Chimeric genes as dominant selectable markers in plant cells.

Area of Science:

• Plant Molecular Biology
• Genetic Engineering
• Biotechnology

Background:

• Opine synthases are plant enzymes resulting from natural Agrobacterium-mediated gene transfer.
• Agrobacteria's Ti plasmids facilitate gene transfer, integration, and expression in plants.
• Nopaline synthase (nos) gene regulatory sequences are active in plant cells.

Purpose of the Study:

• To construct and test chimeric genes for plant transformation.
• To evaluate the use of bacterial antibiotic resistance genes as selectable markers in plants.
• To demonstrate functional expression of foreign genes in plant cells.

Main Methods:

• Chimeric genes were created by combining the nos gene promoter with bacterial aminoglycoside phosphotransferase (APH(3')II) or dihydrofolate reductase (DHFR Mtx) coding sequences.
• Ti plasmids were used as vectors to transfer chimeric genes into tobacco cells.
• Kanamycin and methotrexate were used as selective agents to identify transformed plant cells.

Main Results:

• The chimeric NOS-APH(3')II gene conferred resistance to kanamycin in transformed tobacco cells.
• Transformed cells exhibited APH(3')II phosphorylase activity, confirming gene expression.
• Similar resistance was observed with the NOS-DHFR Mtx gene, indicating functional expression of the methotrexate-insensitive DHFR Mtx enzyme.

Conclusions:

• Chimeric genes utilizing the nos promoter can serve as potent dominant selectable markers in plants.
• Foreign genes can be effectively transferred and functionally expressed in plants using appropriate genetic constructs.
• This approach facilitates the development of genetically modified plants with desired traits.