Preliminary results on desiccation and PVS2-vitrification of 'Carrizo' citrange synthetic seeds as pre-treatments to cryopreservation

Advanced cryopreservation techniques can provide cheap and safe back-ups to traditional ex situ conservation approaches of fruit germplasm, including Citrus species. At the ultra-low temperature of liquid nitrogen (-196°C), plant cells, tissues and organs can be safely stored for theoretically unlimited time. Among the innovative cryoprocedures, storage of synthetic seeds (i.e., encapsulated in vitroderived propagules) in liquid nitrogen has repeatedly proven to be one of the most effective. The technique is a multi-step process, including (i) pre-culturing of the encapsulated explants (generally shoot tips or nodal segments) in sucrose-containing medium, (ii) removal of the freezable water from the cells by desiccation over silica gel or by treatment with highly-concentrated cryoprotectant solutions (such as PVS2), (iii) direct immersion of synthetic seeds in liquid nitrogen, (iv) thawing and (v) conversion of synthetic seeds into plantlets. As a preliminary step towards the optimization of a cryopreservation protocol for 'Carrizo' citrange [Citrus sinensis (L.) Osbeck x Poncirus trifoliata L. Raf.] synthetic seeds, this study investigated the effect on moisture content and regrowth rate of different pre-freezing treatments, i.e., (i) the exposure to increasing sucrose concentrations (0.1, 0.3 and 0.7 M, each one for 24 h), (ii) the desiccation (from one to 7 h), and (iii) the treatment with PVS2 (90 or 180 min, at 0°C). Best result was achieved when sucrose preculture was followed by a 5-hour exposure to silica gel; this treatment induced a consistent reduction of synthetic seed moisture content (MC) (from the initial 82 up to 23%), while the regrowth rate was affected to more limited extent (from 62 to 48%).