Craig Wood | CSIRO Plant Industry

Development of super high oleic oil in safflower

ISPL2012 SEVILLE

Overview What is super high oleic (SHO) oil?

why is SHO a possible industrial oil

Molecular basis of high oleic (HO) safflower

a natural mutation

Molecular basis of further increases in oleic content

use of hairpin RNAi and genetic engineering

Can safflower produce SHO?

What is super oleic oil?

• near pure oleic acid • ~95% oleic acid as a target

• no polyunsaturates • PUFA are reactive • Reduce chemical synthesis efficiencies

• reduced saturates • Increased flux towards 18:1

Triolein

Triolein

SHO is an industrial oil

SHO can be used either:

• Directly →

• as a lubricant and heat-transfer fluids

• Biodegradable

• Stable and liquid at ambient temperatures

• Indirectly →

• processed into azelaic acid (a C9 dicarboxylic acid)

• ‘crack’ 18:1 into two C9 molecules

• Extra double bonds ruin this reaction

• Cosmetics

• Specialized plastics

Why attempt SHO in safflower in Australia?

• Safflower has a simple oil profile

• 18:1, 18:2, 16:0, 18:0

• Non-photosynthetic seed

• Closed flowerhead structure

• Preferred structure

• Not widely grown in Australia

• Not a food crop

• GM segregation is well-established in Australia

• GM-cotton and GM-canola as precedents

• SHO is an easier GM trait for regulatory approval

• SHO is a ‘normal’ oil (18:1) but at very high purity

Why SHO in safflower in Australia?

• Oleic acid is also the starting substrate for other valuable industrial oils and waxes

• Sustainable/renewable bio-economy is a strategic goal for an Australian bio-economy

• Bio-based production of materials and chemicals

• Sustainable plastics from ‘fresh’ oils

• GM-proteins

• Antibodies

• Silk proteins

High oleic safflower

• Low oleic safflower contains about 20% oleic acid

• High-oleic safflower lines have around 75% oleic acid

• Remainder is linoleic (18:2) and saturates (16:0)

• What genes are changed in safflower to create high oleic oil?

• What genes remain unchanged that could contribute to the remaining linoleic acid?

70 20 10 Low Oleic

15 75 High Oleic

Linoleic Oleic Saturates Linolenic

10

Molecular basis of high oleic safflower

• Extensive EST library screening in seeds and vegetative tissue of both low oleic and high oleic genotypes

• Deep sequencing of RNA populations in low oleic and high oleic genotypes

• Biochemical characterisation of candidate genes

70 20 10 Low Oleic

15 75 High Oleic

Linoleic Oleic Saturates Linolenic

10

Gene expression analysis in developing seed of safflower

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At least 11 FAD2 genes in safflower confirmed using southern blots

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Only 3 safflower FAD2 are 18:1 desaturases Others have divergent activities

Only FAD2-1 expression different confirmed using qRT-PCR

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Molecular basis of high oleic safflower

• FAD2-1 genes were sequenced from low oleic safflower and three high oleic varieties (S-317, LeSaf-496 & CW99-OL).

5’ 3’

HIS-1 HIS-3 HIS-2 Low Oleic FAD2-1

HIS-1 HIS-2 High Oleic fad2-1

• All three high oleic varieties had the same mutation in fad2.1

• High oleic safflower first described by Horowitz and Winter (Nature, 1957)

380 aa

Frame shift mutation and dysfunctional 18:1 desaturase

Functional 18:1 desaturase

Can super-high levels of oleic acid be produced by silencing CtFAD2-2? and CtFATB?

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• Large family of FAD2 in safflower • Some expression in seeds is detectable

• Some cross-silencing allowed for in this hairpin design

RNAi-based silencing FAD2-2 and FATB genes in safflower seed

AtOleosin FATB FAD2-2

FATB FAD2-2

hpRNAi-CtFAD2.2-CtFATB

• Specialist technique developed for safflower genetic transformation • Belide et al 2011 (Plant Methods)

• GM shoots are rescued via micro-grafting

• 40 independent lines generated

• Data for elite lines presented here • Mature T3 seed

High oleic safflower transformed with seed-specific hpRNAi-CtFAD2.2-CtFATB

Increasing 18:1 content from 78% to 94%

18:0

16:0

18:1

18:2

HO safflower

SHO safflower

Fatty acid - % of total

18:1 78

18:2 15

16:0 7

18:0 1

Fatty acid - % of total

18:1 94.5

18:2 2.5

16:0 2.0

18:0 1

HO safflower +hpRNAi

High-oleic oils Where does SHO safflower oil rank?

94% SHO Safflower

Linoleic Oleic Saturates Linolenic

HO Soybean 77%

HO Canola 80%

82% HO Sunflower

• Highest oleic

• Lowest linoleic

• Lowest saturates

• Zero linolenic

75% HO Safflower

RA 80% HO Castor

Molecular basis of high and super high oleic acid oils in safflower • Safflower has a large number of FAD2 genes

• Some are classic 18:1 desaturases

• FAD2-1 is mutated in high oleic safflower varieties

• Seed-specific silencing of CtFAD2.2 and CtFATB results in over 94% oleic acid in safflower

Super high oleic safflower trait developed

No developmental drawbacks yet seen in SHO lines…..

Industrial products derived from SHO High value industrial traits

SHO 18:1

RA

DHSA

– C – C – C –

OH

–

– C – C –

C

hydroxylated

cyclic

WAX

ERUCIC

Thank you

CSIRO Plant Industry

Craig Wood

t + 61 2 6246 5065 e craig.wood@csiro.au

ISPL2012 SEVILLE SPAIN

Qing Liu Shijiang Cao Xue-Rong Zhou Stuart Stephan Surinder Singh Allan Green