A Protocol for Accelerating Homozygous Line Screening in Arabidopsis thaliana

Abstract

Obtaining homozygous mutant and transgenic lines is a critical yet time-consuming step in Arabidopsis thaliana research. Conventional breeding procedures require seeds to undergo complete maturation and natural desiccation before harvest, followed by cold stratification to overcome seed dormancy. This process substantially prolongs generation turnover and delays genetic screening. Here, we describe a rapid germination strategy based on the use of partially dehydrated seeds collected approximately 15 days after pollination (DAP). At this developmental stage, embryos have reached physiological maturity, while the seeds have not yet entered deep desiccation-induced dormancy. After surface sterilization and short-term cold treatment (2–3 days at 4 °C), these seeds readily germinate on 1/2 MS medium and develop into normal seedlings. By bypassing the prolonged maturation and dormancy phases associated with naturally dried seeds, this protocol shortens each generation cycle by approximately 1–2 weeks. The method is particularly useful for accelerating the propagation of transgenic materials and the identification of homozygous mutant lines in Arabidopsis. Key features • The time-consuming and tedious screening of transgenic homozygous plants is a bottleneck limiting gene function research. • Provides a simple and effective approach for accelerating homozygous line identification in Arabidopsis thaliana. • Seeds harvested at 15 days after pollination can germinate normally, shortening the screening cycle by approximately 10 days per generation. • Applicable to T-DNA insertion mutants and transgenic lines carrying GFP or selectable markers, without requiring specialized equipment.