Co-expression of flavonoid 3`, 5`-hydroxylase and flavonoid 3`-hydroxylase Accelerates Decolorization in Transgenic Chrysanthemum Petals

The flavonoid 3',5'-hydroxylase (F3',5'H) gene, derived from petunia, was introduced into chrysanthemum tissues by Agrobacterium-mediated genetic transformation. Cotyledon explants were co-cultured with A. tumefaciens LBA 4404 harboring the vector pMBP that carries F3',5'H under the control of the CaMV 35S promoter and nptll as a selectable marker gene. After 72 h of co-cultivation, the explants were placed on an MS medium supplemented with 4 mg $L^{-1}$ BA, 0.1 mg $L^{-1}$ NAA, 400 mg $L^{-1}$ carbenicillin, and 100 mg $L^{-1}$ kanamycin. After 4 weeks, kanamycin-resistant adventitious shoots had developed at a frequency of 6.3%. These shoots were then rooted and acclimatized in potting soil. Integration of F3',5'H into the plant genome was confirmed by Southern blot analysis. Flower buds that had red petals did not differ between the transgenic and the wild-type plants. However, petal color did change from red to bright orange to yellow when the buds developed into fully opened flowers on the transgenics. Spectrometric analysis revealed that the content of flavonoid compounds was more rapidly reduced in the transgenic petals as floral development proceeded. RT-PCR analysis showed that F3',5'H and flavonoid 3'-hydroxylase (F3'H) were expressed simultaneously in the transgenic plants. Therefore, we suggest that this more rapid change in petal color results from 1) competition between levels of transgenic F3',5'H and endogenous F3'H, each of which uses the same substrate in the flavonoid biosynthetic pathway and 2) the intrinsic substrate specificity of chrysanthemum DFR (dihy-droflavonol 4-reductase).