Colorimetric screening of recombinant RNAi vectors without using PCR

Abstract

S New Biotechnology · Volume 25S · September 2009 olus vulgaris L.) is not known. Using immunolocalization, in some species such as Zea mays, Hordeum vulgare and Arabidopsis thaliana Sus showed expression in the basal endosperm transfer cells and in developing embryos. In order to better define the role of Sus in Phaseolus, we have compared the pattern expression of Sus during developmental progression of wild-type and an ethyl methanesulfonate (EMS) mutant embryos. Homozygous embryos isolated from mutant aborted pods were arrested at the globular stage; they present a developmental delay during embryogenesis and lethality in seed growth. Expression profiling of Sus by reverse transcriptase polymerase chain reaction (RT-PCR) showed that the gene was active at low levels in vegetative wild-type tissues such as leaves, flowers, cotyledons and roots. Highest level of expression was observed in seeds and nothing in stems. In situ hybridization studies in wild-type, 3 days after pollination (DAP), show the gene activity in endosperm, suspensor, embryo, but also in a group of cells located in the outer and inner integumentary layer. At 7 and 8 DAP Sus gene product mRNA was detected specifically in suspensor and no signal detect in other tissue. However, at 9 and 12 DAP Sus expression was revealed in suspensor, endothelium layer, and cotyledons, but was absent from the endosperm, outer and inner integumentary layer. These temporal changes in cellular localization of Sus gene during common bean seed development are comparable with those reported in Arabidopsis seed. These data suggested a possible role of Sus in carbon portioning during early to mid stages of seed development, as a process to maintain sink strength. In later stage, Sus play a role within the cotyledon in sucrose utilization. In the EMS mutant plant, disruption in Sus pattern and expression highlight the roles of Sus gene during seed development in common bean. doi:10.1016/j.nbt.2009.06.965