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.     

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     

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.     

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.     

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.     

Insights into the historical biogeography of the date palm (Phoenix dactylifera L.) using geometric morphometry of modern and ancient seeds

Aim The main purpose of this work is to understand the origin, history, historical biogeography and mechanisms of date palm (Phoenix dactylifera L.) domestication. Location Seeds of uncultivated Phoenix individuals from isolated Oman populations, cultivated date palm varieties of various geographical origins and other related Phoenix species were analysed. Additionally, well‐preserved seeds from Egyptian archaeological sites (14th century bc to 8th century ad ) were compared with the morphometric reference model based on the analysis of modern material. Methods Elliptic Fourier transforms (EFT), a morphometric method applied to shape outline analysis, were used to characterize seed shape and to quantify morphological diversity in P. dactylifera and related species. Results Analysis of seed outlines by EFT (1) showed that P. dactylifera can be differentiated from other Phoenix species and (2) enabled the quantification of patterns of shape differentiation in the genus Phoenix at different taxonomic, geographical and chronological levels. Date palm agrobiodiversity, partitioned in distinct morphotypes, appeared to be complex in terms of geographical structure. Allocation of archaeological seeds to different modern Phoenix forms and date palm morphotypes allowed us to reveal ancient forms consumed and/or exploited in Egypt and finally to determine spatial and temporal changes in agrobiodiversity. Main conclusions Based on the morphological diversity quantified in P. dactylifera and related species, we characterized ancestral seed shape features present in uncultivated populations. The geographical distribution pattern of seed shapes points to human dispersal routes that spread cultivation from one or more initial ‘domestication centres’. Finally, this work provides a powerful tool to identify ancient forms as demonstrated by the analysis of well‐preserved Egyptian archaeological seeds, dating from the 14th century bc to the 8th century ad . Results open new and fascinating perspectives on the investigation of the origins and chrono‐geographical fluctuation of date palm agrobiodiversity.     

A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.     

Histocytological analysis of callogenesis and somatic embryogenesis from cell suspensions of date palm (Phoenix dactylifera)

• Background and Aims The date palm is a dioecious perennial species of the Arecaceae for which in vitro micropropagation is essential to ensure the renewal of palm plantations. This study presents a histocytological analysis of the traditional Mauritanian Amsekhsi cultivar beginning from the initial callogenesis and continuing up to the establishment of the cellular embryogenic cell suspensions. The formation of somatic embryos and their development into rooted plants are also described.• Methods Foliar segments of seedlings cultured in the presence of 2,4-D produced primary calli that were chopped to produce fine friable granular calli that subsequently produced cellular suspensions when transferred to liquid medium. The somatic proembryos that developed after removal of the 2,4-D were plated on agar medium where they developed into rooted plants. Thin sections of tissue fragments taken at each stage of the process were stained using Periodic Acid Schiff and Naphthol Blue-Black.• Key Results The first cellular divisions were localized close to the vascular vessels of the leaf. The primary calli were obtained within 2 months. Fine friable granular calli grew quickly after the primary calli were chopped. Individual embryogenic cells were identified that rapidly started to divide and developed into globular proembryos. In addition, in the microcalli, breaking zones appeared in the thick pectocellulosic walls which delimited the pluricellular proembryos. The anatomy of somatic embryos is similar to that of zygotic embryos despite a deficit in the accumulation of intracellular proteins. When rooted with NAA, the vitroplants developed a strong orthotropic taproot.• Conclusions This study contributes to understanding the whole process of somatic embryogenesis, but two specific questions remain to be answered: what factors are involved in the reactivation of the somatic cells at the beginning of the initial callogenesis, and why do the somatic embryos not accumulate proteins in their tissues during maturation?     

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.     

A neutral beta-D-glucan from dates of the date palm,Phoenix dactylifera L

Polysaccharides extracted from Libyan dates with hot water and 0.05 M NaOH were fractionated and purified by ion-exchange and gel-filtration chromatography. According to the methylation and hydrolysis analyses, the results indicate the D-glucan to be linear and to contain both (1-->3)- and (1-->4)-linkages. The anomeric NMR measurements confirm that the sugar residues are beta-glycosidically linked. This is the first report on the isolation of a neutral beta-D-glucan from dates.     

Identification and sequencing of Date-SRY Gene: A novel tool for sex determination of date palm (Phoenix dactylifera L.)

Dioecism has always been an issue in many plant species with its numerous disadvantages, especially in woody trees such as date palms. As one of the most important crops in the Middle Eastern countries, researchers are having problems identifying of sex of the plant in its early stages of development. Hence, proper population stands in the male: female ratio for maintenance is almost impossible in the field for better production. In this study, sex determination of date palm (Phoenix dactilyfera L.) were identified in regions of the Y chromosome (Date-SRY) gene, the pivotal gene that initiates sex determination, using a new technique and thus an economically desirable objective, which will significantly impact profits in seed based cultivations. Partial sequences of the Date-SRY were taken and amplified by nested polymerase chain reaction (PCR). According to the results, the exact sex of date palm was identified in all the tested plants, while amplified regions of the Date-SRY gene closely matched with the human and papaya sequences. In addition, a primer pair was designed to amplify the sequences of the SRY-date gene with confidence that it will identify male date palms. These primer sequences include SRY-date Forward 5′- cggccctctaagtatctgtgcgcaacg-3′ (SRY-date F) and the SRY-date Reverse 5′- gtttgcacttcgaagcagag-3′ (SRY-date R). The complete sequence of the DNA has been registered and deposited in GenBank (BankIt1598036 DPSRY1 KC577225 thenKJ873056).     

Microsatellite markers reveal high genetic diversity in date palm (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) germplasm from Sudan

Genetic diversity in date palm germplasm from Sudan representing 37 female and 23 male accessions was investigated using 16 loci of microsatellite (SSR) primers. Eight female accessions from Morocco were included as reference material. The tested SSR markers showed a high level of polymorphism. A total of 343 alleles were detected at the 16 loci. The number of alleles per marker ranged from 14 to 44 with an average of 21.4 per locus. A high level of expected heterozygosity was observed among Sudan cultivars (0.841), Morocco cultivars (0.820) and male accessions (0.799). The results indicate that the genetic groups of the Sudan cultivars and/or males do not follow a clear geographic pattern. However, the morocco group showed significant differentiation in relation to the Sudan groups, as measured by F (ST) values and genetic distances. The effect of the methods of pollination and cultivar selection on the genetic structure was clearly detected by the weak clustering association that was observed for the majority of accessions originating from Sudan and Morocco as well. This suggests the need for further investigation on the genetic diversity of Sudanese date palm germplasm. A deeper insight will be revealed by a detailed analysis of populations originating from different geographic locations.     

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.     

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.     

Comparative 2-DE proteomic analysis of date palm (Phoenix dactylifera L.) somatic and zygotic embryos

Two-dimensional gel electrophoresis coupled to mass spectrometry has been used to compare the proteome of date palm (Phoenix dactylifera L. cv. Deglet Nour) zygotic and somatic embryos. Proteins were trichloroacetic acid–acetone–phenol extracted, quantified, and resolved by 2-DE in the 5 to 8 pH range. Total protein content and number of resolved spots were higher in zygotic (110 ± 14.5 mg/g DW; 349 spots) than in somatic (70.96 ± 4.8 mg/g DW; 210 spots) embryos. The 2-DE map of both systems showed qualitative (263) and quantitative (72) differences. Statistical analysis of spot intensity was performed by PCA, obtaining two accurate groupings of the samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with average linkage algorithm of the Genesis software package. Sixty-three variable spots were subjected to mass spectrometry analysis, resulting in 23 identifications. Identified proteins were classified in the following functional categories; glycolysis (8 proteins), citrate cycle (1), ATP synthesis (1), carbohydrate biosynthesis (2), amino acids metabolism (1), stress related (4), storage (3), and with no function assigned for three of them. Most of the somatic embryo specific proteins identified belonged to glycolysis pathways, whereas those of the zygotic embryo to storage and stress-related proteins. Differences are discussed in terms of metabolism and biology of both types of embryos.     

An alkali-soluble heteroxylan from seeds of Phoenix dactylifera L

Alkali-soluble polysaccharides, isolated from the seeds of dates, have been investigated using methylation and partial hydrolysis studies. The polysaccharides are shown to contain D-xylose and 4-O-methyl-D-glucuronic acid in a molar ratio of 5:1. An aldobiouronic acid from hemicellulose was characterized, and investigation revealed that the hemicellulose consists of a polymer of (1-->4)-linked D-xylopranosyl residues having branches of D-xylopyranosyl and 4-O-methyl-alpha-D-glucopyranosyluronic acid.     

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.