Showing papers on "Protoplast published in 2016"
TL;DR: It is demonstrated that direct delivery of purified 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.
Abstract: 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.
465 citations
TL;DR: It is demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.
Abstract: Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system.
The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3–17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.
158 citations
TL;DR: A cell-cycle model developed based on observations of different secreting systems, some unpublished results and an extensive literature review is proposed, aiming to understand the processes involved in both the secretory process and the release of secretion products.
75 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: The methods developed in this work remove the cost barrier previously limiting high-throughput screening of genome-editing and gene silencing targets in switchgrass, paving the way for more efficient development of transgenic plants.
Abstract: Key message
A switchgrass protoplast system was developed, achieving a cost reduction of ~1000-fold, a threefold increase in transformation efficiency, and a fourfold reduction in required DNA quantity compared to previous methods.
59 citations
01 Mar 2016
TL;DR: A rice protoplast system is established about 10 years ago, which has been recently used in many laboratories and is useful for protein expression, subcellular localization, bimolecular fluorescence complementation, and co-immunoprecipitation assays.
Abstract: Rice (Oryza sativa) is not only the staple food for half of the world's population but also a model monocot plant for molecular biology studies. Although rice genes have been extensively investigated in the last two decades, the functions of many genes in the rice genome are still not known. One of the rapid and efficient approaches for determining gene function in vivo is protoplast-based transient expression analysis. We established a rice protoplast system about 10 years ago, which has been recently used in many laboratories. This protocol is useful for protein expression, subcellular localization, bimolecular fluorescence complementation, and co-immunoprecipitation assays. © 2016 by John Wiley & Sons, Inc.
48 citations
TL;DR: A simplified method for obtaining high yields and excellent viability of isolated protoplasts from young grapevine leaves is reported and the potential of this highly efficient mesophyll protoplast system for transient gene expression and investigation of the activity of disease resistance proteins in grapevine is illustrated.
Abstract: Plant protoplasts constitute a versatile system for transient gene expression and have been widely used with several plant species for the functional characterization of genes and studies of diverse signaling pathways. However, such a system has not been developed for grapevine (Vitis vinifera L.) due to the challenges of large-scale isolation of viable grapevine protoplasts. Here, we report a simplified method for obtaining high yields and excellent viability of isolated protoplasts from young grapevine leaves. In addition, both the conditions for isolation and transfection of grapevine mesophyll protoplasts were modified, and the system was shown to be suitable for protein expression and studies of protein subcellular localization and protein–protein interactions. In addition, we heterologously and transiently expressed the Arabidopsis thaliana disease resistance protein RPW8.2, which has previously been reported to confer broad-spectrum resistance to several biotrophic pathogens in different plant families, as a fluorescent fusion protein in grapevine protoplasts. We observed that expression of the RPW8.2 fusion protein was induced in response to application of exogenous salicylic acid and following infection by the grapevine downy mildew pathogen, Plasmopara viticola. These results illustrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and investigation of the activity of disease resistance proteins in grapevine.
44 citations
TL;DR: The isolation of viable protoplasts from the fruit flesh of sweet cherry using Hong Deng and their polyethylene glycol-mediated transient transfection using green fluorescent protein (GFP) as a marker gene is reported.
25 citations
TL;DR: Only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive.
22 citations
TL;DR: A protocol has been developed for automation of protoplast isolation and transformation from a 'Bright Yellow' 2 (BY-2) tobacco suspension culture using a robotic platform and was validated with the BY-2 cell culture, which should enable acceleration of crop genomics research.
Abstract: Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a 'Bright Yellow' 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.
22 citations
TL;DR: Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.
Abstract: Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 105 protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5 × 105 Pp ml−1. Protoplast-derived microcalli became visible after 3–4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2–3 weeks, with an overall shoot organogenesis efficiency of 78–93 %. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.
TL;DR: Results showed that 0.4 M mannitol, 4 h enzymolysis time and 1000 rpm for 2 min centrifugal force are the best separation condition for protoplast, and considering a combination of these two factors impacting on protiplast, the best time to digest for 4 h is obtained.
Abstract: The research object of this study is “ML7113” wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. Three main effectors—the concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the FDA staining respectively. Results showed that, with the increasing concentrations of mannitol during 0.2 M - 0.4 M, protoplast yield increases and when the concentration is 0.4 M, the protoplast vitality can be up to 95%; with the extension of enzymolysis time in 2 h to 8 h, protoplast yield reaches a maximum in 6 h, but its vitality achieves the maximum in 4 h; considering a combination of these two factors impacting on protoplast, we obtain the best time to digest for 4 h; meanwhile, with the increasing of the centrifugal force from 500 rpm - 2000 rpm, its comprehensive effect of protoplast vitality and yield is the highest when the centrifugal force is 1000 rpm for 2 min (replicated three times). So 0.4 M mannitol, 4 h enzymolysis time and 1000 rpm for 2 min centrifugal force are the best separation condition.
TL;DR: Plants were regenerated at high efficiency using in vitro micrografting of shoots, roots, and embryos recovered after different somatic hybridizations and exhibited a range of mitochondrial and chloroplastic genome combinations.
Abstract: Somatic embryogenesis and plant regeneration are basic processes for the success of citrus somatic hybridization via protoplast fusion. In many cases, few embryos develop normally and only a small number of plants are recovered. The development of methodologies able to increase the recovery of plants after protoplast fusion experiments it is an important requirement to improve the efficiency of the procedure. Here, plants were regenerated at high efficiency using in vitro micrografting of shoots, roots, and embryos recovered after different somatic hybridizations. Hybridizations were performed using protoplasts isolated from Chios mandarin callus with protoplasts isolated from Clementine mandarin leaves and from Sanguinelli sweet orange callus. Recovered plants were analyzed with flow cytometry and nuclear simple sequence repeat (SSR), mitochondrial InDel, and chloroplast SSR markers to determine genomic structure. One tetraploid cybrid and numerous diploid cybrids were recovered, and these exhibited a range of mitochondrial and chloroplastic genome combinations.
TL;DR: A simple and effective method for isolation and transformation of protoplasts is described, and its application to several plant materials is illustrated.
Abstract: The direct uptake of DNA by naked plant cells (protoplasts) provides an expression system of exception for the quickly growing research in non-model plants, fuelled by the power of next-generation sequencing to identify novel candidate genes. Here, we describe a simple and effective method for isolation and transformation of protoplasts, and illustrate its application to several plant materials.
TL;DR: It is demonstrated that the rubber tree mesophyll protoplast system can be used for functional analysis of immune responses of rubber tree and the dissection of cell signaling pathways.
Abstract: The transient mesophyll protoplast expression system has become a powerful tool for rapid gene functional analysis, and it has been used successfully in several plant species, including model plants Arabidopsis and rice, but not in rubber tree. In this study, we describe an improved method to isolate high quality rubber tree mesophyll protoplasts. The transient expression of exogenous DNA constructs in this mesophyll protoplast system was observed by using green fluorescent protein, and was detected by immunoblot. Furthermore, we used two typical pathogen-associated molecular patterns flg22 and chitin from bacteria and fungi, respectively, as inducers of plant innate immunity responses to detect the immunity response of rubber tree. Our data showed that flg22 and chitin induced activation of mitogen-activated protein kinases, generation and accumulation of reactive oxygen species, and transcription of defense genes. Our study demonstrated that the rubber tree mesophyll protoplast system can be used for functional analysis of immune responses of rubber tree and the dissection of cell signaling pathways. This transient mesophyll protoplast system may be applied to analyse gene function of rubber tree, a perennial plant for which the applicable transformation system was limited.
TL;DR: Improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.
Abstract: The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.
TL;DR: Fatty acid analysis in putative hybrids of O. danica and H. pluvialis confirmed the hybrid origin of these cells and indicated successful genetic recombination between the two microalgal phyla.
Abstract: We carried out interphylum protoplast fusion between Ochromonas danica and Haematococcus pluvialis using polyethylene glycol. Different enzyme mixtures were evaluated to prepare the protoplast of H. pluvialis; however, in the absence of a cell wall, O. danica was not treated with lytic enzymes. Maximum lytic activity of 57% was observed using proteinase K for 90 min. The heterofusants were easily identified by pigmentation of the hybrid colonies on BG11 medium. Fatty acid analysis in putative hybrids of O. danica and H. pluvialis confirmed the hybrid origin of these cells. Accordingly, the characteristic fatty acids of O. danica (C16:2 and C24:0) and H. pluvialis (C16:0 and C18:3n-6) were detected in the hybrid algae. These results indicated successful genetic recombination between the two microalgal phyla.
TL;DR: The morphogenic potential of five Gentiana species of medicinal and ornamental value, i.e. G. kurroo, G. tibetica, was compared using agarose-bead leaf mesophyll protoplast culture and Chromosome counting and stomatal characteristic confirmed the ploidy variation among regenerants.
Abstract: The morphogenic potential of five Gentiana species of medicinal and ornamental value, i.e.G. cruciata, G. kurroo, G. lutea, G. septemfida, and G. tibetica, was compared using agarose-bead leaf mesophyll protoplast culture. Modified Murashige and Skoog medium containing 2.0 mg l−1 1-naphthaleneacetic acid and 0.1 mg l−1 thidiazuron ensured the highest cell division frequency during both protoplast and protoplast-derived cell culture or callus formation. Indirect plant regeneration mainly via the somatic embryogenesis pathway was achieved for G. kurroo and G. tibetica with the greatest efficiency occurring with MS medium supplemented with 1.0 mg l−1 kinetin, 0.5 mg l−1 gibberellic acid, and 80 mg l−1 adenine sulfate. A considerable percentage of autopolyploid and aneuploid cells was detected in callus lines obtained from protoplasts using flow cytometry. The presence of polyploid cells in calli resulted in the regeneration of 85 % polyploid plants of G. kurroo and 14 % polyploids of G. tibetica. Chromosome counting and stomatal characteristic confirmed the ploidy variation among regenerants.
TL;DR: This method combined with SCGE assay represents a new tool for testing the DNA integrity of leaf tissues in higher plants under stress conditions.
Abstract: Abiotic stresses have considerable negative impact on Mediterranean plant ecosystems and better comprehension of the genetic control of response and adaptation of trees to global changes is urgently needed. The Single Cell Gel Electrophoresis assay could be considered a good estimator of DNA damage in an individual eukaryotic cell. This method has been mainly employed in animal tissues, because the plant cell wall represents an obstacle for the extraction of nuclei; moreover, in Mediterranean woody species, especially in the sclerophyll plants, this procedure can be quite difficult because of the presence of sclerenchyma and hardened cells. On the other hand, these plants represent an interesting material to be studied because of the ability of these plants to tolerate abiotic stress. For instance, holm oak (Quercus ilex L.) has been selected as the model plant to identify critical levels of O3 for Southern European forests. Consequently, a quantitative method for the evaluation of cell injury of leaf tissues of this species is required. Optimal conditions for high-yield nuclei isolation were obtained by using protoplast technology and a detailed description of the method is provided and discussed. White poplar (Populus alba L.) was used as an internal control for protoplast isolation. Such a method has not been previously reported in newly fully developed leaves of holm oak. This method combined with Single Cell Gel Electrophoresis assay represents a new tool for testing the DNA integrity of leaf tissues in higher plants under stress conditions.
TL;DR: A detailed protocol for using the Arabidopsis mesophyll protoplasts system to study responses to environmental stress is presented, including preparation of reporter and effector constructs, large scale DNA purification, protoplast isolation, transfection, treatment, and quantification of luciferase-based reporter gene activities.
Abstract: Arabidopsis mesophyll protoplasts can be readily isolated and transfected in order to transiently express proteins of interest. As freshly isolated mesophyll protoplasts maintain essentially the same physiological characteristics of whole leaves, this cell-based transient expression system can be used to molecularly dissect the responses to various stress conditions. The response of stress-responsive promoters to specific stimuli can be accessed via reporter gene assays. Additionally, reporter systems can be easily engineered to address other levels of regulation, such as transcript and/or protein stability. Here we present a detailed protocol for using the Arabidopsis mesophyll protoplast system to study responses to environmental stress, including preparation of reporter and effector constructs, large scale DNA purification, protoplast isolation, transfection, treatment, and quantification of luciferase-based reporter gene activities.
TL;DR: Conclusively, SALP1, a novel membrane protein, is involved in salt tolerance through an ABA-independent signaling pathway in rice through the expression level of OsP5CS gene and balancing ion contents under salt stress.
Abstract: Plasma membrane proteins play critical roles in sensing and responding abiotic and biotic stresses in plants. In the present study, we characterized a previously unknown gene stress associated little protein 1 (SALP1) encoding a plasma membrane protein. SALP1, a small and plant-specific membrane protein, contains only 74 amino acid residues. SALP1 was constitutively expressed in various rice tissues while highly expressed in roots, leaf blade, and immature panicles. Expression analysis indicated that SALP1 was induced by various abiotic stresses and abscisic acid (ABA). Subcellular localization assay indicated that SALP1 was localized on plasma membrane in rice protoplast cells. Overexpressing of SALP1 in rice improved salt tolerance through increasing free proline contents and the expression level of OsP5CS gene, and balancing ion contents under salt stress. Moreover, SALP1 transgenic rice showed reduced sensitivity to ABA treatment, and expression level of SALP1 is not altered by ABI5-like 1 protein. Conclusively, SALP1, a novel membrane protein, is involved in salt tolerance through an ABA-independent signaling pathway in rice.
TL;DR: The PEG-mediated protoplast transformation system was effective for L. lecanii, and the expression of CDEP1 significantly enhanced fungal virulence against cotton aphids.
Abstract: Background Lecanicillium lecanii has been developed as a biopesticide and used in biological control of several agricultural insects. To improve fungal virulence, an optimised polyethylene glycol (PEG)-mediated protoplast transformation system was established for L. lecanii. Pr1A-like cuticle-degrading protease gene (Cdep1) from Beauveria bassiana was transferred into L. lecanii, and its resulting activity against Aphis gossypii was assessed. Results The optimised protoplast generation yielded 2.5 × 10(8) protoplasts g(-1) wet mycelium of fungi, and gave nearly 98% viability and 80% regeneration on plates. Protease activities were increased about fivefold in transformants expressing CDEP1. The median lethal concentration (LC50 ) for transformants expressing CDEP1 was twofold lower than that for the wild type (WT). The median survival time (LT50 ) for transformants expressing CDEP1 was also 14.2% shorter than that for WT, though no significant difference. There were no significant differences in conidial germination as colony growth and conidial yield on plates between transformants expressing CDEP1 and WT. The transformants expressing CDEP1 grew significantly quicker than WT in insects. The transformants expressing CDEP1 were lower in conidial yields on insect cadavers, but insignificantly different from WT. Conclusion The PEG-mediated protoplast transformation system was effective for L. lecanii, and the expression of CDEP1 significantly enhanced fungal virulence against cotton aphids. © 2016 Society of Chemical Industry.
TL;DR: The results are consistent with the conclusion that understanding the genetic variation within parent Trichoderma strains and their corresponding fusants and molecular occurrence of protoplast fusion using RAPD, ISSR and ITS markers is necessary.
Abstract: Protoplast fusions are important tools in strain improvement for bringing genetic recombination and developing hybrid strains in filamentous fungi. These are the powerful techniques for engineering...
TL;DR: A transient increase at 24 h after culture (HAC) in levels of a cytokinin riboside, iPA, suggests a putative novel function in initiating a stem-cell niche in an auxin dependent manner and a sharp rise at 72 HAC of gibberellin GA4, furthermore suggests a function for this hormone during the process of rejuvenation.
Abstract: The ability of plant tissues to retain totipotency despite being fully differentiated has been documented for decades. The transition from mature plant tissue to rejuvenated tissue first requires dedifferentiation of mature tissue, followed by rejuvenation (re-entry into the cell cycle) and somatic embryogenesis. We used a Nicotiana tabacum protoplast-based culture system to elucidate the role played by redox and phytohormone networks during the process of dedifferentiation and rejuvenation. Classical markers of redox homeostasis were measured during the rejuvenation process and lipid peroxidation is proposed as the best marker for indicating recovery of cells from oxidative stress sustained during the process of protoplast preparation and culture, prior to rejuvenation. A transient increase at 24 h after culture (HAC) in levels of a cytokinin riboside, iPA, suggests a putative novel function in initiating a stem-cell niche in an auxin dependent manner. A sharp rise at 72 HAC of gibberellin GA4, furthermore suggests a function for this hormone during the process of rejuvenation. These two key findings are consistent with previously described plant models for lateral root developmental. Therein, iPA could be involved in ‘stem-cell-niche’ initiation. Subsequently, GA4 could be involved in rapidly suppressing this initiation step following the earliest cell divisions, thereby enabling the establishment of this ‘niche’ into a callus-like tissue.
TL;DR: The yields of intracellular polysaccharides and triterpenoids in the second generation and the third generation of the mutants were comparable to that of the first generation, which showed genetic stability of the mutant mutants for the production of poly Saccharide and Triterpenoid.
Abstract: Ganoderma lucidum is a traditional medicinal macrofungus in China, which has two kinds of key bioactive compounds -- polysaccharides and triterpenoids. To improve the polysaccharide and triterpenoid production from G. lucidum, the preparation and regeneration conditions of protoplasts were optimized. This was done by systematic trials with various parameters, and protoplast mutation was subsequently performed. A mycelium that was cultivated for seven days and treated with 0.33 mL of 1% snailase and 0.66 mL of 0.5% cellulase solution for 2.5 h at 30 °C in the presence of osmotic pressure stabilizer mannitol (0.5 mol/L), had the best conditions, in which the resultant protoplasts were 6.40 × 105/mL and the regeneration rate was 6.25%. The resultant protoplasts were subjected to subsequent mutation by lithium chloride or by the combination of lithium chloride and Triton X-100. The highest yields of intracellular polysaccharide and triterpenoid in two mutant strains were 37.50 and 40.81 mg/g, which we...
TL;DR: Efficient collection of fruiting body, basidiospore and protoplast of G. boninense is described, which yielded the highest number of viable protoplasts among all possible combinations tested.
Abstract: Ganoderma boninense, a phytopathogenic white rot fungus had sought minimal genetic characterizations despite huge biotechnological potentials. Thus, efficient collection of fruiting body, basidiospore and protoplast of G. boninense is described. Matured basidiocarp raised under the glasshouse conditions yielded a total of 8.3 x 104 basidiospores/ml using the low speed centrifugation technique. Mycelium aged 3-day-old treated under an incubation period of 3 h in lysing enzyme from Trichoderma harzianum (10 mg/ml) suspended in osmotic stabilizer (0.6 M potassium chloride and 20 mM dipotassium phosphate buffer) yielded the highest number of viable protoplasts (8.9 x 106 single colonies) among all possible combinations tested (regeneration media, age of mycelium, osmotic stabilizer, digestive enzyme and incubation period).
19 Apr 2016
TL;DR: Gurel et al. as mentioned in this paper presented the research to date on sugar beet tissue cultures from a historical point of view, covering the following topics; (i) micropropagation via proliferation of pre-existing (axillary or shoot-tip) meristems, (ii) plant regeneration through development of direct or indirect de novo shoots and somatic embryos from different types of tissues/explants, (iii) haploid plant production via mostly gynogenesis but also androgenesis, protoplast isolation and culture from leaf mesophyll, guard or cell
Abstract: Sugar beet (Beta vulgaris L.) is the most important industrial crop used for sugar production in Europe and one of only two plant sources from which sucrose (i.e., sugar) can be economically produced in the world. Sugarcane and sugar beet contribute to the total sugar production by 77.6% and 22.4%, respectively. Despite its relatively short period of cultivation (ca. 200 years), yield and quality parameters of sugar beet have been significantly improved by conventional breeding methods. However, during the last three decades or so, advanced biotechnological methods including in vitro culture and genetic transformation technologies have been combined with classical breeding programmes, which has then lead to the creation of previously non-existing genotypes. Widely applied techniques such as micropropagation and induction of somaclonal variability have already added new germplasm of improved quality and diversity to the sugar beet breeding pool. Organelle exchange, through symetric or asymetric protoplast fusion, and conversion of gametic cells to embryos (i.e., haploid plant production) are other techniques that can be expected to play an increasing role in the improvement of sugar beet. In addition, 1 Sugar Institute, Department of Plant Breeding, 06930 Etimesgut, Ankara, Turkey. Email: songul_gurel@yahoo.com 2 Abant Izzet Baysal University, Department of Biology, 14280 Bolu, Turkey. Email: gurel_e@ibu.edu.tr * Corresponding author © 2014 by Taylor & Francis Group, LLC 114 Bulbous Plants: Biotechnology conservation of germplasm materials, production of disease-free plants, development of homozygous diploid lines (i.e., double haploids/ diplohaploids) from haploid plants obtained from pollen or ovary cultures and isolation and culture of highly totipotent protoplasts from guard cells have been achieved. However, regeneration protocols for both shoot organogenesis and somatic embryogenesis of sugar beet are not reproducible, usually resulting in low regeneration frequencies. Also, a high degree of genotypic variation, primarily because of its highly heterozygous nature due to outcrossing, is a serious problem for the regeneration success of several sugar beet materials. Thus, with all these respects sugar beet is known to be a recalcitrant species. In this chapter, the research to date on sugar beet tissue cultures will be presented from a historical point of view, covering the following topics; (i) micropropagation via proliferation of pre-existing (axillary or shoot-tip) meristems, (ii) plant regeneration through development of direct or indirect de novo shoots and somatic embryos from different types of tissues/explants, (iii) haploid plant production via mostly gynogenesis but also androgenesis, (iv) protoplast isolation and culture from leaf mesophyll, guard or cell suspension cells, (v) somaclonal variation incurred during indirect shoot organogenesis and somatic embryogenesis from callus and in vitro cell selection to be specifi cally expolited for abiotic stress tolerance, and (vi) long-term storage of sugar beet genetic resources through low-temperature storage and cryo-preservation techniques.
Dissertation•
01 Jan 2016
Patent•
28 Sep 2016
TL;DR: In this paper, a preparation and conversion method of inonotus obliquus protoplast is presented, in which a mixed enzyme solution composed of lywallzyme and driselase is adopted for enzymolysis of an inonoxus hypha membrane and filtered through eight layers of lens wiping paper, filtrate collection and centrifugation are carried out.
Abstract: The invention discloses a preparation and conversion method of inonotus obliquus protoplast. The preparation method comprises the following steps that a mixed enzyme solution composed of lywallzyme and driselase is adopted for enzymolysis of an inonotus obliquus hypha membrane and filtered through eight layers of lens wiping paper, filtrate collection and centrifugation are carried out, and sediment is obtained and is the inonotus obliquus protoplast. The conversion method comprises the steps that plasmid pAN7-1 is induced by PEG/CaC12 to be converted into inonotus obliquus protoplast, and inonotus obliquus containing plasmid pAN7-1 is obtained. According to the inonotus obliquus protoplast obtained through the method, the yield reaches up to 5*107/mL, and the regeneration rate is 7%. The preparation and conversion method lays a foundation for carrying out genetic manipulation modification on the inonotus obliquus in the later period, improving the yield of a triterpene compound and a precursor thereof, then improving the medical value of the inonotus obliquus and meeting market requirements.
Patent•
22 Jun 2016
TL;DR: The extraction method of the tea tree protoplast has the advantages that the obtaining of separating materials is easy, the extraction operation is simple, the number of separated protoplasts is more, the yield is high, the integrity is high and a foundation is laid for the transfection and fusion as mentioned in this paper.
Abstract: The invention relates to an extraction method of tea tree protoplast, and belongs to the technical field of plant protoplasts. The extraction method utilizes tea tree petals as an extraction material, and comprises the following four steps of flower collection, protoplast enzymolysis, releasing and purification. The extraction method of the tea tree protoplast has the advantages that the obtaining of separating materials is easy, the extraction operation is simple, the number of separated protoplasts is more, the yield is high, the integrity is high, and a foundation is laid for the protoplast transfection and fusion.