Showing papers on "Protoplast published in 2010"

Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

Abstract: Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

Protoplast transformation of filamentous fungi.

Abstract: The protoplast method for the transformation of filamentous fungi is described in detail, as is the Restriction Enzyme-Mediated Integration (REMI) procedure for introducing tagged mutations into the fungal genome. A split marker method for generating PCR fragments for targeted integration and deletion of genes of interest is also detailed.

Characterization of the multiple resistance traits of somatic hybrids between Solanum cardiophyllum Lindl. and two commercial potato cultivars.

Abstract: Interspecific somatic hybrids between commercial cultivars of potato Solanum tuberosum L. Agave and Delikat and the wild diploid species Solanum cardiophyllum Lindl. (cph) were produced by protoplast electrofusion. The hybrid nature of the regenerated plants was confirmed by flow cytometry, simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), microsatellite-anchored fragment length polymorphism (MFLP) markers and morphological analysis. Somatic hybrids were assessed for their resistance to Colorado potato beetle (CPB) using a laboratory bioassay, to Potato virus Y (PVY) by mechanical inoculation and field trials, and foliage blight in a greenhouse and by field trials. Twenty-four and 26 somatic hybrids of cph + cv. Agave or cph + cv. Delikat, respectively, showed no symptoms of infection with PVY, of which 3 and 12, respectively, were also resistant to foliage blight. One hybrid of cph + Agave performed best in CPB and PVY resistance tests. Of the somatic hybrids that were evaluated for their morphology and tuber yield in the field for 3 years, four did not differ significantly in tuber yield from the parental and standard cultivars. Progeny of hybrids was obtained by pollinating them with pollen from a cultivar, selfing or cross-pollination. The results confirm that protoplast electrofusion can be used to transfer the CPB, PVY and late blight resistance of cph into somatic hybrids. These resistant somatic hybrids can be used in pre-breeding studies, molecular characterization and for increasing the genetic diversity available for potato breeding by marker-assisted combinatorial introgression into the potato gene pool.

Molecular and morphological characterization of somatic hybrids between Solanum tuberosum L. and S. etuberosum Lindl.

Abstract: Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS13 K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.

Strain improvement of Sporolactobacillus inulinus ATCC 15538 for acid tolerance and production of D-lactic acid by genome shuffling

Abstract: Improvement of acid tolerance and production of D-lactic acid by Sporolactobacillus inulinus ATCC 15538 was performed by using recursive protoplast fusion in a genome shuffling format. The starting population was generated by ultraviolet irradiation, diethyl sulfate mutagenesis, and pH-gradient filter and then, subjected for the recursive protoplast fusion. The concentration of lysozyme, time, and temperature for enzyme treatment were optimized by response surface methodology based on the central composite design. Based on contour plots and variance analysis, the model predicted a maximum Y (multiply protoplasts formation ratio by protoplasts regeneration ratio), 60.4%, and the corresponding above used values were 7.75 mg/ml lysozyme, 1.59 h, and 38°C. A pH-5-resistant recombinant, F3-4, was obtained after three rounds of genome shuffling and its production of D-lactic acid reached 93.4 g/l in a 5 L bioreactor, which was increased by 39.8% and 119% in comparison with that of UV generated strain and the original strain S. inulinus ATCC 15538, respectively. The subculture experiments indicated that F3-4 was genetically stable.

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes

Abstract: Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 107–109 protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments.

Studies of mitochondrial morphology and DNA amount in the rice egg cell

Abstract: In plant vegetative cells, mitochondria are usually small and grain-shaped. In contrast, unusually shaped giant mitochondria (large cup-shaped or long stretched-rod-shaped) appear in the egg cells of geranium, maize, Ipomoea nil, and bracken. In this study, to characterize egg cell mitochondria in rice, we used nonenzymatic manual dissection to isolate unfertilized egg cells of rice and observed the egg cell mitochondria and mitochondrial DNA (mtDNA) simultaneously. These observations showed that the mitochondria in the rice egg cell are small and grain-shaped, unlike the mitochondria in geranium, maize, I. nil, and bracken. Double staining of mitochondria by MitoTracker and mtDNA by SYBR Green I showed that mitochondria in the rice egg cell have a large amount of mtDNA compared with the rice root protoplast. We also used real-time PCR analysis to quantify the mtDNA amount in the rice egg cell. We quantified the copy numbers of four mitochondrial genes per single rice egg cell and rice leaf protoplast. Real-time PCR analysis revealed that the egg cell has more than ten times more copy numbers of all of four genes encoded in the mitochondrial genome compared with the leaf protoplast.

Novel Escherichia coli hybrids with enhanced butanol tolerance

Abstract: Hybrids between Escherichia coli and Lactobacillus brevis were generated via protoplast fusion. Growth kinetics of five hybrid strains and E. coli were used to evaluate the butanol tolerance of the novel strains under different conditions. The hybrid strains tolerated up to 2% (v/v) butanol compared to the 1% (v/v) maximum for E. coli. The growth inhibitory effects of butanol were also significantly less in several of the hybrids compared to E. coli. These results demonstrate the potential use of protoplast fusion to generate butanol-tolerant strains.

Improved techniques for transfecting protoplasts

Abstract: The invention relates to a method for the introduction of one or more molecules of interest in a plant cell protoplast by providing plant cell protoplasts, performing a first transfection of the plant cell protoplast with a composition that is capable of altering the regulation of one or more pathways selected from the group consisting of Mismatch Repair System and Non-Homologous End Joining and/or a composition that is capable of introducing DSBs, performing a second transfection of the plant cell protoplast with one or more molecules of interest such as mutagenic oligonucleotides and allowing the cell wall to form.

Regeneration of somatic hybrids of ginger via chemical protoplast fusion

Abstract: Ginger (Zingiber officinale Rosc.) somatic hybridization was attempted by using polyethylene glycol (PEG)-mediated protoplast fusion. Protoplasts of three ginger cultivars isolated from the embryogenic cell suspensions were fused with each other. The highest binary fusion rate [13.5% in the fusion of ginger ‘Lushan Zhangliang jiang’ + ‘Chenggu Huang Jiang’ (LZ + CH)] was observed with the treatment of 30% PEG6000 for 15 min. The three fusion combinations can efficiently develop into micro-colonies and redifferentiate, but only the fusion of ginger ‘Chenggu Huang Jiang’ + ‘Sichuan Zhugen Jiang’ (CH + SZ) could regenerate plantlets. Approximately 15 months were used for the regeneration of whole plants, and 15 shoots were obtained from the fusion of LZ + CH. Three plantlets were identified as hybrids by using RAPD, and they were all diploids by analysis with flow cytometry.

An efficient transformation system of taxol-producing endophytic fungus EFY-21 ( Ozonium sp. )

Abstract: EFY-21 (Ozonium sp.) is a newly isolated taxol-producing endophytic fungus from Taxus chinensis var. mairei. In this study, an efficient PEG-mediated transformation of EFY-21 was established and conditions for transformation were evaluated. By the optimized enzyme system, mycelium age, digesting temperature and time, over 7 × 107 ml protoplasts were obtained and protoplast regeneration frequency was more than 6%. Plasmid pV2 containing the hygromycin-B phosphotransferase gene driven by a fungal promoter (trpC) was used to transform EFY-21 and 50% PEG with 20 mM Ca2+ was found to be suitable for the transformation. Southern blot analysis revealed that the transforming DNA was successfully integrated into the EFY-21 genome. By the optimized procedure, over two transformants per
g DNA could be obtained. The establishment of efficient transformation system of taxol-producing endophytic fungus enables us to improve taxol production of the fungus by engineering the taxol biosynthetic pathway genes in the future.

Efficient plant regeneration from protoplasts of Kalanchoe blossfeldiana via organogenesis

Abstract: A simple and efficient protocol for plant regeneration from protoplasts of the potted plant Kalanchoe blossfeldiana Poelln. is reported. Mesophyll protoplasts were isolated from axenic leaves after a preculture. The enzymatic digestion of the tissue with a solution containing 0.4% Cellulase Onozuka R-10 and 0.2% Driselase yielded 6.0 × 105 protoplasts per gram fresh weight after density gradient purification. Protoplasts were cultured in the dark at an initial density of 1 × 105 protoplasts per milliliter in a liquid medium with 320 mM mannitol, 130 mM sucrose, 2.3 μM 2,4-dichlorophenoxy acetic acid (2,4-D), 5.4 μM 1-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzyladenine (BA). Cell wall regeneration was observed within 4 days of culture and cell division began after 5–7 days. When cultured in a liquid medium with 5.4 μM NAA and 8.9 μM BA, protoplast-derived colonies proliferated until small visible calli, and adventitious buds appeared after transfer to photoperiod conditions. Developed shoots were rooted on a solid medium supplemented with 0.6 μM indole-3-acetic acid (IAA) and successfully established under greenhouse conditions. The process required 4 months from isolation to rooted plants and the best conditions found gave a plant regeneration efficiency of 6.4 plants per 1 × 105 protoplasts. This is the first protocol reported for plant regeneration from protoplasts for a Crassulaceae family species.

Protoplast Mutation and Genome Shuffling Induce the Endophytic Fungus Tubercularia sp TF5 to Produce New Compounds

Abstract: Tubercularia sp. TF5 is an endophytic fungal strain isolated from the medicinal plant Taxus mairei. Previously, taxol has been detected in the fermentation products of this strain. However, it lost the capability of producing taxol after long-term laboratory culture. Herein, we tried to reactivate the production of taxol by protoplast mutations and genome shuffling. The protoplasts of Tub. sp. TF5 were prepared from its mycelia, and mutated by UV and NTG. The mutant strains regenerated from the mutated protoplasts were selected and classified into four groups on the basis of their phenotypes, the profile of their metabolites analyzed by TLC, MS, and bioassay data. Then, genome shuffling was subsequently carried out with eight mutant strains, with two representatives from each protoplast mutant group, and genome shuffling mutant strains were obtained and screened using the same screening procedure. Although taxol has not been detected in any mutant, two important mutants, M-741 and G-444 were selected for metabolites isolation and determination due to their phenotypes, and differences in TLC analysis result from TF5 and other mutants. Three new sesquiterpenoids, namely tuberculariols A-C (1-3), and a known dihydroisocoumarin (4) were obtained from M-741. Eighteen novel compounds were isolated from G-444, including five new sesquiterpenoids (5-9), two new dihydroisocoumarins (10, 11), one new tetralone (12), together with 10 known compounds (13-20, 1, and 2). The compounds isolated from the M-741 and G-444 were different in structure types and substitutions from those of TF5 (15, 21-29). The results showed, for the first time, that protoplast mutations and genome shuffling are efficient approaches to mining natural products from endophytic fungi. Understanding the mechanisms of unlocking the biosynthesis of new metabolites will facilitate the manipulation of the secondary metabolism in fungi.

Optimization of protoplast isolation protocols from callus of Eurycoma longifolia

Abstract: Protoplast technology offers a unique single cell system that facilitates several aspects of modern biotechnology. In this study, an efficient protocol to isolate the protoplast from callus culture of a valuable medicinal plant in Southeast Asia,Eurycoma longifolia was developed. A range of parameters which influence the isolation of E. longifolia protoplasts were investigated by using “change-one-factor-at-a-time” method. From the results obtained, callus fresh weight (FW) of 0.2 g produced the highest number of viable protoplasts, which was 1.58 ± 0.36 × 104protoplasts/gFW. The highest amount of viable protoplasts (1.75 ± 0.68 × 104protoplasts/gFW) was obtained when the sorbitol concentration was maintained at 0.5 M. The optimum enzyme concentration was found to be 1.5% (w/v) of cellulase and pectinase in which 2.75 ± 1.04 × 104 protoplasts/gFW were isolated. Meanwhile, an incubation period of 3 h with enzyme solution resulted in the maximum yield of protoplasts (5.58 ±1.46 × 104 protoplasts/gFW). Key words: Eurycoma longifolia, callus culture, protoplast, osmoticum, viability.

Recondensation level of repetitive sequences in the plant protoplast nucleus is limited by oxidative stress

Abstract: Protoplast cultures are remarkable examples of plant cell dedifferentiation. The state of dedifferentiation is evidenced by changes in cell morphology, genome organization, as well as by the capability of protoplasts to differentiate into multiple types of cells (depending on the type of the stimulus applied). The first change in the genome structure is connected with large-scale chromatin decondensation, affecting chromocentres involving various types of these repetitive sequences. This paper describes not only the de- and recondensation of satellite DNA type I and 5S rDNA repetitive sequences, but it also compares the recondensation level of chromatin with the levels of oxidative stress which were decreased by using an antioxidant, as well as the capabilities of the antioxidative systems within protoplasts, during the first 72 h of their culture. It is demonstrated that the treatment of protoplasts with ascorbic acid not only decreased the level of oxidative stress but also positively stimulated the expression of the ascorbate peroxidase and catalase. It also led to a greater recondensation of the chromatin (when compared to the untreated protoplasts); in addition, it supported cell proliferation. It is concluded that large-scale genome relaxation is more directly connected with oxidative stress than with large changes in the expression of genes; and further, that its recondensation is related to the start of (as well as the level of) protection by the antioxidative systems.

Clonal Propagation of Cyclamen persicum Via Somatic Embryogenesis

Abstract: Cyclamen (Cyclamen persicum) is an economically important ornamental pot plant with local use as cut flower as well Traditionally, it is propagated via seeds, but interest is given in vegetative propagation of parental lines as well as superior single plants Somatic embryogenesis is an efficient in vitro propagation method for many cyclamen cultivars Starting from ovules of unpollinated flowers, callus is induced and propagated in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-(gamma,gamma-dimethylallylamino)purine (2iP) Transfer to hormone-free medium results in the differentiation of somatic embryos, which afterwards germinate on the same medium These first culture stages take about 6-7 months and are carried out in complete darkness Two to four months after the transfer to light, plantlets develop which can be acclimatized in the greenhouse The regenerated plants are characterized by low percentages of somaclonal variation This protocol has proven useful not only for clonal propagation, but also for artificial seed preparation, cryopreservation, genetic transformation and protoplast regeneration

Dehydroascorbate and glutathione regulate the cellular development of Nicotiana tabacum L. SR-1 protoplasts

Abstract: A Nicotiana tabacum L. SR-1 leaf protoplast system was used to study the effects of dehydroascorbate and glutathione on cellular development. We found that dehydroascorbate is readily taken up by protoplasts and internally reduced to ascorbate. Concomitantly, cell division was inhibited and cell expansion stimulated. In this respect, dehydroascorbate counteracted auxin-mediated leaf protoplast development. In contrast to the effects of dehydroascorbate, glutathione-induced cell dedifferentiation, and this effect is similar to that mediated by high auxin concentrations. We conclude that dehydroascorbate and glutathione affect the auxin-mediated regulation of cellular development. Therefore, the biological role of these compounds extends beyond stress tolerance and defense.

Purification and characterization of an intracellular β-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger

Abstract: Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4.000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular beta-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme had an optimum pH of 5.4 and temperature of 65 degrees C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0-6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product ofglucoside hydrolysis. The K(m) and V(max) values against salicin as substrate were 0.035 mM and 1.7215 micromol min(-1), respectively.

Mutagenesis and inter-specific protoplast fusion between Trichoderma koningii and Trichoderma reesei for biocontrol improvement

Abstract: The present work aimed to apply mutagenesis and inter-specific protoplast techniques of two locally isolated Trichoderma spp. to enhancement their biocontrol abilities against some important plant fungal pathogens which cause root-rot and damping-off diseases that attacking different crops. A combination of UV-light and nitrate treatment induced seven mutants (T. koningii, 3 mutants and T. reesei, 4 mutants). The inter-specific protoplast fusion between T. koningii and T. reesei gave fusion frequency reached 1.25% and thirteen fusants were isolated. In vitro bioassay of the parental Trichoderma spp., their selected mutants and fusants indicated the effective of both T. koningii and T. reesei in suppressive the tested fungal pathogens. On the other hand, most of the selected mutants and fusants showed superiority in their antagonistic activity against these pathogens than their parental strains. the superiority in biological control activities of the selected mutants or fusants than their parents against the tested pathogens may be due to the effect of the genetic treatments (i.e. mutagenesis an protoplasts fusion) on their genetic back ground to be varied that allow to change in genetic control of antifungal metabolites production to be more effective. The results indicated that mutation and protoplast fusion techniques are successful tools to enhance the antagonistic effects of Trichoderma species against several fungal plant pathogens.

A Method for the Isolation of Protoplasts from Grape Berry Mesocarp Tissue

Abstract: As single cell systems, protoplasts have been used in physiological, biochemical and molecular studies aiming towards the investigation, improvement or modification of plants. In grapevine, protoplasts have been isolated from several source tissues but not from grape berry, a major challenge given the uniqueness of grape fruit for human diet and wine production. Also, as the ripe grape berry has long been considered a 'small bag of sugary water' without cell compartmentation and/or membrane integrity, the isolation of intact cells from the mesocarp is of special scientific significance. Protoplasting from grape berry mesocarp cells was achieved with cellulase and pectolyase digestion, followed by differential and gradient centrifugations; however, given the special characteristics of berry tissue, cell wall digestion and protoplast purification were performed in a special environment to maintain their integrity and viability. Light and epifluorescence microscopy revealed the spatial organization of the cytoplasm, where an intricate acidic vacuolar apparatus predominates supporting the idea that berry softening during ripening is not strictly associated with loss in compartmentation and/or membrane integrity. Following the worldwide economical and social importance of wine in modern days, grape berry protoplasts are a major advance for both basic research of fruit ripening and biotechnological applications.

An efficient protoplast isolation and regeneration system in Coprinus comatus

Abstract: An efficient protocol for protoplast isolation and regeneration was first established inCoprinus Comatus. The highest yield of protoplasts was up to 8.9 × 106 cells/ml in digestion solution containing 2.0% lywallzyme and 0.6 M KCl after incubation of four-day-old mycelia at 30°C for 3 h; among which, about 1.4% protoplasts could be regenerated into mycelia after 4 - 6 days of incubation at 25°C in CYM medium with0.6 M mannitol as osmotic stabilizer. The results are beneficial for breeding new cultivars by the methods such as protoplast fusion, mutagenesis as well as transformation. Moreover, the stepwise procedure for protoplast liberation and regeneration could be referred in other species. Key words: Coprinus comatus, mycelia, lywallzyme, protoplast, liberation and regeneration.