Long-term storage of Narcissus anthers and pollen in liquid nitrogen

Summary Three methods for the long-term storage of narcissus pollen were compared; anthers in glass vials held in a desiccator with calcium chloride at 2°C, and polypropylene straws containing either anthers or naked pollen immersed in liquid nitrogen. Pollen from all storage treatments showed 15–16% germination in vitro after 3 days, compared with 27.4% for fresh pollen. Seed set per pod using pollen stored for 3 days was comparable to that of fresh pollen. However, after 351 days, pollen from anthers at 2°C exhibited only 0.1% germination and failed to set seed whereas no further change in germination rate was recorded for pollen from the two liquid nitrogen treatments and seed set was still equivalent to fresh pollen.     

Shortening the generation cycle in faba bean (Vicia faba) by application of cytokinin and cold stress to assist speed breeding

The aim of this study was to reduce the length of the breeding cycle for faba bean by accelerating seed setting. We examined the mode and time of exogenous 6-benzylaminopurine (BAP) (cytokinin) application, and cold treatments and their combinations in two faba bean genotypes. Acropetal node number of pod and seed set and pollen viability were recorded during the experiments. Application of BAP improved pollen germination. The application of 10^–5 M BAP 4 days after flowering increased seed set at the lower nodes. Cold treatment (8/4°C day/night for 2 days) after the onset of flowering induced the formation of more pods and faster pod set compared to the non-cold treatment. The time to first seed was significantly reduced, and pollen viability was increased in plants exposed to cold treatment. Increased pollen viability also showed a significant positive correlation with seed setting. The combinations of 10^–5 BAP and cold treatment together had similar and independent effects. These results will accelerate plant breeding in faba bean by providing additional tools for reducing generation time.     

‘砀山酥梨’花柱可授性及蜜蜂授粉特性的研究

采用半离体培养法和联苯胺-过氧化氢法研究了‘砀山酥梨’花粉萌发特性和柱头可授性。通过对‘酥梨’的最适授粉时间以及7种授粉处理坐果率的研究,探讨其授粉特性。结果表明：开花后每天酥梨柱头活性最佳的时间是11时至15时,柱头最佳授粉时间为开花后第4~5天;‘酥梨’花粉最适萌发温度为25℃,花粉管生长最有利的温度为22~25℃,在相同温度下,花粉萌发率和花粉管长度随时间增加而增加。7种授粉处理‘酥梨’的花朵坐果率中,没有昆虫授粉的‘酥梨’坐果率为0.48%和0.44%,自然授粉和人工授粉坐果率分别为2.83%和13.05%,‘酥梨’ב雪花梨’组蜜蜂授粉的坐果率为32.95%,新鲜花粉和商品花粉坐果率分别为70.2%和13.05%。由此可见,‘酥梨’是异花虫媒果树,蜜蜂可以在‘酥梨’柱头活性最高时进行授粉,从而提高‘酥梨’的坐果率,同时通过对‘酥梨’花器官生理活性和坐果习性的研究,为‘酥梨’的授粉和生产提供基础资料。     

Gamma-irradiation of cacao (Theobroma cacao L.) pollen: Effect on pollen grain viability,germination and mitosis and on fruit set

Summary Theobroma cacao pollen fertilization capability was studied after 0, 50, 100, 200, 500 and 1000 Gy gamma-irradiation. For all irradiation doses, no alteration of pollen grain viability and in vitro germination was observed. In situ, for all doses, pollen tubes penetrated into the styles and reached the ovules 20 hours after pollination. In vitro observations of the pollen grain nuclei after 20 hours incubation showed that pollen irradiation causes inhibition of the division of the generative nucleus. Fruit survival rate 30 days after pollination decreased as irradiation doses increased from 0 to 100 Gy, and over 100 Gy no fruit set was obtained. The possibility of using irradiated pollen as a method for obtaining haploid cacao plantlets is discussed.     

Effect of selection for pollen grain size on various traits in common bean

Summary Selection among microgametophytes usually exploits variation in pollen grain germination. Studies of variation in pollen grain size in common bean (Phaseolus vulgaris L.) suggested that selection for size might lead to changes in sporophytic traits. To determine whether microgametophytic selection based on size would affect pollen grain size in subsequent generations or sporophytic traits that were correlated with pollen grain size, pollen grains from three crosses were separated into two size categories by sieving and then used to pollinate cv. Diacol Calima. Selection resulted in changes in pollen grain diameter for pollen from F₁, F₂ and F₃ plants for all crosses. In vitro germination indicated no differences between vigor of large and small grains, but extraction and sieving reduced germinability. F₁ seed from two of the crosses with size-selected pollen varied in weight according to pollen grain size, but in subsequent generations, the effect disappeared. Both size categories of selected pollen resulted in F₂ progeny with reduced numbers of seeds per pod as compared to controls, suggesting that the size selection process may have resulted in indirect selection for traits reducing seed set. The overall results suggested that genes determining pollen grain size in bean have little or no effect on sporophytic traits such as seed size and seed yield.     

Some consequences of pollen storage in the raspberry (Rubus idaeus L.)

Summary Raspberry pollen was stored at 5°C and –13°C for six months and then tested for its ability to induce pyrene set and the production of viable seedlings, and for its effect upon the segregation of a major gene s. Only pollen stored at –13°C gave a pyrene set and germination percentage adequate to produce sufficient seedlings for a breeding programme. It is suggested that tests of pollen viability in the raspberry should include studies of pollen germination, and the effect of this pollen on pyrene set and seed germination. Possible causes of the loss of viability in the pollen stored at 5°C are discussed.     

Population structure and association mapping of traits related to reproductive development in field pea

Field pea (Pisum sativum) is an important pulse crop globally for human consumption and livestock feed. A panel of 92 diverse pea cultivars was evaluated across nine environments and genotyped using 1536 single nucleotide polymorphisms (SNPs) arranged in a GoldenGate array. Population structure analysis revealed three subpopulations roughly consistent with the cultivar origin. Phenotyping included days to flowering (DTF), duration of flowering (DOF), number of reproductive nodes, number of pods on the main stem, percentage of pods set, percentage of pods retained with seed and pollen germination reduction due to heat stress. Association analyses identified a total of 60 SNPs significantly associated (?log₁₀ p ≥ 4.3) with these seven reproductive development-related traits. Among these 60 marker-trait associations, 33 SNPs were associated with the onset of flowering, 8 SNPs with pod development and 19 SNPs with the number of reproductive nodes. No SNP marker was significantly associated with in vitro pollen germination reduction caused by high temperature stress. We found that 12 SNPs associated with DTF and 2 SNPs associated with DOF overlapped with the SNP markers associated with the number of reproductive nodes. Genomic regions associated with variation for reproductive development-related traits identified in this study provide grounds for future genetic improvement in pea.     

Differential heat sensitivity of two cool-season legumes,chickpea and lentil,at the reproductive stage,is associated with responses in pollen function,photosynthetic ability and oxidative damage

Increasing temperatures are adversely affecting various food crops, including legumes, and this issue requires attention. The growth of two cool-season food legumes, chickpea and lentil, is inhibited by high temperatures but their relative sensitivity to heat stress and the underlying reasons have not been investigated. Moreover, the high-temperature thresholds for these two legumes have not been well-characterised. In the present study, three chickpea (ICCVO7110, ICC5912 and ICCV92944) and two lentil (LL699 and LL931) genotypes, having nearly similar phenology with respect to flowering, were grown at 30/20°C (day/night; control) until the onset of flowering and subsequently exposed to varying high temperatures (35/25, 38/28, 40/30 and 42/32°C; day/night) in a controlled environment (growth chamber; 12 hr/12 hr; light intensity 750 µmol m⁻² s⁻¹; RH-70%) at 108 days after sowing for both the species. Phenology (podding, maturity) was accelerated in both the species; the days to podding declined more in lentil at 35/25 (2.8 days) and 38/28°C (11.3 days) than in chickpea (1.7 and 7.1 days, respectively). Heat stress decreased flowering–podding and podding–maturity intervals considerably in both the species. At higher temperatures, no podding was observed in lentil, while chickpea showed reduction of 14.9 and 16.1 days at 40/30 and 42/32°C, respectively. Maturity was accelerated on 15.3 and 12.5 days at 38/28°C, 33.6 and 34 days at 40/30°C and 45.6 and 47 days at 42/32°C, in chickpea and lentil, respectively. Consequently, biomass decreased considerably at 38/28°C in both the species to limit the yield-related traits. Lentil was significantly more sensitive to heat stress, with the damage—assessed as reduction in biomass, reproductive function-related traits (pollen viability, germination, pollen tube growth and stigma receptivity), leaf traits such as membrane injury, leaf water status, photochemical efficiency, chlorophyll concentration, carbon fixation and assimilation, and oxidative stress, appearing even at 35/25°C, compared with 38/28°C, in chickpea. The expression of enzymatic antioxidants such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and non-enzymatic antioxidants declined remarkably with heat stress, more so in lentil than in chickpea. Carbon fixation (assessed as Rubisco activity) and assimilation (assessed as sucrose concentration, sucrose synthase activity) were also reduced more in lentil than in chickpea, at all the stressful temperatures, resulting in more inhibition of plant biomass (shoot + roots), damage to reproductive function and severe reduction in pods and seeds. At 38/28°C, lentil showed 43% reduction in biomass, while it declined by 17.2% in chickpea at the same time, over the control temperature (30/20°C). At this temperature, lentil showed 53% and 46% reduction in pods and seed yield, compared to 13.4% and 22% decrease in chickpea at the same temperature. At 40/30°C, lentil did not produce any pods, while chickpea was able to produce few pods at this temperature. This study identified that lentil is considerably more sensitive to heat stress than chickpea, as a result of more damage to leaves (photosynthetic ability; oxidative injury) and reproductive components (pollen function, etc.) at 35/25°C and above, at controlled conditions.     

Thresholds,sensitive stages and genetic variability of finger millet to high temperature stress

Finger millet [Eleusine coracana (L.) Gaertn.] is an important coarse cereal crop grown in the arid and semi‐arid regions and often experiences high temperature (HT) stress. The objectives of this research were (i) to quantify effects of season‐long HT stress on physiological and yield traits, (ii) to identify the developmental stages most sensitive to HT stress and (iii) to quantify the genetic variability for HT stress tolerance in finger millet. Research was conducted in controlled environment conditions. HT stress decreased the chlorophyll index, photosystem II activity, grain yield and harvest index. Maximum decrease in number of seeds per panicle and grain yield per plant was observed when stress was imposed during booting, panicle emergence or flowering stages. Maximum genotypic variation was explained by panicle width and number of seeds per panicle at optimum temperature (OT) and grain yield per plant at HT and number of seeds at HT. Based on the stress response and grain yield, tolerant or susceptible genotypes were identified. Finger millet is sensitive to HT stress during reproductive stages, and there was genotypic variability among the finger millet genotypes for number of seeds per panicle and grain yield under HT, which can be exploited to enhance stress tolerance.     

Effect of Abscisic Acid on Pollen Germination and Tube Growth of Maize Genotypes1

As a preliminary step to using gametophytic selection to obtain genotypes of maize, Zea mays L., differing for abscisic acid (ABA) content and/or sensitivity, the influence of ABA on the in vitro germination and tube growth of maize pollen and the presence of genetic variability for these traits was investigated. In a first experiment, pollen samples of three genotypes were germinated on substrates containing increasing amounts of ABA. Low ABA concentration (0.5 νM) significantly increased pollen germination and tube growth, while 500 νM ABA significantly decreased both traits, thus indicating that ABA can influence the growth of maize pollen in vitro. In a second experiment, eleven inbred lines were screened for the in vitro reactivity of their microgametophytes to ABA. The lines differed significantly for the response (% of control) of tube length, but not of pollen germination to ABA.     

Water Deficit during the Reproductive Period of Grass Pea (Lathyrus sativus L.) Reduced Grain Yield but Maintained Seed Size

The grain legume grass pea (Lathyrus sativus L.) is adapted to drought‐prone environments, but the extent and mechanisms of its tolerance are not well understood. In a pot experiment, water deficit was imposed on plants by withholding water from first flowering until predawn leaf water potential (LWP_pd) was ?3.12 MPa. Water deficit reduced dry matter, seed yield, harvest index and water use efficiency by 60 %, 87 %, 67 % and 75 %, respectively, when compared with the controls. Flower production stopped when LWP_pd fell to ?1.8 MPa. At LWP_pd?1.5 MPa, only 25 % of flowers resulted in filled pods (compared with 95 % filled pods in the control) with the rest aborted as flowers (48 %) or pods (27 %). Filled pods had more aborted ovules than controls, resulting in 29 % less seeds per pod. Water deficit reduced pollen viability, germination and the number of pollen tubes reaching the ovary by 13 %, 25 % and 31 %, respectively. Emergence from seeds produced from water‐deficient plants was 21 % less than controls, but seedling shoot dry mass was 18 % higher, in accordance with the 19 % higher seed mass. The sensitivity of flowering to drought limited pod numbers but enabled plants to retain existing pods and develop near‐normal seeds with low β‐N‐oxalyl‐l ‐α‐β‐diaminopropionic acid toxin concentrations. This trait is useful for farming systems reliant on harvested seed for the next crop and in cases where seed size influences the value of the product.     

Cryopreservation of Delphinium pollen at –30 °C

Pollen of garden varieties and species of Delphinium that was cryopreserved at –30 ^°C after 3 hours of air drying and storage on silica gel, had high germination and pollen tube growth in vitroeven after 180-day storage. On the other hand, pollen stored at 25 ^°C showed a marked decrease in the germination rate within 10 days. The best in vitro germination of Delphinium pollen was on a 1% water agar containing 15% sucrose and 0.005 to 0.01% boric acid and at 15 to 20 ^°C. Field pollination with the cryopreserved pollen showed higher fruit and seed set than pollination with pollen stored at 25 ^°C, and was not significantly different from fresh pollen. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effects of high-temperature stress on the germination of pollen grains of upland cotton during square development

The reproductive stage of flowering plants is sensitive to high-temperature stresses. High temperature is a major factor influencing pollen grain viability in upland cotton (Gossypium hirsutum). The objective of this study was to identify the relationship between cotton pollen germination percentage and temperature by assaying the pollen germination of four upland cotton cultivars in vitro at different temperatures during the blooming period. The results showed that in vitro pollen germination percentage was related to the culture temperature of pollen germination and the temperature of the square development process. High temperature affected pollen development and germination, and high-temperature tolerance differed among the cotton cultivars. The pollen germination percentage decreased rapidly with changes in the culture temperature from 30 to 39 °C. A culture temperature of 35 °C might be a critical temperature for the pollen viability transition and could be used to screen cotton cultivars that have pollen grains with high-temperature resistance. Before the high-temperature stage, cultivars with rates of decrease in the percentage of pollen germination of less than 41 % at 35 °C relative to the rates at 30 °C might be considered as high-temperature tolerance cultivars, and cultivars with rates of decrease in the percentage of pollen germination greater than 41 % might be considered as susceptible cultivars. The high-temperature stress for pollen grain germination in vitro was greater than 30 °C, and the high-temperature stress for square development might be greater than 33 °C. Boll retention was significant; it was positively correlated with the pollen germination percentage and negatively correlated with temperature during the high-temperature stage. This study provided a method for rapidly screening cultivars (lines) with high-temperature tolerance pollen in upland cotton breeding.     

Reproductive development response to high daytime temperature in field pea

Flowering plants are highly sensitive to heat stress during reproductive phase, which covers development from floral initiation to seed maturity. The objectives of this study were to diagnose high temperature effects on pollen production and morphology, production of reactive oxygen species (ROS) in pollen grains and ovules in pea cultivar “CDC Golden.” This study also investigated timing and duration of heat exposure at specific developmental stages of floral buds, open flowers and early set pods on flower and pod abortion, seed development and seed yield in “CDC Golden” and a second cultivar “CDC Sage.” The experiments were conducted in growth chambers with two temperature regimes (24/18°C and 35/18°C day/night temperature for 4–14 days) during reproductive development. Heat stress reduced the number of pollen grains per anther, induced smaller pollen grains and increased ROS production in pollen grains, but it did not affect ROS accumulation in ovules and ovule number per ovary. Heat exposure when young floral buds were visible at the first reproductive node was more detrimental to flower retention, seed set, pod development and seed yield compared to heat exposure started later when flowers at the second reproductive node were fully open.     

Susceptibility of Faba Bean (Vicia faba L.) to Heat Stress During Floral Development and Anthesis

Experiments were conducted over 2 years to quantify the response of faba bean (Vicia faba L.) to heat stress. Potted winter faba bean plants (cv. Wizard) were exposed to temperature treatments (18/10; 22/14; 26/18; 30/22; 34/26 °C day/night) for 5 days during floral development and anthesis. Developmental stages of all flowers were scored prior to stress, plants were grown in exclusion from insect pollinators to prevent pollen movement between flowers, and yield was harvested at an individual pod scale, enabling effects of heat stress to be investigated at a high resolution. Susceptibility to stress differed between floral stages; flowers were most affected during initial green‐bud stages. Yield and pollen germination of flowers present before stress showed threshold relationships to stress, with lethal temperatures (t₅₀) ?28 °C and ~32 °C, while whole plant yield showed a linear negative relationship to stress with high plasticity in yield allocation, such that yield lost at lower nodes was partially compensated at higher nodal positions. Faba bean has many beneficial attributes for sustainable modern cropping systems but these results suggest that yield will be limited by projected climate change, necessitating the development of heat tolerant cultivars, or improved resilience by other mechanisms such as earlier flowering times.     

花/蕾低温保存对花椰菜花粉活力及授粉效果的影响

本研究将通过低温(3℃~5℃)干燥处理花椰菜完全开放的花和不同大小的蕾,利用花粉活力测定、花粉萌发观察和杂交结籽率检测等手段分析了低温保存对其花粉活力及授粉效果的影响。研究结果表明,当天开放花的花粉活力和萌发率最高,分别达到91.3%和45.6%,授粉效果也最好,每角果平均可收获11.3粒种子。3℃~5℃干燥储存8 d后花粉活力和萌发率显著降低,储存16 d后花粉杂交结籽率显著降低,不适合作为花粉供体;开花前1 d和2 d的蕾,3℃~5℃干燥储存8 d的花粉活力分别为88.3%和85.2%,萌发率均为32.4%左右,授粉杂交结籽率分别达到9.8和8.3粒种子/角果。随着储存时间的延长,花粉活力及萌发率逐渐降低。3℃~5℃干燥储存32 d的花粉杂交结籽率仍可达到8粒种子/角果。本研究结果为花椰菜杂交一代制种和组合测配的花粉保存提供理论依据。     

Compatibility and incompatibility in witloof-chicory (Cichorium intybus L.). 1. The influence of temperature and plant age on pollen germination and seed production

Summary For the production of inbred lines and F₁ hybrids in witloof-chicory information is wanted on characteristics such as the incompatibility system. These characteristics can only be studied properly if the influence of temperature and physiological status of the plant on pollen germination and seed production is known. Investigations were carried out with 9 self-incompatible (SI) and 6 self-compatible (SC) clones in glasshouses of the IVT phytotron at constant temperatures of 10, 14, 17, 20, 23 and 26°C. In general, in vivo pollen germination percentages were rather low after self pollination with an optimum for germination around 17–20°C. No seeds were formed at the lowest temperature (10°C) while seed production for SC clones was usually (rather) good at higher temperatures. At 26°C seed production in some clones decreased. Both pollen germination and seed production decreased at the end of the flowering period. There was a rather positive relationship at e.g. 17 and 20°C between pollen germination after selfing and seed production. When no pollen germination was observed, no seed formation occurred. When pollen grains did germinate, seed development would not necessarily occur in all cases. So this relationship only enables negative mass selection for SC.     

Prezygotic interspecific hybridization barriers between kenaf (Hibiscus cannabinus L.) and four wild relatives

Use of interspecific hybridization in genetic improvement of kenaf (Hibiscus cannabinus), a multipurpose crop, is limited by low cross‐compatibility. Previous hybridization studies suggest that part of incompatibility barrier is prezygotic. Pollen germination, stigma receptivity and growth of alien pollen on kenaf pistil were studied using wild relatives of kenaf. Four wild and semi‐domesticated species, H. surattensis, H. acetosella, H. radiatus and H. vitifolius, were used as pollen donors in the present experiment. H. radiatus exhibited highest pollen germination (65.89%) on H. cannabinus stigma. For closely related species (H. surattensis, H. acetosella, H. radiatus), stylar incompatibility was noticed, while for distantly related species H. vitifolius, the incompatibility was stigmatic. Number of pollen tubes entering ovule of H. cannabinus pistil was higher for H. radiatus (1.30). Alien pollen growth and penetration of pollen tube through ovule were higher in species sharing same genome of H. cannabinus. High callose deposition was observed in incompatible crosses, suggesting a general mechanism for prezygotic incompatibility in Hibiscus section Furcaria. Callose deposition in incompatible crosses increased with time.     

Cryopreservation of English walnut (Juglans regia L.) pollen

Summary Pollen from eight clones of English walnut (Juglans regia L.) was stored in liquid nitrogen (LN₂) at-196°C for one year. Pollen was monitored for percent germination in vitro before being frozen. Pollen was frozen within two hours of anther dehiscence and subsequently at 24 hr intervals until germination assays indicated that the ability to germinate was lost. Survival after one year was evaluated by determining percent germination in vitro. Freshly collected pollen of only five of the eight clones survived LN₂. By contrast, when the same pollen was held for 24 and 72 hr before freezing, pollen of all eight clones survived. Survival is correlated with moisture content (MC) of the pollen. Pollen samples with MC greater than 7.5% were killed by freezing in LN₂. All pollen samples with MC between 4 and 7.5% survived as did most of the samples with MC between 3.2 and 4%.     

Genetic Variation for Heat Tolerance During the Reproductive Phase in Brassica rapa

We investigated heat tolerance at the reproductive stage in six spring‐type B. rapa accessions and one B. juncea accession as a control. Plants were subjected to two temperature treatments for seven days in controlled environmental rooms, beginning one day before the first open flower on the main stem inflorescence. The high‐temperature treatment ranged from 25 °C to 35 °C during 16 h light and 25 °C during 8 h dark. The control temperature treatment was set at 23 °C during 16 h light and 15 °C during 8 h dark. Soil moisture was maintained at close to field capacity to avoid drought stress. Main stem buds that emerged during the treatment period were tagged, and pod and seed production was recorded at each reproductive node. Leaf temperature depression and leaf conductance increased in the high‐temperature treatment which indicated that plants were not drought stressed. A leafy vegetable type of B. rapa from Indonesia was the most tolerant to high temperature, as defined by its ability to set seed equally well in the control and high‐temperature treatments, followed by an oilseed type from Pakistan. Pollen viability remained above 87 % in all accessions and treatments. We conclude that bud number and length, and pod number produced under high temperatures, might provide a useful preliminary screen for high‐temperature tolerance and that B. rapa may be a valuable source of heat tolerance in canola (B. napus).