SE134:/S2/M1

Sample Set Information

ID	SE134
Title	Unbiased profiling of volatile organic compounds in the headspace of Allium plants using an in-tube extraction device.
Description	Plants produce and emit important volatile organic compounds (VOCs), which have an essential role in biotic and abiotic stress responses and in plant–plant and plant–insect interactions. In order to study the bouquets from plants qualitatively and quantitatively, a comprehensive, analytical method yielding reproducible results is required. We applied in-tube extraction (ITEX) and solid-phase microextraction (SPME) for studying the emissions of Allium plants. The collected HS samples were analyzed by gas chromatography–time-of-flight–mass spectrometry (GC-TOF–MS), and the results were subjected to multivariate analysis. In case of ITEX-method Allium cultivars released more than 300 VOCs, out of which we provisionally identified 50 volatiles. We also used the VOC profiles of Allium samples to discriminate among groups of A. fistulosum, A. chinense (rakkyo), and A. tuberosum (Oriental garlic). As we found 12 metabolite peaks including dipropyl disulphide with significant changes in A. chinense and A. tuberosum when compared to the control cultivar, these metabolite peaks can be used for chemotaxonomic classification of A. chinense, tuberosum, and A. fistulosum.
Authors	Kusano M, Kobayashi M, Iizuka Y, Fukushima A, Saito K.
Reference	BMC Res Notes. 2016 Feb 29;9:133. doi: 10.1186/s13104-016-1942-5.
Comment

PubMed

Sample Information

ID	S2
Title	Rakkyo
Organism - Scientific Name	Allium chinense G.Don
Organism - ID	NCBI taxonomy 130426
Compound - ID
Compound - Source
Preparation	Ten Allium (A.) fistulosum species, six spring onion cultivars, two scallions, and two Japanese-leek cultivars, rakkyo (A. chinense) and Oriental garlic (A. tuberosum), were purchased from a grocer in Kawasaki, Japan or harvested in a Japanese field (see Table 1 and Additional file 2). After removing the roots, a 10-cm length of the sheath and the basal plate of each plant sample were collected and chopped with stainless steel surgical blades (Feather, Tokyo, Japan). Out of the A. fistulosum cultivars, four were grown by applying a method (hilling) similar to that used for growing the leek A. ampeloprasum var. porrum to obtain longer white stems for consumption in Japan (Fig. 1f, g, h, l). Each sample was immediately frozen in liquid nitrogen and kept at −80 °C until use. As the group of samples of Mikata spring onion (class01) was gathered center of the PCA score scatter plot (Fig. 4), this cultivar was chosen as the control.
Sample Preparation Details ID
Comment

Table 1
Allium species used in this study

Class in PCA	Binomial name	Species name	Bland name	Harvested field in Japan
08	Allium chinense	Rakkyo	Young rakkyo	Namegata,Ibaraki
01	Allium fistulosum	Spring onion	Mikata spring onion	Hamamatsu, Shizuoka
02	Allium fistulosum	Green spring onion	Aoi-chan green spring onion	Akitakata, Hiroshima
03	Allium fistulosum	Scallion	Hakata scallion	Hakata, Fukuoka
04	Allium fistulosum	Green spring onion	Green spring onion from Nagareyama chiba	Nagareyama, Chiba
05	Allium fistulosum	White spring onion	White spring onion from Nagano	Nagano
06	Allium fistulosum	Leek	Shimonita leek	Gunma
07	Allium fistulosum	Leek	Shirakami leek	Noshiro, Akita
09	Allium fistulosum	Scallion	Kujo scallion	Nagahama, Gifu
11	Allium fistulosum	Spring onion	Goudo spring onion	Anpachi, Gifu
10	Allium tuberosum	Oriental garlic	Oriental garlic	Nagahama, Shiga
12	Allium fistulosum	Red spring onion	Red spring onion	Tsuruoka, Yamagata

Analytical Method Information

ID	M1
Title	GC-TOF MS
Method Details ID	MS1
Sample Amount	500 μl
Comment

Analytical Method Details Information

ID	MS1
Title	GC-TOF MS
Instrument	Pegasus III TOF mass spectrometer (LECO)
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	Sample preparation procedure The samples were crushed into powder (2 min at 4 °C) in a Mixer Mill MM 311 instrument featuring a grinding jar with a stainless steel screw cap (Restech, Tokyo, Japan) and the frozen powder from each sample (flesh weight, 1 g) was weighed in a 20-ml HS vial (Supelco, MO, USA). For VOC profiling of Allium plants we used a modified method of Tikunov et al. and Kusano et al. Briefly, the 20-ml HS-GC vial (Supelco) containing the frozen powder was closed with a magnetic screw cap (AMR, Tokyo, Japan) for ITEX- and SPME-analysis. Then, 1 ml of 100 mM 2,2′,2′’,2′’’-(ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA)- NaOH water solution (pH 7.5) was added to each vial; the water derived from an Allium sample was considered to be equal to 1 ml. After vortexing, 10 μl of solution containing n-decane (d 22 , 99 %; 50 μM), n-pentadecane (d 32 , 98 %; 50 μM), n-eicosane (d 42 , 98 %; 50 μM) for definition of RI and EPA524.2 fortification solution (20 μg/ml of fluorobenzene, 4-bromofluorobenzene, and 1,2-dichlorobenzene-d4) as ISs was mixed in methanol, then solution was added to each vial as IS. Solid CaCl2 was added to obtain a final concentration of 5 M and the samples were stored overnight at 22 °C. HS collection using the SPME fiber The SPME device for a CTC CombiPAL auto-sampler (CTC Analytics, Zwingen, Switzerland) was purchased from AMR (Tokyo, Japan). We used an SPME fiber comprised of a 65-μm-thick layer of polydimethylsiloxane (PDMS)/divinylbenzene (DVB)-fused silica (FS) fiber/stainless-steel (SS) tube. Before analysis, the fiber was conditioned at 250 °C for 30 s in the injection port of an Agilent 6890 N gas chromatograph (Agilent Technologies, Wilmington, USA) equipped with a 30 m × 0.25 mm inner diameter fused-silica capillary column with a chemically bound 0.25-μl film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA). Collection of volatiles was carried out by inserting the SPME-fiber to the vial and by trapping the VOCs for 20 min at 80 °C under continuous agitation. After HS collection it was placed in the injection port of the gas chromatograph that was coupled to a Pegasus III TOF mass spectrometer (LECO, St. Joseph, USA). The thermodesorption of VOCs occurred for 15 s at 250 °C. HS collection using the ITEX device We used a CTC CombiPAL auto-sampler (PAL COMBI-xt) featuring the ITEX device PAL ITEX-2 option (CTC Analytics). The ITEX procedure was controlled with a PAL Cycle Composer (CTC analytics). We conducted preliminary experiments to choose an appropriate sorbent material from the four materials, Tenax TA, Tenax GR (TGR), Carbosieve SIII (CSIII) and mixed TGR and CSIII (TGR/CSIII), that are commercially available (data not shown). Then, we chose that the sorbent material for the ITEX-2 portion was TGR (80/100 mesh)/CSIII (60/80 mesh). The parameters for HS collection were as described in the Additional file 2. After HS collection, 500 μl of the HS sample were injected into the injection port of the gas chromatograph coupled to the mass spectrometer used for HS collection by ITEX. GC-TOF–MS analysis GC-TOF–MS conditions were as described in the Additional file 2. Data acquisition was on a Pegasus III TOF mass spectrometer (LECO); the acquisition rate was 30 spectra/s in the mass range of a mass-to-charge ratio of m/z = 30–550. Five ISs were used for data normalization.
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