Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences

Area of Science:

• Molecular Biology
• Plant Biotechnology
• Genetics

Background:

• Plastid genomes (plastomes) in plants and algae possess an efficient native homologous recombination pathway.
• Marker genes are often used in plastome engineering but their removal can be challenging.
• Existing methods for marker removal may require multiple transformation steps or sexual crosses.

Purpose of the Study:

• To develop a simple, efficient, and widely applicable method for marker gene excision from plant plastomes.
• To leverage the native homologous recombination pathway for marker-free plastome engineering.
• To enable precise genetic modifications in plastids without leaving residual marker DNA.

Main Methods:

• Utilized DNA direct repeats flanking marker genes within the plastid genome.
• Exploited the native homologous recombination machinery for spontaneous marker excision.
• Employed selection to maintain the marker and ensure homoplasmy, followed by release of selection to allow marker-free plastome accumulation.

Main Results:

• Marker excision frequency is dependent on the length and number of direct repeats, with >600 bp repeats promoting efficient excision.
• A single transformation round is sufficient, eliminating the need for retransformation or sexual crosses for recombinase introduction.
• Marker-free plastomes were successfully isolated in T0 generation shoots and T1 seedlings, demonstrating the method's efficacy.

Conclusions:

• Direct repeat-mediated marker excision is a simple, efficient, and versatile strategy for plastome engineering in plants and algae.
• This technique allows for precise genetic modifications (insertions, mutations, deletions) without extraneous DNA.
• The method facilitates the development of marker-free crops, simplifying downstream applications and commercialization.