Rodenburg K W, de Groot M J, Schilperoort R A, Hooykaas P J
Department of Plant Molecular Biology, Leiden University, Netherlands.
Plant Mol Biol. 1989 Dec;13(6):711-9. doi: 10.1007/BF00016026.
In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3-10-fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts from recalcitrant plant species.
关于根癌土壤杆菌将哪种DNA形式（单链或双链）转移到植物细胞的问题，我们研究了通过电穿孔将单链DNA与双链DNA导入植物原生质体时的行为。为此，我们在M13载体tg130和tg131中克隆了一个带有植物NPTII基因以及CAT基因的构建体。我们发现，两条互补的单链分子在植物原生质体中都产生了大量的CAT活性，这表明单链DNA被植物细胞复制机制转化为双链DNA。出乎意料的是，我们发现单链DNA导致稳定转化（对卡那霉素抗性进行筛选）的频率比双链DNA高3至10倍。这些结果表明，在需要最佳转化频率的实验中，例如使用难转化植物物种的原生质体进行实验时，可以考虑使用单链DNA。
