Jules Bouharmont

Effects of salt stress on growth, mineral nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L) cultivars differing in salinity resistance

Salt-resistant rice cultivars Nona Bokra and IR 4630 exposed at the seedling stage during one or two weeks to 0, 20, 30, 40 or 50 mM NaCl accumulated less Na, Cl, Zn and proline and more K at root and shoot levels than salt-sensitive I Kong Pao and IR 31785. Aiwu, a moderately resistant genotype, exhibited an intermediate behaviour. P transport from root to shoot was inhibited in the most sensitive cultivar IR 31785. Accumulation of Na and Cl and decrease in K content at the shoot level were restricted to the oldest leaves in salt-resistant genotypes while proline accumulated in the youngest leaves in all cultivars. In the presence of NaCl, the osmotic potentials of the roots and of the oldest and youngest leaves were lower in the salt-resistant than in the salt-sensitive genotypes, differences among genotypes increasing with stress intensity. Proline did not appear to be involved in osmotic adjustment in salt-stressed rice plants and the significance of its accumulation is discussed in relation to salinity resistance.

Effects of various salts and of mannitol on ion and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) callus cultures

Mature embryo-derived calli were obtained from three rice (Oryza sativa L.) cultivars differing in salinity resistance at the whole plant level [(I Kong Pao (IKP), (salt-sensitive), Aiwu (moderately resistant) and Nona Bokra (salt-resistant)] and exposed to three iso-osmotic concentrations of NaCl, KCl, Na2SO4, artificial Sea water (ASW) and mannitol. Relative growth rates, ion content and proline accumulation were quantified after 1, 2 and 3 months of stress. hmong salt treatments, KCI was the most detrimental to callus growth in all genotypes and induced a strong increase in Cl content. The NaCl-induced inhibition of growth was lower in calli issued from Nona Bokra than in calli obtained From IKP; Na and CI accumulations as well as internal osmotic potential were lower in Nona Bokra and in Aiwu, thus suggesting a cellular component of salt resistance in these genotypes. Na obvious differences in growth were recorded among genotypes upon mannitol treatment, which appeared more detrimental than NaCl. Proline accumulation was higher in the salt-sensitive cultivar IKP, whatever the nature of the stressing agent: or the stress intensity, and did not appear to be involved in osmotic adjustment. It was concluded that specific ion toxicities are important aspects of salt stress effects on rice cells and chat proline is a symptom of injury in stressed rice calli rather than an indicator of resistance.

Ethylene production by leaves of rice (Oryza sativa L.) in relation to salinity tolerance and exogenous putrescine application

The effects of salt stress on the production of ethylene in leaves of five rice cultivars differing in salinity tolerance were investigated after 15 and 30 days of stress exposure in plants sprayed every 2 days with deionised water or putrescine 100 mu M. NaCl induced an increase in ethylene synthesis which was higher in salt-tolerant than in salt-sensitive cultivars (cvs.), Salt-stress also induced an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) content which was larger in salt-tolerant cvs, whereas a decrease in ACC conversion to ethylene suggested a reduction in ACC oxidase activity. Putrescine increased the growth and the leaf tissue viability (as indicated by the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to formazan) of salt-treated plants in all cvs. This positive effect was associated with an increase in ethylene biosynthesis through an increase in ACC content and a suppression of NaCl-induced inhibition of ACC conversion to ethylene. These data suggest that putrescine could be involved in salinity tolerance in rice.

Selection of Al-resistant plants from a sensitive rice cultivar using somaclonal variation, in vitro and hydroponic cultures

A selection procedure is described that isolated Al-resistant individuals from a sensitive rice (Oryza sativa L.) genotype. Somaclonal variation was used as the only source of variability and selection pressures were applied at both callus and regenerated plant levels. When 10 week-old, embryo-derived calli were submitted to different Al stresses for a period of 20 weeks. After this in vitro stress period, selected calli were grown during 18 weeks, without selection pressure, on regeneration media. Selection pressures were applied on regenerated plants (R0), and 9 R0 plants which produced seeds were selected. The transmission of the Al-resistant character to the R1, R2, R3 and R4 generations was then investigated, and three plant lines which had an increased percentage of Al-resistant plants till the fourth generation of self-pollination, were identified. One was from a callus maintained on an Al-free medium. The efficiency of in vitro selection pressures was therefore debated.

Evaluation of chilling sensitivity in different rice varieties. Relationship between screening procedures applied during germination and vegetative growth

SummaryRice varieties were screened for chilling tolerance during germination and vegetative growth using different techniques. The effects of temperature during germination were investigated from 10 to 25°C. The screening was most effective at 10°C. Time of data collection has to be considered in order to discriminate slow germinating from chilling sensitive varieties. Chilling survival tests (10°C) applied at the 2-leaf stage revealed that different chilling durations brought complementary results: the longer treatments allowed to identify the most tolerant varieties, while shorter treatments allowed to distinguish intermediate from most sensitive rices. Results of chilling tests applied at the 2- and the 8-leaf stages showed a high correlation. Low-temperature electrolyte leakage and low-temperature fluorescence were also highly correlated to each other and to survival tests applied at the same development stage. By contrast, weak correlations were observed between low-temperature germination and plantlet chilling survival. It is concluded that low-temperature electrolyte leakage and low-temperature fluorescence are good indicators of chilling tolerance during the vegetative growth. The possibility to use a chilling screening procedure applied at this stage in order to predict the chilling tolerance at other development stages is discussed.

Improvement of rice callus regeneration in the presence of NaCl

The effects of abscisic acid (37.8 μM), polyethylene glycol (5%), proline (10 mM), tryptophan (490 μM) and indoleacetic acid (5.7 μM) on rice callus regeneration were studied at various doses of NaCl (0, 50 and 100 mM) on three month-old mature embryo-derived callus of two japonica (I Kong Pao and Aiwu) and two indica (IR 2153 and Nona Bokra) rice cultivars differing in salinity tolerance. NaCl strongly decreased the regeneration frequency of all cultivars but slightly increased the survival of regenerated plantlets. Tryptophan stimulated regeneration and increased subsequent survival rates of regenerated plantlets in all cultivars at all NaCl doses. Abscisic acid and polyethylene glycol, though not affecting the final regeneration percentages, delayed regeneration and reduced the mean number of plantlets produced per regenerating callus in all cultivars, as well as rooting ability and survival of regenerated plantlets in indica genotypes. Proline had no marked effect on regeneration, whatever the NaCl dose or cultivar, while indoleacetic acid reduced shoot regeneration and increased root regeneration.

Modulation of plant plasma membrane H+-ATPase by phytotoxic lipodepsipeptides produced by the plant pathogen Pseudomonas fuscovaginae.

Pseudomonas fuscovaginae produces the lipodepsipeptides syringotoxin, fuscopeptin A and fuscopeptin B concurrently. These phytotoxins inhibit acidification of the external medium by fusicoccin-treated rice leaf sheath discs. When tested in vitro on H+-ATPase of rice shoot plasma membranes, syringotoxin and its structural analogue syringomycin, produced by P. syringae pv. syringae, displayed a double effect. At low concentrations they stimulated the ATPase activity of native right-side-out membrane vesicles in a detergent-like manner. At higher concentrations, however, this stimulation was reversed. With membranes treated with the detergent Brij 58, inhibition of ATPase activity was observed at low concentrations of the nonapeptides. The latter effect required the presence of an intact lactone ring formed by the nonapeptide head of these molecules. In contrast, fuscopeptins A and B inhibited enzyme activity regardless of the orientation of the vesicles. These observations were confirmed using plasma membranes from a yeast strain whose own H+-ATPase had been replaced by a single plant H+-ATPase isoform, PMA2, from Nicotiana plumbaginifolia. The kinetics of inhibition induced by the most active compound fuscopeptin B, showed a non-competitive pattern, with a Ki of about 1 microM. The combination of syringotoxin (or syringomycin) with the more hydrophobic fuscopeptins, in amounts with little or no effect, resulted in strong inhibition of the enzyme activity of rice membranes, suggesting a synergistic effect for the two types of toxins.

NaCl-tolerant plants of Poncirus trifoliata regenerated from tolerant cell lines

SummarySalt-tolerant cell lines of citrus rootstock (Poncirus trifoliata cv Pomeroy) were selected by subculturing embryo-derived calli on media containing sublethal concentrations of NaCl (5 and 10 g/l). Selected lines showed a normal growth in the presence of salt at the concentrations used for selection, and salt tolerance persisted after a passage on a salt-free media. Their K+ and Ca2+ content remained higher than in control cells for increasing NaCl concentration in the medium, suggesting a modification of cell membrane permeability as the main cause of NaCl tolerance. Shoots and plants regenerated from selected cell lines showed improved growth and salt tolerance. Calli induced from these plants tolerated a salt concentration of 10 g/l, indicating the persistance of the selected trait.

Origin of Gynogenetic Embryos of Beta-vulgaris L

Haploid plants of Beta vulgaris were obtained by gynogenesis from ovules isolated from male-fertile and annual and biennial male-sterile plants. We show that on a N6 basal medium, supplemented with 2.85 μM IAA and 0.94 μM Kinetin or 0.88 μM BAP, haploids originate directly through embryogenesis. In order to determine the optimal developmental stage of the ovule of Beta vulgaris for gynogenesis, we carried out a histological study of whole ovules from open male-sterile flowers (collected 1 to 5 days after flowering) and unopened male-fertile flowers (collected 1 to 3 days before anthesis). In all cases, the gametophyte appeared completely differentiated. These results suggest that maturity of the gametophyte is reached a few days before anthesis and therefore ovules from unopened flowers are already suitable for plating. A developmental study of the haploid cells of the sugarbeet embryo sac during the first week of in vitro culture showed that the viable gynogenetic embryo originated only from the egg cell.

Use of somaclonal variation and in vitro selection for chilling tolerance improvement in rice

Embryo-derived calli of four rice varieties cultivated at high altitude in Burundi — Facagro 57, Facagro 76, Kirundo 3 and Kirundo 9 — were submitted to different temperature regimes. The percentage of regenerating calli greatly varied depending on variety, length of culture and callus temperature treatment. The reduction of regeneration percentages induced by low temperature was more pronounced in the more sensitive varieties. Regenerated plants (R0) and their progenies in R1, R2 and R3 were cold-screened together with control plants. In all varieties, significantly higher survival rates were obtained in R3 with in vitro plants than with control plants. Such chilling tolerance improvement was not obtained following a massal selection applied during 3 successive generations onto the control plants. In vitro plants regenerated from calli cultivated either at 25 °C, either at 4 °C, were cultivated at different altitudes in Burundi during two successive generations. For most observed traits, the in vitro plants were characterized by lower means, larger variation and higher maximum values than the control plants. The most chilling-tolerant somaclonal families were most usually characterized by extensive differences in fatty acid composition, chilling-induced electrolyte leakage and chlorophyll fluorescence, compared to the varieties they derived from.