Establishment Of Cotton Embryogenic Suspension Cultures And Studies On Programmed Death Of The Suspension Cells

Cell death can be divided into two kinds: programmed cell death (PCD)and necrosis. Programmed cell death (PCD ) is a genetically controlled active process of cell death that functions during development and in response to stress in different biological systems. Necrosis is a kind of cell death with catastrophic damage and the cell has no control over the death process. Animal apoptotic studies show that chromatin condenses, the maintaining of membrane continuity, a condensed cytoplasm, the cleavage of the nuclear DNA into oligonucleosomal sized fragments and enveloped to form apoptotic body are the typical morphological and biochemical features of PCD. The cleavage of the nuclear DNA is reflected in the apperance of DNA ladders consisting of multimers of 140-180 bp by agarose gel electrophoresis, by which PCD can be distinguished from necrosis.It often involves segregation of the dead cells from the living in the researches on programmed cell death mechanisms, the intrinsic death expression programmes exist in every single cell, so the establishments of suspension system consisting of single cells or protoplasts could make the research work more simple, and the suspension cells could provide an ideal research system that is convenient to the control mechanism studies of gene expression and signal transduction in PCD. Recently, more progress was made on the plant cell programmed death, many research works were based on suspension cells.In our studies, two Gossypium hirsutum vars Coker 201 and Ekang9 were selected as the experiment materials.Callus being induced from the hypotyl sections of the sterile cotton seedling were utilized to establish the suspension cell system.Systematic researches on the initial embryogenic callus, the combinations of plant hormones, the components of the media, the cells'growth curve and the cells' senescence during suspension culture reveals that the media MS+2,4-D 0.5 mg/L +KT 0.1 mg/L and MS+2,4-D 0.1 mg/L+KT 0.1 mg/L were suitable for cells proliferation and could produce uniform cells and small cell aggregates with strong viability which were optimal population to study cell growth and its regulation, while the media MS+IBA0.5 mg/L + KT 0.1 mg/L and MS+IBA0.1 mg/L + KT 0.1 mg/L were fit for producing big cell aggregates and embryoes of different developmental stages which could be used to induceand gather embryoes. K+ and inositol were important chemicals for cell proliferation but suppressed somatic embryogenesis of the suspension culture, which could be used to improve and adjust cell growth patterns and viability. In the course of suspension, the fistular cells were substituted by globular cells and small cell aggregates. The growth curve of suspension cells was typical of "S". In the first three days, the cells grew very slowly, from 3 to 15 days the cells proliferated exponentially, and 15days later the ceils proliferation was slowed down. So the suspension culture should be subcultured before the 15th day to sustain the strong cell division competence and cell viability.Experiments were carried out to study the mechanisms of programmed cell death of cotton suspensions during senescene or induced by heatshock, camptothecin, fumonisin Bl and cycloheximide. Cytological study and genomic electrophoresis showed that cotton suspension cells grew well in the MS medium supplemented with 0.1 mg/L 2,4D and 0.1 mg/L KT. Senescence occurred when the cells were unsubcultured. The cells began to lose their viabilities at the 17th day, and at the 21th day oligonucleosomal sized DNA fragments ( DNA ladder) could be detected. Oligonucleosomal sized DNA fragments ( DNA ladder) was the hallmarks of the programmed cell death. Programmed cell death of cotton suspension cells could be induced respectively by some stress factors wich included heatshock (42±3°C for 8 hours), lOjumol/h camptothecin, 20/^mol/L fumonisin Bl and 50mmol/L cycloheximide. Cotton suspension cells which had grown in the MS medium supplemented with 0.1 mg/L 2,4D and 0.1 mg/L KT diferred physiologically from the cells in the MS medium supplemented with 0.1 mg/L IB A and 0.1 mg/L KT, and they responded differentially to the heatshock, lQumol/L camptothecin and 20//mol/L fumonisin Bl, but the response to 50mmol/L cycloheximide was the same.In the establishment of cotton suspension culture , we observed an interesting phenomenon that large-scale cell death occurred when the embryogenic cells were transferred from the medium MS supplemented with IBA 0.5mg/L to frsh MS medium without IBA. Cytological study and genomic DNA electrophoresis showed that this kind of cell death was accompanied by such morphological characters as chromatin condensation, the maintenance of membrane continuity, a condensed cytoplasm and evident DNA fragmentation of multimers of 140-180bp. Inhibitor studies suggested theproteolysis and the caspase-like proteases were involved in cell death. These results supported that cell death caused by withdrawal of exogenous auxins was a kind of PCD. So auxin was involved in the regulation of programmed cell death signal transduction pathways, and might be another plant-specific regulator beside ethylene, abscisic acid and gibberellin in PCD.In the experiment, we also found that large-scale cell death occurred when cotton suspension cells were treated with high concentration of cytokinin (2 mg/L KT or 4 mg/L KT), and the suspension cultures turned brown-black. Agarose gel electrophoresis of genomic DNA showed that apparent DNA fragmentations of multimers of 140-180bp existed in the cells treated with high concentration of cytokinin (2 mg/L KT or 4 mg/L KT). Cytological studies also revealed that this kind of cell death was accompanied by such morphological characters as chromatin condenses, the maintaining of membrane continuity and condensed cytoplasm (shrinkage of the plasma membrane away from cell wall).These results supported that cell death induced by high concentration of cytokinin was a kind of PCD.In the experiment, we also found that cells in different physiological states(UCSA or BAE) and cells in various growth-phases (the exponential growth-phase or the quiescent phase) responded differentially to high concentration of cytokinin. The UCSA cells and the cells in exponential growth-phase were more sensitive to the inducer. It was also shown that the interaction between plant hormones2,4—D and cytokinin could inhibit the occurrence of PCD induced by the high concentration of cytokinin, but the interaction between ABA and cytokinin could not.The toxins secreted from Verticillium dahliae are critical biochemical factors that cause wilt.Treated with toxins from T9 and V*n respectively, both of Coker201 and Ekang 9 suspension cells were caused to death, but the death curves were different, in comparison with others, the death of Ekang 9 suspension cells treated with V*n toxins was faster and earlier. Agarose gel electrophoresis of genomic DNA also showed that evident DNA fragmentation of multimers of 140-180bp (DNA Ladder) existed in the treatment of Ekang 9 to V*n toxins.An reasonable explanation was that an avirulent avr gene existed in the Vsnstrain which corresponded to the resistant R gene in Ekang 9, while in T9 no related avr gene existed. So the V*n could cause HR in Ekang 9suspension cell, but T9 could not.