Designing plants with novel traits using a non-GM methodManash ChatterjeeBenchBio Pvt Ltd.

Competition between breeders, is not any more limited by access to germplasms

Tools for allele engineeringThe identity of the genes controlling the trait

Plant Breeding

Candidate genes

Leader alleles

PhenotypingMutant Pops

Translational research in cropsNovel Allele Discovery (Bench Bio)

3

Sequence Phenotype

Induced Alleles

TILLING:

Engineering Leader Alleles

TILLING

EMS lead to: ~ 100% G:C to A:T transitions

5 % Truncations

64 % Missense changes

31 % Silent mutations

Can Saturation Mutagenesis be an alternative to site directed mutagenesis?

in average, 71 to 81% of the amino acids of the proteins could be mutated.

SAMA57

PLP

Saturation mutagenesis in tomato requires a population of about 100 000 M2 families

SAMA57

PLP

EMS lead to: ~ 100% G:C to A:T transtions

5 % Truncations

64 % Missense changes

31 % Silent mutations

Can Saturation Mutagenesis be an alternative to site directed mutagenesis?

Targeting Induced Local Lesion in Genome (TILLING)

A novel technology that allows rapid selection of mutants

from any gene in any organism

The initial development and testing used Arabidopsis

TILLING now applied to all crops and organisms such as

mouse, fish, flies and worms

Public and Private labs worldwide now engaged in

TILLING

Takes advantage of large amounts of sequence information

available in public databases

Giovannoni J J Plant Cell 2004

Translational research in crops, engineering leader alleles

BBIO FACILITIES

Mutant Population Generation- A specialised activity

CROP TILLING PLATFORM-MUTANT LIBRARIES

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Tomato-Range of phenotypes observed

1Chantreau et al. (2013) BMC Plant Biol.

*

Phenotyping and TILLinG

in Flax (PT-Flax)

‘Tilled’ lignin mutant (cad)

Flax EMS mutant Population-50 to 100 mutants per gene1

1/41kb- a record in diploid

DNA extraction- M2 families

M2 seeds

8 seeds

3 weeks

Taking away

Check on agarose gel (1%)

Qiagen

Dneasy 96 plant kit® gDNA

extraction

EMS

POOL plates

-600 bp

-500 bp

-370 bp

-230 bp

-100 bp

LICOR Gel

ENZ1PCR

PCR amplification

And barcoding

TILLING SUMMARY

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15

Kumar,APK et al,2017,Mol.Breed

INRA-KSU - May 2011

POC: TILLING CANDIDATE GENES IN TOMATO

80 genes in pipe to be screened on

tomato

by TILLING

Carotenoids

contents

Flavonoids

contents

Shelf

life

Fruit colours

Fruit

shape

Fruit

ripening

Architectures

Parthenocarpy

Light

signaling

Brassenosteroids

GA

signalling

Fruit

texture

ACO, ARF13, ARF2b, ARF5, Bli2, COP1, COP10,

COP1like, CRTISO, Cry1a, Cry1b, Cry2, CUL4,

CYP85A3, DELLA, DHS, E8, eIF(iso)4E, eIF4E1,

eIF4E2, eIF4G, FPB5, FRK3, SlDET1, LeDDB1,

LeeIF(iso)4G, LeExp1, LeGR, Lin5, LsMet1, Nam,

PCNA, PG, Rab11, ScE2a, SGNU327258, Sl11F2,

Sl42B19, SlBAK1, SlBRC11, SlBRC12,

SlGID2178, SlGID2277, SlIAA9, SlLAX, SlNAC,

SlNOR, SlPsbS, SlPSKR1, SlTCP3, SlUVI4,

SlWIP, SuSy, ScE2b, SlGRIK, CSN5a, CSN5b,

……………..

NOR : No Ripening

Nac domain protein Long shelf life (LSL) tomato mutants

M82-nor3

M82-nor1

NORmutants

M82 control

M82-nor2

Fruits scored 30 days after harvest

Fruit shelf life: TILLING the NOR gene

increase the carotenoids, flavonoid and lycopene contents in tomato fruits by targeting the Hp2/Det1 complex

Lycopene: antioxidant that reduce the risk of heart attack & prostatecancer

carotenoids and flavonoids: reduce risk of coronary heart disease, certaincancer and age-related diseases

Carotenoid levels in mature green fruits

TILLING Hp2/DET1 in tomato

Pyramiding of TILLING alleles

***

Fruit Firmness

*

Q213Stop

nor KO M82 Control

NOR

EXP

DET1

Disease resistance-Potato virusY on Tomato

Infected Non-infected

VPg : virus-encoded genome linked protein

P1 HC-Pro P3 CI Nia Nib CPVPg AAAAAAA

Translation initiation factor eIF4E

•eIF4E plays an important role in initiation of translation

•CAP binding protein

eIF4E

eIF4G

PABP

5´CAP

3´polyAAAAAA

40S

eIF3

eIF4F

Eukaryotic initiation complex

P1 HC-Pro P3 CI Nia Nib CPVPg AA

VPg

Translation initiation factors: kingpin in plant resistance to RNA viruses

•mo1/ LMV/ Lettuce

•pvr2/ PVY/ Pepper

•sbm1/ PsBMV/ Pea

•rym/BaYMV/ Barley

•Nsv/MNSV/Melon

•Zym/ZYMV/Watermelon

Induced mutations in translation initiation factors:

342pb 822pb 665pb

eIF4E1

342pb 822pb 665pb

eIF4E1

Wild type Sl-eIF4E1 cDNA

Sl-eIF4E1 G1485A cDNA

Exon 1

A

GU AG

Exon 2 Exon 3 Exon 4 Exon 5

Exon 1 Exon 4 Exon 5

Exon 1 Exon 2 GU AG Exon 3 GU AG Exon 4 Exon 5GU AG

MW

730 bp450 bp

1 kb

0.5 kb

G1485A Hm-WT G1485A Hm-WT

Wild type Sl-eIF4E1 cDNA

Sl-eIF4E1 G1485A cDNA

Exon 1

A

GU AG

Exon 2 Exon 3 Exon 4 Exon 5

Exon 1 Exon 4 Exon 5

Exon 1 Exon 2 GU AG Exon 3 GU AG Exon 4 Exon 5GU AG

Wild type Sl-eIF4E1 cDNA

Sl-eIF4E1 G1485A cDNA

Exon 1

A

GU AG

Exon 2 Exon 3 Exon 4 Exon 5

Exon 1 Exon 4 Exon 5

Exon 1 Exon 2 GU AG Exon 3 GU AG Exon 4 Exon 5GU AG

MW

730 bp450 bp

1 kb

0.5 kb

G1485A Hm-WT G1485A Hm-WT

Courtesy: Dr A. Bendahmane, IPS2

TILLING Translation Initiation Factors

342pb 822pb 665pb

eIF4E1

342pb 822pb 665pb

eIF4E1

eIF4E1 Splicing mutant

JO55

WT

e1858Mutant

eIF4Eiso

eIF4E2

e597

Growth rescue of a yeast straindepleted for eIF4E

The eIF4E1 splicing mutant is impaired in cap-binding activity & mRNA translation in yeast

The splicing mutant is immune to PVY (PotatoVirus Y) and PepMoV(Pepper Mottle Virus)

PVY LYE 90V PepMoV

ELISA test:

Courtesy: Dr A. Bendahmane, IPS2

Virus induced gene silencing of SlMlo1 causes enhanced PM resistance, Bai et al, 2008

Example- Powdery Mildew

MLONOR

EXP1

DET1RIN

SGR

CNN

ACO

SP

FT

Other Traits-TILLING key tomato genes in melon

Giovannoni J J Plant Cell 2004

© copyright BenchBio, 2010

Gene A

Gene B

Gene C

Seedless fruits

Forward Genetics-Phenotyping

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Phenotyping M2 families

Seedless tomatoes Full flesh tomato

Phenotyping M2 families

Clone gene later

Forward Genetic Screening for virus resistance

Clone gene later

• BenchBio is set up to do science based contract research and service in plant biotechnology

• Specialising in creating high quality large size Mutant Populations

• Specialising in field phenotyping for novel traits and cloning

• Specialising on discovering induced alleles from plants- mutant-allele discovery platform, M-TILLING/TILLING

• Specialising on discovering natural alleles from plants- natural allele discovery platform, N-TILLING/Eco-TILLING

• Focussed on genes for agricultural and consumer value

• Working on Food, Feed as well as Non-food crops

Summary

Advantages- M-TILLING & N-TILLING

• Non-GMO (non-transgenic)- Work directly in

crops

• Yields a spectrum of alleles

• High throughput and relatively low cost to product

• Significantly less expensive than transgenic

• Less regulatory hurdle to market- 3-4 years faster

in some cases

Mutant population creationR&D SERVICES

Mutant Screening

(TILLING & EcoTILLINGusing enzyme )

Tilling by Illumina

Phenotyping

Upto 100,000 M1 plants

Upto 100,000 M2 families

CROPS:

Field Crops

Ornamentals

Vegetables

Fruit Crops

Medicinal Plants

Biofuel Crops

BBIO Research Services

CRISPR-Promising regulatory passage

Modification of ARGOS8 for higher yield under drought Polyphenol Oxidase (PPO) Knock out / less browning

CRISPR craze & Patent conflicts

USPTO

Interference Proceeding

WON!

CRISPR-Cas9: PATENT CONTROL AND ACCESS & COST

Thanks!

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Contact: mchatterjee@benchbio.com