_A_TransientCompLPPSCPPS-GCMS

Short Name: TransientCompLPPSCPPS-GCMS Assay Class: WET Assay Type: GCMS Title: Transient expression of LPPS and CPPS genes in Nicotiana benthamiana plants Description: The purpose of this assay is to check the transient expression of the LPPS and CPPS genes in Nicotiana benthamiana plants. Two different Agrobacterium strains were assayed and compared the levels of production between them. Volatile monoterpenoid levels were measured by GCMS of samples. pISA Assay creation date: 2021-12-03 pISA Assay creator: RMF Lab manager: DO Sample collection protocol: Between 5 and 6 disks were collected 5 days postinfiltration using a 1.5-2 cm corkborer and snap frozen in liquid nitrogen. Extraction protocol: 50mg of frozen, ground leaf samples were weighed in a 10mL headspace screw-cap vial and stabilized by adding 1mL of 5M CaCl2 and 150 ćL of 500mM EDTA (pH = 7.5), after which they were sonicated for 5 minutes. Volatile compounds were captured by means of headspace solid phase microextraction (HS-SPME) with a 65 ćm polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fiber (Supelco, Bellefonte, PA, USA). Volatile extraction was performed automatically by means of a CombiPAL autosampler (CTC Analytics). Vials were first incubated at 50řC for 10 minutes with 500 rpm agitation. The fiber was then exposed to the headspace of the vial for 20 min under the same conditions of temperature and agitation. Desorption was performed at 250řC for 1 minute (splitless mode) in the injection port of a 6890N gas chromatograph coupled to PEGASUS 4D mass spectrometer (LECO). After desorption, the fiber was cleaned in a SPME fiber conditioning station (CTC Analytics) at 250řC for 5 min under a helium flow. Chromatography protocol: Chromatography was performed on a BPX-35 (30 m, 0.32 mm, 0.25 ćm) capillary column (SGE) with helium as the carrier gas at a constant flow of 2mLxmin-1. The oven conditions started with an initial temperature of 40řC for 2 min, 5řCmin-1 ramp until 250řC, and a final hold at 250řC for 5 minutes. Mass spectrometry protocol: Electron impact ionization (EI), 70 eV ionization energy, m/z range 35-300 at 20 scans/s. Phenodata: ../../phenodata_20200720.txt Featuredata: Creation date: 2020-07-20 Extract ID: $_extr Extraction Method: HS-SPME Date Extraction: 2020-07-20 Derivatization or Labelling: none Date Derivatization or Labelling: 2020-07-20 Derivatized or labeled Extract ID: $_extrD Other Post Extraction Procedures: Storage: Date GC-MS Run: 2020-07-20 GC Instrument: 6890N gas chromatograph (Agilent Technologies) GC Autosampler Model: CombiPAL autosampler (CTC Analytics) GC Column model: BPX-35 (30 m, 0.32 mm, 0.25 ćm) capillary column (SGE) GC Column type: capillary column Guard Column: MS Scan polarity: positive MS Scan mz range: 35-300 MS Instrument: PEGASUS 4D mass spectrometer (LECO) MS Ion source: electron ionization (EI) Mass analyzer: time-of-flight Operator: RMF Notes: Chromatograms were proccesed by means of the ChromaTOF software (LECO). Quantification was made by integrating peak area for the corresponding ion. Ions selected for each compound were the following: QI 69 for lavandulol, QI 81 for chrysanthemol, QI 41 for artemisia alcohol, QI 43 for santolina/yomogi alcohol, QI 43 for santolinatriene/isomyrcenyl acetate. Data: 10.5281/zenodo.6554066