盐胁迫对黄独脱毒苗生长和若干生理生化指标的影响

通过在培养基中添加不同浓度NaCl,探讨盐胁迫对黄独脱毒苗生长及若干生理生化指标的影响。结果表明,在盐胁迫条件下,黄独脱毒苗的生长受到明显抑制,叶片中总叶绿素含量、SOD活性下降,丙二醛含量增加,脯氨酸大量积累;随盐胁迫强度的加大,对试管苗生长及生理生化指标的影响相应加剧;在盐胁迫下,黄独脱毒苗叶片脯氨酸含量与丙二醛含量呈极显著正相关,而与叶绿素含量和SOD活性呈极显著负相关。因此,盐胁迫下叶片中脯氨酸含量的变化可作为黄独脱毒苗受害程度的主要生理鉴定指标。     

应用正交设计法优选黄独脱毒苗快繁培养基

通过正交设计法研究黄独脱毒苗的快繁培养基,考察KT、NAA和2,4-D对黄独脱毒苗快繁的影响。结果表明,黄独脱毒苗快繁的最佳培养基为MS+KT 2.0 mg/L+NAA 0.5 mg/L。     

黄独脱毒苗叶片和茎段再生体系的建立

以黄独茎尖再生苗为试材,研究不同因素对黄独脱毒苗叶片和茎段再生体系的影响,以期对黄独脱毒苗的再生体系进行优化。结果表明,叶片和茎段诱导愈伤组织的最佳培养基是MS+KT 2 mg·L^-1+2,4-D 2 mg·L^-1;叶片和茎段诱导愈伤组织的最佳蔗糖浓度分别为30和50 g·L^-1;叶片和茎段在黑暗中较容易诱导出愈伤组织;叶片和茎段愈伤组织分化的最佳培养基是MS+KT 4 mg·L^-1+NAA 0.1 mg·L^-1;继代2次的叶片和茎段愈伤组织较容易分化;黄独不定芽生根的最佳培养基是1/2MS+IBA 0.1 mg·L^-1+NAA 0.5 mg·L^-1+PP₃₃₃ 1 mg·L^-1。本实验成功建立了黄独脱毒苗叶片和茎段的再生体系,为黄独脱毒苗的工厂化生产奠定了技术基础。     

黄独脱毒苗种质离体保存及其遗传稳定性和病毒变化的初步研究

以黄独脱毒苗带芽茎段为外植体,以MS为基本培养基,采用正交实验和单因子实验方法研究了无机盐水平、蔗糖、甘露醇和植物生长抑制剂PP₃₃₃对黄独脱毒苗离体保存的影响。结果表明,黄独脱毒苗带芽茎段在25±1℃下保存于附加2.0 mg·L^-1 KT、0.5 mg·L^-1 NAA、30 g·L^-1蔗糖、20 g·L^-1甘露醇和4 mg·L^-1 PP₃₃₃的1/4MS培养基上,180 d后其成活率可达90%以上。离体保存后的脱毒苗经形态指标和生理生化指标以及RT-PCR分析测定没有发生遗传变异,也没有检测到PVY病毒。本实验结果为药用植物黄独脱毒苗的种质保存提供了一条简单有效的途径。     

黄独冻后黑暗培养胚性愈伤组织及其再生植株的电镜观察

本研究以黄独为材料,对其冻后黑暗培养胚性愈伤组织及其再生植株进行电镜观察。结果表明:黄独胚性愈伤组织在冻后没有经过一定时间的黑暗培养直接放于正常的光周期下培养,极易使细胞内容物外泄,且造成部分细胞器和细胞核消失或裂解形成空隙,线粒体和高尔基体等细胞器的结构不完整,出现空隙或内容物溢出或断裂现象;而黄独冻后胚性愈伤组织经过3 d的黑暗培养后再置于光周期下培养,受损的细胞较少,细胞排列较紧密,且细胞器降解较少,很少出现空隙等现象,线粒体、高尔基体等细胞器的结构也较为完整。此外,与黄独带芽茎段常温继代再生试管苗相比,黄独胚性愈伤组织经过冻后黑暗培养后,其再生试管苗的根、茎、叶结构没有出现显著变化,且两者的气孔参数和叶绿体数量也均无显著性差异。本试验结果可为植物种质资源超低温保存后的黑暗培养提供了亚显微结构证据。     

环境因素和ABA对葡萄试管苗气孔开闭的影响

黑暗、低温、低湿、高渗等环境因素和ABA处理,虽能降低葡萄试管苗叶气孔开度,但因长时处于饱和湿度和弱光下的气孔保卫细胞发育不良,造成气孔口过度开放,而保卫细胞胀缩变化的幅度,不足以使这种过度开放的气孔口关闭。通过分步炼苗降低试管苗气孔口的开度后,保卫细胞膨压的变化就能使气孔关闭了。试管苗叶气孔在暗中的关闭率,可作为炼苗适合程度的生理指标。     

环境因素和ABA对葡萄试管苗气孔开闭的影响

黑暗、低温、低湿、高渗等环境因素和ＡＢＡ处理,虽能降低葡萄试管苗叶气孔开度,但因长时处于饱和湿度和弱光下的气孔保卫细胞发育不良,造成气孔口过度开放,而保卫细胞胀缩变化的幅度,不足以使这种过度开放的气孔口关闭。通过分步炼苗降低试管苗气孔口的开度后,保卫细胞膨压的变化就能使气孔关闭了。试管苗叶气孔在暗中的关闭率,可作为炼苗适合程度的生理指标。     

珠美海棠试管苗的土壤支撑生根培养和带坨移栽

以粘壤土做培养基支撑物,选用只含有0．5mg·L^-1IBA和15g·L^-1蔗糖的液体培养基,珠关海棠试管苗茎段生根培养的结果显示,试管苗生根率可达到100．0％,明显高于琼脂支撑培养,根长也显著提高,并且长出正常的根毛。土支撑培养生根的珠美海棠试管苗开瓶炼苗21d后带坨移入营养钵中,移栽后不喷雾、并毋需覆盖塑膜、午后空气相对湿度控制在50％-65％,其成活率达到92．2％;在开瓶炼苗21d后,黑暗中叶片气孔的关闭率从26．7％增加到88．5％。     

一氧化氮在乙烯诱导蚕豆气孔关闭中的作用

以蚕豆为材料研究了一氧化氮(nitric oxide,NO)和乙烯对气孔运动的影响。结果表明,10μmol/L的NO供体硝普钠(sodium nitroprusside,SNP)以及0.04%的乙烯能明显诱导蚕豆气孔关闭,并且二者共同处理后,能够增强其促进气孔关闭的作用。乙烯合成抑制剂AVG可以减弱NO诱导气孔关闭的程度,NO清除剂c-PTIO和NR抑制剂NaN3也可减弱乙烯诱导气孔关闭的程度,而一氧化氮合酶(nitric oxide synthase,NOS)抑制剂L-NAME对乙烯诱导气孔关闭的作用不明显。推测,在调控蚕豆气孔关闭过程中,NO可能主要通过NR途径参与乙烯调控气孔关闭过程。     

微管在气孔运动中的作用

用植物微管专一性解聚剂甲基胺草磷(APM)预处理蚕豆(Vicia faba L.)下表皮,再用诱导气孔运动的因子处理,在显微镜下观察气孔孔径的变化。结果显示,用50mg/L APM预处理开放或关闭状态气孔,虽胞质微管被解聚,但气孔孔径没有发生明显的变化,表明胞质微管与开放或关闭状态气孔的维持无关;而去掉APM后,CaCl_2可在4h内诱导气孔关闭,气孔的运动功能又可恢复。进一步的研究表明,开放气孔经APM预处理60min后,再用ABA、Ca~(2 )及暗处理均不能诱导气孔关闭,表明微管可能参与了ABA、Ca~(2 )及暗诱导的气孔关闭过程;关闭气孔经50mg/L APM预处理后,光诱导气孔开度较不经 APM处理的有明显差异,且随着APM预处理时间和浓度的变化,气孔开放程度亦不同,表明微管也参与了光诱导的气孔开放过程。     

KT、2,4-D和NAA对黄独组培苗带芽茎段生长发育的影响

以黄独组培苗带芽茎段为材料,采用植物组织培养和单因子实验法,研究添加KT和2,4-D或KT和NAA组合的MS培养基对黄独带芽茎段再生的影响。结果表明,MS+KT 2.0 mg/L+NAA 0.5 mg/L有利于黄独带芽茎段腋芽萌发和根系生长。     

Elimination of cymbidium mosaic virus and odontoglossum ringspot virus from orchids by meristem culture and thin section culture with chemotherapy

Most commercially cultivated orchid plants are generally infected with cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV). Two methods were used in order to generate virus-free plants: meristem culture and thin section culture with chemotherapy. Meristems (0.10 mm to 1.00 mm) were excised from infected axillary shoots of an infected monopodial orchid hybrid (Mokara Char Kuan ‘Pink’) and cultured in modified Vacin and Went medium. Only larger meristem explants survived and the regenerated plantlets remained virus-infected. In contrast, high percentages of virus-free plantlets were obtained from thin section cultures of infected plantlets and protocorm-like bodies with ribavirin treatment. Interestingly, regenerants from thin section cultures without ribavirin treatment were also found to be free from CyMV and ORSV. All plantlets were tested by enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR).     

蝴蝶兰试管分株快速繁殖研究

以蝴蝶兰无菌幼苗为外植体,在分株增殖培养基(1/2MS+6-BA 3.0mg/L+NAA 0.2mg/L+CM20g/L)上培养30d后,形成幼小丛生植株;将此幼小植株移至生长培养基(1/2MS+6-BA 1.0mg/L+NAA 0.1mg/L)上,30d后便形成具3~4片叶与数条粗壮根的较大植株,移栽成活率达80%以上。     

月光花素对彩叶芋愈伤组织细胞生长和分化的影响

在含有NAA0.2mg/L,2.4-D 0.2mg/L的MS基础培养基中添加月光花素培养彩叶芋愈伤组织,结果显示,低浓度的月光花素能促进愈伤组织生长,高浓度则有明显的抑制作用,最适浓度为1.0~2.0mg/L;培养两个月后,添加适宜浓度的月光花素(0.2~2.0mg/L)的培养基能促进体细胞分化和植株再生,而高浓度(10.0mg/L)处理和对照均未能分化出绿苗。表明适宜浓度的月光花素在合适的生长素剂量的协同作用下,对植物愈伤组织细胞的生长和分化有较强的调节作用。     

Virus Free Garlic (Allium Sativum L.) Plants Obtained by Thermotherapy and Meristem Tip Culture

Virus infection in garlic considerably reduces yield and quality in Argentina. The production of virus free “seed” was attempted by means of thermotherapy and meristem tip culture. A hot water treatment was employed to determine the lethal temperature/time combination for clonal type (c.t.) Blanco cloves. It was established that 50°C × 20 min, 50°C × 15 min and 55°C × 5 min were the limit thermal/time combinations which garlic could withstand. Those treatments were employed followed by meristem tip culture, however, none of the successfully developed plants after culture (only 13 %) were virus-free. Hot air treatments in a growth chamber at 36°C lasting for 30, 40 and 60 days, and at 25°–32° for 30 days in a greenhouse were tested on c.t. Blanco. Cloves kept at room temperature throughout the experiment were employed as controls. In the 25°–32°C treatment, 73% of meristems produced plants and, of these, 33 % were virus free. After 30 and 40 days at 36 °C, 62 % and 67 % of the meristems developed into plantlets, of which respectively 51 % and 50 % were virus-free. Very few meristems (10 %) developed into plants when cloves had been kept at 36°C for 60 days but the resulting plantlets were all virus free. Controls produced 78 % of plants, of which 14 % were virus free. Results of hot air treatments of 36 °C for 40 days performed on c.t. Colorado, Rosado, Paraguayo, Espaol and Hilario Ascasubi were similar to those obtained with c.t. Blanco. In Espaol and Hilario Ascasubi, no virus-free plants were detected among control specimens (no thermotherapy treatment). The only virus (from up to 3 that infected the plants) that persisted in some plants after themotherapy and meristem tip culture was garlic yellow streak.     

原代地鼠肾细胞及狂犬病毒不同培养方式的研究

目的通过比较原代地鼠肾细胞在转瓶、微载体、细胞工厂的3种培养方式的培养效果,为原代地鼠肾细胞选择一种易扩大规模、培养高质量细胞的培养方式,进而提高狂犬病毒的产量。方法消化取得的细胞悬液分别在转瓶、微载体、细胞工厂中培养,通过显微镜观察细胞形态、计数等结果比对培养的差别。结果细胞工厂可以很好地培养原代地鼠肾细胞和狂犬病毒;而细胞在微载体上贴附性差,生长不好。结论实验结果表明细胞工厂可以取代转瓶,用于大规模培养原代地鼠肾细胞扩大狂犬病毒的产量。     

Inactivation or removal of the budded particles of a nuclear polyhedrosis virus of a silkworm, Bombyx mori L. (Lep., Bombycidae)

Effective methods to inactivate or remove budded particles of a nuclear polyhedrosis virus of Bombyx mori (BmNPV) from a cell-cultured media or from host haemolymph that is infected by this virus have been developed. Two types of suspensions containing BmNPV budded virus particles, TC-100 media that cultured BmN4 cells infected by this virus and haemolymph of B. mori larvae infected by this virus, were treated by 6% (w/v) polyethylene glycol (PEG), 0.01% (w/v) chitosan, 0.05% (v/v) linoleic acid (an emulsion), and/or diethylether. Treatment by linoleic acid followed by PEG-precipitation and treatment by diethylether followed by PEG-precipitation were so effective that these treatments suppressed the viral titre of BmNPV-infected larval haemolymph from an original titre (> 10⁹ TCID₅₀ units/ml) to below a detectable limit. These methods are suggested as being potentially useful in an insect factory system; that is, a protein production system utilizing a baculovirus vector and its insect host or cultured cells on a large scale.     

百合不定芽培养脱毒种球生产的研究

百合不定芽培养再生植株的病毒脱毒效果因百合品种和病毒种类而有所差异;‘卡萨布兰卡’百合潜在病毒（LSV）容易脱去,脱毒率达76%,‘魅丽’黄瓜花叶病毒（CMV）能全部脱去。‘卡萨布兰卡’不定芽培养在固体培养基和液体培养基都能很好形成子球;在含有抗病毒剂（10 mg/L DHT）的液体培养基中子球增大显著,LSV脱毒效果理想。无病毒子球移到有防虫网的户外栽培,2年后部分植株被检测出病毒,再感染率因品种而不同,‘卡萨布兰卡’高达73%,麝香百合‘乔治亚’仅17%;无病毒植株‘乔治亚’,香华丽百合和‘卡萨布兰卡’的生长高度要比有病毒植株明显增高。     

Reinitiation of vegetative growth from aseptically cultured terminal floral apex of banana

Terminal floral apices of Musa acuminata cv. Dwarf Cavendish were isolated and cultured in a modified Murashige and Skoog (1962) medium supplemented with N6-benzylamino purine (5 mg/L) and 10% (v/v) coconut water. Under these conditions, the determinate floral buds are transformed into a multiplying vegetative shoot system. Rooted plantlets were obtained by treating shoots with the auxin, naphthaleneacetic acid (1 mg/L), and activated charcoal (0.025%). Practical benefits resulting from these observations, such as the production of virus-free plants and the rapid multiplication of stock material, are discussed.