## 4 Materials and Methods
### Transgenic Overexpression of DZHDZ32 in D. zingiberensis  
The pCXUN-4 XHA vector containing _DZHDZ32_ (pCXUN-_DZHDZ32_-4 XHA) was constructed using a homologous recombination method and subsequently transferred into the _Agrobacterium_ strain EHA105. The hygromycin antibiotic resistance gene present in pCXUN-4 XHA was replaced with a kanamycin resistance gene through homologous recombination. Prior to infecting _D. zingiberensis_, the activated _Agrobacterium_ was transferred into a suspension medium (Supplementary Materials Table S3) and incubated at 28 \({}^{\circ}\)C with shaking at 180 rpm for 3 to 3.5 h. The _Agrobacterium_ suspension was then adjusted to a concentration corresponding to an OD600 value of 0.8. _Agrobacterium_ adjusted to an optimal concentration was utilized for transient expression and genetic transformation. In this transient expression experiment, the suspension was incubated in the dark at 22 \({}^{\circ}\)C for 2 h. Following incubation, it was injected into the abaxial side of young leaves of _D. zingiberensis_. The leaves were then kept in the dark for 12-16 h, followed by exposure to light for 17-21 h before samples were collected. For genetic transformation, the apical and axillary buds of the WT were surface sterilized by immersion in alcohol for 30 s, followed by disinfection in a 2% NaClO solution for 7 min. The buds were then rinsed three times with sterile water. The sterilized buds were immersed in the _Agrobacterium_ suspension and subjected to infection for 30 min. Excess moisture on the surface of the buds was removed using sterile filter paper, and the buds were then placed under a layer of filter paper and dried on a super-clean workbench. The buds were transferred onto a co-cultivation medium overlaid with a layer of filter paper and cultured in the dark at 25 \({}^{\circ}\)C for two days on the co-cultivation medium (Supplementary Materials Table S3). Following this, the buds were washed seven times with sterile water and soaked in a solution containing 400 mg/L of timentin for 30 min. The buds were then transferred to a callus induction and selection medium (Supplementary Materials Table S3). After two weeks, the callus was subcultured onto a callus subculture and selection medium (Supplementary Materials Table S3). Following several weeks, the callus was transferred to a regeneration and selection medium (Supplementary Materials Table S3). After several weeks of growth on the regeneration and selection medium, the antibiotic-resistant shoots were transferred to a rooting medium (Supplementary Materials Table S3). Determination and Analysis of Diosgenin and Its Intermediates Cholesterol and \(\beta\)-Sitosterol  
Diosgenin was extracted from _D. zingiberensis_ following previously described methods [1; 11; 59; 60]. The roots, stems, leaves, and rhizomes from OE lines and WT plants were freeze-dried and ground into a fine powder. A 50 mg aliquot of each sample was subjected to acid hydrolysis with 2 mL of 1 M H\({}_{2}\)SO\({}_{4}\) at 100 \({}^{\circ}\)C for 4 h. After centrifugation at 13,000 rpm for 10 min, the supernatant was discarded. The pellet was washed repeatedly with 1 M NaOH, followed by centrifugation at 13,000 rpm for 10 min each time until the pH of the supernatant reached approximately 7. The resulting precipitate was dried at 55 \({}^{\circ}\)C and reground into powder. One milliliter of isopropanol was added to the powder, followed by shaking at 200 rpm at 50 \({}^{\circ}\)C for 30 min. The mixture was then subjected to ultrasonication for 60 min and centrifuged at 13,000 rpm for 10 min. The supernatant was filtered through a 0.22 \(\upmu\)m membrane and transferred to sample vials for analysis. Quantification was performed using UPLC-MS/MS (Thermo Scientific, Waltham, MA, USA), and data were processed using Xcalibur 4.1 software (Thermo Scientific, Waltham, MA, USA). Three biological replicates were analyzed for each sample. Cholesterol and \(\beta\)-sitosterol were quantified as previously described [4].