Cryopreservation of date palm tissue cultures by vitrification and assessment of genetic stability using ISSR analysis

Abstract

In this work, a successful cryopreservation method of date palm tissue cultures by vitrification was established. Callus and somatic embryos of three date palm cultivars were exposed to dimethylsulfoxide (DMSO) and Plant Vitrification Solution 2 (PVS2) for different durations before plunging into liquid nitrogen for cryoprotection. After freezing phase, the cryopreserved cultures were thawed and cultured on recovery media. Among different times of exposure to DMSO (10%), 60 min gave best results of survival and regrowth of the date palm tissue cultures. Regarding recovery, ‘Zaghlool’ cultivar gave the highest values of callus fresh mass (0.5 g) and growth value (1.00). Also, the highest differentiation percentages (75%) and number of proliferated shootlets (8.50) were recorded with ‘Zaghlool’ cultivar. Concerning PVS2, survival and regrowth gradually increased as exposure time increasing till 30 min and then decreased. Unlike of callus cultures, the survival and regrowth rates of somatic embryos were relatively higher at all PVS2 exposure time. With regard to recovery, the genotype effect took same trend in cryopreservation of callus cultures, since ‘Zaghlool’ was superior in growth parameters in comparison to the other two cultivars. On the other hand, genetic stability of the cryopreserved date palm tissue cultures was assessed using ISSR markers. Among four screened primers, three gave monomorphic bands; all of these banding profiles were similar to those of non-cryopreserved cultures. Generally, the results of ISSR showed high genetic similarity suggesting that cryopreservation using vitrification does not affect genetic stability of date palm tissue cultures. The results of this study demonstrated PVS2 was more effective in cryoprotection of date palm tissue cultures compared with DMSO and cryopreservation by vitrification is a suitable tool for conservation of genetic stable date palm germplasm.