Abscisic acid increases the viability of cryopreserved shoot tips of Lilium pumilum by regulating the osmotic stress and programmed cell death

Abstract

Abstract Lilium pumilum plays an important role in lilium resistance breeding because of its wide distribution and strong stress resistance. It is of great significance to establish an efficient cryopreservation procedure for Lilium pumilum. In this study, the cryopreservation protocol of Lilium pumilum shoot tips by vitrification was established as follows: The 2 months old tissue-culture plants, which outer scales of small bulbs at the base of small plants were removed and only the 1–2 layers scales that encasing the shoot tips were retained were precultured on Murashige Skoog medium (MS) with 0.5M sucrose and 25µM abscisic acid(ABA) for 7 days at 4℃ in the dark; Then, the 2 mm shoot tips were gain under stereoscopic microscope in an ultra-clean workbench, and 10 shoot tips were put into a 1.5ml frozen centrifuge tube and added 1ml Loading solution (MS + 0.4M sucrose + 2M glycerin) for 20min at 25°C; then the Loading solution was removed, precooled PVS2 solution (MS + 0.4M sucrose + 30% glycerol + 15% ethylene glycol + 15%DMSO) was added for 80min at 0℃; The tube was plunged into liquid nitrogen and keeping. When it was needed, the tube was removed from the liquid nitrogen tank, and rapidly thawed in 37 ℃ water bath for 60s; After that, PVS2 solution was removed and shoot tips were washed twice for 10 min each time with unloading solution (MS + 1.2M sucrose) at 25 ℃. Subsequently, the shoot tips were incubated on medium (MS + 30 g·L− 1 sucrose + 6.5 g·L− 1 agar) for regeneration. With this procedure, the shoot tips survival rate was 93.64% and the recovery rate was 63.33%. Further biochemical studies showed that adding exogenous ABA increased the contents of endogenous osmotic regulatory substances (proline, soluble sugar, soluble protein) and reduced the activities of caspase-3-like and caspase-9-like enzyme that characterize programmed cell death (PCD). This suggests that the addition of ABA during preculture phase improved viability of shoot tips by affecting osmotic stress and PCD.