牛耳朵传粉生物学研究

苦苣苔科(Gesneriaceae)报春苣苔属(Primulina Hance)是一个近年来备受关注的类群,其纷繁复杂的物种多样性和属下种间的特有分布引起了分类学家和植物学研究者的极大兴趣。该属除了极少数的物种如牛耳朵[(Primulina eburnea(Hance)Y. Z. Wang)]以外,绝大部分的物种为狭域分布或地方特有种,其分布范围很窄。为了揭示牛耳朵的传粉生物学和繁育系统对其生殖过程和拓殖能力的影响机制,作者系统地研究了牛耳朵的开花物候、花粉与柱头活性、访花昆虫的种类和访花行为、花粉胚珠比、OCI指数和套袋实验结实率,探究其传粉等生殖过程对牛耳朵的广布是否有正面影响。结果表明:牛耳朵的自然花期是3—5月,全花期约45 d,其中盛花期约20 d,单花期6～8 d; 开花后1～2 d花粉活力最强,开花前柱头没有可授性; 花粉胚珠比为537; 杂交指数为5; 去雌套袋、去雄套袋均无法结实,说明本种不存在无融合生殖; 与自然授粉相比,自花授粉结实率略低,异花授粉结实率略高,说明自交亲和; 牛耳朵的主要传粉者是花条蜂(Anthophora florea)和熊蜂(Bombus sp.)。花蜜产量较高、花粉量较大、花粉活力较强等特点,有利于牛耳朵完成传粉和结实的整个繁殖过程。因此,这一结果显然有利于牛耳朵的拓殖进而广布在我国华南至西南地区的喀斯特地区。     

牛耳朵传粉生物学研究

苦苣苔科(Gesneriaceae)报春苣苔属(Primulina Hance)是一个近年来备受关注的类群,其纷繁复杂的物种多样性和属下种间的特有分布引起了分类学家和植物学研究者的极大兴趣。该属除了极少数的物种如牛耳朵[(Primulina eburnea (Hance) Y. Z. Wang)]以外,绝大部分的物种为狭域分布或地方特有种,其分布范围很窄。为了揭示牛耳朵的传粉生物学和繁育系统对其生殖过程和拓殖能力的影响机制,作者系统地研究了牛耳朵的开花物候、花粉与柱头活性、访花昆虫的种类和访花行为、花粉胚珠比、OCI指数和套袋实验结实率,探究其传粉等生殖过程对牛耳朵的广布是否有正面影响。结果表明:牛耳朵的自然花期是3—5月,全花期约45 d,其中盛花期约20 d,单花期6～8 d;开花后1～2 d花粉活力最强,开花前柱头没有可授性;花粉胚珠比为537;杂交指数为5;去雌套袋、去雄套袋均无法结实,说明本种不存在无融合生殖;与自然授粉相比,自花授粉结实率略低,异花授粉结实率略高,说明自交亲和;牛耳朵的主要传粉者是花条蜂(Anthophora florea)和熊蜂(Bombus sp.)。花蜜产量较高、花粉量较大、花粉活力较强等特点,有利于牛耳朵完成传粉和结实的整个繁殖过程。因此,这一结果显然有利于牛耳朵的拓殖进而广布在我国华南至西南地区的喀斯特地区。     

草豆蔻传粉生物学的研究

比较研究了花柱卷曲性植物草豆蔻 Alpinia hainanensis两种表型(上举型和下垂型)的传粉生物学特性。结果表明:草豆蔻的花柱卷曲运动节律与其他已报道的山姜属Alpinia植物基本一致,而下垂型花的花柱卷曲运动明显滞后于上举型花约2 h。草豆蔻的花柱卷曲运动中存在一些不同步的现象,甚至在同一花序上的两朵花的花柱运动节律亦会表现出不一致的现象,但上举型花的花药开裂时间却严格同步,都发生在同类型个体的柱头全部位于花药上方之后进行。草豆蔻上举型花的花蜜分泌量、单花花粉量、花粉/胚珠比率(P/O)均明显比下垂型花多,而两种表型的胚珠数、花蜜糖含量、氨基酸含量无显著差异。在草豆蔻的单花期内不同时间段进行人工授粉,上举型花在其柱头位于花药下方时进行人工授粉,其结实率明显比柱头位于花药上方时人工授粉的处理高,下垂型花则没有显著差异。在自然居群中,草豆蔻的主要传粉者是无垫蜂Ameglla sp.和两种木蜂Xylocopa sp.,但存在传粉者不足而影响结实率的现象。完全套袋、去雄和去雌(去柱头)处理的均不结实,表明草豆蔻中不存在无融合生殖、主动自花授粉和滞后自交的生殖保障现象。而人工自交和异交均具有较高的结实率,表明草豆蔻为自交亲和植物。草豆蔻的繁育系统是具花柱卷曲性运动的异花授粉的交配系统。     

益智传粉生物学的研究

 益智（Alpinia oxyphylla）的花期从2月底至4月下旬;单株花期（花序）约为16～26 d,通常为23～26 d;单花花期一般为1 d。正常情况下,益智和其它山姜属植物一样,具有花柱卷曲性促进异花授粉的机制,表现出两种类型：花柱上举型和花柱下垂型,其花柱卷曲运动的节律与其它已报道的山姜属植物基本一致。但观察发现,当遭遇低温天气时(日最高气温<18℃),单花花期延长为2 d,无论是上举型个体还是下垂型个体,均只表现出一种花型——下垂型：上午开花时花柱弯向上,柱头位于已开裂散发出花粉的花药上方,直到第二天上午6∶30～11∶00间,花柱才陆续地慢慢向下运动,柱头下降至与花药等高或位于花药下方。益智的主要传粉昆虫是蜜蜂（Apidae sp.）、木蜂（Xylocopa sp.）,绝大多数的访花者的访花目的是吸蜜。益智的花蜜分泌量（8.37～15.79 μl）和花蜜含糖量（30.12%～32.83%）较高,花蜜是益智作为传粉者访花的最主要的报酬。实验结果还表明,益智花部中唇瓣对昆虫访花有显著的招引作用;益智的花蜜对蜜蜂的访花频率有显著的影响,对木蜂有一定的招引作用,但并不显著。而花粉（花药）则对昆虫的访花频率影响不大。人工授粉实验结果表明益智存在自交亲和性,无论是上举型或下垂型个体自交和异交均有较高的结实率;人工自交和异交的结实率在上举型植株中存在较大的差异,而在下垂型个体中则差异不明显;去雄套袋、去柱头套袋和完全套袋不授粉等处理均不结实,表明益智不存在无融合生殖现象和自动自花授粉现象。益智的繁育系统是异花授粉交配系统。     

两种广西特有报春苣苔属(苦苣苔科)植物传粉生物学研究

通过野外观察和室内试验,对两种广西特有的桂林小花苣苔和阳朔小花苣苔的传粉生物学进行比较研究。结果表明:两种报春苣苔属植物花期从7月底至8月初;单花花期内花粉具有较高的活性,最高可达92.6%和94.5%;柱头可授性最高时可达90%和95%;花粉/胚珠比率(P/O)分别为110.28±17.45和229.65±18.00;柱头与花药之间存在着空间隔离,可防止自花授粉;在单花花冠裂片张开后第3天,花药开始散粉,但雌蕊在花朵刚开始开放时已伸长,待第3天时柱头已伸长到位于花冠筒口部,高于花药,便于接受异花花粉;该两种植物均不存在无融合生殖现象;两种报春巨苔属植物均高度自交亲和,但很难发生自发的自花授粉,必须依靠外力,因此传粉媒介对于结实率有重要影响,自然条件下基本不发生自花授粉;在自然状态下结实率明显低于两种人工授粉的结实率;小蜂和淡脉隧蜂是目前已知仅见的两种传粉者。     

贯叶连翘的开花动态与繁育系统研究

野外观察贯叶连翘的开花进程和花部形态特征,运用花粉萌发、杂交指数、花粉-胚珠比等方法测定其繁育系统。结果显示:贯叶连翘雌雄异熟,柱头先花药成熟,雌雄蕊无明显异位。单花花期4～5d。仅在开花当日有昆虫传粉,蜜蜂为主要传粉者。花粉在花药开裂1h后活力最大,萌发率达40.10%,花粉在柱头萌发3h后接近子房。根据杂交指数(OCI)推测其繁育系统属于异交,部分自交亲和,有时需要传粉者。花粉-胚珠比(P/O)则表明贯叶连翘的繁育系统为兼性异交。贯叶连翘结实率低,可能与花粉活力,花粉管的生长速度及花粉在柱头的竞争有关。     

濒危植物四药门花的自花授粉

观测了四药门花Loropetalum subcordatum的开花物候、开花动态和访花者; 对其花粉组织化学、花粉胚珠比和花粉活力进行了检测; 通过人工控制授粉实验检测了其繁育系统; 使用扫描电子显微镜(SEM)观察了自花花粉在柱头上的萌发情况并使用荧光显微镜观察了花粉管在花柱中的生长情况。结果表明: 四药门花花期为9月至次年2月, 其中9-10月为开花高峰期; 单花花期4-6 d; 雌蕊先熟, 开花时柱头即有活性, 并持续到花谢; 花药于开花后12-24 h内开裂, 并能直接落置自花柱头; 离体花粉寿命约26 h, 花粉为非淀粉型, 花粉胚珠比为8420±720.86 (n=10); 无花蜜分泌。未观察到任何外来的访花者, 仅见到生活在头状花序中的蓟马Thrips sp., 且未见到蓟马在花间积极迁飞, 表明蓟马不是其有效的传粉者。无处理套网和辅助异株授粉的结实率(5.30%±1.83%/6.67%±1.91%)与自然对照(4.79%±1.45%)差异均不显著(P=0.847/0.616), 扫描电镜观察证明自花花粉能在柱头上顺利萌发, 荧光显微镜观察证明花粉管能在24 h内生长到花柱基部, 说明四药门花是自花传粉, 不存在无融合生殖。四药门花存在雌蕊先熟现象并且仍有异交可能, 说明其祖先类型曾经是异交植物。花谢后子房于次年夏天才开始膨大而果实于10月间开花高峰期后成熟, 说明存在胚发育延迟的现象。本文还讨论了金缕梅科Hamamelidaceae中自花传粉由蝇类传粉演化而来的可能性。     

珍稀濒危植物小花木兰传粉生物学研究

基于定株观测,对小花木兰（Magnolia sieboldii）野生种群和人工栽培种的开花物候进行了研究。结果表明,小花木兰花期为5月中下旬至6月中下旬,花期20d左右,单花花期一般为5～7d,盛花期为6月1～5日;开花过程中花部表型变化明显,花粉在花开时不成熟,但雌蕊已经成熟,花开后雌蕊柄伸长,约20h后下柱头依然湿润,上柱头已萎缩变褐色,再过约40h下柱头也萎缩失去活力．此时花粉成熟,花药裂开;花的柱头接受到的花粉量由下往上依次减少。检测花粉活力、萌发率发现,小花木兰花粉在散发6d后基本失去活力和萌发能力。重力玻片法观测表明,小花木兰为虫媒传粉;花柱的授粉率不高,为65％;柱头上平均花粉量为3．5个;自然状态下结实率低,为13．5％,人工栽培为20．1％;外轮花被片全部去掉,结果率下降至11．3％;去掉雄蕊,结实率下降至10．7％;再辅以人工授粉,结果率上升至25．0％。小花木兰的访花昆虫种类较少,访花频率低,其中蜜蜂和一种蚜科（Aphididae）小昆虫对其传粉影响较大。同花期灯台附（Cornus controversa）等植物对其传粉产生昆虫竞争作用。环境因素如光照、温度、阴雨、海拔等均对其传粉产生一定影响。     

大花黄牡丹的开花特性与繁育系统

2019-2020年观察河南省栾川县引种栽培的西藏特有植物大花黄牡丹(Paeonia ludlowii)开花过程和访花昆虫,计算花粉-胚珠比(P/O)和杂交指数(OCI),检测花粉活力与柱头可授性并结合人工控制授粉试验,探究其开花特性和繁育系统类型。结果表明:(1)大花黄牡丹花朵昼开夜合,花朵随花冠展开下垂,伴花冠闭合回升。(2)花淡香,有花内蜜腺,花瓣与雌蕊子房之间是主要泌蜜部位。(3)2019年群体花期为5月17日～6月19日,持续34 d,为集中开花模式;单花花期(8.95±1.28)d。(4)大花黄牡丹从开花第2天至第6天皆具有较高的花粉活力;开花前1天柱头即具有可授性,持续12 d;花粉活力与柱头可授性最强时期高度重合,共2 d。(5)P/O为119 356.31～731 238.07,OCI为4,繁育系统类型为异交、部分自交亲和、需要传粉者。(6)大花黄牡丹可能有无融合生殖,但不稳定,去雄严重影响结实。(7)河南省栾川县大花黄牡丹主要访花昆虫为意大利蜜蜂(Apis mellifera ligustica)、芦蜂(Ceratina sp.)和隧蜂(Halictus sp.)、灰带管蚜蝇(Eristalis cerealis)和黑带食蚜蝇(Episyphus balteatus),其中意大利蜜蜂访花次数最多。(8)大花黄牡丹侧花比顶花结实稳定,杂交育种时建议在侧花开放第3、4天授粉以获得较高结实率。     

珍稀濒危植物瑶山苣苔开花生物学及繁育系统研究

对仅分布于广西金秀老山自然保护区的国家Ⅰ级保护植物瑶山苣苔进行了开花生物学和繁育系统研究.结果显示:(1)瑶山苣苔花期从8月底至11月初,开花无固定时间.单花开放过程可分为萌动、露白、盛开、凋落4个阶段,且花朵具明显的增大再生长现象.(2)单花、花序、单株水平上的开花物候都表现出开花不同步性,单花花期约5~14 d,单花序花期约11~20 d,单株花期约8~20 d.花粉活力在散粉后6 d内相对较高,但花粉在野外通常于散粉3 d内被昆虫啃食完.(3)柱头在花药散粉时明显高于花药,便于接受异花花粉,柱头在散粉后第4天具可授性,可持续5~6 d.(4)瑶山苣苔杂交指数(OCI)为5,花粉-胚珠比(P/O)为379.64±145.61,繁育系统为兼性异交,自交亲和,传粉过程需要传粉媒介.研究表明,不稳定的传粉环境可能是该物种至濒的主要生殖生物学原因,自发自交"是其在开花期间对不稳定传粉环境的一种适应.     

Discovering the type of seed dormancy and temperature requirements for seed germination of Gentiana lutea L. subsp. lutea (Gentianaceae)

Aims There are a number of mechanisms that regulate germination; among these, seed dormancy, one of the most important, is an adaptative mechanism in plants to promote survival by dispersing germination in space and time until environmental conditions are favourable for germination. The main goals of this study were to determine the temperature requirements for seed dormancy release and germination of Gentiana lutea subsp. lutea, to identify the class and level of seed dormancy and to suggest an optimal germination protocol.Methods Seeds belonging to two different localities were subjected to various pre-treatments, including cold stratification (0 and 5°C), warm stratification (25/10°C) and different combinations of these, and then incubated at a range of constant temperatures (5–25°C) and 25/10°C. Embryo growth during pre-treatments and incubation conditions were assessed at different times by measuring the embryo to seed length ratio (E:S ratio). The final germination percentage (FGP) and the germination rate (t 50) were calculated.Important findings Fleshy mature seeds of G. lutea subsp. lutea have linear underdeveloped embryos. Cold stratification at 0°C was effective in overcoming the physiological dormancy (PD) and promoted embryo growth and subsequent germination. After cold stratification at 0°C, both the root and the shoot emerged readily under a wide range of temperatures. G. lutea subsp. lutea seeds showed an intermediate complex morphophysiological dormancy (MPD). As regards the optimal germination protocol for this taxon, we suggest a period of cold stratification at ca. 0°C followed by seed incubation at 10–20°C. The optimal germination temperatures found for seeds of this taxon, as well as its pre-chilling requirement at 0°C, suggest that it is well adapted to a temperate climate; this behavior highlights an increasing threat from global warming for G. lutea, which could reduce the level of natural emergence in the field, prejudicing also the long-term persistence of the natural populations in Sardinia.     

Corpora lutea in submerged organ cultures at low incubation temperatures

Summary Corpora lutea in submerged organ cultures of mouse ovary retained viability when incubated at 24°C. At harvest, whole corpora lutea appeared uniformly viable, and mitotic figures were occasionally seen among the luteal cells. At 24°C, the observed excellent cytological condition of the corpora lutea was in contrast to the necrosis seen when the explants were incubated at 34°C.     

Corpora lutea in submerged organ cultures at low incubation temperatures

Summary Corpora lutea in submerged organ cultures of mouse ovary retained viability when incubated at 24°C. At harvest, whole corpora lutea appeared uniformly viable, and mitotic figures were occasionally seen among the luteal cells. At 24°C, the observed excellent cytological condition of the corpora lutea was in contrast to the necrosis seen when the explants were incubated at 34°C.     

山羊卵母细胞体外成熟过程中细胞核迁移的研究

本试验应用传统的醋酸地衣红染色法研究了山羊卵母细胞体外成熟过程中细胞核迁移现象,结果发现成熟分裂从GVBD现象起,其细胞核即由卵母细胞的近中央位置逐渐移向细胞表面,之后同源染色体分开,排出第一极体。卵母细胞成熟后,随着时间的延长,细胞核又逐渐远离第一极体端。试验结果采用面积积分法处理,结果为:0~5.7h山羊卵母细胞处于GV期,持续时间为5.7h;5.7~8.9h为DK期,持续时间为3.2h;8.9~15.9h卵母细胞处于MI期,持续7h;15.9~17.5h为AI期,持续1.6h;17.5~22h为MII期,持续4.5h。     

Multiple gene sequences delimit Botryosphaeria australis sp. nov. from B. lutea

Botryosphaeria lutea (anamorph Fusicoccum luteum) most easily is distinguished from other Botryosphaeria spp. by a yellow pigment that is formed in young cultures. This fungus has been reported from a number of cultivated hosts in New Zealand and Portugal. During a survey of Botryosphaeria fungi that occur on native Acacia species in Australia, a yellow pigment was observed in some cultures. These isolates were morphologically similar to B. lutea, but the pigment differed slightly from the one formed by authentic B. lutea isolates. Preliminary data also revealed small differences in ITS rDNA sequence data. The aim of this study was to determine whether these small differences were indicative of separate species or merely variations within B. lutea. Anamorph, teleomorph and culture morphology were compared between B. lutea and Acacia isolates from Australia. Sequence data of two other genome regions, namely the β-tubulin and EF1-α gene and intron regions, were combined with ITS rDNA sequence data to determine the phylogenetic relationship between these isolates. Isolates of B. lutea and those from Australian Acacia species were not significantly different in spore morphology. The yellow pigment, however, was much more distinct in cultures of B. lutea than in cultures from Acacia. There were only a few base pair variations in each of the analyzed gene regions, but these variations were fixed in the two groups in all regions. By combining these data it was clear that B. lutea and the isolates from Acacia were distinct species, albeit very closely related. We, therefore, propose the new epithet B. australis for the fungus from Australia. Botryosphaeria australis also was isolated in this study from exotic Sequoiadendron trees in Australia. Re-analyses of GenBank data in this study showed that B. australis also occurs on other native Australian hosts, namely a Banksia sp. and a Eucalyptus sp., as well as a native Protea sp. in South Africa and on Pistachio in Italy. These records from GenBank have been identified previously as B. lutea. The common occurrence of B. australis on a variety of native hosts across Australia suggests that this fungus is native to this area.     

The conversion of pregnenolone-7alpha-3H and progesterone-4-14C to estrogens by corpora lutea of menstrual cycles and pregnancy.

The metabolism of pregnenolone-7alpha-3H and progesterone-4-14C by human corpora lutea tissue of menstrual cycles and pregnancy was studied. In the incubations, equimolar mixtures of pregnenolone-7alpha-3H and progesterone-4-14C were used as substrates. Three corpora lutea of cycles were used as minced tissue. From those corpora lutea progesterone, 17-hydroxyprogesterone and androstenedione were formed, although no estrogens were formed. One corpus luteum of cycle and one corpus luteum of pregnancy were used as homogenated tissue, and those formed estrone and estradiol as well as the same three delta4-metabolites. The corpus luteum of cycle also formed testosterone. All metabolites including estrogens showed the lower 3H to 14C ratio than the starting ratio. 17-hydroxypregnenolone in only one corpus luteum, and no delta5-metabolites in the other four corpus luteum were identified. It is therefore proposed that the major pathway for estrogen formation in human corpus luteum is pregnenolone yields progesterone yields 17-hydroxyprogesterone yields androstenedione (or testosterone) yields estrone and estradiol.     

The interaction of testosterone and gonadotropins in stimulating estradiol and progesterone secretion by cultures of corpus luteum cells isolated from pigs in early and midluteal phase.

The first objective of this research was to define the capacity of corpora lutea of pig to secrete estradiol in the presence of an androgen substrate which was testosterone. The second objective was to define the synergism between gonadotropic hormones such as LH, FSH, and PRL and testosterone as measured by estradiol and progesterone secretion by two types of porcine luteal cells. Luteal cells were collected from newly forming corpora lutea (0-3 days after ovulation) and from mature corpora lutea (8-10 days after ovulation). After dispersion, luteal cells were suspended in medium M199 supplemented with 10% of calf serum and grown as monolayers at 37 degrees C. Control cultures were grown in medium alone while other cultures were supplemented with either testosterone alone at a concentration of 1 x 10(-7) M or with 10, 100, 500 ng LH plus testosterone, 10, 100, 500 ng FSH plus testosterone or 10, 100, 500 ng PRL plus testosterone. After 2 days of cultivation all cultures were terminated and media were frozen at 20 degrees C for further steroid analysis. Testosterone added to the culture medium in the absence of gonadotropins was without effect on estradiol and progesterone secretion by luteal cells collected in the corpora lutea of the early luteal phase. On the other hand testosterone added to the medium significantly increased progesterone and estradiol secretion by cultured luteal cells collected in the midluteal phase of the cycle. No additive stimulatory action of gonadotropins and testosterone on progesterone secretion was observed in cultures of luteal cells from the early luteal phase but this was not the case in cultures of luteal cells from the midluteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the expression of cytochrome P450c17 associated with ovarian cystic follicles. An immunocytochemical and enzymatic analysis of porcine ovaries.

The steroidogenic activity of normal preovulatory and cystic follicles, and corpora lutea of porcine ovaries was investigated by immunocytochemical and radioenzymatic techniques. Using a specific antibody to porcine cytochrome P450c17, immunocytochemical staining was specifically localized in the theca interna layer of normal follicles and undetectable in the granulosa layer. The theca interna layers of non-luteinized cystic follicles were immunoreactive while those of luteinized follicles were not. Corpora lutea cells were essentially negative. The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase activity was similar in luteinized cystic follicular and corpora lutea tissues, which had 8 times higher activity than found in normal preovulatory follicles. The formation of either corpora lutea or luteinized cysts led to a profound decline (12- to 15-fold) in 17 alpha-hydroxylase and 17,20 lyase activities compared to normal preovulatory follicles. In agreement with these enzyme findings, radioimmunoassays revealed very high levels of progesterone with nearly undetectable levels of androgens in the luteinized cysts. These studies demonstrate the functional similarities between cells of luteinized cysts and those of normal corpora lutea and suggest a pathology associated suppression of P450c17 expression in porcine cystic follicles.     

Identification of Fatty Acids and Aliphatic Hydrocarbons in Sarcina lutea by Gas Chromatography and Combined Gas Chromatography-Mass Spectrometry

The composition and nature of the fatty acids and hydrocarbons of Sarcina lutea were elucidated by gas chromatography and by combined gas chromatography-mass spectrometry. The distribution of fatty acids found in S. lutea showed two families of pairs, or dyads, of saturated monocarboxylic acids (C12-C18) with and without methyl branching. These pairs of fatty acids showed a pattern of iso and anteiso structures for C13, C15, and C17, and iso and normal structures for C12, C14, and C16. Only the C18 showed unsaturation. The distribution of hydrocarbons in the range C22-C29 showed two families of tetrads of unsaturated aliphatic hydrocarbons all showing methyl branching. Each tetrad was composed of four isomers identified as two iso olefins and two anteiso olefins. The only difference between the tetrads pertaining to different families was found in the relative gas chromatographic retention times of the last two components of each group.