Cryopreservation Of Callerya Speciosa Through Droplet-vitrification

Callerya speciosa is a perennial leguminous vine of the Callerya. Germplasm resources preservation of C. speciosa is necessary because it has high medicinal value and market demand, but the wild resources tend to be exhausted. So far, there has been no report about cryopreservation of C.speciosa. Cryopreservation technology is suitable for any plants tissues which can be regenerated, and more than 200 plants have been storaged by this technology. The research of cryopreservation technology about C. speciosa would be beneficial to germplasm long-term preservation and provide technical support for the cryopreservation of C.speciosa. The results would give support for constructing germplasm resources pool and supplying cryopreservation technical reference for other tropical woody vine.In this experiment, the droplet-vitrification cryopreservation technology was performed with axillary buds of C. speciosa as the material. The main results were as following:1. The axillary buds from green plants with 4 weeks seedling age were selected as experimental material. The shoots with axillary buds were cut into fragments with length and diameter 1-2 mm. They were put in LS for 4 h shaking at 150 rpm,25℃ and in darkness. Then the explants were put in PVS2 for 60 min at 0℃under static conditions. Then the materials were kept for at least 15 min in LN. After the above treatment, the explants were unloaded in RS for 15 min at 25℃. They were transferred to semi-solid MS covered with sterile filter paper and cultured for 2 days in darkness. Then the explants were transferred to regeneration medium for 7 days in darkness. Then they were moved to the light conditions for culture, the regeneration rate were up to 60.0%.2.6 genes (codenamed:02_G06,05_C06,06_F06,18-F11,17_H12,18_G06) related to stress tolerance from C. speciosa cDNA library were detected by Real Time PCR technology, the results showed that the expression of these genes had an index increase tendency after cryopreservation and one subculture time.3. Studies on genetic stability of regenerated plants after cryopreservation by ISSR and SSR technology showed there were no different bands, which indicated that DNA level of C.speciosa remained stable after cryopreservation.