Schnorf M, Neuhaus-Url G, Galli A, Iida S, Potrykus I, Neuhaus G
Institute for Plant Sciences, Swiss Federal Institute of Technology, Zürich, Switzerland.
Transgenic Res. 1991 Dec;1(1):23-30. doi: 10.1007/BF02512993.
A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basis layer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l-1 kanamycin or 5 mg l-1 paromomycin. Single resistant cells can be recovered from about 10,000 sensitive cells in one alginate layer. Injection of the neo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.
已建立一种用于注射烟草叶肉原生质体的新培养方法。原生质体被包埋在一层薄薄的藻酸盐中，并从下面的基础层中的培养基获取养分。在藻酸盐层中，原生质体再生为愈伤组织的频率高达80%。包埋的原生质体可用50 mg l-1卡那霉素或5 mg l-1巴龙霉素进行筛选。在一个藻酸盐层中，从约10,000个敏感细胞中可获得单个抗性细胞。将新霉素基因（编码新霉素磷酸转移酶II）注射到藻酸盐层中由原生质体衍生的单个细胞中，可产生对卡那霉素有抗性的菌落，这些菌落可再生成成熟植株。通过酶活性测定表明，这些植株表达新霉素磷酸转移酶。通过对高分子量DNA进行Southern杂交，可证明转基因已整合到植物基因组中。采用这种培养方法，每小时可注射100个细胞。转化频率为2%至20%。在杂交实验中表明，外源基因以孟德尔方式传递给下一代。
