Abstract
Grape (Vitis L.), a highly valued fruit crop, poses significant challenges in genetic transformation and functional characterization of genes. Therefore, there is an urgent need for the development of a rapid and effective method for grape transformation and gene function identification. Here, we introduce a streamlined Agrobacterium-mediated transient transformation system for grape calli. Optimal conditions were established with a leaf-derived callus induction medium; chiefly B5 medium supplemented with 0.05 mg/L NAA, 0.5 mg/L 2,4-D, and 2.0 mg/L KT; and a callus proliferation medium (B5 medium supplemented with 0.5 mg/L NAA and 2.0 mg/L 6-BA), respectively. Notably, GUS enzyme activity peaked (352.96 ± 33.95 mol 4-MU/mg/min) by sonication with Agrobacterium tumefaciens EHA105 and 100 μM AS for 4 min, followed by vacuum infection for 5 min, and co-culture at 25 °C in the dark for 1 day using callus as explants at an optical density (OD600) of 0.8. VaCIPK18 gene was transiently transformed into calli, and transcripts of the gene (endogenous and exogenous) were detected at higher levels than in non-transformed calli (endogenous). Moreover, after 10 days of treatment at 4 °C or −4 °C, the callus net weight of transformed callus was significantly higher than that of the untransformed callus, indicating that the VaCIPK18-overexpressing grape callus could improve cold tolerance. Overall, we establish a simple but effective transient transformation approach for grape callus, which could serve as a useful tool for the rapid assessment of gene function in this important crop.
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Funding
This work is supported by the National Natural Science Foundation of China (grant no. 32060672), the Agricultural Breeding Project of Ningxia Hui Autonomous Region (NXNYYZ202101), and the Ningxia Hui Autonomous Region Key R&D Program (grant number 2023ZDYF0830).
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JW and JZ: performed the experiments, writing—original draft. XH: technical support. KL and YX: revision of the manuscript, validation of results. WX: conceived and designed the research, writing—review and editing.
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Wu, J., Zhang, J., Hao, X. et al. Establishment of an efficient callus transient transformation system for Vitis vinifera cv. ‘Chardonnay’. Protoplasma 261, 351–366 (2024). https://doi.org/10.1007/s00709-023-01901-2
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DOI: https://doi.org/10.1007/s00709-023-01901-2