Development of a PCR-based assay for specific and sensitive detection of Fusarium buharicum from infected okra plant.

Fusarium wilt, caused by the fungus Fusarium buharicum, is an emerging disease of okra in Japan. The disease was first reported in Japan in 2015, causing significant damage to okra seedlings. Due to the potential threat in okra cultivation, the development of an accurate detection method for F. buharicum is needed for the surveillance and management of the disease. In this study, we designed a primer set and developed conventional and nested PCR assays for the specific detection of F. buharicum in infected okra plants and contaminated soil, respectively. We compared the diversity of the translation elongation factor 1 alpha (EF-1α) gene of F. buharicum with 103 other fungal species/isolates to design a species-specific primer. This primer pair successfully amplified approximately 400 bp of PCR product that was only detected in the F. buharicum isolate, not in the other fungal isolates. The developed nested PCR method was highly sensitive and could detect the fungus from a 0.01 fg DNA sample. The primer successfully detected the pathogen in artificially infected plants and soil by conventional and nested PCR, respectively. This is the first report of the development of the F. buharicum-specific primer set and detection assays, which can be used for the specific and sensitive detection of F. buharicum in field samples and for taking early control measures.

Morphological Diversity and Genetic Parameter of Okra (Abelmoschus esculentus L. Moench) Genotypes.

<b>Background and Objective:</b> Considering that the potential for okra as an anti-diabetic is very high, while okra productivity in Indonesia is still low, a plant breeding program through variety development is needed. One of the initial activities that needs to be carried out is the characterization of various genotypes, both quantitative and qualitative characters. This research aimed to obtain information on the diversity of morpho-agronomic characters in okra genotypes. <b>Materials and Methods:</b> The experiment was conducted as a randomized block design, one factor is genotype with three replications. The materials used in this research were 20 okra genotypes. The experimental units used in this research were 60 units. Each experimental unit consists of 10 sample plants. Analysis of quantitative character variations used PKBT-STAT 3.1. Cluster analysis was carried out with PBSTAT-CL 2.1.2 with the Gower dissimilarity and average linkage clustering methods. Furthermore, analysis was carried out using SAS OnDemand for Academics to see the distinguishing characteristics between clusters. <b>Results:</b> There were differences in okra genotypes based on qualitative and quantitative characteristics. The most diverse quantitative character is the yield component, which is the fruit character. Variance in genetic and heritability showed broad and high criteria, respectively. Based on cluster analysis results, okra genotypes were grouped into 3 clusters with a cophenetic distance value of 0.40. Cluster 1 consists of 9 genotypes. Cluster 2 consists of 10 genotypes. Cluster 3 consists of 1 genotype the Red Hill Country genotype. The grouping in cluster analysis was carried out based on leaf width, number of fruits, fruit weight, fruit diameter and carpel thickness character. <b>Conclusion:</b> This diversity of okra germplasm can facilitate plant breeding activities in the future by selecting genotypes to serve as parents according to the objectives carried out.

Genomic and cytogenetic analyses reveal satellite repeat signature in allotetraploid okra (Abelmoschus esculentus).

BACKGROUND: Satellite repeats are one of the most rapidly evolving components in eukaryotic genomes and play vital roles in genome regulation, genome evolution, and speciation. As a consequence, the composition, abundance and chromosome distribution of satellite repeats often exhibit variability across various species, genome, and even individual chromosomes. However, we know little about the satellite repeat evolution in allopolyploid genomes. RESULTS: In this study, we investigated the satellite repeat signature in five okra (Abelmoschus esculentus) accessions using genomic and cytogenetic methods. In each of the five accessions, we identified eight satellite repeats, which exhibited a significant level of intraspecific conservation. Through fluorescence in situ hybridization (FISH) experiments, we observed that the satellite repeats generated multiple signals and exhibited variations in copy number across chromosomes. Intriguingly, we found that five satellite repeats were interspersed with centromeric retrotransposons, signifying their involvement in centromeric satellite repeat identity. We confirmed subgenome-biased amplification patterns of these satellite repeats through existing genome assemblies or dual-color FISH, indicating their distinct dynamic evolution in the allotetraploid okra subgenome. Moreover, we observed the presence of multiple chromosomes harboring the 35 S rDNA loci, alongside another chromosomal pair carrying the 5 S rDNA loci in okra using FISH assay. Remarkably, the intensity of 35 S rDNA hybridization signals varied among chromosomes, with the signals predominantly localized within regions of relatively weak DAPI staining, associated with GC-rich heterochromatin regions. Finally, we observed a similar localization pattern between 35 S rDNA and three satellite repeats with high GC content and confirmed their origin in the intergenic spacer region of the 35 S rDNA. CONCLUSIONS: Our findings uncover a unique satellite repeat signature in the allotetraploid okra, contributing to our understanding of the composition, abundance, and chromosomal distribution of satellite repeats in allopolyploid genomes, further enriching our understanding of their evolutionary dynamics in complex allopolyploid genomes.

Genetic Components Derived Parameters and Heterosis in Okra under Saudi Arabia Conditions.

Four parental genotypes of okra were crossed in complete diallel design to study the direction and extent of relative heterosis and heterobeltiosis for yield and its associated traits for utilization of existing genetic diversity to develop heterotic F1 hybrids in okra. The additive genetic component (D) was significant in all studied traits except average pod weight. Nonadditive (H1 and H2) components were found to be significant in all studied traits. However, the values of the dominant effect (H1) were smaller than the D components for no. of nodes/plant, no. of pods/plant, weight of medium pods, weight of large pods, and total fresh pod yield. The maximum significant MP heterosis in the desirable direction (149.9%) was recorded for the weight of large pods/plot. The maximum significant heterobeltiosis in the desirable direction (120.1%) was recorded for the weight of small pods/plot followed by total fresh pod yield (107.4%), the weight of large pods/plot (104.9%), weight of medium pods/plot (92.1%), average pod weight (51.8%), number of pods/plant (38.4%), and plant height (34.3%). It could be concluded that plant height, average pod weight, and the number of branches could be considered for the development of elite hybrids (heterosis breeding) or inbred lines (pure line selection) in succeeding generations. Therefore, these parameters can be considered for selecting genotypes to improve the pod yield of okra. The superior crosses identified through heterosis analysis were Egyptian Balady × Line 4.1.18 (30.8 ton/ha), Line 4.1.18 × Egyptian Balady (29.8 ton/ha), Dwarf Green Long Pod × Line 4.1.18 (28.3 ton/ha), and Egyptian Balady × Dwarf Green Long Pod (27.6 ton/ha) as these crosses had high performance as well as significant and higher estimates of heterobeltiosis for fruit yield per plant and yield attributing other characters.

Genome architecture and genetic diversity of allopolyploid okra (Abelmoschus esculentus).

The allopolyploid okra (Abelmoschus esculentus) unveiled telomeric repeats flanking distal gene-rich regions and short interstitial TTTAGGG telomeric repeats, possibly representing hallmarks of chromosomal speciation. Ribosomal RNA (rRNA) genes organize into 5S clusters, distinct from the 18S-5.8S-28S units, indicating an S-type rRNA gene arrangement. The assembly, in line with cytogenetic and cytometry observations, identifies 65 chromosomes and a 1.45 Gb genome size estimate in a haploid sibling. The lack of aberrant meiotic configurations implies limited to no recombination among sub-genomes. k-mer distribution analysis reveals 75% has a diploid nature and 15% heterozygosity. The configurations of Benchmarking Universal Single-Copy Ortholog (BUSCO), k-mer, and repeat clustering point to the presence of at least two sub-genomes one with 30 and the other with 35 chromosomes, indicating the allopolyploid nature of the okra genome. Over 130 000 putative genes, derived from mapped IsoSeq data and transcriptome data from public okra accessions, exhibit a low genetic diversity of one single nucleotide polymorphisms per 2.1 kbp. The genes are predominantly located at the distal chromosome ends, declining toward central scaffold domains. Long terminal repeat retrotransposons prevail in central domains, consistent with the observed pericentromeric heterochromatin and distal euchromatin. Disparities in paralogous gene counts suggest potential sub-genome differentiation implying possible sub-genome dominance. Amino acid query sequences of putative genes facilitated phenol biosynthesis pathway annotation. Comparison with manually curated reference KEGG pathways from related Malvaceae species reveals the genetic basis for putative enzyme coding genes that likely enable metabolic reactions involved in the biosynthesis of dietary and therapeutic compounds in okra.

Improving the genetic potential of okra (Abelmoschus esculentus L.) germplasm to tolerate salinity stress.

Okra (Abelmoschus esculentus L.) is the most consumed vegetable worldwide with the potential for diverse ecological adaptation. However, increasing salinization and changing climatic conditions are posing serious threats to the growth, yield, and quality of okra. Therefore, to mitigate increasing soil salinization and ensure sustainable okra production under rapidly changing climatic conditions, evaluation of new okra germplasm to develop salt tolerant cultivars is direly needed. The present study was designed to evaluate the genetic resources of okra genotypes for salt tolerance at growth and reproductive phases. Based on mophological and physio-biochemical responses of plants under stress condition, genotypes were divided into salt tolerant and succeptible groups. The experiment was comprised of 100 okra genotypes and each genotype was grown under control conditions and 6.5 dS m-1 NaCl concentration in a pot having 10 kg capacity. The experiment was conducted in a completely randomized design and each treatment was replicated three times. The results showed vast genetic variability among the evaluated okra germplasm traits like days to emergence, pod length, pod diameter, plant height, stem girth, and other yield-related parameters. Correlation analysis showed a highly significant positive association among the number of leaves at first flower and plant height at first flower.Likewise, pod weight also revealed a highly significant positive relationship for pod weight plant-1, pod length, and K+: Na+. Principal Component Analysis (PCA) revealed that out of 16 principal components (PCs), five components showed more than one eigenvalue and the first six PCs contributed 67.2% of the variation. Bi-plot analysis illustrated that genotypes 95, 111, 133, 99, and 128, under salt stress conditions, exhibited both high yield per plant and salt-tolerant behavior in other yield-related traits. On the basis of all studied traits, a salt susceptible group and a salt-tolerant group were formed. The salt tolerant group comprised of 97, 68, 95, 114, 64, 99, 111, 133, 128, and 109 genotypes, whereas, the salt susceptible group contained 137, 139, 130, 94, and 125 genotypes. Salt-tolerant okra genotypes were suggested to be used in further breeding programs aimed to develop salt tolerance in okra. These insights will empower precision breeding, underscore the importance of genetic diversity, and bear the potential to address the challenges of salt-affected soils while promoting broader agricultural resilience, economic prosperity, and food security.

Morpho-agronomic variability of okra [Abelmoschus esculentus (L.) Moench] genotypes in Dire Dawa, eastern Ethiopia.

A total of 21 okra genotypes were evaluated for 25 morpho-agronomic traits in 2020 at Dire Dawa, Ethiopia in a randomized complete block design with three replications. Analysis of variance showed significant differences at p<0.05 level of significance for all traits. Estimates of genotypic (GCV) and phenotypic (PCV) coefficients of variation range from 9.16 to 42.3% and 9.33 to 44.16%, respectively. Heritability in a broad sense (H2) and genetic advance as a percent of the mean (GAM) ranged from 29.57 to 91.89% and 10.39 to 83.53%, respectively. Estimated variability components (GCV, PCV, H2, and GAM) were high and moderate for all traits except days to 50% emergence 9.33% of GCV and PCV, internode length 9.16% of GCV and green fruit width 29.57% of H2 that were categorized under low. The first four principal component axes (PCA1 to PCA4) accounted for 7.83 to 35.02%, which accounted 74.56% of the total variability with eigenvalues that ranged from 1.95 to 8.75. Genetic distances estimated by Euclidean distance from the 25 traits ranged from 2.33 to 12.56 with a mean of 6.83, standard deviation of 1.8, and a coefficient of variation of 26.46%. The genotypes were grouped into four distinct clusters using the Euclidean distance matrix using UPGMA. Indigenous okra genotypes collected from Ethiopia were more divergent with high genetic distances and had a higher performance for most of the traits including growth, green fruit yield, and seed yield than introduced genotypes. In conclusion, this study showed the presence of variation among genotypes for most of the traits, indicating that selection of genotypes could be effective to develop okra varieties with high green fruit and seed yield through direct selection or crossing.

Selection and Validation of Reference Genes in Different Tissues of Okra (Abelmoschus esculentus L.) under Different Abiotic Stresses.

Okra (Abelmoschus esculentus L.) is a particular vegetable with both edible and medicinal values. However, the expression pattern of the okra reference genes in response to abiotic stress has not been explored. In the present study, 18 potential reference genes were selected from okra in various tissues and abiotic stress conditions, and their expression levels were detected by Real-Time quantitative PCR (RT-qPCR). Their expression stabilities were calculated by four algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). Under cold stress, the most stable genes included GAPC1 and CYP (leaf), CYP and ACT7 (root), HIS6 and GAPC1 (stem), and HIS6 and 60s (different tissues). Under salt stress, EF-1α and UBQ (leaf), EF-1α and UBQ (root), TUA4 and Eif (stem), and HIS6 and Eif (different tissues) were the most stable genes. Under drought stress, UBQ and Eif in the leaf, HIS6 and Eif in the root, TUA4 and HIS6 in the stem, and UBQ and Eif in different tissues were most stably expressed in okra. In addition, complete sequencing results by RefFinder showed that HIS6 and ACT7 in the leaf, HIS6 and Eif in the root, UBC5B and 60s in the stem, and HIS6 and Eif in different tissues, were most the suitable reference genes for okra. Furthermore, AeMYB1R1 transcription factor was used to verify the reliability of RT-qPCR values. In summary, this study was carried out to demonstrate the potential reference genes of okra under abiotic stress, aiming to provide a molecular basis for functional gene analysis and regulatory mechanism research of okra.

Seed production system and adaptability of okra (Abelmoschus esculentus L.) cultivars in Buea, Cameroon.

Okra is grown globally for its nutritional and economic benefits. Okra seeds ensure continuous production of the crop but challenges of poor production, adaptability and management may not allow the seeds to express their full potential. There are two seed production systems in Cameroon; the informal and formal. In Buea, the informal seed system is used by most farmers for seed production/utilization and farmers are reluctant to use hybrid seeds. This study aimed to assess the informal seed system of okra and evaluate the adaptability of seed produce from informal and formal systems in Buea. A survey and a field experiment were carried out. The designs for the survey and field experiment were stratified random sampling and randomized complete block design respectively. Data collection for the survey was done using questionnaires and other data collection instruments, while for the field experiment, data was collected on germination, vegetative growth parameters, incidence and severity of pests / diseases and yield. Data analysis for the survey was done using descriptive statistics, while data from the field experiment was done using a two-way ANOVA test and treatment means compared using the Tukey test at 5% probability. Results from the survey showed that women (60%) were mostly involved in seed production by mass selection from two landraces identified. Preservation of seeds was mostly done with the use of wood ash (58%) and insects were the major postharvest pest (76%). For the field experiment, at 66 DAP, Yellen recorded the highest significant number of leaves (13.417), leaf area (771.4 cm2) and the number of branches (5.64). Clemson spineless recorded the highest significant incidence (89.9%) and severity for pests / diseases while Kirikou and Landrace recorded the least incidence (0.0%) and severity. Kirikou recorded the highest significant yield (6.0 tons/ha), followed by Landrace (5.3 tons/ha). These findings reveal the performance of the landrace and provide reasons why farmers in Buea are reluctant to use hybrid okra seeds. The Landrace should certainly have adaptable genes, coupled with the autogamous nature of okra which encourages inbreeding for homozygous traits, which are dominant in expression compared to heterozygous traits.

Ectopic Expression of AeNAC83, a NAC Transcription Factor from Abelmoschus esculentus, Inhibits Growth and Confers Tolerance to Salt Stress in Arabidopsis.

NAC transcription factors play crucial roles in plant growth, development and stress responses. Previously, we preliminarily identified that the transcription factor AeNAC83 gene was significantly up-regulated under salt stress in okra (Abelmoschus esculentus). Herein, we cloned the nuclear-localized AeNAC83 from okra and identified its possible role in salt stress response and plant growth. The down-regulation of AeNAC83 caused by virus-induced gene silencing enhanced plant sensitivity to salt stress and increased the biomass accumulation of okra seedlings. Meanwhile, AeNAC83-overexpression Arabidopsis lines improved salt tolerance and exhibited many altered phenotypes, including small rosette, short primary roots, and promoted crown roots and root hairs. RNA-seq showed numerous genes at the transcriptional level that changed significantly in the AeNAC83-overexpression transgenic and the wild Arabidopsis with or without NaCl treatment, respectively. The expression of most phenylpropanoid and flavonoid biosynthesis-related genes was largely induced by salt stress. While genes encoding key proteins involved in photosynthesis were almost declined dramatically in AeNAC83-overexpression transgenic plants, and NaCl treatment further resulted in the down-regulation of these genes. Furthermore, DEGs encoding various plant hormone signal pathways were also identified. These results indicate that AeNAC83 is involved in resistance to salt stress and plant growth.

Association mapping for abiotic stress tolerance using heat- and drought-related syntenic markers in okra.

BACKGROUND: Considerable production losses are caused by heat and drought stress in okra. Germplasm evaluation at genetic level is essential for the selection of promising genotypes. Lack of genomic information of okra limits the use of genetic markers. However, syntenic markers of some related family could be used for molecular characterization of major economic traits. METHODS AND RESULTS: Herein, 56 okra genotypes were evaluated for drought and heat tolerance. Sixty-one expressed sequence tags (ESTs) identified for heat and drought tolerance in cotton were searched from literature surveys and databases. The identified ESTs were BLAST searched into okra unigene database. Primers of selected okra unigenes were synthesized and amplified in all genotypes using standard polymerase chain reaction (PCR) protocol. Marker trait association (MTA) of the syntenic unigenes were identified between genotypic and phenotypic data on the basis of linkage disequilibrium Functional syntenic analysis revealed that out of these 61 cotton ESTs 55 had functional homology with okra unigenes. These 55 unigenes were used as markers for further analysis (amplification). Okra genotypes showed significance variations for all the physo-morphological parameters under heat and drought stress. Genotypes Perbhani Karanti, IQRA-III, Selection Super Green, Anmol and Line Bourd performed better under drought stress whereas genotypes Perbhani Karanti, IQRA-III, Green Gold, OK-1501 and Selection Super Green showed heat tolerance. Fifty markers showed amplification in okra. Fifty-six okra genotypes were clustered into three distinct populations. LD analysis has shown most significant linkage between markers Unigene43786 and Unigene3662. MTAs using MLM and GLM models revealed that 23 markers have significant associations (p < 0.05) with different traits under control and stressed conditions. Relative water content is associated with four markers (Unigene10673, Unigene99547, Unigene152901, and Unigene129684) under drought conditions. Whereas, Electrolyte leakage was associated with 3 markers (Unigene109922, Unigene28667 and Unigene146907) under heat stress. CONCLUSION: These identified unigenes may be helpful in the development of drought and heat tolerant genotypes in okra.

Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.).

Quantitative real-time polymerase chain reaction (qRT-PCR) is a sensitive and widely used technique for quantifying gene expression levels, and its accuracy depends on the reference genes used for data normalization. To date, no reference gene has been reported in the nutritious and functional vegetable okra (Abelmoschus esculentus L.). Herein, 11 candidates of reference genes were selected and evaluated for their expression stability in okra in different tissues at different developmental stages by using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Among them, eukaryotic initiation factor 4 alpha (eIF4A) and protein phosphatase 2A (PP2A) showed the highest stability, while TUA5 had the lowest stability. The combined usage of these two most stable reference genes was sufficient to normalize gene expression in okra. Then, the above results were further validated by normalizing the expression of the cellulose synthase gene CesA4. This work provides appropriate reference genes for transcript normalization in okra, which will facilitate subsequent functional gene research on this vegetable crop.

The complete mitochondrial genome of okra (Abelmoschus esculentus): using nanopore long reads to investigate gene transfer from chloroplast genomes and rearrangements of mitochondrial DNA molecules.

BACKGROUND: Okra (Abelmoschus esculentus L. Moench) is an economically important crop and is known for its slimy juice, which has significant scientific research value. The A. esculentus chloroplast genome has been reported; however, the sequence of its mitochondrial genome is still lacking. RESULTS: We sequenced the plastid and mitochondrial genomes of okra based on Illumina short reads and Nanopore long reads and conducted a comparative study between the two organelle genomes. The plastid genome of okra is highly structurally conserved, but the mitochondrial genome of okra has been confirmed to have abundant subgenomic configurations. The assembly results showed that okra's mitochondrial genome existed mainly in the form of two independent molecules, which could be divided into four independent molecules through two pairs of long repeats. In addition, we found that four pairs of short repeats could mediate the integration of the two independent molecules into one complete molecule at a low frequency. Subsequently, we also found extensive sequence transfer between the two organelles of okra, where three plastid-derived genes (psaA, rps7 and psbJ) remained intact in the mitochondrial genome. Furthermore, psbJ, psbF, psbE and psbL were integrated into the mitochondrial genome as a conserved gene cluster and underwent pseudogenization as nonfunctional genes. Only psbJ retained a relatively complete sequence, but its expression was not detected in the transcriptome data, and we speculate that it is still nonfunctional. Finally, we characterized the RNA editing events of protein-coding genes located in the organelle genomes of okra. CONCLUSIONS: In the current study, our results not only provide high-quality organelle genomes for okra but also advance our understanding of the gene dialogue between organelle genomes and provide information to breed okra cultivars efficiently.

Copy number variation of two begomovirus acquired and inoculated by different cryptic species of whitefly, Bemisia tabaci in Okra.

The whitefly, B.tabaci is a major pest of agricultural crops which transmits begomovirus in a species-specific manner. Yellow vein mosaic disease (YVMD) and okra leaf curl disease (OLCD) caused by distinct begomovirus are a major limitation to production of okra in India. In this framework the present investigation reports, for the first time, comparative study of begomovirus species viz. yellow vein mosaic virus (YVMV) and okra enation leaf curl virus (OELCuV) ingested and egested by two cryptic species (Asia I and Asia II 5) of B.tabaci at different time interval using detached leaf assay. A gradual increase of both virus copies were observed with increased feeding exposure in Asia I and Asia II 5. Both the genetic groups of whitefly could acquire the viruses within just 5 minutes of active feeding however, a significant amount of variation was noted in virus uptake by the both. At 24 hours of active feeding Asia II 5 acquired more of YVMV whereas, Asia I ingested more OELCuV. Similarly, the genetic group acquiring higher titre of virus egested higher amount during inoculation period. On the whole, it can be presumed that Asia I is a more effective transmitter of OELCuV whereas, Asia II 5 of YVMV further suggesting increased risk of virus pandemics (both YVMV and OELCuV) in regions where Asia I and Asia II 5 is dominant.

Target-Based Physiological Modulations and Chloroplast Proteome Reveals a Drought Resilient Rootstock in Okra (Abelmoschus esculentus) Genotypes.

As climate changes increase, drought stress is becoming a problem for all major horticultural crops; among them is okra (Abelmoschus esculentus). Despite its superior resilience to heat stress and high nutritional content, it is still underutilized in contrast to other vegetable crops. Moreover, the drought-resistant and drought-sensitive genotypes of okra are also not well known and require further exploration to improve their productivity. To investigate this in more detail, we performed comparative physiological and large-scale chloroplast proteomics on drought-stressed genotypes of okra. We evaluated four major genotypes of okra, viz., NS7774, NS7772, Green Gold, and OH3312 for drought resilient rootstock. The physiological modulations demonstrated a significant change by 50-76% in biomass, net-photosynthetic machinery, water transport, and absorption both in early and late stages of drought stress compared to well-watered crops in all genotypes. Maximum oxidative damage due to drought stress was observed for the genotypes NS7772, Green Gold and OH3312 as depicted by H2O2 and O2- determination. Greater oxidative stress was correlated to lesser antioxidant activity and expression of antioxidant enzymes, such as catalase and ascorbate peroxidase under stress in okra genotypes. The overall photosynthetic pigments, such as total chlorophyll, and total carotenoid content, were also decreased, and stomatal guard cells were disrupted and appeared closed compared to the control for the above three mentioned genotypes, except NS7774. A subsequent tissue-specific proteome analysis of chloroplasts and thylakoids analyzed by BN-PAGE (blue native polyacrylamide gel electrophoresis) revealed either over or under expression of specific proteins, such as ATPase, PSI, PSII core dimer, PSII monomer and ATP synthase. The expression of multiprotein complex proteins, including PSII-core dimer and PSII-core monomer, was slightly higher for the genotype NS7774 when compared to three other genotypes for both 5 and 10 days of drought stress. Further identification of specific proteins obtained in second dimension BN-PAGE provided descriptive detail of seven proteins involved in drought resistance across all genotypes. The identified proteins are majorly involved in photosynthesis under drought stress, suggesting NS7774 as a drought tolerant genotype. Further, the proteomic results were confirmed using Immunoblot by selecting specific protein such as PsaA. Overall, from our physiological modulations and chloroplast proteomics in all genotypes, we summarized NS7774 as a resilient rootstock and the other three genotypes (NS7772, OH3312, and Green Gold) as sensitive ones.

Comparative physiological and full-length transcriptome analyses reveal the molecular mechanism of melatonin-mediated salt tolerance in okra (Abelmoschus esculentus L.).

BACKGROUND: Melatonin, a multifunctional signal molecule, has been reported to play crucial roles in growth and development and stress responses in various plant species. Okra (Abelmoschus esculentus L.) is a food crop with extremely high values of nutrition and healthcare. Recent reports have revealed the protective role of melatonin in alleviating salt stress. However, little is known about its regulatory mechanisms in response to salt stress in okra. RESULTS: In this study, we explored whether exogenous melatonin pretreatment could alleviate salt stress (300 mM NaCl) of okra plants. Results showed that exogenous application of melatonin (50 µM) significantly enhanced plant tolerance to salt stress, as demonstrated by the plant resistant phenotype, as well as by the higher levels of the net photosynthetic rate, chlorophyll fluorescence and chlorophyll content in comparison with nontreated salt-stressed plants. Additionally, melatonin pretreatment remarkably decreased the levels of lipid peroxidation and H2O2 content and scavenged O2•- in melatonin-pretreated plants, which may be attributed to the higher levels of enzyme activities including POD and GR. Moreover, a combination of third- (PacBio) and second-generation (Illumina) sequencing technologies was applied to sequence full-length transcriptomes of okra. A total of 121,360 unigenes was obtained, and the size of transcript lengths ranged from 500 to 6000 bp. Illumina RNA-seq analysis showed that: Comparing with control, 1776, 1063 and 1074 differential expression genes (DEGs) were identified from the three treatments (NaCl, MT50 and MT + NaCl, respectively). These genes were enriched in more than 10 GO terms and 34 KEGG pathways. Nitrogen metabolism, sulfur metabolism, and alanine, aspartate and glutamate metabolism were significantly enriched in all three treatments. Many transcription factors including MYB, WRKY, NAC etc., were also identified as DEGs. CONCLUSIONS: Our preliminary results suggested that melatonin pretreatment enhanced salt tolerance of okra plants for the first time. These data provide the first set of full-length isoforms in okra and more comprehensive insights into the molecular mechanism of melatonin responses to salt stress.

Two different begomovirus species are associated with yellow vein mosaic disease of okra in Sri Lanka.

Yellow vein mosaic disease is the major biotic constraint of okra cultivation in Sri Lanka. Identification and detailed molecular characterization of associated pathogen is needed for effective disease management. The genome of the begomovirus and betasatellite were amplified in symptomatic plant samples using specific degenerate primers. DNA-A genome of twelve isolates representing different locations in Sri Lanka were cloned, sequenced and deposited in GenBank database (Accession No- KX698087- KX698092 and MH455207- MH455212). Size of the complete nucleotide sequences ranged from 2735 to 2786 bp. The genome organization showed characteristics of begomoviruses. The pairwise sequence identity revealed the association of two different begomovirus species. Five of the isolates showed > 91% of sequences identity with Bhendi yellow vein mosaic virus, and the rest of the seven isolates were around 92% of identity with Okra enation leaf curl virus. This is further supported by phylogenetic analysis where both of these group of isolates were in different cluster. Recombination analysis showed the presence of recombinant fragments in the virus isolates associated with okra yellow vein mosaic disease (OYVMD) in Sri Lanka. Attempts to amplify DNA- B were failed in any of the samples tested. However, both type of the begomovirus species associated with betasatellite species, Bhendi yellow vein mosaic betasatellite. The present study has revealed the association of two distinct monopartite begomovirus species, Bhendi yellow vein mosaic virus or Okra enation leaf curl virus, with OYVMD in Sri Lanka.

Bhendi Yellow Vein Mosaic Virus and Bhendi Yellow Vein Mosaic Betasatellite Cause Enation Leaf Curl Disease and Alter Host Phytochemical Contents in Okra.

Whitefly (Bemisia tabaci)-transmitted begomoviruses cause severe diseases in numerous economically important dicotyledonous plants. Okra enation leaf curl disease (OELCuD) has emerged as a serious threat to okra (Abelmoschus esculentus L. Moench) cultivation in the Indian subcontinent. This study reports the association of a monopartite begomovirus (bhendi yellow vein mosaic virus; BYVMV) and betasatellite (bhendi yellow vein mosaic betasatellite; BYVB) with OELCuD in the Mau region of Uttar Pradesh, India. The BYVMV alone inoculated Nicotiana benthamiana and A. esculentus cv. Pusa Sawani plants developed mild symptoms. Co-inoculation of BYVMV and BYVB resulted in a reduced incubation period, an increased symptom severity, and an enhanced BYVMV accumulation by Southern hybridization and quantitative real-time PCR. This is the first study that satisfies Koch's postulates for OELCuD in its natural host. Activities of various antioxidative enzymes were significantly increased in the virus-inoculated okra plants. Differential responses in various biochemical components (such as photosynthetic pigments, phenol, proline, and sugar) in diseased okra plants were observed. This change in phytochemical responses is significant in understanding its impact on virus pathogenesis and disease development.

Cryopreservation of pollen of Abelmoschus moschatus Medik. subsp. moschatus as an aid to overcome asynchronous flowering for wide hybridization with cultivated Okra [A. esculentus (L.) Moench].

BACKGROUND: Asynchronous flowering is one of the major constraints for hybridization between Abelmoschus moschatus subsp. moschatus, a wild species closely related to cultivated okra [A. esculentus (L.) Moench]. Availability of viable pollen is a prerequisite to facilitate breeding in these species. OBJECTIVES: Pollen cryopreservation was attempted in A. moschatus subsp. moschatus, to overcome the asynchronous flowering barrier during wide hybridization with A. esculentus. MATERIALS AND METHODS: Viability of fresh pollen of A. moschatus subsp. moschatus was assessed using acetocarmine and triphenyl tetrazolium chloride (TTC) test and in vitro germination by sitting drop culture method. Pollen of 10 accessions were stored at four temperatures (25, 4, -20 and -196 degree C), in the dark and periodically monitored for viability. The standardized cryopreservation protocol was applied to 24 accessions of A. moschatus subsp. moschatus over three months. In vivo pollen germination of 24 accessions of cryopreserved pollen and its efficacy on fertilizing A. esculentus cv 'Pusa Sawani' were recorded and pollen was utilized for hybridization with A. esculentus. RESULTS: Brewbaker and Kwack medium with 15% sucrose was optimal for in vitro pollen germination. Pollen viability assessed by in vitro germination (60-90 %) was more reliable compared to acetocarmine (90-99 %) and TTC (85-99 %) staining tests. Significant negative correlation was found between pollen germination, storage time and temperature (25, 4 and -20 degree C) in all the accessions. Cryopreserved (-196 degree C) pollen showed significantly higher viability compared to all the other storage conditions, without viability loss. Successful pollination, fruit and seed set was observed in four out of 24 cross combinations attempted. CONCLUSION: The cryopreservation of pollen of A. moschatus subsp. moschatus and its fertilizing ability offers great potential for a successful wide hybridization programme in okra.

Inheritance pattern of okra enation leaf curl disease among cultivated species and its relationship with biochemical parameters.

Okra production in eastern India at present is severely threatened by whitefly-mediated okra enation leaf curl disease (OELCuD). Identification of resistant genotype and understanding the genetic control and biochemical relationship of OELCuD resistance are prerequisite for developing an effective breeding strategy. This study was conducted employing six populations (P1, P2, F1, F2, BC1 and BC2) of two selected (resistant x susceptible (RxS)) crosses. Associationship between severity of OELCuD and biochemical parameters of parents and hybrids at preflowering and flowering stages was studied. Segregation pattern of the genotypes in F2 generation showing OELCuD reaction of two crosses suggested that two duplicate recessive genes was operative for resistance to OELCuD. Generation mean analysis revealed involvement of both additive and nonadditive effects in the inheritance of disease resistance. Hence, postponement of selection in later generations or intermating among the selected segregates followed by one or two generations of selfing to break the undesirable linkage and allow the accumulation of favourable alleles could be suggested for the development of stable resistant genotype against this disease. Higher peroxidase activity and total phenol content in leaf emerged as reliable biochemical markers for early selection of genotype resistant to OELCuD.