Showing papers on "Protoplast published in 2008"

Seaweed protoplasts: status, biotechnological perspectives and needs

Abstract: Protoplasts are living plant cells without cell walls which offer a unique uniform single cell system that facilitates several aspects of modern biotechnology, including genetic transformation and metabolic engineering. Extraction of cell wall lytic enzymes from different phycophages and microbial sources has greatly improved protoplast isolation and their yield from a number of anatomically more complex species of brown and red seaweeds which earlier remained recalcitrant. Recently, recombinant cell wall lytic enzymes were also produced and evaluated with native ones for their potential abilities in producing viable protoplasts from Laminaria. Reliable procedures are now available to isolate and culture protoplasts from diverse groups of seaweeds. To date, there are 89 species belonging to 36 genera of green, red and brown seaweeds from which successful protoplast isolation and regeneration has been reported. Of the total species studied for protoplasts, most belonged to Rhodophyta with 41 species (13 genera) followed by Chlorophyta and Phaeophyta with 24 species each belonging to 5 and 18 genera, respectively. Regeneration of protoplast-to-plant system is available for a large number of species, with extensive literature relating to their culture methods and morphogenesis. In the context of plant genetic manipulation, somatic hybridization by protoplast fusion has been accomplished in a number of economically important species with various levels of success. Protoplasts have also been used for studying foreign gene expression in Porphyra and Ulva. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, bio-chemical synthesis of cell walls etc. This article briefly reviews the status of various developments in seaweed protoplasts research and their potentials in genetic improvement of seaweeds, along with needs that must to be fulfilled for effective realization of the objectives envisaged for protoplast research.

Toward improvements of oomycete transformation protocols.

Abstract: Some of the most important plant pathogens worldwide are oomycetes, and billions of dollars are expended annually to suppress diseases they cause. More efficient disease suppression technologies will be derived from a better understanding of the basic biology of these organisms, but inefficient transformation currently limits basic molecular investigations. Of the various approaches, transformation of protoplasts using polyethylene glycol/calcium chloride remains most successful, but the frequency of stable transformation remains low and inconsistent. Here we report that modifications of a protocol, previously used for Arabidopsis mesophyll cells, successfully releases protoplasts from four different oomycetes (Phytophthora citricola, Phytophthora infestans, Phytophthora sojae, and Pythium aphanidermatum). The protoplasts of all oomycetes were able to take up DNA and regenerate, with protoplast release as well as regeneration being most efficient in P. aphanidermatum. In addition to a good protoplast production system, more effective transformation vectors may improve stable transformation rates. We constructed, and evaluated 17 novel candidate transformation vectors for their ability to drive transient expression of the beta-glucuronidase (GUS) reporter gene in P. infestans and P. aphanidermatum. Five of the newly constructed vectors were also evaluated in P. sojae and P. citricola, and exhibited a similar pattern of transcriptional activity as in P. infestans and P. aphanidermatum. One of the newly constructed vectors, pDBHAMT35G, containing a chimeric promoter, supported the highest GUS expression in P. infestans and P. citricola, and could potentially be useful for future studies.

Protoplast isolation and transient gene expression in switchgrass, Panicum virgatum L.

Abstract: Transient assay systems using protoplasts have been utilized in several plant species and are a powerful tool for rapid functional gene analysis and biochemical manipulations. A protoplast system has not been used in switchgrass (Panicum virgatum L.), even though it is a bioenergy crop that has received considerable attention. Here we report the first protocol to isolate large numbers of viable protoplasts from both leaves and roots of two switchgrass genotypes. Furthermore, we demonstrate transient expression of PEG-mediated DNA uptake in the isolated protoplasts by measuring the activity of β-glucuronidase (GUS) reporter gene driven by either the Cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin 1 promoter. Protoplast transformation with either the 35S or the ubiquitin promoter resulted in an increase in GUS activity compared to the untransformed controls; however, the extent of GUS activity was considerably higher for the ubiquitin promoter than for the 35S promoter. Collectively, our results indicate an efficient protoplast isolation and transient assay system that can be used to facilitate gene expression studies in switchgrass.

Biotechnology Applications for Sugar Beet

Abstract: Sugar beet (Beta vulgaris L.) is an important industrial crop, being one of only two plant sources from which sucrose (i.e., sugar) can be economically produced. Despite its relatively short period of cultivation (ca. 200 years), its yield and quality parameters have been significantly improved by conventional breeding methods. However, during the last two decades or so, advanced in vitro culture and genetic transformation technologies have been incorporated with classical breeding programs, the main aim being the production of herbicide-and salt-tolerant, disease- and pest-resistant cultivars. Among the many applications of in vitro culture techniques, sugar beet has benefited the most from haploid plant production, protoplast culture, and somaclonal variation and in vitro cell selection. Several genetic transformation technologies have been developed, such as Agrobacterium-meditated, PEG-mediated, particle bombardment, electroporation, sonication and somatic hybridization, the first two being the most s...

Production and characterization of somatic hybrids raised through protoplast fusion between edible mushroom strains Volvariella volvacea and Pleurotus florida

Abstract: Twelve somatic hybrid lines were raised through polyethylene glycol-mediated intergeneric protoplast fusion between Volvariella volvacea and Pleurotus florida using a double selection method. Four hybrid lines belonging to two distinct colony morphology types could be maintained in culture. Basidiocarps could be generated from two hybrid lines, one of which showed resemblance to the P. florida parent while the other showed intermediate parental morphology. Hybridity of the fusant lines was established on the basis of colony morphology, mycelial growth rate, hyphal traits, fruit body morphology, isozyme and RAPD analysis. Four isozyme activities were analyzed to provide biochemical criteria for detection of polymorphism among the hybrids and their parents. Using 20 random decamers a total of 203 RAPD bands ranging from 0.09 to 1.6 kb were observed. The UPGMA method of clustering exhibited two major phylogenetic clusters, the first of which comprised of the parents, two fruit body generating hybrid lines and their subsequent fruit body derived lines, while the two non-fruit body generating hybrid lines formed the out group of the first major cluster. Screening of a Pleurotus type hybrid line having high biological efficiency and generation of a temperature tolerant Pleurotus type line through backcross mating between a non-fruit body generating somatic hybrid and V. volvacea parent are the key achievements of this fusion programme.

Changes in energy status of leaf cells as a consequence of mitochondrial genome rearrangement.

Abstract: The MSC16 cucumber (Cucumis sativus L.) mutant with lower activity of mitochondrial Complex I was used to study the influence of mitochondrial metabolism on whole cell energy and redox state. Mutant plants had lower content of adenylates and NADP(H) whereas the NAD(H) pool was similar as in wild type. Subcellular compartmentation of adenylates and pyridine nucleotides were studied using the method of rapid fractionation of protoplasts. The data obtained demonstrate that dysfunction of mitochondrial respiratory chain decreased the chloroplastic ATP pool. No differences in NAD(H) pools in subcellular fractions of mutated plants were observed; however, the cytosolic fraction was highly reduced whereas the mitochondrial fraction was more oxidized in MSC16, as compared to WTc. The NADP(H) pool in MSC16 protoplasts was greatly decreased and the chloroplastic NADP(H) pool was more reduced, whereas the extrachloroplastic pool was much more oxidized, than in WTc protoplast. Changes in nucleotides distribution in cucumber MSC16 mutant were compared to changes found in tobacco (Nicotiana sylvestris) CMS II mitochondrial mutant. In contrast to MSC16 cucumber, the content of adenylates in tobacco mutant was much higher than in tobacco wild type. The differences were more pronounced in leaf tissue collected after darkness than in the middle of the photoperiod. Results obtained after tobacco protoplast fractionating showed that the increase in CMS II adenylate content was mainly due to a higher level in extrachloroplast fraction. Both mutations have a negative effect on plant growth through perturbation of chloroplast/mitochondrial interactions.

Effect of the osmotic conditions during sporulation on the subsequent resistance of bacterial spores.

Abstract: The causes of Bacillus spore resistance remain unclear. Many structures including a highly compact envelope, low hydration of the protoplast, high concentrations of Ca-chelated dipicolinic acid, and the presence of small acid-soluble spore proteins seem to contribute to resistance. To evaluate the role of internal protoplast composition and hydration, spores of Bacillus subtilis were produced at different osmotic pressures corresponding to water activities of 0.993 (standard), 0.970, and 0.950, using the two depressors (glycerol or NaCl). Sporulation of Bacillus subtilis was slower and reduced in quantity when the water activity was low, taking 4, 10, and 17 days for 0.993, 0.970, and 0.950 water activity, respectively. The spores produced at lower water activity were smaller and could germinate on agar medium at lower water activity than on standard spores. They were also more sensitive to heat (97 °C for 5–60 min) than the standard spores but their resistance to high hydrostatic pressure (350 MPa at 40 °C for 20 min to 4 h) was not altered. Our results showed that the water activity of the sporulation medium significantly affects spore properties including size, germination capacity, and resistance to heat but has no role in bacterial spore resistance to high hydrostatic pressure.

Protoplast formation, regeneration and transformation from the taxol-producing fungus Ozonium sp .

Abstract: The effects of some factors on protoplast isolation and regeneration from taxol-producing fungus Ozonium sp. BT2 were investigated, including the enzymolysis time and temperature, the osmotic pressure stabilizer, mycelial incubation time, the culture medium, the culture methods and preprocessing. The mycelia were digested by enzyme combination of 1.5% lywallzyme, 0.5% snailase, 1.5% cellulase and 1.0% lysozyme for 3 h at 30°C, and a high yield of protoplasts (1.31 ◊ 10 8 /g moist mycelia) were obtained. The protoplasts were purified, and then regenerated by different culture methods. The results showed that regeneration frequency was not different. The protoplasts could regenerate on both PDA and Czapek medium with 0.6 M sucrose, 0.6 M sodium chloride and 0.6 M mannitol, respectively. The regeneration rate was about 2.56% under 25°C on Czapek medium using 0.6 M sodium chloride as osmotic pressure stabilizer. Furthermore, transformants were obtained by transforming the protoplasts with plasmid pAN7-1 carrying hygromycin B phosphotransferase gene (hph) conferring hygromycin resistance. This study provides the foundation to develop an engineered strain of taxol-producing fungus by protoplast mutagenesis, fusion and genetic transformation.

NADPH oxidase involvement in cellular integrity

Abstract: NADPH oxidase activity is involved in plant adaptation and development. The reactive oxygen species sourced by NADPH oxidase activity may contribute to wall strength and protoplast volume adjustment. Root hair bulge apices of the NADPH oxidase mutant rhd2/Atrbohc were more robust than the kjk cellulose synthase mutant, but burst more readily than the wild type (WT). Root epidermal wall appeared impaired in rhd2/Atrbohc, as revealed by the number of protoplasts released by wall-degrading enzymes. Root hair bulges of rhd2/Atrbohc burst more than the WT when challenged in situ with hypo-osmotic low ionic strength medium. Inhibition of NADPH oxidase activity with diphenylene iodonium caused WT to phenocopy the rhd2/Atrbohc bursting in response to hypo-osmotic shock. This implicates RHD2/AtRBOHC in softening the cell wall to permit protoplast expansion. Overall, the results point to a role for RHD2/AtRBOHC in contributing to wall strength.

Correction of chlorophyll‐defective, male‐sterile winter oilseed rape (Brassica napus) through organelle exchange: Phenotypic evaluation of progeny

Abstract: Introduction of male-sterile Raphanus sativus cytoplasm into Brassica napus results in a chlorophyll deficiency in the backcrossed male-sterile B. napus line. We describe the apparent correction of this defect by protoplast fusion. enabling somatic recombination and/or elimination of organelles. Methods for fusion of male-sferile and male-fertile protoplasts, and for regeneration of plants are described. Potential recombining ability in somatic fusions is shown to be influenced by the genotypes, with one combination of genotypes resulting in five times more plants than the least successful combination. Of 87 plants that were regenerated. 44 have been analyzed for ploidal level and organellar composition. Two of the 44 plants were found to be male sterile with a green phenotype. The behaviour of these plants was followed during two subsequent seed generations.

Protoplast Isolation and Regeneration from Gracilaria changii (Gracilariales, Rhodophyta)

Abstract: The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.

Expression profiling of the mannuronan c5-epimerase multigenic family in the brown alga laminaria digitata (phaeophyceae) under biotic stress conditions(1).

Abstract: The aim of this study was to profile expression of mannuronan C5-epimerase (ManC5-E) genes of Laminaria digitata (Huds.) J. V. Lamour. under treatment with brown alga cell wall components known to induce defense reaction in this organism. After determining that incubation in the presence of homo-guluronates increased the accumulation of ManC5-E transcripts, we cloned a number of partial ManC5-E genes from nonelicited and elicited specimens. In addition to these fragments, previously identified genes from sporophyte and protoplast cDNA libraries were considered for a clustering analysis. Several groups of ManC5-E genes were observed, including groups containing sequences specific from nonelicited and elicited sporophytes, and sequences isolated from protoplasts. Quantitative real-time PCR experiments were then carried out using genes representative of different groups. Results of transcript accumulation during protoplast regeneration and sporophyte elicitation supported the hypothesis that specific C5-epimerase genes are recruited under pathogen attack, enabling L. digitata to modify its cell wall in response to environmental stimuli.

Molecular characterization of the lectin, bryohealin, involved in protoplast regeneration of the marine alga bryopsis plumosa (chlorophyta)1

Abstract: When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5' untranslated region (UTR) and 201 bp of 3' UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C-terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.

Purification and Characterization of Chitinase A of Streptomyces cyaneus SP-27: An Enzyme Participates in Protoplast Formation from Schizophyllum commune Mycelia

Abstract: A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase and chitinase I, which were isolated from the filtrate, did not form the protoplast by itself while a mixture of them showed protoplast-forming activity. Streptomyces cyaneus SP-27 was isolated based on the productivity of chitinase. The culture filtrate of S. cyaneus SP-27 did not form S. commune protoplasts, but addition of it to alpha-1,3-glucanase of B. circulans KA-304 brought about protoplast-forming activity. Chitinase A isolated from the S. cyaneus SP-27 culture filtrate was more effective than chitinase I of B. circulans KA-304 for the protoplast formation in combination with alpha-1,3-glucanase. The N-terminal amino acid sequence of chitinase A (MW 29,000) has a sequential similarity to those of several Streptomycete family 19 chitinases. Chitinase A adsorbed to chitinous substrate and inhibited the growth of Trichoderma reesei mycelia. Anomer analysis of the reaction products also suggested that the enzyme is a family 19 chitinase.

Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film.

Abstract: Somatic hybridization has been identified as one method for the genetic improvement of roses. The success of somatic hybridization programmes relies to a great extent upon efficient protoplast isolation and culture and selection of heterokaryons. This paper reports the isolation of rose cell suspension protoplasts by direct sucrose flotation and demonstrates their culture using extra thin alginate film. A comparative assessment of the efficiency of conventional culture techniques versus those with extra thin alginate film or thin alginate layer is also presented. A very high plating efficiency (80%) was obtained using thin alginate layer or extra thin alginate film techniques with improved media formulations. Protoplasts of Rosa damascena and R. bourboniana were fused by using polyethylene glycol as fusogen and later immobilized in the thin layer of alginate. The fused protoplasts were tracked on the basis of differential fluorescent staining, and the hybridity of heterokaryons following their development to callus was confirmed by molecular characterization. This novel selection strategy has general applicability and is faster and simpler to perform during somatic hybridization experiments.

Chromosomes are eliminated in the symmetric fusion between Arabidopsis thaliana L. and Bupleurum scorzonerifolium Willd.

Abstract: In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed.

Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

Abstract: This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of micro-colonies with plating efficiencies 3–10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.

Comparison between polyethylene glycol- and polyethylenimine-mediated transformation of Aspergillus nidulans

Abstract: Genetic transformation of many filamentous fungi is carried out by a protocol that utilizes polyethylene glycol (PEG) and calcium ion (Ca2+). This method has remained practically unchanged for more than 20 years, but the roles these molecules play are not definitively understood. To gain a better understanding, we have compared PEG transformation to a protocol using polyethylenimine (PEI) that is the basis for non-viral transfection in mammals and which has a well established molecular model for assisting DNA uptake. Protoplasts of Aspergillus nidulans could be transformed in the presence of Ca2+ with a relatively high ratio of PEI to DNA molecules. By comparing PEI and PEG in terms of interaction with DNA, fungal protoplasts, and response to different transformation conditions, we propose that the role of PEG is most likely to function after transforming DNA is incorporated into protoplasts, rather than the accepted view that it functions outside of the cell. Confirmation that protoplast fusion was not involved in DNA uptake is consistent with this hypothesis.

Characteristics of protoplast inter, intra-fusant and regeneration of antagonistic fungi Trichoderma harzianum and Trichoderma viride

Abstract: A standard method for isolation, fusion and regeneration of protoplasts from Trichoderma harzianum and Trichoderma viride was developed. The protoplasts from T. harzianum and T. viride were isolated using Novozym 234 as lytic enzyme and potassium chloride as osmotic stabilizer. The maximum number of protoplasts (9.37 × 105/ml) was obtained from 16 h old mycelium of T. harzianum and 11.1 × 105/ml for T. viride from 14 h old mycelium at pH 5.5, 28oC for 3 h. The interspecific and intraspecific fusion frequency was determined using 40% polyethylene glycol (PEG) as fusogen. The intrafusants were selected based on their growth, sporulation, pigmentation on chitin and cellulose amended media, where as the interfusants were selected on fungicide resistance as a marker. The protoplast fusion frequency was found to be 1.92% for interspecific fusion. In the case of intraspecific fusion it was about 6.2 and 7.2%, respectively, for T. harzianum and T. viride. The protoplast regeneration frequency of intrafusant was 17% for T. harzianum on chitin medium and 19.2% for T. viride on cellulose medium after two days. The regeneration frequency of 11% for interfusants on fungicide amended medium was observed after three days. The regenerated fusants morphology, growth, sporulation and pigmentation were compared with parental strains.

Collection of proteins secreted from yeast protoplasts in active cell wall regeneration.

Abstract: The yeast cell wall is a dynamic and complex matrix of polysaccharides (glucans, mannans, and chitin), proteins and minor amounts of lipids that isolate the yeast from the extracellular medium, protecting it against osmotic and physical injuries. Removal of this essential structure for cell integrity and viability by controlled enzymatic digestion in an iso-osmotic medium brings about protoplast formation. When yeast protoplasts are incubated in an osmotically stabilized liquid nutrient medium, cell wall precursors are secreted into the culture medium to de novosynthesize the cell wall. During the early stages of the regeneration process of protoplast walls, many wall protein precursors (presumably structural proteins along with remodeling and cross-linking enzymes) are shed into the extracellular medium but not covalently incorporated into the nascent cell wall, intriguingly enabling their easy isolation and solubilization. We have developed a method to collect proteins secreted from yeast protoplasts in active cell wall regeneration under conditions that are suitable for subsequent proteomic analyses. This procedure circumvents some of the troubles intrinsically related to other extraction protocols of cell wall proteins, such as chemical or enzymatic modifications, and poor quality in protein resolution and identification because of linkages to glucan/chitin residues. It further offers a valuable model system to understand how the de novocell wall biosynthesis occurs in the yeast cell or how the yeast cell wall participates in morphogenesis.

Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum.

Abstract: Summary • The role of phosphoinositide-specific phospholipase C (PI-PLC) signaling in the macrotubule-dependent protoplast volume regulation in plasmolyzed root cells of Triticum turgidum was investigated. • At the onset of hyperosmotic stress, PI-PLC activation was documented. Inhibition of PI-PLC activity by U73122 blocked tubulin macrotubule formation in plasmolyzed cells and their protoplast volume regulatory mechanism. In neomycin-treated plasmolyzed cells, macrotubule formation and protoplast volume regulation were not affected. In these cells the PI-PLC pathway is down-regulated as neomycin sequesters the PI-PLC substrate, 4,5-diphosphate-phosphatidyl inositol (PtdInsP2). These phenomena were unaffected by R59022, an inhibitor of phosphatidic acic (PA) production via the PLC pathway. • Taxol, a microtubule (MT) stabilizer, inhibited the hyperosmotic activation of PI-PLC, but oryzalin, which disorganized MTs, triggered PI-PLC activity. Taxol prevented macrotubule formation and inhibited the mechanism regulating the volume of the plasmolyzed protoplast. Neomycin partly relieved some of the taxol effects. • These data suggest that PtdInspP2 turnover via PI-PLC assists macrotubule formation and activation of the mechanism regulating the plasmolyzed protoplast volume; and the massive disorganization of MTs that is carried out at the onset of hyperosmotic treatment triggers the activation of this mechanism.

Protoplast fusion technology for somatic hybridisation in Phaseolus

Abstract: The success of interspecific breeding between Phaseolus vulgaris L. (PV) and the two donor species Phaseolus coccineus L. (PC) or Phaseolus polyanthus Greenm. (PP) requires the utilization of the donor species as female parents. Although incompatibility barriers are post-zygotic, success in such F1 crosses is very limited due to early hybrid embryo abortion. Rescue techniques for globular or early heart-shaped embryos have been improved but hybrid plant regeneration remains very difficult. In this study we describe the use of protoplast fusion techniques within the genus Phaseolus, as an alternative to succeed crosses between PP or PC and PV. Large numbers of heterokaryons have been produced using different genotypes and procedures for fusion, based either on electro-fusion (750 or 1500 V.cm-1), or on the use of a chemical micro-method with polyethylene glycol (PEG 6000) as the fusing agent. Both divisions of heterokaryons and the formation of heterokaryon-derived microcalli were observed.

A new method for producing hybridstrains of the entomopathogenic fungusverticillium lecanii (lecanicillium spp.)through protoplast fusion by usingnitrate non-utilizing (nit) mutants

Abstract: Resumen en: Mycotal and Vertalec are mass-produced fungal strains for insect control. Strain B-2, which was isolated in Japan, has high epiphytic ability on cucumbe...

Development of an effective protoplast fusion system for production of new potatoes with disease and insect resistance using Mexican wild potato species as gene pools

Abstract: Somatic hybridization through protoplast fusion is an important alternative approach for overcoming sexual incompatibility between diploid Solanum species and cultivated potatoes. However, compared with other potato species, methods for protoplast isolation and plant generation for several Mexican wild diploid potato species are not well established. In this study, a systematic procedure was designed for the isolation of a large number of high-quality protoplasts from various Mexican wild species that carry high levels of disease (late blight) and insect [Colorado potato beetle (CPB)] resistance. Using this procedure, an effective potato protoplast fusion system was developed to produce new somatic hybrids between two Mexican, one Argentina wild species, and cultivated potato clones. Regenerated plants and somatic hybrids were obtained at a high frequency from the protoplasts of the diploid wild species and their fused cells with S. tuberosum. Morphological, cytological and molecular marker analyses demon...

Degree of RNA silencing and the ability of a viral suppressor vary depending on the cell species in a protoplast system

Abstract: RNA silencing is a sequence-specific defense mechanism against viruses. As a counterdefense, viruses evolved silencing suppressors to interfere with host silencing. In analyses using protoplasts prepared from cultured cells (BY-2) and mesophyll cells of Nicotiana tabacum and N. benthamiana, viral suppressors differentially functioned in different cell types. This phenomenon has not been discussed in earlier papers on protoplast systems and RNA silencing. In investigations of the cellular activities of viral suppressors and their role in the RNA-silencing pathway, assays with host protoplasts offer many advantages and can complement other in planta assays such as Agrobacterium-mediated transient expression.

Production of Somatic Hybrids between Satsuma Mandarin (Citrus unshiu) and Navel Orange (Citrus sinensis) by Protoplast Fusion

Abstract: We have regenerated altotetraploid plants that are interspecific somatic hybrids between Citrus sinensis Osbeck cv. Yoshida navel orange and Citrus unshiu Marc cv. Okitsu satsuma mandarin. Protoplasts isolated from ‘Yoshida’ leaves were chemically fused with call us-derived protoplasts from ‘Okitsu’. After 6 months of culture, 102 plants were obtained. These hybrids were identified by differential leaf morphology, DNA fluorescence intensity, and DNA analysis. Ploidy analysis via the flow cytometry revealed that 15 of the 102 plants were tetraploids, with the rest being diploids that morphologically resembled their mesophyll parent. SRAP analysis confirmed that 9 of the tetraploid plants were allotetraploid somatic hybrids. These will be utilized as a possible pollen parents for improving seedy citrus cultivars, e.g., ponkan, mandarin, lemon and kumquat, in order to produce triploid seedless hybrids.

Effect of UV-C irradiation on mesophyll protoplasts of Cucumis sativus

Abstract: In the present work we studied the effect of UV-C irradiation on short-term protoplast physiology, with the aim of identifying and assessing parameters which can provide valuable information for asymmetric fusion experiments. Protoplast viability, cell wall regeneration, density of cell suspension and intensity of DAPI signal were followed by using microscopy and by the detection of specific fluorescent or spectroscopic signals in a microplate reader. The control and irradiated mesophyll protoplasts of Cucumis sativus were used for this experiment. In contrast to control cells, viability of irradiated cells significantly decreased. Intensive cell wall regeneration was observed only in control cells, which also showed significantly higher DAPI fluorescence signal. Microscopy for determination of viability by FDA and cell wall regeneration by Calcofluor White were modified for microplate reader instrumentation. These methods are simple, fast and suitable for detection of the effectiveness of UV-C irradiation of cells intended to be used in asymmetric fusion experiments.