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植物组织培养常用英文缩写及词义

植物组织培养常用英文缩写及词义缩写英文名称中文名称A,Ad,Ade adenine 腺嘌呤ABA abscisic acid 脱落酸BA,BAP,6-BA 6-benzyladenine benzy- laminopurine 6-苄腺嘌呤P-CPOA P-chlorophenoxyacetic acid 对-氯苯氧乙酸CCC chlorocholine chloroid 矮壮素CH casein hydrolysate 水解酪蛋白CM coconut milk 椰子乳2,4-D 2,4-dichlorphenoxyacetic acid 2，4-二氯苯氧乙酸2,4-DB 2,4-dichlorphenoxybutyric acid 2，4-二氯苯氧丁酸DNA Deoxyribonucleic acid 脱氧核糖核酸EDTA ethylenediaminetra acetic acid 乙二胺四乙酸GA;GA3gibberellin; gibberellic acid 赤霉素IAA indole-3-acetic acid 吲哚乙酸IBA indole-3-butyric acid 吲哚丁酸-in,vitro 试管内，离体培养-in,vivo 活体内整体培养2-ip;IPA 2-isopantenyl adenine 6-(r,r-dimethylallyl) adenine 异戊烯基腺嘌呤，又6 –（r-二甲基烯丙基）嘌呤KT;Kt;K kinetia 激动素；动力精；糠基腺嘌呤LH lactabumin hydrolysate 水解乳蛋白lx lux 勒克斯m meter 米mg milligram 毫克min minute 分（钟）ml milliliter 毫升mm millimeter 毫米mmol millimole 毫摩尔mol.wt. molecular weight 摩尔重量；分子量NAA naphthalene acetc acid 萘乙酸PBA 6-（苄基氨基）9-（2-四氢吡喃基）-9H-嘌呤PH hydrogen-ion concentration 酸碱度，氢离子浓度ppm part(s)per million 百万分子几；毫克/升PVP polyvinylpyrrolidone 聚乙烯吡咯烷酮RNA ribonucleic acid 核糖核酸rpm(=r/min) 转/分s secend 秒Thidiazuron N-phenyl-N’-1,2,3-thia-diazol-5-ylurea 苯基噻二唑基尿2,4,5-T 2,4,5-trichlorophenoxy acetic acid 2，4，5-三氯苯氧乙酸μm micrometer 微米μmol micromole 微摩尔YE yeast extract 酵母提取物ZT;Zt;Z ziatin 玉米素。

植物组织培养专用英语

植物组织培养专用英语tissue culture 组织培养plant culture 植株培养organ culture 器官培养callus culture 愈伤组织培养cell culture 细胞培养proplast culture 原生质体培养rapid propagation 快速繁殖virus free culture 无毒(苗)培养industrializing propagation 工厂化繁殖cleaning room 清洗室repairing room 准备室inoculation room 接种室culture room 培养室inoculation tools 接种工具forceps 镊子scissors 剪子temperature 温度light intensity 光照强度humidity 湿度penetrating pressure 渗透压medium 培养基inorganic element 无机元素organic compound 有机化合物inose 肌醇amino acid 氨基酸nature compound 天然复合物hormone 激素auxin 生长素indo acetic acid 吲哚乙酸naphthalene acetic acid 萘乙酸indolebutyric acid 吲哚丁酸cytokinnin 细胞激动素kinetin 细胞分裂素zeatin 玉米素agar 琼脂antibiotic material 抗生素antioxide material 抗氧化物active carbon 活性炭growth regulater生长调节剂stock solution 母液inoculation 接种inoculum 接种物maxmal element stock solution 大量元素母液minimal element stock solution微量元素母液iron salt stock solution 铁盐母液sterilization 灭菌消毒fungus-free operationhumid hot sterilization 湿热灭菌dry hot sterilization 干热灭菌ultraviolet rays sterilization 紫外线灭菌high pressure sterilization 高压灭菌filtering sterilization 过滤灭菌burning sterilization 灼烧灭菌fumigation 熏蒸消毒spraying sterilization 喷洒灭菌soaking sterilization 浸泡灭菌inductive phase 诱发期pollution 污染explant外植体。

组织培养

组织培养科技名词定义中文名称：组织培养英文名称：tissue culture定义1：应用无菌操作方法培养生物的离体器官、组织或细胞，使其在人工条件下生长和发育的技术。

应用学科：水产学（一级学科）；水产生物育种学（二级学科）定义2：从机体分离出的组织或细胞在体外人工条件下培养生长的技术。

应用学科：细胞生物学（一级学科）；细胞培养与细胞工程（二级学科）本内容由全国科学技术名词审定委员会审定公布无菌组织培养室植物组织培养概念（广义）又叫离体培养，指从植物体分离出符合需要的组织.器官或细胞，原生质体等，通过无菌操作，在人工控制条件下进行培养以获得再生的完整植株或生产具有经济价值的其他产品的技术。

植物组织培养概念（狭义）指用植物各部分组织，如形成层.薄壁组织.叶肉组织.胚乳等进行培养获得再生植株，也指在培养过程中从各器官上产生愈伤组织的培养，愈伤组织再经过再分化形成再生植物。

目录植物组培发展简史植物组织培养与细胞培养开始于19世纪后半叶，当时植物细胞全能性的概念还没有完全确定，但基于对自然状态下某些植物可以通过无性繁殖产生后代的观察，人们便产生了这样一种想法即能否将植物体的一部分在适当的条件下培养成一个完整的植物体，为此许多植物科学工作者开始了培养植物组织的尝试。

最初的问题仍然是集中在植物细胞有没有全能性和如何使这种全能性表现出来。

1839年Schwann提出细胞有机体的每一个生活细胞在适宜的外部环境条件下都有独立发育的潜能。

1853年trecul利用离体的茎段和根段进行培养获得了愈伤组织，愈伤组织是指一种没有器官分化但能进行活跃分裂的细胞团，但这还不能证明细胞具有全能性，因为由愈伤组织没能再生出完整植物体。

1901年Morgan首次提出一个全能性细胞应具有发育出一个完整植株的能力。

所谓全能性细胞就是指具有完整的膜系统和细胞核的生活细胞，在适宜的条件下可通过细胞分裂与分化，再生出一个完整植株。

White 指出：如果一个给定的有机体的所有细胞都大致相同，并具有全能性，那么在有机体内所观察到的细胞分化必定是这些细胞对有机体内微环境和周围环境的反应。

《组培应用》课件

植物组织培养
通过无菌操作，将植物的器官、组织或细胞等置于人工配置的培养基中，在人工控制的条件下进行培养，以获得新植株的技术。
植物细胞培养
通过无菌操作，将植物细胞置于人工配置的培养基中，在人工控制的条件下进行培养，以获得新植株的技术。
植物离体培养
通过无菌操作，将植物的器官、组织或细胞等从母体上分离下来，置于人工配置的培养基中，在人工控制的条件下进行培养的技术。
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药用植物的生产
总结词
组织培养技术可以用于药用植物的生产，提高产量和品质，降低生产成本。
详细描述
许多药用植物具有较高的经济价值，但由于生长周期长、产量低等原因，难以满足市场需求。通过组织培养技术，可以快速繁殖大量药用植物，提高产量和品质。同时，组织培养技术还可以用于研究药用植物的药理活性成分，为其开发新药提供科学依据。
组培技术的发展历程
1928年
荷兰植物学家Frits Went首次利用无菌操作技术培养出了小植株。
1958年
中国科学家朱光亚等培养出了水稻幼胚愈伤组织。
1902年
德国科学家Hans Cremer首先提出了植物细胞具有全能性的观点。
1934年
美国科学家White培养出了烟草花药愈伤组织。
1978年
3D打印技术的应用
3D打印技术可以用于制造具有复杂结构的组织培养支架，为组培技术的形态发生和器官形成提供了新的可能性。
环境友好型技术的研发
随着环保意识的提高，环境友好型的组培技术成为未来的发展趋势，如利用植物细胞和组织在悬浮培养体系中的大规模培养技术。
组培技术的应用前景
农业和园艺产业

组织培养

注意事项
⑴自来水中含有大量的矿质元素，勿用配制母液； (纯水)
⑵配制大量元素和微量元素母液时，应将每种化合物单独溶解后，再一一混合，同时还应用适量的蒸馏水清洗烧杯内壁，并将清洗后的蒸馏水倒入混合溶液中，这样可以保证每种元素的浓度。混合时要按照教材附表中的顺序进行，最后加入的是氯化钙溶液，这样可以避免发生沉淀。

接（转）苗：无菌条件下，把茎（芽）从母瓶中取出，切分整理后，再接入新的培养基中。
包括取苗、切苗和接苗三步。取苗：先把培养瓶在酒精上烤10s左右，然后解开培养瓶的瓶盖（封口膜），再把瓶口在酒精灯上烤7～10秒；后正式取苗。注意：
⑴取苗时，培养瓶倾斜30度左右，手一定不要过（碰）瓶口。
3 植物生长调节物质母液的配制
植物生长调节物质是培养基的重要组分之一，一般是植物激素类物质，如生长素类的IAA、IBA、NAA，赤霉素类的GA3，细胞分裂素类的2,4-D、KT和6-BA等。大多数生长调节物质难溶于水，因此配制方法也各不相同： ①IAA，IBA和GA3先用少量95%酒精溶解，再加水定容，摇匀后贮于试剂瓶中、贴上标签后，存放在冰箱中；② NAA可用热水或少量95%酒精溶解，再加水定容至所需容积； ③2,4-D可溶于少许1mol/L的NaOH溶液中，然后加水定容； ④KT和6-BA可用少量1mol/L的HCl溶解，再加水定容； ⑤玉米素先溶于少量95%酒精中，然后加水定容.
4. 钙盐母液(10倍)：称取10倍用量的CaCl2· 2H2O 溶于80mL重蒸水中，最后定溶到100mL。 5. 有机物质母液（50倍液）分别称取50倍用量的各种有机物质，依次溶解于 400mL重蒸水中，定容至500mL装入棕色试剂瓶中，存放在冰箱中备用。 6. 生长素如2，4-D、IAA、NAA等。准确称取20mg，先用 2mL95％乙醇溶解，然后加水，定容至20mL，浓度为1mg/mL,放在冰箱内备用。

植物组织培养常用英文缩写及词义

组织培养英文

Plant Cell, Tissue and Organ Culture 64: 145–157, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.145Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineersAlan C. Cassells & Rosario F. CurryDepartment of Plant Science, National University of Ireland, Cork, Ireland (∗ requests for offprints; Fax: +353-21274420; E-mail: a.cassells@ucc.ie)Received 18 April 2000; accepted in revised form 1 December 2000Key words: DNA repair, free radicals, genetic engineering, hyperhydricity, in vitro culture, juvenility, micropropagation, mutation, reactive oxygen species, somaclonal variation, tissue cultureAbstract A number of well deﬁned problems in physiological, epigenetic and genetic quality are associated with the culture of plant cell, tissue and organs in vitro, namely, absence or loss of organogenic potential (recalcitrance), hyperhydricity (‘vitriﬁcation’) and somaclonal variation. These broad terms are used to describe complex phenomena that are known to be genotype and environment dependent. These phenomena affect the practical application of plant tissue culture in plant propagation and in plant genetic manipulation. Here it is hypothesised much of the variability expressed in microplants may be the consequence of, or related to, oxidative stress damage caused to the plant tissues during explant preparation, and in culture, due to media and environmental factors. The characteristics of these phenomena are described and causes discussed in terms of the known effects of oxidative stress on eukaryote genomes. Parameters to characterise the phenomena are described and methods to remediate the causes proposed. Abbreviations: AFLP – ampliﬁed fragment length polymorphism; FISH – ﬂuorescent in situ hybridization; HSP – heat shock proteins; PR-proteins – pathogenesis-related proteins; RFLP – restriction fragment length polymorphism; ROS – reactive oxygen species Introduction Those using tissue culture for multiplication or transformation are concerned to produce microplants that are ‘ﬁt for the purpose’, that is, free of speciﬁed diseases, vigorous, developmentally normal and genetically true-to-type (Cassells, 2000a, b; Cassells et al., 2000). The exceptions are that the market may exploit altered developmental characteristics, e.g. juvenility in herbaceous or woody plants where this results in greater productivity of the microplants when used for cutting production (George, 1993, 1996) or where tissue protocols give earlier ﬂowering (Cassells, 2000a). In general, genotype-dependent, multiplication via buds tends to be the preferred strategy to maintain genetic stability (Figure 1; George, 1993). Proliferation of side shoots from axillary buds, termed ‘nodal culture’ is preferred over proliferation of precocious axillary buds in shoot tip explants. The basis for this is that apical explants may give rise to basal explant callus from which adventitious buds may arise. The latter has been associated in strawberry with genetic variability in the progeny (Jemmali et al., 1997). It is important to mention here that epigenetic and genetic instability in the tissues used for Agrobacterium transformation (somaclonal variation: see Jain et al., 1998), that is expressed in adventitious shoots, may result in chimeral transformants (Cassells et al., 1987), and in somaclonal variation in the background of the transgenic lines (Sala et al., 2000) which may contribute to the silencing of trangenes (Matzke and Matzke, 1998). In human health the importance of oxidative stress has been long recognised in cancer and ageing studies146Figure 1. The methods of micropropagation least likely to produce plants with genetic variation (reproduced with permission; from George, 1993).(Harman, 1956). It is also recognised how complex the underlying mechanisms and processes are (Halliwell and Aruoma, 1993). The cellular mechanisms to manage stress, namely, constitutive and induced production of radical scavengers, free radical and oxidised-protein enzymatic degradation pathways and DNA repair mechanisms are highly conserved in all eukaryotes (Halliwell and Aruoma, 1993; McKersie and Leshem, 1994). Environmental and pathogeninduced stress have been investigated in detail in plants in vivo (Bolwell et al., 1995; Baker and Orlandi, 1995; Doke et al., 1996; McKersie and Leshem, 1994). Stress-like phenomena expressed in vitro and in microplants have been extensively described but less is known about the underlying causal mechanisms (Ziv, 1991; Jain et al., 1998). Both in initiating cultures and in sub-culturing, explant preparation involves wounding of the tissues which is known to cause oxidative stress (Yahraus et al., 1995). Elicitors of oxidative stress e.g. hypochlorite (Wiseman and Halliwell, 1996) and mercuric salts (Patra et al., 1997), are used to surface sterilize theprimary explants. Many factors associated with aberrations in plant tissue culture such as habituation, hyperhydricity (Gaspar, 1998) are caused by oxidative stresses (Keevers et al., 1995), such as high salt (McKersie and Leshem, 1994), water stress (NavariIzzo et al., 1996), mineral deﬁciency (Elstner, 1991), excess metal ions (Caro and Puntarulo, 1996) and possible over exposure to auxin (Droog, 1997). Oxidative stress (Gille and Sigler, 1995; Bartosz, 1997) is deﬁned as an imbalance in the pro- versus anti-oxidant ratio in cells and results in elevated levels of pro-oxidants (ROS: reactive oxygen species; including superoxide, hydrogen peroxide hydroxyl, peroxyl and alkoxyl radicals) (Wiseman and Halliwell, 1996) which can cause cell damage (Sies, 1991). ROS (Figure 2) can react with a spectrum of metabolites, proteins including enzymes, and nucleic acid molecules (Gille and Siegler, 1995). Oxidised enzymes which may be inactivated, are degraded by cytosolic proteinases (Laval, 1996). The inﬂuence of ROS, through altered cell redox potential, on the cell cycle and oxidative damage to both nuclear and organellar147Figure 2. Reactive oxygen species (ROS) produced constitutively in the cell. The upper section shows the natural antioxidants and enzymes used to minimize the toxic effects of ROS. The lower section gives selected examples of the harmful effects of ROS when the pro- and anti-oxidant balance is perturbed in oxidative stress. (MDE, malondialdehyde; HNE, 4-hydroxynonenal).DNA, may result in mutations (Figure 3; Bohr and Dianov, 1999). Oxidative damage in eukaryote cells is expressed in altered hyper- and hypomethylation of DNA (Kaeppler and Phillips, 1993; Tilghman, 1993; Wiseman and Halliwell, 1996; Cerda and Weitzman, 1997; Wacksman, 1997); changes in chromosome number from polyploidy to aneuploidy, chromosome strand breakage, chromosome rearrangements, and DNA base deletions and substitutions (Gille et al., 1994; Czene and Harms-Ringdahl, 1995; Hagege,1995). Such changes could explain, at least in part, the range of variability found in plant cells, tissues and organs in culture and in microplants, namely, recalcitrance including loss of cell competence (Hagege, 1995; Lambe et al., 1997), hyperhydricity (Olmos et al., 1997) and somaclonal variation including epigenetic and genetic variation (Jain et al., 1998; Joyce et al., 1999; Kowalski and Cassells, 1999). The objective of this review is to discuss tissue culture variability, its causes, detection and remediation148 with emphasis on the possible role of oxidative stress in this phenomenon.Aberrations and variation expressed in vitro At the outset it should be recognised that explants, other than buds, from dicot plants have different characteristics to those from monocots, speciﬁcally those of dicots may have a cambium; that there are differences in organogenetic potential between families, genera, species and genotypes; and that different genotypes of a species may show widely different responses (George, 1993, 1996). Further there are differences in the responses of explants from different parts of a plant, which change ontogenetically (George, loc. cit.). Some trends are evident, e.g. increased recalcitrance with advancing age of the cultures (Hagege, 1995) and increased somaclonal variability in microplants with increasing sub-culture number (Brar and Jain, 1998). With a given genotype, wounding of the tissues on cutting (excision), and tissue damage and exposure to sterilants during sterilisation, and suboptimal in vitro factors (Ziv, 1991) are important in relation to genomic damage. So called ‘pre-existing’ genomic diversity at the cell level (D’Amato, 1964; Figure 4) and wound or oxidative damage due to wounding may explain some of the variability subsequently seen in vitro and in the resulting microplants. Possible stress due to unbalanced media, bad culture vessel design and environmental stress may also, or further, contribute to the genetic, epigenetic (developmental) and physiological variability recorded (Ziv, 1991). Wounding or excision per se may be considered both as a trigger for cell division (Sangwan et al., 1992) and as a damaging oxidative burst (Schaaf et al., 1995; Yahraus et al., 1995). As a consequence of the above factors, explants may senesce, fail to respond, undergo cell division and/or produce adventitious organs or somatic embryos. In responding genotypes, the response is generally regulated in a predictable way by manipulation of the auxin to cytokinin ratio and absolute growth regulator concentrations (Skoog and Miller, 1957). In some cases, recalcitrance may be overcome by pulsing in sequence with auxin followed by cytokinin (Christianson and Warnick, 1985). Whether genome variability is ‘pre-existing’, caused by oxidative stress on wounding an/or caused by stress in culture (Figure 4), selection may begin in vitro with the appearance of sectoring in the callus (D’Amato et al., 1980). Cell lineFigure 3. Changes in DNA caused by oxidative stress which can lead to recalcitrance, loss of competence, hyperhydricity and somaclonal variation.selection for in vitro conditions may result in loss of competence; e.g. selection based on ﬁtness of grossly altered genotype(s) may result in the irreversible loss of competence (Hagege, 1995). The main morphological aberration seen in shoots in vitro cultures, both in nodal/bud derived shoots and in adventitious shoots, is hyperhydricity (‘vitriﬁcation’) (Debergh et al., 1992). This term is used to describe aberrant morphology, typically hyperhydrated, translucent tissues and physiological dysfunction in plant tissues in vitro (Ziv, 1991). It is also associated with leaf-tip and bud necrosis. The latter often leads to loss of apical dominance in the shoots and is associated with callusing of the stem base. An important characteristic of this condition is impaired stomatal function which causes problems in establishing microplants (Preece and Sutter, 1991). Morphological variability in plants from in vitro culture may be seen in intrapopulation variability (within a population of adventitiously regenerated plants) (Kowalski and Cassells, 1998) and interpopulation variability (between populations of in vitro plants) (Joyce et al., 1999). The latter may arise when plants are propagated on different media or in culture vessels with different characteristics (Joyce et al., 1999). Intrapopulation variability can be a result of the loss of speciﬁc viruses, including cryptic viruses, from some of the regenerated plants (Matthews, 1991); chimeral breakdown, rearrangement and/or synthesis of unstable chimeral plants (Tilney-Bassett, 1986). In generally, heritable somaclonal variation (Larkin and149Figure 4. Sources of genetic variation in plants obtained through organogenesis in callus cultures (reproduced with permission; from George, 1993).Scowcroft, 1981) has the characteristics of mutation (Anonymous, 1995; Jain et al., 1998), albeit occurring at higher frequency than occurs spontaneously in seed or vegetative propagules (Preil, 1986). It is genotype-dependent and dependent on the pathway of regeneration (Karp, 1991). Epigenetic changes can occur in vitro culture resulting in ‘apparent rejuvenation’ (Pierik, 1990) affecting woody and herbaceous plants (Huxley and Cartwright, 1994; James and Mantell, 1994; Jemmali et al., 1994; Cassells et al., 1999 a, b). Interpopulation variation is usually cryptic, as control populations are not available for comparison; it is recognised in quality differences in plants produced by different protocols or by different micropropagators (GrunewaldtStoker, 1997). Examples of interpopulation variability are populations differing in degree of hyperhydricity or juvenility (Swartz, 1991). While woody plant propagators are familiar with phase change (Howell, 1998), micropropagators of herbaceous plants appear less conscious of this phenomenon but it has implications for disease susceptibility in that polygenic resistance develops as the plant soma matures (Agrios, 1997) and for time to ﬂowering (Howell, 1998). Plants showing prolonged juvenility (epigen-etic/ontogenetic variability) may be more susceptible to damping-off diseases (Agrios, 1997). This is not always the case, as juvenile tissues are reported to have enhanced resistance to fusaric acid (Barna et al., 1995) and Cassells et al. (1991) have shown that potato crops derived from microplants, showing juvenility compared to a tuber-derived crop, were more resistant to potato blight. In vitro plants may have a longer time to ﬂowering compared to those from vegetative propagules (Cassells et al., 1999a). While morphological intrapopulation variability and ontogenetic and physiological variation, expressed in interpopulation variability, are well recognised phenomena in micropropagation, cryptic intrapopulation variation in juvenility has also been detected in adventitiously regenerated plant populations showing genetic variation (Kowalski and Cassells, 1998) suggesting that genetic and epigenetic variability are not necessarily discrete but can occur in the same population. Somaclonal variation is strongly expressed after the microplant population establishment stage as interplant variation in morphological characters. Some of the plants may show characteristics of chimeral breakdown (Tilney-Bassett, 1986). Somaclonal variation has been extensively reviewed in Jain et al. (1998).150Figure 5. Showing the consequences of oxidative stress from induction of host antioxidant defences (repair, heat shock protein induction, pathogenesis related protein induction) to mutation, programmed cell death and uncontrolled cell death. Figure 6. The relationship between stress inducers e.g. medium salt stress, gene activation and the generation of biomarkers for stress remediation and stress damage repair. Examples of damage exposure are ethylene and ethane; of damage are oxidised bases e.g. 8-oxoguanine; of remediation are glutathione and glutathione reductase and of repair, Poly(ADP-ribose) polymerase (see text for further markers).As discussed above, shifts in characters in populations, e.g. physiological or developmental changes, are not readily recognised unless control populations are available (Cassells et al., 1997). These can, however, be visually expressed in loss of apical dominance, leaf number and leaf size and, more importantly in the time to ﬂowering, and yield quality e.g. tuber number and size distribution in potato seed production (Cassells et al., 1999a).Characterisation of epigenetic and genetic changes in microplants pre- and post- establishment Cytometric analysis of callus has shown variability in chromosome number and ploidy in tissue culturederived plants (Geier, 1991; Gupta, 1998). Investigations indicate more chromosome variability in the callus phase than in adventitious shoots (D’Amato et al., 1980), indicating a loss of competence in the more seriously disturbed genomes (Valente et al., 1998). Cell line selection for secondary product formation also shows differences at the metabolite level (Berglund and Ohlsson, 1995). While occasional albino shoots are observed, the expression of morphological variation is difﬁcult to assess in vitro due to variability between shoots due to temporal differences in shoot initiation and because of the limited leaf expansion in in vitro cultures. Variability in both qualitative and quantitative traits has also been reported (Karp, 1991). The latter expressed in increased standard deviations of the character mean (DeKlerk, 1990) and can be quantiﬁed using computerised image analysis (Cassells et al., 1999a). Analysis of DNA-base methylation and various genetic ﬁngerprinting techniques have also been used to conﬁrm and characterise variability in tissue culturederived plants, conﬁrming both morphological and cryptic genetic and epigenetic variability between and within populations (Karp et al., 1998; Cassells et al., 1999b).Current views on the molecular basis of somaclonal variation In recent years plant cell, tissue and organ culture has been developed for applications in plant genetic manipulation (Cassells and Jones, 1995). In this ﬁeld, somaclonal variation has attracted considerable interest as a means of improving crop plants (Jain et al., 1998). Reviews discussed a number of mechanisms to explain somaclonal variation, these included changes in chromosome number, chromosome breakage and rearrangement, DNA ampliﬁcation, point mutations, changes in DNA methylation, changes in organellar DNA, activation of transposons (Frahm et al., 1998; Gupta, 1998; Henry, 1998; Jain et al.,151 1998; Kaeppler et al., 1998). The mechanisms appear to be equally applicable to explaining the basis of variation at the cell and callus level and are similar to the variability resulting from oxidative genome damage and induced mutation. Nagl (1990) has discussed the relationship between stress-induced and ontogenetic changes in plant genomes arguing that plant genomes are inherently ﬂuid. In some genomes, e.g. ﬂax (Schneeberger and Cullis, 1991) and banana (Cullis and Kunert, 2000) there are well characterised genomic instabilities associated with somaclonal variation. (Figure 5). In addition to the above ROS, chemical and physical agents can stimulate lipid peroxidation that can become autocalatytic resulting in the production of organic hydroperoxides (Figure 2). ROS in the presence of iron and copper ions may generate highly mutagenic compounds, e.g. peroxyl radicals and alkoxyl radicals (Koh et al., 1997). The primary response to elevated ROS production (Figure 2) is stimulation of production of antioxidant molecules (radical scavengers) such as ascorbic acid and glutathione in the aqueous phase and alpatocopherol and carotenoids in the lipid phase (Gille and Sigler, 1995) and the activation of antioxidant enzyme systems including superoxide dismutase, catalase and glutathione and ascorbic acid peroxidases (Tsang et al., 1991; Larson, 1995; Smirnoff, 1996). Additional responses involve the activation of heat shock proteins to protect enzymes systems against ROS damage (Burdon, 1993) and of proteases to degrade damaged proteins (Stadtman, 1992). More signiﬁcant is the potential of ROS to cause DNA damage (‘genotoxicity’; Wiseman and Halliwell, 1996). The cell cycle slows or shuts down to minimise the transmission of mutations to daughter cells through mitosis and to facilitate DNA repair (Logemann et al., 1995; Amor et al., 1998; Reichheld et al., 1999) and DNA repair mechanisms, the SOS response, are activated (Laval, 1996; Yamamoto et al., 1997; Vonarx et al., 1998). The outcomes of oxidative stress depend on the balance between pro and anti-oxidants responses. Imbalance may lead to controlled responses (Figure 5) such as induced resistance to pathogens (Ernst et al., 1992), excessive imbalance to cell damage and mutation (Wiseman and Halliwell, 1996), possible programmed cell death (apoptosis) (Polyak et al., 1997) and, in the extreme, to (unprogrammed) cell death (Hippeli and Elstner, 1996). Oxidative stress has been linked to recalcitrance in protoplast culture (Benson and Roubelakis-Angelakis, 1994). Increase in ROS is associated with a range of biotic and abiotic stresses (Gile and Siegler, 1995; Bartosz, 1997). These include salt, drought, heat, and UV-induced stresses. They are also induced by chemical and physical mutagens (Anon, 1977). Their protective and constitutive roles include direct protection against pathogen attack, and involves the role of H2 O2 as a messenger in the induction of host resistance (pathogenesis-related (PR) protein induction) (Lamb and Dixon, 1997). H2 O2 may have a role in xylem formation and other cell-death processes in plants (Howell, 1998). ROS have a role in creatingThe relationship between somaclonal variation and spontaneous mutation The paper of Shepard et al. (1980) on somaclonal variation in potato stimulated interest in the application of this variability in crop improvement but was soon followed by concern about the quality of somaclonal variation and whether it differed qualitatively from spontaneous mutation (Sanford et al., 1984). This issue has been discussed by Karp (1991, 1995) but while it is still controversial, somaclonal variation is used in plant improvement, with induced mutagenesis whose efﬁciency has been improved by exploiting in vitro plant systems (Cassells, 1998). More importantly here, induced mutation and somaclonal variation result in a qualitatively similar, if not quantitatively identical, spectrum of DNA changes (see Figure 3). The issue is whether somaclonal variation and other tissue culture variability are mechanistically like physically-induced mutation and are caused by reactive oxygen species (Anonymous, 1977; 1995; Micke and Donini, 1993).Oxidative stress and mutation Oxidative stress is caused by the unremediated hyperactivity of reactive oxygen species (Figure 2). ROS such as superoxide, hydrogen peroxide and the hydroxyl radical are metabolic intermediates in respiration and photosynthesis and other metabolic activities in plants (see review by Gille and Sigler, 1995; Bartosz, 1997). Their natural cytoplasmic toxicity and genotoxicity is controlled by antioxidants and enzymic pathways in the cell (Hippeli and Elstner, 1996). Various environmental signals (Bartosz, 1997), which lead to an increase in ROS are associated with induced mechanisms to minimise their harmful effects152 variability in the plant genome by activating transposons (Mhiri et al., 1997), inducing polyploidy, chromosome breakage/rearrangements and base mutations (Figure 2). DNA based changes induced by ROS may inhibit methylating enzymes leading to hypomethylation (Cerda and Weitzman, 1997; Wacksman, 1997), e.g. formation of 8-oxoguanine occurs at high frequency which may lead to mismatch at DNA repair (base mutation, e.g. AT–GC changes), similarly for other oxidative base changes. While much of the ROS-induced effects due to wounding may be localised, some ROS can migrate across membranes, e.g. H2 O2 and cause effects directly, or via membrane lipid peroxidation, at the level of DNA in the stressed cells or in neighbouring cells (Figure 2). A gradient of stress damage from the wound area back into the explant may be hypothesised. ROS (and physical mutagens) have been conﬁrmed in animal cells (Halliwell, 1999) as causing the range of epigenetic and genetic changes in DNA that are problematic in plant tissue culture (Figure 3). number and DNA content (Quicke, 1993; Curry and Cassells, 1998). Techniques including ﬂuorescent in situ hybridisation (FISH) (Maluszynska and HeslopHarrison, 1991) and Giemsa banding (Quicke, 1993) are used to look for somatic recombination, including chromosome breakage and rearrangement and may also be used to detect DNA ampliﬁcation and reduction. Changes in DNA base methylation can be investigated using methylation-sensitive restrictions in RFLP and AFLP analysis (Karp et al., 1998) or by PCR of bisulphite modiﬁed DNA (Joyce et al., 1999).Plant hormones and oxidative stress Plant hormones implicated in hyperhydricity (vitriﬁcation) include cytokinins, auxins, and the auxin/cytokinin ratio; gibberellic acid and ethylene (Ziv, 1991; George, 1996). Oxidative stress has been associated with auxin and cytokinin metabolism in Agrobacterium induced tumours (Jia et al., 1996). Ethylene is also strongly linked to oxidative stress (McKersie and Leshem, 1994; Pell et al., 1997). Injury has been shown to activate the oxidation of IAA, while kinetin is reported to be a secondary product of oxidative stress (Barciszewski et al., 1997). In vitro, various media factors have been shown to induce stress, including hormones, and mineral nutrients (Ziv, 1991; George, 1993, 1996) there is evidence that metal toxicities and deﬁciencies may generate ethylene through oxidative stress (Lynch and Brown, 1997). Oxidative stress also affects cytosolic calcium (Price et al., 1994). Oxidative stress has been suggested as a cause of guard cell malfunction (McAnish et al., 1996). Calcium has been implicated in increased stress tolerance (Gong et al., 1997).Parameters used to characterise oxidative stress A number of methods have been used to monitor/characterise oxidative stress (Figure 6). These include measurement of the redox potential (Reichheld et al., 1999), measurement of stress related metabolites e.g. ascorbic acid (Smirnoff, 1996), glutathione (de Vos et al., 1994), hydrogen peroxide (Schreck et al., 1996). Ethylene has been monitored in hyperhydricity (‘vitriﬁcation’) studies (Keevers and Gaspar, 1985) and along with ethane, a marker for lipid peroxidation, has been used to monitor stress in vitro (Cassells et al., 1980; Cassells and Tamma, 1985). Thiobarbituric acid reactive substances are also used to assess lipid peroxidation (Laszczyca et al., 1995). 8oxo-2 -deoxyguanosine (Kasai, 1997; Bialkowski and Olinski, 1999) is considered to be a reliable indicator of genotoxicity as are other bases modiﬁed by ROS (Yamamoto et al., 1997). Enzymes of oxidative metabolism (Mehlhorn, 1990), enzymes associated with the cell cycle (Chiatante et al., 1997), enzymes of the SOS response (Laval, 1996; Wiseman and Halliwell, 1996) e.g. poly(ADP-ribose)-polymerase (Amor et al., 1998), screening for heat-shock proteins (HSP) (Burden, 1993) and PR proteins (Glandorf et al., 1997) have also been used as oxidative stress monitors. Karyotyping, ﬂow cytometry and microdensitometry can be used to measure changes in chromosomeRemediation of oxidative and other tissue culture associated stresses Remediation of oxidative stress can be based on several strategies. Genotypes can be screened for their sensitivity to stress and sensitive genotypes avoided; or they can be bred, mutated or engineered for increased stress tolerance (Gupta et al., 1993). The breeding/genetic manipulation options are both relatively long-term and costly and can only be applied to individual genotypes (Jones and Cassells, 1995). An important consideration where plant tissue culture is used for cloning or transformation is the choice153 of the explant. While mature plant tissue may be polysomatic (D’Amato, 1964), this may not be so in the case of the tissues of young plants in vitro (Curry and Cassells, 1998; Curry and Cassells, unpublished). Selection of explants from the latter may avoid the problem of ‘pre-existing’ variation (Figure 4). The main option, however, is the use of stable pathways of multiplication (Figure 1: George, 1993, 1996); albeit, even with these genetic drift may occur (Jemmali et al., 1997). There is evidence e.g. that protoplasts give rise to greater variability than tissue explants as expressed in the greater variability in plants regenerated from the former (Sree Ramulu et al., 1984). This may reﬂect expression of somatic cell variability (Figure 4) but it also appears to reﬂect the stress experienced in their isolation and regeneration where they are exposed to osmotic stress and the toxic effects of cell wall degrading enzyme preparations (Cassells and Tamma, 1985) and this has implication for their use in transformation via electroporation or biolistics at the protoplast level (Christou, 1995) as opposed to bombardment of apices (Sautter et al., 1995). The immediacy of the oxidative stress caused by excision and manipulation (Yahraus et al., 1995) would suggest that treatment of the explant after excision would be relatively ineffectual. As an alternative, it is suggested that stress be applied to the donor plant (or in vitro microplant) before excision. Under in vivo conditions, it was been shown that stress exposure induces cross-stress tolerance, e.g. UV treatment not only increases tolerance to further UV exposure but also to pathogen induced stress (Ernst et al., 1992), similarly paclobutrazol which is used in vitro and in weaning treatments (George, 1993, 1996) reduces oxidative stress to high light and high temperature (Mahoney et al., 1998). It has been suggested that induction of heat shock proteins be used to protect against oxidative stress (Banzet et al., 1998; Sebehat et al., 1998). Prevention or reduction of oxidative stress damage in vitro may be possible by manipulating hormone and mineral nutrients using the above oxidative stress parameters to monitor the protocols as has been done in the case of protoplasts (Cassells et al., 1980; Cassells and Tamma, 1985). In vitro calli and tissues are more amenable than intact tissues to permeation techniques. Cells, calli and explants can be inﬁltrated and bathed with radical scavengers such as ascorbic acid, mannitol and dimethyl sulphoxide. The stress messenger hydrogen peroxide can be broken down by extracellular catalase. Manipulation of media composition, particularly using simple media formulations in autotrophic or microhydroponic culture (Cassells, 2000a) and modiﬁcation of culture vessel design to facilitate controlled gaseous exchange (Cassells and Walsh, 1998), can greatly inﬂuence microplant resistance to hyperhydricity and improve ontogenic development (Cassells, 2000b).Conclusions It is speculated here that problems underlying the application of plant tissue culture systems in plant cloning (micropropagation) and genetic transformation, namely, recalcitrance, hyperhydricity, poor physiological quality, genetic and epigenetic variation, may have a common basis, at least in part, in oxidative stress-induced damage. This damage is caused by an overwhelming of the antioxidative defenses by primary ROS and secondarily, by the production of ROS by lipid autocatalytic peroxidation (Figure 2). While damaged proteins may be broken down by proteinases, damage is ﬁxed in the DNA. Speciﬁc levels of DNA damage may result in cell death or programmed cell death, other levels in loss of cell competence, altered methylation patterns may result in epigenetic changes, or in mutations (Figure 3). Somaclonal variation shows a similar spectrum of genetic variation to induced mutation; and oxidative stress and irradiation are known to involve ROS. Oxidative stress damage which it is emphasised is genotype dependent, may also operate at the physiological level since ROS have been shown to inﬂuence plant hormones namely, cytokinin, auxin, ethylene metabolism. Oxidative stress also inﬂuences calcium metabolism which in turn is involved in auxin transport, guard cell function and as a secondary messenger. Remediation strategies have been proposed and some makers of oxidative stress listed. It is suggested that induction of heat shock proteins may confer cross tolerance to the stress phenomena encountered as problems in establishing plant tissue cultures. Media and environmental manipulations should be carried out aimed at reducing in vitro stress based on adjusting the media composition and the physical culture environment, paying special attention to hormone stress, mineral composition and water and light stress. Reactive nitrogen species (NOS) not discussed here, can。

组培重点英文老师总结

Microstructure, ultrastructure and function 光镜结构，电镜结构和功能Epithelial Tissue上皮组织：simple squamous epithelium单层扁平上皮（Endothelium内皮，Mesothelium间皮）Simple cuboidal epithelium，Simple columnar epithelium，Pseudostratified ciliated columnar，Stratified squamous epithelium（Nonkeratinised未角质化/ Keratinised），Transitional epitheliumFree surface游离面：Microvilli微绒毛，Cilium纤毛lateral surface侧面：Tight junction，Intermediate junction，Desmosome ，Gap junctionbasal surface基底面：Basement membrane，Plasma membrane infolding质膜内褶，Hemi-desmosome半桥粒Loose Connective tissue: fibroblast成纤维细胞，macrophage 巨噬细胞，plasma cell浆细胞，mast cell肥大细胞；fat cell，undifferentiated mesenchymal cell 未分化的间充质细胞，leukocyte白细胞Collagenous fiber胶原纤维，Elastic fiber弹性纤维，Reticular fiber网状纤维（argyrophilic fiber嗜银纤维）hyaline cartilage透明软骨Bone骨：Osteoprogenitor cell骨祖细胞, Osteoblast成骨细胞，Osteocyte骨细胞, Osteoclast破骨细胞Compact bone骨密质：Circumferential lamellae环骨板，Osteon骨单位(Haversian system) ，Interstitial lamellae间骨板Erythrocyte (Red Blood Cell, RBC)红细胞，Reticulocytes网织红细胞Leukocyte (White Blood Cell, RBC)白细胞Neutrophil中性粒细胞，Eosinophil，basophil，lymphocyte，monocyte，Blood platelet (Thrombocyte)血小板skeletal muscle骨骼肌, cardiac muscle心肌, and smooth muscle平滑肌Sarcomere肌节，Triad三联体，Intercalated disc闰盘Neuron神经元：Nissl body尼氏体，Neurofibril神经原纤维Chemical synapse化学性突触，Neuroglial cell 神经胶质细胞(glial cell)，Myelinated nerve fiber有髓神经纤维Nerve Ending神经末梢Capillary毛细血管（continuous capillary，fenestrated capillary，sinusoidal capillary），Artery动脉，Medium-sized Artery，Large Artery，Vein 静脉，Purkinje fiberMononuclear phagocyte system (MPS)单核吞噬细胞系统，Antigen-presenting cells (APC)抗原呈递细胞,Thymus胸腺：Blood thymus barrier血胸屏障，thymus corpuscle胸腺小体Lymph node 淋巴结:Parenchyma: cortex: superficial cortex浅层皮质+deep cortex=paracortical zone深皮质=副皮质区+ medulla髓质lymphocyte recirculation淋巴细胞再循环,high endothelial venule高内皮微静脉Spleen脾: White pulp白髓:periarterial lymphatic sheath(PALS)动脉周围淋巴鞘+spleniccorpuscle脾小体Marginal zone边缘区Red pulp红随:splenic cord脾索+ splenic sinus脾窦Skin皮肤=epidermis表皮+ dermis真皮Keratinocytes 角质形成细胞Nonkeratinocytes非角质形成细胞: Melanocyte黑素细胞, Langerhans cell朗格汉斯细胞, Merkel cell梅克尔细胞Nitrogenous-hormone secreting cell分泌含氮激素细胞，Steroid-hormone secreting cells分泌类固醇激素细胞Thyroid gland甲状腺: Follicular epithelial cell滤泡上皮细胞, Parafollicular cell (C cell) 滤泡旁细胞Adrenal Gland肾上腺: Cortex:zona glomerulosa球状带+ zona fasciculata束状带 +zona reticularis网状带medullary cells髓质细胞=chromaffin cells嗜铬细胞Hypophysis垂体:Acidophils 嗜酸性细胞，Basophils嗜碱性细胞，chromophobe cells 嫌色细胞Hypophyseal portal system垂体门脉系统, Herring body赫令体,APUD system, Diffuse Neuroendocrine System弥散的神经内分泌系统Esophagus食管:Stomach胃:fundic gland胃底腺:chief cell主细胞，壁细胞……Small intestine小肠:intestinal villi肠绒毛, intestinal glands小肠腺: Paneth cells 帕内特细胞Pancrea胰腺: Exocrine portion外分泌部+ Endocrine portion (pancreas islets, islets of Langerhans) 内分泌部（胰岛）Liver肝:hepatic lobule肝小叶: -central vein中央静脉hepatic plate肝板, liver sinusoid肝血窦, bile canaliculi胆小管, space of Disse / Perisinusoidalspaces窦周隙Portal space门管区Trachea气管:Lung肺: Conducting portion导气部+ Respiratory portion呼吸部Clara cellstype I alveolar cells I型肺泡细胞，blood-air barrier气血屏障+ type II alveolar cells II型肺泡细胞Cornea角膜,Retina视网膜: Visual cells 视细胞(photoreceptors): rodcells and cone cells视杆细胞，视锥细胞macula lutea—central fovea 黄斑-中央凹Crista ampullaris壶腹嵴,Macula utriculi & Macula sacculi 椭圆囊斑，球囊斑, spiral organ螺旋器(Corti organ)Kidney肾:Nephron肾单位:renal corpuscle肾小体+ renal tubule肾小管：Proximal convoluted tubule近曲小管，Distal convoluted tubule远曲小管Filtration barrier/membrane滤过屏障（滤过膜）Juxtaglomerular complex球旁复合体Juxtaglomerular cells球旁细胞+ Macular densa 致密斑+ Extraglomerular mesangial cells球外系膜细胞Testes睾丸:seminiferous tubule 生精小管supporting cell支持细胞=Sertoli cell,testicular interstitial cell 睾丸间质细胞(Leydig cell)blood –testis barrier血睾屏障Ovary卵巢:Primordial follicle原始卵泡, Primary follicle初级卵泡, Secondary follicle次级卵泡, Mature follicle成熟卵泡Ovulation排卵, Corpus luteum黄体Uterus子宫:Menstrual cycle:月经周期。

PlantTissueCulture(组培基础信息)

PLANT TISSUE CULTUREBackgroundPlant research often involves growing new plants in a controlled environment. These may be plants that we have genetically altered in some way or may be plants of which we need many copies all exactly alike. These things can be accomplished through tissue culture of small tissue pieces from the plant of interest. These small pieces may come from a single mother plant or they may be the result of genetic transformation of single plant cells which are then encouraged to grow and to ultimately develop into a whole plant. Tissue culture techniques are often used for commercial production of plants as well as for plant research.Tissue culture involves the use of small pieces of plant tissue (explants) which are cultured in a nutrient medium under sterile conditions. Using the appropriate growing conditions for each explant type, plants can be induced to rapidly produce new shoots, and, with the addition of suitable hormones new roots. These plantlets can also be divided, usually at the shoot stage, to produce large numbers of new plantlets. The new plants can then be placed in soil and grown in the normal manner.Many types of plants are suitable for use in the classroom. Cauliflower, rose cuttings, African violet leaves and carnation stems will all easily produce clones (exact genetic copies) through tissue culture. Cauliflower florets in particular give excellent results since they can be grown into a complete plant in the basic tissue culture media, without the need for additional growth or root hormones. Green shoots are generally observable within three weeks, and roots develop within six weeks.The most important part of this activity, however, is to maintain as sterile an environment as possible. Even one fungal spore or bacterial cell that comes into contact with the growth media will rapidly reproduce and soon completely overwhelm the small plant piece that you are trying to clone.Objectives1. To understand a procedure that is often used to propagate many plants of the same genetic background.2. To understand the importance of sterile techniques.Materials1 Vial of Murashige Skoog (MS) media. (If you wish to make up your owngrowing medium you could use the recipe for the Murashige mediumgiven at the end of this section.)1 L sterile distilled water10 g of agar/L30 g sucrose/L1.5 L or 2 L container in which to prepare the growth mediumsmall amounts of 1M NaOH and 1M HCl to adjust the pH of the media60 flat bottom culture tubes with closures.Glass aquarium or box lined with plasticPlastic sheet to cover the top of the aquariumAdhesive tape10% Bleach in a spray bottle70% alcohol in a spray bottleForceps or tweezersGlovesCutting equipment such as a scalpel blade or razor blade2 bottles of sterile distilled water (purchase at the grocery store)Pressure cookerYour chosen plant (cauliflower, rose, African violet or carnation)paper towel for cutting on or sterile petri dishes if availableBeaker or jar in which to wash the plant materialDetergent-water mixture - 1ml detergent per liter of waterBleach sterilizing solution - dilute commercial bleach (5-6% sodiumhypochlorite) to a final concentration of 1-2% sodium hypochlorite indistilled water in a large beaker or jar.2 or3 beakers or jars of sterile waterA well-lit area away from direct sunlight or use full-spectrum gro-lightsHormones such as BAP (benzylaminopurine) and NAA (naphthalene aceticacid) to stimulate growth and root development,respectively. (Commercial rooting hormone solutions and powders arealso available from hardware stores.)Murashige Minimal Organics Medium recipe(MMOM)Inorganic salts mg/LNH4NO31,650.00KNO31,900.00CaCl2 (anhydrous) 332.20MgSO4 (anhydrous) 180.70KH2PO4170.00Na2EDTA 37.25FeSO4.7H2O 27.80H3BO3 6.20MnSO4.H2O 16.90ZnSO4.H2O 5.37KI 0.83Na2MoO4.2H2O 0.25CuSO4 (anhydrous) 0.016CoCl2 (anhydrous) 0.014Sucrose 30,000.00i-Inositol 100.00 Thiamine.HCl 0.40The pH is adjusted to 5.7 using 0.1 M HCl or NaOH.ProcedurePreparation and sterilization of growing medium (when not provided pre-poured)These steps will make 1 L of growth medium which is enough to prepare about 65 growing tubes.1.Dissolve the MS mixture in about 800 ml of distilled water. Stir thewater continuously while adding the salt mixture. Add 30 g sugar andstir to dissolve. Adjust pH to 5.8 using 1M NaOH or 1M HCl as necessarywhile gently stirring. Add distilled water to make the total volume up to1 L.2.Weigh out 10 grams of agar and add it to the MS solution. Heat thesolution gently while stirring until all the agar has dissolved.3.Pour the still warm medium into the polycarbonate tubes to a depth ofabout 4 cm which will use about 15ml of media per tube.4.Place the tubes (with lids sitting on the tubes but not tightened) in apressure cooker and sterilize for 20 minutes. Cool the pressure cooker, then remove the tubes and tighten the lids. Alternatively the tubes can be placed in boiling water for 30 minutes, but make sure that none ofthe water is able to enter the tubes.NOTE: If you wish to use plants other than cauliflower you need to prepare two different media which contain plant hormones necessary to stimulate development of differentiated tissues. The first one should contain a cytokinin such as BAP which promotes shoot formation and the second one a rooting hormone such as NAA or store bought rooting hormone. To do this, prepare the mixture up until the end of step 2. Keeping the mixture warm so that it does not solidify, divide it equally into two pre-warmed containers. Each container can be used to prepare 30 or so tubes as above. The first container should have BAP added at the rate of 2.0mg/l. The second container should have the NAA hormone added at the rate of 0.1 mg/L. To do this it is necessary to make concentrated solutions of both BAP (2.0mg/ml) and NAA (1.0mg/ml) and filter sterilize them. Add 1ml of the concentrated BAP stock or 100μl of the NAA concentrated stock to each 1 liter of media that you prepare. If you use rooting hormone that is purchased from your local hardware or nursery supply store instead of NAA then just follow the directions before adding to your media.Preparation of a sterile transfer chamber and equipmentA classroom transfer chamber can be made from a clean glass aquarium turned on its side. Scrub the aquarium thoroughly with a 30% bleach solution, makingsure that you wear gloves and do not inhale the fumes. Rinse with sterile distilled water, turn upside down on a clean counter or paper towels and allow to dry. Cut holes in a clean plastic sheet to allow arms to reach into the chamber and reinforce the cut edges with tape if necessary. Tape the clean plastic sheet over the open side of the aquarium making sure that the arm holes are located at a convenient height. Plastic sleeves could also be fitted to these holes if you wish to make it easier to prevent the entry of airborne spores into the chamber. The finished aquarium chamber can be sterilized by spraying with 10% chlorox bleach just prior to each use and drying with sterile paper towel.Wrap the forceps, scalpels, razor blades, paper towel and gloves (rubber or surgical) in aluminum foil, seal with tape and sterilize by processing them in a pressure cooker for twenty minutes. These items can also be sterilized by placing in an oven at 350o F for 15 minutes. You can wrap each item separately or put together a "kit" so that each student will have their own sterile equipment to use.Alternatively the forceps and blades can be sterilized by dipping in 10% bleach and then rinsing in sterile water, or dipping in alcohol and then placing in a flame, although this is not recommended for use in crowded classrooms. If you choose to dip in bleach and rinse in sterile water, it is best if fresh solutions are available for each 3-4 students since the water can easily be contaminated if care is not used. These liquid containers should only be opened once they are inside of the sterile chamber.Plant preparationYour plant material must first be surface sterilized to remove any bacteria or fungal spores that are present. We aim to kill all microorganisms, but at the same time not cause any adverse damage to the plant material.1.Cauliflower should be cut into small sections of florets about 1 cmacross. If using a rose or other cuttings, cut the shoots into about 5 to 7 cm lengths. Whole African violet leaves can also be used.2.Wash the prepared plant material in a detergent-water mixture forabout 20 minutes. If trying hairy plant material scrub with a soft brush (toothbrush). This will help remove fungi etc., and the detergent willhelp wet the material and remove air bubbles that may be trappedbetween tiny hairs on a plant.3.Transfer the washed plant material to the sterilizing chloroxsolution. Shake the mixture for 1 minute and then leave to soak for 10-20 minutes. Carefully pour off the bleach solution using the lid to keepthe plant tissue from coming out and then carefully cap the container.Note 1: At this point, the tissue is considered sterile. All subsequent rinses should be done with sterile water, and all manipulations of the tissue performed with sterile instruments and supplies. Open one container at a time and never leave the lid off of any container longer thannecessary.Note 2: Many students will not fully appreciate the importance of carefully sterilizing explants and so there will be some cultures that become infected with bacterial or fungal growth. If you do not wish to emphasize this aspect of the laboratory students can be provided with plant materials that the instructor has already sterilized prior to use by the class.Transfer of plant material to tissue culture mediumUse the sterile gloves and equipment for all of these steps.1.Place the plant material still in the chlorox bleach sterilizing container,the containers of sterile water, the sterilized forceps and blades, somesterile paper towel to use as a cutting surface and enough tubescontaining sterile medium into the sterile aquarium. The outsidesurfaces of the containers, the capped tubes and the aluminum wrapped supplies should be briefly sprayed with 70% alcohol before moving them into the chamber.2.The gloves can be sprayed with a 70% alcohol solution and hands rubbedtogether to spread the alcohol just prior to placing hands into thechamber. Once students have gloves on and sprayed they must nottouch anything that is outside of the sterile chamber.3.Carefully open the container with the plant material and pour in enoughsterile water to half fill the container. Replace the lid and gently shakethe container to wash tissue pieces (explants) thoroughly for 2-3 minutes to remove the bleach. Pour off the water and repeat the washingprocess 3 more times.4.Remove the sterilized plant material from the sterile water, place onthe paper towel or sterile petri dish. Cut the cauliflower into smallerpieces about 2 to 3 mm across. If using rose cut a piece of stem about 10 mm in length with an attached bud. The African violet leaf can be cutinto small squares about 1-1.5 cm across. Be sure to avoid any tissuethat has been damaged by the bleach, which is apparent by its' palecolor.5.Take a prepared section of plant material in sterile forceps and placeinto the medium in the polycarbonate tube. Cauliflower pieces shouldbe partly submerged in the medium, flower bud facing up. Rose orother cuttings should be placed so that the shoots are level with themedium surface. The African violet leaf pieces should be laid directlyonto the medium surface.6.Replace the cap tightly on the tube.Figure 1: The small explantdevelops callus which thenproduces shoots a few weeksafter being placed into tissueculturemediaGrowing the plants1.The tubes containing plant sections may be placed in a well-lit area ofthe classroom although not in direct sunlight. The shoots will probablygrow more quickly if the explants are placed under fluorescent or grow-lights to provide at least 12 hours of light per day. The aquarium can be used as a growth chamber with the lighting about 8-10" overhead. Thiswill also help maintain a more regular and warm temperature. Ensurethat the temperature does not go over 28o C. New shoots should develop within 2 weeks, and should be well advanced in 3 to 4 weeks. Check the tubes daily and discard any that show signs of infection (beforediscarding first sterilize in the pressure cooker or add bleach into thetube).2.Roots can appear within 6 weeks on cauliflowers. The roses, Africanviolet and other cuttings will need to be moved into rooting media forroots to properly develop. This transfer to the second, rooting mediamust be conducted under the same sterile conditions as at the initiation of the culture. All necessary equipment and the aquarium should be set up as before and properly sterilized.3.Working inside the sterile aquarium chamber, remove the cap from theculture tube. There will usually be several shoots that have arisen from each explant. These shoots should be carefully separated by gentlyremoving the whole explant from the media with sterile forceps andthen separating the shoots by gently pulling them apart using two pairsof forceps. Each shoot should then be placed into a tube of rootingmedia and the bottom of the shoot pushed into the media so that goodcontact is made. The cap is replaced and the shoots are then allowed to grow as in step 1 until roots are formed, usually within 2-3 weeks. Potting the clonesOnce roots are well formed the plants are ready to be transferred into soil.Figure 2:Roots are fully developed prior to moving plants to pots of soil1.Each plant should be carefully removed from its tube of media andplanted into a small pot containing a clean light potting mix. Gentlywash off all the agar medium prior to planting. The plants will still need to be protected at this stage since they are not accustomed to the drier air of the classroom when compared to the moist environment of thetube of media.2.Place all of the pots onto a tray and cover lightly with a plastic dome ortent. Place the plants in an area with 12-16 hours of light (either natural or artificial) but not direct sunlight.3.After a week the cover can be gradually removed and the plantsacclimated to stronger light and drier atmospheric conditions.4.You now have a collection of plants in your classroom that aregenetically exactly the same. You could use these plants to carry outother experimental tests knowing that one of the main variables in theexperiment has been eliminated. Some of these tests could includelooking at plant responses to low light levels, to drought or to saline soil conditions. (see activity 7)Student Activity1. Tissue culture uses a small piece of tissue from a mother plant togrow many new copies of the original plant. What is the term usedto refer to this small piece of tissue?2. What are some of the plants that we might use for tissue culture?3. Why is tissue culture used for propagation of some plants rather thanjust planting seeds?4. What is a sterile environment?5. Why is a sterile environment important in tissue culture?6. How did you or your teacher sterilize the instruments that were usedin this tissue culture activity?7. Could we sterilize the plant tissue in the same manner? Why or whynot?8. What happens if you open your sterile plant container when it is notinside a sterile environment?。

组织培养复习资料

植物组织培养plant tissue culture是指将植物的离体器官、组织、细胞以及去除细胞壁的原生质体，甚至幼小的植株，放在无菌条件下，在人工控制的环境条件下，培养在培养基上对其进行克隆，使其生长、分化并成长为完整植株的过程。

外植体explant用于培养的植物胚胎、器官、组织、细胞和原生质体通常称为外植体。

无菌asepsis是进行组织培养的基本要求，指培养的器皿，器械，培养基，外植体等处于无真菌，细菌，病毒等有害生物状态，以保证外植体在培养器皿中正常生长和发育。

植株培养plant culture对完整植株材料的离体无菌培养。

胚胎培养（embryo culture）从果实或种子子房中国分离出来成熟或未成熟的胚进行离体无菌培养的技术。

器官培养organ culture以植物的根茎叶花果实等器官为外植体的离体无菌培养技术。

细胞培养cell culture 在离体条件下对单个细胞或小的细胞团进行培养并使其增殖的技术) 对植物器官或愈伤组织上分离出的单细胞，花粉单细胞，或很小的细胞团进行培养，形成单细胞无性系或再生植株的技术。

原生质体培养protoplast culture指将植物细胞的细胞壁通过物理或化学的方法去除，然后再进行培养。

原生质体培养的最大用处是体细胞杂交。

初代培养primary culture将外植体进行的第一次培养，称为初代培养。

继代培养subculture组织培养中，外植体或培养物培养一段时间后，为了防止培养的细胞老化，或培养基养分利用完而造成营养不良及代谢物过多积累二产生的毒害的影响，要技术将其转接到新鲜的培养基中继续进行培养，使其能够顺利地增殖、生长、分化，成长完整的植株。

这一过程称为继代培养。

植物离体培养plant culture in vitro由于外植体已脱离了母体，因此植物组织培养又称为植物离体培养。

组织培养tissue culture是对植物体的各部分组织，或对植物器官培养产生的愈伤组织进行培养。

组织培养育苗

56
培养基的配制
水准备母液混合定容
糖
调pH 加入琼脂加热溶解
培养器皿清洗干燥分装封口
高压蒸汽灭菌冷却凝固
培养基配制程序
培养基具体配制操作过程：P21
接种
57
组培实验基本操作技术
器皿和器具的洗涤 P19
1 洗涤剂：无磷中性的肥皂粉洗衣粉和洗洁精 2 常用清洗方法洗涤剂清洗法超声波清洗法
胡萝卜形成层分生组织培养示意图
台湾百合离体培养
大蒜愈伤组织培养
直接器官发生
间接器官发生
体细胞胚胎发生
胚培养
败育
2离体培养体系的建立
供体植物材料的确定
外植体的选择
外植体的接种
外植体的灭菌
外植体的培养
97
常用外植体的种类
茎尖带节茎段节间部茎段叶片和叶柄鳞片
98
培养基配制
移栽
获取外植体
无菌接种
脱分化
诱导愈伤组织的形成
组织培养步骤
试管苗的形成再分化扩大培养
植物组织培养的过程可概括为：
脱分化
再分化根茎叶
外植体愈伤组织
或
胚状体
完整植物
⊙ 茎尖茎段胚及子房等器官做外植体可不经脱分化直接形成试管苗
⊙植物组织培养原理：一是细胞的全能性；二是再生作用;与植物体分离的器官具有恢复完整植株的能力
组培苗经人工炼苗后移栽到驯化苗床上使之适应露地或保护地条件的过程
78
79
80
接种时在近火焰处打开瓶口;使瓶倾斜;以免空气中微生物落入瓶中
正确
错误
整个接种操作应在近火焰处进行;且动作要迅速

植物组织培养技术最新版本

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一、培养基的组成和配制法
1 培养基的成分（1）无机营养物（2）有机物质（3）植物生长刺激物质（4）其它附加物（5）其它对生长有益的未知复合成分：如椰子汁、酵母提取液、麦芽浸出液等。
培养基中如加入0．5～1％的琼脂即为静止培养的固体培养基，否则为悬浮培养的液体培养基。不同植物材料常需要改变配方，如维持生长和诱导细胞分裂和分化的培养基配方就不同，因此配方的种类很多，目前以Ms （Murashige and Skoog）培养基配方为最常用的一种基本培养基，它利于一般植物组织和细胞的快速生长。
.ห้องสมุดไป่ตู้
番茄无菌苗愈伤组织培养
1 培养基配制 2 接种和培养
接种－－在无菌条件下，将灭过菌的材料，经适当切割，转移到培养基上。
.
植物组织培养技术
植物组织培养（plant tissue culture）：是指在无菌条件下，将离体的植物器官（如根、茎、叶、茎尖、花、果实等），组织（如形成层、表皮、皮层、髓部细胞、胚乳等），细胞（如大孢子、小孢子、体细胞等）以及原生质体，在人工控制的环境里培养成完整植株的一门生物学技术。
.
三、选材和灭菌方法
从低等的藻类到苔藓、蕨类、种子植物等高等植物的各类、各部分都可采用作为组织培养的材料，一般裸子植物多采用幼苗、芽、韧皮部细胞，被子植物采用胚、胚乳、子叶、幼苗、茎尖、根、茎、叶、花药、花粉、子房和胚珠等各个部分。
由于植物在自然条件下，表面常被霉菌和细菌污染，故材料必须进行灭菌处理。一般用漂白粉溶液（1～10 ％）、次氯酸钠溶液（0．5～10％）、升汞溶液（0．01％）、乙醇（70％）或过氧化氢（3～10％）等处理后，再用无菌水反复冲洗至净，然后在无菌室内，将所取的组织迅速培养在固体培养基上。在适宜的条件下，受伤组织切口表面不久即能长出一种脱分化的组织堆块，称为愈伤组织（Callus）。

植物组培名词解释

植物组培名词解释广义的植物组织培养（plant tissue culture ）:是指在无菌和人工预知的控制条件下，利用适当的培养基，对离体的植物器官、组织或细胞进行离体培养，使其再生细胞或生长、分化成完整植株的技术。

狭义的植物组织培养指对植物的组织（如分生组织、表皮组织、薄壁组织等）及培养产生的愈伤组织（callus）进行离体培养的技术。

外植体:植物组织培养中，使用的各种器官、组织和细胞统称为外植体(explant)。

愈伤组织:是指外植体因受伤或在离体培养时，其未分化的细胞和已分化的细胞进行活跃的分裂增殖而形成的一种无特定结构和功能的组织。

植物细胞的培养:是指将植物材料从母体植株上切取后，分离成细胞或小细胞团，放在无菌的人工环境条件下使其生长发育的方法。

初代培养:根据培养过程，将从植物体上分离下来的部分进行第一次培养，称为初代培养（primary culture ），以后将培养物转移到新的培养基上继续培养，则统称为继代培养(subculture)。

植物细胞全能性是指一个生活的植物细胞，只要有完整的膜系统和细胞核，它就会有一整套发育成一个完整植株的全部遗传信息，在适当的条件下，这些信息可以表达，再生成一个完整植株。

细胞分化(differentiation):是指生物在个体发育过程中细胞由一般变为特殊的现象，即普通形态结构的细胞，向着不同形态结构的方向发展，从而在机能上也各不相同。

脱分化:由失去分裂能力的细胞回复到分生性状态并进行分裂，形成形成一种高度液泡化的呈无定形态的薄壁细胞即愈伤组织的现象（或过程）称为“脱分化”(dedifferent iation)。

由无分化的愈伤组织的细胞再转变成为具有一定结构，执行一定生理功能的细胞团和组织，进而构成一个完整的植物体或植物器官的现象（或过程），叫做“再分化”(redifferentiation)。

细胞学说(cell theory)：一切生物都是由细胞构成的，细胞是生物体的基本功能单位，细胞只能由细胞分裂而来。

植物组织培养

植物组织培养绪论植物组织培养（Plant tissue culture）广义上是指无菌条件下，在特定的培养基上对离体的植物器官、组织、细胞和原生质体甚至包括完整植株进行培养的技术。

外植体：愈伤组织：一、植物组织的主要特征：（1）在培养容器中进行；（2）无菌培养环境，排除了微生物如真菌、细菌以及害虫等的侵入；（3）各种环境因子如营养因子、激素因子以及光照、温度等物理因子处于人工控制之下，并可达到最适条件。

（4）通常打破了正常的植物发育过程和格局；（5）随着单细胞和原生质体培养技术的发展，对植物显微结构进行操作成为可能。

二、植物组织培养类型：根据不同分类的依据可以分为不同类型。

1、根据培养材料不同分为：（1）完整植株培养（Plant Culture）：对幼苗和较大植株等的培养。

（2）胚胎培养（Embryo Culture）：包括成熟胚、幼胚、子房、胚珠等的培养。

（3）器官培养（Organ Culture）：包括离体根、茎、叶、果实、种子、花器官的培养。

（4）组织培养（Tissue Culture）：如分生组织、薄壁组织、输导组织培养。

（5）细胞培养（Cell Culture）：指对单细胞或较小的细胞团进行培养。

（6）原生质体培养（Protoplast Culture）：指对去掉细胞壁后所获得的原生质体进行培养。

三．植物组培的应用1.植物离体快繁。

2.无病毒苗木培养。

3.培育新品种或创制新物种。

4.次生代谢物生产。

5.植物种质资源的离体保存。

6.人工种子。

第一章1.）实验室包括：准备室，无菌操作室，培养室，温室。

2.）器具的灭菌：1、器具灭菌：培养皿、解剖刀、镊子等用品。

（1）干热灭菌：铝箔包好后，放于恒温干燥箱内，150 ℃保温2小时，成本较高。

（2）高压蒸汽灭菌：高压蒸汽灭菌锅内120 ℃15－20分钟。

（3）灼烧灭菌：接种时，解剖刀及镊子等浸入95％乙醇，然后取出在酒精灯火焰上灼烧杀菌。

2、培养基灭菌：采用高压蒸汽灭菌。

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一~三、绪论和上皮组织
epithelial tissue 上皮组织microvillus 微绒毛cilium 纤毛tight junction 紧密连接intermediate junction 中间连接desmosome 桥粒endothelium 内皮hemidesmosome 半桥粒gap junction 缝隙连接junctional complex 连接复合体plasma membrane infolding 质膜内褶*basement membrane 基膜四、固有结缔组织
loose connective tissue 疏松结缔组织fibroblast 成纤维细胞fibrocyte 纤维细胞macrophage 巨噬细胞plasma cell 浆细胞mast cell 肥大细胞
fat cell 脂肪细胞collagenous fiber 胶原纤维*reticular fiber 网状纤维elastic fiber 弹性纤维五、血液
Plasma 血浆Serum 血清Erythrocyte 红细胞Hemoglobin 血红蛋白Monocyte 单核细胞Neutrophil 中性粒细胞Basophil 嗜碱性粒细胞Eosinophil 嗜酸性粒细胞lymphocyte 淋巴细胞hemopoietic stem cell 造血干细胞*reticulocyte 网织红细胞
六、软骨和骨
cartilage tissue 软骨组织osseous tissue 骨组织bone lamella 骨板*osteon 骨单位osteoblast 成骨细胞osteocyte 骨细胞osteoclast 破骨细胞osteoprogenitor cell 骨祖细胞七、肌组织
muscle fiber 肌纤维skeletal muscle 骨骼肌sarcomere 肌节cardiac muscle 心肌sarcoplasmic reticulum 肌质网transverse tuble 横小管myofibril 肌原纤维thick filment 粗肌丝myosin 肌球蛋白troponin 肌钙蛋白triad 三联体* intercalated disk 润盘八、神经组织
nervous tissue 神经组织multipolar neuron 多极神经元Schwann cell 施万细胞myelinated nerve fiber 有髓神经纤维synapse 突触blood brain barrier 血脑屏障neuroglial cell 神经胶质细胞motor nerve ending 运动神经末梢neurofibril 神经原纤维myelin sheath 髓鞘dendrite 树突axon轴突* Nissl body 尼氏体
十、循环系统
Endocardium 心内膜elastic membrane 弹性膜sinusoid 血窦fenestrated capillary 有孔毛细血管*Purkinje fiber 浦肯野纤维
十一、免疫系统
thymic corpuscle 胸腺小体splenic cord 脾索thymocyte 胸腺细胞diffuse lymphoid tissue 弥散淋巴组织lymphoid nodule 淋巴小结blood -thymus barrier血液-胸腺屏障thymus epithelial cell 胸腺上皮细胞medulla 髓质
* splenic sinus 脾窦white pulp 白髓periarterial lymphatic sheath 动脉周围淋巴鞘
十二、内分泌系统
follicular epithelial cell 滤泡上皮细胞zona glomerulosa 球状带parafollicular cell 滤泡旁细胞zona reticularis 网状带chromaffin cell 嗜铬细胞zona fasiculata 束状带pars distalis 远侧部* Herring body 赫令体十三、消化管
zymogenic cell 胃酶细胞absorptive cell 吸收细胞fundic gland 胃底腺goblet cell 杯状细胞parietal cell 壁细胞small intestinal gland 小肠腺microfold cell 微皱褶细胞surface mucous cell 表面黏液细胞intestinal villus 肠绒毛Paneth cell 帕内特细胞
mucus-bicarbonate barrier 黏液-碳酸氢盐屏障
*intracellular secretory canaliculus 细胞内分泌小管
十四、消化腺
serous acinus 浆液性腺泡hepatic sinusoid 肝血窦
portal area 门管区pancreas islet 胰岛
hepatic lobule 肝小叶bile canaliculus 胆小管hepatocyte 肝细胞fat-storing cell 贮脂细胞
Kupffer cells 库普弗细胞* perisinusoidal space 窦周隙
十五、呼吸系统
terminal bronchiole 终末细支气管alveolar sac 肺泡囊pulmonary alveoli 肺泡alveolar septum 肺泡隔respiratory bronchiole 呼吸性细支气管alveolar duct 肺泡管
* blood-air barrier 气血屏障type Ⅱalveolar cell Ⅱ型肺泡细胞十六、泌尿系统
renal corpuscle 肾小体renal capsule 肾小囊nephron 肾单位renal glomerulus 肾小球juxtaglomerular complex球旁复合体macula densa 致密斑extraglomerular mesangial cell 球外系膜细胞
collecting duct 集合管proximal tubule 近端小管distal tubule 远端小管*filtration barrier 滤过屏障
十七、皮肤
Epidermis 表皮Keratinocyte 角质形成细胞stratum basale 基底层sebaceous gland 皮脂腺sweat gland 汗腺keratohyalin granule 透明角质颗粒* melanocyte 黑素细胞
十八、眼与耳
Retina 视网膜cone cell 视锥细胞
iris 虹膜ciliary body 睫状体membranous labyrinth 膜迷路central fovea 中央凹* spiral organ 螺旋器Cornea 角膜
十九、男性生殖系统
seminiferous tubule 生精小管spermatogonium 精原细胞spermatid 精子细胞Blood-testis barrier 血睾屏障Spermatogenesis 精子发生Spermiogenesis 精子形成Leydig cell 莱氏细胞Sertoli cell 足细胞
* acrosome 顶体
二十、女性生殖系统
Oocyte 卵母细胞Menstruation 月经
zona pellucida 透明带corona radiate 放射冠
ovulation 排卵endometrium 内膜
menstrual cycle 月经周期Primordial follicle 原始卵泡
*corpus luteum 黄体。

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