Transgene-Free, Gene-Edited Cavendish Bananas (Musa acuminata, AAA).

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Crop Science

Background:

• Gene editing offers precise crop modification without foreign DNA integration.
• Consumer acceptance of gene-edited foods is growing.
• Developing transgene-free gene editing in vegetatively propagated crops like bananas presents challenges.

Purpose of the Study:

• To establish an Agrobacterium-based, transgene-free gene editing system for the Cavendish banana.
• To demonstrate the efficiency and applicability of CRISPR/Cas9 for targeted gene modification in bananas.
• To overcome regulatory limitations for gene-edited, vegetatively propagated crops.

Main Methods:

• Utilized a three-tiered selection strategy involving positive and negative antibiotic selection with 5-FC and CODA enzyme.
• Employed CRISPR/Cas9 for transient gene editing targeting carotenoid biosynthesis genes (pds and LCYb).
• Verified transgene-free status using targeted genome sequencing and T-DNA-specific PCR, with whole genome sequencing for confirmation.

Main Results:

• Successfully generated transgene-free, gene-edited Cavendish banana calli and plants.
• Identified edits in 8/32 plants for pds and 34/125 plants for LCYb.
• Achieved an estimated 17.6% to 21.9% efficiency for generating transgene-free, tri-allelic edits.

Conclusions:

• The developed protocol enables efficient, transgene-free gene editing in Cavendish bananas.
• This method facilitates the creation of novel banana cultivars with enhanced disease resistance and agronomic traits.
• The approach addresses regulatory concerns, paving the way for commercialization of gene-edited crops.