AGROBACTERIUM-MEDIATED TRANSFORMATION OF TWO TOMATO CULTIVARS (LYCOPERSICON ESCULENTUM MILL.) CV. SANDRA AND ROCKY

An efficient protocol for Agrobacterium-mediated transformation of tomato cultivars Sandra and Rocky was conducted to examine the possibility of producing transgenic tomato plants cultivars harbouring the nptII gene, conferring kanamycin resistance. To achieve this aim, tomato cotyledon explants were transformed using EHA105 Agrobacterium tumefaciens strain harboring the binary vectors pBI121 which contains Gus gene, and neomycin phosphotransferase II (nptII) as selectable marker gene under the control of a CaMV35S promoter and nopaline synthase (nos) Terminator. Transformant detection was carried out in three distinct ways. First antibiotic selection, Kanamycin at a concentration of100 mgl-1 found to be efficient for this purpose. Second histochemical GUS assay revealed the presence of blue colored zones in a number of shoots and leaves for both in vitro and the greenhouse-grown transgenic plants. Third PCR analysis indicated positive result by showing the fragment for nptII gene in tested transformants, while was absent in non-transgenic control (wild type). On the other hand, the results showed that Sandra cultivar was more efficient for regeneration and subsequently transformation frequency than Rocky cultivar, which record 26.66% of transformation frequency compared with 11.57% in Rocky cultivar.

Novel approaches to circumvent the devastating effects of pests on sugarcane

Sugarcane (Saccharum officinarum L.) is a cash crop grown commercially for its higher amounts of sucrose, stored within the mature internodes of the stem. Numerous studies have been done for the resistance development against biotic and abiotic stresses to save the sucrose yields. Quality and yield of sugarcane production is always threatened by the damages of cane borers and weeds. In current study two problems were better addressed through the genetic modification of sugarcane for provision of resistance against insects and weedicide via the expression of two modified cane borer resistant CEMB-Cry1Ac (1.8 kb), CEMB-Cry2A (1.9 kb) and one glyphosate tolerant CEMB-GTGene (1.4 kb) genes, driven by maize Ubiquitin Promoter and nos terminator. Insect Bio-toxicity assays were carried out for the assessment of Cry proteins through mortality percent of shoot borer Chilo infuscatellus at 2nd instar larvae stage. During V0, V1 and V2 generations young leaves from the transgenic sugarcane plants were collected at plant age of 20, 40, 60, 80 days and fed to the Chilo infuscatellus larvae. Up to 100% mortality of Chilo infuscatellus from 80 days old transgenic plants of V2 generation indicated that these transgenic plants were highly resistant against shoot borer and the gene expression level is sufficient to provide complete resistance against target pests. Glyphosate spray assay was carried out for complete removal of weeds. In V1-generation, 70–76% transgenic sugarcane plants were found tolerant against glyphosate spray (3000 mL/ha) under field conditions. While in V2-generation, the replicates of five selected lines 4L/2, 5L/5, 6L/5, L8/4, and L9/6 were found 100% tolerant against 3000 mL/ha glyphosate spray. It is evident from current study that CEMB-GTGene, CEMB-Cry1Ac and CEMB-Cry2A genes expression in sugarcane variety CPF-246 showed an efficient resistance against cane borers (Chilo infuscatellus) and was also highly tolerant against glyphosate spray. The selected transgenic sugarcane lines showed sustainable resistance against cane borer and glyphosate spray can be further exploited at farmer’s field level after fulfilling the biosafety requirements to boost the sugarcane production in the country.

Identification of regulatory sequences of the 35S promoter and NOS terminator in agricultural products

The pace of scientific and technological progress is steadily growing every day, in this regard much attention is paid to the problems of using genetically modified organisms (GMOs) in agricultural products, since there are risks of negative impact on human health and the environment. Thus, there is a need for constant monitoring of all agricultural products for the content of GMO regulatory sequences. This work is devoted to studying the frequency of occurrence of GMO fragments in the products of agricultural enterprises of the Republic of Tatarstan. The material describes the monitoring of agricultural products for the presence of genetic modification: the 35S promoter of the cauliflower mosaic virus and the NOS terminator – Agrobacterium tumefaciens. Diagnostics was based on real-time polymerase chain reaction using commercial PCR kits. Laboratory work consisted in sample preparation of agricultural products, DNA extraction from received samples, and amplification of genetic material. 1142 samples of agricultural products were analyzed. Based on the studies carried out, the content of the 35S promoter and the NOS terminator was revealed in 18 samples. In most cases, the content of genetic insertions was found in combined and extruded feeds for pigs, poultry, and cattle. This once again increases the relevance of the ongoing research, despite the numerous experiments and scientific discoveries carried out to study GMOs. The results of this study are the reason for further more detailed study of the creation, influence and application of GMOs both in the agricultural sectors and in ecology and molecular biology.

Construct-Specific Loop-Mediated Isothermal Amplification: Rapid Detection of Genetically Modified Crops with Insect Resistance or Herbicide Tolerance

Background Insect resistant and herbicide tolerant genetically modified (GM) events have been approved in many countries. Screening methods could facilitate preliminary testing to check the GM status, which may target control elements, transgenes, and marker genes or construct regions. Among these, methods targeting the construct region, i.e., the junction between two genetic elements of a transgenic cassette are more specific. Objective Loop-mediated isothermal amplification (LAMP) assays targeting three construct regions were developed; between Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac), cry2Ab2 gene and nos terminator (cry2Ab2-tnos), and cp4-epsps gene and nos terminator (cp4epsps-tnos). Method LAMP assays were performed by incubation at constant temperatures for selected targets. Positive amplification was detected as a change in color from orange to green on addition of SYBR® Green dye in visual LAMP and as real-time amplification curves in real-time LAMP. Results These assays showed acceptable specificity and sensitivity. Visual LAMP was found to be sensitive enough to detect as low as 0.005%, equivalent to two target copies. Real-time LAMP assays were able to detect as low as four copies of the target within 40 min, making them suitable for rapid on-site testing for GM organisms (GMO). Practical utility was also verified using spiked test samples. Conclusions These assays could be employed to address some of the biosafety or post-release monitoring issues, as well as to check for approved and unapproved GM events in a country. Highlights LAMP assays targeting three construct regions have been developed, enabling screening for approved or unapproved GMO.

The transcription and export complex THO/TREX contributes to transcription termination in plants

Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3’UTR extensions in several plant genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination.Author summaryProduction of messenger RNAs (mRNAs) involves numerous steps including initiation of transcription, elongation, splicing, termination, as well as export out of the nucleus. All these steps are highly coordinated and failure in any steps has a profound impact on the level and identity of mRNAs produced. The THO/TREX protein complex is associated with nascent RNAs and contributes to several mRNA biogenesis steps, including splicing and export. However, the contribution of the THO/TREX complex to mRNA termination was poorly defined. We have identified a role for two components of the THO/TREX complex, namely the proteins TEX1 and HPR1, in the control of transcription termination in the plant Arabidopsis thaliana. We show that the tex1 and hpr1 mutants have defects in terminating mRNA at the nopaline synthase (NOS) terminator found in T-DNA insertion mutants leading to the transcriptional read-through pass the NOS terminator. We also show that tex1 and hpr1 mutants have defects in mRNA termination at several endogenous genes, leading to the production of 3’UTR extensions. Together, these results highlight a role for the THO/TREX complex in mRNA termination.

Evaluation of a Real Time PCR Assay Method for the Detection of Genetically Modified Organisms in Food Products

The objective of the study was to determine qualitatively by validated Real Time PCR method the occurrence of genetically modified maize and soybean in commercial food products from the Greek market. 70 independent samples were collected, including products from different categories (i.e. cereal based, biscuits and snacks) which declared either corn or soybean on the labelling. The result of the study indicated that 37.1% of maize and soy products (n=70) displayed in the Greek market have detectable levels of genetically modified maize or soy. These products were identified by specific primers and included common GMΟ detection primers for 35S and NOS terminator. Adequate repeatability and reproducibility was demonstrated for the applied Real Time PCR method, as evaluated by intra- and inter-laboratory tests.

A simple and accurate PCR method for detection of genetically modified rice

Background Legislation regulating for labeling and use of genetically modified (GM) crops are increased considerably worldwide in order to health and safety assurance of consumers. For this purpose, a polymerase chain reaction (PCR) method has been developed for detection of GM rice in people’s food diet. Methods In this study, eighty-one non-labeled rice samples were collected randomly from different market sites of Tehran, Iran. In order to analysis, rice genomic DNA was extracted using MBST DNA extraction kit and subsequently, sucrose phosphate synthase (SPS) gene was used to confirm the quality of extracted DNA. Then, cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium nopaline synthase (NOS) terminator were selected as screening targets for detection of GM rice sequences by PCR. Results According to our results, 2 out of 81 (2.4%) samples tested were positive for CaMV 35S promoter while no positive result was detected for NOS terminator. Conclusion The obtained data indicated that this method is capable to identify the GM rice varieties. Furthermore, it can demonstrate the possibility of the presence of GM rice in Tehran’s market, thus putting emphasis on the requirement for developing a precise approach to evaluate this product.

Highly Sensitive GMO Detection Using Real-Time PCR with a Large Amount of DNA Template: Single-Laboratory Validation

Current genetically modified organism (GMO) detection methods allow for sensitive detection. However, a further increase in sensitivity will enable more efficient testing for large grain samples and reliable testing for processed foods. In this study, we investigated real-time PCR-based GMO detection methods using a large amount of DNA template. We selected target sequences that are commonly introduced into many kinds of GM crops, i.e., 35S promoter and nopaline synthase (NOS) terminator. This makes the newly developed method applicable to a wide range of GMOs, including some unauthorized ones. The estimated LOD of the new method was 0.005% of GM maize events; to the best of our knowledge, this method is the most sensitive among the GM maize detection methods for which the LOD was evaluated in terms of GMO content. A 10-fold increase in the DNA amount as compared with the amount used under common testing conditions gave an approximately 10-fold reduction in the LOD without PCR inhibition. Our method is applicable to various analytical samples, including processed foods. The use of other primers and fluorescence probes would permit highly sensitive detection of various recombinant DNA sequences besides the 35S promoter and NOS terminator.