Jean Fallot

Stimulation ofDaucus carota somatic embryogenesis by inhibitors of ethylene synthesis: cobalt and nickel

The effects of Co2+ and Ni2+ on ethylene production and somatic embryogenesis by carrot (Daucus carota L.) cell cultures were studied. At concentrations of 10 μM to 50 μM, CoCl2 effectively inhibited ethylene production by embryogenic cultures and significantly stimulated somatic embryogenesis. The observed increase of embryo number was proportional to the inhibition level of ethylene production. However, CoCl2 had no effect when Ethephon was supplied. Nickel also reduced ethylene production, but to a slightly lesser extent than CoCl2, bringing about a lower increase in the number of somatic embryos. The role of ethylene on somatic embryogenesis is discussed.

Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors, silver and cobalt

The effects of CoCl2, AgNO3 and ethylene released by exogenous 2-chloroethylphosphonic acid (Ethephon), were studied on shoot regeneration from cotyledons of Helianthus annuus cv. E8206R, a poorly regenerative cultivar. Inhibition of ethylene biosynthesis by CoCl2, at concentrations of 20 μK, provoked a substantial enhancement of shoot regeneration (30 %): the control was poorly regenerative. However, CoCl2 had no effect when Ethephon was supplied. Inhibition of ethylene action by AgNO3, at concentrations of 10–25 μM, caused a significant increase in plant regeneration: 25 % instead of 1.2 % in the control. Furthermore, addition of Ethephon to AgNO3-treated tissues failed to reduce the stimulation of shoot regeneration caused by AgNO3. On the basis of these findings, it is suggested that ethylene inhibits the regeneration process from cotyledons of sunflower.

Control of carrot somatic embryogenesis by AgNO3, an inhibitor of ethylene action: Effect on arginine decarboxylase activity

Abstract The effects of AgNO3 and ethylene, released by exogenous supplied 2-chloroethylphosphonic acid (Ethephon), were studied on somatic embryogenesis by carrot (Daucus carota L.) cell suspension cultures. AgNO3, at concentrations of 10–20 μM, caused a 2-fold increase in the number of somatic embryos. At these concentrations AgNO3 did not cause adverse effects on cell survival, medium pH or growth; only a slight increase in ethylene production was observed. However, 1 mg·1−1 or 10 mg·1−1 Ethephon provoked a substantial decrease in the somatic embryo formation: inhibitory rates of 15% and 50% were recorded, respectively. The activity of arginine decarboxylase (ADC) (EC 4.1.1.19), a key enzyme of the polyamine pathway, was significantly stimulated by AgNO3 during the first 4 days of somatic embryogenesis, whereas Ethephon reduced it: 30% inhibition was recorded at 10 mg·1−1 Ethephon. On the basis of these findings, it is suggested that AgNO3 stimulates carrot somatic embryogenesis by inhibiting ethylene action; its implication in the control of ADC activity is discussed.

Eutypine, a phytotoxin produced by Eutypa lata the causal agent of dying-arm disease of grapevine

Abstract A phytotoxic metabolite has been isolated from liquid cultures of Eutypa lata and structurally characterized as 4-hydroxy-3-(3-methylbut-3-en-1-ynyl)benzaldehyde, named eutypine. It was detected by GC-MS and MS-MS analyses in the crude sap and the inflorescences of diseased plants and was not present in healthy material.

Stimulation of somatic embryogenesis and plant regeneration from anther culture of Vitis vinifera cv. Cabernet-Sauvignon

Somatic embryogenesis and subsequent diploid plants have been obtained from anthers of Vitis vinifera Cabernet-Sauvignon, a cultivar so far considered as recalcitrant to in vitro regeneration. Anthers enclosing microspores near the first pollen mitosis were found to be the most responsive. However, from a practical point of view anther length proved to be an easier criterium for determining the optimal physiological anther stage. Calli derived from the anther somatic tissues produced embryoids only when cultured on a medium supplemented with casein hydrolysate. Glutamine and adenine were found to stimulate this embryoid production. Evidence is presented that early removal of cotyledons increases the frequency of normal development of embryoids into plantlets.

Enhancement of shoot regeneration potential by liquid medium culture from mature cotyledons of sunflower (Helianthus annuus L.)

We describe here a liquid culture system for the regeneration of shoots at high frequencies from mature cotyledon tissues of three genotypes of sunflower (Helianthus annuus L.) one of which had previously been found to be recalcitrant to regeneration when cotyledons were cultured on solid medium. Cotyledons were excised from 2-day-old seedlings and incubated in liquid Murashige and Skoogs modified medium supplemented with 5.4 μM naphthaleneacetic acid (NAA) and 4.4 μM benzylaminopurine (BAP). After two weeks in culture, the whole upper surface of regenerating explants was covered with green shootlets. The percentages of regenerating explants of three genotypes varied between 60 and 70%, and the number of shoots per regenerating explant was highly increased. The shootlets were transferred to solid Murashige and Skoogs medium allowing shoot development, then to rooting medium. Rooted plantlets were successfully acclimatized and gave fertile plants. The role of liquid medium culture in the induction of sunflower regeneration is discussed.

Control of quiescence and viability in auxin-deprived pear cells in batch and continuous culture

Abstract The omission of 2,4-D from the culture medium together with an increase in the osmotic potential by means of added mannitol allowed auxin-dependent cells of Passe-Crassane pear to be kept quiescent after a short period of limited increase in cell number (5–6 days). Cell death began to occur after 14–16 days in batch and in a continuous culture system. The ability of the cells to divide again in the presence of 2,4-D remained unchanged up to 9 days after treatment but was rapidly lost thereafter. In such a model auxin-deprivation related changes can be investigated from the biochemical and ultrastructural points of view.

Enzymatic detoxification of eutypine, a toxin from Eutypa lata, by Vitis vinifera cells: partial purification of an NADPH-dependent aldehyde reductase

Abstract. Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata (Pers.: Fr.) Tul., the causal agent of dying arm disease of Vitis vinifera L. (grapevine). Previously, we have shown that eutypine is involved in the development of disease symptoms. In the present study, the effects of V. vinifera cell-suspension cultures on the biological activity of the toxin were investigated. Eutypine was converted by grapevine tissues into a single compound, identified by mass spectrometry and nuclear magnetic resonance as 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl alcohol, designated eutypinol. This compound was found to be non-toxic for grapevine tissues. Unlike eutypine, eutypinol failed to affect the oxidation rate or membrane potential of isolated mitochondria. In grapevine cells, reduction of eutypine into the corresponding alcohol is an NADPH-dependent enzymatic reaction. An enzyme which reduced eutypine was partially purified, over 1000-fold, using a five-step purification procedure. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was found to have a molecular mass of 54–56 kDa. The enzyme exhibited an apparent Km for eutypine of 44 μM, and was active between pH 6.8 and 7.5 with a maximum at pH 7.0. The eutypine reductase activity was improved by Mn2+ and Mg2+ and inhibited by disulfiram and p-hydroxymercuribenzoate. The possible role of the eutypine-detoxification mechanism in the defense reactions of V. vinifera cells is discussed.

A genotype-independent system of regeneration from cotyledons of sunflower (Helianthus annuus L.) The role of ethylene

Abstract A system of regeneration which allows shoot organogenesis, at high frequencies, from mature cotyledon tissues of 30 genotypes of sunflower ( Helianthus annuus L. ) is described. This new two-step protocol included an incubation period of 3 days for cotyledon explants, for 3 days, in liquid Murashige and Skoogs modified medium supplemented by 5.4 μM naphthaleneacetic acid (NAA) and 4.4 μM benzylaminopurine (BAP); then, they were subcultured on the same medium solidified by agar. After 4 weeks of culture, the shoots were transferred to rooting medium, then the rooted plantlets were successfully acclimatized and gave fertile plants. This process of regeneration allowed the triggering of shoot formation in 19 genotypes which had previously been found recalcitrant to regeneration and enhanced the regeneration potential in other poorly regenerative genotypes. The variation of ethylene production by explants directly cultured on solid medium or incubated in liquid medium was studied. The production of this hormone reached a maximum value after 3 days of culture on solid medium while incubation in liquid medium greatly reduced it. The addition of Ethephon to the liquid medium during the incubation period increased ethylene concentration and caused inhibition of shoot formation. On the basis of these findings, it is suggested that the induction of shoot regeneration is controlled by ethylene, so the reduction of ethylene production caused by the period of explant incubation in liquid medium allows regeneration in all genotypes tested. These data suggest that the process of regeneration might be general for sunflower genotypes.

Transport, cytoplasmic accumulation and mechanism of action of the toxin eutypine in Vitis vinifera cells

Summary The mechanism of the toxic action of eutypine, 4-hydroxy-3-(3-methyl-3-butene-l-ynyl) benzaldehyde, a toxin produced by the fungus Eutypa lata , the agent of dying arm disease of the grapevine, was investigated with cell suspension cultures of Vitis vinifera cv. Gamay and l4 C-labelled eutypine. The study of the chemical characteristics of eutypine revealed that the toxin is a weak acid (pKa = 6.2), which also has a lipophilic character (water/octanol partition coefficient of 86). Eutypine was rapidly taken up by the cells and accumulated against a concentration gradient. Uptake showed no saturation at high eutypine concentration, and neither structural analogues of eutypine nor protein modifying reagents had an inhibitory effect on eutypine uptake. These data suggest a mechanism of passive diffusion for eutypine uptake. The eutypine accumulation observed in cells can be pardy explained by an ion trapping mechanism related to the ionization state of the toxin. It has been demonstrated that some eutypine molecules insert into cellular lipids, i.e. cell membranes. Such partition of eutypine in the cells might play a major role in the expression of its toxicity. Eutypine seems to affect the functioning of mitochondria by an uncoupling effect or by inhibiting succinate dehydrogenase activity. These results suggest that eutypine has the same properties as the uncoupling agents that act as mobile proton carriers. The reduction of the energetic charge following eutypine action could explain the symptoms observed in diseased plants.