Plant Science into Practice - NIAB Innovation Farm · China, India, S. Africa ... Puccinia graminis or Puccinia sorghii Susceptible interaction ... Phytate pathway mutants: Maize

Plant Science into Practice

Yield Gains

1982 – 2007, on average 74 kg ha-1yr-1

2010 – 2050, 10 t ha-1 => 20 t ha-1

= 250 kg ha-1yr-1

Cereals of TomorrowWill they be GM?

• My talk

–Where are we now?

–“Finally Wheat”, June 1999

–Why the hold-up with wheat?

–Closing remarks

Where we are now?

UK land area

Is 22 M Ha

Where we are now?

SuccessRoundup-Ready Soybean

Before RR After RR Gains and Losses

Farmer pays $25/acre for weed control

Pays $18/acre +$80m

Seed company sells 70% new crop soya

Sells 100% +$180m

Seed company makes $3 per bag of soya

Makes $5/bag +$70m

Monsanto selling no Roundup on soya

Sells 35 million litres +$300m

Monsanto – no technology fee

Makes $6.50/bag +$220m

Agchem industry – stable market in soya for selective herbicides

Market for selective herbicides devastated

-$850m

Rain Shelter Trial Corn Plot at A Monsanto Research Site

YieldGard Plus with

Roundup Ready

Corn 2

YieldGard Corn Borer with

Roundup Ready Corn 2

+ Force® insecticide

YieldGard® Corn Borer

with Roundup Ready

Corn 2

Roundup

Ready®

Corn 2

HT

Yield = 94 bu/ac

CB Protection

+HT

Yield = 113.7 bu/ac

Soil-applied RW

Protection

+CB Protection

+HT

Yield = 150 bu/ac

RW Protection

+CB Protection

+HT

Yield = 198.1 bu/ac

Gallons of Ethanol

535Pounds of Feed

3,170

Gallons of Ethanol

254Pounds of Feed

1,504

Gallons of Ethanol

307Pounds of Feed

1,819

Gallons of Ethanol

405Pounds of Feed

2,400

Positive Effects of Stress Mitigation Are

Compounded by the Stacking of Biotech Traits

Ag Biotech Driven by Lengthy Product Development

Cycle and Large Investment Process*

Year 0 1 2 4 6 8 93 5 7 10

DiscoveryGene/Trait Identification

Phase IProof of Concept

Phase IIEarly Development

Phase IVRegulatory Submission

• High

throughput

screening

• Model crop

testing

• Gene

optimization

• Crop

transformation

• Trait

development

• Pre-regulatory

data

• Large scale

transformation

• Trait

integration

• Field testing

• Regulatory

data

generation

• Regulatory

submission

• Seed bulk-up

• Pre-marketing

$2-5M(5%)

$5-10M(25%)

$10-15M(50%)

$15-30M(75%)

$20-40M(90%)

On Average:

Time to market: 8-10 years

Total expense: ~$100M

Spendin

g(P

robabilit

y o

f Success

in %

)

* Numbers (time duration, spending, and probability of success) are all estimates. The actual for individual projects could vary.

R&D TIME AND COST

Phase IIIAdvanced Development

Bt/RR2Y(Monsanto)

Low Sat(Monsanto)

Omega-3(Monsanto;

Steandonic Acid)

High

Stearate(Monsanto;

DuPont)

High Beta-

Conglycinin(Monsanto;

DuPont)

Industry Soybean Portfolio*A Steady Pipeline of New Biotech Events Nearly Every Year

Processing:

High Oil Soy(Monsanto)

High Oleic(DuPont)

Liberty Link (Bayer)

□ Agronomic

□ Quality/Food

RR2Y(Monsanto)

201X2009

Modified 7S

Protein FF (DuPont)

High Oleic, Stearate(DuPont)

Low Lin(Syngenta)

Glyphosate &

isoxazole tol. (Bayer)*

Low-

Phytate(DuPont)

Dicamba Tolerant(Monsanto)

Omega-3(EPA/DHA)

DuPont

Feed: High Protein

Soybean(Monsanto; DuPont)

Yield(Monsanto;

Pioneer)

Rust(Monsanto;

Pioneer

Antibody -

containing(against E.

coli 0157:H)

Herbicide tol.: 2,4-D (Dow) and aryloxyphenoxy

propionate herbicides

Disease(Monsanto;

Pioneer)

Soybean Cyst

NematodeMonsanto; Pioneer

GAT/Glyphosate-ALS (Pioneer)

*Estimated commercialization pipeline of soybean biotech events prepared by the American Soybean Association, November 2007.

Where we are now?

• GM crops grown on ~ 150 million hectares – 7x UK land area

– 23 countries, 95% of production in N. & S. America, China, India, S. Africa

• Dominated by – 4 crops

– 3-4 genes, < 10 transgenic events

• High development and regulatory costs

• Trait roadmap for soybean shows the way forward

Two Methods for DNA Transfer

BiolisticsI’d use this for

Functional genomicsChloroplast transformation

AgrobacteriumI’d use this for

Gene CharacterizationCommercial events

Biolistic Transformation

PSII images of wheat leaves three days after inoculation with either

Puccinia graminis or Puccinia sorghii

Susceptible interaction

(Hereward + P. graminis)

Race-specific resistance

Savannah + P. graminis

Non-host resistance

Savannah + P. sorghii

200 m

0 0.5 1.0

PSII

“Massive Attack”

600 NB-LRR and LRR-kinase candidates from riceFull gene amplicons (promoter, CDS, 3’UTR)Long range PCRs

Bombarded in sets of 10 + PMI selectable markerAt least 20 events/set, 4 clonal plants per event

Frequency of R-gene insertion

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8 9 10

Number of R-genes

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6 7 8 9 10

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7 8 9 10

Nu

mb

er

of

ev

en

ts

Number of R-genes

Number of R-genes

Nu

mb

er

of

ev

en

tsN

um

ber

of

ev

en

ts

Set A

Set B

Set C

(b)(c)

+ - 18 21 24 54 56 57 59 61 75 92 Batch 5 Event ID 12731 +/- PCR

Result

A10

20

30

40

50

60

70

80

90

100

+ - 18 21 24 54 56 57 59 61 75 92

B

10

20

30

40

50

60

70

80

90

100

10

20

30

40

50

60

70

80

90

100

Set A

Set B

Set C

Agrobacterium Transformation

SIMJT

Two Methods

© Copyright text

Plant Science into practice

Andy Greenland • 1 September 2010

• Efficiency of wheat transformation– 16-to >30% + efficiency– Continuous method improvement

• Infrastructure– Capacity of growth facilities for donor material– Tissue culture facilities– Plant growth facilities for T0 plants

• >3000 plants to seed per year, based on 3 cycles/chamber

– Seed yield• between 90-200 seed / plant / construct • secure seed storage facility

– Pipeline Management• Analysis and Quality control• Bar-code tracking systems and Oracle database

Phytic acid biosynthesis

Glucose-6-P

Ins(3)P1

InsP5

InsP6Phytic acid

protein storage vacuole

mixed phytate salts

cytoplasm

ZmMRP4

myo-inositol3-phosphate synthase

Inositol polyphosphate kinases

myo-inositolmyo-inositol kinase

Inositol monophosphatase

Pilu et al2009

Phytate pathway mutants:Maize Rice

MIPS lpa1-241 (GM)MIK lpa3 yIPK lpa22-PGK lpa1-1ABC Transporter lpa1-241, (GM) lpa2-1

RNAi – phytic acid biosynthesis

Q-PCR:1-2 copies3-4 copies> 4 copies

Actin promoter RNAi: T1 wheat seed analysis

0

500

1000

1500

2000

2500

3000

3.1

3.2

3.3

3.4

3.5

3.6 3.7 3.8

3.9

3.1

0

3.1

1

3.1

2

3.1

3

4.1

4.2

4.3

4.4

4.5

4.6

4.7

5.1

5.2

5.3

5.4

5.5

5.6

5.7

5.8

5.9

5.1

1

5.1

2

Co

n

line

ng

P/m

g fl

ou

r

Pooled T1 seed average

0

500

1000

1500

2000

2500

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

3.9

3.1

0

3.1

1

3.1

2

3.1

3

4.1

4.2

4.3

4.4

4.5

4.6

4.7

5.1

5.2

5.3

5.4

5.5

5.6

5.7

5.8

5.9

5.1

1

5.1

2

Co

n

Line

ng

P/m

g f

lou

r

Commercial Transformation

Agrobacterium delivery

1000 events per gene

Single gene insertion

No post border integration

Acceptable selectable marker (herbicide

resistance, phosphomannose isomerase, marker segregation)

Ears are inoculated with Agrobacteriumcarrying gene(s) of interestSite of

Agrobacterium delivery

Looking Forward

• Now– Routine for many species but

imprecise

– Finding the right event is labour intensive

• 2nd generation– Predictable performance

– Very high efficiencies

– Multi-gene, large inserts

• 3rd generation (Intelligent Design)– Genome engineering

– Seamless incorporation within breeding programmes

Tissue culture allows the selection and regeneration of transformed plants

HIGH

LOW

PublicAcceptance

Delivery

SLOW

FAST

Why the hold-up with wheat?

Why the hold-up with wheat?

• Uncertainties

– Technical

• Transformation efficiency – solved

• Deployment of “breakthrough genes”– Hybrids may hold the key

– Political

• Unfavourable regulatory climate/lack of commitment to change at senior levels in UK/EU government

– Despite recent Beddington/Kendall comments

• Public acceptance– Most surveys (e.g. FSA) show declining concern for GM

– See http://www.gmo-compass.org/eng/news/stories/

Wheat hybrids

• Commercial proposition–Product differentiation

• Not solely a yield target

–Platform for future delivery of novel traits• Income generation in 6-10 years

Wheat hybrids

o Production poses a number of problemso No suitable sterility mechanism

o Wheat is an inbreeder soo pollen amounts are low

o pollen is heavy and sinks

Strip versus blend planting

Breakthrough Genes

• Sustainability e.g.– Novel durable resistance to key pests (wheat

bulb fly, aphids) and diseases (rusts, Septoria, Fusarium head blight• Quicker, more accurate than alien introgression

– Drought, low N/P input (uptake and use efficiency)

• Dimension changing e.g.– Resistance to take-all in 2nd and 3rd wheat crops

Closing comments

• No remaining technology barriers to commercial transformation in wheat

• F1 hybrids can be a key delivery platform for novel GM traits

• Expect first products in feed wheat varieties as part of an integrated package?

GM Wheat Initiatives at NIAB

• Community Resources– 50 genes to be transformed over 5 years

– Outline proposal submitted to BBSRC BBRF

• Public Good GM Wheat programme– Showcasing beneficial trait genes

– Proposal to Technology Strategy Board

• In-house and collaborative research– Efficiency, multigene, large inserts, new genotypes

– Trait genes; e.g.disease resistance grain size, number and quality, N use, flowering time

The NIAB Transfomation Team

Emma Wallington

Melanie Craze

Sarah Bowden

Ruth Bates

Rhian Howells

Jan Adams