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In cereals, cell suspension culture is an essential source of protoplast. However, over a period of time suspension cultures lose the ability to release protoplasts capable of plant regeneration. In this case, cryopreservation has been investigated as a means of providing stocks of cells for re-initation of embryogenic cell suspension cultures by long term storage of biological tissues at ultra - low temperatures. In this study, dehusked rice seeds were surface sterilized in 30% (v/v) Domestos bleach incubated
onto LS medium supplemented with 2.5 mgrI 2, 4 - D and made semi - solid by the addition of 4grI seakem agarose. Suspension cultures were initiated from friable, globular, embryogenic callus in R2 (cv. Tarom) and AA2 (cvs. Arnol - 3 and Khazar) media, respectively. Cells were transferred to R2 and AA2 media supplemented with 60 grI mannitol 3-4 days before freezing. Cells were harvested on a 45flm nylon mesh and
placed into 2 cm3 polypropylene vials, (0.2 g of cells / vial.) Approximately 0.75 cm3 of
chilled (on iced water), filter sterilized, cryoprotectant mixture [glycerol, 46.0g/1; dimethyl sulphoxide (DMSO), 39.0 g/l; sucrose 342.3 g/l; proline 5.0 g/l] was added to each vial and mixed with the cells. Cells were cryoprotected for 1 h on iced water. Vials were transferred to aluminium cans and the cells frozen in a controlled rate freezer. After one year cells were thawed, by plunging the vials into sterile water (45" c; 1-2 min). The cells were then placed onto double layers of sterile 5.5 cm filter paper discs (Whatman No.1; one disc above the other, per 9 cm petri dish) overlying aliquots ofR2 and AA2 media made semi - solid with Seakem agarose (20-25 ml per dish). The Cells were maintained at (26:i: 1 0 c) in dark and subcultured by transferring cells on the uppermost filter paper discs to fresh AA2/ R2 media as before, 3 d post thaw. Callus and cell suspension cultures were initiated for 3 cultivars (Arnol-3, Tarom and Khazer). Cell suspensions were re-initiated for all cultivars in R2 and AA2 liquid media and
maintained under the same conditions as unfrozen cultures. The post - thaw viability of cells was determined by the reduction of triphenyl tetrazolum chloride (TTC). Protoplasts were isolated from an unfrozen suspension and re-established cell
suspensions. Protoplast yield of re- established cell suspensions were higher than that of unfrozen cells. Generally, protoplast yields increased for - re-established cell suspension cultures as compared with unfrozen control cultures. Shoot regeneration was achieved from protoplasts isolated from cryopreserved re-initiated cell suspension
cultures of all 3 cultivars. In this study, 24 plants for cv. Arnol-3 , 32 plants for cv. Kahzer and 49 Plants for cv. Tarom were produced. Cryopreservation is thus a feasible means for the secure storage of potentially unique, cell suspensions of Iranian Indica and Japanica rices of agronomic importance.