【作者】 宗红；

【导师】 陈运起； 王秀峰；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2007， 硕士

【摘要】 本试验研究了大葱成熟胚组织培养的再生体系、秋水仙碱处理种子诱导多倍体对成活率及种芽变异形态的影响、组织培养与秋水仙碱相结合的诱导方法及存在的问题、诱导过程中愈伤组织的相关生理生化变化。主要结果如下:1以大葱成熟胚为外植体建立再生体系,愈伤组织诱导、不定芽分化、诱导生根的最佳培养基及激素配比分别为: MS+2,4-D2.0 mg/L+BA1.0 mg/L、MS+2,4-D0 mg/L+BA1.5 mg/L、MS培养基。2秋水仙碱处理大葱种子进行多倍体诱导,研究了种子状态、浓度、时间、温度四个因素对出苗率的影响,从种子状态、浓度、时间三个方面对种子发芽后的形态进行了研究。试验表明,萌动种子的出苗率最低、致死率高、种芽的加粗变异率最高,处理时间对加倍效果的作用要大于浓度。经秋水仙碱处理得到了大量加粗变异的种芽,但是,由于受秋水仙碱的毒害,抑制根尖的生长,在后期的生长中逐渐干枯死亡。3深入研究了组织培养与秋水仙碱相结合的多倍体诱导技术。试验采用秋水仙碱加入培养基和秋水仙碱溶液浸泡两种方法处理愈伤组织,从而筛选出最佳的诱导方法和最佳的秋水仙碱处理浓度和时间。经染色体数目鉴定表明,这两种方法都能有效的诱导四倍体细胞(2n=4x=32)的产生(大葱为二倍体植物,正常染色体数为2n=2x=16)。但是,加入培养基法无论在诱导率上还是在操作上都优于液体浸泡法。其中以在培养基中加入0.08%秋水仙碱,处理4d的诱导效果较好,加倍率达26.0%。研究发现,在愈伤组织多倍体诱导过程中,经秋水仙碱处理的愈伤组织,褐化现象极其严重、不定芽分化受阻、诱导率在后期培养过程中有下降的趋势。针对这一问题,试验改变愈伤组织的分化培养基(琼脂、蔗糖、2,4-D、BA),表明,在不添加2,4-D的基础上,适当提高蔗糖的浓度在一定程度上可以减缓褐化现象的发生(60 g/L)、提高芽分化率(40 g/L); BA浓度过高不利于芽分化,应选择适中的浓度,本试验BA浓度为1.5 mg/L时芽分化率较高;琼脂对褐化现象的改善及芽分化率的提高作用不明显。试验得到了2种嵌合体植株,一种为同时具有三倍体和二倍体细胞的植株,另一种为同时具有四倍体和二倍体细胞的植株。对秋水仙碱处理前后大葱愈伤组织SOD、POD、CAT活性的变化进行了初步研究,探讨了多倍体诱导过程中的生理生化变化,研究表明,秋水仙碱处理后,POD、CAT活性变化规律相似,短时间处理,随浓度的增加酶活性增加;长时间处理,随浓度的增加呈先升后降。SOD活性的变化与POD、CAT有所不同,低浓度下活性变化趋缓、不明显,在相对较高浓度,长时间处理下则酶活性增加,这很可能是因为SOD对秋水仙碱的适应性较强,在POD、CAT不能适应条件变化时,SOD仍能保持较高的活性。更多还原

【Abstract】 Four different parts were studied in this paper: the tissue culture of Welsh onion using mature embryos as explants; the influence on seeding rate and aberrance rate with the action of colchicines; the inducing methods and problems with the combination of tissue culture and colchicines; correlative physiological chages in the inducing progress.The main results are as follows.1. An efficient generation system of Welsh onion was established in this experiment, using the mature embryos as explants. It resulted that the best hormone combination for callus induction was MS+2,4-D2.0 mg/L +BA1.0 mg/L and adventitious bud differented best in the medium supplemented with 2,4-D 0mg/L+BA 1.5mg/L. It also showed that adventitious bud could grow well in the medium MS and develop complete plants.2. Seeds were induced using colchicines. The different influences on seeding rate by seed condition ,concentration , periods, temperature were studied. The actions of condition ,concentration , periods to seed aberrance rate were also different. As far as the duding seeds are concerned,it has the lowest seeding rate, the upmost aberrance rate. The induction effect of periods was better than concentration. A great many of aberrance buds were acquired. But ,because of the hurt of colchicines, the growth of root was restrained. Consequently the buds was dying gradually.3. The combination of tissue culture and colchicines was researched deeply. Callus were treated by two methods: adding colchicines into medium and dipping callus in colchicines solution. It resulted that the two methods both could induce tetraploid cells. But the former method is better than the latter one,not only inducing rate but also in the operation progress . Adding 0.08% colchicines into medium 4d could gain the upmost inducing rate 26.0%.In the progress of inducing ,the dying rate of callus was every serious, the growth of adventitious buds were restrained, and the inducing rate would drop in the upper cultivation. To the question, we changed the growth condition of callus(agar,sucrose,2,4-D,BA). It resulted that appropriate increase of sucrose(60g/L) without 2,4-D could reduce dying rate and increase budding rate(40g/L. The experiment acquired two kinds of deviant plants:one with nomal cells and trinalploid cells,the other one with nomal cells and tetraploid cells.The changes of enzyme SOD,POD,CAT after treatments were studied .It resulted that the activity of POD and CAT had simlar changes.In short times, with the increase of concentration, the activity was improved; in longer time, with the increase of concentration, the activity was improved first then dropped. But SOD is very different from POD and CAT. In short times, the activity of SOD changed unconspicuously. In longer times , the activity of SOD was increased. The reason is that the adaptability of SOD is better than POD and CAT. SOD could keep stronger activity in adverse conditions.更多还原