Pyramid Screening: Combining Three Genetic Screens into One Efficient Screen for Shoot Regeneration Mutants in Arabidopsis thaliana

Abstract

Gain-of-function genetic mutants are typically found by creating a non-permissive condition and screening for plants that overcome the stress. Separate genetic screens are conducted for each condition, a potentially time-consuming effort. In severed Arabidopsis thaliana leaves, high light, suboptimal hormone exposure and old age were each independently found to reduce frequency of shoot regeneration. Rather than conducting three separate mutant screens to dissect these three pathways, a laborious process, we hypothesized that we could undertake a single economical screen to retrieve mutations specific for each trait as well as cross-talk alleles between pathways. Instead of creating non-permissive conditions for each of our three traits of interest, we combined the three suboptimal stress conditions such that only when combined was shoot regeneration abolished. No one stress was primarily responsible for loss of our trait, thus ensuring that we could recover mutant alleles in any of the three pathways of interest. Screening of 18,000 mutagenized plants resulted in 12 SHOOTING UP (stu) mutants. Secondary screening revealed that we had recovered alleles that were both specific for a pathway (light, hormones or age) and which acted through multiple pathways. Our approach, which we refer to as pyramid screening, represents an economical method for mutant screening of multiple pathways in parallel (three screens in one) and has the potential to recover alleles that cross-talk between multiple pathways that underlie a complex trait such as organ regeneration. Pyramid screening should be widely applicable across species.