Methyl jasmonate induces oxidative/nitrosative stress and the accumulation of antioxidant metabolites in Phoenix dactylifera L.

OBJECTIVES: The present study aimed to explore the eliciting effects of increasing concentrations (50, 100, and 200 µM) of methyl jasmonate (MeJA). We cultivated actively proliferating buds of Phoenix dactylifera L. cv. Barhee in a temporary immersion system and we monitored the bioactive compound accumulation after 7 days of culture. METHODS: Total phenolic (TPC) and flavonoid (TFC) contents were determined by high-performance liquid chromatography (HPLC), Fourier-transform infrared (FTIR), and radical scavenging activity using DPPH and ABTS assays. We also explored the activity of phenylpropanoid pathway enzymes, namely phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL) and polyphenol oxidase (PPO). RESULTS: Our results revealed that MeJA treatment induced oxidative stress, and at the same time increased the activity of related defense enzymes in a dose-dependent manner. Exogenous application of MeJA at 200 µM increased ROS (two fold), hydrogen peroxide (3.7 fold), nitric oxide (14 fold), MDA (6.3 fold), superoxide dismutase (5.9 fold), catalase (4.4 fold) and guaiacol peroxidase (3.87 fold). Furthermore, the results demonstrated that 200 µM MeJA treatment enhanced the activities of PAL (3.65 fold), TAL (4.35 fold), PPO (threefold) and increased TPC (twofold) and TFC (1.75 fold) contents in buds cultures higher than the control. HPLC analysis showed that buds cultures exposed to 200 µM MeJA accumulated maximum amount of catechin (11 fold), 4-hydroxybenzoic acid (1.48 fold), caffeic acid (2.5 fold) and p-coumaric acid (1.76 fold) and demonstrate antioxidant capacity with the lowest DPPH (114.5 µg ml-1) and ABTS (90.2 µg ml-1) IC50 values on day 7 of culture as compared to the control. The MeJA in the culture medium directly reduced cell viability in a dose dependent manner up to 35% with the highest concentration. CONCLUSION: The results of this study has revealed, for the first time, that MeJA offers a promising potential for the production of phenolic compound in Phoenix dactylifera L. buds.

Extraction, partial purification and characterization of amylase from parthenocarpic date (Phoenix dactylifera): effect on cake quality.

BACKGROUND: Phoenix dactylifera L. plays an important role in social, economic and ecological Tunisian sectors. Some date palms produce parthenocarpic fruit named Sish. The objective of the present study was to extract biomolecules from parthenocarpic fruit by producing value-added products from the fruits. RESULTS: The extraction of amylolytic activity from parthenocarpic fruit (AmyPF) was optimized using Box-Behnken design (BBD). Partial purification of about 250-fold with an activity yield of 47% was achieved. The amylase exhibited a specific activity of 80 U mg-1 protein. The optimum pH and temperature for enzyme activity were 5 and 55 °C respectively. The enzyme was highly active over a wide range of pH (5-10), and significant stabilization was observed at 60 °C. The purified enzyme belongs to the exo type of amylases. Given the economic and industrial relevance of amylases used in the food industry, three different concentrations of AmyPF (0.007, 0.014 and 0.018 U g-1 ) were incorporated into a cake formulation, resulting in a decrease in density, moisture retention and water activity and an increase in hardness. CONCLUSION: The beneficial effect of AmyPF on the technological characteristics of cakes was confirmed by sensory evaluation. © 2017 Society of Chemical Industry.

In silico characterization and Molecular modeling of double-strand break repair protein MRE11 from Phoenix dactylifera v deglet nour.

BACKGROUND: DNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3' to 5' exonuclease and single-stranded DNA endonuclease activities. METHODS: The present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program. RESULTS: The DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn(2+) ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species. CONCLUSIONS: A model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn(2+) and dAMP).

New food from a potato somatic hybrid: nutritional equivalence and safety assessment by a feeding study on rats.

BACKGROUND: Potato tubers from the STBd somatic hybrid line that exhibited improved tolerance to salinity and resistance to fungal and PVY infections were characterised. They were compared for their chemical composition to the Spunta variety produced by conventional agronomic practices. This study aimed to compare nutritional value and safety by feeding rats with STBd or commercial tubers added to the standard diet (20/80 w/w). RESULTS: The analysis of soluble sugar, fat, fibre and ash content of tubers did not reveal any significant differences between the hybrid line and the control Spunta variety. Small differences were observed in dry matter, starch and protein content of hybrid potatoes in comparison to controls. However, all values were within normal ranges reported in the literature. The feeding study on rats showed that overall health, weight gain, food consumption, morphological aspects and weights of organs were comparable between rat groups fed the STBd hybrid and the Spunta variety. CONCLUSION: Taken together, 28 days of consumption of STBd hybrid potato did not exert any adverse effect on rats compared with commercial Spunta potato. The STBd potato line was therefore considered to be as safe for food utilisation as the commercial variety.

Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning.

Oil palm can accumulate up to 90% oil in its mesocarp, the highest level observed in the plant kingdom. In contrast, the closely related date palm accumulates almost exclusively sugars. To gain insight into the mechanisms that lead to such an extreme difference in carbon partitioning, the transcriptome and metabolite content of oil palm and date palm were compared during mesocarp development. Compared with date palm, the high oil content in oil palm was associated with much higher transcript levels for all fatty acid synthesis enzymes, specific plastid transporters, and key enzymes of plastidial carbon metabolism, including phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase. Transcripts representing an ortholog of the WRI1 transcription factor were 57-fold higher in oil palm relative to date palm and displayed a temporal pattern similar to its target genes. Unexpectedly, despite more than a 100-fold difference in flux to lipids, most enzymes of triacylglycerol assembly were expressed at similar levels in oil palm and date palm. Similarly, transcript levels for all but one cytosolic enzyme of glycolysis were comparable in both species. Together, these data point to synthesis of fatty acids and supply of pyruvate in the plastid, rather than acyl assembly into triacylglycerol, as a major control over the storage of oil in the mesocarp of oil palm. In addition to greatly increasing molecular resources devoted to oil palm and date palm, the combination of temporal and comparative studies illustrates how deep sequencing can provide insights into gene expression patterns of two species that lack genome sequence information.

Pistachio (Pistacia vera L.) is a new natural host of Hop stunt viroid.

Besides hop, Hop stunt viroid (HpSVd) infects many woody species including grapevine, citrus, peach, plum, apricot, almond, pomegranate, mulberry and jujube. Here, we report the first detection of HpSVd in pistachio (Pistacia vera L.). Samples corresponding to 16 pistachio cultivars were obtained from a nearby almond collection. From these samples, low molecular weight RNAs were extracted for double polyacrylamide gel electrophoresis, northern-blot analysis and reverse transcription polymerase chain reaction assays. HpSVd was detected in 4 of the 16 pistachio cultivars in the first year and in 6 in the second, being also detected in the almond collection. Examination of the nucleotide sequences of pistachio and almond isolates revealed 13 new sequence variants. Sequences from pistachio shared 92-96 % similarity with the first reported HpSVd sequence (GenBank X00009), and multiple alignment and phylogenetic analyses showed that one pistachio isolate (HpSVdPis67Jabari) clustered with the plum group, whereas all the others clustered with the hop, and the recombinants plum-citrus and plum-Hop/cit3 groups. By identifying pistachio as a new natural host, we confirm that HpSVd is an ubiquitous and genetically variable viroid that infects many different fruit trees cultivated worldwide.

Morphological and biochemical behavior of fenugreek (Trigonella foenum-graecum) under copper stress.

The effects of copper on germination and growth of fenugreek (Trigonella foenum-graecum) was investigated separately using different concentrations of CuSO4. The germination percentage and radical length had different responses to cupric ions: the root growth increased with increasing copper concentration up to 1 mM Cu²âº and was inhibited thereafter. In contrast, the germination percentage was largely unaffected by concentrations of copper below 10 mM. The reduction in root growth may have been due to inhibition of hydrolytic enzymes such as amylase. Indeed, the average total amylolytic activity decreased from the first day of treatment with [Cu²âº] greater than 1 mM. Furthermore, copper affected various plant growth parameters. Copper accumulation was markedly higher in roots as compared to shoots. While both showed a gradual decrease in growth, this was more pronounced in roots than in leaves and in stems. Excess copper induced an increase in the rate of hydrogen peroxide (H2O2) production and lipid peroxidation in all plant parts, indicating oxidative stress. This redox stress affected leaf chlorophyll and carotenoid content which decreased in response to augmented Cu levels. Additionally, the activities of proteins involved in reactive oxygen species (ROS) detoxification were affected. Cu stress elevated the ascorbate peroxidase (APX) activity more than two times at 10 mM CuSO4. In contrast, superoxide dismutase (SOD) and catalase (CAT) levels showed only minor variations, only at 1 mM Cu²âº. Likewise, total phenol and flavonoid contents were strongly induced by low concentrations of copper, consistent with the role of these potent antioxidants in scavenging ROS such as H2O2, but returned to control levels or below at high [Cu²âº]. Taken together, these results indicate a fundamental shift in the plant response to copper toxicity at low versus high concentrations.

Proteomics analysis of date palm leaves affected at three characteristic stages of brittle leaf disease.

Proteomics analysis has been performed in leaf tissue from field date palm trees showing the brittle leaf disease (BLD) or maladie des feuilles cassantes, the main causal agent of the date palm decline in south Tunisia. To study the evolution of the disease, proteins from healthy and affected leaves taken at three disease stages (S1, S2 and S3) were trichloroacetic acid acetone extracted and subjected to two-dimensional gel electrophoresis (5-8 pH range). Statistical analysis showed that the protein abundance profile is different enough to differentiate the affected leaves from the healthy ones. Fifty-eight variable spots were successfully identified by matrix-assisted laser desorption ionization time of flight, 60 % of which corresponded to chloroplastic ones being involved in the photosynthesis electronic chain and ATP synthesis, metabolic pathways implicated in the balance of the energy, and proteases. Changes in the proteome start at early disease stage (S1), and are greatest at S2. In addition to the degradation of the ribulose-1.5-bisphosphate carboxylase oxygenase in affected leaflets, proteins belonging to the photosynthesis electronic chain and ATP synthesis decreased following the disease, reinforcing the relationship between BLD and manganese deficiency. The manganese-stabilizing proteins 33 kDa, identified in the present work, can be considered as protein biomarkers of the disease, especially at early disease step.

Linking Leaf Water Potential, Photosynthesis and Chlorophyll Loss With Mechanisms of Photo- and Antioxidant Protection in Juvenile Olive Trees Subjected to Severe Drought.

The identification of drought-tolerant olive tree genotypes has become an urgent requirement to develop sustainable agriculture in dry lands. However, physiological markers linking drought tolerance with mechanistic effects operating at the cellular level are still lacking, in particular under severe stress, despite the urgent need to develop these tools in the current frame of global change. In this context, 1-year-old olive plants growing in the greenhouse and with a high intra-specific variability (using various genotypes obtained either from cuttings or seeds) were evaluated for drought tolerance under severe stress. Growth, plant water status, net photosynthesis rates, chlorophyll contents and the extent of photo- and antioxidant defenses (including the de-epoxidation state of the xanthophyll cycle, and the contents of carotenoids and vitamin E) were evaluated under well-watered conditions and severe stress (by withholding water for 60 days). Plants were able to continue photosynthesizing under severe stress, even at very low leaf water potential of -4 to -6 MPa. This ability was achieved, at least in part, by the activation of photo- and antioxidant mechanisms, including not only increased xanthophyll cycle de-epoxidation, but also enhanced α-tocopherol contents. "Zarrazi" (obtained from seeds) and "Chemlali" (obtained from cuttings) showed better performance under severe water stress compared to the other genotypes, which was associated to their ability to trigger a higher antioxidant protection. It is concluded that (i) drought tolerance among the various genotypes tested is associated with antioxidant protection in olive trees, (ii) the extent of xanthophyll cycle de-epoxidation is strongly inversely related to photosynthetic rates, and (iii) vitamin E accumulation is sharply induced upon severe chlorophyll degradation.

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.

Optimization of RNA isolation from Brittle Leaf Disease affected date palm leaves and construction of a subtractive cDNA library.

A simple and efficient method was described here for the isolation of high-quality RNA from date palm leaves affected with Brittle Leaf Disease (BLD) and containing high amount of phenolic compounds. The procedure was based on the use of a non-ionic detergent Nonidet-P40 (NP-40), Polyvinylpyrrolidone (PVP), and beta-mercaptoethanol in the extraction buffer in order to isolate cytoplasmic RNA and to prevent the oxidation of phenolic compounds. This method allowed the isolation of intact RNA, suitable for cDNA synthesis and library construction. Differential screening of the subtractive cDNA library from affected leaf RNA led to the identification of some BLD-induced genes.

A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves.

Biological activity during co-composting of sludge issued from the OMW evaporation ponds with poultry manure-Physico-chemical characterization of the processed organic matter.

Olive mill sludge (OMS), a by-product resulting from natural evaporation of olive oil processing effluent, poses a major environmental threat. A current cost-effective practice of OMS management is composting. A mixture of OMS (60%) with poultry manure (PM) was successfully composted for 210 days. During the process, effluents of olive oil mill and confectionary were used to keep moisture at optimal level (40-60%). Biological indicators reflecting stability of the compost (microbial biota respiration and enumeration, and germination index) were analysed for the assessment of the product quality. The composted mixture showed a high microbial activity with a succession of microbial populations depending on the temperature reached during the biodegradation. The pathogen content from PM decreased with composting as did phytotoxic compounds. Phenols and lipids were reduced, respectively, by 40% and 84% while germination index increased with composting progress. Fourier transform infrared (FTIR) spectroscopic analysis revealed that the final compost improved the aromatic content compared to the starting materials, with a decrease in aliphatic groups and a reduction in the easily assimilated components by the microflora acting during the biological process. The final compost was characterized by relatively high organic matter content (26.21%), a low C/N ratio (16.21), an alkaline pH (8.32), a relatively high electrical conductivity (9.21mS/cm) and a high level of nutrients. The germination index for Lepidium sativum L. was 87.71% after 210 days of composting, showing that the final compost was not phytotoxic.

Elimination of polyphenols toxicity from olive mill wastewater sludge by its co-composting with sesame bark.

Olive mill wastes represent a significant environmental problem in Mediterranean areas where they are generated in huge quantities in a short period of time. Their high phenol, lipid and organic acid concentrations turn them into phytotoxic materials, but these wastes also contain valuable resources such as a large proportion of organic matter and a wide range of nutrients that could be recycled. Composting is one of the technologies used for the valorization of this effluent, producing a fertilizer useful for poor soils.The present work deals with the changes that occur in the content of phenolic compounds and the biotoxicity of the oxidized substrate which result from the composting of olive mill wastewater (OMW) sludge with sesame bark. The total organic matter decreased 52.72% while water-soluble phenol degradation decreased 72% after 7 months of processing. Gas chromatography coupled with mass spectroscopy was used to confirm the elimination of polyphenols during composting. Initially, the analysis showed three abundant polyphenolic compounds, one of which was identified as the 4-hydroxyphenyl-ethanol (tyrosol), a well-known antioxidant in OMW. After 7 months of composting, all of the phenolic compounds disappeared. The phytotoxic effects of OMW sludge, assessed by the plant index germination, increased during the composting to reach 80% after 210 days. This trend was confirmed by the correlation between physico-chemical and toxicity parameters. The results obtained confirmed the stability of the compost prepared from OMW sludge with sesame bark and indicated a gradual detoxification as the compost matured.

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.

Antioxidant, Anti-Inflammatory, and Antitumoral Effects of Aqueous Ethanolic Extract from Phoenix dactylifera L. Parthenocarpic Dates.

The aim of this study was to evaluate the antioxidant, the anti-inflammatory, and the antitumoral activities of the aqueous ethanolic extract from Phoenix dactylifera L. parthenocarpic dates. The antioxidant activity was carried using DPPH radical scavenging activity. The result showed that parthenocarpic dates had strongly scavenging activity on DPPH reaching 94% with an IC50 value of 0.15 ± 0.011 mg/mL (p < 0.05). The anti-inflammatory potential was determined by the inhibitory effect of the aqueous ethanolic extract on phospholipase A2 activity as well as on carrageenan-induced paw oedema in mice. The in vitro study showed that the extract inhibited the phospholipase A2 activity with an IC50 value of 130 µg/mL and the in vivo study showed a significantly decrease in the paw oedema after 1 h compared to the control group. Finally, the antiproliferative activity of the aqueous ethanolic extract was assessed by MTT test against MCF-7 and MDA-MB-231 cancer cell lines. This extract was effective in inhibiting MDA-MB-231 and MCF-7 cancer cells growth with IC50 values of 8 and 18 mg/mL, respectively, after 72 h treatment. These results confirm the ethnopharmacological significance of Phoenix dactylifera L. parthenocarpic dates, which could add support for its pharmaceutical use.

Temporary Immersion System for Date Palm Micropropagation.

The temporary immersion system (TIS) is being used with tremendous success for automation of micropropagation of many plant species. TIS usually consists of a culture vessel comprising two compartments, an upper one with the plant material and a lower one with the liquid culture medium and an automated air pump. The latter enables contact between all parts of the explants and the liquid medium by setting overpressure to the lower part of the container. These systems are providing the most satisfactory conditions for date palm regeneration via shoot organogenesis and allow a significant increase of multiplication rate (5.5-fold in comparison with that regenerated on agar-solidified medium) and plant material quality, thereby reducing production cost.

Electrophoresis-Based Proteomics to Study Development and Germination of Date Palm Zygotic Embryos.

Proteomics has become an important and powerful tool in plant biology research. To establish a proteomic reference map of date palm zygotic embryos (ZE), we separated and identified proteins from zygotic embryos during different developmental and germination phases using one, two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteins are extracted with trichloroacetic acid (TCA)/acetone-phenol and resolved by gel electrophoresis. Gel images are captured and analyzed by appropriate software and statistical packages. Quantitative or qualitative variable bands or spots are subjected to MS analysis in order to identify them and correlate differences in the protein profiles with the different stages of date palm zygotic embryo development, maturation, and germination.

Extraction optimization and in vitro and in vivo anti-postprandial hyperglycemia effects of inhibitor from Phoenix dactylifera L. parthenocarpic fruit.

Phoenix dactylifera L. plays an important role in social, economic, and ecological Tunisian sectors. Some date palms produce parthenocarpic fruit named Sish. The aqueous ethanolic extract from P. dactylifera parthenocarpic dates demonstrated a potent inhibition of the enzymes related to type II diabetes. In this work, extraction optimization of amylase inhibitors was carried out using Box-Behnken Design. Bioactivity-guided fractionation of the 70% aqueous ethanol extract was performed to identify the active compounds. The physicochemical results by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed the presence of 13 phenolic compounds. The in vitro study showed that the extract exhibited a more specific inhibitor of α-glucosidase than α-amylase with an IC50 value of 0.6 and 2.5mg/mL, respectively. The in vivo study of this extract effect on the postprandial hyperglycemia activity showed a decrease in plasma glucose levels after 30min stronger than the Acarbose effect. These results confirmed the anti-postprandial hyperglycemia activity of the aqueous ethanolic extract from P. dactylifera parthenocarpic dates, which could lend support for its pharmaceutical use.

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.