Blot Hybridization Analysis Research Articles

Erratum for Angel et al., "Early Diagnosis of Toxoplasmic Encephalitis in AIDS Patients by Dot Blot Hybridization Analysis".

Erratum for Angel et al., "Early Diagnosis of Toxoplasmic Encephalitis in AIDS Patients by Dot Blot Hybridization Analysis".

Identification of the potential matrix protein of invertebrate iridescent virus 6 (IIV6)

Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic virus with a ∼212 kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the IIV6 virion consists of 54 virally encoded proteins. Interactions among the structural proteins were investigated using the yeast two-hybrid system, revealing that the protein of 415R ORF interacts reciprocally with the potential envelope protein 118L and the major capsid protein 274L. This result suggests that 415R might be a matrix protein that plays a role as a bridge between the capsid and the envelope proteins. To elucidate the function of 415R protein, we determined the localization of 415R in IIV6 structure and analyzed the properties of 415R-silenced IIV6. Specific antibodies produced against 415R protein were used to determine the location of the 415R protein in the virion structure. Both western blot hybridization and immunogold electron microscopy analyses showed that the 415R protein was found in virions treated with Triton X-100, which degrades the viral envelope. The 415R gene was silenced by the RNA interference (RNAi) technique. We used gene-specific dsRNA’s to target 415R and showed that this treatment resulted in a significant drop in virus titer. Silencing 415R with dsRNA also reduced the transcription levels of other viral genes. These results provide important data on the role and location of IIV6 415R protein in the virion structure. Additionally, these results may also shed light on the identification of the homologs of 415R among the vertebrate iridoviruses.

Cognitive Impairment in a Complex Family With AAGGG and ACAGG Repeat Expansions in RFC1 Detected by ExpansionHunter Denovo.

We investigated the genetic basis and brain metabolism and blood flow of a Japanese family with spinocerebellar degeneration (SCD), with multiple affected members for 3 generations. After excluding DNA repeat expansion (RE) of common SCD genes by fragment analysis, we performed whole-exome sequencing (WES) and whole-genome sequencing (WGS). Homozygosity mapping was performed using these data. REs were investigated with WGS data using ExpansionHunter Denovo and Expansion Hunter. WES and WGS were unable to identify likely pathogenic variants, and homozygosity mapping failed to narrow down the locus. However, ExpansionHunter Denovo detected REs in intron 2 of the RFC1 gene and led us to the diagnosis of RFC1-related disorders. Subsequent repeat-primed PCR and Southern blot hybridization analyses revealed that 3 of 6 patients and 1 suspected individual had expansions of AAGGG ((AAGGG)exp) and (ACAGG)exp repeats in a compound heterozygous state and 3 had a homozygous (ACAGG)exp. The patients showed a variety of clinical features, including adult-onset ataxia, sensorimotor neuropathy, head tremor, parkinsonism, dystonia, and cognitive impairment. A comparison of previous reports with those of the family in study suggested that motor neuropathy could be a feature of compound heterozygous patients and biallelic (ACAGG)exp patients. Cognitive function tests showed cognitive impairment with a predominance of frontal lobe dysfunction. Examination of MRI, SPECT, and 18F-fluorodeoxyglucose-PET showed clear cortical damage with frontal lobe predominance in 1 case, but no cerebral damage was evident in the other 2 cases. Our report shows the usefulness of WGS and RE detection tools for SCD of unknown cause. The studied family with RFC1-related disorders included patients with (ACAGG)exp and (AAGGG)exp in a compound heterozygous state and was characterized by motor neuropathy. Based on the results of cognitive function tests and imaging studies, 1 patient presented with cognitive impairment due to frontal lobe metabolic changes, but there were also patients who presented with cognitive impairment without apparent cerebral metabolic or blood flow, suggesting that other factors are also associated with cognitive impairment.

Physcomitrium patens pentatricopeptide repeat protein PpPPR_32 is involved in the accumulation of psaC mRNA encoding the iron sulfur protein of photosystem I.

Pentatricopeptide repeat (PPR) proteins are involved in RNA metabolism and also play a role in posttranscriptional regulation during plant organellar gene expression. Although a hundred of PPR proteins exist in the moss Physcomitrium patens, their functions are not fully understood. Here, we report the function of P-class PPR protein PpPPR_32 in P. patens. A transient expression assay using green fluorescent protein demonstrated that the N-terminal region of PpPPR_32 functions as a chloroplast-targeting transit peptide, indicating that PpPPR_32 is localized in chloroplasts. PpPPR_32 knockout mutants grew autotrophically but with reduced protonema growth and the poor formation of photosystem I (PSI) complexes. Quantitative real-time reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in the transcript level of the psaC gene encoding the iron sulfur protein of PSI but no alteration to the transcript levels of other PSI genes. This suggests that PpPPR_32 is specifically involved in the expression level of the psaC gene. Our results indicate that PpPPR_32 is essential for the accumulation of psaC transcript and PSI complexes.

Genetic Characterization of Colistin-Resistant Salmonella enterica ST34 Co-Harbouring Plasmid-Borne mcr-1, bla CTX-M-15 and bla KPC-2 Recovered from a Paediatric Patient in Shenzhen, China.

BackgroundSince 2015, plasmid-borne mcr-1 has been reported in various bacterial strains in the clinical setting globally. However, the transmission mechanisms of this gene in Salmonella are not well defined. This study aimed to characterize the genomic features of a Salmonella enterica ST34 isolate, which carried a mcr-1, mapped to a carbapenemase and extended spectrum β-lactamase encoding gene located on the IncX4 plasmid.MethodsSalmonella enterica was recovered from a diarrheal paediatric patient in Shenzhen, China. Antimicrobial susceptibility testing was performed by using the VITEK 2 system. Drug resistance genes were identified using targeted primers and Sanger sequencing. The transferability and genome location of mcr-1 was determined by performing conjugation, S1-PFGE and Southern blot hybridization analysis. WGS was performed by Illumina MiSeq sequencing and was assembled using the A5-Miseq pipeline, and gene annotation was performed using RAST 2.0. The database Centre for Genomic Epidemiology’s website was used to identify resistance genes and sequence types (STs).ResultsWe found that the isolate was extensively drug resistant and belonging to ST34, carrying an IncX4 plasmid with mcr-1, blaKPC-2 and blaCTX-M-15. We also noticed that genes blaPAO, fosA, catB, the mutation in oprD and mexT (MexEF-OprN efflux regulator), and exotoxin-encoding genes (exoS, exoY and exoT) were associated with resistance and virulence in the genome. In addition, heavy metal resistance genes as silP and silE were determined.ConclusionThis study highlights the potential risk of ST34 of Salmonella enterica serotype Typhimurium carrying multiple drug resistance encoding genes in a single IncX4 plasmid.

Human papillomavirus prevalence and genotype distribution in Vietnamese male patients between 2016 and 2020.

Human papillomavirus (HPV) infection in men is a serious issue because it is associated with genital warts, anogenital cancers, and HPV transmission to their sex partners. This study aimed to investigate the prevalence and genotypes of HPVs in Vietnamese male patients hospitalized with sexually transmitted infection (STI) symptoms between 2016 and 2020 by using polymerase chain reaction and reverse dot blot hybridization analysis. HPV DNA was detected in 191/941 (20.3%) penile cell samples. The HPV patient's mean age was 30.3 in the range of 16- and 69-year-old. The highest HPV prevalence (84.7%) was found in patients between 20- and 39-year-old. A total of 313 HPV genotypes were identified. The multiple-infection rate was 42.9%. The most common high-risk (HR)-HPV genotypes were HPV-16 (8.0%), HPV-51 (7.7%), HPV-52 (4.8%), HPV-56 (4.2%), and HPV-18 (3.8%). Furthermore, HPV-11 and HPV-6 genotypes were the two most common low-risk (LR)-HPV genotypes with the rate of 36.7% and 21.4%, respectively. Notably, HPV-52 was found circulating in Vietnam for the first time. In conclusion, this study results showed that HPV prevalence in Vietnamese male patients was common and diverse. In addition, regarding public health and cancer prevention, the inclusion of the HPV vaccination into the national vaccination program for both men and women is recommended.

Overexpression of chalcone isomerase A gene in Astragalus trigonus for stimulating apigenin

Apigenin is one of the most studied flavonoids and is widely distributed in the plant kingdom. Apigenin exerts important antioxidant, antibacterial, antifungal, antitumor activities, and anti-inflammatory effects in neurological or cardiovascular disease. Chalcone isomerase A (chiA) is an important enzyme of the flavonoid biosynthesis pathway. In order to enhance the apigenin production, the petunia chi A gene was transformed for Astragalus trigonus. Bialaphos survived plants were screened by PCR, dot blot hybridization and RT-PCR analysis. Also, jasmonic acid, salicylic acid, chitosan and yeast extract were tested to evaluate their capacity to work as elicitors for apigenin. Results showed that yeast extract was the best elicitor for induction of apigenin with an increase of 3.458 and 3.9 fold of the control for calli and cell suspension culture, respectively. Transformed cell suspension showed high apigenin content with a 20.17 fold increase compared to the control and 6.88 fold more than the yeast extract treatment. While, transformed T1 calli derived expressing chiA gene produced apigenin 4.2 fold more than the yeast extract treatment. It can be concluded that the highest accumulation of apigenin was obtained with chiA transgenic cell suspension system and it can be utilized to enhancement apigenin production in Astragalus trigonus.

Identification and Molecular Characterization of a Novel Hordeivirus Associated With Yellow Mosaic Disease of Privet (Ligustrum vulgare) in Europe.

Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3′-terminal common region (CR) attached to each segment via a ∼53–71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3′-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal.

Genomic detection of waterborne enteric viruses as water quality indicators in Al-Zarqa River, Jordan.

Al-Zarqa River is the second main tributary to River Jordan after the Yarmouk River. The river flow has been modified by discharge of industrial wastewater and treated domestic water. Concerns about the occurrence of waterborne pathogenic viruses in the surface waters of Al-Zarqa River prompted the analysis of the surface water quality with respect to the presence of enteric viruses. Viruses were concentrated from a total of 33 different water environmental samples including raw sewage, effluent samples and river water collected from and around the river over a period of 11 months. Calculated recovery yields for these concentration methods ranged between 2 and 8%. Polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), nested RT-PCR and southern blotting hybridization analysis were used for the detection of hepatitis A virus, norovirus, astrovirus and human adenovirus 40/41, with the later one being detected in 21 (64%) of the samples that also showed previous positive presence for enteroviruses. To our knowledge, this is the first molecular biology report in Jordan describing the circulation of adenoviruses, which were detected more frequently than enteroviruses in sewage and water samples, and therefore, they can be used as an index for the presence of human pathogenic viruses in water environment.

Application of a dot blot hybridization assay for genotyping Streptococcus uberis from Brazilian dairy herds

Streptococcus uberis is a major cause of environmental mastitis in many regions, and it is associated with clinical and subclinical infections. Although the main source of infection is the environment, reports of strains with a contagious profile have been described. Dot blot hybridization analysis allows the rapid identification of S. uberis population structures within and between herds, and it helps to identify strain diversity as well as possible clonal lineages that directly affect the control of bovine mastitis caused by this pathogen. The aim of this study was to evaluate the diversity of S. uberis isolates obtained from clinical (n = 22) and subclinical (n = 22) cases of mastitis in dairy herds (n = 13) in Brazil over a period of 12 mo. We submitted 44 S. uberis isolates to dot blot hybridization followed by automatic data analysis. We identified 8 different hybridization patterns using genetic markers associated with virulence factors and taxonomy, indicating diversity of S. uberis within the population and suggesting environmental transmission. However, the evidence of identical dot blot patterns in different mammary quarters from the same animal also suggested local contagious transmission. Of the virulence genes evaluated, we found a high prevalence of the genes sua, pauA, and gapC, highlighting the importance of these virulence factors for the adhesion, invasion, and multiplication of S. uberis in subclinical and clinical intramammary infections.

Immunoglobulin and T Cell Receptor β Chain Gene Rearrangement Analysis of Ocular Adnexal Lymphoid Neoplasms: Clinical and Biologic Implications

We investigated the organization of the immunoglobulin heavy chain (IgH) and the T cell receptor beta chain (T beta) gene loci in 20 ocular adnexal and four extraocular lymphoid neoplasms obtained from 18 patients presenting with an ocular adnexal lymphoid neoplasm. Fifteen ocular adnexal and four extraocular lymphoid neoplasms occurring in 13 patients were classified by morphological examination and immunophenotypic analysis as monoclonal B cell lymphomas. Each one of these 19 lymphoid neoplasms exhibited clonal IgH gene rearrangements upon hybridization of EcoRI- or HindIII-digested DNA to a heavy-chain joining region (JH)-specific DNA probe. The bilateral ocular adnexal monoclonal B cell neoplasms occurring simultaneously in two individuals exhibited identical clonal IgH gene rearrangements, which indicated their derivation from an identical B cell clone. The ocular adnexal and the extraocular monoclonal B cell neoplasms occurring in two of three patients also exhibited identical clonal IgH gene rearrangements, which suggested that they too were derived from an identical B cell clone. Five ocular adnexal lymphoid neoplasms were classified by morphological examination and immunophenotypic analysis as benign, polyclonal pseudolymphomas. Three of these five ocular adnexal lymphoid neoplasms exhibited clonal IgH gene rearrangements, which suggested the presence of monoclonal B cell populations that escaped detection by morphological and immunophenotypic examination. None of the 24 pathological samples exhibited clonal T beta gene rearrangements upon hybridization of EcoRI- or BamHI-digested DNA to a T beta gene DNA probe. The results of these studies demonstrate the value of Southern blot hybridization analysis for clonal IgH and T beta gene rearrangements in the diagnosis, classification, and investigation of extranodal lymphoid neoplasms originating and/or presenting in the ocular adnexa.

Moss PPR-SMR protein PpPPR_64 influences the expression of a psaA-psaB-rps14 gene cluster and processing of the 23S-4.5S rRNA precursor in chloroplasts.

Moss PPR-SMR protein PpPPR_64 is a pTAC2 homolog but is functionally distinct from pTAC2. PpPPR_64 is required for psaA gene expression and its function may have evolved in mosses. The pentatricopeptide repeat (PPR) proteins are key regulatory factors responsible for the control of plant organellar gene expression. A small subset of PPR proteins possess a C-terminal small MutS-related (SMR) domain and have diverse roles in plant organellar biogenesis. However, the function of PPR-SMR proteins is not fully understood. Here, we report the function of PPR-SMR protein PpPPR_64 in the moss Physcomitrium patens. Phylogenetic analysis indicated that PpPPR_64 belongs to the same clade as the Arabidopsis PPR-SMR protein pTAC2. PpPPR_64 knockout (KO) mutants grew autotrophically but with reduced protonemata growth and the poor formation of photosystems' antenna complexes. Quantitative reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in transcript levels of the psaA-psaB-rps14 gene cluster but no alteration to transcript levels of most photosynthesis- and non-photosynthesis-related genes. In addition, RNA processing of 23S-4.5S rRNA precursor was impaired in the PpPPR_64 KO mutants. This suggests that PpPPR_64 is specifically involved in the expression level of the psaA-psaB-rps14 gene and in processing of the 23S-4.5S rRNA precursor. Our results indicate that PpPPR_64 is functionally distinct from pTAC2 and is a novel PPR-SMR protein required for proper chloroplast biogenesis in P. patens.

Characterization of horizontal transmission of blueberry latent spherical virus by pollen.

Nicotiana benthamiana plants became infected with blueberry latent spherical virus (BLSV) after pollination with pollen grains produced by BLSV-infected N. benthamiana plants. Interestingly, pollen grains produced by BLSV-infected Vaccinium corymbosum (blueberry), Nicotiana alata, and Petunia × hybrida (petunia) plants also transmitted the virus to healthy N. benthamiana plants after pollination. As seen using aniline blue staining and fluorescence microscopy, pollen grains from BLSV-infected blueberry, N. alata, and petunia plants germinated on stigmas of N. benthamiana, and the pollen tubes penetrated the stigmas in a manner similar to that of N. benthamiana pollen grains on N. benthamiana stigmas. Whole-mount in situ hybridization and chromogenic in situ hybridization analysis showed that infected blueberry and N. benthamiana pollen grains germinated on N. benthamiana stigmas, and virus-containing pollen tubes penetrated the stigmas. Tissue blot hybridization analysis revealed that the initial infection sites were the N. benthamiana stigmas pollinated with infected pollen grains from blueberry and N. benthamiana. In addition, the virus spread from the initial infection sites to the phloem in the stigma and style. Taken together, we suggest that penetrating pollen tubes that harbored the virus results in infection foci in the stigma, and the virus then moves to the vascular tissues in the stigma and style and eventually establishes systemic infection.

Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah, Saudi Arabia.

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.

Studies on the role of IS1216E in the formation and dissemination of poxtA-carrying plasmids in an Enterococcus faecium clade A1 isolate

To analyse the role of IS1216E in the dissemination of the phenicol-oxazolidinone-tetracycline resistance gene poxtA in an Enterococcus faecium clade A1 isolate. MICs were determined by broth microdilution. The poxtA-positive isolate was typed by MLST. The two plasmids were characterized by PCR, conjugation, S1-PFGE, Southern blot hybridization and WGS analysis. The presence of translocatable units (TUs) was examined by PCR and sequencing. Isolate E1077 contains the 217661 bp conjugative plasmid pE1077-217 and the 23710 bp mobilizable plasmid pE1077-23. pE1077-217 harbours erm(B), aac(A)-aph(D), aadE, spw, lsa(E), lnu(B), aphA3 and dfrG, whereas pE1077-23 carries a Tn6657-like transposon containing poxtA and fexB. pE1077-23 was apparently formed by an IS1216E-mediated composite transposon-plasmid fusion event, involving a replicative transposition process. Conjugation experiments showed that pE1077-23 is mobilizable by pE1077-217. Moreover, a novel 31742 bp plasmid, pT-E1077-31, was found in a transconjugant. WGS analysis indicated that pT-E1077-31 was formed by the integration of a Tn6657-derived, IS1216E-based translocatable unit, which carried fexB and poxtA, into a copy of pE1077-23. This study showed the presence of two cointegrate formation events in the formation and spread of a poxtA/fexB-carrying plasmid in E. faecium. One was the integration of a transposon into a plasmid while the other was the integration of a TU into a different site of the same type of plasmid-borne transposon from which it originated. In both events, IS1216E played a major role, suggesting that IS1216E-mediated transposition and translocation processes aid the dissemination and persistence of important antimicrobial resistance genes, such as poxtA, among enterococci.

A simple and rapid protocol for the genetic transformation of Ensete ventricosum

Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

Overexpression of MfSAMS1 Improves Chilling Tolerance in Transgenic Stylo

Stylo [Stylosanthes guianensis (Aublet) Sw.] is an important forage legume and cover crop in tropical and southern subtropical regions. Its growth and survival are largely affected by low temperature in winter in subtropical regions. It is important to improve its chilling tolerance. S‐adenosylmethionine synthetase (SAMS) is the key enzyme catalyzing the formation of S‐adenosylmethionine (SAM), a precursor of polyamines synthesis. To improve chilling tolerance in stylo, MfSAMS1 from Medicago falcata was overexpressed in stylo plants. DNA gel blot hybridization and quantitative reverse transcription PCR analysis showed that MfSAMS1 was inserted into the genomes of transgenic plants and expressed. Compared to the wild‐type, transgenic plants had enhanced SAM and spermidine levels and increased chilling tolerance with higher level of the maximum photochemical efficiency of photosystem II (Fv/Fm) and lower ion leakage after chilling treatment. Polyamine oxidase activity and H2O2 levels were increased in transgenic plants compared with the wild‐type. In addition, higher activities of superoxide dismutase and catalase were observed in transgenic plants than in the wild‐type. The results suggest that overexpression of MfSAMS1 promoted polyamine synthesis and oxidation, which in turn improved H2O2–induced antioxidant protection and protected transgenic plants against chilling stress.Core Ideas Transgenic stylo plants overexpressing MfSAMS1 were produced. Plants showed promoted polyamine synthesis and oxidation. Transgenic plants had increased chilling tolerance with higher antioxidant levels.

Optimisation of Adventitious Shoot Regeneration and Agrobacterium-mediated Transformation in Canna × generalis (Canna Lily)

Canna being ornamental plants has a significant role in agriculture, medical, economy and food industry. Canna has a limited vase life due to the rapid loss of moisture from its perianth. For improving its market value, cuticularisation of the perianth can be achieved by the expression of a heterologous cutin producing gene, using the tissue culture and transformation protocol developed in this study. Efficient, rapid and direct adventitious shoot regeneration was successfully established in Canna × generalis using recalcitrant rhizome explants. The explants were cultured on MS medium supplemented with 6-benzylaminopurine (6-BA), thidiazuron (TDZ), and kinetin. Among the four genotypes taken for tissue culture, the ‘Trinacria variegata’ was the best responding cultivar. And 2 mg · L−1 6-BA or 1.5 mg · L−1 TDZ along with 0.1 mg · L−1 IAA was optimum for their regeneration. The highest regeneration was achieved in ‘Trinacria variegata’ (36%) on 6-BA, 33% on TDZ while kinetin failed to evoke any regenerative responses. Regeneration was enhanced by supplementation of 100 mg · L−1 ascorbic acid (AsA), while, 100 mg · L−1 of l-cysteine or 100 mg · L−1 dithiothreitol (DTT), inhibited regeneration. Shoots were observed to develop 3–5 fibrous roots on MS medium supplemented with 0.5 mg · L−1 indole-3-butyric acid (IBA). The plantlets were transplanted into pots and acclimatised in glasshouse with 100% survival. For transformation of Canna, rhizome explants were co-cultivated for 60 min in Agrobacterium suspension. The explants were washed with 500 mg · L−1 cefotaxime solution, subjected to 100 mg · L−1 kanamycin selection followed by excision of the shoots and culturing them on IBA-supplemented media for root development. Transgene integration in the putative transformants was confirmed by PCR assay and copy number by Southern blot hybridisation analysis.

Overexpression of a NF-YC Gene Results in Enhanced Drought and Salt Tolerance in Transgenic Seashore Paspalum.

Seashore paspalum (Paspalum vaginatum O. Swartz) is an important warm-season turfgrass species. In this study we generated transgenic seashore paspalum overexpressing CdtNF-YC1, a nuclear factor Y transcription factor from hybrid bermudagrass (Cynodon dactylon × Cynodon transvaalensis). DNA blot hybridization and qRT-PCR analysis showed that CdtNF-YC1 was integrated into the genomes of transgenic seashore paspalum plants and expressed. Reduced relative water content (RWC) and survival rate and increased ion leakage were observed in both wild type (WT) and transgenic plants after drought stress, while transgenic plants had higher levels of RWC and survival rate and lower ion leakage than the WT. Maximal photochemical efficiency of photosystem II (Fv/Fm), chlorophyll concentration and survival rate were decreased after salt stress, while higher levels were maintained in transgenic plants than in WT. In addition, an increased Na+ content and decreased or unaltered K+ in leaves and roots were observed after salt treatment, while lower level of Na+ and higher levels of K+ and K+/ Na+ ratio were maintained in transgenic plants than in WT. The results indicated that overexpressing CdtNF-YC1 resulted in enhanced drought and salt tolerance in transgenic plants. Transcript levels of stress responsive genes including PvLEA3, PvP5CS1, PvABI2, and PvDREB1B were induced in response to drought and salt stress, and higher levels were observed in transgenic seashore paspalum than in WT. The results suggest that the enhanced drought and salt tolerance in transgenic seashore paspalum is associated with induction of a series of stress responsive genes as a result of overexpression of CdtNF-YC1.

Petroleum contamination and bioaugmentation in bacterial rhizosphere communities from Avicennia schaueriana.

Anthropogenic activity, such as accidental oil spills, are typical sources of urban mangrove pollution that may affect mangrove bacterial communities as well as their mobile genetic elements. To evaluate remediation strategies, we followed over the time the effects of a petroleum hydrocarbon degrading consortium inoculated on mangrove tree Avicennia schaueriana against artificial petroleum contamination in a phytoremediation greenhouse experiment. Interestingly, despite plant protection due to the inoculation, denaturing gradient gel electrophoresis of the bacterial 16S rRNA gene fragments amplified from the total community DNA indicated that the different treatments did not significantly affect the bacterial community composition. However, while the bacterial community was rather stable, pronounced shifts were observed in the abundance of bacteria carrying plasmids. A PCR-Southern blot hybridization analysis indicated an increase in the abundance of IncP-9 catabolic plasmids. Denaturing gradient gel electrophoresis of naphthalene dioxygenase (ndo) genes amplified from cDNA (RNA) indicated the dominance of a specific ndo gene in the inoculated petroleum amendment treatment. The petroleum hydrocarbon degrading consortium characterization indicated the prevalence of bacteria assigned to Pseudomonas spp., Comamonas spp. and Ochrobactrum spp. IncP-9 plasmids were detected for the first time in Comamonas sp. and Ochrobactrum spp., which is a novelty of this study.