Spinach(Spinacia Oleracea L.) Hairy Root Induction And Establishment Of Efficient Genome Editing System

The plant can produce hairy roots after infected by Agrobacterium rhizgones harboring the Ri plasmids.Spinach(Spinacia oleracea L.)is a common edible vegetable,and its hairy roots system has yet to be reported.In this study Agrobacterium rhizogenes LBA9402 was screened to be suitable for hairy roots induction of spinach.The results showed that the 16% highest induction efficiency was reached when the spinach stem was infected by LBA9402.Hairy roots grew vigorously on SH solid medium without exogenous hormones.The hairy roots of spinach were white and had enormous root hairs.The regeneration plant obtained from the callus redifferentiated from spinach hair roots.In addition,Ri T-DNA and Ti T-DNA carrying rol genes and GFP gene,respectively,was co-transferred into the cells of spinach and produced transgenic hairy roots.PCR analysis and fluorescence microscopy assays showed that rol B gene and GFP gene existed in the hairy roots of spinach and expressed stably.CRISPR/Cas9 is a valuable tool has been extensively used in model plants and crops.Nonetheless,CRISPR/Cas9 has not been successfully used in spinach,a leafy vegetable plant,in spite of the genome sequence of spinach was released.Here,we report the use of CRISPR/Cas9 to precisely edit Spo23361 and Spo10340,the Cellulose Synthase-Like D genes(CSLD)involved in root hair formation in the hairy root of spinach.Four mutation types,replacement,insertion,deletion and combined mutations were observed,among which the deletions accounted for the vast majority,about 64.1%.Mutation rate differed largely among the different targets.7 homozygous/bi-allelic and 8 heterzygous/chemiric mutated lines of Spo23361 were obtained from 15 independent transgenic hairy root lines,all of the 7 homozygous/bi-allelic lines displayed bulking and short root hairs which was a characteristic of CSLD2 mutant.13 heterzygous/chemiric mutated lines,but no homozygous/bi-allelic lines,of Spo10340 were obtained from 15 independent transgenic hairy root lines,all of which showed the same root hairs with normal hairy roots.These results demonstrate that Agrobacterium rhizogenes-mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in spinach,thus paving the way for large-scale genome editing in spinach,which is important for both basic and applied research of spinach.