Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA094162

Local Sample ID:	10_18_2011 M3B
Subject ID:	SU001370
Subject Type:	Plant
Subject Species:	Corylus avellana;Corylus americana
Taxonomy ID:	13451;78632
Genotype Strain:	Corylus avellana;Corylus americana(cv Geneva)

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Subject:

Subject ID:	SU001370
Subject Type:	Plant
Subject Species:	Corylus avellana;Corylus americana
Taxonomy ID:	13451;78632
Genotype Strain:	Corylus avellana;Corylus americana(cv Geneva)

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
10_18_2011 M3B	SA094162	FL013459	460	FeEDDHA (µM)

Collection:

Collection ID:	CO001365
Collection Summary:	Cultures were sampled in triplicate on day 35 of the culture period,
Sample Type:	Plant

Treatment:

Treatment ID:	TR001385
Treatment Summary:	Filbert (C. avellana L. × C. americana Marshall cv. Geneva; Grimo Nut Nursery, Niagara-on-the-Lake, ON, Canada) shoot cultures were provided from germplasm maintained at the Gosling Research Institute for Plant Preservation (GRIPP; University of Guelph, Guelph, ON). Plantlets were grown in GA-7 vessels according to methods previously described (Garrison et al. 2013; Latawa et al. 2016). In brief, cultures were grown on semi-solid modified NCGR-COR medium (Yu and Reed 1995) supplemented with 10 g L-1 myo-inositol, 200 mg L-1 glycine, 100 mg L-1 nicotinic acid, 100 mg L-1 thiamine (PhytoTechnology Laboratories), 17.6 µM benzylaminopurine (BAP; Sigma-Aldrich), 0.014 µM indole-3-butyric acid (IBA; Sigma-Aldrich), 0.29 µM gibberellic acid (GA3; PhytoTechnology Laboratories), and 30 g L-1 glucose with 0, 230, 460 and 690 µM Fe-EDDHA (Sigma, St. Louis, MO) and the pH of the medium was adjusted to 5.7 before autoclaving at 121 ºC for 20 min. Cultures were maintained in a growth room at 23 ± 2oC with a 16 h photoperiod of 40 µmol m-2 s-1 provided by cool-white fluorescent lamps (Osram Sylvania Ltd., Mississauga, ON, Canada).

Treatment ID:

TR001385

Treatment Summary:

Filbert (C. avellana L. × C. americana Marshall cv. Geneva; Grimo Nut Nursery, Niagara-on-the-Lake, ON, Canada) shoot cultures were provided from germplasm maintained at the Gosling Research Institute for Plant Preservation (GRIPP; University of Guelph, Guelph, ON). Plantlets were grown in GA-7 vessels according to methods previously described (Garrison et al. 2013; Latawa et al. 2016). In brief, cultures were grown on semi-solid modified NCGR-COR medium (Yu and Reed 1995) supplemented with 10 g L-1 myo-inositol, 200 mg L-1 glycine, 100 mg L-1 nicotinic acid, 100 mg L-1 thiamine (PhytoTechnology Laboratories), 17.6 µM benzylaminopurine (BAP; Sigma-Aldrich), 0.014 µM indole-3-butyric acid (IBA; Sigma-Aldrich), 0.29 µM gibberellic acid (GA3; PhytoTechnology Laboratories), and 30 g L-1 glucose with 0, 230, 460 and 690 µM Fe-EDDHA (Sigma, St. Louis, MO) and the pH of the medium was adjusted to 5.7 before autoclaving at 121 ºC for 20 min. Cultures were maintained in a growth room at 23 ± 2oC with a 16 h photoperiod of 40 µmol m-2 s-1 provided by cool-white fluorescent lamps (Osram Sylvania Ltd., Mississauga, ON, Canada).

Sample Preparation:

Sampleprep ID:	SP001378
Sampleprep Summary:	Cultures were sampled in triplicate on day 35 of the culture period, accurately weighed (50 mg), and homogenized in 1 mL of 70% ethanol for 30 s (Kontes Pellet Pestle, Fisher Scientific). Samples were centrifuged (16,000 x g) for 3 min to settle particulate matter and the supernatant was filtered (0.1 µm, Ultrafree-MC filtered centrifuge tubes; Millipore, MS, USA) prior to chromatography.

Combined analysis:

Analysis ID	AN002157
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters BEH Acquity C18 (150 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Waters Micromass QTOF Premier
Ion Mode	POSITIVE
Units	Peak Intensity

Chromatography:

Chromatography ID:	CH001575
Chromatography Summary:	Extracts and 70% ethanol blanks (n=3 for each treatment) were separated using a Waters BEH Acquity C18 (2.1 X 150 mm, 1.7 µm) column with the following gradient: 0.1% aqueous formic acid:acetonitrile (0.0-25 min, 95:5-5:95 v/v, 25.01-30.0 min, 95:5 v/v). The flow rate was set to 0.25 mL min-1 for 30 mins at 30 ◦C (Waters Acquity UPLC).
Instrument Name:	Waters Acquity
Column Name:	Waters BEH Acquity C18 (150 x 2.1mm,1.7um)
Column Temperature:	30
Flow Gradient:	0.0 - 10.0 min, : 95:5-5:95 v/v, 10.0-15.0 min, 5:95 v/v, 15.0-20.0min, 5:95-95:5 v/v, 20.0-25.0min, 95:5 v/v)
Flow Rate:	0.25 ml/min
Solvent A:	100% water; 1% formic acid
Solvent B:	100% acetonitrile
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002006
Analysis ID:	AN002157
Instrument Name:	Waters Micromass QTOF Premier
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	A steady flow of leucine enkephalin (Waters 1525 HPLC binary solvent manager, 2 ng mL-1) was used as the internal standard for calibration of the Micromass LCT Premier series ToF-MS (Waters Inc.). Time of flight mass spectrometry was used with previously published optimized conditions (Brown, Murch, et al. 2012; Brown et al. 2012) including: electrospray ionization and positive ion detection in W mode, mass range of 100-1000 amu and a scan time of 0.1s. Data were collected with MassLynx V4.1 and exported via MarkerLynx. Data were processed in Excel™ to align retention times and remove multiply charged ions as described previously (Brown et al. 2012; Brown et al. 2012; Turi and Murch 2013; Turi et al. 2014).
Ion Mode:	POSITIVE