Cryopreservation of Embryonic Axes and Axillary Buds of `Pineapple' Sweet Orange [Citrus sinensis (L.) Osb.] Encapsulated in Alginate Gel

Abstract

Economically, citrus is the second most-important fruit crop grown worldwide; thus germplasm conservation of commercial cultivars, as well as of wild relatives, is essential. Presently, citrus germplasm has been conserved mainly in field genebanks. This approach is helpful; however, it is costly, exposes germplasm to climatic and biological hazards, and is not a long-term conservation system. Cryopreservation (conservation in liquid nitrogen, at –150°C to –196°C) is a technique that can ensure long-term storage of plant material. Attempts to cryopreserve citrus are restricted to a few reports, but the results obtained are encouraging. The basic purpose of this study is to define cryopreservation protocols for embryo axes and axillary buds of `Pineapple' sweet orange using the encapsulation-dehydration method. Embryo axes encapsualted in Na-alginate beads, precultured with high levels of sucrose and dehydrated over silica gel before freezing in liquid nitrogen had 60% survival. No survival was obtained for buds treated the same way, however buds isolated from plants acclimated at 0°C over a 30-day period survived exposure to –20°C when slow cooled at 2°C/hour. Additional experiments will combine cold acclimation, slow cooling and pre-treatment with sugars and other chemical compounds as an attempt to enhance cold hardiness of axillary buds and obtain survival after freezing in liquid nitrogen. Different approaches will be used to increase embryo axes survival rates.