M0221

In this work Arabidopsis thaliana (ecotype Columbia 0) was used as a WT plant. Previously described T‐DNA insertion mutants glcak1‐1 (SALK_076931) and glcak1‐2 (SALK_127949c) were obtained from the Arabidopsis Biological Resource Center and another two mutant lines glcak1‐3 (contains CAS9 nuclease) and glcak1‐4 were created by CRISPR/Cas9 technology. Plants were grown in a growth chamber in pots on standard soil (type ED73) under long‐day conditions with 16‐h light at 150 μmol·m⁻²·s⁻¹. Temperature during the light phase was 23 °C and 18 °C in the dark. For RT‐PCR and labelling experiments seedlings were grown in liquid medium. Seeds were surface sterilised in ethanol and grown in 0.5 × MS (Basal Salt Mixture, Duchefa #M0245; Duchefa, Haarlem, the Netherlands), pH 5.7 (KOH), with 1 g·l⁻¹ sucrose for 10 days. Then the medium was changed to 0.5 × MS (or 0.5 × MS with 0.37 MBq myo‐[2‐³H]‐inositol; final concentration in medium is 0.625 μm) for the next 3 days. For sugar measurements sterile seeds were incubated on 0.5 × MS (without micronutrients; Duchefa #M0221), pH 5.7 (KOH), plates with 0.8% plant agar. Harvested samples were immediately used for experiments or frozen in liquid nitrogen and stored at −80 °C until used.

In vitro plantlets were propagated on “CIP medium” and plain MS medium⁵⁰ ). The CIP medium contains half strength MS salts (Duchefa Biochemie, M0221) supplemented with 30 g/L sucrose, 2.8 g/L gelrite, 2 mg/L calciumpanthotenate, 100 mg/L calciumnitrate, 200 mg/L ascorbic acid and 10 mg/L gibberellic acid, pH was set to 6.12 before autoclavation (b.a.). The MS medium contains MS salts and vitamins (Duchefa Biochemie, M0222) supplemented with 25 g/L sucrose and 2.8 g/L gelrite, pH was set to 6.12 b.a.. Nodal fragments from the in vitro plantlets were excised in a sterile laminar flow bench. This was done by removing the leaves and roots of the plantlet, after which 1 cm stem fragments were cut, each containing one axillary meristem in the middle. Three fragments were transferred to each culture tube and grown in a 24 °C growth room on a 16/8 h light/dark regime with the light being provided with 36 W (cool white)/ 840 Lumilux fluorescent lights. The material was subcultured every 5–6 weeks.
Six weeks after initiation, the number of new nodes was counted. This experiment was initially executed on the following 4 cultivars: TIS, IBA, JEW and TAN. Further propagation of all 10 cultivars was done with the medium that proved to be the most productive.

Wild-type BY-2 (Nicotiana tabacum cv. Bright Yellow-2) cells were grown in Murashige and Skoog (MS) modified medium (basal salt mixture, M0221, Duchefa) at pH 5.6, supplemented with 1 mg l^–1 thiamine-HCl, 0.2 mg l^–1 2,4 dichlorophenylacetic acid, 100 mg l^–1 myo-inositol, 30 g l^–1 sucrose, 200 mg l^–1 KH₂PO₄, and 2 g l^–1 MES. Cell suspensions were maintained under continuous light conditions (200 µE m^–2 s^–1) on a rotary shaker (140 rpm) and diluted (4:80) weekly into fresh medium.

Wild-type BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells and cells of a BY-2 cell line expressing NtRbohD antisense cDNA (gp3) (Simon-Plas et al., 2002 (link)) were grown in Murashige and Skoog (MS) modified medium (basal salt mixture, M0221, Duchefa) at pH 5.6, supplemented with 1mg l⁻¹ thiamine-HCl, 0.2mg l⁻¹ 2,4 dichlorophenylacetic acid, 100mg l⁻¹ myo-inositol, 30g l⁻¹ sucrose, 200mg l⁻¹ KH₂PO₄, and 2g l⁻¹ MES. Chlorophyllian A. thaliana cells (ecotype Columbia) were grown in MS modified medium (including Nitsch vitamins, M0256, Duchefa) at pH 5.7, supplemented with 0.5mg l⁻¹naphthalenacetic acid, 50 µg l⁻¹ kinetin, and 30g l⁻¹ sucrose. Cell suspensions were maintained under continuous light conditions (200 µE m⁻² s⁻¹) on a rotary shaker (140rpm) and diluted weekly (3:80 and 20:100 for tobacco and Arabidopsis, respectively) into fresh medium.

Dehusked seeds of O. sativa cv Nipponbare were surface-sterilized in 70% ethanol for 1 min, then rinsed with sterile distilled water and disinfected by dipping in a 40% bleach solution diluted in distilled water with 0.4% Tween 80 (Sigma-Aldrich P4780-500 ml) for 30 min under gentle agitation at room temperature. The seeds were then rinsed three times with distilled water.
The seeds were sown on square Petri plates (Corning, 431,301; 20 cm × 20 cm) containing 250 ml of Murashige and Skoog (MS/2) solid medium (2.15 g/l of MS medium basal salt mixture (Duchefa Biochemie, M0221), 75 mg/l MS vitamin mixture (Duchefa Biochemie, M0409) and 8 g/l of type II agarose (Sigma-Aldrich, A6877). The Petri plates were then placed vertically in a culture chamber under controlled light (12-h photoperiod, light intensity 500 uEm-2s-1) and temperature (28/25 °C day/night) conditions for 6 days.