Leaf wounding and jasmonic acid act synergistically to enable efficient Agrobacterium-mediated transient transformation of Persea americana

Salazar-González, Jorge A.; Castro-Medina, Manuela; Martínez-Terrazas, Eduardo; Casson, Stuart A.; Urrea-López, Rafael

doi:10.21203/rs.3.rs-1631178/v1

Cited by 3 publications

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Protocol for efficient ginseng transformation

Kim

Park

Hong

et al. 2023

Plant Cell Tiss Organ Cult

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Panax ginseng is a major medicinal crop with pharmaceutical e cacy derived from ginsenoside metabolites. Despite its genome information, the ine ciency of ginseng transformation hinders the study of the molecular mechanism of ginseng plant metabolism. Thus, this protocol aimed to develop an easy and e cient system for ginseng transformation. We established a transformation system using ginseng callus cultured in a liquid medium, which has a higher ratio compared with cotyledon explant. In addition, Agrobacterium tumefaciens has been used for plant transformation. Compared with the LBA4404 strain, C58C1 was inappropriate for ginseng transformation using ginseng callus. We induced and maintained calli in liquid medium and cut them into small pieces before infection. After infection, we selected calli that survived from the antibiotic medium until identi cation of newly growing cells. In β-glucuronidase (GUS) gene assay, the expression of the GUS gene was observed in cells that were newly generated from explants. We treated calli with 0.05 M of MgSO 4 before infection. After MgSO 4 pre-treatment, the transformation e ciency of growing cells around infected callus was increased. Moreover, we constructed and introduced a visible reporter RUBY system to easily identify transformed cells. Using this system, we could identify the cells by a red color with naked eyes. Based on our transformation protocol, the success rate has increased to 77.27% in surviving lines during selection culture. This stable ginseng transformation could facilitate the overexpression and knockout of ginseng lines for functional or synthetic biological studies. Key MessageWe re-designed the ginseng transformation protocol using ginseng calli for explant material, and MgSO 4 pretreatment was conducted before Agrobacterium infection. RUBY reporter was successfully introduced in ginseng.

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Protocol for efficient ginseng transformation

Kim

Park

Hong

et al. 2023

Plant Cell Tiss Organ Cult

View full text Add to dashboard Cite

show abstract

Protocol for efficient ginseng transformation

Kim

Park

Hong

et al. 2022

Preprint

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Panax ginseng is a major medicinal crop with pharmaceutical efficacy derived from ginsenoside metabolites. Despite its genome information, the inefficiency of ginseng transformation hinders the study of the molecular mechanism of ginseng plant metabolism. Thus, this protocol aimed to develop an easy and efficient system for ginseng transformation. We established a transformation system using ginseng callus cultured in a liquid medium, which has a higher ratio compared with cotyledon explant. In addition, Agrobacterium tumefaciens has been used for plant transformation. Compared with the LBA4404 strain, C58C1 was inappropriate for ginseng transformation using ginseng callus. We induced and maintained calli in liquid medium and cut them into small pieces before infection. After infection, we selected calli that survived from the antibiotic medium until identification of newly growing cells. In β-glucuronidase (GUS) gene assay, the expression of the GUS gene was observed in cells that were newly generated from explants. We treated calli with 0.05 M of MgSO4 before infection. After MgSO4 pre-treatment, the transformation efficiency of growing cells around infected callus was increased. Moreover, we constructed and introduced a visible reporter RUBY system to easily identify transformed cells. Using this system, we could identify the cells by a red color with naked eyes. Based on our transformation protocol, the success rate has increased to 77.27% in surviving lines during selection culture. This stable ginseng transformation could facilitate the overexpression and knockout of ginseng lines for functional or synthetic biological studies.

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Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

Basak,

Parajulee,

Dhir

et al. 2024

Plants

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Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters—such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride—were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

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Leaf wounding and jasmonic acid act synergistically to enable efficient Agrobacterium-mediated transient transformation of Persea americana

Cited by 3 publications

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Protocol for efficient ginseng transformation

Protocol for efficient ginseng transformation

Protocol for efficient ginseng transformation

Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

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