Agrobacterium tumefaciens-mediated transformation of Pinus pinea L cotyledons:: an assessment of factors influencing the efficiency of uidA gene transfer

被引:52
作者
Humara, JM
López, M
Ordás, RJ
机构
[1] Univ Oviedo, Dpto Biol Organismos & Sistemas, Unidad Fisiol Vegetal, E-33071 Oviedo, Asturias, Spain
[2] Univ Oviedo, Inst Univ Biotecnol Asturias CNB, CSIC, E-33071 Oviedo, Asturias, Spain
关键词
Agrobacterium tumefaciens; hypersensitive response; Pinus pinea; transformation; sonication-assisted Agrobacterium-mediated transformation;
D O I
10.1007/s002990050709
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
This study is the first report of a protocol for transfer and expression of foreign chimeric genes into cotyledons excised from Pinus pinea L. embryos. Agrobacterium tumefaciens EHA105 harbouring the plasmid p35SGUSint was more infective than LBA4404 or C58 GV3850, as determined by the percentage of cotyledons showing uidA expression. Factors which significantly affected the T-DNA transfer included: (1) preinduction and concentration of bacteria, (2) days of coculture and (3) the wounding procedure applied. More efficient transfer of the uidA gene was achieved growing the bacteria in YEP medium at pH 7, infecting the cotyledons according to the sonication-assisted Agrobacterium-mediated transformation procedure with a bacterial density of 1 (OD600nm) for 5 min, and coculture for 72 h. Using this protocol, 49.7% of the cotyledons showed a diffuse blue staining 7 trays after infection. However, all were necrotic 30 days after inoculation. Since a decrease in bacterial density to 0.01 allowed the recovery of about 4% of cotyledons forming buds 1 month after inoculation, we conclude that the high mortality associated with the infection may be related to the hypersensitive response of the plant to bacterial infection.
引用
收藏
页码:51 / 58
页数:8
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