摘要
菊花(Chrysanthemum morifolium Ramat.)是中国的传统名花,也是世界上最重要的观赏花卉之一。地被菊是菊花适于陆地栽培的一个品种群,其花色丰富,观赏性好,覆盖能力强,具有广阔的应用前景。但由于城市水资源的缺乏和盐渍化的日益严重,限制了地被菊的推广。通过根癌农杆菌介导的方法,将甜菜碱合成相关酶的基因PEAMT转入地被菊基因组中,培育出抗逆性更强的新品种,对地被菊在全国大面积推广,更好的为园林绿化服务具有重要意义。
本研究获得的主要结果如下:
1、建立了地被菊品种‘玉人面’、‘铺地金’的转化受体系统。以MS为基本培养基,采用6-BA和NAA两种激素9种不同配比的组合,从10个地被菊品种中筛选出‘玉人面’、‘铺地金’和‘北林红’3个高频再生品种,再生率分别为98%、96%和96%;‘玉人面’的再生培养基为:MS+6-BA1mg/L+NAA0.1mg/L;‘铺地金’的再生培养基为:MS+6-BA2mg/L+NAA1mg/L;‘北林红’的再生培养基为:MS+6-BA1mg/L+NAA0.5mg/L。同时还研究了不同的外植体类型,继代次数对再生的影响,叶片再生能力最高,但随着继代次数的增加,部分品种叶片再生能力有下降的趋势。研究了‘玉人面’和‘铺地金’对抗生素Kan的敏感性,结果表明7.5mg/L的Kan是菊花品种‘玉人面’叶片外植体筛选培养的临界耐受浓度,10mg/L的Kan是‘铺地金’叶片筛选培养的临界浓度;培养基中浓度为250mg/L的头孢霉素能够很好的抑制农杆菌的生长,且没有影响叶片外植体的再生率和不定芽数,可作为‘玉人面’和‘铺地金’的抑菌性抗生素。
2、建立了地被菊‘玉人面’的遗传转化体系:以预培养12h的叶片为转化材料,用浓度OD600为0.4的携带有PEAMT基因的农杆菌菌液侵染10min,24℃共培养2d,延迟培养4d,用选择压为7.5mg/L的Kan连续筛选直至有不定芽生成。待不定芽长至1cm左右时转入附加20mg/L Kan的1/2MS生根培养基中,继续筛选,并对生根抗性植株进行了PCR和Southern blotting检测,103株生根抗性苗中,有9株呈PCR阳性反应,获得特异性扩增条带,其中4个被检植株产生Southern杂交信号,证明PEAMT基因已整合到地被菊基因组中,外源基因主要是单拷贝插入。
3、从转入PEAMT基因的‘玉人面’株系中,选取生长正常的4个株系及非转
Chrysanthemum morifolium is one of the most famous flowers in China and ornamental species in the world. Ground-cover Chrysanthemum is a superior breeding group of Chrysanthemum morifolium, which applies to land growth in gardens with plenty of colors, wide coverage and excellent ornamental value. However, the application of Ground-cover Chrysanthemum is restricted by the booming problem of lacking water resources and salification in urban areas. Agrobacterium tumefaciens taken as the transformation vector, the spinach's PEAMT gene, which catalyzes the synthesis of glycine betaine, is transferred into the genome of Ground-cover Chrysanthemum. Therefore, this transfer will result in the accumulation of compatible solute--glycine betaine and the enhancement of tolerance in transgenic plants under the stress conditions of drought, salt and low temperature. Furthermore, the transgenic Ground-cover Chrysanthemum can be widely applied into use around the country and serve the gardens.
The main research work and results were as follows:
1. The genetic transformation receptor systems for‘Yurenmian’and‘Pudijin’were established. Using 9 different concentration proportion of 6-BA and NAA which were based on MS culture medium, 3 high efficiently shoot regenerating cultivars, that was‘Yurenmian’,‘Pudijin’and‘Beilinhong’were selected from 10 cultivars of Ground-cover Chrysanthemum, with the regenerating rate being 98%, 96% and 96%. The optimal regeneration medium for‘Yurenmian’was MS+6-BA1mg/L +NAA0.1mg/L, for‘Pudijin’was MS+6-BA2mg/L +NAA1mg/L and for‘Beilinhong’was MS+6-BA1mg/L +NAA0.5mg/L. At the same time, the factors which would influence shoot regeneration such as the different explant types, timing culture in vitro were studied. The results showed that: the leaves’regeneration ability was best, timing culture in vitro influenced the leaves’regeneration ability and the time of keeping high
引文
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