Comparison of different control-pollination techniques for small-flowered eucalypts

Controlled pollination (CP) is a labour-intensive, but useful procedure applied in tree improvement programmes. However, the high costs involved and relatively low seed yields often obtained has, in most cases, necessitated the use of lower quality, open pollinated seed. The aim of the present study was to compare control-pollination methods for combinations among small-flowered eucalypt species. By making crosses within and among Eucalyptus grandis, E. smithii and E. macarthurii, we compared effectiveness, in terms of seed production and level of genetic contamination, of three CP techniques, namely emasculation of bagged flowers and subsequent pollination of receptive stigmas (Conventional method), emasculation and immediate pollination of stigmas with induced receptivity followed by bagging (One Stop Pollination), and pollination of cut styles without emasculation and bagging (Artificially Induced Protogyny). One tree of each species was used as the female in these crosses. Although Artificially Induced Protogyny using ripe and semi-ripe buds produced the highest seeds/flower pollinated in the majority of crosses carried out in this study, the technique, when performed on green buds, resulted in the highest capsule abortion. Molecular analysis using microsatellite markers also revealed that progeny from the Artificially Induced Protogyny method, when using green and semi-ripe buds, were highly contaminated by self- and external pollen. Of the three CP-techniques tested, One Stop Pollination had the lowest genetic contamination. However, this technique also had one of the lowest seed yields, while the Conventional method was intermediate in performance.     

Density-dependent selfing and its effects on seed performance in a tropical canopy tree species, Shorea acuminata (Dipterocarpaceae)

In the conservation and management practices of natural forests, sound reproduction and regeneration form the basis of the maintenance and viability of the tree populations. To obtain and serve biological information for sustainable forest management, we investigated reproductive biology and inbreeding depression in seeds of an important dipterocarp tree species, Shorea acuminata (Dipterocarpaceae), by both field and laboratory experiments. Results of parental analysis of immature and mature seeds showed that selfing rates varied greatly, from 7.6 to 88.4% among eight mother trees, and the mean overall selfing rate was 38.3%. Observed outcrossing events within a 40-ha study plot were predominantly (76.5%) short-distance events with a mating distance (md) ≤ 100 m. Since the selfing rate sharply decreased with increase in the number of flowering conspecifics (i.e., individuals of the same species) within a 100-m radius from the mother trees, the local density of flowering conspecifics appears to be the key factor determining the outcrossing rate in S. acuminata. However, the extremely high selfing rate (88.4%) observed for one tree could not be simply explained by the low local density of flowering conspecifics. Instead, differences in its flowering phenology (its flowering peaked ca. a week earlier than most of the other examined individuals) may have severely limited its receipt of pollen from other conspecifics, and thus promoted selfing. Since there were no significant differences in the proportion of selfed progeny between immature and mature seed stages, there was no evidence of selective abortion of selfed seeds during seed development. However, the seed mass of outcrossed progeny was heavier than that of selfed progeny, and heavier seeds showed higher success rates at germination and seedling establishment. These results suggest that inbreeding depression resulted in reductions in seed mass and may reduce the fitness of selfed seeds in S. acuminata. In addition, the outcrossing rate of S. acuminata was more sensitive to low local conspecific flowering-tree densities than that of a sympatric bee-pollinated dipterocarp species with greater pollination distances. These results suggest that the management of local adult-tree densities is important for avoiding selfing and inbreeding depression in future generations, especially in a species like S. acuminata with predominantly short-distance pollination.     

Floral biology and breeding behavior in the bamboo Dendrocalamus strictus Nees

Floral biology and breeding behavior were studied in the bamboo Dendrocalamus strictus Nees. The inflorescence in this species is a large branching panicle. Fertile florets are intermixed with smaller sterile ones. There are six stamens. The ovary is stipitate and turbinate. The style is long and the stigma is bifid and plumose. Dendrocalamus strictus is typically dichogamous and protogynous. The gynoecium matures 3-4 days before the androecium, effectively preventing self pollination. Flower bloom, which took place over a period of 2 to 3 h, was dependent on air temperature and only occurred between 0600 and 1300 h. Dendrocalamus strictus is anemophilous. Flowers in the male phase were visited by insects. These insects completely neglected the flowers at the female phase. The insects fed on the pollen and were not pollen vectors. When wind was excluded by enclosing the inflorescences in bags there was no seed set, indicating that cross pollination by wind is necessary for fertilization and that parthenocarpy and apomixis are not occurring in this species. Pollen fertility was about 98% as indicated by staining fresh pollen at the time of anther dehiscence and pollen release with Alexander's stain (Alexander 1969). When placed on a modified Brewbaker-Kwak medium containing 1% glucose, the pollen grains germinated well, and the pollen tubes grew to 15-20 times the diameter of mature pollen grains. Although profound protogyny has its disadvantages in times of sporadic flowering, it can be useful in breeding programs because it eliminates the need for emasculation.     

Phenology,pollination and breeding system of <Emphasis Type="Italic">Aegle marmelos</Emphasis> (Linn.) correa (Rutaceae) from India

Studies on reproductive biology are difficult but useful in species like Aegle marmelos (Linn.) Correa which is of considerable socio-economic importance and possess morphogenetic variation and qualities of wider adaptability to different soils. Cytology, phenology, pollination, breeding system and natural regeneration of wild and cultivated trees from India revealed the existence of diploid (2n = 18) and tetraploid trees (2n = 36) in Pachmarhi hills and only diploid trees in Punjab plains and Shiwalik hills. The diploid and tetraploid trees showed normal meiosis and high pollen fertility. Phenological events which included leaf fall, leaf emergence, floral bud break, flowering and fruiting are nearly the same in wild and cultivated trees. Natural pollen transfer in the species was highly efficient. Levels of fruit set following open pollination was quite high and is reduced considerably following hand pollination probably due to some injury caused to stigma during experiments. Inspite of synchronous nature of anther dehiscence and stigmatic receptivity, selfing in a flower was avoided due to herkogamy. Some selfing, however, occurred through geitonogamous mode as bagging of panicles yielded 12.21 ± 0.99 to 14.12 ± 0.91% fruit set. High pollen ovule ratio (9,250–10,600) indicated toward the obligate outbreeding nature. The species suited to insect and wind mode of pollination. High amount of air borne pollen grains deposited on glycerine smeared glass microscopic slides suggested towards the wind mode of pollination. Though flowers are dull coloured a variety of insects visited the flowers due to sweet fragrance and stamen/pollen as food reward. Among insect pollinators, honey bee (Apis dorsata) was the major and legitimate pollinator while the rest were either minor pollinators or mere visitors. Inspite of high fruit/seed set, natural regeneration through seeds was poor as fallen fruits were destroyed by fungal pathogens and white ants. The species also propagated vegetatively through coppices and root suckers. It lacks agamospermy as bagging of emasculated flowers yielded no fruit. It is inferred that ‘bael’ which lacks agamospermy reproduced successfully through gamospermy (xenogamy and geitonogamy) and vegetative mode (coppices and root suckers). We also concluded that tetraploid trees growing in the Pachmarhi hills with large sized fruits possessed better potentialities in horticulture if planted through root suckers or coppices.     

Reproductive biology of the critically endangered tropical tree Talbotiella gentii

Talbotiella gentii is a critically endangered caesalpiniaceous legume endemic to Ghana, restricted in distribution to the margins of the forest zone where there has been extensive fire damage in recent decades. It occurs as isolated, near-monospecific populations, some of which show little seedling regeneration. We studied its reproduction to determine limitations that might inform efforts to prevent its extinction. We report on flowering phenology, pollen dispersal and germination, fruit and seed set, seed size and germination, and seedling survival, using controlled and open pollination. Talbotiella gentii flowers profusely in most years. Although the flowers are coloured and scented, no animal pollinators were observed but pollen can be dispersed by wind. In open pollination, the number of pollen grains on stigmas and their germination was sufficient to fertilise the three ovules, despite competition from heavy stigmatic infestation by fungal spores and hyphae. Fruit set and seed set were low due to high flower losses and fruit abortion during development but ripe seeds showed high germination. Controlled pollination experiments (self, supplemented self, and intra- and inter-population crosses) showed that, while the species is self-compatible, selfing was the least successful treatment in all of the above steps of reproduction, whereas cross-pollination enhanced most steps, the more so for inter-population crosses. Seed size was notably greater in inter-population crosses and was positively correlated with genetic distance of the cross. The larger seeds associated with cross-pollination showed higher germination and seedling survival in the nursery. We conclude that limitations to natural reproduction in T. gentii are principally due to processes between pollen germination and fruit and seed set, including pollen quality (low genetic diversity and inbreeding), fertilisation rates, flower and fruit abortion, and resource limitation. Low seed size in open and self pollination may have implications for seedling establishment. Species fitness might be improved by introducing other genotypes to existing populations, but in situ protection of the populations is essential in the medium term.     

Background pollination in Pinus sylvestris seed orchards

The proportion of background pollen grains in the seed crops of two Pinus sylvestris seed orchards in Central Finland was estimated with the aid of multilocus allozyme markers. The orchards studied were more than 30 years old and in full pollen production. For the bulked seed crops of the seed orchard with southern clones the estimated average of background pollination over four years was 26%. There were statistically significant differences between years. No significant heterogeneity in the degree of background pollination between clones was found. Among single ramets there was significant heterogeneity in the estimated contamination rates, but the variation was not related to position in the orchard. For the seed orchard with northern clones the bulked seed crop was studied only for one year and the level of background pollination was found to be 33 %. These estimates are fairly high, but lower than for many other orchards. Background pollination at this level will cause losses in expected genetic gains. Part of the seeds from northern orchards will not be adapted to the intended area of use.     

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.     

Reduction of pollen contamination in Scots pine seed orchard crop by tent isolation

This study examined the differences in mating system between indoor versus outdoor Scots pine (Pinus sylvestris L.) seed orchard using DNA markers. By analyzing the paternities in seed crops from isolation tents, we found a significant reduction in contamination rate, from 23% in the outdoor open blocks to 0% inside the tents. Increase in selfing rate and a decrease in genetic diversity in the crops were also observed inside the tents. These undesirable effects can be mitigated to some extent by the supplementation of extra pollen genotypes, e.g. selfing rate decreased from 14.4% to 6.6% and the effective number of fathers increased from 6 to 11.4 in the tents without and with supplemental mass pollination, respectively. Our study showed that tent isolation may become an effective rescuer for seed orchards where pollen contamination is severe.     

Floral display and breeding system of Jatropha curcas L.

Plant flowering and breeding characteristics are important for us to understand the reproduction of plant populations. In this paper, we studied the reproduction characteristics of Jatropha curcas in Yuanjiang County (23°36′N, 101°00′E), Yunnan Province. The plant produces flowers in dichasial inflorescences. Normally, the flowers are unisexual, and male and female flowers are produced in the same inflorescence. Only a few male flowers are produced in an inflorescence, and fruits are produced only through pollination between different flowers from the same or different plants. By the treatments of emasculation, bagging and artificial pollination in this experiment, there were few but same fruit set ratios when the inflorescences were emasculated, bagged, or bagged with net, except artificial pollination treatments, which showed that Jatropha curcas could produce fruit through apomixis but not wind pollination. When the inflorescences were unbagged, unemasculated and with free pollination treatments, or bagged, emascu- lated and with artificial cross-pollination treatments, or unbagged, emasculated and with free pollination treatments, there were many fruits produced. It showed that Jatropha curcas shows outcrossing, is self-compatible, and demanding for pollinators. Normally, the male flowers open first and a few flowers bloom in one day in a raceme. These flowers last a long time in bloom. However, a large number of female flowers open from the third to the fifth day, with some female flowers opening first in a few raceme. This shows a tendency to promote xenogamy and minimize geitonogamy.     

Are Phenological Observations Sufficient to Estimate the Quality of Seed Crops from a <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> Open-pollinated Seed Orchard? Consequences for Seed Collections

Reproductive phenology was observed over three years in a Eucalyptus grandis seed orchard in Madagascar to determine the impact of geographic differences of parental selections on the expected genetic composition of seed crops. Pollen flow (observed pollen cloud) was deduced from a paternity assignment based on sampling from one year’s seed production. The two approaches were used to verify whether knowledge of phenology is sufficient to predict the genetic quality of the seeds collected. Despite the high flowering level, with a constant cycle over three years, the results demonstrated wide reproductive phenological differences associated with the parents’ origin, suggesting putative pollination disequilibrium. From both observed and expected pollen clouds, the results showed preferential mating among different provenances, which has consequences for seed crop composition. Phenological observations, which can be made inexpensively, can be used to promote methods of effective seed orchard management in order to improve the genetic quality of seeds.     

Supplemental mass pollination in a seed orchard of Pinus Sylvestris L. investigated by isozyme analyses

Isozyme‐analysis techniques have been applied to test the efficiency of supplemental mass pollination in a seed orchard of Pinus Sylvestris. Mass pollination was done on one single occasion before the general pollen dispersal. The results of isozyme analyses of seed after mass pollination demonstrate that it is possible to introduce desirable genes into the seed orchard crop. Mass pollination without isolation of female strobili gave an average contribution of 4% to the total fertilizations and after isolation of female strobili 26.5%. The main conclusion is that supplemental mass pollination offers very interesting possibilities for breeding, particularly if some kind of isolation of the female flowers is made. It can increase the seed quality as well as improve the breeding value of the seed orchard. More attention should be paid to increasing the efficiency of the controlled pollination. It is suggested that in the future, more effort is invested in improving the technique of mass pollination.     

Evaluating Supplemental Mass Pollination Efficacy in a Lodgepole Pine Orchard in British Columbia using Chloroplast DNA Markers

Supplemental mass pollination (SMP) success in a grafted lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) seed orchard in southern British Columbia was studied by employing four chloroplast DNA (cpDNA) markers to determine fertilization success of 10 pollen parents. SMP was conducted operationally with a bulked pollen mix twice during peak receptivity in the seed orchard. Fertilization success of the 10 SMP parents averaged 16% greater than for wind-pollinated controls in a different section of the orchard. SMP also increased the uniformity of the male contribution in treated seeds.     

Timing of premature acorn abortion in <Emphasis Type="Italic">Quercus serrata</Emphasis> Thunb. is related to mating pattern,fruit size,and internal fruit development

Timing of acorn development and abortion is an important maternal strategy in oak sexual reproduction. To understand the significance of acorn abortion in Quercus serrata, artificial pollination of different mating patterns (outcross, self-, and nonpollination) was performed, and the timing of abortion and the size and internal development of acorns from each mating type were investigated. Acorns were aborted similarly in every pollination treatment until 80 days after pollination. Almost all self- and nonpollinated acorns were rapidly aborted during the period 80–120 days after pollination. During that period, differences in internal fruit development between outcross-pollinated and unsuitably pollinated (self- and nonpolinated) acorns were observed. In addition, cotyledon development and a rapid increase in fruit size were observed in the acorns gained by outcross pollination. The correspondence between the timing of abortion and the timing of rapid size growth and the development of storage organs suggests that this acorn-maintenance strategy may be the consequence of a maternal adaptation that allows better reproductive success under resource limitation.     

Success rate of supplemental mass pollination in a seed orchard of Pinus sylvestris L.

Two experiments (Expt 1, Expt 2) with supplemental mass pollination (SMP) were carried out in a seed orchard of Pinus sylvestris L. in order to assess the biological and operational prospects for SMP. The success of the SMP was studied by means of allozyme markers. In Expt 1 the pollen mix was applied very close to each female strobilus. The estimated success rates for treatments varied between 66% and 84%. When supplemental pollination was done at peak receptivity, one single pollination was enough to obtain high success rates. In Expt 2 the pollen mix was dusted over clusters of receptive strobili on whole‐trees. The estimated success rates for treatments varied between 10% and 23%. The success rates improved when the trees were pollinated on more than one occasion per day during the receptive period. The overall conclusion is that SMP is practical for P. sylvestris seed orchards.     

The pine reproductive process in temperate and tropical regions

Our knowledge of the pine reproductive process is confined to the developmental stages and time relations in a particular species. Lacking in the literature is a comparative analysis from a broader perspective such as pines from temperate versus tropical regions. Also, important information that may have beneficial implications for pine breeding and management purposes may be missed by going with a single generalized reproductive cycle model which is usually derived from well-studied northern temperate species. Detailed developmental analysis of the reproductive process is necessary, particularly for tropical pines where information is limited. Most pines undergo a reproductive cycle that spans three calendar years with two dormant periods. In temperate pines, many of the stages that are initiated by fall generally come to a halt during the winter and development resumes in spring of the following year. In tropical pines, reproductive development appears to be generally a continuous process characterized by a gradual rate of development with indistinct first dormant period. The distinct first dormant period in north temperate pines serves to synchronize pollen release and seed-cone receptivity which results in higher pollination success and seed production. Tropical pines exhibit asynchrony in pollen release and thus has extended pollination period. Relatively less pollen are available which results in lower pollination success and seed set. Interestingly, since asynchrony may enhance cross-pollination, tropical pines might benefit from this through the capture of genetic diversity. The length of the second dormant period is generally the same in temperate and tropical pines which indicates strong genetic control.     

Southern pollen sired more seeds than northern pollen in southern seed orchards established with northern clones of Pinus sylvestris

Abstract

Contamination by southern pollen is a considerable problem in Scots pine (Pinus sylvestris L.) seed orchards established with northern clones. This study investigated whether the contamination was due to the competitive superiority of southern pollen by carrying out competition trials using mixtures of pollen from northern and southern populations of Scots pine. Trials were performed in a southerly seed orchard established with clones originating from northern populations. Seed paternity (siring) was determined through the analysis of allozyme variation. Southern genotypes sired significantly more seeds (76%) than their northern competitors and across all mixed-pollen crosses. Maternal genotype had no effect on seed siring success. The mean flower abortion rate was lower in southern pure-pollen crosses and mixed-pollen crosses than in northern pure-pollen crosses. The results show that local pollen may induce high levels of background pollination in southern seed orchards composed of northern genotype grafts. These results must be taken into account when aiming to produce suitable reforestation material for northern areas.     

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.     

Reduced fertilization capacity of SO2-fumigated Picea omorika-pollen

Dry pollen of Picea omorika was fumigated with 820 μg/m³ SO₂ for 3 , 4 , or 5 days. Few or no adverse effects occurred during subsequent in vitro germination: germination percentage was not affected at all, and reduction of the pollen tube length was only 10% in pollen exposed for 5 days. Nevertheless pollination with fumigated pollen resulted in reduced seed production. 41% fewer seeds per cone were produced, and only 33% were filled seeds, compared to 70% filled seeds of the control. Seed-weight was reduced from 3.6 g per thousand in the control to 3.0 g per thousand, when cones were pollinated with pollen fumigated for 5 days. Similar but less striking effects were found with pollen fumigated for 3 or 4 days.     

Estimates of pollen contamination and selfing in a coastal Douglas-fir seed orchard

Seed orchards are the link from tree breeding to reforestation programs and are theoretically expected to function as closed, perfect populations, ensuring gain and diversity are consistently and predictively delivered as improved seed and seedlings. Seed orchard populations often deviate from panmixia due to fertility variation, reproductive asynchrony, and gene flow, leading to reduced seed crops genetic quality. Here, as a part of multiyear monitoring study, we used DNA fingerprinting (simple sequence repeat markers) to assess a Douglas-fir (Pseudotsuga menziesii) seed orchard's seed crop genetic quality (2009 seed crop). The studied seed crop was produced under ambient temperature (i.e. no reproductive phenology manipulation) and pollination was augmented by pollen from within orchard's pollen donors. DNA fingerprinting of the parental population (66 parents) along with 207 gametophyte (1n) – embryo (2n) pairs of random bulk sample of seed allowed parentage (maternal and paternal) assignment and the direct assessment of pollen contamination (0.18 ± 0.027) and selfing (0.17 ± 0.025) rates as well as parental (pollen, ovule and individual parent) gametic contribution was compared to a previous year's crop (2005). The extended reproductive phenology coupled with variable within-orchard pollen availability has created opportunities for both self and foreign pollen to be successful at various times resulting in the seemingly paradoxical scenario of high selfing and gene flow. These results (2005 and 2009) allowed comparison of seed orchard's crop management practices and are expected to provide scientific foundations to effective seed crops genetic quality improvement.     

人工授粉对杉木种子发芽,形态及生物大分子的影响

应用混合杉木优树花粉进行人工授粉,能明显促进杉木种子发芽、减少涩籽率,提高种子品质;运用微量生化分析方法表明,应用混合优树花粉进行人工授粉所产生的种子种胚中所含生物大分子的量都明显高于用散生木花粉人工授粉所产生的种子,表明应用混合优树花粉进行人工授粉不仅能促进种子萌发,还能提高种子品质。