cDNA cloning of regeneration-specific genes in rice by differential screening of randomly amplified cDNAs using RAPD primers.

A simple method for differential screening of randomly amplified cDNAs using primers for detection of randomly amplified polymorphic DNA (RAPD) has been developed. To detect and clone differentially expressed genes during regeneration, we compared mRNAs from rice calli before the induction of regeneration, 7 days after induction of organogenesis, and 7 days after induction of embryogenesis. The cDNAs were amplified by the polymerase chain reaction (PCR) with a single RAPD primer and were separated by agarose gel electrophoresis. A number of differentially amplified bands were detected. Five of the specific bands were cloned and their expression was analyzed by Northern hybridization. We isolated a cDNA clone which is specific to organogenesis, two clones which are specific to embryogenesis, and two clones which are common to organogenesis and embryogenesis but not present in unorganized calli. Two of the isolated clones are expressed at low levels. Thus, this method is useful for cloning of differentially expressed genes whose transcripts are of low abundance. Expression of one of the embryogenesis-specific cDNA clones, pCRE2, was analyzed in detail. The pCRE2 transcript accumulates transiently in calli after the induction of embryogenesis, and its accumulation in planta was specific to zygotic embryos.