Kazushi Ohishi

Elimination of chrysanthemum stunt viroid from an infected chrysanthemum cultivar by shoot regeneration from a leaf primordium-free shoot apical meristem dome attached to a root tip

In this research we eliminated chrysanthemum stunt viroid (CSVd) from a highly infected chrysanthemum cultivar using a newly established method. ‘Piato’ is one of the most difficult cultivars in which to obtain CSVd-free plants by conventional methods. Leaf primordium-free shoot apical meristems (LP-free SAMs) of ‘Piato’ plants were dissected and attached to CSVd-free chrysanthemum or cabbage root tips. As shown by nested-PCR, CSVd was not detected in some shoots regenerated on both types of root tip. The production rates of CSVd-free plants using chrysanthemum and cabbage root tips were 14% and 3%, respectively. Regeneration of plants from LP-free SAMs of chrysanthemum plants by attaching these SAMs to root tips is an efficient method of generating CSVd-free chrysanthemum plants.

Evaluation of chrysanthemum stunt viroid (CSVd) infection in newly-expanded leaves from CSVd-inoculated shoot apical meristems as a method of screening for CSVd-resistant chrysanthemum cultivars

Summary A total of 199 chrysanthemum (Chrysanthemum × morifolium Ramat.) cultivars were used in screening for resistance to chrysanthemum stunt viroid (CSVd). Plants were inoculated with CSVd by attaching leaf primordia-free, shoot apical meristems to CSVd-infected root tips of a CSVd-sensitive cultivar. CSVd titres in the first and third youngest expanded leaves were analysed after 2 months in culture, and a systematic classification into four groups was conducted. Long-term cultivation records provided by a breeding nursery confirmed our findings of those screened cultivars classified in vitro as “non-detected-type” (i.e., those expected to have a high level of resistance). In addition, of the 15 candidate cultivars selected as “non-detected-type”, four cultivars were shown to have a high level of CSVd-resistance by ex vitro grafting experiments. From the results of our in vitro and ex vitro experiments, the advantages of our newly-developed in vitro-screening system are discussed.