植物研究2026，Vol.46Issue(3)：458-470,13.DOI:10.7525/j.issn.1673-5102.2026.03.007
穿心莲不同材料来源原生质体制备及瞬时转化体系建立
Establishment of a Protoplast Isolation and Transient Transformation System from Different Explant Sources of Andrographis paniculata
摘要
Abstract
This study aimed to optimize the preparation methods of protoplasts from cotyledons and root callus of Andrographis paniculata,and to investigate the key factors affecting their transient transformation,thereby laying a technical foundation for subsequent genetic transformation and gene function research in A.paniculata.The effects of different mannitol concentrations,enzyme concentrations,and enzymatic hydrolysis time on protoplast yield and viability were studied using the enzymatic hydrolysis method.The PEG-mediated transformation method was employed to examine the influence of factors such as PEG concentration and transformation time on transient transformation efficiency,and a transient transformation system for A.paniculata root callus protoplasts was established.The optimal conditions for protoplast preparation from A.paniculata cotyledons were as follows:0.2 g cotyledons were enzymatically hydrolyzed in a solution containing 4.0%cellulase R-10,1.5%macerozyme R-10,and 0.6 mol·L-1 mannitol at 26℃and 55 r·min-1 in darkness for 4 h.Under these conditions,the protoplast yield reached 3.32×10⁵ cells·mL-1,with a viability of 90.73%.For root callus protoplasts,the optimal conditions were:0.4 g 15-day-old callus was enzymatically hydrolyzed in a solution containing 4.0%cellulase R-10,1.0%macerozyme R-10,and 0.8 mol·L-1 mannitol under the same conditions for 9 h,yielding 2.18×106 cells · mL-1 with a viability of 94.19%.In PEG-mediated transient transformation,the optimal conditions were determined to be 40%PEG 4000 treatment for 30 min,10 μg of plasmid DNA,and 0.6 mol·L-1 mannitol.Under these conditions,the transformation efficiency reached 58.03%.Significant differences existed in the optimal preparation conditions for protoplasts derived from different tissues of A.paniculata,emphasizing the need for tissue-specific optimization.This study established an efficient protoplast preparation and transient transformation system for both cotyledons and root callus of A.paniculata,providing a reliable technical platform for future gene function analysis and cell biology research in this medicinal plant.关键词
穿心莲/原生质体/根愈伤组织/子叶/瞬时转化Key words
Andrographis paniculata/protoplast/root callus/cotyledon/transient transformation分类
农业科技引用本文复制引用
丁妹,杨帆,刘安然,杜勤..穿心莲不同材料来源原生质体制备及瞬时转化体系建立[J].植物研究,2026,46(3):458-470,13.基金项目
广东省科技特派员项目(KTP20200137)、广州市重点研发和社会发展科技计划项目(2023B03J1273). （KTP20200137）
