PVX-Cre-mediated marker gene elimination from transgenic plants

Area of Science:

• Plant molecular biology
• Genetic engineering
• Virology

Background:

• Selectable marker genes are crucial for identifying transgenic plants.
• Efficient removal of marker genes is desirable for transgene stability and regulatory compliance.
• Cre-lox recombination is a powerful tool for site-specific DNA manipulation.

Purpose of the Study:

• To develop a transient expression system for Cre recombinase using a plant virus vector.
• To demonstrate the efficient removal of a selectable marker gene (bar) in transgenic *Nicotiana benthamiana*.
• To establish a marker-free plant production method.

Main Methods:

• Transient expression of Cre recombinase via a Potato Virus X (PVX)-based vector in transgenic *lox*-target *Nicotiana benthamiana*.
• Design of a target construct with directly oriented *lox* sites flanking the *bar* gene.
• Observation of GFP expression as an indicator of Cre-mediated recombination and marker gene excision.
• Application of ribavirin to eliminate the PVX-Cre vector from regenerants.

Main Results:

• GFP expression confirmed Cre-mediated excision of the *bar* gene, placing the GFP coding region under the CaMV 35S promoter.
• Efficient systemic infection and recombination were observed in leaves, explants, and T1 progeny.
• PVX-Cre vector elimination was achieved using ribavirin treatment.
• Recombination frequencies ranged from 48% to 82% in regenerated plants.

Conclusions:

• Plant virus vectors can efficiently express Cre recombinase *in vivo*.
• This method provides a viable alternative for producing marker-free transgenic plants.
• The PVX-Cre system offers a controllable and efficient approach for transgene manipulation.