地锦体细胞胚胎发生研究

以地锦幼胚为外植体,接种在附加6 BA2mg·L-1及2,4 D0～4mg·L-1的改良B5培养基中诱导愈伤组织,愈伤诱发率0～100% 对诱导出的愈伤组织采用6 BA和2,4 D不同浓度组合的培养基进行继代,对能稳定继代的愈伤组织,以改良B5为基本培养基进行分化培养,采用L16(44)正交设计,研究6 BA、NAA、AC及LH四因素不同浓度组合对体细胞胚胎发生的影响。结果表明:适宜地锦幼胚体细胞胚胎发生的培养基为:愈伤组织诱导:改良B5 6 BA2 0mg·L-1 2,4 D2 0mg·L-1;愈伤组织继代:改良B5 6 BA2 0mg·L-1 2,4 D1 0mg·L-1;胚状体分化及萌发:改良B5基本培养基。胚状体诱发率33%,成苗率53%,移栽成活率85%。胚状体为外起源发生方式,经球形胚、心形胚、鱼雷形胚、子叶形胚等阶段发育成熟。     

银杏原生质体制备及其融合研究

以银杏品种湖南梅核成熟胚进行组织培养得到的无菌苗和愈伤组织为材料,用不同酶液处理进行原生质体制备,并用PEG法进行原生质体融合实验。结果显示,以2.0%纤维素酶+1.0%果胶酶+5.0mmol/LMES+6mmol/LCacl2+0.6mol/L甘露醇( 号酶液)酶解银杏组织、方法1提取原生质体效果最好,其最高原生质体产率为9.4×107个/g;以17%蔗糖溶液处理得到原生质体质量最好。用40%PEG6000+0.3mol/L钙离子+pH9.5液体处理原生质混合液,银杏原生质体融合率最高,为67%。     

桉树原生质体分离体系建立研究初报

以雷林一号桉及尾叶桉为对象进行了原生质体分离体系建立研究,结果表明：雷林一号桉及尾叶桉胚性愈伤诱导的最适应培养基分别为：Ｂ５＋２,４－Ｄ２．０ｍｇ／Ｌ＋ＫＴ０．８ｍｇ／Ｌ及Ｂ５＋２,４－Ｄ２．０ｍｇ／Ｌ。胚性愈伤继代培养基为：Ｂ５＋２,４－Ｄ０．５～１．０ｍｇ／Ｌ＋ＫＴ０．５ｍｇ／Ｌ。黑暗下３～５天的预培养对愈伤组织上的褐化有明显的抑制作用。雷林一号桉及尾叶桉原生质体分离的最适酶液组合分别为：纤维素酶２％＋离析酶１％＋崩溃酶０．１％;纤维素酶２％＋离析酶１％＋崩溃酶０．０５％。愈伤组织在０．９ｍｏｌ／Ｌ蔗糖溶液中浸泡１ｈ,并于酶解后４ｈ更换一次酶液有利于原生质体产量与活力的提高。淡黄色松散愈伤组织原生质分离效果较其它质地的愈伤组织好。继代后８～１０天愈伤组织作原生质体的分离材料较其它年龄的愈伤组织合适。     

枣树原生质体分离条件的研究

枣树原生质体分离研究是其原生质体培养与融合、外源遗传物质转化等研究的基础工作．用胚性悬浮培养细胞、细粒状胚性愈伤组织、未细切愈伤组织和已细切愈伤组织等４种材料进行原生质体分离．结果表明,枣树胚性悬浮培养细胞是最佳起始材料,用浓度为１０ｇ／Ｌ纤维素酶＋１ｇ／Ｌ离析酶＋ＣＰＷ盐（１３２０ｍｇ／ＬＣａＣｌ２·２Ｈ２Ｏ和１００ｍｇ／ＬＫＨ２ＰＯ４·Ｈ２Ｏ组成的混合液）＋０．７ｍｏｌ／Ｌ甘露醇组成的混合酶液对起始材料进行１６ｈ的酶解,可获得较高的原生质体产量,且原生质体活力较高．     

狭叶薰衣草愈伤组织诱导及原生质体分离研究

探究薰衣草愈伤组织诱导及原生质体分离的最适条件,为薰衣草品种优化和育种工作提供基础数据和理论依据。以薰衣草幼嫩叶片为原材料,采用化学诱导方法诱导薰衣草脱分化形成愈伤组织,利用酶解法促使原生质体游离;结合染色排除法计算原生质体活力。实验结果表明,当MS培养基中加入2,4-D(1.8mg/L)和6-BA(0.5mg/L)时,薰衣草的出愈率最高,可达88%;愈伤组织在纤维素酶和果胶酶的含量分别为1.8%、0.5%的条件下,酶解原生质体的产量最高,可达1.180×10~5个/g,原生质体活力最高,可达78.05%。本实验获得的高效愈伤组织诱导条件,为后续愈伤组织再生体系研究提供资料支持。建立了原生质体分离体系,稳定的获得了高产量、高活力的原生质体。     

地锦和五叶地锦种间杂交不亲和性初步研究

将地锦和五叶地锦杂交,未能获得杂交种。对人工辅助自交和杂交后,花粉萌发和花粉管生长情况进行了荧光显微观测。观察发现：人工辅助自交后,花粉粒可在各自柱头表面正常萌发,花粉管可伸入花柱内。将地锦和五叶地锦杂交时,多数花粉粒不能在柱头表面萌发,或者花粉管呈卷曲、先端膨大、破裂等异常现象。授粉后2～48h的花柱中,未观察到花粉管。由此推断,地锦和五叶地锦之间的不亲和发生在柱头表面。     

五叶地锦扦插育苗技术

五叶地锦,又名爬山虎、长春藤,为葡萄科地锦属落叶藤本植物。枝蔓长度相对无限,老皮暗土褐色,1、2a生成熟茎黄褐色,幼枝粉绿色。多分枝,其上生有多数短而小的卷须。卷须先端有圆形吸盘,能吸附在其它物体上使茎蔓向上生长。叶互生,具有4～10cm长的叶柄。叶片掌状,五深裂,裂片卵状,先端尖锐。叶缘重锐齿。叶表平滑暗绿色,秋后昼夜温差大时呈红色,是优良的室外垂直绿化材料,可使高层建筑充满生机。秋季叶色变红,可使秋景更加丰富。五叶地锦的繁殖可播种、压条和扦插。播种育苗要求技术严格,施业工艺繁琐,幼苗期长,出圃慢,费工费时。压条繁…     

野葛愈伤组织诱导与不定芽分化

采用MS为基本培养基，附加NAA、6-BA和IAA3种激素．诱导野葛不同外植体愈伤组织形成；再将愈伤组织接种在不同浓度6-BA的MS培养基上．分别在光照和黑暗条件下进行不定芽的诱导。结果表明．野葛幼叶、茎段和顶芽在适宜激素的诱导下均能形成愈伤组织，但不同激素组合其愈伤组织诱导率不同。幼叶愈伤组织诱导的最适培养基为MS NAA1．0mg／L 6-BA1．0mg/L．其诱导率为75％．茎段愈伤组织诱导的最适培养基为MS NAA1．0mg／L 6-BA3．0mg／L IAA0．2mg/L，其诱导率为70％。不同外植体形成的愈伤组织其不定芽的分化诱导率不同．茎段愈伤组织在不同浓度6-BA下均能诱导出不定芽；光照有利于芽的分化．在光培养条件下，茎段愈伤组织不定芽平均诱导率为66．7％。     

人心果原生质体分离初探

以人心果幼嫩叶片组织为材料,研究了酶液组合、酶解时间、酶液中甘露醇含量、pH值、温度等因素对其原生质体分离的影响,结果表明:以3%纤维素酶R-10+0.4%果胶酶Y-23为混合酶液,0.6 mol.L-1甘露醇为渗透压稳定剂,pH 5.6的酶液组合,在28℃的黑暗条件下振荡,酶解时间2 h,分离出的原生质体产量最佳,可达到12.4×106个.g-1。     

油桐叶肉细胞原生质体分离及瞬时转化体系的建立

【目的】探索油桐叶肉细胞原生质体分离的最适条件,建立油桐原生质体的遗传转化体系,使在油桐体内研究自身基因的功能成为可能。【方法】以油桐成熟叶片和组培苗幼叶为材料,通过酶解法成功分离得到油桐叶肉细胞的原生质体并确定最适分离条件。在此基础上,以获得的原生质体为受体系统,建立PEG介导的油桐原生质体基因转化方法。【结果】原生质体分离结果表明,酶解时间对原生质体产量和活性的影响最大,其次是纤维素酶浓度,而离析酶浓度和甘露醇浓度对原生质体产量和活性的影响较小。以成熟叶片为材料分离原生质体的最适条件为纤维素酶浓度1.5%、离析酶浓度1%、甘露醇浓度0.6 mol·L-1、酶解时间12 h,以组培苗幼叶为材料分离原生质体的最适条件为纤维素酶浓度2%、离析酶浓度1%、甘露醇浓度0.7 mol·L-1、酶解时间6 h。为了建立油桐原生质体的瞬时转化体系,通过PEG介导法将拟南芥MGT6基因导入到油桐原生质体中,结果发现MGT6蛋白定位于原生质体质膜,与之前报道的研究结果一致,这表明本研究建立的油桐原生质体转化方法可成功将外源基因导入油桐原生质体并使其表达。【结论】建立油桐成熟叶片和组培苗幼叶叶肉细胞原生质体的高效分离方法,综合考虑取材的便利性和对后续原生质体培养的影响,建议以组培苗幼叶为材料分离原生质体,分离条件为纤维素酶浓度2%、离析酶浓度1%、甘露醇浓度0.7 mol·L-1、酶解时间6 h。在分离得到油桐叶肉细胞原生质体的基础上,本研究建立的PEG介导的原生质体遗传转化方法能以油桐叶肉细胞原生质体为受体,高效地将外源基因导入其中并使外源基因表达。本研究结果不仅可促进油桐基础研究的发展,在通过细胞融合和基因工程手段进行油桐种质创新方面也具有重要意义。     

Nitraria sibirica cell suspension culture:establishment,characterization and application

Nitraria sibirica Pall.is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value.The objectives of this study include induction and multiplication of callus,establishment of a suspension cell line,and isolation of protoplasts from cell suspensions.Murashige and Skoog(MS) medium was used for callus induction from mature seeds of N.sibirica.Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mgL~(-1) 6-benzylaminopurine(6-BA) and 1.0 mgL~(-1) 2,4-dichlorophenoxy(2,4-D) acetic acid.Suspension cultures of N.sibirica were initiated by transferring friable calli to the same liquid multiplication medium.Characterization of the suspension culture was assessed based on fresh mass,dry mass,cell viability and p H value of the culture.A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium,including a lag phase,an exponential growth phase,a stationary phase,and a negative acceleration phase.The effect of factors such as pre-plasmolysis,enzyme combination,enzymolysis time and mannitol concentration,on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures.The maximum yield(9.79 9 106 cells/g) and highest viability(79.97%) of protoplast were reached when approximately 1 g of cell suspension(cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 molL~(-1) mannitol mixture solution,cellulose onozuka R-10 2%(w/v),hemicellulose 0.2%,macerozyme R-10 1%,and pectolyase Y-23 0.5%.Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10(P0.05).     

Isolation of protoplast from callus of Populus euphratica and H fluxes across plasma membrane under NaCl stress

We used callus of Populus euphratica Olive to isolate protoplasts, and H fluxes across plasma membrane were investi-gated. The concentration of enzymes for protoplast isolation, e.g. cellulase, pectolyase, macerozyme, hemicellulase, and sorbitol content, incubation time were systemically studied. High yield and viability of protoplast was achieved after 6-8 hours incubation of P. euphratica callus in enzyme solution containing 1.5% (w:v) cellulase R-10, 0.1% (w:v) pectolyase Y-23, 0.2% (w:v) macerozyme R-10, 0.05% (w:v) hemicellulase and 0.75-0.80 mol·L-1 sorbitol. Non-invasively ion selective microelectrode technique was used to access proton fluxes in the absence and presence of NaCl (20 mmol·L-1). Salt-induced transient net H efflux was observed in the plasma membrane of P. euphratica cells. The shift of H flux response to NaCl shock and the relevance to salt tolerance were discussed.     

<Emphasis Type="Italic">Nitraria sibirica</Emphasis> cell suspension culture: establishment,characterization and application

Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L^?1 6-benzylaminopurine (6-BA) and 1.0 mg L^?1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 × 10⁶ cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L^?1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).     

晚松悬浮细胞系的建立和原生质体的分离

植物悬浮细胞系的建立和原生质体的分离已被广泛地应用于生理学、生物化学、细胞学、遗传学及分子生物学的研究 ,它是生物技术中进行原生质体培养、杂交、基因转移、突变系筛选等项研究的较理想手段[1] 。针叶树在这方面的研究起步虽晚 ,但至今已取得令人瞩目的进展[2 ] 。晚松     

Large electro-fused protoplasts ofPopulus alba selected by a micromanipulator: Techniques and some characteristics of cells and their regenerants

An improved method for selection of large electro-fused protoplasts ofPopulus alba by a micromanipulator was developed. The conditions for electric cell fusion treatment were optimized. For the best result, protoplasts with a cell density of 5 × 10⁵/mL were treated with an alternate current (1 MHz, 200 V/cm) and pulsed with a direct current (2 kV/cm) for 100μs in 2.5 mM CaCl₂ and 0.55 M mannitol. The electo-fused protoplasts were cultured in NH₄NO₃-free Murashige and Skoog’s medium containing 0.6 M of mannitol, 0.09 M sucrose, 1μM of 2,4-dichlorophenoxyacetic acid and 0.1μM of benzyladenine, the same medium used for protoplast culture, but at a very low cell density of 5–10 × 10²/mL in a well of a 96-well culture plate. When cell aggregates derived from individual fused protoplasts were transferred to fresh medium with 0, 0.3 or 0.6 M mannitol, large colonies developed. In the shoot differentiation medium, the reaction of the calluses derived from large fused protoplasts towards the growth regulators differed from the non-fused ones. In medium containing 1μM each of naphthalene acetic acid andN-(2-chloro-4-pyridyl)-N′-phenylurea, growth of callus from electro-fused ones was not reduced by much compared to the control, but shoot differentiation was inhibited. Gibberellic acid (0.1–10μM) was beneficial to shoot regeneration; however, irregularly shaped leaves appeared at high gibberellic acid concentrations. Shoots regenerated were rooted in Murashige and Skoog’s medium containing 4μM of indole-3-butyric acid. Some plantlets obtained had a varied morphology. Based on the characteristics of growth, some cells derived from electro-fused protoplasts appear to be physiologically different from the non-fused one.     

不同酶解条件对金边瑞香幼叶原生质体分离的影响

以室内培养的金边瑞香浅黄绿色、质地幼嫩的叶片为材料,探讨了影响其原生质分离的因素。结果表明:对原生质分离效果影响最大的是纤维素酶浓度和酶解时间;采用纤维素酶质量分数为0.2%、甘露醇质量浓度为0.6 mol.L-1、酶解时间为10 h时原生质分离效果最好,原生质体的产量为2.2×105个.g-1。     

胡杨悬浮细胞原生质体游离及质膜单离子通道测定

该文以胡杨 (Populuseuphratica)悬浮细胞为材料 ,利用 1 0 %纤维素酶“onozuka”R 10、0 0 1%果胶酶Y 2 3、0 15 %离析酶R 10和 0 1%半纤维素酶的混合酶液消化细胞壁 ,得到原生质体 .并利用膜片钳细胞贴附技术分别测到质膜内向和外向单通道电流 .通道电流在不同水平上变化 ,表明并非只有一种类型的通道开放 .树木细胞原生质体的分离以及利用膜片钳测定细胞质膜离子通道的成功实践为深入研究木本植物抗盐的细胞学机制奠定了基础     

Selection for salt and drought tolerance in protoplast- and explant-derived tissue cultures of Colt cherry (Prunus avium x pseudocerasus)

Colt cherry (Prunus avium x pseudocerasus) callus cultures were derived from leaf protoplasts, protoplasts of root cell suspension cultures, or by direct culture of leaf and root tissues. Survival of calli cultured on basal proliferation medium containing 25, 50, 100 or 200 mN (millinormal) NaCl, Na(2)SO(4) or KCl, or iso-osmotic (with NaCl) concentrations of mannitol ranged from 1 to 15%. After six transfers on the same medium, surviving cell lines were subjected to three cycles of direct recurrent selection; i.e., in each cycle, they were cultured alternately on basal proliferation medium, and on basal proliferation medium supplemented with NaCl, KCl, Na(2)SO(4) or mannitol. Salt- or mannitol-tolerant cell lines selected in this way had smaller cells than unselected cell lines, and they grew more rapidly and had higher callus and cell survival rates than unselected cell lines when cultured in the presence of salt or mannitol. Cells lines selected for tolerance to one agent (sodium salt, potassium salt or mannitol) showed minimal tolerance to another agent. However, when plants were regenerated from salt- or mannitol-tolerant callus and new cultures derived from them, the new cultures showed tolerance to all of the salts and mannitol. Plant regeneration from the new cultures was not achieved under the conditions that led to the regeneration of the parent plants from callus.