Myo inositol

Two types of basal media prepared using media formulation were explained by Murashige and Skoog (Sigma-Aldrich, St. Louis, MI, USA) containing iron chelated to the disodium salt of EDTA and Gamborg et al. (Duchefa Biochemie, The Netherlands) which containing iron chelated to the mono-sodium salt of EDTA. Briefly, the macronutrients, micronutrients, and vitamins solution was mixed with 0.1 g/L of myo-inositol (Duchefa Biochemie, The Netherlands) and 30 g/L of sucrose (Duchefa Biochemie, The Netherlands). The medium’s pH was adjusted to pH 5.6–5.8 by adding 1 M of either sodium hydroxide (NaOH) and hydrochloric acid (HCl). A total of 3 g/L of Gelrite™ (Duchefa Biochemie, The Netherlands) was added and stirred until completely dissolved. The solution was heated in the microwave before being poured into vials. All the labeled vials were then placed in the autoclave to be sterilized at 121 °C and 1.05 kg/cm² for 20 min The strength of basal media (half, full- and double-strength) was manipulated by either reducing or amplifying the macronutrients, micronutrients, and vitamins. MS with full strength served as a control treatment in this experiment. The data were taken on the number of shoots, length of the shoot (cm), and the number of leaves was recorded after four weeks of incubation.

Seeds of H. reticulatus were provided by Pakan Bazr Company, Isfahan, Iran. H. reticulatus seeds were surface sterilized in 70% (v/v) ethanol and 10% (v/v) NaOCl and then washed three times in sterile water. Afterward, seeds were cultured in MS medium supplemented with 3% (w/v) sucrose, 7.2 g L⁻¹ agar (Duchefa, Haarlem, Netherlands), and 0.1 g L⁻¹ myo-inositol (Duchefa, Netherlands). One week after germinating, cotyledons were isolated as explants.

Wild type Arabidopsis thaliana seeds, ecotype Columbia 0, were kindly supplied by Prof. Dr. Richard Strasser (Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria). When grown in vitro, seeds were surface sterilized in 70% (v/v) ethanol for 2 min, followed by 8 min in 5% (v/v) NaOCl (Sigma-Aldrich, St Louis, MO, USA). Afterwards, the seeds were rinsed eight times with sterile distilled water. The sterilized Arabidopsis seeds were sown in vitro on solid ½ Murashige and Skoog (MS) medium (2.154 g/L MS basal salt [Duchefa Biocheme, Haarlem, The Netherlands], 10 g/L sucrose [Duchefa], 0.1 g/L myo-inositol [Duchefa], 0.5 g/L MES - 2-(N-morpholino)ethanesulfonic acid [Carl Roth, Karlsruhe, Germany], 8 g/L plant tissue culture agar [Duchefa], pH 5.7) supplemented with 10 mg/L phosphinothricin (PPT, Duchefa) or 75 µg/ml kanamycin (Duchefa) as selective agents during selection. Alternatively, Arabidopsis seeds were grown in pots containing commercial soil or individually grown in artificial soil (Jiffy-7, 44 mm Ø). Both plants grown in vitro and in soil were first stratified for 3 days at 4°C in the dark and then transferred to a growth chamber at 21°C with a 16/8 h light/dark photoperiod.