Chee Hark Harn

Director of RampD DeptNongwoo Bio Co

Recent Breeding Technologies for Developing New Germplasms

Intention

-To review breeding technologies available

-To propose a collaboration for helping each other for sharing those technologies and developing new germplasm

-To improve the quality of germplasm and F1 hybrid in Asianseed industry

Germplasm

Germplasmin Nature

Selection out by domestication

Breeding practice

Germplasmavailable

Newgermplasmsdeveloped

Solanaceous Germplasm

It is important to develop new various germplasm by breeding and in order to enhance the quality and diversity of germplasm application of new breeding technologies is inevitable

Intention

-To review breeding technologies available

-To propose a collaboration for helping each other for sharing those technologies and developing new germplasm

-To improve the quality of germplasm and F1 hybrid in Asianseed industry

Germplasm

Germplasmin Nature

Selection out by domestication

Breeding practice

Germplasmavailable

Newgermplasmsdeveloped

Solanaceous Germplasm

It is important to develop new various germplasm by breeding and in order to enhance the quality and diversity of germplasm application of new breeding technologies is inevitable

Germplasm

Germplasmin Nature

Selection out by domestication

Breeding practice

Germplasmavailable

Newgermplasmsdeveloped

Solanaceous Germplasm

It is important to develop new various germplasm by breeding and in order to enhance the quality and diversity of germplasm application of new breeding technologies is inevitable

Solanaceous Germplasm

It is important to develop new various germplasm by breeding and in order to enhance the quality and diversity of germplasm application of new breeding technologies is inevitable

BreedingTechnologies

Molecular Biology(DNA marker)

1990s

1930s

Biotechnology

GenomicsOmics

2000s

OrdinaryBreeding

1980s

Breeding Technologies (Since ~)

Genome Editing(CRISPR)

MutationDH

1960s

2010s

Cultivar A Cultivar BX

Ordinary Breeding Technologies

F1

F2

F3

F4

selection

pedigree selection ampfixation

F5-6yield trial

F7-8local trial

varietyF9-10

Pedigree Selection

Hybrid

F2

F3

F4

segregation

selection

F5

F6-7

x

fixation

F8 line

F1F1 Hybrid Selfing

P1 line

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 line or cultivar (X)X

BC5F1

P1 line X= New line

F1

50

75

875

938

969

984

BC6F1

992

Backcrossing DonorRecurrent

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

Technology

USE (O) or NOT (X)

Korea Multi-nationalcorporationsNWB Top 2-3

Tilling analysis (rays EMS) O O O

Ploidy analysis O O O

DNA marker development for trait

O O O

MAS O O O

MAB O O O

HT GA for MGP O X O

GWAS GBS O X O

Analysis of secondary metabolites O X O

Cell fusion O X O

Genetic transformation ampRisk assessment

O X O

Genome editing O X O

Cisgenesis X X O

Phenotyping analysis X X O

ICT Big data implementation X X O

There are about 200 seed companies in Korea and only a few are able to utilize certain levels of technologies (The rich get richer and the poor get poorer because the new technology is coming out fast)

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

P1 rr

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 RR for PWX

BC5F1

P1 line with RR for PW= New line

F1

BC6F1

donorRecurrent

Rr

Rr

Rr

Rr

Rr

RrSelection by DNA marker

(Rr rr RR)

Rr

RR RR

I Marker-Assisted Selection

RR rr

PepperPW

resistance

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Cultivar A Cultivar BX

Ordinary Breeding Technologies

F1

F2

F3

F4

selection

pedigree selection ampfixation

F5-6yield trial

F7-8local trial

varietyF9-10

Pedigree Selection

Hybrid

F2

F3

F4

segregation

selection

F5

F6-7

x

fixation

F8 line

F1F1 Hybrid Selfing

P1 line

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 line or cultivar (X)X

BC5F1

P1 line X= New line

F1

50

75

875

938

969

984

BC6F1

992

Backcrossing DonorRecurrent

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

Technology

USE (O) or NOT (X)

Korea Multi-nationalcorporationsNWB Top 2-3

Tilling analysis (rays EMS) O O O

Ploidy analysis O O O

DNA marker development for trait

O O O

MAS O O O

MAB O O O

HT GA for MGP O X O

GWAS GBS O X O

Analysis of secondary metabolites O X O

Cell fusion O X O

Genetic transformation ampRisk assessment

O X O

Genome editing O X O

Cisgenesis X X O

Phenotyping analysis X X O

ICT Big data implementation X X O

There are about 200 seed companies in Korea and only a few are able to utilize certain levels of technologies (The rich get richer and the poor get poorer because the new technology is coming out fast)

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

P1 rr

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 RR for PWX

BC5F1

P1 line with RR for PW= New line

F1

BC6F1

donorRecurrent

Rr

Rr

Rr

Rr

Rr

RrSelection by DNA marker

(Rr rr RR)

Rr

RR RR

I Marker-Assisted Selection

RR rr

PepperPW

resistance

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Hybrid

F2

F3

F4

segregation

selection

F5

F6-7

x

fixation

F8 line

F1F1 Hybrid Selfing

P1 line

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 line or cultivar (X)X

BC5F1

P1 line X= New line

F1

50

75

875

938

969

984

BC6F1

992

Backcrossing DonorRecurrent

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

Technology

USE (O) or NOT (X)

Korea Multi-nationalcorporationsNWB Top 2-3

Tilling analysis (rays EMS) O O O

Ploidy analysis O O O

DNA marker development for trait

O O O

MAS O O O

MAB O O O

HT GA for MGP O X O

GWAS GBS O X O

Analysis of secondary metabolites O X O

Cell fusion O X O

Genetic transformation ampRisk assessment

O X O

Genome editing O X O

Cisgenesis X X O

Phenotyping analysis X X O

ICT Big data implementation X X O

There are about 200 seed companies in Korea and only a few are able to utilize certain levels of technologies (The rich get richer and the poor get poorer because the new technology is coming out fast)

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

P1 rr

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 RR for PWX

BC5F1

P1 line with RR for PW= New line

F1

BC6F1

donorRecurrent

Rr

Rr

Rr

Rr

Rr

RrSelection by DNA marker

(Rr rr RR)

Rr

RR RR

I Marker-Assisted Selection

RR rr

PepperPW

resistance

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

P1 line

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 line or cultivar (X)X

BC5F1

P1 line X= New line

F1

50

75

875

938

969

984

BC6F1

992

Backcrossing DonorRecurrent

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

Technology

USE (O) or NOT (X)

Korea Multi-nationalcorporationsNWB Top 2-3

Tilling analysis (rays EMS) O O O

Ploidy analysis O O O

DNA marker development for trait

O O O

MAS O O O

MAB O O O

HT GA for MGP O X O

GWAS GBS O X O

Analysis of secondary metabolites O X O

Cell fusion O X O

Genetic transformation ampRisk assessment

O X O

Genome editing O X O

Cisgenesis X X O

Phenotyping analysis X X O

ICT Big data implementation X X O

There are about 200 seed companies in Korea and only a few are able to utilize certain levels of technologies (The rich get richer and the poor get poorer because the new technology is coming out fast)

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

P1 rr

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 RR for PWX

BC5F1

P1 line with RR for PW= New line

F1

BC6F1

donorRecurrent

Rr

Rr

Rr

Rr

Rr

RrSelection by DNA marker

(Rr rr RR)

Rr

RR RR

I Marker-Assisted Selection

RR rr

PepperPW

resistance

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

Technology

USE (O) or NOT (X)

Korea Multi-nationalcorporationsNWB Top 2-3

Tilling analysis (rays EMS) O O O

Ploidy analysis O O O

DNA marker development for trait

O O O

MAS O O O

MAB O O O

HT GA for MGP O X O

GWAS GBS O X O

Analysis of secondary metabolites O X O

Cell fusion O X O

Genetic transformation ampRisk assessment

O X O

Genome editing O X O

Cisgenesis X X O

Phenotyping analysis X X O

ICT Big data implementation X X O

There are about 200 seed companies in Korea and only a few are able to utilize certain levels of technologies (The rich get richer and the poor get poorer because the new technology is coming out fast)

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

P1 rr

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 RR for PWX

BC5F1

P1 line with RR for PW= New line

F1

BC6F1

donorRecurrent

Rr

Rr

Rr

Rr

Rr

RrSelection by DNA marker

(Rr rr RR)

Rr

RR RR

I Marker-Assisted Selection

RR rr

PepperPW

resistance

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Technology

USE (O) or NOT (X)

Korea Multi-nationalcorporationsNWB Top 2-3

Tilling analysis (rays EMS) O O O

Ploidy analysis O O O

DNA marker development for trait

O O O

MAS O O O

MAB O O O

HT GA for MGP O X O

GWAS GBS O X O

Analysis of secondary metabolites O X O

Cell fusion O X O

Genetic transformation ampRisk assessment

O X O

Genome editing O X O

Cisgenesis X X O

Phenotyping analysis X X O

ICT Big data implementation X X O

There are about 200 seed companies in Korea and only a few are able to utilize certain levels of technologies (The rich get richer and the poor get poorer because the new technology is coming out fast)

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

P1 rr

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 RR for PWX

BC5F1

P1 line with RR for PW= New line

F1

BC6F1

donorRecurrent

Rr

Rr

Rr

Rr

Rr

RrSelection by DNA marker

(Rr rr RR)

Rr

RR RR

I Marker-Assisted Selection

RR rr

PepperPW

resistance

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Recent Breeding Technologies available

Tilling analysis of artificial mutation (rays EMS)

Ploidy analysis using flow-cytometry

DNA marker development for traits

DNA Marker-Assisted Selection (MAS)

DNA Marker-Assisted Backcrossing (MAB)

HT genotyping analysis for molecular genetic purity (MGP) and variety identification

GWAS (genome-wide association study) GBS (genotyping by sequencing)

Analysis of secondary metabolites (phytochemicals) for functional crop

Cell fusion (Protoplast fusion) selecting by markers

Genetic transformation and Risk assessment

Cisgenesis

Genome editing

Phenotyping analysis

ICT big data implementation

P1 rr

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 RR for PWX

BC5F1

P1 line with RR for PW= New line

F1

BC6F1

donorRecurrent

Rr

Rr

Rr

Rr

Rr

RrSelection by DNA marker

(Rr rr RR)

Rr

RR RR

I Marker-Assisted Selection

RR rr

PepperPW

resistance

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

P1 rr

BC2F1

x

BC1F1

BC3F1

BC6F2

BC4F1

BC6F3

x

P2 RR for PWX

BC5F1

P1 line with RR for PW= New line

F1

BC6F1

donorRecurrent

Rr

Rr

Rr

Rr

Rr

RrSelection by DNA marker

(Rr rr RR)

Rr

RR RR

I Marker-Assisted Selection

RR rr

PepperPW

resistance

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Recurrent line

(Elite line)Donor line(Ty-1 Cf-9 Sw-5 Mi)x

x

BC6F1

F1

BC1F1

BC6F3

Rr

rr RR

x

x

Multi disease resistant

tomato selected

by multi-markers

RR

Simultaneous Analysis of Multi-gene Introgression

Dr HR Lee NWB

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

II Marker-Assisted Backcross (MAB)

P1 P2 BC1F1

Target gene

R

D

Less D genome

P1

BC2F1P1BC3F1BC3F2

RTarget gene

D

P1F1

(background selection)

D

One that contains R genome and the target gene

-Using DNA markers that distinguish P1 and P2 and P1 genome background and the target gene X will be selected -It provides an advantage that we can fix a line at BC3F2

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

MAB

Recurrent

BC2F1

BC1F1

Donor XX

F1

P1 P2

BC3F2 X

New line

xBC3F1

ControllerPCR Reader

Chip (48x48)

Chip (96x96)

Chip based SNP analysis

DNA sample

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Recurrent

ⅹBC3F1BC2F1BC1F1

Similarity 63~84

BC2F1- biomark BC3F1-biomarkBC1F1-biomarkDonor

Similarity 84~94 Similarity 95~99

Dr HR Lee NWB

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

(Nuclear Genome Transfer)

NNN N N

+

MSF1 hybrid(super)

MF line(Mediocre)

Fused MF line (new)

Segregation of Heterozygous genome

x

Electro-cell fusionN heterozygote

New germplasm

radiation(γ-Ray)

IOA

III Cell Fusion (Protoplast Fusion cybrids)

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

D FE

CB

A after-fusion B callus induction after-fusion proliferation of callus D E shoot induction F regeneration

Process for carrot regeneration from callus after fusion

A

M Fused carrots

Dr M Jung NWB

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Segregation Pattern of Fused Carrots (C1)

A Phenotypic variation of fused C1 carrots B Pigmentation of carrots 1 elite variety 2 N normal inbred line 3 fused carrot EV elite variety MS male sterile N normal male fertile

BA

1 (EV MS) 2 (N) 3 Fused carrots (N)

Dr M Jung NWB

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

How would you apply the cell fusion technology to Solanaceous crop

Transfer the nucleus of CGMS B-line to A-line in pepper

Seedless pepper-development of tetraploid using fusion-diploid x tetraploid to get triploid

middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Cisgenesis

Reversebreeding

Oligonucleotidedirected

mutagenesis

RNA-dependentDNA methylationAgro-

infiltration

Synthetic genomics

Grafting on GM

rootstock

Genomeediting

New Plant Breeding Technology

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Cisgenesis is the technology that you want to transfer a verygood trait which is a native gene in the wild type or OP toyour breeding line By ordinary crossing it would take a longtime if you use wild type Besides you may have troubles suchas a linkage drag Using GTF technology a whole gene can betransferred

Cisgene is a natural gene coding for a trait from the crop plant itself or from a crossable species which is normally used in conventional breeding Cisgene contains exons and introns and flanking regions such as native promoter and terminator region in a sense orientation

IV Cisgenesis

LineWild type

OP

A whole gene

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

RB LB

KmR

RecLBD CodA-nptIIGene 35S

pMF1000

35S

Dexamethasone(dex) chemically activates the recombinase and excises RS regionRS recombination site RecLBD recombinase

RSRS

RB LB

RecLBD CodA-nptIIGene 35S35S

RSRS

RB

Gene

LB

Genetic Transformation Vector for Cisgenesis

host DNA

host DNA

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

Blight resistant potatoes were developed from wild varietiestransferring three R genes into high yielding varieties Aftertreatment with Phytophthora infestans the normal potatoeshave blight but the cisgenic potatoes are healthy

R S

-available cisgene (a whole gene cloned)-marker free transformation vector

Conditions

-quickly cheaply create new varieties-only beneficial gene transferred-could avoid linkage drags

Advantage of Cisgenesis

Jo et al 2014

xOrdinary breeding

GMO breeding

Cisgenic breeding

7-10 yr

over 10 yr

2-4 yr

Breeding Period Difference among Technologies

Protoplast

CRISPR-Cas9 mediated mutant

RNA guided engineered endonucleases (RGENs)

+

V Genome Editing

NewCrop

1) Transfection2) Mutation3) Culture for regeneration4) Selection by marker

transfectionCas 9 protein

gRNA

CRISPR-Cas9

Ribonucleoproteins (RNPs)DNA

PSY Knockout

CHYB1CHYB2 Knockout

-Beta-carotene content-dark orange

1) Metabolic Engineering for New Germplasm

Ex phytochemical manipulation

Lutein content

Zeaxanthin content

Lycopene content

GE

Dr M Jung NWB

Protoplast

CRISPR-Cas9 mediated mutant

RNA guided engineered endonucleases (RGENs)

+

V Genome Editing

NewCrop

1) Transfection2) Mutation3) Culture for regeneration4) Selection by marker

transfectionCas 9 protein

gRNA

CRISPR-Cas9

Ribonucleoproteins (RNPs)DNA

PSY Knockout

CHYB1CHYB2 Knockout

-Beta-carotene content-dark orange

1) Metabolic Engineering for New Germplasm

Ex phytochemical manipulation

Lutein content

Zeaxanthin content

Lycopene content

GE

Dr M Jung NWB

PSY Knockout

CHYB1CHYB2 Knockout

-Beta-carotene content-dark orange

1) Metabolic Engineering for New Germplasm

Ex phytochemical manipulation

Lutein content

Zeaxanthin content

Lycopene content

GE

Dr M Jung NWB

2) Multiple mutation

Cas 9 protein

gRNA

CRISPR-Cas9

DNA

Cas 9 protein

gRNA

DNA

-Construct multi-deletion-Develop various mutant pools(quality shelf life pestmiddotdisease resistances yield drought tolerance coldheat tolerance color etc)

GE

GE

3) Avoid linkage drag

Donor traitPhenotypedragged

PMMoV(Tm3) resistant Weak vigor amp low fertility

Powdery mildew resistant Smaller amp thicker leaf

BS2 resistant Smaller fruit

GE would break the linkage drag

4) Replacing GMO

By mutating the genes coding enzymes sensitive to herbicide the herbicide-resistant non-GMO could be developed

Herbicide

Enzyme(EPSPS)

Substrate

AA synthesis Plant lives

No AA syn Plant dies

AA synthesis Plant livesEPSPSmutant

GE

Herbicide canrsquot bind to enzyme

ldquoHerbicide ResistantPepper (non-GMO)rdquo

EPSPSblocked

CropGenomeEditing

Targetgene

Characteristics Developer

Corn ZFN IPK Herbicide R Dow

CornMega-

nucleaseundisclosed

Increasedharvest

Benson HillBiosystems

CornMega-

nucleaseundisclosed Increased

StarchAgrivida

CornCRISPR-

Cas9Wx1

Increasedamylopectin

DupontPioneer

Potato TALEN InvDecreased

reduced-sugarsCalyxt

Rice TALENOS11N3 OS8N3

BacteriaBright resistance

Iowa StateUniv

Soybean TALEN FAD2High oleic acid and

low-linolenicCalyxt

Soybean TALEN FAD3High oleic acid and

low-linolenicCalyxt

Wheat TALEN MLOPowdery mildew

resistanceCalyxt

MushroomCRISPR-

Cas9PPO

Browny color resistance

PennsylvaniaState Univ

1stgeneration ZFN 2nd TALEN 3rd CRISPR-Cas9 4th CRISPR-Cpf1

Crops that developed by genome editing technology

The common white button mushroom (Agaricus bisporus) has been modified to resist browning

The mushroom can be cultivated and sold without passing through the agencyrsquos regulatory process mdash making it the first CRISPR-edited organism to receive a green light from the US government April 17 2016

polyphenol oxidase (PPO) knock out

Ordinary B

Molecular B

New B

+

+

Stacking traitsReducing generation

F1 hybridDevelopment

Breeding Tool Box

Selectionafter FT

F1 seedproduction

QC amp QA Sales

Bre

edin

g P

ipelin

e

-Genetic sources for trait pool amp selection-Trait fixation through back-cross-DH line-Mutationhelliphelliphelliphelliphellip

-MAS MAB-Bioinformatics with omics -GWAS GBS-HT genotypinghelliphelliphelliphellip

-New plant breeding technology-Cell fusion -Genome editinghelliphelliphelliphellip

Breeding Pipeline through Technology Combination

Summary

There are new breeding tools available

If we want to develop an elite F1 hybrid we need to set up abreeding pipeline utilizing a series of breeding tools together

However in Asian Seed Industry using all of the technology islimited depending on each companyrsquos capability

For that an open innovation strategy is inevitable among researchscientists in the public and private sector to share geneticresources and breeding tools (a collaboration proposal) toimprove the quality of germplasm and F1 hybrid

Thanks for your attention

3) Avoid linkage drag

Donor traitPhenotypedragged

PMMoV(Tm3) resistant Weak vigor amp low fertility

Powdery mildew resistant Smaller amp thicker leaf

BS2 resistant Smaller fruit

GE would break the linkage drag

4) Replacing GMO

By mutating the genes coding enzymes sensitive to herbicide the herbicide-resistant non-GMO could be developed

Herbicide

Enzyme(EPSPS)

Substrate

AA synthesis Plant lives

No AA syn Plant dies

AA synthesis Plant livesEPSPSmutant

GE

Herbicide canrsquot bind to enzyme

ldquoHerbicide ResistantPepper (non-GMO)rdquo

EPSPSblocked

CropGenomeEditing

Targetgene

Characteristics Developer

Corn ZFN IPK Herbicide R Dow

CornMega-

nucleaseundisclosed

Increasedharvest

Benson HillBiosystems

CornMega-

nucleaseundisclosed Increased

StarchAgrivida

CornCRISPR-

Cas9Wx1

Increasedamylopectin

DupontPioneer

Potato TALEN InvDecreased

reduced-sugarsCalyxt

Rice TALENOS11N3 OS8N3

BacteriaBright resistance

Iowa StateUniv

Soybean TALEN FAD2High oleic acid and

low-linolenicCalyxt

Soybean TALEN FAD3High oleic acid and

low-linolenicCalyxt

Wheat TALEN MLOPowdery mildew

resistanceCalyxt

MushroomCRISPR-

Cas9PPO

Browny color resistance

PennsylvaniaState Univ

1stgeneration ZFN 2nd TALEN 3rd CRISPR-Cas9 4th CRISPR-Cpf1

Crops that developed by genome editing technology

The common white button mushroom (Agaricus bisporus) has been modified to resist browning

The mushroom can be cultivated and sold without passing through the agencyrsquos regulatory process mdash making it the first CRISPR-edited organism to receive a green light from the US government April 17 2016

polyphenol oxidase (PPO) knock out

Ordinary B

Molecular B

New B

+

+

Stacking traitsReducing generation

F1 hybridDevelopment

Breeding Tool Box

Selectionafter FT

F1 seedproduction

QC amp QA Sales

Bre

edin

g P

ipelin

e

-Genetic sources for trait pool amp selection-Trait fixation through back-cross-DH line-Mutationhelliphelliphelliphelliphellip

-MAS MAB-Bioinformatics with omics -GWAS GBS-HT genotypinghelliphelliphelliphellip

-New plant breeding technology-Cell fusion -Genome editinghelliphelliphelliphellip

Breeding Pipeline through Technology Combination

Summary

There are new breeding tools available

If we want to develop an elite F1 hybrid we need to set up abreeding pipeline utilizing a series of breeding tools together

However in Asian Seed Industry using all of the technology islimited depending on each companyrsquos capability

For that an open innovation strategy is inevitable among researchscientists in the public and private sector to share geneticresources and breeding tools (a collaboration proposal) toimprove the quality of germplasm and F1 hybrid

Thanks for your attention

4) Replacing GMO

By mutating the genes coding enzymes sensitive to herbicide the herbicide-resistant non-GMO could be developed

Herbicide

Enzyme(EPSPS)

Substrate

AA synthesis Plant lives

No AA syn Plant dies

AA synthesis Plant livesEPSPSmutant

GE

Herbicide canrsquot bind to enzyme

ldquoHerbicide ResistantPepper (non-GMO)rdquo

EPSPSblocked

CropGenomeEditing

Targetgene

Characteristics Developer

Corn ZFN IPK Herbicide R Dow

CornMega-

nucleaseundisclosed

Increasedharvest

Benson HillBiosystems

CornMega-

nucleaseundisclosed Increased

StarchAgrivida

CornCRISPR-

Cas9Wx1

Increasedamylopectin

DupontPioneer

Potato TALEN InvDecreased

reduced-sugarsCalyxt

Rice TALENOS11N3 OS8N3

BacteriaBright resistance

Iowa StateUniv

Soybean TALEN FAD2High oleic acid and

low-linolenicCalyxt

Soybean TALEN FAD3High oleic acid and

low-linolenicCalyxt

Wheat TALEN MLOPowdery mildew

resistanceCalyxt

MushroomCRISPR-

Cas9PPO

Browny color resistance

PennsylvaniaState Univ

1stgeneration ZFN 2nd TALEN 3rd CRISPR-Cas9 4th CRISPR-Cpf1

Crops that developed by genome editing technology

The common white button mushroom (Agaricus bisporus) has been modified to resist browning

The mushroom can be cultivated and sold without passing through the agencyrsquos regulatory process mdash making it the first CRISPR-edited organism to receive a green light from the US government April 17 2016

polyphenol oxidase (PPO) knock out

Ordinary B

Molecular B

New B

+

+

Stacking traitsReducing generation

F1 hybridDevelopment

Breeding Tool Box

Selectionafter FT

F1 seedproduction

QC amp QA Sales

Bre

edin

g P

ipelin

e

-Genetic sources for trait pool amp selection-Trait fixation through back-cross-DH line-Mutationhelliphelliphelliphelliphellip

-MAS MAB-Bioinformatics with omics -GWAS GBS-HT genotypinghelliphelliphelliphellip

-New plant breeding technology-Cell fusion -Genome editinghelliphelliphelliphellip

Breeding Pipeline through Technology Combination

Summary

There are new breeding tools available

If we want to develop an elite F1 hybrid we need to set up abreeding pipeline utilizing a series of breeding tools together

However in Asian Seed Industry using all of the technology islimited depending on each companyrsquos capability

For that an open innovation strategy is inevitable among researchscientists in the public and private sector to share geneticresources and breeding tools (a collaboration proposal) toimprove the quality of germplasm and F1 hybrid

Thanks for your attention

CropGenomeEditing

Targetgene

Characteristics Developer

Corn ZFN IPK Herbicide R Dow

CornMega-

nucleaseundisclosed

Increasedharvest

Benson HillBiosystems

CornMega-

nucleaseundisclosed Increased

StarchAgrivida

CornCRISPR-

Cas9Wx1

Increasedamylopectin

DupontPioneer

Potato TALEN InvDecreased

reduced-sugarsCalyxt

Rice TALENOS11N3 OS8N3

BacteriaBright resistance

Iowa StateUniv

Soybean TALEN FAD2High oleic acid and

low-linolenicCalyxt

Soybean TALEN FAD3High oleic acid and

low-linolenicCalyxt

Wheat TALEN MLOPowdery mildew

resistanceCalyxt

MushroomCRISPR-

Cas9PPO

Browny color resistance

PennsylvaniaState Univ

1stgeneration ZFN 2nd TALEN 3rd CRISPR-Cas9 4th CRISPR-Cpf1

Crops that developed by genome editing technology

The common white button mushroom (Agaricus bisporus) has been modified to resist browning

The mushroom can be cultivated and sold without passing through the agencyrsquos regulatory process mdash making it the first CRISPR-edited organism to receive a green light from the US government April 17 2016

polyphenol oxidase (PPO) knock out

Ordinary B

Molecular B

New B

+

+

Stacking traitsReducing generation

F1 hybridDevelopment

Breeding Tool Box

Selectionafter FT

F1 seedproduction

QC amp QA Sales

Bre

edin

g P

ipelin

e

-Genetic sources for trait pool amp selection-Trait fixation through back-cross-DH line-Mutationhelliphelliphelliphelliphellip

-MAS MAB-Bioinformatics with omics -GWAS GBS-HT genotypinghelliphelliphelliphellip

-New plant breeding technology-Cell fusion -Genome editinghelliphelliphelliphellip

Breeding Pipeline through Technology Combination

Summary

There are new breeding tools available

If we want to develop an elite F1 hybrid we need to set up abreeding pipeline utilizing a series of breeding tools together

However in Asian Seed Industry using all of the technology islimited depending on each companyrsquos capability

For that an open innovation strategy is inevitable among researchscientists in the public and private sector to share geneticresources and breeding tools (a collaboration proposal) toimprove the quality of germplasm and F1 hybrid

Thanks for your attention

The common white button mushroom (Agaricus bisporus) has been modified to resist browning

The mushroom can be cultivated and sold without passing through the agencyrsquos regulatory process mdash making it the first CRISPR-edited organism to receive a green light from the US government April 17 2016

polyphenol oxidase (PPO) knock out

Ordinary B

Molecular B

New B

+

+

Stacking traitsReducing generation

F1 hybridDevelopment

Breeding Tool Box

Selectionafter FT

F1 seedproduction

QC amp QA Sales

Bre

edin

g P

ipelin

e

-Genetic sources for trait pool amp selection-Trait fixation through back-cross-DH line-Mutationhelliphelliphelliphelliphellip

-MAS MAB-Bioinformatics with omics -GWAS GBS-HT genotypinghelliphelliphelliphellip

-New plant breeding technology-Cell fusion -Genome editinghelliphelliphelliphellip

Breeding Pipeline through Technology Combination

Summary

There are new breeding tools available

If we want to develop an elite F1 hybrid we need to set up abreeding pipeline utilizing a series of breeding tools together

However in Asian Seed Industry using all of the technology islimited depending on each companyrsquos capability

For that an open innovation strategy is inevitable among researchscientists in the public and private sector to share geneticresources and breeding tools (a collaboration proposal) toimprove the quality of germplasm and F1 hybrid

Thanks for your attention

Ordinary B

Molecular B

New B

+

+

Stacking traitsReducing generation

F1 hybridDevelopment

Breeding Tool Box

Selectionafter FT

F1 seedproduction

QC amp QA Sales

Bre

edin

g P

ipelin

e

-Genetic sources for trait pool amp selection-Trait fixation through back-cross-DH line-Mutationhelliphelliphelliphelliphellip

-MAS MAB-Bioinformatics with omics -GWAS GBS-HT genotypinghelliphelliphelliphellip

-New plant breeding technology-Cell fusion -Genome editinghelliphelliphelliphellip

Breeding Pipeline through Technology Combination

Summary

There are new breeding tools available

If we want to develop an elite F1 hybrid we need to set up abreeding pipeline utilizing a series of breeding tools together

However in Asian Seed Industry using all of the technology islimited depending on each companyrsquos capability

For that an open innovation strategy is inevitable among researchscientists in the public and private sector to share geneticresources and breeding tools (a collaboration proposal) toimprove the quality of germplasm and F1 hybrid

Thanks for your attention

Summary

There are new breeding tools available

If we want to develop an elite F1 hybrid we need to set up abreeding pipeline utilizing a series of breeding tools together

However in Asian Seed Industry using all of the technology islimited depending on each companyrsquos capability

For that an open innovation strategy is inevitable among researchscientists in the public and private sector to share geneticresources and breeding tools (a collaboration proposal) toimprove the quality of germplasm and F1 hybrid

Thanks for your attention

Thanks for your attention