2.1 | Study site and test materials
Tissue culture seedlings were selected from Chengdu University of Traditional Chinese Medicine by excising young leaves from the stem apex of the same plant and cultivating them into tissue culture seedlings after surface sterilization, tissue healing, bud clustering, and root induction in the laboratory (E: 103.81°, N: 30.68°).
The culture medium utilized was shell shale sourced from Ximeishan Village (E: 104.54°, N: 30.94°), Shiquan Township, Zhongjiang County, Sichuan Province. The medium was air-dried, passed through a 1-5 mm sieve, cleansed of impurities, sterilized at 180℃ for 3 h in a dry heating process, and cycled twice to eliminate microorganisms and their exudates. After cooling, it was packed in kraft paper and then sterilized again through dry heat at 120℃ for 3-4 h before use. The pH value of the shell shale was 7.78, with an organic matter content of 0.69 g/kg, alkaline nitrogen of 4.2 mg/kg, potassium of 3.79 mg/kg, and phosphorus of 2.23 mg/kg.
The culture device used was a circular polypropylene box with a top diameter of 17.2 cm, a bottom diameter of 11.8 cm, and a height of 9.7 cm. Each box was wrapped in kraft paper and sterilized at high-pressure steam of 0.1MPa and 121℃ for 30 min, and after cooling, filled with 600 g of culture medium.