HSVd elimination from tomato using cryotherapy combined with exogenous HSVd-targeting ds-sRNA Application

Abstract

The development of viroid elimination technology is essential in tomato (Solanum lycopersicum) production. Combining long-term cold therapy and meristem culture is commonly used; however, tomatoes cannot withstand cold. How the Hop stunt viroid (HSVd) affects the growth potential, activities of oxidoreductases and the contents of soluble matters and total chlorophyll was studied. HSVd-targeting double-stranded small RNAs (ds-sRNAs) were synthesized to match different regions of its genome (ds-sRNAs-H1∼H5), and their permeability in plants with HSVd inhibition effects was tested. The HSVd infected tomato shoots were exogenously treated with ds-sRNAs, and the meristems were thereafter taking from treated shoots and processed using leaf primordia-free meristem culture and cryotherapy. HSVd inhibited tomato stem elongation, leaf growth and root development, it elevated the oxidoreductase activity and disturbed the contents of soluble protein, soluble sugar and chlorophyll content. Exogenous ds-sRNAs can penetrate all types of cells to induce HSVd gene silencing in shoot terminals, causing the accumulation of miRNAs produced by HSVd and differed expression of gene silencing-related genes. The shoot terminal ex vivo treated with ds-sRNA which targeting the central conserved region of HSVd genome (H3) gained the highest inhibition of HSVd titer after 36 h of feeding. Leaf primordia-free meristem culture couldn't obtain viroid-free regenerants from ds-RNA-H3 treated meristems, whereas in cryotherapy, meristems soaked in PVS2 for 15 and 20 min resulted viroid elimination of 6.7 % and 10.0 %, 5 and 10 min of PVS2 caused 0 % HSVd elimination. Growth and physiological state of HSVd-eliminated plants recovered to those of healthy controls. The results provide theoretical and technical basis for the sustainable development of the tomato pathogen-free plant industry.