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
10
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
14
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
26
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