Cryopreservation of Chrysanthemum morifolium ( Dendranthema grandiflora Ramat.) using different approaches

Abstract

Chrysanthemum species are grown both as ornamentals and for the production of pyrethrum. Recent increased production and breeding efforts have raised the need for the conservation of valuable germplasm. Chrysanthemum has been cryopreserved by controlled-rate-freezing as early as 1990. We report here deep-freezing of shoot tips of C. morifolium var. Escort by different technical procedures: controlled-rate-freezing, encapsulation/dehydration, ultra-rapid-freezing by the droplet method and vitrification. While vitrification yielded the highest shoot regeneration rates, the very simple droplet method was also successful in this respect. Droplet freezing was successfully performed with nine cultivars. Our results open the door to the successful use of alternative methods if one method fails to cryopreserve a variety. Furthermore, it enables comparative investigations of genetic stability and cyro-injury to be carried out.

Comparison of Cryopreservation Methods of Vegetatively Propagated Crops Based on Thermal Analysis

“…Water content decrease is achieved by preculturing explants with osmotic compounds, air desiccation or vitrification. On bases, vitrification (Sarkar & Naik, 1998), droplet (SchaferMenuhr et al, 1996) and recently vitrification-droplet (Halmagyi et al, 2004;Schafer-Menuhr et al, 1996) methods were developed or adapted for potato.…”

Cryopreservation and genetic stability of Dendranthema grandiflora Tzvelev in vitro cultures

“…Although no further genetic analysis of the variant material was done in that study, the authors concluded that these results should be taken into consideration when cryopreserving chrysanthemum. The same recommendation was done some years later by Halmagyi et al (2004) in their work on cryopreservation of chrysanthemum using different approaches. They suggested that comparative investigations of genetic stability and cryo-injury should be carried out.…”

“…Fukai and Oe (1990) demonstrated that after controlled freezing only a small part of the apex survived and that treatment with DMSO (2 days culture on medium with 5% v/v DMSO, and then incubated for 1h at 0 ºC with 10% DMSO) caused abnormal leaf development. In other cryopreservation works the possible adventitious origin of the recovered shoots is stated (Halmagyi et al 2004, Fukai et al 1991. Hitmi et al (1999aHitmi et al ( , 2000a reported no callus formation in recovered shoot from cryopreserved apices.…”

“…Sakai et al (2000) obtained lower recovery with cv. 'Shuhounochikara' (20%) than Halmagyi et al (2004) with cv. 'Escort' (45%).…”

“…In two of those works, several cryopreservation methods were compared (Halmagyi et al 2004, Sakai et al 2000. Among the ones employed by Halmagyi et al (2004) the droplet ultra-rapid freezing method was tested in nine cultivars, with regeneration ranging from 4 to 70 %, although experiments were not very repetitive. Also in that work, the droplet-vitrification method resulted in the highest shoot regeneration percentage (60%) in the one cultivar more thoroughly studied ('Escort').…”