Hi9835

Due to a high degree of sterility, possibly due to the relatively high temperature inside the greenhouse, only 177 lines of the 400 F₂ plants produced sufficient seeds; those 177 F₃ families were evaluated for salt tolerance in a phytotron with day/night temperature of 29/21 °C and relative humidity of 70%. Seeds were heat treated for 5 d in a convection oven set at 50 °C to break their dormancy, then placed in petri dishes lined with moistened filter papers and incubated at 30 °C for 48 h to germinate. Pre-germinated seeds were sown on styrofoam floats (one seed per hole) with a net bottom floated on distilled water in 11 L plastic trays for 3 days, after which a nutrient solution (Yoshida et al. 1976 ) was used. At 14 d after seeding, salt stress was introduced by adding sodium chloride (NaCl) to an electrical conductivity (EC) of 6 dS m⁻¹ for 3 days followed by EC of 12 dS m⁻¹ until the experiment was terminated (total 21 days). The EC of the culture solution was monitored using an EC meter (HI9835, Hanna Instruments, Romania) and adjusted daily. Two replications were used, with ten individual plants per line evaluated for each replicate. IR29 (sensitive) and FL478 (highly tolerant) (Gregorio et al. 1997 ; Ismail et al. 2007 (link)) were used as checks. The pH of the nutrient solution was adjusted daily to 5.0, and the culture solution was replaced weekly.

Particle size distribution of soil was determined using sieve methods according to [74 ]. Water holding capacity of soil was determined using the gravimetric method, hydraulic conductivity by Darcy’s law, and the soil porosity from the measured values of soil particle and bulk densities calculations [75 ]. Soil electrical conductivity (EC) was measured using HANNA (HI9835) EC meter in 1:2.5 soil/water extract, soil pH (1:2.5 DI water suspension) by Jenway 3505 pH/mV/Temperature Meter and the total carbonate content (expressed as CaCO₃) using the gasometric determination following 6.0 M HCl application [76 ]. Water-soluble cations and anions (1:2.5 DI water extract) were determined using standard methods [76 ]: Na⁺ using a Sherwood, flame photometer (MODEL 360), Ca²⁺, Mg²⁺, and K⁺ using ICP-OES Thermo Scientific™ iCAP™ 7000 Plus Series, CO₃²⁻ and HCO₃⁻ by titration with a standardized H₂SO₄ solution and Cl⁻ by AgNO₃ titration. Total organic elements concentration in soil (C, N, H, and S) was determined using dry combustion method by a Thermo Scientific Flash 2000 analyzer. Available concentrations of PTEs were determined using ICP-OES after extraction by diethylene tri-amine Penta acetic acid (DTPA).