Walter C, Grace L J, Wagner A, White D W R, Walden A R, Donaldson S S, Hinton H, Gardner R C, Smith D R
Forest Research Institute Ltd., Sala St. Rotorua, New Zealand Fax no.: +64-734-75444 E-mail:
AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand, , , , , , NZ.
Plant Cell Rep. 1998 Apr;17(6-7):460-468. doi: 10.1007/s002990050426.
A biolistic particle delivery system was used to genetically transform embryogenic tissue of Pinus radiata. The introduced DNA contained a uidA reporter gene under the control of either the tandem CaMV 35S or the artificial Emu promoter, and the npt II selectable marker controlled by the CaMV 35S promoter. The average number of stable, geneticin-resistant lines recovered was 0.5 per 200 mg fresh weight bombarded tissue. Expression of the uidA reporter gene was detected histochemically and fluorimetrically in transformed embryogenic tissue and in derived mature somatic embryos and regenerated plants. The integration of uidA and npt II genes into the Pinus radiata genome was demonstrated using PCR amplification of the inserts and Southern hybridisation analysis. The expression of both genes in transformed tissue was confirmed by Northern hybridisation analysis. More than 150 transgenic Pinus radiata plants were produced from 20 independent transformation experiments with four different embryogenic clones.
采用生物枪粒子递送系统对辐射松的胚性组织进行遗传转化。导入的DNA包含一个受串联CaMV 35S或人工Emu启动子控制的uidA报告基因，以及一个受CaMV 35S启动子控制的npt II选择标记。每200毫克鲜重轰击组织平均获得0.5个稳定的抗遗传霉素株系。通过组织化学和荧光法在转化的胚性组织、衍生的成熟体细胞胚和再生植株中检测到uidA报告基因的表达。利用插入片段的PCR扩增和Southern杂交分析证明了uidA和npt II基因整合到辐射松基因组中。通过Northern杂交分析证实了这两个基因在转化组织中的表达。利用四个不同的胚性克隆进行了20次独立转化实验，培育出了150多株转基因辐射松植株。
