Showing papers on "Protoplast published in 2009"

Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

Abstract: Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments. In this report, we present a new procedure that we call the Tape-Arabidopsis Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis) are used to make a "Tape-Arabidopsis Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC) to investigate protein-protein interactions in vivo, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr. The protoplasts generated by this new Tape-Arabidopsis Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time.

Genome shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae

Abstract: Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we improved the thermotolerance and ethanol tolerance of an industrial yeast strain SM-3 by genome shuffling while simultaneously enhancing the ethanol productivity. The starting population was generated by protoplast ultraviolet irradiation and then subjected for the recursive protoplast fusion. The positive colonies from the library, created by fusing the inactivated protoplasts were screened for growth at 35, 40, 45, 50 and 55°C on YPD-agar plates containing different concentrations of ethanol. Characterization of all mutants and wild-type strain in the shake-flask indicated the compatibility of three phenotypes of thermotolerance, ethanol tolerance and ethanol yields enhancement. After three rounds of genome shuffling, the best performing strain, F34, which could grow on plate cultures up to 55°C, was obtained. It was found capable of completely utilizing 20% (w/v) glucose at 45–48°C, producing 9.95% (w/v) ethanol, and tolerating 25% (v/v) ethanol stress.

Plant Cell and Tissue Culture - A Tool in Biotechnology: Basics and Application

Abstract: 1 Introduction 2 Historical Developments of Cell and Tissue Culture Techniques 3 Callus Cultures 3.1 Establishment of a Primary Culture from Explants of the Secondary Phloem of the Carrot Root 3.2 Fermenter Cultures 3.3 Immobilized Cell Cultures 3.4 Nutrient Media 3.5 Evaluation of Experiments 3.6 Maintenance of Strains, Cryopreservation 3.7 Some Physiological, Biochemical, and Histological Aspects 4 Cell Suspension Cultures 4.1 Methods to Establish a Cell Suspension 4.2 Cell Population Dynamics 5 Protoplast Cultures 5.1 Production of Protoplasts 5.2 Protoplast Fusion 6 Haploid Techniques 6.1 Application Possibilities 6.2 Physiological and Histological Background 6.3 Methods for Practical Application 6.4 Haploid Plants 7 Plant Propagation-Meristem Cultures, Somatic Embryogenesis 7.1 General Remarks, and Meristem Cultures 7.2 Protocols of Some Propagation Systems 7.2.1 In Vitro Propagation of Cymbidium 7.2.2 Meristem Cultures of Raspberries 7.2.3 In Vitro Propagation of Anthurium 7.3 Somatic Embryogenesis 7.3.1 Basics of Somatic Embryogenesis 7.3.2 Ontogenesis of Competent Cells 7.3.3 Genetic Aspects-DNA Organization 7.3.4 The Phytohormone System 7.3.5 The Protein System 7.3.6 Cell Cycle Studies 7.4 Practical Application of Somatic Embryogenesis 7.5 Artificial Seeds 7.6 Embryo Rescue 8 Some Endogenous and Exogenous Factors in Cell Culture Systems 8.1 Endogenous Factors 8.1.1 Genetic Influences 8.1.2 Physiological Status of 'Mother Tissue' 8.1.3 Growth Conditions of the 'Mother Plant' 8.2 Exogenous Factors 8.2.1 Growth Regulators 8.2.2 Nutritional Factors 8.2.2.1 Improvement of Nutrient Uptake by Transgenic Carrot Cultures 8.3 Physical Factors 9 Primary Metabolism 9.1 Carbon Metabolism 9.2Nitrogen Metabolism 10 Secondary Metabolism 10.1 Introduction 10.2 Mechanism of Production of Secondary Metabolites 10.3 Historical Background 10.4 Plant Cell Cultures and Pharmaceuticals, and Other Biologically Active Compounds 10.4.1 Antitumor Compounds 10.4.2 Anthocyanin Production 10.5 Strategies for Improvement of Metabolite Production 10.5.1 Addition of Precursors, and Biotransformations 10.5.2 Immobilization of Cells 10.5.3 Differentiation and Secondary Metabolite Production 10.5.4 Elicitation 10.5.4.1 Jasmonic Acid 10.5.4.2 Effect of UV on Production of Secondary Metabolites in Cultured Tissues 10.6 Organ Cultures 10.6.1 Shoot Cultures 10.6.2 Root Cultures 10.7 Genetic Engineering of Secondary Metabolites 10.8 Membrane Transport and Accumulation of Secondary Metabolites 10.9 Bioreactors 10.9.1 Technical Aspects of Bioreactor Systems 10.10 Prospects 11 Phytohormones and Growth Regulators 12 Cell Division, Cell Growth, Cell Differentiation 13 Genetic Problems and Gene Technology 13.1 Somaclonal Variations 13.1.1 Ploidy Stability 13.1.2 Some More Somaclonal Variations 13.2 Gene Technology 13.2.1 Transformation Techniques 13.2.1.1 Direct Gene Transfer 13.2.1.2 Agrobacterium-Mediated Gene Transformation 13.2.2 Selectable Marker Genes 13.2.2.1 Reporter Genes 13.2.2.2 Variants of GFP 13.2.3 ss-Glucuronidase (GUS) 13.2.3.1 Procedures for Assay of GUS Gene Expression 13.2.4 Antibiotics Resistance Genes 13.2.5 Elimination of Marker Genes 13.2.5.1 Cre-lox Recombination-Based Systems 13.2.5.2 Ac/Ds System 13.2.5.3 Double Cassette System 13.2.6 Agrobacterium-Mediated Transformation in Dicotyledonous Plants 13.2.6.1 Transgenic Carrot: Potential Source of Edible Vaccines 13.2.6.2 Cell Culture and Transformation Procedures 13.2.6.3 Uses of Transgenes to Increase Host Plant

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Abstract: Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Protoplast isolation and plant regeneration of different genotypes of Petunia and Calibrachoa

Abstract: Protoplasts were isolated from leaves of in vitro-grown shoot cultures of different Petunia and Calibrachoa genotypes by enzyme digestion with 0.6% macerozyme R-10 and 2.0% cellulase R-10. Shoot regeneration was achieved in five out of nine Calibrachoa and three out of four Petunia genotypes. Protoplast yield and frequency of shoot regeneration varied among experiments and genotypes. Among all regenerants, few morphological changes, such as chlorophyll deficiency, variegated leaves, and polyploidization, were observed.

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of Alternaria alternata

Abstract: To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity.

Improvement in cell-bound phytase activity of Pichia anomala by permeabilization and applicability of permeabilized cells in soymilk dephytinization.

Abstract: Aims: Whole cell permeabilization of Pichia anomala to ameliorate the cell-bound phytase activity and usability of permeabilized cells in dephytinization of soymilk. Methods and Results: The cells of P. anomala were subjected to permeabilization using the surfactant Triton X-100 to overcome the permeability barrier and prepare whole cell biocatalysts with high phytase activity. The statistical approach, response surface methodology (RSM) was used to optimize the operating conditions for permeabilization. The treatment of cells with 5% Triton X-100 for 30 min resulted in c. 15% enhancement in cell-bound phytase activity. The shrinkage of protoplast was observed, although cell viability and phytase stability were not significantly altered. The free as well as immobilized permeabilized cells hydrolysed soymilk phytate, and the latter could be reused over four consecutive cycles. Conclusions: Whole cell permeabilization of P. anomala using Triton X-100 led to enhancement in cell-bound phytase activity. The viability and integrity of yeast cells were not significantly affected because of permeabilization. The permeabilized P. anomala cells effectively dephytinized soymilk, and the permeabilized cells immobilized in alginate could be reused because of sustained phytase activity. Significance and Impact of the Study: This is the first report on the use of permeabilized yeast cells for mitigating phytate content of soymilk. Alginate entrapment of permeabilized P. anomala allows reuse of cells for soymilk dephytinization, thus suggesting a potential application in food industry.

Development and evaluation of a storage root-bearing sweetpotato somatic hybrid between Ipomoea batatas (L.) Lam. and I. triloba L.

Abstract: A storage root-bearing somatic hybrid was produced for the first time by protoplast fusion between sweetpotato (Ipomoea batatas (L.) Lam.) cv. Kokei No. 14 and its wild relative I. triloba L. Protoplasts isolated from embryogenic suspension cultures of Kokei No. 14 were fused with petiole protoplasts of I. triloba L. using polyethylene glycol-mediated protocol. Fusion products were cultured in a modified Murashige and Skoog medium containing 0.05 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l−1 kinetin. A total of 176 plants were obtained from 42 out of 134 calluses derived from fused protoplasts, and 91 of these plants were confirmed to be somatic hybrids through peroxidase isozyme, random amplified polymorphic DNA, amplified fragment length polymorphism, and cytological analyses. Upon transfer into soil and grown in the greenhouse and then to the field, 100% survival was observed. A single plant, designated KT1, was found to produce storage roots. Genomic in situ hybridization analysis confirmed presence of chromosomes from both parents and recombinant chromosomes in KT1. Drought tolerance, dry matter content, soluble sugar content, and fertility of this somatic hybrid were evaluated for potential use in sweetpotato breeding.

Isolation, callus formation and plantlet regeneration from mesophyll protoplasts of Tylophora indica (Burm. f.) Merrill: an important medicinal plant

Abstract: Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 × 105 protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 µM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 µmol m−2 S−1 of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.

Intertribal somatic hybrids between Brassica napus and Camelina sativa with high linolenic acid content

Abstract: Intertribal somatic hybrids of Brassica napus and Camelina sativa were developed by protoplast electrofusion. Hybrid identity of the regenerants was determined using flow cytometric analysis of nuclear DNA content and simple sequence repeat (SSR) marker analysis. Three hybrids exhibited specific bands for B. napus and C. sativa. These hybrids showed intermediate leaf, flower and seed morphology compared with the two parental species. The seeds of these three hybrids had a modified fatty acid profile, indicating higher level of linolenic and eicosanoic acids than those of B. napus. Our results suggest that somatic hybridization offers opportunities for transferring entire genomes between B. napus and C. sativa in improving rapeseed breeding.

Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of Robinia pseudoacacia L.

Abstract: A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 105 protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 105 protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and 1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.

Membrane potential changes during pollen germination and tube growth

Abstract: Using methods of quantitative fluorescent microscopy, we studied membrane potential changes during pollen germination and in growing pollen tubes. Two voltage-sensitive dyes were used, i.e., DiBAC4(3), to determine the mean membrane potential values in pollen grains and isolated protoplasts, and Di-4-ANEPPS, to map the membrane potential distribution on the surfaces of the pollen protoplast and pollen tube. We have shown that the activation of the tobacco pollen grain is accompanied by the hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (−108 mV) with respect to the pollen grains (−23 mV) from which they were isolated. We have found the polar distribution of the membrane potential along the protoplast surface and the longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H+-ATPase inhibitor sodium orthovanadate (1 mM) or its activator fusicoccin (1 μM), the longitudinal voltage gradient was modified, but did not disappear. Anion channel blocker NPPB (40 μM) fully discarded the gradient in pollen tubes. The obtained results indicate the hyperpolarization of the plasma membrane during pollen germination and uneven potential distribution on the pollen grain and tube surfaces. An inhibitory analysis of the distribution of the potential in the tube has revealed the involvement of the plasma membrane H+-ATPase and anion channels in the regulation of its value.

Somatic hybrids obtained by asymmetric protoplast fusion between Musa Silk cv. Guoshanxiang (AAB) and Musa acuminata cv. Mas (AA)

Abstract: To attempt to introduce genetic information of disease resistance from Musa acuminata cv. Mas (AA) to Musa silk cv. Guoshanxiang (AAB) and obtain somatic hybrids, we developed an asymmetric protoplast fusion with 20% (w/v) polyethylene glycol (PEG). The protoplasts derived from embryogenic suspension cultural cells of cv. Guoshanxiang (AAB) and cv. Mas (AA) were, respectively treated with 1.5 mM iodoacetamide (IOA) and with ultraviolet light (UV) at an intensity of 50 W/m2 for 120 s. A total of 47 regenerated green plants were obtained and eight of which were survived in greenhouse. Six of the survived plants were identified as hybrids by RAPD analysis and only three hybrids were retained vigorously in field. The hybrid nature of the three plants was further confirmed according to their ISSR (inter-simple sequence repeat) patterns and the results indicated that they were true somatic hybrids. Chromosome analysis revealed that the three hybrids possessed an aneuploid chromosome number (2n = 34).

Protoplast isolation and culture for somatic hybridization of Lupinus angustifolius and L. subcarnosus

Abstract: A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 105 protoplasts g−1 fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.

The heterochromatin as a marker for protoplast differentiation of Cucumis sativus

Abstract: The protoplast cultures of Cucumis sativus in two culture systems were used to study heterochromatin reassembly during dedifferentiation of isolated protoplasts and their subsequent differentiation into calli and proembryos. Here we show that dedifferentiation of the cucumber mesophyll cells is accompanied by a dramatic reduction in size and numbers of nuclear chromocenters. Although chromocenters were newly established during protoplast culture, the measured relative heterochromatin content differed according to the culture system used. Protoplast culture leading to proembryo formation displayed a lower level of relative heterochromatin content than cultures resulting in calli and the relative heterochromatin content reached values close to those estimated for somatic embryos.

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

Abstract: The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO2 overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 106 protoplasts per gram of fresh weight.

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium × hortorum, ‘Panaché Sud’

Abstract: Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm−1 electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm−1 allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l−1 kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l−1 kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.

Improvement in Lupinus luteus (Fabaceae) protoplast culture – stimulatory effect of agarose embedding and chemical nursing on protoplast divisions

Abstract: Comprehensive studies were undertaken to determine optimal conditions for yellow-lupin (Lupinus luteus L.) protoplast culture. The adopted isolation procedure resulted in a high yield of protoplasts obtained from hypocotyls, cotyledons and young leaves. The usefulness of liquid and agarose-solidified media was evaluated in relation to promoting survivability and morphogenetic responses of protoplasts. The influence of different plant-growth regulators and anti-oxidative agents was also examined. Cultivar ‘Parys’ was the most promising material for manipulations. The solidification of medium enhanced the development of cultures initiated from hypocotyls and cotyledons, significantly increasing the division rate. In both liquid and solid media, mitoses were arrested after the initial division and daughter cells failed to divide. An important breakthrough in the development of cultures appeared in media supplemented with 0.1% activated charcoal. Bypassing the mitosis suppression led to the formation of small aggregates from hypocotyl protoplasts. This is the first report on protocolony formation in protoplast cultures of L. luteus. Notable progress achieved here indicates that, although this species is considered as recalcitrant to in vitro manipulations, it is possible to obtain responsive cultures with higher morphogenetic potential and thus provide new material for breeding programs of lupins.

Protoplast culture and fusion between Brassica carinata and Brassica napus.

Abstract: In this study, attempts were made to select in vitro responsible genotypes and to fuse the isolated protoplasts of Brassica carinata and Brassica napus breeding lines (BC DH Dodolla, BC DH -1, BC DH-6 and BN OP-1, BN-SL03/04), obtained from our previous experiments. Combination of three different PEG concentrations (20%, 25% or 30%) and two different treatment durations (15 and 20 min.) were tested. Our experiments identified several genotypes (Brassica carinata DH BC-6 and DH BC-1, Brassica napus DH OP-01) with satisfactory regeneration ability of calli from protoplast cultures. Proper combinations of concentration and treatment time of PEG determined protoplast fusion frequency between genotypes used. Although the 30% PEG solution was evaluated to be the best concentration, large amount of multifusants, unwanted in practical applications, was detected especially in Petri dishes with longer PEG treatment. In general, 25% PEG combined with 20 minutes treatment duration produced satisfactory fusion frequency and good rate of viability was obtained as well.

Identification of protoplast fusion strain Fhhh by randomly amplified polymorphic DNA

Abstract: A functional strain Fhhh was constructed through protoplast fusion of three parental strains (Phanerochaete chrysosporium, Saccharomyces cerevisiae and native bacterium YZ) to improve the degradation efficiency of purified terephthalic acid wastewater. Randomly amplified polymorphic DNA (RAPD) and scanning electron microscope (SEM) analysis were applied to identify and confirm the fusant Fhhh through phenotypic and genetic relationship. The result of SEM analysis demonstrated that the cell shape of fusant Fhhh differed from all three parental strains. RAPD analysis of 40 arbitrary primers generated a total of 1,135 bands. The genetic similarity indices between Fhhh and parental strains Phanerochaete chrysosporium (PC), Saccharomyces cerevisiae (SC) and native bacterium (YZ) were 34.01%, 33.16%, and 35.97%, respectively. The targeted-gene PCR results showed that Fhhh inherited the DNA fragments of mnp and lip genes from parental strain PC and FLO1 gene fragment from parental strain SC. Our results suggested protoplast fusion technique may be considered as a promising technique in environmental pollution control.

Protoplast isolation from Solanum lycopersicum L. leaf tissues and their response to short-term NaCl treatment

Abstract: Protoplasts were isolated from young leaves of tomato (Solanum lycopersicon L cvar Rio Fuego) The optimum conditions for protoplast isolation was established by using 2% cellulose R-10 and 05% macerozyme R-10 dissolved in 04 M sucrose-K3 solution for 12 h cell wall digestion In order to induce salt stress, the mannitol content of the buffer was partially replaced by NaCl to get an isoosmotic incubation solution containing 100 mM NaCl It can be concluded that the number of protoplast in unit volume counted by Burker chamber did not decrease significantly compared to controls due to salt treatment upto 5 hours, but the viability of cells decreased by 55% using fluorescein diacetate staining Hundred mM NaCl simultaneously enhanced the generation of reactive oxygen species in tomato leaf protoplast This means that decreases in fluorescein fluorescence is a good and sensitive parameter for the measurement of Na+-induced decrease in cell viability and cell death in protoplast suspensions

Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

Abstract: A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml−1). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

Somatic Hybridization in the Moss Physcomitrella patens Using PEG- Induced Protoplast Fusion

Abstract: As an alternative to sexual crossing, protoplasts from two strains of moss (Physcomitrella patens) can be hybridized using polyethylene glycol (PEG). Although the efficiency is low, it requires no sophisticated apparatus. Hybrids are readily obtained using complementary auxotrophic mutants or strains with transgenic antibiotic resistance markers. It is now routine to obtain hybrids using transgenic strains that are hygromycinor G418-resistant by selecting hybrids on medium containing both antibiotics.

Separation and culturing method of saussurea involucrate protoplast

Abstract: The invention relates to a separation and culturing method of Sinkiang saussurea protoplast, comprising four steps of culturing of sterile saussurea involucrate seedlings, inducement and differentiation of embryonic callus, separation and purification of plasmogen and culturing of the protoplast which are all applicable to the separation and culturing of the Sinkiang saussurea involucrate protoplast. The invention has simple and easily-operated method and lower requirements on the equipment and culturing conditions; in addition, the enzymolysis method is practical, the conditions for cell wall removal are mild, and the separated protoplast has high yield and strong activity, and is easy for the cultuing of the saussurea involucrate protoplast and a plant regeneration, thus laying the foundations for the culturing and fusing of the protoplast as well as the germplasm resource innovation of the Sinkiang saussurea involucrate and the research of transgenic saussurea involucrate.