2.3 | Seedling cultivation
Under aseptic conditions, test tube seedlings with 3-5 uniform long roots weighing approximately 0.5 g were carefully extracted from the culture flasks, and any adhering agar was eliminated using sterile filter paper. The seedlings were weighed and transferred to culture boxes containing 600 g of shale pellets, 2 plants per box. A total of 96 boxes were planted. Each box was filled with 100 mL of sterile water and 5 mL of sterilized Hoagland’s total nutrient solution at the seedlings’ roots. Each box’s total weight was recorded as a reference value for irrigation. Place on the culture rack in the tissue culture room for cultivation at 25℃/20℃ (day/night) and a light intensity of 3000 lx. Weigh and replace the water every 3 days to maintain 75% WHC of the culture medium, and water the roots of the seedlings with 5 mL of Hoagland’s full nutrient solution each week. After 4 weeks of incubation, the plants were randomly divided into 16 groups of 6 boxes each and watered with the nutrient solution required for the different stress treatment groups. After 3 weeks of culture, observe the morphological characteristics of the plant’s nitrogen and phosphorus deficiency stress and then perform other synergistic stress treatments. The natural precipitation method was used for drought treatment in the drought synergistic stress treatment group. After the WHC dropped to 30%, the 30% WHC was maintained until the end of the treatment. The plants were transferred to an incubator at 42℃/35℃ (day/night) under the same light conditions for the temperature stress treatment. All experiments were completed after 5 days of heat stress.