| The phenomenon of chromosome number variation due to abnormal mitosis commonly exists in somatic cell during plant tissue culture.Researches have shown that in some plants in vitro conditions,the chromosome polyploidization mutation often increases with the time.However,in long-term subculture,polyploidy level composition in citrus callus has remained relatively stable,and some callus still has a strong embryogenesis capacity after subculture for more than 10 years.Former researches presumed that tetraploid cells in diploid callus have a high frequency of apoptosis during subculture,so that the ratio of tetraploid cells maintained relatively stable.To verify the hypothesis,co-culture of diploid 'Guoqing No.1' Satsuma mandarine callus with green fluorescent protein marker and its homologous tetraploid nontransgenic cell lines obtained by low-density embedding has been carried out to study their interactions,with the tetraploid callus cultured alone as control.The main research results are as follows:1.Suspension tetraploid callus co-cultured with diploid callus at different culture stages were used for morphology detection by semi-section and TUNEL assay.Results showed that the apoptosis rate of tetraploid callus both in co-culture and cultured alone increased with time,at different stages of co-culture,the rate in co-culture was always higher than that in cultured alone,and at the 8th,11th and 17th day,a significant difference was found between two cultural conditions(p＜0.05).Additionally,an increasing trend was found in apoptosis rate of tetraploid callus as the overall callus and the proportion of diploid increases.2.Tetraploid callus in suspension co-culture was analyzed by semi-section and nuclear separation,after stained by fluorescent dye DAPI,citrus apoptotic callus cell in suspension culture presented typical characteristics of apoptosis,such as chromatin condensation,nuclei fragmentation,and apoptotic body-like structure. 3.TUNEL assay and cell cycle analysis of both tetraploid and diploid callus cell in the co-culture and culture alone showed that,the apoptosis rate and cell cycle change of diploid callus had no significant difference.But for tetraploid callus cells,the apoptosis rate was significantly higher in co-culture than cultured alone,and a significant retention in the cell cycle at the S phase was found(p＜0.05),indicating that tetraploid cells in the co-culture has more injured cells to enter S-phase and then induced apoptosis.4.In order to study whether direct touch is needed during the interaction between tetraploid and diploid callus cells,we designed a separation co-culture device in which different ploidy callus were separated by 0.45μm filtration membrane,while suspension culture medium could passed through freely,so did chemical substances that secreted by different ploidy calli to ensure their mutual communication.TUNEL assay showed that apoptosis rate of tetraploid callus cell in separation co-culture remained significantly higher than those in cultured alone(p＜0.05),indicating that the interaction between tetraploid and diploid callus cells could occur without direct contact,but with soluble chemicals.5.We cloned three cell cycle-regulatory genes cyclinB2;1,cyclinD3;2,and CDKB2;1,and two apoptosis intermediary genes metacaspase1 and metcaspas2 in 'Guoqing No.1' Satsuma mandarine callus,with the intent to explore whether the cell cycle regulatory proteins and plant apoptosis-related protein play a role in the apoptosis of tetraploid callus cells.The gene expression experiments via real-time PCR method showed that cyclinB2;1,cyclinD3;2 and CDKB2;1 of tetraploid callus is dwon-regnlated in co-culture comparing to that in cultured alone,while expression of metacaspase1 and metcaspas2 increased in co-culture.Moreover,cyclinD3;2 and metcaspase2 gene expression showed significant difference between different culture modes(p＜0.05), indicating that cyclinB2;1,cyclinD3;2 and CDKB2;1 may play a role in the tetraploid cell cycle regulation while metacaspase1 and metcaspas2 possible have a role in tetraploid cells apoptosis.6.We separated protoplasts of tetraploid and diploid callus to study the apoptosis of tetraploid callus on single-cell level.The first time mitosis activity was observed in low-density embedding of tetraploid and diploid protoplasts alone.Results showed that both diploid and tetraploid protoplast underwent their first mitosis on the 7th day.TUNEL detection showed that apoptosis apoptotic positive cells were visible in both single culture and mixed co-culture. |
