STRUCTURAL-CHANGES IN THE PLASTID DNA OF RICE (ORYZA-SATIVA L) DURINGTISSUE-CULTURE

To investigate the rearrangement of the plastid genome during tissue c ulture, DNA from rice callus lines, which had been derived individuall y from single protoplasts isolated from seed or pollen callus (protocl ones), was analyzed by Southern hybridization with rice chloroplast DN A (ctDNA) clones as probes. Among 44 long-term cultured protoclones, m aintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large regi on of the plastid genome. The deletions could be classified into 15 ty pes from their respective sizes and positions. By contrast, no deletio ns were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments r evealed that plastid genomes with deletions in several protoclones wer e organized as head-to-head or tail-to-tail structures. Furthermore, p tDNAs retained during long-term culture all had a common terminus at o ne end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed t he accumulation of starch granules in plastids and amyloplasts in prot oclones in which the plastid genome had undergone deletion. Our observ ations indicated that novel structural changes in the plastid genome a nd morphological changes in the plastid had occurred in rice cells dur ing long-term tissue culture. Moreover, the morphological changes in p lastids were associated with deletions in the plastid genome.