茶壶枣离体多倍体诱导关键技术研究

目的离体多倍体育种是枣树育种的重要方法，对其关键技术的研究有利于建立和完善枣树离体多倍体育种体系，加快枣树多倍体种质的创制。方法以茶壶枣无菌叶片为材料，将预培养不同天数（7 ~ 20 d）的叶片固定后制成石蜡切片，进行细胞学观察以确定适宜的预培养时间。在此基础上，将叶片不定芽再生技术与秋水仙素染色体加倍技术相结合，在叶片预培养8、9、10、11 d后，分别用70和90 mg/L的秋水仙素处理48和72 h，并对处理后得到的不定芽进行流式细胞术倍性检测。结果通过对叶片最佳预培养时间的细胞学研究可以得出，茶壶枣无菌叶片在预培养8 ~ 9 d时，分生细胞的分裂能力最强，且未出现分化现象，在此时期用秋水仙素对叶片进行处理，可大大提高诱导率，并且可以有效避免嵌合体和混倍体的出现。染色体加倍试验发现，在预培养8 d后，用70 mg/L的秋水仙素处理72 h，其诱导率最高，达4.11%，并成功获得8株多倍体植株。通过流式细胞术确定这8株多倍体均为纯合四倍体。结论本研究探索出了将茶壶枣叶片不定芽再生体系与秋水仙素染色体加倍技术相结合，人工诱导茶壶枣多倍体的技术方法。 

Study on key techniques of polyploid induction in Ziziphus jujuba Mill. cv. 'Teapot'

Objective In vitro polyploid breeding is an important method for jujube breeding. The study of its key technologies is conducive to the establishment and improvement of polyploid breeding system and the creation of polyploid germplasm of jujube. Method In this study, the leaves of Ziziphus jujuba Mill. cv. ‘Teapot’ were used as materials, and the leaves of different days (7?20 day) were fixed and then made into paraffin sections for cytological observation. On this basis, the leaf adventitious bud regeneration technology was combined with the colchicine chromosome doubling technique. After the leaves were pre-cultured for 8, 9, 10 and 11 days, they were treated with 70 mg/L and 90 mg/L colchicine for 48 and 72 hours, and the flow cytometry ploidy test was performed on the adventitious buds obtained after treatment. Result Through the cytological study on the optimal pre-culture time of the leaves, it can be concluded that the meristematic cells have the strongest division ability and no differentiation during the pre-culture of 8?9 days. During this period, treatment of leaves with colchicine can greatly increase the induction rate and effectively avoid the appearance of chimeras and mixoploids. The chromosome doubling test found that after 8 days of pre-culture, the induction rate was up to 4.11% after treatment with 70 mg/L colchicine for 72 hours, and 8 polyploid plants were successfully obtained. It was confirmed by flow cytometry that these 8 polyploids were pure tetraploids. Conclusion This study explores the technical method of artificially inducing polyploidy of Ziziphus jujuba Mill. cv. ‘Teapot’ by adventitious bud regeneration technology and colchicine chromosome doubling technique.