Objective  A protocol for tissue culture system was optimized and an in vitro regeneration system suitable for tetraploid Ziziphus jujuba var. spinosa was constructed using same source as the starting material, so as to lay the foundation for the preservation, promotion and utilization of excellent germplasm.

  Method  This study used Z. jujuba var. spinosa germplasm obtained by doubling colchicine as materials, designed experiments and explored the effects of different types and concentrations of plant growth regulators on the proliferation, rooting and adventitious bud regeneration of tetraploid Z. jujuba var. spinosa using tissue culture technology. Additionally, we explored the cytological observation of the process of leaf adventitious shoot regeneration using paraffin sectioning.

  Result  (1) Tetraploid Z. jujuba var. spinosa was grown on stem segments with terminal buds as the explants. The best multiplication medium for tissue culture seedlings of tetraploid germplasm was: 1/2MS + 6-BA 0.5 mg/L + IBA 0.2 mg/L + TDZ 0.5 mg/L + sucrose 30.0 g/L + agar 5.9 g/L, the best rooting medium was 1/2MS + IBA 0.8 mg/L + sucrose 30.0 g/L + agar 5.9 g/L. The rooted seedlings were transplanted to the field after refining, and the survival rate could reach 93.33%. (2) The optimal medium for inducing the in vitro regeneration of adventitious shoots from tetraploid leaves was WPM + TDZ 1.0 mg/L + IBA 0.3 mg/L + sucrose 30.0 g/L + agar 5.9 g/L, in which the leaves could regenerate adventitious shoots by the direct regeneration pathway. (3) The cytological study of the regeneration process of autotetraploid jujube leaves in vitro showed that the regeneration of adventitious shoots originated mainly from thin-walled cells around the vascular bundles of leaf veins, and the meristematic cells were most divisive and undifferentiated at 8−12 d of preculture.

  Conclusion  This study successfully established and optimized the system of tissue culture and in vitro regeneration of tetraploid sour jujube germplasm, and also conducted histological studies on the regeneration process of leaves adventitious bud regeneration, which lays the foundation for the perfection of jujube polyploid system and the subsequent utilization of its polyploid germplasm.