Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Behavior of chloroplasts and chloroplast nuclei during spermatogenesis in the fern,Pteris vittata L.

Summary The fate of the chloroplasts and chloroplast nuclei (cp-nuclei) was followed during spermatogenesis in the fernPteris vittata L. by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI) and by quantitation of chloroplast DNA (cp-DNA) by fluorimetry using a video intensified microscope photon counting system (VIMPICS). The spores were grown on solid medium that contained antheridiogen (An_ptd), and formed an antheridium initial on the protonema cell. The antheridium initial divided and produced 16 spermatocytes and 3 surrounding cells. The chloroplasts in the spermatocytes decreased in volume as cell division was repeated, until finally the volume of each chloroplast was 1/15 of that of the primary chloroplasts. The DNA content of the chloroplasts was also reduced to 1/5 of the original value and when the sperm matured, the fluorescence of cp-DNA disappeared. In the 16-cell spermatocyte, the recognition of the fluorescence of chlorophyll in the chloroplasts with a green excitation filter became difficult. But, the plastids could be observed until the final stage of the sperm. From these observations, it appears that there are two steps in the metamorphosis of chloroplasts during spermatogenesis in the fern. The first step involves the decrease in the volume of chloroplasts, accompanied by reduction of the DNA content, and the second step involves the change of the physical state of chloroplasts to amyloplasts and the disappearance of the cp-DNA from the amyloplasts.     

An immunofluorescence study of mitosis in a mite-pathogen,Neozygites sp. (Zygomycetes: Entomophthorales)

Summary Mitosis in a mite-pathogenic species ofNeozygites (Zygomycetes: Entomophthorales) was investigated by indirect immunofluorescence microscopy using an antibody against -tubulin for visualization of microtubules (MTs). DAPI and rhodamine-conjugated phalloidin were used to stain chromatin and actin, respectively. Salient features of mitosis inNeozygites sp. are (1) a strong tendency for mitotic synchrony in any given cell, (2) conical protrusions at the poles of metaphase and anaphase nuclei revealed by actin staining, (3) absence of astral and other cytoplasmic MTs, (4) a spindle that occupies most of the nuclear volume at metaphase, (5) a spindle that remains symmetrical throughout most of mitosis, (6) kinetochore MTs that shorten during anaphase A, (7) a central spindle that elongates during anaphase B, pushing the daughter nuclei into the cell apices, and (8) interpolar MTs that continue to elongate even after separation of the daughter nuclei. Cortical cytoplasmic MTs are present in a few interphasic and post-cytokinetic cells. The data presented show thatNeozygites possesses features unique to this genus and support the erection of theNeozygitaceae as a separate family in theEntomophthorales.Abbreviations DAPI 4,6-diamidino-2-phenylindole - MT microtubule - SPB spindle pole body     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

Unique positioning of mitochondria in developing microspores and pollen grains inPharbitis nil: mitochondria cover the nuclear surface at specific developmental stages

Summary Changes in the number and distribution of mitochondria in microspores and pollen grains during male gametogenesis inPharbitis nil were examined with Technovit sections stained with 3,3-dihexyloxacarbocyanine iodide. The number of mitochondria per microspore or pollen grain ofP. nil increased constantly and dramatically during male gametogenesis. During this process, mitochondria exhibited characteristic localizations: subpopulations of mitochondria covered the surface of the microspore and vegetative nuclei before and again just after postmeiotic mitosis I (9 and 5 days before flowering, respectively). The mitochondria also surrounded the generative nucleus 2 days after postmeiotic mitosis I (5 days before flowering), although the density of mitochondria on the nuclear surface was lower. Electron microscopy showed that the mitochondria were about 30 nm from the nuclear envelope and that each mitochondrion was located near a nuclear pore. The characteristic localization of mitochondria inP. nil pollen may serve as a model to analyze the mechanisms that control mitochondrial positioning within a cell and interactions between mitochondria and nuclei.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - PM I postmeiotic mitosis I     

Nuclear crystals, lampbrush-chromosome-like structures, and perinuclear cytoskeletal elements associated with nuclear fragmentation in characean internodal cells

Summary This article gives a survey of nucleus-associated structures and inclusions in a diverse range of characean algae includingChara braunii Gm.,Chara corallina Klein ex Willd., em. R.D.W.,Nitella cristata A.Br., em. R.D.W.,Nitella flexilis (L.) Ag.,Nitella furcata (Roxb. ex Bruz.) Ag. em. R.D.W.,Nitella hyalina (DC.) Ag.,Nitella pseudoflabellata A.Br., em. R.D.W.,Nitella pseudoflabellata var.imperialis T.F.A.,Nitella translucens var.axillaris (A.Br.) R.D.W. andNitellopsis obtusa (Desv. in Lois.) J.Gr. Lampbrushchromosome-like structures were found in nuclei ofNitella flexilis andNitellopsis obtusa and seem to be involved in the distribution of genetic material during nuclear fragmentation. Intranuclear tubular crystals of unknown protein composition were present in all species, especially in young, elongating cells, and could be important for establishing the main axis of the nuclei. Spindle-shaped protein crystals that originate in the nucleus and are released into the cytoplasm upon nuclear degeneration were observed in branchlet internodal cells of one population ofNitella flexilis. Perinuclear microtubules were present in all species, but perinuclear actin fibrils were hitherto only found in mostNitella species and inNitellopsis obtusa. None of these nucleus-associated structures seems to be responsible for the formation of constrictions leading to nuclear fragmentation. These constrictions were perpendicular to the main axis of the nucleus and symmetrical in theNitella species but asymmetric inC. braunii, C. corallina, and inNitellopsis obtusa. Statistical analysis of nuclear size, number and constriction sites indicate that fragmentation is a nonsynchronous process independent of the light-dark cycle.Abbreviations CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - LCLS lampbrush chromosome-like structure(s) Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Accumulation of β-tubulin-containing fibres in the cortical domain ofSaccharomyces cerevisiae spheroplasts

Summary The distribution of microtubules inSaccharomyces cerevisiae was studied with the monoclonal antibody (mab) TU-14 against -tubulin. Immunoblotting and immunoprecipitation experiments with a strain overexpressing Tub2p confirmed that the mab TU-14 specifically recognized Tub2p. By immunofluorescence microscopy, the mab TU-14 attached to all known tubulin structures labelled with the standard polyclonal anti--tubulin antibody 206-1. In addition, the mab TU-14 revealed cortical patches in wild-type cells and an abundant network of fibres in the cortex of spheroplasts cultivated in nutrient medium. These cortical fibres seemed to be specific to spheroplasts and suggest that the accumulated Tub2p is predominantly associated with the plasma membrane.     

γ-Tubulin in tobacco pollen tubes: association with generative cell and vegetative microtubules

-Tubulin was localized in tobacco pollen tubes using an antibody raised against a peptide conserved in all known -tubulins. Antibody staining occurs in a primarily punctate pattern along the length of the microtubule bundles in generative cells and along cortical microtubules in the vegetative cytoplasm. During generative cell division, -tubulin is localized in the forming mitotic apparatus. By metaphase, it is present along kinetochore fibers except at their plus ends located at the kinetochores. By telophase, staining is observed in the phragmoplast, where it again avoids the plus ends of microtubules at the cell plate. -Tubulin is also present at the periphery of the sperm nuclei. A patch of intense staining on the distal side of each nucleus marks the site of assembly of a new population of sperm microtubules. No specific fluorescence is present in control pollen tubes treated with preimmune IgG. These localization patterns bear similarities to those seen in somatic cells and in addition may help explain changes in microtubule arrays between generative cells and sperm.     

Microtubule orientation in globular leaflet cells of <Emphasis Type="Italic">Chara inflata</Emphasis>

Chara inflata has globular leaflet cells and cylindrical internodal cells. The morphology of the leaflet cells is different from that of other Characeae. The orientation of cortical microtubules (MTs) in young leaflet and internodal cells of this species was analyzed by immunofluorescence microscopy. MTs with random orientation were observed in leaflet cells, while those relatively transverse to the cell axis were observed in cylindrical internodal cells. In cylindrical leaflet cells in Chara corallina, transverse MTs were observed. These results imply that C. inflata is a morphological mutant lacking a mechanism for orienting cortical MTs transverse in leaflet cells.     

The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cell cycle of the algaScenedesmus quadricauda

Summary DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga,Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them.Abbreviations DAPI 46-diamidino-2-phenylindole - ptDNA DNA in chloroplast nuclei - nucDNA DNA in cell nuclei     

Flow cytometric analysis of genome size variation in cultivated and wildPisum sativum (Fabaceae)

Fifteen cultivars, landraces, and wild accessions ofPisum sativum subspecies, and one accession ofP. abyssinicum were analysed with flow cytometry (DAPI staining) usingP. sativum Kleine Rheinländerin as internal standard. Applying the method of jointly isolating, staining, and measuring nuclei of individual seedlings of test and standard material, it was found that in allP. sativum comparisons G 1 and G 2 peaks were invariably unimodal and symmetric at coefficients of variation mostly less than 2%. This is strong evidence for absence of significant genome size variation in theP. sativum strains analysed. These data are markedly at variance to results of other authors reporting considerable genome size variation withinP. sativum. However, inP. abyssinicum flow cytograms and Feulgen densitometric measurements indicate 4–8% more DNA, at same chromosome number (2n = 14), than inP. sativum. This result demonstrates that genome size variation is indeed existent in the genus and requires further examination.     

Differential distribution of β-pigment-dispersing hormone (β-PDH)-like immunoreactivity in the stomatogastric nervous system of five species of decapod crustaceans

Summary Pigment-dispersing hormone (PDH) acts to disperse pigments within the chromatophores of crustaceans. Using an antibody raised against -PDH from the fiddler crab Uca pugilator, we characterized the distribution of -PDH-like immunoreactivity in the stomatogastric nervous system of five decapod crustaceans: the crabs, Cancer borealis and Cancer antennarius, the lobsters, Panulirus interruptus and Homarus americanus, and the crayfish, Procambarus clarkii. No somata were stained in the stomatogastric ganglion (STG) or the esophageal ganglion in any of these species. Intense PDH-like staining was seen in the neuropil of the STG in P. interruptus only. In all 5 species, cell bodies, processes, and neuropil within the paired circumesophageal ganglia (CGs) showed PDH-like staining; the pattern of this staining was unique for each species. In each CG, the -PDH antibody stained: 1 large cell in C. borealis; 3 small to large cells in C. antennarius; 3–8 medium cells in P. clarkii; 1–4 small cells in H. americanus; and 13–17 small cells in P. interruptus. The smallest cell in each CG in C. antennarius sends its axon, via the inferior esophageal nerves, into the opposite CG; this pair of cells, not labeled in the other species studied, may act as bilateral coordinators of sensory or motor function. These diverse staining patterns imply some degree of evolutionary diversity among these crustaceans. A -PDH-like peptide may act as a neuromodulator of the rhythms produced by the stomatogastric nervous system of decapod crustaceans.     

γ-Tubulin is associated with a cortical-microtubule-organizing zone in the developing guard cells of Allium cepa L.

A key event in the differentiation of elliptically shaped guard cells such as those in Allium is the formation of a radial array of cortical microtubules (Mts) which, by controlling the orientation of wall microfibrils, plays an important role in cell shaping. Previous experiments strongly indicated that the array is nucleated in a zone adjacent to the new ventral wall soon after cytokinesis. In order to further clarify the function of this zone, we performed dual immunolocalizations on Allium guard cells with anti--tubulin, to detect Mts, and an antibody to -tubulin, a protein known to be present at Mt-organizing centers in other species and recently identified in plants as well. -Tubulin antibody stained the cortical zone adjacent to the ventral wall, while little or no fluorescence was present elsewhere along the radial Mt array or at other sites in the cell. The antibody also stained the mitotic poles and phragmoplast in guard mother cells, as it does in other material. No staining was seen when the primary antibody was omitted. The results are consistent with nucleation of the radial array at a cortical-Mt-organizing zone next to the ventral wall, and set the stage for more in-depth studies on the spatial and temporal control of Mt formation in differentiating cells.Abbreviations CLSM confocal laser scanning microscope - FITC fluorescein isothiocyanate - Mt microtubule - MTOC microtubule-organizing center This work was supported by National Science Foundation grant DCB-9019285 to B.A.P., National Institutes of Health (NS30009) and American Cancer Society (CD6255) grants to H.C.J., and a University of Georgia Graduate School Assistantship to B.L. We thank Dr. Mark Farmer and the University of Georgia Center for Advanced Ultrastructural Research for the use of the confocal microscope.     

The essentiality of B chain in stabilizing the structure of the A chain in β1-bungarotoxin fromBungarus multicinctus venom

The dynamic of Trp residue in ₁-bungarotoxin (gb ₁-Bgt), the A chain of ₁-Bgt and phospholipase A₂ (PLA₂) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA₂ is higher than the Trp in ₁-Bgt. The Trp of ₁-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain of ₁-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure of ₁-Bgt was higher than those of the separated A chain and PLA₂. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain in ₁-Bgt.     

Is 2,3-butanedione monoxime an effective inhibitor of myosin-based activities in plant cells?

Summary The effectiveness of 2,3-butanedione monoxime (BDM) as an inhibitor of plant myosins has been investigated. Three myosin-dependent motility phenomena in plants, namely cytoplasmic streaming inChara corallina, light-dependent chloroplast repositioning inElodea sp., and brefeldin A(BFA)-induced Golgi membrane dynamics in wheat (Triticum aestivum L. cv. Kite) roottip cells were investigated. All three processes were inhibited by the sulfhydryl-modifying agent N-ethylmalemide (NEM), indicating the probable involvement of myosin as the motor protein in each case. However, none of these myosin-dependent processes were inhibited by BDM at concentrations as high as 20 mM in some instances. These results therefore question the general usefulness of BDM as an inhibitor of myosin-based activities in plant cells.     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Preferential synthesis of plastid DNA and increased replication of plastids in cultured tobacco cells following medium renewal

During the culture of tobacco BY 2 cells derived from Nicotiana tabacum L. cv. Bright Yellow 2, morphological changes of plastid (pt) nucleoids and their replication were examined by fluorescence microscopy after staining with 46-diamidino-2-phenylindole. Upon transfer to fresh medium, the fluorescence intensity originating from pt nucleoids increased markedly. Copy numbers of ptDNA per cell calculated from the quantitative data by super-sensitive microspectroscopy increased 11-fold within 1 d of culture to reach 11 000, then decreased gradually to 1 000 after one week of culture. Autoradiography by labelling with [³H]thymidine showed that DNA synthesis in plastids occurred exclusively during the first day of culture, whereas nuclear DNA synthesis was observed from the first to the sixth day of culture. Replication of plastids was most frequently observed on the second day. Thereafter the formation of starch granules predominated in plastids up to the fifth day of culture, but the starch granules disappeared in the stationary-phase cells. The meaning of such preferential synthesis of ptDNA upon transfer to fresh medium is discussed in relation to the interaction between plastids and nuclei.Abbreviations pt plastid - DAPI 4,6-diamidino-2-phenylindole     

In vivo studies on the nuclear behavior of the arbuscular mycorrhizal fungusGigaspora rosea grown under axenic conditions

Summary The distribution and fate of nuclei of the arbuscular-my-corrhizal fungusGigaspora rosea during late stages of axenic cultures were studied in fixed cultures by transmitted light, conventional and confocal laser scanning microscopy, and in live cultures with two-photon fluorescence microscopy. Mature specimens not yet showing apical septation displayed oval-shaped nuclei localized in lateral positions of the hypha all along the germ-tube length. Beside these, round-shaped nuclei were found to migrate along the central germ-tube core. Some (rare) germ-tube areas, delimited by septa and containing irregularly shaped, much brighter fluorescent nuclei were also found. Specimens that had just initiated the septation process after germ-tube growth arrest displayed round or oval-shaped nuclei in several portions of the germ tubes. These hyphal areas often alternated with other septa-delimited cytoplasmic clusters which contained distorted, brightly fluorescent nuclei. Completely septated specimens mostly lacked nuclei along their germ tubes. However, highly fluorescent chromatin masses appeared within remnants of cytoplasmic material, often compressed between close septa. Our results provide a first clear picture of the in vivo distribution of nuclei along arbuscular mycorrhizal fungal germ tubes issued from resting spores, and suggest that selective areas of their coenocytic hyphae are under specific, single nuclear control. They indicate as well that random autolytic processes occur along senescingG. rosea germ tubes, probably as a consequence of the absence of a host root signal for mycorrhizal formation. Finally, the data presented here allow us to envisage the fate of nuclei released by the germinating spore after nonsymbiotic fungal growth arrest.Abbreviations AM fungi arbuscular-mycorrhizal fungi - DAPI 4, 6-diamidino-2-phenylindole - FM fluorescence microscopy - CLSM confocal laser scanning microscopy - 2PM two-photon microscopy - PI propidium iodide - PMT photomultiplier tube     

Genome size variation and C-band polymorphism inAlstroemeria aurea, A. ligtu andA. magnifica (Alstroemeriaceae)

Among a total of 43 accessions ofAlstroemeria aurea, A. ligtu andA. magnifica nuclear DNA amounts (2C-values) showed significant intraspecific variation, 1.09, 1.21 and 1.15 fold, respectively, when determined through flow cytometric measurements of fluorescence of propidium iodide (PI) stained nuclei. After staining with another fluorochrome, 4,6-diamidino-2-phenylindole (DAPI), an intraspecific variation of 1.10, 1.11 and 1.12 fold, respectively, was found. C-band polymorphisms were present among and within the accessions of all three species. In some cases only very small differences in C-banding pattern were observed. In other cases, however, differences were more prominent. Besides C-band polymorphism, there were also instances of chromosome length polymorphism, which concerned the total chromosome complement or single chromosomes. The variation in nuclear DNA amount inA. aurea andA. ligtu was more or less continuous, except for one accession ofA. ligtu subsp.simsii. Artificial selection and possibly introgression of chromosomes from other species may have moulded the karyotypes of some of the accessions ofA. aurea, a species that has been under cultivation for more than 160 years. The variation as observed inA. magnifica subsp.magnifica was discontinuous and could be due to a broad species concept.