Department of Horticulture, The Pennsylvania State University, University Park, Pennsylvania 16802.
Plant Physiol. 1985 Mar;77(3):513-9. doi: 10.1104/pp.77.3.513.
Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4 degrees C or 5 hours at 30 degrees C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl(2). After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes.
本文描述了从发育中的玉米胚乳中形成原生质体的方法，以及从这些原生质体中用水分离完整的淀粉体的方法。将胚乳切片在含有纤维素酶和果胶酶的培养基中于 4°C 孵育 5 天或于 30°C 孵育 5 小时后，可获得原生质体。在 Ficoll 密度梯度中纯化后，通过将悬浮液强制通过位于改良一次性注射器下端的 Nitex 网（20 微米）来破裂原生质体。所得滤液铺在 30%、15%和 10%的不连续 Ficoll 密度梯度上。每个 Ficoll 溶液均含有 0.7 摩尔蔗糖、10 毫摩尔精氨酸、10 毫摩尔 dl-二硫苏糖醇、50 毫摩尔 2-（N-吗啉代）乙磺酸（pH5.6）和 2 毫摩尔 CaCl2。在冷处 3 小时后，在 15%的 Ficoll 层中回收了 50%至 93%完整且无细胞质、线粒体和乙醛酸体污染的淀粉体部分。淀粉体完整性通过荧光显微镜和淀粉体膜破裂前后某些淀粉体标记酶的活性来估计。淀粉分支酶、ADP-葡萄糖焦磷酸化酶和亚硝酸盐还原酶被用作淀粉体标记酶。
