In situMechanical Testing of Fully Hydrated Carrots (Daucus carota) in the Environmental SEM

We demonstrate the use of the Environmental Scanning ElectronMicroscope (ESEM) forin situobservation of mechanical testson carrot (Daucus carota)parenchymal tissue. The ESEM toleratesseveral Torr of water vapour in the specimen chamber, thus allowingfully hydrated specimens to be examined at high resolution,but without preparation. Three tests were performed, involvingslicing, tension and compression. The manner in which stresswas distributed in the tissue, crack propagation and cell walldistortion were all observed in real time.Copyright 1998 Annalsof Botany Company. Environmental scanning electron microscopy, carrot,Daucus carota,mechanical testing.     

Stomata and Structure of Tetraploid Apple Leaves cultured in Vitro

Leaves of anther-derived tetraploid apple (Malus pumila Mill.)shoots were examined by low-temperature scanning electron microscopy(LT-SEM). Leaves were serrate and wide with an undulating adaxialsurface due to convex epidermal cells, apparently without crystallineepicuticular wax. Stomata were absent from the adaxial surface,except for the marginal teeth which exhibited 40-60 stomataper leaf; they probably originated from residual mitotic activity.One third of abaxial stomata was occluded by the residual cuticleof the mother guard cell across the stomatal pore which rupturedwhen the stomata became functional. The stomatal index was 7·2(± 1·6) with 60-75 stomata mm^-2, i.e. abaxialstomata of tetraploid leaves expanded in vitro were less frequentthan those in triploid leaves either cultured in vitro (475-575stomata mm^-2) or grown on the tree (320-390 stomata mm^-2) wherethe stomatal index was 21 (± 4). Freeze-fracture transsectionsshowed that the tetraploid in vitro leaves were composed ofa layer of adaxial epidermal cells, followed by a single layerof palisade cells and four to five layers of spongy mesophyllcells and the abaxial layer of epidermal cells, in contrastto juvenile seedling-grown apple leaves in which the two layersof palisade cells comprised the majority (52-60%) of the leafvolume. The same morphological features, such as wide and lesspointed leaves, reduced stomatal density and stomatal index,and increased stomatal size that were previously reported fortree-grown tetraploid leaves were also expressed in vitro. Thus,causes of the stomatal deformation in tissue-cultured Rosaceaeare interpreted to be in part genetic and not purely environmental.Copyright1994, 1999 Academic Press Malus pumila Mill., apple, biotechnology, breeding, cryo-preservation, CO₂, juvenile, low temperature-scanning electron microscopy (LT-SEM), micropropagation, ploidy, stomata, tissue-culture, transpiration     

Importance of the Fixative for Reliable Ultrastructural Preservation of Poikilohydric Plant Tissues. Observations on Dry, Partially, and Fully Hydrated Tissues ofSelaginella lepidophylla

Leaves of desiccated ‘resurrection plants’,Selaginellalepidophylla, were hydrated either through the roots of intactplants or as isolated organs. Air-dry tissue and samples at1, 4, 8 and 24 h (both detached and intact) of hydration wereprepared for electron microscopy using aldehyde fixatives ofdifferent osmotic strengths. Both dry and hydrated tissues werealso prepared using freeze substitution. Significant differencesin the ultrastructural preservation of these different sampleswere noted. There was a direct correlation between the osmolalityof both the fixative and the tissue with the quality of ultrastructuralpreservation. When the osmolality of the fixative was slightly(or even considerably) higher than that of the tissue, optimalpreservation was achieved. Freeze substitution, however, gavethe most faithful preservation of all subcellular compartments,despite the frequent presence of small ice crystals. Additionally,hydration of detached leaves for more than 4 h resulted in swellingdamage of the organelles and cytoplasm, regardless of the fixationprotocol. Broadly interpreted, the results of this study indicate thatan optimal preservation of plant cell and organelle ultrastructurecan be achieved by the use of high osmolality fixatives or,preferably, freeze substitution. These results are also importantin determining the method of hydration of poikilohydric samplesfor physiological studies and for interpretation of functionalchanges as related to the structural condition of the organelles.Copyright1997 Annals of Botany Company Selaginella; fixation; ultrastructure; dry; hydrated     

Effects of Desiccation on some Activated Oxygen Processing Enzymes and Anti-Oxidants in Mosses

The possibility that desiccation tolerance in mosses may bedependent on an ability to process species of activated oxygenwas investigated using the tolerant sand-dune moss Tortula ruraliformis(Besch.) Grout and the sensitive minerotrophic flush speciesDicranella palustris (Dicks.) ex. E. F. Warb (D. squarrosa (Starke)Schp.). Mosses were desiccated in low and high irradiance andresponses of these plants compared to those of dark-desiccatedand hydrated control plants. Both desiccated and undesiccatedplants of T. ruraliformis had a higher superoxide dismutase(SOD) activity than D. palustris, but had similar, or loweractivities of the chloroplastic H₂O₂-processing enzymes peroxidaseand ascorbate peroxidase. In T. ruraliformis, desiccation inboth light and dark led to a significant increase in SOD activity,but did not consistently stimulate the activities of peroxidaseand ascorbate peroxidase. In D. palustris, desiccation in combinationwith irradiance led to a decrease in peroxidase activity, buthad little affect on the activities of other activated oxygen-processingenzymes. Catalase, an extra-chloroplastic enzyme, was up to7-fold more active in hydrated T. ruraliformis than in D. palustris,but desiccation resulted in significant decreases in the activityof this enzyme in both species. Regardless of irradiance level,there was a depletion of the anti-oxidant ascorbic acid in bothspecies when desiccated. Only in T. ruraliformis was there asynthesis of     

Proline Accumulation during Water Stress in Resurrection Plants

In leaves of resurrection plants dried intact, free prolineincreased in nine species and remained unchanged or declinedin two other tolerant species. Infiltration of proline into hydrated resurrection leaves oftwo species (a relatively high accumulator of proline, Borya,and low accumulator, Myrothamnus) prior to dehydration did notimprove the cells' survival rate, and in some cases caused adecrease in survival. These data indicate that the extreme drought tolerance of resurrectionplants is not related per se to proline levels.     

Winterfat (Eurotia lanata(Pursh) Moq.) Seedbed Ecology: Low Temperature Exotherms and Cold Hardiness in Hydrated Seeds as Influenced by Imbibition Temperature

Thermal analyses of freezing events in hydrated lettuce (LactucasativaL.) seeds show a correlation between low temperature exotherms(LTEs) (evidence of ice crystal formation) and seed death. Yet,weather patterns common to the Northern Great Plains of NorthAmerica regularly create conditions where non-dormant seedsof native plants hydrate with snow melt and are subsequentlyexposed to -30 °C or colder conditions. To determine ifsuch weather patterns decimate dispersed seeds, we measuredthe effects of freezing on fully hydrated winterfat (Eurotialanata(Pursh) Moq.) seeds harvested from the Northern Plainsat two USA and one Canadian location. Survival of hydrated seedsto -30 °C at a cooling rate of 2.5 °C h^-1was similarto that of seeds not subjected to cooling, even though botha high temperature exotherm (HTE) and an LTE were observed.Although the LTE was not related to winterfat seed survival,freeze-stressed seeds had reduced germination rates and reducedseedling vigour, particularly for the collection with the lightestseeds. The temperature of LTEs was similar among seed collectionswith a mean of -17.6 °C, but was warmer when the seeds wereimbibed at 0 °C compared to 5, 10 or 20 °C. We founda significant correlation between the HTE and LTE temperatures.The difference and the correlation may be due to the highermoisture content of seeds imbibed at 0 °C. After pericarpremoval, only one exotherm in the range of the LTE was observed.This was also true for the naked embryo. We conclude that anLTE indicates ice formation in the embryo, but that it doesnot signal the death of a winterfat seed.Copyright 1998 Annalsof Botany Company Eurotia lanata(Pursh) Moq.,Krascheninnikovia, Ceratoides,winterfat, exotherm, freezing tolerance, freezing avoidance, seedbed ecology, germination, D50, seedling vigour, seed collection     

The Comportment of the Vegetative Nucleus and Generative Cell in the Pollen and Pollen Tubes of Helleborus foetidus L

It has been reported that in species of Plumbaginaceae, Chenopodiaceae,Cruciferae and Amaryllidaceae a ‘male germ unit’is formed in which the two male gametes remain inter-connected,with one of the pair linked intimately to the vegetative nucleus.In two species the unit has been shown to remain intact in thepollen tube, and some accounts imply that it is polarized inits movement, the vegetative nucleus leading in the tube. Evidence given in this paper indicates that such a unit is unlikelyto be present in Helleborus foetidus L. (Ranunculaceae). Applicationof an optical sectioning technique has shown that at no timeis there a persistent linkage between the generative cell andthe vegetative nucleus in unhydrated, hydrated and germinatingpollen, nor is one present in the early pollen tube. Furthermore,no inter-connections between the two entities were seen in protoplastsfrom living, hydrated and incipiently germinating grains isolatedmechanically in an osmotically balancing medium. Following germination,the vegetative nucleus leaves the grain in advance of the generativecell in most instances, but in the samples examined the generativecell led in about 30 per cent of the tubes. Assembling a polarisedmale germ unit in these circumstances would require (a) theformation of an inter-connection between the vegetative nucleusand the generative cell or one of the gametes derived from itduring passage through the tube, and (b) where the generativecell initially leads in the tube, an exchange in relative positions.It is considered improbable that these conditions could consistentlybe met. Mature, incipiently germinating pollen of H. foetidus releasesa fibrillar component when extruded into suitable media. Websor clusters of fibrils are commonly seen to be associated withboth the vegetative nucleus and the generative cell. The possibilitythat the fibrils are composed of aggregates of microfilamentsis considered. Helleborus foetidus L., pollen germination, generative cell, vegetative nucleus, male germ unit     

Enzyme Treatment as an Aid in the Study of Seed Surface Structures of Solanum Species

Enzyme treatment of seeds before scanning electron microscopyrevealed interesting surface features which are valuable taxonomiccharacters. The seed surface of Solanum aethiopicum (scarleteggplant) was similar to that of its wild progenitor, S. anguivi(= S. indicum pp.). Solanum violaceum (= S. indicum pp.) fromIndia was distinct. These results confirm previous taxonomicstudies based on hybridization and seed protein electrophoresis. Solanaceae, Solanum aethiopicum, Solanum indicum, eggplant, taxonomy, scanning electron microscopy/SEM, seed, testa, microstructure     

Studies of the Surfaces of Desert Plant Seeds: II. Ecological Adaptations of the Seeds of Blepharis persica

A study was made of the role of the hydrated mucilage whicharises from the integumentary hairs of Blepharis seeds and inhibitsgermination where there is an excess of water, and of the anatomicaland physiological basis for this phenomenon. It was found thatthis inhibition was not due to bacterial respiration. The appearanceof the dry mucilage in the unimbibed seeds is described fromscanning electron micrographs. The basic helical framework ofthe secondary wall thickenings and the very unusual second-orderframework of trabeculae in the integumentary hair cells aredescribed.     

Effects of Temperature and Various Red or Far-red Irradiations on Phytochrome- and Gibberellin A3-mediated Germination Control in Partially Hydrated Lettuce Seeds

Dark reversion of phytochrome in partially hydrated lettuceseeds (Lactuca sativa cv. Grand Rapids) is temperature dependent.After initial red irradiation (R) the higher the storage temperature,the higher the dark reversion rate. Following dark moist storage(DMS) at 30 ?C for 15 d none of the seeds receiving initialR germinated, whereas seeds stored at 0 ?C germinated nearlyas well (about 80%) as unstored controls. The half-time fordark reversion at 20 ?C and 30 ?C is 9 d and 3 d respectively.Repeated R treatments given at 5 d intervals during DMS at 20?C and 30 ?C maintained a high germination capacity. With threeor more R treatments the effect of high temperature largelydisappeared. Dark reversion of phytochrome was not observed in partiallyhydrated lettuce seeds receiving continuous red irradiation(cont R) for two or more days. The promotive effect of contR could be reversed at any time with a brief far-red irradiation(FR), indicating that the phytochrome system remained fullyphototransformable. With continuous far-red light (cont FR)the ability of gibberellin A₃ (GA₃) to stimulate germinationdisappeared and response to GA₃ also diminished in cont R followedby FR but at a slower rate indicating the induction of secondarydormancy in these partially hydrated seeds. This induction ofdormancy was retarded by repetitive or cont R but was enhancedby cont FR. The results of this study suggest a role for theaccumulated stable intermediates of phytochrome transformationin partially hydrated seeds with repeated or continuous R treatmentsand different effects of GA₃ and R in the regulation of germination. Key words: Phytochrome, Lactuca sativa, Seed germination, Temperature, Dark reversion of phytochrome, Seed water content     

Constitutive components and induced gene expression are involved in the desiccation tolerance of Selaginella tamariscina

Selaginella tamariscina, one of the most primitive vascularplants, can remain alive in a desiccated state and resurrectwhen water becomes available. To evaluate the nature of desiccationtolerance in this plant, we compared the composition of solublesugars and saturation ratios of phospholipids (PLs) betweenhydrated and desiccated tissues of S. tamariscina using gaschromatography. In this study, differences in gene expressionand ABA contents were also analyzed during dehydration. Theresults revealed that trehalose (at >130 mg g^–1 DW)was the major soluble sugar, and low saturated fatty acid contentin PLs (0.31) was maintained in both hydrated and desiccatedtissues. In addition, the ABA content of S. tamariscina increased3-fold, and genes involved in ABA signaling and cellular protectionwere up-regulated while photosystem-related genes were down-regulatedduring dehydration. The biochemical and molecular findings suggestthat both constitutive and inducible protective molecules contributeto desiccation tolerance of S. tamariscina.     

Variation of Seed-coat Ornamentation in Striga hermonthica (Scrophulariaceae)

The surface features of the seed of the parasitic floweringplant Striga hermonthica were examined with the scanning electronmicroscope. The details of ornamentation were constant on seedsfrom one plant but varied within and between populations andare probably due to out-breeding. The variation was not relatedto geographical origin or to host-preference. Testa patternssimilar to those reported for other Striga species were foundin the samples. Striga hermonthica, Scrophulariaceae: Rhinanthoideae, hemi-parasite, testa, seed-coat, scanning electron microscope, genetic variation     

A Study of 'Phloeotracheids' in Haustoria of Santalaceous Root Parasites using Scanning Electron Microscopy

Tracheary elements within haustoria of six santalaceous rootparasites have been examined by scanning electron microscopywith particular reference to those cells which contain granules.These cells have been called ‘phloeotracheids’ byearly workers. The granules lie free within the lumen of thecell from which a protoplast is absent at maturity. The granulesoccur in short vessel elements and in imperforate cells whichare thought to represent a specialized type of tracheid. Phloeotracheidsare confirmed in Exocarpus bidwilliiand Mida salicifolia andreported for the first time in Buckleya distichophylla, Comandraumbellata, Nestronia umbellataand Pyrularia pubera. The structureof phloeotracheids shown by scanning electron microscopy isdiscussed in relation to previous studies.     

Mucilage in Cacti: Its Apoplastic Capacitance, Associated Solutes, and Influence on Tissue 5

Mucilage content in the stems of four sympatric cactus speciesvaried from none for Ferocactus acanthodes, 19% by dry weightfor Opuntia basilaris, 26% for Opuntia acanthocarpa, and 35%for Echinocereus engelmannii. Although the mucilage differedchemically among the species (the arabinose content ranged from17% to 51% of the sugar monomers), its relative capacitance(change in relative water content per unit change in water potential)remained about 15 Mpa^–1. The relative capacitance of thewater-storage parenchyma averaged 1·04 Mpa^–1 andwas consistent with the mucilage content, being lowest for F.acanthodes and highest for E. engelmannii. Mucilage isolatedfrom hydrated tissue was accompanied by solutes with an osmoticpressure of about 0·2 MPa. Such associated solutes influencethe water-release characteristics of mucilage and hence itsrole as an apoplastic capacitor. In particular, extracellularsolutes can facilitate the release of appreciable mucilage-boundwater to the cells at tissue water potentials occurring duringthe initial phases of drought. Key words: Echinocereus engelmannii, Ferocactus acanthodes, Opuntia acanthocarpa, Opuntia basilaris, water potential isotherms     

Adaptive Significance of a Small Heat Shock/{alpha}-Crystallin Protein (p26) in Encysted Embryos of the Brine Shrimp, Artemia franciscana

Encysted embryos of the primitive crustacean, Artemia franciscana,are among the most resistant of all animal life history stagesto extremes of environmental stress. This resistance has likelybeen acquired during evolutionary adaptation to their harshecological setting. In the laboratory, for example, some ofthese embryos survive more than 6 years of continuous anoxiawhile fully hydrated and at room temperature, a phenomenal capabilitythat can easily be related to their frequent encounters withanoxic conditions in nature. A small heat shock protein belongingto the -crystallin family of proteins, referred to as p26, appearsto play a central role in the stress resistance of these embryos.Our evidence shows that this protein is an important componentin their adaptive repertoire, and vital for the survival ofArtemia in nature.     

Nanoscale Similarities in the Substructure of the Exines ofFagusPollen Grains andLycopodiumSpores

The exine substructure of an angiosperm,Fagus sylvatica(beech)pollen and a pteridophyte,Lycopodium clavatum(a club moss) sporewas investigated by scanning tunnelling microscopy. These pollenand spores, despite their distinct differences in structureand morphology on a micrometre scale, have very similar substructureon a nanometre scale. The substructure appears to consist ofa multi-helix, i.e. a helical chain in turn wound into a helixwith a self-similarity at smaller and smaller length scales.A simple vectorial form is proposed to describe quantitativelythe multi-helical substructure. Copyright 1998 Annals of BotanyCompany Fagus sylvatica,beech,Lycopodium clavatum, club moss, pollen, spores, exine substructure, scanning tunnelling microscopy.     

The Combined Effects of Desiccation and Irradiance on Mosses from Xeric and Hydric Habitats

The combined effects of desiccation and irradiance on the physiologyof the sand dune moss Tortula ruraliformis (Besch.) Grout andthe minerotrophic flush moss Dicranella palustris (Dicks.) Crundw.ex. E. F. Warb (D. squarrosa (Starke) Schp.) were studied. Damageas a result of desiccation in the dark, measured by loss ofprotein and the relative accumulation of thiobarbituric acid(TBA) reactive products (which gives an estimation of lipidperoxidation), was greater in D. palustris. Desiccation alonehad no effect on the total concentrations of chlorophyll andcarotenoids in either species. Water loss resulted in the cessationof measurable photosynthetic oxygen evolution in both species.Respiration was less sensitive to desiccation than was photosynthesis.A combination of irradiance and water stress prevented any recoveryof photosynthesis during subsequent rehydration in D. palustris,but suppressed recovery only marginally (at the highest irradiance)in T. ruraliformis. The loss of protein, chlorophyll, and carotenoids,and lipid peroxidation were all substantially increased in D.palustris desiccated in the light, but these same conditionsresulted in only minimal damage of T. ruraliformis. Continuousexposure to high irradiance was less deleterious to desiccatedthan hydrated T. ruraliformis. The data are discussed in relationto the habitat preferences of the two species, and also in relationto possible causal factors in the initiation of damage. Key words: Desiccation, mosses, oxidative damage, photo-oxidation     

Regeneration of the Root Cap of Zea mays L. and Pisum sativum L.: A Study with the Scanning Electron Microscope

Removal of the root cap from a root apex initiates regenerationof a new cap. The process has been followed using scanning electronmicroscopy. Quantitative data have been obtained for the growthin area of the exposed acroscopic surface of the quiescent centre(QC) and the increase in volume of the regenerating cap tissue.In Zea the surface of the QC shows an initial rapid increasein area followed by a slower increase. In Pisum the surfacearea increases uniformly, a rapid initial phase being absent.Together with observations on the behaviour of an incision atthe exposed surface, the results indicate that in Zea the capnormally imposes a constraint upon radial growth at the acroscopicsurface of the QC; in Pisum the QC appears not to be so constrained.The different responses may be related to the different arrangementsof cells at the apex of the meristem of these two species. Zea mays, Pisum sativum, maize, peao, scanning electron microscopy, root apex, regeneration     

Seed Development in Podocarpus henkelii: an Ultrastructural and Biochemical Study

Biochemical and fine-structural studies were undertaken on theembryo and female gametophyte of seeds of Podocarpus henkeliiduring the 24-week period of post-fertilization growth and development. Although the d. wt of the embryo and female gametophyte showeda steady increase during development, the levels of sugars,amino acids, proteins and lipids differed between seed partsand showed changes during the four sampling intervals. Onlystarch was seen to increase steadily throughout development.Lipid levels were high in the mature embryo and free amino acidsshowed a steady increase until the seeds were shed. At thisstage seeds were fully hydrated, possessed abundant reservesand all organelles appeared fully functional. This was interpretedas part of the development strategy of neotonous (recalcitrant)seeds, namely, the maintenance of full metabolic competencefor continued growth in the absence of development arrest whichfollows drying in orthodox seeds. Podocarpus henkelii, yellow wood, embryo and endosperm growth, neotonous seed, recalcitrance     

Virulence of Agrobacterium on Larix decidua and Their Cellular Interactions as Depicted by Scanning Electron Microscopy

European larch (Larix decidua Mill.) seedlings were inoculatedwith a number of Agrobacterium strains to screen susceptibilityto infection by agrobacteria. Nine of fourteen Agrobacteriumstrains tested were virulent on this conifer species. The attachmentof virulent Agrobacterium to larch seedling tissues was examinedusing scanning electron microscopy. Electron micrographs showedthe attachment specificity of virulent Agrobacterium to larchcells at wound sites in a manner similar to that described forcells of susceptible dicotyledonous angiosperms, indicatinga host-parasite relationship between oncogenic Agrobacteriumspp. and young seedlings of European larch. These unique electronmicrographs provide the first opportunity to document the compatibleinteractions of Agrobacterium and a conifer at the cellularlevel. Further, the evaluation of tumour formation frequencysuggested that the interactions between Agrobacterium and Europeanlarch were affected by wound site, position of inoculation,age of plant tissues, and time of co-culture. Successful infectionof Agrobacterium resulted in genetic transformation of hostcells. Agrobacterium-mediated DNA transfer and expression of bacterialgenes in larch tissues were confirmed by both Southern blotanalysis and opine assay with the transformed tissues. Key words: Agrobacterium, Larix decidua, cellular interaction, transformation, DNA transfer, scanning electron microscopy