|Detail of the metabolite profile
||Hydrolysis non-polar gas chromatography mass spectroscopy (GC-MS)
|Description for analysis method
||A portion of 0.5 mL organic extract was dried under N2 and hydrolysed by adding 0.5 ml 1.25 M HCl in MeOH and incubating for 4 h at 50 ??C. Following hydrolysis, HCl and solvent were evaporated under nitrogen. The sample was resuspended in 30 uL pyridine and derivatized through the addition of 20 uL of a commercial derivatization solution containing MSTFA+1%TMCS (Pierce Biotechnology, Rockford, IL, USA). The sample was incubated for 1 h at 50 ??C to allow though derivatization. The sample was equilibrated to room temperature, transferred to a 200 uL glass insert, and analysed using an Agilent 6890 GC coupled to a 5973 MSD scanning from m/z 50?€?650. Samples were injected at a 1:1 split ratio, and the inlet and transfer line were held at 280 ?C. Separation was achieved with a temperature programme of 80 ??C for 2 min, then ramped at 5 ??C/min to 315 ??C and held for 12 min, a 60 m DB-5MS column (J&W Scientific, 0.25 mm ID, 0.25 lm film thickness) and a constant flow of 1.0 ml/min.
|Species / Cultivar / Tissue
||Medicago sativa / leaf hopper resistance line, Forage Genetics International / Trichome
|Description for biological material
||Trichomes were collected from 80 g F.W. stems of alfalfa plants and pooled together as one replicate for both metabolome and transcriptome experiments. After the first replicate samples were collected, the plants were cut back and grow for 2 weeks. Trichomes collected from these plants were the second replicate samples. And the 3rd replicates samples were collected from the cutting back of the 2nd replicate plants. The semi-quantification will be based on the amount of stems used to collect trichome. After shearing off trichomes, stems were simultaneously collected as a control for metabolite profiling.
||Don Sik Yang, Lloyd W. Sumner. Metabolic Profiling of trichomes from Potato Leafhopper susceptible and resistant alfalfa lines. (unpublished)
Plant Biology Division
The Samuel Roberts NOBLE Foundation, Inc.
Ardmore, OKLAHOMA, U.S.A - 73401.