Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and periphe...Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.展开更多
Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast u...Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.展开更多
文摘Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.
文摘Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.