Influence of Flooding on Net CO2Assimilation, Growth and Stem Anatomy of Annona Species

A series of experiments was conducted to assess net CO₂assimilationand growth responses to waterlogging of grafted and seedlingtrees in the genus Annona. Seedlings of A. glabra, A. muricataandA. squamosa L., and scions of ‘Gefner’ atemoya(A. squamosaxA. cherimola Mill.), ‘49-11’ (‘Gefner’atemoyaxA. reticulata L.), ‘4-5’ (‘Priestley’atemoyaxA. reticulata), A. reticulata grafted onto either A.glabra, A. reticulata orA. squamosa rootstocks were floodedfor up to 60 d. Soil anaerobiosis occurred on the third dayof flooding. Seedlings ofA. glabra and A. muricata, and thescions ‘49-11’, ‘Gefner’ atemoya, andA. reticulata grafted onto A. glabra rootstock were consideredflood tolerant based on their ability to survive and grow inflooded conditions. Scions of the normally flood-sensitive A.reticulata, ‘Gefner’ atemoya, and ‘49-11’tolerated root waterlogging when grafted onto the flood-tolerantspecies, A. glabra. In contrast, flooding of A. squamosa seedlingsand rootstocks, and A. reticulata rootstocks greatly reducedgrowth and net CO₂assimilation rates, and resulted in 20–80%tree mortality. Stem anatomical responses to long-term flooding(12 continuous months) were assessed in seedlings of A. glabraand A. muricata, and trees of ‘49-11’ grafted ontoA. glabra. Flooded trees developed hypertrophied stem lenticels,particularly in A. glabra, and enlarged xylem cells resultingin thicker stems with reduced xylem density. Flooding did notincrease air spaces in pre-existing xylem near the pith or inxylem tissue that was formed during flooding. Thus, flood tolerancedid not involve aerenchyma formation in the stem. Copyright1999 Annals of Botany Company Flood tolerance, net CO₂assimilation, photosynthesis, stem anatomy, shoot growth, anaerobiosis, Annonaceae.     

Regulation of Organ Formation by Cytokinin and Auxin in Lilium Bulbscales Grown In Vitro

Regulation of organ formation by cytokinin and auxin was investigatedin vitro using Lilium auratum Lindl. (wild species habituatedin Japan) and Lilium speciosum Thunb. cv. "Uchida". The interactionof -naphthylacetic acid (NAA) and kinetin on bulbscale or rootdifferentiation was examined. NAA and kinetin showed mainlyindividual but also some synergistic effects. The effects ofbenzyladenine (BA) and kinetin were compared and the resultindicated that BA has a stronger physiological effect on organformation than kinetin and that their effects on Lilium auratumand Lilium speciosum were BA or kinetin-specific. The actionof kinetin on Lilium was affected by sucrose concentration andthe strength of the Murashige and Skoog medium (MS medium),and thus their high concentrations inhibited the kinetin-inducedbulbscale differentiation. Furthermore, a high sucrose levelnegated the kinetin inhibition of root formation, while highMS medium strength in itself inhibited root formation. Morphologicalobservation of bulbscale differentiation induced under a highkinetin level revealed that the new-formed structures are homologousto normally grown bulbs in soil in spite of their particularfeatures. (Received August 3, 1981; Accepted November 2, 1981)     

CYTOGENETIC STUDIES IN THE GENUS TRIBOLIUM (POACEAE: ARUNDINEAE)

The genus Tribolium Desv. consists of nine species, i.e., T. utriculosum (Nees) Renv., T. ciliare (Stapf) Renv., T. echinatum (Thunb.) Desv., T. hispidum (Thunb.) Desv., T. acutiflorum (Nees) Renv., T. obliterum sensu Davidse, T. glomeratum sensu Davidse, T. uniolae (L.f.) Renv., and T. brachystachyum (Nees) Renv. The genus has a basic chromosome number of 6, and from diploid to hexaploid specimens have been examined. Precocious segregation of metaphase I bivalents were observed in four species. Multivalent formation results in unequal chromosome segregation during anaphase I, and several cells with an 11–13 chromosome distribution have been observed. The presence of univalents and anaphase I bridges in all T. brachystachyum specimens suggests a hybrid origin for the species. B-chromosomes were present in specimens from four species. The B-chromosomes are similar to the euchromosomes with the exception that they do not participate in meiosis. The B-chromosomes have a possible isochromosome origin. The cytogenetic evidence presented supports the combination of Plagiochloa and Lasiochloa into Tribolium and indicates that the genus is closely related to Urochlaena, whereas it is not closely related to Prionanthium.     

Electron microscopy of malachite green--glutaraldehyde fixed bacteria.

Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revaling lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.     

Electron Microscopy of Malachite Green— Glutaraldehyde Fixed Bacteria

Malachite green combined with glutaraldehyde has been used recently as a fixative for preserving and revealing lipid complexes in thin sections of eukaryotic cells examined by electron microscopy. When bacteria were prefixed with the above mixture granular electron dense inclusions were revealed in all cultures tested. These inclusions were replaced by electron transparent areas in cells fixed with glutaraldehyde alone. The structures were frequently located near to or within the nucleoid and adjacent to the cell membrane in Gram-negative bacteria and were associated with the nucleoid and mesosomes in Gram-positive species. Polyhydroxybutyrate granules, generally poorly preserved in thin sections of Aquaspirillum serpens, were well preserved by the malachite green-glutaraldehyde fixative. Malachite green complexes were observed outside of the cells in all preparations. Capsules were neither preserved nor stained.     

Effect of estrogen on the affinity of malachite green for staining cardiac lipid inclusions in mice

The effect of estradiol-17-beta on lipids of the ventricular myocardium of mice has been studied with a cytochemical technique in which malachite green was added to glutaraldehyde. This malachite green-glutaraldehyde fixative enhances the visualization of certain phospholipid-related elements. Estrogen induces an affinity of ventricular cardiac lipid inclusions for the cationic dye malachite green. The staining affinity is evidenced only in the estrous female, not in diestrus. In oophorectomized animals, malachite green staining is seen only following estradiol injection, but this effect is blocked by progesterone. In the male, ventricular lipids do not stain, nor do they develop malachite green affinity with estrogen stimulation. These results imply a blockade of the estradiol-mediated dye affinity by progesterone and testosterone. This reinforces the concept of the heart as a target organ for sex steroids and expands the previously described estrogen effects on myocardium.     

Malachite Green: Applications in Electron Microscopy

Incorporation of malachite green into a glutaraldehyde fixative results in enhanced staining of a number of cellular elements. Ribosomes and myofilaments exhibit increased electron density, but cell membranes generally are not stained. In certain tissues, lipid inclusions are uniformly and heavily stained. Other populations of lipid droplets exhibit differential affinity for malachite green, facilitating their division into subclasses. In addition to its function as a dye, malachite green has previously been reported to stabilize lipid elements soluble in aqueous glutaraldehyde. Such a component was observed in the stroma of uterine endometrium. The variety of cell components which exhibit increased contrast after preparation with malachite green suggests that this technique may find widespread application in fine structure studies.     

Malachite green: applications in electron microscopy.

Incorporation of malachite green into a glutaraldehyde fixative results in enhanced staining of a number of cellular elements. Ribosomes and myofilaments exhibit increased electron density, but cell membranes generally are not stained. In certain tissues, lipid inclusions are uniformly and heavily stained. Other populations of lipid droplets exhibit differential affinity for malachite green, facilitating their division into subclasses. In addition to its function as a dye, malachite green has previously been reported to stabilize lipid elements soluble in aqueous glutaraldehyde. Such a component was observed in the stroma of uterine endometrium. The variety of cell components which exhibit increased contrast after preparation with malachite green suggests that this technique may find widespread application in fine structure studies.     

Ultrastructure and Development of Oil Idioblasts in Annona muricata L.

The ultrastructure and development of oil idioblasts in theshoot apex and leaves in Annona muricata L. are described, andthree arbitrary developmental stages are distinguished: cellsin which no additional cell wall layers have been depositedagainst the initial primary cell wall, possessing an electron-translucentcytoplasm and distinct plastids which lack thylakoids (stage1); cells in which a suberized layer has been deposited againstthe primary wall (stage 2, the cytoplasm resembles that of thepreceding stage), and cells in which an additional inner walllayer has been deposited against the suberized layer, whichincreases in thickness with development (stage 3). In this stagean oil cavity is formed, surrounded by the plasmalemma, andattached to a bell-like protrusion of the inner wall layer,the cupule. A complex membranous structure occurs next to thecupule. Smooth tubular endoplasmic reticulum (ER), appearingas linearly arranged tubules, and groups of crystalline bodieswith an almost hexagonal outline are present. The final stagewas further subdivided into three subgroups (a, b, c) basedon the extent of the oil cavity, its contents, and the compositionof the cytoplasm, and increasing thickness of the inner walllayer. The oil is probably synthesized in the plastids, releasedinto the cytoplasm, and then passed through the plasmalemmasurrounding the oil cavity. Oil idioblasts, Annona muricata L., suberized layer, inner wall layer, oil cavity, cupule, smooth tubular ER, crystalline bodies     

Flower and Volatile Oil Ontogeny in Boronia megastigma

The volatile oils of flowers of Boronia megastigma Nees. areimportant commercially but little has been published regardingtheir localization or the dynamics of their synthesis. In thisstudy we examined the histochemistry, anatomy and volatile oilcomposition of the floral organs of plants from a native populationby SEM. Lysigenous glands occurred in all organs except the stigma andandroecium and were associated with the presence of - and ß-pineneand limonene in tissue extracts. These compounds increased inconcentration throughout fruit development. Petals, stamens,staminodes and stigmas were glandular and contained phenolicdeposits, a dense cytoplasm and prominent intercellular spaces.Extracts of these tissues contained dodecanol, ß-iononeand dodecyl acetate, the concentrations of which were greatestat the time of stigma receptivity. The location and timing oftheir synthesis together with their biological activity suggeststhat these volatile oils may have a role in pollination.Copyright1995, 1999 Academic Press Boronia megastigma Nees., Rutaceae, essential oils, volatile oils, reproductive biology, -pinene, ß-pinene, limonene, ß-ionone, dodecyl acetate, heptadecene     

A Test for the Resource Remobilization Hypothesis: Tree Sprouting using Carbohydrates from Above-ground Parts

To test the resource remobilization hypothesis, i.e. the hypothesisthat some trees sprout from root-collars or from the lower partof trunks using resources obtained from above-ground parts ratherthan from resources reserved in their roots, we conducted cuttingexperiments forEuptelea polyandra, a frequently sprouting treespecies with little carbohydrate reserves in its roots,Quercusserrata,a frequently sprouting tree species with large reservesin the roots, andMallotus japonicus, a rarely sprouting treespecies. Trees of each species were cut down in winter leavingtwo kinds of stumps, those approx. 1.5 m in height and thosecut off near the ground. The number and total dry weight ofnewly sprouted shoots per stump were compared between the twotreatments and among the three species at the end of the followinggrowing season. InE. polyandra,both the number and total dryweight of sprouts per stump were very small for both treatmentsand were similar to, or less than, those ofM. japonicus. Onthe other hand,Q. serratasprouted abundantly in both treatments.These results indicate thatE. polyandracannot sprout sufficientlywithout a considerably large volume of above-ground parts orthat additional structures such as foliage and branches maybe necessary for sprouting. We conclude that the resource remobilizationhypothesis is supported for this species.Copyright 1998 Annalsof Botany Company Euptelea polyandraSieb. et Zacc,Quercus serrataThunb,Mallotus japonicus(Thunb.) Muell. Arg., tree sprouting, cutting experiment, resprouter, resource movement, carbohydrate allocation, ground-surface disturbance, root stock, resource remobilization hypothesis.     

Further Investigations of Liposoluble Fluorescent Compounds in Senescing Plant Cells

The accumulation of liposoluble fluorescent compounds with excitationand emission fluorescent maxima in the bands of 350–370nm and 410–440 nm respectively has been observed in thedegrading (the late stationary phase) blue-green algae Anabaenavariabilis K?tz and Anabaena cylindrica Lemm. cells, in thedark-senescing cotyledons from Phaseolus vulgaris L., and inmore than 1-year-old leaves of evergreen plants (Ligustrum japonicumThunb. and Osmanthus fortunei Carr.)- Spectral and chromatographicproperties of the compounds are rather similar to those previouslydescribed in the cells of other senescing plant species. Therole of lipid peroxidation in the formation of fluorescent pigmentsand in the ageing of plant cells is discussed. Key words: Fluorescent pigment, Anabaena sp, Senescence     

Particle beam liquid chromatography-mass spectrometry of triphenylmethane dyes: application to confirmation of malachite green in incurred catfish tissue

Eight triphenylmethane dyes (malachite green, leucomalachite green, gentian violet, leucogentian violet, brilliant green, pentamethyl gentian violet, N′,N′-tetramethyl gentian violet and N′,N″-tetramethyl gentian violet) have been characterized by particle beam liquid chromatography-mass spectrometry. The electron ionization spectra obtained of these dyes by this technique exhibit similar fragmentation, with the formation of phenyl and substituted phenyl radicals, and loss of alkyl groups from the amines. It was observed that the six cationic dyes are reduced in the mass spectrometer source to form the corresponding leuco compounds. This technique was evaluated for the confirmation of malachite green and leucomalachite green in incurred catfish (Ictalurus punctatus) muscle tissue.     

The Galls on Forsythia intermedia Zab.

The galls on Forsythia intermedia Zab. consist of much-branched,root-like structures embedded in corky material which appearsto be sloughed-off from them. These gall ‘roots’have 7 to 14 vascular bundles compared with the 5 of normalroots and contain much more IAA. When galls were incubated in damp chambers the gall ‘roots’extended their growth. These extensions had a root-cap and root-hairsbut also had more vascular bundles than normal and they didnot respond geotropically like normal roots. Two fungi, Gibberella baccata (Wallr.) Sacc. (conid, stat. Fusarlumlateritium Nees.) and Phomopsis dominici Trav. were associatedwith die-back of gall ‘roots’ and two bacteria resemblingCorynebacterium fascians (Til.) Dowson and Agrobacterium tumefaciens(Sm. and Towns.) Conn respectively were isolated from galls.All produced IAA in culture media but their role in the etiologyof the gall, if any, remains in doubt.     

Fatty acids,resin acids and phenols in Pinus muricata

Acetone-soluble extractives of “blue” and “green” strain Pinus muricata D. Don were found to consist of free and “combined” fatty acids, resin acids, and phenols. The composition of the extractives from the two strains was similar though “green” strain P. muricata contained more Δ^8(9),15 isopimaric acid than “blue” strain. This difference may be used to identify these muricata strains if the age of the wood precludes a monoterpene examination.     

Isolation of a malachite green-degrading Pseudomonas sp. MDB-1 strain and cloning of the tmr2 gene

The release of malachite green, a commonly used triphenylmethane dye, into the environment is causing increasing concern due to its toxicity, mutagenicity, and carcinogenicity. A bacterial strain that could degrade malachite green was isolated from the water of an aquatic hatchery. It was identified as a Pseudomonas sp. based on the morphological, physiological, and biochemical characteristics, as well as the analysis of 16S rRNA gene sequence and designated as MDB-1. This strain was capable of degrading both malachite green and leucomalachite green, as well as other triphenylmethane dyes including Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase from MDB-1, was cloned, sequenced and effectively expressed in E. coli. These results highlight the potential of this bacterium for the bioremediation of aquatic environments contaminated by malachite green.     

Floral Development of Three Maesa Species, with Special Emphasis on the Position of the Genus within Primulales

Floral development of three Maesa Forssk. species, M. argenteaWall., M. japonica(Thunb.) Moritzi ex Zoll. and M. perlarius(Lour.)Merr., was studied with a scanning electron microscope. Thedistinctive floral features of the genus, such as presence oftwo persistent bracteoles, perigynous flowers and many-seededfruits, have been used to discuss the systematic position ofthe genus within Primulales. In addition, the development ofcommon stamen-petal primordia, the number, arrangement and positionof ovules and the shape of the anthers provide evidence in favourof placing Maesa in a separate family, Maesaceae, as suggestedby Anderberg and Ståhl (Canadian Journal of Botany, 73:1699–1730, 1995). Copyright 2000 Annals of Botany Company Primulales, Maesa argentea Wall., Maesa japonica(Thunb.) Moritzi ex Zoll., Maesa perlarius(Lour.) Merr., floral ontogeny, SEM     

A comparison of fixatives for the estimation of abundance and biovolume of marine planktonic ciliate populations

The effects of five treatments with four commonly used fixativeson the abundance and cell volume of marine planktonic ciliateswere investigated on a natural community from Plymouth Sound.The fixative treatments were 5% and 9% Bouin's solution, 0.4%acid Lugol's iodine, 1% buffered formaldehyde and 1% glutaraldehyde.The abundance of the aloricate ciliate community varied accordingto fixative treatment, with Lugol's maintaining the greatestcell numbers, followed by Bouin's, glutaraldehyde and formaldehyde.The effects of the fixatives on the abundance of the two mostcommon aloricate taxa, Balanion sp. and Strombidium epidemumwere similar to, and components of, those for the aloricatepopulation as a whole. However, there was little differencebetween the effects of different fixatives on the abundanceof the tintinnid community, or on the abundance of the mostcommon tintinnid, Tintinnopsis nana. Mean cell volumes of Balanionsp., S.epidemum and T.nana were greatest in samples fixed informaldehyde, followed by glutaraldehyde, Lugol's and Bouin's.The mean population volume of the aloricate and tintinnid populationsgenerally reflected this trend with greatest values recordedin formaldehyde- and glutaraldehyde-fixed samples, followedby Lugol's and Bouin's; however, the differential effects ofthe fixatives were not as great. Lugol's therefore appears tobe the most effective fixative tested for the estimation ofciliate abundance and population volume, the latter being afunction of abundance and cell volume. ³Present address: British Antarctic Survey, High Cross, MadingleyRoad, Cambridge CB3 OET, UK     

Studies on the Development of the Air Pores and Air Chambers of Marchantia paleacea: 1. Light Microscopy

The ontogeny of the air pores and air chambers of Marchantiapaleacea begins with the schizogenous development of protodermalintercellular spaces of the initial apertures, and is completedwith the formation of the air pores and giant sub-epidermalair chambers bearing numerous photosynthetic filaments. Intercellularspace formation commences from the thallus surface and proceedsinwards to the first internal layer of cells. The cells amongwhich spaces develop do not originate from one mother cell.Spaces are formed only in the regions of the intersection ofthe anticlinal walls of three, four, or sometimes more successivederivatives of S₁, S₃ and S₄ segments of the apical cell, oneor two of which have been divided periclinally and the restanticlinally. Protodermal intercellular spaces appear in mostor all the corners of these cells, the anticlinal walls of whichexhibit an opposite disposition. The S₁, S₂, S₃ and S₄ segmentsare produced by definite divisions of a five-sided apical celland by a series of divisions give rise to initial cells of theinternal layers of the thallus and initial cells of the protodermaland sub-protodermal layers. The concept of a quiescent apicalcell cannot be accepted, since dividing apical cells have beenobserved, and the pattern of wall disposition of the thallusapex cannot be explained without the active participation ofthe apical cell. The air chambers are apparently of exogenous origin. They resultfrom the broadening of the bottom of the initial apertures bythe coordination of the rate of anticlinal divisions and growthof the protodermal and sub-protodermal cells surrounding theintercellular spaces of the initial apertures. The ontogenyof the pore rings starts at an advanced stage of air chamberformation not from a mother cell but from the cells which surroundthe closed entrance of the air chamber, by a shift of the planeof division from anticlinal to periclinal. Before the periclinaldivisions a new axis of growth perpendicular to the thallussurface is established in the mother cells of the pore. By a polarized growth into the air chamber followed by periclinaldivisions, the cells of the floor form initial cells of thephotosynthetic filaments. The latter divide again to form singleor branched photosynthetic filaments. Marchantia paleacea, air pore, air chamber     

Effects of the Over-Expression of the rolC Gene on Leaf Development in Transgenic Periclinal Chimeric Plants

Using Agrobacterium tumefaciens harboring a vector that carriedthe rolC gene under the control of the 35S RNA promoter of cauliflowermosaic virus, we produced several transgenic plants. Two ofthem were periclinal chimeras with altered leaves that had wrinkleddark green margins and inner pale green regions. One chimericplant had shortened internodes, reduced apical dominance, smallflowers and exhibited male sterility, but the other had a normalphenotype. Analysis of proteins, RNA and DNA indicated thatthe inner pale green tissues consisted of transformed cellswhile the outer dark green tissues were composed of non-transformedcells. Histological analysis indicated that mesophyll cellswere distorted and larger intercellular spaces were presentin the transformed pale green regions. Furthermore, in youngleaves, transformed mesophyll cells were larger than non-transformedcells. However, the normal parts had larger numbers of cellsper unit area than the transformed parts. These observationssuggest that ths expression of 35S-rolC in leaves caused inhibitionof cell division in developing leaves and that the undulatingmargins, composed of non-transformed cells, were a consequenceof the requirement for accommodating more cells in less spacewithin the region of rolC-transformed cells. (Received January 29, 1993; Accepted May 17, 1993)