杨树原生质体分离及电融合技术体系的研究
摘要
本文以南林895杨(Populus×euramericana cv‘Nanlin895’)、转Bt基因南林895杨和银白杨(P. alba)为材料,在建立悬浮细胞系及植株再生的基础上,开展杨树原生质体分离和电融合研究,筛选适合杨树的原生质体分离与纯化、原生质体电融合及杂种筛选和培养等条件、方法。旨在为杨树体细胞杂交种质创新研究提供实验依据。主要研究结果如下:
1.幼嫩茎段较适合诱导愈伤组织。愈伤诱导培养基为:MS+2,4-D 2mg/L。将诱导出的愈伤组织转入愈伤继代培养基:MS+2,4-D 2mg/L+ KT 0.2mg/L,经4~5次继代培养后获得淡黄色、结构疏松的胚性愈伤组织,将其转入液体培养基:MS+2,4-D 2mg/L +NAA 0.2mg/L+KT 0.1mg/L+水解酪蛋白500mg/L,建立杨树悬浮细胞系;
2.南林895杨和转Bt基因南林895杨愈伤组织最适合芽诱导培养基为:MS+6-BA 0.5mg/L+IAA 0.5mg/L;芽增殖培养基为:MS+6-BA 0.5mg/L+IAA 0.2mg/L;生根培养基为:1/2MS(大量元素减半)。银白杨愈伤组织最适合芽诱导培养基为:MS+6-BA 0.5mg/L+KT 0.5mg/L + TDZ 0.002mg/L;芽增殖培养基为:MS+6-BA 0.25mg/L+KT 0.25mg/L+TDZ 0.001mg/L;生根培养基为:1/2MS+IBA 0.2mg/L;
3.杨树叶肉原生质体分离的最佳条件为:酶液:纤维素酶(Cellulase R-10) 1% +纤维素酶(Cellulase RS) 1% +果胶酶(Pectolyase Y-23) 0.5% +甘露醇0.6mol/L,28℃,黑暗条件下54rpm轻摇振荡酶解。南林895杨、转Bt基因南林895杨叶片需酶解10h,而银白杨叶片酶解时间为14h,可获得纯度及活力高的杨树原生质体;
4.杨树悬浮细胞系原生质体分离的最佳条件为:酶液:纤维素酶(Cellulase R-10) 1% +纤维素酶(Cellulase RS) 1% +果胶酶(Pectolyase Y-23) 0.3% +甘露醇0.7mol/L,28℃,黑暗条件下54rpm轻摇振荡酶解10h;
5.杨树原生质体纯化采用2次漂浮离心法,首先800rpm下离心4min,然后400rpm下离心8min,纯化效果较好;
6.杨树原生质体融合较适合的电场参数为:AC强度100 V/cm,AC持续时间40s,DC强度1000V/cm,DC持续时间50μs,4次脉冲,2次循环;
7.融合亲本之一原生质体在融合前经UV照射处理6min,活力降低,细胞停止分裂,与另一亲本融合,结合融合亲本的抗生素标记可作为融合杂种筛选培养的有效方法; 8.原生质体在培养基:改良MS +2,4-D 2mg/L+NAA 0.5 mg/L +KT 0.5 mg/L+葡萄糖0.5mol/L中进行液体浅层培养,培养7d后,观察到了杂种融合体第一次分裂。
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.
1.幼嫩茎段较适合诱导愈伤组织。愈伤诱导培养基为:MS+2,4-D 2mg/L。将诱导出的愈伤组织转入愈伤继代培养基:MS+2,4-D 2mg/L+ KT 0.2mg/L,经4~5次继代培养后获得淡黄色、结构疏松的胚性愈伤组织,将其转入液体培养基:MS+2,4-D 2mg/L +NAA 0.2mg/L+KT 0.1mg/L+水解酪蛋白500mg/L,建立杨树悬浮细胞系;
2.南林895杨和转Bt基因南林895杨愈伤组织最适合芽诱导培养基为:MS+6-BA 0.5mg/L+IAA 0.5mg/L;芽增殖培养基为:MS+6-BA 0.5mg/L+IAA 0.2mg/L;生根培养基为:1/2MS(大量元素减半)。银白杨愈伤组织最适合芽诱导培养基为:MS+6-BA 0.5mg/L+KT 0.5mg/L + TDZ 0.002mg/L;芽增殖培养基为:MS+6-BA 0.25mg/L+KT 0.25mg/L+TDZ 0.001mg/L;生根培养基为:1/2MS+IBA 0.2mg/L;
3.杨树叶肉原生质体分离的最佳条件为:酶液:纤维素酶(Cellulase R-10) 1% +纤维素酶(Cellulase RS) 1% +果胶酶(Pectolyase Y-23) 0.5% +甘露醇0.6mol/L,28℃,黑暗条件下54rpm轻摇振荡酶解。南林895杨、转Bt基因南林895杨叶片需酶解10h,而银白杨叶片酶解时间为14h,可获得纯度及活力高的杨树原生质体;
4.杨树悬浮细胞系原生质体分离的最佳条件为:酶液:纤维素酶(Cellulase R-10) 1% +纤维素酶(Cellulase RS) 1% +果胶酶(Pectolyase Y-23) 0.3% +甘露醇0.7mol/L,28℃,黑暗条件下54rpm轻摇振荡酶解10h;
5.杨树原生质体纯化采用2次漂浮离心法,首先800rpm下离心4min,然后400rpm下离心8min,纯化效果较好;
6.杨树原生质体融合较适合的电场参数为:AC强度100 V/cm,AC持续时间40s,DC强度1000V/cm,DC持续时间50μs,4次脉冲,2次循环;
7.融合亲本之一原生质体在融合前经UV照射处理6min,活力降低,细胞停止分裂,与另一亲本融合,结合融合亲本的抗生素标记可作为融合杂种筛选培养的有效方法; 8.原生质体在培养基:改良MS +2,4-D 2mg/L+NAA 0.5 mg/L +KT 0.5 mg/L+葡萄糖0.5mol/L中进行液体浅层培养,培养7d后,观察到了杂种融合体第一次分裂。
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.
引文
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