Toxicity of Cryoprotectants to Gametophytic Thalli of Red Algae Porphyra yezoensis

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    We assessed the toxicity of cryoprotectant agents (CPAs) to gametophytic thalli of red alga Porphyra yezoensis at room tempera-ture. The CPAs used were: dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GC), 1,2-bu-tanediol (1,2-BD), 1,3-bu-tanediol (1,3-BD), 2,3-butanediol (2,3-BD), 1,3-propanediol (1,3-PD) and propylene glycol (PG). CPA concentrations of 10, 15, 20, 25, 30, 35, 40, 45, and 50% were employed with 30 or 60 s immersion. The toxicity of the eight CPAs to gametophytic thalli of P. yezoensis was in the order: 1,3-BD < DMSO ? 2,3-BD ? PG ? EG < GC < 1,3-PD ? 1,2-BD. All thalli were more sensitive to high CPA concentrations, and most (>75%) thalli survived exposure to 10-25% CPA for 60 s. These data will facilitate selection of the optimal cryoprotectant concentration for cryopreservation of P. yezoensis thalli.


    Red algae , Porphyra yezoensis , Cryoprotectant agents , Toxicity

  • Introduction

    Cryoprotectant agents (CPAs) are chosen because of their useful non-crystallization property of hydrate formation at low temperatures in aqueous solution (Wowk, 2010). How-ever, the toxicity of which is a fundamental limiting factor for the successful cryopreservation of living cells (Fahy, 2010). So far, most reports of CPA toxicities were focused on fin-fishes such as flounder embryos/sperm (Zhang et al., 2005), zebrafish (Zhang and Rawson, 1993; Liu et al., 2001), medaka (Yang et al., 2010), and so on. However, little research on the toxicity of CPAs to macroalgae has been conducted.

    In order to determine the tolerance of gametophytic thalli of Porphyra yezoensis to their toxicity without freezing, we used eight CPAs (sulfoxide, diol and tiol groups) - dimethyl sulfoxide (DMSO), ethylene glycol (ethane-1,2-diol, EG), glycerol (propan-1,2,3-tiol, GC), 1,2-butanediol (1,2-BD), 1,3-butanediol (1,3-BD), 2,3-butanediol (2,3-BD), propylene glycol (1,2-propanediol, PG) and 1,3-propanediol (1,3-PD).

    Materials and Methods

      >  Algal strains and culture conditions

    Gametophytic thalli of P. yezoensis were obtained from TU-1 (Kuwano et al., 1996). Thalli were cultured in a 1 L flask containing Provasoli’s enriched seawater medium (Provasoli, 1968) and pieces of synthetic fiber (3-5 cm in length, 0.25 mm in diameter) at 15℃ and irradiated at 60 ㎛ol photons m-2 s-1 with a 10:14 h light:dark photoperiod for two weeks. Mono-spores were released from thalli attached to the synthetic fi-bers and grew into young thalli of identical genotype.

      >  CPA toxicity test

    Nine concentrations (10, 15, 20, 25, 30, 35, 40, 45 and 50%) of each of the eight CPAs (DMSO, EG, GC, 1,2-BD, 1,3-BD, 2,3-BD, PG and 1,3-PD) were tested. All chemicals were re-agent-grade, and cryoprotectant solutions were prepared in 0.2 ㎛-filtered seawater. Synthetic fibers with attached thalli were cut to 0.5-1 cm lengths and transferred to CPA solutions for 30 or 60 s. Thalli were then stained with 0.1% erythrosine (in seawater) for 20 min and washed with fresh seawater.

    Thalli viability was examined under a light microscope (BX-50; Olympus, Tokyo, Japan). The survival rates were ex-

    pressed as the ratio of the number of living thalli to the total number observed.

    Results and Discussion

    Fig. 1 shows survival of the gametophytic thalli of P. yezoensis according to CPA toxicity. The ability of thalli to survive exposure to CPAs differed, and their tolerance of CPA toxicity tended to decrease with increasing CPA concentra-tion. The thalli showed the highest tolerance to 1,3-BD, and the lowest to 1,2-BD. Glycerol and 1,3-PD were lethal to thalli at 40%, and no thalli survived exposure to 35% 1,2-BD. How-ever, thalli maintained viability (>70%) when exposed to 50% 1,3-BD for 30 s. Therefore, 1,3-BD exhibited the lowest toxic-ity, followed by DMSO, 2,3-BD, PG and 1,2-BD.

    In aqueous solutions, 2,3-BD exhibits glass formation supe-rior to that of other CPA, including 1,2-PD and 1,3-PD (Bau-dot et al., 1996). Shaw et al. (1995) reported that propanediol forms a stable vitreous state on cooling to a greater degree than other CPA in human and mouse embryos. We found that gametophytic thalli of P. yezoensis were highly tolerant of 1,3-BD, even though the determinations were conducted at room temperature.

    Selection of a suitable CPA is important for successful cryopreservation. In general, DMSO, GC and methanol have been widely used in algae (Taylor and Fletcher, 1999). DMSO is the most-commonly used CPA for macroalgae, Porphyra spp. (Kuwano et al., 1996; Liu et al., 2004; Zhou et al., 2007), Gracilaria spp. (van der Meer and Simpson, 1984), and mi-croalgae (Canavate and Lubian, 1994). Glycerol is a polyol with three hydroxyl groups and is a highly hydroscopic, vis-cous, odorless, and sweet-tasting fluid of low toxicity (Fluhr

    et al., 2008). In this study, glycerol was less toxic than DMSO. Gwo et al. (2005) reported low glycerol toxicity in the micro-algae, Nannochloropsis oculata at room temperature.

    CPA toxicity depends on species, developmental stage and storage temperature. In particular, it is known that DMSO is less toxic at 0-5℃ than at higher temperatures (Hubalek, 2003). Therefore, the viability of thalli may be enhanced by low temperature preservation.

    CPA toxicity occurs after permeating into the cell, so the more toxic CPAs are likely to be more membrane permeable, and vice versa. However, greater protection also requires more permeability (Zhang et al., 2005). To date, CPA concentrations before freezing have been considered because they are cryo-protective to cells, but induce cryoinjury in themselves (Fahy, 1986). Moreover, determining the optimum CPA requires a process of trial and error. Therefore, more investigation is re-quired to determine the optimum CPA and its relationship with chemical reactions, temperature, and so on.

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  • [Fig. 1.] Toxicity of different concentrations of cryoprotectants to gametophytic thalli of Porphyra yezoensis illustrated as photograph. DMSO, dimethyl sulfoxide. Scale bars = 50 ㎛.
    Toxicity of different concentrations of cryoprotectants to gametophytic thalli of Porphyra yezoensis illustrated as photograph. DMSO, dimethyl sulfoxide. Scale bars = 50 ㎛.
  • [Fig. 1.] Continued.
  • [Fig. 1.] Continued.