Protein phosphatases 1 and 2A and the regulation of calcium uptake and pollen tube development in Picea wilsonii

To investigate the roles of protein phosphatases 1 and 2A in the development of pollen tubes of Picea wilsonii Mast., pollen grains were cultured in standard medium in the presence and absence of the protein phosphatase inhibitors okadaic acid and calyculin A. At nanomolar concentrations, these compounds blocked pollen tube growth, causing abnormal morphologies of the pollen tubes. Studies with Fluo-3 revealed that the inhibitors reduced the pollen-tube tip-to-base cytoplasmic calcium (Ca(2+)) gradient and arrested extracellular Ca(2+) uptake. The transmission electron microscope observations indicated that the fusion of paramural bodies with plasma membranes occurred frequently in the tip and sub-tip regions of control pollen tubes, but fusion rarely occurred in inhibitor-treated pollen tubes. Staining with aniline blue showed that callose accumulated in the tip regions of inhibitor-treated pollen tubes. Immunolabeling of pollen tubes revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present in the tip region and on the flanks of the sub-tip in normal pollen tubes. In inhibitor-treated pollen tubes, these epitopes existed only in the extreme tip region and at higher concentrations than in control pollen tubes. The esterified pectin recognized by JIM7 was located preferentially at the extreme tip region in normal pollen tubes, but at basal sites in inhibitor-treated tubes. Fourier transform infrared (FTIR) analysis further confirmed the changes in acidic and esterified pectin distributions and their relative contents. These results suggest that protein phosphatase 1 or 2A, or both, are involved in the regulation of Ca(2+) uptake across the plasma membrane, in exocytotic activity and in the biosynthesis of cell wall components, all processes that occur in the tip region of pollen tubes and that control pollen tube development.     

Genetic diversity of Pinus densiflora pollen flowing over fragmented populations during a mating season

We attempted to evaluate the genetic diversity of long-distance transported pollen flowing over fragmented Pinus densiflora populations during a mating season. A P.?densiflora clonal seed orchard, which was located in a rural area where many fragmented populations exist, was selected for pollen capture. Immigrant pollen captured by three clones having different flowering times was regarded as the pollen flowing over fragmented populations during a mating season. The genetic diversity (H _e) values of the immigrant pollen captured by the three clones were high (H _e?>?0.894). The correlation of paternity (r _p) values of the seeds having immigrant parent generated from the three clones were calculated to be negative. From these parameters, the pollen cloud is considered to have maintained high genetic diversity during the mating season. The genetic composition of the pollen cloud showed slight variation. The pollen captured by different trees (i.e., clonal ramets of the three clones) was significantly different based on analysis of molecular variance. Especially, the pollen pools captured by trees planted in the western side of the orchard were significantly different from the gene pool of the surrounding populations. Factors affecting this differentiation could be that the donors of the pollen transported to the orchard vary with time, as well as nonuniform dispersal of the pollen. From these results, the pollen flowing over fragmented P.?densiflora populations is considered to have high genetic diversity, compensating to some extent for fragmentation.     

拟南芥野生型及突变体小孢子发生和发育的比较研究

通过电镜对拟南芥野生型及ｅｐｎ型突变体小孢子发生和发育进一步研究（此突变体的特征是花粉从游离核时期就具有多核、多沟并有分枝的花粉管），值得注意的是突变体在发育至四核花粉母细胞时期，在整个母细胞原生质膜外开始形成小孢子的外壁（与野生型晚期四分体内单倍体小孢子原生质体外形成的外壁相同），此外，在突变体小孢子发生的整个过程中也未见到正常四分体的出现。因此，设想具有两个营养核、两个生殖核的突变体的大花粉直接由非正常的四核花粉母细胞形成，突变体发生缺失的时期在前减数分裂期或减数分裂Ⅱ     

黄牡丹花粉生活力测定方法的比较研究

以黄牡丹的新鲜花粉为试材,利用单因子试验比较了液体培养基中蔗糖浓度、硼、钙、镁、钾对黄牡丹花粉萌发的影响,在此基础上进行了正交试验,比较了蔗糖、H₃BO₃及CaCl₂对黄牡丹花粉萌发的影响;通过对醋酸洋红染色法、I-KI染色法和TTC染色法的比较,寻找快速测定黄牡丹花粉生活力的方法。试验结果表明:蔗糖及H₃BO₃对黄牡丹花粉萌发有极显著影响。在pH值为6.0时,蔗糖150 g·L^-1+H₃BO₃30 mg·L^-1+CaCl₂20 mg·L^-1适宜黄牡丹花粉培养,萌发率为68.7%;纯水培养没有造成花粉原生质体破裂,内含物外流,但萌发率极低,仅为3%;200 g·L^-1以上的高浓度蔗糖溶液和300 mg·L^-1以上的高浓度盐溶液会造成原生质体失水萎缩,质壁分离,这两种情况都抑制花粉萌发;TTC染色法测得的花粉活力率为64.9%,是快速测定黄牡丹花粉生活力的最适染色法。     

柚木花粉离体萌发试验

柚木(Tectona grandis L.f.)是世界名贵的用材树种,天然分布于印度、缅甸、泰国和老挝[1],具有生长快、用途广、纹理美观和价格昂贵的特点,已在热带、南亚热带地区广为引种,是世界上人工林种植面积最大的4个树种之一,也是单位面积产值最高的一个造林树种[2-3]。我国引种柚木已有170多年的     

中国特有种枝毛野牡丹传粉生物学及繁育系统研究

对海南吊罗山国家级自然保护区枝毛野牡丹（Melastoma dendrisetosum）的开花进程,访花昆虫、访花行为、访花频率以及气候条件等进行观察记录,同时检测了花粉活力、柱头可授性、花粉胚珠比、繁育系统,并分析其致濒机制。结果表明：枝毛野牡丹是典型的异型雄蕊植物,5：30—8：30逐步开放,8：30—9：00完全开放,19：00花朵闭合,单花开放时间为12—14 h,群体花期36±2 d,无花蜜。访花昆虫主要有木蜂科、条蜂科、蚁科、食蚜蝇科的昆虫,最有效传粉昆虫为木蜂科昆虫。自然与人工授粉、套袋等试验结果表明,枝毛野牡丹不存在主动自花授粉、无融合生殖的生殖保障现象,为自交亲和的异交种,需要昆虫传粉,枝毛野牡丹繁育系统是兼性自交。枝毛野牡丹相对生殖成功率低仅为0.035,主要限制因子为花粉限制和传粉者限制。     

油杉花粉个体发育与传粉过程

应用常规压片法和整体染色透明法对油杉小孢子的发生和雄配子体的发育过程进行观察,研究其传粉生物学特征.结果表明:油杉小孢子母细胞在1月26日-2月5日进行减数分裂,在减数分裂过程中胞质分裂为同时型,四分体排列方式多为左右对称型;小孢子经过连续4次有丝分裂后,于2月底形成5-细胞型的成熟花粉粒,成熟花粉由两个原叶细胞、一个不育细胞、一个精原细胞和一个管细胞组成,其中两个原叶细胞在花粉成熟时已退化;经TCC法检验,花粉生活力为92.6%;3月1日-8日为油杉传粉期,3日-5日为传粉高峰;在传粉期间,胚珠珠孔端无传粉滴产生,珠孔端耳状结构边缘粘附有花粉.虽然花粉母细胞发育表现出不同步现象,产生少数具3个或4个气囊的异形花粉,但从小孢子的发生及雄配子体发育的整个过程看,未见败育或其他异常现象,能形成大量正常的成熟花粉,因此,花粉个体发育不是影响油杉结籽率低的主要因素,而造成油杉球果结籽率低的主要原因可能在于其传粉过程.     

川榛柱头可授性及花粉管生长特性的研究

以川榛为实验材料,以平榛和平欧杂种榛为对照,采用荧光显微法观察不同环境条件、不同授粉方式、不同花序形态、不同授粉时间柱头上花粉的附着数量、花粉萌发率和花粉管的生长特性,以期确定川榛最佳可授期和授粉方式,掌握花粉萌发所需的适宜温湿度。结果表明:川榛不同花期柱头可授性大小为:盛花期末花期露红期;川榛疏散状花序的可授性优于直簇状,平刷授粉效率优于点授;在加双层湿滤纸的培养皿高湿度条件下,花粉管萌发和生长速度先于温室和田间条件,说明湿润温和的环境更利于川榛花粉的附着、萌发和花粉管的生长;环境温湿度和种质对花粉管萌发具有重要影响,花粉管的生长速度受环境温湿度的影响较大,与种质无关。     

Defensin (TvD1) from Tephrosia villosa exhibited strong anti-insect and anti-fungal activities in transgenic tobacco plants

Plant defensin is a small, cationic, cysteine-rich broad-spectrum antimicrobial peptide with four or five disulfide bridges and has been shown to be a component of the innate immunity system in plants. In the present study, the defensin gene (TvD1) from Tephrosia villosa was overexpressed in tobacco and characterized. Two high-expression (T1, T26) and one low-expression (T13) plant lines were selected through semi-quantitative RT-PCR and used for bioassays along with non-transgenic controls. The high-expression plant line exhibited strong in vivo anti-fungal and anti-feedant activity against the pathogen Rhizoctonia solani and the first and second instar larvae of the Spodoptera litura (F.), the tobacco cutworm, respectively. The low-expression plant line showed a moderate level of tolerance/resistance in the bioassays. The recombinant peptide (rTvD1) exhibited toxicity to tobacco pollen grains in the germination assay, but transgenic plants produced copious fertile pollen and set capsules with viable seeds. The results of this study demonstrate that the single gene (TvD1) effectively controls both fungal and insect pests and, hence, it can be used for crop transformation.     

牡丹远缘杂交新品种及其亲本的花粉形态比较研究

采用光学显微镜及扫描电子显微镜的观察,对4个牡丹亚组间远缘杂交新品种及其亲本的花粉形态进行比较研究。结果表明：当杂交种的遗传组成发生变化时,其花粉形态及外壁纹饰类型必然有一定的改变,但杂交后代中出现的外壁纹饰类型与双亲所具有的类型并无相关性,观察的4个杂交种中除‘夕阳’外,其余均出现了双亲所没有的类型,分别为皱波状、皱波—网状、皱波—粗网状。故仅从花粉形态及外壁纹饰推测牡丹园艺品种的起源还远远不够,必须结合外部形态的观察及细胞学、生化、分子生物学等多方面的依据。此外,对4个亚组间远缘杂交品种及其亲本异常花粉出现的频率统计的结果表明：虽然在亲本中有异常花粉存在,但出现的频率并不高,不会影响到其有性生殖过程;而杂交品种的花粉异常率均超过50%,4个亚组间远缘杂种完全不育。     

Contemporary pollen flow and mating patterns of a subtropical canopy tree Eurycorymbus cavaleriei in a fragmented agricultural landscape

China's subtropical forests have experienced severe deforestation and most native forests have been fragmented into an archipelago-like landscape. The genetic effect of habitat fragmentation depends largely on the level of gene flow within and among population fragments. In the present study, Eurycorymbus cavaleriei, a canopy tree found throughout subtropical China, was selected as a representative insect-pollinated plant species to assess genetic consequence of forest fragmentation. Contemporary pollen dispersal and mating patterns were estimated in two physically isolated stands of E. cavaleriei within fragmented forests using six highly polymorphic microsatellite loci. We found high genetic diversity (H_E = 0.670–0.754) in both adults and offspring in the fragmented agricultural landscape, suggesting that habitat fragmentation did not necessarily erode genetic diversity of E. cavaleriei. Although substantial pollen travelled less than 100 m, paternity analysis revealed that a large amount of long-distance pollination events occurred, with the average pollen dispersal distance being 1107 m and 325 m for the two stands, respectively. Extensive pollen immigration (39.3–42.6%) indicated that there was effective genetic connectivity among E. cavaleriei stands in the fragmented forests. twogener analysis revealed that the exponential power model was the best-fitting dispersal curve with a fat-tailed (b < 1) dispersal feature. The results from a multilocus mating system analysis suggested that a small amount of biparental inbreeding and some correlated mating events occurred in the fragmented forests, which were similar to our parallel findings in the continuous forests of E. cavaleriei. Estimates of pollen pool structure (Φ_FT = 0.128–0.174) indicated large genetic differences between pollen clouds accepted by maternal trees. The number of effective pollen donors (N_ep) in E. cavaleriei, estimated using both mltr (N_ep = 4.2–5.3) and twogener (N_ep = 2.9–3.9) models, was equivalent to the number of effective pollen donors detected in continuous forests of E. cavaleriei. The pollen dispersal and mating patterns detected here indicated that habitat fragmentation did not have a negative impact on pollen movement in E. cavaleriei, possibly due to its generalist pollination system and the resilient foraging behavior of its pollinators in response to changes in landscape structure. The long distances of pollen-mediated gene flow between patches highlight the conservation value of remnant forest fragments in maintaining genetic connectivity at the landscape scale in subtropical China.     

小桐子繁育系统与传粉生态学研究

在野外定位观测小桐子花朵的功能形态特征、开花动态、传粉方式,用杂交指数(OCI)、花粉/胚珠比(P/O)、去雄、套袋、人工授粉等方法分别测定小桐子的繁育系统.结果显示:(1)小桐子是单性花,雌雄同株同序.雄花单花期2 d,雌花单花期5～8 d,雌花直径略大于雄花.(2)雄花开花后9 h内花粉活力较高,24 h后花粉活力明显降低,48 h后花粉基本散失活力;雌花柱头可授性在开花后4 d内最强,5～8 d可授性开始降低,第9天基本失去可授性.(3)传粉方式以虫媒传粉为主,为虫媒植物.(4)繁育系统检测结果为部分自交亲和异交,需要传粉者活动才能完成授粉过程.     

毛白杨花粉败育机制的研究

从细胞遗传学角度较为系统地揭示了毛白杨花粉败育的机制，即主要是毛白杨异质性遗传基础与环境相互作用的结果。（1）在减数分裂中有数目不等的联合程度较差的单价体以及落后染色体出现，这种与异质性相关的染色体行为异常，导致同源染色体向子细胞的不均衡，造成且功能染色体的缺失，从而引起毛白杨一定比率的败育花粉的产生；（2）遗传上的不平衡与温度等环境因子相互作用，进一步引发毛白杨生理乃至结构上的不平衡，花粉母细胞（或孢原细胞）和绒毡层细胞发育异常，从而造成不产粉或产粉较少；环境与基因型互作的差异性导致了花粉败育的年度不稳定性。（3）易位、倒位等染色体结构变异和天然三倍体株系的存在也是造成毛白杨花粉败育的原因。     

Using SSR markers to study the mechanism of 2n pollen formation in Populus × euramericana (Dode) Guinier and P. × popularis

? Using cytological methods and SSR DNA marker analysis, this study revealed the formation mechanisms and the genetic constitutions of the 2n pollen in Populus × euramericana (Dode) Guinier and P. × popularis.

? In P. × euramericana (Dode) Guinier, four abnormalities in microsporogenesis were observed: parallel spindle, fused spindle, tripolar spindle and premature cytokinesis. The first three can lead to first-division restitution (FDR) 2n pollen formation and the last one can form second-division restitution (SDR) 2n pollen. The SSR marker analysis results of parents and their tetraploidy filial generation confirmed that the genetic constitution of 2n pollen produced by P. × euramericana (Dode) Guinier was FDR.

? In P. × popularis, three of these abnormalities were observed: parallel spindle, fused spindle and premature cytokinesis. The SSR marker analysis results showed the genetic constitution of 2n pollen produced by P. × popularis was SDR. Natural 2n female gametes in P. × euramericana (Dode) Guinier are reported for the first time. SSR analysis indicated that natural 2n female gametes of P. × euramericana (Dode) Guinier did exist and were fertile, which could be FDR genetic constitution.

? The results from this study showed a great potential for using 2n gametes to produce polyploid poplar clones, which can be used effectively for polyploid breeding for poplar species in the section Aigeiros.

     

硬头黄竹花的形态与结构研究

[目的]研究硬头黄竹花(Bambusarigidd)的基本形态结构及胚胎发育规律,为其胚胎学积累原始资料,为育种工作打下基础。[方法]通过解剖与形态观察的方法,对硬头黄竹生殖器官、抱子发生及雌雄配子体的发育过程进行解剖观察。[结果]硬头黄竹小穗为假小穗,簇生,基部具潜伏芽,平均长度为3.75 cm,有3~7朵小花。小花内外稈各1片,浆片3枚,雌蕊1枚,雄蕊6枚。雌蕊为三分枝羽毛状柱头。子房长卵圆形,具明显纵向三棱。子房单室,侧膜胎座、倒生胚珠。正常发育的花粉粒为二细胞花粉,花药壁造抱细胞时期为四层药壁,花药成熟时只剩两层药壁。绒毡层为分泌型,花药成熟时,绒毡层完全退化。花药易见发育异常现象,形成不同的败育类型。[结论]硬头黄竹小穗基本结构发育正常,但花药发育后期会大量出现异常现象,这是导致硬头黄竹结实率低的主要原因。     

Effects of transgenic cry1Ab rice pollen on fitness of Propylea japonica (Thunberg)

The transgenic rice lines Kemingdao 1 (KMD1) and Kemingdao 2 (KMD2) contain a synthetic cry1Ab gene derived from Bacillus thuringiensis Berliner and are highly resistant to rice stem borers and foliage-feeding lepidopterans. Propylea japonica (Coleoptera: Coccinellidae) is an important predator of rice insect pests; it also uses rice pollen as a food source under natural conditions. In the present study, the effects of KMD1 and KMD2 pollen expressing Cry1Ab protein on the fitness of P. japonica were assessed in the laboratory. P. japonica larvae and adults were provided with the following four diets: KMD1 pollen with the aphid Myzus persicae, KMD2 pollen with M. persicae, nontransgenic Xiushui 11 (parent variety of KMD1 and KMD2) pollen with M. persicae, and M. persicae only (KMD1–pollen, KMD2–pollen, XS11–pollen, and aphid treatments, respectively). The results showed that the longevity of female adults in the KMD1–pollen treatment was significantly lower than that in the XS11–pollen treatment, but was not significantly different from that in the KMD2–pollen and aphid treatments. Newly emerged males in the KMD2–pollen treatment were evidently less vital than those in the XS11–pollen treatment, but not significantly different from those in the KMD1–pollen and aphid treatments. The development, survival and reproduction indices for the three pollen treatments did not differ significantly from one another. In short, Bt toxin expressed in Bt rice pollen had no evident negative impacts on P. japonica fitness when the pollen was used as a food by this beetle.This revised version was published in March 2005 with corrections to the names of the authors.     

麻竹花粉发育过程观察及其分期

以麻竹为材料,采用醋酸洋红、碘-碘化钾压片以及石蜡切片的观察方法对麻竹花粉发育全过程进行了研究,初步将这一过程划分为8个时期,即小孢子母细胞形成期、小孢子母细胞减数分裂期、小孢子早期、小孢子中期、小孢子晚期、二胞花粉早期、二胞花粉晚期和成熟花粉期,并描述了各个时期的基本特征。麻竹花粉发育过程基本正常,小孢子母细胞减数分裂为连续型,四分体主要为左右对称型,也有四面体形的,成熟的花粉为3-细胞型,具单一萌发孔。     

Pollen dispersal,mating patterns and pollen contamination in an insect-pollinated seed orchard of <Emphasis Type="Italic">Schima superba</Emphasis> Gardn. et Champ.

Pollen flow and mating patterns are the most important factors influencing the genetic structure of insect-pollinated forest trees and are essential parts of genetic management in seed orchards. We investigated pollen flow, the mating system and the level of pollen contamination in a clonal seed orchard of Schima superba Gardn. et Champ. In total, 328 open-pollination progenies coming from 11 mother trees were identified using 13 polymorphic microsatellite loci. A total of 203 full-sib families were identified and were nested among the 11 studied seed donors. The male reproductive success rate from 0.49 to 7.77% for most male parents, with an average of 2.44%. More than 80% of the crosses were found within a distance of 60 m, and the most frequent pollination distance between female parent and male parents was approximately 20 m. Mating system analysis showed that the outcrossing level was very high (t _m  = 1.000, outcrossing rate = 98.5%) in the seed orchard and that there was an average of 2.3 effective pollen donors (N _ep ) per female parent. In addition, the mating success of individual males within neighborhoods was moderately influenced by their fecundity and the direction of their location relative to mother trees. The pollen contamination from outside the seed orchard was high (7.01%). Our findings are valuable for the assessment of seed orchards, and it may be worthwhile to use pollen management strategies to decrease pollen contamination and increase the genetic quality of the seeds produced.     

Effects of selective logging on the mating system and pollen dispersal of Hymenaea courbaril L. (Leguminosae) in the Eastern Brazilian Amazon as revealed by microsatellite analysis

Using nine microsatellite loci, we studied the effects of selective logging on genetic diversity, mating system and pollen dispersal in a population of the tree species Hymenaea courbaril, located in a 546 ha plot in the Tapajós National Forest, Pará State, Brazil. We analyzed 250 offspring (nursery reared seedlings) collected after a logging episode from 14 open-pollinated seed trees. These were compared to 367 seedlings from 20 open-pollinated seed trees previously collected from the pre-logging primary forest. The genetic diversity was significantly lower in the post-logging seed cohort. In contrast to the pre-logging population, significant levels of selfing were detected in the post-logging population (t_m = 0.962, P < 0.05). However, correlated matings were reduced and the effective number of pollen donors almost doubled after harvesting (3.8 against 7.2). Logging also reduced pollen immigration into the plot (from 55% to 38%) and we found no significant correlation between the size of the pollen donors and the number of seeds fathered. Inside the plot, pollen dispersal distance was shorter before logging than after (827 and 952 m, respectively) and the reproductive pollination neighbor area (A_ep) was larger (average of 178 ha). The individual and average variance effective population size within families (ranged from 1.80 to 3.21, average of 2.47) was lower than expected in panmictic populations (N_e = 4). The results indicate that while logging greatly reduced the levels of genetic diversity after logging, it also increased genetic recombination within the population and constrained crossing among related individuals. The results show that low-density tropical tree species such as H. courbaril, when harvested in moderate levels may be resilient to a reduction in the reproductive population and may maintain similar levels of outcrossing and pollen dispersal after logging.     

Short distance pollen movement in a wind-pollinated tree, Quercus lobata (Fagaceae)

Short distance pollen dispersal shapes the local genetic structure of plant populations and determines the opportunity for genetic drift and local selection. In this paper we focus on short distance dispersal (SDD) of pollen in a low-density stand of a savannah oak, Quercus lobata Née. Specifically, we are interested in the proportional contributions of pollen donors, the pollen dispersal kernel that describes local matings, the extent to which wind influences mating success, and the extent to which pollen sources vary within the large canopy of these trees. Using maximum likelihood paternity analysis, we assigned sires for 474 outcrossed progeny of five seed trees, representing 120 of 160 potential mating pairs within a 250 m radius of each focal tree (ca. 20 ha plots). We first established that the effective number of pollen donors for progeny with sires within the plot was about 10 individuals, with average weighted pollination distances of 114.1 m. We estimated 18.5% pollen immigration into the 20 ha plots. We next established that the SDD portion of the dispersal kernel is best described by the exponential power, inverse power, and Weibull functions, all that capture high local dispersal with steep decay. Two of these models suggest that long distance dispersal is abundant, represented by a fat tail, while the Weibull indicates depauperate long distance dispersal, represented by a thin tail. The addition of a directional component corresponding to the predominant wind axis had no meaningful impact on these models. Finally, we established that different parts of an individual tree canopy of Q. lobata sample from the same homogeneous pollen pool showing no bias towards pollen sources near that part of the canopy. Overall findings suggest low-density Q. lobata populations show steep decay of SDD. Policies and ordinances governing the amount of allowable tree removal of savannah oak populations should recommend the preservation of local clusters of adults, as well as some connectivity among clusters.