ICS 65.020 CCS B 16 湖 DB43 南 省 地 方 标 准 DB43/T 1 9 67—2020 浮萍实验室培养技术操作规程 Guideline for Laboratory Culture Technology of Lemna 2020-12-29发布 湖南省市场监督管理局 2021-03-29实施 发 布 DB43/T 1967—2020 目 次 前言························································································································ Ⅲ 1 范围 ····················································································································· 1 2 规范性引用文件 ······································································································ 1 3 术语和定义 ············································································································ 1 3.1 生物量 ············································································································ 1 3.2 无性繁殖体 ······································································································ 1 3.3 无性繁殖群 ······································································································ 1 3.4 叶片 ··············································································································· 1 4 环境条件 ··············································································································· 1 4.1 光照 ··············································································································· 1 4.2 水温 ··············································································································· 1 4.3 pH ·················································································································· 2 5 培养基 ·················································································································· 2 6 仪器设备与器具 ······································································································ 2 6.1 培养设备 ········································································································· 2 6.2 培养器具 ········································································································· 2 6.3 其它设备和工具 ································································································ 2 7 培养过程 ··············································································································· 2 7.1 灭菌 ··············································································································· 2 7.2 培养与扩繁 ······································································································ 2 7.3 保种 ··············································································································· 2 7.4 注意事项 ········································································································· 3 附录 A（资料性） 改进的 Steinberg 培养基的配方····························································· 4 I DB43/T 1967—2020 II DB43/T 1967—2020 前 言 本文件按照 GB/T 1.1—2020 给出的规则起草。 本文件某些内容可能涉及专利。本文件发布机构不承担识别这些专利的责任。 本文件由湖南省农业农村厅提出。 本文件由湖南省农业标准化技术委员会归口。 本文件起草单位：湖南省植物保护研究所,长沙艾格里生物科技有限公司。 本文件主要起草人：刘勇、陈昂、张德咏、张战泓、蒋桂芳、罗杰、罗香文、熊浩、陈武瑛。 III DB43/T 1967—2020 IV DB43/T 1967—2020 浮萍实验室养殖技术操作规程 1 范围 本文件规定了湖南省小浮萍、紫背浮萍等浮萍的室内培养环境条件。 本文件适用于湖南省浮萍实验室培养的管理。 2 规范性引用文件 下列文件中的内容通过文中的规范性引用而构成本文件必不可少的条款。其中，注日期的引用文件， 仅该日期对应的版本适用于本文件；不注日期的引用文件，其最新版本（包括所有的修改单）适用于本 文件。 3 术语和定义 下列术语和定义适用于本文件。 3.1 生物量 Biomass 指一个种群中存活的活体材料的干重，本标准中指生物量的代替品，如叶片数量或叶面积等代表性 测量值即术语“生物量”的代替性测量。 3.2 无性繁殖体 Clone 指通过无性繁殖产生的生物体或细胞。来自同一个无性繁殖体的个体有遗传同一性。 3.3 无性繁殖群 Colony 指相互连着的母体和后代叶片（通常 2～4 片）的集合体。有时也指整个植株。 3.4 叶片 Frond 指浮萍植株的单个“叶状结构” 。它是最小的单位，即个体，有繁殖能力。 4 环境条件 4.1 光照 自然光、白炽灯、荧光灯都可作为室内培养浮萍的光源。将光源固定在距植株 30～50 cm 的上方， 应预防热损伤，且保证光合作用和生长充足的光照。光照强度 6500～10000 Lux。光/暗比为 16/8。 4.2 水温 培养于温度可控的设备或区域中（恒温箱、人工气候箱、恒温室），最佳生长温度为 24±2℃，低 1 DB43/T 1967—2020 温情况下会形成冬芽。 4.3 pH 在 pH6～9 的范围内，能够正常生长。低 pH 对浮萍的生长具有抑制作用，浮萍对 pH 耐受的低限在 5～6 之间。 5 培养基 推荐改进的 Steinberg 培养基（见附录 A），用 HCl 和 NaOH 调整 pH。 6 仪器设备与器具 6.1 培养设备 浮萍应培养于温度和光照可控的设备或区域中，如：恒温箱、人工气候箱、恒温室、高压灭菌锅、 光源、冰箱等。 6.2 培养器具 可以用多种容器，如：三角瓶、表面皿、组织培养瓶等。 6.3 其它设备和工具 超净工作台、照度计、温湿度计、网、筛、接种环、叉子、薄刀片等。 7 培养过程 7.1 灭菌 a） 取样：选取一簇单克隆浮萍，将其分成若干个个体； b） 去根：用灭菌后的刀片去除所有浮萍个体的根； c） 漂洗：在无菌水中剧烈的摇动，冲洗浮萍植株； d） 表面灭菌：0.5％的次氯酸钠溶液中浸泡 1 min e） 漂洗：无菌水或无菌培养液冲洗 2～3 次； f） 接种：在超净工作台中转接到无菌培养液中，确保转接的浮萍个体叶状体底部的囊为绿色。 7.2 培养与扩繁 浮萍在适宜的条件下生长速度很快，通常 7～10 天就会铺满容器并发生重叠，需要将浮萍植株转接 入新鲜的培养基中，整个过程需要无菌操作。具体步骤为： a） 灭菌：培养基和培养容器、操作工具等需 121℃高温高压灭菌，操作环境需紫外灯灭菌 40 min； b） 培养基分装：每个培养器皿 150 mL 培养基，液面高度为 3 cm；