The effect of plant size on vegetative reproduction in a pseudo-annual

The relationship between plant size and vegetative reproduction in clonal plants appears complex because vegetative expansion, growth, and reproduction are not clearly separable in such plants. In pseudo-annuals, which are clonal plants surviving the winter only as seeds and hibernacles produced by the rhizome apices, vegetative growth and reproduction are clearly separate processes so that the relationship between vegetative reproduction and plant size can be studied. We used the pseudo-annual Helianthus x laetiflorus Pers. to study the relationship between plant size and total rhizome biomass, rhizome (hibernacle) biomass, and number of hibernacles. We manipulated resource acquisition of the plants by reducing leaf area (leaf-clipping) and by fertilization, thus affecting plant size. Furthermore, we studied the success of thin and thick hibernacles in terms of future growth and reproduction in a separate experiment. The results showed that vegetative reproduction was positively related to plant size. The ratio between the number of hibernacles and mean hibernacle weight was affected by plant size in such a way that in small plants both number of hibernacles and mean hibernacle weight were reduced to the same extent as compared to those in large plants.However, the size distributions of plants of the next generation growing from thin and thick hibernacles did not differ. It remains unclear therefore why this pseudo-annual species produces thick hibernacles at all.     

Clonal reproduction, vegetative multiplication and habitat colonisation in Tussilago farfara (Asteraceae): A combined morpho-ecological and molecular study

Using a combined morpho-ecological and molecular (amplified fragment length polymorphism (AFLP) fingerprinting) approach, clonal growth, clonal reproduction and the resulting vegetative multiplication were examined in the pioneer species Tussilago farfara (Asteraceae).

In two forest plots in Berlin (Germany), all modules and spacers of the plant were mapped and dug out. The plants showed intense clonal growth with leaf modules connected through long branched rhizomes. The rhizomes were brittle and regularly exhibited rotting zones and breaks. This clonal reproduction can occur rather fast; in a submersed stand in Lanke (Brandenburg, Germany), rhizomes showed signs of disintegration only a few weeks after the modules developed. As a consequence, modules become separated and genets fragment into merigenets.

Using AFLP fingerprints, the relationships of samples from the two dug out plots were assessed: 15 and 18 analysed samples (representing 13 and 16 merigenets) belonged to only two and three distinct genets, respectively. Each of these genets was split into at least two to eleven morphologically independent merigenets, indicating frequent and effective clonal reproduction.

The study highlights the relevance of vegetative multiplication in T. farfara, achieved rapidly through repeated clonal growth and subsequent clonal reproduction. The typical strategy of habitat colonisation and maintenance is described and illustrated for this pioneer species.     

Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle

The use of bioreactors may provide an efficient and economic tool for mass clonal propagation of plants if technical problems can be solved. In this paper, we report the results of experiments aimed at optimising conditions for apple rootstock M26 grown in RITA containers using the temporary immersion principle. We tested different types and sizes of explants, different concentrations of plant growth regulators (BAP, kinetin and IBA) in the multiplication and elongation phases, and medium exchange during the shoot elongation period. The results show that the higher concentrations of cytokinins were required during the shoot multiplication phase, while the lower concentrations were better during the shoot elongation phase. Hyperhydricity was increased with increasing concentration in of cytokinins during both shoot multiplication and shoot elongation phases. The best shoot production in terms of shoot number and shoot quality was obtained using 4.4mol BAP and 0.5mol IBA during the shoot multiplication phase and 1.1mol BAP and 0.25mol IBA during the shoot elongation phase. Medium exchange twice during the shoot elongation phase resulted in higher shoot production compared with no exchange of the medium. However, it also resulted in increased hyperhydricity. Immersion frequency of 16 times per day gave a higher multiplication rate and longer shoots than 8 times per day. The explant size of 0.5cm or 1cm resulted in a significantly higher shoot production rate compared with that of 1.5cm, but shoot length and hyperhydricity were not affected by the explant size. Shoot cultures from the liquid media rooted normally in the RITA containers with more than 90% rooting and the rooted plantlets acclimatised well in the greenhouse.     

Effects of salinity on metabolism and life history characteristics of Daphnia magna

In the Baltic Sea area, the cladoceran Daphnia magna is commonly found in brackish water rockpools and it has been suggested that salinity is one of the niche dimensions that affects the distribution of the species. The salinity tolerance of D. magna was studied both in physiological and life history experiments. The experimental salinities were freshwater, 4S and 8S. The highest respiration and ammonium excretion rates were measured in the freshwater treatment with decreasing respiration and ammonium excretion rates at higher salinities. The lowest O/N ratio (oxygen consumption to ammonium excretion), describing the metabolic status of an organism, was obtained at 8S, although the only significant differences were detected when comparing to 4S treatments. Individual growth rate, reproductive output and population growth rate were highest at 4S. At 8S growth and reproduction were reduced as compared to freshwater and 4S. The life history parameters in the performed experiments indicated higher fitness (expressed as r) as well as more favourable conditions for growth and reproduction at 4S, whereas the O/N ratio was more difficult to interpret and, in this case, gave a less clear picture of the salinity influence.     

Changes in density of chromatin in the meristematic cells ofSinapis alba during transition to flowering

Summary Changes in the density of nuclear chromatin in the shoot apical meristem ofSinapis alba L. during floral transition (floral evocation) are described using Feulgen-stained 2 m thick semi-thin sections and scanning cytophotometric techniques. In both G1 and G2 nuclei the chromatin becomes less heterogeneous and less dense in evoked meristems compared to vegetative meristems. When chromatin is resolved into two fractions the dispersed fraction increases relative to the condensed fraction at evocation. This decondensation process occurs earlier in G1 than in G 2 nuclei. These chromatin changes are presumably closely related to the dramatic stimulation of biosynthetic activity and cell division during floral transition.     

Mictic patterns of the rotifer Brachionus plicatilis Müller in small ponds

Populations of the rotifer Brachionus plicatilis were monitored in three small ponds in a marsh on the Mediterranean coast. Samples were taken approximately every three weeks from July 1992 to November 1993. Salinity, temperature, conductivity, pH and oxygen concentration were measured in the field. Population density was determined from preserved quantitative samples. Individuals were classified as mictic females, amictic females, non-ovigerous females, and males, differentiating between two morphotypes (S and L). From these counts, a level of mixis was calculated. We also determined the proportion of mictic females in natural populations by culturing females isolated from fresh samples. From these data, mictic patterns over time and correlation between levels of mixis and environmental and population parameters were analyzed. From a previous study S and L morphotypes were known to correspond to genetically different clonal groups. Our data showed that reproduction was predominantly parthenogenetic in these clonal groups, but mictic females were found in most samples, the proportion of mictic females ranging from 0 to 29%. The clonal groups showed different patterns of mixis. L clonal group presented a continuous sexual reproductive pattern. In contrast, S clones showed a rather punctuated mictic pattern. A positive correlation between levels of sexual reproduction and population density was found for S and L groups. However, they differed in their density threshold for mictic reproduction. The adaptive meaning of these patterns and their implications in maintaining genetic diversity within and between populations are discussed.     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

Effects of forest age and disturbance on population persistence in the understory herb,Arisaema triphyllum (Araceae)

Long-lived understory herbs experience a highly dynamic forest over space and time, yet can persist for more than a century. To understand how these populations persist, we examined effects of forest age and disturbance on potential sexual reproduction and clonal growth in the sexually labile perennial, Arisaema triphyllum. Potential sexual reproduction (female:male ratio) was significantly greater in the Young and Old-Gap forest states compared with Old, closed-canopy sites, where it was virtually absent. In contrast, clonal growth (estimated by cormlet production) did not differ significantly among the three forest states. Of seven environmental variables measured, only light (positively) and plant density (negatively) contributed significantly to the variation in potential sexual reproduction, while no measured variables contributed significantly to the variation in number of cormlets. The larger sexual reproductive effort (flower+stalk biomass/total biomass) for males in the undisturbed, 100 yr old forest may explain the absence of females in these sites, while the invariant vegetative reproductive effort (cormlet biomass/total biomass) may explain the similarity in average number of cormlets per individual per season across forest states. These results suggest that potential sexual reproduction is resource-limited, while clonal growth may be resource-independent. By maintaining ramet production during unfavorable periods, A. triphyllum populations disperse temporally, waiting for conditions under which sexual reproduction may resume.     

Ultrastructure of Bacillus pulvifaciens

The ultrastructure of vegetative cells and spores of Bacillus pulvifaciens was studied by CTEM and SEM methods. The vegetative cells are rods, 1.6–4.5 m long and 0.4–0.6 m wide, exhibiting typical ultrastructural features of Gram-positive bacteria. The spores are of ellipsoidal shape, 0.6×1.2 m in size, with six longitudinal ribs reaching up to 130 nm in height. There are satelite ribs on both sides of the longitudinal ribs, reaching up to 20 nm in height. Between the longitudinal ribs, additional transversal ribs were observed in SEM. A special tubular layer, separating the outer and inner coat of the spores, was revealed in ultrathin sections. This layer seems to be a typical ultrastructural feature of Bacillus pulvifaciens spores.     

Lateral bud culture of papaya (Carica papaya L.) for clonal propagation

Lateral buds may be preferred to shoot tips for in vitro propagation of papaya because of its unbranched nature. Proliferating shoot cultures from lateral buds appeared extremely compact with shortened internodes and leaf lamina of the cytokinin level (BAP 2 M) reported for multiple shoot production from shoot tips. ZEA (4 M) and 2iP (8 M) although reduced the proliferation rate, resulted in better growth of the shoot from lateral bud. Rooting was observed with IBA 20 M but plantlets so produced remained stunted.     

Structure and dynamics of a <Emphasis Type="Italic">Castanopsis cuspidata</Emphasis> var. <Emphasis Type="Italic">sieboldii</Emphasis> population in an old-growth,evergreen, broad-leaved forest: The importance of sprout regeneration

The population structure and dynamics of Castanopsis cuspidata var. sieboldii were studied to evaluate vegetative and sapling regeneration in an old-growth, evergreen broad-leaved forest exposed to low-severity typhoon disturbances by annual typhoons on the Tsushima Islands in Japan. The density of individuals 5cm d.b.h. was 38.0ha^–1 in 1990; 7.9% were multiple-stemmed individuals. Over the 7-year study period (1990–1997), the number of individuals decreased, although the number of stems increased. Over 30% of apparently dead individuals were reconstituted by sprouting stems. Compared with sprout regeneration, sapling regeneration rarely occurred and was only observed in canopy gaps. Most individuals had at least one sprout shoot (H 30cm, d.b.h. <5cm), and the number and size of sprout shoots increased as the size of the individuals increased. During the study period, larger individuals with stem breakage tended to produce sprout stems. The density of saplings was 1074ha^–1 and they were more abundant in canopy gaps than under closed canopies, but large saplings were very rare even in canopy gaps. The population of C.cuspidata var. sieboldii consisted primarily of single-stemmed individuals with a few multiple-stemmed individuals providing a sprout bank. Larger individuals responded to the low severity typhoon disturbances and formed sprout stems. Although many saplings were observed, regeneration occurred more often by sprout formation than by growth of saplings. Thus, sprout regeneration is an important mode of regeneration, which allows this pioneer-like species to maintain its population in this forest.     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.     

Use of monocharacteristic growth forms and phenological phases to describe and differentiate plant communities in Mediterranean-type ecosystems

The ecomorphological and phenological study was carried out within aMediterranean vegetation context, in Quercus suberforests,which have been substituted by shrublands of Cistus sppwithin two Natural Parks in the south of the Iberian Peninsula. Theecomorphological characters that show meaningful differences between both typesof vegetation are: location of renewal buds, spinescence, stratification,maximum height of the vegetation, organs periodically shed, leaf consistency,leaf tomentosity, leaf size, and life duration of leaves, plant duration,vegetative regeneration after fire, main vegetative growth season, mainflowering season and fruit type. The phenological phases also help to discernbetween forest and shrubland, specially flower bud formation, fruiting, seeddispersal, and the existence of brachyblast vegetative growth and brachyblastleaf shedding. We propose three new indexes based on phenological phases:active period of the species (APS), active period of thecommunity (APC) and reproductive/vegetative activity of thespecies (RVA). The results of their application, in combination with theecomorphological characters, have proved promising in describing vegetation andin clearly differentiating communities. The results also show the existence ofdifferent ecomorphological groups of plants at community level, with consequentecological, historical, phytocoenological and adaptive implications     

Micropropagation and field evaluation of micropropagated plants of turmeric

A protocol was developed for in vitro propagation of turmeric cv `elite' using young vegetative buds from sprouting rhizomes. The shoot buds produced multiple shoots when cultured on MS solid medium supplemented with benzyladenine and 1-naphthalene acetic acid. The effect of various cytokinins on shoot multiplication was studied by culturing the shoot tips on MS liquid medium supplemented with benzyladenine, benzyladenine riboside, kinetin, kinetin riboside, zeatin, 6-,-dimethylallylaminopurine, adenine, adenine sulfate or metatopolin each at 10 M in combination with 1-naphthalene acetic acid (1 M). Significant differences were observed between the treatments. Liquid medium was more favourable than agar medium for shoot multiplication. Among the various concentrations of agar tested, 0.4% and 0.6% were the best and produced the highest number of shoots per explant. Among the different carbohydrates tested, sucrose, fructose, glucose, sugar cubes, maltose, levulose and market sugar were found to be equally effective for shoot multiplication and xylose, rhamnose, lactose and soluble starch were inhibitory. Ninety five percent of the micropropagated plants survived in sterilized soil in paper cups and all of them survived in the field. Among 48 plants, two plants showed variegated leaves on the tillers. The micropropagated plants showed a significant increase in shoot length, number of tillers, number and length of leaves, number of fingers and total fresh rhizome weight per plant when compared with conventionally propagated plants. RAPD analysis of 11 regenerated plants using sixteen 10-mer primers did not show any polymorphism.     

Biology and Egg Morphology of The Dalmatian Barbelgudgeon Aulopyge Huegeli, An Endangered Endemic Species in Croatia

This paper describes patterns of annual and diurnal activity in the endangered endemic cyprinid Aulopyge huegeli from Croatia. The species is largely nocturnal. Spawning in the laboratory begins at temperatures of 20°C. Aulopyge huegeli is a rock and gravel brood hider. The female deposits eggs in various fissures with a special ovipositor. The eggs are whitish and have a diameter between 1.5 and 2mm. The zona radiata is relatively thin (3.2–5.5m) and provides the eggs with the necessary plasticity. The surface is covered with an attaching substance and with short attachment extension around the vegetal pole. This correlates with features of the spawning microhabitats. Aulopyge huegeli has a very small micropyle which consists of a pit with a diameter of 7.5m and a canal with a diameter of 2.5m (micropyle type I).     

In vitro micropropagation of Piper longum – an important medicinal plant

Efficient and rapid tissue culture systems were developed for Piper longum, an important medicinal plant, through shoot tip multiplication and direct regeneration. Multiple shoots were induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.44–22.19 M benzyladenine (BA) and 4.64–13.9 M kinetin (K). Maximum number of shoots were induced with 8.9 M BA and 4.64 M K. Adventitious shoot regeneration from leaf segments was achieved on MS containing 3.6–22.19 M BA along with 3.31–12.4 M picloram (P). Shoot differentiation occurred directly from the leaf bases without intermediale callus formation. Maximum shoot buds were obtained on MS medium with 17.76 M BA and 8.28 M P. Elongated shoots were separated and rooted in MS supplemented with 2.46 M indole butyric acid (IBA). Plantlets, thus developed were established in soil.     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator     

The architecture of Zeylanidium olivaceum (Podostemaceae) inferred from the structure of the primary shoots

Zeylanidium olivaceum (Podostemaceae-Podostemoideae) is the only crustose-rooted species of the genus that still develops prominent primary shoots from the seedling in addition to the secondary (root-borne) shoots forming the clonal plant body. The primary shoots are articulated into an up to 8.5 cm long and 4 mm thick stalk (hypocotyl) and a copiously foliated paint-brush-like shoot which is sympodially branched in the form of a helicoid cyme. The helicoid branching pattern indicates a transversal prophyll position, typical of the dicotyledons, but replaced in most other Podostemoideae by a median prophyll position. The short stems within the leafy head do not separate, but are fused to a dense aggregate (coenosome). Branches are mainly vegetative with a rosette of about 20 elongate subulate leaves. The primary shoots branch in the vegetative stage and thus differ from other Podostemoideae where ramification is confined to the floriferous shoots. The leaves adhere together at the base, forming an apical furrow-like hollow surrounding the shoot tip. The tiny shoot apex is one-layered, radially symmetrical, and develops leaf primordia in a decussate pattern. The erect primary shoots thus differ from the distichously foliated plagiotropic secondary shoots by the decussate phyllotaxis, and by the presence of more than 20 leaves on a shoot as compared to the about six leaves on the vegetative and floriferous secondary shoots. The features observed in the primary shoots are interpreted as primitive as compared to those of the secondary shoots. Z. olivaceum is thus characterised by heterobathmy, i.e., the occurrence of plesiomorphic (primary shoots) and apomorphic features (secondary shoots). The primary shoots exhibit primitive features that apparently have been lost in secondary and primary shoots of most other members of subfamily Podostemoideae.     

Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone