Topic
Protoplast
About: Protoplast is a research topic. Over the lifetime, 5474 publications have been published within this topic receiving 122468 citations.
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TL;DR: Protoplast fusion was investigated as a means of obtaining somatic hybrids between alfalfa, Medicago sativa L., and two sexually incompatible annual species, M. intertexta and M. scutellata, to confirm that the selected calli were nuclear hybrids.
62 citations
TL;DR: CaP nanoparticles could be used as a better transforming vector in plants as compared to the Agrobacterium tumefaciens mediated genetic transformation technique for delivering a transgene into plant cells and protoplast because of high transformation frequency.
Abstract: We have produced ultra-small sized (20–50 nm diameter) calcium phosphate (CaP) nanoparticles encapsulating a reporter gene, pCambia 1301 The material in the nanoparticles is of crystalline nature having a hydroxyapatite structure as revealed from the XRD pattern The maximum loading of pCambia 1301 in the nanoparticles and the pH-dependent dissolution of CaP nanoparticles were studied using gel electrophoresis DNA is highly protected in the cell from cellular nucleases when it is encapsulated into the CaP nanoparticles The transformation efficiency was found to be about 807% compared to 544% by Agrobacterium tumefaciens and only 8% using naked DNA Our results indicate that CaP nanoparticles could be used as a better transforming vector in plants as compared to the Agrobacterium tumefaciens mediated genetic transformation technique In our experiment we presume that the plasmid DNA released from CaP nanoparticles in the cell has, perhaps, been able to enter into the nucleus Transgenic GUS (β-glucuronidase) integrates into the genomic DNA by non-homologous recombination as in the case of Agrobacterium tumefaciens infection Because of high transformation frequency, the method seems to be an attractive option for delivering a transgene into plant cells and protoplast
62 citations
TL;DR: Both the fluorescent signals on the surface of the protoplasts and the Ca(2+) oscillation assayed via the Ca-2+) probe Fluo-3/AM inside the protplasts provided evidence that the potential GABA(B) receptors are present on the plant protoplast membrane.
62 citations
TL;DR: This work provides methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris tissues and provides a high-efficiency and amenable method for leaf mesophll transformation for rapid gene functional characterization studies.
Abstract: Phaseolus 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. Herein, 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. We 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.
62 citations
TL;DR: Isolated mesophyll protoplasts of cassava were induced to regenerate cell walls, divide and form calli via the potato protoplast system previously developed in this laboratory.
62 citations