2017 ASHS Annual Conference

Water deficit is a critical constraint to plant growth and development, and could cause severe crop loss. To develop crops with increased water stress tolerance, it is necessary to identify gene(s) that play important role(s) in water deficit stress response mechanism. We have performed transcriptome analysis of petunia under water stress using high throughput RNA sequencing. The expression of 63 transcription factors (TFs) were upregulated during the early stage of water stress response. Among them, a gene encoding an ethylene responsive transcription factor (PhERF039) was selected for functional analysis in petunia. The objectives of this research were to 1) manipulate the expression level of PhERF039 by over-/under-expression techniques, and 2) determine whether PhERF039 plays a role in the water stress response mechanism. The overexpression driven by 35S promoter was achieved using pICH86988 from Addgene. For generation of knock-out mutant line, CRISPR-Cas9 and Golden Gate cloning systems were used. DNA fragments harboring NPTII, Cas9, and sgRNAs were delivered via Agrobacterium-mediated transformation into petunia leaves. T₀ and homozygous T₁ transgenic plants, and wild type (WT) were evaluated under water deficit stress utilizing an automated irrigation system. The results of the expression and phenotype analyses are presented.