Agrobacterium Tumefaciens Strain Research Articles

Bio-engineered As-Ag-TiO2 nanoparticles enhance the genetic transformation of Pisum sativum L. via proton-coupled electron transfer-dependent alternative protonation

Nanoparticle-assisted biomolecule delivery to improve plant gene transformation and expression is a, cutting-edge approach. In this study, we examined the effects of acetosyringone-doped BENPs on the enhanced genetic transformation of peas. The vir gene inducer acetosyringone was delivered into Agrobacterium via a nano-doping system with TiO2 and Ag NPs. Four BENPs, such as TiO2 (S1), Ag-TiO2 (S2), As-TiO2 (S3), and As-Ag-TiO2 (S4), were green-synthesized from the PLE using hydrothermal and calcination approaches. SEM, HR-TEM-EDAX, SAED, XRD, and XPS physicochemical characterizations ensured the BENPs structure and biomolecule doping. The potential bar transformants were chosen against the MIC of BASTA® using the EHA 105 Agrobacterium tumefaciens strain carrying the pCAMBIA 1304-plasmid. The delivery of nano-doped acetosyringone was studied with various applications, and the typical exogenous acetosyringone delivery in the absence of BENPs was also analyzed as a control experiment. The gus histochemical, PCR, and southern hybridization confirmed that, among the BENPs, S4 at a 4 ppm dose induced the highest gene transformation efficiency (36.33 %) in pea along with three days of co-cultivation, 30 sec of sonication, and 3 min of vacuum infiltration. In BENP-assisted pea gene transformation, S2 at 4 ppm (26.66 %), S3 at 3 ppm (23 %), and S1 at 4 ppm (17.66 %) showed the next three significant gene transformation efficiencies, respectively. The elevated virC and virG gene expression profiles of the S4 BENP-treated Agrobacterium were identified with gene-specific RT-PCR analysis. The PCET activity of S4 was critically confirmed with cyclic voltammetry, which revealed that BENPs might function as an alternative proton supplement system to improve the vir gene induction and transformation of T-DNA. Hence, this alternative PCET-dependent BENP-assisted acetosyringone delivery for Agrobacterium mediated gene transformation exhibits potential value in the development of genetically-modified peas and opens the door to crops for advanced genetic manipulation against various biotic and abiotic stresses.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

Evaluation of antibacterial properties of nisin peptide expressed in carrots

Nisin, derived from Lactococcus lactis, is a well-known natural food preservative. In the present study, the gene of nisin was transformed to carrot by Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing neomycin phosphotransferase II (nptII) gene, peptide signal KDEL, and Kozak sequence. The integration of nisin and nptII transgenes into the plant genome was confirmed by polymerase chain reaction (PCR) and dot blot analysis. The gene expression was also performed by RT-PCR and Enzyme-Linked Immunosorbent Assay. The level of nisin expressed in one gram of transgenic plant ranged from 0.05 to 0.08 μg/ml. The stability of nisin varied in orange and peach juices depending on the temperature on the 70th day. The leaf protein extracted from the transgenic plant showed a significant preservative effect of nisin in peach and orange juice. A complete inhibition activity against Staphylococcus aureus and Escherichia coli in orange juice was observed within 24 h. After 24 h, log 1 and log 2 were obtained in a peach juice containing Staphylococcus aureus and Escherichia coli, respectively. Results of HPLC indicated that Chlorogenic and Chicoric acid compounds were increased in transgenic plants, but this increase was not significant. The study of determining the genetic stability of transgenic plants in comparison with non-transgenic plants showed high genetic stability between non-transgenic plants and transgenic plants. This study confirmed the significant inhibitory effect of nisin protein on gram-positive and gram-negative bacteria.

High-Throughput, Quantitative Screening of Quorum-Sensing Inhibitors Based on a Bacterial Biosensor.

Quorum sensing (QS) is a cell-cell communication mechanism by which bacteria synchronize social behaviors such as biofilm formation and virulence factor secretion by producing and sensing small molecular signals. Quorum quenching (QQ) by degrading signals or blocking signal transmissions has become a promising strategy for disrupting QS and preventing bacterial infection and biofilm formation. However, studies of high-throughput screening and identification approaches for quorum-sensing inhibitors (QSIs) are still inadequate. In this work, we developed a sensitive, high-throughput approach for screening QSIs based on the bacterial biosensor strain Agrobacterium tumefaciens N5 (pBA7P), which contains a traG gene promoter induced by QS signals fused with a promoterless β-lactamase gene reporter. Using this approach, we identified 31 QQ bacteria from ∼2000 soil bacterial isolates, some belonging to the genera Bosea, Cupriavidus, and Flavobacterium that have not been reported previously as QQ bacteria. We also identified four QS inhibitory compounds and one QS signal analogue from ∼5000 small-molecule compounds, which profoundly affected the expression of QS-regulated genes and phenotypes of the pathogenic bacteria. This high-throughput screening system is effective and sensitive for screening of both QQ microbes and small molecules, enabling the discovery of a wide variety of biocompatible compounds.

Efficient genetic transformation of papaya using RNAi CP gene against papaya ringspot virus

Papaya Ring Spot Virus disease is wide spread across papaya growing countries of the world and is one of the major impediments in successful papaya cultivation. Genetically engineered papaya varieties viz., SunUp and Rainbow have already been developed and commercialized in USA using coat protein mediated resistance. However, transgenic papaya conferring resistance to papaya ringspot virus has not been developed in India till date due to lack of suitable genetic transformation protocol for Indian papaya varieties and unavailability of coat protein gene construct for harbouring broad-spectrum resistance. The current study reports an efficient gene delivery mechanism in papaya. Young globular embryos infected for 30 minutes with Agrobacterium tumefaciens strain LBA 4404 harbouring hairpin loop of truncated coat protein gene and subsequently co-cultivated in presence of 100 pM acetosyringone and 1mM spermidine in dark for 72 hours gave rise to independent transgenic events characterized by PCR, dot blot hybridisation and RT-PCR.

Enhancing drought resistance in Pogostemon cablin (Blanco) Benth. through overexpression of ACC deaminase gene using thin cell layer regeneration system.

Pogostemon cablin cultivation faces massive constraints because of its susceptability to drought stress that reduces patchouli propagation and oil yield. The present study has achieved an efficient and rapid direct regeneration system for the transgenic production of P. cablin using Agrobacterium-mediated genetic transformation. To establish an efficient regeneration protocol for fast in-vitro multiplication of patchouli plants, leaf, petiole, and transverse thin cell layer (tTCL) explants were used and inoculated on an MS medium supplemented with different combinations of phytohormones. A comparative study showed a maximum regeneration frequency of 93.30 ± 0.56% per explant was obtained from leaf segments on optimal MS medium fortified with 0.2mg/L BAP and 0.1mg/L NAA. Leaf and petiole explants took 25-35 days to regenerate while tTCL section showed regeneration in just 15-20 days on the same medium. Subsequently, productive genetic transformation protocol OD600 0.6, AS 200µM, 30mg/L kanamycin, and infection time 5 min. was standardized and best-suited explants were infected at optimum conditions from the Agrobacterium tumefaciens (LBA 4404) strain harboring ACC deaminase to generate transgenic P. cablin Benth. (CIM-Samarth) plants. The investigation suggested that the optimized protocol provides a maximum transformation frequency of 42 ± 1.9% in 15-20 days from tTCL. The transgenic plants were shifted to the greenhouse with a 52.0 ± 0.8% survival frequency. A molecular docking study confirmed significant binding affinity of ligand ACC with ACC deaminase at the catalytic site, and ligand interactions showed four H-bonds at the binding pocket with amino acids Cys-196, Val-198, Thr-199, and Gly-200 that validate gene relative expression in transgenic plants. Among all transgenic acclimatized greenhouse-grown patchouli plants, line PT4 showed improved drought resistance under severe water stress as its RWC was 71.7 ± 2.3% to 75.7 ± 2.1% which is greater than the RWC of the control plant, 58.30 ± 0.21%. Analysis of the other physiological indicators, H2O2, chlorophyll content, and ROS result support drought resistance ability. Our study concluded that the first report on P. cablin, tTCL direct regeneration, and standardized transformation protocol created a new opportunity for genetic manipulation to achieve drought-resistant patchouli plants for cultivation in all seasons at the commercial level.

Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy

BackgroundPlant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease.ResultsInitially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1–3 bp deletions and 1 bp insertions adjacent to the PAM site, a long deletion of approximately 161 bp was documented in the T0 generation. Phenotypic analysis of homozygous, ‘transgene-free’ T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants.ConclusionOur results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.

Efficient Agrobacterium mediated genetic transformation of CRISPR/Cas9 construct using cotyledonary node explants of greengram

Efficient Agrobacterium mediated genetic transformation of CRISPR/Cas9 construct using cotyledonary node explants of greengram

Melatonin strongly enhances the Agrobacterium- mediated transformation of carnation in nitrogen-depleted media

With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

Morphogenetic peculiarities of reproductive biology in sugar beet (Beta vulgaris L.) breeding.

This review considers the processes of morphogenesis used in the development of propagation methods and the creation of a new starting material for sugar beet. It has been demonstrated that methods of particulation, in vitro microcloning and cell breeding that reflect non-sexual forms of plant reproduction increase the effectiveness of breeding experiments. The review describes the in vitro culture methods maintaing a tendency in plants for vegetative propagation and stimulating increase in genetic variability of properties when mutagens such as ethyl methanesulfonate, alien genetic structures with mf2 and mf3 bacterial genes in Agrobacterium tumefaciens strains, and selective agents (Сd++ ions and abscisic acid) are incorporated into plant cells. It presents the results of using fluorescent microscopy, cytophotometry, biochemical analysis and determining the level of phytohormones and content of nucleic acids in nuclei for forecasting the seed setting ability. It has demonstrated that long self-pollination of plants causes decrease in fertility of pollen grains, resulting in the sterilization of male gametes and the appearance of pistillody flowers. Self-fertile plants isolated from these lines serve as sterility fixers, while the apomixis elements increased the ovule number, additional embryo sacs and embryos. A role of apomixis in contributing to variability in the onto- and phylogenetic development of plants have been substantiated. The review reflects the morphological features of the in vitro development of sexual and somatic cells in embryos during the formation of seedlings based on floral and vegetative embryoidogeny. Use of the SNP and SSR (Unigenes) molecular-genetic markers having a high polymorphism level has appeared effective to characterize the developed breeding material and hybrid components when carrying out crossings. The study of sugar beet starting materials for the presence of TRs mini-satellite loci making it possible to reveal O-type plants-pollinators (sterility fixing agent) and MS-form plants are of interest for breeding as well. The selected material can be widely used in breeding to produce hybrids, allowing for a 2-3- fold reduction of the development period. The review also discusses the prospects for the development and implementation of new methods and original schemes in sugar beet genetics, biotechnology and breeding.

Construction of an Agroinfectious Clone of a Korean Isolate of Sweet Potato Symptomless Virus 1 and Comparison of Its Infectivity According to Agrobacterium tumefaciens Strains in Nicotiana benthamiana

Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus <i>Mastrevirus</i> (family <i>Geminiviridae</i>) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Korea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) amplicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vector pCAMBIA1303, and agro-inoculated into <i>Nicotiana benthamiana</i> using three <i>Agrobacterium tumefaciens</i> strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The <i>A. tumefaciens</i> strain LBA4404 was the most effective at transferring the SPSMV-1 genome to <i>N. benthamiana</i>. We confirmed the viral replication in <i>N. benthamiana</i> samples through strand-specific amplification using virion-sense- and complementary-sense-specific primer sets.

Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing.

Chickpea is considered recalcitrant to in vitro tissue culture amongst all edible legumes. The clustered, regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based genome editing in chickpea can remove the bottleneck of limited genetic variation in this cash crop, which is rich in nutrients and protein. However, generating stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation protocols. As an attempt to solve this problem, we developed a modified and optimized protocol for chickpea transformation. This study transformed the single cotyledon half-embryo explants using CaMV35S promoter to drive two marker genes (β-glucuronidase gene; GUS and green fluorescent protein; GFP) through binary vectors pBI101.2 and modified pGWB2, respectively. These vectors were delivered in the explants through three different strains of Agrobacterium tumefaciens, viz., GV3101, EHA105, and LBA4404. We found better efficiency with the strain GV3101 (17.56%) compared with two other strains, i.e., 8.54 and 5.43%, respectively. We recorded better regeneration frequencies in plant tissue culture for the constructs GUS and GFP, i.e., 20.54% and 18.09%, respectively. The GV3101 was further used for the transformation of the genome editing construct. For the development of genome-edited plants, we used this modified protocol. We also used a modified binary vector pPZP200 by introducing a CaMV35S-driven chickpea codon-optimized SpCas9 gene. The promoter of the Medicago truncatula U6.1 snRNA gene was used to drive the guide RNA cassettes. This cassette targeted and edited the chickpea phytoene desaturase (CaPDS) gene. A single gRNA was found sufficient to achieve high efficiency (42%) editing with the generation of PDS mutants with albino phenotypes. A simple, rapid, highly reproducible, stable transformation and CRISPR/Cas9-based genome editing system for chickpea was established. This study aimed to demonstrate this system's applicability by performing a gene knockout of the chickpea PDS gene using an improved chickpea transformation protocol for the first time.

Advances in the Transformation of Cyclamen persicum Mill. Through Direct Regeneration Based on an Optimized Kanamycin Selection Scheme.

Gene transfer technology has great value in ornamental plants toward the generation of varieties with new ornate characteristics. In the previous studies through the transformation of cyclamen, hygromycin was mainly used as a selective marker. However, there have been some drawbacks associated with hygromycin usage as a selecting agent. Therefore, in the current study, the optimization of kanamycin concentration in the regeneration media has been considered. Subsequently, the plant transformation using three different in vitro explants from three Cyclamen persicum cultivars using three Agrobacterium tumefaciens strains has been examined. Accordingly, the optimal kanamycin concentrations for regeneration from root and leaf explants were determined as 10 mg/L and for microtuber explants as 30 mg/L. The successful gene transformation in the antibiotic-resistant shoots were examined by PCR and UV-equipped microscopes. The gfp reporter gene transfer resulted in the highest efficiency of transformation (60%) to date, from the leaf explants of cv. Pure White inoculated with Agrobacterium tumefaciens strain LBA4404. In contrast, the lowest gene transfer efficiency (25%) was observed in root explants of cv. Dark Violet and cv. Neon Pink inoculated with strains GV3101 and AGL-1, respectively. The results of the current project are expandable to the subsequent investigations of Cyclamen persicum transformation.

Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus

Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species. The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV. CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

Judicial Opinions 123-127.

Opinion 123 places the epithet of the name Aeromonas punctata on the list of rejected epithets and clarifies the citation of authors of selected names within the genus Aeromonas. Opinion 124 denies the request to place Borreliella on the list of rejected names because the request is based on a misinterpretation of the Code, which is clarified. There are alternative ways to solve the perceived problem. Opinion 125 denies the request to place Lactobacillus fornicalis on the list of rejected names because the provided information does not yield a reason for rejection. Opinion 126 denies the request to place Prolinoborus and Prolinoborus fasciculus on the list of rejected names because a relevant type strain deposit was not examined. Opinion 127 grants the request to assign the strain deposited as ATCC 4720 as the type strain of Agrobacterium tumefaciens, thereby correcting the Approved Lists. These Opinions were ratified by the voting members of the International Committee on Systematics of Prokaryotes.

First Report of Tomato Leaf Curl New Delhi Virus Infecting Several Cucurbit Plants in China

In autumn 2022, a novel and devastating viral disease affecting cucurbits emerged in Ningbo (Zhejiang province), Haimen (Jiangsu province), and Shanghai, China, causing an approximate 650-hectare infestation and resulting in nearly US$15 million in economic losses. The incidence rates of infection reached up to 72.5% on muskmelon (Cucumis melo L. ssp melo), oriental melon (Cucumis melo L. var. agrestis), pumpkin (Cucurbita moschata), luffa (Luffa acutangula), and squash (Cucurbita pepo), and were highly associated with the presence of whitefly (Bemisia tabaci). Infected plants exhibited symptoms such as dwarf stunting, reduced leaf size, leaf chlorotic patches, malformation, fruit deformation, leaf downward rolling, and yellowing (Figure 1). To identify the pathogen, forty cucurbit leaf samples were collected from Haimen (18), Ningbo (19), and Shanghai (3) and tested for cucurbits chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and Begomovirus using RT-PCR or PCR. All samples tested negative for CCYV and CYSDV using species-specific primers; however, 29 out of 40 samples tested positive (see Supplementary Table 1) for Begomovirus using the degenerate primer pairs PA/PB (Deng et al. 1994). PCR products from seven samples, representing different regions and hosts, underwent Sanger sequencing. The nucleotide sequences of these products showed 98.2-99% identity to tomato leaf curl New Delhi virus (ToLCNDV) by BLASTn. Subsequently, the 29 positive cucurbit samples were confirmed using ToLCNDV-specific primer pairs NDVAF/NDVAR and NDVBF/NDVBR (Jyothsna et al. 2013) for DNA-A and DNA-B, respectively. The DNA-A and DNA-B genome sequences of ToLCNDV isolates from Haimen (Haimen4), Ningbo (Ningbo6), and Shanghai (Shanghai1) were obtained using the primer pairs NDVAF/NDVAR, A1961F/A2645R (covering complete DNA-A sequences), NDVBF/NDVBR, and B1613F/B2579R (covering complete DNA-B sequences，see Supplementary Table 2). No amplicon was produced with primer pairs UNA101/UNA102 and beta01/beta02 (Supplementary Table 2) for detecting Alphasatellite and Betasatellite DNAs, respectively. The complete DNA-A genome sequences (2739 bp) of Haimen 4 (accession no. OP585369), Ningbo 6 (accession no. OP585370), and Shanghai 1 (accession no. OP683993) isolates exhibited 99.5-99.6% nucleotide identity to each other, and their highest nucleotide sequence identity (99.3-99.4%) was shared with the DNA-A of ToLCNDV-Zhejiang isolate (accession no. OP356207) from tomato in Zhejiang Province, China. The complete nucleotide sequences (2693 nt) of DNA-B for Haimen 4 (accession no. OP683995), Ningbo 6 (accession no. OP683996), and Shanghai 1 (accession no. OP683994) isolates showed 99.0-99.1% identity to each other, and their highest nucleotide sequence identity (~99.1%) was shared with the DNA-B of ToLCNDV-Zhejiang isolate (accession no. OP356208).All ToLCNDV isolates from mainland China, including the Zhejiang isolate and the three isolates in this study, shared 98.3-98.7% nucleotide sequence identity and 98.2-98.4% with the DNA-A genome of the severe isolate (accession no. HM159454) from tomato in New Delhi, India, and the DNA-B genome of the India:Delhi:Cucumis:2012 isolate from cucumber in New Delhi, India, respectively. However, the genome sequence identities between mainland and Taiwan isolates (accession nos. GU180095 and GU180096) were below 93%, suggesting that mainland China isolates of ToLCNDV are more closely related to the India isolate than to the Taiwan isolate.To fulfill Koch's postulates, infectious clones of the Haimen 4 isolate were constructed and agroinfiltrated into muskmelon, oriental melon, pumpkin, luffa, and squash plants. In brief, two plasmids, containing 1.56-mer DNA-A and 1.4-mer DNA-B genome sequences, were constructed using enzyme digestion and ligation, transformed into Agrobacterium tumefaciens strain GV3101, respectively, and then co-agroinfiltrated into cucurbit plants. Initial symptoms appeared in the new leaves at 7 days post-inoculation (DPI), followed by severe leaf curling, dwarfing, stunting, reduced leaf size, and chlorotic leaf patches at 18 DPI. The presence of DNA-A and DNA-B of ToLCNDV in inoculated plants was confirmed by PCR using primer pairs A1961F/A2645R and B1613F/B2579R, respectively. Collectively, the pathogen of this emerging disease has been identified as ToLCNDV. ToLCNDV was first reported on tomato in India and is now the most predominant and economically significant disease affecting cucurbit and solanaceous crops in Southeast and East Asia, the Middle East, and the Mediterranean Basin (Moriones et al. 2017). In China, ToLCNDV was initially reported on oriental melon in Taiwan (Chang et al. 2010) and subsequently on tomato (Lycopersicon esculentum) in Zhejiang province (Li et al. 2022). To the best of our knowledge, this is the first report of ToLCNDV infecting muskmelon, pumpkin, luffa, and squash in China. Further investigations on the epidemiology of this viral disease in China are needed.

Computational prediction of promotors in Agrobacterium tumefaciens strain C58 by using the machine learning technique.

Promotors are those genomic regions on the upstream of genes, which are bound by RNA polymerase for starting gene transcription. Because it is the most critical element of gene expression, the recognition of promoters is crucial to understand the regulation of gene expression. This study aimed to develop a machine learning-based model to predict promotors in Agrobacterium tumefaciens (A. tumefaciens) strain C58. In the model, promotor sequences were encoded by three different kinds of feature descriptors, namely, accumulated nucleotide frequency, k-mer nucleotide composition, and binary encodings. The obtained features were optimized by using correlation and the mRMR-based algorithm. These optimized features were inputted into a random forest (RF) classifier to discriminate promotor sequences from non-promotor sequences in A. tumefaciens strain C58. The examination of 10-fold cross-validation showed that the proposed model could yield an overall accuracy of 0.837. This model will provide help for the study of promoters in A. tumefaciens C58 strain.

Enhanced Antioxidant and Anticancer Potential of Artemisia carvifolia Buch Transformed with rol A Gene.

Secondary metabolites have been shown to possess a range of biological functions. Flavonoids, due to their ability to scavenge ROS, are famous antioxidants. The plants of Artemisia species are rich sources of flavonoids; however, the amount of these metabolites is less. In the current study, the flavonoid content was detected and then enhanced by genetically modifying the Artemisia carvifolia Buch with Agrobacterium tumefaciens strain GV3101 carrying rol A gene. The transformation of rol A gene was confirmed with PCR and the gene copy number was confirmed by Southern blot analysis. The HPLC analysis revealed the presence of catechin (3.19 ug/mg DW) and geutisic acid (2.22 ug/mg DW) in transformed plants, unlike wild-type plants. In transformed plants, all detected flavonoids (vanillic acid, rutin, catechine, gallic acid, syringic acid, caffeic acid, coumaric acid, geutisic acid, ferulic acid, and cinnamic acid) were increased up to several folds. Real-time qPCR revealed the higher expression levels of the genes for flavonoid biosynthesis enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) in plants transformed with rol A genes, as the expression levels were increased up to 9-20-fold and 2-6-fold, respectively. The rol A transgenic lines T3 and T5 carrying two copies of rol A gene, particularly showed higher expression of both PAL and CHS gene, with the highest expression in T3 line. The transgenic lines demonstrated an average increase of 1.4-fold in the total phenolic content and 1-2-fold in the total flavonoid content as compared to wild-type plants. Total antioxidant capacity and total reducing power were increased up to an average of 1-2-fold and 1.5-2-fold respectively, along with increased free radical scavenging ability. Furthermore, the rol A gene transgenics were found to have much greater cytotoxic capacity than the A. carvifolia wild-type plant against the MCF7, HeLA, and HePG2 cancer cell lines. Current findings show that the rol A gene effectively increases the flavonoid content of A. carvifolia Buch, boosting the plant's capacity as an antioxidant and an anticancer. This is the first-ever report, demonstrating the genetic transformation of Artemisia carvifolia Buch with rol A gene.

A new simple and effective method for PLRV infection to screen for virus resistance in potato

Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.

The Basic Experimental Setup for the Vacuum-assisted, Agrobacterium-mediated Transient Expression in Tomato

Several methods are available for use, to deliver the gene of interest into plants. Among these, Agrobacterium-mediated transformation utilizes binary vector systems to achieve the stable transformation of plants. Alas, this process is labor-intensive and time-consuming as several months are needed to obtain a true transgenic plant. Transient gene expression (e.g., vacuum-infiltration) systems were offered as an alternative over stable transformation, specifically to overcome time-related drawbacks. However, this method requires expensive equipment such as vacuum chambers. In this study, we report a vacuum-infiltration protocol for the transient expression of a reporter gene, modified green fluorescent protein (mGFP), in tomato seedling. With a basic experimental setup (including a custom-built growth chamber) with affordable equipment, we showed that the entire leaf can be infiltrated by applying a mere 200 millibar vacuum for 6 minutes, and thus the transient expression can be achieved in tomato plant, evidenced by PCR-based detection of the T-DNA, detection of mGFP both analytically with SDS-page analysis, and visually by the images acquired by fluorescence microscopy. Furthermore, different Agrobacterium tumefaciens strains were tested for their transformation efficiency, and we showed that LBA4404 was the most effective strain to use in the vacuum-assisted transient expression.