Protoplast Transformation as a Plant-Transferable Transient Expression System

Duarte, Patrícia; Ribeiro, Diana; Carqueijeiro, Inês; Bettencourt, Sara; Sottomayor, Mariana

doi:10.1007/978-1-4939-3393-8_13

Cited by 17 publications

(5 citation statements)

References 16 publications

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“…Transient transformation of protoplasts can be used to quickly transfer a large number of target genes for expression and verification in a short period of time ( Abel and Theologis, 1994 ; Duarte et al., 2016 ). There are many methods for transient transformation, including particle bombardment, microinjection, PEG-mediated transfection, lipofection-mediated transfection and electronic transfection ( Wang Y et al., 2022 ).…”

Section: Introductionmentioning

confidence: 99%

A high-efficiency PEG-Ca2+-mediated transient transformation system for broccoli protoplasts

Yang

Zhao²,

Liu³

et al. 2022

Front. Plant Sci.

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Transient transformation of plant protoplasts is an important method for studying gene function, subcellular localization and plant morphological development. In this study, an efficient transient transformation system was established by optimizing the plasmid concentration, PEG4000 mass concentration and genotype selection, key factors that affect transformation efficiency. Meanwhile, an efficient and universal broccoli protoplast isolation system was established. Using 0.5% (w/v) cellulase R-10 and 0.1% (w/v) pectolyase Y-23 to hydrolyze broccoli cotyledons of three different genotypes for 3 h, the yield was more than 5×106/mL/g, and the viability was more than 95%, sufficient to meet the high standards for protoplasts to be used in various experiments. The average transformation efficiency of the two plasmid vectors PHG-eGFP and CP507-YFP in broccoli B1 protoplasts were 61.4% and 41.7%, respectively. Using this system, we successfully performed subcellular localization of the products of three target genes (the clubroot resistance gene CRa and two key genes regulated by glucosinolates, Bol029100 and Bol031350).The results showed that the products of all three genes were localized in the nucleus. The high-efficiency transient transformation system for broccoli protoplasts constructed in this study makes it possible to reliably acquire high-viability protoplasts in high yield. This research provides important technical support for international frontier research fields such as single-cell sequencing, spatial transcriptomics, plant somatic hybridization, gene function analysis and subcellular localization.

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Section: Introductionmentioning

confidence: 99%

A high-efficiency PEG-Ca2+-mediated transient transformation system for broccoli protoplasts

Yang

Zhao²,

Liu³

et al. 2022

Front. Plant Sci.

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“…They explore the signal transduction and metabolic pathways transiently respond to hormones and stress factors ( Sheen, 2001 ; Hirata et al, 2012 ), answer specific questions related to cell types ( Petersson et al, 2015 ; Denyer et al, 2019 ), and determine the subcellular localization, transport, and interactions of tagged proteins ( Goodman et al, 2004 ; Zhang et al, 2011 ). The versatility of plant protoplasts greatly facilitates modern botany development ( Duarte et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops

et al. 2021

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Versatile protoplast platforms greatly facilitate the development of modern botany. However, efficient protoplast-based systems are still challenging for numerous horticultural plants and crops. Orchids are globally cultivated ornamental and medicinal monocot plants, but few efficient protoplast isolation and transient expression systems have been developed. In this study, we established a highly efficient orchid protoplast isolation protocol by selecting suitable source materials and optimizing the enzymatic conditions, which required optimal D-mannitol concentrations (0.4–0.6 M) combined with optimal 1.2% cellulose and 0.6% macerozyme, 5 μM of 2-mercaptoethanol and 6 h digestion. Tissue- and organ-specific protoplasts were successfully isolated from young leaves [∼3.22 × 106/g fresh weight (FW)], flower pedicels (∼5.26 × 106/g FW), and young root tips (∼7.66 × 105/g FW) of Cymbidium orchids. This protocol recommends the leaf base tissues (the tender part of young leaves attached to the stem) as better source materials. High yielding viable protoplasts were isolated from the leaf base of Cymbidium (∼2.50 × 107/g FW), Phalaenopsis (1.83 × 107/g FW), Paphiopedilum (1.10 × 107/g FW), Dendrobium (8.21 × 106/g FW), Arundina (3.78 × 106/g FW) orchids, and other economically important monocot crops including maize (Zea mays) (3.25 × 107/g FW) and rice (Oryza sativa) (4.31 × 107/g FW), which showed marked advantages over previous mesophyll protoplast isolation protocols. Leaf base protoplasts of Cymbidium orchids were used for polyethylene glycol (PEG)-mediated transfection, and a transfection efficiency of more than 80% was achieved. This leaf base protoplast system was applied successfully to analyze the CsDELLA-mediated gibberellin signaling in Cymbidium orchids. We investigated the subcellular localization of the CsDELLA-green fluorescent protein fusion and analyzed the role of CsDELLA in the regulation of gibberellin to flowering-related genes via efficient transient overexpression and gene silencing of CsDELLA in Cymbidium protoplasts. This protoplast isolation and transient expression system is the most efficient based on the documented results to date. It can be widely used for cellular and molecular studies in orchids and other economically important monocot crops, especially for those lacking an efficient genetic transformation system in vivo.

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“…Wild tobacco (Nicotiana benthamiana) leaf protoplasts were generated by immersing leaf material in a solution containing 1.5% (w/v) Cellulase R10 (Yakult), 0.2 to 0.4% (w/v) Macerozyme R10 (Yakult), 1% (w/v) Hemicellulase (Sigma-Aldrich), 0.4 M mannitol, 20 mM KCl, 20 mM MES (pH 5.7), 10 mM CaCl 2 , and 0.1% (w/v) BSA for 12 h. An equal volume of W5 solution (150 mM NaCl, 125 mM CaCl 2 , 5 mM KCl, and 2 mM MES [pH 5.7]) was added prior to passing the mixture through a 200-mesh sieve. Protoplasts were collected by centrifugation and resuspended in ice-cold W5 (Duarte et al, 2016). Purified plasmids were transferred into these cells using the polyethylene glycol-calcium method with minor modifications (Yoo et al, 2007).…”

Section: Testing Transcription Factor-promoter Interactionsmentioning

confidence: 99%

A PXY-Mediated Transcriptional Network Integrates Signaling Mechanisms to Control Vascular Development in Arabidopsis

et al. 2019

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The cambium and procambium generate the majority of biomass in vascular plants. These meristems constitute a bifacial stem cell population from which xylem and phloem are specified on opposing sides by positional signals. The PHLOEM INTERCALATED WITH XYLEM (PXY) receptor kinase promotes vascular cell division and organization. However, how these functions are specified and integrated is unknown. Here, we mapped a putative PXY-mediated transcriptional regulatory network comprising 690 transcription factor-promoter interactions in Arabidopsis (Arabidopsis thaliana). Among these interactions was a feedforward loop containing transcription factors WUSCHEL HOMEOBOX RELATED14 (WOX14) and TARGET OF MONOPTEROS6 (TMO6), each of which regulates the expression of the gene encoding a third transcription factor, LATERAL ORGAN BOUNDARIES DOMAIN4 (LBD4). PXY signaling in turn regulates the WOX14, TMO6, and LBD4 feedforward loop to control vascular proliferation. Genetic interaction between LBD4 and PXY suggests that LBD4 marks the phloem-procambium boundary, thus defining the shape of the vascular bundle. These data collectively support a mechanism that influences the recruitment of cells into the phloem lineage, and they define the role of PXY signaling in this context in determining the arrangement of vascular tissue.

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Protoplast Transformation as a Plant-Transferable Transient Expression System

Cited by 17 publications

References 16 publications

A high-efficiency PEG-Ca2+-mediated transient transformation system for broccoli protoplasts

A high-efficiency PEG-Ca2+-mediated transient transformation system for broccoli protoplasts

Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops

A PXY-Mediated Transcriptional Network Integrates Signaling Mechanisms to Control Vascular Development in Arabidopsis

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