Simple cryopreservation protocol with an encapsulation technique for tobacco BY-2 suspension cell cultures

Abstract

Tobacco BY-2 cells were successfully cryopreserved by a simple slow prefreezing (equilibrium freezing) method using an encapsulation technique. After the cells were immobilized in alginate gel beads, the beads were treated with cryoprotectant solution (2 M glycerol, 0.4 M sucrose) for 45 min. The beads were then transferred to a laboratory freezer at Ϫ30°C, stored for 2 h, and then immersed in liquid nitrogen. To initiate the regrowth of cells, the beads were warmed in a water bath. Following dilution of cryoprotectant solution, the beads were suspended and cultured in normal medium. With this method, suspension cell cultures were regrown within 7 days. There were no differences in the morphology or growth profiles between cryopreserved cell cultures and the original cell cultures.

Cryopreservation of Plant Genetic Resources

“…In this cryopreservation method, mixtures of glycerol and sucrose or DMSO and sorbitol are used as cryoprotectants Maruyama et al, 2000). In this cryopreservation method, although 'naked' samples are used, Kobayashi et al (2005) utilized cells encapsulated with alginate beads in the suspension cells of tobacco. Fig.…”

Cryopreservation of tobacco BY-2 suspension cell cultures by vitrification with encapsulation

“…In the control experiments, the beads were cultured in mLS medium without any treatment (encapsulated control), and the PVS2-treated beads were transferred to dilution medium without immersion in LN (treated control). The viability of the cells was determined by an Evans blue exclusion method 1 day after thawing (Kobayashi et al 2005). This method is effective in measuring the viability rate of dispersed suspension cells such as BY-2.…”

“…Cryopreservation in liquid nitrogen (LN) provides a reliable method for long-term storage of BY-2 cells and helps to secure against loss of the cell line (Engelmann 2000;Sakai 2002). We recently reported successful cryopreservation of BY-2 cells by a simplified slow prefreezing method combined with an encapsulation technique using alginate gel (Kobayashi et al 2005).…”

“…'Bright Yellow-2' were subcultured from stock cultures preserved by the National BioResource Project (NBRP) of Ministry of Education, Culture, Sports, Science and Technology, Japan, and were maintained in modified Linsmaier and Skoog (mLS) medium (Nagata et al 1992). Cells were collected on day 3 after subculture and encapsulated in alginate gel beads (approximately 4 mm in diameter) at a cell density of 0.2 ml packed cells ml Ϫ1 as described previously (Kobayashi et al 2005). Cryopreservation using the vitrification procedure with encapsulation was performed as described by Hirai and Sakai (1999).…”

Production of phytochemicals by using habituated and long-term cultured cells

“…Plant cells present a problem in that cell lines die out unless they are stored in a freezer or they are subcultured. Although studies on embryogenic cells (Maruyama et al 2000) and suspension cultures (Kobayashi et al 2005;Sung et al 2000) have been attempted, not all cultured cells can be stored in a freezer. Moreover, long-term continuous subculture to maintain a cell culture is necessary for industrial applications.…”