CRYOPRESERVATION OF NUCELLAR CELLS OF NAVEL ORANGE (CITRUS-SINENSIS OSB) BY A SIMPLE FREEZING METHOD

Suspension cultured cells of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) was cryopreserved without using a programmable freezer and dimethyl sulfoxide (DMSO) as a cryoprotectant. The nucellar cells were cryoprotected with a mixture of 2 or 3 M glycerol and 0.4 M sucrose dissolved in Murashige-Tucker basal medium (MT) for 10 min at 25-degrees-C. A cell suspension of about 0.2 ml was loaded into a 0.5-ml transparent straw and frozen spontaneously by placing straws in a freezer at -30-degrees-C for 20-30 min prior to direct immersion into LN. The average rate of survival after thawing, as evaluated by fluorescein diacetate and phenosafranin double staining, was about 90% of non-frozen controls. We also obtained a similar survival rate using a 1.8-ml cryotube when the cell suspension was held at -30-degrees for 40-50 min. The revived cells resumed growth within 3 days and developed into plantlets via embryogenesis. This simple procedure for cryopreservation seems promising for cultured plant cells and meristems.