Transgenic Pinus radiata from Agrobacterium tumefaciens-mediated transformation of cotyledons
Transgenic Pinus radiata from Agrobacterium tumefaciens-mediated transformation of cotyledons
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DOI：
10.1007/s00299-004-0769-z
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发表时间：
2004-07-01
影响因子：
6.2
通讯作者：
Dale, TM
中科院分区：
文献类型：
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作者：
Grant, JE;Cooper, PA;Dale, TM
以市售自由授粉种子为材料，建立了根癌农杆菌介导子叶外植体转化的方法。辐射松是南半球种植面积最大的商业针叶树种。关于该物种转化的报道一直依赖于对来自未成熟胚胎的胚性愈伤组织进行基因轰击。这种方法的主要缺点是能够转化和再生的基因类型数量很少。由于80%以上的辐射松子叶可以用成熟种子的子叶再生，因此将子叶外植体与根癌农杆菌AGL1菌株共培养，其中含有编码新霉素磷酸转移酶II(NptII)基因和β-葡萄糖醛酸酶基因(UidA)的质粒。用遗传素或卡那霉素选择转化芽。共培养后的子叶存活率和选择参数是转化成功的关键因素。在从选择培养基中回收的105个假定转化子中，经聚合酶链式反应分析，70%的转化子整合了nptII基因。由于松针中未知化合物的反应抑制，GUS组织化学方法检测uidA的表达是不可靠的。此外，经PCR和Southern分析鉴定的26个独立转化子中，只有4个包含这两个基因的完整拷贝。剩下的22个转化子似乎有一个截短或重新排列的T-DNA拷贝。这种截断/重排可能是由于花椰菜花叶病毒(CaMV)35S启动子所致。对T-DNA连接点的分析和导入DNA的测序将有助于阐明T-DNA插入的性质，从而有效地针对辐射松的遗传改良。
A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration. Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A. tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (GUS) gene (uidA). Transformed shoots were selected using either geneticin or kanamycin. Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters. Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR. GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles. Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes. The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA. It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.