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.     

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.     

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.     

Optimization of biotin and thiamine requirements for somatic embryogenesis of date palm (Phoenix dactylifera L.)

Summary This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic embryogenesis of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured on hormone-free MS medium containing biotin at 0, 0.1, 1, or 2 mg l⁻¹ combined with thiamine at 0.1, 0.5, 2, or 5 mg l⁻¹. Embryogenic callus weight, number of resultant embryos, and embryo length were significantly influenced by thiamine and biotin concentration. The optimum callus growth treatment consisted of 0.5 mg l⁻¹ thiamine and 2 mg l⁻¹ biotin. This treatment also gave the highest number of embryos. Embryo elongation was greatest at 0.5 or 2 mg l⁻¹ thiamine combined with 1 mg l⁻¹ biotin. Embryos from all treatments germinated and regenerants exhibited normal growth in soil. This study provides an insight into the importance of optimizing various culture medium components to overcome in vitro recalcitrace of date palm.     

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.     

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.     

Regulation of somatic embryogenesis in celery cell suspensions

Embryogenic suspension cultures of celery (Apium graveolens L.) were established from petiole and leaf callus. Suspensions were routinely subcultured in a maintenance medium (with 2.3 M 2,4-D and 0.88 M BA). Somatic embryogenesis occurred in this medium, but was considerably improved in a regeneration medium (2.3 M kinetin, without 2,4-D). Cultures thus maintained, contained embryogenic clumps, aggregated somatic embryos, and few free-floating singular somatic embryos. Addition of mannitol (3–4% w/v) prevented cell lysis, greatly increased the number of singular somatic embryos, improved their normal differentiation, and accelerated torpedo embryo development. Experiments to reveal the nature of the mannitol effect demonstrated that the decreased osmotic potential was an important factor, but not the only one: iso-molar solutions of sucrose alone were not as effective. The mannitol effect could be manifested after a short (2–3 days) exposure period, suggesting a trigger (induction) mechanism. Several pathways of somatic embryogenesis in celery and its regulation by subculturing, with the addition of mannitol, are outlined. Cultures thus maintained resulted in a high rate of normal somatic embryogenesis and production of normal transplantable celery 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     

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.     

Comparative study of somatic embryogenesis from immature and mature embryos and organogenesis from leaf-base of Triticale

Immature and mature zygotic embryos of hexaploid, Triticale var. DT-46 formed an embryogenic callus, with subsequent somatic embryo formation upon subculture to MS (Murashige and Skoog, 1962) or N₆ (Chu et al., 1975) nutrient medium supplemented with various concentrations (9.0–22.5 M) of 2,4-dichlorophenoxyacetic acid (2,4-D). Of the two types of explants, embryogenic tissue from immature embryos responded at a higher frequency, to form somatic embryos over the callus surface. Leaf-base segment cultured on to 2,4-D-containing medium formed a tissue which did not form somatic embryos and instead differentiated into shoot-buds. N₆ medium proved to be more effective than MS in support of somatic embryogenesis or shoot-bud formation. Regeneration of plantlets occurred on 2,4-D-free basal medium. These in vitro-formed plantlets were successfully transferred to soil and set seed.     

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.     

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.     

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.     

Regulation of somatic embryogenesis in celery cell suspensions

The regulation of somatic embryogenesis in celery (Apium graveolens L.) was studied to determine means of increasing its efficiency. Highly embryogenic cell lines were achieved by inducing cell cultures from in vitro plants which were previously regenerated from somatic embryos (secondary cell lines). The early detection of embryogenic potential of new cell lines was found to be regulated by 2,4-dichlorophenoxyacetic acid, mannitol and culture duration. Less frequent subculturing allowed embryogenic potential to be expressed earlier. Increased synchronization of celery somatic embryos was induced by two means: adding abscisic acid to the regeneration medium; and segregating the embryos by sieving them through serial metal mesh screens. When abscisic acid was removed from the growth medium, its effects became quickly transient. Embryos of 1400 µm in size provided the best growth rate and uniformity.     

Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls

Background and AimsThe programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by β-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested.MethodsWe developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action.Key ResultsIn model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide (‘ΔUA–GalA’), taken as diagnostic of PL action. ΔUA–GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA–GalA from higher homologues. The ΔUA–GalA was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA–GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA–GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA–GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol^–1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action.ConclusionsThe results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.     

The occurrence and structure of apparently bisexual flowers in the date palm, Phoenix dactylifera L. (Arecaceae)

Individuals of Phoenix dactylifera L. have expanded pistillodes or pseudocarpels in staminate flowers. These pseudocarpels are located in the centre of the male flowers and are surrounded by stamens. The gynoecium has the characteristic three carpellate arrangement commonly found in female date palm flowers. Pseudocarpels from male flower buds can expand into parthenocarpic fruit. Histology of the expanded pistillodes or pseudotarpels is similar to that of normal carpels from pistillate plants. These pseudocarpels lack ovules. Nutrient medium containing 10 mg 1^-1 of 2,4-dichlorophenoxyacetic acid or p-chlorophenylacetic acid and 0.3% activated neutralized charcoal enhanced the development and outgrowth of the pseudocarpels of cultured male flowers.     

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.     

Genetic structure of the date palm (Phoenix dactylifera) in the Old World reveals a strong differentiation between eastern and western populations

Background and Aims Date palms (Phoenix dactylifera, Arecaceae) are of great economic and ecological value to the oasis agriculture of arid and semi-arid areas. However, despite the availability of a large date palm germplasm spreading from the Atlantic shores to Southern Asia, improvement of the species is being hampered by a lack of information on global genetic diversity and population structure. In order to contribute to the varietal improvement of date palms and to provide new insights on the influence of geographic origins and human activity on the genetic structure of the date palm, this study analysed the diversity of the species.Methods Genetic diversity levels and population genetic structure were investigated through the genotyping of a collection of 295 date palm accessions ranging from Mauritania to Pakistan using a set of 18 simple sequence repeat (SSR) markers and a plastid minisatellite.Key Results Using a Bayesian clustering approach, the date palm genotypes can be structured into two different gene pools: the first, termed the Eastern pool, consists of accessions from Asia and Djibouti, whilst the second, termed the Western pool, consists of accessions from Africa. These results confirm the existence of two ancient gene pools that have contributed to the current date palm diversity. The presence of admixed genotypes is also noted, which points at gene flows between eastern and western origins, mostly from east to west, following a human-mediated diffusion of the species.Conclusions This study assesses the distribution and level of genetic diversity of accessible date palm resources, provides new insights on the geographic origins and genetic history of the cultivated component of this species, and confirms the existence of at least two domestication origins. Furthermore, the strong genetic structure clearly established here is a prerequisite for any breeding programme exploiting the effective polymorphism related to each gene pool.