Pollen Grain Germination Research Articles

Hormonal Signaling in the Progamic Phase of Fertilization in Plants

Pollen–pistil interaction is a basic process in the reproductive biology of flowering plants and has been the subject of intense fundamental research that has a pronounced practical value. The phytohormones ethylene (ET) and cytokinin (CK) together with other hormones such as auxin, gibberellin (GA), jasmonic acid (JA), abscisic acid (ABA), and brassinosteroids (BRs) influence different stages of plant development and growth. Here, we mainly focus on the information about the ET and CK signaling in the progamic phase of fertilization. This signaling occurs during male gametophyte development, including tapetum (TAP) cell death, and pollen tube growth, including synergid programmed cell death (PCD) and self-incompatibility (SI)-induced PCD. ET joins the coordination of successive events in the developing anther, including the TAP development and cell death, anther dehiscence, microspore development, pollen grain maturation, and dehydration. Both ET and CK take part in the regulation of pollen–pistil interaction. ET signaling accompanies adhesion, hydration, and germination of pollen grains in the stigma and growth of pollen tubes in style tissues. Thus, ET production may be implicated in the pollination signaling between organs accumulated in the stigma and transmitted to the style and ovary to ensure successful pollination. Some data suggest that ET and CK signaling are involved in S-RNase-based SI.

PALD encoding a lipid droplet-associated protein is critical for the accumulation of lipid droplets and pollen longevity in Arabidopsis.

Pollen longevity is critical for plant pollination and hybrid seed production, but few studies have focused on pollen longevity. In this study, we identified an Arabidopsis thaliana gene, Protein associated with lipid droplets (PALD), which is strongly expressed in pollen and critical for the regulation of pollen longevity. PALD was expressed specifically in mature pollen grains and the pollen tube, and its expression was upregulated under dry conditions. PALD encoded a lipid droplet (LD)-associated protein and its N terminus was critical for the LD localization of PALD. The number of LDs and diameter were reduced in pollen grains of the loss-of-function PALD mutants. The viability and germination of the mature pollen grains of the pald mutants were comparable with those of the wild-type, but after the pollen grains were stored under dry conditions, pollen germination and male transmission of the mutant were compromised compared with those of the wild-type. Our study suggests that PALD was required for the maintenance of LD quality in mature pollen grains and regulation of pollen longevity.

Definition of a growth medium to evaluate pollen viability in Hemerocallis cultivars

Hemerocallis is a floriferous species, which presents numerous genotypes that are constantly crossed, generating new cultivars. The germination and viability of pollen are influenced both by factors that are intrinsic and extrinsic to the pollen. In view of the above, the purpose of this study was to establish a growth medium for the germination of pollen grains. Cultivar Morgana was used in the experiments performed to establish the growth medium, in which four pH ranges were tested alongside four agar concentrations. For the next test, five sucrose concentrations were tested. Afterwards, four boric acid concentrations were added to the medium, finally followed by four calcium nitrate concentrations. After the definition of the growth medium, the incubation time for pollen grains germination of the species was evaluated. The results were then applied to four cultivars in order to verify the respective germination percentages. It was noted that the highest percentage of in vitro germination occurred with the concentrations of 4 g L−1 agar, 74.6 g L−1 sucrose, 800 mg L−1 boric acid and 590 mg L−1 calcium nitrate, with a pH of 5.74 and ideal incubation time of 3 h. Regina being to cultivar with the highest germination percentage.

The rice phosphate transporter OsPHT1;7 plays a dual role in phosphorus redistribution and anther development.

Inorganic phosphate (Pi) is the predominant form of phosphorus (P) readily accessible to plants, and Pi Transporter 1 (PHT1) genes are the major contributors to root Pi uptake. However, the mechanisms underlying the transport and recycling of Pi within plants, which are vital for optimizing P use efficiency, remain elusive. Here, we characterized a functionally unknown rice (Oryza sativa) PHT1 member barely expressed in roots, OsPHT1;7. Yeast complementation and Xenopus laevis oocyte assay demonstrated that OsPHT1;7 could mediate Pi transport. Reverse-transcription quantitative polymerase chain reaction and histochemical analyses showed that OsPHT1;7 was preferentially expressed in source leaves and nodes. A further fine-localization analysis by immunostaining showed that OsPHT1;7 expression was restricted in the vascular bundle (VB) sheath and phloem of source leaves as well as in the phloem of regular/diffuse- and enlarged-VBs of nodes. In accordance with this expression pattern, mutation of OsPHT1;7 led to increased and decreased P distribution in source (old leaves) and sink organs (new leaves/panicles), respectively, indicating that OsPHT1;7 is involved in P redistribution. Furthermore, OsPHT1;7 showed an overwhelmingly higher transcript abundance in anthers than other PHT1 members, and ospht1;7 mutants were impaired in P accumulation in anthers but not in pistils or husks. Moreover, the germination of pollen grains was significantly inhibited upon OsPHT1;7 mutation, leading to a >80% decrease in seed-setting rate and grain yield. Taken together, our results provide evidence that OsPHT1;7 is a crucial Pi transporter for Pi transport and recycling within rice plants, stimulating both vegetative and reproductive growth.

Studies on size, sterility and germination of pollen grains of certain members of the family Malvaceae

The present study attempted to evaluate the pollen size, percentage of sterility using acetocarmine, percentage of germination in sucrose medium among the members of the family Malvaceae like Malvaviscus arboreus Cav., Sida rhombifolia L., Urena lobata L. Hibiscus furcatus Roxb. Hibiscus rosa-sinensis L. and Gossypium arboreum L. Regarding the size pollen grains of Hibiscus rosa-sinensis showed largest pollen. The smallest pollen grains belong to Urena lobata, L. The pollen sterility using acetocarmine indicated that the pollens of Hibiscus furcatus were completely fertile, while Sida rhombifolia showed highest amount of sterility. The germination of pollen grain in sucrose medium indicated that the pollen grain of all the members studied showed poor germination.

Viability of pollen grains and stigma receptivity in Desert Rose

Abstract Adenium obesum (Forssk.) Roem. & Schult., popularly known as desert rose, has become a valuable ornamental plant. In floriculture, the production of hybrids is prioritized. Hence, knowledge on floral biology and sexual reproduction of the target species is fundamental. The objectives of this study were: (1) to test sucrose concentrations and temperatures for in vitro germination of A. obesum pollen grains; (2) to identify the effect of temperature on the viability of A. obesum pollen grains; and (3) to evaluate the viability of pollen grains and stigma receptivity in pre-anthesis, at flower opening, and 72 h post-flower opening in three accessions of A. obesum. A significant relationship between temperatures and sucrose concentrations was observed in the in vitro germination test. The highest percentage of in vitro germination of pollen grains, 39.81%, was observed at an estimated temperature of 26.05 °C. Desert rose accessions maintained in biochemical oxygen demand (BOD) chambers at 30 °C during a 16-h light photoperiod showed faster flowering, and temperatures ≥ 25 °C induced pollen grain viability percentages above 69%. Temperature is one of the most important abiotic factors, influencing mainly in pollen germination, pollen tube growing and in efficiency fertilization. The ICA-wd accession stood out and can be considered a pollen donor in artificial pollination. The stigmas of flowers were receptive from a day before flower opening until three days after. The two parameters presented above, stigma receptivity and pollen viability, allow inferences about the appropriate time for successful pollination and subsequent fertilization in desert roses.

Viabilidade in vitro de grãos de pólen de jenipapeiro em diferentes meios de cultura

Abstract The objective of this work was to evaluate the viability of genipap (Genipa americana) pollen grain at room temperature in different culture media. The experimental design was completely randomized in a 3x11 factorial arrangement (culture media x incubation times) with four replicates. The number of germinated pollen grains was analyzed at 24-hour intervals up to 288 hours after incubation at room temperature. The culture medium with 100 mg L-1 H3BO3, 80 g L-1 sucrose, and 1.0 g L-1 agar results in a higher to intermediate germination percentage, being the most suitable for studies on the in vitro viability of genipap pollen grains.

Target pollen isolation using automated infrared laser-mediated cell disruption

Single-cell analysis is important to understand how individual cells work and respond at the cell population level. Experimental single-cell isolation techniques, including dilution, fluorescence-activated cell sorting, microfluidics, and micromanipulation, have been developed in recent decades. However, such applications typically require large cell populations and skilled professionals. Additionally, these methods are unsuitable for sequential analysis before and after cell isolation. In this study, we propose a method for target cell isolation using automated infrared laser-mediated disruption of pollen grains in pollen populations. Germination of the target pollen was observed at the same location as that before laser irradiation, and germinated pollen grains were enriched in the cell population. Pollination of laser-irradiated bulk pollen populations also showed that the target pollen preferentially germinated on the stigma. This method is expected to facilitate physiological analyses of target cells at the single-cell level and effectively produce seeds derived from target pollen.

In vitro germination, storage and microscopic studies of pollen grains of four Ocimum species

In the present investigation, pollen morphology and viability of four Ocimum L. species viz. O. sanctum (CIM-Angana), O. basilicum (CIM-Saumya), O. gratissimum, and O. kilimandscharicum were studied. Morphological characters were observed using scanning electron and light microscopy. Pollen of all the four studied Ocimum species have hexacolpate apertures with varied aperture length and width. The exine ornamentation was reticulate and bi-reticulate. O. basilicum has large size (53.9 µm) pollen grains and rest three have medium sized pollen. The viability of pollen was assessed through staining dyes such as 2,5-diphenyl tetrazolium bromide (MTT) and 2,3,5-triphenyl tetrazolium chloride (TTC) and in vitro pollen germination method. O. basilicum possessed maximum viability (92.4%) whereas least viability was found in O. sanctum (38.2%). The maximum pollen germination percentage and tube growth were observed in the semisolid medium gelled with phytagel supplemented with 15% sucrose, 15% PEG, 6.4 mM boric acid and 7.3 mM calcium nitrate. O. basilicum showed the highest germination percentage (90.3%) followed by O. kilimandscharicum (57.1%), O. gratissimum (42.5%) and O. sanctum (25.5%). Pollen of the four Ocimum species were stored at different temperatures viz. 37 °C, + 4 °C, 0 °C, − 20 °C, and − 80 °C for 75 days. The highest pollen germinability of all the four Ocimum species was observed at − 20 °C. This study can be helpful in taxonomic identification of Ocimum species and further utilized in conservation and improvement of different Ocimum species by cross-hybridization breeding programmes.

An appraisal of pollen germination and viability of varied male pollen sources of date palm (Phoenix dactylifera L.)

Date palm is a dioecious fruit tree that permits cross pollination for fruit setting. The germination of viable pollens to fertilize ovule is influenced by environmental cues such as temperature. The germination and viability of pollen grains collected for pollination purpose also varied with the male pollinizer source and the male spathe opening time. An in vitro study performed to determine the percentage of pollen germination at different temperatures (10, 15, 20, 25, and 30 °C) and germination times (4, 8, 12, and 24 h) taken from different male sources and toidentify any variations in the germination and viability of pollen grains collected from spathes opened at different dates (20th February, 1st and 10th March). Pollens from different male sources showed a significant difference regarding germination percentage when incubated at different temperatures and for different germination times. Pollens incubated at 30 (81.07%) and 25°C (78.17%) had the highest germination percentage which decreased to 51.04, 16.23, and 5.90% when incubation temperature dropped to 20, 15, and 10 °C, respectively. Similarly, 71.11% pollens were germinated after 4 h of incubation at 30°C, which was decreased to 63.26 and 25.40% when incubation temperature decreased to 25 and 20°C, respectively after same time interval. Pollen germination and viability were significantly differed when they were collected from spathes opened at three different dates. Early opened spathes had lowest germination (57.58%) and viability percentages (81.19%) than the middle (81.11 and 91.53%) or late (80.71% and 93.05%) opening spathes. It is therefore, concluded that the optimum temperature for date palm pollen grains germination is 25-30°C and at these temperatures maximum pollens germinated within 4 h. Moreover, pollen grains from early opened spathes were less superior compared to the middle or late opening ones.

Reproductive barriers in cassava: Factors and implications for genetic improvement.

Cassava breeding is hampered by high flower abortion rates that prevent efficient recombination among promising clones. To better understand the factors causing flower abortion and propose strategies to overcome them, we 1) analyzed the reproductive barriers to intraspecific crossing, 2) evaluated pollen-pistil interactions to maximize hand pollination efficiency, and 3) identified the population structure of elite parental clones. From 2016 to 2018, the abortion and fertilization rates of 5,748 hand crossings involving 91 parents and 157 progenies were estimated. We used 16,300 single nucleotide polymorphism markers to study the parents' population structure via discriminant analysis of principal components, and three clusters were identified. To test for male and female effects, we used a mixed model in which the environment (month and year) was fixed, while female and male (nested to female) were random effects. Regardless of the population structure, significant parental effects were identified for abortion and fertilization rates, suggesting the existence of reproductive barriers among certain cassava clones. Matching ability between cassava parents was significant for pollen grains that adhered to the stigma surface, germinated pollen grains, and the number of fertilized ovules. Non-additive genetic effects were important to the inheritance of these traits. Pollen viability and pollen-pistil interactions in cross- and self-pollination were also investigated to characterize pollen-stigma compatibility. Various events related to pollen tube growth dynamics indicated fertilization abnormalities. These abnormalities included the reticulated deposition of callose in the pollen tube, pollen tube growth cessation in a specific region of the stylet, and low pollen grain germination rate. Generally, pollen viability and stigma receptivity varied depending on the clone and flowering stage and were lost during flowering. This study provides novel insights into cassava reproduction that can assist in practical crossing and maximize the recombination of contrasting clones.

Decreased anther dehiscence contributes to a lower fertilization rate of rice subjected to shading stress

• The pollination and fertilization processes were impeded by shading stress. • Shading markedly decreased the anther dehiscence rate of rice. • Shading reduced the number of pollen grains and germinated pollen grains per stigma. • Shading significantly decreased the fertilization rate and grain yield of rice. Shading stress has become a worldwide phenomenon and can lead to rice ( Oryza sativa L.) yield loss by decreasing the seed setting percentage (the percentage of filled spikelets to total spikelets per panicle), which depends primarily on spikelet fertility. However, the effect of shading stress on rice spikelet pollination and fertilization processes is not fully understood. Here, a field experiment, spanning two years (2019 and 2020), was conducted using two rice varieties (Huanghuazhan and Fyou 498) grown under full sunlight and 53 % shade. No significant difference in pollen I 2 -KI staining rate was observed between the full sunlight control and the shading treatment in 2019 and 2020. However, shading stress significantly decreased the in vitro pollen germination rate of spikelets that opened 5 d and 8 d after shading in 2020. Shading stress also significantly reduced the anther dehiscence rate of both varieties in both years (except for Huanghuazhan at 2 d after shading in 2019), and a mass of pollen grains remained in the anthers. Together with a reduction in the stigma exsertion rate, shading stress reduced the number of pollen grains per stigma by 29.4–71.2 % and the number of germinated pollen grains per stigma by 9.8–63.2 %. Consequently, shading stress significantly decreased the fertilization rate in these varieties, which contributed to a marked yield loss. We assert that shading stress decreased the fertilization rate by inhibiting key pollination and fertilization processes, particularly anther dehiscence and pollen grain release.

Persistence of the Effects of Se-Fertilization in Olive Trees over Time, Monitored with the Cytosolic Ca2+ and with the Germination of Pollen.

Selenium (Se) is an important micronutrient for living organisms, since it is involved in several physiological and metabolic processes. Biofortification with Se increases the nutritional and qualitative values of foods in Se-deficient regions and increases tolerance to oxidative stress in olive trees. Many studies have shown that Se, in addition to improving the qualitative and nutritional properties of EVO oil, also improves the plant’s response to abiotic stress. This study addressed this issue by monitoring the effects of Se on cytosolic Ca2+ and on the germination of olive pollen grains in oxidative stress. The olive trees subjected to treatment with Na-selenate in the field produced pollen with a Se content 6–8 times higher than the controls, even after 20 months from the treatment. Moreover, part of the micronutrient was organic in selenium methionine. The higher selenium content did not produce toxic effects in the pollen, rather it antagonized the undesirable effects of oxidative stress in the parameters under study. The persistence of the beneficial effects of selenium observed over time in pollens, in addition to bringing out an undisputed adaptability of olive trees to the micronutrient, suggested the opportunity to reduce the number of treatments in the field.

ПРОДУКТИВНІ ЯКОСТІ ГІБРИДІВ РІПАКУ ОЗИМОГО (BRASSICA NAPUS L.)

The content of monosaccharides in flower nectar, nectar productivity, as well as the viability of pollen of hybrids Brassica napus L. Kuga, Dk Expression, Emerald (Smaragd) and Abacus were studied.
 It was found that during the first and second pollen crops the most intensive germination of pollen grains occurred in Emerald (Smarahd) (75 % and 87 %, respectively), and Kuha (80 % and 90 %, respectively) hybrids. In crops of other studied hybrids - Dekalb Expression, Abacus, PT 264 - inhomogeneous pollen grains of different sizes were observed, the percentage of pollen germination was lower. It was found that the average number of monosaccharides is much higher in hybrids Abacus and Kuha (27.5 and 36 mg / 30 flowers, respectively). Kuha (156.8 kg) and Emerald (Smaragd) (81.4 kg) hybrids have the highest nectar fertility. Analysis of monosaccharides content in flower nectar, nectar fertility and pollen viability, as well as the correlation of these indicators allows to predict in the future among the studied hybrids higher productivity of hybrids Kuha and Emerald (Smaragd).

Bumblebees (Bombus terrestris) Improve 'Hass' Avocado (Persea americana) Pollination.

Pollination is limiting for avocado production. We examined whether adding bumblebees (BBs; ca. 10 hives/ha) to conventional honeybees (HB; 5 hives/ha) would improve ‘Hass’ avocado pollination and yields. A preliminary trial (2017/18) in an avocado orchard with four consecutive rows of ‘Hass’ followed by one row of ‘Ettinger’ serving as a pollenizer (20% ‘Ettinger’) showed a considerable increase in ‘Hass’ yield in rows adjacent to (up to 80 m from) the BB hives vs. distant rows (=controls). In 2018/19, the trials were extended to three additional orchards. A significant yield increase was obtained in the BB hive-adjacent trees compared to BB hive-distant ones. Similar results were obtained in 2019/20, in experiments conducted throughout the country. The SNP analysis, to determine the parents of ‘Hass’ fruit at varying distances from the BB hives, showed no differences in the cross-pollination rate (‘Hass’ × ‘Ettinger’). However, pollination rates and the number of germinating pollen grains per stigma decreased with distance from the hives, and correlated to the negative gradient in yield. Taken together, our data suggest that adding BB hives to ‘Hass’ avocado orchards, at ca. 10 hives/ha resulting in 0.5–1.0 BB visits/tree per min, increases pollination and, accordingly, total yield.

Selective Inhibition of Wild Sunflower Reproduction with Mugwort Aqueous Extract, Tested on Cytosolic Ca2+ and Germination of the Pollen Grains.

Wild sunflower (Helianthus annuus L.) is an invasive species widely distributed in several regions of the world, where it shares a large area with domesticated sunflower. The imidazolinone-tolerant sunflower enables the control of problematic weeds (such as Xanthium spp., Brassica spp., wild sunflower) with imidazolinone herbicides (Clearfield® production system) in cultivated sunflower crops, but could facilitate the gene transfer of herbicide resistance, from cultivated sunflower to wild sunflower, generating hard-to-control weed biotypes or herbicide-resistant populations. The development of new practices that involve the selective inhibition of reproduction structures, such as pollen granules, could be an innovative strategy to minimize outcrossing and the origin of weed–crop hybrids in Clearfield® production systems. In this study, the effects of mugwort (Artemisia vulgaris L.) aqueous extract on cytosolic Ca2+ and the germination of pollen grains collected from conventional, wild and IMI-tolerant sunflower were tested. The results showed that mugwort deregulated Ca2+ homeostasis and markedly reduced the germination of conventional and wild sunflower pollen, but not IMI-tolerant pollen. The HPLC analysis revealed the presence of phenolic acids belonging to the hydroxycinnamic and benzoic classes in the mugwort extract. Hydroxycinnamic acids (caffeic and ferulic) deregulated the cytosolic Ca2+ of conventional and wild sunflower pollen, but not those which were IMI-tolerant, similar to mugwort extract. Selective inhibition of wild sunflower pollen in the Clearfield® sunflower crop contributes to a possible new weed management strategy, reducing the wild sunflower reproduction by seed, minimizing the potential risks of outcrossing with the formation of weed–crop hybrids. The Ca2+ selective chelating activity of caffeic or ferulic acids provides elements to be investigated for their possible use as an alternative to mugwort extract.

Ethylene signaling modulates tomato pollen tube growth through modifications of cell wall remodeling and calcium gradient.

Ethylene modulates plant developmental processes including flower development. Previous studies have suggested ethylene participates in pollen tube (PT) elongation, and both ethylene production and perception seem critical at the time of fertilization. The full gene set regulated by ethylene during PT growth is unknown. To study this, we used various EThylene Receptor (ETR) tomato (Solanum lycopersicum) mutants: etr3-ko, a loss-of-function (LOF) mutant; and NR (NEVER RIPE), a gain-of-function (GOF) mutant. The etr3-ko PTs grew faster than wild-type (WT) PTs. Oppositely, NR PT elongation was slower than in WT, and PTs displayed larger diameters. ETR mutations result in feedback control of ethylene production. Furthermore, ethylene treatment of germinating pollen grains increased PT length in etr-ko mutants and WT, but not in NR. Treatment with the ethylene perception inhibitor 1-methylcyclopropene decreased PT length in etr-ko mutants and WT, but had no effect on NR. This confirmed that ethylene regulates PT growth. The comparison of PT transcriptomes in LOF and GOF mutants, etr3-ko and NR, both harboring mutations of the ETR3 gene, revealed that ethylene perception has major impacts on cell wall- and calcium-related genes as confirmed by microscopic observations showing a modified distribution of the methylesterified homogalacturonan pectic motif and of calcium load. Our results establish links between PT growth, ethylene, calcium, and cell wall metabolism, and also constitute a transcriptomic resource.

Flower stages, germination and viability of pollen grains of Annona squamosa L. in tropical conditions

The success in the application of artificial pollination techniques, aspects of floral biology should receive special attention, especially regarding the viability studies of pollen grains. In this sense, two experiments were carried out, aiming to determine the floral stages: floral opening (anthesis), female, male and senescence stages of A. squamosa L flowers under tropical climate conditions, and to evaluate the germination and viability of pollen grains submitted to different storage conditions. In the first experiment, observations and data collection began when flowers were still closed. Readings were taken every two hours for 46 hours until all the flowers reached the senescence stage. For the second, the experimental design was completely randomized, in a 2 x 7 factorial scheme, with 2 storage conditions (environment at 27 ± 2°C and refrigerated at 5 ± 2°C) and 7 pollen grain conditioning times (0, 4, 8, 12, 16, 20 and 24 hours), with 4 repetitions for the analysis of pollen grain germination and 3 repetitions for the analysis of pollen grain viability, and each repetition consisted of one blade. The variables evaluated were percentage of viable and non-viable pollen grains and percentage of germinated and non-germinated pollen grains. For tropical climatic conditions, the flower anthesis (female stage) of the sugar-apple begins at 00:00 h, extending until 12:00h on the same day. The flowers of the tree reach a functionally staminate stage (male stage) from 6 a.m. the next day. Pollen grains stored at room conditions (27 ± 2°C) remain viable for up to 24 hours after collection, showing that storage in a cold environment at low temperatures (5 ± 2°C) is not necessary. Seed pollen grains have a germination percentage of 51.25% when stored under ambient conditions (27 ± 2°C) for up to 4 hours after collection.

Optimization of culture medium for the in vitro germination and histochemical analysis of Passiflora spp. pollen grains

Pollen viability studies are essential for high production of fruits and seeds, as well as for genetic improvement and conservation strategies. The objective of this work was to investigate the most suitable culture medium for in vitro germination of pollen grains and pollen tube growth of 11 wild Passiflora species and to examine the viability by means of histochemical testing with 2% Alexander's solution. This is the first study to provide data on the pollen tube growth of passion fruit. For in vitro germination, two culture media were used, BK and SM, both supplemented with different concentrations of sucrose (5, 10, 15, 20, 25, 30 and 35%). The experimental design was completely randomized in an 11×7 × 2 factorial scheme (species x sucrose concentrations x culture media). Sucrose influenced the germination of pollen grains and the pollen tube growth. P. edmundoi had the highest germination rate (89%) and pollen tube length (2.6 mm) in SM culture medium supplemented with 30% and 15% sucrose, respectively. Regarding the histochemical analysis, all the Passiflora species presented high percentage of viable pollen grains (above 79%). The information obtained in this study allowed identifying the most suitable culture medium for each Passiflora species, helping in the choice of male parents with high potential for fertility, to maximize the potential success of controlled crosses in genetic improvement programs. The results can also support studies of the conservation of pollen grains.

Heat Priming of Lentil (Lens culinaris Medik.) Seeds and Foliar Treatment with γ-Aminobutyric Acid (GABA), Confers Protection to Reproductive Function and Yield Traits under High-Temperature Stress Environments.

Gradually increasing temperatures at global and local scales are causing heat stress for cool and summer-season food legumes, such as lentil (Lens culinaris Medik.), which is highly susceptible to heat stress, especially during its reproductive stages of development. Hence, suitable strategies are needed to develop heat tolerance in this legume. In the present study, we tested the effectiveness of heat priming (HPr; 6 h at 35 °C) the lentil seeds and a foliar treatment of γ-aminobutyric acid (GABA; 1 mM; applied twice at different times), singly or in combination (HPr+GABA), under heat stress (32/20 °C) in two heat-tolerant (HT; IG2507, IG3263) and two heat-sensitive (HS; IG2821, IG2849) genotypes to mitigate heat stress. The three treatments significantly reduced heat injury to leaves and flowers, particularly when applied in combination, including leaf damage assessed as membrane injury, cellular oxidizing ability, leaf water status, and stomatal conductance. The combined HPr+GABA treatment significantly improved the photosynthetic function, measured as photosynthetic efficiency, chlorophyll concentration, and sucrose synthesis; and significantly reduced the oxidative damage, which was associated with a marked up-regulation in the activities of enzymatic antioxidants. The combined treatment also facilitated the synthesis of osmolytes, such as proline and glycine betaine, by upregulating the expression of their biosynthesizing enzymes (pyrroline-5-carboxylate synthase; betaine aldehyde dehydrogenase) under heat stress. The HPr+GABA treatment caused a considerable enhancement in endogenous levels of GABA in leaves, more so in the two heat-sensitive genotypes. The reproductive function, measured as germination and viability of pollen grains, receptivity of stigma, and viability of ovules, was significantly improved with combined treatment, resulting in enhanced pod number (21–23% in HT and 35–38% in HS genotypes, compared to heat stress alone) and seed yield per plant (22–24% in HT and 37–40% in HS genotypes, in comparison to heat stress alone). The combined treatment (HPr+GABA) was more effective and pronounced in heat-sensitive than heat-tolerant genotypes for all the traits tested. This study offers a potential solution for tackling and protecting heat stress injury in lentil plants.