Stability in and correlation between factors influencing genetic quality of seed lots in seed orchard of Pinus tabuliformis Carr. over a 12-year span

Coniferous seed orchards require a long period from initial seed harvest to stable seed production. Differential reproductive success and asynchrony are among the main factors for orchard crops year-to-year variation in terms of parental gametic contribution and ultimately the genetic gain. It is fundamental in both making predictions about the genetic composition of the seed crop and decisions about orchard roguing and improved seed orchard establishment. In this paper, a primary Chinese pine seed orchard with 49 clones is investigated for stability, variation and correlation analysis of factors which influence genetic quality of the seed lots from initial seed harvest to the stable seed production over a 12 years span. Results indicated that the reproductive synchrony index of pollen shedding has shown to be higher than that of the strobili receptivity, and both can be drastically influenced by the ambient climate factors. Reproductive synchrony index of the clones has certain relative stability and it could be used as an indication of the seed orchard status during maturity stage; clones in the studied orchard have shown extreme differences in terms of the gametic and genetic contribution to the seed crop at the orchard's early production phase specifically when they severe as either female or male parents. Those differences are closely related to clonal sex tendency at the time of orchard's initial reproduction. Clonal gamete contribution as male and female parent often has a negative correlation. Clone utilization as pollen, seed or both pollen and seed donors should consider the role it would play in the seed crop; due to numerous factors influencing on the mating system in seed orchards, clonal genetic contribution as male parent is uncertain, and it has major influence on the genetic composition in the seed orchard during the initial reproductive and seed production phase.     

林木种子园花粉污染与防治措施

林木种子园花粉污染是影响种子园种子遗传品质的重要因子,由于野生同种花粉进入种子园而增加遗传损失。本文综述了国内外林木种子园花粉污染研究的概况、污染水平、影响污染水平的因子和减少污染的措施。研究表明：DNA分子标记在分析种子园交配动态和种子园管理效果方面非常有效（例如,cpDNA、RAPD和SSR）;种子园污染水平比预期的更高;花粉产量好像对污染水平没有影响;空间和生殖隔离也没有产生预期的效果。目前,人工辅助授粉和疏伐是减少花粉污染的两种有效措施。为了改善种子园林木的亲本平衡,人工处理技术的使用,例如：赤霉素A4/7处理技术和无性繁殖技术在种子园很受欢迎。污染程度其他方面的研究工作仍在继续,本文提到的基因流的研究。并对我国种子园花粉污染研究和花粉管理提出建议：当分析种子园花粉污染时,涉及到花粉基因型自然的（花粉扩散）、生理（开花时间）和花粉生活力等因素都应该被考虑。     

Mating dynamics of Scots pine in isolation tents

Seed orchards are forest tree production populations for supplying the forest industry with consistent and abundant seed crops of superior genetic quality. However, genetic quality can be severely affected by non-random mating among parents and the occurrence of background pollination. This study analyzed mating structure and background pollination in six large isolation tents established in a clonal Scots pine seed orchard in northern Sweden. The isolation tents were intended to form a physical barrier against background pollen and induce earlier flowering relative to the surrounding trees. We scored flowering phenology inside and outside the tents and tracked airborne pollen density inside and outside the seed orchard in three consecutive pollination seasons. We genotyped 5683 offspring collected from the tents and open controls using nine microsatellite loci, and assigned paternity using simple exclusion method. We found that tent trees shed pollen and exhibited maximum female receptivity approximately 1 week earlier than trees in open control. The majority of matings in tents (78.3 %) occurred at distances within two trees apart (about 5 m). Self-fertilization was relatively high (average 21.8 %) in tents without supplemental pollination (SP), but it was substantially reduced in tents with SP (average 7.7 %). Pollen contamination was low in open controls (4.8–7.1 %), and all tents remained entirely free of foreign pollen. Our study demonstrates that tent isolation is effective in blocking pollen immigration and in manipulating flowering phenology. When complimented with supplemental pollination, it could become a useful seed orchard management practice to optimize the gain and diversity of seed orchard crops.     

Gene flow and mating system in five <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don seed orchards as revealed by analysis of microsatellite markers

We investigated gene flow in five Cryptomeria japonica D. Don seed orchards of two different types (common and miniature) at widely spaced locations using microsatellite markers. The quality of a seed crop is determined by many factors, including pollen contamination from outside sources, self-fertilization, and the proportion of contributions from constituent clones. Contamination rates were found to vary among ramets both within seed orchards (10.0–76.7% in the most variable seed orchard) and among seed orchards (35.0–65.8% on average). Among ramets, there were significant negative correlations between pollen contamination rate and their distance from the orchard edge; among seed orchards, there were significant positive correlations between the pollen contamination rate and the C. japonica forest area nearby. Some proportion of the pollen (10.7% of total contamination) also migrated from parts of the orchards that had not been treated with gibberellin to induce flowering. Self-fertilization rates varied among seed orchards (1.4–4.4% on average), and there were significant positive correlations between self-fertilization rate and the number of ramets per clone in both types of seed orchard. Contributions as pollen donors differed significantly among clones in all seed orchards. The distance between planted ramets, flowering phenology, and relative pollen fecundity may also have contributed to observed differences in paternal contribution. The influence of these factors on genetic potential did not differ greatly between the two types of orchards. This work was supported by grants from the Pioneer Special Studies Program of the Japanese Ministry of Agriculture, Fishery, and Forestry; the Program for Promotion of Basic Research Activities for Innovative Biosciences; and research fellowships from the Japan Society for the Promotion of Science for Young Scientists.     

Contrasted pollen capture mechanisms in Phyllocladaceae and certain Podocarpaceae (Coniferales)

Comparative study shows that Phyllocladus and representative Podocarpaceae differ in the mechanism by which pollen is introduced into the pollen chamber and onto the apex of the nucellus ("pollen capture"). Both types involve a pollination drop, but only in Podocarpaceae is it consistently inverted and in contact with adjacent surfaces. Phyllocladus has functionally nonsaccate pollen (although a vestigial saccus has been claimed); its pollen is wettable and sinks in water. Podocarpaceae (except Saxegorhaea) have saccate pollen, which is nonwettable and floats on water. In Phyllocladus the pollination drop receives the pollen directly and presence of pollen stimulates complete drop withdrawal, which may be a metabolic process. Once pollinated, an ovule does not resecrete a pollination drop. In Podocarpaceae the drop usually receives the pollen indirectly via pollen scavenging and saccate pollen is preferentially captured. The retraction of the drop appears to be the result of evaporation and is presumably nonmetabolic. Drop secretion can be repeated in the presence of pollen. A major consequence of these contrasted mechanisms is that in Phyllocladus the entire contents of the pollination drop are ingested, whereas in Podocarpaceae only that part of the drop that includes saccate pollen is ingested. Because of differences in repeatability of the secretion process, Podocarpaceae are likely to capture more pollen. In neither mechanism does the process favor 'own" pollen. but in Podocarpaceae all but saccate pollen is excluded. We thus have further evidence for differences in pollen capture mechanisms in conifers with a pollination drop, and differences in the behavior of the pollination drop itself.     

Gene flow estimation with microsatellites in a Malagasy seed orchard of Eucalyptus grandis

Eucalyptus grandis has a mixed-mating reproductive system. Malagasy Eucalyptus seed orchards were established 15 years ago with two aims both based on panmixia: open-pollinated seed production and genetic improvement. The panmixia hypothesis has never been confirmed in the seed orchard. From a seedling seed-orchard stand comprising 349 trees and using data obtained with six selected microsatellite markers, paternity analysis was performed for 724 offspring collected on 30 adult trees. Paternity assignment, based on exclusion procedures and likelihood-ratio method, was achieved with high accuracy; the exclusion probability value was 0.997. The outcrossing rate was very high (96.7%). More than 50% of potential male trees (199 out of 349) in the seed orchard contributed to pollination for 440 offspring of 30 progenies (8.6% of the basic population). The pollination rate from outside the seed orchard was high (39.2%), but might be due to the small size of this seed orchard. This study showed that "panmixia-like pollination" can be assumed.     

杉木种子园种子产量及其主导影响因子的分析

 为研究我国杉木种子园产量的主导影响因子及影响效应,调查收集了安徽西田、福建洋口和官庄、广东小坑4个林场杉木（Cunninghamia lanceolata）种子园历年种子产量及与种子生产年份对应的有关气象资料,以开花受粉、幼果发育各个不同时期的20个气象因子与种子产量进行逐步回归分析,探讨我国杉木种子园产量的大小年现象及周期特征。研究结果表明,杉木种子园投产初期大小年现象不显著,投产中、后期大小年现象出现的年度及周期特征完全一致,种子大小年周期一般为2～3年。在同一地区气候条件相似的情况下,即使立地条件、     

Prediction of genetic gain and gene diversity in seed orchard crops under alternative management strategies

Genetic gain and the gene diversity of seed crops from clonal seed orchards were formulated considering genetic selection, fertility variation and pollen contamination, and compared for five different management strategies. Genetic response was studied as a function of orchard management tactics. Management variables included the proportion of clones left after genetic thinning and/or selective seed harvesting. Formulae were derived to calculate gene diversity (expressed as group coancestry or status number) based on the sex ratio in an orchard population. The influence of having different sets of clones serving as seed parents, or pollen parents, or as both, was analysed. In addition, the impact on genetic gain and the gene diversity of seed crops was studied quantitatively as a function of the quantity and quality of gene flow from outside the orchard. The negative impact of fertility variation among orchard genotypes on the gene diversity of the seed crop was quantified. Numerical examples were given to illustrate the impact of orchard management alternatives on genetic gain and gene diversity. The formulae and results of this study can be used for identifying favourable alternatives for the management of seed orchards. Received: 16 December 2000 / Accepted: 13 March 2001     

Performance of microsatellites for parentage assignment following mass controlled pollination in a clonal seed orchard of loblolly pine (Pinus taeda L.)

Mass controlled pollination (MCP), involving large-scale application of pollen on physically isolated female reproductive organs, has been a lower cost alternative to controlled pollination for the commercial production of genetically improved seeds. Nevertheless, rare are the studies that examined the efficacy of operational MCP and no such assessment has been done in loblolly pine to date. The success of MCP was assessed by a microsatellite-based investigation of the realized versus expected parentage of a set of 300 Pinus taeda offspring in 19 families generated in two subsequent rounds of MCP in 2005 and 2006 in a clonal seed orchard in Brazil. Multi-locus combined probability of parentage exclusion both theoretical and realized from actual testing was >99 % for single parent and parent pair testing when using nine or ten markers. Parentage assignments carried out under a maximum likelihood framework revealed a significantly higher success rate of MCP in 2006 (84 %) following technical improvements adopted to minimize pollen contamination and maximize male reproductive success, although significant variability in the correct maternity and full parentage was seen among individual families. The observed patterns of unexpected parentage indicated that this variability likely resulted from mislabeling of clonal ramets of the parents used in the crosses which impacted both maternity and paternity. Preventing pollen contamination will not be sufficient for successful MCP if inaccuracies exist in the identity of the clonal plants that ultimately provide pollen and female strobili, showing that DNA marker auditing and correction of identity of all ramets in a clonal seed orchard should be a standard practice in the operational implementation of MCP.     

Contributions of Pollen Morphology to the Phylogeny of Some Ranalean Families

The hypothesis that gene flow northwards by long distance wind dispersal of pollen is a contributing factor in Scots pine (Pinus sylvestris L.) seed crops in northern Scandinavia (Umeå) was examined. Pollen was observed in the air before local pollen shedding began. This pollen has been shown to germinate and is likely to be viable. This early pollen was probably not of local origin. Wind patterns and the geographic pattern of pollen shedding were such that it is likely that the origin of the early pollen was hundreds of km to the south. Since many of the female strobili are receptive to pollination before local pollen shedding occurred, early non-local pollen has an advantage over later local pollen. Thus, the gene flow caused by pollen migration is likely to be large enough to be of evolutionary significance.     

极小种群野生植物崖柏的生殖物候、传粉及胚胎发育研究

以濒危植物崖柏（Thuja sutchuenensis Franch.）为对象,对其生殖物候、传粉机制进行观察,并采用石蜡切片法对其胚胎发育过程进行研究。结果显示：崖柏于8月分化出大、小孢子叶球,次年3月传粉,为花粉无气囊、具传粉滴、胚珠直立型传粉机制,球果于10月开裂;显微观察发现,传粉期花粉进入珠孔后,贮藏在珠心上方的贮粉室内,同时珠心组织中分化出孢原细胞,进入雌配子体发育阶段,5月中旬,花粉管开始萌发,6月初完成受精,进入胚胎发育阶段,10月初,胚胎发育成熟。研究表明崖柏从大、小孢子叶球形成至种子成熟的整个发育过程中均存在败育,而胚珠败育及雌配子体游离核时期至幼胚发育期间的败育是其生殖障碍的主要原因。本研究获得了崖柏生殖生物学的基础资料,为其人工繁育和制定保护策略提供了重要依据。     

Significance of exine shedding in Cupressaceae-type pollen

In conifers, which have non-saccate Cupressaceae-type pollen, the pollen must land on a pollination drop or be picked up by the pollination drop from the surface of the cone near the ovule before it can be taken into the ovule. After contact with the drop, the pollen intine absorbs moisture from the drop, expands and the exine is shed. In this study the significance of the shedding of the exine is interpreted from experiments in which simulated pollination drops and micropyles were used to determine the movement of pollen and other particles in suspension. The non-expanded pollen, which can be observed upon contact with the pollination drop, sheds the exine, which then functions as a non-elastic particle, while the pollen from which the exine was shed swells and functions as an elastic particle because it is enclosed by the flexible intine. Non-elastic particles are not easily transferred through narrow passages (the micropyle and micropylar canal) and tend to plug these passages. However, elastic particles, such as the swollen pollen, are easily transferred along narrow passages even when non-elastic particles are present. The simulated experiments demonstrate that exine shedding is an important feature in getting pollen through the narrow micropyle and micropylar canal to the nucellus of the ovule.     

Ovulate cone,pollination drop,and pollen capture in Sequoiadendron (Taxodiaceae)

In Sequoiadendron ovules are borne inside the ovulate cone, and pollination drops secreted from these ovules collect pollen. We examined: (1) the relation between ovular position and pollen capture; (2) pollen behavior when in contact with a pollination drop; and (3) ultrastructure of ovules during pollination drop secretion. During wet periods a water sheet forms on the surface of the cone due to bract shape and wettability. Pollination drops persist inside the wetted cone, and pollen capture resumes immediately after drying. Pollen landing on a pollination drop is taken inside the drop and carried into the micropyle when the drop contracts. Several notable ultrastructural features appear in the nucellus, integument, chalaza, and bract lamina during pollination-drop secretion. The abaxial surface of the lamina is covered by a membrane that may contribute to the wettable nature of the surface.     

Pollination drop in Juniperus communis: response to deposited material

BACKGROUND AND AIMS: The pollination drop is a liquid secretion produced by the ovule and exposed outside the micropyle. In many gymnosperms, pollen lands on the surface of the pollination drop, rehydrates and enters the ovule as the drop retracts. The objective of this work was to study the formation of the pollination drop in Juniperus communis, its carbohydrate composition and the response to deposition of conspecific pollen, foreign pollen and other particulate material, in an attempt to clarify the mechanism of pollination drop retraction. METHODS: Branches with female cones close to pollination drop secretion were collected. On the first day of pollination drop exposure, an eyelash mounted on a wooden stick with paraffin was used to collect pollen or silica gel particles, which were then deposited by contact with the drop. Volume changes in pollination drops were measured by using a stereomicroscope with a micrometer eyepiece 3 h after deposition. The volume of non-pollinated control drops was also recorded. On the first day of secretion, drops were also collected for sugar analysis by high-performance liquid chromatography. KEY RESULTS: The pollination drop persisted for about 12 d if not pollinated, and formed again after removal for up to four consecutive days. After pollination with viable conspecific pollen, the drop retracted quickly and did not form again. Partial withdrawal occurred after deposition of other biological and non-biological material. Fructose was the dominant sugar; glucose was also present but at a much lower percentage. CONCLUSIONS: Sugar analysis confirmed the general trend of fructose dominance in gymnosperm pollination drops. Complete pollination drop withdrawal appears to be triggered by a biochemical mechanism resulting from interaction between pollen and drop constituents. The results of particle deposition suggest the existence of a non-specific, particle-size-dependent mechanism that induces partial pollination drop withdrawal. These results suggest that the non-specific response may decrease the probability of pollen landing on the drop, reducing pollination efficiency.     

Wind or insect pollination? Ambophily in a subtropical gymnosperm Gnetum parvifolium (Gnetales)

Gnetum (Gnetales) has long been regarded as insect pollinated due to its range‐restricted distribution in tropical rain forests, where wind pollination is supposed to be detrimental. However, ubiquitous pollen limitation in the tropics might cause transition to anemophily, or even ambophily for reproductive assurance, especially for gymnosperms such as Gnetum, which exhibit various anemophilous syndromes, including absence of petals, greenish color of strobili, dioecy and uniovulate flowers. Pollination treatments applied to Gnetum parvifolium in southern China revealed this rare and endangered species to be pollen limited and incapable of apomixis. Pollen grains of G. parvifolium did not adhere to each other and could be trapped by interception traps near the male and female strobili. Seed set in the netted treatment (anemophily) was significantly higher than in the bagged treatment (apomixis) but lower than open pollination (anemophily and entomophily), indicating that both wind and biotic pollination contributed to seed production of G. parvifolium. The occurrence of ambophily in Gnetum and the prevalence of anemophily in Ehpedra and Welwitschia suggest that wind may also play a role in the pollination of the ancient Gnetales.     

Anatomical observations on the nucellar apex of Wellwitschia mirabilis and the chemical composition of the micropylar drop

Summary In the young ovule of Welwitschia mirabilis the nucellar apex is dome shaped and starch begins to accumulate near the female gametophyte. With the degeneration of the cells of the nucellar apex, a pollen chamber is formed, which contains the micropylar fluid. Starch storage increases considerably in the upper part of the nucellus. Pollen drop emission is not a rhythmic process, and pollination does not produce the rapid withdrawal of droplets. The micropylar drop consists almost entirely of sugars, uronic acids and a very small amount of free amino acids and enzymes. The mechanism of micropylar drop secretion and its probable role in the process of pollination is discussed.This work was supported by a grant from MURST 40%     

Pollination biology of Gnetum (Gnetaceae) in a LOWLAND MIXED DIPTEROCARP forest in Sarawak

Pollination biology of two Gnetum species was studied in a lowland mixed dipterocarp forest in Sarawak, Malaysia. A dioecious shrub species, G. gnemon var. tenerum, flowered in the evening and its strobili emitted a stinking odor. Pollination droplets were secreted from ovules on female strobili and from sterile ovules on male strobili in the evening. These strobili were visited by nectar-seeking moths of Pyralidae and Geometridae. Sticky pollen was attached on proboscides and antennae of these moths. A dioecious woody climber, G. cuspidatum, had cauline strobili on woody stems near the forest floor. Its male strobili emitted a funguslike odor in the evening and secreted nectar on collars that subtended “flower” rings. The strobili were visited by small flies of Lauxaniidae (Diptera), onto the antennae and legs of which pollen was attached. Evaporation of the exposed droplet/nectar of these Gnetum flowers seemed to be minimized by nocturnal flowering near the tropical rain forest floor. Another floral-structural constraint of gymnosperms, i.e., lack of showy petals, was compensated for by floral fragrancy. The prevalence of entomophily in the Gnetales (i.e., Gnetum, Ephedra, and Welwitschia) may suggest that unspecialized insect pollination originated before the divergence between the Gnetales and angiosperms.     

Pollen limitation and seed set associated with year-to-year variation in flowering of Gmelina arborea in a natural tropical forest

The year-to-year variations in flowering, pollen and fruit production in ten Gmelina arborea individuals in a natural forest were observed over a period of five years, 2009?2013, and pollinator visits were observed closely over two years, 2011 and 2012. A pollen supplementation experiment was also undertaken in two years (2011 and 2012) of contrasting flowering levels. Considerable year-to-year variations were observed in flower, pollen and fruit production. The observed variation represented a normal sequence of good-year and poor-year cycling which is most parsimoniously attributed to resource allocation. The average level of pollen production per individual tree in good flowering years oscillated between 6.6 and 9.62 × 10⁸, which in the poor flowering years was between 1.92 and 3.07 × 10⁸. The magnitude of pollen limitation across years was highly significant (p < 0.0001). Fruit set through supplemental pollination was 73% and 96% greater than that of the open-pollinated branches in the year 2011 and 2012, respectively. Pollen limitation, irrespective of the arrival of outcross pollen, can reduce annual seed set in predominantly bee-pollinated trees. However, various other factors may cause flower abortion in addition to pollen limitation. The results of this study would be very valuable to the seed orchard managers and the silviculturists to manage the seed production areas (SPAs) of G. arborea. The study ultimately recommends supplemental pollination in seed orchards of G. arborea for better genetic gain and good seed yield.     

Effects of temperature and pollination site on pollen performance in Betula pendula Roth – evidence for genotype-environment interactions

We studied whether the differences between genetically different pollen donors (Betula pendula Roth clones) with respect to pollen-tube growth rate were consistent under different thermal conditions during pollen germination in vivo and in vitro. We conducted a single-donor hand-pollination experiment with same pollen donors and recipients in a plastic house seed orchard and at an outdoor clone collection. The prevailing daily mean temperature during pollen germination was 13°C higher in the plastic house than outdoors. The pollen-tube growth rate of each pollen donor was additionally determined in vitro on agar medium at five temperatures (10°, 15°, 22°, 30° and 35°C). A significant interaction between paternal clone and pollination site as well as between paternal clone and temperature was found, which provides evidence for genotype-environment interactions. Genotype-environment interactions can have evolutionary significance in maintaining the variation in pollen-tube growth rates. At seed orchards, genotype-environment interactions can cause deviations from the expected genetic composition of the seed crop depending on the prevailing environmental conditions during pollen-tube growth. Received: 24 September 1999 / Accepted: 29 September 1999     

The bumblebee Bombus ardens ardens (Hymenoptera: Apidae) visits white clover in orchards before Oriental persimmon blooms

Flowers on the ground of orchards can provide substantial resources for wild pollinators of orchard trees. Few studies, however, have examined the relative importance of groundcover flowers to orchard pollination by analyzing pollen on the body surface of pollinators. Oriental persimmon trees bloom within the longer blooming period of white clover, which is occasionally found as a flowering plant on the ground of persimmon orchards in Japan. The present study compared the insect species assemblage collected on persimmon flowers with that on clover. Before persimmon bloomed, Bombus ardens ardens and Apis cerana japonica were the major visitors of clover flowers. Once persimmon bloomed, the former was the most abundant bee that visited persimmon flowers over the flowering period. Apis mellifera was captured only on clover flowers. We found numerous clover pollen grains on the body surface of bumblebees captured on persimmon flowers, but far fewer persimmon pollen grains on bees that visited clover. These findings show that B. ardens ardens utilized the clover flowers under the orchards before persimmon bloomed.