omega-3: new opportunities for the canola industry - ag … · omega-3: new opportunities for the...

Omega-3: new opportunities for the canola industry James Petrie | CSIRO

IRC, Saskatoon 2015

www.nuseed.com www.grdc.com.au www.csiro.au

IRC, Saskatoon 2015 | James Petrie, CSIRO | 2

http://www.nuseed.com/

http://www.grdc.com.au/

http://www.csiro.au/

• Marine: long-chain EPA, DPA, DHA (C20, 22)

– Strong health benefits – Microalgal primary production

• Plants: short-chain ALA, SDA (C18) – Limited health benefits – Low conversion, especially to DHA

Long-chain vs short-chain omega-3?


Increasing demand from: • Ageing populations • High-growth economies • Dietary supplement

markets (especially preventative health)

• Pharmaceutical pipeline

Why long-chain omega-3?


Long-chain omega-3 supply vs demand

530

2550

1020 650

[VALUE]

1920 1920

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3500

4000

4500

5000‘0

00 to

nnes

Ocean capacity

GOED and FAO data

Japan model

Mortality model

WHO model

Rx Prescribed Intake (3550mg/day)

Supply Demand models


How do you engineer oil profile?



Genes Combinations

Crop

Plant Transformation Field

DNA Constructs


Genes Combinations

Crop


DNA Constructs

Long-chain omega-3 biosynthesis


DHA biosynthesis organisms



Genes Combinations

Crop


DNA Constructs

21st ISPL July 7-11, 2014

Rapid assessment of gene combinations

Finding the right gene combination


Finding the right gene combination

Australian National Algae Culture Collection



Genes Combinations

Crop


DNA Constructs

A sustainable, land-based platform

Canola: • High quality and healthy oil • Commercially available in

multiple regions • Canada/US/AUS >10M Ha • Decades of breeding and

commercial optimization • Efficient plant, high oil yield



Genes Combinations

Crop


DNA Constructs

• Gene orientation and spacing • Promoter strengths • Promoter-gene combinations • Timing of pathway expression • Avoiding gene silencing

• How do you test the possibilities?

Lots of possible construct designs



Rapid assessment of seed constructs

Testing seed constructs rapidly in leaf


• LEAFY COTYLEDON2 (LEC2) expression triggers embryogenesis • Co-infiltrating a seed construct with LEC2 activates seed

promoters in leaf

Seed Construct Samples

35S mix Minus LEC2 Plus LEC2

SDA 2.0 ± 0 0.9 ± 0.1 1.2 ± 0.1

EPA 0.3 ± 0 - 0.6 ± 0

DPA 2.3 ± 0 - 1.7 ± 0.1

DHA 2.5 ± 0.1 - 2.5 ± 0.2

Progressing from model species to crops

Elements • FP1: B. napus truncated napin • FAE1: A. thaliana Fatty Acid Elongase 1 • Cnl1, Cnl2: L. usitatissimum conlinin

MAR: Matrix attachment regions • TMV transcriptional leader as 5’ UTRs


Progression from research to crops

16:0 18:1 LA ALA SDA EPA DPA DHA

Arabidopsis 8.1 12.5 29.1 18.3 - - - -

- DHA (T3) 10.6 4.6 5.6 31.0 5.3 1.9 1.1 13.3%




Arabidopsis 8.1 12.5 29.1 18.3 - - - -

- DHA (T3) 10.6 4.6 5.6 31.0 5.3 1.9 1.1 13.3%

Camelina 8.0 9.8 18.1 38.2 - - - -

- DHA (T7) 8.8 4.7 8.3 27.8 5.4 6.9 1.6 13.7%




Arabidopsis 8.1 12.5 29.1 18.3 - - - -

- DHA (T3) 10.6 4.6 5.6 31.0 5.3 1.9 1.1 13.3%

Camelina 8.0 9.8 18.1 38.2 - - - -

- DHA (T7) 8.8 4.7 8.3 27.8 5.4 6.9 1.6 13.7%

B. juncea 4.5 45.0 28.1 12.2 - - - -

- DHA (T3) 6.2 12.0 10.2 26.9 3.0 2.3 1.6 17.5%


Optimising the construct for Brassica napus


Testing seed constructs rapidly in canola


• LEAFY COTYLEDON2 (LEC2) expression triggers embryogenesis • Co-transforming a seed construct with LEC2 stimulates somatic

embryogenesis • Embryos are predictive of mature seed function and profile

Stable, multi-gene construct design

Elements • FP1: B. napus truncated napin • FAE1: A. thaliana Fatty Acid Elongase 1 • Cnl1, Cnl2: L. usitatissimum conlinin

MAR: Matrix attachment regions • TMV transcriptional leader as 5’ UTRs



Genes Combinations

Crop


DNA Constructs

Germplasm selection is important

0

5

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25

Copy

num

ber i

n pr

imar

y tr

ansf

orm

ants

Germplasms

Copy number by Germplasm


Germplasm selection is important

0

5

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25

Copy

num

ber i

n pr

imar

y tr

ansf

orm

ants

Germplasms 0

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DHA

%

Germplasms

Copy number by Germplasm T0 DHA by Germplasm (1-2 copy)


Gene dosage in complex constructs

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DHA

%

Copy number by digital PCR

T0 DHA vs Copy number

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DHA

%

Seed Generation

DHA by Generation (1-2 copies)

1 2 3 T0 T1 T2


The new canola oil profile


Parent 1 5.3 44.0 18.6 22.2 - - - -

Parent 2 4.3 72.2 14.0 2.5 - - - -


The new canola oil profile: fish oil-like DHA


Parent 1 5.3 44.0 18.6 22.2 - - - -

Parent 2 4.3 72.2 14.0 2.5 - - - -

Low copy 6.0 28.1 7.0 27.5 4.6 0.5 0.8 12.3%


The new canola oil profile: plus a bit more


Parent 1 5.3 44.0 18.6 22.2 - - - -

Parent 2 4.3 72.2 14.0 2.5 - - - -

Low copy 6.0 28.1 7.0 27.5 4.6 0.5 0.8 12.3%

High copy 5.3 24.4 6.8 25.1 5.9 0.8 1.2 19.3%


The new canola oil profile: is flexible


Parent 1 5.3 44.0 18.6 22.2 - - - -

Parent 2 4.3 72.2 14.0 2.5 - - - -

Low copy 6.0 28.1 7.0 27.5 4.6 0.5 0.8 12.3%

High copy 5.3 24.4 6.8 25.1 5.9 0.8 1.2 19.3%

SDA/DHA 5.0 23.8 9.5 35.0 9.7 0.4 0.0 4.5%


The new canola oil profile: is flexible


Parent 1 5.3 44.0 18.6 22.2 - - - -

Parent 2 4.3 72.2 14.0 2.5 - - - -

Low copy 6.0 28.1 7.0 27.5 4.6 0.5 0.8 12.3%

High copy 5.3 24.4 6.8 25.1 5.9 0.8 1.2 19.3%

SDA/DHA 5.0 23.8 9.5 35.0 9.7 0.4 0.0 4.5%

Total Omega-3: 48.1% Total LC Omega-3: 16.0% Total Omega-6: 7.4% Total LC Omega-6: 0.1% Omega-3:6 ratio: 6.5 : 1 LC Omega-3:6 ratio: 135:1


DHA is targeted to TAG sn-1,3 position(s)


sn-1,3 DHA

sn-2 DHA

DHA synthesis during seed development

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d

Days after flowering

DHA

ALA Total FA


DHA canola seed lipid classes look normal

TAG

DAG

FFA

PC

PE

PA

PI

MGDG

DGDG

Non-lipid

Parent


DHA canola seed lipid classes look normal

TAG

DAG

FFA

PC

PE

PA

PI

MGDG

DGDG

Non-lipid

Parent DHA canola


Genes Combinations

Crop


DNA Constructs


Does it work in the field? Yes



• Field and glasshouse DHA levels are correlated.

• Field trials are ongoing including yield and oil content trials.


R² = 0.027

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Fiel

d Si

te 1

(% D

HA)

Glasshouse (% DHA)



1 Ha of canola at 12% is the DHA yield from 10,000 fish

Thank you

CSIRO Surinder Singh* Allan Green Peter Nichols Pushkar Shrestha Xue-Rong Zhou Nuseed Malcolm Devine Greg Buzza Wenxiang Gao Nelson Gororo Antonio Leonforte Jason McAllister

James Petrie

omega-3: new opportunities for the canola industry - ag … · omega-3: new opportunities for the...

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