Agarwal Sujata, Loar Star, Steber Camille, Zale Janice
Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA.
Methods Mol Biol. 2009;478:105-13. doi: 10.1007/978-1-59745-379-0_6.
A method is described for the floral transformation of wheat using a protocol similar to the floral dip of Arabidopsis. This method does not employ tissue culture of dissected embryos, but instead pre-anthesis spikes with clipped florets at the early, mid to late uninucleate microspore stage are dipped in Agrobacterium infiltration media harboring a vector carrying anthocyanin reporters and the NPTII selectable marker. T1 seeds are examined for color changes induced in the embryo by the anthocyanin reporters. Putatively transformed seeds are germinated and the seedlings are screened for the presence of the NPTII gene based on resistance to paromomycin spray and assayed with NPTII ELISAs. Genomic DNA of putative transformants is digested and analyzed on Southern blots for copy number to determine whether the T-DNA has integrated into the nucleus and to show the number of insertions. The nonoptimized transformation efficiencies range from 0.3 to 0.6% (number of transformants/number of florets dipped) but the efficiencies are higher in terms of the number of transformants produced/number of seeds set ranging from 0.9 to 10%. Research is underway to maximize seed set and optimize the protocol by testing different Agrobacterium strains, visual reporters, vectors, and surfactants.
本文描述了一种利用与拟南芥花浸染法类似的方案对小麦进行花转化的方法。该方法不采用解剖胚胎的组织培养，而是将处于单核小孢子早期、中期至晚期且小花已剪掉的花前穗浸入含有携带花青素报告基因和NPTII选择标记的载体的农杆菌浸染培养基中。检测T1代种子中花青素报告基因在胚胎中诱导产生的颜色变化。将推定转化的种子萌发，基于对巴龙霉素喷雾的抗性筛选幼苗，并通过NPTII酶联免疫吸附测定法进行检测。对推定转化体的基因组DNA进行酶切，并在Southern杂交上分析拷贝数，以确定T-DNA是否已整合到细胞核中，并显示插入的数量。未优化的转化效率为0.3%至0.6%（转化体数量/浸染的小花数量），但就产生的转化体数量/结实种子数量而言，效率更高，范围为0.9%至10%。目前正在进行研究，通过测试不同的农杆菌菌株、视觉报告基因、载体和表面活性剂来最大化结实率并优化方案。
