Immunohistochemical analysis on control of regeneration-pathways in rice calli

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To isolate proteins and gene that control two regeneration-pathways (embryogenesis and organogenesis) in rice calli.1. Four proteins (a, b, c, d) specific to embryogenic calli and two proteins (e, f) specific to regeneration (common in both embryogenic and organogenic calli) were detected by 2 - D PAGE analysis. Partial amino acid sequences of protein a were determined.2. After affinity chromatography of ConA, ten glycoproteins specific to embryogenic calli were detected.3. To isolate rare proteins that control regeneration, immuno-subtraction method was applied. The protein expressed in unorganized calli were subtracted from total proteins of embryogenic or organogenic calli by affinity chromatography using the antiserum against extracts of unorganized calli. Six proteins specific to embryogenic calli, two proteins specific to organogenic calli and a protein specific to regeneration were newly detected.4. To isolate and clone rare genes that are differentially expressed, a novel method of differential screening of random amplified cDNAs with RAPD primers was applied. A number of differentially expressed transcripts in rice calli could be detected and cloned by this method. Northern blot analysis also revealed that the detected differentially expressed transcripts included rare genes and that a gene specific to embryogenic calli was exclusively expressed in zygotic embryos.