外源腐胺对石刁柏愈伤组织胚性能力的影响

石刁柏胚性愈伤组织继代过程中,添加浓度为10 mg·L^-1的外源腐胺能有效地保持愈伤组织的胚胎发生能力,并减少愈伤组织的褐化,但不能提高愈伤组织的体细胞胚的诱导率.腐胺处理过的胚性愈伤组织的内源腐胺含量明显提高.这可能是外源腐胺保持细胞胚性、降低褐化程度的原因.     

烟草叶片愈伤组织形成过程中的细胞程序性死亡

以烟草叶片在MS培养基上诱导的10个不同时期的愈伤组织为材料,通过荧光染色显微观察到细胞核在形态上发生了缩缢变形,DNA Ladder检测也出现了标志细胞程序性死亡特征的DNA片断,表明了愈伤组织形成过程中发生了细胞程序性死亡（programmed cell death, PCD）,而在诱导的第六个时期（13 d）DNA的脱尾现象最为明显,表明这一时期是PCD发生较为活跃的时期。利用RT-PCR法在诱导的前九个时期,即诱导的第3、5、7、9、11、13、15、17、19 d的叶片的愈伤组织中均扩增出了细胞程序性死亡相关Beclin1基因的cDNA片段(678 bp),Northen杂交结果显示,在诱导的第13 d,类Beclin1基因表达较强。通过酶联免疫法测定诱导前14 d叶片中内源激素ABA的含量变化,在诱导的第六时期ABA含量相对达到了峰值,结果表明：在烟草叶片愈伤组织诱导过程中伴随PCD的发生,在此过程中内源激素ABA与Beclin1基因起到了调节作用。     

柑橘愈伤组织倍性变化及其与体细胞胚胎发生能力的关系

柑橘愈伤组织是细胞融合、遗传转化等生物技术研究的良好材料。本实验室通过近20年的工作,先后诱导和保存了近80份不同种类和品种的柑橘愈伤组织。这些愈伤组织在MT基本培养基上继代保存。在长期保存过程中,部分材料仍然保持较好的胚胎发生能力。为研究愈伤组织在继代过程中的染色体变异趋势以及这种变异对胚胎发生能力的影响,本文选择有代表性的35份材料作为试材,在4年中连续3次使用流式细胞仪,对细胞DNA含量进行测定。结果发现,待测的35种愈伤组织均存在部分细胞DNA含量加倍的现象,而且,随着时间的推移,除佩奇橘柚、沙漠蒂甜橙、鲁斯脐橙、印度酸橘等4种愈伤组织的DNA含量加倍的细胞有减少的趋势外,其他愈伤组织的DNA含量加倍的细胞均有增加的趋势。数据分析表明,在待测的35种愈伤组织中,有71．4％的基因型的愈伤组织细胞出现了多倍体细胞增加的趋势。对这35种柑橘愈伤组织进行体细胞胚诱导,发现只有9种愈伤组织具有不同程度的胚胎发生能力,其他26种愈伤组织不能再生。相关性分析表明,柑橘愈伤组织细胞DNA含量增加的细胞比例与体细胞胚胎发生能力之间的相关性较小,相关系数为忙-0．10（P〈0．01）,未达到显著水平。另外,柑橘胚性愈伤组织继代时间与体细胞胚胎发生能力之间的相关性也不显著。     

甘蔗胚性细胞团和愈伤组织某些性质的比较

在培养24天的生长周期中,胚性细胞团的分裂细胞数共出现三次高峰。第一次出现在继代后的第8天,第二次出现在第12天,第三次出现在第18天,其中以第二次高峰期的细胞分裂数量最多。愈伤组织中仅观察到两次高峰,第一次出现在第12天,第二次出现在第18天。细胞分裂数比胚性细胞团少。细胞分裂高峰出现得迟可能愈伤组织比胚性细胞团生长缓慢的一个重要因素。 不同细胞分裂高峰时期胚性细胞团的DNA和蛋白质均比相同时期的愈伤组织含量高,高峰期在第12和第18天,这与内部的DNA和蛋白质含量变化是一致的,这说明胚性细胞团具有较快分裂能力的原因之一。胚性细胞团的过氧化物酶同工酶带也多于愈伤组织,酶带染色较深,表示酶活性高。     

早熟棉体细胞胚胎发生和植株再生体系的建立

以8个早熟或特早熟棉花为材料,通过不同激素组合(1.0 mg/L IBA+0.5 mg/L KT;0.1 mg/L 2,4-D+0.1 mg/L KT)诱导其愈伤组织,为早熟棉花的遗传转化以及基因功能研究奠定基础.结果表明,2种激素组合均能诱导产生4种主要类型的愈伤组织:淡黄色颗粒状愈伤组织,褐化的愈伤组织,翠绿致密的愈伤组织,疯长型愈伤组织.4种愈伤组织转入增殖培养基中前2种愈伤组织能够分化出胚性愈伤组织,胚性愈伤组织再转到分化培养基上培养能够产生胚状体,即体细胞胚;体细胞胚进一步发育成为再生小苗.用该组织培养再生系统,成功地使晋棉5号、中棉27号和辽棉10号等3个早熟棉品种在5～7个月内通过体细胞胚胎分化获得再生小苗.     

枸杞髓组织培养中体细胞胚胎发生与过氧化物酶同工酶分析

枸杞髓组织在 MS+6 - BA0 .1mg/ L+NAA0 .5mg/ L培养基上诱导愈伤组织发生。在 MS+6 - BA0 .1mg/ L+NAA0 .5mg/ L+CH50 0 mg/ L培养基上继代培养 ,再转入 MS+6 - BA2 mg/L +NAA 0 .5mg/ L的分化培养基上进行分化培养。显微观察表明 ,在培养过程中愈伤组织细胞由非胚性细胞转变为胚性细胞 ,直至发育成体细胞胚胎和完整植株 ;电泳结果显示 ,体细胞胚胎发生的各阶段 ,其过氧化物酶同工酶发生相应的变化。     

采用低pH值和适量的玉米素、ABA配合恢复褐化的水稻愈伤组织

在建立水稻体细胞悬浮培养体系的实验中,我们观察到当将胚性愈伤组织从固体培养基转入液体分化培养基进行悬浮培养并去掉2.4-D 时,随着培养时间的延长,愈伤组织往往出现褐化现象。与这种情况类似的是,当将悬浮培养的愈伤组织和成熟胚状体转入固体培养基时,也出现严重的褐化现象。这种褐化现象一般认为是不可逆的,伴随着胚性愈伤组织和成熟胚状体的褐化,胚性愈伤组织逐渐丧失分化能力,成熟胚状体则逐渐死亡。     

常温条件下三倍体毛白杨的愈伤组织诱导和保存

在MS附加 0 .5mg·L-16 BA和 0 .15mg·L-1NAA的培养基上 ,三倍体毛白杨幼叶的愈伤组织诱导率可达 87.6 %。蔗糖浓度大小与愈伤组织增长量成正比 ,与褐变开始时间成反比 ,2 %蔗糖下 ,继代周期可长达 6 1d。每日光照 12h的愈伤组织褐化时间可延长至 82d ;少于 12h ,愈伤组织松散 ,褐变开始时间缩短。愈伤组织开始褐化后 ,再分化时间延长 ,分化率降低。不同继代周期对再生试管苗生根无影响。     

烟草愈伤组织继代培养与分化期间核酸代谢的比较研究

柳叶烟草愈伤组织在分化和芽原基形成期间,DNA 和RNA 含量均高于继代培养物;在芽原基形成后和幼芽生长期间(12天以后),DNA和RNA 含量持续上升,而同期继代培养物巳进入生长静止期,DNA 和RNA 含量基本不变或略有下降。根据RNA 电泳结果还进一步分析了两种愈伤组织培养物各RNA 组分变化与总RNA 含量变化的关系。分化培养物在芽原基形成时有明显升高的RNase 活性峰和持续上升的RNA 合成速率;而此时期继代培养物的RNase 活性及RNA 合成能力均较低;分化愈伤组织的DNA 合成速率在幼芽生长期间仍维持上升趋势,且显著高于同期继代愈伤组织的合成速率。这些结果表明,烟草愈伤组织分化培养物比继代培养物有更旺盛的核酸代谢能力。     

甘蔗原生质体的体细胞胚胎发生

用甘蔗(台糖134)花粉植株叶外植体产生的愈伤组织,作为分离原生质体的材料。原生质体以液体浅层培养的方式培养在修改的 MS 培养基上,经培养后一周内,观察到第一次细胞分裂,约5—6周后,形成了愈伤组织。将胚性细胞团组成的愈伤组织转移到除去或降低2,4-D浓度,但含有 BA 的分化培养基上,约2—3周后,有的愈伤组织发育了胚芽鞘,另一些愈伤组织分化出胚根或根。系统观察了这些原生质体在分裂和愈伤组织形成过程中的体细胞胚胎发生。     

不同基因型玉米的再生能力和胚性与非胚性愈伤组织DNA的差异

研究了细胞分裂素在玉米愈伤组织诱导和植株再生中的作用,结果表明低浓度（０ ．２ ｍｇ／Ｌ） 的细胞分裂素能促进玉米幼胚诱导的愈伤组织再生,６ＢＡ 的效果比ＫＴ更好。不同品种的玉米幼胚诱导的愈伤组织的再生能力差异显著,普甜１ 号和苏玉１ 号的再生频率高达７８ ％ 和７５ ％ ,糯玉米和掖单９ 号仅为１０ ％ 和８ ％ 。植株再生途径也有所不同,普甜１ 号以器官发生为主要途径,苏玉１号则以体胚发生途径为主。经长期继代的愈伤组织失去再生能力,通过ＲＡＰＤ 方法比较发现胚性与非胚性愈伤组织的基因组之间存在差异,说明组织培养过程中愈伤组织的ＤＮＡ 发生了变异。     

AgNO3对橡胶树花药愈伤组织形态及体胚发生的影响

橡胶树的花药愈伤组织在长期继代过程中,胚性易下降甚至丧失;而AgNO3作为乙烯活性抑制剂,被广泛应用于植物组织培养中.该研究以继代培养4 a以上的热研7-33-97花药愈伤组织为材料,在继代培养基中添加2.5 mg·L-1 AgNO3预培养35 d后,观察预培养前后愈伤组织表形及其细胞形态的变化,并设计不同浓度AgNO3及不同处理时间对其进行体胚诱导,90 d后分别统计胚状体总数和正常胚数.结果表明:浅黄色质地柔软的愈伤组织在含AgNO3的培养基上预培养后能转变成鲜黄色易碎愈伤组织,在倒置显微镜下前者大多表现为不规则多边形,细胞内含物较稀薄;而后者则呈圆形或椭圆形,细胞内含物丰富,属于典型的胚性细胞.在体胚诱导的第1个月添加5 mg·L-1 AgNO3能显著促进体胚的发生,AgNO3浓度升至10 mg·L-1时体胚发生受到抑制,且畸形胚的形成率显著增加;在含5 mg·L-1 AgNO3的培养基中培养2个月以上,体胚发育明显受阻,大部分形成畸形胚.该研究结果在一定程度上恢复了橡胶树长期继代花药愈伤组织的胚性能力,并提高了其体胚发生频率,为橡胶树花药胚性愈伤组织长期继代培养过程中胚性的保持提供了参考.     

Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8–10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.     

Programmed cell death during embryogenesis in maize

Programmed cell death (PCD) in plants is considered an integral part of development. Evidence of DNA fragmentation, occurring at specific sites and times during embryo formation in maize (Zea mays L.), was obtained using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labelling (TUNEL) and by genomic DNA ladder detection. During the crucial period of elaboration of the primary shoot and root axis (14-20 d after pollination), TUNEL-positive nuclei are present in the scutellum, coleoptile, root cap and principally in the suspensor. Additional evidence of a form of programmed cell death occurring in these tissues comes from the detection of a DNA ladder. Upon completion of the differentiation process, all embryonic cells are TUNEL-negative, indicating that possible programmed cell death events during maize embryogenesis are confined to structures or organs that do not contribute to the adult plant body.     

渗透胁迫诱导的小麦叶片细胞程序性死亡

用20%PEG6000（-0.63MPa）溶液对小麦（Triticumaestivum）根系进行渗透胁迫,在DNA琼脂糖凝胶电泳图谱上观察到明显的梯状DNA条带,表明PEG处理诱发了DNA核小体间的断裂,从而表现出典型的细胞程序性死亡的生化特征;末端脱氧核糖核酸转移酶介导的3′-OH末端标记法（terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end     

Somatic Embryogenesis and Plantlet Formation of Picea wilsonii Mast. in Ditterent Conditions

Embryogenic callus (translucent callus) was produced from immature zygotic embryos of Picea wilsonii Mast. Subsequently somatic embryogenesis occurred on the brown callus. The somatic embryos could be stimulated to developinto plantlets on the medium without hormone. Young somatic embryos were produced from embryogenic callus in liquid suspension culture, in which suspensor was several or more than ten times the size of the somatic embryo. The somatic embryo showed very similar to zygotic embryos in micro-section and living material.     

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

Summary This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.     

The level of free intracellular zinc mediates programmed cell death/cell survival decisions in plant embryos

Zinc is a potent regulator of programmed cell death (PCD) in animals. While certain, cell-type-specific concentrations of intracellular free zinc are required to protect cells from death, zinc depletion commits cells to death in diverse systems. As in animals, PCD has a fundamental role in plant biology, but its molecular regulation is poorly understood. In particular, the involvement of zinc in the control of plant PCD remains unknown. Here, we used somatic embryos of Norway spruce (Picea abies) to investigate the role of zinc in developmental PCD, which is crucial for correct embryonic patterning. Staining of the early embryos with zinc-specific molecular probes (Zinquin-ethyl-ester and Dansylaminoethyl-cyclen) has revealed high accumulation of zinc in the proliferating cells of the embryonal masses and abrupt decrease of zinc content in the dying terminally differentiated suspensor cells. Exposure of early embryos to a membrane-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine led to embryonic lethality, as it induced ectopic cell death affecting embryonal masses. This cell death involved the loss of plasma membrane integrity, metacaspase-like proteolytic activity, and nuclear DNA fragmentation. To verify the anti-cell death effect of zinc, we incubated early embryos with increased concentrations of zinc sulfate. Zinc supplementation inhibited developmental PCD and led to suppression of terminal differentiation and elimination of the embryo suspensors, causing inhibition of embryo maturation. Our data demonstrate that perturbation of zinc homeostasis disrupts the balance between cell proliferation and PCD required for plant embryogenesis. This establishes zinc as an important cue governing cell fate decisions in plants.     

Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in Arabidopsis

Somatic embryogenesis requires auxin and establishment of the shoot apical meristem (SAM). WUSCHEL ( WUS ) is critical for stem cell fate determination in the SAM of higher plants. However, regulation of WUS expression by auxin during somatic embryogenesis is poorly understood. Here, we show that expression of several regulatory genes important in zygotic embryogenesis were up-regulated during somatic embryogenesis of Arabidopsis. Interestingly, WUS expression was induced within the embryonic callus at a time when somatic embryos could not be identified morphologically or molecularly. Correct WUS expression, regulated by a defined critical level of exogenous auxin, is essential for somatic embryo induction. Furthermore, it was found that auxin gradients were established in specific regions that could then give rise to somatic embryos. The establishment of auxin gradients was correlated with the induced WUS expression. Moreover, the auxin gradients appear to activate PIN1 polar localization within the embryonic callus. Polarized PIN1 is probably responsible for the observed polar auxin transport and auxin accumulation in the SAM and somatic embryo. Suppression of WUS and PIN1 indicated that both genes are necessary for embryo induction through their regulation of downstream gene expression. Our results reveal that establishment of auxin gradients and PIN1-mediated polar auxin transport are essential for WUS induction and somatic embryogenesis. This study sheds new light on how auxin regulates stem cell formation during somatic embryogenesis.