Biallelic Editing of the LOB1 Promoter via CRISPR/Cas9 Creates Canker-Resistant 'Duncan' Grapefruit.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. X. citri subsp. citri causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (Citrus paradisi), which usually contain two alleles of LOB1 and thus, have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBEPthA4-LOBP in grapefruit. Using 'Duncan' grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#DunYao7). X. citri subsp. citri caused canker symptoms on wild-type and nonbiallelic mutant plants but not on #DunYao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #DunYao7, caused canker symptoms on both wild-type and #DunYao7. No off-target mutations were detected in #DunYao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for 'Duncan' grapefruit, paving the way for using disease-resistant varieties to control canker.

Plant-Based Titanium Dioxide Nanoparticles Trigger Biochemical and Proteome Modifications in Triticum aestivum L. under Biotic Stress of Puccinia striiformis.

In this study, we evaluated bioinspired titanium dioxide nanoparticles (TiO2 NPs) that elicited biochemical and proteome modifications in wheat plants under the biotic stress caused by Puccinia striiformis f. sp. tritici (Pst). Biosynthesis of TiO2 NPs was confirmed using UV-Vis spectrophotometry, energy dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy. We found that the nanoparticles with crystalline nature were smaller than 100 nm. The results of FTIR analysis showed the presence of potential functional groups exhibiting O-H, N-H, C-C, and Ti-O stretching. The TiO2 NPs of different concentrations (20, 40, 60, and 80 mg L-1) were exogenously applied to wheat plants under the biotic stress caused by Pst, which is responsible for yellow stripe rust disease. The results of the assessment of disease incidence and percent disease index displayed time- and dose-dependent responses. The 40 mg L-1 TiO2 NPs were the most effective in decreasing disease severity. The bioinspired TiO2 NPs were also evaluated for enzymatic (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), and nonenzymatic metabolites (total proline, phenolic, and flavonoid contents) in wheat plants under stripe rust stress. The 40 mg L-1 TiO2 NPs were effective in eliciting biochemical modifications to reduce biotic stress. We further evaluated the effects of TiO2 NPs through gel- and label-free liquid chromatography-mass spectrometry (LC-MS) proteome analysis. We performed proteome analysis of infected wheat leaves and leaves treated with 40 mg L-1 TiO2 NPs under stripe rust stress. The functional classification of the proteins showed downregulation of proteins related to protein and carbohydrate metabolism, as well as of photosynthesis in plants under biotic stress. An upregulation of stress-related proteins was observed, including the defense mechanisms and primary metabolic pathways in plants treated with 40 mg L-1 TiO2 NPs under stress. The experimental results showed the potential of applying biogenic TiO2 NPs to combat fungal diseases of wheat plants and provided insight into the protein expression of plants in response to biotic stress.

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice.

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.

Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing.

Citrus canker disease affects citrus production. This disease is caused by Xanthomonas citri subsp. citri (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), is translocated from the pathogen to host plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 (EBEPthA4-LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBEPthA4-LOBP of pummelo, which is highly homozygous. However, most commercial citrus cultivars are heterozygous hybrids and more difficult to generate homozygous/biallelic mutants. Here, we employed Cas12a/CBE co-editing method to edit EBEPthA4-LOBP of Hamlin (Citrus sinensis), a commercial heterozygous hybrid citrus cultivar grown worldwide. Binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 was constructed and shown to be functional via Xcc-facilitated agroinfiltration in Hamlin leaves. This construct allows the selection of transgene-free regenerants via GFP, edits ALS to generate chlorsulfuron-resistant regenerants as a selection marker for genome editing resulting from transient expression of the T-DNA via nCas9-mPBE:ALS2:ALS1, and edits gene(s) of interest (i.e., EBEPthA4-LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#HamGFP1 - #HamGFP8), 4 plantlets were transgene-free (#HamNoGFP1 - #HamNoGFP4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#HamNoGFP1, #HamNoGFP2 and #HamNoGFP3) contained biallelic mutations in EBEpthA4, and one line (#HamNoGFP4) had homozygous mutations in EBEpthA4. We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBEPthA4-LOBP in C. sinensis cv. Hamlin, compared to 1.9% mutation efficiency for pummelo in a previous study. Importantly, the four transgene-free plantlets and 3 transgenic plantlets that survived were resistant against citrus canker. Taken together, Cas12a/CBE co-editing method has been successfully used to generate transgene-free canker-resistant C. sinensis cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBEpthA4 of the canker susceptibility gene LOB1.

Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective, environmentally friendly and economic approach for disease control. However, citrus traditional breeding is lengthy and laborious. Here, we develop transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. Canker resistance of the cslob1-edited lines results from both abolishing canker symptoms and inhibiting Xcc growth. The transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS and are exempted from EPA regulation. This study provides a sustainable and efficient citrus canker control solution and presents an efficient transgene-free genome-editing strategy for citrus and other crops.

Conferring of Drought and Heat Stress Tolerance in Wheat (Triticum aestivum L.) Genotypes and Their Response to Selenium Nanoparticles Application.

In this study, the role of selenium nanoparticles (SeNPs, 10 mg·L-1) has been investigated in modulating the negative effects of drought and heat stresses on eight bread wheat (Triticum aestivum L.) genotype seedlings. Those genotypes included Giza-168, Giza-171, Misr-1, Misr-3, Shandweel-1, Sids-1, Sids-12, and Sids-14. The study included six treatments as follows: regular irrigation with 100% Field Capacity (FC) at a temperature of 23 ± 3 °C (T1), drought stress with 60% FC (T2), heat stress of 38 °C for 5 h·day-1 (T3), foliar spray of 10 mg·L-1 of SeNPs only (T4), a combination of drought stress with foliar spray of 10 mg·L-1 of SeNPs (T5), and heat stress with foliar spray of 10 mg·L-1 of SeNPs (T6). The experiment continued for 31 days. Foliar application of SeNPs improved the plant growth, morpho-physiological and biochemical responses, and expression of stress-responsive genes in wheat (T. aestivum L.) seedlings. Overall, morpho-physiological traits such as plant height (PH), shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), and root dry weight (RDW) of wheat genotypes grown under different conditions ranged from 25.37-51.51 cm, 3.29-5.15 g, 0.50-1.97 g, 0.72-4.21 g, and 0.11-1.23 g, respectively. From the morpho-physiological perspective, drought stress had a greater detrimental impact on wheat plants than heat stress, whereas heat stress significantly impacted the expression of stress-responsive genes. Stress responses to drought and heat varied between wheat genotypes, suggesting that different genotypes are more resilient to stress. Exogenous spraying of 10 mg·L-1 of SeNPs improved the photosynthetic pigments, photosynthetic rate, gas exchange, and transpiration rate of wheat plants and enhanced drought and heat tolerance by increasing the activity of antioxidant enzymes including catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) and the expression level of stress-responsive genes. Our results showed that spraying wheat seedlings with 10 mg·L-1 of SeNPs enhanced SOD activity for all genotypes as compared to the control, with the Sids-12 genotype having the highest value (196.43 U·mg-1 FW·min-1) and the Giza-168 genotype having the lowest (152.30 U·mg-1 FW·min-1). The expression of PIP1, LEA-1, HSP70, and HSP90 stress-responsive genes was more significant in tolerant genotypes (Giza-171 and Giza-168) than in sensitive ones (Misr-1 and Misr-3) in response to drought and heat stresses. Under stress conditions, the shoot and root fresh weights, photosynthetic pigment content, stomatal conductance (SC), and transpiration rate (TR) were positively correlated with plant height (PH), while root and shoot dry weights, malondialdehyde (MDA), proline, hydrogen peroxide (H2O2), and APX were negatively correlated. Multivariate analysis and biplot results revealed that genotypes Giza-168, Giza-171, Sids-12, and Sids-14 performed well in both stress situations and were classified as stress-tolerant genotypes. These best genotypes may be employed in future breeding projects as tools to face climate change. This study concluded that various physio-biochemicals and gene expression attributes under drought and heat stress could be modulated by foliar application of SeNPs in wheat genotypes, potentially alleviating the adverse effects of drought and heat stress.

Citrus Protoplast Isolation and Plant Regeneration Through Somatic Embryogenesis.

Protoplasts are an attractive explant source for biotechnological tools widely used on citrus genetic improvement, such as somatic hybridization and direct genetic transformation. These delicate and responsive materials are subjected to cell proliferation induction and differentiation of somatic embryos which further regenerate into entire plants. The isolation of viable protoplasts followed by regeneration of plants through somatic embryogenesis is an important methodology for breeding applications. The methods presented here can also be used as a reference for protoplast work in other species, followed by protocol optimization for different species/genotypes.

Efficacy of Green Cerium Oxide Nanoparticles for Potential Therapeutic Applications: Circumstantial Insight on Mechanistic Aspects.

Green synthesized cerium oxide nanoparticles (GS-CeO2 NPs) have a unique size, shape, and biofunctional properties and are decorated with potential biocompatible agents to perform various therapeutic actions, such as antimicrobial, anticancer, antidiabetic, and antioxidant effects and drug delivery, by acquiring various mechanistic approaches at the molecular level. In this review article, we provide a detailed overview of some of these critical mechanisms, including DNA fragmentation, disruption of the electron transport chain, degradation of chromosomal assemblage, mitochondrial damage, inhibition of ATP synthase activity, inhibition of enzyme catalytic sites, disorganization, disruption, and lipid peroxidation of the cell membrane, and inhibition of various cellular pathways. This review article also provides up-to-date information about the future applications of GS-CeONPs to make breakthroughs in medical sectors for the advancement and precision of medicine and to effectively inform the disease diagnosis and treatment strategies.

Polyamines mitigate the destructive impacts of salinity stress by enhancing photosynthetic capacity, antioxidant defense system and upregulation of calvin cycle-related genes in rapeseed (Brassica napus L.).

Salinity is widespread environmental stress that poses great obstacles to rapeseed development and growth. Polyamines are key plant growth regulators that play a pivotal role in regulating salt tolerance. Rapeseed (Brassica napus L.) seedlings were treated by spermine (Spm) and spermidine (Spd) versus untreated control under salt stress conditions. It was detected that the Spd-treated plants had significantly elevated chlorophyll and proline content and maintained higher photosystem II (PSII) activity than those treated with Spm as well as untreated control under salt-stressed conditions. Similarly, Spd alleviated the devastating effects of NaCl stress on CO2 assimilation and significantly elevated Rubisco activity (ribulose 1,5-bisphosphate carboxylase/oxygenase). The application of Spd also enhanced the activities of different antioxidant enzymes under NaCl stress. It modulated their respective transcription levels, including ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and dehydroascorbate reductase (DHAR). In addition, exogenously sprayed Spd enhanced the polyamine pathway as observed by upregulated transcription of polyamine oxidase (PAO) and diamine oxidase (DAO). The Spd application enhanced expressions of Calvin cycle enzyme related genes such as Rubisco small subunit, Rubisco large subunit, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglyceric acid kinase (PGK), triose-3-phosphate isomerase (TPI), fructose-1,6-bisphosphate aldolase (FBA), sedoheptulose-1,7-bisphosphatase (SBPase), and fructose-1,6-bisphosphate phosphatase (FBPase). Consequently, this study demonstrates that exogenous application of Spd has a valuable role in regulating antioxidant enzyme activity, polyamine pathway, and Calvin cycle enzyme-related genes to alleviate salt stress damage in the plants.

Effect of Inorganic and Organic Nitrogen Sources and Biofertilizer on Murcott Mandarin Fruit Quality.

Mandarin 'Murcott' (Citrus reticulata Blanco) trees aged five years that were grafted onto lemon 'Volkamer' (Citrus volkameriana) rootstock and grown in sandy soil under a drip irrigation system were used in this study during the growing seasons of 2018 and 2019. Ten different fertilization treatments combining inorganic, organic, and biofertilization in a completely randomized block were performed. The results revealed that fertilizing 'Murcott' mandarin trees with 75% of the recommended dose (RD) of nitrogen as inorganic nitrogen (33.5% N) in the form of NH4NO3 + 25% of RD as organic nitrogen in the form of chicken manure (3% N) per tree per year without or with a biofertilizer (Effective Microorganisms, EM1) at 150 mL/tree increased the weight, size, pulp, and peels of mandarin fruit, as well as the fruit juice volume, juice volume/fruit, and vitamin C, but reduced the total acidity in both seasons. However, fertilizing 'Murcott' mandarin trees with 100% of RD as inorganic nitrogen increased the pulp/fruit ratio, and fertilizing with 25% of RD as inorganic nitrogen + 75% of RD as organic nitrogen + biofertilizer EM1 increased the peel/fruit ratio, peel thickness, and fruit firmness. Fertilizing 'Murcott' mandarin trees with 100% organic nitrogen + biofertilizer EM1 increased total soluble solids (TSS) and total sugar contents while producing the lowest nitrate (NO3) percentage in 'Murcott' mandarin fruit compared with trees fertilized with inorganic nitrogen only. The fruit produced by 'Murcott' mandarin trees fertilized with 100% of RD as organic nitrogen with or without biofertilizer EM1 contained higher TSS, total carbohydrates, and sugars and lower nitrate percentages than those fertilized with inorganic nitrogen and biofertilizer EM1. This study contributes to reducing the use of inorganic fertilizers by adding a percentage of an organic fertilizer to obtain a healthy product that contains a lower percentage of NO3, which affects the health of the consumer, and is of high quality and suitable for export.

Morphological and Molecular Characterization of Some Egyptian Six-Rowed Barley (Hordeum vulgare L.).

Barley production is essential in Egypt. In the present study, 15 different six-rowed Egyptian barley cultivars were studied. To differentiate between the different cultivars under study in terms of morphological characteristics and ISSR, molecular characterization reactions were carried out. Moreover, four cultivars (Giza 123, Giza 126, Giza 136, and Giza 138) were selected for further studies using scanning electron microscopy (SEM). Computational analysis of the DNA barcoding sequences of the two plastid markers rbcL and matK was executed, and the results were deposited in the NCBI database. The morphological traits showed low statistical significance among the different cultivars under study via the data collected from two seasons, suggesting that the mean field performance of these Egyptian cultivars may be equal under these conditions. The results showed that the phylogenetic tree was divided into four groups, one of which contained the most closely related genotypes in the genetic distance, including Giza 124, Giza 130, Giza 138, Giza 136, and Giza 137, which converge in the indicative uses of farmers. The seed coat of the studied cultivars was "rugose". The elevation folding of the rugose pattern ranged from 11 ± 1.73 µm (Giza 126) to 14.67 ± 2.43 µm (Giza 123), suggesting variation in seed quality and its uses in feed and the food industry. According to the similarity matrix of ISSR analysis, the highest similarity value (93%) was recorded between Giza 133 and Giza 132, as well as between Giza 2000 and Giza 126. On the other hand, the lowest similarity value (80%) was recorded between Giza 130 and (Giza 133 and Giza 132), indicating that these cultivars were distantly related. Polymorphism information content (PIC) ranged from 0.26 for the primer ISSR UBC 835 to 0.37 for the primers ISSR UBC 814 and ISSR UBC 840. The current study showed that the matK gene is more mutable than the rbcL gene among the tested cultivars.

Melatonin Regulatory Mechanisms and Phylogenetic Analyses of Melatonin Biosynthesis Related Genes Extracted from Peanut under Salinity Stress.

Melatonin improves the tolerance of plants to various environmental stresses by protecting plant cells against oxidative stress damage. The objective of the current study was to determine whether exogenous melatonin (MT) treatments could help protecting peanut (Arachis hypogaea) seedlings against salinity stress. This was achieved by investigating enzymatic and non-enzymatic antioxidant systems and the expression of melatonin biosynthesis related genes in response to salinity stress with or without exogenous MT. The results showed a significant increase in the concentrations of reactive oxygen species (ROS) in peanut seedlings under salinity stress. The exogenous application of melatonin decreased the levels of ROS through the activation of antioxidant enzymes in peanut seedlings under salinity stress. Transcription levels of melatonin biosynthesis related genes such as N-acetylserotonin methyltransferase (ASMT1, ASMT2, ASMT3), tryptophan decarboxylase (TDC), and tryptamine 5-hydroxylase (T5H) were up-regulated with a 150 µM melatonin treatment under salinity stress. The results indicated that melatonin regulated the redox homeostasis by its ability to induce either enzymatic or non-enzymatic antioxidant systems. In addition, phylogenetic analysis of melatonin biosynthesis genes (ASMT1, ASMT2, ASMT3, TDC, T5H) were performed on a total of 56 sequences belonging to various plant species including five new sequences extracted from Arachis hypogaea (A. hypogaea). This was based on pairwise comparison among aligned nucleotides and predicted amino acids as well as on substitution rates, and phylogenetic inference. The analyzed sequences were heterogeneous and the A. hypogaea accessions were primarily closest to those of Manihot esculenta, but this needs further clarification.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

Blood pressure standards for pre-school children in Saudi Arabia.

The prevalence of hypertension (HTN) in children is increasing. Early detection of HTN in childhood may prevent the occurrence of complications in adult age. Blood pressure (BP) varies between populations according to ethnic and environmental factors. Based on these variations, reference norms developed for one particular population may not be applicable to others. Thus, this study aimed to provide age-, gender-, and height-related BP reference standards using oscillometric techniques for pre-school children in Saudi Arabia. A sub-sample of preschool children aged from 2 to 6 years was selected by multi-stage probability sampling of Saudi population. The samples represented Saudi children from the whole country. Data were collected through a house-to-house survey of all selected households in all 13 regions in the country. Oscillometric devices were used to measure the BP. Data were analyzed to study the distribution pattern of systolic (SBP) and diastolic BP (DBP) and to develop reference values based on age, gender, and height. The values for each age and height percentile were compared with the recent (2017) values of the North American children. A total of 2553 Saudi Arabian children (1299 boys and 1254 girls) aged 2-6 years with complete data on age, gender, height, SBP, and DBP were considered for analysis. Values for SBP and DBP were significantly higher in Saudi children than in the North American children. This study adds evidence to the BP variations between populations with influences such as genetic and environmental factors. The need of every population to define its normal BP standards is essential to avoid unnecessary investigations and anxiety in patients and their parents.

Trends in infant nutrition in Saudi Arabia: compliance with WHO recommendations.

BACKGROUND AND OBJECTIVE: The WHO recommends exclusive breastfeeding in the first 6 months of life. Our objective was to evaluate trends in infant nutrition in Saudi Arabia and the degree of compliance with WHO recommendations. SUBJECTS AND METHODS: A nationwide nutritional survey of a sample of Saudi households was selected by the multistage probability sampling procedure. A validated questionnaire was administered to mothers of children less than 3 years of age. RESULTS: Of 5339 children in the sample, 4889 received breast milk at birth indicating a prevalence of initiation of 91.6%. Initiation of breastfeeding was delayed beyond 6 hours after birth in 28.1% of the infants. Bottle feeding was introduced by 1 month of age to 2174/4260 (51.4%) and to 3831/4260 (90%) by 6 months of age. The majority of infants 3870/4787 (80.8%) were introduced to "solid foods" between 4 to 6 months of age and whole milk feedings were given to 40% of children younger than 12 months of age. CONCLUSIONS: The current practice of feeding of Saudi infants is very far from compliance with even the most conservative WHO recommendations of exclusive breastfeeding for 4 to 6 months. The high prevalence of breastfeeding initiation at birth indicates the willingness of Saudi mothers to breastfeed. However, early introduction of complementary feedings reduced the period of exclusive breastfeeding. Research in infant nutrition should be a public health priority to improve the rate of breastfeeding and to minimize other inappropriate practices.

Blood pressure standards for Saudi children and adolescents.

BACKGROUND AND OBJECTIVES: Blood pressure levels may vary in children because of genetic, ethnic and socioeconomic factors. To date, there have been no large national studies in Saudi Arabia on blood pressure in children.Therefore, we sought to establish representative blood pressure reference centiles for Saudi Arabian children and adolescents. SUBJECTS AND METHODS: We selected a sample of children and adolescents aged from birth to 18 years by multi-stage probability sampling of the Saudi population. The selected sample represented Saudi children from the whole country. Data were collected through a house-to-house survey of all selected households in all 13 regions in the country. Data were analyzed to study the distribution pattern of systolic (SBP) and diastolic blood pressure (DBP) and to develop reference values. The 90th percentile of SBP and DBP values for each age were compared with values from a Turkish and an American study. RESULTS: A total of 16 226 Saudi children and adolescents from birth to 18 years were studied. Blood pressure rose steadily with age in both boys and girls. The average annual increase in SBP was 1.66 mm Hg for boys and 1.44 mm Hg for girls. The average annual increase in DBP was 0.83 mm Hg for boys and 0.77 mm Hg for girls. DBP rose sharply in boys at the age of 18 years. Values for the 90th percentile of both SBP and DBP varied in Saudi children from their Turkish and American counterparts for all age groups. CONCLUSION: Blood pressure values in this study differed from those from other studies in developing countries and in the United States, indicating that comparison across studies is difficult and from that every population should use their own normal standards to define measured blood pressure levels in children.

The Impact of Drought Stress on Antioxidant Responses and Accumulation of Flavonolignans in Milk Thistle (Silybum marianum (L.) Gaertn).

Biosynthesis and accumulation of flavonolignans in plants are influenced by different environmental conditions. Biosynthesis and accumulation of silymarin in milk thistle (Silybum marianum L.) were studied under drought stress with respect to the antioxidant defense system at the physiological and gene expression level. The results revealed a reduction in leaf chlorophyll, ascorbic acid, and glutathione contents. In contrast, H2O2, proline, and antioxidative enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR), were increased. These results confirmed that milk thistle undergoes oxidative stress under drought stress. Furthermore, transcription levels of APX, SOD, CAT, 1-Cys-Prx, and PrxQ were significantly increased in milk thistle under drought stress. Overall this suggests that protection against reactive oxygen species and peroxidation reactions in milk thistle are provided by enzymatic and non-enzymatic antioxidants. Flavonolignans from milk thistle seeds after different drought treatments were quantified by high-performance liquid chromatography (HPLC) and showed that severe drought stress enhanced the accumulation of silymarin and its components compared with seeds from the control (100% water capacity). Silybin is the major silymarin component and the most bioactive ingredient of the milk thistle extract. Silybin accumulation was the highest among all silymarin components in seeds obtained from drought-stressed plants. The expression of the chalcone synthase (CHS) genes (CHS1, CHS2, and CHS3), which are associated with the silybin biosynthetic pathway, was also increased during drought stress. These results indicated that milk thistle exhibits tolerance to drought stress and that seed derived from severe drought-stressed plants had higher levels of silymarin.

Trends in the nutritional status of Saudi children.

OBJECTIVE: To evaluate the trend in the nutritional status of Saudi children over a 10-year period. METHODS: The growth data collected between 1993--1994 were compared with those collected between 2004--2005 from all regions of the Kingdom. Both nutritional surveys had a similar design leading to representative samples of Saudi children determined by multistage probability sampling. Similar methodology of measurements of the weight and height were used. The data from the 1994 study, including the third, fifth, fiftieth, ninety-fifth, and the ninety-seventh percentiles, were plotted on the 2005 charts for the weight for age, height for age, weight for height. RESULTS: Compared to the 1994 results, the data of the 2005 study indicate an upward shift of the lower percentiles of the weight for age, and the weight for height, more than height for age, indicating improved nutritional status. However, the upward shift of the higher percentiles for the weight for age, and weight for height in the 2005 survey, indicate increased trend for overweight and obesity. CONCLUSION: There is a demonstrable improvement in the nutritional status of Saudi children, and also tendency toward overweight and obesity over the last decade.

The prevalence of sickle cell disease in Saudi children and adolescents. A community-based survey.

OBJECTIVE: To determine the prevalence and regional distribution of sickle cell disease in Saudi children. METHODS: A sample size of 45,682 children and adolescents from newborn to 19 years of age was selected by multistage random probability sampling of the Saudi households from each of the 13 regions of the country. The study is cross-sectional, community based, and conducted over 2 years from 2004 to 2005. Data, including history and clinical examination were collected with house-to-house survey of all selected households. Data management and analysis was carried out at King Saud University, Riyadh, Saudi Arabia. RESULTS: Sickle cell disease was detected in 108 of 45,682 children and adolescents with a prevalence of 24 per 10,000. The regional distribution of sickle cell disease showed eastern region dominance with a prevalence of 145 per 10,000, followed by the southern region with a prevalence of 24 per 10,000, western region 12 per 10,000, and central region with 6 per 10,000. No cases were found in the northern regions. The male to female ratio was approximately 1:1. CONCLUSION: The results of this national wide community-based survey show a high prevalence of sickle cell disease in the community and the disease is more common in eastern and southern regions of the country. National or regional newborn screening programs for sickle cell disease using hematological tests should be planed. This study shows that the population at risk has an uneven geographical distribution. For this reason, selective rather than universal neonatal screening is likely to be more appropriate in the country.

Consanguinity and major genetic disorders in Saudi children: a community-based cross-sectional study.

BACKGROUND AND OBJECTIVES: There is a high rate of consanguinity in Saudi Arabia; however, information on its relationship with genetic disorders is limited. The objective of this cross-sectional study was to explore the role of consanguinity in genetic disorders. SUBJECTS AND METHODS: The study sample was determined by a multistage probability random sampling procedure. Consanguinity status was obtained during household visits. Primary care physicians performed a history and physical examination of all children and adolescents younger than 19 years, and all cases of genetic diseases were recorded. The chi-square test was used to compare proportions. RESULTS: During the two-year study period (2004-2005), 11 554 of 11 874 (97%) mothers answered the question on consanguinity, and 6470 of 11 554 (56%) were consanguineous. There was no significant association between first-cousin consanguinity and Down syndrome (P=.55). Similarly, there was no significant association with either sickle cell disease (P=.97) or glucose-6-phosphate dehydrogenase deficiency (P=.67) for first-cousin consanguinity. A borderline statistical significance was found for major congenital malformations (P=.05). However, the most significant association with first-cousin consanguinity was congenital heart disease (CHD) (P=.01). Finally, no significant association was found for type 1 diabetes mellitus (P=.92). For all types of consanguinity, similar trends of association were found, with a definite statistically significant association only with CHD (P=.003). CONCLUSION: The data suggest a significant role of parental consanguinity in CHD. However, a relationship between consanguinity and other genetic diseases could not be established. The effect of consanguinity on genetic diseases is not uniform and this should be taken into consideration in genetic counseling.