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     

PCIB an Antiauxin Enhances Microspore Embryogenesis in Microspore Culture of Brassica juncea

An efficient protocol to improve microspore embryogenesis is established in an important oleiferous crop, Brassica juncea (Indian mustard). Colchicine was used for enhancing microspore embryogenesis and also to obtain doubled haploid embryos. Colchicine at high concentrations (>10 mg l⁻¹), for 24 h, proved convenient for direct recovery of diploid embryos. Higher temperature treatment and an antiauxin PCIB (p-chlorophenoxyisobutyric acid) enhanced microspore embryogenesis significantly as compared to colchicine. An increase in temperature from 32°C to 35°C proved very efficient in increasing embryogenesis by 10-fold. The highest embryogenesis rate was obtained when PCIB was added at 35°C in the culture after 1 day of culture initiation. 20 μM PCIB could enhance microspore embryogenesis by 5-fold. Different abnormal shapes of embryos like lemon, banana, flask and fused cotyledons were observed. Both normal and fused cotyledonous embryos showed normal germination when transferred on the B₅ basal medium.     

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).     

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.     

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.     

GIGANTEA is a component of a regulatory pathway determining wall ingrowth deposition in phloem parenchyma transfer cells of Arabidopsis thaliana

Transfer cells are specialised transport cells containing invaginated wall ingrowths that generate an amplified plasma membrane surface area with high densities of transporter proteins. They trans‐differentiate from differentiated cells at sites at which enhanced rates of nutrient transport occur across apo/symplasmic boundaries. Despite their physiological importance, little is known of the molecular mechanisms regulating construction of their intricate wall ingrowths. We investigated the genetic control of wall ingrowth formation in phloem parenchyma transfer cells of leaf minor veins in Arabidopsis thaliana. Wall ingrowth development in these cells is substantially enhanced upon exposing plants to high‐light or cold treatments. A hierarchical bioinformatic analysis of public microarray datasets derived from the leaves of plants subjected to these treatments identified GIGANTEA (GI) as one of 46 genes that are commonly up‐regulated twofold or more under both high‐light and cold conditions. Histological analysis of the GI mutants gi‐2 and gi‐3 showed that the amount of phloem parenchyma containing wall ingrowths was reduced 15‐fold compared with wild‐type. Discrete papillate wall ingrowths were formed in gi‐2 plants but failed to develop into branched networks. Wall ingrowth development in gi‐2 was not rescued by exposing these plants to high‐light or cold conditions. In contrast, over‐expression of GI in the gi‐2 background restored wall ingrowth deposition to wild‐type levels. These results indicate that GI regulates the ongoing development of wall ingrowth networks at a point downstream of inputs from environmental signals.     

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.     

Cytogenetic studies of the intergeneric hybrids between Secale cereale and Elymus caninus,E. brevipes,and E. tsukushiensis (Triticeae: Poaceae)

Summary Integeneric hybridizations were carried out between Secale cereale L. (2n = 14, RR) and three Elymus species, namely, E. caninus (L.) L. (2n = 28, SSHH), E. brevipes (Keng) Löve (2n = 28, SSYY) and E. tsukushiensis Honda (2n = 42, SSHHYY). Chromosome pairing was studied at metaphase I in the parental species and the hybrids. Meiotic configurations of the hybrids were 20.74 1+0.14 II for E. caninus x S. cereale (SHR), 16.35 I+2.17 II+0.09 III for E. brevipes x S. cereale (SYR) and 25.84 I+1.10 II+0.02 III for E. tsukushiensis x S. cereale (SHYR), in addition to some secondary associations in the different hybrids. It is concluded from the study that (1) a certain, different homoeologous relationship exists among S, H and Y genomes in the investigated Elymus species; (2) low homoeology is present between genomes of Elymus (S or H or Y) and rye (R); (3) the Secale genome affects homoeologous chromosome pairing between different genomes in E. brevipes and E. tsukushiensis.     

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.     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

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.     

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.     

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).     

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.