Development of a particle bombardment-mediated transient transformation system for Taxus spp. cells in culture.
In developing and developed nations, plant cell culture systems are used to supply desirable compounds in lieu of chemical synthesis or natural extraction. When plant cell culture systems are unable to meet commercial demand, metabolic engineering offers a method to increase yields. However, to benefit from metabolic engineering approaches, effective transient transformation methods are required to rapidly identify and characterize key regulatory genes before intensive, time-consuming stable transformation efforts can proceed. This paper describes a particle bombardment-mediated transient transformation system for Taxus spp. in cell culture. Optimal parameters were established for the T. cuspidata cell line P991 and the T. canadensis cell line CO93D, resulting in reliable, efficient, transient expression of the firefly luciferase gene under control of the constitutive CaMV 35S promoter. Multiple bombardments and larger gold microcarriers (1.6 vs 1.0 microm in diameter) were particularly effective in increasing luciferase activity and in reducing variation among replicates. This particle bombardment-mediated transformation system was also shown to be capable of transiently expressing the DsRed and beta-glucuronidase reporter genes under the control of the maize ubiquitin and CaMV 35S promoters, respectively. With the ability to transiently transform Taxus spp. cell cultures using a variety of promoters and reporters, characterization of genes related to paclitaxel accumulation in culture can now proceed.
Vongpaseuth
Kham
K
Department of Chemical Engineering, University of Massachusetts-Amherst, 686 N. Pleasant St., Amherst, Massachusetts 01002, USA.
Nims
Ezekiel
E
St Amand
Melissa
M
Walker
Elsbeth L
EL
Roberts
Susan C
SC
D002478:Cells, Cultured; D008845:Microinjections / Q000379:methods*; D010316:Particle Size; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics* / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D020946:Taxus / Q000502:physiology*; D014162:Transfection / Q000379:methods*
D011994:Recombinant Proteins