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.     

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.     

An optimised protocol for plant regeneration from embryogenic suspension cultures of date palm,Phoenix dactylifera L., cv. Deglet Nour

An improved protocol is described for the large-scale micropropagation of an elite date palm ( Phoenix dactylifera L.) cultivar, Deglet Nour. Clonal plants were regenerated from somatic embryos derived from highly proliferating suspension cultures. Friable embryogenic calli were initiated from both leaf and inflorescence explants. Suspension cultures consisting of pro-embryonic masses were established from calli showing a high competency for somatic embryogenesis. The subculture of suspensions in liquid medium enriched with low amounts of plant growth regulators (1 mg l(-1) 2,4-dichlorophenoxyacetic acid with 300 mg l(-1) charcoal) resulted in the differentiation of large numbers of somatic embryos. The productivity of the cultures increased 20-fold (from 10 to 200 embryos per month per 100 mg fresh weight of embryogenic callus) when embryogenic suspensions were used instead of standard cultures on solid media. The overall production of somatic embryos reached 10,000 units per litre per month. Partial desiccation of the mature somatic embryos, corresponding to a decrease in water content from 90% to 75%, significantly improved germination rates (from 25% to 80%). The cutting back of the cotyledonary leaf was also found to stimulate embryo germination. Flow cytometric analysis showed that the micropropagation protocol followed here did not affect the ploidy level of somatic embryo-derived plantlets.     

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.     

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.     

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.     

Effect of pollination intensity on fruit and seed set in cacao (Theobroma cacao L.)

We studied the functional relationship between pollination intensity and fruit survival as well as the number of seeds per pod in the tropical tree Theobroma cacao L. on a Forastero Upper-Amazon clone (UPA 409) in Ivory Coast. Cutting the style 24 h after pollination allowed for counting the number of pollen grains deposited on a stigma without affecting fruit set and seed development. Forty-three pollen grains were necessary to reach 50% of maximum fruit set 28 days after pollination. Above 115 pollen grains, the proportion of developing ovaries reached a maximum of 88% 28 days after pollination and 75% at maturity. With fewer than 238 pollen grains per stigma, there was a close relationship between pollination intensity and number of seeds per pod; the pollenseed ratio increased from 1.61 to 3.81 for PI increasing from 30 to 238 pollen grains. For higher pollination intensities, the average number of seeds per pod reached a maximum of 58. The relationship between pollination intensity and seed content was modelled. Results are consistent with the hypothesis that ovules attracted pollen tubes in a similar way regardless of whether or not they had already been reached by another pollen tube.     

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).     

Comparison of two cacao (Theobroma cacao L.) clones for the effect of pollination intensity on fruit set and seed content

We compared the influence of pollination intensity (PI) on fruit set and seed number per pod in two cacao (Theobroma cacao L.) clones, IFC5 (Forastero Lower-Amazon Amelonado), which is self-compatible and known to produce a high number of seeds per pod under open pollination, and SCA6 (Forastero Upper-Amazon), which is self-incompatible and known to produce a low number of seeds per pod under open pollination. With both clones, PI had a positive effect on fruit set, with the maximum rate requiring more than 150 pollen grains per pod. One-half of the maximum rate of fruit set was reached with 39 pollen grains per pod for SCA6, and 78 for IFC5. With SCA6, a significant positive effect of PI on seed number per pod was also observed, with maximum seed number requiring more than 200 pollen grains per pod. In contrast, seed numbers with IFC5 were approximately equal over the PI range 100–800 pollen grains per pod. Patterns of seed number per pod were compared after: (1) open insect pollination, (2) low-intensity hand pollination and (3) high-intensity hand pollination. The patterns obtained with IFC5 under open pollination showed a peak of 40–50 seeds per pod, whereas the distribution patterns were more even with SCA6. The pattern of seed number under open pollination was similar to that obtained with high-intensity hand pollination for IFC5, and with low-intensity hand pollination for SCA6. We concluded that the high number of seeds per pod observed with IFC5 under natural insect pollination may be explained by a high number of compatible self-pollen grains on the stigma and by a severe drop of low-pollinated flowers eliminating potential lowfilled fruits. With SCA6, however, the number of compatible pollen grains deposited on the stigma was probably low under open pollination, and the flowers required lower pollen quantities to set fruit, which resulted in a high frequency of low seed numbers per fruit. This difference in the capacity to set low-seeded fruits might be considered as an adaptive trait related to the mode of reproduction.     

Effect of water deficit on the cell wall of the date palm (Phoenix dactylifera ‘Deglet nour’, Arecales) fruit during development

Date palm (Phoenix dactylifera) is an important crop providing a valuable nutrition source for people in many countries including the Middle East and North Africa. In recent years, the amount of rain in North Africa and especially in the Tunisian palm grove areas has dropped significantly. We investigated the growth and cell wall remodelling of fruits harvested at three key development stages from trees grown with or without water supply. During development, cell wall solubilization and remodelling was characterized by a decrease of the degree of methylesterification of pectin, an important loss of galactose content and a reduction of the branching of xylan by arabinose in irrigated condition. Water deficit had a profound effect on fruit size, pulp content, cell wall composition and remodelling. Loss of galactose content was not as important, arabinose content was significantly higher in the pectin‐enriched extracts from non‐irrigated condition, and the levels of methylesterification of pectin and O‐acetylation of xyloglucan were lower than in irrigated condition. The lower levels of hydrophobic groups (methylester and O‐acetyl) and the less intensive degradation of the hydrophilic galactan, arabinan and arabinogalactan in the cell wall may be implicated in maintaining the hydration status of the cells under water deficit.     

Effects of parentage,prior fruit set and pollen load on fruit and seed production in Campanula americana L

Summary We conducted a controlled crossing experiment to examine the effects of maternal and paternal parentage, the size of the pollen load, and prior fruit production on the proportion of flowers that set fruit, seed number per fruit and seed weight in a natural population of Campanula americana. Effects due to the maternal parent were large for all measures of fruit and seed production, while the paternal parent had a significant effect only upon mean seed weight. As the number of prior fruits on the maternal plant increased the probability that a flower would produce a mature fruit, the number of seeds per fruit, and total seed weight per fruit all decreased. We found no effect of the size of the pollen loads used in this study on fruit or seed production. These results are consistent with those of other studies that suggest in natural plant populations maternal effects, especially environmental maternal effects, can have an overwhelming effect on fruit and seed production and on seed characteristics.     

Consequence of salinity and excess boron on growth, evapotranspiration and ion uptake in date palm (Phoenix dactylifera L., cv. Medjool)

Patches of common juniper (Juniperus communis L.) shrubs potentially facilitate the formation of fertile islands in heath tundra ecosystems thereby influencing the long-term resilience of these ecosystems. Although the role of juniper in the formation of such ‘islands of fertility’ has been studied in semiarid landscapes, there has been little attention paid to the importance of juniper in other ecosystems. In this study we contrast the soil fertility and rates of N fixation under juniper shrubs with that in open heath tundra in northern Sweden. Plots were established at several individual sites in alpine heath tundra in Northern Sweden and mineral soils to a depth of 10 cm were characterized for available N and P and total C, N, P, Ca, Mg, K, Fe, Mn, Zn, and Cu. Nitrogen fixation rates were measured by acetylene reduction in feather mosses under juniper canopies and contrasted with N fixation in both feather mosses and surface soils in the open heath. Soils under juniper had concentrations of total P greatly in excess of P in open heath, furthermore, juniper islands had the highest concentrations of bioavailable P. Nitrogen fixation rates in the feather moss Pleurozium schreberi (Bird.) Mitt were approximately 150 μmol acetylene reduced m⁻² d⁻¹ under the juniper canopy compared to less than 10 μmol acetylene reduced m⁻² d⁻¹ in the open heath. Feather mosses under the juniper canopy also fixed N at a significantly higher rate (on an aerial basis) than that of surface cores from the open heath that included lichen, mosses, and soil crusts. Juniper facilitates the formation of islands of soil fertility that may in turn facilitate the growth of other plants and positively influence the long term recovery of heath tundra ecosystems following disturbance.     

Effect of biofertilizers and putrescine amine on the physiological features and productivity of date palm (<Emphasis Type="Italic">Phoenix dactylifera,</Emphasis> L.) grown on reclaimed-salinized soil

Key message

Plant growth promoting rhizobia bacteria and mycorrhizae in the presence of putrescine allows and/or enables date palm to increase its adaptation to reclaimed/salinized areas.

Abstract

Amines and biofertilizers play an important role in a plant’s response to adverse environmental conditions including salt and osmotic stress. This study investigates the integration effect of putrescine amine (Put), biofertilizers, and mycorrhizae (My) on the date palm zaghloul genotype irrigated by saline water and grown in reclaimed saline soil. The data collected indicates that selected plant growth promoting rhizobacteria, in the presence of Put, enables the date palm zaghloul genotype to increase its tolerance and adapt to stress conditions in the reclaimed saline soil. Overall, treatments reduced salt-induced oxidative damage in the date palm, resulting in increased productivity and improved fruit quality. The results observed may be a consequence of the increase in photosynthetic pigments, activities of antioxidant enzymes, organic solutes and/or promoting growth substances such as gibberellic acid (GA₃), auxins (IAA) and cytokinin. Moreover, a decrease in the levels of lipid peroxidation and inhibitor substances such as abscisic acid (ABA) may be related. The most effective interaction treatments were seen at 2.5 mM Put due to an increase in the activities of ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD) while a decrease in lipid peroxidation was noticed. The combination of Put with plant growth promoting rhizobacteria (PGPR) at a 5 mM concentration as biofertilizer increased diamine oxidase (DAO) and polyamine oxidase (PAO) activities when compared to the other treatments. The activity of these two enzymes can produce hydrogen peroxide (H₂O₂), which may act in structural defense as a signal molecule and decrease the production of polyamines against salt-induced oxidative damage in date palm. We believe that further investigation is needed to understand the tolerance/adaptation mechanisms in date palm grown under stress condition.