De Buck S, Jacobs A, Van Montagu M, Depicker A
Department of Genetics, Flanders Interuniversity Institute for Biotechnology, Universiteit Gent, Belgium.
Mol Plant Microbe Interact. 1998 Jun;11(6):449-57. doi: 10.1094/MPMI.1998.11.6.449.
In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains. The transformation frequency of unselected A. thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18%. The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used. Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells. Molecular analysis of cocultivated N. tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA. Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants. Remarkably, 10 to 30% of the selected A. thaliana shoots or progenies lost the T-DNA marker they were selected on. As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs. In conclusion, a significant part of the T-DNAs is lost from the transformed cells.
鉴于最近的一项发现，即不同的T-DNA倾向于在单一染色体位置以重复序列的形式连接和整合，我们在拟南芥根外植体和烟草原生质体与两种农杆菌菌株共培养期间，测定了转化和共转化的频率。未选择的拟南芥芽的转化频率低于1%，而共培养的烟草原生质体的转化频率约为18%。共转化频率定义为用第一个T-DNA转化的细胞中含有第二个未选择的T-DNA的频率，无论选择方式、转化频率和所使用的植物系统如何，该频率约为40%且可重复。根据这些结果推断，在64%的转化植物细胞中至少存在两个独立转移的T-DNA。对在非选择性培养基上再生的共培养烟草芽进行分子分析表明，只有少数转化体的T-DNA发生了沉默（2/46）或截短（1/46）。因此，大多数整合的T-DNA在初级转基因植物中表达了其可选择或可筛选的标记。值得注意的是，10%至30%的选择的拟南芥芽或后代失去了它们所选择的T-DNA标记。由于这些再生植株中高频（17%）含有未选择的T-DNA，这些选择的植株可能是由不稳定的、瞬时表达的T-DNA的表达导致的。总之，很大一部分T-DNA从转化细胞中丢失。
