Garlic Sulphur Biochemistry Partner 2: Horticulture Research International Laurence Trueman, Brian...
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Transcript of Garlic Sulphur Biochemistry Partner 2: Horticulture Research International Laurence Trueman, Brian...
Garlic Sulphur Biochemistry
Partner 2:
Horticulture Research International
Laurence Trueman, Brian Thomas, Linda Brown, Brian Smith, & Gareth Griffiths
Workpackage Four
To identify developmental control points for CSO synthesis
Milestones May 2000 - Feb. 2001
• Experimental material planted and harvests begun.
Objective: 2
Strategy: To profile and track sulphur compounds in leaf, leaf
bases, root and clove
• Outline of intended work
• Development of hydroponic system
• Garlic grown to maturity in test system
Report given at Dijon
Experimental material planted...
• Accessions used: Printanor and Messidrome
• Planted first two weeks of December
• Growth media: Hydroponic
• Position: Greenhouse
• Controls: Pot grown garlic – Field grown controls abandoned due to very
wet winter
Harsh Winter conditions in the UK
Growth conditions
• Two full hydroponic systems constructed
• Nutrient medium is a 1/2 strength modified Hewitts solution
Hydroponic verses Pot-grown
Hydroponic verses Pot-grown - Mean (n=3) leaf size (28-3-2001)
0
5
10
15
20
25
30
35
40
45
50
1st leaf 2nd leaf 3rd leaf 4th leaf 5th leaf 6th leaf 7th leaf 8th leaf
Leaf number
Len
gth
(cm
)
Printanor hydroponic
Printanor pot-grown
Messidrome hydroponic
Messidrome pot-grown
Hydroponic verses Pot-grown - Mean leaf size (Hydroponic 26-2-01, Pot 28-3-01)
0
5
10
15
20
25
30
35
40
1st leaf 2nd leaf 3rd leaf 4th leaf 5th leaf
Leaf number
Len
gth
(cm
)
Printanor hydroponic
Printanor pot-grown
Messidrome hydroponic
Messidrome pot-grown
Growth characteristics
• Approximately 2-3 leaves ahead - effectively 31 days
• Little morphological difference although chlorophyl content appears to differ
• Hydroponic garlic sprouted earlier than controls (Printanor 25d, Messidrome 15d)
• Due to higher temperature of hydroponic system?
...and harvests begun Sampling strategy
• At the (visable) emergence of each leaf
• 3 plants (Printanor), 2 plants (Messidrome)
• Equal number pot-grown controls
• Plants divided into leaf base and/or clove, leaf and root material
• Material stored at -40oC whilst awaiting analysis
Sample analysis• Leaf length (ruler)
• Dry weight (freeze drying)
• Soluble protein (bicinchoninic acid assay)
• Chlorophyl content (Arnon assay)
• Total nitrogen (combustion analysis)
• Total sulphur (inductively coupled plasma atomic emission spectrophotometer)
• Cysteine sulphoxide analysis using a (methanol extraction followed by quantification using HPLC)
CSO analysis
Hydroponic garlic Standards
4.271 methiin4.745 ethiin5.531 alliin6.588 propiin
Summary of CSO results
Clove 1 -% of total
Clove 2 -% of total
Clove 3 -% of total
MeanPercentage
Standarderror
Methiin 16.04 18.19 16.21 16.81 ± 1.19
Isoalliin 3.19 2.71 2.93 2.94 ± 0.24
Propiin 2.94 1.89 2.23 2.35 ± 0.54
Alliin 77.83 77.2 78.6 77.88 ± 0.70
Shop bought, hydroponically grown garlic
The two storage organs of garlic
Under extreme conditions garlic plants can form an intermediate bulbing structure
consisting of a swollen leaf base surrounding the meristem
Garlic variety “Solo”Clove 1 -% of total
Clove 2 -% of total
MeanPercentage
Standarderror
Methiin 7.39 4.42 5.91 ± 2.10
Isoalliin 16.60 18.62 17.61 ± 1.43
Propiin 4.63 6.04 5.34 ± 1.00
Alliin 71.38 70.93 71.16 ± 0.32
Comparison of CSO composition hydroponically grown garlic and “Solo”
0
20
40
60
80
100
Methiin Isoalliin Propiin Alliin
Cysteine sulphoxide
perc
enta
ge o
f tot
al
cloves
rings
To identify developmental control points for CSO synthesis
Milestones May 2000 - Feb. 2001
• Experimental material planted and harvests begun
Objective: 2
To identify developmental control points for CSO synthesis
Objective: 2
First year harvests and analysis completed.
Second year experimental material planted
Milestones Feb. 2001 - 2002
To isolate and characterise alliinase cDNA clones
Objective: 3b
• To construct a cDNA library from actively synthesizing tissues
Milestones May 2000 - Feb 2001
Thiosulphinates Pyruvic Ammonia acid
Flavour generation in Allium
S-alk(en)yl-L-Cysteine Sulphoxides
Sulphenic acids + Aminoacrylic acid
Other sulphur-containing flavour compounds
Alliinase
SpontaneousSpontaneous
Two cDNA libraries have been constructed from cv Messidrome
from RNA extracted from leaf and bulb tissue from actively growing
plants
cDNA library Statistics
• Range of insert sizes: 400-3000bp
• Number of indepentent clones– bulb 2.22x106 – leaf 2.28x106
• 1x106 clones from each library have been amplified.
• Aliquots stores at -80oC in 7% DMSO
To isolate and characterise alliinase cDNA clones
Objective: 3b
• To construct a cDNA library from actively synthesizing tissues
Milestones May 2000 - Feb 2001
To isolate and characterise alliinase cDNA clones
Objective: 3b
• cDNA clones encoding alliinase isolated and sequenced
Milestones Feb. 2001 - 2002