Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods

24 p.-4 fig.-6 tab.In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5 % (v/v) NaOCl was found to be most effective, about 97 % of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6- benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdul Aziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number 12-BIO2919-02.Peer reviewe

Bioactivities of Traditional Medicinal Plants in Alexandria

In traditional folklore, medicinal herbs play a vital role in the prevention and treatment of microbial diseases. In the present study, the phenolic profiles of the medicinal plants Asparagus aethiopicus L., Citrullus colocynthis L., Senna alexandrina L., Kalanchoe delagoensis L., Gasteria pillansii L., Cymbopogon citratus, Brassica juncea, and Curcuma longa L. were determined by high-performance liquid chromatography with a diode-array detector method. The results revealed rich sources of important compounds such as robinin in the fruits and leaves of A. aethiopicus; caffeic acid in the tubers of A. aethiopicus and quercitrin in the leaves of G. pillansii. Further, relatively high antioxidant, antibacterial, and antifungal activities were observed in C. colocynthis fruit coat, S. alexandrina pods, and A. aethiopicus leaves, respectively. The relatively higher the bioactivities of plants extracts associated with the phenols in these plants, in particular, the more abundant the phenols. Therefore, it was concluded that the fruit coat of C. colocynthis, pods of S. alexandrina, and leaves of A. aethiopicus might be excellent sources of natural products. These plant extracts also have a wide spectrum of antimicrobial activities that could be used in the pharmaceutical industries and to control diseases

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Artificial microRNA-Based RNA Interference and Specific Gene Silencing for Developing Insect Resistance in Solanum lycopersicum

RNA Interference (RNAi), which works against invading nucleic acids or modulates the expression of endogenous genes, is a natural eukaryotic regulating system, and it works by noncoding smaller RNA molecules. Plant-mediated gene silencing through RNAi can be used to develop plants with insect tolerance at transcriptional or post-transcriptional levels. In this study, we selected Myzus persicae’s acetylcholinesterase 1 gene (Ace 1) as a silencing target to develop transgenic Solanum lycopersicum L. plants’ resistance to aphids. An RNAi plasmid vector containing an artificial microRNA (amiRNA) sequence was engineered and successfully transformed into Jamila and Tomaland, two elite tomato cultivars. A northern blot analysis and PCR were carried out to check the efficacy of Agrobacterium-mediated transformation in T0 transgenic plants. The quantitative PCR data showed a substantial downregulation of the Ace 1 gene in aphids fed in clip cages on T1 transgenic plants. Furthermore, there was a substantial drop in aphid colonies that were fed on T1 transgenic plants of both the cultivars. These findings strongly suggest that transgenic plants that express amiRNA could be an important tool for engineering plants resistant to aphids and possibly for the prevention of viral disease in other plant-infested pests

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents

Natural Control of Weed Invasions in Hyper-Arid Arable Farms: Allelopathic Potential Effect of Conocarpus erectus against Common Weeds and Vegetables

Utilization of plant allelopathic potential to control weed infestations provides an effective, cost-efficient, labor-free, and environmentally acceptable alternative to traditional chemical and mechanical methods. Conocarpus erectus, known as buttonwood, belongs to the Combretaceae family with high contents of phytochemicals and antioxidant activity. There have been no studies on the allelopathic potential of C. erectus. The present study (1) examined the allelopathic potential of C. erectus against selected weeds (Chenopodium murale and Amaranthus viridis) and crops (Solanum lycopersicum and Cucumis sativus) via investigating the growth inhibition ability of its aqueous extract, and (2) identified the potential allelochemicals found in this plant. Aqueous extracts were prepared from leaves, roots, and seeds of C. erectus by immersing the dried powder of the examined plant parts in sterile distilled water for 24 h on a shaker set to 180 rpm. The resulting filtrate was considered as 100% solution, and then dilutions were made to various concentrations (75%, 50%, and 25%). C. erectus leaves and seeds showed the highest rate of inhibition at all concentrations against Chenopodium murale and Amaranthus viridis grown in either Petri dishes or pots. Conversely, all the studied extracts did not show any toxic effects against tomato and cucumber plants grown in pots. In Petri dishes, a slight reduction in growth was observed. HPLC analysis of total phenolic contents in C. erectus methanolic extracts showed that leaves have the highest contents of gallic acid, caffeic acid, and ferulic acid (153.963, 69.135, and 39.801 ppm, respectively). The finding of the current study demonstrated that the part of the plant and the concentration of extraction have a significant effect on phytotoxicity. The positive results of this study might be used to develop environmentally-friendly herbicides for agricultural purposes

Comparative Analysis between Wild and Cultivated Cucumbers Reveals Transcriptional Changes during Domestication Process

The cultivated cucumber (Cucumis sativus L.) was reported to have been developed from a wild cucumber (Cucumis hystrix Chakrav.), nevertheless, these two organisms exhibit noteworthy differences. For example, the wild cucumber is known for its high resistance to different biotic and abiotic stresses. Moreover, the leaves and fruits of the wild cucumber have a bitter taste compared to the cultivated cucumber. These differences could be attributed mainly to the differences in gene expression levels. In the present investigation, we analyzed the RNA-sequencing data to show the differentially expressed genes (DEGs) between the wild and cultivated cucumbers. The identified DEGs were further utilized for Gene Ontology (GO) and pathway enrichment analysis and for identification of transcription factors and regulators. In the results, several enriched GO terms in the biological process, cellular component, and molecular functions categories were identified and various enriched pathways, especially the biosynthesis pathways of secondary products were recognized. Plant-specific transcription factor families were differentially expressed between the wild and cultivated cucumbers. The results obtained provide preliminary evidence for the transcriptional differences between the wild and cultivated cucumbers which developed during the domestication process as a result of natural and/or artificial selection, and they formulate the basis for future genetic research and improvement of the cultivated cucumber

Genetic transformation and siRNA-Mediated gene silencing for aphid resistance in tomato

© The Author(s).We explored the ability of RNA interference (RNAi) to silence the Acetylcholinesterase 1 (Ace 1) gene in aphid Myzus persicae and developed transgenic tomato plants resistant to aphid infestation. Three plasmid constructs, T-449: a single Ace 1 fragment (forward orientation), T-452: two Ace 1 fragments (reverse and forward orientations), and T455: a single inverted Ace 1 fragment, were developed and transformed into two tomato cultivars, Jamila and Tomaland. PCR, northern blotting, and small interfering RNAs (siRNA) analysis were performed to validate the success of Agrobacterium-mediated transformation. The efficiency of transformation was highest for the T-452 construct. In vivo effects of the transformed constructs were confirmed in feeding experiments, and there was significant downregulation of the Ace 1 gene. In addition, an aphid challenge assay was conducted to investigate the siRNA-mediated silencing of the target gene (Ace 1) in the inhibition of fecundity in M. persicae. We found that the plants that were transformed with the T-452 vector had 37.5% and 26.4% lower fecundity at 27 °C in the Jamila and Tomaland, respectively. Our results strongly indicated that the plant-mediated silencing of aphid-RNA might be a robust and effective approach for developing pest and disease resistant in plantsThis project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdul Aziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number 12-BIO2919-02.Peer reviewe

Development of an Efficient and Rapid Micropropagation Protocol for In Vitro Multiplication of <i>Maerua crassifolia</i> Forssk

The difficult propagation of shrub and tree species and their extensive exposure to grazing threaten their abundance and lead to the necessity to find alternative means of propagation for these species. In vitro micropropagation techniques, viz., tissue culture, offer a promising tool for the rapid, cost-effective, and efficient propagation of different plant species. In the current study, a rapid and efficient in vitro multiplication protocol was developed for the micropropagation of Maerua crassifolia Forssk. Our results revealed that Murashige and Skoog (MS) medium with 7.5 µM of 6-benzylaminopurine (BA) and 1.0 µM of 1-naphthaleneacetic acid (NAA) led to the highest shoot formation (13.9 shoots per explant in 85.7% of the cultivated hypocotyls) among all other treatments. The best in vitro root formation was obtained on half-strength MS medium with 1.0 µM of indole 3-butyric acid (IBA) as 94.1% of the cultivated shoots formed 6.8 roots per microshoot on average. Ninety percent of the rooted plantlets were successfully acclimatized and are currently growing in the botanical garden of the Botany and Microbiology Department, King Saud University, Riyadh, Saudi Arabia. The genetic fidelity of the micropropagated plants was authenticated via flow cytometry. The results of the current study explained a simple, cost-effective, and efficient protocol for the micropropagation of the endangered M. crassifolia trees