Procedure For Isolation Of Protoplasts Research Articles

Interspecific hybridization between Ganoderma lingzhi and G. applanatum through protoplast fusion.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum was attempted through polyethylene glycol (PEG) induced fusion technique. The protoplast isolation procedure was simplified, and we obtained a significant number of protoplasts from both Ganoderma species. The number of protoplasts obtained was 5.27 ± 0.31 × 107/mL in G. lingzhi and 5.57 ± 0.49 × 106/mL in G. applanatum. Osmotic stabilizer NaCl (0.4M) at pH 5.8 and enzymolysis time 3.5h have supported high frequency of protoplast regeneration. G. lingzhi and G. applanatum regeneration frequency was 1.73 ± 0.04% and 0.23 ± 0.02%, respectively. 40% of PEG induced high number of protoplast fusion the regeneration frequency was 0.09% on a minimal medium. Two hundred fifty-two fusant colonies were isolated from the following four individual experiments. Among them, ten fusants showed the mycelial morphological difference compared to their parents and other fusant isolates. The fruiting body could be generated on oak sawdust and wheat bran substrate, and a few of them showed recombined morphology of the parental strains. The highest yield and biological efficacy (BE) were recorded in GF248, while least in GF244. The hybridity of the fusant was established based on mycelia, fruiting morphology, and PCR fingerprinting. ISSR and RAPD profile analysis of ten fusants and parents depicted that fusants contained polymorphic bands, which specified the rearrangement and deletion of DNA in the fusants. A Dendrogram was constructed based on the RAPD profile, and the clustering data exhibited two major clusters: cluster I included the G. lingzhi and Cluster II, including the G. applanatum and fusant lines. Total polysaccharide (α, β and total glucan) content was compared with fusants and parental strains. The present study highlighted the efficient methods for protoplast isolation from Ganoderma species. PEG-induced fusants showed high polymorphic frequency index, while the phenotypic characters showed high similarity to G. applanatum. A significant difference was observed in the mushroom yield and its total polysaccharide between the fusants and parental strains.

Isolation of leaf mesophyll protoplasts optimized by orthogonal design for transient gene expression in Catalpa bungei

Catalpa bungei is an important ornamental plant and timber species that originated in China. With the forthcoming C. bungei genome sequence, an efficient and convenient transformation system should be developed. The transient gene expression system using protoplasts is fast and efficient and can be applied to study the functions of genes in C. bungei. In this study, the main factors influencing the isolation of protoplasts from C. bungei were optimized by orthogonal design. The results showed that when the enzymolysis solution contained 3% Cellulase RS (w/v), 2% Macerozyme R-10 (w/v), and 0.4 M mannitol and the enzymolysis time was 8 h, the protoplast yield was 1 × 106 protoplasts/g fresh weight [FW]. The viability of protoplasts was approximately 90 % under optimal enzymolysis conditions. Furthermore, using GFP as the reporter gene, we verified the feasibility of PEG-mediated protoplast transformation of C. bungei. We established an efficient protoplast isolation procedure and feasible transient gene expression system. This methodology, when combined with genetics and omics techniques, will allow further study of the functions of the genes in C. bungei.

A Robotic Platform for High-throughput Protoplast Isolation and Transformation.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a 'Bright Yellow' 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a 'Bright Yellow' 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

Electroporetic transfection of pepper protoplasts with plant potyviruses

Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. Much effort has been expended to study the resistance response of pepper cultivars at whole plant levels but with only limited effort at the cellular level using protoplasts. A pepper protoplast isolation procedure is available but an inoculation procedure is needed that provides consistent and highly efficient infection. An electroporation-based procedure for inoculation of potyviruses was developed using a base procedure developed for Cucumber mosaic virus (CMV). The final parameters identified for efficient potyvirus infection of pepper protoplasts involves two 25ms pulses, 200V each pulse with a 10s interval between pulses. Depending on the method of detection, e.g., ELISA versus RT-PCR, potyvirus RNA inoculum ranged from 10 to 40μg with infection detection occurring with samples of 50,000-100,000 protoplasts.

Tissue degradation and enzymatic activity observed during protoplast isolation in two ornamental Grevillea species

Degradative changes in tissue during protoplast isolation were a contributing factor to low protoplast yields in the saltsensitive Grevillea arenaria (R. Brown) and the salt-tolerant Grevillea ilicifolia (R. Brown). Protein and malondialdehyde content decreased significantly during the protoplast isolation procedure. Acid and neutral proteases were identified, and high acid protease activities were correlated to low protoplast yields. Acid phosphatase, catalase, polyphenol oxidase and lipoxygenase activities increased in both Grevillea species with cell wall digestion. High activities of catalase and low levels of polyphenol oxidase were correlated with high protoplast yields. Levels of acid phosphatase and lipoxygenase were not good indicators of final protoplast yields. The addition of the anti-oxidant, reduced glutathione, and the acid protease inhibitor, pepstatin A, significantly increased protoplast yields. Strategies were identified to minimize deleterious degradative effects during the isolation of protoplasts, including strict pH control, testing a number of cell wall digestion enzymes, and the addition of anti-oxidative metabolites and protease inhibitors.

Isolation of protoplasts for patch-clamp experiments: an improved method requiring minimal amounts of adult leaf or root tissue from monocotyledonous or dicotyledonous plants

For the purpose of patch-clamp studies, a protoplast isolation procedure is presented in which an osmotic shock (changing the osmolarity of the medium) and centrifugation step are omitted to limit mechanical stress. Apart from the reduction of mechanical stress factors, protoplast washing is also limited. Protoplasts have been isolated from different monocotyledonous and dicotyledonous plant species and from different tissues (leaves and roots). The seal success rate in patch-clamp experiments was high (about 85% successful seals showing a seal resistance > 10GΩ). To evaluate the electrogenic viability of the protoplasts, fusicoccin and light responses of the plasma membrane and channel activity were tested. The addition of fusicoccin to the bathing medium caused typical high activation of the proton pump. Switching the light on and off caused transient depolarizations and hyperpolarizations, respectively, matching data reported for mesophyll cells in micro-electrode studies. Whole-cell and single-channel recordings of protoplast plasma membranes isolated from intact tobacco andArabidopsis leaves were comparable with data published for protoplasts from corresponding tissue cultures. It is concluded that our isolation procedure yields protoplasts with electrogenic responses and is therefore suitable for patch-clamp studies in physiological research.

Isolation and characterization of aleurone protoplasts from a malting variety of barley (Hordeum vulgare L.)

A procedure has been developed to isolate protoplasts from mature aleurone layers of the malting variety Alexis and four other barley genotypes. It combines induction of endogenous cell wall degrading enzymes together with use of Onuzuka cellulase R 10 and driselase and results in better yields for two varieties than can be obtained with the huskless variety Himalaya. The viability of the freshly isolated protoplasts is greater than 90% and in spite of the presence of gibberellic acid during isolation procedures, most of the protoplasts are at an early developmental stage, as judged by ultrastructure. Gibberellic acid-induced changes in protoplast structure resemble those reported for Himalaya protoplasts. The protoplasts secrete both α-amylase (EC 3.2.1.1) and (1-3, 1-4)-β-glucanase (EC 3.2.1.73) into the surrounding medium. Transfection studies using a low pI α-amylase promoter to direct chloramphenicol acetyltransferase expression in aleurone protoplasts from Alexis and Himalaya revealed significant differences in their hormone responsiveness. In the absence of hormones, low levels of expression of the reporter enzyme were obtained in Alexis protoplasts, while high levels were characteristic for Himalaya protoplasts. An 8-fold increase in the expression of the reporter gene was induced by supplying the transfected Alexis protoplasts with gibberellin A3, whereas expression in Himalaya protoplasts remained unchanged. When Himalaya protoplasts were isolated from aleurone layers that had not been incubated with GA3 during the initial stages of protoplasting (the classical procedure), the hormone response of the promoter was 2.5-fold. It is thus possible to optimize the aleurone protoplast isolation procedure for different barley genotypes and mutants of interest in studies of transgenic gene expression and hormone induced secretion of proteins from this unique secretory plant tissue.

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.

High-yield isolation of protoplasts from microgram amounts of shoot meristematic tissues and rapid DNA content determination by flow cytometry

This paper describes a novel approach to rapid cell-cycle analysis of shoot meristematic cells. The method involves fixation and disaggregation of meristems into protoplast suspension and flow-cytometric analysis of these protoplasts stained with fluorescent dyes. We have developed a procedure for a high-yield isolation of protoplasts allowing an accurate flow-cytometric analysis with a few micrograms of meristem tissues. We present here determinations of total DNA content of protoplasts stained with propidium iodide in the dicotyledon Sinapis alba, and the monocotyledon Lolium temulentum.

Intact mesophyll protoplasts from Zea mays as a source of phosphoenolpyruvate carboxylase unaffected by extraction: Advantages and limitations

Isolated intact mesophyll protoplasts from Zea mays L. were used as an enzyme source for studying properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1 1 31) just after release from cells into the reaction medium. After the injection of protoplasts into the assay mixture, an initial lag of activity was observed, mainly due to the time necessary for complete disruption of protoplasts by the osmotic shock. The final specific activity obtained was ca 18 μmol mg‐1 of liberated protein min‐1, a value comparable to that usually achieved after arduous purification. Under the assay conditions employed, the chloroplasts were not disrupted and the retention of their proteins, together with the use of purified mesophyll protoplasts, were obviously the reasons for the high specific activity obtained. The activity and properties of phosphoenolpyruvate carboxylase stored in isolated protoplasts were stable for at least 24 h at 5°C. The main difference between the protoplast‐derived and the routinely extracted enzyme was the sensitivity to malate inhibition, which was partially lost in the extracted phosphoenolpyruvate carboxylase; no difference was found in the Km(PEP). The stress imposed by the protoplast isolation procedure diminished the sensitivity of the enzyme to malate inhibition, so that it can be inferred that the real malate sensitivity of pbosphocnolpyruvale carboxylase is even greater and that it is grossly underestimated with routinely extracted enzyme.

Plant Regeneration from Strawberry ( Fragaria x Ananassa) Mesophyll Protoplasts

In vegetatively propagated species with strong heterozygoty and high ploidy levels, plant breeding through somatic cell genetics is of special interest. In strawberry breeding, where plant breeders wish to introduce specific traits such as earliness, winterhardiness and disease resistance into already established cultivars these techniques might be of great value. A prerequisite for somatic hybridization and direct gene transfer is an efficient procedure for isolation of protoplasts and plant regeneration. In this paper we present a method for protoplast isolation and plant regeneration in strawberry, a species which we intend to use in a coming fusion project.

Quantitative characterization of protoplasts and vacuoles from suspension‐cultured cells of Catharanthus roseus

A simple and efficient procedure for isolation of protoplasts and then vacuoles from cultured cells of Catharanthus roseus (L.) G. Don is presented. Protoplasts were disrupted by an osmotic shock and the vacuoles vere purified by flotation on a single‐step gradient. A comparison of the content and concentration of solutes (proteins, sugars, organic acids, alkaloids, mineral ions) in protoplasts and cells showed that massive and selective losses occur for most solutes during protoplast preparation. These are attributed to the osmotic adjustment and changes of membrane permeabilities occurring during plasmolysis. Data concerning the size, yield and purity of the isolated vacuoles are discussed. By analysis of isolated vacuoles, the vacuolar concentration and localization of solutes within protoplasts have been determined. The limits of this latter approach are stressed, however. Some evidence in favour of the selection of a special class of vacuoles during isolation is reported and discussed.

Dimethyl sulfoxide can initiate cell divisions of arrested callus protoplasts by promoting cortical microtubule assembly

A serious problem in the technology of plant cell culture is that isolated protoplasts from many species are reluctant to divide. We have succeeded in inducing consecutive divisions in a "naturally" arrested system-i.e., protoplasts from a hibiscus cell line, which do not divide under standard conditions-and in an artificially arrested system-i.e., colchicine-inhibited callus protoplasts of Nicotiana glutinosa, which do readily divide in the absence of colchicine. In both cases, the reinstallation of a net of cortical microtubules, which had been affected either by colchicine or by the protoplast isolation procedure, resulted in continuous divisions of the formerly arrested protoplasts. Several compounds known to support microtubule assembly in vitro were tested for their ability to promote microtubule assembly in vivo. Best results were obtained by addition of dimethyl sulfoxide to the culture medium. Unlimited amounts of callus could be produced with the dimethyl sulfoxide method from protoplasts which never developed a single callus in control experiments.