[Quick testing-technology of transgenic rice with npt- screen marker gene]

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Developing efficient methods for identifying transgenic plants is crucial for crop improvement.
• The neomycin phosphotransferase (npt) gene is commonly used as a selectable marker in plant transformation.
• Antibiotic resistance assays offer a direct way to screen for successful gene integration.

Purpose of the Study:

• To establish a rapid and accurate system for screening transgenic rice offspring using the npt gene as a selection marker.
• To determine the optimal antibiotic concentrations for selecting transgenic rice at different developmental stages.

Main Methods:

• Japonica rice Zhonghua 9 (ZH9) and transgenic ZH9 (ZH9(R)) were treated with varying concentrations of Kanamycin and G418 antibiotics.
• Detached leaves, seeds, young embryos, and seedlings were tested using optimized G418 concentrations.
• Polymerase Chain Reaction (PCR) was employed to confirm gene presence and linkage.

Main Results:

• G418 proved to be a more effective antibiotic than Kanamycin for selecting transgenic rice with the npt gene.
• Optimal G418 concentrations were determined: 80 mg/L for detached leaves (4 days), 300 mg/L for seeds (7 days), 200 mg/L for young embryos (10 days), and 150 mg/L for seedlings (12 days).
• PCR confirmed the tight linkage between the npt gene and the lysozyme gene.

Conclusions:

• G418 is a highly effective antibiotic for selecting transgenic rice, with specific critical concentrations established for different tissues and developmental stages.
• The developed screening system is convenient, intuitive, and accurate for distinguishing transgenic positive from negative rice plants.
• The npt selectable marker gene is tightly linked with the lysozyme gene in the tested transgenic rice lines.