73. Evaluation of the survival of somatic embryos of Petiveria alliacea L. cryopreserved after different pretreatments with sucrose and PVS2

Abstract

Petiveria alliaceae L. (Phytolacaceae) is a herbaceous plant species, native to humid tropical regions, traditionally used in folk medicine due to its broad spectrum of pharmacological actions, mainly immunomodulatory, anti-inflammatory, cytotoxic and neurotoxic. In Brazil, this species is produced only in home gardens. For this reason, the harvest predatory for use as a drug is causing genetic erosion in natural populations. In this context, biotechnology techniques for crop production are suitable for the production and conservation of germplasm. In vitro culture via somatic embryogenesis, was previously established for this species and the purpose of this study was to evaluate the survival and recovery of embryos cryopreserved after treatment with cryoprotectants. Somatic embryos from leaf explants cultured on MS medium supplemented with PIC (5 mg/l) were subcultured and maintained for one week in 1/2 MS medium with 2% Phytagel in a growth chamber at 30 °C ± 2 °C, with luminous intensity of 46 μM m −2 s −1 and photoperiod of 16 h. Then the embryos were distributed in vials containing MS medium supplemented with different concentrations of sucrose (0.3–0.5–0.7 M). Then the material was submitted to PVS2 cryoprotectant solution at different time periods (0, 15, 30, 45 or 60 min) and then frozen in liquid nitrogen (LN 2 ) for 24 h. In each sample 5–10 embryos were used to evaluate the water content, by the ratio between fresh and dry weight. Thawing was rapid at 40 °C for 2 min in a water bath. The survival of embryos was assessed, after the pre-treatment and after freezing in LN 2 , by testing the TTC (2,3,5 triphenyltetrazolium chloride hydrochloride), by change of color visually detected in embryonic tissues. The recovery was determined by cell proliferation, after 30 days in culture conditions described. Pretreatment with sucrose in embryos caused dehydration proportional to the concentrations tested, and does not affect their survival. Treatment with PVS2 provided a 100% survival, when the embryos were exposed for 15 min, however, exposure for a longer period was lethal to these tissues. The recovery of these embryos was recorded after 30 days of culture by number of embryos observed. Given that there are no previous reports of cryopreservation of Petiveria alliacea germplasm, the optimization and tolerance to dehydration of explants to cryoprotectant solutions are key steps for establishing a vitrification protocol, aiming at long-term conservation of the botanical material, and can benefit the herbal industry.