[Spontaneous and induced programmed cell death in suspension cell cultures of cotton (Gossypium hirsutum L.)].

Cotton suspension cells grew well in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT. Senescence occurred when the cells were unsubcultured. The cells began to lose their viabilities on the 17th day, and on the 21th day oligonucleosomal sized DNA fragments ( DNA ladder) could be detected. Oligonucleosomal sized DNA fragments ( DNA ladder) was the hallmark of the programmed cell death. Programmed cell death of cotton suspension cells could be induced respectively by some stress factors which included heatshock (42+/-3 degrees C for 8 hours), 10 micromol/L camptothecin, 20 micromol/L fumonisin B1 and 50 mmol/L cycloheximide. The cotton suspension cells which grew in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT differred physiologically from the cells in the MS medium supplemented with 0.1 mg/L IBA and 0.1 mg/L KT, and they responded differentially to the heatshock, 10 micromol/L camptothecin and 20 micromol/L fumonisin B1, while the same to 50 mmol/L cycloheximide.

[Construction and analysis of SSH library of Gossypium barbadense upon infection with Verticillium dahliae].

Roots were collected from the seedlings inoculated with pathogen Verticillium dahliae after 2, 4, 8, 12, 24, 48, 72 and 96 hours for total RNA extraction. The cDNAs from the inoculated seedlings were used as the tester and those from the control seedlings as the driver. SSH method was employed to find the differently expressed cDNAs responding to the pathogen. T/A clone library was constructed containing 534 clones. The cDNA inserts were amplified from the bacterial clones directly with M13 primers by PCR. The size of the products ranged 0.2 - 1.2 kb with an average size of 0.5 kb. The SSH products were dotted on nylon filters, and the positive clones were screened by virtual Northern blotting with probes of the two kinds of initiative cDNAs. Totally 78 clones which were up-regulated and putatively involved in the defense response of G. barbadense were identified and sequenced. Sequence similarity searches were performed with the Blastn and Blastx. Most of them showed high or partial homology to genes or ESTs induced by different stresses in Arabidopsis thaliana and other species,such as the pathogenesis-related 10 family of G. hirsumtum and disease resistance-responsive family protein in Arabidopsis thaliana. The results would be helpful to understand the molecular mechanisms of disease response in cotton.

[Withdrawal of exogenous auxin induces programmed cell death of cotton embryogenic suspension cultures].

In the establishment of cotton suspension culture, we had observed an interesting phenomenon that large-scale cell death occurred when the embryogenic cells were transferred from the medium MS supplemented with IBA 0.5 mg/L to fresh 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 140-180 bp. Inhibitor studies suggested the proteolysis and the caspase-like proteases were involved in cell death. These results support that cell death caused by withdrawal of exogenous auxins is a kind of programmed cell death (PCD). So auxin is involved in the regulation of programmed cell death signal transduction pathways, and may be another plant-specific regulator beside ethylene, abscisic acid and gibberellin in PCD.