For the basal thermotolerance (BT) assay, plates containing 7-day-old seedlings were transferred to a new growth chamber (Percival Scientific) with the inside temperature preset to 45°C (HS treatment) or kept in the same growth chamber (22°C; control) and left for 60–75 min. For the acclimated thermotolerance (AT) assay, plates containing 7-day-old seedlings were first exposed to 37°C for 1 h. The plants were then allowed to recover at 22°C for 2 h before being subjected to 45°C or 22°C in a growth chamber (Percival Scientific) for 3 h. In both the BT and AT assays, after HS treatment the plants were allowed to continue to grow at 22°C under LD conditions for 3–4 days before being photographed and examined for survival. Each HS treated glass plate is considered as one biological replicate. In general, at least 30 biological replicates collected from four to five independent experiments were used for survival rate quantification, unless otherwise indicated.
Growth chamber
Manufactured by Percival
Sourced in United States
The growth chamber is a controlled environment enclosure designed for the cultivation and study of plants, microorganisms, or other living organisms. It provides precise control over temperature, humidity, lighting, and other environmental factors to create optimal conditions for growth and development.
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Lab products found in correlation
Benzylaminopurine bap, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Whatman paper, by Cytiva (1 mentions)
Sml0094, by Merck Group (1 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Faststart essential dna green master, by Roche (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Iscript reverse transcription supermix for rt qpcr, by Bio-Rad (1 mentions)
Magenta ga7 boxes, by Bioworld Technology (1 mentions)
Phire plant direct pcr kit, by Thermo Fisher Scientific (1 mentions)
Fluorescent lamps, by Philips (1 mentions)
Murashige skoog medium, by Serva Electrophoresis (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Maxima sybr green fluorescein qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Real time pcr detection system, by Bio-Rad (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Jmp 16, by SAS Institute (1 mentions)
Linsmaier and skoog ls medium, by HiMedia (1 mentions)
Phytagel, by Merck Group (1 mentions)
75 protocols using growth chamber
1
Assessing Plant Thermotolerance Levels
2
Thermotolerance Assay of Arabidopsis Mutants
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For thermotolerance assays, mutant and WT seeds were sterilized and sown on the same plates containing equal volumes of MS; the plates were poured on a leveling table to ensure the same conditions and minimize positional effects. After stratification at 4°C for 3 days, the plates were placed into a growth chamber (Percival) under 22°C and long-day light conditions (16 h light/8 h dark). After 9 days, the plates were transferred directly to a growth chamber (Percival) with the indicated high temperature and the same light conditions as the control for the indicated time; this was followed by a recovery period at 22°C for 4 days, after which the plates were photographed and assessed for survival rates. Survival was defined as the ability to maintain fresh and green leaves and to form new leaves.
For the thermotolerance assay with bikinin treatment, mutant and WT seeds were sterilized and sown on the same MS plates containing 20 μM bikinin (catalog #SML0094; Sigma) or 0.01% DMSO. The growth and heat treatment processes were the same as those described above.
For the thermotolerance assay with bikinin treatment, mutant and WT seeds were sterilized and sown on the same MS plates containing 20 μM bikinin (catalog #SML0094; Sigma) or 0.01% DMSO. The growth and heat treatment processes were the same as those described above.
3
Cultivation of Arabidopsis Mutant Lines
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The seeds of the obtained mutant lines were surface-sterilized by immersion in ethanol and planted on ½ Murashige and Skoog medium solidified with 1% agar. Next, the seeds were stratified at 4 °C for 3 days. After the stratification Petri plates were transferred into growth chambers (Percival Scientific Inc., Perry, IA, USA) and cultivated at 21 °C with a 12-h photoperiod and a photon flux density of 100 µmol m−2 s−1 for 14 days. The seedlings were harvested exactly in the middle of the light or dark period, flash-frozen in liquid nitrogen and homogenized in Retsch mill (Haan, Germany). Plants for histochemical staining were cultivated as described above with the following differences: stratified seeds of Arabidopsis thaliana and transgenic line ARR5::GUS (obtained from The Nottingham Arabidopsis Stock Centre) were cultivated for seven days in a growth chamber (Percival Scientific) at 29 °C with a 12-h photoperiod and a photon flux density of 100 µmol m−2 s−1. On the seventh day the seedlings were harvested exactly in the middle of the light or dark period.
4
Evaluating Plant Growth Promotion by Endophytes
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Plant growth promoting activities were evaluated by inoculating single strains on their host seedlings. First, husk tomato seeds were sterilized with 95% ethanol for 5 min and 20% sodium hypochlorite for 5 min, followed by 5 washes with sterile distilled water. The seeds were germinated and grown in 0.2 × MS medium (Murashine Skoog). Five days after germination, selection of five seedlings with homogeneous growth were transplanted into plates containing MS medium supplemented with 10% NA medium and at the extreme of the plate a 10 µL inoculum of each endophyte bacterium, previously grown on nutrient liquid medium (OD 600 nm = 0.05). The plates were incubated with a photoperiod of 16/8 h day/night in a growth chamber (Percival Scientific). After 7 d of incubation, the total fresh weight, length and weight of the aerial part and roots, and length and number of lateral roots were measured. Chlorophyll content was quantified using an MC-100 Apogee chlorophyll meter. To provide a reference for plant growth-stimulating activity (positive control), two well-studied bacterial strains (Pseudomonas fluorescens strains UM256 and UM270) were included in the analysis for their plant growth-promoting capacities (Hernández-León et al., 2015 ; Rojas-Solis et al., 2016 ).
5
Arabidopsis Endophyte Inoculation Protocol
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Arabidopsis thaliana (ecotype Columbia-0) seeds were surface sterilized by hydrochloric fumigation for 3 h and stratified for 3–5 days at 4°C (Lindsey et al., 2017 (link)). Subsequently, the sterile seeds were grown in vitro on half-strength Linsmaier and Skoog (LS) medium at pH 5.7 with 7% plant agar. Seedlings were grown on vertically oriented plates in a growth chamber (Percival, Iowa, United States) with continuous white light (100 μmol m– 2 s– 1) at 21°C (Qiu et al., 2019 (link)). Prior to inoculation, the 22 selected endophytic isolates were grown for 24 h in LB media with 50 μg tetracycline ml– 1 and 100 μg ampicillin ml– 1 at 28°C. The bacterial cells were centrifuged at 3,500 × g, resuspended in infiltration buffer (10 mM MgSO4, 10 mM MES-KOH pH 5.5), and adjusted to an OD600 of 0.1 in the same buffer. Subsequently, the roots of 7-days old seedlings were inoculated with 2–5 μl of bacterial inoculum per root, being careful not to contaminate shoot tissues, following the protocol for Salmonella enterica and Escherichia coli O157:H7 (Cooley et al., 2003 (link)). Infiltration buffer without bacteria served as the negative control (mock treatment).
6
Generating Transgenic Ceratopteris richardii Plants
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Ceratopteris richardii strain Hn-n40 (link) was used in this study to generate the transgenic plants. Gametophytes were grown on FM plates (pH 6.0) containing 0.5 × MS salts (PhytoTechnology Laboratories) and 0.7% (w/v) agar (Sigma-Aldrich). Sporophytes were formed on fertilized gametophytes and were transferred to soil, typically after 3–4 weeks of fertilization. Both gametophytes and young sporophytes were grown under continuous light at 28 °C. Adult sporophytes were grown in the LILY greenhouse facility at Purdue for harvesting spores.
Ceratopteris calli were induced from young sporophytes (shoot tips or fronds) on the callus induction medium (pH 5.8) that contains 1 × MS salts (PhytoTechnology Laboratories), 2% (w/v) sucrose, 1 mg/L benzylaminopurine (BAP), and 0.7% agar (Sigma–Aldrich). Calli were cultivated under continuous light at 28 °C in the growth chamber (Percival).
Ceratopteris calli were induced from young sporophytes (shoot tips or fronds) on the callus induction medium (pH 5.8) that contains 1 × MS salts (PhytoTechnology Laboratories), 2% (w/v) sucrose, 1 mg/L benzylaminopurine (BAP), and 0.7% agar (Sigma–Aldrich). Calli were cultivated under continuous light at 28 °C in the growth chamber (Percival).
7
Whitefly Development on TYLCV-Resistant Genotypes
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The time required for B. tabaci adult development was studied on TYLCV-resistant and -susceptible genotypes. For this purpose, 6-week-old plants maintained in 15 x 14 cm pots in a growth chamber (Percival, Perry, IA) at 26± 0.1°C (mean ± SD) with 14:10 h (L:D) photoperiod were used. Three plants per cultivar were used, and three clip-cages containing two adult females were attached to each plant allowing an oviposition period of 48 hours. Once adult whiteflies and cages were removed, six eggs in each cage were randomly selected and monitored daily until adult emergence.
8
Growth Conditions for Potato and Tobacco
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Potato (Solanum tuberosum ssp. andigena 7540 and S. tuberosum cv. Désirée) and tobacco (Nicotiana tabacum cv. Petite Havana) were used as model systems in this study. In vitro cultures of all the plants were maintained at 22±1 °C with light intensity of 300 mmol m−2 s−1 in a growth incubator (Percival Scientific, USA) with either a long day (LD; 16 h light, 8 h dark) or short day (SD; 8 h light, 16 h dark) photoperiod, depending on the experimental treatment. Soil-grown plants of potato and tobacco were maintained at 300 μmol m−2 s−1 light intensity with 22±1 °C day temperature and 20 °C night temperature in a growth chamber (Percival Scientific, USA) under either a LD or SD photoperiod, depending on the experimental treatment.
9
Arabidopsis Growth Protocol from Seed to Maturity
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Seeds were sown on 0.5× Murashige and Skoog medium (pH 5.7) containing 1% agar and 0.05% MES. Following 48 h of stratification (4°C, dark), seedlings were grown at room temperature (~23°C) under 24 h light for 7–10 d before being transplanted to soil. The soil used was Sunshine SMB-238 (SunGro Horticulture, Agawam, MA) supplemented with 10-10-10 fertilizer and Marathon pesticide following the manufacturer's instructions. Plants were grown until maturity in a growth chamber (Percival Scientific, Perry, IA) under a long-day photoperiod (16 h light at 20°C/8 h dark at 18°C, 70% humidity, and ~125 μmol m−2 s−1 light intensity).
10
Comparative Plant Growth and Sampling
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Arabidopsis thaliana WT ecotype Columbia-0 and epiRIL12 plants from the eighth generation [43 (link)] were grown in soil under a 16h/8h (light/dark) cycle after 2 days at 4°C for stratification. Florets and 1–2 cm green siliques were harvested 3 days to 2 weeks after pollination, respectively. Oryza sativa ssp. japonica cv. Nipponbare rice plants were cultivated in a growth chamber (Percival, USA) under a 12h light-dark cycle (12h-28°C/12h-26°C) and with a relative humidity of 80% during the day and 70% during the night. The light intensity varied gradually in 40 min at the beginning and end of the day. Grain material was harvested 3 to 5 to 15 days after pollination for the immature and mature stage, respectively. Seeds were germinated in the dark on a humid Whatman paper for 5 days before harvest. Dissection of seeds was performed under the binocular on mature seeds. Callus material was previously described [21 (link)].
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