Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts

We have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one-dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell-free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters. We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse-width time-of-flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15-55 micron (r2 greater than 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia. These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.     

Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Isolation,sorting, and characterization of uni- and binucleate tapetal protoplasts from anthers of normal and Texas cytoplasmic male-sterileZea mays L.

Summary A cytological study of Texas cytoplasmic male sterile (Tcms) and normal (N) anther tapetal protoplasts ofZea mays was undertaken to determine whether there were any differences prior to Tcms male cell abortion not noted in previous published studies. Squash preparations, tapetal protoplast separation via flow cytometry, image analysis, and electron microscopy were utilized. Chemically preserved tapetal protoplasts from both lines were prominently angular in shape and typically smaller than any other cell type in the anthers. The tapetum from both lines consisted of a mixture of uninucleate and binucleate protoplasts. The Tcms tapetum consistently had a higher proportion of binucleate protoplasts during all stages of microsporogenesis prior to abortion. The size of Tcms uniand binucleate tapetal protoplasts was more variable than the N tapetal protoplasts and was largest during the microspore stage when male cells abort. Tapetal nuclear size in both lines was less variable. Uni- and binucleate tapetal protoplasts from each line could be separated from the other anther cells and from each other by filtration and then by flow cytometry, based on intensity of nuclear fluorescence. These results suggest that Tcms uninucleate tapetal protoplasts have a higher level of DNA than N uninucleate tapetal protoplasts. Both fluorescence microscopy and electron microscopy confirmed pure populations of intact uni- and binucleate tapetal protoplasts using flow cytometry. The results from this study indicate that the methodology presented here could be used for a variety of further studies to better understand the cellular and molecular basis of male sterility in maize, and in other taxa, where the tapetum is the primary target that leads to male sterility.Abbreviations AO acridine orange - Bi binucleate protoplast - D dyad - DAPI 4,6-diamidino-2-phenylindole - FC flow cytometry - M meiocyte - MI microspore - MMC mithramycin - N normal anther tapetal protoplast - PI propidium iodide - PS protoplast sorting - RT room temperature - SM sporogenous mass - Tems Texas cytoplasmic male sterile anther tapetal protoplast - Uni uninucleate protoplast     

Analysis of viability and cell types of macroalgal protoplasts using flow cytometry

The ability to rapidly distinguish viable sub-populations of cells within populations of macroalgal protoplast isolations was demonstrated using flow cytometry. Viable protoplasts from Ulva sp. and Porphyra perforata J. Ag. were distinguished from non-viable protoplasts based on differential fluorescein accumulation. The identities of cortical and epidermal protoplasts from Macrocystis pyrifera (L.) C. Ag. were inferred based on light-scattering and chlorophyll a autofluorescence. Three cell types could be distinguished among protoplasts released from thalli of P. perforata based on chlorophyll a and phycoerythrin autofluorescence. Mixed protoplast populations of Ulva sp. and P. perforata were also discernable based on relative chlorophyll a and phycoerythrin autofluorescence. The ability to screen heterogenous protoplast populations rapidly, combined with the cell sorting capabilities of many flow cytometers, should prove valuable for seaweed biotechnology.     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。     

Development of a quantitative method for determination of the optimal conditions for protoplast isolation from cultured plant cells

An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 × 10³ (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 × 10³ (number/ml min)] and Wasabia japonica [kv = 14.2 × 10³ (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed.     

Protoplast liberation and regeneration in the ascomycete Hypomyces ochraceus (Pers.) Tul

A rapid and convenient method for producing protoplasts from 3 d old mycelium of the ascomycete Hypomyces ochraceus is described. The procedure involves a Helix pomatia enzyme preparation and sucrose (20%) for stabilization. Pretreatment with disulfide bond reducing agents reduced the amount of viable protoplasts. Formation of protoplasts and different stages of regeneration were observed by phase contrast microscopy. There was only one type of true regeneration from protoplasts to hyphae in 15-30% gelatine medium by direct forming a germ tube from the original protoplast. Cytological events and physiological conditions are discussed.     

Coulter counter cell size determination of protoplasts fromArabidopsis thaliana PIP1b aquaporin antisense lines under iso- and hypo-osmotic conditions

Summary Water uptake ofArabidopsis thaliana protoplasts was measured after transfer into hypo-osmotic conditions. The time-dependent swelling of protoplast populations was monitored by a Coulter counter device. In order to ascertain the contribution of the plasma membrane intrinsic protein 1b (PIP1b) to the membrane's water permeability, protoplasts of five different plant lines that were transformed with a PIP1b antisense construct were compared to controls. The size distribution of 5 independent protoplast preparations provided similar results for control and antisense lines under iso-osmolar conditions. After transfer into hypo-osmotic conditions, a time difference for the swelling of protoplasts from the different sources was observed. The sizes of control protoplasts changed in less than 20 s, which indicates high water influx rates. In contrast, the protoplast populations obtained from 5 different antisense plants took about 75 s to reach a steady-state cell size distribution. The difference in time by a factor of about 3 confirms the significance of the aquaporin PIP1b for the water permeability of plant plasma membranes and the cellular water transport.     

Age dependence of cowpea protoplasts for uptake of spermidine and infectibility by alfalfa mosaic virus

Cowpea protoplasts were prepared from plants of different ages and examined for their ability to take up polyamines and for their infectibility by alfalfa mosaic virus. A lag period of 20 h was necessary before the onset of rapid polyamine uptake; the occurrence of this rapid uptake depended on the age of the leaves used for protoplast preparation. The percentage of infection of cowpea protoplasts by alfalfa mosaic virus, and the amount of virus produced also depended on the age of the plants used for protoplast preparation. In contrast, the uptake of amino acids was rapid in all cowpea protoplasts tested.     

Factors governing the flow cytometric analysis and sorting of large biological particles

We have investigated the suitability of large flow cell tips for the flow cytometric analysis and sorting of large biological particles, including plant cells (pollen) and protoplasts. Using flow tips ranging in diameter from 79-204 micron, we have optimized conditions for the establishment of a stable hydrodynamic flow leading to accurate droplet production. We describe instrument modifications required for large particle sorting and demonstrate the use of these experimental conditions for the sorting to high purity of pollen and viable plant protoplasts possessing diameters as large as 95 micron. Our experiments have revealed a complex interaction among sorting efficiency, particle diameter, flow cell tip diameter and bimorphic crystal drive frequency. This interaction can be satisfactorily explained in terms of interference effects owing to phase differences between the particle-induced disturbance and the undulation driven by the bimorphic crystal.     

Factors influencing transient gene expression in electroporated tall fescue protoplasts

For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase gene was introduced by electroporation. In a time-course study of transient gene expression, GUS activity peaked at 24 h after electroporation. Among the different field strength conditions tested, maximum GUS activity was observed at 750 V/cm. Increases in the amount of plasmid DNA to 80 μg/ml led to increased GUS activity. GUS activities increased in linear fashion with increasing protoplast densities up to 2 × 10⁶/ml. Age of suspension cells from which protoplasts were derived influenced transient expression with maximum GUS activity obtained in 3- and 5-day-old suspensions. These results show that monocot and dicot protoplasts respond similarly in electroporation.     

Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans

A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from ecch species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion.Abbreviations AC alternating current - DC direct current     

Protoplast isolation and regeneration in Streptomyces clavuligerus

The regeneration of streptomycete protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. Reports of studies on the regeneration of protoplasts from Streptomyces clavuligerus are limited and for this reason the experiments described in this paper were carried out. An investigation of protoplast formation and cytology was made to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency were isolated from mycelium, grown in a two-stage culture system (without glycine), using lysozyme dissolved in a sucrose osmoticum containing 1% bovine serum albumin. The latter promoted improved protoplast viability. A systematic survey was made of the components of regeneration medium R5, previously used for S. clavuligerus, and other potentially advantageous components and conditions, in an attempt to raise the regeneration frequency of the protoplasts. An improved regeneration medium (R6) and protocol which supported higher and more consistent levels of regeneration of S. clavuligerus protoplasts resulted from these experiments. These improved procedures for protoplast isolation and regeneration proved to be suitable for other streptomycete species.     

Callus formation from isolated sunflower (Helianthus annuus) mesophyll protoplasts

Mesophyll protoplasts of the cultivated sunflower,Helianthus annuus, have been consistently found not to divide or regenerate calli, despite the efforts of several groups. In the present report, we describe the conditions for donor plant culture, protoplast isolation, and their culture that were suitable for repeated regeneration of green, nodular, vigorously growing calli from isolated sunflower mesophyll protoplasts. The best conditions for protoplast isolation employed the use of both CAYLA cellulase and CAYLA pectinase. Culture conditions were not much different from those established earlier for sunflower hypocotyl protoplasts. The most startling observation was the great variability of division frequencies between experiments even under strictly controlled, identical experimental conditions. This finding points to an important influence of a variable in the physiological state of the donor plant which is difficult to control.     

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126

AIMS: Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. METHODS AND RESULTS: Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. CONCLUSIONS: The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.     

Barley scutellum protoplasts: Isolation, culture and plant regeneration

Many applications of cereal protoplast culture systems are still limited by the difficulties of regeneration from suspension cells which are the usual protoplast source. The objective of the present study therefore was to investigate the conditions for the development of a culture system for protoplasts capable of plant regeneration isolated directly from immmature scutella of barley. The procedure developed involves a two-stage pre-culture of scutellar tissue, followed by vacuum infiltration with cell wall degrading enzymes and the culture of alginate-embedded protoplasts. The pre-culture of the scutella and the co-cultivation of protoplasts with nurse cells were the most important factors for the success of the culture system, but several other parameters affecting protoplast yield, viability and sustained division were identified, including the developmental stage of the embryo, the use of cold conditioning periods during pre-culture, the composition of the pre-culture and protoplast culture medium, and the embedding matrix. Protoplasts isolated from scutellar tissues of barley cvs Dissa, Clipper, Derkado and Puffin were capable of sustained division in culture. Macroscopic protoplast-derived tissues were obtained in all cultivars, except ev. Puffin, and fertile plants were regenerated from cvs Dissa and Clipper 3–4 months after protoplast isolation. The procedure described provides a novel approach for the isolation of totipotent protoplasts in barley which avoids the need for suspension cultures.     

Controlled cell wall regeneration for efficient microinjections of Nicotiana tabacum var. Carlson protoplasts

Nicotiana tabacum var. Carlson protoplast culture conditions were modified to contain a cell wall inhibitor, 2,6-dichlorobenzonitrile, in order to delay cell wall regeneration and to allow efficient nuclear and cytoplasmic microinjections. Under modified conditions, the protoplast preparations appeared healthier as compared to the control protoplasts and showed no resistance at all during microinjection. Furthermore, the duration of protoplast microinjection was extended for up to 3–4 days. In order to set up nuclear microinjections, the nuclei of these protoplasts were stained either before or after immobilization without any adverse effect on their mitotic activity. Successful cytoplasmic microinjections were demonstrated by injecting Alfalfa mosaic virus (AMV) RNA, which resulted in viral infection of 14% of the injected protoplasts.Abbreviations AMV Alfalfa Mosaic virus - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - DB 2,6-dichlorobenzonitrile - LR lissamine rhodamine - NAA 1-naphthalene-acetic acid     

Freezing behavior of free protoplasts of winter rye

Free protoplasts prepared from the epicotyls of nonhardened rye seedlings were subjected to fast and slow freezing on a microscope-adapted thermoelectric stage. During rapid freezing to ?12 °C, ice formation occurred inside the protoplasts causing lethal disruption of cell and membrane organization. Under slow freezing to ?12 °C, ice formation occurred outside the protoplast with accompanying dehydration and contraction of the protoplast. Complete rehydration and recovery of the protoplasts occurred upon thawing after slow freezing. Free protoplasts therefore afford a new system for the study of mechanisms of plant cell freezing injury and resistance free of the complications presented by a cell wall.     

The influence of culture conditions on mitotic activity in protoplasts derived fromPisum root cortical explants

Summary Protoplast cultures were prepared from explants of the roots of seedling peas. In defined, synthetic media these cultures were mitotically active. A variety of culture conditions were investigated and the influences of these conditions on the mitotic activity of the protoplasts were observed. Marked inhibition of mitoses were observed after exposure to high light intensities, and in the absence of a proper exogenous supply of hormones. The protoplasts showed extreme sensitivity to the nature and concentration of the exogenous hormone supply. The protoplasts were mitotically active at low population densities (6,000–8,000 protoplasts/ ml of medium). They did not divide in cultures in which glucose was supplied as a carbohydrate source, but divided actively in cultures in which sucrose (2%) was supplied. The responses to temperature and pH were similar to most protoplast systems which have been reported. The definition of a wide variety of optimal culture conditions resulted in high mitotic activity in protoplast cultures with low population densities.