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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Novel Plastid-Nuclear Genome Combinations Enhance Resistance to Citrus Canker in Cybrid Grapefruit.

Host disease resistance is the most desirable strategy for control of citrus canker, a disease caused by a gram-negative bacterium Xanthomonas citri subsp. citri. However, no resistant commercial citrus cultivar has been identified. Cybridization, a somatic hybridization approach that combines the organelle and nuclear genomes from different species, was used to create cybrids between citrus canker resistant 'Meiwa' kumquat (Fortunella crassifolia Swingle snym. Citrus japonica Thunb.) and susceptible grapefruit (Citrus paradisi Macfad) cultivars. From these fusions, cybrids with grapefruit nucleus, kumquat mitochondria and kumquat chloroplasts and cybrids with grapefruit nucleus, kumquat mitochondria and grapefruit chloroplasts were generated. These cybrids showed a range of citrus canker response, but all cybrids with kumquat chloroplasts had a significantly lower number of lesions and lower Xanthomonas citri subsp. citri populations than the grapefruit controls. Cybrids with grapefruit chloroplasts had a significantly higher number of lesions than those with kumquat chloroplasts. To understand the role of chloroplasts in the cybrid disease defense, quantitative PCR was performed on both cybrid types and their parents to examine changes in gene expression during Xanthomonas citri subsp. citri infection. The results revealed chloroplast influences on nuclear gene expression, since isonuclear cybrids and 'Marsh' grapefruit had different gene expression profiles. In addition, only genotypes with kumquat chloroplasts showed an early up-regulation of reactive oxygen species genes upon Xanthomonas citri subsp. citri infection. These cybrids have the potential to enhance citrus canker resistance in commercial grapefruit orchards. They also serve as models for understanding the contribution of chloroplasts to plant disease response and raise the question of whether other alien chloroplast genotypes would condition similar results.

Metabolically speaking: Possible reasons behind the tolerance of 'Sugar Belle' mandarin hybrid to huanglongbing.

Huanglongbing (HLB) is currently considered the most destructive disease of citrus. Since its spread to the Americas, HLB has killed millions of trees and caused a sharp decline in production in many citrus growing regions. With the continuous spread of HLB disease in Florida and worldwide, there is an urgent need for the development of commercial citrus cultivars with a strong tolerance to HLB. Interestingly, field observations showed that some of the recently released mandarin hybrids such as 'Sugar Belle' were tolerant to HLB. In this study, we investigated the volatile and non-volatile metabolites of greenhouse-grown 'Sugar Belle' mandarin and four of its ancestors in order to understand why 'Sugar Belle' mandarin is relatively tolerant to HLB. Leaf volatiles were directly extracted with hexane and analyzed using gas chromatography-mass spectrometry (GC-MS). Leaf polar metabolites were extracted with a mixture of methanol:water (1:1, v/v), derivatized to their trimethylsilyl ethers, and analyzed using GC-MS. Forty-seven volatile compounds and forty-two polar metabolites were detected in 'Sugar Belle' mandarin leaves and its ancestors. 'Sugar Belle' was high in several volatiles such as α-thujene, para-cymene, γ-terpinene, thymol, ß-elemene, and (E)-ß-caryophyllene. Some of these volatiles, especially thymol, ß-elemene, and (E)-ß-caryophyllene are known for their anti-microbial activity. In addition, 'Sugar Belle' mandarin was the highest in synephrine, benzoic acid, ferulic acid, caffeic acid, chiro-inositol, fructose, glucose, threonic acid, saccharic acid, and galactaric acid, and the second in threonine, malic acid, and myo-inositol compared to the ancestors. Phenolic compounds such as benzoic, ferulic, and caffeic acids may act as antibacterial agents, whereas others like sugar alcohols may protect 'Sugar Belle' mandarin from stress during pathogen attack. The tolerance of 'Sugar Belle' and other newly released mandarin hybrids should be further evaluated using greenhouse controlled studies. If tolerance of these hybrids is confirmed, they could be used to replace the traditionally susceptible cultivars.

Somatic Embryogenesis: Still a Relevant Technique in Citrus Improvement.

The genus Citrus contains numerous fresh and processed fruit cultivars that are economically important worldwide. New cultivars are needed to battle industry threatening diseases and to create new marketing opportunities. Citrus improvement by conventional methods alone has many limitations that can be overcome by applications of emerging biotechnologies, generally requiring cell to plant regeneration. Many citrus genotypes are amenable to somatic embryogenesis, which became a key regeneration pathway in many experimental approaches to cultivar improvement. This chapter provides a brief history of plant somatic embryogenesis with focus on citrus, followed by a discussion of proven applications in biotechnology-facilitated citrus improvement techniques, such as somatic hybridization, somatic cybridization, genetic transformation, and the exploitation of somaclonal variation. Finally, two important new protocols that feature plant regeneration via somatic embryogenesis are provided: protoplast transformation and Agrobacterium-mediated transformation of embryogenic cell suspension cultures.

Regional variation in prevalence of overweight and obesity in Saudi children and adolescents.

BACKGROUND/AIMS: There are limited data on regional variation of overweight and obesity in the Kingdom of Saudi Arabia. Therefore, the aim of this report is to explore the magnitude of these variation in order to focus preventive programs to regional needs. SETTING AND DESIGN: Community-based multistage random sample of representative cohort from each region. PATIENTS AND METHODS: the study sample was cross-sectional, representative of healthy children and adolescents from 2 to 17 years of age. Body mass index (BMI) was calculated according to the formula (weight/height²). The 2000 center for disease control reference was used for the calculation of prevalence of overweight and obesity defined as the proportion of children and adolescents whose BMI for age was above 85 th and 95 th percentiles respectively, for Northern, Southwestern and Central regions of the Kingdom. Chi-square test was used to assess the difference in prevalence between regions and a P value of <0.05 was considered significant. RESULTS: The sample size was 3525, 3413 and 4174 from 2-17 years of age in the Central, Southwestern and Northern regions respectively. The overall prevalence of overweight was 21%, 13.4% and 20.1%, that of obesity was 9.3%, 6% and 9.1% in the Central, Southwestern and Northern regions respectively indicating a significantly-lower prevalence in the Southwestern compared to other regions (P<0.0001). CONCLUSIONS: This report revealed significant regional variations important to consider in planning preventive and therapeutic programs tailored to the needs of each region.