Vito S. Polito

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy, immunofluorescence, and rhodamine-phalloidin

SummaryThe structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.

Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation

Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants.

Spatial and temporal organization of actin during hydration, activation, and germination of pollen inPyrus communis L.: a population study

SummaryDynamics of F-actin organization during activation and germination ofPyrus communis (pear) pollen was examined using rhodaminephalloidin. Prior to activation, the rhodamine-phalloidin labelling pattern appeared as circular profiles in the peripheral cytoplasm of the vegetative cell and as coarse granules around the vegetative nucleus. In activated pollen, parallel arrays of cortical F-actin were aligned circumferentially, along the polar axis in non-apertural areas of the pollen grain, and at 45° to 90° to the polar axis beneath the apertures. Some pollen also showed fluorescent granules or fusiform bodies dispersed throughout the cytoplasm, but as the number of such pollen diminished with prolonged incubation, these are being considered as intermediate patterns. In later stages, the filaments became organized as interapertural bundles traversing the three apertures. However, prior to emergence of the pollen tube, labelling became confined to a single aperture. In germinated pollen grains, actin microfilaments are aligned more or less axially with respect to the axis of the developing pollen tube.The granular labelling pattern seen around the vegetative nucleus prior to pollen activation also became clearly filamentous with pollen activation; this filamentous pattern persisted until germination when it was replaced by cables that aligned longitudinally with respect to the emerging tube axis.The results demonstrate that the organization of actin undergoes considerable changes in the period preceding pollen germination and that microfilament polarization is achieved before pollen germination.

Response to chilling stress in plant cells II. Redistribution of intracellular calcium

SummaryWe have investigated the possibility that the rapid low temperature effects upon cyclosis and subcelluar structure might be due to a breakdown in compartmentation of intracellular calcium, leading to an increase in cytoplasmic Ca2+. Changes in fluorescence of chlortetracycline (CTC), a probe for membrane-bound Ca2+ were monitored in the corners of individual trichome cells (effective spot size ca. 800 square microns) with the aid of a Zeiss epifluorescence microscope linked to a Zeiss Zonax analyzing system. A consistent decrease in signal was observed as cells of chilling-sensitiveLycopersicon esculentum Mill. (cv.Ace) were cooled below their threshold temperature for chilling sensitivity. On rewarming, as the temperature rose above the chilling threshold, there was an increase in fluorescent signal. In contrast, trichomes ofDigitalis purpurea (chilling-resistant) showed no such changes. The uncoupling agent, CCCP, and the Ca2+-chelator, EGTA, induced marked decreases in the fluorescent signal in cells from both species. A more direct approach to testing the hypothesis was to examine the effect of modulating cytoplasmic Ca2+ with the aid of the Ca2+ -ionophore A 23187 and a Ca2+ concentration series in EGTA buffer. Above 10−8 M external free Ca2+, streaming began to be inhibited, full inhibition occurring at 5 x 10−6M Ca2+. The strand structure started to disappear when the Ca2+ rose above 10−7M. Disappearance of strands was accompanied by an increase in the number of cells with vesiculated cytoplasm, an effect analogous to that of chilling temperatures on these cells. The phenothiazines, trifluoperazine and chlorpromazine (10−5M) had similar effects. However but such effects were not seen with R 24571 or N(6-aminohexyl)5-chloro-1-napthalenesulfonamide (W 7) until concentrations were reached that orders of magnitude above their IC50 for calmodulin.

Membrane-associated calcium during pollen grain germination: a microfluorometric analysis

SummaryPear (Pyrus communis L.) pollen was germinated and grown in hanging drop cultures containing phenothiazine drugs, trifluoperazine and chlorpromazine, potent inhibitors of the Ca2+-calmodulin complex. Responses for the two drugs were similar: at 1.0–2 10.0 ΜM pollen germination and tube growth were inhibited. Inhibition of tube growth was not uniform; at 10.0 ΜM growth of about half of those tubes which had germinated was inhibited while the remaining half of the population grew normally. This bimodal distribution of tube growth was noted, but to a lesser extent, at lower concentrations of the drugs as well. Microfluorometric analysis of membrane calcium using chlorotetracycline, a fluorescent chelate probe for membrane calcium, revealed that upon pollen grain hydration there was a sharp decrease in the concentration of calcium associated with membranes and that membrane calcium became localized primarily at the periphery of the newly hydrated pollen grain. Prior to germination there was a reloading of calcium onto membranes in the region of the germination aperture through which the pollen tube would emerge. The release of Ca2+ from membrane sites upon hydration was partially inhibited by treatment with 10 ΜM trifluoperazine. Distribution of membrane calcium in populations of the inhibitor-treated pollen grains was not bimodal however; approximately half of the population had CTC-fluorescence emissions exceeding the maximum value found in the control population. These results suggest that there is a maximum concentration of membrane-associated calcium consistent with normal pollen germination and tube growth and that phenothiazines interfere with the unloading of Ca2+ from membrane sites.

In vitro germination and storage of English walnut pollen

Abstract A method for in vitro germination and tube growth of English walnut ( Juglans regia L.) pollen is described. The medium contained 20% sucrose, 1.0 mM CaCl 2 and 0.16 mM boric acid solidified with 0.65% agar, and was suitable for freshly collected pollen from each of the 21 clones tested. Pollen collection and storage conditions were evaluated using pollen germination on this medium as an indicator. There was considerable variation in results among the clones examined, but in general the results indicated that pollen collection should be made when catkins begin to shed pollen in the field; germination of pollen from catkins forced in the laboratory was generally low. The ability of pollen to germinate declined rapidly when held at ambient conditions. Storage in closed vials at −20°C resulted in complete loss of pollen germination ability by 3 months for some clones and by 8 months for all clones tested. When the relative humidity of the −20°C storage environment was maintained near 30%, germinability was retained, although at reduced levels, for up to 1 year. Cryopreservation using liquid nitrogen (−196°C) was satisfactory for pollen of 2 cultivars which retained the ability to germinate and grow in vitro, but not for 2 others which did not.

Differential cold sensitivity of pollen grain germination in two Prunus species

SummaryPollen grains from selected cutivars of almond [Prunus dulcis (Mill.) D. A. Webb] and peach (Prunus persicaBatsch L.) were exposed to a range of temperatures between 1°C and 34°C on a thermogradient plate. Pollen germination at temperatures below 9°C was conspicuously greater in almond than peach. Miximal germination percentages were attained at about 16°C (almond) and 23°C (peach). The two species did not differ in their capacity for pollen tube elongation over a broad range of temperatures. Maximal pollen tube elongation occurred at temperatures 5°C to 8°C higher than maximal pollen germination. Species affiliation appeared to be of much greater consequence than chilling requirement or bloom date of the sporophyte in predicting gametophytic response to temperature.

Initiation and development of pistillate flowers in Actinidia chinensis

Abstract Initiation and development of pistillate flowers in Actinidia chinensis Planch, cultivar ‘Hayward’ (kiwifruit, Chinese gooseberry) was followed using light and scanning electron microscopy. Results indicate that dormant buds contain undifferentiated primordia in the leaf axils. These primordia remain undifferentiated until shortly before bud break, approximately two months before full bloom, when the primordia become trilobed as bracts and lateral flower primordia are initiated. The lateral flowers often abort in this cultivar. Acropetal development of the terminal flower proceeds rapidly. Five to seven sepals are initiated, followed by a whorl of five to seven petals. Stamen initiation is centripetal and the anthers develop to produce inviable pollen. The central portion of the floral apex is converted into a large compound pistil with numerous hollow styles, each terminated by an open stigma.

Genetic diversity of Pistachio (Pistacia vera, Anacardiaceae) Germplasm based on Randomly Amplified Polymorphic DNA (RAPD) markers

We used Randomly Amplified Polymorphic DNA (RAPD) markers to examine patterns of relatedness among 29 pistachio (Pistacia vera L.) cultivars and accessions. These included 13 cultivars that we had previously described, and an additional 16 items from the USDA National Clonal Germplasm Repository/Davis comprising cultivars and land races originating further east of the cultivars described previously, and material from wild P. vera stands in or near the putative center of origin for pistachio in South Central Asia. The results show high levels of polymorphism in the species emphasizing the importance of preservation of the remaining wild stands of P. vera. Analyses support the concept that cultivars in use west of the Zagros-Caucasus ranges likely originate from a limited germplasm base. The newly examined cultivated material shows greater genetic diversity, consistent with the hypothesis that pistachio cultivation originated in or near South Central Asia. Results also indicate that for at least two cases, material identified differently in two collections are the same clones, thus illustrating the value of molecular marker techniques in describing and maintaining germplasm collections for clonally propagated species.RésuméEn este trabajo se ha empleado la técnica del ADN polimórfico amplificado al azar (RAPD) para examinar la similitud genética entre 29 cultivares y accesiones de pistachero (Pistacia vera L). Este material incluye 13 cultivares considerados en un trabajo anterior y 16 nuevas accesiones del Banco Nacional de Germoplasma Clonal del USDA en Davis que incluyen cultivares y razas locales de regiones más orientales que las consideradas anteriormente así como material procedente de basques naturales de P. vera situados en las proximidades del presunto centro de origen del pistachero. Los resultados obtenidos muestran un alto grado de polimorfismo, lo que indica la necesidad de conservar el germoplasma de P. vera todavía existente en estado natural. Además se confirma la hipótesis de que los cultivares procedentes del oeste de la zona Caucásica y del Zagros se originaron a partir de una base genética limitada. El nuevo material estudiado muestra una mayor diversidad genética lo que corrobora la idea de que el cultivo del pistachero se inició en Asia Central. Al menos en dos de los casos estudiados, el material identificado en dos colecciones diferentes como distintos genotipos, en realidad se trata del mismo clon, lo que demuestra la utilidad de los marcadores moleculares en la descripción y mantenimiento de colecciones de germoplasma en especies de reproducción vegetativa.

In dry pear (Pyrus communis L.) pollen, membranes assume a tightly packed multilamellate aspect that disappears rapidly upon hydration

SummaryUltrastructural details of dry (7% moisture content) and hydratedPyrus communis L. pollen are revealed following freezesubstitution preparation for electron microscopy. Dry pollen is characterized by tightly packed, multilamellate membranous profiles found in association with plasma membrane, vesicles, ER, dictyosomes and some double-membrane bound organelles. Dry pollen also shows unit-membrane bound, densely osmiophilic bodies often with tightly packed multilamellations contained within and, at times, in their bounding membranes. These features are not evident in hydrated pollen. Results suggest that multilamellate membranes form as the plasma membrane, vesicles, ER, and double-membrane bound organelles undergo dehydration, and that upon hydration they rapidly resume normal unilamellate structure.