Freezone 2.5 l freeze dryer

The chlorophyll content index (CCI) of the functional leaves was measured at 1 to 7 DOR and 1 to 7 DOR using a SPAD (soil and plant analysis development) analyzer (SPAD-502 Chlorophyll Meter, Konica Minolta, Tokyo, Japan). The relative CCI of each leaf, represented by the SPAD value, can be used to study the effect of waterlogging on leaf yellowing in cowpea genotypes associated with nitrogen remobilization and leaf senescence. Three readings were collected from each cowpea genotype’s top-most fully expanded trifoliate and averaged.
Five representative cowpea plants (from each treatment/replications/genotype) were harvested on 3 DOW, 7 DOW, 3 DOR, and 7 DOR to obtain growth data on the effects of waterlogging stress. The plant component, fresh mass (FM), was measured using a weighing scale from all plants. Plant FM samples were lyophilized using a FreeZone 2.5 L freeze dryer (Labconco Corp., Kansas City, MO, USA) to determine the dry mass (DM) and percent dry mass (%DM). The cowpea’s relative water content (RWC) was determined as per the method of Barrs and Weatherley [69 (link)] with minor modifications. The RWC value is estimated as ((FM − DM/TM − DM) × 100). TM is the turgid mass, determined by soaking the FM of five replicated plants per treatment per genotype in distilled water and then obtaining the weight after 24 h.

Thymus, liver, spleen, heart, lung and tumour tissues isolated from the NSD and HSD groups were weighed (wet weight), frozen at −80 °C for 24 h and then freeze-dried using a Labconco FreeZone 2.5 L freeze dryer (Table Model, USA) for 48 h (dry weight [DW]). The difference between the wet weight and DW was tissue water content. We then put the tissues in 50% HNO₃ for 48 h and incubated them at 190 °C for 12 h. Finally, we dissolved the tissues in 1% HNO₃ and examined Na⁺ and K⁺ content by atomic adsorption spectrometry (HITACHI180-80) and Cl⁻ content by titration with 0.1 N silver nitrate (Model Titrando, German Metrohm).

Six representative cucumber plants (from each treatment/rep/cultivar) were harvested at the end of the 10-day experiment to obtain morphological performance of the impact of waterlogging stress. Plant’s phenotypic data of leaf number (LN), leaf area (LA), and leaf fresh mass (FM) were evaluated for each treatment combination. LA was measured using the LI-3100 leaf-area meter (Li-Cor Bioscience, Lincoln, NE). Plant component FM was measured from all plants by using a weighing scale. The sample of the plant FM was lyophilized using a FreeZone 2.5 L Freeze Dryer (Labconco Corp., Kansas City, MO, United States) to determine the DM and percent dry mass (%DM).

GSLs were extracted as previously reported [26 (link),28 (link)]. The plant material was divided into root, stem, leaf, and flower for Sisymbrium officinale and root, stem, and siliquae for S. orientale. The samples were freeze-dried using FreeZone 2.5 L freeze-dryer (Labconco, Kansas City, MO, USA) and ground to a fine powder, from which 100 mg were extracted for 5 min at 80 °C in 2 × 1 mL MeOH/H₂O (70:30 v/v) to inactivate the endogenous myrosinase. DEAE-Sephadex A-25 anion-exchange resin (10 g, GE Healthcare) was mixed with 125 mL of ultrapure water, and the resulting mixture was stored in a refrigerator (4 °C). Each extract (1 mL) was loaded onto a mini-column filled with 0.5 mL of DEAE-Sephadex A-25 anion-exchange resin solution (1 cm height × 0.5 cm diameter) and conditioned with 25 mM acetate buffer (pH 5.6). After washing the column with 70% MeOH and 1 mL of ultrapure water, the optimal conditions for desulfation were set by adding a buffer solution. Each mini-column was loaded with 20 μL (0.35 U/mL) of purified sulfatase and left to stand for 18 h at room temperature. The desulfoGSLs were then eluted with 1.5 mL of ultra-pure H₂O, lyophilized and diluted to 1 mL. The samples were stored at −20 °C until further analysis by HPLC-DAD-MS/MS.