Project Title: Postharvest stress manipulation in Tenderstem® broccoli – Examining the potential for stress-adaptative responses to promote sustainable quality retention
Data collected 01/06/2023 - 28/02/2024
Ewan Gage, Research Fellow, Plant Science Research Group, Cranfield University 
Funding for this work was provided through the Horticulture Quality and Food Loss Network under Biotechnology and Biological Sciences Research Council grant BB/T010819/1 and Engineering and Physical Sciences Research Council project EP/V042548/1

Methods
2. Materials and methods
2.1. Plant Material and Postharvest Treatments
Tenderstem® broccoli florets cut to commercial standards (15 – 20cm) were sourced from a commercial grower (Sussex, United Kingdom) were received within 24 h of harvest on 8th of August 2022. Following selection for uniformity of size, colour, and maturity, florets were submerged in solutions of i) 0, 10, 100 and 1000 µmol L-1 MeJA with 0.1% Tween-20 (v/v) or ii) 2.5, 5 and 7.5 mmol L-1 H2O2 in deionised water, or iii) a deionised water control  for 1 h, before air drying at 20°C for 3 h, adapting the approaches of Duarte-Sierra et al. (2022) and Luo et al., 2018) . Florets were then stored at 5 and 10 °C for 8 days in darkness at 95% relative humidity. Each replicate consisted of four florets and were assessed in triplicate. Samples of flower buds of consistent maturity were collected every 48 h, and were snap frozen with liquid nitrogen. Samples for RNA analysis were maintained at -80°C until extraction, whilst samples for biochemical analysis were freeze dried at -50 °C under vacuum before powdering and storage at -40 °C. 

2.2. Colour analysis
Florets were imaged using a Lumenera Infinity 3-9UR high-definition digital camera with charge-coupled device colour sensor (Lumenera Corporation, ON, Canada). Samples were photographed inside a Photo-e-Box plus 1419 set at D65 (6500 K) (Or-tec, California, USA) under fluorescent light tubes (back and sides) to provide consistent lighting. Image analysis was conducted using ImageJ software (version 1.53t, National Institutes of Health, Bethesda, USA) according to Schneider et al. (2012). RGB values were determined for three 2 cm diameter circles per floret (Figure 1). RGB values were converted into CIELab colour space prior to determination of hue angle (°h = arctg (b*/a*)) and chroma (C* = (a*2+b*2)1/2) according to Voss (1992). 

2.3. Pigments
Chlorophyll a (Chla), chlorophyll b (Chlb) and total carotenoids were quantified in triplicate following a modification of Wellburn (1994). Freeze-dried powder (50 mg) was extracted using 4.5 ml 80% (v/v) acetone on ice in darkness for 15 min before centrifugation at 8000 x g for 10 min at 4 °C. Absorbance was recorded at 663 nm, 645 nm and 470 nm using an Infinite 200 Pro plate reader (Tecan Austria GmbH, Grödig, Austria) with five reads per well. Quantification was made as follows: Chla = 12.25A663 – 2.79A645; Chlb = 21.50A645 – 5.10A663; Total Carotenoids = (1000A470 – 1.82Chla – 85.02Chlb)/198.

2.4. Organic acids
Citric, maleic and ascorbic acids were quantified following Crespo et al.  (2010) with some modifications. Freeze-dried powder (150 mg) were extracted in 3% metaphosphoric acid for 10 min on ice before filtration (0.2 µm polypropylene filters, Millipore, UK). Extracts were analysed by High Performance Liquid Chromatography (HPLC) fitted with diode array detector (DAD, 1200 Series, G1315B, Agilent Technology, UK) set at 210 nm. Separation was achieved using a Prevail organic acid column, 4.6 mm x 250 mm, 5 µM particle size (Hichrom, UK) using 25 Mm KH2PO4 adjusted to pH 2.5 with a metaphosphoric acid mobile phase. Extracts were quantified using external standards (Sigma-Aldrich, UK). 

2.5. Gene expression
Lipoxygenase (BoLOX2) and Oxophytodienoate Reductase (BoOPR3) were taken as candidates for activity of the JA pathway, while Vegetative Storage Protein 2 (BoVSP2) was used as an indicator of downstream JA activity and oxidative stress responses (Tytgat et al., 2013). Pheophytinase (BoPPH) was used as an indicator of vegetative senescent activity as this has been identified previously as the leading catabolic enzyme in broccoli (Aiamla-or et al., 2012). RNA extraction from snap-frozen floret samples was performed using Rneasy Plant Mini Kit (Qiagen, UK), including on-column DNAse treatment. RNA integrity was verified using agarose gel electrophoresis and spectrophotometry (260/280 nm ratio). cDNA synthesis was performed using Superscript™ II RT (Invitrogen, UK) before RT-qPCR was performed on a CFX96™ RT-PCR system (Bio-Rad, UK) using SsoAdvanced™ Universal SYBR® Green (Bio-rad, UK) and gene specific primers. Primers were designed using PrimerQuest™ (Integrated DNA Technologies), specific primer sequences and targets are given in Supplementary Information 1. qPCR analysis was performed for triplicate biological replicates (taken from separate RNA extractions), each with triplicate technical replicates, and were normalised against β-Actin using the ΔΔCt method. 

Published Paper
http://dx.doi.org/10.1080/14620316.2024.2449032.