Dhingra Amit, Daniell Henry
Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, USA.
Methods Mol Biol. 2006;323:245-62. doi: 10.1385/1-59745-003-0:245.
Chloroplast genetic engineering offers a number of unique advantages, including high-level transgene expression, multigene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to other important plant species. For example, Arabidopsis may be an ideal model system for chloroplast functional genomics. The employment of chloroplast transformation technology in Arabidopsis has been hampered by the lack of an efficient and reproducible protocol that provides fertile chloroplast transgenic plants. Transformation of the Arabidopsis chloroplast genome was achieved via organogenesis but the efficiency was at least a 100-fold lower than in tobacco and had the drawback of polyploidy in the leaf tissue that resulted in sterile transgenic plants. This problem can be overcome by adapting procedures that are now available to regenerate plants from both diploid and tetraploid explants via callus. In addition, it is feasible to regenerate Arabidopsis via somatic embryogenesis. Recent breakthroughs in highly efficient plastid transformation of recalcitrant crops such as cotton and soybean have opened the possibility of engineering Arabidopsis plastid genome via somatic embryogenesis. Therefore, protocols of recent improvements in tissue culture, DNA delivery, and the novel vector designs are provided here in order to achieve highly efficient plastid transformation in Arabidopsis.
叶绿体基因工程具有许多独特的优势，包括高水平的转基因表达、在单次转化事件中进行多基因工程、通过母系遗传实现转基因隔离、不存在基因沉默、位置效应和多效性效应以及不需要的外源DNA。已有40多个转基因通过烟草叶绿体基因组稳定整合并表达，以赋予所需的农艺性状或高水平表达疫苗抗原和生物制药。尽管取得了如此重大的进展，但这项技术尚未扩展到其他重要的植物物种。例如，拟南芥可能是叶绿体功能基因组学的理想模型系统。由于缺乏能提供可育叶绿体转基因植物的高效且可重复的方案，拟南芥中叶绿体转化技术的应用受到了阻碍。通过器官发生实现了拟南芥叶绿体基因组的转化，但效率至少比烟草低100倍，并且存在叶组织多倍体的缺点，导致转基因植物不育。通过采用现有的从二倍体和四倍体外植体通过愈伤组织再生植物的程序，可以克服这个问题。此外，通过体细胞胚胎发生再生拟南芥也是可行的。棉花和大豆等难转化作物在高效质体转化方面的最新突破，为通过体细胞胚胎发生对拟南芥质体基因组进行工程改造开辟了可能性。因此，本文提供了组织培养、DNA导入和新型载体设计方面近期改进的方案，以便在拟南芥中实现高效的质体转化。
