Development of genetically modified chrysanthemums resistant to Chrysanthemum stunt viroid

Abstract

Chrysanthemum (Dendranthema X grandiflorum) belonging to the family Asteraceae has been cultivated for more than 3,000 years in Asia and Europe. It is currently one of the most popular flowers and also important for the worldwide floriculture industry. Viroids are the smallest pathogens composed of circular RNAs and cause infectious diseases in the plant host. So far, two viroids including Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) infecting chrysanthemum has been reported. Especially, CSVd infection leads to serious damage in chrysanthemum production with following symptoms like bleached leaves, stunting, and reduced level of anthocyanin. Therefore, it is desirable to make genetically modified (GM) chrysanthemums resistant to CSVd. To engineer viroid resistant chrysanthemums, RNAi mediated silencing system using partial RNA sequences of the viroid was applied. Four different vectors containing different sense/antisense RNA sequences of CSVd were constructed and used for Agrobacterium mediated transformation. Initially, a total of 39 transgenic lines carrying the individual CSVd RNA sequence were finally obtained. To test resistance against CSVd infection, 60 out of 145 GM chrysanthemum plants were inoculated with CSVd by the sap inoculation. Relative resistance level was determined by semi-quantitative RT-PCR using total RNAs extracted from both inoculated and upper systemic leaves and CSVd specific primers. Of 16 tested GM chrysanthemums, nine GM chrysanthemum lines showed very strong resistance against CSVd infection and/or replication. In general, GM chrysanthemums grow uniformly and are much healthier than non-GM chrysanthemums. This study is the first report which develops GM chrysanthemums resistant to CSVd using CSVd sense/antisense RNAs.