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.

Comparison of a Biosensor and a Commercial UV-Visible Method for Measuring Hexavalent Chromium in Liquid Medium

The objective of the research was to contrast two methods for the quantification of hexavalent chromium. The first method is the biosensor that from the gene transformation of the cells of Escherichia coli, was incorporated by electroporation the plasmid pTOP Blunt V2, synthesized with luxA genes that provides luminescence through the catalytic activity of the luciferase top and chr genes that give the bacteria resistance to chromium. The second method is the application of the UV-visible colorimetric technique. Chromium was analysed at different concentrations, from 0.05 mg l−1 (maximum allowable limit for human consumption); 0.1 mg l−1; 0.2 mg l−1; 0.4 mg l−1; 0.8 mg l−1 and 1 mg l−1 with 5 replicates, subsequent to this, the two methods of chromium analysis were applied in river samples, thus obtaining that the biosensor in concentrations of 2x106 CFU of E. coli, has a margin of error of 1.4%, a result derived from the coefficient of determination of the absorbance of chromium, unlike the UV-visible method with the colorimetric equipment, which presented a reading error of 3.9%.

Gene Transformation by Chitosan Nanoparticle to Enhance Fatty Acid Production in Zea mays (L.)

Zea mays is an important crop and an essential source of fatty acids. Hence, increasing and adding new fatty acids led to the enhancement of these properties. Transformation of external Acetyl-CoA gene (Aco) can enhance fatty acid components, as ACo is expressed into Acetyl-CoA carboxylase (ACCase) enzyme, which is the first essential step in the fatty acid production process. Chitosan nanoparticles are safe and fast polymer nanoparticles that are applied for gene transformation. Conventional PCR was performed for the detection of the ACo gene in both transgenic and nontransgenic maize lines. The results confirm the presence of the gene in the transgenic lines and absence in non-transgenic lines. The Gas chromatography-mass spectrometry (GC-MS) analysis for fatty acid contents in transgenic and non-transgenic maize lines showed an increase in fatty acid contents in transgenic lines compared to non-transgenic ones. Besides, the transgenic maize’s lines produced extra new fatty acids not found in non-transgenic ones.

Comparative genomics of three Colletotrichum scovillei strains and genetic analysis revealed genes involved in fungal growth and virulence on chili pepper

Colletotrichum scovillei is a virulent pathogen and the dominant species causing anthracnose of chili pepper in many Asian countries. Three strains of this pathogen, Coll-524, Coll-153 and Coll-365, show varied virulence on chili pepper fruit. Among the three strains, Coll-365 showed significant defects in growth and virulence. To decipher the genetic variations among these strains and identify genes contributing to growth and virulence, in this study, comparative genomic analysis and gene transformation to verify gene function were applied. The genomes of the three strains were sequenced and Coll-524 had 1.3% and 1.5% more genes than Coll-153 and Coll-365, respectively. Compared to Coll-524 and Coll-153, Coll-365 had numerous gene losses including 33 effector genes that are distributed in different scaffolds and a cluster of 14 genes in a 34-kb genomic fragment. Through gene transformation, three genes in the 34-kb fragment were identified to have functions in growth and/or virulence of C. scovillei. Gene 15019 encoding a protein related to phospholipase A2-activating protein enhanced the growth of Coll-365. A combination of 15019 with one transcription factor gene 15022 and one C6 zinc finger domain-containing protein gene 15029 was found to enhance the pathogenicity of Coll-365. Introduction of gene 15215, which encodes a LysM domain-containing protein, into Coll-365 caused a reduction in the germination rate of Coll-365. In conclusion, the higher virulent strain Coll-524 had more genes and encoded more pathogenicity related proteins and transposable elements than the other two strains, which may contribute to the high virulence of Coll-524. In addition, the absence of the 34-kb fragment plays a critical role in the defects of growth and virulence of strain Coll-365.

Antibiotic Resistance Gene Transformation and Ultrastructural Alterations of Lettuce (Lactuca sativa L.) Resulting from Sulfadiazine Accumulation in Culture Solution

The research herein explored the possible mechanism of toxicity of the antibiotic sulfadiazine (SD) and the related antibiotic resistance gene transformation in lettuce by systematically investigating its growth responses, ultrastructural changes, and antibiotic resistance gene transformation via solution culture experiments. The results showed that SD mainly accumulated in the roots of lettuce at concentrations ranging from 6.48 to 120.87 μg/kg, which were significantly higher than those in leaves (3.90 to 16.74 μg/kg). Lower concentrations of SD (0.5 and 2.0 mg/L) in the culture nutrient solution exerted little effect on lettuce growth, while at SD concentrations higher than 10 mg/L, the growth of lettuce was significantly inhibited, manifesting as shorter root length and lower dry matter yield of whole lettuce plants. Compared with that for the control group, the absolute abundance of bacteria in the root endophyte, rhizosphere, and phyllosphere communities under different concentrations of SD treatment decreased significantly. sul1 and sul2 mainly accumulated in the root endophyte community, at levels significantly higher than those in the leaf endophyte community. Studies of electrolyte leakage and ultrastructural characteristics of root and leaf cells indicated that lettuce grown in culture solutions with high SD concentrations suffered severe damage and disintegration of the cell walls of organs, especially chloroplasts, in leaves. Furthermore, the possible mechanism of SD toxicity in lettuce was confirmed to start with the roots, followed by a free flow of SD into the leaves to destroy the chloroplasts in the leaf cells, which ultimately reduced photosynthesis and decreased plant growth. Studies have shown that antibiotic residues have negative effects on the growth of lettuce and highlight a potential risk of the development and spread of antibiotic resistance in vegetable endophyte systems.

Modeling Agrobacterium-Mediated Gene Transformation of Tobacco (Nicotiana tabacum)—A Model Plant for Gene Transformation Studies

The multilayer perceptron (MLP) topology of an artificial neural network (ANN) was applied to create two predictor models in Agrobacterium-mediated gene transformation of tobacco. Agrobacterium-mediated transformation parameters, including Agrobacterium strain, Agrobacterium cell density, acetosyringone concentration, and inoculation duration, were assigned as inputs for ANN–MLP, and their effects on the percentage of putative and PCR-verified transgenic plants were investigated. The best ANN models for predicting the percentage of putative and PCR-verified transgenic plants were selected based on basic network quality statistics. Ex-post error calculations of the relative approximation error (RAE), the mean absolute error (MAE), the root mean square error (RMS), and the mean absolute percentage error (MAPE) demonstrated the prediction quality of the developed models when compared to stepwise multiple regression. Moreover, significant correlations between the ANN-predicted and the actual values of the percentage of putative transgenes (R2 = 0.956) and the percentage of PCR-verified transgenic plants (R2 = 0.671) indicate the superiority of the established ANN models over the classical stepwise multiple regression in predicting the percentage of putative (R2 = 0.313) and PCR-verified (R2= 0.213) transgenic plants. The best combination of the multiple inputs analyzed in this investigation, to achieve maximum actual and predicted transgenic plants, was at OD600 = 0.8 for the LB4404 strain of Agrobacterium × 300 μmol/L acetosyringone × 20 min immersion time. According to the sensitivity analysis of ANN models, the Agrobacterium strain was the most important influential parameter in Agrobacterium-mediated transformation of tobacco. The prediction efficiency of the developed model was confirmed by the data series of Agrobacterium-mediated transformation of an important medicinal plant with low transformation efficiency. The results of this study are pivotal to model and predict the transformation of other important Agrobacterium-recalcitrant plant genotypes and to increase the transformation efficiency by identifying critical parameters. This approach can substantially reduce the time and cost required to optimize multi-factorial Agrobacterium-mediated transformation strategies.

Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis

For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.

Metabolic Analysis Reveals Cry1C Gene Transformation Does Not Affect the Sensitivity of Rice to Rice Dwarf Virus

Metabolomics is beginning to be used for assessing unintended changes in genetically modified (GM) crops. To investigate whether Cry1C gene transformation would induce metabolic changes in rice plants, and whether the metabolic changes would pose potential risks when Cry1C rice plants are exposed to rice dwarf virus (RDV), the metabolic profiles of Cry1C rice T1C-19 and its non-Bt parental rice MH63 under RDV-free and RDV-infected status were analyzed using gas chromatography–mass spectrometry (GC-MS). Compared to MH63 rice, slice difference was detected in T1C-19 under RDV-free conditions (less than 3%), while much more metabolites showed significant response to RDV infection in T1C-19 (15.6%) and in MH63 (5.0%). Pathway analysis showed biosynthesis of lysine, valine, leucine, and isoleucine may be affected by RDV infection in T1C-19. No significant difference in the contents of free amino acids (AAs) was found between T1C-19 and MH63 rice, and the free AA contents of the two rice plants showed similar responses to RDV infection. Furthermore, no significant differences of the RDV infection rates between T1C-19 and MH63 were detected. Our results showed the Cry1C gene transformation did not affect the sensitivity of rice to RDV, indicating Cry1C rice would not aggravate the epidemic and dispersal of RDV.