ArticleBMC biotechnology2016
Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.
Article in BMC biotechnology, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 39 papers.
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39 citing papers in PubMed, 79 citations in OpenAlex.
- Efficient isolation and transient transformation of cowpea protoplasts.BMC plant biology · 2026Article
- Optimization of CRISPR/Cas9-mediated CtPDS knockout in guar protoplasts.The plant genome · 2026Article
- Glow with the Flow: Reproducible Analysis of Transiently Transformed Protoplasts Using Dual Fluorescent Reporters in R.bioRxiv : the preprint server for biology · 2025Article
- A viral small interfering RNA-host plant mRNA pathway modulates virus-induced drought tolerance by enhancing autophagy.The Plant cell · 2024Article
- Strigolactone induces D14-dependent large-scale changes in gene expression requiring SWI/SNF chromatin remodellers.The Plant journal : for cell and molecular biology · 2024Article
- Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328).Plant methods · 2024Article
- Cell size differences affect photosynthetic capacity in a Mesoamerican and an Andean genotype ofFrontiers in plant science · 2024Article
- First Report on Mesophyll Protoplast Isolation and Regeneration System for thePlants (Basel, Switzerland) · 2023Article
- Isolation, Purification, and Application of Protoplasts and Transient Expression Systems in Plants.International journal of molecular sciences · 2023Review
- From Petri Dish to Field: Plant Tissue Culture and Genetic Engineering of Oats for Improved Agricultural Outcomes.Plants (Basel, Switzerland) · 2023Review
- Establishment of protoplasts transient expression system in Pinellia ternata (Thunb.) Breit.Biotechnology letters · 2023Article
- A molecular toolkit to boost functional genomic studies in transformation-recalcitrant vegetable legumes.Horticulture research · 2023Article
- Isolation of Adhirons Specific for Plant Protoplast Membrane Biomarkers Is Simplified by Phagemid Design.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera.Plant methods · 2022Article
- DNA Free CRISPR/DCAS9 Based Transcriptional Activation System forPlants (Basel, Switzerland) · 2022Article
- Genetic engineering of novel yellow color african violet (Saintpaulia ionantha) produced by accumulation of Aureusidin 6-O-glucoside.Biological procedures online · 2022Article
- Establishing an efficient protoplast transient expression system for investigation of floral thermogenesis in aroids.Plant cell reports · 2022Article
- An efficientFrontiers in plant science · 2022Article
- Gene introduction approaches in chloroplast transformation and its applications.Journal, genetic engineering & biotechnology · 2021Review
- Leaf infiltration in plant science: old method, new possibilities.Plant methods · 2021Review
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Authors and funding
5 authors at 3 institutions in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundPhaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris.
resultsHerein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies.
conclusionsWe present simple and efficient methodologies for protoplast isolation from multiple P. vulgaris tissues. We also provide a high-efficiency and amenable method for leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT leaf disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools for P. vulgaris research.
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