【作者】 张静；

【导师】 施季森；

【作者基本信息】 南京林业大学 ， 林木遗传育种， 2007， 硕士

【摘要】 东方百合杂种系(Oriental hybrids)和麝香百合杂种系(Longiflorum hybrids)是目前国内外百合切花市场上供应的两个主要热销品系,具有较高的观赏价值和经济价值。本研究以东方百合西伯利亚(Lilium siberia)和麝香百合(Lilium longiflorum)为材料,从组织培养诱导体细胞染色体加倍和有性阶段诱导2n花粉二条途径对百合多倍体诱变育种进行了研究,以期为创造百合人工三倍体奠定技术和种质基础。建立百合组织培养体系是多倍体诱导的前提试验,结果表明:激素浓度及配比是百合鳞片芽诱导的主要影响因子,东方百合西伯利亚的最适芽诱导培养基为MS+蔗糖40g/L+6-BA1.0mg/L+NAA 0.5mg/L;最适壮苗及生根培养基为MS+蔗糖80g/L +NAA 0.3 mg/L,15d后生根率可达100%,鳞茎肥大且长势旺盛。蔗糖浓度对鳞茎壮苗和生根的影响重大,但过高的蔗糖浓度又会使培养基渗透压过高从而导致鳞茎出现萎蔫和死亡。西伯利亚百合体细胞染色体数目为2n=2x=24。其核型公式为2n=2x=24=4m+2t+14st+4sm(SAT),属于3B型。试验采用物理和化学方法对百合体细胞进行了加倍诱导。物理方法处理后发现效果不明显,仅出现了一些表型上的变异。化学方法采用共培养和直接浸泡二种方式。结果发现,直接浸泡效果较共培养好。经共培养的方式处理后得到2株嵌合体;浸泡方式处理后除获得36株嵌合体外,在浸泡时间为48h,秋水仙素浓度为0.02%和0.05%的处理中分别出现了1株和3株染色体加倍的鳞茎。对这4株鳞茎扩大繁殖并继代3次后得到的18株鳞茎,其中四倍性细胞(2n=4x=48)均达到了90%以上,可以确定为稳定的同源四倍体。在对四倍体和二倍体进行细胞学比较分析显示,四倍体植株的叶下表皮气孔密度和保卫细胞大小与二倍体植株相比差异均为极显著。因此,我们认为浸泡处理时间以48h为佳,秋水仙素浓度以0.02%和0.05%为佳。利用麝香百合花蕾诱导2n花粉的试验中发现,百合花蕾长度和花粉母细胞所处的分裂期具有对应关系,是估计花粉母细胞发育时期较为可靠的指标。且同一花蕾花粉囊里的花粉母细胞大都处于同一分裂相期。花粉母细胞染色体在间期复制一次后,经历连续的两次分裂,最后形成具有单倍染色体数目的花粉粒,属于连续型胞质分裂。当花蕾长度为1cm～2.5cm时花粉母细胞分裂处于旺盛期,2n花粉的有效诱导时期为花蕾长度为0.5～2.4cm。麝香百合花蕾经脱脂棉包裹和注射秋水仙素溶液处理后,发现花蕾组织较百合鳞片等体细胞组织对机械损伤和药品处理更为敏感,注射处理的方法较脱脂棉包裹处理对花蕾的伤害要轻。用0.1%的秋水仙素溶液注射百合花蕾3次后,产生了直径较大的2n花粉粒。光学显微镜下观察其颜色较深,体积约为正常n花粉的2倍,其与正常n花粉的比率为8.41%。更多还原

【Abstract】 Oriental hybrids and Longiflorum hybrids are very important in lily cut flowers on the world market. They act as ornamental and commercial function with high economical value. So choosing two of them, Lilium siberia and Lilium Longiflorum as materials, we studied on the somatic tissue and pollen chromosome doubling through tissue culture and buds treatment. In this way, we can found the technical and material resources ground for creating artifitial lily triploid.Founding the tissue culture system is one of the preconditions for the induction of lily somatic tissue polyploids. The result of tissue culture shows: the most appropriate medium for bulb scale germination of L.siberia is MS + sucrose 4% + BA 1.0mg/L + NAA 0.5mg/L medium. The concentration and mixture ratio of hormone are two of the important factors in bud inducing. The concentration of sucrose is the most important factor in bulblets growth, and the most appropriate medium for rooting induction and sound seedling of bulblets is MS + sucrose 8% + NAA 0.3mg/L.Knowing the diploid chromosome number and karyrotype of L.siberia is another precondition for its somatic tissue polyploids induction. Through the observation of mitosis course of L.siberia, we get the conclusion that the diploidy chromosome number of L.siberia is 2n=2x=24, its karyotype formula is 2n=2x=24=4m + 2t +14st + 4sm(SAT), belonging to 3B type. Then, We use physical and chemical methods to induce L.siberia somatic tissue polyploids, and in the end we find: the effect of physical treatment is not obvious because we just get some phynotype variation bulblets but no polyploidy cells after the evaluation of chromosome number. Polyploidy induction by chemical methods is carried out with colchicine by tow ways, that is cultured on the medium with colchicine and marinating in the solution of colchicine. By the way of cultured on the medium with colchicine, we only get 2 mixoploids, their cells some are diploids and others are tetraploids, and we have not find pure tetraploids. The best concentration of colchicine are 0.02% and 0.05%, and the best time of its treatment is 48h by marinating way. After these treatments, we get 4 tetrapliods in all, the rates of tetraploidy cells are all above 90% in their 18 offsprings. After the cytological comparation of diploids and tetrasploids leaves, we find the difference of stomatal density and size both are significant.In the study of 2n pollen induction of L.Longiflorum, we first studied on the meiosis course of its pollen mother cells, and the result shows: There is relationship between flower buds length and PMC developmental periods. When the flower bud length is 1.0cm～2.5cm, the PMC is of the vigorous development. PMCs are in the same stage of the same anther. The chromosomes of PMC duplicate once and then go through dividing twice. In the end, one PMC will become four pollens. Because there is no cell plate forming in the telophaseI, it belongs to successive cytokinesis. The best time for 2n pollen induction is when the length of flower buds is 0.5～2.4cm. On the ground of this, we treated the L.Longiflorum flower buds by two methods to induce 2n pollens: injection with colchicine solution and wraping with pledget sinked by colchicine solution. The former method is more suitable to lily flower buds than the latter one because lily flower buds are too sensitive to bear the high physical and chemical hurt of wraping way. After injection 0.1% colchicine solution three times into lily flower buds, we get some much bigger and deeper color pollens, they are regarded as 2n pollens.更多还原