Yuan Guoliang, Islam Md Torikul, Tuskan Gerald A, Yang Xiaohan
Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Bio Protoc. 2025 Feb 20;15(4):e5214. doi: 10.21769/BioProtoc.5214.
Gene stacking, the process of introducing multiple genes into a single plant to enhance desired traits, is essential for plant genetic improvement through both conventional breeding and genetic transformation. In general, transformation-based gene stacking can be achieved through either co-transformation to simultaneously introduce multiple genes or sequential multi-round transformation. While co-transformation is generally faster and more efficient than sequential multi-round transformation, it often requires two selectable marker genes, which confer resistance to antibiotics, for selecting transgenic events. However, in most cases, there is only one best selectable marker gene for a specific plant species or genotype. Also, it is harder to optimize the concentrations of two antibiotics for co-transformation than using one antibiotic for selecting transgenic events. To overcome this challenge, we recently developed an innovative split selectable marker system for plant co-transformation, allowing the use of one selectable marker gene to select transgenic events. This method involves constructing two binary vectors, each carrying a subset of genes of interest and a partial fragment of the selectable marker gene, which is connected to a partial intein fragment. Following -mediated co-transformation, plants harboring both binary vectors are selected using a single antibiotic, such as kanamycin. This split-marker system can be used to co-transform multiple genes into both herbaceous and woody plants, accelerating genetic improvement of polygenic traits or integrative improvement of multiple traits to simultaneously increase crop yield and quality. Key features • Developed an intein-mediated split selectable marker system for efficient gene stacking in plants. • Utilizes a single antibiotic for identifying transgenic events, simplifying the selection process of co-transformation compared to traditional methods. • Applicable to both herbaceous and woody plant species for co-transforming multiple genes. • Enhances scalability and feasibility of gene stacking in plant genetic engineering and crop improvement initiatives.
基因堆叠是将多个基因导入单个植物以增强所需性状的过程，对于通过传统育种和遗传转化进行植物遗传改良至关重要。一般来说，基于转化的基因堆叠可以通过共转化同时导入多个基因或通过连续多轮转化来实现。虽然共转化通常比连续多轮转化更快、更有效，但它通常需要两个赋予抗生素抗性的选择标记基因来筛选转基因事件。然而，在大多数情况下，对于特定的植物物种或基因型，只有一个最佳的选择标记基因。此外，与使用一种抗生素筛选转基因事件相比，优化两种抗生素用于共转化的浓度更加困难。为了克服这一挑战，我们最近开发了一种用于植物共转化的创新型分裂选择标记系统，允许使用一个选择标记基因来筛选转基因事件。该方法涉及构建两个二元载体，每个载体携带感兴趣基因的一个子集和选择标记基因的一个部分片段，该片段与一个部分内含肽片段相连。通过农杆菌介导的共转化后，使用单一抗生素（如卡那霉素）筛选含有两个二元载体的植物。这种分裂标记系统可用于将多个基因共转化到草本植物和木本植物中，加速多基因性状的遗传改良或多个性状的综合改良，以同时提高作物产量和品质。关键特性：• 开发了一种内含肽介导的分裂选择标记系统，用于在植物中高效进行基因堆叠。• 使用单一抗生素鉴定转基因事件，与传统方法相比简化了共转化的筛选过程。• 适用于草本和木本植物物种，用于共转化多个基因。• 增强了植物基因工程和作物改良计划中基因堆叠的可扩展性和可行性。
