Monsanto Agricultural Company, 700 Chesterfield Village Parkway, 63198, St. Louis, MO, USA.
Plant Cell Rep. 1990 Oct;9(6):335-9. doi: 10.1007/BF00232864.
Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 1∶1 ratio to 65% at a 100∶1 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.
已研究了利用聚乙二醇（PEG）介导的 DNA 摄取程序影响玉米原生质体稳定转化和共转化频率的因素。发现原生质体平板培养条件、预处理缓冲液组成、PEG 浓度和 DNA 浓度都很重要。当用环状质粒 DNA 转化时，载体 DNA 没有益处。线性化质粒 DNA 的作用在实验中不一致，并且可能取决于载体 DNA 的存在。通过增加未选择：选择 DNA 的比例，非连锁标记基因（潮霉素磷酸转移酶）的功能共转化增加，从 1∶1 比例的 39%变化到 100∶1 比例的 65%。在最佳条件下，每输入 100 万个原生质体可回收多达 300 个转化的愈伤组织。该方案简单、廉价且有效，适用于需要大量稳定转化或共转化细胞系的玉米研究。
