Protoplast isolation of callus in Echinacea augustifolia

Zhu, Lian; Wang, Bochu; Zhou, Jing; Lingxi, Chen; Chuan-yun, Dai; Duan, Chaojun

doi:10.1016/j.colsurfb.2005.05.002

Cited by 18 publications

(10 citation statements)

References 17 publications

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“…Results in Figure 1B are represented as 100% viability for the control (0 h) which is composed of freshly isolated protoplasts at high yields without cell breakage or osmotic shrinkage, and a 20% relative loss in viability at 24 h when protoplasts are incubated at RT. Longer time periods (>24 h) resulted in a significant loss in protoplast viability as seen in other investigations ( Liqing et al, 2005 ; Zhang and Wong, 2011 ). Protoplasts were thus prepared immediately prior to experimentation.…”

Section: Resultssupporting

confidence: 77%

Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

2015

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Lipopolysaccharide (LPS) from Gram-negative bacteria is recognized as a microbe-associated molecular pattern (MAMP) and not only induces an innate immune response in plants, but also stimulates the development of characteristic defense responses. However, identification and characterization of a cell surface LPS-receptor/binding site, as described in mammals, remains elusive in plants. As an amphiphilic, macromolecular lipoglycan, intact LPS potentially contains three MAMP-active regions, represented by the O-polysaccharide chain, the core and the lipid A. Binding site studies with intact labeled LPS were conducted in Arabidopsis thaliana protoplasts and quantified using flow cytometry fluorescence changes. Quantum dots (Qdots), which allow non-covalent, hydrophobic labeling were used as a novel strategy in this study and compared to covalent, hydrophilic labeling with Alexa 488. Affinity for LPS-binding sites was clearly demonstrated by concentration-, temperature-, and time-dependent increases in protoplast fluorescence following treatment with the labeled LPS. Moreover, this induced fluorescence increase was convincingly reduced following pre-treatment with excess unlabeled LPS, thereby indicating reversibility of LPS binding. Inhibition of the binding process is also reported using endo- and exocytosis inhibitors. Here, we present evidence for the anticipated presence of LPS-specific binding sites in Arabidopsis protoplasts, and furthermore propose Qdots as a more sensitive LPS-labeling strategy in comparison to the conventional Alexa 488 hydrazide label for binding studies.

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Section: Resultssupporting

confidence: 77%

Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

2015

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“…The time that was needed for digestion varied depending on the tissue source, and this variation could be due to the changing cell wall composition across tissues. While isolating hypocotyls and nodule protoplasts, the preplasmolysis and osmolarity of the enzyme solution had a significant effect on the protoplast yield [ 35 , 36 ]. Generally, protoplasts burst in hypotonic solution and collapse in hypertonic solution [ 37 , 38 ] and in the present study, 9–13 % mannitol imposed appropriate osmotic pressure.…”

Section: Discussionmentioning

confidence: 99%

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis

et al. 2016

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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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0283-8) contains supplementary material, which is available to authorized users.

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“…In summary, this simpli ed method provides researchers an alternative way to isolate rice mitochondria. As the protoplasts of different plants are accessible [33][34][35], the mitochondria extraction method can be applied to other plants without preparing abundant plant materials. Compared to the traditional mitochondria extraction methods, this mitochondria isolation method does not include the expensive reagents, instruments and complicated steps.…”

Section: Discussionmentioning

confidence: 99%

A Simplified Method to Isolate Rice Mitochondria

Huang

et al. 2020

Preprint

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Background: Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers participate in the studies of plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, traditional plant mitochondria extraction methods are time-consuming and complicated operation of ultra-centrifuge with the expensive reagent. To develop a more rapid and convenient method for isolation of plant mitochondria, in this study we established a simplified method to isolate rice mitochondria efficiently for further study.Results: To isolate rice mitochondria, the cell wall was first dispelled by enzymolysis to obtain the protoplast which is similar to the animal cell. Then the rice mitochondria were isolated with a modified method basing on the animal mitochondria isolation protocol. The extracted mitochondria were next detected on DNA level and protein level to rule out the contamination of nucleus and chloroplasts. Furthermore, we examined the physiological status and characters of the isolated mitochondria, including the integrity of mitochondria, mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria were remained intact for further studies.Conclusion：The combination of plant protoplasts isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability, and can be broadly applied in the researches on plant mitochondria.

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Protoplast isolation of callus in Echinacea augustifolia

Cited by 18 publications

References 17 publications

Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis

A Simplified Method to Isolate Rice Mitochondria

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