## 2 Results
### The Optimization of Genetic Transformation Methods for D. zingiberensis  
This study established a more efficient and rapid genetic transformation system (Figure 3), with the advancement providing a more effective genetic transformation approach for the study of diosgenin biosynthesis and other secondary metabolite production in _Dioscorea_ species. Genetic transformation within the _Dioscorea_ genus has long been a challenge, with most functional gene studies relying on heterologous expression systems or unstable transformation methods [11; 43]. Reports of stable genetic transformation using _Dioscorea_ species as host plants are limited [23; 24; 25]. The main obstacles include low transformation efficiency and a prolonged period from _Agrobacteriumium_ infection to the regeneration of complete transgenic plants (Table 1), which have hindered in-depth functional genomics research in this genus. Figure 3: The time course of genetic transformation in _D. zingiberensis._ (**a**) After _Agrobacteriumium_ infection of buds, explants were transferred to a co-cultivation medium, then to a callus induction and selection medium, and subsequently to a callus subculture and selection medium. The dedifferentiation and callus formation status at 7, 14, 28, and 35 DAI are shown. (**b**) Calli were moved to a regeneration and selection medium for shoot induction. Images show regenerated shoots at 63, 77, and 91 DAI. (**c**) Regenerated shoots were transferred to a rooting medium for root development, with images taken at 105 and 119 DAI. In our system, we achieved a transformation success rate of 46.8%, with transgenic plants regenerated in just four months (Figure 3). The buds of _D. zingiherensis_ are infected with _Agrobacteriumum_ carrying the _DZHDZ32_ gene and then transferred to a co-cultivation medium for co-cultivation. Day 0 is the day when _Agrobacteriumum_ infection (DAI) occurs. After 2 days of co-cultivation with the _Agrobacteriumum_, the bacterium is washed off, and the buds are transferred to a callus induction and selection medium to induce callus formation. By day 7 (7 DAI), noticeable tissue swelling can be observed, which becomes more pronounced by day 14 (14 DAI). At this point, the buds are transferred to a callus subculture and selection medium (which does not contain 2,4-D) to continue callus induction. Once the callus reaches the 35 DAI stage (Figure 3), it can be transferred to a regeneration and selection medium and placed under light. Approximately 4 weeks later, regenerated shoot points can be observed. When the regenerated shoots reach 91 DAI (Figure 3), they can be transferred to a rooting medium to induce rooting. About 2 weeks later (105 DAI), substantial regenerated roots can be observed, and after 4 weeks (119 DAI), the regenerated roots are fully developed. In the genus _Dioscorea_, transformation efficiencies vary greatly across different species. Specifically, the genetic transformation of _D. zingiherensis_ is exceptionally difficult. Nevertheless, our research group has made significant breakthroughs in this area. Building on these advances, we have significantly reduced the time required while achieving higher transformation efficiency. Apical and axillary buds of _D. zingiherensis_ were used as explants due to their low contamination rate--only 1%--which is markedly lower than that observed in tissues such as seeds, flowers, or leaves. This low contamination rate provided a solid foundation for subsequent transformation experiments. During transformation, the addition of 2,4-D (2,4-dichlorophenoxyayacetic acid) promoted dedifferentiation but inhibited redifferentiation of the tissues. In the early dedifferentiation stage, high concentrations of 2,4-D promoted callus formation from the buds. Once the callus was established, explants were transferred to a subculture medium containing NAA (naphthaleneacetic acid) and 6-BA (6-benzylaminopurine) but lacking 2,4-D to relieve the inhibitory effect and promote shoot regeneration.