Visual selection allows immediate identification of transgenic rice calli efficiently accumulating transgene products.
In genetic transformation systems, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. However, simultaneous use of the gene encoding green fluorescent protein (GFP) and an antibiotic resistance gene facilitates the selection process, since it allows visible selection of transformed cells. Here, we report the development of a visual selection system for transformed cells using a GFP marker without selection against antibiotics after Agrobacterium-mediated transformation in rice. Both GFP protein levels and GFP fluorescence in calli isolated by visual selection were higher than in calli selected on hygromycin (Hyg), suggesting that transgenic calli hyper-accumulating GFP were efficiently obtained by selection using GFP fluorescence itself rather than Hyg resistance. Furthermore, gfp transcripts in calli isolated by visual selection were more abundant than under Hyg selection; in contrast, transcript levels of hpt in calli selected visually were comparable to those obtained under Hyg selection. These results suggest that there was no correlation between hpt and gfp expression levels, despite the fact that they are aligned in tandem on an integrated locus after selection by either GFP fluorescence or Hyg resistance. This fact indicates that positional effects can influence the expression of each transgene differently, even when they are located in tandem at the same locus. In summary, based on our results, we discuss a model system for rice cell culture transformation for the production of recombinant proteins using visual selection.
Saika
Hiroaki
H
Plant Genetic Engineering Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Toki
Seiichi
S
D002934:Cinnamates / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000378:metabolism; D008856:Microscopy, Fluorescence; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes
D002934:Cinnamates; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C026273:hygromycin A