Du Ningxia, Pijut Paula M
Department of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center (HTIRC), Purdue University, 715 West State Street, West Lafayette, IN, 47907, USA.
Plant Cell Rep. 2009 Jun;28(6):915-23. doi: 10.1007/s00299-009-0697-z. Epub 2009 Apr 3.
A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were transformed in the presence of 100 microM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l(-1) was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with 13.3 microM 6-benzylaminopurine, 4.5 microM thidiazuron, 50 mg l(-1) adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse. This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash.
开发了一种绿梣（Fraxinus pennsylvanica）下胚轴外植体的遗传转化方案。使用携带二元载体pq35GR的根癌农杆菌菌株EHA105对绿梣下胚轴进行转化，该二元载体包含新霉素磷酸转移酶（nptII）和β-葡萄糖醛酸酶（GUS）融合基因以及一个增强型绿色荧光蛋白基因。预培养的下胚轴外植体在含有100微摩尔乙酰丁香酮的条件下，通过90秒超声处理加10分钟真空渗透进行转化。使用20毫克/升的卡那霉素筛选转化细胞。不定芽在添加了13.3微摩尔6-苄基腺嘌呤、4.5微摩尔噻二唑脲、50毫克/升硫酸腺嘌呤和10%椰子水的Murashige和Skoog培养基上再生。通过中间愈伤组织阶段从下胚轴切段产生GUS和聚合酶链反应（PCR）阳性芽。通过PCR确认GUS阳性芽中GUS和nptII基因的存在，并通过Southern印迹法测定PCR阳性芽中nptII基因的拷贝数。三株转基因小植株在温室中驯化。这种利用下胚轴的转化和再生系统为根癌农杆菌介导的绿梣转化奠定了基础。目前正在进行研究，使用含有苏云金芽孢杆菌Cry8Da蛋白的构建体对绿梣进行遗传转化。
