Spad 502 meter

Relative chlorophyll quantification in SPAD units was achieved using a non-destructive chlorophyll SPAD-502 meter (Minolta). For protein extraction, samples were freeze-dried for 72 h then ground to a fine powder. Soluble proteins were extracted in a buffer containing 20 mM citrate, 160 mM Na₂HPO₄ (pH 6.8), and a pinch of polyvinylpyrrolidone by a 15 min incubation step at 1500 rpm. After a 30 min centrifugation step at 4 °C, proteins from the supernatant were quantified using the Bradford reagent with BSA as the standard.

Chlorophyll meter readings were obtained at five growth stages including tillering (TL), stem elongation (SE), panicle initiation (PI), booting (BT), and heading (HD) using a SPAD-502 meter (Minolta Camera Co., Osaka, Japan). Chlorophyll meter readings were measured from the four uppermost fully expanded leaves of ten randomly selected plants from each plot. Five evenly spaced chlorophyll meter readings, avoiding leaf midribs, were taken per leaf at each growth stage and averaged as the mean chlorophyll meter readings of the leaf. Then, the chlorophyll meter readings of top four leaves for every experimental plot were averaged to minimize the influences of leaf position on the plant and sampling location on the leaf on chlorophyll meter readings.

In peach trees, the assessment of the leaf re-greening was carried out weekly, by measuring the leaf Chl concentration in the 40 labeled shoots (20 treated and 20 untreated with Fe) in each of the four trees. Leaf Chl was estimated in every leaf using a SPAD 502 meter (Minolta Co., Osaka, Japan), measuring in the midst of the distal treated and basal untreated areas (two measurements each). In the non-fertilized, control leaves, measurements were also made in the distal and basal leaf parts. Values shown are means ± SE (n = 4 trees, using the averages of the 20 leaves measured in each tree). The time course of the changes in the SPAD values was expressed as the relative increment at each measurement time compared to the initial values before the first application.
In sugar beet, the re-greening effect was assessed daily by estimating the leaf Chl concentration with the SPAD device in four different leaves per plant. In each leaf, four measurements were made in the distal treated area and four more in the basal untreated area. In the unfertilized control leaves, measurements were also made in the distal and basal leaf parts. Values shown are means ± SE (n = 4 plants, using the average of 4 leaves per plant). The time course of the changes in the SPAD values was expressed as relative increments with respect to the initial values, as indicated above for peach leaves.

To measure the amount of chlorophyll in tomatoes, we used a SPAD-502 meter (Konica Minolta, Inc.) [171 (link)]. Readings were performed on the first true leaves of two-week-old Bonny Best plants treated with 50 mL of a 30 mM trehalose solution or water. The data represent two biological replicates with ten plants per treatment per replicate.

At 0, 10, 20, and 30 DAT, the SPAD (Soil Plant Analysis Development) units were measured with a portable SPAD-502^® meter (Minolta; Tokyo, Japan). To determine the concentrations of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids), the methodology described by Sumanta et al. [87 ] was used. Fifty mg of plant material were taken and macerated with 10 mL of 95% (v/v) ethanol. The samples were placed in 15 mL Falcon tubes and centrifuged at 10,000 rpm in a refrigerated centrifuge (Eppendorf 5424; Eppendorf, Germany) for 5 min at 4 °C, and the supernatant was recovered in a new Falcon tube. From the supernatant obtained, 0.5 mL was taken and mixed with 4.5 mL of 95% ethanol. In these samples, the absorbance was measured in a spectrophotometer (Jenway 6715 UV/Vis; Staffordshire, UK) at 470, 649, and 664 nm. The concentrations of the photosynthetic pigments were determined with the following formulas:


The total chlorophyll concentration was the sum of chlorophylls a and b.

Plant height and basal stem diameter were measured at the end of the experiment with a tape measure and a caliper. After the final fruit harvest, the plants were collected, bagged, and placed in an oven (Novatech, model HS45-AIA, Murrieta, CA, USA) at 70 °C for 72 h. The total dry weight of the plant was recorded, including pruned leaves, leaves, stems, and roots. The SPAD index (SPAD-502 meter, Minolta, Japan) was recorded at intervals of 15 days throughout the duration of the study. Two healthy recently mature leaves were selected per experimental unit, and on each leaf three randomly chosen spots were sampled to obtain the SPAD value.