Insulated piggyBac vectors for insect transgenesis

BACKGROUND: Germ-line transformation of insects is now a widely used method for analyzing gene function and for the development of genetically modified strains suitable for pest control programs. The most widely used transposable element for the germ-line transformation of insects is piggyBac. The site of integration of the transgene can influence gene expression due to the effects of nearby transcription enhancers or silent heterochromatic regions. Position effects can be minimized by flanking a transgene with insulator elements. The scs/scs' and gypsy insulators from Drosophila melanogaster as well as the chicken β-globin HS4 insulator function in both Drosophila and mammalian cells. RESULTS: To minimize position effects we have created a set of piggyBac transformation vectors that contain either the scs/scs', gypsy or chicken β-globin HS4 insulators. The vectors contain either fluorescent protein or eye color marker genes and have been successfully used for germ-line transformation of Drosophila melanogaster. A set of the scs/scs' vectors contains the coral reef fluorescent protein marker genes AmCyan, ZsGreen and DsRed that have not been optimized for translation in human cells. These marker genes are controlled by a combined GMR-3xP3 enhancer/promoter that gives particularly strong expression in the eyes. This is also the first report of the use of the ZsGreen and AmCyan reef fluorescent proteins as transformation markers in insects. CONCLUSION: The insulated piggyBac vectors should protect transgenes against position effects and thus facilitate fine control of gene expression in a wide spectrum of insect species. These vectors may also be used for transgenesis in other invertebrate species

Tension band wiring of fracture distal end of clavicle: Favorable clinical outcome among the patients

Background: A fracture of the clavicle is common traumatic injury. It can be classified into middle third, medial third, and distal third fractures. Around 10–15% of clavicle fractures occur in the distal third. The ideal method of fixation in distal third clavicle fracture remains controversial till date. Aims and Objectives: The clinical outcome of the patients having fracture of the distal end clavicle using rigid fixation with tension band wiring (TBW) with Kirshner wires and SS wire to be assessed instead of other conventional procedures. Materials and Methods: Eight patients of Neer type II closed distal-third clavicle fracture of <3 weeks of duration were included in our study. The fractures were reduced by open reduction and fixed with TBW with two K-wires and SS wire. Results: The mean average age of patients was 41.87 years. All fractures united clinically and radiologically. The mean average time of union was 12 weeks. There was two case of hardware prominence, no fracture related complications. One case of superficial infection was found in study. All of them regained near normal range of motion, and the mean average constant Murley score for distal clavicle fracture at the end of 1 year was 88. At the end of 1 year follow-up, all patients had reached their pre-injury performance levels. Conclusion: There was an encouraging result with TBW in distal third clavicle fracture in our study. This technique is simple with lesser need of expertise and is also cost effective

Speed breeding in growth chambers and glasshouses for crop breeding and model plant research

‘Speed breeding’ (SB) shortens the breeding cycle and accelerates crop research through rapid generation advancement. SB can be carried out in numerous ways, one of which involves extending the duration of plants’ daily exposure to light, combined with early seed harvest, to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. In this protocol, we present glasshouse and growth chamber–based SB approaches with supporting data from experimentation with several crops. We describe the conditions that promote the rapid growth of bread wheat, durum wheat, barley, oat, various Brassica species, chickpea, pea, grass pea, quinoa and Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale up plant numbers for single-seed descent (SSD). In addition, instructions are provided on how to perform SB on a small scale in a benchtop growth cabinet, enabling optimization of parameters at a low cost

A Review of sensor technology for in-field phosphate monitoring

With the increasing awareness of fertilizer effects on environmental and soil quality, soil tests are being conducted. In-field phosphate sensors have been a topic of heavy research since the early 1980s. It provides sustainable alternatives to more time consuming and costly laboratory analyses. Phosphate sensors can be used in agricultural and site-specific crop management to optimize application of fertilizers and minimize contamination of rainfall runoff. The main limitation in the development of phosphate sensors is selectivity. Another drawback in the development of phosphate sensors is lifetime. Further research is needed to address these problems and make in-field phosphate sensors more widely available.8 page(s

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low β-L-ODAP grass pea lines

Background: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. Results: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for β-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled β-L-ODAP) allowing accurate quantification of β-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any β-L-ODAP in these species. The LCMS method was also used to quantify β-L-ODAP accurately in different tissues of grass pea. Conclusions: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and β-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of β-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for β-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-β-L-ODAP genotypes

TILLING by Sequencing (TbyS) for targeted genome mutagenesis in crops

TILLING (Targeting Induced Local Lesions in Genomes) by Sequencing (TbyS) refers to the application of high-throughput sequencing technologies to mutagenised TILLING populations as a tool for functional genomics. TbyS can be used to identify and characterise induced variation in genes (controlling traits of interest) within large mutant populations, and is a powerful approach for the study and harnessing of genetic variation in crop breeding programmes. The extension of existing TILLING platforms by TbyS will accelerate crop functional genomics studies, in concert with the rapid increase in genome editing capabilities and the number and quality of sequenced crop plant genomes. In this mini-review, we provide an overview of the growth of TbyS and its potential applications to crop molecular breeding

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low β-L-ODAP grass pea lines

Grass pea ( Lathyrus sativus ) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties

Delivery of Apoplastic Extracellular Vesicles Encapsulating Green-Synthesized Silver Nanoparticles to Treat Citrus Canker

The citrus canker pathogen Xanthomonas axonopodis has caused severe damage to citrus crops worldwide, resulting in significant economic losses for the citrus industry. To address this, a green synthesis method was used to develop silver nanoparticles with the leaf extract of Phyllanthus niruri (GS-AgNP-LEPN). This method replaces the need for toxic reagents, as the LEPN acts as a reducing and capping agent. To further enhance their effectiveness, the GS-AgNP-LEPN were encapsulated in extracellular vesicles (EVs), nanovesicles with a diameter of approximately 30–1000 nm naturally released from different sources, including plant and mammalian cells, and found in the apoplastic fluid (APF) of leaves. When compared to a regular antibiotic (ampicillin), the delivery of APF-EV-GS-AgNP-LEPN and GS-AgNP-LEPN to X. axonopodis pv. was shown to have more significant antimicrobial activity. Our analysis showed the presence of phyllanthin and nirurinetin in the LEPN and found evidence that both could be responsible for antimicrobial activity against X. axonopodis pv. Ferredoxin-NADP+ reductase (FAD-FNR) and the effector protein XopAI play a crucial role in the survival and virulence of X. axonopodis pv. Our molecular docking studies showed that nirurinetin could bind to FAD-FNR and XopAI with high binding energies (−10.32 kcal/mol and −6.13 kcal/mol, respectively) as compared to phyllanthin (−6.42 kcal/mol and −2.93 kcal/mol, respectively), which was also supported by the western blot experiment. We conclude that (a) the hybrid of APF-EV and GS-NP could be an effective treatment for citrus canker, and (b) it works via the nirurinetin-dependent inhibition of FAD-FNR and XopAI in X. axonopodis pv

Grasspea

Grasspea is an important food security crop, especially under severely adverse environmental conditions such as prolonged drought. The crop has a long history, but its cultivation has declined probably due to the presence of β-ODAP, a neurotoxin, which can cause a severe disease in people and domestic animals when grasspea is the main source of nutrition in an unbalanced diet. Grasspea, and the people who depend on it, can therefore benefit from the removal of this specific compound. A wide range of genetic and genomic tools are now available which can facilitate this development. The purpose of this article is to describe genetic and genomic resources and their specific features in grasspea.AE, NE, PE, and AS were supported by the John Innes Centre Institute Development Grant, the Biotechnology and Biological Sciences Research Council (BBSRC) Detox Grass pea project (BB/L011719/1), the BBSRC SASSA UPGRADE project (BB/R020604/1), and the BBSRC Institute Strategic Programme (BBS/E/J/000PR9799). NE gratefully acknowledges the support of an Institute Strategic Fellowship from The John Innes Centre. These authors thank Cathie Martin for her support and many useful discussions. MCVP would like to acknowledge financial support by Fundação para a Ciência e Tecnologia (FCT), Portugal, through the research unit GREEN-IT (UID/04551/2020). DR was supported by AEI projects AGL2017-82907-R and PID2020-11468RB-100. JM and PN were financially supported by the Czech Academy of Science (RVO: 60077344). SK acknowledges support of the BBSRC SASSA UPGRADE project (BB/R020604/1) and a grant from Templeton World Charity Foundation, Inc. XH acknowledges support from the China Agriculture Research System of MOF and MARA-Food Legumes (CARS-08-Z4).Peer reviewe