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题名: 新疆一枝蒿离体培养再生体系建立及多倍体诱导研究
作者: 唐晓义
答辩日期: 2009-06
导师: 王晓军
专业: 有机化学
授予单位: 中国科学院研究生院
授予地点: 北京
学位: 硕士
关键词: 药用植物 ; 新疆一枝蒿 ; 秋水仙素 ; 多倍体
摘要: 新疆一枝蒿(Artemisia rupestris L.)又名岩蒿,属菊科蒿属多年生草本植物。药用全草,具有祛风活血、散瘀消肿、健胃消食、清热解毒、抗过敏、解蛇毒等功能,用于治疗毒蛇咬伤、荨麻疹、消化不良、感冒、肝炎、过敏性疾病等症,为常用维吾尔药材之一。为了提高新疆一枝蒿药材的产量和品质,我们利用组织培养技术进行了新疆一枝蒿多倍体育种的研究,旨在选育出有效成分含量高的优良品种。现将实验结果总结如下:1. 新疆一枝蒿离体快速繁殖体系的建立。结果显示:新疆一枝蒿无菌苗茎段在MS+6-BA 1.0 mg·L-1+NAA 0.05 mg·L-1培养基上诱导出的丛生芽长势健壮,芽增殖系数可达11。在不加激素的MS培养基上再生芽生根率可达100%。2. 新疆一枝蒿的多倍体诱导与鉴定。采用茎段浸泡法和培养基培养法对新疆一枝蒿进行了化学诱导,形态学观察结果显示:茎段浸泡法的诱导效果比培养基培养法好,其中以0.2%秋水仙素浸泡茎段24 h的处理效果最好,变异率达27.3%。染色体鉴计数和流式细胞仪检测结果均表明我们获得了四倍体和异倍性嵌合体。采取切割不定芽并诱导不定芽丛生的方法对异倍性嵌合体进行分离,流式细胞仪检测结果显示分离得到了四倍体植株,表明采取该方法来分离异倍性嵌合体是可行的。四倍体继代植株的倍性鉴定结果表明诱变获得的四倍体植株倍性具有较好的稳定性。3. 新疆一枝蒿试管苗的移栽。结果显示:草炭土比蛭石和珍珠岩的混合基质更适合作为新疆一枝蒿的试管苗移栽基质。移栽后的7 d内存活率可达100%,但7 d到14 d之间,由于实验环境中温度、湿度无法控制稳定,部分试管苗叶片会枯萎,逐渐茎部也会枯萎以至死亡,存活率仅为38%。4. 四倍体植株和二倍体植株的形态学的初步观察。结果显示:四倍体植株和二倍体植株相比,四倍体植株茎干粗壮,节间长度缩短,根粗壮,叶片厚且颜色深绿。四倍体植株和二倍体植株气孔大小分别为:34.7 ± 0.2 × 29.6 ± 0.2μm 和22.8 ± 0.2 × 19.4 ± 0.2 μm(长×宽),气孔显著增大;四倍体植株和二倍体植株的气孔密度分别为:84.3 ± 4.8 mm-2和120.5 ± 5.7 mm-2,气孔密度显著下降。 5. 四倍体植株和二倍体植株的HPLC指纹图谱和一枝蒿酮酸含量的分析。对瓶内生长9个月的新疆一枝蒿植株及其亲本二倍体进行了HPLC指纹图谱比对,并对二者中所含一枝蒿酮酸的含量进行了测定。HPLC指纹图谱比对结果显示:四倍体植株和二倍体植株均存在原药材指纹图谱的中的4个特征峰,如蒙花苷、一枝蒿酮酸、金腰素乙和洋艾素。一枝蒿酮酸的含量测定结果显示:四倍体植株的一枝蒿酮酸含量明显高于二倍体的,增加约55.8%。
英文摘要: Artemisia rupestris L., belongs to the genus Artemisia of family Compositae, and is a well-known traditional Chinese medicinal plant in Xinjiang used for detoxification, anti-hypersusceptibility, antitumor, antibacterial, antivirus and protecting liver. A study on the breeding of polyploid of Artemisia rupestris L. by tissue culture was done aims at developing superior varieties of Artemisia rupestris L.. The experimental results are summarized as follows: 1. The establishment of the technique and methods of tissue culture of Artemisia rupestris L.. The results indicated adventitious shoots were obtained on shoots explants with an average of 11 shoots per shoot explant on MS medium supplemented with 1.0 mg•L-1 6-BA and 0.05 mg•L-1NAA and grew vigorously. There was 100% rooting percentage on MS medium. 2. Polyploids were induced and determinated from Artemisia rupestris L.. Polyploids were induced by two methods, soaking shoot segments in colchicine solution and adding colchicine in the culture medium, respectively. The results of morphology comparison showed that the soaking-treatment increased the efficiency of plant variation than the other methods. As to method of soaking-treatment, the optimal condition of treatment was 0.2% colchicine for 24 h and the variation percentage reached 27.3%. The results of chromosome counting and flow cytometry analysis showed that as well as generating tetraploids, these techniques generated cytochimera in Artemisia rupestris L.. The cytochimera were seperated by this way, the shoots of cytochimera were cut into single shoots and then were induced adventitious shoots formation. The flow cytometry analysis of ploidy level revealed tetraploid shoots were obtained from cytochimera. This technique can be employed when a genome-doubling agent generates cytochimera but no tetraploid. 3. The tube plantlets were transplanted. The results indicated turfy soil was more suitable for Artemisia rupestris L. than the medium contained ermiculite and perlite. There was 100% survival rate during 7 days after transplanting, but there was only 38% survival rate in thirty days’ time because that some of tube plantlets went to die due to the instability of temperature and humidity. 4. The morphology comparison of tetraploid and diploid was made. The results demonstrated that tetraploid plants possessed darker green and thicker leaves, stronger stem and larger root than the diploid ones. In this study, the average size of stomata in diploid planlets was 22.8 ± 0.2 × 19.4 ± 0.2 µm(length × width), and that of tetraploid plantlet was 34.7 ± 0.2 × 29.6 ± 0.2 µm, respectively. The average density of stomata in tetraploid plantlets was significantly lower than that of dipliod plantlets. These morphology characteristics could be strong indicators of change in ploidy level. 5. In order to evaluate the medicinal value of polyploid plants of Artemisia rupestris L., leaf samples of tetraploids growing for 9 months in the flask were extracted and analyzed by HPLC. The results showed that tetraploid and diploid both have 4 characteristic peaks including rupestonic acid, linarin, chrysosplenetin B and artemetin. And tetraploid plants showed higher productivity of rupestonic acid than the control. These tetraploid plants will be screened for a longer period of time and used in breeding to obtain superior new varieties for the commercial production.
内容类型: 学位论文
URI标识: http://ir.xjipc.cas.cn/handle/365002/3566
Appears in Collections:资源化学研究室_学位论文

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作者单位: 中国科学院新疆理化技术研究所

Recommended Citation:
唐晓义. 新疆一枝蒿离体培养再生体系建立及多倍体诱导研究[D]. 北京. 中国科学院研究生院. 2009.
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