Pongamia pinnata is a legume plant which has great potential to be used as a biofuel feedstock. Conventional propagation of P. pinnata was found to be inefficient for mass propagation. Employing plant tissue culture techniques for micropropagation and further plant improvement of P. pinnata will be the right path to fulfill future challenges in biofuel production. This study aimed to establish a plant regeneration system for potential micropropagation and genetic manipulation of P. pinnata in future. In vitro nodal explants were used and Woody Plant Medium (WPM) containing 30 µM 6-benzylaminopurine (BAP) and 1 mM phloroglucinol (PG) was able to induce higher frequency of multiple shoot buds compared to other media investigated in this study. For shoot regeneration study, WPM containing 15 µM of zeatin and 1 mM PG was able to induce longer shoots while rooting of the regenerated shoots was enhanced by WPM supplemented with indole-3-butyric acid (IBA) in combination with silver thiosulphate (STS). A simple and effective acclimatisation protocol was established with very high survival frequency of regenerated plantlets. Root nodulation of the successfully acclimatised plants was also observed. In short, multiple shoot buds were successfully induced, regenerated and rooted in vitro. The rooted plantlets were successfully acclimatised and grown healthily. It was concluded that a successful plant regeneration protocol of P. pinnata was achieved for potential application in micropropagation and genetic manipulation.
We report the complete genome sequence of Agrobacterium leguminum strain EL101, isolated from the tree bark of Khaya senegalensis in Kampar, Perak, Malaysia, obtained using Q20+ Nanopore Sequencing chemistry. The assembled genome has a total length of 5,324,685 bp, comprising a circular chromosome, a linear chromid, and two non-Ti circular plasmids.
Parotid abscess is uncommon in neonates. It is frequently related to prematurity, prolonged gavage feeding and dehydration. We report a case of a late preterm infant who developed the classical manifestation of unilateral acute Staphylococcus aureus suppurative parotitis progressing to formation of abscess which responded to surgical drainage and antibiotic therapy.
This study investigates how field practices in handling and applying pesticides influence the long-term patterns of professional agricultural operators' exposure to pesticides. It presents the first use of a comprehensive pesticide application dataset collected on behalf of the European Food Safety Authority with 50 operators selected to cover arable and orchard cropping systems in Greece, Lithuania and the UK. Exposure was predicted based on the harmonised Agricultural Operator Exposure Model (AOEM) and compared with Acceptable Operator Exposure Levels (AOELs). The amount of pesticides handled by individual operators across a cropping season was largest in the UK arable and orchard systems (median 580 and 437kg active substance, respectively), intermediate for the arable systems in Greece and Lithuania (151 and 77kg, respectively), and smallest in the Greek orchard system (22kg). Overall, 30 of the 50 operators made at least one application within a day with predicted exposure greater than the AOEL. The rate of AOEL exceedance was greatest in the Greek cropping systems (8 orchard operators, 2.8-16% of total applications; 7 arable operators, 1.1-14% of total applications), and least for the Lithuanian arable system (2 operators, 2.9-4.5% of total applications). Instances in Greece when predicted exposure exceed the AOEL were strongly influenced by the widespread use of wettable powder formulations (>40% of the total pesticide active substance handled for 11 of the 20 Greek operators). In contrast, the total area of land treated with an active substance on a single day was more important in the UK and Lithuania (95th percentile observed value was 132 and 19haday-1 for UK arable and orchard systems, respectively). Study findings can be used to evaluate current assumptions in regulatory exposure calculations and to identify situations with potential risk that require further analysis including measurements of exposure to validate model estimations.
In childhood acute lymphoblastic leukaemia (ALL), cytogenetics play an important role in diagnosis, allocation of treatment and prognosis. Conventional cytogenetic analysis, involving mainly karyotyping in our experience, has not been successful in a large proportion of cases due to inadequate metaphase spreads and poor chromosome morphology. Our aim is to develop a highly sensitive and specific method to screen simultaneously for the four most frequent fusion transcripts resulting from specific chromosomal translocations, namely, both the CML- and ALLtype BCR-ABL transcripts of t(9;22), E2A-PBX1 transcript of t(1;19), the MLL-AF4 transcript of t(4;11) and TEL-AML1 (also termed ETV6-CBFA2) of the cryptic t(12;21). A multiplex reverse transcription polymerase chain reaction protocol (RT-PCR) was developed and tested out on archival bone marrow samples and leukaemia cell lines. In all samples with a known translocation detected by cytogenetic techniques, the same translocation was identified by the multiplex-PCR assay. Multiplex RT-PCR assay is an effective, sensitive, accurate and cost-effective diagnostic tool which can improve our ability to accurately and rapidly risk-stratify patients with childhood ALL.
Occupational exposure to pesticide mixtures comprising active substance(s) and/or co-formulant(s) with known/possible endocrine-disrupting activity was assessed using long-term activity records for 50 professional operators representing arable and orchard cropping systems in Greece, Lithuania, and the UK. Exposure was estimated using the harmonised Agricultural Operator Exposure Model, and risk was quantified as a point of departure index (PODI) using the lowest no observed (adverse) effect level. Use of substances with known/possible endocrine activity was common, with 43 of the 50 operators applying at least one such active substance on more than 50% of spray days; at maximum, one UK operator sprayed five such active substances and 10 such co-formulants in a single day. At 95th percentile, total exposure was largest in the UK orchard system (0.041 × 10-2 mg kg bw-1 day-1) whereas risk was largest in the Greek cropping systems (PODI 0.053 × 10-1). All five cropping systems had instances indicating potential for risk when expressed at a daily resolution (maximum PODI 1.2-10.7). Toxicological data are sparse for co-formulants, so combined risk from complex mixtures of active substances and co-formulants may be larger in reality.
This study investigated changes over 25 years (1987-2012) in pesticide usage in orchards in England and Wales and associated changes to exposure and risk for resident pregnant women living 100 and 1000 m downwind of treated areas. A model was developed to estimate aggregated daily exposure to pesticides via inhaled vapour and indirect dermal contact with contaminated ground, whilst risk was expressed as a hazard quotient (HQ) based on estimated exposure and the no observed (adverse) effect level for reproductive and developmental effects. Results show the largest changes occurred between 1987 and 1996 with total pesticide usage reduced by ca. 25%, exposure per unit of pesticide applied slightly increased, and a reduction in risk per unit exposure by factors of 1.3 to 3. Thereafter, there were no consistent changes in use between 1996 and 2012, with an increase in number of applications to each crop balanced by a decrease in average application rate. Exposure per unit of pesticide applied decreased consistently over this period such that values in 2012 for this metric were 48-65% of those in 1987, and there were further smaller decreases in risk per unit exposure. All aggregated hazard quotients were two to three orders of magnitude smaller than one, despite the inherent simplifications of assuming co-occurrence of exposure to all pesticides and additivity of effects. Hazard quotients at 1000 m were 5 to 16 times smaller than those at 100 m. There were clear signals of the impact of regulatory intervention in improving the fate and hazard profiles of pesticides used in orchards in England and Wales over the period investigated.
Human milk bank is a source of human milk supply in many neonatal intensive care units. However, there are some hospitals without this facility because of financial or religious impediments, such as the Muslim community.
Brassinosteroid Insensitive 1 (BRI1)-Associated Kinase I (BAK1) has been reported to interact with BRI1 for brassinosteroid (BR) perception and signal transduction that regulate plant growth and development. The aim of this study is to investigate the functions of a rice OsBAK1 homologue, designated as OsI-BAK1, which is highly expressed after heading. Silencing of OsI-BAK1 in rice plants produced a high number of undeveloped green and unfilled grains compared to the untransformed plants. Histological analyses demonstrated that embryos were either absent or retarded in their development in these unfilled rice grains of OsI-BAK1 RNAi plants. Down regulation of OsI-BAK1 caused a reduction in cell number and enlargement in leaf bulliform cells. Furthermore, transgenic rice plants overexpressing OsI-BAK1 were demonstrated to have corrugated and twisted leaves probably due to increased cell number that caused abnormal bulliform cell structure which were enlarged and plugged deep into leaf epidermis. The current findings suggest that OsI-BAK1 may play an important role in the developmental processes of rice grain filling and leaf cell including the bulliform cells.
Broad host range (BHR) expression vector is a vital tool in molecular biology research and application. Currently, most of the plasmid vectors used in Agrobacterium spp. are binary vectors that are designed for plant transformation, and very few are designed for expressing transgenes in Agrobacterium spp. Class 1 integrons are common genetic elements that allow for the efficient capture and expression of antibiotic resistance genes, especially in Gram-negative bacteria. One of its compound promoters, PcS + P2, was used in this study and has been reported to be the strongest class 1 integron constitutive promoter; it is referred to as "integron promoter" (P int) henceforth. Herein, we created two versions of isopropyl-d-thiogalactopyranoside (IPTG)-inducible promoters by substituting and/or inserting lacO sequence(s) into P int. These inducible promoters, which possess different degrees of stringency and inducibility, were used to construct two broad host range expression vectors (pWK102 and pWK103) based on the versatile pGREEN system. This allows them to be stably maintained and replicated in both Escherichia coli and Agrobacterium tumefaciens. Functional validation of these vectors was performed by the expression of the reporter gene, superfolder green fluorescent protein (sfGFP), which was cloned downstream of these promoters. Due to the strong induction and tunable expression of a transgene located downstream to the inducible integron promoter, these vectors may be useful for heterologous gene expression in both E. coli and A. tumefaciens, thus facilitating recombinant protein production and genetic studies in Gram-negative bacteria.
SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-023-03507-0.
In Malaysia, bananas (Musa spp.) are the second most cultivated fruit and the fourth most cultivated fruit in terms of export revenue. In October 2018, about 5.0 out of 6.6 hectares of a banana plantation located in Teluk Intan, Malaysia, was impacted by an outbreak of banana disease. The onset of bacterial wilt symptoms is characterized by initial leaf wilting, followed by the subsequent withering of the entire plant during later stages, fruit stalk and fruit pulp discoloration, fruit rotting, and pseudostem necrosis. The diseased banana's symptomatic pseudostems and fruit pulps were surface-sterilised in 70% ethanol for 30 s, followed by 2% NaClO for 3 min, rinsed three times in sterilised water, and cut into small pieces approximately 5 mm2 in size. The tissues were macerated in a sterilised 0.85% NaCl solution for 5 min, and the resulting suspension was streaked onto nutrient agar, followed by incubation at 28°C for 2 days. After incubation, bacterial colonies with five unique morphological characteristics were observed. Two colonies of each unique morphological type were randomly chosen and subjected to preliminary bacterial identification by 16S rRNA gene sequencing. Based on BLASTn analysis, the five unique morphological types of bacteria were preliminarily identified as Enterobacter cloacae, Citrobacter farmeri, Klebsiella variicola, Kosakonia radicincitans, and Phytobacter ursingii. Previous reports identified K. variicola and K. radicincitans as banana pathogens, but Malaysia has yet to report the former. The amplified partial 16S rDNA sequences of both K. variicola isolates (designated as UTAR-BC1 and UTAR-BC2; GenBank accession numbers: PP531448 and PP531460, respectively), which were chosen to be the focus of this study, exhibited complete similarity to each other and were 100% identical (1426/1426 identity and 1420/1420 identity, respectively) to K. variicola (CP026013.1). To verify the identity of the bacterial isolate, three housekeeping genes, namely, infB(PP538994), rpoB (PP538995), and gyrB (PP538996) of UTAR-BC1, were amplified, sequenced, and subjected to multilocus phylogenetic analysis via the neighbour-joining method (1,000 bootstrap values). Phylogenetic analysis revealed that UTAR-BC1 belongs to the K. variicola clade. A pathogenicity assay of UTAR-BC1 was conducted on 4-month-old healthy banana plantlets (cv. Nangka) using the pseudostem injection method (Tripathi et al., 2008). First, UTAR-BC1 was grown overnight in nutrient broth and then adjusted to 108 CFU/ml in a sterile 10 mM MgCl2 solution. A total volume of 100 µL of the bacterial suspension was injected into the pseudostem of five healthy banana plantlets via a syringe with a needle. Control plants were mock-inoculated with a sterile 10 mM MgCl2 solution. The experiments were replicated thrice and inoculated plants were maintained at room temperature with natural sunlight and humidity, which resembled the field conditions. Two months after inoculation, all of the UTAR-BC1 inoculated spots of banana plantlets showed severe necrosis, while the banana leaves showed symptoms of wilted appearance, whereas the control plants remained symptomless. The reisolated pathogen from 90% of the symptomatic pseudostems and leaf blades shares the same morphological and molecular features as UTAR-BC1, thus fulfilling Koch's postulates. Previously, K. variicola has been reported to be a banana pathogen causing rhizome rot in India (Loganathan et al., 2021), plantain soft rot in Haiti (Fulton et al. 2020), and sheath rot and bulb rot in China (Sun et al., 2023; Jiang et al., 2024). To the best of our knowledge, this is the first report of bacterial wilt disease in bananas attributed to K. variicola in Malaysia. This finding will facilitate the surveillance of K. variicola as an emerging pathogen in banana plants in this region, thereby safeguarding the country's food security and promoting socio-economic growth.
Background: Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles.
Results: We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images.
Conclusions: Our protocol should be adaptable for monitoring activation for other plant small GTPases and protein-protein interactions for other FRET sensors in various plant cells.