【作者】 王虹；

【导师】 张金凤；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2004， 硕士

【摘要】 科罗拉多冷杉组织培养技术的研究为其以后的组培快繁和其它生物技术奠定技术的基础。本文对科罗拉多冷杉组织培养技术进行了研究，包括外植体的表面灭菌、最适宜外植体的筛选和最佳启动培养基、继代培养基、生根培养基等的筛选。 研究结果表明：(1) 本试验以70％酒精和0.1％升汞为消毒剂，筛选适宜的外植体灭菌时间。种子(去除外种皮)用0.1％升汞表面处理8分钟，污染率较低(1.11％)；苗尖用0.1％升汞表面处理2分钟，污染率(1.11％)和褐化率较低(1.11％)。 (2) 以各种源科罗拉多冷杉成熟胚、子叶、子叶—下胚轴、下胚轴、苗尖、带子叶顶芽为外植体，只有成熟胚、苗尖、带子叶顶芽获得了无菌培养体系。 (3) 成熟胚不定芽诱导的培养基为1／2MS+2.7mg／L6-BA，种源间存在差异，只有4号种源成熟胚有不定芽发生，诱导率为23.33％；苗尖启动培养基为MS或MS+0.1mg／L6-BA，种源间差异显著，各种源苗尖启动率在80.0％～93.3％范围之内；胚萌发培养基为DCR，各种源成熟胚萌发率在83.33％～98.33％范围之内；成熟胚愈伤组织诱导培养基为SH+1.8mg／L2，4-D+1.2mg／L6-BA。愈伤组织诱导需要黑暗培养。 (4) 不定芽不易伸长，随着培养时间的延长易干枯或褐化死亡；愈伤组织在继代2～3次后，褐化死亡。这是本试验遇到的困难，也将是今后研究过程中要解决的难点。苗尖的腋芽诱导培养基为MS+0.3mg／L6-BA+0.1 mg／LIBA，从诱导出的腋芽数来看，8号＞12号；从苗高看，12号＞10号。带子叶顶芽的分化培养基为SH+0.4～0.5mg／L6-BA+0.3mg／LIBA，各种源带子叶顶芽的分化率在40.00％～76.67％范围内。诱导出的腋芽和针叶基潜伏芽均可在MS培养基上进行伸长培养，2～3月后，可以长成1～2cm无根苗。此时健壮的无根苗可以转入生根培养基中进行诱导生根。 (5) 科罗拉多冷杉无根试管苗生根困难，经过一年多的研究，目前仅513号种源有根发生，生根培养基为SH+0.5mg／L6-BA+0.5mg／LIBA+0.5mg／LNAA。 以上培养基均添加蔗糖30g／L(生根培养基20g／L)，琼脂6.5g／L，pH值5.8左右。培养温度24℃±2℃，每天光照12～14小时，光照强度1200～1600lx。 本试验采用科罗拉多冷杉成熟胚、苗尖、带子叶顶芽为外植体，成功地得到了无根试管苗，但是诱导无根试管苗生根很困难，需要进一步研究。更多还原

【Abstract】 The techniques research about the tissue culture of Abies concolor will lay a foundation for its vegetative propagation and application of other biotechnology. In this thesis, the technology about tissue culture of Abies concolor was studied in detail. The whole technique system included sterilization, optimal explants selection and the optimal media selection for initiation, proliferation, rooting.The results were as follows:(1) Different disinfectant times were tested with the 70% alcohol and 0.1% HgCl2. For seeds, disinfested in 0.1% HgCl2 for 8 minutes was the best and its pollution rate was the lowest (1.11%). For shoot tips, disinfested in 0.1% HgCl2 for 2 minutes was the best and its pollution rate and browning rate were the lowest.(2) Mature embryos, cotyledons, cotyledon-hypocotyls, hypocotyls, shoot tips, terminal buds from mature embryos of different provenances of Abies concolor were taken as explants. Mature embryos, shoot tips and terminal buds were the suitable explant materials.(3) The optimal induction medium of adventitious buds consisted of one-half strength MS medium and 2.7mg/L 6-BA. The results indicated that there were differences among various provenances. Adventitious buds had achieved from mature embryos of the provenance 4 of which induction frequency reached 23.33%. The suitable initiation media of shoot tips were MS medium or MS medium containing 0.1mg/L 6-BA.Significant differences were detected among provenances, the initiation frequency ranged from 80.0% to 93.3%. The best germination medium of mature embryos was DCR medium. The germination frequency ranged from 83.33% to 98.33%. The callus induction was successfully taken place in darkness on the SH medium containing 1.8mg/L 2,4-Dand 1.2mg/L6-BA.(4) Adventitious buds elongate difficultly and browned to death with the culture time gone by. It is a difficult problem that callus would browned to death, after it wastransferred 2 to 3 times. The selected induction medium of axillary buds was MS medium containing 0.3mg/L 6-BA and 0.1mg/L IBA. The shoot tips of provenance 8 produced more axillary buds than provenance 12. The height of shoot tips of provenance 12 was higher than that of provenance 8. The differentiation medium of terminal buds was SH medium containing 0.4~0.5mg/L 6-BA and 0.3mg/L IBA. The differentiation frequency ranged from 40.00% to 76.67%. The axillary buds and dormant buds could elongate 1-2 cm height on MS medium.(5) The shoots of Abies concolor were difficult to root. At present, the roots were only achieved from shoots of provenance 513. The rooting medium was SH medium containing 0.5mg/L 6-BA, 0.5mg/L IBA and 0.5mg/L NAA.All media contained 30g/L sucrose (rooting medium contained 20g/L) and 6.5g/L agar. The pH of all media was adjusted to 5.8 prior to autoclaving. The temperature in the tissue culture room was adjusted at 24C +2C, with an illumination time of 12-14 hours and illumination intensity 1200~1600Lx.Shoots were achieved from mature embryos, shoot tips, terminal buds, but which were difficult to root and further work was needed.更多还原