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.