Showing papers on "Protoplast published in 2013"

First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus Glomus intraradices.

Abstract: Arbuscular mycorrhizal (AM) symbiosis is known to stimulate plant drought tolerance. However, the molecular basis for the direct involvement of AM fungi (AMF) in plant water relations has not been established. Two full-length aquaporin genes, namely GintAQPF1 and GintAQPF2, were cloned by rapid amplification of cDNA 5'- and 3'-ends from an AMF, Glomus intraradices. Aquaporin localization, activities and water permeability were examined by heterologous expression in yeast. Gene expression during symbiosis was also analyzed by quantitative real-time polymerase chain reaction. GintAQPF1 was localized to the plasma membrane of yeast, whereas GintAQPF2 was localized to both plasma and intracellular membranes. Transformed yeast cells exhibited a significant decrease in cell volume on hyperosmotic shock and faster protoplast bursting on hypo-osmotic shock. Polyethylene glycol (PEG) stimulated, but glycerol inhibited, the aquaporin activities. Furthermore, the expression of the two genes in arbuscule-enriched cortical cells and extraradical mycelia of maize roots was also enhanced significantly under drought stress. GintAQPF1 and GintAQPF2 are the first two functional aquaporin genes from AMF reported to date. Our data strongly support potential water transport via AMF to host plants, which leads to a better understanding of the important role of AMF in plant drought tolerance.

Characterization of Amoeboaphelidium protococcarum, an Algal Parasite New to the Cryptomycota Isolated from an Outdoor Algal Pond Used for the Production of Biofuel

Abstract: Mass culture of algae for the production of biofuels is a developing technology designed to offset the depletion of fossil fuel reserves. However, large scale culture of algae in open ponds can be challenging because of incidences of infestation with algal parasites. Without knowledge of the identity of the specific parasite and how to control these pests, algal-based biofuel production will be limited. We have characterized a eukaryotic parasite of Scenedesmus dimorphus growing in outdoor ponds used for biofuel production. We demonstrated that as the genomic DNA of parasite FD01 increases, the concentration of S. dimorphus cells decreases; consequently, this is a highly destructive pathogen. Techniques for culture of the parasite and host were developed, and the endoparasite was identified as the Aphelidea, Amoeboaphelidium protococcarum. Phylogenetic analysis of ribosomal sequences revealed that parasite FD01 placed within the recently described Cryptomycota, a poorly known phylum based on two species of Rozella and environmental samples. Transmission electron microscopy demonstrated that aplanospores of the parasite produced filose pseudopodia, which contained fine fibers the diameter of actin microfilaments. Multiple lipid globules clustered and were associated with microbodies, mitochondria and a membrane cisternae, an arrangement characteristic of the microbody-lipid globule complex of chytrid zoospores. After encystment and attachment to the host cells, the parasite injected its protoplast into the host between the host cell wall and plasma membrane. At maturity the unwalled parasite occupied the entire host cell. After cleavage of the protoplast into aplanospores, a vacuole and lipids remained in the host cell. Amoeboaphelidium protococcarum isolate FD01 is characteristic of the original description of this species and is different from strain X-5 recently characterized. Our results help put a face on the Cryptomycota, revealing that the phylum is more diverse than previously understood and include some of the Aphelidea as well as Rozella species and potentially Microsporidia.

Progress in plant protoplast research

Abstract: In this review we focus on recent progress in protoplast regeneration, symmetric and asymmetric hybridization and novel technology developments. Regeneration of new species and improved culture techniques opened new horizons for practical breeding in a number of crops. The importance of protoplast sources and embedding systems is discussed. The study of reactive oxygen species effects and DNA (de)condensation, along with thorough phytohormone monitoring, are in our opinion the most promising research topics in the further strive for rationalization of protoplast regeneration. Following, fusion and fragmentation progress is summarized. Genomic, transcriptomic and proteomic studies have led to better insights in fundamental processes such as cell wall formation, cell development and chromosome rearrangements in fusion products, whether or not obtained after irradiation. Advanced molecular screening methods of both genome and cytoplasmome facilitate efficient screening of both symmetric and asymmetric fusion products. We expect that emerging technologies as GISH, high resolution melting and next generation sequencing will pay major contributions to our insights of genome creation and stabilization, mainly after asymmetric hybridization. Finally, we demonstrate agricultural valorization of somatic hybridization through enumerating recent introgression of diverse traits in a number of commercial crops.

Characterization of the Early Events Leading to Totipotency in an Arabidopsis Protoplast Liquid Culture by Temporal Transcript Profiling

Abstract: The molecular mechanisms underlying plant cell totipotency are largely unknown. Here, we present a protocol for the efficient regeneration of plants from Arabidopsis thaliana protoplasts. The specific liquid medium used in our study leads to a high rate of reentry into the cell cycle of most cell types, providing a powerful system to study dedifferentiation/regeneration processes in independent somatic cells. To identify the early events in the establishment of totipotency, we monitored the genome-wide transcript profiles of plantlets and protoplast-derived cells (PdCs) during the first week of culture. Plant cells rapidly dedifferentiated. Then, we observed the reinitiation and reorientation of protein synthesis, accompanied by the reinitiation of cell division and de novo cell wall synthesis. Marked changes in the expression of chromatin-associated genes, especially of those in the histone variant family, were observed during protoplast culture. Surprisingly, the epigenetic status of PdCs and well-established cell cultures differed, with PdCs exhibiting rare reactivated transposons and epigenetic changes. The differentially expressed genes identified in this study are interesting candidates for investigating the molecular mechanisms underlying plant cell plasticity and totipotency. One of these genes, the plant-specific transcription factor ABERRANT LATERAL ROOT FORMATION4, is required for the initiation of protoplast division.

Endogenous nitric oxide generation in protoplast chloroplasts

Abstract: NO generation is studied in the protoplast chloroplasts. NO, ONOO − and ROS (O and H 2 O 2 ) are generated in chloroplasts. Nitric oxide synthase-like protein appears to be involved in NO generation. Nitric oxide stimulates chlorophyll biosynthesis and chloroplast differentiation. The present study was conducted to better understand the process of NO generation in the leaf chloroplasts and protoplasts. NO, peroxynitrite and superoxide anion were investigated in the protoplasts and isolated chloroplasts using specific dyes, confocal laser scanning and light microscopy. The level of NO was highest after protoplast isolation and subsequently decreased during culture. Suppression of NO signal in the presence of PTIO, suggests that diaminofluorescein-2 diacetate (DAF-2DA) detected NO. Detection of peroxynitrite, a reaction product of NO and superoxide anion, further suggests NO generation. Moreover, generation of NO and peroxynitrite in the chloroplasts of wild-type Arabidopsis and their absence or weak signals in the leaf-derived protoplasts of Atnoa1 mutants confirmed the reactivity of DAF-2DA and aminophenyl fluorescein to NO and peroxynitrite, respectively. Isolated chloroplasts also showed signal of NO. Suppression of NO signal in the presence of 100 μM nitric oxide synthase inhibitors [l-NNA, Nω-nitro-l-arginine and PBIT, S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea] revealed that nitric oxide synthase-like system is involved in NO synthesis. Suppression of NO signal in the protoplasts isolated in the presence of cycloheximide suggests de novo synthesis of NO generating protein during the process of protoplast isolation. Furthermore, the lack of inhibition of NO production by sodium tungstate (250 μM) and inhibition by l-NNA, and PBIT suggest involvement NOS-like protein, but not nitrate reductase, in NO generation in the leaf chloroplasts and protoplasts.

Protoplast fusion for crop improvement and breeding in China

Abstract: Protoplast fusion offers an opportunity for circumventing barriers to sexual reproduction and allows for gene transfer of nuclear and cytoplasmic genomes to enrich the gene pool of cultivated species. Moreover, protoplast fusion effectively generates novel germplasm for elite breeding of conventional crosses and promotes crop improvement in existing cultivars. Over the past few decades, protoplast fusion in China has considerably progressed not only for food crops but also for economic plants. In this review, we present and discuss the development of somatic hybrids in wheat, rapeseed, citrus, and cotton, some of which are already in use or have potential for future commercial use in China. For example, an elite salt- and drought-resistant line, Shangrong No. 3, has already been registered as a commercial wheat cultivar. Some other hybrids have been found to have disease resistance as well as modified fatty acids, high oil and protein contents, novel cytoplasmic male sterility, and numerous other desirable agronomic characteristics that are useful for further breeding.

Introgression of bacterial wilt resistance from eggplant to potato via protoplast fusion and genome components of the hybrids

Abstract: Bacterial wilt resistant somatic hybrids were obtained via protoplast fusion between potato and eggplant and three types of nuclear genomes were identified in the hybrids through GISH and SSR analysis. Cultivated potato (Solanum tuberosum L.) lacks resistance to bacterial wilt caused by Ralstonia solanacearum. Interspecific symmetric protoplast fusion was conducted to transfer bacterial wilt resistance from eggplant (S. melongena, 2n = 2x = 24) into dihaploid potato (2n = 2x = 24). In total, 34 somatic hybrids were obtained, and of these, 11 rooted and were tested for genome components and resistance to race 1 of R. solanacearum. The hybrids exhibited multiple ploidy levels and contained the dominant nuclear genome from the potato parent. Three types of nuclear genomes were identified in the hybrids through genomic in situ hybridization (GISH) and simple sequence repeat (SSR) analysis, including (1) the potato type of the tetraploids in which eggplant chromosomes could not be detected by GISH but their nuclear DNA was confirmed by SSR, (2) the biased type of the hexaploids in which the chromosome dosage was 2 potato:1 eggplant, and (3) the chromosome translocation type of the mixoploids and aneuploids that was characterized by various rates of translocations of nonhomologous chromosomes. Cytoplasmic genome analysis revealed that mitochondrial DNA of both parents coexisted and/or recombined in most of the hybrids. However, only potato chloroplast DNA was retained in the hybrids speculating a compatibility between cpDNA and nuclear genome of the cell. The pathogen inoculation assay suggested a successful transfer of bacterial wilt resistance from eggplant to the hybrids that provides potential resistance for potato breeding against bacterial wilt. The genome components characterized in present research may explain partially the inheritance behavior of the hybrids which is informative for potato improvement.

Changes of DNA methylation and hydroxymethylation in plant protoplast cultures.

Abstract: Cytosine methylation patterns in higher eukaryotes are important in gene regulation Along with 5-methylcytosine (5-mC), a newly discovered constituent of mammalian DNA, 5-hydroxymethylcytosine (5-hmC), is the other modified base in higher organisms In this study we detected 5-hmC in plant protoplast DNA and demonstrated its increasing content during the first 72 hrs of protoplast cultivation In contrast to 5-hmC, the amount of 5-mC decreased during protoplast cultivation It was also found that 5-hmC did not primarily arise as a product of oxidative DNA damage following protoplast culture

Differentiation of Protoplasts and of Transformed Plant Cells

Abstract: Differentiation of protoplasts and of transformed plant cells , Differentiation of protoplasts and of transformed plant cells , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Proteome-based profiling of hypercellulase-producing strains developed through interspecific protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis.

Abstract: Thirty heterokaryons, formed by protoplast fusion of Aspergillus nidulans and Aspergillus tubingensis, were selected on the basis of their ability to grow on 2-deoxyglucose (0.2 %, w/v) and intermediate spore color. These heterokaryons were studied for cellulase production using shake flask and solid substrate cultures at 40 °C. Fusants 51 and 28 exhibited appreciably higher levels of endoglucanase, cellobiohydrolase, β-glucosidase, and FPase activities when compared with parental strains. Employing proteomic-based approaches, the differential expression of proteins in secretome of fusants and parental strains were analyzed using two-dimensional electrophoresis. The expression of some of the proteins in the fusants was found to be up/downregulated. The upregulated proteins in the fusant 51 were identified by liquid chromatography–mass spectroscopy as endoxylanase, endochitinase, β-glucosidase, as well as hypothetical proteins. The cellulases produced by fusants 28 and 51 showed improved saccharification of alkali treated rice straw when compared with the parental strains.

Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco

Abstract: It is known that protoplasts derived from either leaves or suspension cultures of a citrus genotype vary greatly in their regeneration capacities; however, the underlying physiological mechanisms are not well known. In this study, oxidative stress and antioxidant systems during in vitro culture of callus-derived protoplasts and leaf mesophyll-derived protoplasts of Ponkan (Citrus reticulata Blanco) were analyzed to gain insights into observed physiological differences. Morphological observations using light microscopy and scanning microscopy have shown that new cell wall materials appeared within 2–3 days, and the integrate cell walls were regenerated approximately after 6 days of culture of the callus protoplasts, whereas no cell wall formation was observed in the mesophyll protoplasts after culture. During the culture, higher levels of H2O2 and malondialdehyde were detected in the mesophyll protoplasts as compared with the callus ones. On the contrary, the callus protoplasts possessed higher activities of antioxidant enzymes (SOD, POD and CAT) and larger amount of glutathione and ascorbic acid (at one time point) than the mesophyll protoplasts during the culture process. The current data indicate that the mesophyll and callus protoplasts displayed remarkable difference in the degree of oxidative stress and the antioxidant systems, suggesting that high levels of antioxidant activities might play an important role in the regeneration of protoplasts.

Asymmetric Somatic Plant Hybridization: Status and Applications

Abstract: To create asymmetric somatic hybrids, the genome of the so-called donor protoplast is fragmented prior to protoplast fusion. As a result, only a limited amount of the donor genome is transferred to the fusion product. This technique can circumvent some commonly observed problems related to symmetric fusion and offers a practical breeding tool for asexual hybridization. Genomes are typically fragmented by irradiation, microprotoplast production or application of metabolic inhibitors such as iodoacetamide. Irradiation and microprotoplast production fragment the nuclear genome, whereas iodoacetamide inactivates the cytoplasmic genome. It can therefore be used to introduce cytoplasmic male sterility, an important practical application. For hybrid verification and genome characterization, molecular markers and cytogenetic techniques are applied. This review highlights and discusses progress made during the last decade in spermatophytes asymmetric protoplast fusion.

A Protoplast Transformation System for Gene Deletion and Complementation in Sclerotinia sclerotiorum

Abstract: Protoplast transformation is an important technique for establishing a mutation library and determining gene function for Sclerotinia sclerotiorum and other plant pathogenic fungi. In this study, we determined the effect of various conditions on preparation of protoplasts for transformation. These conditions included the age of the culture providing the hyphae to be digested; enzyme composition, buffer solution and concentration; and digestion time and temperature. The optimum conditions for preparing protoplasts were as follows: 10 ml of enzyme solution (1.5% lysing enzyme in 0.8 m mannitol and citric acid-sodium citrate buffer) reacting with 0.1 g of hyphae (cultured for 36 h) at 30°C for 2.5 h. The transformation efficiency was 60–85 transformants per microgram of DNA. In addition, an expression vector for gene complementation was constructed, and an additional dominant selectable marker (neomycin) was demonstrated. To verify the reliability of the expression vector, we constructed and transformed the complementation vector of Shk1 for gene complementation based on the Shk1 deletion mutant △Shk1. The results showed that the expression level and biological phenotypes of Shk1 were restored in the complementary strain △Shk1+Shk1. The techniques and procedures described will improve our ability to study gene function in S. sclerotiorum and are likely applicable to other plant pathogens.

Protoplast fusion between white and brown oyster mushrooms

Abstract: Genetic crossing of white oyster mushroom (Pleurotus floridae) to introduce longer storage life trait can only be done within individuals in this particular species. However, longer storage life trait is possessed by brown oyster mushroom (Pleurotus cystidiosus). Therefore, a protoplast fusion experiment between white and brown oyster mushrooms was conducted to obtain an oyster mushroom strain showing high productivity and long storage life. The experiment was done at the biology laboratory of the University of Al Azhar Indonesia from May 2008 to August 2009. Protoplast fusion was done by isolating protoplast from 5-day old monokaryotic mycelia grown in potato dextrose broth (PDB). Around 3.15 x 105 protoplasts ml-1 were harvested using mixture of cellulase Onozuka R-10 (1%) and macerozyme R-10 (1%) from brown oyster mushroom with 80.61% viability. Similarly, 3.71 x 105 protoplasts ml-1 were harvested using lysing enzyme (2%) from white oyster mushroom with 83.68% viability. Protoplast fusions were conducted using 40% PEG6000 for 10 minutes. The candidate fusants were then screened using minimum regeneration media (MRM). There were 22 colonies grew on MRM media and four colonies (FS1, FS2, FS3, and FS4) showed clamp connection as well as primordia formation to be chosen as candidate fusants. However, isozyme studies using malate dehydrogenase and acid phosphatase as marker enzymes confirmed that only FS1 and FS2 were the hybridized products. The two colonies showed different mycelia growth patterns and hyphae sizes, fruit body morphology and productivity compared to their parents. These two fusants, however, did not indicate the presence of longer storage life trait as expected despite a higher productivity achieved by FS1. In this study, the protoplast fusion only yielded higher productivity strain of mushroom with different colors without any changes in storage life.

Response of carrot protoplasts and protoplast-derived aggregates to selection using a fungal culture filtrate of Alternaria radicina

Abstract: Protoplasts isolated from three accessions of cultivated carrot and 5-day-old protoplast-derived aggregates were subjected to selection to identify somaclonal variants with enhanced tolerance to the fungal disease black rot incited by Alternaria radicina. Different concentrations [1, 2, 3.5, 5, 10, 20, 35 and 50 % (v/v)] of a fungal culture filtrate (FCF) from 2-week-old liquid cultures of A. radicina were used. Protoplasts and aggregates were subjected to short-term selection for a period of 10 days. All FCF concentrations added to the cultures on the day of isolation decreased protoplast survival frequency and plating efficiency, while FCF applied 5 days later inhibited cell divisions in 5–50 % concentrations. The responses of protoplasts to the treatment were genotype dependent. Most R0 plants were regenerated in all accessions from cell lines grown with 1 % FCF, while only a few plants were produced from 2 to 3.5 % FCF-treated cultures of ‘Dolanka’ and the breeding line ‘9304B’, respectively. Nineteen-percent of putative stress-tolerant regenerants were tetraploids, while only 5 % tetraploids were observed in the control. The incidence of unique random amplified polymorphic DNA fragments indicating possible chromosomal rearrangements was low and did not differ among regenerants after selection and those derived from the control. Mobilization of miniature inverted repeat transposable elements was not observed. Some R0 individuals regenerated both from FCF-treated and untreated cultures showed lower susceptibility to A. radicina in a laboratory assay in comparison to control plants grown from seed. Regenerants from FCF-treated cultures showed lower frequency of flowering plants and a higher rate of male sterility. Pollen viability of the putative stress-tolerant regenerants varied over a wide range (6–98 %), independently of in vitro selection conditions. Our data suggest that A. radicina FCF may be feasible for the in vitro selection to generate plants with superior phenotypic performance against A. radicina.

Relation between Amino Acids Profiles and Recalcitrancy of Cell Growth or Salt Tolerance in Tissue and Protoplast Cultures of Three Mangrove Species, Avicennia alba , Bruguiera sexangula , and Sonneratia alba

Abstract: Amino acids profiles were investigated in tissues, cultured cells, i.e. callus or suspension cells, and their protoplasts of three mangrove species, Avicennia alba, Bruguiera sexangula, and Sonneratia alba. Original tissues of cultured cells of three mangrove species were cotyledons and hypocotyls, leaves, and cotyledons, respectively. In protoplasts isolated from cultured cells, glutamine and alanine were the major amino acids. Different contents of glycine, proline and serine were observed among protoplasts of three mangrove species. Large differences in the major amino acids were found among cultured cells and their protoplasts while no difference was found between callus and suspension cells independent of additional salt in culture medium. Protoplasts of original tissues, young leaves and cotyledons, contained alanine and glutamine and/or asparagine. In suspension cells of B. sexangula, total contents of amino acids were low while their protoplasts showed similar value as of other samples. Protoplasts of leaf and cotyledons of A. alba and cotyledons of A. lanata, A. marina and S. alba were also investigated. The total contents of amino acids and their profiles might be related to the recalcitrance for the growth and salt tolerance or halophilic nature of cells and basal media used for the maintenance of cell cultures or protoplast cultures of the mangrove species. This is the first report on callus induction from hypocotyls of A. alba.

An Effective Procedure for In Vitro Culture of Eleusine coracana (L.) and Its Application

Abstract: Efficient protocols for callus production, plantlet regeneration, protoplast isolation, and micronucleation of finger millet (Eleusine coracana (L.) Gaertn.) were developed. White nodulated calli were formed on medium with N6 macrosalts, MS microsalts, 2.4-dichlorophenoxyacetic acid (2 mg L−1), kinetin (0.4 mg L−1), 1-naphthalene acetic acid (2 mg L−1), and certain additives. It was found that appropriate supplementation leads to formation of numerous shoots. Healthy rooted plantlets formed on hormone-free media. Although different tested additives had no significant effect on percentage of callus formation, it affected callus quality that further dictated plant-forming capacities. Seedlings were better source tissues for protoplasts isolation compared to callus cultures. About protoplasts were isolated from one gram of seedling coleoptyles. Microcolonies were visible after 20–25 days' incubation on KM8p medium supplemented with glutamine (100 mg L−1) and proline (500 mg L−1). Here we also present a procedure of an efficient induction of micronuclei after chlorpropham (10 μM) and cytochalasin-B (20 μM) seedlings treatment with subsequent microprotoplasts isolation. This technique is discussed for the transfer of alien chromosomes and genes from finger millet by microprotoplast-mediated chromosome transfer.

Halophilic and salts tolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba

Abstract: The effects of sea salts, NaCl, KCl, MgCl2, MgSO4, and CaCl2, on the growth of protoplast cultures of two mangrove species, Sonneratia alba and Avicennia alba, were investigated using 96-well culture plates. Plants of these two species naturally grow at the seaward side of a mangrove forest. Cotyledon protoplasts of S. alba showed halophilic nature to NaCl, KCl, and MgCl2 at low concentrations (10–50 mM) when cultured in Murashige and Skoog’s (MS) medium containing 0.6 M mannitol. CaCl2 at a concentration higher than 25 mM was inhibitory to cell growth. On the other hand, in protoplast culture of A. alba suspension cells, which were induced from cotyledon tissues, in the modified amino acid (mAA) medium containing 1.2 M sorbitol, tolerance to NaCl, MgCl2 and MgSO4 were observed at a wide range of concentrations up to 400 mM. CaCl2 was always inhibitory for cell divisions in A. alba, but stimulatory for spherical enlargement of cells. However, no difference in cell enlargement was observed among other salts. Similarity and difference in reactivity to salts between protoplasts and suspension cells from our previous studies were discussed in relation to the site of salt tolerance or halophilic adaptation within mangrove cells. For protoplast cultures, the site(s) for response of S. alba and A. alba are located in the cytoplasm and/or the cell membrane.

In vivo localization in Arabidopsis protoplasts and root tissue.

Abstract: In eukaryotic cells, a large number of proteins are transported to their final destination after translation by a process called intracellular trafficking. Transient gene expression, either in plant protoplasts or in specific plant tissues, is a fast, flexible, and reproducible approach to study the cellular function of proteins, protein subcellular localizations, and protein-protein interactions. Here we describe the general method of protoplast isolation, polyethylene glycol-mediated protoplast transformation and immunostaining of protoplast or intact root tissues for studying the localization of protein in Arabidopsis.

Protoplast mutation breeding method for improving cordycepin content of cordyceps militaris

Abstract: The invention relates to a protoplast mutation breeding method for improving the cordycepin content of cordyceps militaris. The method comprises the following steps of activated culture of bacterial strains, solid culture of mycelia, liquid mycelia culture, mycelia enzymolysis, protoplast nitrosoguanidine (NTG) mutation, protoplast regeneration culture, regenerated bacteria subculture, fermentation culture, filtering, further washing of mycelia, measurement of cordycepin content in the mycelia, preservation of the bacteria strain with high cordycepin content, measurement of the genetic stability and the like. Due to the adoption of the method, the cordycepin content of the cordyceps militaris can be increased.

Method for preparing and regenerating phomopasis asparagi protoplast

Abstract: The invention discloses a method for preparing and regenerating a phomopasis asparagi protoplast, which relates to the technical field of fungus protoplast preparation and regeneration in cell engineering. The method comprises the following detailed operation steps of: (1) preparing a phomopasis asparagi conidium suspension; (2) preparing a fresh mycelium; (3) performing enzymolysis on mycelium cell walls; (4) separating the protoplast; and (5) regenerating the protoplast. The method is easy to operate, low in requirements on equipment and short in enzymolysis time and regeneration period, and effectively enhances the efficiency of preparation and regeneration of the protoplast.

Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

Abstract: This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL−1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30 × 105 protoplasts g−1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.

Bimolecular Fluorescence Complementation (BIFC) Protocol for Rice Protoplast Transformation

Abstract: [] After the plant cells are removed the cell walls by digestive enzyme, the plant protoplasts still have good cell activity. The protoplasts can be used to transiently express proteins of target genes in living plant cells through polyethylene glycol (PEG) mediated transformation. The purpose of this method is to employ the rice protoplasts and Green fluorescent protein (GFP) as an experimental system to observe the protein interactions in vivo. Meanwhile a 505~530 nm emission filter is used in confocal microscope to eliminate the interference of the autofluorescence from plant cells. The phenomenon of plant cell body spontaneous fluorescence can be eliminated by confocal observation.