Chitosan nanoparticles as a new technique in gene transformation into different plants tissues

The utilization of chitosan nanoparticles is a novel technique for gene transformation into plant tissues. It takes a few minutes to transform gene to plant. UidA gene was detected in <i>Escherichia coli</i> (K12 strain) using polymerase chain reaction analysis by UidA-specific primers. The gene was transformed into the explants of two different plant species (<i>Solanum tuberosum</i> and <i>Paulownia tomentosa</i>). These plants have different natures as crop and woody plants respectively. Therefore, they have different abilities to express the UidA gene. The gene is expressed into blue color in plant tissues due to the formation and expression of the GUS enzyme. The transformation of the UidA gene was detected morphologically by the formation of blue color; and molecular using PCR. Chitosan nanoparticles were characterized by UV/Visible spectroscope and photographing with a transmission electron microscope (TEM). As a result of this research, it is suggested that chitosan nanoparticles be used in gene transformation into plant tissues. Because it is safe, quick, and inexpensive, as well as biocompatible and biodegradable.

Stability of Transgene Inheritance in Progeny of Field-Grown Pear Trees over a 7-Year Period

Breeding woody plants is a very time-consuming process, and genetic engineering tools have been used to shorten the juvenile phase. In addition, transgenic trees for commercial cultivation can also be used in classical breeding, but the segregation of transgenes in the progeny of perennial plants, as well as the possible appearance of unintended changes, have been poorly investigated. We studied the inheritance of the uidA gene in the progeny of field-grown transgenic pear trees for 7 years and the physical and physiological parameters of transgenic seeds. A total of 13 transgenic lines were analyzed, and the uidA gene segregated 1:1 in the progeny of 9 lines and 3:1 in the progeny of 4 lines, which is consistent with Mendelian inheritance for one and two transgene loci, respectively. Rare and random deviations from the Mendelian ratio were observed only for lines with one locus. Transgenic seeds’ mass, size, and shape varied slightly, despite significant fluctuations in weather conditions during cultivation. Expression of the uidA gene in the progeny was stable. Our study showed that the transgene inheritance in the progeny of pear trees under field conditions occurs according to Mendelian ratio, does not depend on the environment, and the seed vigor of transgenic seeds does not change.

The Prevalence of Uropathogenic Escherichia coli Strains among Outpatients with Urinary Tract Infection in Zakho Hospitals-Zakho City, Duhok Province/ Iraq

This study involved the prevalence of uropathogenic Escherichia coli (UPEC) among outpatients of UTI attending three major hospitals in Zakho city. Four hundred urine samples were collected from patients of UTI of both sexes and different ages (≤ 1 year to over 50 years), during the period from July 2018 until January 2019. All urine samples were analyzed by conventional bacteriological method for the presence of Escherichia coli (E. coli), while molecular method was used for the presence of species-specific uidA gene in the isolated E. coli. Out of 400 samples, 141 (35.25%) were infected with UPEC from enrolled patients. The rate was higher in females than males (90.78% vs 9.22%), respectively. In both sexes, the age group 41-50 years in both sexes showed the highest rate (46.67%) of infection, and statistically this rate of infection was significant (p< 0.013) among both sexes and various age groups. Furthermore, in all ages, married patients showed slightly higher prevalence than un-married one (38% vs 32.5%), but this difference was statistically non-significant (p>0.05%). The rate of UTI was higher among urban inhabitants (40.56%) than others. During the months of the year, the peak (90.48%) in both sexes was during December while the lowest rates (13.64%) was during January.

Eszett (ẞ; ß)-Glucuronidase, a False Positive of Beta (β)-Glucuronidase: Focus on Plant Genetic Transformation

The Escherichia coli beta (β)-glucuronidase gene ( GUS ), coded for by the uidA gene, is a popular reporter gene in plant genetic transformation experiments. As a result of a typographic-type error, leading to confusion between Eszett (uppercase ẞ; lowercase ß), a German special character, and Greek lowercase beta (β), some published papers claimed to have used Eszett (ẞ/ß)-glucuronidase, which does not exist. Attention was paid to the 114 false positive entries, i.e., ẞ/ß-glucuronidase, that were detected on PubMed on July 6, 2021. From the 114 entries, 81 (71.1%) were in papers in the field of plant science. After screening 79 of the full texts, the error was quantified in the article’s location. The error was detected in 100% of abstracts on PubMed and also in 100% of the abstracts on the original journal/publisher websites, while 62.0% of papers had this error in the text (once or multiple times). The origin of these errors is unclear. Given that there are approximately 4000, 1100 and 10,600 hits for this false positive on sciencedirect.com, Springer Link, and Google Scholar, respectively, the quantification of this error based on PubMed suggests that a large and thorough quantitative post-publication analysis of papers claiming erroneously to have used non-existent Eszett (ẞ/ß)-glucuronidase is needed. Importantly, where possible, those errors should be corrected.

TRANSIENT EXPRESSION OF REPORTER GENES IN CULTIVARS OF Amaranthus caudatus L.

Local cultivars of A. caudatus: Helios and Karmin were used as plant material. Amaranth is a new pseudocereal introduced in Ukraine. The plant biomass of amaranth is used in medicine, food industry and cosmetology industry. Aim. The purpose of the work was to identify the optimal conditions for the transient expression of reporter genes in Amaranthus caudatus cultivars. Methods. Biochemical and microscopy methods were used in the following work. Seedlings and adult plants of different age were infiltrated with agrobacterial suspensions separately (genetic vector pCBV19 with a uidA gene and genetic vector pNMD2501 with a gfp gene in Agrobacterium tumefaciens GV3101 strain). Results. Transient expression of the uidA and gfp genes was obtained in amaranth plants after conduction series of experiments. The most intensive transient expression of gfp and uidA genes was observed in seedlings infiltrated at the age of 1 day. The maximum fluorescence of the GFP protein was observed on 5th–6th days. Conclusions. It was shown that the cultivar Helios was more susceptible to agrobacterial infection than the cultivar Karmin. The effectiveness of Agrobacterium mediated transformation was from 16% to 95% for the Helios cultivar and from 12% to 93% for the Karmin cultivar. The obtained results indicate that the studied amaranth cultivars can potentially be used for obtaining transient expression of target genes and synthesizing target proteins in their tissues in the future.

How to drive phloem gene expression? A case study with preferentially expressed citrus gene promoters

New approaches for developing disease-resistant genetically modified organisms have included specific targets for gene expression to enhance the chances for pathogen control. Gene expression driven by phloem-derived Citrus sinensis gene promoters could be evaluated and compared with the expression induced by a strong constitutive promoter in the same tissue, leading to the production of transgenic sweet oranges potentially more resistant to diseases caused by phloem-limited bacteria. ‘Carrizo’ citrange [ (Poncirus trifoliataL.) Raf. x Citrus sinensis (L.) Osbeck] was transformed, via Agrobacterium tumefaciens, with the binary vector pCAMBIA2301 bearing the uidA gene (ß-glucuronidase) driven by the CaMV35S constitutive promoter (CaMV35S::uidA) or by the CsPP2.B1 (CsPP2.B1::uidA) or by the CsVTE2 (CsVTE2::uidA) citrus promoters. In vitro regenerated shoots were grafted onto ‘Rangpur’ lime (C. limonia Osbeck). The genetic transformation was confirmed by Southern blot analyses. uidA gene expression was evaluated by RT-qPCR, and gene histolocalization controlled by these three promoters was accessed by X-GLUC treated stem sections. uidA gene expression exhibited by tissue-specific promoters was overall lower than from the constitutive promoter CaMV35; however, constructs driven by tissue-specific promoters may lead to expression in restricted tissues. CsPP2.B1 and CsVTE2 promoters can be considered adequate for the utilization in gene constructs aiming disease resistance.

Detection of Escherichia albertii in urinary and gastrointestinal infections in Kermanshah, Iran

Background Escherichia albertii (E. albertii) is a Gram-negative and facultative anaerobe bacterium. In recent years, the bacterium has been isolated from the feces of people with gastroenteritis as a pathogen that causes diarrhea. Due to insufficient information on the phenotypic and biochemical characteristics of E. albertii, it is difficult to distinguish it from other species of the Enterobacteriaceae family. This is especially prevalent in the pathotypes of Escherichia coli (E. coli). Moreover, in clinical laboratories, it is mistakenly identified as E. coli or even Hafnia alvei (H. alvei). This study was performed for the first time in Iran to identify E. albertii by PCR method from a sample of urinary and gastrointestinal infections obtained from clinical laboratories in Kermanshah, which were distinguished as E. coli. Methods In this study, 60 urinary samples and 40 fecal samples that were identified as E. coli by phenotypic and biochemical methods in clinical laboratories. The samples were re-evaluated in the first step in terms of specific phenotypic and biochemical characteristics of E. coli. Subsequently, DNA was extracted from the isolates by the phenol method. Then, two lysP and mdh genes were detected for E. albertii and the uidA gene for E. coli by PCR using specific primers pairs. Results The results obtained from phenotypic and biochemical tests indicated that all samples were consistent with E. coli characteristics. However, findings from PCR showed that out of a total of 100 samples, specific genes of E. coli were identified in 6 samples (6%) and uidA gene in 94 remaining samples (94%). Of these 6 samples, 5 samples were urinary tract infections, and only one was a gastrointestinal infection. Conclusion The findings of this study show that E. albertii can be considered as one of the causes of urinary and gastrointestinal infections that are mistakenly identified in clinical laboratories as E. coli.Therefore, the use of molecular methods for accurate and definitive diagnosis of bacteria can be useful.

Molecular Characterization of Escherichia Coli Isolates from Food Animals

The present study was carried out for isolation and molecular characterization of Escherichia coli from faecal and meat samples of food animals viz. cattle, pigs and poultry. A total of 66 E. coli isolates were recovered from 420 samples of different food animals and further confirmed by PCR targeting E. coli specific uidA gene. These isolates were sensitive to chloramphenicol and ciprofloxacin and resistant to ampicillin and cloxacillin. Out of 66 isolates, 42 were typed into 13 different ‘O’ serogroups, 13 untypable and remaining 11 were identified as rough. Serogroup O84, O101, O118, O120 and O147 were predominant and serogroup O118 was found to be common in the samples of all 3 species of food animals. Five (7.57%) and 3 (4.54%) of E. coli isolates were found to harbor virulent genes, stx2 and est, respectively. Twenty representative E. coli isolates selected randomly from 20 different locations were subjected to molecular typing by PCR targeting Repetitive Extragenic Palindromic (REP) sequences. The region specific molecular types of E. coli could not be detected by using REP-PCR based discrimination.

The Rooting of Stem Cuttings and the Stability of uidA Gene Expression in Generative and Vegetative Progeny of Transgenic Pear Rootstock in the Field

Adventitious rooting plays an important role in the commercial vegetative propagation of trees. Adventitious root formation is a complex biological process, but knowledge of the possible unintended effects induced by both the integration/expression of transgenes and in vitro conditions on the rooting is limited. The long-term stability of transgene expression is important both for original transformants of woody plants and its progeny. In this study, we used field-grown pear rootstock GP217 trees transformed with the reporter ß-glucuronidase (uidA) genes with and without intron and re-transformed with the herbicide resistance bar gene as model systems. We assessed the unintended effects on rooting of pear semi-hardwood cuttings and evaluated the stability of transgene expression in progeny produced by generative (seedlings) and vegetative (grafting, cutting) means up to four years. Our investigation revealed that: (1) The single and repeated transformations of clonal pear rootstocks did not result in unintended effects on adventitious root formation in cuttings; (2) stability of the transgene expression was confirmed on both generative and vegetative progeny, and no silenced transgenic plants were detected; (3) yearly variation in the gene expressions was observed and expression levels were decreased in extremely hot and dry summer; (4) the intron enhanced the expression of uidA gene in pear plants approximately two-fold compared to gene without intron. The current study provides useful information on transgene expression in progeny of fruit trees under natural environmental conditions.