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Green fluorescent protein (GFP) has been used extensively as a novel non-invasive reporter in the investigation of issues such as promoter function, transformation marker recognition, and sub-cellular localization analysis. A plant-optimized synthetic GFP modified via the replacement of the serine to threonine at position 65 [sGFP(S65T)] has achieved high levels of expression with no toxic side-effects in many plants. It would be useful to develop a non-invasive selection system based only upon GFP expression levels. A trapping vector was constructed using an sGFP(S65T) and a quantitative fluorescence imaging system was used for non-invasive screening of trapped tobacco cultured cells. Putative trapped lines could be identified using this system. Moreover, the GFP products from the candidate trapped lines could be quantitatively visualized directly via SDS-PAGE using the fluorescence imaging system. We conclude that the non-invasive selection system described here could be a powerful new tool for plant biotechnology.

We have focused on possible stronger promoters in the chloroplast: those of psbA encoding D1 protein of photosystem II reaction center, 16S rDNA in rrn operon, the bacterial fused promoter tac, and the bacteriophage T7 gene φ10 in combination with transgenic T7 RNA polymerase (RNAP). Arabidopsis plants were made transgenic in the nuclear genome with the construct of a chimeric gene for T7 RNAP fused to a chloroplast transit peptide at its N-terminus placed under the control of CaMV 35S promoter. We have transiently expressed gene for β-glucuronidase (GUS) under control of the above promoters in the Arabidopsis chloroplast followed by particle bombardment. Expression in the chloroplast but not in the nucleus was confirmed histochemically and by treatment with α-amanitin. T7 promoter was the strongest among the examined promoters in the Arabidopsis chloroplast, being applicable to higher expression of foreign genes in the chloroplast with managed expression of T7 RNAP.