植物激素和抗生素调节的转基因火炬松愈伤组织的生长和分化

包含质粒载体pBI121的农杆菌菌株LBA4404被用于转化火炬松的成熟合子胚。质粒载体pBI121含有胭脂碱合成酶基因的启动子驱动的新霉素磷酸转移酶基因（npt Ⅱ)和花椰菜花叶病毒的35S启动子驱动的GUS基因。器官发生的转基因愈伤组织和转基因的再生植株已经获得，并经GUS组织化学染色、聚合酶链式反应和Southern杂交分析证实。植物激素（BA/IBA）和抗生素对转基因愈伤组织的生长和分化的影响被研究。500mg/L羧苄青霉素和2mg/LBA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加54.2%，分化增加45.7%。500mg/L Claforan和2mg/L BA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加40.8%，分化增加38.7%。高浓度的植物激素和抗生素降低了转基因愈伤组织的生长和分化。实验结果有助于建立一个高效的农杆菌介导的火炬松遗传转化系统，也有助于未来针叶树的遗传转化研究。

Growth and Differentiation of Transgenic Callus Regulated by Phytohormones and Antibiotics in Transformation of Loblolly Pine

Mature zygotic embryos of loblolly pine (Pinus taeda L. )were transformed by Agrobacterium tumefaciens strain LBA 4404 harbouring the plasmid pBI121 which carried the selectable marker gene, neomycin phosphotransferase Ⅱ (nptⅡ) controlled by the promoter of the nopaline synthase gene, and the uidA reporter gene, encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Organogenic transgenic calli and transgenic regenerated plantlets were produced on selection medium containing 15mg/L kanamycin, and confirmed by GUS histochemical staining, polymerase chain reaction (PCR), and southern blot analysis. Influences of phytohormone (BA/IBA) and antibiotics on growth and differentiation of organogenic transgenic calli were investigated. Of the phytohormone (BA/IBA) and antibiotics administered, 500mg/L carbenicillin combined with 2 mg/L BA and 0.5mg/L IBA (BA/IBA=4) resulted in a 54.2% higher increase in the growth of transgenic calli as well as in the differentiation of transgenic calli, which was 45.7% more than that of control on the 6th week of culture. Claforan at 500 mg/L combined with 2 mg/L BA and 0.5mg/L IBA resulted in a 40.8% increase in the growth of transgenic calli, and 38.7% increase in the frequency of transgenic calli forming adventitious shoots compared with the control. The growth and differentiation of transgenic calli of loblolly pine was reduced preferentially by higher BA/IBA (BA/IBA=8), as well as high con-centration of antibiotics (carbenicillin and claforan, 550mg/L each). But it was observed that 450mg/L and 500mg/L carbenicillin and claforan caused an increase in growth and differentiation of transgenic calli. These results suggested that the establishment of an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine was also dependent on the regulation of phytohormone and antibiotic on growth and differentiation of transgenic calli. This work could be useful for the future studies of genetic transformation of conifers.