园艺植物生物技术第四章课件

1、Plant cell engineering应用细胞生物学和分子生物学方法，借助工程学的试验方法和技术，在细胞水平上研究改造园艺植物遗传特性和生物学特性，以获得特定的细胞、细胞产品或园艺植物新品种的科学。Main contentsPlant cell culture Protoplast Isolation and Fusion1. Plant cell culture Haberlandt (Austria) : Cultivate isolated plant cells in vitro on an artificial medium -Concept： 指对植物器官或愈伤组织上分离出来

2、的单细胞(或小细胞团)进行培养，形成单细胞无性系或再生植物的技术。Significance：（1）研究细胞生理代谢过程及各种影响因子；（2）用于植物品种的改良；（3）用于植物次生代谢物质的生产。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Cu

3、lture methodsPlate 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

4、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。1.1.3 Influencing factors of single cell culturea. Medium：特殊成分，植物激素，pHb. Cell density：平板临界密度103个/mLc. Environmental conditions ：25，1%CO21.2. 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 cel

5、l 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

6、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 cyclei. 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

7、 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. 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 contin

8、uous culture 开放型 Closed continuous culture 封闭型 1.2.3 Requirements for good suspension culture systema. 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。2. Protoplast Isolation and Fusion原生质体的游离与融合2.1. Protoplast Isolation Methodsmechanical methodsenzymatic method

9、s2.1.1 Mechanical methodApplication rangelarge 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

10、 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 demonstr

11、ated the possibility of enzymatic isolation of protoplasts from higher plants. Influencing factors (1)Physiological state of tissue and cell materialProtoplasts have been isolated from a variety of tissues and organs including leaves, petioles, shoot apices, roots, fruits, coleoptiles, hypocotyls, s

12、tem, 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) EnzymesThe release of protoplasts is very much dependent on the nature and composition of enzymes used to d

13、igest 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 wallPectin: a component of middle lamella th

14、at 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 lamellaCellulase 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 dep

15、endent 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 m

16、ust 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, glucos

17、e, fructose, galactose or sucrose.Ionic substances离子型物质: KCl、CaCl2,MgSO4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: Fluoresc

18、ein 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

19、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 s

20、tudies光合作用研究(6) Culture techniquesAgar 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

21、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200050001x的光强下培养。原生质体培养的一般温度范围是2028，建议原生质体培养基的pH范围是5559，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

22、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