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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
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