Drought, as an abiotic stressor, globally limits cereal productivity, leading to early aging of leaves and lower yields. The expression of the isopentenyl transferase (IPT) gene, which is involved in cytokinin (CK) biosynthesis, can delay drought-induced leaf senescence. In this study, the Agrobacterium Isopentenyl transferase (IPT) gene was introduced into two local hexaploid wheat cultivars, NR-421 and FSD-2008. The expression cassette was developed containing the IPT gene under transcriptional regulation of the stress-inducible promoter 'Dehydrin,’ sourced from Hordeum vulgare. The gene expression cassette was assembled in pSB219M, a modified transformation vector for monocots, equipped with both an antibiotic (spectinomycin) and an herbicide selection marker (BASTA). Initial screening of transgenic plants involved BASTA selection (2 and 3 mg/L) and was subsequently confirmed through PCR analysis. The transformation efficiencies of NR-421 and FSD-2008 were 0.4

Promoters play an important role in controlling the expression of genes in transgenic plants. Inducible promoters regulated by stressful conditions are valuable tools for the temporal expression of stress-tolerant genes. Exploring new promoters aims to fine-tune gene expression. Overexpression of a gene in transgenic research using strong and constitutive promoters is always an energy barrier to the cell and can lead to gene silencing, making mild expression preferable, especially when controlling a cascade of genes using transcriptional factors. Although generalized promoters are available, their tissue specificity and controlled level of induction remains questionable. Inducible promoters express genes only when coded proteins are needed under specific conditions. Therefore, exploration of stress inducible promoters and determining their level of expression is deeply desired in transgenic research for a desired level of gene expression. In this study, three monocot stress-inducible promoters controlling the dehydrin gene in Hordeum vulgare, HSP101 in Oryza sativa, and ZmPIS in Zea mays were isolated and compared for transient beta-glucuronidase (GUS) production under different stress conditions. The transient expression of each promoter was analyzed by GUS histochemical staining and fluorometric assays after mimicking drought and salinity stresses using polyethylene glycol (PEG-6000), abscisic acid (ABA), and 200mM NaCl salt treatments, respectively. The results indicated that drought stress induced by PEG-6000 caused highest expression of GUS by all the three promoters followed by ABA and salt stress was the least to induce these promoters. Furthermore, PlantCARE and PLACE analyses of the promoter DNA sequences highlighted various cis-regulatory elements, such as DREB, MYB, ABRE, and WRKY, as key motifs common to these stress-inducible promoters. These studies indicate that dehydrin, HSP101, and ZmPIS promoters respond to drought from the highest to moderate levels in sequential order. It was concluded that all three promoters reported here responded well to drought stress.

Addressing food security is a priority in developing countries. This study aimed to improve wheat yield by overexpressing the TaNF-YB4 transcription factor, which is involved in carbon assimilation and stress tolerance. An expression cassette for TaNF-YB4 was developed in a modified wheat transformation vector (pSB219) and examined through transient expression in Nicotiana tabacum, followed by Agrobacterium-mediated transformation of wheat variety FSD-2008. T0 transgenic plants were propagated to obtain T3 generation PCR-positive plants. Transgene expression was assessed in PCR-verified T2 plants using RT-PCR and qRT-PCR at six weeks postgermination. qRT-PCR analysis using the Delta Delta CT method indicated higher TaNF-YB4 expression in transgenic lines than in the wild-type control plants. Improved agronomic and phenotypic traits were observed with a 6-36 % increase in 1000-grain weight in the selected transgenic lines. Root architecture assessments demonstrated enhanced root length, surface area, and projected area in transgenic lines compared with wild-type plants. Additionally, notable variances in total chlorophyll, protein, and sugar content levels were observed between the transgenic lines and control plants, demonstrating statistical significance with a p-value <= 0.05. This study indicates that low-level constitutive expression of TaNF-YB4 can enhance wheat yield, presenting a viable strategy for improving wheat productivity.

The failure of breeding strategies has caused scientists to shift to other means where the new approach involves exploring the microbiome to modulate plant defense mechanisms against Cotton Leaf Curl Disease (CLCuD). The cotton microbiome of CLCuD-resistant varieties may harbor a multitude of bacterial genera that significantly contribute to disease resistance and provide information on metabolic pathways that differ between the susceptible and resistant varieties. The current study explores the microbiome of CLCuD-susceptible Gossypium hirsutum and CLCuD-resistant Gossypium arboreum using 16 S rRNA gene amplification for the leaf endophyte, leaf epiphyte, rhizosphere, and root endophyte of the two cotton species. This revealed that Pseudomonas inhabited the rhizosphere while Bacillus was predominantly found in the phyllosphere of CLCuV-resistant G. arboreum . Using salicylic acid-producing Serratia spp . and Fictibacillus spp . isolated from CLCuD-resistant G. arboreum, and guided by our analyses, we have successfully suppressed CLCuD in the susceptible G. hirsutum through pot assays. The applied strains exhibited less than 10% CLCuD incidence as compared to control group where it was 40% at 40 days post viral inoculation. Through detailed analytics, we have successfully demonstrated that the applied microbes serve as a biocontrol agent to suppress viral disease in Cotton.

Renewable fuel resources can provide an alternative to meet the energy demands and compensate the diminishing resources of fossil fuels. Lignocellulosic biomass from non -crop plants can provide an alternative for the production of biofuels. However, presence of lignin in lignocellulosic biomass is a limiting factor to achieve industrial objectives. Industrial methods for the removal of lignin from lignocellulosic biomass are cumbersome, inefficient and expensive. The objective of this study was to produce lignocellulosic biomass with low lignin contents by down regulation of endogenous Cinnamoyl CoA Reductase (CCR) in Eucalyptus camaldulensis. Lignin downregulation was accomplished through RNAi. Putative transgenic plants were screened for the presence of transgene through polymerase chain reaction (PCR). CCR expression profiling, lignin estimation and effects of lignin down regulation leading to morphological changes were studied in transgenic and control E. camaldulensis plants. Real time Reverse transcriptase PCR for expression profiling of endogenous CCR gene revealed down regulation of CCR RNA transcripts from 72% to 80% in transgenic as compared to control and wild type plants. CCR enzyme activity was significantly reduced in transgenic plant as compared to control plant. Lignocellulosic biomass from stem of transgenic plants showed reduction in Klason lignin to 25.5% in transgenic plants as compared to wild type E. camaldulensis. Transgenic plants showed-28% height reduction and-53% stem girth reduction in comparison to untransformed control plants. Transformation efficiency of 0.13% was observed after successful transformation, regeneration, selection, molecular screening and expression profiling of the transgenic plants. This study confirmed role of CCR downregulation leading to lignin reduction in E. camaldulensis through RNAi for development of lignocellulosic biomass with low lignin contents.

BACKGROUND:Gossypium arboreum is a cotton crop native to tropical and subtropical regions that are naturally resistant to cotton leaf curl virus (CLCuV). However, its cultivation is unfavorable due to the lower quality and shorter fiber length of cotton when compared to the market leading G. hirsutum. Plasma membrane intrinsic protein 2 (PIP2) is an aquaporin responsible for the transport of water and small molecules across cellular membranes. This fluid transport influences cell elongation and cotton fibre development. Hence, increased PIP2 expression may yield plants with enhanced fiber qualities including length.METHODS AND RESULTS:To test this hypothesis, G. arboreum was transformed with a PIP2 gene construct (35SCpPIP2) using the Agrobacterium-mediated shoot apex cutting method. Relative expression of the CpPIP2 gene in transgenic plants increased up to 35-fold when compared with non-transgenic controls. Transgenic plants displayed a corresponding increase of staple length (up to 150%) when compared with non-transgenic controls. Transgene integration was examined using FISH and karyotyping and revealed the presence of a single transgene located on chromosome 6.CONCLUSION:Since G. arboreum is naturally whitefly and CLCuV resistant, this improvement of fiber length evidenced for CpPIP2 transgenic plants renders their crop production more economically viable.

Abiotic stresses and emerging climate change patterns are forecasted to be the biggest challenge to food security. Salt and drought are the critical abiotic stresses responsible for the wheat yield gap with irrigated and fertile lands. In this study, the Hordeum vulgare NHX1 gene, which encodes for vacuolar Na+/H+ antiporter, was transformed in two wheat varieties, FSD-2008 and Galaxy. The HvNHX1 gene expression cassette was developed under a constitutive viral promoter (2X CaMV35S). The construct was assembled in pSB219, a monocot transformation vector containing the herbicide tolerance gene (bar). The transgenic plants were initially screened by two rounds of BASTA selection (2 mg/L and 3 mg/L). PCR later confirmed the putative transgenics. The transformation efficiency was estimated to be 0.4% for Galaxy and 0.2% for FSD-2008, respectively. Expression analysis of the NHX1 gene in T2 transgenics and non-transgenic controls through qRT-PCR revealed a 12 fold higher expression of the transgene in Galaxy and onefold higher expression in FSD-2008. Under salt stress, the transgenic lines displayed increased chlorophyll content, reduced electrolyte leakage, and higher relative water content in their leaves than in the control plants. Moreover, under stress conditions (200 mM NaCl), the transgenic lines yielded higher biomass and seed weight than non-transgenic controls. The results demonstrated that the constitutive expression of the HvNHX1 gene in wheat resulted in better grain yield than parent lines. Additionally, the bar gene co-transformed with the HvNHX1 confers herbicide (BASTA) resistance in salt-tolerant wheat transgenics.

Two variants of RuBisCO promoter from zea mays were selected to generate hybrid promoter by using bioinformatics tools. Sequence analysis of both RuBisCO promoter variants revealed several critical cis regulatory elements and transcription factors binding sites within the promoter region. Various regulatory motifs related to constitutive expression were located in RuBisCO promoter fragments. Analysis of cis -regulatory regions has paved way to design synthetic promoters. Both variants were separately cloned in TA vector (pTZ57R/T) and then joined to get the complete hybrid promoter (Rub-H). Hybrid RuBisCO promoter was further cloned in expression vector pGR1. Transient GUS assay revealed that hybrid promoter exhibited endosperm specific expression in wheat. From the study it is demonstrated that hybrid promoter (Rub-H) may be used to derive constitutive expression in monocots. The present work provides an important insight in the designing of hybrid monocot promoters to improve various traits in crops without facing IPR issues. It is expected that complete understanding of the regulatory regions and transcription factors in the regulatory regions would help in designing new synthetic/ hybrid promoters for tissue specific or constitutive expression of transgenes.

The eukaryotic gene expression is controlled by a regulatory region called promoter. Many plant promoters have been characterized for regulatory motifs. There are three types of plant promoters i.e. inducible, constitutive and tissue specific on basis of regulatory motifs. Plant sources have been searched for isolation of strong promoters that are being utilized in molecular biology research. The researchers need to address IPR issues for utilizing the strong patented promoters for the expression of their transgenes. The identification and characterization of strong dicot promoter is necessary for the expression of transgenes by native researchers to evaluate their artificial gene. The promoters isolated from viral sources have some limitations. The dicot promoter sequence of ?-tubulin (?-TbSt) was explored and isolated from potato. The ?-TbSt promoter sequence consists of light responsive, hormonal responsive and stress responsive elements. Motifs having responsive elements were identified in ?-TbSt promoter. The ?-TbSt promoter is highly constitutive promoter.

Precision in genome editing has a long history of adaptation from zinc finger nucleases to transcription activator-like effector nucleases, LAGLIDADG homing endonucleases, oligonucleotide-directed mutagenesis, and finally CRISPR/Cas technology. The bacterial-derived adaptive immune system has been exploited to artificially mimic the CRISPR/Cas9-based genome editing into other eukaryotic organisms. This robust and simple technology requires only two key components (sgRNA and Cas9) for precise editing, but this versatile tool has been struggling with an issue of off-site targeting. Initially, seed region of SgRNA has been a focal point to improve specificity, and several rules to develop efficient gRNAs were identified. Those rules were valid for simpler organisms but major specificity problems were concerned with complex organisms, that is, plants with higher ploidy levels. Different algorithms and machine learning models are based on different experimentally validated datasets that predict best if CRISPR-experimental conditions of querier data also matched with initial datasets used in building those models. The good bioinformatic tools are designed on a large amount of CRISPR-edited NGS data and support multiple genomes to predict an efficient sgRNA design by maximizing on-target and minimizing off-target scores. This paper describes both sgRNA designing rules and the available bioinformatics tools for efficient sgRNA design.

RNA interference (RNAi) is a conserved phenomenon in eukaryotes which silence the expression of invading nucleic acids in a sequence-specific manner. The small interfering RNAs (siRNAs) laid down the foundation for RNAi technology, which has greatly been used in the downregulation of transgenes, transposons, and viruses infecting plants. Advancements in next-generation sequencing technologies generated a huge amount of DNA sequence data for thousands of living organisms that necessitated the development of bioinformatics for appropriate analysis of the available data. Dozens of publicly available web resources are available nowadays that provide access to the genomics, transcriptomics, siRNA, and microRNA databases. Downregulation of gene expression through RNAi is used by the living organisms to inhibit the gene expression both at transcriptional and translational levels. The reduction or inhibition of gene expression within the eukaryotic cells utilizes RNA-induced silencing complex which is guided by small RNA molecules [21–24 nucleotide (nt)]. This RNAi machinery is present in all eukaryotes and acts as the host defense mechanism against invading nucleic acids including viruses, transgenes, and transposons. The technology can be used in vitro through transgenic approaches. The RNAi-based gene silencing has the only drawback that sometimes it leads to off-target gene silencing that can be minimized by careful selection of DNA sequences. Bioinformatics tools and online databases are greatly helpful to overcome these shortcomings. This chapter describes the basic mechanisms of RNAi as the introductory information and mainly focuses on the utilization of different bioinformatics tools and online databases for effective and target-specific utilization of RNAi technology.

Bt cotton expressing Cry1Ac is being cultivated in Pakistan. It has been observed that pink bollworm may have developed resistance against single Bt gene (Cry1Ac). For durable resistance, insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct was developed. There was a need to characterize NIBGE-1601 event for intellectual property rights protection. The Presence of NIBGE Cry1Ac and NIBGE Cry2Ab genes was checked in NIBGE-1601 cotton plants through PCR, while there was no amplification using primers specific for Monsanto events (MON531, MON15985, MON1445). Using genome walking technology, NIBGE-601 event has been characterized. Event-specific primers of NIBGE-1601 were designed and evaluated to differentiate it from other cotton events mentioned above. NIBGE-1601 event detection primers are highly specific, therefore, can detect NIBGE 1601 event at different conditions using single or multiplex PCR. In the qualitative PCR, using NIBGE-1601 event specific primers, 0.05 ng was the limit of detection for NIBGE-1601double gene cotton genomic DNA. Thus event characterization and development of event-specific diagnostics will help in breeding new cotton varieties resistant to cotton bollworms.

A single transcription factor is known to coordinate expression of a set of metabolites in a biochemical pathway; its use therefore can be a functional strategy in generating plants with desired traits.Triticum aestivum Dof1(TaDof1) transcription factor is mainly associated with improved nitrogen assimilation in plants. In the current research, the transgenic wheat overexpressingTaDof1transcription factor, under a constitutive promoter, was developed byAgrobacterium-mediated transformation. The two elite wheat cultivars (Galaxy and Faisalabad-2008) were selected for transformation study. The T(0)plants were subjected to screening using selection medium containing herbicide BASTA. PCR results confirmed that only 8 out of 31 plants possessed the completeTaDof1cassette. A transformation efficiency of 0.46% for Galaxy and 0.08% for Faisalaad-2008 was obtained. The quantitative RT-PCR was performed on T(1)plants grown under nitrogen-limiting conditions. A substantial rise in the expression of citrate synthase (CS), isocitrate dehydrogenase (ICDH), phosphoenolpyruvate carboxylase (PEPC), and pyruvate kinase (PK) genes regulated byTaDof1was observed after 4 weeks of nitrogen stress in T(1)plants. The maximum fold increase of 464 was recorded for ICDH. Our findings indicate a cooperative modification of nitrogen and carbon metabolisms since they are intimately linked together. Overexpression ofTaDof1in wheat resulted in a significant increase in various agronomic traits. Furthermore, various physiological and biochemical markers (chlorophyll, protein, and soluble sugar contents) exhibited a profound change inTaDof1transgenic plants in comparison with wild type plants. The results clearly depict the merits of employing transcription factors in engineering plant metabolisms.

Plasma membrane intrinsic proteins (PIP1) are the most common integral membrane proteins belong to a larger family of intrinsic aquaporin proteins. They are member of aquaporin gene family and have gained importance as highly expressed genes in plants. In this study, the promoter of aquaporin PIP1 gene was identified, analyzed and retrieved from high throughput genomic sequence (HTGS) database. The cis-acting regulatory elements, transcription start sites and transcription factor binding sites of selected promoter were identified through different bio-informatics tools. Many light responsive, phytohormone, stress and defense related cis-regulatory elements were detected in PIP1 promoter region indicating its role as a constitutive promoter. The PIP1 promoter was isolated from Solanum tuberosum. It was initially cloned in TA vector (pTZ57R/T) and later transferred to plant expression binary vectors, pGR1 and pGA482 for transient and stable expression studies in tobacco. The GUS expression results of PIP1 promoter in different tobacco tissues showed its functional importance in regulating gene expression in a constitutive manner. Further, it was concluded that the PIP1 aquaporin promoter is constitutively expressed with a strength equivalent to CaMV 2x35S promoter. These findings indicated the significance of isolated promoter for genetic engineering of plants for crop improvement.

Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, however, there is a major concern that bollworms may develop resistance. Here we have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA482-12R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Both genes are expressed in transgenic cotton plants and is likely to help breeders in developing new cotton cultivars by incorporating these genes in cotton lines having no Bt genes or expressing Cry1Ac gene (Mon 531). Positive transgenic cotton was identified by PCR using specific primers for the amplification of both Cry1Ac and Cry2Ab genes. Cry1Ac and Cry2Ab expression was confirmed with an immunostrip test and quantified using ELISA that showed significant spatio-temporal expression of Cry2Ab ranging from 3.28 to 7.72 µg/g of the tissue leaf. Insect bioassay with army worm ( Spodoptera litura ) was performed to check the efficacy of NIBGE (National Institute for Biotechnology and Genetic Engineering) double gene transgenic cotton plants and up to 93% insect mortality was observed.

Decreased iron and zinc bioaccessibility, caused by the anti-nutrient phytic acid, is one of the leading reasons for micronutrient deficiency-related disorders. Biofortification of wheat with phytase gene to enhance iron and zinc bioaccessibility appears to be a fitting solution for this problem, especially in developing countries where most of the population belongs to the lower economic sector. However, societal views on crops, particularly crops that are genetically modified (GM) to express a new trait, needs to be changed. Risk assessment of GM crops can play a crucial role in fostering positive public perception, since it is imperative to ensure safety before allowing human consumption. The present study performed compositional and morphological risk assessment of T3 and T4 generations of phytase transgenic wheat by comparing their biochemical and morphological traits. Transgenic plants were analysed for their carbohydrate, protein, starch and phytic acid content along with iron bioaccessibility, zinc bioaccessibility and phytase enzyme activity. Morphological traits studied included plant height, seed number, seed weight and spike number. No significant differences were observed for carbohydrate, protein, starch content and for morphological traits; however, a significant increase was observed in phytase activity as well as iron and zinc bioaccessibility, which correlated with a significant decrease in phytic acid. These results demonstrate that phytase transgenic wheat is as native as local wheat varieties and can potentially increase iron and zinc bioaccessibility.

Cotton the most important fiber crop is facing a major threat due to a viral disease caused by cotton leaf curl virus (CLCuV). The cotton specie, Gossypium arboreum is resistant to this disease. Cotton scientists are working to find the key genes in G. arboreum that confer resistance against cotton leaf curl disease (CLCuD). Current research work is an effort to find some potential biotic stress related resistance genes from G. arboreum and the their evaluation against CLCuV infection utilizing functional genomics approaches. Leaf cDNA library was constructed from field grown G. arboreum which was further utilized to identify and isolate clones involved in resistance against CLCuD. The clone sequences were exploited to establish expressed sequence tags (EST). The EST represented some important biotic stress resistance genes like lipoxygenase, cytochrome P450, CPMMV like coat protein, serine threonine kinase, a RGA, lipid transfer protein and ubiquitin conjugating enzyme E2. As cotton is a fiber crop so some trichome development genes like aquaporin, arabinogalactans and cellulose synthase were also found. Lipoxygenases are known to be involved in apoptosis and biotic and abiotic stress responses in plants. Here the members of LOX are identified in biotic stress resistant G. arboreum. G. arboreum genome encode 13 LOX proteins. The G. arboreum LOXs are validated based on protein alignment studies. This is the first report wherein number of LOXs are identified in cotton which may help to better understand the apoptosis and responses to biotic and abiotic stresses in naturally resistant G. arboreum.

Bacterial plasmids carry genes that code for additional traits such as osmoregulation, CO2 fixation, antibiotic and heavy metal resistance, root nodulation and nitrogen fixation. The main objective of the current study was to identify plasmid-conferring osmoregulatory genes in bacteria isolated from rhizospheric and non-rhizospheric soils of halophytes (Salsola stocksii and Atriplex amnicola). More than 55% of halophilic bacteria from the rhizosphere and 70% from non-rhizospheric soils were able to grow at 3 M salt concentrations. All the strains showed optimum growth at 1.5-3.0 M NaCl. Bacterial strains from the Salsola rhizosphere showed maximum (31%) plasmid elimination during curing experiments as compared to bacterial strains from the Atriplex rhizosphere and non-rhizospheric soils. Two plasmid cured strains Bacillus HL2HP6 and Oceanobacillus HL2RP7 lost their ability to grow in halophilic medium, but they grew well on LB medium. The plasmid cured strains also showed a change in sensitivity to specific antibiotics. These plasmids were isolated and transformed into E. coli strains and growth response of wild-type and transformed E. coli strains was compared at 1.5-4 M NaCl concentrations. Chromosomal DNA and plasmids from Bacillus filamentosus HL2HP6 were sequenced by using high throughput sequencing approach. Results of functional analysis of plasmid sequences showed different proteins and enzymes involved in osmoregulation of bacteria, such as trehalose, ectoine synthetase, porins, proline, alanine, inorganic ion transporters, dehydrogenases and peptidases. Our results suggested that plasmid conferring osmoregulatory genes play a vital role to maintain internal osmotic balance of bacterial cells and these genes can be used to develop salt tolerant transgenic crops.