Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H₂O₂ to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H₂O₂ content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H₂O₂ and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H₂O₂ by 3,3′-diaminobenzidine (DAB) staining suggested that H₂O₂ could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H₂O₂-based method. Moreover, HyB induced overproduction of H₂O₂ in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

Pumpkins (Cucurbita moschata), valued for their nutritional, medicinal, and economic significance, face threats from Meloidogyne incognita, a critical plant-parasitic nematode. This study extensively examines the impact of M. incognita on the growth, physiological, and biochemical responses of C. moschata. We demonstrate that M. incognita infection leads to significant growth impairment in C. moschata, evidenced by reduced plant height and biomass, along with the significant development of nematode-induced galls. Concurrently, a pronounced oxidative stress response was observed, characterized by elevated levels of hydrogen peroxide and a significant increase in antioxidant defense mechanisms, including the upregulation of key antioxidative enzymes (superoxide dismutase, glutathione reductase, catalase, and peroxidase) and the accumulation of glutathione. These responses highlight a dynamic interaction between the plant and the nematode, wherein C. moschata activates a robust antioxidant defense to mitigate the oxidative stress induced by nematode infection. Despite these defenses, the persistence of growth impairment underscores the challenge posed by M. incognita to the agricultural production of C. moschata. Our findings contribute to the understanding of plant–nematode interactions, paving the way for the development of strategies aimed at enhancing resistance in Cucurbitaceae crops against nematode pests, thus supporting sustainable agricultural practices.

Duckweed (Lemna aequinoctialis) is one of the smallest flowering plants in the world. Due to its high reproduction rate and biomass, duckweeds are used as biofactors and feedstuff additives for livestock. It is also an ideal system for basic biological research and various practical applications. In this study, we attempt to establish a micropropagation technique and Agrobacterium-mediated transformation in L. aequinoctialis. The plant-growth regulator type and concentration and Agrobacterium-mediated transformation were evaluated for their effects on duckweed callus induction, proliferation, regeneration, and gene transformation efficiency. Calli were successfully induced from 100% of explants on Murashige and Skoog (MS) medium containing 25.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μM thidiazuron (TDZ). MS medium containing 4.5 μM 2,4-D and 2.0 μM TDZ supported the long-lasting growth of calli. Fronds regenerated from 100% of calli on Schenk and Hildebrandt (SH) medium containing 1.0 μM 6-benzyladenine (6-BA). We also determined that 200 μM acetosyringone in the cocultivation medium for 1 day in the dark was crucial for transformation efficiency (up to 3 ± 1%). Additionally, we propose that both techniques will facilitate efficient high-throughput genetic manipulation in Lemnaceae.

Aquaponic systems are made up of hydroponic beds and recycled aquaculture systems. The significant elements that determine how effectively an aquaponic system operates are the nitrogen cycle (nitrification) and the phosphorus cycle (phosphate). Because some research indicates that aquaponics systems are primarily deficient in phosphorus, phosphate-enriched nutrients were added to raise the phosphorus levels. During an eight-week experimental period, the effects of water quality parameters and microbiology, animal and plant growth performance, chlorophyll compounds in lettuce, and the bacterial community were analyzed. Phosphate concentration (1.604 ± 1.933 mg L⁻¹) and ammonia-oxidizing bacteria (AOB) (1.19 × 10² ± 1.30 CFU mL⁻¹) give significant positive reactions to the added nutrients. However, the prawn survival rate (17.00 ± 0.63%) showed a significantly negative response to nutrition modification containing phosphate, and the percentage of bacterial pathogens became more dominant (pathogen 40.51%; N_bacteria 35.05%; probiotics 24.44%). This study shows that adding phosphate increases phosphorus levels in an aquaponics system and changes the microbial community and species growth performance.