Cryopreservation of Vitis vinifera via Droplet- vitrification

 In the present study, two Egyptian grapevine cultivars (Red romy and Ghariby) and a 
Chinese variety (Cabernet sauvignon) were successfully cryopreserved by droplet vitrification. 
Axillary shoot tips were excised from two months old plantlets cultured on solidified ½MS medium 
with 0.5mg benzyladenine, 3% sucrose and 0.7% agar (pH 5.8) at 25 ◦C, under a 12 h light/12 h 
dark photoperiod with a light intensity of 40 μE m–2 s–1. For vitrification, excised shoot tips were 
precultured on half strength MS solidified medium supplemented with 0.1 M sucrose for 3 days in 
darkness and then treated with a mixture of 2 M glycerol and 0.4 M sucrose (LS solution) for 20 
min at 25 ◦C. Shoot tips were then dehydrated with half-strength PVS2 vitrification solution (30% 
(w/v) glycerol, 15% (w/v) ethylene glycol, 15 % dimethylsulfoxide and 0.4 M sucrose in MS basal 
medium, for 30 min. This was followed by full strength PVS2 for (25min, 50 min or 60 min) at 0 ◦C 
before direct immersion in liquid nitrogen. The results showed that the mean percentage of survived 
shoot tips was not significantly different among the three genotypes, i.e., 66.67, 54.72 and 58.06% 
for Cabernet sauvignon, Red romy, and Ghariby, respectively. Also, the mean number of shoot tips 
regrowth was not significantly different, i.e. 57.50, 50.50 and 50.50 for Cabernet sauvignon, Red 
romy and Ghariby, respectively. The optimal duration of dehydration with PVS2 for survival and 
regrowth was 50 min. Ten ISSR primers were used for assessing the stability of the cryopreserved 
genotypes compared to the noncryopreserved. A negligible percentage of polymorphism was 
detected in the two cultivars Red romy (6.52%)   and Ghariby (2.26%) with no morphological 
changes after cryopreservation. While, the cryopreserved plantlets of the cultivar Cabernet 
sauvignon did not exhibit any variability at the morphological or molecular levels compared to the 
control (noncryopreserved plantlets).