5.3.22
TRANSGENIC CHRYSANTHEMUM EXPRESSING DOUBLE-STRANDED SPECIFIC RIBONUCLEASE (PAC 1) IS RESISTANT TO CHRYSANTHEMUM STUNT VIROID

T OGAWA, T HORI, M TSUKAHARA, M YOSHIOKA, M KAKITANI, I ISHIDA and T TOGURI

Central Laboratories for Key Technology, Kirin Brewery Co. Ltd, 1-13-5 Fukuura, Yokohama 236, Japan

Background and objectives
Viroids are the smallest infectious agents that cause serious diseases in higher plants. It is difficult to control viroid disease, because factors essential for their survival in host plants are also essential for the host itself. Transgenic potato plants expressing yeast-derived double-stranded RNA-specific ribonuclease (pac1) showed resistance to potato spindle tuber viroid (PSTVd) [1]. We applied this pac1-mediated viroid resistance system to the chrysanthemum-CSVd combination.

Materials and methods
Transformation using in vitro-grown chrysanthemum (D. grandiflore cv. Reagan) was carried out by the Agrobacterium-mediated leaf disc method. Chrysanthemum stunt viroid (CSVd) was kindly provided by T. Sano (Hirosaki University, Japan). The resistance of transgenic pac1 chrysanthemum against CSVd was evaluated by mechanical inoculation of low molecular-weight RNA which was extracted from chrysanthemum leaves infected with CSVd. Detection of CSVd in the inoculated plants was carried out by imprint-hybridization or microplate hybridization methods.

Results and conclusions
Accumulation of CSVd was not detected in about half of the plants in some transgenic lines 50 days after inoculation, but was detected in all the control plants. Moreover, the accumulation of CSVd in transgenic plants, which was detected in the leaves, was much lower than that in the control plants. Plants propagated by cuttings from transgenic plants which escaped CSVd-infection were free of CSVd. These results showed that transgenic chrysanthemum expressing pac1 is resistant to CSVd. This system will be widely applicable to the protection of viroid disease in various plants.

References
1. Sano T, Nagayama A, Ogawa T et al., 1997. Nature Biotechnology 15, 1290-1294.