High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts

Gene constructs that contained the -glucuronidase (GUS) gene under the control of a pollen-specific Zm13 promoter from maize and a LAT52 promoter from tomato were introduced by electroporation into pollen protoplasts isolated from bicellular pollen grains of Lilium longiflorum. After 20 h in culture, the pollen protoplasts exhibited the apparent expression of GUS in a fluorometric assay. The GUS activity induced under the control of the Zm13 promoter was over 10 000 times higher than activity in the control (with no DNA or without electroporation). By contrast, the GUS gene was nearly silent in the lily microspore protoplasts and generative cell protoplasts. The GUS activity driven by the Zm13 and LAT52 promoters was also detected by a cytochemical assay. The frequency of blue-staining pollen protoplasts was about 70% in the case of the Zm13 promoter. The efficiency of gene transfer by electroporation was much higher than by particle bombardment. This protoplast-specific electroporation system is suitable for rapid and reliable examination of pollen-specific promoters, being as good as the particle bombardment system.     

秋水仙素诱变离体卷丹多倍体的研究

用0.15%秋水仙素附加2.00%二甲基亚砜诱变离体培养的卷丹小鳞茎,避光条件下摇床诱导,用组织培养结合不定芽诱导技术获得了多倍体苗,并对多倍体染色体数目进行鉴定。结果显示,诱导96 h效果最好,变异率达到54.29%。细胞学观察发现,对照为三倍体、非整倍体和极少数单倍体细胞组成的嵌合体,诱变出的4棵变异株细胞分别为染色体数目由53~72条的不同比例构成,属于典型的非整倍的异倍型嵌合体。诱变株与对照植株间幼苗叶形指数、气孔密度及气孔大小等特征差异比较明显。     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

超临界CO2萃取百合花挥发油的工艺研究

本文介绍了超临界CO2萃取百合花中挥发油的提取分离工艺,重点研究了超临界CO2萃取压力、温度、时间对出油率的影响。正交试验结果表明：影响超临界CO2萃取的主要因素为C3〉A2〉B2（A为萃取压力,B为萃取温度,C为萃取时间）;最佳工艺参数：SC-CO2萃取压力为18MPa,温度为50℃,时间为90min,流量为25L／min,所得百合花挥发油的出油率高达2．92％。     

Pollination in Lilium sargentiae (Liliaceae) and the first confirmation of long‐tongued hawkmoths as a pollinator niche in Asia

The geographical distribution of pollinators is an important factor determining the biogeographical pattern of floral evolution and diversification. In Africa, a guild of plant species has converged in a floral syndrome for pollination by long‐tongued hawkmoths (predominantly Agrius convolvuli). It is hypothesized that such floral convergence could track the geographical distribution of long‐tongued hawkmoths, so it may not be confined to Africa. We investigated the pollination biology of Lilium sargentiae E. H. Wilson, which is endemic to China and exhibits traits suggestive of long‐tongued hawkmoth pollination. Lilium sargentiae was visited by A. convolvuli as well as small beetles (Nitidulidae) but pollination was mostly or totally effected by the moth. It was consistent with other hawkmoth‐pollinated plants in terms of floral tube length, nectar traits, tepal reflectance, and scent composition. We present the first experimental evidence for the hypothesis proposed above and for hawkmoth pollination in the widespread and ornamentally and economically important genus Lilium L. in a natural habitat. Our findings imply that long‐tongued hawkmoths (especially the extremely widespread A. convolvuli) as a distinctive pollinator niche may have underlain the evolution of long‐tubed (>8 cm) flowers across different continents.     

百合品种间的数量分类研究

根据 4 8个形态学性状 ,应用聚类分析将 9个不同来源的百合品种分为 3个类群 :亚洲系、东方系和麝香系。并显示了东方系和麝香系亲缘关系较近 ,符合北美百合协会对百合品种的分类与百合的育种历史、演化关系。文中将 4 8个形态学性状数据集划分为营养器官性状与生殖器官性状两个数据子集。单独采用营养器官性状子集进行聚类分析时 ,出现较大的差异 ,而对生殖器官性状子集的分析结果较为合理     

岷江百合类萌发素蛋白基因LrGLP2的克隆及表达特性分析

类萌发素蛋白是植物中普遍存在的一类可溶性糖蛋白,在植物抗逆胁迫中起着重要作用。依据岷江百合编码GLP的EST序列设计引物,采用快速扩增cDNA末端技术,从岷江百合犯ilium regale Wilson）克隆得到一个新的GLＰt基因的全长eDNA序列,命名为LrGLP2。LrGLP2全长cDNA为921bp,含有654bp的开放阅读框,49bp5’非编码区以及218bp3’UTR,编码217个氨基酸的蛋白质。LrGLP2编码蛋白质与已知植物GLPs家族成员间的同源性和聚类分析表明LrGLP2与来源于水稻（Oryza sativa）、节节麦似egilopstauschii）、葡萄（Vitis vinifera）中的GLPs具有较高的相似性。qRT-PCR分析显示,LrGLP2在岷江百合正常生长发育的根中有一定量的表达,而在茎和叶中几乎检测不到表达量。水杨酸、茉莉酸以及H202处理均不同程度抑制LrGLP2的转录水平,但乙烯处理能明显诱导LrGLP2的表达。此外,岷江百合接种尖孢镰刀菌（Fusari—umoxysporumfsp．tiliO后,LrGLP2在接种后2h表达迅速上调,12h表达量急剧上升,至24h表达量达到最大值,之后表达量下降,可见￡rGLP2参与岷江百合对尖孢镰刀菌的防卫反应。     

秦巴山区野生百合(Lilium spp.)的育种应用研究

为探索秦巴山区野生百合资源在百合育种中的应用方向及途径, 获得具有其独特遗传背景的育种材料,将秦巴山区野生百合的抗病毒及独特观赏特性等优良遗传性状逐渐渗到栽培品种中去。该研究借助切割柱头杂交及胚抢救技术,选取6种秦巴山区野生百合［岷江百合(Lilium gegale)、宜昌百合(L. leucanthum)、山丹（L. pumilum）、野百合（L. brownii）、宝兴百合（L. duchartrei）、川百合（L. davidii）］以及亚洲百合（Asiatic hybrids,AA）品种‘Elite’、东方百合（Oriental hybrids,OO）品种‘Sorbonne’、‘Siberia’和‘Marlon’、OT百合（Oriental × Trumpet hybrids,OT）品种‘Yelloween’、‘Serano’、‘Corel′door’进行了32组共计263朵花的（品）种间杂交,并针对膨大变软的果实剥离可供离体培养的胚及胚囊进行胚抢救。结果表明：（1）不同杂交组合坐果率、胚获得及萌发率呈现出较大差异,综合坐果率为11.4%,对30个膨大的果实中共计38个可供离体培养的胚及胚囊进行胚抢救,有7株最终萌发。（2）以野生百合为父母本的12组杂交组合中,6组获得了膨大果实,得到6株杂交后代。（3）栽培百合做母本,野生百合做父本的20组杂交组合中,共9组获得了膨大果实,除‘Elite’ × 山丹可直接收获种子外,共得到1株杂交后代。（4）以岷江百合及宜昌百合为亲本的远缘杂交TT × AA及回交OT × TT成功获得杂种后代。（5）秦巴山区6种野生百合在远缘杂交中获得育种后代的几率存在较大差异,宜昌百合和岷江百合获得后代几率较高,宝兴百合获得后代几率较低,野百合未获得后代。以上结果表明,岷江百合和宜昌百合为母本的TT × AA杂交和宝兴百合为父本的TT × AA杂交以及岷江百合和宜昌百合为父本的OT回交,为三种百合的育种利用提供了新途径,野百合的育种应用途径需要继续探索。     

细叶百合的生物量和营养分配

 以栽培的2年生细叶百合（Lilium pumilum）为材料,于2000年的生长季从蕾期至种子成熟期进行6次取样,对其各器官生物量和氮、磷元素的配置进行了动态研究。结果表明,细叶百合虽然以种子繁殖为主,但在整个生长季用于生殖器官的生物量投资的比例并不很大,大量干物质分配到地下器官鳞茎中（平均为60.17%）;茎、叶的生物量分配比例仅次于鳞茎;雄蕊生物量分配比例明显高于雌蕊。在叶萌动及展叶初期植株全氮百分含量最高;从春季萌动至秋季果实成熟,叶中的氮呈逐渐递减的趋势;茎和生殖器官的全氮含量在蕾期最大;生殖器官与叶、鳞茎的全氮含量相关显著。磷在生殖器官的含量较高,这与磷在植物有性生殖过程中的重要作用相一致;生殖器官与茎的全磷含量相关显著。地下器官全氮、全磷随季节变化有增多的趋势;地上各器官全氮、全磷相关显著,随季节变化有明显减少的趋势。     

病毒侵染对西伯利亚百合DNA甲基化的影响

采用基于AFLP的甲基化敏感扩增多态性(MSAP)技术,用10对引物对侵染百合花叶病毒和丛簇病毒的西伯利亚百合植株和无毒植株进行DNA甲基化水平和模式分析.结果发现,西伯利亚百合无毒植株和病毒侵染植株的平均甲基化水平分别为40.1%和31.5%;平均全甲基化率分别为13.0%和9.7%;半甲基化率分别为27.1%和21.8%.研究表明,百合DNA甲基化多以半甲基化的形式存在;病毒侵染导致百合植株DNA甲基化水平降低,且对整体甲基化水平、全甲基化水平和半甲基化水平均产生了影响;说明病毒侵染百合后植株出现的症状在一定程度上与DNA甲基化存在关联.