【作者】 刘丽丽；

【导师】 王淑杰；

【作者基本信息】 吉林大学 ， 农业生物环境与能源工程， 2013， 硕士

【摘要】 红豆杉植物体内含有强活性的抗癌物质紫杉醇，对人体多种常见的癌症均有良好的疗效。目前野生红豆杉资源已濒临灭绝，因此，为了避免破坏野生资源，国内外诸多商家开始大面积建立红豆杉人工林，由此引发了植物界科学家对其育苗技术的探索。植物组织培养在植物育苗中应用越来越广泛。目前，关于东北红豆杉(taxuscuspidata Sied. et Zucc.)组培快繁技术的研究相对较少，尚处于初级阶段。因此，本研究以东北红豆杉为材料，对最佳外植体的选择、不同培养阶段培养基配方、培养基的简化及微扦插环境进行研究，最终建立东北红豆杉工厂化育苗技术体系并进行成本预算，为实现东北红豆杉工厂化育苗提供理论和技术支持。研究结论如下：1）在初代培养中，通过不同外植体、培养基种类、激素、简化培养等对东北红豆杉芽萌动的影响试验，得出东北红豆杉组培育苗最佳外植体为2~3cm半木质化茎尖；简化前最佳培养基配方为B5+琼脂0.6%+蔗糖2%+IBA3mg/L+AC0.1%+农用链霉素0.4g/L，可以使愈伤组织诱导率达95%以上，愈伤量适中，芽萌动率达95%以上，芽萌动系数高，萌动时间短（6d）；简化后培养基最佳配方为1/2B5+琼脂0.6%+白砂糖2%+IBA3mg/L+AC0.1%+农用链霉素0.4g/L，并对芽的萌动无不良影响；培养容器可采用塑料杯代替三角瓶；通过对培养基成分及培养容器的简化节约成本大约为94.4%，其中培养容器约占93.7%。2）在继代培养中，通过不同种类培养基、不同激素配方、继代周期、简化培养对芽增殖的影响试验，得出东北红豆杉组培育苗继代培养最佳培养基配方为MS+琼脂0.8%+蔗糖3%+IBA2mg/L+BA0.05mg/L+AC0.1%+农用链霉素0.4g/L，简化后最佳培养基配方为1/2MS+琼脂0.8%+白砂糖3%+IBA2mg/L+BA0.05mg/L+AC0.1%+农用链霉素0.4g/L，简化后培养基对芽增殖无不良影响；培养容器仍采用塑料杯代替三角瓶；通过培养基及培养容器的简化节约成本大约为93.4%，其中培养容器约占92.5%。3）在微扦插试验中，以东北红豆杉当年生半木质化小枝条作为试验材料，对微扦插环境及基质配方进行了研究。初步筛选出较优的微扦插环境，即光照时间为16h/d（光强为1000lux）、温度为25℃、空气湿度为80%~85%；本研究采用有下界约束的单形格子混料设计对微扦插基质配方进行优化，因素包括沙子、苔藓、蛭石、山皮土，在非人工控制环境条件下（实验室内）使其生根，最终确定，东北红豆杉微扦插最佳基质配方为苔藓：蛭石：山皮土=1:4:5，可使生根率接近100%。4）通过以上试验的研究成果，建立了东北红豆杉工厂化组培育苗技术体系，包括初代培养、继代培养、微扦插生根3个部分，并对工厂化育苗成本进行了初步预算，得出平均每生产一株东北红豆杉组培苗所需成本约为0.9691元，其中人工费最高，约占50%，为东北红豆杉工厂化组培育苗提供参考。更多还原

【Abstract】 The strongly active anti-cancer paclitaxel existing in Taxus plants has a good effecton controlling various types of common human cancers. Because Taxus has been on theverge of extinction, many domestic and foreign businessmen has established large areasof artificial forests of Taxus, in order to avoid the damage of wildlife resources, and thistrend has triggered an exploration of Taxus breeding techniques. Plant tissue culture hasbeen applied more and more widely in plant breeding techniques. Currently, there is littleresearch for rapid propagation technology of Taxus cuspidata tissue culture, and it is stillin the primary stage. Therefore, in this research, Taxus cuspidata was chosen as materials,and the selection the optimum explants, the medium composition in different culturestages, the simplification of the culture medium and the micro-cutting environment werestudied. Finally, the factory breeding technology system of Taxus cuspidata wasestablished and its cost budget was estimated, in order to provide the theoretical andtechnical support for the realization of Taxus cuspidata factory seedling. Conclusions areas follows:1) In the primary culture stage, the study focused on the effects on bud sprouting ofTaxus cuspidata of different types of explants, medium, hormone composition, simplifiedculture and so on. The results showed that the optimum explants of Taxus cuspidata werethose with2~3cm semi-lignified shoot tips. Before simplification, the optimum mediumcomposition was B5+agar0.6%+sucrose2%+IBA3mg/L+AC0.1%+streptomycin0.4g/L, which could insure more than95%callus induction rate, moderate callus volume,more than95%bud sprouting rate, larger bud sprouting coefficient and shorter time ofbud sprouting (6d). After simplification, the optimum composition was1/2B5+agar0.6%+sugar2%+IBA3mg/L+AC0.1%+streptomycin0.4g/L, which made bud sprouting rateup to89%with no remarkable negative effect on bud sprouting. Plastic cups were used toreplace triangle bottles. It would save the cost at about94.4%in which culture containersaccounts for about93.7%by means of the simplification of culture medium componentsand culture containers.2) In the subculture stage, the study focused on the effects on the shoot proliferationof Taxus cuspidata of different types of medium, hormone composition, subculture cycle,and simplified culture. The results showed that the optimum composition was MS+agar0.8%+sucrose3%+IBA2mg/L+BA0.05mg/L+0.1%AC+streptomycin0.4g/L beforesimplification, whereas after simplification, the optimum composition was1/2MS+agar0.8%+sugar3%+IBA2mg/L+BA0.05mg/L+AC0.1%+streptomycin0.4g/L, and ithad no negative effect on shoot proliferation. Plastic cups were still used to replace triangle bottles. It would save the cost at about93.4%in which culture containersaccounts for about92.5%by means of the simplification of culture medium componentsand culture containers.3) In the micro-cutting experiments, several small half lignification branches ofTaxus cuspidata were taken as experimental materials in order to research themicro-cutting environment and the matrix composition. The illumination time was16h/days (light intensity was1000lux), temperature was25℃, air humidity was80%~85%,which were initially screened as better micro-cutting environment. Simplex latticemixture design bounded by lower limit was applied to optimize matrix composition ofmicro-cutting, including sands, moss, vermiculite, hillside soil. The roots were developedunder nonartificial control condition (laboratory). The results showed that the optimummatrix composition of micro-cutting was moss: vermiculite: hillside soil=1:4:5and therooting rate was nearly100%.4) Based on the research stated above, the factory cultivation seedling technologysystem of Taxus cuspidata was established, including primary culture, subculture, androoting of micro-cutting three parts. The initial cost budget estimation of factorycultivation seedling showed that the cost was0.9691yuan each tissue cultivate seedlingin which labor accounted for50%. It would provide reference for factory cultivationseedling of Taxus cuspidata.更多还原