一种复叶耳蕨愈伤组织和悬浮细胞的快速繁殖方法
申请公布号:CN104263691A
申请号:CN201410462973.7
申请日期:2014.09.12
申请公布日期:2015.01.07
发明人:杨存
分类号:C12N5/04(2006.01)I;C12N5/02(2006.01)I
主分类号:C12N5/04(2006.01)I
地址:210046 江苏省南京市栖霞区尧化街道甘家边108号1幢701室
摘要:本发明研究了一种复叶耳蕨愈伤组织和悬浮细胞的快速繁殖方法,包括无菌叶片的获得、愈伤组织的诱导、悬浮细胞的培养等步骤。本发明可以快速获得大量的复叶耳蕨细胞,操作简单可控,细胞繁殖率高,生长周期短,可以无限度的提供制药原料,有助于复叶耳蕨药用成份的有效开发和利用。
主权项: 一种复叶耳蕨愈伤组织和悬浮细胞的快速繁殖方法,包括无菌叶片的获得、愈伤组织的诱导、悬浮细胞的培养等,主要步骤如下:(1)取复叶耳蕨的幼嫩叶片,按照常规的消毒方法对其进行灭菌处理;(2)将步骤(1)灭菌处理过的复叶耳蕨叶片接入培养基配方为Nitsch+TDZ0.2mg/L+IBA1‑3mg/l+10%椰乳诱导培养基中进行愈伤组织诱导培养,附加蔗糖30g/L,琼脂6.5g/L,PH5‑6,光照1000lx,温度15℃;(3)取步骤(2)诱导出来的愈伤组织放入液体培养基Nitsch+2,4‑D0.1‑0.2mg/L+TDZ0.1‑0.2mg/L+EDTA+消泡剂进行悬浮细胞培养,附加蔗糖30g/L,PH5.8,光照1500lx,温度15℃;(4)取步骤(3)悬浮培养出来的细胞接入固体平板,筛选次生代谢物质含量高的细胞株。
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