Gurel Songul, Gurel Ekrem, Miller Tamara I, Lemaux Peggy G
Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA.
Methods Mol Biol. 2012;847:109-22. doi: 10.1007/978-1-61779-558-9_10.
Successful efforts describing in vitro culturing, regeneration, and transformation of grain sorghum were reported, using particle bombardment, as early as 1993, and with Agrobacterium tumefaciens in 2000. Reported transformation efficiencies via Agrobacterium routinely range from 1 to 2%. Recently, such efficiencies via Agrobacterium in several plant species were improved with the use of heat and centrifugation treatments of explants prior to infection. Here, we describe the successful use of heat pretreatment of immature embryos (IEs) prior to Agrobacterium inoculation to increase routine transformation frequencies of a single genotype, P898012, to greater than 7%. This reproducible frequency was calculated as numbers of independently transformed IEs, confirmed by PCR, western, and DNA hybridization analysis, that produced fertile transgenic plants, divided by total numbers of infected IEs.
早在1993年就有报道称，利用粒子轰击成功实现了高粱的体外培养、再生和转化；2000年又报道了利用根癌农杆菌实现上述目标。通过根癌农杆菌介导的转化效率通常在1%至2%之间。最近，通过在感染前对外植体进行加热和离心处理，提高了几种植物通过根癌农杆菌介导的转化效率。在此，我们描述了在接种根癌农杆菌之前对未成熟胚（IEs）进行热预处理的成功应用，该方法可将单一基因型P898012的常规转化频率提高到7%以上。这个可重复的频率是通过PCR、western和DNA杂交分析确认产生可育转基因植株的独立转化IEs数量除以感染IEs的总数来计算的。
