| In this paper, Populus×euramericana cv'Nanlin895', transgenic Populus×euramericana cv'Nanlin895'with Bt gene and P.abla were used as experiment materials, on the base of the establishment of suspension culture and plant regeneration, we studied protoplast isolation and protoplast electofusion in poplar, selected the optimum conditions and methods for the protoplast isolation, protoplast purification, protoplast eletrofusion, protoplast culture and selecting the somatic hybrids. The main results described as follows:1. The plantlet twigs were feasible to induce callus. The medium for callus inducement was MS+2,4-D 2mg/L. The induced calli were transferred into the medium for callus subculture MS+2,4-D 2mg/L+ KT 0.2mg/L. The calli become light yellow and texture short after 4-5 subculture periods, which were used to establish cell suspension culture of poplar with the liquid medium MS+2,4-D 2mg/L +NAA 0.2mg/L+KT 0.1mg/L+CH 500mg/L;2. The medium MS+6-BA 0.5mg/L+IAA 0.5mg/L is the optimum medium for bud inducement of Populus×euramericana cv'Nanlin895'and transgenic Populus×euramericana cv'Nanlin895'with Bt gene. The medium for subculture multiplication is MS+6-BA 0.5mg/L+IAA 0.2mg/L. The medium for rooting is 1/2MS; The optimum medium for bud inducement of P.alba is MS+6-BA 0.5mg/L+KT 0.5mg/L + TDZ 0.002mg/L. The medium for subculture multiplication is MS+6-BA 0.25mg/L+KT 0.25mg/L+TDZ 0.001mg/L. The medium for rooting is 1/2MS+IBA 0.2mg/L;3. The optimum method of isolating mesophyll protoplasts of poplar was established. Leaf sections were incubated in a enzyme solution containing Cellulase R-10 1% , Cellulase RS 1% ,Pectolyase Y-23 0.5%,Mannitol 0.6mol/L, in the dark at 28℃on a rotary shaker at 54rpm. The isolation time of Populus×euramericana cv'Nanlin895'and transgenic Populus×euramericana cv'Nanlin895'with Bt gene is 10 hours, and the isolation time of P.alba is 14 hours;4. The optimum method of isolating cell suspension protoplasts of poplar was established. Cells were incubated in a enzyme solution containing Cellulase R-10 1% , Cellulase RS 1% ,Pectolyase Y-23 0.3%,Mannitol 0.7mol/L, in the dark at 28℃on a rotary shaker at 54rpm for 10 hours;5. The protoplasts of poplar were purified by centrifugation at 800 rpm for 4min, and then at 400rpm for 8min;6. The optimum electric parameters for protoplast fusion in poplar were alternating current (AC) 100V/cm, duration 40s; direct current (DC) 1000V/cm, 4times, duration 5μs, 2 cycles; 7. After irradiation with UV light for 6min, the viability of one parental protoplast became low and cell stopped dividing, together with the antibiotic marker of parental, could be used as a method for the selection of the somatic hybrids;8. The fused protoplasts of poplar were cultured in the medium modified MS +2,4-D 2mg/L+NAA 0.5 mg/L +KT 0.5 mg/L+ sucrose 0.5mol/L by the means of liquid-thin layer culture. The first division was observed in 8 days. |
