Tunisian date-palm germplasm is characterized by an important genetic diversity since more than 250 ecotypes have been identified in the continental and coastal oasises of the country. However, these important phytogenetic resources are threatened either by severe genetic erosion or plagues. In order to molecularly characterize the local germplasm, we have been interested by the development of Random Amplified Microsatellite Polymorphism (RAMPO) and Amplified Fragment Length Polymorphism (AFLP) methods to generate molecular markers. Our results showed that using both markers systems, the Tunisian date-palm germplasm is characterized by a large and continuous genetic diversity. In addition and due to the greater number of markers per assay, AFLP technique seems to be more informative than RAMPO method. In fact, 186 and 428 polymorphic bands were detected using RAMPO and AFLP primers, respectively. Moreover, AFLP markers were found most polymorphic with the highest average PIC value (0.7). In addition, independent and combined cluster analyses of the RAMPO and AFLP fragments showed that cultivars are clustered independently from both trees sex and geographical origin.

The chemical composition (moisture, Brix, proteins, acidity, total polyphenol, Na, K, Ca, Mg, Fe, Cu, Zn, Mn, Ni and Cr, etc.) were determined in cactus fruits extracts belonging to many Opuntia species sampled from a Tunisian collection. Analyzed species exhibited clear differences in their fruit contents. Fruit extract derived from Opuntia ficus-indica is especially interesting because of its high content of total sugars, K and alpha-Tocopherol. The physicochemical characterization of prickly pears clearly evidenced its high nutritional potential.

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.

The present study report a protocol for the efficient in vitro propagation of kenaf (Hibiscus cannabinus L., an industrial crop having high cellulosic fiber content) on hormone free MS medium using the shoot apex and nodal explants. Shoot tips and nodes were isolated from 15 days old seedlings cultivated on MS medium. Different combinations and concentrations of auxin/cytokinin were used and added to the MS medium to assess the shoot and root induction of theses explants. Several subcultures were drived in order to enhance the multiplication rate. Healthy and well developed in vitro propagated shoots were transferred for acclimatization under greenhouse conditions in pots filled with different substrates (sand + compost or perlite). Our results showed that shoots could elongate and root within 4–6 weeks on MS basal medium without any callus formation. However, addition of growth regulators to the MS medium leaded to a decrease in shoot and root induction rates. Indeed, the highest shoot regeneration frequency (90.5%) was obtained on MS control medium. Elongated shoots were transferred onto the same hormone free MS medium using five subcultures where the multiplication rate reached the highest value (3.66) at the fifth and last step. The in vitro rooted plantlets were acclimatized in greenhouse and successfully transplanted to natural conditions with 70% survival.

The genetic diversity organization and evaluation of relationships within Tunisian date-palm cultivars were assessed using the random amplified microsatellite polymorphism (RAMPO) and amplified fragment length polymorphism (AFLP) methods. 18 combinations of random/ISSR primers and six AFLP primers combinations were tested with DNAs isolated from 40 date-palm cultivars. Our results show that using both markers systems, the Tunisian date-palm germplasm is characterized by a large and continuous genetic diversity. In addition, due to the greater number of markers per assay, the AFLP technique seems to be more informative than the RAMPO method. In fact, 186 and 428 polymorphic bands were detected using RAMPO and AFLP primers, respectively. Moreover, AFLP markers were found most polymorphic with the highest average PIC value (0.7) and marker index (50.54). In addition, independent as well as combined analyses of the cluster analyses of the RAMPO and AFLP fragments showed that cultivars are clustered independently from the sex of trees or else their geographical origin. On the other hand, based on Pearson and Spearman correlation between RAMPO and AFLP distance, matrices were positive and highly significant. This result indicates good congruence between these two molecular markers. The opportunity of the designed methods is discussed with the molecular characterization of genotypes in order to enhance the conservation and the improvement of the local date-palm germplasm.

We used simple sequence repeat markers and 25 morphological characters to characterize 18 Tunisian fig (Ficus carica L.) cultivars.Morphological traits suggested a high level of variation in the germplasm.Principal component analysis (PCA) differentiated the studied cultivars.In the derived dendrogram the cultivars clustered independently of their geographical origin and sex of trees.Simple sequence repeat (SSR) markers were used to compare genetic polymorphism with the observed phenotypic variation.Using six microsatellite primers, 39 alleles and 59 genotypes were identified.The high values of polymorphism information content (PIC), ranging from 0.67 to 0.85, confirmed the effectiveness of microsatellite analysis for determining molecular polymorphism and characterizing the studied cultivars.Multilocus genotyping unambiguously distinguished all the cultivars.The ability of each type of feature to differentiate cultivars of this crop is discussed.

Researchers have successfully micropropagated several cultivars of date palm (Phoenix dactylifera L.) using in vitro tissue culture techniques based on somatic embryogenesis and shoot organogenesis. However, major hindrances exist in scale-up including low productivity of cultures, synchronization of embryogenesis, conversion of matured somatic embryos, low multiplication rates of de novo shoots and high production cost of plantlets. Recently, tremendous success has been achieved in automation of micropropagation steps of many plant species using liquid culture systems. This achievement constitutes an alternative for resolving all the preceding problems. For example, yield of cotyledonary somatic embryos produced in suspension cultures were 17-fold greater than on agar-solidified medium. The transfer of shoot clusters in temporary immersion bioreactor clearly improved the yield of regenerated shoots 5.5-fold in comparison with that regenerated on agar-solidified medium. The aim of this chapter is to critically outline the potential of liquid culture systems (suspension culture and temporary immersion system, TIB) to large scale and automation of date palm micropropagation. The principles, advantages and disadvantages of these methods are also described.

Plant response to high salinity was assessed using DDRT-PCR on salt stressed and control barley plants (accession Kebelli II). The expression profiles showed approximately 2000 bands, out of these, 134 were up-regulated and 89 were repressed by salt stress. 13 fragments up regulated under saline stress were then reamplified, cloned and sequenced. Sequence analysis revealed that some of them present homologies with proteins in other species that are involved in cell wall structure and turnover such as serine carboxypeptidase II. A number of identified clones encoded putative stress response proteins such as NADP-dependant glyceraldehyde dehydrogenase and wound-induced protein. In addition, some of them encode gene related to resistance to anion flow-related proteins. The responses of these genes to salinity were confirmed using Northern blot or reverse Northern hybridization experiments.

Over recent decades date palm (Phoenix dactylifera L.) polymorphism and genetic diversity have been studied extensively, bringing about a revolution in this area of research. Molecular markers have been successfully designed and data have proved their efficiency in the genome assessment of this palm. In fact, these now constitute an important tool for many date palm investigations. Tunisian date palm germplasm is characterized by high genetic diversity because more than 250 cultivars have been identified. This local germplasm is, however, seriously threatened by severe genetic erosion due to diverse biotic and abiotic stresses. Evaluation of polymorphism and genetic diversity has become a prerequisite to establishment of a research program aimed at rational germplasm conservation. For that objective, research work has focused on the development of phenotypic and biochemical characterization of Tunisian date palms. Moreover, DNA-based methods have been successfully realized which permit establishment of cultivar identification keys, as well as to determine the genetic relationships among them. Data are discussed in relation to the opportunities presented by designed markers in the improvement and breeding of date palm.

Twenty one date palm (Phoenix dactylifera L.) accessions originated from different date palm groves were studied to assess the overall degree of polymorphism, detect similarities between genotypes and assess important agronomic traits. Thirty vegetative and reproductive variables were scored and subjected to multivariate analysis. Results showed a considerable phenotypic diversity among local date palm germplasm. Principal component analysis (PCA) revealed that characters related to leaflets length and spine, fruit and seed sizes accounted for a large proportion of the observed variability. Cluster analysis (CA) showed a typically continuous phenotypic diversity among date palm accessions and little associations between accessions from same geographic origin. Also, some heterogeneity within accessions that received the same denomination was evidenced. (C) 2010 Elsevier B.V. All rights reserved.

The genetic variation and relationships among 12 local barleys and the varieties Martin and Manel were evaluated using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR). A high level of polymorphism was found with both RAPD and SSR markers and the mean polymorphism information content (PIC) values were 0.477 and 0.533 for RAPD and SSR markers, respectively. In RAPD analyses, 69 out of 93 bands (74%) were polymorphic. The number of alleles ranged from 4 to 10 per primer, with an average of 6.2 per primer. The RAPD-based genetic dissimilarity (RAPD-GD) ranged from 0.114 to 0.933, with the mean of 0.523. In SSR analyses, a total of 43 alleles were detected, among which 39 alleles (90.7%) were polymorphic. The number of alleles per primer ranged from 2 to 4 with an average of 2.87 alleles per SSR primer. The SSR-derived genetic dissimilarity (SSR-GD) ranged from 0.423 to 0.910, with the mean of 0.665. SSR was better than RAPD in detecting genetic diversity among the barley accessions. A poor correlation (r = 0.193) was found between both sets of genetic similarity data, suggesting that both sets of markers revealed unrelated estimates of genetic relationships. Key words: Barley, RAPD markers, SSR markers, genetic diversity.

Juices made from fruits of 30 Tunisian accessions of pomegranate were studied for their organic acids, sugars, and anthocyanin contents, using high performance liquid chromatography. Among the detected organic acids, malic acid was the major one (>50%) followed by citric acid (>22%), while among sugars, fructose and glucose were most present in pomegranate juice contributing 53.9 and 43.4% of the total sugar content, respectively. The total anthocyanin content ranged from 9–115 mg per litre of juice with the following ranges of the six compounds found: cyanidin-3,5-diglucoside (3.1–74.4 mg/L), delphinidin-3-glucoside (0.7–22.0 mg/L), cyanidin-3-glucoside (0.8–21.0 mg/L), pelargonidin-3-glucoside (0.5–16.1 mg/L), pelargonidin-3,5-diglucoside (0.0–11.8 mg/L), and delphinidin-3,5-diglucoside (0.0–5.4 mg/L). Based on the analyzed parameters, cluster analysis allowed grouping cultivars into two main clusters. One was made of sour cultivars and the second of the sweet ones. Principle component and cluster analyses suggested that the composition of the pomegranate fruits is determined by cultivar rather than cultivation location.

In the present work, we report the development of 11 microstallite markers (SSR) for Punica granatum. Evaluated on a set of 27 pomegranate accessions sampled in Tunisia, they displayed 25 alleles, with number of alleles per locus ranging between 1 and 4, and an observed heterozygosity from 0.037 and 0.592. This set of SSR markers can be very useful for studies dealing with genetic diversity assessment of germplasm, with cultivars/varieties fingerprinting and pedigree analysis of this economically important fruit species.

The genetic diversity among Tunisian pomegranate cultivars has been investigated. Using universal primers, the random amplified polymorphic DNA (RAPD) method was used to generate banding profiles from a set of twelve cultivars. Data was then computed with appropriate programs to construct a dendrogram illustrating the relationships between the studied cultivars. Our data proved the efficiency of the designed method to examine the DNA polymorphism in this crop since the tested primers are characterized by a collective resolving power of 12.83. In addition, the cluster analysis has exhibited a parsimonious tree branching independent from the geographic origin of the cultivars. In spite of the relatively low number of primers and cultivars, RAPD constitutes an appropriate procedure to assess the genetic diversity and to survey the phylogenetic relationships in this crop.

The trnL (UAA) intron and the intergenic spacer between the 3′ exon of trnL (UAA) and trnF (GAA) sequences were used as genetic markers for differentiating Ficus carica cultivars and establishing refined genetic relationships. The study was based on 20 fig cultivars, collected from south and centre of Tunisia. Since, the intron was thought to be more variable among close relatives than is the chloroplast spacer. The size of these non-coding regions varied from 554 to 589 and from 989 to 1022 bases pairs for the intron and the combined sequences correspondingly. The average of GC content was 33.9% and 34.6% in the intron and the combined intron and spacer respectively. High values of A+T contents were detected in both data sets and may explain the high proportions of transversions founded. The observed variation pattern of plastid DNA provides evidence of an important genetic diversity. The overall transition/transversion bias (R) was 0.202 in the intron and 0.27 in the combined regions. The RI index of 0.592 indicates that these combined sequences have clearly more homoplasy then the intron (RI=0.705) and spacer (RI=0.777) sequences separately. Phylogenetic trees were generated based on maximum parsimony (MP) and neighbor-joining (NJ) analysis of the chloroplast sequences data. Results proved that a typically continuous genetic diversity characterizes the local fig germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical or tree sex correspondence. Although the level of apparent diversity is considerable, we may conclude that non-coding regions of chloroplast genome provide a new and practical opportunity to evaluate genetic diversity and to discriminate fig cultivars. Revealed cytoplasmic DNA markers are reliable to elaborate a molecular data base to conduct management and breeding programs on local fig germplasm.

Hen we report the nucleotide variation in two non-coding regions of the chloroplast DNA (cpDNA) to construct a possible evolutionary scenario in Ficus carica L Our results sugE est the occurrence of haplotypic and nucleotide diversity with a large variation level of chloroplast non-coding regions Furthermore our results demonstrated an explicit rejection of the null hypothesis that within F carica the intron trnL and the spacer trnL-trnF evolved under a strictly neutral model of molecular evolution Although recent population expansion could serve as one alternative explanation for the detected excess of singleton our results imply a positive selection and the genetic hitchhiking effect is unlikely Parameters performed supported scenario of sweep selection and recent expansion of F carica across Tunisia Our results indicate that both positive selection and demographic histories have jointly contributed to the observed patterns of nucleotide diversity and haplotypes structure Based on the results we characterize the fig resources and provide several suggestions for effective conservation and improvement programs (C) 2010 Elsevier Ltd All rights reserved

Plant regeneration through somatic embryogenesis from young leaf explants (5–10 mm long) adjacent to the apex of 5–6 year old offshoots of Tunisian date palm (Phœnix dactylifera L.), cultivar Boufeggous was successfully achieved. Factors affecting embryogenic callus initiation, including plant growth regulators and explant size, were investigated. The highest induction frequencies of embryogenic calli occurred after 6–7 months on MS medium supplemented with 10 mg l−1 2,4-D and 0.3 mg l−1 activated charcoal. The subculture of these calli onto maintenance medium resulted in the formation of proembryos. Fine chopping and partial desiccation (6 and 12 h) of embryogenic calli with proembryos prior to transfer to MS medium supplemented with 1 mg l−1 ABA stimulated the rapid maturation of somatic embryos. Maturated somatic embryo yield per 0.5 g FW of embryogenic callus was 51 embryos with an average maturation time of 55 days. This was increased to 422 with finely chopped callus, and 124 and 306 embryos following 6 and 12 h desiccation treatments, respectively. The average time to maturation for these 3 treatments was 35, 43 and 38 days, respectively. Subsequent substitution of ABA in MS medium with 1 mg l−1 NAA resulted in the germination and conversion of 81% of the somatic embryos into plantlets with normal roots and shoots. The growth of regenerated somatic plants was also monitored in the field.