Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Developing efficient transformation systems is crucial for crop improvement.
• Traditional selectable markers often involve antibiotics or herbicides, raising environmental and regulatory concerns.
• The phosphomannose isomerase (PMI) gene offers a non-antibiotic, non-herbicidal alternative for plant selection.

Purpose of the Study:

• To establish and optimize an Agrobacterium-mediated transformation system for sorghum using the mannose selection system.
• To evaluate the efficacy of the Escherichia coli phosphomannose isomerase (PMI) gene as a selectable marker in sorghum.
• To assess the transformation efficiency and Mendelian segregation of transgenes in sorghum.

Main Methods:

• A dual-marker plasmid containing the manA (PMI) selectable marker and sgfp reporter gene was constructed under the ubi1 promoter.
• Immature sorghum embryos were transformed using Agrobacterium tumefaciens, followed by selection on mannose-containing media.
• Southern blot, Western blot, and chlorophenol red (CPR) assays were used to confirm gene integration, expression, and marker feasibility.

Main Results:

• A total of 167 transgenic sorghum plants were generated from two genotypes (C401 and Pioneer 8505) using mannose selection.
• Green fluorescence protein (GFP) expression was observed in embryoids and shoots, confirming successful transformation.
• Transformation efficiencies of 2.88% for Pioneer 8505 and 3.30% for C401 were achieved, exceeding previous reports.

Conclusions:

• The mannose selection system utilizing the PMI gene is effective and efficient for sorghum transformation.
• This method provides a viable alternative to antibiotic or herbicidal selection, producing transgenic plants without undesirable agents.
• Transgenes segregated in a Mendelian fashion in the T1 generation, indicating stable inheritance.