Measurement of serum IgE antibodies against Japanese cedar pollen (Cryptomeria japonica) in Japanese monkeys (Macaca fuscata) with pollinosis.

IgE antibodies against allergens of Japanese cedar (Cryptomeria japonica, CJ) pollen in the serum of seven Japanese monkeys (Macaca fuscata) with pollinosis were measured by fluorometric indirect enzyme-linked immunosorbent assay (ELISA). All of the monkeys were found to have specific IgE to the crude pollen antigen. The specific IgE levels were well correlated with those determined by the Pharmacia CAP system. IgE antibodies were then assayed with two kinds of purified allergens (Cry j I and Cry j II) by the ELISA. We found that five monkeys had specific IgE to both allergens, although the other two had IgE only to Cry j I or Cry j II; there is different immune responsiveness to the two major allergens in the monkeys.     

Seasonal changes of humoral and cellular immune responses to Japanese cedar (Cryptomeria japonica) pollen allergens in Japanese monkeys (Macaca fuscata) with pollinosis

The natural occurrence of Japanese cedar [Cryptomeria japonica (CJ)] pollinosis has been reported in Japanese monkeys (Macaca fuscata). The present study was designed to investigate seasonal changes in immunological reactions to CJ pollen allergens in monkeys with CJ pollinosis. Blood samples were collected from six monkeys with CJ pollinosis before and after CJ pollen season. Seasonal changes in specific IgE and IgG to major allergens (Cry j 1 and Cry j 2) were observed before and after CJ pollen season. The humoral responses decreased significantly before CJ pollen and increased after CJ pollen season. Similar seasonal changes in peripheral blood mononuclear cells proliferative responses to CJ allergens were observed before and after CJ pollen season. These humoral and cellular immune responses might serve as a biomarker for assessing new immunotherapies for monkeys with pollinosis.     

Oral immunotherapy against a pollen allergy using a seed-based peptide vaccine

Peptide immunotherapy using dominant T-cell epitopes is safer and more effective than conventional immunotherapy for the treatment of immunoglobulin E (IgE)-mediated allergic diseases. When allergenic T-cell epitope peptides are expressed in the edible part of transgenic plants, successful mucosal immune tolerance to these allergens may be attainable by the consumption of these plants. In this study, we generated transgenic rice seed that accumulated high concentrations (about 60 microg per grain) of polypeptide consisting of seven dominant human T-cell epitopes derived from the Japanese cedar pollen allergens, Cry j 1 and Cry j 2, in the endosperm. Oral administration of these transgenic rice seeds to B10.S mice before or after they were immunized with Cry j 1 holoprotein reduced not only their T-cell proliferative response to Cry j 1, but also their serum IgE levels, proving the efficacy of oral immunotherapy for the treatment of pollinosis.     

Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens,Cry j 1 and Cry j 2, against Japanese cedar pollinosis

Transgenic rice accumulating the modified major Japanese cedar pollen allergens, Cryptomeria japonica 1 (Cry j 1) and Cryptomeria japonica 2 (Cry j 2), which were deconstructed by fragmentation and shuffling, respectively, in the edible part of the seed was generated by transformation of a good‐tasting rice variety, ‘Koshihikari’. These modified cedar pollen antigens were deposited in ER‐derived protein bodies (PB‐I), which are suitable for delivery to the mucosal immune system in gut‐associated lymphoid tissue when orally administered because antigens bioencapsulated in PB‐I are resistant against hydrolysis by intestinal enzymes and harsh environments. Mice fed transgenic seeds daily for three weeks and then challenged with crude cedar pollen allergen showed marked suppression of allergen‐specific CD4⁺ T‐cell proliferation, IgE and IgG levels compared with mice fed nontransgenic rice seeds. As clinical symptoms of pollinosis, sneezing frequency and infiltration of inflammatory cells such as eosinophils and neutrophils were also significantly reduced in the nasal tissue. These results imply that oral administration of transgenic rice seeds containing the structurally disrupted Cry j 1 and Cry j 2 antigens, serving as universal antigens, is a promising approach for specific immunoprophylaxis against Japanese cedar pollinosis.     

Variation in Ambrosia pollen concentration in Southern and Central Poland in 1982–1999

The aim of the study was to investigate theAmbrosia pollen concentrations inselected Polish cities and for Kraków torelate it to some meteorological factors.Sampling was carried out in Kraków in1982–1997 and in Rabka in 1992–1996 with theuse of the gravimetric method. In Zakopane,Kraków, Ostrowiec witokrzyski,Warszawa and Pozna in 1995–1996 both thegravimetric and volumetric methods (Burkardtrap) were employed. In Kraków themonitoring has been performed since 1994 usingthe volumetric method. The results show theragweed pollen presence in August and Septemberwith the tendency to appear more frequently inAugust in some years. In Kraków (1994–1999)Ambrosia pollen was found either in thelast two weeks of August or in the first twoweeks of September which seems to be a regularand repeatable pattern every two years.Seasonal fluctuations of Ambrosia pollenconcentration do not show a clear increasingtendency except at Warszawa and Ostrowiecwitokrzyski in 1996 and at Krakówin 1999. Percentage of Ambrosia pollen inannual sums of total pollen is very low anddoes not exceed 1% except at Ostrowiecwitokrzyski in 1996 (1.2%) and atKraków in 1999 (2%). For Kraków theanalysis of some meteorological factors (Tmax,Tmin, precipitation, wind direction) wasperformed. High temperature and lack of rain orlow precipitation correlate well with ragweedpollen concentrations. During the Ambrosia pollen seasons ESE, E, S, SE, WSW, SWwind directions prevailed which could suggest along-distance transport from Ukraine, the CzechRepublic, Slovakia and also from Hungary, one ofthe most ragweed-polluted countries.     

Pectate Lyase Pollen Allergens: Sensitization Profiles and Cross-Reactivity Pattern

BackgroundPollen released by allergenic members of the botanically unrelated families of Asteraceae and Cupressaceae represent potent elicitors of respiratory allergies in regions where these plants are present. As main allergen sources the Asteraceae species ragweed and mugwort, as well as the Cupressaceae species, cypress, mountain cedar, and Japanese cedar have been identified. The major allergens of all species belong to the pectate lyase enzyme family. Thus, we thought to investigate cross-reactivity pattern as well as sensitization capacities of pectate lyase pollen allergens in cohorts from distinct geographic regions.MethodsThe clinically relevant pectate lyase pollen allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1 were purified from aqueous pollen extracts, and patients´ sensitization pattern of cohorts from Austria, Canada, Italy, and Japan were determined by IgE ELISA and cross-inhibition experiments. Moreover, we performed microarray experiments and established a mouse model of sensitization.ResultsIn ELISA and ELISA inhibition experiments specific sensitization pattern were discovered for each geographic region, which reflected the natural allergen exposure of the patients. We found significant cross-reactivity within Asteraceae and Cupressaceae pectate lyase pollen allergens, which was however limited between the orders. Animal experiments showed that immunization with Asteraceae allergens mainly induced antibodies reactive within the order, the same was observed for the Cupressaceae allergens. Cross-reactivity between orders was minimal. Moreover, Amb a 1, Art v 6, and Cry j 1 showed in general higher immunogenicity.ConclusionWe could cluster pectate lyase allergens in four categories, Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1, respectively, at which each category has the potential to sensitize predisposed individuals. The sensitization pattern of different cohorts correlated with pollen exposure, which should be considered for future allergy diagnosis and therapy.     

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.     

Release behavior of small sized daughter allergens from Cryptomeria japonica pollen grains during urban rainfall event

In Japan, Cryptomeria japonica pollen (with diameter ~30 μm) is scattered during each spring season. Daughter allergenic particles, which are smaller in size than their parent pollen grain and are abundant in fine particles (the particle sizes < 1.1 μm, PM_1.1), are released in the atmosphere. The daughter allergenic particles of pollen can be transported in the urban atmosphere for a long period of time after their release. In particular, the daily variation delays in the peaks of allergenic Cry j 1 concentrations compared with the peaks of airborne parent pollen counts were observed in high levels during 1 or 2 sunny days after rainfall. In addition, long range transportation of Asian dusts (ADS) from the East Asian continent was also found during the pollen scattering seasons in Japan. Therefore, the interaction between pollen and air pollutants, including ADS, should be of concern. Thus, in this study, the morphological change of Cryptomeria japonica pollen and the elution behavior of its allergenic contents (Cry j 1) were investigated. Our results confirmed the existence of fine daughter allergen particles, which are clearly differ from the parent pollen grains in size. Fine allergenic particles in atmosphere were increased, while coarse allergenic particles were decreased on sunny days after rainfall. However, the correlation between the mass concentrations of fine particles and mass levels of Cry j 1 in coarse particles (the particle sizes > 7.0 μm) was poor. The possible reason may be pollen burst at high humidity before rainfall. Additionally, Cry j 1 contents were emitted from the so-called Ubisch body, which contains allergenic Cry j 1 abundantly when pollen was in contact with rainfall. In particular, we found that 60% of allergenic Cry j 1 contents released in air polluted rainfall contained Ca²⁺ ion derived from road dust and ADS. Therefore, rainfall should be a main factor to induce transition of pollen allergenic contents to fine particles. In conclusion, allergenic particles which are small sized and translated into fine particles by rainfall can be inhaled into the lower respiratory tract and contribute to the hypersensitivity of asthma.     

Bacillus thuringiensisδ-Endotoxin Proteins Show a Correlation in Toxicity and Short Circuit Current Inhibition Against Helicoverpa zea

Pesticidal activity of Bacillus thuringiensisδ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A, was determined by using the force-feeding bioassay method to 4^th instar larvae of Helicoverpa zea. H. zea was susceptible to Bt toxins in the order Cry1Ac > Cry1Ab > Cry1Aa > Cry2A with 63.60, 89.04, 159.65, and 375.78 ng/larvae respectively. The abilities of selected Bacillus thuringiensis toxins to inhibit short circuit current (I_SC) in midgut epithelia of H. zea were also investigated by voltage clamp assay. The voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short circuit current (I_SC) by activated toxin. A Cry1Aa toxin dilution of 33.3 and 500 ng/ml resulted in inhibition of I_SC of −2.29 μA/min (lag time 15 min) and −4.48 μA/min (lag time, 2 min) respectively. The Cry1Ab dilution of 25 ng/ml inhibited I_SC to −1.39 μA/min, a lag time of 14 min, and 333.3 ng/ml dilution resulted in decay of I_SC−2.49 μA/min, lag time 1 min respectively. The Cry1Ac lower dilution 16.7 ng/ml inhibited I_SC to −1.39 μA/min, lag time 4 min, and a high dilution 333.3 ng/ml decay I_SC to −2.44 μA/min, lag time 1 min. The inhibition of I_SC (−1.10 μA/min, lag time 25) at lower dilution (33.3 ng/ml) and high dilution (500 ng/ml), decay (−2.38 μA/min, lag time 5 min), showed a correlation between toxin concentration and inhibitory response with Cry2A toxin. The lag time decreased with increasing concentration of toxin applied, which is additional evidence of dose response besides direct correlation of toxicity assays and I_SC. Received: 7 April 2000 / Accepted: 2 May 2000     

The role of allergenic proteins Pla a 1 and Pla a 2 in the germination of Platanus acerifolia pollen grains

Platanus acerifolia (Aiton) Willdenow is a plane tree, widely grown as an ornamental tree in many cities of the United States and Western Europe, which has become an important source of airborne allergens in our cities. The aim of the present study is to immunolocalize the major allergens in the pollen grain and to examine their potential function in the fertilization process. Observations were made in mature and hydrated, activated pollen of P. acerifolia for 5, 15, 30 min and 2 h in the germination medium. Specimens were fixed using freezing protocols for transmission electron microscopy (TEM). For immunogold labelling, cryosections and resin-embedded ultrathin sections were incubated using rabbit antisera against the purified pollen allergens Pla a 1 and Pla a 2. Elution of P. acerifolia allergens took place after 5 min of pollen incubation in buffered medium. Intense labelling of Pla a 1 and Pla a 2 was detected after pollen exudates were released. In pollen grains, Pla a 1 was predominantly localized in concentric cisternae of the endoplasmic reticulum (ER), situated between the vegetative nucleus and the generative cell, and was released from pollen grains 5 min after hydration; cytoplasmic localization decreased 15 min after hydration. In pollen grains, glycoprotein Pla a 2 was abundant in association with Golgi cisternae and vesicles situated in the apertural periphery of the mature pollen grains. Pla a 2 proteins were also detected in ER and in the generative cell wall. Immunolabelling of Pla a 2 decreased 5 min after pollen hydration but was again intense after 15–30 min in germination medium, presumably as a consequence of renewed expression and glycosylation of this protein. Pla a 1 belongs to a new class of allergens related to proteinaceous invertase and pectin methyl esterase inhibitors (PII, PMEI) which could be involved in membrane protection and pectin de-esterification control during pollen hydration. Pla a 2 has an exopolygalacturonase (PG) enzymatic activity consistent with pollen-stigma adhesion mechanisms or compatibility systems. Moreover, the expression of Pla a 2 found 15–30 min after hydration might contribute to pollen-tube growth and the modification of transmitting tissue cell walls. The abundant production and elution of Pla a 1 and Pla a 2 proteins may alter the environment in which pollen tube elongation occurs, thus promoting a potential crosstalk between the pollen and the gynoecium.     

Development of transgenic rice seed accumulating a major Japanese cedar pollen allergen (Cry j 1) structurally disrupted for oral immunotherapy

Rice seed-based edible vaccines expressing T-cell epitope peptides derived from Japanese cedar major pollen allergens have been used to successfully suppress allergen-specific Th2-mediated immunoglobulin E (IgE) responses in mouse experiments. In order to further expand the application of seed-based allergen-specific immunotherapy for controlling Japanese cedar pollinosis, we generated transgenic rice plants that specifically express recombinant Cry j 1 allergens in seeds. Cry j 1 allergens give low specific IgE-binding activity but contain all of the T-cell epitopes. The allergens were expressed directly or as a protein fusion with the major rice storage protein glutelin. Fusion proteins expressed under the control of the strong rice endosperm-specific GluB-1 promoter accumulated in rice endosperm tissue up to 15% of total seed protein. The fusion proteins aggregated with cysteine-rich prolamin and were deposited in endoplasmic reticulum-derived protein body I. The production of transgenic rice expressing structurally disrupted Cry j 1 peptides with low IgE binding activity but spanning the entire Cry j1 region can be used as a universal, safe and effective tolerogen for rice seed-based oral immunotherapy for cedar pollen allergy in humans and other mammals.     

Immunocytochemical localization of Cry j 1, the major allergen of<Emphasis Type="Italic"> Cryptomeria japonica</Emphasis> (Taxodiaceae) in<Emphasis Type="Italic"> Cupressus arizonica</Emphasis> and<Emphasis Type="Italic"> Cupressus sempervirens</Emphasis> (Cupressaceae) pollen grains

Immunocytochemical localization of cross-reactive antigens to Cry j 1, the major allergen of Cryptomeria japonica, in the pollen grains of Cupressus arizonica and Cupressus sempervirens, was conducted using transmission electron microscopy. Mature and activated pollen grains were fixed for immunocytochemistry using conventional and freezing protocols. Sections containing the samples were incubated with anti-Cry j 1 monoclonal antibody (mAb) (KW-S91). Cross-reactive antigens to Cry j 1 were detected in the mature pollen grains of Cupressus arizonica and Cupressus sempervirens, and abundant gold particles were seen in the orbicules and wall, and in the Golgi, nucleus, and inclusions in reserve materials. After 5 min of activation, labelling noticeably decreased. From 15 min to 48 h, the cytoplasm exhibits a new labelling pattern, with gold markers being associated with protein storage vacuoles. The decrease in cross-reactive antigens during the first 5 min of activation could be due to a rapid release of proteins recognized by Cry j 1 mAb during pollen attachment in the pollination droplet. Our results show that the content of allergenic proteins is unstable, displaying variation relative to the progress of germination in Cupressus sempervirens and Cupressus arizonica pollen grains. The ability to do so may be viewed as an adaptive strategy of Cupressaceae pollen grains to maximize their biosynthetic efficiency.     

Intranasal sensitization of Japanese cedar pollen by the co-administration of low doses of cholera toxin but not its recombinant B subunit to mice

We evaluated the effects of cholera toxin (CT) and the B subunit of cholera toxin (CTB) on the intranasal sensitization of Japanese cedar pollen (JCP) in mice. JCP suspended in phosphate-buffered saline was administered into the nostrils of mice in combination with varying doses of CT or recombinant CTB(r-CTB) once a week for 5 weeks. Antibody responses specific to sugi basic protein (SBP) were monitored by ELISA for seven weeks. The sensitization of JCP alone did not induce IgG1, IgG2b, IgG2a, IgE or IgA. In contrast, sensitization of JCP in combination with CT (JCP/CT) elicited the prominent production of SBP-specific IgG1 and low levels of IgG2b and IgG2a on Day 49. IgE production was detected only in the serum of mice which were treated with JCP/CT, and not under any other protocol. Using spleen cells from these mice, cytokine production was examined by ELISA in culture supernatants after they had been stimulated in vitro with major cedar pollen allergens, Cry j 1, Cry j 2 or SBP. Notable responses were an increase of IFN-gamma as well as IL-4 in JCP/CT-sensitized cells stimulated with Cry j 2, but not in those stimulated with Cry j 1. No significant differences were detected in IL-5 production among the experimental groups. Histopathological examination, however, showed that eosinophil infiltration was evident in the nasal mucosa of the JCP/CT-sensitized mice following challenge with JCP/CT, but weak with BSA/CT or CT alone. Thus, the immunological and histological analyses indicated that the co-administration of a low dose of CT in combination with JCP allows the induction of pollen-allergic states in mice.     

Size distribution of allergenic Cry j 2 released from airborne <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> pollen grains during the pollen scattering seasons

Japanese cedar (Cryptomeria japonica) pollinosis is one of seasonal allergic rhinitis that mainly occurs in Japan. The pollinosis is caused by two main kinds of allergenic proteins called Cry j 1 and Cry j 2 which exist in Cryptomeria japonica pollen. In our previous study, we reported that the size-segregated of airborne fine allergenic Cry j 1 and morphological change of Cry j 1 due to the contact with rainfall. However, the study on airborne allergenic Cry j 2 in different particle sizes has not been identified until now. Therefore, the main aim of this study is to investigate the size distribution and scattering behavior of allergenic Cry j 2. The Cry j 2 particles were collected and determined in different particle sizes at the urban sampling points during the most severe pollination season of 2012 in Saitama, Japan. After the size-segregated Cry j 2 allergenic particles were collected using an Andersen high-volume (AHV) atmospheric sample, the airborne Cry j 2 concentrations were determined with a surface plasmon resonance (SPR) method. At the same time, the airborne Cryptomeria japonica pollens were also counted by the Durham pollen sampler. The higher concentrations of the allergenic Cry j 2 were detected even in particle sizes equal to or less than 1.1 μm (PM_1.1) than other particle sizes. The airborne particles ranges from 0.06 to 11 μm were also collected by a low-pressure impactor (LPI) atmospheric sampler. After that, the concentrations of Cry j 2 allergenic particles in fine particle sizes were measured by the SPR method either. With the help of this study, we have confirmed the existence of fine daughter allergenic particles, which clearly differ from the parent pollen grains in size, especially after the rainy days. It is possible that the daughter allergenic species will be released from the fractions of cell wall and burst pollen grains. We concluded that rainwater was one of the important factors that affects the release of pollen allergenic proteins of both Cry j 1 and Cry j 2 from the parent pollen grains.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Nisin-induced expression of recombinant T cell epitopes of major Japanese cedar pollen allergens in Lactococcus lactis

Applied Microbiology and Biotechnology - Japanese cedar pollinosis is a seasonal allergic disease caused by two major pollen allergens: Cry j 1 and Cry j 2 antigens. To develop an oral vaccine to...     

Deposition of a recombinant peptide in ER-derived protein bodies by retention with cysteine-rich prolamins in transgenic rice seed

A 7Crp peptide composed of seven major human T cell epitopes derived from the Japanese cedar pollen allergens Cry j 1 and Cry j 2 is an ideal tolerogen for peptide immunotherapy against Japanese cedar pollinosis. To maximize the accumulation level of the 7Crp peptide in transgenic rice seed, we tested endosperm specific promoters and intracellular localizations suitable for stable accumulation. A 7Crp peptide carrying the KDEL ER retention signal directed by the 2.3-kb promoter of the glutelin GluB-1, which contains a signal peptide, accumulated at the highest level of about 60 μg/grain. Notably, the 7Crp peptide predominantly accumulated in ER-derived protein bodies irrespective of the presence of various sorting signals or expression as a fusion protein with glutelin. We attribute this abnormal pattern of accumulation to the formation of disulfide bonds between the 7Crp peptide and cysteine-rich (Cys-rich) prolamin storage proteins. Furthermore, the formation of these aggregates induced the chaperone proteins BiP and PDI as an ER stress response.     

Isolation of Allium pollen protoplasts

Studies on protoplasts isolation were carried out with mature pollen grains of 29 samples of species of Allium aflatunense, A. cepa, A. fistulosum, A. karataviense, A. longicuspis, A. nutans, A. odorum, A. sativum and A. schoenoprasum. Surface sterilized pollen grains drifted from crushed anthers were incubated in an enzyme solution containing 1% (w/v) cellulase Onozuka R-10, 1% (w/v) Macerozyme R-10, 0,5 mol l^-1 sucrose and the basal salts of Nitsch medium. Protoplasts were released within 3 to 120 min, either from the pollen grain, through a slightly disturbed germination pore (narrow aperture), or through a wider aperture, when the exine surrounding the germination pore was disturbed. For the first time, protoplasts were obtained from 13 genotypes of 6 Allium species, at a rate of 1 to 30% of the digested intact pollen grains, depending on the genotype.     

Response of in vitro pollen germination and pollen tube growth of groundnut (Arachis hypogaea L.) genotypes to temperature

Air temperatures of greater than 35 °C are frequently encountered in groundnut‐growing regions, especially in the semi‐arid tropics. Such extreme temperatures are likely to increase in frequency under future predicted climates. High air temperatures result in failure of peg and pod set due to lower pollen viability. The response of pollen germination and pollen tube growth to temperature was quantified in order to identify differences in pollen tolerance to temperature among 21 groundnut genotypes. Plants were grown from sowing to harvest in a poly‐tunnel under an optimum temperature of 28/22 °C (day/night). Pollen was collected at anther dehiscence and was exposed to temperatures from 10° to 47·5 °C at 2·5 °C intervals. The results showed that a modified bilinear model most accurately described the response to temperature of percentage pollen germination and maximum pollen tube length. Genotypes were found to range from most tolerant to most susceptible based on both pollen characters and membrane thermostability. Mean cardinal temperatures (T_min, T_opt and T_max) averaged over 21 genotypes were 14·1, 30·1 and 43·0 °C for percentage pollen germination and 14·6, 34·4 and 43·4 °C for maximum pollen tube length. The genotypes 55‐437, ICG 1236, TMV 2 and ICGS 11 can be grouped as tolerant to high temperature and genotypes Kadiri 3, ICGV 92116 and ICGV 92118 as susceptible genotypes, based on the cardinal temperatures. The principal component analysis identified maximum percentage pollen germination and pollen tube length of the genotypes, and T_max for the two processes as the most important pollen parameters in describing a genotypic tolerance to high temperature. The T_min and T_opt for pollen germination and tube growth, rate of pollen tube growth were less predictive in discriminating genotypes for high temperature tolerance. Genotypic differences in heat tolerance‐based on pollen response were poorly related (R² = 0·334, P = 0·006) to relative injury as determined by membrane thermostability.