Chromosome numbers, meiotic behavior, and pollen viability of species of Vriesea and Aechmea genera (Bromeliaceae) native to Rio Grande do Sul, Brazil

Chromosome number, meiotic behavior, and pollen viability were analyzed in 15 species of two genera, Vriesea and Aechmea, native to Rio Grande do Sul, Brazil. This study is the first cytogenetic analysis of these taxa. The chromosome numbers are all n = 25, consistent with the proposed base number of x = 25 for Bromeliaceae. All examined taxa displayed regular bivalent pairing and chromosome segregation at meiosis. Observed meiotic abnormalities include univalents in metaphase I; missing or extra chromosomes and precocious division of centromeres in metaphase II; laggards in telophase I and anaphase II/telophase II. The high pollen viability (>88%) reflects a regular meiosis.     

Microsporogenesis in Brachiaria brizantha (Poaceae) as a selection tool for breeding

The genus Brachiaria comprises more than 100 species and is the single most important genus of forage grass in the tropics. Brachiaria brizantha, widely used in Brazilian pastures for beef and dairy production, is native to tropical Africa. As a subsidy to the breeding program underway in Brazil, cytological studies were employed to determine the chromosome number and to evaluate microsporogenesis in 46 accessions of this species available at Embrapa Beef Cattle (Brazil). Thirty-four accessions presented 2n = 36; seven had 2n = 45, and five had 2n = 54 chromosomes. Based on the higher level of chromosome association observed in diakinesis, in tetra-, penta-, and hexavalents, respectively, it was concluded that they are derived from x = 9; consequently, these accessions are tetra- (2n = 4x = 36), penta- (2n = 5x = 45), and hexaploids (2n = 6x = 54). The most common meiotic abnormalities were irregular chromosome segregation due to polyploidy. Chromosome stickiness, abnormal cytokinesis, non-congressed bivalents in metaphase I and chromosomes in metaphase II, and chromosome elimination were recorded at varying frequencies in several accessions. The mean percentage of meiotic abnormalities ranged from 0.36 to 95.76%. All the abnormalities had the potential to affect pollen viability by generating unbalanced gametes. Among the accessions, only the tetraploid ones with less than 40% of abnormalities are suitable as pollen donors in intra- and interspecific crosses. Currently, accessions with a high level of ploidy (5 and 6n) cannot be used as male genitors in crosses because of the lack of sexual female genitors with the same levels of ploidy.     

Pollen sources used by species of Meliponini (Hymenoptera: Apidae) along the Rio Negro channel in Amazonas,Brazil

This work analyses the pollen stored by Meliponini bees whose nests were sampled along the Rio Negro channel, Amazonas, Brazil, during the final phase of the rainy season, which is also the time of pollen scarcity. From a total of 51 samples studied, 78 pollen types were identified, of which 39 were determined to species level. The pollen types were from 70 genera and 42 families. Based on the pollen records, a clustering analysis was performed, as well as the calculation of the trophic overlap index. The study demonstrates that the plants used by the bees do not depend on them for pollination in most cases; it rather suggests scarcity of pollen resources from the plants adapted to the pollination of Meliponini, and emphasises the ability of such bees to switch to new available resources. This work indicates a list of important plants for meliponiculture suitable for cultures near meliponaries.     

Viability and longevity of pollen from transgenic and nontransgenic tall fescue (Festuca arundinacea) (Poaceae) plants

Pollen is an important vector of gene flow in plants, particularly for outcrossing species like tall fescue. Several aspects of pollination biology were investigated using pollen from transgenic and nontransgenic plants of tall fescue (Festuca arundinacea Schreb.), the most important forage species worldwide of the Festuca genus. To effectively assess in vitro pollen viability in tall fescue, an optimized germination medium (0.8 mol/L sucrose, 1.28 mmol/L boric acid and 1.27 mmol/L calcium nitrate) was developed. Treatment with relatively high temperatures (36° and 40°C) and high doses of UV-B irradiation (900-1500 μW/cm(2)) reduced pollen viability, while relative humidity did not significantly influence pollen viability. Viability of pollen from transgenic progenies (T1 and T2) was similar to that from seed-derived control plants. Pollen from primary transgenics (T0) and primary regenerants (R0) had various levels of viability. Hand pollination using the primary regenerants and transgenics revealed that no seed set could be obtained when pollen viability was lower than 5%. Pollen from transgenic progenies and nontransgenic control plants could survive up to 22 h under controlled conditions in growth chamber. However, under sunny atmospheric conditions, viability of transgenic and nontransgenic pollen reduced to 5% in 30 min, with a complete loss of viability in 90 min. Under cloudy atmospheric conditions, pollen remained viable up to 240 min, with about 5% viability after 150 min. This report is the first on pollen viability and longevity in transgenic forage grasses and could be useful for risk assessment of transgenic plants.     

Changes of pollen viability of ornamental plants after long-term preservation in a cryopreservation pollen bank

This study determined the changes in pollen viability of 102 species/cultivars of ornamental plants (affiliated to 32 genera of 14 families) following long-term liquid nitrogen storage in a cryopreservation pollen bank. The goal was to provide information on the safety and stability of pollen cryopreservation technology. Fresh pollen at the time of storage was used as the control, and the study examined the pollen viability of ornamental plants cryopreserved for 8, 9, or 10 years. The results show that pollen of the 102 species/cultivars in the cryopreservation pollen bank retained viability ranging from 1% to 58%, After long-term storage there were changes in viability: 11.76% (12 species/cultivars) had increased viability, 16.67% (17 species/cultivars) had stable viability, and the viability of 71.57% (73 species/cultivars) showed a decreasing trend.     

Selection for tolerance to copper during pollen formation in Mimulus guttatus Fischer ex DC

In Mimulus guttatus, copper tolerance is determined largely by a single gene and is expressed in both the sporophyte and microgametophyte. This study explores the extent to which selection during pollen formation affects copper tolerance in the sporophytic generation. Two sets of plants heterozygous for copper tolerance, produced by reciprocal crosses between different copper-tolerant or sensitive families, and the plant on which the original observations were based, were cloned and grown in control or copper-supplemented solutions. Pollen viability and the number of tolerant progeny produced in backcrosses to sensitive plants were compared. In addition, the effect of copper treatment on pollen viability in vitro was compared for plants tolerant, sensitive and heterozygous for copper tolerance. The extent to which in vitro pollen viability decreased in response to copper treatment corresponded to the copper tolerance of the pollen source. When grown with added copper, four of the five plants showed significant reductions in pollen viability, ranging from 18% to 48% of control values. The reductions in pollen viability were correlated with an increase in tolerant progeny (r= 0.679, p=0.004). Increases in tolerant progeny could be large, ranging from 119% to 170% of that of controls, but were usually smaller than was predicted from the reductions in viable pollen. In addition, plants derived from reciprocal crosses differed significantly in the extent to which pollen viability was decreased and sporophytic tolerance increased. Thus, while selection during pollen formation could increase sporophytic tolerance, sporophytic factors, perhaps including cytoplasmic or epigenetic ones, moderated the effectiveness of pollen selection for copper tolerance.     

Cytogenetic and molecular characterization of morphologically variable Sisyrinchium micranthum (Iridaceae) in southern Brazil

Sisyrinchium micranthum is native to southern Brazil and shows a wide range of morphological variation in this region. This study was performed to compare individuals of this species in terms of traits such as morphology, chromosome number, meiotic behaviour, pollen viability and genetic diversity using inter simple sequence repeat markers. First, general morphological aspects were evaluated and plants were classified into three morphological categories (CI, CII and CIII) to analyse the cytogenetic and genetic data. Three chromosome numbers corresponding to three ploidies were found: 2n = 2x = 16, 2n = 4x = 32 and 2n = 6x = 48. All morphological types showed regular meiotic behaviour and high meiotic index values and pollen viability. CII is the most frequent in southern Brazil. The most common haploid chromosome number, n = 8, has not been reported previously in this species. The percentage of polymorphic loci ranged from 6.45 to 61.29% for each accession. Molecular and cytogenetic analyses indicate that some S. micranthum accessions may have more than one ploidy. This study represents the first effort to characterize this taxonomically complex species based on cytogenetic and genetic aspects. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 350–364.     

Maytenus obtusifolia Mart. (Celastraceae): a tropical woody species in a transitional evolutionary stage of the gynodioecy–dioecy pathway

‘Gynodioecy–dioecy’ is one of the pathways by which dioecy can arise from hermaphroditism. Studies on sex determination and development of gynodioecious systems have focused on temperate and/or annual species. Little is known about the evolutionary dynamics of gynodioecy and dioecy in perennial tropical species, where these systems have more frequently evolved. Maytenus obtusifolia is an abundant species in restingas in southeastern Brazil. The sexual system of M. obtusifolia was investigated by studying the floral structure and reproductive biology. We considered the sexual system as an intermediate step in the pathway gynodioecy-dioecy. The characterization of the hermaphrodite morph was complex, because of a gradient of variation in floral morphology and reproductive characteristics (sizes of the style and stigmatic surface, pollen viability, embryo sacs containing hypertrophied synergids, and fruit set). This variation leads to different proportions of functional male and female flowers among hermaphrodite plants and is responsible for the different levels of reproductive success. Female reproductive success and pollen viability were negatively correlated with the hermaphrodite morph (r = ?0.67). The higher fruiting intensity and fruit/flower ratio of females (41 %) compared to hermaphrodites (2 %) and the fact that female plants produce more and better-quality seeds support the female compensation. We suggest that female sterility may be linked to the set of changes in the carpels. The differences in the quality and quantity of pollen grains of hermaphrodite plants, and the similar individual rates of pollen viability observed for three consecutive flowering events, may indicate a relationship with nuclear cytoplasmic sex determination.     

Effect of enhanced UV-B radiation on pollen quantity,quality, and seed yield in Brassica rapa (Brassicaceae)

We conducted three experiments to examine the influence of ultraviolet-B radiation (UV-B; 280–320 nm) exposure on reproduction in Brassica rapa (Brassicaceae). Plants were grown in a greenhouse under three biologically effective UV-B levels that simulated either an ambient stratospheric ozone level (control), 16% (“low enhanced”), or 32% (“high enhanced”) ozone depletion levels at Morgantown, WV, USA in mid-March. In the first experiment, we examined whether UV-B level during plant growth influenced in vivo pollen production and viability, and flower production. Pollen production and viability per flower were reduced by ≈50% under both enhanced UV-B levels relative to ambient controls. While plants under high-enhanced UV-B produced over 40% more flowers than plants under the two lower UV-B treatments, whole-plant production of viable pollen was reduced under high-enhanced UV-B to 17% of that of ambient controls. Whole-plant production of viable pollen was reduced under low-enhanced UV-B to 34% of ambient controls. In the second experiment, we collected pollen from plants under the three UV-B levels and examined whether source-plant UV-B exposure influenced in vitro pollen germination and viability. Pollen from plants under both enhanced-UV-B treatments had initially lower germination and viability than pollen from the ambient level. After in vitro exposure to the high-enhanced UV-B levels for 6 h, viability of the pollen from plants grown under ambient UV-B was reduced from 65 to 18%. In contrast, viability of the pollen from plants grown under both enhanced UV-B treatments was reduced to a much lesser extent: only from ≈43 to 22%. Thus, ambient source-plant pollen was more sensitive to enhanced UV-B exposure. In the third experiment, we used pollen collected from source plants under the three UV-B levels to fertilize plants growing under ambient-UV-B levels, and assessed subsequent seed production and germination. Seed abortion rates were higher in plants pollinated with pollen from the enhanced UV-B treatments, than from ambient UV-B. Despite this, seed yield (number and mass) per plant was similar, regardless of the UV-B exposure of their pollen source. Our findings demonstrate that enhanced UV-B levels associated with springtime ozone depletion events have the capacity to substantially reduce viable pollen production, and could ultimately reduce reproductive success of B. rapa.     

Melissopalynology in an area of Atlantic Forest (northeast region,Brazil)

Apis mellifera is a bee that was introduced to Brazil and has adapted very well to the climate conditions and vast diversity of plants that exist in the country. In the northeast region of Brazil, beekeepers make use of the association between bees and plants by selling various bee products, notably honey. One way to identify species visited by bees in an area is by the pollen in its products. Based on this, 16 samples of honey were analysed, which were collected over a period of two years and obtained from an apiary in the Atlantic Forest biome in the municipality of Entre Rios (Bahia). In addition, climatic data (precipitation and temperature) of the region were obtained for the months sampled. The average temperature of the region during the collection months varied from 22 to 28 °C. The highest precipitation recorded in the region was 133.7 mm³ and the lowest was 0.3 mm³. Seventy pollen types were found. The family Fabaceae was notable, with ten pollen types, of which Mimosa pudica was the most important with a high frequency of occurrence and distribution. The Eucalyptus (Myrtaceae) pollen type also had a high frequency of occurrence and distribution. In addition, pollen types corresponding to species that supply nectar to bees were identified, which contributes to the large diversity of bee plants for Apis mellifera in the study area.     

Pollinator preference and pollen viability mediated by flower color synergistically determine seed set in an Alpine annual herb

Gentiana leucomelaena manifests dramatic flower color polymorphism, with both blue‐ and white‐flowered individuals (pollinated by flies and bees) both within a population and on an individual plant. Previous studies of this species have shown that pollinator preference and flower temperature change as a function of flower color throughout the flowering season. However, few if any studies have explored the effects of flower color on both pollen viability (mediated by anther temperature) and pollinator preference on reproductive success (seed set) in a population or on individual plants over the course of the entire flowering season. Based on prior observations, we hypothesized that flower color affects both pollen viability (as a function of anther temperature) and pollen deposition (as a function of pollinator preference) to synergistically determine reproductive success during the peak of the flowering season. This hypothesis was tested by field observations and hand pollination experiments in a Tibetan alpine meadow. Generalized linear model and path analyses showed that pollen viability was determined by flower color, flowering season, and anther temperature. Anther temperature correlated positively with pollen viability during the peak of the early flowering season, but negatively affected pollen viability during the peak of the mid‐ to late flowering season. Pollen deposition was determined by flower color, flowering season (early, or mid‐ to late season), and pollen viability. Pollen viability and pollen deposition were affected by flower color that in turn affected seed set across the peak of the flowering season (i.e., when the greatest number of flowers were being pollinated). Hand pollination experiments showed that pollen viability and pollen deposition directly influenced seed set. These data collectively indicate that the preference of pollinators for flower color and pollen viability changed during the flowering season in a manner that optimizes successful reproduction in G. leucomelaena. This study is one of a few that have simultaneously considered the effects of both pollen viability and pollen deposition on reproductive success in the same population and on individual plants.     

Effects of salinity on seed set in rice

Salinity reduces fertility in rice (Oryza sativa L.), but little is known of the underlying cause(s). In order to determine the relative importance of pollen viability and stigmatic receptivity for seed setting, plants of the rice cultivar IR36 were treated with ‘artificial’ sea water (0,10, 25 or 5Omol^?3 with respect to NaCl) from 1 month after germination until the main tiller flowered. An increase in the salinity in the medium resulted in a decrease in the number of fertile florets and in the viability of pollen as determined both by pollen germination and by pollen staining with the tetrazolium salt 3-(4,5-dimethyl-ithyazolyl)-2,5-diphenyl monotetrazolium bromide. In order to assess the effects of salt on stigmas, seed production was measured for salt-grown and non-salt-grown female plants pollinated with viable pollen (from plants grown in the absence of salt). The percentage of seed set was reduced by 38% when the female plants were grown in 1Omol m^?3 Na and by 72% at 25mol m^?3 Na: no seed setting was recorded for plants grown in 50mol m^?3 Na. Comparisons between crosses involving male and female parents grown at different salinities indicated that effects on the female plants dominated those on pollinator plants. Mineral analysis of leaves of different ages showed that there was a gradient of K concentration from leaf to leaf which was opposite to that of Na and Cl at all levels of applied salinity: K was maximal in the flag leaf, where Na and Cl were minimal. Analysis also revealed that there was an increase in the concentrations of Na and Cl and a decrease in the concentration of K in the pollen grains and stigmas of plants subjected to saline conditions. Correlations between the concentration of Na and Cl in pollen and pollen staining and pollen germination in vitro suggest that Na and Cl perse were responsible for the poor viability. The change in ionic concentrations in pollen and stigmas was much larger than that in the younger leaves, and in particular very much larger than that in the lemmas and paleas.     

The Effect of Water Stress on Floral Characters, Pollination and Seed Set in White Clover (Trifolium repens L.)

White clover plants were subjected to either short-term developingwater stress or long-term stable levels of water deficit. Thehort-term stress reduced plant water status to about –2·0MPa over 16 d. The long-term stress was less severe, but wassustained for several weeks. Long-term water stress promotedthe production of inflorescences. However, water stress alsoincreased floret abortion and the premature death of whole flowerheads. The number of ovules per floret was decreased by waterstress. The most striking effect of both long- and short-term waterdeficit was to reduce pollen viability measured with the fluorochromaticassay. This was not an artefact of assay conditions. The pollenfrom water-stressed flower heads was not reversibly dehydrated;it did not score at similar viability to controls after incubationin conditions which hydrate pollen. In addition, the pollenfrom water-stressed plants lost viability more rapidly thanpollen from well-watered plants after removal from the flowerhead. The consequences of reduced pollen viability on seed set wereinvestigated by hand-crossing within and between groups of plantsmaintained for several weeks at three levels of water supply.Flower heads pollinated with pollen from water-stressed plantsset fewer seeds per floret than those pollinated with controlpollen. Key words: Trifolium repens, white clover, water stress, floral characters, seed set     

Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.     

Cytomixis and meiotic abnormalities during microsporogenesis are responsible for male sterility and chromosome variations in Houttuynia cordata

Houttuynia cordata (Saururaceae) is a leaf vegetable and a medicinal herb througout much of Asia. Cytomixis and meiotic abnormalities during microsporogenesis were found in two populations of H. cordata with different ploidy levels (2n = 38, 96). Cytomixis occurred in pollen mother cells during meiosis at high frequencies and with variable degrees of chromatin/chromosome transfer. Meiotic abnormalities, such as chromosome laggards, asymmetric segregation and polyads, also prevailed in pollen mother cells at metaphase of the first division and later stages. They were caused by cytomixis and resulted in very low pollen viability and male sterility. Pollen mother cells from the population with 2n = 38 showed only simultaneous cytokinesis, but most pollen mother cells from the population with 2n = 96 showed successive cytokinesis; a minority underwent simultaneous cytokinesis. Cytomixis and irregular meiotic divisions appear to be the origin of the intraspecific polyploidy in this species, which has large variations in chromosome numbers.     

The estimation of pollen viability in rice

Methods for testing pollen viability in rice were evaluatedby comparing staining with aniline blue in lactophenol, twotetrazolium salts and fluorescein diacetate with germinationin an in vitro culture medium. Staining with thiazolyl blue(MTT) was nicely correlated with germination. Under non-saline conditions, rice pollen was only viable fora short period of time, with an approximately 50% loss of viabilitywithin 20 min of shedding. When plants were salinized at eitherpanicle initiation or the booting stage, pollen viability wasreduced. Key words: Rice, Oryza sativa, pollen viability, salinity     

Evidence of apomixis in hemisexual dogroses, Rosa section Caninae

All members of Rosa section Caninae, dogroses are polyploid and characterized by their unbalanced meiosis, which in most cases leads to a pronounced morphological influence from the maternal parent. In a previous investigation on a pair of reciprocal crosses between two species in this section, Rosa dumalis and R. rubiginosa (2n=35), nine offspring plants (approximately 10%) did not receive any of the 21 RAPD markers present in the respective pollen parent. This was interpreted as a possible occurrence of apomixis. These nine plants have now been subjected to a further study with additional markers. Thirteen new RAPD markers showed the same result as in the previous investigation: none of the nine plants inherited any of the pollen donor markers. The reproducibility of the RAPD markers was checked by mixing DNA samples to obtain a series of artificial hybrids between the two parent plants. Twelve RAPD markers gave the expected result, whereas one marker appeared only 50% of the time. In addition, pollen viability, mean number of seeds per hip, mean seed weight, and mean weight of fruit flesh per hip have been studied on the four progeny groups: R. dumalis×R. rubiginosa plants which received pollen donor markers (PM plants), R. dumalis×R. rubiginosa plants which did not receive any pollen donor markers (NPM plants), R. rubiginosa×R. dumalis PM plants and R. rubiginosa×R. dumalis NPM plants. A canonical discriminant analysis based on these four reproductive characters separated the four progeny groups. There were significant differences between the two PM groups in all investigated characters, and also between the PM and the NPM groups in pollen viability. The result from the RAPD markers together with the differences in pollen viability between the PM and NPM progeny groups is taken as an indication that apomixis occurs within the Caninae section. Received: 13 October 1999 / Revision accepted: 28 January 2000     

Pollen viability and limitation of seed production in a population of the circumpolar cushion plant, Silene acaulis (Caryophyllaceae)

Pollen viability among genders and limitation of female seed production in a natural trioecious population of the circumpolar cushion plant Silene acaulis was examined. Pollen viability was estimated by an in vitro pollen germination experiment. Both male and hermaphrodite flowers displayed large variation in pollen viability (0–53% in hermaphrodite and 0–54% in male flowers). There was a significant difference between genders in pollen viability: male plants had on average higher pollen viability than hermaphrodite plants. Resource and pollen limitation of seed production was studied by an experiment consisting of three treatments; (I) hand-pollination and removal of all other flowers on the cushion, (II) hand-pollination without removal of other flowers, and (III) open pollination without removal of flowers. Hand-pollination increased seed production, whereas removal of flowers had no effect on seed production. Abortion of pollinated ovules during seed development and seed mass did not differ among treatments. To control for effect of fruit number on seed production, data from naturally pollinated individuals was used. There was a positive correlation between both total number of seeds and fruit number, mean seed number per fruit and fruit number, respectively. These results indicate that seed production of 5. acaulis is mainly limited by pollen availability whereas resource competition between fruits is not important as a limiting factor. The possible role of male quality differences between genders and pollen limitation of seed production for maintenance of trioecious reproductive systems is discussed.     

Evaluation of Viability,Shedding Pattern,and Longevity of Pollen from Genetically Modified (GM) Herbicide-tolerant and Wild-type Zoysiagrass (<Emphasis Type="Italic">Zoysia japonica</Emphasis> Steud.)

Knowledge about pollen viability is important when evaluating the risk of genetically modified (GM) plants. Here, staining via iodine potassium iodide (IKI) or triphenyl tetrazolium chloride (TTC) could not distinguish between live and dead pollen from Zoysia japonica. Therefore, to obtain a reliable assessment of such viability and longevity, we developed an optimum germination medium containing 20% sucrose and 50 ppm H₃BO₃. Pollen grains transferred to the germination medium at about 1000 hours had a germination rate of >90%. Pollen was most predominantly shed at approximately 1000 hours, with viability declining to nearly 0% at 1200 hours. All germinability was lost within 150 min when stored at 25°C. No significant difference was found between GM and non-GM plants in their pollen viability or longevity.     

Extensive citrus triploid hybrid production by 2x?×?4x sexual hybridizations and parent-effect on the length of the juvenile phase

The citrus fresh market demands the production of seedless citrus fruits, as seedy fruits are not accepted by consumers. The recovery of triploid plants has proven to be the most promising approach to achieve this goal, since triploids have very low fertility, are generally seedless and do not induce seeds in other cultivars by cross pollination. Triploid plants can be recovered by 2x?×?4x sexual hybridization. In this work, we present an effective methodology to recover triploid plants from 2x?×?4x hybridizations based on in vitro embryo rescue, ploidy level analysis by flow cytometry and genetic origin of triploid plants. The pollen viability of diploid and tetraploid citrus genotypes was analyzed by comparing the pollen germination rate in vitro. The pollen viability of tetraploid (doubled-diploid) genotypes is generally reduced but sufficient for successful pollination. Triploid embryos were identified in normal and undeveloped seeds that did not germinate under greenhouse conditions. The influence of parents and environmental conditions on obtaining triploid plants was analyzed and a strong interaction was noted between the parents and environmental conditions. The parental effect on the length of the juvenile phase was also demonstrated through observations of a large number of progeny over the last 15?years. The juvenile phase length of the triploid hybrids obtained with 'Fortune' mandarin as female parent and tetraploid 'Orlando' tangelo as male parent was shorter than the juvenile phase obtained with a clementine as female parent and tetraploids of 'Nova', 'W. Leaf' and 'Pineapple' male parents. Key message Effective methodology to recover citrus triploid plants from 2x?×?4x sexual hybridizations and the parental effect on the length of the juvenile phase.