Micropropagation by Somatic Embryogenesis of Coffea Species and Cryopreservation

咖啡物种体细胞胚胎发生的微繁殖和冷冻保存

• 批准号: 04660015
• 负责人: YASUDA Takeshi
• 金额: $ 1.28万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04660015/
• 关键词: Somatic embryo; Coffee; Cryopreservation; Cytokinin; Protoplast; Tropical Tree; Coffea; 凍結保存

The embryogenic callus and somatic embryo of Coffea arabica and C.canephora can be induced by cytokinin alone from the section of young leaf of mature trees. The cytokinin induced callus has a high embryogenic potetial and is very useful for clonal propagation. As cytokinin, 2-isopentenyl-adenine(2iP)is the best, and benzyladenin(BA), zeatin and kinetin also are effective for somatic embryogenesis from coffee leaf slices. Additional auxins act inhibitory effect on somatic embryogenesis. Protoplast were prepared from embryogenic calluses from C.arabica and canephora and cultured on Gelrite medium with cytokinin as a plant regulator. Somatic embryos were regenerated from protoplasts. These simplified method for preparation and culture of protoplasts is very useful for cell fusion and genetic engeering. Somatic embryos of Coffea were successfully cryopreserved after being exposed to a series of preparative steps ; precultured on media with increasing concentration of sucrose, encapsulated in alginate beads, dehydrated up to 30% and pluged into liquid nitrogen.

阿拉比卡咖啡和中果咖啡的胚性愈伤组织和体细胞胚可以单独用细胞分裂素从成熟树的幼叶切片中诱导。细胞分裂素诱导的愈伤组织具有很高的胚胎发生潜力，对于克隆繁殖非常有用。作为细胞分裂素，2-异戊烯基腺嘌呤(2iP)是最好的，苯甲基腺嘌呤(BA)、玉米素和激动素也对咖啡叶切片的体细胞胚胎发生有效。额外的生长素对体细胞胚胎发生具有抑制作用。原生质体由阿拉比卡和中果的胚性愈伤组织制备，并在以细胞分裂素作为植物调节剂的 Gelrite 培养基上培养。体细胞胚胎由原生质体再生。这些简化的原生质体制备和培养方法对于细胞融合和遗传工程非常有用。经过一系列准备步骤后，咖啡的体细胞胚胎被成功冷冻保存；在蔗糖浓度增加的培养基上预培养，封装在藻酸盐珠中，脱水至30%并插入液氮中。

项目成果

TAHARA: "Formation of somatic embryos from protoplasts of Coffea arabica" Hort Science. 29(印刷中). (1994)

田原：“阿拉比卡咖啡原生质体的形成”Hort Science 29（出版中）。

山口 康之: "植物分子・細胞工学マニュアル" 講談社サイエンティフィック, 228 (1992)

山口康之：《植物分子和细胞工程手册》讲谈社科学，228（1992）

安田武司: "組織培養のepoch-making2.コーヒーの組織培養" 植物細胞工学. 5. 53-57 (1993)

安田武：“组织培养划时代2.咖啡组织培养”植物细胞工程5. 53-57 (1993)。

HATANAKA: "Direct regrowth of encapsulated somatic embryos of coffee(coffea canephora)after cooling in liquid nitrogen" Cryo-Letters. 15. 47-52 (1994)

HATANAKA：“咖啡（coffea canephora）的封装体细胞胚胎在液氮中冷却后直接再生”Cryo-Letters。

HATANAKA: "Direct regrowth of encapsulated somatic embryos of coffee(Coffea canephora) after cooling in liquid nitrogen" Cryo-Letters. 15. 47-52 (1994)

HATANAKA：“咖啡（Coffea canephora）的封装体细胞胚胎在液氮中冷却后直接再生”Cryo-Letters。