Approach strategies and application of metabolomics to biotechnology in plants.

Metabolomics refers to the technology for the comprehensive analysis of metabolites and low-molecular-weight compounds in a biological system, such as cells or tissues. Metabolites play an important role in biological phenomena through their direct involvement in the regulation of physiological mechanisms, such as maintaining cell homeostasis or signal transmission through protein-protein interactions. The current review aims provide a framework for how the integrated analysis of metabolites, their functional actions and inherent biological information can be used to understand biological phenomena related to the regulation of metabolites and how this information can be applied to safety assessments of crops created using biotechnology. Advancement in technology and analytical instrumentation have led new ways to examine the convergence between biology and chemistry, which has yielded a deeper understanding of complex biological phenomena. Metabolomics can be utilized and applied to safety assessments of biotechnology products through a systematic approach using metabolite-level data processing algorithms, statistical techniques, and database development. The integration of metabolomics data with sequencing data is a key step towards improving additional phenotypical evidence to elucidate the degree of environmental affects for variants found in genome associated with metabolic processes. Moreover, information analysis technology such as big data, machine learning, and IT investment must be introduced to establish a system for data extraction, selection, and metabolomic data analysis for the interpretation of biological implications of biotechnology innovations. This review outlines the integrity of metabolomics assessments in determining the consequences of genetic engineering and biotechnology in plants.

Influence of Genetically Modified Soybean Expressing Epidermal Growth Factor on Arthropod Biodiversity.

The field study was undertaken to examine the potential for adverse effects of transgenic soybean expressing bioactive human epidermal growth factor (with tolerance to the herbicide glufosinate, PPT) on the abundance and diversity of plant-dwelling arthropods by comparing with those of a non-GM parental cultivar, Gwangan soybean. Field surveys of soybean fields were carried out over two consecutive years, 2016 and 2017 at Ochang and Jeonju, Korea. The number of captured individuals associated with either of EGF and Gwangan soybean plants increased in 2017 compared with 2016 in both Ochang and Jeonju. During the survey period, the diversity and richness of the occurred insects and arachnids increased, dominance decreased, and the evenness of the insects remained static. The insects of Hymenoptera Order occurred most often comprised 25.4% of total captured insect pests. On the contrary, natural enemy from Hymenoptera Order and other insects from Diptera Order occurred more frequently (29.9% and 19.0%, respectively) in both the survey regions during the study periods. The score from PROXSCAL multidimensional scaling using combined data showed that the occurrence of insects and arachnids were separated due to their cultivation regions and years, irrespective of soybean cultivars. Consequently, the results indicated that there happened no notable change in the composition of arthropod communities in soybean agroecosystem due to GM event in soybean expressing EGF.

Interspecific Hybridization of Transgenic Brassica napus and Brassica rapa-An Overview.

In nature, interspecific hybridization occurs frequently and can contribute to the production of new species or the introgression of beneficial adaptive features between species. It has great potential in agricultural systems to boost the process of targeted crop improvement. In the advent of genetically modified (GM) crops, it has a disadvantage that it involves the transgene escaping to unintended plants, which could result in non-specific weedy crops. Several crop species in the Brassica genus have close kinship: canola (Brassica napus) is an ancestral hybrid of B. rapa and B. oleracea and mustard species such as B. juncea, B. carinata, and B. nigra share common genomes. Hence, intraspecific hybridization among the Brassica species is most common, especially between B. napus and B. rapa. In general, interspecific hybrids cause numerous genetic and phenotypic changes in the parental lines. Consequently, their fitness and reproductive ability are also highly varied. In this review, we discuss the interspecific hybridization and reciprocal hybridization studies of B. napus and B. rapa and their potential in the controlled environment. Further, we address the fate of transgenes (herbicide resistance) and their ability to transfer to their progenies or generations. This could help us to understand the environmental influence of interspecific hybrids and how to effectively manage their transgene escape in the future.

A Review of the Unintentional Release of Feral Genetically Modified Rapeseed into the Environment.

Globally, the cultivation area of genetically modified (GM) crops is increasing dramatically. Despite their well-known benefits, they may also pose many risks to agriculture and the environment. Among the various GM crops, GM rapeseed (Brassica napus L.) is widely cultivated, mainly for oil production. At the same time, B. napus possesses a number of characteristics, including the ability to form feral populations and act as small-seeded weeds, and has a high potential for hybridization with other species. In this review, we provide an overview of the commercialization, approval status, and cultivation of GM rapeseed, as well as the status of the feral rapeseed populations. In addition, we highlight the case studies on the unintentional environmental release of GM rapeseed during transportation in several countries. Previous studies suggest that the main reason for the unintentional release is seed spillage during transport/importing of rapeseed in both GM rapeseed-cultivating and -non-cultivating countries. Despite the fact that incidents of unintentional release have been recorded often, there have been no reports of serious detrimental consequences. However, since rapeseed has a high potential for hybridization, the possibilities of gene flow within the genus, especially with B. rapa, are relatively significant, and considering their weedy properties, effective management methods are needed. Hence, we recommend that specific programs be used for the effective monitoring of environmental releases of GM rapeseed as well as management to avoid environmental and agricultural perturbations.

An Overview of Near Infrared Spectroscopy and Its Applications in the Detection of Genetically Modified Organisms.

Near-infrared spectroscopy (NIRS) has become a more popular approach for quantitative and qualitative analysis of feeds, foods and medicine in conjunction with an arsenal of chemometric tools. This was the foundation for the increased importance of NIRS in other fields, like genetics and transgenic monitoring. A considerable number of studies have utilized NIRS for the effective identification and discrimination of plants and foods, especially for the identification of genetically modified crops. Few previous reviews have elaborated on the applications of NIRS in agriculture and food, but there is no comprehensive review that compares the use of NIRS in the detection of genetically modified organisms (GMOs). This is particularly important because, in comparison to previous technologies such as PCR and ELISA, NIRS offers several advantages, such as speed (eliminating time-consuming procedures), non-destructive/non-invasive analysis, and is inexpensive in terms of cost and maintenance. More importantly, this technique has the potential to measure multiple quality components in GMOs with reliable accuracy. In this review, we brief about the fundamentals and versatile applications of NIRS for the effective identification of GMOs in the agricultural and food systems.

Compositional equivalence assessment of insect-resistant genetically modified rice using multiple statistical analyses.

The safety of transgenic Bt rice containing bacteria-derived mCry1Ac gene from Bacillus thuringiensis (Bt) was assessed by conducting field trials at two locations for two consecutive years in South Korea, using the near-isogenic line comparator rice cultivar ('Ilmi', non-Bt rice) and four commercial cultivars as references. Compositional analyses included measurement of proximates, minerals, amino acids, fatty acids, vitamins, and antinutrients. Significant differences between Bt rice and non-Bt rice were detected; however, all differences were within the reference range. The statistical analyses, including analysis of % variability, analysis of similarities (ANOISM), similarity percentage (SIMPER) analysis, and permutational multivariate analysis of variance (PERMANOVA) were performed to study factors contributing to compositional variability. The multivariate analyses revealed that environmental factors more influenced rice components' variability than by genetic factors. This approach was shown to be a powerful method to provide meaningful evaluations between Bt rice and its comparators. In this study, Bt rice was proved to be compositionally equivalent to conventional rice varieties through multiple statistical methods.

Comparison of the seed nutritional composition between conventional varieties and transgenic soybean overexpressing Physaria FAD3-1.

BACKGROUND: PfFAD3 transgenic soybean expressing omega-3 fatty acid desaturase 3 of Physaria produces increased level of α-linolenic acid in seed. Composition data of non-transgenic conventional varieties is important in the safety assessment of the genetically-modified (GM) crops in the context of the natural variation. RESULTS: The natural variation was characterized in seed composition of 13 Korean soybean varieties grown in three locations in South Korea for 2 years. Univariate analysis of combined data showed significant differences by variety and cultivation environment for proximates, minerals, anti-nutrients, and fatty acids. Percent variability analysis demonstrated that genotype, environment and the interaction of environment with genotype contributed to soybean seed compositions. Principal component analysis and orthogonal projections to latent structure discriminant analysis indicated that significant variance in compositions was attributable to location and cultivation year. The composition of three PfFAD3 soybean lines for proximates, minerals, anti-nutrients, and fatty acids was compared to a non-transgenic commercial comparator (Kwangankong, KA), and three non-transgenic commercial varieties grown at two sites in South Korea. Only linoleic and linolenic acids significantly differed in PfFAD3-1 lines compared to KA, which were expected changes by the introduction of the PfFAD3-1 trait in KA. CONCLUSION: Genotype, environment, and the interaction of environment with genotype contributed to compositional variability in soybean. PfFAD3-1 soybean is equivalent to the conventional varieties with respect to these components. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Discrimination of Transgenic Canola (Brassica napus L.) and their Hybrids with B. rapa using Vis-NIR Spectroscopy and Machine Learning Methods.

In recent years, the rapid development of genetically modified (GM) technology has raised concerns about the safety of GM crops and foods for human health and the ecological environment. Gene flow from GM crops to other crops, especially in the Brassicaceae family, might pose a threat to the environment due to their weediness. Hence, finding reliable, quick, and low-cost methods to detect and monitor the presence of GM crops and crop products is important. In this study, we used visible near-infrared (Vis-NIR) spectroscopy for the effective discrimination of GM and non-GM Brassica napus, B. rapa, and F1 hybrids (B. rapa X GM B. napus). Initially, Vis-NIR spectra were collected from the plants, and the spectra were preprocessed. A combination of different preprocessing methods (four methods) and various modeling approaches (eight methods) was used for effective discrimination. Among the different combinations, the Savitzky-Golay and Support Vector Machine combination was found to be an optimal model in the discrimination of GM, non-GM, and hybrid plants with the highest accuracy rate (100%). The use of a Convolutional Neural Network with Normalization resulted in 98.9%. The same higher accuracy was found in the use of Gradient Boosted Trees and Fast Large Margin approaches. Later, phenolic acid concentration among the different plants was assessed using GC-MS analysis. Partial least squares regression analysis of Vis-NIR spectra and biochemical characteristics showed significant correlations in their respective changes. The results showed that handheld Vis-NIR spectroscopy combined with chemometric analyses could be used for the effective discrimination of GM and non-GM B. napus, B. rapa, and F1 hybrids. Biochemical composition analysis can also be combined with the Vis-NIR spectra for efficient discrimination.

Comparative evaluation of nutritional compositions between transgenic rice harboring the CaMsrB2 gene and the conventional counterpart.

As a part of a safety assessment of new transgenic crops, compositional equivalence studies between transgenic crops with non-transgenic comparators are almost universally required. This study was conducted to compare nutritional profiles of proximate composition, and fatty acid, amino acid, mineral, and vitamin contents, and anti-nutrients, between transgenic drought-tolerant Agb0103 rice harboring the pepper methionine sulfoxide reductase B2 gene CaMsrB2 and the parental rice cultivar, 'Ilmi' as a non-transgenic control. Both transgenic and non-transgenic rice were grown and harvested in 2 different locations. Proximate compositions of moisture, starch, protein, lipid, and ash content of Agb0103 rice were similar to parental non-transgenic rice. There were no differences between transgenic and non-transgenic rice with respect to the whole nutritional composition, except for minor locality differences for a few nutritional components. Agb0103 rice with improved resistance to drought is nutritionally equivalent to the parental rice cultivar.

The novel, actin-like protein Tact3 is expressed in rodent testicular haploid germ cells.

Mouse testis actin-like proteins 1 and 2 (mTact1 and mTact2), which are expressed in murine haploid germ cells, have been described previously. Here, we report the cloning and characterization of a third actin-like protein from rat, rat testis actin-like protein 3 (rTact3). The complete cDNA of the rTact3 gene was approximately 3.7 kb in length, and its corresponding amino acid sequence consisted of 1219 amino acids. The rTact3 gene lacks introns, similar to mTact1 and mTact2. The 356 C-terminal amino acids of rTact3 showed 43% homology with mTact1, whereas the 863 N-terminal amino acids did not show any significant homology. Northern blot analysis revealed that rTact3 mRNA was expressed only in adult rat testes and not during the prepubescent stage. In situ hybridization revealed that rTact3 was expressed exclusively during round and elongated spermatids maturation stages in rat testes. Immunohistochemical experiments using antibodies raised against a synthetic peptide showed that the expression of the rTact3 protein was also restricted in round and elongated spermatids, specifically in the head and acrosome of mature rat sperm. The 5'-flanking region of the mTact3 gene was found to contain a TATA-box motif as well as two putative CREB/c-Jun and five C/EBP motifs. mTact3 promoter activity was enhanced in a dose-dependent manner by the transfection of CREB, c-Jun, or C/EBP in NIH3T3 cells. These results suggest that Tact3 proteins might play an important role in rodent germ-cell development.

Expression of PAT and NPT II proteins during the developmental stages of a genetically modified pepper developed in Korea.

Estimation of the protein levels introduced in a biotechnology-derived product is conducted as part of an overall safety assessment. An enzyme-linked immunosorbent assay (ELISA) was used to analyze phosphinothricin acetyltransferase (PAT) and neomycin phosphotransferase II (NPT II) protein expression in a genetically modified (GM) pepper plant developed in Korea. PAT and NPT II expression levels, based on both dry weight and fresh weight, were variable among different plant generations and plant sections from isolated genetically modified organism (GMO) fields at four developmental stages. PAT expression was highest in leaves at anthesis (11.44 µg/gdw and 2.17 µg/gfw) and lowest in roots (0.12 µg/gdw and 0.01 µg/gfw). NPT II expression was also highest in leaves at anthesis (17.31 µg/gdw and 3.41 µg/gfw) and lowest in red pepper (0.65 µg/gdw and 0.12 µg/gfw). In pollen, PAT expression was 0.59-0.62 µg/gdw, while NPT II was not detected. Both PAT and NPT II showed a general pattern of decreased expression with progression of the growing season. As expected, PAT and NPT II protein expression was not detectable in control pepper plants.

Molecular analysis of bacterial community structures in paddy soils for environmental risk assessment with two varieties of genetically modified rice, Iksan 483 and Milyang 204.

The impacts of planted transgenic rice varieties on bacterial communities in paddy soils were monitored using both cultivation and molecular methods. The rice field plot consisted of eighteen subplots planted with two genetically modified (GM) rice and four non-GM rice plants in three replicates. Analysis with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes revealed that the bacterial community structures were quite similar to each other in a given month, suggesting that there were no significant differences in bacterial communities between GM and non- GM rice soils. The bacterial community structures appeared to be generally stable with the seasons, as shown by a slight variation of microbial population levels and DGGE banding patterns over the year. Comparison analysis of 16S rDNA clone libraries constructed from soil bacterial DNA showed that there were no significant differences between GM and non-GM soil libraries but revealed seasonal differences of phyla distribution between August and December. The composition profile of phospholipid fatty acids (PLFA) between GM and non-GM soils also was not significantly different to each other. When soil DNAs were analyzed with PCR by using primers for the bar gene, which was introduced into GM rice, positive DNA bands were found in October and December soils. However, no bar gene sequence was detected in PCR analysis with DNAs extracted from both cultured and uncultured soil bacterial fractions. The result of this study suggested that, in spite of seasonal variations of bacterial communities and persistence of the bar gene, the bacterial communities of the experimental rice field were not significantly affected by cultivation of GM rice varieties.