INTRODUCTION: Genetic transformation, combined with genome editing strategies, has provided essential insights into plant biology and revolutionized crop improvement. MicroTom ( Solanum lycopersicum ‘MicroTom’) is widely used for functional characterization due to its short life cycle and clear genetic background. However, traditional tissue culture-dependent genetic transformation systems for MicroTom are constrained by low efficiency, long durations, and highly trained individuals. Therefore, developing a rapid and efficient tissue culture-independent genetic transformation system is necessary. RATIONALE: Agrobacterium-mediated transformation is the most widely used method for gene transfer in plants. During this process, wounded plant cells secrete phenolic compounds that induce Agrobacterium cells to transfer and integrate foreign DNA into plant chromosomes. In this study, we established a novel transformation protocol using two-week-old MicroTom seedlings as recipients. After removing the apical cotyledons and true leaves, the wounded hypocotyls were directly inoculated with an A. tumefaciens suspension. Based on the somatic cell reprogramming mechanism triggered by wounding signaling, the wound hypocotyls formed calli and regenerated adventitious shoots, accompanied by the integration of foreign DNA. RESULTS: Two-week-old seedlings with hypocotyl diameters exceeding 1.5 mm were optimal for Agrobacterium infection. After a 2-day preculture in darkness, the hypocotyls were infected with A. tumefaciens suspension (OD ₆₀₀=0.6) for 10 min and then cocultured in darkness at 90% relative humidity for 3 d. Callus differentiation was observed at the hypocotyl ends at 10 days post-inoculation (dpi), and adventitious shoots regenerated at 20 dpi. Mature T ₀-generation seeds could be harvested within 4–5 months post-inoculation. Approximately 87.6% of the wounded hypocotyls regenerated adventitious shoots at 20 dpi, and PCR analysis confirmed that 28.6% of the regenerated shoots contained the foreign gene. Through antibiotic screening combined with an EGFP reporter system, the stable expression rate of the foreign gene in T ₁-generation lines reached 73.5%. CONCLUSION: Compared to conventional tissue culture-dependent systems, the established in planta transformation in this study offers improved transformation efficiency, a shorter transformation cycle, and simplified nonsterile operational procedures. This system provides a robust platform for functional genomics studies and significantly lowers technical barriers in tomato molecular breeding.