Studies On Somatic Embryogenesis And Germplasm Cryopreservation Of Litchi

Litchi (Litchi chinesis Sonn) belongs to Sapindaceae family and is a subtropical fruit known for its delicious flavors. As there are difficulties in litchi vitro culture, only few studies reported on somatic embryogenesis and germplasm Cryopreservation. Therefore, development of biological technique is limited. This experiment was conducted on somatic embryogenesis and germplasm Cryopreservation of litchi by vitrification. The aim is to establish procedure of anther somatic embryogenesis and suspension cells Cryopreservation, and to provide foundation for biological technique as well. The main research results are as follows:1. A series of experiments have been carried out on anther somatic embryogenesis. Anthers were inoculated on MS medium containing 2.0mg/L 2, 4-D, 0.2mg/L NAA and supplemented with sucrose 50g/L. After 2 months, the embryogenic calli were obtained. Then it was transferred to the MS medium containing 0.5mg/l KT, 0.1mg/L NAA, and 0.5%AC for embryoids differentiation. About a month later, hundreds of embryoids formed. These embryoids were transferred to the MS medium containing 1.0mg/LBA, 1.0mg/L KT, 0.5mg/LNAA, 500mg/L CH, and supplemented with sucrose 50g/L. Cultured for 2-3 months, the regenerated plantlets were obtained. The system was established for somatic embryogenesis of litchi by anther culture.2. Preservation of embryogenic calli was studied. Embryogenic calli grew well on MS medium containing 1.0mg/L 2, 4-D, 0.2mg/L NAA. Long-term preservation can be achieved by combined solid and liquid medium culture alternatively. Now somatic embryogenesis is preserved for a year by above culture manner. At the same time embryogenic calli had vigorous growth with embryogenic characteristics.3. Embryogenic suspension cells were used to study Cryopreservation by vitrification, it optimized Cryopreservation procedure: take 3- to 5-day-age embryogenic suspension cells and precultured for 2 days with 0.4mol/L glucitol, then pretreated with 60%PVS2 for 15 minutes at 25 癈 and dehydrated with PVS2 for 15-20 minutes at 0℃. Above suspensions were plunged into liquid nitrogen directly and conserved for 24 hours and then rapidly thawed in a water bath at 40℃. They were washed twice with 1.2mol/L sucrose solution. TTC analysis and recovery growth were employed. Relative survival rate reached 27.1%.4. Ultrastructural changes in cryopreserved litchi embryogenic suspension cells by vitrification were assessed using transmission electron microscopy. It was founded that the plasmolysis became severe during the precultivation, dehydration, and cooling. A few cells wall, cell membrane and nucleus envelope were lethally injured during the cooling process, while other cells were basically similar to control.