dedifferentiated cell lines (embryogenic and non-embryogenic),
however, are still preserved using controlled rate slow freezing
[14–16]. The method described in this chapter has been applied
at first for the cryopreservation of Nicotiana tabacum BY2 cell line
at the German Collection of Microorganisms and Cell Cultures
(DSMZ). Later the method was successfully applied to more than
17 different cell lines with slight modifications. Many parameters
such as the osmotic conditions and duration of the culturing prior
to cryopreservation (i.e., pre-culture), type and concentration of
cryoprotectant, and the freezing program have to be adjusted for
each specific cell line. In Subheading 4 of this chapter, parameters
applied for different cell lines are listed (see Notes 1–12). Embryogenic cultures normally contain more clumps than
non-embryogenic cell lines and require longer times for
pre-culturing and incubation with cryoprotectants. On the other
hand, they often show a higher general stress level and exhibit a
better drought resistance [17], which are key properties for survival
of cryopreservation. Both embryogenic and non-embryogenic cell
cultures can be preserved by controlled rate freezing [14]. Droplet
vitrification [18] and encapsulation/dehydration [19] have been
applied more often for embryogenic cultures than for
non-embryogenic cell lines [18, 20]. Since these methods are
technically completely different from controlled rate freezing,
they are not described here. Vitrification has been only rarely
applied to cell cultures, and we refer to Reinhoud et al. [21] for a
simple and efficient vitrification method. In this chapter, we
describe a typical procedure for cryopreservation of plant cell cultures using a controlled rate freezing approach.
2 Materials
1. Normal/standard liquid culture/growth medium (prepared as
usual).
2. Calcium-free growth medium containing 3% sodium alginate
(alginate completely dissolves only after autoclaving) (see Note
4).
3. Calcium-free growth medium.
4. Growth medium containing 100 mM calcium chloride.
5. Growth medium containing 0.9 M sorbitol for osmotic
pre-culturing (see Note 5).
6. Petri dishes containing normal growth medium.
7. Dimethyl sulfoxide (Me 2 SO) (see Note 7).
8. The following materials have to be wrapped in aluminum foil
and sterilized: culture jars (Erlenmeyer flasks for routine cultivation on a gyratory shaker), three suction filtration flasks
640
Heinz Martin Schumacher et al.
however, are still preserved using controlled rate slow freezing
[14–16]. The method described in this chapter has been applied
at first for the cryopreservation of Nicotiana tabacum BY2 cell line
at the German Collection of Microorganisms and Cell Cultures
(DSMZ). Later the method was successfully applied to more than
17 different cell lines with slight modifications. Many parameters
such as the osmotic conditions and duration of the culturing prior
to cryopreservation (i.e., pre-culture), type and concentration of
cryoprotectant, and the freezing program have to be adjusted for
each specific cell line. In Subheading 4 of this chapter, parameters
applied for different cell lines are listed (see Notes 1–12). Embryogenic cultures normally contain more clumps than
non-embryogenic cell lines and require longer times for
pre-culturing and incubation with cryoprotectants. On the other
hand, they often show a higher general stress level and exhibit a
better drought resistance [17], which are key properties for survival
of cryopreservation. Both embryogenic and non-embryogenic cell
cultures can be preserved by controlled rate freezing [14]. Droplet
vitrification [18] and encapsulation/dehydration [19] have been
applied more often for embryogenic cultures than for
non-embryogenic cell lines [18, 20]. Since these methods are
technically completely different from controlled rate freezing,
they are not described here. Vitrification has been only rarely
applied to cell cultures, and we refer to Reinhoud et al. [21] for a
simple and efficient vitrification method. In this chapter, we
describe a typical procedure for cryopreservation of plant cell cultures using a controlled rate freezing approach.
2 Materials
1. Normal/standard liquid culture/growth medium (prepared as
usual).
2. Calcium-free growth medium containing 3% sodium alginate
(alginate completely dissolves only after autoclaving) (see Note
4).
3. Calcium-free growth medium.
4. Growth medium containing 100 mM calcium chloride.
5. Growth medium containing 0.9 M sorbitol for osmotic
pre-culturing (see Note 5).
6. Petri dishes containing normal growth medium.
7. Dimethyl sulfoxide (Me 2 SO) (see Note 7).
8. The following materials have to be wrapped in aluminum foil
and sterilized: culture jars (Erlenmeyer flasks for routine cultivation on a gyratory shaker), three suction filtration flasks
640
Heinz Martin Schumacher et al.