Improving Agrobacterium tumefaciens-mediated genetic transformation for gene function studies and mutagenesis in cucumber (Cucumis sativus)

Authors: LIU, HANQIANG - University Of Wisconsin; ZHAO, JIANYU - University Of Wisconsin; CHEN, FEIFAN - University Of Wisconsin; WU, ZHIMING - Hebei Academy Of Agriculture; TAN, JUNYI - University Of Wisconsin; NGUYEN, NHIEN HAO - University Of Wisconsin; CHENG, ZHIHUI - Northwest A&f University; Weng, Yiqun

Submitted to: Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/2023
Publication Date: 2/27/2023
Citation: Liu, H., Zhao, J., Chen, F., Wu, Z., Tan, J., Nguyen, N., Cheng, Z., Weng, Y. 2023. Improving Agrobacterium tumefaciens-mediated genetic transformation for gene function studies and mutagenesis in cucumber (Cucumis sativus). Genes. 14(3), 601. https://doi.org/10.3390/genes14030601.
DOI: https://doi.org/10.3390/genes14030601

Interpretive Summary: We are in the post-genomics era. Many genes are being cloned, but their functions need to be characterized. New gene editing technologies also provided a power tool for crop improvement. The Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for study of gene functions and crop improvement in cucumber. However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting shoot regeneration and overall transformation efficiency in cucumber including varieties, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, as well as antibiotics for selection of positive transformants. We show that, in general, Asian long cucumbers had higher shoot regeneration efficiency than US pickling or slicing cucumbers. Cotyledon and hypocotyl from germinating seeds had similar shoot regeneration efficiency, which also depends on germination. Transformation with the Agrobacterium strain AGL1 yielded higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective for selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants in Poinsett 76 slicing and H19 pickling cucumbers. Screening among transgenic plants identified novel, inheritable mutants for fruit size/shape, leaf color/shape, sex expression or plant architecture suggesting T-DNA insertion as a potential source of mutagenesis. We developed a protocol for Agrobacterium-mediated transformation in cucumber which could be used as the baseline for further improvement of cucumber transformation. This work should be of valuable to cucumber researchers and breeders.

Technical Abstract: In the post-genomics era, Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber, Cucumis sativus L. However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting shoot regeneration and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, as well as selective agents. We show that, in general, North China cucumbers exhibited higher shoot regeneration efficiency than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledon or hypocotyl had similar shoot regeneration efficiency, which was also affected by the harvest time of explants. Transformation with the Agrobacterium strain AGL1 yielded higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in screening out non-transformants or chimeras during shoot regeneration (at 100 mgL-1) or rooting (at 50 mgL-1) . Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene in Poinsett 76 and H19 or RNAi lines for the CsAPRR2 gene in Poinsett 76 cucumber line with the highest transformation efficiency around 1% (1 positive transgenic plant from 100 seeds). Screening among T1 transgenic plants identified novel, inheritable mutants for fruit size/shape, leaf color/shape, sex expression or plant architecture suggesting T-DNA insertion as a potential source of mutagenesis. The protocol for Agrobacterium-mediated transformation in cucumber from this study could be used as the baseline for further improvement of cucumber transformation.