Microbeams of heavy charged particles.

We have established a single cell irradiation system, which allows selected cells to be individually hit with defined number of heavy charged particles, using a collimated heavy-ion microbeam apparatus at JAERI-Takasaki. This system has been developed to study radiobiological processes in hit cells and bystander cells exposed to low dose and low dose-rate high-LET radiations, in ways that cannot be achieved using conventional broad-field exposures. Individual cultured cells grown in special dishes were irradiated in the atmosphere with a single or defined numbers of 18.3 MeV/amu 12C, 13.0 MeV/amu 20Ne, and 11.5 MeV/amu 40Ar ions. Targeting and irradiation of the cells were performed automatically at the on-line microscope of the microbeam apparatus according to the positional data of the target cells obtained at the off-line microscope before irradiation. The actual number of particle tracks that pass through cell nuclei was detected with prompt etching of the bottom of the cell dish made of ion track detector TNF-1 (modified CR-39), with alkaline-ethanol solution at 37 degrees C for 15-30 minutes. Using this system, separately inoculated Chinese hamster ovary cells, confluent normal human fibroblasts, and single plant cells (tobacco protoplasts) have been irradiated. These are the first studies in which single-ion direct hit effect and the bystander effect have been investigated using a high-LET heavy particle microbeam.     

Irradiation of mammalian cultured cells with a collimated heavy-ion microbeam

As the first step for the analysis of the biological effect of heavy charged-particle radiation, we established a method for the irradiation of individual cells with a heavy-ion microbeam apparatus at JAERI-Takasaki. CHO-K1 cells attached on a thin film of an ion track detector, CR-39, were automatically detected under a fluorescence microscope and irradiated individually with an 40Ar13+ ion (11.5 MeV/nucleon, LET 1260 keV/microm) microbeam. Without killing the irradiated cells, trajectories of irradiated ions were visualized as etch pits by treatment of the CR-39 with an alkaline-ethanol solution at 37 degrees C. The exact positions of ion hits were determined by overlaying images of both cells and etch pits. The cells that were irradiated with argon ions showed a reduced growth in postirradiation observations. Moreover, a single hit of an argon ion to the cell nucleus resulted in strong growth inhibition. These results tell us that our verified irradiation method enables us to start a precise study of the effects of high-LET radiation on cells.     

System of cell irradiation with a defined number of heavy ions (III).

A single cell irradiation system has been developed at JAERI-Takasaki to study radiobiological processes in single-ion-hit mammalian cells and bystander cells, in ways that cannot be achieved using conventional broad field exposures. Individual mammalian cultured cells are irradiated in the atmosphere on the cell dish, the bottom of which is made of ion-track-detector CR-39, with a single or defined numbers of 13.0 MeV/amu 20Ne and 11.5 MeV/amu 40Ar ions. Targeting and irradiation of the cells are performed automatically at the on-line microscope of the microbeam apparatus according to the positional data of the target cells obtained at the off-line microscope before irradiation. Using this system, Chinese hamster ovary (CHO-K1) cells were irradiated with counted number of 20Ne and 40Ar ions. Thereafter, the growth of the cells was observed individually and repeatedly during post-irradiation incubation. The cells hit by a single 40Ar ion on their nucleus showed strong growth inhibition. Meanwhile, the cells in the irradiated dish but not hit by the ion (bystander cells) showed limited cell growth. This might be a bystander effect caused by heavy ion hit cell co-existing in the same dish.     

Targeted irradiation of Mammalian cells using a heavy-ion microprobe

The existing focusing heavy-ion microprobe at the Gesellschaft für Schwerionenforschung in Darmstadt (Germany) has been modified to enable the targeted irradiation of single, selected cells with a defined number of ions. With this setup, ions in the range from helium to uranium with linear energy transfers (LETs) up to approximately 15,000 keV/microm can be positioned with a precision of a few micrometers in the nuclei of single cells that are growing in culture on a thin polypropylene film. To achieve this accuracy, the microbeam traverses a thin vacuum window with minimal scattering. Electron emission from that window is used for particle detection. The cells are kept in a specially designed dish that is mounted directly behind the vacuum window in a setup allowing the precise movement and the imaging of the sample with microscopic methods. The cells are located by an integrated software program that also controls the rapid deflection and switching of the beam. In this paper, the setup is described in detail together with the first experiments showing its performance. We describe the ability of the microprobe to reliably hit randomly positioned etched nuclear tracks in CR-39 with single ions as well as the ability to visualize the ion hits using immunofluorescence staining for 53BP1 as a marker of DNA damage in the targeted cell nuclei.     

Initial yields of DNA double-strand breaks and DNA Fragmentation patterns depend on linear energy transfer in tobacco BY-2 protoplasts irradiated with helium, carbon and neon ions

The ability of ion beams to kill or mutate plant cells is known to depend on the linear energy transfer (LET) of the ions, although the mechanism of damage is poorly understood. In this study, DNA double-strand breaks (DSBs) were quantified by a DNA fragment-size analysis in tobacco protoplasts irradiated with high-LET ions. Tobacco BY-2 protoplasts, as a model of single plant cells, were irradiated with helium, carbon and neon ions having different LETs and with gamma rays. After irradiation, DNA fragments were separated into sizes between 1600 and 6.6 kbp by pulsed-field gel electrophoresis. Information on DNA fragmentation was obtained by staining the gels with SYBR Green I. Initial DSB yields were found to depend on LET, and the highest relative biological effectiveness (about 1.6) was obtained at 124 and 241 keV/microm carbon ions. High-LET carbon and neon ions induced short DNA fragments more efficiently than gamma rays. These results partially explain the large biological effects caused by high-LET ions in plants.     

Induction of a bystander chromosomal damage of He-ion microbeams in mammalian cells.

We report here a bystander effect in chromosomal damage using He-ion microbeam. Human-hamster hybrid cells were irradiated with a precision He-ion microbeam generated by the Columbia microbeam system. When 20% of the cells were exposed to single He ion, the incidence of cells with chromatid-type breaks detected with the PCC technique was covered wide range from 0 to 6 breaks per cell. In contrast, the distribution showed a mixed two-peak pattern, such as non-exposed and all-cell exposed patterns, under the condition of assuming no bystander effect by treating with an effective inhibitor of cell-cell communication. These findings provide clear evidence that single He-ion irradiated cells can induce bystander chromosomal alterations in neighboring cells not directly hit by He ion.     

PADC nuclear track detector for ion spectroscopy in laser-plasma acceleration

The transparent polymer polyallyl-diglycol-carbonate (PADC), also known as CR-39, is widely used as detector for heavy charged particles at low fluence. It allows for detection of single protons and ions via formation of microscopic tracks after etching in NaOH or KOH solutions. PADC combines a high sensitivity and high specificity with inertness towards electromagnetic noise. Present fields of application include laser-ion acceleration, inertial confinement fusion, radiobiological studies with cell cultures, and dosimetry of nuclear fragments in particle therapy. These require precise knowledge of the energy-dependent response of PADC to different ion species. We present calibration data for a new type of detector material, Radosys RS39, to protons (0.2–3 MeV) and carbon ions (0.6–12 MeV). RS39 is less sensitive to protons than other types of PADC. Its response to carbon ions, however, is similar to other materials. Our data indicate that RS39 allows for measuring carbon ion energies up to 10 MeV only from the track diameters. In addition, it can be used for discrimination between protons and carbon ions in a single etching process.     

Effects of abscisic acid on K+ channels in Vicia faba guard cell protoplasts

Potassium channels were resolved in Vicia faba guard cell protoplasts by patch voltage-clamp. Whole-cell currents and single K+ channels had linear instantaneous current-voltage relations, reversing at the calculated Nernst potential for K+. Whole cell K+ currents activated exponentially during step depolarizations, with half-activation times of 400-450 msec at +80 mV and 90-110 msec at +150 mV. Single K+ channel conductance was 65 +/- 5 pS with a mean open time of 1.25 +/- 0.30 msec at 150 mV. Potassium channels were blocked by internal Cs+ and by external TEA+, but they were insensitive to external 4-aminopyridine. Application of 10 microM abscisic acid increased mean open time and caused long-lasting bursts of channel openings. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology.     

A focused scanning vertical beam for charged particle irradiation of living cells with single counted particles

The Surrey vertical beam is a new facility for targeted irradiation of cells in medium with singly counted ions. A duo-plasmatron ion source and a 2 MV Tandem? accelerator supply a range of ions from protons to calcium for this beamline and microscope endstation, with energy ranges from 0.5 to 12 MeV. A magnetic quadrupole triplet lens is used to focus the beam of ions. We present the design of this beamline, and early results showing the capability to count single ions with 98% certainty on CR-39 track etch. We also show that the beam targeting accuracy is within 5 μm and selectively target human fibroblasts with a <5 μm carbon beam, using γ-H2AX immunofluorescence to demonstrate which cell nuclei were irradiated. We discuss future commissioning steps necessary to achieve submicron targeting accuracy with this beamline.     

Selectivity to K+ and Na+ of protoplast fractions isolated from different regions of Aspergillus nidulans hyphae

The selectivity to K+ and Na+ of protoplast samples representing cytoplasm isolated from different regions of the hyphal filament of Aspergillus nidulans was investigated. Concentrations of both ions contained in successive protoplast fractions were measured. During lytic digestion, protoplasts were released first from apical regions and subsequently from progressively older regions of hyphae. A low K+/Na+ ratio was found in protoplasts containing primarily apical cytoplasm and a high K+/Na+ ratio was found in protoplasts originating from older regions of hyphae. The ratios were the same whether MgSO4 or mannitol was used as stabilizer. Absolute concentrations of both ions were higher in protoplasts of apical origin. Protoplasts stabilized in mannitol lost more ions than those stabilized in MgSO4 over an 8 h incubation period. Na+ losses were higher from apical protoplasts whereas K+ losses were higher from protoplasts liberated from older regions of hyphae. The addition of divalent metal cations (1.5 mM-Mn2+ or Mg2+) reduced losses of Na+ from protoplasts but did not affect loss of K+. Data obtained using protoplast samples were related to those obtained for intact mycelium. Absolute losses of both ions from mycelium were lower than for protoplasts but when compared on a protein basis the data suggested that protoplasts possess properties similar to those of intact mycelium in terms of K+ and Na+ selectivity.     

水稻原生质体培养及植株再生的研究

由粳稻77-170品系及籼稻品种IR-50的细胞悬浮培养物游离的原生质体,用琼脂糖包埋于RY-2培养基中,发生了持续分裂。前者植板率达2.5％以上,二者最后都再生出植株。对游离和培养方法做了如下改进:1)采用两步法,即先用果胶酶,再用果胶酶和纤维素酶的混合酶进行游离,可避免原生质体发生融合并获得高质量的原生质体;2)悬浮细胞培养基中加入ABA有利于原生质体的存活和分裂;3)琼脂糖包埋培养可大大提高植板率;4)用较高渗透压的培养基培养原生质体再生的细胞团及愈伤组织,可提高植株再生频率。由于这两个品种(系)的培养物都已继代一年半之久,再生植株均为白化苗。这是迄今第一个由籼稻原生质体再生植株的报道。     

激光诱导金盏菊原生质体融合方法初探

本文简述运用激光微束诱导金盏菊(Calendula Officinali L.)叶肉细胞原生质体融合的方法和初步结果,并就激光诱导植物原生质体融合的条件进行初步讨论。     

Isolation of protoplasts from somatic embryos of carrot

Protoplasts were isolated enzymatically from synchronously induced globular somatic embryos from a carrot suspension culture. Among the macerating enzymes tested, Driselase was the most effective for release of protoplasts from embryos. A higher medium osmolarity was required for the isolation of protoplasts from embryos than from undifferentiated cells. Protoplasts from embryos were smaller than protoplasts from undifferentiated cells. On step gradients of Ficoll, protoplasts from embryos gave one major band. Protoplasts from undifferentiated cells gave two major bands, one lighter and the other heavier than the protoplasts from embryos.     

Regeneration of diploid and tetraploid plants from callus-derived protoplasts of Agapanthus praecox ssp. orientalis (Leighton) Leighton

protoplasts was developed for the Liliaceous ornamental plant, Agapanthus praecox ssp. orientalis (Leighton) Leighton `Royal Purple Select' (2n=2x=32).Viable protoplasts were routinely isolated from leaf-derived embryogenic calluses with yields of 0.8 to 1.5x10 protoplasts per g FW of calluses. Protoplasts started to divide 5 to 7 days after isolation, and protoplast-derived colonies consisting of 50 to 100 cells were obtained after 1 month. A plating efficiency of 0.8% was obtained after 2 months of culture using a gellan gum-solidified medium containing 1 mg 1^-1 each of PIC and BA under continuous illumination. Protoplastderived calluses produced somatic embryos at a frequency of 46.7 % on PGR-free medium, whereas 68.3 % of the calluses regenerated adventitious shoots on a medium containing 1 mg 1^-1 BA. Somatic embryos and adventitious shoots developed into plantlets, which were successfully transplanted to pots. Flow cytometric analysis and chromosome observation revealed that both diploid and tetraploid plants were regenerated from protoplasts.     

A simple,versatile feeder layer system for Brassica oleracea protoplast culture

Protoplasts from cauliflower (Brassica oleracea ssp. botrytis) and broccoli (ssp. italica) leaves and hypocotyls were successfully cultured on membrane filters over a feeder layer of cells from a B. campestris suspension culture. Cells from rice, tomato and tobacco suspensions were not as effective as the B. campestris cells. Plants were recovered from protoplasts of previously recalcitrant Brassica genotypes. Protoplasts cultured in low numbers (10–100) on the feeder layer divided and formed colonies capable of plant regeneration, as did fused protoplasts.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - PCV packed cell volume     

Reuse of enzymes for isolation of protoplasts

Summary A method has been developed for the reuse of cell wall digesting enzymes to isolate protoplasts from actively-growing suspension cultures of plant cells. Protoplasts could be satisfactorily prepared as many as three times using the same enzyme mixture without any loss in yield or viability of the isolated protoplasts. The yields of nuclei isolated from protoplasts prepared with used enzyme solution were comparable to those obtained with fresh enzymes.     

Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean

Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid BA|Benzyladenine - BA Benzyladenine     

Plant cell growth responds to external forces and the response requires intact microtubules

Microfibril deposition in most plant cells is influenced by cortical microtubules. Thus, cortical microtubules are templates that provide spatial information to the cell wall. How cortical microtubules acquire their spatial information and are positioned is unknown. There are indications that plant cells respond to mechanical stresses by using microtubules as sensing elements. Regenerating protoplasts from tobacco (Nicotiana tabacum) were used to determine whether cells can be induced to expand in a preferential direction in response to an externally applied unidirectional force. Additionally, an anti-microtubule herbicide was used to investigate the role of microtubules in the response to this force. Protoplasts were embedded in agarose, briefly centrifuged at 28 to 34g, and either cultured or immediately prepared for immunolocalization of their microtubules. The microtubules within many centrifuged protoplasts were found to be oriented parallel to the centrifugal force vector. Most protoplasts elongated with a preferential axis that was oriented 60 to 90 degrees to the applied force vector. Protoplasts treated transiently with the reversible microtubule-disrupting agent amiprophos-methyl (applied before and during centrifugation) elongated but without a preferential growth axis. These results indicate that brief biophysical forces may influence the alignment of cortical microtubules and that microtubules themselves act as biophysical responding elements.     

Physiological properties and cytological features of protoplasts prepared fromChlorella saccharophila

Summary Protoplasts were prepared from cells ofChlorella saccharophila by treatment with a mixture of pectinase and cellulase. The yield of protoplasts is dependent upon the culture conditions prior to cell wall digestion. In thin section chemically-fixed protoplasts were without wall remnants at the surface of the plasma membrane. Of particular interest is the relationship between the Golgi apparatus and a nuclear envelope-endoplasmic reticulum continuum. Protoplasts have a photosynthetic capacity lying between 70 and 80% of that of normal cells, but show the same response towards CO₂ concentration and DCMU inhibition. Protoplasts also respond to changes in the osmolarity of the surrounding medium in accordance with the Boylevan't Hoff equation as if they are an osmometer. The nonosmotic volume (NOV) was calculated.Abbreviations GA Golgi apparatus - ER endoplasmic reticulum - NE nuclear envelope - PM plasma membrane - N nucleus - S starch - M mitochondria - V vacuole     

The isolation, culture and regeneration of Petunia leaf protoplasts

Methods are described for the enzymatic release of protoplasts from leaves of Petunia hybrida and for the utilization of protoplasts in studies in plant developmental biology. As a result of spontaneous fusion during cell wall degradation of leaf material, fresh preparations can contain a high proportion of multinucleate protoplasts. This level can be dramatically reduced by a gradual plasmolysis of the material prior to enzyme incubation.Leaf protoplasts maintained in liquid media are seen to undergo cell wall synthesis, “budding,” and limited regenerated cell division sometimes associated with anthocyanin production. Under such conditions, multinucleate cells are formed as a result of mitosis without cytokinesis.Protoplasts, plated out in a fully defined medium, undergo cell wall synthesis followed by sustained progeny cell division with eventual cell colony production. Cell colonies, derived from individual mesophyll protoplasts, grow rapidly upon subculture, to produce callus capable of shoot differentiation and ultimately whole plant formation. Protoplasts isolated from varieties of P. hybrida were found to differ in their cultural requirements.