The nature of callose produced during self-pollination inSecale cereale

The response to incompatible (self) pollination in rye (Secale cereale L.) includes the rapid deposition in the germinating pollen grain and pollen tube of a substance that stains with aniline blue, resorcin blue and calcofluor, and in these respects resembles callose. This substance has been isolated and analysed by acid hydrolysis and methylation as well as specific enzyme hydrolysis. It contains a glucan component with 1,4--glucosidic and 1,3--glucosidic linkages within the same linear chains. The proportion of 1,4-to 1,3-glucosidic linkages in the preparation is 779.Abbreviation DMSO dimethylsulphoxide     

Molecular analysis of mitochondrial DNA from rye (Secale cereale L.)

Summary Molecular characterization of mitochondrial (mt) DNA of rye (Secale cereale L.), free of significant amounts of contaminating chloroplast (cp) DNA, was initiated using the open-pollinated cultivar Halo as a source of mtDNA. Based on the compilation of data from restriction patterns, the molecular size of the mtDNA was estimated to be 410 Kb and its buoyant density was determined as 1.705 g/ml. Southern hybridization, using labelled cp genes (P700 and ribulosebiphosphate-carboxylase large subunit), indicated the presence of cpDNA-homologous regions on putative mtDNA fragments. Mt DNAs of inbred lines with fertile and cytoplasmic male sterile (CMS) Pampa cytoplasm were also analysed. Whereas the restriction patterns of mtDNAs of Halors and the fertile line turned out to be identical, Pampa mtDNA showed a unique restriction pattern, indicating (as in most other CMS systems) the involvement of mtDNA rearrangements in the expression of male sterility in rye. All 3 mtDNAs investigated contain regions homologous to the plasmid S1 of the CMS-S cytoplasm of Maize (Zea mays), as indicated by hybridization experiments. In Pampa cytoplasm the S-homologous sequence is located within a rearranged region of mtDNA.     

Microspore embryogenesis and the development of a double haploidy protocol for cow cockle (Saponaria vaccaria)

Factors affecting microspore embryogenesis of cow cockle (Saponaria vaccaria) were evaluated including donor plant growing conditions, genotype, bud size, density, medium composition, and culture conditions. Of the two donor plant (day/night) temperature regimes evaluated (10/5°C and 20/15°C), plants grown at 20/15°C were the most embryogenic. An embryogenic frequency of greater than 350 embryos/100 buds was observed in the most embryogenic genotype, cv. ‘White Beauty’. Buds from 3–9 mm in length were evaluated for their embryogenic potential; buds that were 4–7.9 mm produced the most embryos/100 buds. Of all the media compositions evaluated, NLN medium with 15% sucrose resulted in the most embryos. Cow cockle microspores required an initial period of 32°C for 3 days for production of microspore-derived embryos (MDEs).     

Heterochromatin and silver banding of rye (Secale cereale,Gramineae) chromosomes

Air-dried chromosomes of rye when stained with aqueous silver nitrate show differential banding patterns. In addition to staining the NOR sites, the silver nitrate stains all regions of constitutive heterochromatin, as identified by Giemsa C-banding, as well as a number of small interstitial regions. However, the heterochromatin on the B chromosome is not stained by the silver method. This is proposed as a rapid and reliable banding method.     

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.     

Rye B chromosome behaviour at first and second pollen mitosis and its relationship with anther maturity

Summary Rye plants carrying B chromosomes with different nuclear and cytoplasmic constitutions have been analyzed at first pollen mitosis. No differences of B chromosome behaviour have been detected. It has been concluded that non-disjunction and preferential distribution are processes controlled by Bs themselves.At second pollen anaphase, B laggards have been observed. Both non-disjunction and B laggards occurred with higher frequency in younger anthers.     

Comparisons of the hybrids Hordeum chilensexH. vulgare,H. chilensexH. bulbosum,H. chilensexSecale cereale and the amphidiploid of H. chilensexH. vulgare

Summary Diploid hybrids between Hordeum chilense and three other species, namely H. vulgare, H. bulbosum and Secale cereale, are described together with the amphidiploid of H. chilensexH. vulgare. Both the diploid hybrid and the amphidiploid of H. chilensexH. vulgare were chromosomally unstable, H. chilensexH. bulbosum was less so, while H. chilensexS. cereale was stable. Differential amphiplasty was found in all combinations. No homoeologous pairing was found in the Hordeum hybrids but in H. chilensexS. cereale there was chromosome pairing both within the two genomes and between the genomes.     

Mapping seed storage protein loci Sec-1 and Sec-3 in relation to five chromosomal rearrangements in rye (Secale cereale L.)

Summary Linkage relationships were established between the secalin loci, Sec 1 (40-K gamma and omega secalins, homologous to the wheat gliadins) and Sec 3 (HMW = high-molecular-weight secalins, homologous to the wheat HMW glutenin subunits), and five chromosomal rearrangements involving chromosome 1R of rye (Secale cereale L.). These were: interchanges T273W (1RL/5RS), T306W (1RS/5RL), and T850W (1RS/ 4RL), Robertsonian centromere split Rb1RW and the interchanged Robertsonian split Rb2R/248W. The analysis established the linkage relationships between the secalin loci and the breakpoints of the rearrangements, in addition to the quantitative effects of the rearrangements on the linkage. Sec-1 is located in the satellite at a position at least 2.5 cMorgan from the proximal border of the terminal C-band, and about 30 cMorgan from the nucleolar organizing region (NOR). The locus is also physically closer to the terminal C-band than to the NOR, but not as much as corresponds with the map distances. Similarly, the physical distance between Sec-3 and the centromere is greater than corresponds with the recombination frequency (0%–9%). Although overall recombination in 1RL remains the same, recombination between the centromere and Sec-3 is greatly reduced in the Robertsonian split combined with the interchange. This is not the case with the single Robertsonian split.     

Microspore cultures as donor tissue for the initiation of embryogenic cell suspensions in barley

We have initiated embryogenic cell suspension cultures of barley (Hordeum vulgare L.) Igri from isolated microspore cultures. Data were obtained on the time required for establishment, frequency of establishment, i.e. number of calluses out of the total number of initiations giving rise to suspensions, and embryogenic capacity of the suspension cultures. For comparison, establishment of embryogenic cell suspensions from callus derived from immature zygotic embryos of Igri, Dissa and Golden Promise was also carried out. The results revealed that embryogenic suspension cultures were established in half the time and with a seven-fold higher frequency from microspore cultures than from zygotic embryo-derived calluses. The suspension cultures were still capable of embryo formation after two years. However, only albino plantlets were regenerated. For comparison, long term callus cultures derived from microspores, anthers and zygotic embryos were established. From the anther and zygotic embryo-derived callus cultures green plants were continuously regenerated, whereas the microspore-derived callus cultures lost this ability after the second subculture.     

Stress as the major signal controlling the developmental fate of tobacco microspores: towards a unified model of induction of microspore/pollen embryogenesis

Specific stress treatments (sucrose starvation, alone or combined with a heat shock) applied to isolated tobacco (Nicotiana tabacum L.) microspores irreversibly blocked normal gametophytic development and induced the formation of embryogenic cells, which developed subsequently into pollen-derived embryos by culture at 25°C in a sugar-containing medium. A cold shock at 4°C did not inhibit microspore maturation in vitro and did not induce cell division activity, even when combined with a starvation treatment. In the absence of sucrose, microspores isolated in the G1 phase of the cell cycle replicated their DNA and accumulated in G2. Late microspores underwent miotosis during the first day of culture which resulted in a mixed population of bicellular pollen grains and uninucleate microspores, both embryogenic. After the inductive stress treatments the origin of the first multicellular structures, formed in the sugar-containing medium, could be traced to divisions of the microspore cell or divisions of the vegetative cell of bicellular pollen, indicating that the symmetry of microspore mitosis in vitro is not important for embryogenic induction. These results represent a step forward towards a unified model of induction of embryogenesis from microspores/pollen which, within a relatively wide developmental window, are competent to deviate from normal gametophytic development and initiate the alternative sporophytic programme, in response to specific stress signals.Abbreviation DAPI 4,6-diamidino-2-phenylindole We acknowledge the help of Monica Boscaiu and Zarko Hrzenjak with the artwork, and Michaela Braun-Mayer for growing the tobacco plants. This project was financed by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung, grant S6003-BIO.     

Epidermal derivatives as xylem elements and transfer cells: a study of the host-parasite interface in two species of Triphysaria (Scrophulariaceae)

Summary Haustoria ofTriphysaria pusilla andT. versicolor subsp.faucibarbata from a natural habitat were analysed by light and electron microscopy. The keel-shaped edge of the secondary haustorium generally splits the epidermis and cortex of the host root parallel to the root axis, and penetrates to the host vascular tissue. Anticlinally elongated epidermal cells of the haustorium constitute most of the host/parasite interface. Some of these epidermal cells are divided by oblique cell walls. Some of their oblique daughter cells as well as some undivided epidermal cells differentiate into xylem elements. Single epidermal cells occasionally intrude into the vascular tissue of the host and individual host cells can be invaded. The surface area of the plasmalemma in parasitic parenchymatous interface cells is increased by the differentiation of wall labyrinths characteristic of transfer cells and by the development of membrane-lined cytoplasmic tubules or flattened sacs which become embedded in the partly lignified interface cell-wall. Mycorrhizal fungal hyphae enter the xylem bridge in some haustoria. Implications of these observations for the function of the haustorium are discussed.     

Development of microsporesin vivo andin vitro inBrassica napus L.

Summary Populations of highly homogeneous uninucleate and binucleate microspores ofBrassica napus cv. Topas were obtained by bud selection and percoll fractionation. The development of the uninucleate and the binucleate microspores in culture was compared to thosein vivo using the fluorochrome DAPI to stain DNA. The major developmental pathway of the uninucleate microsporesin vitro resulted in embryo formation. The characteristic of this pathway was that the first division produced two diffusely stained nuclei and subsequent divisions gave rise to a multinucleate embryoid. The second pathway which occurred in a small number of the uninucleate microspores led to callus formation. The majority of the binucleate microsporesin vitro followed the developmental pattern of their counterpartsin vivo and were not embryogenic. The embryogenic binucleate microspores produced embryos through the divisions of the vegetative nucleus.Plant Research Centre Contribution # 1147     

Composition of the cell walls of Nicotiana alata Link et Otto pollen tubes

Cell walls isolated from pollen of Nicotiana alata germinated in vitro contain glucose and arabinose as the predominant monosaccharides. Methylation analysis and cytochemical studies are consistent with the major polysaccharides being a (13)--D-glucan (callose) and an arabinan together with small amounts of cellulose. The cell walls contain 2.8% uronic acids. Alcian blue stains the pollen-tube walls intensely at the tip, indicating that acidic polysaccharides are concentrated in the tip. Synthetic aniline-blue fluorochrome is specific primarily for (13)--D-glucans and stains the pollen-tube walls, except at the tip. Protein (1.5%), containing hydroxyproline (2.4%), is present in the cell wall.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Abnormal structure of protophloem sieve-element cell wall in colchicine-treated roots of Triticum aestivum L.

The structural aberrations of the cell walls of protophloem sieve elements (PSEs) in roots of wheat (Triticum aestivum L. cv. Maris Huntsman) caused by the anti-microtubule drug colchicine were investigated by electron microscopy. The initial effect of the drug on cell wall development was found to be an exceptionally rough wall surface, presumably caused by an uncontrolled fusion of Golgi vesicles with the plasma membrane. Cellulose microfibrils, which in normal PSEs are aligned transversely to the long axis and parallel to the cortical microtubules, in colchicine-treated PSEs display a predominant longitudinal orientation. The pattern of wall development is disturbed by deposition of wall material also within the sieve pores of the sieve-pore/plasmodesmata complexes, resulting in evenly thickened walls instead of the normal uneven layers, and in narrowing the sieve pores to the size of plasmodesmata. In prolonged and continuous colchicine treatment, PSEs develop unusual wall ingrowths projecting deeply into the cytoplasm, creating an extraordinary cell type not found in normal roots. The results confirm the view of microtubule involvement in the proper deposition and orientation of cellulose microfibrils, and in the normal patterning of the cell wall thickenings of differentiating PSEs.Abbreviations c colchicine-treated - PSE protophloem sieve element The author is grateful to Dr. B. Galatis, Dr. P. Apostolakos and Dr. C. Katsaros, Institute of General Botany, University of Athens, Greece, for helpful discussions and suggestions, and for the generous gift of the colchicine used here. This work was carried out in the Department of Botany, University of Thessaloniki, Greece, while observations were also made in the Lehrstuhl für Zellenlehre, University of Heidelberg, Germany, and in the Department of Botany, University of Georgia, USA. The author is thankful to Prof. E. Schnepf (Zellenlehre, Heidelberg, Germany) and Prof. B.A. Palevitz (Department of Botany, University of Athens, Ga., USA), for generously providing access to their equipment and facilities. The work was financially supported in part by the Stiftung Volkswagenwerk and by the Research Committee, University of Thessaloniki (No 7537).     

Microtubule organization in the differentiating transfer cells of the placenta inLilium spp.

Summary Placental cells in the ovarian transmitting tissue ofLilium spp. are organized as transfer cells with inbuddings facing the ovarian locule. A detailed analysis of microtubule (MT) organization during development of these polarized cells is reported here. Formation of wall projections occurs at the apical part of the cell starting on the day of anthesis, and a fully mature secretion zone is found four days after anthesis. MTs are organized into distinct cortical and central arrays. The cortical array undergoes a unique transition at anthesis. MTs in the basal half of the cell remain in longitudinal bundles while in the apical half of the cell their longitudinal orientation is replaced by a transverse alignment. One day after anthesis, these transverse bundles become a meshwork of short, randomly organized MTs, while MTs in the basal half of the cell retain their longitudinal alignment. The realignment of MTs in the apical half of the cell coincides with the deposition of the secondary cell wall. The central array is composed of short, randomly arranged strands of MTs in the cytoplasm between the nucleus and the apical and basal periclinal walls of the cell. This array first appears as solitary strands in the apical part of the cell one day before anthesis. The central array extends during development and is eventually seen in the basal half of the cell. We propose that MTs in the cortical region near the apical wall act as templates for the deposition of cellulose microfibrils in the secondary cell wall. MTs in the central array in these transfer cells may be involved in the trafficking of vesicles and/or positioning of organelles near the secretion zone.Abbreviations MT microtubule - daa day after anthesis - dba day before anthesis     

Pollen germination and tube growth in rye (Secade cereale L.) homozygous and heterozygous for reciprocal translocations

Summary To contribute to the knowledge of the role of reciprocal translocations in rye, a component of fertility was estimated by comparing germination and pollen tube growth in homozygous and heterozygous plants for reciprocal translocations. The results obtained indicate that there are no differences in germination and pollen tube growth rate when homozygous and heterozygous plants as a whole are compared. However, there are significant differences in pollen tube growth between plants carrying different translocations. This suggests that the chromosome constitution of a plant is relevant to these fitness-estimating parameters together with its particular genetic background.     

Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH

The regulation of developmental pathways in cultured microspores of tobacco (Nicotiana tabacum L) and snapdragon (Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25°C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0–8.5 for 4–6 days at 25 °C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of ¹⁴C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development.     

Electron-translucent angular areas in developing tapetum cell walls and pollen grains ofTilia platyphyllos

Summary InTilia platyphyllos, the anther tapetal cell walls undergo significant modifications from the tetrad stage onwards. During the tetrad stage the inner tangential and radial parts of the tapetal walls begin to dissolve, while the distal parts swell. After the tetrad stage, the distal and outer radial tapetal cell walls become covered by a thick, irregular, highly electron-dense, polysaccharide layer. Striking features of the maturing tapetal walls (microspore stage and later) are electron-translucent, structureless, unstainable angular areas of variable dimensions. Similar electron-translucent areas occur in the exine arcades and apertures, but also isolated in the locular fluid ofT. platyphyllos. Electron-translucent areas, that are also found in the exine arcades and tapetal cells of other angiosperms, can be interpreted as the products of poorly understood metabolic processes.