Staminodes evolution and in vitro development innovation in date palm (Phoenix dactylifera L.)

The in vitro cultivation of date palm staminodes (vestigial stamens) at different stages of female floral ontogenesis confirms the persistence at an immature state of such organs at all the floral differentiation stages. This is evidenced even in fully mature female flowers. Our study revealed the advanced developmental patterns of these rudimentary structures, which bear diverse morphogenetic potentialities. In vitro cultivation of staminodes provides new opportunities for in vitro regeneration of date palm. Such developmental processes were found to be modulated by the stage of floral differentiation, which closely reflected the level of staminode maturity. Development was also impacted by the composition and concentration in plant growth regulators (NAA, BAP and 2,4-D) of the culture media. The large morphogenetic plasticity of the staminodes disposed them to evolutionary variations of the date palm reproduction system. The practical benefits (micropropagation) and the fundamental interests (evolutionary process) of our investigation are discussed.     

The date palm (Phoenix dactylifera L.) micropropagation using completely mature female flowers

This study describes an efficient and reproducible protocol for in vitro date palm propagation using mature female flowers. It focuses on the promising proliferation capacity exhibited by a number of female flower tissues taken at the final developmental stage. This capacity resided in the ability to preserve minuscule zones in a juvenile state located at the floral organ armpits (sepals and petals). The originality of this method lies in the possibility of propagation of very rare varieties, particularly the genotypes that exist in only one copy without the excision of the plant mother, the source of the tissue collected to be cultivated, which was not the case for all previous methods. The findings revealed that 2,4-D at 1mg/l, most of the varieties tested showed reactivity. The success of this technique was also noted to depend on the concurrent control of various factors pertaining mainly to the hormonal composition of the culture medium and the appropriate time of tissue transfer, which depends on the proliferation state as well as the culture period. This study describes the nature of the proliferation from the mature female flowers and their outcome, particularly those at the origin of embryogenic and budding strains and discusses the advantages of this novel multiplication method as compared to the currently available ones.     

Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.)

Peroxidase from date palm (Phoenix dactylifera L.) leaves was purified to homogeneity and characterized biochemically. The enzyme purification included homogenization, extraction of pigments followed by consecutive chromatographies on DEAE-Sepharose and Superdex 200. The purification factor for purified date palm peroxidase was 17 with 5.8% yield. The purity was checked by SDS and native PAGE, which showed a single prominent band. The molecular weight of the enzyme was approximately 55 kDa as estimated by SDS–PAGE. The enzyme was characterized for thermal and pH stability, and kinetic parameters were determined using guaiacol as substrate. The optimum activity was between pH 5–6. The enzyme showed maximum activity at 55 °C and was fairly stable up to 75 °C, with 42% loss of activity. Date palm leaves peroxidase showed K_m values of 0.77 and 0.045 mM for guaiacol and H₂O₂, respectively. These properties suggest that this enzyme could be a promising tool for applications in different analytical determinations as well as for treatment of industrial effluents at low cost.     

Proteomic analysis of the development and germination of date palm (Phoenix dactylifera L.) zygotic embryos

By using a comparative proteomic approach (2‐DE coupled to MS/MS), the development, maturation, and germination of date palm zygotic embryos, have been studied. Proteins were trichloroacetic acid (TCA)–acetone–phenol extracted and resolved by 2‐DE in the 5–8 pH range. The total protein content and the number of spots resolved increased from early (12 weeks after pollination (WAP); 68.96 mg/g DW: 207 spots) to late (17 WAP; 240.85 mg/g DW: 261 spots) stages, decreasing upon germination (from 120.8 mg/g DW: 273 spots in mature embryos to 26.35 mg/g DW: 87 spots in 15 days after germination). Up to 194 spots showed qualitative or quantitative differences between stages. Statistical analysis of spot variation was performed by PCA, obtaining a more accurate grouping of the samples and determining the most discriminant spots. Samples were also clustered based on Pearson distance and Ward's minimum distance. Sixty‐five variable spots were subjected to MS analysis, resulting in 21 identifications. The identified proteins belong to the following functional categories: enzymes of glycolysis, tricarboxylic acid cycle, and carbohydrate biosynthesis, protein translation, storage (glutelin), and stress‐related proteins. The evolution pattern of the functional groups was examined and discussed in terms of metabolism adaptation to the different embryogenic and germination stages.     

Methylation-sensitive amplification polymorphism in date palms (Phoenix dactylifera L.) and their off-shoots

DNA methylation plays an important role in the regulation of gene expression in eukaryotes. In this study, the extent and patterns of DNA methylation were assessed in date palm mother-plants and their off-shoots using the methylation-sensitive amplified polymorphism (MSAP) technique. Three types of bands were generated using 12 pairs of primers. Type I were present in both ECOR I + HPA II and ECOR I + MSP I lanes; type II were present in ECOR I + HPA II lanes, but not in ECOR I + MSP I lanes; and type III bands were present in ECOR I + MSP I lanes, but not in ECOR I + HPA II lanes. The total numbers of these three types of bands were 782, 55, and 34, respectively. Among these three types of bands, the polymorphic bands were, respectively, 37, 10, and 0. The distribution of polymorphic bands among mother-plants and off-shoots suggests the methylation variation was present in both the mother-plants and off-shoots. Forty- four out of these 47 polymorphic bands show clear difference between mother-plant and off-shoots: 38 were present only in off-shoots and 6 in both mother-plants and off-shoots. Compared to methylation status in mother-plants, the methylation variation during off-shoot growth of date palm can be characterized as a process involving primarily de-methylation. Hypomethylation of DNA in off-shoots, compared with mother-plants, reflects the marked expression of this molecular feature, which may be related to gene expression during off-shoot development. The methylation or de-methylation status of specific loci in the mother-plants and their off-shoots were probably random events.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.     

Molecular phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting

Genetic diversity among 13 different cultivars of date palm (Phoenix dactylifera L.) of Saudi Arabia was studied using random amplified polymorphic DNA (RAPD) markers. The screening of 140 RAPD primers allowed selection of 37 primers which revealed polymorphism, and the results were reproducible. All 13 genotypes were distinguishable by their unique banding patterns produced by 37 selected primers. Cluster analysis by the unweighted paired group method of arithmetic mean (UPGMA) showed two main clusters. Cluster A consisted of five cultivars (Shehel, Om-Kobar, Ajwa, Om-Hammam and Bareem) with 0.59–0.89 Nei and Li's coefficient in the similarity matrix. Cluster B consisted of seven cultivars (Rabeeha, Shishi, Nabtet Saif, Sugai, Sukkary Asfar, Sukkary Hamra and Nabtet Sultan) with a 0.66–0.85 Nei and Li's similarity range. Om-Hammam and Bareem were the two most closely related cultivars among the 13 cultivars with the highest value in the similarity matrix for Nei and Li's coefficient (0.89). Ajwa was closely related with Om-Hammam and Bareem with the second highest value in the similarity matrix (0.86). Sukkary Hamra and Nabtet Sultan were also closely related, with the third highest value in the similarity matrix (0.85). The cultivar Barny did not belong to any of the cluster groups. It was 34% genetically similar to the rest of the 12 cultivars. The average similarity among the 13 cultivars was more than 50%. As expected, most of the cultivars have a narrow genetic base. The results of the analysis can be used for the selection of possible parents to generate a mapping population. The variation detected among the closely related genotypes indicates the efficiency of RAPD markers over the morphological and isozyme markers for the identification and construction of genetic linkage maps.Communicated by H.F. Linskens     

Evaluation of extraction methods for the identification of proteins from date palm (Phoenix dactylifera L.) seed and flesh

Date fruits are well known to be very nutritious. Nevertheless, the protein contents of the fruit, particularly the seed and flesh, are still understudied, largely due to their difficult physical characteristics. This study was conducted to compare three different protein extraction methods which were the trichloroacetic acid (TCA)-acetone (TCA-A), phenol (Phe), and TCA-acetone-phenol (TCA-A-Phe), and to perform proteomic analysis on date palm seed and flesh. Phe extraction method showed the highest protein yields for both seed (8.26?mg/g) and flesh (1.57?mg/g). Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Phe, and TCA-A-Phe extraction methods were shown to be efficient in removing interfering compounds and gave well-resolved bands over a wide range of molecular weights. Following liquid chromatography–tandem mass spectrometry analysis, about 50–64% of extracted proteins were identified with known functions including those involved in glycolysis, Krebs cycle, defense, and storage. Phe protein extraction method was proven to be the optimal method for date flesh and seed.     

Structure and biochemistry of endosperm breakdown in date palm (Phoenix dactylifera L.) seeds

Summary The zone of endosperm breakdown in the germinated date seed (Phoenix dactylifera L.) is a narrow area immediately adjacent to the surface of the enlarging cotyledon, or haustorium. The zone width is correlated with the amount of cell division in the adjacent region of the haustorium. The sequence of endosperm breakdown is: 1. protein bodies vacuolate, 2. storage cell walls become electron-transparent immediately adjacent to the protoplast of each endosperm cell, 3. all remaining cytoplasm and lipid bodies disappear, and 4. the remaining cell walls become electron-transparent and collapse against the haustorium surface. Two cell wall hydrolases are present—endo-mannanase (EC3.2.1.78) and -mannosidase (EC3.2.1.25). -mannosidase is detectable in the endosperm before germination. At germination, the major portion of activity is found in the softened endosperm. -mannanase is only detectable from germination and there is always hundreds of fold greater activity in the softened endosperm than elsewhere. Proteinase is detectable in trace amounts at germination in the softened endosperm but is also found in the haustorium at later stages. Isolated haustoria, incubated in extracted ivory nut (Phytelephas macrocarpa) mannan in buffer, cause no mannan breakdown. Haustoria, incubated in a solution of locust bean galactomannan, cause no decrease in galactomannan viscosity. Our observations suggest that although haustoria probably regulate mannan breakdown in the endosperm, they do not seem to secrete the hydrolytic enzymes concerned.     

Effect of GA3 and 2,4-D foliar application on the anatomy of date palm (Phoenix dactylifera L.) seedling leaf

Two concentrations (10-5M and 10-3M) of both GA3 and 2,4-D were used as foliar spray to evaluate the response of date palm (Phoenix dactylifera L.) cv. Khedri seedlings. They affected some of the anatomical characteristics of the first leaf emerging after the beginning of the spray. The high concentration of GA3 increased the size of the midrib and its vascular bundle numbers. Both low and high concentrations of 2,4-D inhibited the formation of the midrib. 2,4-D in both low and high concentrations decreased the number of vessels in both protoxylem and metaxylem and also decreased their diameters, where as GA3 in low and high concentrations have less effect on the number of vessels and its diameters. GA3 in high concentration increased the number of vascular bundles in 1mm long of the leaf blade, while 2,4-D in low and high concentrations decreased their numbers. 10-3M of 2,4-D increased the size and layers of special hypodermal cells.     

Nuclear microsatellite markers for the date palm (Phoenix dactylifera L.): characterization and utility across the genus Phoenix and in other palm genera

A (GA)_n microsatellite‐enriched library was constructed and 16 nuclear simple sequence repeat (SSR) loci were characterized in Phoenix dactylifera. Across‐taxa amplification and genotyping tests showed the utility of most SSR markers in 11 other Phoenix species and the transferability of some of them in Elaeis guineensis, 11 species of Pritchardia, Pritchardiopsis jeanneneyi and six species of Astrocaryum. The first to be published for P. dactylifera, these new SSR resources are available for cultivar identification, pedigree analysis, germplasm diversity as well as genetic mapping studies.     

SCoT-marker analysis of Oryzaephilus surinamensis L. (Coleoptera: Silvanidae) and stored date kernels of Phoenix dactylifera (L.) fumigated with ozone and phosphine gases

Oryzaephilus surinamensis, a common grain product pest around the world, particularly causing great damage and losses to the date crops available in the storage. It has been traditionally resisted by using organic pesticides despite suspicions of their harmful effects on dates. This study aimed at the evaluation of the usage of ozone gas as an effective pesticide and also focuses on the safety alternatives provided by the respective method as compared to the usage of phosphine as a pesticide. While studying the ozone effects, the O. surinamensis was exposed to the gas for a variety of time scales (2, 4 and 6 h), distinctly for every life stages of the insect such as egg, larva and adults, infested the Phoenix dactylifera L date palm species present in the Siwa oasis. The obtained results showed that the adult insects had high resistance to ozone gas, while the larvae and egg stages were less resistant. The reduction rate of vital insect was 100% in all stages. Start codon analysis of DNA also showed that there are some SCoT primers can be identified and differentiated between the different treatments and control. On the other hand, the percentage of polymorphisms in insects was 50% while in date kernels 25% by six SCoT primers used. This study highlighted higher efficacy and safety regarding the usage of ozone gas in effectively controlling the manifestation of O. Surinamensis and thereby reducing the loss of stored date crops as compared to the phosphine.     

Discovery of the human homolog of sex-determining region (SRY) gene in dioecious plants

Sex determination in the early developmental stages of dioecious crops is economically-beneficial. During this study, a human homology of SRY gene was successfully identified in dioecious crops. SRY gene sequences of date palm and jojoba were submitted to GenBank under the accession numbers KC577225 and MK991776, respectively. This is the first report regarding the novel sex-determination methodology of four dioecious plants (jojoba, date palm, papaya, and pistachios). SRY sex gene was found in all the tested dioecious plant and human samples. This novel approach is simple and of significant importance for breeders. It facilitates the unambiguous selection of jojoba and date palm female plants at an early age and reduces the plantation cost of cultivating non-productive male plants. This is a rapid sex-determination technique for dioecious plants and mammals at an early stage. This technique specifically targets the SRY sequence that has been comprehensively investigated in humans. The kit development for the SRY-based sex determination of various crops is in progress.     

Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.)

Background

Clp/Hsp100 chaperones are involved in protein quality control. They act as independent units or in conjunction with a proteolytic core to degrade irreversibly damaged proteins. Clp chaperones from plant chloroplasts have been also implicated in the process of precursor import, along with Hsp70 chaperones. They are thought to pull the precursors in as the transit peptides enter the organelle. How Clp chaperones identify their substrates and engage in their processing is not known. This information may lie in the position, sequence or structure of the Clp recognition motifs.

Results

We tested the influence of the position of the transit peptide on the interaction with two chloroplastic Clp chaperones, ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). The transit peptide of ferredoxin-NADP⁺ reductase was fused to either the N- or C-terminal end of glutathione S-transferase. Another fusion with the transit peptide interleaved between two folded proteins was used to probe if AtClpC2 and AtClpD could recognize tags located in the interior of a polypeptide. We also used a mutated transit peptide that is not targeted by Hsp70 chaperones (TP1234), yet it is imported at a normal rate. The fusions were immobilized on resins and the purified recombinant chaperones were added. After a washing protocol, the amount of bound chaperone was assessed. Both AtClpC2 and AtClpD interacted with the transit peptides when they were located at the N-terminal position of a protein, but not when they were allocated to the C-terminal end or at the interior of a polypeptide.

Conclusions

AtClpC2 and AtClpD have a positional preference for interacting with a transit peptide. In particular, the localization of the signal sequence at the N-terminal end of a protein seems mandatory for interaction to take place. Our results have implications for the understanding of protein quality control and precursor import in chloroplasts.     

Physicochemical and Functional Properties of Typical Tunisian Drink: Date Palm Sap (Phoenix dactylifera L.)

The present work aimed to study the physicochemical characteristics and the functional properties of the male date palm sap (Phoenix dactylifera L.). The surface properties at the air–water interface were studied on the basis of the drop volume method. Foaming properties (foam capacity and stability) were evaluated using bubbling method by optical and conductimetric measurements (foamscan). Composition analysis revealed a high sugar content (92.29% w/w dry matter basis) with dominance of sucrose. Sap contains also 5.14% w/w of proteins and 2.57% w/w of ash. Proteins are probably the source of the surface activity and the observed foam power. At higher contents of dry matter, lyophilized sap solutions showed Newtonian comportment and improved surface activity and foam properties. Results present some interesting functional properties that suggest to deepen research particularly in sap proteins.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

In vitro plant regeneration and ethylmethanesulphonate (EMS) uptake in somatic embryos of date palm (Phoenix dactylifera L.)

The effect of the auxins dicamba (3,6-dichloro-2-methoxybenzoic acid) and picloram (4-amino-3,5,6-trichloropicolinic acid) on callus growth and embryogenesis in Phoenix dactylifera L. was investigated. Maximum callus fresh weight was obtained in nutrient medium enriched with 200 µm picloram. Somatic embryogenesis and subsequent plant regeneration was achieved following transfer of such calli to hormone-free medium. Germination of the somatic embryos was influenced by treatment with the chemical mutagen ethylmethanesulphonate (EMS). Uptake of the labelled mutagen ([¹⁴C]EMS) by the somatic embryos increased with increased incubation time. Presence of dimethyl sulphoxide (DMSO) as a carrier agent during mutagenic treatment was necessary for efficient mutagen uptake.     

An improved method for micropropagation and regeneration of date palm (Phoenix dactylifera L.)

We developed a novel large-scale micropropagation pathway for date palm (Phoenix dactylifera L.) based on organogenesis. We obtained organogenic stems from shoot tip explants of the Moroccan date palm cultivar Najda, and investigated shoot proliferation from these organogenic stems in vitro on various media; Beauchesne medium (BM) and Murashige and Skoog medium (MS) at full-strength, half-strength, and one-third-strength, containing various concentrations (0, 0.25, 0.5, and 1 mg/L) of 2-naphthoxyacetic acid (NOAA) and kinetin. The optimal medium during the multiplication phase was half-strength Murashige and Skoog medium (MS/2) supplemented with 0.5 mg/L NOAA and 0.5 mg/L kinetin (23.5 morphologically superior shoots per explant, with low vitrification rates). For the shoot elongation phase, shoots were transferred to the same proliferation medium, or to MS or MS/2 media without plant growth regulators (PGRs). Shoots elongated rapidly and showed a high rate of root formation on media supplemented with PGRs. For example, on MS/2 medium containing 1 mg/L NOAA and 1 mg/L kinetin, the average shoot length was 15.1 cm, the average number of roots per shoot was 6.2, and their average length was 3.4 cm. On PGR-free media, shoots were shorter with wider and greener leaves, and had fewer roots. The plantlets were transferred to a greenhouse for acclimation. The survival rate after 2 months was related to the medium used during the elongation phase; >90 % of shoots that were cultured on PGR-free media survived, while there was a poor survival rate of shoots that had been cultured on media containing PGRs.