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Tracking organelle activities through efficient and stable root genetic transformation system in woody plants.

ABSTRACT: Due to the protracted transgenic timeline and low efficiency in stable genetic transformation of woody plants, there has been limited exploration of real-time organelle imaging within stable transgenic woody plant cells. Here, we established an efficient in vivo genetic transformation system for woody plants using an Agrobacterium rhizogenes-mediated approach. This system was successfully validated in multiple perennial woody species. Using citrus as a model, we introduced organelle-targeted fluorescent reporters via genetic transformation and investigated their subcellular localization and dynamics using advanced imaging techniques, such as confocal microscopy and live-cell imaging. Moreover, we subjected transgenic MT-GFP-labeled mitochondria in root cells to stress conditions simulating agricultural adversities faced by fruit crops. The stress-induced experiments revealed notable alterations in mitochondrial morphology. Our study contributes novel insights into membrane trafficking processes, protein localization dynamics, and cellular physiology in woody plants, while also providing stable and efficient genetic transformation methods for perennial woody species.

SUBMITTER: Gong J

PROVIDER: S-EPMC10831326 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Tracking organelle activities through efficient and stable root genetic transformation system in woody plants.

Gong Jinli J Chen Yishan Y Xu Yanna Y Gu Miaofeng M Ma Haijie H Hu Xiaoli X Li Xiaolong X Jiao Chen C Sun Xuepeng X

Horticulture research 20231126 1

Due to the protracted transgenic timeline and low efficiency in stable genetic transformation of woody plants, there has been limited exploration of real-time organelle imaging within stable transgenic woody plant cells. Here, we established an efficient <i>in vivo</i> genetic transformation system for woody plants using an <i>Agrobacterium rhizogenes</i>-mediated approach. This system was successfully validated in multiple perennial woody species. Using citrus as a model, we introduced organell ...[more]

PMID: 38304333

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Project description:Poison ivy (Toxicodendron radicans) is best known for causing exasperating allergenic delayed-contact dermatitis symptoms that last for weeks on persons who have contacted the plant. Urushiols are alkylcatechols produced by poison ivy responsible for causing this dermatitis. While urushiol chemical structures are well known, the metabolic intermediates and genes responsible for their biosynthesis have not been experimentally validated. A molecular genetic characterization of urushiol biosynthesis in poison ivy will require stable genetic transformation and subsequent regeneration of organs that retain the capacity synthesize urushiol. To this end, Agrobacterium rhizogenes was used to generate hormone-independent poison ivy hairy root cultures. Optimal conditions for hairy root formation were skotomorphic poison ivy hypocotyls prick-inoculated with A. rhizogenes, and preferential propagation of cultures with an atypical clumpy hairy root growth habit. The origin of the poison ivy accession used for A. rhizogenes prick-inoculation did not affect the initial formation of calli/hairy root primordia, but rather significantly influenced the establishment of long-term hormone-independent hairy root growth. A. rhizogenes harboring a recombinant T-DNA binary plasmid with an intron-containing Firefly Luciferase gene produced stable transgenic hairy root lines expressing luciferase activity at high frequency. Poison ivy hairy root lines produced significantly lower steady-state urushiol levels relative to wild-type roots, but higher urushiol levels than a poison ivy undifferentiated callus line with undetectable urushiol levels, suggesting that urushiol biosynthesis requires intact poison ivy organs. The lower urushiol levels in poison ivy hairy root lines facilitated the first identification of anacardic acid metabolites initially in hairy roots, and subsequently in wild-type roots as well. This study establishes a transformation hairy root regeneration protocol for poison ivy that can serve as a platform for future reverse-genetic studies of urushiol biosynthesis in poison ivy hairy roots.

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Dataset Information

Tracking organelle activities through efficient and stable root genetic transformation system in woody plants.

Publications

Tracking organelle activities through efficient and stable root genetic transformation system in woody plants.

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