Tian L-N, Charest P J, Séguin A, Rutledge R G
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S, P.O. Box 3800, Sainte-Foy, Quebec G1V 4C7, Canada e-mail:
Natural Resources Canada, Canadian Forest Service, Science Branch, 580 Booth St., 7th Floor, Ottawa, Ontario K1A 0E4, Canada, , , , , , CA.
Plant Cell Rep. 2000 Mar;19(4):358-362. doi: 10.1007/s002990050740.
Using particle bombardment of mature somatic embryos followed by the induction of secondary embryogenesis in the presence of hygromycin, we produced over 90 lines of transgenic embryonal masses expressing β-glucuronidase from two genotypes of black spruce. Transformation efficiencies of up to 7% (1 transgenic line per 14 embryos bombarded) were achieved by extending the period of selection from 8 to 12 weeks. Proliferation of transformed embryonal masses in the presence of hygromycin had no effect on either embryogenicity or embryo maturation. Southern blot hybridization and PCR amplification confirmed the presence of the hygromycin phosphotransferase gene in genomic DNA. The expression of the β-glucuronidase gene in the needles of regenerated seedlings support the potential for long-term transgene expression in spruce.
通过对成熟体细胞胚进行粒子轰击，随后在潮霉素存在下诱导次生胚发生，我们从两种黑云杉基因型中产生了90多个表达β - 葡萄糖醛酸酶的转基因胚性团系。通过将选择期从8周延长至12周，转化效率达到了7%（每轰击14个胚获得1个转基因系）。在潮霉素存在下转化胚性团的增殖对胚性或胚成熟均无影响。Southern杂交和PCR扩增证实了基因组DNA中潮霉素磷酸转移酶基因的存在。再生幼苗针叶中β - 葡萄糖醛酸酶基因的表达支持了云杉中转基因长期表达的潜力。
