In-site and Ex-site Date Palm Exposure to Heavy Metals Involved Infra-Individual Biomarkers Upregulation.

As a tree of considerable importance in arid regions-date palm, Phoenix dactylifera L. survival in contaminated areas of Sfax city has drawn our attention. Leaf samples of the plants grown in the study area showed high levels of cadmium (Cd), copper (Cu), and chromium (Cr). On the basis of this finding, the cellular mechanisms that explain these metal accumulations were investigated in controlled conditions. After four months of exposure to Cd, Cr, or Cu, high bioconcentration and translocation factor (TF>1) have been shown for date palm plantlets exposed to Cd and low TF values were obtained for plantlets treated with Cr and Cu. Moreover, accumulation of oxidants and antioxidant enzyme activities occurred in exposed roots to Cu and Cd. Secondary metabolites, such as polyphenols and flavonoids, were enhanced in plants exposed at low metal concentrations and declined thereafter. Accumulation of flavonoids in cells may be correlated with the expression of the gene encoding Pdmate5, responsible for the transport of secondary metabolites, especially flavonoids. Other transporter genes responded positively to metal incorporation, especially Pdhma2, but also Pdabcc and Pdnramp6. The latter would be a new candidate gene sensitive to metallic stress in plants. Expressions of gene coding metal chelators were also investigated. Pdpcs1 and Pdmt3 exhibited a strong induction in plants exposed to Cr. These modifications of the expression of some biochemical and molecular based-markers in date palm helped to better understand the ability of the plant to tolerate metals. They could be useful in assessing heavy metal contaminations in polluted soils and may improve accumulation capacity of other plants.

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.

Transcriptome assembly and abiotic related gene expression analysis of date palm reveal candidate genes involved in response to cadmium stress.

Date palm in Tunisia is of major economic importance but are also factors of social, environmental and economic stability. An annotated assembly of the transcriptome of cultivar Deglet Nour was reported. RNA was isolated from plant Cd-contaminated leaves, and 37,049 unique Illumina RNA-seq reads were used in a transcriptome assembly. The draft transcriptome assembly consists of 6789 contigs and 17.285 singletons with a means length of 858 bp and 1.042 bp, respectively. The final assembly was functionally annotated using Blast2GO software, allowing the identification of putative genes controlling important agronomic traits. The annotated transcriptome data sets were used to query all known Kyoto Encyclopedia of Genes and Genomes pathways. The most represented molecular functions and biological processes were nucleotide binding and transcription, transport and response to stress and abiotic and biotic stimuli. A prediction of the genes interaction network was proposed by selecting corresponding functionally similar genes from Arabidopsis datasets, downloaded by GeneMANIA version 2.1. Several Cd-responsive genes expression was monitored in in vitro isolated explant of Cd stressed Deglet Nour. Some chelators encoding genes were upregulated confirming in silico findings. Genes encoding HMs transporters in date palm showed expression enhancement more pronounced after 20 days of exposure. P. dactylifera transcriptome provides a valuable resource for future functional analysis of candidate genes involved in metal stress response.

Citrus limon from Tunisia: Phytochemical and Physicochemical Properties and Biological Activities.

Natural plant extracts contain a variety of phenolic compounds which are assigned various biological activities. Our work aims to make a quantitative and qualitative characterization of the Zest (ZL) and the Flesh (FL) of lemon (Citrus limon), to valorize the pharmacological uses of lemon, by evaluating in vitro activities (DPPH, free radical scavenging and reducing power). The antibacterial, antifungal, and antiproliferative activities were sought in the ability of Citrus limon extracts to protect DNA and protein. We found that the ZL contains high amounts of phenolics responsible for the important antioxidant properties of the extract. However, the FL is richer in flavonoids than the ZL. The FL extract was also found to be more effective than the ZL in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals. We also concluded that the FL extract exhibited potent antibacterial activity unlike ZL. Analysis by LC/MS-MS identified 6 compounds (Caffeoyl N-Tryptophan, Hydroxycinnamoyl-Oglucoside acid, Vicenin 2, Eriocitrin, Kaempferol-3-O- rutinoside, and Quercetin-3-rutinoside). These preliminary results showed that Citrus limon has antibacterial and antioxidant activity in vitro. It would be interesting to conduct further studies to evaluate the in vivo potential in an animal model.

Ceratonia siliqua honeys from Morocco: Physicochemical properties, mineral contents, and antioxidant activities.

Physicochemical properties, main mineral content, and antioxidant activity were determined for eight floral carob honeys collected from different geographical regions of Morocco. Moroccan honeys showed good chemical and nutritional qualities, fulfilling the criteria described in the standard codex for honey. The percentages obtained for ashes were (0.13-0.69%), electrical conductivity (0.36-1.35 mS/cm), water content (17.30-22.80%), pH (4.17-5.05), free acidity (11.0-42.50 meq/kg), lactone acidity (4.0-16.50 meq/kg), and for total acidity (16.50-59.50 meq/kg). In addition, minerals such as K, Na, Mg, Cu, Zn, and Ca of honey samples were determined and potassium was the major mineral in all samples. The antioxidant activities based on the free radical scavenging, reducing power, and total antioxidant activity were investigated, and the antioxidant capacity of the honey samples was correlated with their biochemical constituents such as total phenol and flavonoids content, and the best antioxidant capacity was confirmed by the honey from Taounate.

In Vitro Cryopreservation of Date Palm Caulogenic Meristems.

Cryopreservation is the technology of choice not only for plant genetic resource preservation but also for virus eradication and for the efficient management of large-scale micropropagation. In this chapter, we describe three cryopreservation protocols (standard vitrification, droplet vitrification, and encapsulation vitrification) for date palm highly proliferating meristems that are initiated from vitro-cultures using plant growth regulator-free MS medium. The positive impact of sucrose preculture and cold hardening treatments on survival rates is significant. Regeneration rates obtained with standard vitrification, encapsulation-vitrification, and droplet-vitrification protocols can reach 30, 40, and 70%, respectively. All regenerated plants from non-cryopreserved or cryopreserved explants don't show morphological variation by maintaining genetic integrity without adverse effect of cryogenic treatment. Cryopreservation of date palm vitro-cultures enables commercial tissue culture laboratories to move to large-scale propagation from cryopreserved cell lines producing true-to-type plants after clonal field-testing trials. When comparing the cost of cryostorage and in-field conservation of date palm cultivars, tissue cryopreservation is the most cost-effective. Moreover, many of the risks linked to field conservation like erosion due to climatic, edaphic, and phytopathologic constraints are circumvented.

Indirect Somatic Embryogenesis of Date Palm Using Juvenile Leaf Explants and Low 2,4-D Concentration.

This chapter describes an efficient protocol for large-scale micropropagation of date palm. Somatic embryo-derived plants are regenerated from highly proliferating suspension cultures. Friable embryogenic callus is initiated from juvenile leaves using slightly modified Murashige and Skoog (MS) medium supplemented with 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures consisting of proembryonic masses are established from highly competent callus for somatic embryogenesis using half-strength MS medium enriched with 0.1 mg/L 2,4-D and 300 mg/L activated charcoal. The productivity of cultures increased 20-fold when embryogenic cell suspensions were used instead of standard protocols on solidified media. The overall production of somatic embryos mostly exceeds 10,000 units per liter per month. Partial desiccation of mature somatic embryos, corresponding to a decrease in water content from 90 down to 75%, significantly improved germination rates.

Effect of the phosphogypsum amendment of saline and agricultural soils on growth, productivity and antioxidant enzyme activities of tomato (Solanum lycopersicum L.).

The objective of this study was to investigate the effects of phosphogypsum (PG) amendment on the physiochemical proprieties of saline and agricultural soils along with the growth, productivity and antioxidant enzyme activities of tomato plants ( Solanum lycopersicum L.) grown on the amended soils under controlled conditions. Obtained results showed that the amendment of saline soil (H) by PG induced a decrease in pH as well as in electrical conductivity. However, for the non saline soil (MC), there was a decrease in pH associated with an increase in electrical conductivity. For both soils, PG amendment led to an increase in Calcium (Ca) and sodium (Na), and a decrease in potassium (K) in plant tissues. Cadmium (Cd), Zinc (Zn) and Chromium (Cr) contents in different parts of plants increased in proportion with PG concentration in the soils. Apart from Cd, all the analyzed metals in tomato fruit were found to be below the recommended maximum allowable concentration (MAC). Our results showed that PG application, at doses not exceeding 20%, seems to be beneficial for growth, photosynthetic activity and productivity of tomato plants as well as in decreasing salinity of saline soils. In these conditions, the use of PG could be a promising project for the rehabilitation of marginalized and saline ecosystems with either ornamental or non-fruit species. For both soils, a significant accumulation of MDA in shoots was detected, reflecting cell membrane damage especially when the PG amendment reached 20%. Beyond 20 and 40% PG, tomato plants developed an enzymatic antioxidant defense system in response to salinity and heavy metal stress. However, at 80% PG, enzymes activities were significantly inhibited.

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.

Multiple bud cultures of 'Barhee' date palm (Phoenix dactylifera) and physiological status of regenerated plants.

Adventitious bud clusters of date palm 'Barhee' were successfully established from juvenile leaves (<1cm) using reduced amounts of 2,4-D (0.2mgL(-1)) to limit the risk of somaclonal variation. An average of 8.4 adventitious buds per explant were obtained. Histological examination showed that the superficial cell layers of leaves had the highest caulogenic capacity. High sucrose concentration (70gL(-1)) was used for the conversion of initial buds to multiple bud clusters. The promoting effect of temporary immersion on shoot proliferation was found to be significant when compared to cultivation on solid media. Elongation of shoots was also better using a thin film of PGR-free liquid medium instead of a solid medium. Anatomical observations indicated that roots from vitroplants were potentially functional at various developmental stages. However, only 12-month-old vitroplants were found to be physiologically able to control transpirational vapor loss. Additionally, the photochemical activity of photosystem II in these vitroplants was close to that measured in plants that were already acclimatized. As a result, 83.3% of regenerated plants were successfully acclimatized. No phenotypic variation was observed among more than 500 adventitious bud-derived plants. All regenerants survived after field transplantation. We found that the production of adventitious bud clusters in small bioreactors was able to provide an efficient micropropagation system for date palm cv. 'Barhee'. An in vitro hardening step was a prerequisite for the successful transfer of vitroplants in soil.

In vitro hermaphrodism induction in date palm female flower.

This study explores and reports on the gain brought to the morphogenetic aptitude of female date palm inflorescences through in vitro hermaphrodism induction. It investigates the main factors involved in the process of sex modification through hormonal induction, such as the floral developmental stage and hormone combination and concentration. It demonstrates that the vestigial stamens (staminodes) of female date palm flowers display a new and high capacity to proliferate under particular in vitro conditions, without blocking carpel's development, leading to morphologically typical hermaphrodite flowers. This de novo activation of repressed stamens was found to occur rapidly. The isolated pollen mother cells appear in the obtained anther's locules and undergo an ordinary microsporogenesis process. The data show that hermaphrodism induction depended heavily on both the growth regulators applied and the flower's developmental stage. They also confirm the early theory that suggests that dioecious plants come from a hermaphrodite ancestor. Such hermaphrodism control can provide new prospects and opportunities for the investigation of the in vitro self-fertilization process. It can also be useful in improving the understanding of the genetic mechanism involved in sex organ development in date palm.