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.     

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.     

Genetics of the viability of pollen grain produced at low temperatures inLycopersicon Mill.

Summary The different viabilities of pollen produced at low temperatures in intra- and inter-specific crosses of tomato were studied. Cultivars Red Top, Moneymaker, and Marroqui were crossed with cultivar E-15 and these four cultivars were hybridized with lines of the wild speciesL. pimpinellifolium PE-13,L. parviflorum PE-52,L. pennellii PE-47, andL. hirsutum PE-37 and PE-41. A six-generation family of the Moneymaker x PE-47 cross was obtained to carry out a more detailed genetical study of pollen grain viability at low temperatures. Pollen grain viability was evaluated during the winter via acetocarmine staining. When the parents were compared with their F₁, the intra-specific tomato crosses showed dominance to better-quality pollen, theL. esculentum x L. pimpinellifolium inter-specific crosses showed positive heterosis, while the crosses ofL. esculentum with the tolerant speciesL. pennellii andL. hirsutum showed intermediate inheritance. However, in theL. esculentum x L. pennellii family, the dominance and the non-allelic interactions (homozygosis x homozygosis) were the most important factors, so that dominance to better viability at low temperatures appeared to be the general mode of inheritance. Genetical control of pollen grain viability at low temperatures seemed to be polygenetic.     

Cytochemical investigation of long-term stored maize pollen

Summary Maize pollen quality was investigated after long-term storage both in a refrigerator and in liquid nitrogen by a combination of viability tests and cytochemical methods. Determination of the activities of a number of enzymes involved in important metabolic pathways was carried out. Quinone formation was also studied, as some products of secondary metabolism affect pollen grain viability. One year of pollen storage in liquid nitrogen had little effect on the activities of oxidoreductases and hydrolases and had no significant effect on pollen grain viability evaluated by acetocarmine, neutral red and acridine organe. Only the FCR test showed slightly decreased viability. After one and two years of storage in a refrigerator, pollen grain viability, tested using acetocarmine, neutral red and acridine orange, did not change substantially. Simultaneously the FCR test showed a considerable decrease in pollen grain viability. Long-term storage in a refrigerator resulted in the loss of cytochrome oxidase activity and rise of alcohol dehydrogenase, lactate dehydrogenase, peroxidase and polyphenoloxidase activities as well as of quinone formation.Abbreviations ADH Alcohol dehydrogenase - DOPA L-3,4-Dihydroxyphenylalanine - FCR Fluorochromatic reaction - FDA Fluorescein diacetate - GDH Glutamate dehydrogenase - IDH Isocitrate dehydrogenase - LDH Lactate dehydrogenase - NADI reaction with -NAphthol and DImethyl-p-phenylenediamine hydrochloride - 6-PGDH 6-phosphogluconate dehydrogenase     

Variation in pollen viability in the onion (Allium cepa L.)

Summary The pollen viability of onions in a glasshouse was recorded from May to October 1975, using the fluorescein test. The average viability was 60–95% for most of this period but fell to less than 1% during the last two weeks of August. There was great variation in pollen viability between anthers within a flower and between flowers within a head. Attempts to induce pollen inviability by low temperature treatments at various stages of inflorescence development were unsuccessful. Low levels of pollen inviability appear to be a characteristic feature of onions, but the high level of inviability which was found both in this and in a previous season was associated particularly with the August period.     

Cryopreservation of pollen from two rose cultivars

Summary An investigation was undertaken on the storage characteristics of pollen collected from two English rose cultivars. A rapid decline in viability was observed in pollen stored at +4° C and –20° C, whereas the viability of pollen, stored at ultra-low temperature (–196° C), remained constant. Cryopreserved pollen was shown to retain its ability for fertilisation. The effects of the stage of flower development and anther dehiscence were assessed on both pre-and post-cryopreservation viabilities. Successful long-term storage of pollen will facilitate hybridisation of rose species and cultivars that do not flower synchronously.     

Viability and storage of bromeliad pollen

Several bromeliad species from two different subfamilies, were used to develop a reliable method to evaluate pollen viability. Pollen germination on a medium containing 20% sucrose, 0.001%H₃BO₃ and 0.5% agar was comparable to germination on a compatible stigma. Maximum germination was reached within 2 to 10 hours depending on the species. Based on this test, six species were considered as being good pollen donors with germination percentages between 49%and 83%. Furthermore, pollen from these species and cultivars could be stored in liquid nitrogen (–196 ^°C) without a considerable loss of viability. For all species, a dehydration period of 4 hours prior to cryopreservation and a rehydration period of 1 hour after cryostorage were essential. Greenhouse humidity influenced anther moisture content and cryostorability. This revised version was published online in July 2006 with corrections to the Cover Date.     

Storage of broccoli pollen in liquid nitrogen

Summary Broccoli pollen which had been stored in liquid nitrogen retained its viability, but seed produced using this pollen rapidly lost its germinability. Plants raised from this seed, and their progenies, gave no indication of genetic damage resulting from the low temperature treatment of pollen.     

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.     

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.     

A procedure suitable for in vitro pollen selection inBrassica oleracea

Summary Experiments to determine whetherBrassica oleracea pollen could effect fertilisation following incubation in liquid culture medium are reported. Pollen was incubated in vitro, collected by centrifugation and used to pollinate compatible pistils. While retaining viability and the capacity to germinate such pollen was unable to penetrate the stigma papillae. However, incubated pollen produced functional tubes following pollination of styles from which the stigmas had been removed. These tubes grew through the style to the ovary and viable seed was obtained. The potential application of this procedure in pollen selection is discussed.     

Freeze preservation of gladiolus pollen

Viability and fertility profiles of cryopreserved gladiolus pollen from 7 cultivars have shown that it is possible to use cryogenic methods for conservation and management of the haploid gene pool in this species. There was no decline in pollen viability (in vitro) levels after 1 and 10 years of cryogenic storage. Field pollinations with cryogenic stored pollen induced capsule and seed set in varying capacities. Long-term cryogenic storage of gladiolus pollen could enhance breeding efficiency through better management of the haploid gene pool resources. Pollen parents could be made available throughout the breeding programme, ensuring guaranteed supply at the time of peak stigma receptivity. A pollen cryobank facility established for this species would increase genetic diversity conservation at the haploid stage.IIHR Contribution No. 112/93     

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     

The processing of coconut pollen

Results of experiments on collection, viability testing, storage and dispatch of coconut pollen are given, and their relevance to the breeding of coconuts is briefly discussed.Pollen was obtained in large quantities following oven-drying of male flowers at 40° C. Viability decreased as drying time increased but viable grains were obtained in greater numbers after two days than after drying for a single day.Germination of pollen in vitro was better at 30° C and 35° C than at other temperatures tried; media with high gelatine concentrations (30%) seemed superior to those with less gelatine.For normal breeding purposes storage of pollen for two or three months is adequate. At low temperature, reliable storage in sealed ampoules for at least this period was obtained after further drying pollen over silica gel. Some samples retained good viability for considerably longer. Most samples stored over silica gel were still viable after 5 months, though viability was low. Over damp CaCl₂ considerable reduction in viability occurred during the first month but there was little further reduction at least up to 7 months after collection, and all samples were still viable after 18 months. Freeze-dried pollen in ampoules under vacuum had up to 40% viability after storage for one year.At room temperature, viability of pollen kept at controlled humidities over sulphuric acid solutions, though retained for longer periods than in an uncontrolled atmosphere, was only reliably maintained for about three weeks; some samples remained viable for longer periods. Freeze-drying was found to greatly increase the longevity of pollen kept at room temperature; freeze-dried pollen used in a trial shipment to New Guinea retained viability for four months and was successfully used in a number of controlled pollinations.     

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.     

The shriveling of pollinated pistils as an aid to rapid determination of Chrysanthemum pollen viability

Summary Shriveling of pistils observed one day after pollinating the dise florets of Chrysanthemum is normally followed by a good seed set 8 weeks later. When shriveling fails to appear, no seeds will be formed.It is suggested to use the easily visible pistil shriveling as a rapid in vivo test of pollen viability.     

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.     

Pollen and pollination experiments. VI. Heat resistance of pollen

Summary Pollen of dry apple, pear, lily and rose pollen was heated up to 48 h at a range of temperatures. About half or more than half of the pollen grains survived 48 h at 40 C, 24 h at 50 C, 8 16 h at 60 C. 4 8 h at 70 C, more than one hour at 80 C. and between 10 and 20 min at 90 C. Presumably, pollen able to withstand low humidity is also heat resistant, a property which may be usable to make pollen virus free through heat treatment and perhaps to overcome incompatibility.     

Use of irradiated pollen as mentor pollen to induce self-fertilization of two self-incompatible Upper Amazon cacao clones

Summary Two self-incompatible Upper Amazon cacao clones, T85/799 and T79/501, were pollinated with compatible Amelonado pollen subjected to varying doses of gamma irradiation (10–100 Gy). The proportion of flat non-viable beans to fully formed, viable beans in the pods increased with an increase in dosage of gamma rays. At 60 Gy all the beans produced were flat and non-viable, beyond this dosage fruit set was zero. Pollinating the self-incompatible cacao clones with a 1 : 1 mixture of compatible mentor pollen irradiated at 60 Gy and normal self pollen produced a mixture of flat, non-viable beans and fully-formed viable beans. Similar experiments using irradiated pollen with a marker gene suggested that the fully-formed viable beans resulted from selfing. Increasing the proportion of the radiation-treated compatible pollen in the mixture increased the number of fully-formed beans. However, when compatible pollen which had been treated either at 80 Gy or with temperatures of 35° C, 40° C and 45° C for periods of five, ten and fifteen minutes in factorial combination were mixed with self pollen, no successful pollinations were achieved. Pollen viability tests indicated that, whilst pollen treated at 60 Gy were about 50% viable, those treated at either 80 Gy or with temperatures of 35–45° C were mostly not viable. This suggests that, to overcome the incompatibility in cacao, the tubes of the mentor pollen grains used should at least grow into the style. The possible causes for overcoming the self-incompatibility in cacao are discussed.     

Storage of maize (Zea mays L.) pollen at −196°C in liquid nitrogen

Summary The water content of pollen has a decisive influence on its storability in liquid nitrogen. Pollen with an initial high water content cannot be stored successfully at extremely low temperatures, so a certain degree of drying must be carried out before storage. Provided the viability of the pollen is not significantly reduced during drying, the pollen remains viable and fertile when kept at –196°C.