Halotolerant Endophytic Bacteria Regulate Growth and Field Performance of Two Durum Wheat Genotypes with Contrasting Salinity Tolerance Potential.

Soil salinity hampers durum wheat plant growth and development at various stages. The detrimental effects of salinity on plant cellular and physiological processes necessitate strategies to alleviate its negative impact and improve overall crop yield. This study investigates the efficacy of plant growth-promoting rhizobacteria (PGPR) bacteria inoculation in mitigating salinity stress on two durum wheat genotypes with contrasting degrees of salinity tolerance (Tamaroi, salt-sensitive and Line 5004, salt-tolerant) under greenhouse and field conditions. For this purpose, two halotolerant-PGPR strains, Pseudomonas jordanii strain G34 and Oceanobacillus jordanicus strain GSFE11, were utilized for the inoculation. For the greenhouse experiment, the two selected genotypes were subjected to salinity at the flag leaf stage with continuous irrigation with a Hoagland solution supplemented with 50 mM NaCl. Field experiments were conducted across two locations with contrasting salinity levels over two growing seasons. At the end of both experiments, various parameters including total weight, spike weight, grain weight, spike number, spikelet number, grains per spike and thousand kernel weight were measured. The halotolerant PGPRs, P. jordanii strain G34 and O. jordanicus strain GSFE11, proved effective in alleviating salinity-induced adverse effects and enhancing growth under greenhouse and field conditions. However, bacterial inoculation significantly improved growth in the salt-sensitive genotype and such effects were not observed in the tolerant genotype, emphasizing genotype-specific responses. Notably, inoculation with O. jordanicus increased Na+ and Ca2+ uptake in the salt-tolerant "Line 5004" without hindering growth, suggesting one of its potential mechanisms for salt tolerance. This research demonstrates the potential of halotolerant-PGPR inoculation in enhancing durum wheat production in saline environments, but also underscores the importance of understanding genotype-specific responses for tailored interventions.

Secondary Metabolites Profiling, Antimicrobial and Cytotoxic Properties of Commiphora gileadensis L. Leaves, Seeds, Callus, and Cell Suspension Extracts.

Commiphora gileadensis L. is an important endangered medicinal plant that belongs to the family Burseraceae. In this study, C. gileadensis callus culture was established successfully using mature leaves as explants cultured on Murashige and Skoog (MS) media supplemented with 24.50 µM of indole butyric acid (IBA) and 2.22 µM 6-Benzylaminopurine (BAP) (callus induction media). The obtained callus was maintained on MS medium supplemented with 16.11 µM naphthalene acetic acid (NAA) in combination with 6.66 µM BAP, which resulted in a substantial increase in callus fresh and dry weights. The cell suspension culture was established successfully using liquid callus induction media supplemented with 3.0 mg·L-1 proline. Thereafter, the chemical constituents of different C. gileadensis methanolic extracts (callus, cell suspension, leaves, and seeds) were profiled, and their cytotoxic and antimicrobial properties were investigated. The LC-MS GNPS analyses were applied for chemical profiling of the methanolic plant extracts, and several natural products were identified, including flavonols, flavanones, and flavonoids glycosides, with two unusual families that included puromycin, 10-hydroxycamptothecin, and justicidin B. The methanolic extracts have shown selective antimicrobial and cytotoxic properties against different microbes and cancer cell lines. For instance, leaf extract showed the highest zone of inhibition for Staphylococcus aureus, while cell suspension culture was effective against Staphylococcus epidermidis and Staphylococcus aureus. All extracts showed selective activity against A549 cell lines for the cytotoxicity assay, while the leaf extract had a broad cytotoxic effect against all tested cell lines. This study revealed that C. gileadensis callus and cell suspension cultures can be employed to increase the in vitro formation of biologically active compounds that may have cytotoxicity and antibacterial action against different cancer cell lines and bacterial species. Further studies are required to isolate and identify such constituents that corroborate the observed activities.

In Vitro Cultures and Volatile Organic Compound Production in Chiliadenus montanus (Vhal.) Brullo.

Callus and microshoot cultures were established for Chiliadenus montanus (Vhal.) Brullo. (Asteraceae), a medicinal plant known for producing volatile organic compounds (VOCs). Callus induction was achieved successfully by culturing leaf explants on full-strength Murashige and Skoog medium (MS) supplemented with 2.2 µM 2, 4-dichlorophenoxy acetic acid (2,4-D) and 6.9 µM kinetin (Kin). Successful direct shoot regeneration was achieved using nodal explants cultured onto half-strength MS media supplemented with 1.4 µM Gibberellic Acid (GA3) and 4.4 µM 6-Benzylaminopurine (BAP). Indirect microshoots were successfully regenerated using callus cultured on MS media supplemented with 8.8 µM BAP, 2.2 µM Zeatin, and 1.4 µM GA3 followed by culturing on MS media supplemented with 8.8 µM BAP and 0.5 µM naphthalene acetic acid (NAA). Using wild plant aerial parts, callus and microshoots samples, VOCs were extracted successfully using Headspace Solid-Phase Micro-Extraction (HS-SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). In wild plant extracts, sesquiterpene hydrocarbons were found to be predominant with the following principal components: Alloaromadendrene (11.92%), trans-Cadina-1(6),4-diene (7.54%), and α-caryophyllene (6.77%). The analysis of in vitro microshoots revealed high levels of oxygenated monoterpenes with cis-Myrtanol (16.62%), and ß-Cyclocitral (14.3%) as the main components. Callus extract was dominated by monoterpene hydrocarbons and the main compounds identified were (Z)-ß-Ocimene (22.27%), p-Cymene (15.13%), and α-pinene (13.78%). In conclusion, an efficient in vitro production system of VOCs in C. montanus was established that can be used in the future for boosting their production without endangering wild plants.

Genome-Wide Identification and Analysis of the MADS-Box Gene Family in American Beautyberry (Callicarpa americana).

The MADS-box gene family encodes a number of transcription factors that play key roles in various plant growth and development processes from response to environmental cues to cell differentiation and organ identity, especially the floral organogenesis, as in the prominent ABCDE model of flower development. Recently, the genome of American beautyberry (Callicarpa americana) has been sequenced. It is a shrub native to the southern region of United States with edible purple-colored berries; it is a member of the Lamiaceae family, a family of medical and agricultural importance. Seventy-eight MADS-box genes were identified from 17 chromosomes of the C. americana assembled genome. Peptide sequences blast and analysis of phylogenetic relationships with MADS-box genes of Sesame indicum, Solanum lycopersicum, Arabidopsis thaliana, and Amborella trichopoda were performed. Genes were separated into 32 type I and 46 type II MADS-box genes. C. americana MADS-box genes were clustered into four groups: MIKCC, MIKC*, Mα-type, and Mγ-type, while the Mß-type group was absent. Analysis of the gene structure revealed that from 1 to 15 exons exist in C. americana MADS-box genes. The number of exons in type II MADS-box genes (5-15) greatly exceeded the number in type I genes (1-9). The motif distribution analysis of the two types of MADS-box genes showed that type II MADS-box genes contained more motifs than type I genes. These results suggested that C. americana MADS-box genes type II had more complex structures and might have more diverse functions. The role of MIKC-type MADS-box genes in flower and fruit development was highlighted when the expression profile was analyzed in different organs transcriptomes. This study is the first genome-wide analysis of the C. americana MADS-box gene family, and the results will further support any functional and evolutionary studies of C. americana MADS-box genes and serve as a reference for related studies of other plants in the medically important Lamiaceae family.

Differential expression of SlKLUH controlling fruit and seed weight is associated with changes in lipid metabolism and photosynthesis-related genes.

The sizes of plant organs such as fruit and seed are crucial yield components. Tomato KLUH underlies the locus fw3.2, an important regulator of fruit and seed weight. However, the mechanism by which the expression levels of KLUH affect organ size is poorly understood. We found that higher expression of SlKLUH increased cell proliferation in the pericarp within 5 d post-anthesis in tomato near-isogenic lines. Differential gene expression analyses showed that lower expression of SlKLUH was associated with increased expression of genes involved in lipid metabolism. Lipidomic analysis revealed that repression of SlKLUH mainly increased the contents of certain non-phosphorus glycerolipids and phospholipids and decreased the contents of four unknown lipids. Co-expression network analyses revealed that lipid metabolism was possibly associated with but not directly controlled by SlKLUH, and that this gene instead controls photosynthesis-related processes. In addition, many transcription factors putatively involved in the KLUH pathway were identified. Collectively, we show that SlKLUH regulates fruit and seed weight which is associated with altered lipid metabolism. The results expand our understanding of fruit and seed weight regulation and offer a valuable resource for functional studies of candidate genes putatively involved in regulation of organ size in tomato and other crops.

Genome Wide Association Mapping of Spot Blotch Resistance at Seedling and Adult Plant Stages in Barley.

Barley spot blotch (SB) caused by Cochliobolus sativus is one of the major constrains to barley production in warmer regions worldwide. The study was undertaken to identify and estimate effects of loci underlying quantitative resistance to SB at the seedling and adult plant stages. A panel of 261 high input (HI-AM) barley genotypes consisting of released cultivars, advanced breeding lines, and landraces, was screened for resistance to SB. The seedling resistance screening was conducted using two virulent isolates from Morocco (ICSB3 and SB54) while the adult plant stage resistance was evaluated at two hot spot locations, Faizabad and Varanasi, in India under artificial inoculation using a mixture of prevalent virulent isolates. The HI-AM panel was genotyped using DArT-Seq high-throughput genotyping platform. Genome wide association mapping (GWAM) was conducted using 13,182 PAV and 6,311 SNP markers, for seedling and adult plant resistance. Both GLM and MLM model were employed in TASSEL (v 5.0) using principal component analysis and Kinship Matrix as covariates. Final disease rating and Area Under Disease Progress Curve (AUDPC) were used for the evaluation of adult stage plant resistance. The GWAM analysis indicated 23 QTL at the seedling stage (14 for isolate ICSB3 and 9 for isolate SB54), while 15 QTL were detected at the adult plant stage resistance (6 at Faizabad and 9 at Varanasi) and 5 for AUDPC based resistance at Varanasi. Common QTL at seedling and adult plant stages were found across all barley chromosomes. Seedling stage QTL explained together 73.24% of the variance for seedling resistance to isolate ICSB3 and 49.26% for isolate SB54, whereas, QTL for adult plant stage resistance explained together 38.32%, 44.09% and 26.42% of the variance at Faizabad and Varanasi and AUDPC at Varanasi, respectively. Several QTL identified in this study were also reported in previous studies using bi-parental and association mapping populations, corroborating our results. The promising QTL detected at both stages, once validated, can be used for marker assisted selection (MAS) in SB resistance barley breeding program.

Assessment of Genetic Diversity among Pleurotus spp. Isolates from Jordan.

Pleurotus is considered an important genus that belongs to the family Pleurotaceae and includes the edible King Oyster mushroom (Pleurotus eryngii). In the present study, 19 Pleurotus isolates were collected from two locations in the north of Jordan (Tell ar-Rumman and Um-Qais). The morphological characteristics among collected isolates revealed that there was a morphological similarity among the collected isolates. Nucleotide sequence analysis of the internal transcribed spacer (ITS1⁻5.8S rDNA⁻ITS4 region) and 28S nuclear large subunit (nLSU) in the ribosomal DNA gene of the isolated stains showed that all of them share over 98% sequence similarity with P. eryngii. Genetic diversity among the collected strains was assessed using inter simple sequence repeat (ISSR) analysis using 18 different primer pairs. Using this approach, 141 out of 196 bands obtained were considered polymorphic and the highest percentage of polymorphism was observed using primer UBC827 (92.3%) with an overall Polymorphism Information Content (PIC) value of 70.56%. Cluster analysis showed that the Jordanian Pleurotus isolates fall into two main clades with a coefficient of similarity values ranging from 0.59 to 0.74 with a clear clustering based on collection sites. The results of the present study reveal that molecular techniques of ISSR and rDNA sequencing can greatly aid in classification and identification of Pleurotus spp. in Jordan.

Genome Wide Association Mapping of Seedling and Adult Plant Resistance to Barley Stripe Rust (Puccinia striiformis f. sp. hordei) in India.

Barley stripe rust is caused by Puccinia striiformis f.sp. hordei, (Psh), occurs worldwide, and is a major disease in South Asia. The aim of this work was to identify and estimate effects of loci underlying quantitative resistance to rust at seedling and adult plant stages. HI-AM panel of 261 barley genotypes consisting of released cultivars from North and South America, Europe, Australia, advanced breeding lines, and local landraces from ICARDA barley program were screened at seedling and adult plant stages for resistance to Psh. Seedling resistance was evaluated with the five prevalent Psh races in India. Screening for the adult plant stage resistance was also performed in two different locations by inoculating with a mixture of the five races used for seedling screeing. The panel was genotyped using DaRT-Seq high-throughput genotyping platform. The genome-wide association mapping (GWAM) showed a total of 45 QTL located across the seven barley chromosomes for seedling resistance to the five races and 18 QTL for adult plant stage resistance. Common QTL for different races at seedling stage were found on all chromosomes except on chromosome 1H. Four common QTL associated with seedling and adult plant stage resistance were found on chromosomes 2, 5, and 6H. Moreover, one of the QTL located on the long arm of chromosome 5H showed stable effects across environments for adult plant stage resistance. Several QTL identified in this study were also reported before in bi-parental and association mapping populations studies validating current GWAM. However 15 new QTL were found at adult plant stage on all chromosomes except the 4H, explaining up to 36.79% of the variance. The promising QTL detected at both stages, once validated, can be used for MAS in Psh resistance breeding program globally.

In Vitro Preservation of Transgenic Tomato (Solanum lycopersicum L.) Plants Overexpressing the Stress-Related SlAREB1 Transcription Factor.

In vitro preservation of transgenic tomato lines overexpressing the stress-responsive transcription factor SlAREB1 was studied by using slow growth and cryopreservation techniques. Slow growth preservation was performed by using different concentrations of sucrose (0, 100, 200, 300 mm) and abscisic acid (0, 4, 8, 12 µm) in Murashige and Skoog (MS) media, while cryopreservation was conducted by using encapsulation dehydration, V-cryoplates and seeds. Significant differences were observed between tested lines grown on MS media supplemented with 200 mm sucrose where transgenic lines overexpressing SlAREB1 showed improved growth when compared with negative control. The addition of abscisic acid (ABA) to the preservation media affected negatively transgenic lines growth and development when compared with ABA-free media. In encapsulation dehydration, non-cryopreserved transgenic lines overexpressing SlAREB1 pretreated in 0.8 M sucrose for 1 day and subjected to different dehydration periods showed significantly higher survival percentages when compared with negative control. For V-cryoplates technique, cryopreserved transgenic lines overexpressing SlAREB1 treated in 0.3 M sucrose for 3 days with or without cold acclimatization showed significantly higher survival percentages when compared with the negative control. Seed cryopreservation was performed successfully with a clear reduction in germination percentage in transgenic lines overexpressing high levels of SlAREB1. In conclusion, transgenic tomato lines overexpressing SlAREB1 were found to improve tolerance against different abiotic stresses associated with different in vitro preservation protocols.

The Role of Microsatellites in Streptophyta Gene Evolution.

Microsatellites form hotspot regions for recombination. In this research, we investigated whether genic microsatellites can be responsible for generating new genes by enhancing crossover between gene containing microsatellites and other genomic regions. We tested our hypothesis on 33,531 UniGene entries containing microsatellites. Each sequence was divided into microsatellites upstream and downstream fragments, and each pair of sequences was compared to study the microsatellites effect. The candidate pairs of genes are supposed to share a high similar fragment in one side of the microsatellites, while the other fragments should be completely different. This in silico approach detected 448 valid pairs of sequences in which both of them showed semi-resemblance nature. The synteny analysis for the detected sequences against 55 plant genomes indicated low representation of them across plant kingdom. Our results will add a body of knowledge toward understanding the role of microsatellites in gene evolution.

Over-expression of SlSHN1 gene improves drought tolerance by increasing cuticular wax accumulation in tomato.

Increasing cuticular wax accumulation in plants has been associated with improving drought tolerance in plants. In this study, a cDNA clone encoding the SlSHN1 transcription factor, the closest ortholog to WIN/SHN1 gene in Arabidopsis, was isolated from tomato plant. Expression analysis of SlSHN1 indicated that it is induced in response to drought conditions. The over-expression of SlSHN1 in tomato under the control of the constitutive CaMV 35S promoter produced plants that showed mild growth retardation phenotype with shiny and dark green leaves. Scanning electron microscopy showed that the over-expression of SlSHN1 in tomato resulted in higher cuticular wax deposition on leaf epidermial tissue when compared to non-transformed plants. Expression analysis in transgenic lines over-expressing SlSHN1 indicated that several wax-related synthesis genes were induced. Transgenic tomato plants over-expressing SlSHN1 showed higher drought tolerance when compared with wild type plants; this was reflected in delayed wilting of transgenic lines, improved water status and reduced water loss rate when compared with wild type plants. In conclusion, the SlSHN1 gene can modulate wax accumulation and could be utilized to enhance drought tolerance in tomato plant.

An efficient in vitro-inoculation method for Tomato yellow leaf curl virus.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV) is a member of the family Geminiviridae, genus Begomovirus. To test the infectivity of TYLCV in tomato plants, an improved protocol for inoculation of in vitro-cultured tomato plants was developed. RESULTS: A TYLCV isolate was cloned, sequenced and used to construct a 1.8-mer infectious clone. Three weeks old microshoots of TYLCV-susceptible tomato plants were inoculated with Agrobacterium tumefaciens harboring the infectious clone for the TYLCV isolate. After two weeks, the TYLCV symptoms started to appear on the in vitro-inoculated plants and the symptoms became more severe and pronounced eight weeks post-inoculation. The method was used efficiently to uncover the resistance mechanism against TYLCV in Solanum habrochaites accession LA 1777, a wild tomato known for its high resistance to whitefly and TYLCV. CONCLUSIONS: The reported in vitro-inoculation method can be used to screen tomato genotypes for their responses to TYLCV under controlled conditions and it will be a useful tool for better understanding of the TYLCV biology in tomato plants.