Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1-4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg 1(-1) thiamine, 0.2 mg 1(-1) biotin, 0.2 mg 1(-1) pyridoxine, 0.25 mg 1(-1) 6-benzylaminopurine (BAP), 0.5 mg 1(-1) gibberellic acid (GA(3)) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10-30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g 1(-1) agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal. 67.73% for cv. Fairy and 57.57% for cv. Maidy.