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.     

In vitro germination and viability of buckwheat (Fagopyrum esculentum Moench) pollen

An in vitro method for the germination of common buckwheat pollen was developed. Pollen grains were successfully germinated in an artificial medium consisting of 0.2 g each of MnSO₄, Ca(NO₃)2.4H₂O and KNO₃, 0.04 g H₃BO₃, 15 g sucrose and 30 g polyethylene glycol (molecular weight approximately 20,000) dissolved in 100 ml of double distilled water. The viability of pollen was assessed by in vivo and in vitro germination tests at 20 °C and 25 °C over a 38 h time period. Pollen grains were collected and germinated at 4 h intervals from freshly harvested flowers grown under 16 h day length and a constant temperature. Maximum pollen viability was found 2 h and 6 h after first light when plants were maintained at 25 °C and 20 °C, respectively. Viability, as measured by germination percentage, was similar at both temperature regimes. Some pollen remained viable for approximately 34 to 38 h in intact flowers, but all pollen lost viability in less than an hour when stored at room temperature without humidity control. This revised version was published online in July 2006 with corrections to the Cover Date.     

‘曹州’木瓜传统栽培类型及特异种质花粉生活力研究

为研究‘曹州’木瓜不同栽培类型及特异种质花粉生活力的差异,用离体培养法测定10个‘曹州’木瓜花粉生活力,探索不同种质、不同花性间花粉生活力的差异。结果表明：不同种质木瓜花粉萌发速度存在一定的差异,‘玉兰’、‘剩花’、‘细皮’、‘豆青’、‘常青’、‘小狮子头’、‘矮化’木瓜花粉检测最佳时间为培养3 h,‘佛面’、‘大狮子头’和‘梨形’木瓜为培养6 h。完全花和败育花花粉生活力差异不显著;‘曹州’木瓜花粉生活力较好,萌发率值集中在80%左右,种质间存在显著差异,最低萌发率为42.9%,最高萌发率为86.1%。     

Allotetraploid hybrids produced by protoplast fusion for seedless triploid <Emphasis Type="BoldItalic">Citrus</Emphasis> breeding

In connection with crop improvement strategies for marginal regions it has been proposed to increase the outcrossing rate in barley which would presuppose high and persistent pollen viability to ensure successful cross fertilisation. The present study was designed to investigate the viability of mature pollen from extruded and non-extruded anthers of cultivated and wild barley (Hordeum vulgare ssp vulgare and ssp spontaneum, respectively) in comparison with the closely related but obligatory outcrossing species H. bulbosum. Pollen viability (PV) was assessed employing the p-phenylenediamine-peroxidase-test on pollen derived from spikes or anthers immediately after collection and after treatment at 20, 30 and 40 ^∘C for 1, 2, 4, 8 and 16 h. The latter treatment was interposed after 8 h by a 10 h period of darkness at 12 ^∘C and thus called 26 h treatment, consecutively. Initial PV was high with 98% across all genotypes and even at 40 ^∘C did not fall below 80% after 8 h. After the 26 h treatment, PV of two H. vulgare genotypes originating from semi-arid regions and of H. bulbosum fell below 60% while the other genotypes retained a PV of > 80%. Viability of pollen of extruded anthers in H. vulgare ssp. spontaneum was on average slightly but significantly lower than PV of non-extruded anthers but still remained above 90%, even after the 26 h treatment at 40 ^∘C. Pollen viability of the outcrossing species H. bulbosum ranged on a very similar level as PV of H. vulgare. Results indicate that pollen of H. vulgare retains a sufficiently high level of viability to ensure successful cross-fertilisation over a period of at least 26 h even at high temperatures of up to 40 ^∘C.     

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%.     

Establishment of reliable methods of in vitro pollen germination and pollen preservation of Brassica rapa (syn. B. campestris)

A simple and reliable method for evaluating the viability of Brassica pollen was established in which the in vitro germination rate of pollen was adopted as the index of the viability of pollen grains. Pollen grains were preincubated in an atmosphere in which the relative humidity (RH) was fixed to 52% or 66% at 20 °C for 5 hours. They were cultured for 16 hours at 25 °C in a liquid Kwack's medium (1964) supplemented with 20% sucrose, and the pH was adjusted to 8.0. They were then observed under a microscope and the number of germinating and unchanged pollen grains were counted. The germination rate of pollen was improved and stabilized by preincubation and the use of a high pH medium. More than 90% of the freshly harvested pollen grains of Brassica rapa (syn. B. campestris) germinated constantly in these conditions Undehisced anthers were collected from flowers at anthesis and dehydrated by incubation at 20 °C for 16–24 hours in an atmosphere where the RH was fixed to 15% or 32%. They were put into a plastic vial and preserved in a freezer at -20 °C. The germination percentage of the preserved pollen was scored at intervals during preservation. The germination rate of the pollen grains preserved at -20°C for 1 year was higher than 50% and the pollen proved to be efficient for seed set. Most of the seeds germinated normally. This revised version was published online in August 2006 with corrections to the Cover Date.     

Storage of sugarbeet pollen

Summary To develop the technology for long-term pollen preservation, sugarbeet pollen was collected from plants grown in the greenhouse and in the field, and was stored 1 day to 1 year at 5, -18, and -196°C. Pollen containing about 12% moisture was successfully stored in liquid nitrogen (LN₂) up to 1 year; this pollen effected fertilization of male-sterile flowers as well as freshly collected pollen. Germination of the resultant seed was good and not different from seed from fresh pollinations. Pollen stored at -18°C for 1 year did not result in as much seed set as fresh pollen, and 1 year at 5°C was essentially lethal. In vitro pollen germination served as a post-storage viability measure, provided the pollen was hydrated before germination. The methods tested in these experiments provided a relatively simple, reliable, and inexpensive means for preservation of sugarbeet pollen for breeding purposes and for preservation of genetic resources.Joint contribution of the Agricultural Research Service, USDA, and the Beet Sugar Development Foundation.     

Hybrid Tea-rose pollen. I. Germination and storage

Summary Germination and storage trials were carried out with pollen of several rose varieties. The pollen grains germinated well in a 15% sucrose solution with 40 ppm boric acid. Staining the pollen with a 0.1% tetrazolium solution and standardizing the degree of colour at which the pollen grains are counted as viable, provided a good viability estimate, simpler to carry out than in vitro germination. Germination capacity and staining ability of the pollen were greatly impeded-about halved-by dehydration during storage in desiccators at low humidity. This effect could be corrected by humidifying the pollen beforehand for about one hour, though this pre-treatment increased the percentage of germinated pollen grains more than the percentage stained. There was no difference between the two percentages in fresh or in deep-frozen pollen.Pollen stored at 1°C and high relative humidity soon lost its germination capacity: between 0 and 20% humidity a considerable proportion of the pollen remained viable for 9 months and longer. Storage for the same period in vacuum-sealed glass tubes at –24°C maintained viability as well or better and would probably prolong it further. Some of the cold-stored pollen induced a reasonable seed set after one year, a low seed set was obtained even after two years of storage at 1°C and low humidity.     

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.     

Storage of avocado pollen

Summary Avocado pollen was stored at a range of temperatures and relative humidities (RH) for up to one year and the pollen was tested for viability in vivo.Pollen stored for one month was capable of germination on the stigma and penetrating the ovule when stored at 4°C with <1,23,55 and 75% r.h. and at -196°C with 0% r.h. Most pollen samples stored at 25 and -15°C at a range of r.h. would germinate on the stigma but none would penetrate the ovule.After one year of storage, pollen at 4°C and <1 and 23% r.h. would germinate on the stigma but would not penetrate the ovule. There was no germination of pollen stored at 4°C and 55 and 75% r.h. Only pollen stored at -196°C and 0% r.h. would penetrate the ovule, but thawing and refreezing once during the year destroyed the viability.     

Cross compatibility between Abelmoschus esculentus and A. moschatus

Interspecific cross compatibility between cultivated and wild okra (Abelmoschus esculentus and A. moschatus) and pollen tube growth behaviour in the crosses among a local cultivar of A. esculentus, A. moschatus and their F₁s were studied. Fruit set was observed in all the crosses except one and seed setting was absent in two of the crosses which set fruit. All seed produced were shrivelled but F₁ plants were obtained from two crosses where cultivated okra was used as the seed parent. The F₁ plants were perennial in nature with very low pollen viability and seed set. A high percentage of pollen germination and profuse pollen tube penetration in the style were observed in the cross A. esculentus × A. moschatus but low pollen tube penetration with abnormal pollen tubes was observed in the reciprocal cross. The number of pollen tubes was very low but they appeared to be normal in the backcross A. esculentus × F₁, but were generally abnormal in the reciprocal cross. Both pre- and postzygotic barriers seemed to occur in crosses between the two species. The present studies indicate that these barriers can be overcome and desirable characters from A. moschatus transferred to cultivated okra using conventional hybridisation techniques. This revised version was published online in July 2006 with corrections to the Cover Date.     

Breeding tomato for pollen tolerance to low temperatures by gametophytic selection

Haploid selection for traits related to pollen cold tolerance in tomato was performed in segregating populations derived from a Lycopersicon esculentum × L. pennellii hybrid. BC₁ populations were obtained by combining normal and low temperature treatments on two stages of pollen development: pollen formation, and germination and pollen tube growth. F₁ hybrids were cultivated under low and normal temperatures and their pollen was used to pollinate L. esculentum plants at low and normal temperatures. The four BC₁ populations obtained were tested for the quality and quantity of pollen produced at low temperatures. The population obtained by cold treatment at both stages had a significantly improved pollen germination ability at low temperatures. The two other coldselected BC₁ populations showed no differences compared with the unselected BC population. A second cycle of pollen selection, corresponding to BC₂, was applied in order to test its persistence in the subsequent generations and the possibility to further improve the character. This second cycle showed no improvement although some plants retained the high pollen germination ability at low temperatures that was observed in the first cycle. Hence, gametophytic selection of some characters related with tomato pollen performance may be feasible, at least for the first cycle of selection.     

桃花粉活性检测和离体萌发特性的研究

果树花粉活力直接影响授粉、受精乃至座果。为了解果树品种的花粉生活力,给引种栽培和杂交育种工作创造有利条件,以桃花粉为试验材料,采用染色方法检测花粉的活性。试验结果表明,含有蓝墨水的花粉培养基,花粉萌发率可以达到64.67%,与对照相比差异不明显,因此该方法比较适合于快速检测花粉活性;固体培养基培养2个品种桃花粉,萌发率比液体培养基培养分别高25%和20%。另外温度、花粉密度、培养基中蔗糖及硼酸浓度都对花粉萌发率和花粉管长度有影响,在温度为24℃、1粒花药/100 μL、10%的蔗糖或100 mg/L的硼酸的条件下,均有利于花粉萌发和花粉管生长。     

Comparison of different pollen viability assays to evaluate pollen fertility of potato dihaploids

Summary The main obstacle in breeding potato (Solanum tuberosum L.) dihaploids is the severe limitation of male fertility. To determine pollen viability assays that correlate well to fertility in crosses, results of five different pollen viability assays were compared by correlation analysis with fruit and seed set characters in test crosses, and to pollen tube growth in situ (PL-test). The methods used were: staining the pollen cells with carmino acetic acid (CAA-test); in vitro pollen germination (PG-test); and detection of pollen staining rates after incubation with fluoresceine diacetate (FDA-test), 2-3-5-triphenyle tetrazolium chloride (TTC-test), and 5-bromo-4-chloro-3-indolyle--galactoside (X-Gal-test).The results of test crosses and pollen tube growth in situ correlated with the results of all other assays with the following ranking, from highest to lowest: enzyme activity assays (X-Gal-test, FDA-test, TTC-test), in vitro pollen germination (PG-test), and pollen staining by CAA. The newly developed X-Gal-test for monitoring -galactosidase activity showed the least variation of all assays investigated. Thus, this highly reproducible simple procedure is recommended for male fertility screening.Abbreviations B/F Berries obtained per 100 flowers - CAA Carmino acetic acid - FDA Fluoresceine diacetate - PG Pollen germination rate in vitro - PL Pollen tube growth in situ - S/B Seeds per berry - S/F Seeds per pollinated flower - TTC 2-3-5-triphenyle tetrazolium chloride - X-Gal 5-bromo-4-chloro-3-indolyle--galactoside     

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.     

美国梾木(Cornus spp.)花粉活力及萌发特性

为了了解美国梾木花粉活力及萌发特性,采用扫描电镜技术对13个美国梾木无性系花粉形态进行观测与方差分析,并采用荧光显微技术和TTC染色法对无性系2号花粉萌发特性及花粉活力进行观察。结果显示:(1)不同无性系间花粉大小差异不显著,但外壁纹饰差异较大,极面沟距的变异系数最高且遗传最为稳定。(2)花序开花率为75%时花粉活力最高,整花4℃条件下短期保存花粉活力较高。(3)授粉后,多数花粉可在柱头上正常萌发,72 h后花粉管即可进入子房。研究认为,花粉外壁及极面沟距可作为鉴定美国梾木无性系的形态指标,美国梾木花粉活力及萌发特性是抗逆及丰产杂交组合的重要保证。     

Pollen fertility among BC2 offspring of Impatiens interspecific hybrids of New Guinea and Indonesian ancestry

BC_T2 seedlings, derived from the cross Tangeglow (I. hawkeri Bull. × I. aurantiaca Teysm.) × 7851-1 (I. hawkeri Bull. × I. platypetala Lindl.) with 7851-1 as recurrent parent, were tested for presence of pollen fertility. Of 59 BC₂ seedlings, 11 were capable of in vitro pollen germination and also of effecting fertilization and subsequent seed set as pollen parent when crossed with Tangeglow as the seed-parent tester. The Spearman's rank correlation coefficient [r_s] of + 0.63 indicated a moderately good correlation of seedling ranking, based on pollen germination percent in vitro and seed set in vivo. Pollen germination of the 11 pollen-fertile BC₂ seedlings varied from a mean low of 4% to a mean high of 41%. The 3 highest pollen-germinating BC₂ seedlings also had the highest seed sets, but siblings showed a wide range in pollen-germinating ability. The best pollen germination for a seedling was 8% in the BC₁ and 41% in the BC₂. Pollen fertility in the BC₂ is discussed in relation to using interspecific hybridization in an Impatiens breeding program. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Pollen storage at low temperature as a procedure for the improvement of cold tolerance in spring rape, Brassica napus L.

The possibility of selecting spring rape for cold tolerance at the mature pollen grain stage was studied by investigating the effects of pollen storage at low temperatures on the quality of pollen grains and on the cold tolerance of the plants generated from them. Pollen treatments of F¹ hybrids affected fertilization ability much more than viability and even after 10 days storage at 3 or 10°C the pollen germination percentage was reasonably high. Pollen storage for 7 or 10 days at 3 or 10°C significantly increased the cold tolerance of F² seed germination, with 3°C being more effective. Pollen storage for a shorter time had no effect upon the number of resulting genotypes tolerant to low temperature. This approach may be successfully applied in plant breeding to enrich segregating plant populations with cold-tolerant genotypes.     

中国石蒜花粉活力测定研究

为了解中国石蒜的花粉活力及其最佳体外萌发条件,分别采用TTC染色法和离体萌发法对中国石蒜的新鲜花粉的活力进行了测定。pH 7.0,5%TTC在35℃条件下染色结果显示99%的中国石蒜花粉具有活力。对影响中国石蒜花粉萌发和花粉管伸长的3个因素（蔗糖、硼酸和氯化钙）进行单因素实验,发现三者的最适浓度分别为蔗糖150 g/L、硼酸60 mg/L、氯化钙50 mg/L。3种浓度的培养液中,中国石蒜的花粉萌发率分别为36.7%、77.6%和44.2%。在中国石蒜花粉萌发最适蔗糖浓度(150 g/L)的基础上,正交设计硼酸和氯化钙浓度,实验结果表明,其最适浓度与单因素实验相同,但花粉萌发率达87%,花粉管伸长量达897 μm,远远高于单因素实验的结果,表明三者协同作用,促进中国石蒜花粉的萌发和花粉管的伸长。上述实验证实中国石蒜花粉具有较高的活力。为中国石蒜与其他石蒜属植物的种间杂交可行性提供了实验依据。