Abstract: INTRODUCTION: Succulent plants have high added value with high morphological diversity and strong stress resistance. However, the industrial production of these plants encounters obstacles such as the challenge of low propagation efficiency and small seed quantities. Tissue culture technology offers a novel solution to overcome these challenges. However, significant differences in the culture medium used between different species and the difficulty of cross group generalization of tissue culture parameters have increased the difficulty of establishing rapid propagation systems. RATIONALE: Therefore, the three succulent plants with diverse colors and shapes ( Echeveria ‘Pink Rubby’, Crassula fusca, and Disocactus flagelliformis) from two families and three genera were selected as experimental materials in this study. The universal issue culture system and freezing section techniques for succulent plants were be established, including procedures for disinfecting leaf/stem explants, differentiating adventitious buds, rooting adventitious buds (using traditional and open tissue culture methods), and transplanting. A frozen section technique to facilitate the analysis of anatomical structural differences between tissue culture plantlets and wild seedlings. RESULTS: We found that the optimal medium for leaf/stem differentiation adventitious bud was MS supplemented with 3.0 mg·L^–1 6-BA and 0.8 mg·L^–1 NAA, achieving an induction rate of over 95%. For adventitious bud rooting, the optimal medium of MS with 4.0 mg·L^–1 IBA, 2.0 mg·L^–1 NAA, and 0.4 mg·L^–1 KT, further enhanced with an additional 0.3 mg·L^–1 S206 in open tissue culture, resulted in a rooting rate exceeding 90%. The optimal universal transplanting substrate was a mix of grass charcoal soil and sand at a ratio of 2:1 by volume, resulting in a survival rate exceeding 99%. In addition, we found that the epidermal thickness of tissue cultured plantlets of three succulent plants were significantly thinner than that of wild seedlings. Only the vascular bundle area of Disocactus flagelliformis was much smaller than that of wild seedlings, but the number of vascular bundles of tissue cultured plantlets was significantly higher than that of wild seedlings. The frozen section technique was established uses conventional glue embedding, offering low cost, ease of operation, and universality. CONCLUSION: Our findings offer theoretical underpinning for establishing an efficient different species propagation system and frozen section technique. This study provides practical references for the rapid propagation and transplantation domestication of succulent plants.

Key words: Echeveria ‘Pink Rubby’, Crassula fusca, Disocactus flagelliformis, tissue culture, anatomical structure