Stacked traits have become an important trend in the current development of genomically modified crops. The bidirectional promoter can not only prevent the co-suppression of multigene expression, but also increase the efficiency of the cultivation of transgenic plants with multigenes. In Gossypium hirsutum, Ghrack1 and Ghuhrf1 are head-to-head gene pairs located on chromosome D09. We cloned the 1429-bp intergenic region between the Ghrack1 and Ghuhrf1 genes from Gossypium hirsutum. The cloned DNA fragment GhZU had the characteristics of a bidirectional promoter, with 38.7% G+C content, three CpG islands and no TATA-box. Using gfp and gus as reporter genes, a series of expression vectors were constructed into young leaves of tobacco. The histochemical GUS (Beta-glucuronidase) assay and GFP (green fluorescence protein) detection results indicated that GhZU could drive the expression of the reporter genes gus and gfp simultaneously in both orientations. Furthermore, we transformed the expression vectors into Arabidopsis and found that GUS was concentrated at vigorous growth sites, such as the leaf tip, the base of the leaves and pod, and the stigma. GFP was also mainly expressed in the epidermis of young leaves. In summary, we determined that the intergenic region GhZU was an orientation-dependent bidirectional promoter, and this is the first report on the bidirectional promoter from Gossypium hirsutum. Our findings in this study are likely to enhance understanding on the regulatory mechanisms of plant bidirectional promoters.

The safety assessment and control of stacked transgenic crops is increasingly important due to continuous crop development and is urgently needed in China. The genetic stability of foreign genes and unintended effects are the primary problems encountered in safety assessment. Omics techniques are useful for addressing these problems. The stacked transgenic maize variety 12–5 × IE034, which has insect-resistant and glyphosate-tolerant traits, was developed via a breeding stack using 12–5 and IE034 as parents. Using 12–5 × IE034, its parents (12–5 and IE034), and different maize varieties as materials, we performed proteomic profiling, molecular characterization and a genetic stability analysis. Our results showed that the copy number of foreign genes in 12–5 × IE034 is identical to that of its parents 12–5 and IE034. Foreign genes can be stably inherited over different generations. Proteomic profiling analysis found no newly expressed proteins in 12–5 × IE034, and the differences in protein expression between 12 and 5 × IE034 and its parents were within the range of variation of conventional maize varieties. The expression levels of key enzymes participating in the shikimic acid pathway which is related to glyphosate tolerance of 12–5 × IE034 were not significantly different from those of its parents or five conventional maize varieties, which indicated that without selective pressure by glyphosate, the introduced EPSPS synthase is not has a pronounced impact on the synthesis of aromatic amino acids in maize. Stacked-trait development via conventional breeding did not have an impact on the genetic stability of T-DNA, and the impact of stacked breeding on the maize proteome was less significant than that of genotypic differences. The results of this study provide a theoretical basis for the development of a safety assessment approach for stacked-trait transgenic crops in China.

Cotton is one of the world’s important cash crops and occupies a significant position in agricultural production and the national economy. However, insect pests severely affect the growth, yield and quality of cotton. To ensure high and stable cotton yields, the cultivation of insect-resistant transgenic cotton via transgenic technology can not only effectively reduce the impact of chemical pesticides on crops but also exert excellent control effects against pests such as cotton bollworms. In this study, the plant expression vector pC2300-VEC harboring the target genes epsps, cry1Ac and vip3A was introduced into the genome of the recipient cotton cultivar CCRI 24 via Agrobacterium-mediated transformation. The obtained transgenic cotton plants were subjected to the identification of target genes and target traits, and the insect-resistant transgenic cotton line BrsC35 was ultimately obtained. PacBio sequencing combined with conventional molecular characterization methods was used to analyze its insertion site, copy number and other characteristics, providing a new germplasm for insect-resistant transgenic cotton.

Alfalfa (Medicago sativa L.) is a vital global forage crop. Transgenic technology promises enhanced yield and quality, but requires rigorous environmental risk assessment, particularly regarding pollen-mediated gene flow, for which standardized protocols are lacking. Based on an optimized in vitro culture medium, this study developed a method to assess alfalfa pollen viability. Using a single-factor experimental design, key assessment parameters were established at 1/4/8 h and 20/30/40 °C. A comparative analysis revealed no significant difference (p > 0.05) in pollen viability between the transgenic line SA6-8 and its non-transgenic parent “ZM-1” within this evaluation system. This result indicates that the genetic modification did not impact the pollen viability of SA6-8. By establishing this in vitro germination-based pollen viability assessment system and comparatively analyzing pollen viability between transgenic alfalfa and its non-transgenic parent under diverse environmental conditions, our approach provides crucial insights for optimizing transgenic alfalfa planting strategies and strengthening biosafety review protocols.

Rice (Oryza sativa L.) is a vital staple crop, and the environmental risk assessment of transgenic varieties is crucial for formulating biosafety policies. Rice pollen grains are spherical, with an average diameter of 40.03 ± 2.75 μm. This study established a standardized protocol for in vitro pollen germination by first optimizing key culture conditions. A single-factor experimental design identified the optimal medium composition as 150 g/L sucrose, 40 mg/L boric acid, 20 mg/L calcium chloride, 10 mg/L monopotassium phosphate, and 10 mg/L magnesium sulfate. The ideal germination temperature was determined to be 31 ± 1 °C, with no germination observed below 16 °C or above 40 °C. Pollen germination rates declined significantly within 5 min post-isolation and ceased completely after 30 min. Building on this optimized protocol, a standardized evaluation method was developed, defining key assessment conditions at temperatures of 25/31/37 °C and post-isolation times of 0/5/15 min. Under these defined conditions, the pollen viability of glyphosate-resistant transgenic rice G2-6 was compared to its non-transgenic recipient ZH11. No significant differences were found at any tested time–temperature combination (p > 0.05). This work establishes a practical and reproducible standard for transgenic rice pollen assessment, offering a scientific basis for evidence-based biosafety regulation and policy-making.

Vitamin B₆ is a water-soluble vitamin that is essential for all living organisms in their life activities. Among its forms, pyridoxal 5’-phosphate (PLP) is the primary metabolically active form of Vitamin B₆, which usually plays a crucial role in the metabolism of proteins, fatty acids, and carbohydrates. To date, although the molecular functions of genes involved in vitamin B₆ biosynthesis, including Pdx1, Pdx2, Pdx3, and Sos4, have been reported in various plants, no studies have yet explored the functions of NtPDX1 and NtPDX2 in tobacco. This study used the Nicotiana tabacum L. as material to clone the CDS sequence of the NtPDXs. Through bioinformatics analysis, we predicted the phylogenetic relationships and functions of these genes; the subcellular localization of NtPDX2 was found to be in the cytoplasmic structures. By conducting both constitutive overexpression and homozygous knockout studies of the NtPDX2, we observed a significant increase in vitamin B₆ content in the stem tissues of overexpressing plants (up to 150%), while knockout plants showed a decrease to 60%. This led to changes in agronomic traits such as plant height and stem thickness in tobacco plants. The overexpressing plants exhibited a significant increase in height (100.93 cm) and stem thickness (13.64 cm), whereas the knockout plants were shorter in height (73.10 cm) and had thinner stems (10.83 cm). By integrating transcriptome sequencing technology with molecular biology methods, we aim to elucidate the molecular mechanisms underlying the role of NtPDX2 in tobacco growth and development, thereby providing new genetic resources and a theoretical foundation for the cultivation of new tobacco varieties with superior quality for flue-cured tobacco.