Analysis of the rolC Promoter Region lnvolved in Somatic Embryogenesis-Related Activation in Carrot Cell Cultures'

In cell cultures of carrot (Daucus carota l.), somatic embryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D. Previous analysis of transgenic carrot cells containing the 5' noncoding sequence of the Ri plasmid rolC and a structural gene for bacterial 0-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somatic embryogenesis. In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium. We also analyzed the cis region of the rolC promoter that is responsible for somatic embryogenesis-related activation (SERA), namely relatively low 0glucuronidase (CUS) activity in calli and proembryogenic masses (PEM) and high CUS activity in heartand torpedo-stage embryos. When the -255-bp region of the rolC gene was used, SERA was retained. Interna1 deletions within this -255-bp region did not alter SERA by the rolC promoter. Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35s core region (-90 to +6 bp), it conferred relatively low CUS activity in calli and PEM but high CUS activity in heart and torpedo embryos. When -848to -255-bp or -255to -94bp fragments of the rolC promoter were fused to the same CaMV 35s core region, CUS activity patterns were not related to somatic embryogenesis. These results suggest that the combination of severa1 regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures.