A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability

Lei, Rong; Qiao, Wenjie; Hu, Fan; Jiang, Hongshan; Shuifang, Zhu

doi:10.1016/j.mex.2014.11.004

Cited by 28 publications

(25 citation statements)

References 27 publications

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“…Growth, directionality and compatibility was observed for Brachypodium seeds on all three PDMS punched molds and the results were in line with the previous reports conducted with Nicotiana and Arabidopsis [14,19,20]. After several experiments, we concluded that after 4 days of vernalization and 2 DAG seedling stage, the seedling had to be inserted in the correct orientation in the chip to make it grow along the length of the narrow 1mm channel.…”

Section: Discussionsupporting

confidence: 90%

Through the Looking Glass: Real Time Imaging in Brachypodium Roots and Osmotic Stress Analysis

Khan¹,

Elitaş²,

Yüce³

et al. 2018

Preprint

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To elucidate dynamic developmental processes in plants, live tissues and organs have to be visualized frequently and for long time periods. The development of roots is studied in depth at a cellular resolution not only to comprehend the basic processes fundamental to maintenance and pattern formation but also study stress tolerance adaptation in plants. Despite technological advancements, maintaining continuous access to samples and simultaneously preserving their morphological structures and physiological conditions without causing damage presents hindrances in the measurement, visualization and analyses of growing organs including plant roots. We propose a preliminary system which integrates the optical real-time visualization through light microscopy with a liquid culture which enables us to image at the tissue and cellular level horizontally growing Brachypodium roots every few minutes and up to 24 hours. We describe a simple setup which can be used to track the growth of the root as it grows including the root tip growth and osmotic stress dynamics. We demonstrate the system’s capability to scale down the PEG-mediated osmotic stress analysis and collected data on gene expression under osmotic stress.

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Section: Discussionsupporting

confidence: 90%

Through the Looking Glass: Real Time Imaging in Brachypodium Roots and Osmotic Stress Analysis

Khan¹,

Elitaş²,

Yüce³

et al. 2018

Preprint

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“…Physically, this means that the energy barrier for nanoparticle transport across protoplast membrane is lower, as reflected in the lower critical zeta potential value for nanoparticle penetration into protoplast interior compared to that required to traverse chloroplast double lipid bilayers ( Figure ). Furthermore, without the protection of plant cell wall, the protoplast membrane is known to be delicate and easily ruptured due to external handling such as electroporation and bombardment of nanoparticles . This was not accounted for by the existing LEEP model, which only predicts the minimum surface charge that a nanoparticle of certain size has to possess in order to traffic past the membrane.…”

Section: Resultsmentioning

confidence: 99%

“…The centrifugation and resuspension process was repeated three times to achieve high purity of viable protoplasts. The viability of protoplasts was determined by staining the protoplasts with FDA, as described elsewhere . The total protoplasts yield was determined by a hemocytometer.…”

Section: Methodsmentioning

confidence: 99%

Rational Design Principles for the Transport and Subcellular Distribution of Nanomaterials into Plant Protoplasts

Lew

Wong

Kwak

et al. 2018

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107

139

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The ability to control the subcellular localization of nanoparticles within living plants offers unique advantages for targeted biomolecule delivery and enables important applications in plant bioengineering. However, the mechanism of nanoparticle transport past plant biological membranes is poorly understood. Here, a mechanistic study of nanoparticle cellular uptake into plant protoplasts is presented. An experimentally validated mathematical model of lipid exchange envelope penetration mechanism for protoplasts, which predicts that the subcellular distribution of nanoparticles in plant cells is dictated by the particle size and the magnitude of the zeta potential, is advanced. The mechanism is completely generic, describing nanoparticles ranging from quantum dots, gold and silica nanoparticles, nanoceria, and single-walled carbon nanotubes (SWNTs). In addition, the use of imaging flow cytometry to investigate the influence of protoplasts' morphological characteristics on nanoparticle uptake efficiency is demonstrated. Using DNA-wrapped SWNTs as model nanoparticles, it is found that glycerolipids, the predominant lipids in chloroplast membranes, exhibit stronger lipid-nanoparticle interaction than phospholipids, the major constituent in protoplast membrane. This work can guide the rational design of nanoparticles for targeted delivery into specific compartments within plant cells without the use of chemical or mechanical aid, potentially enabling various plant engineering applications.

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“…The protocol for apple protoplast preparation was similar to that for the grapevine, except for the addition of hemicellulase to the cell-wall digestion enzyme solution (1–2%). The viability and density of grape and apple protoplast were determined using a haemocytometer, by staining the protoplast with fluorescein diactetate (FDA) as described elsewhere (Lei et al, 2015). At least two biological replications and three technical replication sets were used to optimize and measure enzyme concentration and protoplast yield.…”

Section: Methodsmentioning

confidence: 99%

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

et al. 2016

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The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.

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A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability

Abstract: Graphical abstract

Cited by 28 publications

References 27 publications

Through the Looking Glass: Real Time Imaging in Brachypodium Roots and Osmotic Stress Analysis

Through the Looking Glass: Real Time Imaging in Brachypodium Roots and Osmotic Stress Analysis

Rational Design Principles for the Transport and Subcellular Distribution of Nanomaterials into Plant Protoplasts

DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

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