摘要
本研究以杨树优良无性系为材料,选择组培苗叶片和茎段诱导愈伤组织,建立杨树悬浮细胞系并植株再生,进而筛选原生质体分离的最佳条件;在此基础上,应用PEGΚ高Ca~(2+)高pH法开展杨树原生质体成对融合,为创造杨树新种质提供依据。主要结论如下:
1 不同基因型材料的愈伤组织诱导率几乎没有差异。2,4-D浓度介于1mg/L和3mg/L之间,经过5次调控继代的愈伤组织,形成的愈伤组织由色泽淡黄而鲜艳的小颗粒组成,生长速度快,适宜建立胚性悬浮细胞系。
2 胚性愈伤组织转入液体培养基时,杨树种不同建立稳定的悬浮细胞系所需的时间相差不大。以MS培养基为基本培养基,附加1.0-2.0mg/L的2,4-D,悬浮细胞培养起始密度以40ml的液体培养基加入鲜重4g悬浮细胞,5~8d左右继代一次有利于杨树胚性悬浮细胞系的建立和保持。
3 在悬浮培养条件下,杨树细胞先形成细胞团,再由一些胚性细胞团发育形成一个或几个胚状体。诱导胚状体发生时较高浓度的BA可诱导胚性愈伤组织产生较多的胚状体,低浓度的BA+NAA则可促进胚状体伸长。
4 酶液的成分组成与浓度对原生质体的游离有明显影响。本实验采用1.0%纤维素酶Cellulase RS、0.5%果胶酶Pectolyase Y23、0.5%半纤维素酶Hemicellulase和1.0%离析酶Macerozyme R-10组合酶液来游离原生质体,其中添加0.6mol/L甘露醇Mannitol、1470mg/LCaCl_2·2H_2O和95mg/L KH_2PO_4,pH值为5.8。
5 不同材料和不同取材时间对原生质体产量和活力有显著影响:采用继代3d的悬浮细胞游离原生质体所得的产量最高;试管苗则采用继代25d的叶片能获得最高的产量,而且原生质体活力也最高;悬浮细胞游离原生质体得率和活力均明显高于试管苗叶片游离的原生质体。酶解时间对原生质体产量和活力也存在影响:酶解8h的悬浮细胞原生质体产量基本上达到最高产量,酶解10h后,有些原生质体开始破裂,原生质体活力下降。渗透势对游离原生质体的影响则不明显。
6 PEG—高Ca~(2+)高pH法诱导原生质体融合研究结果表明,35%的PEG浓度能使原生质体融合获得较高的杂种融合率,同时原生质体不易破裂,是杨树PEG—高pH高钙法诱导原生质体融合的适宜浓度。
In this paper, calli were induced from the blades and stems of sterile seedlings of the poplars. Suspension cultures were established. The factors influencing protoplasts isolation were studied. The poplar protoplast fusion was also studied, by use of the polyethylene glycol (PEG) method. The main results described as follows:
1 There were almost no differences in the ratios of callus induction among different poplar of materials used in the experiments. With the concentration of l~3mg/L 2,4-D and after five subculture periods, the calli were vivid light yellow granules and grew fast, which was ideal to establish suspension culture.
2 It was showed that there were no significant differences in the time needed to establish suspension culture among different Populus species. Suspension in the liquid MS medium combined with 2,4-D 1.0~2.0mg L -1, KT 0.2mg L-1, with the starting density of 4g fresh suspension cell per 40mL liquid medium, and a subculturing period per 5~8d, were the best for establishment and maintaince of suspension culture.
3 Under the condition of suspension culture, cell cluster were formed first, and then one or several embryoids were induced from embryogenic cultures. High concentration of BA can induce more embryoids than low concentration, and low concentration of BA combined with NAA promoted the growth of embryoids.
4 Four enzymes:1.0% Cellulase RS, 0.5% Pectolyase Y23, 0.5% Hemicellulase, andl.0% Macerozyme R-10 combined with 0.6mol/L Mannitol, 1470mg/L CaCl2 2H2O, and 95mg/L KH2PO4,pH value 5.8, were applied to isolate protoplasts. It was indicated that components and concentrations of enzyme distinctly influenced the results of protoplasts isolation.
5 It was showed that different materials and different subculture time distinctly influenced the yield and vitality of protoplasts: after suspension cells subcultured for 3 days, the yield of the protoplasts was the highest. And the yield of the protoplasts isolated from the leaves of sterile seedlings after subculturing for 25 days was the highest and the protoplasts showed the strongest vitality. Higher yield and stronger vitality of the protoplasts isolated from suspension cells than from the leaves of sterile seedlings were observed. The results also showed that the isolation time affected the yield and vitality of the protoplasts. With 8 hours isolation, the protoplasts yield of suspension cells was almost the highest. Some protoplasts began to decomposed and the vitality began to decrease after 10 hours isolation. No significant influences of osmatic pressure to the protoplasts isolation were observed.
6 The results of the fusion of the selected protoplasts induced by Polyethylene glycol (PEG) showed that a relatively high fusion probability could be obtained when the protoplasts were induced by the concentration of 35% PEG and the protoplasts were not prone to decomposed.
引文
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