**2.1.1 Slow programmed freezing (also known as "prefreezing")**

Slow programmed freezing was a major cryopreservation method for plant genetic resources until the 1980's. The procedure in this method is shown in Fig. 1.

Fig. 1. The protocol of slow programmed freezing (from Kumu et al., 1983).

Plant genetic resources (cells and tissues) were packed in cryotube or straw, and cryoprotectants were added. In this method, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glucose were utilized as cryoprotectants. In many cases, these were used independently, but Finkle & Ulrich (1979) reported that the regrowth percentage of germplasm after cryopreservation was higher when mixing cryoprotectans in sugar cane cells. Packed specimens were gradually cooled from -20 oC to -100 oC using a programmable freezer or ethanol baths. Processing which freezes cryoprotectant in a tube artificially is performed near -7~-8 oC in the middle of the freezing. In this treatment, ice is made to form out of a cell under gradual cooling. Intracellular moisture penetrates a plasma membrane, and arrives at the surface of the ice besides a cell, and freezes. This is called 'extracellular freezing'. Intracellular moisture is removed and a cytoplasm is contracted by 'extracellular freezing'. Kindly refer to the book of Kartha (1985) to understand the principle of this phenomenon. After making specimens freeze to a predetermined freezing-temperature, they are immersed in LN. The freezing-temperature is arranged by -40 oC in many species. Cryopreserved tubes are warmed using hot water (40 oC) for 1~2 min, and cryoprotectants

Slow programmed freezing was a major cryopreservation method for plant genetic

**2.1 Cryopreservation method of plant genetic resourses** 

In this section, I introduce cryopreservation procedures by using figures.

resources until the 1980's. The procedure in this method is shown in Fig. 1.

Fig. 1. The protocol of slow programmed freezing (from Kumu et al., 1983).

Plant genetic resources (cells and tissues) were packed in cryotube or straw, and cryoprotectants were added. In this method, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glucose were utilized as cryoprotectants. In many cases, these were used independently, but Finkle & Ulrich (1979) reported that the regrowth percentage of germplasm after cryopreservation was higher when mixing cryoprotectans in sugar cane cells. Packed specimens were gradually cooled from -20 oC to -100 oC using a programmable freezer or ethanol baths. Processing which freezes cryoprotectant in a tube artificially is performed near -7~-8 oC in the middle of the freezing. In this treatment, ice is made to form out of a cell under gradual cooling. Intracellular moisture penetrates a plasma membrane, and arrives at the surface of the ice besides a cell, and freezes. This is called 'extracellular freezing'. Intracellular moisture is removed and a cytoplasm is contracted by 'extracellular freezing'. Kindly refer to the book of Kartha (1985) to understand the principle of this phenomenon. After making specimens freeze to a predetermined freezing-temperature, they are immersed in LN. The freezing-temperature is arranged by -40 oC in many species. Cryopreserved tubes are warmed using hot water (40 oC) for 1~2 min, and cryoprotectants

**2.1.1 Slow programmed freezing (also known as "prefreezing")** 

are removed from a tube. After rewarming, samples are moved from the cryotube, and recultured. The cooling rate in this method is important. It differs from 0.5 oC/min to 50 oC/min with plant species and the size of the plant germplasm. However, in the case of the freezing speed of 2 oC/min or more, the regrowth after preservation tends to fall (Sugawara & Sakai, 1974; Uemura & Sakai, 1980). The disadvantage of this method is that there are many species for which the prefreezing method is not utilized at all. In addition, there are plant tissues which freeze to death partially, and cases in which the decrease in subsequent viability induced also exists (Grout & Henshaw, 1980; Haskins & Kartha, 1980).

#### **2.1.2 Slow unprogrammed freezing (also known as "simple freezing")**

This cryopreservation method was reported using samples of several species in the early 1990's. The advantage of this method is that researchers can cryopreserve without a special programmable freezer, compared with slow programmed freezing.

The slow unprogrammed freezing is shown in Fig. 2. Plant tissues are added to the tube containing cryoprotectants. Tubes are treated for about 10 min at room temperature (25 oC), and are kept at -30 oC for 30~120 min. They are then immersed in LN thereafter. Cryopreserved tubes are warmed using hot water (40 oC) for 1~2 min, and cryoprotectants are removed from a tube. After rewarming, samples are moved from the cryotube, and recultured. In this cryopreservation method, mixtures of glycerol and sucrose or DMSO and sorbitol are used as cryoprotectants (Sakai et al., 1991; Niino et al., 1992; 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. 2. The protocol of slow unprogrammed freezing (from Sakai et al., 1991).

Cryopreservation of Plant Genetic Resources 443

5.7~5.8, but without plant growth regulators. After unloading, samples are removed from

Conponent (g/L) PVS1 PVS2 PVS3

Glycerol 220.0 300.0 500.0

Sucrose 136.9 500.0

Table 1. Components of major plant vitrification solutions. Components of three plant vitrification solutions are referred from previous reports (Uragami et al., 1989; Sakai et al.,

The encapsulation-vitrification method was reported first by Matsumoto et al. (1995) using shoot apices of *Wasabia japonica*, and then spread all over the world. The advantage of this method is that regrowth of plant germplasm after cryopreservation is markedly increased by encapsulating plant samples with alginate beads. The encapsulation of plant germplasms makes for less damage to samples during vitrification procedures (loading treatment and dehydration treatment). On the other hand, due to encapsulation-dehydration, treatment time becomes long compared with that of vitrification and the cryopreservation operation

The procedure for encapsulation-vitrification is shown in Fig. 4. Plant tissues are immersed in the calcium-free liquid medium supplemented with 0.4 mol/L sucrose, 30.0 g/L sodium alginate and glycerol (1.0~2.0 mol/L). The mixture (including a plant cell or tissue) was added drop by drop to the liquid medium containing 0.1 mol/L calcium chloride, forming beads about 5 mm in diameter. The above-mentioned liquid mediums (30.0 g/L sodium alginate and 0.1 mol/L calcium chloride) were adjusted by pH 5.7, but without plant growth regulators. Encapsulated specimens are added to the culture bottle containing LS for osmoprotection. Beads in the bottles are osmoprotected for 16 hours at room temperature (25 oC). LS is the liquid culture medium in which sucrose (0.75~0.8 mol/L) and the glycerol (2.0 mol/L) were contained. After loading, LS is removed from a bottle, and PVS is added newly for the dehydration of plant tissues. The same as with vitrification, the dehydration

After dehydration of PVS, encapsulated samples are moved to a cryotube containing fresh PVS, and immersed in LN. Cryopreserved tubes are warmed using hot water (40 oC) for 1~2 min, and the vitrification solution is removed from the tube. After removal of the solution, unloading solution (supplemented with 1.2 mol/L sucrose; pH 5.7) is added to a tube, and cryoprotectants are removed from plant tissues for 30 min at 25 oC. After unloading,

Ethylene Glycol 150.0 150.0

Dimethyl Sulfoxide (DMSO) 70.0 150.0

Propylene Glycol 150.0

Sorbitol 91.1

becomes complicated (for example, encapsulation).

using PVS is performed at 0 oC in light of the toxicity to plant cells.

samples are moved from the cryotube, and recultured.

the cryotube, and recultured.

1990; Nishizawa et al., 1993).

**2.1.4 Encapsulation-vitrification** 
