Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic hybridization in higher plants.

Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two non-allelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco.     

Somatic hybridization in higher plants

Summary Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two nonallelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976.     

The inheritance of diastatic power and alpha-amylase contents in spring barley

Summary Nuclei were isolated from various types of donor protoplasts and were transferred into receptor protoplasts in numerous combinations. Five percent uptake was achieved under conditions which did not interfere with viability and subsequent culture of receptor protoplasts. Methodological investigations on nuclei uptake were carried out with cereal and tobacco protoplasts. To look for biological proof of integration and replication of transferred nuclear genes, two complementing, chlorophyll-deficient, light-sensitive mutants of tobacco were used as sources of nuclei and receptor protoplasts. Ca. 5.5 × 10⁷ receptor protoplasts were cultured following transplantation experiments involving these complementing mutants and about 1.8 × 10⁷ of the resulting calli were subjected to selective conditions which discriminate against the parental types. No nuclear hybrids were detected, although in control experiments somatic hybrids were obtained by protoplast fusion. Some explanations for failure of nuclear hybrid formation are discussed together with other possible approaches for selective somatic combination of plant cell genophores.     

Fine structure of fusion products from soybean cell culture and pea leaf protoplasts

Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.Abbreviations PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy Supported by National Research Council of Canada, Grant A6304     

Procedure for isolating micronuclei from rat kangaroo cultured cells containing individualized chromosomes

Micronuclei are small interphase nuclei containing part of the genome; the DNA content of the smallest micronuclei is equivalent to one chromosome. For analysis by biochemical method and by cytofluorometry of interphase micronuclei containing a single chromosome, several isolation and purification procedures were tested and checked by fluorescent microscopy using the DNA dye Hoechst 33 342 and electron microscopy. Micronucleation of rat kangaroo epithelial cells was induced by colchicine treatment for three days. Micronuclei were isolated in a low ionic strength buffer containing collagenase, with concomitant mechanical shocks. Eighty % of the micronuclei were released after 3 to 7 min, with minimum nuclear breakage. Subsequent filtration through several polycarbonate filters 12, 8 and 5 micron in diameter enabled purification of the smallest micronuclei without aggregates or debris. Micronuclear morphology was well preserved, as shown by electron microscope observations. Therefore, we established the optimal conditions allowing gentle mass isolation of individual micronuclei of cultured PtK1 cells, compatible with flow cytometry analysis.     

Electron microscopic study of plant-animal cell fusion

Summary Avian erythrocytes and protoplasts isolated from mesophyll cells of tobacco plants were suspended in 1% protease, agglutinated with polyethylene glycol (PEG) and subsequently fused upon elution of the PEG. The fusion reaction was monitored by scanning (SEM) and transmission (TEM) electron microscopy. SEM studies showed a marked difference in the topography of agglutinated cells. During, and subsequent to fusion, the markedly different surfaces of the two cell types became homogeneous and lines of demarcation between the cells were no longer visible. TEM revealed that adhesion occurred over the entire membrane area between agglutinated cells. Incipient fusion was evidenced by the appearance of vacuoles at the intermembrane surfaces. During initial elution of the PEG, cytoplasmic channels between erythrocytes and protoplasts were evident. With continued elution of the PEG, starch-containing plant chloroplasts and starch grains were seen within erythrocytes and homogenous erythrocyte cytoplasm was present inside plant protoplasts. Cytoplasmic mixing between the two cell types occurred within 3 hours of elution. The frequency of interkingdom fusion was estimated to be 0.5–1%.     

Fusion Experiments of Isolated Generative Cells in Several Angiosperm Species

The generative cells used for fusion experiments were isolated from pollen grains of Zephyranthes candida and Lycoris radiata by “2-step osmotic shock” and from those of Hippeastrum vittata, Hemerocallis minor and Iris tectorum by “weak enzyme treatment” as reported previously. Using PEG method, fusions have been successfully induced between generative cells of the same species mentioned above, between generative cells of Z. candida and L. radiata, between generative cells and petal protoplasts in L. radiata, and between generative cells of L. radiata and hypocotyl protoplasts of Brassica napus. In all cases either homokaryons or heterokaryons could be obtained. Fusion of nuclei was observed sometimes in homokaryons of generative cells in L. radiata. The generative nuclei in fusion products could be well identified by labelling the generative cells before fusion with DAPI. FDA test demonstrated that most of the fusion products were viable. Factors affecting fusion efficiency including cell density, PEG concentration, duration of PEG treatment and effect of calcium ions were studied in fusion of generative cells in Z. candida. Our experiments indicate that isolated generative cells are likely to be deprived of cell wails and may be regarded as a special kind of protoplasts for direct fusion experiments.     

DNA binding and uptake by nuclei isolated from plant protoplasts: factors affecting DNA binding and uptake

DNA binding and uptake by nuclei isolated from soybean (Glycine max L. Merr.) protoplasts were investigated using radioactive homogeneous DNA prepared from soybean cells. DNA binding to nuclei was found to decrease drastically with increased incubation time. Total uptake and acid-precipitable uptake reached a maximum after 20 minutes of incubation. Optimum DNA binding and uptake occurred at pH 6 and the process was enhanced by increasing the incubation temperature to 40 C. Salmonella typhimurium DNA and poly ([dA-dT]-[dA-dT]) competitively inhibited DNA binding whereas calf thymus DNA was less competitive; however, Micrococcus lysodeikticus DNA stimulated DNA binding and tobacco mosaic virus RNA had no effect. DNA binding and uptake was enhanced by addition of Mg ions, Ca ions, poly-l-lysine, and ATP. Increasing amounts of EDTA appeared to decrease DNA binding. Pronase strongly inhibited DNA binding and uptake.     

Karyogamy after Electrofusion of Single Egg and Sperm Cell Protoplasts from Maize: Cytological Evidence and Time Course

In maize, in vitro fusion of isolated male gametes with isolated egg cell protoplasts can be induced by electric pulses. Until now, karyogamy has not been demonstrated. In this study, we cytologically examined fusion products fixed at different times after electrofusion with phase contrast microscopy and transmission electron microscopy. We obtained a precise timetable from 23 samples studied during the first 3 hr. The sperm nucleus was integrated within the egg cell protoplast, migrated toward the egg cell nucleus, and fused with it within 1 hr, as demonstrated by ultrastructural observations, three-dimensional reconstructions of nuclei, and subsequent nuclear volume estimates. Fusion of nuclei occurred before zygotic mitosis, as is the case in vivo. These findings demonstrate karyogamy during in vitro fertilization of maize.     

Rapid-growth responses of corn root segments: Effect of citrate-phosphate buffer on elongation

Summary Chloroplasts from the alga, Vaucheria dichotoma (L.) Ag., are taken up into protoplasts of carrot (Daucus carota L.) during polyethylene-glycol treatment. Since chloroplasts are found with equal frequency in uni- and multinucleate protoplasts, chloroplast uptake does not depend on protoplast fusion. However, higher frequencies of chloroplast uptake are observed when experimental conditions favor greater aggregation of protoplasts. The intracellular localization of chloroplasts is confirmed by electron microscopy, and it is shown that the chloroplasts, once within the protoplasts, are not surrounded by a limiting membrane of carrot origin.     

Uptake of isolated plant chromosomes by plant protoplasts

For mass isolation of plant metaphase chromosomes, cultured cells of wheat (Triticum monococcum) and parsley (Petroselinum hortense) were synchronized by hydroxyurea and colchicine treatment. This synchronization procedure resulted in high mitotic synchrony, especially in suspension cultures of parsley in which 80% of the cells were found to be at the metaphase stage. Mitotic protoplasts isolated from these synchronized cell cultures served as a source for isolation of chromosomes. The described isolation and purification method yielded relatively pure chromosome suspension. The uptake of the isolated plant chromosomes into recipient wheat, parsley, and maize protoplasts was induced by polyethylene-glycol treatment. Cytological studies provided evidences for uptake of plant chromosomes into plant protoplasts.Abbreviations PEG polyethylene glycol - HU hydroxyruea - C colchicine - HUC hydroxyurea and colchicine - CIM chromosome isolation medium - TCM Tris chromosome medium     

The development of branched silk gland nuclei

Nuclei in the giant polyploid silk gland cells of Calpodes ethlius grow by endomitosis and can develop hundreds of branches during larval life. The shape of the these nuclei is characteristic for each region of the gland. We have found shape to be correlated with arrangement of the nuclear matrix. Scanning electron microscopy showed nuclear matrices with shapes similar to those of feulgen stained nuclei. Profiles of isolated matrices seen by transmission electron microscopy had filaments aligned parallel to the long axis of nuclear branches. DNA stained by Hoechst had a similar parallel alignment within the branches. Nuclear shape may be maintained by a small number of components, since electrophoretic analysis showed only a few abundant polypeptides in the matrix fraction. Silk gland nuclei have some of the same nuclear matrix antigens found in smaller, more regularly shaped, eukaryote nuclei.     

Distribution of cortical microtubules in tobacco protoplasts. An immunofluorescence microscopic and ultrastructural study

Summary The arrangement of cortical microtubules in tobacco protoplasts is described using the following techniques: 1. Transmission electron microscopy (TEM) of thin sections of whole protoplasts, 2. TEM of negatively stained protoplast ghosts, and 3. Indirect immunofluorescence microscopy of protoplast ghosts. Ghosts were prepared by attaching freshly isolated protoplasts to glass coverslips or formvar/carbon-coated grids with poly-L-lysine and then bursting them either osmotically or by detergent treatment in the presence of a microtubule stabilizing buffer. Osmotic bursting of protoplasts yielded large pieces of plasma membrane with attached microtubules. These preparations proved very useful for measuring the density and length of cortical microtubules. Detergent treatment dissolved the plasma membrane and altered the distribution of cortical microtubules.     

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.     

大豆细胞核的分离及其超显微结构的研究

本文通过对影响大豆细胞核分离诸因素的研究,介绍一种快速、简便的核分离方法。采用这种方法分离出的细胞核产量高,光镜,电镜观察证明核制品形态好,纯度较高,核膜较完整。     

Ultrastructural characterization and three-dimensional reconstruction of isolated maize (Zea mays L.) egg cell protoplasts

Summary Isolated egg cell protoplasts ofZea mays L., inbred line A 188, have been studied at the transmission electron microscope level. Their preparation for electron microscopy has been performed by embedding in ultra-low gelling agarose as a preliminary step. Five isolated egg cell protoplasts were serially ultrathin sectioned and studied in detail. One of these protoplasts was reconstructed in three dimensions to provide additional information on its structure. After enzymatic digestion and microdissection, isolated egg cells are true, highly vacuolized protoplasts. The structure of their organelles agrees with in situ observations, indicating an ultrastructural intactness after isolation: the mitochondria are polymorphic, form reticulate networks, and have well developed cristae; the plastids contain starch grains; and the spherical nucleus is euchromatic. As in situ, the organelles of the isolated egg cell protoplasts are aggregated near the nucleus. The complete picture provided by this work should serve as a comparative base for studies on in vitro fertilization products.     

A method for high-frequency intergeneric fusion of plant protoplasts

Summary Protoplasts of Vicia hajastana Grossh. obtained from suspension-culture cells and Pisum sativum L. obtained from leaves adhered tightly to each other in concentrated solutions of high-molecular-weight polyethylene glycol (PEG). The adhesion occurred non-specifically between the free protoplasts from the same species as well as from the different species and genus. It was enhanced by enrichment of the PEG solution with calcium. Very few heteroplasmic fusions occurred during the period when the protoplasts were incubated in the PEG solution. However, many heterokaryons (up to 10%) were formed soon after the PEG solution was diluted out. The same phenomena were also observed in protoplasts from suspension-culture cells of Glycine max L. and from leaves of Hordeum vulgare L. Vicia and soybean protoplasts obtained from cultured cells regenerated cell walls and underwent sustained cell division after such treatment. Some Vicia-pea heterokaryons divided once. Over 10% of the soybean-barley hybrids divided in 7 days. Some divided 4–5 times and formed small clusters of cells in 10 days. The hybrids were recognizable because they contained chloroplasts from the leaf protoplast and exhibited morphological characters typical of the chlorophyll-less cells. None of the protoplasts from pea and barley leaves, either with or without PEG treatment, underwent cell division during the period of observation. The mechanism of adhesion and fusion of the protoplasts has been discussed.National Research Council (Canada) No. 13732.     

Endocytosis and intracellular localisation of type 1 ribosome-inactivating protein saporin-s6

Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from basic sites in HeLa nuclei after intoxication with saporin-S6.