Agri-Biotechnology Research Laboratory Nippon Paper Industries Co., Ltd., Kita-ku, Tokyo, Japan.
Plant Cell Rep. 2012 Jan;31(1):225-35. doi: 10.1007/s00299-011-1159-y. Epub 2011 Oct 19.
Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.
蓝桉是最重要的纸浆用人工林硬木之一。然而，其低转化频率阻碍了该物种与有用基因的遗传工程。我们发现，带顶芽的下胚轴切段具有最高的再生能力，并且已经开发出一种使用这些材料的高效农杆菌介导的转化方法。然后，我们将一个耐盐性基因，即带有 GUS 报告基因的细菌胆碱氧化酶基因（codA），导入蓝桉。来自带顶芽下胚轴的转基因芽再生的最高频率为 7.4%，四次实验的平均频率为 4.0%，比不带顶芽的下胚轴高 12 倍。使用约 10000 个外植体，通过 GUS 分析证实了超过 250 个再生芽为转化体。对 100 个伸长芽的 Southern blot 分析证实了稳定转化体的成功生成。对 44 个选定的转基因株系进行了甜菜碱积累的研究，发现其甜菜碱水平比非转基因对照高出 1 到 12 倍。通过在 1000ppm CO2 富集下进行自养生根法，成功地对 16 个转基因株系进行了生根。将 16 个转基因株系的完整植株移植到盆栽土壤中，并在生长室中正常生长。它们对 300mM NaCl 表现出耐盐性。我们的系统的要点是使用带顶芽的外植体作为材料，在选择培养基上抑制顶芽的伸长，并从顶芽相对的一侧再生转基因芽。这种方法也可能解决其他重要植物的转化问题。
