Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 99

Other than a report of low temperature storage of cherimoya pollen (Lora et al., 2006), there has been little activity with respect to cryopreservation of this and related species. Thinh (1997) described cryopreservation by vitrification to c.20 tropical monocotyledonous plants. This protocol with some variation has been tested with in vitro-grown shoot tips of cherimoya ‘Fino de Jete’ with limited success. Several cryoprotectors have been assayed and the best results were obtained with the pretreatment of hypocotyl segments (2 mm) with MS medium (Murashige and Skoog, 1962) supplemented with 0.4M glycerol and 0.3M sucrose during 3 days. This solution was replaced with a sterile solution of PVS-2 (Sakai et al., 1990, 1991) consisting in MS medium plus 3.26 M glycerol, 2.42 M ethylene glycol, 1.9 M DMSO and 0.4 M sucrose (pH 5.7) and maintained on ice. After 1 h in this solution, the explants were transferred to cryovials and immersed in liquid nitrogen (LN) for 3 h. Material was thawed by plunging the cryovials in water at 25°C for 3 min. The PVS-2 solution was then eliminated by rinsing 2× with a solution of MS medium with 1.2 M sucrose. After that, the hypocotyl explants were dried in sterile filter paper in a laminar flow cabinet and transferred aseptically to MS medium supplemented with 0.7 μM BA, 3% sucrose, 0.8% agar and 100 mg/l cefotaxime. After bud sprouting, shoots were incubated for 6 h in MS medium supplemented with 5.37 μM NAA + 0.29 μM GA3 + 4.4 μM BA for shoot development. Shoots were then transferred to micropropagation medium (Padilla and Encina, 2004) (see Section 3, Micropropagation). A low recovery rate (15%) was obtained.

An encapsulation–dehydration procedure based on the procedure for shoot tips of sugarcane (Gonzalez-Arnao and Engelmann, 2006) was attempted with cherimoya (Encina and Carmona, unpublished data). The optimum time and method of dehydration was determined. A moisture content c.25% was obtained when ten explants were kept for 3 h in a container with 50 g of silica gel.