Shohael A M, Chakrabarty D, Yu K W, Hahn E J, Paek K Y
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju, South Korea.
J Biotechnol. 2005 Nov 4;120(2):228-36. doi: 10.1016/j.jbiotec.2005.06.010. Epub 2005 Aug 10.
Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.
在添加了1 mg l(-1) 2,4-二氯苯氧乙酸（2,4-D）的Murashige和Skoog（MS）基础培养基上培养无梗五加叶外植体，诱导出胚性愈伤组织，而胚胎成熟不需要添加植物生长调节剂。添加1 mg l(-1) 2,4-D可通过防止胚胎成熟来维持胚性培养。在含有4 mg l(-1)赤霉素（GA(3)）的MS培养基上实现了最佳的胚胎萌发和小植株发育。低强度MS培养基（1/2和1/3强度）比全强度MS培养基在生产具有发育良好的茎和根的正常小植株方面更有效。这些植株成功移栽到土壤中。利用胚性愈伤组织建立悬浮培养体系，用于后续在生物反应器中生产体细胞胚。将10 g胚性细胞（鲜重）接种到含有2 l无植物生长调节剂的MS培养基的3 l气球型鼓泡生物反应器（BTBB）中，收获了121.8 g不同发育阶段的成熟体细胞胚，可通过过滤分离。子叶体细胞胚萌发，转移到含有2 l添加4 mg l(-1) GA3的MS培养基的3 l BTBB中后转化为小植株。高效液相色谱分析表明，与体细胞胚的不同阶段相比，田间种植植株叶片中的总刺五加苷含量显著更高。然而，刺五加苷B的含量在萌发胚中最高。与其他发育阶段相比，萌发胚中刺五加苷E和刺五加苷E1的含量也更高。这一结果表明，通过体细胞胚的生物反应器培养可以实现无梗五加生物量的高效大规模生产，并可作为药用原料来源。
