Abstract:Sugarcane (Saccharum sp. hybrid co. 71/210) calluses were derived from the young leaves and cultured on solid N6 medium containing 2 mg/l 2,4-D. The callus subcultures were used as the experiments of cryopreservation in liquid nitrogen (–196℃). Some factors effecting the viability and survival of cryopreserved materials were investigated in order to establish a good technological system of germplasm conservation. The present experimental results indicated that the 10th to 15th day callus subcultures showed higher viability and survival after cryopreservation at –196℃ (Fig. 1), and so they are suitable materials used as cryopreservation. The effects of cryoprotectants tested on the survival and regeneration of sugarcane calluses after freezing and storage in liquid nitrogen were shown in Fig. 2 and Table 1. The protective role of DMSO used alone is not significant. However, the combination of DMSO with sugars and sugar alcohols markedly improved survival and regeneration of calluses. Especially, the mixture of 10% DMSO and 0.5 mol/1 sorbitol is more effective than that of DMSO with the other sugars: glucose, sucrose, mannitol and maltose etc. But, the addition of polyethylene glycol 6000 to the mixtures of DMSO and sugars did not produce a clear enhancement of protective effectiveness. The effectiveness of the slow-freezing method that the specimens were cooled at the rate of 1℃/min from 0℃ to –40℃, kept at –40℃ for 1–3 hrs, and then put into liquid nitrogen is much better than that obtained by the other way that the samples were not kept at terminal temperature (–40℃) for a period or that of the rapid-freezing method etc. (Fig. 3 and Table 2). Experiments for comparison of 4 thawing methods were carried out. The results indicated that the effectiveness of thawing with tap water is as good as that in a 40℃ water bath (Fig. 4). Studying the recovery growth conditions of the calluses after cryopreservation, we found that the illumination is unfavourable for recovery of growth. The percentage of survival is higher and the rate of growth is much faster in culture in dark than under light (Table 3). By use of the combination of various favourable factors mentioned above, the survival percentage of sugarcane calluses after cryopreservation achieved over 90%; and the calluses stored for six months could be normally differentiated into a great deal of new plantlets (Plate I, Figs. 3 and 4).