Social Control On Chromosome Number Variation By Cell Apoptosis Pathway In Citrus Callus

Variations in chromosomes number ubiquitously occur in plant tissue culture. Citrus callus has strong regenerability, and can be preserved for long period of years without loss of regeneration capability. Cytological examination of single-protoplast derived cell lines showed that chromosome number variations arose spontaneously from mitotic irregularities, and appeared at times when maintained in vitro. Paradoxically, ploidy level of long-term-cultured citrus callus remained stable, leading to producing true-to-type plants. It appeared that tetraploid cells underwent programmed cell death following their emergence.To corroborate this hypothesis, diploid cells isolated from GFP-transgene callus and tetraploid cells were stimultaneously cultured in liquid MT medium. Aggregations of tetraploid cells were obtained and prepared for microscopic observations, in which some cells showed typical cell apoptosis. Chromatin condensation and margination around the nuclear membrane have been observed, as well as the condensation of protoplast away from the cell wall. Boundary obviously existed between the apoptotic cells and normal viable cells. Further research was done to elucidate the frequency of cell apoptosis in tetraploid cells cultured with diploid cells at different proportions and for different periods. It showed that tetraploid cells underwent more apoptosis when they were cultured along with diploid cells for 2 to 7 days than cultured alone. When the tetraploid cells predominated in the mixed population, they were prone to die. The frequency of apoptosis increased with culture time, which was significantly high 2 days after culture. TUNEL (TdT-mediated dUTP Nick-End Labeling) assay showed that the positive rate of PCD was up to 36.9% after co-culture for 7 days, higher than that based on cytological observation, due to the DNA damage that occurred earlier than the condensation of nucleus.To investigate the interaction between cells with different ploidy, they were cultured in the same medium in a container separated by a filter membrane with pore diameter of 0.22μm to avoid direct contact. No significant difference of apoptosis rate was observed between tetraploids cultured with diploids and those cultured alone for 4 days, indicating that the interaction requires direct contact between cells under this growth condition.