Feedback-insensitive anthranilate synthase gene as a novel selectable marker for soybean transformation

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Concerns exist regarding the escape of antibiotic- or herbicide-resistant transgenes from transgenic crops.
• Plant-origin selectable marker genes present a safer alternative for plant transformation.
• The anthranilate synthase gene (ASA2) is involved in tryptophan biosynthesis.

Purpose of the Study:

• To evaluate the efficacy of a feedback-insensitive anthranilate synthase gene (ASA2) from tobacco as a selectable marker for soybean transformation.
• To confirm the integration, expression, and inheritance of the ASA2 transgene in soybean.
• To investigate the potential for inter-species complementation of anthranilate synthase activity.

Main Methods:

• Agrobacterium-mediated transformation of soybean mature embryo axis tissue.
• Selection of transformed cells using the tryptophan analogue 5-methyltryptophan (5-MT).
• Molecular analyses including Southern blot, Northern blot, and PCR to confirm gene integration, expression, and inheritance.

Main Results:

• Successful integration of the ASA2 gene into the soybean genome was confirmed by Southern blot.
• The ASA2 gene was expressed in leaf tissue, leading to a significant increase (59-123%) in free tryptophan levels compared to wild-type soybean.
• Mendelian inheritance of the transgene was observed in the T1 progeny.

Conclusions:

• The feedback-insensitive ASA2 gene functions effectively as a selectable marker gene in soybean transformation.
• The tobacco ASA2 gene can complement the function of soybean n-subunits, forming an active heterologous enzyme.
• This study highlights the potential of plant-derived selectable markers for developing safer transgenic crops.