Micropropagation by Somatic Embryogenesis of Coffea Species and Cryopreservation

细胞分裂素可在成熟树幼叶上单独诱导小咖啡和canephora的胚性愈伤组织和体细胞胚。细胞分裂素诱导的愈伤组织具有较高的胚胎发生潜力，对克隆繁殖非常有用。2-异戊基腺嘌呤（2iP）作为细胞分裂素效果最好，苯ladenin（BA）、玉米素和细胞分裂素对咖啡叶切片的体细胞胚胎发生也有较好的促进作用。附加生长素对体细胞胚胎发生有抑制作用。以小阿拉比卡和canephora的胚性愈伤组织为材料制备原生质体，并在以细胞分裂素为植物调节剂的Gelrite培养基上培养。体细胞胚由原生质体再生。这些简化的原生质体制备和培养方法对细胞融合和基因工程具有重要意义。经过一系列的准备步骤，成功地将咖啡体细胞胚胎冷冻保存；在蔗糖浓度增加的培养基上预培养，包封在海藻酸珠中，脱水至30%，然后插入液氮中。

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.