Hormone interplay in the regulation far-red-responsive stem elongation in tomato 4 149 Table 4.2. Chemicals information used in this chapter. Molecular weight CAS Stock solution Working solution GA3 346.4 77-06-5 0.2 M 25, 50, 100 μM IAA 175.2 87-51-4 0.1 M 5, 10, 25, 50, 100 μM Brassinolide 480.7 72962-43-7 0.1 M 25, 50, 100 μM Naphthylphthalamic acid (NPA) 291.3 132-66-1 0.1 M 25, 50, 125, 250 μM 4-Biphenylboronic acid (BBo) 198.0 5122-94-1 0.1 M 100, 200 μM PEO IAA 293.3 6266-66-6 0.1 M 25, 50,100 μM Paclobutrazol 293.8 76738-62-0 0.5 M 25, 50,100 μM Brassinazole 327.8 280129-83-1 0.5 M 25, 50,100 μM 4.4.5 Microscopy Handmade sections were obtained from the middle of internode, then they were stained with Safrinin O (0.01% w/v) for 5 minutes and put back into 70% ethanol and imaged using a Leica M205 fluorescence microscope. The collected images were measured using ImageJ (Schindelin et al., 2012) . The cell length data were obtained from longitudinal sections, cell layer number/thickness data were obtained from cross sections. We measure cross section for at least three angles for these features. 4.4.6 Extraction and Quantification of IAA through Liquid Chromatography-Tandem Mass Spectrometry To extract IAA from leaf and internode, approximately 20 mg of snap-frozen leaf material was used per sample. The tissue was finely powdered at -80°C by manual grinding. The powdered samples were subjected to extraction using 1 mL of cold methanol containing [phenyl 13C6]-IAA (at a concentration of 0.1 nmol/mL) as an internal standard, following a previously established protocol. After extraction, the samples were filtered using a 0.45 mm Minisart SRP4 filter and analyzed on the same day. The IAA content was assessed using a Waters Xevo TQs tandem quadrupole mass spectrometer, as detailed in previous descriptions from literature (Küpers et al., 2023). 4.4.7 RNA extraction and cDNA synthesis In the first qPCR experiment plants were either placed in WL or in R/FR=0.2. In the second qPCR treatment plants were either treated with GA, IAA, or GA + IAA, BR, IAA+GA+BR and placed in WL or treated with a mock and placed in either WL or R/FR = 0.2. We used four biological replicates, consisting of 8-12 plants, for one biological replicate,
RkJQdWJsaXNoZXIy MTk4NDMw