2.1. Materials and Growth Conditions of Plants

Based on our unpublished data, four cotton genotypes were used, including BX014 and LuYuan343 (LP tolerant), FJA and DLNTDH (LP sensitive). Hydroponically cultured plants were grown at the Cotton Research Institute of the Chinese Academy of Agricultural Sciences, Anyang, China.

Healthy seeds were surface sterilized with ethanol (70% v/v) for 10 min and disinfected with 3% sodium hypochlorite (v/v) for 20 min. The seeds were then rinsed five times with distilled water. Both cotton genotypes were incubated in a growth chamber in a combination of sand and vermiculite (w/w). This study was conducted using nutrient-solution hydroponics. The seedlings were transplanted into 8 L plastic boxes after germination. The plants were grown under natural light in a greenhouse at 25/20 °C (day/night) temperatures and 60% humidity. Each genotype was sown in seven replicates for each P treatment, each plant as a repeat. The seedlings were treated with various nutrient supplies at the two true-leaf stages. After transplantation, the seedlings were provided with 1/2-strength Hoagland solution for one week, followed by full-strength Hoagland solution until the end of the experiment. The plants were provided with 100 mL of dH₂O daily, to replenish the water lost by transpiration. Each plant’s location was changed randomly each week to counteract the impact of the positioning [31].

The conventional nutrient solution was as follows: 0.1 mmol/L EDTA·Fe·Na, 1 mmol/L MgSO₄·7H₂O, 2 μmol/L ZnSO₄·7H₂O, 46 μmol/L H₃BO₃, 4 μmol/L MnCl₂·4H₂O, 0.3 μmol/L CuSO₄·5H₂O, 0.12 μmol/L (NH₄)₆Mo₇O₂₄·4H₂O, 2.5 mmol/L Ca (NO₃)₂·4H₂O, and combinations of three different P concentrations, including 15 µM and 50 μM (low P) and 500 μM (normal P), supplied as KH₂PO₄ in hydroponic culture. The nutrient solutions were continuously aerated with an electric pump, the airflow rate was 2 L/min, and the pH was maintained at 5.8 ± 0.5. The nutrient solutions were changed every seven days and aerated. The whole test period was 28 days, and the plants showed obvious signs of P deficiency; seven plants from each treatment were collected for further physiological measurements.