Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia

Petunia (Petunia x hybrida Vilm.) is an important horticultural crop conserved in the National Genebank of China. Here, a droplet-vitrification cryopreservation protocol was developed for petunia shoot tips. An orthogonal array experiment and additional one-factor experiments were performed to optimize key variables, including the age of in vitro plants, the concentration of sucrose in the preculture solution, the preculture duration, the duration of osmoprotection (2.0 M glycerol and 0.4 M sucrose), the length of exposure to and concentration of plant vitrification solution 2 (30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.4 M sucrose), and the recovery medium. By using the combined results of the orthogonal and one-factor experiments, the droplet-vitrification procedure for petunia cultivar Niu 2 was formulated efficiently and effectively. The highest regrowth levels were obtained using the following procedure: shoot tips were dissected from in vitro plantlets that were 20 d old, precultured in MS liquid medium with 0.2 M sucrose solution for 2 d, incubated with osmoprotection solution for 30 min at 25A degrees C, cryoprotected with PVS2 for 30 min at 0A degrees C, and rapidly immersed in liquid nitrogen. Cryopreserved shoot tips were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25A degrees C and regrown on solidified MS basal medium with half concentration of NH4NO3, KH2PO4, KNO3, and sucrose. Regrowth levels were as high as 80%. No morphological changes were observed and amplification of 15 simple sequence repeats revealed no genetic alterations after cryopreservation.