园艺植物生物技术第四章.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,Main contents,Plant cell culture,Protoplast Isolation and Fusion,1.,Plant cell culture,Haberlandt,(Austria):,Cultivate isolated plant cells,in vitro,on an artificial medium-,Basic procedures of,enzymolysis method,取叶片，表面消毒,撕掉下表皮，切成小块,加入酶解液（,0.5%,果胶酶,+0.8%,甘露醇,+1%,硫酸葡聚糖钾）,摇床上保温培养,b.,Isolation of single cell from,callus tissue,（,1,）离体器官,（,2,）愈伤组织,（,3,）继代培养,2,个月,（,4,）转至液体培养基震荡培养,（,5,）悬浮细胞,（,6,）无菌过滤，离心,（,7,）单细胞,对分离得到的单个细胞进行培养，诱导其分裂增殖，形成细胞团，再通过细胞分化形成芽、根等器官或胚状体，直到长成完整植株的技术。,1.1.2 Methods of single cell culture,Culture methods,Plate culture,平板培养,Nurse culture,看护培养,Microculture,微室培养,Conditioned medium culture,条件培养基培养,a.,Plate culture,:,分散的植物细胞接种于含薄层固体培养基的培养皿内的培养过程称为平板培养法。,方法是将经机械破碎过筛或酶,(,纤维素酶及果胶酶等,),消化分散纯化的细胞，经计数及稀释，接种到加热熔化而刚冷却至,35,左右的固体培养基中充分混匀，倾入培养皿中，石蜡密封，于,25,培养，约,20,天后细胞即可生长成团。统计生长情况。,b.Nurse culture,：,从悬浮培养物或脆弱愈伤组织上用针或玻璃毛细管分离单细胞，每个细胞放在一个方形滤纸片的上表面，而滤纸片放在活跃生长的愈伤组织上进行培养的方法,。,c.,Microculture,:,悬浮单细胞接种于凹玻片或玻璃环与盖玻片组成的微室内的固体培养基中的培养方法。,1.1.3 Influencing factors of single cell culture,a.,Medium,：,特殊成分，植物激素，,pH,b.,Cell density,：,平板临界密度,10,3,个,/mL,c.,Environmental conditions,：,25,，,1%CO,2,1.2.,cell suspension culture,细胞的悬浮培养,是指将游离的植物细胞按一定的细胞密度，悬浮在液体培养基中进行培养的方法。,适宜于大规模培养，细胞工程产业的技术基础,.,1.2.1,Procedures of cell suspension culture,愈伤组织,1g,愈伤,/10ml,培养基,150,、,250ml,三角瓶,（,30,、,70ml,培养基）,120rpm,25,黑暗培养,换液,1,次,/3d,以防粘稠,蔗糖梯度离心或过滤法,(,淘汰过大或生理状态不好的细胞团,),继代培养,80rpm,（悬浮细胞系稳定以后）,继代时间为,1,周时（原悬浮液：新培养基,=1,：,4,）,悬浮愈伤组织形成及植株再生,愈伤组织要求：松散性好，增殖快，再生能力强。其外观一般是鲜艳的乳白或淡黄色，呈细小颗粒状，松散易碎。,适于悬浮培养,适于再生植株,a.Batch culture,分批培养,分批培养是在一个封闭的系统中进行细胞培养，当培养物增加到一定量时，需继代培养，即取出少量培养物转接于新鲜培养液中。,1.2.2 Types of cell suspension culture,The cells in culture exhibit the following five phases of a growth cycle,i.Lag phase,where cells prepare to divide.,ii.Exponential phase,where the rate of cell division is highest.,iii.Linear phase,where cell division slows but the rate of cells expansion increases.,iv.Deceleration phase,where the rates of cell division and elongation decreases.,v.Stationary phase,where the number and size of cells remain constant.,b.,Semi-continuous culture,半连续培养,是利用培养罐等装置进行细胞大量培养的一种方式。在半连续培养时，当培养容器内细胞增殖到一定数量后，倒出一半细胞悬浮液于另一培养罐等容器，再分别加入新鲜培养基继续培养。半连续培养能够重复获得大量均一的培养细胞供进一步利用。,c.,Continuous culture,连续培养,是用一定容积的非密闭系统进行细胞培养的方法，在培养过程中不断注入新鲜培养基，而使营养物连续得到补充，细胞生长和增殖连续进行，同时有等体积的培养物排除系统。,Open continuous culture,开放型,Closed continuous culture,封闭型,1.2.3 Requirements for good suspension culture system,a.,悬浮培养物分散性好，细胞团较小，一般由几十个以下的细胞组成。,b.,均一性好，细胞形状和细胞团大小大致相同。,c.,生长迅速，悬浮细胞的量一般,2-3,天甚至更短时间便可增加一倍。,1.3 Application of cell culture,Production of secondary metabolites,辣椒素、花青素,Selection of,mutant,突变体的选择,Polyploid inducement,诱导多倍体,生产人工种子,2.Protoplast Isolation and Fusion,原生质体的游离与融合,2.1.Protoplast Isolation,Methods,mechanical methods,enzymatic methods,2.1.1,Mechanical method,Application range,large and highly vacuolated cells of storage tissues,onion bulb scales,radish root and beet root tissue,Disadvantages,(i)It is restricted to certain tissues which have large vacuolated cells.,(ii)Yield of protoplasts is generally very low.Protoplasts from less vacuolated and highly meristematic cells do not show good yield.,(iii)The method is tedious and laborious.,(iv)Viability of protoplasts is low because of the presence of substances released by damaged cells.,2.1.2,Enzymatic method,Cocking在1960年证实了用酶从高等植物中分离出原生质体的可能性。,Cocking in 1960 demonstrated the possibility of enzymatic isolation of protoplasts from higher plants.,Influencing factors,(1)Physiological state of tissue and cell material,Protoplasts have been isolated from a variety of tissues and organs including leaves,petioles,shoot apices,roots,fruits,coleoptiles,hypocotyls,stem,embryos,microspores,callus,and cell suspension cultures of a large number of plantspecies.,分离材料包括叶片、叶柄、芽尖、根、果实、胚芽鞘、胚轴、茎、胚芽、花粉粒、愈伤组织和细胞悬浮培养物等。植物幼苗或新生枝的完全伸展叶片的叶肉组织是分离原生质体的最方便、最合适的植物材料。,(2),Enzymes,The release of protoplasts is very much dependent on the nature and composition of enzymes used to digest the cell wall.,原生质体的释放在很大程度上取决于用于消化细胞壁的酶的性质和组成。,Three primary components of the cell wall:cellulose,hemicellulose and pectin substances.,细胞壁主要成分,:,纤维素、半纤维素和果胶类物质,。,Cellulose and hemicelluloses:the components of primary and secondary structure of cell wall,Pectin:a component of middle lamella that joins the cells.,纤维素和半纤维素是细胞壁初生结构和次生结构的成分，而果胶类物质是连接细胞的胞间层的成分。,Pectinase:mainly degrades the middle lamella,Cellulase and hemicellulase:digest the cellulosic and hemicellulosic components of the cell wall,respectively.,果胶酶主要是降解中层，而纤维素酶和半纤维素酶分别用于消化细胞壁的纤维素和半纤维素成分。,The activity of the enzyme is pH dependent and is generally indicated by the manufacturer.,pH:4.7-6.0.,Temperature:25-30,Treatment time:30min-20h,(3)Osmoticum,渗透剂,Protoplasts released directly into standard cell culture medium will burst.Hence,in isolating protoplasts,the wall pressure that is mechanically supported by cell wall must be replaced with an appropriate osmotic pressure in the protoplast isolation mixture and also later in the culture medium.,原生质体直接释放到标准培养基中会破裂，因此，在分离原生质体期间，对原来由细胞壁机械维持的压力用原生质体分离混合液以及后来培养基的适当渗透压代替。,低,(,或负,),渗透势常是由于加入各种离子或非离子型溶质产生的。,Non-ionic substances,非离子型物质,:carbohydrates,mannitol,sorbitol,glucose,fructose,galactose or sucrose.,Ionic substances,离子型物质,:KCl,、,CaCl,2,MgSO,4,最常用的渗透剂,:Mannitol,sorbitol,(4),Protoplast purification,原生质体的纯化,酶消化后得到的混合物含有亚细胞碎片、未消化的细胞、破碎的和完好的原生质体。对这个混合物通过过滤,(filtration),、离心,(centrifugation),和漂洗,(washing),联合的方法进行纯化。,(5),Protoplast viability and density,原生质体的活力和密度,Methods:,Fluorescein diacetate(FDA)staining method,荧光素二乙酸,(FDA),染色法,：,FDA,是一种积累在活原生质体质膜中的染料，能被荧光显微镜检测出来。当在细胞膜中积累荧光素二乙酸时，,5min,内活的、完整的原生质体就发出黄绿色光。,Phenosafranine staining,酚藏花红染色法：,使用酚藏花红时的终浓度为,0.01,，它只能使无生命力的原生质体被染成红色，活的原生质体不能被酚藏花红染色。,Calcofluor White(CFW)staining,荧光增白剂,(CFW),染色法,Exclusion of Evans blue dye by intact membranes,完整细胞膜的排斥,Evans,蓝染料法,Observations on cyclosis or protoplast streaming as a measure of active,metabolism,观察胞质环流法检测有活力的代谢,Variation of protoplast size with osmotic changes,随渗透改变的原生质体体积变化,Oxygen uptake measured by an oxygen electrode which indicates respiratory me tabolism,用氧电极测放氧从而检测代表活力的呼吸代谢,Photosynthetic studies,光合作用研究,(6),Culture techniques,Agar culture,琼脂培养法,Liquid culture,液体培养法,Liquid droplet method,小液滴培养法,Hanging droplet method,悬滴培养法,Feeder layer,饲喂层法,Co-culturing,联合培养法,(7),Culture medium,培养基成分,培养原生质体和培养植物细胞的营养需要是极其相似的。用于培养植物细胞的矿质盐的成分已经被调整到使之满足培养原生质体的特殊需要。,甘露醇和山梨糖醇是用于维持渗透压最常用的化合物,(8),Environmental factors,环境因子,在黑暗中或微弱光下先培养几天，然后再将培养物转移到约,2000,50001x,的光强下培养。原生质体培养的一般温度范围是,20,28,，建议原生质体培养基的,pH,范围是,5,5,5,9,，这个,pH,范围的培养效果看起来很好。,2.2 Protoplast development,原生质体发育,2.2.1 Cell wall formation,细胞壁形成,2.2.2 Growth,division and plant regeneration,生长、分裂和植株再生,2.3,Somatic hybridization,体细胞杂交,Concept,离体条件下，通过分离的体细胞原生质体融合产生杂合体，再通过其异核体的发育形成杂种植株的技术被称为体细胞杂交。,体细胞杂交包含原生质体融合（,fusion of protoplasts,）,、杂种细胞的选择（,selection of hybrid cells,）,、杂种植株的鉴定（,identification of hybrid plants,）,原生质体融合是基因组不同的两个原生质体的融合，可以通过自发融合（,spontaneous fusion,）,和诱导融合（,induced fusion,）,的方法实现。,2.3.1 Protoplast fusion,原生质体融合,(1),Spontaneous fusion method,自然融合法,(2),Induced fusion method,诱导融合法,Treatment with sodium nitrate,硝酸钠处理法,Calcium ions at high pH,高,pH,钙离子,Polyethylene glycol method,聚乙二醇法,Electrofusion,电融合,原生质体融合产物的鉴定和恢复是基于对一般可见特征的观察，杂种细胞显示出隐性突变的遗传互补和体外生长要求的生理学互补。,2.3.2 Identification and selection of hybrid cells,杂种细胞的鉴定和选择,(1),Chlorophyll deficiency complementation,叶绿素缺陷互补,(2),Auxotroph complementation,营养缺陷型互补,(3),Complementation of resistance markers,抗性标记互补,(4),Use of metabolic inhibitors,代谢抑制剂的使用,(5),Use of visual characteristics,肉眼可见特征的应用,Use of morphologically distinct cells,用形态学特征区别细胞,Use of petal protoplasts with leaf protoplasts,用花瓣原生质体形象化鉴定杂种,Fluorescent labeling,荧光标记,The growth pattern of hybrid callus is different from either parental line,杂种愈伤组织生长型与任何一个亲本系都不同,Differences in the morphology of callus,愈伤组织形态上的差异,(6),Compound selection system,综合选择体系,(1),Morphology,形态学,(2),Isoenzyme analysis,同工酶分析,(3),Chromosomalconstitution,染色体组成,(4),Molecular techniques,分子技术,(5),Genetic characterization,遗传鉴定,2.3.3 Verification and characterization of somatic hybrids,体细胞杂种的鉴定与特性,