Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation...

Area of Science:

• Plant Biotechnology
• Crop Science
• Genetics and Genomics

Background:

• Maize (Zea mays) genetic transformation is genotype-dependent and inefficient, particularly for tropical lines.
• Existing transformable maize lines are often temperate-adapted, limiting field applications for tropical agriculture.
• Ectopic expression of morphogenic regulators (MRs) offers a potential solution for universal maize transformation.

Purpose of the Study:

• To develop and apply a successful genome editing (GE) protocol for agronomically relevant tropical maize lines.
• To assess the efficiency and genotype dependency of MR-based GE in diverse tropical maize varieties.
• To utilize CRISPR/Cas9 for targeted gene knockout and verify edits in tropical maize.

Main Methods:

• Employed an Agrobacterium-mediated transformation protocol using a CRISPR/Cas9 construct targeting the VIRESCENT YELLOW-LIKE (VYL) gene.
• Tested the protocol on five agronomically relevant tropical maize lines, including those with SNPs at the target site.
• Verified VYL gene mutations in protoplasts and analyzed indel inheritance in subsequent generations.

Main Results:

• Achieved efficient GE in three out of five tropical maize lines, with transformation efficiencies up to 6.63%.
• Confirmed targeted VYL gene knockout via indels in 97% of events, with successful inheritance.
• Observed off-target mutations in the VYL paralog VYL-MODIFIER, potentially linked to WUSCHEL (WUS) MR expression.

Conclusions:

• Demonstrated successful and efficient genome editing in agronomically important tropical maize genotypes.
• The MR-based approach overcomes genotype dependency, expanding GE-amenable maize lines.
• This advancement facilitates broader application of GE for crop improvement in tropical regions.