OJIP protocol was performed on fully developed, mature leaves of plants after 45 days of growth. The samples were dark-adapted for 20 min. A Fluorpen FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic) was used for measuring the OJIP transients. Different biophysical and phenomenological parameters related to PSII status [53 ] was investigated by JIP-test according to the protocol described by [20 (link)]. Parameters derived from the OJIP protocol provides information on the energy fluxes of light absorption (ABS) and trapping (TR) of the excitation energy and electron transport (ETO) per reaction center (RC), which are described on the Supplementary Table S1 .
Fluorpen fp 100 max
Manufactured by Photon Systems Instruments
Sourced in Czechia
The Fluorpen FP 100-MAX is a handheld fluorometer designed for rapid and accurate measurement of fluorescence in various samples. The device features a compact and durable design, with a high-sensitivity photodetector and a user-friendly interface. The Fluorpen FP 100-MAX is capable of measuring a wide range of fluorescent parameters, making it a versatile tool for various applications.
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16 protocols using fluorpen fp 100 max
1
Chlorophyll Fluorescence OJIP Analysis
2
Photosynthesis and Fluorescence Measurements
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The net photosynthetic rate, transpiration rate and stomatal conductance were determined in situ using an LCi Portable Photosynthesis System (ADC BioScientific, Hoddesdon, the United Kingdom) with the following conditions in the measurement chamber: air temperature at 25°C, ambient CO2 concentration at 550±50 μL L-1, air flow rate at 205±30 μmol s-1, and irradiance at 300 μmol m-2 s-1 [25 (link)].
The polyphasic rise of the chlorophyll fluorescence transient (OJIP) was measured at the upper surface of the dark-adapted (20 min) leaves in situ with the portable fluorometer FluorPen FP100max (Photon System Instruments, Brno, Czech Republic) as described in [26 (link)]. The parameters of the JIP test (seeS1 Table ) were calculated according to the theory of energy flow in the photosynthetic electron-transport chain [29 , 30 ]. The relative variable fluorescences WOI, WOJ, WOK and WIP (i.e., normalizations of the whole fluorescence transients) and the difference kinetics ΔWOJ and ΔWOK (as the differences between the drought-stressed and control plants) were also calculated according to [31 ] and their graphical representation was utilised to obtain further information on the primary photosynthetic processes.
The chlorophyll a and b contents and total carotenoids were determined spectrophotometrically [32 ] in the N,N-dimethylformamide extracts prepared as described in [33 (link)].
The polyphasic rise of the chlorophyll fluorescence transient (OJIP) was measured at the upper surface of the dark-adapted (20 min) leaves in situ with the portable fluorometer FluorPen FP100max (Photon System Instruments, Brno, Czech Republic) as described in [26 (link)]. The parameters of the JIP test (see
The chlorophyll a and b contents and total carotenoids were determined spectrophotometrically [32 ] in the N,N-dimethylformamide extracts prepared as described in [33 (link)].
3
Photosynthetic Efficiency Evaluation
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The OJIP transients of dark-adapted (20 min) plants were measured using a Fluorpen FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic) at 2, 4, 8, 12, 16, and 21 dpi. The OJIP protocol was used to investigate biophysical and phenomenological parameters related to plant stress and photosystem II (PSII) status (listed in Supplementary Table 1 Supplementary Table 1
4
Chlorophyll a Fluorescence and Pigment Analysis
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Chlorophyll a Fluorescence emission measurements were carried out on three leaves per treatment using a portable fluorometer (FluorPen FP100max, Photon System Instruments, Brno, Czech Republic), equipped with a light sensor (Photon System Instruments, Brno, Czech Republic). The basal fluorescence signal, Fo, was induced by an internal LED blue light (1–2 μmol photons m−2 s−1) on 30′ dark-adapted leaves. The maximal fluorescence signal in the dark, Fm, was determined by a saturating light pulse of 3000 μmol photons m−2 s−1 of 1s. The PSII maximal photochemical efficiency, Fv/Fm, was calculated as the ratio (Fm − Fo)/Fm. After fluorescence measurements, the same leaves (three per treatment) were collected to determine total chlorophylls (a + b), total carotenoids (x + c), a/b and (a + b)/(x + c) ratios. Fresh samples (10 mg) were extracted using ice-cold 100% acetone and centrifuged (Labofuge GL, Heraeus Sepatech, Hanau, Germany) at 5000 rpm for 5 min. The absorbance of supernatants was measured by spectrophotometer (Cary 100 UV-VIS, Agilent Technologies, Santa Clara, CA, USA) at 470, 645, and 662 nm. The pigment concentration was determined using Lichtenthaler equations [45 (link)] and expressed in mg g−1 of fresh weight (mg g−1 FW). All the measurements were performed after 27 days of growth.
5
Chlorophyll Fluorescence Transient Analysis
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Fast induction of fluorescence transient (the so-called OJIP protocol) was performed on fully developed, mature leaves of plants after 45 days of growth. The samples were dark-adapted for 20 min. A Fluorpen FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic) was used for measuring the OJIP transients. Different biophysical and phenomenological parameters related to PSII status49 were investigated by JIP-test according to the protocol described by50 (link).
The maximum quantum yield of PSII (Fv/FM) and Performance index per absorbed light (PIABS) was calculated using the Eqs. (1 ) and (2 ), respectively:
The maximum quantum yield of PSII (Fv/FM) and Performance index per absorbed light (PIABS) was calculated using the Eqs. (
6
Stomatal Dynamics and Photosynthetic Efficiency in Carnation
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On the 8th day of vase life at 2 h after exposure to different light treatments, stomatal opening was determined using a nail polish replica method on the lower epidermis of the second lateral leaflets from the apex (abaxial side) as described by Aliniaeifard and Van Meeteren (2016) (link). The nail polish layer was separated with a strip of transparent sticky tape and pasted on a glass slide and observed using a light microscope (BH-323, Olympus, Tokyo, Japan). Pore aperture of more than 6 μm was considered as open stomata, less than 3 μm as closed, and between 3 and 6 μm as semi-closed. The polyphasic chlorophyll a fluorescence (OJIP) transients were determined using a Fluorpen FP 100-MAX (Photon Systems Instruments, Drásov, Czechia) on young fully expanded carnation leaves after 20 min dark adaptation (Strasser, and Strasser, 1995 ) according to the JIP test (Strasser et al., 2000 ) at 1, 4, 8, and 12 days following onset of the trial. The measurement of transient fluorescence was induced by a saturating light of 3000 μmol m–2 s–1 PPFD. Three leaves of each cut carnation stem were used for each replicate. The parameters obtained from this protocol were calculated according to Kalhor et al. (2018) (link).
7
Chlorophyll Fluorescence Analysis of Walnut Drought Stress
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In the second experiment, chlorophyll fluorescence parameters of 60 selected extreme families (very drought tolerant to very sensitive) were measured under severe stress (24 days after drought) and recovery (two weeks after re-irrigation). Polyphasic Chl a fluorescence transients (OJIP-test) were measured using a portable fluorometer (Fluorpen FP 100-MAX, Photon Systems Instruments, Drasov, Czech Republic) in the middle part of the sapling in young fully-expanded walnut leaflets with 3 replicates for each treatment (control or drought) after 20 min dark adaptation. To fully ensure that all PSII centers are open, plants were allowed to dark-adapt overnight, and the lights were extinguished in the greenhouse until measurements were concluded pre-dawn (between 1 and 5 a.m.). The fluorescence measurements were taken by a saturating light of ~3000 μmol m−2 s−1. Fluorescence intensities were recorded at four time points: 50 μs (O), 2 ms (J), 60 ms (I), and maximum fluorescence at around 1 s (P). Measurements related to the OJIP test were calculated based on the approaches described by Strasser et al. (2000, 2004) [13 ,29 ]. The definition of the measured parameters and detailed calculation formulas are listed in Table 1 . More details of the OJIP-test are given in the supplementary file.
8
Chlorophyll Dynamics in Stressed Plants
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Chlorophyll content and fluorescence were measured on both control and stress plants. Data were collected at two development points—LFE1 and LFE3—always at the same time of day (09:00). Measurements were performed using a FluorPen FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic). Fluorescence transients for chlorophyll-a were recorded in the center of the completely spent leaf (second leaf from the top). Leaves were allowed to adjust to the darkness for 30 min before measurements were made using leaf clamps provided by the manufacturer. Leaves were then exposed to a pulse of saturating light at an intensity of 3000 μmol m−2 s−1, and all studied parameters were analyzed. Nine replicates were analyzed for each cultivar and treatment (three leaves from three plants/treatment). The parameters analyzed in this study are listed in Table 2 .
9
Measuring Saffron Leaf Chlorophyll Fluorescence
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Attached, young, fully developed leaves of saffron plants were used for measuring the polyphasic Chl a fluorescence (OJIP) transients 60 days after flowering, using a Fluorpen FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic) following 20 min of dark adaptation. The parameters obtained from the OJIP protocol were calculated according to previous studies [19 (link),20 (link),21 (link),22 ]. Basic and calculated parameters and their formula are shown in Table 1 .
10
Leaf Gas Exchange and Chlorophyll Fluorescence
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Leaf gas-exchange and chlorophyll "a" fluorescence emission measurements were carried out on 2 well-exposed and fully expanded leaves per 15 plants during the veraison phase of the two growing seasons (2017) (2018) . Net CO 2 assimilation rate (P n ) and stomatal conductance (g s ) were performed by means of a portable infra-red gas-analyzer (LCA 4; ADC, BioScientific, Hoddesdon, United Kingdom) equipped with a broad-leaf PLC (cuvette area 6.25 cm 2 ). Chlorophyll "a" fluorescence emission was measured using a portable FluorPen FP100 Max fluorometer with a light sensor (Photon System Instruments, Brno, Czech Republic). A blue LED internal light of 1-2 µmol photons m 2 s -1 was used to induce the ground fluorescence F 0 on 30 dark adapted leaves. A saturating light pulse of 3.000 µmol photons m 2 s -1 was applied to induce the maximal fluorescence level in the dark, F m . The following parameters were considered: the maximum PSII photochemical efficiency (F v /F m ) calculated as (F m -F 0 )/F m , the quantum yield of PSII linear electron transport ( PSII ) and non-photochemical quenching (NPQ) (Genty et al., 1989; (link)Bilger and Björkman, 1990) (link). The measurements in the light were conducted from 12:00 to 14:00 pm under environmental Photosynthetic Photon Flux Density (PPFD) ranging between 1,800 and 2,300 photons m 2 s -1 .
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