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 Feb 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 ( )

Hydroponic verses Pot-grown - Mean leaf size (Hydroponic , Pot )

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 -40 o C 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 methiin ethiin alliin propiin

Summary of CSO results 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”

Comparison of CSO composition hydroponically grown garlic and “Solo”

To identify developmental control points for CSO synthesis Milestones May Feb 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

To isolate and characterise alliinase cDNA clones Objective: 3b To construct a cDNA library from actively synthesizing tissues Milestones May 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 Spontaneous

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: bp Number of indepentent clones –bulb 2.22x10 6 –leaf 2.28x10 6 1x10 6 clones from each library have been amplified. Aliquots stores at -80 o C in 7% DMSO

To isolate and characterise alliinase cDNA clones Objective: 3b To construct a cDNA library from actively synthesizing tissues Milestones May Feb 2001

To isolate and characterise alliinase cDNA clones Objective: 3b cDNA clones encoding alliinase isolated and sequenced Milestones Feb