Assessment of Genomic Diversity and Selective Pressures in Crossbred Dairy Cattle of Pakistan.

Improving the low productivity levels of native cattle breeds in smallholder farming systems is a pressing concern in Pakistan. Crossbreeding high milk-yielding holstein friesian (HF) breed with the adaptability and heat tolerance of Sahiwal cattle has resulted in offspring that are well-suited to local conditions and exhibit improved milk yield. The exploration of how desirable traits in crossbred dairy cattle are selected has not yet been investigated. This study aims to provide the first overview of the selective pressures on the genome of crossbred dairy cattle in Pakistan. A total of eighty-one crossbred, thirty-two HF and twenty-four Sahiwal cattle were genotyped, and additional SNP genotype data for HF and Sahiwal were collected from a public database to equate the sample size in each group. Within-breed selection signatures in crossbreds were investigated using the integrated haplotype score. Crossbreds were also compared to each of their parental breeds to discover between-population signatures of selection using two approaches: cross-population extended haplotype homozygosity and fixation index. We identified several overlapping genes associated with production, immunity, and adaptation traits, including U6, TMEM41B, B4GALT7, 5S_rRNA, RBM27, POU4F3, NSD1, PRELID1, RGS14, SLC34A1, TMED9, B4GALT7, OR2AK3, OR2T16, OR2T60, OR2L3, and CTNNA1. Our results suggest that regions responsible for milk traits have generally experienced stronger selective pressure than others.

A source of resistance against yellow mosaic disease in soybeans correlates with a novel mutation in a resistance gene.

Yellow mosaic disease (YMD) is one of the major devastating constraints to soybean production in Pakistan. In the present study, we report the identification of resistant soybean germplasm and a novel mutation linked with disease susceptibility. Diverse soybean germplasm were screened to identify YMD-resistant lines under natural field conditions during 2016-2020. The severity of YMD was recorded based on symptoms and was grouped according to the disease rating scale, which ranges from 0 to 5, and named as highly resistant (HR), moderately resistant (MR), resistant (R), susceptible (S), moderately susceptible (MS), and highly susceptible (HS), respectively. A HR plant named "NBG-SG Soybean" was identified, which showed stable resistance for 5 years (2016-2020) at the experimental field of the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, a location that is a hot spot area for virus infection. HS soybean germplasm were also identified as NBG-47 (PI628963), NBG-117 (PI548655), SPS-C1 (PI553045), SPS-C9 (PI639187), and cv. NARC-2021. The YMD adversely affected the yield and a significant difference was found in the potential yield of NBG-SG-soybean (3.46 ± 0.13a t/ha) with HS soybean germplasm NARC-2021 (0.44 ± 0.01c t/ha) and NBG-117 (1.12 ± 0.01d t/ha), respectively. The YMD incidence was also measured each year (2016-2020) and data showed a significant difference in the percent disease incidence in the year 2016 and 2018 and a decrease after 2019 when resistant lines were planted. The resistance in NBG-SG soybean was further confirmed by testing for an already known mutation (SNP at 149th position) for YMD in the Glyma.18G025100 gene of soybean. The susceptible soybean germplasm in the field was found positive for the said mutation. Moreover, an ortholog of the CYR-1 viral resistance gene from black gram was identified in soybean as Glyma.13G194500, which has a novel deletion (28bp/90bp) in the 5`UTR of susceptible germplasm. The characterized soybean lines from this study will assist in starting soybean breeding programs for YMD resistance. This is the first study regarding screening and molecular analysis of soybean germplasm for YMD resistance.

Genetic insights into crossbred dairy cattle of Pakistan: exploring allele frequency, linkage disequilibrium, and effective population size at a genome-wide scale.

Linkage disequilibrium (LD) affects genomic studies accuracy. High-density genotyping platforms identify SNPs across animal genomes, increasing LD evaluation resolution for accurate analysis. This study aimed to evaluate the decay and magnitude of LD in a cohort of 81 crossbred dairy cattle using the GGP_HDv3_C Bead Chip. After quality control, 116,710 Single Nucleotide Polymorphisms (SNPs) across 2520.241 Mb of autosomes were retained. LD extent was assessed between autosomal SNPs within a 10 Mb range using the r2 statistics. LD value declined as inter-marker distance increased. The average r2 value was 0.24 for SNP pairs < 10 kb apart, decreasing to 0.13 for 50-100 kb distances. Minor allele frequency (MAF) and sample size significantly impact LD. Lower MAF thresholds result in smaller r2 values, while higher thresholds show increased r2 values. Additionally, smaller sample sizes exhibit higher average r2 values, especially for larger physical distance intervals (> 50 kb) between SNP pairs. Effective population size and inbreeding coefficient were 150 and 0.028 for the present generation, indicating a decrease in genetic diversity over time. These findings imply that the utilization of high-density SNP panels and customized/breed-specific SNP panels represent a highly favorable approach for conducting genome-wide association studies (GWAS) and implementing genomic selection (GS) in the Bos indicus cattle breeds, whose genomes are still largely unexplored. Furthermore, it is imperative to devise a meticulous breeding strategy tailored to each herd, aiming to enhance desired traits while simultaneously preserving genetic diversity.

Genome-Wide Identification and Analysis of the Hsp40/J-Protein Family Reveals Its Role in Soybean (Glycine max) Growth and Development.

The J-protein family comprises molecular chaperones involved in plant growth, development, and stress responses. Little is known about this gene family in soybean. Hence, we characterized J-protein genes in soybean, with the most highly expressed and responsive during flower and seed development. We also revealed their phylogeny, structure, motif analysis, chromosome location, and expression. Based on their evolutionary links, we divided the 111 potential soybean J-proteins into 12 main clades (I-XII). Gene-structure estimation revealed that each clade had an exon-intron structure resembling or comparable to others. Most soybean J-protein genes lacked introns in Clades I, III, and XII. Moreover, transcriptome data obtained from a publicly accessible soybean database and RT-qPCR were used to examine the differential expression of DnaJ genes in various soybean tissues and organs. The expression level of DnaJ genes indicated that, among 14 tissues, at least one tissue expressed the 91 soybean genes. The findings suggest that J-protein genes could be involved in the soybean growth period and offer a baseline for further functional research into J-proteins' role in soybean. One important application is the identification of J-proteins that are highly expressed and responsive during flower and seed development in soybean. These genes likely play crucial roles in these processes, and their identification can contribute to breeding programs to improve soybean yield and quality.

Deciphering host-pathogen interaction during Streptomyces spp. infestation of potato.

Potato crop, currently, is the staple food crop of about 1.3 billion global population. Potato is attaining even more admiration globally day by day owing to its public acceptability. However, potato sustainable production is distinctly challenged by multiple factors like diseases, pests and climate change etc. Among diseases, common scab is one of the prime threats to potato crop due to its soil-borne nature and versatility in phytotoxins' secretion. Common scab is caused multiple number of phytopathogenic streptomyces strains. Despite extensive research programs, researchers are still unable to identify a significant solution to this threat that is proliferating exceptional rate across the globe. To develop feasible remedies, adequate information regarding host-pathogen interaction should be available. This review possesses insights on existing pathogenic species, the evolution of novel pathogenic streptomyces spp. and phytotoxins produced by the pathogenic strains. Furthermore, which type of physiological, biochemical and genetic activities occur during pathogen's infestation of the host are also canvassed.

Employing template-directed CRISPR-based editing of the OsALS gene to create herbicide tolerance in Basmati rice.

Rice (Oryza sativa) is one of the primary food crops which contributes major portion of daily calorie intake. It is used as model crop for various genome editing studies. Basmati rice was also explored for establishing non-homologous end joining-based genome editing. But it was not clear whether homology-directed repair (HDR)-based genome editing can be done in Basmati rice. The current study was designed to establish HDR-based genome editing in Basmati rice to develop herbicide tolerance. There is severe weed spread when rice is grown via direct planted rice method in various countries to save labour and water resources. Therefore, the use of herbicides is necessary to control weeds. These herbicides can also affect cultivated rice which creates the need to develop herbicide-tolerant rice. In current study, we introduced a point mutation in Acetolactate Synthase gene to convert tryptophan to leucine at position 548. For this purpose, different constructs for HDR were tested with different RNA scaffold and orientation of repair templates. Out of four different architectures, the one having repair template identical to the target DNA strand precisely edited the target site. We successfully established template-directed CRISPR-Cas9 system in Super Basmati rice by detecting desired substitutions at the target site in Acetolactate Synthase locus. Moreover, this editing of Acetolactate Synthase gene resulted in the production of herbicide tolerance in Super Basmati rice. This study suggests that such type of HDR system can be used to precisely edit other genes for crop improvement.

Physiological responses induced by phospholipase C isoform 5 upon heat stress in Arabidopsis thaliana.

Plant's perception of heat stress involves several pathways and signaling molecules, such as phosphoinositide, which is derived from structural membrane lipids phosphatidylinositol. Phospholipase C (PLC) is a well-known signaling enzyme containing many isoforms in different organisms. In the present study, Phospholipase C Isoform 5 (PLC5) was investigated for its role in thermotolerance in Arabidopsis thaliana. Two over-expressing lines and one knock-down mutant of PLC5 were first treated at a moderate temperature (37 °C) and left for recovery. Then again exposed to a high temperature (45 °C) to check the seedling viability and chlorophyll contents. Root behavior and changes in 32Pi labeled phospholipids were investigated after their exposure to high temperatures. Over-expression of PLC5 (PLC5 OE) exhibited quick and better phenotypic recovery with bigger and greener leaves followed by chlorophyll contents as compared to wild-type (Col-0) and PLC5 knock-down mutant in which seedling recovery was compromised. PLC5 knock-down mutant illustrated well-developed root architecture under controlled conditions but stunted secondary roots under heat stress as compared to over-expressing PLC5 lines. Around 2.3-fold increase in phosphatidylinositol 4,5-bisphosphate level was observed in PLC5 OE lines upon heat stress compared to wild-type and PLC5 knock-down mutant lines. A significant increase in phosphatidylglycerol was also observed in PLC5 OE lines as compared to Col-0 and PLC5 knock-down mutant lines. The results of the present study demonstrated that PLC5 over-expression contributes to heat stress tolerance while maintaining its photosynthetic activity and is also observed to be associated with primary and secondary root growth in Arabidopsis thaliana.

Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance.

Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F1 generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.

Smart breeding approaches in post-genomics era for developing climate-resilient food crops.

Improving the crop traits is highly required for the development of superior crop varieties to deal with climate change and the associated abiotic and biotic stress challenges. Climate change-driven global warming can trigger higher insect pest pressures and plant diseases thus affecting crop production sternly. The traits controlling genes for stress or disease tolerance are economically imperative in crop plants. In this scenario, the extensive exploration of available wild, resistant or susceptible germplasms and unraveling the genetic diversity remains vital for breeding programs. The dawn of next-generation sequencing technologies and omics approaches has accelerated plant breeding by providing the genome sequences and transcriptomes of several plants. The availability of decoded plant genomes offers an opportunity at a glance to identify candidate genes, quantitative trait loci (QTLs), molecular markers, and genome-wide association studies that can potentially aid in high throughput marker-assisted breeding. In recent years genomics is coupled with marker-assisted breeding to unravel the mechanisms to harness better better crop yield and quality. In this review, we discuss the aspects of marker-assisted breeding and recent perspectives of breeding approaches in the era of genomics, bioinformatics, high-tech phonemics, genome editing, and new plant breeding technologies for crop improvement. In nutshell, the smart breeding toolkit in the post-genomics era can steadily help in developing climate-smart future food crops.

Development of event-specific detection method for identification of insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct.

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.

Comparison of in Vitro and in Planta Toxicity of Vip3A for Lepidopteran Herbivores.

Agricultural pest infestation is as old as domestication of food crops and contributes a major share to the cost of crop production. In a transgenic pest control approach, plant production of Vip3A, an insecticidal protein from Bacillus thuringiensis, is effective against lepidopteran pests. A synthetic Vip3A gene was evaluated for efficacy against Spodoptera litura Fabricius (Lepidoptera: Noctuidae; cotton leafworm), Spodoptera exigua Hübner (Lepidoptera: Noctuidae; beet armyworm), Spodoptera frugiperda Smith (Lepidoptera: Noctuidae; fall armyworm), Helicoverpa armigera Hübner (Lepidoptera: Noctuidae; cotton bollworm), Helicoverpa zea Boddie (Lepidoptera: Noctuidae; corn earworm), Heliothis virescens Fabricius (Lepidoptera: Noctuidae; tobacco budworm), and Manduca sexta L. (Lepidoptera: Sphingidae; tobacco hornworm) in tobacco. In artificial diet assays, the concentration required to achieve 50% mortality was highest for H. zea followed by H. virescens > S. exigua > H. armigera > M. sexta > S. frugiperda > S. litura. By contrast, in bioassays with detached leaves from Vip3A transgenic tobacco, the time until 50% lethality was M. sexta > H. virescens > S. litura > H. zea > H. armigera > S. exigua. There was no significant correlation between the artificial diet and transgenic plant bioassay results. Notably, the two insect species that are best-adapted for growth on tobacco, M. sexta and H. virescens, showed the greatest time to 50% mortality on Vip3A-transgenic tobacco. Together, our results suggest that artificial diet assays may be a poor predictor of Vip3A efficacy in transgenic plants, lepidopteran species vary in their sensitivity to Vip3A in diet-dependent manner, and host plant adaptation of the targeted herbivores should be considered when designing transgenic plants for pest control.

Precise CRISPR-Cas9 Mediated Genome Editing in Super Basmati Rice for Resistance Against Bacterial Blight by Targeting the Major Susceptibility Gene.

Basmati rice is famous around the world for its flavor, aroma, and long grain. Its demand is increasing worldwide, especially in Asia. However, its production is threatened by various problems faced in the fields, resulting in major crop losses. One of the major problems is bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xoo hijacks the host machinery by activating the susceptibility genes (OsSWEET family genes), using its endogenous transcription activator like effectors (TALEs). TALEs have effector binding elements (EBEs) in the promoter region of the OsSWEET genes. Out of six well-known TALEs found to have EBEs in Clade III SWEET genes, four are present in OsSWEET14 gene's promoter region. Thus, targeting the promoter of OsSWEET14 is very important for creating broad-spectrum resistance. To engineer resistance against bacterial blight, we established CRISPR-Cas9 mediated genome editing in Super Basmati rice by targeting 4 EBEs present in the promoter of OsSWEET14. We were able to obtain four different Super Basmati lines (SB-E1, SB-E2, SB-E3, and SB-E4) having edited EBEs of three TALEs (AvrXa7, PthXo3, and TalF). The edited lines were then evaluated in triplicate for resistance against bacterial blight by choosing one of the locally isolated virulent Xoo strains with AvrXa7 and infecting Super Basmati. The lines with deletions in EBE of AvrXa7 showed resistance against the Xoo strain. Thus, it was confirmed that edited EBEs provide resistance against their respective TALEs present in Xoo strains. In this study up to 9% editing efficiency was obtained. Our findings showed that CRISPR-Cas9 can be harnessed to generate resistance against bacterial blight in indigenous varieties, against locally prevalent Xoo strains.

Hazards of nitrogen fertilizers and ways to reduce nitrate accumulation in crop plants.

In modern agriculture, farm produce accumulates a lot of nitrates that can reach toxic levels owing to the unfair use of nitrogen fertilizers, cultural methods, farming policies in multiple areas of the world, thereby increasing concerns about the availability of hygienic food supply and environmental hazards. Over the past few decades, global interest in achieving greater output through intensive fertilization has been a growing trend. The fertilizer based on urea or ammonium mainly yields ammonium, which is then transformed to nitrate through the oxidation process that is biologically mediated. Nitrate tends to accumulate differently in distinct crop plants and distinct components of agricultural commodities based on species, crop variety, genetic history, environmental circumstances, harvest phase, post-harvest storage conditions, agronomic variables, nature, and fertilizer application rate. The current article highlights various factors that could directly or indirectly contribute to the accumulation of nitrates in different parts of crop plants and discusses strategies to minimize the accumulation of nitrates in farm produce, thus ensuring healthy food supply and protecting the environment from the accumulation of nitrates.

Genome Editing Technologies for Rice Improvement: Progress, Prospects, and Safety Concerns.

Rice (Oryza sativa) is an important staple food crop worldwide; to meet the growing nutritional requirements of the increasing population in the face of climate change, qualitative and quantitative traits of rice need to be improved. Stress-tolerant crop varieties must be developed with stable or higher yields under stress conditions. Genome editing and speed breeding have improved the accuracy and pace of rice breeding. New breeding technologies including genome editing have been established in rice, expanding the potential for crop improvement. Recently, other genome editing techniques such as CRISPR-directed evolution, CRISPR-Cas12a, and base editors have also been used for efficient genome editing in rice. Since rice is an excellent model system for functional studies due to its small genome and close syntenic relationships with other cereal crops, new genome-editing technologies continue to be developed for use in rice. In this review, we focus on genome-editing tools for rice improvement to address current challenges and provide examples of genome editing in rice. We also shed light on expanding the scope of genome editing and systems for delivering homology-directed repair templates. Finally, we discuss safety concerns and methods for obtaining transgene-free crops.

A critical look on CRISPR-based genome editing in plants.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing, derived from prokaryotic immunity system, is rapidly emerging as an alternative platform for introducing targeted alterations in genomes. The CRISPR-based tools have been deployed for several other applications including gene expression studies, detection of mutation patterns in genomes, epigenetic regulation, chromatin imaging, etc. Unlike the traditional genetic engineering approaches, it is simple, cost-effective, and highly specific in inducing genetic variations. Despite its popularity, the technology has limitations such as off-targets, low mutagenesis efficiency, and its dependency on in-vitro regeneration protocols for the recovery of stable plant lines. Several other issues such as persisted CRISPR activity in subsequent generations, the potential for transferring to its wild type population, the risk of reversion of edited version to its original phenotype particularly in cross-pollinated plant species when released into the environment and the scarcity of validated targets have been overlooked. This article briefly highlights these undermined aspects, which may challenge the wider applications of this platform for improving crop genetics.

Investigating the In Vitro Regeneration Potential of Commercial Cultivars of Brassica.

In vitro regeneration is a pre-requisite for developing transgenic plants through tissue culture-based genetic engineering approaches. Huge variations among different genotypes of the genus Brassica necessitate the identification of a set of regeneration conditions for a genotype, which can be reliably used in transformation experiments. In this study, we evaluated the morphogenesis potential of four commercial cultivars (Faisal canola, Punjab canola, Aari canola, Nifa Gold) and one model, Westar, from four different explants namely cotyledons, hypocotyls, petioles and roots on three different Brassica regeneration protocols, BRP-I, -II and -III. The regeneration efficiency was observed in the range of 6-73%, 4-79.3%, 0-50.6%, and 0-42.6% from cotyledons, petioles, hypocotyls and roots, respectively, whereas, the regeneration response in terms of average shoots per explant was found to be 0.76-10.9, 0.2-3.2, 0-3.4 and 0-2.7 from these explants. Of the commercial varieties tested, almost all varieties showed poorer regeneration than Westar except Aari canola. In comparison to Westar, its regeneration frequency from cotyledons was up to 7.5-fold higher on BRP-I, while it produced up to 21.9-fold more shoots per explant. Our data show that the explant has strong influence on the regeneration response, ranging from 24% to 92%. While the growth of commercial cultivars was least affected by the regeneration conditions provided, the effect on Westar was twice that of the commercial cultivars. After determining the optimal explant type and regeneration conditions, we also determined the minimum kanamycin concentration levels required to selectively inhibit the growth of untransformed cells for these cultivars. Regenerated shoots of Aari canola could be successfully grown to maturity within 16-18 weeks, with no altered phenotype noted and normal seed yields obtained. Therefore, the commercial variety, Aari canola, could be a good candidate for future genetic transformation studies.

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests.

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.

Expression patterns of cp4-epsps gene in diverse transgenic Saccharum officinarum L. genotypes.

Glyphosate, a functional analogue of phosphoenolpyruvate (PEP), blocks the shikimate pathway by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) through interference with the conversion of (shikimate-3-phosphate) S3P and PEP to 5-enolpyruvylshikimate-3-phosphate (EPSP) and subsequently leads to plant death. This metabolic pathway possesses great potential to be used for development of herbicide resistant transgenic crops and here in this study, we wanted to check the expression potential of CP4-EPSPS gene in various sugarcane genotypes. A synthetic version of CP4-EPSPS gene synthesized commercially, cloned in pGreen0029 vector, was transformed into regenerable embryogenic calli of three different sugarcane cultivars HSF-240, S2003US-778 and S2003US-114 using biolistic gene transfer approach for comparative transcriptional studies. Transgenic lines screened by PCR analysis were subjected to Southern hybridization for checking transgene integration patterns. All the tested lines were found to contain multiple (3-6) insert copies. Putative transgenic plants produced the CP4-EPSPS protein which was detected using immunoblot analysis. The CP4-EPSPS transcript expression detected by qRT-PCR was found to vary from genotype to genotype and is being reported first time. In vitro glyphosate assay showed that transformed plants were conferring herbicide tolerance. It is concluded that different cultivars of sugarcane give variable expression of the same transgene and reasons for this phenomenon needs to be investigated.

In-planta expression of insecticidal proteins provides protection against lepidopteran insects.

The development of advanced biotechnological control strategies opens a new era of environment friendly pest management. The current study is part of such an effort, in which we developed a control strategy based on gene pyramiding that confers broad-spectrum resistance against lepidopteran (Helicoverpa armigera and Spodoptera litura) and hemipteran (Myzus persicae, Phenacoccus solenopsis, and Bemisia tabaci) insect pests. Previously, we reported a double gene construct expressing Hvt and lectin in tobacco (Nicotiana tabacum) plants under phloem specific promoters which confers resistance against hemipteran insects. Here we extended our studies by evaluating the advanced generation of these tobacco plants expressing hvt-lectin against lepidopteran insects. Tobacco plants expressing both toxins were tested against H. armigera and S. litura. Insect bioassay results showed 100% mortality of H. armigera within 48-72 hours and 100% mortality of S. litura within 72-96 hours. Our results suggest that the use of both toxins as a gene pyramiding strategy to control both lepidopteran and hemipterans insects on commercial basis to reduce the use of chemical pesticides.

Paediatric duodenal injury complicated by common bile duct rupture due to blunt trauma: a multispecialist approach.

We report a case of late presenting duodenal perforation with common bile duct rupture secondary to blunt handlebar trauma in an 11-year-old boy. The patient presented with upper abdominal wall ecchymosis, pain and vomiting. He was discharged after 24 hours with resolving symptoms. However, the boy presented 2 days later febrile with signs of peritonitis. CT indicated duodenal perforation, which was confirmed during laparotomy where common bile duct rupture was also demonstrated. Primary repair of the duodenum was undertaken. Here, decompression was achieved with a nasogastric tube proximal to the injury and T-tube duodenostomy distally. Common bile duct repair was achieved over a biliary stent. This case represents a rare subset of duodenal injury for which there is a paucity of evidence for optimal surgical management, particularly in the paediatric setting. This operative plan will guide surgeons and junior doctors in managing complicated cases like this in future.