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Vacuum infiltration enhances the Agrobacterium-mediated transient transformation for gene functional analysis in persimmon (Diospyros kaki Thunb.)

Mo, Rongli, Yang, Sichao, Zhang, Qinglin, Xu, Liqing, Luo, Zhengrong
Scientia horticulturae 2019 v.251 pp. 174-180
Diospyros kaki, agroinfiltration, enzymes, genotype, leaves, monitoring, persimmons, proanthocyanidins, proliferating cell nuclear antigen, reporter genes, seedlings, sonication, staining
An Agrobacterium-mediated transient transformation system was established for gene functional analysis in persimmon (Diospyros kaki Thunb.) utilizing sonication followed by vacuum infiltration of seedlings in vitro. The optimal vacuum conditions for agroinfiltration of persimmon were evaluated by monitoring expression of GUS reporter gene. The technique was further applied to evaluate five different genotypes. Among the genotypes examined in vitro, ‘Xiaoguo-tianshi’ (Chinese PCNA type) produced the highest expression levels of both GUS and GFP, and was the most ideal genotype. Furthermore, the developed system was successfully utilized for DkLAR (leucoanthocyanidin reductase) gene transient silencing. DMACA (4- dimethylaminocinnamaldehyde) staining and the Folin-Ciocalteau assay revealed that the proanthocyanidins (PAs) levels was markedly decrease in ‘Xiaoguo-tianshi’ and ‘Mopanshi’ (Chinese PCA type) leaves. These results confirmed that DkLAR plays an important role in the accumulation of PAs in persimmon. In conclusion, based on the Agrobacterium-mediated vacuum infiltration, we developed a reliable and highly efficient transient transformation system that can be used for gene functional analysis through the seedlings in vitro.