Shoot recovery and genetic integrity of Chrysanthemum morifolium shoot tips following cryopreservation by droplet-vitrification

Abstract

We reported here an efficient, widely applicable droplet-vitrification cryopreservation for shoot tips of Chrysanthemum morifolium. Nodal segments, each being 0.5cm in length and containing one bud positioned on nodes 3–7, were taken from 6 weeks old stock shoots and cultured on a shoot maintenance medium (SMM) for 12 days to promote bud elongation. Shoot tips (2.0mm in size) containing 5–6 leaf primordia were excised from elongated buds and precultured on Murashige and Skoog medium (MS) containing 0.5M sucrose for 1 day. Precultured shoot tips were loaded, dehydrated with PVS2 for 30min at 0°C and then transferred onto droplets containing 2.5μl PVS2 on aluminum foils (2cm×0.8cm), prior to a direct immersion in liquid nitrogen (LN) for 1h. Thawed shoot tips were post-cultured on shoot recovery medium containing MS supplemented with 0.05mgL−1 GA3 in the dark for 3 days and then transferred on the same medium under standard culture conditions for shoot recovery. The droplet-vitrification procedure resulted in the highest (83%) and lowest (43%) shoot regrowth rates for C. morifolium ‘Japanese Red’ and ‘Xizi Qiuzhuang’, with an average rate of 68% in six C. morifolium genotypes tested. Histological observations showed that the pattern and percentage of surviving cells were similar in cryopreserved shoot tips of these two genotypes. No polymorphic bands were detected by simple sequence repeats (SSR) and ploidy levels analyzed by flow cytometry (FCM) were maintained in plantlets regenerated from cryopreserved shoot tips of the two genotypes.