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1、第一章 文献综述Chapter 1: Literature Review藠头（Allium chinensis G. Don），英文名rakkyo，又名薤头、荞头、火葱、野韭等，属百合科（Liliaceae）葱属（Allium）形成小鳞茎的多年生宿根草本植物，主要食用嫩叶和鳞茎,系中国南方特有的稀缺蔬菜品种之一，属辛香类蔬菜。藠头的叶片绿色、细长、中空，有大葱样辛辣气味，叶鞘基部膨大形成长卵或纺锤形鳞茎。藠头作二年生栽培，分蘖力强。其鳞茎的加工产品质地洁白，口感脆嫩，具防暑、开胃、助消化等保健功效。盐渍、甜酸藠头深受消费者欢迎，并出口东南亚及日本、韩国等国（文惠玲等，2003）。在中医中，藠头
2、的干燥鳞茎又称为薤白（祝之友，2000），其味辛、苦，性温，无毒，具有宽胸、理气等功效，用于治疗胸闷刺痛、肺气喘急等疾病已有2000多年的历史。现代医学研究发现，薤白还具有抑菌消炎、解痉平喘、抗血小板凝聚、抗氧化、抗肿瘤、降低血脂、抗动脉粥样硬化的作用（张卿等，2003）。藠头既可食用又能入药，具有多种药理作用和保健的功能，是复合型功能食品，被誉为“菜中灵芝”。Allium chinensis G. Don, with the English name of rakkyo, is also known as allium bakeri, scallion, shallot and allium
3、 ramosum. It belongs to the category of Allium genus of Liliaceae family, and is a kind of perennial herb with cloves. The main parts of this plant for food are leaves and bulbs. It is one of the unique scarce vegetable varieties in South China, and it is classified as spicy vegetable. The leaves of
4、 Allium chinensis G. Don are green-colored, slender and cannular. It has spicy odor like that of a green Chinese onion. The leaf sheath base enlarges so as to form long ovoid or spindle-shaped bulb. It is biennially cultivated, and has a strong tillering capacity. The processing products of its bulb
5、s are white in texture and crisp and tender in taste, having health care effects such as sunstroke prevention, appetite promotion, and digestion aid. Salted and sweet & sour Allium chinensis G. Don products are well received by customers, and are exported to South Asia, Japan, South Korea, etc. (Wen
6、 Huiling, 2003). In traditional Chinese medicine, the dry bulb of Allium chinensis G. Don is also called allium macrostemon (Zhu Zhiyou, 2000) with pungent and bitter taste, and wild nature, which is free from toxicity. It has the functions such as comforting the chest and regulating the flow of vit
7、al energy. It has been used in the treatment of diseases like chest tightness and twinge, lung dyspnea for more than 2000 years. According to the findings of modern medical research, allium macrostemon is also capable of inhibiting bacteria, eliminating inflammation, relieving spasm, calming breathi
8、ng, resisting platelet aggregation, oxidation and tumors, reducing blood fat, and fighting atherosclerosis (Zhang Qing, et al.). Allium chinensis G. Don is not only edible but also capable of being taken for medicine purpose. It has multiple pharmacological effects and health care functions, and is
9、a kind of compound functional food. Therefore, it is hailed as “ganoderma lucidum of vegetables”.藠头原产于我国，已有3700多年的种植历史，先秦时已广为利用。以江西、湖南、湖北、广西、云南、四川、贵州等省（区）栽培较多，目前在江西新建、云南开远以及武汉江夏梁子湖等地区有大面积植。在浙江和西藏还有野生种群（陈静芬，1989）。俄罗斯、朝鲜、日本都有分布和少量栽培（柳荣成，1995）。Allium chinensis G. Don is originated from China, and has a
10、 planting history over 3700 years. It was widely used as early as Pre-Qin Dynasty. It has been mostly planted in Jiangxi, Hunan, Hubei, Guangxi, Yunnan, Sichuan and Guizhou. Currently, it is planted with large areas in regions such as Xinjian in Jiangxi, Kaiyuan in Yunnan, and Jiangxia Liangzi Lake
11、in Wuhan. Also, there are wild population in Zhejiang and Xizang (Chen Jingfen, 1989). Allium chinensis G. Don is also distributed in Russia, North Korea and Japan with limited planting quantity (Liu Rongcheng, 1995).藠头栽培种多为三倍体、四倍体等多倍性植株（2n=4x=32，2 n = 3 x =24）(利容千，1989；杜武峰等，1993)。虽然在自然条件下能够开花，但因其小饱
12、子败育空瘪，不能形成精子；大饱子及雌配子体发育异常，不能形成正常胚囊结构，故花而不实（董庆华等，1997）。因此，生产上仍然采用营养繁殖，不仅用种量大，繁殖系数低，而且种藠的贮藏和运输增加成本，限制了许多名优品种的大面积推广。营养繁殖还使病毒积累导致种性退化严重，产量和品质下降，严重影响其经济价值和食用性，给生产消费和出口创汇带来了严重的障碍，是目前生产上需要解决的问题。The cultivated species of Allium chinensis G. Don are mainly polyploidy plants like triploid and tetraploid (2n=4
13、x=32，2 n=3 x=24) (Li Rongqian, 1989; Du Wufeng, et al., 1993). Although it can blossom under the natural condition, its small spores abort and are empty and cannot form sperms; the development of big spores and female gametophytes is abnormal, and no normal embryo sac structure can be formed; as a r
14、esult, it can only blossom, but is unable to fruit (Dong Qinghua, et al., 1997). Therefore, vegetative propagation is still adopted in production, which is large in seed consumption and low in reproduction coefficient. Furthermore, the storage and transportation of the seeds of Allium chinensis G. D
15、on will add cost which restricts the large-area promotion of many famous cultivated varieties. Also, vegetative propagation may facility the accumulation of virus, causing the serious degradation of the seed nature, the reduction of output and quality, which will severely influence its economic valu
16、e and edibility and bring serious obstacles for the production consumption and earning of foreign exchange through export. It is at present a problem required to be solved in the production.利用组织培养方法获得无毒或少毒再生植株，并在短时间内快速增殖，获得大量组培苗是解决这些问题的有效途径。最近几十年，组织培养技术在葱属植物的离体培养及研究中发挥了巨大作用，取得了令人瞩目的成绩，但目前利用组织培养技术获得藠
17、头再生植株，国内外对这方面的研究很少, 仅有鳞茎盘（傅德明等，2006；Xu等，2008）、茎尖（黄钊等，2006）组织培养成功的报道。因此，利用组织培养技术建立一套较为完整、系统的藠头脱毒、快繁体系，对其育种、种资创新将具有重要价值。It is an effective approach to handle these problems by utilizing tissue culture method to obtain non-toxic or low-toxic regenerated plants and realize rapid propagation within a sho
18、rt period of time so as to obtain a large quantity of plantlets. In recent decades, tissue culture technology has played a significant role in the in vitro culture and research of allium plants, and remarkable accomplishments have been reached. However, at present, the researches on the utilization
19、of tissue culture technology in obtaining Allium chinensis G. Don regenerated plants at home and abroad are still insufficient. There are only reports concerning the success of tissue culture in basal plate (Fu Deming, et al., 2006; Xu, et al., 2008) and stem tip (Huang Zhao, et al., 2006). Therefor
20、e, to establish a set of relatively complete and systematic Allium chinensis G. Don detoxification and rapid propagation system through the utilization of tissue culture technology will be essential to the seed cultivation and seed resource innovation.1 藠头的概况1. Profile of Allium chinensis G. Don1.1特
21、征特性1.1 Characteristics藠头株高 4555cm。叶丛生基叶数枚，叶较细，呈三角形中空，有不明显的35棱，长4050cm，粗1.41.6cm，绿色，叶鞘长7cm。鳞茎由一个茎瓣组成，分蘖力强。一个鳞茎栽植后可心分生成1020个鳞茎。外皮紫色或淡黄色，肉白色。鳞茎纵径45 cm，横径2.5cm左右，单个鳞茎质量10.515.5g，生长期九个月左右。伞形花序，顶生，通常含有1625朵淡紫色小花。The plant height of Allium chinensis G. Don is from 45 to 55cm. Its leaves grow in tuft with s
22、everal base leaves. The leaves are relatively slender with triangle hollowness. It has inconspicuous 3 to 5 edges with the length of 40-50cm, the thickness of 1.4-1.6cm and the length of leaf is 7cm. Bulb is composed of a pedical valve with strong tillering capacity. One bulb can be divided to gener
23、ate 10 to 20 bulbs after planting. The outer skin is purple or light yellow and fresh color. The vertical diameter of the bulb is 4 to 5cm while the horizontal diameter is about 2.5cm. The weight of a single bulb is 10.5 to 15.5g. Its growth period is about nine months. As an acrogens, it has the um
24、brella-shaped inflorescence with 16 to 25 light purple flowers in general.藠头适应性强，喜冷凉，生长适宜温度为 1519，30以上则休眠越夏，10以下生长缓慢，不能忍受0以下的长时间低温。藠头属长日照作物，要求较弱的光照强度，耐荫。在中国，6月开始形成鳞茎， 810月份开花，9月份鳞茎成熟（王夫玉等，2002）。藠头对土壤和阳光的需求并不严格，各种土壤都能种植，但以排水良好的疏松山地及砂壤土为好。抗病力强，吸肥力强。适宜于间作和在果园内套种（周华光等，2007）。Allium chinensis G. Don has s
25、trong adaptability, and favors cool environment. The suitable growth temperature is from 15 to 19. If the temperature surpasses 30, it will be dormant to go through the summer. If the temperature is below 10, its growth will slow down. It can not stand low temperature below 0 for a long time. Allium
26、 chinensis G. Don is a long-day crop which needs relatively weak illumination strength with shade tolerance. In China, it begins the formation of bulbs in June and blossoms from August to October. In September, the bulbs are mature (Wang Fuyu, et al., 2002). Allium chinensis G. Don does not have ver
27、y strict requirements for soil and sunlight. It can be planted in various soils. However, loose hill field and sandy soil with good drainage are preferred. It has strong disease resistance and fertility absorption capability, which is suitable to intercropping and interplanting in the fruit garden (
28、Zhou Huaguang, et al., 2007).1.2营养成分1.2 Nutritional Components生长成熟的藠头中每100克鳞茎含水分约87.9克，碳水化合物8.0克，蛋白质1.6克，纤维素1.2克，富含糖、蛋白质、胡萝卜素、维生素C、16种氨基酸及多种药理成分。此外还含有蒜氨酸、琥珀酸及挥发油等。Mature Allium chinensis G. Don contain approximately 87.9g moisture, 8.0g carbohydrate, 1.6g protein and 1.2g cellulose in every 100g of
29、bulbs. Also, it is rich in sugar, protein, carotene, vitamin C, 16 amino acids and multiple pharmacological compositions. Besides, it contains alliin, succinic acid, volatile oil and so on.藠头含铁量甚高，不仅高居葱蒜类之首，而且远较一般蔬菜高，因而有利于机体血红蛋白的合成，免疫功能的提高。所含挥发物质增进食欲，帮助消化，增强吸收功能，具有食疗的功效。经过腌渍后的藠头，颗粒整齐，金黄发亮，香气浓郁，肥嫩脆糯，
30、鲜甜而微带酸辣，早在清朝年间就被选为贡品。食之能具有增食欲、助消化、解疫气、健脾胃、解油腻和醒酒的功能，是佐餐的佳品。俗称：“餐前饭后食六颗、不打郎中门前过”。Allium chinensis G. Don has very high iron content, not only ranking first in the allium plants but also leading the common vegetables. Therefore, it is beneficial to the synthetization of body hemoglobin and also the im
31、provement of immune function. The volatile matters contained can promote appetite, help digestion, enhance absorption function, and have the effect of food therapy. Salted Allium chinensis G. Don has neat grains which have shiny golden color. Also, it has full aroma, and it tastes fresh, crisp and g
32、lutinous. Allium chinensis G. Don with fresh, sweet and slightly sour and spicy taste was selected as tribute in Qing Dynasty. It can promote appetite, help digestion, eliminate phytophthora gas, care the spleen and the stomach, get rid of grease and remove the effects of alcohol, and is thus regard
33、ed as the fine product to go with staple food. As the saying goes, “eat six granules (of Allium chinensis G. Don) before and after the meal keeps a doctor away.”1.3功能成分1.3 Functional Components藠头的干燥鳞茎称为薤白，其功能成分中已分离到挥发油（Kameoka等，1984；彭军鹏等，1994）、含氮化合物（彭军鹏等，1995）、皂苷类化合物（Kuroda 等，1995；Peng 等，1996a；Peng
34、等，1996b）、长链脂肪酸、棕榈酸、大蒜糖以及前列腺素A1和B1（孙启良，1991）等主要物质。.其中挥发油成分主要为含硫化合物，占50%以上（姜勇等，1999），含有强烈抑制血小板聚集活动的甲基烯丙基三硫(MATs)（吴雁等，1993；彭军鹏等，1994）。腺苷（彭军鹏等，1995）以及甾体皂苷 (彭军鹏等，1996)。The dry bulb of Allium chinensis G. Don is called allium macrostemon whose functional ingredients have been isolated into the following m
35、ain materials: volatile oil (Kaneoka, et al., 1984; Peng Junpeng, et al., 1994), nitrogen compounds (Peng Junpeng, et al., 1995), saponins compounds (Kuroda, et al., 1995; Peng, et al., 1996a; Peng, et al., 1996b), long-chain fatty acid, palmitic acid, garlic sugar and prostaglandin A1 and B1 (Sun Q
36、iliang, 1991). Among the above materials, the volatile component is mainly sulfur compounds, which takes up over 50% (Jiang Yong, et al., 1999). It also contains MATs which can strongly restrain the platelet aggregation (Wu Yan, et al., 1993; Peng Junpeng, et al., 1994). Also, adenosine (Peng Junpen
37、g, et al., 1995) and steroid saponin (Peng Junpeng et al., 1996) are included.孟松等（2005）从藠头中分离了抗菌性的活性物质，主要成分为甲基烯丙基二硫化物，它对多种细菌和真菌的生长有抑制作用，特别是对白色念珠菌(Candida albicans)。孙运军等（2004）确证了藠头中抑菌的活性物质对大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subfilis)、痢疾志贺菌(Shigella dysenteriae)和伤寒沙门氏菌(Salmonella typhimufium)等多种细菌
38、和真菌的明显抑制作用。同时发现藠头的活性物质对肝癌(HepG-2) 细胞和海拉(Hela)细胞均有抑制作用。Meng Song, et al. (2005) have isolated antimicrobial active substances from Allium chinensis G. Don with the main component of methyl allyl disulfide which has the inhibiting effect on the growth of multiple bacteria and funguss, especially for C
39、andida albicans. Sun Yunjun, et al. (2004) have confirmed that the antimicrobial active substances contained in Allium chinensis G. Don have obvious inhibiting effects on multiple bacteria and funguses such as Escherichia coli, Bacillus subfilis, Shigella dysenteriae and Salmonella typhimufium. Also
40、, they have discovered that the active substance in Allium chinensis G. Don have inhibiting effects on HepG-2 cells and Hela cells.2.葱属植物的组织培养2. Tissue Culture of Allium Plants葱属 (Allium) 属百合科 (Liliaceae) 植物，约有500个种，我国有7080种。其中大葱 (A. fistulosum L.) 、洋葱 (A. cepa L.) 、大蒜(A. sativum L.)和韭菜 (A. tuberosu
41、m Rottler. ex Spr.) 是普遍栽培的重要蔬菜。胡葱(Aascalonicum L.)、藠头 (A. chinensis G. Don.) 、韭葱 (A. porrum L.) 、细香葱 (A. schoenoprasum L.) 等也是很好的香辛类蔬菜。一些野生种亦可食用，如山韭 (A. senescens var. minor) 、阿尔泰葱 (A. altaicum Pall) 、 沙葱 (A. mongolicum Rege1) 等。Allium belongs to the category of liciaceae. There are about 500 variet
42、ies of allium plants in the world, in which China has 70 to 80 varieties. Among all the allium plants, A. fistulosum L., A. cepa L., A. sativum L., and A. tuberosum Rottler. ex Spr are important vegetables commonly planted. Aascalonicum L., A. chinensis G. Don, A. porrum L. and A. schoenoprasum L. a
43、re also very good spicy vegetables. Meanwhile, some wild species such as A. senescens var. minor, A. altaicum Pall and A. mongolicum Rege1 are edible, too. 葱属植物的离体再生可经过两条途径：器官发生途径和胚状体发生途径。器官发生途径是指离体的组织或细胞在组织培养的条件下形成不定芽、根和花芽等器官的过程。胚状体发生是指体细胞胚来自于单个体细胞，其发生过程与有性的合子胚相似。葱属植物的离体培养已有不少报道，但多集中在洋葱、蒜、葱和韭等少数几种植
44、物，涉及茎尖、根尖分生组织、器官培养，试管受精和胚培养，以及原生质体培养和融合等。In vitro regeneration of allium plants can be reached through two approaches: organogenesis approach and embryogenesis approach. Organogenesis approach refers to the process in which the in vitro tissues or cells form organs such as the adventitious bud, root
45、 and bud under the condition of tissue culture. Embryogenesis has the similar process with that of sexual zygotic embryos, and the somatic embryos are from single somatic cells. There are already many reports on the in vitro culture of allium plants, but the focus is mainly for a minority of plants
46、such as onion, garlic, green onion and leek, involving stem tip, root-tip meristem, organ culture, in vitro fertilization, embryo culture and protoplast culture and fusion.2.1分生组织培养2.1 Meristem Culture葱属植物的茎尖分生组织是离体培养的优良外植体材料。茎尖0.40.6 mm范围内为无病毒区，作外植体进行培养可成功地获得无病毒再生植株，并能有效的保持再生植株的遗传稳定性。The stem-tip m
47、eristem of the allium plants is an excellent explant material for in vitro culture. It is the non-toxic area within the scope of 0.4 to 0.6mm of the stem tip. Take it as the explant to culture, non-toxic regenerated plants can be successfully obtained, which can effectively keep the genetic stabilit
48、y of the regenerated plants.Havranek（1973）以大蒜茎尖为外植体，获得了第一批无病毒植株。陈世儒等（1991）以紫皮蒜和白皮蒜为供试材料，切取0.20.9 mm、带一个或不带叶原基的大蒜茎尖分生组织，接种在MS+2.0 mg/L 6-BA+0.6 mg/L NAA的培养基上，新梢增殖和根的分化状态良好。徐培文等（1991）试验表明，用热处理（37 ）带23个叶原基的大蒜茎尖30d后，茎尖易剥离，组培苗成活率高，脱毒效果好。试验还筛选出了最佳诱芽和诱根培养基，分别是：B5+3.0 mg/L 6-BA+0.1 mg/L NAA + 3.0 mg/L AD+0.
49、5 mg/L KT；1/2B5+0.01 mg/L 6-BA +0.05 mg/L NAA，由此建立了高效的大蒜脱毒快繁体系。Nagakubo等（1993）采用“茎尖培养-芽增殖-试管内诱导小鳞茎”的方法适当缩短了大蒜脱毒进程。30个月内的繁殖系数达512。李昌华等（1995）用山西的5个地方大蒜栽培种进行茎尖离体培养，获得无病毒蒜种。结果表明，茎尖大小为0.20.5 mm，不定芽增殖培养基以B5或MS附加1.02.0 mg/L KT和0.010.1 mg/L NAA，效果最佳。高山林等（2000）以大蒜茎尖分生组织为外植体，筛选出最佳的培养基。诱导愈伤组织的最适培养基为：MS+0.2 mg/L 6-BA+0.5 mg/L NAA，月生长率1270倍；诱导丛生芽的最适培养基为：B5+0.5 mg/L 6-BA+0.2 mg/L IAA，丛生芽繁殖系数高达255倍，技术上达到了快速繁殖规模生产的要求。张寒霜等（2004）发现大蒜茎尖培养以B5+0.1 mg/L NAA+3.0 mg/L 6-BA+20 g/L glucose（葡萄糖）培养基较好，茎尖越小，脱
