Genome-wide identification of CCO gene family in cucumber (Cucumis sativus) and its comparative analysis with A. thaliana.

Carotenoid cleavage oxygenase (CCO) is an enzyme capable of converting carotenoids into volatile, aromatic compounds and it plays an important role in the production of two significant plant hormones, i.e., abscisic acid (ABA) and strigolactone (SL). The cucumber plant genome has not been mined for genomewide identification of the CCO gene family. In the present study, we conducted a comprehensive genome-wide analysis to identify and thoroughly examine the CCO gene family within the genomic sequence of Cucumis sativus L. A Total of 10 CCO genes were identified and mostly localized in the cytoplasm and chloroplast. The CCO gene is divided into seven subfamilies i.e. 3 NCED, 3 CCD, and 1 CCD-like (CCDL) subfamily according to phylogenetic analysis. Cis-regulatory elements (CREs) analysis revealed the elements associated with growth and development as well as reactions to phytohormonal, biotic, and abiotic stress conditions. CCOs were involved in a variety of physiological and metabolic processes, according to Gene Ontology annotation. Additionally, 10 CCO genes were regulated by 84 miRNA. The CsCCO genes had substantial purifying selection acting upon them, according to the synteny block. In addition, RNAseq analysis indicated that CsCCO genes were expressed in response to phloem transportation and treatment of chitosan oligosaccharides. CsCCD7 and CsNCED2 showed the highest gene expression in response to the exogenous application of chitosan oligosaccharides to improve cold stress in cucumbers. We also found that these genes CsCCD4a and CsCCDL-a showed the highest expression in different plant organs with respect to phloem content. The cucumber CCO gene family was the subject of the first genome-wide report in this study, which may help us better understand cucumber CCO proteins and lay the groundwork for the gene family's future cloning and functional investigations.

Enhancing the quality of staple food crops through CRISPR/Cas-mediated site-directed mutagenesis.

MAIN CONCLUSION: The enhancement of CRISPR-Cas gene editing with robust nuclease activity promotes genetic modification of desirable agronomic traits, such as resistance to pathogens, drought tolerance, nutritional value, and yield-related traits in crops. The genetic diversity of food crops has reduced tremendously over the past twelve millennia due to plant domestication. This reduction presents significant challenges for the future especially considering the risks posed by global climate change to food production. While crops with improved phenotypes have been generated through crossbreeding, mutation breeding, and transgenic breeding over the years, improving phenotypic traits through precise genetic diversification has been challenging. The challenges are broadly associated with the randomness of genetic recombination and conventional mutagenesis. This review highlights how emerging gene-editing technologies reduce the burden and time necessary for developing desired traits in plants. Our focus is to provide readers with an overview of the advances in CRISPR-Cas-based genome editing for crop improvement. The use of CRISPR-Cas systems in generating genetic diversity to enhance the quality and nutritional value of staple food crops is discussed. We also outlined recent applications of CRISPR-Cas in developing pest-resistant crops and removing unwanted traits, such as allergenicity from crops. Genome editing tools continue to evolve and present unprecedented opportunities to enhance crop germplasm via precise mutations at the desired loci of the plant genome.

Heterologous expression of cry1Ia12 insecticidal gene in cotton encodes resistance against pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae); an alternate insecticidal gene for insect pest management.

BACKGROUND: Cotton is continuously exposed to sucking and chewing insect pest pressure since emergence to harvesting. Pink bollworm (Pectinophora gossypiella) has become major chewing insect pest to reduce the cotton yield and results in bad lint quality even in transgenic crops. The efficiency of insecticidal genes has been compromised due to extensive utilization of transgenic crops. METHODS AND RESULTS: The present study was conducted to evaluate the efficacy of an alternate cry1Ia12 insecticidal gene against pink bollworm (PBW) in cotton. Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA2300 expression vector containing cry1Ia12 gene under the control of 35S CaMV was used to transform a local cotton cultivar GS-01. The various molecular analyses revealed the transgene integration and expression in primary transformants. Among five selected transgenic plants, tcL-08 showed maximum (16.06-fold) mRNA expression of cry1Ia12 gene whereas tcL-03 showed minimum (2.33-fold) expression. Feeding bioassays of 2nd and 3rd instar pink bollworm (PBW) larvae on immature cotton bolls, flowers and cotton squares revealed up to 33.33% mortality on tcL-08 while lowest mortality (13.33%) was observed in tcL-03 and tcL-15. Furthermore, the average weight and size of survived larvae fed on transgenic plants was significantly lesser than the average weight of larvae survived on non-transgenic plants. CONCLUSIONS: The present study suggests the cry1Ia12 gene as an alternate insecticidal gene for the resistance management of cotton bollworms, especially PBW.

Characterization and survival of broad-spectrum biocontrol agents against phytopathogenic fungi.

The current study intended to isolate, characterize and identify biocontrol bacteria possessing broad-spectrum antifungal activity from the phyllosphere of different crops including maize, wheat and potato and to assess their growth-promoting activity. In this study 14/113 biocontrol bacteria showed antifungal activity. Bacterial isolates M11 and M33 from maize out of 113 were re-selected on the basis of their strong (more than 50%) broad spectrum antifungal activity after their assessment against four economically important phytopathogenic fungi including Alternaria alternata, Rhizoctonia solani, Fusarium oxysporum and Fusarium verticillioides. The isolates were further assessed for plant growth promoting traits, i.e., indole-3-acetic acid production, phosphate solubilization, production of cellulase, microbial volatile compounds, hydrogen cyanide and siderophores. All fourteen isolates showed positive results for the production of indole-3-acetic acid hormone and cellulase enzyme, 10 isolates were positive for hydrogen cyanide production; siderophores production was observed in 7 isolates while 5 isolates showed ability to solubilize inorganic phosphate. Microbial volatile compounds were only synthesized by M11 and M33, which were identified as Bacillus amyloliquefaciens and Bacillus subtilis respectively by 16S rRNA gene sequencing. The survival study revealed that biocontrol bacteria B. amyloliquefaciens and B. subtilis have the ability to survive in cost effective molasses containing carrier material up to a three-month period.

Genetic architecture of Al3+ toxicity tolerance in rice F2:3 populations determined through QTL mapping.

Aluminum (Al3+) toxicity is one of the factors limiting crop production in acidic soils. Identifying quantitative trait loci (QTLs)/genes for tolerance to Al3+ toxicity at seed germination can aid the development of new tolerant cultivars. The segregating population derived from Pak Basmati (Indica) × Pokkali (Indica) was used for mapping QTLs linked with tolerance to Al3+ toxicity ranging from 0 to 20 mM at pH 4 ± 0.2 at germination. The favorable alleles for all new QTLs were analyzed based on germination traits, i.e., final germination percentage (FG%), germination energy (GE), germination speed (GS), germination index (GI), mean germination time (MGT), germination value (GV), germination velocity (GVe), peak value of germination (GPV), and germination capacity (GC), and growth traits, such as root length (RL), shoot length (SL), total dry biomass (TDB) and germination vigor index (GVI). The phenotypic evolution showed transgressive variations. For genome-wide mapping, 90 polymorphic SSRs with 4 gene-specific markers and Win QTL Cart were used for QTL analysis. In all, 35 QTLs for germination and 11 QTLs for seedling growth were detected in distinct chromosomal regions by composite interval mapping (CIM), and multiple interval mapping (MIM) confirmed the pleiotropy at region RM128 on chromosome 1. Based on our genetic mapping studies, the genes/QTLs underlying tolerance to Al3+ toxicity could differ for both the germination and seedling stages in segregated populations. The QTLs identified in this study could be a source of new alleles for improving tolerance to Al3+ toxicity in rice.

Resistance to Chilo infuscatellus (Lepidoptera: Pyraloidea) in transgenic lines of sugarcane expressing Bacillus thuringiensis derived Vip3A protein.

Sustainable agriculture requires management of insect pests through resistance development. The biological potential of Cry toxins and Vip protein, derived from Bacillus species, is widely recognized in this context. The identification, evaluation of new insecticidal protein genes with different mode of action and entomotoxicity against sugarcane stem borer (Chilo infuscatellus) is important to overcome evolved insect resistance. In this study, we reported the generation of transgenic sugarcane lines expressing Vip3A toxin driven by polyubiquitin promoter for resistance against sugarcane stem borer. The V0 transgenic sugarcane plants were initially characterized by GUS histochemical staining, PCR and Southern blot assays that confirmed genetic transformation of twelve independent sugarcane lines. Variable transgene expression was found among transgenic sugarcane lines when revealed through Realtime quantitative PCR (RT-qPCR) with highest in S10 line while minimum was observed in V5 line. A similar expression pattern was observed in transgenic sugarcane lines for Vip3A protein concentration which ranged from 5.35 to 8.89 µg/mL. A direct correlation was observed between the Vip3A protein and Vip3A transgene expression in the transgenic sugarcane lines. In in-vitro insect bioassay on V1, Vip3A transgenic sugarcane lines exhibited high resistance to C. infuscatellus with upto 100% mortality compared to the control sugarcane line. Our findings suggest that a single copy insertion of Vip3A gene in transgenic sugarcane lines render them resistant to borer and these lines can be potentially used for generation of insect resistant transgenic sugarcane and could also be employed in gene pyramiding with Bt toxin to prolong resistance.

Advances in exogenous RNA delivery techniques for RNAi-mediated pest control.

Climate change imposes a great threat to world food security and encourages insect pest proliferation and spreading. Because of these challenges, identifying novel biotechnology pest management and its applications is inevitable. RNA interference (RNAi) is a gene regulatory process used for the maintenance and regulation of host defences against invading viruses. Nevertheless, it is widely used for the analysis of gene function. In recent years, the potential use of RNA interference (RNAi) as a tool for manipulating crop traits, as well as an alternative for crop protection, has undergone outstanding developments. In this review, we describe some genes involved in insect dsRNA uptake and discuss the reasons for varying RNAi response in insect pests, emphasizing the presence of nucleases and double-stranded RNA binding protein. We explore recent breakthroughs in innovative dsRNA delivery for efficient and effective knockdown in insect pests. Conclusively, topical delivery of dsRNA combined with a nanoparticle complex holds great potential for RNAi-mediated pest control.

A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane.

RNA interference (RNAi) is commonly used to produce virus tolerant transgenic plants. The objective of the current study was to generate transgenic sugarcane plants expressing a short hairpin RNAs (shRNA) targeting the coat protein (CP) gene of sugarcane mosaic virus (SCMV). Based on multiple sequence alignment, including genomic sequences of four SCMV strains, a conserved region of ~ 456 bp coat protein (CP) gene was selected as target gene and amplified through polymerase chain reaction (PCR). Subsequently, siRNAs2 and siRNA4 were engineered as stable short hairpin (shRNA) transgenes of 110 bp with stem and loop sequences derived from microRNA (sof-MIR168a; an active regulatory miRNA in sugarcane). These transgenes were cloned in independent RNAi constructs under the control of the polyubiquitin promoter. The RNAi constructs were delivered into two sugarcane cultivars 'SPF-234 and NSG-311 in independent experiments using particle bombardment. Molecular identification through PCR and Southern blot revealed anti-SCMV positive transgenic lines. Upon mechanical inoculation of transgenic and non-transgenic sugarcane lines with SCMV, the degree of resistance was found variable among the two sugarcane cultivars. For sugarcane cultivar NSG-311, the mRNA expression of the CP-SCMV was reduced to 10% in shRNA2-transgenic lines and 80% in shRNA4-transgenic lines. In sugarcane cultivar SPF-234, the mRNA expression of the CP-SCMV was reduced to 20% in shRNA2-transgenic lines and 90% in shRNA4 transgenic lines, revealing that transgenic plants expressing shRNA4 were almost immune to SCMV infection.

Mini Review - Epigenetics: Quest for no-escape to HIV, a persistent pathogen.

Acquired Immune Deficiency Syndrome (AIDS) is a disease infection mix, which is primarily because of 'deficient' immune system. Human Immune-deficiency Virus (HIV) makes the immune system susceptible to many infections by infiltrating it. Many researchers believe that HIV is a mutated form of Simian Immune-deficiency Virus (SIV). After being clinically discovered in 1981 in America, it is said to have caused 36 million deaths. Treatment of AIDS has been a 'burning ' issue ever since its discovery. There is no cure for AIDS! Although, Recombinant Transcriptase Inhibitors (RTis) are being considered a major treatment against HIV that can not only lessen the effect of HIV but also can prolong the life of HIV positive patients. More recent advancement includes 'transplantation of transgenic stem cells' in HIV positive patients. As latency of HIV provirus in host genome is the preeminent weapon of this virus against RTis that compel it to hide from host immune system and a persistent pathogen thereof. Thus, epigenetic activation of latent provirus pool by methyl inhibitors along with non¬toxic chemical drugs seems to be a more promising treatment to avoid the burden of lifelong RTI.

Genome-wide in-silico analysis of ethylene biosynthesis gene family in Musa acuminata L. and their response under nutrient stress.

Ethylene is a gaseous phytohormone involved in plants' growth and developmental processes, including seed germination, root initiation, fruit ripening, flower and leaf senescence, abscission, and stress responses. Ethylene biosynthesis (EB) gene analysis in response to nitrogen (N) and potassium (K) stress has not yet been conducted in Musa acuminata (banana) roots. The genome mining of banana (Musa acuminata L.) revealed 14 putative 1-aminocyclopropane-1-carboxylate synthase (ACS), 10 1-aminocyclopropane-1-carboxylate oxidase (ACO), and 3 Ethylene overproducer 1 (ETO1) genes. ACS, ACO, and ETO1 proteins possessed amino acid residues ranging from 422-684, 636-2670, and 893-969, respectively, with molecular weight (Mw) ranging from 4.93-7.55 kD, 10.1-8.3 kD and 10.1-10.78 kD. The number of introns present in ACS, ACO, and ETO1 gene sequences ranges from 0-14, 1-6, and 0-6, respectively. The cis-regulatory element analysis revealed the presence of light-responsive, abscisic acid, seed regulation, auxin-responsive, gibberellin element, endosperm-specific, anoxic inducibility, low-temperature responsiveness, salicylic acid responsiveness, meristem-specific and stress-responsive elements. Comprehensive phylogenetic analyses ACS, ACO, and ETO1 genes of Banana with Arabidopsis thaliana revealed several orthologs and paralogs assisting in understanding the putative functions of these genes. The expression profile of Musa acuminata genes in root under normal and low levels of nitrogen and potassium shows that MaACS14 and MaACO6 expressed highly at normal nitrogen supply. MaACS1 expression was significantly upregulated at low potassium levels, whereas, MaACO6 gene expression was significantly downregulated. The functional divergence and site-specific selective pressures on specific gene sequences of banana have been investigated. The bioinformatics-based genome-wide assessment of the family of banana attempted in the present study could be a significant step for deciphering novel ACS, ACO, and ETO1 genes based on genome-wide expression profiling.

Host-induced silencing of the CpCHI gene resulted in developmental abnormalities and mortality in maize stem borer (Chilo partellus).

RNAi-based insecticides for crop protection have witnessed rapid improvement over the years. However, their potential to efficiently control maize stem borer (Chilo partellus) pests has remained underexplored. In this study, double-stranded C. partellus chitinase (dsCHI) toxicity was investigated in C. partellus larvae. Furthermore, we developed transgenic maize lines expressing dsRNA targeted against C. partellus chitinase transcripts and performed detached leaf insect feeding bioassays. Our results revealed that C. partellus chitinase transcript expression was significantly downregulated by 57% and 82% in the larvae. Larvae exhibited various phenotypic distortion levels across developmental stages, and 53% mortality occurred in transgenic fed larvae compared to those fed on nontransgenic leaves. In conclusion, we have identified the C. partellus chitinase gene as a potential target for RNAi-mediated control and demonstrated that oral delivery via bacteria and plant-mediated delivery are viable means of achieving C. partellus RNAi-mediated control.

Genome scale analysis of 1-aminocyclopropane-1-carboxylate oxidase gene family in G. barbadense and its functions in cotton fiber development.

A class of proteins, 1-aminocyclopropane-1-carboxylate oxidase (ACO), is required in the final step of production of ethylene from its immediate precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Despite the crucial and regulatory role of ACO gene family in the fiber development, it has not been thoroughly analyzed and annotated in G. barbadense genome. In the present study, we have identified and characterized all isoforms of ACO gene family from genomes of Gossypium arboreum, G. barbadense, G. hirsutum and G. raimondii. Phylogenetic analysis classified all ACO proteins into six distinct groups on the basis of maximum likelihood. Gene locus analysis and circos plots indicated the distribution and relationship of these genes in cotton genomes. Transcriptional profiling of ACO isoforms in G. arboreum, G. barbadense and G. hirsutum fiber development exhibited the highest expression in G. barbadense during early fiber elongation. Moreover, the accumulation of ACC was found highest in developing fibers of G. barbadense in comparison with other cotton species. ACO expression and ACC accumulation correlated with the fiber length in cotton species. Addition of ACC to the ovule cultures of G. barbadense significantly increased fiber elongation while ethylene inhibitors hindered fiber elongation. These findings will be helpful in dissecting the role of ACOs in cotton fiber development and pave a way towards genetic manipulations for fiber quality improvement.

Management of mung bean leaf spot disease caused by Phoma herbarum through Penicillium janczewskii metabolites mediated by MAPK signaling cascade.

Vigna radiata L., an imperative legume crop of Pakistan, faces hordes of damage due to fungi; infecting host tissues by the appressorium. The use of natural compounds is an innovative concern to manage mung-bean fungal diseases. The bioactive secondary metabolites of Penicillium species are well documented for their strong fungi-static ability against many pathogens. Presently, one-month-old aqueous culture filtrates of Penicillium janczewskii, P. digitatum, P. verrucosum, P. crustosum, and P. oxalicum were evaluated to check the antagonistic effect of different dilutions (0, 10, 20, … and 60%). There was a significant reduction of around 7-38%, 46-57%, 46-58%, 27-68%, and 21-51% in Phoma herbarum dry biomass production due to P. janczewskii, P. digitatum, P. verrucosum, P. crustosum, and P. oxalicum, respectively. Inhibition constants determined by a regression equation demonstrated the most significant inhibition by P. janczewskii. Finally, using real-time reverse transcription PCR (qPCR) the effect of P. Janczewskii metabolites was determined on the transcript level of StSTE12 gene involved in the development and penetration of appressorium. The expression pattern of the StSTE12 gene was determined by percent Knockdown (%KD) expression that was found to be decreased i.e. 51.47, 43.22, 40.67, 38.01, 35.97, and 33.41% for P. herbarum with an increase in metabolites concentrations viz., 10, 20, 30, 40, 50 and 60% metabolites, respectively. In silico studies were conducted to analyze the role of Ste12 a transcriptional factor in the MAPK signaling pathway. The present study concludes a strong fungicidal potential of Penicillium species against P. herbarum. Further studies to isolate the effective fungicidal constituents of Penicillium species through GCMS analysis and determination of their role in signaling pathways are requisite.

Expression of Chitinase and shRNA Gene Exhibits Resistance to Fungi and Virus.

With the increasing global population, saving crops from diseases caused by different kinds of bacteria, fungi, viruses, and nematodes is essential. Potato is affected by various diseases, destroying many crops in the field and storage. In this study, we developed potato lines resistant to fungi and viruses, Potato Virus X (PVX) and Potato Virus Y (PVY), by inoculating chitinase for fungi and shRNA designed against the mRNA of the coat protein of PVX and PVY, respectively. The construct was developed using the pCAMBIA2301 vector and transformed into AGB-R (red skin) potato cultivar using Agrobacterium tumefaciens. The crude protein extract of the transgenic potato plant inhibited the growth of Fusarium oxysporum from ~13 to 63%. The detached leaf assay of the transgenic line (SP-21) showed decreased necrotic spots compared to the non-transgenic control when challenged with Fusarium oxysporum. The transgenic line, SP-21, showed maximum knockdown when challenged with PVX and PVY, i.e., 89 and 86%, while transgenic line SP-148 showed 68 and 70% knockdown in the PVX- and PVY-challenged conditions, respectively. It is concluded from this study that the developed transgenic potato cultivar AGB-R showed resistance against fungi and viruses (PVX and PVY).

Biocontrol rhizobacteria enhances growth and yield of wheat (Triticum aestivum) under field conditions against Fusarium oxysporum.

The current study aimed to identify the survival of bio-control bacteria with antifungal activity against Fusarium oxysporum and assess their growth promoting activity in wheat crop field conditions. To evaluate the fungicidal activities of isolated bacteria using the dual culture method, both qualitative and quantitative bioassays were performed. Plant Growth Promoting activities such as Indole 3-Acetic Acid (IAA), phosphate solubilization, Hydrogen cyanide (HCN), and Siderophore production were assessed for three biocontrol bacterial isolates (BCB 07, BCB16, and BCB 83) out of 180 with 70% antagonistic activity against Fusarium oxysporum. Chitinase, protease, and cellulase interaction in isolates was also tested. BCB16 was selected as it had 70% antagonist activity against F. oxysporum but also had the highest PGPR (Plant Growth Promoting Rhizobacteria) traits when compared to the other two isolates. BCB16 was also tested for survival in talc powder and in wheat crop field conditions. Even after 4 months in talc powder, the survival rate remained stable. In a wheat crop field, BCB16 reduced the disease incidence of Fusarium oxysporum by 54.38%. When compared to fungus alone treatment, BCB16 increased average yield by 57% alone and 32% in challenged conditions. BCB16 was identified molecularly using the 16s rRNA gene. Bacillus amyloliquefaciens shared 97% of the deduced sequence. The sequence was submitted to genbank and assigned the accession number OM333889. Bacillus amyloliquefaciens has the potential to be used in the field as an alternative to synthetic fungicides against Fusarium oxysporum.

Isolation and characterization of biocontrol bacteria.Molecular Identification of efficient biocontrol bacteria.Survival of biocontrol bacteria in talc powder as carrier material.The Bacillus amyloliquefaciens has plant growth-promoting characteristics.B. amyloliquefaciens reduced the disease incidence of F. oxysporum by 57% in field trials.

Consequences of Drought Stress Encountered During Seedling Stage on Physiology and Yield of Cultivated Cotton.

Survival of living organisms depends on the availability of water resources required for agriculture. In the current scenario of limited water resources, it is our priority to maximise the yield potential of crops with a minimum supply of available water. In this study, we evaluated seven cultivated varieties of Gossypium hirsutum (FH-114, FH-152, FH-326, FH-492, FH-942, VH-327 and FH-NOOR) for their tolerance, yield potential and fibre quality under water shortages. We also studied the effect of drought stress on osmoregulation, chlorophyll content, antioxidant (peroxidase and catalase) activity, lipid peroxidation and secondary metabolite accumulation in the varieties under study. It was revealed that three varieties (FH-114, FH-152 and VH-327) exhibited a lower stress susceptibility index and more tolerance to drought stress. All the varieties demonstrated enhanced proline and malondialdehyde content, but no significant change in chlorophyll content was observed under limited water supply. Antioxidant activity offered by catalase and phenolic content was enhanced in FH-492 whilst peroxidase activity increased in FH-114 and FH-326. Phenolic content was highest in FH-942 and decreased significantly in the remaining varieties. Ginning outturn of the cotton varieties increased in VH-327 (19.8%) and FH-326 (3.7%), was not affected in FH-114 and FH-492 and was reduced in FH-152, FH-942 and FH-NOOR. All cotton varieties tested showed an increase in micronaire thickness when exposed to drought stress as early as the seedling stage. This study highlights the evaluation and screening of cotton varieties for their response to drought stress in terms of yield and fibre quality when exposed to water shortages during plant development and can help in devising irrigation plans.

Silencing a Myzus persicae Macrophage Inhibitory Factor by Plant-Mediated RNAi Induces Enhanced Aphid Mortality Coupled with Boosted RNAi Efficacy in Transgenic Potato Lines.

Myzus persicae causes considerable losses to crops as a major pest. The damage is direct by feeding and also partly indirect because it vectors plant viruses. The currently available control strategies rely on unsafe and nonecofriendly chemical pesticide applications. Plant-mediated RNA interference (RNAi) has emerged as a powerful tool in crop protection from insect pests. Aphid salivary proteins are essential for phloem feeding and act as mediators of the complex interactions between aphids and their host plants. We documented the efficacy of dsRNA directed against macrophage inhibitory factor (MIF1) of M. persicae to induce aphid mortality and gene silencing through the generation of transgenic potato lines. A binary construct harbouring dsMIF1 driven by the CaMV35S promoter was introduced into the local potato variety 'AGB-white' by Agrobacterium-mediated transformation. PCR and Southern blotting validated the transgene presence and genomic integration in seven transgenic potato lines. An in vitro detached leaf assay revealed a significantly high aphid mortality of 65% in the transgenic potato line sDW-2, while the aphid mortality was 77% in the sDW-2 transgenic line during the in planta bioassay in comparison with 19% aphid mortality in the control nontransgenic potato line. A significantly high silencing effect was observed in the mRNA expression of MIF1, which was reduced to 21% in aphids fed on the transgenic potato line sDW-2. However, variable knockdown effects were found among six other transgenic potato lines, ranging from 30 to 62%. The study concluded that plant-mediated silencing of aphid RNA induces significant RNAi in M. persicae, along with enhanced aphid mortality.

Expression of ice recrystallization inhibition protein in transgenic potato lines associated with reduced electrolyte leakage and efficient recovery post freezing injury.

Potato (Solanum tuberosum L.) is considered to be frost-susceptible as short spells of frost can reduce the tuber yield and quality. Ice recrystallization inhibition (IRI) protein helps prevent growth of ice crystals in the cell apoplast during frost and help prevent damage associated with freezing stress. In this study, we investigated the in planta potential of Lolium perenne derived IRI3 transgene in improving the tolerance of transgenic potato lines for freezing stress. The codon optimized IRI3 transgene was introduced into potato cultivar Diamant through Agrobacterium mediated transformation. Three transgenic potato lines were successfully generated which were confirmed for transgene insertion and genomic integration by polymerase chain reaction and Southern blot. It was evident that the IRI3 transcript decreased in initial 24 h of cold stress treatment while the IRI3 mRNA expression up regulated in subsequent hours of cold treatment with maximum increase to 20 folds at 96 h post stress. A similar trend was also revealed in ion-leakage assay which showed that during cold stress, the transgenic potato lines depicted reduced ion leakage of 14-22% as compared to non-transgenic control plants. Further, the generated transgenic potato lines were tolerant to the frost spell in quarantine field conditions as compared to the non-transgenic potato lines. Additionally, the transgenic lines exhibited efficient recovery post frost injury in field conditions. The biochemical profiles of chlorophyll, proline and higher levels of antioxidant enzyme (superoxide dismutase, Catalase) activity and malondialdehyde levels showed that despite the phenotypic impact of low temperature, the transgenic potato lines quickly adjusted to maintain their cellular homeostasis post freezing stress by increasing the antioxidant defenses. This study suggests that up regulation of IRI3 transcript and regulatory network of cold stress response in transgenic potato lines improve frost tolerance and help stabilize yield in cultivated potato.

Effectiveness of community health workers involvement in smoking cessation programme: A systematic review.

BACKGROUND: Sustainable Development Goals (SDG) has set the target to reduce premature mortalities from non-communicable diseases (NCDs) by one-third. One of the ways to achieve this is through strengthening the countries' implementation of the World Health Organization Framework Convention on Tobacco Control (WHO FCTC). Community health workers (CHWs) involvement has shown promising results in the prevention of NCDs. This systematic review is aimed at critically evaluating the available evidence on the effectiveness of involving CHWs in smoking cessation. MATERIALS AND METHODS: We systemically searched PubMed and CENTRAL up to September 2019. We searched for published interventional studies on smoking cessation interventions using the usual care that complemented with CHWs as compared to the usual or standard care alone. Our primary outcome was abstinence of smoking. Two reviewers independently extracted data and assessed study risks of bias. RESULT: We identified 2794 articles, of which only five studies were included. A total of 3513 smokers with 41 CHWs were included in the studies. The intervention duration range from 6 weeks to 30 months. The studies used behavioral intervention or a combination of behavioral intervention and pharmacological treatment. Overall, the smoking cessation intervention that incorporated involvement of CHWs had higher smoking cessation rates [OR 1.95, 95% CI (1.35, 2.83)]. Significant smoking cessation rates were seen in two studies. CONCLUSION: Higher smoking cessation rates were seen in the interventions that combined the usual care with interventions by CHWs as compared to the usual care alone. However, there were insufficient studies to prove the effectiveness. In addition, there was high heterogeneity in terms of interventions and participants in the current studies.

Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus.

Chilo partellus is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in C. partellus larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank ELF as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However, Tubulin and HSP70 were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of Chitinase and Acetylcholinesterase were normalized with the four most stable reference genes (ELF, HSP70, V-ATPase and Tubulin) and the least stable reference gene (18S and HSP70) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in C. partellus.