Protoplast isolation from bamboo leaves
Protoplast isolation from bamboo leaves
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DOI：
10.5511/plantbiotechnology.27.353
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发表时间：
2010-01-01
影响因子：
1.6
通讯作者：
Kobayashi, Mikio
中科院分区：
文献类型：
--
作者：
Hisamoto, Yoko;Kobayashi, Mikio
从四种竹种的叶子中分离出每 0.3 克鲜重大于 2.0 x 10(6) 个细胞的原生质体：一种草本竹、少花石竹和三种木本竹、毛竹、竹竹和竹。为了无菌原生质体分离，将折叠的叶子在70％乙醇中灭菌，并从叶鞘中取出内部组织。将叶材料在含有 0.8 M 甘露醇（pH 5.8，32 摄氏度）和酶混合物的改良怀特培养基溶液中孵育 4 小时，其中木本竹含有 3% Macerozyme R-10、2.5% Meicelase 和 2% 纤维素酶 Onozuka，而 L. pauciflora 则分别含有 2.5%、2.5% 和 1% 这些酶。通过双层 Miracloth 漏斗过滤原生质体，然后在分析前用 0.6 M 至 0.4 M 含甘露醇的 White 培养基（pH 5.8）洗涤。 FDA 染色测定原生质体活力平均为 83%。
Protoplasts with a yield greater than 2.0 x 10(6) cells per 0.3 g fresh weight were isolated from leaves of four bamboo species: an herbaceous bamboo, Lithachne pauciflora, and three woody bamboos, Phyllostachys meyeri, Sasa jotanii, and Bambusa vulgaris. For aseptic protoplast isolation, folded leaves were sterilized in 70% ethanol, and the internal tissues were drawn out from the leaf sheath. The leaf material was incubated for 4 h in a modified White's medium solution containing 0.8 M mannitol ( pH 5.8, 32 degrees C) and an enzyme mixture containing 3% Macerozyme R-10, 2.5% Meicelase, and 2% Cellulase Onozuka for woody bamboos, and 2.5%, 2.5%, and 1% of these enzymes, respectively, for L. pauciflora. Protoplasts were filtered through a double-layered Miracloth funnel and then washed with 0.6 M to 0.4 M mannitol-containing White's medium ( pH 5.8) prior to analysis. Protoplast viability averaged 83% as determined by FDA staining.