Mutator plant organelle DNA polymerase for elevated chloroplast and mitochondrial mutation rates in crops

Overview

This technology leverages a mutator Plant Organelle DNA Polymerase (POP) to elevate mutation rates in chloroplasts and mitochondria. Because POP is the sole DNA polymerase responsible for replicating DNA in these organelles, an error-prone variant introduces genetic diversity into a previously intractable genetic compartment. This enables the selection of new traits specified by organelle genes, including herbicide resistance and tolerance to photoinhibition. The approach is applicable to all crops, and because the mutator POP transgene can be crossed out, the resulting improved plants can be transgene-free.

Technical specifications

• Mutator POP isolation: An in vitro bacterial screening assay was used to isolate a mutator tobacco POP deficient in both proofreading and polymerization accuracy.
• Error rate: The mutator POP exhibits an error rate approximately 140-fold higher than wild-type POP.
• Genetic behavior: The mutator POP is semi-dominant to wild-type POP, an important feature for in planta application.
• Trait targets: Chloroplast genes governing herbicide resistance and photoinhibition can be targeted for mutation and selection.
• Selection methods: Positive selection agents (such as antibiotics and herbicides targeting chloroplast gene products) and pigment-deficient phenotypes can be used to identify mutant chloroplast genomes.
• Transgene-free output: The nuclear-encoded mutator POP can be crossed out, yielding transgene-free crops with improved chloroplast genomes.

Technology readiness level

The mutator POP has been validated through an in vitro bacterial screening assay and was selected as a 2020 breakthrough article in Nucleic Acids Research for its applications to plant organelle genome manipulation. Future validation involves expressing the mutator POP in tobacco chloroplasts using an inducible promoter, isolating transgenic plants, and testing for elevated chloroplast mutation rates using positive selection screens and shoot regeneration assays on selective media. Chloroplast genomes in resistant shoots will be sequenced to characterize mutation frequency, number, and distribution. The projected timeline for this validation phase is approximately one year.