The 4Fs of cotton: genome editing of cotton for fiber, food, feed, and fuel to achieve zero hunger.

Cotton is globally known for its high-priority cellulose-rich natural fiber. In addition to providing fiber for the textile industry, it is an important source material for edible oil, livestock feed, and fuel products. Global warming and the growing population are the major challenges to the world's agriculture and the potential risks to food security. In this context, improving output traits in cotton is necessary to achieve sustainable cotton production. During the last few years, high throughput omics techniques have aided in identifying crucial genes associated with traits of cotton fiber, seed, and plant architecture which could be targeted with more precision and efficiency through the CIRPSR/Cas-mediated genome editing technique. The various CRISPR/Cas systems such as CRISPR/Cas9, CRISPR/nCas9, and CRISPR/Cas12a have been employed to edit cotton genes associated with a wide range of traits including fiber length, flowering, leaf colour, rooting, seed oil, plant architecture, gossypol content, somatic embryogenesis, and biotic and abiotic stresses tolerance, highlighting its effectiveness in editing the cotton genome. Thus, CRISPR/Cas-mediated genome editing has emerged as a technique of choice to tailor crop phenotypes for better yield potential and environmental resilience. The review covers a comprehensive analysis of cotton phenotypic traits and their improvement with the help of the latest genome editing tools to improve fiber, food, feed, and fuel-associated genes of cotton to ensure food security.

Cross-regulation of cytoskeleton and calcium signaling at plant-pathogen interface.

During plant-pathogen interactions, cytoskeleton and calcium signaling work independently as well as in coordination with each other for developing preformed and induced defense responses. A cell wall (CW) - plasma membrane (PM) - cytoskeleton (CS) continuum is maintained by coordination of cytoskeleton and calcium signaling. The current review is focused on the current knowledge of cytoskeleton­calcium cross-regulation during plant-pathogen interactions. Implications of recent technological developments in the existing toolkit that can address the outstanding questions of cytoskeleton­calcium coordination plant immunity are also discussed.

Phylogenomic curation of Ovate Family Proteins (OFPs) in the U's Triangle of Brassica L. indicates stress-induced growth modulation.

The Ovate Family Proteins (OFPs) gene family houses a class of proteins that are involved in regulating plant growth and development. To date, there is no report of the simultaneous functional characterization of this gene family in all members of U's Triangle of Brassica. Here, we retrieved a combined total of 256 OFP protein sequences and analyzed their chromosomal localization, gene structure, conserved protein motif domains, and the pattern of cis-acting regulatory elements. The abundance of light-responsive elements like G-box, MRE, and GT1 motif suggests that OFPs are sensitive to the stimuli of light. The protein-protein interaction network analysis revealed that OFP05 and its orthologous genes were involved in regulating the process of transcriptional repression through their interaction with homeodomain transcription factors like KNAT and BLH. The presence of domains like DNA binding 2 and its superfamily speculated the involvement of OFPs in regulating gene expression. The biotic and abiotic stress, and the tissue-specific expression analysis of the RNA-seq datasets revealed that some of the genes such as BjuOFP30, and BnaOFP27, BolOFP11, and BolOFP10 were highly upregulated in seed coat at the mature stage and roots under various chemical stress conditions respectively which suggests their crucial role in plant growth and development processes. Experimental validation of prominent BnaOFPs such as BnaOFP27 confirmed their involvement in regulating gene expression under salinity, heavy metal, drought, heat, and cold stress. The GO and KEGG pathway enrichment analysis also sheds light on the involvement of OFPs in regulating plant growth and development. These findings have the potential to serve as a forerunner for future studies in terms of functionally diverse analysis of the OFP gene family in Brassica and other plant species.

De novo transcriptome assembly of Dalbergia sissoo Roxb. (Fabaceae) under Botryodiplodia theobromae-induced dieback disease.

Dalbergia sissoo Roxb. (Shisham) is a timber-producing species of economic, cultural, and medicinal importance in the Indian subcontinent. In the past few decades, Shisham's dieback disease caused by the fungus Botryodiplodia theobromae has become an evolving issue in the subcontinent endangering its survival. To gain insights into this issue, a standard transcriptome assembly was deployed to assess the response of D. sissoo at the transcriptomic level under the stress of B. theobromae infection. For RNA isolation, the control and infected leaf tissue samples were taken from 1-year-old greenhouse-grown D. sissoo plants after 20 days of stem-base spore inoculation. cDNA synthesis was performed from these freshly isolated RNA samples that were then sent for sequencing. About 18.14 Gb (Giga base) of data was generated using the BGISEQ-500 sequencing platform. In terms of Unigenes, 513,821 were identified after a combined assembly of all samples and then filtering the abundance. The total length of Unigenes, their average length, N50, and GC-content were 310,523,693 bp, 604 bp, 1,101 bp, and 39.95% respectively. The Unigenes were annotated using 7 functional databases i.e., 200,355 (NR: 38.99%), 164,973 (NT: 32.11%), 123,733 (Swissprot: 24.08%), 142,580 (KOG: 27.75%), 139,588 (KEGG: 27.17%), 99,752 (GO: 19.41%), and 137,281 (InterPro: 26.72%). Furthermore, the Transdecoder detected 115,762 CDS. In terms of SSR (Simple Sequence Repeat) markers, 62,863 of them were distributed on 51,508 Unigenes and on the predicted 4673 TF (Transcription Factor) coding Unigenes. A total of 16,018 up- and 19,530 down-regulated Differentially Expressed Genes (DEGs) were also identified. Moreover, the Plant Resistance Genes (PRGs) had a count of 9230. We are hopeful that in the future, these identified Unigenes, SSR markers, DEGs and PRGs will provide the prerequisites for managing Shisham dieback disease, its breeding, and in tree improvement programs.

LbCas12a mediated suppression of Cotton leaf curl Multan virus.

Begomoviruses are contagious and severely affect commercially important fiber and food crops. Cotton leaf curl Multan virus (CLCuMuV) is one of the most dominant specie of Begomovirus and a major constraint on cotton yield in Pakistan. Currently, the field of plant genome editing is being revolutionized by the CRISPR/Cas system applications such as base editing, prime editing and CRISPR based gene drives. CRISPR/Cas9 system has successfully been used against biotic and abiotic plant stresses with proof-of-concept studies in both model and crop plants. CRISPR/Cas12 and CRISPR/Cas13 have recently been applied in plant sciences for basic and applied research. In this study, we used a novel approach, multiplexed crRNA-based Cas12a toolbox to target the different ORFs of the CLCuMuV genome at multiple sites simultaneously. This method successfully eliminated the symptoms of CLCuMuV in Nicotiana benthamiana and Nicotiana tabacum. Three individual crRNAs were designed from the CLCuMuV genome, targeting the specific sites of four different ORFs (C1, V1 and overlapping region of C2 and C3). The Cas12a-based construct Cas12a-MV was designed through Golden Gate three-way cloning for precise editing of CLCuMuV genome. Cas12a-MV construct was confirmed through whole genome sequencing using the primers Ubi-intron-F1 and M13-R1. Transient assays were performed in 4 weeks old Nicotiana benthamiana plants, through the agroinfiltration method. Sanger sequencing indicated that the Cas12a-MV constructs made a considerable mutations at the target sites of the viral genome. In addition, TIDE analysis of Sanger sequencing results showed the editing efficiency of crRNA1 (21.7%), crRNA2 (24.9%) and crRNA3 (55.6%). Furthermore, the Cas12a-MV construct was stably transformed into Nicotiana tabacum through the leaf disc method to evaluate the potential of transgenic plants against CLCuMuV. For transgene analysis, the DNA of transgenic plants of Nicotiana tabacum was subjected to PCR to amplify Cas12a genes with specific primers. Infectious clones were agro-inoculated in transgenic and non-transgenic plants (control) for the infectivity assay. The transgenic plants containing Cas12a-MV showed rare symptoms and remained healthy compared to control plants with severe symptoms. The transgenic plants containing Cas12a-MV showed a significant reduction in virus accumulation (0.05) as compared to control plants (1.0). The results demonstrated the potential use of the multiplex LbCas12a system to develop virus resistance in model and crop plants against begomoviruses.

The changing landscape of agriculture: role of precision breeding in developing smart crops.

Food plants play a crucial role in human survival, providing them essential nutrients. However, traditional breeding methods have not been able to keep up with the demands of the growing population. The improvement of food plants aims to increase yield, quality, and resistance to biotic and abiotic stresses. With CRISPR/Cas9, researchers can identify and edit key genes conferring desirable qualities in agricultural plants, including increased yield, enhanced product quality attributes, and increased tolerance to biotic and abiotic challenges. These modifications have enabled the creation of "smart crops" that exhibit rapid climatic adaptation, resistance to extreme weather conditions and high yield and quality. The use of CRISPR/Cas9 combined with viral vectors or growth regulators has made it possible to produce more efficient modified plants with certain conventional breeding methods. However, ethical and regulatory aspects of this technology must be carefully considered. Proper regulation and application of genome editing technology can bring immense benefits to agriculture and food security. This article provides an overview of genetically modified genes and conventional as well as emerging tools, including CRISPR/Cas9, that have been utilized to enhance the quality of plants/fruits and their products. The review also discusses the challenges and prospects associated with these techniques.

Comparative transcriptomic and evolutionary analysis of FAD-like genes of Brassica species revealed their role in fatty acid biosynthesis and stress tolerance.

BACKGROUND: Fatty acid desaturases (FADs) are involved in regulating plant fatty acid composition by adding double bonds to growing hydrocarbon chain. Apart from regulating fatty acid composition FADs are of great importance, and are involved in stress responsiveness, plant development, and defense mechanisms. FADs have been extensively studied in crop plants, and are broadly classed into soluble and non-soluble fatty acids. However, FADs have not yet been characterized in Brassica carinata and its progenitors. RESULTS: Here we have performed comparative genome-wide identification of FADs and have identified 131 soluble and 28 non-soluble FADs in allotetraploid B. carinata and its diploid parents. Most soluble FAD proteins are predicted to be resided in endomembrane system, whereas FAB proteins were found to be localized in chloroplast. Phylogenetic analysis classed the soluble and non-soluble FAD proteins into seven and four clusters, respectively. Positive type of selection seemed to be dominant in both FADs suggesting the impact of evolution on these gene families. Upstream regions of both FADs were enriched in stress related cis-regulatory elements and among them ABRE type of elements were in abundance. Comparative transcriptomic data analysis output highlighted that FADs expression reduced gradually in mature seed and embryonic tissues. Moreover, under heat stress during seed and embryo development seven genes remained up-regulated regardless of external stress. Three FADs were only induced under elevated temperature whereas five genes were upregulated under Xanthomonas campestris stress suggesting their involvement in abiotic and biotic stress response. CONCLUSIONS: The current study provides insights into the evolution of FADs and their role in B. carinata under stress conditions. Moreover, the functional characterization of stress-related genes would exploit their utilization in future breeding programs of B. carinata and its progenitors.

Genomic diversity of aquaporins across genus Oryza provides a rich genetic resource for development of climate resilient rice cultivars.

BACKGROUND: Plant aquaporins are critical genetic players performing multiple biological functions, especially climate resilience and water-use efficiency. Their genomic diversity across genus Oryza is yet to be explored. RESULTS: This study identified 369 aquaporin-encoding genes from 11 cultivated and wild rice species and further categorized these into four major subfamilies, among which small basic intrinsic proteins are speculated to be ancestral to all land plant aquaporins. Evolutionarily conserved motifs in peptides of aquaporins participate in transmembrane transport of materials and their relatively complex gene structures provide an evolutionary playground for regulation of genome structure and transcription. Duplication and evolution analyses revealed higher genetic conservation among Oryza aquaporins and strong purifying selections are assisting in conserving the climate resilience associated functions. Promoter analysis highlighted enrichment of gene upstream regions with cis-acting regulatory elements involved in diverse biological processes, whereas miRNA target site prediction analysis unveiled substantial involvement of osa-miR2102-3p, osa-miR2927 and osa-miR5075 in post-transcriptional regulation of gene expression patterns. Moreover, expression patterns of japonica aquaporins were significantly perturbed in response to different treatment levels of six phytohormones and four abiotic stresses, suggesting their multifarious roles in plants survival under stressed environments. Furthermore, superior haplotypes of seven conserved orthologous aquaporins for higher thousand-grain weight are reported from a gold mine of 3,010 sequenced rice pangenomes. CONCLUSIONS: This study unveils the complete genomic atlas of aquaporins across genus Oryza and provides a comprehensive genetic resource for genomics-assisted development of climate-resilient rice cultivars.

The SHERLOCK Platform: An Insight into Advances in Viral Disease Diagnosis.

Persistence and prevalence of microbial diseases (pandemics, epidemics) is the most alarming threats to the human resulting in huge health and economic losses. Rapid detection and understanding of the disease dynamics by molecular biotechnology tools allow for robust reporting, treatment and control of diseases. As per WHO, the optimal diagnostic approach should be quick, specific, sensitive, without a stringed instrument, and low cost. The drawbacks of traditional detection techniques promote the use of CRISPR-mediated nucleic acid detection methods such as SHERLOCK as detection method. It takes advantage of the unexpected in vitro features of CRISPR-Cas system to develop field-deployable sensitive detection tools. Previously, CRISPR-mediated diagnostic methods have extensively been reviewed particularly for SARS-COV-2 detection, but it fails to provide the insight into advances of this technique. This study is the first attempt to review the advances of SHERLOCK approach as diagnostic tool for viral diseases detection. Variations of SHERLOCK mechanism for improved efficiency are discussed. Particularly integrated SHERLOCK approaches in terms of extraction-free assay and Bluetooth-enabled detection are reviewed to access their feasibility for the development of simpler and cost-effective diagnostic toolkits. Insight in to perks and limitations of diagnostic methods indicates its potential as ultimate diagnostic instrument for disease management.

A Novel Detection Refinement Technique for Accurate Identification of Nephrops norvegicus Burrows in Underwater Imagery.

With the evolution of the convolutional neural network (CNN), object detection in the underwater environment has gained a lot of attention. However, due to the complex nature of the underwater environment, generic CNN-based object detectors still face challenges in underwater object detection. These challenges include image blurring, texture distortion, color shift, and scale variation, which result in low precision and recall rates. To tackle this challenge, we propose a detection refinement algorithm based on spatial-temporal analysis to improve the performance of generic detectors by suppressing the false positives and recovering the missed detections in underwater videos. In the proposed work, we use state-of-the-art deep neural networks such as Inception, ResNet50, and ResNet101 to automatically classify and detect the Norway lobster Nephrops norvegicus burrows from underwater videos. Nephrops is one of the most important commercial species in Northeast Atlantic waters, and it lives in burrow systems that it builds itself on muddy bottoms. To evaluate the performance of proposed framework, we collected the data from the Gulf of Cadiz. From experiment results, we demonstrate that the proposed framework effectively suppresses false positives and recovers missed detections obtained from generic detectors. The mean average precision (mAP) gained a 10% increase with the proposed refinement technique.

Ln2Te6O15 (Ln = La, Gd, and Eu) "Anti-Glass" Phase-Assisted Lanthanum-Tellurite Transparent Glass-Ceramics: Eu3+ Emission and Local Site Symmetry Analysis.

The presence of lanthanide-tellurite "anti-glass" nanocrystalline phases not only affects the transparency in glass-ceramics (GCs) but also influences the emission of a dopant ion. Therefore, a methodical understanding of the crystal growth mechanism and local site symmetry of doped luminescent ions when embedded into the precipitated "anti-glass" phase is crucial, which unfolds the practical applications of GCs. Here, we examined the Ln2Te6O15 "anti-glass" nanocrystalline phase growth mechanism and local site symmetry of Eu3+ ions in transparent GCs produced from 80TeO2-10TiO2-(5 - x)La2O3-5Gd2O3-xEu2O3 glasses, where x = 0, 1, 2. A crystallization kinetics study identifies a unique crystal growth mechanism via a constrained nucleation rate. The extent of "anti-glass" phase precipitation and its growth in GCs with respect to heat-treatment duration is demonstrated using X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM) analysis. Qualitative analysis of XRD confirms the precipitation of both La2Te6O15 and Gd2Te6O15 nanocrystalline phases. Rietveld refinement of powder X-ray diffraction patterns reveals that Eu3+ ions occupy "Gd" sites in Gd2Te6O15 over "La" sites in La2Te6O15. Raman spectroscopy reveals the conversion of TeO3 units to TeO4 units with Eu2O3 addition. This confirms the polymerizing role of Eu2O3 and consequently high crystallization tenacity with increasing Eu2O3 concentration. The measured Eu3+ ion photoluminescence spectra revealed its local site symmetry. Moreover, the present GCs showed adequate thermal cycling stability (∼50% at 423 K) with the highest activation energy of around 0.3 eV and further suggested that the present transparent GCs would be a potential candidate for the fabrication of red-light-emitting diodes (LEDs) or red component phosphor in W-LEDs.

Founder transformants of cotton (Gossypium hirsutum L.) obtained through the introduction of DS-Red, Rec, Rep and CRISPR/Cas9 expressing constructs for developing base lines of recombinase mediated gene stacking.

Cotton being the major fiber crop across the world is exposed to numerous biotic and abiotic stresses. Genetic transformation of cotton is vital to meet the world's food, feed and fiber demands. Genetic manipulation by randomly transferring the genes emanate variable gene expression. Targeted gene insertion by latest genome editing tools results in predictable expression of genes at a specified location. Gene stacking technology emerged as an adaptive strategy to combat biotic and abiotic stresses by integrating 2-3 genes simultaneously and at a specific site to avoid variable gene expression at diverse locations. This study explains the development of cotton's founder transformants to be used as a base line for multiple gene stacking projects. We introduced Cre and PhiC31 mediated recombination sites to specify the locus of incoming genes. CRISPR-Cas9 gene was integrated for developing CRISPR based founder lines of cotton. Cas9 gene along with gRNA was integrated to target Rep (replication) region of cotton leaf curl virus. Replication region of virus was specifically targeted to diminish further proliferation and preventing the virus to develop new strains. To successfully develop these primary transformants, a model transformation system has been optimized with the red color visualization (DS-Red). Following red color transformation system, three baselines with recombination specified site (Rec), targeted replication region (Rep) and Cas9 founder lines have been developed. These founder transformants are useful for developing recombinase mediated and CRISPR/Cas9 based originator lines of cotton. Moreover, these transformants will set up a base system for all the recombinase mediated gene stacking projects.

Optimization of Regeneration and Agrobacterium-Mediated Transformation Protocols for Bi and Multilocular Varieties of Brassica rapa.

The regeneration of the high-yielding multilocular types has not been attempted, although successful regeneration and transformation in brassica have been done. Here, we report efficient regeneration and transformation protocols for two B. rapa genotypes; UAF11 and Toria. The B. rapa cv UAF11 is a multilocular, non-shattering, and high-yielding genotype, while Toria is the bilocular type. For UAF11 8 shoots and for Toria 7 shoots, explants were observed on MS supplemented with 3 mg/L BAP + 0.4 mg/L NAA + 0.01 mg/L GA3 + 5 mg/L AgNO3 + 0.75 mg/L Potassium Iodide (KI), MS salt supplemented with 1 mg/L IBA and 0.37 mg/L KI produced an equal number of roots (3) in UAF11 and Toria. For the establishment of transformation protocols, Agrobacterium-mediated floral dip transformation was attempted using different induction media, infection time, and flower stages. The induction medium III yielded a maximum of 7.2% transformants on half-opened flowers and 5.2% transformants on fully opened flowers in UAF11 and Toria, respectively, with 15 min of inoculation. This study would provide the basis for the improvement of tissue culture and transformation protocols in multilocular and bilocular Brassica genotypes.

Characterization of new COBRA like (COBL) genes in wheat (Triticum aestivum) and their expression analysis under drought stress.

BACKGROUND: The COBL genes encode a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. Recently identified COBRA genes are supposed as a key regulator of the orientation of cell expansion in the root indicating that COBRA gene family members are likely to be important players at the plasma membrane-cell wall interface. METHODS AND RESULTS: Five COBL gene namely, TaCOBL 1, TaCOBL 2, TaCOBL 3, TaCOBL 4 and TaCOBL 5 were identified using database search and domain predictions. Chromosomal location of each gene was mapped on karyotype. Structure of genes, promoter analysis and phylogenetic analysis were performed using different bioinformatics tools. Set of novel SNPs were also predicted. Gene ontologies were analyzed, and the processes and pathways were identified in which COBRA genes were involved. The molecular weight all the cobra proteins was in range of 50-75 KDa with 429-461 amino acid residues. The COBL genes were mapped on homeologous groups 2, 4, 5, 6 and 7. Gene ontology analysis revealed that TaCOBL genes were involved in cellulose microfibril organization, mucilage biosynthetic process involved in seed coat development, plant-type cell wall biogenesis plant-type cell wall cellulose biosynthetic process, seed coat development and seed development. Three drought responsive cis-elements (WRKY, ABRE and DRE) were found nearby COBL genes The qRT-PCR revealed TaCOBL genes are drought responsive and can be further explored to understand their role in drought tolerance in wheat. CONCLUSION: The comprehensive annotation and expression profiling of COBL genes revealed that all five COBL genes are drought response. The promoter cis-regulatory element analysis revealed that COBL genes had stress related WRKY, ABRE and DRE cis-regulatory elements. This evidence suggest that TaCOBL genes are involved in drought stress tolerance.

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.

Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants.

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

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.

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.

Development of DNA barcodes for selected Acacia species by using rbcL and matK DNA markers.

Acacia species are very important tree species in tropical and subtropical countries of the World for their economic and medicinal benefits. Precise identification of Acacia is very important to distinguish the invasive species from rare species however, it is difficult to differentiate Acacia species based on morphological charcters. In addition, precise identification is also important for wood charcterization in the forest industry as these species are declining due to illegal logging and deforestation. To overcome thsese limitations of morphological identification, DNA barcoding is being used as an efficient and quick approach for precise identification of tree species. In this study, we selected two chloroplast and plastid base DNA markers (rbcL and matK) for the identification of five selected tree species of Acacia (A. albida, A. ampliceps, A. catechu, A. coriacea and A. tortilis). The genomic DNA of the selected Acacia species was extracted, amplified through PCR using specific primers and subsequently sequenced through Sanger sequencing. In matK DNA marker the average AT nucleotide contents were higher (59.46%) and GC contents were lower (40.44%) as compared to the AT (55.40%) and GC content (44.54%) in rbcL marker. The means genetic distance K2P between the Acacia species was higher in matK (0.704%) as compared to rbcL (0.230%). All Acacia species could be identified based on unique SNPs profile. Based on SNP data profiles, DNA sequence based scannable QR codes were developed for accurate identification of Acacia species. The phylogenetic analysis based on both markers (rbcL and matK) showed that both A. coriacea and A. tortilis were closely related with each other and clustered in the same group while other two species A. albida and A. catechu were grouped together. The specie A. ampliceps remained ungrouped distantly, compared with other four species. These finding highlights the potential of DNA barcoding for efficient and reproducible identification of Acacia species.