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ABSTRACT Day neutrality, or insensitivity to photoperiod (day length), is an important domestication trait in many crop species. Although the oilseed cropC. sativahas been cultivated since the Neolithic era, day‐neutral accessions have yet to be described. We sought to leverage genetic diversity in existing germplasms to identifyC. sativaaccessions with low photoperiod sensitivity for future engineering of this trait. To do so, we quantified variation in hypocotyl length across 161C. sativaaccessions of 4‐day‐old seedlings grown in long‐day and short‐day conditions as a high‐throughput approximation of variation in the photoperiod response. Soil‐grown adult plants from selected accessions also showed variation in the response to day length in several traits; however, the responses in seedling and adult traits were not correlated, suggesting complex mechanistic underpinnings. Although RNA‐seq experiments of the reference accession Licalla identified several differentially regulatedArabidopsissyntelogs involved in photoperiod response and development, includingCOL2,FT,LHY, andWOX4,expression of these genes in the accessions did not correlate with differences in their photoperiod sensitivity. Taken together, we show that all tested accessions show some degree of photoperiod response and that this trait is likely complex, involving several and separable seedling and adult traits.
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Critical parameters for robust Agrobacterium ‐mediated transient transformation and quantitative promoter assays in Catharanthus roseus seedlings
Abstract 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 ofAgrobacterium‐infiltration parameters on the transient transformation efficiency ofCatharanthus roseusseedlings. 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 theAgrobacteriumgrowth 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.Agrobacteriumin exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in ourAgrobacterium‐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 rawRenillaluciferase (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 ofAgrobacteriuminfiltration inC. roseusseedlings will facilitate the study of this important medicinal plant and will expand the application ofAgrobacterium‐mediated transformation methods in other plant species.
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- Award ID(s):
- 2031237
- PAR ID:
- 10578849
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Plant Direct
- Volume:
- 8
- Issue:
- 6
- ISSN:
- 2475-4455
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/pld3.596
