Male-sterile Trees Research Articles

Sexual interference revealed by joint study of male and female pollination success in chestnut.

Most seed plants produce both pollen and ovules. In principle, pollen export could interfere with pollen import through self-pollination, resulting in ovule usurpation and reduced fruit set. Evidence for such interference exists under experimental settings but its importance under natural conditions is unknown. To test for sexual interference in nature, it is necessary to study together mating system, through paternity analyses, and fruit set, the proportion of flowers giving seeds or fruits. We developed a new model combining both processes, using chestnut (Castanea) as case study. We carried out a paternity analysis in an intensively studied plot of 273 trees belonging to three interfertile chestnut species and including a range of individuals with more or less functional stamens, resulting in a large data set of 1924 mating events. We then measured fruit set on 216 of these trees. Fruit set of male-fertile trees was much lower than that of male-sterile trees. Our process-based model shows that pollen is not limiting in the study site and hence cannot account for reduced fruit set. It also indicates that self-pollination is high (74%) but selfing rate is low (4%). Self-pollen is less competitive than cross-pollen, reducing sexual interference, but not sufficiently, as many ovules end up being self-fertilized, 95% of which abort before fruit formation, resulting in the loss of 46% of the fruit crop. These results suggest that the main cause of reduced reproductive potential in chestnut is sexual interference by self-pollen, raising questions on its evolutionary origins.

Revisiting pollination mode in chestnut (Castanea spp.): an integrated approach

ABSTRACT Wind, insects, or both? The pollination mode of chestnuts, an important genus of nut-producing forest trees of the Fagaceae family, is still unclear. We revisit this old question using an integrated approach, focusing on cultivated Castanea sativa trees and hybrids in South-western France. We first conducted a large-scale insect isolation experiment. We then monitored 16 trees, focusing on flowering phenology, flower abundance and insect visits. Half of these trees are male-sterile, helping explore the role of pollen in insect attraction. Finally, we characterized the pollination syndrome of chestnuts and contrasted it with that of wind-pollinated oaks using original and published data. Chestnut female flowers have erect styles resembling stamens from male flowers, a probable case of intersexual mimicry. The tree’s unusual phenology includes two peaks of pollen production. Pollinator exclusion experiments demonstrated a predominant role of insects in chestnut pollination. Flowering trees attract large numbers of beetles, bees and flies. In contrast, the few insects seen on female flowers (66 in 32 h of observation, <2% of the total) were mostly beetles. Compared to male-fertile trees, male-sterile trees attract fewer insects overall but their female flowers are more frequently visited and they have higher fruit set. All chestnut flower traits examined, such as the tiny pollen grains and the huge rate of pollen production, resulting in the highest pollen/ovule ratio ever reported in plants, are compatible with a beetle pollination syndrome. The high uncertainty of this pollination mode and its convergence with wind pollination explain the pervading confusion regarding chestnut pollination.

Marker-Assisted Selection of Trees with MALE STERILITY 1 in Cryptomeria japonica D. Don

The practical use of marker-assisted selection (MAS) is limited in conifers because of the difficulty with developing markers due to a rapid decrease in linkage disequilibrium, the limited genomic information available, and the diverse genetic backgrounds among the breeding material collections. First, in this study, two families were produced by artificial crossing between two male-sterile trees, ‘Shindai11’ and ‘Shindai12’, and a plus tree, ‘Suzu-2’ (Ms1/ms1) (S11-S and S12-S families, respectively). The segregation ratio between the male-sterile and male-fertile trees did not deviate significantly from the expected 1:1 ratio in either family. These results clearly suggested that the male-sterile gene of ‘Shindai11’ and ‘Shindai12’ is MALE STERILITY 1 (MS1). Since it is difficult to understand the relative positions of each marker, due to the lack of a linkage map which all the closely linked markers previously reported are mapped on, we constructed a partial linkage map of the region encompassing MS1 using the S11-S and S12-S families. For the S11-S and S12-S families, 19 and 18 markers were mapped onto the partial linkage maps of the MS1 region, respectively. There was collinearity (conserved gene order) between the two partial linkage maps. Two markers (CJt020762_ms1-1 and reCj19250_2335) were mapped to the same position as the MS1 locus on both maps. Of these markers, we used CJt020762 for the MAS in this study. According to the MAS results for 650 trees from six prefectures of Japan (603 trees from breeding materials and 47 trees from the Ishinomaki natural population), five trees in Niigata Prefecture and one tree in Yamagata Prefecture had heterozygous ms1-1, and three trees in Miyagi Prefecture had heterozygous ms1-2. The results obtained in this study suggested that ms1-1 and ms1-2 have different geographical distributions. Since MAS can be used effectively to reduce the labor and time required for selection of trees with a male-sterile gene, the research should help ensure that the quantity of breeding materials will increase to assist future tree-breeding efforts.

Comprehensive analysis of small RNAs expressed in developing male strobili of Cryptomeria japonica.

Deep sequencing of small RNAs (sRNAs) in developing male strobili of second-generation offspring originating from a nuclear genic male sterile tree of Cryptomeria japonica were performed to characterize sRNA populations in the male strobili at early pollen developmental stages. Comparing to sequences of microRNA (miRNA) families of plant species and sRNAs expressed in the reproductive organs of representative vascular plants, 37 conserved miRNA families were detected, of which eight were ubiquitously expressed in the reproductive organs of land plant species. In contrast, miR1083 was common in male reproductive organs of gymnosperm species but absent in angiosperm species. In addition to conserved miRNAs, 199 novel miRNAs candidates were predicted. The expression patterns of the obtained sRNAs were further investigated to detect the differentially expressed (DE) sRNAs between genic male sterile and fertile individuals. A total of 969 DE sRNAs were obtained and only three known miRNA families were included among them. These results suggest that both conserved and species-specific sRNAs contribute to the development of male strobili in C. japonica.

A simple allele-specific PCR marker for identifying male-sterile trees: Towards DNA marker-assisted selection in the Cryptomeria japonica breeding program

The number of people in Japan suffering from Cryptomeria japonica pollinosis has risen considerably since the 1970s as the area planted with this species has increased. In order to reduce the amount of pollen dispersed, breeding programs using trees with male-sterile genes have been implemented. We have constructed partial linkage maps surrounding a male sterility gene (ms-1) in four families of C. japonica to facilitate this process. The marker most closely linked to ms-1 was different in the four mapping families: gSNP00438, gSNP01452, estSNP00083, and estSNP01228 in the TO13S family (3.1 cM from ms-1); gSNP05835 and gSNP06239 in the S3T67 family (2.0 cM from ms-1); gSNP05835 in the F1N4 family (1.5 cM from ms-1); and gSNP06239 in the T5 family (4.2 cM from ms-1). This is probably mainly due to genetic differences between the parents used to produce the mapping families. However, in all four families, the accuracy with which male-sterile trees could be identified using the closest markers was more than 96.0 %. These results suggested that marker-assisted selection of male-sterile trees within a given family is feasible using the closest flanking markers to the ms-1 locus. We also developed an allele-specific PCR marker for identifying male-sterile trees in the TO13S family from which male-sterile seedlings are produced. Allele-specific PCR using three primer combinations produced two clear fragments, which could be easily separated by agarose gel electrophoresis: one fragment with a molecular weight of 410 bp, which was present in all samples and could thus be used as a positive control, and another of lower molecular weight (196 bp), which was specific for male-sterile trees. This marker makes it possible to carry out a simple and economical PCR assay for the detection of the SNP linked to the target gene without the need to use fluorescent labels. This study shows how a simple allele-specific PCR marker for an important major gene in a forest tree species can be developed using information from a high-density linkage map. In addition, our results will facilitate the first application of MAS (marker assisted selection) in conifers because the male sterility in C. japonica has several advantages and may be one of the best examples for MAS in conifers.

The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don.

BackgroundHigh-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (ms1) for MAS in C. japonica, a species that is important in Japanese afforestation but which causes serious social pollinosis problems.ResultsWe constructed a high-density saturated genetic linkage map for C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM.ConclusionsUsing the high-density map, we located the ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity.

Cytological study of a male-sterile Cryptomeria japonica that does not release microspores from tetrads

To determine the mechanism of male-sterility Cryptomeria japonica tree Shindai3, the process of microspore development was observed under light and fluorescence microscopy. Microspore development in the Shindai3 was normal until the tetrad stage, but separation of the microspores from the tetrads was not observed even after callose had been degraded. In contrast to the microspore stage in a male-fertile tree, amorphous substances were observed around tetrads in the Shindai3, and the amount of the amorphous substance increased from the middle of November to the middle of December. The substances are hypothesized to prevent the separation of individual microspores from tetrads by inducing adhesion between microspore cells within the tetrad or enclosing the tetrads. The form of the tetrads in the Shindai3 was maintained until just before the pollen dispersal season, but they ultimately degenerated. The results of this study indicate that the mechanism causing male sterility in Shindai3 differs from that previously reported for other male-sterile trees of C. japonica.

Advanced generation peach-almond hybrids as seedling rootstocks for almond: first year growth and potential pollenizers for hybrid seed production

For decades, seedling peaches have been used as the standard rootstock in California almond orchards. Vigorous, deep rooted trees are needed in almond orchards for maximum yields and to withstand the annual tree-shaking at harvest. Currently, researchers are actively evaluating rootstocks for almonds in field trials and in various screening protocols. In this study, seedling rootstocks, obtained from male-sterile advanced generation peach-almond (PEAL) hybrid mother trees, were compared with ‘Nemaguard’ peach seedlings for emergence in the nursery row, trunk caliper at propagation time, and end of season dormant above ground tree weight. Seedling emergence was affected significantly (P ≤ 0.05) by seed source, as was trunk caliper and end of season dormant above ground tree weight. Trunk caliper and dormant above ground tree weight were also affected significantly (P ≤ 0.05) by planting year. Results obtained in this study demonstrate the enhanced first year growth from seedlings of advanced generation PEAL hybrids, as compared to ‘Nemaguard’ seedlings. Seedling emergence in the rootstock bed was not affected significantly by planting year. Advanced generation PEAL hybrid seedlings were ready for June-budding at an earlier date compared to ‘Nemaguard’ seedlings, providing the potential for larger-sized finished nursery stock by the end of the growing season. Due to the male-sterile status of the advanced generation PEAL mother trees, bloom periods of several root-knot nematode resistant rootstock cultivars were examined for their degree of synchronicity with the mother trees. The examined rootstocks and mother trees varied in both chill hour and post-chill heat requirements necessary to effect bloom. ‘Flordaguard’ peach rootstock began bloom in advance of the male-sterile mother trees, whereas the bloom period of ‘Tsukuba No. 4’ occurred well after, suggesting they would not be effective synchronous pollenizers for consistent hybrid seed production. Based on more limited flowering period data, better bloom synchronicity was achieved with a Tsukuba No. 4 X Flordaguard hybrid.

Plus tree of Cryptomeria japonica D. Don with a heterozygous male-sterility gene

To find plus tree clones of Cryptomeria japonica that are heterozygous for a male-sterility gene (Aa), we crossed a homozygous male-sterile tree (aa) with 63 clones. Male sterility in this case is controlled by a recessive allele at a single gene locus and is expressed only in homozygotes. All F1 seedlings obtained by crossing the male-sterile mother tree and 62 out of the 63 clones produced pollen. In contrast, F1 seedlings obtained from the crossing between the male-sterile mother tree and a plus tree clone, Ohara 13, produced 64 male-sterile individuals and 52 fertile individuals. The segregation ratio fitted the expected 1:1 ratio according to a chi-square test. These results clearly demonstrate that the Ohara 13 clone is heterozygous for a male-sterility gene.

Study of sugi (Cryptomeria japonica D. Don) with male-sterility gene using CAPS markers

The frequency of open-pollinated male-sterile seedlings from a male-sterile mother tree in Cryptomeria japonica D. Don was determined in 765 open-pollinated seedlings. Pollen was not formed by 29 out of the 765 seedlings. To find pollen donor candidates for 29 male-sterile seedlings, a male-sterile tree was crossed with five individuals in the neighborhood of the mother tree. Analysis of microspores in male flowers of F1 seedlings revealed that one of the five individuals had a heterozygous male-sterility gene. Paternity tests were conducted on the 29 male-sterile seedlings using eight kinds of CAPS markers in C. japonica. The pollen parents of 29 male-sterile seedlings were not only a tree in the neighborhood of the mother tree, but also other trees. Since it was found that the pollen parents of four of the male-sterile seedlings were not in the study plot, it was inferred that C. japonica trees that possess the male-sterility gene grow in other places and may be widely distributed.

Inheritance of male sterility in apricot

Progenies (total of 1,114 seedlings) from crosses representing all possible genotypic combinations between 4 male-fertile and 1 male-sterile apricot parents were scored for the male sterility trait. Crosses between putative heterozygous normal cultivars yielded 25% of male-sterile seedlings, which supports a previous hypothesis that male sterility is controlled by a recessive allele of one nuclear locus. Crosses between those parents and putative homozygous normal cultivars did not produce any male-sterile tree. Finally, the proportion of male-sterile progeny in crosses between a male-sterile and two male-fertile cultivars depended on the genotype of the male parent. When it was heterozygous approximately 50% of the progeny was sterile, whereas when a homozygous fertile parent was used, no male-sterile progeny was obtained. These results confirm a previously proposed model, in which the male sterility trait in apricot is controlled by a single recessive gene.

Inheritance of the Trait of Male Sterility in Cryptomeria japonica

Matings of male-sterile C. japonica and fertile elite C. japonica, as well as backcross seedlings of male-sterile trees, were carried out to clarify the genetic trait of male sterility of the C. japonica. The seeds from male-sterile C. japonica were germinated in an incubator and grown them in the greenhouse between 1994 and 1997. The seedlings were treated with 100 ppm gibberellin at early July 1994 and early July 1995 to promote the formation of male flowers. In the middle of January 1995, the male flowers of all seedlings were examined under the microscope to confirm the production of pollen. In January 1996, pollen did not developed in to the male flowers from the seedlings between the fertiled elite and backcross C. japonica. In January 1997, all seedlings of elite C. japonica produced pollen in their male flowers; however, pollen did not developed in 55 out of 120 backcrossed seedlings. This evidence suggests that the heredity pattern of male sterility in C. japonica is nuclear male sterility controlled by a pair of recessive genes.

Chloroplast-DNA variation in cultivated and wild olive (Olea europaea L.)

Polymorphism in the lengths of restriction fragments of the whole cpDNA molecule was studied in cultivated olive and in oleaster (wild olive) over the whole Mediterranean Basin. Seventy two olive cultivars, 89 very old trees cultivated locally, and 101 oleasters were scored for ten endonucleases. Moreover, maternal inheritance of cpDNA in olive was shown by analysing the progeny of a controlled cross between two parents which di!ered in their cpDNA haplotypes. In the whole species, three site- and three length-mutations were observed, corresponding to "ve distinct chlorotypes. The same chlorotype (I) was predominant in both oleasters and cultivated olive trees, con"rming that these are closely related maternally. Three other chlorotypes (II, III and IV) were observed exclusively in oleaster material and were restricted either to isolated forest populations or to a few individuals growing in mixture with olive trees possessing the majority chlorotype. An additional chlorotype (V) was characterised by three mutations located in distinct parts the cpDNA molecule but which were never observed to occur separately. This chlorotype, more widely distributed than the other three, in both cultivated and wild olive, and occurring even in distant populations, was observed exclusively in male-sterile trees showing the same speci"c pollen anomaly. However, in the present study, no evidence was provided for a direct relationship between the occurrence of the cpDNA mutations and male sterility. It is suggested that the large geographic distribution of chlorotype V may be related to the high fruit production usually observed on male-sterile trees. These may be very attractive for birds which are fond of olive fruit and spread the stones e$ciently. Probably for the same reason, people preserved male-sterile oleasters for long periods and, in several places, used male-sterile cultivars over large areas.

Cytological and Genetical Studies on Male Sterility in Cryptomeria japonica D. Don

Genetic male sterility is a useful trait in plant breeding, especially in angiosperm crops such as corn, onion and carrot. We found a male sterile sugi (Cryptomeria japonica D. Don) tree in Toyama, Japan. Pollen of sugi is one of the major causes of pollinosis in Japan. We carried out this research in an attempt to make clear the characteristics and inheritance of this male sterility. Microsporogenesis of the male sterile tree proceeded meiosis, however, the microspores collapsed after they were separated from pollen tetrads in locules, resulting in complete male sterility. Most likely, ethylene evolution was responsible for male sterility expression. Full seed setting in the male sterile tree indicated normal macrosporogenesis. Seeds obtained from crossing between male sterile and normal lines showed relatively high level of germination and their seedlings grew vigorously. The somatic chromosome numbers of 241 germinated seeds, derived from the male sterile tree, were mostly 22, euploid. These results indicated that male sterile tree was different from other similar previously reported trees with low pollen fertility, resulting from triploid or trisomics. Probably, male sterility in sugi is either nuclear genetic male sterility or cytoplasmic male sterility.

Observations on Inheritance of Male Sterility in Apricot

Male sterility is defined as the deviant condition in normally bisexual plants when no viable pollen is formed (Frankel and Galun, 1977). Male sterility has been exploited as an effective tool to aid hybrid seed production in many crops. Using this trait, seed producers can transform one inbred from a planned hybridization into a “female” line, thereby ensuring seed of hybrid origin on the male-sterile parent (Janick, 1972). However, male sterility is an undesirable characteristic in scion cultivars of Prunus to be used for fruit production because this trait would restrict yield in large monocultural production blocks. From the viewpoint of an apricot breeding program, male sterility is a characteristic to be avoided (Scott and Weinberger, 1944). A review of apricot (Prunus armeniaca L.) pollen fertility (Burgos, 1991) indicated that only two cases of male-sterile apricot cultivars (‘Arrogance’ and ‘Colorao’) have been described (Garcia et al., 1988). In this paper we show where several male-sterile trees were observed in progenies from controlled hybridizations in P. armeniaca, and we propose a preliminary inheritance model of the trait. Nine different crosses with a total of 378 trees were scored for the male sterility trait. Male-sterile anthers can be distinguished visually from normal fertile anthers during the bloom period. Shrunken, discolored anthers are indicative of male-sterile pollen and provide a sharp contrast to the swollen, yellow appearance of normal pollen-fertile anthers (Connors, 1927). Several samples of shrunken, discolored anthers were chosen randomly to

Pollination biology of Prunus mahaleb L.: deferred consequences of gender variation for fecundity and seed size

Abstract This study describes the reproductive biology of Prunus mahaleb, a rosaceous treelet, in a southeastern Spanish population. The species is gynodioecious with 55.4% of the plants being male-fertile and 44.6% presenting non-functional, shrunken anthers with no pollen, and behaving as functional females. Individual trees produced the same flower morph in four consecutive study years. Fruit set in bagged inflorescences was absent in male-sterile trees and was very reduced in male-fertiles. Open-pollinated flowers of male-fertiles showed greater fruit set (x= 29.1%) than male-steriles (x = 25.3%). Average fruit set in three experimental treatments (control, selfed, and crossed) were 29.05%, 41.9% and 38.6%, respectively, for male-fertile trees; those for male-steriles were 25.3%, 0% and 39.2%, respectively. Seeds from male-sterile plants were heavier (71.33 mg) than those of male-fertile plants (66.05 mg) but did not differ in germination ability. Male-fertile and male-sterile trees differ significantly in average nectar concentration (73.5% and 55.9%, respectively) and nectar volume secreted/flower and day (0.142 μl and 0.171 μl, respectively). A total of 41 species of insect flower visitors were recorded. Calliphorid and tachinid flies (41.97% of total visits), and andrenid bees (30.30%), were the most frequent visitors. The diversity of insect visitors was greater in male-fertile trees. Bees and flies accounted for 50.5% and 49.5%, respectively of total visits to male-fertile trees. Flies were far more frequent than bees at male-sterile flowers (76.9% and 23.1%, respectively). Male-sterile trees received higher average visitation (47 insects/census) than male-fertile trees (32 insects/census). Individual trees showed a relative constancy of the fecundity rankings between years. Fruit production was significantly higher in male-sterile trees, with a four-year average of 6558 fruits, in contrast with male-fertile trees which yielded 4670 fruits. This 1.77-fold difference in favour of male-sterile trees over male-fertiles was not compensated by the greater fruit set of the latter. This is attributable to the lower outcrossing rate of male-fertiles, estimated as 52.68% from seed mass data. In addition, greater seed mass of female progeny, and higher visitation rate by insects might explain the maintenance of this polymorphism. These results have far-reaching implications for the demography and seed-dispersal ecology of this endozoochorus species.

Pollination biology of Prunus mahaleb L.: deferred consequences of gender variation for fecundity and seed size

This study describes the reproductive biology of Prunus mahaleb, a rosaceous treelet, in a southeastern Spanish population. The species is gynodioecious with 55.4% of the plants being male-fertile and 44.6% presenting non-functional, shrunken anthers with no pollen, and behaving as functional females. Individual trees produced the same flower morph in four consecutive study years. Fruit set in bagged inflorescences was absent in male-sterile trees and was very reduced in male-fertiles. Open-pollinated flowers of male-fertiles showed greater fruit set (x̄= 29.1%) than male-steriles (x̄ = 25.3%). Average fruit set in three experimental treatments (control, selfed, and crossed) were 29.05%, 41.9% and 38.6%, respectively, for male-fertile trees; those for male-steriles were 25.3%, 0% and 39.2%, respectively. Seeds from male-sterile plants were heavier (71.33 mg) than those of male-fertile plants (66.05 mg) but did not differ in germination ability. Male-fertile and male-sterile trees differ significantly in average nectar concentration (73.5% and 55.9%, respectively) and nectar volume secreted/flower and day (0.142 μl and 0.171 μl, respectively). A total of 41 species of insect flower visitors were recorded. Calliphorid and tachinid flies (41.97% of total visits), and andrenid bees (30.30%), were the most frequent visitors. The diversity of insect visitors was greater in male-fertile trees. Bees and flies accounted for 50.5% and 49.5%, respectively of total visits to male-fertile trees. Flies were far more frequent than bees at male-sterile flowers (76.9% and 23.1%, respectively). Male-sterile trees received higher average visitation (47 insects/census) than male-fertile trees (32 insects/census). Individual trees showed a relative constancy of the fecundity rankings between years. Fruit production was significantly higher in male-sterile trees, with a four-year average of 6558 fruits, in contrast with male-fertile trees which yielded 4670 fruits. This 1.77-fold difference in favour of male-sterile trees over male-fertiles was not compensated by the greater fruit set of the latter. This is attributable to the lower outcrossing rate of male-fertiles, estimated as 52.68% from seed mass data. In addition, greater seed mass of female progeny, and higher visitation rate by insects might explain the maintenance of this polymorphism. These results have far-reaching implications for the demography and seed-dispersal ecology of this endozoochorus species.

Gynodioecy and Male Sterility in Eucalyptus leucoxylon F. Muell. (Myrtaceae)

The breeding system of Eucalyptus leucoxylon was investigated using crosses, microscopy, and isozyme analysis. In the study population, 57% of trees were male sterile because microspores were produced by meiosis and formed exines; however, they contained little starch, lacked intines, and had collapsed by anthesis, surrounded by incompletely degenerated tapetum. Hermaphrodite trees produced bisexual flowers, which were protandrous. Anthers dehisced in the bud and 93% of pollen was deposited on the upper style and lower stigma as the filaments extended; only 7% remained on the anthers. Hermaphrodite flowers set low levels of autogamous and assisted selfed seed, compared with open pollinations. Counts of pollen tubes in open-pollinated pistils of each morph showed that female flowers received less pollen than hermaphrodite flowers, but despite this, there was no difference in open-pollinated seed set between the two morphs. Multilocus estimates of outcrossing rates (t̂) from male-sterile trees showed values approaching complete outcrossing (0.96), while hermaphrodite trees showed significant levels of inbred seed in open-pollinated seed crops (0.83) but still maintained a higher outcrossing rate than reported for most other eucalypts. This study has shown that E. leucoxylon maintains high levels of outbreeding, despite the deposition of self pollen on the stigma in the secondary pollen presentation mechanism; it may achieve this through protandry, pollen removal, male sterility, and late-acting self-incompatibility.

Some Outstanding New Chestnut Hybrids I.

GRAVES, ARTHUR HARMOUNT. (Conn. Agric. Expt. Station, New Haven, Conn.)Some outstanding new chestntt hybrids II. Bull. Torrey Bot. Club 89: 161-172, 1962. Three new chestnut hybrids are described which are to serve as the ortets of their respective clones: (1) Toumey Chestnut, a tall, erect tree, rapid-growing but not especially prolific; leaves with prominent shoulder below each tooth, pubescence both stellate and simple; bur slightly flattened, nut plump, squarish, flavor indifferent; (2) Hamden Chestnut, a handsome, ornamental, spreading, late-flowering tree with large, thickish, glossy leaves, aiid a moderate supply of large nuts; (3) Dwarf Japanese-American Chestnut, a dwarf, male-sterile tree suitable for a small area, a prolific bearer, with leaves showing lepidote glanids beneath, nuts fairly sweet. These trees are all blight-resistant, and hardy in zone V of Rehder.

STUDIES IN CACAO

SUMMARY Previous investigations on the pollination of cacao are reviewed. It is pointed out that the consensus of opinion is in favour of the pollinating agent being small insects, either thrips, aphides or ants. Direct proof of natural crossing was obtained by examination of flowers on a male sterile tree, in which 9 per cent. of the flowers were found to be pollinated. Daily examination of flowers artificially pollinated, flowers attended by ants and aphides, and flowers with no attendant insects, indicated that the majority of cacao flowers are never pollinated under Trinidad conditions. Adequate pollination took place in 35 per cent. of flowers attended by ants and aphides, and is only 6 per cent of the controls. It is concluded that ants and aphides are the chief pollinating agents in the field under examination. Elimination of crawling insects demonstrated the presence of another pollinating agent, probably a night flying insect which pollinated about 1 per cent. of flowers.