ROS-induced oxidative stress is closely related to pollen deterioration following cryopreservation

Oxidative stress induced by excessive accumulation of reactive oxidative species (ROS) during cryopreservation is thought to be one factor contributing to cryodamage of biological materials. To explore the role of oxidative stress in the cryopreservation of plant pollen, germination, ROS, and malondialdehyde (MDA) levels of pollen from 20 ornamental plant species were compared before and after cryopreservation. The results showed that the germinability of cryopreserved pollen from 13 out of the 20 species was not significantly different from that of fresh pollen (group 1), only one increased significantly, while the other six declined significantly (group 2). The MDA content in cryopreserved pollen from nine species in group 1 showed no significant difference from that of fresh pollen, while four species in group 2 rose significantly. This suggested that pollen viability and MDA levels were negatively correlated. ROS generation in cryopreserved pollen from nine species in group 1 was unchanged compared to fresh pollen, while five species in group 2 increased significantly. This suggested that pollen viability was negatively correlated with ROS generation. Additionally, both ROS and MDA levels in pollen from four species in group 2 increased significantly. In conclusion, pollen from most species possesses some cryostorage tolerance, but some species are severely damaged by cryostorage. Oxidative stress induced by the cryostorage in liquid nitrogen (LN) may be a key factor for the decreased viability in pollen following cryopreservation.     

桃花粉低温和超低温保存方法比较研究

桃(Prunus persica(L.)Batsch)是我国重要的无性繁殖作物种质资源,目前主要保存于3个国家无性繁殖作物种质圃。随着以茎尖、花粉、休眠芽为保存载体的超低温保存技术的发展,超低温保存已成为无性繁殖作物重要备份保存方式。本研究以15份桃种质花粉为研究对象,开展含水量、回湿处理和保存温度(4℃低温保存和液氮超低温保存)对保存后花粉离体萌发率的影响研究。研究结果:明确了桃种质花粉超低温保存的含水量;揭示了回湿处理对部分桃种质花粉超低温保存产生显著影响;超低温保存后花粉离体萌发率最高可达83%;4℃低温保存和超低温保存比较研究结果表明,超低温保存4年后14份桃种质花粉离体萌发率仍可保持30%以上,11份桃种质花粉离体萌发率与保存前花粉离体萌发率相比无显著变化甚至显著提高,而4℃低温保存的花粉离体萌发率降至0。该研究为国家种质库建立花粉规模化超低温保存提供技术支撑。     

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

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

Monitoring indoor pollen over two years

The present study monitored a private apartment for pollen that had settled on the floor. Pollen was sampled and analysed from two rooms over a period of two years. The study showed that pollen accumulations indoors are dominated by wind-pollinated species reflecting the current flowering season. The adjacent vegetation and the cleaning activities influenced the pollen content. Pollen from zoophilous plants mostly originated from ornamental plants within the flat. Similarities and differences between the two rooms and the two years were observed. These findings have implications for forensic investigations as well as allergological research.     

蓝花丹开花生物学观测及花粉储存研究

对四川引种栽培的蓝花丹（Plumbago auriculata Lam.）花粉与柱头形态、花部特征及开花物候进行了系统观测,并对其花粉的采集时间与储存温度进行了初步研究。结果显示：（1）两种类型花粉和柱头形态均具有较大差异,短花柱型（S型）花粉的极轴长P值和赤道轴长E值均显著大于长花柱型（L型）花粉,L型柱头则明显长于S型,且两种花粉的纹饰和柱头的瘤状突起物形态也明显不同;（2）蓝花丹具有较高的开花同步性指数（为0.89）,但相对开花强度不高,属于中等强度;（3）蓝花丹两种花型在花蕾半开放时采收花粉,其活力最高,L型花粉活力可达85.24%±4.22%,S型可达87.74%±2.95%;（4）L型与S型花粉分别于25℃干燥0.5 h和1 h后再低温储存,其活力保持更好;（5）干燥后的花粉在-86℃条件下保存效果最好,储存30 d后L型花粉活力高达66.51%±0.85%、S型达69.07%±1.57%。本研究表明蓝花丹二型植株在生殖资源分配上存在明显差异,这种差异一方面导致花粉大小和形态及其所需干燥时间的不同,另一方面导致二型花柱头表现出明显不同的特征,这些差异性的结构是否参与了蓝花丹自交不亲和反应的识别过程,还有待进一步研究。此外,较低的开花强度会造成虫媒传粉困难,这可能是蓝花丹自然结实率极低的重要因素之一。     

Cryopreservation and long-term storage of pear germplasm

Summary Germplasm collections of vegetatively propagated crops are usually maintained as plants in fields or potted in greenhouses or screened enclosures. Safety duplication of these collections, as duplicate plants or separate collections, is costly and requires large amounts of space. Cryopreservation techniques which were recently developed for long-term storage of pear germalasm may offer an efficient alternative to conventional germplasm collection maintenance. Pear (Pyrus L.) germplasm may now be stored as seeds (species), dormant buds or pollen from field-grown trees, or shoot tips fromin vitro-grown plants (cultivars). Pear germplasm may now be cryopreserved and stored for long periods (> 100 yr) utilizing slow-freezing or vitrification ofin vitro-grown shoot-tips. Dormant bud freezing, pollen, and seed cryopreservation of other lines are being developed to complete the base collection forPyrus. This cryopreserved collection provides base (long-term) storage for the field-grown pear germplasm collection at the National Clonal Germplasm Repository, Corvallis, Oregon. Based on a presentation at the 1997 Congress on In Vitro Biology held in Washington, D.C., June 14–18, 1997.     

Effects of soil fertility and mycorrhizal infection on pollen production and pollen grain size of Cucurbita pepo (Cucurbitaceae)

The effects of soil fertility (two levels of soil nitrogen and two levels of soil phosphorus) and mycorrhizal infection on pollen production and pollen grain size were studied in two cultivars of the common zucchini (Cucurbita pepo). Overall, soil fertility and mycorrhizal infection had significant effects on traits affecting the male function of plants (staminate flower production, pollen production per flower and pollen grain size). There were also differences between the cultivars for these male traits in all three experiments. In addition, pollen grain size decreased toward the end of the growing season. In the mycorrhiza experiment, both phosphate concentration per pollen grain and total phosphate content per anther were greater but not significantly greater in the mycorrhizal plants than in the non-mycorrhizal plants. A significant negative relationship between pollen production and pollen grain size was found in the mycorrhiza and soil phosphorus experiments, indicating that there was a trade-off between pollen production and pollen size. This study is the first to show that mycorrhizal infection has an effect on male function (pollen production and size) in addition to the well-documented effects on female function (fruit/seed production and size).     

Declining viability and lipid degradation during pollen storage

Declining viability of pollen during storage at 24° C in atmospheres of 40% relative humidity (RH) and 75% RH was studied, with special emphasis on lipid changes. Pollens of Papaver rhoeas and Narcissus poeticus, characterized by a high linolenic acid content, were compared with Typha latifolia pollen which has a low linolenic acid content. The rationale behind this was to answer the question of whether lipid peroxidation is involved in the rapid viability loss and reduced membrane integrity of, in particular, the unsaturated-lipid pollen types. Viability and membrane integrity degraded more rapidly at 75% RH than at 40% RH. All pollen species showed deesterification of acyl chains of lipids but no detectable peroxidation at both RH levels. Considerable amounts of lipid-soluble antioxidants were detected that did not degrade during storage. Free fatty acids and lysophospholipids were formed during storage, the effects of which on membranes are discussed. These degradation products were very prominent in the short-lived Papaver pollen. The loss of viability does coincide with phospholipid deesterification. A significant decrease of the phospholipid content occurred at 75% RH, but not at 40% RH. Based on compositional analyses of phospholipids and newly formed free fatty acids, it was concluded that the deesterification of acyl chains from the lipids occurred at random. We suggest that, due to the low water content of the pollen, free radicals rather than unspecific acyl hydrolases are involved in the deesterification process.     

不同贮藏温度下大别山五针松花粉活力的变化

大别山五针松( Pinus dabeshanensis C. Y. Cheng et Y. W. Law)为松科( Pinaceae)松属( Pinus Linn.)植物,自然种群数量极少,目前仅发现在安徽省岳西县大王沟海拔900~1300 m阴坡和半阴坡有相对集中的分布,种群规模计200余株,且多为成年个体,林下幼苗极少,自然更新困难,为中国特有珍稀树种之一[1]。大别山五针松常在每年3月份至4月份形成花芽,5月中下旬花粉成熟并散发,花粉具气囊,此时雌球花张开接受传粉,球果翌年9月成熟[2]。据作者近年的调查,大别山五针松种子败育率较高,且种子质量较低,这也是该种类濒危的重要原因之一。     

Effects of soil phosphorus on pollen production,pollen size,pollen phosphorus content,and the ability to sire seeds in Cucurbita pepo (Cucurbitaceae)

To determine the effects of soil phosphorus on pollen production, pollen grain size, phosphate concentration per pollen grain, and the siring ability of pollen, two cultivars of the common zucchini (Cucurbita pepo) were grown under two soil phosphorus conditions in an experimental garden. Overall, soil phosphorus availability had a significant effect on reproductive output through the female function and on traits affecting the male function of plants (staminate flower production, pollen production per flower, and pollen grain size). In addition, pollen produced by plants in the high phosphorus soils had a higher phosphate concentration than pollen produced by plants in the low phosphorus soils. A pollen mixture experiment revealed that pollen produced by plants in the high phosphorus treatment sired significantly more seeds than pollen produced by plants in the low phosphorus treatment. This study showed that growing conditions such as soil phosphorus can influence the size of a pollen grain and its chemical composition, which, in turn, can affect its ability to sire mature seeds.     

Reproductive isolation and hybrid pollen disadvantage in Ipomopsis

One cause of reproductive isolation is gamete competition, in which conspecific pollen has an advantage over heterospecific pollen in siring seeds, thereby decreasing the formation of F1 hybrids. Analogous pollen interactions between hybrid pollen and conspecific pollen can contribute to post-zygotic isolation. The herbaceous plants Ipomopsis aggregata and I. tenuituba frequently hybridize in nature. Hand-pollination of I. aggregata with pollen from F1 or F2 hybrids produced as many seeds as hand-pollination with conspecific pollen, suggesting equal pollen viability. However, when mixed pollen loads with 50% conspecific pollen and 50% hybrid pollen were applied to I. aggregata stigmas, fewer than half of the seeds had hybrid sires. Such pollen mixtures are frequently received if plants of the two species and F1 and F2 hybrids are intermixed, suggesting that this advantage of conspecific over hybrid pollen reduces backcrossing and contributes to reproductive isolation.     

Conservation of endemic species from the Russian Far East using seed cryopreservation

A seed response to cryopreservation has been studied in 11 endemic plant species from the Russian Far East to determine the possibility of their long-term storage. It has been shown that the cryogenic treatment does not kill seeds. The viability of 10 species did not decrease (or even increased) after their cryogenic storage. We have not revealed any deviations in the development of plants germinated from seeds stored at an extremely low temperature (−196°C). To monitor germination after long-term storage, procedures of seed germination under laboratory conditions have been developed. The results of this study contribute to the creation of low-temperature seed banks, able to conserve and restore a floral biological diversity.     

Polyester and nylon powders used as pollen diluents preserve pollen germination and tube growth in controlled pollinations

Pollen acquisition for seed production, breeding programs and supplemental pollination can be costly and difficult. The identification of dry particulates for use as pollen diluents would facilitate the use of limited amounts of pollen and aid in accurate pollen application and dispersion. Four powders - Rilsan ES, polyester, wheat flour, and Lycopodium spores - were evaluated as pollen diluents using petunia as a model system. Diluents were combined with petunia pollen at a 5:1 (v/v) ratio. Two types of studies were conducted: (1) storage studies evaluated the viability of pollen combined and held with diluent for different durations; and (2) in vivo studies evaluated pollen tube growth in the styles of flowers pollinated with pollen-diluent mixtures. Pollen germination was not affected when stored as pollen-diluent mixtures for 4 days. Slight detrimental effects on pollen germination were observed after 6 days storage with Rilsan ES powders. Pollination with all the pollen-diluent mixtures resulted in fewer pollen tubes growing in the style compared to controls diluted with heat-killed pollen instead of diluent powders. Lycopodium-pollen mixtures were the most inhibitory, providing only 8% of the tube numbers observed in controls. Pollen mixed with polyester powders, Rilsan ES powders or wheat flour had tube numbers ranging from 47 to 61% of the control, but still had 175 or more pollen tubes per style, which would be sufficient for high rates of seed set in petunia. Wheat flour-pollen mixtures tended to clump and degrade pollen flow. Rilsan ES and polyester were identified as two promising pollen diluent powders that can facilitate accurate metering and distribution of pollen, produce large numbers of pollen tubes, and maintain pollen viability under storage.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

Ripe pollen carbohydrate changes in Trachycarpus fortunei: the effect of relative humidity

Pollen of the palm Trachycarpus fortunei was kept at 25°C and relative humidities (RH) of 20, 55 and 98%. Changes in viability, water content and carbohydrates were measured over 2–17 days. Water content remained almost constant at 20 and 50% RH and increased dramatically at 98%. Pollen viability and germination rate remained almost constant over 14 days at 20% RH and decreased to about 2% after 7–9 days at 55% and to even less at 98% RH. Although the three experimental conditions were constant, qualitative and quantitative variations in pollen carbohydrates were recorded, even after pollen had lost its viability. The quantities of mono-, di- and polysaccharides varied with the period of pollen storage at the various RH. The greatest changes in glucose, fructose and sucrose content were recorded at 55 and 98% RH. At these relative humidities, maximum glucose and fructose content and minimum sucrose content occurred at maximum water content. Starch was not present in mature pollen but appeared and peaked after 7–9 days of pollen storage at 55 and 98%. Appearance of starch coincided with an increase in pectin content. PAS-positive cytoplasmic polysaccharides showed an increasing trend at 20% RH. A relation was found between pollen viability, water content and monosaccharide content. Pollen viability and germination capacity remained high at 20% RH for 14 days. At this relative humidity, pollen water, glucose and fructose contents remained almost constant, while sucrose reached its maximum value. The fluctuations of more complex carbohydrates (starch, pectins and PAS-positive cytoplasmic polysaccharides) were less easy to interpret. Changes observed under experimental conditions could simulate processes occurring in nature during pollen presentation and dispersal.     

Transgenic ryegrasses (Lolium spp.) with down-regulation of main pollen allergens

Ryegrass pollen (Lolium species) is a widespread source of air-borne allergens and is a major cause of hayfever and seasonal allergic asthma, which affect approximately 25% of the population in cool temperate climates. The main allergens of ryegrass pollen are the proteins Lol p 1 and Lol p 2. These proteins belong to two major classes of grass pollen allergens to which over 90% of pollen-allergic patients are sensitive. The functional role in planta of these pollen allergen proteins remains largely unknown. Here we describe the generation and analysis of transgenic plants with reduced levels of the main ryegrass pollen allergens, Lol p 1 and Lol p 2 in the most important worldwide cultivated ryegrass species, L. perenne and L. multiflorum. These transgenic plants will allow the study of the functional role in planta of these pollen proteins and the determination of potential for development of hypo-allergenic ryegrass cultivars.     

Pollen viability and pollen vigor

Summary Investigations were carried out to correlate pollen viability, assessed on the basis of a fluorochromatic reaction (FCR) test, with pollen vigor, assessed on the basis of the time taken for in vitro germination in pollen grains subjected to high humidity (>95% RH) and temperature (38 °C) or storage stress of Nicotiana tabacum, Agave sp., Tradescantia virginiana, and Iris sp. Both high RH and temperature, as well as storage stresses, affected pollen vigor before affecting pollen viability. The results are discussed in the light of available data on the viability and vigor of stressed pollen and of aged seeds. The need for consideration of pollen vigor, particularly in stored pollen, the inadequacy of the methods presently used, and some of the methods suitable to assess pollen vigor are elaborated.     

Cryopreservation of <Emphasis Type="Italic">Citrus</Emphasis> anthers in the National Crop Genebank of China

Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. The aim of this study was to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. The optimal warming, rehydration, and in vitro germination medium formulations affected the germination levels after pollen cryopreservation, with species- and cultivar-dependent effects. The Citrus anthers were dehydrated to the moisture content of 5–14% before LN exposure and warmed at 25 (cryopreserved Citrus anthers with a moisture content of lower than 10%) or 37°C (a moisture content of 10% or higher), then rehydrated, and cultured on medium with 150-g L^?1 sucrose, 0.1-g L^?1 boric acid, 1.0-g L^?1 calcium nitrate, 0.1-g L^?1 potassium nitrate, 0.3-g L^?1 magnesium sulfate, and 10-g L^?1 agar. After 2 yr of storage, in vitro germination levels of Citrus pollen after cryopreservation were significantly higher (> 22% for all ten cultivars) than those of samples that were stored at 4°C (0%). In vitro germination levels of pollen from six of ten cultivars after cryopreservation remained relatively high after 2 yr of storage (38–93%). The highest viability of 93% was obtained for C. cavaleriei ‘2–3’. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers.     

Loss of viability of tomato pollen during long-term dry storage is associated with reduced capacity for translating polyamine biosynthetic enzyme genes after rehydration

The possibility that a loss of pollen viability during dry storage in a freezer is caused by the reduced pollen capacity to enhance polyamine biosynthetic enzyme activity after rehydration was investigated using pollen grains of tomato (Solanum lycopersicum=Lycopersicon esculentum) stored at -30 degrees C under dry conditions for up to 42 months. Pollen grains showed normal germinability for at least 12 months in storage, but those stored for longer than 24 months exhibited a significant reduction in germinability and fruit-setting ability. This age-dependent reduction in pollen viability coincided with the extent to which the pollen lost the capacity to increase arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) activities and polyamine contents upon rehydration. Immunoblot analysis indicated that the capacity of pollen to translate ADC and SAMDC mRNAs was impaired in accordance with the loss of viability. Also, the capacity to synthesize proteins in general decreased with the increase in storage duration. The addition of 1 mM putrescine, spermidine, or spermine to incubation medium promoted germination, impregnation of pollen grains with 1 mM spermidine restored fertilization ability, and the addition of 1 mM spermidine to incubation medium promoted protein synthesis exclusively in pollen grains which had been stored for a long time. These results indicate that the reduction in viability of tomato pollen during long-term dry storage in a freezer involves a decline in the capacity to enhance gene translation for polyamine biosynthetic enzymes upon rehydration.     

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

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