Formation of multiplex lamellae by equilibrium slow freezing of cortical parenchyma cells of mulberry and its possible relationship to freezing tolerance

Summary Cortical parenchyma cells of mulberry (Morus bombycis Koidz. cv. Goroji) become extremely cold hardy in winter and can tolerate equilibrium freezing below –30 °C and subsequent immersion into liquid nitrogen. We show in this ultrastructural study that, in these extremely cold hardy cortical parenchyma cells of mulberry collected in winter, initiation of freezing at –5 °C resulted in the formation of multiplex lamellae (MPL) that completely covered the area beneath the plasma membrane. The MPL were produced by fusion of pre-existing vesicular endoplasmic reticulum (ER), via a reticular ER network. The completed MPL were composed of a parallel array of sheet-like ER cisternae. This structural reorganization of the ER was completed within 10 min upon freezing at –5 °C and was quickly reversed upon thawing. The same structural reorganization of the ER was produced by osmotic dehydration of the cortical tissues with a 2.7 osmol sorbitol solution at 20 °C. Thus, the structural reorganization of the ER upon freezing was, in fact, produced by dehydration. In winter samples, the formation of MPL with the initiation of freezing completely inhibited close apposition of membranes upon deep freezing that has been reported to be a cause of freezing injury via the production of ultrastructural changes in the plasma membrane. Similar but more or less incomplete MPL were produced by freezing or osmotic dehydration in cortical parenchyma cells collected in spring and autumn, and these MPL partly inhibited close apposition of membranes. MPL were not produced in the cells of mulberry collected in summer and close apposition of membranes occurred upon deep freezing. We speculate that the formation of MPL with the initiation of freezing might play a specific role in inhibiting the close apposition of membranes due to the specific nature of the cisternal membranes and might, consequently, be responsible for the high freezing tolerance of winter cells.     

Changes in leaf ultrastructure and carbohydrates inArabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation

Summary We studied cell ultrastructure and carbohydrate levels in the leaf tissue ofArabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation. Freezing tolerance of the leaves from 26 day old plants was determined after 48 h and 10 days at 4°C. Acclimation treatment of 48 h decreased the lethal freezing temperature from –5.7°C to –9.4°C. Freezing tolerance was not altered further by acclimation at 4 °C for 10 days. Ultrastructural changes in the parenchyma cells were evident after 6 to 24 h of cold acclimation. The plasma membrane showed signs of extensive turnover. Evidence of membrane invaginations and sequestering of membrane material was observed. In addition, numerous microvesicles, paramural bodies, and fragments of endoplasmic reticulum were noticed in the vicinity of plasma membrane. Modifications in the structure of cell membranes were evident after 5 days of exposure to low temperature. Small, darkly stained globules were seen on the plasma membrane, tonoplast, chloroplast envelope membrane, mitochondrion outer membrane, dictyosome cisternae membrane, and microvesicle membrane. As far as we are aware, this type of membrane modification has not been described previously in plant cells exposed to low temperature. We propose to call these structures membraglobuli. Acclimation treatment also increased the concentrations of soluble sugars and starch. These observations suggest that cold acclimation inA. thaliana induces changes in both plasma membrane properties and carbohydrate composition.     

Water relations and mucopolysaccharide increases for a winter hardy cactus during acclimation to subzero temperatures

Summary Thickness, relative water content (RWC), osmotic pressure, water potential isotherms, and mucopolysaccharide content were measured for the photosynthetic chlorenchyma and the water-storage parenchyma of the winter hardy cactus, Opuntia humifusa, after shifting from day/night air temperatures of 25° C/15° C to 5° C/–5° C. After 14 d at 5° C/–5° C, the average fraction of water contained in the symplast decreased from 0.92 to 0.78, the water potential of saturated (fully hydrated) tissue was essentially unchanged, but the osmotic pressure of saturated tissue decreased (by 0.15 MPa for the chlorenchyma and 0.12 MPa for the water-storage parenchyma). After 7 weeks at 5° C/–5° C, tissue thickness was reduced by 61% for the chlorenchyma and 65% for the water-storage parenchyma, and the RWC decreased by 42% and 68%, respectively; these changes contributed to an osmotic pressure increase of 0.55 MPa for the chlorenchyma and 0.34 MPa for the water-storage parenchyma. During the 7 week acclimation to low temperature, mucopolysaccharide increased by 114% for the chlorenchyma and by 89% for the water-storage parenchyma. The water potential of the extracted mucopolysaccharide was relatively constant for an RWC between 1.00 and 0.30, decreasing abruptly below 0.30. Changes in water relations parameters and in mucopolysaccharide content during low-temperature acclimation may reduce water efflux from the cells, and thus reduce damage due to rapid dehydration during extracellular freezing.     

Ultrastructural changes in suspension culture cells of Panicum maximum during cryopreservation

A Panicum maximum cell suspension was used to study ultrastructural changes during cryopreservation. Pregrowing the cells in mannitol caused reduction in the vacuolar volume by redistribution of the large central vacuole into a number of smaller vesicles. Invaginations were formed in the plasma membrane of the cells, to accommodate the reduced cell volume. Swelling of organelles occurred during different stages of cryopreservation. The cisternae of the endoplasmic reticulum dilated and formed vesicles. Although some damage was apparent, organelles were still recognizable in cells frozen slowly and freeze-fixed at –10°C. The cells were able to repair such damage within two days in culture, and regained their normal appearance. Cells frozen slowly without any cryoprotection, and cells frozen rapidly by direct immersion into liquid nitrogen after cryoprotection, were lethally damaged by destruction of membranous structures. Osmiophilic granules were found along the plasma membrane of lethally damaged cells, indicating that their formation is a consequence of freeze damage, rather than a mechanism to prevent injury.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide     

Loading process of sugars into cabbage petiole and asparagus shoot apex cells by incubation with hypertonic sugar solutions

Summary The freezing tolerance of cabbage petioles and asparagus shoot apexes was increased by preincubation with 0.8 M sugar solutions. In cabbage petioles with an initial freezing tolerance of –3 °C (temperature for 50% cell survival), as determined by both electrolyte leakage and fluorescein diacetate vital staining, the freezing tolerance was increased to –13 °C by incubation with sorbitol solutions for 3 h. In meristematic cells of asparagus shoot apexes with an initial freezing tolerance of –7.5 °C, as determined by fluorescein diacetate vital staining, the freezing tolerance was increased to –30 °C by incubation with 0.8 M sugar solutions for 3 h, although other cells in the shoot apexes were killed by higher freezing temperatures. During incubation of both cabbage petioles and asparagus shoot apexes with sugar solutions, sugars were intracellularly taken up by osmotically induced fluid-phase endocytotic vesicles, as indicated by comovement of Lucifer Yellows carbohydrazide (LYCH) observed with a confocal laser scanning microscope. The amounts of intracellularly taken up sugars increased concomitantly with the formation of endocytotic vesicles depending on the time of incubation in parallel with a gradual increase of freezing tolerance. However, the endocytotic vesicles and their contents were retained not only after prolonged incubation after maximum freezing tolerance had been achieved but also after recovery of these tissue cells to isotonic conditions or after freeze-thawing. These results suggest that although sugars are intracellularly taken up by endocytotic vesicles, they might be sequestered within vesicles, casting doubt on their protective role to the plasma membranes as a main site of freezing injury. The pretreatment with 1 mMp-chloromercuribenzenesulfonic acid (PCMBS), an inhibitor of sugar transport, reduced the amounts of intracellular sugar uptake without affecting the formation of endocytotic vesicles, suggesting that sugars were, at least partly, taken up by sugar transporters. In the pretreatment with PCMBS, the freezing tolerance of incubated tissues with sugar solutions was significantly reduced, although addition of PCMBS per se did not affect survival. These results suggest that sugars taken up by sugar transporters, rather than sugars taken up by endocytotic vesicles, are mainly responsible for the increased freezing tolerance of cabbage petioles and asparagus shoot apexes. Furthermore, we aimed to study the occurrence of fluid-phase endocytosis with LYCH in an isotonic condition. Our results indicated that uptake of LYCH by fluid-phase endocytotic vesicles was not detected microscopically in isotonic condition, although LYCH was spectrofluorimetrically taken up in isotonic condition. Spectrofluorimetric uptake of LYCH was inhibited by addition of probenecid, an anion transport inhibitor. These results suggest that in cabbage petioles and asparagus shoot apexes, LYCH is taken up by anion transport but not by fluid-phase endocytosis in isotonic condition, and uptake of LYCH by fluid-phase endocytosis is restricted to occur only in hypertonic condition.Abbreviations CLSM confocal laser scanning microscope - FDA fluorescein diacetate - LYCH Lucifer Yellow carbohydrazide - PCMSB p-chloromercuribenzenesulfonic acid - T_EL50 temperature at which 50% electrolyte leakage occurred     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.     

Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice

Pieces excised from leaf bases and laminae of seedlings of Triticum aestivum L. cv. Lennox were slowly frozen, using a specially designed apparatus, to temperatures between 2° and 14° C. These treatments ranged from non-damaging to damaging, based on ion-leakage tests to be found in the accompanying report (Pearce and Willison 1985, Planta 163, 304–316). The frozen tissue pieces were then freeze-fixed by rapidly cooling them, via melting Freon, to liquid-nitrogen temperature. The tissue was subsequently prepared for electron microscopy by freeze-etching. Ice crystals formed during slow freezing would tend to be much larger than those formed during subsequent freeze-fixation. Ice crystals surrounding the excised tissues were much larger in the frozen than in the control tissues (the latter rapidly freeze-fixed from room temperature). Large ice crystals were present between cells of frozen laminae and absent from controls. Intercellular spaces were infrequent in control leaf bases and no ice-filled intercellular spaces were found in frozen leaf bases. Intracellular ice crystals were smaller in frozen tissues than in controls. It is concluded that all ice formation before freeze-fixation was extracellular. This extracellular ice was either only extra-tissue (leaf bases), or extra-tissue and intercellular (laminae). Periplasmic ice was sometimes present, in control as well as slowly frozen tissues, and the crystals were always small; thus they were probably formed during freeze-fixation rather than during slow freezing. The plasma membrane sometimes showed imprints of cell-wall microfibrils. These were less abundant in leaf bases at 8° C than in controls, and were present on only a minority of plasma membranes from laminae. Therefore, extracellular ice probably did not compress the cells substantially, and changes in cell size and shape were possibly primarily a result of freezing-induced dehydration. Fine-scale distortions (wrinkles) in the plasma membrane, while absent from controls, were present, although only rarely, in both damaged and non-damaged tissues; they were therefore ice-induced but not directly related to the process of damage.     

Reproductive biology of Stictosiphonia hookeri (Rhodomelaceae,Rhodophyta) from Argentina,Chile, South Africa and Australia in laboratory culture

The red alga Stictosiphonia hookeri is epilithic in shaded habitats of the upper intertidal zone from 30 to 55° S. Thalli of this species from Argentina, Chile, South Africa and Australia, usually without reproductive structures when collected, all developed tetrasporangia in culture. Although good vegetative growth occurred in all nine isolates at 20–25 °C, 12:12 light: dark cycle, 10–30 µmol photons m^–2 s^–1, none reproduced in these conditions except one isolate from Australia. At 15 °C the four South African (34 °S) isolates developed tetrasporangial stichidia, and three completed a Polysiphonia-type life history. Gametophytes were unisexual or bisexual. At 15 °C one isolate from Chile (36 °S) formed tetrasporangia, but sporelings were not viable. At 10 °C isolates from Argentina and Chile (53 °S and 54 °S) formed tetrasporangia; however, only the Chile isolate completed a Polysiphonia-type life history with unisexual gametophytes. The temperature required to induce sporogenesis correlates with the range of water and air temperatures in the natural habitats of each isolate. In irradiances >50 µmol m^–2 s^–1 the thalli became yellow- brown within two weeks because of phycobiliprotein loss, but this did not impair growth or reproduction. The Argentina and Chile isolates were resistant to freezing in seawater for at least two days, showing no cell damage. The protein cuticle of the outer cell wall is repeatedly shed in culture. This may serve to minimize the attachment of epiphytes in the field.     

A freeze-etch study of the effects of extracellular freezing on cellular membranes of wheat

Seedlings of Triticum aestivum L. cv. Lennox were grown in different environments to obtain different hardiness. Pieces of laminae and leaf bases were slowly cooled to sub-zero temperatures and the damage caused was assessed by an ion-leakage method. Comparable pieces of tissue were slowly cooled to temperatures between 2° and-14°C and were then freeze-fixed and freeze-etched. Membranes generally retained their lamellar structures indicated by the abundance of typical membrane fracture faces in all treatments, and some membrane fracture faces had patches which lacked the usual scattering of intramembranous particles (IMP). These IMP-free areas were present in the plasma membrane of tissues given a damaging freezing treatment, but were absent from the plasma membrane of room-temperature controls, of supercooled tissues, and of tissues given a non-damaging freezing treatment. The frequency of IMP-free areas and the proportion of the plasma membrane affected increased with increasing damage. In the most damaged tissue (79% damage; leaf bases exposed to-8°C), 20% of the plasma membrane was IMP-free. The frequencies of IMP at a distance from the IMP-free areas were unaffected by freezing treatments. There was a patchy distribution of IMP in other membranes (nuclear envelope, tonoplast, thylakoids, chloroplast envelope), but only in the nuclear envelope did it appear possible that their occurrence coincided with damage. The IMP-free areas of several membranes were sometimes associated together in stacks. Such membranes lay both to the outside and inside of the plasma membrane, indicating that at least some of the adjacent membrane fragments arose as a result of membrane reorganization induced by the damaging treatment. Occasional views of folded IMP-free plasma membrane tended to confirm this conclusion. The following hypothesis is advanced to explain the damage induced by extracellular freezing. Areas of plasma membrane become free of IMP, probably as a result of the freezing-induced cellular dehydration. The lipids in these IMP-free patches may be in the fluid rather than the gel phase. The formation of these IMP-free patches, especially in the plasma membrane, initiates or involves proliferation and possibly fusion of membranes, and during or following this process, the cells become leaky.Abbreviations EF exoplasmatic fracture face - IMP intramembranous particles - PF protoplasmatic fracture face     

Encapsulation of eggs of the encyrtid wasp, Metaphycus swirskii, by the hemispherical scale, Saissetia coffeae: Effects of host age and rearing temperature

The combined effects of host age and rearing temperature upon the encapsulation of eggs of Metaphycus swirskii Annecke & Mynhardt (Hymenoptera: Encyrtidae) by the hemispherical scale, Saissetia coffeae (Walker) (Homoptera: Coccidae), were determined. Encapsulation incidence at 16, 20, 24 and 28°C increased with the age of the host, being 5.5–19.6% in 3rd instar nymphs, 45.4–74.2% in young female scales and 88.8–100% in preovipositing female scales. Within each host physiological age range, encapsulation frequency was not materially influenced by most of the temperatures. At 32°C, regardless of host age, S. coffeae encapsulated 99.5 to 100% of parasitoid eggs. Complete immunity to successful parasitism by M. swirskii occurred at 20, 24, 28 and 32°C in preovipositing female scales and at 32°C in 3rd-instar nymphs and young female scales of S. coffeae.

---

Résumé Cette étude a déterminé les effets combinés de l'âge de l'hôte et de la température sur l'encapsulation des oeufs de M. swirskii Annecke & Mynhardt (Hym. Encyrtidae) par S. coffeae Walker (Homopt. Coccidae). La fréquence d'encapsulation à 16, 20, 24 et 28°C a augmenté avec l'âge de l'hôte, 5,5–19,6% dans les larves de 3ème stade, 45,4–74,2% chez les jeunes femelles et 88,8–100% chez les femelles avant la ponte. Dans chaque gamme d'âge physiologique de l'hôte, la fréquence d'encapsulation n'est pas influencée par la plupart des températures. A 32°C, quel que soit l'âge de l'hôte, S. coffeae a encapsulé 99,5–100% des oeufs du parasitoïde. Une immunité complète au parasitisme par M. swirskii a été obtenue à 20, 24, 28 et 32°C pour les femelles de cochenilles dans la période précédant la ponte, et à 32°C pour les larves de 3ème stade et les jeunes femelles de S. coffeae.

     

Cheese whey: an alternative growth and protective medium for<Emphasis Type="Italic"> Rhizobium loti</Emphasis> cells

Cheese whey (CW)-based growth medium efficiently protects Rhizobium loti cells during freezing and desiccation and can maintain their growth in a manner similar to that of traditional mannitol-based medium (YEM). The cheese-whey-based medium (CW) improved viability when used to re-suspend cell pellets kept at –20 °C and –80 °C and resulted in the survival of over 90% of the cells. Moreover, bacterial pellets obtained from cells grown in CW withstand desiccation better than cells grown in YEM. Survival was over 60% after 30 days at 4 °C. No differences were observed in nodulation efficiency between YEM-grown and CW-grown cells. Fast protein liquid chromatography (FPLC) protocols are presented for total protein profile analyses of sweet and acid cheese whey.In memoriam of Sylvio Cortina Vicepresident of Fundación COREPRO     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

Freezing injury in cold-acclimated and unhardened spinach leaves

Spinach plants (Spinacia oleracea L.) were frost-hardened by cold-acclimation to 1° C or kept in an unhardy state at 20°/14° C in phytotrons. Detached leaves were exposed to temperatures below 0°C. Rates of photosynthetic CO₂ uptake by the leaves, recorded after frost treatment, served as a measure of freezing injury. Thylakoid membranes were isolated from frost-injured leaves and their photosynthetic activities tested. Ice formation occurred at about-4° to-5° C, both in unhardened and cold-acclimated leaves. After thawing, unhardened leaves appeared severely damaged when they had been exposed to-5° to-8° C. Acclimated leaves were damaged by freezing at temperatures between-10° to-14° C. The pattern of freezing damage was complex and appeared to be identical in hardened and unhardened leaves: 1. Inactivation of photosynthesis and respiration of the leaves occurred almost simultaneously. 2. When the leaves were partly damaged, the rates of photosynthetic electron transport and noncyclic photophosphorylation and the extent of light-induced H⁺ uptake by the isolated thylakoids were lowered at about the same degree. The dark decay of the proton gradient was, however, not stimulated, indicating that the permeability of the membrane to-ward protons and metal cations had not increased. 3. As shown by partial reactions of the electron transport system, freezing of leaves predominantly inhibited the oxygen evolution, but photosystem II and photosystem I-dependent electron transport were also impaired. 4. Damage of the chloroplast envelope was indicated by a decline in the percentage of intact chloroplasts found in preparations from injured leaves. The results are discussed in relation to earlier studies on freezing damage of thylakoid membranes occurring in vitro.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES 2(N-morpholino) ethane sulfonic acid     

Photodynamic Damage to Yeast Subcellular Organelles Induced by Elevated Levels of Endogenous Protoporphyrin IX

The 2,2"-dipyridyl-induced accumulation of protoporphyrin IX in Saccharomyces cerevisiae cells was shown to be accompanied by the photoinhibition of cell respiration and the enhancement of the photoinduced permeability of plasma membranes to the fluorescent dye primuline. The visible-light illumination (at 400–600 nm) of the mitochondria and plasma membranes isolated from yeast cells with a high level of endogenous protoporphyrin IX intensified lipid peroxidation in these subcellular organelles. Comparative studies showed that the rad 52 mutant cells, which are deficient in the postreplicative recombinational DNA repair system, are considerably more sensitive to the inactivating action of visible light than are the wild-type cells and the rad 3 mutant cells, which are deficient in the excision DNA repair system. The contribution of photodynamic damage to the yeast subcellular organelles to the lethal photodynamic effect is discussed.     

Behavioural variations among strains of Trichogramma spp.: Adaptability to field-temperature conditions

In order to select a candidate strain of Trichogramma sp. for inundative releases against lepidopteran pests in cabbage field-crops in the Netherlands, the parasitization activity of a collection of 60 different Trichogramma spp. strains was studied during 2 h and 24 h exposures at 12°C. Activity and parasitism varied significantly among strains and the two characteristics were not correlated, suggesting the action of two differentially temperature-dependent mechanisms influencing both characteristics. Native strains were characterized by a low activity at 12°C, making their usefulness for inundative releases doubtful. In three strains tested at 12, 17, 20, 25 and 30°C, activity increased linearly with temperature, until nearly all females parasitized at 20 or 25°C. Parasitism increased with increasing temperature to a maximum at 20–25°C and declined at 30°C. Handling time decreased asymptotically with increasing temperature. The results of this study suggest that Trichogramma strains vary in adaptability to low temperature, making this characteristic a useful criterion for evaluation of candidate strains.

---

Résumé Afin de sélectionner une souche de Trichogramma sp. (Hymenoptera, Trichogrammatidae) candidate pour des lâchés inondatifs contre les Lépidoptères pestes de chou aux Pays-Bas, l'activité de parasitisation d'une collection de 60 souches différentes de Trichogramma spp. a été étudiée à 12°C pendant des durées d'exposition de 2 h et 24 h. L'activité proportionnelle et le degré de parasitisation des femelles varient significativement selon des souches et ne sont par corrélés. Ceci suggère l'action des deux mécanismes distincts, dépendant de la température et contrôlant independamment chacun de ces processus. Les souches indigènes sont caractérisées par une faible activité à 12°C, mettant en doute leur utilisation pour les lâchés inondatifs. Dans 3 souches testées à 12, 17, 20, 25 et 30°C, l'activité augmente linéairement avec la température, jusqu'a ce que quasi toutes les femelles parasitent les oeufs hôtes à 20°C et 25°C. Le taux de parasitisation augmente avec la température atteignant un maximum à 20–25°C puis diminue à 30°C. Le temps de manipulation d'un oeuf hôte diminue asymptotiquement lorsque la température croît. Les résultats de cette étude suggèrent que la capacité d'adaptation aux températures basses varie selon les souches de Trichogramma. Cette caractéristique constitue un critère utile pour l'évaluation de souches candidates potentielles.

     

Laboratory studies on aestivation in the cabbage root fly (Delia radicum)

Laboratory experiments at 16°–30°C showed that the cabbage root fly can only be induced into aestivation during the early part of the pupal stage. The relationship between the percentage of pupae entering aestivation (y) and the average daily temperature (T) was the same under both constant and alternating temperatures. The percentage of pupae entering aestivation at a particular temperature was given by y=13T–255. An additional 13% of the pupae entered aestivation for each degree rise between 20° and 27°C. There was no constant period of arrested development following the induction of aestivation. Aestivating pupae started to develop into flies as soon as the temperature fell below 20°C. The reinduction of aestivation occurred more readily than the initial induction. Insect survival was not reduced after a month of aestivation. The effects of aestivation on forecasting the time of the second (summer) generation of flies are discussed.

---

Étude au laboratoire de l'estivation de la mouche du Chou (Delia radicum)

Résumé Au laboratoire, à 10°–30°C, l'induction de l'estivation ne peut être obtenue qu'avec des pupes formées depuis peu.Le rapport pourcentage de pupes entrant en estivation (y) température journalière moyenne (T) a été le même, que la température soit constante ou alternée. La relation entre la température et ce pourcentage est donnée par y=13 T–255. Le pourcentage de pupes entrant en estivation a augmenté de 13% pour chaque élévation d'un degré entre 20° et 27°C. L'estivation une fois induite, la période d'arrêt de développement n'est pas constante. Dès que la température est devenue inférieure à 20°C, la formation d'imagos a commencé dans les pupes en estivation. Une nouvelle estivation a été plus facile à provoquer que l'estivation initiale. Le taux de survie des insectes n'avait pas diminué après un mois d'estivation. La discussion examine l'utilisation des conséquences de l'estivation dans la prédiction de la seconde génération d'adultes: génération d'été.

     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

Freeze tolerance of soil chytrids from temperate climates in Australia

Very little is known about the capacity of soil chytrids to withstand freezing in the field. Tolerance to freezing was tested in 21 chytrids isolated from cropping and undisturbed soils in temperate Australia. Samples of thalli grown on peptone–yeast–glucose (PYG) agar were incubated for seven days at −15 °C. Recovery of growth after thawing and transferring to fresh medium at 20 °C indicated survival. All isolates in the Blastocladiales and Spizellomycetales survived freezing in all tests. All isolates in the Chytridiales also survived freezing in some tests. None of the isolates in the Rhizophydiales survived freezing in any of the tests. However, some isolates in the Rhizophydiales recovered growth after freezing if they were grown on PYG agar supplemented with either 1 % sodium chloride or 1 % glycerol prior to freezing. After freezing, the morphology of the thalli of all isolates was observed under LM. In those isolates that recovered growth after transfer to fresh media, mature zoosporangia were observed in the monocentric isolates and resistant sporangia or resting spores in the polycentric isolates. Encysted zoospores in some monocentric isolates also survived freezing. In some of the experiments the freezing and thawing process caused visible structural damage to the thalli. The production of zoospores after freezing and thawing was also used as an indicator of freeze tolerance. The chytrids in this study responded differently to freezing. These data add significantly to our limited knowledge of freeze tolerance in chytrids but leave many questions unanswered.     

Ultrastructural damage due to freezing followed by thawing in shoot meristem and leaf mesophyll cells of tall fescue (Festuca arundinacea Schreb.)

Tillers of Festuca arundinacea Schreb. were subjected to-8°C in a bath of methylated spirits for three-quarters of an hour. They were thawed at room temperature and some material taken from the shoot apical meristem and leaf blade for electron microscopy. Similar material was taken from control plants for electron microscopy. Nine tillers subjected to-8°C and thawed subsequently failed to regrow. Nine control tillers regrew. All the treated meristem cells and about half the treated leaf mesophyll cells were extensively altered. Their nuclei were contracted, organelles were swollen or partly disrupted, plasmalemma and nuclear membranes were broken or absent and vacuoles were sometimes disrupted. Strongly osmiophilic material accumulated in the vicinity of membranes. About half the leaf mesophyll cells differed from the control mesophyll cells only in having more spherosomes and narrower thylakoids. Parallels with other ultrastructural studies of stress damage and the indications the results give of possible primary damaging events are discussed.Abbreviations ER endoplasmic reticulum - G golgi body - M mitochondrion - Mb microbody - N nucleus - NM nuclear membrane - No nucleolus - P plasmatemma - Pg plastoglobuli - Pp proplastid - Pr polysomes - S spherosome - SOM strongly osmiophilic material - T tonoplast - Th thylakoids - V vacuole     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.