Establishment of recombinant Catharanthus roseus stem cells stably overexpressing ORCA4 for terpenoid indole alkaloids biosynthesis

Yang, Yibo; Ding, Liuyu; Zhou, Ying; Guo, Zhishan; Yu, Rongmin; Zhu, Jianhua

doi:10.1016/j.plaphy.2023.02.039

Cited by 1 publication

(1 citation statement)

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“…VIGS in C. roseus leaf tissue has been pivotal in identifying TIA biosynthesis enzymes and regulatory factors (Carqueijeiro et al, 2015 ; Cruz et al, 2020 ; Liscombe & O'Connor, 2011 ; Yamamoto et al, 2021 ). Increasingly, Agrobacterium infiltration of C. roseus cotyledons, leaf tissue, and flower tissue has been used to overexpress transcription factors and monitor their effects on transcript levels or promoter activity, thus allowing rapid screening of transcription factor function (Carqueijeiro et al, 2021 ; Colinas et al, 2021 ; Colinas & Goossens, 2022 ; Di Fiore et al, 2004 ; Koudounas et al, 2022 ; Kumar et al, 2015 ; Kumar et al, 2018 ; Kumar et al, 2020 ; Mortimer et al, 2020 ; Pan et al, 2019 ; Schweizer et al, 2018 ; Van Moerkercke et al, 2016 ; Yang et al, 2023 ).…”

Section: Introductionmentioning

confidence: 99%

Critical parameters for robust Agrobacterium‐mediated transient transformation and quantitative promoter assays in Catharanthus roseus seedlings

Cole‐Osborn,

Meehan,

Lee‐Parsons

2024

Plant Direct

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Agrobacterium‐mediated transient expression methods are widely used to study gene function in both model and non‐model plants. Using a dual‐luciferase assay, we quantified the effect of Agrobacterium‐infiltration parameters on the transient transformation efficiency of Catharanthus roseus seedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre‐ and post‐infiltration dark incubation and is less sensitive to the Agrobacterium growth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven‐ to eight‐fold while a dark incubation pre‐ and post‐infiltration increased transformation efficiency by five‐ to 13‐fold. Agrobacterium in exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in our Agrobacterium‐infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6‐fold in raw firefly luciferase (FLUC) and raw Renilla luciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation of Agrobacterium infiltration in C. roseus seedlings will facilitate the study of this important medicinal plant and will expand the application of Agrobacterium‐mediated transformation methods in other plant species.

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