Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C₄ and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C₄/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C₄/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each experimental condition. Furthermore, different explant sources, Agrobacterium tumefaciens strains, and regeneration and antibiotic selection media were tested in various combinations to optimise a protocol for stable genetic transformation of P. oleracea. Altogether, we provide essential tools for functional gene analysis in the context of C₄/CAM photosynthesis, including an efficient RNA isolation method, preferred reference genes for RT-qPCR normalisation for a range of experimental conditions, and a protocol to produce P. oleracea stable transformants using A. tumefaciens.

_{C 4}和景天酸代谢 (CAM) 光合作用在单个生物体中的发生以前被认为是一种有趣的光合作用好奇心，最近已成为未来生物技术应用的信息来源。在 C ₄ /CAM 兼性物种中，Portulaca oleracea L. 已被用作 C ₄生化和基因表达分析的模型/CAM 在现场和实验室条件下。在目前的工作中，我们专注于开发分子工具以促进该物种的功能基因组学研究，从 RNA 分离方案的优化到稳定遗传转化的方法。对 RNA 提取程序的 11 种变体进行了测试，并比较了 RNA 的数量和质量。此外，7 个样本集包含来自激素和非生物胁迫处理、不同植物器官、叶片发育阶段和亚种的总 RNA，用于在 12 个参考基因中选择最稳定的参考基因，用于每个实验条件的 RT-qPCR 分析。此外，不同的外植体来源，根癌农杆菌菌株、再生和抗生素选择培养基以各种组合进行测试，以优化用于P. oleracea稳定遗传转化的方案。_{总而言之，我们为 C 4} /CAM 光合作用背景下的功能基因分析提供了必要的工具，包括高效的 RNA 分离方法、用于一系列实验条件的 RT-qPCR 标准化的首选参考基因，以及生产P. oleracea的方案使用A. tumefaciens 的稳定转化体。