Preliminary Results on Desiccation and PVS2-Vitrification of 'Carrizo' Citrange Synthetic Seeds as Pre-Treatments to Cryopreservation

Abstract

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 degrees 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 vitro-derived 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 degrees 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%).