Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions

Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 M IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration percentage. High auxin concentrations (6.0 or 11.0 M) inhibited bud differentiation, especially when explants were cultured in darkness. On the other hand, low auxin levels and photoperiod improved shoot development. Excised shoots were induced to form roots by transfer to hormone-free MS medium with macronutrients at half strength. The obtained plantlets were ultimately grown in the greenhouse.Abbreviations BA benzyladenine - BM basal medium - IAA indoleacetic acid - MS Murashige & skoog - NAA -naphthaleneacetic acid     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Sublethal effects of aldicarb on the behaviour of Aphis fabae and two clones of Myzus persicae and on the transmission of beet mosaic virus by these aphids

In laboratory experiments treatment of sugar-beet plants with aldicarb stimulated the mobility of Aphis fabae and two clones of Myzus persicae which were susceptible (S) and resistant (R) to carbamate-based insecticides, respectively. On the other hand, the number of aphids probing and the total number of probes made was reduced, and hence the transmission of beet mosaic virus (BMV) was restricted. In outdoor experiments the spread of BMV from aldicarb-treated plants by naturally infesting aphids was also restricted. The number of infected plants decreased with increasing distance from the sources of infection.

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Résumé Des plantes de betterave traitées au laboratoire avec de l'aldicarbe ont stimulé la mobilité d'Aphis fabae et de deux clones de Myzus persicae, l'un sensible et l'autre résistant à des insecticides contenant des carbamates. Par ailleurs, le nombre de pucerons en train de sonder les feuilles ainsi que le nombre total de sondages ont été réduits et ainsi la transmission du virus de la mosaïque de la betterave (BMV) a été limitée. Dans des expériences à l'extérieur, la vitesse de propagation de BMV par des pucerons sur des plantes traitées à l'aldicarbe a été aussi plus limitée. Le nombre de plantes contaminées diminuait avec la distance de la source de contamination.

     

The effect of glucosinolates on responses of young Phyllotreta nemorum larvae to non-host plants

All recorded host plants of Phyllotreta nemorum L. (Coleoptera: Chrysomelidae) contain glucosinolates and belong to the plant families Brassicaceae (Cruciferae), Resedaceae and Capparaceae. The acceptability of 56 plant species from 28 other plant families (non-hosts) for young larvae has been studied in the laboratory. None of these species were fully acceptable for initiations of leaf mines when intact untreated leaves were presented, and only one species, Malva silvestris L. (Malvaceae), was partially acceptable. The acceptability of some species increased when leaf discs were presented instead of intact leaves; but the highest percentages of mine initiations occurred in leaf discs treated with the glucosinolate, sinigrin. A stimulatory effect of sinigrin could be demonstrated in experiments with 7 plant species: Papaver dubium L., Papaver rhoeas L., Fumaria officinalis L., Malva silvestris L., Pisum sativum L., Campanula latifolia L. and Lactuca sativa L. The majority of species remained unacceptable even after treatment with glucosinolates.The main causes for these differences between plant species are supposed to be differences in contents of deterrents and/or other stimulants for mine initiation. These possibilities are discussed in relation to the content of allelochemicals in acceptable plants and the position of these plants in botanical classifications.

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Zusammenfassung Phyllotreta nemorum L. ist ein oligophager Erdfloh, der an Cruciferen und anderen Glukosinolat-haltigen Pflanzenarten gebunden ist. Die Imagines fressen Löcher in die Blätter und die Weibchen legen ihre Eier in den Boden. Die Larven sind Blattminierer. Nach dem Schlüpfen im Boden klettern sie an die Pflanzen hoch, und die Einbohrung und der Anfang der Minierung erfolgt in eines der unteren Blätter der Wirtspflanze.Die Wirkung von Glukosinolaten auf die Einbohrung von Junglarven in Pflanzenarten, die keine natürliche Inhalt von Glukosinolaten haben, ist in Laborexperimenten untersucht worden. 56 Pflanzenarten aus 28 Familien wurden präsentiert teils als unbehandelte Blätter und teils als Glukosinolatbehandelte Blattscheiben. Unbehandelte Blätter von nicht-Glukosinolathaltigen Arten waren immer unbefriedigend für die Larven. Nur in eine Art, Malva silvestris L. war die Frequenz der Einbohrung ein bisschen höher als 10%. Eine signifikante Erhöherung der Anzahl eingebohrten Larven nach der Sinigrin-behandlung erfolgte in 7 Pflanzenarten: Papaver dubium L., P. rhoeas L., Fumaria officinalis L., Malva silvestris L., Pisum sativum L., Campanula latifolia L. und Lactuca sativa L. Doch blieben die meisten Pflanzenarten (84%) auch nach der Sinigrin-Behandlung unbesiedelt.Pflanzenarten, die nach der Sinigrin-Behandlung nicht besiedelt werden enthalten vielleicht frasshemmende Stoffe, oder ihnen fehlen noch weitere Frass-stimulierende Stoffe. Diese Möglichkeiten werden diskutiert in Zusammenhang mit den Inhalt von Allelochemikalien in besiedelten Pflanzenarten und mit ihrer taxonomischer Position.

     

A comparison of in vitro flowers to in vivo flowers of haploid and diploidNicotiana tabacum L.

Thin cell layers (TCLs) were cultured from inflorescences of diploid (2n=4x=48) and haploid (2n=2x=24)Nicotiana tabacum L. "Samsun" and the subsequent flowers formed in vitro were then compared to in vivo flowers. Plants derived from TCLs possessed flowers that were typical of their seed or androgenetically-derived counterparts, whereas de novo flowers from TCLs were abnormal when compared to their counterparts. The TCLs of haploid plants produced more flower buds than diploid TCLs, and did so in a shorter period of time. In vitro flowers and anthers at both ploidy levels were considerably smaller than the in vivo flowers; in vitro flowers also had variable numbers of anthers and pistils. The embryogenic capacity of anthers taken from in vivo diploid flowers was 5 times greater than that of in vitro diploid or haploid anthers. In vivo haploid anthers produced no embryoids, whereas in vitro haploid anthers did produce embryoids. Observations of mitotic cells in root tips of plants derived from anther cultures of in vitro haploid flowers revealed a mixoploid nature. Diploid meiosis was regular and haploid meiosis was irregular regardless of the origin (in vitro or in vivo) of the flowers.Supported by state Hatch funds.     

Leaf age and larval performance of the leaf beetle Paropsis atomaria

ABSTRACT.

• 1 Larval performance of the leaf beetle Paropsis atomaria Oliver was determined for larvae raised on both new and mature leaves of Eucalyptus blakelyi Maiden. Larvae were transferred to mature leaves at different ages; control larvae stayed on new leaves through all instars.
• 2 Only larvae reared on new leaves through the third instar survived to pupate on mature leaves; developmental time was prolonged by 20% and pupal weight was reduced by 50% in these larvae compared with larvae reared entirely on new leaves. Almost all larvae died when transferred to mature leaves as first, second or third instars.
• 3 Low survival and slow development on mature leaves was mainly due to failure by larvae to feed. Larvae palpated leaves and could discriminate among leaf ages immediately, without biting into the leaf tissue.
• 4 New leaves had higher concentrations of oil and tannins than old leaves, while there were no significant differences in nitrogen concentrations in the two types of leaves. Mature leaves were more than 3 times tougher than new leaves, in terms of g mm^?2 of penetrometer force.
• 5 In drought years E. blakelyi may not produce sufficient new leaves to supply specialist herbivores with their preferred food resource. We infer that drought years reduce P. atomaria larval performance significantly, and influence the population dynamics of the insect.

     

Stomatal response to leaf water potential in almond trees under drip irrigated and non irrigated conditions

Almond plants (Amygdalus communis L. cv. Garrigues) were grown in the field under drip irrigated and non irrigated conditions. Leaf water potential () and leaf conductance (g₁) were determined at three different times of the growing season (spring, summer and autumn). The relationships between and g₁ in both treatments showed a continuous decrease of g₁ as decreased in spring and summer. Data from the autumn presented a threshold value of (approx. –2.7 MPa in dry treatment, and approx. –1.4 MPa in wet treatment) below which leaf conductance remained constant.     

Stomatal sensing of the environment

The effects of environmental factors on stomatal behaviour are reviewed and the questions of whether photosynthesis and transpiration eontrol stomata or whether stomata themselves control the rates of these processes is addressed. Light affects stomata directly and indirectly. Light can act directly as an energy source resulting in ATP formation within guard cells via photophosphorylation, or as a stimulus as in the case of the blue light effects which cause guard cell H⁺ extrusion. Light also acts indirectly on stomata by affecting photosynthesis which influences the intercellular leaf CO₂ concentration ( C _i). Carbon dioxide concentrations in contact with the plasma membrane of the guard cell or within the guard cell acts directly on cell processes responsible for stomatal movements. The mechanism by which CO₂ exerts its effect is not fully understood but, at least in part, it is concerned with changing the properties of guard cell plasma membranes which influence ion transport processes. The C _i may remain fairly constant for much of the day for many species which is the result of parallel responses of stomata and photosynthesis to light. Leaf water potential also influences stomatal behaviour. Since leaf water potential is a resultant of water uptake and storage by the plant and transpirational water loss, any factor which affects these processes, such as soil water availability, temperature, atmospheric humidity and air movement, may indirectly affect stomata. Some of these factors, such as temperature and possibly humidity, may affect stomata directly. These direct and indirect effects of environmental factors interact to give a net opening response upon which is superimposed a direct effect of stomatal circadian rhythmic activity.