Large deletions in chloroplast DNA of rice calli after long-term culture

Alteration in chloroplast DNA (ctDNA) in long-term cultures of calli derived from rice seeds were investigated using a BamHI-1 fragment of rice ctDNA as a probe. Among 10 callus lines cultured for 36 months, two lines (T36-1 and T36-2) were found to have large deletions (76 kb and 63 kb, respectively) in the chloroplast genome, while none of the same callus lines cultured for 6 months showed any ctDNA alteration. When T36-1 and T36-2 were cultured on MS maintenance medium for an additional six months (becoming T42-1 and T42-2, respectively), the ctDNA of T42-2 underwent a further deletion of 0.5 kb. Maps of deleted ctDNA molecules were constructed using fifteen hybridization probes. Based on the restriction maps of the deleted ctDNAs, the T36-1 and T42-1 callus lines lost two large inverted repeats, together with the intervening small single copy region. The T36-2 and T42-2 callus lines lost the rbcL gene and most of the large single copy region. The only region that these two ctDNA molecules have in common is an approximately 18 kb region that includes various psb and trn genes. These results suggest that this region of the rice chloroplast genome contains at least one functional replication origin of rice ctDNA. Regenerated plantlets were obtained from long-term cultured calli of T36-2, indicating that the deletion of a part of the ctDNA did not affect shoot differentiation from the calli. Southern hybridization patterns of the regenerated plantlets were almost the same as those of the callus lines. The mechanisms responsible for deletion of ctDNA in the callus cells are discussed.