Abdelwahed Ghorbel

Water stress induced changes in the leaf lipid composition of four grapevine genotypes with different drought tolerance

To dissect differences in both lipid accumulation and composition and the role of these modifications during drought stress, four grapevine cultivars exhibiting differential tolerance to drought were subjected to water shortage. Tolerant cultivars, Kahli Kerkennah and Cardinal, exhibited higher leaf water potential (Ψw), and lower lipid peroxidation compared to the sensitive cultivars Guelb Sardouk and Superior Seedless during stress. Total lipid amounts increased during stress only in the leaves of the tolerant cultivars. Drought induced increases in the ratios digalactosyldiacylglycerol/monogalactosyldiacylglycerol and phosphatidylcholine/phoshatidylethanolamine of almost all the drought stressed cultivars. Moreover, the overall analysis of the composition of fatty acids revealed that a linolenic acid was prevalent in grapevine and the unsaturation level of lipids increased under water stress in all the cultivars. Specific adjustments in the lipid composition during stress could compromise stress tolerance.

Genetic diversity in melon (Cucumis melo L.): Anevaluation of African germplasm

The genetic diversity among 126 exotic (108) andreference array (RA) melon (Cucumismelo L.) accessions (18) was assessed byvariation at 49 random amplified polymorphic DNA marker bands(putative loci) using 29 10-mer primers. Africanaccessions of unknown melon market classes were compared to the RAaccessions from a broad range of C.melo subsp. melo groups(Cantalupensis, Conomon, Inodorus and Flexuosus). Althoughdifferences in groupings occurred after multidimensional scaling andcluster analysis, both analyses placed African accessions into twogroups, which were separate from RA groupings. One African group of33 accessions containing accessions from Zimbabwe (5),Zambia (24), Mali (1), one of two Senegalaccessions and two of three South African accessions examined. Thesecond group, which consisted of 67 accessions containing collectionsfrom Egypt (40), Tunisia (6), Libya(13), Morocco (1), Algeria (2),Ethiopia (1), Niger (1), Sierra Leone(1), S. Africa (1), Zambia (1) andZimbabwe (1). Depending on the multivariate analysistechnique employed, accessions from Kenya, Senegal and Ghana formedeither unique groupings or were grouped with accessions(Cantalupensis) from the RA. Both analyses indicate thatthe genetic differences inherent between the African gene pools isassociated with the geographic proximity of African countries(northern vs. central-southern Africa) in thegermplasm array examined. Moreover, these data indicate that thegenetic diversity of U.S. and European commercial RA germplasm(Cantalupensis and Inodorus) could be enhanced by theintroduction of genetic variation from African accessions, and thatit would be advantageous to acquire more accessions from thisgeographically and ecologically varied region to ensure the retentionof existing genetic diversity.

Identification et caractérisation d'un gène de réponse à la déshydratation « rd22 » chez la vigne (Vitis vinifera L.)

To identify and isolate genes related to abiotic stresses (salinity and drought) tolerance in grapevine, a candidate gene approach was developed and allowed isolating a full-length cDNA of rd22 gene from the Cabernet Sauvignon variety. The latter, named Vvrd22, is a dehydration-responsive gene that is usually induced by the application of exogenous ABA. Details of the physicochemical parameters and structural properties (molecular mass, secondary structure, conserved domains and motives, putative post-translational modification sites...) of the encoded protein have also been elucidated. The expression study of Vvrd22 was carried out at the berry growth stages and at the level of plant organs and tissues as well as under both drought and salt stresses. The results showed that Vvrd22 is constitutively expressed at a low level in all analyzed tissues. Moreover, salt stress induced Vvrd22 expression, particularly for the tolerant variety (Razegui), contrary to the sensitive one (Syrah), which did not display any expression variation during the stress, which means that Vvrd22 is involved in salt stress response and that its expression level depends on regulatory mechanisms that are efficient only for the tolerant variety. On the other hand, under drought stress, Vvrd22 is induced in an identical manner for both tolerant and sensitive varieties. In addition, stress signal molecules such as ABA (lonely applied or in combination with sucrose) induced Vvrd22 expression, even at a low level. A minimal knowledge about the role and the functionality of this gene is necessary and constitutes a prerequisite condition before starting and including Vvrd22 in any program of improvement of grapevines abiotic stress tolerance.

Isolation and expression analysis of salt induced genes from contrasting grapevine (Vitis vinifera L.) cultivars.

Salt stress adversely affects the growth of grapevine plants. In order to understand the molecular basis of salt stress response in grapevine plants, suppression subtractive hybridization (SSH) and microarray based screening approaches were combined. Two leaf-specific subtractive cDNA libraries were constructed from grapevine plants subjected to a moderate, incremental salt stress treatment. SSH were performed 6h and 24h after NaCl peaked at 100mM using cDNAs prepared from leaves of a salt tolerant cultivar (Razegui) as testers and cDNAs from unstressed leaves as drivers. Then, a pre-screened subset of cDNA clones from these SSH libraries were used to construct a Vitis vinifera cDNA array, in order to verify the expression changes of the genes upon salt treatment. Expression profiles were compared between the salt tolerant and a susceptible cultivar (Syrah) under both control conditions and after salt stress treatment. Seven cDNA clones were identified which were up-regulated by salt stress in two independent growth experiments and confirmed by RNA blot analysis. The transcript expression patterns of the selected genes differed between the contrasting grapevine cultivars tested with respect to stress-regulation. The possible relationship of individual cDNAs with salinity tolerance mechanisms is discussed.

Genetic diversity in African cucumber (Cucumis sativus L.) provides potential for germplasm enhancement

Genetic diversity among 26 cucumber (Cucumis sativus L. var. sativus) accessions from five African countries [Algeria (1), Egypt (21), Ethiopia (2), Kenya (1), and Libya (1)] present in the U.S. National Plant Germplasm System (NPGS) were examined by assessing variation at 71 polymorphic random amplified polymorphic DNA (RAPD) loci. Genetic distances (GD; simple matching coefficient) were estimated among these African accessions and a reference array (RA) of 21 accessions representative of the genetic variation in cucumber. GD among African accessions ranged between 0.41 and 0.97. GD among accessions in the reference array ranged between 0.36 and 0.88. Multivariate analysis identified three distinct groupings (1–3) of African accessions; Group 1 contained 21 accessions (Egypt, Ethiopia and Libya), Group 2 consisted of two accessions (Kenya, Algeria), and Group 3 possessed three accessions (Egypt). These groupings were distinct from each other (P > 0.001). Accessions in Group 1 differed genetically from all other accessions examined (P > 0.01), and accessions in Groups 2 and 3 were uniquely associated with several RA accessions. While GD among accessions in Group 1 ranged between 0.52 and 0.90, distances among Group 2 accessions varied between 0.93 and 0.97. The GD between the two accessions in Group 3 was 0.65. An accession from Syria (PI 181874) and from one Turkey (PI 199383) were genetically more similar to accessions in Group 1 than to other accessions in the RA. Likewise, accessions in Group 2 were genetically similar to two RA accessions from China and a European glasshouse cucumber line, and Group 3 accessions showed genetic affinities with the U.S. market class cultivar Dasher II. Data suggest that some Egyptian accessions (Group 1) possess unique genetic variation, that this germplasm has potential for broadening the genetic base of commerical cucumber, and that further collection of African germplasm is likely to enhance genetic diversity of cucumber in NPGS.

Population genetic structure analysis in endangered Hordeum vulgare landraces from Tunisia: Conservation strategies

Genetic markers have been employed in combination with morphological characters to identify patterns of population structure in 13 barley landrace populations from Tunisia. These endangered barley populations are grown by few local farmers in low-input farming systems. Based on 117 random amplified polymorphic DNA markers and 34 morphological traits, variance analyses indicated that most of the variation is partitioned within rather than between populations. Inbreeding index, gene flow values and cluster analysis revealed also significant differentiation between all populations. Gene flow decreased rapidly as the geographic distance increased. This may imply that seed exchange between farmers was limited to a regional scale. The lower correlation between the Euclidean distance matrices based on morphological and molecular data suggests that both data are comparably important to generate an unbiased picture of differentiation trends. Our findings support the required setting up of conservation strategies for Hordeum vulgare L. landraces from Tunisia. Key words: Barley landrace germplasm, RAPD, morphology, population differentiation analysis, conservation.Pink dombeya (Dombeya burgessiae) was tested for its potential as a flowering potted plant, using the growth retardant Cycocel® (2-chloroethyl)-trimethylammonium chloride. The treatments included a control, 0.5, 1, 2 and 3 mg/L of Cycocel® per pot and were applied when seedlings reached 7 - 8 cm in height. All treatments significantly reduced plant height. The plants treated with 0.5 mg/L were only marginally shorter than the control, while the height of plants treated with 1, 2 and 3 mg/L Cycocel®, were greatly reduced. Increased Cycocel® concentrations decreased plant width in all treatments, as compared with the control. At all applied concentrations, the deterioration of foliage greenness was observed. The highest concentration of Cycocel® (3 mg/L) resulted in cycocel-induced chlorosis. This was followed by the 2, 1 and 0.5 mg/L respectively, all showing symptoms of chlorosis; however to a lesser extent. Cycocel® treated plants exhibited greener foliage in the new leaves formed in the weeks after application compared with the control. It is important to mention that the new leaves formed in the weeks after application were not affected, and were in fact darker green with the higher concentrations of Cycocel® treatments.The fresh and dry weights of plants severely decreased with the increased Cycocel® concentrations. The highest concentration (0.3% a.i.) caused the largest reduction, with plants in this treatment only weighing 38% of the total fresh weight of the control and 35% of the total dry weight.

Molecular biology and transport properties of grapevine Na+/H+ antiporter

Na+/H+ antiporters are involved in the transport of sodium and hydrogen ions across membranes and contribute in pH regulation of actively metabolizing cells. They play a primary role in homeo-stasis and are found in every biological kingdom, from bacteria to humans to higher plants. In plants, vacuolar Na+/H+ antiporters use the proton electrochemical gradient generated by the vacuolar H+-translocating enzymes, H+-ATPase, and H+-PPiase to couple the downhill movement of H+ with the uphill movement of Na+. Moreover, it has been shown that they compartmentalize Na+ into the vacuoles for detoxification and improve consequently the salt tolerance in yeasts and plants. Recently, genes encoding these Na+/H+ antiporters have been identified and studied using a molecular genetic approach in the model systemsArabidopsis or Saccharomyces cerevisiae. We describe here the identification, cloning, molecular characterization and functional properties in yeast heterologous system of a vacuolar Na+/H+antiporter from grapevine. To identify a Na+/H+ antiporter from grapevine we applied a candidate gene approach. A 1.83-kb genomic sequence adjacent to the VvNHX1 gene was isolated using the thermal asymmetric interlaced-PCR. Histochemical localization of β-glucuronidase gene (GUS) activity was directed by VvNHX1 promoter-GUS fusion in transgenic Arabidopsis. To determine the subcellular localization of the VvNHX1 protein by heterologous expression in yeast and transient expression in onion epidermal cells, chimera constructions were prepared using a modified green fluorescent protein mGFP6. An RT-PCR approach was used to examine the VvNHX1 mRNA levels in different organs and tissues of grapevine plants. To assess VvNHX1 transport properties, VvNHX1 was expressed in the nhx1 mutant TY001 (that lacks the endogenous Nhx1 Na+/H+ antiporter) and the rates of H+-coupled transport was measured by fluorescence quenching. Rates of cation-dependent proton movements in vacuoles isolated from yeast expressing VvNHX1 were measured.

Somatic embryogenesis and plantlet regeneration from immature anthers of Opuntia ficus-indica

Summary An in vitro protocol has been developed for callus induction, somatic embryogenesis, and plant regeneration in the Barbary fig (Opuntia ficus-indica) cultivars ‘Gialla’ and ‘Moore’. Immature anthers cultivated in the dark on induction medium which consisted of Chée and Pool medium containing 2.0 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.5 mg l–1 thidiazuron (TDZ), produced primary nodular and greenish calli within 6 – 8 weeks. Periodic transfer of nodular calli on induction medium in the dark induced the formation of compact embryogenic masses after 4 – 5 months of cultivation, with induction rates of 58.97 ± 1.10% and 61.15 ± 1.16% for ‘Moore’ and ‘Gialla’, respectively. Various distinct stages were observed during the development of somatic embryos on induction medium, including pro-embryogenic, globular, heart-, torpedo-, and cotyledonary-shaped somatic embryos, as well as secondary somatic embryos. Further development of somatic embryos was accomplished using half-strength Murashige and Skoog medium containing 1% (w/v) activated charcoal under a 16-h photoperiod. Bipolarity of the somatic embryos was confirmed by environmental scanning electron microscopy. Regenerated plantlets with well-developed cladodes and roots were transferred to a greenhouse and acclimatised successfully.

Pathotype and microsatellite analyses reveal new sources of resistance to barley scald in Tunisia

We examined the variation and relationships between pathogenicity and a microsatellite-based haplotype in 79 Tunisian Rhynchosporium secalis isolates that were collected from the most commonly cultivated barley populations in Tunisia, Rihane cv. and local landraces, with the goal of finding genes that might be used to monitor resistance to scald. Isolates could be classified into three distinct virulence groups based on artificial inoculation of 19 differential cultivars with known scald resistance genes. The resistance gene BRR2 carried by the Astrix differential cultivar appeared to be the most effective in Tunisia. Pathotypes sampled from the Rihane host were more virulent than those sampled from local barley landraces. Because some differential cultivars that carried the same resistance genes showed different reaction patterns to 48 of the isolates, we postulated that other unknown resistance gene(s) specific to Tunisian isolates may be prevalent and could be used in Tunisian barley breeding programs. Microsatellite fingerprinting allowed the detection of 11 alleles linked to the virulence and pathogenic identification of 52% of the tested isolates. Thus, microsatellite analysis may provide a rapid tool for pathogen detection, without an inoculation step that requires long incubation periods before ultimate disease assessment.