The B. cinerea strain used was CECT2100 (Spanish Type Culture Collection, Universitat de València). It was routinely cultured on potato dextrose agar (PDA) supplemented with lyophilized tomato leaves at 24°C. Conidia were collected from 4-week-old cultures by scraping surface plates and washing with sterile water, then filtering through cotton to remove debris. Conidia were washed with sterile water twice (5000 rpm, 10 min, 25°C), quantified with a hemacytometer, resuspended in water, and adjusted to the final concentration. When conidia were used to inoculate plants (E. lathyris or Arabidopsis), they were resuspended in Gamborg's B5 medium (Duchefa, the Netherlands) supplemented with 10 mM sucrose and 10 mM potassium phosphate at pH 6 (Benito et al., 1998 ). P. cucumerina (Ton and Mauch-Mani, 2004 (link)) was grown on half-strength PDA at 24°C. Conidia were collected from 4-week-old cultures following the same protocol used for B. cinerea conidia, resuspended in water, and adjusted to the final concentration.
Gamborg s b5 medium
Manufactured by Duchefa Biochemie
Sourced in Netherlands
Gamborg's B5 medium is a cell culture medium formulated for the growth and maintenance of plant cells and tissue. It provides a balanced combination of essential nutrients, vitamins, and growth factors required for plant cell culture applications.
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5 protocols using gamborg s b5 medium
1
Culturing and Inoculation Protocols for Fungal Pathogens
2
Protoplast Isolation from Nicotiana attenuata
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Seeds of Utah 39 th inbred line were germinated on Petri dishes with Gamborg's B5 medium including vitamins (Duchefa Biochemie, NL), as described previously (Krügel et al., 2002) . Plants were grown under 16 h light/8 h dark conditions at 26°C with ± 2°C variation. Protoplasts were isolated from approximately 60 corolla limbs of N. attenuata at ZT 8, ZT 12, and ZT 16 of the first day of flower opening. Corolla limbs were excised by scalpel and immediately immersed in a plant culture dish containing 25 mL of cell-wall-degrading enzyme mixtures (Yoo et al., 2007) . After 5 minutes of vacuum treatment, tissues were shaken at 20 rpm on an orbital shaker for 30 min in the dark, and the cell suspension was filtered onto a Petri dish using a 40 μm cell strainer (Corning, USA). The protoplast suspensions were centrifuged at 100 g for 5 min, and the resulting pellets were resuspended and washed with 10 % (w/v) mannitol solution. Cells were washed, centrifuged again, and finally filtered with 40 μm Flowmi tip strainer (Bel-Art SP Scienceware, USA). Cell concentration and viability were calculated using 0.2 % trypan blue on Cellometer Auto 2000 (Nexcelom Bioscience, USA). All samples showed a viability over 70 %. The entire protoplasting procedure was completed in 2 h.
3
Optimal Conditions for Arabidopsis and Marchantia Growth
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Arabidopsis thaliana seeds were sterilized in 70% ethanol for 10-15 min and subsequently washed with 100% ethanol, after which they were left to dry in sterile conditions. For all experiments, the seeds were stratified in the dark for 2 days at 4°C before being placed in the growth room. Plants were grown in vitro under long-day conditions (16-h light/8-h dark, Lumilux Cool White lm, 50 to 70 µmol m -2 s -1 ) at 21°C on solidified half-strength Murashige and Skoog (MS) medium (2.151 g/L, 10 g/L sucrose, and 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES), adjusted to pH 5.7 with 1 M KOH and 8 or 10 g/L plant agar). For analysis of root or shoot phenotypes, plants were grown vertically or horizontally, respectively. The treatments with MG132 were performed at a concentration of 100 µM for 24 h.
Marchantia polymorpha accession Takaragaike-1 (Tak-1) plants were grown under continuous light conditions at 21°C on solidified half-strength Gamborg's B5 medium (Duchefa Biochemie) with 10 g/L plant agar.
Marchantia polymorpha accession Takaragaike-1 (Tak-1) plants were grown under continuous light conditions at 21°C on solidified half-strength Gamborg's B5 medium (Duchefa Biochemie) with 10 g/L plant agar.
4
Inbred N. attenuata Ecotype Cultivation
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The 31st inbred generation of the N. attenuata “Utah” ecotype, originally collected from a native population at a field site located in Utah (USA) was used in the experiments. Additionally, for the amplification of the TRV California isolate, healthy seedlings of spinach, pepper and N. benthamiana were used. Wild-type seeds of N. attenuata were surface sterilized and germinated on Gamborg’s B5 medium (Duchefa, www.duchefa-biochemie.com ) as previously described [53 ]. After 10 days the seedlings were carefully removed from the agar and transferred to soil in small Teku plastic pots (www.poeppelmann.com ) in the glasshouse. Again, after 10 days in Teku pots, seedlings were transferred to 1 L pots in soil in a growth chamber. Growth chamber conditions were temperatures of 22 °C, 26 °C, 28 °C, 30 °C and 32 °C, relative humidity of 65% and the duration of 16/8 h of day and night cycle and light intensity of 135–195 μMols− 1 m− 2 PAR.
5
Optimal Conditions for Arabidopsis and Marchantia Growth
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Arabidopsis thaliana seeds were sterilized in 70% ethanol for 10-15 min and subsequently washed with 100% ethanol, after which they were left to dry in sterile conditions. For all experiments, the seeds were stratified in the dark for 2 days at 4°C before being placed in the growth room. Plants were grown in vitro under long-day conditions (16-h light/8-h dark, Lumilux Cool White lm, 50 to 70 µmol m -2 s -1 ) at 21°C on solidified half-strength Murashige and Skoog (MS) medium (2.151 g/L, 10 g/L sucrose, and 0.5 g/L 2-(N-morpholino) ethanesulfonic acid (MES), adjusted to pH 5.7 with 1 M KOH and 8 or 10 g/L plant agar). For analysis of root or shoot phenotypes, plants were grown vertically or horizontally, respectively. The treatments with MG132 were performed at a concentration of 100 µM for 24 h.
Marchantia polymorpha accession Takaragaike-1 (Tak-1) plants were grown under continuous light conditions at 21°C on solidified half-strength Gamborg's B5 medium (Duchefa Biochemie) with 10 g/L plant agar.
Marchantia polymorpha accession Takaragaike-1 (Tak-1) plants were grown under continuous light conditions at 21°C on solidified half-strength Gamborg's B5 medium (Duchefa Biochemie) with 10 g/L plant agar.
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