【作者】 胡彦；

【导师】 刘玉军；

【作者基本信息】 北京林业大学 ， 植物学， 2010， 博士

【摘要】 紫苏属(genus Perilla)植物系唇形科(Lamiaceae),野芝麻亚科(subfamily Lamioideae),塔花族(tribe Saturejeae),紫苏亚族(subtribe Perillinae)单种属一年生自花授粉植物,原产于东亚地区。紫苏作为油料、蔬菜和中药作物广泛栽培于中国、日本、韩国等国家。紫苏是国家卫生部首批颁布的药食兼用的60种物品之一,它含有多种具有生理活性的化学成分,具有独特的药用价值和食用价值。现代药理学研究表明,紫苏叶片提取物表现出抗过敏,抗炎症,预防肿瘤的功效；紫苏油具有减少胆固醇、预防肿瘤、并改善与年龄有关的认知障碍等保健功能。因此,研究和开发利用我国的紫苏资源,具有巨大的开发潜力和广阔市场前景。本研究利用光学显微镜和扫描电镜观察紫苏属植物表皮细胞及花粉粒的形态特征,探讨这些特征在紫苏属植物分类学上的意义。利用现代成分分析技术,对紫苏属植物5个变种有代表性的试材叶片挥发油的含量和成分、种子油脂肪酸的成分进行了测定和评价,探讨了吹扫捕集技术在紫苏挥发油成分分析上的应用；对用秋水仙碱诱导产生多倍体紫苏的育种方法进行了研究,取得了如下结论。1.根据裴鉴和李锡文主要的分类学观点,从我国7个省、市、自治区收集到的34份紫苏试材包括了紫苏属植物的5变种,即野生紫苏变种．frutescens(Linn.)Britt. var. acuta (Thunb.) Kudo.,耳齿紫苏变种P. frutescens (Linn.) Britt. var. auriculato-dentata C. Y. Wu et Hsuan ex H. W. Li.,白苏变种P. frutescens (Linn.) Britt. var. frutescens,回回苏变种P. frutescens (Linn.) Britt. var. crispa (Benth.) H. W. Li.和紫苏变种P. frutescens (Linn.) Britt. var. arguta (Benth.) Hand.-Mazz.。5个变种试材之间在叶片的大小、叶片的形态、叶片的颜色和植株的高度等方面存在差异,它们在生育期,紫苏籽颜色,粒径,平均千粒重,单株产量等方面也有区别。2.采用扫描电镜对紫苏属植物21份花粉进行观察,结果表明,该属花粉为单花粉,异极,辐射对称,花粉粒的形态呈近扁球形至扁球形。在每一份试材中,6沟花粉最常见,偶见8沟花粉。根据花粉粒的外壁纹饰是否具有连续的覆盖层,21份试材的花粉粒可分为两类：(1)14份试材的花粉粒具不规则的网状纹饰；(2)7份试材的花粉粒具连续的覆盖层,无穿孔。花粉粒外壁纹饰的样式、花粉粒的大小,结合其它的分类学证据,证明我们将紫苏属植物分为一种5变种的分类方法是恰当的。3．在光学显微镜下,紫苏属植物15份试材叶表皮细胞的形状呈不规则形、不规则多边形；表皮细胞的垂周壁样式呈较平直、浅波状、波状或深波状。气孔器主要分布于下表皮,上表皮偶见气孔器分布；气孔器类型多为横列型,偶见无规则形。在扫描电镜下,气孔器呈圆形,椭圆形等形态,保卫细胞对称排列,气孔缝形态多种多样。紫苏属植物表皮细胞和气孔器的形态的差异性对紫苏属植物种下变种的划分无意义,但对变种下栽培品种的划分具有重要的意义。4.采用水蒸气蒸馏法研究了从国内收集的8份紫苏试材挥发油的含量。结果表明,在紫苏芳香油积累的高峰期,紫苏试材芳香油的差异性大,最高者可达0.212%,最低者仅为0.065%。采用气相色谱—质谱联用技术(GC／MS)对超临界CO2萃取法(SFE-CO2)所提取的紫苏属植物叶片挥发油化学成分及其含量进行分析。结果表明：8份紫苏属植物试材中共检测出45种组分,但共有的成分仅有5种,这说明紫苏属植物叶片挥发油组分在不同试材之间存在较大的差异。5.采用吹扫捕集技术对紫苏不同变种叶片中的挥发性成分进行了富集,以气质联用仪(GC/MS)进行鉴定。结果表明,分别属于紫苏属植物三个变种的4份试材之间叶片挥发油的组分和相对含量差异巨大。将采用上述测定方法测得的实验数据与相同试材但采用超临界CO2萃取GC/MS分析所得到的挥发油的组分相比较,发现采用吹扫捕集的方法测定紫苏挥发油的成分是一种快速、经济、有效的测定方法。6.采用化学法萃取和GC／MS法,对紫苏属植物5个变种的10份试材种子的含油量及其脂肪酸组成进行了研究。结果表明,紫苏属植物10份试材间种子的含油量介于33.49%～42.58%之间；10份试材种子油中共检测出6种脂肪酸组分：棕榈酸、亚油酸、亚麻酸、硬脂酸,10-十八碳烯酸和油酸,其中前4种为10份试材的共有成分。10份试材种子油中α-亚麻酸含量介于71.75%～80.06%之间,不饱和脂肪酸的含量介于88.80%-92.82%之间。7.本研究用不同浓度的秋水仙素水溶液处理紫苏幼苗茎尖的生长点,得到了紫苏突变体植株,经过5年的筛选、鉴定,获得了5个具有潜在开发价值的紫苏多倍体新品系。在电镜或光镜下,与二倍体亲本比较,紫苏同源多倍体品系的花粉粒变大,气孔器明显变大,气孔指数变小；并且多倍体紫苏品系具有植株变矮,叶片变厚,叶色发生了变化,生育期延长,种子变大,种子结实率降低,种子千粒重变大,单株产量降低的特点。诱导产生的同源4倍体叶片挥发油的成分与其2倍体亲本相比,可产生新的组分和缺失某些组分,和共有成分含量的增加或者降低。两个同源4倍体之间叶片挥发油的组分与相对含量也存在较大的差异。更多还原

【Abstract】 The monotypic genus Perilla belongs to the family Lamiaceae, subfamily Lamioideae, tribe Saturejeae, subtribe Perillinae. It is self-pollinating and originates in East Asia. As a type of oil and vegetable crop, as well as traditional Chinese medicinal plants, the Perilla plants are wildly cultivated throughout China, Japan, and South Korea. Perilla is in the list of products, which serves as both food and medicine, announced by the China Ministry of Health. It contains many kinds of bio-active substances. These bio-active substances have unique medicinal and nutritional values. Modern pharmacological studies have shown that leaf extracts of perilla plants have antiallergic, anti-inflammatory, and tumor-preventing effects, etc. These health benefits, which also include reduction of cholesterol levels and improvement of age-related learning deficiencies, have been attributed to perilla oil. As such, research on development and utilization of perilla resources in China, has great potential and broad market prospects. In this paper, the morphological features of pollen grains and micromorphological features of leaf epidermis of samples of genus perilla were examined using scanning electron microscopy (SEM) and light microscopy (LM), to determine whether detailed features of the pollen grains and leaf epidermis could be used for classification. The contents and compositions of volatile oil from perilla leaves, oil contents and fatty acid compositions in seeds of the genus Perilla have been determined and evaluated in this study by modern chemical analysis methods. The method that purges and traps extraction with GC-MS determination of volatile oil compositions in leaves of Perilla genus has also been studied. Moreover, the study also focused on the methods of colchicine-induced polyploid breeding of Perilla. The results are as follows:1. According to Pei and Li’s taxonomic opinions,34 perilla samples collected across seven provinces in China were classified into five varieties of one species, namely, P. frutescens (Linn.) Britt. var. acuta (Thunb.) Kudo., P. frutescens (Linn.) Britt. var. auriculato-dentata C. Y. Wu et Hsuan ex H. W. Li., P. frutescens (Linn.) Britt. var. frutescens, P. frutescens (Linn.) Britt. var. crispa (Benth.) H. W. Li. and P. frutescens (Linn.) Britt. var. arguta (Benth.) Hand.-Mazz. The size, shape and color of leaves, color of flowers and height of plants samples of the five varieties of one species of genus perilla are diverse. Their growth period, seed color, seeds diameter, average 1000-grain weight, average yield per plant are diverse too.2. Pollen morphology of 21 samples of Perilla L. was examined under scanning electron microscope (SEM). The results showed that the pollen grains are monads, heteropolar, radiosymmetric and suboblate to oblate in shape. Most of the pollen grains were hexacolpate, with a few octocolpate grains in each sample. Based on whether they had continuous tecta on the ornamentations, the pollen grains from the 21 samples were classified into two categories:(i) 14 with irregular reticulates; and (ii) seven with continuous tecta with no perforations. The ornamentation pattern and size of pollen grains jointly provide evidence that it is appropriate to use them to classify the genus Perilla into five varieties of one species.3. Under light microscopy (LM), the leaf epidermal cells of 15 samples of genus perilla are usually irregular or irregular polygonal in shape while their anticlinal wall patterns are often slightly straight, sinuolate, sinuous or sinuate. The stomatal apparatuses are usually diacytic while sometimes anomocytic and usually found on abaxial side. Shapes of stomatal apparatus of 15 perilla samples such as round or elliptic were observed with SEM. Guard cell of stomatal apparatus had a symmetric order, while variation existed in the opening of the stomatal apparatus. These various leaf epidermal cells features and stomatal apparatus shape had little taxonomical significance among five varieties of one species. However, they might be useful in distinguishing cultivars of a particular variety of genus perilla.4. The content of aromatic oil in 8 samples of genus perilla collected from China was also studied using steam distillation. The result shows that the content of aromatic oil in different samples at the stage of peak of accumulative dynamics are variant, the highest one can reach at 0.212 percent and the lowest one is only 0.065 percent. Gas chromatography-mass spectrometry (GC/MS) was used to analyse and compare the chemical components with their contents of volatile oils extracted from same 8 samples of genus perilla by Supercritical CO2 fluid extraction (SFE-CO2). In overall,45 kinds of volatiles were identified from leaves of the eight samples, but only five were common among the eight samples. The results showed that the components of volatile oils in leaves were distinctly different among eight samples, which belong to five varieties of the genus perilla.5. Volatiles from leaves of different varieties in the genus perilla were extracted using the purge and trap technique and analyzed with GC/MS. The results showed that the components and the relative contents of volatiles oils in leaves were distinctly different among four samples belonging to three varieties of the genus Perilla. The experimental data measured by methods mentioned above were compared with the volatile oil components of the same samples but extracted with the method of supercritical CO2 flow and analyzed with GC/MS. The result showed that this method is a rapid and effective one for determining volatile compounds in leaves of genus perilla.6. The oil contents and fatty acid compositions of ten seed samples of five varieties of the genus perilla were studied using methods of chemical extraction and GC/MS analyses. The results show that oil contents of the ten samples ranged from 33.49% to 42.58%. According to the GC/MS analysis results, the fatty acid compositions of the seed oils were simple. In all the 10 samples, only six kinds of fatty acids were detected, namely palmitic, linoleic,α-linolenic, stearic,10-octadecenoic and oleic acids. Among them, the first four were common constituents of the ten samples. It was clear that the relative percentages of a-linolenic acid in ten samples ranged from 71.75% to 80.06%, the content of unsaturated fatty acids in total fatty acids ranged from 88.80% to 92.82%.7. Cotton was dipped into the aqueous solution with different concentrations of colchicine, and then put onto the stem tip of perilla seedling for 24 hours, after which polyploidy variant plants were achieved. Five polyploid lines of perilla have been obtained through five years screening and identification of variant strains. Compared with diploid control, five polyploid lines of perilla had bigger pollen grains, bigger stomatal apparatus and lower density of stomata under SEM or LM. Furthermore, compared to the corresponding diploid controls, polyploid lines had lower plant height, thicker leaves, varied leave color, delayed growth period, bigger seeds, lower fertility, increased 1000-grain weight and lower yield of per plant. Compared with the volatile oil components in leaves of their diploid parents, two polyploid lines of perilla contains some new components or lack of certain ingredients, or changes in relative contents of some common components they both contain. The components and relative contents of volatile oil in the leaves between two polyploid lines of perilla also have great differences.更多还原