Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line
Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line
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DOI：
10.1007/s00299-014-1656-x
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发表时间：
2014-10-01
影响因子：
6.2
通讯作者：
Zhao, Zuo-Yu
中科院分区：
文献类型：
--
作者：
Cho, Myeong-Je;Wu, Emily;Zhao, Zuo-Yu
描述了一种改进的农杆菌介导的转化方案，用于具有优化的培养基修饰和AGL1的顽固性商业玉米良种自交系。这些改进可应用于其他商业自交系。本研究描述了使用最佳共培养、静息和选择培养基，在顽固商业玉米精英自交系 PHR03 中显着改进的农杆菌介导的转化方案。在静息和选择培养基中使用绿色再生组织培养基成分、高铜和6-苄氨基嘌呤，显着提高了转化频率。在静息培养基中使用葡萄糖通过提高未成熟胚胎的组织诱导率、组织存活和组织增殖进一步增加转化频率。因此，在共培养、静息和选择培养基中葡萄糖、铜和细胞分裂素的最佳组合使用农杆菌菌株 LBA4404 在 PHR03 的转化中使 T-0 植物水平从 2.6% 显着提高到十倍。此外，我们评估了四种不同的农杆菌菌株 LBA4404、AGL1、EHA105 和 GV3101 的转化频率和事件质量。 AGL1 在 T-0 植物水平上的转化频率最高，高达 57.1%。然而，与 LBA4404 相比，AGL1 导致质量较低的事件（定义为没有农杆菌 T-DNA 主链的转基因单拷贝）（30.1% vs 25.6%）。我们建议这些改进可以应用于其他顽固的商业玉米自交系。
An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds.This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T-0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T-0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.