Role of peroxynitrite in programmed cell death induced in self-incompatible pollen

Reactive oxygen species and NO are involved in the signaling pathway of programmed cell death (PCD). Information concerning the role of these molecules in self-incompatible pollination is scarce especially in non-model species studied in vivo. We recently reported that in the olive tree, compatible and self-incompatible pollen have different levels of reactive oxygen and nitrogen species and that PCD is induced in self-incompatible pollen. Levels of O₂^.- and NO are higher in pollen after self-incompatible pollination than after compatible pollination. The presence of these reactive species was concomitant with the presence of peroxynitrite. Similar results were obtained on pollen-germination experiments both in vivo and in vitro. These data, together with observations made after treating pollinated flowers with scavengers, suggest that peroxynitrite plays a role in PCD induced after self-incompatible pollination and we propose here a model to describe the way in which it might work.     

Fluorescence and scanning electron microscopic study on self-incompatibility in distylous Averrhoa carambola L.

Various combinations of intermorph, selfing and intramorph pollinations were carried out in Averrhoa carambola and the pollinated pistils were observed under fluorescence and scanning electron microscopes in time-course experiments. In both compatible and incompatible pollinations, similar behavior of pollen germination and penetration was observed in the first 4 h after pollination. In compatible intermorph pollination, pollen tubes were found at the base of the transmitting tract of the style at 8 h and 24 h after pollination in both the pin and thrum morphs. With thrum flowers, selfing resulted in pollen tubes being uniformly arrested at the junction between the stigmatic and stylar tissues. Penetration of pollen tubes into the upper portion of style was observed in thrum intramorph pollination and, when the second member was treated as the gynoecial tissue under the same pollination, penetration of tubes was further enhanced. Pin flowers, on selfing, resulted in pollen tube penetration farther down the style than was the case with thrum selfing. Intramorph pollination of pin morph behaved in a similar manner to selfing and was not affected by genotypes. Beside the stamen-style dimorphism, the receptive surface of the cob stigma was larger in pin than thrum flowers. While pin pollen was round, thrum pollen was oblong in shape with pin to thrum ratio on the polar axis being 1.2 and on the equatorial axis 0.8. The stigma of pin morph belonged to the dry type, while that of the thrum resembled the wet type.     

Disorganization of F-actin cytoskeleton precedes vacuolar disruption in pollen tubes during the in vivo self-incompatibility response in Nicotiana alata

Background and Aims The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. Methods The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. Key Results Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. Conclusions The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes.     

Nectarless flowers with deep corolla tubes in Pedicularis: does long pistil length provide an arena for male competition?

Pollinator‐mediated selection does not seem to have a direct influence on the evolution of a long corolla tube in a nectarless flower. We hypothesized that the long pistil length of the nectarless flower with a deep corolla tube provided an opportunity for male competition. Pedicularis siphonantha, a nectarless and partially self‐incompatible lousewort with substantial variation in corolla tube length, was used to test the hypothesis. We tested whether and how corolla tube length affected seed production per capsule and seed germination rate with different pollination treatments. Flowers were hand‐pollinated with pollen from one self donor and one outcross donor and mixed pollen grains consisting of equal amounts from the two donor types, respectively. Additionally, seeds from open‐pollinated flowers with different corolla tube lengths were collected separately for measurement of germination rate. Pollination treatment and corolla tube length significantly affected number of seeds per capsule. Moreover, a significant positive relationship between seeds per capsule and corolla tube length was found when mixed hand pollination was conducted. Seeds of self hand‐pollinated flowers had a lower germination rate than those from outcross‐pollinated flowers. Under open pollination, seeds from flowers with longer corolla tubes tended to have higher germination rate. In P. siphonantha, outcross pollen may have a higher probability of contributing to the next generation when transferred to flowers with longer corolla tubes. The pistil length, therefore, should be seen as a female choice mechanism, which provides an arena for male‐to‐male competition. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 526–532.     

Pollen tubes enter neighbouring ovules by way of receptacle tissue,resulting in increased fruit-set in Sagittaria potamogetifolia Merr

Using fluorescence microscopy, deposition of pollen on stigmas and pollen tube growth in the gynoecium of Sagittaria potamogetifolia Merr., a monoecious species with an apocarpous gynoecium, were observed. The maximum rate of pollination averaged 83.9 +/- 4.7 %, and the number of pollen grains per stigma ranged from zero to 30. Pollen tubes grew through one stigma to the base of the ovary at almost the same speed, but generally only one of the pollen tubes then turned towards the ovule and finally entered the nucellus through the micropyle. The other pollen tubes grew through the ovary base and the receptacle tissue into ovules of adjacent carpels whose stigmas were not pollinated or which had been pollinated later. This phenomenon is termed pollen tube 'reallocation' by the authors. To verify the direct effect of the phenomenon on fruit set, artificial pollination experiments were conducted in which two or more pollen grains were placed onto only one stigma in each gynoecium; frequently more than one fruitlet was obtained from each flower treated. The reallocation of pollen tubes among pistils in the gynoecium could effect fertilization of ovules of unpollinated pistils and lead to an increase in sexual reproduction efficiency. It would, to some extent, also increase pollen tube competition among pistils of the whole gynoecium.     

Influence of plant reproductive systems on the evolution of hummingbird pollination

Many hummingbird‐pollinated plant species evolved from bee‐pollinated ancestors independently in many different habitats in North and South America. The mechanisms leading to these transitions are not completely understood. We conducted pollination and germination experiments and analyzed additional reproductive traits in three sister species pairs of which one species is bee‐ and the other hummingbird‐pollinated. All hummingbird‐pollinated species showed higher seed set and germination rates in cross‐pollinated than in self‐pollinated flowers. In the self‐compatible, bee‐pollinated sister species this difference did not exist. As expected, seed set and germination rate were higher after cross‐pollination in the largely self‐incompatible genus Penstemon independently of the pollination syndrome. However, the bird‐pollinated species produce only half of the amount of ovules and pollen grains per flower compared to the bee‐pollinated sister species. This indicates that hummingbird pollination is much more efficient in self‐incompatible populations because hummingbirds waste less pollen and provide higher outcrossing rates. Therefore, hummingbird pollination is less resource costly. Overall, we suggest that hummingbirds may increase the reproductive success compared to bees, influencing the evolution of hummingbird pollination in ecosystems with diverse bee assemblages.     

Analysis of stylar self-incompatibility competence by use of heat induced inactivation

Summary Immersion of Lilium longiflorum pistils in 49 °C water for increasing durations of 1,2,3, or 4 minutes immediately prior to incompatible pollination resulted in a correspondingly progressive decrease in the stylar self-incompatibility competence, as determined from the lengths attained by pollen tubes during 48 hours growth in the styles at 24 °C. Neither pistils remaining on the plant nor those detached from the plant which were immersed after anthesis in 49 °C water for 5 minutes regained self-incompatibility competence during a 48 hour incubation at 24 °C prior to incompatible pollination. Heat treatment of detached pistils as early as 39 hours prior to bud anthesis also resulted in an inactivation of stylar self-incompatibility competence when incompatible pollination was made at 24 hours after anthesis. Experiments utilizing heat treatment of partial lengths of detached whole styles revealed that pollen tubes which have grown through as much at 45 millimeters of either a physiologically incompatible or compatible portion of the style are still capable of shifting to either a higher growth rate or lower growth rate upon entry into respectively either a physiologically compatible or incompatible portion of the style.     

Self‐pollination and parthenocarpic ability in developing ovaries of self‐incompatible Clementine mandarins (Citrus clementina)

This study aimed to determine if self‐pollination is needed to trigger facultative parthenocarpy in self‐incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). ‘Marisol’ and ‘Clemenules’ mandarins were selected, and self‐pollinated and un‐pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self‐pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time‐course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA₁ and GA₄), auxin (IAA) and abscisic acid (ABA) content in the ovary. ‘Clemenules’ showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9 days after pollination in both cultivars. Self‐pollination did not stimulate parthenocarpy, whereas both un‐pollinated and self‐pollinated ovaries set fruit regardless of the cultivar. On the other hand, ‘Marisol’ un‐pollinated flowers showed greater parthenocarpic ovary growth than ‘Clemenules’ un‐pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of ‘Marisol’ paralleled higher levels of GA₁ in the ovary (+34% at anthesis). ‘Marisol’ ovary also showed higher hexoses and starch mobilization, but lower ABA levels (?64% at anthesis). Self‐pollination did not modify carbohydrates or GA content in the ovary compared to un‐pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination‐independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self‐incompatible mandarins is constitutively regulated.     

Fruit characteristics, seed production and pollen tube growth in the wild chilli pepper Capsicum flexuosum

Fruits formed after different pollination regimes (flowers hand pollinated, unpollinated, and open pollinated) and the seeds obtained were characterized in the wild chilli pepper Capsicum flexuosum Sendtn. Pollen tube development in vivo and ovary growth were also analyzed. Seedless fruits and empty seeds were abundant among the fruits from hand pollinated and open pollinated flowers, while no more than one seed with embryo was found in a low percentage of fruits from such pollination treatments. Parthenocarpic fruits were formed from unpollinated flowers. Pollen tube growth was arrested in the upper third of the style for almost all pollen tubes except for a single one that may continue elongating occasionally. The ovary size increased continuously after pollination, even without fertilization. The sum of the evidence registered may help to explain the low number of seeds with embryo harvested, the abundance of seedless fruits formed from pollinated flowers (possibly parthenocarpic), and the high rate of parthenocarpic fruits formed from unpollinated flowers.     

Pollination biology of Indotristicha ramosissima (Podostemaceae: Tristichoideae)

Floral morphology, phenology and mode of pollination have been studied in Indotristicha ramosissima (Wt.) van Royen. Although the plants are submerged, self-pollination (autogamy) occurs above water. This is aided by considerable elongation of the pedicel (20 mm) prior to pollination. The filaments of the stamens also elongate rapidly (∼6.5 mm/h) before and after pollination. The flowers are typically trimerous. Each anther contains 1743 ± 187 pollen grains. These are spherical, multiporate, 3-celled and ∼97% fertile at the time of shedding. Germination of pollen on the stigma and the growth of pollen tubes have been traced in both naturally and manually (self- as well as cross-) pollinated pistils. The pollen:ovule ratio is ∼72:1 and the ovule: seed ratio is ∼2:1. Mature fruits are 8 or 9 ribbed and open by 2 or 3 longitudinal slits that release ∼32 seeds.     

Seed set after pollination with in-vitro-matured,isolated pollen of Triticum aestivum

Summary Immature pollen of two varieties of Triticum aestivum, at the stage right after the first pollen mitosis, was isolated from individual anthers and cultured in microcultures of microliter droplets. In a specifically designed medium, some of the pollen grains developed to maturity. These were applied to excised stigmas on agar, where they produced pollen tubes. Application to flowers in vivo led to seed set. Pollen was matured in vitro from a variety that produced a different protein banding pattern on SDS-PAGE as compared to the variety that was pollinated. The protein banding in the produced seeds showed the hybrid pattern, demonstrating that the seeds were not produced by self-pollination in this in-breeding species but by pollination with the in-vitro-matured pollen.     

EFFECTS OF POLLINATION TIMING ON FRUITING IN ASCLEPIAS SPECIOSA TORR. (ASCLEPIADACEAE)

A series of hand-pollination experiments was performed to test the hypothesis that pollination timing determines which flowers produce pods in Asclepias speciosa, a species with few ovaries maturing into fruit. A comparison of fruiting of different aged ovaries showed that throughout anthesis, pistils are equally receptive to pollen tube penetration and growth. Pollen declines in vigor as flowers age as indicated by comparisons of different aged pollen both in vitro and in vivo. However, the overall effect of this decline on fruiting in the field is lessened by a simultaneous decline during anthesis in the rate of pollinium removal by insect pollinators. Within an umbel, earlier pollinated flowers are more likely to form mature pods than flowers pollinated 2 days later, apparently as a result of within umbel competition between developing pods for limited resources. The longer the delay between pollinations within umbels, the more mature pods form, probably as a result of a decline in intensity of competition between pods. Pollinations during the afternoon result in fewer mature pods and seeds than morning and evening pollinations, although fruiting is not dependent on humidity or temperature at the time of pollination. Differences in mature pod production between treatments were not significant for any experiment indicating that ecological or evolutionary factors other than pollination timing have the greatest effect on fruiting.     

Pollination-induced ethylene promotes the early phase of pollen tube growth in Petunia inflata

In Petunia inflata, a species with gametophytic self-incompatibility, pollination triggers two phases of ethylene production by the pistil, the first of which peaks 3 hours after pollination with compatible or incompatible pollen. To investigate the physiological significance of the first phase of ethylene production, pollinated flowers were treated with 2,5-norbornadiene (NBD), an inhibitor of ethylene action. Treatment with NBD reduced pollen tube growth in a dose-dependent manner during the first six hours after pollination; however, pollen tube growth was insensitive to NBD if the treatment was applied 6 hours or more after pollination. Simultaneous application of exogenous ethylene substantially offset the inhibitory effects of NBD in flowers pollinated for 4 hours. Another inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), also produced a strong inhibition of pollen tube growth during the first six hours of pollination. The experiments with 1-MCP pretreatment indicate that pistil tissues are the primary target of the pollination-induced ethylene.     

Changes in proteins and peroxidases induced by compatible pollination in the ovary of Nicotiana tabacum L. ahead of the advancing pollen tubes

Proteins and peroxidases produced by the ovules and placenta of tobacco (Nicotiana tabacum L.) in response to compatible pollination were analyzed by two-dimensional polyacrylamide gel electrophoresis and by enzyme staining in flat-bed native isoelectric focusing gels. For two-dimensional gels, ovaries were sampled at 36 h after pollination, at which time pollen tubes have penetrated much of the length of the style but have not yet entered the ovary. At least 11 major proteins from pollinated ovaries had no detectable counterparts in unpollinated ovaries. These showed a range of molecular mass and pI. For peroxidase isozyme assays, ovaries were sampled at 0, 12, 24, 36 and 48 h after pollination. At 45–50 h, pollen tubes were beginning to enter the top of the ovary but could still be separated from the ovules and placenta during sampling. Ovules and placentae from unpollinated pistils showed only one form of peroxidase, whereas those from pollinated pistils showed additional isozymes at pH 5.4 and pH 10.0. Both new isozymes increased in staining intensity over the first 36 h after pollination. At 48 h, however, the acidic peroxidase had continued to increase, while the basic component had declined so as to be barely detectable. The observations are discussed in relation to accumulating evidence that some form of pollination-induced signal reaches the ovary ahead of the advancing pollen tubes. The nature of this signal and possible involvement of peroxidases are also briefly discussed.     

Sunny-side up: flower heliotropism as a source of parental environmental effects on pollen quality and performance in the snow buttercup, Ranunculus adoneus (Ranunculaceae)

Floral traits affect mating success via their influence on the microenvironment in which sexual reproduction occurs as well as their impact on pollinator attraction. Here we investigate the importance of flower heliotropism as a source of parental environmental effects on pollen quality and performance. Flowers of the snow buttercup, Ranunculus adoneus, closely track the sun's rays. We experimentally restrained flowers to test for effects of heliotropism on pollen quality and performance after pollination. When equivalent amounts of pollen were transferred to recipient pistils, pollen from solar-tracking donor flowers exhibited a 32% advantage in germination compared to pollen from stationary (tethered) donor flowers. By the end of anthesis, pistils of tracking flowers contained 40% more germinating pollen grains and 44% more pollen tubes midway down the style than pistils of stationary ones. Solar tracking had no direct effect on pollen tube growth. The greater amount of germinating pollen in tracking flowers accounted for the treatment effect on pollen tube density. A survey of pollen receipt and pollen germination in naturally tracking flowers indicated that solar tracking primarily affects pollen tube density by promoting pollen germination rather than pollen deposition. We conclude that flower heliotropism, by enhancing the paternal environment for pollen development and the maternal environment for pollen germination, represents a source of positive parental environmental effects on pollen performance in snow buttercups.     

Floral Biology, Pollination, Pistil Receptivity, and Pollen Tube Growth of Teak (Tectona grandis Linn f.)

Teak flowers are weakly protandrous and pollen is shed withina few hours of flower opening. Pollen is tricolpate and 29 µmin diameter. The papillate stigma is of the wet type and isreceptive from 1100–1300h. The style is hollow throughoutits length. Nectar and pollen are the major floral rewards forpollinators. The major pollinators areCeratina sp. which carryteak pollen on most parts of their bodies, especially the specializedhair structures (scopal brushes) on the tibia. The most effectivepollination period in terms of flowers pollinated and pollenper flower is between 0900 and 1300h. At 1300h the number ofpollen per flower is the highest, ranging from 1–36 (average7). Pollen tubes grow very fast. Within 2 h after pollination8% of the pollen tubes have reached the micropylar end of theovule and pollen tubes first enter the embryo sac at 8 h. Onlyone to two pollen tubes enter the micropyles of a flower. Although78% of flowers were pollinated in open-pollination, the lowfruit set (3.5%) suggests that there are factors other thanpollination limiting fruit set. The main factor appears to bea high amount of selfing, and self-incompatibility occurs whenpollen tubes are arrested at the lower portion of the ovary. Tectona grandis ; floral biology; pollen tube growth; pollination; receptivity; pollinators     

Breeding systems in Cyrtanthus (Amaryllidaceae): variation in self‐sterility and potential for ovule discounting

• Breeding systems of plants determine their reliance on pollinators and ability to produce seeds following self‐pollination. Self‐sterility, where ovules that are penetrated by self‐pollen tubes that do not develop into seeds, is usually considered to represent either a system of late‐acting self‐incompatibility or strong early inbreeding depression. Importantly, it can lead to impaired female function through ovule or seed discounting when stigmas receive mixtures of self and cross pollen, unless cross pollen is able to reach the ovary ahead of self pollen (‘prepotency’). Self‐sterility associated with ovule penetration by self‐pollen tubes appears to be widespread among the Amaryllidaceae.
• We tested for self‐sterility in three Cyrtanthus species – C. contractus, C. ventricosus and C. mackenii – by means of controlled hand‐pollination experiments. To determine the growth rates and frequency of ovule penetration by self‐ versus cross‐pollen tubes, we used fluorescence microscopy to examine flowers of C. contractus harvested 24, 48 and 72 h after pollination, in conjunction with a novel method of processing these images digitally. To test the potential for ovule discounting (loss of cross‐fertilisation opportunities when ovules are disabled by self‐pollination), we pollinated flowers of C. contractus and C. mackenii with mixtures of self‐ and cross pollen.
• We recorded full self‐sterility for C. contractus and C. ventricosus, and partial self‐sterility for C. mackenii. In C. contractus, we found no differences in the growth rates of self‐ and cross‐pollen tubes, nor in the proportions of ovules penetrated by self‐ and cross‐pollen tubes. In this species, seed set was depressed (relative to cross‐pollinated controls) when flowers received a mixture of self and cross pollen, but this was not the case for C. mackenii.
• These results reveal variation in breeding systems among Cyrtanthus species and highlight the potential for gender conflict in self‐sterile species in which ovules are penetrated and disabled by pollen tubes from self pollen.

     

Flower reshaping in the transition to hummingbird pollination in Loasaceae subfam. Loasoideae despite absence of corolla tubes or spurs

Many angiosperm lineages present transitions from bee to hummingbird pollination. The flower design in most of these lineages includes either corolla tubes or nectar spurs, structures that commonly experienced an elongation with the acquisition of hummingbird pollination. It is proposed that this increases the fit between the bird head and flower structures, and isolates or partially blocks bees from the interaction. But can this transition occur if the ancestral flower design lacks tubes or spurs? Here we focus on the transition from bee to hummingbird pollination in the Loasaceae subfamily Loasoideae. Loasoideae flowers have radial corollas with separated petals; therefore, they do not display corolla tubes nor nectar spurs. These flowers also present a whorl of nectar scales and staminodes, unique to the subfamily, which is involved in flower–pollinator fit and in nectar harvesting. To explore flower shape adaptation to hummingbird pollination, we tested for correspondence between pollinators and flower shape in Loasoideae. In order to achieve this, we first compared the evolutionary history of flower phenotype and pollination mode, and then used stochastic character mapping and geometric-morphometric variables in a comparison of alternative evolutionary models. The results of our study suggest that the transition from bee to bird pollination was accompanied by changes in the shape of the staminodial complex, along with the evolution of relatively closed corollas. Moreover, while bird pollination seems to be the end point in the evolution of pollination syndromes in many angiosperm lineages, rodent pollinated flowers probably evolved from ancestral bird pollinated flowers in Loasoideae. Our findings suggest that the evolution of bird pollinated flowers from ancestral bee pollinated flowers does not require the presence of corolla tubes or spurs, and can take place as long as the flower design includes structures participating in flower–pollinator fit.     

Pollen tube reallocation in two preanthesis cleistogamous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae)

Pollen deposition on stigmas and pollen tube growth in two apocarpous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae), were examined with fluorescence microscopy. The reallocation of pollen tubes among pistils was observed in both species. The percentage of pollinated stigmas per flower was only 22.0% in R. rostratum and 51.0% in S. guyanensis, though the seed/ovule ratios are higher than 65% in both species. The number of pollen grains on each single stigma ranged from 0 to 96 in R. rostratum, and from 0 to 125 in S. guyanensis. When more than one pollen grain deposited on a stigma, all pollen tubes grew to the ovary, but only one of them turned towards the ovule and finally entered the nucleus. The other tubes grew through the receptacle tissue into ovules of adjacent carpels whose stigmas were unpollinated or pollinated later. The intercarpellary growth of pollen tubes could be a mechanism to increase the efficiency of sexual reproduction in an apocarpous gynoecium with low pollination on the pistils.     

In vitro culture of unfertilized ovules in carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.)

To induce development of isolated carrot ovules, flowers were pollinated with pollen from different species. Effects of pollen origin, medium composition, and light conditions were evaluated for 13 carrot cultivars. The highest efficiency of development was recorded following pollination of carrot flowers with parsley pollen. The highest frequency of embryo formation (0.84%) was obtained for cv. ‘Flamanka’; whereas the highest frequency of callus formation (1.56%) was obtained for cv. ‘Karlena’. Medium supplemented with indole-3-acetic acid (IAA) promoted embryo development (0.63%), while supplementation with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) promoted callus development (1.34%). Placement of isolated ovules in the dark stimulated embryo development. Embryos and calli thus obtained were regenerated into plants. Flow cytometry revealed that 97.7% regenerants were diploid. Based on a phosphoglucoisomerase (PGI) isozyme assay, 45.9% diploid regenerants were homozygous. Pollen tube growth of parsley, parsnip, celery, and cabbage in carrot pistils were studied using the aniline blue fluorescent method. The pollen tube growth was inhibited in transmitting tissues of the pistils.