A protocol for Agrobacterium-mediated transformation of Kalancho blossfeldiana with a flavonoid 3,5 hydroxylase (F35H) gene

In the present investigation, explants from Kalanchoe blossfeldiana were used for gene transformation. The young leaves were inoculated with Agrobacterium tumefaciens LBA4404 strain with a binary vector plasmid pArtblue containing F3'5'H gene under control of CaMV35S promoter and nptII selectable marker gene. After inoculation, the explants were transferred to the co-cultivation medium. They were then transferred to the selection medium containing kanamycin and were sub-cultured every two weeks. Leaves of the putative transgenic shoots that survived in the selection medium were used in reverse transcription polymerase chain reaction (RT-PCR) analysis to detect gene expression. The RT-PCR analysis showed the presence of 550 bp F3'5'H amplification products and had an expression of F3'5'H  gene. Plants with the introduced F3'5'H gene produced totally pale red flowers. Key words: Kalanchoe blossfeldiana, Agrobacterium-mediated transformation, young leaf, F3'5'H gene, reverse transcription-polymerase chain reaction (RT-PCR).