To develop a procedure for regenerating plants directly from pyrenes, as an alternative to in vitro embryo culture, germination studies were done on maté tree, Ilex paraguariensis, seeds using aseptic conditions. The effect of the endosperm, the woody endocarp and cold treatment on embryo development and germination were examined by culturing isolated embryos, intact, acid-scarified and bisected pyrenes from mature fruits. The results show that maté tree plantlets may be obtained by in vitro culture of bisected pyrenes on solidified (0.65% agar) quarter-strength Murashige and Skoog medium containing 3% sucrose. It is recommended to pre-culture the pyrenes at 4 ± 2°C to obtain a higher germination rate and more vigorous seedlings. This is the first report of the in vitro culture of Ilex pyrenes. Compared to the culture of isolated embryos this procedure shortens the duration of the embryo culture technique and minimizes damage to the young embryos.

An in vitro culture protocol was developed that increased the germination percentage and decreased the lag time to germination for Ilex dumosa R. pyrenes as a tool for replacing the laborious task of embryo rescue technique. This method involves transversely cutting surface-sterilized pyrenes with a scalpel blade, then placing the micropylar one-third end with the rudimentary embryo (≈0.25 mm long) on solidified (agar 0.65%) quarter-strength salts and vitamins of Murashige and Skoog, 1962 medium with 3% sucrose, and incubating in a growth room at 27 ± 2 °C with a 14-h photoperiod (116 μmol·m−2·s−1). Most of the cut pyrenes (greater than 50%) germinated within the first month after inoculation and achieved maximum germination (≈70%) in 2 months compared with whole pyrenes, which began to germinate 3 months after sowing and required more than 8 months for maximum germination (37%). Moreover, the germination percentage of cut pyrenes was significantly higher than the germination of isolated embryos (34%). Thus, the cut pyrenes culture is a simpler and more effective technique than embryo rescue. Easily, on average, a trained operator is able to culture ≈1000 cut pyrenes per day instead of ≈100 isolated embryos.

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Tropical Ilex species have recalcitrant seeds. This chapter describes protocols for long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. microdonta, I. integerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos are aseptically removed from the seeds and precultured (7 days) in the dark at 27±2°C on solidified quarter-strength Murashige and Skoog medium with 3% sucrose and 0.1 mg/L zeatin. The embryos are then encapsulated in 3% calcium alginate beads and pretreated at 24-h intervals in liquid medium supplemented with progressively increasing sucrose concentrations (0.5, 0.75, and 1 M). The beads are dehydrated for 5 h with silica gel to 25% water content (fresh weight basis) and then placed in sterile 5-mL cryovials. Then the beads are either plunged rapidly in liquid nitrogen where they are kept for 1 h (rapid cooling), or cooled at 1°C/min to -30°C and then immersed in liquid nitrogen for 1 h (slow cooling). After cryopreservation, the beads are rewarmed by immersion of the cryovials for 1 min in a water bath at 30°C. Finally, the beads are transferred onto culture medium (1/4MS, 3% sucrose, and 0.1 mg/L zeatin, solidified with 0.8% agar) and incubated in a growth room at 27±2°C under a 14-h light (116 μmol/m2/s) and 10-h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on the species and the treatment) were obtained with the cryopreserved embryos.