Towards the development and practical application of crops using
new plant breeding techniques (NPBTs) such as genome editing(Provisional Translation)

New Plant Breeding Technique Study Group
September 2015

Contents
I. Introduction

1

II. Trend of R&D and Regulations on NPBTs Overseas
1. Trend of R&D(1) Genome editing using artificial restriction enzyme(2) Oligonucleotide-directed mutagenesis(3) Cisgenesis/intragenesis(4) RNA-dependent DNA methylation(5) Grafting using GM rootstock(6) Reverse breeding(7) Agro-infiltration(8) Seed Production Technology (SPT) process
2. Status of Consideration on Regulatory Framework (abbreviation)(1) EU(2) USA(3) Australia and New Zealand(4) Other international trends

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III. Examples of R&D in Japan and Considerations on the Effects on Biological Diversity
1. Techniques to Remove the Introduced Transgenes during the Breeding Process(1) Accelerated breeding of fruit plants using early flowering genes(2) Recurrent selection of inbreeding crops such as rice(3) Considerations on the effects on biological diversity
2. Genome Editing using Artificial Restriction Enzyme(1) Outline of R&D(2) Considerations on the effects on biological diversity

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IV. Points to Be Considered toward Future R&D and Practical Application
1. Promotion of Related R&D
2. Initiatives towards Promoting the Social Understanding(1) Appropriate response to GM regulations(2) Methods to provide the nation with information and to communicate(3) Promotion of international harmonization pertaining to regulatory framework

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References38

Terminology

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List of Members of the New Plant Breeding Technique Study Group46

History of Deliberations47

I. Introduction
Breeding of crops has a long history of humans modifying wild or native species for many years
for the purpose of realizing improved production or stable supply of food. Starting from the simple
processes of humans selecting plants with useful traits (hereinafter referred to as “mutants”) foundby chance in nature and reproducing them, breeding of crops has gradually evolved into much moreadvanced methods. Nowadays, the most mainstream of breeding techniques is breeding by crossing
where desirable traits are accumulated into the cultivated crops by crossing such mutants, and thedevelopment and use of new mutation breeding techniques are progressing where mutants are
artificially developed by means of radiation or chemicals. The development of breeding techniques
and untiring efforts on breeding have greatly contributed to the improved agricultural productivity,
stable supply of safer and higher-quality food, as well as realizing food security throughout the world.
It is widely known nowadays through the development of molecular biology in recent years thatthe principle of developing mutants basically relies on the genes in organisms, that the main substanceof genes is DNA, and that the combinations of bases (adenine (A), thymine (T), cytosine (C) andguanine (G)) constituting DNA (hereinafter referred to as “base sequence information”) control thetraits of organisms.
In recent years, new highly-sophisticated techniques have been developed to select and grow new
agriculturally-useful varieties more effectively. One of them is DNA marker assisted selection, where
the base sequence information of genes involved with traits useful for agricultural production is
identified by decoding the genomic 1 information of crops (e.g., rice) and comparing it with thevarious mutants that exist in nature, and such information is used as a marker in the selection of usefulvarieties. Such newly developed techniques are about to be applied to breeding of various crops
including rice and vegetables, and application of such molecular biological knowledge to the field ofcrop breeding in the future is expected to drastically increase the breeding speed and to significantly
reduce the development cost. Development of breeding techniques to draw out the maximum
potential of crops is essential for addressing some globally important food production issues including
the necessity to increase the food production associated with the increasing global population and thefight against the global warming. Utilization of molecular biology to that end seems just a matter ofcourse in this era.

1

Refers to a group of genetic information that is necessary for organisms to live. For example, the DNA of riceconsists of about 0.4 billion base pairs and that of humans consists of about 3 billion base pairs.

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In Europe and the USA, in addition to the DNA marker assisted selection, progress has been madeon the development of “new plant breeding techniques (NPBTs 2)” that enable accurate and efficient
introduction of traits useful for agricultural production, where genetic modification technologies areapplied to a part of the breeding process of conventional breeding by crossing or mutation breeding.
As described in Sections II and III below, there are various techniques for NPBTs, including:(1) those aiming at improving the efficiency of developing mutants by conventional mutation
breeding (e.g., genome editing, oligonucleotide-directed mutagenesis (ODM)); and,(2) those aiming at shortening the breeding term for conventional methods such as breeding bycrossing (e.g., accelerated breeding of fruit plants, agro-infiltration).
The characteristic common to these techniques is that while genetic modification (GM)technologies are applied to a part of the breeding process, the final crop products can be free fromtransgenes used for genetic modification and therefore there is a possibility that plants equivalent tosuch engineered plants can be developed through the selection from the natural diversity or throughconventional breeding by crossing, mutation breeding, etc.
For that reason, when such crops become commercialized, it will become difficult to differentiatethem from crops developed by conventional breeding techniques and there arises a question whether
regulations on genetically modified organisms (GMOs) shall be applied to such crops. Currently,
discussions are being held overseas on the regulatory framework for NPBTs under GM regulations
concerning the food and feed safety and the environmental impact (Food Standards Australia New
Zealand, 2013; European Food Safety Authority, 2012a; 2012b; Lusser et al., 2011).
When genetic modification technologies were used for crop breeding, the common purpose was tobestow traits that cannot be acquired by natural crossing or conventional breeding techniques, forinstance introduction of an insect killing trait possessed by microbe into a crop.
Such a crop possesses a transgene originating from microbe etc. and new substances expressed by
the transgene, becoming an organism that humans have never consumed or cultivated before; there isa possibility of the crop causing unexpected harmful effects on human health or wildlife. Therefore,
from the viewpoint of preventing such harmful effects, national regulatory frameworks have been set
to require safety assessment for each individual case and only those with confirmed safety andapproval by the Government can be cultivated, imported, etc. Currently, such a system of ex-ante
regulations is widely adopted in various countries in the world.

2

New plant Breeding Techniques-2-The Ministry of Agriculture, Forestry and Fisheries (MAFF) of Japan is now accelerating thedevelopment of new innovative varieties that generate “strength” in Japan’s agricultural and livestock
products as part of its policy to realize “Aggressive Agriculture, Forestry and Fisheries”, and NPBTs
may become important key techniques to realize the goal, along with the DNA marker assisted
selection.
Additionally, amid the situation where the transformation of agriculture, forestry and fisheries
industries into growing industries is placed as an important political task for the entire Japanese
Government, the “Comprehensive Strategy on Science, Technology and Innovation 2015 (Cabinet
Decision on June 19, 2015) 3” indicates the “development of next-generation breeding systems suchas NPBTs” as one of the priority R&D initiatives to be promoted in the future, and the relevant
ministries are jointly promoting R&D under the Strategic Innovation Promotion Program (SIP) 4.
However, on practically applying the results of such research, the important task in Japan is how
to develop social understanding of crops and foods developed by NPBTs at the commercialization,
while there still remain strong concerns among Japanese consumers and producers over the safety ofcrops and foods to which GM techniques are applied.
Additionally, while they have already started discussions on the regulatory framework for NPBTs
under GM regulations in the EU etc., we also need to promote collection and analysis of scientificknowledge and experience that can form a basis of such discussions, and to hold discussions withdiverse stakeholders in the future taking into account the situation of NPBT R&D overseas and thestatus of consideration on the regulations over NPBT in other countries. Further, there is a necessity
to promote activities to internationally share such scientific findings towards achieving international
harmonization on the regulatory framework for NPBTs and genetically modified organisms.
Based on such background, in October 2013, a study group consisting of experts was establishedwithin the Agriculture, Forestry and Fisheries Research Council Secretariat, aiming at gathering
domestic and overseas information on NPBTs, compilation of scientific findings on the effects on the
biological diversity, proper promotion of related R&D, smooth social implementation of research
results, etc.
The main duty of the Study Group was to investigate what kinds of breeding techniques currently
exist as NPBTs, to assess and analyze how the crops developed by taking advantage of thetechnological characteristics etc. of NPBTs are addressed under the current GM regulations (Food34

http://www8.cao.go.jp/cstp/sogosenryaku/index.html
http://www8.cao.go.jp/cstp/gaiyo/sip/-3-

Sanitation Act, Feed Safety Act and the Cartagena Act) in advance, and thereby to promote therelevant R&D in accordance with the current regulations.
However, during the considerations at the Study Group, it was indicated that (1) many NPBTs
apply the latest findings of molecular biology to breeding of crops and further technical improvementand development in the future are expected, (2) in Japan, scientific assessment of GM techniqueapplied foods on their safety and effects on the biological diversity is in principle carried out on acase-by-case basis, taking into account the properties etc. of the engineered crops or food (product)
instead of individual breeding techniques (process), and (3) assessment of food safety etc. must be
done by experts in each field.
To that end, the Study Group decided only to provide scientific opinions on effects on the biologicaldiversity that is the expertise of the Study Group members, using some R&D for which MAFF aims
at practical application in the future as case studies (Chapter III).
The Study Group hopes that R&D at MAFF shall properly comply with regulations such as the
Cartagena Act etc. in the future based on the points to be considered that were summarized in thisReport (Chapter IV). The Study Group also hopes investigations and discussions will be enhanced byvarious experts including food safety experts by continuously collecting and analyzing scientificfindings, obtaining information on relevant regulations overseas and by promoting international
dialogue towards the harmonization of regulatory framework etc., through cooperation andcollaboration with the relevant regulatory authorities including food safety aspects.
Additionally, social implementation of leading-edge science and technology such as NPBTs
essentially requires promotion of interactive mutual communication with a wide variety of relevant
people from the R&D stages, and it is important to reflect the voices of anticipation, anxiety andconcerns from the nation on the R&D and the process of practical application. In that sense, the Study
Group strongly desires that the release of this Report triggers a fostering of better understanding of
the technical characteristics and regulatory issues of NPBTs by the general public and commencementof broad discussions on NPBTs and GMOs in Japan on a wide range of topics, including how NPBTs
shall be utilized in the future for the purpose of revitalizing the agriculture, forestry and fisheries
industry, improving the quality of life for the nation, and solving the global issues that humans may
encounter in the future.

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1. Trend of R&D
In the EU, the “New Techniques Working Group (NTWG 5)” consisting of scientists etc. wasestablished under the European Commission (EC) in 2007 in response to a consideration request
by the Netherlands, and deliberations have been made on the regulatory framework for NPBTs.
As part of the deliberations, in 2011, the European Commission, Joint Research Centre (JRC),
Institute for Prospective Technological Studies released a report entitled “New plant breeding
techniques. State-of-the art and prospects for commercial development 6”. According to this report,
currently the development and practical application have progressed for 7 techniques (techniques(1) to (7) below) centering on research institutions and private companies in the US and Europe,and:
1. The EU leads the world in the number of related scientific papers issued and the USA leadsin the number of patent applications;
2. As a result of a questionnaire survey given to major biotechnology enterprises, NPBTs havebeen adopted by commercial breeders and the most advanced crops could reach the stage ofcommercialization in the short to medium term (2-3 years) if the crops produced by using
these techniques are not classified as GMOs.
3. All techniques are undergoing preparations for practical application, placing emphasis on
higher efficiency rather than conventional breeding and substantial reduction in the cost fordeveloping new products. Crops resulting from most of the techniques cannot be distinguishedfrom conventionally bred crops and detection is therefore not possible.
In 2012, the Food Standards Australia New Zealand (FSANZ) established a scientific panel 7 forthe purpose of obtaining expert advice on the handling of such breeding techniques under the GM
regulations. During the course of its workshops held to date, a total of 12 techniques have been
introduced in addition to the 6 breeding techniques (e.g., genome editing) that were deliberated in

5

New Techniques Working Group
Lusser et al. “New plant breeding techniques. State-of-the art and prospects for commercial development”,
http://ipts.jrc.ec.europa.eu/publications/pub.cfm?id=4100
7 http://www.foodstandards.gov.au/consumer/gmfood/Pages/New-plant-breeding-techniques-in-the-spotlight.aspx
6-5-

the EU, including the hybrid maize seed production technology (SPT 8 , technique (8) below)
practically applied by the US DuPont Pioneer 9.
NPBTs discussed in the EU and Australia/NZ are outlined below.
(1) Genome editing using artificial restriction enzyme
It is known that mutants like so-called “bud variations” are generated due to mutation of thegene (DNA) in plants caused by various natural effects such as UV. Nowadays, mutation breeding
to artificially induce mutation by utilizing radiation or chemicals is practically applied and used inbreeding and improving of crops. In such mutation breeding, mutation of DNA randomly occurs
on the chromosome of plants (hereinafter referred to as “on the genome”), and the possibility ofmutation occurring to the gene (DNA) involved with the target trait of breeding and improving is
extremely low. For that reason, it used to take many years for humans to find useful varieties (Abeet al., 2013).
Lately, “artificial restriction enzymes” that allow for accurate cleavage of specific base sequence
domains of target genes (DNA) have been developed, and it has become possible to arbitrarily
induce gene modification (deletion, substitution or insertion) to the targeted domain on a genome(genome editing).
By applying the genome editing to plant breeding, it would be possible to arbitrarily modify aendogenous gene that affects the flower color, plant height, etc. and thereby develop new
innovative varieties in a short period of time.
There are two representative methods of genome editing using artificial restriction enzyme; one
is Zinc Finger Nuclease (ZFN) and the other is Transcription Activator Like Effector Nuclease(TALEN). Both of the proteins consist of “DNA binding domain” to identify the specific basesequence information on the genome and “DNA cleavage domain (Fok I)” with restriction enzyme
activity.
The mechanism of these techniques is that when the relevant DNA binding domain binds to thehomologous section of double-stranded DNA of the organism (host) and the DNA cleavage domain
has formed a dimer, the double-stranded DNA of the host is cleaved (Osakabe and Osakabe, 2013;
Engstrom et al., 2009) (Fig. 1).

8

Seed Production Technology, https://www.pioneer.com/CMRoot/pioneer/about_global/our_research/enabling_technologies/enabling_technologies_sheets/tech_spt_2014.pdf
9 While the report of FSANZ introduced it as a technique of “Pioneer Hi-Bred International”, this report follows
the material by MAFF, http://www.s.affrc.go.jp/docs/commitee/diversity/130326/pdf/sankou3.pdf

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DNA
cleavagedomainDNAbinding
domainA. ZFN cleaves a
specific sequence on
the chromosome

B. Accurate DNA repair

When an error occurs during DNA repair…
C. Deletion ofa baseOriginal sequence(no mutation)

Base deletion-type mutation

Fig. 1. Schematic of DNA cleavage by an artificial restriction enzyme (ZFN).
Additionally, recently, a technique called CRISPR 10/Cas system has been developed, where the
DNA binding domain utilizes not a protein but RNA (Sakuma, 2013).
The chance of the same base sequence existing on genome can be calculated as 1/(418)(maximum of 1 in 70 billion), for instance, if the number of bases in the identified sequence ofDNA binding domain is 18 (Fig. 1). Considering that plant genomes consist of billions of basepairs, as long as special attention is paid to the specificity of the base sequence of DNA binding
domain at the time of designing the artificial restriction enzyme, it can be said that the possibilityof cleaving non-targeted base sequence on the genome (so-called “off-target”) is extremely low(Hsu et al., 2013).
Usually double-strand DNA breaks are swiftly repaired in the cell of the host, and there are three
types of genome editing that takes advantage of DNA repair (Ezura and Osawa, 2013; Porteus,2009) (Fig. 2):
10

Clustered Regularly Interspaced Short Palindromic Repeats-7-1. Expects an error occurring during the repair and random mutation (base substitution, insertionor deletion) occurring for one or a few bases (Site-Directed Nuclease 1 (SDN-1));
2. Systematically induces mutation for 1 or a few bases by artificially synthesizing a short DNA
fragment (template) that is homologous to the target base sequence and introducing it along withan artificial restriction enzyme at the time of cleaving (SDN-2); and,3. Forms a special DNA fragment at a specific domain on the genome by introducing a long DNA
fragment containing a gene of several thousand base pairs not originating from a compatible
same or related varieties (transgene) in a form sandwiched by sequences homologous to thetarget sequence (SDN-3).
(ZFN-2)(ZFN-1)
Expression cassette of
ZFN gene(ZFN-3)DNA fragment with homology to theIntroduction targeted sequences but containing

Introduction

mutations ( : one or several bases)

DNA fragments with homology to target

Introduction sequences but harboring useful exogenous
sequence (several kilobases)* In higher plants, it is necessary
for targeted mutagenesis toinsert ZFN expression
cassettes into the plant
genome, since the frequencies
of targeted mutagenesis isquite low. The lines in whichtransgenes (ZFN expression
cassettes) are segregated-out
can be selected in theprogenies.

Crop cell
Expression
Loss*Cleave thetarget geneDNA

Target gene↑↑↑
Repaired via
homologous
recombination(HR) between
target geneand introduced
DNA fragment

Double strand breaks(DSBs) caused by
ZFN are processedby endogenous DNA
repair mechanisms

Several kbp↑
Useful exogenous
genes areinserted into thetargeted gene via
HR - mediated
DNA repair
mechanism

Fig. 2. Types of genome editing.
While ZFN and TALEN enable induction of such mutation to the gene (DNA) of animals bydirectly introducing the ZFN/TALEN protein (artificial restriction enzyme) into the cell, different
methods are used for plant cells. For plants, usually, (1) the protein is transiently expressed byincorporating the gene into a vector often used for GM techniques, or (2) the gene (gene that
expresses artificial restriction enzyme) is incorporated into the crop genome, the artificial
restriction enzyme is expressed, mutation is induced, and then the relevant gene is removed bybackcrossing with conventional varieties or other method.

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(2) Oligonucleotide-directed mutagenesis
Oligonucleotide-directed mutagenesis (ODM 11) is a technique to induce mutation to endogenous
gene of organism (crop) artificially, similar to genome editing above, and has a history of R&D
over nearly 30 years.
The process of ODM is to synthesize an oligonucleotide 12 or a short single-stranded DNA
fragment (length of 20-30 bases) that is homologous to the target base sequence but has mutationof about 1 base and to directly introduce it into a cell by particle bombardment etc. (Ezura andOsawa, 2013) (Fig. 3).
Plant Cell
Introduction

Oligonucleotide (20-100bp) homologous to thetarget gene to which
partial mutation (* several
bases) is introduced

Specific domain of target
gene

Binds to the specific domain

Mutation is induced

Fig. 3. Outline of ODM.
In ODM, mutation of about 1 base is induced to the target sequence on the genome, resulting ina change in the pattern for expressing the relevant gene. Such a mutation can occur naturally, andthe mutation-induced crop cannot be distinguished from those selected through natural diversity or
those developed by the conventional breeding techniques.

11
12

Oligonucleotide-Directed Mutagenesis
A short sequence of DNA or RNA of around 20-100 base pairs-9-Using ODM, canola, maize, etc. with herbicide resistance have been developed to date. However,its low mutation rate is the bottleneck of its practical application.
(3) Cisgenesis/intragenesis
Cisgenesis is a method to introduce a gene of the same or related compatible species (cis-gene)to crops using GM technologies. Its presupposition is no introduction of genes or DNA fragmentsfrom any other species (Ezura and Osawa, 2013; Holme et al., 2013).
In the conventional breeding by crossing, attempts to introduce a specific gene (e.g., pest
resistance gene) from a wild relatives often result in causing harmful effects to other useful traits(e.g., yield, quality) possessed by the cultivars, which requires backcrossing with the cultivars after
the introduction process. This backcrossing has to be repeated many times and over a long term.
Additionally there is a difficulty in backcrossing vegetative propagation crops such as fruit plants,
potato and sugar cane. In such occasions, cisgenesis may be an effective method of breeding.
Intragenesis also uses the same or related species as the source of genes in a similar manner ascisgenesis. However, intragenesis is used to control the expression amount of a specific gene bypartially modifying the promoter 13 or terminator 14 that constitutes the gene (Ezura and Osawa,
2013; Holme et al., 2013) (Fig. 4).

13
14

A part of gene to which an RNA polymerase binds and starts gene transcription
A part of gene which indicates cessation of gene transcription- 10 -

Cisgenesis

Intragenesis
Reconstructing the combinationof promoter etc.

Species A or related species

Species A or related species
Useful geneSpecies A

Species A
Exon

Clone thegene andligate to avectorIntroduction

Introduction

PromoterDNA

Clone themreconstructgene andligate to avectorDNA

Terminator

A gene reconstruct from sameor related species is introduced

A gene of the same or related
species is introduced** There is a possibility of multiple genes randomly introduced to the hostgenome and the remaining T-DNA border sequence of the vector.

The genotypic composition is different from species A

The genotypic composition is basically the same as species A

Fig. 4. Outline of cisgenesis and intragenesis.
In both techniques, cisgenes are introduced into plants by particle bombardment 15 etc. When
the Agrobacterium method 16 is used, however, attention must be paid on the possibility of bordersequences at both edges of vector’s T-DNA domain (base sequence originating from
Agrobacterium) being contained on the crop genome (European Food Safety Authority, 2012a).
(4) RNA-dependent DNA methylation
RNA-dependent DNA methylation (RdDM 17 ) is a technique to methylate 18 some bases(cytosine) of domains involved with expression of specific endogenous gene (e.g., promoter) andthereby to control the expression of the relevant gene without altering the base sequence on crop
genome (Ezura and Osawa, 2013).

15

http://www.nias.affrc.go.jp/gmogmo/FAQ/app/J6.html
http://www.nias.affrc.go.jp/gmogmo/FAQ/app/J1.html
17 RNA-dependent DNA methylation
18 Refers to the addition of a methyl group to the C5 position of the pyrimidine ring of cytosine (C) constituting thepromoter sequence domain. Expression of the gene is suppressed due to the methylation.
16- 11 -

It is known that such a mechanism of RdDM is the same as a phenomenon that naturally takes
place on a daily basis in the cells of organisms. For instance, in some varieties of morning glory
the expressions of flower color synthesis genes vary depending on the domains of the petal, andsome parts of petals exhibit irregular patterns (e.g., white blotches). It has become known recently
that such a phenomenon is induced by RdDM (Suzuki, 2011).
To use RdDM in breeding, one shall construct a DNA fragment that is homologous to a part ofthe sequence of the promoter domain of the gene whose expression is to be suppressed (with its
base sequence repeatedly sequenced in the opposite direction), and introduce the DNA fragment
into a crop by using a vector.
In the cell of the crop, double-stranded RNA (dsRNA 19) transcribed from the DNA fragment is
produced, and the dsRNA is then decomposed into low-molecular-weight single-stranded RNAs(siRNAs 20) through the biological reaction in the cell. It is said that the siRNA acts on the promoter
domain of endogenous gene, inducing methylation of some bases (Pooggin, 2013; Chinnusamy etal., 2009; O. Mathieu and Bender, 2004).
In RdDM, there may be two cases: the DNA fragment is transiently expressed in the cell without
being integrated into the genome (disappears thereafter) and therefore is not to be inherited toprogenies; and the DNA fragment is integrated into the genome and is to be inherited (Fig. 5).

19
20

double stranded RNA
small interfering RNA- 12 -(Case where siRNA-producing gene is not introduced toDNA)(Case where siRNA-producing gene is introduced to DNA)siRNA isproduced fromthe gene ofvectorIntroduction

siRNA isproduced fromthe introduced
gene

Incorporated
to DNAsiRNA-producing
gene

Homologous sequence of thepromoter domain that controls theexpression of gene etc. (target)*

 “DNA methylation-inducing
protein”* takes up the siRNA

Protein involved with the virus
resistance of plant. Multiplicationof virus is suppressed by taking
up the virus (RNA) and inducing
methylation.

Methylation (inactivation)
 siRNA attaches to the homologous
sequence of the target

 “DNA-methylating protein”
methylates the target sequencesiRNA-producing geneis lostThe methylated (inactivated)state is lost after several
generations.

The methylated (inactivated)state is stably inherited toprogenies.

Fig. 5. Outline of RNA-dependent DNA methylation.
Even in the case where the DNA fragment is not inherited to progenies, the methylated state of
the promoter is inherited for several generations at least. Therefore, it is possible to suppress theexpression of specific endogenous gene without modifying the base sequence of the host genome.
(5) Grafting using GM rootstock
Grafting is a technique to artificially joint together a rootstock and a scion which are different
plants from each other. Currently, grafting is generally used for fruit plants and vegetables (e.g.,
eggplant, tomato, cucumber).
The effects and purpose of grafting include 1) control tree vigor to make the fruiting age earlier
or to improve yield, 2) dwarfing of trees to improve the efficiency of farm work, and 3) enablingof repeated cultivation by using a rootstock that is resistant to soil diseases. For instance, when a
GM rootstock with resistance to a specific soil pest is developed and a scion of non-GM variety isgrafted to the rootstock, it would become possible to avoid the effects of the soil pest withoutchanging the quality etc. of the produce harvested from the scion (Koepke and Dhingra, 2013;
Ezura and Osawa, 2013) (Fig. 6).

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Ordinaryvarieties

Scion (non-GM plant)

HarvestRootstock (GM plant)
GM varieties
resistant to soil pest

Fig. 6. Outline of grafting.
Generally, materials are transported from a rootstock to a scion through sieve tubes, and thetransgenes (e.g., disease resistance gene) integrated into the genome of the rootstock do not migrate
to the scion. However, it has become known recently that specific proteins or part of RNA producedin the rootstock are also propagated to the scion through the sieve tubes and control the strength,
flowering time, etc. of the scion (Notaguchi et al., 2008; Harada, 2010).
(6) Reverse breeding
Currently, F1 hybrid (F1) is the most mainstream in breeding of maize and vegetables for the
purpose of developing new varieties with traits such as high yield by taking advantage of thecharacteristics of heterosis 21.
F1 plants are bred through the process of devoping the parental lines with pairing geneticallyhomologous chromosomes (homozygotes 22 ), crossing them and selecting the descendent lines
21 A phenomenon where the first crossed offspring exhibits superior traits in plant size, resistance to disease or theenvironment, etc. compared to the parental lines
22 Those with exactly the same genes (base sequence) inherited from each parent on certain genetic loci

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where the effects of heterosis is expressed (heterozygotes 23). Reverse breeding is a technique torestore the parental lines (homozygotes) from the F1 plants (heterozygotes) by retracing this process.
The specific procedure to restore the parental lines starts with obtaining a gamete (1n (pollen))that is the same as the chromosome of the target parent lines (2n, homozygotes). First, in orderto prevent recombination among chromatids during the meiosis period, a DNA fragment with abase sequence homologous to the endogenous gene involved with the relevant recombination (withthe repeated sequence in the opposite direction) is developed and introduced into a crop.
Once introduced, double-stranded RNA (dsRNA) originating from the DNA fragment is
produced inside the cell. Through RNA interference 24, expression of the endogenous gene involved
with recombination during the meiosis period is interfered with, resulting in suppressedinterchromosomal recombination. The thereby produced gamete (1n (pollen)) has exactly the same
chromosomes originating from the parents. The parental lines (2n) could be restored by doublingof the chromosomes. (Ezura and Osawa, 2013; Dirks et al., 2009; Matzke and Birchler, 2005) (Fig.
7).
(Case where dsRNA- or siRNA-producing gene is not introduced to DNA)

Introduction

dsRNA- orsiRNA-producing
geneSuperior
hybrid line

Chromosome(hetero)MeiosisRNA interferencedsRNA or siRNA is
produced from thegene of vector

Gene involved with homologous
recombination during the meiosis
period is suppressed by dsRNA or
siRNA

dsRNA
or
siRNA

Introduced gene is lost

Pollen (haploid)
Doubling, redifferentiation

Restoration of homozygous
plant (parental line)

Crossing
Superior hybrid line

Fig. 7. Outline of reverse breeding.

23 Those with different alleles on certain genetic loci inherited from parents
24A method to suppress the expression of a certain gene through artificial production of double-stranded RNA in

the plant body by taking advantage of the phenomenon where mRNA with a base sequence complementary to
double-stranded RNA gets decomposed- 15 -

However, in the parental line group there are some plants where the DNA fragment introduced
to suppress the interchromatid recombination still remains on their genome. Therefore, plants
without the relevant DNA remaining must be selected by PCR etc. to be used as the parental line.
(7) Agro-infiltration
Agro-infiltration is a technique to locally infect a plant with Agrobacterium (bacteria) to which
specific genes are incorporated, and to select plants with disease resistance, pest resistance, etc. by
examining the expression of the relevant gene (level of the exhibited symptoms).
Agrobacterium is soil bacterium that infects the root or stem of beans and vegetables in natureand produces lumps called crown gall. Since Agrobacterium possesses the Ti plasmid 25 that can
introduce DNA into the genome of plant, it is used as a vector (transporter) for developing GM
crops.
When developing a GM crop, usually, cultured cells etc. are infected with Agrobacterium andthe target gene is introduced into the genome of the plant through the Ti plasmid. In agroinfiltration process, however, only a particular organ of the plant body such as the leaf (limited to
non-reproductive tissues) is infected. Therefore the target gene exists either in a free state in thecell without being integrated to the genome or, even if with being integrated to the genome, limited
to the relevant organ (e.g., leaf) (Ezura and Osawa, 2013; Ohadi and Rasouli et al., 2013).
By applying this technique, for instance when examining and selecting lines resistant to a
specific viral disease from line groups obtained by normal crossbreeding, it becomes possible toestimate the existence of disease resistance by examining the disease symptoms (the level of theexpression of the relevant protein) on the leaf etc. that are infected with Agrobacterium, in whicha gene of the protein produced by the disease virus is incorporated (Fig. 8).

25 A plasmid that is possessed by Agrobacterium and involved with the formulation of crown gall. It consists of the

T-DNA domain that will be introduced into the chromosome of infected cells and the Vir domain that are involved
with cutting out or incorporating the T-DNA domain into the host cell.

- 16 -(Case where recombinant gene is not inherited to the next generation)

Inoculate GM microbe having
genes involved with disease of
crop* and make the leaf locallyinfected* Especially for the cases such as if a genederived from pathogenic virus is
introduced, etc.









Disease-resistant plant does notexhibit symptoms

Select and cultivate healthy(disease-resistant) plant
Since it is a local infection to the leaf, theGM microbe does not remain in seeds.

Fig. 8. Outline of agro-infiltration.
Infection with Agrobacterium is usually local and virus-like spreading of it throughout the plantis unlikely to occur. Therefore, it is considered that the genes originating from Agrobacterium do
not remain in the body of a thereby examined and selected plant, as long as the infected domain(e.g., leaf) is removed.
As another method of agro-infiltration, there is a method targeting the reproductive tissues suchas flowers. (floral dip). In this case, a transgene (aforementioned gene that codes the viral diseaseprotein) is incorporated into the genome of reproductive cells, and the progenies are GMOs.
(8) Seed Production Technology (SPT) process
The SPT process is a technique developed by the US DuPont Pioneer to effectively produce F1
hybrid maize seeds, and is already practically applied in the US.

- 17 -

Production of F1 hybrid seeds of maize is generally carried out by crossing different parents.
However, since one plant has both the tassel florets and ear florets, the tassel florets of female
parents are usually cut (emasculation) to prevent inbreeding.
In order to omit the emasculation process, DuPont Pioneer developed a system that enables
effective production of “SPT-maintainer lines” and “seeds for producing F1 (non-GM)” by
introducing a pollen sterility gene etc. into the line for producing F1 seeds (homozygous). The SPTmaintainer lines have the recombinant genes heterozygously, and inbreeding the lines allows toefficiently produce seeds of the SPT-maintainer lines and the seeds for producing F1 which are
male sterile (Fig. 9).
Technique to multiply abundant seeds from male sterility (pollen can’t be produced) lines
where seed multiplication by inbreeding is difficult
1. Introductionof SPT gene

SPT gene(1) Restorer gene
(Expressed on
formulation of pollen)

2. Mechanismof SPT gene
expression in
the malesterility
maize line

(1) Pollen become
formulated at thestamen(2) Pollen sterility gene
(Expressed during pollen
maturation phase)(3) Gene that
expresses redcolor

3 genes are
introduced as a set
into one of thechromosomes

Chromosome(2) Pollen (haploids) that possess the SPT gene are sterilized
Pollen maturation

PollenPollen (haploids) that do not possess the SPT gene remain fertile

Meiosis3. Multiplication of SPT-maintainer line and seeds for producing F1♀ (with SPT)

♂ (with SPT)

Seeds are not formed♀ (with SPT)

♂ (without SPT)

♀ (without SPT)

Seeds formation (red)GM♂ (with SPT)

Seeds are not formed

Used as SPT-maintainer line

♀ (without SPT)

♂ (without SPT)

Seeds formation
Non-GM

Used for crossing F1

Fig. 9. Outline of SPT.
With the pollen sterility gene, a gene that expresses a red fluorescent protein in the grain is also
introduced. Therefore, even if the seeds that possess those genes get mixed in the F1 hybrid seeds,
such grains with remaining transgenes can be identified by the color through a high-performancecolor sorting machine and removed 26.
26

http://www.mhlw.go.jp/stf/shingi/2r9852000002tccm-att/2r9852000002tcwe.pdf- 18 -

2. Status of Consideration on Regulatory Framework (abbreviation)
Regulatory framework for crops developed by NPBTs may vary country to country, dependingon the range of organisms covered by the current GM regulations adopted by the country. Even if
such crops may not be subject to GM regulations at the moment, the fact is that cutting-edge GM
techniques are being used. Differences between countries in the concept on how carefully thesecrops shall be managed in the viewpoints of food safety and effects on the biological diversity may
affect the future application of regulations on NPBTs and crops engineered by NPBTs.
From such viewpoints, mainly by scientist groups, overseas countries have summarized
scientific opinions on the framework for NPBTs and their products under the GM regulations
through examination of the characteristics of each breeding technique and comparison withconventional breeding techniques.
The study group has discussed with those information from overseas countries partially from
URL listed as described below.
(1) EUEASAC (European Academies Science Advisory Council) "Planting the Future"(2013)
http://www.easac.eu/fileadmin/Reports/Planting_the_Future/EASAC_Planting_the_Future_FULL_REPORT.pdf

European Commission "Directive 2001/18/EC" (2001)
http://www.biosafety.be/gb/dir.eur.gb/del.rel./2001_18/2001_18_tc.html

EFSA (European Food Safety Authority)
http://www.efsa.europa.eu/

EFSA "Scientific opinion addressing the safety assessment of plants developed through cisgenesis
and intragenesis" (2012)
http://www.efsa.europa.eu/en/efsajournal/pub/2561.htm

EFSA "Scientific opinion addressing the safety assessment of plants developed using Zinc Finger
Nuclease 3 and other Site-Directed Nucleases with similar function" (2012)
http://www.efsa.europa.eu/en/efsajournal/pub/2943.htm

EPSO (European Plant Science Organisation) "Crop Genetic Improvement Technologies" (2015)
http://www.epsoweb.org/file/2038

Greenpeace "Open letter to the Commission on new genetic engineering methods" (2015)
http://www.greenpeace.org/eu-unit/en/Publications/2015/Open-letter-to-the-Commission-on-new-geneticengineering-methods/- 19 -

JRC (Jount Research Centre) "Workshop on comparative regulatory approaches for new plant
breeding techniques" (2011)
http://ipts.jrc.ec.europa.eu/presentations/NPBT.cfm

North American Agricultural Biotechnology Council "New DNA-Editing Approaches: Methods,
Applications and Policy for Agriculture" (2014)
http://nabc.cals.cornell.edu/Publications/Reports/pubs_reports_26.html

Official Journal of the European Union "Regulation (EC) No.1829/2003 of the European
Parliament and of the Council of 22 September 2003 on Genetically Modified Food and Feed"(2003)
http://ec.europa.eu/food/food/animalnutrition/labelling/Reg_1829_2003_en.pdf

Official Journal of the European Union "Directive 2009/41/EC of the European Parliament and of
the Council" (2009)http://eur-lex.europa.eu/LexUriServ/ LexUriServ.do?uri=OJ:L:2009:125:0075:0097:EN:PDF(2) USA
Cibus
http://www.cibus.com/

EPA (Environmental Protect Agency)
http://www.epa.gov/

FDA (Food and Drug Administration)
http://www.fda.gov/

FDA "Food from Genetically Engineered Plants"
http://www.fda.gov/Food/FoodScienceResearch/GEPlants/default.htm

Health Canada "Novel Food Information – Cibus Canola Event 5715 (Imidazolinone andSulfonylurea Herbicide Tolerant)"
http://www.hc-sc.gc.ca/fn-an/gmf-agm/appro/canola-5715-eng.php

USDA (United States Department of Agriculture)
http://www.aphis.usda.gov/

USDA "Biotecnology"
https://www.aphis.usda.gov/wps/portal/aphis/ourfocus/biotechnology

USDA APHIS (Animal and Plant Health Inspection Service) "Docket No. Aphis-2012-0067" (2014)
http://www.aphis.usda.gov/brs/fedregister/BRS_20141110b.pdf

White House "Executive Order 13563; Improving Regulation and Regulatory Review" (2011)https://www.whitehouse.gov/sites/default/files/omb/inforeg/eo12866/eo13563_01182011.pdf(3) Australia and New Zealand- 20 -

FSANZ (Food Standards Australia New Zealand)
http://www.foodstandards.gov.au/

FSANZ "New plant breeding techniques workshops" (2012, 2013)
http://www.foodstandards.gov.au/consumer/gmfood/Pages/New-plant-breeding-techniques-in-thespotlight.aspx

OGTR (Office of the Gene Technology Regulator)
http://www.ogtr.gov.au/

Scion
http://www.scionresearch.com/(4) Other international trends
APEC (Asia-Pacific Economic Cooperation) "2010 APEC Ministerial Meeting on Food Security"(2010)
http://www.apec.org/Meeting-Papers/Ministerial-Statements/Food-Security/2010_food.aspx

OECD (Organisation for Economic Co-operation and Development) "Consensus Documents for
the Work on Harmonisation of Regulatory Oversight in Biotechnology"
http://www.oecd.org/science/biotrack/consensusdocumentsfortheworkonharmonisationofregulatoryoversighti
nbiotechnology.htm- 21 -

III. Examples of R&D in Japan and Considerations on the Effects on Biological Diversity
During the advancing R&D and initiatives towards practical application related to NPBTs mainlyin the US and Europe, in Japan, such initiatives were taken only at a limited number of universities
and research institutions. Full-fledged R&D on NPBTs in Japan had to wait for the announcement of
the FY2013 commissioned project of MAFF, “Project to Develop the Next-Generation Production
Basic Technologies of Agricultural and Livestock Products using Genomic Information” 27(hereinafter referred to as “NG Genome Project”). 28
Therefore, the Study Group decided to summarize the general ways of thinking on the effect on
biological diversity of NPBT, regarding three techniques promoted for practical use by MAFF in the
project above as case studies.
Regarding the regulatory framework for NPBTs pertaining to food safety, the Study Group decided
not to include it in the scope of its deliberations since that will be separately deliberated at the “Expert
Committee on Genetically Modified Food” 29 established under the Newly Developed Food
Investigating Group, Food Sanitation Subcommittee, Pharmaceutical Affairs and Food Sanitation
Council, Ministry of Health, Labor and Welfare.
The Cartagena Act (Article 2, Paragraph 2 30) defines a GMO subject to the regulation. One of themis an organism that possesses nucleic acid, or a replicated product thereof, obtained through use of atechnology, as stipulated by the ordinance of the competent ministries, for the processing of nucleic
acid extracellularly. Therefore, the Study Group decided to:(1) deliberate the possibility of a foreign nucleic acid used for gene recombination or the replicatedproduct thereof remaining in the plant; and,(2) summarize the general ways of thinking of adverse effects on the biological diversity throughthe comparison with crops produced by conventional breeding techniques, or considerations on
the possibility of unintended mutation occurring, etc., since there are rules to exclude organisms

27 http://www.s.affrc.go.jp/docs/project/information/h25/zisedai.htm
28 Such R&D on NPBTs are transferred to the Strategic Innovation Promotion Program (SIP) of the Cabinet Office,where R&D is comprehensively and strategically progressed for a wider range of techniques and plants throughthe collaboration by the relevant ministries.
Reference URL: http://www8.cao.go.jp/cstp/gaiyo/sip/keikaku/9_nougyou.pdf
29 http://www.mhlw.go.jp/stf/shingi/2r98520000008fcs.html#shingi148834
30 http://www.bch.biodic.go.jp/english/law.html- 22 -

that fall in the category of so-called “self-cloning” and “natural occurrence” from organisms
subject to the regulations (Article 2, Paragraph 1 of the enforcement regulations of the Act 31).
1. Techniques to Remove the Introduced Transgenes during the Breeding Process(1) Accelerated breeding of fruit trees using early flowering genes
Seedlings of fruit trees such as citrus usually take 5-10 years to flower and fruit, which is a major
obstacle to the development of new varieties. Especially when introducing useful traits (e.g.,disease resistance) that exist in related wild species into commercial varieties, in order to remove
unnecessary traits (defective traits) that exist in the related wild species to ensure the useful traits
originally possessed by commercial varieties, backcrossing with commercial varieties must be
repeated for many generations.
For instance, for apple (Malus × domestica Borkh.), there is a case where the apple scab 32
resistance gene (Vf gene) originating from a related varieties Malus floribunda 821 was introduced
to the apple cultivar (variety name: ‘Goldrush’), and it is said that it took 7 generations over 68
years to achieve the desired result (‘Goldrush’) after crossing ‘Rome Beauty’ and Malus floribunda
821 in 1926.
In response to such a situation, in 1999, the FT (Flowering locus T) gene of thale-cress(Arabidopsis thaliana) was discovered by the group of Prof. Araki of Kyoto University, and it was
confirmed in 2007 that the FT protein expressed by the gene is a plant hormone (florigen) thatinduces flower bud formation of plants (Corbesier et al., 2007; Tamaki et al., 2007). Applying thisresearch result, currently, under the NG Genome Project, the National Agriculture and Food
Research Organization, Institute of Fruit Tree Science is working on the development ofaccelerated breeding for citrus using this FT gene (Endo et al., 2005; Endo et al., 2009).
1) Development of accelerated breeding by introduction of CiFT gene
Citrus tristeza virus (CTV) 33 infects citrus such as Satsuma mandarin (Citrus unshu) and causes
major damage, while it is known that CTV resistance gene exists in a related species, trifoliateorange (Poncirus trifoliata).

31 http://www.bch.biodic.go.jp/english/law.html
32 Disease of apple, pear, etc. caused by some types of filamentous fungi where black spots emerge on the fruit orleaves33 Citrus tristeza virus- 23 -

To introduce the resistance gene into citrus through cross breeding, crossing with trifoliateorange is to be made first, the hybrid seedling with CTV resistance and relatively good traits are to
be selected, and selected plants are to be crossed with a citrus variety in order to remove thedefective traits of trifoliate orange. This process needs to be repeated to achieve plants with thedesired outcome.
However, assuming backcrossing needs to be performed for about 7 generations as was the caseof the apple scab resistance gene, since it takes about 7-10 years (per generation) for hybrid
seedlings to reliably flower and fruit, introgression of CTV resistance gene would require over halfa century (7 generations × 7-10 years).
To that end, in this project research, CiFT gene 34, a homologue 35 of the FT gene, was introduced
into trifoliate orange by the Agrobacterium method (Endo et al., 2005; Endo et al., 2009), which
was then crossed with hyuganatsu (Citrus tamurana;cultivars). The hybrid seedling successfully
flowered and fruited about 2 years after planting.

If the hybrid seedling is repeatedly crossed with hyuganatsu (a commercial variety) about 7 times,
a commercial variety resembling hyuganatsu with resistance to CTV will be obtained in about 10
years according to the estimation. Additionally, during the process of selecting the hybrids, use of
DNA markers that widely cover the genome of commercial varieties may further improve theefficiency of the selection, and it is prospected that the breeding period would be eventually
reduced to about 5-7 years. Plants having CTV resistance gene without having the CiFT gene will
segregate in the hybrid progenies; hyuganatsu plants resistant to CTV free from transgene (CiFT
gene) will be obtained by selection using the DNA marker assisted selection.
2) Accelerated breeding using Apple Latent Spherical Virus (ALSV) vector
Even when a virus infects a plant, the virus RNA itself is not usually incorporated into the plant
genome. Therefore, when a plant is infected by the virus with incorporated FT gene, the FT protein
can induce early flowering in the plant without the FT gene incorporated into the plant genome.

34
35

Citrus Flowering locus T
Gene that is considered homologous- 24 -

Prof. Yoshikawa of Iwate University is developing a flowering promotion technique for apple
by using one of the latent viruses, “Apple Latent Spherical Virus (ALSV 36)”.
Specifically, it was confirmed that when cotyledon of apple seedling (during the germinating
period) was inoculated and infected with an FT gene-incorporated ALSV (FT-ALSV) throughparticle bombardment, FT protein was expressed during the seedling stage of apple, and that flower
buds were initiated and bloomed in about 2 months (7-8 leaf stage). It was demonstrated that crosspollinated seeds of apple can be obtained in 1 year by pollinating the thereby obtained pollen to
the normal pistil of apple flowers. Since ALSV rarely infects pollen or seeds, the recombinant virus
will not be found in the next generation apple seedlings. (Yoshikawa, 2012; Suzuki, 2011)

Apple latent virus to which early
flowering gene is introduced

Fruit trees usually take about 10 years to flower and fruit, but by
having seeds infected with a virus to which the early flowering geneis introduced, it is possible to make it flower and fruit within 1 year
via pot culturing in an contained greenhouse.

Infection

PollenVirus does
not remain in
the pollen

Seeds
1 cycle per year

Grow
new varietiesin 7-10 years(♂)

×
9 months
after infection

6 months
after infection

50 daysafter infection

New varieties
Latent virus (GM virus)
does not remain(♀)

Fig. 11. Outline of applying flowering promotion technique by FT-ALSV.
In the experiment above, the flowering rate of seedlings was about 30%, and flowering occurred
only once. In order to improve the flowering properties, a technique (RNA interference) to suppress
the expression of flowering suppressor endogenous gene of apple is also currently in progress using
separately developed ALSV that is incorporated with a flowering suppressor gene (Terminal flower
1 (TFL1) gene) (Sasaki et al., 2011; Igarashi et al., 2009).
When apple seedlings were infected with ALSV to which both the FT and TFL 1 genes areincorporated, apple trees flowered at a rate of more than 90% and exhibited the characteristic ofever-flowering, meaning it has become possible to collect pollen throughout the year.
36

Apple Latent Spherical Virus- 25 -

This technique is applicable to various crops including other fruits, soybean and vegetables.
Development of accelerated breeding for grapes, pears, etc. is currently in progress at Iwate
University etc. under the Strategic Innovation Promotion Program (SIP) of the Cabinet Office,aiming at practical application with one crossing generation being reduced to one year or less.
(2) Recurrent selection of inbreeding crops such as rice
Usually, breeding by crossing two varieties is used for inbreeding crops 37 like rice. However,this technique has a limitation in the number of usable breeding material (genetic resources), andincorporation of various genes like those related to the yield ability (genes involved with thenumber of spikelet, photosynthetic ability, biomass amount, etc.) requires repeated crossing withvarious breeding material. As a result, development of ultra-high yielding varieties etc. in the future
is considered to take a substantial amount of time.
Amid such a situation, taking into account the fact that the unit crop of maize (outbreeding
crop 38 ) has been steadily increased through breeding and improving over years, the National
Agriculture and Food Research Organization, Institute of Crop Science (NICS) and the National
Institute of Agrobiological Sciences (NIAS) undertook the development of a breeding system forapplying the recurrent selection, which is generally used for breeding maize nowadays, toinbreeding crops such as rice to break through the limitation in their unit crop.

37 Refers to plants where fertilization mainly occurs between the pollen and the egg cell of the same flower (orbetween the male flower and the female flower of the same plant for unisexual flowers); examples are rice, wheat,and soybeans
38 Refers to plants where fertilization mainly occurs between flowers from different plants; examples are maize andbuckwheat- 26 -

Rice (breeding by crossing)

Maize (recurrent selection)[Artificial crossing]
Emasculated by manuallycutting the flower (glume) of
rice

Outbreed[Artificial crossing][Artificial crossing]♀

♀

(Emasculated) Inbreed (Emasculated) Inbreed
and select(High quality) and select10 years
10 years(High yield)♂

(High yield)(Disease-resistant)(Recurrent)

Male parent (♂)(Disease-resistant)Inbreed andselect10 years(Recurrent)

Female parent (♀)(Emasculated)

Female parent (♀)(Drought-resistant)(High quality)

InbreedArtificial
crossing

Lines with higher yield and excellence in disease
resistance etc. are selected and fed back as parent
plants. By repeating this, useful genes areaccumulated.

SelectNew varieties where superior
traits are accumulated

New varieties where superior
traits are accumulated

Accumulation of various genes involved withyield ability etc. requires many years.

It is possible to accumulate various genes involved withyield ability etc. in a short period of time.

Fig. 12. Difference between the breeding technique for maize and that for rice.
Recurrent selection of maize is a method to accumulate genes that exist in maize by repeatedly
crossing and inbreeding various breeding materials with different genetic backgrounds and thereby
to breed an ideal parental group for producing hybrid with accumulated genes involved with yield,
quality, environmental stress resistance, disease/pest resistance, etc. (Fig. 12).
At NICS and NIAS, a dominant male sterility gene etc. for making rice efficiently outbreed isfirst introduced to rice, and plants that have those genes heterozygously (trait transformants) areselected. As the pollen of the transformants becomes sterilized (i.e. outbreeding), by cultivating
various breeding materials with different genetic backgrounds around them, outbred plants (F1) areautomatically obtained. About half of the progenies obtained inherit the male sterility trait (Fig.
14).

- 27 -

As a herbicide resistance gene (positive trait gene) and a conditional lethal (negative marketgene) are individually incorporated into the cassette of the male sterility gene cassette introduced
to the relevant rice (Tanaka, 2010) (Fig. 13), among the progenies:(1) positive selection (e.g., herbicide treatment) results in selecting about half of the survived
rice that inherited the male sterility trait; and,(2) negative selection results in selecting plants without possessing the transgenes of the malesterility gene etc. (about half of all plants).
Constitutive
promoter

Anther-specificpromoter

Herbicide
resistance gene

Herbicide resistancegene cassetteInducible
promoter

Male sterility gene

Lethal gene(e.g., ribonuclease)(e.g., ribonuclease)

Male sterility gene
cassette

Inducible apoptosis
gene cassetteFig. 13. Schematic illustration of introduced gene cassette.
New transgene free varieties with superior yield are to be produced by repeating such selection
as crossing and fixation of the important traits in the negative selection (Tanaka, 2010).

- 28 -

Outbred
seeds
Theoretically male sterility plants (Msms) and non-malesterility plants (fertile) (msms) emerge with a segregation
ratio of 1:1

Select through herbicide
resistanceSelect through inducible
leathality

Male sterility plant(Msms)

Fertile plant(msms) No transgeneconfined
field
Select superior plant

Select with
progressing genetic
fixation throughinbreeding

Selection

Gather seeds from selected male sterility
plants, and mix

Fixedvarieties(Tanaka, 2010 [modified by the Study Group])

Fig. 14. Outline of recurrent selection for inbreeding crops such as rice.
NICS and NIAS are planning to establish such a breeding system in the next 5 years or so andto produce and supply new promising varieties (lines) one after another.
(3) Considerations on the effects on biological diversity
1) Possibility of transgenes used for recombination or their copies remaining
For either technique, at the initial stage of breeding, plants, in which transgenes (e.g., CiFT gene,
TFL 1 gene, pollen sterility gene) or recombinant viruses (ALSV) remain, emerge.
Therefore, during the initial stage of breeding where crossing and selection are carried out(experimental research stage), appropriate use in accordance with the existing Cartagena Act (e.g.,
application for the approval to use in a closed system or to test at an confined field) is required.
Meanwhile, since the final commercial product can be selected from those with no relevanttransgenes remaining (Null Segregant), it may be exempted from the regulations of the existing
Cartagena Act if the absence of transgenes is confirmed by an appropriate method including PCR,Southern hybridization, and the next-generation sequencer.

- 29 -

Therefore, it is necessary for developers to provide the regulatory authorities with the detailed
information (e.g., what kind of crossing and selection processes have been used to breed the final
commercialized products) in advance and to receive judgment on the applicability of regulations
to their future products.
2) Comparison with conventional breeding and possibility of unintended mutagenesis
Transgenes and recombinant viruses used for the breeding techniques above are used to improvethe efficiency of breeding in crossing or selection process, and it is essential to confirm that therelevant transgenes do not remain in the final commercialized varieties. Then, such newly
developed crops (new varieties) can be regarded as those produced by conventional breeding
techniques.
Additionally, in breeding of crop plants, usually, there is a process to eliminate undesirable
plants (plants with defective traits) from the line groups engineered. As a result, only the plants
with good qualities and ease of growth become the selected varieties.
From the above understanding, if it is confirmed that transgenes are not included in the final
crop, the crop can be regarded the same as those produced by conventional breeding such as
crossing. Therefore, the Study Group considered that there are no matters that call for specificconcerns over the effects on biological diversity.

- 30 -

2. Genome Editing using Artificial Restriction Enzymes(1) Outline of R&D
Regarding SDN-1 that is on the extension line of conventional mutation breeding, while it allows
for destruction of the action of genes by causing deletion of several bases after cleaving the target
genes, it is not adequate for creating mutants where the base sequence on the genome is alteredonly for one base (Single Nucleotide Polymorphism (SNP 39)).
To that end, NIAS is promoting the development of techniques to accurately induce mutation to
specific genes of crop genome for one to several bases only (point mutation) using SDN-2.
Additionally, currently, effective expression of artificial restriction enzymes in plant bodies
unavoidably requires incorporating artificial restriction enzyme genes (transgenes) onto the plant
genes. Therefore, NIAS is also working on the development of a technique to completely remove
transgenes by taking advantage of piggyBac, one of the transposons 40 of insects, after mutation(deletion, substitution or insertion for one to several bases) is induced to target genes.
By applying these techniques to breed crops, it will become possible in the future to suppress
genes that generate allergens in rice or those that generate toxic substances in potato (e.g.,
glycoalkaloids) and to develop varieties with valuable novel phenotypes in a very short period oftime, such as feed rice that is rich in a specific amino acid, sweeter crop that is rich inoligosaccharide, tomato with higher rate of flowering and higher yield, parthenocarpic tomato andcapsicum that ripen even under low temperature in winter, grass that is easily digested by livestock,and pollen-free Japanese cedar.

39
40

Single Nucleotide Polymorphism
DNA (base sequence) whose location on the genome is transportable within a cell

- 31 -

(2) Considerations on the effects on biological diversity
1) Possibility of transgenes used for recombination or their copies remaining
For both SDN-1 and SDN-2, usually, an artificial restriction enzyme gene (transgene) will beincorporated into the plant genome by means of Agrobacterium etc. Therefore, until the transgene
is completely removed by carrying out, for instance, backcrossing with non-GM variety afterward,
appropriate use in accordance with the existing Cartagena Act shall be required.
In SDN-2, a short guiding DNA fragment is introduced into a cell at the time of DNA repair
after cleavage and the repaired sequence on the genome is artificially controlled. As a result a basesequence that is the same as the guiding DNA fragment is inserted into the produced crop.
Therefore, these crops may be regarded as GMOs subject to the regulations of the Cartagena Act.
However, SDN-2 is to induce artificial substitution or insertion to a single or several-base nucleic
acid, and a similar mutant may also be produced by SDN-1 albeit low in possibility. Additionally,a similar level of deletion, substitution or insertion of bases can occur in selection from natural
diversity, conventional mutation breeding, etc.
Furthermore, in the existing Cartagena Act, even GM techniques have been used to develop
them, the organisms where the same substitution of nucleic acid can occur under natural conditions(so-called “natural occurrence”) can be exempted from its regulations.
Therefore, there is a possibility that organisms produced by SDN-1 can be exempted from theexisting Cartagena Act regulations. For SDN-2, it is important for the R&D side to actively gather
and analyze related information (e.g., characteristics of mutated trait and whether the same mutants
can be produced by other techniques), submit them to the regulatory authorities and ask for
judgment on the applicability of regulations on a case-by-case basis.
For proving that the artificial restriction enzyme genes (relevant transgenes) are surely removed,
it is required to carry out analysis using PCR, Southern hybridization, next-generation sequencer,
etc. and to receive confirmation by the regulatory authorities.
2) Comparison with conventional breeding and possibility of unintended mutagenesis
While both SDN-1 and SDN-2 induce deletion, substitution or insertion of bases to the specificdomain of crop genome, such mutation for several bases occurs in selection from natural diversity- 32 -

or via conventional breeding by crossing, mutation breeding, etc. in an unintended form (Abe etal., 2013).
Additionally, it is considered that the chance of unintended mutation occurring is lower, and thatthe risk on biological diversity etc. is reduced more than conventional mutation breeding byradiation, chemicals, etc. causing random mutation on multiple locations on the genome sinceSDN-1 and SDN-2 allow arbitrary modification of the target genes only.
Scientific knowledge on base deletion, substitution or insertion that can occur naturally or by
conventional breeding techniques is still limited, therefore gathering and accumulation of more
knowledge and findings are required. However, at least for SDN-1 and SDN-2 where mutations
are induced at several-base levels, such mutations are considered to occur by conventional mutation
breeding as well.
Further, as described in II-1-(1) (page 7), the possibility of off-target (which triggers unintended
mutation) is expected to be extremely low for both techniques, and even if unintended mutation
occurred to traits other than the intended trait by accidentally cleaving the domain other than thetarget gene, it is also expected that such plants are usually removed through the selection processof breeding.
From the above understanding, regarding SDN-1 and SDN-2 that induce mutation to several
bases, if it is confirmed that no artificial restriction enzyme (transgene) is included in the final
products, the products can be regarded the same as those produced by conventional mutation
breeding. Therefore, the Study Group considered that there are no specific concerns over the effects
on biological diversity.
However, at this stage, it is expected that there may be cases where sufficient knowledge is notavailable on the characteristics of mutated target genes, the expression mechanism of the intended
trait, etc. In such cases, it is appropriate for developers to provide the regulatory authorities withthe related information (e.g., detailed information on the breeding process, characteristics of thetarget mutated gene in the crop, introduced trait etc.) in advance so that the risk pertaining to theeffects on biological diversity can be appropriately predicted, and to receive scientific assessment
from experts as necessary.

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IV. Points to Be Considered toward Future R&D and Practical Application
1. Promotion of Related R&D
In the situation of advancing globalization of agricultural product trading, the urgent task toradically strengthen the international competitiveness of Japan’s agriculture is the development of
epoch-making agricultural technologies that will bring breakthroughs to the agriculture in Japan.
Especially, in the field of agricultural breeding, epoch-making grain varieties etc. are developed
one after another by utilizing GM techniques based on the US, and the cost reduction by reducing
the amount of agrochemicals used and the increase of yield have been achieved. On the contrary,in Japan, along with the increasing difficulty in cultivating GMOs domestically, the options oftechniques usable for breeding main crops including rice are limited, and it takes many years todevelop new varieties.
Regarding vegetables and flowering plants, for which personal breeding is actively performed,
Japan’s high quality products are highly valued overseas, and the export of seedlings has beensteadily increasing in recent years, especially to Asia. However, it has become difficult to obtain
useful plant genetic resources from overseas as breeding materials, and there is a concern overstagnancy spreading in the development of new varieties.
NPBTs are techniques that basically draw out the maximum potential of agriculturally usefulgenetic traits that exist in the same or related species, which may efficiently improve the quality,
functionality, yield, etc. of crops, and thereby NPBTs enable creation of unprecedented epochmaking varieties in a short period of time. In case the crops engineered by NPBTs do not possesstransgenes at the end and have the same gene composition as crops produced by conventional
breeding, risks on the food/feed safety and the effects on biological diversity can be suppressed.
Additionally, NPBTs may allow for reduction of development cost involved with the compliancewith GM regulations, so there is a possibility for Japanese private companies etc. to apply NPBTs
to breeding and improving various crops including vegetables and flowering plants.
To that end, while paying attention to the trend of rapidly advancing R&D in the US and Europe,
Japan should consider NPBTs as part of its next-generation breeding techniques and make strategic
investment into R&D, from basic R&D to applied R&D.

- 34 -

Especially, the “Comprehensive Strategy on Science, Technology and Innovation 2015 41 ”
decided by the Cabinet in June 2015, indicates the development of next-generation breeding
systems such as NPBTs as one of the priority R&D initiatives to be promoted in Japan, and therelevant ministries are jointly promoting R&D under the Strategic Innovation Promotion Program(SIP) of the Cabinet Office. Under the SIP, the government (research institutions under MAFF,
MEXT, etc.), academia (universities) and industry (related private enterprises such as those in thebreeding industry) shall collaborate in actively implementing initiatives in the development ofJapanese NPBTs, conversing them into intellectual properties, creating innovative varieties ofvarious plants including vegetables and flowers as well as grains through application of NPBTs,
enhancing regulatory science, promoting direct dialogue with citizens for developing science
communication, etc.
2. Initiatives towards Promoting the Social Understanding(1) Compliance with GM regulations
In NPBTs, GM techniques are used and crops introduced with transgenes are handled duringcertain points of the breeding process. Therefore, appropriate handling in accordance with theexisting Cartagena Act 42 is required during the breeding process.
With regard to cultivating the resultant novel engineered varieties or using them for food or feed,prior consultations with the regulatory authorities is necessary since there remains the possibility
that GM regulations in accordance with the Cartagena Act 43, Food Sanitation Act 44, or Feed Safety
Act 45 apply respectively.
On making judgment on the applicability of such GM regulations, the judgement criteria maybe (1) whether the transgene used for recombination remains, and (2) whether the same crop canbe selected from natural diversity or developed by conventional breeding techniques. Therefore, itis necessary for developers to actively provide the regulatory authorities with the related
information (e.g., detailed information on the breeding process, characteristics of the target mutated41

http://www8.cao.go.jp/cstp/sogosenryaku/2015.html
http://www.lifescience.mext.go.jp/bioethics/anzen.html#kumikae43
http://www.maff.go.jp/j/syouan/nouan/carta/about/sop_eng.html
44 http://www.mhlw.go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shokuhin/idenshi/index.html
45 MAFF website: http://www.maff.go.jp/j/syouan/tikusui/siryo/index.html
Food and Agricultural Materials Inspection Center website: http://www.famic.go.jp/ffis/feed/sub1_tuti.html
42- 35 -

genes in the crop, introduced trait etc.) so that the risk pertaining to food and feed safety and theeffects on biological diversity can be appropriately predicted. It is also necessary to assuredly andthoroughly conduct prior consultations. Such activities will become the first step towards fostering
the social understanding of NPBTs.
(2) Methods to provide public with information and to communicate with it
Since many NPBTs use the latest molecular biological findings, in order to socially implement
such cutting-edge techniques, it is important to promote interactive communications with various
interested parties from the R&D stages, provide explanations on the meaning, contents, etc. of
R&D in an easy-to-understand manner, and to reflect the voices of anticipation, anxiety andconcerns from such parties on the R&D and the process of practical application. Additionally,
considering the lingering social anxiety over the agricultural products and food utilizing GM
techniques, the key point will be how to successfully disseminate information and communicate ina convincing manner that, (1) development of crops that are resistant to various environmental
stresses and are superior in yield is an urgent task to address the issues including the global
environmental changes and food shortage, and use of NPBTs that drastically increase the speed ofcrop breeding is extremely important, (2) similar plants can be selected from natural diversity ordeveloped by conventional breeding techniques, etc., along with showing the actually developed
crop plants (new varieties), while promoting the development of epoch-making novel varieties thatbring tangible benefits to the farmers and consumers in Japan.
To that end, it is important to continue to collect related scientific findings and formulate
opinions about the effects on biological diversity etc. at scientific panels like the Study Group, andfoster the sense of trust in the nation on the use of NPBTs through promoted communications witha broader range of knowledgeable persons, consumer groups, mass media, producers, industry, etc.
based on such scientific opinions, establishing easy – to understand guidance.
(3) Promotion of international harmonization pertaining to regulatory framework
Currently each country or region is considering the regulatory framework for NPBTs, and there
is a possibility that differences among the frameworks could cause confusion in the trade of
agricultural products in the future. More specifically, Japan imports a large amount of crops fromthe US and other countries, and it will be inevitable to coordinate and have discussions with trading
partners on the regulatory framework.

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It will be important to accelerate the formulation of scientific opinions etc. in Japan and tocontribute to sharing such scientific information and views, and thereby international
harmonization pertaining to regulatory framework for NPBTs at suited opportunities such as theOECD-WG in the future.

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Terminology
Agrobacterium method
A gene introduction method taking advantage of the principle of soil bacterium Agrobacterium
incorporating its genes into plants in nature.
Specifically, this technique uses the characteristic of Agrobacterium incorporating a part (section
called “T-DNA”) of transporter DNA (plasmid) into plant cells. A target gene can be introduced
to the chromosome (genome) of plant cells by removing the virulence gene, incorporating a gene
involved with an agriculturally useful trait there, and inserting it into a plant cell.
CRISPR/Cas system
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is an artificial
restriction enzyme technique using the acquired immune mechanisms for phages or plasmids ofprokaryotes (e.g., bacteria) where the system to cleave and eliminate extraneous DNA is applied.
Different from ZFN or TALEN, the CRISPR/Cas system identifies the base sequence
information on the genome (DNA binding domain) by RNA instead of protein, and designing of
the artificial restriction enzyme is easier than ZFN or TALEN.
Breeding by crossing
A breeding technique to cross genetically different varieties to develop crossbreed groups withvarious mutations and to select and grow plants with superior traits among them. It is a common
technique used for breeding various crops including rice.
Southern hybridization
A technique to detect DNA, which was devised by Edwin Southern.
DNA fragments with various lengths cut by restriction enzymes are put through agarose gelelectrophoresis, and the existence of the target DNA is defined by the positions of DNA fragments
segregated on the gel.
Next-generation sequencer
A method to determine a substantial amount of base sequences by simultaneously analyzing
millions of randomly cut DNA fragments. It enables a wide range of analysis including wholegenome sequence, epigenome analysis (e.g., analysis of DNA methylation state) and transcriptome
analysis (analysis of RNA as transcript).
DNA marker assisted selection- 43 -

As described in Page 1, a breeding technique to use the base sequence information near the
specific gene involved with an agriculturally useful trait as the marker (DNA marker) and toefficiently select plants that possess that base sequence.
For instance, breeding and selection of new varieties superior in disease resistance by
conventional breeding methods can be achieved only by cultivating a large amount of crossbred
plants at experimental fields and by investigating the disease symptoms. However, by using DNA
markers, disease-resistant plants can be selected in a short period of time only by extracting andanalyzing DNA of leaf etc.
Mutation breeding
A breeding technique to artificially induce mutation to the gene on plant genome by irradiation,
chemical treatment, etc. and to select crops with superior agricultural traits from such plants.
For instance, in Tottori Prefecture which is a major producer of pears, a variety named“Nijisseiki” has been widely grown, but this variety is weak against black spot, and the disease
prevention cost has long been a major issue. Then, a new varieties named “Gold Nijisseiki” which
has black spot resistant isselected by NIAS (radiation breeding field), and Gold Nijisseiki now

accounts for approximately 40% of pears grown in Tottori Prefecture.
Transcription Activator Like Effecter Nuclease (TALEN)
An artificial restriction enzyme technique where the infection mechanism of plant pathogen
Xanthomonas is applied.
Similar to ZFN (Fig. 1 on Page 7), TALEN is an artificial restriction enzyme consisting of “DNAcleaving domain (Fok I)” with restriction enzyme activity and “DNA binding domain” (TALE)
that identifies specific base sequence information on genome. At the DNA binding domain, while
ZFN identifies the sequence by 3-base units, TALEN identifies by 1-base units.
Particle bombardment
One of the methods to introduce DNA into cells.
DNA is introduced by dusting fine particles of gold or tungsten with DNA and injecting theminto cells by means of helium gas pressure etc. Generally, this method is used for plants with a low
gene introduction rate by the Agrobacterium method (e.g., maize).
PCR method
PCR stands for Polymerase Chain Reaction. It is a method to exponentially multiply DNA byrepeating the reaction to replicate a specific domain of DNA using two DNA fragments (primers)that sandwich the specific domain of DNA and enzyme that are involved with DNA replication- 44 -(polymerase). The PCR method is established as the basic technique of gene research for various
purposes including determination of gene sequence and quantification of genes.
It has now become possible to confirm the existence of DNA (gene) even if the amount of target
DNA contained in the analysis specimen is extremely small (about 10 copies).

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List of Members of the New Plant Breeding Technique Study Group

Name

Position

Specialty

Professor, Faculty of Life and Environmental
Ryo Ohsawa

Sciences, the University of Tsukuba

Plant breeding

Professor, Faculty of Life and Environmental
Hiroshi Kamada*

Sciences, Gene Research Center, theUniversity of Tsukuba

Molecular biology

Professor, Department of General Systems
Masakazu ShimadaStudies, Graduate School of Arts andConservation

Sciences, the University of Tokyo

ecology

Professor, Department of Regional andMasashi Tachikawa

Environmental Science, College ofInternational GMO

Agriculture, Ibaraki University

policyVice Chairman of Working Group on
Masahiro Nakagawara

Harmonisation of regulatory Oversight in
Biotechnology, OECD

Plant breeding

Head of Ecological Genetics Research
Nobuyoshi Nakajima

Section, Center for Environmental Biology
and Ecosystem Studies, National Institute for

Plant physiology

Environmental Studies, Japan

Akihiro Hino

Vice General Manager of the Central

Genetic

Laboratory, Nippon Flour Mills Co., Ltd.

biochemistry, GM
detection technology

Note: Member Nakagawara was elected as the Chairman through mutual election* The late Professor Hiroshi Kamada participated as a member until March 2014

- 46 -

History of Deliberations○ 1st meeting: October 4, 2013 (Fri)<Agenda>(1) About the Trend surrounding NPBTs(2) About the Immediate Directionality of Considerations(3) Other○ 2nd meeting: October 29, 2013 (Tue)<Agenda>(1) About the Operating Status of Self-Cloning and Natural Occurrence, etc.
(2) Case Study about NPBTs(3) Other<Details of Case Study>(1) Fruit Plant Accelerated Breeding using Early Flowering Gene
(2) Application of Recurrent Selection to Inbreeding Crops such as Rice○ 3rd meeting: November 27, 2013 (Wed)<Agenda>(1) Case Study about NPBTs(2) About the Interim Report(3) Other<Details of Case Study>
Mutation Breeding using Artificial Restriction Enzyme etc.
○ 4th meeting: December 19, 2013 (Thu)<Agenda>(1) About the Overseas Trend Investigation Report(2) About the Consideration on the Development and Practical Application of Crops using NPBTs(Interim Report)(3) Other○ 5th meeting: March 25, 2014 (Tue)<Agenda>(1) About the Results of OECD Working Group on the Harmonisation of Regulatory Oversight in
Biotechnology and Workshops on NPBTs(2) About the Consideration on the Development and Practical Application of Crops using NPBTs(Interim Report)(3) About the Strategic Innovation Promotion Program (Cross-ministerial Strategic Innovation
Promotion Program: SIP)

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(4) Other○ 6th meeting: March 27, 2015 (Wed)<Agenda>(1) About the Recent Trend in Japan and Overseas(2) About the Study Group Report(3) Other○ 7th meeting: July 22, 2015 (Wed)<Agenda>(1) About the Report of APEC-HLPDAB(2) About the Final Report (Draft)(3) Other- 48 -