Plants are often exposed to biotic and abiotic stresses that affect plant growth, development, and productivity. Drought is an important abiotic stress that has a particularly serious impact on plant growth and development. We transformed rice with PsGPD using Agrobacterium-mediated transformation. We generated independent PsGPD-homozygous transgenic rice plants selected as single copy/intergenic lines by the TaqMan copy number assay and by T-DNA flanking sequences. These transgenic rice plants showed improvement of drought tolerance compared to wild-type plants under drought condition. RNA sequencing analysis showed that 2,992 genes were transcriptionally affected by the PsGPD transgene or drought treatment. In total, 145 genes were modulated by the PsGPD transgene before and after drought treatment. Among these candidate genes, 4 were up- and downregulated in all four comparisons. Several genes, including Os04t0576900, Os03t0629800, and Os04t0518400 (OsPAL7), were involved in tetrapyrrole synthesis. Os09t0522200 (DREB1A), an important component in hormone signal transduction, is a transcription factor (TF) gene that plays vital roles in stress responses. We partially characterized the functions of PsGPD in the drought stress response and the role of major TFs in the drought tolerance mechanism. These genes will be useful targets for both future research and the breeding of drought tolerance in rice.

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

Citations

Citations to this article as recorded by  

• Molecular Characterization of CRISPR-Cas9-Edited Rice Across Generations and Associated Technical Challenges in Nucleotide Editing Tracing
  Yang Qin, Sang Dae Yun, Hye Lin Kim, Je Yeon Choi, Myung-Ho Lim, Sung Aeong Oh, Soon Ki Park
  Plant Breeding and Biotechnology.2025;[Epub]     CrossRef
• Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri
  Julie Thomy, Frederic Sanchez, Marta Gut, Fernando Cruz, Tyler Alioto, Gwenael Piganeau, Nigel Grimsley, Sheree Yau
  Cells.2021; 10(3): 664.     CrossRef
• Comparative transcriptome profiling of different tissues from beta-carotene-enhanced transgenic soybean and its non-transgenic counterpart
  Yang Qin, Hee-Jong Woo, Kong-Sik Shin, Myung-Ho Lim, Seong-Kon Lee
  Plant Cell, Tissue and Organ Culture (PCTOC).2020; 140(2): 341.     CrossRef