Wetten Andy C, Thomas Jean-Luc, Wagiran Alina, Chiurugwi Tinashe
School of Biological Sciences, University of Reading, Reading, UK.
Methods Mol Biol. 2012;847:255-65. doi: 10.1007/978-1-61779-558-9_22.
A transgenesis programme has been developed for Impatiens balsamina that will allow elucidation of the roles played by individual genes in the flower reversion phenomenon shown by this model species. The lack of explants exhibiting adventitious shooting in I. balsamina hinders Agrobacterium-based transformation, but the multiple shoots that arise from cotyledonary nodes present a suitable target for biolistics. These tissues can be disrupted by the helium blast effect associated with conventional biolistic devices, so we have utilised modifications to the PDS 1000/He equipment originally developed for transformation of fragile insect tissues. By loading microcarriers on to a rigid, rather than flexible, macrocarrier, the blast effect is largely eliminated, and the use of a focussing nozzle allows the bombardment to be concentrated on the target tissues. This approach reduces waste of plasmid DNA and gold microcarriers and achieves transfection at lower, less disruptive helium pressures than would otherwise be necessary to efficiently penetrate below the shoot epidermis and generate heritable transgenic lines.
已为凤仙花开发了一个转基因程序,这将有助于阐明该模式物种所表现出的花逆转现象中单个基因所起的作用。凤仙花中缺乏表现出不定芽形成的外植体,这阻碍了基于农杆菌的转化,但子叶节产生的多个芽为生物弹道学提供了合适的靶标。这些组织会被与传统生物弹道装置相关的氦气爆炸效应破坏,因此我们对最初为脆弱昆虫组织转化而开发的PDS 1000/He设备进行了改进。通过将微载体加载到刚性而非柔性的宏载体上,爆炸效应在很大程度上得以消除,并且使用聚焦喷嘴可使轰击集中在靶组织上。这种方法减少了质粒DNA和金微载体的浪费,并在比有效穿透芽表皮以下并产生可遗传转基因系所需的更低、破坏性更小的氦气压力下实现转染。