Induction and characterization of micronuclei in plant cells. Perspectives for micronucleus-mediated chromosome transfer.

In this thesis, micronucleation in plant cells has been investigated and systems for isolation and transfer of organelles have been established.The discovery, described in chapter two, that the phosphoric amide herbicide amiprophos-methyl induced micronuclei at a high frequency in cell suspensions of N.plumbaginifolia, has opened the possibility to develop a microcell-mediated chromosome transfer system analogous to that in mammalian cell lines. In mammalian cells, micronuclei are induced by prolonged exposure of cells to spindle toxins (colchicine, Colcemid), resulting in up to 60% micronucleated cells (Matsui et al., 1982). Micronucleated cells are isolated by the "shake-off' method, and subjected to high speed centrifugation, which results in fractionation of the cells into microcells, containing micronulei with one or a few chromosomes. Subsequently, microcells are fused to the recipient cells. The transferred chromosomes were found to remain intact and mitotically stable (Fournier, 1982). This technique has hitherto not been available for plant cells or protoplasts, due to the lack of efficient procedures to induce micronuclei. Gamma-irradiation is now often used in the construction of monochromosomal addition lines by somatic hybridization (Bates et al., 1987), to induce chromosome damage which promotes chromosome elimination from one of the fusion partners. As has been pointed out in the introduction (chapter one), ionizing radiation induces chromosome rearrage ments, deletions and insertions (Menczel et al., 1982). From research on mammalian cells, it is known that these phenomena occur with a lower frequency after microcell-mediated chromosome transfer (Fournier, 1981). If microprotoplasts would become available for plant genetic manipulation, transfer of a limited number of chromosomes by microprotoplast fusion would offer an alternative to the use of gamma-irradiation. With the finding that APM induces micronuclei at high frequency in plants, transfer of low numbers of chromosomes after micronucleation can now be tested for use in plant genetic manipulation. The APM treatment was found to be reversible, as was demonstrated by washing the cell suspension cultures free from APM. After washing, normal growth and cell division were soon resumed, with some abnormal, multipolar spindles in the first division after washing. This observation is in good agreement with the the reversible inhibition of microtubule polymerization by APM (Falconer and Seagull, 1987). This low cytotoxicity makes APM a useful tool in the induction of micronuclei in plants.The flow cytometric analysis of the nuclear DNA content of APM-treated cel suspension cultures of N.plumbaginifolia, revealed the presence of many micronuclei with a DNA content equivalent to one metaphase chromosome (which consists of both sister chromatids). Similar observations have been made in micronucleated rat kangaroo cells after treatment with Colcemid (Sekiguchi et al., 1978). Sorting of the micronuclei on the basis of the fluorescence of ethidium-bromide, followed by analysis of the DNA content by Feulgen staining (chapter three), shows that it is possible to separate micronuclei on the basis of their DNA content by flowcytometry, like it has been shown for isolated plant metaphase chromosomes. Chromosome identification is sometimes possible with isolated metaphase chromosomes (de Laat and Blaas, 1984; Conia et al., 1987a; 1987b). Identification of chromosomes present in a particular micronucleus is not possible. This is due to different degrees of chromosome decondensation in the micronuclei (which influences the fluorescence signal of the fluorochrome -DNA complex by quenching), and due to the various combinations of chromosomes in micronuclei containing more than one metaphase chromosome. This is illustrated by the DNA histograms of isolated micronuclei in chapter two, which lack the specific chromosome peaks, present in metaphase chromosome preparations (chapter four). When micronuclei are present in large numbers, the overall DNA histogram will show no appreciable contribution of a particular type of chromosome combination in micronuclei, since chromosome grouping appears to be a random process, as was shown by the analysis of the number of micronuclei per cell in chapter two, and by cytological data in chapter two and three. Furthermore, the reduction of the number of micronuclei per micronucleated cell, which appears to be the consequence of fusion of micronuclei into a lobed restitution nucleus, gives rise to even more combinations of chromosomes.The processes, involved in the formation of micronuclei, are studied in chapter three and four. The effects of the anti-microtubular herbicides APM, oryzalin and the alkaloid colchicine, used for metaphase arrest and induction of micronuclei in mammalian cells, on the mitotic index and micronueleus formation are compared. The disruption of the spindle by direct inhibition of microtubule assemble is responsible for the accumulation of cells at metaphase. The concentrations of the inhibitors required for complete metaphase arrest, vary from 3 μM for APM and oryzalin to 500 μM for colchicine, as a consequence of differences in binding specificity (Hertel et al., 1980; Dustin 1984). The differences in the percentage of ball metaphases indicate specific effects of the above mentioned inhibitors on chromosome scattering. Apart from the disruption of the microtubules, APM and oryzalin have been shown to influence the accumulation of calcium in the mitochondria (Hertel et al., 1981). Moreover, oryzalin disturbs the active excretion of calcium by the plasma membrane. These combined effects result in an increased cytoplasmic calcium concentration (Hertel et al., 1980), which will be higher after oryzalin treatment than after APM treatment, due to the reduction of active calcium excretion by oryzalin. Our 'data suggest that the APM or oryzalin induced increase of the cytoplasmic calcium concentration is involved in both formation and fusion of micronuclei. Colchicine, which does not influence the cytoplasmic calcium concentration, is not effective in the induction of micronuclei. The higher cytoplasmic calcium levels after oryzalin treatment, would increase the fusogenic properties of the nuclear membranes, which would explain why micronuclei exist for a shorter time after oryzalin treatment as compared to APM treatment. This hypothesis will be tested in future experiments by treatments with the calcium ionophore A23187 in combination with the calcium-specific chelator ethyleneglycolbis- (2-aminoethylether)-N,N'-tetra acetic acid (EGTA), with simultaneous measurements of the cytoplasmic calcium concentrations with the new calcium specific fluorochromes Fluo-3 and Rhod-2 (Haugland, 1989).In order to obtain both large numbers of micronucleated cells, and large numbers of micronuclei per micronucleated cell, theeffect of DNA synthesis inhibitors was investigated. The results in chapter five show, that a considerable increase in the number of micronucleated cells can be achieved by HU or APH treatments, and that the time at which micronuclei appear can be controlled. The results further indicate that metaphases have to be exposed to APM for at least 12h, before micronucleation occurs, and that their lifetime is in the same order. These data demonstrate that it is possible to manipulate the conditions of the treatments in order to obtain either a high yield of metaphase chromosomes, or a high yield of micronuclei, with little contamination by micronuclei or chromosomes, respectively. In this way, it becomes possible to determine the moment at which the number of micronuclei per cell is at its maximal value.The isolation and characterization of microprotoplasts from micronucleated suspension cells is described in chapter six. Data obtained from DNA content measurements and flow cytometry demonstrate the presence of up to 40% of subprotoplasts with a DNA content less than the G1-level of the APM treated suspension cells. This indicates that genome fractionation has occurred, and the data on the FDA-staining show that most of the subprotoplasts still possess an intact plasma membrane, since FDA can not be retained by vacuolar membranes only (Lesney, 1986). The viability of the microprotoplasts and other types of subprotoplasts is indicated by the successful culture after gradient fractionation. As it is impossible to measure the DNA-content of microprotoplasts in a non-destructive way, no preselection could be performed to use only microprotoplasts for fusion. In a mass fusion system, the smallest microcells will be the least likely to fuse when electrofusion is used, because their small diameter will prevent alignment and membrane breakdown, which are both related to particle diameter (Zimmermann et al., 1982). Individual selection and fusion could overcome this problem (Koop et al., 1983). This control is essential for the efficient application of microprotoplasts, since the DNA content per microprotoplast will depend upon the DNA content per micronucleus in the cell suspension. Microprotoplast fusion will result in transfer of a part of the total number of chromosomes, directly followed by spontaneous chromosome elimination when two distantly related species are fused, since chromosome elimination seems to be directed by genome dose effects (Graves, 1984; Gilissen et al., 1989). Sofar no successful fusion experiments have been performed, which makes it impossible at the moment to comment on the usefulness of microprotoplasts in chromosome transfer. However, fusion experiments with karyoplasts indicate that it is possible to perform fusions in a controlled way (Spangenberg et al., 1987).In addition to the microprotoplast fusion, microinjection was developed for transfer of organelles and micronuclei. Glass needles with a large orifice (5pM) were prepared, along with a pressure system, based on the application of mercury. With the injection system, described in chapter seven, it is possible to suck donor material from a donor protoplast, and inject this directly into the recipient. The data on the complementation of the albino tobacco by injection of mature green chloroplasts or etiolated plastids, indicate that protoplasts can survive the injection treatment, and that the injected plastids can be replicated by the recipient. In this way, the organelles to be transferred are not subjected to damaging isolation procedures and they can be preselected visually. Selective transfer of organelles offers a number of advantages when compared to fusion techniques, or transfer of isolated genes. One of the advantages is the protective nature of the membranes associated with chloroplasts, mitochondria and nuclei. Although structural integrity and functionality has been demonstrated for isolated chloroplasts and mitochondria, it is not known whether isolated organelles are still physiologically intact. The isolation of intact nuclei from plant cells has also been described, with data indicating their structural integrity, as well as their ability to transfer genes into recipient protoplasts (Saxena et al., 1986). Transfer of marker genes does not necessarily implicate the functional integrity of isolated nuclei, since transfer of marker genes can be achieved by uptake of isolated genomic DNA. Preliminary results obtained from experiments with microinjection of micronuclei, indicate that it is possible to remove micronuclei from the donor by suction. Sofar, transfer into a recipient has not been achieved. The kanamycine- resistance, which was introduced into N.plumbaginifolia by transformation with Agrobacterium tumefaciens , will be used as selectable marker after transfer of micronuclei. The transfer of chromosomes will be tested with species specific repetitive DNA probes, which are able to discriminate between the donor genome N.plumbaginifolia and the recipient (either Lycopersicon esculentum or Solanum tuberosum ) . Several probes with the required specificity have already been characterized, from a series of highly repetitive sequences, isolated from N.plumbaginifolia (data not shown).With the methods, described in this thesis, the transfer of chromosomes via micronuclei has come within reach. Future work will focus on achieving transfer, and study the fate of the introduced micronuclei. This should provide an answer whether micronuclei can be used as chromosome carriers in plants, as has already been shown in mammalian somatic cell genetics