or transiently expressed in vivo
• after binding of a specific DNA sequence a double strand break (DSB) is induced
• the DSB is repaired by the cellular repair machinery
• during repair point mutations, indels or homologous recombination (HR) occurr
The repair by HR can be used for efficient gene targeting
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5‘------CATCGGCTCTTCCATATGACGT ACTGTGCTCAACACCAAACAGA------3
Nuclease technology
3‘------GTAGCCGAGAAGGTATACTG CATGACACGAGTTGTGGTTTGTCT------5
Nuclease technology includes meganucleases, ZFN and TALENs
Repair of the DSB via
NHEJ produces a non-GMO whereas the integration of new DNA by HR results in a GMO (ever?)
HR (0,01%)
Integration/Excision
binding domain
binding domain
NHEJ (>99%)
PM Indels
ZFN1 and 2 = non-GMO
= restriction domain
homologous DNA
- + HR (1%)
ZFN3 = GMO
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New techniques & GMO Legislation
Working group: Techniques to be excluded from GMO legislation by Annex IB
(2001/18/EC) and Annex II part A (2009/41/EC):
ODM, ZFN-1 and -2; Grafting with non-GM scion;
Offspring of plants subjected to Agro-infiltration “sensu stricto”;
RdDM subjected plants without heritable change of their DNA
(working group interpretation: methylation alone is not a heritable change)
Methylation is a heritable phenotypic variation but not a genetic variation
Offspring from reverse breeding
Techniques which are within the scope of GMO legislation by Annex IA Part 1 (2001/18/EC):
ZFN-3, Cis- and Intragenesis; Grafting with GM scion;
Agro-infiltration “sensu stricto” (subjected plants) or floral dip (also offspring);
RdDM subjected plants with integrated foreign DNA
All intermediate organisms containing recombinant DNA
The techniques ZFN-3 and Cisgenesis (and combinations) might be excluded if the resulting organism is similar to self cloning
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Safety aspects - EFSA opinions:
Cis- and Intragenesis
Similar hazards with cisgenesis compared to conventional breeding but novel hazards can arise with intragenesis
ZFN-3
Similar but less hazards with ZFN-3 and transgenic plants
New techniques & GMO Legislation
The EFSA guidances for risk assessment of food and feed and
genetically modified plants (EFSA 2010 and 2011) are fully
applicable for the evaluation of plants resulting from Cis-/
Intragenesis and ZFN-3.
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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)
Since other nucleases with a similar function to ZFN are considered in this opinion the term
site-directed nuclease 3 (SDN-3) is used to describe the technique rather than ZFN-3
specifically.
The EFSA GMO Panel compared the hazards associated with plants produced by the
SDN-3 technique with those obtained by conventional plant breeding techniques and by
currently used transgenesis. With respect to the genes introduced, the SDN-3
technique does not differ from transgenesis or from the other genetic modification
techniques currently used, and can be used to introduce transgenes, intragenes or
cisgenes. The main difference between the SDN-3 technique and transgenesis is that
the insertion of DNA is targeted to a predefined region of the genome. Therefore, the
SDN-3 technique can minimise hazards associated with the disruption of genes and/or
regulatory elements in the recipient genome. Whilst the SDN-3 technique can induce
off-target changes in the genome of the recipient plant these would be fewer than those
occurring with most mutagenesis techniques. Furthermore, where such changes occur
they would be of the same types as those produced by conventional breeding
techniques.
New techniques & GMO Legislation
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APHIS has taken steps to refine and limit its regulatory authority to only plants that are plant pests, were created with plant pests, or used a plant pest in the method to create the plant.
2004: Cibus Global, HT canola, ODM; APHIS: no authority to regulate products created by mutagenesis techniques
2006: maize ZFN-1,2 deletion technology, Dow AgroScience; APHIS: no regulated article
2011: HT Kentucky bluegrass, Scotts; APHIS: no regulated article
Decisions made relative to specific cases, but indication: APHIS will not regulate new breeding technologies in and of themselves
Situation US: USDA-APHIS
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2011: EPA issued a Notice of Proposed Rule-Making: cisgenic PIPs (plant incorporated protectants) are exempted from registration
FDA does not formally approve the new food/feed to be safe per se; they indicate it is not materially different from the unmodified version with respect to composition or safety as it is „as safe as its non-modified counterparts“
Situation US: EPA & FDA
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Photo:
Anke
Schiemann
Thank you very much for your attention
Those who want the world to continue as it is, do not want the world to continue
