Outline of the Report by
the New Plant Breeding Technique Study Group- Towards the development and practical application ofcrops using new plant breeding techniques (NPBTs)
such as genome editing -September 2015
Agriculture, Forestry and Fisheries
Research Council Secretariat

機密性○情報○○限り

History of Plant Breeding and Improving

 Breeding of crops has a long history of humans modifying wild or native species. In recent years, the development
and use of breeding techniques such as crossing and mutation have greatly contributed to the improvement ofagricultural productivity and the stable supply of food, etc.
Ancient

ModernSeveral decades ago

Present(History of breeding)Selection of useful species (mutants) from nature
Crossing of useful species(Use of radiation, chemicals) Artificial induction of mutation
Elucidation of genetic information (genome)
Use of genetic modification technique
Use of genome editing

Glycine soja

Soybeans“Nature gives successive variations; man adds
them up in certain directions useful to him”
“The Origin of Species” (C. Darwin,
1859)

Japanese wild radish

Japanese radish

1Breeding History of Koshihikari and Akitakomachi
1870～1930

1930～1980

1980～1984

These are names of rice varieties.
The varieties written in black are the ones developed in Japan.
愛国

銀坊主
農林8号
朝日

農林22号

上州

器量好

農林6号

撰一

Koshihikari

Cold tolerance
Good taste

森多早生

大場
愛国

陸羽20号

亀の尾

亀の尾4号

農林1号
陸羽132号

北支太米（China）

Akitakomachi
Ｔadukan （Philippines）

実験系統

農林8号
戦捷（China）

田戦捷

器量好、朝日

早生朝
日2号

真珠

北真１号

農林8号

農林23号

農林6号
597

Zenith （USA）

奥羽292号
越南43号

東山41号
実験系統

Rice blast
resistance etc.

大系437

農林41号
東北71号
2

Conventional breeding It is widely known nowadays that the principle of breeding basically relies on the genes in organisms, and that the
combinations of four bases constituting DNA control the traits of any organism.

Gene involved with diseaseresistance (base sequence)• Disease-resistant• Good taste

A C G T G C T A T

Chromosome

Cell
Chromosome• High yield• Relatively good taste• Disease-resistant

T G C A C G A T A

A C A G A A G G T

T G T C T T C C A• Good taste• Disease-resistant• High yield

3

Gene expression in plant Genome size and gene number on genome ofsome organisms

Chromosome
Nucleus(million base pairs)

Number of
genes

Rice (Japonica)

400

32,000

Maize

2,200

45,000

Human

3,200

20,000

Drosophilamelanogaster180

15,000Escherichia coli
(K12 strain)

5

4,000

OrganismDNA

Gene(Part of DNA)

Non-coding
sequence

TAG • • • • •AT C • • • • •Non-coding
sequence

Coding sequence

A T G TG G T T AAG C • • • • •TA CAC C AAT T C G • • • • •

Met

Trp

Leu

Ser

•••••

CCC•••••
GGG • • • • •Amino acid

Genome size

ProteinPhysiological reactions or phenotype4

Decoding the Genome Information of Rice
 Recently, genes involved with traits useful for agricultural production have been identified one after another bydecoding the genomic information of crops (e.g., rice).

 Examples of genes identified in rice Genetic blueprint for breeding purpose (rice)

qSW5, Seed width Nat genet (2008)

2

4

5

6

7

8

9

10

11

12

Good taste

Gn1, Seed number Science (2005)

Hd1

SSI, SSIII, Starch synthesis Plant Physiol(2006, 2007)

PAIR1, Meiosis Plant Cell (2004)
MEL1, Germ cell formulation Plant Cell (2007)

Pit

qLTG3-1, Low temperature germinability PNAS
(2008)
d1, Plant height PNAS (1999)
d11, Plant height Plant Cell (2005)

High

pi21

Xa1, Bacterial blight-resistance PNAS (1998)
Pib, Rice blast resistance Plant J (1999)
OsRac1, Disease resistance PNAS (2006)
WRKY45, Rice blast and bacterial blightresistance Plant Cell (2007)
RACK1A, Disease resistance Plant Cell (2008)

OsBR6ox, Brassinosteroid biosynthesis
Plant Physiol (2002)
P450, Gibberellin biosynthesis
Plant Physiol (2004)

pi21, Rice blast resistance Science (2009)

LOG, Number of stems Nature (2007)
OsGA2ox1, Plant type
Nature Biotech (2003)

Spl7, Heat stress resistance PNAS (2002)
qUVR10, UV resistance Genetics (2005)

SNORKEL1, SNOKEL2,
Ethylene response Nature (2009)

OsDREB1A-D, OsDREB2A,
Drought, high-salt and cold resistance
Plant J (2003)
Lsi1, Lsi2, Silicic acid absorption
Nature (2006) (2007)

Partial modification ofmaterial by Ishige (2013.7.19)

Bph10(t)Stva

Brown planthopper resistancebph11(t)Ability to ripen
under high temp.

High

Low

Rice blast resistanceCadmium
content

RaisesLowersCold tolerance
Lsi6, Silicic acid absorption
Plant Cell (2008)

Stvb

Bph16(t)gid1, Gibberellin response Nature (2005)
gid2, Gibberellin response Science (2003)

RMS, Cytoplasm male sterility PNAS (2009)

Piz, Pi9Low

MSP1, Germ cell differentiation Plant Cell (2003)

POLLUX, CYCLOPS,
Mycorrhizal fungi symbiosis Plant Cell
(2008)

3

Gn1

qSH1, Seed shattering Science (2006)

Hd1, Heading date Plant Cell (2000)
Hd6, Heading date PNAS (2001)
Hd3a, Heading date PCP (2002)
Ehd1, Heading date Genes Dev (2004)
Ehd2, Heading date Plant Physiol (2008)

1

Grain number

Rice stripe virus resistanceField resistance against rice blast

Brown planthopper resistanceHeading date (becomes late)

Yielding ability

True resistance against rice blast

Cold resistanceHeading date (becomes early)

External appearance

Rice stripe virus resistance

Taste

Low temperature germinability

Cadmium content

Note: Ellipses written on the chromosome shown above are some of the genes
identified by their function.

5

Example of Creating New Species by DNA Marker Assisted Selection
 Under such a situation, in recent years, a new technique called DNA marker assisted selection was developed, which allowed to
identify base sequence information of genes involved with traits useful for agricultural production and to use such information as amarker in the selection of a new useful varieties. This technique is applied for breeding of various crops including rice andvegetables.
 For instance, to breed blast-resistant rice, conventional techniques required many years of experiments including actual cultivationof various crossed varieties and examination of their disease resistance. However, by using DNA marker, rice with disease
resistance can be examined and selected in a short period of time simply by extraction of DNA from the leaves.
 Breeding record of blast-resistance rice “Tomohonami”
Kinki No.15(Norin No.8)
Kinki No.9
(Norin No.6)

1

3

5

4

6

7

8

9

10

11

12

Koshihikari(Etsu-nan No.17)
Norin No.1
(Hokuriku No.4)
Chubu No.125(Tomohonami)

Kanto No.79

40-11(Kiho)

2

Norin No.22(Kinki No.34)

Moritawase
Riku-u No.132

 Gene structure of genome (chromosome) of “Tomohonami”

Mineasahi(Chubu No.17)
Inakei IL946

Sensho-type

Sensho(Mazuokabo, upland rice)

Mineasahi-type

Koshihikari-type

Location of pi21, gene with field resistance against rice blast
Koshihikari(Estu-nan No.17)

Koshihikari
While it has been known that a strong rice blast resistance gene (pi21) exists inupland rice, introducing it by crossing into a rice variety normally grown in Japan(paddy rice) would lead to worsen the taste of the rice as pi21 is highly linked togenes involved with taste.
Therefore, the National Institute of Agrobiological Sciences and the Aichi
Agricultural Research Center jointly solved this problem by taking advantage ofDNA marker. They crossed paddy rice Mineasahi with the upland rice Sensho
and backcrossed the descendants with Mineasahi, then they selected a plantwith only the rice blast resistance gene (pi21) (Inakei IL946).

Tomohonami

Rice growth state in an area with frequent rice blast infection

6

Characteristics of New Plant Breeding Techniques (NPBTs)
 In addition to DNA marker assisted selection, progress has been made on the development of “new plant breeding
techniques (NPBTs)” in some countries that enable accurate and efficient introduction of traits useful for agricultural
production, where genetic modification technologies are applied to a part of the breeding process of the conventional
breeding.
 There are various techniques for NPBTs, including (1) those aiming at improving the efficiency of conventional mutation
breeding and (2) those aiming at shortening the breeding term for conventional breeding by crossing, etc.
 The common characteristic to these techniques is that while genetic modification technologies are applied to a part ofthe breeding process, the final crop products are free from transgenes used for genetic modification. Therefore, someof the crops produced by NPBTs can be considered the same as those selected from the natural diversity or producedby conventional breeding.
 For this reason, when such crops become commercialized, we will have difficulty differentiating them from crops
produced by conventional breeding techniques. There arises a question of whether regulations on genetic modification
shall be applied to such crops. Currently, in several countries, discussions are being held on the regulatory framework
for NPBTs under GM regulations concerning the food and feed safety and the environmental impact.
Techniques aiming at improving theefficiency of conventional mutation
breeding

Techniques aiming at shortening thebreeding term of conventional breeding bycrossing, etc.

 Genome editing (e.g., ZFN, TALEN,CRISPR/Cas) Oligonucleotide-directed mutagenesis(ODM)
 RNA-dependent DNA methylation (RdDM)

 Cisgenesis/Intragenesis
 Grafting using genetically modified rootstock Agro-infiltration
 Accelerated breeding of fruit plants utilizing
early flowering genes
 Recurrent selection of inbreeding crops suchas rice

Other techniques

 Reverse breeding Seed Production
Technology (SPT)7

Conventional Use of Genetic Modification Technologies in Breeding Conventionally, 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. For instance insecticide
genes that form microbes have been introduced into some crops.

 Method for creating genetically modified crops
Microbe, etc.

 Mechanism of insect-resistant (Bt) maize

Useful gene
Insects like pyralidDNA

Cloned geneand ligation

Cell of crop

1. In alkaline condition in its midgut,
Bt protein are partially digested
and peptides remain.

Introduce

Vector(Courier DNA)DNA

Bt protein that
selectively acts
on specificinsects isexpressed

2. The peptide (key) binds to thereceptor (key hole) ofintestinal cells.

3. The intestinal cells aredestroyed, becoming unable
to absorb nutrients.

Genetically modified crop

Transgene

Dies of starvation

Bt gene found in soil bacteria is
introduced into the genome of maize8Framework of Regulations on Genetically Modified Crops in Japan

Securing theopportunityof selection

Framework for ensuring thesafetySelling

Distribution, processing

Commercial
cultivation
Importing

Developed andproductized overseas

Contained field testingContained green
house

Laboratory

Flow of development andcommercialization

 GMOs possess transgenes originating from microbes etc. and new substances expressed by the transgenes, becoming
organisms that humans have never eaten or cultivated before. Therefore GMOs have a possibility of unexpected adverse effects
on human health or wildlife.
 in order to prevent such harmful effects, regulations have been established requiring the Japanese government to conduct asafety assessment for each individual case. Only those approved as safe by the government can be cultivated, imported, etc..

Assessment of Effects on Biological Diversity (Cartagena Act) Approval of cultivation or importing in Japan

Food safety assessment (Food Sanitation Act)
Feed safety assessment (Feed Safety Law) Approval of import/distribution as food/feed

Labeling regulations (Food Sanitation Act and JAS Act) Formulation of labeling standards
 Confirmation of labeling at distribution stage
9

Background of Establishing a New Plant Breeding Technique Study Group







The Ministry of Agriculture, Forestry and Fisheries (MAFF) is now accelerating the development of new innovative varieties that generate“advantages” in Japanese agricultural and livestock products as a part of its policy to realize “Aggressive Agriculture, Forestry and Fisheries”.
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 Japanese government, the “Comprehensive Strategy on Science, Technology and Innovation 2015”
developed in June 2015 indicates the development of next-generation breeding systems (e.g., NPBTs) as a priority R&D initiative, andrelevant ministries are jointly promoting R&D under the Strategic Innovation Promotion Program (SIP).
On practical application of the results of such research, the important task in Japan is how to develop social understanding, because there
still remain strong concerns among consumers over the safety of crops and food to which GM techniques are applied.
Additionally, in case in which discussions on the regulatory framework for NPBTs under GM regulations started in the EU or elsewhere, theJapanese Government needs to promote the collection and analysis of scientific knowledge and experience that can form a basis fordiscussions on NPBTs, and to hold discussions with diverse stakeholders in the future. Further, there is a necessity to conduct activities
towards international regulatory harmonization.
Based on such background, in October 2013, a study group consisting of expert scientists was established within the Agriculture, Forestry
and Fisheries Research Council Secretariat, for gathering domestic and overseas information on NPBTs, compilation of scientific findings on
the effect on the biological diversity, proper promotion of related R&Ds, smooth social implementation of research results, etc.
Name of expert
scientists
Ryo Osawa

Hiroshi Kamada

Position
Professor, Faculty of Life and Environmental Sciences, the University of Tsukuba(Member of the Committee on Assessment of Adverse Effect on Biological Diversity, Ministry of the Environment (MOE) andMAFF)
Professor, Faculty of Life and Environmental Sciences, Gene Research Center, the University of Tsukuba
Member of the Expert Committee on Genetically Modified Foods, Food Safety Commission of Japan(Member of the Committee on Assessment of Adverse Effect on Biological Diversity, MOE and MAFF)

Specialty
Thremmatology
Molecular biology, GM risk
communication

Masakazu Shimada

Professor, Department of General Systems Studies, Graduate School of Arts and Sciences, the University of Tokyo(Member of the Committee on Assessment of Adverse Effect on Biological Diversity, MOE and MAFF)

Conservation ecology

Masashi Tachikawa

Professor, Department of Regional and Environmental Science, College of Agriculture, Ibaraki University

International GMO policy

Masahiro Nakagawara

Vice Chairman of Working Group on Harmonisation of regulatory Oversight in Biotechnology, OECD

Thremmatology

Nobuyoshi Nakajima

Akihiro Hino

Head of Ecological Genetics Research Section, Center for Environmental Biology and Ecosystem Studies, National Institute forEnvironmental Studies, Japan
Plant physiology(Member of the Committee on Assessment of Adverse Effect on Biological Diversity, MOE and MAFF)
Genetic biochemistry, GM
Vice General Manager of the Central Laboratory, Nippon Flour Mills Co., Ltd.
detection technology, risk
(Member of the Committee on Assessment of Adverse Effect on Biological Diversity, MOE and MAFF)
communication

10

Trend of Research and Development Overseas[Related patents][Scientific publications] According to “New plant breeding techniques. State-of-the art and prospects for commercial development” reported by the European
Commission, Joint Research Centre, Institute for Prospective Technological Studies in 2011.
(1) EU leads the world in the number of related scientific papers and the USA leads the world in the number of patent applications.
(2) As a result of a questionnaire survey given to major biotechnology enterprises, NPBTs “have been adopted by commercial
breeders, and the most advanced crops could reach the stage of being commercialization in the short medium term (2-3
years),” if the crops produced by these techniques are not classified as GMOs.
(3) All techniques are undergoing preparations for practical application, placing emphasis on the fact that in the final commercial
products there is an absence of transgenes which is used for the developments and only transiently exist in the plants during thebreeding process. Also, it is focused those higher efficiency than conventional breeding and substantial reduction in the cost fordeveloping new products.
Othercountries
12.1%
Asia
12.6%

EU-27
42.6%

North
America
32.7%

Asia
7.0%

Other
countries
5.1%

North
America
50.3%

EU-27
37.6%

SectorN. publications%

Academia

143

77%

Industry

24

13%

Joint

19

10%

Total

186

SectorN. patents%

Academia

27

20%

Industry

104

76%

Joint

6

4%

Total

137

Source: Material from a lecture by Dr. Emilio Rodríguez Cerezo of JRC, Institute for Prospective
Technological Studies (Feb 2014)
Note: All the data is for the period from year 1991 to 2011.

11

NPBT: Case 1 Genome Editing using Artificial Restriction Enzyme (ZFN)
 Lately, “artificial restriction enzymes” that allow for accurate cleavage of specific base sequence domains of target genes (DNA) havebeen developed, and it has become possible to arbitrarily induce gene modification (deletion, substitution or insertion) to the targeteddomain on a genome (genome editing).
 By applying the genome editing to plant breeding, it would be possible to arbitrarily modify an endogenous gene that affects the flower
color, plant height, etc. and thereby develop new innovative varieties in a short period of time.
 The chance of the same base sequence existing on a genome is calculated, for instance, as 1/(4^18) (maximum of 1 in 70 billion), if
the number of bases in the DNA binding domain is 18. Considering that plant genomes consist of billions of base pairs, as long as
special attention is paid to the specificity of the base sequence of the DNA binding domain at the time of designing the artificial
restriction enzyme, the possibility of cleavage on the non-targeted base sequence (so-called “off-target”) is extremely low.
Plant cell
Expression cassette of ZFN* gene
Introduction into plant
cells via Agrobacterium
mediated system etc.

Nuclease domain

DNA binding domain

Expression
Fokl

3

2

1

Vector--CGC CGC CGC CAC CGTA GGA GGA GGA GCC---GCG GCG GCG GTG GCAT CCT CCT CCT CGG--※ZFN Zinc Finger Nuclease

Fokl
Specific gene sequence is cleaved by artificial
restriction enzyme

3

2

1

--CGCCGCCGCCACCGTAGGAGGAGGAGCC---GCGGCGGCGGTGGCATCCTCCTCCTCGG--

When an error occurs during DNA repair…
Repaired precisely--CGCCGCCGCCAC C--GCGGCGGCGGTG GGTAGGAGGAGGAGCC-CATCCTCCTCCTCGG--

no mutation

Insertion, deletion, or substitution is
integrated into targeted sequences--CGCCGCCGCCAC C--GCGGCGGCGGTG GGTAGGAGGAGGAGCC-CATCCTCCTCCTCGG--

mutation(suppression of the targeted gene, etc.)

12Type of DNA Mutation by ZFN(ZFN-1)(ZFN-2)(ZFN-3)Useful gene＋

Expression cassette of
ZFN gene

DNA fragment with homology to theIntroduction targeted sequences but containing
mutations ( : one or several bases)

Introduction＋

DNA fragments with homology to the target

Introduction sequences but harboring useful exogenous
sequence are inserted (several kilo bases)

Crop cell
Artificial
restriction enzyme(protein)

Expression

DNA

Cleave thetargeted gene

Loss*

Targeted gene↑↑↑
Repaired via
homologous
recombination
betweentargeted geneand introduced
DNA fragment

Double strand breaks
caused by ZFN areprocessed byendogenous DNA
repair mechanisms

Mutation is induced* 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.

Mutation is induced↑

Several kbp

Useful exogenous
sequence areinserted into thetargeted gene via
homologous
recombination
mediated DNA
repair mechanism

Gene is introduced

13

Comparison between Conventional GM Techniques and Genome Editing
Directionality of breeding application

Technical advantage

Genome editing

Conventional GM
techniques

Speed

Able to introduce usefulgenes of different species(e.g., microbes) wherecrossing is difficult○

Accuracy○

Range ofvariation◎

1) Addressing global environmental changes (e.g.,
global warming, desertification)2) Smartization of agriculture by introducing diseaseand pest-resistance3) Production of useful substances (e.g., drugs) incrops

Able to modify sequence
information of an organism
targeting a specific nucleic
acid sequence (gene)

1) Addressing the needs for longevity by developing
crops rich in functional ingredients

◎

◎

○

2) Improving the self-sufficiency rate by developing
rice high in feed nutritive value3) Development of new species with various traits
that have not been actualized (e.g., disease- andpest-resistance)

etc.
14

NPBT: Case 2 Cisgenesis and Intragenesis
 Cisgenesis is a method to introduce a gene of the same or related compatible varieties (cis-gene) to crops using
GM technologies. It may be an effective method to breed vegetative propagation crops such as fruit plants,
potatoes and sugar cane.
 Intragenesis also uses the same or related varieties as the source of gene in a similar manner as cisgenesis.
However, intragenesis is used to control the expression amount of a specific gene by partially modifying thepromoter or terminator that constitute the gene.
Cisgenesis

Intragenesis
Same or related species of species A

Same or related species of species A

Changing the combination ofpromoter etc.

Useful geneSpecies A

Species A
Exon

Take thegene out andconnect to avectorIntroduction

Introduction

PromoterDNA

Take themout, create
gene and
connect toa vector

DNA

Terminator

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 basically the same as species A

A gene created from same or
related species is introduced

The genotypic composition is different from species A

15

Case 3 Agro-infiltration

Case 4 Grafting using GM rootstock Agro-infiltration is a technique to locally infect a plant with
Agrobacterium (bacteria) to which specific genes areincorporated, and, then, to select plants with disease
resistance etc. by examining the expression of the relevantgene.
 Genes originating from the Agrobacterium do not remain in
the body of the selected plants, as long as the infecteddomain (e.g., leaf) is removed.

 When a GM rootstock resistant to a specific soil pest isdeveloped and a non-GM varieties is grafted to therootstock, it is possible to cultivate crops without changing
the quality of harvest from the scion with avoiding the effectsof the soil pest.
 Generally, transgenes (e.g., disease resistance gene)
integrated into the genome of rootstock do not migrate to thescion.

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 unculturable pathogenic virus
is introduced. etc.







Ordinaryvarieties



GMO ornon-GMO?

Scion (non-GM plant)

HarvestRootstock (GM plant)
Disease-resistant plant does notexhibit symptoms

GMO ornon-GMO?

GM varieties
resistant to soil pest

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

16Consideration on Effects on Biological Diversity, etc.
 The Cartagena Act defines a GMO as “an organism that possesses nucleic acid, or a replicated product thereof, obtained through useof a technology, as stipulated in 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 GM or its replicated product thereof remaining in the plant (“possessed”
or not); and,
(2) Summarize the general ways of thinking on adverse effects on the biological diversity through the comparison with crops created byconventional breeding techniques, since there are rules to exclude organisms obtained through so-called “self-cloning” and “natural
occurrence” from organisms subject to the regulation (enforcement ordinance of the Act).

 Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms (Act No. 97 of 2003)(Definitions)
Article 2(2) In this Act, “living modified organism” shall mean an organism that possesses nucleic acid, or a replicated product thereof, obtained through use of
the any of the following technologies:(i) Those technologies, as stipulated in the ordinance of the competent ministries, for the processing of nucleic acid extracellularly(ii) Those technologies, as stipulated in the ordinance of the competent ministries, for fusing of the cells of organisms belonging to different
taxonomical families.
 Enforcement Ordinance of the Act (MOF, MEXT, MHLW, MAFF, METI & MOE Ordinance No. 1 of 2003)
Article 2 Technologies stipulated in the ordinance of the competent ministries under Article 2 paragraph 2 subparagraph 1 of the Act shall be thetechnology for processing nucleic acid extracellularly for the purpose of introducing the nucleic acid into cells, viruses or viroids to transfer or
replicate the nucleic acid, while excluding those mentioned in the following:(i) Technology for processing by using, as nucleic acid to be introduced into cells, only the nucleic acid shown in the following:
A. The nucleic acid of living organism belonging to the same species as that of the living organism which the cells originate from
B. The nucleic acid of living organism belonging to the species that exchanges nucleic acid with the species of the living organism which thecells originate from in natural conditions(ii) Technology for processing by using, as nucleic acid to be introduced into viruses or viroids, only the nucleic acid of viruses or viroids thatexchanges nucleic acid with the viruses or viroids in natural conditions

17

Example of NPBT Development in Japan (1)– Accelerated breeding of fruit trees utilizing early flowering genes – Seedlings of fruit trees usually take 5-10 years to flower and fruit, which is a major obstacle to the development of
new varieties.
 For instance, when the apple scab resistance gene (Vf gene) was introduced to apple cultivar (Rome Beauty) froma related varieties (Malus floribunda), it took 7 generations over 68 years to achieve the desired result (Gold Rush).

(6) Crossed in 1972

Malus floribunda(5)`Rome Beauty’

(4)
(3)
(2)
Prima
Released in1970Gold Rush Released in 1994

(1) Crossed in 1926
821

 A certain quality was finallyachieved after 7 generations over
68 years

 If the generation can be shortened to 1-2 years, breeding will advance drastically18

– Accelerated breeding of fruit trees utilizing early flowering genes – (contd.) In 1999, the FT gene that controls the flowering of plants was discovered by Prof. Araki et al. of Kyoto University.
Prof. Yoshikawa of Iwate University developed a flowering promotion technique for apple by introducing the FT gene by using oneof the latent viruses, “Apple Latent Spherical Virus (ALSV)”.
 This technique is applicable to various crops including other fruits, soybean and vegetables. Development of accelerated breeding
techniques 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 is reduced to one year or less.

Apple latent virus to which early
flowering gene is introduced

Fruit plants 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 isolated greenhouse.

Infection

PollenVirus does
not remain in
the pollen

Seed
1 cycle per year

Grow
new species
in 7-10 years(♂)

×
9 months
after infection

6 months
after infection

50 daysafter infection

New varieties
Latent virus (GM virus)
does not remain

(♀)

19

Example of NPBT Development in Japan (2)– Recurrent selection of inbreeding crops such as rice – Usually, rice is bred by crossing two varieties. However, this technique has a limitation in the number of usable breeding materials, and it
takes a substantial amount of time to improve the agricultural traits such as yield ability that involve multiple genes related.
 The National Agriculture and Food Research Organization, Institute of Crop Science, etc. undertook the development of a breeding
system for applying the recurrent selection, which is generally used for breeding outbreeding crop such as maize nowadays, toinbreeding crops such as rice (utilization of male sterility gene to make rice to outbreed), taking into account the fact that the unit crop
yield of maize has been steadily improved over years through recurrent selection.
 Transition in unit crop of maize (US) and rice (Japan)

 Difference in breeding techniques between maize and rice

(Kg/10a)
Rice (breeding by crossing)

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

Female parent (♀)(Emasculated)

Male parent (♂)(Disease-resistant)Inbreed andselect10 years[Artificial crossing][Artificial crossing]♀

♀

(High yield)

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

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

Inbreed400
Artificial
crossing

200

0
1930

(Disease-resistant)(Recurrent)

800Outbreed(Recurrent)

1000

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

Select

1950

1970

1990

2010

Note: Green is the actual unit crop of maize in the US (USDA) and red is that of rice in Japan.

New species where superior
traits are accumulated

New species 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.

20Consideration on Effects of Abovementioned Cases on Biological Diversity(1) Possibility of transgenes remaining At the initial stage of breeding (experimental research
stage), plants emerge in which transgenes orrecombinant viruses remain.

Appropriate use based on the Cartagena Act(application for the approval to use in a closed systemor to test at an isolated field) is required Final commercial varieties can be selected from thosewith no relevant transgenes remaining (Null Segregant)(2) Comparison with conventional breeding, etc.
 Transgenes and recombinant viruses are used to improvethe efficiency of cross breeding or selection process, but
no transgenes and other related DNA remain in the final
commercial varieties.

 Therefore, newly produced crops (new varieties) can be
regarded as the ones created by conventional breeding.
 In breeding of crop plants, usually, there is a process toeliminate undesirable plants from the line groups
engineered. As a result, only the plants with good qualities
and ease of growth are selected as commercial varieties.

There is a possibility of being exempted from
Cartagena Act regulations if it is confirmed to be freefrom transgenes by appropriate methods such as PCR

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. Therefore, the Study Group considered that there
are no matters that call for specific concerns over the effects on biological diversity.
21

Genome Editing utilizing Artificial Restriction Enzyme
 Genome editing is an epoch-making mutation breeding techniques that allows for inducing mutation to specific genes in crops. However,
the induction of mutation requires introduction of a transgene that produces an artificial restriction enzyme into the genome of the crop.
 The National Institute of Agrobiological Sciences is developing techniques to completely remove artificial restriction enzyme genes
introduced to the genome after mutagenesis, a method to improve the occurrence rate of mutation (utilization of SDN-2), etc.
 It will become possible to suppress genes that generate allergens in rice or toxic substances in potatoes (e.g., glycoalkaloids). It will also
be able to develop feed rice rich in a specific amino acid, sweeter crop rich in oligosaccharide, tomato with high rate of flowering andhigh yield, tomato having parthenocarpy, tomato and capsicum that ripen even under low temperature in winter, grass that is easily
digested by livestock, pollen-free Japanese cedar, etc., in a very short period of time.
Targeted mutating type (SDN-1)

Targeted substituting type (SDN-2)+

Expression cassette of ZFN gene
Introduction

Template DNA fragment
Introduction

Crop cell
Artificial
restriction enzyme(protein)

Expression

DNA

Cleave thetarget gene

Loss

Target gene↑↑
Desired mutation is
introduced into the target
gene via homologous
recombination
using a template DNA

Double strand breaks
caused by ZFN areprocessed by endogenous
DNA repair mechanisms
Mutation is induced

Mutation is induced

22

Consideration on Effects of Abovementioned Cases on Biological Diversity(1) Possibility of transgenes remaining(2) Comparison with conventional breeding, etc.

 Usually, an artificial restriction enzyme gene (transgene) will beincorporated into the plant genome.

 SDN-1 or 2 induces deletion, substitution or insertion of bases.
Such mutations of several bases occurs naturally or inconventional breeding such as mutation breeding unintendedly.

Until the artificial restriction enzyme gene (transgene) iscompletely removed, appropriate use based on the Cartagena
Act is required.
 (With transgene removing,) deletion, substitution, or insertionof bases can occur in nature or conventional mutation
breeding. In the existing Cartagena Act, “natural occurrence”
is exempted from the regulations.

 Compared to conventional mutation breeding, SDN-1 and 2
allow arbitrary modification of the target gene only, so there is asmaller chance of unintended mutation occurrence, whichmeans they may reduce the risk of effects on biological diversityetc.
 Mutation of gene that controls seed shattering of rice
The 612th base of first
chromosome of rice is substituted
Nipponbare
Kasalath(1) SDN-1: There is a possibility to be exempted from theCartagena Act regulations.
(2) SDN-2: It is important for developers to request for
judgment on a case-by-case basis, with submitting related
information including the characteristics of mutated trait to
the regulatory.

ATTTCA
ATTGCA

Picture on the left:
Left: Nipponbare (Japonica)
Right: Kasalath (Indica)
Source: Saeko Konishi et al., Science (2006)(Institute of the Society for Techno-Innovation of Agriculture, Forestry, and Fisheries)

From the above understanding, regarding SDN-1 and SDN-2 that induce mutation to several bases, if it is finally confirmed that no artificial
restriction enzyme is included in the final crops, the crops can be regarded as the same as those produced by conventional breeding
techniques. Therefore, the Study Group considered that there are no specific concerns over the effects on biological diversity.
However, currently, there may be cases where sufficient knowledge is not available on the mutated target genes. In such cases, it isappropriate to provide the regulatory authorities with the related information in advance and to receive scientific consideration from experts
as necessary.
23

Promotion of Future R&D
 In Japan, along with the increasing difficulty in practical cultivating GMOs domestically, the options of techniques usable for breeding
main crops such as rice are limited, and it takes many years to develop new varieties.
Regarding vegetables and flowering plants, personal breeding is actively performed and the export of seedlings is steadily increasing inrecent 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 over stagnancy in the development of new varieties.
 NPBTs are techniques that can draw out the maximum potential of agriculturally useful genetic traits that exist in the same or related
species, efficiently improve the quality, functionality, yield, etc. of crops, and thereby enable creation of unprecedented epoch-making
varieties in a short period of time.
Since the engineered crops do not possess transgenes at the end and may have the same gene organization as crops produced byconventional 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 compliance with GM regulations, and there is a possibility for Japanese privatecompanies etc. to apply NPBTs to breeding and improving various crops including vegetables and flowering plants.
 Scale of global seedling market (2009) Regulatory compliance cost for insect-resistant maize
Cost categories
Preparation for hand-off of events into regulatory
Molecular characterization
Compositional assessment
Animal performance and safety studies
Protein production and characterization
Protein safety assessment
Nontarget organism studies
Agronomic and phenotypic assessment
Production of tissues
ELISA development, validation and expression analysis
EPA expenses for PIPs (e.g., environmental use permit)
Environmental fate studies
EU import (detection methods, fees)
Canada costs
Stewardship
Toxicology (90-day rat) – when done
Facility & management overhead costs

Range of cost incurred ($)
20,000 50,000
300,000 1,200,000
750,000 1,500,000
300,000 845,000
162,000 1,725,000
195,000 853,000
100,000 600,000
130,000 460,000
680,000 2,200,000
415,000 610,000
150,000 715,000
32,000 800,000
230,000 405,000
40,000 195,000
250,000 1,000,000
600,000 4,500,000
71,850 538,875

Total

7,060,000

Source: Nature Biotechnology, Volume 25, Number 5, May 2007

- 15,440,000(unit: million US dollars)

3%

8%
27%

10%
Global total:
36,500

26%
26%

Asia

N. America

EU

S. America

AfricaOther

Source: Website of the Society of American Archivists

24Promotion of Future R&D
 Under the Strategic Innovation Promotion Program (SIP) of the Cabinet Office, the government (research institutions under MAFF,
MEXT, etc.), academia (universities) and industry (related private enterprises such as those in the breeding industry) shall collaborate inactively implementing initiatives in the development of Japanese NPBTs, converting them into intellectual properties, creating innovative
varieties of various plants including grains, vegetables and flowers through application of NPBTs, enhancing regulatory science andpromoting direct dialogue with the citizens with developing science communication, etc.
 R&D period: FY2014-2018<Promotion Committee>

 Fiscal budget: 810 million yen (FY2015)
 Outline of the Program
1 Improvement and development of new plant breeding techniques (NPBTs)
 To improve genome editing techniques such as TALEN and CRISPR/Cas9 sothat they become applicable to various plants including rice and vegetables
 To newly develop Japan’s genome editing
 To improve the early flowering technique for apples so that it becomes applicableto other fruit plants and trees

Cabinet Office
PD (Chair), Government Agencies(Space Strategy Office, Food Safety
Commission, MIC, National Tax
Agency, MEXT, MAFF, METI,
Meteorological Agency, MOE andCabinet Office), Research Institute(BRAIN and JST) and External
Experts

2 Development of epoch-making agricultural and fishery products
 To develop ultra-high-yield rice varieties
 To develop vegetables (e.g. tomato) rich in functional ingredients
 To develop tuna suited for culturing

JST

PD

MAFF

NARO Bio-oriented
Technology Research
Collaboration
Advancement Institution(BRAIN)
Publicinvitation

3 Scientific research on social implementation
 To develop techniques to prove there are no transgenes and other related DNA
remaining To accumulate scientific findings on natural mutation and artificial mutation at thenucleic acid level To establish methods to provide citizen with relevant information and promote
communication

Universities

Research
Institutes

Companies

Researchgroups25

Initiatives towards Promoting Social Understanding<Compliance with GM regulations> Appropriate control based on the Cartagena Act is required in R&D stages, because foreign genes have been introduced toagricultural products during their breeding process even if transiently.
 Applicants are required to consult with competent authorities prior to cultivation of new varieties and prior to its use in foods
and feeds, so as to make sure whether the final products is subjected to the regulations. .
<The way of providing information and communicating with the nations> It is important to promote interactive communication with various interested parties from the R&D stages and to reflect thevoices of anticipation, anxiety and concerns from such parties on the R&D and the process of practical application, as many
NPBTs use the latest molecular biological findings.
 Additionally, considering the lingering social anxiety over the agricultural products and food utilizing GM techniques, the key
point will be how to disseminate information and communicate in a convincing manner on:(1) The significance of introducing NPBTs for increasing the speed of plant breeding to address the issues including the global
environmental changes and food shortage; and(2) Similar plants can be produced naturally or through conventional breeding techniques.
These information sharing should be along with showing the actual crop plants (new species). The development of new epochmaking species that brings tangible benefits to the farmers and consumers in Japan should be promoted.
 For the reason above, it is important to continue to steadily collect related scientific findings and formulate opinions about theeffects on biological diversity etc., and promote dialogue with various interested parties including a broad range of experts,
consumer organization, mass media, farmers, industries etc. so as to generate a support and trust from a wide society.
<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 differencesamong the countries/regions could cause confusion in the trade of agricultural products in the future.
 International harmonization of regulatory oversight and scientific view in NPBTs in fora such as OECD-WG is important, as wellas accelerating the activities such as the formulation of scientific opinions in Japan.

26