Geneticallly enginereed trehalose accumulation improves cryopreservation tolerance of chrysanthemum (Dendranthema grandiflorum kitam.) shoot-tips

Shoot-tips isolated from two transgenic lines of chrysanthemum (Dendranthema grandiflorum Kitam.) var. Indianapolis in vitro plantlets with induced capacity to biosynthesize trehalose, and from a non-transformed line, were subjected to cryopreservation using a vitrification procedure. After dissection, apices were precultured on semi-solid MS medium with 0.3 M sucrose for 4 days, loaded in a 0.4 M sucrose + 2 M glycerol solution for 20-30 min and exposed to PVS2 or PVS3 vitrification solutions for 0, 20, 40 or 60 min at room temperature prior to rapid immersion in liquid nitrogen. The highest shoot regeneration after cryopreservation was obtained with exposure to either PVS solution for 40 min. Plant regeneration from cryopreserved shoot-tips ranged between 48 percent and 67 percent for transgenic lines and between 33 percent and 36 percent for non-transgenic lines. No polymorphic loci were detected in plantlets regenerated from cryopreserved and non-cryopreserved shoot-tips with RAPD techniques using eight primers that amplified 101 monomorphic loci.