学校代码:10564 学 号:2014200219 分 类 号:Q94 密 级: 硕 士 学 位 论 文 二倍体与其加倍四倍体紫锥菊的性状比较和杂交育种研究 周 娟 指 导 教 师: : 杨跃生 研究员 学 院 名 称 : 生命科学学院 专 业 名 称 : 植物学 答辩委员会主席: 廖文波 教授 中国·广州 2019 年 6 月 万方数据 华南农业大学 学位论文原创性声明 本人郑重声明:所呈交的学位论文是本人在导师的指导下独立进行研究所取得的研究成果。除了文中特别加以标注引用的内容外,本论文不包含任何其他个人或集体已经发表或撰写的作品成果。对本文的研究做出重要贡献的个人和集体,均已在文中以明确方式标明。本人完全意识到本声明的法律结果由本人承担。 作者签名: 日期: 学位论文版权使用授权书 本学位论文作者完全了解学校有关保留、使用学位论文的规定,即:研究生在校攻读学位期间论文工作的知识产权单位属华南农业大学。学校有权保存并向国家有关部门或机构送交论文的复印件和电子版,允许学位论文被查阅或在校园网上发布并供校内师生和与学校有共享协议的单位浏览(除在保密期内的涉密论文外);学校可以公布学位论文的全部或部分内容,可以允许采用影印、缩印或其它复制手段保存、汇编学位论文。本人电子文档的内容和纸质论文的内容相一致。 作者签名: 日期: 导师签名: 日期: 学位论文提交同意书 本学位论文符合国家和华南农业大学关于研究生学位论文的相关规定,达到学位授予要求,同意提交。 导师签名: 日期: 学科带头人签名: 日期: 万方数据 I 摘 要 紫锥菊(Echinacea purpurea (L.) Moench)原产于北美洲,具有较高的药用价值和观赏价值,目前世界各地区均有引种。另一方面,多倍体植物常常具有生物量大和抗性强等优点,而三倍体是倍数最低的多倍体,在多数情况下表现优于更高倍数的多倍体。本研究通过对 3 个紫锥菊二倍体株系及其加倍的四倍体进行种植比较,观察其加倍效应,并在此基础上,以不同株系的二倍体和四倍体作为亲本进行杂交,根据杂交结果对各亲本组合进行评价。 在 3 个株系的二倍体及其对应的四倍体组培苗中,所有的四倍体都比其对应的二倍体表现出叶柄更长,叶片变大,数量增加,颜色加深;根粗短,须根减少,侧根增多。田间栽培期间则表现出四倍体株高降低、花序数减少、花盘高度增加,并保持了叶片颜色更深更厚、叶片表面更为粗糙的特点。加倍延迟了紫锥菊的物候期,使营养生长期相对更长,但头状花序由花蕾期到管状花完全开放,直至花序枯萎的天数并没有显著差异。在广州地区栽培的一年生二倍体紫锥菊一年有两次花期,而四倍体只有一次。栽培过程中还观察到加倍导致舌状花着生状态的改变,例如,柱头出现 3 叉、4 叉和花药为 6 枚等特殊性状。在细胞学鉴定方面,四倍体的叶片气孔显著增大、单位面积密度减少。二倍体和四倍体在以上这些方面的特征差异可作为多倍体紫锥菊鉴定的前期标准。 通过碘-碘化钾法、醋酸洋红法、离体萌发法和授粉结实法对上述 3 个二倍体紫锥菊花粉活力进行鉴定,发现不同的鉴定方法,其活力大小有所差异。使用碘-碘化钾进行染色,仅有少数花粉颜色发生较明显变化,其余颜色相近不容易分辨,因此该方法并不适用于紫锥菊花粉活力的测定。醋酸洋红法、离体萌发法和授粉结实法鉴定花粉活力的结果,在这三个株系的排序上完全一致。但离体培养和授粉结实法受培养基、温度和环境等因素的影响较为复杂,所得结果略低于醋酸洋红法,特别是操作较为繁琐。因此,可认为醋酸洋红法是鉴定紫锥菊花粉活力最为合适的方法。对于紫锥菊种子的萌发,赤霉素能起到一定的作用,较为有效的条件为 200 mg/L 赤霉素浸泡处理 4 h。 对二倍体和四倍体紫锥菊植株根部药用成分的比较,测定了 3 种主要药用成分含量,所有样品中都检测到少量的绿原酸和咖啡酸,菊苣酸含量均最高。不过,以菊苣酸含量为比较标准,不同株系的情况有很大的差别。其中最早萌发新芽的株系的四倍万方数据 II 体的菊苣酸含量高于二倍体;取样时尚未萌发新芽的株系的四倍体和二倍体的菊苣酸的含量几乎不存在差异;取材时二倍体植株已萌发新芽而四倍体植株尚未萌芽的株系的二倍体菊苣酸的含量显著高于四倍体。由此可以看出,菊苣酸的含量不仅存在基因型的差别,也可能受到生长发育阶段的影响。 以 3 个四倍体紫锥菊株系为母本,3 个二倍体紫锥菊为父本进行不同株系间的杂交,共有 6 个杂交组合,其中 3 个杂交组合获得了杂交后代。从对杂交种子的倍性鉴定结果可知,四倍体和二倍体杂交可获得较高比例的三倍体,此外还有一些四倍体和非整倍体。 关键词:紫锥菊;多倍体;杂交育种;药用成分 万方数据 III Comparison of Diploids Purple Coneflower (Echinacea purpurea (L.) Moench) with Their Induced Tetraploids on Several Traits and the Study of Cross Breeding Zhou Juan (College of Life Sciences, South China Agricultural University, Guangzhou 510642, China) Abstract: Echinacea purpurea (L.) Moench is native to North America. Because of its high medicinal and ornamental value, E. purpurea has been introduced for cultivation in various regions of the world. On the other hand, polyploid plants have the advantages of large biomass and strong resistance, and among them, the triploid possesses the least sets of chromosomes while performs better than other ploidy levels in most cases. This study compared three E. purpurea tetraploids lines with three diploid lines from which the tetraploid lines were induced on several traits, and investigated the crosses between diploids and tetraploids of different lines. Among the in vitro grown plantlets, all the tetraploids had longer petioles, more bigger and thicker leaves with darker color; had shorten but thicker roots, reduced number of small roots while increased number of branching roots. In the field, the plant height and the number of inflorescence were reduced while the height of the flower disk was significantly increased in tetraploid plants. At the same time, the traits of dark color and thick leaves were kept, and the leaf surface was even rougher. The phenological period was delayed evidently in tetraploid E. purpurea, leading to a longer vegetative growth period. However, there was no significant difference in the duration of time from flower bud stage to withering stage. In Guangzhou area, diploid E. purpurea could flower twice for the first cultivation year, while tetraploid could flower only once. Some changes of the traits have all so been observed in the chromosome doubled tetraploid, such as in the state of the lingual flowers, the appearance of 3- and 4-pronged stigmas, and one more anther in the flower. In terms of cytological observation, the size of stomata on the leaves of the tetraploid plants was significantly increased and the density per unit area was decreased. The differences in biological characteristics between diploid and tetraploid plants can be 万方数据 IV used as criteria at the early stage for identifying the ploidy level of E. purpurea. The vigor of pollen grains of the three diploids was compared. Only a small portion of the pollen grains from the diploid plants changed their color evidently after stained with iodine-potassium iodide, and the most were not easily to judge. Investigation of the vigor of pollen grains by the three other methods, acetic acid magenta, in vitro germination and pollination yielded consistent results. Considering that in vitro culture and pollination method were much more cumbersome, acetic acid magenta should be the first choice. In another experiment, diploid E. purpurea seeds were treated with gibberellin water solution and it was found that the most effective method was treating the seeds with 200 mg/L gibberellin for 4 h. The contents of three main medicinal compounds in the roots of diploid and tetraploid E. purpurea were measured. The content of cichoric acid was always the highest and the other two, chlorogenic acid and caffeic acid were of much smaller amount in all samples. However, with the cichoric acid content as an indicator, large differences were observed among the three lines. In the line that resumed growth after winter first with shoots, the cichoric acid content was higher in tetraploid than in diploid; in the line that the diploid and tetraploid plants were still in dormancy, there was no significant difference in the content of cichoric acid between the two ploidy level plants; in the line that diploid plants had resumed growth with shoots while tetraploid plants were still in dormancy, the cichoric acid content in diploid was significantly higher than tetraploid. Therefore, it might be concluded that the content of cichoric acid was not only determined by the genotypes, but also influenced by the growing stages. Three tetraploid E. purpurea plants were used as the female parent, and three diploid were used as the male parent for crossing between different lines. There were six cross combinations totally, but only three combinations yielded seeds From the results of chromosome counting, it was clear that most of the seeds were triploid, and a small portion of tetraploid and aneuploid could also be obtained. Key words: Echinacea purpurea (L.) Moench; Polyploid; Cross breeding; Medicinal compound 万方数据 V 缩略词及中英文对照表 缩略词 英文名称 中文名称 ABA Abscisic acid 脱落酸 BA 6-Benzyladenine 6-苄基腺嘌呤 cm Centimeter 厘米 d Day 天 h Hour 小时 HPLC High-performance liquid chromatography 高效液相色谱 IBA Indol-3-butyric acid 吲哚乙酸 Lx Lux 勒克斯(光照强度) mg Milligram 毫克 mg/g Milligram/Gram 毫克/克 mg/L Milligram/liter 毫克/升 mg/ml Milligram/Milliliter 毫克/毫升 min Minute 分钟 ml Milliliter 毫升 MS Murashige and Skoog’s medium (1962) MS 培养基 NAA Naphthaleneacetic acid 萘乙酸 TDZ Thidiazuron 噻二唑苯基脲 μL Microliter 微升 μm Micrometer 微米 万方数据 VI 目 录 1 前言 .................................................................................................................................... 1 1.1 紫锥菊的研究背景 ......................................................................................................... 1 1.1.1 紫锥菊地理分布和生物学特性 .................................................................................. 1 1.1.2 紫锥菊化学成分及药理作用研究进展 ...................................................................... 1 1.2 紫锥菊的育种研究 ......................................................................................................... 2 1.2.1 紫锥菊组织培养研究进展 .......................................................................................... 2 1.2.2 紫锥菊多倍体育种 ...................................................................................................... 4 1.2.3 紫锥菊杂交育种 .......................................................................................................... 5 1.2.4 紫锥菊的栽培管理及病虫害概况 .............................................................................. 6 1.3 社会效益与市场前景 ......................................................................