EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus Seedlings

Mortensen, Samuel; Cole, Lauren F.; Bernal-Franco, Diana; Sathitloetsakun, Suphinya; Cram, Erin J.; Lee‐Parsons, Carolyn W. T.

doi:10.1007/978-1-0716-2349-7_18

Cited by 3 publications

(8 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…C. roseus var. Little Bright Eye seeds were surface sterilized as previously described (Mortensen et al, 2022 ). Seeds were carefully spread across the surface of the media without pushing them into the media; pushing seeds into the media can slow or inhibit germination (Figure S1 ).…”

Section: Methodsmentioning

confidence: 99%

“…Seedlings were vacuum infiltrated with A. tumefaciens GV3101 (pMP90) as previously described (Mortensen et al, 2022 ; Mortensen, Bernal‐Franco, et al, 2019 ). All experiments were infiltrated with two Agrobacterium strains in a 1:1 ratio at a final OD 600 = .4 (OD 600 = .2 per strain).…”

Section: Methodsmentioning

confidence: 99%

“…For all experiments except those testing Agrobacterium growth conditions (Figure 2 ), a streak of colonies grown on solid media for 3 days was used to inoculate a 10 mL culture of LB with antibiotics (Gent and Kan) and grown overnight. The Agrobacterium was then induced and prepared for infiltration as previously described (Mortensen et al, 2022 ; Mortensen, Bernal‐Franco, et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

“…We significantly improved transformation efficiency by employing vacuum infiltration and optimizing multiple parameters, such as seedling age, Agrobacterium final optical density at 600 nm (OD 600 ), number of days post‐infiltration prior to harvest, the use of a constitutively active VirG gene, and the use of silencing suppressors (Mortensen, Bernal‐Franco, et al, 2019 ). With this improved EASI method, we studied ORCA3 activation and ZCT1 repression of the STR promoter and STR expression; STR promoter activity and transcript levels increased similarly with ORCA3 overexpression via EASI, highlighting the utility of EASI for studying both promoter activity and transcript levels (Mortensen et al, 2022 ). This EASI method was also used to study ZCT1 promoter activity (Mortensen, Weaver, et al, 2019 ).…”

Section: Introductionmentioning

confidence: 97%

“…Our group has spent years developing a transient expression method in C. roseus seedlings known as the efficient Agrobacterium ‐mediated seedling infiltration (EASI) method (Mortensen et al, 2022 ; Mortensen, Bernal‐Franco, et al, 2019 ). The EASI method uses vacuum infiltration to introduce Agrobacterium tumefaciens into C. roseus seedlings.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Cole‐Osborn,

Meehan,

Lee‐Parsons

2024

Plant Direct

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%