Cryopreservation of Ilex dumosa (Aquifoliaceae) germplasm.

Abstract

Most of the subtropical Ilex species have recalcitrant seeds and therefore, not suitable for long-term preservation using conventional seed storage methods. Thus, the germplasm of Ilex spp. is maintained in the field as ex situ genebanks. This work describes experiments demonstrating the feasibility of long-term conservation of I. dumosa through cryopreservation of both whole seeds and zygotic rudimentary embryos. Lately, this species has received greater attention from plant breeders because, besides the quality of its leaves for making the stimulatory beverage named 'maté' with less caffeine than the ones from I. paraguariensis, the plants are resistant to some pests. For cryopreservation of zygotic embryos and apical shoot-tips, the explants were aseptically removed, encapsulated in 3% calcium alginate beads and pregrown for 24 h intervals in liquid medium enriched with progressively increasing sucrose concentrations (0.5, 0.75 and 1 M). The beads were then dehydrated with silica gel to 25% water content and plunged rapidly in liquid nitrogen. The beads were rewarmed by immersion in a water bath at 30°C. Finally, the beads were transferred onto culture medium (1/4 MS, 3% sucrose, 0.1 mg L-1 zeatin, solidified with 0.8% agar) and incubated in a growth room at 27°C under a 14 h photoperiod (116 μmolm-2 s-1). By culturing cryopreserved embryos, as much as 42% of them produced plants. However, no plants were recovered when apical shoot-tips were cryopreserved. In addition, a procedure for cryopreservation of mature intact seeds of I. dumosa by desiccation with silica gel and rapid freezing was developed. Up to 40% of the cryopreserved seeds produced plants when they were cultivated in vitro in an appropriate culture medium.