Suja George

SUMMARY: The development of marker‐free transgenic plants is essential to address biosafety concerns and facilitate regulatory approval. Co‐transformation strategies involving separate T‐DNAs for the gene of interest and selectable marker gene offer a clean approach but are often hampered by linked integration and vector backbone incorporation. In this study, we designed and evaluated a series of double T‐DNA vectors with varying intervening sequence lengths and orientations to determine their impact on co‐transformation efficiency and integration patterns in different plant species. Our results showed that shorter spacer regions increased the likelihood of linked T‐DNA integration, while an ~3 kb intervening region minimized this risk. Contrary to previous findings, inverse orientation of T‐DNAs with respect to each other in the vector significantly increased the frequency of linked and closely spaced integrations compared to tandem arrangements. Co‐transformation efficiency and integration outcomes varied across species and transformation methods, with Arabidopsis exhibiting higher rates of linked integration possibly due to germline transformation via floral dip, in contrast to somatic cell transformation in tobacco, lettuce, and tomato. Incorporation of a GFP reporter gene within the intervening region enabled easy identification of unlinked integration events in the T0 generation, reducing downstream screening efforts. Marker‐free plants were successfully recovered in the T1 generation, confirming the effectiveness of this approach. These findings emphasize the importance of T‐DNA design, orientation, and target cell type in optimizing co‐transformation strategies for generating marker‐free transgenic plants.

Selectable marker genes (SMGs) are essential for identifying transgenic plants but raise concerns regarding biosafety, regulatory compliance, and public acceptance. In this study, we used a CRISPR/Cas9-based strategy to eliminate the SMG from transgenic tobacco plants. Leaf discs from plants carrying DsRED (SMG) and aminoglycoside phosphotransferase (gene of interest, GOI) were re-transformed with a CRISPR vector containing four gRNAs designed to target both flanking regions of the SMG cassette. Approximately 20% of the regenerated shoots exhibited loss of red fluorescence, and PCR and sequencing analyses confirmed that about half of these carried a smaller amplicon, indicating a successful SMG excision efficiency of around 10%. Mutation analysis further revealed the presence of small indels at gRNA target sites, in addition to the deletion of SMG cassette. Quantitative real-time PCR (qPCR) analysis confirmed the absence of DsRED expression in SMG-deleted lines, while the Cas9 and GOI remained actively expressed. The SMG-free plants displayed normal growth, flowering, and seed production, indicating CRISPR marker excision had no adverse effects on plant development and fertility. In addition, Cas9-free, marker-free transgenic plants were recovered through segregation in T1 generation. This approach is adaptable to various transgenic plant species and provides a practical solution for generating marker-free transgenic crops, thereby enhancing their acceptance and commercialization.

The indiscriminate use of antibiotics in agriculture has raised concerns about antibiotic residues in food products, necessitating robust analytical methods for detection and quantification. In this study, our primary aim was to develop a robust and advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology specifically designed for the accurate quantification of ticarcillin degradation products in tomato leaves. The choice of ticarcillin as the target analyte stems from its frequent use in agriculture and the potential formation of degradation products, which can pose a threat to food safety. The use of tomatoes as the target sample matrix in this study is justified by their significance in human diets, their widespread cultivation, and their suitability as a model for assessing antibiotic residue dynamics in diverse agricultural environments. By optimizing the MS/MS parameters, the study successfully demonstrates the practicality and reliability of the employed LC-MS/MS method in accurately assessing ticarcillin degradation product (Thiophene-2-Acetic acid and Thiophene-3-Acetic acid) levels. The chromatographic separation was achieved using a specialized column, ensuring high resolution and sensitivity in detecting analytes. Multiple reaction monitoring (MRM) data acquisition was employed to enhance the selectivity and accuracy of the analysis. The developed method exhibited excellent linearity and precision, meeting the stringent requirements for antibiotic residue analysis in complex matrices. Key outcomes of this study include the successful identification and quantification of ticarcillin and its degradation products in tomato leaves, providing crucial insights into the fate of this antibiotic in agricultural settings. The methodology’s applicability was further demonstrated by analyzing real-world samples, highlighting its potential for routine monitoring and ensuring food safety compliance. In summary, our study constitutes a noteworthy advancement in the domain of antibiotic residue analysis, offering a reliable method for quantifying ticarcillin degradation products in tomato leaves. The optimized parameters and MRM-based LC-MS/MS approach enhance the precision and sensitivity of the analysis, opening up opportunities for further studies in the assessment of antibiotic residues in agricultural ecosystems.

Efficient regeneration of transgenic plants from explants after transformation is one of the crucial steps in developing genetically modified plants with desirable traits. Identification of novel plant growth regulators and developmental regulators will assist to enhance organogenesis in culture. In this study, we observed enhanced shoot regeneration from tomato cotyledon explants in culture media containing timentin, an antibiotic frequently used to prevent Agrobacterium overgrowth after transformation. Comparative transcriptome analysis of explants grown in the presence and absence of timentin revealed several genes previously reported to play important roles in plant growth and development, including Auxin Response Factors (ARFs), GRF Interacting Factors (GIFs), Flowering Locus T (SP5G), Small auxin up-regulated RNAs (SAUR) etc. Some of the differentially expressed genes were validated by quantitative real-time PCR. We showed that ticarcillin, the main component of timentin, degrades into thiophene acetic acid (TAA) over time. TAA was detected in plant tissue grown in media containing timentin. Our results showed that TAA is indeed a plant growth regulator that promotes root organogenesis from tomato cotyledons in a manner similar to the well-known auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). In combination with the cytokinin 6-benzylaminopurine (BAP), TAA was shown to promote shoot organogenesis from tomato cotyledon in a concentration-dependent manner. To the best of our knowledge, the present study reports for the first time demonstrating the function of TAA as a growth regulator in a plant species. Our work will pave the way for future studies involving different combinations of TAA with other plant hormones which may play an important role in in vitro organogenesis of recalcitrant species. Moreover, the differentially expressed genes and long noncoding RNAs identified in our transcriptome studies may serve as contender genes for studying molecular mechanisms of shoot organogenesis.