Cryopreservation of in vitro almond shoot tips by vitrification

Shoot tips of two almond scion cultivars, 'Ne Plus Ultra' and 'Nonpareil 15-1', and one almond/peach hybrid rootstock were successfully cryopreserved using a one-step vitrification technique. Three week old in vitro cultures were cold-hardened at 4 degrees C on the multiplication medium (Murashige and Skoog for 'Ne Plus Ultra' and the hybrid rootstock; Almehdi and Parfitt for 'Nonpareil 15-1') for three weeks. Shoot tips, 2-2.5 mm long, were excised and precultured for 1 d at 4 degrees C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25 degrees C for 45 min for the almond scion cultivars and 60 min for the hybrid rootstock, and then stored under liquid nitrogen (LN) for at least 3 d. After rapid thawing at 30 degrees C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH4NO3 or (NH4)(2)SO4. Shoot regeneration was usually observed within 2-3 weeks. Survival after LN, recorded as the percentage of shoot tips that produced at least one new shoot four weeks after thawing, was 87.5, 60.0 and 72.5% for 'Ne Plus Ultra','Nonpareil 15-1' and the hybrid rootstock respectively. The one-step vitrification method is a promising simple technique for cryopreserving almond scion and rootstock shoot tips from in vitro cultures.