Plant regeneration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids

Leaf explants of Paphiopedilum phiIippinense hybrids (hybrid PH59 and PH60) directly formed adventitious shoots from wound regions within 1 month, when cultured on modified Murashige and Skoog medium (1/2-strength macro- and full-strength micro-elements) free of plant growth regulator in darkness. The combinations of 2,4-dichlorophenoxyacetic acid ((2,4-D) acid (0, 4.52 and 45.25 M) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) (0, 0.45, 4.54 and 22.71 M) were used to test their effects on direct shoot bud formation from two types of explants (1.5-cm long intact leaf explants and 0.5-cm long leaf segment explants). In hybrid PH59, 4.54 M TDZ increased mean numbers of shoots per explant with leaf segment explants. In hybrid PH60, 4.52 M 2,4-D plus 0.45 M TDZ promoted direct shoot bud formation from leaf segment explants. In addition, three treatments (4.52 M 2,4-D, 22.71 M TDZ, 4.52 M 2,4-D plus 4.54 M TDZ) gave a higher response than control on mean numbers of shoots per explant with intact leaf explants. Healthy plantlets each with one to three roots were obtained from leaf-derived shoots after transfer onto a hormone-free medium for 22 months. These plantlets were acclimatized in a greenhouse and grew well with 100% survival rate.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

Storage,mobilization and interrelations of starch,sugars, protein and fat in the ray storage tissue of poplar trees

The seasonal pattern in starch, various sugars, protein, and fat, and their interrelationship, has been followed in 3-year-old branch wood of poplar trees (Populus x canadensis Moench robusta) under natural site conditions. The deposition of starch, protein and fat proceeds at different times. Starch accumulates from May until October, fat mainly during the summer months, and protein when the leaves are yellowing in September and October. The maximum concentrations in the branch wood were 15–18 g starch, 6–9 g protein, 4–8 g fat, 10–15 g sucrose, and up to 30 g total sugars per milligram dry weight (DW). During starch deposition periods no increased sucrose level is found in the tissue. The maximum daily starch deposition rate was 0.2–0.4 g starch/day/mg DW of wood. During starch hydrolysis in late autumn and winter, a dramatic increase in sucrose and its galactosides is measured (up to 15–27 g/mg DW in total). In early spring, before budbreak, the concentrations of these sugars diminishes sharply. In contrast to this clear-cut starch-to-sugar conversion in autumn no significant starch-to-fat conversion is detected. An elevated content of free glycerol, however, is found in winter. In spring, starch and storage protein are mobilized completely, or almost completely, in poplar twig wood. A noteworthy pool of maltose is found transiently during autumn (up to 8 g/mg DW) and again in spring. The results demonstrate that the individual storage materials, e.g. starch, protein, and fat, are accumulated fairly independently in the wood storage parenchyma. Tissue sugar levels, in contrast, appear to be closely related to the seasonal variations in starch content, on the one hand, and to the acclimation and deacclimation of the cells, on the other. The interrelations of the storage materials and sugars are discussed.     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

Cytological analysis on the distribution and origin of the alien chromosome pair conferring blue aleurone color in several European common wheat (Triticum aestivum L.) strains

Summary Meiotic chromosome pairing and Giemsa C-banding analyses in crosses of several European blue-grained wheat strains with Chinese Spring double ditelosomic and other aneuploid lines showed that Triticum aestivum Blaukorn strains Berlin, Probstdorf, Tschermak, and Weihenstephan are chromosome substitutions, in which the complete wheat chromosome 4A pair is replaced, whereas the strains Brünn and Moskau are 4B substitutions. The alien chromosome pair in all of these strains is an A genome chromosome (4A) from diploid Triticum monococcum or T. boeoticum not present in common tetraploid and hexaploid cultivated wheats. The Blaukorn strain Weihenstephan W 70a86 possesses, in addition to a rye chromosome pair 5R compensating for the loss of part of chromosome 5D, a 4A/5DL translocation replacing chromosome pair 4B of wheat.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Plantlet regeneration from callus initiated from flower buds in the wild species Allium senescens var. minor

Callus regeneration was observed from flower buds of Allium senescens var. minor inoculated in BDS, MS or B5 medium supplemented with 4.4 M benzyladenine alone or in combination with 4.5 M 2,4-dichlorophenoxy-acetic acid (2,4-d), with 2,4-d and kinetin (4.5 M/4.6 M) or with 5.3 M naphthaleneacetic acid. Ovules enlarged initially but the embryogenic tissue degenerated as callus development progressed from the nectar regions of the petals. Shoot buds and leaf primordia developed from the meristematic protuberances that originated from the surface of the callus. BDS medium with 4.5 M 2,4-d and 13.3 M BA was most suitable for shoot multiplication. The regenerated shoots were rooted in respective liquid medium without any growth regulators and successfully transferred to soil with 90% survival rate.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid     

The tryptic peptide composition of the beta chains of hemoglobins A and B of the domestic cat (Felis catus)

Summary The tryptic peptides from the ^A and ^B chains of cat hemoglobins A and B have been isolated and the amino acid compositions determined. Differences between the two chains were found in two peptides,T-1 (GlySer) andT-14 (AsnSer and LysArg). The GlySer and LysArg substitutions are placed at-1 and-144 respectively from earlier work, and the third substitution, AsnSer at-139 is suggested from this work. In addition, the presence of a blocked amino terminus in ^B has been confirmed. Tentative sequences constructed by homology with known-chain structures suggest the occurrence of substitutions at _{1 1} contacts in ^A and ^B that may be functionally significant. There are at least 18 differences in amino acid composition between cat ^A and dog-chains and 22 differences between cat ^A and normal adult human-chains.     

Memorization and delayed expression of regulatory messages in plants

Plantlets of Bidens pilosus L., considered to be basically symmetrical, can be lateralized (A/B) by being administered a symmetry-breaking signal such as puncturing one of the plant cotyledons. The induced asymmetry remains latent as long as the plants have not been made permissive, i.e. as long as the plant apex is left functioning. When the apex has been removed (plant decapitation), the latent asymmetry is expressed by one of the cotyledonary buds (a/b) statistically beginning to elongate before the other. The interval of time between delivering the symmetry-breaking signal and making the plant permissive is the memorization-time, t. Memorization can be quantified by using a precedence index, q, the values of which range from 0 (no detectable asymmetry with regard to bud growth) to ±1 (bud growth perfectly asymmetric in favour of either bud b or a). Even for memorization times, t, up to 14 d, q-values up to 0.4 (or even larger) are observed. Various experimental characteristics (e.g. light, temperature, presence or absence of the root system) but not the plant age can affect the q-values, at the moment when the treatments are performed, at least in the range of 6 to 25 d. Combining several puncturing treatments either increases or decreases the q-values, depending on the nature of these treatments and the time-intervals, t, between them. Symmetrically removing both cotyledons in the minutes following the puncturing of one of them does not significantly alter the results, which means that the symmetry-breaking message is rapidly transported and memorized within the plant. Non-traumatic asymmetrical treatments (droplets of saline solutions, light-gradients) can also act as symmetry-breaking signals and be memorized. Plants other than Bidens are likely to possess similar memorization ability, although the q-values observed up to now have not been very large.     

Cell multiplication and ultrastructure ofMicrasterias denticulata (Desmidiaceae) grown under salt stress

Cells ofMicrasterias denticulata Bréb. were kept in nutrient solution of high osmolality (salt stress) for four weeks. In a special cell multiplication test it was established that cell division is gradually inhibited at increasing salt concentrations and totally arrested at the highest concentration (26 mosm/kg). Recovery studies proved that even cells from the highest concentration range start dividing immediately after being placed in aqua bidest. thus indicating the full reversibility of the inhibiting effect. — Cells of the highest concentration range show marked ultrastructural changes. Besides an enormous accumulation of starch and oil bodies and a condensed appearance of the ground plasma, a reduction of mitochondria, ER and the Golgi-system is found. The most striking effect occurs on the vacuolar system which appears extremely reduced and condensed. The cell wall is thickened by the formation of an additional cell wall layer with a spongy electron microscopical appearance. Through the cell wall many droplets of a probably fat-like substance are excreted. — In summary, salt stress induces growth-inhibited akinete cells in the sense ofFritsch; these can be reactivated by decreasing the salt concentration. The salt-induced akinete state seems to be an ecological adaption to unfavourable conditions rather than a degeneration of the cells.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.23. 12. 1988     

Die Homologie des Perigons der Zingiberaceen Ein Beitrag zur Morphologie und Phylogenie des Monokotylen-Perigons

Nicolaia elatior is used as an example to demonstrate that the mucronate tepals ofZingiberaceae correspond to hypsophylls (bracts) consisting of a leaf sheath and a rudimentary Oberblatt (= leaf petiole + lamina) represented by the mucro. Evidence for this interpretation is furnished by all available criteria: leaf sequence (exhibiting a complete continuum of forms from foliage leaves over cata- and hypsophylls to the tepals), nervature, and ontogeny.The present conception is compared with the well-founded thesis ofLeinfellner that the perigone ofLiliaceae is derived from the androecium. The different morphological status of the perigone in both families is not regarded as the result of different phylogenetic origin, but as a manifestation of morphogenetic transgressions from one phyllome category to an adjacent one: In theLiliaceae the perigone is under a strong morphogenetic influence of the androecium, and therefore displays staminal characters, in theZingiberaceae it is under the dominating influence of the extrafloral region, and thus appears as a hypsophyllous structure. If this assumption of a morphologically oscillating perigone is correct, it will be fundamentally impossible to demonstrate unequivocally the phylogenetic origin of the monocotyledonous perigone.

Im wissenschaftlichen Werk Prof. Dr.Walter Leinfellners steht an erster Stelle die Morphologie der Blütenorgane. Als sein dankbarer Schüler möchte ich ihm aus Anlaß seines 70. Geburtstages die folgende Studie zu einem Thema zueignen, das ihn wie mich gleichermaßen angesprochen hat und schon Gegenstand der Forschungsarbeit des Jubilars war: die Homologie des Monokotylen-Perigons.     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

Sialyltransferases of developing rat brain

The activity of four different sialyltransferases acting on N- or O-linked chains of glycoproteins was studied in brains of 19 days-old embryos, 1 day-old newborns and adult rats. By using asialofetuin, fetuin andN-acetyllactosamine as acceptors, it has been possible to measure independently the following enzyme activities: CMP-NeuAc:Gal1-3GalNac (2–3)-sialyltransferase (EC 2.4.99.4), CMP-NeuAc:Gal1-4GlcNAc (2–3)-sialyltransferase (EC 2.4.99.6), CMP-NeuAc:Gal1-4GlcNAc (2–6)-sialyltransferase (EC 2.4.99.1) and CMP-NeuAc:NeuAc2-3Gal1-3GalNac (2–6)-sialyltransferase (EC 2.4.99.7). The specific activity of the first three enzymes which act on asialylated acceptors showed a 2.6-fold decrease in a parallel manner after ontogenic development, while the activity of NeuAc2-3Gal1-3GalNac (2–6)-sialyltransferase was four times lower in adult than in embryonic brain, showing a stronger dependence on ontogenic development. Despite the higher level of sialyltransferases able to act on glycoproteins, in fetal brain these glycoproteins do not contain a higher amount of sialic acid.Abbreviations HPLC high performance liquid chromatography - N-CAM neural cell adhesion molecule     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Direct plant regeneration from leaf explants of Plumbago species

Direct plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige and Skoog (MS) medium supplemented with 6.7 M 6-benzylaminopurine (BA), 1.4 M indole-3-acetic acid (IAA), 370 M adenine sulfate (Ads) and 3% (w/v) sucrose. The shoot initials developed within 2–3 weeks on the leaf margin as well as from the cut surface of the leaf. High frequency shoot-bud regeneration was achieved on similar medium in subsequent subcultures. The semi-mature leaves produced more shoot-buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi-mature explants produced shoot-buds per leaf explant within 4 weeks of culture. Shoots rooted on half-strength basal MS medium supplemented with 1.2 M indole-3-butyric acid (IBA) and 2% (w/v) sucrose; approximately 90% of the in vitro raised plantlets survived in the greenhouse. The regenerated plantlets looked morphologically similar to the mother plants. This protocol might be useful for genetic improvement programs.     

The addition of Dasypyrum villosum (L.) Candargy chromosomes to durum wheat (Triticum durum Desf.)

Summary Six monosomic addition lines were produced in which different Dasypyrum villosum (L.) Candargy chromosomes were added to the chromosome complement of Triticum durum Desf. cv. Creso. Each added alien chromosome was found to have a specific effect on plant morphology and fertility. Transmission rate varied widely (from 7.5 to 27.7%) among the six univalent chromosomes. Different monotelosomic addition plants derived by a relatively high frequency of chromosome misdivision were isolated. The addition lines should be useful for studying Dasypyrum chromosome homoeology and the introduction of alien variation into durum and common wheats.Research supported by a grant from the Italian Research Council for Finalized Project IPRA. Sub-project Plant Breeding, Paper No. 1095     

Saponin-like substances inhibit α-galactosidase production in the endosperm of fenugreek seeds

When fenugreek (Trigonella foenum-graecum L.) endosperms plus testa (endosperms), which had been isolated from 5-h-imbibed seeds, were incubated for at least 2 h under germination conditions, they leaked substances which, like exogenous abscisic acid (ABA), inhibited the production of fenugreek endosperm -galactosidase. However, unlike ABA, 8 h treatment with these inhibitors had no effect on fenugreek endosperms which had been isolated from 15-h-imbibed seeds and leached for 2 h. This indicated that either their inhibitory action was on processes which were related to the production of -galactosidase and had been completed by this time, or that there might be factors present which inactivate these inhibitors. It was also concluded that the action of the endosperm leachate could not be attributed to the presence of ABA. The activity of the leachate decreased when it originated from endosperms imbibed for periods longer than 25 h and thin-layer chromatography (TLC) of extracts from these endosperms showed decreased contents of the leachable inhibitors as imbibition proceeded. From the seed leachate, which had a TLC pattern and inhibitory action similar to that of the endosperm, were isolated three substances which, when applied to endosperms, inhibited the production of -galactosidase activity. According to their chromatographic behaviour and their reaction with specific reagents, there are strong indications that these substances are saponins. These diffusible saponin-like substances were located in both endosperm and perisperm and their physiological role is discussed.Abbreviations ABA abscisic acid - PEG polyethylenglycol - TLC thin-layer chromatography We wish to thank the Alexander S. Onasis Public Benefit Foundation for a grant to K.Z. and Dr. J.S.G. Reid (University of Stirling, Scotland) for a kind gift of fenugreek seeds.     

Involvement of β 1,4 galactosyltransferase 1 and Galβ 1→4GlcNAc groups in human hepatocarcinoma cell apoptosis

1,4 galactosyltransferase 1 ( 1,4GT1) synthesizes Gal 14GlcNAc groups in N-linked sugar chains of animal glycoproteins, which have been demonstrated to play an important role in many biological events, including sperm-egg interaction, cell migration and mammalian embryonic development. In this study, the mRNA level of 1,4GT1 was found to increase greatly during the 7721 hepatocarcinoma cells apoptosis induced by cycloheximide. Ricinus Communis Agglutinin-I staining indicated generous increase of Gal 14GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of 1,4GT1, which also led to the increased Gal 14GlcNAc groups on the transfected cell surface. All the observations suggested that 1,4GT1 and Gal 14GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.     

The chromosomal locations and linkage relationships of the structural genes for the prolamin storage proteins (secalins) of rye

Summary Rye secalins are a polymorphic mixture of polypeptides which are classified into four major groups. Previous studies have shown that the structural genes for two of the groups (the -secalins and 40K -secalins) are located on the short arm of chromosome 1R and those for a third group (the high molecular weight secalins) on the long arm of the same chromosome. Analysis of F₂ grain from crosses between inbred lines of S. cereale shows that the structural genes for the -secalins (designated Sec 1) and the high molecular weight secalins (designated Sec 3) are loosely linked (40.8 ±3.76% recombination, 57.4 ± 11.30 cM). Analysis of wheat rye addition lines shows that the structural genes for the 75K -secalins are present on chromosome 2R. This locus is provisionally designated Sec 2. These genes are probably derived from those for the 40K -secalins by duplication, divergence and translocation. Analysis of secalin fractions from wild species of rye shows that all contain 75K -secalins, indicating that the duplication and divergence, if not the translocation, occurred before speciation of the genus.