The transgenic Arabidopsis thaliana lines (Col-0 background) expressing either VND7-VP16-GR or VP16-GR were those described by [12 (link)]. The VND7-VP16-GR and VP16-GR seedlings were grown under continuous light at 22 °C for 7 d on Murashige and Skoog medium (Wako, Osaka, Japan) containing 1% (w/v) sucrose (nacalai tesque, Kyoto, Japan), 0.05 (w/v) MES (nacalai tesque), and 0.6% (w/v) Gellan gum (Wako), adjusted to pH 5.7.
Murashige and skoog medium
Manufactured by Fujifilm
Sourced in Japan
Murashige and Skoog (MS) medium is a commonly used culture medium for plant tissue culture. It provides a balanced nutrient solution containing essential macro and micronutrients, vitamins, and growth regulators required for the growth and development of plant cells, tissues, and organs in vitro.
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8 protocols using murashige and skoog medium
1
Arabidopsis thaliana Vascular Development
2
Arabidopsis Seed Sterilization and DNA Extraction
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The seeds of Arabidopsis thaliana cv. Columbia (2n = 10) were sterilized in 1 mL of 10% (v/v) kitchen bleaching solution (KAO, Tokyo, Japan) for 30 min, rinsed several times with distilled water, vernalized at 4 °C for 2 to 3 days, and germinated on 0.5× Murashige and Skoog medium (Fujifilm-Wako, Tokyo, Japan) supplemented with 0.5% agar for 10–15 days at 25 °C. DNA was extracted from seedlings in DNA Suisui buffer (RIZO Inc., Tsukuba, Japan) and precipitated using ethanol.
3
Hydroponic Growth of Arabidopsis
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Arabidopsis Columbia (Col-0) was used in this study. Arabidopsis plants were grown in a hydroponic system. Seeds were sown on half-strength Murashige and Skoog medium (Wako Pure Chemical, Osaka, Japan) supplemented with 0.8% agar and grown under a cycle of 8 h of light and 16 h of dark with a photon flux density of 120 μ mol m−2 s−1 at 22 °C. Ten-day-old seedlings were grown on a sheet of a polyethylene raft through holes (1 cm in diameter) floating over a 1500-fold diluted liquid fertilizer (Hyponex). The fertilizer was changed weekly, and their roots were soaked in the fertilizer under the light conditions described above. Unless otherwise stated, four-week-old plants were used.
4
Isolation of Arthrobacter from Tobacco Rhizosphere
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Seeds of N. tabacum cv. Burley 21 (tobacco) were provided by Japan Tobacco, Inc. (Tokyo, Japan). Tobacco seeds were surface sterilized with 70% ethanol (EtOH) for 1 min and 1% sodium hypochlorite (NaClO) for 10 min and rinsed five times with sterile water. Plants were germinated on Murashige and Skoog medium (Wako Pure Chemical Industries) supplemented with 0.8% agar. Plants were grown for 2 weeks in a cultivation room set at 28°C under a light/dark (16/8-h) photoperiod. Tobacco seedlings were then transferred to plastic pots filled with soil collected at the KUAS and grown for 12 weeks in a greenhouse. A 10-week-old plant after transplanting was subjected to reisolation of Arthrobacter. The plants were fertilized every week using Hyponex (Hyponex Japan, Osaka, Japan).
The rhizosphere and rhizoplane soils were collected using 250 ml of phosphate-buffered saline (PBS) after removing the loosely attached soil on the roots by gentle shaking, as described previously (6 (link)). The roots after sonication were washed with tap water and surface sterilized with 70% EtOH for 1 min and 1% NaClO. The surface-sterilized roots were then washed five times with sterile water and stored at −80°C until DNA extraction. The remaining root tissues were immediately subjected to bacterial isolation.
The rhizosphere and rhizoplane soils were collected using 250 ml of phosphate-buffered saline (PBS) after removing the loosely attached soil on the roots by gentle shaking, as described previously (6 (link)). The roots after sonication were washed with tap water and surface sterilized with 70% EtOH for 1 min and 1% NaClO. The surface-sterilized roots were then washed five times with sterile water and stored at −80°C until DNA extraction. The remaining root tissues were immediately subjected to bacterial isolation.
5
Isolation of Rice and Arabidopsis Protoplasts
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Rice and Arabidopsis protoplasts were isolated, as described by Kuroha et al. (2018) (link), with minor modifications. Rice (O. sativa L., Nipponbare) plants were grown in plastic boxes with 1/2 Murashige and Skoog medium (Wako, Osaka, Japan) and 0.35% gellan gum (Wako) under white light conditions at 30 °C for 10 days. Arabidopsis (Arabidopsis thaliana Columbia-0) plants were grown in pots containing fertilized soil under white light condition at 23 °C for 14 days. Rice leaf sheaths were used to isolate rice protoplasts, and Arabidopsis leaves were used to isolate Arabidopsis protoplasts. After enzymatic digestion of each strip, the protoplasts were released by filtering through a 57 µM nylon mesh, followed by adjusting approximately 4×106 cells mL−1 with 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.7) containing 0.4 M Mannitol and 15 mM MgCl2 for polyethylene glycol (PEG)-mediated transformation.
6
Arabidopsis Ecotype Columbia Mutant Analysis
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Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used as the wild type. The T-DNA insertional mutants used in this study included erdj3a-1 (SALK_103280), erdj3a-2 (SALK_027193), erdj3b-1 (SALK_113364), erdj3b-2 (SALK_055599), p58 ipk -1 (SALK_140273), and p58 ipk -2 (SALK_080901; Yamamoto et al., 2008) . Seeds were either sown in soil or surface sterilized with chlorine gas and sown on Murashige and Skoog medium (Wako) containing 0.7% (w/v) agar and 1% (w/v) Suc. The Arabidopsis plants were grown at 22°C or 29°C under continuous light (50 mmol m 22 s 21 ).
7
Arabidopsis thaliana Growth Conditions
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Arabidopsis thaliana ecotype C24, obtained from the Arabidopsis Biological Resource Center (ABRC; https://abrc.osu.edu/ ), was used as the wild-type plant in this study. The AlSRKb-FLAG+AlSCRb A. thaliana transformants were constructed as described in Yamamoto et al. (2014) (link). Seeds were surface-sterilized with 20% bleach and sown on Murashige and Skoog medium (FUJIFILM Wako, Osaka, Japan) containing 0.7% (w/v) agar and 1% (w/v) sucrose. All A. thaliana plants were grown at a constant temperature of 23 °C under a long-day photoperiod (16 h light/8 h dark; light intensity 100 mmol photons m−2 sec−1). The relative humidity was not controlled.
8
Seedling Transcriptome Analysis Pipeline
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Sterilized seeds were sown on 1.5% (w/v) agar plates containing half-strength Murashige and Skoog medium (Wako, Japan) supplemented with 2% sucrose and 0.05% MES-KOH (pH 5.7). Plates were wrapped with aluminum foil and incubated at 4°C for a few days. Then, plates were vertically placed in a growth chamber at 22°C under continuous fluorescent light.
Seedlings were collected after 3, 5, and 7 days of incubation, immediately frozen in liquid nitrogen, and ground to a fine powder using a pestle and mortar. Total RNA was extracted from the powder using the NucleoSpin RNA Plant Kit (Takara; http://www.takara-bio.co.jp/), according to the manufacturer's protocol.
Seedlings were collected after 3, 5, and 7 days of incubation, immediately frozen in liquid nitrogen, and ground to a fine powder using a pestle and mortar. Total RNA was extracted from the powder using the NucleoSpin RNA Plant Kit (Takara; http://www.takara-bio.co.jp/), according to the manufacturer's protocol.
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