Cytotoxic and Antioxidant Effects of Phoenix dactylifera L. (Ajwa Date Extract) on Oral Squamous Cell Carcinoma Cell Line.

AIM: The aim of the current study is to investigate the antioxidant and apoptotic potential of Ajwa date flesh (ADF) and Ajwa date pit (ADP) extract on human squamous cell carcinoma cell line (HSC-2). METHOD: ADF and ADP were extracted with a solvent extraction method using hexane, acetone, and ethanol, which were then subjected to antioxidant assay by 2,2-diphenyl-1-picrylhydrazyl (DPPH). HSC-2 cells were then treated with different concentrations of ADF and ADP extract for 24, 48, and 72 hours. MTT assay was performed to assess the antiproliferative effect, and Annexin V-FITC was used for the detection of cellular apoptosis. RESULTS: Acetone extracts of ADF and ADP had the highest radical scavenging and antioxidant activities followed by the ethanolic extracts, whereas ADP appeared to have significantly higher antioxidant effects than ADF. MTT assay demonstrated that acetone extracts of ADF and ADP were significantly cytotoxic against HSC-2 cells in a dose- and time-dependent manner. The half inhibitory concentration (IC50) of ADF was found to be 8.69 mg/ml at 24 h, and the maximum cell growth inhibition was observed at 50 mg/ml. The IC50 for the ADP was found to be 0.97 mg/ml at 24 h, and the maximum cell growth inhibition was observed at 5 mg/ml. Statistical analysis of the flow cytometry assay showed that the treatment with ADF and ADP extracts had a significant apoptotic effect which occurred in a dose-dependent manner. HSC-2 cells were seen in the late apoptotic stage with higher doses of ADF and ADP extract. ADP extract demonstrated higher apoptotic activity than ADF extract. In addition, combined treatment of ADF and ADP was also performed on HSC-2 cells which demonstrated higher apoptotic activity when compared to the single extract. CONCLUSION: Ajwa date fruit has a promising cytotoxic effect by inhibiting the growth and proliferation of OSCC cells and inducing cell death by apoptosis.

A preliminary study on mycobiota and ochratoxin a contamination in commercial palm dates (Phoenix dactylifera).

The occurrence of mycotoxins and mycotoxigenic fungi in palm dates has not been thoroughly documented. The aims of the present study were to identify the mycobiota present in commercial date samples (n = 19), to determine the ability of the isolated fungi to produce mycotoxins, and to determine and quantify the presence of OTA in date fruits. The majority of products originated from Tunisia (n = 14) and Algeria (n = 3). The dominant fungal species were Aspergillus niger, Aspergillus tubingensis and Aspergillus flavus which were most frequently found in premium quality and organic produce, produced without chemical preservatives. OTA was found in only one sample at a concentration of 0.75 µg/kg, as determined by HPLC with fluorescence detection. Although this preliminary study did not find elevated levels of OTA, its presence in one out of 19 samples indicates that palm dates for human consumption require continuous and stringent control, in order to prevent contaminated produce from entering the market.

Fucoidans of Moroccan Brown Seaweed as Elicitors of Natural Defenses in Date Palm Roots.

Fucoidans from Moroccan brown seaweed Bifurcaria bifurcata and Fucus spiralis were tested for their elicitor activity after their purification and complete characterization. The fucoidans of B. bifurcata (BBF) and of F. spiralis (FSF) were extracted and purified then characterized by infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and size exclusion chromatography. The results show that BBF and FSF are mainly sulfated with 45.49 and 49.53% (w/w) sulfate, respectively. Analysis of neutral sugars determined by gas chromatography-mass spectrometry showed that FSF and BBF were mainly composed of 64% and 91% fucose and 20% and 6% galactose, respectively, with a few other sugars such as glucose (8% in FSF), rhamnose (1% in BBF) and mannose (8% in FSF and, 2% in BBF). The eliciting activity of these sulfated polysaccharides in stimulating the natural defenses of the date palm was evaluated through the activity of phenylalanine ammonia-lyase (PAL), and the increase in phenols and lignin content in the roots. The results obtained clearly show that the two fucoidans early and intensely stimulate the natural defenses of the date palm after 24 h of treatments. This remarkable elicitor effect seems to be linked to the sulfated groups compared to non-sulfate alginates extracted from the same algae. These results open promising perspectives for a biological control approach against date palm diseases.

Elicitor-Induced Production of Biomass and Pharmaceutical Phenolic Compounds in Cell Suspension Culture of Date Palm (Phoenix dactylifera L.).

Plants that synthesize bioactive compounds that have high antioxidant value and elicitation offer a reliable in vitro technique to produce important nutraceutical compounds. The objective of this study is to promote the biosynthesis of these phenolic compounds on a large scale using elicitors in date palm cell suspension culture. Elicitors such as pectin, yeast extract (YE), salicylic acid (SA), cadmium chloride (CdCl2), and silver nitrate (AgNO3) at 50, 100, and 200 mg/L concentrations are used. The effects of elicitors on cell culture were determined in terms of biomass [packed cell volume (PCV), fresh and dry weight], antioxidant activity, and phenolic compounds (catechin, caffeic acid, kaempferol, apigenin) were determined using high-performance liquid chromatography (HPLC). Results revealed that enhanced PCV (12.3%), total phenolic content [317.9 ± 28.7 mg gallic acid equivalents (GAE)/100 g of dry weight (DW)], and radical scavenging activity (86.0 ± 4.5%) were obtained in the 50 mg/L SA treated cell culture of Murashige and Skoog (MS) medium. The accumulation of optimum catechin (26.6 ± 1.3 µg/g DW), caffeic acid (31.4 ± 3.8 µg/g DW), and kaempferol (13.6 ± 1.6 µg/g DW) was found in the 50 mg/L SA-treated culture when compared to the control. These outcomes could be of great importance in the nutraceutical and agronomic industries.

Hyperspectral Reflectance of Light-Adapted Leaves Can Predict Both Dark- and Light-Adapted Chl Fluorescence Parameters, and the Effects of Chronic Ozone Exposure on Date Palm (Phoenix dactylifera).

High-throughput and large-scale measurements of chlorophyll a fluorescence (ChlF) are of great interest to investigate the photosynthetic performance of plants in the field. Here, we tested the capability to rapidly, precisely, and simultaneously estimate the number of pulse-amplitude-modulation ChlF parameters commonly calculated from both dark- and light-adapted leaves (an operation which usually takes tens of minutes) from the reflectance of hyperspectral data collected on light-adapted leaves of date palm seedlings chronically exposed in a FACE facility to three ozone (O3) concentrations (ambient air, AA; target 1.5 × AA O3, named as moderate O3, MO; target 2 × AA O3, named as elevated O3, EO) for 75 consecutive days. Leaf spectral measurements were paired with reference measurements of ChlF, and predictive spectral models were constructed using partial least squares regression. Most of the ChlF parameters were well predicted by spectroscopic models (average model goodness-of-fit for validation, R2: 0.53-0.82). Furthermore, comparing the full-range spectral profiles (i.e., 400-2400 nm), it was possible to distinguish with high accuracy (81% of success) plants exposed to the different O3 concentrations, especially those exposed to EO from those exposed to MO and AA. This was possible even in the absence of visible foliar injury and using a moderately O3-susceptible species like the date palm. The latter view is confirmed by the few variations of the ChlF parameters, that occurred only under EO. The results of the current study could be applied in several scientific fields, such as precision agriculture and plant phenotyping.

Use of Alginate Extracted from Moroccan Brown Algae to Stimulate Natural Defense in Date Palm Roots.

Our study aimed to search for seaweed polysaccharides able to stimulate date palm defense mechanisms. Extraction, purification, characterization, and elicitor activity of sodium alginate (FSSA and BBSA) from Moroccan brown seaweeds Fucus spiralis and Bifurcaria bifurcata were investigated. FSSA and BBSA were characterized by proton nuclear magnetic resonance spectroscopy (1H-NMR) and size exclusion chromatography (HPLC-SEC). The mannuronic acid/guluronic acid (M/G) ratio of FSSA was M/G= 0.92 indicating that FSSA contained 48% and 52% of mannuronic and guluronic acids respectively, and the M/G ratio of BBSA was 0.47 indicating that BBSA contained 32% and 68% of mannuronic and guluronic acids respectively. Elicitor activity of FSSA and BBSA was carried out by developing an innovative study model on the date palm. The elicitor capacities were evaluated by investigating phenolic metabolism including phenylalanine ammonia-lyase (PAL) activity and total polyphenol content in seedling roots of date palm maintained in alginates solution (FSSA and BBSA) at different concentrations. The results obtained show that the PAL activity and the phenolic compound content were significantly stimulated with 1 mg.mL-1 of FSSA and BBSA; after 1 day of treatment with FSSA, and after 12 hours of treatment with BBSA. These results show clearly those alginates extracted from Moroccan brown algae induced in date palm roots the stimulation of natural defense mechanisms.

Effect of alkaline and sonication pretreatments on the rumen degradability of date palm seeds.

The main objective of this research was to evaluate the effects of chemical treatment and sonication (ultrasound) processing on the fiber composition and rumen degradability of date palm seeds (DPS). In the first trial, the effects of incubation or sonication in 4% sodium hydroxide (NaOH) on DPS fiber content and ruminal degradability were evaluated. Relative to untreated seeds, the ruminal degradability of DPS neutral detergent fiber (NDF) and organic matter (OM) increased (P < 0.05) for the treated seeds and were highest (P < 0.05) for the sonicated seeds. Relative to untreated seeds, the hemicellulose and lignin content were lower (P < 0.05) for the sonicated seeds, while the cellulose content was higher (P < 0.05) for the incubated seeds. In the second trial, the effects of subjecting DPS to different sonication times (5, 10, 20, and 30 min) were evaluated. The degradability of seeds' NDF and OM were greater (P < 0.05) for the sonicated than unsonicated seeds. The highest NDF degradability was seen after 30 min sonication, whereas the OM degradability was not affected by sonication time (P > 0.05). In the third trial, the effects of subjecting DPS to sonication in different NaOH solutions (1%, 2%, 4% NaOH) were evaluated. Relative to untreated seeds, the rumen degradability of seeds' NDF and OM increased with all NaOH concentrations but was highest (P < 0.05) with the 4% NaOH. In conclusion, our results showed that treating DPS with 4% NaOH increased the seeds' ruminal degradability, and subjecting DPS to sonication further improved their degradability in the rumen.

Silicon-mediated alleviation of combined salinity and cadmium stress in date palm (Phoenix dactylifera L.) by regulating physio-hormonal alteration.

We investigated the physio-molecular effects of separate and combined cadmium (Cd; 200 µM) and salinity (NaCl; 100 mM) stress on date palm during silicon (Si; 1.0 mM) applications. The results showed that exogenous Si led to significant improvements in plant growth, as well as physiology when compared with non-Si-treated seedling under stressed (Cd/NaCl) conditions. Interestingly, Si application led to lower metal (Cd) uptake and enhanced plant macronutrient uptake under combined stress, in turn, alleviating the combined salinity- and Cd-induced oxidative stress by lowering the lipid peroxidation rate, and peroxidase and catalase activities. Furthermore, ascorbate peroxidase level and the cytosolic Cu/Zn superoxide dismutase expression were significantly enhanced by Si application under combined stress. We further analyzed the effect of Si on modulation of stress-related hormonal crosstalk. Si markedly downregulated endogenous salicylic acid, jasmonic acid, and abscisic acid under NaCl stress and combined NaCl-Cd stress. However, during Cd toxicity alone, Si showed varying accumulation of these phytohormones. The results suggest that hindering the Cd uptake and enhancing silicon accumulation ultimately led to improvement of biomass and efficiency of the antioxidant system for alleviating combined stress. Moreover, higher transcript accumulation of PROLINE TRANSPORTER 2 and GAPDH and downregulation of ABA RECEPTOR by Si treatment under combined stress in date palm seedlings indicate the stress-ameliorative role of Si. The study provides evidence of the positive influence of Si on alleviating the combined toxicity of Cd and NaCl in date palm and can be further extended for field trials in Cd- and salinity-affected areas.

Metabolomic analysis of date palm seedlings exposed to salinity and silicon treatments.

Silicon is known to promote plant growth as well as stress tolerance of plants. The current study was undertaken to assess the growth promoting effect of silicon on date palm seedling development as well as its ability to abate some of the negative effects of salinity. In this study, date palm seedlings were treated with silicon and sodium chloride salts, and the effect of these salts on some physiological parameters of the plants was determined. In addition, a global nontargeted metabolomics analysis was performed for the leaf and root tissues using liquid chromatography-mass spectrometry (LC-MS). The results showed that under non-stress conditions, silicon treatment enhanced the growth of the date palm seedlings, however, under salinity, silicon slightly mitigates the negative effects of salt stress on the date palm seedlings although it enhances the potassium accumulation under this condition. The global metabolomics analysis has identified a total of 1,101 significant differentially accumulated (p, q ≤ 0.05) metabolites in leaves and roots under silicon, salinity or their combination. A differential pairwise metabolic profile comparison revealed the accumulation of distinct metabolites in response to silicon and salinity treatments such as antioxidant compounds pyridoxine, cepharanthine, allithiamine, myristic acid and boldine; osmoregulators such as mucic acid; along with the accumulation of detoxification intermediates such as S-D-lactoylglutathione, beta-cyano-L-alanine and gamma-glutamyl-conjugates. In addition, histochemical analyses revealed that application of silicon significantly (p ≤ 0.05) enhanced the formation of the Casparian strip. Identification of the differentially accumulated metabolites could offer an insight into how silicon is able to promote growth and salinity tolerance in date palms.

Impact of Antioxidants on in vitro Rooting and Acclimatization of Two Egyptian Dry Date Palm Cultivars.

BACKGROUND AND OBJECTIVES: Date palm (Phoenix dactylifera L.) is propagated vegetatively via offshoots, which is limited by either the offshoots numbers produced from a superior selected plant, or the occurrence of these offshoots only during the juvenile phase of the date palm life cycle. As a result, the in vitro propagation could be considered an alternative technique for large scale propagation of date palm. Obtaining well-developed root system is considered the most important step in establishment of reliable protocol for successful production of date palm and subsequently successful acclimatization of the in vitro derived plants. The aim of this study is to obtain a well-developed root system through using different antioxidants, with detecting the similarity between the in vitro derived plants and the mother plants using RAPD, long RAPD and ISSR techniques. MATERIALS AND METHODS: Individual plantlets obtained from maturation of somatic embryos in vitro of about 5-7 cm in length with 2-3 leaves were used as plant material. Plantlets were cultured on half strength MS liquid medium supplemented with 0.5 mg L-1 thiamine-HCl+2.0 mg L-1 glycine+0.1 mg L-1 biotin+40 g L-1 sucrose+ 0.1mg L-1 NAA with different concentrations from either AgNO3 or citric or ascorbic acids (0.0, 0.5, 1, 2 mg L-1). Growth development, root number and root length (cm) were evaluated at the end of the second subculture (12 weeks). Data were reported as Mean±Standard deviation (SD). Data were subjected to one way-analysis of variance (p<0.05). Results were processed by Excel (2010). RESULTS: Among the different antioxidants with different concentrations used, generally it was found that 2 mg L-1 of each agent gave the highest values of growth development, roots number and roots length. However, using 2 mg L-1 AgNO3 gave the best results with all parameters. Regarding the response of date palm cultivar, it was remarkable that Bartamoda showed relatively better results than Sakkoty cultivar. According to PCR reactions, the results of RAPD, long RAPD and ISSR profile of tissue culture-derived plantlets grown on a medium supplemented with 2 mg L-1 AgNO3 obviously revealed high similarity to mother plants. CONCLUSION: It could be concluded that using 2 mg L-1 AgNO3 gave the best results for growth development, root numbers and length of the two cultivars but Bartamoda showed relatively better results than Sakkoty cultivar. The tissue culture-derived plantlets on this medium (2 mg L-1 AgNO3) revealed high similarity to mother plant as a result to RAPD, long RAPD and ISSR profiles.

Identification of a dioxin-responsive oxylipin signature in roots of date palm: involvement of a 9-hydroperoxide fatty acid reductase, caleosin/peroxygenase PdPXG2.

Dioxins are highly hazardous pollutants that have well characterized impacts on both animal and human health. However, the biological effects of dioxins on plants have yet to be described in detail. Here we describe a dioxin-inducible caleosin/peroxygenase isoform, PdPXG2, that is mainly expressed in the apical zone of date palm roots and specifically reduces 9-hydroperoxide fatty acids. A characteristic spectrum of 18 dioxin-responsive oxylipin (DROXYL) congeners was also detected in date palm roots after exposure to dioxin. Of particular interest, six oxylipins, mostly hydroxy fatty acids, were exclusively formed in response to TCDD. The DROXYL signature was evaluated in planta and validated in vitro using a specific inhibitor of PdPXG2 in a root-protoplast system. Comparative analysis of root suberin showed that levels of certain monomers, especially the mono-epoxides and tri-hydroxides of C16:3 and C18:3, were significantly increased after exposure to TCDD. Specific inhibition of PdPXG2 activity revealed a positive linear relationship between deposition of suberin in roots and their permeability to TCDD. The results highlight the involvement of this peroxygenase in the plant response to dioxin and suggest the use of dioxin-responsive oxylipin signatures as biomarkers for plant exposure to this important class of xenobiotic contaminants.

Effect of edible co-polymers coatings using γ-irradiation on Hyani date fruit behavior during marketing.

The present work introduces a preparation of coating fruits film from natural biodegradable materials with evaluation of its efficiency in keeping the quality of fresh date fruits. Triple blend (Tb) which involved PVA, chitosan (Cs) and tannic (TA) acids was studied in preservation of Rutab (Hyani) date. Antimicrobial characters besides decay of fruits during a cold storage were determined. The blend solutions were exposed to the γ-irradiation (5.0 to 20 kGy) before casting or use. The effects of polymer composition and irradiation dose on the mechanical and thermo-mechanical properties were studied. The obtained results showed that γ-irradiation and the addition of tannic acid (TA) increased the mechanical properties of the films and the shelf-life of Rutab (Hyani) date during the marketing period (12 ±â€¯2 °C, 98%, RH) from one week to one month of marketing period for consumers with accepted freshness and quality.

Insight into the expression variation of metal-responsive genes in the seedling of date palm (Phoenix dactylifera).

Phytochelatin synthase and metallothionein gene expressions were monitored via qPCR in order to investigate the molecular mechanisms involved in Cd and Cr detoxification in date palm (Phoenix dactylifera). A specific reference gene validation procedure using BestKeeper, NormFinder and geNorm programs allowed selection of the three most stable reference genes in a context of Cd or Cr contamination among six reference gene candidates, namely elongation factor α1, actin, aldehyde dehydrogenase, SAND family, tubulin 6 and TaTa box binding protein. Phytochelatin synthase (pcs) and metallothionein (mt) encoding gene expression were induced from the first days of exposure. At low Cd stress (0.02 mM), genes were still up-regulated until 60th day of exposure. At the highest metal concentrations, however, pcs and mt gene expressions decreased. pcs encoding gene was significantly up-regulated under Cr exposure, and was more responsive to increasing Cr concentration than mt encoding gene. Moreover, exposure to Cd or Cr influenced clearly seed germination and hypocotyls elongation. Thus, the results have proved that both analyzed genes participate in metal detoxification and their expression is regulated at transcriptional level in date palm subjected to Cr and Cd stress. Consequently, variations of expression of mt and pcs genes may serve as early-warning biomarkers of metal stress in this species.

Biochemical and Genetical Responses of Phoenix dactylifera L. to Cadmium Stress.

The cadmium (Cd), a heavy metal, causes toxicity, which leads to hampering the growth and development of the plant. The molecular and biochemical approaches were used for the investigation of antioxidant system response and genotoxicity in date palm (Phoenix dactylifera L.) cv. Sagai in pot experiment having Cd. The root length was more affected than the shoot length as more accumulation of Cd occurs in roots. Fresh weights of root and shoot were reduced significantly in treated plants as compared to the control. The proline content was increased at low concentration of Cd (300 µM-CdCl2) than the medium and high concentrations (600 and 900 µM-CdCl2), respectively. The thiobarbituric acid reactive substances (TBARS) content was increased at 600 and 900 µM-CdCl2 compared to the plants treated at 300 µM-CdCl2 and controls. Antioxidant enzymatic assay was performed under Cd stress and compared with control plants. The catalase (CAT) and superoxide dismutase (SOD) activities were found to be high in plants treated with CdCl2 at 300 µM compared to at 600 and 900 µM-CdCl2, respectively. The genotoxicity of Cd was assessed using the inter-simple sequence repeat (ISSR) marker where all treated and control plants were clustered into three main groups based on genetic similarity. P. dactylifera plants were found to be more divergent at high Cd stress as compared to control and plants treated at low concentration of Cd.

Effect of AgNO3 and BAP on Root as a Novel Explant in Date Palm (Phoenix dactylifera cv. Medjool) Somatic Embryogenesis.

BACKGROUND AND OBJECTIVE: Somatic embryogenesis techniques are used for cloning a wide range of varieties of date palms around the world. The aim of the present study was to develop an efficient method with the lowest cost and the greatest potential to obtain in vitro plantlets of date palm cv. Medjool. Also, produce embryogenic callus and somatic embryos without using 2,4-dichlorophenoxyacetic acid (2,4-D). METHODOLOGY: In this study, produced plantlets through somatic embryogenesis were used in vitro roots as explant cultured on Murashige and Skoog (MS) media containing three level of Silver Nitrate (AgNO3) (0, 3 and 6 mg L-1) plus two level of 6-benzylaminopurine (BAP) (0 and 2 mg L-1) plus 0.1 mg L-1 1-naphthylacetic acid (NAA) for callus induction. After 12 weeks of culture, callus induction and after 16 weeks, production of embryogenic callus and embryos were occurred from root explants. RESULTS: According to the results, medium containing 2 mg L-1 BAP and 3 mg L-1 silver nitrate+0.1 mg L-1 NAA showed the highest amount of embryogenic callus fresh weight (1.38 g). This treatment also cause the highest number and length of embryos by production of 90.04 embryogenic callus with length of 11.18 mm. On the other hand, shoots were appeared from germinated embryos and white roots began to appear within 8 weeks. Medium contains 3 mg L-1 BAP and 0.1 mg L-1 NAA with average of 12.27 cm shoot length and 15.48 cm root length was the best. Control treatment had the lowest average shoot (3.71 cm) and root (5.03 cm) length. CONCLUSION: This study showed that certain concentration of silver nitrate and BAP has stimulating effect on growth of produced embryonic callus from root segments of Medjool cultivar of date palm.

In Vitro Conservation of Date Palm Tissue Cultures.

In vitro technology offers a potential solution for the conservation of date palm germplasm. Slow growth induced by low temperature allows storage from several months up to few years. Otherwise, cryopreservation is suitable for long-term in vitro conservation, at between -79 and -196 °C. This chapter describes a protocol for cold storage at 5 °C and cryopreservation of date palm tissue cultures. For cold storage, 70% of shoot buds remain healthy after storing for 12 months at 5 °C, and callus cultures remain fully viable after 12 months of storage. For cryopreservation of embryogenic cultures using dehydration by air, apparently, 20 min air drying is the best for cryopreservation. Among different types of sugars used as osmotic agents in pre-culture medium, 1 M sucrose is the best for the survival of cryopreserved cultures. However, exposure of embryogenic cultures to vitrification solution for 60 min at 0 °C gives the highest percentage of survival and conversion to plantlets.

Cryopreservation of Date Palm Pro-Embryonic Masses Using the D Cryo-plate Technique.

In this chapter, we describe a cryopreservation (liquid nitrogen, -196 °C) protocol developed for long-term storage of date palm pro-embryonic masses (PEMs), which uses the recently established D cryo-plate technique. Clumps of PEMs (3-5 mm in size) were dissected from PEM cultures and placed on pretreatment medium containing 171 g/L sucrose for 3 days. Clumps were placed in the wells of aluminum cryo-plates in which they were made to adhere using droplets of 3% calcium alginate. PEMs were treated for 20 min with a loading solution containing 184 g/L glycerol and 136.8 g/L sucrose. They were then dehydrated for 90-120 min in the air current of a laminar airflow cabinet and immersed directly in liquid nitrogen. For rewarming, the cryo-plates holding the PEMs were immersed for 15 min in an unloading solution containing 410.4 g/L sucrose. The PEMs were then detached from the cryo-plates, placed for 3 days in the dark on posttreatment medium containing 102.6 g/L sucrose, and transferred on recovery medium under light conditions. Using this protocol, 74.6 and 95.8% recovery were achieved with the PEMs of the two cultivars tested, Sukkari and Sultany.

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.

In Vitro Conservation of Date Palm Shoot-Tip Explants and Callus Cultures Under Minimal Growth Conditions.

Date palm fruit production has great economic significance for many countries. There is a fundamental necessity to conserve valuable date palm germplasm, but there are various problems with in vivo and ex situ conservation. In vitro storage has several advantages over conventional germplasm conservation methods. The in vitro technique offers a developed method of slow-growth storage, which is considered as an alternate solution for short- and medium-term storage of date palm germplasm under controlled conditions. Minimal growth conditions for germplasm conservation are generally achieved by reducing growth rate through modification of environmental growing conditions and culture, by using low temperatures, and the addition of growth retardants and osmotic agents. This chapter describes a protocol for short-term in vitro conservation of date palm shoot-tip and callus cultures under slow-growth storage conditions, using sucrose as an osmotic agent and abscisic acid (ABA) as a growth retardant at 15 °C for 12 months.

In Vitro Conservation of Date Palm Somatic Embryos Using Growth-Retardant Conditions.

In vitro conservation is carried out to maintain disease-free genetic materials, in a small area, protected against pests, insects, soil problems (alkaline, acidic, excess salinity, lack of organic matter, too dry, or too wet), climatic changes, and high-multiplication potential. A requirement of successful in vitro conservation is that the plants can be regenerated into complete plants rapidly when desired. The current work describes in vitro propagation and conservation techniques employing slow-growth conditions of date palm somatic embryo cultures. Clusters of somatic embryos resulting from an indirect micropropagation protocol are conserved in MS culture medium supplemented with an osmotic agent (sucrose at 90 g/L) combined with a growth-retardant hormone (abscisic acid) at 2 mg/L incubated at low temperature (18 °C) and low light intensity (10 µmol/m2/s). The survival and plant recovery rates are recorded after 10 months. Date palm somatic embryo clusters can be conserved for up to 10 months without subculture with high survival rates. Successfully conserved somatic embryos multiply and germinate to regenerate plants with well-developed shoots and roots, which survive acclimatization and field transfer.