Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots.
An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar "Blue Uniform" was more responsive than "White Uniform". Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by beta-glucuronidase and ELISA analyses, respectively.
Sriskandarajah
Sridevy
S
Department of Agricultural Sciences, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, 1871, Copenhagen, Frederiksberg C, Denmark. srs@kvl.dk
Frello
Stefan
S
Jørgensen
Kirsten
K
Serek
Margrethe
M
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D029422:Campanulaceae / Q000235:genetics* / Q000254:growth & development / Q000382:microbiology; D003470:Culture Media / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics; D007612:Kanamycin / Q000494:pharmacology; D018520:Plant Shoots / Q000235:genetics / Q000254:growth & development / Q000382:microbiology; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000254:growth & development / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D036226:Seedlings / Q000235:genetics / Q000254:growth & development / Q000382:microbiology; D013045:Species Specificity; D014170:Transformation, Genetic / Q000235:genetics*
D003470:Culture Media; D007612:Kanamycin; D005966:Glucuronidase