Establishment of a Split-Root System

A commercial cotton (Gossypium hirsutum cv ‘K836’), which was developed by our lab, was used in the experiments. One kilogram of cotton fuzz seeds was placed in a plastic container, treated with 100 mL of concentrated sulfuric acid, and stirred for 1 to 2 min for acid delinting. The acid-delinted seeds were then washed six times and sun dried for 2 to 3 d. The seeds were sown at 2 cm depth in plastic boxes (60 cm × 45 cm × 15 cm) containing sterilized wet sand. The boxes were placed in a greenhouse with a light/dark regime of 16/8 h, a light intensity of 400 μmol m⁻² s⁻¹ photosynthetically active radiation, and a temperature of 30°C ± 2°C. Seedlings were thinned to 100 plants per box at full emergence. At the two-true-leaf stage, uniform seedlings were carefully removed from the sand and washed with distilled water. Split-root systems were established by grafting with these seedlings as described by Kong et al. (2012, 2017). Briefly, a slash (/)-shaped incision was made with a blade on the hypocotyl 2 cm below the two cotyledons, leaving about one-third of the hypocotyl tissues intact. The top of the rootstock was cut to form a deep triangle (△) at the same position of the hypocotyl from another seedling. The triangle section was then inserted into the slash incision of the plant and firmly wrapped with Parafilm. Grafted seedlings were transferred into plastic boxes containing aerated nutrient solution and sprayed with water to prevent wilting. The nutrient solution was topped up with deionized water as required and renewed weekly. The solution consisted of 1.25 mm Ca(NO₃)₂, 1.25 mm KNO₃, 0.5 mm MgSO₄, 0.25 mm NH₄H₂PO₄, 0.05 mm EDTA-FeNa, 10 μm H₃BO₃, 0.5 μm ZnSO₄, 0.1 μm CuSO₄, 0.5 μm MnSO₄, and 0.0025 μm (NH₄)₆Mo₇O₂₄ and was adjusted to pH 6 with KOH. When a new leaf emerged from the grafted seedling 2 weeks after grafting, the Parafilm was removed. Grafted seedlings with two uniform split-root systems were selected and allowed to grow in a greenhouse under a 14/10-h (light/dark) photoperiod at 30°C/26°C and relative humidity of 60%/80% for 30 d. Nutrient solutions were renewed daily. Strong seedlings with uniform split roots were selected for further study.