Protoplastculture（原生质体培养）.ppt

Chapter 3Protoplast culture Somatic hybridization Part 1 Protoplast culture Part 2 Somatic hybridization Wen-Wu Guo（郭文武教授） Part 1: Protoplast culture （原生质体培养） Definition of Protoplast Protoplast (原生质体): a naked cell without cell wall Sub-protoplast (亚原生质体)：incomplete protoplast with or without nucleus Nucleo-plast (核质体)：nuclear protoplast with little cytoplasm (由原生质膜和薄层细胞质包围细胞核形成的小原生质体，即小原生质体) (Miniprotoplast) Micro-protoplast（微原生质体）: protoplast with one or a few chromosomes Cytoplast (胞质体): enucleated protoplast (不含细胞核而仅含部分胞质的原生质体) Cytoplast isolation in Citrus Xu et al., 2006 Progress in PP research Key achievements in plant PP research 1880, protoplast was first used by Hanstein 1960, PP isolation from tomato root by enzyme incubation by Cocking 1971, mesophyll PP plant regeneration in tobacco by Takebe 1981, embryogenic PP plant regeneration by Vardi in Isreal 1985, First rice PP plant regeneration by Fujimura 1986, single PP culture plant regeneration in Rapeseed by Spangenberg Fact sheet in plant PP research In Horticultural crops, citrus ranks first in PP research PP plant regeneration succeeded in citrus, persimmon, banana, apple, pear, grape, kiwi, etc PP regeneration succeeded in over 320 plant species belonging to 49 families 146 genera Tendency: model plants to staple economic plants; annual to perennial plants PP plant regeneration is possible from almost all kinds of higher plants 原生质体研究的意义 没有细胞壁的包裹，质膜直接和外界环境进行接触，是一种优良的单细胞体系，是获得单细胞无性系和选育突变体的优良起始材料 作为基因工程的受体材料等 植物原生质培养应用的最广泛，成果最显著的是在体细胞杂交方面的应用 A scheme of plant protoplast manipulation Plants Suspension cells Pre-treatment enzymolysis Purification Viability test Culture and regeneration Protoplast transformation GFP as the visual marker Reference: Guo WW et al. 2005. Plant Cell Reports 24: 482-486 Protoplast fusion 一、原生质体分离 基础材料准备 预处理与酶解 原生质体活力测定 原生质体收集与纯化 原生质体分离的材料准备 无菌试管苗叶片 上胚轴和子叶 培养细胞 预处理及酶解 酶 渗透压 调节剂 酶解 酶 来 源 生产厂家 纤维素酶类 Onozuka R–10 绿色木霉 Yakult Honsha Co. Ltd.,