Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of β-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Flower colour variation in the montane plant Gentiana lutea L. (Gentianaceae) is unrelated to abiotic factors

Background: Flower colour variation among populations may result from the spatial variation of selective agents. The structure of phenotypic variation informs on the ecological processes related to this variation. Variation in floral traits is mainly attributed to variation in the pollinator fauna, while variation in vegetative traits is usually linked to abiotic factors or herbivores.

Aims: We investigated the geographical variation of flower colour (and correlated traits) in Gentiana lutea and the relationship with the variation in abiotic factors.

Methods: Phenotypic variation (flower colour, petal length, petal width, stalk length, leaf length, flower number, petal number and number of basal leaves) was assessed in 429 plants of 12 populations located at north-west Iberia. Additionally, we obtained data on the geographical coordinates, elevation, temperature, rainfall and radiation for each population.

Results: Populations mostly differed in flower colour, from orange to yellow from west to the east. Abiotic factors were unrelated to variation in either floral or vegetative traits.

Conclusions: Phenotypic variation among G. lutea populations does not result from adaptation to environmental factors. Other factors, such as historical events or selective pressure exerted by biotic interactions, might explain the flower colour variation in G. lutea along the Cantabrian Range.     

秦艽与小秦艽光合日变化的研究

运用光合作用测定系统,对野生秦艽(Gentiana macrophylla Pall．)和小秦艽(G．dahurica Fisch．)的开花期的光合特性进行研究。结果表明,2种秦艽的净光合速率、蒸腾速率、气孔导度日变化曲线都呈双峰型,有明显的光合“午休”现象。小秦艽的光合速率、蒸腾速率、气孔导度照著高于秦艽,分别高出2．7 μmolCO2·m-2·s-1、1．5 mmol·m-2·s-1和140．7 mmol．m-2·s-1而叶温则低于秦艽2．8C。相关分析表明,开花期的温度、光照对2种秦艽的光合都有一定影响,蒸腾速率、气孔导度调节光合作用,而引起2种秦艽光合速率降低的主要因素为非气孔因素。     

4种秦艽属植物不同器官中4种环烯醚萜苷成分含量的比较分析

利用HPLC法测定了秦艽(Gentiana macrophylla Pall.)、粗茎秦艽(G.crassicaulis Duthie ex Burk.)、麻花秦艽(G.straminea Maxim.)和小秦艽(G.dahurica Fisch.)根、茎、叶和花中4种环烯醚萜苷成分(包括马钱苷酸、獐牙菜苦苷、龙胆苦苷和獐牙菜苷)的含量,并对来源于不同产地秦艽和小秦艽不同器官4种环烯醚萜苷成分的含量进行了比较。结果显示:4种秦艽属植物不同器官4种环烯醚萜苷成分含量差异明显,来源于3个产地(陕西太白、甘肃西河乡和马狭)的秦艽和2个产地(青海互助和陕西麟游)的小秦艽不同器官中4种环烯醚萜苷含量也均具有明显差异。4种秦艽属植物根、茎、叶和花中龙胆苦苷和马钱苷酸总量分别为质量分数5.996%～10.869%、0.310%～4.065%、0.235%～4.138%和0.545%～5.591%;根中獐牙菜苦苷和獐牙菜苷的质量分数分别为0.516%～0.953%和0.042%～0.210%,茎中分别为0.173%～0.383%和0.031%～1.700%,叶中分别为0.068%～0.684%和0.020%～3.208%,花中分别为0.460%～0.832%和0.138%～3.827%。粗茎秦艽各器官龙胆苦苷和马钱苷酸总量均最高,小秦艽各器官龙胆苦苷和马钱苷酸总量均最低,总体上,秦艽和粗茎秦艽中龙胆苦苷和马钱苷酸总量高于小秦艽和麻花秦艽;小秦艽茎、叶和花中獐牙菜苷含量均最高,而秦艽茎和叶及粗茎秦艽花中獐牙菜苷含量均最低。4种秦艽属植物根部龙胆苦苷和马钱苷酸含量均明显高于茎、叶和花,獐牙菜苦苷在根和花中的积累较多,獐牙菜苷在花中的含量均相对最高。产自青海互助的小秦艽茎、叶和花中獐牙菜苷含量均最高,质量分数分别为2.884%、5.215%和7.321%。研究结果表明:4种环烯醚萜苷成分含量不但与植物种类和器官有关,而且与产地和采样时间也有关。     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morpho-logy always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermedi-ate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence compar-ison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) inter-genic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we exam-ined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species.     

麻花艽和管花秦艽( 龙胆科) 之间自然杂交类型的分子验证

野外考察发现麻花艽( Gentiana straminea Maxim. ) 和管花秦艽( G. siphonantha Maxim . ex Kusn. ) 同域分布时存在大量形态位于二者之间的个体。经形态变异研究后发现它们可能是这两个物种之间的杂交后代。对两个亲本种以及假设杂交群体共55 个个体的核糖体ITS 序列和叶绿体trnS-G 序列的分析结果表明:中间形态个体是麻花艽和管花秦艽的自然杂交后代。此外分析了两个亲本种以及杂交群内个体间trnS-G和ITS 序列的变异状况以及分子标记结果与形态鉴定不一致的可能原因; 指出可能是杂交诱导的叶绿体基因组重组以及早期物种分化中的谱系筛选不彻底等原因造成了亲本种群体内序列变异的多样化。     

粗茎秦艽与西藏秦艽(龙胆科)种间的形态学、染色体与分子界定

粗茎秦艽Gentiana crassicaulis Duthie ex Burk和西藏秦艽G. tibetica King ex Hook. f.的主要区别是前者花冠小、檐部蓝色, 后者花大、檐部紫褐色; 但二者在西藏东南部地区的形态界限不清楚, 具有较多的中间形态过渡类型。本文以该地区居群作为主要研究对象, 并以云南和西藏其他地区的典型形态类群为对照, 比较研究了两个物种间的形态学、染色体和分子标记界定。共研究了10个居群69个个体的trnS-G和ITS序列、6个居群26株植株的染色体倍性和54份标本的形态变异。结果表明, 花冠大小与染色体倍性密切相关。云南等地分布的粗茎秦艽均为二倍体(2n=26), 花冠较小; 西藏西部的西藏秦艽均为四倍体(2n=52), 花冠较大。而西藏东南部的形态过渡类型也为四倍体, 花冠较大, 且其中的部分植株花颜色变浅, 类似西藏秦艽。但母系与双亲遗传背景的分子标记都表明西藏东南部居群与典型形态特征上的粗茎秦艽聚为一支, 应属于粗茎秦艽; ITS序列上也未发现杂合位点。这些分子证据都不支持近期杂交渗入造成形态过渡性的假设。综合分析表明多倍化造成了粗茎秦艽种内形态特征的变异, 其四倍体个体与四倍体西藏秦艽在形态特征上的趋同, 最终导致了二者在西藏东南部地区形态界限的模糊。但是, 分子证据可以很好地区分这种多倍化造成的形态趋同。我们的研究结果还表明, 综合利用分子、染色体和广义形态证据来解决近缘种之间种范围的界定具有更大的优势。此外, 还讨论了粗茎秦艽不同染色体倍性的分布以及该高山物种可能的起源与扩散途径。     

中国广西唇柱苣苔属(苦苣苔科)一新种——黄花牛耳朵

报道了在广西发现的苦苣苔科唇柱苣苔属Chirita一新种,即黄花牛耳朵C.lutea Yan Liu & Y.G.Wei.黄花牛耳朵的体态和叶形与牛耳朵(C.eburnea)相似,但前者聚伞花序俯垂,花冠黄色,花药与退化雄蕊先端密被白色绵毛.     

Occurrence of the rotifer,Habrotrocha cf. rosa Donner,in the purple pitcher plant,Sarracenia purpurea L., (Sarraceniaceae) along the eastern seaboard of North America

Sarracenia purpurea L. plants were studiedin seven bogsalong the eastern seaboard of the United States fromNew Jersey to Georgia. Water sampled from 225 leaveswas found to contain several invertebrates. A singlebdelliod rotifer, a form of Habrotrocha cf. rosaDonner very similar to that described by Bateman(1987) in S. purpurea in western Newfoundland,was identified in 70% of the leaves. Investigationsare in progress to examine the possibility that thisstrong association indicates H. cf. rosa isspecific to S. purpurea.     

A barcode analysis of the genus Lobophora (Dictyotales,Phaeophyceae) in the western Atlantic Ocean with four novel species and the epitypification of L. variegata (J.V. Lamouroux) E.C. Oliveira

In the western Atlantic Ocean, the brown algal genus Lobophora is currently represented by a single species, L. variegata, with a type locality designated by Lamouroux as ‘Antilles’. In this study, we used molecular-assisted alpha taxonomy (MAAT) to assess species diversity of Lobophora in Bermuda, the Florida Keys, St. Croix and Guadeloupe (Lesser Antilles). Using cox1 and cox3 sequences as barcode markers, five species of Lobophora, four of them novel, were delineated, all previously having been identified in the area as L. variegata. Our morphological and habitat studies, made possible by abundant sampling, have revealed unique characters for each of these western Atlantic species, including distinct cellular arrangements, as well as different depth ranges for certain species. Observations made from Lamouroux’s holotype of Dictyota variegata (= Lobophora variegata) allowed us to assess the anatomy of this species, which enabled us to easily align this early taxon to one of our genetic species from the western Atlantic. As the type was unavailable for genetic analysis, we selected a recent St. Croix (Virgin Is., Antilles) specimen as the epitype to support it with molecular sequence data.     

Scope for genetic enhancement of the parasitisation potential of four native strains of Trichogrammatoidea sp. nr. lutea Girault (Hymenoptera: Trichogrammatidae) in Kenya

In response to emerging interest in commercial mass production of Trichogramma for Helicoverpa armigera biocontrol in eastern Africa, laboratory experiments were undertaken to assess the scope for genetic enhancement of the parasitisation potential of native strains of the local common trichogrammatid species, Trichogrammatoidea sp. nr. lutea. Four promising strains (ex-Kilifi – Kilifi District, ex-Kwa Chai – Kibwezi District, ex-Rarieda – Bondo District and ex-Ebuhayi, Kakamega District) were tested for cross-mating in reciprocal combinations with focus on fecundity and progeny female ratio. While all the crosses resulted in F1 progeny of both sexes, significant differences were observed between homogamic and reciprocal heterogamic crosses in fecundity, progeny production, proportion of female progeny and adult longevity. Among all the crosses, the cross between ex-Rarieda strain females and ex-Kilifi strain males resulted in progeny that was significantly superior in fecundity and progeny female ratio. Conversely, Kilifi strain females crossed to males from ex-Rarieda strain gave rise to progeny with relatively low fecundity and female ratio. There were significant differences between homogamic crosses and most reciprocal heterogamic crosses in the major biological attributes. Genotypic and phenotypic variance-covariance matrices generated for six life-history traits showed high positive correlations for most traits in both inbred (P<0.05) and reciprocal heterogamic crosses (P<0.05 and P<0.001). Fecundity and number of female offspring were the most important factors in the heterogamic crosses. The results confirmed the scope for genetic enhancement through inter-strain crossing for improving the field impact potential of T. sp. nr. lutea being targeted for commercial mass production.     

黄花牛耳朵(苦苣苔科)的传粉生物学

通过野外观察和繁育系统的实验,对黄花牛耳朵(Chirita lutea Yan Liu et Y.G.wei)的传粉生物学进行了研究.结果表明,黄花牛耳朵的花期从7月初至8月底,单株花期约35～47 d,单花化期约6～10 d,花的开放无固定的时间.在花期内花粉活性约80%,柱头可授性约75%～90%.花粉/胚珠比率(P/O)为1 215.73±266.13.柱头在花药散粉时已生长至花筒口部,明显高于花药,便于接受异花花粉.黄花牛耳朵不存在无融合生殖,高度白交亲和,但较难发生自动的白花授粉,产生种子主要依靠传粉媒介.自然授粉的结实率明显低于人工授粉的结实率,存在传粉限制,蜜蜂(Apidae sp.)、方头泥蜂(Crabro sp.)、无垫蜂(Ameglla sp.)是主要的传粉者.     

光果甘草与乌拉尔甘草清除活性氧能力的比较

采用化学发光法测定了光果甘草（Glycyrrhiza glabra L.）与乌拉尔甘草（G.uralensis Fisch.）中总黄酮和总皂甙对3种重要活性氧（O2^-、OH和H2O2）的清除能力,其中光果甘草对活性氧的清除能力超过乌拉尔甘草;黄酮类化合物对3种活性氧的清除能力强于皂甙类化合物。因此,若以清除活性氧为应用目标,应尽量使用光果甘草。     

Polytene chromosome maps in four species of tsetse flies Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans (Diptera: Glossinidae): a comparative analysis

Photographic polytene chromosome maps from pupal trichogen cells of four tsetse species, Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans were constructed and compared. The homology of chromosomal elements between the species was achieved by comparing banding patterns. The telomeric and subtelomeric chromosome regions were found to be identical in all species. The pericentromeric regions were found to be similar in the X chromosome and the left arm of L1 chromosome (L1L) but different in L2 chromosome and the right arm of L1 chromosome (L1R). The L2 chromosome differs by a pericentric inversion that is fixed in the three species, G. pallidipes, G. morsitans morsitans and G. m. submorsitans. Moreover, the two morsitans subspecies appeared to be homosequential and differ only by two paracentric inversions on XL and L2L arm. Although a degree of similarity was observed across the homologous chromosomes in the four species, the relative position of specific chromosome regions was different due to chromosome inversions established during their phylogeny. However, there are regions that show no apparent homology between the species, an observation that may be attributed to the considerable intra—chromosomal rearrangements that have occurred following the species divergence. The results of this comparative analysis support the current phylogenetic relationships of the genus Glossina.     

秦艽及其近缘种植物资源在我国的分布

秦艽是我国大宗中药材之一,在传统中医药理论中有重要的应用价值。近年来伴随秦艽药用价值研究的不断深入,其需求量不断增加,致使秦艽野生资源日趋减少。通过野外考察和资料查阅,系统阐述了秦艽组植物资源在我国的分布概况,并详细介绍了药典规定品种的具体分布,从而为合理利用和保护秦艽资源,开发新资源提供参考。     

Paraboea guilinensis L. Xu & Y. G. Wei,a new species of Gesneriaceae from Guangxi,China

A new species of Gesneriaceae, Paraboea guilinensis L. Xu & Y. G. Wei, is described from Guangxi, China. The new species is similar to P. crassifolia (Hemsl.) Burtt in its habit and leaf form, but differs by its leathery leaves, glabrous peduncles, pedicels and calyx, distinctly bilabiate corollas, and capsules not spirally twisted.     

Pollination ecology of Gentiana siphonantha (Gentianaceae) and a further comparison with its sympatric congener species

In this study, we studied pollination ecology of Gentiana siphonantha (Gentianaceae), a late-autumn flowering alpine perennial in the northeastern Qinghai-Tibetan Plateau for two years for the first time. We also aimed to compare the pollination differentiation between this species and sympatrically distributed G. straminea that has a close phylogenetic origin but is flowering early. Flowers of G. siphonantha are characteristic of di-chogamy and herkogamy and this floral development prevents occurrence of autonomous self-pollination. This implication was confirmed by the breeding experiments, since no seed was produced when flowers were isolated. However, this gentian proved to be highly self-compatible when geitonogamous selfing was artificially induced. Each individual plant of this species has an average of 14.6 flowers ranging from 4 to 31, at both staminate and pistillate stages with a ratio of 1.2:1 in full anthesis. Both floral development and breeding experiments suggested that pollen vectors were indispensable for successful seed sets of this species. A great variety of insects were observed to visit this species, but the most common and only legitimate pollinator is Bumbus sushikini. A statistic observation suggested that this pollinator showed no preference to either staminate or pistillate flowers and visited them at random. We further calculated the frequency of their visits between and within individual plants. Among the pollinators’ bouts, the proportions of geitonogamous visits within an individual plant occupy about 87.8%. Such a case implied that geitonogamous selfing prevails in this species in spite of floral dichogamy and her-kogamy that were suggested to promote outcrossing. Compared with sympatric G. straminea, the total floral longevity and the male and female duration of this species are shorter. However, the number of flowers of each individual plant and branch increased when in full anthesis. It is interesting that both closely related species shared the same pollinator despite their distinct difference in flower morphology. This finding is inconsistent with the previous hypothesis that both flower color and corolla tube depth have coevolved with different pollinators during speciation and formation of reproductive isolation. Both visit frequencies of the individual flower and geitonoga-mous visits within the individual plant are higher in G. siphonantha than in G. straminea. This difference may result from their different inflorescence designs that actively act upon behaviors of pollinator. Although these two species differed in flowering phenology, their flowering stages overlapped for a few days, suggesting incomplete pollination isolation between them.     

离子注入加速棉花陆海杂种农艺性状遗传稳定性的研究

利用３０ＫｅＶ的Ｎ＾＋离子束注入陆地棉和海岛棉的花粉及杂交Ｆ１种子,分析了杂种后代农艺性状遗传稳定性的变化。结果表明：Ｎ＾＋离子辐照能显著地加速杂种后代的遗传稳定性,Ｆ４Ｍ４代性状稳定度可达９０％左右。Ｆ１种子辐照后代农艺性状多偏向陆地棉,而且花粉辐照杂交后代农艺性状表现出偏母遗传。同时以＾６０Ｃｏγ射线效应为对照,初步探讨了Ｎ＾＋离主入加速遗传稳定的机制。