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- Author or Editor: Natalia R. Dolce x
- HortScience x
This research aimed at evaluating the desiccation tolerance and ability to withstand cryostorage of intact seeds of seven South American Ilex species, and comparing different methodologies for in vitro germination of fresh and cryostored seeds. Intact seeds were silica gel–desiccated from 2 to 14 hours, placed in cryovials, and immersed in liquid nitrogen (LN). Survival was assessed through in vitro germination of intact seeds, bisected seeds, or isolated embryos. Seeds of the seven Ilex species (Ilex brasiliensis (Sprengel) Loes., Ilex brevicuspis R., Ilex dumosa var. dumosa R., Ilex integerrima (Vell. Conc.) R., Ilex paraguariensis A. St. Hil., Ilex pseudoboxus R., and Ilex theezans R.) tolerated desiccation to ≈6% moisture content (MC) and could be successfully cryopreserved when MC decreased between 6.4% and 8.4% depending on the species, before immersion in LN. In addition, it was established as the optimal condition for in vitro seed germination of the seven Ilex species. A simple and cost-effective cryogenic procedure (which did not require the use of cryoprotectants or sophisticated facilities) was defined for seeds of seven Ilex species, which provides a new alternative for safe long-term preservation of Ilex germplasm.
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.
Pollen storage is of great importance for plant breeding and production besides an efficient means for preservation of haploid gene pool of plant genetic resources and rare or endangered species. Pollinia of Cohniella cepula were stored over 1 year at 4, −20, −70, and −196 °C. Fertilizing ability of fresh and stored (30 to 360 days) pollinia was determined by the fruit and seed formation for each treatment, as well as by the seed viability, in vitro seed germination, and seedling growth. Pollinia stored at −70 and −196 °C showed high fertilizing ability (94.4% to 100.0%) even 1 year after collection, revealing no significant differences with fresh pollinia. Seeds from all treatments showed high viability (91.2% to 94.3%) through the 2,3,5-triphenyltetrazolium chloride (TTC) reduction assay and high in vitro germination (91.7% to 97.3%). Thus, successful ultracold storage of C. cepula pollinia was feasible without any desiccation, cryoprotection, or precooling treatment before placing into an ultra freezer (−70 °C) or immersing in liquid nitrogen (LN) (−196 °C).