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Establishment of rapeseed (Brassica napus L.) cotyledon transient transformation system for gene function analysis

Zhi-Yan Zhang, Guan-Ying Li, Jie-Li Wang, Xiao-Juan Guo, Zheng Wang and Xiao-Li Tan

Rapeseed (Brassica napus) is second leading oil crop worldwide, due to its importance, genomes of its relative Brassica rapa (AA genome), Brassica oleracea (CC genome), and itself AACC have been sequenced successively. Functional genomic study of rapeseed is urgently needed to elucidate the molecular mechanisms of its yield, quality, and stress resistance. Rapeseed is recalcitrant to genetic transformation compared with other dicots. Therefore, we seek to develop a simple and efficient transient transformation system in rapeseed cotyledons. The beta-glucuronidase (GUS) reporter gene was employed to evaluate the transient expression levels. Agrobacterium strains, densities of agrobacteria cell suspension, and the infiltrated time were optimized. To verify the reliability of this system,subcellular localization and bimolecular fluorescence complementation (BiFC) were used. We found that the A. tumefaciens strain GV3101 was the suitable strain for transformation of the rapeseed cotyledons. Among the different incubation times, four days after infiltration with Agrobacterium GV3101 cells (OD600=0.8) provided the highest frequency of transformation. Subcellular localization and BiFC assays demonstrated a highly efficient transient transformation system in rapeseed cotyledons. The cotyledon transit expression system is efficient and fast and could accelerate functional genomics studies in B. napus

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