Western white pine (Pinus monticola Dougl.) reproduction: II. Fertilisation and cytoplasmic inheritance

Fertilisation and proembryo development are described from transmission electron micrographs emphasising the origin and fate of the maternal and paternal mitochondria and plastids. During central cell and egg development mitochondria migrate toward the nuclei, forming a perinuclear zone consisting predominantly of maternal mitochondria and polysomes. At the same time, maternal plastids transformed and at fertilisation are excluded from the neocytoplasm. The pollen tube releases two sperm nuclei into the egg with cytoplasm from the generative cell and the tube cell. The leading sperm nucleus fuses with the egg nucleus and a small number of paternal mitochondria and plastids are taken into the perinuclear zone. The second sperm nucleus degenerates. As the zygote nucleus undergoes mitosis followed by free nuclear division and nuclear migration to the chalazal end of the archegonium, maternal and paternal organelles intermingle within the neocytoplasm. The result is paternal inheritance of plastids and biparental, but predominantly maternal, inheritance of mitochondria. This pattern is consistent within the Pinaceae but differs from some other conifer families. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Segregation and linkage relationships of isoenzymes in shortleaf pine (Pinus echinata Mill.)

 Segregation and linkage relationships were analyzed between 28 isoenzyme loci in ten natural stands representing much of the natural range of Pinus echinata Mill. (shortleaf pine). A total of 203 possible two-locus combinations were tested. Three linkage groups were revealed in this study at a linkLOD of 4.0. The first linkage group (A) consisted of Pgi and Adh-1; Gdh, Idh, Skdh-2, G6pd-2 and Aco were mapped to the second linkage group (B); the third group (C) had 2 loci: Mdh-2 and Mdh-3. A moderate linkage between Mnr-2 and Dia-2 and weak linkages between Mnr-1 and Dia-1, and Got-2 and 6pgd-2 were also detected. The significance of these results in shortleaf pine is discussed and compared to linkage maps previously reported in other conifers, including pines. Received: 7 April 1997 / Accepted: 25 April 1997     

Linkage mapping and genome length in eastern white pine (Pinus strobus L.)

 Haploid linkage analysis of eastern white pine, Pinus strobus L., was carried out using mainly RAPD markers and microsatellite, or simple-sequence-repeat, markers. Ninety one loci mapped to 12 linkage groups of three or more markers. The resulting framework genome map, the first for a soft pine species, contained 69 markers. The map covered 58% of the estimated genome length of 2071 cM(K), with a 95% confidence interval of 1828–2242 cM(K). A systematic comparison of linkage data from eastern white pine, longleaf pine (P. palustris Mill.) and maritime pine (P. pinaster Ait.), gave genome-length estimates for all three species very close to either 2000 cM(K) or 2600 cM(H), depending on whether the Kosambi(K) or Haldane(H) map functions, respectively, were employed. Differences among previous pine genome-length estimates were attributed to the divergent criteria used in the methods of estimation, and indicate the need for the adoption of uniform criteria when performing genome-length estimates. Current data suggest that members of the two pine subgenera, which diverged during the late Mesozoic era, have highly conserved rates of recombination. Received: 5 January 1997/Accepted: 24 January 1997     

Climate-tree-growth relationships of Scots pine stands (Pinus sylvestris L.) exposed to soil dryness

 Dendroclimatological techniques were used to assess the impact of climatic factors on radial tree growth (total ring-width and latewood-width) of stunted Scots pine trees (Pinus sylvestris L.) exposed to soil dryness and nutrient deficiency on a dolomite substrate. The response of eight scattered populations representing various habitats, yet influenced by the same regional climate was investigated. Total ring-width and latewood-width were dated, standardized and several chronology statistics, which estimate the chronology signal strength and the potential climate signal in the series, were determined. Dendroclimatic analysis comprised evaluation of event/pointer years as well as response function elements. Response function analysis indicates that at most sites wide rings are significantly associated with high precipitation in April to June and cool conditions in May of the current year, and high precipitation in August to September of the preceding year. Latewood chronologies show a lower climate signal at all sites. Limited water availability causes a homogeneous relationship of radial tree growth to climate at all habitats, though site characteristics (slope magnitude, slope aspect, soil depth, vegetation cover) differ substantially. Cluster analysis of negative event/pointer years suggests that within the study area stands react to extreme climatic events depending on susceptibility to soil dryness, which is primarily determined by site topography. Received: 5 January 1998 / Accepted: 22 April 1998     

Variation in chloroplast single-sequence repeats in Portuguese maritime pine (Pinus pinaster Ait.)

Genetic variation in 12 Pinus pinaster (maritime pine) populations spanning most of the distribution range of the species in Portugal was evaluated using six polymorphic chloroplast microsatellite (cpSSR) loci. Thirty-two haplotypes were found. There were indications of very weak differentiation among populations (Weir’s θcoefficient, 0.023), and the R _ST value, derived from the stepwise mutation model (SMM), was not significantly different from zero. The pattern, in which similarities in allele size, in base pairs, do not contribute to the genetic structure, may be due to the recent mixing of genetic material from different stands through plantations. Overall, a high level of haplotypic variation within populations was detected. Using the SMM estimator (mean genetic distance of individuals within populations, D ² _sh –––) we divided the populations into two groups, with above and below average values. The first group contained 5 populations, mainly from the central part of the country, which possess, in general, high levels of haplotypic diversity. Among them, 2 populations were divergent from the others based on the pair-wise Nei’s distance. The results indicate that there is no discernible geographic genetic pattern for the Portuguese populations of P. pinaster investigated. The history of expansion of the species range in Portugal during the twentieth century (mainly due to human activity) and extensive gene flow among populations associated with the expansion could explain this finding. Received: 15 February 2000 / Accepted: 14 April 2000     

Primers designed to amplify a mitochondrial nad1 intron in ponderosa pine, Pinus ponderosa, limber pine, P. flexilis, and Scots pine, P. sylvestris

The b/c intron of the mitochondrial nad1 gene, was sequenced to characterize the indel region of ponderosa pine, Pinus ponderosa. The sequence in ponderosa pine was aligned with the sequence in Scots pine, Pinus sylvestris, to design seven primers that are useful for sequencing and for revealing size variation in amplified fragments in ponderosa pine, Scots pine, and limber pine, Pinus flexilis. These primers reveal variability in all three species, and the pattern of variability within ponderosa pine is described by a preliminary survey. The indel region of ponderosa pine contains three distinct elements with lengths of 31, 32, and 34 bp. Received: 1 March 2000 / Accepted: 14 April 2000<@head-com-p1a.lf>Communicated by P.M.A. Tigerstedt     

Ethanol synthesis, nitrogen, carbohydrates, and growth in tissues from nitrogen fertilized Pseudotsuga menziesii (Mirb.) Franco and Pinus ponderosa Dougl. ex Laws. seedlings

 Seedlings of Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, and ponderosa pine, Pinus ponderosa Dougl. ex Laws., were grown in a controlled environment and fertilized with nutrient solutions containing 150 ppm (+N), or 0 ppm nitrogen (−N). These treatments greatly altered seedling growth, and the concentrations of N and carbohydrates in their tissues. Metabolically active tissues, such as roots, incubated with a limited supply of O₂ became hypoxic faster and synthesized more ethanol than less active tissues, such as needles. All tissues that were incubated for 4 h in N₂ synthesized ethanol. Needles incubated in N₂ and light had much lower quantities of ethanol than needles in N₂ and dark, suggesting that O₂ from photosynthetsis limited internal anoxia. Most tissues from +N seedlings synthesized greater quantities of ethanol in N₂ anoxia than tissues from −N seedlings, probably because they were able to produce more enzymes with a greater availability of N. However, this increase in ethanol synthesis between N treatments was most pronounced in the phloem. Ethanol and soluble sugar concentrations were negatively related in needles and positively related in roots of N+ seedlings, but not −N seedlings. Starch concentrations had no effect on the amount of ethanol produced by any tissue. Regardless of N treatments, all tissues from ponderosa pine produced more N₂-induced ethanol than Douglas-fir, in part because its tissues contained different concentrations of soluble sugars and N as a consequence of phenological differences between the species. However, ponderosa pine tissues may also maintain greater quantities of anaerobic enzymes, or their isozymes than Douglas-fir. Received: 22 February 1998 / Accepted 23 June 1998     

Variations in maturation strains and root shape in root systems of Maritime pine (Pinus pinaster Ait.)

 In order to determine if different types of wood were being laid down in the root system of Maritime pine (Pinus pinaster Ait), in response to wind loading, longitudinal residual maturation strains (LRMS), indicating the existence of mechanical stress in developing wood cells, were measured in the trunk and lateral roots. Two age groups of trees (5- and 13-year- old) were compared. LRMS were greater in the trunk and roots of 13-year-old trees than in 5-year-old trees. This phenomenon may be due to increased competition between older trees. LRMS in leeward roots of both age-groups were positive i.e. the wood cells had developed under compression, as also occurs in reaction wood of gymnosperms. As leeward roots are placed under compression during tree sway, an abnormal type of wood may form in the roots in order to counteract the increased stress. In other roots, the strains were negative i.e. the cells had developed under tension, as occurs in normal wood. In the roots of younger trees, LRMS were also positive nearer the stem, thus indicating that wood formation may also be influenced by bending stresses experienced in this zone. In addition to LRMS measurements, radial growth in roots was examined in order to determine the influence of mechanical loading on secondary growth. In older trees, there was a significant increase of 34% in woody growth below the biological centre, compared to that above. This eccentricity is unlike that found in most other tree species, where secondary growth is usually greater on the upper side of the root. However, Maritime pine has a tap root, which will alter the pattern of stress within the root system. Under wind loading, a concentration of mechanical stress will develop at the bases of the stem, lateral roots and tap root. Received: 7 July 1997 / Accepted: 11 December 1997     

Identification of quantitative trait loci influencing annual height- and diameter-increment growth in loblolly pine (Pinus taeda L.)

 Quantitative trait loci (QTLs) for annual height- and diameter-increment growth were estimated in each of two three-generation loblolly pine pedigrees. Annual height-increment was measured in three consecutive years in the BASE pedigree and in four consecutive years in the QTL pedigree. Annual diameter-increment was measured only in the QTL pedigree for two consecutive years. An interval mapping-approach was used to estimate the number of QTLs, the magnitude of QTL effects, and their position on genetic linkage maps. Thirteen different height-increment and eight different diameter-increment QTLs were detected, suggesting that the these traits are, at least in part, controlled by a few genes of large effect. Little evidence was found for the expression of individual QTLs in multiple years or in multiple genetic backgrounds. These results were discussed in terms of the power of the experiment and their consequences for marker-assisted breeding. Received: 12 July 1998/Accepted: 5 August 1998     

Isolation, genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine ( Pinus monticola Dougl. ex. D. Don.)

Western white pine ( Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust ( Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.     

Genetic mapping of expressed sequence tag polymorphism (ESTP) markers in loblolly pine (Pinus taeda L.)

The development and mapping of genetic markers based upon expressed sequence tag polymorphisms (ESTPs) in loblolly pine (Pinus taeda L.) are reported. The new markers were generated by PCR-amplification of loblolly pine genomic DNAs with primers designed from sequenced cDNAs. The cDNA libraries were constructed from RNAs expressed in the needles of loblolly pine seedlings or in the xylem from young trees. DNA polymorphisms were identified by analyzing the amplified products for differences in fragment size or restriction sites, or by examining mobility differences using denaturing gradient gel electrophoresis (DGGE). DGGE revealed more DNA polymorphisms than the other two methods. Fifty six ESTPs were mapped using either of two mapping populations and positioned onto a loblolly pine consensus genetic map. Unlike many other markers commonly used in forestry, ESTPs can be used as orthologous markers for comparative mapping, to map genes of known function, or to identify candidate genes affecting important traits in loblolly pine. Received: 10 April 2000 / Accepted: 13 July 2000     

Chloroplast phylogeography and evolution of highly polymorphic microsatellites in lodgepole pine ( Pinus contorta)

We employed a novel set of six highly polymophic chloroplastic simple sequence repeat (cpSSR) loci to investigate the phylogeography of lodgepole pine (Pinus contorta Dougl. Ex. Loud.), and to examine aspects of the evolutionary process operating on these repetitive DNA sequences. Chloroplast haplotypes of 500 trees, sampled throughout the range of lodgepole pine, were determined. We found a marked association of genetic distance with physical distance within the scale of 0 to 1,000 km, but no association beyond that range. Likewise, geographic clustering was observed only among recent clades in a dendrogram. These phylogeographic patterns are consistant with a rapid rangewide expansion (”big-bang”) followed by recent, local population differentiation (”galaxy formation”). In support of this expansion, coalescent simulations of the genealogical process gave a long-term effective population size in the low thousands, and a time to common ancestry of about 1,500 generations (12,000 years), consistent with a post-Pleistocene population expansion as documented by previous pollen-sediment analyses. Two lines of evidence (mapping mutational events onto a phylogeny, and evaluation of observed versus expected gene diversity) suggest that five of the cpSSR loci evolve primarily by a stepwise model of evolution of single repeat changes (but with a small proportion of changes involving two or more repeats), and the coalescent simulations point to a mutation rate of about 10^–3. Received: 7 December 2000 / Accepted: 17 May 2001     

X-ray microanalytical studies of mineral localization in the needles of white pine (Pinus strobus L.)

Eastern white pine (Pinus strobus L.) shoots from mature trees were collected from two sites of contrasting soil pH: the Glendon campus of York University in Toronto, Ontario (pH 6.7 at 40 cm); and Muskoka near Huntsville, Ontario (pH 4.2 at 40 cm). Needles of ages 1-3 years were removed from the shoots, and the percentage of ash and silica was determined for all ages. Other needles were frozen in liquid nitrogen and kept in a cryo-biological storage system before x-ray microanalysis. Percentages of ash and silica were higher in the needles from Muskoka. Ash and silica increased with needle age for trees from the Muskoka site, but less so at the Toronto site. Of the 13 elements (Na, Mg, Al, Si, P, S, Cl, K, Ca, Mn, Fe, Cu and Zn) detected by microanalysis, Mn, Fe, Cu and Zn were detected in small amounts in the epidermis, endodermis and transfusion tissue (the layer of tracheids and parenchyma immediately surrounding the vascular bundles), and K, P, S and Cl were almost ubiquitous in distribution. Sodium was occasionally detected in the transfusion tissue, and magnesium was concentrated in the endodermal cells. The epidermal walls, transfusion tissue and endodermis were major sites of calcium localization. Silicon was concentrated in the extreme tips of the needles in all tissues, but particularly in the transfusion tissue, and more so in the Muskoka samples. Microanalysis revealed a higher Al content in the Muskoka needles, that Al was concentrated in the needle tips and that the transfusion tissues were major sites of accumulation.     

Genetic variation and migration pathways of maritime pine (Pinus pinaster Ait) in the Iberian peninsula

The genetic variability and migration pathways of Pinus pinaster after glaciation in the Iberian peninsula was studied by means of 18 loci from 12 natural populations of the species. The analysis showed the existence of three groups of populations with different levels of diversity and patterns of recolonization. The southern Iberian group displays a high level of diversity, with a stepping-stone model of variation. The presence of rare alleles in this group and their position in the phylogenetic tree suggest the existence of refugia during glaciations in this zone. The eastern Iberian group also has high levels of diversity but is clearly separate from the first group based on their genetic distances. The Atlantic group displays a low level of diversity that could be interpreted as a rapid recolonization of the entire area by the Eastern group that has not yet developed to a divergence in this area. The southern Iberian range is indicated to be the dispersal centre of the species after the last glaciation. Received: 15 February 1999 / Accepted: 30 April 1999     

Identification of QTLs influencing wood property traits in loblolly pine (Pinus taeda L.). I. Physical wood properties

Physical wood property traits for loblolly pine (Pinus taeda L.) were analyzed for the presence of quantitative trait loci (QTLs) in a three-generation outbred pedigree. These traits include wood specific gravity (wsg), volume percentage of latewood (vol%), and microfibril angle (mfa). Phenotypic data were collected for rings 2–11 for wsg and vol%, and rings 3, 5 and 7 for mfa. Both earlywood and latewood were analyzed for each trait. An interval mapping approach designed for an outbred pedigree was used to estimate the number of QTLs, the magnitude of QTL effects, and their genomic position. Nine unique QTLs were detected for composite traits (average of all rings) for wsg, five for vol%, and five for mfa. The majority of these QTLs were verified by analyses of individual-ring traits. Additional QTLs for each trait were also detected by these individual-ring analyses. Most QTLs for wsg were specific to either earlywood or latewood, whereas each QTL for mfa was detected for both earlywood and latewood. Before these QTLs are utilized in a breeding program, they should be verified in larger experiments and in different genetic and environmental backgrounds. Received: 15 February 2000 / Accepted: 31 March 2000     

In vitro differentiation of plantlets from embryonic explants of lodgepole pine (Pinus contorta Dougl. ex Loud.)

A protocol is described for plantlet formation in juvenile tissues of Pinus contorta. Shoots were induced on embryonic, cotyledonary and hypocotyl explants cultured on a defined medium supplemented with cytokinin. The concentration of salts, vitamins and cytokinin (benzylamino purine) in the medium, as well as different temperature regimes, strongly influenced the frequency of bud formation. Differentiation of shoot primordia and their subsequent development was also markedly affected by cytokinin exposure times. Bud development and elongation were enhanced by elimination of the phytohormone, reducing the strength of mineral salts, vitamins and sucrose in the medium, as well as by the inclusion of charcoal. Rooting was induced by treating the shoots with a sterilized rooting powder containing indole-butyric acid and culturing them in agar-solidified medium containing reduced mineral salts, vitamins, sucrose and charcoal. The number of chromosomes and their structure were found to be normal in the regenerated plantlets.     

Genetic variability and structure of natural and domesticated populations of Caribbean pine (Pinus caribaea Morelet)

 Isozyme analysis of seed samples derived from natural and managed populations of the tropical pine Pinus caribaea vars ‘bahamensis’ and ‘caribaea’ was used to assess population genetic structure in its native range and to detect changes occurring during early domestication of the species. Baseline data from natural populations of the two varieties showed that populations sampled as seed are characterized by high gene diversity (mean H_e=0.26) and a low level of inbreeding ( mean F_is=0.15). A UPGMA tree of genetic relatedness among populations indicates that the two varieties represent distinct evolutionary units. Within each variety there is significant differentiation among populations, and this is greater for the more fragmented populations of var ‘bahamensis’ (F_st=0.08) than for var ‘caribaea’ (F_st=0.02). Seed from a seed orchard population of var ‘caribaea’ established within its natural range showed no change in genetic diversity but did show a reduced inbreeding coefficient (F_is=0.09) compared with its progenitor populations, suggesting a decrease in selfing and/or biparental inbreeding. A bulked seed sample from an exotic plantation of var ‘bahamensis’ in Australia displayed a large increase in the inbreeding coefficient (F_is=0.324) compared with that found in natural populations, possibly due to elevated self-fertilization. Finally, a bulked seed sample from an exotic plantation population of var ‘caribaea’ from China showed enhanced genetic diversity, an increase in the inbreeding coefficient and more linkage disequilibrium than its presumed progenitor populations. It was also genetically divergent from them. RFLP analysis of chloroplast DNA variation in the Chinese sample suggested that seeds of the related taxa P. elliottii and P. taeda, or seeds derived from hybridization with these taxa growing in the seed production area, had been included in the seed crop during harvesting. We conclude that monitoring of appropriate genetic markers may be an effective means of identifying potentially deleterious genetic changes occurring during forest tree domestication. Received: 10 August 1998 / Accepted: 8 September 1998     

Saturation mapping of a major gene for resistance to white pine blister rust in sugar pine

 The molecular basis of resistance to diseases in plants can be better understood if the genes coding for resistance can be cloned. The single major dominant gene (R) that confers resistance to the white pine blister rust fungus (Cronartium ribicola Fisch.) in sugar pine (Pinus lambertiana Dougl.) has been previously mapped. The objectives of the present study were to saturate the region flanking R with tightly linked markers and to construct genetic maps for each of four individual seed trees. Bulked segregant analysis (BSA) and haploid segregation analysis were employed to identify random amplified polymorphic DNA (RAPD) markers linked to R. Automated PCR analysis was used to assay 1115 primers with susceptible and resistant DNA pools from each of four seed trees (8920 PCR reactions). Thirteen RAPD loci were identified that were linked to R. The linkage analyses programs JoinMap 1.4 and Mapmaker 2.0 were used to order RAPD loci relative to R and to construct maps for each of the individual seed trees. Two seed trees, 5701 and 6000, had a large number of tightly linked markers flanking R. These trees will be used in subsequent high-resolution mapping experiments to identify very tightly linked markers to facilitate the eventual cloning of R. Received: 1 May 1998 / Accepted: 13 July 1998     

Identification of QTLs influencing wood property traits in loblolly pine ( Pinus taeda L.). II. Chemical wood properties

Chemical wood property traits were analyzed for the presence of quantitative trait loci (QTLs) in a three-generation outbred pedigree of loblolly pine (Pinus taeda L.). These traits were assayed using pyrolysis molecular beam mass spectrometry and include mass spectrum peak intensities associated with carbohydrates, α-cellulose and hemicellulose sugars, and lignin. Models for projection to latent structures (PLS) were used to also estimate the chemical composition of cell walls (i.e., α-cellulose, galactan and lignin) from mass spectrum data using multivariate regression. Both earlywood and latewood fractions from the fifth annual ring were analyzed for each trait. An interval mapping approach designed for an outbred pedigree was used to estimate the number of QTLs, the magnitude of QTL effects, and their genomic position. Eight unique QTLs influencing cell wall chemistry were detected from multiple peak intensities and/or PLS estimates using the one- and two-QTL models. Significant differences in chemical contents were observed among the populations from North Carolina vs Oklahoma, and results from QTL×environment analyses suggest that QTLs interact with environmental location. QTLs should be verified in larger experiments and in different genetic and environmental backgrounds. QTL mapping will help towards eventually identifying genes having a major effect on chemical wood properties. Received: 19 January 2001 / Accepted: 31 May 2001     

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis)

Summary. In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles – plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine. Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, People’s Republic of China.