Recipient Somatic Embryogenesis System Promotes CRISPR/Cas Genome Editing

Abstract

Somatic embryogenesis (SE) is an important type of plant regeneration, which can be used as a recipient system of genome editing. It is vital to optimize the SE system to improve the mutated somatic cells regenerated into fertile plants after transformation. 2,4-D was used as inducer of somatic embryogenesis from mature seeds. Arabidopsis wild type Col-0 and ABA biosynthesis double mutant <i>nced6 / 9 </i>were grown in 15°C and 25°C respectively, to investigate the effect of sampled plants growth conditions on the SE rate of their seeds and to optimize SE protocol. On the other hand, construct Pbi121-U6-26-Cas9-sgRNA-ELF6-FastR was used for genome editing. The positive seeds after floral dip were separated into two parts: some for sowing and the rest seeds went though SE induction. The efficiency of gene editing was compared by sequencing. Our results show that the SE rate of <i>nced6 / 9</i> seeds from 25°C was significantly lower than that from 15°C. When transgenic positive seeds were sown directly, the target sequence was not found to be edited; However, when seeds went though somatic embryogenesis protocol, gene editing were found around PAM sequence, suggesting that SE system could improve gene editing after transformation. The sampled plants grown at low temperature (15°C) may affect SE ability of their seeds by ABA level. The mature seed somatic embryogenesis system can improve the efficiency of gene editing.