Ogawa Yoichi, Shirakawa Makoto, Koumoto Yasuko, Honda Masaho, Asami Yuki, Kondo Yasuhiro, Hara-Nishimura Ikuko
Kazusa Unit, Honda Research Institute Japan (HRI-JP), Kisarazu, Chiba, 292-0818, Japan,
Plant Cell Rep. 2014 Jul;33(7):1161-72. doi: 10.1007/s00299-014-1605-8. Epub 2014 Apr 4.
A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.
开发了一种简单可靠的用于柳枝稷的农杆菌介导转化方法。使用该方法，可以产生许多携带多个目标基因的转基因植物，而不会出现未转化的逃逸植株。柳枝稷（Panicum virgatum L.）是一种很有前途的用于生物能源的生物质作物。为了以简单的方式获得携带多基因性状的转基因柳枝稷植株，通过构建基于Gateway的二元载体、优化转化条件和开发一种新的选择方法，建立了农杆菌介导的转化方法。构建了携带bar选择标记基因的多轮Gateway兼容目的二元载体pHKGB110，以便在一次转化中引入多个目标基因。两个报告基因表达盒GUSPlus和gfp分别构建在两个入门载体上，然后通过连续的LR反应引入到pHKGB110的单个T-DNA区域。携带所得二元载体pHKGB112的根癌农杆菌EHA101和颖果来源的紧密胚性愈伤组织用于转化实验。延长共培养7天，然后在含有美罗培南的培养基上培养，提高了转化效率，且农杆菌不会过度生长，然而，头孢噻肟或替门汀对其没有抑制作用。此外，在生根阶段，通过将假定转化的芽直接浸入除草剂Basta溶液中几秒钟（称为“除草剂浸渍法”），完全消除了未转化的逃逸芽。还证明，超过90%的bar阳性转化体携带了从pHKGB112传递的两个报告基因。这种简单可靠的转化方法，结合了一种新的选择技术和基于多轮Gateway的二元载体的使用，将适合于产生大量携带多个基因的转基因株系。
