Developing a robust in vivo hairy root system for assessing transgene expression and genome editing efficiency in papaya

Abstract

Papaya is one of the most important fruits in tropical and subtropical countries. However, genetic improvement has had limited success to date due to time-consuming and complex transformation and regeneration technologies, as well as a lack of reproducible and efficient transient gene expression assays. Here, we report the development of a highly efficient Rhizobium rhizogenes-based in vivo hairy root system for evaluating transgene expression and activity including CRISPR/Cas gene editing reagents in the Vietnamese papaya cultivar Linhan. To optimize the papaya transformation parameters, we introduced the R. rhizogenes strain K599 into papaya hypocotyls at 1-, 5- and 10-mm below the cotyledon nodes by a needle using 5-, 7- and 10-day old seedlings and then monitored the frequency of hairy root formation at 18 days post infection. We found that the age of the seedlings and the distance of the infection site from the cotyledon node were inversely correlated with the efficacy of hairy root induction, being 5-day-old plants and 1-mm distance the best parameters. The etablished protocol was then employed to investigate transformation frequency using the gus reporter gene. Of the tested hairy roots, 47.22% were positive for GUS staining, which indicates a high level of transgene transfer and stability. Finally, we introduced a dual guide RNA CRISPR/Cas9 cassette targeting eukaryotic translation initiation factor isoform 4E (eIF(iso)4E) gene into papaya by R. rhizogenes and then screened for gene editing events by heteroduplex analysis and Sanger sequencing. Our analysis revealed that 50% of induced roots contained the expected mutations in the eIF(iso)4E gene, which makes our system ideal for testing transgene activity prior making stable transgenic papaya lines.