【目的】建立并优化农杆菌介导的杉木(Cunninghamia lanceolata)组培苗瞬时遗传转化体系。【方法】以增殖培养的杉木845B无性系组培苗为受体材料,采用携带GUS的pCAMBIA1301载体进行瞬时转化,先后进行正交试验及系列递进优化试验,根据GUS染色、GUS酶活性测定及GUS的表达量筛选最佳转化条件。【结果】通过正交试验系统建立杉木瞬时转化体系,所选5个关键因素的影响程度从大到小依次为共培养时间 > 侵染液浓度 > 侵染时间 > 乙酰丁香酮(AS)浓度 > 渗透压;最优组合条件为3%(质量分数,下同)蔗糖、200 μmol/L AS、侵染液A₆₀₀为1.2,侵染3 h(1.5 h时稀释至A₆₀₀为 0.8),共培养48 h。递进优化试验进一步发现:①不稀释侵染液(A₆₀₀ 为1.2)直接侵染2.0 h 可获最佳效果[酶活160 pmol/(μg·min)],表达量提升109倍;②EHA105菌株转化效率显著高于其他菌株[酶活514 pmol/(μg·min)],表达量提升213倍;③25 min真空处理(相对真空度-60 kPa)及23 ℃共培养能够优化转化效果[酶活112、166 pmol/(μg·min)],表达量分别提升76和97倍。【结论】成功建立了一种简便高效的杉木845B无性系组培苗瞬时遗传转化体系,方法流程为①制备含3%蔗糖、200 μmol/L AS的重悬液。②使用该重悬液重悬根癌农杆菌EHA105菌体,制备成A₆₀₀ = 1.2的侵染液。③使用该侵染液侵染2.0 h(期间辅以25 min的抽真空处理)。④23 ℃共培养48 h。本研究为杉木的基因功能研究提供了实践平台。

【Objective】Cunninghamia lanceolata, a pivotal conifer species in subtropical forestry, plays a vital role in timber supply, ecological restoration, and carbon sequestration. However, the lack of an efficient transient genetic transformation system has long impeded functional genomics research, restricting the exploration of gene functions associated with growth regulation, stress adaptation, and wood formation in this economically and ecologically important species. This study aims to establish and systematically optimize an agrobacterium-mediated transient genetic transformation system for C. lanceolata clone 845B tissue-cultured seedlings, thereby constructing a reliable technical platform for gene functional analysis.【Method】Proliferated tissue-cultured seedlings of the elite clone 845B were employed as receptor materials, and the pCAMBIA1301 vector harboring the β-glucuronidase (GUS) reporter gene was utilized for transient transformation. A multi-stage optimization strategy integrating orthogonal array design and sequential single-factor experiments was implemented to identify optimal transformation conditions. The orthogonal experiment focused on five critical parameters, co-cultivation duration, bacterial suspension density (A₆₀₀), infection time, acetosyringone (AS) concentration, and osmotic pressure, to evaluate their relative impacts on transformation efficiency, with assessments based on GUS staining, GUS enzyme activity quantification, and GUS gene expression analysis.【Result】Orthogonal analysis revealed a hierarchical order of the five factors’ influence: co-cultivation duration > bacterial suspension density > infection time > AS concentration > osmotic pressure. The initial optimal combination derived from the orthogonal design comprised 3% (mass fraction, same below) sucrose (osmotic regulator), 200 μmol/L AS (vir gene inducer), an agrobacterium suspension with A₆₀₀ of 1.2, a two-stage infection protocol (3 h total, with dilution to A₆₀₀ 0.8 at 1.5 h), and 48 h of co-cultivation. Subsequent single-factor optimization further refined the protocol. First, eliminating the dilution step and maintaining an undiluted bacterial suspension (A₆₀₀=1.2) for 2.0 h of continuous infection achieved peak performance, with GUS enzyme activity reaching 160 pmol/(μg·min) and GUS gene expression increasing by 109-fold compared to suboptimal conditions. Second, strain selection demonstrated that Agrobacterium tumefaciens EHA105, a hypervirulent strain, significantly outperformed other tested strains, yielding a GUS enzyme activity of 514 pmol/(μg·min) and a 213-fold increase in GUS expression, underscoring the critical role of strain virulence in transformation efficiency. Third, physical enhancement measures were evaluated: 25 min of vacuum infiltration at a relative vacuum degree of -60 kPa improved GUS activity to 112 pmol/(μg·min) and elevated expression by 76-fold, while adjusting the co-cultivation temperature to 23 ℃ further enhanced GUS activity to 166 pmol/(μg·min) and increased expression by 97-fold, indicating that precise control of physical parameters (vacuum and temperature) is essential for optimizing agrobacterium-plant interactions. Based on these systematic optimizations, the finalized efficient transient transformation protocol for C. lanceolata clone 845B is as follows: (1) prepare a resuspension buffer containing 3% sucrose and 200 μmol/L AS. (2) Resuspend Agrobacterium tumefaciens EHA105 cells in this buffer to an A₆₀₀ of 1.2 to prepare the infection solution. (3) Infect tissue-cultured seedlings with the infection solution for 2.0 h, supplemented with 25 min of vacuum infiltration (-60 kPa) during the infection process. (4) Co-cultivate the infected seedlings at 23 ℃ for 48 h.【Conclusion】This study successfully established a simple, efficient, and reproducible agrobacterium-mediated transient genetic transformation system for C. lanceolata. The protocol overcomes key technical bottlenecks in conifer transformation, including low agrobacterium infectivity, poor plant material compatibility, and suboptimal co-cultivation conditions. The systematic optimization of parameters (bacterial density, infection duration, AS concentration, strain selection, vacuum infiltration, and temperature) provides a comprehensive framework for adapting transient transformation to other conifer species. The developed system will significantly accelerate functional genomics research in C. lanceolata, enabling rapid validation of gene functions related to growth regulation, disease resistance, drought tolerance, and wood property modification. Furthermore, it lays a solid technical foundation for molecular breeding of elite C. lanceolata varieties, contributing to the sustainable development of subtropical forestry.