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Senescence is the terminal phase in the development of a plant, plant organ or plant tissue. Flower senescence, for example, occurs after pollination when the petals no longer serve a useful purpose, and for certain flowers is accompanied by a climacteric rise in ethylene production. In carnation, this climacteric surge in ethylene formation is temporally coincident with inrolling of the petals, which is the first morphological manifestation of petal senescence (Thompson et al.,1982). Petal inrolling is thought to reflect loss of cell turgor attributable to membrane leakiness. Indeed, there is growing evidence that a progressive decline in membrane selective permeability and the ensuing loss of intracellular compartmentation are characteristic features of both flower and leaf senescence (Thompson et al., 1998).

Increasing flower’s longevity by blockage of the onset of senescence has economical importance. Currently, STS (silver thiosulfate complex anion), is being used as an anti-senescence preservative in the cut-flower industry. But, STS contains a heavy metal ion (Ag+) and has a potential environmental hazard. Therefore, it is desired to have alternatives to STS, which are safe for organisms and have a less risk for environmental pollution. DPSS is a novel preservative for cut carnation flowers, which were developed recently [1] and has just come to the market in Japan. Its activity to retard carnation flower senescence is compatible to or more than that of STS. DPSS is safe for organisms including mammals, because its oral and dermal administration causes no acute toxicity in mice and exerts no mutagenic effects by Ames test.

A range of compounds bind to the putative ethylene receptor. Some are agonists and mimic the effects of ethylene; some are antagonists and block ethylene action. In the past few years some particularly effective blocking agents for the ethylene receptor have been discovered. They block the receptor for up to 12 days at 25 C when provided in a single exposure and for much longer periods if exposure is repeated at 8–10 days intervals. A 24-hour exposure to 0.5 nl.l1 1-methylcyclopropene (1-MCP) protects banana fruit and carnation flowers from the effects of ethylene, but about 40 nl.l-1 is required to prevent ethylene-induced inhibition of pea seedling growth and abscission in mung beans. The reason for this large concentration difference is unknown. Other cyclopropene compounds block banana ripening for shorter periods of time (3, 5, 7 or 12 days) but require higher treatment concentrations than 1-MCP.

In many flowers pollination accelerates ethylene biosynthesis and developmental changes observed during the natural senescence of unpollinated flowers. A burst of ethylene production from the stigma/ style is often the first detectable post-pollination event. We are interested in the nature of the pollen-pistil interaction that induces ethylene biosynthesis and the role of ethylene in triggering subsequent post-pollination phenomena including ovary development and senescence of the corolla. In carnations we have identified a pollination responsive ACC synthase that is up regulated by 1 hour after pollination in styles. We have demonstrated that the regulation of this gene by pollination is independent of ethylene action, but that ethylene action within the pollinated style is required to initiate subsequent post-pollination events in the ovary and petals. Using Petunia hybrida as a model we have shown that pollen-borne ACC is synthesized in the haploid pollen grain by a pollen specific ACC synthase gene. Using transgenic plants that fail to accumulate ACC in their pollen we have shown that pollination-induced ethylene production and ACC synthase mRNA accumulation in the style is not dependent on ACC in the pollen grain. With the identification of pollination responsive ACC synthase genes and the use of pollen that is deficient in potential elicitors it should be possible to identify the primary pollination signal in Petunia and carnation flowers.

The most significant action of low oxygen on the senescence of detached plant organs is the retardation of the onset of the climacteric rise in ethylene evolution. We have observed that this aspect of low oxygen is saturable, in that for oxygen to delay the climacteric rise in ethylene evolution in apples and carnation flowers its concentration must be reduced below about 6.078 kPa. In cut carnation flowers, 4.5% oxygen delays the accumulation in mRNA of both ACC-oxidase and -synthase by about 6–7 days beyond that found in air samples. In contrast, low oxygen enhances the activity of alcohol dehydrogenase (ADH). In flowers kept under 1.52 kPa O2 there is no accumulation of ACC-synthase or -oxidase transcripts for 17 days, the duration of the experiment. The data thus indicate that there is a mechanism that responds to low oxygen by retarding the synthesis of enzymes associated with normal senescence while at the same time enhancing the synthesis of anoxic proteins, yet without inducing anaerobic fermentation. Preliminary results indicate that a heme-containing protein may be involved in the “sensing” of the concentration of oxygen.

As one of the major contributors to the cut-flower market and a commercial leader in terms of number of stems sold worldwide (Jensen, Malter 1995), carnation (Dianthus caryophyllus L.) has been an important target for the breeding of new varieties with novel characteristics. Although new carnation varieties are continuously being produced through classical breeding, their high heterozygosity and limited gene pool, and a lack of knowledge regarding their genetic makeup, severely restrict such breeding programs (Woodson 1991). Thus, the possibility of genetically transforming carnation, as well as other cut-flower species, via direct gene transfer is quite attractive (reviewed by Zuker et al. 1998).

Ethylene is involved in many developmental processes including the senescence of petals in ethylene sensitive flower species such as carnation, orchids and Petunia. The mode of action of ethylene in petal senescence may be its effect on the expression of numourous genes, among them genes coding for enzymes directly involved in cell death and genes encoding ethylene biosynthetic enzymes or components of the ethylene perception and signal transduction route(s). In carnation, a range of different senescence-related (SR) genes were isolated by Woodson and co-workers. Expression of most of these genes increased during the rise in ethylene production while treatment with the ethylene inhibitor 2,5-norbornadiene generally was inhibitory, indicating that expression is controlled by ethylene [1]. Based on homology studies with other known proteins, putative roles of some of the proteins in petal senescence is expected. Among the carnation SR genes, besides from the ethylene biosynthetic genes ACC synthase and ACC oxidase, the following activities have been reported: beta-glucosidase, beta-galactosidase, glutathione-S-transferase, carboxyphosphonoenolpyruvate mutase and thiol protease [1 and references therein]. Although some of these enzymes may be directly or indirectly involved in the processes leading to petal senescence, definite proof of their function e.g in transgenic plants, is lacking.

Fusarium oxysporum f. sp. dianthi (FOD), incitant of vascular wilt, is the most important pathogen on carnation, where it causes severe losses in all growing areas. The use of resistant carnation cultivars still provides the most effective solution to this problem and indexing is routinely adopted for the obtainment of pathogen-free propagative material, (Baayen and Kleijn, 1989; Garibaldi and Gullino, 1987). Eight physiological races or pathotypes of FOD have been reported (Garibaldi, 1975), and knowledge of the distribution of pathotypes is a basic prerequisite for choosing of suitable resistant varieties in each growing area.

The work presented in the oral paper comprises the development of a regeneration and transformation system for carnation (Dianthus caryophyllus L.) [1]. One of the more critical factors in the genetic transformation is the ability to regenerate a mature plant from a single cell. Therefore much attention was paid to shoot regeneration of carnation. Explants from leaves were used and adventitious shoot formation was developed at the bases of the explants. It was possible to generate adventitious shoots at the transition region from stem to leaf: the leaf base. The position of leaves on the plant appeared to be important for subsequent regeneration. The youngest leaves (leaf position 1) showed the highest regeneration percentage. The shoot regeneration procedure was efficient, 65 % of the explants showed regeneration with an average of 10 shoots per explant. Regenerants from leaf explants did not show aberrant plant growth. The shoot regeneration procedures were applicable for a wide range of cultivars.

1-Methylcyclopropene (1-MCP), formerly designated as Sis-X, has been shown to be an effective inhibitor of ethylene responses in carnation flowers in either the light or the dark. The binding appears to be to the receptor and to be “permanent”. A 6 h treatment at 2.5 nl 1-1 is sufficient to protect against ethylene, and 0.5 nl 1-1 is sufficient if exposure is for 24 h. As carnation flowers age, a little higher concentration appears to be needed. Most of the natural increase in ethylene production during senescence is prevented by treatment with 1-MCP. A closely related compound, methylenecyclopropane shows ethylene activity. A tritium labelled 1-MCP (60 mCi mmol-1) has been prepared. A higher specific activity is needed for more critical studies.

It has become something of an established fact that plants cultured in vitro from tissue explants have less cuticular waxes than normal plants. This appears plausible, for in the virtually water vapour-saturated atmosphere of the culture vessel they don’t ‘need’ a good water barrier on their surface, and thus wax synthesis is more or less superfluous. When plantlets are removed from the culture vessels after some weeks and exposed to the greenhouse atmosphere for further cultivation, rapid desiccation and eventually death occur frequently and cause great economic losses at this stage. It was found that with carnation plantlets the survival rate after removal from the culture vessels was 25 times higher if the plantlets had formed glaucous leaves in vitro, as compared to the majority of plantlets which had formed non-glaucous leaves [33]. From findings such as this, the general conclusion has been made that high cuticular water loss contributes substantially to the desiccation problem, in addition to transpiration through non-functional stomata and water uptake problems due to underdeveloped or absent roots.

Morphological and physiological disorders in leaves are often observed under culture conditions intended to promote rapid bud proliferation. High water potential in the medium and relative humidity in the culture vessel, elevated levels of sucrose and growth regulators and the presence of certain ions in unfavorable ratios, as well as low light intensity induce anomalous leaf anatomy and morphology termed ‘vitrification’. Abnormal shoot morphogenesis has been termed hyperhydration, glassiness, succulence and translucence, depending on the severity of the phenomenon. These descriptions are based on visual characterizations and depend on the external morphology of the studied species [12, 24, 33]. In vitro plants having abnormal leaves function poorly ex vitro due to inadequate photosynthetic activity and high rates of water loss which lead eventualy to plant desiccation [3, 7, 9, 32, 34, 37]. The major causes for poor plant survival are underdeveloped mesophyll, poor cuticular wax formation and malfunctioning guard cells [9,27, 34, 35]. Stomata from vitreous leaves remained open and did not respond to darkness, abscisic acid (ABA), high Ca2+ and hypertonic solutions — stimuli which usually cause stomatal closure [3, 22, 29, 34, 35]. Ziv et al. [37] reported that the defect in stomatal function resided in the guard cell walls which, unlike the protoplasts, did not contract in hypertonic solution. The mechanical properties of the guard cell walls in the stomatal apparatus are determined by the cell wall structure and composition [22]. Since both hyperhydration and guard cell malfunction result from changes in the cell walls, this research investigated the relationship between leaf hyperhydration and stomatal deformation and malfunction with the purpose of using stomatal function as a simple, more accurate parameter to evaluate vitrification.

Saprophytic Fusarium spp., isolated from Fusarium-suppressive soils, are active as biocontrol agents against several Fusarium wilts. Since perfect stages rarely occur in nature, intra- or interspecific crosses through protoplast fusion may provide new strains with enhanced biocontrol activity by combining different desirable traits from two or more parental strains. A protoplast fusion was accomplished between two strains (parental strains A, resistant to benomyl and B, sensitive to benomyl, orangepigmented) of antagonistic F.oxysporum and over 100 hybrids were selected by the inheritance of colour and benomyl resistance markers from the parents. Several fusants were tested in comparison with their parental strains against F.oxysporum f.sp. dianthi on potted carnation plants. A remarkable variability was observed with respect to the biocontrol activity of the hybrids: some of them were able to effectively reduce carnation wilt incidence when inoculated as conidial suspensions by the root dipping method at transplant, while others did not show any biological control capability.

The newly discovered solid form of carbon — C 60 (buckminsterfullerene) when applied either as colloidal solution or in liposomes significantly reduces ethylene evolution from senescing legume [Pisum sativum and Vicia faba] foliage and from cut carnation (Dianthus caiyophyllus)flowers, the liposomes being twice as effective as colloidal solutions. C 60 mode of action is attributed to ethylene adsorption stemming from the interaction between the vast C 60 surface area — 1,317 m2/gr — and the affinity of its carbon atoms for the π component in the ethylene double bond.

The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased synthesis of the phytohormone ethylene. This ethylene serves to initiate and regulate the processes of programmed organ death. We have isolated and cloned mRNAs which encode the ethylene biosynthetic pathway enzymes 1aminocyclopropane-l-carboxylate (ACC) synthase and ACC oxidase. These cDNAs have been used to study the regulation of ethylene biosynthesis during flower petal senescence. The increase in ethylene production and onset of petal senescence are associated with dramatic changes in gene expression. Several senescence-related (SR) mRNAs have been isolated and used to study specific changes in gene expression during flower petal senescence. In several cases, the increase in SR mRNA abundance occurs concomitant with the ethylene climacteric. Indeed, treatment of flowers with inhibitors of ethylene synthesis or action prevents petal senescence and the accumulation of SR mRNAs. Sequence analysis of SR mRNAs has revealed homologies with other known proteins, which in some cases points to putative roles of these genes in the regulation of petal senescence. In order to elucidate the molecular mechanisms involved in the regulation of SR gene expression, we have initiated an analysis of the cis-elements responsible for developmental and hormonal gene regulation during floral senescence.

Cell and tissue death is an usual symptom in plant diseases caused by pathogenic fungi; in incompatible plant-fungus interactions the death of one or few cells generally is associated with plant resistance and the growth restricted of the potential pathogen. Moreover, while in incompatible interactions between plants and obligate biotrophs cell death explains host resistance, in the case of all other fungi host cell death cannot be a limiting factor of the growth of pathogen, but it could be a biological signal for the elicitation of effective defense mechanisms in plants. It was reported that some substances of fungal origin show necrogenous activity correlated to the elicitation of defense responses in plants; in opposite, there are other plantfungus systems where no correlation has been found between host cell death and disease resistance. In our previous papers we showed that thermostable substance(s) (elicitor) have been extracted from mycelial cell walls of Fusarium oxvsporum f.sp. dianthi race 2 (Fod2), which induce(s) synthesis of Phenylalanine Ammonia-Lyase, organic-soluble compounds and phytoalexins in callus cultures derived from carnation cultivars resistant to Fod2, but not in callus cultures derived from susceptible cvs. In the present work we show that the elicitor induces cell death of in vitro carnation cells derived from the cultivar Pulcino, resistant, but not in those from the cultivar Corrida, susceptible. About the 38 % of cells of Pulcino die just 8 hours after eliciting treatment - about the 55% after 12 hours -, when assessing cell viability by fluorescein acetate method. A cell line from Pulcino cells has been selected without mutagenesis, which does not die following eliciting treatment and it has been called sensitive-minus (Pulcino s.m.). Explants of callus of Pulcino s.m., when inoculated with Fod2, are extensively colonized as well as explants of callus of Corrida. To date, we cannot be sure whether and how cell death is involved in activing the mechanisms which retard the fungal colonization of callus cultures. But the fact that the sensitive-minus cell line from Pulcino contemporaneously is altered both in elicited cell death response and in the colonization by Fod2, suggests to us that cell death could be one of the first signals between carnation cells and hyphae for triggering further biochemical defense responses.

Advances in in vitro cultivation of plant tissues have opened up new ways to study host-pathogen interactions and to increase resistance to disease by genetic improvement. Many parasitic fungi, both obligate and non-obligate, have been cultivated on calli, cells or protoplasts derived from host and/or non-host plants. The object of these studies has usually been to ascertain the response of in vitro tissue culture to fungi, and to verify the correspondence of the results (with respect to resistance/susceptibility) with those obtained with the entire plant inoculated with the pathogen or with a non-pathogen.

Ethylene regulates the processes of petal senescence in both carnation and petunia. To investigate the relationship between transcriptional regulation of ethylene production and petal senescence, we have cloned genes involved in ethylene biosynthesis from the petals of these species. Using the polymerase chain reaction we have isolated a cDNA clone encoding l-aminocyclopropane-1-carboxylic acid (ACC) synthase from petunia. Heterologous screening of a senescing petal cDNA library provided an ACC synthase encoding clone from carnation. Increased levels of the mRNAs hybridizing to these cDNA clones is observed in climacteric petals and can be correlated with the onset of senescence. A cDNA clone encoding ACC oxidase was isolated by differential screening of a senescing carnation petal library. Antisense expression of ACC oxidase in transgenic carnation plants inhibits petal inrolling, increasing the post harvest life of the flowers.

Synthetic oligonucleotides homologous to conserved sequences of ACC synthase were used to prime the synthesis and amplification of fragments of about 1000 base pairs by PCR in samples of cDNA to RNA isolated from senescing carnation flowers. Two putative ACC synthase PCR clones were isolated of which one was identical to the sequence of a carnation ACC synthase cDNA clone (CARACC3) recently isolated by Park et al. (Plant Mol. Biol. 18:377–386, 1992), the other clone (CARAS1) was approximately 66% homologous to CARACC3. For both ACC synthase clones, specific oligonucleotides were synthesized. Using PCR we were now able to distinguish between the two ACC synthase mRNAs in samples of total RNA isolated from different flower parts. The occurrance of both messengers was stimulated by aging and by treatment with ethylene in a tissue-specific way.

Antagonism between fluorescent pseudomonads and plant pathogens is well established as a mechanism of disease suppression. Accordingly, Pseudomonas fluorescens strain WCS417 was demonstrated to suppress Fusarium wilt in a moderately resistant cultivar of carnation, grown in rockwool (Van Peer et al., 1990). Since disease suppression did not occur when a susceptible cultivar was used, it was suggested that mechanisms other than for example iron competition are involved as well. The hypothesis that Pseudomonas fluorescens strain WCS417 can induce resistance was postulated and tested.

Three related carnation cultivars (Ginevra, Epomeo and Chinera) were less sensitive to exogenous ethylene than cv. White Sim. The concentration to give 50% of the maximum response (H5O) after a 24 h treatment was 0.21 μl 1−1 in cv. White Sim and 0.32, 0.48, and 0.65 μl 1−1, in the cvs. Ginevra, Epomeo, and Chinera, respectively.

Wilt disease of carnation (Dianthus caryophyllus L.) is caused by the vascular pathogen Fusarium oxysporum f.sp. dianthi. Due the polygenic nature of the resistance of the plant to race 2 of this pathogen (Fod2) a range of cultivars can be found with varying resistance levels. Part of the plant defence probably resides in the production of phenolic compounds; some of which possess fungistatic properties (phytoalexins). Previously it has been shown that the capacity of carnation cultivars to produce phenolic compounds in response to infection with Fod2 correlates with the level of resistance to this fungus. Here we address the question to the role of cell wall components of Fod2 in the elicitation of the synthesis of these phenolic compounds in planta.

Two species of spider mite, namely the two-spotted spider mite (Tetranychus urticae Koch) and the carmine spider mite (Tetranychus cinnabarinus (Boisduval)), are serious pests of plants grown under glass or polythene. Plants damaged include cucumber, dwarf French bean, pepper, strawberry, tomato, carnation, chrysanthemum and rose; also arum lily, hydrangea, orchids and many pot plants as well as stone fruits and vines.

Caterpillars of several species of tortrix moth can cause damage to strawberry. By far the most important in Britain are the strawberry tortrix (Acleris comariana (Lienig & Zeller)), the straw-coloured tortrix (Clepsis spectrana (Treitschke)) and the dark strawberry tortrix (Olethreutes lacunana (Denis & Schiffermuller)), but damage is also caused locally by other species including the carnation tortrix (Cacoecimorpha pronubana) (Hubner)) and the flax tortrix (Cnephasia asseclana (Denis & Schiffermuller)).

Cultivars of tomatoes, cucumbers, lettuce and peppers have been bred for resistance to one or more pathogens. Some tomato and cucumber cultivars have resistance to a wide range of diseases. Resistance has been transient in many cases and a succession of cultivars with new genes or new combinations of resistance genes has been necessary to maintain control. There has been a number of notable exceptions and these have included durable resistance to such pathogens as Fulvia fulva and tomato mosaic virus. With lettuce the resistance situation is complicated by the occurrence of fungicide resistant pathotypes. There are no strains of Agaricus bisporus purposely bred for disease resistance.

Among the numerous factors that modulate photosynthetic activity (such as light, temperature, CO2 and O2 concentrations…) the partitioning and utilisation of the photosynthates within the plant (i.e. the interactions of “source” and “sink”) is often regarded as an over–riding factor. (1.2). In this context, it was reported that addition of sucrose or glucose in the external medium of chlorophyllous higher plant cell culture inhibited photosynthesis and chloroplast development (3,4,5) and that maximal rate of photosynthesis were correlated with low cellular sucrose /glucose content (6). In order to precise the cascad of events leading to the photosynthesis inhibition we studied the effects of glucose feeding on photosynthesis and respiration of photoautotrophic suspension culture of carnation cells. The different gas exchange processes occuring in the light were discriminated using 18O and 13C isotopes and a mass spectrometry technique.

Low temperature (2–5°C) treatment of bean (Phaseolus vulgaris L.) leaf discs and carnation (Dianthus caryophyllus L.) flowers resulted in virtual cessation of ethylene production. Returning tissues to higher temperatures (20–25°C) stimulated rapid and high levels of ethylene production in bean leaf discs, while there was a substantial lag period in the non-wounded carnation flowers. Low temperature exposure prevented the production of the ethylene synthesis intermediate 1-aminocyclopropane-l-carboxylic acid (ACC). In bean leaf tissue, there was rapid ACC synthesis on warming and conversion to ethylene. Consideration is given to the nature of the mechanism preventing ACC synthesis at low temperature, the process whereby ACC production may be induced within 10 min of warming and the nature of the mechanism whereby low temperature treated tissues may store the potential for high level, ‘overshoot’ ethylene production which is expressed only on warming. All these factors have a potential to influence the post-harvest characteristics of commodities that have been coolstored after harvest. The changes induced by coolstorage of carnation flowers are considered in detail.

A membrane-bound enzymatic conversion of 1 — aminocyclopropane — 1 — carboxylic acid (ACC) to ethylene was recently demonstrated in carnation petals by Mayak et al. (1). They also reported the presence of a cytoplasmic inhibitor of this reaction. The aim of this work was to characterize this inhibitor.

Since the identification of 1-aminocyclopropane-1-carboxylic acid (ACC) as the immediate precursor of ethylene biosynthesis (1,2), the nature of the reaction converting it to ethylene has drawn considerable attention. Most of our knowledge of this reaction comes from studies using intact tissues as model systems (for a review see ref. 3). On the basis of the findings of these studies, it was suggested that membranes are involved in the reaction.

A CO2 laser-based photoacoustic method was used to determine the early changes in ethylene production during senescence of carnation (Dianthus caryophyllus) and emasculated orchid (Phalaenopsis) flowers. Individual flowers were placed in small cuvettes (80 ml) and flushed with purified air (flow rate 0.9 1/h). The effluent was directed through a photoacoustic cell for determination of ethylene. The laser photoacoustic equipment consisted of a line tunable CO2 laser in conjunction with a single-pass resonant acoustic cell. The minimum detection limit of the system for ethylene was 0.03 nl/1.

The metabolism of silver thiosulphate in carnation petals was investigated. The thiosulphate moiety was converted into the sulphate anion, with an accompanying evolution of hydrogen sulphide. The silver ion was associated with polypeptides of membranes which are mainly of intra-cellular origin.

Genetic (interclonal) variability for flower colour and three quantitative characters (number of flowers, flowering date, plant height) was analyzed in clones of the mediterranean carnation (Dianthus Caryophyllus L.) cv. Corrida obtained from cuttings,meristems (control and irradiated with 2500r and 5000r of X-rays)and petal culture in vitro. Flower colour variations, probably resulting from deletions were present in all cases but plants derived from cuttings,their frequency increasing with X-ray dosage. X-rays and petal culture also increased heritability estimates for the characters plant height and flowering date but not for number of flowers.