Hokkaido Agriculture Research Centre, National Agriculture and Food Research Organization Toyohira-ku, Sapporo, 062-8555, Japan.
Biotechnology Development Laboratories, KANEKA CORPORATION, Takasago, 6768688, Japan.
Sci Rep. 2017 Sep 13;7(1):11443. doi: 10.1038/s41598-017-11936-0.
The currently favoured method for wheat (Triticum aestivum L.) transformation is inapplicable to many elite cultivars because it requires callus culture and regeneration. Here, we developed a simple, reproducible, in planta wheat transformation method using biolistic DNA delivery without callus culture or regeneration. Shoot apical meristems (SAMs) grown from dry imbibed seeds were exposed under a microscope and subjected to bombardment with different-sized gold particles coated with the GFP gene construct, introducing DNA into the L2 cell layer. Bombarded embryos were grown to mature, stably transformed T plants and integration of the GFP gene into the genome was determined at the fifth leaf. Use of 0.6-µm particles and 1350-psi pressure resulted in dramatically increased maximum ratios of transient GFP expression in SAMs and transgene integration in the fifth leaf. The transgene was integrated into the germ cells of 62% of transformants, and was therefore inherited in the next generation. We successfully transformed the model wheat cultivar 'Fielder', as well as the recalcitrant Japanese elite cultivar 'Haruyokoi'. Our method could potentially be used to generate stable transgenic lines for a wide range of commercial wheat cultivars.
目前，小麦（Triticum aestivum L.）转化的首选方法不适用于许多优良品种，因为它需要愈伤组织培养和再生。在这里，我们开发了一种简单、可重复的体内小麦转化方法，使用生物弹道 DNA 传递，无需愈伤组织培养或再生。从干吸胀种子中生长的茎尖分生组织（SAM）在显微镜下暴露，并通过不同大小的金颗粒进行轰击，这些金颗粒涂有 GFP 基因构建体，将 DNA 导入 L2 细胞层。轰击后的胚胎生长为成熟的、稳定转化的 T 植株，并在第五叶确定 GFP 基因的整合。使用 0.6-µm 颗粒和 1350-psi 压力可显著提高 SAM 中瞬时 GFP 表达和第五叶中转基因整合的最大比例。转基因整合到 62%的转化体的生殖细胞中，因此可以遗传到下一代。我们成功地转化了模式小麦品种“Fielder”，以及难以转化的日本优良品种“Haruyokoi”。我们的方法有可能用于生成广泛的商业小麦品种的稳定转基因系。
