Abandoned agriculture soil can be recultivated by promoting biological phosphorus fertility when amended with nano-rock phosphate and suitable bacterial inoculant.

In semi-arid regions, post-restoration vegetation recovery on abandoned agricultural lands often fails due to inherently low organic matter content and poor soil fertility conditions, including phosphorus (P). As such, amending these soils with controlled release P fertilizer, especially with suitable P solubilizing bacteria (PSB) may promote plant growth and productivity by stimulating biological P fertility. To this aim, a pot study was performed to evaluate the agronomic potential of maize and soil biological P pools, using encapsulated (ENRP) and non-encapsulated (NRP) nano-rock phosphate as the P fertilizer source, on reclaimed agricultural soil in the presence and absence of PSB inoculant. The experiment was setup following a 3 × 2 factorial arrangement with four replicates. Without PSB, NRP treatment showed marginal positive effects on plant growth, P nutrition and P use efficiency (PUE) compared to control treatment. Although larger gains with NRP treatment were more noticeable under PSB inoculation, ENRP was the most convenient slow-release P fertilizer, increasing plant growth, P nutrition and grain yield compared to all treatments. Importantly, PSB inoculation with ENRP resulted in significantly higher increase in soil CaCl2-P (8.91 mg P kg soil-1), citrate-P (26.98 mg P kg soil-1), enzyme-P (18.98 mg P kg soil-1), resin-P (11.41 mg P kg soil-1), and microbial-P (18.94 mg P kg soil-1), when compared to all treatment combinations. Although a decrease in soil HCl-P content was observed with both types of P fertilizer, significant differences were found only with PSB inoculation. A significant increase in soil biological P pools could be due to the higher specific area and crystalline structure of nano materials, providing increased number of active sites for PSB activity in the presence of biobased encapsulated shell. Furthermore, the increase in PSB abundance, higher root carboxylate secretions, and decreased rhizosphere pH in response to nano-structured P fertilizer, implies greater extension of rhizosphere promoting greater P mobilization and/or solubilization, particularly under PSB inoculated conditions. We conclude that cropping potential of abandoned agricultural lands can be enhanced by the use of nano-rock phosphate in combination with PSB inoculant, establishing a favorable micro-environment for higher plant growth and biochemical P fertility.

A manipulative interplay between positive and negative regulators of phytohormones: A way forward for improving drought tolerance in plants.

Among different abiotic stresses, drought stress is the leading cause of impaired plant growth and low productivity worldwide. It is therefore essential to understand the process of drought tolerance in plants and thus to enhance drought resistance. Accumulating evidence indicates that phytohormones are essential signaling molecules that regulate diverse processes of plant growth and development under drought stress. Plants can often respond to drought stress through a cascade of phytohormones signaling as a means of plant growth regulation. Understanding biosynthesis pathways and regulatory crosstalk involved in these vital compounds could pave the way for improving plant drought tolerance while maintaining overall plant health. In recent years, the identification of phytohormones related key regulatory genes and their manipulation through state-of-the-art genome engineering tools have helped to improve drought tolerance plants. To date, several genes linked to phytohormones signaling networks, biosynthesis, and metabolism have been described as a promising contender for engineering drought tolerance. Recent advances in functional genomics have shown that enhanced expression of positive regulators involved in hormone biosynthesis could better equip plants against drought stress. Similarly, knocking down negative regulators of phytohormone biosynthesis can also be very effective to negate the negative effects of drought on plants. This review explained how manipulating positive and negative regulators of phytohormone signaling could be improvised to develop future crop varieties exhibiting higher drought tolerance. In addition, we also discuss the role of a promising genome editing tool, CRISPR/Cas9, on phytohormone mediated plant growth regulation for tackling drought stress.

Jasmonic acid: a key frontier in conferring abiotic stress tolerance in plants.

Abiotic stresses are the primary sources of crop losses globally. The identification of key mechanisms deployed and established by plants in response to abiotic stresses is necessary for the maintenance of their growth and persistence. Recent discoveries have revealed that phytohormones or plant growth regulators (PGRs), mainly jasmonic acid (JA), have increased our knowledge of hormonal signaling of plants under stressful environments. Jasmonic acid is involved in various physiological and biochemical processes associated with plant growth and development as well as plant defense mechanism against wounding by pathogen and insect attacks. Recent findings suggest that JA can mediate the effect of abiotic stresses and help plants to acclimatize under unfavorable conditions. As a vital PGR, JA contributes in many signal transduction pathways, i.e., gene network, regulatory protein, signaling intermediates and enzymes, proteins, and other molecules that act to defend cells from the harmful effects of various environmental stresses. However, JA does not work as an independent regulator, but acts in a complex signaling pathway along other PGRs. Further, JA can protect and maintain the integrity of plant cells under several stresses by up-regulating the antioxidant defense. In this review, we have documented the biosynthesis and metabolism of JA and its protective role against different abiotic stresses. Further, JA-mediated antioxidant potential and its crosstalk with other PGRs have also been discussed.

Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus.

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3-5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20-40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60-70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

An Outlook on Global Regulatory Landscape for Genome-Edited Crops.

The revolutionary technology of CRISPR/Cas systems and their extraordinary potential to address fundamental questions in every field of biological sciences has led to their developers being awarded the 2020 Nobel Prize for Chemistry. In agriculture, CRISPR/Cas systems have accelerated the development of new crop varieties with improved traits-without the need for transgenes. However, the future of this technology depends on a clear and truly global regulatory framework being developed for these crops. Some CRISPR-edited crops are already on the market, and yet countries and regions are still divided over their legal status. CRISPR editing does not require transgenes, making CRISPR crops more socially acceptable than genetically modified crops, but there is vigorous debate over how to regulate these crops and what precautionary measures are required before they appear on the market. This article reviews intended outcomes and risks arising from the site-directed nuclease CRISPR systems used to improve agricultural crop plant genomes. It examines how various CRISPR system components, and potential concerns associated with CRISPR/Cas, may trigger regulatory oversight of CRISPR-edited crops. The article highlights differences and similarities between GMOs and CRISPR-edited crops, and discusses social and ethical concerns. It outlines the regulatory framework for GMO crops, which many countries also apply to CRISPR-edited crops, and the global regulatory landscape for CRISPR-edited crops. The article concludes with future prospects for CRISPR-edited crops and their products.

Identification of cysteine-rich receptor-like kinase gene family in potato: revealed StCRLK9 in response to heat, salt and drought stresses.

The investigation into cysteine-rich receptor-like kinases (CRLKs) holds pivotal significance as these conserved, upstream signalling molecules intricately regulate fundamental biological processes such as plant growth, development and stress adaptation. This study undertakes a comprehensive characterisation of CRLKs in Solanum tuberosum (potato), a staple food crop of immense economic importance. Employing comparative genomics and evolutionary analyses, we identified 10 distinct CRLK genes in potato. Further categorisation into three major groups based on sequence similarity was performed. Each CRLK member in potato was systematically named according to its chromosomal position. Multiple sequence alignment and phylogenetic analyses unveiled conserved gene structures and motifs within the same groups. The genomic distribution of CRLKs was observed across Chromosomes 2-5, 8 and 12. Gene duplication analysis highlighted a noteworthy trend, with most gene pairs exhibiting a Ka/Ks ratio greater than one, indicating positive selection of StCRLKs in potato. Salt and drought stresses significantly impacted peroxidase and catalase activities in potato seedlings. The presence of diverse cis -regulatory elements, including hormone-responsive elements, underscored their involvement in myriad biotic and abiotic stress responses. Interestingly, interactions between the phytohormone auxin and CRLK proteins unveiled a potential auxin-mediated regulatory mechanism. A holistic approach combining transcriptomics and quantitative PCR validation identified StCRLK9 as a potential candidate involved in plant response to heat, salt and drought stresses. This study lays a robust foundation for future research on the functional roles of the CRLK gene family in potatoes, offering valuable insights into their diverse regulatory mechanisms and potential applications in stress management.

miRNAs for crop improvement.

Climate change significantly impacts crop production by inducing several abiotic and biotic stresses. The increasing world population, and their food and industrial demands require focused efforts to improve crop plants to ensure sustainable food production. Among various modern biotechnological tools, microRNAs (miRNAs) are one of the fascinating tools available for crop improvement. miRNAs belong to a class of small non-coding RNAs playing crucial roles in numerous biological processes. miRNAs regulate gene expression by post-transcriptional target mRNA degradation or by translation repression. Plant miRNAs have essential roles in plant development and various biotic and abiotic stress tolerance. In this review, we provide propelling evidence from previous studies conducted around miRNAs and provide a one-stop review of progress made for breeding stress-smart future crop plants. Specifically, we provide a summary of reported miRNAs and their target genes for improvement of plant growth and development, and abiotic and biotic stress tolerance. We also highlight miRNA-mediated engineering for crop improvement and sequence-based technologies available for the identification of miRNAs associated with stress tolerance and plant developmental events.

Developing drought-smart, ready-to-grow future crops.

Breeding crop plants with increased yield potential and improved tolerance to stressful environments is critical for global food security. Drought stress (DS) adversely affects agricultural productivity worldwide and is expected to rise in the coming years. Therefore, it is vital to understand the physiological, biochemical, molecular, and ecological mechanisms associated with DS. This review examines recent advances in plant responses to DS to expand our understanding of DS-associated mechanisms. Suboptimal water sources adversely affect crop growth and yields through physical impairments, physiological disturbances, biochemical modifications, and molecular adjustments. To control the devastating effect of DS in crop plants, it is important to understand its consequences, mechanisms, and the agronomic and genetic basis of DS for sustainable production. In addition to plant responses, we highlight several mitigation options such as omics approaches, transgenics breeding, genome editing, and biochemical to mechanical methods (foliar treatments, seed priming, and conventional agronomic practices). Further, we have also presented the scope of conventional and speed breeding platforms in helping to develop the drought-smart future crops. In short, we recommend incorporating several approaches, such as multi-omics, genome editing, speed breeding, and traditional mechanical strategies, to develop drought-smart cultivars to achieve the 'zero hunger' goal.

Putting CRISPR-Cas system in action: a golden window for efficient and precise genome editing for crop improvement.

The daunting task of feeding an ever-growing population is an immense challenge for the contemporary scientific community, especially in view of the rapidly changing climate throughout the world. Amidst these threatening crises, we witness rapid development in genome editing (GE) technologies, revolutionizing the field of applied genomics and molecular breeding. Various GE tools have been developed during the last two decades, but the CRISPR/Cas system has most recently made a significant impact on crop improvement. The major breakthroughs of this versatile toolbox are genomic modifications like single base-substitutions, multiplex GE, gene regulation, screening mutagenesis, and enhancing the breeding of wild crop plants. Previously, this toolbox was used to modify genes related to significant traits such as biotic/abiotic resistance/tolerance, post-harvest traits, nutritional regulation, and to address self-incompatibility analysis-related challenges. In the present review, we have demonstrated the functional dynamics of CRISPR-based GE and its applicability in targeting genes to accomplish novel editing of crops. The compiled knowledge will provide a solid foundation for highlighting the primary source for applying CRISPR/Cas as a toolbox for enhancing crops, to achieve food and nutritional security.

Potential Targets for CRISPR/Cas Knockdowns to Enhance Genetic Resistance Against Some Diseases in Wheat (Triticum aestivum L.).

Wheat is one of the most important food crops worldwide. Even though wheat yields have increased considerably in recent years, future wheat production is predicted to face enormous challenges due to global climate change and new versions of diseases. CRISPR/Cas technology is a clean gene technology and can be efficiently used to target genes prone to biotic stress in wheat genome. Herein, the published research papers reporting the genetic factors corresponding to stripe rust, leaf rust, stem rust, powdery mildew, fusarium head blight and some insect pests were critically reviewed to identify negative genetic factors (Susceptible, S genes) in bread wheat. Out of all reported genetic factors related to these disease, 33 genetic factors (S genes) were found as negative regulators implying that their down-regulation, deletion or silencing improved disease tolerance/resistance. The results of the published studies provided the concept of proof that these 33 genetic factors are potential targets for CRISPR/Cas knockdowns to improve genetic tolerance/resistance against these diseases in wheat. The sequences of the 33 genes were retrieved and re-mapped on the latest wheat reference genome IWGSC RefSeq v2.1. Phylogenetic analysis revealed that pathogens causing the same type of disease had some common conserved motifs and were closely related. Considering the significance of these disease on wheat yield, the S genes identified in this study are suggested to be disrupted using CRISPR/Cas system in wheat. The knockdown mutants of these S genes will add to genetic resources for improving biotic stress resistance in wheat crop.

Exploring the Genetic Diversity and Population Structure of Wheat Landrace Population Conserved at ICARDA Genebank.

Landraces are considered a valuable source of potential genetic diversity that could be used in the selection process in any plant breeding program. Here, we assembled a population of 600 bread wheat landraces collected from eight different countries, conserved at the ICARDA's genebank, and evaluated the genetic diversity and the population structure of the landraces using single nucleotide polymorphism (SNP) markers. A total of 11,830 high-quality SNPs distributed across the genomes A (40.5%), B (45.9%), and D (13.6%) were used for the final analysis. The population structure analysis was evaluated using the model-based method (STRUCTURE) and distance-based methods [discriminant analysis of principal components (DAPC) and principal component analysis (PCA)]. The STRUCTURE method grouped the landraces into two major clusters, with the landraces from Syria and Turkey forming two clusters with high proportions of admixture, whereas the DAPC and PCA analysis grouped the population into three subpopulations mostly according to the geographical information of the landraces, i.e., Syria, Iran, and Turkey with admixture. The analysis of molecular variance revealed that the majority of the variation was due to genetic differences within the populations as compared with between subpopulations, and it was the same for both the cluster-based and distance-based methods. Genetic distance analysis was also studied to estimate the differences between the landraces from different countries, and it was observed that the maximum genetic distance (0.389) was between the landraces from Spain and Palestine, whereas the minimum genetic distance (0.013) was observed between the landraces from Syria and Turkey. It was concluded from the study that the model-based methods (DAPC and PCA) could dissect the population structure more precisely when compared with the STRUCTURE method. The population structure and genetic diversity analysis of the bread wheat landraces presented here highlight the complex genetic architecture of the landraces native to the Fertile Crescent region. The results of this study provide useful information for the genetic improvement of hexaploid wheat and facilitate the use of landraces in wheat breeding programs.

Engineering broad-spectrum resistance to cotton leaf curl disease by CRISPR-Cas9 based multiplex editing in plants.

Advances in genome editing technologies have tremendous potential to address the limitations of classical resistance breeding. CRISPR-Cas9 based gene editing has been applied successfully in plants to tolerate virus infections. In this study, we successfully tested CRISPR-Cas9 system to counteract cotton leaf curl disease (CLCuD) caused by whitefly transmitted cotton leaf curl viruses (CLCuVs). We also analyzed the ability of CLCuV to escape the Cas9 endonuclease activity. Targeting overlapping genes of most prevalent CLCuVs with three gRNAs resulted in virus interference, as validated by low virus titer. Furthermore, multiplex CRISPR-Cas9 construct simultaneously targeting six genes of CLCuV, was found more effective to interfere with virus proliferation compared to targeting single region individually. Additionally, transgenic N. benthamiana plants expressing multiple gRNAs simultaneously showed enhanced tolerance against CLCuV infection when compared to wild-type plants. T7 Endonuclease-I (T7EI) assay, showing indels in the CLCuV genome, confirmed the occurrence of double strand breaks (DSBs) in DNA at target sequence induced by Cas9 endonuclease. We observed that targeting CLCuV genome at multiple sites simultaneously resulted in better interference, also with inefficient recovery of altered virus molecules. Next, we tested multiplex construct in cotton to interfere CLCuV infection. We found significant decrease in virus accumulation in cotton leaves co-infiltrated with multiplex cassette and virus compared to cotton leaves infiltrated with virus only. The results demonstrate future use of CRISPR-Cas9 system for engineering virus resistance in crops. Moreover, our results also advocate that resistance to mixed virus infections can be engineered using multiplex genome editing.

Genome-Wide Characterization of Glutathione Peroxidase (GPX) Gene Family in Rapeseed (Brassica napus L.) Revealed Their Role in Multiple Abiotic Stress Response and Hormone Signaling.

Plant glutathione peroxidases (GPXs) are the main enzymes in the antioxidant defense system that sustain H2O2 homeostasis and normalize plant reaction to abiotic stress conditions. To understand the major roles of the GPX gene family in rapeseed (Brassica napus L.), for the first time, a genome-wide study identified 25 BnGPX genes in the rapeseed genome. The phylogenetic analysis discovered that GPX genes were grouped into four major groups (Group I-Group IV) from rapeseed and three closely interrelated plant species. The universal investigation uncovered that the BnGPXs gene experienced segmental duplications and positive selection pressure. Gene structure and motifs examination recommended that most of the BnGPX genes demonstrated a comparatively well-maintained exon-intron and motifs arrangement within the identical group. Likewise, we recognized five hormones-, four stress-, and numerous light-reactive cis-elements in the promoters of BnGPXs. Five putative bna-miRNAs from two families were also prophesied, targeting six BnGPXs genes. Gene ontology annotation results proved the main role of BnGPXs in antioxidant defense systems, ROS, and response to stress stimulus. Several BnGPXs genes revealed boosted expression profiles in many developmental tissues/organs, i.e., root, seed, leaf, stem, flower, and silique. The qRT-PCR based expression profiling exhibited that two genes (BnGPX21 and BnGPX23) were suggestively up-regulated against different hormones (ABA, IAA, and MeJA) and abiotic stress (salinity, cold, waterlogging, and drought) treatments. In short, our discoveries provide a basis for additional functional studies on the BnGPX genes in future rapeseed breeding programs.

Controlling Geminiviruses before Transmission: Prospects.

Whitefly (Bemisia tabaci)-transmitted Geminiviruses cause serious diseases of crop plants in tropical and sub-tropical regions. Plants, animals, and their microbial symbionts have evolved complex ways to interact with each other that impact their life cycles. Blocking virus transmission by altering the biology of vector species, such as the whitefly, can be a potential approach to manage these devastating diseases. Virus transmission by insect vectors to plant hosts often involves bacterial endosymbionts. Molecular chaperonins of bacterial endosymbionts bind with virus particles and have a key role in the transmission of Geminiviruses. Hence, devising new approaches to obstruct virus transmission by manipulating bacterial endosymbionts before infection opens new avenues for viral disease control. The exploitation of bacterial endosymbiont within the insect vector would disrupt interactions among viruses, insects, and their bacterial endosymbionts. The study of this cooperating web could potentially decrease virus transmission and possibly represent an effective solution to control viral diseases in crop plants.

Fresh and composted industrial sludge restore soil functions in surface soil of degraded agricultural land.

A field study was conducted to test the potential of 5-year consecutive application of fresh industrial sludge (FIS) and composted industrial sludge (CIS) to restore soil functions at surface (0-15cm) and subsurface (15-30cm) of the degraded agricultural land. Sludge amendments increased soil fertility parameters including total organic carbon (TOC), soil available nitrogen (SAN), soil available phosphorus (SAP) and soil available potassium (SAK) at 0-15cm depth. Soil enzyme activities i.e. dehydrogenase (DHA), ß-glucosidase (BGA) and alkaline phosphatase (ALp) were significantly enhanced by FIS and CIS amendments in surface soil. However, urease activity (UA) and acid phosphatase (ACp) were significantly reduced compared to control soil. The results showed that sludge amendments significantly increased microbial biomass nitrogen (MBN) and microbial biomass phosphorus (MBP) at both soil depth, and soil microbial biomass carbon (MBC) only at 0-15cm depth. Significant changes were also observed in the population of soil culturable microflora (bacteria, fungi and actinomycetes) with CIS amendment in surface soil suggesting persistence of microbial activity owing to the addition of organic matter source. Sludge amendments significantly reduced soil heavy metal concentrations at 0-15cm depth, and the effect was more pronounced with CIS compared to unamended control soil. Sludge amendments generally had no significant impact on soil heavy metal concentrations in subsoil. Agronomic viability test involving maize was performed to evaluate phytotoxicity of soil solution extract at surface and sub-surface soil. Maize seeds grown in solution extract (0-15cm) from sludge treated soil showed a significant increase of relative seed germination (RSG), relative root growth (RRG) and germination index (GI). These results suggested that both sludge amendments significantly improved soil properties, however, the CIS amendment was relatively more effective in restoring soil functions and effectively immobilizing wastewater derived heavy metals compared to FIS treatment.